Biodiversity and Phylogeny of Myxosporean Parasites (Cnidaria, Myxozoa) Infecting Fish and Annelids in Portuguese Estuaries Sónia Rocha

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Biodiversity and phylogeny of myxosporean parasites Sónia Rocha. .ICBAS (Cnidaria, Myxozoa) infecting fish and annelids in Portuguese estuaries D 2019

Biodiversity and phylogeny of myxosporean parasites (Cnidaria, Myxozoa) infecting fish and annelids in Portuguese estuaries Sónia Rocha

INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR SÓNIA RAQUEL OLIVEIRA ROCHA

BIODIVERSITY AND PHYLOGENY OF MYXOSPOREAN PARASITES (CNIDARIA, MYXOZOA) INFECTING FISH AND ANNELIDS IN PORTUGUESE ESTUARIES

Tese de Candidatura ao grau de Doutor em Ciências Biomédicas, submetida ao Instituto de Ciências Biomé- dicas de Abel Salazar da Universidade do Porto.

Instituição de acolhimento – Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), Universidade do Porto

Orientador – Doutora Graça Maria Figueiredo Casal Categoria – Professora Auxiliar Afiliação – Instituto Universitário de Ciências da Saúde - CESPU

Coorientador – Doutor Carlos José Correia de Azevedo Categoria – Professor Catedrático Jubilado Afiliação – Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto

Coorientador – Doutor Pedro Nuno Simões Rodrigues Categoria – Professor Associado com Agregação Afiliação – Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto

Esta tese é dedicada ao meu querido pai José Rocha, ao meu avô António Oliveira e à minha sogra Angelina Oliveira. É na memória do vosso amor e da vossa força que encontro a inspiração para viver plenamente a minha vida.

Agradecimentos

Entre doutorandas, por vezes ouvi dizer que elaborar uma tese de doutoramento é mais difícil do que ter um filho. Tendo a experiência de ambos posso afirmar que, de igual forma, é necessária a colaboração de uma autêntica “aldeia” para que o esforço seja bem-sucedido e os objetivos propostos alcançados. Como tal, não posso deixar de agradecer a todos que, de forma direta ou indireta, me apoiaram e motivaram nesta jornada. À minha orientadora, Professora Doutora Graça Casal, pela orientação e confiança que deposita no meu trabalho, e pela liberdade que me concedeu na tomada de decisões, permitindo-me evoluir como profissional. Ao meu co-orientador, Professor Catedrático Jubilado Carlos Azevedo, por me contagiar com o seu gosto e entusiasmo inigualável pela parasitologia. Obrigada pela disponibilidade e confiança com que me recebeu. Obrigada por todo o apoio, conselhos sábios e carinho com que me brindou ao longo dos últimos quase 10 anos. Ao meu co-orientador, Professor Doutor Pedro Rodrigues, por ter aceite co-orientar o meu projeto de doutoramento. Obrigada pelas correções e sugestões que contribuíram para a elaboração desta tese. Ao Professor Doutor Alexandre Lobo da Cunha e Professor Doutor Eduardo Rocha, que não sendo meus orientadores, me orientaram e apoiaram sempre que possível. Muito obrigada pela amabilidade com que me receberam, deixando que eu fizesse do Departamento de Microscopia uma segunda casa. Ao Professor Doutor Carlos Antunes e restantes membros do Aquamuseu do Rio Minho, pelo esforço incansável na coleta de peixe e oligoquetas do Rio Minho, sem o qual não teria sido possível realizar a maior parte do trabalho integrante desta tese. À Professora Doutora Maria João Santos, pela sua simpatia e boa-vontade em auxiliar- me sempre que necessário, mesmo em questões de última hora. Ao meu colega, Doutor Luís Filipe Rangel, pelo auxílio prestado e pelas longas conversas telefónicas que, em várias ocasiões, me ajudaram a decidir aspetos cruciais da minha tese. À Ângela, por todas as horas que dispensou comigo em viagens, amostragens, processamentos, e tantas mais tarefas que nem consigo enumerar. Digo, e não tenho dúvidas, que sem ti jamais teria conseguido terminar esta tese. Por isso, parabéns a nós duas! À Elsa, por ser uma segunda mãe para mim, sempre disponível a ajudar-me e, mais importante, a dar-me conselhos que considero valiosos e que tenho a certeza me serão úteis por toda a vida. Obrigada pela preciosa ajuda nos processamentos e nos cortes infindáveis, os quais tantas vezes foram frustrados, mas que sempre me ajudou a percepcionar com positivismo.

À Célia, por todo o apoio que me dá nas mais diversas “frentes” da minha vida, por seres uma excelente profissional e colega de trabalho, e acima de tudo por seres uma amiga inestimável. Obrigada por me ouvires e por estares sempre presente quando preciso. À Fernanda, minha companheira de viagens, por ser essa luz brilhante cheia de vida que me animou em tantas ocasiões, dando-me força para continuar, mesmo quando a “coisa estava preta”. À Cláudia, pelas sessões de fotos das tainhas e por todos os momentos de descontração em que nos rimos de tudo e de nada. À Rute, pela pessoa generosa que é, tendo-se sempre prontificado a auxiliar no que fosse necessário, mesmo não tendo tempo para executar as suas próprias tarefas, e mesmo não tendo ido ainda de lua-de-mel. À Ana Paula, pelas horas passadas à “cata da minhoca”, sempre com uma boa- disposição capaz de transformar horas verdadeiramente fastidiosas em momentos de diversão. À Paula, por funcionar como um EndNote orgânico e pela sua infindável boa vontade em ajudar, mesmo nas tarefas mais ingratas. À Susana, Ana Maria, Raquel, Tânia, Maria João, David e Tito, por todos os momentos em que, de uma forma ou de outra, me ajudaram e permitiram que desabafasse com eles as minhas frustrações, tendo recebido em resposta um positivismo revigorante. A toda a minha família e amigos, pelo apoio incondicional que me dão em todos os aspetos da minha vida, e por aturarem todos os meus problemas, alguns mais objetivos, outros simplesmente existenciais. À minha mãe, por ser uma força da vida e me ensinar que o caminho se faz sempre para a frente. Os meus sucessos são também o produto daquilo em que me moldaste. Obrigada pelas incontáveis vezes que tiveste que tomar conta do teu amado netinho para que eu pudesse ficar a trabalhar até mais tarde, ou pudesse estar “agarrada” horas a fio a um computador. Ao Miguel, por ser o meu maior pilar em tudo. Obrigada pelas revisões, pelos esquemas, pelas medidas, pelas planches, pelas formatações, pelas horas, pela vida que dedicas a mim e à nossa pequena família. Sem ti eu não teria terminado esta tese, sem ti eu não reconheceria a minha vida, sem ti eu já não seria eu. E ao meu Afonso José, que não me ajudou em nada na tese, só dificultou, mas a quem eu amo mais do que tudo na minha vida. És a minha luz e razão de fazer mais e ir mais além.

Financial support

The authors acknowledge the financial support provided by the “Fundação para a Ciência e Tecnologia” - FCT (Lisbon, Portugal) within the scope of the Ph.D. fellowship grant attributed to S. Rocha (SFRH/BD/92661/2013) through the programme QREN-POPH/FSE; as well as the Engº António de Almeida Foundation (Porto, Portugal).

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Publications

According to the relevant national legislation, the author declares that this thesis includes data that as either been published or is currently submitted for publication. As a doctoral candidate, the author was responsible for the scientific design and execution of the experimental work, the analysis and interpretation of data and writing of the original papers indicated below.

Rocha, S., Rangel, L.F., Castro, R., Severino, R., Azevedo, C., Santos, M.J. and Casal, G. (2016). Ultrastructure and phylogeny of Ceratomyxa diplodae (Myxosporea: Ceratomyxidae), from gall bladder of European seabass Dicentrarchus labrax. Diseases of Aquatic Organisms 121, 117‒128. doi: 10.3354/dao03049

Rocha, S., Rangel, L.F., Castro, R., Severino, R., Azevedo, C., Santos, M.J. and Casal, G. (2019). The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis. Journal of Invertebrate Pathology 160,33‒42. doi: 10.1016/j.jip.2018.12.001

Rocha, S., Azevedo, C., Oliveira, E., Alves, Â., Antunes, C., Rodrigues, P. and Casal, G. (2019). Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular redescription of the cryptic species Myxobolus exiguus. Parasitology 146, 479‒496. doi: 10.1017/s0031182018001671

Rocha, S., Alves, Â., Fernandes, P., Antunes, C., Azevedo, C. and Casal, G. (2019). Description of a new actinosporean prompts union of the raabeia and echinactinomyxon collective groups (Cnidaria, Myxozoa), due to overlap in actinospore morphology. Diseases of Aquatic Organisms (In Press)

Rocha, S., Alves, Â., Antunes, C., Azevedo, C. and Casal, G. (2019). Molecular data infers the involvement of a marine aurantiactinomyxon in the life cycle of the myxosporean parasite Paramyxidium giardi (Cnidaria, Myxozoa). Parasitology (In Press)

Rocha, S., Alves, Â., Antunes, C., Fernandes, P., Azevedo, C. and Casal, G. (2019). Characterization of sphaeractinomyxon types (Cnidaria, Myxozoa) from marine and freshwater oligochaetes in a Portuguese estuary, with the demise of the endocapsa collective group. Folia Parasitologica (In Press)

Rocha, S., Casal, G., Alves, Â., Antunes, C., Rodrigues, P. and Azevedo, C. (2019). Myxozoan (Cnidaria, Myxozoa) biodiversity in mullets (Teleostei, Mugilidae) unravels hyperdiversification of Myxobolus (Cnidaria, Myxosporea). Parasitology Research (In Press)

Rocha, S., Azevedo, C., Alves, Â., Antunes, C. and Casal, G. (2019). Morphological and molecular characterization of myxobolids (Cnidaria, Myxozoa) infecting cypriniforms (Actinopterygii, Teleostei) endemic to the Iberian Peninsula. Parasite (Under review)

Rocha, S., Rangel, L.F., Casal, G., Azevedo, C., Rodrigues, P. and Santos, M.J. (2019). Molecular-based inferences confirm the involvement of sphaeractinomyxon in the life cycle of mugiliform-infecting Myxobolus species (Cnidaria, Myxosporea). (To be submitted)

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Abstract

Myxozoans are a highly diversified group of cnidarian endoparasites with a complex life cycle that alternates between two stages: a myxosporean stage that develops in a vertebrate intermediate host, mainly fish; and an actinosporean stage that develops in an invertebrate definitive host, namely an oligochaete or polychaete. For a long time, interest in myxozoans has been mostly associated with their recognized economic importance, given that some species are known to cause emerging diseases in wild and reared populations, with negative impact in fisheries and aquaculture industries. More recently, the unraveling of their extraordinary evolutionary history boosted interest in the group, given that their interactions probably represent important drivers of the evolution of parasitism in early diverging metazoans. Moreover, the increasing number of publications describing new species suggests that myxozoan biodiversity is greatly underestimated and that these parasites might be major ecological players in aquatic ecosystems. Despite fishing and aquaculture constituting major economic activities in Portugal, a review of the available literature showed that few studies have targeted the myxozoan community inhabiting the extensive aquatic resources of this country. The present thesis aimed to tackle this issue by pursuing myxozoan surveys in three Portuguese estuaries, namely those of the Rivers Minho, Douro and Alvor, from which little or no information was previously available. Fishes and annelids were sampled and screened for the detection of myxozoan development in tissues and internal cavities. The results revealed a previously unknown rich diversity of this cnidarian group, with a total of 21 myxosporean species (17 new) and 15 actinosporean stages (13 new) being reported from fishes and oligochaete hosts, respectively. With the exception of Ceratomyxa auratae from the gall bladder of gilthead seabream Sparus aurata Linnaeus, 1758 in the Alvor estuary, all other new species records refer to members of the family Myxobolidae found infecting fish in the Minho estuary. Myxobolus arcasii n. sp., M. duriensis n. sp. and Thelohanellus paludicus n. sp. are described from three distinct species of Iberian endemic cypriniforms, respectively from the “bermejuela” Achondrostoma arcasii (Steindachner, 1866), the Northern straight-mouth nase Pseudochondrostoma duriense (Coelho, 1985) and from the Southern Iberian spined-loach Cobitis paludica (de Buen, 1930). The remaining 13 species, all belonging to the genus Myxobolus, were found to infect either the thinlip grey mullet Chelon ramada (Risso, 1827), the thicklip grey mullet Chelon labrosus (Risso, 1827) or the flathead grey mullet Mugil cephalus Linnaeus, 1758. The previously known species Ceratomyxa diplodae Lubat et al., 1989, Myxobolus pseudodispar Gorbunova, 1936, M. exiguus Thélohan, 1895 and Ellipsomyxa mugilis (Sitjà-Bobadilla and Alvarez-Pellitero, 1993) were also observed among the fish specimens analysed. New species were described and taxonomically allocated based on the combined

analysis of several criteria, including myxospore morphology, host specificity, site of infection and molecular data of the SSU rDNA gene. Comprehensive re-descriptions were also provided for C. diplodae Lubat et al., 1989 sensu Sitjà-Bobadilla and Alvarez-Pellitero, 1993 infecting European seabass Dicentrarchus labrax Linnaeus, 1758, and M. exiguus infecting C. ramada, given that the scarcity of data provided in their original descriptions led them to become potentially cryptic species complexes. Considering the enormous amount of Myxobolus spp. found in C. ramada and other mullet hosts, a detailed and critical review of all mugiliform- infecting myxobolids is further provided and will certainly prove to be extremely useful for establishing reliable taxonomic comparisons in future species descriptions of the group. Overall, the unreliability of using only morphological criteria for species differentiation, as well as for the distinction of actinosporean types and collective groups, was reinforced. Thirteen new types are described from the raabeia (1 type), aurantiactinomyxon 81 type) and sphaeractinomyxon (11 types) collective groups, but without the usage of molecular data could have been easily misidentified amongst themselves and in relation to previously known types. Moreover, the recognition of single types displaying overlap of morphological features that in the past have been used for the differentiation of distinct collective groups, lead to the demise of the echinactinomyxon, endocapsa and tetraspora collective groups. The first are united with raabeia and the two latter transferred to sphaeractinomyxon. Phylogenetic analyses of the the SSU rDNA gene provided insight into the evolutionary patterns and drivers of the myxosporean groups analysed, either giving support or discrediting previously known assumptions. The phylogenetic analysis of C. auratae refuted the previously proposed common ancestry and geographically driven evolution of sparid-infecting Ceratomyxa spp., while that of C. diplodae questioned the strict host specificity that it is generally accepted for the members of the genus Ceratomyxa, namely by reinforcing the potentially cryptic nature of this species. Overall, the molecular data currently available for Ceratomyxa was shown to be insufficient to unravel the evolutionary paths in place. In turn, the phylogenetic analysis of cypriniform- and mugiliform-infecting myxobolids revealed clustering patterns that mostly reflected the evolutionary radiation of their hosts. While the formation of several leuciscid- and cyprinid-infecting subclades revealed that myxobolids entered different families of the order Cypriniformes multiple times during their evolution, the clustering of all mugiliform-infecting Myxobolus spp. within a single clade suggested a monophyletic origin of this group, establishing a clear parallelism to the monophyly of the order Mugiliformes. The positioning of all sphaeractinomyxon types with available molecular data (including former endocapsa and tetraspora) within this monophyletic clade of mugiliform- infecting Myxobolus, revealed a potential correlation between these morphotypes as counterparts of a common life cycle. This hypothesis was ultimately confirmed by the genetic matches obtained between Myxobolus spp. newly reported from the Minho estuary and three

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of the marine sphaeractinomyxon types found in the Douro estuary. The congruence of this novel myxosporean/actinosporean association contradicts the overall reported lack of agreement between myxosporean genera and actinosporean morphotypes. The evolutionary success of this association is evidenced by the hyperdiversification of Myxobolus in mullet hosts, which is suggested to correlate with the ecological plasticity of mullets, as well as with the effectiveness of the Myxobolus and sphaeractinomyxon spore morphotypes in promoting dissemination and the invasion of their respective hosts. A life cycle inference was further demonstrated between the aurantiactinomyxon type found infecting the marine oligochaete Tubificoides pseudogaster (Dahl, 1960) in the Minho estuary and the eel-infecting Paramyxidium giardi (Cépède, 1906) Freeman and Krist- mundsson, 2018. In light of the associations established in these newly recognized life cycles, the presence of a single actinospore type in both freshwater and marine oligochaete hosts could be clarified by their usage of a migratory fish as vertebrate host; thus strengthening the contention that the acquisition of fish as second hosts was crucial in enabling myxosporeans to cross environmental barriers and conquer new habitats. Acknowledging the diversity of oligochaete species identified as hosts for the new actinosporean types in all three estuaries, as well as in previous studies, the family Naididae Ehrenberg, 1828 is suggested to have played a preponderant role in the settlement and evolution of myxosporeans in estuarine and marine habitats. This apparently successful parasite/host relationship is hypothesized to correlate primarily with the cosmopolitan nature and high availability of naidids in aquatic environments worldwide. Nonetheless, the body of knowledge currently available for myxosporean-annelid interactions is patchy and requires for future studies to recognize the abiotic and biotic factors shaping these relationships. In conclusion, this work reinforces the importance of investing in the continuous research of the unexplored biodiversity of myxosporeans, not only in estuaries, but in aquatic ecosystems in general. This will certainly allow the identification of new life cycle associations and factors mediating myxosporean-host interactions, which are fundamental for recognizing the origin, diversification and evolutionary patterns of these ancient group of parasites – the oldest among metazoans and an example of evolutionary success.

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Resumo

As espécies do grupo Myxozoa são endoparasitas cnidários altamente diversificados e possuidores de um ciclo de vida complexo, durante o qual ocorre alternância entre dois estádios de vida: um estádio de mixosporídio que se desenvolve num hospedeiro intermediário vertebrado, comumente um peixe; e um estádio de actinosporídio que se desenvolve num hospedeiro definitivo invertebrado, nomeadamente um oligoqueta ou um poliqueta. Durante muito tempo, o interesse no estudo dos mixozoários esteve, principalmente, associado à sua reconhecida importância económica, uma vez que este grupo engloba várias espécies causadoras de parasitoses emergentes em populações de peixe selvagem e de cultivo, tendo, por isso, impacto negativo nas indústrias de pesca e de aquicultura. Mais recentemente, a descoberta da extraordinária história evolutiva deste grupo incrementou o interesse científico no seu estudo, por revelar as suas interações como sendo potencialmente representativas dos fatores que impulsionaram a evolução do parasitismo entre metazoários primitivos. Também, a publicação contínua de numerosos trabalhos que descrevem novas espécies, demonstra que a biodiversidade dos mixozoários se encontra muito subestimada, e que estes parasitas podem assumir grande importância na consciencialização de questões ecológicas existentes em ecossistemas aquáticos. Não obstante a indústria pesqueira e de aquicultura constituírem atividades económicas importantes em Portugal, uma revisão da literatura disponível demonstrou que poucos estudos tiveram como alvo reconhecer a comunidade mixozoária que habita os extensos recursos aquáticos do nosso país. A presente tese pretendeu abordar esta questão por efetuar pesquisa de mixozoários em três estuários portugueses, nomeadamente, nos respeitantes aos rios Minho e Douro e ria de Alvor, dos quais pouca ou nenhuma informação estava previamente disponível. Neste contexto, peixes e anelídeos foram amostrados para a deteção de infeção por parasitas mixozoários nos tecidos e cavidades internas. Os resultados revelaram uma biodiversidade rica e previamente desconhecida deste grupo de cnidários, com um total de 21 espécies da classe Myxosporea (17 novas) e 15 estádios de actinosporídeos (13 novos) aqui reportados de peixes e oligoquetas, respetivamente. Excetuando a ocorrência de Ceratomyxa auratae na vesícula biliar da dourada Sparus aurata Linnaeus, 1758 proveniente do estuário da Ria de Alvor, todos os outros registos efetuados de novas espécies, referem-se a membros da família Myxobolidae encontrados em peixes amostrados do estuário do Rio Minho. Myxobolus arcasii n. sp., M. duriensis n. sp. e Thelohanellus paludicus n. sp. são descritos a partir de três espécies distintas de cipriniformes endémicos da Península Ibérica, respetivamente, do peixe ruivaco Achondrostoma arcasii (Steindachner, 1866), da boga Pseudochondrostoma duriense (Coelho, 1985) e do verdemã do Norte Cobitis paludica (de Buen, 1930). As restantes 13 espécies, todas pertencentes ao

género Myxobolus, são descritas a partir de infeções em diferentes espécies de tainhas, nomeadamente, Chelon ramada (Risso, 1827), Chelon labrosus (Risso, 1827) e Mugil cephalus Linnaeus, 1758. As espécies Ceratomyxa diplodae Lubat et al., 1989, Myxobolus pseudodispar Gorbunova, 1936, M. exiguus Thélohan, 1895 e Ellipsomyxa mugilis (Sitjà- Bobadilla e Alvarez-Pellitero, 1993) foram também registadas entre os espécimes de peixes analisados. A descrição e posicionamento taxonómico das novas espécies foi realizada com base na análise combinada de vários critérios, incluindo morfologia dos mixosporos, especificidade do hospedeiro, sítio de infeção e informação molecular do gene da pequena subunidade ribossomal. Re-descrições detalhadas foram realizadas para as espécies C. diplodae Lubat et al., 1989 sensu Sitjà-Bobadilla e Alvarez-Pellitero, 1993 a partir de infeções no robalo Dicentrarchus labrax Linnaeus, 1758 e M. exiguus a partir de infeções na tainha C. ramada, dado que a escassez dos dados fornecidos nas suas descrições originais levou a que se tornassem complexos de espécies, potencialmente crípticas. Considerando a enorme biodiversidade de espécies do género Myxobolus encontrada nas espécies de tainha analisadas, e mais particularmente, em C. ramada, uma revisão detalhada e crítica de todos os mixobolídios parasitas de peixes mugiliformes é fornecida e certamente será extremamente útil para o estabelecimento de comparações taxonómicas em futuras descrições de espécies do grupo. Em geral, as comparações taxonómicas efetuadas, reforçam a falibilidade do uso de critérios morfológicos para estabelecer diferenciação entre espécies, bem como para a distinção entre estádios de actinosporídeos e grupos coletivos. Treze novos tipos dos grupos coletivos raabeia (1 tipo), aurantiactinomyxon (1 tipo) e sphaeractinomyxon (11 tipos) são descritos de oligoquetas de água-doce e marinhas, mas é demonstrado que, sem o uso de técnicas moleculares, poderiam ter sido facilmente identificados erradamente entre si, e em relação a tipos previamente conhecidos. Também, o reconhecimento de tipos singulares que, simultaneamente, exibem características morfológicas que são comumente utilizadas para diferenciar grupos coletivos distintos, é dado como motivo para a invalidação dos grupos coletivos echinactinomyxon, endocapsa e tetraspora. Os primeiros são unidos aos raabeia e, os dois últimos, incluídos entre os membros do grupo coletivo sphaeractinomyxon. Análises filogenéticas do gene da pequena subunidade ribossomal revelaram padrões evolutivos e condutores da evolução dos grupos de mixosporídeos analisados, apoiando ou desacreditando hipóteses anteriores. A análise filogenética da espécie C. auratae refutou a ancestralidade comum previamente proposta para as espécies do género Ceratomyxa que infetam esparídeos, bem como a suposta relevância da geografia como condutor da evolução deste grupo. Por sua vez, o posicionamento filogenético de C. diplodae, questiona a estrita especificidade ao hospedeiro que é geralmente aceite para os membros do género

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Ceratomyxa, nomeadamente, por reforçar a natureza potencialmente críptica desta espécie. Em geral, a informação molecular atualmente disponível para o género Ceratomyxa demonstra-se insuficiente para permitir o reconhecimento de trajetórias evolutivas e dos seus elementos condutores. Por sua vez, a análise filogenética dos mixobolídios parasitas de cipriniformes e de mugiliformes, revelou padrões filogenéticos que espelham a radiação evolutiva dos seus hospedeiros. A formação de várias subclades contendo separadamente espécies que parasitam leuciscídeos e ciprinídeos, revela que durante a sua evolução, os mixobolídios adquiriram múltiplas vezes os membros destas famílias e, potencialmente outras da ordem Cypriniformes, como hospedeiros vertebrados. Já o agrupamento de todos os Myxobolus que infetam tainhas numa única clade, sugere a origem monofilética deste grupo, estabelecendo um claro paralelismo à monofilia da ordem dos Mugiliformes. O posicionamento de todos os tipos de sphaeractinomyxon com dados moleculares disponíveis (incluindo antigos endocapsa e tetraspora) dentro desta clade monofilética de Myxobolus que infetam tainhas, revelou uma potencial correlação entre estes morfotipos, como contrapartes de um ciclo de vida comum. Esta hipótese foi confirmada pelo estabelecimento de correspondências genéticas entre as espécies do género Myxobolus descritas do estuário do Rio Minho, e três dos sphaeractinomyxon marinhos encontrados em oligoquetas do estuário do Douro. Reconhecida pela primeira vez neste trabalho, a congruência da associação entre estes morfotipos contradiz a falta de correspondência que é, geralmente, reportada entre os diferentes géneros de mixosporídeos e grupos coletivos de actinosporídeos. O sucesso evolutivo desta associação é evidenciado pela hiperdiversificação do género Myxobolus em hospedeiros mugilídeos, a qual se sugere correlacionar com a plasticidade ecológica desta família de peixes, bem como com a elevada eficácia dos morfotipos do género Myxobolus e do grupo coletivo sphaeractinomyxon em promover a disseminação e processos de invasão aos respetivos hospedeiros. A descrição do tipo de aurantiactinomyxon encontrado no epitélio intestinal da oligoqueta marinha Tubificoides pseudogaster (Dahl, 1960) do estuário do Rio Minho permitiu ainda reconhecer o ciclo de vida do mixosporídio da enguia Paramyxidium giardi (Cépède, 1906) Freeman e Kristmundsson, 2018, por se verificar a existência de correspondência genética entre estes dois estádios de vida. As associações estabelecidas pelos ciclos de vida demonstrados neste trabalho, permitiram compreender que a presença de um único tipo de actinosporídeo tanto em oligoquetas de água doce, como marinhas, é resultante do uso de um peixe migratório como hospedeiro vertebrado. Consequentemente, reforça-se a alegação de que a aquisição de peixes como hospedeiros secundários foi crucial para que os mixosporídeos pudessem ultrapassar barreiras ambientais e conquistar novos habitats.

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Com base na diversidade das espécies de oligoquetas identificadas como hospedeiras dos tipos de actinosporídeos encontrados nos três estuários analisados neste trabalho, bem como em estudos anteriores, sugere-se um papel preponderante da família Naididae Ehrenberg, 1828 na colonização e evolução dos mixosporídeos em ecossistemas estuarinos e marinhos. O sucesso desta relação parasita/hospedeiro parece correlacionar-se, primariamente, com a natureza cosmopolita e a elevada disponibilidade dos naidídeos em ambientes aquáticos de todo o mundo. No entanto, a informação presentemente disponível para as interações entre os mixosporídeos e os seus hospedeiros anelídeos é extremamente reduzida, e requer que estudos futuros se foquem em reconhecer os fatores abióticos e bióticos que as moldam. Concluindo, este trabalho reforça a importância do investimento na investigação contínua da biodiversidade inexplorada destes organismos, não só nos estuários, mas nos ecossistemas aquáticos em geral. Isto seguramente permitirá identificar novas associações de ciclos de vida e fatores mediadores das interações dos mixosporídeos aos hospedeiros, considerados fundamentais para o reconhecimento da origem, diversificação e padrões evolutivos destes parasitas metazoários, que são os mais antigos à face da terra e um exemplo de sucesso evolutivo.

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Table of Contents

Agradecimentos………………………………………………………………………………………. v Financial support……………………………………………………………………………………..vii Publications…………………………………………………………………………………………....ix Abstract………………………………………………………………………………………………...xi Resumo………………………………………………………………………………………………. xv Chapter I - General Introduction……………………………………………………………………..1

Myxozoa………………………………………………………………………………………. 3 Historical Overview…………………………………………………………………………...4 Taxonomic system and main criteria used for classification……………………………..6 Myxosporean diversity and morphotypes…………………………………………………..8 Myxosporean life cycles…………………………………………………………………….12 Myxosporean development………………………………………………………………...15 Drivers of myxosporean evolution…………………………………………………………18 Phylogenetic reconstruction………………………………………………………………..21 Background in Portugal……………………………………………………………………..25 References………………………………………………………………………………….. 29 Chapter II - Study aims and approach…………………………………………………………….53 Study aims……………………………………………………………………………………55 Study approach……………………………………………………………………………...55 Chapter III - Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae)…………………………………………………………………………………………….. 57 Abstract……………………………………………………………………………………… 59 Introduction…………………………………………………………………………………..59 Materials and methods……………………………………………………………………...61 Results………………………………………………………………………………………..63 Discussion……………………………………………………………………………………67 Acknowledgments………………………………………………………………………….. 73 References………………………………………………………………………………….. 74 Chapter IV - Ultrastructure and phylogeny of Ceratomyxa diplodae (Myxosporea: Ceratomyxidae), from gall bladder of European seabass Dicentrarchus labrax………………79 Abstract……………………………………………………………………………………… 81 Introduction…………………………………………………………………………………..81 Materials and methods……………………………………………………………………...83 Results………………………………………………………………………………………..85

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Discussion……………………………………………………………………………………88

Acknowledgments…………………………………………………………………………...94

References…………………………………………………………………………………...94 Chapter V - The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis………………….101 Abstract……………………………………………………………………………………..103 Introduction…………………………………………………………………………………103 Materials and methods…………………………………………………………………….105 Results………………………………………………………………………………………107 Discussion…………………………………………………………………………………. 114 Acknowledgments………………………………………………………………………… 119 References………………………………………………………………………………… 120 Chapter VI - Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular re-description of the cryptic species Myxobolus exiguus…………………………………………………………………….....127 Abstract……………………………………………………………………………………..129 Introduction…………………………………………………………………………………129 Materials and methods…………………………………………………………………….131 Results………………………………………………………………………………………133 Discussion…………………………………………………………………………………. 138 Acknowledgments………………………………………………………………………… 151 References………………………………………………………………………………… 151 Chapter VII - Description of a new actinosporean prompts union of the raabeia and echinactinomyxon collective groups (Cnidaria, Myxozoa), due to overlap in actinospore morphology………………………………………………………………………………………….163 Abstract……………………………………………………………………………………..165 Introduction…………………………………………………………………………………165 Materials and methods…………………………………………………………………….167 Results………………………………………………………………………………………170 Discussion…………………………………………………………………………………. 177 Acknowledgments………………………………………………………………………… 188 References………………………………………………………………………………… 188 Chapter VIII - Molecular data infers the involvement of a marine aurantiactinomyxon in the life cycle of the myxosporean parasite Paramyxidium giardi (Cnidaria, Myxozoa)………………195 Abstract……………………………………………………………………………………..197 Introduction…………………………………………………………………………………197

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Materials and methods…………………………………………………………………….200 Results………………………………………………………………………………………201 Discussion…………………………………………………………………………………. 205 Acknowledgments………………………………………………………………………… 210 References………………………………………………………………………………… 210 Chapter IX - Characterization of sphaeractinomyxon types (Cnidaria, Myxozoa) from marine and freshwater oligochaetes in a Portuguese estuary, with the demise of the endocapsa collective group……………………………………………………………………………………..219 Abstract……………………………………………………………………………………..221 Introduction…………………………………………………………………………………221 Materials and methods…………………………………………………………………….223 Results………………………………………………………………………………………225 Discussion…………………………………………………………………………………. 234 Acknowledgments………………………………………………………………………… 239 References………………………………………………………………………………… 240 Chapter X - Myxozoan (Cnidaria, Myxozoa) biodiversity in mullets (Teleostei, Mugilidae) unravels hyperdiversification of Myxobolus (Cnidaria, Myxosporea)…………………………245 Abstract……………………………………………………………………………………..247 Introduction…………………………………………………………………………………247 Materials and methods…………………………………………………………………….250 Results………………………………………………………………………………………253 Discussion…………………………………………………………………………………. 276 Acknowledgments………………………………………………………………………… 286 References………………………………………………………………………………… 286 Chapter XI - Morphological and molecular characterization of myxobolids (Cnidaria, Myxozoa) infecting cypriniforms (Actinopterygii, Teleostei) endemic to the Iberian Peninsula……….. 295 Abstract……………………………………………………………………………………..297 Introduction…………………………………………………………………………………297 Materials and methods…………………………………………………………………….299 Results………………………………………………………………………………………301 Discussion…………………………………………………………………………………. 311 Acknowledgments………………………………………………………………………… 318 References………………………………………………………………………………… 318 Chapter XII - Molecular-based inferences confirm the involvement of sphaeractinomyxon in the life cycle of mugiliform-infecting Myxobolus species (Cnidaria, Myxosporea)…………..327 Abstract……………………………………………………………………………………..329 Introduction…………………………………………………………………………………329

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Materials and methods…………………………………………………………………….332 Results………………………………………………………………………………………334 Discussion…………………………………………………………………………………. 340 Acknowledgments………………………………………………………………………… 345 References………………………………………………………………………………… 345 Chapter XIII - General Discussion………………………………………………………………..355 Myxosporean biodiversity…………………………………………………………………357 Taxonomic descriptions and species re-descriptions………………………………….363 Phylogeny…………………………………………………………………………………..366 Life cycle inferences and novel myxosporean/actinosporean associations…………369

Myxosporean-annelid interactions………………………………………………………..371 References………………………………………………………………………………… 373 Chapter XIV - Conclusion and Future Perspectives…………………………………………….383 Conclusion and Future Perspectives…………………………………………………….385 References………………………………………………………………………………… 389

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Chapter I

General Introduction

Chapter I | General Introduction

Myxozoa

Myxozoans are cnidarian endoparasites that mainly infect aquatic invertebrates and vertebrates as definitive and intermediate hosts, respectively. Diverse and widely distributed, these parasites are important components of ecosystems, and have been previously estimated to represent ca. 18% of the presently known cnidarian diversity (Zhang, 2011). However, the increasing number of publications describing new species, clearly indicates that myxozoan diversity has been greatly underestimated and may surpass that of its free-living relatives (Okamura et al., 2015). Interest in this group of parasites has been intrinsically linked to their considerable ecological, economic and even medical importance. Some species are known to cause emerging diseases that impact wild and reared populations (e.g. Ceratonova shasta, Enteromyxum leei, Henneguya ictaluri, Myxobolus cerebralis, Kudoa thyrsites and Tetracapsuloides bryosalmonae), with significant economic losses having been reported in aquaculture and fishery industries due to impaired growth, decreased body condition, loss of breeders, stock depletion and decreased marketability of fish carcasses, in addition to the costs associated with treatments and disinfection (e.g. Diamant et al., 1994; Kent et al., 1994b; Pote et al., 2000; Yanagida et al., 2004; Foott et al., 2007; Hallett and Bartholomew, 2012; Henning et al., 2013; Sarker et al., 2015; Marshall et al., 2016; Kotob et al., 2017). Human health may be challenged by these parasites when raw infected fish are consumed by immunocompromised individuals (McClelland et al., 1997; Boreham et al., 1998; Lebbad and Willcox, 1998; Moncada et al., 2001; Kawai et al., 2012; Iwashita et al., 2013). The recent recognition of myxozoans as a radiation of endoparasitic cnidarians further increased interest in the group, as their interactions likely represent important drivers of the evolution of parasitism in early diverging metazoans. During their evolutionary history, myxozoans became miniaturized, incurred great morphological simplification and evolved complex life cycles by engaging in sophisticated interactions with their invertebrate and vertebrate hosts. The acquisition of vertebrates as intermediate hosts, in particular, was crucial for the successful diversification of myxozoans, as it facilitated alternative transmission and dispersion strategies that were decisive in the conquest of new habitats (Holzer et al., 2018). Fish are the most common intermediate hosts of myxozoans, but amphibians, reptiles, birds and mammals can also be infected by these parasites (Lom and Dyková, 2006). Some fish– parasitic myxozoans further evolved into hyperparasites of platyhelminth endo- and ectoparasites of fish (e.g. Overstreet, 1976; Siau, et al., 1981; Freeman and Shinn, 2011), and a muscle-dwelling Kudoa species was found in an octopus (Yokoyama and Masuda, 2001). Thus, the information currently available already demonstrates the distinct evolutionary success of this ancient parasitic group, which continued study may yet provide fundamental

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Chapter I | General Introduction insight into metazoan endoparasitism.

Historical Overview

The first report of a myxozoan parasite was performed by Jurine (1825), who discovered cysts developing in the musculature of whitefish, Coregonus fera Jurine, 1825, in Lake Léman. Several descriptions followed without taxonomic allocation of these parasites (see Shulman, 1990 and references therein), which Müller named 'psorosperms' (Müller, 1841). It was only in the early 1880s that Otto Bütschli assigned myxozoans to Sporozoa, as the subclass Myxosporidia. The Sporozoa comprised a diverse group of spore-forming unicellular parasites of animals, further including the Microsporidia Sprague, 1977 [currently recognized as fungi associated with Cryptomycota (Weiss and Becnel, 2014)] and members of the protist phylum Apicomplexa Levine, 1970. Despite some structural characters giving support to their protist nature (see Marques, 1987; Lom and Dyková, 1997), recognition of their multicellularity prompted Štolc (1899) to propose the inclusion of myxozoans within Metazoa. Several subsequent studies gave additional support to this classification, namely by showing the presence of metazoan-related structures (e.g. septate and adherens-type cell junctions) in myxozoans (e.g. Emery, 1909; Ikeda, 1912; Weill, 1938). The study of Weill (1938) was particularly important since it demonstrated the similarity existent between myxozoan polar capsules and cnidarian nematocysts. This led the author to propose myxozoans as cnidarians, potentially related to the larval stages of Polypodium hydriforme Ussov, 1885, an enigmatic cnidarian parasite of the oocytes of sturgeon and paddlefish (Acipenseridae). In 1970, Grassé attributed Myxozoa with the status of a phylum within the Metazoa, following what had previously been proposed by Lom (1969). Despite having been received with skepticism, this classification was ultimately confirmed in the 1990s, when sequencing of the SSU rDNA gene revealed myxozoans as highly modified metazoans that suffered extreme morphological simplification due to the acquisition of a endoparasitic life-style (Smoothers et al., 1994; Siddall et al., 1995; Schlegel et al., 1996). The recognition of their affinity to major metazoan groups, however, remained uncertain until very recently. Early molecular studies, based mainly on analyses of the SSU rDNA gene, usually pointed Myxozoa as the sister taxon to P. hydriforme, whenever the latter was included in the analysis. Nonetheless, the position of this clade (named Endocnidozoa) was unreliable, appearing either placed as the sister clade to Bilateria or nested within Cnidaria, depending on taxon sampling, alignment, optimization method, and the characters considered (Smothers et al., 1994; Siddall et al., 1995; Kim et al., 1999; Siddall and Whiting, 1999; Zrzavý, 2001; Zrzavý and Hypša, 2003; Evans et al., 2008, 2010). The hypothesis that myxozoans were derived cnidarians, probably related to

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Chapter I | General Introduction

Medusozoa, received broad support from several studies showing morphological and functional homology between myxozoan polar capsules and cnidarian nematocysts (e.g. Weill et al.,1938; Lom, 1990; Siddall et al., 1995; Cannon and Wagner, 2003). However, it was argued that nematocyst-like structures could have evolved prior to the divergence of bilaterians and cnidarians or even have arisen independently within these groups (Jiménez-Guri et al., 2007). The inclusion of the bizarre Buddenbrockia plumatellae Schröder, 1910 within Myxozoa fueled controversy, as its morphological features provided conflicting evolutionary signals. The classification of this rare vermiform-like endoparasite within Myxozoa was based on its morphological and biological similarities to the members of the then recently established class Malacosporea Canning et al., 2000 [specifically to the causative agent of Proliferative Kidney Disease Tetracapsuloides bryosalmonae (Monteiro et al., 2002)], and included the use of a freshwater bryozoan as host and the presence of “nematocyst-like” polar capsules in both the epidermis and infective spores, as well as of typical septate junctions. Its worm-like body and triploblastic organization, however, was more comparable to that of nematodes, despite B. plumatellae lacking a recognizable nervous system, gut and external sense organs. Also, the presence of four well-defined blocks of longitudinal muscles running its entire length, allowed this parasite to undergo nematode-like bending movements in the host's coelomic cavity, instead of the retractive and peristaltic movements commonly reported in cnidarians (Pickens, 1988). These mixed features led authors to suggest B. plumatellae as being representative of the missing link in the evolution of myxozoans from a bilaterian ancestor (Canning et al., 2002; Okamura and Canning, 2003). The characterization of bilaterian-like Hox genes in this species gave additional support to the bilaterian affinity of myxozoans (Anderson et al., 1998). Nonetheless, these results were later discredited by Jiménez-Guri et al. (2007), who further argued that the four blocks of muscle in B. plumatellae were radially distributed like in cnidarians, making it a tetraradial worm with a single axis of symmetry. In more recent years, comprehensive multigene analyses (e.g. Jiménez-Guri et al., 2007; Nesnidal et al., 2013; Feng et al., 2014; Chang et al., 2015; Holzer et al., 2018) and the identification of synapomorphic genes between Myxozoa and Cnidaria (e.g. Feng et al., 2014; Shpirer et al., 2014, 2018; Holland et al., 2011) finally confirmed the myxozoan origin within Cnidaria. Holzer et al. (2018) further suggested that Myxozoa and P. hydriforme represent not one but two independent routes to endoparasitism in the Cnidaria, consequently invalidating the “Endocnidozoa”. Thus, after a long period of controversy, myxozoans are now widely accepted as highly diversified cnidarian parasites. Another important breakthrough in myxozoan history was the unravelling of their two- stage life cycles, which involve alternation between a definitive invertebrate host and an intermediate vertebrate host. Prior to this groundbreaking discovery by Wolf and Markiw

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Chapter I | General Introduction

(1984), the different stages developing to produce spores in the fish hosts (myxospores) and in the annelid hosts (actinospores) were regarded as separate entities belonging to independent groups of parasites – the classes Myxosporea Bütschli, 1881 and Actinosporea Noble, 1980, respectively. This classification was mainly based on their presence in different host groups, but also in the distinctiveness of the morphological features found between myxospores and actinospores. Actinospores were first discovered by Antonin Štolc (1899), who described hexactinomyxon, synactinomyxon and triactinomyxon morphotypes infecting tubificid oligochaetes collected from the Vltava River in Czech Republic. This author further named the newly found group Actinomyxidia and, despite the homology found between the polar capsules of these organisms and those of myxozoans, placed it within the Dicyemida (parasites of the renal appendages of cephalopods). Nonetheless, this classification did not find support in subsequent studies that overall considered actinosporeans to be more-closely related to myxozoans (see Marques, 1984 and references therein). In 1970, Grassé placed actinosporeans within the phylum Myxozoa, where it remained as a sister taxon to Myxosporea, until recognition that actinospores and myxospores are, in fact, alternate stages of a common life cycle. Relying on the evidence provided by Wolf and Markiw (1984) and subsequent studies of experimental transmission (e.g. El-Matbouli and Hoffmann, 1989, 1993; Ruidisch et al., 1991; El-Matbouli et al., 1992a, b; Grossheider and Körting, 1992; Kent et al., 1993; Yokoyama et al. 1993a), Kent et al. (1994a) proposed the demise of the class Actinosporea, further suggesting that its generic names be retained as collective-group names, used as vernacular designations for typing actinospores developing in annelid hosts. Thus, taxa described solely on the basis of actinospores are currently referred to as types within the different collective groups.

Taxonomic system and main criteria used for classification

Currently, two classes are recognized within the unranked subphylum Myxozoa: Malacosporea and Myxosporea. The former is quite small, comprising only four species (including Buddenbrockia plumatallae) within a single order and family (Fig. 1), characterized by having retained primitive cnidarian features (e.g. epithelia and muscles). Malacosporeans infect the body cavities of freshwater bryozoans – their definitive hosts – to produce mala- cospores within spherical inactive sacs or elongated vermiform stages that, upon release into the water column, will then infect a fish as intermediate vertebrate host. In turn, the class Myxosporea is very large, currently comprising more than 2,200 species distributed among 17 families and 64 genera (Fig. 1). This number is expected to represent just a small fragment of the real biodiversity of this group, which underwent substantial radiation during their

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Chapter I | General Introduction

Figure 1. Updated taxonomy of Myxozoa.

evolutionary trajectory. Overall, myxosporeans are characterized by their derived features (e.g. lack of tissues and formation of complex spores). They utilize annelids (oligochaetes, polychaetes and sipunculids) as definitive hosts, and are able to use a broader array of vertebrate groups as intermediate hosts. Despite mainly infecting freshwater and marine fish, a few of these parasites have also been described from amphibians, reptiles, and even homeotherms (waterfowl and shrews) (Lom and Dyková, 2006). The taxonomy of myxozoans is largely based on spore morphology and morphometry, following early classification systems of the group (Kudo, 1933; Tripathi, 1948; Shulman,

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Chapter I | General Introduction

1959). Other features traditionally used for classifying myxozoan taxa include identification of the host species and habitat, tissue of infection, and even the characterization of vegetative stages. The implementation of molecular tools to the study and classification of myxozoans, however, has shown that the reliable differentiation of taxa, especially at the species- and genus-level, must require the inclusion of molecular data due to the artificiality of morphological criteria. For instance, the genera Ellipsomyxa, Myxidium, Zschokkella and Sigmomyxa have convergent morphotypes that hinder correct identification of known and new species (see Fiala, 2006; Bartošová et al., 2009; Fiala and Bartošová, 2010; Liu et al., 2010; Rocha et al., 2013b; Fiala et al., 2015b). Acknowledging that myxospore morphology-based taxonomy is mostly inconsistent with phylogenetic studies based on molecular markers (more commonly the SSU rDNA gene), numerous taxonomic revisions have been performed in the past few decades in order to resolve poly- or paraphyletic taxa. For instance, the families Pentacapsulidae, Hexacapsulidae and Septemcapsulidae were suppressed and their genera synonymized with Kudoa of the family Kudoidae, upon molecular evidence that the number of polar capsules constituted an artificial criterion for discriminating between these families (Whipps et al., 2004). The genus Kudoa remained polyphyletic only due to the inclusion of two Sphaerospora sequences, including S. dicentrarchi, which were ultimately synonymized with the genus, currently monophyletic (see Casal et al., 2019). Another example is that of the genus Polysporoplasma, suppressed due to clustering of its type species within the Sphaerospora sensu stricto lineage (Bartošová et al., 2013). The genus Leptotheca was also suppressed and its members synonymized with Ceratomyxa and Sphaerospora (Gunter and Adlard, 2010). In the other way around, for instance, the family Myxobilatidae was resurrected in order to encompass the phylogenetically related genera Acauda, Hoferellus and Myxobilatus, formerly ranked in different families (Whipps, 2011). Several new genera have also been erected to incorporate species that, whilst being morphologically similar to the morphotypes of other genera, can be differentiated on the basis of phylogenetic data and other biological data [e.g. Ceratonova and Paramyxidium (Atkinson et al., 2014; Freeman and Kristmundsson, 2018)]. Despite these advances, many myxozoan taxa remain poly- or paraphyletic; a situation that is expected to progressively be altered by the exponential increase of available molecular data.

Myxosporean diversity and morphotypes

Myxosporeans are characterized by the production of spores in both life cycle stages, as these structures are necessary for achieving transmission between vertebrate and invertebrate hosts. Spores are multicellular, comprised by two to seven external valve cells that enclose one to many infectious amoeboid cells (sporoplasms), and one to many polar capsules. The

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Chapter I | General Introduction latter are intracellular organelles, each containing an eversible polar tubule that, upon release, attaches to the host’s surface, allowing the sporoplasms (or their secondary cells – sporoplasm germ cells) to invade the host and begin infection. Despite sharing these main morphological features, myxospores and actinospores exhibit distinctive morphotypes (Lom and Dyková, 2006). Myxospores have a thick wall composed by hardened valve cells that unite along a conspicuous suture and, after release from the host's body, sink to the sediments, where they can remain infectious for annelids for months or even years due to their resistance (Hoffman and Putz, 1969; Hoffman and Markiw, 1977; El-Matbouli and Hoffmann, 1991; Lom and Dyková, 2006; Koel et al., 2010). Conversely, actinospores are short-lived and usually have inflatable valvular processes for increased floatability in the water column, so as to enhance contact with potential fish hosts (Yokoyama et al., 1993b; Xiao and Desser, 1998a; Lom and Dyková, 2006). As previously mentioned, different myxospore morphotypes are classified into distinct myxosporean genera, distinguished based on morphological features such as the number and shape of valves, presence or absence of surface ornamentations and/or caudal processes, position of the suture line, and number and position of the polar capsules (Fig. 2; Lom and Dyková, 2006). Overall, morphotypes of the family Myxobolidae (specifically belonging to the genera Henneguya, Myxobolus and Thelohanellus) are the most commonly reported from freshwater habitats, comprising about 50% of the myxosporean diversity described to date. Together, members of this family account for more than 1,100 species, the majority of which is histozoic in fish hosts. Few species are coelozoic and even fewer have been reported to occur in amphibian hosts (see Eiras, 2002; Eiras et al., 2005, 2014; Lom and Dyková, 2006;

Figure 2. Schematic drawings representative of main myxospore morphotypes. (A) Zschokkella; (B) Myxobolus; (C) Thelohanellus; (D) Henneguya; (E) Kudoa; (F) Chloromyxum; (G) Ceratomyxa; (H) Ellipsomyxa; (I) Myxidium; (J) Sphaerospora.

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Chapter I | General Introduction

Eiras and Adriano, 2012; Zhang et al., 2013). Other main myxospore morphotypes are those of the genera Ceratomyxa, Chloromyxum, Kudoa, Myxidium, Zschokkella, Sphaeromyxa and Sphaerospora; the successful evolution of which is demonstrated by their multiple origin in marine and freshwater lineages due to convergent events (Fiala and Bartošová, 2010). The remaining morphotypes represent solely about 10% of myxosporean biodiversity, being distributed among ca. 50 genera that have been rarely reported (Lom and Dyková, 2006; Fiala et al., 2015a). In turn, actinospores are generally characterized by having triradiate symmetry and valves that inflate osmotically upon release into the environment, producing valvular processes that diverge in different directions to reduce sinking. Three polar capsules and numerous sporoplasms are located anteriorly to the valvular processes. Differentiation between morphotypes is, therefore, mainly based on the shape and size of the actinospores' body and valvular processes, shape and relative position of the polar capsules, and number of secondary cells in the sporoplasm (Fig. 3). According to these criteria, actinosporean morphotypes are grouped into 21 collective groups (antonactinomyxon, aurantiactinomyxon, echinactinomyxon, endocapsa, guyenotia, helioactinomyxon, hexactinomyxon, hungactinomyxon, neoactinomyxum, ormieractinomyxon, pseudotriactinomyxon, raabeia, saccimyxon, seisactinomyxon, siedleckiella, sphaeractinomy-

Figure 3. Schematic drawings representative of some actinospore morphotypes. (A) Aurantiactinomyxon; (B, C) Neoactinomyxum as observed in apical and lateral view, respectively; (D) Raabeia; (E) Synactinomyxon; (F) Tetractinomyxon; (G) Triactinomyxon.

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Chapter I | General Introduction xon, synactinomyxon, tretractinomyxon, tetraspora, triactinomyxon and unicapsulactinomyxon), some of which were erected as genera of the former class Actinosporea (e.g. hexactinomyxon, raabeia, sphaeractinomyxon and triactinomyxon) (Lom and Dyková, 2006), while others were created in order to comprise distinct morphotypes described in more recent decades (e.g. endocapsa, saccimyxon, seisactinomyxon, tetraspora and unicapsulactinomyxon) (Hallett et al., 1999; Hallett and Lester, 1999; Rangel et al., 2011; Milanin et al., 2017; Atkinson et al., 2019). Despite sphaeractinomyxon and tetraspora sharing the same morphotype, the second was established based on the lower number of actinospores that it produces per pansporocyst: tetraspora forms groups of only four actinospores, while all others develop in groups of eight (Hallett and Lester, 1999). The validity of the usage of this criterion to differentiate between the sphaeractinomyxon and tetraspora collective groups was recently discussed by Rangel et al. (2016a). An interesting feature that some morphotypes further evolved in order to increase flutuability and enable dispersion over longer distances is the formation of spore nets. Actinospores of the antonactinomyxon, hungactinomyxon, ormieractinomyxon, siedleckiella and synactinomyxon collective groups do no exit the hosts body as isolated units, but rather as nets of eight spores attaching to each other by the tips of their valvular processes (Lom and Dyková, 2006). About 200 actinosporean types have been described to date (Lom and Dyková, 2006), but this number is expected to increase given its discrepancy to the number of known myxosporeans. Overall, the limited body of knowledge currently available for actinosporeans is mostly likely due to the little direct commercial and recreational value of their annelid hosts. In fact, research on this group only gathered momentum after Wolf and Markiw (1984) demonstrated actinosporeans as life cycle stages of potentially pathogenic myxosporeans. Moreover, their typically low prevalence of infection demands an enormous effort in collecting and examining potential hosts (McGeorge et al., 1997; Xiao and Desser, 1998a, b; Özer et al., 2002; Oumouna et al., 2003; Hallett et al., 2003; Eszterbauer et al., 2006; Marcucci et al., 2009; Rosser et al., 2014). Thus far, aurantiactinomyxon, echinactinomyxon, neoactinomyxum, raabeia and triactinomyxon morphotypes are, undoubtedly, the most commonly reported from freshwater environments worldwide (Lom and Dyková, 2006). In turn, the few studies conducted in brackish/marine environments identify the sphaeractinomyxon and tetractinomyxon morphotypes as the most common in marine oligochaetes and polychaetes, respectively (see Marques, 1984; Hallett et al., 1997, 1998, 1999; Køie et al., 2004, 2007, 2008, 2013; Karlsbakk and Køie, 2012; Rangel et al., 2009, 2016a).

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Chapter I | General Introduction

Myxosporean life cycles

The complexity of myxozoan life cycles was not appreciated until the causative agent of whirling disease, the salmonid-infecting Myxobolus cerebralis, was shown to use the freshwater oligochaete Tubifex tubifex in its life cycle (Wolf and Markiw, 1984). Prior to this revolutionary discovery, experimental transmission of myxozoans was rarely successful. Uspenskaya (1957) and Halliday (1976) reported that spores of Myxobolus cerebralis became infective after “ageing” in the sediment for about 4 months. Molnár (1979) showed transmission of Myxobolus pavlovskii from the gills of silver carp Hypophthalmichthys molitrix, but only when muddy water was used. Overall, these findings correlated parasite infectivity with muddy substrates, but the infectious agent remained unknown. The discovery of Wolf and Markiw thus represented a milestone in myxozoan research, showing that the little success obtained in early experiments was probably due to the presence of potential oligochaete hosts in the mud placed in the fish tanks. Since then, many other studies have presented direct and indirect evidence for the two-stage life cycle of myxozoans, with the parasites alternating between a definitive invertebrate host and an intermediate vertebrate host (see Eszterbauer et al., 2015). Despite some experimental studies have reported successful direct fish-to-fish transmission of myxosporeans, most relied on the deliberate, invasive transfer of parasitic stages between fish specimens (e.g. Johnson, 1980; Kent and Hedrick, 1985; Molnár and Kovács-Gayer, 1986; Körting et al., 1989; Diamant, 1997; Moran et al., 1999; Sitjà-Bobadilla et al., 2007; Estensoro et al., 2010), which can be considered as a form of “transplantation” rather than “direct transmission”. Thus far, only species of the genus Enteromyxum have been unequivocally shown as capable of direct fish-to-fish transmission, either by ingestion of infected tissues, cohabitation with infected specimens, or exposure to contaminated effluents. Nevertheless, the possibility that these species also have a “natural” invertebrate host cannot be excluded (see Eszterbauer et al., 2015 and references therein). In the same manner, direct transmission between annelid hosts has never been detected but vertical transmission from mother to daughter during paratomy has been demonstrated to occur (Morris and Adams, 2006; Atkinson and Bartholomew, 2009) Since 1984, dozens of myxosporean life cycle have been elucidated, either through holistic transmission studies or DNA sequence match between myxosporean and actinosporean counterparts (see Eszterbauer et al., 2015). Holistic transmission experiments are extremely laborious and time-consuming, namely due to difficulties in acquiring and maintaining specific hosts under laboratory conditions. Moreover, while some success has been obtained in transmitting the parasitic infection from fish to annelids (e.g. El-Mansy and Molnár, 1997a,b; El-Mansy et al., 1998; Molnár et al., 1999; Székely et al., 1999, 2001, 2002; Eszterbauer et al., 2000; Rácz et al., 2004), attempts of infecting fish with viable actinospores

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Chapter I | General Introduction have mostly failed (e.g. Székely et al., 2001). The implementation of molecular tools to the study of myxosporean life cycles has further discredited the reliability of this methodology, having proved that erroneous associations may occur as a result of mixed infections (Holzer et al., 2004; Atkinson and Bartholomew, 2009; Marton and Eszterbauer, 2011; Székely et al., 2014). Consequently, life cycle studies now mostly rely on DNA match, specifically of the SSU rDNA gene, for inferring correspondence between myxosporean and actinosporean stages. The vast majority of known myxosporean life cycles refer to species that parasitize potadromous, catadromous or anadromous fish in freshwater habitats; this includes mainly myxobolids of the genera Myxobolus, Henneguya and Thelohanellus, but also species of the genera Ceratonova, Chloromyxum, Hoferellus, Myxidium, Myxobilatus, Paramyxidium, Parvicapsula, Sphaerospora and Zschokkella (e.g. Styer et al., 1991; Grossheider and Körting, 1992; Benajiba and Marques, 1993; Kent et al., 1993; Yokoyama et al., 1993a; Bartholomew et al., 1997, 2006; Lin et al., 1999; Eszterbauer et al., 2000, 2006; Holzer et al., 2004, 2006; Kallert et al., 2005; Atkinson and Bartholomew, 2009; Marton and Eszterbauer, 2011; Székely et al., 2014). Fewer studies provide information for myxosporeans that parasitize fish in brackish/marine habitats, with a total of 8 life cycles reported among species of the genera Ceratomyxa, Ellipsomyxa, Gadimyxa, Kudoa, Ortholinea and Sigmomyxa(Køie et al., 2004, 2007, 2008, 2013; Rangel et al., 2009, 2015, 2016b, 2017; Karlsbakk and Køie, 2012). Overall, oligochaetes are the invertebrate hosts most commonly used by myxosporeans in freshwater habitats, while polychaetes appear to be the hosts of choice in brackish/marine habitats (Køie et al., 2004, 2007, 2008; Rangel et al., 2009, 2011, 2016b; Karlsbakk and Køie, 2012). Nonetheless, there are a few known exceptions: the triactinomyxon stages of Ortholinea auratae Rangel et al., 2014 and O. labracis Rangel et al., 2017 infect the marine oligochaetes Limnodriloides agnes Hrabĕ, 1967 and a Capitella spp., respectively (Rangel et al., 2015, 2017), while the tetractinomyxon stages of Ceratonova shasta (Noble, 1950) and Parvicapsula minibicornis Kent et al., 1977 use the freshwater polychaete Manayunkia speciosa Leidy, 1859 as invertebrate host (Bartholomew et al., 1997, 2006). Even though only a small fraction of myxosporean life cycles have been resolved, the information so far acquired demonstrates that there is no obvious correlation between myxospore and actinospore morphotypes (Fig. 4). In fact, several different myxospore morphotypes have been shown to share the same actinospore morphotype. For instance, the species Ceratomyxa auerbachi, Ceratonova shasta, Ellipsomyxa gobii, E. mugilis, Gadimyxa atlantica, Parvicapsula minibicornis, Sigmomyxa sphaerica and Sphaerospora dicentrarchi all have tetractinomyxon counterparts developing in polychaete hosts (Bartholomew et al., 1997, 2006; Køie et al., 2004, 2007, 2008; Karlsbakk and Køie, 2012; Rangel et al., 2009; 2016b). In the same manner, species of the genera Chloromyxum, Henneguya, Hoferellus and Thelohanellus have been correlated with actinospores of the aurantiactinomyxon morphotype

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Chapter I | General Introduction

Figure 4. Schematic drawing depicting the lack of congruence between myxosporean genera and actinosporean morphotypes: species of the genus Myxobolus have been paired with actinosporean types belonging to the aurantiactinomyxon, raabeia and triactinomyxon collective groups. developing in oligochaete hosts (e.g. Styer et al., 1991; Grossheider and Körting, 1992; Székely et al., 1998; Lin et al., 1999; Holzer et al., 2004; Eszterbauer et al., 2006; Zhao et al., 2016, 2017). These associations suggest that myxospores underwent greater morphological differentiation than actinospores during their evolution, probably as the result of adaptation to a broader array of tissues and hosts in different habitats. The production of distinct morphotypes (myxospores and actinospores) within the same life cycle evidences the considerable plasticity of myxosporean spore design and is hypothesized to correlate with optimizing transmission between vertebrate and invertebrate hosts (Fiala et al., 2015a). In fact, studies have shown that a single actinosporean genotype may produce more than one phenotype in the invertebrate host, probably representing designs that are intended for distinc fish hosts (Hallett et al., 2002; Holzer et al., 2004; Eszterbauer et al., 2006; Zhao et al., 2016). Despite the growing interest in myxosporean life cycles, several actinospore morphotypes (e.g. hexactinomyxon, hungactinomyxon and sphaeractinomyxon), as well as many myxosporean genera (e.g. Ceratomyxa, Sphaeromyxa and Sphaerospora), have never been linked to any specific counterpart, neither by experimental transmission nor by DNA match. The acquisition of increased knowledge on this subject will almost certainly prove to be

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Chapter I | General Introduction important in the understanding of myxosporean phylogenetic relationships and evolutionary drivers.

Myxosporean development

The processes involved in the transmission and sequential development of myxosporeans within their vertebrate and invertebrate hosts were the focus of several studies during the past century (e.g. Ikeda, 1912; Janiszewska, 1957; Sitjà-Bobadilla and Alvarez- Pellitero, 1993; El-Matbouli et al., 1995; El-Matbouli and Hoffmann, 1998; Kent et al., 2001; Meaders and Hendrickson, 2009; Rangel et al., 2009, 2011, 2012; Morris and Freeman, 2010; Morris, 2010, 2012). Nonetheless, some doubts remain, especially regarding the parasite’s development in the invertebrate host. Development within these hosts involves the successive occurrence of schizogony, gametogony and sporogony, generally following a common pattern regardless of the actinospore morphotype being produced, site of infection and host species (see Fig. 5). However, different studies have lead to alternative views on specific developmental stages. According to El-Matbouli and Hoffmann (1998) and Kent et al. (2001), the actinosporean stage of Myxobolus cerebralis begins when an permissive annelid species ingests the myxospores settled in the sediment. In the lumen of the annelid gut, the polar tubules extrude from the polar capsules, anchoring the myxospore to the host’s cells. The valves then open along the suture line, allowing the infectious agent – the binucleated sporoplasm – to penetrate the intestinal epithelium and initiate the schizogony phase. Both diploid nuclei undergo several divisions originating a multinucleate cell. The latter can divide to produce additional multinucleate cells or incur plasmotomy generating numerous uninucleate cells that fuse together to form binucleate cells. The gametogony phase begins with karyogamy of the binucleated cells giving rise to tetranucleated cells. Plasmotomy follows to produce a set of four cells that form the early pansporocyst, in which two somatic cells envelop two generative cells. The latter are differentiated based on size and designated α (small) and β (large) cells (Janiszewska, 1957). These cells undergo three mitotic divisions and one meiotic division, resulting in 16 haploid gametocytes and 16 polar bodies. While polar bodies are expulsed, each α gametocyte fuses with a β gametocyte to produce 8 diploid zygotes within the pansporocyst. The two enveloping somatic cells can also undergo mitotic divisions during these processes. Zygotes engage in sporogony by incurring two mitotic divisions to form sporoblasts with four cells; one cell is located centrally and the others peripherally. Peripheral cells undergo another mitotic division in order to produce one valvogenic cell and one capsulogenic cell each. In turn, the cell that is positioned centrally forms the sporoplasmic cell through a process of

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Chapter I | General Introduction

Figure 5. Schematic drawing of the life cycle of Myxobolus cerebralis, depicting the development of the myxosporean (1–16) and actinosporean stages (17–30) in the vertebrate and invertebrate host, respectively (adapted from Kent et al., 2001). 1. Actinospore attaches to the fish, allowing the sporoplasm to penetrate the host’s body. 2. Secondary cells of the sporoplasm divide by endogeny. 3–13. Presporogonic development; the replication of vegetative stages allows the infection to spread throughout the host’s body in order to reach the specific site of sporulation. 14–16. Sporogonic development. Multicellular myxospores are produced within vegetative bodies – plasmodia. 17. Mature myxospores released from the fish host are ingested by the annelid host, allowing the sporoplasm to enter the gut epithelium. 18–20. Schizogony phase. Binucleate cells are produced. 21–26. Gametogony phase. Early pansporocysts are formed by two somatic cells enveloping two generative cells. The latter undergo three mitotic divisions and one meiotic division, resulting in 16 haploid gametocytes and 16 polar bodies. Gametocytes fuse to form 8 zygotes within the pansporocyst. 27–28. Sporogony phase. Multicellular actinospores are produced within the pansporocysts. 29–30. Mature actinospores are released from the annelid host and inflate in order to enter in contact with the fish host and complete the life cycle.

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Chapter I | General Introduction endogenous cleavage, in which an inner cell (sporoplasm germ) is formed within a vegetative cell. Mitotic divisions of the inner cell give rise to a variable number of sporoplasm germs. Valvogenic cells grow thinner and adhere together, surrounding the capsulogenic cells and a portion of the sporoplasm. The latter remains naked within the pansporocyst until the final number of sporoplasm germs is reached. At the end of sporogony, 8 actinospores are usually contained within each pansporocyst, except for some rare exceptions (see Rangel et al., 2016a). Recent studies, however, propose that the first developmental stage in the annelid host is the binucleated cell, which results directly from the binucleate sporoplasm of the myxospore. Mitosis of the binucleated cell gives rise to numerous daughter cells that proliferate within the host’s body. The involvement of multinucleated schizogonic stages and uninucleated cells, precursor to the binucleate cells in actinosporean development, is refuted through evidence of misinterpretation with microsporidian co-infections (Morris and Freeman, 2010; Morris, 2012). This is supported by the lack of observation of schizogonic stages in other studies. Morris (2012) further challenges the fusing of α and β cells. According to this author, only β cells are gametic and haploid, while α cells are somatic and diploid. After the generative cells (α and β) of the initial pansporocyst divide mitotically three times, only β cells undergo meiosis twice, resulting in the production of eight haploid germ cells and 16 polar bodies. Instead of fusing, each α cell engulfs one β cell, thus forming 8 sporoblasts within the pansporocysts. The α cell then divides to originate three sporogonic cells that surround the germ cell (β cell). Mitosis of the sporogonic cells give rise to six daughter cells, three of which become the valvocapsulogenic cells, while the other three cells fuse together around the germ cell to originate the actinospore sporoplasm (sporoplasmogenesis). Valvocapsulogenic cells divide once more to produce one capsulogenic cell and one valvogenic cell each. Lastly, the valvogenic cells expand to envelop both the capsulogenic and sporoplasmic cells. In the vertebrate host, two distinct phases are accepted to occur during myxosporean development: a presporogonic phase and sporogony (see Fig. 5). Actinospores discharged from the annelid’s gut enter in contact with the fish tegument, subsequently extruding their polar tubules to allow the sporoplasm to penetrate the host’s body. Portals of entry have been shown to mostly include the gills, skin and buccal cavity (e.g. Yokoyama and Urawa, 1997; Antonio et al., 1999; Holzer et al., 2003). Infection by Henneguya ictaluri occurs via the intestine (Belem and Pote, 2001), demonstrating that the consumption of infected annelids may also serve as a route of fish infection by some myxosporeans. Presporogonic development begins with the sporoplasm undergoing endogenous cleavage to form a cell-in-cell stage that is characteristic of Myxozoa. Numerous rapid mitotic divisions of the secondary cell lead to the formation of a parasitic aggregate that compresses the nucleus of the primary cell. The secondary cells then undergo endogenous divisions to

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Chapter I | General Introduction produce new cell-doublets with and inner tertiary cell. These cells-doublets ultimately rupture the primary cell, followed by the subsequent rupture of the host’s cell, and become free in the extracellular space, allowing the infection to spread deeper and throughout the host’s body. Upon reaching the specific site of infection, the cell-doublets initiate the sporogonic development by giving rise to vegetative stages named plasmodia or pseudoplasmodia. These structures mostly result from the primary cell growth accompanied by multiple karyogamies that produce numerous vegetative nuclei. In turn, the secondary cell (former tertiary cell) divides to produce numerous generative cells. In pseudoplasmodia, there is only one vegetative nucleus and sporogony is direct, occurring through the aggregation of generative cells to produce sporogonic cells (valvogenic, capsulogenic and sporoplasmogenic) that will maturate to form just one or two myxospores, according to the development being monosporic or disporic. In large polysporic plasmodia, sporogony is mediated by the formation of pansporoblasts. Each pansporoblast is comprised by one generative cell that envelops one or two other generative cells depending if the development is monosporic or disporic. The outer cell constitutes the pericyte responsible for nutrient mediation, while the inner cells are sporogonic and divide to give rise to the valvogenic, capsulogenic and sporoplasmogenic cells. The formation of polysporic plasmodia is more common and can occur in both coelozoic or histozoic species; i.e. that develop in the body cavities or in the tissues of the host, respectively. Pseudoplasmodia are more rarely reported and are characteristic of coelozoic species. Besides representing sporogonic structures, pseudoplasmodia and plasmodia are active feeding phases, with pinocytosis being widely reported in histozoic species (e.g. Current, 1979; Current et al., 1979; Azevedo et al., 2011), and peripheral projections for increased surface observed in coelozoic species (e.g. Sitjà-Bobadilla and Alvarez-Pellitero, 1993, 2001; Canning et al., 1999; Rocha et al., 2011; Azevedo et al., 2013; Rangel et al., 2014). The processes mediating the development and maturation of the valvogenic, capsulogenic and sporoplamogenic cells in order to produce the valves, polar capsules and sporoplasm, respectively, are overall similar between different myxosporean taxa and have been detailed in several studies (e.g. Weidner and Overstreet, 1979; Desser et al., 1983; Stehr, 1986; Lom and Dyková, 1992; Canning et al., 1999; Özer and Wootten, 2001; Alvarez-Pellitero et al., 2002; Ali et al., 2003, 2007).

Drivers of myxosporean evolution

The evolutionary paths of endoparasites are greatly influenced by interactions with their hosts and, therefore, can be expected to reflect evolutionary processes of co-speciation and host-switch, as well as environmental adaptations aiming to optimize dispersion and

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Chapter I | General Introduction transmission strategies. Accordingly, drivers of myxosporean diversification have been found among biotic factors associated with host colonization and abiotic factors associated with the environment in which the spores must disperse and persist in order to effectively perpetuate the parasite's life cycle (Fiala et al., 2015a). These drivers are mirrored at different scales in myxosporean phylogeny and provide important information regarding the origin and evolution of these parasites. Up until very recently, host habitat was regarded as the main factor responsible for myxosporean evolutionary trends. Early phylogenetic analyses suggested some correspondence between major myxosporean clades and the vertebrate host environment and, therefore, divided Myxosporea into two main lineages – freshwater and marine (Kent et al., 2001), with a third main grouping being the taxon-rich Sphaerospora sensu stricto clade (Fiala, 2006; Jirků et al., 2007; Bartošová et al., 2013). Since then, multiple species were reported as exceptions to this major division, including Ceratonova shasta and several representatives of the genera Chloromyxum, Henneguya, Myxobolus, Ortholinea, Parvicapsula and Sphaeromyxa, among others (e.g. Fiala and Dyková, 2004; Jones et al., 2004; Diamant et al., 2004; Fiala, 2006; Carriero et al., 2013; Rangel et al., 2014, 2017; Rocha et al., 2014; Fiala et al., 2015c). Nevertheless, the division of myxosporeans according to the host’s habitat being freshwater or marine remained widely accepted, based on the idea that the migratory pattern of the fish hosts would most likely allow these parasites to transition between freshwater, brackish and marine habitats (Fiala, 2006). A notion supported by the evidence of host-shift between freshwater and marine environments in multiple occasions during myxosporean evolution (e.g. Fiala and Bartošová, 2010). On the other hand, based on information gained from a limited number of life cycles, Holzer et al. (2007) suggested that the main evolutionary signal driving myxosporean radiation was the invertebrate host type, considering that this character was mirrored in large-scale myxozoan phylogenies, as well as SSU rDNA secondary structure. Myxosporeans parasitize oligochaetes and polychaetes in both freshwater and marine environments. Oligochaetes are more abundant than polychaetes in freshwater environments, whereas polychaetes are more common than oligochaetes in marine environments (see Alexander et al., 2015). Accordingly, the major division in myxosporean phylogeny was hypothesized to separate between species that infect oligochaetes or polychaetes, rather than the aquatic environment of the vertebrate host being freshwater or marine. Support to this view was given by a few life cycle studies that correlated the species phylogenetic placement as exceptions within the major freshwater or marine lineages with the invertebrate host being an oligochaete or polychaete (Bartholomew et al., 1997, 2006; Rangel et al., 2015, 2017). Thus, the inclusion of marine species such as Ortholinea auratae and O. labracis in the freshwater lineage could be explained by their use of marine oligochaetes as definitive hosts, rather than

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Chapter I | General Introduction by the migratory nature of their vertebrate hosts – the gilthead seabream Sparus aurata and the European seabass Dicentrarchus labrax, respectively (Rangel et al., 2015, 2017). In the same manner, the positioning of the freshwater species Ceratonova shasta and Parvicapsula minibicornis within the marine lineage could be inferred to result from their use of the freshwater polychaete Manayunkia speciosa as invertebrate host (Bartholomew et al., 1997, 2006). Ultimately, a very recent and comprehensive co-phylogenetic study by Holzer et al. (2018) provided evidence that the main division in myxosporean lineages is predominantly defined by the invertebrate host type (oligochaetes vs. polychaetes) and less by their environment (freshwater vs. marine), without exceptions. Following this invertebrate host and freshwater/marine separation, divergences within major myxosporean lineages appear to be mostly related to the tissue of infection in the intermediate vertebrate host, thus recognizing tissue tropism as an important driver of myxosporean evolution. Accordingly, both the oligochaete- and polychaete-infecting (freshwater/marine) lineages have well-defined clades comprising exclusively coelozoic species (in the gall bladder or urinary tract) or histozoic species (Andree et al., 1999; Eszterbauer, 2004; Holzer et al., 2004; Whipps et al., 2004; Fiala, 2006; Fiala and Bartošová, 2010). On the other hand, myxospore morphology has been widely demonstrated to be of minor importance in the higher-level phylogenetic relationships of myxosporeans. Despite species with the same morphotype predominating in some clades of both lineages (e.g. Ceratomyxa, Kudoa and Myxobolus), most genera are polyphyletic due to convergency events having led to the arise of similar morphologies multiple times during myxosporean evolution (e.g. Chloromyxum, Kudoa, Myxidium, Sphaerospora and Zschokkella) (Hervio et al., 1997; Andree et al., 1999; Eszterbauer and Székely, 2004; Holzer et al., 2004; Fiala, 2006; Fiala and Bartošová, 2010). The evolutionary signals driving species clustering within the most representative clades of the main myxosporean lineages have been object of recent studies. Radiation within the Myxobolus clade has been widely shown to correlate with the vertebrate host group (order and family) more than with tissue tropism (Carriero et al., 2013). This type of clustering is not supported in other clades possibly because they have scarce molecular data available for species that have more diversified biological features, thus hindering recognition of radiation patterns related to the vertebrate host. Nonetheless, similar host-related clustering has been reported at a finer-scale from other myxosporean clades (e.g. Alama-Bermejo et al., 2011). More recently, the co-phylogenetic analyses performed by Patra et al. (2018) revealed significant phylogenetic congruence between “true” sphaerosporids and their vertebrate hosts. This is unsurprising given that the acquisition of fish as second hosts led to the rapid diversification of myxosporeans. Understanding the co-evolutionary history of these parasites and their intermediate hosts, however, may prove hard considering that their reciprocal

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Chapter I | General Introduction adaptation is received as a “mixed signal” of invertebrate and vertebrate co-phylogeny (Holzer et al., 2018). Lastly, geographic clustering at a smaller scale has further been demonstrated within some myxosporean clades (e.g. Whipps et al., 2003; Henderson and Okamura, 2004; Whipps and Kent, 2006; Alama-Bermejo et al., 2011). As previously mentioned, abiotic drivers also played a part in the evolution of myxosporeans but remain poorly understood. These drivers relate to the environmental conditions that spores face in order to achieve successful dispersion and transmission. As such, spores that must endure long dormancy periods in the sediments have evolved hardened valves, while spores that need to linger in the water column in order to potentially meet an adequate host have evolved characters that reduce sinking rates (Fiala and Bartošová, 2010; Fiala et al., 2015a; Kodádková et al., 2015).

Phylogenetic reconstruction

Phylogenetically, Myxosporea split into three well-supported lineages: an oligochaete- infecting lineage that mainly comprises species reported from freshwater habitats, a polychaete-infecting lineage that mostly contains species reported from marine habitats, and the Sphaerospora sensu stricto lineage with unknown invertebrate hosts (Fiala and Bartošová, 2010; Bartošová et al., 2013; Kodádková et al., 2015; Holzer et al., 2018). The position of the latter is uncertain; it mostly appears as sister/basal to all myxosporeans, but has also been assigned as sister to the freshwater lineage or to the marine lineage (Holzer et al., 2007; Jirků et al., 2007; Bartošová et al., 2009, 2013; Karlsbakk and Køie, 2009; Kodádková et al., 2015). It constitutes a monophyletic clade, comprising only Sphaerospora species (including the type species S. elegans) characterized by possessing long inserts in their rDNA genes and that are mostly coelozoic in the excretory system of marine and freshwater fish and amphibians (Holzer et al., 2007; Jirků et al., 2007; Bartošová et al., 2013; Patra et al., 2018). The only exceptions are Sphaerospora fugu (Tun et al., 2000) and S. molnari Lom et al., 1983, which are histozoic in the intestinal and gill epithelium of their vertebrate hosts, respectively (see Lom et al., 1983; Tun et al., 2000). Several other species of Sphaerospora, termed Sphaerospora sensu lato, cluster independently within the oligochaete- and polychaete-infecting lineages. Both these lineages comprise a great variety of myxosporean morphotypes that cluster according to tissue tropism and other fine-scale evolutionary signals, as previously mentioned. The polychaete-infecting lineage (former marine lineage) presently includes nine distinct clades: the Bipteria vetusta clade, the Ceratomyxa clade, the gall bladder clade, the urinary bladder clade, the Ceratonova clade, the Enteromyxum clade, the Gastromyxum clade, the Monomyxum clade, and the clade of multivalvulids (Fig. 6). The earliest divergent clade is that of Bipteria vetusta from the gall bladder of rabbit fish Chimaera monstrosa (Kodádková et al.,

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Figure 4. Scheme depicting the main clades composing the oligochaete- and polychaete-infecting lineages, with the Sphaerospora sensu stricto lineage forming a sister/basal clade to all other myxosporeans. Based on personal phylogenies of the SSU rDNA gene using mamixum likelihood, and on the tree topology presented in Fiala et al. (2015b).

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2015). Following split of this sublineage, phylogenetic analyses suggest the taxon-rich Ceratomyxa clade as the first basal branch of the polychaete-infecting lineage. The latter comprises gall bladder-infecting myxosporeans belonging almost exclusively to the genus Ceratomyxa, with the exception of the sphaerosporid Palliatus indecorus and the sinuolineidid Myxodavisia bulani (Fiala, 2006; Gunter et al., 2009; Alama-Bermejo et al., 2011; Fiala et al., 2015c). The remaining non-ceratomyxid species that are coelozoic in the gall bladder of marine fish constitute the gall bladder clade, which includes representatives of Auerbachia, Coccomyxa, Ellipsomyxa, Myxidium, Sigmomyxa, Sinuolinea and Zschokkella (see Heiniger and Adlard, 2014; Fiala et al., 2015c). The urinary bladder clade is also rich in morphotypes, and accounts for almost all myxosporean species that infect the urinary bladder of marine myxosporeans, including representatives of Gadimyxa, Latyspora, Parvicapsula, Schulmania, Sinuolinea, Sphaerospora and Zschokkella (a few exceptions cluster within the oligochaete- infecting lineage) (see Bartošová et al., 2011; Kodádková et al., 2014; Fiala et al., 2015c). Overall, the positioning of the gall bladder clade and urinary bladder clade, as well as that of the Ceratonova clade, have been reported as unreliable and dependent of the phylogenetic method and marker used (see Fiala et al., 2015b and references therein). The Ceratonova clade comprises solely two congeners histozoic in the intestine of their fish hosts (Atkinson et al., 2014; Fiala et al., 2015c). The remaining histozoic clades are also presently monophyletic and considered to be the most derived within the polychaete-infecting lineage. The Enteromyxum, Gastromyxum and Monomyxum clades are species-poor, while the most derived clade, the clade of multivalvulids, is taxon-rich. Members of both the Enteromyxum and Gastromyxum clades infect the digestive tract of marine fish hosts; the first occur mostly in the intestinal epithelium, while the second have the cardiac glands of the stomach wall as site of infection (see Diamant et al., 1994; Padrós et al., 2001; Palenzuela et al., 2002; Yanagida et al., 2004; Freeman and Kristmundsson, 2015). In turn, the Monomyxum clade encompasses only two hyperparasitic species that infect the parenchymal tissues of gill monogeneans from marine fish (Freeman and Kristmundsson, 2015). Finally, the clade of multivalvulids comprises all sequences available for the order Multivalvulida, dividing into two monophyletic sister clades: the Kudoa clade and the Unicapsula clade (Fiala et al., 2015c; Freeman and Kristmundsson, 2015; Kodádková et al., 2015). The monophyly of the Kudoa clade is quite recent and results from the taxonomic revision and inclusion of Sphaerospora dicentrarchi and another unnamed Sphaerospora species in the genus Kudoa (see Casal et al., 2019). The most representative clades of the oligochaete-infecting lineage (former freshwater lineage) are the hepatic biliary clade, the freshwater Chloromyxum clade, the Myxidium lieberkuehni clade, the Paramyxidium clade, the urinary clade and the Myxobolus clade (Fig.

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6). The hepatic biliary clade comprises representatives of a great variety of myxosporean genera (e.g. Chloromyxum, Cystodiscus, Myxidium, Soricimyxum, Sphaeromyxa and Zschokkella) that parasitize the hepatic biliary system of taxonomically distinct hosts, including freshwater and marine fish, amphibians, reptiles, waterfowl and terrestrial insectivorous animals. Overall, phylogenetic relationships within this clade are poorly-resolved, with the species that infect non-fish hosts mostly appearing scattered within fish-infecting taxa (Hartigan et al., 2012; Kristmundsson and Freeman, 2013). Marine taxa are represented by several Zschokkella species and a monophyletic Sphaeromyxa subclade sharing a common ancestor with Myxidium coryphaenoidium and Myxidium baueri (Kristmundsson and Freeman, 2013). Also contained within the hepatic biliary clade is Myxobolus spirosulcatus, the only myxobolid known to cluster outside the Myxobolus clade (Yokoyama et al., 2010). In turn, the Chloromyxum, Paramyxidium and Myxidium lieberkuehni clades are less taxon-rich. The first is monophyletic, comprising a few species of the genus that infect freshwater teleosts (Bartošová and Fiala, 2011; Jirků et al., 2011). The Paramyxidium clade is also monophyletic, comprising a few species reported from various tissues of fish belonging to the superorder Elopomorpha, but also numerous sequences generated from actinospores of the aurantiactinomyxon, echinactinomyxon, raabeia and synactinomyxon collective groups that most likely represent a hidden diversity of unidentified myxosporeans (Freeman and Kristmundsson, 2018). This clade is sister to the Myxidium lieberkuehni clade, which comprises species from the excretory system and gall bladder of freshwater fish hosts, including the kidney-infecting species Sphaerospora oncorhynchi and a few Chloromyxum spp. that form a well-supported subclade (Freeman and Kristmundsson, 2018). The great majority of freshwater urinary-infecting species, however, are positioned within the taxonomically heterogenic urinary clade, which includes members of the genera Acauda, Chloromyxum, Hoferellus, Myxidium, Myxobilatus, Ortholinea and Zschokkella. Ortholinea spp. parasitize the urinary bladder of marine fish and, therefore, were considered an exception to clustering according to the vertebrate host environment. As previously mentioned, recognition of the involvement of oligochaete hosts in the life cycle of O. auratae and O. labracis gave support to the phylogenetic division of myxosporeans according to the invertebrate host type (Rangel et al., 2015, 2017). Phylogenetic studies either resolve the urinary clade within or as sister to the Myxobolus clade (e.g. Fiala, 2006; Holzer et al., 2007; Jirků et al., 2007; Whipps, 2011; Karlsbakk et al., 2017; Rangel et al., 2017; Freeman and Kristmundsson, 2018). The latter clade is the most derived and largest within the myxosporean phylogenetic tree, including numerous species of the taxon-rich genera Myxobolus and Henneguya, together with representatives of Thelohanellus, Cardimyxobolus, Hennegoides and Unicauda (Carriero et al., 2013; Li and Sato, 2014; Shin et al., 2014). The species Chloromyxum careni from the renal system of amphibians also clusters

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Chapter I | General Introduction within the oligochaete-infecting lineage, possibly as an independent sublineage (Jirků et al., 2011). A well-supported clade of marine elasmobranch-infecting Chloromyxum species further clusters in a basal position to the oligochaete-infecting lineage, suggesting these species as being representative of the chloromyxid ancestor that invaded freshwaters (Fiala and Dyková, 2004; Fiala, 2006; Azevedo et al., 2009; Gleeson and Adlard, 2012; Rocha et al., 2013a). In fact, the basal phylogenetic positioning of the marine elasmobranch-infecting Chloromyxum clade, together with that of chimaera-infecting Bipteria vetusta in relation to all other clades of the polychaete-infecting lineage, and of ceratomyxids infecting sharks in relation to all its congeners, suggest that a common evolutionary ancestor of both freshwater and marine myxosporeans parasitized Chondrichthyes (Kodádková et al., 2015; Holzer et al., 2018). In addition, polychaetes have been shown to be the oldest known invertebrate hosts of myxosporeans (Holzer et al., 2018). Thus, the predominantly marine nature of ancestral vertebrate and invertebrate hosts gives support to the marine origin of myxosporeans (Holzer et al., 2018). Considering that the earliest splits in myxosporean phylogenies are coelozoic and that the most derived clades are histozoic, it has been further hypothesized that the ancestors of both lineages were coelozoic (Fiala and Bartošová, 2010). As new evidences emerge, it becomes obvious that the acquisition of further molecular data is crucial in order to fully understand the origin, evolutionary path and phylogenetic relationships of myxosporeans, the oldest metazoan parasites on Earth and an example of evolutionary success.

Background in Portugal

To the best of our knowledge, about 21 myxosporean species have been reported from fishes inhabiting Portuguese rivers and estuaries, with further 10 species known from marine hosts in coastal waters. Species of the genus Myxobolus have been the most commonly reported from freshwater stretches, while in the brackish waters of downstream estuaries, the myxosporean community appears to be composed by a broader diversity of genera, including members of Ceratomyxa, Hofferellus, Kudoa, Myxobilatus, Ortholinea, Paramyxidium and Zschokkella (Table 1). Despite no observation of Ellipsomyxa mugilis (Sitjà-Bobadilla e Alvarez-Pellitero, 1993) myxospores has been made in Portuguese grey mullets, this species is also known to occur in the Aveiro estuary, considering that its tetractinomyxon life cycle counterpart was found developing in the coelomic cavity of the marine polychaete Hediste diversicolor (Müller, 1776) in this geographic area (see Table 2). The life cycles of Ortholinea auratae Rangel et al., 2015 from gilthead seabream Sparus aurata Linnaeus, 1758, and Ortholinea labracis Rangel et al., 2017 and Kudoa dicentrarchi (Sitjà-Bobadilla and Alvarez-Pellitero, 1992) Casal et al., 2019 from European seabass Dicentrarchus labrax (Linnaeus, 1758) have also been elucidated through means of DNA

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Summary of myxosporean species reported from Portugal. from species reported myxosporeanof Summary

Table

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actinosporean types reported in Portugal. in actinosporeanreported types

Summary of of Summary

Table 2 Table

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Chapter I | General Introduction match between corresponding myxosporean and actinosporean SSU rDNA sequences. Both Ortholinea species have triactinomyxon counterparts that develop in the intestinal epithelium of marine oligochaetes: actinospores of O. auratae were found infecting Limnodriloides agnes Habrě, 1967 in European seabass ponds of a fish farm in the Alvor estuary, while the actinospores of O. labracis were reported from an undetermined species of the genus Tectidrilus Erséus, 1982 occurring in the gilthead seabream ponds of the same fish farm (Rangel et al., 2015, 2017). As in Ellipsomyxa mugilis, the actinosporean development of K. dicentrarchi takes place in the coelomic cavity of a marine polychaete (in this case an undetermined species of the genus Capitella) to produce actinospores of the tetractinomyxon collective group (Rangel et al., 2016b). In addition, 12 actinosporean types with unknown myxosporean stages have been reported in Portugal: the Sphaeractinomyxon types 1 to 10 of Rangel et al. (2016a) from the coelomic cavity of marine oligochaetes inhabiting the brackish waters of the Aveiro estuary; the Synactinomyxon of Székely et al. (2005) from the intestinal epithelium of the freshwater oligochaete Tubifex tubifex in the River Sousa; and the Unicapsulactinomyxon type of Rangel et al. (2011) from the coelomic cavity of the marine polychaete Diopatra neapolitana in the Aveiro estuary (Table 2). The myxosporean species reported from mainland coastal areas, as well as from the Azores and Madeira archipelagos, are distributed among the genera Alatospora, Bipteria, Ceratomyxa, Chloromyxum and Kudoa (see Table 1). Reports of Ceratomyxa are the most abundant, however, it is likely that the Ceratomyxa sp. reported by Santos et al. (2009) from the gall bladder of Aphanopus carbo in the mainland coast off Sesimbra and in the Azores and Madeira archipelagos is conspecific with Ceratomyxa tenuispora Kabata, 1960, a species previously described from the same site of infection and fish host in the Madeira archipelago (Costa et al., 1996; Casal et al., 2007). None of the marine species thus far reported from Portugal has its full life cycle known.

References

Afonso-Dias, I., Kalavati, C., MacKenzie, K. and MacKenzie, K.S. (2007). Three new species of Myxosporea (Bivalvulida: Ceratomyxidae: Alatasporidae) from the gall bladders of anglerfishes Lophius spp. (Teleostei: Lophiidae) in the Northeast Atlantic Ocean. Zootaxa 1466, 35–46. Alama-Bermejo, G., Raga, J.A. and Holzer, A.S. (2011). Host-parasite relationship of Ceratomyxa puntazzi n. sp. (Myxozoa: Myxosporea) and sharpsnout seabream Diplodus puntazzo (Walbaum, 1792) from the Mediterranean with first data on ceratomyxid host specificity in sparids. Veterinary Parasitology 182, 181–192.

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Alexander, J.D., Kerans, B.L., El-Matbouli, M., Hallett, S.L. and Stevens, L. (2015). Annelid- Myxosporean Interactions. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 217–234. Ali, M.A., Abdel-Baki, A.S., Sakran, T., Entzeroth, R. and Abdel-Ghaffar, F. (2003). Light and electron microscopic studies of Myxobolus stomum n. sp. (Myxosporea: Myxobolidae) infecting the blackspotted grunt Plectorhynicus gaterinus (Forsskal, 1775) in the Red Sea, Egypt. Parasitology Research 91, 390–397. Ali, M.A., Abdel-Baki, A.S., Sakran, T., Entzeroth, R. and Abdel-Ghaffar, F. (2007). Myxobolus lubati n. sp. (Myxosporea: Myxobolidae), a new parasite of haffara seabream Rhabdosargus haffara (Forsskal, 1775), Red Sea, Egypt: a light and transmission electron microscopy. Parasitology Research 100, 819–827. Alvarez-Pellitero, P., Molnár, K., Sitjà-Bobadilla, A. and Székely, C. (2002). Comparative ultrastructure of the actinosporean stages of Myxobolus bramae and M. pseudodispar (Myxozoa). Parasitology Research 88, 198–207. Anderson, C.L., Canning, E.U. and Okamura, B. (1998). A triploblast origin for Myxozoa? Nature 392, 346–347. Andree, K.B., Székely, C., Molnár, K., Gresoviac, S.J. and Hedrick, R.P. (1999). Relationships among members of the genus Myxobolus (Myxozoa: Bilvalvidae) based on small subunit ribosomal DNA sequences. Journal of Parasitology 85, 68–74. Antonio, D.B., El-Matbouli, M. and Hedrick, R.P. (1999). Detection of early developmental stages of Myxobolus cerebralis in fish and tubificid oligochaete hosts by in situ hybridization. Parasitology Research 85, 942–944. Atkinson, S.D. and Bartholomew, J.L. (2009). Alternate spore stages of Myxobilatus gasterostei, a myxosporean parasite of three-spined sticklebacks (Gasterosteus aculeatus) and oligochaetes (Nais communis). Parasitology Research 104, 1173– 1181. Atkinson, S.D., Foott, J.S. and Bartholomew, J.L. (2014). Erection of Ceratonova n. gen. (Myxosporea: Ceratomyxidae) to encompass freshwater species C. gasterostea n. sp. from threespine stickleback (Gasterosteus aculeatus) and C. shasta n. comb. from salmonid fishes. Journal of Parasitology 100, 640–645. Atkinson, S.D., Hallett, S.L., Diaz-Morales, D., Bartholomew, J.L. and de Buron, I. (2019). First myxozoan infection (Cnidaria: Myxosporea) in a marine polychaete from North America and erection of actinospore collective group Saccimyxon. Journal of Parasitology 105, 252–262. Azevedo, C., Casal, G., Garcia, P., Matos, P., Teles-Grilo, L. and Matos, E. (2009). Ultrastructural and phylogenetic data of Chloromyxum riorajum sp. nov. (Myxozoa), a

Page | 30

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parasite of the stingray Rioraja agassizii in Southern Brazil. Diseases of Aquatic Organisms 85, 41–51. Azevedo, C., Casal, G., Marques, D., Silva, E. and Matos, E. (2011). Ultrastructure of Myxobolus brycon n. sp. (Phylum Myxozoa), parasite of the piraputanga fish Brycon hilarii (Teleostei) from Pantanal (Brazil). Journal of Eukaryotic Microbiology 58, 88–93. Azevedo, C., Lom, J. and Corral, L. (1989). Ultrastructural aspects of Myxidium giardi (Myxozoa, Myxosporea) parasite of the european eel Anguilla anguilla. Diseases of Aquatic Organisms 6, 55–61. Azevedo, C., Videira, M., Casal, G., Matos, P., Oliveira, E., Al-Quraishy, S. and Matos, E. (2013). Fine structure of the plasmodia and myxospore of Ellipsomyxa gobioides n. sp. (Myxozoa) found in the gallbladder of Gobioides broussonnetii (Teleostei: Gobiidae) from the lower Amazon river. Journal of Eukaryotic Microbiology 60, 490–496. Bartholomew, J.L., Atkinson, S.D. and Hallett, S.L. (2006). Involvement of Manayunkia speciosa (Annelida: Polychaeta: Sabellidae) in the life cycle of Parvicapsula minibicornis, a myxozoan parasite of Pacific salmon. Journal of Parasitology 92, 742– 748. Bartholomew, J.L., Whipple, M.J., Stevens, D.G. and Fryer, J.L. (1997). The life cycle of Ceratomyxa shasta, a myxosporean parasite of salmonids, requires a freshwater polychaete as an alternate host. Journal of Parasitology 83, 859–868. Bartošová, P. and Fiala, I. (2011). Molecular evidence for the existence of cryptic species assemblages of several myxosporeans (Myxozoa). Parasitology Research 108, 573– 583. Bartošová, P., Fiala, I. and Hypša, V. (2009). Concatenated SSU and LSU rDNA data confirm the main evolutionary trends within myxosporeans (Myxozoa: Myxosporea) and provide an effective tool for their molecular phylogenetics. Molecular Phylogenetics and Evolution 53, 81–93. Bartošová, P., Fiala, I., Jirků, M., Cinková, M., Caffara, M., Fioravanti, M.L., Atkinson, S.D., Bartholomew, J.L. and Holzer, A.S. (2013). Sphaerospora sensu stricto: taxonomy, diversity and evolution of a unique lineage of myxosporeans (Myxozoa). Molecular Phylogenetics and Evolution 68, 93–105. Bartošová, P., Freeman, M.A., Yokoyama, H., Caffara, M. and Fiala, I. (2011). Phylogenetic position of Sphaerospora testicularis and Latyspora scomberomori n. gen. n. sp. (Myxozoa) within the marine urinary clade. Parasitology 138, 381–393. Belem, A.M. and Pote, L.M. (2001). Portals of entry and systemic localization of proliferative gill disease organisms in channel catfish Ictalurus punctatus. Diseases of Aquatic Organisms 48, 37–42. Benajiba, M.H. and Marques, A. (1993). The alternation of actinomyxidian and myxosporidian

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sporal forms in the development of Myxidium giardi (parasite of Anguilla anguilla) through oligochaetes. Bulletin of the European Association of Fish Pathologists 13, 100–103. Boreham, R.E., Hendrick, S., O'Donoghue, P.J. and Stenzel, D.J. (1998). Incidental finding of Myxobolus spores (Protozoa: Myxozoa) in stool samples from patients with gastrointestinal symptoms. Journal of Clinic Microbiology 36, 3728–3730. Canning, E.U., Curry, A., Anderson, C.L. and Okamura, B. (1999). Ultrastructure of Myxidium trachinorum sp. nov. from the gallbladder of the lesser weever fish Echiichthys vipera. Parasitology Research 85, 910–919. Canning, E.U., Tops, S., Curry, A., Wood, T.S. and Okamura, B. (2002). Ecology, development and pathogenicity of Buddenbrockia plumatellae Schröder, 1910 (Myxozoa, Malacosporea) (syn. Tetracapsula bryozoides) and establishment of Tetracapsuloides n. gen. for Tetracapsula bryosalmonae. Journal of Eukaryotic Microbiology 49, 280– 295. Cannon, Q. and Wagner, E. (2003). Comparison of discharge mechanisms of cnidarian cnidae and myxozoan polar capsules. Reviews in Fisheries Science 11, 185–219. Carriero, M.M., Adriano, E.A., Silva, M.R., Ceccarelli, P.S. and Maia, A.A. (2013). Molecular phylogeny of the Myxobolus and Henneguya genera with several new South American species. PLoS One 8, e73713. Casal, G., Costa, G. and Azevedo, C. (2007). Ultrastructural description of Ceratomyxa tenuispora (Myxozoa), a parasite of the marine fish Aphanopus carbo (Trichiuridae), from the Atlantic coast of Madeira Island (Portugal). Folia Parasitologica 54, 165–171. Casal, G., Soares, E.C., Rocha, S., Silva, T.M., Santos, E.L., Nascimento, R., Oliveira, E. and Azevedo, C. (2019). Description of a new myxozoan Kudoa eugerres n. sp. and reclassification of two Sphaerospora sensu lato species. Parasitology Research 118, 1719–1730. Chang, E.S., Neuhof, M., Rubinstein, N.D., Diamant, A., Philippe, H., Huchon, D. and Cartwright, P. (2015). Genomic insights into the evolutionary origin of Myxozoa within Cnidaria. Proccedings of the National Academy of Sciences 112, 14912–14917. Costa, G., Eiras, J.C., Chubb, J., MacKenzie, K. and Berland, B. (1996). Parasites of the black scabbard fish, Aphanopus carbo Lowe, 1839 from Madeira. Bulletin of the European Association of Fish Pathologists 16, 13–16. Costa, G., Lom, J., Andrade, C. and Barradas, R. (1998). First report of Ceratomyxa sparusaurati (Protozoa: Myxosporea) and the occurrence of epitheliocystis in cultured sea bream, Sparus aurata L. from Madeira. Bulletin of the European Association of Fish Pathologists 18, 165–167. Cruz, C., Ferreira, S. and Saraiva, A.M. (1998). Ocorrência de Myxobolus cyprini em

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Chondrostoma polylepis em Portugal. Acta Parasitológica Portuguesa 5, 63–66. Cruz, C., Saraiva, A. and Ferreira, S. (2000). Preliminary observations on Myxobolus sp. from cyprinid fishes in Portugal. Bulletin of the European Association of Fish Pathologists 20, 65–69. Cruz, C., Vaz, A. and Saraiva, A. (2003). Occurrence of Kudoa sp. (Myxozoa) in Trachurus trachurus L. (Osteichthyes) in Portugal. Parasite 10, 165–167. Cruz e Silva, M.P. and Grazina-Freitas, M.S. (1984). Kudoa sp. em músculo de pescada (Merluccius merluccius L.). Repositório de Trabalho do Laboratório Nacional de Investigação Veterinária 16, 151–154. Current, W.L. (1979). Henneguya adiposa Minchew (Myxosporida) in the channel catfish: ultrastructure of the plasmodium wall and sporogenesis. Journal of Protozoology 26, 209–217. Current, W.L., Janovy, J.J. and Knight, S.A. (1979). Myxosoma funduli Kudo (Myxosporida) in Fundulus kansae: ultrastructure of the plasmodium wall and of sporogenesis. Journal of Protozoology 26, 574–583. Desser, S.S., Molnár, K. and Weller, I. (1983). Ultrastructure of sporogenesis of Thelohanellus nikolsii Akhmerov, 1955 (Myxozoa: Myxosporea) from the common carp, Cyprinus carpio. Journal of Parasitology 69, 504–518. Diamant, A. (1997). Fish-to-fish transmission of a marine myxosporean. Diseases of Aquatic Organisms 30, 99–105. Diamant, A., Lom, J. and Dyková, I. (1994). Myxidium leei n. sp., a pathogenic myxosporean of cultured sea bream Sparus aurata. Diseases of Aquatic Organisms 20, 137–141. Diamant, A., Whipps, C.M. and Kent, M.L. (2004). A new species of Sphaeromyxa (Myxosporea: Sphaeromyxina: Sphaeromyxidae) in devil firefish, Pterois miles (Scorpaenidae), from the northern Red Sea: morphology, ultrastructure, and phylogeny. Journal of Parasitology 90, 1434–1442. Eiras, J.C. (2002). Synopsis of the species of the genus Henneguya Thélohan, 1892 (Myxozoa: Myxosporea: Myxobolidae). Systematic Parasitology 52, 43–54. Eiras, J.C. and Adriano, E.A. (2012). A checklist of new species of Henneguya Thélohan, 1892 (Myxozoa: Myxosporea: Myxobolidae) described between 2002 and 2012. Systematic Parasitology 83, 95–104. Eiras, J.C., Molnár, K. and Lu, Y.S. (2005). Synopsis of the species of Myxobolus Bütschli, 1882 (Myxozoa: Myxosporea: Myxobolidae). Systematic Parasitology 61, 1–46. Eiras, J.C., Zhang, J. and Molnár, K. (2014). Synopsis of the species of Myxobolus Bütschli, 1882 (Myxozoa: Myxosporea: Myxobolidae) described between 2005 and 2013. Systematic Parasitology 88, 11–36. El-Mansy, A. and Molnár, K. (1997a). Development of Myxobolus hungaricus (Myxosporea:

Page | 33

Chapter I | General Introduction

Myxobolidae) in oligochaete alternate hosts. Diseases of Aquatic Organisms 31, 227– 232. El-Mansy, A. and Molnár, K. (1997b). Extrapiscine development of Myxobolus drjagini Akhmerov, 1954 (Myxosporea: Myxobolidae) in oligochaete alternative hosts. Acta Veterinaria Hungarica 45, 427–438. El-Mansy, A., Molnár, K. and Székely, C. (1998). Development of Myxobolus portucalensis Saraiva & Molnár, 1990 (Myxosporea: Myxobolidae) in the oligochaete Tubifex tubifex (Müller). Systematic Parasitology 41, 95–103. El-Matbouli, M., Fischer-Scherl, T. and Hoffmann, R.W. (1992a). Present knowledge on the life cycle, taxonomy, pathology, and therapy of some Myxosporea spp. important for freshwater fish. Annual Review of Fish Diseases 2, 367–402. El-Matbouli, M., Fischer-Scherl, T. and Hoffmann, R.W. (1992b). Transmission of Hoferellus carassii Achmerov, 1960 to goldfish Carassius auratus via an aquatic oligochaete. Bulletin of the European Association of Fish Pathologists 12, 54–56. El-Matbouli, M. and Hoffmann, R.W. (1989). Experimental transmission of two Myxobolus spp. developing bisporogeny via tubificid worms. Parasitology Research 75, 461–464. El-Matbouli, M. and Hoffmann, R.W. (1991). Effects of freezing, aging, and passage through the alimentary canal of predatory animals on the viability of Myxobolus cerebralis spores. Journal of Aquatic Animal Health 3, 260–262. El-Matbouli, M. and Hoffmann, R.W. (1993). Myxobolus carassii Klokaceva, 1914 also requires an aquatic oligochaete, Tubifex tubifex, as an intermediate host in its life cycle. Bulletin of the European Association of Fish Pathologists 13, 189–192. El-Matbouli, M. and Hoffmann, R.W. (1998). Light and electron microscopic studies on the chronological development of Myxobolus cerebralis to the actinosporean stage in Tubifex tubifex. International Journal for Parasitology 28, 195–217. El–Matbouli, M., Hoffmann, R.W. and Mandok, C. (1995). Light and electron microscopic observations on the route of the triactinomyxon-sporoplasm of Myxobolus cerebralis from epidermis into rainbow trout cartilage. Journal of Fish Biology 46, 919–935. Emery, C. (1909). I missosporidii sono Protozoi? Monitore Zoologico Italiano 22, 247. Estensoro, I., Redondo, M.J., Alvarez-Pellitero, P. and Sitjà-Bobadilla, A. (2010). Novel horizontal transmission route for Enteromyxum leei (Myxozoa) by anal intubation of gilthead sea bream Sparus aurata. Diseases of Aquatic Organisms 92, 51–58. Eszterbauer, E. (2004). Genetic relationship among gill-infecting Myxobolus species (Myxosporea) of cyprinids: molecular evidence of importance of tissue-specificity. Diseases of Aquatic Organisms 58, 35–40. Eszterbauer, E., Atkinson, S., Diamant, A., Morris, D., El-Matbouli, M. and Hartikainen, H. (2015). Myxozoan life cycles: practical approaches and insights. In Okamura, B., Gruhl,

Page | 34

Chapter I | General Introduction

A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 175–198. Eszterbauer, E., Marton, S., Rácz, O.Z., Letenyei, M. and Molnár, K. (2006). Morphological and genetic differences among actinosporean stages of fish-parasitic myxosporeans (Myxozoa): difficulties of species identification. Systematic Parasitology 65, 97–114. Eszterbauer, E. and Székely, C. (2004). Molecular phylogeny of the kidney-parasitic Sphaerospora renicola from common carp (Cyprinus carpio) and Sphaerospora sp. from goldfish (Carassius auratus auratus). Acta Veterinaria Hungarica 52, 469–478. Eszterbauer, E., Székely, C., Molnár, K. and Baska, F. (2000). Development of Myxobolus bramae (Myxosporea: Myxobolidae) in an oligochaete alternate host, Tubifex tubifex. Journal of Fish Diseases 23, 19–25. Evans, N.M., Holder, M.T., Barbeitos, M.S., Okamura, B. and Cartwright, P. (2010). The phylogenetic position of Myxozoa: exploring conflicting signals in phylogenomic and ribosomal data sets. Molecular Biology and Evolution 27, 2733–2746. Evans, N.M., Lindner, A., Raikova, E.V., Collins, A.G. and Cartwright, P. (2008). Phylogenetic placement of the enigmatic parasite, Polypodium hydriforme, within the Phylum Cnidaria. BMC Evolutionary Biology 8, 139. Feng, J.M., Xiong, J., Zhang, J.Y., Yang, Y.L., Yao, B., Zhou, Z.G. and Miao, W. (2014). New phylogenomic and comparative analyses provide corroborating evidence that Myxozoa is Cnidaria. Molecular Phylogenetics and Evolution 81, 10–18. Fiala, I. (2006). The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. International Journal for Parasitology 36, 1521–1534. Fiala, I. and Bartošová, P. (2010). History of myxozoan character evolution on the basis of rDNA and EF-2 data. BMC Evolutionary Biology 10, 228. Fiala, I., Bartošová-Sojková, P., Okamura, B. and Hartikainen, H. (2015a). Adaptive Radiation and Evolution within the Myxozoa. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 69–84. Fiala, I., Bartošová-Sojková, P. and Whipps, C.M. (2015b). Classification and phylogenetics of Myxozoa. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 85–110. Fiala, I. and Dyková, I. (2004). The phylogeny of marine and freshwater species of the genus Chloromyxum Mingazzini, 1890 (Myxosporea: Bivalvulida) based on small subunit ribosomal RNA gene sequences. Folia Parasitologica 51, 211–214. Fiala, I., Hlavničková, M., Kodádková, A., Freeman, M.A., Bartošová-Sojková, P. and Atkinson, S.D. (2015c). Evolutionary origin of Ceratonova shasta and phylogeny of the marine myxosporean lineage. Molecular Phylogenetics and Evolution 86, 75–89.

Page | 35

Chapter I | General Introduction

Foott, J.S., Stone, R. and Nichols, K. (2007). Proliferative kidney disease (Tetracapsuloides bryosalmonae) in Merced River Hatchery juvenile Chinook salmon: mortality and performance impairment in 2005 smolts. California Fish and Game 93, 57–76. Freeman, M.A. and Kristmundsson, Á. (2015). Histozoic myxosporeans infecting the stomach wall of elopiform fishes represent a novel lineage, the Gastromyxidae. Parasites & Vectors 8, 517. Freeman, M.A. and Kristmundsson, Á. (2018). Studies of Myxidium giardi Cépède, 1906 infections in Icelandic eels identifies a genetically diverse clade of myxosporeans that represents the Paramyxidium n. g. (Myxosporea: Myxidiidae). Parasites & Vectors 11, 551. Freeman, M.A. and Shinn, A.P. (2011). Myxosporean hyperparasites of gill monogeneans are basal to the multivalvulida. Parasites & Vectors 4, 220. Gilman, M.M. and Eiras, J.C. (1998). Kudoa sp. (Myxosporea: Multivalvulida) infecting Sardina pilchardus (Walb., 1792) off the Portuguese coast. Research and Reviews in Parasitology 58, 135–137. Gleeson, R.J. and Adlard, R.D. (2012). Phylogenetic relationships amongst Chloromyxum Mingazzini, 1890 (Myxozoa: Myxosporea), and the description of six novel species from Australian elasmobranchs. Parasitology International 61, 267–274. Grazina-Freitas, M.S., Cruz e Silva, M.P. and Ventura, M.T. (1986). Patologia de peixes marinhos. Casos estudados no Laboratório Nacional de Investigação Veterinária. Repositório de Trabalho do Laboratório Nacional de Investigação Veterinária 18, 61– 66. Grossheider, G. and Körting, W. (1992). First evidence that Hoferellus cyprini (Doflein, 1898) is transmitted by Nais sp. Bulletin of the European Association of Fish Pathologists 12, 17–20. Gunter, N. and Adlard, R. (2010). The demise of Leptotheca Thélohan, 1895 (Myxozoa: Myxosporea: Ceratomyxidae) and assignment of its species to Ceratomyxa Thélohan, 1892 (Myxosporea: Ceratomyxidae), Ellipsomyxa Køie, 2003 (Myxosporea: Ceratomyxidae), Myxobolus Bütschli, 1882 and Sphaerospora Thélohan, 1892 (Myxosporea: Sphaerosporidae). Systematic Parasitology 75, 81–104. Gunter, N.L., Whipps, C.M. and Adlard, R.D. (2009). Ceratomyxa (Myxozoa: Bivalvulida): robust taxon or genus of convenience? International Journal for Parasitology 39, 1395– 1405. Hallett, S.L. and Bartholomew, J.L. (2012). Myxobolus cerebralis and Ceratomyxa shasta. In Woo, P.T.K. and Buchmann, K. (eds). Fish parasites: pathobiology and protection. CABI, Oxfordshire, United Kingdom. Hallett, S.L., Atkinson, S.D. and El-Matbouli, M. (2002). Molecular characterisation of two

Page | 36

Chapter I | General Introduction

aurantiactinomyxon (Myxozoa) phenotypes reveals one genotype. Journal of Fish Diseases 25, 627–631. Hallett, S.L., Atkinson, S.D., Schol, H. and El-Matbouli, M. (2003). Characterisation of two novel types of hexactinomyxon spores (Myxozoa) with subsidiary protrusions on their caudal processes. Diseases of Aquatic Organisms 55, 45–57. Hallett, S.L., Erséus, C. and Lester, R.J.G. (1997) Actinosporea from Hong Kong marine oligochaeta. In Morton, B. (ed). Proccedings of the eight international marine biological workshop: the marine flora and fauna of Hong Kong and Southern China. Hong Kong University Press, Hong Kong, pp. 1–7. Hallett, S.L., Erséus, C. and Lester, R.J.G. (1999). Actinosporeans (Myxozoa) from marine oligochaetes of the Great Barrier Reef. Systematic Parasitology 44, 49–57. Hallett, S.L. and Lester, R.J.G. (1999). Actinosporeans (Myxozoa) with four developing spores within a pansporocyst: Tetraspora discoidea n. g. n. sp. and Tetraspora rotundum n. sp. International Journal for Parasitology 29, 419–427. Hallett, S.L., O'Donoghue, P.J. and Lester, R.J.G. (1998). Structure and development of a marine actinosporean, Sphaeractinomyxon ersei n. sp. (Myxozoa). Journal of Eukaryotic Microbiology 45, 142–150. Halliday, M.M. (1976). The biology of Myxosoma cerebralis: the causative organism of whirling disease of salmonids. Journal of Fish Biology 9, 339–357. Hartigan, A., Fiala, I., Dyková, I., Rose, K., Phalen, D.N. and Slapeta, J. (2012). New species of Myxosporea from frogs and resurrection of the genus Cystodiscus Lutz, 1889 for species with myxospores in gallbladders of amphibians. Parasitology 139, 478–496. Heiniger, H. and Adlard, R.D. (2014). Relatedness of novel species of Myxidium Bütschli, 1882, Zschokkella Auerbach, 1910 and Ellipsomyxa Køie, 2003 (Myxosporea: Bivalvulida) from the gall bladders of marine fishes (Teleostei) from Australian waters. Systematic Parasitology 87, 47–72. Henderson, M. and Okamura, B. (2004). The phylogeography of salmonid proliferative kidney disease in Europe and North America. Proceedings of the Royal Society B: Biological Sciences 271, 1729–1736. Henning, S.S., Hoffman, L.C. and Manley, M. (2013). A review of Kudoa-induced myoliquefaction of marine fish species in South Africa and other countries. South African Journal of Science 109, 1–5. Hermida, M., Cruz, C. and Saraiva, A. (2013). Parasites as biological tags for stock identification of blackspot seabream, Pagellus bogaraveo, in Portuguese northeast Atlantic waters. Scientia Marina 77, 607–615. Hermida, M., Saraiva, A. and Cruz, C. (2008). Metazoan parasite community of a European eel (Anguilla anguilla) population from an estuary in Portugal. Bulletin of the European

Page | 37

Chapter I | General Introduction

Association of Fish Pathologists 28, 35–40. Hervio, D.M.L., Kent, M.L., Khattra, J., Sakanari, J., Yokoyama, H. and Devlin, R.H. (1997). Taxonomy of Kudoa species (Myxosporea), using a small-subunit ribosomal DNA sequence. Canadian Journal of Zoology 75, 2112–2119. Hoffman, G.L. and Markiw, M.E. (1977). Control of whirling disease (Myxosoma cerebralis): use of methylene blue staining as a possible indicator of effect of heat on spores. Journal of Fish Biology 10, 181–183. Hoffman, G.L. and Putz, R.E. (1969). Host susceptibility and the effect of aging, freezing, heat, and chemicals on spores of Myxosoma cerebralis. The Progressive Fish-Culturist 31, 35–37. Holland, J.W., Okamura, B., Hartikainen, H. and Secombes, C.J. (2011). A novel minicollagen gene links cnidarians and myxozoans. Proceedings of the Royal Society B: Biological Sciences 278, 546–553. Holzer, A.S., Bartošová-Sojková, P., Born-Torrijos, A., Lövy, A., Hartigan, A. and Fiala, I. (2018). The joint evolution of the Myxozoa and their alternate hosts: a cnidarian recipe for success and vast biodiversity. Molecular Ecology 27, 1651–1666. Holzer, A.S., Sommerville, C. and Wootten, R. (2003). Tracing the route of Sphaerospora truttae from the entry locus to the target organ of the host, Salmo salar L., using an optimized and specific in situ hybridization technique. Journal of Fish Diseases 26, 647–655. Holzer, A.S., Sommerville, C. and Wootten, R. (2004). Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences. International Journal for Parasitology 34, 1099–1111. Holzer, A.S., Sommerville, C. and Wootten, R. (2006). Molecular identity, phylogeny and life cycle of Chloromyxum schurovi Shul'man & Ieshko 2003. Parasitology Research 99, 90–96. Holzer, A.S., Wootten, R. and Sommerville, C. (2007). The secondary structure of the unusually long 18S ribosomal RNA of the myxozoan Sphaerospora truttae and structural evolutionary trends in the Myxozoa. International Journal for Parasitology 37, 1281–1295. Ikeda, I. (1912). Studies on some sporozoan parasites of sipunculoids. I. The life history of a new actinomyxidian Tetractinomyxon intermedium g. et sp. nov. Archiv für Protistenkunde 25, 240–272. Iwashita, Y., Kamijo, Y., Nakahashi, S., Shindo, A., Yokoyama, K., Yamamoto, A., Omori, Y., Ishikura, K., Fujioka, M., Hatada, T., Takeda, T., Maruyama, K. and Imai, H. (2013). Food poisoning associated with Kudoa septempunctata. Journal of Emergency Medicine 44, 943–945.

Page | 38

Chapter I | General Introduction

Janiszewska, J. (1957). Actinomyxidia II: new systematics, sexual cycle, description of new genera and species. Zoologica Poloniae 8, 3–34. Jiménez-Guri, E., Philippe, H., Okamura, B. and Holland, P.W. (2007). Buddenbrockia is a cnidarian worm. Science 317, 116–118. Jirků, M., Bartošová, P., Kodádková, A. and Mutschmann, F. (2011). Another chloromyxid lineage: molecular phylogeny and redescription of Chloromyxum careni from the Asian horned frog Megophrys nasuta. Journal of Eukaryotic Microbiology 58, 50–59. Jirků, M., Fiala, I. and Modrý, D. (2007). Tracing the genus Sphaerospora: rediscovery, redescription and phylogeny of the Sphaerospora ranae (Morelle, 1929) n. comb. (Myxosporea, Sphaerosporidae), with emendation of the genus Sphaerospora. Parasitology 134, 1727–1739. Johnson, K.A. (1980). Host susceptibility, histopathologic and transmission studies on Ceratomyxa shasta, a myxosporidan parasite of salmonid fish. Fish Pathology 14, 183– 184. Jones, S., Prosperi-Porta, G., Dawe, S., Taylor, K. and Goh, B. (2004). Parvicapsula minibicornis in anadromous sockeye (Oncorhynchus nerka) and coho (Oncorhynchus kisutch) salmon from tributaries of the Columbia River. Journal of Parasitology 90, 882– 885. Jurine, L.L. (1825). Histoire des poissons du Lac Léman. Mémoires de la Société de Physique et d'Histoire Naturelle de Genève 3, 133–235. Kallert, D.M., Eszterbauer, E., El-Matbouli, M., Erséus, C. and Haas, W. (2005). The life cycle of Henneguya nuesslini Schuberg & Schröder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore. Journal of Fish Diseases 28, 71–79. Karlsbakk, E. and Køie, M. (2009). Bipteria formosa (Kovaleva et Gaevskaya, 1979) comb. n. (Myxozoa: Myxosporea) in whiting Merlangius merlangus (Teleostei: Gadidae) from Denmark. Folia Parasitologica 56, 86–90. Karlsbakk, E. and Køie, M. (2012). The marine myxosporean Sigmomyxa sphaerica (Thelohan, 1895) gen. n., comb. n. (syn. Myxidium sphaericum) from garfish (Belone belone (L.)) uses the polychaete Nereis pelagica L. as invertebrate host. Parasitology Research 110, 211–218. Karlsbakk, E., Kristmundsson, Á., Albano, M., Brown, P. and Freeman, M.A. (2017). Redescription and phylogenetic position of Myxobolus aeglefini and Myxobolus platessae n. comb. (Myxosporea), parasites in the cartilage of some North Atlantic marine fishes, with notes on the phylogeny and classification of the Platysporina. Parasitology International 66, 952–959. Kawai, T., Sekizuka, T., Yahata, Y., Kuroda, M., Kumeda, Y., Iijima, Y., Kamata, Y., Sugita- Konishi, Y. and Ohnishi, T. (2012). Identification of Kudoa septempunctata as the

Page | 39

Chapter I | General Introduction

causative agent of novel food poisoning outbreaks in Japan by consumption of Paralichthys olivaceus in raw fish. Clinical Infectious Diseases 54, 1046–1052. Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffmann, R.W., Khattra, J., Hallett, S.L., Lester, R.J., Longshaw, M., Palenzeula, O., Siddall, M.E. and Xiao, C. (2001). Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic Microbiology 48, 395–413. Kent, M.L. and Hedrick, R.P. (1985). PKX, the causative agent of proliferative kidney disease (PKD) in Pacific salmonid fishes and its affinities with the Myxozoa. Journal of Protozoology 32, 254–260. Kent, M.L., Margolis, L. and Corliss, J.O. (1994a). The demise of a class of protists - taxonomic and nomenclatural revisions proposed for the protist phylum Myxozoa Grassé, 1970. Canadian Journal of Zoology 72, 932–937. Kent, M.L., Margolis, L., Whitaker, D.J., Hoskins, G.E. and McDonald, T.E. (1994b). Review of Myxosporea of importance in salmonid fisheries and aquaculture in British Columbia. Folia Parasitologica 41, 27–37. Kent, M.L., Whitaker, D.J. and Margolis, L. (1993). Transmission of Myxobolus arcticus Pugachev and Khokhlov, 1979, a myxosporean parasite of Pacific salmon, via a triactinomyxon from the aquatic oligochaete Stylodrilus heringianus (Lumbriculidae). Canadian Journal of Zoology 71, 1207–1211. Kim, J., Kim, W. and Cunningham, C.W. (1999). A new perspective on lower metazoan relationships from 18S rDNA sequences. Molecular Biology and Evolution 16, 423– 427. Kodádková, A., Bartošová-Sojková, P., Holzer, A.S. and Fiala, I. (2015). Bipteria vetusta n. sp. - an old parasite in an old host: tracing the origin of myxosporean parasitism in vertebrates. International Journal for Parasitology 45, 269–276. Kodádková, A., Dyková, I., Tyml, T., Ditrich, O. and Fiala, I. (2014). Myxozoa in high Arctic: survey on the central part of Svalbard archipelago. International Journal for Parasitology: Parasites and Wildlife 3, 41–56. Koel, T.M., Kerans, B.L., Barras, S.C., Hanson, K.C. and Wood, J.S. (2010). Avian piscivores as vectors for Myxobolus cerebralis in the Greater Yellowstone ecosystem. Transactions of the American Fisheries Society 139, 976–988. Køie, M., Karlsbakk, E., Einen, A.C. and Nylund, A. (2013). A parvicapsulid (Myxozoa) infecting Sprattus sprattus and Clupea harengus (Clupeidae) in the Northeast Atlantic uses Hydroides norvegicus (Serpulidae) as invertebrate host. Folia Parasitologica 60, 149– 154. Køie, M., Karlsbakk, E. and Nylund, A. (2007). A new genus Gadimyxa with three new species (Myxozoa, Parvicapsulidae) parasitic in marine fish (Gadidae) and the two-host life

Page | 40

Chapter I | General Introduction

cycle of Gadimyxa atlantica n. sp. Journal of Parasitology 93, 1459–1467. Køie, M., Karlsbakk, E. and Nylund, A. (2008). The marine herring myxozoan Ceratomyxa auerbachi (Myxozoa: Ceratomyxidae) uses Chone infundibuliformis (Annelida: Polychaeta: Sabellidae) as invertebrate host. Folia Parasitologica 55, 100–104. Køie, M., Whipps, C.M. and Kent, M.L. (2004). Ellipsomyxa gobii (Myxozoa: Ceratomyxidae) in the common goby Pomatoschistus microps (Teleostei: Gobiidae) uses Nereis spp. (Annelida: Polychaeta) as invertebrate hosts. Folia Parasitologica 51, 14–18. Körting, W., Kruse, P. and Steinhagen, D. (1989). Development of “Csaba cells” in experimentally infected Cyprinus carpio. Angewandte Parasitologie 30, 185–188. Kotob, M.H., Gorgoglione, B., Kumar, G., Abdelzaher, M., Saleh, M. and El-Matbouli, M. (2017). The impact of Tetracapsuloides bryosalmonae and Myxobolus cerebralis co- infections on pathology in rainbow trout. Parasites & Vectors 10, 442. Kristmundsson, Á. and Freeman, M.A. (2013). Sphaeromyxids form part of a diverse group of myxosporeans infecting the hepatic biliary systems of a wide range of host organisms. Parasites & Vectors 6, 51. Kudo, R. (1933). A taxonomic consideration of Myxosporidia. Transactions of the American Microscopical Society 52, 195–216. Lebbad, M. and Willcox, M. (1998). Spores of Henneguya salminicola in human stool specimens. Journal of Clinical Microbiology 36, 1820–1820. Li, Y.C. and Sato, H. (2014). Two novel myxosporean species (Myxosporea: Bivalvulida), Myxobolus marumotoi n. sp. and Cardimyxobolus japonensis n. sp., from the dark sleeper, Odontobutis obscura, in Japan. Parasitology Research 113, 1371–1381. Lin, D., Hanson, L.A. and Pote, L.M. (1999). Small subunit ribosomal RNA sequence of Henneguya exilis (class Myxosporea) identifies the actinosporean stage from an oligochaete host. Journal of Eukaryotic Microbiology 46, 66–68. Liu, Y., Whipps, C.M., Gu, Z.M. and Zeng, L.B. (2010). Myxobolus turpisrotundus (Myxosporea: Bivalvulida) spores with caudal appendages: investigating the validity of the genus Henneguya with morphological and molecular evidence. Parasitology Research 107, 699–706. Lom, J. (1969). Notes on the ultrastructure and sporoblast development in fish parasitizing myxosporidian of the genus Sphaeromyxa. Zeitschrift für Zellforschung und Mikroskopische Anatomie 97, 416–437. Lom, J. (1990). Myxozoa. In Margulis, L., Corliss, J.O., Melkonian, M. and Chapman, D.J. (eds). Handbook of the Protoctista; the structure, cultivation, habits and life histories of the eukaryotic microorganisms and their descendants exclusive of animals, plants and fungi. Jones and Bartlett Publishers, Boston, pp. 36–52. Lom, J. and Dyková, I. (1992). Myxosporidia (Phylum Myxozoa). Protozoan Parasites of

Page | 41

Chapter I | General Introduction

Fishes. Developments in Aquaculture and Fisheries Science, Vol. 26. Elsevier, Amsterdam, Netherlands, pp. 159‒235. Lom, J. and Dyková, I. (1997). Ultrastructural features of the actinosporean phase of myxosporea (Phylum myxozoa): a comparative study. Acta Protozoologica 36, 83–103. Lom, J. and Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53, 1–36. Lom, J., Dyková, I., Pavlásková, M. and Grupcheva, G. (1983). Sphaerospora molnari sp.nov. (Myxozoa: Myxosporea), an agent of gill, skin and blood sphaerosporosis of common carp in Europe. Parasitology 86, 529–535. Marcucci, C., Caffara, M. and Goretti, E. (2009). Occurrence of actinosporean stages (Myxozoa) in the Nera River system (Umbria, central Italy). Parasitology Research 105, 1517–1530. Marques, A. (1984). Contribution a la connaissance des Actinomyxidies: ultrastructure, cycle biologique, systématique. Ph.D. Thesis. Université des Sciences et Techniques de Languedoc, Montepellier, France, pp. 218. Marques, A. (1987). La sexualité chez les Actinomyxidies: étude chez Neoactinomyxon eiseniellae (Ormières et Frézil, 1969), Actinosporea Noble, 1980; Myxozoa, Grassé, 1970. Annales des Sciences Naturelles (Zoologie) Paris 8, 81–101. Marshall, W.L., Sitjà-Bobadilla, A., Brown, H.M., MacWilliam, T., Richmond, Z., Lamson, H., Morrison, D.B. and Afonso, L.O. (2016). Long-term epidemiological survey of Kudoa thyrsites (Myxozoa) in Atlantic salmon (Salmo salar L.) from commercial aquaculture farms. Journal of Fish Diseases 39, 929–946. Marton, S. and Eszterbauer, E. (2011). The development of Myxobolus pavlovskii (Myxozoa: Myxobolidae) includes an echinactinomyxon-type actinospore. Folia Parasitologica 58, 157–163. McClelland, R.S., Murphy, D.M. and Cone, D.K. (1997). Report of spores of Henneguya salminicola (Myxozoa) in human stool specimens: possible source of confusion with human spermatozoa. Journal of Clinical Microbiology 35, 2815–2818. McGeorge, J., Sommerville, C. and Wootten, R. (1997). Studies of actinosporean myxozoan stages parasitic in oligochaetes from the sediments of a hatchery where Atlantic salmon harbour Sphaerospora truttae infection. Diseases of Aquatic Organisms 30, 107–119. Meaders, M.D. and Hendrickson, G.L. (2009). Chronological development of Ceratomyxa shasta in the polychaete host, Manayunkia speciosa. Journal of Parasitology 95, 1397– 1407. Menezes, J. (1984). A case of massive cutaneous myxobolosis in wild mullet. Boletim do Instituto Nacional de Investigação das Pescas 12, 71–73.

Page | 42

Chapter I | General Introduction

Menezes, J., Vigário, A.M., Moledo, A. and Pita, G. (1990). Recurso sardinha: parasitose com incidência económica. Pesca e Navegação 97, 22–24. Milanin, T., Atkinson, S.D., Silva, M.R.M., Alves, R.G., Maia, A.A.M. and Adriano, E.A. (2017). Occurrence of two novel actinospore types (Cnidaria: Myxosporea) in Brazilian fish farms, and the creation of a novel actinospore collective group, Seisactinomyxon. Acta Parasitologica 62, 121–128. Molnár, K. (1979). Myxobolus pavlovskii (Achmerov, 1954) (Myxosporidia) infection in the silver carp and bighead. Acta Veterinaria Academiae Scientiarum Hungaricae 27, 207– 216. Molnár, K., El-Mansy, A., Székely, C. and Baska, F. (1999). Development of Myxobolus dispar (Myxosporea: Myxobolidae) in an oligochaete alternate host, Tubifex tubifex. Folia Parasitologica 46, 15–21. Molnár, K., Eszterbauer, E., Marton, S., Székely, C. and Eiras, J.C. (2012). Comparison of the Myxobolus fauna of common barbel from Hungary and Iberian barbel from Portugal. Diseases of Aquatic Organisms 100, 231–248. Molnár, K. and Kovács-Gayer, E. (1986). Experimental induction of Sphaerospora renicola (Myxosporea) infection in common carp (Cyprinus carpio) by transmission of SB- protozoans. Journal of Applied Ichthyology 2, 86–94. Moncada, L.I., López, M.C., Murcia, M.I., Nicholls, S., León, F., Guío, O.L. and Corredor, A. (2001). Myxobolus sp., another opportunistic parasite in immunosuppressed patients? Journal of Clinical Microbiology 39, 1938–1940. Moran, J.D.W., Whitaker, D.J. and Kent, M.L. (1999). Natural and laboratory transmission of the marine myxozoan parasite Kudoa thyrsites to Atlantic salmon. Journal of Aquatic Animal Health 11, 110–115. Morris, D.J. (2010). Cell formation by myxozoan species is not explained by dogma. Proceedings of the Royal Society B: Biological Sciences 277, 2565–2570. Morris, D.J. (2012). A new model for myxosporean (Myxozoa) development explains the endogenous budding phenomenon, the nature of cell within cell life stages and evolution of parasitism from a cnidarian ancestor. International Journal for Parasitology 42, 829–840. Morris, D.J. and Adams, A. (2006). Transmission of freshwater myxozoans during the asexual propagation of invertebrate hosts. International Journal for Parasitology 36, 371–377. Morris, D.J. and Freeman, M.A. (2010). Hyperparasitism has wide-ranging implications for studies on the invertebrate phase of myxosporean (Myxozoa) life cycles. International Journal for Parasitology 40, 357–369. Müller, J. (1841). Über Psorospermien. Archiv für Anatomie, Physiologie und Wissenschaftliche Medicin 5, 477–496.

Page | 43

Chapter I | General Introduction

Nesnidal, M.P., Helmkampf, M., Bruchhaus, I., El-Matbouli, M. and Hausdorf, B. (2013). Agent of whirling disease meets orphan worm: phylogenomic analyses firmly place Myxozoa in Cnidaria. PLoS One 8, e54576. Okamura, B. and Canning, E.U. (2003). Orphan worms and homeless parasites enhance bilaterian diversity. Trends in Ecology & Evolution 18, 633–639. Okamura, B., Gruhl, A. and Bartholomew, J.L. (2015). An Introduction to myxozoan evolution, ecology and development. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 1–20. Oumouna, M., Hallett, S., Hoffmann, R. and El-Matbouli, M. (2003). Seasonal occurrence of actinosporeans (Myxozoa) and oligochaetes (Annelida) at a trout hatchery in Bavaria, Germany. Parasitology Research 89, 170–184. Overstreet, R.M. (1976). Fabespora vermicola sp. n., the first myxosporidan from a platyhelminth. Journal of Parasitology 62, 680–684. Özer, A. and Wootten, R. (2001). Ultrastructural observations on the development of some actinosporean types within their oligochaete hosts. Turkish Journal of Zoology 25, 199– 216. Özer, A., Wootten, R. and Shinn, A.P. (2002). Survey of actinosporean types (Myxozoa) belonging to seven collective groups found in a freshwater salmon farm in Northern Scotland. Folia Parasitologica 49, 189–210. Padrós, F., Palenzuela, O., Hispano, C., Tosas, O., Zarza, C., Crespo, S. and Alvarez- Pellitero, P. (2001). Myxidium leei (Myxozoa) infections in aquarium-reared Mediterranean fish species. Diseases of Aquatic Organisms 47, 57–62. Palenzuela, O., Redondo, M.J. and Alvarez-Pellitero, P. (2002). Description of Enteromyxum scophthalmi gen. nov., sp. nov. (Myxozoa), an intestinal parasite of turbot (Scophthalmus maximus L.) using morphological and ribosomal RNA sequence data. Parasitology 124, 369–379. Patra, S., Bartošová-Sojková, P., Pecková, H., Fiala, I., Eszterbauer, E. and Holzer, A.S. (2018). Biodiversity and host-parasite cophylogeny of Sphaerospora (sensu stricto) (Cnidaria: Myxozoa). Parasites & Vectors 11, 347. Pickens, P.E. (1988). Systems that control the burrowing behavior of a sea-anemone. Journal of Experimental Biology 135, 133–164. Pote, L.M., Hanson, L.A. and Shivaji, R. (2000). Small subunit ribosomal RNA sequences link the cause of Proliferative Gill Disease in channel catfish to Henneguya n. sp. (Myxozoa: Myxosporea). Journal of Aquatic Animal Health 12, 230–240. Rácz, O.Z., Székely, C. and Molnár, K. (2004). Intraoligochaete development of Myxobolus intimus (Myxosporea: Myxobolidae), a gill myxosporean of the roach (Rutilus rutilus).

Page | 44

Chapter I | General Introduction

Folia Parasitologica 51, 199–207. Rangel, L.F., Azevedo, C., Casal, G. and Santos, M.J. (2012). Ultrastructural aspects of Ellipsomyxa mugilis (Myxozoa: Ceratomyxidae) spores and developmental stages in Nereis diversicolor (Polychaeta: Nereidae). Journal of Parasitology 98, 513–519. Rangel, L.F., Castro, R., Rocha, S., Cech, G., Casal, G., Azevedo, C., Székely, C., Cavaleiro, F. and Santos, M.J. (2016a). Description of new types of sphaeractinomyxon actinospores (Myxozoa: Myxosporea) from marine tubificid oligochaetes, with a discussion on the validity of the tetraspora and the endocapsa as actinospore collective group names. Parasitology Research 115, 2341–2351. Rangel, L.F., Castro, R., Rocha, S., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2016b). Tetractinomyxon stages genetically consistent with Sphaerospora dicentrarchi (Myxozoa: Sphaerosporidae) found in Capitella sp. (Polychaeta: Capitellidae) suggest potential role of marine polychaetes in parasite's life cycle. Parasitology 143, 1067–1073. Rangel, L.F., Cech, G., Székely, C. and Santos, M.J. (2011). A new actinospore type Unicapsulactinomyxon (Myxozoa), infecting the marine polychaete, Diopatra neapolitana (Polychaeta: Onuphidae) in the Aveiro Estuary (Portugal). Parasitology 138, 698–712. Rangel, L.F., Rocha, S., Borkhanuddin, M.H., Cech, G., Castro, R., Casal, G., Azevedo, C., Severino, R., Székely, C. and Santos, M.J. (2014). Ortholinea auratae n. sp. (Myxozoa, Ortholineidae) infecting the urinary bladder of the gilthead seabream Sparus aurata (Teleostei, Sparidae), in a Portuguese fish farm. Parasitology Research 113, 3427– 3437. Rangel, L.F., Rocha, S., Casal, G., Castro, R., Severino, R., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2017). Life cycle inference and phylogeny of Ortholinea labracis n. sp. (Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm. Journal of Fish Diseases 40, 243– 262. Rangel, L.F., Rocha, S., Castro, R., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2015). The life cycle of Ortholinea auratae (Myxozoa: Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete. Parasitology Research 114, 2671–2678. Rangel, L.F., Santos, M.J., Cech, G. and Székely, C. (2009). Morphology, molecular data, and development of Zschokkella mugilis (Myxosporea, Bivalvulida) in a polychaete alternate host, Nereis diversicolor. Journal of Parasitology 95, 561–569. Rocha, S., Casal, G., Al-Quraishy, S. and Azevedo, C. (2013a). Morphological and molecular characterization of a new myxozoan species (Myxosporea) infecting the gall bladder of

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Raja clavata (Chondrichthyes), from the Portuguese Atlantic Coast. Journal of Parasitology 99, 307–317. Rocha, S., Casal, G., Al-Quraishy, S. and Azevedo, C. (2014). Morphological and ultrastructural redescription of Chloromyxum leydigi Mingazzini, 1890 (Myxozoa: Myxosporea), type species of the genus, infecting the gall bladder of the marine cartilaginous fish Torpedo marmorata Risso (Chondrichthyes: Torpedinidae), from the Portuguese Atlantic Coast. Folia Parasitologica 61, 1–10. Rocha, S., Casal, G., Garcia, P., Matos, E., Al-Quraishy, S. and Azevedo, C. (2014). Ultrastructure and phylogeny of the parasite Henneguya carolina sp. nov. (Myxozoa), from the marine fish Trachinotus carolinus in Brazil. Diseases of Aquatic Organisms 112, 139–148. Rocha, S., Casal, G., Matos, P., Matos, E., Dkhil, M. and Azevedo, C. (2011). Description of Triangulamyxa psittaca sp. nov. (Myxozoa: Myxosporea), a new parasite in the urinary bladder of Colomesus psittacus (Teleostei) from the Amazon River, with emphasis on the ultrastructure of plasmodial stages. Acta Protozoologica 50, 327–338. Rocha, S., Casal, G., Rangel, L., Severino, R., Castro, R., Azevedo, C. and Santos, M.J. (2013b). Ultrastructural and phylogenetic description of Zschokkella auratis sp. nov. (Myxozoa), a parasite of the gilthead seabream Sparus aurata. Diseases of Aquatic Organisms 107, 19–30. Rosser, T.G., Griffin, M.J., Quiniou, S.M., Greenway, T.E., Khoo, L.H., Wise, D.J. and Pote, L.M. (2014). Molecular and morphological characterization of myxozoan actinospore types from a commercial catfish pond in the Mississippi delta. Journal of Parasitology 100, 828–839. Ruidisch, S., El-Matbouli, M. and Hoffmann, R.W. (1991). The role of tubificid worms as an intermediate host in the life cycle of Myxobolus pavlovskii (Akhmerov, 1954). Parasitology Research 77, 663–667. Santos, M.J. (1996). Observations on the parasitofauna of wild sea bass (Dicentrarchus labrax L.) from Portugal. Bulletin of the European Association of Fish Pathologists 16, 77–79. Santos, M.J., Saraiva, A., Cruz, C., Eiras, J.C., Hermida, M., Ventura, C. and Soares, J.P. (2009). Use of parasites as biological tags in stock identification of black scabbardfish, Aphanopus carbo Lowe, 1839 (Osteichthyes: Trichiuridae) from Portuguese waters. Scientia Marina 73, 55–62. Saraiva, A. and Chubb, J.C. (1989). Preliminary observations on the parasites of Anguilla anguilla (L.) from Portugal. Bulletin of the European Association of Fish Pathologists 9, 88–89. Saraiva, A., Cruz, C. and Ferreira, S. (2000). Studies on Myxidium rhodei Léger, 1905 (Myxozoa: Myxosporea) on Chondrostoma polylepis from River Ave, North Portugal.

Page | 46

Chapter I | General Introduction

Bulletin of the European Association of Fish Pathologists 20, 106–110. Saraiva, A. and Eiras, J.C. (1996). Parasite community of european eel Anguilla anguilla (L.) in the river Este, northern Portugal. Research and Reviews in Parasitology 56, 179– 183. Saraiva, A., Eiras, J.C. and Cruz, C. (1998). Fenómenos histopatológicos em peixes provocados por doenças de etiologia parasitária. Acta Parasitologica Portuguesa 5, 67–72. Saraiva, A. and Molnár, K. (1990). Myxobolus portucalensis n. sp. in the fins of the European eel Anguilla anguilla (L.) in Portugal. Revista Ibérica de Parasitología 50, 31–35. Sarker, S., Kallert, D.M., Hedrick, R.P. and El-Matbouli, M. (2015). Whirling disease revisited: pathogenesis, parasite biology and disease intervention. Diseases of Aquatic Organisms 114, 155–175. Schlegel, M., Lom, J., Stechmann, A., Bernhard, D., Leipe, D., Dyková, I. and Sogin, M.L. (1996). Phylogenetic analysis of complete small unit ribosomal RNA coding region of Myxidium lieberkuehni: evidence that Myxozoa are Metazoa and related to the Bilateria. Archiv für Protistenkunde 147, 1–9. Shin, S.P., Nguyen, V.G., Jeong, J.M., Jun, J.W., Kim, J.H., Han, J.E., Baeck, G.W. and Park, S.C. (2014). The phylogenetic study on Thelohanellus species (Myxosporea) in relation to host specificity and infection site tropism. Molecular Phylogenetics and Evolution 72, 31–34. Shpirer, E., Chang, E.S., Diamant, A., Rubinstein, N., Cartwright, P. and Huchon, D. (2014). Diversity and evolution of myxozoan minicollagens and nematogalectins. BMC Evolutionary Biology 14, 205. Shpirer, E., Diamant, A., Cartwright, P. and Huchon, D. (2018). A genome wide survey reveals multiple nematocyst-specific genes in Myxozoa. BMC Evolutionary Biology 18, 138. Shulman, S.S. (1959). New system of myxosporidia. Trudy Karelsk. fil. AN SSSR, 14, Voprosy parazitol. Karelii 33–47. Shulman, S.S. (1990). Myxosporidia of the USSR. Russian Translations Series, 1st edition. Routledge, United Kingdom, pp. 643. Siau, Y., Gasc, C. and Maillard, C. (1981). Premières observations ultrastructurales d’une myxosporide appartenant au genre Fabespora, parasite de trématode. Protistologica 17, 131–137. Siddall, M.E., Martin, D.S., Bridge, D., Desser, S.S. and Cone, D.K. (1995). The demise of a phylum of protists: phylogeny of Myxozoa and other parasitic cnidaria. Journal of Parasitology 81, 961–967. Siddall, M.E. and Whiting, M.F. (1999). Long-Branch abstractions. Cladistics 15, 9–24.

Page | 47

Chapter I | General Introduction

Sirin, C., Santos, M.J. and Rangel, L.F. (2018). Morphological and molecular analyses of Bipteria lusitanica n. sp. in wild white seabream, Diplodus sargus (Linnaeus, 1758) in Portugal. Parasitology Research 117, 2035–2041. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (2001). Leptotheca sparidarum n. sp. (Myxosporea: Bivalvulida), a parasite from cultured common dentex (Dentex dentex L.) and gilthead sea bream (Sparus aurata L.) (Teleostei: Sparidae). Journal of Eukaryotic Microbiology 48, 627–639. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1993). Zschokkella mugilis n. sp. (Myxosporea, Bivalvulida) from mulltes (Teleostei, Mugilidae) of Mediterranean waters – light and electron-microscopic description. Journal of Eukaryotic Microbiology 40, 755–764. Sitjà-Bobadilla, A., Diamant, A., Palenzuela, O. and Alvarez-Pellitero, P. (2007). Effect of host factors and experimental conditions on the horizontal transmission of Enteromyxum leei (Myxozoa) to gilthead sea bream, Sparus aurata L., and European sea bass, Dicentrarchus labrax (L.). Journal of Fish Diseases 30, 243–250. Smothers, J.F., von Dohlen, C.D., Smith Jr., L.H. and Spall, R.D. (1994). Molecular evidence that the myxozoan protists are metazoans. Science 265, 1719–1721. Stehr, C. (1986). Sporogenesis of the myxosporean Kudoa paniformis Kabata & Whitaker, 1981 infecting the muscle of the Pacific whiting, Merluccius productus (Ayres). Journal of Fish Diseases 9, 493–504. Štolc, A. (1899). Actinomyxidies, nouveau groupe de Mésozoaires parent des Myxosporidies. Bulletin International de l'Académie des Sciences de Bohème 22, 1–12. Styer, E.L., Harrison, L.R. and Burtle, G.J. (1991). Communications: experimental production of Proliferative Gill Disease in channel catfish exposed to a myxozoan-infected oligochaete, Dero digitata. Journal of Aquatic Animal Health 3, 288–291. Székely, C., Borkhanuddin, M.H., Cech, G., Kelemen, O. and Molnár, K. (2014). Life cycles of three Myxobolus spp. from cyprinid fishes of Lake Balaton, Hungary involve triactinomyxon-type actinospores. Parasitology Research 113, 2817–2825. Székely, C., Eiras, J.C. and Eszterbauer, E. (2005). Description of a new synactinomyxon type from the River Sousa, Portugal. Diseases of Aquatic Organisms 66, 9–14. Székely, C., El-Mansy, A., Molnár, K. and Baska, F. (1998). Development of Thelohanellus hovorkai and Thelohanellus nikolskii (Myxosporea : Myxozoa) in oligochaete alternate hosts. Fish Pathology 33, 107–114. Székely, C., Molnár, K., Eszterbauer, E. and Baska, F. (1999). Experimental detection of the actinospores of Myxobolus pseudodispar (Myxosporea: Myxobolidae) in oligochaete alternate hosts. Diseases of Aquatic Organisms 38, 219–224. Székely, C., Molnár, K. and Rácz, O. (2001). Complete developmental cycle of Myxobolus pseudodispar (Gorbunova) (Myxosporea: Myxobolidae). Journal of Fish Diseases 24,

Page | 48

Chapter I | General Introduction

461–468. Székely, C., Rácz, O., Molnár, K. and Eszterbauer, E. (2002). Development of Myxobolus macrocapsularis (Myxosporea: Myxobolidae) in an oligochaete alternate host, Tubifex tubifex. Diseases of Aquatic Organisms 48, 117–123. Tripathi, Y.R. (1948). Some new Myxosporidia from Plymouth with a proposed new classification of the order. Parasitology 39, 110–118. Tun, T., Yokoyama, H., Ogawa, K. and Wakabayashi, H. (2000). Myxosporeans and their hyperparasitic microsporeans in the intestine of emaciated tiger puffer. Fish Pathology 35, 145–156. Uspenskaya, A.V. (1957). The ecology and spreading of the pathogen of Myxosoma cerebralis of trout in the fish ponds of the Soviet Union. In Petrushevski, G.K. (ed). Parasites and diseases of fish. Bulletin of the Institute of Freshwater Fisheries, Leningrad. Ventura, M.T. and Paperna, I. (1984). Histopathology of Myxidium giardi Cépède, 1906 infection in European eels, Anguilla anguilla L., in Portugal. Aquaculture 43, 357–368. Weidner, E. and Overstreet, R.M. (1979). Sporogenesis of a myxosporidan with motile spores. Cell and Tissue Research 201, 331–342. Weill, R. (1938). L’interprétation des Cnidosporidies et la valeur taxonomique de leur cnidome. Leur cycle comparé à la phase larvaire des Narcomeduses cuninides. Travaux de la Station Zoologique de Wimereaux 13, 727–744. Weiss, L.M. and Becnel, J.J. (2014). Microsporidia: Pathogens of Opportunity, First edition. Wiley Blackwell, Oxford, UK, pp. 728. Whipps, C.M. (2011). Interrenal disease in bluegills (Lepomis macrochirus) caused by a new genus and species of Myxozoan. Journal of Parasitology 97, 1159–1165. Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215–219. Whipps, C.M., Grossel, G., Adlard, R.D., Yokoyama, H., Bryant, M.S., Munday, B.L. and Kent, M.L. (2004). Phylogeny of the multivalvulidae (Myxozoa: Myxosporea) based on comparative ribosomal DNA sequence analysis. Journal of Parasitology 90, 618–622. Whipps, C.M. and Kent, M.L. (2006). Phylogeography of the cosmopolitan marine parasite Kudoa thyrsites (Myxozoa: Myxosporea). Journal of Eukaryotic Microbiology 53, 364– 373. Wolf, K. and Markiw, M.E. (1984). Biology contravenes taxonomy in the Myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science 225, 1449– 1452. Xiao, C. and Desser, S.S. (1998a). Actinosporean stages of myxozoan parasites of

Page | 49

Chapter I | General Introduction

oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of triactinomyxon and raabeia. Journal of Parasitology 84, 998–1009. Xiao, C. and Desser, S.S. (1998b). Actinosporean stages of myxozoan parasites of oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of echinactinomyxon, neoactinomyxum, aurantiactinomyxon, guyenotia, synactinomyxon and antonactinomyxon. Journal of Parasitology 84, 1010–1019. Yanagida, T., Nomura, Y., Kimura, T., Fukuda, Y., Yokoyama, H. and Ogawa, K. (2004). Molecular and morphological redescriptions of enteric myxozoans, Enteromyxum leei (formerly Myxidium sp. TP) and Enteromyxum fugu comb. n. (syn. Myxidium fugu) from cultured tiger puffer. Fish Pathology 39, 137–143. Yokoyama, H. and Masuda, K. (2001). Kudoa sp. (Myxozoa) causing a post-mortem myoliquefaction of North-Pacific giant octopus Paroctopus dofleini (Cephalopoda: Octopodidae). Bulletin of the European Association of Fish Pathologists 21, 266–268. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1993a). Involvement of Branchiura sowerbyi (Oligochaeta, Annelida) in the transmission of Hoferellus carassii (Myxosporea, Myxozoa), the causative agent of kidney enlargement disease (KED) of goldfish Carassius auratus. Fish Pathology 28, 135–139. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1993b). Some biological characteristics of actinosporeans from the oligochaete Branchiura sowerbyi. Diseases of Aquatic Organisms 17, 223–228. Yokoyama, H. and Urawa, S. (1997). Fluorescent labelling of actinospores for determining the portals of entry into fish. Diseases of Aquatic Organisms 30, 165–169. Yokoyama, H., Yanagida, T., Freeman, M.A., Katagiri, T., Hosokawa, A., Endo, M., Hirai, M. and Takagi, S. (2010). Molecular diagnosis of Myxobolus spirosulcatus associated with encephalomyelitis of cultured yellowtail, Seriola quinqueradiata Temminck & Schlegel. Journal of Fish Diseases 33, 939–946. Zhang, J.Y., Gu, Z.M., Kalavati, C., Eiras. J.C., Liu, Y., Guo, Q.Y. and Molnár, K. (2013). Synopsis of the species of Thelohanellus Kudo, 1933 (Myxozoa: Myxosporea: Bivalvulida). Systematic Parasitology 86, 235–256. Zhang, Z.Q. (2011). Animal biodiversity: an outline of higher-level classification and taxonomic richness. Zootaxa 3148, 7–12. Zhao D, Borkhanuddin, M.H., Wang, W., Liu, Y., Cech, G., Zhai, Y. and Székely, C. (2016). The life cycle of Thelohanellus kitauei (Myxozoa: Myxosporea) infecting common carp (Cyprinus carpio) involves aurantiactinomyxon in Branchiura sowerbyi. Parasitology Research 115, 4317–4325. Zhao, D.D., Zhai, Y.H., Liu, Y., Wang, S.J. and Gu, Z.M. (2017). Involvement of aurantiactinomyxon in the life cycle of Thelohanellus testudineus (Cnidaria:

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Chapter I | General Introduction

Myxosporea) from allogynogenetic gibel carp Carassius auratus gibelio, with morphological, ultrastructural, and molecular analysis. Parasitology Research 116, 2449–2456. Zrzavý, J. (2001). The interrelationships of metazoan parasites: a review of phylum- and higher-level hypotheses from recent morphological and molecular phylogenetic analyses. Folia Parasitologica 48, 81–103. Zrzavý, J. and Hypša, V. (2003). Myxozoa, Polypodium, and the origin of the Bilateria: the phylogenetic position of “Endocnidozoa” in light of the rediscovery of Buddenbrockia. Cladistics 19, 164–169.

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Chapter II

Study aims and approach

Chapter II | Study aims and approach

Study aims

Considering the ecological and economic importance of myxosporeans, this thesis aimed to provide novel information regarding the biodiversity of these parasites in Portuguese estuaries. In this context, its main objectives were the following:

I. Identify myxosporean species developing in fish hosts;

II. Identify actinosporean stages developing in annelid hosts;

III. Characterize both myxosporean and actinosporean stages using morphological, morphometric and molecular data, as well as ultrastructural data when necessary;

IV. Expand the available Myxosporea genetic database in order to allow unequivocal species identification;

V. Clarify potential life cycles through DNA match of myxosporean and actinosporean SSU rDNA sequences;

VI. Study the phylogenetic relationships of myxosporean species in order to infer possible evolutionary patterns;

VII. Review taxonomic issues in light of updated morphological and molecular analyses.

Study approach

In order to pursue the objectives proposed in this thesis, myxosporean surveys were conducted in three study areas: the Alvor estuary near “Portimão”; the Minho estuary near the villages of “Vila Nova de Cerveira” and “Caminha”; and the Douro estuary near “Cabedelo”. In the past few years, our research group had gain access to the diversity of myxosporeans inhabiting the Alvor estuary by studying the occurrence of this group in reared European seabass Dicentrarchus labrax (Linnaeus, 1758) and gilthead seabream Sparus aurata Linnaeus, 1758, as well as in annelids collected from the earth ponds in which specimens of these two species are grown separately. A work and study that was further developed in this thesis. In turn, the performance of myxosporean surveys in the River Minho was pioneer, given the absolute lack of prior information regarding the occurrence of this group of parasites in the

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Chapter II | Study aims and approach vertebrate and invertebrate communities inhabiting this water resource. Samplings of fish and annelids were performed from a freshwater location in the upper estuary, near the village of “Vila Nova de Cerveira”, and from the brackish waters of the lower estuary, near the village of “Caminha”. Fish samples included both endemic and native fish species. Endemic species were represented by specimens of the “bermejuela” Achondrostoma arcasii (Steindachner, 1866) (Cypriniformes, Leuciscidae), the Northern straight-mouth nase Pseudochondrostoma duriense (Coelho, 1985) (Cypriniformes, Leuciscidae), and the Southern Iberian spined-loach Cobitis paludica (de Buen, 1930) (Cypriniformes, Cobitidae, Cobitinae). Native fish were represented by three distinct species of grey mullets, namely thinlip grey mullet Chelon ramada (Risso, 1827), thicklip grey mullet Chelon labrosus (Risso, 1827) and flathead grey mullet M. cephalus. With regard to annelids, the establishment of these two ecologically distinct sampling sites allowed the collection of species belonging to intrinsically different invertebrate communities. In the River Douro, only annelids were analysed, in an attempt to provide support for the myxosporean/actinosporean correlations that were hypothesized from early results obtained from the Alvor and Minho estuaries. Overall, the myxosporean and actinosporean stages detected in the fish and annelids examined, were analysed using combined microscopic and molecular tools. Light microscopy observations were complemented with studies of transmission electron microscopy when necessary. Molecular analyses were based on the sequencing of the SSU rDNA gene, given that it constitutes the molecular marker that is more commonly used for reconstructing myxosporean phylogeny. Phylogenetic analyses were performed in order to recognize evolutionary patterns and drivers of myxosporean evolution.

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Chapter III

Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae)

This chapter was adapted from:

Chapter III | Description of Ceratomyxa auratae n. sp.

Abstract

A new myxosporean parasite is described from the gall bladder of the gilthead seabream Sparus aurata in a Southern Portuguese fish farm, with basis on light and transmission electron microscopy, as well as in molecular procedures. In the bile, young and mature mono- to disporic plasmodia were elliptical and presented smooth surface membranes. Crescent- shaped myxospores measured 6.7 ± 0.7 (5.3–7.6) μm in length and 27.0 ± 3.0 (19.7–31.2) μm in thickness. The myxospore wall was constituted by two symmetrical valves united along a slightly curved suture line, each presenting a lateral projection with a rounded end. Two equal- sized subspherical polar capsules, measuring 3.6 ± 0.2 (2.9–3.8) μm in length and 3.5 ± 0.3 (2.9–3.8) μm in width, were located at the same level, each displaying a polar tubule coiled in 5 turns. Molecular analysis of the SSU rDNA gene confirmed the parasite as a new member of the genus Ceratomyxa, making this the fourth report of Ceratomyxa from the gall bladder of S. aurata in the Iberian Peninsula. This reinforces the assumption that species richness of ceratomyxids in South European sparids is high, but the phylogenetic analysis performed disagrees with the existence of a common ancestor for Ceratomyxa species infecting sparid hosts, as well as with clustering according to geographical location. The main Ceratomyxa clade is not monophyletic due to the inclusion of Palliatus indecorus and Pseudoalatospora kovalevae; a situation that will probably be resolved by the taxonomic revision of these genera.

Introduction

Several members of the family Sparidae Rafinesque, 1810 are commercially important for both wild fisheries and aquaculture in South European countries, with the gilthead seabream Sparus aurata Linnaeus, 1758 being a significant example. Despite the economic importance of this fish species for Portuguese fish farms, there are only two recent studies that provide information regarding the myxosporean parasites infecting the gilthead seabream in Portuguese aquaculture facilities. Rocha et al. (2013) reported Zschokkella auratis infecting the gall bladder of this host, and Rangel et al. (2014) reported Ortholinea auratae infecting the urinary bladder and posterior kidney. Elsewhere another eight species of Myxosporea have been reported to infect this host, some of which are associated with increased morbidity and mortality rates. Kudoa iwatai, which is a systemic species (Diamant et al., 2005); Enteromyxum leei (formerly Myxidium leei) in the intestinal tract (Diamant et al., 1994); a Henneguya sp. in the gills and heart (Bahri et al., 1996); Sphaerospora sparidarum (formerly Leptotheca sparidarum) (Sitjà-Bobadilla and Alvarez-Pellitero, 2001) and Sphaerospora sparis (formerly Polysporoplasma sparis) (Sitjà-Bobadilla et al., 1992; Sitjà-Bobadilla and Alvarez-Pellitero, 1995; Bartošová et al., 2013) in the kidney; Ceratomyxa sparusaurati (Sitjà-Bobadilla et al.,

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Chapter III | Description of Ceratomyxa auratae n. sp.

1995), Ceratomyxa sp. 1 ex S. aurata and Ceratomyxa sp. 2 ex S. aurata (Alama-Bermejo et al., 2011), all in the gall bladder. The genus Ceratomyxa Thélohan, 1892 constitutes one of the largest myxosporean taxa, comprising about 300 species, the great majority of which are coelozoic in the gall bladder of marine teleosts, exceptionally appearing in elasmobranchs. Five species have been described from the freshwater environment, but only Ceratomyxa hongtzensis infects a strictly freshwater host, the bagrid catfish Pelteobagrus eupogon (Eiras, 2006; Lom and Dyková, 2006). Ceratomyxa have elongated, crescent-shaped or arcuate myxospores with valves that are frequently conical, exceeding in length the axial diameter of the spore. Myxospores contain two subspherical polar capsules and a binucleate sporoplasm or, more rarely, two uninucleate sporoplasms (Lom and Dyková, 1992, 2006). The majority of Ceratomyxa spp. appear to be relatively harmless, probably due to their coelozoic development. Few reports document the occurrence of pathological damage to host tissues, especially in less studied non-commercial fish species (Gunter et al., 2009). An example of a pathogenic Ceratomyxa spp. is C. sparusaurati, which despite being coelozoic in the gall bladder of S. aurata, causes serious histopathological damages that are associated with increased mortalities (Palenzuela et al., 1997). Despite spore morphology constituting a useful tool for species comparison, phylogenetic studies show that it is not the most reliable criterion for recognizing myxosporean taxonomy, further revealing that the type of aquatic environment, tissue tropism and host relatedness are more phylogenetically informative criteria (Kent et al., 2001; Eszterbauer, 2004; Holzer et al., 2004; Fiala, 2006; Carriero et al., 2013; Rocha et al., 2013). For the molecular analysis of Ceratomyxa, GenBank provides the SSU rDNA sequences of a significant number of species that, however, are all coelozoic in teleosts exclusively inhabiting brackish/marine environments. The only exception is the SSU rDNA sequence of Ceratomyxa diamanti, which was obtained from the gall bladder of a catadromous teleost, Polydactylus macrochir, during its freshwater rearing in the Brisbane River, Queensland, Australia (Gunter et al., 2009). No molecular data are available for Ceratomyxa spp. infecting freshwater hosts; at least not since the genus Ceratonova was erected to encompass Ceratonova shasta syn. Ceratomyxa shasta (Noble 1950) as type species, and Cn. gasterostea as its only congener (Atkinson et al., 2014). Ceratonova shasta constituted a long-standing taxonomic outlier to all other Ceratomyxa spp. due to its histozoic development in the intestinal epithelium, its freshwater life cycle, and its phylogenetic distance (Gunter et al., 2009; Atkinson and Bartholomew, 2010; Atkinson et al., 2014) The present study seeks to describe the fourth ceratomyxid species found infecting the gall bladder of S. aurata, providing a reliable comparative basis for future parasitological surveys concerning this parasite genus and fish host.

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Chapter III | Description of Ceratomyxa auratae n. sp.

Materials and methods

Fish and parasite sampling

Between June 2012 and June 2014, two hundred and sixty specimens of the gilthead seabream S. aurata Linnaeus, 1758 (Teleostei: Sparidae) were obtained from a fish farm in the Alvor estuary, near the Atlantic coast (37° 08′ N, 08° 37′ W), Portimão, Algarve, Portugal. The fish were collected from monthly stocks for commercialization, so they were slaughtered in accordance with the animal ethical laws stipulated for the aquaculture industry in Portugal and then shipped to the laboratory within thermic containers filled with chipped ice. Upon arrival, the dissections of fish were performed for the parasitological survey of several organs and tissues, with preliminary microscopic observations revealing myxosporean plasmodia and myxospores parasitizing the gall bladder of some specimens. The bile of the parasitized fish was collected and prepared for light microscopy, transmission electron microscopy (TEM) and molecular procedures.

Light microscopy and morphological analysis of myxospores

The parasitic stages in the bile were examined and photographed using a Zeiss Axiophot microscope (Grupo Taper, Sintra, Portugal), equipped with a Zeiss Axiocam digital camera Icc3. Axiovision 4.6 software (Grupo Taper, Sintra, Portugal) was used for image analysis. Morphometry was determined from fresh material, in accordance with Lom and Arthur (1989). All measurements include the mean value ± standard deviations (S.D.), range of variation and number of myxospores measured (range, n).

Transmission electron microscopy

Free myxospores and plasmodia were isolated from the bile and fixed in 5% glutaraldehyde buffered in 0.2 M sodium cacodylate (pH 7.4) for 20–24 h, washed in the same buffer, and post-fixed in 2% osmium tetroxide also buffered with 0.2 M sodium cacodylate (pH 7.4) for 3–4 h. All these steps were performed at 4 °C. The samples were then dehydrated in an ascending graded series of ethanol, followed by embedding using a series of oxide propylene and Epon mixtures, ending in EPON. Semithin sections were stained with methylene blue-Azure II. Ultrathin sections were double-contrasted with uranyl acetate and lead citrate, and then examined and photographed using a JEOL 100CXII TEM (JEOL Optical, Tokyo, Japan), operating at 60 kV.

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Chapter III | Description of Ceratomyxa auratae n. sp.

DNA extraction, amplification and sequencing

Free myxospores and plasmodia were obtained from the bile of four infected specimens and then fixed and preserved in absolute ethanol at 4 °C. Genomic DNA extraction was performed using a GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, USA), following the manufacturer's instructions. The DNA was stored in 50 μl of TE buffer at −20 °C until further use. The SSU rDNA gene was amplified and sequenced using both universal primers and myxosporean-specific primers (Table 1). PCRs were performed in 50 μl reactions using 10 pmol of each primer, 10 nmol of each dNTP, 2.5 mM of MgCl2, 5 μl 10 × Taq polymerase buffer, 1.5 units Taq DNA polymerase (NZYTech, Lisbon, Portugal), and 3 μl (approximately 100–150 ng) of genomic DNA. The reactions were run on a Hybaid PxE Thermocycler (Thermo Electron Corporation, Milford, Massachusetts, USA), with initial denaturation at 95 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 53 °C for 45 s, and 72 °C for 90 s. The final elongation step was performed at 72 °C for 7 min. Five-μl aliquots of the PCR products were electrophoresed through a 1% agarose 1× Tris-acetate-EDTA buffer (TAE) gel stained with ethidium bromide. PCR products were purified using the ExoFast method, in which an enzymatic cleanup that eliminates unincorporated primers and dNTPs is performed with Exonuclease I (Escherichia coli) and FastAP Thermosensitive (SAP). The PCR products from different regions of the SSU rDNA gene were sequenced directly. The sequencing reactions were performed using a BigDye Terminator v1.1 from the Applied Biosystems kit (Applied Biosystems, Carlsbad, California, USA), and were run on an ABI3700 DNA analyzer (Perkin-Elmer, Applied Biosystems, Stabvida, Oeiras, Portugal).

Table 1. Polymerase chain reaction primers used for the amplification and sequencing of the SSU rDNA gene of Ceratomyxa auratae n. sp.

Name Sequence (5’-3’) Paired with Source 18E CTG GTT GAT CCT GCC AGT CeratR1, MYX4R Hillis and Dixon, 1991 MyxospecF TTC TGC CCT ATC AAC TTG TTG MYX4R, 18R Fiala, 2006 MYX4F GTT CGT GGA GTG ATC TGT CAG 18R Present study CeratR1 CCA ATG TCT GGA TTG GGT A 18E Present study MYX4R CTG ACA GAT CAC TCC ACG AAC 18E, MyxospecF Hallett and Diamant, 2001 18R CTA CGG AAA CCT TGT TAC G MyxospecF, MYX4F Whipps et al., 2003

Distance and phylogenetic analysis

The forward and reverse segments sequenced were manually aligned with ClustalW (Thompson et al., 1994) in MEGA 5 software (Tamura et al., 2011), and ambiguous bases were clarified using corresponding ABI chromatograms. To determine the phylogenetic

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Chapter III | Description of Ceratomyxa auratae n. sp. position of the new sequence of Ceratomyxa among its closest relatives sequenced to date, 61 myxosporean SSU rDNA sequences were obtained from GenBank and analysed. Thirty of these sequences belonged to species of the genus Ceratomyxa and were chosen according to highest similarity score, and so were Palliatus indecorus (DQ377712) and Pseudoalataspora kovalevae (JX467675). Tetracapsuloides bryosalmonae (U70623) and Buddenbrockia plumatellae (AY074915) were selected as outgroup. The remaining SSU rDNA sequences used in the analysis were selected according to aquatic environment and organ of infection. Phylogenetic and molecular evolutionary analyses were conducted using MEGA 5.05. Alignments were performed using ClustalW, with an opening gap penalty of 10 and a gap extension of four for both paired and multiple alignments (Tamura et al., 2011). The phylogenetic analysis was performed using maximum likelihood (ML), neighbor- joining (NJ) and maximum parsimony (MP) methodologies. For ML, the general time reversible substitution model with 4 gamma-distributed rate variation among sites was performed. For NJ, Kimura 2-parameter as substitution model with gamma distribution (shape parameter = 1.4) was performed. For MP, the close neighbor interchange (CNI) heuristic option with a search factor of 1 and random initial trees addition of 500 replicates was performed. All positions with less than 95% site coverage were eliminated from all trees, resulting in a total of 872 positions in the final dataset. The bootstrap consensus tree was inferred from 500 replicates for ML, NJ and MP. Distance estimation was performed for a second alignment of the SSU rDNA sequences clustering together with the parasite described here and the other three Ceratomyxa spp. that occur in the gall bladder of S. aurata, resulting in a total of 867 positions in the final dataset. This analysis was also carried out in MEGA 5.05, using the Kimura-2 parameters model distance matrix for transitions and transversions, with all gaps and missing data were eliminated.

Results

Characterization of Ceratomyxa auratae n. sp. (Figs. 1‒3)

Taxonomic placement Phylum Myxozoa Grassé, 1970; Class Myxosporea Bütschli, 1881; Order Bivalvulida Shulman, 1959; Family Ceratomyxidae Doflein, 1899; Genus Ceratomyxa Thélohan, 1892

Morphological description and taxonomic summary Light microscopy: Subspherical to elliptical plasmodia, in different stages of maturation, as well as mature myxospores were observed floating freely in the bile (Fig. 1A, B).

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Chapter III | Description of Ceratomyxa auratae n. sp.

Figure 1. Light and transmission electron micrographs of Ceratomyxa auratae n. sp. infecting the gall bladder of Sparus aurata. (A) Young and mature plasmodia (Pl) and myxospores (S) observed in DIC optics. (B) Mature plasmodia (Pl), each containing a crescent-shaped myxospore, as observed in DIC optics. (C) Ultrathin section of young and subspherical plasmodia containing developing myxospores (S) and displaying the cytoplasm riddled with vesicles (V) and lipidic droplets (Li). (D) Ultrathin section of a mature elliptical plasmodium (Pl) containing a myxospore (S). (E) Ultrathin section of a plasmodium (Pl) showing two capsulogenic cells (CC) in different developmental stages: one displaying the capsular primordium constituted by a nucleus (N) and rough endoplasmic reticulum (RER), and the other, more developed, showing the polar tubule (PF) coiled within the globular structure of the maturing polar capsule. (F) Ultrastructural detail of the capsular primordium evidencing some of its constituents: the nucleus (N), the rough endoplasmic reticulum (RER) and mitochondria (Mt). (G) Globular capsular primordium (Cp) extending into an external tubule (arrow), which will latter invert and coil along the inner wall of the polar capsule, originating the polar tubule.

Myxospores are elongated and crescent-shaped with rounded ends, 6.7 ± 0.7 (5.3–7.6) μm

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Chapter III | Description of Ceratomyxa auratae n. sp. long (n = 16) and 27.0 ± 3.0 (19.7–31.2) μm thick (n = 18). Two equal-sized subspherical polar capsules, 3.6 ± 0.2 (2.9–3.8) μm long (n = 19) and 3.5 ± 0.3 (2.9–3.8) μm wide (n = 19), are located at the same level at the myxospores' anterior pole (Fig. 1A, B). Ultrastructure: Young plasmodia subspherical and mature plasmodia elliptical, all presenting a smooth surface membrane and the cytoplasm riddled with mitochondria and vesicles containing granular material of different electronic densities, as well as some lipidic droplets and large vacuoles. Sporogenic development is mono- to disporic (Fig. 1C, D). The earliest stages of sporogenesis observed were young sporoblasts comprised by two large and single nucleated valvogenic cells surrounding the developing sporoplasmogenic cell and capsulogenic cells (Figs. 1E–G, 2A, B). At the beginning of its differentiation, each capsulogenic cell formed a capsular primordium composed by a cylindrical formation of microtubules surrounded by rough endoplasmic reticulum and numerous mitochondria, and containing a single nucleus (Fig. 1E, F). In more advanced stages, the capsular primordium assumed a globular shape and extended into an external tubule (Fig. 1G). The inversion and coiling of this tubule along the inner wall of the globular shape originates the polar tubule that characterizes the polar capsules (Fig. 2C, D). Myxospores constituted by two symmetrical valves, thick and smooth, united along a slightly curved suture line (Fig. 2E) and each presenting a lateral projection with a rounded end (Figs. 1A, B, 2B). Within, two polar capsules displaying a double-layered wall, comprised by a thinner outer electron-dense layer and a thicker inner electron-lucent layer, and containing a dense and homogeneous matrix where the polar tubule coils in 5 turns (Fig. 2A, C, D). The sporoplasm, located at the posterior pole, presented two nuclei and many electron-dense sporoplasmosomes. A semischematic drawing of a myxospore in sutural view is presented in Fig. 3, also incorporating ultrastructural features. Type host: The gilthead seabream Sparus aurata Linnaeus, 1758 (Teleostei: Sparidae). Type locality: Alvor estuary, near the Atlantic coast (37° 08′ N, 08° 37′ W), Portimão, Algarve, Portugal. Site of infection: within the gall bladder. Prevalence: 3.8% (ten infected in a total of 260 examined). Pathogenicity: Collected and analysed fish did not present external symptoms of infection or disease, neither was mortality recorded from the stock used. Type material: One glass slide with semithin sections of plasmodia containing several myxospores of the hapantotype deposited in the Type Slide Collection of the Laboratory of Animal Pathology at the Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2015.7. Identical sequences of the SSU rDNA gene were obtained from samples belonging to four infected specimens; a total of 1,846 bp were deposited in GenBank (Accession number KP765721). Etymology: “auratae” derives from the specific epithet of the host species.

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Chapter III | Description of Ceratomyxa auratae n. sp.

Figure 2. Transmission electron micrographs of Ceratomyxa auratae n. sp. infecting the gall bladder of Sparus aurata. (A) Developing myxospore showing two valvogenic cells (VC) surrounding two capsulogenic cells (CC). Each of the latter exhibits a reminiscent nucleus (N). Also notice one of the two nuclei (*) of the sporoplasmogenic cell. (B) Plasmodium (Pl) containing two myxospores (S), one of which displays the sporoplasmogenic cell (SPC) and its nucleus (*) appearing adjacent to one of the capsulogenic cells (CC). Notice the nucleus (arrow) of one of the two valvogenic cells. (C) Capsulogenic cell (CC) displaying its nucleus (N) and the almost matured polar capsule in longitudinal section. Notice the stopper (arrow) located at the apex of the latter, and the polar tubule (PF) coiling within. (D) Longitudinal section of a polar capsule displaying its double-layered wall (arrowheads) capped by a stopper (arrow) at the apex and containing a homogenous matrix (*) where the polar tubule (PF) coils. (E) Ultrastructural detail of the two valves united along a slightly curved suture line (arrowheads).

Molecular analysis

Four partial sequences of the SSU rDNA gene were obtained from parasitic material collected from four infected host individuals. These sequences were identical and a consensus DNA sequence with 1,846 bp was deposited in GenBank (accession number KP765721). To determine the phylogenetic position of the new sequence obtained here and its relation to closely related taxa, a total of 63 SSU rDNA sequences were retrieved from GenBank, 30 of which refer to Ceratomyxa spp., and included those with higher BLAST scores and maximum identity. ML, NJ, and MP phylogenetic trees constructed for the selected SSU rDNA sequences

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Chapter III | Description of Ceratomyxa auratae n. sp.

revealed the parasite clustering with all other Ceratomyxa spp. analysed (Fig. 4). The formation of this large Ceratomyxa clade, marine and coelozoic, appears supported by bootstrap values Figure 3. Schematic drawing of a myxospore of Ceratomyxa auratae n. of 96% for ML, 97% for NJ and sp., depicting the internal and external organizations described in the text. 86% for MP. Also clustering within this clade are P. kovalevae and P. indecorus. Other marine myxosporeans that are coelozoic in the gall bladder (Sphaeromyxa kenti, S. zaharoni, S. hellandi, Zschokkella sp. PS030203 and Z. auratis) do not cluster within the previous clade, but rather with freshwater myxosporeans that are also coelozoic in the gall bladder (Sphaerospora sp. 2 EE-2004, Zschokkella sp. DDI-2008 and Z. parasiluri). Also considered in the phylogenetic analysis, the freshwater and histozoic parasite Cn. shasta and its congener, Cn. gasterostea, appear related to a clade containing marine myxosporeans that are histozoic (Enteromyxum scophthalmi, E. leei, K. ogawai, K. iwatai, K. hypoepicardialis and K. leptacanthae) or that infect the excretory system (Zschokkella sp. 4 IF-2006, Z. lophii and Sinuolinea phyllopteryxa) rather than to the clade containing freshwater species that are histozoic (Myxobolus pyramidis, M. pellicides, M. bibullatus, Henneguya rhinogobii and H. pseudorhinogobii) or that infect the excretory system of their fish hosts (Myxobilatus gasterostei, Ortholinea auratae, Zschokkella sp. AH2003, Myxidium giardi and Sphaerospora oncorhynchi). Pairwise comparisons among the SSU rDNA sequences that cluster together with the parasite described here, or that share the same organ in the gilthead seabream, resulted in percentages of similarity lower than 90%. The only exception is Ceratomyxa gurnardi, with a percentage of identity of 92% (Table 2).

Discussion

The characteristics of the myxosporean species described here are consistent with those defined for the genus Ceratomyxa according to traditional criteria, namely in terms of myxospore morphology (Lom and Dyková, 2006; Atkinson et al., 2014). Nevertheless, phylogenetic studies show that this criterion is not the most reliable for myxosporean differentiation, which should include molecular analyses, typically of the SSU rDNA gene (Kent et al., 2001; Eszterbauer, 2004; Holzer et al., 2004; Fiala, 2006; Bartošová et al., 2009; Fiala and Bartošová, 2010; Carriero et al., 2013; Rocha et al., 2013). In fact, the use of molecular tools has recently led to the erection of a new ceratomyxid taxon, the genus Ceratonova, in order to more appropriately incorporate species that, while being morphologically very

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Chapter III | Description of Ceratomyxa auratae n. sp.

Table 2. Percentage of similarity for the small subunit rDNA sequences of Ceratomyxa species that cluster together with Ceratomyxa auratae n. sp., as well as for those that also infect the gall bladder of Sparus aurata.

ID Species GenBank 1 2 3 4 5 6 7 8 9 10 11 12

(1) Ceratomyxa auratae n. sp. (KP765721) -

(2) Ceratomyxa gurnardi (JQ071439) 92.0 -

(3) Ceratomyxa hallettae (FJ204248) 89.9 89.3 -

(4) Ceratomyxa cyanosomae (JX971424) 89.4 88.8 91.5 -

(5) Ceratomyxa sp. 2 ex S. aurata (JF820293) 89.3 88.0 88.4 87.0 -

(6) Ceratomyxa gleesoni (EU729693) 89.0 88.4 90.2 88.7 85.3 -

(7) Ceratomyxa sparusaurati (AF411471) 88.9 87.7 88.6 87.2 99.1 85.1 -

(8) Ceratomyxa ostorhinchi (JX971425) 88.5 88.9 91.2 96.0 86.8 88.9 86.8 -

(9) Ceratomyxa thalassomae (EU045332) 88.3 89.1 90.9 95.8 86.0 89.0 86.7 95.7 -

(10) Ceratomyxa rueppellii (JX971423) 88.3 88.8 94.4 91.0 86.4 90.1 85.9 90.5 91.0 -

(11) Ceratomyxa robertsthomsoni (FJ204253) 87.6 87.9 93.8 90.6 85.5 89.8 85.1 90.1 90.2 98.1 -

(12) Ceratomyxa sp. 1 ex S. aurata (FJ820292) 87.2 86.0 87.2 85.1 96.9 84.1 97.7 84.7 83.5 83.9 83.0 - similar to Ceratomyxa, are phylogenetically distant (Atkinson et al., 2014). Molecular comparison between Ceratomyxa spp. is restricted by the general lack of molecular data available for more diversified species of this genus, specifically in terms of host taxonomy, aquatic environment and geographic location. As such, the morphological criterion regains its importance as a taxonomic tool for species differentiation. Acknowledging the widely accepted phylogenetic criteria of tissue tropism, aquatic environment and host affinity for Myxosporea (Kent et al., 2011; Eszterbauer, 2004; Holzer et al., 2004; Fiala, 2006; Bartošová et al., 2009; Fiala and Bartošová, 2010; Carriero et al., 2013; Rocha et al., 2013), the morphological comparison performed in this study first took into consideration all the Ceratomyxa spp. that are coelozoic in the gall bladder of sparid hosts (and more specifically those previously described from the gall bladder of S. aurata) (Table 3). No significant morphometric and morphologic resemblance to Ceratomyxa auratae n. sp. was found. Further morphological comparison to other Ceratomyxa infecting the gall bladder of brackish/marine teleosts, especially those without molecular data, also revealed no gross similarity to C. auratae n. sp., despite the fact that many reports are outdated and lack reliable data for comparative purposes (for instance, Ceratomyxa herouardi and Ceratomyxa coris are without measurements) (Eiras, 2006). Overall, the results presented here are consistent with previous studies (Kent et al., 2001; Fiala, 2006; Gunter et al., 2009), through supporting the existence of a close phylogenetic relationship among all Ceratomyxa species. Now that the position of Cn. shasta has been resolved by the erection of its own genus within the family Ceratomyxidae (Atkinson et al., 2014), the remaining exceptions to monophyly in the Ceratomyxa clade are P. indecorus and P. kovalevae. The placement of P. indecorus within the large Ceratomyxa clade was noticed by Fiala (2006) and, since then, was supported by other phylogenetic studies (Gunter

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Chapter III | Description of Ceratomyxa auratae n. sp.

spp. that infect the gall bladder of sparid hosts. SL, myxospore length; ST, myxospore thickness; PCL, polar capsule capsule polar PCL, thickness; myxospore ST, length; myxospore SL, hosts. sparid of bladder gall the infect that spp.

Ceratomyxa

Morphological comparison between comparison Morphological

Table 3. Table in givenµm. are coils.Measurements tubulepolar capsule length; polarwidth; PTc, PCW,

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Chapter III | Description of Ceratomyxa auratae n. sp.

et al., 2009; Alama-Bermejo et al., 2011). Lom and Dyková (2006) describe Palliatus species as coelozoic in the gall bladder of marine fish, characterized by myxospores that are subspherical with an anteriorly prominent sutural ridge, and enveloped in a membranaceus veil that in non-mature spores is twisted in two cords around the myxospore. The genus Palliatus Kovaleva and Dubina, 1979 currently belongs to the family Sphaerosporidae Davis, 1917 but several studies have suggested that a taxonomic revision of this group may be required. Shulman et al. (1997) proposed the erection of a new taxon, the family Palliatidae, for this genus. Conversely, Gunter et al. (2009) believed that this genus would be more appropriately assigned to the family Ceratomyxidae, based both on the molecular analysis of the SSU rDNA gene, and on tissue tropism (members of Sphaerosporidae mainly infect the urinary system of their hosts, while all Palliatus spp. infect the gall bladder). Considering the phylogenetic placement of P. indecorus in our study and, without disregarding tissue tropism and aspects of the generic morphology, it seems correct that this genus should be attributed to the family Ceratomyxidae. Nevertheless, further SSU rDNA sequences from Palliatus, particularly from the type species Palliatus mirabilis Shulman et al., 1979 are necessary before the taxonomic position of this genus is clarified. In turn, the SSU rDNA sequence of P. kovalevae constitutes the only molecular data available for the genus Pseudoalatospora Kovaleva and Gaevskaya, 1983, which Lom and Dyková (1992) describe as comprising myxosporean parasites that are coelozoic in the gall bladder of marine fish, with myxospores that are extremely elongated in the plane perpendicular to the central straight sutural line and display membranaceus projections adhering along their posterior half. These projections are doubled to form parachute-like pockets. The genus Pseudoalatospora currently belongs to the family Alatosporidae Shulman et al., 1979 but shares similar morphological characters with Ceratomyxa, differing only in the presence/absence of delicate alate processes. According to Kalavati et al. (2013), these alate processes are fragile and difficult to observe, raising the possibility that some Pseudoalatospora spp. may be erroneously assigned to the genus Ceratomyxa. Nevertheless, taking into account the close phylogenetic relationship of P. kovalevae to Ceratomyxa spp. revealed in this study, as well as the shared tissue

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Chapter III | Description of Ceratomyxa auratae n. sp.

Figure 4. Maximum likelihood tree of the SSU rDNA sequence of Ceratomyxa auratae n. sp. and other selected myxozoan species. The numbers on the branches are bootstrap confidence levels on 500 replicates corresponding to ML/NJ/MP trees. There was a total of 872 positions in the final dataset. GenBank accession numbers in parentheses after the species name; scale is given under the tree.

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Chapter III | Description of Ceratomyxa auratae n. sp. tropism and faint morphological differences between these genera, its species would probably be more adequately assigned within the same genus, namely within the family Ceratomyxidae, which represents a stronger taxon than the family Alatosporidae. The latter contains only another genus, Alatospora Shulman et al., 1979, which also contains species that are coelozoic in the gall bladder of marine fish and display myxospore morphology highly similar to Pseudoalatospora and, therefore, also to Ceratomyxa, but lacks molecular data to corroborate phylogenetic similarity to ceratomyxids. Thus, further sequencing of both Pseudoalatospora and Alatospora will probably lead to the demise of the family Alatosporidae, with subsequent assignment of its species to the genus Ceratomyxa. Ceratonova shasta clusters with its only congener, Cn. gasterostea, and together form a subclade positioned within a clade containing marine myxosporeans that are histozoic or infect the excretory system, and that is sister to the large Ceratomyxa clade. This position was constant in all tree topologies and supports the idea that Ceratonova spp. represent an independent lineage of ceratomyxids, as suggested by Fiala (2006) for Cn. shasta. It is also noticed that Ceratonova spp. constitute exceptions to the Myxosporea main phylogenetic division into a marine clade and a freshwater clade. Considering the phylogenetic setting within the large clade of Ceratomyxa, it is noted that C. auratae n. sp. does not cluster within the subclade containing other Ceratomyxa spp. sequenced from the gall bladder of sparid hosts [C. sparusaurati, C. puntazzi, Ceratomyxa sp. 1 S. aurata, Ceratomyxa sp. 2 S. aurata, Ceratomyxa sp. ex Diplodus annularis (Palenzuela et al., 2002; Alama-Bermejo et al., 2011)], but rather within a sister subclade containing Ceratomyxa spp. that infect fish hosts of the families Triglidae (C. gurnardi), Apogonidae (C. rueppellii, C. ostorhinchi and C. cyanosomae), Serranidae (C. gleesoni), Lethrinidae (C. hallettae) and Labridae (C. thalassomae) of the order Perciformes (Heiniger et al., 2008; Gunter and Adlard, 2009; Gunter et al., 2009; Heiniger and Adlard, 2013; Sobecka et al., 2013), as well as of the family Mugilidae (Ceratomyxa robertsthomsoni) of the order Mugiliformes (Gunter et al., 2009). Ceratomyxa arabica, which was also reported from the gall bladder of a sparid fish host, Acanthopagrus bifasciatus, in the Arabian Gulf in Saudi Arabia (Al-Qahtani et al., 2015), also clusters in a separate subclade. This placement disagrees with previous studies that suggest a common ancestor for Ceratomyxa species infecting sparid hosts, as well as their clustering according to a geographical location (Alama-Bermejo et al., 2011), since C. auratae n. sp. clusters closely with species described from Australian waters (Heiniger et al., 2008; Gunter and Adlard, 2009; Gunter et al., 2009; Heiniger and Adlard, 2013) and C. gurnardi from the Atlantic Ocean off Scotland (Sobecka et al., 2013). The inclusion of C. gurnardi in this clade is, however, dubious since its sequence is rather small (949 bp) in comparison to all the other ceratomyxid SSU rDNA sequences used in the phylogenetic analysis, which account for at least 1,300 bp. Overall, this type of clustering suggests that

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Chapter III | Description of Ceratomyxa auratae n. sp. radiation with host taxa and geographical location do not drive the overall phylogeny of Ceratomyxa. With the exception of C. mylionis, C. arabica and C. acanthopagri, all other Ceratomyxa spp. infecting the gall bladder of sparid hosts have been described from the North Atlantic and Mediterranean waters bathing South European countries, as seen in Table 3. Ceratomyxa auratae n. sp. is the ninth species of its genus described from sparid hosts in South European waters, and the fourth from the gall bladder of S. aurata in the Iberian Peninsula. The description of this new ceratomyxid reinforces the assumption that species richness of Ceratomyxa in South European sparids is high and that a single sparid host species can be infected by more than a single species of Ceratomyxa (Gunter and Adlard, 2009; Alama- Bermejo et al., 2011), with or without the occurrence of co-infection. On the other hand, molecular systematics have shown that most marine Ceratomyxa are restricted to a single host (Gunter et al., 2009; Heiniger and Adlard, 2008, 2013), so reports based on morphology alone of several species of this genus from multiple hosts suggest that a significant number of known species are yet to be described. For instance, Ceratomyxa diplodae was originally described from D. annularis (Lubat et al., 1989) and later from Dicentrarchus labrax (Sitjà-Bobadilla and Alvarez-Pellitero, 1993) and Diplodus puntazzo (Katharios et al., 2007), but no molecular data were provided in any of these reports. Therefore, it is probable that the morphological variations existent between C. diplodae descriptions are not host dependent but inter-specific and represent further species richness in South European sparids. The ultrastructural study of the sporogony of C. auratae n. sp. revealed asynchronous development, with both young and mature plasmodia simultaneously present in infected gall bladders. The ultrastructural features of the plasmodial development described here are congruent with those generally recognized in coelozoic myxosporeans, i.e. the size and shape of the plasmodia vary according to the stage of the sporogonic development (Sitjà-Bobadilla and Alvarez-Pellitero, 1993, 2001; Rangel et al., 2011, 2014). However, the differentiation of peripheral projections does not occur, and the morphometric and morphological alterations incurred by the plasmodia appear associated with the growth of the myxospores, rather than with nutritional purposes. Also, although no long-term study was performed on the fish specimens parasitized by C. auratae n. sp., no gross ultrastructural damages to the gall bladder epithelium were observed, and clinical signs of infection or disease were absent, indicating that this species does not affect gravely the gilthead seabream.

Acknowledgments

This work was financially supported by FCT (Lisbon, Portugal), within the scope of the project DIRDAMyx, reference FCOMP-01-0124-FEDER-020726/FCT-PTDC/MAR/116838/

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Chapter III | Description of Ceratomyxa auratae n. sp.

2010 and the Ph.D. fellowship grants attributed to S. Rocha (SFRH/BD/92661/2013) and L. Rangel (SFRH/BD/82237/2011), through the programme QREN-POPH/FSE; the Eng. António de Almeida Foundation (Porto, Portugal); and the project EUCVOA (NORTE-07-0162-FEDER- 000116) (Portugal). This work complies with the current laws of the country where it was performed.

References

Alama-Bermejo, G., Raga, J.A. and Holzer, A.S. (2011). Host-parasite relationship of Ceratomyxa puntazzi n. sp. (Myxozoa: Myxosporea) and sharpsnout seabream Diplodus puntazzo (Walbaum, 1792) from the Mediterranean with first data on ceratomyxid host specificity in sparids. Veterinary Parasitology 182, 181–192. Al-Qahtani, H.A., Mansour, L., Al-Quraishy, S. and Abdel-Baki, A.S. (2015). Morphology, phylogeny and seasonal prevalence of Ceratomyxa arabica n. sp. (Myxozoa: Myxosporea) infecting the gallbladder of Acanthopagrus bifasciatus (Pisces: Sparidae) from the Arabian Gulf, Saudi Arabia. Parasitology Research 114, 465–471. Atkinson, S.D. and Bartholomew, J.L. (2010). Disparate infection patterns of Ceratomyxa shasta (Myxozoa) in rainbow trout (Oncorhynchus mykiss) and Chinook salmon (Oncorhynchus tshawytscha) correlate with internal transcribed spacer-1 sequence variation in the parasite. International Journal for Parasitology 40, 599–604. Atkinson, S.D., Foott, J.S. and Bartholomew, J.L. (2014). Erection of Ceratonova n. gen. (Myxosporea: Ceratomyxidae) to encompass freshwater species C. gasterostea n. sp. from threespine stickleback (Gasterosteus aculeatus) and C. shasta n. comb. from salmonid fishes. Journal of Parasitology 100, 640–645. Bahri, S., Hassine, O.K.B. and Marques, A. (1996). Henneguya sp. (Myxosporea, Bivalvulida) infecting the gills of wild gilthead sea bream Sparus aurata L., from the coast of Tunisia. Bulletin of the European Association of Fish Pathologists 16, 51–53. Bartošová, P., Fiala, I. and Hypša, V. (2009). Concatenated SSU and LSU rDNA data confirm the main evolutionary trends within myxosporeans (Myxozoa: Myxosporea) and provide an effective tool for their molecular phylogenetics. Molecular Phylogenetics and Evolution 53, 81–93. Bartošová, P., Fiala, I., Jirků, M., Cinková, M., Caffara, M., Fioravanti, M.L., Atkinson, S.D., Bartholomew, J.L. and Holzer, A.S. (2013). Sphaerospora sensu stricto: taxonomy, diversity, and evolution of a unique lineage of myxosporeans (Myxozoa). Molecular Phylogenetics and Evolution 68, 93–105. Carriero, M.M., Adriano, E.A., Silva, M.R.M., Ceccarelli, P.S. and Maia, A.A.M. (2013). Molecular phylogeny of the Myxobolus and Henneguya genera with several new South

Page | 74

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American species. PLoS One 8, e73713. Diamant, A., Lom, J. and Dyková, I. (1994). Myxidium leei n. sp., a pathogenic myxosporean of cultured sea bream Sparus aurata. Diseases of Aquatic Organisms 20, 137–141. Diamant, A., Ucko, M., Paperna, I., Colorni, A. and Lipshitz, A. (2005). Kudoa iwatai (Myxosporea: Multivalvulida) in wild and cultured fish in the Red Sea: redescription and molecular phylogeny. Journal of Parasitology 91, 1175–1189. Eiras, J.C. (2006). Synopsis of the species of Ceratomyxa Thélohan, 1892 (Myxozoa: Myxosporea: Ceratomyxidae). Systematic Parasitology 65, 49–71. Eszterbauer, E. (2004). Genetic relationship among gill-infecting Myxobolus species (Myxosporea) of cyprinids: molecular evidence of importance of tissue-specificity. Diseases of Aquatic Organisms 58, 35–40. Fiala, I. (2006). The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. International Journal for Parasitology 36, 1521–1534. Fiala, I. and Bartošová, P. (2010). History of myxozoan character evolution on the basis of rDNA and EF-2 data. BMC Evolutionary Biology 10, 228. Georgévitch, J. (1916). Note sur les myxosporidies des poissons de la baie de Vilefranche et de Monaco. Bulletin de l'Institut océanographique de Monaco 322, 1–7. Gunter, N.L. and Adlard, R.D. (2008). Bivalvulidan (Myxozoa: Myxosporea) parasites of damselfishes with description of twelve novel species from Australia's Great Barrier Reef. Parasitology 135, 1165–1178. Gunter, N.L. and Adlard, R.D. (2009). Seven new species of Ceratomyxa from the gallbladders of serranid fishes from the Great Barrier Reef, Australia. Systematic Parasitology 73, 1–11. Gunter, N.L. and Adlard, R.D. (2010). The demise of Leptotheca Thélohan, 1895 (Myxozoa: Myxosporea: Ceratomyxidae) and assignment of its species to Ceratomyxa Thélohan, 1892 (Myxosporea: Ceratomyxidae), Ellipsomyxa Køie, 2003 (Myxosporea: Ceratomyxidae), Myxobolus Bütschli, 1882 and Sphaerospora Thélohan, 1892 (Myxosporea: Sphaerosporidae). Systematic Parasitology 75, 81–104. Gunter, N.L., Whipps, C.M. and Adlard, R.D. (2009). Ceratomyxa (Myxozoa: Bivalvulida): robust taxon or genus of convenience? International Journal for Parasitology 39, 1395– 1405. Hallett, S.L. and Diamant, A. (2001). Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia. Diseases of Aquatic Organisms 46, 197–212. Heniniger, H. and Adlard, R.D. (2013). Molecular identification of cryptic species of Ceratomyxa Thélohan, 1892 (Myxosporea: Bivalvulida) including the description of

Page | 75

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eight novel species from apogonid fishes (Perciformes: Apogonidae) from Australian waters. Acta Parasitologica 58, 342–360. Heiniger, H., Gunter, N.L. and Adlard, R.D. (2008). Relationships between four novel ceratomyxid parasites from the gall bladders of labrid fishes from Heron Island, Queensland, Australia. Parasitology International 57, 158–165. Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. The Quarterly Review of Biology 66, 411–453. Holzer, A.S., Sommerville, C. and Wootten, R. (2004). Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences. International Journal for Parasitology 34, 1099–1111. Kalavati, C. and MacKenzie, K. (1999). The genera Ceratomyxa Thélohan, 1892, Leptotheca Thélohan, 1895 and Sphaeromyxa Thélohan, 1892 (Myxosporea: Bivalvulida) in gadid fish of the Northeast Atlantic. Systematic Parasitology 43, 209–216. Kalavati, C., MacKenzie, K., Collins, C., Hemmingsen, W. and Brickle, P. (2013). Two new species of myxosporean parasites (Myxosporea: Bivalvulida) from gall bladders of Macruronus magellanicus Lönnberg, 1907 (Teleostei: Merlucciidae). Zootaxa 3647, 541–554. Katharios, P., Garaffo, M., Sarter, K., Athanassopoulou, F. and Mylonas, C.C. (2007). A case of high mortality due to heavy infestation of Ceratomyxa diplodae in sharpsnout sea bream (Diplodus puntazzo) treated with reproductive steroids. Bulletin of the European Association of Fish Pathologists 27, 43–47. Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffmann, R.W., Khattra, J., Hallett, S.L., Lester, R.J., Longshaw, M., Palenzeula, O., Siddall, M.E. and Xiao, C. (2001). Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic Microbiology 48, 395–413. Lom, J. and Arthur, J.R. (1989). A guideline for the preparation of species descriptions in Myxosporea. Journal of Fish Diseases 12, 151–156. Lom, J. and Dyková, I. (1992). Myxosporidia (Phylum Myxozoa). Protozoan Parasites of Fishes. Developments in Aquaculture and Fisheries Science, Vol. 26. Elsevier, Amsterdam, Netherlands, pp. 159‒235. Lom, J. and Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53, 1–36. Lubat, V., Radujkovic, B., Marques, A. and Bouix, G. (1989). Parasites de poissons marins du Montenegro: myxosporidies. Acta Adriatica 30, 31–50. Palenzuela, O., Redondo, M.J. and Alvarez-Pellitero, P. (2002). Description of Enteromyxum scophthalmi gen. nov., sp. nov. (Myxozoa), an intestinal parasite of turbot (Scophthalmus maximus L.) using morphological and ribosomal RNA sequence data.

Page | 76

Chapter III | Description of Ceratomyxa auratae n. sp.

Parasitology 124, 369‒379. Palenzuela, O., Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1997). Ceratomyxa sparusaurati (Protozoa: Myxosporea) infections in cultured gilthead sea bream Sparus aurata (Pisces:Teleostei) from Spain: aspects of the host–parasite relationship. Parasitology Research 83, 539–548. Rangel, L.F., Rocha, S., Borkhanuddin, M.H., Cech, G., Castro, R., Casal, G., Azevedo, C., Severino, R., Székely, C. and Santos, M.J. (2014). Ortholinea auratae n. sp. (Myxozoa, Ortholineidae) infecting the urinary bladder of the gilthead seabream Sparus aurata (Teleostei, Sparidae), in a Portuguese fish farm. Parasitology Research 113, 3427– 3437. Rocha, S., Casal, G., Matos, P., Matos, E., Dkhil, M. and Azevedo, C. (2011). Description of Triangulamyxa psittaca sp. nov. (Myxozoa: Myxosporea), a new parasite in the urinary bladder of Colomesus psittacus (Teleostei) from the Amazon River, with emphasys on the ultrastructure of plasmodial stages. Acta Protozoologica 50, 327–338 Rocha, S., Casal, G., Rangel, L., Severino, R., Castro, R., Azevedo, C. and Santos, M.J. (2013). Ultrastructural and phylogenetic description of Zschokkella auratis sp. nov. (Myxozoa), a parasite of the gilthead seabream Sparus aurata. Diseases of Aquatic Organisms 107, 19–30. Shulman, S.S., Donets, Z.S. and Kovaleva, A.A. (1997). Class Myxosporidia of the world fauna, vol. 1. Saint-Petersburg: Nauka. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1993). Light and electron microscopical description of Ceratomyxa labracis n. sp. and a redescription of C. diplodae (Myxosporea: Bivalvulida) from wild and cultured Mediterranean Sea bass Dicentrarchus labrax (L.) (Teleostei: Serranidae). Systematic Parasitology 26, 215– 223. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1995). Light and electron microscopic description of Polysporoplasma n. g. (Myxosporea: Bivalvulida), Polysporoplasma sparis n. sp. from Sparus aurata (L.) and Polysporoplasma mugilis n. sp. from Liza aurata. Euroupean Journal of Protistology 31, 77–89. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (2001). Leptotheca sparidarum n. sp. (Myxosporea: Bivalvulida), a parasite from cultured common dentex (Dentex dentex L.) and gilthead sea bream (Sparus aurata L.) (Teleostei: Sparidae). Journal of Eukaryotic Microbiology 48, 627–639. Sitjà-Bobadilla, A., Franco-Sierra, A. and Alvarez-Pellitero, P. (1992). Sphaerospora (Myxosporea: Bivalvulida) infection in cultured gilthead sea bream, Sparus aurata L.: a preliminary report. Journal of Fish Diseases 15, 339–343. Sitjà-Bobadilla, A., Palenzuela, O. and Alvarez-Pellitero, P. (1995). Ceratomyxa sparusaurati

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Chapter III | Description of Ceratomyxa auratae n. sp. n. sp. (Myxosporea: Bivalvulida), a new parasite from cultured gilthead seabream (Sparus aurata L.) (Teleostei: Sparidae): light and electron microscopic description. Journal of Eukaryotic Microbiology 42, 529–539. Sobecka, E., Szostakowska, B., Zietara, M.S. and Wiecaszek, B. (2013). Morphological and molecular characterization of Ceratomyxa gurnardi sp. n. (Myxozoa: Ceratomyxidae) infecting the gall bladder of the grey gurnard Eutrigla gurnardus (L.) (Scorpaeniformes, Triglidae). Parasitology Research 112, 731–735. Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28, 2731–2739. Thélohan, P. (1894). Recherches sur les myxosporidies. Bulletin biologique de la France et de la Belgique 5, 100–394. Thompson, J.D., Higgins, D.G. and Gilson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673– 4680. Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J.G., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from Eastern Australia: Kudoa thyrsites from Mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites sp. nov. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215– 219.

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Chapter IV

Ultrastructure and phylogeny of Ceratomyxa diplodae (Myxosporea: Ceratomyxidae), from gall bladder of European seabass Dicentrarchus labrax

This chapter was adapted from:

Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae

Abstract

The myxosporean parasite Ceratomyxa diplodae Lubat et al., 1989 sensu Sitjà-Bobadilla and Alvarez-Pellitero, 1993, originally described from the annular seabream Diplodus annularis in the Adriatic Sea, has subsequently been reported from several other sparid hosts, and also a moronid fish, the European seabass Dicentrarchus labrax from the Mediterranean Sea. In this study, molecular identity and additional morphological data are given for this parasite infecting the gall bladder of D. labrax in a southern Portuguese fish farm. In the bile, disporic plasmodia were spherical to subspherical with a smooth surface membrane. Most myxospores were crescent-shaped, 5.1 ± 0.5 (4.8−6.7) μm long and 21.9 ± 1.0 (20.4−23.9) μm thick, with a few being more arcuate, 5.7 ± 0.4 (5.3−6.3) μm long and 17.3 ± 1.0 (16.3−19.1) μm thick. The wall consisted of two symmetrical valves united along a slightly curved suture line, and moderately tapering to rounded ends. Two spherical polar capsules measuring 2.9 ± 0.3 (2.5−3.4) μm in diameter, each containing a polar tubule forming 8 to 9 coils organized in two rows. Host species, tissue tropism, and myxospore morphology supported species identification. Phylogenetic analyses of the SSU rDNA sequence positioned the parasite among most sparid-infecting Ceratomyxa spp., suggesting the existence of a common ancestor for these species. The acquisition of molecular data from infections of C. diplodae in its original host and in other sparids is essential in order to ascertain if the morphological and biological variations found among reports of this parasite are intra- or inter-specific.

Introduction

Several members of the class Myxosporea Bütschli, 1881 are recognized as pathogenic agents of fish, known to cause losses in aquaculture (Diamant, 1992; Sitjà-Bobadilla and Alvarez-Pellitero, 1993a; Breton and Marques, 1995; Kent et al., 2001; Fioravanti et al., 2004; Yokoyama et al., 2012). The European seabass Dicentrarchus labrax Linnaeus, 1758 (Teleostei: Moronidae) is an important commercial fish cultured in the European North Atlantic and Mediterranean area (FAO, 2014). As such, the study and rapid diagnostic assessment of the parasites infecting this fish species, including myxosporeans, assumes an important role for the maintenance of its production. In the above-mentioned geographic areas, 6 myxosporean parasites are known to infect D. labrax: Sphaerospora testicularis Sitjà-Bobadilla and Alvarez-Pellitero, 1990, which intensively infects the testes and causes castration (Alvarez-Pellitero and Sitjà-Bobadilla, 1993a; Rigos et al., 1999); S. dicentrarchi Sitjà-Bobadilla and Alvarez-Pellitero, 1992, which parasitizes the connective tissue, reducing the growth rate and increasing the possibility of secondary infections (Sitjà-Bobadilla and Alvarez-Pellitero, 1992; Rigos et al., 1999; Fioravanti et al., 2004); Enteromyxum leei Diamant et al., 1994, which

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae causes enteritis associated with mass mortalities in several fish species, including D. labrax (Fioravanti et al., 2006); Ortholinea labracis Rangel et al. 2016, recently described from the urinary bladder and posterior kidney (Rangel et al., 2016); Ceratomyxa diplodae Lubat et al., 1989 and C. labracis Sitjà-Bobadilla and Alvarez-Pellitero, 1993, which infect the gall bladder (Alvarez-Pellitero and Sitjà-Bobadilla, 1993b; Sitjà-Bobadilla and Alvarez-Pellitero, 1993b). In the Indo-Pacific region, Kudoa iwatai Egusa and Shiomitsu, 1983 has also been reported infecting the muscle of D. labrax in correlation with cases of food poisoning (Diamant et al., 2005; Matsukane et al., 2011; Suzuki et al., 2012). Ceratomyxa spp. are considered relatively harmless, either because these parasites induce minimum tissue damage or, more likely, because no studies have investigated their pathogenicity. Consequently, little information is available on C. labracis and C. diplodae, besides their original descriptions (Lubat et al., 1989; Sitjà-Bobadilla and Alvarez-Pellitero, 1993b). Ceratomyxa Thélohan, 1892 is one of the largest genera of Myxosporea, comprising about 300 species, most of which are coelozoic in the gall bladder of marine teleosts. Traditionally, the differentiation between Ceratomyxa spp., as well as Myxosporea in general, was achieved through comparative morphological criteria, such as myxospore dimensions, shape and size of the lateral processes, and number of coils of the polar tubule. Species reports were solely based on light microscopy (LM) observations and schematic line drawings, leading to unreliable descriptions and hampering the recognition of some morphologically cryptic species (Lom and Dyková, 1992, 2006; Eiras, 2006; Heiniger and Adlard, 2013). In the case of Ceratomyxa, its elevated number of species and dubious separation from the genus Leptotheca Thelohán, 1895 have further hampered taxonomic comparisons. In the past decade, the implementation of molecular analyses has allowed the resolution of old taxonomic inaccuracies, through the establishment of informative biological correlates to myxosporean phylogeny. These include the type of aquatic environment, tissue tropism, and host relatedness, but not spore morphology due to its plasticity and the limited number of defining characters that can be observed using LM (Kent et al., 2001; Eszterbauer, 2004; Holzer et al., 2004; Fiala, 2006; Heiniger et al., 2008; Bartošová et al., 2009; Fiala and Bartošová, 2010; Carriero et al., 2013; Rocha et al., 2013). Gunter and Adlard (2010) proposed the abolition of the genus Leptotheca, with the reassignment of its species to Ceratomyxa, Sphaerospora Thélohan, 1982, Ellipsomyxa Køie, 2003, and Myxobolus Bütschli, 1882, mainly on the basis of tissue tropism. More recently, the genus Ceratonova Atkinson et al., 2014 was erected to encompass Ceratonova shasta syn. Ceratomyxa shasta (Noble 1950), a long- standing taxonomic outlier to all other Ceratomyxa spp. due to its histozoic development in the intestinal epithelium, its freshwater life cycle, and its phylogenetic distance (Gunter et al., 2009; Atkinson and Bartholomew, 2010; Atkinson et al., 2014). Nonetheless, myxospore morphology cannot be disregarded, particularly in the case of Ceratomyxa spp., for which molecular

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae comparisons are hindered by the general lack of molecular data available for more diversified members of the genus (Rocha et al., 2015). Considering the importance of having reliable morphological and molecular data for the proper identification and rapid diagnosis of myxosporean species, this study provides a morphological, ultrastructural, and molecular re-description of C. diplodae, based on material collected from the gall bladder of D. labrax sampled from a southern Portuguese fish farm.

Materials and Methods

Fish and parasite sampling

Specimens (n = 141) of European seabass Dicentrarchus labrax Linnaeus, 1758 were obtained between June 2012 and June 2013 from a fish farm in the Alvor estuary, near the Atlantic coast (37° 08’ N, 08° 37’ W), Portimão, Algarve, Portugal. Monthly fish collections were performed from batches intended for commercialization. Collected specimens were slaughtered in accordance with the animal ethical laws stipulated for the aquaculture industry in Portugal and then shipped to the laboratory in insulated containers filled with chipped ice. Upon arrival, fish specimens were dissected and a parasitological examination of several organs and tissues was performed. LM revealed infection of the gall bladder with the myxosporean parasite Ceratomyxa diplodae. In some specimens, co-infection with C. labracis was observed. As such, infected bile containing solely, or mainly, young developmental stages was not used for further microscopic and molecular procedures. Parasitic material was chosen based on the morphological distinction of mature myxospores, which are not cryptic, since C. labracis possesses very thin and long lateral processes, overall displaying very different morphological aspects from C. diplodae.

Morphological and ultrastructural analysis of myxospores

For LM, the parasitic stages in the bile were examined and photographed using a Zeiss Axiophot microscope (Grupo Taper, Sintra, Portugal), equipped with a Zeiss Axiocam digital camera Icc3. Axiovision 4.6 software (Grupo Taper, Sintra, Portugal) was used for image analysis. Morphometry was determined from fresh material (Lom and Arthur, 1989). All measurements include the mean ± SD, range of variation, and number of spores measured (range, n). For transmission electron microscopy (TEM), samples of bile infected with C. diplodae were fixed in 5% glutaraldehyde buffered in 0.2 M sodium cacodylate (pH 7.4) for 20 to 24 h, washed in the same buffer, and post-fixed in 2% osmium tetroxide also buffered with 0.2 M

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae sodium cacodylate (pH 7.4) for 3 to 4 h. All of these steps were performed at 4°C. The samples were then dehydrated in an ascending graded series of ethanol, followed by embedding using a series of oxide propylene and EPON mixtures, ending in EPON. Semithin sections were stained with methylene blue-Azure II. Ultrathin sections were double-contrasted with uranyl acetate and lead citrate, and then examined and photographed using a JEOL 100 CXII TEM (JEOL Optical), operating at 60 kV.

DNA extraction, amplification, and sequencing

Samples of bile infected with C. diplodae were obtained from 3 fish specimens and preserved separately in absolute ethanol at 4 °C. Genomic DNA extraction was performed using a GenEluteTM Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, USA), following the manufacturer’s instructions. The DNA was stored in 50 μl of TE buffer at −20 °C until further use. The SSU rDNA gene was amplified and sequenced using both universal primers and myxosporean-specific primers (Table 1). PCRs were performed in 50 μl reactions using 10 pmol of each primer, 10 nmol of each dNTP, 2.5 mM of MgCl2, 5 μl of 10× Taq polymerase buffer, 1.5 units of Taq DNA polymerase (NZYTech, Lisbon, Portugal), and 3 μl (approximately 100−150 ng) of genomic DNA. The reactions were run on a Hybaid PxE Thermocycler (Thermo Electron Corporation, Milford, Massachusetts, USA), with initial denaturation at 95 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 53 °C for 45 s, and 72 °C for 90 s. The final elongation step was performed at 72 °C for 7 min. Aliquots (5 μl) of the PCR products were electrophoresed through a 1% agarose 1× Tris-acetate- EDTA buffer (TAE) gel stained with ethidium bromide. PCR products were purified using the ExoFast method, in which an enzymatic cleanup that eliminates unincorporated primers and dNTPs is performed with Exonuclease I (E. coli) and FastAP Thermosensitive (SAP). PCR products from different regions of the SSU rDNA gene were sequenced directly. The sequencing reactions were performed using a BigDye Terminator v1.1 from the Applied Biosystems Kit (Applied Biosystems, Carlsbad, California, USA), and were run on an ABI3700 DNA analyser (Perkin-Elmer, Applied Biosystems, Stabvida, Oeiras, Portugal).

Distance and phylogenetic analysis

The forward and reverse segments sequenced were manually aligned with ClustalW in MEGA 6.06 software (Thompson et al., 1994; Tamura et al., 2013), and ambiguous bases were clarified using corresponding ABI chromatograms. To determine the phylogenetic position of the parasite amongst its closest relatives sequenced to date, 55 myxosporean SSU

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae rDNA sequences were chosen from GenBank according to the highest similarity score: 52 belonging to the genus Ceratomyxa, Palliatus indecorus Shulman et al., 1979 (DQ377712), Pseudoalataspora kovalevae Kalavati et al., 2013 (JX467675), and Myxodavisia bulani Fiala et al., 2015 (KM273030). Zschokkella lophii Freeman et al., 2008 (DQ301509), Kudoa iwatai Egusa and Shiomitsu, 1983 (AY514038), and Myxidium incurvatum Thélohan, 1892 (DQ377708) were selected as outgroups. Phylogenetic and molecular evolutionary analyses were conducted using MEGA 6.06. Alignments were performed using ClustalW, with an opening gap penalty of 10 and a gap extension of 4 for both paired and multiple alignments (Tamura et al., 2013); ambiguous regions were eliminated manually. Phylogenetic analyses included maximum likelihood (ML), neighbor-joining (NJ), and maximum parsimony (MP) methodologies. For ML, the general time reversible substitution model with 4 gamma-distributed rate variation among sites was performed. For NJ, a Kimura 2-parameter substitution model with a gamma distribution (shape parameter = 1.4) was performed. For MP, the close neighbor interchange heuristic option was selected, with a search factor of 1 and addition of random initial trees, and 500 replicates were performed. All positions with less than 100% site coverage were eliminated from all trees, resulting in a total of 1082 positions in the final dataset. The bootstrap consensus tree was inferred from 500 replicates for ML and MP, and 1000 replicates for NJ. Distance estimation was also carried out in MEGA 6.06, using the p-distance model with all ambiguous positions removed for each sequence pair.

Table 1. Polymerase chain reaction primers used for the amplification and sequencing of the SSU rDNA gene of Ceratomyxa diplodae.

Name Sequence (5’-3’) Position Paired with Source

18e CTG GTT GAT CCT GCC AGT 1 CeratR1, MYX4R Hillis and Dixon, 1991

MyxospecF TTC TGC CCT ATC AAC TTG TTG 312 MYX4R, 18r Fiala, 2006

MYX4F GTT CGT GGA GTG ATC TGT CAG 1300 18r Rocha et al., 2015

CeratR1 CCA ATG TCT GGA TTG GGT A 420 18e Rocha et al., 2015

MYX4R CTG ACA GAT CAC TCC ACG AAC 1300 18e, MyxospecF Hallett and Diamant, 2001

18r CTA CGG AAA CCT TGT TAC G 1832 MyxospecF, MYX4F Whipps et al., 2003

Results

Our parasitological survey revealed the presence of several myxosporean parasites that are known to infect Dicentrarchus labrax in other geographical areas. Sphaerospora dicentrarchi was observed infecting the connective tissue of several organs; S. testicularis was observed in the testes of fully matured males; and, less frequently, both Ceratomyxa diplodae and C. labracis were observed in the gall bladder, being co-infective in some of the analysed

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae specimens. Also resulting from this parasitological survey was the description of Ortholinea labracis from the urinary bladder and posterior kidney. Infections by Enteromyxum leei and Kudoa iwatai were not observed.

Ceratomyxa diplodae Lubat et al., 1989 sensu Sitjà-Bobadilla and Alvarez-Pellitero, 1993

Diagnosis: Plasmodia in different stages of maturation, as well as mature myxospores, were observed floating free in the bile (Fig. 1A−D). Myxospores crescent-shaped, 5.1 ± 0.5 (4.8−6.7) μm long and 21.9 ± 1.0 (20.4−23.9) μm thick (n = 15) (Fig. 1B); occasionally more arcuate, 5.7 ± 0.4 (5.3−6.3) μm long and 17.3 ± 1.0 (16.3−19.1) μm thick (n = 6) (Fig. 1C); total length 5.7 ± 0.5 (4.8−6.7) μm and total thickness 20.0 ± 2.5 (16.3−24.0) μm (n = 21). Abnormal myxospores with 3 valves and 3 polar capsules were occasionally observed (Fig. 1D). Two equally sized spherical polar capsules measuring 2.9 ± 0.3 (2.5−3.4) μm in diameter (n = 30) (Fig. 1B,C). Description: Plasmodia spherical to subspherical, with smooth cellular membrane and presenting disporic development. Cytoplasmic contents included mitochondria, vesicles containing granular material of different electronic density, and lipidic globules. The earliest stages of sporogony observed were developing sporoblasts (Fig. 1E,F). The two valves of the myxospores were roughly equal, smooth, and united along a slightly curved suture line, moderately tapering to rounded ends. Two polar capsules located at the same level at the myxospores’ anterior pole, each containing a polar tubule forming 8 to 9 coils organized in two rows. The polar capsule wall was double-layered, comprised by a thinner outer electron-dense layer and a thicker inner electron-lucent layer, and containing a dense and heterogeneous matrix (Fig. 1G−J). Sporoplasm binucleate and containing electron-dense sporoplasmosomes (Fig. 1K). A semischematic drawing of a myxospore in sutural view is presented in Fig. 2. Prevalence: 7.1% (10 infected in a total of 141 examined during a one year sampling). Pathogenicity: The collected and analysed fish did not present external symptoms of infection or disease, and neither was mortality recorded from the stock used. Vouchers: One glass slide with semithin sections of plasmodia containing several myxospores was deposited in the Type Slide Collection of the Laboratory of Animal Pathology at the Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, with the reference CIIMAR 2016.11. Sequences: One SSU rDNA sequence with a total of 1,819 bp, deposited in GenBank with the accession number KX099691. Molecular analysis: ML, NJ, and MP phylogenetic trees revealed the parasite clustering within the large Ceratomyxa clade, more specifically in a subclade comprising Ceratomyxa spp. from sparids and siganids. This subclade was supported by strong bootstrap values in all

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae

Figure 1. Light and transmission electron micrographs of Ceratomyxa diplodae infecting the gall bladder of Dicentrarchus labrax. (A) Aggregate of spherical to subspherical plasmodia (Pl) presenting smooth cellular membrane and containing developing sporoblasts (Sb), observed under differential interference contrast (DIC) optics. (B) Mature crescent-shaped myxospore, as observed under DIC optics. (C) Occasional more arcuate myxospore, as observed under DIC optics. (D) Rare aberrant myxospore displaying three valves and three polar capsules, as observed under DIC optics. (E) Ultrathin section showing a subspherical plasmodium containing two developing sporoblasts (Sb). (F) Ultrathin section depicting two mature myxospores surrounded by an aggregate of plasmodia (Pl) containing developing sporoblasts (Sb). Notice the vegetative nucleus (*) in one of the plasmodia. (G) Oblique longitudinal section of a mature myxospore displaying one of its two polar capsules (PC), and allowing recognition of the extrusion pores (arrows) through which the polar tubules exit, located near the suture line (arrowheads). (H) Oblique section of a rare aberrant myxospore showing two of its three polar capsules. (I) Ultrastructural detail of the slightly curved suture line (arrowheads) uniting the two valves. (J) Longitudinal section of a polar capsule showing the polar tubule (PF) coiled along its double-layered wall (arrowheads) and capped at the apex by a stopper (arrow). (K) Ultrastructure of the binucleate sporoplasm. phylogenetic analyses. Ceratomyxa labracis (AF411472), which also infects the gall bladder of D. labrax, never grouped with C. diplodae in this subclade, and displayed an unresolved

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae positioning in the overall tree topology, despite consistently clustering together with C. verudaensis Fiala et al., 2015 (KM273027) from the gall bladder of Scorpaena porcus (Linnaeus, 1758). Also clustering within the large Ceratomyxa clade were Pseudoalatospora kovalevae (JX467675), Palliatus indecorus (DQ377712) and Myxodavisia bulani (KM273030) (Fig. 3). Pairwise comparisons among the SSU rDNA sequences closely related to C. diplodae revealed greatest similarity to an unnamed Ceratomyxa sp. (JF820293), C. puntazzi (JF820290), Ceratomyxa sp. (JF820291), C. sparusaurati (AF411471), and Ceratomyxa sp. (JF820292), with percentages of identity higher than 95%. C. labracis (AF411472) showed only 89.9% identity to C. diplodae (Table 2).

Figure 2. Schematic drawing of Ceratomyxa diplodae depicting the internal and external organization of the myxospores: common crescent-shaped type (left) and rare more arcuate type (right).

Discussion

At present, it is widely accepted that the resolution of taxonomic and phylogenetic issues within the class Myxosporea can only result from the combined consideration of both morphological and molecular characters. For many years, species reports were solely based on LM observations, sometimes leading to poor descriptions that now warrant validation through the use of more accurate analyses. Consequently, in the past few years, several species have been re-described and, in some cases, taxonomically revised according to the molecular input (Gunter and Adlard, 2010; Liu et al., 2013; Atkinson et al., 2014; Rocha et al., 2014). In this study, the combined use of LM, TEM, and molecular procedures allowed the re- description of Ceratomyxa diplodae from the gall bladder of Dicentrarchus labrax in a southern Portuguese fish farm. This species was originally described from the gall bladder of the annular seabream Diplodus annularis (Teleostei, Sparidae) in Adriatic waters (Lubat et al., 1989). The description lacked photographs of the parasite and did not present a comparative discussion to the morphological traits of other Ceratomyxa, thus making future identifications of the

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae

Table 2. Comparison between the SSU rDNA sequences closely related to Ceratomyxa diplodae: percentage of identity (upper right) and nucleotide difference (lower left).

ID Species GenBank pb (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

(1) Ceratomyxa diplodae KX099691 1819 - 97.1 97.1 96.6 96.6 96.4 91.7 91.3 91.3 89.9

(2) Ceratomyxa sp. JF820293 1743 50 - 98.5 98.5 98.6 97.1 91.5 91.5 91.2 89.9

(3) Ceratomyxa puntazzi JF820290 1708 50 26 - 98.4 97.7 97.0 91.8 91.9 91.6 89.7

(4) Ceratomyxa sp. JF820291 1766 60 26 27 - 98.2 96.9 91.7 91.6 91.3 89.5

(5) Ceratomyxa sparusaurati AF411471 1741 59 23 37 31 - 98.2 91.5 91.2 91.0 90.1

(6) Ceratomyxa sp. JF820292 1769 64 50 52 54 31 - 90.7 90.8 90.6 88.9

(7) Ceratomyxa sp. DQ333431 1732 143 141 133 138 146 155 - 94.6 93.6 90.3

(8) Ceratomyxa barnesi FJ204245 1445 125 121 116 121 121 132 74 - 96.7 90.7

(9) Ceratomyxa sp. DQ333429 1705 147 145 133 143 151 154 108 45 - 90.6

(10) Ceratomyxa labracis AF411472 1763 173 169 167 174 170 186 166 128 158 - parasite difficult. Moreover, Lubat et al. (1989) apparently did not deposit any museum specimens (type material). Later, Sitjà-Bobadilla and Alvarez-Pellitero (1993b) identified C. diplodae from the gall bladder of D. labrax, based on myxospore morphometry, proximity of the sampling areas, and relatedness of the host families. Their re-description improved the morphological characterization and provided some LM micrographs of the myxospores, as well as a referenced holotype. C. diplodae was further reported occurring from the gall bladder of the cultured sparids common dentex Dentex dentex Linnaeus, 1758 and sharpsnout seabream Diplodus puntazzo (Rigos et al., 1997; Katharios et al., 2007). Identification was based on myxospore morphometry, geographic location, and host relatedness. The lack of molecular data supporting species identification, combined with the morphological and biological disparities among these reports, namely in terms of host species and tissue tropism, question their reliability. Although broad host specificity is a known trait of several myxosporean species (Hoffman et al., 1965; Diamant et al., 2006; Jirků et al., 2006), molecular systematics suggest that the genus Ceratomyxa is mostly host specific (Gunter and Adlard, 2008, 2009; Gunter et al., 2009; Alama-Bermejo et al., 2011; Heiniger and Adlard, 2013), so that the molecular re-description of several species reported from multiple hosts on a morphological basis will probably expand the genus further. Taking this into consideration, it is possible to assume that the morphological and biological variations between reports of C. diplodae may be inter-specific, rather than intraspecific (Rocha et al., 2015). Alama-Bermejo et al. (2011) attempted to sequence C. diplodae from its original host, D. annularis, but found yet another morphologically distinct Ceratomyxa species; this is in agreement with several studies that have reported the occurrence of infection by multiple ceratomyxids in some fish hosts (Gunter and Adlard, 2009; Heiniger and Adlard, 2013; Rocha et al., 2015). In addition to the above mentioned records of C. diplodae from the

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae

Figure 3. Maximum likelihood tree of the SSU rDNA sequence of Ceratomyxa diplodae (underlined) and other selected myxozoan species (white boxes: species described from sparid hosts; grey type: non-ceratomyxid species clustering with the Ceratomyxa clade). Numbers on the branches are bootstrap confidence levels corresponding to maximum likelihood/neighbor-joining/maximum parsimony trees. The final data set included a total of 1082 positions. GenBank accession numbers are provided in parentheses.

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae gall bladder of sparids, 9 other species and 3 records of Ceratomyxa exist from fish hosts of the family Sparidae, mostly in the Mediterranean and North Atlantic coasts off southern European countries (Table 3). In the present study, the identification of C. diplodae was achieved through morphological comparison to the features reported by Sitjà-Bobadilla and Alvarez-Pellitero (1993b), as the latter was performed from the same host and constitutes the most reliable account of the parasite species. The myxospores studied here presented similar measurements to those reported in the description from D. labrax, further coinciding with the observation of different shapes: myxospores are frequently crescent-shaped, occasionally arcuate, with the rare occurrence of abnormal forms with 3 valves and 3 polar capsules. The only difference found between this and the previous report is the number of polar tubule coils, which are 3 to 4 according to the observations of Sitjà-Bobadilla and Alvarez-Pellitero (1993b). Here, the combined use of LM and TEM revealed the polar tubule forming 8 to 9 coils organized in 2 rows. It is possible that, having relied solely on LM observations, the previous description considered only one of the two polar tubule rows, thus reporting a smaller number of coils. The occurrence of aberrant spores with 3 valves and 3 polar capsules has been documented for other species of the genus, such as C. bassoni Abdel-Ghaffar et al., 2008, C. protopsettae Fujita, 1923, and C. gurnardi Sobecka et al., 2013 (Cho et al., 2004; Abdel-Ghaffar et al., 2008; Sobecka et al., 2013). Co-infections of C. diplodae and C. labracis were observed in infected specimens, with the parasites being differentiated on the basis of morphological and molecular information. Our comparative study further took into consideration the morphological and molecular traits of all other Ceratomyxa spp. known to infect sparid fish hosts (Tables 2 and 3). Comparison to C. arcuata Thélohan, 1892 relied on the morphological data provided in its original description from the sparid fish Pagellus bogaraveo (Brünnich, 1768), since its SSU rRNA sequence was obtained from non-sparid fish hosts. Ceratomyxa pallida Thélohan, 1894, C. mylionis (Ishizaki, 1960), and C. acanthopagri (Zhao and Song, 2003) are without molecular data, but differ morphologically from C. diplodae. Lastly, C. herouardi Georgévitch, 1916 was described without measurements and, as molecular data is not available, comparison to this species is difficult. Although the genus Ceratomyxa represents one of the most cohesive myxosporean lineages, the internal phylogenetic relationships of its species remains unclear. Fiala et al., (2015) resolved 5 subclades within the main Ceratomyxa clade: (1) basal subclade comprising M. bulani and Ceratomyxa leatherjacketi Fiala et al., 2015; (2) subclade containing the species sequenced from elasmobranchs; (3) subclade in which Palliatus indecorus clusters with Ceratomyxa sp. 2 and C. synaphobranchi Fiala et al., 2015; (4) subclade comprising some sub-tropical and tropical species from the Pacific and Indian Oceans; (5) most derived taxon- rich subclade containing the majority of species with unresolved deeper nodes. The

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae

here available), given in μm. in given available), here

spp. spp. reported from the gall bladder of sparid hosts. SL: myxospore length; ST: myxospore thickness; PCL:

Ceratomyxa

to to other

Ceratomyxa diplodae

Comparison Comparison of

Table 3. (w means (range) SD ± are coils. polar Measurements PTc: tubule polarcapsule width; polarcapsule PCW: length;

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae phylogenetic analysis performed here agrees with these findings and further acknowledges the phylogenetic position of P. kovalevae in a poorly resolved group of the most derived subclade, closely related to C. pantherini Gunter et al. 2010, C. anko Freeman et al., 2008, and C. appendiculata Thélohan, 1892. Molecular systematics reveals that the Ceratomyxa clade is not monophyletic due to the inclusion of P. indecorus, P. kovalevae, and M. bulani; this condition will probably be resolved by the taxonomic revision of these species, as suggested in previous studies (Fiala, 2006; Gunter et al., 2009; Fiala et al., 2015; Rocha et al., 2015). The SSU rDNA sequence obtained here for C. diplodae also clustered within the most derived subclade, forming a well-supported group with most of the Ceratomyxa spp. that have been sequenced from sparid hosts (C. puntazzi, C. sparusaurati, the two Ceratomyxa sp. from S. aurata, and a Ceratomyxa sp. from Diplodus annularis), and closely related to C. barnesi and two other Ceratomyxa spp. from siganid hosts. The other sparid-infecting Ceratomyxa spp. included in the analysis (C. arabica, C. auratae and C. arcuata) clustered separately, within different groups of the subclade. The inclusion of C. diplodae from the moronid fish host D. labrax among most of the sparid-infecting Ceratomyxa suggests the existence of a recent common ancestor for these species, which is further supported by geographic clustering (all these species were described from the Mediterranean area and North Atlantic off Portugal). Notwithstanding, geography has been shown not to correlate well with Ceratomyxa evolution (Gunter et al., 2009; Heiniger and Adlard, 2013; Rocha et al., 2015), and this is supported by the present phylogenetic analysis, in which species from several different locations clustered together. For instance, C. auratae from the North Atlantic off Portugal, as well as C. arabica from Saudi Arabia, clustered together with species from Australian waters. At this point, molecular data from infections of C. diplodae in its sparid hosts would be valuable to confirm those fish species as hosts for the parasite. Information on the pathogenic action of coelozoic myxosporeans is very sparse, and the majority of Ceratomyxa spp. are considered to be relatively harmless. The few exceptions usually produce lesions characterized by vacuolization, deformation, and necrosis of the gall bladder epithelial cells, as described for the infections of Ceratomyxa

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Chapter IV | Ultrastructure and phylogeny of Ceratomyxa diplodae spp. in D. labrax, C. puntazzi in D. puntazzo and C. dehoopi Reed et al., 2007 in Clinus superciliosus (Linnaeus, 1758) (see Alvarez-Pellitero and Sitjà-Bobadilla, 1993b; Reed et al., 2007; Alama-Bermejo et al., 2011). In the case of C. diplodae, Katharios et al. (2007) reported high mortalities of D. puntazzo due to heavy infestations of the parasite in potentially immunosuppressed specimens, and Merella et al. (2005) correlated C. diplodae with the enlargement of the gall bladder in fish intensively infected with the polyopisthocotylean Atrispinum salpae (Parona and Perugia, 1890). Nonetheless, it cannot be disregarded that the latter studies were subject to a lack of reliable characters for species diagnosis, as previously mentioned. In our study, mortality was not reported from the sampling stocks, and infected specimens of D. labrax did not display symptoms of disease. Gross pathology of the gall bladder was not observed, demonstrating that only low-intensity infections were recorded. Therefore, histopathological studies of highly parasitized individuals may produce different results.

Acknowledgements

This work was financially supported by FCT (Lisbon, Portugal), within the scope of the project DIRDAMyx, reference FCOMP-01-0124-FEDER-020726 / FCT- PTDC/MAR/116838/- 2010, and the PhD fellowship grants attributed to S.R. (SFRH/BD/92661/2013) and to L.F.R. (SFRH/ BD/ 82237/2011) through the program QREN-POPH/FSE; and by the Eng.º António de Almeida Foundation (Porto, Portugal). Additional support came from the Structured Program of R&D&I INNOVMAR − (Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035), Research Line INSEAFOOD (Innovation and valorization of seafood products: meeting local challenges and opportunities), within the R&D Institution CIIMAR (Interdisciplinary Centre of Marine and Environmental Research), founded by the Northern Regional Operational Programme (NORTE 2020), through the European Regional Development Fund (ERDF). This work complies with the current laws of Portugal.

References

Abdel-Ghaffar, F., Ali, M.A., Al-Quraishy, S., Al-Rasheid, K., Al-Farraj, S., Abdel-Baki, A.S. and Bashtar, A.R. (2008). Four new species of Ceratomyxa Thélohan 1892 (Myxozoa: Myxosporea: Ceratomyxidae) infecting the gallbladder of some Red Sea fishes. Parasitology Research 103, 559−565. Alama-Bermejo, G., Raga, J.A. and Holzer, A.S. (2011). Host-parasite relationship of Ceratomyxa puntazzi n. sp. (Myxozoa: Myxosporea) and sharpsnout seabream

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Diplodus puntazzo (Walbaum, 1792) from the Mediterranean with first data on ceratomyxid host specificity in sparids. Veterinary Parasitology 182, 181−192. Al-Qahtani, H.A., Mansour, L., Al-Quraishy, S. and Abdel-Baki, A.S. (2015). Morphology, phylogeny and seasonal prevalence of Ceratomyxa arabica n. sp. (Myxozoa: Myxosporea) infecting the gallbladder of Acanthopagrus bifasciatus (Pisces: Sparidae) from the Arabian Gulf, audi Arabia. Parasitology Research 114, 465–471. Alvarez-Pellitero, P. and Sitjà-Bobadilla, A. (1993a). Pathology of Myxosporea in marine fish culture. Diseases of Aquatic Organisms 17, 229−238. Alvarez-Pellitero, P. and Sitjà-Bobadilla, A. (1993b). Ceratomyxa spp. (Protozoa: Myxosporea) infections in wild and cultured sea bass, Dicentrarchus labrax, from the Spanish Mediterranean area. Journal of Fish Biology 42, 889−901. Atkinson, S.D. and Bartholomew, J.L. (2010). Disparate infection patterns of Ceratomyxa shasta (Myxozoa) in rainbow trout (Oncorhynchus mykiss) and Chinook salmon (Oncorhynchus tshawytscha) correlate with internal transcribed spacer-1 sequence variation in the parasite. International Journal of Parasitology 40, 599−604. Atkinson, S.D., Foott, J.S. and Bartholomew, J.L. (2014). Erection of Ceratonova n. gen. (Myxosporea: Ceratomyxidae) to encompass freshwater species C. gasterostea n. sp. from three-spine stickleback (Gasterosteus aculeatus) and C. shasta n. comb. from salmonid fishes. Journal of Parasitology 100, 640−645. Bartošová, P., Fiala, I. and Hypša, V. (2009). Concatenated SSU and LSU rDNA data confirm the main evolutionary trends within myxosporeans (Myxozoa: Myxosporea) and provide an effective tool for their molecular phylogenetics. Molecular Phylogenetics and Evolution 53, 81−93. Breton, A.L. and Marques, A. (1995). Occurrence of an histozoic Myxidium infection in two marine cultured species: Puntazzo puntazzo C. and Pagrus major. Bulletin of the European Association of Fish Pathologists 15, 210−212. Carriero, M.M., Adriano, E.A., Silva, M.R., Ceccarelli, P.S. and Maia, A.A. (2013). Molecular phylogeny of the Myxobolus and Henneguya genera with several new South American species. PLoS One 8, e73713. Cho, J.B., Kwon, S.R., Kim, S.K., Nam, Y.K. and Kim, K.H. (2004). Ultrastructure and development of Ceratomyxa protopsettae Fujita, 1923 (Myxosporea) in the gallbladder of cultured olive flounder, Paralichthys olivaceus. Acta Protozoologica 43, 241−250. Diamant, A. (1992). A new pathogenic histozoic Myxidium (Myxosporea) in cultured gilthead sea bream Sparus aurata L. Bulletin of the European Association of Fish Pathologists 12, 64−66. Diamant, A., Ucko, M., Paperna, I., Colorni, A. and Lipshitz, A. (2005). Kudoa iwatai (Myxosporea: Multivalvulida) in wild and cultured fish in the Red Sea: redescription and

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molecular phylogeny. Journal of Parasitology 91, 1175−1189. Diamant, A., Ram, S. and Paperna, I. (2006). Experimental transmission of Enteromyxum leei to freshwater fish. Diseases of Aquatic Organisms 72, 171−178. Eiras, J.C. (2006). Synopsis of the species of Ceratomyxa Thélohan, 1892 (Myxozoa: Myxosporea: Ceratomyxidae). Systematic Parasitology 65, 49−71. Eszterbauer, E. (2004). Genetic relationship among gill-infecting Myxobolus species (Myxosporea) of cyprinids: molecular evidence of importance of tissue-specificity. Diseases of Aquatic Organisms 58, 35−40. FAO (Food and Agriculture Organization of the United Nations) (2014). The state of world fisheries and aquaculture 2014. FAO, Rome. Fiala, I. (2006). The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. International Journal of Parasitology 36, 1521−1534. Fiala, I. and Bartošová, P. (2010). History of myxozoan character evolution on the basis of rDNA and EF-2 data. BMC Evolutionary Biology 10, 228. Fiala, I., Hlavnicková, M., Kodádková, A., Freeman, M.A., Bartošová-Sojková, P. and Atkinson, S.D. (2015). Evolutionary origin of Ceratonova shasta and phylogeny of the marine myxosporean lineage. Molecular Phylogenetics and Evolution 86, 75−89. Fioravanti, M.L., Caffara, M., Florio, D., Gustinelli, A. and Marcer, F. (2004). Sphaerospora dicentrarchi and S. testiculares (Myxozoa: Sphaerosporidae) in farmed European seabass (Dicentrarchus labrax) from Italy. Folia Parasitologica 51, 208−210. Fioravanti, M.L., Caffara, M., Florio, D., Gustinelli, A. and Marcer, F. (2006). A parasitological survey of European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) cultured in Italy. Veterinary Research Communications 30, 249−252. Georgévitch, J. (1916). Note sur les myxosporidies des poisons de la baie de Vilefranche et de Monaco. Bulletin de l'Institut océanographique de Monaco 322, 1−7. Gunter, N.L. and Adlard, R.D. (2008). Bivalvulidan (Myxozoa: Myxosporea) parasites of damselfishes with description of twelve novel species from Australia’s Great Barrier Reef. Parasitology 135, 1165−1178. Gunter, N.L. and Adlard, R.D. (2009). Seven new species of Ceratomyxa Thélohan, 1892 (Myxozoa) from the gall-bladders of serranid fishes from the Great Barrier Reef, Australia. Systematic Parasitology 73, 1−11. Gunter, N. and Adlard, R. (2010). The demise of Leptotheca Thélohan, 1895 (Myxozoa: Myxosporea: Ceratomyxidae) and assignment of its species to Ceratomyxa Thélohan, 1892 (Myxosporea: Ceratomyxidae), Ellipsomyxa Køie, 2003 (Myxosporea: Ceratomyxidae), Myxobolus Bütschli, 1882 and Sphaerospora Thélohan, 1892 (Myxosporea: Sphaerosporidae). Systematic Parasitology 75, 81−104. Gunter, N.L., Whipps, C.M. and Adlard, R.D. (2009). Ceratomyxa (Myxozoa: Bivalvulida):

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robust taxon or genus of convenience? International Journal of Parasitology 39, 1395−1405. Hallett, S.L. and Diamant, A. (2001). Ultrastructure and smallsubunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia. Diseases of Aquatic Organisms 46, 197−212. Heiniger, H. and Adlard, R.D. (2013). Molecular identification of cryptic species of Ceratomyxa Thélohan, 1892 (Myxosporea: Bivalvulida) including the description of eight novel species from apogonid fishes (Perciformes: Apogonidae) from Australian waters. Acta Parasitologica 58, 342−360. Heiniger, H., Gunter, N.L. and Adlard, R.D. (2008). Relationships between four novel ceratomyxid parasites from the gall bladders of labrid fishes from Heron Island, Queensland, Australia. Parasitology International 57, 158−165. Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and phylogenetic interference. The Quarterly Review of Biology 66, 411−453. Hoffman, G.L., Putz, R.E. and Dunbar, C.E. (1965). Studies on Myxosoma cartilaginis n. sp. (Protozoa-Myxosporidea) of centrarchid fish and a synopsis of Myxosoma of North American freshwater fishes. Journal of Protozoology 12, 319−332. Holzer, A.S., Sommerville, C. and Wootten, R. (2004). Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences. International Journal of Parasitology 34, 1099−1111. Jirků, M., Bolek, M.G., Whipps, C.M., Janovy, J., Kent, M.L. and Modry, D. (2006). A new species of Myxidium (Myxosporea: Myxidiidae), from the western chorus frog, Pseudacris triseriata triseriata, and Blanchard’s cricket frog, Acris crepitans blanchardi (Hylidae), from eastern Nebraska: morphology, phylogeny, and critical comments on amphibian Myxidium taxonomy. Journal of Parasitology 92, 611−619. Kalavati, C. and MacKenzie, K. (1999). The genera Ceratomyxa Thélohan, 1892, Leptotheca Thélohan, 1895 and Sphaeromyxa Thélohan, 1892 (Myxosporea: Bivalvulida) in gadid fish of the Northeast Atlantic. Systematic Parasitology 43, 209−216. Katharios, P., Garaffo, M., Sarter, K., Athanassopoulou, F. and Mylonas, C.C. (2007). A case of high mortality due to heavy infestation of Ceratomyxa diplodae in sharpsnout sea bream (Diplodus puntazzo) treated with reproductive steroids. Bulletin of the European Association of Fish Pathologists 27, 43−47. Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffmann, R.W., Khattra, J., Hallett, S.L., Lester, R.J., Longshaw, M., Palenzeula, O., Siddall, M.E. and Xiao, C. (2001). Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic Microbiology 48, 395−413.

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Liu, Y., Whipps, C.M., Gu, Z.M., Huang, M.J., He, C., Yang, H.L. and Molnar, K. (2013). Myxobolus musseliusae (Myxozoa: Myxobolidae) from the gills of common carp Cyprinus carpio and revision of Myxobolus dispar recorded in China. Parasitology Research 112, 289−296. Lom, J. and Arthur, J.R. (1989). A guideline for the preparation of species descriptions in Myxosporea. Journal of Fish Diseases 12, 151−156. Lom, J. and Dyková, I. (1992). Myxosporidia (Phylum Myxozoa). Protozoan Parasites of Fishes. Developments in Aquaculture and Fisheries Science, Vol. 26. Elsevier, Amsterdam, Netherlands, pp. 159‒235. Lom, J. and Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53, 1−36 . Lubat, J., Radujkovic, B., Marques, A. and Bouix, G. (1989). Parasites des poissons marins du Montenegro: Myxosporidies. Acta Adriatica 30, 31−50. Matsukane, Y., Sato, H., Tanaka, S., Kamata, Y. and Sugita-Konishi, Y. (2011). Kudoa iwatai and two novel Kudoa spp., K. trachuri n. sp. and K. thunni n. sp. (Myxosporea: Multivalvulida), from daily consumed marine fish in western Japan. Parasitology Research 108, 913−926. Merella, P., Cherchi, S., Salati, F. and Garippa, G. (2005). Parasitological survey of sharpsnout seabream Diplodus puntazzo (Cetti, 1777) reared in sea cages in Sardinia (western Mediterranean). Bulletin of the European Association of Fish Pathologists 25, 140−147. Rangel, L.F., Rocha, S., Casal, G., Castro, R., Severino, R., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2016). Life cycle inference and phylogeny of Ortholinea labracis n. sp. (Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm. Journal of Fish Diseases 40, 243−262. Reed, C.C., Basson, L., Van As, L.L. and Dyková, I. (2007). Four new myxozoans (Myxosporea: Bivalvulida) from intertidal fishes along the south coast of Africa. Folia Parasitologica 54, 283−292. Rigos, G., Grigorakis, K., Christophilogiannis, P., Nengas, I. and Alexis, M. (1997). Ceratomyxa spp. (Myxosporea) infection in cultured common dentex from Greece. Bulletin of the European Association of Fish Pathologists 17, 174−176. Rigos, G., Christophilogiannis, P., Yiagnisi, M., Andriopoulou, A., Koutsodimou, M., Nengas, I. and Alexis, M. (1999). Myxosporean infections in Greek mariculture. Aquaculture International 7, 361−364. Rocha, S., Casal, G., Rangel, L., Severino, R., Castro, R., Azevedo, C. and Santos, M.J. (2013). Ultrastructural and phylogenetic description of Zschokkella auratis sp. nov. (Myxozoa), a parasite of the gilthead seabream Sparus aurata. Diseases of Aquatic

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Organisms 107, 19−30. Rocha, S., Casal, G., Al-Quraishy, S. and Azevedo, C. (2014). Morphological and ultrastructural redescription of Chloromyxum leydigi Mingazzini, 1890 (Myxozoa: Myxosporea), type species of the genus, infecting the gall bladder of the marine cartilaginous fish Torpedo marmorata Risso (Chondrichthyes: Torpedinidae), from the Portuguese Atlantic coast. Folia Parasitologica 61, 1−10. Rocha, S., Casal, G., Rangel, L., Castro, R., Severino, R., Azevedo, C. and Santos, M.J. (2015). Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae). Parasitology International 64, 305−313. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1992). Light and electron microscopic description of Sphaerospora dicentrarchi n. sp. (Myxosporea: Sphaerosporidae) from wild and cultured sea bass, Dicentrarchus labrax L. Journal of Protozoology 39, 273−281. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1993a). Pathologic effects of Sphaerospora dicentrarchi Sitjà-Bobadilla and Alvarez-Pellitero, 1992 and S. testicularis Sitjà- Bobadilla and Alvarez-Pellitero, 1990 (Myxosporea: Bivalvulida) parasitic in the Mediterranean sea bass Dicentrarchus labrax L. (Teleostei: Serranidae) and the cell- mediated immune reaction: a light and electron microscopy study. Parasitology Research 79, 119−129. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1993b). Light and electron microscopical description of Ceratomyxa labracis n. sp. and a redescription of C. diplodae (Myxosporea: Bivalvulida) from wild and cultured Mediterranean sea bass Dicentrarchus labrax (L.) (Teleostei: Serranidae). Systematic Parasitology 26, 215−223. Sitjà-Bobadilla, A., Palenzuela, O. and Alvarez-Pellitero, P. (1995). Ceratomyxa sparusaurati n. sp. (Myxosporea, Bivalvulida), a new parasite from cultured gilthead seabream (Sparus aurata L.) (Teleostei, Sparidae)—light and electron microscopic description. Journal of Eukaryotic Microbiology 42, 529–539. Sobecka, E., Szostakowska, B., Zietara, M.S. and Wiecaszek, B. (2013). Morphological and molecular characterization of Ceratomyxa gurnardi sp. n. (Myxozoa: Ceratomyxidae) infecting the gallbladder of the grey gurnard Eutrigla gurnardus (L.) (Scorpaeniformes, Triglidae). Parasitology Research 112, 731−735. Suzuki, J., Murata, R., Sadamasu, K. and Kai, A. (2012). Cases of food poisoning caused possibly by Kudoa spp. in the Tokyo metropolitan area. International Area Studies Review 33, 153−155. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30:

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2725−2729. Thélohan, P. (1894). Recherches sur les myxosporidies. Bulletin biologique de la France et de la Belgique 5, 100−394. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673−4680. Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J.G., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from Eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites sp. nov. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215−219. Yokoyama, H., Grabner, D. and Shirakashi, S. (2012). Transmission biology of the Myxozoa. In Carvalho, D.E. (ed). Health and environment in aquaculture. InTech, Rijeka, pp. 1– 42.

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Chapter V

The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis

This chapter was adapted from:

Chapter V | Potential life cycle role of sphaeractinomyxon

Abstract

Three new types of sphaeractinomyxon (Cnidaria, Myxozoa) are described from the coelomic cavity of the marine oligochaete Tubificoides insularis, collected from the Alvor estuary, Algarve, Portugal. Another known type is also registered from this location and host: Sphaeractinomyxon type 10 of Rangel et al. (2016), which was originally described from the marine oligochaete Tubificoides pseudogaster in the Aveiro estuary, Portugal. Phylogenetic analyses revealed the case isolates and all other available SSU rDNA sequences of sphaeractinomyxon clustering within a clade containing Myxobolus spp. that infect mullets, thus suggesting that this collective group plays a role in the life cycle of mugiliform-infecting myxobolids. Also clustering within this clade were all types of tetraspora and endocapsa, calling into question the distinctiveness of these collective groups. Acknowledging a previous work showing that the pansporocysts of sphaeractinomyxon produce a variable number of actinospores, we suggest that the tetraspora collective group be deemed invalid and its types transferred to sphaeractinomyxon. In turn, endocapsa requires validation through the description of new types truly differentiating them from sphaeractinomyxon.

Introduction

Following the pioneering study of Wolf and Markiw (1984), many other reports have presented direct and indirect evidence for the existence of two stages in the life cycle of Myxosporea: a myxosporean stage that develops in a vertebrate host, mainly in fish; and an actinosporean stage that occurs in an invertebrate host (Eszterbauer et al., 2015). This discovery boosted the number of descriptions of actinosporean stages worldwide, so that currently there are more than 200 types of actinosporeans attributed to different collective groups, which are vernacular designations of the genera of the former Class Actinosporea, as settled by Kent et al. (1994). Surveyed from fish farms (e. g. Eszterbauer et al., 2006; Rosser et al., 2014; Xi et al., 2015; Rangel et al., 2017) and natural waters (e. g. Xiao and Desser, 1998; Hallett et al., 1999; Székely et al., 2014; Rangel et al., 2016a), most actinosporeans were previously described on the basis of morphological characteristics. This practice has been shown to greatly hamper exact identification, as some types display great morphological variability (Hallett et al., 2004; Eszterbauer et al., 2006), and others of the same collective group overlap in terms of measurements (Rangel et al., 2016a). Currently, studies rely on a combination of morphological features and molecular data for the unambiguous characterization of actinospores (e.g. Rosser et al., 2014; Xi et al., 2015; Rangel et al., 2016a, 2017).

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Chapter V | Potential life cycle role of sphaeractinomyxon

The sphaeractinomyxon collective group encompasses 20 types described from freshwater and marine oligochaetes. Six types were described prior to the discovery of Wolf and Markiw (1984): Sphaeractinomyxon stolci in 1904, by Caullery and Mesnil; Sphaeractinomyxon gigas in 1923, by Granata; Sphaeractinomyxon danicae in 1938, by Georgevitch; Sphaeractinomyxon ilyodrili in 1940, by Jirovec; Sphaeractinomyxon amanieui in 1963, by Puytorac; and Sphaeractinomyxon rotundum in 1984, by Marques (Caullery and Mesnil, 1904, 1905; Puytorac, 1963; Marques, 1984). Another four types were described in the years that followed: Sphaeractinomyxon types 1 and 2 of Hallett et al. (1997); Sphaeractinomyxon ersei (Hallett et al., 1998); and Sphaeractinomyxon leptocapsula (Hallett et al., 1999). More recently, another 10 types were added to the group, from oligochaetes inhabiting the Aveiro estuary in Portugal: 7 types from Limnodriloides agnes Hrabě, 1967 and 3 from Tubificoides pseudogaster (Dahl, 1960) (Rangel et al., 2016a). Of the above, S. ersei and the 10 types described by Rangel et al. (2016a) have information for the SSU rDNA gene available in the NCBI database. Currently, about 60 myxozoan species have their complete life cycle inferred through DNA sequence matches of the different parasite stages, or known through holistic transmission studies (Eszterbauer et al., 2015). Nonetheless, several collective groups have never been directly linked to their myxosporean counterparts. Phylogenetic analyses reveal that the clade of myxobolids comprises most of the actinosporean morphotypes represented in the NCBI database from oligochaetes in freshwater and marine environments (Hallett et al., 1999; Kent et al., 2001; Holzer et al., 2004; Eszterbauer et al., 2006; Milanin et al., 2017; Rangel et al., 2017). Consequently, most known life cycles refer to species of myxobolids (Myxobolus, Henneguya and Thelohanellus) infecting fish and oligochaetes in freshwater environments (e. g. Styer et al., 1991; Kent et al., 1993; Lin et al., 1999; Eszterbauer et al., 2000, 2006; Kallert et al., 2005; Marton and Eszterbauer, 2011; Székely et al., 2014); with fewer descriptions for members of other myxosporean genera (Ceratomyxa, Ceratonova, Chloromyxum, Ellipsomyxa, Gadimyxa, Hofferellus, Myxidium, Myxobilatus, Ortholinea, Parvicapsula, Sigmomyxa, Sphaerospora, and Zschokkella), especially in brackish/marine environments (Grossheider and Körting, 1992; Benajiba and Marques, 1993; Yokoyama et al., 1993a; Bartholomew et al., 1997, 2006; Holzer et al., 2004, 2006; Køie et al., 2004, 2007, 2008; Atkinson and Bartholomew, 2009; Rangel et al., 2009, 2017; Karlsbakk and Køie, 2012). This study provides morphological and molecular data for three new types of sphaeractinomyxon found infecting the marine oligochaete Tubificoides insularis (Stephenson, 1923) in the Alvor estuary, Algarve, Portugal. The phylogenetic setting of this collective group is presented, and the potential role that it plays in the life cycle of mugiliform-infecting myxobolids is discussed.

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Chapter V | Potential life cycle role of sphaeractinomyxon

Materials and methods

Sampling and morphological characterization

Between April 2013 and March 2014, samples of mud were collected monthly at low tide in one site of the Alvor estuary (37° 08′ N, 08° 37′ W), Portimão, Algarve, Portugal. In the laboratory, oligochaetes were collected from the mud and were held at room temperature, individually distributed into 48-well plates containing salt water. A stereo microscope was used to determine the release of actinospores from the sampled oligochaetes in the following days. After this time period, all specimens were individually examined under a light microscope for the detection of actinosporean stages in internal tissues and cavities. As waterborne actinospores were not observed, prevalence of infection was determined from the specimens displaying actinosporean infection upon microscopic observation. Developmental stages and free actinospores were examined and photographed using a Zeiss Axiophot microscope (Grupo Taper, Sintra, Portugal) equipped with a Zeiss AxioCam Icc3 digital camera. AxioVision 4.6.3 software (Grupo Taper, Sintra, Portugal) was used in image analysis. Morphology and morphometry were characterized using fresh material, in accordance to Lom et al. (1997). Measurements included the mean value ± standard deviation (SD), range of variation, and number of measured actinospores (n).

Molecular characterization

Genomic DNA was extracted only from oligochaetes displaying mature infections, i.e. containing mature actinospores, using a GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, USA). The DNA was stored in 50 μl of TE buffer at −20 °C until further use. The SSU rDNA gene of the actinospores was amplified using both universal and myxosporean-specific primers: the 5′-end with the primers 18E (5′-CTG GTT GAT CCT GCC AGT-3′) (Hillis and Dixon, 1991) and ACT3r (5′-ATT GTT CGT TCC ATG-3′) (Hallett and Diamant, 2001); the 3′-end with the primers ACT3f (5′-CAT GGA ACG AAC AAT3′) (Hallett and Diamant, 2001) and 18R (5′-CTA CGG AAA CCT TGT TAC G-3′) (Whipps et al., 2003); and the overlapping sequence by pairing the primer ACT5f (5′-TGT GCC TTG AAT AAA T-3′) (this study) with the 18R primer. PCRs were performed in 50 µl reactions using 10 pmol of each primer, 10 nmol of each dNTP, 2.5 mM of MgCl2, 5 µl 10 × Taq polymerase buffer, 1.5 units Taq DNA polymerase (NZYTech, Lisbon, Portugal), and 3 µl (approximately 100–150 ng) of genomic DNA. The reactions were run on a Hybaid PxE Thermocycler (Thermo Electron Corporation, Milford, Massachusetts, USA), with initial denaturation at 95 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 53 °C for 45 s, and 72 °C for 90 s. The final elongation step was

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Chapter V | Potential life cycle role of sphaeractinomyxon performed at 72 °C for 7 min. In turn, the 16S rDNA gene of mitochondrial DNA (mtDNA) of the oligochaete host was amplified using the universal primers 16sar-L (5′-CGC CTG TTT ATC AAA AAC AT-3′) and 16sbr-H (5′-CCG GTC TGA ACT CAG ATC ACG T-3′) (Palumbi et al., 2002). PCRs were carried out according to the conditions previously mentioned for the actinospores. Five-µl aliquots of the PCR products were electrophoresed through a 1% agarose 1 × tris-acetate-EDTA buffer (TAE) gel stained with ethidium bromide. PCR products were purified using a single-step enzymatic clean-up that eliminates unincorporated primers and dNTPs by ExoFast method. Sequencing reactions were performed using a BigDye Terminator v1.1 from the Applied Biosystems Kit (Applied Biosystems, Carlsbad, California, USA), and were run on an ABI3700 DNA analyzer (Perkin-Elmer, Applied Biosystems, Stabvida, Oeiras, Portugal).

Phylogenetic analysis

The partial SSU rDNA sequences obtained for each case isolate were aligned separately in MEGA 6.06, allowing the construction of the respective assembled sequences. The dataset used for phylogenetic analyses was chosen in order to represent the myxosporean freshwater lineage, emphasizing the different actinosporean collective groups that cluster within this lineage, and further incorporating all taxa with known life cycle in freshwater environments. The SSU rDNA sequence available for the myxosporean stage of Myxobolus djragini (AF085179) was not included due to uncertainty in its reliability (Molnár, 2011). The final dataset comprised a total of 140 SSU rDNA sequences, and included Chloromyxum riorajum (FJ2624481), Chloromyxum clavatum (JQ793641), Chloromyxum leydigi (DQ377710) as outgroup. Sequences were aligned using the software MAFFT version 7 available online, and posteriorly manually edited in MEGA 6.06. Phylogenetic analyses using the maximum likelihood algorithm were conducted in MEGA 6.06, using the general time reversible substitution model with four gamma-distributed rate variation among sites, with bootstrap confidence values calculated from 1000 replicates. Bayesian inference analyses were performed using MrBayes v3.2.6 (Ronquist and Huelsenbeck, 2003). The general time reversible model with gamma-shaped rate variations across sites (Invgamma) (GTR + I + Γ) was used, in accordance to the ModelTest algorithm of the software. Posterior probability distributions were generated using the Markov Chain Monte Carlo (MCMC) method, with four chains running simultaneously for 1 million generations, and every 100th tree sampled. A second alignment was performed in MAFFT version 7 for the sequences of both myxosporean and actinosporean stages comprising the mugiliform-infecting clade. Distance estimation was performed for the latter alignment, using the p-distance model and pairwise

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Chapter V | Potential life cycle role of sphaeractinomyxon deletion, with all ambiguous positions removed for each sequence pair in MEGA 6.06.

Results

Myxozoan survey, overall prevalence of infection and host identification

In total, 789 oligochaetes were examined during the myxozoan survey, of which 760 were identified as Tubificoides insularis, and the remaining 29 as Limnodriloides agnes. Three of the 29 specimens (10.3%) of L. agnes were infected with early developmental stages of undetermined actinosporean collective groups. Of the 760 T. insularis specimens, 46 (6.1%) were infected with developmental stages of the sphaeractinomyxon collective group. Other actinosporean collective groups were not observed infecting this host species. Identification of the infected oligochaetes containing fully matured actinospores was confirmed by a comprehensive combination of morphological features and molecular data of the 16S mitochondrial DNA (mtDNA). The identical sequences obtained from infected oligochaetes were assembled into a single consensus sequence and deposited in the NCBI database under the accession number MK085118. Host sequences presented high similarity to those available in the NCBI database for the lineages of T. insularis, (99.8% to lineage I and 97.8–98.3% to lineage II), with values lower than 90.0% of similarity to all other annelid sequences. Identification was fully supported by the morphological features of the oligochaete hosts, e. g. the presence of cuticular papillation, bifid setae and hair chaetae (Brinkhurst, 1985). Overall the highest prevalence of sphaeractinomyxon infection was registered during autumn (6.6%; 31 infected in 470 oligochaetes examined between October and December), followed by summer (5.4%; 8 infected in 149 examined between July and September), and spring (5.0%; 7 infected in 141 examined hosts between April and June). We were not able to determine the prevalence of infection during the winter months (January to March), because specimens of T. insularis were not present in the mud samples collected during that time period. Molecular analysis of the parasitesˈ SSU rDNA gene distinguished between four different types of sphaeractinomyxon, so that three novel types are described here, and a record of a previously known type is also presented.

Characterization of three novel sphaeractinomyxon types (Cnidaria, Myxozoa), and record of a previously known type

Sphaeractinomyxon type 1 (Figs. 1A–B, 2A) Description: Mature actinospores spherical in apical and lateral view, measuring 18.0 ±

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1.1 (16.6–20.8) µm in length (n = 26), 18.6 ± 1.2 (16.9–20.8) µm in width (n = 27), and 19.4 ± 0.8 (18.0–20.5) µm in diameter (n = 10). Three pyriform polar capsules, 5.2 ± 0.5 (4.2–5.8) µm long (n = 11) and 3.4 ± 0.3 (2.8–3.9) µm wide (n = 11), located at the centre of the actinospores, each containing a polar tubule exhibiting 3 coils. Sporoplasm with 200 or more secondary cells. Pansporocysts presented synchronous development, each containing 8 actinospores in equivalent stages. Mature actinospores were not observed exiting the host body. Type host: Tubificoides insularis (Stephenson, 1923) (Annelida, Oligochaeta). Type locality: Alvor estuary, near the Atlantic coast (37° 08′ N, 08° 37′ W), Portimão, Algarve, Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: Overall prevalence of infection 0.8% (6 infected in a total of 760 hosts examined); 2.1% during spring (3 infected in 141 examined between April and June); 1.3% during summer (2 infected in 149 examined between July and September); 0.2% during autumn (1 infected in 470 examined between October and December); no hosts were recorded during winter (between January and March). Type material: Series of phototypes of the hapantotype, deposited together with a representative DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2018.20. Remarks: Morphometry was determined from mature actinospores in two infected hosts. Individual measurements of the two isolates were identical, so no significant morphometric variation was recorded. The identical partial sequences obtained for the SSU rDNA gene of the isolates were assembled into a single sequence of 1,984 bp, which was deposited in the NCBI database under the accession number MH017876. This sequence did not match any SSU rDNA sequences currently available for myxozoans, and was most similar to those comprising the clade of mugiliform-infecting myxobolids (Fig. 4). Distance estimation revealed highest percentage of similarity to Sphaeractinomyxon type 8 of Rangel et al. (2016a) (KU569317) (93.9%), and Triactinomyxon of Székely et al. (2007) (DQ473515) (93.7%).

Sphaeractinomyxon type 2 (Figs. 1C–D, 2B) Description: Mature actinospores spherical in apical and lateral view, measuring 28.2 ± 1.2 (26.6–30.2) µm in length (n = 6), 29.5 ± 1.0 (28.4–31.1) µm in width (n = 6), and 29.8 ± 1.3 (27.8–32.2) µm in diameter (n = 17). Three pyriform polar capsules, 7.0 ± 0.5 (6.4–7.6) µm long (n = 6) and 5.1 ± 0.3 (4.8–5.7) µm wide (n = 6), located at the centre of the actinospores, each containing a polar tubule exhibiting 3 to 4 coils. Sporoplasm with 200 or more secondary cells. Pansporocysts presented synchronous development, each containing 8 actinospores in equivalent stages. Mature actinospores were not observed exiting the host body.

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Figure 1. Light micrographs showing mature actinospores of the new types of sphaeractinomyxon found in the coelomic cavity of Tubificoides insularis. (A–B) Sphaeractinomyxon type 1 in lateral (A) and apical (B) view. (C–D) Sphaeractinomyxon type 2 in lateral (C) and apical (D) view. (E) Sphaeractinomyxon type 3 in lateral view. (F–G) Sphaeractinomyxon type 3 in apical view. Same actinospore observed in different focal planes.

Figure 2. Schematic drawings depicting the mature actinospores of the new types of sphaeractinomyxon found in the coelomic cavity of Tubificoides insularis, as observed in lateral (top) and apical (bottom) view. (A) Sphaeractinomyxon type 1. (B) Sphaeractinomyxon type 2. (C) Sphaeractinomyxon type 3.

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Type host: Tubificoides insularis (Stephenson, 1923) (Annelida, Oligochaeta). Type locality: Alvor estuary, near the Atlantic coast (37° 08′ N, 08° 37′ W), Portimão,

Algarve, Portugal.

Site of infection: Throughout the coelomic cavity. Prevalence: Overall prevalence of infection 0.3% (2 infected in a total of 760 hosts examined); 0.0% during spring (0 infected in 141 examined between April and June); 0.0% during summer (0 infected in 149 examined between July and September); 0.4% during autumn (2 infected in 470 examined between October and December); no hosts were recorded during winter (between January and March). Type material: Series of phototypes of the hapantotype, deposited together with a representative DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2018.21. Remarks: Morphometry was determined from one of the two infected hosts, in which fully matured actinospores could be measured. The partial sequences obtained for the SSU rDNA gene of the isolates from the two hosts differed in two positions of the 1,986 bp assembled sequence: in position 29 and 1554, which are represented by Y (T/C). The assembled sequence was deposited in the NCBI database under the accession number MH017877. This sequence did not match the SSU rDNA sequences currently available for myxozoans and was most similar to those comprising the clade of mugiliform-infecting myxobolids (Fig. 4). Distance estimation revealed highest percentage of similarity to Sphaeractinomyxon type 9 of Rangel et al. (2016a) (KU569318) (96.8%), and Triactinomyxon of Székely et al. (2007) (DQ473515)

(95.8%). All others presented values of percentage of similarity lower than 95.0%

Sphaeractinomyxon type 3 (Figs. 1E–B, 2C, 3) Description: Mature actinospores triangular in apical view and subspherical in lateral view, measuring 20.6 ± 0.7 (19.1–21.6) µm in length (n = 17), 29.1 ± 1.1 (28.1–31.1) µm in width (n = 17), and 28.9 ± 0.8 (27.6–30.2) µm in diameter (n = 17). Three pyriform polar capsules, 6.2 ± 0.4 (5.6–7.1) µm long (n = 28) and 4.8 ± 0.3 (4.5–5.4) µm wide (n = 28), located at the centre of the actinospores, each containing a polar tubule exhibiting 4 coils. Sporoplasm with 200 or more secondary cells. Pansporocysts presented synchronous development, each containing 8 actinospores in equivalent stages. Mature actinospores were not observed exiting the host body. Development: The first developmental stages observed were binucleated cells (Fig. 3A), which divided twice to form a group of four cells: two somatic cells that formed the pansporocyst wall, and two generative cells that were enclosed within this structure (Fig. 3B, C). The two generative cells underwent mitotic divisions and one meiotic division to produce a

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Chapter V | Potential life cycle role of sphaeractinomyxon final set of 16 haploid cells (Fig. 3D–F), which united two by two to form 8 diploid zygotes inside the pansporocyst (Fig. 3G). Zygotes then underwent mitotic divisions to form immature actinospores, each comprised of a sporoplasmic cell connected to three valvogenic cells that enveloped three capsulogenic cells. The valvogenic and capsulogenic cells occupied a more

Figure 3. Developmental stages of Sphaeractinomyxon type 3 in the coelomic cavity of Tubificoides insularis. (A– F) Gametogamy phase: (A) Binucleated cell; (B) Binucleated cell in cellular division; (C) Initial pansporocyst with 2 inner cells; (D–F) Pansporocysts with four, six, and 16 inner cells, respectively. (G–M) Sporogony phase: (G) Pansporocyst with 8 zygotes undergoing cellular divisions; (H) Pansporocyst showing the valvogenic and capsulogenic cells (*) located centrally, while the sporoplasmic cells (SC) lean against the pansporocyst wall; (I) Pansporocyst showing the actinospores' involucres centrally located and formed by three valvogenic cells (VC) surrounding three internal capsulogenic cells (CC). The sporoplasmic cells (SC) lean against the pansporocyst wall; (J) Same as I, but with the actinospores' involucres and sporoplasmogenic cells more developed; (K) Isolated actinospore involucre from a ruptured pansporocyst showing the thin valvogenic cells (VC) more expanded than in the previous stage, as well as three central capsulogenic cells with fully formed polar capsules (PC); (L) Pansporocyst containing eight immature actinospores. Note that at this stage the sporoplasmic cells have already entered their corresponding involucres. (M) Ruptured pansporocyst containing mature actinospores (one of the eight actinospores has already exited the pansporocyst).

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Chapter V | Potential life cycle role of sphaeractinomyxon central position inside the pansporocyst, while the sporoplasmic cell was located adjacent to the pansporocyst wall (Fig. 3H). As the actinospores developed, the valvogenic cells expanded, the capsulogenic cells turned into the polar capsules, and the sporoplasmic cell increased in size, while multiplying the number of secondary cells inside. The development of the sporoplasmic cells occurred outside the involucre formed by the valvogenic and capsulogenic cells; it was only at the end of sporogony that the sporoplasmic cells entered or were enclosed by the corresponding valvogenic cells (Fig. 3I–L). Finally, a set of 8 mature actinospores was observed inside each pansporocyst (Fig. 3M). Type host: Tubificoides insularis (Stephenson, 1923) (Annelida, Oligochaeta). Type locality: Alvor estuary, near the Atlantic coast (37° 08′ N, 08° 37′ W), Portimão, Algarve, Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: Overall prevalence of infection 0.3% (2 infected in a total of 760 hosts examined); 0.0% during spring (0 infected in 141 examined between April and June); 1.3% during summer (2 infected in 149 examined between July and September); 0.0% during autumn (0 infected in 470 examined between October and December); no hosts were recorded during winter (between January and March). Type material: Series of phototypes of the hapantotype, deposited together with a representative DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2018.22. Remarks: Morphometry was determined from mature actinospores observed in both infected hosts. Individual measurements were identical between both isolates, so no significant morphometric variation was recorded. The identical partial sequences obtained for the SSU rDNA gene of the two isolates were assembled into a single sequence comprised by 2,024 bp, which was deposited in the NCBI database under the accession number MH017878. This sequence did not match any of the SSU rDNA sequences currently available for myxozoans, being most similar to those comprising the clade of mugiliform-infecting myxobolids (Fig. 4). Distance estimation revealed highest percentage of similarity to Sphaeractinomyxon type 3 of Rangel et al. (2016a) (KU569312) (90.6%). All others presented values of percentage of similarity lower than 90.0%.

Sphaeractinomyxon type 10 of Rangel et al. (2016a) in the Alvor estuary Description: Mature actinospores spherical in apical and lateral view, measuring 22.7 ± 1.4 (21.7–23.7) µm in length (n = 2), 25.1 ± 1.1 (24.2–25.9) µm in width (n = 2), and 24.2 ± 0.2 (24.0–24.3) µm in diameter (n = 2). Three pyriform polar capsules, 5.6 ± 0.2 (5.4–5.8) µm long (n = 2) and 3.9 ± 0.2 (3.7–4.1) µm wide (n = 2), located at the centre of the actinospores, each

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Figure 4. Tree topology resultant from the Bayesian analysis of 140 SSU rDNA sequences representative of the myxosporean freshwater lineage and including all taxa with known life cycles in this aquatic environment. Numbers

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at the nodes represent Bayesian posterior probabilities/maximum likelihood bootstrap values; dashes represent a different branching for the maximum likelihood tree or a bootstrap support value under 50. Taxa in blue parasitize marine fish, with sequences from the present study in bold; taxa in red parasitize anadromous or catadromous fish hosts. In the case of complete life cycle, the actinosporean stage is given after the corresponding myxosporean stage; ET stands for life cycle proven by experimental transmission, while MI stands for life cycle proven by molecular inference. Main clades are enclosed in boxes; final host groups of the Myxobolus clade are designated by vertical lines. containing a polar tubule exhibiting 3 coils. Sporoplasm with 200 or more secondary cells. Pansporocysts presented synchronous development, each containing 8 actinospores in equivalent stages. Mature actinospores were not observed exiting the host body. Type host: Tubificoides pseudogaster (Dahl, 1960) (Annelida, Oligochaeta). Other hosts: Tubificoides insularis (Stephenson, 1923) (Annelida, Oligochaeta). Type locality: Aveiro estuary (40° 40′ N, 08° 45′ W), Portugal. Other localities: Alvor estuary, near the Atlantic coast (37° 08′ N, 08° 37′ W), Portimão, Algarve, Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: Overall prevalence of infection in T. insularis collected from the Alvor estuary was 0.3% (2 infected in a total of 760 oligochaetes examined); 0.7% during spring (1 infected in 141 examined between April and June); 0.0% during summer (0 infected in 149 examined between July and September); 0.2% during autumn (1 infected in 470 examined between October and December); no hosts were recorded during winter (between January and March). Remarks: The sequences obtained for the SSU rDNA gene of both isolates were an exact match to that available for Sphaeractinomyxon type 10 of Rangel et al. (2016a) in its type host, T. pseudogaster, from the Aveiro estuary, Portugal. Distance estimation revealed highest percentage of similarity to Sphaeractinomyxon type 8 of Rangel et al. (2016a) (KU569317) (93.1%), Sphaeractinomyxon type 1 (this study) (92.9%), and Sphaeractinomyxon type 2 (this study) (92.7%). Significant morphometric variation was not recorded in relation to the original description of this type.

Discussion

The morphological features of the parasites studied here support their identification as members of the sphaeractinomyxon collective group, with molecular analysis further distinguishing between four different types. Three constitute new types that are described here, while the other type was identified as Sphaeractinomyxon type 10 of Rangel et al. (2016a), previously described from the Aveiro estuary (Portugal), around 500 km North from our sampling location in the Alvor estuary. All of these types, new and known, develop within the

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Chapter V | Potential life cycle role of sphaeractinomyxon coelomic cavity of their oligochaete hosts, following the pattern that has been commonly described for members of the sphaeractinomyxon collective group (e.g. Hallett et al., 1998). Parasitic development inside the host is asynchronous, with early stages appearing alongside mature actinospores throughout the coelomic cavity. In turn, sporogony is synchronous within the pansporocysts, with 8 actinospores in the same developmental stage. The development of sphaeractinomyxon type 3 differed from the other types in the conspicuous triangular shape of the immature and mature actinospores. Identification of oligochaete hosts as Tubificoides insularis was achieved through the combined analyses of morphologically distinctive characters and molecular data of the 16S locus. This combined approach is helpful for non-oligochaete taxonomists to correctly identify annelid hosts, which is an important descriptive character. In the case of sphaeractinomyxon, Rangel et al. (2016a) suggested that this collective group has host specificity at the species- level, because the 10 types described from the Aveiro estuary were restricted to their specific type species. In fact, most sphaeractinomyxon types have also been reported exclusively from their type hosts; such are the cases of S. amanieui from Baltidrilus costatus (Claparède, 1863), S. gigas from Limnodrilus hoffmeisteri Claparède, 1862, S. ilyodrili from Potamothrix prespaensis (Hrabĕ, 1931), Sphaeractinomyxon type 1 of Hallett et al. (1997) from Aktedrilus mortoni Erséus, 1984, and Sphaeractinomyxon type 2 of Hallett et al. (1997) from Ainudrilus geminus Erséus, 1990 (Hallett et al., 1997; Marques, 1984; Puytorac, 1963). S. danicae was originally described from an unidentified species of Eiseniella Michaelsen, 1900, and later reported from Eiseniella tetraedra (Savigny, 1826), a species that may very well be its type host (Marques, 1984). Similarly, the only available reports for S. rotundum and S. leptocapsula are their original descriptions, each performed from an unidentified species of the family Naididae Ehrenberg, 1828 (Marques, 1984; Hallett et al., 1999). Thus, having been reported from more than one host species, S. ersei and S. stolci supposedly constitute the only exceptions to the strict host specificity of the collective group (Marques, 1984; Hallett et al., 1998, 1999). S. stolci was simultaneously reported from three species of the family Naididae, namely Clitellio arenarius Savigny, 1820, Tubificoides benedii (d'Udekem, 1855), and an unidentified Tubifex sp.; however, considering that all of these reports were performed without the use of molecular tools, it is more probable that this type constitutes an assemblage of a few types that share morphometric similarity. In the same manner, the occurrence of S. ersei in unidentified tubificids from Heron Island is uncertain, as there was no molecular comparison to the parasitic material in the type host Doliodrilus diverticulatus Erséus, 1984. As such, the occurrence of Sphaeractinomyxon type 10 of Rangel et al. (2016a) in T. insularis constitutes the first confirmed exception to the strict host specificity of the collective group, as the sequences obtained in this study were an exact match to those obtained from the type material in T. pseudogaster. Ascertaining the true degree of host specificity of sphaeractinomyxon, as

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Chapter V | Potential life cycle role of sphaeractinomyxon well as of other actinosporean collective groups, is certainly an important task for future research, as it will help to reliably identify both known and new types. Turning to consider the prevalence of infection determined in this study, it is noted that individual values were lower than 1% for all types described here, which is congruent with previous studies of other types of sphaeractinomyxon, as well as of types of other actinosporean collective groups (e. g. Hallett et al., 1999; Székely et al., 2007; Rangel et al., 2011, 2016a; Xi et al., 2015; Milanin et al., 2017). The overall prevalence of infection of the sphaeractinomyxon collective group in the sample of oligochaetes examined here from the Alvor estuary is significantly lower than that reported by Rangel et al. (2016a) for the same type of myxozoan survey in the Aveiro estuary (18.7%). The pattern of seasonal variation, however, is identical to the one reported in that study, as overall prevalence of infection is similar between spring, summer and autumn. Other works reported higher prevalence of infection during spring and summer but, contrary to this study, considered only individual hosts displaying actinospore release (e.g. Yokoyama et al., 1993b; El-Mansy et al., 1998a, 1998b; Xiao and Desser, 1998; Oumouna et al., 2003), and, therefore, might have disregarded immature infections possibly present in the autumn months. Rangel et al. (2016a) showed that sphaeractinomyxon types can be present in the oligochaete hosts throughout the year, with maturation of the actinospores and subsequent release taking place mostly during spring and summer months. In this study, it was not possible to obtain information regarding the overall prevalence of infection of sphaeractinomyxon during winter, as few oligochaete specimens, and none of the genus Tubificoides, were present in the mud samples collected between January and March. This was probably due to the heavy precipitation in the Alvor estuary during that time period. Several studies have shown that oligochaete availability in aquatic ecosystems is influenced by fluctuations of ecological and biophysical parameters (e.g. water temperature, precipitation, salinity, eutrophication, and others), which occur naturally, or are directly or indirectly related to anthropogenic factors (Cole et al., 2002; Silva et al., 2006; Burgmer et al., 2007; Daufresne et al., 2007; Durance and Ormerod, 2007; Lin and Yo, 2008; Cunha et al., 2011; Armendáriz et al., 2012). As such, it is likely that infection by these sphaeractinomyxon types can occur during less rainy winters, if or when the oligochaete host is available, as shown for other sphaeractinomyxon types (Rangel et al., 2016a). The information gathered in the past few years reveals that there is no obvious agreement between actinosporean morphotypes and myxosporean genera (Eszterbauer et al., 2015). In fact, our phylogenetic analysis shows that the clade of myxobolids comprises most of the actinosporean morphotypes represented in the NCBI database (antonactinomyxon, aurantiactinomyxon, echinactinomyxon, endocapsa, helioactinomyxon, hexactinomyxon, hungactinomyxon, neoactinomyxum, raabeia, sphaeractinomyxon, seisactinomyxon, synactinomyxon, tetraspora, and triactinomyxon) from both freshwater and marine

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Chapter V | Potential life cycle role of sphaeractinomyxon oligochaetes (e. g. Hallett et al., 1999; Kent et al., 2001; Holzer et al., 2004; Eszterbauer et al., 2006; Milanin et al., 2017; Rangel et al., 2017). Thus far, triactinomyxon are, undoubtedly, the most common counterparts for myxobolids, being involved in the life cycle of several Myxobolus spp. that infect Cypriniformes and Salmoniformes (e.g. Wolf and Markiw, 1984; Székely et al., 2014), as well as that of Myxobolus portucalensis from the anguiliform host Anguilla anguilla (El-Mansy et al., 1998c), and Henneguya nuesslini from salmoniform fish hosts (Kallert et al., 2005). Aurantiactinomyxon types have been associated with the life cycles of Thelohanellus and Myxobolus spp. that infect Cypriniformes (Eszterbauer et al., 2006; Molnár et al., 2010), as well as of Henneguya from siluriform fish hosts (Styer et al., 1991; Lin et al., 1999), and we show that they possibly play a role in the life cycle of perciform- and characiform-infecting myxobolids (Fig. 4). Neoactinomyxum types have also been directly linked to Thelohanellus (Xi et al., 2015), and are here shown as possible counterparts to the life cycles of perciform-infecting myxobolids (Fig. 4). Raabeia and echinactinomyxon stages have been linked to the life cycle of myxobolids that infect Cypriniformes (Yokoyama et al., 1995; Molnár et al., 1999; Holzer et al., 2004; Eszterbauer et al., 2006; Marton and Eszterbauer, 2011), with raabeia also possibly playing a role in the life cycle of siluriform- infecting myxobolids (Fig. 4, Rosser et al., 2014). In turn, antonactinomyxon has only been linked to the life cycle of Chloromyxum auratum (Atkinson et al., 2007), and never to a myxobolid counterpart, while members of the endocapsa, helioactinomyxon, hexactinomyxon, hungactinomyxon, sphaeractinomyxon, seisactinomyxon, synactinomyxon, and tetraspora collective groups have never been linked directly to the life cycle of any myxosporean, neither by experimental transmission nor by DNA matching. In the case of sphaeractinomyxon, up until recently, the only SSU rDNA sequence available for this collective group was that of S. ersei from the marine oligochaete D. diverticulatus in Moreton Bay, Australia. The work of Rangel et al. (2016a) significantly elevated the number of available sequences through the molecular description of 10 new types from the marine oligochaetes L. agnes and T. pseudogaster in the Aveiro estuary, Portugal. All of these SSU rDNA sequences were incorporated in our phylogenetic analyses of the case isolates, which revealed all members of the sphaeractinomyxon collective group consistently clustering alongside several myxosporean species described from mullets worldwide. Acknowledging recent studies that demonstrate vertebrate host affinity as the strongest evolutionary signal for myxobolids (e. g. Carriero et al., 2013; Rocha et al., 2014a, 2014b), this phylogenetic setting suggests that sphaeractinomyxon types from marine oligochaetes play a role in the life cycle of mugiliform- infecting myxobolids. Other actinosporean stages clustering within the mugiliform-infecting clade were: Tetraspora discoidea, also from the marine oligochaete D. diverticulatus in Moreton Bay, Australia (Hallett and Lester, 1999); Endocapsula rosulata from the marine oligochaete Heterodrilus keenani Erséus, 1981 in Heron Island, Australia (Hallett et al., 1999);

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Chapter V | Potential life cycle role of sphaeractinomyxon and the Endocapsa and Triactinomyxon of Székely et al. (2007), both reported from a single specimen of the freshwater oligochaete Psammoryctides albicola (Michaelsen, 1901) in the Euphrates River, Syria (Székely et al., 2007). As previously mentioned, triactinomyxon types are involved in the life cycles of several myxobolids, but tetraspora and endocapsa are yet to be linked to specific myxosporean stages. The validity of these two collective groups was recently questioned (Rangel et al., 2016a), due to the lack of morphological characters truly differentiating them from sphaeractinomyxon. Tetraspora differ from sphaeractinomyxon by presenting actinospores that develop in groups of 4 within the pansporocysts (Hallett and Lester, 1999); however, this character may be too variable to establish distinct collective groups, as shown by Rangel et al. (2016a). Also, according to Lom and Dyková (2006), actinosporean stages should be classified on the basis of actinospore morphology, because developmental stages lack reliable differentiating characters. Considering the phylogenetic analyses here performed for these collective groups, we suggest that tetraspora be deemed invalid, with its two types transferred to sphaeractinomyxon: T. discoidea becomes Sphaeractinomyxon type A of Hallett and Lester (1999), and T. rotundum becomes Sphaeractinomyxon type B of Hallett and Lester (1999). The endocapsa collective group was erected to encompass actinospores that differ from sphaeractinomyxon only by its “submerged” polar capsules and by the presence of irregular “processes in the form of swellings” that do not change when in contact with water. The subject of the validity of this group was debated in a previous work (Rangel et al. 2016a), and in light of the phylogenetic analyses here performed, we maintain that the existence of endocapsa requires corroboration. In general, the information available for the life cycles of myxosporeans mainly refers to species that use freshwater oligochaetes as invertebrate hosts; this includes parasites of potadromous, anadromous, and catadromous fish hosts (e. g. Benajiba and Marques, 1993; Atkinson and Bartholomew, 2009; Székely et al., 2014). The few studies concerning the marine environment suggested polychaetes as the hosts of choice for myxosporeans infecting marine and oceanodromous fish hosts (Køie et al., 2004, 2007, 2008; Rangel et al., 2009, 2011, 2016b; Karlsbakk and Køie, 2012). Nonetheless, there are few known exceptions: the triactinomyxon stages of both Ortholinea auratae and O. labracis have marine oligochaetes as invertebrate hosts (Rangel et al., 2015, 2017); while the tetractinomyxon stages of Ceratonova shasta and Parvicapsula minibicornis infect the freshwater polychaete Manayunkia speciosa Leidy, 1859 (Bartholomew et al., 1997, 2006). The phylogenetic positioning of these species as exceptions to the division of Myxosporea into a major freshwater clade and a major marine clade, support the contention that the main evolutionary signal for myxosporeans is the type of invertebrate host, rather than the aquatic environment of the vertebrate host. In fact, the existence of an oligochaete lineage and a polychaete lineage was first noted by Holzer et al. (2007) and has received further support by recent studies (Fiala et al., 2015c; Rangel et al.,

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2017; Holzer et al., 2018). Our phylogenetic analysis also supports this pattern, as the case isolates and all other SSU rDNA sequences available for types of sphaeractinomyxon, endocapsa, and tetrapora cluster consistently within the so-called freshwater lineage, despite having all been reported from marine oligochaetes [with the exception of the Endocapsa of Székely et al. (2007)]. Furthermore, the Triactinomyxon of Roubal et al. (1997) (AF306792), which was reported from a marine oligochaete also clusters within the major freshwater clade, with highest affinity to species of the genus Henneguya (Fig. 4). In conclusion, being catadromous in nature, mullets are the temporary hosts for myxosporean species that may potentially develop in oligochaetes or polychaetes, be it either in freshwater, brackish or marine environments. Thus far, Ellipsomyxa mugilis is the only myxosporean with its life cycle reported from a mullet fish host in a marine environment, having a tetractinomyxon counterpart that develops in the polychaete Hediste diversicolor (O.F. Müller, 1776) (Rangel et al., 2009). In light of the phylogenetic analysis here presented, a task for future myxozoan surveys is to sample mullets, as this fish group comprises the most probable hosts for the myxobolid counterparts of available sphaeractinomyxon types. It would also be interesting to gather molecular information from reported freshwater sphaeractinomyxon types, although we believe they would cluster among their marine relatives, following the example of the Endocapsa of Székely et al. (2007). Thus far, only triactinomyxon, sphaeractinomyxon (including former Tetraspora), and endocapsa have been reported from marine oligochaetes, while tetractinomyxon is the only collective group to have been described from both freshwater and marine polychaetes. This reduced diversity might reflect the lack of studies concerning marine oligochaetes, as well as polychaetes (both marine and freshwater), but on the other hand, could represent correlations of the actinosporean morphotypes to evolutionary signals that are not yet perceivable.

Acknowledgements

This work was supported by the Foundation for Science and Technology (Lisbon, Portugal) within the scope of the Ph.D. fellowship grant attributed to S. Rocha (SFRH/BD/92661/2013) through the programme QREN-POPH/FSE; the Eng° António de Almeida Foundation (Porto, Portugal); the Structured Program of R&D&I INNOVMAR – Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER000035, namely within the Research Line INSEAFOOD Innovation and valorization of seafood products: meeting local challenges and opportunities, within the R&D Institution CIIMAR (Interdisciplinary Centre of Marine and Environmental Research), supported by the Northern Regional Operational Programme (NORTE2020), through the European Regional Development Fund (ERDF); and the Deanship of Scientific

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Research at King Saud University (ISPP no. 0067).

References

Armendáriz, L., Ocón, C. and Capítulo, A.R. (2012). Potential responses of oligochaetes (Annelida, Clitellata) to global changes: experimental fertilization in a lowland stream of Argentina (South America). Limnologica 42, 118–126. Atkinson, S.D. and Bartholomew, J.L. (2009). Alternate spore stages of Myxobilatus gasterostei, a myxosporean parasite of three-spined sticklebacks (Gasterosteus aculeatus) and oligochaetes (Nais communis). Parasitology Research 104, 1173– 1181. Atkinson, S.D., Hallett, S.L. and Bartholomew, J.L. (2007). The life cycle of Chloromyxum auratum (Myxozoa) from goldfish, Carassius auratus (L.), involves an antonactinomyxon actinospore. Journal of Fish Diseases 30, 149–156. Bartholomew, J.L., Atkinson, S.D. and Hallett, S.L. (2006). Involvement of Manayunkia speciosa (Annelida: Polychaeta: Sabellidae) in the life cycle of Parvicapsula minibicornis, a myxozoan parasite of Pacific salmon. Journal of Parasitology 92, 742– 748. Bartholomew, J.L., Whipple, M.J., Stevens, D.G. and Fryer, J.L. (1997). The life cycle of Ceratomyxa shasta, a myxosporean parasite of salmonids, requires a freshwater polychaete as an alternate host. Journal of Parasitology 83, 859–868. Benajiba, M.H. and Marques, A. (1993). The alternation of actinomyxidian and myxosporidian sporal forms in the development of Myxidium giardi (parasite of Anguilla anguilla) through oligochaetes. Bulletin of the European Association of Fish Pathologists 13, 100–103. Brinkhurst, R.O. (1985). A further contribution to the taxonomy of the genus Tubificoides Lastockin (Oligochaeta: Tubificidae). Canadian Journal of Zoology 63, 400–410. Burgmer, T., Hillebrand, H. and Pfenninger, M. (2007). Effects of climate-driven temperature changes on the diversity of freshwater macroinvertebrates. Oecologia 151, 93–103. Carriero, M.M., Adriano, E.A., Silva, M.R.M., Ceccarelli, P.S. and Maia, A.A.M. (2013). Molecular phylogeny of the Myxobolus and Henneguya genera with several new South American species. PLoS One 8, e73713. Caullery, M. and Mesnil, F. (1904). Sur un type nouveau (Sphaeractinomyxon stolci n. g. n. sp.) d' Actinomyxidies, et son développement. Comptes Rendus des Séances de la Société de Biologie et de ses Filiales 56, 408–410. Caullery, M. and Mesnil, F. (1905). Recherches sur les Actinomyxidies. I. Sphaeractinomyxon stolci. Archiv für Protistenkunde 6, 272–308.

Page | 120

Chapter V | Potential life cycle role of sphaeractinomyxon

Cole, L., Bardgett, R.D., Ineson, P. and Adamson, J.K. (2002). Relationships between enchytraeid worms (Oligochaeta), climate change, and the release of dissolved organic carbon from blanket peat in northern England. Soil Biology and Biochemistry 34, 599– 607. Cunha, M.R., Paterson, G.L.J., Amaro, T., Blackbird, S., Stigter, H.C., Ferreira, C., Glover, A., Hilário, A., Kiriakoulakis, K., Neal, L., Ravara, A., Rodrigues, C.F., Tiago, Á. and Billett, D.S.M. (2011). Biodiversity of macrofaunal assemblages from three Portuguese submarine canyons (NE Atlantic). Deep-Sea Research Part II 58, 2433–2447. Daufresne, M., Bady, P. and Fruget, J.F. (2007). Impacts of global changes and extreme hydroclimatic events on macroinvertebrate community structures in the French Rhône River. Oecologia 151, 544–559. Durance, I. and Ormerod, S.J. (2007). Climate change effects on upland stream macroinvertebrates over a 25-year period. Global Change Biology 13, 942–957. El-Mansy, A., Székely, C. and Molnár, K. (1998a). Studies on the occurrence of actinosporean stages of fish myxosporeans in a fish farm of Hungary, with the description of triactinomyxon, raabeia, aurantiactinomyxon and neoactinomyxon types. Acta Veterinaria Hungarica 46, 259–284. El-Mansy, A., Székely, C. and Molnár, K. (1998b). Studies on the occurrence of actinosporean stages of myxosporeans in Lake Balaton, Hungary, with the description of triactinomyxon, raabeia and aurantiactinomyxon types. Acta Veterinaria Hungarica 46, 437–450. El-Mansy, A., Molnár, K. and Székely, C. (1998c). Development of Myxobolus portucalensis Saraiva and Molnár, 1990 (Myxosporea: Myxobolidae) in the oligochaete Tubifex tubifex (Müller). Systematic Parasitology 41, 95–103. Eszterbauer, E., Atkinson, S., Diamant, A., Morris, D., El-Matbouli, M. and Hartikainen, H. (2015). Myxozoan life cycles: practical approaches and insights. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 175–198. Eszterbauer, E., Marton, S., Rácz, O.Z., Letenyei, M. and Molnár, K. (2006). Morphological and genetic differences among actinosporean stages of fish-parasitic myxosporeans (Myxozoa): difficulties of species identification. Systematic Parasitology 65, 97–114. Eszterbauer, E., Székely, C., Molnár, K. and Baska, F. (2000). Development of Myxobolus bramae (Myxosporea: Myxobolidae) in an oligochaete alternate host, Tubifex tubifex. Journal of Fish Diseases 23, 19–25. Fiala, I., Bartošová-Sojková, P., Okamura, B. and Hartikainen, H. (2015). Classification and phylogenetics of Myxozoa. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing,

Page | 121

Chapter V | Potential life cycle role of sphaeractinomyxon

Switzerland, pp. 85–110. Grossheider, G. and Körting, W. (1992). First evidence that Hoferellus cyprini (Doflein, 1898) is transmitted by Nais sp. Bulletin of the European Association of Fish Pathologists 12, 17–20. Hallett, S.L., Atkinson, S.D., Erséus, C. and El-Matbouli, M. (2004). Molecular methods clarify morphometric variation in triactinomyxon spores (Myxozoa) released from different oligochaete hosts. Systematic Parasitology 57, 1–14. Hallett, S.L. and Diamant, A. (2001). Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland. Australia. Diseases of Aquatic Organisms 46, 197–212. Hallett, S.L., Erséus, C. and Lester, R.J. (1999). Actinosporeans (Myxozoa) from marine oligochaetes of the Great Barrier Reef. Systematic Parasitology 44, 49–57. Hallett, S.L., Erséus, C. and Lester, R.J.G. (1997). Actinosporea from Hong Kong marine oligochaeta. In Morton, B. (ed). Proceedings of the eight international marine biological workshop: the marine flora and fauna of Hong Kong and Southern China. Hong Kong University Press, Hong Kong, pp. 1–7. Hallett, S.L. and Lester, R.J. (1999). Actinosporeans (Myxozoa) with four developing spores within a pansporocyst: Tetraspora discoidea n.g. n.sp. and Tetraspora rotundum n.sp. International Journal for Parasitology 29, 419–427. Hallett, S.L., O’Donoghue, P.J. and Lester, R.J.G. (1998). Structure and development of a marine actinosporean, Sphaeractinomyxon ersei n. sp. (Myxozoa). Journal of Eukaryotic Microbiology 45, 142–150. Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. The Quarterly Review of Biology 66, 411–453. Holzer, A.S., Bartošová-Sojková, P., Born-Torrijos, A., Lövy, A., Hartigan, A. and Fiala, I. (2018). The joint evolution of the Myxozoa and their alternate hosts: a cnidarian recipe for success and vast biodiversity. Molecular Ecology 27, 1651–1666. Holzer, A.S., Sommerville, C. and Wootten, R. (2004). Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences. International Journal for Parasitology 34, 1099–1111. Holzer, A.S., Sommerville, C. and Wootten, R. (2006). Molecular identity, phylogeny and life cycle of Chloromyxum schurovi Shul'man & Ieshko 2003. Parasitology Research 99, 90–96. Holzer, A.S., Wootten, R. and Sommerville, C. (2007). The secondary structure of the unusually long 18S ribosomal RNA of the myxozoan Sphaerospora truttae and structural evolutionary trends in the Myxozoa. International Journal for Parasitology 37,

Page | 122

Chapter V | Potential life cycle role of sphaeractinomyxon

1281–1295. Kallert, D.M., Eszterbauer, E., El-Matbouli, M., Erséus, C. and Haas, W. (2005). The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore. Journal of Fish Diseases 28, 71–79. Karlsbakk, E. and Køie, M. (2012). The marine myxosporean Sigmomyxa sphaerica (Thélohan, 1895) gen. n., comb. n. (syn. Myxidium sphaericum) from garfish (Belone belone (L.)) uses the polychaete Nereis pelagica L. as invertebrate host. Parasitology Research 110, 211–218. Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffmann, R.W., Khattra, J., Hallett, S.L., Lester, R.J., Longshaw, M., Palenzeula, O., Siddall, M.E. and Xiao, C. (2001). Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic Microbiology 48, 395–413. Kent, M.L., Margolis, L. and Corliss, J.O. (1994). The demise of a class of protists - taxonomic and nomenclatural revisions proposed for the protist phylum Myxozoa Grassé, 1970. Canadian Journal of Zoology 72, 932–937. Kent, M.L., Whitaker, D.J. and Margolis, L. (1993). Transmission of Myxobolus arcticus Pugachev and Khokhlov, 1979, a myxosporean parasite of Pacific salmon, via a triactinomyxon from the aquatic oligochaete Stylodrilus heringianus (Lumbriculidae). Canadian Journal of Zoology 71, 1207–1211. Køie, M., Karlsbakk, E. and Nylund, A. (2007). A new genus Gadimyxa with three new species (Myxozoa, Parvicapsulidae) parasitic in marine fish (Gadidae) and the two-host life cycle of Gadimyxa atlantica n. sp. Journal of Parasitology 93, 1459–1467. Køie, M., Karlsbakk, E. and Nylund, A. (2008). The marine herring myxozoan Ceratomyxa auerbachi (Myxozoa: Ceratomyxidae) uses Chone infundibuliformis (Annelida: Polychaeta: Sabellidae) as invertebrate host. Folia Parasitologica 55, 100–104. Køie, M., Whipps, C.M. and Kent, M.L. (2004). Ellipsomyxa gobii (Myxozoa: Ceratomyxidae) in the common goby Pomatoschistus microps (Teleostei: Gobiidae) uses Nereis spp. (Annelida: Polychaeta) as invertebrate hosts. Folia Parasitologica 51, 14–18. Lin, D., Hanson, L.A. and Pote, L.M. (1999). Small subunit ribosomal RNA sequence of Henneguya exilis (class Myxosporea) identifies the actinosporean stage from an oligochaete host. Journal of Eukaryotic Microbiology 46, 66–68. Lin, K.J. and Yo, S.P. (2008). The effect of organic pollution on the abundance and distribution of aquatic oligochaetes in an urban water basin. Taiwan. Hydrobiologia 596, 213–223. Lom, J. and Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53, 1–36. Lom, J., McGeorge, J., Feist, S.W., Morris, D. and Adams, A. (1997). Guidelines for the uniform characterisation of the actinosporean stages of parasites of the phylum Myxozoa.

Page | 123

Chapter V | Potential life cycle role of sphaeractinomyxon

Diseases of Aquatic Organisms 30, 1–9. Marques, A. (1984). Contribution a la connaissance des Actinomyxidies: ultrastructure, cycle biologique, systématique. Ph.D. Thesis. Université des Sciences et Techniques de Languedoc, Montepellier, France, pp. 218. Marton, S. and Eszterbauer, E. (2011). The development of Myxobolus pavlovskii (Myxozoa: Myxobolidae) includes an echinactinomyxon-type actinospore. Folia Parasitologica 58, 157–163. Milanin, T., Atkinson, S.D., Silva, M.R., Alves, R.G., Maia, A.A. and Adriano, E.A. (2017). Occurrence of two novel actinospore types (Cnidaria: Myxosporea) in Brazilian fish farms, and the creation of a novel actinospore collective group, Seisactinomyxon. Acta Parasitologica 62, 121–128. Molnár, K. (2011). Remarks to the validity of Genbank sequences of Myxobolus spp. (Myxozoa, Myxosporidae) infecting Eurasian fishes. Acta Parasitologica 56, 263–269. Molnár, K., El-Mansy, A., Székely, C. and Baska, F. (1999). Development of Myxobolus dispar (Myxosporea: Myxobolidae) in an oligochaete alternate host. Tubifex tubifex. Folia Parasitologica 46, 15–21. Molnár, K., Marton, S., Székely, C. and Eszterbauer, E. (2010). Differentiation of Myxobolus spp. (Myxozoa: Myxobolidae) infecting roach (Rutilus rutilus) in Hungary. Parasitology Research 107, 1137–1150. Oumouna, M., Hallett, S.L., Hoffmann, R.W. and El-Matbouli, M. (2003). Seasonal occurrence of actinosporeans (Myxozoa) and oligochaetes (Annelida) at a trout hatchery in Bavaria, Germany. Parasitology Research 89, 170–184. Palumbi, S., Martin, A., Romano, S., McMillan, W.O., Stice, L. and Grabowski, G., (2002). The Simple Fools Guide to PCR, Version 2.0. University of Hawaii, Honolulu. http:// palumbi.stanford.edu/SimpleFoolsMaster. Puytorac, P.D. (1963). L'ultrastructure des cnidocystes de l'Actinomyxidae: Sphaeractinomyxon amanieui sp. nov. Comptes Rendus de l'Académie des Sciences Paris 256, 1594–1596. Rangel, L.F., Castro, R., Rocha, S., Cech, G., Casal, G., Azevedo, C., Székely, C., Cavaleiro, F. and Santos, M.J. (2016a). Description of new types of sphaeractinomyxon actinospores (Myxozoa: Myxosporea) from marine tubificid oligochaetes, with a discussion on the validity of the tetraspora and the endocapsa as actinospore collective group names. Parasitology Research 115, 2341–2351. Rangel, L.F., Castro, R., Rocha, S., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2016b). Tetractinomyxon stages genetically consistent with Sphaerospora dicentrarchi (Myxozoa: Sphaerosporidae) found in Capitella sp. (Polychaeta: Capitellidae) suggest potential role of marine polychaetes in parasite's life

Page | 124

Chapter V | Potential life cycle role of sphaeractinomyxon

cycle. Parasitology 143, 1067–1073. Rangel, L.F., Cech, G., Székely, C. and Santos, M.J. (2011). A new actinospore type Unicapsulactinomyxon (Myxozoa), infecting the marine polychaete, Diopatra neapolitana (Polychaeta: Onuphidae) in the Aveiro estuary, Portugal. Parasitology 138, 698–712. Rangel, L.F., Rocha, S., Casal, G., Castro, R., Severino, R., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2017). Life cycle inference and phylogeny of Ortholinea labracis n. sp. (Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm. Journal of Fish Diseases 40, 243– 262. Rangel, L.F., Rocha, S., Castro, R., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2015). The life cycle of Ortholinea auratae (Myxozoa: Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete. Parasitology Research 114, 2671–2678. Rangel, L.F., Santos, M.J., Cech, G. and Székely, C. (2009). Morphology, molecular data, and development of Zschokkella mugilis (Myxosporea, Bivalvulida) in a polychaete alternate host, Nereis diversicolor. Journal of Parasitology 95, 561–569. Rocha, S., Casal, G., Garcia, P., Matos, E., Al-Quraishy, S. and Azevedo, C. (2014a). Ultrastructure and phylogeny of the parasite Henneguya carolina sp. nov. (Myxozoa), from the marine fish Trachinotus carolinus in Brazil. Diseases of Aquatic Organisms 112, 139–148. Rocha, S., Casal, G., Velasco, M., Alves, A., Matos, E., Al-Quraishy, S. and Azevedo, C. (2014b). Morphology and phylogeny of Thelohanellus marginatus n. sp. (Myxozoa: Myxosporea), a parasite infecting the gills of the fish Hypophthalmus marginatus (Teleostei: Pimelodidae) in the Amazon River. Journal of Eukaryotic Microbiology 61, 586–593. Ronquist, F. and Huelsenbeck, J.P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Rosser, T.G., Griffin, M.J., Quiniou, S.M., Greenway, T.E., Khoo, L.H., Wise, D.J. and Pote, L.M. (2014). Molecular and morphological characterization of myxozoan actinospore types from a commercial catfish pond in the Mississippi delta. Journal of Parasitology 100, 828–839. Roubal, F.R., Hallett, S.L. and Lester, R.J.G. (1997). First record of triactinomyxon actinosporean in marine oligochaete. Bulletin of the European Association of Fish Pathologists 17, 83–85. Silva, G., Costa, J.L., Almeida, P.R. and Costa, M.J. (2006). Structure and dynamics of a benthic invertebrate community in an intertidal area of the Tagus estuary, western

Page | 125

Chapter V | Potential life cycle role of sphaeractinomyxon

Portugal: a six year data series. Hydrobiologia 555, 115–128. Styer, E.L., Harrison, L.R. and Burtle, G.J. (1991). Communications: Experimental production of proliferative gill disease in channel catfish exposed to a myxozoan-infected oligochaete, Dero digitata. Journal of Aquatic Animal Health 3, 288–291. Székely, C., Borkhanuddin, M.H., Cech, G., Kelemen, O. and Molnár, K. (2014). Life cycles of three Myxobolus spp. from cyprinid fishes of Lake Balaton, Hungary involve triactinomyxon-type actinospores. Parasitology Research 113, 2817–2825. Székely, C., Hallett, S.L., Al-Samman, A. and Dayoub, A. (2007). First description of myxozoans from Syria: novel records of hexactinomyxon, triactinomyxon and endocapsa actinospore types. Diseases of Aquatic Organisms 74, 127–137. Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215–219. Wolf, K. and Markiw, M.E. (1984). Biology contravenes taxonomy in the Myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science 225, 1449– 1452. Xi, B.W., Zhou, Z.G., Xie, J., Pan, L.K., Yang, Y.L. and Ge, X.P. (2015). Morphological and molecular characterization of actinosporeans infecting oligochaete Branchiura sowerbyi from Chinese carp ponds. Diseases of Aquatic Organisms 114, 217–228. Xiao, C. and Desser, S.S. (1998). The oligochaetes and their actinosporean parasites in Lake Sasajewun, Algonquin Park. Ontario. Journal of Parasitology 84, 1020–1026. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1993a). Involvement of Branchiura sowerbyi (Oligochaeta, Annelida) in the transmission of Hoferellus carassii (Myxosporea, Myxozoa), the causative agent of kidney enlargement disease (KED) of goldfish Carassius auratus. Fish Pathology 28, 135–139. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1993b). Some biological characteristics of actinosporeans from the oligochaete Branchiura sowerbyi. Diseases of Aquatic Organisms 17, 223–228. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1995). Myxobolus cultus n. sp. (Myxosporea: Myxobolidae) in the goldfish Carassius auratus transformed from the actinosporean stage in the oligochaete Branchiura sowerbyi. Journal of Parasitology 81, 446–451.

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Chapter VI

Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular redescription of the cryptic species Myxobolus exiguus

This chapter was adapted from:

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Abstract

Mullets inhabit a wide range of habitats from tropical to temperate regions and play a critical role in their ecosystems. This commercially important fish group constitutes a significant source of food in several geographic regions, and the production of some species for consumption is an increasing trend. About 64 myxosporean species have been reported in mullets, some of which are cryptic, as is the case of Myxobolus exiguus and M. muelleri. This paper provides, for the first time, a detailed and critical revision of the data available for myxobolids reported in mullets, determining the species that have bona fide mugiliform fish hosts, in accordance with the original species descriptions, the available molecular data and the currently accepted taxonomic and phylogenetic criteria. Phylogenetic analyses using Bayesian inference and maximum likelihood methodologies suggest that the evolutionary history of myxobolids with bona fide mugiliform fish hosts reflects that of its vertebrate hosts, while reinforcing known evolutionary factors and old systematic issues of the clade of myxobolids. A comprehensive morphological, ultrastructural and molecular re-description is also provided for the cryptic species M. exiguus, from infections in the visceral peritoneum of the thinlip grey mullet Chelon ramada in the River Minho, Portugal.

Introduction

The order Mugiliformes comprises a single family, Mugilidae Cuvier, 1829, which contains about 70 species, distributed worldwide, commonly known as mullets. The great majority of mullets are highly euryhaline, inhabiting tropical and temperate habitats that include rivers, estuaries, coastal areas and seas (Hotos and Vlahos, 1998; Cardona, 2001; Durand et al., 2012). Due to their omnivorous nature and benthic feeding strategy, mullets are able to feed on a great variety of materials including epiphytic algae, insects, annelids, crustaceans, mollusks and even detritus. As a result of their ecological plasticity, this family plays an important role in the ecosystem, namely by contributing to the flow of energy and matter from the lower to the upper levels (Cardona, 2001; Laffaille et al., 2002; Almeida, 2003; Zetina- Rejón et al., 2003). At the commercial level, the importance of mugilids depends on the geographic region and whether they are cultured for gathering roe or for food consumption. Nonetheless, the world production of mullets is an increasing trend, both in fishery and aquaculture industries (Crosetti and Cataudella, 1995; Saleh, 2006). Several studies have been conducted on the protozoan and metazoan microorganisms parasitizing mullets worldwide (e.g. Merella and Garippa, 2001; Bahri et al., 2003; Fioravanti et al., 2006; Yurakhno and Ovcharenko, 2014; Özer and Kirca, 2015; Sarabeev, 2015). According to Yurakhno and Ovcharenko (2014), this fish group accounts for the description of

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus about 64 myxosporean species from genera of the families: Sphaeromyxidae Lom and Noble, 1984; Sphaerosporidae Davis, 1917; Myxidiidae Thélohan, 1892; Myxobilatidae Shulman, 1953 [the genus Ortholinea was recently transferred to this family, and the family Ortholineidae dismantled (Karlsbakk et al., 2017)]; Sinuolineidae Shulman, 1959; Alatosporidae Schulman, Kovaleva and Dubina, 1979; Chloromyxidae Thélohan, 1892; Kudoidae Meglitsch, 1960 and Myxobolidae Thélohan, 1892. The latter family is the largest within Myxozoa Grassé, 1970, namely due to the species-richness of the genera Myxobolus Bütschli, 1882 and Henneguya Thélohan, 1892. Worldwide, the genus Myxobolus comprises over 850 species, the majority of which are histozoic in freshwater fish, less frequently infecting hosts from estuarine and marine environments. Few species present coelozoic development and even fewer have been reported to occur in amphibian hosts. On its turn, the genus Henneguya comprises about 200 species that mostly infect freshwater fish, with the exception of ca. 35 species that are known to occur in marine hosts (e.g. Lom and Dyková, 1992, 2006; Eiras, 2002; Eiras et al., 2005, 2014; Eiras and Adriano, 2012; Khlifa et al., 2012; Li et al., 2012; Azevedo et al., 2014; Rocha et al., 2014a; Özer et al., 2016a). Traditionally, the taxonomy of myxosporeans was mainly based on spore morphology, and its association with a particular host and organ of infection. However, molecular analyses have been showing that the comparison of spore morphological traits is insufficient for classifying myxosporeans, both at the genus and species level (e.g. Fiala, 2006; Bartošová et al., 2009; Fiala and Bartošová, 2010; Liu et al., 2010). In the case of myxobolids, the artificiality of the morphological criterion hampers identification at the species level, since many species share similar spore shape and size, while others present significant intraspecific variations (Lom, 1987; Mitchell, 1989; El-Matbouli et al., 1992). Also, although most species are acknowledged to be host and tissue restricted (Molnár, 1994), others have been reported to indiscriminately infect a wide range of hosts and tissues (e.g. Forró and Ezsterbauer, 2016). Taxonomic comparisons are further challenged by the paucity of reliable data from most original descriptions, which relied solely on light microscopy and schematic line drawings (e.g. Lubat et al., 1989), with few studies using transmission electron microscopy for ultrastructural characterization. Consequently, several species have been identified as potentially cryptic (Easy et al., 2005; Ferguson et al., 2008; Atkinson et al., 2015), thus warranting authentication through the use of currently accepted taxonomic criteria, i.e. combined spore morphology, host specificity, tissue specificity and molecular data. Considering all of the above, this study provides, for the first time, a detailed and critical revision of the data available for myxobolids reported in mullets, evaluating the reliability of these reports through the comprehensive and careful analysis of original species descriptions, available molecular data and currently accepted taxonomic and phylogenetic criteria. Myxobolids which occurrence is considered to be reliable in mullets are herein referred to as having bona fide mugiliform fish hosts. A

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus morphological, ultrastructural and molecular re-description is further performed for the cryptic species Myxobolus exiguus Thélohan, 1895 from infections in the visceral peritoneum of the thinlip grey mullet Chelon ramada in the River Minho, Portugal.

Materials and methods

Fish and parasite sampling

Between March 2015 and January 2018, 18 specimens of the thinlip grey mullet C. ramada (Risso, 1827) (Teleostei and Mugiliformes) were captured from the River Minho (41° 56′ N, 08° 45′ W), Vila Nova de Cerveira, Portugal. Specimens were transported live to the laboratory and, prior to dissection, anesthetized with ethylene glycol monophenyl ether until dead. The parasitological survey of several organs and tissues was performed at both the macro- and microscopic levels. Cysts and parasitized tissues were prepared for light microscopy, transmission electron microscopy and molecular procedures.

Light microscopy and morphological examination

Parasitized tissues were examined and photographed using a Leitz-Dialux 20 microscope, equipped with a differential interference contrast (DIC) optics. Morphometry was determined from fresh material (Lom and Arthur, 1989). All measurements include the mean value ± standard deviation (S.D.), range of variation and number of spores measured (range, n).

Transmission electron microscopy

Fragments of parasitized tissue were fixed in 5% glutaraldehyde buffered in 0.2 M sodium cacodylate (pH 7.2) for 20–24 h, washed in the same buffer and post-fixed in 2% osmium tetroxide also buffered with 0.2 M sodium cacodylate (pH 7.4) for 3–4 h. All these steps were performed at 4 °C. The samples were then dehydrated in an ascending graded series of ethanol, followed by embedding using a series of oxide propylene and Epon mixtures, ending in EPON. Semithin sections were stained with methylene blue- Azure II. Ultrathin sections were double-contrasted with uranyl acetate and lead citrate, and then examined and photographed using a JEOL 100 CXII TEM (JEOL Optical, Tokyo, Japan), operating at 60 kV.

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

DNA extraction, amplification and sequencing

Fragments of parasitized tissue were obtained from three fish specimens and preserved in 80% ethanol at 4 °C. Genomic DNA extraction was performed using a GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, USA), following the manufacturer’s instructions. The DNA was stored in 50 µL of TE buffer at −20 °C until further use. The SSU rDNA gene was amplified and sequenced using both universal and myxosporean-specific primers (Table 1). Polymerase chain reactions (PCRs) were performed in 50 µL reactions using 10 pmol of each primer, 10 nmol of dNTPs, 2 mM of MgCl2, 5 µL 10× Taq polymerase buffer, 2.5 units Taq DNA polymerase (NZYTech, Lisbon, Portugal) and approximately 50–100 ng of genomic DNA. The reactions were run on a Hybaid PxE Thermocycler (Thermo Electron Corporation, Milford, Massachusetts, USA), with initial denaturation at 95 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 53 °C for 45 s and 72 °C for 90 s. The final elongation step was performed at 72 °C for 7 min. Five microlitre aliquots of the PCR products were electrophoresed through a 1% agarose 1× tris-acetate-EDTA buffer (TAE) gel stained with ethidium bromide. PCR products were purified using the ExoFast method, in which an enzymatic clean-up that eliminates unincorporated primers and dNTPs is performed with Exonuclease I (Escherichia coli) and FastAP Thermosensitive (SAP). The PCR products from different regions of the SSU rDNA gene were sequenced directly. The sequencing reactions were performed using a BigDye Terminator v1.1 from the Applied Biosystems Kit (Applied Biosystems, Carlsbad, California, USA), and were run on an ABI3700 DNA analyser (Perkin-Elmer, Applied Biosystems, Stabvida, Oeiras, Portugal).

Table 1. Polymerase chain reaction primers used for the amplification and sequencing of the SSU rDNA gene.

Name Sequence (5’-3’) Position Paired with Source

18e CTG GTT GAT CCT GCC AGT 1 ACT3r, MYX4R Hillis and Dixon, 1991

ACT3f CAT GGA ACG AAC AAT 900 18r Hallett and Diamant, 2001

MYX4F GTT CGT GGA GTG ATC TGT CAG 1300 18r Rocha et al., 2015

ACT3r ATT GTT CGT TCC ATG 900 18e Rocha et al., 2014a

MYX4R CTG ACA GAT CAC TCC ACG AAC 1300 18e Hallett and Diamant, 2001

18r CTA CGG AAA CCT TGT TAC G 1832 ACT3f, MYX4F Whipps et al., 2003

Distance estimation and phylogenetic analysis

The partial sequences obtained for the case isolate were aligned in MEGA 6.06, allowing the construction of the parasite's assembled SSU rDNA sequence, with a total of 2,013 bp. In

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus order to calculate distance estimation, a dataset was created solely for the SSU rDNA sequence of the case isolate and all other SSU rDNA sequences available for Myxobolus spp. that have been reported from mullets. This includes M. bramae Reuss, 1906, M. branchialis (Markevitsch, 1932) Landsberg and Lom, 1991 and M. rotundus Nemeczek, 1911, despite these species having not been sequenced from hosts of the order Mugiliformes. Of the several SSU rDNA sequences available in GenBank for M. rotundus, only one (FJ851447) was chosen to represent this species in the dataset, considering that all sequences were identical among each other, with the exception of one (AY165179) that is misidentified (Zhang et al., 2010) and, therefore, was not included. Similarly, two SSU rDNA sequences of M. bramae (AF085177 and AF507968) are available in GenBank but, despite having both been obtained from infections occurring in the cypriniform host Abramis brama Linnaeus, 1758, only one is considered valid (AF507968), thus having been included in the dataset. Sequences were aligned using software MAFFT version 7 available online, and distance estimation was performed in MEGA 6.06, with the p-distance model and all ambiguous positions removed for each sequence pair. For phylogenetic analyses, the dataset was widened to encompass other representatives of the clade of myxobolids. The final dataset comprised of a total of 93 SSU rDNA sequences, and included Chloromyxum riorajum (FJ2624481), Myxidium lieberkuehni (X76638) and Sphaerospora oncorhynchi (AF201373) as the outgroup. Sequences were aligned using software MAFFT version 7 available online, and posteriorly manually edited in MEGA 6.06. Phylogenetic trees were calculated from the sequence alignments using maximum likelihood (ML) and Bayesian inference (BI). Models of nucleotide substitution were evaluated using MEGA 6.06. The general time reversible substitution model with estimates of invariant sites and gamma distributed among site rate variation (GTR+I+Γ) was chosen as the best suited model for the dataset, and was used in both ML and BI analyses. ML analyses were also conducted in MEGA 6.06 (Tamura et al., 2013), with bootstrap confidence values calculated from 1000 replicates. BI analyses were performed using MrBayes v3.2.6 (Ronquist and Huelsenbeck, 2003), with posterior probability distributions generated using the Markov Chain Monte Carlo method, with four chains running, simultaneously, for 500000 generations, and every 100th tree sampled.

Results

Revised description and taxonomic summary of M. exiguus (Figs. 1–3)

Diagnosis: Cysts whitish and spherical, about 1 mm in diameter, located adjacent to the peritoneum lining the viscera. Mature myxospores subspherical in valvular view and ellipsoidal

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus in sutural view, measuring 9.3 ± 0.6 (8.4–10.7) μm in length and 8.2 ± 0.5 (7.6–8.9) μm in width (n = 25). Valves are smooth presenting several markings near the suture line. Two pyriform equal-sized polar capsules located side by side at the myxospores’ anterior pole, 4.8 ± 0.2 (4.4–5.3) μm long and 2.8 ± 0.3 (2.2–3.1) μm wide (n = 25), each containing an isofilar polar tubule forming five coils (Fig. 1A, B).

Figure 1. Light micrographs of Myxobolus exiguus infecting the peritoneum of Chelon ramada in the River Minho. (A) DIC micrograph showing some free fresh mature myxospores, subspherical in valvular view and ellipsoidal in sutural view, and containing two polar capsules. (B) Free fresh mature myxospore displaying several markings near the suture line. (C) Semithin section of the periphery of a cyst evidencing the vacuolated ectoplasm (arrow) adhering to loose connective tissue (*), where some fibroblasts (F) are observed.

Ultrastructural description: Cysts’ wall with cytoplasmic expansions forming ladder-like junctions that strongly adhere to the mesothelial cells of the peritoneum. Detachment of cytoplasmic portions of mesothelial cells by insertion of the cysts’ wall expansions into connective tissue. Several fibroblasts widely separated by bundles of collagen fibres in loose connective tissue (Figs. 1C, 2A–C). Cysts’ ectoplasm highly vacuolated and devoid of cytoplasmic organelles (Figs. 1C, 2C); endoplasm rich in mitochondria, vegetative nuclei and containing all sporogonic stages. Sporogony asynchronous and centripetal: generative cells and young sporoblasts located at the cysts’ periphery; immature and mature myxospores at the centre, each within a vacuole-like structure (Figs. 1C, 2B, D). Myxospores wall thin and smooth, comprised of two symmetrical valves adhering together along a straight suture line. Polar capsules with a double-layered wall formed by an outer thin electron-dense layer and an inner thick electron-lucent layer. Polar tubule coils in an electron-dense homogenous matrix (Fig. 2D–F). Cap-like structure at the apex of polar capsule, directed at the corresponding extrusion pore. Extrusion pores near the suture line, corresponding to the portions of the valves with diminished thickness (Fig. 2G). Sporoplasm at the myxospores’ posterior pole, with two nuclei and several sporoplasmosomes randomly distributed in a heterogeneous matrix (Fig. 2D, E). Morphology of the myxospores is represented in a schematic drawing (Fig. 3) depicting

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Figure 2. Transmission electron micrographs of Myxobolus exiguus infecting the peritoneum of Chelon ramada in the River Minho. (A) Periphery of a cyst (C) adhering to a mesothelial cell near its nucleus (N), and reaching the loose connective tissue (LCT), where fibroblasts (F) are observed widely separated by bundles of collagen fibres. (B) Periphery of a cyst displaying numerous generative nuclei (Gn) and forming cytoplasmic expansions (arrows) that strongly adhere to the mesothelial cells (Mc) and reach the LCT. (C) Detailed aspect of the cytoplasmic expansions forming ladder-like junctions (arrows) that connect the cyst (C) to the mesothelial cells (Mc). Notice the numerous vacuoles (Vs) occupying the cyst’s ectoplasm. (D) Longitudinal section of a myxospore in valvular view, located within a vacuole-like structure (*), and displaying its two polar capsules (PC) and binucleate sporoplasm (Sp). (E) Longitudinal section of a myxospore in sutural view, depicting the number of polar tubule (PF) coils, as well as some sporoplasmosomes (Sps) randomly distributed in the sporoplasm. (F) Transverse section of a myxospore showing its two valves united along a straight suture line (arrowheads), and its two PCs presenting a double-layered wall (arrow) that surrounds an electron-dense matrix (*) and a coiled polar tubule (PF). (G) Longitudinal oblique section of a polar capsule displaying its cap-like structure (arrow) in continuity with the valve’s extrusion pore (*), located near the suture line (arrowheads).

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus the ultrastructural features described here. Type host: the thinlip grey mullet C. ramada (Risso, 1827) (Teleostei, Mugiliformes). Site of infection: the visceral peritoneum. Prevalence: three infected in 18 specimens analysed (16.7%). Type locality: France (Vivier-sur-Mer, Marseille, Banyuls). Other localities: Tunisia (Ichkeul Lake); Portugal (River Minho). Pathogenicity: long-term pathological assessments were not performed but collected and analysed fish did not present evident external symptoms of infection or disease. Vouchers: one glass slide containing semi-thin sections of the hapantotype was deposited in the Type Slide Collection of the Laboratory of Animal Pathology at the Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2018.19. Sequences: one assembled SSU rDNA sequence with a total of 2,013 bp and GenBank accession no. MH236070.

Molecular comparison of the case isolate to other Myxobolus spp. reported from mullets

Pairwise comparisons between the SSU rDNA sequence obtained here and all others available in GenBank for Myxobolus spp. reported from mugiliform fish hosts (Table 2) revealed the case isolate presenting 100% of similarity to two sequences: one identified as M. exiguus (AY129317), and the other identified as M. muelleri Bütschli, 1882 (AY129314), with these also sharing 100% of similarity between each other. The other SSU rDNA sequences of M. Figure 3. Schematic drawing depicting the exiguus (AY129316) and M. muelleri (AY129313) ultrastructural organization of a myxospore of obtained from infections in mugiliform fish hosts Myxobolus exiguus in sutural view. followed, with 99.4% of similarity to the case isolate, and 100% of similarity between each other. The two remaining available sequences of M. muelleri (AY325284 and DQ439806), which correspond to infections of this species in the cypriniform fish host Squalius cephalus (Linnaeus, 1758), shared 97.0% of similarity between each other, but differed significantly from their conspecific sequences available from mugiliform fish hosts, as well as from those of M. exiguus and the sequence in study, with percentages of identity that varied between 72.2 and 73.4%. All other species resulted in percentages of similarity lower than 90%.

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Phylogenetic positioning of M. exiguus and other myxobolids reported from mugiliform fish hosts

BI and ML analyses resulted in similar topologies, with some entropy in the middle of the tree, namely due to the unstable positioning of the subclade comprising SSU rDNA sequences from myxobolids that infect marine perciform fish hosts. The phylogenetic

analyses performed here re-

spp. infecting mugilids: percentage of identity (top diagonal) and nucleotide nucleotide and diagonal) (top identity of percentage mugilids: infecting spp.

vealed the case isolate clustering to form a well suppor-

Myxobolus ted clade together with most of the SSU rDNA sequences available for myxobolid species described from mugiliform fish hosts: M. exiguus (AY129316 and AY129317); M. muelleri (AY129313 and AY129314); M. parvus Schul- man, 1962 (KX242161); M.

episquamalis Egusa et al., NA sequences of the case isolate and all other other all and isolate case the of sequences NA D 1990 (KC733437); M. bizerti Bahri and Marques, 1996

(AY129318); M. ichkeulensis . Bahri and Marques, 1996 (AY129315); Myxobolus sp. Kim et al., 2013 (KC733438);

M. spinacurvatura Maeno et

Comparison between the SSU r SSU the between Comparison

al., 1990 (AF378341); and

able 2 able

difference (bottom diagonal) difference(bottom T also with members of the

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus sphaeractinomyxon, endocapsa and triactinomyxon collective groups. Exceptions to the mugiliform-infecting clade are the other two SSU rDNA sequences available for M. muelleri (DQ439806 and AY352284), as well as those of M. bramae (AF507968), M. branchialis (JQ388887) and M. rotundus (FJ851447), which cluster among cypriniform-infecting myxobolids (Fig. 4).

Discussion

Overview of mugiliform-infecting myxobolids

In this paper, we summarize the data available for myxobolids that have mugiliforms as bona fide fish hosts (Tables 3 and 4), with measurements from the original descriptions (whenever given) and updated scientific names for host species (according to FishBase). The vast majority of mugiliform-infecting Myxobolus species are registered from the genus Mugil Linnaeus, 1758, with the flathead grey mullet M. cephalus Linnaeus, 1758 accounting for an astonishing number of 19 species infecting several of its organs in coastal waters of the Mediterranean Sea, Atlantic Ocean, Indian Ocean and North Pacific Ocean; while white mullet M. curema Valenciennes, 1836 accounts for two species from the Atlantic coast off Senegal, and lebranche mullet M. liza Valenciennes, 1836 for one species in Brazilian waters. The genus Chelon Artedi, 1793 accounts for 12 species: four from several organs of golden grey mullet C. auratus (Risso, 1810) (syn. Liza aurata) in the Mediterranean Sea and North Atlantic Ocean; three from thicklip grey mullet C. labrosus in European and North African waters; two from several organs of the leaping mullet C. saliens (Risso, 1810) [syn. L. saliens (Risso, 1810)] in Eurasian coastal waters; one from goldspot mullet C. parsia (Hamilton, 1822) in India; one from tade grey mullet C. planiceps (Valenciennes, 1836), also in India; and one from C. ramada (Risso, 1827) [syn. L. ramada (Risso, 1827)] in Europe and North Africa. Seven species have been reported from hosts of the genus Planiliza Whitley, 1945: four from the gills and gut of large-scale mullet P. macrolepis (Smith, 1846) [syn. C. macrolepis (Smith, 1846)] in India; and three from several organs of so-iuy mullet P. haematocheila (Temminck and Schlegel, 1845) [syn. L. haematocheila (Temminck and Schlegel, 1845)] in Eurasian coastal waters. Other hosts accounting for a single species are: the corsula Rhinomugil corsula (Hamilton, 1822), the yellowtail mullet Sicamugil cascasia (Hamilton, 1822) and the squaretail mullet Ellochelon vaigiensis (Quoy and Gaimard, 1825), all from in India; the hornlip mullet Oedalechilus labiosus (Valenciennes, 1836) in the Red Sea off Egypt; and the bluespot mullet Moolgarda seheli (Forsskål, 1775) off Thailand. On its turn, only two species of Henneguya have been reported from the gills and brain of M. cephalus off Senegal (Table 4). Some of these parasite species have been reported, and even originally described, from more than one

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Figure 4. Tree topology resulting from the Bayesian analysis of 93 SSU rDNA sequences representative of the clade of myxobolids. Numbers at the nodes are Bayesian posterior probabilities/ML bootstrap values; asterisks represent full support in both methodologies; dashes represent a different branching for the ML tree or a bootstrap support value under 50. Bold taxa correspond to species that have been reported from mugiliform fish hosts, with the invalid sequences of M. muelleri contained within square brackets. The SSU rDNA sequence obtained in this study for M. exiguus is marked with a dark grey box. Final host groups are indicated by vertical lines.

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus mugiliform fish host; it is the case of M. achmerovi Schulman, 1966, M. anili Sarkar, 1989, M. cheni Schulman, 1962, M. exiguus and M. parvus. Also, some have been indiscriminately reported from several organs and tissues; it is the case of M. achmerovi, M. adeli, M. cephalis, M. dasguptai, M. parvus and M. spinacurvatura (Table 3). The great biodiversity of Myxobolus spp. parasitizing mugiliform fish hosts reflects not only the species-richness of this myxosporean genus, which is the most common in freshwater environments, but probably correlates with the migratory patterns and feeding strategies of mullets. The catadromous nature of mullets allows these fish species to move into freshwater and brackish environments, thus increasing risk of exposure to typically freshwater parasites, such as Myxobolus spp. Also, being benthic feeders, mullets have increased proximity to infected annelids and, therefore, are more prone to contact with waterborne actinosporean stages. The high number of Myxobolus spp. parasitizing M. cephalus in particular, might suggest that this species possesses higher susceptibility than other mullets to myxosporean infection, but most likely simply reflects the higher number of parasitological surveys that have been conducted in this fish species, as a result of its economic importance in fisheries and aquaculture. Most mugiliform-infecting species are without molecular data, so that their reports (original and subsequent) have been solely based on morphological traits, which molecular- based systematics reveal are artificial for the reliable description of myxobolids (e.g. Fiala, 2006; Bartošová et al., 2009; Fiala and Bartošová, 2010; Liu et al., 2010). Thus, the legitimacy of these species, and their occurrence in the several reported sites of infection and hosts, must be evaluated through the use of molecular tools; a task that it might prove more difficult than expected, not only due to the frequent occurrence of co-infections (e.g. Molnár et al., 2006), but also due to the vague boundary between intraspecific and interspecific variability of myxobolids. For instance, our molecular analysis shows that the SSU rDNA sequence provided by Molnár et al. (2006) for M. muelleri displays only 97.0% of similarity to its conspecific sequence by Eszterbauer (2004), while it also shares a similar percentage of identity (97.0–97.5%) to the SSU rDNA sequences of M. arrabonensis (KP025680), M. bliccae (HM138772) and M. bramae (AF507968). Similarly, high values of intraspecific variability have been reported for different isolates of M. koi (3.0%), M. flavus (1.9%), H. corruscans (2.3%) and H. maculosus (1.9%) (Camus and Griffin, 2010; Carriero et al., 2013). On the other hand, very low interspecific variability has been reported between M. pseudodispar, M. musculi and M. cyprini (0.3–0.6%); M. pendula and M. pellicides (0.4%); M. fryeri and M. insidiosus (0.5%); M. intramusculi and M. procerus (2.1%); M. paksensis and M. cycloides (2.4%); and M. szentendrensis and M. intimus (2.8%) (Kent et al., 2001; Molnár et al., 2002; Easy et al., 2005; Ferguson et al., 2008; Cech et al., 2015). Considering all of the above, it is clear that the reliable classification of myxobolids can only result from the comprehensive evaluation of biological, morphological and molecular features. Another problem that researchers face when

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus studying myxobolids, as well as myxosporeans in general, is the amount of unpublished, incomplete, erroneous and confusing data in GenBank. Thus, it is important to recognize and avoid the use of poor records. In fact, Molnár (2011) identified and deemed invalid several SSU rDNA sequences of Myxobolus spp. In this study, it is further acknowledged that some SSU rDNA sequences of M. exiguus are erroneously attributed to M. muelleri (AY129313 and AY129314), as are the cases of the SSU rDNA sequences of M. turpisrotundus, M. toyamai and M. cutanei, erroneously designated as M. rotundus (AY165179), T. toyamai (HQ338729) and U. caudatus (JQ388890), respectively.

Assessment of the legitimacy of mugiliform fish as hosts for cryptic species, with the re-description of M. exiguus

The great majority of myxobolids are host specific (Molnár, 1994), with few having been recognized to infect a wide range of hosts belonging to the same taxonomic family or order. For instance, M. pseudodispar has been shown to infect a wide range of cypriniforms (Molnár et al., 2002; Forró and Ezsterbauer, 2016) and, in the same manner, M. cerebralis is known to parasitize several species of salmonids (El-Matbouli et al., 1999; Hoffman, 1999; Hedrick et al., 2001; Ferguson et al., 2008). Similarly, most myxobolids have well-defined sites of infection (Molnár, 1994; 2002; Molnár et al., 2006), but several species have been indiscriminately reported from multiple organs, either due to misidentifications, or to the parasite’s specificity to a given tissue. For instance, the plasmodia of M. diaphanus develop in the connective tissue of several different organs of the banded killifish Fundulus diaphanus (Lesueur, 1817) (Cone and Easy, 2005). As such, indicating specific tissue tropism, rather than just the organ of infection, is necessary for the correct characterization of myxobolids, and myxosporeans in general. Furthermore, recent phylogenetic studies have consistently shown vertebrate host group as the strongest evolutionary signal for myxobolids (e.g. Ferguson et al., 2008; Carriero et al., 2013; Rocha et al., 2014a), followed by the aquatic environment of the host species and tissue tropism (Kent et al., 2001; Eszterbauer, 2004; Holzer et al., 2004; Fiala, 2006). Overall, about 38 species and four records of Myxobolus have been performed from mugiliform fish hosts worldwide (Table 3 and 5; Naidenova et al., 1975; Donets, 1979; Ibragimov, 1987; Yurakhno and Maltsev, 2002; Yurakhno, 2004; Eiras et al., 2007, 2014), with C. ramada being host to M. exiguus and M. muelleri (Thélohan, 1895; Siau, 1978; Lubat et al., 1989; Bahri et al., 2003). Both these species are cryptic, having been described from a wide range of tissues and hosts. The original description of M. exiguus simultaneously reported the parasite from the stomach epithelium, pyloric caeca, kidney and spleen of the mugilids C. labrosus and C. ramada in France, and from the gills of the cypriniform fish host A. brama (Linnaeus, 1758), with basis on a single schematic line drawing and some spores’

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

S: smaller; L: larger. Measurements are means ± SD (range) (when available), givenavailable),µm. (when in means are larger. Measurements (range) SD ± smaller; L: S:

coils;

mugiliform fish hosts. SL: myxospore length; SW: myxospore width; ST: myxospore thickness; PCL: polar polar PCL: thickness; myxospore ST: width; myxospore SW: length; myxospore SL: hosts. fish mugiliform

tubule

bonafide

spp. with with spp.

c: number of polar c:of number

Myxobolus

Summary of data available for for available data of Summary

Table 3 Table capsule width; capsuleP PCW: polar length;

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

measurements (Thélohan, 1895). Since then, this parasite was reported from several other cypriniform and mugiliform fish hosts inhabiting freshwater across Europe and the Mediterranean Sea, including: Alburnus alburnus (Linnaeus, 1758), Leuciscus aspius (Linnaeus, 1758), Blicca bjoerkna (Linnaeus, 1758), Chondrostoma nasus (Linnaeus, 1758), L. idus (Lin- naeus, 1758), Pelecus cultratus (Linnaeus, 1758), Ru- tilus rutilus (Linnaeus, 1758), Scardinius erythrophthalmus (Linnaeus, 1758), C. auratus, C. saliens and Mugil cephalus (Kudo, 1919; Siau, 1978; Lubat et al., 1989; Bahri et al., 2003). Bahri et al. (2003) sequenced the SSU rDNA gene of this parasite (AY129316, AY129317) using samples obtained from the intestine of C. ramada from the Ichkeul Lake, Tunisia. However, these authors also provided two SSU rDNA sequences for M. muelleri (AY129313 and AY129314) obtained from the mesenteric vessels of C. ramada, which our analysis shows, are equal to the ones provided for M. exiguus. According to the phylogenetic analysis performed by Bahri et al. (2003), the sequences obtained for M. muelleri and M. exiguus differed solely by three nucleotide substitutions, with the myxospores exhibiting subtle morphological differences and undergoing sporogony in different organs (M. muelleri in mesenteric vessels and M. exiguus in the intestine). However, acknowledging that C. ramada should not be considered as a bona fide host for M. muelleri, it seems probable that Bahri et al. (2003) sequenced the SSU rDNA gene of the same parasite, M. exiguus, from the visceral peritoneum, which lines the intestine and double-folds to form the mesentery attaching to the gastrointestinal organs. Molecular comparison of the SSU rDNA sequence obtained here to those obtained by Bahri et al. (2003) identified the parasite in study as M. exiguus. This identification is corroborated by the specificity of the site of infection and host species, but

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

also by the morphological characters of the myxospores, which dimensions are congruent with those provided in

the original description of M. exiguus by Thélohan coils; S: smaller; smaller; S: coils; (1895), as well as those provided by Bahri et al. (2003)

[albeit being slightly bigger, and presenting fewer polar tubule tubule coils (five) than those (six to seven) described for the myxospores collected from the mesenteric vessels and misidentified as M. muelleri]. Bahri et al. (2003)

further described the valves as smooth, with 10–12

c: number of polar polar of number c: T markings appearing near the suture line, as it was also observed in this study. Considering that M. exiguus was originally described on the basis of a single schematic line drawing, and that its morphometrics were obtained from myxospores that most probably belong to different Myxobolus spp., as shown by the several tissues of infection and hosts accounted for in the original description, this paper aims to present a comprehensive morphological and molecular re-description of this myxobolid species. Chelon ramada is here suggested as

the type host for M. exiguus, not only because it is among

the host species included in the original description, but

also because the parasite gained its molecular identity

mugiliform fish hosts. SBL: myxospore body length; SBW: myxospore body width; SBT: myxospore body body myxosporeSBT: width; bodymyxospore SBW: length; bodymyxospore SBL: hosts. fish mugiliform

from infections in the visceral peritoneum of this mullet

species. Thus, other fish species, namely cypriniforms, bonafide should be disregarded as bona fide hosts for M. exiguus.

Similarly, the visceral peritoneum is suggested as type

spp. with spp.with

tissue, so that other tissues and organs of infection, such as the gills, should also be disregarded as sites of

Henneguya infection for M. exiguus. On its turn, M. muelleri, type species of the genus, was originally described from the gills of several cypriniforms, without indication of a type host, and since then reported from several different tissues and organs

in a great number of fish hosts from Eurasia and North L: total length of myxospore; LCA: length of caudal appendages; PCL: polar capsule length; PCW: polar capsule width; P width; capsule polar PCW: length; capsule polar PCL: appendages; caudal of length LCA: myxospore; of length total L:

America, including: the kidney and ovaries of Phoxinus

Summary of data available for available data of Summary

. phoxinus (Linnaeus, 1758); the eyes of Symphodus me-

lops (Linnaeus, 1758) and A. alburnus; the gills of Zingel

Table 4 Table ST thickness; ingivenavailable),µm. means are Measurements(range) SD ± (when L: larger.

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus asper (Linnaeus, 1758), Barbus barbus (Linnaeus, 1758), R. rutilus and Lota lota (Linnaeus, 1758); the pseudobranches of Cottus gobio (Linnaeus, 1758); and the gills, fins, eyes, mesentery, intestine, gall bladder, urinary bladder, liver, kidney, spleen, gonads, heart and muscle of M. cephalus, C. auratus, C. saliens and C. ramada (Kudo, 1919; Shulman, 1984; Lom and Dyková,1992; Bahri et al., 2003; Molnár et al., 2006; Umur et al., 2010; Yurakhno and Ovcharenko, 2014). Considering the currently accepted evolutionary signals of Myxosporea, namely the importance of host affinity for myxobolids (Carriero et al., 2013), it is clear that M. muelleri constitutes a species-complex, comprising several species that are phenotypically similar and, therefore, have been misidentified. This is further supported by the astonishing variation in the shape and size of the myxospores of M. muelleri between reports. In their taxonomic revision of the genus Myxobolus, Landsberg and Lom (1991) suggested settling S. cephalus as the type host for M. muelleri. Eszterbauer (2004) provided a SSU rDNA sequence (AY325284) for the parasite sampled from the gills of the chub S. cephalus from the River Danube, Hungary; but it was Molnár et al. (2006) who characterized M. muelleri by providing a comprehensive morphological and molecular re-description of the species from samples obtained from the gills, as well as from the swimbladder of S. cephalus in Hungary (DQ439806). Bahri et al. (2003) had supposedly sequenced M. muelleri from the mesenteric vessels of C. ramada from the Ichkeul Lake, Tunisia. Nonetheless, our molecular analyses show that the sequences obtained from the mugilid fish host (AY129313 and AY129314) display lower percentage of identity (72.2–73.4%) than those obtained by Eszterbauer (2004) and Molnár et al. (2006), revealing that the parasite infecting C. ramada is not M. muelleri, but M. exiguus, as previously stated. Thus, our study agrees with Molnár (2011) in that the SSU rDNA sequences obtained by Bahri et al. (2003) for M. muelleri should be deemed invalid. We further suggest disregarding C. ramada and other mugilids has bona fide hosts for M. muelleri, as well as gadids, percids and scorpaenids. Analysing the legitimacy of mugiliform fish as hosts for other Myxobolus spp. reported from mullets, some species require obvious attention, as their original descriptions were performed from cypriniforms. Myxobolus acutus (Fujita, 1912) Landsberg and Lom, 1991, originally S. acuta, was first described from the gills of Carassius auratus gibelio in Japan, and later reported from the scales of M. cephalus and P. haematocheila from several Russian Rivers, and from the Sea of Japan (Landsberg and Lom, 1991; Eiras et al., 2005; Yurakhno and Ovcharenko, 2014). Myxobolus bramae was originally described from the gills of A. brama in Russia, and later reported from a wide range of organs and tissues of M. cephalus in the Black Sea, including the gills, skin, fins, heart, muscle, mouth, esophagus, intestine, swim bladder, liver, gall bladder, spleen and kidney (Iskov, 1989; Eiras et al., 2005; Yurakhno and Ovcharenko, 2014). Both Andree et al. (1999) and Eszterbauer (2004) deposited an SSU rDNA sequence for this parasite obtained from the gills of its type host in Hungary (AF085177 and

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

AF507968, respectively), which turned out shared very low percentage of similarity between each other. Considering that the common bream is the host for several other gill-infecting Myxobolus spp. in Hungary (Molnár and Székely, 1999), Eszterbauer (2004) and Ferguson et al. (2008) suggested that the samples used by Andree et al. (1999) were probably contaminated by myxospores of another species and the corresponding SSU rDNA sequence was ultimately deemed invalid (Molnár, 2011). Myxobolus rotundus, which was also originally described from the gills of A. brama in Germany, as well as from the gudgeon Gobio gobio (Linnaeus, 1758), was later reported from the gills, heart and other internal organs of C. auratus in the Black Sea (Donets, 1979; Iskov, 1989; Eiras et al., 2005). This parasite ultimately had its SSU rDNA gene characterized from infections in its type tissue and host (Székely et al., 2009). Myxobolus branchialis, originally Myxosoma branchialis, was first described from the gills of B. barbus in Ukraine, but then reported from the gills, kidney and spleen of M. cephalus, C. auratus and C. saliens in the Caspian Sea and Black Sea (Schulman, 1966; Ibragimov, 1987; Iskov, 1989; Eiras et al., 2005). Molnár et al. (2012) gave molecular identity to the parasite upon its re-description from the gills of common barbel and Iberian barbel Luciobarbus bocagei (Steindachner, 1864) in Hungary and Portugal. Finally, M. circulus (Achmerov, 1960) Landsberg and Lom, 1991, originally Myxosoma circulus, was first described from the gills of Cyprinus carpio Linnaeus, 1758 in Russia, being later reported from the gills, fins, muscle and kidney of M. cephalus in the Black Sea (Naidenova et al., 1975; Iskov, 1989; Yurakhno, 2004; Eiras et al., 2005). Given the molecular trends accepted for myxobolids (Andree et al., 1999; Kent et al., 2001; Eszterbauer, 2004; Fiala, 2006; Carriero et al., 2013), it is highly unlikely for a cypriniform-infecting species to parasitize members of the order Mugiliformes. Thus, we suggest disregarding mullets as legitimate hosts for all these species, which should be considered restricted to their original hosts, and others proven through means of molecular tools (as is the case of M. branchialis) (Table 5). For the same reason, we suggest disregarding the cyprinid C. carpio haematopterus as a bona fide host for M. achmerovi Schulman, 1966, which original description was performed from the gills, fins and mesentery of M. cephalus and P. haematocheila (Eiras et al., 2005; Yurakhno and Ovcharenko, 2014).

Ultrastructure of M. exiguus

Ultrastructural studies of the plasmodial and sporogonic development can provide valuable supplementary information for the distinction of individual species and, more importantly, for understanding host–parasite interactions (Hallett and Diamant, 2001; Rocha et al., 2013; 2014b). Current (1979) further suggested that certain ultrastructural differences in the plasmodium wall may partially correlate with the degree of pathogenicity of the parasite. In general, the wall of cyst-forming myxosporeans is structured similarly between different

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

, species and genera,

et al. with few variations that probably result from the

(Molnár physical and biological conditions of the tissue/organ of infection, as well as the host

immune response (Lom ± SD (range) (when available), given given available), (when (range) SD ± and Dyková, 1992; Hal- lett and Diamant, 2001; Rocha et al., 2013). Most histozoic species present a smooth plasmodial wall, with pinocytosis or phagocytosis being widely accepted as the processes sup- plying the nutrients ne-

cessary for the parasi- coils; S: smaller; L: larger. Measurements are means are Measurements larger. smaller;L: S: coils; te’s development (Cur-

rent and Janovy, 1976; tubule Mitchell, 1977; Current, 1979; Current et al., 1979; Casal et al., 2006;

c: number of polar polar of number c: Azevedo et al., 2011). T The ultrastructural featu-

res of the plasmodial de-

spp. spp. erroneouslyfrom mugiliformreported fish hosts. myxospore SL:SW: myxospore length; myxospore width; ST:thickness;

velopment of M. exiguus description description and molecular identification of the parasite from its original site of infection and host species in Hungary - is similar to that of other Myxobolus histozoic myxosporeans only in that pinocytotic activity is evidenced by the large number of vacuoles occupying the

ectoplasmic layer. Its

Summary of Summary for data valid

*Data *Data from the morphological re

plasmodial wall, howe-

006).

PCL: polar capsule length; PCW: polar capsule width; width; P capsule polar PCW: length; capsule PCL:polar in µm. 2 Table5 ver, displays an irregular

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus outline, with peripheral projections expanding the parasite–host interface, probably for optimizing nutrient intake. This feature has been reported for few other histozoic species, e.g. M. insignis (Azevedo et al., 2013) and M. filamentum (Naldoni et al., 2015), since the differentiation of peripheral projections is common to the plasmodial development of coelozoic species (see Sitjà-Bobadilla and Alvarez-Pellitero, 1993; 2001; Rocha et al., 2011). Overall, the ultrastructural study performed revealed significant unique features of the plasmodial development of M. exiguus, namely in its attachment to the mesothelial cells. In turn, the sporogony of M. exiguus is essentially similar to that of other myxobolids with centric asynchronous development (e.g. Current, 1979; Current et al., 1979; Naldoni et al., 2015), in that the ectoplasm appears highly vacuolated and devoid of cytoplasmic organelles, while the endoplasm is riddled with organelles and different developmental stages of the parasite: generative cells and developing sporoblasts at the periphery, and immature myxospores at the centre.

Phylogenetic analysis

The phylogenetic analysis performed here is congruent with previously published cladograms (e.g. Kent et al., 2001; Fiala, 2006; Ferguson et al., 2008; Carriero et al., 2013; Rocha et al., 2014a), in that it shows the vertebrate host group as the most relevant evolutionary signal for myxobolids, with tissue tropism and aquatic environment playing less conspicuous roles. Accordingly, all SSU rDNA sequences available for myxobolids with legitimate mugiliform fish hosts are shown here clustering together to form a well-supported subclade of the clade of myxobolids. Members of the sphaeractinomyxon, endocapsa and triactinomyxon collective groups that probably play a role in the life cycles of myxobolid species from mullet hosts also cluster within this subclade. In turn, the valid SSU rDNA sequences available for M. bramae, M. branchialis, M. muelleri and M. rotundus, which were obtained from their cypriniform type hosts, all cluster within the clade comprising cypriniform-infecting myxobolids. This emphasizes the incongruence of reporting these species from mugiliform fish hosts, as well as the artificiality of using morphological characters for species identification. The fallibility of morphology as an evolutionary signal for myxobolids has been well reported in several studies (e.g. Fiala, 2006; Bartošová et al., 2009; Fiala and Bartošová, 2010; Liu et al., 2010). For instance, traditional taxonomy separates the genera Myxobolus and Henneguya according to the absence or presence of caudal appendages, respectively (Lom and Dyková, 2006). Nonetheless, phylogenetic analyses have consistently shown the convergent evolution of caudal appendages (Fiala and Bartošová, 2010; Liu et al., 2010), revealing that this morphological trait bares little insight into the relationships of myxobolids. In fact, abnormal spore extensions have been reported for some Myxobolus spp. (Mitchell, 1989; Cone and

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Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Overstreet, 1997; Bahri, 2008; Liu et al., 2010, 2013, 2014, 2015; Zhang et al., 2014), including M. bizerti from the gills of M. cephalus, and M. exiguus (misidentified as M. muelleri, as previously mentioned) from the mesenteric vessels of C. ramada (e.g. Longshaw et al., 2003; Eiras et al., 2005; Kaur and Singh, 2010; Camus et al., 2017). It has been suggested that the origins and radiations of myxosporean parasites probably reflect the evolution of their fish hosts (e.g. Carriero et al., 2013; Kodádková et al., 2015). Evolutionary phylogenies of fish reveal that the order Mugiliformes is monophyletic in relation to its sister taxa, despite the polyphyly and/ or paraphyly that takes place at the genera-level, due to systematics based in poorly informative anatomical characters (Durand et al., 2012). The phylogenetic analyses performed here supports the co-evolutionary history of myxosporeans and their vertebrate hosts, as it shows all legitimate mugiliform-infecting myxobolids clustering together to form a monophyletic well-supported subclade within the clade of myxobolids. In the future, it would be interesting to unravel the significance that this co-evolutionary history had in the adaptive strategies of myxosporeans to different micro- and macroenvironments.

Acknowledgements

This work was financially supported by FCT (Lisbon, Portugal) within the scope of the Ph.D. fellowship grant attributed to S. Rocha (SFRH/BD/92661/2013) through the programme QREN-POPH/ FSE; and the Eng° António de Almeida Foundation (Porto, Portugal). Ethical standards. The work developed in this study was performed in accordance with European ethical standards.

References

Abdel-Baki, A.A.S. (2011). Light and electron microscopic studies on Myxobolus egyptica sp. nov. (Myxozoa, Myxosporea), infecting the hornlip mullet Oedalechilus labiosus from the Red Sea. Acta Parasitologica 56, 255–262. Almeida, P.R. (2003). Feeding ecology of Liza ramada (Risso, 1810) (Pisces, Mugilidae) in a south-western estuary of Portugal. Estuarine, Coastal and Shelf Science 57, 313–323. Andree, K.B., Székely, C., Molnár, K., Gresoviac, S.J. and Hedrick, R.P. (1999). Relationships among members of the genus Myxobolus (myxozoa: Bilvalvidae) based on small subunit ribosomal DNA sequences. Journal of Parasitology 85, 68 –74. Atkinson, S.D., Bartošová-Sojková, P., Whipps, C.M. and Bartholomew, J.L. (2015). Approaches for characterising myxozoan species. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer

Page | 151

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

International Publishing, Switzerland, pp. 111–123. Azevedo, C., Casal, G., Marques, D., Silva, E. and Matos, E. (2011). Ultrastructure of Myxobolus brycon n. sp. (phylum Myxozoa), parasite of the piraputanga fish Brycon hilarii (Teleostei) from Pantanal (Brazil). Journal of Eukaryotic Microbiology 58, 88–93. Azevedo, C., São Clemente, S.C., Casal, G., Matos, P., Oliveira, E., Al-Quraishy, S. and Matos, E. (2013). Ultrastructure of the plasmodial development of Myxobolus insignis (Myxozoa), infecting the Amazonian fish Semaprochilodus insignis (Prochilodontidae). Acta Protozoologica 52, 91–97. Azevedo, C., Rocha, S., Matos, P., Matos, E., Oliveira, E., Al-Quraishy, S. and Casal, G. (2014). Morphology and phylogeny of Henneguya jocu n. sp. (Myxosporea, Myxobolidae), infecting the gills of the marine fish Lutjanus jocu. European Journal of Protistology 50, 185–193. Bahri, S. (2008). Abnormal forms of Myxobolus bizerti and Myxobolus mulleri (Myxosporea: Bivalvulida) spores with caudal appendages. Bulletin of the European Association of Fish Pathologists 28, 252–255. Bahri, S. and Marques, A. (1996). Myxosporean parasites of the genus Myxobolus from Mugil cephalus in Ichkeul lagoon, Tunisia: description of two new species. Diseases of Aquatic Organisms 27, 115–122. Bahri, S., Andree, K.B. and Hedrick, R.P. (2003). Morphological and phylogenetic studies of marine Myxobolus spp. from mullet in Ichkeul Lake, Tunisia. Journal of Eukaryotic Microbiology 50, 463–470. Bartošová, P., Fiala, I. and Hypša, V. (2009). Concatenated SSU and LSU rDNA data confirm the main evolutionary trends within myxosporeans (Myxozoa: Myxosporea) and provide and effective tool for their molecular phylogenetics. Molecular Phylogenetics and Evolution 53, 81–93. Camus, A.C. and Griffin, M.J. (2010). Molecular characterization and histopathology of Myxobolus koi infecting the gills of a koi, Cyprinus carpio, with an amended morphological description of the agent. Journal of Parasitology 96, 116–124. Camus, A.C., Dill, J.A., Rosser, T.G., Pote, L.M. and Griffin, M.J. (2017). Myxobolus axelrodi n. sp. (Myxosporea: Myxobolidae) a parasite infecting the brain and retinas of the cardinal tetra Paracheirodon axelrodi (Teleostei: Characidae). Parasitology Research 116, 387–397. Cardona, L. (2001). Non-competitive coexistence between Mediterranean grey mullet: evidence from seasonal changes in food availability, niche breadth and trophic overlap. Journal of Fish Biology 59, 729–744. Carriero, M.M., Adriano, E.A., Silva, M.R., Ceccarelli, P.S. and Maia, A.A. (2013). Molecular phylogeny of the Myxobolus and Henneguya genera with several new South American

Page | 152

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

species. PLoS One 8, e73713. Casal, G., Matos, E. and Azevedo, C. (2006). A new myxozoan parasite from the Amazonian fish Metynnis argenteus (Teleostei, Characidae): light and electron microscope observations. Journal of Parasitology 92, 817–821. Cech, G., Borzák, R., Molnár, K. and Székely, C. (2015). Three new species of Myxobolus Bütschli, 1882 (Myxozoa: Myxobolidae) infecting the common nase Chondrostoma nasus (L.) in the River Danube. Systematic Parasitology 92, 101–111. Cone, D.K. and Easy, R.H. (2005). Supplemental diagnosis and molecular taxonomy of Myxobolus diaphanus (Fantham, Porter et Richardson, 1940) (Myxozoa) parasitizing Fundulus diaphanus (Cyprinodontiformes) in Nova Scotia, Canada. Folia Parasitologica 52, 217–222. Cone, D.K. and Overstreet, R. (1997). Myxobolus mississippiensis n. sp. (Myxosporea) from gills of Lepomis macrochirus in Mississippi. Journal of Parasitology 83, 122–124. Crosetti, D. and Cataudella, S. (1995). Grey mullet culture. In Nash, C.E. (ed). World Animal Science 34B: Production of Aquatic Animals. Elsevier, Burlington, MA, USA, pp. 271– 288. Current, W.L. (1979). Henneguya adiposa Minchew (Myxosporida) in the channel catfish: ultrastructure of the plasmodium wall and sporogenesis. Journal of Protozoology 26, 209–217. Current, W.L. and Janovy, J.J. (1976). Sporogenesis in Henneguya exilis infecting the channel catfish: an ultrastructural study. Protistologica 13, 157–167. Current, W.L., Janovy, J.J. and Knight, S.A. (1979). Myxosoma funduli Kudo (Myxosporida) in Fundulus kansae: ultrastructure of the plasmodium wall and of sporogenesis. Journal of Protozoology 26, 574–583. Das, M.K. (1996). Myxozoan and urceolarid ciliate parasites of wild and cultured Liza parsia in deltaic West Bengal. Journal of the Inland Fisheries Society of India 28, 46‒56. Donets, Z.S. (1979). The zoogeographical analysis of Myxosporidia in the USSR southern water reservoirs. Evolution and ecology of Sporozoa and Cnidosporidia. Trudy Zoologicheskogo Instituta, Akademiia nauk, SSSR, pp. 65–90. Dorothy, K.P. and Kalavati, C. (1992). Two new myxosporean parasites of the mullet Liza macrolepis (Smith). Uttar Pradesh Journal of Zoology 12, 15–19. Durand, J.D., Shen, K.N., Chen, W.J., Jamandre, B.W., Blel, H., Diop, K., Nirchio, M., Garcia de Leon, F.J., Whitfield, A.K., Chang, C.W. and Borsa, P. (2012). Systematics of the grey mullets (Teleostei: Mugiliformes: Mugilidae): molecular phylogenetic evidence challenges two centuries of morphology-based taxonomy. Molecular Phylogenetics and Evolution 64, 73–92. Easy, R.H., Johnson, S.C. and Cone, D.K. (2005). Morphological and molecular comparison

Page | 153

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

of Myxobolus procerus (Kudo, 1934) and M. intramusculi n. sp. (Myxozoa) parasitizing muscles of the trout-perch Percopsis omiscomaycus. Systematic Parasitology 61, 115– 122. Egusa, S., Maeno, Y. and Sorimachi, M. (1990). A new species of Myxozoa, Myxobolus episquamalis sp. n. infecting the scales of the mullet, Mugil cephalus L. Fish Pathology 25, 87–91. Eiras, J.C. (2002). Synopsis of the species of the genus Henneguya Thélohan, 1892 (Myxozoa: Myxosporea: Myxobolidae. Systematic Parasitology 52, 43–54. Eiras, J.C. and Adriano, E.A. (2012). A checklist of new species of Henneguya Thélohan, 1892 (Myxozoa: Myxosporea, Myxobolidae) described between 2002 and 2012. Systematic Parasitology 83, 95–104. Eiras, J.C. and D’Souza, J. (2004). Myxobolus goensis n. sp. (Myxozoa, Myxosporea, Myxobolidae), a parasite of the gills of Mugil cephalus (Osteichthyes, Mugilidae) from Goa, India. Parasite 11, 243–248. Eiras, J.C., Molnár, K. and Lu, Y.S. (2005). Synopsis of the species of Myxobolus Bütschli, 1882 (Myxozoa: Myxosporea: Myxobolidae). Systematic Parasitology 61,1–46. Eiras, J.C., Abreu, P.C., Robaldo, R. and Pereira Júnior, J. (2007). Myxobolus platanus n. sp. (Myxosporea, Myxobolidae), a parasite of Mugil platanus Günther, 1880 (Osteichthyes, Mugilidae) from Lagoa dos Patos, RS, Brazil. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 59, 895–898. Eiras, J.C., Zhang, J. and Molnár, K. (2014). Synopsis of the species of Myxobolus Bütschli, 1882 (Myxozoa: Myxosporea, Myxobolidae) described between 2005 and 2013. Systematic Parasitology 88, 11–36. El-Matbouli, M., Fischer-Scherl, T. and Hoffmann, R.W. (1992). Present knowledge on the life cycle, taxonomy, pathology, and therapy of some Myxosporea spp. important for freshwater fish. Annual Review of Fish Diseases 2, 367–402. El-Matbouli, M., Hoffmann, R.W., Schoel, H., McDowell, T.S. and Hedrick, R.P. (1999). Whirling disease: host specificity and interaction between the actinosporean stage of Myxobolus cerebralis and rainbow trout Oncorhynchus mykiss. Diseases of Aquatic Organisms 35,1–12. Eszterbauer, E. (2004). Genetic relationship among gill-infecting Myxobolus species (Myxosporea) of cyprinids: molecular evidence of importance of tissue-specificity. Diseases of Aquatic Organisms 58, 35–40. Fall, M., Kpatcha, K.P., Diebakate, C., Faye, N. and Toguebaye, B.S. (1997). Observations on myxosporidia (Myxozoa) of the genus Myxobolus parasites of Mugil cephalus (Pisces, Teleostei) from Senegal. Parasite 4, 173–180. Faye, N., Kpatcha, T.K., Fall, M. and Toguebaye, B.S. (1997). Heart infections due to

Page | 154

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

myxosporean (Myxozoa) parasites in marine and estuarine fishes from Senegal. Bulletin of the European Association of Fish Pathologists 17, 115–117. Faye, N., Kpatcha, T.K., Debakate, C., Fall, M. and Toguebaye, B.S. (1999). Gill infections due to myxosporean (Myxozoa) parasites in fishes from Senegal with description of Myxobolus hani sp. n. Bulletin of the European Association of Fish Pathologists 19, 14– 16. Ferguson, J.A., Atkinson, S.D., Whipps, C.M. and Kent, M.L. (2008). Molecular and morphological analysis of Myxobolus spp. of salmonid fishes with the description of a new Myxobolus species. Journal of Parasitology 94, 1322–1334. Fiala, I. (2006). The phylogeny of Myxosporea (Myxozoa) based on small sub-unit ribosomal RNA gene analysis. International Journal for Parasitology 36, 1521–1534. Fiala, I. and Bartošová, P. (2010). History of myxozoan character evolution on the basis of rDNA and EF-2 data. BMC Evolutionary Biology 10, 228. Fioravanti, M.L., Caffara, M., Florio, D., Gustinelli, A., Marcer, F. and Quaglio, F. (2006). Parasitic diseases of marine fish: epidemiological and sanitary considerations. Parassitologia 48, 15–18. Forró, B. and Eszterbauer, E. (2016). Correlation between host specificity and genetic diversity for the muscle-dwelling fish parasite Myxobolus pseudodispar: examples of myxozoan host-shift? Folia Parasitologica 63, 019. Haldar, D.P., Samal, K.K. and Mukhopadhyaya, D. (1996). Studies on the protozoan parasites of fishes in Orissa: eight species of Myxobolus Bütschli (Myxozoa: Bivalvulida). Journal of the Bengal Natural History Society 16, 3–24. Hallett, S.L. and Diamant, A. (2001). Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia. Diseases of Aquatic Organisms 46, 197–212. Hedrick, R.P., McDowell, T.S., Mukkatira, K., Georgiadis, M.P. and MacConnell, E. (2001). Susceptibility of three species of anadromous salmonids to experimentally induced infections with Myxobolus cerebralis, the causative agent of whirling disease. Journal of Aquatic Animal Health 13, 43–50. Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. The Quaterly Review of Biology 66, 411–453. Hoffman, G.L. (1999). Parasites of North American Freshwater Fishes, 2nd edition. Comstock Publishing Associates, Ithaca, NY, pp. 576. Holzer, A.S., Sommerville, C. and Wootten, R. (2004). Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences. International Journal for Parasitology 34, 1099–1111.

Page | 155

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Hotos, G.N. and Vlahos, N. (1998). Salinity tolerance of Mugil cephalus and Chelon labrosus (Pisces: Mugilidae) fry in experimental conditions. Aquaculture 167, 329–338. Ibragimov, S.R. (1987). Forming of the Parasites Fauna of Mullets in the Caspian Sea. Institute of Zoology of AS, Baku, Aserbaijan SSR, pp. 1–14. Iskov, M.P. (1989). Myxosporidia (Myxosporea). In Markevitch, A.P. and Schulman, S.S. (eds). Fauna Ukrainy, Vol. 37. Naukowa Dumka, Kiev, pp. 212. Iversen, E.S., Chitty, N. and Van Meter, N. (1971). Some Myxosporidia from marine fishes in South Florida. Journal of Protozoology 18, 82–86. Karlsbakk, E., Kristmundsson, A., Albano, M., Brown, P. and Freeman, M.A. (2017). Redescription and phylogenetic position of Myxobolus aeglefini and Myxobolus platessae n. comb. (Myxosporea), parasites in the cartilage of some North Atlantic marine fishes, with notes on the phylogeny and classification of the Platysporina. Parasitology International 66, 952–959. Kaur, H. and Singh, H.S. (2010). Two new species of Myxobolus (myxosporea, Bivalvulida) from the Indian major carp Labeo rohita Hamilton, 1822. Protistology 6, 264–270. Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffmann, R.W., Khattra, J., Hallett, S.L., Lester, R.J., Longshaw, M., Palenzeula, O., Siddall, M.E. and Xiao, C. (2001). Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic Microbiology 48, 395–413. Khlifa, S., Miller, T.L., Adlard, R.D., Faye, N. and Sasal, P. (2012). Henneguya mauritaniensis n. sp. (Myxozoa) from the arterial bulb of Pagrus caeruleostictus (Valenciennes, 1830) off Mauritania. Parasitology Research 111, 1287–1294. Kim, W.S., Kim, J.H. and Oh, M.J. (2013). Morphologic and genetic evidence for mixed infection with two Myxobolus species (Myxozoa: Myxobolidae) in gray mullets, Mugil cephalus, from Korean waters. The Korean Journal of Parasitology 51, 369–373. Kodádková, A., Bartošová-Sojková, P., Holzer, A.S. and Fiala, I. (2015). Bipteria vestuta n. sp. – an old parasite in an old host: tracing the origin of myxosporean parasitism in vertebrates. International Journal for Parasitology 45, 269–276. Kpatcha, T.K., Faye, N., Diebakate, C., Fall, M. and Toguebaye, B.S. (1997). New myxosporidian species of the genus Henneguya Thélohan, 1895 (Myxozoa, Myxosporea) parasites of marine fishes from Senegal. Light and electron microscopic studies. Annales des Sciences Naturelles Zoologie et Biologie Animale 18, 81–91. Kudo, R.R. (1919). Studies on Myxosporidia. A synopsis on genera and species of Myxosporidia. Illinois Biological Monographs 5, 1–265. Laffaille, P., Feunteun, E., Lefebvre, C., Radureau, A., Sagan, G. and Lefeuvre, J.C. (2002). Can thin-lipped mullet directly exploit the primary and detritic production of European macrotidal salt marshes? Estuarine, Coastal and Shelf Sciences 54, 729–736.

Page | 156

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Landsberg, J.H. and Lom, J. (1991). Taxonomy of the genera of the Myxobolus/Myxosoma group (Myxobolidae: Myxosporea), current listing of species and revision of synonyms. Systematic Parasitology 18, 165–186. Li, Y.C., Sato, H., Kamata, Y., Ohnishi, T. and Sugita-Konishi, Y. (2012). Three novel myxobolid species of genera Henneguya and Myxobolus (myxosporea: Bivalvulida) from marine fish in Japan. Parasitology Research 111, 819–826. Liu, Y., Whipps, C.M., Gu, Z.M. and Zeng, L.B. (2010). Myxobolus turpisrotundus (Myxosporea: Bivalvulida) spores with caudal appendages: investigating the validity of the genus Henneguya with morphological and molecular evidence. Parasitology Research 107, 699–706. Liu, Y., Whipps, C.M., Gu, Z.M., Huang, M.J., He, C., Yang, H.L. and Molnár, K. (2013). Myxobolus musseliusae (Myxozoa: Myxobolidae) from the gills of common carp Cyprinus carpio and revision of Myxobolus dispar recorded in China. Parasitology Research 112, 289–296. Liu, Y., Whipps, C.M., Nie, P. and Gu, Z. (2014). Myxobolus oralis sp. n. (Myxosporea: Bivalvulida) infecting the palate in the mouth of gibel carp Carassius auratus gibelio (Cypriniformes: Cyprinidae). Folia Parasitologica 61, 505–511. Liu, X.H., Zhang, J.Y., Batueva, M.D. and Voronin, V.N. (2015). Supplemental description and molecular characterization of Myxobolus miyarii Kudo, 1919 (Myxosporea: Myxobolidae) infecting intestine of Amur catfish (Silurus asotus). Parasitology Research 115, 1547–1556. Lom, J. (1987). Myxosporea: a new look at long-known parasites of fish. Parasitology Today 3, 327–332. Lom, J. and Arthur, J.R. (1989). A guideline for the preparation of species descriptions in Myxosporea. Journal of Fish Diseases 12, 151–156. Lom, J. and Dyková, I. (1992). Myxosporidia (Phylum Myxozoa). Protozoan Parasites of Fishes. Developments in Aquaculture and Fisheries Science, Vol. 26. Elsevier, Amsterdam, Netherlands, pp. 159‒235. Lom, J. and Dyková, I. (1994). Studies on Protozoan parasites of Australian fishes. European Journal of Protistology 30, 431–439. Lom, J. and Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53, 1–36. Longshaw, M., Frear, P. and Feist, S.W. (2003). Myxobolus buckei sp. n. (Myxozoa), a new pathogenic parasite from the spinal column of three cyprinid fishes from the United Kingdom. Folia Parasitologica 50, 251–262. Lubat, J., Radujkovic, B., Marques, A. and Bouix, G. (1989). Parasites des poissons marins du Montenegro: myxosporidies. Acta Adriatica 30, 31–50.

Page | 157

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Maeno, Y., Sorimachi, M., Ogawa, K. and Egusa, S. (1990). Myxobolus spinacurvatura sp. n. (Myxosporea: Bilvalvulida) parasitic in deformed mullet, Mugil cephalus. Fish Pathology 25, 37–41. Maeno, Y., Sorimachi, M., Ogawa, K. and Kearn, G.C. (1995). Myxobolus spirosulcatus n. sp. (Myxosporea, Bivalvulida) infecting the bile duct of the yellowtail Seriola quinqueradiata from Japan. Systematic Parasitology 31, 189–193. Maíllo-Bellón, P.A., Marques, A. and Gracia-Royo, M.P. (2011). Myxosporean infection of grey mullet in the Ebro Delta: identification and ultrastructure of Myxobolus ichkeulensis Bahri and Marques, 1996 infecting the gills of Mugil cephalus L. Acta Protozoologica 50, 65–69. Merella, P. and Garippa, G. (2001). Metazoan parasites of grey mullets (Teleostea: Mugilidae) from the Mistras Lagoon (Sardinia, western Mediterranean). Scientia Marina 65, 201– 206. Mitchell, C.W. (1977). Myxosporidia. In Krier, J.P. (ed). Parasitic Protozoa. Academic Press, New York, San Francisco and London, pp. 115–157. Mitchell, L.G. (1989). Myxobolid parasites (Myxozoa: Myxobolidae) infecting fishes of western Montana, with notes on histopathology, seasonality, and intraspecific variation. Canadian Journal of Zoology 67, 1915–1922. Molnár, K. (1994). Comments on the host, organ and tissue specificity of fish myxosporeans and on the types of their intrapiscine development. Parasitologia Hungarica 27, 5–20. Molnár, K. (2002). Site preference of myxosporean spp. on the fins of some Hungarian fish species. Diseases of Aquatic Organisms 52, 123–128. Molnár, K. (2011). Remarks to the validity of Genbank sequences of Myxobolus spp. (Myxozoa, Myxosporidae) infecting Eurasian fishes. Acta Parasitologica 56, 263–269. Molnár, K. and Székely, C. (1999). Myxobolus infection of the gills of common bream (Abramis brama L.) in Lake Balaton and in the Kis-Balaton reservoir, Hungary. Acta Veterinaria Hungarica 47, 419–432. Molnár, K., Eszterbauer, E., Székely, C., Dán, Á. and Harrach, B. (2002). Morphological and molecular biological studies on intramuscular Myxobolus spp. of cyprinid fish. Journal of Fish Diseases 25, 643–652. Molnár, K., Marton, S., Eszterbauer, E. and Székely, C. (2006). Comparative morphological and molecular studies on Myxobolus spp. infecting chub from the River Danube, Hungary, and description of M. muellericus sp n. Diseases of Aquatic Organisms 73, 49–61. Molnár, K., Székely, C., Hallett, S.L. and Atkinson, S.D. (2009). Some remarks on the occurrence, host-specificity and validity of Myxobolus rotundus Nemeczek, 1911 (Myxozoa: Myxosporea). Systematic Parasitology 72, 71–79.

Page | 158

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Molnár, K., Eszterbauer, E., Marton, S., Székely, C. and Eiras, J.C. (2012). Comparison of the Myxobolus fauna of common barbel from Hungary and Iberian barbel from Portugal. Diseases of Aquatic Organisms 100, 231–248. Naidenova, N.N., Schulman, S.S. and Donets, Z.S. (1975). Type Protozoa, class Myxosporidia. In Guide of Parasites of Vertebrates of Black and Azov Seas. Naukova Dumka, Kiev, pp. 20–50. Naldoni, J., Zatti, S.A., Capodifoglio, K.R., Milanin, T., Maia, A.A., Silva, M.R. and Adriano, E.A. (2015). Host-parasite and phylogenetic relationships of Myxobolus filamentum sp. n. (Myxozoa: Myxosporea), a parasite of Brycon orthotaenia (Characiformes: Bryconidae) in Brazil. Folia Parasitologica 62, 014. Narasimhamurti, C.C. and Kalavati, C. (1979). Myxosoma lairdi n. sp. (Protozoa: Myxosporidia) parasitic in the gut of the estuarine fish, Liza macrolepis Smith. Proceedings of the Indian Academy of Science 88, 269–272. Narasimhamurti, C., Kalavati, C. and Saratchandra, B. (1980). Myxosoma microspora n. sp. (Myxosporidia: Protozoa) parasitic in the gills of Mugil cephalus L. Journal of Fish Biology 16, 345–348. Özak, A.A., Demirkale, I. and Cengizler, I. (2012). Two new records of Myxobolus Bütschli, 1882 (Myxozoa, Myxosporea, Myxobolidae) species from Turkey. Turkish Journal of Zoology 36, 191–199. Özer, A. and Kirca, D.Y. (2015). Parasite fauna of the grey mullet Mugil cephalus L. 1758, and its relationship with some ecological factors in Lower Kizilirmak Delta located by the Black Sea, Turkey. Journal of Natural History 49, 933–956. Özer, A., Gürkanli, C.T., Özkan, H., Acar, G., Ciftci, Y. and Yurakhno, V. (2016b). Molecular characterization and morphological aspects of Myxobolus parvus (Myxozoa) from Liza saliens (Mugilidae) off the Turkish Black Sea coasts. Parasitology Research 115, 3513–3518. Özer, A., Özkan, H., Gurkanli, C.T., Yurakhno, V. and Ciftci, Y. (2016a). Morphology, histology and phylogeny of Henneguya sinova sp. nov. (Myxobolidae: Myxozoa) infecting gills of Parablennius tentacularis in the Black Sea, Turkey. Diseases of Aquatic Organisms 118, 207–215. Rocha, S., Casal, G., Matos, P., Matos, E., Dkhil, M. and Azevedo, C. (2011). Description of Triangulamyxa psittaca sp. nov. (Myxozoa; Myxosporea), a new parasite in the urinary bladder of Colomesus psittacus (Teleostei) from the Amazon River, with emphasis on the ultrastructure of plasmodial stages. Acta Protozoologica 50, 327–338. Rocha, S., Casal, G., Rangel, L., Severino, R., Castro, R., Azevedo, C. and Santos, M.J. (2013). Ultrastructural and phylogenetic description of Zschokkella auratis sp. nov. (Myxozoa), a parasite of the gilthead seabream Sparus aurata. Diseases of Aquatic

Page | 159

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Organisms 107, 19–30. Rocha, S., Casal, G., Garcia, P., Matos, E., Al-Quraishy, S. and Azevedo, C. (2014a). Ultrastructure and phylogeny of the parasite Henneguya carolina sp. nov. (Myxozoa), from the marine fish Trachinotus carolinus in Brazil. Diseases of Aquatic Organisms 112, 139–148. Rocha, S., Casal, G., Al-Quraishy, S. and Azevedo, C. (2014b). Morphological and ultrastructural redescription of Chloromyxum leydigi Mingazzini, 1890 (Myxozoa: Myxosporea), type species of the genus, infecting the gall bladder of the marine cartilaginous fish Torpedo marmorata Risso (Chondrichthyes: Torpedinidae), from the Portuguese Atlantic Coast. Folia Parasitologica 61, 1–2. Rocha, S., Casal, G., Rangel, L., Castro, R., Severino, R., Azevedo, C. and Santos, M.J. (2015). Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae). Parasitology International 64, 305–313. Ronquist, F. and Huelsenbeck, J.P. (2003). Mrbayes3: Bayesian phylogenetic inference under mixed models. Bioinformatics (Oxford, England) 19, 1572–1574. Saleh, M.A. (2006). Cultured Aquatic Species Information Programme (CASIP). Mugil cephalus. Rome, Italy: FAO. Sarabeev, V. (2015). Helminth species richness of introduced and native grey mullets (Teleostei: Mugilidae). Parasitology International 64, 6–17. Sarkar, N.K. (1989). Myxobolus anili sp. nov. (Myxozoa: Myxosporea) from a marine teleost fish Rhinomugil corsula Hamilton. Proceedings of the Zoological Society of Calcutta 42, 71–74. Sarkar, N.K. (1999). Some new Myxosporidia (Myxozoa: Myxosporea) of the genera Myxobolus Bütschli, 1882, Unicapsula Davis, 1942, Kudoa Meglitsch, 1947, Ortholinea Shulman, 1962 and Neoparvicapsula Gajevskaya, Kovaleva and Shulman, 1982. Proceedings of the Zoological Society of Calcutta 52, 38–48. Shulman, S.S. (1966). Myxosporidia of the USSR. Moscow-Leningrad: Nauka, pp. 504. Shulman, S.S. (1984). Parasitic Protozoa. In Bauer, O.N. (ed). Key to the Parasites of Freshwater Fauna of USSR. Nauka, Leningrad, pp. 428. Siau, Y. (1978). Contribution à la connaissance des Myxosporidies: étude de Myxobolus exiguus Thélohan, 1895 (Cytologie, Cycle, Actions sur l’hôte, Epidémiologie). Thèse d’État, Université des Sciences et Techniques du Languedoc, Montpellier, pp. 153. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1993). Zschokkella mugilis n. sp. (Myxosporea: Bivalvulida) from mullets (Teleostei: Mugilidae) of Mediterranean waters: light and electron microscopic description. Journal of Eukaryotic Microbiology 40, 755–764.

Page | 160

Chapter VI | Myxobolids from Mugiliformes and re-description of M. exiguus

Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (2001). Leptotheca sparidarum n. sp. (Myxosporea: Bivalvulida), a parasite from cultured common dentex (Dentex dentex L.) and gilthead sea bream (Sparus aurata L.) (Teleostei: Sparidae). Journal of Eukaryotic Microbiology 42, 627–639. Székely, C., Hallett, S.L., Atkinson, S.D. and Molnár, K. (2009). Complete life cycle of Myxobolus rotundus (Myxosporea: Myxobolidae), a gill myxozoan of common bream Abramis brama. Diseases of Aquatic Organisms 85, 147–155. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30, 2725– 2729. Thélohan, P. (1895). Recherches sur les Myxosporidies. Bulletin Scientifique de la France et de la Belgique 4, 100–394. Umur, S., Pekmezci, G.Z., Beyhan, Y.E., Gurler, A.T. and Acici, M. (2010). First record of Myxobolus muelleri (Myxosporea: Myxobolidae) in flathead grey mullet Mugil cephalus (Teleostei, Mugilidae) from Turkey. Ankara Üniversitesi Veteriner Fakültesi 57, 205– 207. U-taynapun, K., Penprapai, N., Bangrak, P., Mekata, T., Itami T. and Tantikitti, C. (2011). Myxobolus supamattayai n. sp. (Myxosporea: Myxobolidae) from Thailand parasitizing the scale pellicle of wild mullet (Valamugil seheli). Parasitology Research 109, 81–91. Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215–219. Yemmen, C., Ktari, M.H. and Bahri, S. (2012). Parasitofauna of some mugilid and soleid fish species from Tunisian lagoons. Acta Adriatica 52, 173–182. Yurakhno, V.M. (2004). The fauna of myxosporeans (Protozoa: Myxosporea) of fishes in the Black Sea and its seasonal and interannual aspects of variability. In Nigmatullin, C.M. (ed). Sovremennyje Problemy Parazitologii, Zoologii i Ekologii. KGTU, Kaliningrad, pp. 160–171. Yurakhno, V.M. and Maltsev, V.N. (2002). New data on myxosporeans of mullets in the Atlantic Ocean basin. Ekologiya Morya 61, 39–42. Yurakhno, V.M. and Ovcharenko, M.O. (2014). Study of Myxosporea (Myxozoa), infecting worldwide mullets with description of a new species. Parasitology Research 113, 3661– 3674. Zetina-Rejón, M.J., Arreguín-Sánchez, F. and Chávez, E.A. (2003). Trophic structure and flows of energy in the Huizache–Caimanero lagoon complex on the Pacific coast of Mexico. Estuarine, Coastal and Shelf Science 57, 803–815.

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Zhang, J.Y., Wang, J.G., Li, A.H. and Gong, X.N. (2010). Infection of Myxobolus turpisrotundus sp. n. in allogynogenetic gibel carp, Carassius auratus gibelio (Bloch), with revision of Myxobolus rotundus (s. l.) Nemeczek reported from C. auratus auratus (L.). Journal of Fish Diseases 33, 625–638. Zhang, J.Y., Al-Quraishy, S. and Abdel-Baki, A.A. (2014). The morphological and molecular characterization of Myxobolus khaliji n. sp. (Myxozoa: Myxosporea) from the double bar seabream Acanthopagrus bifasciatus (Forsskal, 1775) in the Arabian Gulf, Saudi Arabia. Parasitology Research 113, 2177–2183.

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Chapter VII

Description of a new actinosporean prompts union of the raabeia and echinactinomyxon collective groups (Cnidaria, Myxozoa), due to overlap in actinospore morphology

This chapter was adapted from:

Chapter VII | Union of raabeia and echinactinomyxon collective groups

Abstract

A new actinosporean is morphologically and molecularly described from the intestinal epithelium of the freshwater oligochaete Ilyodrilus templetoni in the upper estuary of the River Minho, Northern Portugal. Mature actinospores resembled both echinactinomyxon and raabeia types, emphasizing the already known lack of a clear boundary between these two collective groups. Historically, raabeia and echinactinomyxon types have been differentiated solely based on the shape of the valvular processes being curved or straight, respectively. Our observations, however, show that this morphological character is too variable for distinguishing between these two collective groups, since the actinospores of the raabeia described here displayed valvular processes that could either be straight, downward or upward curved. Several similar cases can be found in the available literature. Considering this overlap in actinospore morphology, we propose that echinactinomyxon be deemed invalid and its types be included in raabeia, as the latter constitutes the oldest group among the two. Known echinactinomyxon types, however, should not be renamed as raabeia, as this would create unnecessary confusion. Accordingly, a more comprehensive definition of the raabeia collective group is provided. Phylogenetic analyses reveal polyphyletic clustering of raabeia/echinactinomyxon types among members of the myxosporean suborders Variisporina and Platysporina, reiterating the lack of agreement between actinosporean morphotypes and myxosporean genera. The new type described here specifically clusters within the Paramyxidium clade, alongside other SSU rDNA sequences of raabeia, echinactinomyxon, aurantiactinomyxon and synactinomyxon. Considering that most Paramyxidium spp. parasitize Anguilla anguilla (Linnaeus, 1758), future myxozoan surveys in the River Minho should include this species.

Introduction

Actinospores were first reported by Štolc (1899), who described these organisms as parasites related to myxosporeans, but representing an independent taxonomic entity – the class Actinosporea. During the decades that followed, few studies provided information regarding the morphology, ecology and systematics of actinosporeans, possibly because they were thought to have little economic significance. Also, because prevalence of infection is typically low, actinosporean surveys demand a considerable effort in collecting and examining potential hosts. Research on this field only gathered momentum after Wolf and Markiw (1984) demonstrated that actinosporeans corresponded to fish-parasitic myxosporean stages developing in annelids. Since then, several other studies have produced experimental and molecular evidence for the alternation of two myxosporean life cycle stages: a myxosporean

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Chapter VII | Union of raabeia and echinactinomyxon collective groups stage that mainly develops in fish; and an actinosporean stage that develops in an oligochaete or polychaete (Eszterbauer et al., 2015). Relying on evidences provided by initial studies of experimental transmission (El-Matbouli and Hoffmann, 1989; Ruidisch et al., 1991; El-Matbouli et al., 1992; Grossheider and Körting, 1992; El-Matbouli and Hoffmann, 1993; Kent et al., 1993; Yokoyama et al., 1993a), Kent et al. (1994) proposed the demise of the class Actinosporea and further suggested that its genera be used as vernacular designations for typing actinospores developing in annelid hosts. About 200 actinosporean types are currently comprised within ca. 20 collective groups (Lom and Dyková, 2006), however, this number is expected to increase given its discrepancy to the much larger number of known myxosporeans. It is therefore vital that actinosporeans be properly characterized, so that they may be accurately identified in the future. Furthermore, annelids have been shown to be the definitive and most ancient hosts of myxosporeans (Holzer et al., 2018); thus, recognizing the morphological, biological and phylogenetic patterns of actinosporeans is most certainly important for clarifying systematics and understanding the evolution of Myxosporea. For a long time, actinosporean reports were solely based on light microscopy observations and schematic drawings, leading to poor initial descriptions that lacked reliable data for comparative purposes. Acknowledging this problem, Lom et al. (1997a) provided guidelines for the uniform characterization of actinosporeans, following the morphological and morphometric traits of the actinospores and other developmental stages. The implementation of molecular methodologies to the study of these parasites, however, has revealed that morphological criteria are unreliable for the identification and differentiation of actinosporeans. In fact, studies show that several types display significant morphological variability among isolates (Hallett et al., 2004; Eszterbauer et al., 2006; Atkinson et al., 2009), while different types of the same collective group overlap in terms of measurements (Rangel et al., 2016). Nowadays, descriptions have come to rely on a combination of morphological and molecular features for the unambiguous characterization of new and known types (e.g. Rosser et al., 2014; Xi et al., 2015, 2017; Rangel et al., 2016; Rocha et al., 2019a). Among the earlier reports of actinosporeans are the studies performed by Janiszewska (1955, 1957), in which the raabeia and echinactinomyxon collective groups were erected as genera of the former class Actinosporea. To date, ca. 34 types of raabeia have been reported, four of which are directly involved in the life cycles of fish-infecting myxosporeans, more specifically Myxobolus cultus Yokoyama et al., 1995, Myxobolus dispar Thélohan, 1895, Myxobolus lentisuturalis Dyková et al., 2002 and Myxidium truttae (Léger, 1930) (see Yokoyama et al., 1995; Molnár et al., 1999; Holzer et al., 2004; Eszterbauer et al., 2006; Caffara et al., 2009). About 25 types lack molecular data, including the type that was experimentally shown to be the actinosporean counterpart of M. dispar (see Molnár et al., 1999). The remaining 9 types have SSU rDNA sequences available in GenBank, acquired

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Chapter VII | Union of raabeia and echinactinomyxon collective groups either from their original descriptions (Caffara et al., 2009; Borkhanuddin et al., 2014) or from later reports (Kent et al., 2001; Holzer et al., 2004; Eszterbauer et al., 2006; Hallett et al., 2006; Xi et al., 2013). In turn, the echinactinomyxon collective group encompasses about 23 types, two of which were shown to be involved in the life cycle of Myxobolus pavlovskii (Achmerov, 1954) (see Marton and Eszterbauer, 2011) and Myxobolus wulii (Wu and Li, 1986) Landsberg and Lom, 1991 (see Xi et al., 2015). Only the two latter types, Echinactinomyxon radiatum and the Echinactinomyxon types 1 and 5 of Özer et al. (2002) have molecular data available in GenBank. In this study, a myxozoan survey was performed in freshwater and marine oligochaete communities inhabiting a Northern Portuguese River. Among the different collective groups recorded, raabeia was represented by one new type that is morphologically and molecularly described herein from a freshwater oligochaete of the upper estuary. The validity of using the shape of the valvular processes as the sole morphological character for distinguishing between raabeia and echinactinomyxon is discussed considering the available literature, the overlap in actinospore morphology, and the phylogenetic data currently available. Consequently, the unification of echinactinomyxon and raabeia types within the latter collective group is proposed.

Materials and methods

Sampling sites and morphological characterization

Between 2015 and 2016, mud collections were performed from two distinct sites of the River Minho estuary, northwest Portugal. One sampling site was located in the upper estuary, close to the fyke-nets stationed near Vila Nova de Cerveira (41° 56′ N, 08° 45′ W), while the other sampling site was located in the lower estuary, near Caminha (41° 52' N, 08° 50' W). A Van Veen grab sediment sampler with an area of 500 cm2 and a maximum capacity of 5,000 cm3 was used to collect mud from the sampling location in the upper estuary, while mud from the lower estuary was collected manually at low tide. In the laboratory, oligochaetes were isolated from the mud and kept individually in 12-well plates at 4 ºC. This temperature was used solely to optimize oligochaete preservation; water temperature at both sampling sites varied throughout the year, being lowest during winter months (January to March) and highest during summer months (July to September). In the upper estuary, water temperature ranges from 10 ºC in the winter to a maximum of 19.5 ºC in the summer; while in the lower estuary, the water thermal amplitude is smaller, ranging from 10.9 ºC in the winter to 16 ºC in the summer (unpublished data). Well plates were either filled with fresh- or brackish water (15‰ salinity), in accordance to the sampling site from which the oligochaetes were obtained. Salinity

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Chapter VII | Union of raabeia and echinactinomyxon collective groups values at the fyke-nets are generally below 0.5‰, with a slight increase during the summer period. In turn, salinity values in the lower estuary range between 15 and 40‰ throughout the year (Dias et al., 2016). Following the collection of oligochaetes from the mud samples, which generally took 3 to 4 days to perform, each specimen was examined using a light microscope for the detection of actinosporean stages in internal tissues and cavities. Developmental stages and free actinospores were examined and photographed using an Olympus BX41 light microscope (Olympus, Japan). Morphometry was determined from fresh actinospores, according to the guidelines provided by Lom et al. (1997a). Measurements are given as mean value ± standard deviation (range of variation), followed by the number of actinospores measured (n).

Transmission electron microscopy

Partial portions of infected oligochaetes displaying mature actinospores were fixed in 3% glutaraldehyde buffered in 0.2 M sodium cacodylate (pH 7.4) for 4 h. A wash was performed in the same buffer prior to post-fixation in 2% osmium tetroxide also buffered with 0.2 M sodium cacodylate for 1 h. Both fixations were performed at 4 ºC. An ascending graded series of ethanol was used to dehydrate the samples. Infiltration was achieved using a series of propylene oxide and EPON mixtures, and was followed by embedding in EPON. Semithin sections (~1 µm thick) were stained with methylene blue-Azure II, while ultrathin sections (50‒ 70 nm) were double-contrasted using uranyl acetate and lead citrate. Ultrathin sections were observed and photographed with an electron microscope JEOL 100CXII TEM (JEOL Ltd., Tokyo) operated at 60 kV and equipped with a Gatan Inc. camera (model 830 J01w44) with DigitalMicrograph (version 230.542.0) software (Gatan Inc., USA).

Molecular characterization

Genomic DNA was extracted from whole or partial portions of oligochaetes displaying myxozoan infection, using the GenEluteTM Mammalian Genomic DNA Miniprep Kit (Sigma- Aldrich, St Louis, USA) according to the manufacturer’s instructions. Amplification of the SSU rDNA gene of the actinospores was performed using universal and myxosporean-specific primers (Table 1). PCRs were carried out in 50 µl reaction mixtures comprising 3 µl (approximately 100–150 ng) of extracted DNA, 10 pmol of each primer, 10 nmol of each dNTP,

2.0 mM of MgCl2, 5 µl 10× Taq polymerase buffer, 1.5 units Taq DNA polymerase (NZYTech, Lisbon, Portugal) and 36.5 µl of water. The PCR reactions were run on a Hybaid PxE Thermocycler (Thermo Electron Corporation, Waltham, Massachusetts, USA), with initial denaturation at 95 ºC for 3 min, followed by 35 cycles of 94 ºC for 45 sec, 53 ºC for 45 sec,

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Table 1. Polymerase chain reaction primers used for the amplification and sequencing of the SSU rDNA gene.

Name Sequence (5’-3’) Paired with Source

18E CTG GTT GAT CCT GCC AGT MyxospecR, MYX4R Hillis and Dixon, 1991

MyxospecF TTC TGC CCT ATC AAC TTG TTG MYX4R, 18R Fiala, 2006

MYX4F GTT CGT GGA GTG ATC TGT CAG 18R Rocha et al., 2015

MyxospecR CAA CAA GTT GAT AGG GCA GAA 18E Fiala, 2006

MYX4R CTG ACA GAT CAC TCC ACG AAC 18E, MyxospecF Hallett and Diamant, 2001

18R CTA CGG AAA CCT TGT TAC G MyxospecF, MYX4F Whipps et al., 2003 and 72 ºC for 90 sec. The final elongation step was performed at 72 ºC for 7 min. In turn, the 16S mitochondrial DNA (mtDNA) gene of the oligochaete hosts was amplified with the primers 16sar-L (5’-CGC CTG TTT ATC AAA AAC AT-3’) and 16sbr-H (5’-CCG GTC TGA ACT CAG ATC ACG T-3’) (Palumbi et al., 2002), using the conditions and PCR profile previously mentioned for the actinospores. The PCR products were electrophoresed through a 1% agarose 1× tris-acetate-EDTA buffer (TAE) gel stained with ethidium bromide. Amplified DNA was purified using Puramag™ magnetic beads coated with carboxylic acid groups (MCLAB, San Francisco, California, USA). Sequencing reactions were performed with the same primers used for amplification, using a BigDye Terminator v3.1 Cycle Sequencing Kit from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, EUA), and were run on an ABI3700 DNA analyzer from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, EUA).

Phylogenetic analysis

The partial sequences obtained for each infected oligochaete specimen were separately assembled into corresponding consensus SSU rDNA sequences in MEGA7 (Kumar et al., 2016). All consensus sequences obtained were aligned using the software MAFFT version 7 available online and compared in MEGA7, in order to identify possible correspondences amongst them. A dataset was generated for the type in study, according to the highest similarity scores obtained using BLAST search and, consequently, included all known Paramyxidium spp. and closely related sequences of the aurantiactinomyxon, echinactinomyxon, raabeia and synactinomyxon collective groups. More distantly related SSU rDNA sequences of echinactinomyxon and raabeia types were further included in this dataset, which was aligned using the software MAFFT version 7 available online. Distance estimation was calculated in MEGA7, with the p-distance model and all ambiguous positions removed for each sequence pair. For phylogenetic analyses, representatives of the main subclades of the myxosporean freshwater lineage were further included in the dataset; Chloromyxum leydigi Mingazzini, 1890

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(DQ377710) and Chloromyxum riorajum Azevedo et al., 2009 (FJ624481) were selected as outgroup. In total, 85 SSU rDNA sequences were aligned using the software MAFFT version 7 available online, and posteriorly manually edited in MEGA7. Phylogenetic trees were obtained using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP). BI analyses were conducted in MrBayes v3.2.6 (Ronquist and Huelsenbeck, 2003). The modeltest algorithm of the software showed the general time reversible model with gamma- shaped rate variations across sites (Invgamma) (GTR+I+Γ) as the best-fit evolutionary model for BI analyses. Posterior probability distributions were generated using the Markov Chain Monte Carlo (MCMC) method. Four chains runned simultaneously for 500,000 generations, with burn-in set at 25%, and trees sampled every 100 generations. ML and MP were conducted in MEGA7, with bootstrap confidence values calculated from 500 replicates. ML analyses were performed using the General Time Reversible model with Gamma distributed rate and Invariant sites (GTR + G + I), selected on the basis of the lowest score of Bayesian Information Criterion (BIC) and corrected Akaike Information Criterion (AIC). MP analyses were obtained using the Subtree-Pruning-Regrafting algorithm with a search level of 1 and random initial tree addition of 10 replicates.

Results

Myxozoan survey

During this study, 4,593 oligochaete specimens were collected from the River Minho estuary and screened for myxozoan infection: 4,016 from the location near the fyke-nets in the upper estuary, and 577 from the lower estuary near Caminha. Thirteen actinosporean types were morphologically and molecularly differentiated from a total of 61 oligochaetes displaying myxozoan infection; these could be assigned to 4 collective groups: sphaeractinomyxon (6 types), aurantiactinomyxon (4 types), synactinomyxon (2 types), and raabeia (1 type). Four freshwater tubificoid naidids [Limnodrilus hoffmeisteri Claparède, 1862, Ilyodrilus templetoni (Southern, 1909), a Potamothrix sp. and Psammorycti- des barbatus (Grube, 1861)] were identified as the host oligochaetes in the upper estuary, while the marine oligochaete Tubificoides pseudogaster (Dahl, 1960) was the only species found to be infected in the lower estuary. The description of the raabeia type is presented herein; descriptions of the remaining types will be published elsewhere. Actinospores of the raabeia collective group were found developing in the intestinal epithelium of 15 specimens out of the above-mentioned 61 oligochaetes displaying myxozoan infection. The DNA sequence of raabeia obtained from these specimens was identical throughout their entire length, which varied from 1,972 bp to 2,066 bp. Five additional

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Chapter VII | Union of raabeia and echinactinomyxon collective groups specimens displaying only young morphologically unidentifiable developmental stages were also determined to be infected by this genotype. Thus, a new raabeia type is characterized herein, based on actinospore morphology and molecular data of the SSU rDNA gene acquired from a total of 20 infected specimens. Infection by this type was present throughout the year in the upper estuary but was never detected in the oligochaete specimens collected from the lower estuary. Combined morphological and molecular data of the 16S mitochondrial DNA gene allowed identification of all host specimens of the new raabeia type as belonging to the freshwater oligochaete species Ilyodrilus templetoni.

Characterization of a novel Raabeia type (Cnidaria, Myxozoa) (Figs. 1‒4)

Description: Mature actinospores cylindrical and elongated in lateral view (Fig. 1A), with 3 equally sized valvular processes originating just below the actinospore body without curving, each displaying a single ovoid nucleus near the base. Valvular processes either straight, upward or downward curved, and gradually tapering toward pointed tips (Fig. 1B‒H). Actinospore body 23.9 ± 1.3 (22.0‒26.1) µm long (n = 30) and 12.4 ± 0.8 (11.3‒14.0) µm wide (n = 30); valvular processes 108.0 ± 3.5 (101.7‒113.6) µm long (n = 30) and 6.2 ± 0.5 (5.4‒ 7.3) µm wide (n = 30). Three prominent polar capsules protruding from the anterior portion of the actinospores, pyriform and equally sized, 5.1 ± 0.4 (4.4‒6.0) µm long (n = 30) and 3.6 ± 0.3 (2.9‒4.0) µm wide (n = 30), each containing a polar tubule displaying 5 to 6 longitudinal coils (Fig. 1D). Sporoplasm containing ca. 12 secondary cells. Actinospore morphology is depicted in a schematic drawing (Fig. 2). Ultrastructure of the sporogonic stage: Pansporocysts in different developmental stages located in the intestinal epithelium (Fig. 3). Disorganization of the intestinal epithelium was observed near late pansporocysts, with aspects of invasion of the coelomic cavity (Fig. 4A). Early pansporocysts formed by thin somatic cells surrounding 8 zygotes that display few cellular differentiation (Fig. 4A, B). Late pansporocysts containing developing sporoblasts formed by 3 valvogenic cells that surround 3 capsulogenic cells at the anterior portion (Fig. 4C‒E) and one sporoplasmic cell at the posterior portion (Fig. 4F, G). Each capsulogenic cell differentiates into a polar capsule comprised by a double-layered wall and an internal polar tubule coiling longitudinally (Fig. 4D). At the apex of each polar capsule, a conical structure, designated stopper, protrudes from the valvogenic cells and is aligned with a permanent open pore in the actinospore involucre (Fig. 4E). Valvogenic cells differentiate into long and flattened valvular processes, telescopically folded, and devoid of cytoplasmic content (Fig. 4F, G). Sporoplasm containing several electron-dense secondary cells (Fig. 4H). At the end of sporogony, pansporocysts contain 8 mature actinospores. Type host: Ilyodrilus templetoni (Southern, 1909) (Annelida, Oligochaeta).

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Figure 1. Light micrographs of the actinospores of the new type of raabeia found infecting the freshwater oligochaete Ilyodrilus templetoni in the upper estuary of the River Minho: (A) Ruptured pansporocyst showing the 8 actinospores that it previously contained; (B) Actinospores in lateral view; (C) Actinospore in apical view; (D) Detailed aspect of the three polar capsules protruding from the apex of the actinospore body, as observed in apical view; (E‒H) Actinospores evidencing the shape of the valvular processes varying from straight and “rigid” (F) to upward curved (E, G) and downward curved (H).

Type locality: The upper estuary of the River Minho, near Vila Nova de Cerveira (41° 56′ N, 08° 45′ W), Portugal. Site of infection: The intestinal epithelium.

Prevalence: 0.5% (20 infected in a total of 4,016 oligochaetes examined from the upper estuary).

Deposition of material: Series of phototypes deposited together with a representative

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Figure 2. Schematic drawings depicting mature actinospores of the new type of raabeia found infecting the freshwater oligochaete Ilyodrilus templetoni in the upper estuary of the River Minho estuary, as observed in lateral view. The valvular processes either appeared straight (A), upward curved (B) or downward curved (C).

Figure 3. Semithin sections evidencing the asynchronous pansporocyst development of the new type of raabeia in the intestinal epithelium of the freshwater oligochaete Ilyodrilus templetoni from the upper estuary of the River Minho: (A, B) Intestinal epithelium (IE); intestinal lumen (IL); early pansporocyst (EP); late pansporocyst (LP).

DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2019.26. Molecular data: One SSU rDNA sequence with a total of 2,066 bp, representative of the identical consensus sequences separately obtained from the parasitic material in the intestinal

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Figure 4. Transmission electron micrographs of the sporogonic development of the new type of raabeia found infecting the freshwater oligochaete Ilyodrilus templetoni in the upper estuary of the River Minho: (A) Zygote cells (asterisk) inside an early pansporocyst (EP); intestinal epithelium (IE); intestinal lumen (IL); (B) Early pansporocyst containing zygotes and displaying the somatic cells (SC) comprising its periphery; (C) Transverse section of the apex of the actinospore body, showing the valvogenic cells (VC) surrounding three capsulogenic cells (CC), each containing an almost fully matured polar capsule (PC); (D, E) Detailed aspects of the polar capsules (PC) showing the polar tubule (PT) coiling along the inner wall and the conical stopper (*) at the apex, near the suture line (double arrows). Notice the nuclei (Nu) of the capsulogenic cells; (F) Posterior portion of the body of two actinospores evidencing the sporoplasmic cell (S) containing several secondary electron-dense cells; nuclei (Nu); valvular processes telescopically folded (arrows); (G) Transverse section of the posterior portion of the actinospore body showing the suture line (double arrows) formed by the union of the valvogenic cells, the sporoplasmic cell (S) and the valvular processes telescopically folded (arrows); (H) Longitudinal section of a mature actinospore body showing the sporoplasm (S) and 2 of the 3 polar capsules (PC) surrounded by the valves (V).

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Chapter VII | Union of raabeia and echinactinomyxon collective groups epithelium of 20 infected oligochaetes, deposited in GenBank with the accession no. MK550896. Remarks: Morphometry was determined from mature actinospores observed in 15 out of the 20 hosts that displayed infection by this type. Morphological comparison revealed no gross similarity to raabeia and echinactinomyxon types that are without molecular data of the SSU rDNA gene (see Table 2). Despite the overall measurements of the actinospores falling within the large morphometric range of the Echinactinomyxon type of Marcucci et al. (2009), the latter differs by having smaller polar capsules and larger valvular processes that form four short branches at the distal end. In terms of shape, the actinospores in our study best resemble those of Echinactinomyxon radiatum, however differing from the latter in several morphometric aspects and molecular data. The SSU rDNA sequence of the parasite did not match any of the myxozoan sequences currently available in GenBank. Distance estimation revealed closest proximity to the sequences of Paramyxidium, aurantiactinomyxon, echinactinomyxon, raabeia and synactinomyxon comprising the Paramyxidium clade (Fig. 5), with similarity values that ranged between 89.3% to P. magi (MH414927) and 82.1% to P. bulani (MH414929).

Phylogenetic analysis

Phylogenetic analyses produced similar tree topologies irrespective of the methodology used. Raabeia and echinactinomyxon types appeared distributed among the different clades represented in the tree topology, spanning over myxosporean genera belonging to the bivalvulid suborders Variisporina and Platysporina (Fig. 5). The Raabeia type described here clustered among Variisporina, more specifically within the well-supported Paramyxidium clade, at the basis of a group comprising the Raabeia type 4 of Özer et al. (2002), the Echinactinomyxon type 1 of Özer et al. (2002), several members of the aurantiactinomyxon and synactinomyxon collective groups, and most Paramyxidium spp. The only sequence of the Paramyxidium clade not included in this group was Paramyxidium bulani (MH414929), which occupied the most basal positioning to all others (including the new Raabeia type). The SSU rDNA sequence of Echinactinomyxon radiatum also clustered among Variisporina, namely within the Myxidium lieberkuehni clade, which formed a well-supported sister group to a clade of Chloromyxum spp. Together these formed a robustly supported sister clade to the Paramyxidium. The Platysporina, represented in the three topology by the clade of myxobolids and the heterogenic freshwater urinary clade, formed a well-supported sister clade to this grouping of the Paramyxidium, Myxidium lieberkuehni and Chloromyxum clades. Most raabeia and echinactinomyxon types clustered among Platysporina, specifically within the clade of myxobolids, being conspecific with Myxobolus dispar, M. pavlovskii, M. wulii, M. cultus and M. lentisuturalis, or appearing alongside other members of this genus and sequences of

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Figure 5. Bayesian Inference tree topology of the SSU rDNA sequences of 83 selected myxosporeans, rooted to Chloromyxum leydigi and C. riorajum. Numbers at the nodes represent BI posterior probabilities and ML/MP bootstrap values; asterisks indicate full support in all three methodologies; dashes represent poorly resolved nodes or a different branching pattern in the ML and MP tree topologies. Sequences of raabeia and echinactinomyxon types are presented in bold; with the sequence of new type marked white within a dark grey box.

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Henneguya. Lastly, the Raabeia counterpart of Myxidium truttae clustered together with the Raabeia type 1 of Oumouna et al. (2003) within a clade comprising representatives of several genera of Variisporina (e.g. Cystodiscus, Myxidium, Sphaeromyxa and Zschokkella) that infect the hepatic biliary system, and that is basal to all others included in the tree topology. The Raabeia type B of Xiao and Desser (1998a) appeared positioned alone at the basis of this latter clade.

Amended definition of raabeia Janiszewska, 1955

Actinospore body spherical to subspherical, ellipsoidal or cylindrical, with three polar capsules protruding from the apex. Three elongated valvular processes arise from the actinospore body without a style, being straight or curved, tapering to a single sharp point or forming multiple branches at the distal end (see some examples in Fig. 6). Known as counterparts in the life cycles of Myxobolus cultus, M. dispar, M. lentisuturalis, M. pavlovskii, M. wulii and Myxidium truttae. The original type is Raabeia gorlicensis Janiszewska, 1955 from the freshwater oligochaete Tubifex tubifex in Poland. Thus far, 58 types have been identified from this collective group [35 described as raabeia (including the type presented herein) and 23 originally described as members of echinactinomyxon], and all have a freshwater oligochaete as host. A summary of all raabeia/echinactinomyxon types is provided in Table 2.

Discussion

A new actinosporean type is morphologically and molecularly described herein from the intestinal epithelium of the freshwater oligochaete Ilyodrilus templetoni (Southern, 1909) inhabiting the upper estuary of the River Minho in Portugal. Molecular analyses determined infection by this type in 20 individuals, 15 of which displayed mature actinospores. Intraspecific variation was not detected among the SSU rDNA sequences obtained from infected specimens, as these were identical throughout their entire length. This agrees with the typical range of intraspecific variation that is commonly reported for myxosporeans, and that can be as low as 0% and as high as 3.6% (Schlegel et al., 1996; Andree et al., 1999; Ferguson et al., 2008). The prevalence of infection determined in this study (0.5%) also agrees with the commonly low prevalences (< 1%) reported for raabeia and echinactinomyxon types occurring in the wild (Xiao and Desser, 1998a, b; Negredo and Mulcahy, 2001; Székely et al., 2002). Few actinosporean surveys have detected higher values of prevalence of infection in the environment and attribute the difference to the re-examination of oligochaetes for extended periods of time (Yokoyama et al., 1993b; El-Mansy et al., 1998a, b; Marcucci et al., 2009), which was not performed in this study.

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Chapter VII | Union of raabeia and echinactinomyxon collective groups

Desser Desser

and

of Koprivnikar of

’

Raabeia type Raabeia

‘

c: number of polar tubule coils; SCn: number of secondary

inomyxon. SBL: actinospore body length; SBW: actinospore body width; LVP: length length LVP: width; body actinospore SBW: length; body actinospore SBL: inomyxon.

Summary of data available for raabeia and echinactinomyxon types, with the new type described here presented in bold. The The bold. in presented here described type new the with types, echinactinomyxon and raabeia for available data of Summary

; ET: experimental transmission; MI: molecular inference. Measurements are means ± SD (range) (when available), given in given (range) SD µm. means areavailable), (when ± Measurements inference. molecular MI: transmission; experimental ; ET:

Table 2. Table raabeia/echinact of features the with comply not does it that given included, not (2002)was of valvular processes; WVP: width of valvular processes; PCL: polar capsule length; PCW: polar capsule width; P cells

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Chapter VII | Union of raabeia and echinactinomyxon collective groups

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Chapter VII | Union of raabeia and echinactinomyxon collective groups

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Chapter VII | Union of raabeia and echinactinomyxon collective groups

le le Despite molecular studies sho- and

wing that morphological criteria are

an life cycle. life an highly unreliable for the differentiation Echinactinomyxon Echinactinomyxon and identification of actinosporeans

(e.g. Hallett et al. 2004, Eszterbauer et surements given here are surements given here are

ided in the body text. text. body in the ided al. 2006, Caffara et al. 2009, Xi et al.

collected from collected a goldfish farm in

found that the that found

2015), these remain as the sole form of comparison to the many known types

B. B. sowerbyi that are without available molecular

The The Echinactinomyxon type 2 of Negredo

information. Consequently, reliable

Holzer et al. (2004) al. et Holzer

Özer et Özer al. mea The (2002).

differrential diagnoses are difficult to owever,

H be performed, especially in relation to . .

early descriptions that provide insuffi-

2000)

, cient information for comparative pur-

There There is this in the of measurements disparity type the text between body

tten

poses. In this study, some morpholo-

Woo

and gical similarities to known types could

2009) from 2009) stages young developmental in be found; however, correspondence

Mulcahy Mulcahy (2001).

(see Özer Özer (see between types cannot be performed

and

with any confidence, given their distant

This type was originally by described Janiszewska (1964) as a member of the triactinomyxon collective 5

geographical habitats and different host species. There is no doubt that molecular studies are required in order

to identify known types from potential

Özer et al. (2002) morphologically identified their Echinactinomyxon type Özer 5 their the et as Echinactinomyxon identified al.to one previously morphologically reported be (2002) the cyc life 6

new isolates in different hosts or

geographic locations.

(1984). share less than 50% of their SSU rDNA sequences, demonstrating that they are not alternate stages of the same myxospore same the of stages alternate not are they that demonstrating sequences, rDNA SSU their of 50% than less share

Actinosporean types are current- tly classified into ca. 20 collective

S. truttae S. groups, most of which correspond to Scherl et al., 1986 by means of experimental transmission experimental of means by 1986 al., et Scherl

- former genera distinguished on the especially in the length of the valvular processes and size of the polar capsules. The measurements given here are those prov those are here given measurements The capsules. polar the of size and processes valvular the of length the in especially

basis of actinospore morphology. The

Fischer

Özer Özer et al. (2002) identified this type as potentially being identical to the Raabeia type of McGeorge et al. (1997).

3 actinospores of the type described

and the myxospores of of myxospores the and herein resembled both members of echinactinomyxon and raabeia, thus

emphasizing the lack of a clear

There is some disparity in the measurements of the polar capsules of these types between the There polar in some is these capsules of disparity types between bybody the text of and the measurements provided table

2 Sphaerospora truttae Sphaerospora distinctive boundary between these

two collective groups. Raabeia actino- Özer Özer et al. identified type this (2002) to as being the identical potentially type Raabeia 1 of Negredo

A SSU fourth rDNA sequence is available in for GenBank this type (AB121146), obtained by et Caffara al. ( spores are defined as having three

Bologna, Italy. Bologna, those provided in the body text. Mulcahy (2001) was also by reported the authors as bearing great similarity to E. radiatum. but group was by Marques tolater assigned echinactinomyxon of counterpart (2002) al. et Özer of 5 type (2002), al. et Özer by provided table and 1 7

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Chapter VII | Union of raabeia and echinactinomyxon collective groups long, pointed and curved valvular processes that arise from the “epispore” (now an outdated term) without a style (Janiszewska, 1955). Echinactinomyxon actinospores differ from the latter only in the rigidity of their valvular processes, which are characterized as being “spiny, straight, rigid” (Janiszewska, 1957). Our observations, however, clearly show that the shape of the valvular processes is too variable to allow distinction between raabeia and echinactinomyxon. Having observed hundreds of mature actinospores of the raabeia type described here from a total of 15 individual oligochaetes, it was possible to discern that the shape of the valvular processes varied from straight and “rigid” to being upward or downward curved. The ultrastructural study performed here further evidence that the valvular processes are devoid of cytoplasmic content that could sustain a permanent rigidity of these structures. Our observations are in accordance with previous transmission electron microscopy studies of raabeia and echinactinomyxon actinospores that report the valvular processes lacking cytoplasmic architecture, therefore appearing telescopically folded within the pansporocysts (see Lom et al., 1997b; Özer and Wootten, 2001; Marcucci et al., 2009). The difficult distinction between members of these two groups was first discussed by Hallett et al. (2006), who proposed that differential diagnoses involving either raabeia or echinactinomyxon types should include members of the other collective group. Indeed, some studies have since then followed this suggestion (e.g. Marcucci et al., 2009; Xi et al., 2015). While reviewing the literature, we found many cases in which the differentiation of actinospores as belonging to either the raabeia or the echinactinomyxon collective group was performed subjectively by the authors. For instance, Molnár et al. (1999) identified the actinosporean counterpart of M. dispar as belonging to the raabeia collective group. This identification was based on the branching pattern formed at the distal end of the valvular processes being similar to that previously reported for Raabeia furciligera. Nonetheless, the authors themselves stated that most actinospores displayed straight valvular processes resembling echinactinomyxon, with only a few having slightly curved processes. Similarly, the schematic representations provided for Raabeia furciligera by Marques (1984) suggest that the actinospores of this type also varied in the format of the valvular processes, which could be straight or slightly curved. In fact, several other raabeia types have valvular processes that only curve slightly at the distal end or almost do not curve (see Fig. 6; McGeorge et al., 1997; El-Mansy et al., 1998a; Özer et al., 2002; Oumouna et al., 2003; Hallett et al., 2006; Borkhanuddin et al., 2014). In the other way around, several echinactinomyxon types have valvular processes that curve slightly (see Fig. 6; Marques, 1984; Xiao and Desser, 1998b; Özer and Wootten, 2000; Xi et al., 2015). For instance, the Echinactinomyxon type C of Xiao and Desser (1998b) was described as occasionally displaying slightly curved valvular processes and is depicted that way in the schematic drawing provided in the description. Similarly, the valvular processes of the Echinactinomyxon type of Özer and Wootten (2000), erroneously reported as the life cycle

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Chapter VII | Union of raabeia and echinactinomyxon collective groups

Figure 6. Schematic drawings of several raabeia and echinactinomyxon types redrawn from their original descriptions and showing the shape of the valvular processes as being too variable to allow differentiation between these two collective groups: (A) Raabeia type 1 of Borkhanuddin et al. (2014); (B) Raabeia type 2 of Hallett et al. (2006); (C, D) Raabeia furciligera Janiszewska and Krzton, 1973; (E) Raabeia type 2 of Özer et al. (2002); (F) Echinactinomyxon type C of Xiao and Desser (1998b); (G) Echinactinomyxon type of Özer and Wootten (2000); (H) Echinactinomyxon type CZ of Xi et al. (2015); (I) Echinactinomyxon astilum (Janiszewska, 1964) Marques, 1984.

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Chapter VII | Union of raabeia and echinactinomyxon collective groups counterpart of Sphaerospora truttae Fischer-Scherl et al., 1986, were curved at the distal end (see Özer and Wootten, 2000; Özer et al., 2002; Holzer et al., 2004). Several studies further report the valvular processes of both echinactinomyxon and raabeia types forming branches at the distal end, as mentioned above. This morphological feature has been suggested to reflect a biological adaptation of the parasite to lotic environments, as it allows the formation of large actinospore aggregates that are more likely to intercept the host fish by floating in the water column or adhering to submerged material (Marcucci et al., 2009). This trait was first described from the actinospores of Raabeia furciligera (Janiszewska and Krztón, 1973), and since then reported for the raabeia counterpart of M. dispar (see Molnár et al. 1999), Echinactinomyxon type of Székely et al. (2002), Raabeia types 1, 2, 4, 5 and 6 of Özer et al. (2002) and Echinactinomyxon type of Marcucci et al. (2009), further blurring the distinction between these two groups. Considering all the above, it can be inferred that the morphological variability of the valvular processes is not discriminatory between collective groups, but rather it is representative of phenotypic variations within a given type. In fact, the occurrence of morphological variability among actinospores of the same type is not a novelty, with molecular studies having demonstrated that phenotypic differences can occur between isolates that share the same genotype (see Hallett et al., 2002; Eszterbauer et al., 2006). In light of the above, we suggest that echinactinomyxon be deemed invalid and its types be included in raabeia, as the latter constitutes the oldest group among the two. Known echinactinomyxon types, however, should not be renamed as raabeia, as this would create unnecessary confusion. Raabeia is henceforth defined as having a spherical to subspherical, ellipsoidal or cylindrical actinospore body with three polar capsules protruding from the apex. Three elongated valvular processes arise from the actinospore body without a style, being straight or curved, and tapering to a single sharp point or forming multiple branches at the distal end. It is further noted that the morphology of the type described by Koprivnikar and Desser (2002) does not comply with either the former or revised definition of the raabeia collective group, as its valvular processes do not tapper before branching. Consequently, its inclusion in a more suitable collective group is required, pending further investigation and acquisition of morphological and molecular data. Previous studies suggest that raabeia and echinactinomyxon have host specificity (Xiao and Desser, 1998b; Molnár et al., 1999). The few molecular data thus far acquired for their members concurs with this assumption, as no type has been reported to occur in more than one oligochaete host species. Congruently, the new type of raabeia described in this study was never detected in oligochaete hosts other than I. templetoni. Overall, the Echinactinomyxon type of Özer and Wootten (2000) and the Raabeia type 1 of El-Mansy et al. (1998a) constitute the only exceptions to the reported strict host specificity of these groups. The first was primarily described from Lumbriculus variegatus (Müller, 1774), but also Tubifex

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Chapter VII | Union of raabeia and echinactinomyxon collective groups tubifex (Müller, 1774), while the second was simultaneously reported from T. tubifex and Branchiura sowerbyi Beddard, 1892 (see El-Mansy et al., 1998a; Özer and Wootten, 2000). However, the occurrence of these two types of actinospores in their respective reported hosts requires validation via molecular data. About 34 types of raabeia and 23 types of echinactinomyxon are described in the literature, and all have a freshwater oligochaete as host. Accordingly, in this study, infection by raabeia was not detected in the marine oligochaetes examined from the lower estuary of the River Minho. The great majority of types belonging to these groups have been identified from infections occurring in individuals of T. tubifex and L. hoffmeisteri; with fewer types having been described from individuals of B. sowerbyi, Dero digitata (Müller, 1774), L. variegatus, Isochaetides michaelseni Lastočkin, 1937 and Rhyacodrilus komarovi Timm, 1990 (see Table 2). Either due to difficulties in the identification of the host species or because the actinospores were isolated from water, several other types lack information regarding their type host (see McGeorge et al., 1997; El-Mansy et al., 1998a; Özer et al., 2002; Oumouna et al., 2003; Hallett et al., 2006). A few others have their oligochaete hosts identified only up to the genus- or family-level (see Marques, 1984; Bellerud, 1993; Békési et al., 2002; Marcucci et al., 2009; Marton and Eszterbauer, 2011). With the exception of L. variegatus (Lumbriculidae) and an ocnerodrilid oligochaete reported to host the Raabeia type of Békési et al. (2002), all previously mentioned host species belong to the family Naididae. In this study, another species belonging to this family, I. templetoni, is further recognized as a potential host for raabeia types. The broad use of naidids as definitive hosts suggests that the family Naididae played a preponderant role in the establishment of these parasites in distinct freshwater habitats and geographic locations. To date, members of raabeia, and echinactinomyxon alike, have mostly been linked to the life cycle of Myxobolus spp. that infect Cypriniformes (Yokoyama et al., 1995; Molnár et al., 1999; Holzer et al., 2004; Eszterbauer et al., 2006; Marton and Eszterbauer, 2011); however, the Raabeia type of McGeorge et al. (1997) was specifically identified as the actinosporean counterpart of Myxidium truttae (Holzer et al., 2014). Congruently, the phylogenetic analysis performed in this study shows raabeia/echinactinomyxon types spanning over myxosporean genera that belong to the bivalvulid suborders Variisporina and Platysporina (see Fig. 5). This clustering pattern suggests their involvement in the life cycles of taxonomically distant species, thus strengthening the contention that there is no obvious agreement between actinosporean morphotypes and myxosporean genera (Eszterbauer et al., 2015; Rocha et al., 2019a). The type described here specifically clusters within the Paramyxidium clade, alongside other members of the collective group, as well as types of aurantiactinomyxon and synactinomyxon. The recently erected genus Paramyxidium Freeman and Kristmundsson, 2018 encompasses a total of four species that were described from

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Chapter VII | Union of raabeia and echinactinomyxon collective groups infections taking place in European eel Anguilla anguilla (Linnaeus, 1758) (Anguilliformes) and Indo-Pacific tarpon Megalops cyprinoides (Broussonet, 1782) (Elopiformes) (Freeman and Kristmundsson, 2018). Several orders of fish integrate the vertebrate community inhabiting the River Minho, but Elopiformes is not included. In turn, the order Anguilliformes is represented solely by A. anguilla. Thus far, myxozoan surveys in the River Minho have only targeted mugiliform fish, with Myxobolus exiguus constituting the sole species reported from this geographic area, parasitizing the visceral peritoneum of the thinlip grey mullet Chelon ramada (Risso, 1827) (Rocha et al., 2019b). Future research in this geographic location should, therefore, aim to survey other fish species, including European eel A. anguilla, as potential hosts of the Raabeia type described here.

Acknowledgments

The authors thank Miguel Pereira for his iconographic assistance. This work was financially supported by the Foundation for Science and Technology (Lisbon, Portugal) within the scope of the Ph.D. fellowship grant attributed to S. Rocha (SFRH/BD/92661/2013) through the programme QREN-POPH/FSE; and the Engº António de Almeida Foundation (Porto, Portugal).

References

Andree, K.B., Székely, C., Molnár, K., Gresoviac, S.J. and Hedrick, R.P. (1999). Relationships among members of the genus Myxobolus (Myxozoa: Bilvalvidae) based on small subunit ribosomal DNA sequences. Journal of Parasitology 85, 68–74. Atkinson, S.D., Hallett, S.L., Díaz-Morales, D., Bartholomew, J.L. and de Buron, I. (2019). First myxozoan infection (Cnidaria: Myxosporea) in a marine polychaete from North America and erection of actinospore collective group Saccimyxon. Journal of Parasitology (In Press). Békési, L., Székely, C. and Molnár, K. (2002). Atuais conhecimentos sobre Myxosporea (Myxozoa), parasitas de peixes. Um estágio alternativo dos parasitas no Brasil. Brazilian Journal of Veterinary Research and Animal Science 39, 271‒276. Bellerud, B.L. (1993). Etiological and epidemiological factors effecting outbreaks of proliferative gill disease on Mississippi channel catfish farms. Ph.D. Dissertation. College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi, pp. 164. Borkhanuddin, M.H., Cech, G., Molnár, K., Nemeth, S. and Székely, C. (2014). Description of raabeia, synactinomyxon and neoactinomyxum developing stages of myxosporeans

Page | 188

Chapter VII | Union of raabeia and echinactinomyxon collective groups

(Myxozoa) infecting Isochaetides michaelseni Lastokin (Tubificidae) in Lake Balaton and Kis-Balaton Water Reservoir, Hungary. Systematic Parasitology 88, 245‒259. Caffara, M., Raimondi, E., Florio, D., Marcer, F., Quaglio, F. and Fioravanti, M.L. (2009). The life cycle of Myxobolus lentisuturalis (Myxozoa: Myxobolidae), from goldfish (Carassius auratus auratus), involves a Raabeia-type actinospore. Folia Parasitologica 56, 6‒12. Dias, E., Morais, P., Cotter, A.M., Antunes, C. and Hoffman, J.C. (2016). Estuarine consumers utilize marine, estuarine and terrestrial organic matter and provide connectivity among these food webs. Marine Ecology Progress Series 554, 21–34. El-Mansy, A., Székely, C. and Molnár, K. (1998a). Studies on the occurrence of actinosporean stages of fish myxosporeans in a fish farm of Hungary, with the description of triactinomyxon, raabeia, aurantiactinomyxon and neoactinomyxon types. Acta Veterinaria Hungarica 46, 259‒284. El-Mansy, A., Székely, C. and Molnár, K. (1998b). Studies on the occurrence of actinosporean stages of myxosporeans in Lake Balaton, Hungary, with the description of triactinomyxon, raabeia and aurantiactinomyxon types. Acta Veterinaria Hungarica 46, 437‒450. El-Matbouli, M., Fischer-Scherl, T. and Hoffmann, R.W. (1992). Transmission of Hoferellus carassii Achmerov, 1960 to goldfish Carassius auratus via an aquatic oligochaete. Bulletin of the European Association of Fish Pathologists 12, 54‒56. El-Matbouli, M. and Hoffmann, R. (1989). Experimental transmission of two Myxobolus spp. developing bisporogeny via tubificid worms. Parasitology Research 75, 461‒464. El-Matbouli, M. and Hoffmann, R.W. (1993). Myxobolus carassii Klokaceva, 1914 also requires an aquatic oligochaete, Tubifex tubifex as an intermediate host in its life cycle. Bulletin of the European Association of Fish Pathologists 13, 189‒192. Eszterbauer, E., Atkinson, S., Diamant, A., Morris, D., El-Matbouli, M. and Hartikainen, H. (2015). Myxozoan life cycles: practical approaches and insights. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp XIII, 441. Eszterbauer, E., Marton, S., Rácz, O.Z., Letenyei, M. and Molnár, K. (2006). Morphological and genetic differences among actinosporean stages of fish-parasitic myxosporeans (Myxozoa): difficulties of species identification. Systematic Parasitology 65, 97‒114. Ferguson, J.A., Atkinson, S.D., Whipps, C.M. and Kent, M.L. (2008). Molecular and morphological analysis of Myxobolus spp. of salmonid fishes with the description of a new Myxobolus species. Journal of Parasitology 94, 1322–1334. Fiala, I. (2006). The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. International Journal of Parasitology 36, 1521‒1534. Freeman, M.A. and Kristmundsson, A. (2018). Studies of Myxidium giardi Cépède, 1906

Page | 189

infections in Icelandic eels identifies a genetically diverse clade of myxosporeans that represents the Paramyxidium n. g. (Myxosporea: Myxidiidae). Parasites & Vectors 11, 551. Grossheider, G. and Körting, W. (1992). First evidence that Hoferellus cyprini (Doflein, 1898) is transmitted by Nais sp. Bulletin of the European Association of Fish Pathologists 12,17‒20. Hallett, S.L., Atkinson, S.D. and El-Matbouli, M. (2002). Molecular characterisation of two aurantiactinomyxon (Myxozoa) phenotypes reveals one genotype. Journal of Fish Diseases 25, 627‒631. Hallett, S.L., Atkinson, S.D., Erséus, C. and El-Matbouli, M. (2004). Molecular methods clarify morphometric variation in triactinomyxon spores (Myxozoa) released from different oligochaete hosts. Systematic Parasitology 57, 1‒14. Hallett, S.L., Atkinson, S.D., Erséus, C. and El-Matbouli, M. (2006). Myxozoan parasites disseminated via oligochaete worms as live food for aquarium fishes: descriptions of aurantiactinomyxon and raabeia actinospore types. Diseases of Aquatic Organisms 69, 213‒225. Hallett, S.L. and Diamant, A. (2001). Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia. Diseases of Aquatic Organisms 46, 197‒212. Hallett, S.L., O´Donoghue, P.J. and Lester, R.J.G. (1998). Structure and development of a marine actinosporean Sphaeractinomyxon ersei n. sp. (Myxozoa). Journal of Eukaryot Microbiology 45, 142‒150. Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. The Quarterly Review of Biology 66, 411‒453. Holzer, A.S., Bartošová-Sojková, P., Born-Torrijos, A., Lövy, A., Hartigan, A. and Fiala, I. (2018). The joint evolution of the Myxozoa and their alternate hosts: A cnidarian recipe for success and vast biodiversity. Molecular Ecology 27, 1651‒1666. Holzer, A.S., Sommerville, C. and Wootten, R. (2004). Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences. International Journal of Parasitology 34, 1099–1111. Janiszewska, J. (1955). Actinomyxidia. Morphology, ecology, history of investigations, systematics, development. Acta Parasitologica Polonica 2, 405–437. Janiszewska, J. (1957). Actinomyxidia II: new systematics, sexual cycle, description of new genera and species. Zoologica Poloniae 8, 3–34. Janiszewska, J. and Krztón, M. (1973). Raabeia furciligera sp. n. (Cnidosporidia, Actinomyxidia) from the body cavity of Limnodrilus hoffmeisteri Claparède, 1862. Acta

Chapter VII | Union of raabeia and echinactinomyxon collective groups

Protozoologica 12, 165‒167. Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffmann, R.W., Khattra, J., Hallet, S.L., Lester, R.J., Longshaw, M., Palenzeula, O., Siddall, M.E. and Xiao, C. (2001). Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic Microbiology 48, 395‒413. Kent, M.L., Margolis, L. and Corliss, J.O. (1994). The demise of a class of protists - taxonomic and nomenclatural revisions proposed for the protist phylum Myxozoa Grassé, 1970. Canadian Journal of Zoology 72, 932‒937. Kent, M.L., Whitaker, D.J. and Margolis, L. (1993). Transmission of Myxobolus arcticus Pugachev and Khokhlov, 1979, a myxosporean parasite of Pacific salmon, via a triactinomyxon from the aquatic oligochaete Stylodrilus heringianus (Lumbriculidae). Canadian Journal of Zoology 71, 1207‒1211. Koprivnikar, J. and Desser, S.S. (2002). A new form of raabeia-type actinosporean (Myxozoa) from the oligochaete Uncinais uncinata. Folia Parasitologica 49, 89‒92. Kumar, S., Stecher, G. and Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution 33, 1870‒ 1874. Lom, J. and Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53, 1‒36. Lom, J., McGeorge, J., Feist, S.W., Morris, D. and Adams, A. (1997a). Guidelines for the uniform characterisation of the actinosporean stages of parasites of the phylum Myxozoa. Diseases of Aquatic Organisms 30, 1‒9. Lom, J., Yokoyama, H. and Dyková, I. (1997b). Comparative ultrastructure of Aurantiactinomyxon and Raabeia, actinosporean stages of myxozoan life cycles. Archiv für Protistenkunde 148, 173‒189. Marcucci, C., Caffara, M. and Goretti, E. (2009). Occurrence of actinosporean stages (Myxozoa) in the Nera River system (Umbria, central Italy). Parasitology Research 105, 1517‒1530. Marques, A. (1984). Contribution à la connaissance des Actinomyxidies: ultrastructure, cycle biologique, systématique. Ph.D. Thesis, Université des Sciences et Techniques de Languedoc, Montepellier, France Marton, S. and Eszterbauer, E. (2011). The development of Myxobolus pavlovskii (Myxozoa: Myxobolidae) includes an echinactinomyxon-type actinospore. Folia Parasitologica 58, 157‒163. McGeorge, J., Sommerville, C. and Wootten, R. (1997). Studies of actinosporean myxozoan stages parasitic in oligochaetes from the sediments of a hatchery where Atlantic salmon harbour Sphaerospora truttae infection. Diseases of Aquatic Organisms 30, 107‒119.

Page | 191

Chapter VII | Union of raabeia and echinactinomyxon collective groups

Molnár, K., El-Mansy, A., Székely, C. and Baska, F. (1999). Development of Myxobolus dispar (Myxosporea : Myxobolidae) in an oligochaete alternate host, Tubifex tubifex. Folia Parasitologica 46, 15‒21. Negredo, C. and Mulcahy, M.F. (2001). Actinosporean infections in oligochaetes in a river system in southwest Ireland with descriptions of three new forms. Diseases of Aquatic Organisms 46, 67‒77. Oumouna, M., Hallett, S., Hoffmann, R. and El-Matbouli, M. (2003). Seasonal occurrence of actinosporeans (Myxozoa) and oligochaetes (Annelida) at a trout hatchery in Bavaria, Germany. Parasitology Research 89, 170‒184. Özer, A., Wootten, R. (2000). The life cycle of Sphaerospora truttae (Myxozoa : Myxosporea) and some features of the biology of both the actinosporean and myxosporean stages. Diseases of Aquatic Organisms 40, 33‒39. Özer, A. and Wootten, R. (2001). Ultrastructural observations on the development of some actinosporean types within their oligochaete hosts. Turkish Journal Of Zoology 25, 199‒216. Özer, A., Wootten, R. and Shinn, A.P. (2002). Survey of actinosporean types (Myxozoa) belonging to seven collective groups found in a freshwater salmon farm in Northern Scotland. Folia Parasitologica 49, 189‒210. Palumbi, S., Martin, A., Romano, S., McMillan, W.O., Stice, L. and Grabowski, G. (2002). The Simple Fools Guide to PCR, Version 2.0. University of Hawaii, Honolulu. Rangel, L.F., Castro, R., Rocha, S., Cech, G., Casal, G., Azevedo, C., Székely, C., Cavaleiro, F. and Santos, M.J. (2016). Description of new types of sphaeractinomyxon actinospores (Myxozoa: Myxosporea) from marine tubificid oligochaetes, with a discussion on the validity of the tetraspora and the endocapsa as actinospore collective group names. Parasitology Research 115, 2341‒2351. Rocha, S., Azevedo, C., Oliveira, E., Alves, Â., Antunes, C., Rodrigues, P. and Casal, G. (2019b). Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular redescription of the cryptic species Myxobolus exiguus. Parasitology 146, 479‒496. Rocha, S., Casal, G., Rangel, L., Castro, R., Severino, R., Azevedo, C. and Santos, M.J. (2015). Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae). Parasitology International 64, 305‒313. Rocha, S., Rangel, L.F., Castro, R., Severino, R., Azevedo, C., Santos, M.J. and Casal, G. (2019a). The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides

Page | 192

Chapter VII | Union of raabeia and echinactinomyxon collective groups

insularis. Journal of Invertebrate Pathology 160, 33‒42. Ronquist, F. and Huelsenbeck, J.P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572‒1574. Rosser, T.G., Griffin, M.J., Quiniou, S.M.A., Greenway, T.E., Khoo, L.H., Wise, D.J. and Pote, L.M. (2014). Molecular and morphological characterization of myxozoan actinospore types from a commercial catfish pond in the Mississippi delta. Journal of Parasitology 100, 828‒839. Ruidisch, S., El-Matbouli, M. and Hoffmann, R.W. (1991). The role of tubificid worms as an intermediate host in the life cycle of Myxobolus pavlovskii (Akhmerov, 1954). Parasitology Research 77, 663‒667. Schlegel, M., Lom, J., Stechmann, A., Bernhard, D., Leipe, D., Dyková, I. and Sogin, M.L. (1996). Phylogenetic analysis of complete small unit ribosomal RNA coding region of Myxidium lieberkuehni: evidence that Myxozoa are Metazoa and related to the Bilateria. Archiv für Protistenkunde 147, 1–9. Štolc, A. (1899). Actinomyxidies, nouveau groupe de Mesozoaires parent des Myxosporidies. Bulletin international de l'Académie des sciences de Bohême 12, 1–12. Styer, E.L., Harrison, L.R. and Burtle, G.J. (1992). Six new species of actinomyxids from Dero digitata. International Workshop on Myxosporea, October 6–8, České Budĕjovice, Czech Republic (abstract only) Székely, C., Urawa, S. and Yokoyama, H. (2002). Occurrence of actinosporean stages of myxosporeans in an inflow brook of a salmon hatchery in the Mena River System, Hokkaido, Japan. Diseases of Aquatic Organisms 49, 153‒160. Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215‒219. Wolf, K. and Markiw, M.E. (1984). Biology contravenes taxonomy in the Myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science 225, 1449‒ 1452. Xi, B.W., Li, P., Liu, Q.C., Chen, K., Teng, T. and Xie, J. (2017). Description of a new Neoactinomyxum type actinosporean from the oligochaete Branchiura sowerbyi Beddard. Systematic Parasitology 94, 73‒80. Xi, B.W., Zhang, J.Y., Xie, J., Pan, L.K., Xu, P. and Ge, X.P. (2013). Three actinosporean types (Myxozoa) from the oligochaete Branchiura sowerbyi in China. Parasitology Research 112, 1575‒1582. Xi, B.W., Zhou, Z.G., Xie, J., Pan, L.K., Yang, Y.L. and Ge, X.P. (2015). Morphological and molecular characterization of actinosporeans infecting oligochaete Branchiura

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Chapter VII | Union of raabeia and echinactinomyxon collective groups

sowerbyi from Chinese carp ponds. Diseases of Aquatic Organisms 114, 217‒228. Xiao, C. and Desser, S.S. (1998a). Actinosporean stages of myxozoan parasites of oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of triactinomyxon and raabeia. Journal of Parasitology 84, 998‒1009. Xiao, C.X. and Desser, S.S. (1998b). Actinosporean stages of myxozoan parasites of oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of echinactinomyxon, neoactinomyxum, aurantiactinomyxon, guyenotia, synactinomyxon and antonactinomyxon. Journal of Parasitology 84, 1010‒1019. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1993a). Involvement of Branchiura sowerbyi (Oligochaeta, Annelida) in the transmission of Hoferellus carassii (Myxosporea, Myxozoa), the causative agent of kidney enlargement disease (KED) of goldfish Carassius auratus. Fish Pathology 28, 135‒139. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1993b). Some biological characteristics of actinosporeans from the oligochaete Branchiura sowerbyi. Diseases of Aquatic Organisms 17, 223‒228. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1995). Myxobolus cultus n. sp. (Myxosporea: Myxobolidae) in the Goldfish Carassius auratus transformed from the actinosporean stage in the oligochaete Branchiura sowerbyi. Journal of Parasitology 81, 446‒451.

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Chapter VIII

Molecular data infers the involvement of a marine aurantiactinomyxon in the life cycle of the myxosporean parasite Paramyxidium giardi (Cnidaria, Myxozoa)

This chapter was adapted from:

Chapter VIII | Life cycle inference of Paramyxidium giardi

Abstract

An aurantiactinomyxon type is described from the marine naidid Tubificoides pseudogaster (Dahl, 1960), collected from the lower estuary of a Northern Portuguese River. This type constitutes the first of its collective group to be reported from Portugal, and only the fourth described from a marine oligochaete worldwide. Extensive morphological comparisons of new aurantiactinomyxon isolates to all known types without available molecular data are proposed to be unnecessary, given the artificiality of the usage of morphological criteria for actinosporean differentiation and the apparent strict host specificity of the group. Recognition of naidid oligochaetes as the hosts of choice for marine types of aurantiactinomyxon and other collective groups, suggests that the family Naididae played a preponderant role in the myxosporean colonization of estuarine communities. Molecular analyses of the type in study further infer its involvement in the life cycle of Paramyxidium giardi (Cépède, 1906) Freeman and Kristmundsson, 2018, a species that infects the kidney of European eel Anguilla anguilla (Linnaeus, 1758) and that has been reported globally, including from Portuguese waters. The low intraspecific difference registered in relation to Icelandic isolates of P. giardi (0.6%) is hypothesized to result from the emergence of genotypically different subspecies due to geographic isolation.

Introduction

Myxosporeans are parasitic cnidarians that mainly infect aquatic vertebrates and invertebrates. The involvement of annelids in the life cycle of myxosporeans was first revealed by Wolf and Markiw (1984), upon their discovery that the salmonid-infecting species Myxobolus cerebralis Hofer, 1903 has a life cycle stage that develops in the freshwater oligochaete Tubifex tubifex (Müller, 1774). Since then, several studies have provided direct and indirect evidence for a two-stage life cycle of these parasites, in which a myxosporean stage that develops in a fish alternates with and actinosporean stage that develops in an oligochaete or polychaete (see Eszterbauer et al., 2015). Both life cycle stages are characterized by the production of spores necessary to achieve dissemination and transmission between vertebrate and invertebrate hosts. Spores are multicellular, comprised by two to several external valve cells that form a wall surrounding one to many polar capsules, and one to many sporoplasms. Despite sharing these main morphological features, the myxospores produced in the fish host and the actinospores produced in the annelid host exhibit highly distinct morphotypes (Lom and Dyková, 2006). Currently, actinospore morphotypes are divided into ca. 20 collective groups; the differentiation and classification of distinct types within these groups being traditionally based

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Chapter VIII | Life cycle inference of Paramyxidium giardi on morphological criteria. The latter include the shape and size of the actinospores’ body and valvular processes, shape and position of the polar capsules, and number of secondary cells in the sporoplasm (Lom et al., 1997; Lom and Dyková, 2006). Actinospores of the aurantiactinomyxon collective group, specifically, are defined as having three stout, semicircularly curved, leaf-like valvular processes attached to an ellipsoidal body with protruding polar capsules at the apex, and containing a sporoplasm with many secondary cells (Lom and Dyková, 2006). In the past few decades, however, the implementation of molecular tools to the study of actinosporeans has revealed that morphological criteria are unreliable for performing differential diagnoses between types (e.g. Hallett et al., 2002, 2004; Eszterbauer et al., 2006; Xi et al., 2015; Rangel et al., 2016a; Zhao et al., 2016). Congruently, actinospores descriptions are currently based on the combined analysis of morphological and molecular data. Initially, life cycle studies relied solely on data acquired through means of experimental transmission between vertebrate and invertebrate hosts. The complicated logistics and time- consuming nature of this methodology, however, hinders the achievement of results. Moreover, its reliability has been discredited by the usage of molecular tools, which shows that mixed and non-spore forming infections are common and may lead to erroneous associations (Holzer et al., 2004; Atkinson and Bartholomew, 2009; Marton and Eszterbauer, 2011; Székely et al., 2014). Consequently, life cycle studies now mainly rely on sequencing of the SSU rDNA gene for inferring correspondence between myxosporean and actinosporean stages. To date, near 60 myxosporean life cycles have been elucidated (see Eszterbauer et al., 2015). Most refer to myxobolid species (Myxobolus, Henneguya and Thelohanellus) that infect fish and oligochaetes in freshwater environments (Myxobolus, Henneguya and Thelohanellus) (e.g. Styer et al., 1991; Kent et al., 1993; Lin et al., 1999; Eszterbauer et al., 2000, 2006, 2015; Kallert et al., 2005; Marton and Eszterbauer, 2011; Székely et al., 2014), with fewer studies providing information pertaining to the life cycles of myxosporean genera such as Ceratomyxa, Ceratonova, Chloromyxum, Ellipsomyxa, Gadimyxa, Hofferellus, Myxidium, Myxobilatus, Ortholinea, Paramyxidium, Parvicapsula, Sigmomyxa, Sphaerospora and Zschokkella (Grossheider and Körting, 1992; Benajiba and Marques, 1993; Yokoyama et al., 1993a; Bartholomew et al., 1997, 2006; Holzer et al., 2004, 2006; Køie et al., 2004, 2007, 2008; Atkinson and Bartholomew, 2009; Rangel et al., 2009, 2017; Karlsbakk and Køie, 2012). In marine environments, polychaetes appear to be the hosts of choice (Køie et al., 2004, 2007, 2008; Rangel et al., 2009, 2011, 2016b), with a few known exceptions occurring in estuarine waters, including Portuguese Rivers (Bartholomew et al., 1997, 2006; Rangel et al., 2015, 2017). Overall, few studies have aimed to investigate myxosporean biodiversity in invertebrate communities of this geographic region, with a total of 20 actinosporean types described from the raabeia (1 type), triactinomyxon (2 types), tetractinomyxon (2 types), sphaeractinomyxon

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(13 types), synactinomyxon (1 type) and unicapsulaticnomyxon (1 type) collective groups (Székely et al., 2005; Rangel et al., 2009, 2011, 2015, 2016a, b, 2017; Rocha et al., 2019a, b). Apart from the Raabeia type of Rocha et al. (2019a) and the Synactinomyxon type of Székely et al. (2005), which were described from the freshwater oligochaetes Ilyodrilus templetoni (Southern, 1909) and Tubifex tubifex, respectively, all the other above-mentioned types were found infecting marine oligochaetes or polychaetes in downstream estuaries. Both the triactinomyxon and tetractinomyxon types have been paired with their respective myxosporean counterparts, through the positive match of SSU rDNA sequences. The triactinomyxon types, one reported from the oligochaete Limnodriloides agnes Habrě, 1967 and the other from an undetermined species of the genus Tectidrilus Erséus, 1982, were molecularly identified as the species Ortholinea auratae Rangel et al., 2015 and O. labracis Rangel et al., 2017, which parasitize the urinary bladder of gilthead seabream Sparus aurata Linnaeus, 1758 and European seabass Dicentrarchus labrax (Linnaeus, 1758), respectively (Rangel et al., 2015, 2017). In turn, both tetractinomyxon types were described from marine polychaetes, Hediste diversicolor (Müller, 1776) and an undetermined species of Capitella Blainville, 1828, and molecularly shown to be involved in the life cycle of Ellipsomyxa mugilis (Sitjà-Bobadilla and Alvarez-Pellitero, 1993) from the gall bladder of grey mullets and Sphaerospora dicentrarchi Sitjà-Bobadilla and Alvarez-Pellitero, 1992 from systemic infections in D. labrax, respectively (Rangel et al., 2009, 2016b). To our best knowledge, further 17 myxosporean species have been reported from fishes inhabiting Portuguese rivers and estuaries, including Paramyxidium giardi (Cépède, 1906) Freeman and Kristmundsson, 2018 (see Eiras, 2016). The myxosporean species Paramyxidium giardi was originally described as a member of the genus Myxidium, from infections in the kidney of European eel Anguilla anguilla (Linnaeus, 1758) in France (Cépède, 1906). Over time, numerous reports of this species were performed from infections occurring in multiple organs of anguillid eels worldwide (Ishii, 1915; Fujita, 1927; Hine, 1975, 1978, 1980; Copland, 1981; Treasurer and Cox, 1997), including specimens of A. anguilla from Portuguese estuaries (Ventura and Paperna, 1984; Azevedo et al., 1989; Saraiva and Eiras, 1996; Saraiva and Chubb, 1989; Hermida et al., 2008). In 1993, Benajiba and Marques further identified the actinosporean counterpart of P. giardi as belonging to the aurantiactinomyxon collective group. The authors used cysts from the kidney of infected A. anguilla to experiment infection on samples of Tubifex that were reported to include non- parasitized specimens, having been collected from a previously studied area (presumably in France). Recent phylogenetic analyses corroborate the potential involvement of aurantiactinomyxon, as well as of raabeia and synactinomyxon types, in the life cycle of Paramyxidium spp. (Freeman and Kristmundsson, 2018; Rocha et al., 2019a). Nonetheless, molecular data has not been acquired that confirms the association between this genus and

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Chapter VIII | Life cycle inference of Paramyxidium giardi any of the above-mentioned collective groups. In this study, microscopic and molecular tools are used to describe an aurantiactinomyxon type found infecting the intestinal epithelium of the marine oligochaete Tubificoides pseudogaster (Dahl, 1960) in the lower estuary of the River Minho, Portugal. This type constitutes the first of its collective group to be reported from Portuguese waters, and only the fourth described from a marine oligochaete. Its involvement in the life cycle of Paramyxidium giardi is molecularly inferred by comparison of the newly obtained SSU rDNA sequence to that available in GenBank from Icelandic isolates used in the species re- description.

Materials and methods

Sampling sites and morphological characterization

As part of a two-year myxozoan survey conducted in the River Minho between 2015 and 2016, mud was collected at low tide from the lower estuary, near the village of Caminha (41° 52' N, 08° 50' W). In the laboratory, oligochaetes were collected from the mud and placed individually in 12-well plates containing brackish water (15‰ salinity) and kept at 4 ºC. Salinity was chosen according to the value range (15 to 40‰) generally registered for this parameter in the lower estuary throughout the year (see Dias et al., 2016). All oligochaetes were individually examined using the light microscope for the detection of actinosporean stages in internal tissues and cavities. Developmental stages and free actinospores were examined and photographed using an Olympus BX50 light microscope (Olympus, Japan). Morphometry was determined from fresh material, in accordance to Lom et al. (1997). Measurements include the mean value ± standard deviation (SD), range of variation, and number of measured actinospores (n).

DNA extraction, amplification and sequencing

Genomic DNA from infected oligochaetes was extracted using the GenEluteTM Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, USA), following the manufacturer’s instructions. The DNA was stored in 50 μl of TE buffer at -20 ºC. The SSU rDNA gene of the actinospores was amplified using both universal and myxosporean-specific primers: the 5’-end by pairing the primer 18E (5’-CTG GTT GAT CCT GCC AGT-3’) (Hillis and Dixon, 1991) with the primers MyxospecR (5’-CAA CAA GTT GAT AGG GCA GAA-3’) (Fiala, 2006) and MYX4R (5’-CTG ACA GAT CAC TCC ACG AAC-3’) (Hallett and Diamant, 2001), and the 3’-end by pairing the primers MyxospecF (5’-TTC TGC CCT ATC AAC TTG TTG-3’)

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(Fiala, 2006) and MYX4F (5’-GTT CGT GGA GTG ATC TGT CAG-3’) (Rocha et al., 2015) with the primer 18R (5’-CTA CGG AAA CCT TGT TAC G-3’) (Whipps et al., 2003). In turn, the 16S mitochondrial DNA (mtDNA) gene of the oligochaete host was amplified using the universal primers 16sar-L (5’-CGC CTG TTT ATC AAA AAC AT-3’) and 16sbr-H (5’-CCG GTC TGA ACT CAG ATC ACG T-3’) (Palumbi et al., 2002). All PCRs were performed in 50 µl reactions using 10 pmol of each primer, 10 nmol of each dNTP, 2.0 mM of MgCl2, 5 µl 10× Taq polymerase buffer, 1.5 units Taq DNA polymerase (NZYTech, Lisbon, Portugal), and 3 µl (approximately 100–150 ng) of genomic DNA. The reactions were run on a Hybaid PxE Thermocycler (Thermo Electron Corporation, Milford, Massachusetts, USA), with initial denaturation at 95 ºC for 3 min, followed by 35 cycles of 94 ºC for 45 s, 53 ºC for 45 s, and 72 ºC for 90 s. The final elongation step was performed at 72 ºC for 7 min. Five-µl aliquots of the PCR products were electrophoresed through a 1% agarose 1× tris-acetate-EDTA buffer (TAE) gel stained with ethidium bromide. PCR products were purified using Puramag™ magnetic beads coated with carboxylic acid groups (MCLAB, San Francisco, California, USA). Sequencing reactions were performed with the same primers used for amplification, using a BigDye Terminator v3.1 Cycle Sequencing Kit from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, EUA), and were run on an ABI3700 DNA analyzer from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, EUA).

Sequence assembly and distance estimation analysis

The SSU rDNA sequence of the parasite was assembled in MEGA7 (Kumar et al., 2016), by aligning the partial sequences obtained for each PCR fragment. A dataset was generated according to the highest similarity scores obtained using BLAST search, and included all known Paramyxidium spp., as well as closely related sequences of the aurantiactinomyxon, raabeia and synactinomyxon collective groups. All SSU rDNA sequences available for more distantly related aurantiactinomyxon types were also included in the dataset. Sequences were aligned using MAFFT version 7 available online, and distance estimation was calculated in MEGA7, with the p-distance model and all ambiguous positions removed for each sequence pair.

Results

Myxozoan survey, prevalence of infection and host identification

During this study, 577 oligochaetes were isolated from the mud collected in the lower estuary of the River Minho. Myxozoan infections were determined in a total of 9 specimens, in

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Chapter VIII | Life cycle inference of Paramyxidium giardi all of which it was possible to observe mature actinospores alongside younger developmental stages. Five specimens displayed infection in the coelomic cavity by members of the sphaeractinomyxon collective group. The remaining 4 specimens displayed actinosporean development in the intestinal epithelium: two by a type of the aurantiactinomyxon collective group, and the other two by a type of the synactinomyxon collective group. The description of the aurantiactinomyxon type is presented here; descriptions of the remaining types will be published elsewhere. All 9 oligochaete specimens displaying myxozoan infection were identified as belonging to the marine species Tubificoides pseudogaster (Dahl, 1960). Identification was based on combined morphological features and molecular data of the 16S mitochondrial DNA (mtDNA). Infected oligochaetes looked alike, having similar dimensions, bifid setae and a body tegument devoid of papillae. Sequences of the 16S mtDNA were 98‒100% similar to those available for the lineage II of this species, as reported by Kvist et al. (2010).

Characterization of the novel Aurantiactinomyxon type (Cnidaria, Myxozoa)

Description: Developmental stages of the gametogamy and sporogony phases developing in the intestinal epithelium (Fig. 1). Youngest developmental stages observed were binucleated cells (Fig. 2A). The latter divided twice to produce a set of four cells that formed the initial pansporocyst, comprised by two somatic cells surrounding two generative inner cells (Fig. 2B, C). Three successive mitotic divisions and one meiotic division of the generative cells produced a final set of 16 haploid cells within the pansporocysts (Fig. 2D‒F). At the end of gametogamy, these cells united two by two to form 8 diploid zygotes (Fig. 2G). Subsequent mitotic divisions of the zygotes originated 8 immature actinospores, each comprised by a sporoplasmic cell connected to three valvogenic cells and three capsulogenic cells. The valvogenic cells surrounded the capsulogenic cells and appeared positioned centrally, while

Figure 1. Light micrographs showing pansporocysts of the new type of aurantiactinomyxon developing in the intestinal epithelium of the marine oligochaete Tubificoides pseudogaster from the lower estuary of the River Minho.

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Figure 2. Light micrographs of some developmental stages of the new type of aurantiactinomyxon found infecting the marine oligochaete Tubificoides pseudogaster in the lower estuary of the River Minho. (A‒F) Gametogamy phase: (A) Binucleated cell; (B) Binucleated cell in cellular division; (C) Initial pansporocyst with two involucre cells and two inner cells; (D‒F) Pansporocysts with four, 14 and 16 inner cells, respectively. (G‒I) Sporogony phase: (G) Pansporocyst with 8 zygotes undergoing cellular divisions; (H) Pansporocyst showing the valvogenic and capsulogenic cells located centrally (*), while the sporoplasmic cells (SC) lean against the pansporocyst wall; (I) Pansporocyst containing 8 immature actinospores. Notice the capsulogenic cells; (J, K) Mature actinospores in apical view, evidencing the 3 spherical polar capsules protruding from the anterior end, and the rounded tips of the 3 equally sized leaf-like valvular processes; (L) Mature actinospore as observed in lateral view. corresponding sporoplasmic cells leaned against the pansporocyst’s wall. Sporoplasmic cells increased in size and multiplied the number of secondary cells prior to migrating into the involucre formed by the valvogenic cells (Fig. 2H‒I). At the end of sporogony, 8 mature actinospores were observed within each pansporocyst. Actinospore body subspherical, 14.4 ± 0.6 (13.6‒15.9) µm long (n = 25) and 12.7 ± 0.7 (11.3‒13.3) µm wide (n = 25), entirely embraced by 3 equally sized valvular processes, leaf-like with rounded tips, 22.4 ± 2.4 (18.1‒ 27.6) µm long (n = 47) and 15.5 ± 0.9 (13.3‒17.0) µm wide at the base (n = 25). Three equally sized spherical polar capsules protruding from the anterior end of the actinospores, 2.6 ± 0.3 (1.9‒3.5) µm in diameter (n = 53), each containing a polar tubule exhibiting 4 to 5 coils (Figs.

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2J‒L and 3). Sporoplasm containing an undetermined number of secondary cells. Type host: Tubificoides pseudogaster (Dahl, 1960) (Annelida, Oligochaeta). Type locality: The lower estuary of the River Minho, near Caminha (41° 52' N, 08° 50' W), Portugal. Site of infection: The intestinal epithelium. Prevalence: 0.3% (2 infected in a total of 577 oligochaetes examined). Deposition of material: Series of phototypes deposited together with a representative DNA sample in the Type Ma- terial Collection of the Laboratory of Animal Pathology, Interdisciplinary Cen- tre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2019.44. Molecular data: One SSU rDNA Figure 3. Schematic drawing depicting a mature actinospore of the new type of aurantiactinomyxon found gene sequence with a total of 2,027 bp, infecting the marine oligochaete Tubificoides pseudogas- deposited in GenBank with the accession ter in the lower estuary of the River Minho, as observed no. MK635346. The latter is represent- in lateral (A) and apical (B) view. tative of two identical sequences that

were separately obtained from the actinosporean developmental stages in the intestinal epithelium of two infected oligochaetes. Remarks: Morphometry was determined from mature actinospores observed in both infected hosts. Morphometric variation between individual measurements was not recorded. Comparison to the ca. 39 known aurantiactinomyxon types without available molecular data revealed some morphometric similarity to the actinospores of the Aurantiactinomyxon type of McGeorge et al. (1997), Aurantiactinomyxon type of Xiao and Desser (1998) and Aurantiactinomyxon type 2 of Özer et al. (2002). Nonetheless, these types parasitize different oligochaete hosts in freshwater habitats of distant geographic locations (Canada and Scotland), further differing from the one in study in specific morphological aspects of the actinospores. Both the actinospores of the Aurantiactinomyxon type of McGeorge et al. (1997) and of the Aurantiactinomyxon type 2 of Özer et al. (2002) are spherical instead of subspherical, with the first further having thinner valvular processes, and the second being

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Chapter VIII | Life cycle inference of Paramyxidium giardi generally bigger. In turn, the Aurantiactinomyxon type of Xiao and Desser (1998) has pyriform polar capsules instead of spherical, and a slightly smaller actinospore body. It should be noted that no gross similarity was determined in relation to the three marine aurantiactinomyxon types reported in the literature (Hallett et al., 1997), all of which are without available molecular data. In turn, morphological comparison to the freshwater Aurantiactinomyxon type of Benajiba and Marques (1993), previously reported to be the life cycle counterpart of P. giardi, was strongly hampered by the paucity of morphometric data given in its original description. Molecular comparison could be performed only in relation to 20 freshwater types with SSU rDNA sequences available from either their original descriptions, follow-up studies, or from the myxosporean species determined to be their life cycle counterparts through experimental transmission. The results showed significant genetic differences of the SSU rDNA sequences in all cases (ranging from 1.4% to as high as 24.8%). In turn, a high similarity value (99.4%) was obtained in relation to the sequence of Paramyxidium giardi available in GenBank (MH414925) from infections in the kidney of European eel A. anguilla. These two sequences differ in a total of 9 nucleotide positions over 2,020 bp. Overall, the parasite was more related to other members of the genus Paramyxidium, as well as to actinosporean types known to be phylogenetically associated with the latter, namely raabeia, synactinomyxon and aurantiactinomyxon (similarity values ranged from 98.6% to as low as 81.6%). All other myxosporean sequences available in GenBank shared less than 80% similarity with the new Aurantiactinomyxon type.

Discussion

The majority of known actinosporean stages have been described based solely on morphological criteria. The implementation of molecular tools to the study of myxosporeans, however, has revealed that species having morphologically similar actinospores may be only distantly related based on SSU rDNA sequence data. In the other way around, species having actinospores with divergent morphology may be closely related (Hallett et al., 2002, 2004; Eszterbauer et al., 2006; Xi et al., 2015; Rangel et al., 2016a; Zhao et al., 2016). In the case of aurantiactinomyxon, for instance, previous studies showed that distinct morphotypes can, in fact, share the same genotype (see Hallett et al., 2002; Eszterbauer et al., 2006; Zhao et al., 2016). Consequently, the morphological characterization of actinospores is important but not enough for the description of new types, making the acquisition of molecular data indispensable for the identification of actinosporean stages. Our study strengthens this contention, given that without the use of molecular data, the Aurantiactinomyxon type described here could have been easily misidentified with the Aurantiactinomyxon type 1 of Negredo and Mulcahy (2001), as these types share a strong morphological resemblance

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(Negredo and Mulcahy, 2001; Negredo et al., 2003). Despite morphological comparison to types without molecular data having revealed significant similarity to the Aurantiactinomyxon type of McGeorge et al. (1997), Aurantiactinomyxon type of Xiao and Desser (1998) and Aurantiactinomyxon type 2 of Özer et al. (2002), correspondence to any of these types could never be established with confidence, given their different hosts, habitats and geographical origins. In addition to the known unreliability of the usage of morphological criteria for type differentiation (see Hallett et al., 2002; Eszterbauer et al., 2006; Zhao et al., 2016), aurantiactinomyxon appear to have strict host specificity, considering that no type has been molecularly proven to infect more than a single annelid species. Thus, we suggest that extensive morphological comparisons to all types that are without molecular data and have distinct oligochaete hosts are unnecessary. Actinospores of new isolates should only be identified either by DNA match or through a comprehensive morphological and biological comparison to known types sharing the same annelid host, regardless of there being high morphological similarity to other types. Morphological features and genetic data identified the host species of the new Aurantiactinomyxon type as being Tubificoides pseudogaster. This annelid species is known to host several types of sphaeractinomyxon in Portuguese estuaries (this study; Rangel et al., 2016a) and is further reported here to host infection by a member of the synactinomyxon collective group. Despite this significant biodiversity, concomitant infections were never observed in individual specimens. This is congruent with the very low infection rates commonly reported in wild oligochaete populations (e.g. Hallett et al., 2001; Negredo and Mulcahy, 2001; Atkinson and Bartholomew, 2009). In the same manner, the low value of individual prevalence of infection determined in this study (0.3%) agrees with those previously reported for aurantiactinomyxon types (e.g. Xiao and Desser, 1998; Xi et al., 2015; Zhao et al., 2016; Milanin et al., 2017). Actinospore development was further demonstrated to follow the pattern typically reported for members of the aurantiactinomyxon collective group; development within the host was asynchronous, but sporogony within the pansporocysts was synchronous. Multiple layers of pansporocysts were present in the intestinal epithelium, partially protruding into the coelomic cavity (e.g. Marcucci et al., 2009; Zhao et al., 2017). Almost all aurantiactinomyxon types reported in the literature infect oligochaete species belonging to the family Naididae Ehrenberg, 1828 [currently includes the members of the former Tubificidae (Erséus et al., 2008)], be it in freshwater or marine environments. Aurantiactinomyxon pavinsis constitutes the only exception, having been widely reported to infect the freshwater lumbriculid Stylodrilus heringianus Claparède, 1862 (see Marques, 1984; Oumouna et al., 2003; Holzer et al., 2004; Marcucci et al., 2009). A few types have their oligochaete hosts identified only up to the genus- or family-level, but all infect naidids (see Marques, 1984; Grossheider and Körting, 1992; Benajiba and Marques, 1993), while the

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Aurantiactinomyxon types 7 and 11 of El-Mansy et al. (1998), Aurantiactinomyxon type 1 of Oumouna et al. (2003) and Aurantiactinomyxon type 1 of Hallett et al. (2006) constitute the only members of the collective group that lack host information. Up until this study, only the three aurantiactinomyxon types described by Hallett et al. (1997) were known to occur in the marine environment, parasitizing the naidids Limnodriloides toloensis Erséus, 1984, Pacifidrilus vanus (Erséus, 1984) and P. darvelli (Erséus, 1984). The species Tubificoides pseudogaster is, therefore, identified here as another member of the family Naididae serving as host for the aurantiactinomyxon collective group in marine environments. To date, few other actinosporean types have been described from marine oligochaetes and all to parasitize naidids. These marine oligochaete-infecting types mostly belong to the sphaeractinomyxon collective group, with few representatives belonging to endocapsa and triactinomyxon. The 13 distinct types of sphaeractinomyxon recently reported from Portuguese estuaries are hosted by T. pseudogaster, its congener T. insularis (Stephenson, 1923), and Limnodriloides agnes Hrabĕ, 1967 (see Rangel et al., 2016b; Rocha et al., 2019b). Worldwide, the remaining known marine sphaeractinomyxon types were also reported from naidids, belonging to the genera Ainudrilus Finogenova, 1982, Aktedrilus Knöllner, 1935, Bathydrilus Cook, 1970, Doliodrilus Erséus, 1984, Heronidrilus Erséus and Jamieson, 1981, Limno- driloides Pierantoni, 1903 and Thalassodrilides Brinkhurst and Baker, 1979 (Hallett et al., 1997, 1998, 2001; Hallett and Lester, 1999). Similarly, the three marine types included within the endocapsa collective group were reported from naidids of the genera Heronidrilus, Heterodrilus Pierantoni, 1902 and Thalassodrilides (Hallett et al., 1999, 2001). Lastly, to our best knowledge there are 5 marine triactinomyxon types described in the literature from naidids of the subfamily Limnodriloidinae Erséus, 1982 (including Limnodriloides and Tectidrilus spp.) (see Roubal et al., 1997; Hallett et al., 2001; Rangel et al., 2015, 2017). Overall, this suggests that the family Naididae played a preponderant role in the establishment and evolution of myxosporeans in invertebrate communities of estuarine and marine habitats. Recently, a comprehensive co-phylogenetic study by Holzer et al. (2018) evidenced that oligochaetes and polychaetes are the most ancient hosts of myxosporeans. As such, it can be assumed that the composition, ecology, spatial distribution and susceptibility of these hosts is crucial for myxosporean evolution and diversification. The Naididae is probably the most diverse and cosmopolitan oligochaete family, being present in all biogeographic regions, including sub-Antarctic islands (Timm and Martin, 2015). Its involvement in myxosporean settlement and evolution, therefore, most likely relates to the high availability of its species in different aquatic habitats worldwide. Conjectures to other potential factors possibly influencing the reported range of myxosporean infections in naidids are difficult to be performed based on the limited information currently available for annelid-myxosporean interactions, especially in marine habitats. Future research should, therefore, aim to provide insight on the host-,

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Chapter VIII | Life cycle inference of Paramyxidium giardi parasite-, and environmental-related factors that influence myxosporean infection in annelid hosts. The acquisition of further information on this subject will, most certainly, prove to be relevant in improving our knowledge of myxosporean evolution. Nowadays, the recognition of myxosporean life cycles is mainly based on DNA match between myxosporean and actinosporean counterparts. This molecular approach for establishing life cycle connections relies on the typically significant interspecific variability, and limited intraspecific variability, of the SSU rDNA gene of myxosporeans (Køie et al., 2004; Atkinson and Bartholomew, 2009). In some cases, however, it can be difficult to distinguish between different species and different geographic genotypes of one single species. Molecularly inferred life cycles range from 99.4% to 100% similarity between the SSU rDNA sequences of myxosporean and actinosporean counterparts (see Eszterbauer et al., 2015). These values agree with the intraspecific variability generally accepted for myxosporeans, which can range between 0‒3.6%, but is usually lower than 1% (Atkinson et al., 2015 and references therein). In this study, comparative molecular analysis of the SSU rDNA gene revealed the Aurantiactinomyxon type infecting T. pseudogaster sharing 99.4% of similarity with P. giardi, thus inferring its involvement in the life cycle of this myxosporean pathogen. The low genetic difference obtained (0.6%) is most likely representative of intraspecific variability due to the different geographic origin of the biological material, as previously suggested in the life cycle studies of Myxidium truttae Léger, 1930, Myxobolus cultus Yokoyama et al., 1995 and Thelohanellus kitauei Egusa and Nakajima, 1981 (see Holzer et al., 2004; Eszterbauer et al., 2006; Xi et al., 2013; Zhao et al., 2016). In these cases, the emergence of genotypically different subspecies was hypothesized to be the outcome of geographic isolation, with subsequent evolutionary adaptation to distinct environmental factors, and possibly different invertebrate hosts. In order to confirm our assumption of intraspecific variability between geographic isolates of P. giardi, a myxozoan survey on A. anguilla from the River Minho is being prepared and will aim to acquire molecular information from myxospores. This will allow to verify if genetic variation also occurs within a given geographic location and/or between life cycle counterparts. In this context, acquiring information from molecular markers other than the SSU rDNA gene may prove necessary in order to either confirm or refute the observations of relatedness performed here. Similarly, it would be interesting to sample marine oligochaetes from Icelandic Rivers where infections by P. giardi are known to occur. The information thus far available for myxosporean life cycles demonstrates that there is no obvious correspondence between actinosporean and myxosporean morphotypes. Accordingly, phylogenetic analyses show most collective groups spanning distinct myxosporean suborders, as is the case of aurantiactinomyxon (e.g. Rocha et al., 2019b). Members of aurantiactinomyxon have been implicated in the life cycles of species belonging

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Chapter VIII | Life cycle inference of Paramyxidium giardi to the genera Chloromyxum, Henneguya, Hoferellus, Myxobolus, Myxidium and Thelohanellus (Styer et al., 1991; Grossheider and Körting, 1992; Benajiba and Marques, 1993; Lin et al., 1999; Holzer et al., 2004; Eszterbauer et al., 2006; Molnár et al., 2010). Our results further broaden the array of genera associated with the aurantiactinomyxon collective group, by molecularly inferring the involvement of the type described here in the life cycle of Paramyxidium giardi. This finding is corroborated by the experimental transmission study performed by Benajiba and Marques (1993), who also identified the actinosporean counterpart of P. giardi as an aurantiactinomyxon type, but developing in a freshwater tubificid. Accordingly, Freeman and Kristmundsson (2018) suggested freshwater oligochaetes as the most probable annelid hosts of Paramyxidium spp., considering that known fish hosts [European eel and Indo-Pacific tarpon Megalops cyprinoides (Broussonet, 1782)], were sampled from freshwater habitats. Nonetheless, both these species are migratory fish that spawn at sea producing leptocephalic larvae that migrate into inland waters to grow. Taking into account that a significant portion of their lives is spent in fresh- and brackish waters, it is possible that infection is acquired from marine oligochaetes inhabiting estuarine habitats, as our results suggest in the case of P. giardi. During our myxozoan survey, molecular evidence was also found for the development of a single type of sphaeractinomyxon in both a freshwater oligochaete species of the genus Potamothrix Vejdovský and Mrázek, 1903 and T. pseudogaster, suggesting that the myxosporean counterpart of this type uses a fish host that is not entirely restricted to freshwater, most probably a grey mullet (unpublished results). Congruently, the possibility of P. giardi having acquired both marine and freshwater oligochaetes as hosts cannot be disregarded, considering that the ecology of the European eel allows it to contact with intrinsically distinct invertebrate communities throughout its life. The genus Paramyxidium Freeman and Kristmundsson, 2018 encompasses species that, whilst being morphologically similar to Myxidium, differ by having histozoic development in various tissues of fish from Elopomorpha (see Freeman and Kristmundsson, 2018). The European eel is the sole representative of Elopomorpha in Portuguese waters. Thus far, it has been recognized to host four myxosporean species in this geographic location, including P. giardi from infections that occur in multiple organs (Ventura and Paperna, 1984; Azevedo et al., 1989; Saraiva and Chubb, 1989; Saraiva and Eiras, 1996; Hermida et al., 2008). The study by Freeman and Kristmundsson (2018), however, showed that P. giardi is not systemic, and that while myxospore morphology is conserved and, therefore, unreliable for identification at the species-level, the site of infection appears to be an important diagnostic feature for the group. As such, it is expected that the myxozoan survey of A. anguilla in Portuguese Rivers will lead to the discovery of other Paramyxidium spp.

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Acknowledgments

The authors acknowledge Professor Eduardo Rocha and the Laboratory of Histology ofthe Institute of Biomedical Sciences, University of Porto, for the usage of the Olympus BX50 light microscope, as well as Miguel Pereira for his iconographic assistance. This work was financially supported by the Foundation for Science and Technology (Lisbon, Portugal) within the scope of the Ph.D. fellowship grant attributed to S. Rocha (SFRH/BD/92661/2013) through the programme QREN-POPH/FSE; and the Engº António de Almeida Foundation (Porto, Portugal).

References

Atkinson, S.D. and Bartholomew, J.L. (2009). Alternate spore stages of Myxobilatus gasterostei, a myxosporean parasite of three-spined sticklebacks (Gasterosteus aculeatus) and oligochaetes (Nais communis). Parasitology Research 104, 1173‒ 1181. Atkinson, S.D., Bartošová-Sojková, P., Whipps, C.M. and Bartholomew, J.L. (2015). Approaches for characterising Myxozoan species. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan evolution, ecology and development. Switzerland: Springer International Publishing, pp. 111‒123. Azevedo, C., Lom, J. and Corral, L. (1989). Ultrastructural aspects of Myxidium giardi (Myxozoa, Myxosporea), parasite of the European eel Anguilla anguilla. Diseases of Aquatic Organisms 6, 55‒61. Bartholomew, J.L., Atkinson, S.D. and Hallett, S.L. (2006). Involvement of Manayunkia speciosa (Annelida : Polychaeta : Sabellidae) in the life cycle of Parvicapsula minibicornis, a myxozoan parasite of Pacific salmon. Journal of Parasitology 92, 742‒ 748. Bartholomew, J.L., Whipple, M.J., Stevens, D.G. and Fryer, J.L. (1997). The life cycle of Ceratomyxa shasta, a myxosporean parasite of salmonids, requires a freshwater polychaete as an alternate host. Journal of Parasitology 83, 859‒868. Benajiba, M.H. and Marques, A. (1993). The alternation of actinomyxidian and myxosporidian sporal forms in the development of Myxidium giardi (parasite of Anguilla anguilla) through oligochaetes. Bulletin of the European Association of Fish Pathologists 13, 100‒103. Cépède, C. (1906). Myxidium giardi Cépède, et la prétendue immunité des Anguilles a l’ègard des infections myxosporidiennes. Comptes Rendus des Séances de la Société de Biologie et de ses Filiales 6, 170–173.

Page | 210

Chapter VIII | Life cycle inference of Paramyxidium giardi

Copland, J.W. (1981). The occurrence and distribution of Myxidium giardi Cépède, 1906 in wild and cultured European eels, Anguilla anguilla L., in England. Journal of Fish Diseases 4, 231–242. Dias, E., Morais, P., Cotter, A.M., Antunes, C. and Hoffman, J.C. (2016). Estuarine consumers utilize marine, estuarine and terrestrial organic matter and provide connectivity among these food webs. Marine Ecology Progress Series 554, 21–34. Eiras, J.C. (2016). Parasites of marine, freshwater and farmed fishes of Portugal: a review. Brazilian Journal of Veterinary Parasitology 25, 259‒278. El-Mansy, A., Székely, C. and Molnár, K. (1998). Studies on the occurrence of actinosporean stages of fish myxosporeans in a fish farm of Hungary, with the description of triactinomyxon, raabeia, aurantiactinomyxon and neoactinomyxon types. Acta Veterinaria Hungarica 46, 259‒284. Erséus, C., Wetzel, M.J. and Gustavsson, L. (2008). ICZN rules – a farewell to Tubificidae (Annelida, Clitellata). Zootaxa 1744, 66‒68. Eszterbauer, E., Atkinson, S., Diamant, A., Morris, D., El-Matbouli, M. and Hartikainen, H. (2015). Myxozoan life cycles: practical approaches and insights. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan evolution, ecology and development. Switzerland: Springer International Publishing, pp. 175‒198. Eszterbauer, E., Marton, S., Rácz, O.Z., Letenyei, M. and Molnár, K. (2006). Morphological and genetic differences among actinosporean stages of fish-parasitic myxosporeans (Myxozoa): difficulties of species identification. Systematic Parasitology 65, 97‒114. Eszterbauer, E., Székely, C., Molnár, K. and Baska, F. (2000). Development of Myxobolus bramae (Myxosporea : Myxobolidae) in an oligochaete alternate host, Tubifex tubifex. Journal of Fish Diseases 23, 19‒25. Fiala, I. (2006). The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. International Journal for Parasitology 36, 1521–1534. Freeman, M.A. and Kristmundsson, A. (2018). Studies of Myxidium giardi Cépède, 1906 infections in Icelandic eels identifies a genetically diverse clade of myxosporeans that represents the Paramyxidium n. g. (Myxosporea: Myxidiidae). Parasites and Vectors 11, 551. Fujita, T. (1927). Studies on Myxosporidia of Japan. Journal of the Faculty of Agriculture of the Hokkaido Imperial University 16, 229–247. Grossheider, G. and Körting, W. (1992). First evidence that Hoferellus cyprini (Doflein, 1898) is transmitted by Nais sp. Bulletin of the European Association of Fish Pathologists 12, 17‒20. Hallett, S.L., Atkinson, S.D. and El-Matbouli, M. (2002). Molecular characterisation of two aurantiactinomyxon (Myxozoa) phenotypes reveals one genotype. Journal of Fish

Page | 211

Chapter VIII | Life cycle inference of Paramyxidium giardi

Diseases 57, 1‒14. Hallett, S.L., Atkinson, S.D., Erséus, C. and El-Matbouli, M. (2004). Molecular methods clarify morphometric variation in triactinomyxon spores (Myxozoa) released from different oligochaete hosts. Systematic Parasitology 57, 1‒14. Hallett, S.L., Atkinson, S.D., Erséus, C. and El-Matbouli, M. (2006). Myxozoan parasites disseminated via oligochaete worms as live food for aquarium fishes: descriptions of aurantiactinomyxon and raabeia actinospore types. Diseases of Aquatic Organisms 69, 213‒225. Hallett, S.L. and Diamant, A. (2001). Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia. Diseases of Aquatic Organisms 46, 197‒212. Hallett, S.L., Erséus, C. and Lester, R.J. (1997). Actinosporea from Hong Kong marine oligochaeta. In Morton, B. (ed). Proceedings of the eight international marine biological workshop: the marine flora and fauna of Hong Kong and Southern China. Hong Kong: Hong Kong University Press, pp. 1‒7. Hallett, S.L., Erséus, C. and Lester, R.J. (1999). Actinosporeans (Myxozoa) from marine oligochaetes of the Great Barrier Reef. Systematic Parasitology 44, 49‒57. Hallett, S.L., Erséus, C., O’Donoghue, P.J. and Lester, R.J.G. (2001). Parasite fauna of australian marine oligochaetes. Memoirs of the Queensland Museum 46, 555‒576. Hallett, S.L. and Lester, R.J. (1999). Actinosporeans (Myxozoa) with four developing spores within a pansporocyst: Tetraspora discoidea n. g. n. sp. and Tetraspora rotundum n. sp. International Journal of Parasitology 29, 419‒427. Hallett, S.L., O’Donoghue, P.J. and Lester, R.J.G. (1998). Structure and development of a marine actinosporean, Sphaeractinomyxon ersei n. sp. (Myxozoa). Journal of Eukaryotic Microbiology 45, 142‒150. Hermida, M., Saraiva, A. and Cruz, C. (2008). Metazoan parasite community of a European eel (Anguilla anguilla) population from an estuary in Portugal. Bulletin of the European Association of Fish Pathologists 28, 35‒40. Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. The Quarterly Review of Biology 66, 411‒453. Hine, P.M. (1975). Three new species of Myxidium (Protozoa: Myxosporidia) parasitic in Anguilla australis Richardson, 1848 and A. dieffenbachii Gray, 1842 in New Zealand. Journal of the Royal Society of New Zealand 5, 153–161. Hine, P.M. (1978). Variations in the spores of Myxidium zealandicum Hine, 1975 (Protozoa: Myxosporidia). New Zealand Journal of Marine and Freshwater Research 12, 189–195. Hine, P.M. (1980). A review of some species of Myxidium Bütschli, 1882 (Myxosporea) from

Page | 212

Chapter VIII | Life cycle inference of Paramyxidium giardi

eels (Anguilla spp.). The Journal of Protozoology 27, 260–267. Holzer, A.S., Bartošová-Sojková, P., Born-Torrijos, A., Lövy, A., Hartigan, A. and Fiala, I. (2018). The joint evolution of the Myxozoa and their alternate hosts: A cnidarian recipe for success and vast biodiversity. Molecular Ecology 27, 1651‒1666. Holzer, A.S., Sommerville, C. and Wootten, R. (2004). Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences. International Journal of Parasitology 34, 1099‒1111. Holzer, A.S., Sommerville, C. and Wootten, R. (2006). Molecular identity, phylogeny and life cycle of Chloromyxum schurovi Shul'man & Ieshko 2003. Parasitology Research 99, 90‒96. Ishii, S. (1915). Myxosporidiosis of the Japanese eel. Zoological Magazine of Tokyo 27, 372– 382. Kallert, D.M., Eszterbauer, E., El-Matbouli, M., Erséus, C. and Haas, W. (2005). The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore. Journal of Fish Diseases 28, 71‒79. Karlsbakk, E. and Køie, M. (2012). The marine myxosporean Sigmomyxa sphaerica (Thélohan, 1895) gen. n., comb. n. (syn. Myxidium sphaericum) from garfish (Belone belone (L.)) uses the polychaete Nereis pelagica L. as invertebrate host. Parasitology Research 110, 211‒218. Kent, M.L., Whitaker, D.J. and Margolis, L. (1993). Transmission of Myxobolus arcticus Pugachev and Khokhlov, 1979, a myxosporean parasite of Pacific salmon, via a triactinomyxon from the aquatic oligochaete Stylodrilus heringianus (Lumbriculidae). Canadian Journal of Zoology 71, 1207‒1211. Køie, M., Karlsbakk, E. and Nylund, A. (2007). A new genus Gadimyxa with three new species (Myxozoa, Parvicapsulidae) parasitic in marine fish (Gadidae) and the two-host life cycle of Gadimyxa atlantica n. sp. Journal of Parasitology 93, 1459‒1467. Køie, M., Karlsbakk, E. and Nylund, A. (2008). The marine herring myxozoan Ceratomyxa auerbachi (Myxozoa: Ceratomyxidae) uses Chone infundibuliformis (Annelida: Polychaeta: Sabellidae) as invertebrate host. Folia Parasitologica 55, 100‒104. Køie, M., Whipps, C.M. and Kent, M.L. (2004). Ellipsomyxa gobii (Myxozoa: Ceratomyxidae) in the common goby Pomatoschistus microps (Teleostei: Gobiidae) uses Nereis spp. (Annelida: Polychaeta) as invertebrate hosts. Folia Parasitologica 51, 14–18. Kumar, S., Stecher, G. and Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33, 1870– 1874. Kvist, S., Sarkar, I.N. and Erséus, C. (2010). Genetic variation and phylogeny of the cosmopolitan marine genus Tubificoides (Annelida: Clitellata: Naididae: Tubificinae).

Page | 213

Chapter VIII | Life cycle inference of Paramyxidium giardi

Molecular Phylogenetics and Evolution 57, 687–702. Lin, D., Hanson, L.A. and Pote, L.M. (1999). Small subunit ribosomal RNA sequence of Henneguya exilis (class Myxosporea) identifies the actinosporean stage from an oligochaete host. Journal of Eukaryotic Microbiology 46, 66‒68. Lom, J. and Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53, 1–36. Lom, J., McGeorge, J., Feist, S.W., Morris, D. and Adams, A. (1997). Guidelines for the uniform characterisation of the actinosporean stages of parasites of the phylum Myxozoa. Diseases of Aquatic Organisms 30, 1‒9. Marcucci, C., Caffara, M. and Goretti, E. (2009). Occurrence of actinosporean stages (Myxozoa) in the Nera River system (Umbria, central Italy). Parasitology Research 105, 1517‒1530. Marques, A. (1984). Contribution à la connaissance des Actinomyxidies: ultrastructure, cycle biologique, systématique. PhD Thesis, Université des Sciences et Techniques de Languedoc, Montepellier, France, pp. 218. Marton, S. and Eszterbauer, E. (2011). The development of Myxobolus pavlovskii (Myxozoa: Myxobolidae) includes an echinactinomyxon-type actinospore. Folia Parasitologica 58, 157‒163. McGeorge, J., Sommerville, C. and Wootten, R. (1997). Studies of actinosporean myxozoan stages parasitic in oligochaetes from the sediments of a hatchery where Atlantic salmon harbour Sphaerospora truttae infection. Diseases of Aquatic Organisms 30, 107‒119. Milanin, T., Atkinson, S.D., Silva, M.R., Alves, R.G., Maia, A.A. and Adriano, E.A. (2017). Occurrence of two novel actinospore types (Cnidaria: Myxosporea) in Brazilian fish farms, and the creation of a novel actinospore collective group, Seisactinomyxon. Acta Parasitologica 62, 121‒128. Molnár, K., Marton, S., Székely, C. and Eszterbauer, E. (2010). Differentiation of Myxobolus spp. (Myxozoa: Myxobolidae) infecting roach (Rutilus rutilus) in Hungary. Parasitology Research 107, 1137‒1150. Negredo, C., Dillane, E. and Mulcahy, M.F. (2003). Small subunit ribosomal DNA characterization of an unidentified aurantiactinomyxon form and its oligochaete host Tubifex ignotus. Diseases of Aquatic Organisms 54, 229‒241. Negredo, C. and Mulcahy, M.F. (2001). Actinosporean infections in oligochaetes in a river system in southwest Ireland with descriptions of three new forms. Diseases of Aquatic Organisms 46, 67‒77. Oumouna, M., Hallett, S.L., Hoffmann, R.W. and El-Matbouli, M. (2003). Seasonal occurrence of actinosporeans (Myxozoa) and oligochaetes (Annelida) at a trout hatchery in Bavaria, Germany. Parasitology Research 89, 170‒184.

Page | 214

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Özer, A., Wootten, R. and Shinn, A.P. (2002). Survey of actinosporean types (Myxozoa) belonging to seven collective groups found in a freshwater salmon farm in Northern Scotland. Folia Parasitologica 49, 189‒210. Palumbi, S., Martin, A., Romano, S., McMillan, W.O., Stice, L. and Grabowski, G. (2002). The Simple Fools Guide to PCR, Version 2.0. University of Hawaii, Honolulu. http://palumbi.stanford.edu/SimpleFoolsMaster. Rangel, L.F., Castro, R., Rocha, S., Cech, G., Casal, G., Azevedo, C., Székely, C., Cavaleiro, F. and Santos, M.J. (2016a). Description of new types of sphaeractinomyxon actinospores (Myxozoa: Myxosporea) from marine tubificid oligochaetes, with a discussion on the validity of the tetraspora and the endocapsa as actinospore collective group names. Parasitology Research 115, 2341‒2351. Rangel, L.F., Castro, R., Rocha, S., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2016b). Tetractinomyxon stages genetically consistent with Sphaerospora dicentrarchi (Myxozoa: Sphaerosporidae) found in Capitella sp. (Polychaeta: Capitellidae) suggest potential role of marine polychaetes in parasite's life cycle. Parasitology 143, 1067‒1073. Rangel, L.F., Cech, G., Székely, C. and Santos, M.J. (2011). A new actinospore type Unicapsulactinomyxon (Myxozoa), infecting the marine polychaete, Diopatra neapolitana (Polychaeta: Onuphidae) in the Aveiro estuary, Portugal. Parasitology 138, 698‒712. Rangel, L.F., Rocha, S., Casal, G., Castro, R., Severino, R., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2017). Life cycle inference and phylogeny of Ortholinea labracis n. sp. (Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm. Journal of Fish Diseases 40, 243‒ 262. Rangel, L.F., Rocha, S., Castro, R., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2015). The life cycle of Ortholinea auratae (Myxozoa: Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete. Parasitology Research 114, 2671‒2678. Rangel, L.F., Santos, M.J., Cech, G. and Székely, C. (2009). Morphology, molecular data, and development of Zschokkella mugilis (Myxosporea, Bivalvulida) in a polychaete alternate host, Nereis diversicolor. Journal of Parasitology 95, 561‒569. Rocha, S., Casal, G., Rangel, L., Castro, R., Severino, R., Azevedo, C. and Santos, M.J. (2015). Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae). Parasitology International 64, 305‒313. Rocha, S., Rangel, L.F., Castro, R., Severino, R., Azevedo, C., Santos, M.J. and Casal, G.

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(2019). The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis. Journal of Invertebrate Pathology 160, 33‒42. Roubal, F.R., Hallett, S.L. and Lester, R.J.G. (1997). First record of triactinomyxon actinosporean in marine oligochaete. Bulletin of the European Association of Fish Pathologists 17, 83‒85. Saraiva, A. and Chubb, J.C. (1989). Preliminary observations on the parasites of Anguilla anguilla (L.) from Portugal. Bulletin of the European Association of Fish Pathologists 9, 88‒89. Saraiva, A. and Eiras, J.C. (1996). Parasite community of european eel Anguilla anguilla (L.) in the river Este, northern Portugal. Research and Reviews in Parasitology 56, 179‒ 183. Styer, E.L., Harrison, L.R. and Burtle, G.J. (1991). Communications: Experimental production of proliferative gill disease in channel catfish exposed to a myxozoan-infected oligochaete, Dero digitata. Journal of Aquatic Animal Health 3, 288‒291. Székely, C., Borkhanuddin, M.H., Cech, G., Kelemen, O. and Molnár, K. (2014). Life cycles of three Myxobolus spp. from cyprinid fishes of Lake Balaton, Hungary involve triactinomyxon-type actinospores. Parasitology Research 113, 2817‒25. Székely, C., Eiras, J.C. and Eszterbauer, E. (2005). Description of a new synactinomyxon type from the River Sousa, Portugal. Diseases of Aquatic Organisms 66, 9‒14. Timm, T. and Martin, P.J. (2015). Clitellata: Oligochaeta. In Thorp JH and Rogers DC (eds). Thorp and Covich’s Freshwater Invertebrates (4th edition). USA: Academic Press, pp. 529‒549. Treasurer, J.W. and Cox, D. (1997). The occurrence of Myxidium giardi Cépède, in cultured eels, Anguilla anguilla L., in West Scotland. Bulletin of the European Association of Fish Pathologists 17, 171–173. Ventura, M.T. and Paperna, I. (1984). Histopathology of Myxidium giardi Cépède, 1906 infection in European eels, Anguilla anguilla L., in Portugal. Aquaculture 43, 357‒368. Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215‒219. Wolf, K. and Markiw, M.E. (1984). Biology contravenes taxonomy in the Myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science 225, 1449‒ 1452.

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Xi, B.W., Zhang, J.Y., Xie, J., Pan, L.K., Xu, P. and Ge, X.P. (2013). Three actinosporean types (Myxozoa) from the oligochaete Branchiura sowerbyi in China. Parasitology Research 112, 1575‒1582. Xi, B.W., Zhou, Z.G., Xie, J., Pan, L.K., Yang, Y.L. and Ge, X.P. (2015). Morphological and molecular characterization of actinosporeans infecting oligochaete Branchiura sowerbyi from Chinese carp ponds. Diseases of Aquatic Organisms 114, 217‒228. Xiao, C.X. and Desser, S.S. (1998). Actinosporean stages of myxozoan parasites of oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of echinactinomyxon, neoactinomyxum, aurantiactinomyxon, guyenotia, synactinomyxon and antonactinomyxon. Journal of Parasitology 84, 1010‒1019. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1993a). Involvement of Branchiura sowerbyi (Oligochaeta, Annelida) in the transmission of Hoferellus carassii (Myxosporea, Myxozoa), the causative agent of kidney enlargement disease (KED) of goldfish Carassius auratus. Fish Pathology 28, 135‒139. Zhao, D., Borkhanuddin, M.H., Wang, W., Liu, Y., Cech, G., Zhai, Y. and Székely, C. (2016). The life cycle of Thelohanellus kitauei (Myxozoa: Myxosporea) infecting common carp (Cyprinus carpio) involves aurantiactinomyxon in Branchiura sowerbyi. Parasitology Research 115, 4317‒4325. Zhao, D.D., Zhai, Y.H., Liu, Y., Wang, S.J. and Gu, Z.M. (2017). Involvement of aurantiactinomyxon in the life cycle of Thelohanellus testudineus (Cnidaria: Myxosporea) from allogynogenetic gibel carp Carassius auratus gibelio, with morphological, ultrastructural, and molecular analysis. Parasitology Research 116, 2449‒2456.

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Chapter IX

Characterization of sphaeractinomyxon types (Cnidaria, Myxozoa) from marine and freshwater oligochaetes in a Portuguese estuary, with the demise of the endocapsa collective group

This chapter was adapted from:

Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa

Abstract

Six types of sphaeractinomyxon are reported from the coelomic cavity of oligochaetes collected from the River Minho estuary in Northern Portugal. Four new types are morphologically and molecularly described from freshwater species belonging to the genera Psammoryctides Hrabe, 1964 and Potamothrix Vejdovský and Mrázek, 1902 in the upper estuary, thus significantly increasing the number of known freshwater sphaeractinomyxon. In the lower estuary, Sphaeractinomyxon types 8 and 10 of Rangel et al. (2016a) are recorded infecting the marine oligochaete Tubificoides pseudogaster (Dahl, 1960). A single specimen of T. pseudogaster further displayed infection by one of the four new types found in the upper estuary, suggesting the involvement of sphaeractinomyxon in the life cycles of myxosporean species that infect migratory fish hosts. The acquisition of these second hosts is proposed to have allowed the myxosporean counterparts of sphaeractinomyxon to cross environmental barriers and conquer new habitats. Phylogenetic analyses of the SSU rDNA gene reveal the four new types clustering within the monophyletic clade of mugiliform-infecting myxobolids, thus strengthening the previously proposed involvement of the sphaeractinomyxon collective group in the life cycles of this specific group of myxosporeans. Endocapsa types also cluster within this clade, having actinospores that differ from those of sphaeractinomyxon only in the presence of “valvular swellings” that do not change when in contact with water. In this study, however, one type was found displaying actinospores with and without “valvular swellings” in the same oligochaete specimen. This overlap in actinospore morphology is given as ground for the demise of the endocapsa collective group.

Introduction

Infection of aquatic oligochaetes by actinospores was first reported by Štolc (1899), who described Synactinomyxon tubificis, Triactinomyxon ignotum and Hexactinomyxon psammo- ryctis developing in tubificids collected from the River Vltava in Czech Republic. In the years that followed this discovery, the acquisition of knowledge pertaining to actinosporean infections was relatively limited, not only due to low prevalence of infection, but possibly also because they were thought to have little direct economic significance. In 1984, however, Wolf and Markiw demonstrated that the life cycle of Myxobolus cerebralis Hofer, 1903, an economically important myxosporean parasite of salmonid fishes, developed triactinomyxon actinospores in the gut epithelium of the oligochaete Tubifex tubifex (Müller, 1774). This discovery lead to the demise of the class Actinosporea (see Kent et al., 1994), but simultaneously boosted interest in the study of actinospores as life cycle stages of Myxosporea. Up until the invalidation of Actinosporea, six sphaeractinomyxon had been described: S.

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa stolci, S. gigas, S. danicae, S. ilyodrili, S. amanieui and S. rotundum (Caullery and Mesnil, 1904; Granata, 1923; Georgevitch, 1938; Jirovec, 1940; Puytorac, 1963; Marques, 1984). These types were named following the binomial nomenclature system and described mainly based on the morphological characters of their actinospores, which were represented in schematic drawings. In the years that followed, Sphaeractinomyxon types 1 and 2 of Hallett et al. (1997), Sphaeractinomyxon ersei (Hallett et al., 1998) and Sphaeractinomyxon leptocapsula (Hallett et al., 1999) were described from marine oligochaetes in Hong Kong and Australia, but only S. ersei had its SSU rDNA gene sequenced. More recently, 13 sphaeractinomyxon types were morphologically and molecularly described from marine oligochaetes in two Portuguese estuaries (Rangel et al., 2016a; Rocha et al., 2019b). Overall, the implementation of molecular methodologies to the study of sphaeractinomyxon types has revealed a higher biodiversity than expected based on morphology-based criteria. In fact, Rangel et al. (2016a) showed that actinospore measurements overlap between different types of this collective group, so that the usage of molecular tools is indispensable for the description of new types. Phylogenetic analyses of the SSU rDNA gene further disclosed a possible involvement of the sphaeractinomyxon collective group in the life cycle of mugiliform-infecting myxobolids (Rocha et al., 2019b), which remains to be proved by either molecular inference or experimental transmission studies. In the late 90’s, two other collective groups were erected in order to encompass actinospores that differed from sphaeractinomyxon in specific aspects of their morphology and sporogonic development. The tetraspora collective group comprised types that while displaying the typical morphology of sphaeractinomyxon, developed in groups of four within the pansporocysts, instead of the usual groups of eight. However, recent studies acknowledged this character as being too variable to establish distinction between the two collective groups. Consequently, tetraspora was deemed invalid and its two types were transferred to sphaeractinomyxon: Sphaeractinomyxon types A and B of Hallett and Lester (1999), formerly classified as Tetraspora discoidea and Tetraspora rotundum, respectively (Rocha et al., 2019b). These types were described from Australian marine oligochaetes based solely on actinospore morphology (Hallett and Lester, 1999), but the first has since then been molecularly reported from its type host. In turn, endocapsa was erected to encompass actinospores that differed from sphaeractinomyxon only in the presence of “valvular swellings” that did not change when in contact with water (Hallett et al., 1999). The validity of this collective group was questioned in recent studies (Rangel et al., 2016a; Rocha et al., 2019b). Nonetheless, it remains valid and currently comprises four types: Endocapsa rosulata and Endocapsa stepheni from the marine oligochaete Heterodrilus cf. keenani Erséus, 1981 in Australia (Hallett et al., 1999, 2001); Endocapsa type 1 of Hallett et al. (2001) from immature tubificids, also in Australia; and Endocapsa type of Székely et al. (2007) from the freshwater

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa oligochaete Psammoryctides albicola (Michaelsen, 1901) in Syria. Molecular data of the SSU rDNA gene is available for both Endocapsa rosulata and the Endocapsa type of Székely et al. (2007). This study proposed to investigate the biodiversity of actinosporean stages infecting freshwater and marine oligochaetes in a Northern Portuguese River, from where only one myxosporean species was previously reported (see Rocha et al., 2019a). Among the different collective groups recorded, sphaeractinomyxon was represented by two known and four new types that are here morphologically and molecularly described. The morphological variability found among the actinospores belonging to one of the new types is given as ground for the demise of the endocapsa collective group.

Materials and methods

Sampling sites and morphological characterization

Between 2015 and 2016, collections of mud were performed from two sites in the River Minho, northern Portugal: one site was located in the upper estuary, close to the fyke-nets stationed near the village of “Vila Nova de Cerveira” (41° 56′ N, 08° 45′ W); and the other was located in the lower estuary, near the village of “Caminha” (41° 52' N, 08° 50' W). Mud from the upper estuary was collected using a Van Veen grab sediment sampler with an area of 500 cm2 and a maximum capacity of 5000 cm3, while mud from the lower estuary was manually collected at low tide. In the laboratory, oligochaetes were isolated from the mud and kept at 4 ºC, individually placed into 12-well plates either containing dechlorinated freshwater or brackish water (15‰ salinity), in accordance to the sampling site from which the mud was obtained. Salinity values at the fyke-nets in the upper estuary are generally bellow 0.5‰, increasing slightly during dry summer periods. In turn, salinity values in the lower estuary range between 15 and 40‰ throughout the year (see Dias et al., 2016). All specimens were examined using the light microscope for the detection of actinosporean stages in internal tissues and cavities. Developmental stages and free actinospores were observed and photographed using an Olympus BX41 light microscope (Olympus, Japan). Morphometry was determined from fresh material, in accordance to Lom et al. (1997). Measurements include the mean value ± standard deviation (SD), range of variation, and number of measured actinospores (n). All measurements are in micrometres.

Molecular methods

Genomic DNA from infected oligochaetes was extracted using the GenEluteTM

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa

Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, USA), following the manufacturer’s instructions. The DNA was stored in 50 μl of TE buffer at -20 ºC until further use. The SSU rDNA gene of the actinospores was amplified using both universal and myxosporean-specific primers: the 5’-end by pairing the primer 18E (5’-CTG GTT GAT CCT GCC AGT-3’) (Hillis and Dixon, 1991) with ACT3r (5’-ATT GTT CGT TCC ATG-3’) (Rocha et al., 2014) and MYX4R (5’-CTG ACA GAT CAC TCC ACG AAC-3’) (Hallett and Diamant, 2001); and the 3’-end by pairing the primers ACT3f (5’-CAT GGA ACG AAC AAT-3’) (Hallett and Diamant, 2001) and MYX4F (5’-GTT CGT GGA GTG ATC TGT CAG-3’) (Rocha et al., 2015) with 18R (5’-CTA CGG AAA CCT TGT TAC G-3’) (Whipps et al., 2003). PCRs were performed in 50 µl reactions using 10 pmol of each primer, 10 nmol of each dNTP, 2.0 mM of MgCl2, 5 µl 10× Taq polymerase buffer, 1.5 units Taq DNA polymerase (NZYTech, Lisbon, Portugal), and 3 µl (approximately 100–150 ng) of genomic DNA. The reactions were run on a Hybaid PxE Thermocycler (Thermo Electron Corporation, Milford, Massachusetts, USA), with initial denaturation at 95 ºC for 3 min, followed by 35 cycles of 94 ºC for 45 sec, 53 ºC for 45 sec, and 72 ºC for 90 sec. The final elongation step was performed at 72 ºC for 7 min. In turn, the 16S rRNA gene of the oligochaete hosts was amplified using the universal primers 16sar-L (5’- CGC CTG TTT ATC AAA AAC AT-3’) and 16sbr-H (5’-CCG GTC TGA ACT CAG ATC ACG T-3’) (Palumbi et al., 2002). PCRs were carried out according to the conditions previously mentioned for the actinospores. Five-µl aliquots of the PCR products were electrophoresed through a 1% agarose 1× tris-acetate-EDTA buffer (TAE) gel stained with ethidium bromide. Amplified DNA was purified using Puramag™ magnetic beads coated with carboxylic acid groups (MCLAB, San Francisco, California, USA). Sequencing reactions were performed with the same primers used for amplification on a BigDye Terminator v3.1 Cycle Sequencing Kit from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, EUA), and were run on an ABI3700 DNA analyzer from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, EUA).

Phylogenetic analyses

The partial SSU rDNA sequences of each case isolate were aligned using MEGA7 (Kumar et al., 2016) for the construction of assembled sequences. A dataset was generated according to the highest similarity scores obtained using BLAST search. Accordingly, all sphaeractinomyxon types with available SSU rDNA sequences were incorporated in the dataset, as well as closely-related myxosporean and actinosporean stages, i.e. all Myxobolus spp. thus far reported from mullet hosts, Endocapsa rosulata, Endocapsa type of Székely et al. (2007) and Triactinomyxon type of Székely et al. (2007). Sequences were aligned using MAFFT version 7 available online, and distance estimation was calculated in MEGA7, with the

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa p-distance model and all ambiguous positions removed for each sequence pair. For phylogenetic analyses, other representatives of the clade of myxobolids were included in the dataset, as well as Myxidium lieberkuehni Bütschli, 1882 (X76638) as outgroup species. The final dataset comprised 62 SSU rDNA sequences that were aligned using the software MAFFT version 7 available online, and posteriorly manually edited in MEGA7. Phylogenetic trees were constructed using maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI). ML and MP analyses were conducted in MEGA7 with bootstrap confidence values calculated from 500 replicates. ML analyses were performed based on the General Time Reversible model with Gamma distributed rate and Invariant sites (GTR + G + I) selected on the basis of the lowest score of Bayesian Information Criterion (BIC) and corrected Akaike Information Criterion (AIC) with the MEGA package. MP analyses were obtained using the Subtree-Pruning-Regrafting algorithm with a search level of 1 and random initial tree addition of 10 replicates. BI analyses were performed in MrBayes v.3.2.6 (Ronquist and Huelsenbeck, 2003), using the general time reversible model with gamma-shaped rate variations across sites (Invgamma) (GTR+I+Γ). Posterior probability distributions were generated using the Markov Chain Monte Carlo (MCMC) method, with four chains running simultaneously for 500,000 generations. Burn-in was set at 25%, and trees were sampled every 100 generations to compile the majority rule consensus tree.

Results

Myxozoan survey and overall prevalence of infection of sphaeractinomyxon

During this study, 4,593 oligochaete specimens were isolated from the sediments collected from the River Minho: 4,016 from the sampling site near the fyke-nets in the upper estuary, and 577 from the lower estuary near “Caminha”. Myxozoan infection was found in a total of 61 oligochaetes, with the parasites’ developmental stages being either located in the intestinal epithelium or in the coelomic cavity. The actinospores and developmental stages occurring in the intestinal epithelium belonged to the aurantiactinomyxon (4 types), synactinomyxon (2 types) and raabeia (1 type) collective groups. In turn, the actinosporean stages developing in the coelomic cavity were all identified as belonging to the sphaeractinomyxon collective group and are reported here. In total, six different types of sphaeractinomyxon could be distinguished: four constitute new types that are described here, while the other two are known types recorded for the first time in the study area. Identification and characterization of these types was based on both the morphological traits of mature actinospores (whenever present in the infected oligochaetes) and molecular data of the SSU rDNA gene. Young developmental stages could

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa only be identified based on molecular data. Overall prevalence of infection of sphaeractinomyxon in the River Minho estuary was determined to be 0.2% (10 infected in a total of 4,593 oligochaetes examined); 0.1% in the upper estuary (5 infected in a total of 4,016 oligochaetes examined); and 0.9% in the lower estuary (5 infected in a total of 577 oligochaetes examined). Infected oligochaetes were identified through the combinaned analysis of morphological features and molecular data of the 16S mitochondrial DNA (mtDNA). In the upper estuary, the freshwater “tubificoid naidids” Limnodrilus hoffmeisteri Claparède, 1862, Ilyodrilus templetoni (Southern, 1909), Psammoryctides barbatus (Grube, 1861), and a Potamothrix sp. were identified as the species hosting myxozoan infection. In the lower estuary, only the marine oligochaete Tubificoides pseudogaster (Dahl, 1960) was found to be infected. Infection by sphaeractinomyxon, specifically, was determined in specimens of P. barbatus (1 type), Potamothrix sp. (2 types), and T. pseudogaster (3 types). Only one of the infected oligochaetes in the upper estuary could not be identified, because it was immature and died in the well plate, showing clear signs of degradation.

Characterization of four novel Sphaeractinomyxon types (Cnidaria, Myxozoa)

Sphaeractinomyxon type 1 (Figs. 1A‒D, 3A, B) Description: Mature actinospores spherical in apical view and ellipsoidal in lateral view, measuring 26.2 ± 1.1 (24.3‒27.6) in length (n = 6), 24.9 ± 1.4 (22.3‒26.7) in width (n = 8), and 24.3 ± 0.7 (23.2‒24.9) in diameter (n = 6). Some actinospores exhibiting swellings located laterally in three regions equidistant apart. At the centre, three polar capsules, pyriform and symmetric, 5.5 ± 0.5 (4.8‒6.2) long (n = 10) and 4.1 ± 0.3 (3.6‒4.7) wide (n = 23), each containing a polar tubule displaying 5 longitudinal coils. Sporoplasm with hundreds of secondary cells. Host: Undetermined. Locality: The upper estuary of the River Minho, near “Vila Nova de Cerveira” (41° 56′ N, 08° 45′ W), Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: 0.02% (1 infected in a total of 4,016 oligochaetes examined from the sampling site in the upper estuary). Deposition of material: One glass slide of the hapantotype deposited in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR2019.48. Molecular data: One SSU rDNA sequence with a total of 2,005 bp, assembled from the identical partial sequences obtained from the parasitic material in the coelomic cavity of a

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa

Figure 1. Light micrographs of new types of sphaeractinomyxon infecting oligochaetes in the River Minho estuary. (A‒D) Sphaeractinomyxon type 1 from an undetermined oligochaete species in the upper estuary: mature actinospores without “valvular swellings”, as observed in apical (A) and lateral (B) view; mature actinospores with “valvular swellings”, as observed in apical (C) and lateral (D) view. (E‒G) Sphaeractinomyxon type 2 infecting Psammoryctides barbatus (Grube, 1861) in the upper estuary: pansporocyst showing five of the eight actinospores developing within (E); group of mature actinospores, as observed after rupture of a pansporocyst (F); mature actinospore in apical view (G). single infected specimen, and deposited in GenBank with the accession no. MK418446. Remarks: Morphometry was determined from mature actinospores observed in a single infected host. Morphological comparison revealed no gross similarity to sphaeractinomyxon types that are without available molecular data, i.e. S. stolci, S. gigas, S. danicae, S. ilyodrili, S. amanieui, S. rotundum, S. leptocapsula, Sphaeractinomyxon types 1 and 2 of Hallett et al. (1997), and Sphaeractinomyxon type B of Hallett and Lester (1999). Morphological similarity was also not found in relation to the two endocapsa types that lack molecular data, i.e. E. stepheni and Endocapsa type 1 of Hallett et al. (2001). The sequence obtained for the parasite did not match any of the SSU rDNA sequences currently available for myxozoans, being most similar to those comprising the clade of mugiliform-infecting myxobolids (Fig. 5). Distance estimation revealed highest percentage of similarity to a Myxobolus sp. reported from the gills, intestine and tail of Mugil cephalus Linnaeus, 1758 from the Mediterranean Sea off Northern Israel (MF118765) (97.8%), Sphaeractinomyxon type 3 reported in this study (MK418448) (95.8%), and Sphaeractinomyxon type 2 of Rocha et al. (2019b) (MH017877) (95.2%). All others presented similarity values lower than 95.0%, including the Endocapsa type of Székely et al. (2007) (DQ473516) (93.6%) and Endocapsa rosulata (AF306791) (89.2%).

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Sphaeractinomyxon type 2 (Figs. 1E‒G, 3C) Description: Mature actinospores spherical in apical view and ellipsoidal in lateral view, measuring 44.6 ± 1.1 (43.4‒45.9) in length (n = 6), 53.7 ± 2.1 (51.3‒56.7) in width (n = 6), and 52.7 ± 4.2 (47.6‒57.9) in diameter (n = 6). At the centre, three polar capsules, pyriform and symmetric, 9.4 ± 0.6 (8.6‒10.6) long (n = 13) and 8.2 ± 0.3 (7.8‒8.7) wide (n = 25), each containing a polar tubule with an undetermined number of coils. Sporoplasm with hundreds of secondary cells. Host: Psammoryctides barbatus (Grube, 1861) (Annelida, Oligochaeta). Locality: The upper estuary of the River Minho, near “Vila Nova de Cerveira” (41° 56′ N, 08° 45′ W), Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: 0.05% (2 infected in a total of 4,016 oligochaetes examined from the sampling site in the upper estuary). Deposition of material: Series of phototypes of the hapantotype deposited together with a representative DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR2019.49. Molecular data: One SSU rDNA sequence with a total of 2,002 bp, representative of two identical consensus sequences separately obtained from the parasitic material in the coelomic cavity of two infected specimens, deposited in GenBank with the accession no. MK418447. Remarks: Morphometry was determined from one of the two infected hosts, in which fully matured actinospores could be measured. Morphological comparison revealed no gross similarity to sphaeractinomyxon and endocapsa types that are without available molecular data. The obtained SSU rDNA sequence did not match any sequence currently available for myxozoans, being most similar to those comprising the clade of mugiliform-infecting myxobolids (Fig. 5). Distance estimation revealed highest percentage of similarity to a Myxobolus sp. reported from the gills, intestine and tail of M. cephalus from the Mediterranean Sea off Northern Israel (MF118765) (95.1%); all others presented similarity values lower than 95.0%. The actinosporean type determined to be genetically more similar to the one in study was the Sphaeractinomyxon type 9 of Rangel et al. (2016a) (KU569318) (94.1%).

Sphaeractinomyxon type 3 (Figs. 2A‒D, 3D) Description: Mature actinospores spherical to angular in apical view and subspherical in lateral view, measuring 20.5 ± 1.0 (18.6‒23.0) in length (n = 25), 21.3 ± 0.8 (19.7‒22.7) in width (n = 25), and 21.7 ± 1.2 (19.4‒23.6) in diameter (n = 20). At the centre, three polar capsules, pyriform and symmetric, 4.8 ± 0.4 (4.1‒5.5) long (n = 25) and 3.6 ± 0.2 (3.2‒4.0) wide (n = 25), each containing a polar tubule displaying 4 longitudinal coils. Sporoplasm with

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Figure 2. Light micrographs of new types of sphaeractinomyxon infecting oligochaetes in the River Minho estuary. (A‒D) Sphaeractinomyxon type 3 found infecting an unidentified Potamothrix sp. in the upper estuary: developmental stages as observed within the coelomic cavity (A) and outside the host's body (B); group of actinospores (C); immature actinospores displaying “valvular swellings” (D). (E‒I) Sphaeractinomyxon type 4 infecting Tubificoides pseudogaster (Dahl, 1960) in the lower estuary: developmental stages in the coelomic cavity (E, F); group of actinospores as observed after rupture of the pansporocyst (G); mature actinospores in apical view (H); mature actinospore in lateral view (H). hundreds of secondary cells. Host: Unidentified “tubificoid naidid” of the genus Potamothrix Vejdovský and Mrázek, 1903 (Annelida, Oligochaeta). Locality: The upper estuary of the River Minho, near “Vila Nova de Cerveira” (41° 56′ N, 08° 45′ W), Portugal.

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa

Site of infection: Throughout the coelomic cavity. Prevalence: 0.02% (1 infected in a total of 4,016 oligochaetes examined from the sampling site in the upper estuary). Deposition of material: Series of phototypes of the hapantotype deposited together with a representative DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR2019.50. Molecular data: One SSU rDNA sequence with a total of 1,971 bp, assembled from the identical partial sequences obtained from the parasitic material in the coelomic cavity of a single infected specimen, and deposited in GenBank with the accession no. MK418448. Remarks: Morphometry was determined from mature actinospores observed in a single infected host. Morphological comparison to sphaeractinomyxon and endocapsa types that are without available molecular revealed some morphometric similarity to S. leptocapsula. The latter, however, differs from the type described here in the triangular shape of its actinospores (see Hallett et al., 1999). The sequence obtained for the parasite did not match any of the SSU rDNA sequences currently available for myxozoans, being most similar to those comprising the clade of mugiliform-infecting myxobolids (Fig. 5). Distance estimation revealed highest percentage of similarity to a Myxobolus sp. reported from the gills, intestine and tail of M. cephalus from the Mediterranean Sea off Northern Israel (MF118765) (98.8%), and to the Sphaeractinomyxon type 1 reported in this study (MK418446) (95.8%). All others presented similarity values lower than 95.0%.

Sphaeractinomyxon type 4 (Figs. 2E‒I, 3E) Description: Mature actinospores triangular in apical view and ellipsoidal in lateral view, measuring 16.4 ± 0.8 (15.3‒17.7) in length (n = 25), 19.9 ± 0.7 (18.7‒21.3) in width (n = 25), and 19.7 ± 0.6 (18.7‒20.7) in diameter (n = 25). At the centre, three polar capsules, pyriform and symmetric, 4.5 ± 0.4 (4.0‒5.3) long (n = 25) and 3.2 ± 0.2 (3.0‒3.3) wide (n = 25), each containing a polar tubule displaying 3 to 4 longitudinal coils. Sporoplasm with hundreds of secondary cells. Hosts: Unidentified “tubificoid naidid” of the genus Potamothrix Vejdovský and Mrázek, 1903 (Annelida, Oligochaeta), and Tubificoides pseudogaster (Dahl, 1960) (Annelida, Oligochaeta). Locality: The River Minho estuary: upper estuary near “Vila Nova de Cerveira” (41° 56′ N, 08° 45′ W) and lower estuary near “Caminha” (41° 52' N, 08° 50' W), Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: Overall prevalence of infection 0.04% (2 infected in a total of 4,593 oligochaetes examined from the River Minho estuary): 0.02% in the upper estuary (1 infected

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Figure 3. Schematic drawings depicting mature actinospores of the new types of sphaeractinomyxon found infecting the coelomic cavity of freshwater and marine oligochaetes in the estuary of the River Minho, as observed in apical (left) and lateral (right) view. (A) Sphaeractinomyxon type 1, actinospores without “valvular swellings”. (B) Sphaeractinomyxon type 1, actinospores displaying “valvular swellings”. (C) Sphaeractinomyxon type 2. (D) Sphaeractinomyxon type 3. (E) Sphaeractinomyxon type 4. in a total of 4,016 oligochaetes examined); 0.2% in the lower estuary (1 infected in a total of 577 oligochaetes examined). Deposition of material: Series of phototypes of the hapantotype deposited together with a representative DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR2019.51. Molecular data: One SSU rDNA sequence with a total of 2,025 bp, representative of two identical consensus sequences separately obtained from the parasitic material in the coelomic

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa cavity of two infected specimens, and deposited in GenBank with the accession no. MK418449. Remarks: Morphometry was determined from one of the two infected hosts (T. pseudogaster), in which fully matured actinospores could be measured. Morphological comparison revealed no gross similarity to sphaeractinomyxon and endocapsa types that are without available molecular data. The sequence obtained for the parasite did not match any of the SSU rDNA sequences currently available for myxozoans, being most similar to those comprising the clade of mugiliform-infecting myxobolids (Fig. 5). Distance estimation revealed highest percentage of similarity to a Myxobolus sp. reported from the gills, intestine and tail of M. cephalus from the Mediterranean Sea off Northern Israel (MF118765) (98.8%); all others presented similarity values lower than 95.0%. The actinosporean type determined to be genetically more similar to the one in study was the Sphaeractinomyxon type 1 also reported here (MK418446) (94.7%).

Record of two previously known sphaeractinomyxon types

Sphaeractinomyxon type 8 of Rangel et al. (2016a) (Fig. 4A‒C) Description: Mature actinospores spherical in apical and lateral view, measuring 18.4 ± 0.3 (18.1‒18.7) in length (n = 25), 19.4 ± 0.5 (18.4‒20.0) in width (n = 25), and 19.3 ± 0.3 (18.7‒20.0) in diameter (n = 25). At the centre, three polar capsules, pyriform and symmetric, 4.3 ± 0.4 (3.2‒5.2) long (n = 25) and 3.1 ± 0.2 (2.9‒3.2) wide (n = 25), each containing a polar tubule displaying 2 to 3 longitudinal coils. Sporoplasm with hundreds of secondary cells. Host: Tubificoides pseudogaster (Dahl) (Annelida, Oligochaeta). Localities: The Aveiro estuary (40° 40’ N, 08° 45’ W), and the lower estuary of the River Minho near “Caminha” (41° 52' N, 08° 50' W); both in Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: 0.2% (1 infected in a total of 577 oligochaetes examined from the sampling site in the lower estuary). Remarks: The SSU rDNA gene sequence obtained for the case isolate comprised 1,959 bp and was an exact match to that available for Sphaeractinomyxon type 8 of Rangel et al. (2016a) from its original description in the coelomic cavity of T. pseudogaster collected from the Aveiro estuary, Portugal. Significant morphometric variation was not recorded in relation to the original description (see Rangel et al., 2016a).

Sphaeractinomyxon type 10 of Rangel et al., 2016a (Fig. 4D‒F) Description: Mature actinospores spherical in apical view and ellipsoidal in lateral view, measuring 22.7 ± 0.7 (21.3‒24.0) in length (n = 26), 26.1 ± 0.7 (25.3‒27.0) in width (n = 26),

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Figure 4. Light micrographs of the known types of sphaeractinomyxon found infecting Tubificoides pseudogaster (Dahl, 1960) in the lower estuary of the River Minho. (A‒C) Sphaeractinomyxon type 8 of Rangel et al. (2016a): developmental stages in the coelomic cavity (A); pansporocyst showing three mature actinospores in lateral view (B); mature actinospore in apical view (C). (D‒F) Sphaeractinomyxon type 10 of Rangel et al. (2016a): pansporocyst containing eight mature actinospores (D); mature actinospores in lateral view (E); mature actinospore in apical view (F). and 25.8 ± 0.6 (25.0‒27.0) in diameter (n = 30). At the centre, three polar capsules, pyriform and symmetric, 4.8 ± 0.2 (4.7‒5.3) long (n = 24) and 3.6 ± 0.2 (3.3‒4.0) wide (n = 30), each containing a polar tubule displaying 3 longitudinal coils. Sporoplasm with hundreds of secondary cells. Hosts: Tubificoides pseudogaster (Dahl) (Annelida, Oligochaeta), and Tubificoides insularis (Stephenson) (Annelida, Oligochaeta). Localities: The Aveiro estuary (40° 40’ N, 08° 45’ W), the Alvor estuary near the Algarve Atlantic coast (37º 08’ N, 08º 37’ W), and the lower estuary of the River Minho near “Caminha” (41° 52' N, 08° 50' W); all in Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: 0.5% (3 infected in a total of 577 oligochaetes examined from the sampling site in the lower estuary). Remarks: The SSU rDNA sequences obtained for this type in three infected host specimens comprised ~2,010 bp. These were an exact match to the sequence available for the Sphaeractinomyxon type 10 of Rangel et al. (2016a) from its original description in the coelomic cavity of the same host, T. pseudogaster, in the Aveiro estuary, Portugal. Significant

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa morphometric variation was not recorded in relation to the original description of this type, nor to its report from Tubificoides insularis (Stephenson, 1923) in the Alvor estuary, near the Algarve Atlantic coast (see Rangel et al., 2016a; Rocha et al., 2019b).

Phylogenetic analyses

ML, BI and MP analyses produced similar tree topologies and revealed all new SSU rDNA sequences obtained in this study clustering within the clade of mugiliform-infecting myxobolids (Fig. 5), alongside all Myxobolus spp. thus far reported from mullet hosts, as well as all other types of the sphaeractinomyxon and endocapsa collective groups with available molecular data. The triactinomyxon type of Székely et al. (2007) (DQ473515) was also positioned within this clade.

Discussion

In this study, actinosporean infection by members of the sphaeractinomyxon collective group was found in the coelomic cavity of both freshwater and marine oligochaetes inhabiting the River Minho estuary. Four new types are described here, while two others were identified as being Sphaeractinomyxon types 8 and 10 of Rangel et al. (2016a). These two latter types were originally described from specimens of T. pseudogaster collected from the Aveiro estuary (Portugal) (Rangel et al., 2016a), about 180 km South from our sampling location in the River Minho. The Sphaeractinomyxon type 10 of Rangel et al. (2016a) was further reported to occur in a wetland system in Western Algarve (see Rocha et al., 2019b), which is located about 700 km South from our sampling location. The implementation of molecular tools to the study of actinosporean stages has been revealing that the morphological characterization of actinospores is important but insufficient for the description of new types (see Hallett et al., 2002, 2004; Eszterbauer et al., 2006). In the case of sphaeractinomyxon, studies show that actinospore measurements overlap between molecularly different types that may occur in the same oligochaete host species (see Rangel et al., 2016a). As such, the new and known types reported here were described using a combination of morphological and molecular data. Although the actinospores of the four new types differed amongst each other in their overall morphometry, a high morphological similarity was found between the actinospores of the new Sphaeractinomyxon type 4 and those of the Sphaeractinomyxon type 8 of Rangel et al. (2016a) (Table 1). Both these types were observed developing in the coelomic cavity of specimens of T. pseudogaster collected from the lower estuary of the River Minho and, therefore, could only be distinguished through comparison of their respective SSU rDNA sequences. Consequently, our results strengten the contention that

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa

Figure 5. Tree topology resulting from the maximum likelihood analysis of the SSU rDNA sequences of 62 selected myxosporeans, rooted to Myxidium lieberkuehni. Numbers at the nodes represent ML bootstrap values/BI posterior probabilities/MP bootstrap values; asterisks represent full support in all methodologies; dashes represent poorly resolved nodes or a different branching pattern. New SSU rDNA sequences are in bold; SSU rDNA sequences of former endocapsa are underlined. the acquisition of molecular information is indispensable for both the characterization of new types and the proper identification of known types.

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa

Table 1. Actinospore morphometry of the sphaeractinomyxon types reported here from oligochaetes in the River Minho, Portugal. SL: actinospore length in lateral view; SW: actinospore width in lateral view; SD: actinospore diameter in apical view; PCL: polar capsule length; PCW: polar capsule width; PTc: number of polar tubule coils. Measurements are means ± SD (range), given in µm.

sphaeractinomyxon types SL SW SD PCL PCW PTc

Type 1 (this study) 26.2 ± 1.1 (24.3‒27.6) 24.9 ± 1.4 (22.3‒26.7) 24.3 ± 0.7 (23.2‒24.9) 5.5 ± 0.5 (4.8‒6.2) 4.1 ± 0.3 (3.6‒4.7) 5

Type 2 (this study) 44.6 ± 1.1 (43.4‒45.9) 53.7 ± 2.1 (51.3‒56.7) 52.7 ± 4.2 (47.6‒57.9) 9.4 ± 0.6 (8.6‒10.6) 8.2 ± 0.3 (7.8‒8.7) ‒

Type 3 (this study) 20.5 ± 1.0 (18.6‒23.0) 21.3 ± 0.8 (19.7‒22.7) 21.7 ± 1.2 (19.4‒23.6) 4.8 ± 0.4 (4.1‒5.5) 3.6 ± 0.2 (3.2‒4.0) 4

Type 4 (this study) 16.4 ± 0.8 (15.3‒17.7) 19.9 ± 0.7 (18.7‒21.3) 19.7 ± 0.6 (18.7‒20.7) 4.5 ± 0.4 (4.0‒5.3) 3.2 ± 0.2 (3.0‒3.3) 3‒4

Type 8 of Rangel et al. (2016a) 18.4 ± 0.3 (18.1‒18.7) 19.4 ± 0.5 (18.4‒20.0) 19.3 ± 0.3 (18.7‒20.0) 4.3 ± 0.4 (3.2‒5.2) 3.1 ± 0.2 (2.9‒3.2) 2‒3

Type 10 of Rangel et al. (2016a) 22.7 ± 0.7 (21.3‒24.0) 26.1 ± 0.7 (25.3‒27.0) 25.8 ± 0.6 (25.0‒27.0) 4.8 ± 0.2 (4.7‒5.3) 3.6 ± 0.2 (3.3‒4.0) 3

Oligochaete hosts were also identified through the combined analysis of morphological traits and molecular information of the 16S locus. Recognition at the species-level, however, was only achieved for the freshwater oligochaete Psammoryctides barbatus, identified as the host of Sphaeractinomyxon type 2 in the upper estuary, and the marine oligochaete T. pseudogaster, identified as the host of the new Sphaeractinomyxon type 4 and Sphaeractinomyxon types 8 and 10 of Rangel et al. (2016a) in the lower estuary. In turn, the oligochaete hosts of Sphaeractinomyxon types 3 and 4 in the upper estuary could only be recognized as belonging to the same unidentified species of the genus Potamothrix. Lastly, it was not possible to morphologically and molecularly identify the oligochaete species infected with Sphaeractinomyxon type 1, because the specimen in question was immature and died in the well plate, showing clear signs of degradation. Despite more than one type being registered here from the same Potamothrix sp. in the upper estuary, as well from T. pseudogaster in the lower estuary, mixed infections were not observed. This is congruent with previous studies that reported several different actinosporean types from a single oligochaete species, but never taking place simultaneously in the same individual (e.g. El-Mansy et al., 1998; Xi et al., 2013, 2015; Rosser et al., 2014). In fact, a considerable diversity of sphaeractinomyxon types have been reported to infect T. pseudogaster, T. insularis and Limnodriloides agnes Hrabĕ, 1967 in Portuguese estuaries, but concomitant infections were never recorded (see Rangel et al., 2016a; Rocha et al., 2019b). Of the 25 types of sphaeractinomyxon described in the literature, only four have been reported to infect oligochaetes associated with freshwater environments: S. danicae from an Eiseniella sp., probably E. tetraedra (Savigny, 1826); S. gigas from Limnodrilus hoffmeisteri Claparède, 1862; S. ilyodrili from Potamothrix prespaensis (Hrabĕ, 1931); and S. rotundum from unidentified tubificids at Latour-bas-Elne and Villeneuve de la Raho, France (Marques, 1984). Our study considerably increases the number of sphaeractinomyxon reported from freshwater, and further suggests a susceptibility of Potamothrix spp. to infection by

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa sphaeractinomyxon. In brackish/marine environments, species of the genera Tubificoides Lastočkin, 1937 and Limnodriloides Pierantoni, 1903 appear to be the more susceptible to infection by sphaeractinomyxon, thus far having been each recognized as hosts for 7 different types of this collective group in Portuguese estuaries (see Rangel et al., 2016a; Rocha et al., 2019b). Nonetheless, myxozoan surveys targeting brackish/marine invertebrate communities in other geographic locations may come to reveal other susceptible oligochaete taxa. Most sphaeractinomyxon types have been reported exclusively from a single host species. Up until this study, sphaeractinomyxon type 10 of Rangel et al. (2016a) constituted the only confirmed exception to a strict host specificity of the collective group, having molecular evidence of its development in the coelomic cavity of both T. pseudogaster and T. insularis (see Rocha et al., 2019b). Despite S. stolci and S. ersei having also been reported from more than one oligochaete species, their identification in different hosts was based solely on morphological criteria and, therefore, remains uncertain. As such, the occurrence of Sphaeractinomyxon type 4 in two distinct oligochaete species of the family Naididae Ehrenberg, 1828, more specifically in a freshwater Potamothrix sp. inhabiting the upper estuary and in the marine oligochaete T. pseudogaster inhabiting the lower estuary, confirms that sphaeractinomyxon types are not necessarily restricted to a single host species. Moreover, the presence of this type in both a freshwater and a brackish habitat reinforces the potential involvement of the sphaeractinomyxon collective group in the life cycle of myxosporean species that infect migratory fish hosts; probably mullets, as hypothesized by Rocha et al. (2019b). Holzer et al. (2018) showed that myxozoans diversified massively after entering fish as second hosts, given that the acquisition of this vertebrate group enabled alternative transmission and dispersion strategies that were crucial in the conquest of new habitats. Mullets are catadromous, meaning that they are born in saltwater and then migrate into freshwater, where they grow before returning to the ocean to spawn. In costal and estuarine ecosystems, where these fishes spend a considerable portion of their lives, a zonal distribution of species has been shown to occur according to salinity gradients. Some species, such as the thinlip grey mullet Chelon ramada (Risso, 1827), have a higher adaptability to low salinities and water pollution, thus being able to migrate into the upper estuary (Torricelli et al., 1981; Cardona, 2006). In this context, it can be hypothesized that the acquisition of mullets as second hosts would have allowed the myxosporean counterparts of sphaeractinomyxon to potentially cross environmental barriers and conquer new habitats, whenever a susceptible host could be found in the invertebrate community. The presence of Sphaeractinomyxon type 10 of Rangel et al. (2016a) in three Portuguese estuaries (Rangel et al., 2016a; Rocha et al., 2019b) further corroborates the crucial role of the vertebrate host in the geographic dissemination of myxosporean parasites. Individual values of prevalence if infection were determined to be lower than 1% for all

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa types reported here. These values are in accordance with those reported in previous studies for other types of sphaeractinomyxon, as well as for types of other actinosporean collective groups (e.g. Hallett et al., 1999; Székely, et al. 2007; Rangel et al., 2011, 2016b; Xi et al., 2015; Milanin et al., 2017; Rocha et al., 2019b). In turn, the overall prevalence of infection of sphaeractinomyxon determined in this study was significantly lower than that reported in previous myxozoan surveys of Portuguese estuaries (see Rangel et al., 2016a; Rocha et al., 2019b), which can be correlated to the sampling sites that were chosen in the River Minho. Other works collected oligochaetes solely from lower estuaries, while our efforts were divided between two sampling sites, one of which being located in freshwater. Indeed, overall prevalence of infection was noted to be significantly higher in the lower estuary than in the upper estuary, which is consistent with the involvement of sphaeractinomyxon in the life cycles of migratory fish hosts. As previously mentioned, migratory fish spend considerable portions of their lives in brackish environments and, therefore, are prone to acquire myxozoan infection from the invertebrate communities settled in the lower estuaries. Thus, further sampling of oligochaetes from the lower estuary of the River Minho will probably reveal a higher biodiversity of the sphaeractinomyxon collective group. Considering that the sphaeractinomyxon types described here are expected to be counterparts in the life cycles of myxosporean parasites, future research in the River Minho and other Portuguese estuaries should also aim to sample potential fish hosts, namely mullets. To date, Myxobolus exiguus constitutes the only myxosporean parasite that has been reported from the River Minho, more specifically from the visceral peritoneum of the thinlip grey mullet C. ramada (Rocha et al., 2019a). The phylogenetic analysis performed in this study showed the new types described here, both freshwater and marine, clustering alongside all other members of the sphaeractinomyxon collective group within the mugiliform-infecting clade of myxobolids (Fig. 5), in accordance with the study of Rocha et al. (2019b). Following the demise of the tetraspora collective group and reassignment of its types to sphaeractinomyxon, only three other actinosporean stages cluster within this mugiliform-infecting clade: Endocapsula rosulata, Endocapsa type of Székely et al. (2007), and Triactinomyxon type of Székely et al. (2007). The endocapsa collective group was erected to encompass actinospores that differ morphologically from sphaeractinomyxon only by having irregular “processes in the form of swellings” that do not change when in contact with water (Hallett et al., 1999). The microscopic observations performed in this study, however, revealed the presence of actinospores with and without “valvular swellings” in the coelomic cavity of the oligochaete host displaying infection by the new Sphaeractinomyxon type 1; therefore, questioning the validity of the usage of this morphological criterion for distinguishing between sphaeractinomyxon and endocapsa. Immature actinospores displaying “valvular swellings” were further observed as part of the development of the other types described here (see Fig. 2D,E). This is congruent with the

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa observations of Rangel et al. (2016a), who reported immature sphaeractinomyxon actinospores often displaying “valvular swellings” like those described for endocapsa. These authors also acknowledged that dehydration of mature actinospores caused the sporoplasm to concentrate centrally, leading to the formation of “swellings”. A more in-depth look at the available literature further evidences the tenuous distinction existent between the sphaeractinomyxon and endocapsa collective groups. For instance, Hallett et al. (1999) reported actinospores reminiscent of the original type, Endocapsa rosulata, but without “valvular swellings” (typical sphaeractinomyxon by default), developing in oligochaetes collected from the same geographic locality. Similarly, the single lobe-like swelling of Endocapsa stepheni was reported to be visible in some actinospores in situ (Hallett et al., 1999). Overall, the figures provided in the description of the Endocapsa type of Székely et al. (2007) show degradation of the actinospores and, therefore, do not give support to the group. We have also found that the actinospores of Sphaeractinomyxon gigas were reported to form a double trigonal pyramid with rounded faces, which can be part of an ellipse (Marques, 1984). Thus, it is can be stated that the presence/absence of “valvular swellings” in the actinospores constitutes an artificial morphological character for differentiation between sphaeractinomyxon and endocapsa. The validity of the endocapsa collective group is further questioned by phylogenetic analyses of the SSU rDNA gene that, similar to that provided in this study, show all endocapsa and sphaeractinomyxon types with available molecular data consistently clustering together within a myxobolid clade previously suggested to have a monophyletic origin (Rocha et al., 2019a, b). Considering all the above, we suggest that endocapsa be deemed invalid and its types transferred to sphaeractinomyxon: Endocapsa rosulata becomes Sphaeractinomyxon type 1 of Hallett et al. (1999), Endocapsa stepheni becomes Sphaeractinomyxon type 2 of Hallett et al. (1999), the Endocapsa type of Hallett et al. (2001) becomes the Sphaeractinomyxon type of Hallett et al. (2001), and the Endocapsa type of Székely et al. (2007) becomes Sphaeractinomyxon type of Székely et al. (2007). The evolution of myxozoans and their invertebrate hosts has been shown to be highly congruent, namely because oligochaetes and polychaetes are the definitive and most ancient hosts of this parasitic group (Holzer et al., 2018). As such, future comprehensive revisions of some actinosporean collective groups may be necessary in order to more acurately reflect current phylogenetic and morphological information.

Acknowledgments

The authors acknowledge the iconographic assistance given by Miguel Pereira. The work here presented was financially supported by FCT (Lisbon, Portugal) within the scope of

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Chapter IX | Description of new sphaeractinomyxon prompts demise of endocapsa the Ph.D. fellowship grant attributed to S. Rocha (SFRH/BD/92661/2013) through the programme QREN-POPH/FSE; and the Engº António de Almeida Foundation (Porto, Portugal). It complies with the current laws of the country in which it was performed.

References

Cardona, L. (2006). Habitat selection by grey mullets (Osteichthyes: Mugilidae) in Mediterranean estuaries: the role of salinity. Scientia Marina 70, 443–455. Caullery, M. and Mesnil, F. (1904). Sur un type nouveau (Sphaeractinomyxon stolci n. g. n. sp.) d'Actinomyxidies, et son développement. Comptes Rendus des Seances de la Societe de Biologie et de Ses Filiales 56, 408‒410. Dias, E., Morais, P., Cotter, A.M., Antunes, C. and Hoffman, J.C. (2016). Estuarine consumers utilize marine, estuarine and terrestrial organic matter and provide connectivity among these food webs. Marine Ecology Progress Series 554, 21–34. El-Mansy, A., Székely, C. and Molnár, K. (1998). Studies on the occurrence of actinosporean stages of fish myxosporeans in a fish farm of Hungary, with the description of triactinomyxon, raabeia, aurantiactinomyxon and neoactinomyxon types. Acta Veterinaria Hungarica 46, 259‒284. Eszterbauer, E., Marton, S., Rácz, O.Z., Letenyei, M. and Molnár, K. (2006). Morphological and genetic differences among actinosporean stages of fish-parasitic myxosporeans (Myxozoa): difficulties of species identification. Systematic Parasitologica 65, 97‒114. Georgevitch, J. (1938). Sur Sphaeractinomyxon danicae n. sp., Actinomyxidae parasite d'un Oligochète du lac d'Ochrida. Comptes Rendus Mathematique Academie des Sciences, Paris 207, 250‒251. Granata, L. (1923). Richerche sugli Attinomissidi. III. Sphaeractinomyxon gigas n. sp. Monitore Zoologico Italiano 34, 64‒68. Hallett, S.L., Atkinson, S.D. and El-Matbouli, M. (2002). Molecular characterisation of two aurantiactinomyxon (Myxozoa) phenotypes reveals one genotype. Journal of Fish Diseases 25, 627‒631. Hallett, S.L., Atkinson, S.D., Erséus, C. and El-Matbouli, M. (2004). Molecular methods clarify morphometric variation in triactinomyxon spores (Myxozoa) released from different oligochaete hosts. Systematic Parasitologica 57, 1‒14. Hallett, S.L. and Diamant, A. (2001). Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia. Diseases of Aquatic Organisms 46, 197‒212. Hallett, S.L., Erséus, C. and Lester, R.J.G. (1997). Actinosporea from Hong Kong marine

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oligochaete. In Morton, B. (Ed). Proceedings of the eight international marine biological workshop: the marine flora and fauna of Hong Kong and Southern China. Hong Kong University Press, Hong Kong, pp. 1‒7. Hallett, S.L., Erséus, C. and Lester, R.J.G. (1999). Actinosporeans (Myxozoa) from marine oligochaetes of the Great Barrier Reef. Systematic Parasitologica 44, 49‒57. Hallett, S.L., Erséus, C., O’Donoghue, P.J. and Lester, R.J.G. (2001). Parasite fauna of australian marine oligochaetes. Memoirs of the Queensland Museum 46, 555‒576. Hallett, S.L. and Lester, R.J.G. (1999). Actinosporeans (Myxozoa) with four developing spores within a pansporocyst: Tetraspora discoidea n.g. n.sp. and Tetraspora rotundum n.sp. International Journal of Parasitology 29, 419‒427. Hallett, S.L., O’Donoghue, P.J. and Lester R.J.G. (1998). Structure and development of a marine actinosporean, Sphaeractinomyxon ersei n. sp. (Myxozoa). Journal of Eukaryotic Microbiology 45, 142‒150. Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. The Quarterly Review of Biology 66, 411‒453. Holzer, A.S., Bartošová-Sojková, P., Born-Torrijos, A., Lövy, A., Hartigan, A. and Fiala, I. (2018). The joint evolution of the Myxozoa and their alternate hosts: a cnidarian recipe for success and vast biodiversity. Molecular Ecology 27, 1651‒1666. Jirovec, O. (1940). Zur Kenntnis einiger in Oligochäten parasitieren den Protisten : II. Ueber Sphaeractinomyxon ilyodrili n. sp. und Neoactinomyxon globosum Granata 1922. Archiv für Protistenkunde 94, 212‒223. Kent, M.L., Margolis, L. and Corliss, J.O. (1994). The demise of a class of protists - taxonomic and nomenclatural revisions proposed for the protist phylum Myxozoa Grassé, 1970. Canadian Journal of Zoology 72, 932‒937. Kumar, S., Stecher, G. and Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution 33, 1870‒ 1874. Lom, J., McGeorge, J., Feist, S.W., Morris, D. and Adams, A. (1997). Guidelines for the uniform characterisation of the actinosporean stages of parasites of the phylum Myxozoa. Diseases of Aquatic Organisms 30, 1‒9. Marques, A. (1984). Contribution a la connaissance des Actinomyxidies: ultrastructure, cycle biologique, systématique. Ph.D. Thesis. Université des Sciences et Techniques de Languedoc, Montepellier, France, pp. 218. Milanin, T., Atkinson, S.D., Silva, M.R., Alves, R.G., Maia, A.A. and Adriano E.A. (2017). Occurrence of two novel actinospore types (Cnidaria: Myxosporea) in Brazilian fish farms, and the creation of a novel actinospore collective group, Seisactinomyxon. Acta Parasitologica 62, 121‒128.

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Palumbi, S., Martin, A., Romano, S., McMillan, W.O., Stice, L. and Grabowski, G. (2002). The Simple Fools Guide to PCR, Version 2.0. University of Hawaii, Honolulu. Puytorac P.D. 1963: L'ultrastructure des cnidocystes de l'Actinomyxidae: Sphaeractinomyxon amanieui sp. nov. Comptes Rendus Mathematique Academie des Sciences, Paris 256, 1594‒1596. Rangel, L.F., Castro, R., Rocha, S., Cech, G., Casal, G., Azevedo, C., Székely, C., Cavaleiro, F. and Santos, M.J. (2016a). Description of new types of sphaeractinomyxon actinospores (Myxozoa: Myxosporea) from marine tubificid oligochaetes, with a discussion on the validity of the tetraspora and the endocapsa as actinospore collective group names. Parasitology Research 115, 2341‒2351. Rangel, L.F., Castro, R., Rocha, S., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2016b). Tetractinomyxon stages genetically consistent with Sphaerospora dicentrarchi (Myxozoa: Sphaerosporidae) found in Capitella sp. (Polychaeta: Capitellidae) suggest potential role of marine polychaetes in parasite's life cycle. Parasitology 143, 1067‒1073. Rangel, L.F., Cech, G., Székely, C. and Santos, M.J. (2011). A new actinospore type Unicapsulactinomyxon (Myxozoa), infecting the marine polychaete, Diopatra neapolitana (Polychaeta: Onuphidae) in the Aveiro estuary, Portugal. Parasitology 138, 698‒712. Rocha, S., Azevedo, C., Oliveira, E., Alves, Â., Antunes, C., Rodrigues, P. and Casal, G. (2019a). Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular redescription of the cryptic species Myxobolus exiguus. Parasitology 146, 479‒496. Rocha, S., Casal, G., Garcia, P., Matos, E., Al-Quraishy, S. and Azevedo, C. (2014). Ultrastructure and phylogeny of the parasite Henneguya carolina sp. nov. (Myxozoa), from the marine fish Trachinotus carolinus in Brazil. Diseases of Aquatic Organisms 112, 139–148. Rocha, S., Casal, G., Rangel, L., Castro, R., Severino, R., Azevedo, C. and Santos, M.J. (2015). Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae). Parasitology International 64, 305−313. Rocha, S., Rangel, L.F., Castro, R., Severino, R., Azevedo, C., Santos, M.J. and Casal, G. (2019b). The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis. Journal of Invertebrate Pathology 160, 33‒42. Ronquist, F. and Huelsenbeck, J.P. (2003). MrBayes 3: Bayesian phylogenetic inference under

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mixed models. Bioinformatics 19, 1572‒1574. Rosser, T.G., Griffin, M.J., Quiniou, S.M., Greenway, T.E., Khoo, L.H., Wise, D.J. and Pote, L.M. (2014). Molecular and morphological characterization of myxozoan actinospore types from a commercial catfish pond in the Mississippi delta. Journal of Parasitology 100, 828‒839. Štolc, A. (1899). Actinomyxidies, nouveau groupe de Mesozoaires parent des Myxosporidies. Bulletin international de l'Académie des Sciences de Bohême 12, 1–12. Székely, C., Hallett, S.L., Al-Samman, A. and Dayoub, A. (2007). First description of myxozoans from Syria: novel records of hexactinomyxon, triactinomyxon and endocapsa actinospore types. Diseases of Aquatic Organisms 74, 127‒137. Torricelli, P., Tongiorgi, P. and Almansi, P. (1981). Migration of grey mullet fry into the Arno river: Seasonal appearance, daily activity, and feeding rhythms. Fisheries Research 1, 219–234. Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215‒219. Wolf, K. and Markiw, M.E. (1984). Biology contravenes taxonomy in the Myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science 225, 1449‒ 1452. Xi, B.W., Zhang, J.Y., Xie, J., Pan, L.K., Xu, P. and Ge, X.P. (2013). Three actinosporean types (Myxozoa) from the oligochaete Branchiura sowerbyi in China. Parasitology Research 112, 1575‒1582. Xi, B.W., Zhou, Z.G., Xie, J., Pan, L.K., Yang, Y.L. and Ge, X.P. (2015). Morphological and molecular characterization of actinosporeans infecting oligochaete Branchiura sowerbyi from Chinese carp ponds. Diseases of Aquatic Organisms 114, 217‒228.

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Chapter X

Myxozoan biodiversity in mullets (Teleostei, Mugilidae) unravels hyperdiversification of Myxobolus (Cnidaria, Myxosporea)

This chapter was adapted from:

Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts

Abstract

Mullets are ecologic and commercially important fish species. Their ubiquitous nature allows them to play critical roles in freshwater and marine ecosystems, but also makes them more vulnerable to diseases and parasitic infection. In this study, a myxozoan survey was performed on three species of mullet captured from a northeast Portuguese river. The results disclose a high biodiversity, specifically due to the hyperdiversification of Myxobolus. Thirteen new species of this genus are described based on microscopic and molecular procedures: 7 from the thinlip grey mullet Chelon ramada, 2 from the thicklip grey mullet Chelon labrosus, and 4 from the flathead grey mullet Mugil cephalus. Myxobolus exiguus and Ellipsomyxa mugilis are further registered from their type host C. ramada, as well as 6 more myxospore morphotypes that possibly represent distinct Myxobolus species. Overall, the results obtained clearly show that the number of host-, site- and tissue-specific Myxobolus spp. is much higher than what would be expected in accordance to available literature. This higher biodiversity is therefore discussed as either being the result of the usage of poor discriminative criteria in previous studies, or as being a direct consequence of the biological and ecological traits of the parasite and of its vertebrate and invertebrate host communities. Bayesian inference, maximum likelihood and maximum parsimony analyses position the new species within a clade comprising all other Myxobolus spp. that infect mugiliform hosts, thus suggesting that this parasitic group has a monophyletic origin. Clustering of species in relation to the host genus is also revealed and strengthens the contention that the evolutionary history of mugiliform- infecting Myxobolus reflects that of its vertebrate hosts. In this view, the hyperdiversification of Myxobolus in mullet hosts is hypothesized to correlate with the processes of speciation that led to the ecological plasticity of mullets.

Introduction

The family Mugilidae, whose members are commonly known as mullets, is one of the most ubiquitous among euryhaline teleosts, containing about 70 species that are distributed in marine, brackish and freshwater habitats of tropical, subtropical and temperate regions worldwide (Hotos and Vlahos, 1998; Cardona, 2001; Durand et al., 2012; Nelson et al., 2016). The ecological plasticity of mullets is not restricted to habitat, as their omnivorous nature and benthic feeding strategy allows them to feed on a great variety of materials, including epiphytic algae, insects, annelids, crustaceans, mollusks, and even detritus (Cardona, 2001; Laffaille et al., 2002; Almeida, 2003). Consequently, these fishes carry out decisive roles in their ecosystems, namely in the energy and matter flow from the lower to the upper levels, but are also more vulnerable to diseases and parasitic infection (Paperna and Overstreet, 1981;

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Laffaille et al., 2002; Almeida, 2003; Zetina-Rejón et al., 2003; Rocha et al., 2019a). In turn, the commercial importance of mullets varies with geographic location; nonetheless, its global production for human consumption is an increasing trend (Oren, 1981; Crosetti and Cataudella, 1995; Saleh, 2006). In the Mediterranean, these fishes have been considered an important food source since the Roman period (Crosetti, 2016). The production of mullets depends on the acquisition of fry or fingerlings from natural stocks, which contributes to an increasing interest in studying the parasitic community infecting wild populations. Myxozoans are parasitic cnidarians that diversified extensively in aquatic ecosystems, mainly using fish as their temporary vertebrate hosts (Holzer et al., 2018). Considering that many species of this group represent ecologic and economic threats to wild and reared fish populations, studying myxozoan biodiversity in mullets is crucial for the sustainability of natural stocks and increment of aquaculture production. During the past century, several studies have aimed to provide information regarding the myxozoan community parasitizing mullets, with ca. 80 species of the class Myxosporea Bütschli, 1881 thus far described from these hosts in different geographic locations. Of these, about 38 are species of the family Myxobolidae Thélohan, 1892, predominantly of the genus Myxobolus Bütschli, 1882, while the remaining are distributed among genera of the families: Alatasporidae Schulman et al., 1979; Ceratomyxidae Doflein, 1899; Chloromyxidae Thélohan, 1892; Kudoidae Meglitsch, 1960; Myxidiidae Thélohan, 1892; Myxobilatidae Shulman, 1953 [currently includes the genus Ortholinea, as a result of the dismantling of the family Ortholineidae (Karlsbakk et al., 2017)]; Sinuolineidae Shulman, 1959; Sphaeromyxidae Lom and Noble, 1984; and Sphaerosporidae Davis, 1917 (see Yurakhno and Ovcharenko, 2014; Barreiro et al., 2017; Yang et al., 2017; Rocha et al., 2019a). In the Iberian Peninsula, 11 Myxobolus spp. have been reported to occur in mugilid hosts, mostly from the Mediterranean waters off the Spanish Eastern coast. Myxobolus adeli (Isjumova, 1964) Yurakhno and Ovcharenko, 2014 was originally described from the Iberian Peninsula, parasitizing the digestive tract, swim bladder, gills, and muscles of golden grey mullet Chelon auratus (Risso, 1810) in the Ebro Delta and Santa Pola Bay, Spain (see Yurakhno and Ovcharenko, 2014). Myxobolus exiguus Thélohan, 1895 was morphologically re-described from the visceral peritoneum of thinlip grey mullet Chelon ramada (Risso, 1827) in a northern Portuguese River (Minho) (Rocha et al., 2019a), having been identified based on molecular information of the SSU rDNA gene. In turn, Myxobolus episquamalis Egusa et al., 1990, Myxobolus ichkeulensis Bahri and Marques, 1996, Myxobolus nile Negm-Eldin et al., 1999, and Myxobolus rohdei Lom and Dyková et al., 1994, were reported to be present in this geographic region solely on the basis of myxospore morphological traits (see Yurakhno and Ovcharenko, 2014). The remaining 5 Myxobolus spp. are unnamed, having been recently molecularly described from Mugil cephalus Linnaeus, 1758 in the Spanish Mediterranean

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts coast (Sharon et al., 2019). In addition to these Myxobolus species, Ellipsomyxa mugilis Sitjà-Bobadilla and Alvarez- Pellitero, 1993, Kudoa trifolia Holzer et al., 2006, Kudoa unicapsula Yurakhno et al., 2007, Sphaerospora mugilis Sitjà-Bobadilla and Alvarez-Pellitero, 1995, and a species of the genus Alataspora Schulman et al., 1979 were also originally described from mullets inhabiting Spanish Mediterranean coastal waters (see Sitjà-Bobadilla and Alvarez-Pellitero, 1993, 1995; Holzer et al., 2006; Yurakhno et al., 2007; Yurakhno and Ovcharenko, 2014). Yurakhno and Ovcharenko (2014) further reported the occurrence of Enteromyxum leei Diamant et al., 1994, Sphaeromyxa sabrazesi Laveran and Mesnil, 1900, Sphaerospora dicentrarchi Sitjà-Bobadilla and Alvarez-Pellitero, 1992, and Zschokkella admiranda Yurakhno, 1993 in mullets from this geographic location, having identified these species based on the morphological traits of their myxospores. Thus, with the exception of the occurrence of M. exiguus in C. ramada from the River Minho (Rocha et al., 2019a), and of actinosporean stages of E. mugilis infecting the polychaete Hediste diversicolor Müller, 1776 in the Aveiro estuary (Rangel et al., 2009), to our best knowledge nothing more is known about the myxozoan community infecting mullets in the Atlantic coastal waters of Portugal. The River Minho marks the boundary between northern Portugal and Spain. It originates in “Serra da Meira”, in the province of Lugo (Spain) and runs more than 300 km to drain into the Atlantic Ocean at the Portuguese northwest coast near the village of “Caminha”. This freshwater ecosystem has great ecologic and economic importance at a regional level; however, its biodiversity is threatened by the synergetic effects of habitat loss, introduction of non-native species, climate change, impoundments and river regulations, fishing activities and pollution. Mullets are no exception, being subjected to these natural and anthropogenic pressures. Also, despite their relatively regionally low commercial value, mullets in the River Minho are commercially exploited, providing a source of income for local fishermen (Sousa et al., 2008; Mota et al., 2016). Overall, the River Minho is home to four species of mullets: the golden grey mullet C. auratus, the thinlip grey mullet C. ramada, the thicklip grey mullet Chelon labrosus (Risso, 1827) and the flathead grey mullet M. cephalus. Co-habitation is made possible by the capability of mugilid species to utilize different energy sources (Crosetti and Cataudella, 1995). In coastal and estuarine systems, the salinity gradients also allow a zonal distribution of mugilids (Cardona, 2006) and a day and night turnover of different species has been reported to further support co-habitation (Torricelli et al., 1981). Consequently, all four species may be present throughout the year in the River Minho with highest incidence during the summer period. However, it is C. ramada that gets caught more often in upstream locations, due to its high adaptability to low salinities and water pollution (Cardona, 2006). In this study, a myxozoan survey conducted on mullets captured from the River Minho disclosed a high biodiversity of Myxobolus, with a total of thirteen new species described using

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts microscopic and molecular tools. Phylogenetic analyses using Bayesian inference and maximum likelihood methodologies revealed the monophyletic origin of mugiliform-infecting myxobolids and corroborated the evolutionary hyperdiversification of Myxobolus within mullet hosts.

Materials and methods

Fish sampling and myxozoan survey

Between 2013 and 2018, trimestral samplings of fishes were performed from fyke-nets located in the River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Fish samples included species of the orders Anguilliformes, Atheriniformes, Cypriniformes, Perciformes, Pleuronectiformes and Mugiliformes. Mugilids were represented by specimens of the thinlip grey mullet Chelon ramada (Risso, 1827) [n = 22; total length 20.3 ± 11.4 (10.5– 41.2) cm; weight 137 ± 201 (9–601) gr], the thicklip grey mullet Chelon labrosus (Risso, 1827) [n = 3; total length 44.0 ± 2.1 (42.5–45.5) cm; weight 884 ± 13 (875–893) gr], and the flathead grey mullet Mugil cephalus Linnaeus, 1758 [n = 10; total length 40.6 ± 6.8 (33.5–53.0) cm; weight 759 ± 376 (440–1582) gr]. Fish were transported live to the laboratory and anesthetized with ethylene glycol monophenyl ether (Merck, Germany) at 1ml/L. Upon dissection, fish specimens were surveyed for the presence of myxozoan parasites in several organs and tissues, specifically: brain; eye and ocular cavity; tegument and scales; gills and opercular cavity; skeletal muscle; heart; liver; gall bladder; spleen; gonads; swim bladder; kidney; urinary bladder; and digestive tube.

Microscopic analysis and morphological examination

All collected cysts and parasitized tissues were photographed using an Olympus BX50 light microscope (Olympus, Japan) in order to determine the morphology and morphometry of the myxospores from fresh material, following the guidelines provided by Lom and Arthur (1989). Co-infections were primarily determined microscopically through the morphometric traits of the myxospores. Tissue samples did not proceed for molecular analyses whenever evidence of infection by more than a single morphological type of myxospore was found, except if these belonged to morphologically distinguishable myxozoan genera (e.g. Myxobolus and Ellipsomyxa). All measurements herein provided include the mean value ± standard deviation (S.D.), range of variation and number of myxospores measured (range, n).

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts

DNA extraction, amplification and sequencing

Samples of cysts or fragments of parasitized tissue were fixed and preserved in 80% ethanol at 4 ºC. Genomic DNA was extracted from samples without microscopic evidence of co-infection by members of the same genus. Extractions were performed using a GenEluteTM Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, USA), according to the manufacturer’s instructions. The SSU rDNA gene was amplified and sequenced using both universal and myxosporean-specific primers (Table 1), so that all partial sequences overlapped in several regions. PCRs were performed in 50 µl reactions using 10 pmol of each primer, 10 nmol of dNTPs, 2 mM of MgCl2, 5 µl 10× Taq polymerase buffer, 2.5 units Taq DNA polymerase (NZYTech, Lisbon, Portugal), and approximately 50–100 ng of genomic DNA. PCRs were run on a Hybaid PxE Thermocycler (Thermo Fisher Scientific, Waltham, Massachusetts, EUA), with initial denaturation at 95 ºC for 3 min, followed by 35 cycles of 94 ºC for 45 sec, 53 ºC for 45 sec, and 72 ºC for 90 sec. The final elongation step was performed at 72 ºC for 7 min. Five- µl aliquots of the PCR products were electrophoresed through a 1% agarose 1× tris-acetate- EDTA buffer (TAE) gel stained with ethidium bromide. PCR products were purified using Puramag™ magnetic beads coated with carboxylic acid groups (MCLAB, San Francisco, California, USA). The PCR products from different regions of the SSU rDNA gene were sequenced directly. Sequencing reactions were performed using a BigDye Terminator v3.1 Cycle Sequencing Kit from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, EUA), and were run on an ABI3700 DNA analyzer from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, EUA).

Sequence assembly, distance estimation, and phylogenetic analysis

The partial sequences obtained for each case isolate were aligned in MEGA 7 (Kumar et al., 2016) for the construction of the respective assembled SSU rDNA sequences. Tissue samples were regarded as unreliable, or as possible co-infections, whenever the obtained partial sequences incorporated overlapping peaks, low quality peaks, or suspect base-calling errors in regions other than at the extremities. A preliminary alignment was performed for the numerous novel assembled SSU rDNA sequences, using software MAFFT version 7 available online. Sequences determined to be identical between different case isolates were used to calculate prevalence of infection. For distance estimation, datasets were generated according to the highest similarity scores obtained using BLAST search. One dataset included the SSU rDNA sequences of the

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts

Table 1. Polymerase chain reaction primers used for the amplification and sequencing of the SSU rDNA gene.

Name Sequence (5’-3’) Paired with Source MyxospecR, ACT2r, 18E CTG GTT GAT CCT GCC AGT Hillis and Dixon 1991 ACT3r, MYX4R MyxospecF TTC TGC CCT ATC AAC TTG TTG MYX4R, 18R Fiala 2006

ACT2f CCT GGT CCG AAC ATC CGA AGG ATA C 18R This study

ACT3f CAT GGA ACG AAC AAT 18R Hallett and Diamant 2001

ACT5f TGT GCC TTG AAT AAA T 18R Rocha et al. 2019b

MYX4F GTT CGT GGA GTG ATC TGT CAG 18R Rocha et al. 2015

MyxospecR CAA CAA GTT GAT AGG GCA GAA 18E Fiala 2006

ACT2r GTA TCC TTC GGA TGT TCG GAC CAG G 18E This study

ACT3r ATT GTT CGT TCC ATG 18E Rocha et al. 2014

MYX4R CTG ACA GAT CAC TCC ACG AAC 18E, MyxospecF Hallett and Diamant 2001 MyxospecF, ACT2f, 18R CTA CGG AAA CCT TGT TAC G Whipps et al. 2003 ACT3f, MYX4F

13 new Myxobolus spp. described here, as well as all others available for Myxobolus spp. that have bona fide mugiliform fish hosts (see Rocha et al., 2019a), and those of phylogenetically related actinosporean stages, i.e. sphaeractinomyxon, endocapsa, and the Triactinomyxon of Székely et al. 2007 (DQ473515). Another dataset comprised all available SSU rDNA sequences for species of the genus Ellipsomyxa. Sequences were aligned using MAFFT version 7 available online, and distance estimation was calculated in MEGA 7, with the p- distance model and all ambiguous positions removed for each sequence pair. For the phylogenetic analysis of the new Myxobolus spp., only the SSU rDNA sequences of myxosporean stages were kept. The final dataset comprised a total of 24 SSU rDNA sequences, including Myxobolus cerebralis (U96492) and Myxobolus wulii (KP642131) as outgroup. Sequences were aligned using software MAFFT version 7 available online, and manually edited in MEGA 7. Phylogenetic trees were calculated using Bayesian inference (BI), maximum-likelihood (ML) and maximum parsimony methodologies. ML and ML analyses were conducted in MEGA 7, with bootstrap values calculated from 1,000 replicates. For ML, the general time reversible substitution model with estimates of invariant sites and gamma distributed among site rate variation (GTR + I +G) was chosen as the best suited model, based on the lowest score of Bayesian Information Criterion (BIC) and corrected Akaike Information Criterion (AIC) with the MEGA package. For MP, the Subtree-Pruning-Regrafting algorithm was used, with a search level of 1 and random initial tree addition of 10 replicates. BI analyses were performed using MrBayes v3.2.6 (Ronquist and Huelsenbeck 2003); posterior probability distributions were generated using the Markov Chain Monte Carlo method, with four chains running simultaneously, for 500 000 generations, and every 100th tree sampled.

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts

Results

Collected and analysed fish did not display obvious external signs of infection or disease. Macro- and microscopic analysis for the detection of myxozoan parasites revealed the presence of myxospores in multiple organs and tissues, appearing either disseminated or contained within plasmodia or cysts. Myxospores were all morphologically identified as belonging to the genus Myxobolus (Myxosporea, Myxobolidae), with the exception of a species of the genus Ellipsomyxa (Myxosporea, Ceratomyxidae) parasitizing the gall bladder of C. ramada. Another myxosporean species belonging to an undetermined genus was also found in the intestine of several specimens of C. ramada. The data acquired for this latter species remains, at this point, insufficient for its proper characterization. All 22 specimens of C. ramada displayed infection by Myxobolus spp. in at least one of the organs and tissues analysed, with co-infections by members of this genus taking place in the gills, spleen, gall bladder, kidney, and digestive tube (see Table 2). Myxosporean infection was never detected in the tegument, scales, brain, eyes, gonads and swim bladder of this fish host. A total of 6 specimens displayed further cysts developing attached to the visceral peritoneum, which morphological and molecular analysis identified as M. exiguus [data published in Rocha et al. (2019a)]. At least three different morphotypes of Myxobolus were observed either forming plasmodia or appearing disseminated in the digestive tube, mostly in the intestine. Co-infection by two or three of these morphotypes was microscopically determined in 9 out of the 11 specimens displaying infection in the digestive tube. Contamination with myxospores of M. exiguus due to rupture of microscopic cysts in the visceral peritoneum was further registered in specimens #12 and #20 (see Table 2). In total, only the parasitized intestinal samples belonging to specimens #9 and #19 were without microscopic signs of co-infection and, therefore, proceeded for molecularly analysis (see Table 2). The partial SSU rDNA sequences obtained from the intestinal samples of specimen #19, however, incorporated many overlapping peaks and suspect base-calling errors, suggesting them as being unreliable. Thus, only the data obtained from specimen #9 was used to describe here a new Myxobolus species from the intestine of C. ramada. Two morphologically distinguishable myxospores of Myxobolus were observed developing in 5 out of the 10 specimens displaying kidney infection (see Table 2). Despite being without microscopic evidence of co-infection, samples belonging to specimens #1 and #3 could not be used for molecular procedures due to the very low intensity of the infection, associated with obvious aspects of lysis of the myxospores. Molecular analysis of kidney samples belonging to specimens #12, #14 and #16 originated 100% identical SSU rDNA partial sequences, allowing the description of a new Myxobolus species from this organ.

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts

M. M. hepatobiliaris

n. sp.; Mh:

M. M. muscularis

n. n. sp.;Mm:

spp. 1 to 6. 6. spp. 1 to

infection was molecularly determined. determined. molecularly was infection

. . pharyngobranchialis

Co 3

M Myxobolus

examined, examined, as determined by light microscopic observations. PI: overall prevalence of

n. Mp:sp.

6: unidentified unidentified 6:

M C. C. ramada

–

M. M. ramadus n. sp.; Mh?/Mren?/Mc?: myxospores consistent with the morphology of the species described here from the speciesthe from here described of morphology the myxospores the consistent with Mh?/Mren?/Mc?: sp.; n.

n. sp.; Mr:

M. cerveirensis M.

M. M. adiposus

n. sp.; Mc: n.

infection with a myxosporean species of an undetermined genus. genus. undetermined an of species a myxosporean with infection

Co 2

M. renalisM.

per organ examined; Ma:

; Mren: ; infection infection in the different organs of the 22 specimens of

mugilis Ellipsomyxa

Myxobolus M. exiguus M.

Myxobolus

. in question, but which identity could not be molecularly confirmed; M1confirmed; molecularly be not could which inidentity but question,

infection with infection

Table 2 infection of n. Me: sp.; organ 1

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In the same manner, two species of Myxobolus were observed forming cysts in the gills of C. ramada (see Table 2). One species formed whitish and filamentous cysts developing in the gill lamellae of all 4 specimens with infection in this organ, while a second species formed brownish and filamentous cysts in the afferent artery of the gill filaments of only specimens #11 and #16. The cysts developing in the gill lamellae reached macroscopic dimensions (2-3 mm in length) and could be easily excised and isolated, allowing the species to be described here. In turn, the species developing in the afferent artery of the gill filaments could not be described as a result of the microscopic size of its cysts. Attempts to isolate them were not successful, since the SSU rDNA partial sequences obtained from respective DNA extractions incorporated overlapping peaks and suspect base-calling errors. In the gall bladder, two morphotypes of Myxobolus were also observed, one having myxospores overall bigger than the other: the bigger myxospores appeared floating free in the bile, while the smaller myxospores formed plasmodia in the gall bladder wall. These two Myxobolus spp., together with myxospores of the genus Ellipsomyxa, were simultaneously observed in specimen #20. Specimens #12, #14, #18 and #19 displayed co-infection between Ellipsomyxa and the Myxobolus having bigger myxospores; specimen #17 displayed only the bigger myxospores of Myxobolus; and specimen #15 had only the smaller myxospores of Myxobolus (see Table 2). Apart from the gall bladder sample of specimen #20, all other samples were molecularly analysed and showed no evidence of co-infection between Myxobolus. The species having smaller myxospores is described here for the first time, while the species having bigger myxospores was identified as being M. exiguus. The myxospores of Ellipsomyxa were identified as belonging to the species E. mugilis. Lastly, the infections detected in the spleen of specimens #13 and #18 were promptly regarded as co-infections, due to microscopic observations revealing the presence of two morphologically distinguishable morphotypes of Myxobolus (see Table 2). In summary, regarding C. ramada, 7 Myxobolus spp. are described here: 4 from sites in which co-infection was registered, and three from sites of infection that were without microscopic or molecular evidence of co-infection. The latter included one species from the adipose tissue in the ocular cavity and urinary bladder; another from the pharyngobranchial organ in the opercular cavity; and the last from the skeletal and heart muscle (Figs. 1–3 and Table 3). Light photomicrographs and measurements are also provided for the unidentified Myxobolus morphotypes that appeared as co-infective in the gills, spleen, kidney, and digestive tube (Fig. 4 and Table 4). In the case of C. labrosus, one of the three specimens examined displayed macroscopic cysts attached to the visceral peritoneum, while the other two specimens displayed only plasmodia developing in the urinary bladder. Microscopic and molecular analyses showed no evidence of co-infection, so that both these species of Myxobolus are described here from C.

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts labrosus (Fig. 5 and Table 3). Regarding the myxozoan survey performed on M. cephalus, only infection by Myxobolus spp. was detected in 5 out of the 10 specimens examined. Infection was determined to occur in the gills, gall bladder, urinary bladder and intestine. Overall prevalence of infection was: 30.0% in the gall bladder (3 infected in 10 specimens examined); 20.0% in the urinary bladder and intestine (2 infected in 10 specimens examined); and 10.0% in the gills (1 infected in 10 specimens examined). Microscopic and molecular analyses showed no evidence of co- infection between isolates of the same organ, but revealed significant differences among isolates of different organs, so that 4 Myxobolus species are described here from M. cephalus (Fig. 6 and Table 3). Schematic drawings depicting the overall morphology of the myxospores in valvular view are provided for the 13 new Myxobolus spp. described herein (Fig. 7). Morphometric and biological data are summarized in Table 3, which further provides GenBank accession numbers and type material references for all the new species. Type material in the form of a series of phototypes and representative DNA samples of the hapantotypes was deposited in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal.

Description of new species

Myxobolus ramadus n. sp. Diagnosis: Cysts whitish and filamentous, about 2-3 mm in length, located in the gill lamellae. Myxospores spherical to subspherical in valvular view and ellipsoidal in sutural view, with two pyriform equally sized polar capsules located side by side at the anterior pole, and a small iodinophilous vacuole randomly located in the sporoplasm (Figs. 1A, B, 7A and Table 3). Type host: The thinlip grey mullet Chelon ramada (Risso, 1827) (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Prevalence: Four infected in 22 specimens examined (18.2%). Etymology: The specific epithet “ramadus” refers to the host species. Molecular data: One SSU rDNA sequence with a total of 2,021 bp, representative of two identical sequences that were separately assembled from the partial results obtained from macroscopic cysts in the gills of two infected specimens. Remarks: Morphometry was determined from mature myxospores observed in all infected hosts. Individual measurements were identical between all case isolates, so no significant morphometric variation was recorded. Myxospores are morphologically similar to those of the unidentified Myxobolus sp. 3 in the spleen of this host, however differing by being

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Figure 1. Light micrographs of Myxobolus spp. infecting the thinlip grey mullet Chelon ramada in the River Minho. A–B. Myxospores of M. ramadus n. sp. as observed after rupture of a cyst in the gill lamellae. C–D. Myxospores of M. pharyngobranchialis n. sp. appearing disseminated in the denticulate pharyngeal pad of the pharyngobranchial organ, after rupture of a plasmodium. E–G. Myxospores of M. muscularis n. sp. as observed in the heart (E) and skeletal muscle (F), and in detail (G).

significantly thicker (see Fig. 4C, D and Table 4). Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed some morphometric similarity to M. episquamalis, M. exiguus, Myxobolus hani Faye et al., 1999 and Myxobolus parsi Das, 1996. The parasite in study, however, differs morphologically from all these species by lacking markings near its suture line. Moreover, the myxospores of M. parsi are bigger in terms of length and width, while those of M. episquamalis are narrower, being oval instead of subspherical or spherical (see Egusa et al. 1990; Das 1996; Faye et al. 1999; Rocha et al. 2019a). Differentiation from M. episquamalis and M. exiguus is further ascertained by comparison of molecular data, given that distance estimation resulted in similarity values lower than 90.0% to all SSU rDNA sequences analysed, including M. episquamalis (88.8%) and M. exiguus (86.6%). Thus, this parasite is suggested as a new species, herein named Myxobolus ramadus n. sp.

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Myxobolus pharyngobranchialis n. sp. Diagnosis: Polysporic plasmodia located in the denticulate pharyngeal pad of the pharyngobranchial organ. Myxospores ellipsoidal in valvular and sutural view, with 6 to 8 markings near the suture line. Two pyriform equally sized polar capsules located side by side at the anterior pole (Fig. 1C, D, 7B and Table 3). Type host: The thinlip grey mullet Chelon ramada (Risso, 1827) (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Prevalence: Five infected in 22 specimens examined (22.7%). Etymology: The specific epithet “pharyngobranchialis” refers to the organ of infection in which the parasite was observed. Molecular data: One SSU rDNA sequence with a total of 1,993 bp, representative of five identical sequences that were separately assembled from the partial results obtained from plasmodia in the pharyngobranchial organ of five infected specimens. Remarks: Morphometry was determined from mature myxospores observed in all infected hosts. Individual measurements were identical between all case isolates, so no significant morphometric variation was recorded. Myxospores are morphometrically similar to those of the unidentified Myxobolus sp. 3 from the spleen and Myxobolus sp. 4 from the kidney (see Fig. 4C–F and Table 4). The latter, however, differs by having oval myxospores that are significantly thinner and overall have longer polar capsules than those being described here. In turn, having the same shape and similar morphometric range, it is possible that the myxospores of the unidentified Myxobolus sp. 3 from the spleen belong to the parasite in study. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed some morphometric similarity to M. episquamalis, M. exiguus, M. hani and Myxobolus sp. of Kim et al. (2013b). Differentiation from both M. hani and Myxobolus sp. of Kim et al. (2013b) can be performed based on specific morphological traits; the myxospores of M. hani are significantly shorter and have a subspherical shape, rather than the ellipsoidal shape reported here, while those of Myxobolus sp. of Kim et al. (2013b) are larger with smaller polar capsules (see Faye et al. 1999; Rocha et al., 2019a). In turn, differentiation from M. episquamalis and M. exiguus is mainly based on the molecular comparison of SSU rDNA sequences, considering that few distinctive morphological traits exist between these species and the parasite being described. Overall, the myxospores of M. episquamalis differ from those in study only in their oval shape, while those of M. exiguus display a higher number of polar tubule coils and fewer number of sutural markings. Distance estimation resulted in similarity values lower than 96.0% to all SSU rDNA sequences analysed, including M. episquamalis (86.9%), M. exiguus (92.6%) and Myxobolus sp. of Kim et al. (2013b) (86.0%). Thus, this parasite is suggested as a new species, herein named Myxobolus pharyngobranchialis n. sp.

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Myxobolus muscularis n. sp. Diagnosis: Polysporic plasmodia located in the fibres of the heart (Fig. 1E) and skeletal muscle (Fig. 1F). Myxospores oval in valvular view and ellipsoidal in sutural view, displaying 8 to 10 markings surrounding the posterior half of the suture line. Two pyriform equally sized polar capsules located side by side at the anterior pole (Figs. 1G, 7C and Table 3). Type host: The thinlip grey mullet Chelon ramada (Risso, 1827) (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Prevalence: Three infected in 22 specimens examined (13.6%): 2 specimens with infection in the heart (9.1%), and 2 with infection in the skeletal muscle (9.1%), meaning that one of the 3 specimens displayed infection by this species in both the skeletal and heart muscle. Etymology: The specific epithet “muscularis” refers to the site of infection of the parasite. Molecular data: One SSU rDNA sequence with a total of 2,009 bp, representative of four identical sequences that were separately assembled from the partial results obtained from plasmodia in four tissue samples (2 from the heart muscle, and the other two from the skeletal muscle), belonging to a total of three infected specimens. Remarks: Morphometry was determined from mature myxospores observed in both infected hosts. Individual measurements were identical between both case isolates, so no significant morphometric variation was recorded. Myxospores display some morphometric similarity to the unidentified Myxobolus sp. 3 from the spleen and the unidentified Myxobolus sp. 4 from the kidney (see Fig. 4C–F and Table 4), however differing in significant morphological traits. The myxospores of the unidentified Myxobolus sp. 3 from the spleen are ellipsoidal instead of oval, while those of the unidentified Myxobolus sp. 4 from the kidney are more elongated with bigger polar capsules. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed the parasite displaying some morphological similarity with Myxobolus anili Sarkar, 1989, Myxobolus cheni Schulman, 1962, M. episquamalis, M. exiguus, M. hani and M. parsi. The myxospores of M. anili, M. exiguus and M. hani, however, differ from those in study in terms of shape (M. anili is ellipsoidal, while both M. exiguus and M. hani are subspherical). Moreover, the myxospores of M. anili are significantly longer and larger, while those of M. parsi are much thicker, with their length and width falling close the superior limit of the morphometric range reported here (see Sarkar, 1989; Faye et al., 1999; Rocha et al., 2019a). On the other way around, M. cheni has a smaller morphometric range that overlaps with that of the parasite in study at its inferior limit; therefore, the myxospores of M. cheni are generally smaller than those being described here (see Rocha et al., 2019a). Overall, highest resemblance was found in relation to M. episquamalis, if considered that it shares the same oval shape, markings near the suture line, and morphometric range (see Egusa et al., 1990;

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given ingivenµm.

spp. spp. here. described SL: myxospore length; SW:myxospore width; myxospore ST: thickness;

Myxobolus

site of infection and morphometry of the new

location,

Host, Host,

Table 3 (range), meansare Measurements coils. SD ± tubule of width; number polar PTc: polar capsule length;PCL:polar capsule PCW:

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Rocha et al., 2019a). Differentiation from this species was only possible through the molecular comparison of SSU rDNA sequences. Distance estimation resulted in similarity values lower than 95.0% to all SSU rDNA sequences analysed, including M. exiguus (90.7%) and M. episquamalis (88.7%). Thus, this parasite is suggested as a new species, herein named Myxobolus muscularis n. sp.

Myxobolus adiposus n. sp. Diagnosis: Clusters of myxospores forming plasmodia attached to the adipose tissue surrounding the optic nerve in the ocular cavity (Fig. 2A), and in the urinary bladder (Fig. 2B). Myxospores spherical, displaying 12 to 14 markings near the suture line. Two pyriform equally sized polar capsules located side by side at the anterior pole (Figs. 2C, 7D and Table 3). Type host: The thinlip grey mullet Chelon ramada (Risso, 1827) (Teleostei, Mugilidae). Other hosts: The flathead grey mullet Mugil cephalus Linnaeus, 1758 (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Other localities: The Mediterranean Sea off Northern Israel. Prevalence: Five infected in 22 specimens examined (22.7%): 5 specimens with infection in the ocular cavity (22.7%), two of which also displaying infection in the urinary bladder (18.2%). Etymology: The specific epithet “adiposus” refers to the site of infection of the parasite being the adipose tissue. Molecular data: One SSU rDNA sequence with a total of 2,000 bp, representative of seven identical sequences that were separately assembled from the partial results obtained from plasmodia in seven samples of adipose tissue (5 from the ocular cavity, and the other two from the urinary bladder), belonging to a total of five infected specimens. Remarks: Morphometry was determined from mature myxospores observed in all infected hosts. Individual measurements were identical between case isolates, so no significant morphometric variation was recorded. Having spherical myxospores, this parasite differs significantly from all others reported here. In the same manner, no significant similarity was found in relation to all other Myxobolus spp. morphologically reported from mullet hosts (see Rocha et al., 2019a). Distance estimation, however,

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts revealed 99.8% of similarity to the SSU rDNA sequence of a Myxobolus sp. (MF118765) that was recently reported from infections in the gills, intestine, and tail of M. cephalus from the Mediterranean Sea off Northern Israel (isolate IsraelMS gipt) (Sharon et al., 2019), and this small divergence led us to consider them as being of the same species. All other SSU rDNA sequences included in the analysis showed similarity values lower than 95.0% to the parasite in study. Considering all the above, this parasite is suggested as a new species, herein named Myxobolus adiposus n. sp., with C. ramada as type host and the River Minho as type locality.

Myxobolus hepatobiliaris n. sp. Diagnosis: Microscopic cysts in the liver (Fig. 2D) and gall bladder wall (Fig. 2E). Myxospores ellipsoidal in valvular and sutural view, displaying 6 to 8 markings near the suture line. Two pyriform equally sized polar capsules located side by side at the anterior pole (Figs. 2 F, 7E and Table 3). Type host: The thinlip grey mullet Chelon ramada (Risso, 1827) (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Prevalence: Two infected in 22 specimens examined (9.1%): one specimen with infection in the liver (4.5%), and another with infection in the gall bladder wall (4.5%). Etymology: The specific epithet “hepatobiliaris” refers to the organs of infection in which the parasite was observed. Molecular data: One SSU rDNA sequence with a total of 1,962 bp, representative of two identical sequences that were separately assembled from the partial results obtained from microscopic cysts in two tissue samples (one from the liver, and another from the gall bladder), belonging to a total of two infected specimens. Remarks: Morphometry was determined from mature myxospores observed in both infected hosts. Individual measurements were identical between case isolates, so no significant morphometric variation was recorded. Myxospores are morphometrically similar to those of the unidentified Myxobolus sp. 1 from the gills, with slighter resemblance to those of the unidentified Myxobolus sp. 2 from the spleen (see Fig. 4A–D and Table 4). Nonetheless, the myxospores of the latter differ in being overall bigger, while the myxospores of the unidentified Myxobolus sp. 1 from the gills are oval shaped instead of ellipsoidal, moreover being generally larger than those described here. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed some morphological similarity to Myxobolus macrolepi Dorothy and Kalavati, 1992, Myxobolus mugauratus (Pogoreltseva, 1964) Landsberg and Lom, 1991 and Myxobolus parvus Schulman, 1962. The myxospores of M. macrolepi, however, have a significantly wider morphometric range and higher number of polar tubule coils (6 to 7 against the 4 reported here), while those of both M. mugauratus and

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Figure 2. Light micrographs of Myxobolus spp. infecting the thinlip grey mullet Chelon ramada in the River Minho. A–C. Myxospores of M. adiposus n. sp. forming clusters in the adipose tissue of the ocular cavity (A), appearing disseminated after rupture of a plasmodium in the urinary bladder (B), and as observed in detail (C). D–F. Myxospores of M. hepatobiliaris n. sp. forming microscopic cysts in the liver (D), developing in the gall bladder (E), and as observed in detail (F).

M. parvus have notably bigger polar capsules. The myxospores of M. parvus are also larger than those of the parasite in study (see Rocha et al., 2019a). Distance estimation showed a high similarity to the Myxobolus species described here from the kidney of C. ramada (97.3%), with all other SSU rDNA sequences included in the analysis resulting in similarity values lower than 92.0%, including M. parvus (89.3%). Thus, this parasite is suggested as a new species, herein named Myxobolus hepatobiliaris n. sp.

Myxobolus renalis n. sp. Diagnosis: Microscopic cysts developing in the kidney (Fig. 3A). Myxospores ellipsoidal to subspherical in valvular view and ellipsoidal in sutural view, displaying 8 to 10 markings near the suture line. Two pyriform equally sized polar capsules located side by side at the anterior pole (Figs. 3B, C, 7F and Table 3). Type host: The thinlip grey mullet Chelon ramada (Risso, 1827) (Teleostei, Mugilidae).

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Figure 3. Light micrographs of Myxobolus spp. infecting the thinlip grey mullet Chelon ramada in the River Minho. A–C. Myxospores of M. renalis n. sp. forming microscopic cysts in the kidney (A), appearing disseminated after rupture of a cyst (B), and as observed in detail (C). D–F. Myxospores of M. cerveirensis n. sp. forming plasmodia in the intestine (D), appearing disseminated after rupture of a plasmodium (E), and as observed in detail (F).

Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Prevalence: Three specimens molecularly confirmed to be infected by this parasite in 22 specimens examined (13.6%). Etymology: The specific epithet “renalis” refers to the organ of infection in which the parasite was observed. Molecular data: One SSU rDNA sequence with a total of 1,976 bp, representative of three identical sequences that were separately assembled from the partial results obtained from microscopic cysts in kidney samples of three infected specimens. Remarks: Morphometry was determined from mature myxospores observed in the three host specimens molecularly confirmed to be infected by this parasite. Individual measurements were identical between case isolates, so no significant morphometric variation was recorded. Myxospores are morphometrically similar to those of the unidentified Myxobolus sp. 2 from the spleen of C. ramada (see Fig. 4C, D and Table 4), and possibly belong to the same species, despite the myxospores in the spleen being overall more elongated than those described here. Although some morphometric similarity could also be found in relation to the unidentified

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Myxobolus sp. 1 from the gills (see Fig. 4A, B and Table 4), the myxospores of the latter differ in being oval shaped instead of ellipsoidal to subspherical. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed some morphometric similarity to M. macrolepi, M. parvus and Myxobolus supamattayai U-taynapun et al., 2011. The parasite in study differs from M. macrolepi in having myxospores with stricter morphometric range and fewer number of polar tubule coils (4 against the 6 to 7 turns of M. macrolepi). In turn, the myxospores of M. parvus have longer polar capsules than those in study, while M. supamattayai myxospores are larger (see Rocha et al., 2019a). Differentiation of the parasite in study from these two latter species, however, is mainly based on the molecular comparison of SSU rDNA sequences. Distance estimation showed a high similarity to the Myxobolus species described here from the liver and gall bladder of C. ramada (97.3%), with all other SSU rDNA sequences analysed resulting in similarity values lower than 92.0%, including M. parvus (89.7%) and M. supamattayai (84.1%). Thus, this parasite is suggested as a new species, herein named Myxobolus renalis n. sp.

Myxobolus cerveirensis n. sp. Diagnosis: Polysporic plasmodia located in the intestine (Fig. 3D). Myxospores ellipsoidal in valvular and sutural view, with at least 6 markings surrounding the posterior half of the suture line. Two pyriform equally sized polar capsules located side by side at the anterior pole (Figs. 3E, F, 7G and Table 3). Type host: The thinlip grey mullet Chelon ramada (Risso, 1827) (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Prevalence: One specimen molecularly confirmed to be infected by this parasite in 22 specimens examined (4.5%). Etymology: The specific epithet “cerveirensis” refers to the type locality of the parasite in the River Minho, which is near “Vila Nova de Cerveira”. Molecular data: One SSU rDNA sequence with a total of 1,976 bp, assembled from the identical partial sequences obtained from plasmodia in the intestinal sample of a single infected specimen. Remarks: Morphometry was determined from mature myxospores observed in a single infected host. Myxospores without significant overall morphometric similarity to the unidentified species reported here from C. ramada. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed some morphometric similarity to M. episquamalis and M. hani. The myxospores of the latter can be differentiated from those in study by their subspherical shape and larger width. In turn, the myxospores of M. episquamalis differ only in their oval shape, considering that the measurements of the parasite in study are well within the

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Figure 4. Light micrographs of the unidentified Myxobolus spp. infecting the thinlip grey mullet Chelon ramada in the River Minho. A–B. Myxospores of Myxobolus sp. 1 as observed after rupture of a microscopic cyst in the afferent artery of the gill filaments (A), and in detail (B). C–D. Myxospores of Myxobolus sp. 2 (smaller) and Myxobolus sp. 3 (bigger) appearing disseminated amongst each other in the spleen. E–F. Myxospores of Myxobolus sp. 4 disseminated in the kidney after rupture of a plasmodium. Notice the presence of the smaller myxospores of M. renalis n. sp. (arrows) among those of Myxobolus sp. 4. G–H. Myxospores of Myxobolus sp. 5 as observed after rupture of a plasmodium located in the intestinal wall. I. Myxospore of Myxobolus sp. 6 as observed disseminated in the intestinal wall.

morphometric range reported for this species (see Egusa et al., 1990; Faye et al., 1999; Rocha et al., 2019a). Despite the morphological likeness, distance estimation showed low similarity to M. episquamalis (86.7%), as well as to all other SSU rDNA sequences analysed, which did not surpass 92.0% of similarity. Thus, this parasite is suggested as a new species, herein named Myxobolus cerveirensis n. sp.

Myxobolus peritonaeum n. sp. Diagnosis: Cysts yellowish, subspherical to filamentous, and measuring about 1-2 mm, attached to the visceral peritoneum. Myxospores oval in valvular view, displaying 8 to 10 markings near the suture line. Two pyriform equally sized polar capsules located side by side at the anterior pole (Figs. 5A, B, 7H and Table 3). Type host: The thicklip grey mullet Chelon labrosus (Risso, 1827) (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal.

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Table 4. Site of infection and morphometry of the unidentified Myxobolus myxospores observed in the thinlip grey mullet Chelon ramada from the River Minho, Portugal. SL: myxospore length; SW: myxospore width; ST: myxospore thickness; PCL: polar capsule length; PCW: polar capsule width; PTc: number of polar tubule coils; L: larger; S: smaller. Measurements are means ± SD (range), given in µm.

Myxobolus spp. Site of infection SL SW ST PCL PCW PTc

Myxobolus sp. 1 Gill filaments 6.9 ± 0.3 (6.3–7.3) 5.9 ± 0.5 (4.7–6.7) 4.2 ± 0.2 (4.0–4.7) 3.1 ± 0.2 (2.7–3.3) 1.8 ± 0.2 (1.3–2.0) – (n = 40) (n = 40) (n = 40) (n = 45) (n = 45)

Myxobolus sp. 2 Spleen 7.1 ± 0.4 (6.5–8.0) 5.5 ± 0.3 (5.1–6.0) 4.3 ± 0.3 (3.8–4.7) 3.1 ± 0.3 (2.6–3.6) 2.1 ± 0.2 (1.6–2.4) 3–4 (n = 25) (n = 25) (n = 21) (n = 25) (n = 25)

Myxobolus sp. 3 Spleen 8.9 ± 0.4 (7.9–9.6) 7.9 ± 0.4 (7.1–8.5) 5.5 ± 0.3 (5.2–6.1) 4.5 ± 0.2 (4.0–5.0) 2.7 ± 0.2 (2.5–3.2) 6 (n = 25) (n = 25) (n = 9) (n = 25) (n = 25)

Myxobolus sp. 4 Kidney 9.6 ± 0.3 (9.0–10.0) 6.7 ± 0.3 (6.3–7.3) 5.5 ± 0.2 (5.3–6.0) 4.9 ± 0.2 (4.7–5.3) 3.0 ± 0.2 (2.7–3.3) 5–6 (n = 30) (n = 30) (n = 8) (n = 50) (n = 50)

Myxobolus sp. 5 Intestine 7.8 ± 0.4 (7.2–8.8) 7.5 ± 0.4 (6.5–8.5) 5.5 ± 0.3 (5.1–6.0) L 4.7 ± 0.4 (3.9–5.4) L 3.3 ± 0.2 (3.0–3.8) L 8 (n = 30) (n = 30) (n = 26) (n = 25) (n = 25) S 6 S 3.6 ± 0.2 (3.3–4.2) S 2.1 ± 0.2 (1.6–2.4) (n = 25) (n = 25)

Myxobolus sp. 6 Intestine 15.0 ± 0.4 (14.7–15.7) 13.7 ± 0.4 (13.3–14.7) – 7.9 ± 0.5 (6.7–8.7) 5.3 ± 0.4 (4.7–6.0) – (n = 8) (n = 8) (n = 16) (n = 16)

Prevalence: One infected in three specimens examined (33.3%). Etymology: The specific epithet “peritonaeum” refers to the site of infection of the parasite. Molecular data: One SSU rDNA sequence with a total of 1,976 bp, assembled from the identical partial sequences obtained from cysts excised from the visceral peritoneum of a single infected specimen. Remarks: Morphometry was determined from mature myxospores observed in cysts collected from a single infected host. Myxospores without significant overall morphometric similarity to the unidentified species reported here from C. ramada. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed the parasite in study having high morphological similarity to M. episquamalis. Although M. exiguus and M. mugchelo have also been found in the peritoneum of C. labrosus, the myxospores in study are overall smaller than those of M. exiguus, while being larger than those of M. mugchelo (see Rocha et al., 2019a). Despite the morphological likeness to M. episquamalis, distance estimation showed low similarity to this species (88.0%), as well as to all other SSU rDNA sequences analysed, which did not surpass 96.0% of similarity. Thus, this parasite is suggested as a new species, herein named Myxobolus peritonaeum n. sp.

Myxobolus labrosus n. sp. Diagnosis: Polysporic plasmodia located in the urinary bladder. Myxospores oval in valvular view and ellipsoidal in sutural view, displaying about 8 markings near the suture line.

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Figure 5. Light micrographs of Myxobolus spp. infecting the thicklip grey mullet Chelon labrosus in the River Minho. A–B. Myxospores of M. peritonaeum n. sp. as observed after rupture of a macroscopic cyst attached to the visceral peritoneum. C–D. Myxospores of M. labrosus n. sp. forming plasmodia in the urinary bladder (C), and as observed in detail (D).

Two pyriform equally sized polar capsules located side by side at the slightly pointed anterior pole (Figs. 5C, D, 7I and Table 3). Type host: The thicklip grey mullet Chelon labrosus (Risso, 1827) (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Prevalence: Two infected in three specimens examined (66.7%). Etymology: The specific epithet “labrosus” refers to the specific epithet of the host species. Molecular data: One SSU rDNA sequence with a total of 1,965 bp, representative of two identical sequences that were separately assembled from the partial results obtained from plasmodia in the urinary bladder samples of two infected specimens. Remarks: Morphometry was determined from mature myxospores observed in both infected hosts. Individual measurements were identical between case isolates so no significant morphometric variation was recorded. Myxospores without significant overall morphometric similarity to the unidentified species reported here from C. ramada. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed some morphometric similarity to M. anili, Myxobolus bankimi Sarkar, 1999, M. exiguus, and Myxobolus sp. of Kim et al. (2013b) (see Rocha et al., 2019a). Morphological differentiation between the parasite in study and most of the above-mentioned species is mainly based on their myxospores’ shape not being oval [M. exiguus myxospores are subspherical, while those of M. anili and Myxobolus sp. of Kim et al. (2013b) are ellipsoidal] (see Sarkar, 1989; Kim et al., 2013b; Rocha et al., 2019a). Only M. bankimi was described to have ovoid to elongated ovoid shaped myxospores that closely resemble those reported here (see Sarkar, 1999). Nonetheless, the myxospores

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts of the latter species are generally longer with shorter polar capsules, additionally lacking the sutural markings that characterize the myxospores of the parasite in study. Differentiation from M. exiguus and Myxobolus sp. of Kim et al. (2013b) was further ascertained through molecular comparison of respective SSU rDNA sequences. Distance estimation showed low similarity to M. exiguus (93.2%) and Myxobolus sp. of Kim et al. (2013b) (86.0%), as well as to all other SSU rDNA sequences analysed, which did not surpass 96.0% of similarity. Thus, this parasite is suggested as a new species, herein named Myxobolus labrosus n. sp.

Myxobolus mugiliensis n. sp. Diagnosis: Cysts whitish, subspherical to filamentous, about 1-2 mm in length, located in the gill lamellae. Myxospores ellipsoidal in valvular and sutural view, displaying 8 to 10 markings near the suture line. Two pyriform equally sized polar capsules located side by side at the anterior pole (Figs. 6A, B, 7J and Table 3). Type host: The flathead grey mullet Mugil cephalus Linnaeus, 1758 (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Other localities: The Spanish Mediterranean coast, and possibly the Samsun coasts of the Black Sea in Turkey. Prevalence: One infected in 10 specimens examined (10.0%). Etymology: The specific epithet “mugiliensis” refers to the name of the genus of the host species. Molecular data: One SSU rDNA sequence with a total of 1,972 bp, assembled from the identical partial sequences obtained from cysts excised from the gills of a single infected specimen. Remarks: Morphometry was determined from mature myxospores observed in cysts collected from a single infected host. Myxospores without significant overall morphometric similarity to the unidentified species reported here from C. ramada. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed significant similarity to M. rohdei, Myxobolus spinacurvatura Maeno et al., 1990, Myxobolus sp. II of Yemmen et al. (2012), and Myxobolus sp. of Kim et al. (2013b). The parasite in study, however, differs from most of these species in specific morphological aspects. For instance, the myxospores of M. rohdei are thinner with fewer polar tubule coils (3 to 4 in comparison to the 5 coils reported here), those of Myxobolus sp. II of Yemmen et al. (2012) are larger and spherical in shape, while those of Myxobolus sp. of Kim et al. (2013b) have a wider morphometric range and overall smaller polar capsules (see Rocha et al., 2019a). In turn, morphological differentiation from M. spinacurvatura is hard to be performed, given that the measurements of the

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts myxospores in study are overall within the morphometric range reported for this species. Despite the morphological likeness, distance estimation showed low similarity to M. spinacurvatura (92.8%), while revealing 100% and 99.9% of similarity to the SSU rDNA sequences of a Myxobolus sp. deposited in GenBank under the accession numbers MF118767 and MH392320. Both these sequences were obtained from infections in the gills of M. cephalus: the first from specimens originating from wild catches in the Spanish Mediterranean coast (isolate Spain1-g) (Sharon et al., 2019), and the second from specimens originating from the Samsun coasts of the Black Sea in Turkey (unpublished data in GenBank). All other SSU rDNA sequences included in the analysis showed similarity values lower than 98.0%. Considering all the above, this parasite is suggested as a new species, herein named Myxobolus mugiliensis n. sp., with the River Minho as type locality (Table 3).

Figure 6. Light micrographs of Myxobolus spp. infecting the flathead grey mullet Mugil cephalus in the River Minho. A–B. Myxospores of M. mugiliensis n. sp. as observed after rupture of a macroscopic cyst in the gill lamellae (A), and in detail (B). C–D. Myxospores of M. vesicularis n. sp. forming plasmodia in the gall bladder wall (C), and as observed in detail (D). E–F. Myxospores of M. urinaris n. sp. forming plasmodia in the urinary bladder wall (E), and as observed in detail (F). G–H. Myxospores of M. galaicoportucalensis n. sp. appearing disseminated after rupture of a plasmodium in the intestinal wall (G), and as observed in detail (H).

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Myxobolus vesicularis n. sp. Diagnosis: Polysporic plasmodia located in the gall bladder wall. Myxospores ellipsoidal in valvular and sutural view, displaying 10 to 12 markings near the suture line. Two pyriform equally sized polar capsules located side by side at the anterior pole (Figs. 6C, D, 7K and Table 3). Type host: The flathead grey mullet Mugil cephalus Linnaeus, 1758 (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Other localities: The Spanish Mediterranean coast. Prevalence: Three infected in 10 specimens examined (30.0%). Etymology: The specific epithet “vesicularis” refers to the organ of infection in which the parasite was observed. Molecular data: One SSU rDNA sequence with a total of 1,943 bp, representative of three identical sequences that were separately assembled from the partial results obtained from plasmodia in the gall bladder wall tissue of three infected specimens. Remarks: Morphometry was determined from mature myxospores observed in all three infected hosts. Individual measurements were identical between case isolates, so no significant morphometric variation was recorded. Myxospores without significant overall morphometric similarity to the unidentified species reported here from C. ramada. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed the parasite falling within the morphometric range of M. spinacurvatura, while further sharing some morphological similarity with Myxobolus sp. II of Yemmen et al. (2012). The myxospores of the latter, however, differ from those in study in their spherical shape, smaller width and thinner polar capsules (see Yemmen et al. 2012; Rocha et al., 2019a). In turn, differentiation from M. spinacurvatura could only be achieved based on molecular data of the SSU rDNA gene. Distance estimation revealed the parasite in study displaying only 92.0% of similarity to M. spinacurvatura, while sharing 99.9% of similarity to a Myxobolus sp. (MF118772) recently reported from infections in the gills and tail of M. cephalus from wild catches in the Spanish Mediterranean coast (isolate Spain5-i) (Sharon et al., 2019). The occurrence of infection in three distinct organs corroborates the histological findings of Sharon et al. (2019) that showed the parasite having specificity towards the connective tissue. Considering all the above, this parasite is suggested as a new species, herein named Myxobolus vesicularis n. sp., with the connective tissue as site of infection and the River Minho as type locality (Table 3).

Myxobolus urinaris n. sp. Diagnosis: Polysporic plasmodia located in the urinary bladder. Myxospores ellipsoidal

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts in valvular view, with two pyriform equally sized polar capsules located side by side at the anterior pole (Figs. 6E, F, 7L and Table 3). Type host: The flathead grey mullet Mugil cephalus Linnaeus, 1758 (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal. Prevalence: Two infected in 10 specimens examined (20.0%). Etymology: The specific epithet “urinaris” refers to the organ of infection in which the parasite was observed. Molecular data: One SSU rDNA sequence with a total of 1,958 bp, representative of two identical sequences that were separately assembled from the partial results obtained from plasmodia in the urinary bladder samples of two infected specimens. Remarks: Morphometry was determined from mature myxospores observed in both infected hosts. Individual measurements were identical between case isolates, so no significant morphometric variation was recorded. Myxospores without significant overall morphometric similarity to the unidentified species reported here from C. ramada. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed some morphometric similarity to M. exiguus, M. rohdei and Myxobolus sp. of Kim et al. (2013b). The myxospores of M. rohdei can be differentiated from those of the parasite in study based on their bigger size and fewer number of polar tubule coils (see Rocha et al., 2019a). In turn, morphological differentiation from M. exiguus and Myxobolus sp. of Kim et al. (2013b) is solely based on the absence of sutural markings in the myxospores in study, given that these three species share highly similar morphometric ranges (see Kim et al., 2013b; Rocha et al., 2019a). The newness of the parasite described here, however, is confirmed by distance estimation analysis, which revealed similarity values lower than 98.0% to all SSU rDNA sequences analysed, including those of M. exiguus (85.9%) and Myxobolus sp. of Kim et al. (2013b) (94.0%). Thus, this parasite is suggested as a new species, herein named Myxobolus urinaris n. sp.

Myxobolus galaicoportucalensis n. sp. Diagnosis: Polysporic plasmodia located in the intestine. Myxospores ellipsoidal in valvular and sutural view, displaying 12 to 14 markings near the suture line. Two pyriform equally sized polar capsules located side by side at the anterior pole (Figs. 6G, H, 7M and Table 3). Type host: The flathead grey mullet Mugil cephalus Linnaeus, 1758 (Teleostei, Mugilidae). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near “Vila Nova de Cerveira”, Portugal.

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Figure 7. Schematic drawings of the myxospores of the 13 new Myxobolus spp. described here from mullets in the River Minho, as observed in valvular view. A. M. ramadus n. sp. B. M. pharyngobranchialis n. sp. C. M. muscularis n. sp. D. M. adiposus n. sp. E. M. hepatobiliaris n. sp. F. M. renalis n. sp. G. M. cerveirensis n. sp. H. M. peritonaeum n. sp. I. M. labrosus n. sp. J. M. mugiliensis n. sp. K. M. vesicularis n. sp. L. M. urinaris n. sp. M. M. galaicoportucalensis n. sp.

Other localities: The Spanish Mediterranean coast. Prevalence: Two infected in 10 specimens examined (20.0%). Etymology: The specific epithet “galaicoportucalensis” refers to the parasite’s type locality – the River Minho – marking the boundary between Portugal and Galicia (Spain). Molecular data: One SSU rDNA sequence with a total of 1,975 bp, representative of two identical sequences that were separately assembled from the partial results obtained from plasmodia in the intestinal samples of two infected specimens. Remarks: Morphometry was determined from mature myxospores observed in both infected hosts. Individual measurements were identical between case isolates, so no significant morphometric variation was recorded. Myxospores without significant overall morphometric similarity to the unidentified species reported here from C. ramada. Comparison to all other Myxobolus spp. previously reported from mullet hosts revealed the parasite falling within the morphometric range of M. spinacurvatura, while further sharing some morphological

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts similarity with Myxobolus sp. II of Yemmen et al. (2012). The myxospores of the latter differ from those in study in being generally shorter and having thinner polar capsules (see Rocha et al., 2019a). Differentiation from M. spinacurvatura could not be performed based on the morphological traits of the myxospores; however, distance estimation showed the parasite in study sharing only 92.3% of similarity with this species. This analysis further revealed 100% and 99.9% of similarity to the sequences of a Myxobolus sp. (MF118763, MF118768 and MF118766) that was recently reported from infected specimens of M.cephalus caught from the Spanish Mediterranean coast: MF118763 from the scales (isolate Spain7-s); MF118768 from the muscle (isolate Spain1-m); and MF118766 from the tail (Spain1-t) (Sharon et al., 2019). Considering all the above, this parasite is suggested as a new species, herein named Myxobolus galaicoportuclaensis n. sp., with the River Minho as type locality (Table 3). Still regarding distance estimation, a 99.6% similarity was further calculated in relation to the SSU rDNA sequence of Sphaeractinomyxon type 2 of Rangel et al., 2016. Despite this high percentage of similarity, nucleotide comparison among the sequences revealed the few differing nucleotides bearing significance for species differentiation, given that they are spread throughout the sequence, with their positioning being consistent between all identical partial sequences. All other SSU rDNA sequences included in the analysis displayed similarity values lower than 98.0% to the parasite in study.

Occurrence of known species in the thinlip grey mullet Chelon ramada

Myxobolus exiguus Thélohan, 1895 This species was recently re-described from macroscopic cysts developing in the visceral peritoneum of C. ramada captured from this same study area (see Rocha et al., 2019a). During this myxozoan survey, myxospores of M. exiguus could also be morphologically and molecularly identified in smears of the gall bladder (Fig. 8A, B) and intestine (Fig. 8C), as a result of contamination due to the rupture of microscopic cysts in the visceral peritoneum.

Ellipsomyxa mugilis (Sitjà-Bobadilla and Alvarez-Pellitero, 1993) Developmental stages of E. mugilis were found parasitizing the gall bladder of C. ramada, with a prevalence of infection of 22.7% (5 infected in 22 specimens examined). Disporic plasmodia and mature myxospores were observed floating free in the bile (Fig. 8D– G). Young plasmodia appeared polymorphic, with an irregular cellular membrane due to the presence of pseudopodia throughout (Fig. 8D), while mature plasmodia appeared subspherical with smooth cellular membrane, each containing two immature myxospores (Fig. 8E). Mature myxospores were ellipsoidal, measuring 5.8 ± 0.2 (5.5–6.0) µm (n = 12) in length, 9.0 ± 0.7

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(6.7–9.7) µm (n = 30) in width, and 12.0 ± 0.9 (11.1–14.0) µm (n = 30) in thickness. The suture line was laterally shifted and slightly curved, separating the two asymmetric valves. Two subspherical equally sized polar capsules, 4.0 ± 0.2 (3.5–4.0) µm long (n = 16) and 3.0 ± 0.3 (2.7–3.9) µm wide (n = 40), opened subterminally and in opposite directions, each containing a polar tubule coiled in 6 turns (Fig. 8F, G). One SSU rDNA sequence with a total of 1,720 bp was deposited in GenBank with the accession no. MK193812. The latter is representative of five identical sequences that were separately assembled from the partial results obtained from plasmodia and myxospores in the gall bladder of five infected specimens. A voucher constituted by a series of phototypes and representative DNA sample of the hapantotype was deposited in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2019.40. The morphological features of the plasmodia and myxospores were overall congruent with those previously reported by Sitjà-Bobadilla and Alvarez-Pellitero (1993), upon the original

Figure 8. Light micrographs of known myxosporean species infecting the thinlip grey mullet Chelon ramada in the River Minho. A–C. Myxospores of Myxobolus exiguus observed in smears of the gall bladder (A, B) and of the intestine (C), as a result of contamination due to the rupture of microscopic cysts in the visceral peritoneum. D–G. Disporic plasmodia and mature myxospores of Ellipsomyxa mugilis, coelozoic in the gall bladder: young plasmodia polymorphic, with cellular membrane irregular due to the presence of pseudopodia (D); mature plasmodia subspherical with smooth cellular membrane (E); mature myxospores floating free in the bile (F), and as observed in detail (G).

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts species description of E. mugilis, albeit our observations showing the polar tubule coiled in 6 turns instead of 5. Molecular comparison further confirmed the identity of the parasite, which shared 99.8–100% of similarity with the three SSU rDNA sequences available for E. mugilis in GenBank. High similarity values were further obtained to the available SSU rDNA sequences of Ellipsomyxa syngnathi Køie and Karlsbakk, 2009 (99.8%) and Ellipsomyxa gobii Køie, 2003 (99.6%), whereas all other Ellipsomyxa spp. displayed lower than 97.4% similarity.

Phylogenetic analysis

ML, BI and MP analyses revealed the SSU rDNA sequences of the new species of Myxobolus clustering among all other congeners thus far molecularly reported from mullet hosts, forming a well-supported clade of mugiliform-infecting Myxobolus spp. (Fig. 9). The species described here from C. ramada and C. labrosus specifically clustered among all others that infect hosts of the genus Chelon (except for M. ramadus n. sp.), to form a well-supported subclade. All Myxobolus spp. that infect hosts of the genus Mugil, including those described here, also clustered together to form another well-supported subclade sister to the former. The SSU rDNA sequence of M. ramadus n. sp. appeared positioned alone at the basis of the two latter subclades. Nonetheless, the most basal position of the clade was occupied by the sole molecular representative of Myxobolus spp. that infect hosts of the genus Crenimugil, M. supamattayai.

Discussion

Myxozoan survey and description of new species

The reliable description of Myxosporea is currently accepted as being the result of the combined analysis of several criteria, namely myxospore morphology, host specificity, tissue of infection and molecular data (see Atkinson et al., 2015). Considering that phylogenetic studies widely show the vertebrate host group as the most relevant evolutionary signal for myxobolids (Ferguson et al., 2008; Carriero et al., 2013; Rocha et al., 2014), the species comparisons of Myxobolus performed in this study only took into consideration congeners reported from mugiliform fish hosts. Tissue tropism also constitutes an important evolutionary signal for myxobolids and other myxosporeans in general (see Eszterbauer, 2004; Holzer et al., 2004; Ferguson et al., 2008). Nonetheless, the site of infection could not be used as an informative character for species comparison, considering that specific tissues of infection were not determined in this study, and neither is this type of information included in most descriptions of mugiliform-infecting Myxobolus. Future studies should, therefore, be careful to

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Figure 9. Tree topology resulting from the maximum likelihood analysis of mugiliform-infecting Myxobolus spp., rooted to Myxobolus cerebralis (U96492) and Myxobolus wulii (KP642131). Number at the nodes are ML bootstrap values/BI posterior probabilities/MP bootstrap values; dashes represent a different branching pattern or a bootstrap support value under 50. The new Myxobolus spp. described in this study are marked using bold. Host groups are indicated using coloured boxes.

provide histological data for determining exact sites of infection. Overall, differentiation of the new Myxobolus spp. described here, amongst each other and in relation to congeners previously reported from mullets, was mainly based on molecular comparisons of SSU rDNA sequences. Morphology-based comparisons, however, remained necessary in order to establish differentiation from the many mugiliform-infecting Myxobolus spp. that are without available molecular data. Although frequently necessary, the comparison of morphological traits for the identification of myxosporeans must be carried out cautiously. Phylogenetic studies have shown before that morphology-based criteria are unreliable for differentiation at the species- level. In the case of Myxobolus, the artificiality of the morphological criteria is evidenced by the numerous species of this genus that share similar myxospore shape and size, despite being molecularly different (see Atkinson et al., 2015). In this study, differentiation of M. mugiliensis n. sp., M. vesicularis n. sp. and M. galaicoportucalensis n. sp. from M. spinacurvatura could have not been performed without the help of molecular data, given that all these species have

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts ellipsoidal myxospores with sutural markings, similar morphometric range and number of polar tubule coils. In the same manner, differentiation between M. peritonaeum n. sp. and M. episquamalis was only made possible by comparison of their respective SSU rDNA sequences. Thus, it can be stated that, without molecular input, these species could have been easily identified erroneously, consequently giving origin to species complexes. Molecular comparison of SSU rDNA sequences were further crucial in allowing differentiation between the several new species described here. For instance, M. hepatobiliaris n. sp. and M. renalis n. sp. from C. ramada share nearly identical myxospore morphology, however differing in 2.7% of their SSU rDNA sequences. Similarly, the myxospores of M. vesicularis n. sp. and M. galaicoportucalensis n. sp. from M. cephalus are indistinguishable but differ in 5.7% of their SSU rDNA sequences. High values of intraspecific variability have been reported for different isolates of a few Myxobolus spp. [e.g. Myxobolus flavus Carriero et al., 2013 (1.9%), and Myxobolus koi Kudo, 1919 (3.0%)] (Camus and Griffin, 2010; Carriero et al., 2013). In the cases shown here, however, the possibility of high intraspecific variability was disregarded, considering that more than one SSU rDNA sequence was obtained for most of these species, being 100% identical amongst different isolates. Overall, the new species described here showed high values of interspecific variability amongst each other, as well as to all other mugiliform-infecting congeners and actinosporean related sequences. Distance estimation revealed interspecific variability higher than 4% for the new Myxobolus spp. found infecting C. ramada and M. cephalus, and higher than 2% for those found in C. labrosus. Lower values of genetic difference (0.1%) and exact matches were obtained for M. adiposus n. sp., M. mugiliensis n. sp., M. vesicularis n. sp. and M. galaicoportucalensis n. sp. in relation to the SSU rDNA sequences of Myxobolus spp. that were mainly obtained from infected M. cephalus captured from the Spanish Mediterranean coast (see Sharon et al., 2019). The lowest value of genetic difference calculated here and that possibly represents small interspecific variability was that of M. galaicoportucalensis n. sp. and the Sphaeractinomyxon type 2 of Rangel et al., 2016, which differed solely in 0.4% of their SSU rDNA sequences, corresponding to a total of 8 different nucleotides. Despite this low value, sequence alignments showed that the position of the differing nucleotides bore significance for species differentiation, thus indicating it as possibly being representative of small interspecific variability. This is corroborated by the 100% of similarity that was calculated between the different geographic isolates of M. galaicoportucalensis n. sp. myxospores. On the other hand, it is also possible that this low genetic difference can be explained by ongoing processes of speciation in the vertebrate host due to host-shift and recombination of different lineages in annelid hosts (see Forró and Eszterbauer, 2016), which would make it representative of intraspecific variability. Considering that life cycle inferences through molecular data should be performed cautiously, the genetic difference found between these

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Chapter VI | Hyperdiversification of Myxobolus spp. in mullet hosts two sequences is here prudently maintained as being representative of interspecific variability until further data arises. This result, however, reinforces the involvement of the sphaeractinomyxon collective group in the life cycles of mugiliform-infecting myxobolids, as hypothesized by Rocha et al. (2019b). Most mugiliform-infecting Myxobolus spp. were originally described prior to the implementation and vulgarization of molecular procedures, consequently being without available molecular data. In fact, of the ca. 38 species of Myxobolus reported to infect mullets, only 8 have molecular data available, plus a few SSU rDNA sequences belonging to unnamed Myxobolus spp. that infect M. cephalus (Kim et al., 2013b; Sharon et al., 2019). Of the above mentioned 8 species, only Myxobolus bragantinus Cardim et al., 2018 and M. supamattayai were originally described through combined morphological and molecular features (U- taynapun et al., 2011; Cardim et al., 2018). In turn, Myxobolus bizerti Bahri and Marques, 1996, M. episquamalis, M. exiguus, M. ichkeulensis and M. spinacurvatura were given molecular identity in subsequent reports from their original sites of infection and type hosts (Bahri et al., 2003; Rocha et al., 2019a). Only M. parvus was sequenced from a mullet species other than its type host. The molecular data available for this species was obtained from infections in the gills and kidney of leaping mullet Chelon saliens (Risso, 1810) from the Turkish coast of the Black Sea (Özer et al. 2016), despite it having been originally described from M. cephalus and so-iuy mullet Planiliza haematocheila (Temminck and Schlegel, 1845) off China (see Eiras et al. 2005). The occurrence of M. parvus in C. saliens therefore requires molecular validation through sequencing of the parasite from its type site of infection and host. Similarly, several other Myxobolus spp. have been reported from more than one mullet host on the basis of myxospore morphology (see Rocha et al., 2019a). Up until this study, however, none had been molecularly confirmed to infect more than a single mullet species. Myxobolus adiposus n. sp. now constitutes the sole exception, having molecular data available from infections that take place in C. ramada and M. cephalus. This confirms that host specificity of mugiliform-infecting Myxobolus is not restricted to a single host, with some species possibly having a cosmopolitan nature. In this context, providing molecular data for the many mugiliform-infecting Myxobolus spp. that have not been sequenced, specifically from their original site of infection and type host, constitutes an important task for future researches targeting myxozoans in mullets. The acquisition of this information is essential for both the reliable description of new Myxobolus spp. from these fishes, and for the recognition of the true host range of previously established species that have been reported from more than one mullet host. Similarly, several mugiliform-infecting Myxobolus have been indiscriminately reported from a wide array of sites of infection (see Rocha et al., 2019a). Despite some species of this genus having been recognized to develop in different organs [e.g. Myxobolus diaphanus (Fantham et al., 1940), Myxobolus cuneus Adriano et al., 2006 and Myxobolus cordeiroi

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Adriano et al., 2009], this wide array of infection sites results from their specificity to the connective tissue (see Cone and Easy, 2005; Adriano et al., 2006, 2009). In this study, M. adiposus n. sp. is described from the adipose tissue in the ocular cavity and urinary bladder, having further been molecularly reported by Sharon et al. (2019) from infections in the gills, intestine and tail. Myxobolus muscularis n. sp. is also described from the heart and skeletal muscle based on sequencing of the SSU rDNA gene from its different sites of infection in C. ramada. While the myxospores of M. adiposus n. sp. were significantly easy to morphologically differentiate from all others observed in this study due to their spherical shape and symmetric polar capsules, those of M. muscularis n. sp. shared significant morphological similarity with the myxospores of M. pharyngobranchialis n. sp. and M. episquamalis, and could only be distinguished on the basis of molecular data. Thus, the validity of the occurrence of a single species in different organs should be based on sequencing of the SSU rDNA gene or other adequate molecular markers, rather than on myxospore morphology. Taking into consideration all the above, the necessity of using molecular tools for the myxozoan survey of mullets is undisputable and will most certainly increase the biodiversity of Myxobolus spp. known from these fish hosts. Molecular approaches should, however, be mindful of the possibility of co-infections, so as to not produce assembled chimeric sequences. In this study, the occurrence of co-infection was determined in several of the specimens of C. ramada examined. In fact, 6 other potentially different Myxobolus spp. were further observed in this host: Myxobolus sp. 1 forming filamentous microscopic cysts in the afferent artery of the gill filaments; Myxobolus sp. 2 and Myxobolus sp. 3 appearing disseminated in the spleen; Myxobolus sp. 4 disseminated or forming plasmodia in the kidney; and Myxobolus sp. 5 and Myxobolus sp. 6 forming plasmodia and appearing disseminated in the intestine, respectively. Molecular characterization of these Myxobolus was not possible due to the lack of parasites isolates (see Table 2). The myxospores of the two unidentified morphotypes occurring in the intestine differ significantly from all others observed in this study, as well as from those of previously known species, therefore, potentially representing two new species. In turn, the myxospores of the Myxobolus sp. 1 in the gills and the Myxobolus sp. 2 infecting the spleen are morphometrically very similar amongst each other, as well as to those of M. hepatobiliaris n. sp. and M. renalis n. sp., which despite sharing the overall same myxospore morphometry were distinguished through means of molecular procedures. A similar situation was noticed between the myxospores of the Myxobolus sp. 3 in the spleen, the Myxobolus sp. 4 in the kidney, M. muscularis n. sp. and M. pharyngobranchialis n. sp., which are also morphometrically similar amongst each other, albeit the two latter having been demonstrated to be molecularly different. Considering these cases, it can be hypothesized that the acquisition of isolated biological material belonging to the unidentified morphotypes in the gills, spleen and kidney may come

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Chapter VI | Hyperdiversification of Myxobolus spp. in mullet hosts to reveal yet four other potential new species parasitizing C. ramada, regardless of their overall morphological similarity. It should be noted, however, that the measurements of Myxobolus sp. 3 and Myxobolus sp. 4 are also similar to those of M. exiguus, so that the possibility of co- infection in the spleen and kidney due to rupture of microscopic cysts in the visceral peritoneum cannot be disregarded. During this myxozoan survey, myxospores of M. exiguus could be morphologically and molecularly identified in smears of the gall bladder and intestine, as a result of contamination. In turn, myxospores resembling M. adeli, M. episquamalis, M. ichkeulensis, M. nile, and M. rohdei were never observed, despite these species having been reported from mugilids in the Western Mediterranean off Spain, based on morphological criteria of the myxospores (see Yurakhno and Ovcharenko, 2014). Only two myxozoan species not belonging to the genus Myxobolus were observed in this study, both occurring in C. ramada: the coelozoic Ellipsomyxa mugilis forming disporic plasmodia attached to the gall bladder wall, or floating in the bile alongside free mature myxospores; and a species belonging to an unidentified genus, histozoic in the intestinal wall. Ellipsomyxa mugilis was originally described as Zschokkella mugilis from the gall bladder of the mugilids C. ramada, C. saliens, and M. cephalus in the Western Mediterranean off Spain (Sitjà-Bobadilla and Alvarez-Pellitero, 1993). Since then, this parasite was further reported from C. saliens off the Mediterranean coast of Tunisia (Thabet et al., 2016), as well as from actinosporean stages developing in the polychaete Hediste diversicolor in the Portuguese Atlantic coast, more specifically in the Aveiro estuary (Rangel et al., 2009), which is located about 175 km south from the estuary of the River Minho. In this study, the identification of E. mugilis was based on both myxospore morphology and molecular data of the SSU rDNA gene. Besides providing confirmation of species identity, molecular comparisons further revealed low genetic difference to the SSU rDNA sequences of E. syngnathi (0.2%) and E. gobii (0.4%). This low interspecific variability agrees with previously reported values (see Køie and Karlsbakk, 2009; Thabet et al., 2016). Currently, these three species are distinguished based on morphological details of the myxospores, and narrow vertebrate host specificity. Ellipsomyxa mugilis and E. gobii further differ in actinospore morphometry (Køie, 2000; Køie et al., 2004; Køie and Karlsbakk, 2009; Rangel et al., 2009). A more comprehensive microscopic and molecular analysis of these three species is obviously necessary in order to either reinforce their differentiation or to reveal then as being cryptic. In turn, the data acquired for the non-myxobolid parasite infecting the intestine of C. ramada is, at this point, insufficient for genus determination and species characterization, so that further studies will be required. The myxosporean species Kudoa trifolia, K. unicapsula, Sphaerospora mugilis, and a species of the genus Alataspora were also originally described from mullet hosts in the Western Mediterranean off Spain (Sitjà-Bobadilla and Alvarez-Pellitero, 1993, 1995; Holzer et al., 2006; Yurakhno et al., 2007; Yurakhno and Ovcharenko, 2014), with Yurakhno and

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Ovcharenko (2014) further reporting the occurrence of Enteromyxum leei, Sphaeromyxa sabrazesi, Sphaerospora dicentrarchi and Zschokkella admiranda in mugilids of this geographic location, based on morphological criteria. Despite the geographic proximity, none of these species were found infecting the specimens of mullets examined here from the River Minho, which can possibly be related to differences in the composition of the invertebrate communities inhabiting the Western Mediterranean Sea and the Portuguese estuaries and coastal waters of the North Atlantic Ocean.

Species-richness of Myxobolus in mullets

About 38 species of Myxobolus have been reported to infect mullets worldwide (Marcotegui and Martorelli, 2017; Cardim et al., 2018; Rocha et al., 2019a). This study adds 13 new species to this list from infections found in C. ramada, C. labrosus and M. cephalus captured from the River Minho, in Northwest Portugal. The occurrence of unidentified myxospores in several organs of C. ramada, potentially belonging to new species, suggests that this already high biodiversity can, in fact, be increased by further pursuing myxozoan surveys in mullet hosts. Prior to this study, only M. exiguus and M. mugchelo had been reported to occur in C. ramada, among other fish hosts. After being originally described by Thélohan (1895) from the stomach epithelium, pyloric caeca, kidney, and spleen of C. ramada and C. labrosus, as well as from the gills of the cyprinid Abramis brama (Linnaeus, 1758), M. exiguus was subsequently reported from a wide range of sites of infection and host species, clearly indicating it as a species complex. To resolve this issue, a recent study re-described M. exiguus from infections taking place in C. ramada captured from the River Minho, with this mullet species being settled as type species, and the visceral peritoneum as type tissue (Rocha et al., 2019a). The data presented here, which stems from the myxozoan survey from which the re-description of M. exiguus was performed, revealed the parasite occurring in all specimens of C. ramada examined posteriorly. This indicates that the prevalence of infection of this parasite in C. ramada is probably higher than that reported in its re-description, as microscopic cysts in the visceral peritoneum may pass unnoticed or be easily confused as a co-infection in organs of the visceral cavity. In turn, M. mugchelo was originally described from the mesentery of C. labrosus from Italy, and recently reported from the intestinal wall of C. ramada from the same geographic location (Eiras et al., 2005; Ovcharenko et al., 2017). In this study, myxospores displaying the petite morphometric features reported for M. mugchelo were not observed in both C. ramada and C. labrosus, suggesting that this species may be absent in this specific geographic region. Nonetheless, it cannot be disregarded that only a few specimens of C. labrosus were examined

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Chapter VI | Hyperdiversification of Myxobolus spp. in mullet hosts in this study, so that further myxozoan surveys can come to disclose the presence of M. mugchelo in the River Minho. Still regarding C. labrosus, the description of M. peritonaeum n. sp. and M. labrosus n. sp. increases to 5 the number of Myxobolus spp. thus far reported from this mullet species, which is also the host for M. parenzani developing in the gills, as well as a possible host for M. exiguus, both reported on the basis of myxospore morphometry (see Rocha et al., 2019a). Consequently, the molecular data presented here for M. peritonaeum n. sp. and M. labrosus n. sp. constitutes the first available in GenBank for Myxobolus infections occurring in C. labrosus. Mugil cephalus constitutes the mullet species from which a higher number of Myxobolus spp. have been reported, now accounting for ca. 23 species of this genus. It has been suggested that the Myxobolus-richness in M. cephalus probably reflects the higher number of myxozoan surveys that have been conducted on this mullet species, as a result of its economic importance for fisheries and aquaculture industries in several geographic locations (see Rocha et al., 2019a). Our study concurs with this assumption, given that myxozoan survey of C. ramada revealed a biodiversity of Myxobolus significantly higher than that expected upon consideration of the literature available. In comparison, Myxobolus-richness in C. labrosus was significantly low, but again, it cannot be disregarded that only a few specimens of this mullet species were examined, so that future myxozoan surveys targeting C. labrosus may increase its Myxobolus biodiversity. In the past years, several studies have aimed to provide information regarding the biodiversity of myxozoans infecting mullets in specific geographic locations (e.g. Bahri and Marques, 1996; Bahri et al., 2003; U-taynapun et al., 2011; Kim et al., 2013a, b; Yurakhno and Ovcharenko, 2014; Özer et al., 2016; Thabet et al., 2016; Barreiro et al., 2017; Yang et al., 2017). Nonetheless, this study constitutes that from which a higher biodiversity of Myxobolus is reported. This probably relates to the careful morphological and molecular analyses that were performed here for species characterization, with no “pre-identification” at the species- level being performed based on myxospore morphology and morphometry. For instance, Özer et al. (2016) examined more than 200 specimens of leaping mullet C. saliens obtained from the Sinop coast of the Black Sea in a period of just over a year, but only identified infections by myxospores of M. parvus in several organs. Despite providing an SSU rDNA sequence from the parasite in the gills and kidney, reports of this species in the gall bladder and lower jaw of C. saliens were solely based on myxospore morphology, with the authors even acknowledging some morphological difference of the myxospores observed in the gall bladder. Similarly, Yurakhno and Ovcharenko (2014) examined ca. 450 specimens of C. auratus captured from the Mediterranean, Black and Azov Seas during the course of two years, and described solely M. adeli from the intestine, pyloric caeca, esophagus, stomach, swim bladder, gills and muscles, on the basis of myxospore morphometry. The broad array of organs displaying

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Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts infection in both these cases, however, suggest that more than one Myxobolus spp. could have been present in the specimens analysed. Thus, the contrasting biodiversity of Myxobolus reported from mullets in different geographic localities may be essentially consequential to the use of poor discriminative criteria for identification at the species-level. Nonetheless, U- taynapun et al. (2011) examined near 150 specimens of bluespot mullet Moolgarda seheli (Forsskål, 1775) captured from the Thailand coast of the Andaman Sea during the course of two years, and described only the blackish cysts of M. supamatayai developing in the skin, having found no microscopic signs of myxozoan infection in the other potential organs analysed. In this case, low biodiversity is probably related to the biological and ecological traits of the parasite and host vertebrate and invertebrate communities. Oligochaetes and polychaetes are the definitive and most ancient hosts of myxosporeans (Holzer et al., 2018); thus, it is reasonable to assume that the composition, spatial distribution, and susceptibility of the invertebrate community is determinant for the successful establishment and diversification of myxosporeans in different geographic locations. Euryhaline hosts, such as anadromous and catadromous fishes, migrate between freshwater and marine habitats, passing through brackish waters during different periods of their lifetime. This capability of adaptation to different gradients of salinity allows migratory fish to interact with intrinsically different vertebrate and invertebrate communities, therefore making them potential temporary hosts for a wide array of myxozoan species that may develop in freshwater or marine annelids (e.g. Bartholomew et al., 1997, 2006; Rangel et al., 2017). Mullets are catadromous, they spawn in saltwater and then migrate into freshwater as juveniles, where they grow into adults before migrating back into the ocean. As such, mullets are potential temporary hosts for myxozoan species developing in oligochaetes or polychaetes, regardless being in freshwater, brackish or marine environments (Rocha et al., 2019b). Species of the genera Myxobolus, Henneguya Thélohan, 1892 and Thelohanellus Kudo, 1933 are the most commonly reported from freshwater habitats, less frequently occurring in brackish and marine habitats. These genera are known to display life cycles that involve freshwater oligochaetes, and possibly also marine oligochaetes, as invertebrate hosts (Eszterbauer et al., 2015; Rocha et al., 2019b). In this context, the lower biodiversity of Myxobolus reported in previous studies that targeted mullets in brackish/marine waters is probably consequential to the low availability of adequate invertebrate hosts for members of this myxosporean genus in those areas. In fact, this may also explain the significant variation of Myxobolus-richness that is reported here from C. ramada, C. labrosus and M. cephalus. In this study, the thinlip grey mullet C. ramada was undoubtedly the mullet species that displayed highest rate of Myxobolus infection, and by a significantly elevated number of different species. In accordance, it also constitutes that which more frequently is captured in upstream locations,

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Chapter VI | Hyperdiversification of Myxobolus spp. in mullet hosts as a result of its high adaptability to low salinities and water pollution (Cardona, 2006); this being the reason why specimens of C. ramada were more frequently caught in our study area. Therefore, it can be hypothesized that the species that are able to tolerate lower salinity gradients are more prone to infection by myxobolids, as they interact more broadly with upstream benthic communities. Several studies have shown that species composition of annelid communities in Rivers and coastal waters is influenced by environmental factors, including salinity gradients (see Pfannkuche, 1980; Pascar-Gluzman and Dimentman, 1984; Moroz, 1994; Seys et al., 1999; Schenková and Helešic, 2006; Krodkiewska, 2007; Armendáriz et al., 2011; Rosa et al., 2015; Kang et al., 2017). Future researches might aim to correlate the spatial distribution of mugiliform-infecting Myxobolus with that of its invertebrate hosts, and also to the migratory patterns of potential mullet hosts. Nonetheless, it will first be necessary to recognize full life cycles of these mugiliform-infecting Myxobolus, namely through the identification of invertebrate hosts; so that research in this field should aim to provide novel information on this subject.

Phylogenetic analysis

The phylogenetic analysis performed here is congruent with previously published phylograms of mugiliform-infecting Myxobolus spp. (e.g. Rocha et al., 2019a). Following the main phylogenetic trend of myxobolids to group in accordance with the vertebrate host taxonomic order (Ferguson et al., 2008; Carriero et al., 2013; Rocha et al., 2014), the SSU rDNA sequences of the new Myxobolus spp. described here cluster among congeners that have bona fide mugiliform fish hosts (see Rocha et al., 2019a), to form a well-supported clade of mugiliform-infecting Myxobolus spp. (Fig. 9). The inner topology of this clade further reveals tendency for species clustering in relation to host genus. Accordingly, the Myxobolus spp. that infect mullets of the genus Chelon form a well-supported subclade, sister to another well- supported subclade comprising species that infect mullets of the genus Mugil. Being the sole molecular representative of Myxobolus spp. that infect members of the genus Crenimugil, M. supamattayai stands alone, occupying the most basal position of the mugiliform-infecting Myxobolus clade. Myxobolus ramadus n. sp. constitutes the only exception to species clustering according to the host genus, appearing positioned alone at the basis of the two subclades separately comprising Myxobolus spp. that infect hosts of the genera Chelon and Mugil. This phylogenetic placement of Myxobolus ramadus n. sp. shows that parasite host- switch has taken place between mugiliform genera. A contention that is further corroborated by the occurrence of M. adiposus n. sp. in M. cephalus, as reported by Sharon et al. (2019). Phylogenetic studies have demonstrated that the origin and radiations of myxozoans reflect the evolution of their hosts (Carriero et al., 2013; Kodádková et al., 2015; Holzer et al.,

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2018; Patra et al., 2018). Despite the co-evolutionary history of this parasitic group and its vertebrate hosts being received as a “mixed signal” of invertebrate and vertebrate cophylogeny due to the more ancient reciprocal adaptation of myxozoans and their definitive invertebrate hosts, the acquisition of vertebrates as alternate hosts was crucial for species diversification (Holzer et al., 2018). The phylogenetic analysis presented in this study agrees with this contention by revealing hyperdiversification of Myxobolus after entering mugiliform fish as alternate hosts. This high biodiversity probably reflects the processes of speciation that have led to the great ecological plasticity of mugiliform fish, which allows them to migrate and live, even if temporarily, in intrinsically distinct habitats, consequently entering in contact and feeding from a great variety of living organisms and materials. The placement of all new Myxobolus spp. described here within the previously known clade of mugiliform-infecting Myxobolus further supports the monophyletic origin of this group. Nonetheless, the results obtained in this study clearly show that the number of host-, site- and tissue-specific species in mullets is probably much higher than expected based on hitherto available literature, so that future myxozoan surveys may come to reveal other lineages of Myxobolus that evolved from distinct entries into mugiliform fish hosts.

Acknowledgements

The authors acknowledge Professor Eduardo Rocha and the Laboratory of Histology of the Institute of Biomedical Sciences, University of Porto, for the usage of the Olympus BX50 light microscope, as well as Miguel Pereira for his iconographic assistance. This study was financially supported by FCT (Lisbon, Portugal) within the scope of the Ph.D. fellowship grant attributed to S. Rocha (SFRH/BD/92661/2013) through the programme QREN-POPH/FSE; and the Engº António de Almeida Foundation (Porto, Portugal). It complies with the current laws of the country in which it was performed.

References

Adriano, E.A., Arana, S., Alves, A.L., Silva, M.R., Ceccarelli, P.S., Henrique-Silva, S. and Maia, A.A. (2009). Myxobolus cordeiroi n. sp., a parasite of Zungaro jahu (Siluriformes: Pimelodidae) from Brazilian Pantanal: morphology, phylogeny and histopathology. Veterinary Parasitology 162, 221–229. Adriano, E.A., Arana, S. and Cordeiro, N.S. (2006). Myxobolus cuneus n. sp. (Myxosporea) infecting the connective tissue of Piaractus mesopotamicus (Pisces: Characidae) in Brazil: histopathology and ultrastructure. Parasite 13, 137–142. Almeida, P.R. (2003). Feeding ecology of Liza ramada (Risso, 1810) (Pisces, Mugilidae) in a

Page | 286

Chapter VI | Hyperdiversification of Myxobolus spp. in mullet hosts

south-western estuary of Portugal. Estuarine, Coastal and Shelf Science 57, 313–323. Armendáriz, L.C., Capítulo, A.R. and Ambrosio, E.S. (2011). Relationships between the spatial distribution of oligochaetes (Annelida, Clitellata) and environmental variables in a temperate estuary system of South America (Río de la Plata, Argentina). New Zealand Journal of Marine and Freshwater 45, 263‒279. Atkinson, S.D., Bartošová-Sojková, P., Whipps, C.M. and Bartholomew, J.L. (2015). Approaches for characterising myxozoan species. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan evolution, ecology and development. Springer International Publishing, Switzerland, pp. 111‒123. Bahri, S., Andree, K.B. and Hedrick, R.P. (2003). Morphological and phylogenetic studies marine Myxobolus spp. from mullet in Ichkeul Lake, Tunisia. Journal of Eukaryotic Microbiology 50, 463‒470. Bahri, S. and Marques, A. (1996) Myxosporean parasites of the genus Myxobolus from Mugil cephalus in Ichkeul lagoon, Tunisia: description of two new species. Diseases of Aquatic Organisms 27, 115‒122. Barreiro, L., Caamaño, R., Cabaleiro, S., Ruiz de Ocenda, M.V. and Villoch, F. (2017). Ceratomyxosis infection in cultured striped red mullet (Mullus surmuletus Linnaeus, 1786) broodstock. Aquaculture International 25, 2027‒2034. Bartholomew, J.L., Atkinson, S.D. and Hallett, S.L. (2006). Involvement of Manayunkia speciosa (Annelida: Polychaeta: Sabellidae) in the life cycle of Parvicapsula minibicornis, a myxozoan parasite of Pacific salmon. Journal of Parasitology 92, 742‒ 748. Bartholomew, J.L., Whipple, M.J., Stevens, D.G. and Fryer, J.L. (1997). The life cycle of Ceratomyxa shasta, a myxosporean parasite of salmonids, requires a freshwater polychaete as an alternate host. Journal of Parasitology 83, 859‒868. Camus, A.C. and Griffin, M.J. (2010). Molecular characterization and histopathology of Myxobolus koi infecting the gills of a koi, Cyprinus carpio, with an amended morphological description of the agent. Journal of Parasitology 96, 116‒124. Cardim, J., Silva, D., Hamoy, I., Matos, E. and Abrunhosa, F. (2018). Myxobolus bragantinus n. sp. (Cnidaria: Myxosporea) from the gill filaments of the redeye mullet, Mugil rubrioculus (Mugiliformes: Mugilidae), on the eastern Amazon coast. Zootaxa 4482, 177-187. Cardona, L. (2001). Non-competitive coexistence between Mediterranean grey mullet: evidence from seasonal changes in food availability, niche breadth and trophic overlap. Journal of Fish Biology 59, 729‒744. Cardona, L. (2006). Habitat selection by grey mullets (Osteichthyes: Mugilidae) in Mediterranean estuaries: the role of salinity. Scientia Marina 70, 443–455.

Page | 287

Chapter X | Hyperdiversification of Myxobolus spp. in mullet hosts

Carriero, M.M., Adriano, E.A., Silva, M.R., Ceccarelli, P.S. and Maia, A.A. (2013). Molecular phylogeny of the Myxobolus and Henneguya genera with several new South American species. PLoS One 8, e73713. Cone, D.K. and Easy, R.H. (2005). Supplemental diagnosis and molecular taxonomy of Myxobolus diaphanus (Fantham, Porter et Richardson, 1940) (Myxozoa) parasitizing Fundulus diaphanus (Cyprinodontiformes) in Nova Scotia, Canada. Folia Parasitologica 52, 217‒222. Crosetti, D. (2016). Current state of grey mullet fisheries and culture. In Crosetti, D. and Blaber, S. (eds). Biology, ecology and culture of grey mullets (Mugilidae). CRC Press, Boca Raton, Florida, USA, pp. 398‒450. Crosetti, D. and Cataudella, S. (1995). Grey mullet culture. In Nash, C.E. (ed). World Animal Science 34B: Production of Aquatic Animals. Elsevier Burlington, Massachusetts, USA, pp. 271–288. Das, M.K. (1996). Myxozoan and urceolarid ciliate parasites of wild and cultured Liza parsia in deltaic West Bengal. Journal of the Inland Fisheries Society of India 28, 46‒56. Durand, J.D., Shen, K.N., Chen, W.J., Jamandre, B.W., Blel, H., Diop, K., Nirchio, M., Garcia de León, F.J., Whitfield, A.K., Chang, C.W. and Borsa, P. (2012). Systematics of the grey mullets (Teleostei: Mugiliformes: Mugilidae): molecular phylogenetic evidence challenges two centuries of morphology-based taxonomy. Molecular Phylogenetics and Evolution 64, 73‒92. Egusa, S., Maeno, Y. and Sorimachi, M. (1990). A new species of Myxozoa, Myxobolus episquamalis sp. n. infecting the scales of the mullet, Mugil cephalus L. Fish Pathology 25, 87‒91. Eiras, J.C., Molnár, K. and Lu, Y.S. (2005). Synopsis of the species of Myxobolus Bütschli, 1882 (Myxozoa: Myxosporea: Myxobolidae). Systematic Parasitology 61, 1‒46. Eszterbauer, E. (2004). Genetic relationship among gill-infecting Myxobolus species (Myxosporea) of cyprinids: molecular evidence of importance of tissue-specificity. Diseases of Aquatic Organisms 58, 35‒40. Eszterbauer, E., Atkinson, S., Diamant, A., Morris, D., El-Matbouli, M. and Hartikainen, H. (2015). Myxozoan life cycles: practical approaches and insights. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan evolution, ecology and development. Springer International Publishing, Switzerland, pp. 175‒198. Faye, N., Kpatcha, T.K., Debakate, C., Fall, M. and Toguebaye, B.S. (1999). Gill infections due to myxosporean (Myxozoa) parasites in fishes from Senegal with description of Myxobolus hani sp. n. Bulletin of the European Association of Fish Pathologists 19, 14‒ 16. Ferguson, J.A., Atkinson, S.D., Whipps, C.M. and Kent, M.L. (2008). Molecular and

Page | 288

Chapter VI | Hyperdiversification of Myxobolus spp. in mullet hosts

morphological analysis of Myxobolus spp. of salmonid fishes with the description of a new Myxobolus species. Journal of Parasitology 94, 1322‒1334. Fiala, I. (2006). The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. International Journal of Parasitology 36, 1521‒1534. Forró, B. and Eszterbauer, E. (2016). Correlation between host specificity and genetic diversity for the muscle-dwelling fish parasite Myxobolus pseudodispar: examples of myxozoan host-shift? Folia Parasitologica 63, 019. Hallett, S.L. and Diamant, A. (2001). Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia. Diseases of Aquatic Organisms 46, 197‒212. Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. The Quarterly Review of Biology 66, 411‒453. Holzer, A.S., Bartošová-Sojková, P., Born-Torrijos, A., Lövy, A., Hartigan, A. and Fiala, I. (2018). The joint evolution of the Myxozoa and their alternate hosts: A cnidarian recipe for success and vast biodiversity. Molecular Ecology 27, 1651‒1666. Holzer, A.S., Blasco-Costa, I., Sarabeev, V.L., Ovcharenko, M.O. and Balbuena, J.A. (2006). Kudoa trifolia sp. n. - molecular phylogeny suggests a new spore morphology and unusual tissue location for a well-known genus. Journal of Fish Diseases 29, 743‒755. Holzer, A.S., Sommerville, C. and Wootten, R. (2004). Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences. International Journal of Parasitology 34, 1099‒1111. Hotos, G.N. and Vlahos, N. (1998). Salinity tolerance of Mugil cephalus and Chelon labrosus (Pisces: Mugilidae) fry in experimental conditions. Aquaculture 167, 329‒338. Kang, H., Bae, M-J., Lee, D-S., Hwang, S-J., Moon, J-S. and Park, Y-S. (2017). Distribution patterns of the freshwater oligochaete Limnodrilus hoffmeisteri influenced by environmental factors in streams on a Korean nationwide scale. Water 9, 921. Karlsbakk, E., Kristmundsson, A., Albano, M., Brown, P. and Freeman, M.A. (2017). Redescription and phylogenetic position of Myxobolus aeglefini and Myxobolus platessae n. comb. (Myxosporea), parasites in the cartilage of some North Atlantic marine fishes, with notes on the phylogeny and classification of the Platysporina. Parasitology International 66, 952‒959. Kim, W.S., Kim, J.H., Jang, M.S., Jung, S.J. and Oh, M.J. (2013a). Infection of wild mullet (Mugil cephalus) with Myxobolus episquamalis in Korea. Parasitology Research 112, 447‒451. Kim, W.S., Kim, J.H. and Oh, M.J. (2013b). Morphologic and genetic evidence for mixed infection with two Myxobolus species (Myxozoa: Myxobolidae) in grey mullets, Mugil

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cephalus, from Korean waters. Korean Journal of Parasitology 51, 369‒373. Kodádková, A., Bartošová-Sojková, P., Holzer, A.S. and Fiala, I. (2015). Bipteria vetusta n. sp. - an old parasite in an old host: tracing the origin of myxosporean parasitism in vertebrates. International Journal of Parasitology 45, 269‒276. Køie, M. (2000). First record of an actinosporean (Myxozoa) in a marine polychaete annelid. Journal of Parasitology 86, 871‒872. Køie, M. and Karlsbakk, E. (2009). Ellipsomyxa syngnathi sp. n. (Myxozoa, Myxosporea) in the pipefish Syngnathus typhle and S. rostellatus (Teleostei, Syngnathidae) from Denmark. Parasitology Research 105, 1611‒1616. Køie, M., Whipps, C.M. and Kent, M.L. (2004). Ellipsomyxa gobii (Myxozoa: Ceratomyxidae) in the common goby Pomatoschistus microps (Teleostei: Gobiidae) uses Nereis spp. (Annelida: Polychaeta) as invertebrate hosts. Folia Parasitologica 51, 14‒18. Krodkiewska, M. (2007). The distribution of Potamothrix bavaricus (Oeschmann, 1913) (Oligochaeta) in anthropogenic freshwater habitats of an industrialised area (Upper Silesia, Poland). Limnologica 37, 259‒263. Kumar, S., Stecher, G. and Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution 33, 1870‒ 1874. Laffaille, P., Feunteun, E., Lefebvre, C., Radureau, A., Sagan, G. and Lefeuvre, J.C. (2002). Can thin-lipped mullet directly exploit the primary and detritic production of European macrotidal salt marshes? Estuarine, Coastal and Shelf Science 54, 729‒736. Lom, J. and Arthur, J.R. (1989). A guideline for the preparation of species descriptions in Myxosporea. Journal of Fish Diseases 12, 151‒156. Marcotegui, P. and Martorelli, S. (2017). Myxobolus saladensis sp. nov., a new species of gill parasite of Mugil liza (Osteichthyes, Mugilidae) from Samborombón Bay, Buenos Aires, Argentina. Iheringia Série Zoologia 107, e2017026. Moroz, T.G. (1994). Aquatic Oligochaeta of the Dnieper-Bug Estuary system. Hydrobiologia 278, 133‒138. Mota, M., Rochard, E. and Antunes, C. (2016). Status of the diadromous fish of the Iberian Peninsula: past, present and trends. Limnetica 35, 1‒18. Nelson, J.S., Grande, T.C. and Wilson, M.V.H. (2016). Fishes of the World, 5th edn. John Wiley & Sons, Hoboken, New Jersey, USA, pp. 752. Oren, O.H. (1981). Aquaculture of Grey Mullets International Biological Programme. Vol. 26. Cambridge University Press, Cambridge, UK, pp. 507. Ovcharenko, M., Dezfuli, B.S., Castaldelli, G., Lanzoni, M. and Giari, L. (2017). Histological and ultrastructural study of Myxobolus mugchelo (Parenzan, 1966) with initial histopathology survey of the Liza ramada host intestine. Parasitology Research 116,

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1713‒1721. Özer, A., Gürkanli, C.T., Özkan, H., Acar, G., Ciftci, Y. and Yurakhno, V. (2016). Molecular characterization and morphological aspects of Myxobolus parvus (Myxozoa) from Liza saliens (Mugilidae) off the Turkish Black Sea coasts. Parasitology Research 115, 3513‒3518. Paperna, I., Overstreet RM (1981) Parasites and diseases of mullets (Mugilidae). In Oren, O.H. (ed). Aquaculture of grey mullets. Cambridge University Press, Cambridge, pp. 1‒19. Pascar-Gluzman, C. and Dimentman, C. (1984). Distribution and habitat characteristics of Naididae and Tubificidae in the inland waters of Israel and the Sinai Peninsula. Hydrobiologia 115, 197–205. Patra, S., Bartošová-Sojková, P., Pecková, H., Fiala, I., Eszterbauer, E. and Holzer, A.S. (2018). Biodiversity and host-parasite cophylogeny of Sphaerospora (sensu stricto) (Cnidaria: Myxozoa). Parasites and Vectors 11, 347. Pfannkuche, O. (1980). Distribution, abundance and life cycles of Oligochaeta from the marine hygropsammal with special reference to the Phallodrilinae (Tubificidae). International Review of Hydrobiology 65, 835‒848. Rangel, L.F., Rocha, S., Casal, G., Castro, R., Severino, R., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2017). Life cycle inference and phylogeny of Ortholinea labracis n. sp. (Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm. Journal of Fish Diseases 40, 243‒ 262. Rangel, L.F., Santos, M.J., Cech, G. and Székely, C. (2009). Morphology, molecular data, and development of Zschokkella mugilis (Myxosporea, Bivalvulida) in a polychaete alternate host, Nereis diversicolor. Journal of Parasitology 95, 561‒569 Rocha, S., Azevedo, C., Oliveira, E., Alves, Â., Antunes, C., Rodrigues, P. and Casal, G. (2019a). Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular redescription of the cryptic species Myxobolus exiguus. Parasitology 146, 479‒496. Rocha, S., Casal, G., Garcia, P., Matos, E., Al-Quraishy, S. and Azevedo, C. (2014). Ultrastructure and phylogeny of the parasite Henneguya carolina sp. nov. (Myxozoa), from the marine fish Trachinotus carolinus in Brazil. Diseases of Aquatic Organisms 112, 139‒148. Rocha, S., Casal, G., Rangel, L., Castro, R., Severino, R., Azevedo, C. and Santos, M.J. (2015). Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae). Parasitology International 64, 305−313. Rocha, S., Rangel, L.F., Castro, R., Severino, R., Azevedo, C., Santos, M.J., and Casal, G.

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(2019b). The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis. Journal of Invertebrate Pathology 160, 33–42. Ronquist, F. and Huelsenbeck, J.P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Rosa, B.F.J.V., Martins, R.T. and Alves, R.G. (2015). Distribution of oligochaetes in a stream in the Atlantic Forest in southeastern Brazil. Brazilian Journal of Biology 75, 1–7. Saleh, M.A. (2006). Cultured aquatic species information programme (CASIP): Mugil cephalus. FAO, Rome, Italy. Sarkar, N.K. (1989). Myxobolus anili sp. nov. (Myxozoa: Myxosporea) from a marine teleost fish Rhinomugil corsula Hamilton. Proceedings of the Zoological Society Calcutta 42, 71‒74. Sarkar, N.K. (1999). Some new Myxosporidia (Myxozoa: Myxosporea) of the genera Myxobolus Butschli, 1882, Unicapsula Davis, 1942, Kudoa Meglitsch, 1947, Ortholinea Shulman, 1962 and Neoparvicapsula Gajevskaya, Kovaleva and Shulman, 1982. Proceedings of the Zoological Society Calcutta 52, 38‒48. Schenková, J. and Helešic, J. (2006). Habitat preferences of aquatic oligochaeta (Annelida) in the Rokytná River, Czech Republic – A small highland stream. Hydrobiologia 564, 117– 126. Seys, J., Vincx, M. and Meire, P. (1999). Spatial distribution of oligochaetes (Clitellata) in the tidal freshwater and brackish parts of the Schelde estuary (Belgium). Hydrobiologia 406, 119–132. Sharon, G., Ucko, M., Tamir, B. and Diamant, A. (2019). Co-existence of Myxobolus spp. (Myxozoa) in gray mullet (Mugil cephalus) juveniles from the Mediterranean Sea. Parasitology Research 118, 159–167. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1993). Zschokkella mugilis n. sp. (Myxosporea, Bivalvulida) from mulltes (Teleostei, Mugilidae) of Mediterranean waters - light and electron-microscopic description. Journal of Eukaryotic Microbiology 40, 755–764. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1995). Light and electron microscopic description of Polysporoplasma n. g. (Myxosporea: Bivalvulida), Polysporoplasma sparis n. sp. from Sparus aurata (L), and Polysporoplasma mugilis n. sp. from Liza aurata L. European Journal of Protistology 31, 77–89. Sousa, R., Dias, S.C., Guilhermino, L. and Antunes, C. (2008). Minho River tidal freshwater wetlands: threats to faunal biodiversity. Aquatic Biology 3, 237–250. Thabet, A., Tlig-Zouari, S., Al Omar, S.Y. and Mansour, L. (2016). Molecular and morphological characterisation of two species of the genus Ellipsomyxa Køie, 2003 (Ceratomyxidae)

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from the gall-bladder of Liza saliens (Risso) off Tunisian coasts of the Mediterranean. Systematic Parasitology 93, 601–611. Thélohan, P. (1895). Recherches sur les Myxosporidies. Bulletin biologique de la France et de la Belgique 4, 100–394. Torricelli, P., Tongiorgi, P. and Almansi, P. (1981). Migration of grey mullet fry into the Arno river: Seasonal appearance, daily activity, and feeding rhythms. Fisheries Research 1, 219–234. U-taynapun, K., Penprapai, N., Bangrak, P., Mekata, T., Itami, T. and Tantikitti, C. (2011). Myxobolus supamattayai n. sp. (Myxosporea: Myxobolidae) from Thailand parasitizing the scale pellicle of wild mullet (Valamugil seheli). Parasitology Research 109, 81–91. Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215‒219. Yang, C., Zhou, Y., Zhao, Y., Huang, W. and Huang, C. (2017). Erection of Unicapsulocaudum mugilum gen. et sp. nov. (Myxozoa: Ceratomyxidae) based on its morphological and molecular data. Journal of Natural History 51, 457–467. Yemmen, C., Ktari, M.H. and Bahri, S. (2012). Parasitofauna of some mugilid and soleid fish species from Tunisian lagoons. Acta Adriatica 52, 173–182. Yurakhno, V.M. and Ovcharenko, M.O. (2014). Study of Myxosporea (Myxozoa), infecting worldwide mullets with description of a new species. Parasitology Research 113, 3661– 3674. Yurakhno, V.M., Ovcharenko, M.O., Holzer, A.S., Sarabeev, V.L. and Balbuena, J.A. (2007). Kudoa unicapsula n. sp (Myxosporea: Kudoidae) a parasite of the Mediterranean mullets Liza ramada and L. aurata (Teleostei : Mugilidae). Parasitology Research 101, 1671–1680. Zetina-Rejón, M.J., Arreguı́n-Sánchez, F. and Chávez, E.A. (2003). Trophic structure and flows of energy in the Huizache–Caimanero lagoon complex on the Pacific coast of Mexico. Estuarine, Coastal and Shelf Science 57, 803–815.

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Chapter XI

Morphological and molecular characterization of myxobolids (Cnidaria, Myxozoa) infecting cypriniforms (Actinopterygii, Teleostei) endemic to the Iberian Peninsula

This chapter was adapted from:

Rocha, S., Azevedo, C., Alves, Â, Antunes, C. and Casal, G. (2019). Morphological and molecular characterization of myxobolids (Cnidaria, Myxozoa) infecting cypriniforms (Actinopterygii, Teleostei) endemic to the Iberian Peninsula. Parasite (Under Review)

Chapter XI | Myxobolids from endemic Iberian cypriniforms

Abstract

The Iberian Peninsula provides a unique freshwater ecosystem for native and endemic cypriniforms to thrive. Despite these fishes being hosts for multiple myxobolids worldwide, few researches have been performed in this geographic location. In this study, the examination of three Iberian endemic cypriniforms showed that myxosporean richness in the Iberian Peninsula is underestimated, with three new and one known myxobolid species being reported based on morphological and molecular data. Myxobolus arcasii n. sp. is described from the kidney and gonads of the bermejuela Achondrostoma arcasii, M. duriensis n. sp. from the gills of the Northern straight-mouth nase Pseudochondrostoma duriense, and Thelohanellus paludicus n. sp. from the intestine of the Southern Iberian spined-loach Cobitis paludica. Myxobolus pseudodispar Gorbunova, 1936 is further reported from several organs of P. duriense, and from the spleen of A. arcasii. The occurrence of M. pseudodispar in Iberian endemic species suggests that host-shift followed its co-introduction with central European leuciscids into this geographic location. Several other myxobolids originally described from barbels in central Europe have also been reported from the Iberian endemic cypriniform Luciobarbus bocagei. Nonetheless, excepting M. musculi, the identification of these myxobolids in L. bocagei is here shown to be dubious and require molecular confirmation. Phylogenetic analyses reveal Myxobolus arcasii n. sp. and M. duriensis n. sp. clustering within different lineages of leucisicid-infecting species, showing that myxobolids entered Leuciscidae as hosts multiple times during their evolution. Constituting the first myxobolid reported from the family Cobitinae, Thelohanellus paludicus n. sp. stands alone in the tree topology.

Introduction

The family Myxobolidae Thélohan, 1892 is the largest among Myxozoa, specifically due to the species-richness of the genera Myxobolus Bütschli, 1881, Henneguya Thélohan, 1892 and Thelohanellus Kudo, 1933. Species belonging to these three genera are the most commonly reported from freshwater habitats, less frequently occurring in brackish and marine habitats. Of the about 1,200 known species of myxobolids, a significant amount has been described from fish hosts of the order Cypriniformes (Eiras, 2002; Eiras et al., 2005, 2014; Eiras and Adriano, 2012; Zhang et al., 2013). The latter constitutes the largest group of freshwater fishes worldwide, being distributed across Europe, Asia, Africa and North America (Saitoh et al., 2011; Eschmeyer and Fong, 2018). Despite having highly diversified lifestyles, cypriniforms are restricted to freshwater and, therefore, can naturally expand their distribution only through the direct connection of habitats (Saitoh et al., 2011; Stout et al., 2016). The historical shaping of continental lands and inland waters thus influenced the evolution and

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Chapter XI | Myxobolids from endemic Iberian cypriniforms radiation of this fish group (Kumazawa and Nishida, 2000; Saitoh et al., 2011), which accounts for numerous species that are endemic to specific geographic locations. The Iberian Peninsula has one of the greatest European percentages of endemism, not only due to the isolation caused by the Pyrenees and the Straits of Gibraltar, but also due to its complex fluvial network, which comprises a high number of independent river basins, in which freshwater communities are strongly isolated, and structured according to the orographic and climatic peculiarities of the region (Hernando and Soriguer, 1992; Doadrio, 2001; Clavero et al., 2004). The majority of the native freshwater fish species in this geographic region belong to the order Cypriniformes, more specifically to the families Cyprinidae Rafinesque, 1815, Cobitidae Swainson, 1838, and Nemacheilidae Regan, 1911; with few representatives of the orders Acipenseriformes, Anguilliformes, Atheriniformes, Clupeiformes, Cyprinidontiformes, Petromyzontiformes, and Salmoniformes. Endemic species are found among representatives of the orders Cypriniformes and Cyprinidontiformes (Hernando and Soriguer, 1992; Filipe et al., 2009), and include the “bermejuela” Achondrostoma arcasii (Steindachner, 1866) (Cypriniformes, Leuciscidae), the Northern straight-mouth nase Pseudochondrostoma duriense (Coelho, 1985) (Cypriniformes, Leuciscidae), and the Southern Iberian spined-loach Cobitis paludica (de Buen, 1930) (Cypriniformes, Cobitidae, Cobitinae). These three species are currently classified as vulnerable according with IUCN criteria and face massive conservation threats, namely due to the loss of spawning habitats and feeding grounds caused by severe anthropogenic changes of freshwater ecosystems. These include not only pollution and intense alterations, but also the introduction and spread of non-native species (Aparicio et al, 2000; Doadrio, 2001; Doadrio et al., 2011). The acquisition of knowledge pertaining to parasites of native and endemic fish species is of major importance for gaining awareness of ecological issues in freshwater ecosystems, as parasites are useful indicators of the health of wild fish populations, as well as of habitat quality (e.g. Marcogliese, 2005; Hudson et al., 2006; Hernandez et al., 2007; Vidal-Martinez et al., 2010; Simková et al., 2012). Nonetheless, and despite the recognized importance of myxozoans as fish pathogens worldwide (Lom and Dyková, 1992, 2006; Kent et al., 2001; Yokoyama et al., 2012), few studies concern the myxozoan community infecting native and endemic freshwater fishes of the Iberian Peninsula. In the present study, microscopic and molecular descriptions are provided for three new myxobolids infecting cypriniforms endemic to the Iberian Peninsula. The muscle-dwelling species, Myxobolus pseudodispar Gorbunova, 1936, is further reported from two of the endemic fish species analysed, revealing that host-shift occurred following its co-introduction with central Eurpean leuciscids into the Iberian Peninsula. Overall, the myxobolid biodiversity currently known from cypriniform hosts in this geographic area is shown to be underestimated.

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Chapter XI | Myxobolids from endemic Iberian cypriniforms

Materials and Methods

Fish sampling, myxozoan survey, and morphological analysis

Between 2013 and 2017, trimestral samplings of fish were performed from fyke-nets located in the River Minho (41º 56’ N, 08º 45’ W), near the border village of “Vila Nova de Cerveira”, Portugal. The River Minho marks the boundaries between northern Portugal and the Spanish autonomous community of Galicia. It originates in “Serra da Meira”, in the province of Lugo (Spain), and runs more than 300 km to drain into the Atlantic Ocean at the Portuguese northwest coast near the village of “Caminha”. Fish samples included specimens of three cypriniform species endemic to the Iberian Peninsula: Achondrostoma arcasii (Steindachner, 1866) (n = 5; total length 13.2 ± 1.9 [9.9–14.4] cm; weight 33.2 ± 13.8 [10.0–47.0] gr); Pseudochondrostoma duriense (Coelho, 1985) (n = 15; total length 22.5 ± 8.0 [9.3–33.0] cm; weight 137.8 ± 103.0 [6.0–349] gr); and Cobitis paludica (de Buen, 1930) (n = 27; total length 9.2 ± 1.0 [7.0–11.2] cm; weight 5.2 ± 2.5 [2.0–12.0] gr). Specimens were transported live to the laboratory and, prior to dissection, anesthetized with ethylene glycol monophenyl ether (Merck, Germany) at 1ml/L. Several organs and tissues were macro- and microscopically examined for the presence of myxozoan parasites. Cysts and myxospores were photographed using an Olympus BX41 light microscope (Olympus, Japan). Morphometry was determined from fresh material, according to the guidelines provided by Lom and Arthur (1989). All measurements include the mean value ± standard deviation (S.D.), range of variation and number of myxospores measured (range, n). Prevalence of infection includes the mean value and interval confidence values.

DNA extraction, amplification, and sequencing

Cysts and fragments of tissues containing myxospores were preserved in absolute ethanol at 4 ºC. Genomic DNA extraction was performed using a GenEluteTM Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, USA), following the manufacturer’s instructions. The SSU rDNA gene was amplified using both universal and myxosporean-specific primers: the 5’-end bu pairing the primer 18E (5’-CTG GTT GAT CCT GCC AGT-3’) (Hillis and Dixon, 1991) with the primers MyxospecR (5’-CAA CAA GTT GAT AGG GCA GAA-3’) (Fiala, 2006), ACT3r (5’-ATT GTT CGT TCC ATG-3’) (Rocha et al., 2014) and MYX4R (5’-CTG ACA GAT CAC TCC ACG AAC-3’) (Hallett and Diamant, 2001); and the 3’-end by pairing the primers MyxospecF (5’-TTC TGC CCT ATC AAC TTG TTG-3’) (Fiala, 2006), ACT3f (5’-CAT GGA ACG AAC AAT-3’) (Hallett and Diamant, 2001) and MYX4F (5’-GTT CGT GGA GTG

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ATC TGT CAG-3’) (Rocha et al., 2015) with the primer 18R (5’-CTA CGG AAA CCT TGT TAC G-3’) (Whipps et al., 2003). PCRs were performed in 50 µl reactions using 10 pmol of each primer, 10 nmol of each dNTP, 2.0 mM MgCl2, 5 µl 10× Taq polymerase buffer, 2.5 units Taq DNA polymerase (NZYTech, Lisbon, Portugal), and approximately 50–100 ng of genomic DNA. The reactions were run on a Hybaid PxE Thermocycler (Thermo Electron Corporation, Milford, Massachusetts, USA), with initial denaturation at 95 ºC for 3 min, followed by 35 cycles of 94 ºC for 45 sec, 53 ºC for 45 sec, and 72 ºC for 90 sec. The final elongation step was performed at 72 ºC for 7 min. Five-µl aliquots of the PCR products were electrophoresed through a 1% agarose 1× tris-acetate-EDTA buffer (TAE) gel stained with ethidium bromide. PCR products were purified using Puramag™ magnetic beads coated with carboxylic acid groups (MCLAB, San Francisco, California, USA). The PCR products from different regions of the SSU rDNA gene were sequenced directly. The sequencing reactions were performed using a BigDye Terminator v3.1 Cycle Sequencing Kit from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and were run on an ABI3700 DNA analyzer from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Sequence assembly, distance estimation, and phylogenetic analysis

The partial sequences obtained for the different case isolates were aligned and assembled in MEGA 6.06 (Tamura et al., 2013). In order to calculate distance estimation, newly generated sequences were submitted to BLAST search (NCBI) for retrieving the SSU rDNA sequences with highest similarity score. Other sequences belonging to congeners reported to infect the same host, or closely related hosts, were also included in the analysis, except for those which were deemed invalid by Molnár (2011), as are the cases of the sequences of Myxobolus impressus (AF507970) and Myxobolus dogieli (EU003977, EU003978). The selected sequences were then aligned using the software MAFFT version 7 available online, and distance estimation was performed in MEGA 6.06, with the p-distance model and all ambiguous positions removed for each sequence pair. For phylogenetic analysis, the dataset was widened to encompass the SSU rDNA sequences of other cypriniform-infecting myxobolids. Sequences belonging to species reported from the Iberian Peninsula were included, i.e. M. branchialis (Markevitsch, 1932), M. branchilateralis Molnár et al., 2012, M. cutanei Alvarez-Pellitero and González-Lanza, 1985, M. leuciscini González-Lanza and Alvarez-Pellitero, 1985, M. musculi Keysselitz, 1908, M. pfeifferi Thélohan, 1895, M. pseudodispar Gorbunova, 1936 and M. tauricus Miroshnichenko, 1979. The final dataset comprised a total of 67 SSU rDNA sequences, plus Myxidium lieberkuehni (X76638) and Zschokkella auratis (KC849425) as outgroup. Alignments were

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Chapter XI | Myxobolids from endemic Iberian cypriniforms performed using the software MAFFT version 7 available online, and posteriorly manually edited in MEGA 6.06. Phylogenetic trees were calculated from the sequence alignments using maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI). The general time reversible substitution model with estimates of invariant sites and gamma distributed among site rate variation (GTR+I+Γ) was used in both ML and BI analyses, in accordance to the modeltest algorithms of the software. BI analyses were performed using MrBayes v3.2.6 (Ronquist and Huelsenbeck, 2003), with posterior probability distributions generated using the Markov Chain Monte Carlo (MCMC) method, with four chains running simultaneously for 1 million generations, and every 100th tree sampled. MP trees were obtained using the Subtree- Pruning-Regrafting algorithm with a search level of 1 and random initial tree addition of 10 replicates. Both ML and MP analyses were conducted in MEGA 6.06, with bootstrap confidence values calculated from 500 replicates.

Results

Myxozoan survey and overall prevalence of infection

Collected and analysed fish specimens did not present obvious external symptoms of infection or disease. Macro- and microscopic analysis of 13 different organs revealed the presence of myxospores, disseminated or contained within cysts, in the gills, muscle, spleen, gonads, kidney, stomach, and intestine of several specimens. All myxospores were morphologically identified as belonging to the family Myxobolidae (phylum Cnidaria Hatschek, 1888, subphylum Myxozoa). Only one out of the five specimens of Achondrostoma arcasii analysed was simultaneously infected with two morphotypes of the genus Myxobolus. One morphotype formed cysts that were present in both the gonads and kidney, while the other appeared disseminated in the spleen. Individual prevalence of infection of both morphotypes was 20.0% (1 infected in 5 specimens analysed). Molecular analysis of the SSU rDNA gene confirmed the presence of two distinct Myxobolus spp. infecting A. arcasii, with the morphotype in the gonads and kidney being described here as a new species, and the one occurring in the spleen being identified as M. pseudodispar Gorbunova, 1936. In turn, 14 out of the 15 specimens of Pseudochondrostoma duriense analysed were infected by at least one of two Myxobolus morphotypes: one formed cysts in the gills, while the other appeared disseminated in the muscle, spleen, liver, kidney, stomach and intestine. Overall prevalence of infection of Myxobolus in P. duriense was 93.3%; 53.3% (8 infected in 15 specimens analysed) for the morphotype infecting the gills, and 86.7% (13 infected in 15 specimens analysed) for the morphotype appearing disseminated in several tissues (see Table

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Chapter XI | Myxobolids from endemic Iberian cypriniforms

1). Molecular analysis of the SSU rDNA gene confirmed the presence of two Myxobo- lus spp. infecting P. duriense, with the morphotype occurring in the gills being described here as a new species, and the other also being identified as M. pseudodispar. Concerning the myxozoan survey performed on Cobitis paludica, only one representative of the genus Thelohanellus was found infecting the intestine of a single specimen and is described herein as a new

species.

. .

examined, examined, as determined by light microscopic observations. PI: overall prevalence of infection

Morphological and molecular characterization of

P. P. duriense myxobolids

M. pseudodispar M. Myxobolus arcasii n. sp.

(Figs. 1A–C and 2A)

n. sp.; Mp: sp.; n.

Diagnosis: Microscopic plas-

modia of variable shapes and

infection infection in the organs of

M. duriensis M. sizes in the hematopoietic tissue of the kidney (Fig. 1A),

Myxobolus and in the undifferentiated tissue of the gonads (Fig. 1B). Myxospores subspheri-

cal in valvular view and

per organ examined; Md:examined;per organ

ellipsoidal in sutural view, 9.7

Presence/absence Presence/absence of

. ± 0.5 (9.3–10.7) µm long (n =

Myxobolus 15), 8.1 ± 0.2 (8.0–8.7) µm

Table 1 of

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Chapter XI | Myxobolids from endemic Iberian cypriniforms wide (n = 15) and 6.5 ± 0.2 (6.3–6.7) µm (n = 8) thick. Valves smooth with 6 to 8 markings near the suture line. Two pyriform equally sized polar capsules located side by side at the myxospores' anterior pole, 3.9 ± 0.3 (3.3–4.3) µm long and 3.0 ± 0.2 (2.7–3.3) µm wide (n = 15). Polar tubule forming 6 (rarely 7) coils (Fig. 1C). Overall morphology is depicted in a schematic drawing representative of a myxospore in valvular view (Fig. 2A). Type host: Achondrostoma arcasii (Steindachner, 1866) (Cypriniformes, Leuciscidae) (common names: “bermejuela”, “ruivaco”, “panjorca” or “bogardo”). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near the village of “Vila Nova de Cerveira”, Portugal. Site of infection: The hematopoietic tissue of the kidney, and undifferentiated tissue of the gonads. Prevalence: 20.0% (one infected in five specimens examined). Type specimens: One glass slide containing semi-thin sections of the hapantotype was deposited in the Type Slide Collection of the Laboratory of Animal Pathology at the Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2019.41. Molecular data: One SSU rDNA gene sequence with a total of 1,970 bp and GenBank accession no. MK053784. Etymology: The specific epithet “arcasii” derives from the specific epithet of the host species. Remarks: Comparison of the parasite to other Myxobolus spp. reported from fishes of genera closely related to Achondrostoma Robalo et al., 2007 revealed highest morphometric similarity to M. gallaicus Iglesias et al., 2001, M. leuciscini, M. bramae Reuss, 1906 and M. szentendrensis Cech et al., 2015 (Table 2). The three latter species, however, can be readily distinguished from the parasite in study based on molecular data of the SSU rDNA gene. In turn, differentiation from M. gallaicus is established based on morphological traits; myxospores of M. gallaicus are slender with longer polar capsules and a higher number of polar tubule coils. Further comparison to the remaining Myxobolus spp. previously reported from more distantly related leuciscid genera revealed some morphometric similarity of the parasite to M. hyborhynchi Fantham et al., 1939 from bluntnose minnow Pimephales notatus (Rafinesque, 1820) in Canada, as well as to M. schuberti Li and Desser, 1985 and M. siddalli Salim and Desser, 2000 from common shiner Luxilus cornutus (Mitchill, 1817) in Canada (see Eiras et al., 2005). Differentiation from M. siddalli could be performed by molecular comparison of respective SSU rDNA sequences. Despite lacking molecular data for comparison, M. hyborhynchi differs from the parasite in study in having significantly thinner polar capsules, while M. schuberti myxospores and polar capsules are generally longer and display fewer number of polar filament coils. Overall, distance estimation retrieved values of similarity

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Chapter XI | Myxobolids from endemic Iberian cypriniforms

Figure 1. Light micrographs of wetmounts of the new myxobolid species infecting Iberian endemic cypriniforms. A– B. Cysts of Myxobolus arcasii n. sp. in the hematopoietic tissue of the kidney, and in the undifferentiated tissue of the gonads of Achondrostoma arcasii, respectively. C. Myxospore of Myxobolus arcasii n. sp. evidencing the two polar capsules in which the polar tubule coils. D–E. Myxospores of Myxobolus duriensis n. sp. in the primary gill filaments of Pseudochondrostoma duriense, evidencing the polar tubule coiling within the polar capsules, and the iodinophilous vacuole in the sporoplasm. F–G. Myxospores of Thelohanellus paludicus n. sp. in the intestinal epithelium of Cobitis paludica, showing the polar tubule coiling within the polar capsule, and the two conspicuous iodinophilous vacuoles in the sporoplasm.

consistently lower than 95.0%, including to M. leuciscini (91.5%), M. bramae (88.9%), M. szentendrensis (80.8%), and M. siddalli (91.0%). Considering all the above, this parasite is suggested as a new species, herein named Myxobolus arcasii n. sp.

Myxobolus duriensis n. sp. (Figs. 1D–E and 2B) Diagnosis: Microscopic cysts, spherical to subspherical, in the primary gill filaments. Myxospores subspherical in valvular view and ellipsoidal in sutural view, with smooth valves,

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Chapter XI | Myxobolids from endemic Iberian cypriniforms

13.5 ± 0.3 (13.0–14.0) µm long (n = 20), 9.0 ± 0.5 (8.0–9.7) µm wide (n = 20) and 7.6 ± 0.3 (7.3–8.0) µm (n = 13) thick. Two pyriform equally sized polar capsules located side by side at the myxospores' anterior pole, 4.9 ± 0.3 (4.3–5.3) µm long and 3.4 ± 0.1 (3.3–3.7) µm wide (n = 25). Polar tubule forming 6 coils. One, more rarely two, small iodinophilous vacuoles in the sporoplasm (Fig. 1D–E). Overall morphology is depicted in a schematic drawing representative of a myxospore in valvular view (Fig. 2B). Type host: The Northern straight-mouth nase Pseudochondrostoma duriense (Coelho, 1985) (Cy- priniformes, Leuciscidae) (common name: “boga”). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near the village of “Vila Nova de Cerveira”, Portugal. Site of infection: The primary gill filaments. Prevalence: 53.3% (8 infected in 15 specimens examined). Type specimens: One glass slide containing semi-thin sections of the hapantotype was deposited in the Type Slide Collection of the Laboratory of Animal Pathology at the Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2019.42. Molecular data: One SSU rDNA gene sequence Figure 2. Schematic drawings of the new with a total of 1,983 bp, deposited in GenBank with the myxobolid species found infecting Iberian accession no. MK053783. The latter is representative endemic cypriniforms; myxospores drawn in of four identical sequences that were separately valvular view. A. Myxobolus arcasii n. sp. B. Myxobolus duriensis n. sp. C. Thelohanellus assembled from the partial results obtained from cysts paludicus n. sp. in the gills of four infected specimens. Etymology: The specific epithet “duriensis” derives from the specific epithet of the host species. Remarks: Comparison of the parasite in study to all other Myxobolus spp. previously described from leuciscid hosts, and more specifically to those reported from fishes of genera closely related to Pseudochondrostoma Robalo et al., 2007 (Table 2), revealed significant

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Chapter XI | Myxobolids from endemic Iberian cypriniforms

. . SL: myxospore length; SW:

ils; S: smaller; L: larger. Measurements from

es to belonging closely related genera

from fish

spp. spp. reported

Myxobolus

n. n. sp. to other

M. M. duriensis

n. sp. and

M. M. arcasii

Comparison Comparison of

Table 2 myxospore width; ST: myxospore thickness; PCL: polar capsule length; PCW: polarcapsule width; PTc: number of polartubule co meansasgiven host µm, available). areinfections(range) SD ± in in(when type the

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Chapter XI | Myxobolids from endemic Iberian cypriniforms

differences be it either in the morphology of

the myxospores or molecular data of the SSU

. . rDNA gene. Closest morphometric resemblance was determined in relation to M. chernovae (Chernova, 1970) Landsberg and

Lom, 1991, M. compressus Kudo, 1934, M. (Molnár et al., 2007) et al., (Molnár fanthami (Fantham et al., 1939) Landsberg and Lom, 1991, M. orbitalis (Fantham et al.,

nár et al., 2006). al., et nár 1939) Landsberg and Lom, 1991, M.

parallelipticoides (Fantham et al., 1939) is fish host in Hungary in Hungary host fish is Landsberg and Lom, 1991 and M. pfrille (Fantham et al., 1939) Landsberg and Lom,

1991, all of which were described from fish described from th from described - hosts belonging to other leuciscid genera in distant geographic locations. M. chernovae was described from roach Rutilus rutilus (Linnaeus, 1758) in Russia; M. compressus from River shiner Notropis blennius (Girard, 1856) in the USA; M. fanthami and M. orbitalis from common shiner Luxilus cornutus in

Canada; and M. parallelipticoides and M.

was morphologically and molecularly re molecularly and morphologically was

pfrille from finescale dace Chrosomus neogaeus (Cope, 1867), also in Canada (see M. leuciscini M. Eiras et al., 2005). Despite sharing some

, since since , morphometric similarity with the parasite in study, the myxospores of M. chernovae are larger and display shorter polar capsules. In

turn, those of M. compressus, M. fanthami Squalius cephalus Squalius and M. orbitalis have significantly thinner polar capsules, with the two latter further

description and molecular identification of the parasite from its original site of infection and host species in Hungary (Mol Hungary in species host and infection of site its original from the parasite of identification molecular and description being generally bigger. The myxospores of M. - parallelipticoides and M. pfrille also display thinner polar capsules and have a significantly wider morphometric range than those in study. Distance estimation revealed

highest similarity to M. szentendrensis Data from the morphological re morphological the from Data

Measurements from the original description in in description the original from Measurements (96.2%), with all other SSU rDNA sequences

1 2

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Chapter XI | Myxobolids from endemic Iberian cypriniforms included in the analysis retrieving similarity values lower than 95.0%. Thus, this parasite is suggested as a new species, herein named M. duriensis n. sp.

Thelohanellus paludicus n. sp. (Figs. 1F–G and 2C) Diagnosis: Plasmodia not observed. Individual myxospores disseminated in the intestinal epithet-lium. Myxospores ellipsoidal with a slightly more pointed anterior pole, with smooth valves, 14.4 ± 0.5 (14.0–15.3) µm long (n = 15) and 8.4 ± 0.3 (8.0–8.7) µm wide (n = 15). Single pyriform polar capsule, 6.7 ± 0.3 (6.0–7.0) µm long and 3.9 ± 0.1 (3.7–4.0) µm wide (n = 15), positioned slightly to the left of the medial plane in valvular view. Polar tubule forming 5 to 6 coils. Two to three conspicuous iodinophilous vacuoles in the sporoplasm (Fig. 1F–G). Overall morphology is depicted in a schematic drawing representative of a myxospore in valvular view (Fig. 2C). Type host: The Southern Iberian spined-loach Cobitis paludica (de Buen, 1930) (Cypriniformes, Cobitidae, Cobitinae) (common name: “verdemã do Norte”). Type locality: The River Minho (41º 56’ N, 08º 45’ W), near the village of “Vila Nova de Cerveira”, Portugal. Site of infection: The intestine. Prevalence: 3.7% (one infected in 27 specimens examined). Type specimens: One glass slide containing semi-thin sections of the hapantotype was deposited in the Type Slide Collection of the Laboratory of Animal Pathology at the Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2019.43. Molecular data: One SSU rDNA gene sequence with a total of 1,602 bp and GenBank accession no. MK053786. Etymology: The specific epithet “paludicus” derives from the specific epithet of the host species. Remarks: Currently, there are no reports of Thelohanellus spp. infecting fishes of the genus Cobitis Linnaeus, 1758, nor of any other fish genera in the Iberian Peninsula. Comparison to the three congeners reported from fish of the subfamily Cobitinae Swainson, 1838 worldwide, revealed T. paludicus n. sp. differing not only in host species and geographic location, but also in myxospore morphometry (Table 3). The myxospores and polar capsules of T. paludicus n. sp. are larger than those of T. acuminatus Akhmerov, 1955, a species that was originally described from the gills of common carp Cyprinus carpio Linnaeus, 1758 in Far- East Russia, and further reported from the pond loach Misgurnus anguillicaudatus (Cantor, 1842). In turn, despite T. paludicus n. sp. being overall morphometrically similar to T. misgurni (Kudo, 1919) Kudo, 1933, the latter was described from the gall bladder of M. anguillicaudatus in Tokyo and displays thinner myxospores. Finally, the measurements of T. paludicus n. sp.

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Chapter XI | Myxobolids from endemic Iberian cypriniforms

are within the wide morphometric range reported for T. pyriformis (Thélohan, 1892) Kudo, 1933, however, the high morphological variability found among the various reports of the latter species from different tissues and organs of several cyprinids across Europe [including the weatherfish Misgurnus fossilis (Linnaeus, 1758)], suggest it as length; length; SW: myxospore width; ST: a possible species complex. Molecular comparison to these three species is not possible due to the lack of available information. Overall, distance

arger. Measurementsarger. given are in µm, as means estimation revealed T. paludicus n. sp. without significant percentage of similarity to any of the SSU rDNA sequences presently available for its congeners and other myxobolids in general.

Myxobolus pseudodispar Gorbunova, 1936 This cosmopolitan species was identified from infected tissue samples of both P. duriense and A. arcasii, based on the morphometric aspects of the myxospores (i.e. asymmetrical shape and polar capsules different in size), broad range of sites of infection, and molecular data of the SSU

spp. spp. from cyprinid fish hosts of the subfamily Cobitinae. SL: myxospore rDNA gene. Infection by this parasite was determined to occur in the muscle, spleen, liver, kidney, stomach and intestine of 13 out of 15

Thelohanellus Thelohanellus specimens of P. duriense analysed (86.7%), as well as in the spleen of a single individual out of the five specimens of A. arcasii analysed (20.0%).

Large, elongated plasmodia were observed

n. n. sp. to other

in the muscle, while disseminated myxospores

appeared located in the stomach and intestine, and

T. T. paludicus in melanomacrophage centres of the spleen and renal parenchyma (Fig. 3). Myxospores were ellipsoidal with smooth valves, asymmetric, 10.9 ±

0.4 (10.0–11.3) µm long (n = 30) and 7.1 ± 0.3 (6.7–

Comparison Comparison of

7.3) µm wide (n = 30). Two unequal polar capsules

were pointed laterally to the left of the medial plane

Table Table 3. myxospore thickness; PCL: polar capsulelength; PCW: polar capsule width; PTc: numberof polartubule coils; S: smaller; L: l (range) SD ± available). (when

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Chapter XI | Myxobolids from endemic Iberian cypriniforms in valvular view. The polar tubule coiled obliquely in 4 (rarely 5) turns (Fig. 3). In total, two partial SSU rDNA gene sequences belonging to this species were deposited in GenBank, under the accession no. MK053785 and MK024332. The first comprised a total of 1,961 bp, being representative of the identical partial sequences obtained from the muscle, spleen, liver, kidney, stomach, and intestine of different infected specimens of P. duriense; while the second comprised 1,206 bp and was obtained from infected samples of spleen belonging to a single specimen of A. arcasii. These sequences shared 99.2% of similarity amongst each other, accounting for 9 nucleotide substitutions in a comparison established from 1,206 bp. Distance estimation revealed similarity values ranging between 94.6–97.3% to Hungarian isolates of M. pseudodispar from the type host Rutilus rutilus, 94.2–98.7% to isolates from rudd Scardinius erythrophthalmus (Linnaeus, 1758), 96.2–98.2% to isolates from freshwater bream Abramis brama (Linnaeus, 1758), 95.2–97.9% to isolates from white bream Blicca bjoerkna (Linnaeus, 1758), and 96.3–96.5% to isolates from bleak Alburnus alburnus

Figure 3. Light micrographs of myxospores of Myxobolus pseudodispar Gorbunova, 1936 infecting the connective tissue of several organs of Iberian endemic cypriniforms. A. Myxospore evidencing the overall asymmetry of its morpholofical features. B. Infection in the spleen of Achondrostoma arcasii. C–E. Infection in the kidney, digestive tube, and muscle of Pseudochondrostoma duriense, respectively.

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Chapter XI | Myxobolids from endemic Iberian cypriniforms

(Linnaeus, 1758).

Phylogenetic analysis

Bayesian inference and maximum likelihood analyses generated highly similar topologies, with some entropy in the middle of the tree due to the ML lower bootstrap support values of some branches (Fig. 4). The phylogenetic placement of the 5 new SSU rDNA sequences in study was consistent between phylograms: Myxobolus arcasii n. sp. and M. duriensis n. sp. clustered in separate branches, but within subclades comprising myxobolids that infect leuciscid hosts; Thelohanellus paludicus n. sp. occupied a basal position to several subclades of myxobolids that infect leuciscid and cyprinid hosts; while both sequences obtained for M. pseudodispar clustered among the SSU rDNA sequences of other muscle- dwelling parasites of leuciscids and cyprinids worldwide, more specifically with a conspecific sequence from a leuciscid host in Hungary (KU340979). Overall, species sequenced from hosts belonging to Leuciscidae, Cyprinidae and Labeoninae grouped to form multiple distinct subclades within the tree topology.

Discussion

Characterization and identification of the myxobolid species

Currently, it is widely accepted that reliable descriptions of myxosporeans can only be the outcome of the combined analysis of several criteria, i.e. myxospore morphology, host and tissue specificity, and molecular data (Easy et al., 2005; Ferguson et al., 2008; Atkinson et al., 2015). This is especially true for distinguishing between myxobolids, as myxospores of congener species share great morphological similarity amongst each other, making it essential for descriptions to include additional characters, namely of molecular nature. The great majority of myxobolids, however, were described mostly based on myxospore morphology (Eiras, 2002; Eiras et al., 2005, 2014; Eiras and Adriano, 2012; Zhang et al., 2013). Thus, despite its artificiality, morphology-based comparisons remain necessary for establishing differentiation between the many species that are without molecular data. Acknowledging that phylogenetic studies widely show the vertebrate host group as the most relevant evolutionary signal for myxobolids (Ferguson et al., 2008; Carriero et al., 2013; Rocha et al., 2014), the morphological comparisons performed in this study only took into consideration congeners reported from closely-related cypriniform species. Myxobolus arcasii n. sp. and M. duriensis n. sp. are described from Achondrostoma arcasii and Pseudochondrostoma duriense, respectively. In terms of phylogeny, the genera

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Chapter XI | Myxobolids from endemic Iberian cypriniforms

Figure 4. Tree topology resulting from the ML analysis of 67 SSU rDNA sequences of cypriniform-infecting myxobolids, plus Myxidium lieberkuehni (X76638) and Zschokkella auratis (KC849425) as outgroup. Numbers at the nodes are ML bootstrap values/BI posterior probabilities/MP bootstrap values; asterisks represent full support

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Chapter XI | Myxobolids from endemic Iberian cypriniforms

in all three methodologies; dashes represent a different branching of the BI/MP tree or a bootstrap support value under 50. Dark grey boxes evidence sequences belonging to species that have been reported from the Iberian Peninsula, including the five new sequences presented in this study; the family or sub-family of the vertebrate host is indicated using vertical lines.

Achondrostoma and Pseudochondrostoma are closely related to each other, as well as to the genera Chondrostoma Agassiz, 1832, Iberochondrostoma, Protochondrostoma and Parachondrostoma, representing six lineages of Leuciscidae that, up until recently, were all comprised within Chondrostoma (Robalo et al., 2007). Considering this, the morphological comparison performed here for M. arcasii n. sp. and M. duriensis n. sp. took into consideration the ca. 118 Myxobolus spp. previously described from leuciscid fishes (Eiras et al., 2005, 2014; Molnár et al., 2006, 2014; Cech et al., 2015; Liu et al., 2016; Atkinson et al., 2017), with emphasis on those reported from hosts belonging to the genera Achondrostoma, Chondrostoma and Pseudochondrostoma (see Table 2). To our best knowledge, myxozoan parasites have never been reported from Iberochondrostoma, Protochondrostoma and Parachondrostoma. Of the ca. 23 Myxobolus spp. previously reported from Achondrostoma, Chondrostoma and Pseudochondrostoma, only a few were originally described from these three genera: M. gallaicus from Pseudochondrostoma polylepis (Steindachner, 1864); M. leuciscini simultaneously from P. polylepis, A. arcasii and chub Squalius cephalus (Linnaeus, 1758); and M. arrabonensis Cech et al., 2015, M. chondrostomi Donec, 1962, M. paksensis Cech et al., 2015 and M. szentendrensis from the common nase Chondrostoma nasus (Linnaeus, 1758). Several others were either originally described from hosts of more distant leuciscid genera [M. albovae Krasilnikova in Shulman, 1966; M. bliccae Donec and Tozyyakova, 1984; M. bramae; M. donecae Kashkovski, 1969; M. lobatus (Nemeczek, 1911) Landsberg and Lom, 1991; M. macrocapsularis Reuss, 1906; M. muelleri Bütschli, 1882; and M. pseudodispar] (see Table 2), or from fish species of other cypriniform families (M. carassii Klokacheva, 1914; M. caudatus Gogebashvili, 1966; M. circulus Akhmerov, 1960; M. cyprini Doflein, 1898; M. ellipsoides Thélohan, 1892; M. dispar Thélohan, 1895; and M. musculi) prior to being reported from several other cypriniforms, including the leuciscids C. nasus and P. polylepis (Kudo, 1919; Donec and Shulman, 1984; González-Lanza and Alvarez-Pellitero, 1985; Shulman, 1988; Lom and Dyková, 1992; Iglesias et al., 2001; Eiras et al., 2005). Also included in this list was Myxobolus impressus Miroshnichenko, 1980, simultaneously reported from both the leuciscid Squalius cephalus and the cyprinid Barbus barbus in Ukraine (Miroshnichenko, 1980). Considering that most Myxobolus spp. are known to be host specific, i.e. they infect only a single or several closely related fish species (Molnár, 1994; Molnár et al., 2002, 2006, 2011; Blazer et al., 2004; Eszterbauer, 2004), it is unlikely that M. carassii, M. caudatus, M. circulus, M. cyprini, M. ellipsoides, M. dispar and M. musculi infect fishes of the family Leuciscidae, for

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Chapter XI | Myxobolids from endemic Iberian cypriniforms which these species were not included in Table 2. Myxobolus exiguus Thélohan, 1895, which was also reported to occur in Chondrostoma nasus and other Eurasian cyprinids (Shulman, 1988), was not considered for morphological comparison, since a recent study disregarded cypriniforms as bona fide hosts for this species, now restricted to Mugiliformes (see Rocha et al., 2019). Overall, species comparisons showed M. arcasii n. sp. and M. duriensis n. sp. differing significantly from all other species reported from leuciscids, be it either in the morphology of the myxospores or molecular data of the SSU rDNA gene. Albeit M. leuciscini and M. arcasii n. sp. sharing A. arcasii as their original host and there being few morphometric differences between their myxospores, these species could be readily differentiated based on molecular data. Myxobolus leuciscini was originally reported from three distinct hosts, and later sequenced from only Squalius cephalus (Molnár et al., 2007). Thus, it can be suggested that, as originally described by González-Lanza and Alvarez-Pellitero (1985), M. leuciscini probably comprised several morphologically identical species, including M. arcasii n. sp. Thelohanellus paludicus n. sp. is the first myxosporean species described from a fish host of the genus Cobitis, as well as the first Thelohanellus spp. reported from the Iberian Peninsula. Consequently, morphological comparisons to this species were established in relation to the three congeners reported from fish genera of the subfamily Cobitinae worldwide: T. acuminatus, T. misgurni and T. pyriformis (see Table 3). Of the three latter, only T. misgurni was originally described from a host of the subfamily Cobitinae (M. anguillicaudatus), while T. acuminatus and T. pyriformis were originally described from hosts of the subfamilies Cyprininae and Tincinae, respectively. Again, it seems unlikely that both these species can infect members of the subfamily Cobitinae. In fact, the high morphological variability found among the various reports of T. pyriformis from different tissues and organs of several cyprinids across Europe suggest it as a possible species complex, despite its statute as type species of the genus Thelohanellus. As T. acuminatus, T. misgurni and T. pyriformis have not been sequenced from any reported host, molecular comparison to T. paludicus n. sp. is impossible; nonetheless, the latter was shown to differ significantly from these three species in both biological and morphological traits. The study performed here further provides the first combined morphological and molecular report of M. pseudodispar from the spleen of A. arcasii, and from the muscle, spleen, kidney and digestive tube of P. duriense. Identification of the parasite was based on both the morphological features of the myxospores and molecular data of the SSU rDNA gene. Despite our study showing some genetic diversity between the sequences obtained here from A. arcasii, P. duriense and those currently available in GenBank for this species, these values agreed with previous molecular studies that address the high intraspecific variability found among different isolates of M. pseudodispar. This myxobolid was recently hypothesized to constitute a cryptic species complex, with genetic diversity explained by host-shift followed by

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Chapter XI | Myxobolids from endemic Iberian cypriniforms ongoing processes of speciation in secondary hosts, and recombination of different lineages in oligochaete hosts (see Forró and Eszterbauer, 2016).

Phylogenetic analysis

The phylogenetic analysis performed in this study revealed the three new species described here clustering among other cypriniform-infecting myxobolids. This agrees with previous studies that show the vertebrate host group as a relevant evolutionary signal for myxobolids (Ferguson et al., 2008; Carriero et al., 2013; Rocha et al., 2014). Myxobolus arcasii n. sp. and M. duriensis n. sp. specifically cluster among other species that infect leuciscids, but within separate clades, suggesting that myxobolids entered Leuciscidae as hosts multiple times during their evolution. In turn, Thelohanellus paludicus n. sp. stands positioned alone at the basis of the leuciscid-infecting clade that contains M. duriensis n. sp. This positioning reflects the absence of molecular data belonging to more closely related congeners, as T. paludicus n. sp. constitutes the first myxobolid molecularly reported from cypriniforms of the family Cobitinae. Lastly, the SSU rDNA sequences of M. pseudodispar, obtained from infections in A. arcasii and P. duriense, cluster together with their conspecific sequences from Hungarian isolates (here represented by a single SSU rDNA sequence, KU340979 in Fig. 4). Overall, our phylogenetic analyses show that species infecting the same host family cluster together, but not necessarily within the same lineage. Thus, it can be suggested that myxobolids entered different cypriniform’ families (e.g. Cyprinidae and Leuciscidae) multiple times during their evolution. Tissue tropism-related phylogenetic clustering has also been commonly reported for myxobolids, as well as myxosporeans in general (see Molnár and Eszterbauer, 2015 and references therein). Accordingly, the phylogenetic analyses performed during this study placed M. duriensis n. sp. among other gill-infecting myxobolids, as well as M. pseudodispar among other muscle-dwelling parasites. Nonetheless, M. arcasii n. sp. was also shown clustering among several gill-infecting myxobolids, despite its development taking place in the hematopoietic tissue of the kidney, and in the undifferentiated tissue of the gonads. It may be that M. arcasii n. sp. and these gill-infecting species are evolutionarily related by having tropism to tissues with high rates of regeneration and demanding nutrient-intake; however, it is also likely that the positioning of M. arcasii n. sp. reflects the influence of one or more stronger evolutionary signals. Tissue tropism is most likely an important fine-scale evolutionary signal for myxobolids, however, the lack of information regarding the specific tissue of development of most species hampers recognition of its real influence in the evolution of these parasites. Furthermore, it cannot be disregarded that the evolutionary signals currently accepted as having played a preponderant role in the evolution of myxobolids are based on “mixed signals”

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Chapter XI | Myxobolids from endemic Iberian cypriniforms of invertebrate and vertebrate co-phylogeny (Holzer et al., 2018). Thus, the discernment of phylogenetic patterns related to the invertebrate host may reveal other evolutionary signals that were decisive in the evolution of these parasites.

Myxobolid biodiversity in endemic Iberian cypriniforms

A review of the available literature found 12 myxosporean species that have been reported from cypriniform hosts in the Iberian Peninsula. Among these, 11 belonged to the genus Myxobolus: M. branchilateralis, originally described from the gills of the common barbel Barbus barbus (Linnaeus, 1758) in Hungary, and simultaneously reported from Luciobarbus bocagei (Steindachner, 1864) in Portugal; M. branchialis, originally described from the gills of B. barbus in Ukraine, and later reported from the same fish host in Hungary and L. bocagei in Portugal; M. cutanei from the scales of L. bocagei in both Spain and Portugal; M. gallaicus and M. leuciscini from the gills of the Iberian nase P. polylepis in Spain; M. muelleri, a species complex that gained identity through its morphological and molecular re-description from the gills of chub Squalius cephalus, but possibly occurs in several other reported cypriniform hosts, including A. arcasii and P. polylepis in Spain; M. impressus Miroshnichenko, 1980, originally described from the fins and gills of B. barbus and S. cephalus in Ukraine, and later reported from the gills of P. polylepis in Spain; M. musculi, a muscle parasite of B. barbus in Hungary and L. bocagei in Portugal; M. pfeifferi, originally described from the connective tissue of several organs of B. barbus, and later reported from L. bocagei in Portugal; M. pseudodispar from the muscle of roach Rutilus rutilus and several other cyprinids in Europe, including P. polylepis and S. cephalus in Portugal; and M. tauricus, originally described from the gills, fins and muscles of the Crimean barbel Barbus tauricus Kessler, 1877 in Ukraine, and later reported from the pin bones and fins of B. barbus in Hungary and L. bocagei in Portugal (see Thélohan, 1895; Alvarez-Pellitero and González-Lanza, 1985; González-Lanza and Alvarez- Pellitero, 1985; Alvarez-Pellitero, 1989; Cruz et al., 2000; Saraiva et al., 2000; Eiras et al., 2005, 2014; Igleisas et al., 2001; Molnár et al., 2006, 2012). The remaining non-myxobolid species, Myxidium rhodei Lèger, 1905, was reported from the kidney of P. polylepis and S. cephalus in Spain (Alvarez-Pellitero, 1989). Cypriniforms are mostly restricted to freshwater and can naturally expand their distribution only through the direct connection of habitats (Saitoh et al., 2011; Stout et al., 2016). The translocation of known host species into new geographic areas, therefore, plays an important role in the dissemination and establishment of cypriniform-infecting parasites. The common carp Cyprinus carpio, for instance, is largely recognized as being responsible for numerous co-introductions and co-invasions of different parasitic groups worldwide, including myxozoans (e.g. Molnár, 2009; Oros et al., 2011; Vilizzi et al., 2014; Smit et al., 2017; Scholz

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Chapter XI | Myxobolids from endemic Iberian cypriniforms et al., 2018). Reported cases of co-introduction with C. carpio include the myxobolid species Thelohanellus hovorkai Akhmerov, 1960 and T. nikolskii Akhmerov, 1955, both originally from the Amur basin and later introduced to Hungary (Molnár, 1982, 2009). In the same manner, the human-mediated translocation of the silver carp Hypophthalmichthys molitrix (Valenciennes, 1844) and bighead carp Hypophthalmichthys nobilis (Richardson, 1845) to Hungary from Estearn Asia in the early 1960s, was reported to have led to the co-introduction of Myxobolus pavlovskii (Akhmerov, 1954) Landsberg and Lom, 1991 (see Molnár, 1979; Eiras et al., 2005; Marton and Eszterbauer, 2011). In this study, the occurrence of M. pseudodispar in Iberian endemic cypriniforms evidences another case of parasite/host co-introduction. Myxobolus pseudodispar is one of the most common muscle-dwelling parasites of leuciscids in Europe, having been originally described from roach Rutilus rutilus, and since then reported from several other species, including rudd Scardinius erythrophthalmus, freshwater bream Abramis brama, common bleak Alburnus alburnus and white bream Blicca bjoerkna (Molnár et al., 2002; Forró and Eszterbauer, 2016). All these fish species were introduced into the Iberian Peninsula during the 20th century (Hernando and Soriguer, 1991; Kottelat and Freyhof, 2007), where M. pseudodispar was reported to occur in several organs of the endemic species A. arcasii and P. polylepis, and the native species S. cephalus (González-Lanza and Alvarez-Pellitero, 1985; Cruz et al., 2000). In this study, M. pseudodispar is further reported from the endemic species P. duriense, therefore adding a new host record to this parasite in the Iberian Peninsula. Despite strict host specificity being frequently reported among myxobolids (Molnár, 1994; Blazer et al., 2004; Eszterbauer, 2004; Molnár et al., 2006, 2011, 2012; Molnár and Eszterbauer, 2015), several species of Myxobolus, including M. pseudodispar, M. bliccae and M. macrocapsularis, have been reported to have host-shifted between members of the cypriniform family Leuciscidae (Molnár et al., 2006, 2011; Forró and Eszterbauer, 2016). Considering this, it is possible that M. pseudodispar was co-introduced into the Iberian Peninsula with different leuciscid hosts in multiple occasions. Nonetheless, further research on endemic and exotic species is necessary in order to understand the origin and dispersion of M. pseudodispar in the Iberian Peninsula; a task that will certainly prove difficult given the continuous introduction of potential cypriniform hosts into this geographical region during the past century (Hernando and Soriguer, 1992; Kottelat and Freyhof, 2007). The acquisition of this knowledge would, moreover, be relevant for understanding how the cryptic speciation of M. pseudodispar relates to its adaptation to distinct micro- and macroenvironments. Similarly to M. pseudodispar, several others of the above-mentioned Myxobolus spp. reported from Iberian endemic cypriniforms were originally described from central and eastern European species; such are the cases of M. branchialis, M. branchilateralis, M. musculi, M. pfeifferi and M. tauricus, originally described from either B. barbus or B. tauricus and, since

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Chapter XI | Myxobolids from endemic Iberian cypriniforms then, reported from the Iberian endemic species Luciobarbus bocagei. Nonetheless, the occurrence of most of these species in L. bocagei has not been corroborated by molecular data. Reports of M. branchialis and M. branchilateralis from L. bocagei lack molecular data for comparison to the SSU rDNA sequences available from Hungarian isolates in B. Barbus. In the other way around, M. pfeifferi was sequenced from L. bocagei but never from its original host B. barbus. Molnár et al. (2012) provided molecular data for M. tauricus from both Hungarian isolates in B. barbus and Portuguese isolates in L. bocagei; however, given the significant genetic variability found between geographical isolates, the authors themselves suggested that they were possibly dealing with two distinct species. Thus, Myxobolus musculi constitutes the only species which identification from infections in L. bocagei was molecularly substantiated through means of comparison to the data available in GenBank from isolates belonging to other geographical areas. Nonetheless, the high genetic variability found between Hungarian isolates of M. musculi in B. barbus and Portuguese isolates in L. bocagei, as well as between isolates belonging to the same geographic location, suggests that future cross- infection experiments are necessary to confirm putative host-shift of this species, especially since there is no record of the introduction of B. barbus into the Iberian Peninsula. Overall, the study performed here reveals that myxosporean richness in the Iberian Peninsula is underestimated, with three new and one known species being described from endemic cypriniforms. As such, it is suggested that myxozoan research in this geographic region be expanded to target a broader array of endemic, native and non-native species. New myxozoan surveys of L. bocagei, specifically, are necessary in order to ascertain the identity of the myxobolids previously reported from this host, as it is plausible that some will constitute new species records.

Acknowledgments

This work was financially supported by FCT (Lisbon, Portugal) within the scope of the Ph.D. fellowship grant attributed to S. Rocha (SFRH/BD/92661/2013) through the programme QREN-POPH/FSE, and the Engº António de Almeida Foundation (Porto, Portugal). The authors would like to thank the iconographic assistance given by Miguel Pereira. The authors declare to have no conflicts of interest.

References

Alvarez-Pellitero, P. (1989). Myxidium rhodei (Protozoa: Myxozoa: Myxosporea) in cyprinid fish from the Duero basin (NW) Spain. Diseases of Aquatic Organisms 7, 13–16.

Page | 318

Chapter XI | Myxobolids from endemic Iberian cypriniforms

Alvarez- Pellitero, P. and González-Lanza, C. (1985). Studies on Myxobolus spp. of Barbus barbus bocagei from the river Esla (León, NW Spain). Angewandte Parasitologie 26, 3–12. Aparicio, E., Vargas, M.J., Olmo, J.M. and de Sostoa, A. (2000). Decline of native freshwater fishes in a Mediterranean watershed on the Iberian Peninsula: a quantitative assessment. Environmental Biology of Fishes 59, 11–19. Atkinson, S.D. and Banner, C.R. (2017). A novel myxosporean parasite Myxobolus klamathellus n. sp. (Cnidaria: Myxosporea) from native blue chub (Gila coerulea) in Klamath Lake, Oregon. Parasitology Research 116, 299–302. Atkinson, S.D., Bartošová-Sojková, P., Whipps, C.M. and Bartholomew, J.L. (2015). Approaches for characterising myxozoan species. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan evolution, ecology and development. Springer International Publishing, Switzerland, pp. 111‒123. Blazer, V.S., Densmore, C.L., Schill, W.B., Cartwright, D.D. and Page, S.J. (2004). Comparative susceptibility of Atlantic salmon, lake trout and rainbow trout to Myxobolus cerebralis in controlled laboratory exposures. Diseases of Aquatic Organisms 58, 27– 34. Carriero, M.M., Adriano, E.A., Silva, M.R., Ceccarelli, P.S. and Maia, A.A. (2013). Molecular phylogeny of the Myxobolus and Henneguya genera with several new South American species. PLoS One 8, e73713. Cech, G., Borzák, R., Molnár, K. and Székely, C. (2015). Three new species of Myxobolus Bütschli, 1882 (Myxozoa: Myxobolidae) infecting the common nase Chondrostoma nasus (L.) in the River Danube. Systematic Parasitology 92, 101–111. Clavero, M., Blanco-Garrido, F. and Prenda, J. (2004). Fish fauna in Iberian Mediterranean river basins: biodiversity, introduced species and damming impacts. Aquatic Conservation: Marine and Freshwater Ecosystems 14, 575–585. Cruz, C., Saraiva, A. and Ferreira, S. (2000). Preliminary observations on Myxobolus sp from cyprinid fish in Portugal. Bulletin of the European Association Fish Pathologists 20, 65– 69. Doadrio, I. (2001). Atlas y libro rojo de los peces continentales de España. Madrid: Ministerio de Medio Ambiente. Doadrio, I., Perea, S., Garzón-Heydt, P. and González, J.L. (2011). Ictiofauna Continental Española. Bases para su seguimiento. DG Medio Natural y Política Forestal, MARM., Madrid, 616 pp. Donec, Z.S. and Shulman, S.S. (1984). Knidosporidii (Cnidosporidia). In Bauer, O.N. (ed). Key to the determination of parasites of freshwater fishes of the USSR. Nauka, Leningrad, pp. 88–251.

Page | 319

Chapter XI | Myxobolids from endemic Iberian cypriniforms

Easy, R.H., Johnson, S.C. and Cone, D.K. (2005). Morphological and molecular comparison of Myxobolus procerus (Kudo, 1934) and M. intramusculi n. sp. (Myxozoa) parasitising muscles of the trout-perch Percopsis omiscomaycus. Systematic Parasitology 61, 115– 122. Eiras, J.C. (2002). Synopsis of the species of the genus Henneguya Thélohan, 1892 (Myxozoa: Myxosporea: Myxobolidae). Systematic Parasitology 52, 43–54. Eiras, J.C. and Adriano, E.A. (2012). A checklist of new species of Henneguya Thélohan, 1892 (Myxozoa: Myxosporea, Myxobolidae) described between 2002 and 2012. Systematic Parasitololy 83, 95–104. Eiras, J.C., Molnár, K. and Lu, Y.S. (2005). Synopsis of the species of Myxobolus Bütschli, 1882 (Myxozoa: Myxosporea: Myxobolidae). Systematic Parasitology 61, 1–46. Eiras, J.C., Zhang, J. and Molnár, K. (2014). Synopsis of the species of Myxobolus Bütschli, 1882 (Myxozoa: Myxosporea, Myxobolidae) described between 2005 and 2013. Systematic Parasitology 88, 11–36. Eschmeyer, W.N. and Fong, J.D. Catalog of fishes. Species by family/subfamily. (http://researcharchive.calacademy.org/research/ichthyology/catalog/SpeciesByFamil y.asp). Electronic version accessed 10 August 2018. Eszterbauer, E. (2004). Genetic relationship among gill-infecting Myxobolus species (Myxosporea) of cyprinids: molecular evidence of importance of tissue-specificity. Diseases of Aquatic Organisms 58, 35–40. Ferguson, J.A., Atkinson, S.D., Whipps, C.M. and Kent, M.L. (2008). Molecular and morphological analysis of Myxobolus spp. of salmonid fishes with the description of a new Myxobolus species. Journal of Parasitology 94, 1322–1334. Fiala, I. (2006). The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. International Journal of Parasitology 36, 1521–1534. Filipe, A.F., Araújo, M.B., Doadrio, I., Angermeier, P.L. and Collares-Pereira, M.J. (2009). Biogeography of Iberian freshwater fishes revisited: the roles of historical versus contemporary constraints. Journal of Biogeography 36, 2096–2110. Forró, B. and Eszterbauer, E. (2016). Correlation between host specificity and genetic diversity for the muscle-dwelling fish parasite Myxobolus pseudodispar: examples of myxozoan host-shift? Folia Parasitologica 63, 019. González-Lanza, C. and Alvarez-Pellitero, P. (1985). Myxobolus spp. of various cyprinids from the river Esla (León, NW Spain). Description and population dynamics. Angewandte Parasitologie 26, 71–83. Hallett, S.L. and Diamant, A. (2001). Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago

Page | 320

Chapter XI | Myxobolids from endemic Iberian cypriniforms

analis (Sillaginidae) from the coast of Queensland, Australia. Diseases of Aquatic Organisms 46, 197–212. Hernandez, A.D., Bunnell, J.F. and Sukhdeo, M.V. (2007). Composition and diversity patterns in metazoan parasite communities and anthropogenic disturbance in stream ecosystems. Parasitology 134, 91–102. Hernando, J.A. and Soriguer, M.C. (1992). Biogeography of the freshwater fish of the Iberian Peninsula. Limnetica 8, 243–253. Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. The Quarterly Review of Biology 66, 411–453. Holzer, A.S., Bartošová-Sojková, P., Born-Torrijos, A., Lovy, A., Hartigan, A. and Fiala, I. (2018). The joint evolution of the Myxozoa and their alternate hosts: a cnidarian recipe for success and vast biodiversity. Molecular Ecology 27, 1651–1666. Hudson, P.J., Dobson, A.P. and Lafferty, K.D. (2006). Is a healthy ecosystem one that is rich in parasites? Trends in Ecology and Evolution 21, 381–385. Iglesias, R., Paramá, A., Alvarez, F., Leiró, J. and Sanmartín, M.L. (2001). Myxobolid parasites infecting the gills of the Iberian nase, Chondrostoma polylepis (Steindachner), in Galicia (NW Spain), with a description of Myxobolus gallaicus sp. nov. Journal of Fish Diseases 24, 125–134. Jirsa, F., Konecny, R., Frank, C. and Sures, B. (2011). The parasite community of the nase Chondrostoma nasus (L. 1758) from Austrian rivers. Journal of Helminthology 85, 255– 262. Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffmann, R.W., Khattra, J., Hallett, S.L., Lester, R.J., Longshaw, M., Palenzeula, O., Siddall, M.E. and Xiao, C. (2001). Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic Microbiology 48, 395–413. Kottelat, M. and Freyhof, J. (2007). Handbook of European Freshwater Fishes. Publications Kottelat, Cornol and Freyhof, Berlin, p. 646. Kudo, R. (1919). Studies on Myxosporidia. A synopsis on genera and species of Myxosporidia. Illinois Biological Monographs 5, 1–265. Kudo, R. (1933). A taxonomic consideration of Myxosporidia. Transactions of the American Microscopical Society 52, 195–216. Kumazawa, Y. and Nishida, M. (2000). Molecular phylogeny of osteoglossoids: a new model for Gondwanian origin and plate tectonic transportation of the Asian arowana. Molecular Biology and Evolution 17, 1869–1878. Liu, X.H., Voronin, V.N., Dudin, A.S. and Zhang, J.Y. (2016). Morphological and molecular characterization of Myxobolus mucosus sp. n. (Myxosporea: Myxobolidae) with

Page | 321

Chapter XI | Myxobolids from endemic Iberian cypriniforms

basifilamental sporulation in two cyprinid fishes, Rutilus rutilus (L.) and Leuciscus leuciscus (L.) in Russia. Parasitology Research 115, 1297–1304. Lom, J. and Arthur, J.R. (1989). A guideline for the preparation of species descriptions in Myxosporea. Journal of Fish. Diseases 12, 151–156. Lom, J. and Dyková, I. (1992). Protozoan Parasites of Fishes. In Developments in Aquaculture and Fisheries Science. Elsevier, Amsterdam, 26, p. 315. Lom, J. and Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53, 1–36. Longshaw, M., Frear, P.A. and Feist, SW. (2005). Descriptions, development and pathogenicity of myxozoan (Myxozoa : Myxosporea) parasites of juvenile cyprinids (Pisces : Cyprinidae). Journal of Fish Diseases 28, 489–508. Marcogliese, D.J. (2005). Parasites of the superorganism: are they indicators of ecosystem health? International Journal of Parasitology 35, 705–716. Marton, S. and Eszterbauer, E. (2011). The development of Myxobolus pavlovskii (Myxozoa: Myxobolidae) includes an echinactinomyxon-type actinospore. Folia Parasitologica 58, 157–163. Miroshnichenko, A.I. (1980). Myxobolus impressus n. sp., a new myxosporidian (Cnidosporidia, Myxosporidia) from freshwater fish in the Crimea. Biologicheskie Nauki 9, 38–39. Molnár, K. (1979). Myxobolus pavlovskii (Achmerov, 1954) (Myxosporidia) infection in the silver carp and bighead. Acta veterinaria Academiae Scientiarum Hungaricae 27, 207– 216. Molnár, K. (1982). Biology and histopathology of Thelohanellus nikolskii Achmerov, 1955 (Myxosporea, Myxozoa), a protozoan parasite of the common carp (Cyprinus carpio). Zeitschrift für Parasitenkunde 68, 269–277. Molnár, K. (1994). Comments on the host, organ and tissue specificity of fish myxosporeans and on the types of their intrapiscine development. Parasitologia Hungarica 27, 5–20. Molnár, K. (2009). Data on the parasite fauna of the European common carp Cyprinus carpio carpio and Asian common carp Cyprinus carpio haematopterus support an Asian ancestry of the species. AACL Bioflux, 391–400. Molnár, K. (2011). Remarks to the validity of Genbank sequences of Myxobolus spp. (Myxozoa, Myxosporidae) infecting Eurasian fishes. Acta Parasitologica 56, 263–269. Molnár, K., Cech, G. and Székely, C. (2011). Histological and molecular studies of species of Myxobolus Bütschli, 1882 (Myxozoa: Myxosporea) in the gills of Abramis, Blicca and Vimba spp. (Cyprinidae), with the redescription of M. macrocapsularis Reuss, 1906 and M. bliccae Donec & Tozyyakova, 1984. Systematic Parasitology 79, 109–121.

Page | 322

Chapter XI | Myxobolids from endemic Iberian cypriniforms

Molnár, K. and Eszterbauer, E. (2015). Specificity of infection sites in vertebrate hosts. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 295–313. Molnár, K., Eszterbauer, E., Guti, C.F. and Székely, C. (2014). Two new Myxobolus spp. (Myxozoa: Myxobolidae) from white bream, Blicca bjoerkna (Linnaeus, 1758) developing in basifilamental location of gills. Acta Protozoologica 53, 277–285. Molnár, K., Eszterbauer, E., Marton, S., Székely, C. and Eiras, J.C. (2012). Comparison of the Myxobolus fauna of common barbel from Hungary and Iberian barbel from Portugal. Diseases of Aquatic Organisms 100, 231–248. Molnár, K., Eszterbauer, E., Székely, C., Dán, Á. and Harrach, B. (2002). Morphological and molecular biological studies on intramuscular Myxobolus spp. of cyprinid fish. Journal of Fish Diseases 25, 643–652. Molnár, K., Marton, S., Eszterbauer, E. and Székely, C. (2006). Comparative morphological and molecular studies on Myxobolus spp. infecting chub from the River Danube, Hungary, and description of M. muellericus sp. n. Diseases of Aquatic Organisms 73, 49–61. Molnár, K., Marton, S., Eszterbauer, E. and Székely, C. (2007). Description of Myxobolus gayerae sp. n. and re-description of M. leuciscini infecting European chub from the Hungarian stretch of the river Danube. Diseases of Aquatic Organisms 78, 147–153. Molnár, K. and Székely, C. (1999). Myxobolus infection of the gills of common bream (Abramis brama L.) in Lake Balaton and in the Kis-Balaton reservoir, Hungary. Acta Veterinaria Hungarica 47, 419–432. Oros, M., Král'ová-Hromadová, I., Hanzelová, V., Bruňanská, M. and Orosová, M. (2011). Atractolytocestus huronensis (Cestoda): A new invasive parasite of common carp in Europe. In Sanders, J.D. and Peterson, S.B. (eds). Carp: Habitat, Management and Diseases. Nova Science Publishers, UK, p. 215. Robalo, J.I., Almada, V.C., Levy, A. and Doadrio, I. (2007). Re-examination and phylogeny of the genus Chondrostoma based on mitochondrial and nuclear data and the definition of 5 new genera. Molecular Phylogenetics and Evolution 42, 362–372. Rocha, S., Azevedo, C., Oliveira, E., Alves, Â., Antunes, C., Rodrigues, P. and Casal, G. (2019). Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular redescription of the cryptic species Myxobolus exiguus. Parasitology 146, 479–496. Rocha, S., Casal, G., Garcia, P., Matos, E., Al-Quraishy, S. and Azevedo, C. (2014). Ultrastructure and phylogeny of the parasite Henneguya carolina sp nov (Myxozoa), from the marine fish Trachinotus carolinus in Brazil. Diseases of Aquatic Organisms 112, 139–148.

Page | 323

Chapter XI | Myxobolids from endemic Iberian cypriniforms

Rocha, S., Casal, G., Rangel, L., Castro, R., Severino, R., Azevedo, C. and Santos, M.J. (2015). Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae). Parasitology International 64, 305–313. Ronquist, F. and Huelsenbeck, J.P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Saitoh, K., Sado, T., Doosey, M.H., Bart, J.H.L., Inoue, J.G., Nishida, M., Mayden, R.L. and Miya, M. (2011). Evidence from mitochondrial genomics supports the lower Mesozoic of South Asia as the time and place of basal divergence of cypriniform fishes (Actinopterygii: Ostariophysi). Zoological Journal of the Linnean Society 161, 633–662. Saraiva, A., Cruz, C. and Ferreira, S. (2000). Studies of Myxidium rhodei Leger, 1905 (Myxozoa : Myxosporea) on Chondrostoma polylepis from River Ave, North Portugal. Bulletin of the European Association of Fish Pathologists 20, 106–110. Scholz, T., Šimková, A., Razanabolana, J.R. and Kuchta, R. (2018). The first record of the invasive Asian fish tapeworm (Schyzocotyle acheilognathi) from an endemic cichlid fish in Madagascar. Helminthologia 55, 84–87. Shulman, S.S. (1988). Myxosporidia of the USSR. Amerind Publishing Co., New Delhi, p. 631. Šimková, A., Navrátilová, P., Dávidová, M., Ondračková, M., Sinama, M., Chappaz, R., Gilles, A. and Costedoat, C. (2012). Does invasive Chondrostoma nasus shift the parasite community structure of endemic Parachondrostoma toxostoma in sympatric zones? Parasites and Vectors 5, 200. Smit, N.J., Malherbe, W. and Hadfield, K.A. (2017). Alien freshwater fish parasites from South Africa: diversity, distribution, status and the way forward. International Journal for Parasitology: Parasites and Wildlife 6, 386–401. Stout, C.C., Tan, M., Lemmon, A.R., Lemmon, E.M. and Armbruster, J.W. (2016). Resolving Cypriniformes relationships using an anchored enrichment approach. BMC Evolutionary Biology 16, 244. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30, 2725– 2729. Thélohan, P. (1895). Recherches sur les Myxosporidies. Bulletin Scientifique de la France et de la Belgique 4, 100–394. Vidal-Martinez, V.M., Pech, D., Sures, B., Purucker, S.T. and Poulin, R. (2010). Can parasites really reveal environmental impact? Trends in Parasitology 26, 44–51. Vilizzi, L., Tarkan, A. and Ekmekçi, F. (2014). Parasites of the common carp Cyprinus carpio L., 1758 (Teleostei: Cyprinidae) from water bodies of Turkey: updated checklist and review for the 1964–2014 period. Turkish Journal of Zoology 39, 545–554.

Page | 324

Chapter XI | Myxobolids from endemic Iberian cypriniforms

Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215–219. Yokoyama, H., Grabner, D. and Shirakashi, S. (2012). Transmission biology of the Myxozoa. In Carvalho, D.E., David, G.S. and Silva, R.J. (eds). Health and environment in aquaculture. InTechOpen. Zhang, J.Y., Gu, Z.M., Kalavati, C., Eiras, J.C., Liu, Y., Guo, Q.Y. and Molnár, K. (2013). Synopsis of the species of Thelohanellus Kudo, 1933 (Myxozoa: Myxosporea: Bivalvulida). Systematic Parasitology 86, 235–256.

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Chapter XII

Molecular-based inferences confirm the involvement of sphaeractinomyxon in the life cycle of mugiliform-infecting Myxobolus species (Cnidaria, Myxosporea)

This chapter was adapted from:

Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

Abstract

Four new types of sphaeractinomyxon (Cnidaria, Myxozoa) are morphologically and molecularly described from the coelomic cavity of a marine Baltridilus sp. (Annelida, Oligochaeta) collected from the River Douro estuary, northern Portugal. Morphological overlap between the new types reinforce the artificiality of using solely morphology-based criteria for distinguishing between members of the same collective group, especially when developing in the same oligochaete species. The occurrence of multiple types in a single Baltidrilus sp. reveals the members of this naidid genus as permissive hosts for sphaeractinomyxon in brackish waters, further strengthening the contention that the family Naididae Ehrenberg, 1828 played a preponderant role of in the myxosporean colonization of estuarine communities. Moreover, the lack of concomitant infections in this host species, as well as in other oligochaetes that appear to be highly permissive to sphaeractinomyxon infections (e.g. Tubificoides pseudogaster, Tubificoides insularis and Limnodriloides agnes), is suggested to reflect the influence of host-, parasite-, and environmental-related factors in myxosporean- annelid interactions. Molecular analyses matched the SSU rDNA sequences of three new types with those of mugiliform-infecting Myxobolus spp. previously reported from the River Minho estuary in northern Portugal and from the Spanish Mediterranean coast. Recognition of these life cycle inferences confirm the previously hypothesized involvement of the sphaeractinomyxon collective group in the life cycle of this specific group of myxobolids. The functionality of the Myxobolus and sphaeractinomyxon morphotypes are suggested to have contributed for the successful evolutionary hyperdiversification of the genus Myxobolus in mullets.

Introduction

The class Myxosporea Bütschli, 1881 encompasses highly-reduced, obligate cnidarian parasites that mainly parasitize aquatic vertebrates and invertebrates (Lom and Dyková, 2006). Myxosporean life cycles are complex and typically involve horizontal transmission between a myxosporean stage that develops in fish and an actinosporean stage that develops in annelid worms (see Eszterbauer et al., 2015). Although the myxosporean stage can also develop in other vertebrate groups, such as amphibians, reptiles, waterfowl and terrestrial mammals, the complete life cycle of these species is presently unknown. Two distinct types of spores are produced during the myxosporean life cycle and constitute the means through which transmission is achieved between the intermediate vertebrate hosts and the definitive invertebrate hosts. Myxospores are produced as the result of myxosporean development in the fish, being shed into the aquatic environment via urine, faeces, cyst rupture or after death

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon of the host. Following release, the myxospores settle in the water column to encounter a viable oligochaete or polychaete for actinosporean development. In the annelid worm, actinospores are produced within pansporocysts, typically developing in the intestinal epithelium or coelomic cavity, before passing out into the aquatic environment. Upon contact with water, the valves of the actinospores inflate, so that they become buoyant and drift passively in the water column to encounter a susceptible fish host, thus giving continuity to the life cycle (Kent et al., 2001; Lom and Dyková, 2006). To date, only ca. 60 myxosporean life cycles have been resolved and most involve freshwater species that use oligochaetes as invertebrate hosts (see Eszterbauer et al., 2015). In turn, the fewer studies pertaining to the life cycle of marine myxosporeans suggest polychaetes as the hosts of choice in marine environments (Køie et al., 2004, 2007, 2008; Rangel et al., 2009, 2011, 2016a; Karlsbakk and Køie, 2012). Nonetheless, a few exceptions are known to occur in estuarine waters: the triactinomyxon stages of Ortholinea auratae Rangel et al., 2015 and O. labracis Rangel et al., 2017, as well as the aurantiactinomyxon stage of Paramyxidium giardi (Cépède, 1906) Freeman and Kristmundsson, 2018, were reported to develop in marine oligochaetes (Rangel et al., 2015, 2017; Rocha et al., 2019a); while the tetractinomyxon stages of Ceratonova shasta and Parvicapsula minibicornis were reported to use the freshwater polychaete Manayunkia speciosa Leidy, 1859 as invertebrate host (Bartholomew et al., 1997, 2006). Overall, the limited body of knowledge available for myxosporean life cycles results from the difficulties found in pairing myxosporean and actinosporean counterparts. Initially, life cycle studies relied on information obtained through experimental infections, which besides being laborious and time-consuming, resulted in the establishment of associations that, in some cases, were ultimately discredited by the usage of molecular tools (e.g. Holzer et al., 2004; Atkinson and Bartholomew, 2009; Marton and Eszterbauer, 2011; Székely et al., 2014). As such, life cycle studies are now mainly based on DNA match between myxosporean and actinosporean counterparts. This more reliable and simpler method, however, requires the finding and molecular analysis of actinosporean stages in potential annelid hosts, which in itself constitutes a difficult task. At present, only ca. 200 types of actinospores are known, compared to the more than 2,200 myxosporean species that have been characterized based on the myxospores produced in the vertebrate host. The lower number of actinospore descriptions is not only consequential to the little commercial and recreational value of their annelid hosts, which discouraged interest in the group prior to the discovery of its involvement in the life cycle of fish-pathogenic myxosporeans (Wolf and Markiw, 1984), but also to their typically low prevalence of infection and several other host- and parasite-related factors that make sampling infected annelids an exceptional challenge (see Milanin et al., 2017 and references therein).

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

Even though only a small fraction of myxosporean life cycles are known, the information thus far acquired demonstrates that there is no obvious correlation between myxospore and actinospore morphotypes. For example, species of the genus Myxobolus have been associated with aurantiactinomyxon, triactinomyxon and raabeia [including former echinactinomyxon (Rocha et al., 2019c)] counterparts (Eszterbauer et al., 2015; Rocha et al., 2019f). In fact, phylogenetic analyses show that the clade of myxobolids comprises most of the actinosporean morphotypes represented in the NCBI database, namely antonactinomyxon, aurantiactinomyxon, helioactinomyxon, hexactinomyxon, hungactinomyxon, neoactinomyxum, raabeia, sphaeractinomyxon, seisactinomyxon, synactinomyxon and triactinomyxon (Eszterbauer et al., 2006; Hallett et al., 1999; Holzer et al., 2004; Kent et al., 2001; Milanin et al., 2017; Rangel et al., 2017; Rocha et al., 2019f). Types of helioactinomyxon, hexactinomyxon, hungactinomyxon, sphaeractinomyxon, seisactinomyxon and synactinomyxon, however, have never been linked directly to the life cycle of any given species, neither by experimental transmission nor by DNA matching. Recently, the members of the sphaeractinomyxon collective group were hypothesized to play a role in the life cycle of mugiliform-infecting myxobolids with basis on phylogenetic data (Rocha et al., 2019f). The sphaeractinomyxon collective group currently encompasses 32 types (including former tetraspora and endocapsa representatives) reported from either freshwater or marine oligochaetes (Marques, 1984; Hallett et al., 1997, 1998, 1999, 2001; Hallett and Lester, 1999; Rangel et al., 2016b; Rocha et al., 2019b, f). More than half of these types were recently described from Portuguese estuaries: 10 types were reported by Rangel et al. (2016b) infecting marine oligochaetes [either Limnodriloides agnes Hrabě, 1967 or Tubificoides pseudogaster (Dahl, 1960)] in the Aveiro estuary; 3 types were reported by Rocha et al. (2019f) from the marine oligochaete Tubificoides insularis (Stephenson, 1923) in the Alvor estuary; and other 4 types were found infecting freshwater oligochaetes of the genus Potamothrix Vejdovský and Mrázek, 1903 and Psammoryctides barbatus (Grube, 1861), as well as the marine oligchaete T. pseudogaster, in the River Minho estuary (see Rocha et al., 2019b). The molecular identification of Sphaeractinomyxon type 10 of Rangel et al. (2016b) and Sphaeractinomyxon type 4 of Rocha et al. (2019b) in more than a single oligochaete species, has revealed that sphaeractinomyxon types do not have strict host specificity. The broader host range of the Sphaeractinomyxon type 4 of Rocha et al. (2019b), in particular, was further proposed to correlate with the biological features of the vertebrate host. Considering that this type uses both freshwater and marine oligochaetes as definitive hosts, Rocha et al. (2019b) suggested its myxosporean counterpart to parasitize a migratory fish; probably a mullet, as previously hypothesized by Rocha et al. (2019f). In this study, myxosporean surveys were performed on oligochaetes collected from another Portuguese estuary, more specifically from the lower estuary of the River Douro near

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

Porto, Northern Portugal. The results reinforce the already recognized rich biodiversity of the sphaeractinomyxon collective group in brackish waters, as four new types are morphologically and molecularly described from a marine oligochaete of the genus Baltridilus Timm, 2013. Molecular analyses matched the SSU rDNA sequences of three of these types with those of mugiliform-infecting Myxobolus spp., thus confirming the involvement of the sphaeractinomyxon morphotype in the life cycle of this specific group of myxobolids. The features of the Myxobolus and sphaeractinomyxon morphotypes are discussed to have allowed the hyperdiversification of the genus Myxobolus in mullets, by optimizing transmission between hosts.

Materials and Methods

Sampling and morphological characterization

Between September 2014 and April 2015, sporadic samplings of mud were performed at low tide from a single site (“Cabedelo”) in the lower estuary of the River Douro (41° 08' 32" N, 08° 39' 41" W), northern Portugal. The mud was transported to the laboratory and analysed for the manual collection of oligochaetes, which were kept separately in 48-well plates containing saltwater at room temperature, for an approximate period of two weeks. During this time period, a stereo microscope was used in order to determine actinospore release into the well-plates. Posteriorly, all oligochaete specimens were examined under a light microscope for the detection of actinospores and other developmental stages in the coelomic cavity and tissues. As waterborne actinospores were never observed, prevalence of infection was calculated from specimens in which actinosporean development was microscopically detected and molecularly confirmed. Actinospores were examined and photographed using a Zeiss Axiophot microscope (Grupo Taper, Sintra, Portugal), equipped with a Zeiss AxioCam Icc3 digital camera and the AxioVision 4.6.3 software (Grupo Taper, Sintra, Portugal) for image analysis. Morphology and morphometry were determined using fresh material, in accordance to the recommendations given by Lom et al. (1997). Measurements include the mean value ± standard deviation (SD), range of variation, and number of measured actinospores (n).

Molecular characterization

Infected oligochaetes were kept separately in absolute ethanol until used for molecular analysis. Genomic DNA was extracted using the GenEluteTM Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, USA) and following the manufacturer’s instructions. The

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

DNA was stored in 50 μl of TE buffer at -20 ºC until further use. A nested PCR was performed in order to amplify the SSU rDNA gene of the actinospores. The first round of PCR used the universal eukaryotic primers ERIB1 and ERIB10 (Table 1) in 25 µl reactions comprised of 0.25 µl of 10 pmol of each primer, 0.2 mM of each dNTP, 2.5 mM of MgCl2, 2.5 µl of 10× Taq polymerase buffer, 1.25 units Taq DNA polymerase (NZYTech, Lisbon, Portugal), 2 µl (approximately 100–150 ng) of genomic DNA, and 18 µl of water. The reactions were run on a Bio-Rad MJ Mini Gradient Thermal Cycler, with initial denaturation at 95 ºC for 3 min, followed by 35 cycles of 95 ºC for 1 min, 55 ºC for 1 min, and 72 ºC for 2 min. The final elongation step was performed at 72 ºC for 7 min. The second round of PCR was performed using as template 1 µl of the first round PCR and specific myxosporean primers (Table 1). The reaction mixtures were the same as for the first round PCR, except for the usage of 0.5 µl of 10 pmol of each primer. Amplification was achieved using an initial denaturation at 95 ºC for 3 min, followed by 35 cycles of 94 ºC for 45 s, 53 ºC for 45 s, and 72 ºC for 90 s, and a final elongation step at 72 ºC for 7 min. In turn, the 16S mitochondrial DNA (mtDNA) of the oligochaete hosts was amplified using the universal primers 16sar-L and 16sbr-H (Table 1). PCRs were carried out in a single reaction using the conditions previously described for the second round PCR of the actinospores with 2 µl of extracted genomic DNA as template. Aliquots (5 µl) of the PCR products were electrophoresed through a 1% agarose 1× tris- acetate-EDTA buffer (TAE) gel stained with GreenSafe Premium (NZYTech, Lisbon, Portugal). PCR products were purified using Puramag™ magnetic beads coated with carboxylic acid groups (MCLAB, San Francisco, California, USA). Sequencing reactions were performed with the same primers used for amplification (Table 1), using a BigDye Terminator v3.1 Cycle Sequencing Kit from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and were run on an ABI3700 DNA analyzer from AppliedBiosystems (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Sequence assembly and distance estimation analysis

The partial SSU rDNA sequences obtained for each infected oligochaete were aligned separately in MEGA7 (Kumar et al., 2016), allowing the construction of assembled sequences that were then compared amongst each other in order to assertively calculate the prevalence of infection of each actinospore type reported here. A dataset of close relatives was retrieved from GenBank according to the highest similarity scores obtained using BLAST search; it included all species of mugiliform-infecting myxobolids, as well as members of the sphaeractinomyxon collective group. Sequence alignments were performed using MAFFT version 7 available online, and distance estimation was calculated in MEGA7, using the p- distance model with all ambiguous positions removed for each sequence pair.

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

Table 1. Polymerase chain reaction primers used for actinospore and oligochaete DNA amplification and sequencing.

Results

Myxozoan survey and host recognition

During this study, a total of 720 oligochaete specimens were collected from the lower estuary of the River Douro, northern Portugal. Three morphologically distinct oligochaete species were represented in the mud samples analysed, but myxozoan infection was only detected in the most abundant: an unidentified species of the genus Baltidrilus Timm, 2013 that composed ca. 70% of the totality of oligochaetes examined. All infected specimens shared the same morphological features, which were generally congruent with those described for Baltidrilus costatus (Claparède, 1863), i.e. presence of dorsal palmate chaetae between segments III and XIII, with bifid chaetae in the remaining segments, and absence of hair chaetae and cuticular papillation (Brinkhurst, 1971; Timm, 2013). Comparative analysis of the 16S mtDNA sequences obtained from each case isolate revealed them to be identical amongst each other, further sharing 99.1% of similarity to the sequences of B. costatus currently available in GenBank (AY340460 and AY885609). The latter sequence (AY885609) is identified as Tubificoides pseudogaster but, according to Kvist et al. (2010), was misidentified and belongs to B. costatus. In total, 10 specimens of Baltidrilus sp. displayed myxozoan infection by members of the sphaeractinomyxon collective group, corresponding to an overall prevalence of infection of 1.4%. Infection by other actinosporean collective groups was not found in the oligochaete samples analysed. Specimens displaying sphaeractinomyxon infection were detected throughout the entire sampling period, but waterborne actinospores were never observed in the well-

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon plates. Mature actinospores and other developmental stages were present in the coelomic cavity, having asynchronous development. In turn, the actinospore development within the pansporocysts was synchronous. Molecular analysis of the parasites' SSU rDNA gene allowed distinction between four different types of sphaeractinomyxon that are described here for the first time.

Characterization of four novel Sphaeractinomyxon types (Cnidaria, Myxozoa)

Sphaeractinomyxon type 1 (Figs. 1A–C, 2A) Description: Mature actinospores rounded or slightly angular in apical view and rounded to slightly oblong in lateral view, measuring 17.6 ± 0.7 (16.0–18.9) µm in length (n = 12), 18.9 ± 0.8 (17.8–19.9) µm in width (n = 12), and 19.3 ± 0.6 (18.4–20.5) µm in diameter (n = 13). Three polar capsules positioned centrally, pyriform and symmetric, 4.4 ± 0.3 (3.8–4.9) µm long (n = 14) and 3.5 ± 0.2 (3.1–3.8) µm wide (n = 14), each comprising a polar tubule displaying 2–3 longitudinal coils. Sporoplasm containing many secondary cells. Pansporocysts with synchronous development, each containing 8 actinospores in equivalent stages. Type host: Species of the genus Baltidrilus Timm, 2013 (Oligochaeta, Naididae). Type locality: “Cabedelo” (41° 08' 32" N, 08° 39' 41" W), lower estuary of the River Douro, northern Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: 0.3% (2 infected in a total of 720 oligochaetes examined). Type material: Series of phototypes of the hapantotype deposited together with a representative DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR2019.52. Molecular data: One SSU rDNA sequence with a total of 2,014 bp, deposited in GenBank with the accession no. MN037506. The latter is representative of two identical sequences that were separately obtained from the actinosporean developmental stages in the coelomic cavity of two infected oligochaetes. Remarks: Morphometry was determined from mature actinospores observed in both infected hosts, among which morphometric variation was not registered. Morphological comparison showed no gross similarity between the parasite in study and the 12 types of sphaeractinomyxon that lack molecular data, i.e. S. stolci, S. gigas, S. danicae, S. ilyodrili, S. amanieui, S. rotundum, S. leptocapsula, Sphaeractinomyxon types 1 and 2 of Hallett et al. (1997), Sphaeractinomyxon type B of Hallett and Lester (1999), Sphaeractinomyxon type 2 of Hallett et al. (1999), and Sphaeractinomyxon type of Hallett et al. (2001). In turn, distance estimation analysis revealed 100% of similarity to the SSU rDNA sequences of Myxobolus

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon mugiliensis Rocha et al., 2019 parasitizing the gills of flathead grey mullet Mugil cephalus Linnaeus, 1758 in the River Minho estuary (northern Portugal) (MK203082) and in the Spanish Mediterranean coast (MF118767) (see Rocha et al., 2019e; Sharon et al., 2019). A similarity value of 99.8% was further determined in relation to a third SSU rDNA sequence of M. mugiliensis that is available from infections in M. cephalus from the Samsun coasts of the Black Sea in Turkey (MH392320). All other SSU rDNA sequences included in the analysis resulted in similarity values lower than 98.0%. Sphaeractinomyxon types genetically more similar were the Sphaeractinomyxon type 2 of Rangel et al. (2016a) (KU569311) (97.5%) and the Sphaeractinomyxon type 2 reported in this study (MN037503) (96.6%).

Sphaeractinomyxon type 2 (Figs. 1D, 2B) Description: Mature actinospores subspherical in apical and lateral view, measuring 18.1 ± 0.7 (17.0–19.6) µm in length (n = 19), 19.1 ± 0.5 (17.7–19.8) µm in width (n = 19), and 19.6 ± 0.7 (18.0–20.9) µm in diameter (n = 17). Three polar capsules positioned centrally, pyriform and symmetric, 4.7 ± 0.5 (4.1–5.4) µm long (n = 12) and 3.3 ± 0.2 (2.9–3.7) µm wide (n = 12), each comprising a polar tubule displaying 2–3 longitudinal coils. Sporoplasm containing many secondary cells. Pansporocysts with synchronous development, each containing 8 actinospores in equivalent stages. Type host: Species of the genus Baltidrilus Timm, 2013 (Oligochaeta, Naididae). Type locality: “Cabedelo” (41° 08' 32" N, 08° 39' 41" W), lower estuary of the River Douro, northern Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: 0.3% (2 infected in a total of 720 oligochaetes examined). Type material: Series of phototypes of the hapantotype deposited together with a representative DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR2019.53. Molecular data: One SSU rDNA sequence with a total of 2,020 bp, deposited in GenBank with the accession no. MN037503. The latter is representative of two identical sequences that were separately obtained from the actinosporean developmental stages in the coelomic cavity of two infected oligochaetes. Remarks: Morphometry was determined from mature actinospores observed in both infected hosts, among which morphometric variation was not registered. Morphological comparison showed no gross similarity to the 12 types of sphaeractinomyxon that lack molecular data. In turn, distance estimation revealed 99.8% and 99.6% of similarity to the SSU rDNA sequences of a Myxobolus sp. (MF118773 and MF118771) reported from the muscle and kidney of M. cephalus in the Spanish Mediterranean coast (see Sharon et al., 2019). The

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

Figure 1. Light micrographs showing mature actinospores of the new types of sphaeractinomyxon found infecting the coelomic cavity of Baltidrilus sp. in the lower estuary of the River Douro. (A‒C) Sphaeractinomyxon type 1 in lateral (A, B) and apical (C) view. (D) Pansporocyst of the Sphaeractinomyxon type 2 showing 8 actinospores in different perspectives. (E, F) Sphaeractinomyxon type 3 in lateral (E) and apical (F) view. (G, H) Sphaeractinomyxon type 4 in lateral (G) and apical (H) view. small genetic variability found in relation to these sequences corresponds to a total of three and five nucleotide differences, respectively, and is considered here as being representative of intraspecific variability. All other SSU rDNA sequences included in the analysis resulted in similarity values lower than 98.0%. Sphaeractinomyxon types genetically more similar were the Sphaeractinomyxon type 2 reported in this study (MN037506) (96.6%) and the Sphaeractinomyxon type 1 of Rangel et al. (2016a) (KU569311) (95.8%).

Sphaeractinomyxon type 3 (Figs. 1E–F, 2C) Description: Mature actinospores spherical in apical view and lateral view, measuring 21.6 ± 1.2 (19.1–23.7) µm in length (n = 30), 22.6 ± 0.9 (20.7–25.2) µm in width (n = 30), and 23.0 ± 0.8 (21.7–24.9) µm in diameter (n = 19). Three polar capsules positioned centrally, pyri-

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

Figure 2. Schematic drawings depicting mature actinospores of the new types of sphaeractinomyxon found infecting the coelomic cavity of Baltidrilus sp. in the lower estuary of the River Douro, as observed in lateral (left) and apical (right) view. (A) Sphaeractinomyxon type 1. (B) Sphaeractinomyxon type 2. (C) Sphaeractinomyxon type 3. (D) Sphaeractinomyxon type 4. form and symmetric, 5.8 ± 0.4 (4.9–7.1) µm long (n = 34) and 4.4 ± 0.3 (3.7–5.0) µm wide (n = 34), each comprising a polar tubule displaying 2–3 longitudinal coils. Sporoplasm containing many secondary cells. Pansporocysts with synchronous development, each containing 8 actinospores in equivalent stages. Type host: Species of the genus Baltidrilus Timm, 2013 (Oligochaeta, Naididae). Type locality: “Cabedelo” (41° 08' 32" N, 08° 39' 41" W), lower estuary of the River Douro, northern Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: 0.3 % (2 infected in a total of 720 oligochaetes examined). Type material: Series of phototypes of the hapantotype deposited together with a representative DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR2019.54. Molecular data: One SSU rDNA sequence with a total of 2,023 bp, deposited in GenBank with the accession no. MN037504. The latter is representative of two identical sequences that were separately obtained from the actinosporean developmental stages in the coelomic cavity of two infected oligochaetes. Remarks: Morphometry was determined from mature actinospores observed in both infected hosts, among which morphometric variation was not registered. Morphological comparison to the 12 types of sphaeractinomyxon that lack molecular data revealed some

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon morphometric similarity to S. leptocapsula and S. rotundum. The actinospores of S. leptocapsula, however, are narrower than those in study, further differing by being triangular in apical view and ellipsoidal to broad pyriform in lateral view (see Hallett et al., 1999). In turn, despite the actinospores of S. rotundum sharing the spherical shape of the type in study, they differ in being narrower and having bigger polar capsules (see Marques, 1984). The sequence obtained for the parasite did not match any of the SSU rDNA sequences currently available for myxozoans. Distance estimation showed closest similarity to all other sphaeractinomyxon types and Myxobolus spp. reported from mullets, namely Sphaeractinomyxon type 2 of Rocha et al. (2019f) (MH017877) (97.2%), Sphaeractinomyxon type 9 of Rangel et al. (2016a) (KU569318) (96.5%), and a Myxobolus sp. reported from the gills, intestine and tail of M. cephalus from the Mediterranean Sea off Northern Israel (MF118765) (96.0%). All other SSU rDNA sequences included in the analysis retrieved similarity values lower than 96.0%.

Sphaeractinomyxon type 4 (Figs. 1G–H, 2D) Description: Mature actinospores spherical in apical view and ellipsoidal in lateral view, measuring 24.5 ± 1.8 (23.6–27.2) µm in length (n = 4), 26.5 ± 1.2 (25.3–28.1) µm in width (n = 5), and 26.2 ± 0.9 (24.6–27.5) µm in diameter (n = 8). Three polar capsules positioned centrally, pyriform and symmetric, 7.2 ± 0.6 (6.2–7.9) µm long (n = 8) and 5.5 ± 0.2 (5.3–5.9) µm wide (n = 8), each comprising a polar tubule displaying 2–3 longitudinal coils. Sporoplasm containing many secondary cells. Pansporocysts with synchronous development, each containing 8 actinospores in equivalent stages. Type host: Species of the genus Baltidrilus Timm, 2013 (Oligochaeta, Naididae). Type locality: “Cabedelo” (41° 08' 32" N, 08° 39' 41" W), lower estuary of the River Douro, northern Portugal. Site of infection: Throughout the coelomic cavity. Prevalence: 0.6 % (4 infected in a total of 720 oligochaetes examined). Type material: Series of phototypes of the hapantotype deposited together with a representative DNA sample in the Type Material Collection of the Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR2019.55. Molecular data: One SSU rDNA sequence with a total of 1,954 bp, deposited in GenBank with the accession no. MN037505. The latter is representative of four identical sequences that were separately obtained from the actinosporean developmental stages in the coelomic cavity of four infected oligochaetes. Remarks: Morphometry was determined from one of the four infected hosts, in which fully matured actinospores could be measured. Morphological comparison to the 12 types of sphaeractinomyxon that lack molecular data revealed some morphometric similarity to the

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Sphaeractinomyxon type B of Hallett and Lester (1999). Nonetheless, the latter can be distinguished from the type in study based on the morphology of its polar capsules, which are smaller and have a higher number of polar tubule coils, as well as in its original host and type locality. Distance estimation analysis revealed the parasite sharing 99.8% of similarity with the sequence of Myxobolus labrosus Rocha et al., 2019 from the urinary bladder of thicklip grey mullet Chelon labrosus (Risso, 1827) in another Portuguese northern River (MK203081) (see Rocha et al., 2019e). The small genetic variability found between these sequences corresponds to a total of three nucleotide differences and is considered here as being representative of intraspecific variability. All other SSU rDNA sequences included in the analysis retrieved similarity values lower than 95.0%. Sphaeractinomyxon types genetically more similar were the Sphaeractinomyxon type 1 of Rocha et al. (2019b) (MK418446) (94.2%) and the Sphaeractinomyxon type 1 of Rocha et al. (2019f) (MH017876) (94.0%).

Discussion

During this study, four distinct types of sphaeractinomyxon were found infecting a marine species of the genus Baltidrilus in the lower estuary of the River Douro, Northern Portugal. Their development in the coelomic cavity of infected specimens followed the pattern that has been commonly reported for members of the sphaeractinomyxon collective group (e.g. Caullery and Mesnil, 1905; Hallett et al., 1998; Rangel et al., 2016b; Rocha et al., 2019f), i.e. generalized asynchrony of the overall parasitic development and synchrony of the sporogonic phase within the pansporocysts. The low individual values of prevalence of infection determined for these types were also congruent with those previously reported for other types of sphaeractinomyxon in Portuguese estuaries and other geographic locations (e.g. Hallett et al., 1999; Rangel et al., 2016b; Rocha et al., 2019b, f). In turn, the value obtained for the overall prevalence of infection of the collective group differed significantly from the values previously reported for the same type of myxozoan survey in the Aveiro (18.7%), Alvor (6.1%) and Minho estuaries (0.2%) (see Rangel et al., 2016b; Rocha et al., 2019b, f). Although little is known about myxosporean-annelid interactions in general, these value differences most likely reflect the complexity and variability of the biotic and abiotic factors that shape the ecosystem and naturally influence the encounter rates between myxospores and permissive annelid hosts. Differentiation between the four new sphaeractinomyxon types was mainly based on molecular data of the SSU rDNA gene, considering the widely reported artificiality of using morphological criteria for distinguishing between members of sphaeractinomyxon (Rangel et al., 2016b; Rocha et al., 2019b, f) and other actinosporean collective groups in general (e.g. Hallett et al., 2002, 2004; Eszterbauer et al., 2006; Caffara et al., 2009; Xi et al., 2015). Indeed, the types 1 and 2 reported here could only be differentiated through comparison of their

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon respective SSU rDNA sequences, given that their measurements considerably overlapped amongst each other. This reinforces the inaccuracy of using solely morphology-based criteria for distinguishing between types of the same collective group, especially when developing in the same oligochaete species. Morphological comparisons were only performed in relation to the 12 types of sphaeractinomyxon that lack molecular data. Among these, S. amanieui was also originally described from an oligochaete host of the genus Baltidrilus, specifically Baltidrilus costatus. Notwithstanding, none of the types described here bared resemblance to the actinospores of S. amanieui, which are reported to measure 50–60 µm in diameter (see Puytorac, 1963). Furthermore, the identification of the host species of S. amanieui as B. costatus is dubious, given that this parasite was reported from freshwater [near Clermont- Ferrand, France] and, while B. costatus is euryhaline, it is commonly associated with brackish and marine coastal waters (see Sweeney, 2016; Knight-Jones, 2017; Marszewska et al., 2017). In this study, the identification of oligochaete hosts was performed based on the combined analysis of morphological traits and molecular data of the 16S mtDNA gene. Despite all infected specimens displaying high morphological and molecular similarity to the naidid Baltidrilus costatus, identification was performed only up the genus-level. As non-oligochaete taxonomists, we felt unsure in determining whether the genetic distance found between the sequences obtained here and those available in GenBank for B. costatus results from intra- or interspecific variability and, therefore, conservatively identified all infected specimens as belonging to a Baltidrilus sp. Rocha et al. (2019a) suggested a preponderant role of the family Naididae Ehrenberg, 1828 in the myxosporean colonization of estuarine communities, given that naidids appear to be hosts of choice for marine myxosporeans in brackish waters. The host identification performed in this study strengthens this contention, as it identifies naidids of the genus Baltidrilus as permissive hosts for myxosporeans in estuarine habitats. In general, the occurrence of more than one actinosporean type in a given oligochaete species is not an uncommon feature among the different collective groups. For instance, El- Mansy et al. (1998) reported more than a dozen types of aurantiactinomyxon and neoactinomyxum parasitizing the freshwater oligochaete Branchiura sowerbyi in a Hungarian fish farm. Similarly, Xiao and Desser (1998a, b) described types of aurantiactinomyxon, neoactinomyxum, raabeia and triactinomyxon infecting the freshwater species Limnodrilus hoffmeisteri Claparède, 1862 in natural waters of the Algonquin Park, Ontario; while Rosser et al. (2014) reported types of the aurantiactinomyxon, helioactinomyxon, raabeia and triactinomyxon collectives groups parasitizing specimens of the freshwater oligochaete Dero digitata (Müller, 1774) in a commercial catfish pond in the Mississippi Delta. In Portuguese estuaries, several different types of sphaeractinomyxon have also been reported to occur in the same host species: 8 types were reported from Tubificoides pseudogaster, 4 types from

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Tubificoides insularis, and 3 types from Limnodriloides agnes (Rangel et al., 2016b; Rocha et al., 2019b, f). In this study, further 4 types are described from a single Baltidrilus sp., therefore suggesting a susceptibility of this oligochaete to acquire myxozoan infection by members of the sphaeractinomyxon collective group. Despite the rich biodiversity of sphaeractinomyxon reported from the above-mentioned oligochaete species, concomitant infections were never registered. This is in accordance with previous studies that reported multiple actinosporean types from the same oligochaete species, but never occurring simultaneously in a single individual (e.g. El-Mansy et al., 1998; Xi et al., 2013, 2015; Rosser et al., 2014). Again, this absence of mixed infections in oligochaete species that appear to be highly permissive to actinosporean invasion and development is most likely the outcome of specific host-, parasite- and environmental-related factors. Considering the relevance of these factors in the successful dissemination, establishment and evolution of myxosporeans, future research should aim to provide insight into their diversity and influence on myxosporean-annelid interactions. Most known myxosporean life cycles refer to Myxobolus spp. that have freshwater oligochaetes as invertebrate hosts. Members of this genus have been directly linked to aurantiactinomyxon, triactinomyxon and raabeia counterparts (see Eszterbauer et al., 2015), with phylogenetic analyses further revealing possible correspondences to types of the antonactinomyxon, helioactinomyxon, hexactinomyxon, hungactinomyxon, neoactinomyxum, sphaeractinomyxon, seisactinomyxon and synactinomyxon collective groups (Eszterbauer et al., 2006; Hallett et al., 1999; Holzer et al., 2004; Kent et al., 2001; Milanin et al., 2017; Rangel et al., 2017; Rocha et al., 2019f). While some of these actinosporean morphotypes are known to be involved in the life cycle of other myxosporean genera [e.g. an antonactinomyxon type is the counterpart of Chloromyxum auratum (Atkinson et al., 2007); neoactinomyxum types have been identified as counterparts for Chloromyxum, Hoferellus and Thelohanellus spp. (Yokoyama et al., 1993a; Holzer et al., 2006; Xi et al., 2015)], others have never been linked to any given myxosporean species. It is the case of sphaeractinomyxon types, whose consistent clustering within the monophyletic clade of mugiliform-infecting myxobolids, led Rocha et al. (2019f) to suggest their potential involvement in the life cycle of Myxobolus spp. that parasitize mullets. To our best knowledge, 51 species of Myxobolus have been reported from mugilid hosts worldwide (see Marcotegui and Martorelli, 2017; Cardim et al., 2018; Rocha et al., 2019d, e). Prior to this study, none had its full life cycle known, neither by experimental transmission studies nor by DNA match of corresponding myxosporean and actinosporean stages. Thus, the molecular comparisons of the SSU rDNA gene performed here allowed recognition of the first life cycle inferences of mugiliform-infecting Myxobolus spp., further confirming the involvement of sphaeractinomyxon types as actinosporean counterparts for this myxosporean genus. The sequence obtained for Sphaeractinomyxon type 1 was an exact match to those of

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M. mugiliensis from the gills of M. cephalus in the River Minho estuary and in the Spanish Mediterranean coast (Rocha et al., 2019e; Sharon et al., 2019), further sharing 99.8% of similarity with a conspecific sequence available from infected M. cephalus in the Samsun coasts of the Black Sea in Turkey (unpublished data in GenBank). Values of 99.8% of similarity were also obtained for Sphaeractinomyxon types 2 and 4 in relation to the SSU rDNA sequences of a Myxobolus sp. from the muscle and kidney of M. cephalus in the Spanish Mediterranean coast (Sharon et al., 2019), and M. labrosus from the urinary bladder of C. labrosus in the River Minho estuary (Rocha et al., 2019e), respectively. The low genetic distance (0.2%) determined between these sequences is well within the range of intraspecific variability that is generally accepted for myxosporeans, which can vary from 0‒3.6% but is usually lower than 1% (see Atkinson et al., 2015 and references therein). Of the above-mentioned 51 mugiliform-infecting Myxobolus spp., a total of 20 were reported to occur in the Iberian Peninsula, parasitizing mullets that were either captured from the estuary of the River Minho in northern Portugal (Rocha et al. 2019d, e), or from the Spanish Mediterranean coast (Yurakhno and Ovcharenko, 2014; Sharon et al., 2019). Of these, only Myxobolus adeli (Isjumova, 1964) Yurakhno and Ovcharenko, 2014, Myxobolus nile Negm- Eldin et al., 1999 and Myxobolus rohdei Lom and Dyková et al., 1994 are without molecular data and, therefore, cannot be molecularly compared to sphaeractinomyxon sequences. Considering that this study elevates to 21 the number of sphaeractinomyxon types molecularly described from Portuguese estuaries, and that each one of these types is presumably counterpart to a mugiliform-infecting Myxobolus sp., it can be affirmed that the biodiversity of this myxosporean genus in mullets is nothing less than astonishing. According to Rocha et al. (2019e), the successful evolution and diversification of Myxobolus in mullet hosts probably correlates with the processes of speciation that led to the ecological plasticity of these fishes. Mullets are amongst the most ubiquitous representatives of euryhaline teleosts, being able to inhabit marine, brackish and freshwater habitats in tropical, subtropical and temperate regions worldwide (Hotos and Vlahos, 1998; Cardona, 2001; Durand et al., 2012; Nelson et al., 2016). Moreover, their omnivorous nature and benthic feeding strategy allows them to feed on a great variety of living organisms and materials (Cardona, 2001; Laffaille et al., 2002; Almeida, 2003). Thus, the acquisition of these highly diversified fishes as hosts most likely allowed Myxobolus spp. to conquer new geographic habitats, with subsequent adaptation to distinct invertebrate communities and environmental- related factors. In this context, it cannot be disregarded the role that functional spore morphology played in the successful colonization of new habitats. Longshaw and Feist (2005) proposed that actinospore morphology may be a function of the environment and that each actinospore type will not necessarily have a specific corresponding myxospore stage. Phylogenetic analyses widely agree with this assumption,

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Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon mostly showing actinosporean morphotypes spanning over several different myxosporean taxa (e.g. Eszterbauer et al., 2006; Fiala et al., 2015; Xi et al., 2015; Rangel et al., 2017; Rocha et al., 2019c, f). Thus far, sphaeractinomyxon types appear to be an exception, consistently clustering only with Myxobolus spp. that infect mullet hosts. The apparent evolutionary success of this specific actinospore-myxospore relationship is probably determined by the functionality of their morphotypes, which are highly adapted to the ecology of their respective hosts and, therefore, promote effective transmission of invasive stages. Despite sharing main morphological features, myxospores and actinospores exhibit a wide range of distinctive morphotypes (Lom and Dyková, 2006). While myxospores are resistant and mostly adapted for settling, actinospores are short-lived and generally have inflatable valvular processes that provide floatability in order to increase encounter rates with permissive fish hosts (Yokoyama et al., 1993b; Xiao and Desser, 1998a; Lom and Dyková, 2006). As benthic feeders, mullets already have increased proximity to potentially infected annelids and, therefore, are more prone to contact with actinospores (Rocha et al., 2019d). Moreover, the consumption of infected annelids should not be disregarded as a viable route for mullets to acquire myxosporean infection. Although the gills, skin and buccal cavity constitute the main portals of entry for myxosporeans in the vertebrate host (e.g. Yokoyama and Urawa, 1997; Antonio et al., 1999; Holzer et al., 2003), infection via the intestine has also been demonstrated (Belem and Pote, 2001). In this context, the limited floatability of the sphaeractinomyxon morphotype, as suggested by its lack of valvular processes, shows that this collective group probably evolved to infect bottom dwelling fish, as is the case of mullets. In turn, most oligochaetes (including Tubificoides, Baltidrilus and other “naidids in general) are deposit feeders that selectively consume sediment particles, digesting the associated bacteria and organic matter (Brinkhurst et al., 1972; Rodriguez et al., 2001; Jeuniaux, 2012). Considering that the consumption of myxospores is perhaps the only invasion route for annelids (see Alexander et al., 2015), the host’s trophic ecology is expected to exert selection on myxospore morphotype. The species richness of the genus Myxobolus clearly reveals the evolutionary success of its morphotype for achieving transmission and infection in permissive oligochaete hosts. Besides being negatively buoyant due to their spherical shape and reduced contact surface, the myxospores of Myxobolus have been reported to be adapted for deposition with small particles, thus increasing the likelihood of consumption by permissive deposit-feeding annelids (Lemmon and Kerans, 2001; Kerans and Zale, 2002). As more life cycles are resolved and additional myxospore-actinospore morphotype correlations are acknowledged, future researches might aim to study morphotype evolution in the context of their interactions with susceptible hosts and abiotic related factors.

Acknowledgments

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The authors thank Thais Oliveira and Ricardo Castro for the mud collection and sampling of oligochaetes, and Miguel Pereira for his assistance in the iconographic work. This work was supported by the Foundation for Science and Technology (Lisbon, Portugal) within the scope of the Ph.D. fellowship grant attributed to S. Rocha (SFRH/BD/92661/2013) through the programme QREN-POPH/FSE; the Engº António de Almeida Foundation (Porto, Portugal); and the Structured Program of R&D&I INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER- 000035, namely within the Research Line INSEAFOOD Innovation and valorization of seafood products: meeting local challenges and opportunities, within the R&D Institution CIIMAR (Interdisciplinary Centre of Marine and Environmental Research), supported by the Northern Regional Operational Programme (NORTE2020), through the European Regional Development Fund (ERDF).

References

Alexander, J.D., Kerans, B.L., El-Matbouli, M., Hallett, S.L. and Stevens, L. (2015). Annelid- myxosporean interactions. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan evolution, ecology and development. Springer International Publishing, Switzerland, pp. 217‒234. Almeida, P.R. (2003). Feeding ecology of Liza ramada (Risso, 1810) (Pisces, Mugilidae) in a south-western estuary of Portugal. Estuarine, Coastal and Shelf Science 57, 313–323. Antonio, D.B., El-Matbouli, M. and Hedrick, R.P. (1999). Detection of early developmental stages of Myxobolus cerebralis in fish and tubificid oligochaete hosts by in situ hybridization. Parasitology Research 85, 942–944. Atkinson, S.D. and Bartholomew, J.L. (2009). Alternate spore stages of Myxobilatus gasterostei, a myxosporean parasite of three-spined sticklebacks (Gasterosteus aculeatus) and oligochaetes (Nais communis). Parasitology Research 104, 1173‒ 1181. Atkinson, S.D., Bartošová-Sojková, P., Whipps, C.M. and Bartholomew, J.L. (2015). Approaches for characterising myxozoan species. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan evolution, ecology and development. Springer International Publishing, Switzerland, pp 111‒123. Atkinson, S.D., Hallett, S.L. and Bartholomew, J.L. (2007). The life cycle of Chloromyxum auratum (Myxozoa) from goldfish, Carassius auratus (L.), involves an antonactinomyxon actinospore. Journal of Fish Diseases 30, 149‒156.

Page | 345

Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

Barta, J. R., Martin, D. S., Liberator, P. A., Dashkevicz, M., Anderson, J. W., Feighner, S. D., Elbrecht, A., Perkins-Barrow, A., Jenkins, M. C., Danforth, H. D., Ruff, M.D. and Profous-Juchelka, H. (1997). Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences. Journal of Parasitology 83, 262-271. Bartholomew, J.L., Atkinson, S.D. and Hallett, S.L. (2006). Involvement of Manayunkia speciosa (Annelida : Polychaeta : Sabellidae) in the life cycle of Parvicapsula minibicornis, a myxozoan parasite of Pacific salmon. Journal of Parasitology 92, 742‒ 748. Bartholomew, J.L., Whipple, M.J., Stevens, D.G. and Fryer, J.L. (1997). The life cycle of Ceratomyxa shasta, a myxosporean parasite of salmonids, requires a freshwater polychaete as an alternate host. Journal of Parasitology 83, 859‒868. Belem, A.M. and Pote, L.M. (2001). Portals of entry and systemic localization of proliferative gill disease organisms in channel catfish Ictalurus punctatus. Diseases of Aquatic Organisms 48, 37–42. Brinkhurst, R.O. (1971). A guide for the identification of British aquatic Oligochaeta, 2o Ed. Freshwater Biological Association. Brinkhurst, R., Kaushik, N.K. and Chua, K.E. (1972). Interspecific interactions and selective feeding by tubificid oligochaetes. Limnology and Oceanography 17, 122–133. Caffara, M., Raimondi, E., Florio, D., Marcer, F., Quaglio, F. and Fioravanti, M.L. (2009). The life cycle of Myxobolus lentisuturalis (Myxozoa: Myxobolidae), from goldfish (Carassius auratus auratus), involves a Raabeia-type actinospore. Folia Parasitologica 56, 6‒12. Cardim, J., Silva, D., Hamoy, I., Matos, E. and Abrunhosa, F. (2018). Myxobolus bragantinus n. sp. (Cnidaria: Myxosporea) from the gill filaments of the redeye mullet, Mugil rubrioculus (Mugiliformes: Mugilidae), on the eastern Amazon coast. Zootaxa 4482, 177-187. Cardona, L. (2001). Non-competitive coexistence between Mediterranean grey mullet: evidence from seasonal changes in food availability, niche breadth and trophic overlap. Journal of Fish Biology 59, 729‒744. Caullery, M. and Mesnil, F., (1905). Recherches sur les Actinomyxidies. I. Sphaeractinomyxon stolci. Archiv für Protistenkunde 6, 272‒308. Durand, J.D., Shen, K.N., Chen, W.J., Jamandre, B.W., Blel, H., Diop, K., Nirchio, M., Garcia de León, F.J., Whitfield, A.K., Chang, C.W. and Borsa, P. (2012). Systematics of the grey mullets (Teleostei: Mugiliformes: Mugilidae): molecular phylogenetic evidence challenges two centuries of morphology-based taxonomy. Molecular Phylogenetics and Evolution 64, 73‒92. El-Mansy, A., Székely, C. and Molnár, K. (1998). Studies on the occurrence of actinosporean

Page | 346

Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

stages of fish myxosporeans in a fish farm of Hungary, with the description of triactinomyxon, raabeia, aurantiactinomyxon and neoactinomyxon types. Acta Veterinaria Hungarica 46, 259‒284. Eszterbauer, E., Atkinson, S., Diamant, A., Morris, D., El-Matbouli, M. and Hartikainen, H. (2015). Myxozoan life cycles: practical approaches and insights. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 175‒198. Eszterbauer, E., Marton, S., Rácz, O.Z., Letenyei, M. and Molnár, K. (2006). Morphological and genetic differences among actinosporean stages of fish-parasitic myxosporeans (Myxozoa): difficulties of species identification. Systematic Parasitology 65, 97‒114. Fiala, I., Bartošová-Sojková, P., Okamura, B. and Hartikainen, H. (2015). Classification and phylogenetics of Myxozoa. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 85‒110. Hallett, S.L., Atkinson, S.D. and El-Matbouli, M. (2002). Molecular characterisation of two aurantiactinomyxon (Myxozoa) phenotypes reveals one genotype. Journal of Fish Diseases 25, 627‒631. Hallett, S.L., Atkinson, S.D., Erséus, C. and El-Matbouli, M. (2004). Molecular methods clarify morphometric variation in triactinomyxon spores (Myxozoa) released from different oligochaete hosts. Systematic Parasitology 57, 1‒14. Hallett, S.L. and Diamant, A. (2001). Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia. Diseases of Aquatic Organisms 46, 197‒212. Hallett, S.L., Erséus, C. and Lester, R.J. (1999). Actinosporeans (Myxozoa) from marine oligochaetes of the Great Barrier Reef. Systematic Parasitology 44, 49‒57. Hallett, S.L., Erséus, C. and Lester, R.J.G. (1997). Actinosporea from Hong Kong marine oligochaeta. In Morton, B. (ed). Proceedings of the eight international marine biological workshop: the marine flora and fauna of Hong Kong and Southern China. Hong Kong University Press, Hong Kong, pp. 1‒7. Hallett, S.L., Erséus, C., O’Donoghue, P.J. and Lester, R.J.G. (2001). Parasite fauna of australian marine oligochaetes. Memoirs of the Queensland Museum 46, 555‒576. Hallett, S.L. and Lester, R.J. (1999). Actinosporeans (Myxozoa) with four developing spores within a pansporocyst: Tetraspora discoidea n. g. n. sp. and Tetraspora rotundum n. sp. International Journal of Parasitology 29, 419‒427. Hallett, S.L., O’Donoghue, P.J. and Lester, R.J.G. (1998). Structure and development of a marine actinosporean, Sphaeractinomyxon ersei n. sp. (Myxozoa). Journal of

Page | 347

Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

Eukaryotic Microbiology 45, 142‒150. Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. The Quarterly Review of Biology 66, 411‒453. Holzer, A.S., Sommerville, C. and Wootten, R. (2003). Tracing the route of Sphaerospora truttae from the entry locus to the target organ of the host, Salmo salar L., using an optimized and specific in situ hybridization technique. Journal of Fish Diseases 26, 647–655. Holzer, A.S., Sommerville, C. and Wootten, R. (2004). Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences. International Journal of Parasitology 34, 1099‒1111. Holzer, A.S., Sommerville, C. and Wootten, R. (2006). Molecular identity, phylogeny and life cycle of Chloromyxum schurovi Shul'man & Ieshko 2003. Parasitology Research 99, 90‒96. Hotos, G.N. and Vlahos, N. (1998). Salinity tolerance of Mugil cephalus and Chelon labrosus (Pisces: Mugilidae) fry in experimental conditions. Aquaculture 167, 329‒338. Jeuniaux, C. (2012). Nutrition and digestion. In Florkin, M. and Scheer, B.T. (eds). Chemical Zoology IV: Annelida, Echiura, and Sipuncula. New York and London: Academic Press, pp. 69‒92. Karlsbakk, E. and Køie, M. (2012). The marine myxosporean Sigmomyxa sphaerica (Thélohan, 1895) gen. n., comb. n. (syn. Myxidium sphaericum) from garfish (Belone belone (L.)) uses the polychaete Nereis pelagica L. as invertebrate host. Parasitology Research 110, 211‒218. Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffmann, R.W., Khattra, J., Hallett, S.L., Lester, R.J., Longshaw, M., Palenzeula, O., Siddall, M.E. and Xiao, C. (2001). Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic Microbiology 48, 395‒413. Kerans, B.L. and Zale, A.V. (2002). The ecology of Myxobolus cerebralis. American Fisheries Society Symposium 29, 145–166. Knight-Jones, P., Knight-Jones, E.W., Mortimer-Jones, K., Nelson-Smith, A., Schmelz, R.M. and Timm, T. (2017). Annelids (Phylum Annelida). In Hayward, P.J. and Ryland, J.S. (eds). Handbook of the marine fauna of North-west Europe, 2nd edition. United Kingdom: Oxford University Press, pp. 165‒270. Køie, M., Karlsbakk, E. and Nylund, A. (2007). A new genus Gadimyxa with three new species (Myxozoa, Parvicapsulidae) parasitic in marine fish (Gadidae) and the two-host life cycle of Gadimyxa atlantica n. sp. Journal of Parasitology 93, 1459‒1467. Køie, M., Karlsbakk, E. and Nylund, A. (2008). The marine herring myxozoan Ceratomyxa auerbachi (Myxozoa: Ceratomyxidae) uses Chone infundibuliformis (Annelida:

Page | 348

Chapter XII | Life cycle of mugiliform-infecting Myxobolus spp. involve sphaeractinomyxon

Polychaeta: Sabellidae) as invertebrate host. Folia Parasitolgica 55, 100‒4. Køie, M., Whipps, C.M. and Kent, M.L. (2004). Ellipsomyxa gobii (Myxozoa: Ceratomyxidae) in the common goby Pomatoschistus microps (Teleostei: Gobiidae) uses Nereis spp. (Annelida: Polychaeta) as invertebrate hosts. Folia Parasitologica 51, 14‒18. Kumar, S., Stecher, G. and Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution 33, 1870‒ 1874. Kvist, S., Sarkar, I.N. and Erséus, C. (2010). Genetic variation and phylogeny of the cosmopolitan marine genus Tubificoides (Annelida: Clitellata: Naididae: Tubificinae). Molecular Phylogenetics and Evolution 57, 687–702. Laffaille, P., Feunteun, E., Lefebvre, C., Radureau, A., Sagan, G. and Lefeuvre, J.C. (2002). Can thin-lipped mullet directly exploit the primary and detritic production of European macrotidal salt marshes? Estuarine, Coastal and Shelf Science 54, 729‒736. Lemmon, J.C. and Kerans, B.L. (2001). Extraction of whirling disease myxospores from sediments using the plankton centrifuge and sodium hexametaphosphate. Intermountain Journal of Sciences 7, 57–62. Lom, J. and Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53, 1‒36. Lom, J., McGeorge, J., Feist, S.W., Morris, D. and Adams, A. (1997). Guidelines for the uniform characterisation of the actinosporean stages of parasites of the phylum Myxozoa. Diseases of Aquatic Organisms 30, 1‒9. Longshaw, M. and Feist, S.W. (2005). The role of morphology and ecology in the transmission of actinospore types (Myxozoa) in freshwater ecosystems. EAFP 12th International Conference, “Diseases of Fish and Shellfish”, Copenhagen, 11–16 September 2005. Book of Abstracts, P.13.10, p. 195. Marcotegui, P. and Martorelli, S. (2017). Myxobolus saladensis sp. nov., a new species of gill parasite of Mugil liza (Osteichthyes, Mugilidae) from Samborombón Bay, Buenos Aires, Argentina. Iheringia. Série Zoologia 107, e2017026. Marques, A. (1984). Contribution a la connaissance des Actinomyxidies: ultrastructure, cycle biologique, systématique. Ph.D. Thesis. Université des Sciences et Techniques de Languedoc, Montepellier, France, pp. 218. Marszewska, L., Dumnicka, E. and Normant-Saremba, M. (2017). New data on benthic Naididae (Annelida, Clitellata) in Polish brackish waters. Oceanologia 59, 81‒84. Marton, S., Eszterbauer, E. (2011). The development of Myxobolus pavlovskii (Myxozoa: Myxobolidae) includes an echinactinomyxon-type actinospore. Folia Parasitologica 58, 157‒163. Milanin, T., Atkinson, S.D., Silva, M.R., Alves, R.G., Maia, A.A. and Adriano, E.A. (2017).

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Occurrence of two novel actinospore types (Cnidaria: Myxosporea) in Brazilian fish farms, and the creation of a novel actinospore collective group, Seisactinomyxon. Acta Parasitologica 62, 121‒128. Nelson, J.S., Grande, T.C. and Wilson, M.V.H. (2016). Fishes of the World, 5th edn. John Wiley & Sons, Hoboken, New Jersey, USA, pp 752. Palumbi, S., Martin, A., Romano, S., McMillan, W.O., Stice, L. and Grabowski, G. (2002). The Simple Fools Guide to PCR, Version 2.0. University of Hawaii, Honolulu. http://palumbi.stanford.edu/SimpleFoolsMaster Puytorac, P.D. (1963). L'ultrastructure des cnidocystes de l'Actinomyxidae: Sphaeractinomyxon amanieui sp. nov. Comptes rendus de l'Académie des Sciences, Paris 256, 1594‒1596. Rangel, L.F., Castro, R., Rocha, S., Cech, G., Casal, G., Azevedo, C., Székely, C. Cavaleiro, F. and Santos, M.J. (2016b). Description of new types of sphaeractinomyxon actinospores (Myxozoa: Myxosporea) from marine tubificid oligochaetes, with a discussion on the validity of the tetraspora and the endocapsa as actinospore collective group names. Parasitology Research 115, 2341‒2351. Rangel, L.F., Castro, R., Rocha, S., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2016a). Tetractinomyxon stages genetically consistent with Sphaerospora dicentrarchi (Myxozoa: Sphaerosporidae) found in Capitella sp. (Polychaeta: Capitellidae) suggest potential role of marine polychaetes in parasite's life cycle. Parasitology 143, 1067‒1073. Rangel, L.F., Cech, G., Székely, C. and Santos, M.J. (2011). A new actinospore type Unicapsulactinomyxon (Myxozoa), infecting the marine polychaete, Diopatra neapolitana (Polychaeta: Onuphidae) in the Aveiro estuary, Portugal. Parasitology 138, 698‒712. Rangel, L.F., Rocha, S., Casal, G., Castro, R., Severino, R., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2017). Life cycle inference and phylogeny of Ortholinea labracis n. sp. (Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm. Journal of Fish Diseases 40, 243‒ 262. Rangel, L.F., Rocha, S., Castro, R., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2015). The life cycle of Ortholinea auratae (Myxozoa: Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete. Parasitology Research 114, 2671‒2678. Rangel, L.F., Santos, M.J., Cech, G. and Székely, C. (2009). Morphology, molecular data, and development of Zschokkella mugilis (Myxosporea, Bivalvulida) in a polychaete alternate host, Nereis diversicolor. Journal of Parasitology 95, 561‒569.

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Rocha, S., Alves, Â., Antunes, C., Azevedo, C. and Casal, G. (2019a). Molecular data infers the involvement of a marine aurantiactinomyxon in the life cycle of the myxosporean parasite Paramyxidium giardi (Cnidaria, Myxozoa). Parasitology (In Press). Rocha, S., Alves, Â., Antunes, C., Fernandes, P., Azevedo, C. and Casal, G. (2019b). Characterization of sphaeractinomyxon types (Cnidaria, Myxozoa) from marine and freshwater oligochaetes in a Portuguese estuary, with the demise of the endocapsa collective group. Folia Parasitologica (In Press) Rocha, S., Alves, Â., Fernandes, P., Antunes, C., Azevedo, C. and Casal, G. (2019c). Description of a new actinosporean prompts union of the raabeia and echinactinomyxon collective groups (Cnidaria, Myxozoa), due to overlap in actinospore morphology. Diseases of Aquatic Organisms (In Press). Rocha, S., Azevedo, C., Oliveira, E., Alves, Â., Antunes, C., Rodrigues, P. and Casal, G. (2019d). Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular redescription of the cryptic species Myxobolus exiguus. Parasitology 146, 479‒496. Rocha, S., Casal, G., Alves, Â., Antunes, C., Rodrigues, P. and Azevedo, C. (2019e). Myxozoan (Cnidaria, Myxozoa) biodiversity in mullets (Teleostei, Mugilidae) unravels hyperdiversification of Myxobolus (Cnidaria, Myxosporea). Parasitology Research (In Press) Rocha, S., Casal, G., Rangel, L., Castro, R., Severino, R., Azevedo, C. and Santos, M. J. (2015). Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae). Parasitology International 64, 305-313. Rocha, S., Rangel, L.F., Castro, R., Severino, R., Azevedo, C., Santos, M.J. and Casal, G. (2019f). The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis. Journal of Invertebrate Pathology 160, 33‒42. Rodriguez, P., Martinez-Madrid, M., Arrate, J.A. and Navarro, E. (2001). Selective feeding by the aquatic oligochaete Tubifex tubifex (Tubificidae, Clitellata). Hydrobiologia 463, 133–140. Rosser, T.G., Griffin, M.J., Quiniou, S.M., Greenway, T.E., Khoo, L.H., Wise, D.J. and Pote, L.M. (2014). Molecular and morphological characterization of myxozoan actinospore types from a commercial catfish pond in the Mississippi delta. Journal of Parasitology 100, 828‒839. Sharon, G., Ucko, M., Tamir, B. and Diamant, A. (2019). Co-existence of Myxobolus spp. (Myxozoa) in gray mullet (Mugil cephalus) juveniles from the Mediterranean Sea.

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Parasitology Research 118, 159–167. Sweeney, P. (2016). The current status of aquatic oligochaetes in Ireland. Bulletin of the Irish Biogeographical Society 40, 3‒17. Székely, C., Borkhanuddin, M.H., Cech, G., Kelemen, O. and Molnár, K. (2014). Life cycles of three Myxobolus spp. from cyprinid fishes of Lake Balaton, Hungary involve triactinomyxon-type actinospores. Parasitology Research 113, 2817‒2825. Timm, T. (2013). Baltidrilus nom. nov., a substitute name for the genus Heterochaeta Claparède, 1863 (Annelida, Clitellata, Tubificidae) non Heterochaeta Westwood, 1843 (Insecta, Mantodea, Mantidae). Zootaxa 3734, 499–500. Whipps, C.M., Adlard, R.D., Bryant, M.S., Lester, R.J., Findlay, V. and Kent, M.L. (2003). First report of three Kudoa species from eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus). Journal of Eukaryotic Microbiology 50, 215‒219. Wolf, K. and Markiw, M.E. (1984). Biology contravenes taxonomy in the Myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science 225, 1449‒ 1452. Xi, B.W., Zhang, J.Y., Xie, J., Pan, L.K., Xu, P. and Ge, X.P. (2013). Three actinosporean types (Myxozoa) from the oligochaete Branchiura sowerbyi in China. Parasitology Research 112, 1575‒1582. Xi, B.W., Zhou, Z.G., Xie, J., Pan, L.K., Yang, Y.L. and Ge, X.P. (2015). Morphological and molecular characterization of actinosporeans infecting oligochaete Branchiura sowerbyi from Chinese carp ponds. Diseases of Aquatic Organisms 114, 217‒228. Xiao, C. and Desser, S.S. (1998a). Actinosporean stages of myxozoan parasites of oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of triactinomyxon and raabeia. Journal of Parasitology 84, 998‒1009. Xiao, C.X. and Desser, S.S. (1998b). Actinosporean stages of myxozoan parasites of oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of echinactinomyxon, neoactinomyxum, aurantiactinomyxon, guyenotia, synactinomyxon and antonactinomyxon. Journal of Parasitology 84, 1010‒1019. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1993a). Involvement of Branchiura sowerbyi (Oligochaeta, Annelida) in the transmission of Hoferellus carassii (Myxosporea, Myxozoa), the causative agent of kidney enlargement disease (KED) of goldfish Carassius auratus. Fish Pathology 28, 135‒139. Yokoyama, H., Ogawa, K. and Wakabayashi, H. (1993b). Some biological characteristics of actinosporeans from the oligochaete Branchiura sowerbyi. Diseases of Aquatic Organisms 17, 223–228. Yokoyama, H. and Urawa, S. (1997). Fluorescent labelling of actinospores for determining the

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portals of entry into fish. Diseases of Aquatic Organisms 30, 165–169. Yurakhno, V.M. and Ovcharenko, M.O. (2014). Study of Myxosporea (Myxozoa), infecting worldwide mullets with description of a new species. Parasitology Research 113, 3661– 3674.

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Chapter XIII

General Discussion

Chapter XIII | General Discussion

Myxosporean biodiversity

Myxosporeans have more than 2,200 species described to date (Lom and Dyková, 2006), representing about 18% of the cnidarian biodiversity currently known, a percentage that is probably underestimated and likely to increase due to the numerous taxonomic publications that continue to describe new species (Zhang, 2011; Okamura et al., 2015). In turn, only ca. 200 actinosporean types have been described thus far, but this number is also expected to increase, given its discrepancy to the number of known myxosporean species and increased interest in recognizing life cycle connections. Acknowledging the above, and further considering the devastating effects that myxosporeans can have in wild and reared fish populations, this thesis aimed to provide novel data regarding the myxosporean community infecting fish and annelids in Portuguese estuaries from which little or no information was previously available. Overall, the results obtained considerably increased the myxosporean biodiversity known from Portuguese estuarine habitats, with a total of 22 myxosporean species and 15 actinosporean stages being reported from fishes and oligochaete hosts, respectively (Table 1). In the Alvor estuary, two species belonging to the genus Ceratomyxa Thélohan, 1982 were found: Ceratomyxa auratae Rocha et al., 2015 infecting the gilthead seabream Sparus aurata Linnaeus, 1758, and Ceratomyxa diplodae Lubat et al., 1989 sensu Sitjà-Bobadilla and Alvarez-Pellitero, 1993 infecting the European seabass Dicentrarchus labrax Linnaeus, 1758 (Table 1). Ceratomyxa auratae constituted the ninth species of the genus to be described from sparid hosts in South European waters, and the fourth from the gall bladder of S. aurata in the Iberian Peninsula. As such, its description reinforced both the assumptions that species richness of Ceratomyxa in South European sparids is high, and that the same sparid can be infected by more than a single species of this genus (Gunter and Adlard, 2009; Alama-Bermejo et al., 2011). Considering that molecular systematics reveal Ceratomyxa as being mostly host specific (Gunter and Adlard, 2008, 2009; Gunter et al., 2009; Alama-Bermejo et al., 2011; Heiniger and Adlard, 2013), it was further suggested that the resolution of potential species complexes would probably increase this already high biodiversity. The re-description of C. diplodae contributed towards this aim, as it provided firsthand molecular data for a parasite that has been morphologically reported from several sparid and non-sparid hosts (see Lubat et al., 1989; Sitjà-Bobadilla and Alvarez-Pellitero, 1993; Rigos et al., 1997; Katharios et al., 2007). In the estuary of the River Minho, the genus Myxobolus was the most commonly observed among the fish species analysed, accounting for 14 species and 6 distinct morphotypes infecting mullets, plus 3 species parasitizing the Iberian endemic cypriniforms Achondrostoma arcasii (Steindachner, 1866) and Pseudochondrostoma duriense (Coelho,

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Table 1. Summary of myxosporean species and actinosporean types found in Portuguese estuaries during the present thesis.

Species Host Site of infection Biotype

Myxosporean Ceratomyxa auratae Rocha et al., 2015 Sparus aurata Gall bladder Brackish species

Ceratomyxa diplodae Lubat et al., 1989 Dicentrarchus labrax Gall bladder Brackish

sensu Sitjà-Bobadilla and Alvarez-Pellitero, 1993

Actinosporean Sphaeractinomyxon type 1 of Rocha et al. (2019f) Tubificoides insularis Coelomic cavity Brackish types

Sphaeractinomyxon type 2 of Rocha et al. (2019f) Tubificoides insularis Coelomic cavity Brackish

Alvor estuary Alvor

Sphaeractinomyxon type 3 of Rocha et al. (2019f) Tubificoides insularis Coelomic cavity Brackish

Sphaeractinomyxon type 10 of Rangel et al. (2016) Tubificoides insularis Coelomic cavity Brackish

Myxosporean Myxobolus exiguus Thélohan, 1895 Chelon ramada Visceral peritoneum Brackish species

Myxobolus ramadus n. sp. Rocha et al., 2019 Chelon ramada Gill lamellae Brackish

Myxobolus pharyngobranchialis n. sp. Rocha et al., 2019 Chelon ramada Pharyngobranchial organ Brackish

Myxobolus adiposus n. sp. Rocha et al., 2019 Chelon ramada Adipose tissue Brackish

Myxobolus muscularis n. sp. Rocha et al., 2019 Chelon ramada Skeletal and heart muscle Brackish

Myxobolus hepatobiliaris n. sp. Rocha et al., 2019 Chelon ramada Liver and gall bladder Brackish

Myxobolus renalis n. sp. Rocha et al., 2019 Chelon ramada Kidney Brackish

Myxobolus cerveirensis n. sp. Rocha et al., 2019 Chelon ramada Intestine Brackish

Myxobolus peritoneum n. sp. Rocha et al., 2019 Chelon labrosus Visceral peritoneum Brackish

Myxobolus labrosus n. sp. Rocha et al., 2019 Chelon labrosus Urinary bladder Brackish

Myxobolus mugiliensis n. sp. Rocha et al., 2019 Mugil cephalus Gill lamellae Brackish

Myxobolus vesicularis n. sp. Rocha et al., 2019 Mugil cephalus Connective tissue Brackish

Minho estuary Minho Myxobolus urinaris n. sp. Rocha et al., 2019 Mugil cephalus Urinary bladder Brackish

Myxobolus galaicoportucalensis n. sp. Rocha et al., 2019 Mugil cephalus Intestine Brackish

Ellipsomyxa mugilis (Sitjà-Bobadilla and Chelon ramada Gall bladder Brackish Alvarez-Pellitero, 1993)

Myxobolus arcasii n. sp. (Chapter XI) Achondrostoma arcasii Kidney and undifferentiated Freshwater tissue of the gonads

Myxobolus duriensis n. sp. (Chapter XI) Pseudochondrostoma duriense Primary gill filaments Freshwater

Myxobolus pseudodispar Gorbunova, 1936 Achondrostoma arcasii Spleen Freshwater

Pseudochondrostoma duriense Muscle, spleen, liver, Freshwater kidney, stomach and intestine

Thelohanellus paludicus (Chapter XI) Cobitis paludica Intestine Freshwater

Actinosporean Raabeia type of Rocha et al. (2019c) Ilyodrilus templetoni Intestinal epithelium Freshwater types

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Sphaeractinomyxon type 1 of Rocha et al. (2109b) Undetermined Coelomic cavity Freshwater

Sphaeractinomyxon type 2 of Rocha et al. (2019b) Psammoryctides barbatus Coelomic cavity Freshwater

Sphaeractinomyxon type 3 of Rocha et al. (2019b) Potamothrix sp. Coelomic cavity Freshwater

Sphaeractinomyxon type 4 of Rocha et al. (2019b) Potamothrix sp.; Coelomic cavity Freshwater Tubificoides pseudogaster Brackish

Sphaeractinomyxon type 8 of Rangel et al. (2016) Tubificoides pseudogaster Coelomic cavity Brackish

Sphaeractinomyxon type 10 of Rangel et al. (2016) Tubificoides pseudogaster Coelomic cavity Brackish

Aurantiactinomyxon type of Rocha et al. Tubificoides pseudogaster Intestinal epithelium Brackish (2019a)/Paramyxidium giardi (Cépède, 1906) Freeman and Kristmundsson, 2018

Actinosporean Sphaeractinomyxon type 1 of Rocha et al. (Chapter Baltidrilus sp. Coelomic cavity Brackish types XII)/Myxobolus mugiliensis

Sphaeractinomyxon type 2 of Rocha et al. (Chapter Baltidrilus sp. Coelomic cavity Brackish XII)/Myxobolus sp. Sharon et al., 2019

Sphaeractinomyxon type 3 of Rocha et al. (Chapter XII) Baltidrilus sp. Coelomic cavity Brackish

Douro estuary Douro Sphaeractinomyxon type 4 of Rocha et al. (Chapter Baltidrilus sp. Coelomic cavity Brackish XII)/Myxobolus labrosus

1985). The available literature shows that the family Myxobolidae is the most taxon-rich among myxosporeans, with species of the genera Henneguya, Myxobolus and Thelohanellus being the mostly reported in freshwater habitats (Eiras, 2002; Eiras et al., 2005, 2014; Lom and Dyková, 2006; Eiras and Adriano, 2012; Zhang et al., 2013). Thus, the species-richness of Myxobolus found in the Minho estuary probably relates to the fact that most samples were performed in fish collected from fyke-nets positioned in a freshwater estuarine location. Other than these, a single Thelohannelus species was described from the Southern Iberian spined- loach Cobitis paludica (de Buen, 1930), and Ellipsomyxa mugilis (Sitjà-Bobadilla and Alvarez- Pellitero, 1993) was reported from the gall bladder of the thinlip grey mullet Chelon ramada (Risso, 1827). The fact that only a single Thelohanellus species, and none Henneguya, were found in the fish specimens collected from the fyke-nets (Table 1), is probably explained by yet undetermined biotic and abiotic factors. In fact, contrarily to the Myxobolus and Thelohanellus morphotypes, which are optimized for ingestion by deposit feeding annelids (Lemmon and Kerans, 2001; Kerans and Zale, 2002), Henneguya morphotypes are thought to have prolonged suspension in the water column due to their long valvular processes. This feature potentially facilitates consumption by filter feeder annelids (Alexander et al., 2015), which may not be present in the invertebrate community of the Minho upper estuary or not be permissive to myxosporean infection. Cypriniformes constitute the largest group of freshwater fishes worldwide (Eschmeyer and Fong, 2018) and, therefore, are the hosts for the great majority of known myxobolid species (see Eiras, 2002; Eiras et al., 2005, 2014; Eiras and Adriano, 2012; Zhang et al., 2013).

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As such, it was anticipated that myxosporean biodiversity in the endemic cypriniforms analysed would include at least some members of Myxobolidae, such as Myxobolus and Thelohanellus, a expectation confirmed in this thesis for the “bermejuela” A. arcasii, the Northern straight- mouth nase P. duriense, and the Southern Iberian spined-loach C. paludica (see Table 1). This results prompted us to review the myxobolid biodiversity reported in the literature from Iberian endemic cypriniforms, which prior to this thesis, accounted for a total of 11 Myxobolus spp. registered from either Luciobarbus bocagei (Steindachner, 1864), the Iberian nase Pseudochondrostoma polylepis (Steindachner, 1864), and Achondrostoma arcasii (see Chapter XI). The occurrence of most of these Myxobolus species in the above-mentioned Iberian endemic cypriniforms, however, was found to be dubious and require molecular confirmation. Considering that myxobolids cluster phylogenetically according to the vertebrate host group (namely family and order) (Ferguson et al., 2008; Carriero et al., 2013; Rocha et al., 2014), it was suggested that species originally described from fish hosts belonging to distinct cypriniform families (e.g. Myxobolus muelleri Bütschli, 1882) were probably erroneously identified in these hosts. Furthermore, several of the species reported to infect L. bocagei were originally described from central and eastern European cypriniforms, namely barbel Barbus barbus (Linnaeus, 1758) and Crimean barbel Barbus tauricus Kessler, 1877, of which there is no record of introduction into the Iberian Peninsula. Thus, the results presented in this thesis established that myxosporean biodiversity in Iberian endemic cypriniforms is both underestimated and poorly interpreted. Regarding the reports previously performed from L. bocagei, it was suggested that potentially new Myxobolus spp. await description from this host, based on their molecular differentiation from morphologically undistinguishable congeners in central and eastern European barbels. On the other hand, the previously reported presence of Myxobolus pseudodispar Gorbunova, 1936 in the Iberian Peninsula was confirmed in this thesis, as the parasite was morphologically and molecularly identified from infected specimens of A. arcasii and P. duriense (Table 1). Its occurrence in P. duriense, specifically, added a new host record to this muscle-dwelling parasite, common among cypriniforms of the family Leuciscidae (Molnár et al., 2002; Forró and Eszterbauer, 2016). Considering that its original host (roach Rutilus rutilus), and other reported hosts, were all introduced into the Iberian Peninsula during the 20th century (Hernando and Soriguer, 1992; Kottelat and Freyhof, 2007), M. pseudodispar was here suggested to have entered this geographic region, possibly multiple times, as the result of parasite/host co-introduction. The occurrence of host-shift was supported by the genetic diversity found between the Myxobolus pseudodispar SSU rDNA sequences obtained from A. arcasii, P. duriense and others previously available in GenBank from central European hosts. This strengthened the contention that M. pseudodispar constitutes a cryptic species

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Chapter XIII | General Discussion complex, as previously hypothesized by Forró and Eszterbauer et al. (2016). A considerable species diversity of Myxobolus has also been reported from mullets worldwide and was here summarized in Rocha et al. (2019d). As part of their catadromous nature, mullets spend a significant portion of their lives in estuarine habitats, moving between fresh- and brackish waters. One of the factors allowing the co-habitation of different species in the same estuarine habitat is their zonal distribution according to salinity gradients (Cardona, 2006). As a result of its higher adaptability to low salinities and water pollution, Chelon ramada migrates further into freshwater than other mullets (Torricelli et al., 1981; Cardona, 2006); the reason why specimens belonging to this species were more frequently caught in the fyke-nets. Accordingly, it was in C. ramada that highest rate of Myxobolus infection was found, and by a significantly elevated number of different species (see Table 1). Moreover, the presence of unidentified myxospores in several organs, potentially belonging to new species, suggested that this already high biodiversity can, in fact, be increased by further pursuing myxozoan surveys in this host. The occurrence of Myxobolus infection in mullets that are less tolerant to freshwater, such as the thicklip grey mullet Chelon labrosus and the flathead grey mullet Mugil cephalus, showed that the acquisition of this vertebrate group as secondary hosts allowed myxobolids to move and settle in brackish habitats, whenever a permissive host could be found within downstream invertebrate communities. In the other way around, the occurrence of Ellipsomyxa mugilis in several specimens of C. ramada demonstrates how this host carries infections acquired from the marine invertebrate communities into freshwater habitats. Although the presence of E. mugilis in Portuguese waters was known due to its actinosporean counterpart having been described from the marine polychaete Hediste diversicolor in the Aveiro estuary (Rangel et al., 2009), this thesis provided the first report of its myxospores in a vertebrate host from the River Minho geographic region. Turning to consider the biodiversity of actinosporean stages reported during this thesis, it is noted that a total of 11 new types and 2 known types were reported from the sphaeractinomyxon collective group, with only one raabeia and one aurantiactinomyxon types being further described. This higher abundance of sphaeractinomyxon does not reflect any specific parasite-, host- or environmental-related factor, but rather a continued attempt to confirm the life cycle connection that was here hypothesized between this collective group and mugiliform-infecting Myxobolus spp. In fact, further three aurantiactinomyxon and two synactinomyxon types were observed parasitizing annelids in the River Minho (see Rocha et al., 2019b, c), and are intended for publication in a near future. Prior to this thesis, 20 sphaeractinomyxon types were known in the literature: 14 parasitizing marine oligochaetes, and the remaining 6 parasitizing freshwater oligochaetes (Caullery and Mesnil, 1904, 1905; Puytorac, 1963; Marques, 1984; Hallett et al., 1997, 1998, 1999; Rangel et al., 2016). The present thesis significantly increased this number by adding

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16 representatives to this collective group, either through the description of novel types or through the revision of known types (see Chapter XII; Rocha et al., 2019b, c, f). The three new types and four new types reported from the Alvor and Douro estuary, respectively, were all found parasitizing the coelomic cavity of marine hosts: the first in Tubificoides insularis (Stephenson, 1923), and the second in an unidentified species of the genus Baltidrilus Timm, 2013. The Sphaeractinomyxon type 10 of Rangel et al. (2016) was also observed in specimens of T. insularis from the Alvor estuary (see Table 1). Having been originally described from Tubificoides pseudogaster in the Aveiro estuary (Rangel et al., 2016), this type constituted the first confirmed exception to the strict host specificity of the collective group. In turn, the new types reported from the Minho estuary were all found in freshwater oligochaetes, namely Psammoryctides barbatus (Grube, 1861) and an unidentified species of the genus Potamothrix Vejdovský and Mrázek, 1903. Again, the finding of freshwater sphaeractinomyxon in the River Minho probably relates to the fact that most sediment samples from this geographic location were collected near the fyke-nets in the upper estuary. The survey of annelids from the lower estuary of the River Minho revealed the presence of Sphaeractinomyxon types 8 and 10 of Rangel et al. (2016), but also the Sphaeractinomyxon type 4 from Potamothrix in the upper estuary, infecting the marine oligochaete Tubificoides pseudogaster. This finding further reinforced the involvement of sphaeractinomyxon in the life cycle of myxosporeans that parasitize migratory fish, namely mullets, as it was early hypothesized in this thesis. Furthermore, it confirmed that sphaeractinomyxon do not have strict host specificity, thus contradicting earlier reports of the collective group (e.g. Rangel et al., 2016). Although only one raabeia type was found in the upper estuary of the River Minho (Table 1), its description significantly increased the known diversity of raabeia, since it led to the invalidation of the echinactinomyxon collective group, with subsequent inclusion of its types in raabeia. In turn, the Aurantiactinomyxon type found infecting Tubificoides pseudogaster in the lower estuary of this River was determined to be the life cycle counterpart of Paramyxidium giardi (Cépède, 1906) Freeman and Kristmundsson, 2018, demonstrating that further myxosporean diversity can be found in the River Minho. In the same manner, the remaining actinosporean types reported in this thesis are also expected to be associated with the life cycles of myxosporean species that are presently unknown or that are without available molecular data. Although myxosporeans infect several vertebrate groups, the great majority of species have been described from fish hosts (Lom and Dyková, 2006); as such, the continued survey of fishes from the above-mentioned estuaries is necessary and will most likely increase the biodiversity that is reported here, potentially allowing the establishment of further life cycle connections.

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Taxonomic descriptions and species re-descriptions

The allocation of organisms into hierarchical taxonomic groupings is central to understating their biology, diversity and evolution. Traditionally, the taxonomy of myxosporeans was based on myxospore morphology and biology, following early classification systems (Kudo, 1933; Tripathi, 1948; Shulman, 1959) that were established prior to the discovery of the involvement of actinosporeans as intra-annelid life cycle stages of these parasites (Wolf and Markiw, 1984). For much of their history, myxosporean genera and species were described based on the morphological features of mature myxospores and developmental stages, host specificity and site of infection. The implementation of molecular tools to the study of this parasitic cnidarians, however, as shown that the usage of morphology- based criteria for taxonomic purposes is mostly artificial (Fiala, 2006; Bartošová et al., 2009; Fiala and Bartošová, 2010; Liu et al., 2010), given that differences in myxospore morphology may not be associated with significant genetic divergences. Moreover, studies have shown that myxospore shape and size can change as they mature, so that a high level of natural morphological and morphometric variation can occur within or between hosts (Lom and Dyková, 1995; Holzer et al., 2013). This demonstrates that the application of new taxonomic approaches, namely of molecular nature, are necessary in order to assess the validity of existing taxa, facilitate the description of new taxa, and resolve known cryptic species. During this thesis, 17 new myxosporean species belonging either to the genera Ceratomyxa, Myxobolus or Thelohanellus were described, and two cryptic species were given comprehensive re-descriptions (Table 1). The acquisition of molecular data from parasite isolates proved to be crucial for all studies. New myxosporean species were described and taxonomically classified based on the combined analysis of several criteria; these included myxospore morphology, host specificity, site of infection, and SSU rDNA molecular data. Considering the above-mentioned plasticity of myxospore morphology, species differentiation from previously reported congeners was, whenever possible, based on molecular comparisons. Nonetheless, comparison of morphological traits had to be performed in relation to the large number of species that remain without molecular data. In a few cases, most notoriously among mugiliform-infecting Myxobolus spp., myxospore morphologic and morphometric overlap was observed between molecularly distinct species. For instance, M. mugiliensis n. sp., M. vesicularis n. sp. and M. galaicoportucalensis n. sp. from C. ramada were noted to share similar morphological and morphometric features with M. spinacurvatura Maeno et al., 1990 from M. cephalus, but could be easily differentiated from this species based on SSU rDNA molecular data. The same for M. peritoneum n. sp. from C. labrosus and M. episquamalis Egusa et al., 1990 from M. cephalus. The species comparisons performed in this

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Chapter XIII | General Discussion study, therefore, highlighted the problems of using morphology-based criteria alone and reinforced the necessity of using molecular tools for differentiation at the species-level. Early taxonomic studies mostly relied on light microscopy and drawings for describing new myxosporeans. Consequently, several cryptic species have been recognized and warrant validation using currently accepted taxonomic criteria (Easy et al., 2005; Ferguson et al., 2008; Atkinson et al., 2015). In this thesis, two cryptic species were identified and re-described: Ceratomyxa diplodae Lubat et al., 1989 sensu Sitjà-Bobadilla and Alvarez-Pellitero, 1993 and Myxobolus exiguus Thélohan, 1895. Both parasites were studied using updated microscopy and molecular procedures that allowed unequivocal identification of morphological and molecular features. It was also shown how ultrastructural studies, besides being useful for recognizing specific morphological and developmental characters, can aid in determining specific tissues of infection and potential pathogenic actions of the parasite, as well as in understanding host-parasite interactions. Nonetheless, considering the relevance of tissue tropism as a driver of myxosporean evolution (Ferguson et al., 2008; Carriero et al., 2013; Rocha et al., 2014), future taxonomic studies should provide histological data as a requirement. Acknowledging that there is much entropy in species reports from mullet hosts, this thesis further contributed to the literature with a detailed and critical review of mugiliform- infecting myxobolids. In this review, the reliability of species reports from mullets was determined through the careful analysis of original species descriptions, available molecular data, and currently accepted taxonomic and phylogenetic criteria. The result was a comprehensive summary of all myxobolids with bona fide mugiliform fish hosts, which will certainly prove to be extremely useful for establishing reliable taxonomic comparisons in future species descriptions of the group, as was further confirmed in the present thesis. Like their myxosporean counterparts, the majority of known actinosporean stages have also been described based almost solely on actinospore morphology, with molecular methodologies, however, increasingly revealing the usage of morphological criteria as being unreliable for type differentiation and identification. In the same manner that significant morphological variability has been reported between isolates belonging to the same type (Hallett et al., 2004; Eszterbauer et al., 2006; Atkinson et al., 2009), molecularly distinct types were shown to be morphologically undistinguishable amongst each other (Rangel et al., 2016). Several of the sphaeractinomyxon types described during this thesis could not be differentiated based on morphology alone, especially as they were reported from the same oligochaete host. The Sphaeractinomyxon type 4 of Rocha et al. (2019b) and Sphaeractinomyxon type 8 of Rangel et al. (2016) developing in the coelomic cavity of Tubificoides pseudogaster from the lower estuary of the River Minho, shared highly similar morphological traits and could only be distinguished through molecular comparison of their respective SSU rDNA sequences. The

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Chapter XIII | General Discussion

Sphaeractinomyxon types 1 and 2 reported from a marine species of the genus Baltidrilus in the lower estuary of the River Douro (Chapter XII) also displayed significant morphometric overlap, however differing molecularly. Similarly, the Aurantiactinomyxon shown to be the life cycle counterpart of P. giardi shared a strong morphological resemblance with the Aurantiactinomyxon type 1 of Negredo and Mulcahy (2001), and could have easily been misidentified as such, if not considering the genetic difference found between their respective SSU rDNA sequences. In addition, the actinospores of the Raabeia type described from Ilyodrilus templetoni (Southern, 1909) in the upper estuary of the River Minho, when displaying straight valvular processes, highly resembled Echinactinomyxon radiatum, being differentiated from this type mainly on the basis of molecular data. Thus, the actinosporean descriptions performed in this thesis firmly reinforced the inaccuracy of using solely morphology-based criteria for distinguishing between actinosporean types. Further considering the apparently strict host specificity of the aurantiactinomyxon and raabeia collective groups, it was suggested that extensive morphological comparisons to all types of the same collective group that are without molecular data and have distinct oligochaete hosts are unnecessary. Thus, it was proposed that new isolates be only identified either by DNA match or through a comprehensive morphological and biological comparison to known types sharing the same annelid host. A major contribution of this thesis to actinospore classification was the revision and invalidation of the echinactinomyxon, endocapsa and tetraspora collective groups. Traditio- nally, the echinactinomyxon collective group differed from raabeia only in the rigidity of its valvular processes, characterized as being “spiny, straight, rigid” (Janiszewska, 1957). The valvular processes of raabeia were, in turn, defined as being pointed and curved (Janiszewska, 1955). The usage of this morphological criterion, however, proved to be inappropriate for classifying the Raabeia type of Rocha et al. (2019c) described in this thesis, given that the shape of the actinospores’ valvular processes was too variable to allow distinction between the two collective groups. The ultrastructural study of this type further revealed these morphological structures as being devoid of cytoplasmic content that could sustain a permanent rigidity. Upon review of the available literature, several cases were found in which actinospore types were subjectively classified as belonging to either the echinactinomyxon or raabeia collective groups. Considering this, invalidation of the echinactinomyxon collective group was proposed, with its types being transferred to raabeia, as the latter constituted the oldest group among the two. Accordingly, an amended definition of the raabeia collective group is provided in this thesis, which further supplies a comprehensive summary of all known types presently included within the latter, immensely useful for future type descriptions. The invalidation of tetraspora and endocapsa was similarly based on the variability of the morphological and developmental characters used for establishing differentiation between these two collective groups, but also in the phylogenetic analysis of available SSU rDNA

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Chapter XIII | General Discussion sequences. Both tetraspora and endocapsa produced actinospore morphotypes that were highly similar to sphaeractinomyxon, differing from the latter in specific details: tetraspora actinospores developed in groups of 4 (instead of 8) within the pansporocysts (Hallett and Lester, 1999); while endocapsa were described as having irregular “processes in the form of swellings” that did not change when in contact with water (Hallett et al., 1999). Rangel et al. (2016), however, reported several sphaeractinomyxon types developing a variable number (1 to 8) of actinospores within the pansporocysts. In turn, the Sphaeractinomyxon type 1 described from the Minho upper estuary in this thesis (Rocha et al., 2019b), was shown to display actinospores with and without “valvular swellings”. A review of the available literature corroborated the tenuous boundary existent between these two collective groups and sphaeractinomyxon. Moreover, phylogenetic analyses of the SSU rDNA gene revealed that all endocapsa, tetraspora and sphaeractinomyxon types with available molecular data consistently cluster with each other, specifically within a myxobolid clade that was suggested to have a monophyletic origin (Rocha et al., 2019d, e, f). Considering all the above, this thesis proposed the invalidation of both endocapsa and tetraspora, with their types being renamed as sphaeractinomyxon, and a more inclusive definition of this collective group being provided in order to reflect more accurately the current morphological and phylogenetic information.

Phylogeny

During the past few decades, phylogenetic studies have provided crucial information for unravelling the origin and evolutionary history of myxosporeans. The continuous addition of new molecular data from species of this cnidarian group has allowed the progressive recognition of important evolutionary patterns and drivers. In this thesis, phylogenetic studies were conducted on genera belonging to both the oligochaete- and polychaete-infecting lineages [former freshwater/marine lineages (see Holzer et al., 2018)]. The outcome of these analyses have, not only, reinforced known assumptions, but most importantly, disclosed novel information regarding specific groups. Phylogenetic analyses of the polychaete-infecting lineage targeted specifically the inner topology of the Ceratomyxa sub-lineage, a taxon-rich clade that has been reported to comprise all sequenced Ceratomyxa spp., but also the sphaerosporid Palliatus indecorus Shulman et al., 1979 and the sinuolineidid Myxodavisia bulani Fiala et al., 2015 (Fiala 2006; Gunter et al., 2009; Alama-Bermejo et al., 2011; Fiala et al., 2015). In accordance, the SSU rDNA sequences obtained for both C. auratae and C. diplodae clustered within the latter clade, which was further shown to include Pseudoalatospora kovalevae Kalavati et al., 2013 as another exception to its monophyly. The inner topologies obtained were, in general, congruent with those reported in previous studies (e.g. Fiala et al., 2015). Nonetheless, the positioning of C. auratae,

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Chapter XIII | General Discussion specifically, provided phylogenetic evidence that contradicted the previously proposed common ancestry of sparid-infecting Ceratomyxa spp. (see Alama-Bermejo et al., 2011), given that this species formed a well-supported clade with Australian congeners reported from fish hosts belonging to the families Triglidae, Apogonidae, Serranidae, Lethrinidae, Labridae and Mugilidae. This clustering further showed that geography does not drive the overall phylogeny of sparid-infecting Ceratomyxa spp., as it had also been hypothesized by Alama-Bermejo et al. (2011). In turn, the phylogenetic positioning of C. diplodae from the moronid fish host D. labrax, amongst most Mediterranean sparid-infecting congeners, revealed that these species have a recent common ancestor. This supported the cryptic nature of C. diplodae by suggesting that host-switch occurred at some point, and that this species might be incurring speciation in distinct hosts. Thus, sequencing of this parasite from reported sparid hosts [Diplodus annularis, Diplodus puntazzo and Dentex dentex (Lubat et al., 1989; Rigos et al., 1997; Katharios et al., 2007)] is necessary in order to ascertain if the morphological and biological variations found among reports are intra- or inter-specific. Previous phylogenetic studies have suggested that the non-monophyly of the Ceratomyxa clade would ultimately be resolved by the taxonomic revision of Palliatus and Myxodavisia (see Fiala, 2006; Gunter et al., 2009; Fiala et al. 2015). Similar conclusions could be drawn in this thesis regarding the inclusion of P. kovalevae within this clade. Considering that the genera Pseudoalatospora Kovaleva and Gaevskaya, 1983 and Ceratomyxa display very little morphological differences, and share the same host habitat and tissue tropism, the phylogenetic positioning of P. kovalevae reinforces the notion that a taxonomic revision of this genus is required. The same was proposed for the sister genus Alatospora Shulman et al., 1979, which despite being without molecular data, also comprises species that are coelozoic in the gall bladder of marine fish and produce myxospores morphologically very similar to both Pseudoalatospora and Ceratomyxa. Overall, it was hypothesized that further sequencing of Pseudoalatospora and Alatospora spp. will most likely prompt the demise of the family Alatosporidae, with subsequent reassignment of its species to the genus Ceratomyxa. One of the main goals of the phylogenetic analyses of the oligochaete-infecting lineage performed in this thesis was to provide insight into myxosporean/actinosporean associations. The more comprehensive datasets used in Rocha et al., (2019c, f) revealed the SSU rDNA sequences of oligochaete-infecting actinosporeans spanning over myxosporean genera of the bivalvulid suborders Variisporina and Platysporina, thus strengthening the view that there is no obvious agreement between actinosporean morphotypes and myxosporean genera. The only exceptions were the SSU rDNA sequences available for sphaeractinomyxon types (including former endocapsa and tetraspora), which consistently clustered within the subclade of mugiliform-infecting Myxobolus spp. Considering that published phylograms widely show the vertebrate host group as the most relevant driver of myxobolid evolution (Ferguson et al.,

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Chapter XIII | General Discussion

2008; Carriero et al., 2013; Rocha et al., 2014), the involvement of sphaeractinomyxon in the life cycle of mugiliform-infecting Myxobolus spp. was hypothesized. The strength of this actinosporean/myxosporean association was reinforced by all phylogenetic analyses performed posteriorly for these groups, as all new Myxobolus spp. from mullets and sphaeractinomyxon types reported in this thesis also clustered within the above-mentioned clade. In turn, the Raabeia type of Rocha et al. (2019c) reported from the Minho upper estuary clustered within the Paramyxidium clade, with no specific affinity to any given sequence belonging to this group. The Aurantiactinomyxon type found in the Minho lower estuary is also positioned within this clade, given that it is conspecific with Paramyxidium giardi. It should be further noted that the clustering of the new marine sphaeractinomyxon types, and Aurantiactinomyxon type from Tubificoides pseudogaster, among freshwater species, supported the recently proposed phylogenetic division of myxosporeans into an oligochaete-infecting lineage and polychaete-infecting lineage, rather than a freshwater lineage and marine lineage (Holzer et al., 2018). Considering that most species descriptions performed in this thesis targeted members of the family Myxobolidae, other phylogenetic analyses were focused in the clade of myxobolids, more specifically in the clades of mugiliform- and cypriniform-infecting species. Overall, the topologies obtained followed the main phylogenetic trend that has been recognized for the family Myxobolidae, with species clustering according to the vertebrate host group (namely taxonomic order and family) (Ferguson et al., 2008; Carriero et al., 2013; Rocha et al., 2014). Tissue tropism-related clustering has also been reported to occur among myxobolids, and myxosporeans in general (see Molnár and Eszterbauer et al., 2015 and references therein). Nonetheless, its relevance as a fine-scale evolutionary driver of myxobolid evolution was not broadly analysed in this thesis, given that histology was not performed for determining specific tissues of infection, and neither is this type of information available for most species. Currently, it is widely accepted that the origin and radiations of myxozoans reflect the evolution of their hosts (Carriero et al., 2013; Kodádková et al., 2015; Holzer et al., 2018; Patra et al., 2018). Regarding cypriniform-infecting myxobolids, the formation of several leuciscid- and cyprinid-infecting subclades in the tree topologies revealed that these parasites entered different families of the order Cypriniformes multiple times during their evolution. Contrarily, and in a clear parallelism to the monophyly of the order Mugiliformes, all mugiliform-infecting Myxobolus spp. described here clustered within a single clade, providing evidence for their monophyletic origin, and further reinforcing previous assumptions of their correlation to sphaeractinomyxon. Moreover, the hyperdiversification of Myxobolus in mullet hosts, as reported in this thesis, was proposed to reflect the processes of speciation that lead to the recognized ecological plasticity of this fish group. Nonetheless, it cannot be ignored that,

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Chapter XIII | General Discussion despite the considerable input provided in this thesis, the molecular data currently available for mugiliform-infecting Myxobolus is most likely representative of a minor fraction of the true biodiversity of these species. As such, future studies may reveal other lineages of Myxobolus from distinct origins in mugiliform fish hosts.

Life cycle inferences and novel myxosporean/actinosporean associations

Prior to this thesis, only two of the ca. 60 myxosporean life cycles resolved in the literature were known to involve a marine oligochaete as invertebrate host. The triactinomyxon counterpart of Ortholinea auratae Rangel et al., 2014, a parasite from the urinary bladder of gilthead seabream S. aurata in the Alvor estuary (Southern Portugal), was reported to develop in the intestinal epithelium of the marine oligochaete Limnodriloides agnes Hrabĕ, 1967 (see Rangel et al., 2015). Its congener, Ortholinea labracis Rangel et al., 2017, a urinary bladder parasite of European seabass D. labrax in the same Portuguese estuary, was similarly reported to produce triactinomyxon actinospores in the intestinal epithelium of a marine oligochaete belonging to the genus Tectidrilus Erséus, 1982 (see Rangel et al., 2017). The present thesis triples the number of myxosporean life cycles that involve marine oligochaetes, further providing novel information for previously unknown life cycle associations. Molecular analyses of the SSU rDNA gene determined the Aurantiactinomyxon type developing in T. pseudogaster as the actinosporean life cycle counterpart of Paramyxidium giardi, a parasite that was recently re-described from infections in the kidney of European eel Anguilla anguilla (Linnaeus, 1758) from Icelandic Rivers (Freeman and Kristmunsson, 2018). This myxosporean/actinosporean association confirmed the observations of Benajiba and Marques (1993), who had previously suggested that the actinosporean counterpart of Myxidium giardi (now revised as P. giardi) belonged to the aurantiactinomyxon collective group. Life cycle inferences were further performed for the Sphaeractinomyxon types 1, 2 and 4 found infecting a Baltidrilus sp. in the Douro estuary, which were molecularly determined to be the counterparts of Myxobolus spp. that infect the flathead grey mullet Mugil cephalus and thicklip grey mullet Chelon labrosus in the Minho estuary and in the Spanish Mediterranean coast. This confirmed the previously hypothesized involvement of sphaeractinomyxon in the life cycle of mugiliform-infecting Myxobolus spp., establishing a myxosporean/actinosporean association that was previously unknown. Prior to this thesis, sphaeractinomyxon types had never been linked to any given myxosporean species, and neither was the life cycle of any mugiliform-infecting Myxobolus reported in the literature. Genetic matches between mugiliform-infecting Myxobolus spp. and their sphaeractinomyxon counterparts were determined from values of percentage of similarity that

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Chapter XIII | General Discussion ranged from 99.8% to a 100%. In turn, the life cycle of P. giardi was molecularly inferred from a 99.4% of similarity to the sequence of the Aurantiactinomyxon described from the Minho lower estuary. The low genetic difference (0.6%) is within the intraspecific variability range generally accepted for myxosporeans (see Atkinson et al., 2015 and references therein), and was suggested to result from the emergence of genotypically different subspecies due to geographic isolation, with subsequent evolutionary adaptation to distinct biotic and abiotic factors. Although a similar value of genetic difference was determined between the SSU rDNA sequences of Myxobolus galaicoportucalensis n. sp. and the Sphaeractinomyxon type 2 of Rangel et al. (2016), a 100% of similarity was observed between the geographical isolates of M. galaicoportucalensis n. sp. myxospores. Consequently, the low genetic difference found between these sequences was prudently maintained as being representative of interspecific variability, until data arises that can confirm or refute these findings. In their experimental transmission study, Benajiba and Marques (1993) identified the oligochaete host of P. giardi as a freshwater tubificid. Freeman and Kristmundsson (2018) also hypothesized freshwater oligochaetes as the most probable annelid hosts of Paramyxidium spp., considering that known vertebrate hosts [European eel and Indo-Pacific tarpon Megalops cyprinoides (Broussonet, 1782)] were sampled from freshwater habitats. Nonetheless, the morphological and molecular data acquired in this thesis assertively identified the invertebrate host of P. giardi as the marine oligochaete Tubificoides pseudogaster. Molecular evidence also pointed for the development of the Sphaeractinomyxon type 4 of Rocha et al. (2019b) in both a freshwater oligochaete of the genus Potamothrix and the marine oligochaete T. pseudogaster. These findings supported the notion that the acquisition of fishes as second hosts enabled myxosporeans with alternative transmission and dispersion strategies that were crucial in the conquest of new habitats, as previously suggested by Holzer et al. (2018). Both European eel, Indo-Pacific tarpon and mullets are migratory fish that transition between saltwater and freshwater at some point during their life time. As such, these species should be regarded as potential temporary hosts for myxosporeans species developing in oligochaetes or polychaetes, regardless being in freshwater, brackish or marine environments (Rocha et al., 2019f). In this thesis, it was suggested that the capacity of fish to transition between brackish and freshwater has allowed their myxosporeans parasites to cross environmental barriers and conquer new habitats, whenever a permissive host could be found in the invertebrate community. In the case of mullets, specifically C. ramada, their considerable high tolerance to lower salinity gradients, and subsequent broader interaction with upstream benthic communities, was hypothesized to be the main evolutionary catalyst for their species-richness of Myxobolus. Nonetheless, the role that other biotic and abiotic factors played in the establishment of this apparently highly successful host/parasite association cannot be

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Chapter XIII | General Discussion disregarded. In fact, confirmation of the involvement of sphaeractinomyxon in the life cycle of mugiliform-infecting Myxobolus spp., showed the hyperdiversification of this genus in mullet hosts to reflect not only the ecological plasticity of the vertebrate host group, as previously mentioned, but also the evolutionary and functional success of the spore morphotypes promoting dissemination and transmission, as will be elaborated next. Moreover, the presence of Sphaeractinomyxon type 10 of Rangel et al. (2016) in three Portuguese estuaries (see Rangel et al., 2016; Rocha et al., 2019b, f) further corroborated the vertebrate host-mediated geographic dissemination of myxosporean parasites, thus emphasizing the preponderant role of the vertebrate host in the colonization of new habitats. Although the evolutionary diversification of myxospore and actinospore morphotypes has seldom been approached in the literature, it is likely that they were somehow shaped by their adaptation to biotic and abiotic factors. While some morphotypes appear to be adapted for settling, others display features that facilitate floating in the water column (Lom and Dyková, 2006; Alexander et al., 2015). In this thesis, the congruence of the relationship established between the Myxobolus and sphaeractinomyxon morphotypes constituted the first exception to the widely acknowledged lack of correspondence between myxosporean and actinosporean morphotypes. The apparent evolutionary success of this specific myxospore/actinospore association was hypothesized to correlate with the functional design of their morphotypes, which are optimized for promoting dissemination and host invasion. Lacking the inflatable valvular processes that are commonly reported among other actinosporean groups, sphaeractinomyxon were suggested to be adapted for transmitting infection to benthic feeders, such as mullets. In turn, the negative buoyance of the Myxobolus myxospores, associated with their capability to adhere to small particles and bacteria (Lemon and Kerans, 2001), was suggested to increase the likelihood of consumption by deposit feeders, such as Tubificoides, Baltidrilus and other naidid oligochaetes in general (Brinkhurst et al., 1972; Rodriguez et al., 2001; Jeuniaux, 2012). As the body of knowledge concerning myxosporean/actinosporean associations increases, it becomes clear that the abiotic and biotic factors mediating host/parasite interactions should be the core of future studies. A information that will be useful for understanding both the evolution and ecology of these parasites, subsequently enabling research of more effective control measurements in the future.

Myxosporean-annelid interactions

Myxosporean-annelid interactions have been scarcely studied, especially in brackish/marine environments. Most actinosporean stages have been described from freshwater oligochaetes, with fewer marine types mainly reported from polychaetes instead. This differential use of oligochaetes/polychaetes according to environment is probably based

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Chapter XIII | General Discussion on group availability, as polychaetes are less common than oligochaetes in freshwater environments, whereas oligochaetes are less common than polychaetes in marine environments (see Alexander et al., 2015 and references therein). The broader diversity of annelids inhabiting estuarine habitats, however, apparently allows myxosporeans to find permissive hosts among marine oligochaetes (e.g. Hallett et al., 1997; Roubal et al., 1997; Rangel et al., 2015, 2016) and freshwater polychaetes (e.g. Bartholomew et al., 1997, 2006). In this context, the present thesis increases considerably the number of actinosporean types described from marine oligochaetes, given that only the Raabeia type of Rocha et al. (2019c) and three Sphaeractinomyxon types of the Minho estuary (Rocha et al., 2019b) were exclusively reported from freshwater oligochaetes. In total, three marine naidids (Tubificoides pseudogaster, T. insularis and a Baltidrilus sp.) were determined to serve as permissive hosts for the development of sphaeractinomyxon types. Tubificoides pseudogaster further hosted the Aurantiactinomyxon counterpart of P. giardi in the River Minho. Acknowledging that the few other actinosporean types described in the literature from marine oligochaetes also use naidid hosts, the present thesis hypothesized that the family Naididae Ehrenberg, 1828 [currently includes formed Tubificidae (Erséus et al., 2008)] played a preponderant role in the settlement and evolution of myxosporeans in estuarine and marine habitats. In the same manner, the freshwater oligochaetes hosting the Raabeia type of Rocha et al. (2019c) and the Sphaeractinomyxon types reported here from the Minho upper estuary also belonged to the family Naididae. Despite a few members of the family Lumbriculidae Vejdovský, 1884 being hosts for actinosporean types in freshwater habitats, the broader usage of naidids in this environment agrees with the previous hypothesis and shows that this annelid family was also important in the establishment of myxosporeans in freshwater habitats. The factors determining what appears to be a successful co- evolutionary relationship between these organisms are unknown but it could be speculated to correlate primarily with the broad diversity and cosmopolitan nature of naidids. Further conjectures were difficult to perform given the limited body of information currently available for myxosporean-annelid interactions. The overall development of the different actinosporean types in their oligochaete hosts, followed the pattern commonly reported among actinosporeans, be it either in the coelomic cavity or intestinal epithelium (e.g. Hallett et al., 1998; Marcucci et al., 2009). Although it was not a specific aim of this thesis to study the sequential stages of actinosporean development, it should be mentioned that multinucleated schizogonic stages were never observed. Instead, the first developmental stages detected in infected oligochaetes were binucleated cells. This agrees with the findings of Morris and Freeman (2010) and Morris (2012), who refuted the widely accepted involvement of multinucleated schizogonic stages and uninucleated cells in

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Chapter XIII | General Discussion actinosporean development by suggesting their misinterpretation from co-infective microsporidian stages. Individual values of prevalence of infection were below 1% and, therefore, were in agreement with the values usually reported for other actinosporean types occurring in the wild (e.g. Xiao and Desser, 1998 a, b; Hallett et al., 1999; Xi et al., 2015; Rangel et al., 2016; Milanin et al., 2017). On the other hand, the values calculated for the overall prevalence of infection of the sphaeractinomyxon collective group in the estuaries analysed significantly differed amongst each other. Also, despite several actinosporean types having been found to parasitize the same oligochaete species (e.g. T. pseudogaster, T. insularis, Baltidrilus sp.), concomitant infections were never observed. Many similar cases can be found in the literature (e.g. El- Mansy et al., 1998; Xiao and Desser, 1998 a, b; Rosser et al., 2014; Rangel et al., 2016). Overall, these differences in prevalence of infection, was well as the absence of mixed infections in oligochaete species that appear to be highly permissive for actinosporean development, were hypothesized to reflect the complexity and variability of the biotic and abiotic factors that influence myxospore viability and dispersal, host encounter, invasion and proliferation (reviewed in Alexander et al., 2015).

References

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minibicornis, a myxozoan parasite of Pacific salmon. Journal of Parasitology 92, 742– 748. Bartholomew, J.L., Whipple, M.J., Stevens, D.G. and Fryer, J.L. (1997). The life cycle of Ceratomyxa shasta, a myxosporean parasite of salmonids, requires a freshwater polychaete as an alternate host. Journal of Parasitology 83, 859–868. Bartošová, P., Fiala, I. and Hypša, V. (2009). Concatenated SSU and LSU rDNA data confirm the main evolutionary trends within myxosporeans (Myxozoa: Myxosporea) and provide an effective tool for their molecular phylogenetics. Molecular Phylogenetics and Evolution 53, 81–93. Benajiba, M.H. and Marques, A. (1993). The alternation of actinomyxidian and myxosporidian sporal forms in the development of Myxidium giardi (parasite of Anguilla anguilla) through oligochaetes. Bulletin of the European Association of Fish Pathologists 13, 100–103. Brinkhurst, R., Kaushik, N.K. and Chua, K.E. (1972). Interspecific interactions and selective feeding by tubificid oligochaetes. Limnology and Oceanography 17, 122–133. Cardona, L. (2006). Habitat selection by grey mullets (Osteichthyes: Mugilidae) in Mediterranean estuaries: the role of salinity. Scientia Marina 70, 443–455. Carriero, M.M., Adriano, E.A., Silva, M.R., Ceccarelli, P.S. and Maia, A.A. (2013). Molecular phylogeny of the Myxobolus and Henneguya genera with several new South American species. PLoS One 8, e73713. Caullery, M. and Mesnil, F. (1904). Sur un type nouveau (Sphaeractinomyxon stolci n. g. n. sp.) d'Actinomyxidies, et son développement. Comptes Rendus des Seances de la Societe de Biologie et de Ses Filiales 56, 408‒410. Caullery, M. and Mesnil, F. (1905). Recherches sur les Actinomyxidies. I. Sphaeractinomyxon stolci. Archiv für Protistenkunde 6, 272–308. Easy, R.H., Johnson, S.C. and Cone, D.K. (2005). Morphological and molecular comparison of Myxobolus procerus (Kudo, 1934) and M. intramusculi n. sp. (Myxozoa) parasitising muscles of the trout-perch Percopsis omiscomaycus. Systematic Parasitology 61, 115‒ 122. Eiras, J.C. (2002). Synopsis of the species of the genus Henneguya Thélohan, 1892 (Myxozoa: Myxosporea: Myxobolidae). Systematic Parasitology 52, 43–54. Eiras, J.C. and Adriano, E.A. (2012). A checklist of new species of Henneguya Thélohan, 1892 (Myxozoa: Myxosporea: Myxobolidae) described between 2002 and 2012. Systematic Parasitology 83, 95–104. Eiras, J.C., Molnár, K. and Lu, Y.S. (2005). Synopsis of the species of Myxobolus Bütschli, 1882 (Myxozoa: Myxosporea: Myxobolidae). Systematic Parasitology 61, 1–46. Eiras, J.C., Zhang, J. and Molnár, K. (2014). Synopsis of the species of Myxobolus Bütschli,

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1882 (Myxozoa: Myxosporea: Myxobolidae) described between 2005 and 2013. Systematic Parasitology 88, 11–36. El-Mansy, A., Székely, C. and Molnár, K. (1998). Studies on the occurrence of actinosporean stages of fish myxosporeans in a fish farm of Hungary, with the description of triactinomyxon, raabeia, aurantiactinomyxon and neoactinomyxon types. Acta Veterinaria Hungarica 46, 259‒284. Erséus, C., Wetzel, M.J. and Gustavsson, L. (2008). ICZN rules – a farewell to Tubificidae (Annelida, Clitellata). Zootaxa 1744, 66‒68. Eschmeyer, W.N. and Fong, J.D. Catalog of fishes. Species by family/subfamily. (http://researcharchive.calacademy.org/research/ichthyology/catalog/SpeciesByFamil y.asp). Electronic version accessed 10 August 2018. Eszterbauer, E., Marton, S., Rácz, O.Z., Letenyei, M. and Molnár, K. (2006). Morphological and genetic differences among actinosporean stages of fish-parasitic myxosporeans (Myxozoa): difficulties of species identification. Systematic Parasitology 65, 97–114. Ferguson, J.A., Atkinson, S.D., Whipps, C.M. and Kent, M.L. (2008). Molecular and morphological analysis of Myxobolus spp. of salmonid fishes with the description of a new Myxobolus species. Journal of Parasitology 94, 1322–1334. Fiala, I. (2006). The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. International Journal of Parasitology 36, 1521–1534. Fiala, I. and Bartošová, P. (2010). History of myxozoan character evolution on the basis of rDNA and EF-2 data. BMC Evolutionary Biology 10, 228. Fiala, I., Hlavničková, M., Kodádková, A., Freeman, M.A., Bartošová-Sojková, P. and Atkinson, S.D. (2015). Evolutionary origin of Ceratonova shasta and phylogeny of the marine myxosporean lineage. Molecular Phylogenetics and Evolution 86, 75–89. Forró, B. and Eszterbauer, E. (2016). Correlation between host specificity and genetic diversity for the muscle-dwelling fish parasite Myxobolus pseudodispar: examples of myxozoan host-shift? Folia Parasitologica 63, 019. Freeman, M.A. and Kristmundsson, Á. (2018). Studies of Myxidium giardi Cépède, 1906 infections in Icelandic eels identifies a genetically diverse clade of myxosporeans that represents the Paramyxidium n. g. (Myxosporea: Myxidiidae). Parasites & Vectors 11, 551. Gunter, N.L. and Adlard, R.D. (2008). Bivalvulidan (Myxozoa: Myxosporea) parasites of damselfishes with description of twelve novel species from Australia's Great Barrier Reef. Parasitology 135, 1165–1178. Gunter, N.L. and Adlard, R.D. (2009). Seven new species of Ceratomyxa from the gallbladders of serranid fishes from the Great Barrier Reef, Australia. Systematic Parasitology 73, 1–11.

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Chapter XIII | General Discussion

Gunter, N.L., Whipps, C.M. and Adlard, R.D. (2009). Ceratomyxa (Myxozoa: Bivalvulida): robust taxon or genus of convenience? International Journal for Parasitology 39, 1395– 1405. Hallett, S.L., Atkinson, S.D., Erséus, C. and El-Matbouli, M. (2004). Molecular methods clarify morphometric variation in triactinomyxon spores (Myxozoa) released from different oligochaete hosts. Systematic Parasitology 57, 1‒14. Hallett, S.L., Erséus, C. and Lester, R.J. (1997). Actinosporea from Hong Kong marine oligochaeta. In Morton, B. (ed). Proceedings of the eight international marine biological workshop: the marine flora and fauna of Hong Kong and Southern China. Hong Kong: Hong Kong University Press, pp. 1‒7. Hallett, S.L., Erséus, C. and Lester, R.J. (1999). Actinosporeans (Myxozoa) from marine oligochaetes of the Great Barrier Reef. Systematic Parasitology 44, 49‒57. Hallett, S.L., Erséus, C., O’Donoghue, P.J. and Lester, R.J.G. (2001). Parasite fauna of australian marine oligochaetes. Memoirs of the Queensland Museum 46, 555‒576. Hallett, S.L. and Lester, R.J. (1999). Actinosporeans (Myxozoa) with four developing spores within a pansporocyst: Tetraspora discoidea n. g. n. sp. and Tetraspora rotundum n. sp. International Journal of Parasitology 29, 419‒427. Hallett, S.L., O´Donoghue, P.J. and Lester, R.J.G. (1998). Structure and development of a marine actinosporean Sphaeractinomyxon ersei n. sp. (Myxozoa). Journal of Eukaryot Microbiology 45, 142‒150. Heiniger, H. and Adlard, R.D. (2013). Molecular identification of cryptic species of Ceratomyxa Thélohan, 1892 (Myxosporea: Bivalvulida) including the description of eight novel species from apogonid fishes (Perciformes: Apogonidae) from Australian waters. Acta Parasitologica 58, 342−360. Hernando, J.A. and Soriguer, M.C. (1992). Biogeography of the freshwater fish of the Iberian Peninsula. Limnetica 8, 243–253. Holzer, A.S., Bartošová, P., Pecková, H., Tyml, T., Atkinson, S., Bartholomew, J., Sipos, D., Eszterbauer, E. and Dyková, I. (2013) ‘Who’s who’ in renal sphaerosporids (Bivalvulida: Myxozoa) from common carp, Prussian carp and goldfish—molecular identification of cryptic species, blood stages and new members of Sphaerospora sensu stricto. Parasitology 140:46–60 Holzer, A.S., Bartošová-Sojková, P., Born-Torrijos, A., Lovy, A., Hartigan, A. and Fiala, I. (2018). The joint evolution of the Myxozoa and their alternate hosts: a cnidarian recipe for success and vast biodiversity. Molecular Ecology 27, 1651–1666. Janiszewska, J. (1955). Actinomyxidia. Morphology, ecology, history of investigations, systematics, development. Acta Parasitologica Polonica 2, 405–437. ~ Janiszewska, J. (1957). Actinomyxidia II: new systematics, sexual cycle, description of new

Page | 376

Chapter XIII | General Discussion

genera and species. Zoologica Poloniae 8, 3–34. Jeuniaux, C. (2012). Nutrition and digestion. In Florkin, M. and Scheer, B.T. (eds). Chemical Zoology IV: Annelida, Echiura, and Sipuncula. New York and London: Academic Press, pp. 69‒92. Katharios, P., Garaffo, M., Sarter, K., Athanassopoulou, F. and Mylonas, C.C. (2007). A case of high mortality due to heavy infestation of Ceratomyxa diplodae in sharpsnout sea bream (Diplodus puntazzo) treated with reproductive steroids. Bulletin of the European Association of Fish Pathologists 27, 43–47. Kerans, B.L. and Zale, A.V. (2002). The ecology of Myxobolus cerebralis. American Fisheries Society Symposium 29, 145–166. Kodádková, A., Bartošová-Sojková, P., Holzer, A.S. and Fiala, I. (2015). Bipteria vetusta n. sp. - an old parasite in an old host: tracing the origin of myxosporean parasitism in vertebrates. International Journal for Parasitology 45, 269–276. Kottelat, M. and Freyhof, J. (2007). Handbook of European Freshwater Fishes. Publications Kottelat, Cornol and Freyhof, Berlin, p. 646. Kudo, R. (1933). A taxonomic consideration of Myxosporidia. Transactions of the American Microscopical Society 52, 195–216. Lemmon, J.C. and Kerans, B.L. (2001). Extraction of whirling disease myxospores from sediments using the plankton centrifuge and sodium hexametaphosphate. Intermountain Journal of Sciences 7, 57–62. Liu, Y., Whipps, C.M., Gu, Z.M. and Zeng, L.B. (2010). Myxobolus turpisrotundus (Myxosporea: Bivalvulida) spores with caudal appendages: investigating the validity of the genus Henneguya with morphological and molecular evidence. Parasitology Research 107, 699–706. Lom, J. and Dyková, I. (1995). New species of the genera Zschokkella and Ortholinea (Myxozoa) from the Southeast Asian teleost fish, Tetraodon fluviatilis. Folia Parasitologica 42, 161–168. Lom, J. and Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitologica 53, 1–36. Lubat, J., Radujkovic, B., Marques, A. and Bouix, G. (1989). Parasites des poissons marins du Montenegro: Myxosporidies. Acta Adriatica 30, 31−50. Marcucci, C., Caffara, M. and Goretti, E. (2009). Occurrence of actinosporean stages (Myxozoa) in the Nera River system (Umbria, central Italy). Parasitology Research 105, 1517–1530. Marques, A. (1984). Contribution à la connaissance des Actinomyxidies: ultrastructure, cycle biologique, systématique. Ph.D. Thesis, Université des Sciences et Techniques de Languedoc, Montepellier, France.

Page | 377

Chapter XIII | General Discussion

Milanin, T., Atkinson, S.D., Silva, M.R.M., Alves, R.G., Maia, A.A.M. and Adriano, E.A. (2017). Occurrence of two novel actinospore types (Cnidaria: Myxosporea) in Brazilian fish farms, and the creation of a novel actinospore collective group, Seisactinomyxon. Acta Parasitologica 62, 121–128. Molnár, K. and Eszterbauer, E. (2015). Specificity of infection sites in vertebrate hosts. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 295–313. Molnár, K., Eszterbauer, E., Székely, C., Dán, Á. and Harrach, B. (2002). Morphological and molecular biological studies on intramuscular Myxobolus spp. of cyprinid fish. Journal of Fish Diseases 25, 643–652. Morris, D.J. (2012). A new model for myxosporean (Myxozoa) development explains the endogenous budding phenomenon, the nature of cell within cell life stages and evolution of parasitism from a cnidarian ancestor. International Journal for Parasitology 42, 829–840. Morris, D.J. and Freeman, M.A. (2010). Hyperparasitism has wide-ranging implications for studies on the invertebrate phase of myxosporean (Myxozoa) life cycles. International Journal for Parasitology 40, 357–369. Negredo, C. and Mulcahy, M.F. (2001). Actinosporean infections in oligochaetes in a river system in southwest Ireland with descriptions of three new forms. Diseases of Aquatic Organisms 46, 67‒77. Okamura, B., Gruhl, A. and Bartholomew, J.L. (2015). An Introduction to myxozoan evolution, ecology and development. In Okamura, B., Gruhl, A. and Bartholomew, J.L. (eds). Myxozoan Evolution, Ecology and Development. Springer International Publishing, Switzerland, pp. 1–20. Patra, S., Bartošová-Sojková, P., Pecková, H., Fiala, I., Eszterbauer, E. and Holzer, A.S. (2018). Biodiversity and host-parasite cophylogeny of Sphaerospora (sensu stricto) (Cnidaria: Myxozoa). Parasites & Vectors 11, 347. Puytorac, P.D. (1963). L'ultrastructure des cnidocystes de l'Actinomyxidae: Sphaeractinomyxon amanieui sp. nov. Comptes Rendus de l'Académie des Sciences Paris 256, 1594–1596. Rangel, L.F., Castro, R., Rocha, S., Cech, G., Casal, G., Azevedo, C., Székely, C., Cavaleiro, F. and Santos, M.J. (2016). Description of new types of sphaeractinomyxon actinospores (Myxozoa: Myxosporea) from marine tubificid oligochaetes, with a discussion on the validity of the tetraspora and the endocapsa as actinospore collective group names. Parasitology Research 115, 2341–2351. Rangel, L.F., Rocha, S., Casal, G., Castro, R., Severino, R., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2017). Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Page | 378

Chapter XIII | General Discussion

(Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm. Journal of Fish Diseases 40, 243– 262. Rangel, L.F., Rocha, S., Castro, R., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2015). The life cycle of Ortholinea auratae (Myxozoa: Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete. Parasitology Research 114, 2671–2678. Rangel, L.F., Santos, M.J., Cech, G. and Székely, C. (2009). Morphology, molecular data, and development of Zschokkella mugilis (Myxosporea, Bivalvulida) in a polychaete alternate host, Nereis diversicolor. Journal of Parasitology 95, 561–569. Rigos, G., Grigorakis, K., Christophilogiannis, P., Nengas, I. and Alexis, M. (1997). Ceratomyxa spp. (Myxosporea) infection in cultured common dentex from Greece. Bulletin of the European Association of Fish Pathologists 17, 174−176. Rocha, S., Alves, Â., Antunes, C., Azevedo, C. and Casal, G. (2019a). Molecular data infers the involvement of a marine aurantiactinomyxon in the life cycle of the myxosporean parasite Paramyxidium giardi (Cnidaria, Myxozoa). Parasitology (In Press) Rocha, S., Alves, Â., Antunes, C., Fernandes, P., Azevedo, C. and Casal, G. (2019b). Characterization of sphaeractinomyxon types (Cnidaria, Myxozoa) from marine and freshwater oligochaetes in a Portuguese estuary, with the demise of the endocapsa collective group. Folia Parasitologica (In Press) Rocha, S., Alves, Â., Fernandes, P., Antunes, C., Azevedo, C. and Casal, G. (2019c). Description of a new actinosporean prompts union of the raabeia and echinactinomyxon collective groups (Cnidaria, Myxozoa), due to overlap in actinospore morphology. Diseases of Aquatic Organisms (In Press). Rocha, S., Azevedo, C., Oliveira, E., Alves, Â., Antunes, C., Rodrigues, P. and Casal, G. (2019d). Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular redescription of the cryptic species Myxobolus exiguus. Parasitology 146, 479‒496. Rocha, S., Casal, G., Alves, Â., Antunes, C., Rodrigues, P. and Azevedo, C. (2019e). Myxozoan (Cnidaria, Myxozoa) biodiversity in mullets (Teleostei, Mugilidae) unravels hyperdiversification of Myxobolus (Cnidaria, Myxosporea). Parasitology Research (In Press) Rocha, S., Casal, G., Garcia, P., Matos, E., Al-Quraishy, S. and Azevedo, C. (2014). Ultrastructure and phylogeny of the parasite Henneguya carolina sp. nov. (Myxozoa), from the marine fish Trachinotus carolinus in Brazil. Diseases of Aquatic Organisms 112, 139–148. Rocha, S., Rangel, L.F., Castro, R., Severino, R., Azevedo, C., Santos, M.J. and Casal, G.

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(2019f). The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis. Journal of Invertebrate Pathology 160, 33‒42. Rodriguez, P., Martinez-Madrid, M., Arrate, J.A. and Navarro, E. (2001). Selective feeding by the aquatic oligochaete Tubifex tubifex (Tubificidae, Clitellata). Hydrobiologia 463, 133–140. Rosser, T.G., Griffin, M.J., Quiniou, S.M., Greenway, T.E., Khoo, L.H., Wise, D.J. and Pote, L.M. (2014). Molecular and morphological characterization of myxozoan actinospore types from a commercial catfish pond in the Mississippi delta. Journal of Parasitology 100, 828–839. Roubal, F.R., Hallett, S.L. and Lester, R.J.G. (1997). First record of triactinomyxon actinosporean in marine oligochaete. Bulletin of the European Association of Fish Pathologists 17, 83–85. Shulman, S.S. (1959). New system of myxosporidia. Trudy Karelsk. fil. AN SSSR, 14, Voprosy parazitol. Karelii 33–47. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1993). Light and electron microscopical description of Ceratomyxa labracis n. sp. and a redescription of C. diplodae (Myxosporea: Bivalvulida) from wild and cultured Mediterranean Sea bass Dicentrarchus labrax (L.) (Teleostei: Serranidae). Systematic Parasitology 26, 215– 223. Torricelli, P., Tongiorgi, P. and Almansi, P. (1981). Migration of grey mullet fry into the Arno river: Seasonal appearance, daily activity, and feeding rhythms. Fisheries Research 1, 219–234. Tripathi, Y.R. (1948). Some new Myxosporidia from Plymouth with a proposed new classification of the order. Parasitology 39, 110–118. Wolf, K. and Markiw, M.E. (1984). Biology contravenes taxonomy in the Myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science 225, 1449‒ 1452. Xiao, C. and Desser, S.S. (1998a). Actinosporean stages of myxozoan parasites of oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of triactinomyxon and raabeia. Journal of Parasitology 84, 998–1009. Xiao, C. and Desser, S.S. (1998b). Actinosporean stages of myxozoan parasites of oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of echinactinomyxon, neoactinomyxum, aurantiactinomyxon, guyenotia, synactinomyxon and antonactinomyxon. Journal of Parasitology 84, 1010–1019. Xi, B.W., Zhou, Z.G., Xie, J., Pan, L.K., Yang, Y.L. and Ge, X.P. (2015). Morphological and

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molecular characterization of actinosporeans infecting oligochaete Branchiura sowerbyi from Chinese carp ponds. Diseases of Aquatic Organisms 114, 217‒228. Zhang, J.Y., Gu, Z.M., Kalavati, C., Eiras. J.C., Liu, Y., Guo, Q.Y. and Molnár, K. (2013). Synopsis of the species of Thelohanellus Kudo, 1933 (Myxozoa: Myxosporea: Bivalvulida). Systematic Parasitology 86, 235–256. Zhang, Z.Q. (2011). Animal biodiversity: an outline of higher-level classification and taxonomic richness. Zootaxa 3148, 7–12.

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Chapter XIV

Conclusion and Future Perspectives

Chapter XIV | Conclusion and Future Perspectives

Conclusion and Future Perspectives

Despite the rapid progress and considerable advances attained in recent decades, our knowledge of myxosporean biology, ecology and evolution remains fragmented. As new discoveries are made and the available information increases, namely through the usage of molecular tools, more questions arise about the evolutionary history and functional role of myxosporeans in aquatic and terrestrial ecosystems. In order to tackle these major issues, however, it is first necessary to resolve old problematics and increase the basis of diversity and life cycle connections currently available for this group of parasitic cnidarians. In this thesis, novel information was provided that enabled some insight into the biodiversity, life cycles, host-parasite interactions, phylogeny and evolution of myxosporeans. The biodiversity of this group known from Portuguese estuaries and from specific vertebrate and invertebrate host groups was revised and significantly increased through the description of new species and actinosporean stages. Taxonomic issues were addressed through the re- description of known species complexes, and the validity of previously established actinosporean collective groups was evaluated according to novel morphological and molecular data. Full life cycles were resolved through the establishment of newly discovered myxosporean/actinosporean associations, and an attempt was made to provide some background for myxosporean-annelid interactions. Evolutionary patterns, origin and trajectories were analysed for specific groups, either confirming or refuting previously reported evolutionary drivers of myxosporean radiation. Future aims and tasks can be devised based on the information acquired and provide interesting topics and perspectives for researchers working with myxosporeans. Overall, the sheer amount of myxosporean species and actinosporean stages reported during this thesis demonstrated that myxosporean biodiversity in Portuguese estuaries is clearly underrated. Therefore, a continued effort is required in carrying out myxozoan surveys in fish and annelids and should be extended to other estuaries and coastal regions. Some fish species and families were identified as being probable hosts for multiple myxosporean species and, therefore, should be targeted specifically. The addition of Ceratomyxa auratae to the already high species-richness of Ceratomyxa spp. known to infect gilthead seabream Sparus aurata Linnaeus, 1758, as well as other South European sparids, revealed the need to further explore myxosporean biodiversity in this host family. The gilthead seabream is the most important mariculture species in the Mediterranean Sea, representing an important source of income for Portuguese fisheries and aquaculture industries. Considering that several studies have analysed the myxosporean community infecting this fish species in different South European locations (e.g. Sitjà-Bobadilla et al., 1992, 1995; Diamant et al., 1994; Sitjà-Bobadilla and Alvarez-Pellitero, 1995, 2001; Bahri et

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Chapter XIV | Conclusion and Future Perspectives al., 1996; Alama-Bermejo et al., 2011), but never reported C. auratae amongst their findings, there is a clear need to replicate the myxozoan survey of S. aurata in different geographic localities, including Portuguese estuaries other than the Alvor. Regarding Ceratomyxa diplodae Lubat et al., 1989, future research should aim to provide molecular data for this species in previously reported sparid hosts, including the original host – the annular seabream Diplodus annularis (Linnaeus, 1758). Acquiring this information is crucial to either confirm or refute the cryptic nature of C. diplodae, given that the phylogenetic positioning of the sequence obtained here from infections in the moronid Dicentrarchus labrax, among sparid-infecting Ceratomyxa spp., suggests that host-switch occurred between members of the families Sparidae and Moronidae. Iberian endemic cypriniforms were also shown to be the potential hosts of a hidden biodiversity of freshwater myxosporeans, namely belonging to the family Myxobolidae. Thus, myxozoan research in this geographic region should be expanded to target a broader array of endemic fish species. The myxozoan survey of Luciobarbus bocagei (Steindachner, 1864), specifically, is necessary in order to validate or refute the identity of the myxobolids previously reported from this host, given the probability that they have been misidentified. The acquisition of knowledge pertaining to the myxosporean biodiversity parasitizing Iberian endemic species will further provide background for future studies aiming to recognize the impact that human-mediated host/parasite co-introductions have on native ecosystems. In this thesis, Myxobolus pseudodispar Gorbunova, 1936 was hypothesized to have entered the Iberian Peninsula, possibly multiple times, through means of its co-introduction with central European leuciscids, with processes of host-shift allowing it to acquire endemic fish as hosts. Nonetheless, in order to fully understand the origin and dispersion pattern of this myxobolid in the Iberian Peninsula, further research is required and should target other endemic leuciscids, as well as exotic species. Moreover, considering the recognized cryptic status of M. pseudodispar (see Forró and Eszterbauer, 2016), the acquisition of this knowledge may constitute an important steppingstone in our understanding of the processes that lead to myxosporean speciation and how they relate to distinct micro- and macroenvironmental pressures. Acknowledging the phylogenetic setting and life cycle connection established in this thesis between mugiliform-infecting Myxobolus spp. and sphaeractinomyxon types, mullets were identified as being the most probable vertebrate hosts for all members of this collective group. Molecular data is available for the 21 sphaeractinomyxon types reported from freshwater and marine oligochaetes in Portuguese estuaries, but only three types were inferred to close the life cycle of known mugiliform-infecting Myxobolus spp. As such, further myxozoan surveys of mullets are necessary and will most likely considerably increase the already high biodiversity of Myxobolus parasitizing this host group. Examination of thinlip grey mullet Chelon

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Chapter XIV | Conclusion and Future Perspectives ramada, specifically, is required in order to describe and taxonomically allocate the several myxospore morphotypes that were co-infective in the gills, spleen, kidney and digestive tube. In turn, the life cycle connection established between a member of the aurantiactinomyxon collective group and Paramyxidium giardia (Cépède, 1906) Freeman and Kristmundsson, 2018, confirmed the presence of this myxosporean species in Portuguese waters, most likely infecting its original host – the European eel Anguilla Anguilla (Linnaeus, 1758). Previous studies reported P. giardi developing in multiple organs of European eel in Portuguese estuaries, based solely on myxospore morphological traits (Ventura and Paperna, 1984; Azevedo et al., 1989; Saraiva and Chubb, 1989; Saraiva and Eiras, 1996; Hermida et al., 2008). Nonetheless, it was recently demonstrated that the sporogonic development of P. giardi is restricted to the kidney, and that the site of infection constitutes an important diagnostic feature for members of the genus Paramyxidium (Freeman and Kristmundsson, 2018). Consequently, future surveys of A. anguilla in Portuguese Rivers are required in order to ascertain the true biodiversity of Paramyxidium spp. parasitizing this fish host. Moreover, the molecular analysis of the myxospores of P. giardi in this geographic locality is required in order to evaluate the genetic diversity found here between the myxosporean and actinosporean counterparts of this species, which was suggested to represent the emergence of genotypically different subspecies due to geographic isolation. Research on this host may also help identify the myxospore counterpart of the Raabeia type described in this thesis, given that the latter clusters among species that mainly parasitize European eel, within the currently monophyletic Paramyxidium clade. Still regarding the myxosporean biodiversity found in the fish analysed, it should be highlighted that the occurrence of multiple species in a single host specimen evidences that histological studies are required in order to determine specific tissues of infection. The acquisition of this information is essential for both species identification and the evaluation of potential pathogenic effects and, more importantly, it is crucial for understanding the relevance of tissue tropism as a driver of myxosporean evolution and radiation. Considering this, future research in the field should aim to provide histological data for the new species described in this thesis, as well as for other potentially new species. Concerning the myxozoan surveys performed on annelids, an immediate task for the near future is the description of the synactinomyxon and other aurantiactinomyxon types that were found infecting freshwater and marine oligochaetes in the River Minho. Prior to this thesis, few studies had reported myxosporean development in marine oligochaetes (Hallett et al., 1997, 1998, 1999, 2001; Roubal et al., 1997; Hallett and Lester, 1999; Rangel et al., 2015, 2016, 2017). However, the richness of actinosporean stages found here parasitizing this annelid group in estuarine habitats shows that future research should target the invertebrate community of the lower estuaries, as the diversity of these ecosystems apparently leads to the

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Chapter XIV | Conclusion and Future Perspectives establishment of more varied myxosporean-annelid interactions. Taking into account the diversity of invertebrate hosts identified during this thesis, as well as in previous studies (Hallett et al., 1997, 1998, 1999, 2001; Roubal et al., 1997; Hallett and Lester, 1999; Rangel et al., 2015, 2016, 2017), the oligochaetes of the family Naididae were suggested to have played a preponderant role in the colonization and diversification of myxosporeans in estuarine habitats. Although this apparent successful parasite/host relationship could be hypothesized to correlate with the cosmopolitan nature and high availability of naidids in aquatic environments worldwide, the body of knowledge presently available for myxosporean-annelid interactions is patchy and allows for few conjectures that are mostly speculative. Thus, there is a pressing need for myxosporean research to start focusing on the biotic and abiotic factors that shape myxosporean-annelid interactions. These factors most likely influence not only host choice, but also spore viability, host encounter, invasion and proliferation, and were suggested in this thesis to be represented in the low values of prevalence of infection and lack of concomitant infections in highly permissive oligochaete hosts. In this context, spore functionality and its correlation with host ecology is another issue that requires investigation. Considering that oligochaetes and polychaetes are the definitive and most ancient hosts of myxosporeans (Holzer et al., 2018), the unravelling of the factors mediating myxosporean- annelid interactions is further expected to shed relevant insight into myxosporean evolution, as these will most likely constitute important drivers of species diversification. Obviously, the acquisition of this knowledge constitutes a future grand challenge for myxosporean researchers, given that it requires the characterization of host ecological niches, and the recognition of the evolution, composition, spatial distribution, ecology and susceptibility of estuarine annelid communities. Despite the co-evolutionary history of myxosporeans and its vertebrate hosts being obscured by the more ancient reciprocal adaptation of these parasites to their invertebrate hosts, the work performed in this thesis strengthened the contention that the acquisition of fish as secondary hosts was crucial for species diversification, given that it allowed the conquest of new habitats. Thus, future research might also aim to correlate the spatial distribution and evolutionary patterns of myxosporeans with the biological and ecological features of their vertebrate hosts. Mullets and the European eel, as demonstrated in this thesis, constitute good study subjects for this aim, as they are prone to acquire and carry myxosporean infection between different habitats and geographic locations, but others should be explored as well. In this thesis, the hyperdiversification of Myxobolus spp. in mullets was analysed from a phylogenetic point view and correlated with the ecological plasticity of mullets, as well as with the functionality of the spore morphotypes promoting dissemination and invasion. It would be interesting for future studies to analyse the immune responses that mediate myxosporean

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Chapter XIV | Conclusion and Future Perspectives elimination and subsequent disease resistance in these highly parasitized hosts. Although mullets have relatively low commercial value, they might constitute good study models for the future development of disease control measures. In general, the issues and tasks outlined here will, undoubtedly, be time-consuming and demand a continuous effort in increasing the biodiversity of myxosporean species and actinosporean stages described through well-supported microscopic and molecular methodologies. Nonetheless, the pursuit of these challenges will certainly be rewarding and continue to reveal the astonishing biological, ecological and evolutionary intricacies of myxosporeans, while opening new research windows for enthusiasts of parasitology, ecology, evolution, immunology, and other scientific areas.

References

Alama-Bermejo, G., Raga, J.A. and Holzer, A.S. (2011). Host-parasite relationship of Ceratomyxa puntazzi n. sp. (Myxozoa: Myxosporea) and sharpsnout seabream Diplodus puntazzo (Walbaum, 1792) from the Mediterranean with first data on ceratomyxid host specificity in sparids. Veterinary Parasitology 182, 181–192. Azevedo, C., Lom, J. and Corral, L. (1989). Ultrastructural aspects of Myxidium giardi (Myxozoa, Myxosporea), parasite of the European eel Anguilla anguilla. Diseases of Aquatic Organisms 6, 55‒61. Bahri, S., Hassine, O.K.B. and Marques, A. (1996). Henneguya sp. (Myxosporea, Bivalvulida) infecting the gills of wild gilthead sea bream Sparus aurata L., from the coast of Tunisia. Bulletin of the European Association of Fish Pathologists 16, 51–53. Diamant, A., Lom, J. and Dyková, I. (1994). Myxidium leei n. sp., a pathogenic myxosporean of cultured sea bream Sparus aurata. Diseases of Aquatic Organisms 20, 137–141. Forró, B. and Eszterbauer, E. (2016). Correlation between host specificity and genetic diversity for the muscle-dwelling fish parasite Myxobolus pseudodispar: examples of myxozoan host-shift? Folia Parasitologica 63, 019. Freeman, M.A. and Kristmundsson, Á. (2018). Studies of Myxidium giardi Cépède, 1906 infections in Icelandic eels identifies a genetically diverse clade of myxosporeans that represents the Paramyxidium n. g. (Myxosporea: Myxidiidae). Parasites & Vectors 11, 551. Hallett, S.L., Erséus, C. and Lester, R.J.G. (1997) Actinosporea from Hong Kong marine oligochaeta. In Morton, B. (ed). Proccedings of the eight international marine biological workshop: the marine flora and fauna of Hong Kong and Southern China. Hong Kong University Press, Hong Kong, pp. 1–7. Hallett, S.L., Erséus, C. and Lester, R.J.G. (1999). Actinosporeans (Myxozoa) from marine

Page | 389

Chapter XIV | Conclusion and Future Perspectives

oligochaetes of the Great Barrier Reef. Systematic Parasitology 44, 49–57. Hallett, S.L., Erséus, C., O’Donoghue, P.J. and Lester, R.J.G. (2001). Parasite fauna of australian marine oligochaetes. Memoirs of the Queensland Museum 46, 555‒576. Hallett, S.L. and Lester, R.J.G. (1999). Actinosporeans (Myxozoa) with four developing spores within a pansporocyst: Tetraspora discoidea n. g. n. sp. and Tetraspora rotundum n. sp. International Journal for Parasitology 29, 419–427. Hallett, S.L., O'Donoghue, P.J. and Lester, R.J.G. (1998). Structure and development of a marine actinosporean, Sphaeractinomyxon ersei n. sp. (Myxozoa). Journal of Eukaryotic Microbiology 45, 142–150. Hermida, M., Saraiva, A. and Cruz, C. (2008). Metazoan parasite community of a European eel (Anguilla anguilla) population from an estuary in Portugal. Bulletin of the European Association of Fish Pathologists 28, 35–40. Holzer, A.S., Bartošová-Sojková, P., Born-Torrijos, A., Lövy, A., Hartigan, A. and Fiala, I. (2018). The joint evolution of the Myxozoa and their alternate hosts: A cnidarian recipe for success and vast biodiversity. Molecular Ecology 27, 1651‒1666. Rangel, L.F., Castro, R., Rocha, S., Cech, G., Casal, G., Azevedo, C., Székely, C., Cavaleiro, F. and Santos, M.J. (2016). Description of new types of sphaeractinomyxon actinospores (Myxozoa: Myxosporea) from marine tubificid oligochaetes, with a discussion on the validity of the tetraspora and the endocapsa as actinospore collective group names. Parasitology Research 115, 2341‒2351. Rangel, L.F., Rocha, S., Casal, G., Castro, R., Severino, R., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2017). Life cycle inference and phylogeny of Ortholinea labracis n. sp. (Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm. Journal of Fish Diseases 40, 243‒ 262. Rangel, L.F., Rocha, S., Castro, R., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M.J. (2015). The life cycle of Ortholinea auratae (Myxozoa: Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete. Parasitology Research 114, 2671‒2678. Roubal, F.R., Hallett, S.L. and Lester, R.J.G. (1997). First record of triactinomyxon actinosporean in marine oligochaete. Bulletin of the European Association of Fish Pathologists 17, 83–85. Saraiva, A. and Chubb, J.C. (1989). Preliminary observations on the parasites of Anguilla anguilla (L.) from Portugal. Bulletin of the European Association of Fish Pathologists 9, 88–89. Saraiva, A. and Eiras, J.C. (1996). Parasite community of european eel Anguilla anguilla (L.) in the river Este, northern Portugal. Research and Reviews in Parasitology 56, 179–

Page | 390

Chapter XIV | Conclusion and Future Perspectives

183. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (1995). Light and electron microscopic description of Polysporoplasma n. g. (Myxosporea: Bivalvulida), Polysporoplasma sparis n. sp. from Sparus aurata (L.) and Polysporoplasma mugilis n. sp. from Liza aurata. Euroupean Journal of Protistology 31, 77–89. Sitjà-Bobadilla, A. and Alvarez-Pellitero, P. (2001). Leptotheca sparidarum n. sp. (Myxosporea: Bivalvulida), a parasite from cultured common dentex (Dentex dentex L.) and gilthead sea bream (Sparus aurata L.) (Teleostei: Sparidae). Journal of Eukaryotic Microbiology 48, 627–639. Sitjà-Bobadilla, A., Franco-Sierra, A. and Alvarez-Pellitero, P. (1992). Sphaerospora (Myxosporea: Bivalvulida) infection in cultured gilthead sea bream, Sparus aurata L.: a preliminary report. Journal of Fish Diseases 15, 339–343. Sitjà-Bobadilla, A., Palenzuela, O. and Alvarez-Pellitero, P. (1995). Ceratomyxa sparusauratin. sp. (Myxosporea: Bivalvulida), a new parasite from cultured gilthead seabream (Sparus aurata L.) (Teleostei: Sparidae): light and electron microscopic description. Journal of Eukaryotic Microbiology 42, 529–539. Ventura, M.T. and Paperna, I. (1984). Histopathology of Myxidium giardi Cépède, 1906 infection in European eels, Anguilla anguilla L., in Portugal. Aquaculture 43, 357–368.

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