Nota prévia Na elaboração desta dissertação, e nos termos do número 2 do Artigo 4º do Regulamento Geral dos Terceiros Ciclos de Estudos da Universidade do Porto e do Artigo 31º do D.L. 74/2006, de 24 de Março, com a nova redação introduzida pelo D.L. 230/2009, de 14 de Setembro, foi efetuado o aproveitamento total de um conjunto coerente de trabalhos de investigação já publicados ou submetidos para publicação em revistas internacionais indexadas e com arbitragem científica, os quais integram alguns dos capítulos da presente tese. Tendo em conta que os referidos trabalhos foram realizados no âmbito do Projeto DirdaMyx - Dinâmica de Infecção em Robalo e Dourada de Aquacultura por Myxozoa (FCT referência: PTDC/MAR/116838/2010) com a colaboração de outros autores, o candidato esclarece que, em todos eles, participou ativamente na sua conceção, na parte prática e obtenção de dados, na análise e discussão de resultados, bem como na escrita, estruturação de artigos, e ainda no processo de submissão para publicação a revistas da especialidade. Em todos os artigos que integram a tese Luis Rangel é primeiro autor. FCUP i Acknowledgements Acknowledgements The success of this work had the contribution of many people, and institutions, to which I address my thanks. In particular: To my supervisor Professor Maria João Santos (University of Porto, Faculty of Sciences, Portugal) that over the years has guided and advised me in my scientific career, being an valuable and permanent reference. To Dr. Csaba Székely, Dr. Cech Gabor and Hafiz (Hungarian Academy of Sciences, Budapest, Hungary) for their valuable collaboration and advice. To Ricardo Severino, for the huge effort in collecting fishes, worms and mud in the fish farm and for welcomed me in his laboratory. To Ricardo Castro, which has not spared efforts in the exhaustive sampling of annelids, and gave all the help that made this work a success. To Professor Carlos Azevedo, Professor Graça Casal, and Sónia Rocha (University of Porto, Abel Salazar Institute for the Biomedical Sciences, Portugal), for all the advice, help and collaboration, especially in molecular biology and electron microscopy. My thanks also to the technicians Elsa Oliveira and Ângela Alves. To my colleagues, Francisca Cavaleiro, Miguel Ramos, Joana Marques, Margarida Hermida, Joana Costa, not only for the companion and friendship, but also for the scientific collaboration. And to all others that directly or indirectly gave some contribution for the success of this work. Finally, I would like to thank my family for their unconditionally support, in good times and bad times. This research was partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT—Foundation for Science and Technology, under the project BPEst-C/MAR/LA0015/2013 and the project DIRDAMyx, reference FCOMP-01- 0124-FEDER-020726/FCT-PTDC/MAR/116838/2010. Partially supported by 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 FCUP ii Acknowledgements Centre of Marine and Environmental Research), supported by the Northern Regional Operational Programme (NORTE2020), through the European Regional Development Fund (ERDF). This research was supported by Programa Operacional Potencial Humano (POPH) - Quadro de Referência Estratégico Nacional (QREN), Fundo Social Europeu (FSE) and the Portuguese Ministério de Educação e Ciência through a PhD fellowship (Fundação para a Ciência e a Tecnologia, FCT - SFRH/BD/82237/2011). This work was developed at: FCUP iii Abstract Abstract Overfishing exerts an enormous pressure on marine resources and, consequently, it is urgent to develop alternative ways to meet the world's food requirements. One such alternative is fish farming which, in recent years, has experienced a great increase in production. However, fish farming can only succeed if it is economically viable. Amongst the problems that threaten the economic viability of the fish farming industry, disease and parasites are the most relevant. Thus, the study and development of the body of knowledge on fish diseases and parasites are crucial. Amongst the most frequent parasites in fish farms, with tendency to increase in the near future, are the myxozoans. These parasites can have very adverse effects and cause major losses in fish production. However, there is still a huge gap in the body of knowledge on this particular group of parasites, especially in what concerns the life cycle. This unsolved problem leads the main aims of the present thesis work, which includes the survey and elucidation of the life cycles of myxozoan parasites infecting farmed fishes with great economic value, moreover, the European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata), in Algarve, and the actinospores survey in the fish farm tanks and in wild environment, in Algarve and Aveiro Estuary. While conducting this work, two new species of myxosporeans were described: Ortholinea auratae n. sp., isolated from the European seabass, and Ortholinea labracis n. sp., isolated from the gilthead seabream, both from the Algarve fish farm. Both species were found infecting the urinary bladder and, less frequently, the kidneys of their hosts. The life cycles of these two species were elucidated. Actinosporean stages of Ortholinea auratae n. sp. developed in the intestinal epithelium of Limnodriloides agnes oligochaetes, producing triactinomyxon actinospores. Ortholinea labracis n. sp. developed in the intestinal epithelium of Tectidrilus sp. oligochaetes, also producing triactinomyxons actinospores. Both invertebrate hosts were collected from the mud of the fish farm tanks. The life cycle of Sphaerospora dicentrarchi, a systemic parasite of the European seabass, was also elucidated. This result was achieved after finding a new type of tetractinomyxon actinospore in the coelomic cavity of Capitella sp., a capitelid polychaete collected in European seabass farming tanks in the Algarve fish farm. While analysing the 18S ribosomal RNA gene sequences of both polychaete actinospore and FCUP iv Abstract fish myxospore, it was possible to determine a 99.9% similarity. Therefore, it is possible to infer that they represent the two S. dicentrarchi life cycle stages. Myxosporean surveyed in annelids from estuaries revealed new types of actinospores with implications for the taxonomy and differentiation of actinospore collective groups. From Alvor Estuary (Algarve), a new type of echinactinomyxon was described from the coelomic cavity of the capitelid polychaete Heteromastus filiformis. Previously, these types of actinospore were always described from freshwater oligochaetes, and its presence in marine polychaetes is here recorded for the first time. Also, the 18S ribosomal RNA gene sequence was found to exhibit 94% of similarity with marine Sphaeromyxa species. In phylogenetic terms, Sphaeromyxa species cluster within myxosporeans of the freshwater lineage. From Aveiro Estuary, 10 new types of sphaeractinomyxon were described from the coelomic cavity of two oligochaete hosts (L. agnes and Tubificoides pseudogaster). Morphological and development analysis raised questions about the validity of the endocapsa and tetraspora actinospore collective groups. Developmental stages from the invertebrate hosts were studied by ligth microscopy and transmission electron microscopy. The most significant result was the record of the internalization or engulfment of one cell by another in myxosporeans, leading to secondary cells origin, as it was previously demonstrated for the malacosporeans. Keywords: Parasites, Myxozoa, life cycles, actinospores, aquaculture, European seabass, Dicentrarchus labrax, gilthead seabream, Sparus aurata, Annelida, Ortholinea auratae, Ortholinea labracis, echinactinomyxon, triactinomyxon, sphaeractinomyxon, tetractinomyxon, Algarve, Aveiro Estuary, Portugal FCUP v Resumo Resumo O excessivo esforço de pesca tem exercido uma enorme pressão nos recursos marinhos e criado a necessidade de desenvolver outras formas alternativas para suprir as necessidades alimentares mundiais. Uma dessas alternativas é a piscicultura que, nos últimos anos, tem tido um grande crescimento. No entanto, a piscicultura só pode evoluir se for economicamente viável. De entre os inúmeros problemas que a piscicultura tem de enfrentar e resolver, as parasitoses são os que mais danos podem causar numa produção piscícola. Assim, é importante conhecer e estudar as possíveis parasitoses que podem afectar essa produtividade. Um grupo de parasitas muito frequente nas pisciculturas, e com tendência para aumentar a sua prevalência, são os parasitas Myxozoa. Estes parasitas podem ter efeitos muito nefastos e causar grandes prejuízos. No entanto, ainda existe um grande hiato no seu conhecimento, principalmente em relação ao seu ciclo de vida. É neste contexto que surge esta tese, que teve como objectivo, o recenseamento dos parasitas Myxozoa e o estudo do seu ciclo de vida, em peixes de cultura com grande importância económica, como é o caso do robalo (Dicentrarchus labrax) e da dourada (Sparus aurata), numa piscicultura do Algarve. E ainda, o recenseamento de actinosporos em anelídeos de tanques de cultura de peixes