Chapter 1. General Introduction

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 da piscicultura e em ambiente de estuário no Algarve e na Ria de Aveiro. No decurso do trabalho, foram descritas duas novas espécies de mixosporídeos. Uma espécie no robalo (Ortholinea auratae n. sp.) e outra na dourada (Ortholinea labracis n. sp.) na piscicultura do Algarve. Ambas as espécies infectam a bexiga urinária e, com menor prevalência, os rins. Os seus ciclos de vida foram desvendados. A fase actinosporea de Ortholinea auratae n. sp. desenvolve-se no epitélio intestinal de oligoquetas Limnodriloides agnes, produzindo actinosporos do tipo triactinomyxon. Ortholinea labracis n. sp. desenvolve-se no epitélio intestinal de oligoquetas Tectidrilus sp., produzindo igualmente triactinomyxons. Os hospedeiros invertebrados de ambas as espécies foram recolhidos no lodo dos tanques onde as duas espécies de peixe são cultivadas. Foi também finalmente determinado o ciclo de vida do parasita sistémico Sphaerospora dicentrarchi, do robalo. Este resultado foi possível após a descrição de um novo tipo de tetractinomyxon, na cavidade celómica de um poliqueta capitelídeo (Capitela sp.), recolhido em tanques de cultura do robalo na piscicultura do Algarve. FCUP vi Resumo

Por análise molecular, com base na sequenciação do gene 18S RNA ribossómico dos parasitas do poliqueta e do robalo, obteve-se uma semelhança de 99,9% entre as sequências, inferindo-se assim, o seu ciclo de vida. A amostragem de mixosporídeos, em anelídeos recolhidos em ambientes de estuário, também permitiu descobrir novos tipos de actinosporos, com implicações na taxonomia e na diferenciação de grupos colectivos de actinosporos. Numa espécie de poliquetas capitelídeos (Heteromastus filiformis), recolhidos no estuário do Alvor (Algarve), foi descrito um novo tipo de echinactinomyxon. Esta descoberta representa uma novidade, pois este tipo de actinosporo só estava descrito em oligoquetas dulciaquícolas. A análise da sua sequência 18S RNA ribossómico, mostra que este actinosporo tem 94% de semelhança com espécies de Sphaeromyxa, um mixosporídeo marinho que, no entanto, agrupa filogeneticamente com os parasitas da linhagem de água doce. Em duas espécies de oligoquetas (L. agnes e Tubificoides pseudogaster), recolhidos na Ria de Aveiro, foram descritos 10 tipos novos de sphaeractinomyxon, todos eles da cavidade celómica. A análise da sua morfologia e desenvolvimento levanta questões sobre a validade dos tipos de actinosporos tetraspora e endocapsa. Neste trabalho foram analisados e estudados os estádios de desenvolvimento dos actinosporos nos seus hospedeiros invertebrados, quer ao nível da microscopia optica, quer ao nível da ultraestrutura. Um dos resultados mais relevantes é a constatação da existência, nos mixosporídeos, de um processo de internalização de células, durante a formação de células secundárias, como já foi demonstrado para os malacosporídeos.

Palavras-chave: Parasitas, Myxozoa, ciclos de vida, actinosporos, piscicultura, robalo, Dicentrarchus labrax, dourada, Sparus aurata, Annelida, Ortholinea auratae, Ortholinea labracis, echinactinomyxon, triactinomyxon, sphaeractinomyxon, tetractinomyxon, Algarve, Ria de Aveiro, Portugal

FCUP vii Scientific Papers

Scientific Papers

This thesis includes eight scientific papers, already published, in publication process or accepted for publication, in international journals (ISI) and concerning part of the results obtained during the experimental work.

1) Rangel, L. F., Gibson, D. I. and Santos, M. J. (2014). Synopsis of the species of the genus Myxobilatus Davis, 1944 (Myxozoa: Myxosporea: Myxobilatidae). Systematic Parasitology 87, 187–198. doi:10.1007/s11230-013-9467-y.

2) 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. doi:10.1645/GE-3005.1.

3) 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. doi:10.1007/s00436-014-4008-4.

4) 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. doi:10.1007/s00436-015-4472-5.

5) 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 (In Press). doi:10.1111/jfd.12508.

FCUP viii Scientific Papers

6) Rangel, L. F., Castro, R., Rocha, S., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M. J. (2016). 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 (In Press). doi: 10.1017/S0031182016000512.

7) Rangel, L. F., Rocha, S., Castro, R., Severino, R., Casal, G. and Santos, M. J. (2015). A new type of Echinactinomyxon (Myxozoa), infecting a marine polychaete, Heteromastus filiformis (Polychaeta: Capitellidae) in the Alvor Estuary (Portugal). Microscopy and Microanalysis 21 (Suppl. s5), 85–86. doi:10.1017/ S1431927615014233.

8) 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 (In Press). doi: 10.1007/s00436-016-4983-8.

FCUP ix Table of Contents

Table of Contents

Acknowledgements ...... i

Abstract ...... iii

Resumo ...... v

Scientific Papers ...... vii

Table of Contents ...... ix

Index of Tables ...... xiii

Index of Figures ...... xvii

Chapter 1 ...... 1

General Introduction ...... 1

1.1. Myxozoan parasites ...... 3

1.2. Historical background of Myxozoa ...... 5

1.3. Myxosporean life cycles ...... 6

1.4. Myxosporean development ...... 9

1.5. Fish hosts and myxosporean parasites ...... 12

1.6. Study Aims...... 18

Chapter 2 ...... 19

Synopsis of the species of the genus Myxobilatus Davis, 1944 (Myxozoa: Myxosporea: Myxobilatidae)...... 19

2.1. Abstract ...... 21

2.2. Introduction ...... 23

2.3. Materials and methods ...... 24

2.4. Results ...... 25

2.5. Final comments ...... 36

2.6. Acknowledgements ...... 37 FCUP x Table of Contents

Chapter 3 ...... 39

Ultrastructural aspects of Ellipsomyxa mugilis (Myxozoa: Ceratomyxidae) spores and developmental stages in Nereis diversicolor (Polychaeta: Nereidae) ...... 39

3.1. Abstract ...... 41

3.2. Introduction ...... 43

3.3. Material and methods ...... 43

3.4. Results ...... 44

3.5. Discussion ...... 47

3.6. Acknowledgments ...... 52

Chapter 4 ...... 53

Ortholinea auratae n. sp. (Myxozoa, Ortholineidae) infecting the urinary bladder of the gilthead seabream Sparus aurata (Teleostei, Sparidae), in a Portuguese fish farm .... 53

4.1. Abstract ...... 55

4.2. Introduction ...... 57

4.3. Material and methods ...... 58

4.4. Results ...... 61

4.5. Discussion ...... 66

4.6. Acknowledgments ...... 70

Chapter 5 ...... 71

The life cycle of Ortholinea auratae (Myxozoa: Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete ...... 71

5.1. Abstract ...... 73

5.2. Introduction ...... 75

5.3. Material and methods ...... 76

5.4. Results ...... 77

5.5. Discussion ...... 81

5.6. Acknowledgments ...... 84 FCUP xi Table of Contents

Chapter 6 ...... 85

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 ...... 85

6.1. Abstract ...... 87

6.2. Introduction ...... 89

6.3. Material and methods ...... 91

6.4. Results ...... 94

6.5. Discussion ...... 106

6.6. Acknowledgments ...... 112

Chapter 7 ...... 113

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 ...... 113

7.1. Abstract ...... 115

7.2. Introduction ...... 117

7.3. Material and methods ...... 118

7.4. Results ...... 119

7.5. Discussion ...... 122

7.6. Acknowledgments ...... 126

Chapter 8 ...... 127

A new type of Echinactinomyxon (Myxozoa), infecting a marine polychaete, Heteromastus filiformis (Polychaeta: Capitellidae) in the Alvor Estuary (Portugal) .... 127

8.1. Abstract ...... 129

8.2. Introduction ...... 131

8.3. Material and methods ...... 131

8.4. Results ...... 131

8.5. Discussion ...... 133 FCUP xii Table of Contents

8.6. Acknowledgments ...... 134

Chapter 9 ...... 135

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 ...... 135

9.1. Abstract ...... 137

9.2. Introduction ...... 139

9.3. Material and methods ...... 140

9.4. Results ...... 142

9.5. Discussion ...... 153

9.6. Acknowledgments ...... 156

Chapter 10 ...... 157

Concluding Remarks...... 157

10.1. Final notes ...... 159

10.2. Future Research ...... 163

References ...... 165

FCUP xiii Index of Tables

Index of Tables

Chapter 1

General introduction

Table 1.1 – Current taxonomy of Myxozoa according to Fiala et al. (2015)...... 4

Table 1.2 – Actinospore collective groups and the number of life cycles elucidated for each group...... 7

Chapter 2

Synopsis of the species of the genus Myxobilatus Davis, 1944 (Myxozoa: Myxosporea: Myxobilatidae)

Table 2.1 – Metrical data from the spores of Myxobilatus spp...... 27

Chapter 4

Ortholinea auratae n. sp. (Myxozoa, Ortholineidae) infecting the urinary bladder of the gilthead seabream Sparus aurata (Teleostei, Sparidae), in a Portuguese fish farm

Table 4.1 – Primers used for PCR and sequencing...... 59

Table 4.2 – Ortholinea spp. characterized by the presence of surface ridges and described from the excretory system of freshwater and marine fish. SL: spore length; SW: spore width; ST: spore thickness; PCL: polar capsule length; PCW: polar capsule width; nFC: number of polar filament coils. Measurements are given in µm...... 69

Chapter 5

The life cycle of Ortholinea auratae (Myxozoa: Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete

Table 5.1 Primers used for DNA amplification and sequencing...... 77 FCUP xiv Index of Tables

Table 5.2 – Morphological comparison of the triactinospores of Ortholinea auratae to all other described marine triactinospores (the mean±SD with the range in parentheses; in μm)...... 82

Chapter 6

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

Table 6.1 – Polymerase chain reaction primers used for the amplification and sequencing of the SSU rRNA gene of Ortholinea labracis n. sp. and the 16S gene of the oligochaete host...... 93

Table 6.2 – Ortholinea spp. characterized by the presence of surface ridges and described from the excretory system of marine fish. PC: polar capsule; SL: spore length; SW: spore width; ST: spore thickness; PCL: polar capsule length; PCW: polar capsule width; PFC: number of polar filament coils. Measurements are given in µm...... 104

Table 6.3 – Morphological comparison between the triactinospores linked to Ortholinea labracis n. sp. and all other described marine triactinospores (the mean±SD with the range in parentheses; in μm)...... 105

Chapter 7

Table 7.1 – Primers used for DNA amplification and sequencing the host Capitella sp. and the myxozoa Sphaerospora dicentrarchi...... 119

FCUP xv Index of Tables

Chapter 9

Table 9.1 – Primers used in actinospores and oligochaete DNA amplification and sequencing...... 141

Table 9.2 – Spore morphology of sphaeractinomyxon types from Limnodriloides agnes (types 1–7) and Tubificoides pseudogaster (types 8–10)...... 149

Table 9.2 – Pairwise genetic differences between the 18S rDNA sequences of the ten new sphaeractinomyxon types...... 151

FCUP xvi Index of Tables

FCUP xvii Index of Figures

Index of Figures

Chapter 1

General introduction

Fig. 1.1 – Some examples of actinospores. Redrawn from the original illustrations. a) Tetractinomyxon of Ellipsomyxa mugilis (Rangel et al., 2009). b) Neoactinomyxon (Negredo and Mulcahy, 2001). c) Echinactinomyxon (Negredo and Mulcahy, 2001). d) Synactinomyxon (Özer et al., 2002). e) Siedleckiella (Székely et al., 2003). f) Aurantiactinomyxon (Negredo and Mulcahy, 2001). g) Triactinomyxon of Myxobolus rotundus (Székely et al., 2009). h) Unicapsulactinomyxon (Rangel et al., 2011). Spores not to scale...... 8

Fig. 1.2 – Ellipsomyxa mugilis life cycle representation in the polychaete Hediste diversicolor (adapted from Rangel et al., 2009). a) Myxospores of E. mugilis. b) Binucleated cells in H. diversicolor coelom. c) Tetranucleated cell in plasmotomy. d) Early pansporocyst with two enveloping cells and two internal cells. e) Pansporocyst with four internal cells (2α and 2β). f) Pansporocyst with 10 internal cells (8α and 2β). g) Pansporocyst with 16 internal cell. h) Pansporocyst with eight sporoblasts resulting from the two zygote mitotic divisions. i) Pansporocyst with eight mature spores. k) Free mature actinospores. b–g) Gametogony phase, h–j) Sporogonic phase...... 9

Fig. 1.3 – Life cycle and development of myxosporeans (adapted from Kent et al., 2001). 1-16: Myxosporean development in the fish hosts. 17-30: Actinosporean development in the annelid hosts. 1: Actinospore infects the fish host. 2: Sporoplasm secondary cell divides by endogeny. 3-13: Presporogonic or extrasporogonic vegetative replication. 14-16: Sporulation and formation of myxospores. 17: Actinospore infect he annelid host. 18-20: Schizogony stages. The final binucleated cells with an α and β nuclei. 21-26: Gametogony stages. Internal cells undergo three mitotic and one meiotic division. 24-25: Gametes fuse resulting in eight zygotes. 27-29: Sporogony stages. Mature spores are released. 30: Mature actinospore enter in contact with a verebrate host...... 10

Fig. 1.4 – Freshwater myxosporean development model proposed by Morris (adapted from Morris, 2012). 1: Infection of oligochaete by myxospore sporoplasm. 2: FCUP xviii Index of Figures

Proliferation of binucleated cells. 3-4: Two binucleated cells fuse and form a tetranucleated stage. Fertilisation. 5: Tetranucleate cell plasmotomy forming four diploid cells. 6: Pansporocyst with two internal cells, one somatic cell (α) and one gametic cell (β). 7-8: The α and β cells divide originating eight α cels and eight β cells. The β cells undergo meiosis, releasing 16 polar bodies. 9: Diplod somatic cells (α) attaches to germ cells (β). 10-11: Somatic diploid cells divide and surround the haploid germ cell, forming the sporoblast. 12: Sporogonic and germ cells divide by mitosis. 13: Sporogonic cells rearrange to form sporoplasmogenic cells (grey cells) surrounding germ cells, and valvocapsulogenic cells (clear cells). 14: Sporoplasmogenic cells fuse and form the sporoplasm. 15-17: Valvocapsulogenic cells divide producing three valvogenic and three capsulogenic cells. 18-19: Germ cells divide within sporoplasm. 20: mature actinospore. Black spots=diploid nuclei; white spots=haploid nuclei...... 12

Fig. 1.5 – Myxosporean parasites of European seabass (Dicentrarchus labrax). a) Myxospores of Sphaerospora dicentrarchi in the intestinal epithelium. b) Myxospores of Sphaerospora testicularis in the gonad c) Myxospore of Ceratomyxa diplodae free in the bile. d) Myxospore of Ceratomyxa labracis free in the bile. Scale bar = 10 µm. ... 13

Fig. 1.6 – Myxosporean parasites of gilthead seabream (Sparus aurata). a) Myxospore of Sphaerospora sparis in the kidney. b) Myxospore of Zschokkella auratis free in the bile. c) Myxospores of Ceratomyxa auratae free in the bile. Scale bar = 10 µm...... 16

Chapter 2

Synopsis of the species of the genus Myxobilatus Davis, 1944 (Myxozoa: Myxosporea: Myxobilatidae)

Fig. 2.1 – Line drawings of spores of Myxobilatus spp., redrawn from the original illustrations. All scale-bars: 10 µm. 1, M. accessobranchialis; 2, M. anguillaris; 3, M. anteronippus; 4, M. asymmetricus; 5, M. baicalensis [a, original description in Dogiel and Bogolepova (1957); b, redescription from Zaika, 1964 in Shulman (1966)]; 6, M. carassii; 7, M. caudalis; 8, M. cheni; 9, M. convexus; 10, M. cotti; 11, M. fossilis; 12, M. fragilicaudus; 13, M. gasterostei; 14, M. gobii [a, from Evlanov (1981); b, from Lom (1986)]; 15, M. hemiculteri; 16, M. legeri [a, from Cépède (1905); b, from Shulman (1962)]; 17, M. mastacembeli; 18, M. medius [a, from Thélohan (1892); b, from Shulman (1962)]; 19, M. mictosporus [a, from Kudo (1920); b, from Davis (1944)] .... 31

FCUP xix Index of Figures

Fig. 2.2 – Line drawings of spores of Myxobilatus spp., redrawn from the original illustrations. All scale-bars: 10 µm. 20, M. minutus; 21, M. nostalgicus; 22, M. notopterus; 23, M. noturi; 24, M. odontamblyopusi; 25, M. ohioensis; 26, M. paragasterostei; 27, M. platessae; 28, M. polymorphus; 29, M. pseudorasborae; 30, M. rupestris; 31, M. schulmani; 32, M. semotilii; 33, M. sichuanensis; 34, M. sinipercae; 35, M. synodontis; 36, M. varicorhini; 37, M. wisconsinensis; 38, M. yukonensis; 39, M. yunnanensis ...... 35

Chapter 3

Ultrastructural aspects of Ellipsomyxa mugilis (Myxozoa: Ceratomyxidae) spores and developmental stages in Nereis diversicolor (Polychaeta: Nereidae)

Fig. 3.1 – Early and gametogony developmental stages of Ellipsomyxa mugilis. a) Tetranucleated cell showing three nuclei in this level. Dense bodies and vacuoles are also present. b) Four-cell stage preceding the formation of the pansporocyst. The cells (asterisks) are joined together by desmosomelike junctions (arrows). c) Formation of the pansporocyst. Two cells (asterisks) are enveloping two internal cells (only one internal cell is visible in this level). d) Initial stage of gametogony. The two enveloping cells (asterisks) form the pansporocyst wall, enclosing the generative cells. Abbreviations: Db, Dense body; Mi, mitochondrion N, nucleus; Nc, nucleolus; Va, vacuole. Arrowheads, rough endoplasmic reticulum. All scale bars = 2 mm...... 46

Fig. 3.2 – Sporogony developmental stages of Ellipsomyxa mugilis. a) Longitudinal transverse section of a pansporocyst constituted by two enveloping cells, connected by desmosome-like junctions (arrows), showing four sporoblasts in the initial stages of sporogony. b) Magnification of a detail of Fig. 2a showing two sporoblasts. In the bottom sporoblast, one central cell is enveloping the other, which will became the first secondary cell in the future spore’s sporoplasm cell. In the top sporoblast, there is only one central cell, already with a secondary cell inside. c) A sporoblast with two central cells in which one have almost enveloped the other. d) Two capsulogenic cells surrounded by two valvogenic cells (asterisks), showing the capsular primordium (arrows) and two large lipid droplets. e) A capsulogenic cell showing a capsular primordium and the external tube (arrow) in cross section. f) A spore showing two secondary cells and one somatic nucleus of the sporoplasm cell. The enveloping valvogenic cells are present (arrows). Abbreviations: C1–C2, Central cells in sporoblast; FCUP xx Index of Figures

CP, capsular primordium; L, lipid droplets; N, nucleus; P, primary cell; Pa, pansporocyst cell; S, secondary cell; Sp, sporoplasm cell. All scale bars = 2 mm. .... 48

Fig. 3.3 – Sporogony developmental stages of Ellipsomyxa mugilis. a) Cross section of a spore at the sporoplasm cell level showing three valvogenic cells connected by three cell junctions (arrows). b) Cross section of three capsulogenic cells with a straight-line boundary between them. Two polar capsules are visible showing the start of the polar filament (arrows) coiling inside the capsules. The cytoplasm shows signs of lysis with lighter areas (double asterisks). c) Longitudinal section of two capsulogenic cell. This picture also shows a sinuous curve boundary between the capsulogenic cells. d) Oblique spore showing a cellular junction in the valvogenic cells (arrowhead) and a space (asterisk) between the polar capsule wall and the capsulogenic cell cytoplasm. The polar filaments (arrows) are already coiled inside the polar capsule. The cytoplasm of the capsulogenic cell shows signs of lysis with lighter areas (double asterisks). e) The longitudinal section of polar capsule of a mature spore showing the polar filament with eight coils (arrows) and the core zone filled with lipid droplets. This picture also shows a space (asterisk) between the polar capsule wall and the capsulogenic cell cytoplasm. f). Longitudinal section of a spore showing pseudopodia-like projections (arrows) of the posterior part of the sporoplasm cell connected to the valvogenic cell by a cell junction. Abbreviations: CC, capsulogenic cell; L, lipid droplets; N, nucleus; Pa, pansporocyst cell; PC, polar capsule; S, secondary cell; Sp, sporoplasm cell. All scale bars = 2 mm...... 49

Fig. 3.4 – Schematic drawing of a polar capsule of Ellipsomyxa mugilis actinospore and surrounding structure organization...... 50

Chapter 4

Ortholinea auratae n. sp. (Myxozoa, Ortholineidae) infecting the urinary bladder of the gilthead seabream Sparus aurata (Teleostei, Sparidae), in a Portuguese fish farm

Fig. 4.1 – Plasmodia and spores of Ortholinea auratae n. sp. from the urinary bladder of Sparus aurata as observed using the differential interference contrast optics. a) Cluster of plasmodia. b) Young plasmodium displaying a highly irregular cellular membrane, due to the presence of numerous peripheral projections. c) Mature disporic plasmodium containing a disporic pansporoblast. d) Mature polysporic plasmodium FCUP xxi Index of Figures containing many developing spores. e) Mature spores in valvular view and one in sutural view (arrow). f) Mature spore in valvular view, displaying a visible intercapsular process...... 62

Fig. 4.2 – Light and transmission electron micrographs of Ortholinea auratae n. sp. from the urinary bladder of Sparus auratus. a) Semi-thin section of plasmodia (Pl) containing different sporogenic stages of development and displaying highly irregular cellular membranes (arrows). Notice the sporoblast (Sb) contained within an immature plasmodium as well as the developing spores (S) within more mature plasmodia. b) Ultrathin section of a young plasmodium (Pl) displaying vegetative nuclei (N) and numerous mitochondria (mt) as well as a highly irregular cellular membrane due to the presence of peripheral projections (arrows). Notice the glycocalyx-like sheet (arrowheads) covering the cellular membrane and peripheral projections. c) Detailed aspect of the periphery of a plasmodium (Pl) evidencing the glycocalyx-like sheet covering its cellular membrane (arrowheads)...... 63

Fig. 4.3 – Transmission electron micrographs of Ortholinea auratae n. sp. from the urinary bladder of Sparus auratus. a) Plasmodium displaying different sporogenic stages of development, namely, several generative cells (GC), a developing sporoblast (Sb) containing five nuclei, and a mature spore (S) as well as a vegetative nucleus (N) and numerous mitochondria (mt). b) Early sporoblast formed by an outer cell, the pericyte (asterisk), surrounding a sporogenic cell (SC) with a distinct nucleus (N). c) Mature plasmodium showing two spores contained within a disporous pansporoblast (asterisk). Notice the polar capsules (PC) in longitudinal and transverse section as well as the sporoplasm (Sp) containing several sporoplasmossomes (Sps). d) The spore’s wall (Wa) was comprised by two shell valves uniting along a straight suture line (arrow) and enveloped in a glycocalyx-like sheet (arrowheads). e) Detailed aspect of the glycocalyx-like sheet (arrowheads) enveloping the spore (S) wall. Notice the cellular membrane of the plasmodium (Pl) also covered by a glycocalyx-like sheet (arrows). f) Transverse section of a polar capsule, displaying its double-layer wall (arrowheads) containing a heterogeneous matrix (asterisk) and an electron-dense polar filament (PF)...... 64

Fig. 4.4 – Scanning electron micrographs of Ortholinea auratae n. sp. from the urinary bladder of Sparus auratus. a) Spore in frontal valvular view, allowing recognition of the surface pattern formed by the ridges. b) Spore in slightly oblique sutural view, evidencing the suture line (arrowheads) and the organization of the surface ridges. . 65 FCUP xxii Index of Figures

Fig. 4.5 – Schematic drawing of the spore of Ortholinea auratae n. sp...... 65

Fig. 4.6 – Maximum parsimony tree of the SSU rDNA sequence of Ortholinea auratae n. sp. and other selected myxozoan species. The numbers on the branches are bootstrap confidence levels on 100 replicates for MP and 500 replicates for NJ/ML trees. There were a total of 896 positions in the final dataset. GenBank accession numbers are in parentheses after the species name; scale is given under the tree. .. 68

Chapter 5

The life cycle of Ortholinea auratae (Myxozoa: Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete

Fig. 5.1 – Light microscopy photographs of the parasitic development of Ortholinea auratae infecting the intestinal epithelium of Limnodriloides agnes. a) Fresh triactinospore. b) Triactinospore showing its polar capsules (PC) and sporoplasm (Sp). c) Free sporoplasmic secondary cells (SSC), as a result of the polar filaments (PF) extrusion from the polar capsules (PC). d) Semithin cut of a heavily infected oligochaete, showing several pansporocysts (asterisks) developing in the intestinal epithelium...... 79

Fig. 5.2 – Transmission electron micrographs of the parasitic development of Ortholinea auratae infecting the intestinal epithelium of Limnodriloides agnes. a) Gametogonic stage: a pansporocyst formed by two enveloping cells (Pa) united by cellular junctions (arrows) and surrounding four generative cells (asterisk) rich in mitochondria (Mt). The cytoplasm of the enveloping cells shows mitochondria and large vesicles (Vs). b) Sporogonic stage: pansporocyst showing three developing spores. The three capsulogenic cells (CC) appear totally or partially involved by the three valvogenic cells (asterisk). The latter are united by cell junctions (arrowheads). The sporoplasmogenic cell (Sp), showing a secondary cell (SC) and its nucleous (N), is connected to the set of valvogenic cells through a small apical zone (arrows). c) Sporoblasts displaying three capsulogenic cells (CC) with its capsular primordia (PCP) not yet totally involved by the valvogenic cells; see the cellular junctions (arrowheads) and the terminal ends (arrows). The sporoplasmogenic cell (Sp) is still outside the triactinospore involucre. d) Cross section of the apical part of a developing triactinospore showing the three capsulogenic cells (CC) containing numerous FCUP xxiii Index of Figures mitochondria (Mt) in their cytoplasm. Two capsulogenic cells displaying the polar filament coiled within the maturing body of the polar capsules, while the third still displays the external tube (ET) that is precursor to the polar filament. These cells are involved by the valvogenic cells, which appear united by cellular junctions (arrows). e) Detailed aspect of a capsulogenic cell showing its cytoplasm containing large mitochondria (Mt) and an almost matured polar capsule (PC) presenting its polar filament coiled within. f) Detail of the sporoplasm (Sp) displaying numerous secondary cells (SC) and mitochondria (Mt)...... 80

Fig. 5.3 – Schematic drawing of the spores and life cycle of Ortholinea auratae. a) Triactinospore. Scale bar=100 μm. b) Myxospore in valvular and sutural view. Scale bar=10 μm. c) Schematic illustration of the life cycle...... 81

Chapter 6

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

Fig. 6.1 – Plasmodia and spores of Ortholinea labracis n. sp. from the urinary bladder of Dicentrarchus labrax, as observed using the differential interference contrast microscopy. a) Cluster of young and mature plasmodia, highly polymorphic. b) Round mature plasmodium displaying smooth cellular membrane. c) Elongated plasmodia containing developing myxospores at one end, which is larger and extends into numerous peripheral projections (arrow). d) Mature disporic plasmodium containing a disporous pansporoblast. e) Round mature plasmodium displaying numerous peripheral projections at one portion of the cellular membrane (arrows). f) Mature myxospores in valvular view and one in sutural view (arrow)...... 96

Fig. 6.2 – Transmission electron micrographs of Ortholinea labracis n. sp. from the urinary bladder of Dicentrarchus labrax. a) Young plasmodium displaying vegetative nuclei (N) and numerous mitochondria (Mt), vesicles (V) and lipidic globules (Li). Notice the irregularity of the cellular membrane due to the presence of peripheral projections (arrow). b) Round plasmodium containing different stages of the sporogenic development, namely a generative cell (GC), developing sporoblasts (Sb), and mature myxospores (S). Notice the smooth cellular membrane (arrows). c) Elongated plamodium displaying a developing disporoblast (*). Notice the surface ridges on the FCUP xxiv Index of Figures myxospores’ valves (arrows). d) Sporoblast showing its almost fully developed polar capsules (PC), and one of the two sporoplasmic nuclei (N). e) Longitudinal section of a polar capsule showing its apex (arrowhead) located near the suture line. f) Oblique transverse section of the two polar capsules displaying their double-layered wall (arrowheads) containing a heterogeneous matrix (*), and the polar filament (PF) coiling within. g) Tangencial section of the anterior pole of a myxospore allowing recognition of one of the two extrusion pores (arrow), located close to the suture line (arrowheads). h) Ultrastructural detail of the two valves united along a straight suture line (arrowheads). Notice the surface ridges ornamenting the valves...... 97

Fig. 6.3 – Light microscopy photographs of the parasitic development of Ortholinea labracis n. sp. infecting the intestinal epithelium of the oligochaete host. a) Binucleated cell. b) Initial pansporocyst of the gametogamy stage, with two involucre cells and two inner cells. c) Pansporocyst with four inner cells. d) Pansporocyst with 10 inner cells. e) Pansporocyst of the sporogony stage with eight zygotes starting their cellular division. f) Almost mature pansporocyst with eight spores inside. g) Free actinospore after release from the oligochaete gut...... 99

Fig. 6.4 – Transmission electron micrographs of the parasitic development of Ortholinea labracis n. sp. infecting the intestinal epithelium of the oligochaete host. a) Zygote cells inside a pansporocyst with large nuclei (N) and clusters of mitochondria (Mt). b) A developing spore in the stage of four cells (asterisks) united by cell junctions. c, d) Detailed aspect of a developing actinospore showing the sporoplasmic cell (Sp) containing several electron-dense secondary cells (SC) and being surrounded by the valvogenic cells (VC). e) Transversel section of the capsulogenic cells (asterisks) of an actinospore showing mitochondria (Mt) in the citoplasm and the polar capsules (PC) with the polar filaments (PF) inside. f) Longitudinal section of a polar capsule (PC) showing the conical structure (asterisk) protruding from the valvogenic cells (arrowheads)...... 100

Fig. 6.5 – Schematic drawings of the spores and the inferred life cycle of Ortholinea labracis n. sp. a) Triactinomyxon actinospore. b) Myxospore in valvular and sutural view. c) Schematic illustration of the inferred life cycle of Ortholinea labracis n. sp...102

Fig. 6.6 – Maximum likelihood tree of the SSU rRNA sequence of Ortholinea labracis n. sp. and other selected myxozoan species. The numbers on the branches are bootstrap FCUP xxv Index of Figures confidence levels on 500 replicates. There were a total of 1473 positions in the final dataset. GenBank accession numbers in parentheses after the species name...... 108

Chapter 7

Fig. 7.1 – Photomicrographs of developmental stages and free actinospores of Sphaerospora dicentrarchi in the coelomic cavity of the capitellid polychaete Capitella sp., from fresh material. a) Initial binucleated rounded cell. b) Initial pansporocyst with two internal cells (arrow). c) Pansporocyst in the gametogamy phase with several inner cells. d) Set of four spores in different stages of development, released from burst pansporocysts. e) Pansporocyst in the sporogony stage with eight spores inside. f) Set of four mature free spores, two of them showing an extension of the valvogenic cells (right side) and other two without it (left side). Scale bars = 10 µm...... 120

Fig. 7.2 – Schematic illustration of the inferred life cycle of Sphaerospora dicentrarchi...... 121

Fig. 7.3 – Graphical comparison of the morphometrics of Sphaerospora dicentrarchi myxospores from different sampling locations. Traces=range; Bullets=mean value. Measures in µm. (References for Spain: Sitjà-Bobadilla and Álvarez-Pellitero, 1992; Italy: Mladineo, 2003; Aveiro: Santos, 1998; Algarve: Present study)...... 122

Chapter 8

A new type of Echinactinomyxon (Myxozoa), infecting a marine polychaete, Heteromastus filiformis (Polychaeta: Capitellidae) in the Alvor Estuary (Portugal)

Fig. 8.1 – Mature pansporocyst in the intestinal ephitelium of Heteromastus filiformis as seen with DIC...... 132

Fig. 8.2 – Mature free Echinactinomyxon spore as seen with DIC...... 132

FCUP xxvi Index of Figures

Fig. 8.3 – Maximum likelihood tree for the SSU rRNA sequence of the new type of Echinactinomyxon here described and other selected myxosporeans. GenBank accession numbers after the species name; scale given under the tree...... 133

Chapter 9

Fig. 9.1 – Spores of sphaeractinomyxon types found in the coelomic cavity of Limnodriloides agnes (a–n) and Tubificoides pseudogaster (o–r). a) Sphaeractinomyxon type 1 filling the coelomic cavity of L. agnes. b) Sphaeractinomyxon type 1 in lateral view. c) Sphaeractinomyxon type 1 in apical view. d) Sphaeractinomyxon type 2 in apical and lateral view. e) Sphaeractinomyxon type 3 in lateral view. f) Sphaeractinomyxon type 3 in apical view. g) Sphaeractinomyxon type 4 in lateral view. h) Sphaeractinomyxon type 5 in apical view. i) Sphaeractinomyxon type 5 in lateral view. j) Sphaeractinomyxon type 6 in apical and lateral view inside a bisporous pansporocyst. k) Developmental stages of sphaeractinomyxon type 6 inside a gonad cavity. l) Sphaeractinomyxon type 7 in apical view. m) Sphaeractinomyxon type 7 in lateral view. n) Polar capsules of a sphaeractinomyxon type 7 smashed spore exhibiting the polar filaments coiled longitudinally. o) Sphaeractinomyxon type 8 in apical and lateral view. p) Sphaeractinomyxon type 9 in apical and lateral view inside a pansporocyst. q) Sphaeractinomyxon type 10 in lateral view. r) Sphaeractinomyxon type 10 in apical view. Scale bars=20 µm...... 146

(double asterisks) leaning against the pansporocyst wall. m) Pansporocyst with a more advanced stage of sporogony that will produce two final spores. Two sporoplasmic cells (double asterisks) against the pansporocyst wall, two involucres formed by three valvogenic cells surrounding three inner capsulogenic cells (asterisks) and some elongated cells with unknown function are visible. n) Pansporocyst in the same sporogony stage as the one in the previous figure but in a type of sphaeractinmyxon that will produce four final spores. o) Pansporocysts in a slightly more advanced sporogony stage than the previous two figures, but producing eight final spores. The capsulogenic cells are already forming the polar capsules. p) Pansporocyst in a more advanced sporogony stage were the majority of the spores have already the sporoplasmic cells inside the spore involucre, but two sporoplasmic cells (double asterisks) are still outside their spore involucre (asterisks). q) Pansporocyst with eight immature spores. r) Some immature spores outside a busted pansporocyct accompanied by a solitaire mature spore on the right side. s-u) Pansporocysts with mature spores from three different types of sphaeractinomyxon. One type developing two spores (s), another type developing four spores (t) and, finally a type developing eight spores (u). Note the presence of four vegetative cells (asterisks) around the four mature spores in (t). Scale bars=20 µm...... 152

FCUP xxviii Index of Figures

FCUP 1 Chapter 1. General Introduction

Chapter 1

General Introduction

FCUP 2 Chapter 1. General Introduction

FCUP 3 Chapter 1. General Introduction

1.1. Myxozoan parasites

Myxozoa Grassé, 1970 is a subphylum of microscopic endoparasites of vertebrate and invertebrate hosts that comprises about 2,200 species (Lom and Dyková, 2006; Yokoyama et al., 2012). Currently, this subphylum is divided in two classes: Malacosporea Canning, Curry, Feist, Longshaw and Okamura, 2000, and Myxosporea Bütschli, 1881 (Table 1.1). Malacosporeans are found in body cavities of freshwater bryozoans, and malacospores are produced inside spherical sacs or elongate worms stages from where they are released to the water column where they may infect a fish host (Canning et al., 2000; Lom and Dyková, 2006; Bartošová-Sojková et al., 2014; Hartikainen et al., 2014). Presently, only three species are described, Buddenbrockia plumatellae Schröder, 1910; Buddenbrockia allmani Canning, Curry, Hill and Okamura, 2007 and Tetracapsuloides bryosalmonae Canning, Curry, Feist, Longshaw and Okamura, 1999. The life cycle of T. bryosalmonae is the only one fully demonstrated experimentally and by molecular features. Tetracapsuloides bryosalmonae infects the bryozoan Fredericella sultana Blumenbach, 1779 and the kidneys of salmonids fishes, causing the proliferative kidney disease known as PKD (Lom and Dyková, 2006; Bartošová-Sojková et al., 2014). Myxosporeans are the most speciose group within Myxozoa. These parasites infect mainly freshwater and marine fishes (Lom and Dyková, 2006), but they were also found to infect other vertebrate hosts like birds, amphibians, reptiles and mammalians (Friedrich et al., 2000; Eiras, 2005; Garner et al., 2005; Lom and Dyková, 2006; Prunescu et al., 2007; Bartholomew et al., 2008; Dyková et al., 2011; Jirků et al., 2011). Annelids are identified as the invertebrate hosts (Ikeda, 1912; Lom and Dyková, 2006; Yokoyama et al., 2012; Eszterbauer et al., 2015). Even the sipunculids, in which tetractinomyxons were originally described (Ikeda, 1912), are now placed within Annelida by phylogenetical analysis (Struck et al., 2007; Weigert et al., 2014). Alternation of the life cycle between a vertebrate and an invertebrate host was experimentally demonstrated or inferred by molecular analysis for 47 species (Eszterbauer et al., 2015). The majority of the few life cycles known so far involve freshwater species, and only six are related to the marine environment (Køie et al., 2013; Eszterbauer et al., 2015).

FCUP 4 Chapter 1. General Introduction

Table 1.1 – Current taxonomy of Myxozoa according to Fiala et al. (2015a). Phylum Cnidaria Unranked subphylum Myxozoa Class Malacosporea Order Malacovalvulida Family Saccosporidae Buddenbrockia, Tetracapsuloides

Class Myxosporea Order Bivalvulida Suborder Variisporina Family Sphaeromyxidae Sphaeromyxa Family Myxidiidae Myxidium, Zschokkella, Enteromyxum, Sigmomyxa, Soricimyxum, Cystodiscus Family Ortholineidae Ortholinea, Neomyxobolus, Cardimyxobolus, Triangula, Kentmoseria Family Sinuolineidae Sinuolinea, Myxodavisia, Myxoproteus, Bipteria, Paramyxoproteus, Neobipteria, Schulmania, Noblea, Latyspora Family Fabesporidae Fabespora Family Ceratomyxidae Ceratomyxa, Meglitschia, Ellipsomyxa, Ceratonova Family Sphaerosporidae Sphaerospora, Wardia, Palliatus Family Myxobilatidae Myxobilatus, Acauda, Hoferellus Family Chloromyxidae Chloromyxum, Caudomyxum, Agarella Family Coccomyxidae Coccomyxa, Auerbachia, Globospora Family Alatosporidae Alatospora, Pseudoalatospora, Renispora Family Parvicapsulidae Parvicapsula, Neoparvicapsula, Gadimyxa Suborder Platysporina Family Myxobolidae Myxobolus, Spirosuturia, Unicauda, Dicauda, Phlogospora, Laterocaudata, Henneguya, Hennegoides, Tetrauronema, Thelohanellus, Neothelohanellus, Neohenneguya, Trigonosporus Order Multivalvulida Family Trilosporidae Trilospora, Unicapsula Family Kudoidae Kudoa Family Spinavaculidae Octospina Incertae sedis in Multivalvulida: Trilosporoides

FCUP 5 Chapter 1. General Introduction

1.2. Historical background of Myxozoa

The first reference to a myxozoan was made by Jurine in 1825, with the description of cysts in the muscles of the salmonid Coregonus fera Jurine, 1825, from the Léman lake, located between France and Switzerland (Jurine, 1825). Several other descriptions followed without any taxonomic allocation for these parasites. In 1841, Müller named these parasites as 'Psorospermien' (Psorosperms). This name, without taxonomic value, was also used by other workers that followed (Gurley, 1894; Shulman, 1966). Officially, the classification for Myxozoa began when Otto Bütschli erected the order Myxosporidea, included in the Sporozoa class (Bütschli, 1882). Myxobolus muelleri and Myxidium lieberkuhnii were the first myxosporeans species described. Since then, the number of described species has increased considerably (see Lom and Dyková, 2006). The discovery and description, in 1899, of the first actinospore types – i.e. synactinomyxon, hexactinomyxon and triactinomyxon – is attributed to Antonin Štolc, who isolated them from specimens of oligochaetes collected in Vltava River in Czech Republic. However, Štolc placed them within Dicyemida (parasites of the renal appendages of cephalopods) in a group named Actinomyxidia. In 1900, Mrázek disagreed from Štolc's opinion and considered these parasites more close to Myxosporidea, especially to Ceratomyxa species (Caullery and Mesnil, 1905). Caullery and Mesnil (1904) described a new type of actinospore, sphaeractinomyxon, from marine oligochaetes, with a very distinct morphology from the other actinospores and Ceratomyxa species, and raised the idea that Actinomyxidia should be considered a sister group of the myxosporeans (Caullery and Mesnil, 1905; Marques, 1984). Finally, in 1970, Grassé classified myxosporeans and actinosporeans in the phylum Myxozoa, within the class Myxosporea, for the vertebrate myxozoan parasites and the class Actinosporea, for the invertebrate myxozoan parasites. Meanwhile, Myxozoa taxonomy had undergone two important changes after it was established as a phylum. First, the discovery that myxosporeans alternate their life cycle in vertebrate and invertebrate hosts, developing actinosporean stages in the latter (Wolf and markiw, 1984), lead to the suppression of the class Actinosporea, and since then, its genera became collective group names (Kent et al., 1994). Second, myxozoans, considered for a long time as protozoans, on the basis of molecular and ultrastructural studies, were finally recognized as metazoan organisms (Smothers et al., 1994). Initial molecular studies placed the Myxozoa close to nematodes (Smothers et al., 1994; Schlegel et al., 1996) but more accurate studies could demonstrate a FCUP 6 Chapter 1. General Introduction closer association to Cnidaria, as a sister taxon of the parasitic cnidarian Polypodium hydriforme Ussow, 1887 (Siddall et al., 1995; Chang et al., 2015; Okamura and Gruhl, 2015).

1.3. Myxosporean life cycles

For many years, the origin of the fish myxosporean infection was an unknown process. Several attempts to directly infect naïve fishes, with spores extracted or collected from infected fishes, were usually unsuccessful (Molnár, 1979). In some cases the infection occurred several months after the direct infection attempt, when the aquaria hold mud as a substrate (Molnár, 1979). This led several authors to propose the need for the spores to 'age' in the mud before they could became infectious (Hoffman and Putz, 1969; Molnár, 1979). Besides, the constant presence of the infectious agent in the environmental waters led several other authors to suggest the possible existence of an intermediate host (see Yokoyama et al., 1991). Finally, Wolf and Markiw (1984) demonstrated the alternation of the life cycle of Myxobolus cerebralis (Hofer, 1903) in salmonid fishes and oligochaete worms, with the production of triactinomyxon actinospores in the latter. Direct fish–to–fish transmission of myxosporeans was sucesseful in few cases (Molnár, 1984; Diamant, 1997; Moran et al., 1999; Sitjà- Bobadilla et al., 2007; Estensoro et al., 2010; Eszterbauer et al., 2015), but they are considered facultative transmission modes (Morris, 2012), which implies that an invertebrate host must also exist for these species. There are 18 recognized actinospore collective groups (Table 1.2; Fig. 1.1), each one comprising from few to dozens of different types of actinospores. Some types are released from the invertebrate hosts as individual spores (aurantiactinomyxon, echinactinomyxon, endocapsa, guyenotia, hexactinomyxon, neoactinomyxum, pseudotriactinomyxon, raabeia, sphaeractinomyxon, tetraspora, tetractinomyxon, triactinomyxon and unicapsulactinomyxon), while others are released in a set of eight spores attached to each other by the tip of the processes, forming nets (antonactinomyxon, hungactinomyxon, ormieractinomyxon, siedleckiella and synactinomyxon). Since the discovery of the two-host life cycle (Wolf and Markiw, 1984), experimental infections studies were performed with few results, because they represent a laborious and time consuming method (Székely et al., 1999, 2001). For instance, some success was obtained in infecting oligochaetes with myxospores (El- Mansy and Molnár, 1997a,b; El-Mansy et al., 1998; Molnár et al., 1999a,b; Székely et FCUP 7 Chapter 1. General Introduction al., 1999, 2001, 2002; Eszterbauer et al., 2000, 2015; Rácz et al., 2004), but there is a great percentage of failure in infecting fishes with actinospores (Székely et al., 1999, 2001). The experimental process also has the risk of erroneous species identification or mismatch; for instance, by experimental infection, the echinactinomyxon type 5 of Özer and Wotten (2000) was considered to be the alternate stage of Sphaerospora truttae Fischer-Scherl, El-Matbouli and Hoffmann, 1986. However, while using molecular tools, Holzer et al. (2004) proved the mismatch of the two stages. Another example that showed wrong results involves Myxobolus pavlovskii (Akhmerov, 1954). By experimental infection, this parasite was considered to develop in Tubifex tubifex (Müller, 1774), producing hexactinomyxon actinospores (Ruidisch et al., 1991) but, according to Marton and Eszterbauer (2011), on basis of molecular data, this myxosporean infects Limnodrilus oligochaetes, producing echinactinomyxon actinospores. Because of the constrains found in experimental infections, and also the difficulties to survey actinospores in nature, only less than 50 species had their life cycles elucidated (Eszterbauer et al., 2015).

Table 1.2 – Actinospore collective groups and the number of life cycles elucidated for each group. Collective group name and author Number of Life cycles: Genus (number of species) Antonactinomyxon Janiszewska, 1957 1: Chloromyxum (1) Aurantiactinomyxon Janiszewska, 1952 11: Chloromyxum (1), Hoferellus (2), Myxidium (1), Thelohanellus (4), Henneguya (3)

Echinactinomyxon Janiszewska, 1957 1: Myxobolus (1) Endocapsa Hallett et al., 1999 0 Guyenotia Naville, 1930 0 Hexactinomyxon Štolc, 1899 0 Hungactinomyxon Rácz et al., 2005 0 Neoactinomyxum Granata, 1922 2: Chloromyxum (1), Hoferellus (1) Ormieractinomyxon Marques, 1984 0 Pseudotriactinomyxon Hallett et al., 2003 0 Raabeia Janiszewska, 1955 4: Myxobolus (3), Myxidium (1) Siedleckiella Janiszewska, 1953 1: Zschokkella (1) Sphaeractinomyxon Caullery and Mesnil, 1904 0 Synactinomyxon Štolc, 1899 0 Tetractinomyxon Ikeda, 1912 7: Ceratonova (1), Parvicapsula (1), Ceratomyxa (1), Ellipsomyxa (1), Gadimyxa (1), Sigmomyxa (1), Zschokkella (1) Tetraspora Hallett and Lester, 1999 0 Triactinomyxon Štolc, 1899 21: Myxobolus (19), Henneguya (1), Myxobilatus (1) Unicapsulactinomyxon Rangel et al., 2011 0

FCUP 8 Chapter 1. General Introduction

Most of the life cycles elucidated so far involve freshwater myxosporeans and oligochaetes as invertebrate hosts (Eszterbauer et al., 2015). There are two exceptions, Ceratonova shasta (Noble, 1950) and Parvicapsula minibicornis Kent, Whitaker and Dawe, 1997, two freshwater myxosporeans infecting salmonid fishes that use as invertebrate host, a freshwater polychaete, Manayunkia speciosa (Leidy, 1859) (Bartholomew et al., 1997, 2006). In the marine environment, there are six myxosporeans species with their life cycles elucidated, involving polychaetes as invertebrate hosts. Ellipsomyxa gobii Køie, 2003, from the gallbladder of Pomatoschistus microps (Krøyer, 1838), infects Nereis spp. (Køie et al., 2004); Ellipsomyxa mugilis (Sitjà-Bobadilla and Álvarez-Pellitero, 1993), a parasite of the gallbladder of mugilids fishes, infects Hediste diversicolor (O. F. Müller, 1776) (Rangel et al., 2009) (Fig. 1.2); Gadimyxa atlantica Køie, Karlsbakk and Nylund, 2007, from the kidney and urinary bladder of Gadus morhua Linnaeus, 1758, infects Spirorbis spp. (Køie et al., 2007); Sigmomyxa sphaerica Karlsbakk and Køie, 2012, from the gallbladder of Belone belone (Linnaeus, 1761), infects Nereis pelagica Linnaeus, 1758 (Karlsbakk and Køie, 2012); Ceratomyxa auerbachi (Noble, 1950), from the gallbladder of Clupea harengus Linnaeus, 1758 infects Chone infundibuliformes Krøyer, 1856 (Køie et al., 2008); and Parvicapsula spp., from the FCUP 9 Chapter 1. General Introduction kidneys of Sprattus sprattus (Linnaeus, 1758), and C. harengus infect Hydroides norvegicus Gunnerus, 1768 (Køie et al., 2013).

Fig. 1.2 – Ellipsomyxa mugilis life cycle representation in the polychaete Hediste diversicolor (adapted and rearranged from Rangel et al., 2009). a) Myxospores of E. mugilis. b) Binucleated cells in H. diversicolor coelom. c) Tetranucleated cell in plasmotomy. d) Early pansporocyst with two enveloping cells and two internal cells. e) Pansporocyst with four internal cells. f) Pansporocyst with 10 internal cells. g) Pansporocyst with 16 internal cell. h) Pansporocyst with eight young sporoblasts resulting from two zygote mitotic divisions. i) Pansporocyst with eight young spores. j) Pansporocyst with eight mature spores. k) Free mature actinospores. b–g) Gametogony phase, h–j) Sporogonic phase.

1.4. Myxosporean development

The complete life cycle stages (Fig. 1.3) were described for M. cerebralis infecting salmonids (El-Matbouli and Hoffmann, 1998; Kent et al., 2001). In the fish, upon contact with the infectious actinospore, the actinosporean sporoplasm penetrates and migrates intercellularly in the fish integument epidermis or gill epithelium. Then, the sporoplasmic cell disintegrates and its internal cells invade the fish tissues. After several multiplications, a plasmodium is produced, initiating the sporogony phase, which ends with the formation of myxospores. The latter will be ingested by the annelid worms. Here, the spore cells are disintegrated, and the binucleated sporoplasm then penetrates the intestinal epithelium cells and initiates a schizogony phase. The sporoplasm nuclei undergo multiple divisions producing a multinucleate cell. These FCUP 10 Chapter 1. General Introduction cells can divide and form additional multinucleated cells, or divide by plasmotomy and produce uninucleate cells, which fuse together to form binucleate cells. The gametogony stage initiates with the binucleated cells becoming tetranucleated cells by nuclear divisions and, by plasmotomy, divides forming a set of four cells, two of them involving the other two, forming the early pansporocyst. The two internal cells usually are designated by α and β cells. These cells undergo three mitotic divisions and one meiotic division, resulting in 16 haploid gametocytes and 16 polar bodies. The α cells then fuse with β cells, forming eight zygotes. The enveloping cells can also undergo mitotic divisions. In the sporogony stage, the zygotes undergo two mitotic divisions forming sporoblasts with four cells. Three peripheral cells divide to form three capsulogenic cells and three valvogenic cells. The fourth centrally located cell undergoes divisions to form the sporoplasmic cell. Finally, the valvogenic cells expand and involve the capsulogenic and sporoplasm cell, completing the spore formation.

Fig. 1.3 – Life cycle and development of myxosporeans (adapted from Kent et al., 2001). 1-16: Myxosporean development in the fish hosts. 17-30: Actinosporean development in the annelid hosts. 1: Actinospore infects the fish host. 2: Sporoplasm secondary cell divides by endogeny. 3-13: Presporogonic or extrasporogonic vegetative replication. 14-16: Sporulation and formation of myxospores. 17: Actinospore infect the annelid host. 18-20: Schizogony stages. The final binucleated cells with an α and β nuclei. 21-26: Gametogony stages. Internal cells undergo three mitotic and one meiotic division. 24-26: Gametes fuse resulting in eight zygotes. 27-29: Sporogony stages. Mature spores are released. 30: Mature actinospore enter in contact with a vertebrate host.

FCUP 11 Chapter 1. General Introduction

More recent studies have questioned the existence of some stages and the route of some processes during the actinosporean stage. Multinucleated schizogonic stages, previously described as the first stage developing in the annelid host (El- Matbouli and Hoffmann, 1998) was considered to be confused with microsporidean parasites stages (Morris and Freeman, 2010). Uninucleated cells, that should precede the formation of the binucleate cells, were also considered an erroneous interpretation of previous workers (Morris and Freeman, 2010). According to Morris (2012), in the annelid host, the first developmental stage is the binucleated cell, which results directly from the binucleate sporoplasm of the myxospore. This cell multiplies by mitose and the resulting daughter cells migrate throught the annelid body. In the model proposed by Morris (2012) for the freshwater myxosporeans (Fig. 1.4), the fertilisation occurs when two binucleated cells fuse to form a diploid binucleate stage that then undergoes karyogamy resulting in a tetranucleated stage. Also in this model, in the early pansporocyst, the β cells are gametic and the α cells are somatic. After three mitotic divisions of α and β cells, the β cells undergo meiosis twice, producing 16 polar bodies. The net result is eight haploid germ cells and eight diploid somatic cells. Contrary to the anterior model, α and β cells do not fuse together; instead, the α cell unite to a β cell, and then, the α cell divides to originate three sporogonic cells surrounding the germ cell (β cell). The three sporogonic cells divide and three daughter cells became valvocapsulogenic cells, and the other three daughter cells fuse together around the germ cell, originating the actinospore sporoplasm (sporoplasmogenesis). The three valvocapsulogenic cells then divide, originating three capsulogenic cells and three valvogenic cells, which expand and envelop the capsulogenic and sporoplasm cells. Myxozoa organisms have unique characteristics, and one of the most striking characteristics of Myxozoa is the cell-in-cell condition, i.e., the existence of secondary and tertiary cells whose origin constitute the Myxozoa dogma (Lom and Dyková, 2006; Morris, 2010). The formation of these cells was explained as a phenomenon of endogenous budding (Lom and Dyková, 1992b; Lom and Dyková, 2006), in which the future secondary cell is formed when one nucleus, resulting from a nuclear division of the nucleus of the primary cell, is surrounded by endoplasmic reticulum, which later becomes the cellular membrane of the secondary cell (Lom and Dyková, 1992b). More recently, Morris and Adams (2007) showed that the sporoplasm cells of T. bryosalmonae spores acquire the secondary cells by an engulfment process (or internalization) of another cell. This led Morris (2010) to suggest that primary cells acquire the first secondary cell by an engulfment process, with possible variations between species. This secondary cell can then produce, by mitosis, more secondary cells inside the primary cell. FCUP 12 Chapter 1. General Introduction

Fig. 1.4 – Freshwater myxosporean development model proposed by Morris for the actinospore phase (adapted from Morris, 2012). 1: Infection of oligochaete by myxospore sporoplasm. 2: Proliferation of binucleated cells. 3-4: Two binucleated cells fuse and form a tetranucleated stage. Fertilisation. 5: Tetranucleate cell plasmotomy forming four diploid cells. 6: Pansporocyst with two internal cells, one somatic cell (α) and one gametic cell (β). 7-8: The α and β cells divide originating eight α cels and eight β cells. The β cells undergo meiosis, releasing 16 polar bodies. 9: Diplod somatic cells (α) attaches to germ cells (β). 10-11: Somatic diploid cells divide and surround the haploid germ cell, forming the sporoblast. 12: Sporogonic and germ cells divide by mitosis. 13: Sporogonic cells rearrange to form sporoplasmogenic cells (grey cells) surrounding germ cells, and valvocapsulogenic cells (clear cells). 14: Sporoplasmogenic cells fuse and form the sporoplasm. 15-17: Valvocapsulogenic cells divide producing three valvogenic and three capsulogenic cells. 18-19: Germ cells divide within sporoplasm. 20: mature actinospore. Black spots=diploid nuclei; white spots=haploid nuclei.

1.5. Fish hosts and myxosporean parasites

Fish aquaculture production grows every year worldwide, and reached 66.6 million tonnes in 2012, according to FAO (2014). In Europe, fish production amounts to 2.8 million tonnes. Portuguese aquaculture is also increasing and, in 2013, the total production reached 9,955 tonnes. European seabass, Dicentrarchus labrax (Linnaeus, 1758), is the most important commercial fish cultured in South European countries, especially in the North Atlantic and Mediterranean area. Gilthead seabream, Sparus aurata Linnaeus, 1758, is another important fish, intensively cultured in the Mediterranean Sea. The production of European seabass and gilthead seabream in 2013, in Portugal, was 455 and 1,201 tonnes, respectively (INE, 2015; FAO, 2014). FCUP 13 Chapter 1. General Introduction

1.5.1. European seabass Dicentrarchus labrax (Linnaeus, 1758), Moronidae

European seabass is a demersal fish that can sustain variation of temperatures (eurythermic) and salinity (euryhaline). Specimens were recorded to attain a maximum length and weight of 103 cm and 12 kg, respectively. It is found from Norway to Morocco, and in the Canary Islands and Senegal (Smith, 1990). In summer months, it inhabits coastal waters, in estuaries and lagoons, entering occasionally in rivers. In winter, European seabass migrates to offshore. It has only one breeding season, starting in winter, in the Mediterranean Sea, and ending in June in the Atlantic populations (Smith, 1990; FAO, 2013). European seabass is an opportunistic fish (Pickett and Pawson, 1994). Adults are predators feeding on fishes, prawns, crabs and cuttlefish, while juveniles feed mainly on crustaceans and, in a small portion, annelids (Pickett and Pawson, 1994; Cabral and Costa, 2001; Laffaille et al., 2001; Hampel et al., 2005). Nevertheless, in some localities they feed mainly on polychaetes, like capitellids and annelids (Pickett and Pawson, 1994; Hampel et al., 2005; Martinho et al., 2008). Seven myxosporeans species were described infecting the European seabass (Fig. 1.5).

Sphaerospora dicentrarchi Sitjà-Bobadilla and Álvarez-Pellitero, 1992 (Fig. 1.5a), is a systemic histozoic parasite, and it is also found in Dicentrarchus punctatus (Bloch, 1792) (Sitjà-Bobadilla and Álvarez-Pellitero, 1992; Xavier et al., 2013). The parasite infects the connective tissue of several organs, with a special preference for FCUP 14 Chapter 1. General Introduction the gallbladder and the intestine. The distribution area for this parasite includes the North Atlantic, Mediterranean Sea and Adriatic Sea. Prevalence of infection reaches 100% in wild populations and 84% in culture conditions, and is stable throughout the year, only with slight increase in spring and summer (Sitjà-Bobadilla and Álvarez- Pellitero, 1992, 1993c; Santos, 1998; Mladineo, 2003; Fioravanti et al., 2004, 2006; Merella et al., 2006; Mladineo et al., 2010). Sphaerospora dicentrarchi does not cause many harm to the fish hosts, but under stress conditions it can enable secondary infections. On the other hand, the infection in the gallbladder and intestine can disturb the normal digestive function of the intestine, and thus cause growth problems (Rigos et al., 1999). Sphaerospora testicularis Sitjà-Bobadilla and Álvarez-Pellitero, 1990 (Fig. 1.5b), is a coelozoic parasite inhabiting the testes, especially the lumen of seminiferous tubules of male fishes (Sitjà-Bobadilla and Álvarez-Pellitero, 1993b). The parasite is reported from the Mediterranean Sea. Prevalence of infection reaches 3% in wild populations (Sitjà-Bobadilla and Álvarez-Pellitero, 1990) and 75% in culture conditions (Sitjà-Bobadilla and Àlvarez-Pellitero, 1990, 1993c; Fioravanti et al., 2004, 2006). Sphaerospora testicularis sporogonic development is linked to gonad maturation of the male fish and, accordingly, prevalence is higher in the European seabass breeding season (Sitjà-Bobadilla and Álvarez-Pellitero, 1993c). External signs of disease include the presence of whitish or orange-yellowish nodules or spots. Near the end of the breeding season, the parasite can cause severe lesions and tissue destruction, with little or non spermatozoid production (Sitjà-Bobadilla and Álvarez-Pellitero, 1990), leading to focal necrosis and parasitic castration (Álvarez-Pellitero and Sitjà-Bobadilla, 1993b). Ceratomyxa diplodae Lubat, Radujkovic, Marques and Bouix, 1989 (Fig. 1.5c) is a coelozoic parasite inhabiting the gallbladder and bile (Sitjà-Bobadilla and Álvarez- Pellitero, 1993a), and is reported from the Mediterranean Sea, North Atlantic and Adriatic Sea. Prevalence of infection reaches 10% in wild populations, and 27% in culture conditions (Álvarez-Pellitero and Sitjà-bobadilla, 1993a; Santos, 1998; Fioravanti et al., 2006; Merella et al., 2006). The parasite is more prevalent from autumn to spring in the Mediterranean Sea (Álvarez-Pellitero and Sitjà-bobadilla, 1993a), and in the spring in the North Atlantic coast (Santos, 1998). When in heavy infections, the parasite spreads to other organs like the intestine, swimming bladder, pancreas and mesentery. Usually, no external sign of infection is visible in the fish. Nevertheless, C. diplodae can cause histopathologic lesions in the gallbladder, tissue necrosis and inflammation of the sub epithelial connective tissue, and it also can affect the neighbour pancreatic tissue (Álvarez-Pellitero and Sitjà-Bobadilla, 1993b). FCUP 15 Chapter 1. General Introduction

Ceratomyxa labracis Sitjà-Bobadilla and Álvarez-Pellitero, 1993 (Fig. 1.5d), is a coelozoic parasite inhabiting the gallbladder and bile (Sitjà-Bobadilla and Álvarez- Pellitero, 1993a), and is reported from the Mediterranean Sea and North Atlantic. Prevalence of infection reaches 14% in wild populations and 70% in culture conditions (Álvarez-Pellitero and Sitjà-bobadilla, 1993a; Santos, 1998; Fioravanti et al., 2006; Merella et al., 2006). The parasite is more prevalent in winter in the Mediterranean Sea (Álvarez-Pellitero and Sitjà-bobadilla, 1993a) and in spring in the North Atlantic coast (Santos, 1998). When in heavy infection, it can spread to other organs, having the same histopathology described for the previous species C. diplodae (Álvarez-Pellitero and Sitjà-bobadilla, 1993a, b). Enteromyxum leei (Diamant, Lom and Dyková, 1994) is an enteric histozoic parasite of gilthead seabream (Diamant et al., 1994), and is reported from the Mediterranean Sea. European seabass seem to be an occasional host, especially in culture conditions, with a prevalence of infection of 4% (Fioravanti et al., 2006). Kudoa iwatai Egusa and Shiomitsu, 1983 is a histozoic parasite inhabiting the trunk muscle and many other internal organs of the European seabass and the gilthead seabream, and is reported from Japan and the Northern Red Sea (Israel). In the type host, Oplegnathus punctatus (Temminck and Schlegel, 1844), the parasite causes flesh liquefaction but, in European seabass, it does not cause any visible reactions (Diamant et al., 2005). Myxobilatus sp. is a coelozoic parasite found in the kidney and urinary bladder. This parasite was reported from Aveiro Estuary. Prevalence reaches 40% in wild populations, and during the spring season (Santos, 1998).

1.5.2. Gilthead seabream, Sparus aurata Linnaeus, 1758, Sparidae

Gilthead seabream is a demersal fish, and can record maximum length and weight of 70 cm and 17 kg, respectively. It is found in Eastern Atlantic coasts, Mediterranean Sea, and Black Sea (Bauchot and Hureau, 1990). Gilthead seabream is a euryhaline and eurythermal species, which allow it to habit marine and brackish waters, like lagoons and estuaries, used especially when juvenile. They breed in open sea, from October to December, and the juveniles migrate toward the coastal warmer waters in spring. This species is a protandrous hermaphrodite, maturing first as a male within one to two years, and then as a female within two to three years (Bauchot and Hureau, 1990; FAO, 2005). In lagoons, juvenile's of gilthead seabream feed mainly on amphipods and small crustaceans and, as they grow, start to include copepods, decapods, and later, annelids, crustaceans and fishes. In the sea, as older fishes, feed FCUP 16 Chapter 1. General Introduction essentially on gastropods and bivalves, polychaete and decapods, and rarely predate on other fishes (Rosecchi, 1987; Pita et al., 2002). Eight myxosporeans species were described infecting the gilthead seabream (Fig. 1.6).

Sphaerospora sparis (Sitjà-Bobadilla and Álvarez-Pellitero, 1995) (Fig. 1.6a) is a histozoic parasite inhabiting the kidney of the gilthead seabream (Sitjà-Bobadilla et al., 1992; Sitjà-Bobadilla and Álvarez-Pellitero, 1995), and is reported from the Mediterranean Sea, North Atlantic and Adriatic Sea. Prevalence of infection reaches 100% in culture conditions. The parasite is more prevalent in spring, in the North Atlantic coasts. Sphaerospora sparis seems to reduce the fish growth and may cause some mortality (Sitjà-Bobadilla and Álvarez-Pellitero, 1995; Palenzuela et al., 1999; Mladineo, 2003; Fioravanti et al., 2006; Mladineo et al., 2010). Zschokkella auratis Rocha, Casal, Rangel, Severino, Castro, Azevedo and Santos, 2013 (Fig. 1.6b) is a coelozoic parasite inhabiting the gallbladder floating free in the bile, and is reported from the Atlantic coast, Algarve (Portugal). Prevalence of infection reaches 12%. No external sign of diseases is visible (Rocha et al., 2013). Ceratomyxa auratae Rocha, Casal, Rangel, Castro, Severino, Azevedo and Santos, 2015 (Fig. 1.6c) is a coelozoic parasite inhabiting the gallbladder, and is reported from the Atlantic coast, Algarve (Portugal). Prevalence of infection reaches 4% in cultured fishes. No external sign of disease is visible, and the parasite seems not to cause lesions or mortality (Rocha et al., 2015). Ceratomyxa sparusaurati Sitjà-Bobadilla, Palenzuela and Álvarez-Pellitero, 1995 is a coelozoic parasite inhabiting the gallbladder and bile of gilthead seabream (Sitjà-Bobadilla et al., 1995), and is reported from the Mediterranean Sea, North Atlantic and Adriatic Sea. Prevalence of infection reaches 60% in culture conditions FCUP 17 Chapter 1. General Introduction

(Sitjà-Bobadilla et al., 1995; Palenzuela et al., 1997; Costa et al., 1998; Mladineo, 2003; Fioravanti et al., 2006; Merella et al., 2006). Infection with this parasite is persistent in the Spanish Mediterranean coast, without much variation in seasonal prevalence (Palenzuela et al., 1997), while in the Adriatic Sea the parasite is more prevalent in spring (Mladineo, 2003). In light infections no external sign is visible but, in heavy infections, the parasite causes hyperplasia of the gallbladder and a distension of the fish abdomen, which may result in the death of the fish (Palenzuela et al., 1997). Henneguya sp. is a histozoic parasite inhabiting the gill filaments and the heart of the gilthead seabream (Bahri et al., 1996; Caffara et al., 2003), and is reported from Tunisia, Italy and Sardinia. Prevalence of infection reaches 4% in wild populations, and 11% in cultured fish (Bahri et al., 1996; Caffara et al., 2003; Merella et al., 2006). Infected fishes show gill pallor but the heart infection have no visible external signs; nevertheless, the location of the infection in the heart has the potential to cause harm (Caffara et al., 2003). Sphaerospora sparidarum (Sitjà-Bobadilla and Álvarez-Pellitero, 2001) is a coelozoic parasite from Dentex dentex (Linnaeus, 1758) and gilthead seabream, inhabiting the kidney tissues and ureters, and is reported from the occidental Mediterranean Sea (Sitjà-Bobadilla and Álvarez-Pellitero, 2001). Prevalence of infection reaches 19% in culture conditions for gilthead seabream (Sitjà-Bobadilla and Álvarez-Pellitero, 2001). No external sign of disease is visible. Sphaerospora sparidarum trophozoites flatten, vacuolize and atrophy the renal epithelium and, in heavy infections, may cause hyperplasia in the kidney tubules, the glomerulus and the Bowman's space (Sitjà-Bobadilla and Álvarez-Pellitero, 2001). Enteromyxum leei (Diamant, Lom and Dyková, 1994) is an enteric histozoic parasite (Diamant et al., 1994), and is reported from the Mediterranean Sea. Prevalence of infection reaches 72% in culture conditions (Diamant et al., 1994; Fioravanti et al., 2006). This parasite infiltrates the intestinal epithelium, causing emaciation and mortality (Diamant, 1992). European seabass can also be infected with E. leei; nevertheless, it seems to be an occasional host, with a prevalence of infection of 4% (Fioravanti et al., 2006). Kudoa iwatai Egusa and Shiomitsu, 1983 is a histozoic parasite inhabiting the trunk muscle and many other internal organs of the gilthead seabream and the European seabass, and is reported from the Japan and the Northern Red Sea (Israel). Prevalence of infection reaches 44% in culture conditions for gilthead seabream. In the type host, Oplegnathus punctatus (Temminck and Schlegel, 1844), the parasite causes flesh liquefaction, but in gilthead seabream and European seabass, it does not cause any visible reactions (Diamant et al., 2005). FCUP 18 Chapter 1. General Introduction

1.6. Study Aims

The general aim of this thesis is to study myxozoan parasites in farmed fishes and annelids, and to elucidate their life cycles for known and new found species. Specific aims were as follows:

1) To conduct a survey of myxozoan parasites in farmed fishes, particularly in the European seabass and gilthead seabream in aquaculture facilities in Algarve (Portimão).

2) To conduct a survey of myxozoan parasites in annelids from aquaculture facilities in Algarve (Portimão) and from the wild environment in Aveiro Estuary.

3) To characterize the morphology, ultrastructure, genetics, morphometrics and histopathology of all new myxozoan species or actinospore types eventually found.

4) To characterize all developmental stages and site of infection for all new actinospore types found.

5) To characterize the myxozoans found for the 18S ribosomal RNA gene, as a method for rapid identification and diagnoses of the fish farm pathologies.

6) To match the 18S ribosomal RNA gene for the myxozoans found in fishes and annelids and, eventually, elucidate the life cycle of the fish farm parasites.

7) To characterize the ultrastructure of the actinosporean phase of the species Ellipsomyxa mugilis, from the polychaete Hediste diversicolor.

8) To characterize a Myxobilatus species reported in European seabass from Aveiro Estuary.

FCUP 19 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

Chapter 2

Synopsis of the species of the genus Myxobilatus Davis, 1944 (Myxozoa: Myxosporea: Myxobilatidae)

This chapter has been adapted from:

Rangel, L. F., Gibson, D. I. and Santos, M. J. (2014). Synopsis of the species of the genus Myxobilatus Davis, 1944 (Myxozoa: Myxosporea: Myxobilatidae). Systematic Parasitology 87, 187–198. doi:10.1007/s11230-013-9467-y.

FCUP 20 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

FCUP 21 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

2.1. Abstract

A synopsis of the species of Myxobilatus Davis, 1944 (Myxozoa: Myxosporea: Myxobilatidae) is presented. Thirty-nine nominal species are included. The major characteristics and an illustration are given for each species based on the original records.

FCUP 22 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

FCUP 23 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

2.2. Introduction

Species of the myxozoan genus Myxobilatus Davis, 1944 are mainly parasites of the kidneys, urinary ducts and urinary bladder of freshwater, and occasionally marine or brackish-water, fishes. According to Lom and Dyková (2006), the main characteristics of the species of the genus are as follows: Spores elongate and pointed anteriorly. Shell valves often with fine ridges which extend posteriorly into two caudal appendages. Polar capsules pyriform, positioned perpendicular to sutural plane. Binucleate sporoplasm may contain iodinophilous vacuole. Coelozoic trophozoites, small or large, disporic or polysporic, with pansporoblast formation, occur throughout entire length of urinary system. Rarely histozoic. Type-species: Myxobilatus gasterostei (Parisi, 1912). Myxobilatus was erected by Davis (1944) when he revised Henneguya Thélohan, 1892 and redistributed its species over three genera, i.e. the amended Henneguya (sensu stricto) and two new genera, Unicauda Davis, 1944 and Myxobilatus. The separation of species was based on the spore flattening plane in relation to the sutural plane and on the nature of the caudal processes. In species of Henneguya and Unicauda, the spores are flattened in a plane parallel to the sutural plane, and the shell valves are extended into two caudal processes of a similar nature (Henneguya) or into only a single caudal process of a different nature (Unicauda). In Myxobilatus, the spores are flattened in a plane perpendicular to the sutural plane, and the shell valves are extended by two caudal processes of a similar nature which are separated throughout their entire length. Davis (1944) also considered spores of Myxobilatus spp. to be bilaterally symmetrical, i.e. the spores are flattened on one side (ventral) and convex on the other (dorsal), when examined in valvular view. To the new genus Myxobilatus, Davis (1944) transferred five species from Henneguya, H. gasterostei Parisi, 1921, H. mictosporus Kudo, 1920, H. ohioensis Herrick, 1941, H. rupestris Herrick, 1941 and H. wisconsinensis Mavor and Strasser, 1916, and also included two new species, M. caudalis Davis, 1944 and M. asymmetricus Davis, 1944. Other species have subsequently been transferred to Myxobilatus, and many new species have been described by numerous authors. Some of these, however, do not entirely fit the original diagnosis; for example, the full separation of the two caudal processes throughout their entire length (Dogiel and Bogolepova, 1957; Siau, 1971; Li and Desser, 1985) and the spore asymmetry in valvular view (inter alia Guilford, 1965; Arthur and Margolis, 1975; Nie and Li, 1992). FCUP 24 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

Myxobilatus was originally included in the family Myxobolidae Thélohan, 1892, but Shulman (1953) subsequently erected the family Myxobilatidae Shulman, 1953 for this genus alone. However, in a taxonomic review of the Myxosporidia by Lom and Noble (1984), the Myxobilatidae was not recognised and Myxobilatus was transferred to the family Sphaerosporidae Davis, 1917, a decision which also was followed later by Lom and Dyková (2006) in a revision of the Myxozoa. Nevertheless, in a recent phylogenetic study based on molecular data, Whipps (2011) supported the restoration of the family Myxobilatidae to accommodate not only species of Myxobilatus but also those of Hoferellus Berg, 1898 and Acauda Whipps, 2011. The inclusion of Myxobilatus and Hoferellus within the Myxobilatidae had in fact already been proposed by Molnár (1988), when he noted great similarities between these two genera, not only in their morphology but also in terms of their development and seasonality.

2.3. Materials and methods

For this synopsis, the original descriptions of each species were consulted. Bibliographic material was collected in the library of the Natural History Museum, London, and from the internet when available on-line. The Web of Knowledge (Zoological Record) was also consulted. Data on each species of Myxobilatus, arranged alphabetically, are presented in two ways: the characteristics of the species, such as spore shape or host(s), are given in the main body of the text, whereas the morphometric data for the spores are presented in Table 2.1 for a ready comparison. A number is attributed to each species and used throughout the text, table and figures for utility. Each species is illustrated by a copy of the original image. For some species, with incomplete original descriptions or with less than informative original illustrations, we included data from subsequent redescriptions. Note that some authors have used the expression spore ‘width’, possibly referring to the thickness of the spore. Other authors have used other expressions, such as ‘wide’ (Mukhopadhyay and Haldar, 2003), ‘breadth’ (Davis, 1944; Qadri and Kumari, 1965; Susha and Janardanan, 1994) and ‘greatest width’ (Dogiel and Bogolepova, 1957), or have not even made it clear whether they are referring to ‘width’ or ‘thickness’ (Thélohan, 1892; Lom, 1986). For these latter cases, in Table 2.1, we have considered these measurements in the width column and labelled them with an asterisk. FCUP 25 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

The measurement values in the text and Table 2.1 are expressed in micrometres and include the range and the mean value ± standard deviation when available, in parentheses. We are aware that the accuracy of some of these measurements is beyond the limit of resolution of the light microscope, but these are as given in the original descriptions. Abbreviations: AN, accession number in GenBank; CA, caudal appendage; HE, host environment; IV, iodinophilous vacuole; LSB, length of spore body; PC, polar capsule; PFC, polar filament coils; SI, site of infection; SL, sutural line; SV, shell valve; TH, typehost; TL, type-locality; TLS, total length of spore; TSB, thickness of spore body; WSB, width of spore body.

2.4. Results

The results of our investigation brought to light 39 nominal species which have been attributed to Myxobilatus. They are described below in alphabetical order, metrical data of their spores are presented in Table 2.1 and they are illustrated in Figs. 2.1–2.39. Note that, in some cases, not all of the relevant data were given in the original descriptions.

Myxobilatus spp.: basic data

1. M. accessobranchialis Obiekezie and Okaeme, 1987. Spore ovoidal in sutural view and plano-convex in valvular view, asymmetrical; PC pyriform, unequal; PFC 5–6; SV with 4–5 fine striations; SL straight and distinct; CA equal, fully divided and slightly divergent; IV present; histozoic, disporous; SI accessory breathing organs; TH Heterobranchus bidorsalis Saint-Hilaire (Clariidae); TL Kainji Lake, New Bussa, Nigeria; HE freshwater.

2. M. anguillaris Mukhopadhyay and Haldar, 2003. Spore ovoid but rounded in end view, asymmetrical; PC elongate, equal; PFC 4–5; SV smooth; CA long; IV present; histozoic, polysporous; SI basal portion of gills; TH Taenioides anguillaris (Linnaeus) (Gobiidae); TL Canning, West Bengal, India; HE estuarine.

3. M. anteronippus Basu, Modak and Haldar, 2012. Spore lenticular in valvular view, with slightly rounded anterior and almost tapered posterior end, asymmetrical; PC FCUP 26 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944 pyriform, unequal, with nipple- or knob-like structure present at anterior extremity of each PC; PFC in larger PC 6–7, PFC in smaller PC 4–5; SV smooth; SL straight and indistinct; CA equal, long and tapered; IV present; histozoic, disporous; SI fins (pectoral, dorsal and caudal) and operculum; TH Taenioides cirratus (Blyth) (Gobiidae); TL Canning (22°20'N, 88°40'E), West Bengal, India; HE estuarine.

4. M. asymmetricus Davis, 1944. Spores taper sharply toward anterior end, which is slightly rounded; PC pyriform, unequal; SV with longitudinal striations parallel to sutural line; SL visible, may take spiral course posteriorly; CA usually strongly curved; IV absent; coelozoic, polysporous; SI urinary bladder; TH Sander vitreus (Mitchill) (Percidae); TL Mississippi River at Fairport, Iowa, USA; HE freshwater.

5. M. baicalensis (Dogiel in Dogiel and Bogolepova, 1957) Shulman, 1966 (= Henneguya baicalensis). Spore oblong-oval, with narrowed anterior end; PC elongate, equal; CA forked or fused together; coelozoic, disporous to polysporous; SI urinary bladder; TH Asprocottus herzensteini Berg (Abyssocottidae); TL Lake Baikal, Russia; HE freshwater. According to Shulman (1962), spore asymmetrical; PC pyriform; SV smooth.

6. M. carassii Nie and Li, 1992. Spore elongate-oval in valvular view, symmetrical; PC pyriform, equal; SV with 6–9 parallel striations; SL straight, conspicuous; CA smooth, straight, bifurcate; coelozoic; SI urinary bladder and ureter; TH Carassius auratus auratus (Linnaeus) (Cyprinidae); TL Huama Lake, Hubei Province, China; HE freshwater.

7. M. caudalis Davis, 1944. Spore ovoid in sutural view, elliptical in valvular view, with truncated anterior end, asymmetrical; PC pyriform, equal; SV with longitudinal striations; SL readily visible; CA exceptionally long; IV present; coelozoic, disporous and polysporous; SI urinary bladder; TH Aplodinotus grunniens Rafinesque (Sciaenidae); TL Mississippi River at Fairport, Iowa, USA; HE freshwater.

8. M. cheni Ma in Chen and Ma, 1998. Spores oval in valvular view, oval or elliptical in sutural view; PC pyriform, equal; SV with striations; SL straight; CA equal, short; IV present; coelozoic; SI urinary bladder; TH Schizothorax davidi (Sauvage) (Cyprinidae); TL Mabian River, Mabian County, Sichuan Province, China; HE freshwater.

FCUP 27 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

2.6)

2.0)

3.2);

1.7);

1.7)

(2.0

1.9)

3.0)

3.3)

3.2)

4.9) × 2.2 ± 0.2 × ± 0.2 2.2 4.9)

5.0) × 1.8 ± 0.2 (1.5 × ± 0.2 1.8 5.0)

6.7) × 2.8 ± 0.3 (2.4 0.3 2.8 × ± 6.7)

6.0) × 1.6 ± 0.1 (1.5 0.1 1.6 × ± 6.0)

3.3) × 1.6 ± 0.3 (0.8 ± 0.3 × 3.3)1.6

2.7

3.5

2.5

3.9) × (1.5 1.7 3.9)

4.2) × (2.4 2.5 4.2)

6.9) × (2.0 2.8 6.9)

6.0) × 2.5 6.0)

4.0) × 1.6 4.0)

5.6) × (2.4 2.8 5.6)

5.6 × 5.6 2.5

9.0 × 9.0 3.0

7.5

3.6 × 3.6 2.0

5.5

(4.8

6 × 3

3.7 (3.1

3.7 (3.6

5.0

7.5

6.4

6.3 (4.5

5.6

3.3 (2.8

5.5 (4.8

4.53.0 ×

2.4

4.6

L: 10.5 × 4.0; S: 7.5 × 7.5L:4.0 ×10.5 4.0; S:

S: 4.3 (4.0 ± 0.2

L:0.5 5.9 (5.1 ±

2.7 (1.7 ± 0.6

S: 4.6 (4.2 ± 0.3

L:0.2 5.7 (5.5 ±

Polar capsule size (length × width)size ×Polar (length capsule

33)

40.5)

98.4)

26.6)

22.5

37.3

16.6

19.5

33.5

32.4 (26.7

58.9 (30.0

22.3 (15.6

9.9

31.0 (30 ± 1.2

spore

Total length of

22)

9.1)

30.0)

84.0)

13.0)

28)

24.7)

30.0

38.0

29.0

8.5

± 1.5 (4.2 ± 1.5

35 (up to 40)35 to (up

(15

8.0

26.3

22.5

18.0 (13.2

46.0 (10.8

19.0

10.5 (6.4

28.0

(19.9

22.5 ± 1.6

7.0

20.0 (19 ± 0.7

appendages

Length caudal of

7.2)

3.1)

7.2)

6.4)

7.5

6.5

6.2

6.0

6.1 (6.0

8.0

6.3 (6.0

6.8 (6.7 ± 0.5

3.0 (2.8 ± 0.1

body

Thickness of spore Thickness spore of

6.9)

4.2)*

4.6)

6.2)*

7.2)

9.0)*

7.2)

spp.

7.5

6.4

8.2

5.5

7.5*

5.1

4.8 (4.6

6.0

6.7 (5.4

7.0

6.0

4.3

6.5 (5.9

6.2 (4.8

7.3

5.0

5.6

10*

6.5 (6.1 ± 0.2

3.7 (3.3 ± 0.4

4.5 (4.4 ± 0.1

body

Widthspore of

7.5)

Myxobilatus

13.2)

18.0)

14.4)

13.6)

12.5)

13.5)

12.5)

15.0

14.0

12.5

12.6

(9.2

9.3

13.5

9 (8.5

11.5

12.2 (11.4

11.2

17.8 (13.2

12.9 (9.8

8.2

11.9

11.4

(11.4

12.5 ± 0.7

6.2 (5.0 ± 0.7

(11.0

11.5 ± 0.6

body

Length spore of

Metrical data from the spores of of spores the from data Metrical

–

M. mictosporus M.

M. mediusM.

M. mastacembeliM.

M. legeri M.

M. hemiculteriM.

M. gobii M.

M. gasterosteiM.

M. fragilicaudus M.

M. fossilis M.

M. cotti M.

M. convexusM.

M. cheni M.

M. caudalis M.

M. carassii M.

M. baicalensis M.

M. asymmetricusM.

M. anteronippusM.

M. anguillarisM.

M. accessobranchialis M.

Species

1 No.

Table 2.1 2.1 Table

FCUP 28 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

3.1)

2.5)

5.6)

3.2)

2.0)

3.3)

7.2) × 2.6 ± 0.3 (2.1 ± 0.3 × 7.2)2.6

3.9) × 2.1 ± 0.1 (1.9 ± 0.1 × 3.9)2.1

2.4

2.1

2.8

7.0); S: 4.7 (4.2 4.7 7.0); S:

4.8) × (1.6 2.3 4.8)

4.9) × 2.4 4.9)

3.0) × (1.0 1.5 3.0)

5.4) × 2.5 5.4)

4.7) × (2.7 3.0 4.7)

4.8 × 4.8 1.8

6.9 × 6.9 1.7

6.5 × 6.5 2.6

3.6 × 3.6 2.5

8.4 × 8.4 2.8

3.2 × 3.2 1.6

(4.8

4.1 (3.6

6.5 (5.6 ± 0.5

3.52.5 ×

3.6

4.02.7 ×

4.8

2.5 (2.0

5.2

5.0

3.0

7.0

4.8

3.4 (3.0 ± 0.3

4.8 (4.2

L: (5.6 6.1

4.2 (3.6

3.0

Polar capsule size (length × width)size ×Polar (length capsule

36.2)

93.9)

34.3)

19.0)

59.2)

28.0)

39.2)

87)

36.5

75.7 (52.0

26.2 (16.7 ± 4.5

30.7 (27.2

16.5 (12.5

64 (49

23.5

31.0

48.6 (35.2

19.2 (18.6

33.3 (30.8

Total spore length of

24.9)

22.9)

75.5)

19.0)

46.4)

29.5)

(17.9

13.2)

74)

9.5)

22.5

12.8

14.3

58.6 (32.8

14.3 (5.6 ± 3.9

9.6

17.8 (14.4

9.1

52.8 (35

19.5

26.8

36.5 (23.2

20.6 ± 1.9

10.3 (7.2

24.7 (22.4

8.5 (7.0

11.8

appendages

Length caudal of

for more details) for more

6.9)

6.4)

4.8)

8.0)

6.4)

7.2)

7.0

6.5

5.5

4.8

6.0 (5.6

5.9 (5.3 ± 0.6

6.6

4.6 (4.2

6.0

4.7

6.7 (4.8

5.6 (4.8

6.3 (6.0

3.5

spore body

Thickness of

7.0)

6.3)

5.6)

4.8)

6.0)

8.0)

6.4)

7.2)

5.6)

8.8)*

0.6 (4.9 0.6

4.0

5.4

7.0

5.2 (4.8

5.9 ±

7.0

4.8 (4.7

5.0 (4.5

3.5

6.4 (5.6

4.8

5.6

5.9 (4.8

5.2 (4.8 ± 0.4

6.6 (6.0

4.5 (4.2

8.1 (7.7

body

Widthspore of

14.3)

10.3)

18.4)

15.3)

13.6)

14.4)

10.0)

10.8)

9.8)

12.3)

16.0

11.2

14.0

10.0

12.0

17.1 (16.0

12.3 (9.5 ± 1.4

11.5

9.4

12.9 (12.8

9.0 (7.5

13.6

11.2 (9.6

9.8

10.0

11.5

12.6

12.1 (11.2

9.0 (8.1 ± 0.5

8.9 (7.2

9.3 (8.4

11 (9.8

Lengthbody spore of

opusi

nsinensis

: L, larger polar capsule; S, smaller polar capsule polar smaller S, capsule; polar :larger L,

Continued.

–

M. yunnanensis M.

M. yukonensis M.

M. wisco M.

M. varicorhiniM.

M. synodontisM.

M. sinipercaeM.

M. sichuanensisM.

M. semotiliiM.

M. schulmaniM.

M. rupestrisM.

M. pseudorasborae M.

M. polymorphusM.

M. platessaeM.

M. paragasterostei M.

M. ohioensis M.

M. odontamblyM.

M. noturi M.

M. notopterusM.

M. nostalgicus M.

M. minutusM.

Species

No.

* Measures given originally under different designations of width (see ‘Material and methods’ and ‘Material (see width of designations different under originally given * Measures Abbreviations Table 2.1 2.1 Table

FCUP 29 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

9. M. convexus Jurakhno 1991 (emend.). Spores oval, with very thick walls and convex valves; PC pyriform, equal; SV smooth; CA long; coelozoic; SI urinary bladder; TH Parablennius tentacularis (Brünnich) (Blenniidae); TL off Batiliman, Ukraine, Black Sea; HE marine.

10. M. cotti Guilford, 1965. Spore ovoid in frontal and side view, anterior end papillate, symmetrical; PC pyriform, equal; PFC: 3–4; SV with 6–8 striations; SL distinct; CA long, curved, divergent at distal end; IV present; coelozoic, polysporous; SI urinary bladder; TH Cottus bairdii Girard (Cottidae); TL Green Bay, Lake Michigan, USA; HE freshwater.

11. M. fossilis Susha and Janardanan, 1994. Spore lanceolate in sutural view, elliptical in valvular view, symmetrical; PC pyriform, equal; PFC: 6–7; SV with striations; SL straight, with distinctly raised sutural ridge; CA equal, long, tapered; IV present; coelozoic, polysporous; SI urinary bladder; TH Heteropneustes fossilis (Bloch) (Heteropneustidae); TL Valapad, Thrissur District, Kerala State, India; HE freshwater.

12. M. fragilicaudus R. Shulman in Shulman and Kulemina, 1969. CA long, thin, fragile; coelozoic; SI ureters; TH Cottus gobio Linnaeus (Cottidae); TL Volga Basin (Lake Seliger), Russia; HE freshwater.

13. M. gasterostei (Parisi, 1912) Davis 1944 (= Henneguya gasterostei). Spore oval in frontal view, asymmetrical; PC pyriform, equal; SV with longitudinal striations; CA tapered, bifurcate; IV present; coelozoic, di- or tetrasporous; SI kidney; TH Gasterosteus aculeatus Linnaeus (Gasterosteidae); TL Lago di Garda, Italy; HE freshwater; AN: EU861210, AY495703, AJ582063 and EU861209 (actinospore).

14. M. gobii Evlanov, 1981. Spore oval, with narrow tapering anterior pole, symmetrical; PC pyriform, equal; SV with faint longitudinal striations; coelozoic; SI kidney (urinary tubules); TH Gobio gobio (Linnaeus) (Cyprinidae); TL Vishtynetskoye Lake, Kaliningrad Region, Russia; HE freshwater. Redescription by Lom (1986) from G. gobio in Czech Republic: Spore oviform; PC tear-shaped, equal; PFC: 6–7; SV with 12 fine longitudinal striations; CA long, straight, gradually tapering; LSB 9.5–11.4 (10.2), WSB 5.6–6.2 (6), CA length 11–19 (18), PC length 4.2–5.0 (4.5), PC width 2.0−2.9 (2.2).

FCUP 30 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

15. M. hemiculteri Li, 1996. Spore ellipsoidal in valvular view, spindle-shaped in sutural view, symmetrical; PC pyriform, equal; SV with 5–6 parallel striations; SL straight, thin; CA straight, bifurcate in middle; IV present; coelozoic, di- or tetrasprorous; SI ureter and kidney; TH Hemiculter leucisculus (Basilewsky) (Cyprinidae); TL Liangzi Lake, Hubei Province, China; HE freshwater.

16. M. legeri (Cépède, 1905) Davis, 1944 (= Henneguya legeri). Spore ovoid, symmetrical; CA very thin, usually bifurcate; coelozoic; SI urinary bladder; TH Barbatula barbatula (Linnaeus) (Nemacheilidae); TL Isère, Rhône-Alpes Region, France; HE freshwater. According to Shulman (1962, 1966, 1984): SV smooth; PC pyriform, equal; PC length 2.8–3.5, PC width 2.

17. M. mastacembeli Qadri and Kumari, 1965. Spore pyriform or elongate, with blunt, narrow anterior end and broad, rounded or flattened posterior end; PC pyriform, equal; CA fully divided; IV present; histozoic, disporous; SI intestine; TH Mastacembelus armatus (Lacépède) (Mastacembelidae); TL Hyderabad (17°21'N, 78°28'E), Andhra Pradesh, India; HE freshwater. According to Arthur and Margolis (1975): spore symmetrical; SV smooth.

18. M. medius (Thélohan, 1892) Shulman, 1966 (= Henneguya media). SV with striations; coelozoic, polysporous; SI kidney; TH Gasterosteus aculeatus Linnaeus; also in Pungitius pungitius (Linnaeus) (Gasterosteidae); TL France; HE freshwater. Redescription by Shulman (1962): Spore oval, symmetrical; PC pyriform, equal; SV with longitudinal striations; CA short, thick, usually fused; LSB 10.0–13.0, WSB 5.0-7.5, TSB 7.9, CA length 12.0–15.0, TSL 24.0–30.0, PC length 2.0–2.5.

19. M. mictosporus (Kudo, 1920) Davis, 1944 (= Henneguya mictospora). Spore broad spindle-shaped, with attenuated anterior end; PC pyriform, equal; SV with 6–8 longitudinal striations; CA long; IV present; coelozoic, monosporous (rare), disporous to polysporous; SI urinary bladder; TH Lepomis cyanellus Rafinesque; also in L. humilis (Girard) and Micropterus salmoides (Lacépède) (Centrarchidae); TL Stony Creek, Illinois, USA; HE freshwater. According to Davis (1944): spore asymmetrical.

FCUP 31 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

FCUP 32 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

20. M. minutus Evdokimova, 1977. Spore extremely small, pointed anteriorly; PC pyriform, equal; SV smooth; CA relatively thick; coelozoic; SI gallbladder; TH Paralichthys patagonicus Jordan (Paralichthyidae); TL Patagonian Shelf, Southwest Atlantic (off Argentina); HE marine.

21. M. nostalgicus Lom, 1986. Spore oviform; PC tear-shaped, equal; PFC 5–6; SV smooth; SL without distinct elevated ridge; CA slender, slightly curved, set apart at their base; coelozoic; SI kidney (renal tubules); TH Tinca tinca (Linnaeus) (Cyprinidae); TL Vrbový at Brzotín near Rožňava, Slovakia; HE freshwater.

22. M. notopterus Kalavati and Vaidehi, 1996. Spore elongate-oval, with slightly pointed anterior end, symmetrical; PC pyriform, unequal; PFC 3–4; SV smooth; SL straight; CA bifurcate, straight, thin, long; IV absent; coelozoic, monosporous; SI gallbladder; TH Notopterus notopterus (Pallas) (Notopteridae); TL Chilka Lake (19°28'- 19°54'N, 85°06'-85°35'E), Odisha State, India; HE estuarine.

23. M. noturi Guilford, 1965. Spore ovoid in frontal and side views, symmetrical; PC pyriform, equal; PFC: 4–6; SV with 8 striations; SL conspicuous; CA equal, short, tapered and curved; coelozoic, polysporous; SI urinary bladder; TH Noturus gyrinus (Mitchill) (Ictaluridae); TL Green Bay, Lake Michigan, USA; HE freshwater.

24. M. odontamblyopusi Basu and Haldar, 2004. Spore oval in valvular view, lanceolate in sutural view, with pointed anterior and posterior ends, asymmetrical; PC pyriform, equal; PFC 5–6; SV with faint longitudinal striations; SL straight; CA equal, long, fully divided, tapered; IV absent; histozoic, disporous; SI gill filaments; TH Odontamblyopus rubicundus (Hamilton) (Gobiidae); TL Canning (22°20'N, 88°40'E), West Bengal, India; HE estuarine.

25. M. ohioensis (Herrick, 1941) Davis, 1944 (= Henneguya ohioensis). Spore elliptical, asymmetrical; PC pyriform, equal; SV with fine, longitudinal striations; CA divergent, broad at base, gradually tapering towards extremity; coelozoic, disporous; SI urinary bladder; TH Lepomis gibbosus (Linnaeus) (Centrarchidae); TL Lake Erie, Ohio, USA; HE freshwater.

26. M. paragasterostei Zaika, 1965. Spore symmetrical; SV with striations; coelozoic, disporous to polysporous; SI urinary bladder and kidneys; TH Leuciscus baicalensis (Dybowski) (Cyprinidae), also in Cyphocottus megalops (Gratzianov) FCUP 33 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

(Scorpaeniformes), Batrachocottus baicalensis (Dybowski), Leocottus kesslerii (Dybowski) and Cottocomephorus grewingkii (Dybowski) (Cottocomephoridae); TL Lake Baikal, Russia; HE freshwater.

27. M. platessae (Bazikalova, 1932) Shulman and Shulman-Albova, 1953 (= Henneguya platessae). Spore asymmetrical; PC pyriform, equal; coelozoic, disporous to polysporous; SI urinary bladder; TH Pleuronectes platessa Linnaeus (Pleuronectidae); TL Barents Sea; HE marine. Redescription by Shulman and Shulman-Albova (1953): Spore asymmetrical; LSB 9.0–9.5, WSB 3.4–3.6, TSB 4.8-5.1, CA length 17.5–22.2, TLS 27–32, PC length 4.0–4.2.

28. M. polymorphus Nie and Li, 1992. Spore rounded in apical view with 4 symmetrical protrusions; PC pyriform, equal; SV with 7 slightly curved striations; SL in sutural view and another conspicuous line in valvular view; CA bifurcate, thin, long; coelozoic; SI urinary bladder; TH Siniperca chuatsi (Basilewsky) (Percichthyidae); TL Huama Lake, Hubei Province, China; HE freshwater.

29. M. pseudorasborae Shulman, 1962. Spore pyriform, symmetrical; PC pyriform, equal; SV smooth; coelozoic, mono- to tetrasporous; SI urinary bladder; TH Pseudorasbora parva (Temminck and Schlegel) (Cyprinidae); TL Amur River Basin, Russia; HE freshwater.

30. M. rupestris (Herrick, 1941) Davis, 1944 (= Henneguya rupestris). Spore elliptical and slightly truncated at anterior end in sutural view, asymmetrical; PC pyriform, equal; PFC 4–6; SV with 5–8 faint, longitudinal striations; CA long, very slender, nearly straight; IV present; coelozoic, polysporous; SI urinary bladder; TH Ambloplites rupestris (Rafinesque) (Centrarchidae); TL Lake Erie, Ohio, USA; HE freshwater.

31. M. schulmani Mytenev, 1975. Spore elongate, diamond-shaped, with constriction in middle and anterior end significantly narrowed, symmetrical; PC pyriform, equal; SV slightly striated longitudinally; CA relatively short, with distinct basal swellings; SI kidney; TH Pungitius pungitius (Linnaeus) (Gasterosteidae); TL Pyalitsa, Murmansk, Kola Peninsula, Russia; HE freshwater.

32. M. semotilii Li and Desser, 1985. Spore small, ovoid to spindle-shaped, round in cross-section, symmetrical; PC pyriform, equal; PFC 4–5; SV with 6 longitudinal striations; CA short, straight, usually fused, occasionally bifurcate; IV present; FCUP 34 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944 coelozoic, disporous; SI ureters; TH Semotilus atromaculatus (Mitchill) (Cyprinidae); TL Lake Opeongo, Ontario, Canada; HE freshwater.

33. M. sichuanensis Ma in Chen and Ma, 1998. Spore diamond-shaped, symmetrical; PC pyriform, equal; SV smooth; SL straight; CA long, curved; IV present; coelozoic; SI urinary bladder, ureter; TH Carassius auratus auratus (Linnaeus) (Cyprinidae); TL Qujiang River, Quxian, Sichuan Province, China; HE freshwater.

34. M. sinipercae (Dogiel and Akhmerov in Akhmerov, 1960) Shulman, 1966 (= Henneguya sinipercae). Spore oval, asymmetrical; PC pyriform, equal; SV with light, wavy striations; coelozoic, di- to polysporous; SI urinary bladder; TH Siniperca chuatsi (Basilewsky) (Percichthyidae); TL Amur River Basin, Russia; HE freshwater.

35. M. synodontis Siau, 1971. Spore ovoid, symmetrical; SL conspicuous; CA single; IV present; histozoic; SI gills; TH Synodontis ansorgii Boulenger (Mochokidae); TL Porto Novo Lagoon, Djassin, Benin; HE freshwater.

36. M. varicorhini Kandilov, 1963. Spore ovoid, with tapered posterior and anterior ends, symmetrical; PC pyriform, equal; SV smooth; IV present; coelozoic; SI urinary bladder; TH Capoeta capoeta capoeta (Güldenstädt) (Cyprinidae); TL Kura River Basin, Azerbaijan; HE freshwater.

37. M. wisconsinensis (Mavor and Strasser, 1916) Davis, 1944 (= Henneguya wisconsinensis). Spore ovoid, symmetrical; PFC 5; CA bifurcate; coelozoic, polysporous; SI urinary bladder; TH Perca flavescens (Mitchill) (Percidae); TL Lake Mendota, Wisconsin, USA; HE freshwater.

38. M. yukonensis Arthur and Margolis, 1975. Spore lenticular, with tapering anterior end in sutural and valvular view, symmetrical; PC pyriform, equal; PFC 4–5; SV with 5−7 very fine striations; SL straight; CA only slightly divergent distally; IV absent; coelozoic, mono-, di- and trisporous; SI kidney tubules and urinary bladder; TH Cottus cognatus Richardson (Cottidae); TL Aishihik Lake,Yukon Territory, Canada; HE freshwater.

39. M. yunnanensis Ma, Wang and Cai, 1986. Spore diamond-shaped, asymmetrical; PC oval, equal; SV with 4–5 striations; SL straight; CA long; IV present; coelozoic; SI FCUP 35 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944 urinary bladder; TH Carassius auratus auratus (Linnaeus) (Cyprinidae); TL Qilu Lake, Tonghai County (24°02'N, 102°24'E), Yunnan Province, China; HE freshwater.

Figs. 2.2 – Line drawings of spores of Myxobilatus spp., redrawn from the original illustrations. All scale-bars: 10 µm. 20, M. minutus; 21, M. nostalgicus; 22, M. notopterus; 23, M. noturi; 24, M. odontamblyopusi; 25, M. ohioensis; 26, M. paragasterostei; 27, M. platessae; 28, M. polymorphus; 29, M. pseudorasborae; 30, M. rupestris; 31, M. schulmani; 32, M. semotilii; 33, M. sichuanensis; 34, M. sinipercae; 35, M. synodontis; 36, M. varicorhini; 37, M. wisconsinensis; 38, M. yukonensis; 39, M. yunnanensis

FCUP 36 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

Life-cycle of Myxobilatus spp.

Of all the species in this genus, only M. gasterostei has had its life-cycle elucidated. The myxospores of this species are released from the fish host, Gasterosteus aculeatus, and infect the oligochaete host, Nais communis Piquet. The triactinomyxon actinospores released from this invertebrate host then re-infect the fish (Atkinson and Bartholomew, 2009). This life-cycle was determined by Atkinson and Bartholomew (2009) with the aid of molecular studies, as these authors were able to match the SSU rDNA sequences of myxospores obtained from the fish with those from the triactinomyxon actinospores obtained from the oligochaete. The triactinomyxon of M. gasterostei, according to Atkinson and Bartholomew (2009), has the following features: LSB 31.5–51.0 (38.0 ± 5.6), WSB 11.6–17.6 (13.8 ± 1.7), CA length 215.3–316.7 (257.7 ± 23.8), CA width 10.4–16.9 (13.3 ± 1.5), spore axis length 104.0–125.3 (113.8 ± 6.3), spore axis width 10.0–18.6 (14.6 ± 2.2), PC length 5.3–7.1 (6.5 ± 0.5), PC width 3.0–5.0 (4.1 ± 0.6), PFC 5–6, sporoplasm with c.512 germ cells. AN: EU861209.

2.5. Final comments

This synopsis includes data from the original descriptions of the 39 nominal species of Myxobilatus. These are typically coelozoic parasites of the urinary system, but some species infect other organs, such as the gallbladder (2 species), and other species are histozoic in the gills (3), the accessory breathing organ (1), the fins (1) and the intestine (1). They infect a wide range of hosts, but are especially prevalent in Cypriniformes and Perciformes (60% of species). The majority occur in freshwater hosts (32 species), with a small number parasitising fishes in marine (3 species) and brackishwater (4 species) environments. The spores of Myxobilatus are elongate and/or ovoid in shape. In valvular view, they may be symmetrical or asymmetrical, i.e. the plane of flattening, perpendicular to the suture line, is more pronounced on one side of the spore than the other, which is usually more convex, resulting in an asymmetrical shape. This character was described by Davis (1944) when he erected the genus; however, only 14 species are currently described as asymmetrical, 19 are symmetrical and for six species this information is lacking. Almost two-thirds of the species have their shell valves ornamented in the form of longitudinal striations and the remainder have smooth shell valves. FCUP 37 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

The presence of an iodinophilous vacuole was originally a diagnostic character of the genus (Davis, 1944). However, although most species do have this feature, there are four species described without it and such data are missing for 18 species. Another diagnostic character used by Davis (1944) is the presence of two caudal appendages separated along their entire length. However, some of the species now attributed to the genus do not have this feature; for example, M. synodontis is described as having only a single caudal appendage and two species, M. baicalensis and M. semotilii, are described as having caudal appendages fused along part of their length, i.e. resembling a single bifurcate appendage. There is a discrepancy between the redescriptions of M. legeri given by Shulman (1962, 1966, 1984) and the illustrations of the spores from the host Blicca bjoerkna (Linnaeus). The shell valves were described as smooth, but the drawings show longitudinal lines representing striations (Fig. 16b). The only reference by Cépède (1905) to the spore shell valves in the original description was a comment that they were wrinkled and fragile (Fig. 16a). Lom (1986) also redescribed this species from the cyprinid hosts Rutilus rutilus (Linnaeus) and Scardinius erythrophthalmus (Linnaeus) in the Czech Republic, and included a drawing of a spore from each host, both with striated shell valves. However, he also considered the possibility that these redescriptions represent different species. For future descriptions of species of Myxobilatus, it is very apparent that the guidelines given by Lom and Arthur (1989) should be followed comprehensively. The caudal appendages need to be described in detail, including the symmetry of the spore in valvular view and the striations on the shell valves. All measurements that Lom and Arthur (1989) recommend need to be recorded, with special care taken in relation to their orientation; for example, in the distinction between the width and thickness of the spore body. It is also increasingly apparent, that molecular data should become an integral part of this process.

2.6. Acknowledgements

This work was financially supported by the FCT, within the scope of the project DIRDAMyx, reference FCOMP-01-0124-FEDER-020726/FCT-PTDC/MAR/116838/ 2010, (Portugal) and Pest-C/MAR/LA0015/2013; the PhD fellowship grant SFRH/BD/82237/2011 to L. Rangel and Sabbatical fellowship grant BSAB-1239/2012 to M. J. Santos. Dr Larisa Poddubnaya kindly helped with the acquisition of some Russian literature. FCUP 38 Chapter 2. Synopsis of the species of the genus Myxobilatus Davis, 1944

FCUP 39 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor

Chapter 3

Ultrastructural aspects of Ellipsomyxa mugilis (Myxozoa: Ceratomyxidae) spores and developmental stages in Nereis diversicolor (Polychaeta: Nereidae)

This chapter has been adapted from:

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. doi:10.1645/GE-3005.1.

FCUP 40 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor

FCUP 41 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor

3.1. Abstract

The ultrastructure of the spores and developmental stages of Ellipsomyxa mugilis in Nereis diversicolor were studied by transmission electron microscopy. The ultrastructure features and the developmental stages show many similarities with the general pattern described for other actinospores. However, several new features are definitely worth noting. For example, tetranucleated cells precede the formation of the initial pansporocyst, which preserves the two original enveloping cells until the end of sporogony. In the initial stages of sporogony, the future sporoplasm cell acquires the first secondary cell by an engulfment process. In the final stage of sporogony, spores are formed by a sporoplasm with two secondary cells and one somatic nucleus, and the polar capsule has a polar filament with a helicoidal arrangement possessing 7–8 coils.

FCUP 42 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor

FCUP 43 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor

3.2. Introduction

Myxozoans are microscopic metazoan parasites, whose known life cycles often alternate between vertebrate and invertebrate hosts (Lom and Dyková, 2006). That is the case of Ellipsomyxa mugilis Sitjà-Bobadilla and Álvarez-Pellitero, 1993 (Køie and Karlsbakk, 2009), which is a coelozoic parasite in both phases of its life cycle. The actinospore phase of E. mugilis occurs in the nereidid polychaete Nereis diversicolor, where it is found in the coelomic cavity (Rangel et al., 2009). Its myxospore phase occurs in mugilid fishes’ gallbladder and bile (Sitjà-Bobadilla and Álvarez-Pellitero, 1993e). Lom and Dyková (1992a, 1997) showed that both phases of the myxozoan life cycle—myxospore and actinospore—share many common features, namely, pansporoblast formation, polar capsule structure and morphogenesis, the presence of sporoplasmosomes in the sporoplasms, and cell-in-cell organization, among others. Ultrastructure studies of myxozoan parasites are important, not only for describing the morphology and the sporogenesis of spores (Lom and Dyková, 1996; Okamura et al., 2002; Morris and Adams, 2007), but also for better understanding the life cycle, transmission processes, and pathogenicity of different species (Cuadrado et al., 2008). The ultrastructure studies also help to elucidate aspects of myxozoan taxonomy and phylogeny (Redondo et al., 2003). Nevertheless, ultrastructural studies of the actinospore and developmental stages are still scarce (see Marques, 1982; Lom and Dyková, 1992a; Trouillier et al., 1996; Lom and Dyková, 1997; El-Matbouli and Hoffmann, 1998; Hallett et al., 1998; Álvarez-Pellitero et al., 2002; Oumouna et al., 2002; Morris, 2010). For E. mugilis, the myxospore ultrastructure has already been characterized, when first described from mullets in Mediterranean waters (Sitjà- Bobadilla and Álvarez-Pellitero, 1993e), but nothing is known for the actinospore stages. The aim of the present work is to describe the ultrastructure of the spores and developmental stages of the parasite E. mugilis in the coelomic cavity of N. diversicolor and to investigate the cell-in-cell condition, already described as typical of myxozoans (Lom and Dyková, 2006) and recently questioned by Morris (2010).

3.3. Material and methods

Ellipsomyxa mugilis spores and developmental stages were obtained in the coelomic fluid from three infected specimens of N. diversicolor collected in the Aveiro Estuary, FCUP 44 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor

Portugal (40°40'N, 8°45'W). The worms (n=138) were sampled in March and June 2009, and the parasites were identified in accordance with the description provided by Rangel et al. (2009). Fresh infected tissues were fixed in 3% glutaraldehyde with 0.2 M sodium cacodylate buffer (pH = 7.4) at 4 °C for 10 h, washed overnight at 4 °C with the same buffer, and post-fixed in 2% OsO4 buffered with the same solution for 3 hr at 4 °C. After dehydration in an ascending graded ethanol series and propylene oxide, the material was embedded in Epon. Ultrathin sections were double stained with aqueous uranyl acetate and lead citrate and then observed using a JEOL 100CXII TEM operated at 60 kV.

3.4. Results

Ultrathin sections of infected tissues exhibited several spores and developmental stages of E. mugilis in N. diversicolor. The earliest stages found were tetranucleated cells, as observed in a set of ultrathin serial sections. However, because these four nuclei are located in different planes, only three of them can be simultaneously shown in a single section and corresponding photo (Fig. 3.1a). They have several elongated mitochondria, with a few cristae, dense bodies, vacuoles, and some vesicles. The subsequent stages observed were the four-cell stages (joined together by desmosome-like junctions) and the formation of the initial pansporocyst (Fig. 3.1b). In the latter, two peripheral cells (somatic cells) began to envelope the other two internal cells (generative cells), which had a more rounded form (Fig. 3.1c). Both types of cells had large nuclei, with more, or less, central and compact nucleoli. The mitochondria were much more conspicuous in the internal cells, and they had the same features as those described for the tetranucleated cells. Both cell types, peripheral and internal, had dense bodies in their cytoplasm, but they were more conspicuous in the peripheral cells. The peripheral cells had long cisternae of rough endoplasmic reticulum (RER), following the contour of the cellular membranes (Figs. 3.1b–c). In the internal cells, the cisternae of RER seemed to go around the other organelles, in a 'zig-zag' fashion (Fig. 3.1b). Until the end of the gametogony, the pansporocysts were formed only by the two initial peripheral cells, connected by desmosome-like junctions (Fig. 3.1d). It was not possible to observe either zygote formation or the four-cell first sporoblast stage. The first sporogony stages observed in the ultrathin sections were sporoblasts with six or seven visible cells (Fig. 3.2a). Five or six cells, connected by cell junctions, were visible around one, or two, more internal cells. The cells of this stage FCUP 45 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor were characterized by having mitochondria with the same features previously described and RER cisternae. Some dense bodies were also visible, but they were even scarcer when compared with previous developmental stages. In some sporoblasts, with two central cells, one of them was in the process of enveloping the other one (Figs. 3.2b–c). In another sporoblast, there was only one central cell, and, inside it, a secondary cell could be seen (Fig. 3.2b). Both situations were observed in a pansporocyst’s single section (Fig. 3.2a). Capsulogenesis started with the formation of a capsular primordium extended by an external tubule (Figs. 3.2d–e). Around the external tubule there were microtubules. In the capsular primordium wall, there were two layers of different density, one external electron-dense layer followed by an electron lucent layer and a homogeneous electron dense core (Fig. 3.2e). In a more advanced stage of development, the spores already had the configuration of mature spores (Fig. 3.2f). The three capsulogenic cells were located at one edge, with their polar capsules positioned in a triangular fashion immediately below the sporoplasm cell; all of these cells were enveloped by the three valvogenic cells (Figs. 3.2f, 3.3a). In a set of ultrathin serial sections the three capsulogenic cells were observed juxtaposed. Under a cross-sectional view, such a junction follows a more, or less, straight line (Fig. 3.3b), while, in a longitudinal view, the cellular membranes of the capsulogenic cells form a sinuous curve, like the perimeter of the pieces in a puzzle (Fig. 3.3c). During the maturation process, the capsulogenic cells showed signs of some degeneration of its cytoplasm (Figs. 3.3b, d), which was visible by the presence of lighter areas presenting aspects of lysis. The polar capsule wall of more mature spores is formed in two layers, one external electron-dense layer, externally delimited by a thin membrane, and a more internal electron lucent layer (Figs. 3.3d, e, 3.4). In the central region of the polar capsule is present a polar filament and materials with different densities, which are, most probably, lipid droplets (Fig. 3.3e). In many sections, a space delimited by a membrane was observed between the polar capsule wall and the capsulogenic cell cytoplasm (Figs. 3.3d, e). The polar filament had a helicoidal arrangement inside the polar capsule. It began with six coils in newly formed polar capsules and ended with seven or eight coils in more mature capsules (Fig. 3.3e). The polar capsule opens to the exterior near the base of the polar filament, which is inverted inside the polar capsule. In this zone, there was an electron-dense structure with a fibrilar appearance (Fig. 3.3e) functioning like a plug, and, in many sections, we observed a cell junction between the valvogenic cells (Fig. 3.3d).

FCUP 46 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor

The valvogenic cells are flattened when the spore matures, becoming reduced to a small amount of cytoplasm and organelles. At this maturation stage, the valvogenic cells completely surrounded the capsulogenic cells and the sporoplasm cell. The sporoplasm cells, of maturating and mature spores, possessed two secondary cells and one somatic nucleus (Fig. 3.2f). Their cytoplasm and organelles had a more electron-dense appearance than the other cell types. The most conspicuous organelles were mitochondria, but some cytoplasmic inclusions were also visible. Other organelles frequently observed in the sporoplasm cell were FCUP 47 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor sporoplasmosomes. These organelles were electron dense, resembling tiny rods and scattered all over the sporoplasm cytoplasm. In the posterior part of the spore, the sporoplasm cell established cell junctions with the valvogenic cells by pseudopodia-like projections (Fig. 3.3f). During the entire spore development period, the pansporocyst is formed by only two cells. The cell junction between the two pansporocyst cells corresponds to the constriction zone that was visible with ordinary light microscopy (Fig. 3.2a).

3.5. Discussion

The present E. mugilis ultrastructure study in the host N. diversicolor confirmed the general development sequence described for this species by ordinary light microscopy (Rangel et al., 2009). The ultrastructure observations, developmental stages, and spores of E. mugilis agree, in general terms, with the features described so far for the few other actinospores studied to date. For example, we also noticed the existence of tetranucleated cells preceding the formation of the initial pansporocyst (El-Matbouli and Hoffmann, 1998), the initial pansporocyst formation by two external cells enveloping two internal cells (Lom and Dyková, 1992a, 1997; El-Matbouli and Hoffmann, 1998), the formation of polar capsules from a capsular primordium with external tubuli (Lom and Dyková, 1992a, 1997; El-Matbouli and Hoffmann, 1998), and the general structure of mature actinospores (Lom and Dyková, 1992a, 1997; El-Matbouli and Hoffmann, 1998; Hallett et al., 1998; Álvarez-Pellitero et al., 2002). Moreover, the details reported here, e.g., as for mitochondria, reveal strong similarity with those already described for the myxospore of E. mugilis ultrastructure characterized by Sitjà-Bobadilla and Álvarez-Pellitero (1993e). Mitochondria of the actinospore developmental stages, and of the myxospore plasmodia, presented the same size, elongated shape, and few cristae irregularly distributed. However, some differences and additional details are worth discussion. The ultrastructure of the four-cell stage, preceding the formation of the initial pansporocyst, was previously illustrated for triactinomyxon- and aurantiactinomyxon- type spores (Lom and Dyková, 1992a, 1997), for the raabeia-type spore of Myxobolus cultus (Lom and Dyková, 1997), and for the triactinomyxon of Myxobolus cerebralis (El- Matbouli and Hoffmann, 1998). These authors reported that, contrary to the internal cells, only the peripheral cells that form the pansporocyst possess several dense bodies and phagosomes. However, we now observed that the internal cells of E. mugilis also have dense bodies and phagosomes. FCUP 48 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor

Fig. 3.2 – Sporogony developmental stages of Ellipsomyxa mugilis. a) Longitudinal transverse section of a pansporocyst constituted by two enveloping cells, connected by desmosome-like junctions (arrows), showing four sporoblasts in the initial stages of sporogony. b) Magnification of a detail of Fig. 3.2a showing two sporoblasts. In the bottom sporoblast, one central cell is enveloping the other, which will became the first secondary cell in the future spore’s sporoplasm cell. In the top sporoblast, there is only one central cell, already with a secondary cell inside. c) A sporoblast with two central cells in which one have almost enveloped the other. d) Two capsulogenic cells surrounded by two valvogenic cells (asterisks), showing the capsular primordium (arrows) and two large lipid droplets. e) A capsulogenic cell showing a capsular primordium and the external tube (arrow) in cross section. f) A spore showing two secondary cells and one somatic nucleus of the sporoplasm cell. The enveloping valvogenic cells are present (arrows). Abbreviations: C1–C2, Central cells in sporoblast; CP, capsular primordium; L, lipid droplets; N, nucleus; P, primary cell; Pa, pansporocyst cell; S, secondary cell; Sp, sporoplasm cell. All scale bars = 2 mm.

FCUP 49 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor

Fig. 3.4 – Schematic drawing of a polar capsule of Ellipsomyxa mugilis actinospore and surrounding structure organization.

For the stage that characterizes the spores, Lom and Dyková (1992a, 1997) refer to the existence of a plug of moderate electron-dense material in the mouth of the polar capsule. This plug is topped by a cone that also contains an electron dense substance. The cone is covered by a layer of less dense material, a layer of fibers, or even by a layer of short microtubules. The polar capsule of E. mugilis has a similar plug, made of a denser material and covered by a fibrilar structure, but the cone shape described by Lom and Dyková (1992a, 1997) was not observed. In this zone of E. mugilis, the valvogenic cells have some cell junctions, showing the existence of a pore (Fig. 3.3d). In our observed ultrathin sections, the plug did not protrude between the valvogenic cells, as described by Lom and Dyková (1997) and by Álvarez-Pellitero et al. (2002) for triactinomyxon of Myxobolus bramae and Myxobolus pseudodispar. The polar filament has a helicoidal arrangement in E. mugilis with seven or eight coils. The number of coils seen in TEM sections here replaces the five or six coils previously revealed by light microscopy (Rangel et al., 2009) and probably reported from immature spores or from spores in which not all coils were easily visible. The number of somatic nuclei in the sporoplasm cell seems to be variable among the different species. Lom and Dyková (1992a), studying Triactinomyxon legeri, and Hallett et al. (1998), studying the triactinomyxon of M. bramae and M. psudodispar, refer to two somatic nuclei in the sporoplasm cell, whereas El-Matbouli and Hoffmann (1998) refer to the existence of a single somatic nucleus in the sporoplasm cell of triactinomyxon of M. cerebralis. Likewise, in E. mugilis, the sporoplasm cell has only one somatic nucleus. Other organelles observed included mitochondria, dense bodies, and sporoplasmosomes. In the posterior region, the sporoplasm cell had pseudopodia- FCUP 51 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor like projections, which were connected by cell junctions to the valvogenic cells (Fig. 3.3f). Pseudopodia-like projections of the sporoplasm cells seem to be a common feature (Lom and Dyková, 1997; Hallet et al., 1998; Álvarez-Pellitero et al., 2002), but the cell junctions between them and valvogenic cells have not yet been described. During the entire development of E. mugilis spores, the pansporocyst wall consisted of only two cells, connected by desmosome-like junctions. The constriction zone of the pansporocyst observed with light microscopy (Rangel et al., 2009) corresponds to this cell junction zone reported in the present study. The cell-in-cell condition, typical of myxozoans, has been explained as a phenomenon of endogenous budding (Lom and Dyková, 2006), in which the secondary cell is formed when one nucleus of the primary cell is surrounded by endoplasmic reticulum, which later becomes the cellular membrane of the secondary cell. More recently, Morris and Adams (2007) showed that the sporoplasm cells of spores from Tetracapsuloides bryosalmonae (malacosporean) develop inside Fredericella sultana and acquire the secondary cells by an engulfment process (or internalization) of another cell. This led Morris (2010) to suggest that primary cells acquire the first secondary cell by an engulfment process, with possible variations between species. This secondary cell can then produce, by mitosis, more secondary cells inside the primary cell. In ultrathin sections of early sporoblasts of E. mugilis, with six or seven visible cells, we observed some sporoblasts with two central cells, in which one is enveloping the other (Figs. 3.2a–c); in other sporoblasts, we saw only one central cell with a secondary cell already inside (Figs. 3.2a, b). Accordingly, the engulfment of cells seems to be the most suitable explanation for the origin of the first secondary cell in the E. mugilis actinospore. Nevertheless, the results in our study do not agree with the existence of what Morris (2010) reported as valvocapsulogenic (VC) cells and sporoplasmogenic (SP) cells in the pathway presented in his Figure 9 (pathway B), for the origin of the sporoplasm in the aurantiactinomyxon-type spore. In this pathway, in an early sporoblast formed by three sporogonic cells surrounding a germ cell, the three sporogonic cells divide, and the resulting six cells reorganize to form three VC cells and three SP cells. The latter cells fuse and internalize the germ cell, which has also divided, forming a sporoplasm with two internal cells. The VC cells then divide to form the three valvogenic cells and the three capsulogenic cells. In E. mugilis, after the central cell had acquired a secondary cell (Figs. 3.2a, b) by a likely envelopment process (Fig. 3.2c), it remains surrounded by more than three cells (four or five cells visible in the ultrathin sections). Thus, the process of secondary cells formation described by Morris (2010), with the intervention of SP cells, does not seem to occur in E. mugilis. FCUP 52 Chapter 3. Ultrastructural aspects of Ellipsomyxa mugilis in Nereis diversicolor

In conclusion, actinospore developmental stages of E. mugilis show many similarities to the development and ultrastructure features with the few actinospores already described. However, it remarkably differs in the formation of the first secondary cell of the sporoplasm. The present observations support the work of Morris (2010) in that endogenous budding does not occur in myxozoan developmental biology, as previously stated by Lom and Dyková (2006).

3.6. Acknowledgments

The authors thank the Portuguese Foundation for Science and Technology (FCT) for funding this work under the Pluriannual Program attributed to the CIIMAR–CIMAR Associated Laboratory, the Ph.D. fellowship grant SFRH/BD/82237/2011 to L. Rangel, and Project PTDC/MAR/116838/2010.

FCUP 53 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

Chapter 4

Ortholinea auratae n. sp. (Myxozoa, Ortholineidae) infecting the urinary bladder of the gilthead seabream Sparus aurata (Teleostei, Sparidae), in a Portuguese fish farm

This chapter has been adapted from:

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. doi:10.1007/s00436-014-4008-4

FCUP 54 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

FCUP 55 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

4.1. Abstract

A new myxosporean species, Ortholinea auratae n. sp., is described from the gilthead seabream, Sparus aurata Linnaeus, 1758 (Teleostei, Sparidae) from a fish farm in Algarve, Portugal. Plasmodia and spores were found in the urinary bladder and, less frequently, in the posterior kidney. Plasmodia were polymorphic, presenting an irregular cellular membrane due to the presence of several peripheral projections, which in turn were covered by a glycocalyx-like sheet. Mature spores were subspherical in valvular view and ellipsoidal in sutural view, measuring 9.0±0.3 (8.2–10.1) μm in length, 8.3±0.4 (7.5–9.1) μm in width, and 7.2±0.5 (6.3–8.4) μm in thickness. The two valves comprising the spores displayed an intricate pattern of surface ridges and were also enveloped by a glycocalyx-like sheet. Two subspherical polar capsules, 3.2±0.1 (2.9-3.6) μm long and 2.7±0.1 (2.4–2.9) μm wide, were located at the anterior pole and displayed divergent orientation. The polar filament coiled in three to four turns. The comprehensive analysis of the parasite’s ultrastructural observations and molecular data for the small subunit (SSU) ribosomal DNA (rDNA) gene identify O. auratae n. sp. as a new species, clustering together with other myxosporeans infecting the excretory system to form a subclade of the main freshwater clade.

FCUP 56 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

FCUP 57 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

4.2. Introduction

The gilthead seabream, Sparus aurata Linnaeus, 1758, is a teleost fish of great economic value in the Mediterranean and Portuguese coasts. Several studies have been conducted on the microorganisms parasitizing this species, mainly in the Mediterranean coast (Fioravanti et al., 2006; Merella et al., 2006; Mladineo et al., 2010); however, in Portugal, only one study from the archipelago of Madeira concerns this subject (Costa et al., 1998). Nine myxozoan species have been described infecting the gilthead seabream: Zschokkella auratis (Rocha et al., 2013), Ceratomyxa sparusaurati (Sitjà‐Bobadilla et al., 1995), Ceratomyxa sp. 1 ex S. aurata and Ceratomyxa sp. 2 ex S. aurata (Alama- Bermejo et al., 2011), all from the gallbladder; Sphaerospora sparidarum (Sitjà- Bobadilla and Álvarez-Pellitero, 2001), and Sphaerospora sparis (Sitjà-Bobadilla et al., 1992; Sitjà-Bobadilla and Álvarez-Pellitero, 1995; Bartošová et al., 2013) from the kidney; Enteromyxum leei (Diamant et al., 1994) from the intestinal tract; Henneguya sp. (Bahri et al., 1996) from the gills and heart; and Kudoa iwatai (Diamant et al., 2005), which is a systemic species. Resulting from a parasitological survey conducted in sparid fishes from a southern Portuguese fish farm, a new species belonging to the genus Ortholinea Shulman, 1962 was detected infecting the urinary bladder of S. aurata. This constitutes the first record of a myxosporean parasite described from this organ in the gilthead seabream as well as the first reference of an Ortholinea species from the Portuguese coast. The genus Ortholinea includes about 20 species, mostly coelozoic in the excretory system, with the exception of four species: Ortholinea visakhapatnamensis, which infects the visceral peritoneum (Padma Dorothy and Kalavati, 1993); Ortholinea percotti, which infects the gills and fins (Akhmerov, 1960); and Ortholinea asymmetrica and Ortholinea australis, which infect the gallbladder (Lom et al., 1992; Kovalyova et al., 1993). Ortholinea species usually infect marine fishes; only Ortholinea africanus is known to parasitize a freshwater host, the Nile tilapia Oreochromis niloticus (Abdel- Ghaffar et al., 2008). Spores are described as spherical or subspherical, slightly flattened parallel to the sutural plane or pointed posteriorly, and containing two subspherical to pyriform polar capsules and a binucleate sporoplasm (Lom and Dyková, 2006), revealing the lack of precise characteristics for morphological differentiation. In fact, phylogenetic studies have shown that spore morphology constitutes a somewhat artificial criterion for the discernment of true myxosporean FCUP 58 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata taxonomy, further revealing aquatic environment, tissue tropism, and host phylogenetic proximity as more reliable criteria for acknowledging the phylogenetic and evolutionary issues of Myxosporea (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). However, in the case of Ortholinea, GenBank provides sequences for the small subunit (SSU) ribosomal DNA (rDNA) of only three species, hindering the molecular analysis of this genus and giving new need to morphological comparison. The present study seeks to address this gap in the knowledge by providing a morphological, ultrastructural, and molecular description of the Ortholinea species found infecting the urinary bladder of S. aurata.

4.3. Material and methods

Between June 2012 and May 2013, 124 specimens of S. aurata were collected from a fish farm facility located at the Alvor Estuary, Atlantic coast (37° 8′ N, 8° 37′ W), Portimão, Algarve, Portugal. The fish were kept fresh on ice and transported to the laboratory, where dissection was performed and followed by the parasitological survey of various internal tissues and organs. The gills, fins, and eyes were also examined. Infected samples were examined and photographed using a Zeiss Axiophot microscope (Grupo Taper, Sintra, Portugal), equipped with a Zeiss AxioCam Icc3 digital camera. AxioVision 4.6 software (Grupo Taper) was used for the image analysis. Spore’s morphometry was determined from fresh material, in accordance to Lom and Arthur (1989). All measurements include the mean value±standard deviations (SDs), range of variation, and number of spores measured (range, n). Infected samples of urine and urinary bladder tissue were collected and prepared for electron microscopy and molecular procedures. For transmission electron microscopy, small fragments of the urinary bladder containing plasmodia and spores were fixed in 5 % glutaraldehyde buffered in 0.2 M sodium cacodylate (pH=7.4) for 24 h at 4 °C, washed overnight in the same buffer, and then postfixed in 2 % osmium tetroxide with the same buffer for 4 h. After dehydration in an ascending ethanol series ending in propylene oxide, the samples were embedded in EPON. Semi-thin sections were stained with methylene blueazure II. Ultrathin sections were double contrasted with uranyl acetate and lead citrate and were observed and photographed using a JEOL 100CXII TEM operated at 60 kV. FCUP 59 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

For scanning electron microscopy, free spores were isolated from infected urine and fixed in 5 % glutaraldehyde buffered in 0.2 M sodium cacodylate (pH=7.4) for 20 h at 4 °C, washed in the same buffer at the same temperature, and postfixed in 2 % osmium tetroxide with the same buffer for 3 h. The samples were then dehydrated in an ascending ethanol series, critical point dried, coated with a gold-palladium alloy (60 %), and observed and photographed in a JSM-6301 F SEM operated at 15 kV. For the molecular analysis, DNA was extracted from spores preserved in ethanol using a DNeasy™ tissue kit (animal tissue protocol; Qiagen, Germany) according to the manufacturer’s instructions. For the amplification of the SSU rDNA, nested PCR was performed. For the first PCR, a set of universal eukaryotic primers was used (Table 4.1). The total volume of the PCR reaction was 25 μl and comprised 2 μl of genomic DNA, 5 μl of 1 mM deoxynucleotides (dNTPs) (MBI Fermentas), 0.25 μl of each primer, 2.5 μl of 10× Taq buffer (MBI Fermentas), 0.1 μl of Taq polymerase (2 U; MBI Fermentas), and 15 μl of water. The following profile was used to amplify the SSU rDNA region: an initial denaturation step at 95 °C for 3 min; 35 cycles at 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min; and termination with an extension period of 72 °C for 7 min, before storage at 4 °C. Second PCR was performed using myxosporean-specific primers (Table 4.1). PCRs were conducted with a volume of 50 μl consisting of 1 μl of amplified DNA, 10 μl of 1 mM dNTPs (MBI Fermentas), 0.5 μl of each primer, 5 μl of 10× Taq buffer (MBI Fermentas), 0.2 μl of Taq polymerase (2 U; MBI Fermentas), and 33 μl of water. Amplification was carried out using the following profile: 95 °C for 3 min; 35 cycles at 95 °C for 50 s, 50 °C for 50 s, and 72 °C for 1 min 40 s; and termination with an extension period of 72 °C for 7 min, before storage at 4 °C.

Table 4.1 – Primers used for PCR and sequencing.

Primer Sequence (5'–3') Application Source

ERIB1 ACC TGG TTG ATC CTG CCA G 1st round PCR Barta et al. (1997)

ERIB10 CTT CCG CAG GTT CAC CTA CGG 1st round PCR Barta et al. (1997)

Myx1F GTG AGA CTG CGG ACG GCT CAG 2nd round PCR Hallett and Diamant (2001)

2nd round PCR and Eszterbauer and Székely SphR GTT ACC ATT GTA GCG CGC GT sequencing (2004)

MC5 CCT GAG AAA CGG CTA CCA CAT CCA Sequencing Molnár et al. (2002)

MB5r ACC GCT CCT GTT AAT CAT CAC C Sequencing Eszterbauer (2004)

MB5f GAT GAT TAA CAG GAG CGG TTG G Sequencing Eszterbauer (2004)

ACT1fr TTG GGT AAT TTG CGC GCC TGC TGC C Sequencing Hallett and Diamant (2001)

FCUP 60 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

PCR cycles were run in a PTC-200 thermocycler (MJ Research). The PCR products were electrophoresed through a 1 % agarose 1× Tris-acetate-EDTA (TAE) buffer gel stained with 1% ethidium bromide. Amplified DNA was purified using the EZ-10 Spin Column PCR Purification Kit (Bio Basic Inc.). Purified PCR products were sequenced with the primers listed in Table 4.1, using the ABI BigDye Terminator v3.1 Cycle Sequencing Kit with an ABI 3100 Genetic Analyzer. Phylogenetic analysis was performed using the obtained SSU rDNA sequence of the isolate and other myxosporean SSU rDNA sequences retrieved from GenBank, namely, those presenting the highest similarity score in Basic Local Alignment Search Tool (BLAST). Also included in the analysis were all Ortholinea SSU rDNA sequences available in GenBank as well as the SSU rDNA sequences of the other myxosporean species that have been described from the gilthead seabream and other representative species of myxosporean phylogeny. Tetracapsuloides bryosalmonae and Buddenbrockia plumatellae were selected as out-group species. To ensure the accuracy of the analysis and to maintain a high tree resolution, sequences with less than 1,000 bp were not used, avoiding loss of information due to shortening of the aligned sequences and the appearance of numerous gaps (Liu et al., 2010). Alignments were performed with ClustalW in MEGA 5.05 software (Tamura et al., 2011), with an opening gap penalty of 10 and a gap extension of 4 for both paired and multiple alignments, resulting in a total of 2,481-bp informative characters in the final dataset. Subsequent phylogenetic and molecular evolutionary analyses were conducted using MEGA 5.05, using maximum parsimony (MP), neighborjoining (NJ), and maximum likelihood (ML) methodologies. For MP, the close neighbor-interchange heuristic option with a search factor of 1 and random initial tree addition of 500 replicates was performed. For NJ, a Kimura two-parameter substitution model with gamma distribution (shape parameter=1.4) was performed. For ML, the general time- reversible substitution model with four gamma-distributed rate variations among sites was performed. All positions with less than 95 % site coverage were eliminated from all trees, resulting in a total of 896 positions in the final dataset. The bootstrap consensus tree was inferred from 100 replicates for MP and 500 replicates for NJ and ML. The second alignment was performed for the SSU rDNA sequence of the case isolate and the SSU rDNA sequences presenting the highest similarity score in BLAST (clustering in the same clade of the case isolate), resulting in a total of 2,129-bp positions in the final dataset. Distance estimation was carried out in MEGA 5.05, using the Kimura two-parameter model distance matrix for transitions and transversions, with all ambiguous positions removed for each sequence pair.

FCUP 61 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

4.4. Results

The examination of S. aurata specimens revealed the presence of a coelozoic myxosporean parasite in the urinary bladder (Fig. 4.1). Plasmodia and mature spores were observed floating free in the urine and, in more intense infections, covering the inner wall of the urinary bladder (Fig. 4.1a). Infection was also found in the terminal portion of the posterior kidney. Prevalence of infection was determined at 51.6% (64 infected in 124 examined specimens). No symptoms or macroscopic signs of infection were observed.

Morphologic description

Plasmodia were polymorphic, the most common shape being rounded (Fig. 4.1a–d), and displayed an irregular cellular membrane due to the presence of several long and thin peripheral projections (Figs. 4.1b and 4.2a–c), which appeared covering the entire surface of these structures or projecting from only a portion of the plasmodial membrane. A glycocalyx-like sheet enveloped the entire surface of the plasmodia, including the peripheral projections (Figs. 4.2b, c and 4.3e). The ectoplasmic margin was transparent and void of organelles, while the endoplasm was denser and granular, presenting several vegetative nuclei, vesicles, and numerous mitochondria (Figs. 4.1c, d and 4.2b, c). Plasmodia were polysporous (Fig. 4.1d) and, less frequently, disporous (Fig. 4.1c). Plasmodia containing the earliest stages of sporogenic development ranged between 15.0–42.9 × 14.2–34.4 μm (n=30), while mature plasmodia, containing developing sporoblasts and immature spores (Fig. 4.3a–c), ranged between 24.3−55.4 × 22.1–39.6 μm (n=10). Mature spores were subspherical in valvular view (Fig. 4.1e, f) and ellipsoidal in sutural view (Fig. 4.1e), measuring 9.0±0.3 μm (8.2–10.1; n=62) in length, 8.3±0.4 μm (7.5–9.1; n=42) in width, and 7.2±0.5 μm (6.3–8.4; n=26) in thickness. The two valves comprising the spore’s wall were united along a straight and evident suture line and presented an intricate pattern of surface ridges (Fig. 4.4a, b), with few variations between spores. Each valve displayed a minimum of 19 ridges. The sutural ridge was visible and flanked on each side by three to four parallel circular extrasutural ridges. At the center of the valves, the ridges coiled concentrically (Fig. 4.4b) or instead formed a set of extrasutural central ridges running longitudinally and/or diagonally (Fig. 4.4a). Similar to the plasmodia cellular membrane, the spore’s wall also appeared enveloped by a glycocalyx-like sheet (Fig. 4.3d, e). FCUP 62 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

Fig. 4.1 – Plasmodia and spores of Ortholinea auratae n. sp. from the urinary bladder of Sparus aurata as observed using the differential interference contrast optics. a) Cluster of plasmodia. b) Young plasmodium displaying a highly irregular cellular membrane, due to the presence of numerous peripheral projections. c) Mature disporic plasmodium containing a disporic pansporoblast. d) Mature polysporic plasmodium containing many developing spores. e) Mature spores in valvular view and one in sutural view (arrow). f) Mature spore in valvular view, displaying a visible intercapsular process.

Spores contained two equal subspherical polar capsules, 3.2±0.1 μm long (2.9−3.6; n=60) and 2.7±0.1 μm wide (2.4–2.9; n=64), presenting divergent orientation and opening to opposite sides of the suture line. A conspicuous intercapsular process was observed (Fig. 4.1e, f). The polar capsules displayed a double-layered wall, comprised of a thin outer electron-dense layer and a larger inner electron-lucent layer surrounding a dense and slightly heterogenous matrix (Fig. 4.3c, f). The polar filament coiled in three to four turns and, when extruded, measured 23.6±1.1 μm (22.2–25.1; n=6) in length. The sporoplasm was binucleated, contained several electrondense sporoplasmossomes, and lacked an iodinophilous vacuole (Fig. 4.3c).

FCUP 63 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

The ultrastructural features here described are represented in a schematic drawing (Fig. 4.5), providing better perception of the spore’s morphology.

Taxonomic summary

Parasite species: Ortholinea auratae n. sp. (Ortholineidae). Type host: S. aurata Linnaeus, 1758 (Teleostei, Sparidae). Site of infection: Urinary bladder and terminal portion of the posterior kidney. Type locality: Alvor Estuary, Atlantic coast (37°08′N, 8°37′W), Portimão, Algarve, Portugal. Prevalence: Rate of 51.6 % (64 out of 124). Pathology: No symptoms or macroscopic signs of infection were evident. Etymology: The specific epithet auratae refers to the piscine host S. aurata. Materials deposited: Slides containing semi-thin sections of mature plasmodia and spores were deposited in our institution collection CIIMAR, PT, reference: CIIMAR 2013.2

FCUP 64 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

FCUP 65 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

Fig. 4.4 – Scanning electron micrographs of Ortholinea auratae n. sp. Fig. 4.5 – Schematic drawing of the from the urinary bladder of Sparus auratus. a) Spore in frontal valvular spore of Ortholinea auratae n. sp. view, allowing recognition of the surface pattern formed by the ridges. b) Spore in slightly oblique sutural view, evidencing the suture line (arrowheads) and the organization of the surface ridges.

Phylogenetic analyses

The resulting sequences for the SSU rDNA gene, obtained from plasmodia and spores collected from three infected fish specimens, were sequenced and deposited in GenBank under the accession numbers KF703856, KF703857, and KF703858. The three sequences obtained were identical and based on 1,546, 1,660, and 1,689 bp, respectively. Pairwise distances among the SSU rDNA sequences showed the highest similarity to Acauda hoffmani (89.7 %), Myxobilatus gasterostei (85.8 %), Hoferellus gilsoni (79.3 %), Zschokkella sp. AH2003 (77.7 %), and Ortholinea orientalis (79.9 %). MP/NJ/ML analyses of the SSU rDNA sequences placed the parasite within the excretory system clade of the major freshwater clade, alongside the sequences of A. hoffmani, H. gilsoni, M. gasterostei, Zschokkella sp. AH2003, and O. orientalis, with a bootstrap support value of 67 % for MP, 84 % for NJ, and 55 % for ML (Fig. 4.6). These five myxosporean species infect the kidney, urinary bladder, and ureters of freshwater fish, with the exception of O. orientalis, which infects the ureters of two marine fish hosts, Clupea harengus Linnaeus, 1758 and Sprattus sprattus Linnaeus, 1758. The remaining two sequences of Ortholinea available in GenBank, Ortholinea sp. AL-2006, and Ortholinea sp. HS-2012, which were described from the urinary bladder and kidney of the marine fish Siganus rivulatus Forsskål and Niebuhr (1775), respectively, cluster together at the basis of the freshwater excretory system clade. The freshwater histozoic clade is represented by Myxobolus and Henneguya: one subclade histozoic in the gills, one subclade histozoic in internal organs, and one subclade histozoic in the FCUP 66 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata nervous system of anadromous Salmoniformes. The coelozoic freshwater clade is represented by freshwater Zschokkella, except the marine Z. auratis, and two Sphaerospora species that infect the kidney of their hosts. Forming a more ancient clade at the basis of the main freshwater clade are Chloromyxum leydigi and Chloromyxum clavatum. The marine excretory system clade is represented by members of Zschokkella, Sphaerospora, and one Sinuolinea species; the marine histozoic clade is represented by Enteromyxum and the marine coelozoic clade by Ceratomyxa, Zschokkella, and Ellipsomyxa.

4.5. Discussion

In acknowledging the difficulties inherent to the differentiation of genera belonging to the family Ortholineidae (Lom and Noble, 1984; Lom and Dyková, 2006), the present study uses combined morphological, ultrastructural, and molecular features to describe the myxosporean parasite infecting the urinary bladder of S. aurata. Considering that the gilthead seabream inhabits both brackish and marine habitats and that molecular studies suggest tissue tropism as a phylogenetic trend for myxosporeans (Eszterbauer, 2004; Holzer et al., 2004; Fiala, 2006; Freeman et al., 2008; Bartošová et al., 2011), only the records of Ortholinea from the excretory system, both freshwater and marine, were considered for morphological comparison. Also, several studies report surface ridges as a reliable feature for morphological differentiation (Lom and Dyková, 1993, 2006; Bartošová and Fiala, 2011), including for members of Ortholinea (Ali, 2000, 2009); therefore, species with smooth valves were not compared. Among the 20 known Ortholinea species, there are only five species with similar spore dimensions that infect the excretory system and have surface ridges, differing from O. auratae n. sp. in several aspects of the morphometry (Table 4.2) as well as in the ultrastructural detail of the surface ridges, polar capsules, and suture line. For instance, Ortholinea macruri and Ortholinea striateculus present pyriform polar capsules; Ortholinea undulans also presents pyriform polar capsules, as well as an undulating suture line; and Ortholinea fluviatilis displays a slightly undulating suture line (Meglitsch, 1970; Kovalyova et al., 1993; Su and White, 1994; Lom and Dyková, 1995). Turning to consider the ultrastructure of the sporogenic development, O. auratae n. sp. displays pansporoblast formation, thus following what it seems to be the tendency for Ortholinea species, despite the few studies concerning this subject (Padma Dorothy and Kalavati, 1993; Lom and Dyková, 1995; Sarkar, 1999; Ali, 2000; Moshu and Trombitsky, 2006). Only O. asymmetrica is known to form spores directly FCUP 67 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata from the generative cells (Kovalyova et al., 1993). The plasmodial development of O. auratae n. sp. is similar to those usually recognized in coelozoic myxosporeans, since it is characterized by variations in size and the differentiation of peripheral projections, supposedly for the improvement of nutritional intake (Sitjà-Bobadilla and Álvarez- Pellitero, 1993e, 2001; Rocha et al., 2011). On the other hand, the glycocalyx-like sheet covering the plasmodia, as well as the spores, constitutes a rather distinctive feature, which purpose remains unknown, but may be involved in cellular strengthening towards the host immune response as well as the physical and biological conditions of the organ of infection (Rocha et al., 2013). Molecular comparison between members of the genus Ortholinea is hampered by the scarcity of data available in GenBank, which provides sequences for the SSU rDNA of only three Ortholinea species. O. auratae n. sp. showing higher phylogenetic similarity to species of other genera, rather than to the few of its own genus, not only reveals the pronounced lack of data but also demonstrates the significant influence of other phylogenetic criteria compared to the morphologic criterion. Concerning the molecular based division of myxosporean into the major freshwater and marine clades (Kent et al., 2001; Fiala, 2006; Bartošová et al., 2009), the Ortholinea species here analyzed, including O. auratae n. sp., constitute exceptions for this main division. It is not a novelty for marine species to cluster within the major freshwater clade, as it occurs for several species of the genera Henneguya, Chloromyxum, Ceratomyxa, Myxobolus, Myxidium, Parvicapsula, and Sphaeromyxa (Fiala, 2006; Azevedo et al., 2009; Rocha et al., 2013) as well as for freshwater species to cluster within the major marine clade, as is the case of Ceratomyxa shasta (Kent et al., 2001; Fiala, 2006; Fiala and Bartošová, 2010). There are several possible explanations for these exceptions, the main being the migratory pattern of the fish host (namely, for parasites infecting anadromous fishes) (Kent et al., 2001; Fiala, 2006) and the myxosporean evolution between aquatic environments as inferred with basis on morphologic characters combined with molecular data (Fiala and Bartošová, 2010; Bartošová et al., 2011). Tissue tropism also constitutes a criterion influencing myxosporean phylogeny (Eszterbauer, 2004; Holzer et al., 2004; Fiala, 2006; Freeman et al., 2008; Bartošová et al., 2011; Dyková et al., 2013) and is followed by O. auratae n. sp., which clusters together with other species that infect the excretory system. In fact, the analysis here presented supports this phylogenetic trend throughout, excepting only two species, Sphaerospora elwhaiensis and Sphaerospora sp. Hungary EE-2004, which infect the kidney of their hosts (Eszterbauer and Székely, 2004; Jones et al., 2011), but cluster within the freshwater coelozoic clade. FCUP 68 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

FCUP 69 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

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Meglitsch

Su and WhiteSu and

Kovalyova Kovalyova

Lomand Dyková

(

Abdel

Present study

Source

–

—

4 (rarely 5)

nFC

.1 .1

3.0)

3.1)

3.3)

3.9)

2.9)

–

re given in µm. in given re

(2.0

2.2

(2.8

2.9

2.6

(2.8

3.1

(2.3

2.9 ± 0.2

(2.4

2.7 ± 0

PCW

4.0)

3.6)

3.3)

3.9)

3.6)

3.9

–

(2.0

2.9

(3.4

3.5

3.3

(2.8

3.1

(2.3

2.9 ± 0.2

(2.9

3.2 ± 0.1

PCL

ory system of freshwater ory system and of marine freshwater fish. SL: SW: spore spore length; width;

8.0)

4.6)

8.4)

7.0

–

(5.0

6.3

—

6.5

6.8

(3.9

4.1 ± 0.4

(6.3

7.2 ± 0.5

9.0)

10.4)

8.0)

8.5)

9.1)

9.0

–

(6.0

7.4

(8.9

10.0

8.0

(7.3

7.8

(6.9

7.7 ± 0.3

(7.5

8.3 ± 0.4

10.0)

10.5)

8.4)

8.5)

10.1)

10.6

–

(7.0

8.3

(9.1

9.3

(7.9

8.3

(6.9

7.7 ± 0.2

(8.2

9.0 ± 0.3

Zealand

New New

Australia

Namibia

Asia

Egypt

Portugal

Location

and and

Caulopsetta scapha

novaezelandiae

Peltorhamphns

Leptatherina presbyteroides

Coelorinchusfasciatus

Tetraodon fluviatilis

Oreochromis niloticus

Sparus aurata

Hosts

spp. characterized by characterized the spp. ridges of surface presence and from the described excret

spp.

n. sp.n.

Ortholinea

–

O. undulans O.

O. striateculus O.

O. macruriO.

O. fluviatilis O.

O. africanus O.

O. auratO.

Ortholinea

Table Table 4.2 a Measurements coils. filament polar of number nFC: width; capsule polar PCW: length; capsule polar PCL: thickness; ST: spore

FCUP 70 Chapter 4. Ortholinea auratae n. sp. from the gallbladder of Sparus aurata

More recently, molecular studies have further renewed the contention that host phylogenetic proximity also influences species clustering, therefore playing an important role in myxosporean phylogeny (Carriero et al., 2013; Moreira et al., 2013). The molecular analysis here performed concurs with species clustering by host family and order, especially in the clades containing Henneguya and Myxobolus species, probably because these two genera possess more adequate comparative molecular data. In the case of Ortholinea, the few molecular data available are insufficient to provide information on this evolutionary signal. Although Ortholinea sp. AL-2006 and Ortholinea sp. HS-2012 cluster together, having both been described from a fish host of the family Siganidae, these species are, in fact, parasites of the same host species, S. rivulatus, making illations at the family level void of significance. Overall, the paucity of reliable molecular data for most myxosporean genera cannot be ignored when substantiating hypotheses, especially for poorly studied genera such as Ortholinea, for which the acquisition of additional data is crucial.

4.6. Acknowledgments

This work was financially supported by FCT (Portugal), within the scope of the project DIRDAMyx, reference FCOMP-01-0124-FEDER-020726/FCT-PTDC/MAR/116838/ 2010 and the Ph.D. fellowship grant SFRH/BD/82237/2011 to L. Rangel; the Eng. António de Almeida Foundation (Portugal); and partially by the grants of the Hungarian Government, managed by the National Development Agency and financed by the Research and Technology Innovation Fund (KTIA AIK-12-1-2013-0017) and OTKAK100132. This work complies with the current laws of the country where it was performed.

FCUP 71 Chapter 5. The life cycle of Ortholinea auratae

Chapter 5

The life cycle of Ortholinea auratae (Myxozoa: Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete

This chapter has been adapted from:

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. doi:10.1007/s00436-015- 4472-5

FCUP 72 Chapter 5. The life cycle of Ortholinea auratae

FCUP 73 Chapter 5. The life cycle of Ortholinea auratae

5.1. Abstract

Actinospores released from the marine oligochaete Limnodriloides agnes inhabiting a Southern Portuguese fish farm are molecularly recognized as developmental stages of the life cycle of Ortholinea auratae, a myxosporean parasite that infects the urinary bladder of Sparus aurata. The molecular analysis of the 18S rRNA gene reveals a similarity of 99.9 to 100 % of the actinospores analyzed to the myxospores of O. auratae. The actinospores belong to the triactinomyxon morphotype and occur in groups of eight within pansporocysts that develop in the intestinal epithelium of the oligochaete host. This is the first record of a myxosporean using an oligochaete as its invertebrate host in the marine environment.

FCUP 74 Chapter 5. The life cycle of Ortholinea auratae

FCUP 75 Chapter 5. The life cycle of Ortholinea auratae

5.2. Introduction

Since Wolf and Markiw (1984) revealed the involvement of an invertebrate host in the life cycle of Myxobolus cerebralis, several other studies have aimed to clarify the complete life cycles of myxosporean parasites. Initially, these studies relied solely in experimental infections. This methodology is, however, laborious and time consuming (Székely et al., 2014), and the reliability of its results has been discredited by the use of molecular tools (Holzer et al., 2004; Marton and Eszterbauer, 2011). Nowadays, both the description of myxosporean parasites and the understanding of its complex life cycles rely on molecular methodologies (Eszterbauer et al., 2006; Caffara et al., 2009; Karlsbakk and Køie, 2012; Køie et al., 2013; Székely et al., 2014). Although there are about 2310 myxosporean parasites described (Lom and Dyková, 2006; Morris, 2012), only about 40 have their complete life cycle elucidated (Lom and Dyková, 2006; Yokoyama et al., 2012; Székely et al., 2014). Of these, six correspond to myxosporean parasites inhabiting the marine environment, all of which use polychaetes as their invertebrate hosts: Ellipsomyxa gobii Køie, 2003 and E. mugilis (Sitjà-Bobadilla and Álvarez-Pellitero, 1993e) infect Nereis spp. (Køie et al., 2004; Rangel et al., 2009); Gadimyxa atlantica Køie, Karlsbakk and Nylund, 2007 infects species of Spirorbis (Køie et al., 2007); Ceratomyxa auerbachi (Noble, 1950) infects Chone infundibuliformis Krøyer 1856 (Køie et al., 2008); Sigmomyxa sphaerica Karlsbakk and Køie 2012 infects Nereis pelagica Linnaeus, 1758 (Karlsbakk and Køie, 2012); and Parvicapsula spp. infect Hydroides norvegicus Gunnerus, 1768 (Køie et al., 2013). Species of the myxosporean genus Ortholinea are mainly parasites of the kidneys, urinary ducts, and urinary bladder of fish hosts, mostly marine. Although they infect fish species belonging to several taxonomic orders, there are more descriptions among the Perciformes (Sarkar, 1999; Ali, 2000; Abdel-Ghaffar et al., 2008; Karlsbakk and Køie, 2011). Ortholinea auratae Rangel et al., 2014 was recently described infecting the urinary bladder and posterior kidney of the gilthead seabream, Sparus aurata Linnaeus, 1758, with a prevalence of infection of 51.6 % (Rangel et al., 2014). The study here presented not only discloses the first complete life cycle of a parasite of the genus Ortholinea, as well as of the family Ortholineidae, as it also constitutes the first record of a myxosporean using an oligochaete as its invertebrate host in the marine environment.

FCUP 76 Chapter 5. The life cycle of Ortholinea auratae

5.3. Material and methods

An oligochaete survey was conducted in a fish farm located at the Alvor Estuary, Atlantic coast (37°8'N, 8°37'W), Portimão, Algarve, Portugal. Oligochaetes were collected from mud samples of the ponds containing European seabass and gilthead seabream, as well as from the water outlet ditches, and from an area of the Alvor Estuary that is located downstream to the fish farm. The collected oligochaetes were kept individually in cell well plates containing salt water, and a stereo microscope was used to examine the specimens for the release of actinospores during the following days. Fresh preparations of all oligochaetes, releasing and not releasing actinospores, were performed using water, and examined using a light microscope to detect the presence of actinospores in the internal tissues and coelomic cavity, as well as to register possible co-infections. The identification of host species was performed by morphological examination of preserved specimens of oligochaetes in 70 % ethanol by Dr. Rüdiger Schmelz, from the ECT Oekotoxikologie and Dr. Enrique Martínez-Ansemil from the University of Coruña (personal communication). 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) was used for the image analysis. Morphology and morphometry were obtained from fresh material, in accordance to Lom et al. (1997). All measurements include the mean value±standard deviations (SDs), range of variation, and number of actinospores measured (range, n). For transmission electron microscopy (TEM), infected oligochaetes were fixed in 5% glutaraldehyde buffered in 0.2M sodium cacodylate (pH=7.4) for 24 h at 4 °C, washed for 12 h in the same buffer, and then postfixed in 2% osmium tetroxide with the same buffer for 4 h. The samples were then dehydrated in an ascending ethanol series, followed by embedding using a series of propylene oxide and EPON mixtures, ending in EPON. Semi-thin sections were stained with methylene blue-Azure II. Ultrathin sections were double-contrasted with uranyl acetate and lead citrate and were observed and photographed using a JEOL 100CXII TEM operated at 60 kV. For the molecular analysis, free actinospores and infected oligochaetes were preserved in absolute ethanol. Genomic DNA was extracted using a GenElute™ Mammalian Genomic DNA Miniprep Kit, following the manufacturer's instructions. Additionally, genomic DNA was also extracted from myxospores of O. auratae collected from the urinary bladder of infected specimens of S. aurata sampled from the FCUP 77 Chapter 5. The life cycle of Ortholinea auratae same fish farm. The parasites' SSU ribosomal RNA (rRNA) gene was amplified using both universal primers and myxosporean-specific primers (Table 5.1). For the oligochaete host DNA, the universal primers 16sar-L and 16sbr-H (Palumbi et al., 2002) were used (Table 5.1). PCRs were performed in 50 μl reactions using 10 pmol of each primer, 10 nmol of each dNTP, 2.5 mM MgCl2, 5 μl of 10× Taq polymerase buffer (Finnzymes), 1.5 units of Taq DNA polymerase, and approximately 100–150 ng of genomic DNA. The reactions were run on a Hybaid PxE Thermocycler, 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- microliter aliquots of the PCR products were electrophoresed through a 1 % agarose 1× tris-acetate-EDTA buffer (TAE) gel stained with ethidium bromide. The sequencing reactions were performed using a BigDye Terminator v1.1 from the Applied Biosystems Kit and were run on an ABI3700 DNA analyzer (Perkin-Elmer, Applied Biosystems).

Table 5.1 Primers used for DNA amplification and sequencing.

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

18e CTG GTT GAT CCT GCC AGT MyxospecR, ACT3r Hillis and Dixon (1991)

MyxospecF TTC TGC CCT ATC AAC TTG TTG ChloromyxR1, 18r Fiala (2006)

MyxospecR CAA CAA GTT GAT AGG GCA GAA 18e Fiala (2006) Hallett and Diamant ACT3f CAT GGA ACG AAC AAT 18r (2001) Hallett and Diamant ACT3r ATT GTT CGT TCC ATG 18e (2001) MYX4F GTT CGT GGA GTG ATC TGT CAG 18r Rocha et al. (2015)

ChloromyxR1 CCT TCC GTC AAT TCC TTT AAG MyxospecF Azevedo et al. (2009)

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

16sar-L CGC CTG TTT ATC AAA AAC AT 16sbr-H Palumbi et al. (2002)

16sbr-H CCG GTC TGA ACT CAG ATC ACG T 16sar-L Palumbi et al. (2002)

5.4. Results

A total of 878 specimens of the same oligochaete species were microscopically examined for myxosporean infection, with 16 (1.8 %) presenting parasitic developmental stages in the intestinal epithelium and releasing actinospores of the triactinomyxon morphotype. The prevalence of infection varied according to the collection site, being highest in the ponds from the fish farm that contained gilthead seabream (16.0 %) and lowest in the ponds containing European seabass (1.5 %). No infection was detected in the oligochaetes collected from the water outlet ditches and FCUP 78 Chapter 5. The life cycle of Ortholinea auratae directly from the Alvor Estuary. All infected oligochaetes were immature individuals. The parasitic developmental stages were located in the oligochaete intestinal epithelium (Fig. 5.1d), causing hyperplasia, which appeared more evident in the most advanced stages of development. The oligochaete host was morphologically identified as Limnodriloides agnes Hrabě, 1967 (Dr. Rüdiger and Dr. Martínez, personal communication). The BLAST search for the 18S (KR025870) and 16S (KR025871) rDNA confirmed that the host belonged to the genus Limnodriloides. The microscopic observations performed revealed the actinospores displaying the anchor form that is typical of the triactinomyxon morphotype (Figs. 5.1a and 5.3a). Free mature triactinospores did not form nets and presented three prominent pyramidal polar capsules (Fig. 5.1b) that measured 5.2±0.3 μm (4.4–5.8; n=62) in length and 3.6 ±0.2 μm (3.1–4.2; n=62) in width. Each polar capsule contained a polar filament coiling in five to six coils, helicoidally arranged, and measuring 37.9±5.0 μm (29.0–45.8; n=12) in length when extruded. The triactinospores body measured 49.9±5.2 μm (40.9–62.2; n=30) in length and 12.6±0.9 μm (10.9–14.4; n=44) in width. The sporoplasms observed displayed at least 188 secondary cells (Fig. 5.1b, c). The triactinospores presented a style, 83.1±9.0 μm (67.9–97.8; n=29) long and 13.2±1.3 μm (11.0–16.9; n=46) wide, and from its base departed three caudal processes, slightly curved upward and measuring 215.1±29.6 μm (141.0–287.6; n=114) in length and 13.3±1.3 μm (10.2−18.0; n=108) in width near its base. The caudal processes formed an angle of 120.1°±17.4 (90.0–150.0; n=37) to the style axis. The total length of the triactinospores was 136.2±9.4 μm (115.2–152.6; n=47) with a total span of 435.9±56.7 μm (268.4−512.7; n=33). The triactinospores developed in groups of eight inside pansporocysts formed by two somatic cells united by cell junctions (Fig. 5.2a). The internal cell membranes of the somatic cells showed a greater abundance of stereocilia in the areas surrounding the cell junctions (Fig. 5.2a, c). In the cytoplasm, electron-dense mitochondria with few cristae and numerous vesicles, many of the phagosoma type, were observed. At the initial stages of sporogony, the valvogenic cells start to surround the capsulogenic cells, keeping the majority of the sporoplasm free. The latter remains connected to the valvogenic cells by a small apical zone (Fig. 5.2b), until the valvogenic cells completely surround and close both the capsulogenic cells and the sporoplasm within an involucre. During this process, the cytoplasm of the valvogenic cells, initially rich in mitochondria and with large nuclei with eccentric nucleoli, loses organelles almost until the cells become reduced to the two cytoplasmic membranes. Before the complete enclosing by the valvogenic cells, the polar capsules start to develop inside the capsulogenic cells with the formation of the capsular primordium FCUP 79 Chapter 5. The life cycle of Ortholinea auratae

(Fig. 5.2c). At this stage, the cells are still large with a large nucleus and with a cytoplasm rich in mitochondria, several vesicles and other cell organelles (Fig. 5.2b, c). The formation of the polar capsules is asynchronous (Figs. 5.2d, e). More mature capsulogenic cells has full developed polar capsules and a cytoplasm with several large mitochondria with a very typical matrix (Fig. 5.2e).

The sporoplasm displayed electron-dense mitochondria and numerous vesicles. Its development and the formation of secondary cells start even before the sporoplasm is enclosed by the valvogenic cells. Initially, the secondary cells are large with numerous mitochondria and large nuclei with nucleoli. As they mature, the secondary cells increase in number but decrease in size and become very electron-dense (Fig. 5.2f). When completely mature, the triactinospores are released into the gut lumen, being posteriorly discharged into the water column.

FCUP 80 Chapter 5. The life cycle of Ortholinea auratae

Fig. 5.2. – Transmission electron micrographs of the parasitic development of Ortholinea auratae infecting the intestinal epithelium of Limnodriloides agnes. a) Gametogonic stage: a pansporocyst formed by two enveloping cells (Pa) united by cellular junctions (arrows) and surrounding four generative cells (asterisk) rich in mitochondria (Mt). The cytoplasm of the enveloping cells shows mitochondria and large vesicles (Vs). b) Sporogonic stage: pansporocyst showing three developing spores. The three capsulogenic cells (CC) appear totally or partially involved by the three valvogenic cells (asterisk). The latter are united by cell junctions (arrowheads). The sporoplasmogenic cell (Sp), showing a secondary cell (SC) and its nucleous (N), is connected to the set of valvogenic cells through a small apical zone (arrows). c) Sporoblasts displaying three capsulogenic cells (CC) with its capsular primordia (PCP) not yet totally involved by the valvogenic cells; see the cellular junctions (arrowheads) and the terminal ends (arrows). The sporoplasmogenic cell (Sp) is still outside the triactinospore involucre. d) Cross section of the apical part of a developing triactinospore showing the three capsulogenic cells (CC) containing numerous mitochondria (Mt) in their cytoplasm. Two capsulogenic cells displaying the polar filament coiled within the maturing body of the polar capsules, while the third still displays the external tube (ET) that is precursor to the polar filament. These cells are involved by the valvogenic cells, which appear united by cellular junctions (arrows). e) Detailed aspect of a capsulogenic cell showing its cytoplasm containing large mitochondria (Mt) and an almost matured polar capsule (PC) presenting its polar filament coiled within. f) Detail of the sporoplasm (Sp) displaying numerous secondary cells (SC) and mitochondria (Mt).

FCUP 81 Chapter 5. The life cycle of Ortholinea auratae

Sequencing of the PCR products obtained for the myxospores and the actinospores of O. auratae resulted in the construction of two consensus rRNA sequences comprised by 2046 bp (KR025868) and 2031 bp (KR025869), respectively. These two sequences present a similarity of 99.9 %, thus identifying the two myxosporean stages as belonging to the same species, and thus closing its life cycle (Fig. 5.3). The SSU rRNA sequence obtained for the actinospores also presents a similarity percentage of 100, 99.9, and 100 % to the other sequences of O. auratae available in GenBank: KF703856, KF703857, and KF703858, respectively.

5.5. Discussion

This study revealed actinospores of the triactinomyxon morphotype as developmental stages of the life cycle of O. auratae, a myxosporean parasite that infects the urinary bladder and posterior kidney of S. aurata. The comparative analysis of the SSU rRNA sequences obtained for the triactinospores developing in the oligochaete L. agnes and for the myxospores of O. auratae revealed a percentage of similarity (99.9 to 100 %) that confirmed them as alternating stages in the life cycle of O. auratae. Thus far, only three actinospores of the triactinomyxon morphotype have been described from the marine environment, all without molecular data reliable for comparative purposes. Thus, its comparison to the triactinospores here described can only be based in morphological aspects, which differ significantly, showing no correspondence between them. Overall, the triactinospores of Ortholinea auratae FCUP 82 Chapter 5. The life cycle of Ortholinea auratae present smaller measurements than the triactinospores type 1 and type 2 described by Hallett et al. (2001). Despite its length agreeing with the dimensions of the triactinospores described by Roubal et al. (1997), they differ by its shorter caudal processes (Table 5.2).

Table 5.2 – Morphological comparison of the triactinospores of Ortholinea auratae to all other described marine triactinospores (the mean±SD with the range in parentheses; in μm) Character Ortholinea Triactinomyxon Triactinomyxon Triactinomyxon auratae type of Roubal et type 1 of Hallett et type 2 of Hallett al., 1997 al., 2001 et al., 2001

Spore length 136.2±9.4 125.0 236 ~346 (115.2–152.6) (96.0–142.0)ab (208–268)

Spore body length 49.9±5.2 — 34 — (40.9–62.2) (32–44)

Spore body width 12.6±0.9 (7.7–21.6)a — ~38 (10.9–14.4)

Caudal processes 215.1±29.6 138.0 296 ~517 length (141.0–287.6) (94.0–185.0)ab (240–360)

Caudal processes 120.1±17.4 (90.0–100.0)b 90 — angle with style (90.0–150.0)

Polar capsules 5.2±0.3 4.0 (8–11) ~8 length (4.4–5.8) (3.0–5.0)ab

Source Present study aRoubal et al. Hallett et al. (2001) Hallett et al. (1997); bHallett et (2001) al. (2001)

Actinospores of the triactinomyxon morphotype are commonly involved in the life cycle of freshwater myxosporeans that use oligochaetes as invertebrate hosts (Lom and Dyková, 2006; Székely et al., 2014). This morphotype is mostly associated with the life cycles of Myxobolus species in general (Lom and Dyková, 2006; Székely et al., 2009; Molnár et al., 2010; Szekély et al., 2014), and more specifically to the life cycle of Henneguya nuesslini (Kallert et al., 2005a) and Myxobilatus gasterostei (Atkinson and Bartholomew, 2009). Thus far, the studies seeking to address the life cycles of myxosporeans seem to maintain the correlation between the morphotypes of actinospores and myxposporean genera in both the marine and the freshwater environments. For instance, actinospores of the tetractinomyxon morphotype have been associated with the life cycles of species of Ellipsomyxa (Køie et al., 2004; Rangel et al., 2009), Gadimyxa (Køie et al., 2007), Parvicapsula (Køie et al., 2013), Sigmomyxa (Karlsbakk and Køie, 2012), and Ceratomyxa (Køie et al., 2008) in the FCUP 83 Chapter 5. The life cycle of Ortholinea auratae marine environment. In the freshwater environment, these correlations seem to be maintained, at least in the case of Ceratomyxa (Bartholomew et al., 1997) and Parvicapsula (Bartholomew et al., 2006). The study here presented reveals an association between the genus Ortholinea and the actinospores of the triactinomyxon morphotype in the marine environment, which may or not be maintained in the freshwater environment. The only certainty is that each actinosporean morphotype can be involved in the life cycle of several myxosporean genera. The triactinospores of O. auratae develop within the intestinal epithelium of the invertebrate host, as is typical of this morphotype (Lom and Dyková, 1992a; Yokoyama et al., 2012; Székely et al., 2014). The parasitological survey conducted in this study revealed parasitic infection only in immature oligochaetes, strengthening the idea that actinosporean development impairs the host growth, as suggested by previous studies that correlate these parasites with the sequester of nutrients necessary for the development of gametes (Stevens et al., 2001; Shirakashi and El-Matbouli, 2009). Moreover, the occurrence of tissue hyperplasia possibly leads to a reduction in the nutrient absorption capacity of the intestinal epithelium (Gilbert and Granath, 2003), thus damaging the hosts' sexual development. The morphological examination of the oligochaete hosts performed by two experts (Dr. Rüdiger and Dr. Martinez, personal communication) revealed that it was the species L. agnes, and its DNA sequence is presented here for the first time. These worms are cosmopolitan oligochaetes of reduced dimensions, reaching a maximum of 10 to 12 mm long and 0.4 mm in diameter, which can be found in marine and brackish waters and subtidal habitats, often in muddy sands (Erséus, 1992), as in this study. These small worms burrowed in the mud sediment must be infected by O. auratae when feeding on detritic particles contaminated by O. auratae myxospores deposited in the sediment. The gilthead seabream, on the other hand, must become infected by O. auratae mainly by the floating triactinospores released into the water column through the oligochaete's gut. Nevertheless, since the annelids, mainly polychaetes, are part of the juvenile's gilthead seabream diet (Rosecchi, 1987; Pita et al., 2002), this can also be a possible path for the fish infection. The overall prevalence of infection of O. auratae in oligochaetes was 1.8 %, with some variation according to the sampling sites. The prevalence of infection was higher in oligochaetes collected from the gilthead seabream ponds, as expected since these are the vertebrate hosts of O. auratae. The presence of infected oligochaetes in the ponds containing European seabass can be explained by the occasional entry of gilthead seabream and other fish species through the ponds water outlet ditches. Also, the ponds are used interchangeably in the production of both species; thus, FCUP 84 Chapter 5. The life cycle of Ortholinea auratae considering that many studies provide evidence to the persistence of myxosporean infection in its invertebrate hosts (Gilbert and Granath, 2001), it is feasible that oligochaetes infected with O. auratae are present in the ponds containing European seabass, with lower prevalence of infection comparatively to the ponds containing gilthead seabream. So far, only six life cycles of marine myxosporean parasites have been elucidated, all with polychaetes as invertebrate hosts (Køie et al., 2004; Køie et al., 2007; Køie et al., 2008; Rangel et al., 2009; Karlsbakk and Køie, 2012; Køie et al., 2013). Despite various morphotypes of actinospores had been described from marine oligochaetes (Caullery and Mesnil, 1904; Puytorac, 1963; Hallett et al., 1997, 1998, 1999; Hallett and Lester, 1999), this study constitutes the first description of a marine myxosporean life cycle involving an oligochaete host.

5.6. Acknowledgments

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 "PEst- C/MAR/LA0015/2013" the project DIRDAMyx, reference FCOMP-01-0124-FEDER- 020726/FCT-PTDC/MAR/116838/2010, and the Ph.D. fellowship grant SFRH/BD/82237/2011 attributed to L. Rangel and the Ph.D. fellowship grant SFRH/BD/92661/2013 attributed to S. Rocha through the programme POPH/FSE QREN; and the project EUCVOA (NORTE-07-0162-FEDER-000116) (Portugal). This work complies with the current laws of the country where it was performed.

FCUP 85 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Chapter 6

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

This chapter has been adapted from:

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 (In Press). doi:10.1111/jfd.12508.

*) These authors shared the first authorship.

FCUP 86 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

FCUP 87 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

6.1. Abstract

Ortholinea labracis n. sp. is described and its life cycle is inferred from a Southern Portuguese fish farm, with basis on microscopic and molecular procedures. This myxosporean parasite infects the urinary bladder of the European seabass Dicentrarchus labrax, and the intestinal epithelium of a marine oligochaete of the genus Tectidrilus. Myxospores subspherical in valvular view and ellipsoidal in sutural view, measuring 7.6 ± 0.3 (6.8‒8.7) µm in length, 7.2 ± 0.2 (6.7‒7.7) µm in width and 6.5 ± 0.4 (5.8‒7.7) µm in thickness. Two polar capsules, 3.0 ± 0.2 (2.6‒3.4) µm long and 2.4 ± 0.1 (2.0‒2.9) µm wide, located at the same level, but with divergent orientation and opening to opposite sides of the suture line. Sequencing of the SSU rRNA gene revealed a similarity of 100% between the analyzed myxospores and triactinomyxon actinospores. The phylogenetic setting of Ortholinea labracis n. sp. shows subgrouping in correlation with tissue tropism, but identifies this parasite as another exception to the main division of Myxosporea into the main freshwater and marine lineages.

FCUP 88 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

FCUP 89 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

6.2. Introduction

Myxosporea Bütschli, 1881 is a class of microscopic endoparasites characterized by a complex two-host life cycle that alternates between vertebrate hosts, mainly fishes, and invertebrate hosts, such as annelids and sipunculids. Parasites within this group are responsible for severe losses in pisciculture, either by causing high mortalities, or by leading to chronic diseases that reduce the commercial value of fish stocks (Diamant, 1992; Sitjà-Bobadilla and Álvarez-Pellitero, 1993b; Le Breton and Marques, 1995; Kent et al., 2001; Fioravanti et al., 2004; Yokoyama et al., 2012). Moreover, the growing importance of fisheries and aquaculture industries worldwide has led to an increasing interest in studies focusing on systematics and ecology of these parasites. The European sea bass, Dicentrarchus labrax (Linnaeus, 1758), is the most important commercial fish cultured in Southern Europe, especially in the North Atlantic and the Mediterranean area (FAO, 2014). Six myxosporean species are known to occur in this host worldwide: Sphaerospora testicularis Sitjà-Bobadilla and Álvarez-Pellitero, 1990 causing castration in males due to intense infection of the testes (Álvarez-Pellitero and Sitjà-Bobadilla, 1993b; Rigos et al., 1999); Sphaerospora dicentrarchi Sitjà-Bobadilla and Álvarez-Pellitero, 1992, a systemic parasite that infects the connective tissue and that can reduce the growth rate and facilitate secondary infections by other opportunistic infectious organisms (Sitjà-Bobadilla and Álvarez-Pellitero, 1992; Rigos et al., 1999; Fioravanti et al., 2004); Ceratomyxa diplodae Lubat, Radujkovic, Marques and Bouix, 1989 and Ceratomyxa labracis Sitjà-Bobadilla and Álvarez-Pellitero, 1993, infecting the gallbladder (Álvarez-Pellitero and Sitjà-Bobadilla, 1993a); Kudoa iwatai Egusa and Shiomitsu, 1983, infecting the muscle and several other organs (Diamant et al., 2005); and Enteromyxum leei Diamant, Lom and Dyková, 1994, an enteric parasite (Fioravanti et al., 2006). Wolf and Markiw (1984) first unravelled the complexity of the myxozoan life cycle upon the discovery that the salmonid-infecting parasite Myxobolus cerebralis Hofer, 1903 has an actinosporean counterpart developing in the freshwater oligochaete Tubifex tubifex (Müller, 1774). Since then, several studies have aimed at the elucidation of myxosporean life cycles, mainly through experimental infections between fish and invertebrate hosts (Kent et al., 1993; Yokoyama et al., 1995; Székely et al., 2001, 2002, 2009; Kallert et al., 2005a, 2005b). However, these experiments were very laborious, time-consuming and often produced questionable results (Holzer et al., 2004; Rácz et al., 2004; Marton and Eszterbauer, 2011; Székely et al., 2014). Having FCUP 90 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp. been proved more accurate and reliable in matching myxosporean and actinosporean counterparts, molecular methodologies are nowadays the main tool for inferring myxozoan life cycles (Atkinson and Bartholomew, 2009; Karlsbakk and Køie, 2012; Køie et al., 2013; Székely et al., 2014). Despite the fact that the class Myxosporea presently comprises more than 2300 species, only less than 50 have complete known or inferred life cycles (Lom and Dyková, 2006; Yokoyama et al., 2012; Székely et al., 2014) and, from these, only seven relate to the marine environment: Ellipsomyxa gobii Køie, 2003 from the gallbladder of Pomatoschistus microps (Krøyer, 1838), infects Nereis spp. (Køie et al., 2004); Ellipsomyxa mugilis (Sitjà-Bobadilla and Álvarez-Pellitero, 1993), a parasite of the gallbladder of mugilids fishes, infects Hediste diversicolor (O. F. Müller, 1776) (Rangel et al., 2009); Gadimyxa atlantica Køie, Karlsbakk and Nylund, 2007, from the kidney and urinary bladder of Gadus morhua Linnaeus, 1758, infects Spirorbis spp. (Køie et al., 2007); Sigmomyxa sphaerica Karlsbakk and Køie, 2012, from the gallbladder of Belone belone (Linnaeus, 1761), infects Nereis pelagica Linnaeus, 1758 (Karlsbakk and Køie, 2012); Ceratomyxa auerbachi (Noble, 1950), from the gallbladder of Clupea harengus Linnaeus, 1758 infects Chone infundibuliformes Krøyer, 1856 (Køie, Karlsbakk and Nylund, 2008); Parvicapsula spp., from the kidneys of Sprattus sprattus (Linnaeus, 1758), and C. harengus infect Hydroides norvegicus Gunnerus, 1768 (Køie et al., 2013); and Ortholinea auratae Rangel, Rocha, Borkhanuddin et al., 2014, from the urinary bladder of Sparus aurata Linnaeus, 1758 infects Limnodriloides agnes Hrabě, 1967 (Rangel et al., 2015b). All these have an actinospore counterpart of the tetractinomyxon type and infect brackish/marine polychaetes, with the exception of O. auratae. The latter was recently described from the urinary bladder of reared S. aurata, and its report constituted the first reference of the genus Ortholinea Shulman, 1962 in Portugal (Rangel et al., 2014). The inferred life cycle of O. auratae involve the marine oligochaete L. agnes, with actinospores of the triactinomyxon type developing in the intestinal epithelium of the invertebrate (Rangel et al., 2015b). The elucidation of myxosporean life cycles is important in order to fully understand the biology, ecology and seasonality of these parasites, especially in cultured environments, where they can cause significant economic losses to fish production, as noted above. Also, the annelid phase is the most promising for possible life cycle break, which could be an important biological control measure for several parasitic diseases. In this study, the life cycle of a new species of the genus Ortholinea Shulman, 1962 infecting the urinary bladder of D. labrax and the intestinal epithelium of the marine oligochaete Tectidrilus sp., is inferred and its sores are described in FCUP 91 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp. morphological and molecular terms on the basis of material isolated during a parasitological survey conducted in a Southern Portuguese fish farm.

6.3. Material and methods

Fish collection and sampling of myxosporean stages

Specimens of D. labrax were collected from a fish farm located in the Alvor Estuary, near the Atlantic coast (37° 8′ N, 8° 37′ W), Portimão, Algarve, Portugal. Monthly collections of about 12 fish each were performed between June 2012 and June 2013 (n = 167). After this time period, punctual samplings were performed between 2013 and 2015 (n = 41). Prevalence of infection was, however, calculated using a sample size of 155 fish, referring to the total number of specimens from which the urinary bladders were effectively isolated and analyzed. Spring 2012 - 7, Summer 2012 – 8, Autumn 2012 – 25, Winter 2013 – 40, Spring 2013 – 34, Summer 2013 – 5, Autumn 2013 – 4, Summer 2014 – 11, Winter 2015 – 9, Spring 2015 – 12. Fish were kept fresh on ice and transported to the laboratory, where dissection was performed. Afterwards, various internal tissues and organs were surveyed for parasites.

Annelid collection and sampling of actinosporean stages

The oligochaete survey was conducted in specimens from the same fish farm at the Alvor Estuary. Oligochaetes were collected from mud samples of the ponds containing European seabass and gilthead seabream, S. aurata, as well as from the water outlet ditches, and from an area of the Alvor Estuary located downstream to the fish farm. The collected oligochaetes were kept individually in 48-well cell culture plates containing salt water and a stereo microscope was used to examine the specimens for the release of actinospores during one to two weeks, since these marine oligochaetes are difficult to maintain in small wells for long periods of time. When the oligochaetes started to perish, fresh preparations of all oligochaetes, releasing and not releasing actinospores, were performed using salt water, and examined with a light microscope to detect the presence of actinospores in the internal tissues and coelomic cavity.

FCUP 92 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Light microscopy and morphological analysis

Samples infected with myxozoan parasites were examined and photographed using a Zeiss Axiophot microscope (Grupo Taper, Sintra, Portugal), equipped with a Zeiss Axiocam digital camera Icc3. Axiovision 4.6.3 software (Grupo Taper) was used for image analysis. The morphometry of myxospores and actinospores was characterized from fresh material, in accordance to Lom and Arthur (1989) and Lom et al. (1997). All measurements include the mean value ± standard deviations (S.D.), range of variation (range) and number of spores measured.

Transmission electron microscopy

Infected fish urine and parasitized oligochaetes were fixed in 5% glutaraldehyde buffered in 0.2 M sodium cacodylate (pH=7.4) for 20–24 h, washed in the same buffer, and postfixed 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.

DNA extraction, amplification and sequencing

Infected fish urine and parasitized oligochaetes were fixed and 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 DNA was stored in 50 μl of TE buffer at -20 ºC until further use. The SSU rRNA gene was amplified using both universal primers and myxosporean-specific primers (Table 6.1). For the oligochaete host DNA, the universal primers 16sar-L and 16sbr-H (Palumbi et al., 2002) were used (Table 6.1). PCRs were performed in 50 µl reactions using 10 pmol of each primer, 10 nmol of each dNTP, 2.5 mM 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), with initial denaturation at 95 ºC for 3 min, followed by 35 cycles of 94 FCUP 93 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

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 a single-step enzymatic cleanup that eliminates unincorporated primers and dNTPs by ExoFast method. The PCR products from different regions of the SSU rRNA gene were sequenced directly. The sequencing reactions were performed using a BigDye Terminator v1.1 from the Applied Biosystems Kit (Applied Biosystems, Carlsbad, California), and were run on an ABI3700 DNA analyzer (Perkin-Elmer, Applied Biosystems, Stabvida, Oeiras, Portugal).

Table 6.1 – Polymerase chain reaction primers used for the amplification and sequencing of the SSU rRNA gene of Ortholinea labracis n. sp. and the 16S gene of the oligochaete host.

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

18e CTG GTT GAT CCT GCC AGT ACT3r, ChloromyxR1 Hillis and Dixon (1991)

MXF TTC TGC CCT ATC AAC TTG TTG ChloromyxR1, 18r Fiala (2006)

ACT3f CAT GGA ACG AAC AAT 18r Hallett and Diamant (2001)

ACT3r ATT GTT CGT TCC ATG 18e Hallett and Diamant (2001)

ChloromyxR1 CCT TCC GTC AAT TCC TTT AAG 18e, MXF Azevedo et al. (2009)

18r CTA CGG AAA CCT TGT TAC G MXF, ACT3f Whipps et al. (2003)

16sar-L CGC CTG TTT ATC AAA AAC AT 16sbr-H Palumbi et al. (2002)

16sbr-H CCG GTC TGA ACT CAG ATC ACG T 16sar-L Palumbi et al. (2002)

Distance and phylogenetic analysis

Phylogenetic and molecular evolutionary analyses were conducted using MEGA 5.05. The SSU rRNA sequences of the parasite were aligned in ClustalW, together with 96 other myxosporean sequences retrieved from GenBank, including those with highest similarity score, which were O. auratae (GenBank accession no. KR025869), Acauda hoffmani Whipps, 2011 (HQ913566), Myxobilatus gasterostei (Parisi, 1912) (EU861210), Zschokkella sp. AH2003 (AJ581918), Hoferellus gilsoni (Debaisieux, 1925) (AJ582062), Chloromyxum schurovi Shul'man and Ieshko, 2003 (AJ582007), Ortholinea orientalis (Shulman and Shulman-Albova, 1953) (HM770873), Ortholinea saudi Abdel-Baki, Soliman, Saleh, et al., 2015 (JX456461), Ortholinea sp. AL-2006 (DQ333433), and a myxosporean parasite of the triactinomyxon type (KF263540). Tetracapsuloides bryosalmonae (Canning, Tops, Curry et al., 2002) (U70623) and Buddenbrockia plumatellae Schröder, 1910 (AY074915) were selected as outgroup. FCUP 94 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Alignments were ran with an opening gap penalty of 10 and a gap extension of four for both paired and multiple alignments (Tamura et al., 2011), and positions containing gaps and missing data were manually removed. Phylogenetic analyses were performed using the maximum likelihood (ML) method, performed with the general time reversible substitution model with 4 gamma- distributed rate variation among sites. A total of 1473 positions were used in the final dataset, and the bootstrap consensus tree was inferred from 500 replicates. Distance estimation was performed in MEGA 5.05, using the p-distance model with all ambiguous positions removed for each sequence pair.

6.4. Results

From the 155 urinary bladders that were isolated and analysed, 17 presented infection by a myxosporean parasite that, in some cases, was also found infecting in the terminal portion of the posterior kidney. Total prevalence of infection (sensu Bush et al., 1997) was of 11.0%. The parasite presented coelozoic development, with plasmodia and myxospores floating free in the urine. No symptoms or macroscopic signs of infection were observed in parasitized hosts. A total of 1900 oligochaetes belonging to five different species were collected and examined for myxosporean infection. Two species were the most frequent: L. agnes (n = 878), host of the triactinomyxon stages of O. auratae; and a Tectidrilus sp. (n = 972), infected with a new triactinomyxon type lodged in the intestinal epithelium and causing hyperplasia. The latter species was collected from the Alvor Estuary (n = 782) and European seabass ponds (n = 190), but infection was registered solely from the ponds, from which 18 infected worms (9.5% of prevalence of infection) were collected in the months of March and April. The host's 16S rRNA gene was sequenced (KU696552) and a BLAST search found no correspondence with previous 16S rRNA sequences in GenBank. The closest species were Tectidrilus verrucosus (Cook, 1974) (AY885630) and Tectidrilus bori (Righi and Kanner, 1979) (AY885631), with a maximum similarity score of 88.6%.

Morphological characterization of myxospores (Figs. 6.1-6.2)

Light microscopy: Young and mature plasmodia highly polymorphic, round, elongated or very irregular. Dimensions vary greatly as a result of the polymorphic nature of these FCUP 95 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp. structures (Fig. 6.1a). Cellular membrane also highly irregular, almost smooth or with thin peripheral projections. The latter occupy the entire plasmodial surface or are located only at a portion (Figs. 6.1b‒e). Plasmodia polysporic (Figs. 6.1b‒c, e), rarely disporic (Fig. 6.1d), simultaneously containing different stages of the sporogenic development. Mature myxospores subspherical in valvular view and ellipsoidal in sutural view, measuring 7.6 ± 0.3 (6.8‒8.7; n=94) µm in length, 7.2 ± 0.2 (6.7‒7.7; n=67) µm in width and 6.5 ± 0.4 (5.8‒7.7; n=44) µm in thickness. Two equal-sized subspherical polar capsules, 3.0 ± 0.2 (2.6‒3.4; n=74) µm long and 2.4 ± 0.1 (2.0–2.9; n=74) µm wide, located at the same level at the anterior half of the myxospores but with divergent orientation and opening to opposite sides of the suture line. Polar filament coiled in four to five turns (Fig. 6.1f; Table 6.2). Ultrastructure: In earliest stages of sporogenesis, the endoplasm appears dense and granular, presenting several vegetative nuclei, mitochondria, vesicles, lipidic globules and other reserve bodies (Fig. 6.2a). In later stages of sporogenesis, generative cells and disporous pansporoblasts in different stages of differentiation occupy the larger portion of the endoplasm. The ectoplasm, transparent and void of organelles, is smooth or projects to form thin pseudopodia of various dimensions in length (Figs. 6.2b‒c). Sporoblasts constituted by two valvogenic cells united along a suture line and surrounding two capsulogenic cells and one sporoplasmogenic cell (Fig. 6.2d). Mature myxospores display an ornamented wall surrounding two polar capsules and one sporoplasm. Wall composed by two symmetric valves united along a straight suture line and presenting an intricate pattern of surface ridges (Figs. 6.2c, h). Polar capsules constituted by a double-layered wall, a thinner outer electron-dense layer and a thicker inner electron-lucent layer, containing the dense and heterogeneous matrix in which the polar filament coils. The extrusion pores of the polar filaments are located at opposite sides of the suture line in the myxospores anterior pole (Figs. 6.2e−g). The sporoplasm, located at the posterior half of the myxospores, displays two nuclei and several electron-dense sporoplasmosomes.

FCUP 96 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

FCUP 97 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Fig. 6.2 – Transmission electron micrographs of Ortholinea labracis n. sp. from the urinary bladder of Dicentrarchus labrax. a) Young plasmodium displaying vegetative nuclei (N) and numerous mitochondria (Mt), vesicles (V) and lipidic globules (Li). Notice the irregularity of the cellular membrane due to the presence of peripheral projections (arrow). b) Round plasmodium containing different stages of the sporogenic development, namely a generative cell (GC), developing sporoblasts (Sb), and mature myxospores (S). Notice the smooth cellular membrane (arrows). c) Elongated plamodium displaying a developing disporoblast (*). Notice the surface ridges on the myxospores’ valves (arrows). d) Sporoblast showing its almost fully developed polar capsules (PC), and one of the two sporoplasmic nuclei (N). e) Longitudinal section of a polar capsule showing its apex (arrowhead) located near the suture line. f) Oblique transverse section of the two polar capsules displaying their double-layered wall (arrowheads) containing a heterogeneous matrix (*), and the polar filament (PF) coiling within. g) Tangencial section of the anterior pole of a myxospore allowing recognition of one of the two extrusion pores (arrow), located close to the suture line (arrowheads). h) Ultrastructural detail of the two valves united along a straight suture line (arrowheads). Notice the surface ridges ornamenting the valves. FCUP 98 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Morphological characterization and ultrastructure of actinospores (Figs. 6.3‒6.4)

The first visible developmental stages were binucleated cells (Fig. 6.3a). The gametogamy stage started with the formation of the initial pansporocyst containing two generative cells within (Fig. 6.3b). These generative cells then divided into four cells (Fig. 6.3c), six cells, 10 cells (Fig. 6.3d) and finally 16 cells, which then fused together resulting in a pansporocyst containing eight zygotes (Fig. 6.4a). The sporogony stage started with the division of the zygotes (Fig. 6.3e). The formation of actinospores resulted from successive cellular divisions giving rise to cells that differentiate into valvogenic, capsulogenic and sporoplasmic cells (Figs. 6.4b‒f). The initial development of the sporoplasm happens outside the spore involucre, being later surrounded by the valvogenic cells during maturation. Initially, the secondary cells are large, but as the sporoplasm matures, it becomes filled with many smaller secondary electron-dense cells (Figs. 6.4c‒d). In the apices of the polar capsules, a conical structure protruding from the valvogenic cells creates a permanent open pore in the actinospore involucre for each polar capsule (Fig. 6.4f). At the end of the sporogony, the pansporocysts contain eight mature actinospores (Fig. 6.3f). All developmental stages occur in the intestinal epithelium of the oligochaete host, with mature free actinospores (Fig, 6.3g) being later released into the intestinal lumen, and consequently excreted to the water column. Free triactinospores (Fig. 6.3g) do not form nets. Triactinospores with three prominent pyramidal polar capsules (Figs. 6.3f‒g, 6.4f) measuring 5.9±0.4 (4.8–6.6; n=37) µm in length and 3.9±0.2 (3.4–4.5; n=37) µm in width. Polar filament helicoidally arranged into five coils (Fig. 6.4e) and measuring 37.8±3.3 (33.9–42.5; n=5) µm in length when fully extruded. Actinospore body 56.3±8.6 (40.9–70.8; n=19) µm long and 12.8±0.8 (11.7–14.8; n=20) µm wide. Style measuring 90.7±16.0 (61.9–112.4; n=19) µm in length and 15.0±1.8 (12.0–19.9; n = 22) µm in width; from the base extending three long caudal processes slighted curved upwards, 191.8±29.3 (133.3-244.7; n=65) µm long and 15.8±2.3 (11.8–22.3; n=59) µm wide near the base. Caudal processes forming an angle of 128.2°±14.9 (95.0–160.0; n=25) to the style axis. Actinospore total length 148.0±12.6 (119.2–168.7; n=23) µm with a total span of 390.7 ± 43.5 (272.2−464.9; n=21) µm. Number of sporoplasmic secondary cells undetermined, but presumed to be more than 200.

FCUP 99 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

FCUP 100 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

FCUP 101 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Taxonomic summary

Class Myxosporea Bütschli, 1881 Order Bivalvulida Shulman, 1959 Family Ortholineidae Lom and Noble, 1984 Genus Ortholinea Shulman, 1962

Parasite species: Ortholinea labracis n. sp. (Myxosporea: Ortholineidae) Type host: European seabass, Dicentrarchus labrax Linnaeus, 1758 (Teleostei: Moronidae). The actinosporean stage develops in an oligochaete host of the genus Tectidrilus. Site of infection: plasmodia and myxospores in the urinary bladder and terminal portion of the posterior kidney of the fish host. Actinosporean stages in the intestinal epithelium of the oligochaete. Type locality: Alvor Estuary, near the Atlantic coast (37º 08’ N, 08º 37’ W), Portimão, Algarve, Portugal. Prevalence: 11% (17 out of 155). Pathogenicity: collected and analyzed fish did not present external symptoms of infection or disease, neither was mortality recorded from the stock used. Etymology: the specific epithet 'labracis' derives from the specific epithet of the host species. Type material: Two glass slides containing semithin sections of myxospores and actinospores of the hapantotype and one vial with an oligochaete infected with triactinomyxon stages in absolute ethanol deposited in the Slide Collection and Vial Collection of CIIMAR, at the Laboratory of Animal Pathology at the Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal, reference CIIMAR 2016.13 and CIIMAR 2016.14, and CIIMAR 2016.V1 respectively. Two SSU rRNA gene sequences, one referring to parasitic material of the myxosporean stage, with a total of 2055 bp and GenBank accession no. KU363830, and the other from the actinosporean stage, with a total of 2054 bp and GenBank accession no. KU363831. One 16S rRNA gene sequence of the oligochaete host, with a total of 527 bp, and GenBank accession no. KU696552.

Morphological comparison

Of the about 22 known Ortholinea spp., seven are morphologically similar to Ortholinea labracis n. sp., with basis on organ of infection, presence of ornamental ridges and overall myxospore dimensions, however differing in several morphometric aspects and FCUP 102 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp. ultrastructural details (Table 6.2). Compared to Ortholinea labracis n. sp., Ortholinea fluviatilis Lom and Dyková, 1995 and Ortholinea undulans (Meglitsch, 1970) have similar morphology but differ in having undulating suture lines and in the pyriform shape of the polar capsules of the latter; O. orientalis has bigger polar capsules and a triangular myxospore body; Ortholinea polymorpha (Davis, 1917) has smaller pyriform polar capsules; Ortholinea antipae Moshu and Trombitsky, 2006 and Ortholinea clupeidae Aseeva, 2000 are less wide and both have triangular myxospore bodies; and O. auratae has a bigger myxospore body. The triactinomyxons of Ortholinea labracis n. sp. are distinct from all other marine triactinomyxon types described so far (Table 6.3). The length of the actinospores and of the polar capsules is larger than that of O. auratae and the triactinomyxon type described by Roubal, Hallett and Lester (1997), while being shorter than that of triactinomyxons type 1 and type 2 described by Hallett et al. (2001).

Molecular analysis and phylogeny

Sequencing of the PCR products obtained from the myxospores and actinospores resulted in the assembly of two consensus SSU rRNA sequences comprised by 2055 bp (KU363830) and 2054 bp (KU363831), respectively. Comparison between these two sequences revealed a similarity of 100%, thus implying the analyzed stages as probable being representative of the life cycle of the parasite in study (Fig. 6.5).

FCUP 103 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

The phylogenetic analysis revealed the parasite consistently clustering together with O. auratae (KR025869), O. orientalis (HM770873), A. hoffmani (HQ913566), M. gasterostei (EU861210), Zschokkella sp. AH2003 (AJ581918), H. gilsoni (AJ582062), C. schurovi (AJ582007), and a myxosporean parasite of the triactinomyxon type (KF263540), to form a clade with strong bootstrap support values. Ortholinea saudi (JX456461) and Ortholinea sp. AL-2006 (DQ333433) clustered together in all analyses but with unresolved position in the overall tree topology. In some topologies, these species clustered within the Ortholinea clade, basal to the Myxobolus clade; in others, these two species and the main Ortholinea clade formed separate subclades of the Myxobolus clade, with the main Ortholinea clade appearing sister to the subclade containing Myxobolus that infect Salmoniformes; and in others, O. saudi and Ortholinea sp. AL-2006 together formed a clade basal to the main Ortholinea clade and the Myxobolus clade. The low bootstrap values supporting these topologies demonstrate that the phylogenetic positioning of O. saudi (JX456461) and Ortholinea sp. AL-2006 (DQ333433) is not resolved by the molecular data currently available. Pairwise comparisons among these sequences revealed highest similarity of the sequences in study to O. auratae (95.7%) and A. hoffmani (91%), with all others resulting in percentages of identity lower than 90%. The lowest percentages of identity were those of O. saudii (67.2%) and Ortholinea sp. AL-2006 (64.1%). The best SSU-based tree topology obtained showed the Ortholinea clade located within the main freshwater clade and sister to the Myxobolus clade, while other marine species that infect the excretory system clustered to form a clade within the main marine clade. This marine urinary bladder group comprises two minor subclades: the Parvicapsula subclade, which includes Gadimyxa spp. and S. testicularis; and the Zschokkella subclade, which includes the type species Zschokkella hildae Auerbach, 1910, as well as Sinuolinea spp. and Latyspora spp. In most analyses, the marine urinary clade appeared as the most derived group of the marine lineage, with the Ceratomyxa clade being the most basal group. Among them, the marine histozoic clade, comprised by Kudoa spp. and Enteromyxum spp., and the marine gallbladder clade, represented by Ellipsomyxa spp., Cocomyxa jirilomi Diamant, Lipshitz and Ucko, 2007, Zschokkella neopomacentri Thélohan, 1892 and Myxidium incurvatum Diamant and Palenzuela, 2005. The relationships between the clades of the marine lineage were, however, topologically unstable and dependent on the alignment and phylogenetic analysis performed. On the other hand, the inner topology of the freshwater lineage was consistent in all analyses, with the exception of the phylogenetic placement of O. saudi and Ortholinea sp. AL-2006, as previously mentioned. FCUP 104 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

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Meglitsch

Davis

(

Shulman

(

Lom

Aseeva

Rangel

Trombitsky Trombitsky

Moshu Moshu

Present study

Source

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3.0)

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2.9)

SL: spore length; SW: spore width; SW: SL:spore spore width; length;

2.5

–

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2.2

2.0

‒

(2.8

3.1

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1.8‒2.5

(2.6‒3.4)

3.0±0.2

PCL

PC: polar capsule; PC: polar capsule;

8.0)

8.4)

3.0

–

(5.0

6.3

–

5.1

6.8

2.5

(6.3

7.2±0.5

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(5.8‒7.7)

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–

9.0)

8.0)

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6.3

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(6.0

7.4

10.0)

8.0 (7.0

7.5‒7.6

(7.3

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shape

Pyriform

Spherical

Subspherical

Pyriform

Subsperical

Spherical

Subsperical

shape

Subspherical

Spherical

triangular

Subspherical to

Ellipsoidal

triangular

Subspherical to

Subspherical

Nearly triangular

Subspherical

Spore

Zealand

New New

States

United

Russia

Asia

Russia

Portugal

Ukraine

Portugal

Location

and

le length; PCW: polar capsule width; PFC: number of polar filament coils. Measurements are given in µm. in given are Measurements coils. filament polar of number PFC: width; capsule polar PCW: le length;

spp. characterized by te presence of surface ridges and described from the excretory system marine fish. of system from the ridges described excretory te surface and by of spp. presence characterized

novaezeelandiae

Peltorhamphus

Arnoglossusscapha

Opsanus tau

Clupeapallasii

Tetraodon fluviatilis

Clupea pallasii pallasii Clupeapallasii

Sparus aurata

Alosa tanaica

Dicentrarchuslabrax

Hosts

spp.

n. sp.n.

Ortholinea

–

labracis

O. undulans O.

O. polymorphaO.

O. orientalis O.

O. fluviatilis O.

O. clupeidaeO.

O. auratae O.

O. antipaeO.

Ortholinea ST: spore thickness; PCL: polar capsu polar PCL: thickness; ST: spore Table 6.2 Table 6.2

FCUP 105 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

)

2001

(

. . 2001

. .

et al et

et al

Hallett

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100.0)

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n. n. sp. and all other described marine triactinospores (the mean±SD with the range in

–

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Hallett

Roubal

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4.0

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138.0

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125.0

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Triactinomyxon

)

512.7)

287.6)

–

150.

–

152.6)

–

45.8)

18.0)

16.9)

97.8) 97.8)

14.4)

62.2)

Ortholinea Ortholinea labracis

–

2015

(

4.2)

5.8)

. .

–

(90.0

(141.0

(115.2

(10.9

(40.9

(4.4

et al

Rangel

435.9±56.7(268.4

37.9±5.0(29.0

3.6±0.2(3.1

5.2±0.3

120.1±17.4

13.3±1.3(10.2

215.1±29.6

13.2±1.3(11.0

83.1±9.0(67.9

12.6±0.9

49.9±5.2

136.2±9.4

Ortholineaauratae

n. sp. n.

464.9)

–

160.0)

244.7)

168.7)

–

112.4)

42.5)

22.3)

19.9)

–

14.8)

70.

–

4.5)

6.6)

–

43.5 (272.2 43.5

Present study

390.7 ±

37.8±3.3(33.9

3.9±0.2(3.4

5.9±0.4(4.8

128.2(95.0 14.9 ±

15.8±2.3(11.8

191.8±29.3(133.3

15.0±1.8(12.0

90.7±16.0(61.9

12.8±0.8(11.7

56.3±8.6(40.9

148.0±12.6(119.2

Ortholinealabracis

Morphological Morphological comparison between the triactinospores linked to

–

processes length processes

3 3

Source

Span

Polarlength filament

Polar capsulesPolarwidth

Polar capsules length capsulesPolar

with style with

Caudalangle processes

Caudalwidth processes

Caudal

Stylewidth

Stylelength

Sporewidth body

Sporelength body

Sporelength

Character parentheses; in in parentheses; Table Table

FCUP 106 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Regarding the actinosporean stages included in the phylogenetic analyses performed, the main freshwater clade comprised the vast majority of known actinospore types (antonactinomyxon, aurantiactinomyxon, echinactinomyxon, endocapsa, guyenotia, hexactinomyxon, hungactinomyxon, neoactinomyxum, raabeia, sphaeractinomyxon, synactinomyxon, tetraspora, triactinomyxon), some of which are represented in figure 6.6 and mostly cluster within the Myxobolus clade. The main marine clade encompassed the totality of representatives of only two actinospore types: tetractinomyxon and unicapsulactinomyxon (Fig. 6.6).

6.5. Discussion

The characteristics of the myxosporean species here described are consistent with those defined for the genus Ortholinea, according to traditional morphological and biological criteria: species coelozoic in the urinary system of marine fishes, having spherical or subspherical myxospores that are slightly flattened parallel to the sutural plane or pointed posteriorly, and that contain two subspherical to pyriform polar capsules and a binucleate sporoplasm (Lom and Dyková, 2006). Not all of the about 22 known species of Ortholinea are true to this traditional outline, with O. visakhapatnamensis Padma Dorothy and Kalavati, 1993 having been described from the peritoneum (Padma Dorothy and Kalavati, 1993), Ortholinea percotti Dogiel and Akhmerov, 1960 from the gills and fins (Akhmerov, 1960), and Ortholinea asymmetrica Kovalynova, Velev and Vladov, 1993 and Ortholinea australis Lom, Rhode and Dyková, 1992 from the gallbladder (Lom et al., 1992; Kovalyova et al., 1993). Also, Ortholinea africanus Abdel-Ghaffar, El-Toukhy, Al-Quraishy et al., 2008 is coelozoic in the urinary bladder, but infects a freshwater host, the Nile tilapia Oreochromis niloticus (Linnaeus, 1758) (Abdel-Ghaffar et al., 2008). This could indicate that the taxonomic placement and differentiation of Ortholinea spp. according to morphologic criteria is artificial, especially considering that myxosporean phylogeny based on molecular data of the SSU rRNA gene has revealed the aquatic environment and organ of infection as stronger evolutionary signals than myxospore morphology (Andree et al., 1999; Kent et al., 2001; Eszterbauer, 2004; Holzer et al., 2004; Fiala, 2006). Nonetheless, it cannot be disregarded the possibility that these species might have been improperly assigned to the genus Ortholinea, as most were described solely on the basis of line schematic drawings, with the exception of O. australis and O. africanus, and all lack the designation of an hapantotype (Akhmerov, 1960; Lom et al., 1992; Kovalyova et al., FCUP 107 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

1993; Padma Dorothy and Kalavati, 1993, Abdel-Ghaffar et al., 2008). A thorough molecular approach to this genus, and more specifically to its type species, Ortholinea divergens (Thélohan, 1895) from Lipophrys pholis (Linnaeus, 1758), is expected to result in the taxonomic revision of some of its species. Unfortunately, the few molecular data available for the genus Ortholinea is insufficient to ascertain its taxonomy and phylogeny, as only four SSU rRNA gene sequences are provided in GenBank, all of which refer to species fitting the traditional outline of the genus: Ortholinea sp. Al-2006 from Siganus rivulatus Forsskål and Niebuhr, 1775 off Israel (unpublished data); O. orientalis from C. harengus and S. sprattus off Denmark (Karlsbakk and Køie, 2011); O. auratae from S. aurata in the South Atlantic coast of Portugal (Rangel et al., 2014); and O. saudii from S. rivulatus in the Red Sea, Saudi Arabia (Abdel-Baki et al., 2015). Resulting from this scarcity of molecular data, the differentiation and taxonomic placement of Ortholinea spp. remains highly dependent on the comparison of morphological features. Upon consideration of all these criteria, the morphological and molecular features of the parasite here described support its identification as a new species of the genus Ortholinea, herein named Ortholinea labracis n. sp. The plasmodial development of Ortholinea labracis n. sp. in the urinary bladder of the fish host is characterized by variations in size and shape, as well as by the differentiation of peripheral projections that mainly extend from just one portion of the cellular membrane. These developmental processes are commonly observed in coelozoic species and are thought to be implicated in the improvement of nutritional intake (Sitjà-Bobadilla and Álvarez-Pellitero, 1993e, 2001; Rocha et al., 2011). The microscopic study performed also revealed the asynchrony of the sporogenic development, as both young and mature plasmodia were simultaneously present in infected urinary bladders, and ultrastructural observations depicted plasmodia concurrently containing generative cells, sporoblasts and mature myxospores. The myxospores of Ortholinea labracis n. sp. develop within disporous pansporoblasts, following what it seems to be the tendency for this genus (Padma Dorothy and Kalavati, 1993; Lom and Dyková, 1995; Sarkar, 1999; Ali, 2000; Moshu and Trombitsky, 2006), with the exception of the direct sporogenesis reported for O. asymmetrica (Kovalyova et al., 1993). Despite the lack of a long term study performed on parasitized fish specimens, no gross ultrastructural damages to the urinary bladder epithelium were observed, and clinical signs of infection or disease were absent, indicating that Ortholinea labracis n. sp. does not affect the health integrity of the European seabass.

FCUP 108 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Fig. 6.6 – Maximum likelihood tree of the SSU rRNA sequence of Ortholinea labracis n. sp. and other selected myxozoan species. The numbers on the branches are bootstrap confidence levels on 500 replicates. There were a total of 1473 positions in the final dataset. GenBank accession numbers in parentheses after the species name. FCUP 109 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Concerning the parasitic stages in the invertebrate host, Ortholinea labracis n. sp. follows the developmental path commonly described for triactinomyxon types (see El-Matbouli and Hoffmann, 1998), with pansporocysts containing eight developing actinospores that, upon maturation, are released into the intestinal lumen of the oligochaete host. The ultrastructural features of the actinospores include the presence of a conical structure located in the apex of the polar capsules. This structure is commonly observed in several actinospore types (Lom and Dyková, 1997), for instance, having been described from sphaeractinomyxon (Puytorac, 1963), neoactinomyxum (Marques, 1984), triactinomyxon (Lom and Dyková, 1992a) and other types (see Lom and Dyková, 1997). This feature can also be found in some myxospores, like Myxobolus cotti El-Matbouli and Hoffmann, 1987 (El-Matbouli et al., 1990). Myxosporean life cycles involving triactinomyxon types are frequently described for freshwater fish and oligochaete hosts (Yokoyama et al., 2012; Székely et al., 2014) but, in the marine environment, only the inferred life cycle of O. auratae is known to involve a triactinomyxon type (Rangel et al., 2015b). The present study proposes a second marine life cycle of this myxosporean genus that also involves the development of triactinomyxon actinospores within an oligochaete definitive host. The Limnodriloidinae subfamily encompasses oligochaete species, generally, with oesophageal diverticula, reduced number of setae, absence of hair setae, and a smooth ('transparent') body wall, among other characters (Erséus, 1982, 1991). However, the genus Tectidrilus was established by Erséus (1982) to accommodate species of Limnodriloidinae that have the body wall covered by papillae in the posterior region of the body. Morphologically, the oligochaete worms serving as hosts for Ortholinea labracis n. sp. share the characters described for Limnodriloidinae, and have the body wall covered by papillae in its posterior region as described for Tectidrilus genus. Genetically, these worms are most similar to Tectidrilus species and also to other Limnodriloidinae oligochaetes. Aquaculture land tanks appear to be ideal environments for the propagation of myxosporean parasites and the development of related chronic infections, since both vertebrate and invertebrate hosts are present, allowing a continuous infectious cycle. The myxospores of Ortholinea labracis n. sp. are released with the fish urine into the water column and probably settle in the muddy sediment, where infection of the invertebrate hosts occurs through contact with the tegument or by ingestion. On the other hand, the high floatability of the triactinomyxons probably allows them to rise in the water column and enter in contact with the tegument or gills of the fish host, as demonstrated for M. cerebralis life cycle (El-Matbouli et al., 1999). Here, another FCUP 110 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp. probable infection path for the fish can be the ingestion of infected worms, considering that the European seabass is an opportunistic predator (Pickett and Pawson, 1994). In this case, infection most likely takes place in juveniles, since annelids are considered negligible as food items for adult fish (Spitz et al., 2013). The molecular analyses performed were unable to assertively resolve the phylogeny of the genus Ortholinea due to the unstable positioning of O. saudii and Ortholinea sp. AL-2006, so that this taxon can either be paraphyletic or polyphyletic. Nevertheless, considering that O. saudii and Ortholinea sp. AL-2006 strongly cluster together, while displaying very low percentage of identity to all other Ortholinea spp., it is reasonable to assume that the genus Ortholinea will probably turn out to be polyphyletic, with homoplasic processes explaining the convergent evolution of phenotypes. Also interfering with the phylogenetic positioning of these species might be the relatively small size of the SSU rRNA sequence of O. saudii (831 bp) in comparison to all other Ortholinea SSU rRNA sequences used in the phylogenetic analyses, which account for at least 1950 bp. Ortholinea labracis n. sp. clusters alongside all other sequenced Ortholinea spp., but also A. hoffmani, M. gasterostei, Zschokkella sp. AH2003, H. gilsoni, C. schurovi, as well as a myxosporean parasite of the triactinomyxon type (KF263540), to form a clade of the freshwater lineage. Despite the lack of knowledge regarding tissue tropism and host of the latter, all other species comprised within the Ortholinea clade infect the kidney, urinary bladder and ureters of their fish hosts, be it either in the freshwater, brackish or marine environment (Holzer et al., 2004, 2006; Atkinson and Bartholomew, 2009; Karlsbakk and Køie, 2011; Whipps, 2011; Rangel et al., 2014; Abdel-Baki et al., 2015). Considering the molecular trend of dividing Myxosporea into a major freshwater clade and a major marine clade (Kent et al., 2001; Fiala, 2006; Bartošová et al., 2009), all sequenced Ortholinea spp., including Ortholinea labracis n. sp., are regarded as exceptions to this main division, as a result of their brackish/marine habitat. Several marine species of the genera Henneguya, Chloromyxum, Myxobolus, Myxidium, Parvicapsula, Zschokkella and Sphaeromyxa are also known exceptions to this main division (Fiala, 2006; Azevedo et al., 2009; Rocha et al., 2013) and, in the same manner, some freshwater species consistently cluster within the major marine clade, as is the case of Ceratonova shasta (Noble, 1950) (Bartholomew et al., 1997; Kent et al., 2001; Fiala, 2006; Fiala and Bartošová, 2010). The main theories explaining these exceptions have been the migratory pattern of the fish host, which in some cases can transition between freshwater, brackish and marine environments, as well as the acknowledgement that some myxosporean species underwent adaptation to different aquatic habitats during their evolution (Fiala and FCUP 111 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp.

Bartošová, 2010; Bartošová et al., 2011). Nonetheless, the increased acquisition of molecular data regarding actinosporean stages over the past few years and, more importantly, the inference of several myxosporean life cycles (specifically in the marine environment), raises some new insights on this matter. Most of the molecular data available for actinosporean stages in GenBank belong to the triactinomyxon, tetractinomyxon, aurantiactinomyxon, neoactinomyxum and raabeia types, with few entries for echinactinomyxon, hexactinomyxon, endocapsa, tetraspora, guyenotia, antonactinomyxon, hungactinomyxon, sphaeractinomyxon and unicapsulactinomyxon. All of these actinospore types have been sequenced from oligochaete hosts, freshwater or marine (Hallett et al., 1999; Kent et al., 2001; Holzer et al., 2004; Eszterbauer et al., 2006), with the exception of the tetractinomyxon and unicapsulactinomyxon types, which have been sequenced from polychaete hosts (Køie et al., 2004; Rangel et al., 2009, 2011). Phylogenetic analyses reveal that all the representatives of the two latter actinospore types (the tetractinomyxon of C. auerbachi, C. shasta, E. gobii, E. mugilis, S. sphaerica, Parvicapsula minibicornis Kent, Whitaker and Dawe, 1997 and G. atlantica; and the unicapsulactinomyxon of Enteromyxum spp.) cluster within the major marine clade, while the actinospore types that infect oligochaetes all cluster within the major freshwater clade (Fiala et al., 2015a). Acknowledging this relationship between the invertebrate host and the species phylogenetic placement brings forth the idea that the main division in Myxosporea phylogeny should be between species infecting oligochaetes or polychaetes, rather than the aquatic environment of the vertebrate host being freshwater or marine (Holzer et al., 2007). The inferred life cycle and phylogenetic analysis of Ortholinea labracis n. sp., as well as that of O. auratae, support this contention, as both have actinosporean counterparts of the triactinomyxon type that develop in marine oligochaetes (Rangel et al., 2015b). Also giving strength to the existence of an oligochaete lineage and a polychaete lineage are the life cycles of C. shasta and P. minibicornis. Both these species infect salmonid fish hosts in freshwater habitats of North America, and use the freshwater polychaete Manayunkia speciosa Leidy, 1859 as their invertebrate host (Bartholomew et al., 1997, 2006); this possibly being the reason determining their phylogenetic placement as exceptions within the marine lineage. Nonetheless, the information currently available for the life cycles of marine myxosporeans that cluster within the freshwater lineage (i.e. other Ortholinea, Sphaeromyxa and several Zschokkella, Myxidium and Sphaerospora), is insufficient to allow the re-definition of the main evolutionary signal driving the phylogeny of Myxosporea. Furthermore, there is no information regarding the morphology and definitive hosts of actinosporean FCUP 112 Chapter 6. Life cycle inference and phylogeny of Ortholinea labracis n. sp. counterparts of myxosporean species infecting vertebrate host groups other than fish (Fiala et al., 2015a). Tissue tropism and host relatedness are the other evolutionary signals that molecular studies recognize as influencing the phylogeny of myxosporeans (Eszterbauer, 2004; Holzer et al., 2004; Fiala, 2006; Bartošová et al., 2009, 2011; Fiala and Bartošová, 2010; Carriero et al., 2013; Moreira et al., 2014). The analysis here performed support the phylogenetic trend of tissue tropism throughout, with Ortholinea labracis n. sp. clustering together with other species from the excretory system. The influence of host relatedness is impactful in the Myxobolus clade (Carriero et al., 2013; Moreira et al., 2014), here divided into three subclades that represent myxobolids infecting Cypriniformes, Mugiliformes and Salmoniformes, but its importance as an evolutionary signal for other myxosporean taxa is still debatable. In any case, the few molecular data available for Ortholinea hampers the perception of the importance that this evolutionary signal may display for this taxon.

6.6. Acknowledgments

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 “PEst-C/MAR/LA0015/2013” the project DIRDAMyx, reference FCOMP-01- 0124-FEDER-020726/FCT-PTDC/MAR/116838/2010, and the Ph.D. fellowship grant SFRH/BD/82237/2011 attributed to L. Rangel and the Ph.D. fellowship grant SFRH/BD/92661/2013 attributed to S. Rocha through the programme POPH/FSE QREN, and the fellowship grants attributed to F. Cavaleiro and R. Castro through the project DIRDAMyx, reference FCOMP-01-0124-FEDER-020726/FCT- PTDC/MAR/116838/2010; and the project EUCVOA (NORTE-07-0162-FEDER- 000116) (Portugal). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The authors declare that they have no conflict of interest.

FCUP 113 Chapter 7. Life cycle of Sphaerospora dicentrarchi

Chapter 7

This chapter has been adapted from:

Rangel, L. F., Castro, R., Rocha, S., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F. and Santos, M. J. (2016). 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 (In Press). doi: 10.1017/S0031182016000512.

FCUP 114 Chapter 7. Life cycle of Sphaerospora dicentrarchi

FCUP 115 Chapter 7. Life cycle of Sphaerospora dicentrarchi

7.1. Abstract

Known life cycles of myxosporean parasites have two hosts, but very few life cycles have been disclosed, especially in the marine environment. Sphaerospora dicentrarchi Sitjà-Bobadilla and Álvarez-Pellitero, 1992 is a systemic parasite from the European seabass, Dicentrarchus labrax (Linnaeus, 1758), a highly valuable commercial fish. It affects its health, leading to aquaculture production losses. During 2013 and 2014, an actinospore survey was conducted in a total of 5942 annelids collected from a fish farm in Algarve and from the Aveiro Estuary, in Portugal. A new tetractinomyxon actinospore was found in a capitellid polychaete, belonging to the genera Capitella collected at the fish farm. The tetractinomyxons were pyriform measuring 11.1±0.7 µm in length and 7.2±0.4 µm in width, and presented three rounded polar capsules measuring 2.4±0.3 µm in diameter. The molecular analysis of the 18S rRNA gene sequences from the tetractinomyxons revealed a similarity of 100% with the DNA sequences deposited in the GenBank from S. dicentrarchi myxospores collected from the European seabass and the spotted seabass in the same fish farm and 99.9% similarity with the DNA sequence obtained from the myxospores found infecting the European seabass in the Aveiro Estuary. Therefore, the new tetractinomyxons are inferred to represent the actinospore phase of the S. dicentrarchi life cycle.

FCUP 116 Chapter 7. Life cycle of Sphaerospora dicentrarchi

FCUP 117 Chapter 7. Life cycle of Sphaerospora dicentrarchi

7.2. Introduction

Sphaerospora dicentrarchi Sitjà-Bobadilla and Álvarez-Pellitero, 1992 is a systemic histozoic parasite of the European seabass, Dicentrarchus labrax (Linnaeus, 1758), and spotted seabass, Dicentrarchus punctatus (Bloch, 1792) (Sitjà-Bobadilla and Álvarez-Pellitero, 1992; Xavier et al., 2013), found infecting the connective tissue of the majority of organs in its hosts, with a particular preference for the gallbladder and the intestine (Sitjà-Bobadilla and Álvarez-Pellitero, 1992). It causes chronic infections and losses in European seabass fish farm production, by making the host fish more susceptible to other pathogenic opportunistic infections (Fioravanti et al., 2004). This parasite has a wide distribution in the Mediterranean Sea and the Iberia Peninsula, with high prevalences of infection (38-100%) in the European seabass (Sitjà-Bobadilla and Álvarez-Pellitero, 1992, 1993c; Santos, 1998; Mladineo, 2003; Fioravanti et al., 2004, 2006; Merella et al., 2006; Mladineo et al., 2010). Sphaerospora dicentrarchi is a member of the class Myxosporea Bütschli, 1881, a group of microscopic endoparasites whose known life cycles involve alternation between vertebrates, mainly a fish, and invertebrates, mainly an annelid (Lom and Dyková, 2006; Morris, 2012; Yokoyama et al., 2012). There is still a remarkable lack of knowledge on the life cycles of the majority of the myxosporean species described, especially of those inhabiting the marine environment, where they are known only for seven species (Køie et al., 2004, 2007, 2008, 2013; Rangel et al., 2009, 2015b; Karlsbakk and Køie, 2012). For the freshwater species, the most usual invertebrate host is an oligochaete (Lom and Dyková, 2006; Yokoyama et al., 2012; Székely et al., 2014), but in the marine environment the polychaetes have been reported as invertebrate hosts. The infections by actinospores can be found in marine polychaetes of the families Sabellidae (Køie et al., 2008), Nereididae (Køie, 2000; Rangel et al., 2009; Karlsbakk and Køie, 2012), Serpulidae (Køie, 2002; Køie et al., 2007, 2013), Onuphidae (Rangel et al., 2011) and Capitellidae (Rangel et al., 2015a). The most frequent actinospores in polychaetes, found until now, are tetractinomyxons. The two exceptions are the unicapsulactinomyxon (Rangel et al., 2011) and the echinactinomyxon (Rangel et al., 2015a) actinospores. Because the annelid form is considered the most vulnerable comprehensive knowledge of the identity and habitat requirements of the annelid host may offer a better management avenue for controlling disease in fish farms, potentially preventing economic losses. In the present study, actinospore stages genetically consistent with S. dicentrarchi were found in a capitellid polychaete invertebrate host. FCUP 118 Chapter 7. Life cycle of Sphaerospora dicentrarchi

7.3. Material and methods

Polychaetes were sampled monthly and surveyed for actinospores from February 2013 to March 2014 in two different regions of Portugal. One survey was conducted in a fish farm located in the south of the country, at the Alvor Estuary, Atlantic coast (37°8′N, 8°37′W), Portimão, Algarve, in which polychaetes were collected from the mud of ponds, producing European seabass and gilthead seabream, Sparus aurata (Linnaeus, 1758), as well as from the water outlet ditches, and from an area of the Alvor Estuary that’s located downstream to the fish farm. The other survey was conducted around 400km north in a wild environment, in the Aveiro Estuary, Atlantic coast (40°40′N, 8°45′W). In both sample locations the water salinity was around 35 ‰. In estuaries the mud was collected during the low tide, and in the fish farm tanks the mud was collected either from recently empty tanks or with the help of a core tube in water filled tanks. The mud was brought to the laboratory and the polychaetes were collected by hand with the aid of plastic pipettes from the mud spread over dissection plates. Sphaerospora dicentrarchi myxospores were obtained from cultured European seabass in the Algarve fish farm for morphological study, and from wild seabass specimens (caught in the Aveiro Estuary by professional anglers) for molecular study. During the parasitological survey, the polychaete worms were examined by cutting off a piece of their posterior body segments and squashed on a microscopy slide. The coelomic fluid and the tissues were screened for myxosporean parasites under an optical microscope with a 200-400x magnification. Specimens of European seabass were dissected and their gallbladder and intestine were screened for S. dicentrarchi. Free actinospores and myxospores were examined and photographed using a Zeiss Axiophot microscope (Grupo Taper, Sintra, Portugal) with Nomarski differential interference contrast, equipped with a Zeiss AxioCam Icc3 digital camera. AxioVision 4.6.3 software (Grupo Taper) was used for the image analysis. Morphology and morphometry were obtained from fresh material, in accordance to Lom and Arthur (1989), Lom et al. (1997) and Rangel et al. (2009). All measurements included mean values, ± standard deviation (S.D.) and range. For the molecular analysis, myxospores, actinospores, and infected polychaetes were preserved in absolute ethanol. Genomic DNA was extracted using a GenEluteTM Mammalian Genomic DNA Miniprep Kit, following the manufacturer’s instructions. The parasites’ SSU ribosomal RNA (rRNA) gene was amplified using both FCUP 119 Chapter 7. Life cycle of Sphaerospora dicentrarchi universal primers and myxosporean-specific primers (Table 7.1). For the polychaete host DNA, the universal primers 16sar-L and 16sbr-H (Palumbi et al., 2002) were used (Table 7.1). PCRs were performed in 50 μl reactions using 0.01 pM of each primer, 10 mM of each dNTP, 2.5 mM MgCl2, 5 μl of 10× Taq polymerase buffer (Finnzymes), 1.5 units of Taq DNA polymerase, and approximately 100−150 ng of genomic DNA. The reactions were run on a Hybaid PxE Thermocycler, 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. The sequencing reactions were performed using a BigDye Terminator v1.1 from the Applied Biosystems Kit, and were run on an ABI3700 DNA analyzer (Perkin-Elmer, Applied Biosystems).

Table 7.1 – Primers used for DNA amplification and sequencing the host Capitella sp. and the myxozoa Sphaerospora dicentrarchi.

Primer Sequence (5'–3') Source Myxozoa R1 CCT TCC GTC AAT TCC TTT AAG Azevedo et al. (2009) MyxospecF TTC TGC CCT ATC AAC TTG TTG Fiala (2006) MyxospecR CAA CAA GTT GAT AGG GCA GAA Fiala (2006) ACT3f CAT GGA ACG AAC AAT Hallett and Diamant (2001) ACT3r ATT GTT CGT TCC ATG Hallett and Diamant (2001) Annelida 16sar-L CGC CTG TTT ATC AAA AAC AT Palumbi et al. (2002) 16sbr-H CCG GTC TGA ACT CAC ATC ACG T Palumbi et al. (2002) Both 18e CTG GTT GAT CCT GCC AGT Hillis and Dixon (1991) 18r CTA CGG AAA CCT TGT TAC G Whipps et al. (2003)

7.4. Results

A total of 4275 and 1667 polychaetes worms were examined from the Algarve fish farm and the Aveiro Estuary, respectively. They amounted to about 40 different species grouped in 21 polychaete families. From the Aveiro Estuary, several nereidids Hediste diversicolor (O.F. Müller, 1776) were infect with tetractinomyxon stages consistent with Ellipsomyxa mugilis (Sitjà-Bobadilla and Álvarez-Pellitero, 1993c) actinospores. From the Algarve fish farm samples, one capitellid worm was infected by multiple actinospore FCUP 120 Chapter 7. Life cycle of Sphaerospora dicentrarchi stages in the coelomic cavity (Fig. 7.1), showing no external signs of the infection. The actinospores were morphologically identified as belonging to the tetractinomyxon collective group.

Considering only the capitellids worms, 916 and 121 worms were examined from de Algarve fish farm and the Aveiro Estuary, respectively. The infected worm was found only in the sample of capitellids from European seabass ponds, in March 2014. Considering only this location, one worm was infected in 520 (0.2% of prevalence of infection). While considering the entire sample of capitellids from the Algarve fish farm, one worm was infected in 916 (0.1%). From Aveiro Estuary none of the 121 capitellid specimens collected were infected. FCUP 121 Chapter 7. Life cycle of Sphaerospora dicentrarchi

The tetractinomyxons were found in a polychaete identified as belonging to the family Capitellidae by morphology. After a BLAST search, the 18S rRNA sequence obtained for the polychaete host (GenBank accession number KT970640) presented a similarity percentage of 99% to Capitella sp. (FN421417) and 98% to Capitella teleta Blake et al., 2009 (U67323) and Capitella capitata (Fabricius, 1780) (JF509728). The 16S rRNA sequence (KT970641) presented a similarity percentage of 91% to C. teleta (JF509722). The other two species do not have 16S rRNA sequences deposited in the GenBank, while the remaining polychaetes with sequences presented a similarity below 80%. The tetractinomyxons were found developing inside pansporocysts in groups of eight (Fig. 7.1). The free spores were pyriform with three apical rounded polar capsules (Figs. 7.1f and 7.2). Spores (n=12) measured 11.1±0.7 (10.2–12.4) µm in length and 7.2±0.4 (6.9–8.0) µm in width. The spore body measured 6.9±0.2 (6.5–7.1) µm in length. The polar capsules measured 2.4±0.3 (2.0–3.0) µm in diameter. The polar filaments and the secondary cells in the sporoplasms were not visible. Many spores had a small extension of the valvogenic cells while this character was absent in others (Figs. 7.1f and 7.2).

Fig. 7.2 – Schematic illustration of the inferred life cycle of Sphaerospora dicentrarchi.

The myxospores of S. dicentrarchi (n=14) from European seabass of the Algarve fish farm measured 4.9±0.2 (4.5–5.4) µm in length, 3.4±0.2 (3.1–3.6) µm in width, and 5.3±0.3 (4.9–5.8) µm in thickness. The pyriform polar capsules measured 1.8±0.2 (1.4–2.1) µm in length and 1.3±0.1 (1.1–1.5) µm in width (Fig. 7.3). FCUP 122 Chapter 7. Life cycle of Sphaerospora dicentrarchi

The sequencing of the 18S rRNA gene of S. dicentrarchi from the myxospores collected from European seabass of the Aveiro Estuary, and from the capitellid tetractinomyxons collected in the Algarve fish farm resulted in a sequence with 1666 bp (KT970638) and 1672 bp (KT970639) respectively. They differ only in one nucleotide position. Taking as reference the myxospore DNA sequence, the nucleotides differ in the position 1448 where a guanine is replaced by an adenine in the actinospore DNA sequence, so both sequences were 99.9% similar. In the GenBank there are three sequences for S. dicentrachi. One sequence from Italy (AY278564) unpublished, with 844 bp, incorporates many errors in the sequence 5' and 3' extremities, which make it unreliable for comparison purposes. The other two sequences, from D. labrax (KC516864) and D. punctatus (KC516865) were obtained in the same Algarve fish farm. These two later sequences are smaller (569 bp for both) compared to the sequences of DNA from both myxospore (1666 bp) and actinospore (1672 bp) from this study, and the portion where they align were 100% identical.

7.5. Discussion

This study identifies a new marine tetractinomyxon actinospore from a capitellid polychaete, Capitella sp., and presents molecular data which allow inferring a life cycle match between these actinospores stages and the myxospores of S. dicentrarchi. This is the seventh inferred marine life cycle involving polychaetes and this type of actinospore. From the nine different tetractinomyxon actinospores described in the literature, the pyriform actinospore of S. dicentrarchi is morphologically more similar to E. mugilis FCUP 123 Chapter 7. Life cycle of Sphaerospora dicentrarchi

(Rangel et al., 2009) and Ellipsomyxa gobii Køie, 2003 (Køie, 2000; Køie et al., 2004), but with a smaller size in all characters. The others tetractinomyxon morphotypes described have a tetrahedral form (Ikeda, 1912; Bartholomew et al., 1997) or are more spherical or subspherical (Bartholomew et al., 2006; Køie, 2002; Køie et al., 2007, 2013; Karlsbakk and Køie, 2012). All measures of the myxospores of S. dicentrarchi are within the range of the original description (Fig. 7.3), with the exception of the spore width, which has some variation, but does not diverge significantly. Width and thickness measurements are prone to some variation, according to the spore orientation, especially in these very small spores. Some doubts were raised about the validity of the S. dicentrarchi 18S rRNA gene sequence deposited in GenBank (AY278564) from Italy (Morris and Adams, 2008), especially because it clustered with the Kudoa species (Eszterbauer and Székely, 2004; Fiala, 2006), but a posterior study which analysed the 28S rRNA gene and the combined 18S + 28S rRNA data, confirmed the phylogenetic position of S. dicentrarchi (Bartošová et al., 2009, 2011). A more recent study, which included the 18S and the 28S rRNA genes, also reconfirmed the 18S rRNA sequence for S. dicentrarchi in two different hosts, D. labrax and D. punctatus, from Algarve (Xavier et al., 2013). In the present study, a more extended 18S rRNA sequence for S. dicentrarchi was obtained from spores of both vertebrate and invertebrate hosts, strengthening even more the validity of the molecular data. Discussion about the position of this species in the myxozoan phylogeny are common in recent phylogenetic studies, as they all place consistently S. dicentrarchi among the Kudoa species clade (Diamant et al., 2005; Morris and Adams, 2008; Bartošová et al., 2009, 2011). This led several authors to question the position of S. dicentrarchi among the Sphaerospora (Diamant et al., 2005; Fiala, 2006; Bartošová et al., 2011) due mainly to the unique characters that differentiate them from other Sphaerospora species, like very small spores with binucleate sporoplasm in a histozoic 'bag-like' polysporic plasmodia and the ultrastructural detail of overlapping shell valves (Sitjà-Bobadilla and Álvarez-Pellitero, 1992) which is more typical in Kudoa species (Dyková et al., 2009). Therefore, these authors suggested that S. dicentrarchi may be a two-valved Kudoa species. Sphaerospora dicentrarchi is phylogenetically distant from Sphaerospora species sensu stricto (Bartošová et al., 2011), so we cannot generalize that the now inferred life cycle of S. dicentrarchi involving polychaetes and tetractinomyxon actinospores to those other species of Sphaerospora. On the other hand, the close FCUP 124 Chapter 7. Life cycle of Sphaerospora dicentrarchi affinity of this species to the Kudoa marine clade (Fiala, 2006) suggests that polychaetes may be candidate hosts for Kudoa species. The cosmopolitan worm species, C. capitata, has been found to consist of several distinct cryptic species (Grassle and Grassle, 1976; Méndez et al., 2000; Blake, 2009; Blake et al., 2009). Among them, we find the species C. teleta, recently described by Blake et al. (2009). These worms are considered to be a group of opportunistic species, inhabiting organically-enriched sediment (Tsutsumi, 1990; Tsutsumi et al., 1990), like the ones we find in fish farm ponds. The worm found to be infected with S. dicentrarchi in this study, was a Capitella species, most probably belonging to this Capitella species complex. So, for the land fish farm ponds where European seabass are reared, it is natural to find this worm host, possibly perpetuating the S. dicentrarchi infection. Also, this means that estuaries are good environments for these species, and since wild European seabass use this environment for reproduction (Costa, 1988; Laffaille et al., 2001; Martinho et al., 2008) both hosts can be easily found in the same location. The high occurrence of infection of S. dicentrarchi in the fish host (Sitjà- Bobadilla and Álvarez-Pellitero, 1992, 1993c; Santos, 1998; Mladineo, 2003; Fioravanti et al., 2004, 2006; Merella et al., 2006; Mladineo et al., 2010) contrast with the low prevalence of infection in the invertebrate host. However, this difference of prevalences between the vertebrate and the invertebrate hosts is common to the other myxosporean species. For instance, E. mugilis can reach a level of infection of 70% in mugilids fishes (Sitjà-Bobadilla and Álvarez-Pellitero, 1993c); however, only 0.5% of the polychaete worms are infected (Rangel et al., 2009). Generally, prevalence of infection of actinospores in the invertebrate hosts is very low, ranging from 0.1 to 4% (Yokoyama et al., 2012). Polychaetes in particular, have prevalences more variable, ranging from 0.3 to 17% (Rangel et al., 2011; Karlsbakk and Køie, 2012), but they are more frequently around 1 to 8% (Køie et al., 2004, 2008, 2013; Køie, 2005; Rangel et al., 2009, 2015a). Nevertheless, because the tetractinomyxons in this study were found in a host belonging to a species complex, the prevalence of infection may also be underestimated, since not all Capitella species in this species complex may serve as host for these actinospores. Sphaerospora dicentrarchi tetractinomyxons develop in the polychaetes coelomic cavity. This is the most typical site of infection in the invertebrate host for this type of actinospore (Ikeda, 1912; Køie, 2000, 2002; Bartholomew et al., 2006; Rangel et al., 2009; Karlsbakk and Køie, 2012; Køie et al., 2013), with the exception of Ceratonova shasta (Noble, 1950) tetractinomyxons which develops in the epidermal FCUP 125 Chapter 7. Life cycle of Sphaerospora dicentrarchi tissue (Bartholomew et al., 1997). However, the infection was underestimated, since only the posterior part of the worms was surveyed for parasite infection. It is not clear how the coelozoic tetractinomyxons are released from the invertebrate coelomic cavity to infect the vertebrate host. Several processes have been suggested (Ikeda, 1912; Køie, 2000, 2002; Bartholomew et al., 2006; Rangel et al., 2009). One possible way is through the pores in the invertebrate tegument, like the gonopores (Køie, 2002; Bartholomew et al., 2006) or the nephridiopores (Ikeda, 1912). One other possible way is the release of the actinospores after the death of the worm host either for reproduction (Rangel et al., 2009) or by natural death (Ikeda, 1912; Køie, 2000). Finally, it was also suggested that the vertebrate host may be infected by ingesting infected worms (Køie, 2000). The tetractinomyxons of S. dicentrarchi are smaller compared to the spores of E. gobii (Køie, 2000) and E. mugilis (Rangel et al., 2009) and have a closer size to other tetractinomyxons with a tethraedric or spherical form. Their smaller size turns them more suitable for gonopore or nephridiopore release mechanisms. The Capitella species complex worms in spite of having a short lifespan, approximately one year (Warren, 1976; Martin and Bastida, 2006), reproduce several times during the year (Warren, 1976; Martin and Grémare, 1997; Martin and Bastida, 2006) which can facilitate the exit of tetractinomyxons through the polychaete gonopores. Another possible mechanism of infection for S. dicentrarchi can be the ingestion of infected worms. It was suggested that S. dicentrarchi can infect young fish orally, first infecting hematopoietic organs and then spread via the bloodstream and became a chronic infection in older fishes (Sitjà-Bobadilla and Álvarez-Pellitero, 1993c). The crustaceans are the main food items for juvenile European seabass, while annelids are a very small portion on their diet (Pickett and Pawson, 1994; Cabral and Costa, 2001; Laffaille et al., 2001; Hampel et al., 2005). However, in some localities the annelids, mainly polychaetes like capitellids and nereidids can be important (Pickett and Pawson, 1994; Hampel et al., 2005; Martinho et al., 2008), due to the opportunistic character of this fish (Pickett and Pawson, 1994). In adult European seabass, the use of annelids as prey is considered negligible (Spitz et al., 2013). Diamant (1997) suggested that in the marine environment, fish-to-fish transmission of myxosporean parasites could be the model, nevertheless the recent findings of actinospores matching genetically with fish myxospores (Køie et al., 2004, 2007, 2008, 2013; Karlsbakk and Køie, 2012; Rangel et al., 2009, 2015b) and the present data allow us to infer that two host life cycles may also be an important mechanism of transmission for marine myxosporeans. However, there are many thousands of marine myxosporeans species known so far and the life cycles have only been inferred for fewer than 10 species. FCUP 126 Chapter 7. Life cycle of Sphaerospora dicentrarchi

7.6. Acknowledgments

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 “PEst-C/MAR/LA0015/2013”, and the project DIRDAMyx, reference FCOMP- 01-0124-FEDER-020726/FCT-PTDC/MAR/116838/2010, both to M. J. S., and the Ph.D. fellowship grant SFRH/BD/82237/2011 attributed to L. Rangel and the Ph.D. fellowship grant SFRH/BD/92661/2013 attributed to S. Rocha through the programme POPH/FSE QREN, and the fellowship grants attributed to F. Cavaleiro and R. Castro through the project DIRDAMyx, reference FCOMP-01-0124-FEDER-020726/FCT- PTDC/MAR/116838/2010. This work complies with the current laws of the country where it was performed.

FCUP 127 Chapter 8. A new type of Echinactinomyxon infecting a marine polychaete, Heteromastus filiformis

Chapter 8

A new type of Echinactinomyxon (Myxozoa), infecting a marine polychaete, Heteromastus filiformis (Polychaeta: Capitellidae) in the Alvor Estuary (Portugal)

This chapter has been adapted from:

Rangel, L. F., Rocha, S., Castro, R., Severino, R., Casal, G. and Santos, M. J. (2015). A new type of Echinactinomyxon (Myxozoa), infecting a marine polychaete, Heteromastus filiformis (Polychaeta: Capitellidae) in the Alvor Estuary (Portugal). Microscopy and Microanalysis 21 (Suppl. s5), 85–86. doi:10.1017/S1431927615014233

FCUP 128 Chapter 8. A new type of Echinactinomyxon infecting a marine polychaete, Heteromastus filiformis

FCUP 129 Chapter 8. A new type of Echinactinomyxon infecting a marine polychaete, Heteromastus filiformis

8.1. Abstract

A new type of echinactinomyxon is described from the intestinal epithelium of the marine polychaete Heteromastus filiformis (Claparède, 1864). The actinospores measured 17.6 μm in length and 9.1 μm in width. The caudal processes were straight, measuring 86.4 μm in length and 4.9 μm in width, and formed an angle of 125-140° to the actinospore body axis. Three pyriform polar capsules measured 3.2 by 2.2 μm. The SSU rRNA sequence obtained was most similar to Sphaeromyxa species that infect the gallbladder of marine fishes, clustering within the main freshwater lineage.

FCUP 130 Chapter 8. A new type of Echinactinomyxon infecting a marine polychaete, Heteromastus filiformis

FCUP 131 Chapter 8. A new type of Echinactinomyxon infecting a marine polychaete, Heteromastus filiformis

8.2. Introduction

The study of Actinosporea (a former class of the Myxozoa) was conducted for many years without the knowledge that they represented the definitive stage of the life cycle of myxosporean parasites (Wolf and Markiw, 1984; Kent et al., 2001; Lom and Dyková, 2006). The discovery of the life cycle of Myxobolus cerebralis altered this perspective (Wolf and Markiw, 1984), leading to the elucidation of several other life cycles in the past few years (Kent et al., 2001; Lom and Dyková, 2006). Most studies aim at the life cycles of freshwater Myxozoa infecting oligochaete hosts, while in the marine environment there are many species of Myxozoa whose life cycles are yet to unravel. In this work we present a new type of marine actinospore of the collective group Echinactinomyxon.

8.3. Material and methods

Polychaetes were collected in the Alvor Estuary, Portimão, Algarve, downstream to a fish farm. Specimens were examined using differential interference contrast (DIC) optics. Measurements of actinospores were taken from fresh material. Infected polychaetes were processed for molecular procedures, namely sequencing of the SSU rRNA gene. For inferring phylogenetic relationships, the Maximum Likelihood (ML) method was performed in MEGA 5.05, using Kimura 2-parameter as substitution model with uniform rates.

8.4. Results

From the collected sample, 13 polychaetes belonging to the family Capitellidae were identified as Heteromastus filiformis (Claparède, 1864). Of these, one was infected with actinospores belonging to the Echinactinomyxon collective group. The developmental stages were located in the intestinal epithelium (Fig. 8.1). The actinospores (Fig. 8.2) measured 17.6 μm in length and 9.1 μm in width. The caudal processes were straight, measuring 86.4 μm in length and 4.9 μm in width, and formed an angle of 125-140° to the actinospore body axis. The three pyriform polar capsules measured 3.2 by 2.2 μm. The SSU rRNA sequence obtained was most similar to Sphaeromyxa hellandi FCUP 132 Chapter 8. A new type of Echinactinomyxon infecting a marine polychaete, Heteromastus filiformis

DQ377693, DQ377692, DQ377701 (94%) and Sphaeromyxa lycodi KC524734 (91%). The ML tree revealed the Echinactinomyxon type here described clustering together with Sphaeromyxa species that infect the gallbladder of marine fishes (Fig. 8.3).

Fig. 8.1 – Mature pansporocyst in the intestinal ephitelium of Heteromastus filiformis as seen with DIC.

Fig. 8.2 – Mature free Echinactinomyxon spore as seen with DIC.

FCUP 133 Chapter 8. A new type of Echinactinomyxon infecting a marine polychaete, Heteromastus filiformis

8.5. Discussion

This study constitutes the first record of an actinospore of the collective group Echinactinomyxon infecting a polychaete in the marine environment, since all other previously recorded Echinactinomyxon types infect freshwater oligochaetes. Furthermore, freshwater Echinactinomyxon have been, thus far, implicated in the life cycle of Myxobolus (Marton and Eszterbauer, 2011; Holzer et al., 2004), while the type here described presents greater affinity to marine species of the genus Sphaeromyxa. A recent myxozoan phylogeny reveals the latter clustering within the coelozoic clade of the main freshwater clade (Fiala and Bartošová, 2010).

FCUP 134 Chapter 8. A new type of Echinactinomyxon infecting a marine polychaete, Heteromastus filiformis

8.6. Acknowledgments

This work was financially supported by FCT (Portugal), within the scope of the project DIRDAMyx, reference FCOMP-01-0124-FEDER-020726/FCT-PTDC/MAR/116838/ 2010, the Ph.D. fellowship grant SFRH/BD/82237/2011 attributed to L. Rangel, and the Ph.D. fellowship grant SFRH/BD/92661/2013 attributed to S. Rocha.

FCUP 135 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Chapter 9

This chapter has been adapted from:

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 (In Press). doi: 10.1007/s00436-016-4983-8.

FCUP 136 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

FCUP 137 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

9.1. Abstract

Ten new types of sphaeractinomyxon actinospores are morphologically and molecularly described from the coelomic cavity of two marine oligochaete hosts, Limnodriloides agnes Hrabě, 1967 and Tubificoides pseudogaster (Dahl, 1960), from Aveiro Estuary, Portugal. The smallest sphaeractinomyxon type measured 17 µm (length) × 19 µm (width) × 19 µm (apical diameter), whereas the largest type measured 61 µm × 76 µm × 80 µm. While considering the 10 types of sphaeractinomyxon, it was found that the number of spores developing inside pansporocysts varied between one, two, four and eight. The total prevalence of infection was of 19% for the two host species, with a maximum recorded for spring and summer (25–26%). While considering each type of sphaeractinomyxon individually, it was found that the prevalence values ranged between 0.3 and 1.7%. All described sphaeractinomyxons were most similar to Myxobolus species. The validity of the tetraspora and endocapsa collective group names is discussed.

FCUP 138 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

FCUP 139 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

9.2. Introduction

The discovery and description, in 1899, of the first actinospore types – i.e. synactinomyxon, hexactinomyxon, and triactinomyxon – is attributed to Antonin Štolc, who isolated them from specimens of oligochaetes collected in Vltava River in Czech Republic (Caullery and Mesnil, 1905). A few years later, in 1904 the first sphaeractinomyxon actinospore, designated with a binomial name, i.e. Sphaeractinomyxon stolci, was described from marine oligochaetes (Caullery and Mesnil, 1904, 1905). Since then, several other sphaeractinomyxons were described: Sphaeractinomyxon gigas in 1923, by Granata; Sphaeractinomyxon danicae in 1923, by Georgevitch; Sphaeractinomyxon ilyodrili in 1940, by Jirovec (Marques, 1984); Sphaeractinomyxon amanieui (Puytorac, 1963); and Sphaeractinomyxon rotundum (Marques, 1984). Until this time, all actinospore types were considered to represent legitimate species. They were classified in a separate class, named Actinosporea, and named following the binomial nomenclature system. However, in 1984, Wolf and Markiw were able to demonstrate the alternation of the life cycle of Myxobolus cerebralis in an oligochaete, involving the formation of triactinomyxon actinospores. The class Actinosporea was then extinct and the genera of actinosporeans became collective group names (Kent et al., 1994). In the following, new types of sphaeractinomyxon were described: sphaeractinomyxon types 1 and 2 (Hallett et al., 1997), Sphaeractinomyxon ersei (Hallett et al., 1998), and Sphaeractinomyxon leptocapsula (Hallett et al., 1999). Two new actinospore collective group names, tetraspora and endocapsa, were erected to encompass the new types of sphaeractinomyxons exhibiting some variations in its characters (Hallett and Lester, 1999; Hallett et al., 1999). Tetraspora actinospores differ from sphaeractinomyxon in the number of spores it develops inside pansporocysts, exclusively, i.e., four spores instead of the usual eight spores. Two types of tetraspora were described, Tetraspora discoidea and Tetraspora rotundum (Hallett and Lester, 1999). Endocapsa actinospores differ from sphaeractinomyxon in having small irregular valvular expansions and not protruding polar capsules. Four types of endocapsa were described, Endocapsa rosulata and Endocapsa stepheni (Hallett et al., 1999), endocapsa type 1 (Hallett et al., 2001), and endocapsa type of Székely et al. (2007). FCUP 140 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Several life cycles have been demonstrated by experimental infection or inferred by molecular biology involving actinospore types from about half of the actinospore collective groups (Eszterbauer et al., 2015). Until now, sphaeractinomyxon actinospores were never associated with a life cycle of any identified myxosporean, but phylogenetically sphaeractinomyxons are associated with marine Myxobolus species (Kent et al., 2001). The body of knowledge on myxosporeans and the group's taxonomy are still based in the myxosporean phase and myxospore morphology, despite the fact that molecular biology has demonstrated a real need for new ways to approach the myxosporean classification (Fiala, 2006; Fiala et al., 2015b). The study of actinospores is still very insipient, especially in what concern the marine species, and surely they will have a crucial role in the future classification of the Myxosporea Bütschli, 1881 class. This study is intended to represent a contribution for that goal, adding 10 new marine sphaeractinomyxon actinospores, morphologically and molecularly described from two species of oligochaete hosts.

9.3. Material and methods

Actinospore sampling and morphological study

From 2013 to 2014, an actinospore survey was conducted in 651 oligochaete specimens from Aveiro Estuary (40°40′N, 8°45′W), Portugal. Oligochaetes were collected from the mud at low tide and kept individually in cell well plates containing salt water. A stereo microscope was used to examine the specimens for the release of actinospores during the following days. All specimens were posteriorly examined individually under a light microscope (×200-×400 of magnification) with a drop of salt water and gently pressured by a lamella, to detect the presence of actinospores in the coelomic cavity. 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) 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 deviations (SDs), range of variation, and number of measured actinospores.

FCUP 141 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Molecular characterization

Genomic DNA from actinospores and oligochaete hosts was extracted using the GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich), following the manufacturer’s instructions. The 18S ribosomal RNA (rRNA) gene of actinospores was PCR-amplified with the universal eukaryotic primers ERIB1 and ERIB10 (Table 9.1). PCR was carried out in a 25-µl reaction volume, using 2 µl of extracted genomic DNA, 0.5 µl of 10 mM deoxyribonucleotide triphosphates (dNTPs; nzyTech), 0.25 µl of 10 pmol of each primer, 2.5 µl of 10× Taq DNA polymerase buffer, 1.25 µl of 50 mM

MgCl2, 1.25 U of Taq DNA polymerase (nzyTech), and 18 µl of water. The reactions were run on a Bio-Rad - MJ Mini Gradient Thermal Cycler, with initial denaturation at 95 °C for 3 min, followed by 35 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. The final elongation step was performed at 72 °C for 7 min. This was followed by a nested PCR, using as a template 1 µl of the initial PCR and specific myxosporeans primers (Table 9.1). The PCR mixture reaction was the same as for the first PCR, except for the 0.5 µl of 10 pmol of each primer. The nested PCR cycle had an 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 1:30 min, and a final elongation at 72 °C for 7 min. Concerning the oligochaete hosts, the 16S rRNA gene was PCR-amplified using the universal primers 16sar-L and 16sbr-H (Table 9.1). The PCR was carried out in a single reaction in the same conditions as for the actinospore DNA nested PCR, but using 2 µl of extracted genomic DNA. All PCR products were electrophoresed through a 1 % agarose 1× Tris- acetate-EDTA buffer (TAE) gel stained with GreenSafe Premium (nzyTech). The PCR amplification products were purified and sequenced by STABVida (Portugal).

Table 9.1 – Primers used in actinospores and oligochaete DNA amplification and sequencing. Name Sequence (5’-3’) Pared with Source

ERIB1 ACC TGG TTG ATC CTG CCA G ERIB10 Barta et al. (1997) ERIB10 CTT CCG CAG GTT CAC CTA CGG ERIB1 Barta et al. (1997) 18e CTG GTT GAT CCT GCC AGT ACT3r, Myx4r, ACT1r Hillis and Dixon (1991) 18r CTA CGG AAA CCT TGT TAC G MyxospecF, ACT3f Whipps et al. (2003) MyxospecF TTC TGC CCT ATC AAC TTG TTG ACT1r, 18r Fiala (2006) ACT3f CAT GGA ACG AAC AAT ACT1r, 18r Hallett and Diamant (2001) ACT3r ATT GTT CGT TCC ATG 18e Hallett and Diamant (2001) Myx4r CTG ACA GAT CAC TCC ACG AAC 18e Hallett and Diamant (2001) ACT1r AAT TTC ACC TCT CGC TGC CA 18e, MyxospecF Hallett and Diamant (2001) 16sar-L CGC CTG TTT ATC AAA AAC AT 16sbr-H Palumbi et al. (2002) 16sbr-H CCG GTC TGA ACT CAG ATC ACG T 16sar-L Palumbi et al. (2002)

FCUP 142 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Staden Package software (pregap4 and gap4) version 2.0.0 (Staden et al., 2000) was used to assist the 16S rRNA and 18S rRNA consensus sequences assembling. Similarities between sequences (pairwise p-distance) were calculated using MEGA 5 software (Tamura et al., 2011). Consensus sequences were submitted to a standard nucleotide BLAST search for close relatives from NCBI (http://blast.ncbi.nlm.nih.gov).

9.4. Results

During the survey, 651 oligochaetes were examined for parasites. From these, 122 (18.7%) were found infected with actinospores identified as representatives of sphaeractinomyxon collective group. All examined oligochaete hosts looked alike, with similar dimensions, bifid setae, and a body tegument devoid of papillae. However, molecular biology has demonstrated the existence of two distinct oligochaete species. After submitting the 16S rRNA gene sequence from both oligochaete species to a BLAST search, one consensus sequence with 537 bp, obtained from nine specimens (GenBank accession number KU569321), was found to be 99.8% similar to Limnodriloides agnes Hrabě, 1967 (KR025871), while the other, with 527 bp, obtained from five specimens (KU569320), was 98.8% similar to Tubificoides pseudogaster (Dahl, 1960) (HM459968), specifically, Tu. pseudogaster from the lineage II, as reported by Kvist et al. (2010). Accordingly, the prevalence of infection was calculated considering the total sample of both oligochaetes. Sphaeractinomyxons were found in oligochaetes throughout the year. Seasonal prevalence was 8.1% for winter (January–March; n=211), 26.3% for spring (April–June; n=137), 19.5% for summer (July–September; n=123), and 25.0% for autumn (October−December; n=180). From the total number of infected oligochaetes, 30.3% had only initial developmental stages and/or immature spores, especially in the autumn season, with a maximum of 40.0%; 5.7% had co-infections with another type of sphaeractinomyxon, and, in one case, with a triactinomyxon type. The individual prevalence of infection for each type of actinospore was very low, ranging from 0.3 to 1.7%, even considering that it must be underestimated, because they were calculated comprising two different hosts.

FCUP 143 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Taxonomy and morphology of studied actinospores

Sphaeractinomyxon type 1 (new type) Figs. 9.1a-c and Table 9.2

Description: Mature spores spherical in apical and lateral view. Spores (n=36) 17.2±1.5 (15.2–20.5) µm in length, 18.8±1.3 (16.7–21.7) µm in width, and 18.8±0.9 (16.9–21.0) µm in diameter; three pyriform polar capsules 4.7±0.4 (3.8–5.8) µm in length and 3.4±0.3 (2.7–3.8) µm in width. Polar filaments exhibiting three to four longitudinal coils. Sporoplasm having many secondary cells. Spores developing in number of 8 inside pansporocysts. Type host: Limnodriloides agnes Hrabě, 1967 Type locality: Aveiro Estuary, Portugal Site of infection: Coelomic cavity Prevalence of infection: 1.7% (11 out of 651) GenBank accession no.: KU569310 Remarks: The sequences in GenBank most similar to this type were Triactinomyxon sp. SH-2006 (DQ473515) and Endocapsa sp. SH-2006 (DQ473516), with 91% of similarity, followed by Myxobolus exiguus Thélohan, 1895 (AY129317), Myxobolus muelleri Bütschli, 1882 (AY129314), and Myxobolus episquamalis Egusa et al., 1990 (JF810537), with 90% of similarity.

Sphaeractinomyxon type 2 (new type) Fig. 9.1d and Table 9.2

Description: Mature spores spherical to angular in apical and lateral view. Spores (n=10) 23.3±0.8 (22.1–24.8) µm in length, 28.4±1.0 (26.2–29.8) µm in width, and 28.6±1.2 (26.6–31.1) µm in diameter; three pyriform polar capsules 5.9±0.2 (5.7–6.0) µm in length and 4.1±0.2 (4.0–4.3) µm in width. Polar filaments exhibiting three to four longitudinal coils. Sporoplasm having many secondary cells. Spores developing in number of 8 inside pansporocysts. Type host: Limnodriloides agnes Hrabě, 1967 Type locality: Aveiro Estuary, Portugal Site of infection: Coelomic cavity Prevalence of infection: 0.5% (3 out of 651) GenBank accession no.: KU569311 FCUP 144 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Remarks: The sequences in GenBank most similar to this type were Myxobolus sp. WSK-2013 (KC733438) and S. ersei (AF306790), with 94% of similarity, and Myxobolus ichkeulensis Bahri and Marques, 1996 (AF378337), with 93% of similarity.

Sphaeractinomyxon type 3 (new type) Figs. 9.1e, f and Table 9.2

Description: Mature spores angular in apical view and spherical to ellipsoidal in lateral view. Spores (n=16) 30.5±1.8 (27.9–33.4) µm in length, 33.6±2.2 (28.4–38.0) µm in width, and 33.9±0.8 (32.5–35.3) µm in diameter; three pyriform polar capsules 7.5±0.7 (6.5–8.3) µm in length and 6.2±0.4 (5.8–7.0) µm in width. Polar filaments exhibiting three longitudinal coils. Sporoplasm having many secondary cells. Spores developing in number of 4 inside pansporocysts. Type host: Limnodriloides agnes Hrabě, 1967 Type locality: Aveiro Estuary, Portugal Site of infection: Coelomic cavity Prevalence of infection: 0.5% (3 out of 651) GenBank accession no.: KU569312 Remarks: The sequences in GenBank most similar to this type were E. rosulata (AF306791), with 91% of similarity, and M. exiguus (AY129317) and M. muelleri (AY129314), with 90% of similarity.

Sphaeractinomyxon type 4 (new type) Fig. 9.1g and Table 9.2

Description: Mature spores spherical to angular in apical view and spherical to slightly ellipsoidal in lateral view. Spores (n=19) 33.2±2.8 (28.1–36.7) µm in length, 36.5±2.3 (32.4–42.6) µm in width, and 37.9±2.2 (34.1–40.3) µm in diameter; three pyriform polar capsules 7.8±0.4 (7.0–8.7) µm in length and 6.6±0.4 (5.8–7.1) µm in width. Polar filaments exhibiting two to three longitudinal coils. Sporoplasm having many secondary cells. Spores developing in number of 4 inside pansporocysts. Type host: Limnodriloides agnes Hrabě, 1967 Type locality: Aveiro Estuary, Portugal Site of infection: Coelomic cavity Prevalence of infection: 0.8% (5 out of 651) GenBank accession no.: KU569313 FCUP 145 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Remarks: The sequences in GenBank most similar to this type were Endocapsa sp. SH-2006 (DQ473516), with 89% of similarity, and M. episquamalis (JF810537), M. exiguus (AY129317), and M. muelleri (AY129314), with 91% of similarity.

Sphaeractinomyxon type 5 (new type) Figs. 9.1h, i and Table 9.2

Description: Mature spores angular in apical view and ellipsoidal in lateral view. Spores (n=10) 42.5±2.1 (38.8–45.6) µm in length, 54.8±2.7 (50.0–59.5) µm in width, and 56.0±2.4 (52.9–58.8) µm in diameter; three pyriform polar capsules 9.1±0.3 (8.8–9.6) µm in length and 7.6±0.3 (7.0–7.9) µm in width. Polar filaments exhibiting two to three longitudinal coils. Sporoplasm having many secondary cells. Spores developing in number of 4 inside pansporocysts. Type host: Limnodriloides agnes Hrabě, 1967 Type locality: Aveiro Estuary, Portugal Site of infection: Coelomic cavity Prevalence of infection: 0.6% (4 out of 651) GenBank accession no.: KU569314 Remarks: The sequences in GenBank most similar to this type were M. exiguus (AY129317) and E. rosulata (AF306791), with 90% of similarity, and M. episquamalis (JF810537), with 89% of similarity.

Sphaeractinomyxon type 6 (new type) Figs. 9.1j, k and Table 9.2

Description: Mature spores angular in apical view and ellipsoidal in lateral view. Spores (n=20) 51.5±5.2 (46.0–56.9) µm in length, 64.5±3.3 (61.6–69.2) µm in width, and 62.1±2.8 (56.7–68.4) µm in diameter; three pyriform polar capsules 10.2±0.4 (9.9–10.9) µm in length and 8.9±0.8 (7.5–9.7) µm in width. Polar filaments exhibiting two to three longitudinal coils. Sporoplasm having many secondary cells. Spores developing in number of 2 to 4 inside pansporocysts. Type host: Limnodriloides agnes Hrabě, 1967 Type locality: Aveiro Estuary, Portugal Site of infection: Coelomic cavity Prevalence of infection: 0.8% (5 out of 651) GenBank accession no.: KU569315

FCUP 146 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

FCUP 147 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Remarks: The sequences in GenBank most similar to this type were M. episquamalis (JF810537), M. exiguus (AY129317), and M. muelleri (AY129314), with 90% of similarity, and E. rosulata (AF306791), with 87% of similarity.

Sphaeractinomyxon type 7 (new type) Figs. 9.1l-n and Table 9.2

Description: Mature spores angular in apical view and ellipsoidal in lateral view. Spores (n=10) 60.9±5.6 (55.0–68.7) µm in length, 75.6±6.2 (64.7–85.8) µm in width, and 80.3±5.1 (71.8–91.4) µm in diameter; three pyriform polar capsules 10.6±0.9 (9.4–11.9) µm in length and 9.7±0.4 (9.2–10.1) µm in width. Polar filaments exhibiting two to three longitudinal coils. The sporoplasm having many secondary cells. Spores developing in number of 1 to 4 inside pansporocysts. Type host: Limnodriloides agnes Hrabě, 1967 Type locality: Aveiro Estuary, Portugal Site of infection: Coelomic cavity Prevalence of infection: 1.2% (8 out of 651) GenBank accession no.: KU569316 Remarks: The sequences in GenBank most similar to this type were E. rosulata (AF306791), Myxobolus bizerti Bahri and Marques 1996 (AY129318), M. exiguus (AY129317), and M. muelleri (AY129314), all with 90% of similarity.

Sphaeractinomyxon type 8 (new type) Fig. 9.1o and Table 9.2

Description: Mature spores spherical in apical and lateral view. Spores (n=21) 8.1±0.9 (17.0–21.3) µm in length, 18.6±0.8 (17.7–21.1) µm in width, and 18.1±1.0 (17.1–20.0) µm in diameter; three pyriform polar capsules 4.7±0.2 (4.3–5.2) µm in length and 3.3±0.2 (3.0–3.5) µm in width. Polar filaments exhibiting two to three longitudinal coils. The sporoplasm having many secondary cells. Spores developing in number of 8 inside pansporocysts. Type host: Tubificoides pseudogaster (Dahl, 1960) Type locality: Aveiro Estuary, Portugal Site of infection: Coelomic cavity Prevalence of infection: 0.3% (2 out of 651) GenBank accession no.: KU569317 FCUP 148 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Remarks: The sequences in GenBank most similar to this type were Triactinomyxon sp. SH-2006 (DQ473515) and Endocapsa sp. SH-2006 (DQ473516), with 94% of similarity, and M. exiguus (AY129317) and M. muelleri (AY129314), with 93% of similarity.

Sphaeractinomyxon type 9 (new type) Fig. 9.1p and Table 9.2

Description: Mature spores spherical in apical and lateral view. Spores (n=21) 20.9±1.0 (19.0–23.4) µm in length, 22.0±1.5 (20.4–26.9) µm in width, and 22.6±1.2 (20.7–26.5) µm in diameter; three pyriform polar capsules 5.6±0.4 (4.8–6.0) µm in length and 4.3±0.3 (3.9–4.8) µm in width. Polar filaments exhibiting two to three longitudinal coils. The sporoplasm having many secondary cells. Spores developing in number of 8 inside pansporocysts. Type host: Tubificoides pseudogaster (Dahl, 1960) Type locality: Aveiro Estuary, Portugal Site of infection: Coelomic cavity Prevalence of infection: 0.9% (6 out of 651) GenBank accession no.: KU569318 Remarks: The sequences in GenBank most similar to this type were Triactinomyxon sp. SH-2006 (DQ473515), with 96% of similarity, Endocapsa sp. SH- 2006 (DQ473516), with 94% of similarity, and M. exiguus (AY129317) and M. muelleri (AY129314), with 93% of similarity.

Sphaeractinomyxon type 10 (new type) Figs. 9.1q, r and Table 9.2

Description: Mature spores spherical in apical view and spherical to slightly ellipsoidal in lateral view. Spores (n=47) 22.3±1.1 (19.3–24.9) µm in length, 24.0±1.7 (19.6–27.4) µm in width, and 24.3±1.6 (21.2–27.5) µm in diameter; three pyriform polar capsules 5.2±0.3 (4.5–6.2) µm in length and 4.0±0.2 (3.4–4.5) µm in width. Polar filaments exhibiting three longitudinal coils. Sporoplasm having many secondary cells. Spores developing in number of 8 inside pansporocysts. Type host: Tubificoides pseudogaster (Dahl, 1960) Type locality: Aveiro Estuary, Portugal Site of infection: Coelomic cavity Prevalence of infection: 0.8% (5 out of 651) FCUP 149 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

GenBank accession no.: KU569319 Remarks: The sequences in GenBank most similar to this type were Triactinomyxon sp. SH-2006 (DQ473515), with 93% of similarity, and Endocapsa sp. SH-2006 (DQ473516), M. exiguus (AY129317), and M. muelleri (AY129314), with 92% of similarity.

Table 9.2 – Spore morphology of sphaeractinomyxon types from Limnodriloides agnes (types 1–7) and Tubificoides pseudogaster (types 8–10).

Spore type Spore length Spore width Spore diameter Polar capsule Polar capsule length width

1 17.2±1.5 18.8±1.3 18.8±0.9 4.7±0.4 3.4±0.3 (15.2–20.5) (16.7–21.7) (16.9–21.0) (3.8–5.8) (2.7–3.8)

2 23.3±0.8 28.4±1.0 28.6±1.2 5.9±0.2 4.1±0.2 (22.1–24.8) (26.2–29.8) (26.6–31.1) (5.7–6.0) (4.0–4.3)

3 30.5±1.8 33.6±2.2 33.9±0.8 7.5±0.7 6.2±0.4 (27.9–33.4) (28.4–38.0) (32.5–35.3) (6.5–8.3) (5.8–7.0)

4 33.2±2.8 36.5±2.3 37.9±2.2 7.8±0.4 6.6±0.4 (28.1–36.7) (32.4–42.6) (34.1–40.3) (7.0–8.7) (5.8–7.1)

5 42.5±2.1 54.8±2.7 56.0±2.4 9.1±0.3 7.6±0.3 (38.8–45.6) (50.0–59.5) (52.9–58.8) (8.8–9.6) (7.0–7.9)

6 51.5±5.2 64.5±3.3 62.1±2.8 10.2±0.4 8.9±0.8 (46.0–56.9) (61.6–69.2) (56.7–68.4) (9.9–10.9) (7.5–9.7)

7 60.9±5.6 75.6±6.2 80.3±5.1 10.6±0.9 9.7±0.4 (55.0–68.7) (64.7–85.8) (71.8–91.4) (9.4–11.9) (9.2–10.1)

8 18.1±0.9 18.6±0.8 18.1±1.0 4.7±0.2 3.3±0.2 (17.0–21.3) (17.7–21.1) (17.1–20.0) (4.3–5.2) (3.0–3.5)

9 20.9±1.0 22.0±1.5 22.6±1.2 5.6±0.4 4.3±0.3 (19.0–23.4) (20.4–26.9) (20.7–26.5) (4.8–6.0) (3.9–4.8)

10 22.3±1.1 24.0±1.7 24.3±1.6 5.2±0.3 4.0±0.2 (19.3–24.9) (19.6–27.4) (21.2–27.5) (4.5–6.2) (3.4–4.5)

The spores and corresponding developmental stages were always found in the coelomic cavity, for all described actinospore types (Fig. 9.1a); in some cases, they were also found inside reproductive structure cavities (Fig. 9.1k). Spore development was asynchronous, as all different developmental stages were observed simultaneously in a same individual. Nevertheless, spore development inside each individual pansporocyst was usually synchronous, even though asynchronous development was also observed in some rare cases. FCUP 150 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

After observing the developmental stages of the several new sphaeractinomyxon types, it is possible to infer the successive steps in spore development (Fig. 9.2). The first identifiable stage corresponded to binucleated cells (Fig. 9.2a). The next stage is a set of four cells (Figs. 9.2b, c), which then create the initial pansporocyst with two somatic cells forming the pansporocyst walls and another two generative cells inside the pansporocyst (Fig. 9.2d). The two internal cells start to divide in three (Fig. 9.2e) and four internal cells (Fig. 9.2f). Two such internal cells then divide further two times, resulting in stages with six (Fig. 9.2g) and then 10 smaller cells (Fig. 9.2h), while the other two larger cells only then start to divide (Fig. 9.2i). In the end of the gametogamy, a pansporocyst with 16 morphologically indistinct cells is found (Fig. 9.2j). The gametogamy ends with the formation of zygotes (Fig. 9.2k). The gametogamy stage had similar developmental stages in all different types of described actinospore; however, the sporogony stage was found to differ to a little extent among the different types, according to the final number of spores formed in each pansporocyst. In the beginning of the sporogony, for the actinospore types with the development of eight spores, pansporocysts with a set of eight cells centrally located and eight sporoplasmic cells leaning against the pansporocyst wall were found (Fig. 9.2l). The centrally located cells divide, forming eight involucres formed by three valvogenic cells surrounding three capsulogenic cells (Fig. 9.2o). As the spores develop, the sporoplasmic cells migrate inside the spore involucre (Fig. 9.2p). When immature (Figs. 9.2q, r), the spores look larger compared to mature spores, and their appearance can give the false idea of spores with expansions (Fig. 9.2r). For the actinospore types with the development of two or four spores, pansporocysts with two (Fig. 9.2m) or four (Fig. 9.2n) involucres and the same amount of sporoplasmic cells leaning against the pansporocyst wall were found. In these stages, some elongated cells are also observed, whose function or purpose can only be speculated, and which seem to correspond to vegetative cells that do not develop to form spores. In the end of the sporogony, it is possible to observe pansporocysts with different numbers of mature spores, according to the different actinospore types (Figs. 9.2s, t, u). In pansporocysts with less than eight mature spores, it was possible to observe some small vegetative cells, as the example in Fig. 9.2t, where four vegetative cells are easily seen between the four mature spores. These vegetative cells were also observed in mature pansporocysts of the sphaeractinomyxon type 1 in oligochaetes heavily infected. This type of actinospore usually develops eight spores per pansporocyst, but several pansporocysts had a smaller number of spores and the missing spores were compensated by the presence of these vegetative cells.

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The mature spores were never seen exiting the worm body, not by the intestine either by the gonophores or the nephridiopores, even in heavily infected worms. In some cases, when the worm host body was excessively pressured by a cover slip, the spores free in the coelom could break through the intestine epithelium and invade its lumen, but this was not a common occurrence.

Table 9.3 – Pairwise genetic differences between the 18S rDNA sequences of the 10 new sphaeractinomyxon types.

Type 1 Type 2 Type 3 Type 4 Type 5 Type 6 Type 7 Type 8 Type 9 Type 10 Type 1 (KU569310) Type 2 0.143 (KU569311) Type 3 0.112 0.130 (KU569312) Type 4 0.109 0.125 0.057 (KU569313) Type 5 0.112 0.138 0.098 0.098 (KU569314) Type 6 0.109 0.131 0.099 0.100 0.080 (KU569315) Type 7 0.109 0.131 0.072 0.052 0.099 0.110 (KU569316) Type 8 0.076 0.131 0.107 0.104 0.115 0.109 0.114 (KU569317) Type 9 0.079 0.134 0.106 0.102 0.110 0.109 0.112 0.057 (KU569318) Type 10 0.082 0.133 0.113 0.104 0.116 0.120 0.118 0.058 0.061 (KU569319)

Genetic distances between the 10 sphaeractinomyxon new types were calculated and are presented in Table 9.3. The lowest genetic differences found were between sphaeractinomyxon type 4 and type 7 (5.2%), while the highest respected sphaeractinomyxon type 1 and type 2 (14.3%). A BLAST search for the 10 sphaeractinomyxon new types found no match in the GenBank. The most similar species were Myxobolus species (89–94%), especially M. exiguus (AY129317), M. muelleri (AY129314), Myxobolus sp. (KC733438), M. ichkeulensis (AF378337), Myxobolus spinacurvatura Maeno, Sorimachi, Ogawa and Egusa, 1990 (AF378341), M. episquamalis (JF810537), and M. bizerti (AY129318). They also had similarities with some actinospore types in GenBank (86–96%): S. ersei (AF306790), Triactinomyxon sp. SH-2006 (DQ473515), E. rosulata (AF306791), Endocapsa sp. SH-2006 (DQ473516), Te. discoidea (AF306793) and Raabeia TGR-2014 (KF263539).

FCUP 152 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Fig. 9.2 –Developmental stages of the sphaeractinomyxons in the coelomic cavity of Limnodriloides agnes and Tubificoides pseudogaster. a) A binucleated cell. b, c) Binucleated cells dividing into two and four cells. d) Initial pansporocyst with two enveloping cells and two internal cells. e, f) Pansporocysts with three and four internal cells. g, h) Pansporocysts in which two internal cells divided successively two times originating a final set of eight smaller cells while two other larger cells rest undivided. i) Pansporocysts in which the lager undivided cells from the anterior stages started their division. j) Final stage of gametogamy with a pansporocysts with 16 inner cells. k) Pansporocyst in the beginning of the sporogony showing eight zygotes. l) Pansporocyst with eight set of cells in a more central area and sporoplasmic cells (double asterisks) leaning against the pansporocyst wall. m) Pansporocyst with a more advanced stage of sporogony that will produce two final spores. Two sporoplasmic cells (double asterisks) against the pansporocyst wall, two involucres formed by three valvogenic cells surrounding three inner capsulogenic cells FCUP 153 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

(asterisks) and some elongated cells with unknown function are visible. n) Pansporocyst in the same sporogony stage as the one in the previous figure but in a type of sphaeractinmyxon that will produce four final spores. o) Pansporocysts in a slightly more advanced sporogony stage than the previous two figures, but producing eight final spores. The capsulogenic cells are already forming the polar capsules. p) Pansporocyst in a more advanced sporogony stage were the majority of the spores have already the sporoplasmic cells inside the spore involucre, but two sporoplasmic cells (double asterisks) are still outside their spore involucre (asterisks). q) Pansporocyst with eight immature spores. r) Some immature spores outside a busted pansporocyct accompanied by a solitaire mature spore on the right side. s-u) Pansporocysts with mature spores from three different types of sphaeractinomyxon. One type developing two spores (s), another type developing four spores (t) and, finally a type developing eight spores (u). Note the presence of four vegetative cells (asterisks) around the four mature spores in (t). Scale bars=20 µm.

9.5. Discussion

Oligochaete worms in Aveiro Estuary were found infected with 10 new types of sphaeractinomyxon actinospore (species), duplicating the number of the strictly described sphaeractinomyxons (Marques, 1984; Hallett et al., 2001). The new types of sphaeractinomyxon can be distinguished in morphological terms by the size of the spores and polar capsules, within each oligochaete species host. One of the main conclusions that can be drawn from the results of this study is the strict specificity of the different sphaeractinomyxon types to the oligochaete host species. Each specific sphaeractinomyxon type was never found in both species of oligochaetes L. agnes and Tu. pseudogaster. This great specificity makes the morphological comparison between sphaeractinomyxons infecting different host species useless. From the 16 sphaeractinomyxon types (10 sphaeractinomyxon, 2 tetraspora and 4 endocapsa) described in the literature, none was reported to infect L. agnes or Tu. pseudogaster. Another conclusion is the need to use molecular biology in future descriptions of these types of spores from different species of oligochaete hosts, since spore measurements can overlap in many different sphaeractinomyxon types. Therefore, in ecological studies, besides spore's measures, the host oligochaete species is also an important character to identify the sphaeractinomyxon type without the use of molecular biology, and eventually locality can also play an important role in species separation. The developmental stages observed in this study follow the same path described for the sphaeractinomyxon types, since the first development description made by Caullery and Mesnil (1905). The major differences between these actinospores and the others types is the fact that sphaeractinomyxons can develop a different number of spores (from one to eight) inside pansporocysts, while all other described actinospore types have always eight spores. FCUP 154 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Main differentiating characteristics between the different sphaeractinomyxon types, besides the number of spores developing inside the pansporocysts, are the size and form of the spores. The form and number of the developing spores seem to be related with the size of the spores. The spore form changes from round in apical and lateral view, in the smaller sphaeractinomyxon types, to more angular in apical view and ellipsoidal in lateral view, in the larger sphaeractinomyxon types. The spore shape change is not only a species-specific character but also an environmental influenced character. For instance, spores observed under the pressure of a cover slip tend to become more angular as the pressure increases; accordingly, researchers should have some caution when observing these types of spores in a microscope slide. Spore size is also related with the number of spores developing inside the pansporocyst. The smaller sphaeractinomyxon types develop in number of 8 inside pansporocysts, while the median size sphaeractinomyxon types develop in number of 2 to 4 spores inside pansporocysts, and the larger sphaeractinomyxon types develop in number of 1 to 2 inside pansporocysts. This character seems to be not only typical for each species but also an environmentally influenced character. For instance, in heavily infected worms, the number of mature spores inside the pansporocysts was smaller than expected; in these cases, the missing spores were 'replaced' by small vegetative cells, suggesting that the lack of space or excessive pressure exerted in the worm coelomic cavity had some kind of inhibiting effect during the spore's development. None of the infected oligochaetes was found to release sphaeractinomyxons from the coelomic cavity. There is some literature reporting the presence of this type of spores in the intestinal lumen of their hosts (Hallett et al., 1998, 1999, 2001), but, in this study, the spores where only observed in the lumen of the intestine when excessive pressure was applied to the coverslip over the oligochaete under microscopy observation. This suggests that the presence of spores inside the intestine lumen is likely accidental, and not a normal exiting route. Overall prevalence of infection of all new types of sphaeractinomyxon, for the two host species, was 19% and could reach a maximum of 26% in spring, with a minimum in winter. Parasite infection was found throughout the year, but the spring and summer months seem to be more appropriate for the dissemination of these types of actinospores since, in autumn, there is a great number of worms with only developmental stages and immature spores. Nevertheless, individually, the prevalence of infection was low, which is consistent with the prevalence values reported in the literature (Yokoyama et al., 2012). FCUP 155 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes

Molecular biology could demonstrate a close proximity of the 10 new types of sphaeratinomyxon to Myxobolus species. The same is true for the other sphaeractinomyxon, tetraspora, and endocapsa types in GenBank. The number of spores developing inside pansporocysts was used as argument for the erection of tetraspora as a new actinospore collective group (Hallett and Lester, 1999); nevertheless, these authors considered that, in morphological terms, these spores represented typical sphaeractinomyxons, only developing in a set of four spores. In face of the results obtained in this study, no reason is found to justify the existence or creation of new collective groups for sphaeractinomyxons based in this character. Endocapsa collective group, erected by Hallett et al. (1999), is another group of actinospores similar to the sphaeractinomyxons and, in the same way, these authors state that, by default, the endocapsa can be considered as sphaeractinomyxons. The major differences described are the 'submerged' polar capsules and the presence of irregular 'processes in the form of swellings'. In the work of Hallett et al. (1999), several forms of different swellings are drawn, and an important detail is described by these authors: the swellings do not change when in contact with water, contrary to what happens to the other actinospore types with typical processes. Besides the later record, there is another more recent record for the endocapsa type in Lake Assad (Euphrates River), Syria (Székely et al., 2007), which is the only freshwater endocapsa isolated until now. Unfortunately, however, spore illustrations in that work do not help to support the endocapsa actinospores, as depicted spores have the aspect of being degraded. We cannot dispute this collective group without observing the described spores, but, in the course of this study, it was possible to observe more than 100 infected oligochaetes and thousands of mature spores ranging from 15 to 91 µm in size, and their developmental stages, and had never found spores with swellings or processes. However, many immature spores, very similar to the description made for the endocapsa actinospores, were observed with submerged polar capsules and a kind of swellings but, side by side, we also observed mature spores with its typical sphaeractinomyxon form within the same host (for instance, see Fig. 9.2r). Another important detail was the modifications suffered by spores, especially when they started to dehydrate, in which the sporoplasm concentrates more in the center of the spores, and the corners of the more angular spores start to appear as swellings. In conclusion, the endocapsa collective group needs a validation by new records that can indisputably confirm them as a legitimated collective group. On the other hand, it is mandatory to considerer if the number of spores developing inside pansporocysts is reason enough to divide the sphaeractinomyxon actinospores in new FCUP 156 Chapter 9. New types of sphaeractinomyxon actinospores from marine tubificid oligochaetes and different collective groups (for instance, tetraspora for four spores, dispora for two spores, unispora for one spore, etc.). In our opinion, that there is no need for such separation.

9.6. Acknowledgments

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 “PEst-C/MAR/LA0015/2013” the project DIRDAMyx, reference FCOMP-01- 0124-FEDER-020726/FCT-PTDC/MAR/116838/2010, and the Ph.D. fellowship grant SFRH/BD/82237/2011 attributed to L. Rangel and the Ph.D. fellowship grant SFRH/BD/92661/2013 attributed to S. Rocha through the programme POPH/FSE QREN, and the fellowship grants attributed to F. Cavaleiro and R. Castro through the project DIRDAMyx, reference FCOMP-01-0124-FEDER-020726/FCT- PTDC/MAR/116838/2010, and the Hungarian OTKA K 100132 grant. We thank for the identification of the oligochaetes for Dr. Rüdiger Schmelz. This work complies with the current laws of the country where it was performed.

FCUP 157 Chapter 10. Concluding Remarks

Chapter 10

Concluding Remarks

FCUP 158 Chapter 10. Concluding Remarks

FCUP 159 Chapter 10. Concluding Remarks

10.1. Final notes

Major aims proposed for this thesis were successfully accomplished and has made important contributions to the knowledge of the marine myxozoan parasites. Some final notes are worth to discuss.

- The myxozoan parasites survey conducted in the fish farm in Algarve revealed two new Ortholinea species, O. auratae, in gilthead seabream (Chapter 4), and O. labracis, in European seabass (Chapter 6), both infecting the urinary bladder of their hosts. These are the first references of Ortholinea species for Portugal, and represent a new geographical record for the genus. Ortholinea species are typical coelozoic parasites of the excretory system of fishes (Lom and Dyková, 2006). There are only two reports on the effects of these parasites on the fish host. Ortholinea polymorpha (Davis, 1917), infecting the urinary bladder of Opsanus tau (Linnaeus, 1766), causes cellular lesions but does not show external signs of the disease (Davis, 1917). Ortholinea australis Lom, Rohde and Dyková, 1992, which exceptionally infects the gallbladder and biliary ducts of Australian sparids fishes, can cause, in massive infections, hepatic disorder or stagnation of the bile outflow (Lom et al., 1992). For the two Ortholinea species found in this work, no external signs of disease were visible (Chapters 4 and 6). Nevertheless, myxosporean infections are known to facilitate secondary infections with health losses and growth delays of fish (Fioravanti et al., 2004). This is an important contribution because it shows how favorable the fish farm environment is for the spread of diseases, and to know all possible risks related to the fish farming is an important management tool.

- One new type of echinactinomyxon actinospore was found in the intestinal epithelium of a marine capitelid polychaete, H. filiformis, collected from the Alvor Estuary, downstream to the fish farm (Chapter 8). This is an interesting finding because, until now, echinactinomyxons were always found in the intestinal epithelium of freshwater oligochaetes (Marques, 1984; Özer et al., 2002; Marcucci et al., 2009; Marton and Eszterbauer, 2011), and one type was associated to the life cycle of the freshwater parasite Myxobolus pavlovskii (Akhmerov, 1954) (Marton and Eszterbauer, 2011). The finding of this type of echinactinomyxon in a polychaete host is not only interesting for itself, but also because the SSU rRNA gene sequence phylogenetically places this FCUP 160 Chapter 10. Concluding Remarks actinospore among Sphaeromyxa species. Sphaeromyxa constitutes an exception to the marine/freshwater myxosporean lineage (Fiala, 2006). These parasites cluster within the freshwater gallbladder infecting clade (Fiala, 2006; Bartošová et al., 2009). This new type of echinactinomyxon breaks the notion that the life cycle of freshwater lineage myxosporeans uses oligochaetes and the marine lineage myxosporeans use polychaetes as invertebrate hosts (Fiala et al., 2015a).

- Actinosporeans survey in annelids in the Aveiro Estuary (Chapter 9), disclosed 10 new types of sphaeractinomyxon in two oligochaete hosts (L. agnes and T. pseudogaster), which doubled the number of sphaeractinomyxons previously described (Marques, 1984; Hallett et al., 2001). Morphological and developmental studies of these actinospores helped to clarify the characters that define this type of actinospores (Caullery and Mesnil, 1904, 1905; Lom and Dyková, 2006). Sphaeractinomyxons types, exhibiting some variant characters, were attributed to two new actinospore types: tetraspora, for spores developing in number of four inside pansporocysts (Hallett and Lester, 1999); and endocapsa, for spores with irregular valvogenic cells swellings and 'submerged' polar capsules (Hallett et al., 1999). Nevertheless, the present work shows that the number of spores developing inside pansporocysts range from one to eight spores, and that this character is not only species specific, but also it may be environmentally influenced. For these reasons, tetraspora actinospore types should be considered as typical sphaeractinomyxon actinospores (Chapter 9). Concerning the endocapsa collective group, during the present work, mature sphaeractinomyxons that could match the endocapsa description were never found, but, on the other hand, immature spores, in their final maturation stages could indeed resemble very closely, the description of endocapsa spores. This raised several doubts on the validity of this actinospore collective group, which can only be clarified with new records (Chapter 9).

- One general conclusion that can be drawn from these results is the fact that myxosporeans apparently have no preference between oligochaetes or polychaetes, independently of the environment where they live. This work found both groups of marine annelids to be infected and to participate, as hosts, in the life cycle of myxosporeans (Chapter 5, 6, 7, 8 and 9). It would not be surprising if the annelids were the preferred hosts for myxosporeans. Sipunculids, found to be infected with actinosporean stages (Ikeda, 1912), are now phylogenetically placed within the Annelida (Struck et al., 2007; Weigert et al., 2014). This means that the actinospore phase of myxosporeans is now known to use exclusively annelids as invertebrate hosts. FCUP 161 Chapter 10. Concluding Remarks

- Another important contribution was the elucidation of three new life cycles of myxosporeans from the marine environment (Chapter 5, 6 and 7). By using molecular tools, it was possible to match the 18S ribosomal RNA sequences of O. auratae (Chapter 5), O. labracis (Chapter 6) and S. dicentrarchi (Chapter 7) to two triactinomyxons (in oligochaetes) and one tetractinomyxon (in polychaetes), respectively. Ortholinea spp. life cycles also represent a new record, because these are the first life cycles involving marine oligochaetes, and also the first described life cycle for this myxosporean genus. Triactinomyxons were already described in the marine environment, in the intestinal epithelium of Australian Duridrilus sp., Limnodriloides sp. and unidentified tubificids (Roubal et al., 1997; Hallett et al., 2001), but they were never associated to a fish myxosporean. On the other hand, tetractinomyxon were always found in polychaetes, and are the actinosporean stage for myxosporean species from diverse genera like Ellipsomyxa (Køie et al., 2004; Rangel et al., 2009), Gadimyxa (Køie et al., 2007), Ceratomyxa (Køie et al., 2008), Sigmomyxa (Karlsbakk and Køie, 2012), Parvicapsula (Bartholomew et al., 2006; Køie et al., 2013), and Ceratonova (Bartholomew et al., 1997).

- Studies on myxosporean life cycles, using experimental infections, are not only laborious and time consuming (Székely et al., 2014), but also prone to mismatches or erroneous interpretation (Holzer et al., 2004; Marton and Eszterbauer, 2011). Using molecular tools, specifically the SSU rRNA gene, is a very convenient and practical method to prove or not a match between a myxospore from a vertebrate host and an actinospore from an invertebrate host, like it was demonstrated in the present work (Chapters 5, 6 and 7). The molecular tools also proved their usefulness in the identification of the invertebrate hosts, if not to the species level, at least to the genus level. Molecular tools have also some problems like the sequencing of the wrong parasites co-infecting the same host, or the failure in amplifying the SSU rRNA of the parasite of interest. Nevertheless, these problems seem easier to overcome with the rapid evolving of the genetic technology. However, experimental infections can be used to complement the life cycles study, especially in what concerns the processes and mechanisms of infection in fish and annelids.

- This work helped to clarify some ultrastructural aspects of the developmental stages and mature actinospores. The ultrastructural characterization was very detailed in the study of the tetractinomyxons of E. mugilis (Chapter 3), and it was complemented with more details from the triactinomyxons of O. auratae (Chapter 5) and O. labracis FCUP 162 Chapter 10. Concluding Remarks

(Chapter 6). Ultrastructural details included (Chapter 3) the demonstration of the existence of tetranucleated cells prior to the formation of the pansporocysts. Another finding was the existence of dense bodies and phagosomes in both internal cells and enveloping cells that will form the initial pansporocyst, contrary to what was described for other types (Lom and Dyková, 1992a, 1997; El-Matbouli and Hoffmann, 1998). Pseudopodia-like projections of the sporoplasm cells seem to be a common feature in actinospores (Lom and Dyková, 1997; Hallet et al., 1998; Álvarez-Pellitero et al., 2002) but, in this work, it was possible to show, for the first time, the existence of cell junctions between the sporoplasm cell through the pseudopodia-like projections and the valvogenic cells (Chapter 3). Ultrastrutural studies also helped to clarify the number of polar filament coils within E. mugilis polar capsules, from the five or six coils described (Rangel et al., 2009) to seven or eight coils in the ultrathin sections (Chapter 3).

- One important observation made in this work related to the central dogma of myxozoan cells. The origins of the secondary cell were explained by a process of endogenous budding (Lom and Dyková, 1992b). However, in recent studies involving the malacosporean T. bryosalmonae, the formation of the secondary cells was explained as an engulfment process (or internalization) of another cell, and after that, the internalized cell would multiply by mitoses (Morris and Adams, 2007; Morris, 2010). Ultrathin sections of young sporoblasts of E. mugilis exhibited a set of cells in which one central cell was enveloping (or engulfing) another cell and, in the end of the process, one cell had a secondary cell within its cytoplasm (Chapter 3). This result confirms, for the myxosporeans, the same phenomena reported by Morris and Adams (2007) for the malacosporeans. This gives some support to Morris (2010, 2012) model for the origins of the secondary cells in the sporoplasm, but do not corroborate the existence of the valvocapsulogenic cells, described for the freshwater myxosporeans.

- One aim of this work was the description of the myxosporean species found in the kidneys and urinary bladder of European seabass from Aveiro Estuary and identified as a Myxobilatus species. For that aim, a synopsis of all described Myxobilatus species was prepared (Chapter 1). Thirty-nine nominal species of Myxobilatus were included. Unfortunately, all European seabass surveyed in the Aveiro Estuary and the Algarve fish farm were not infected by this parasite, leaving this task for future research.

FCUP 163 Chapter 10. Concluding Remarks

10.2. Future Research

The successful of this work incentives the establishment of further aims to complement and improve the findings of the present study.

- Finding new myxosporean species infecting European seabass and the gilthead seabream, two fish species exhaustively studied in the Mediterranean Sea showed the need to replicate the myxozoan survey in different localities and geographic regions, with special emphases with the North Atlantic coasts.

- Myxosporean parasites of different species infecting so many organs in one single fish host must have a detrimental effect in the growth and health of the fishes, so detailed histopathologic studies are needed to evaluate the level of damage caused not only by each individual species, but also the combined effect of several parasite species.

- It is indisputable the importance of annelids in the life cycle of myxosporean parasites, therefore the search for the actinosporean stages of the myxosporeans parasites infecting European seabass, gilthead seabream and many other fish species, for which the life cycle is still unknown, must continue. Only the full knowledge of the life cycle of a parasite will allow the development and implement of methodologies that enables the infection cycle break and try to maximize the fish farms productivity.

- For the European seabass there is a need to describe the reported Myxobilatus species in the kidneys and urinary bladder found in fishes collected from Aveiro Estuary in 1998, by Santos.

- The experimental infection of oligochaetes by the new Myxozoa species now recorded will allow us to definitively confirm the life cycles inferred in the present work, and probably to better understand the environmental conditions of the annelid infection.

FCUP 164 Chapter 10. Concluding Remarks

FCUP 165 References

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