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

IN ST IT U T O D E C IÊ N C IA S B IO M É D IC A S A B E L S A L A Z A R

Sónia

Rocha

.

Biodiversity

and

phylogeny

of

myxosporean

parasites

(Cnidaria,

Myxozoa)

infecting

fish

and

annelids

in

P

ortuguese

estuaries

Biodiversity

and

phylogeny

of

myxosporean

parasites

(Cnidaria,

Myxozoa)

infecting

fish

and

annelids

in

P

ortuguese

estuaries

Sónia

Rocha

Biodiversity and phylogeny of myxosporean

parasites (Cnidaria, Myxozoa) infecting fish

and annelids in Portuguese estuaries

Sónia Rocha

D

D

.ICB

AS

201

9

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

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

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

Orientador – Doutora Graça Maria Figueiredo Casal Categoria – Professora Auxiliar

Afiliação – Instituto Universitário de Ciências da Saúde - CESPU

Coorientador – Doutor Carlos José Correia de Azevedo Categoria – Professor Catedrático Jubilado

Afiliação – Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto

Coorientador – Doutor Pedro Nuno Simões Rodrigues Categoria – Professor Associado com Agregação

Afiliação – Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto

Esta tese é dedicada ao meu querido pai José Rocha, ao meu avô António Oliveira e à minha sogra Angelina Oliveira.

É na memória do vosso amor e da vossa força que encontro a inspiração para viver plenamente a minha vida.

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Agradecimentos

Entre doutorandas, por vezes ouvi dizer que elaborar uma tese de doutoramento é mais difícil do que ter um filho. Tendo a experiência de ambos posso afirmar que, de igual forma, é necessária a colaboração de uma autêntica “aldeia” para que o esforço seja bem-sucedido e os objetivos propostos alcançados. Como tal, não posso deixar de agradecer a todos que, de forma direta ou indireta, me apoiaram e motivaram nesta jornada.

À minha orientadora, Professora Doutora Graça Casal, pela orientação e confiança que deposita no meu trabalho, e pela liberdade que me concedeu na tomada de decisões, permitindo-me evoluir como profissional.

Ao meu co-orientador, Professor Catedrático Jubilado Carlos Azevedo, por me contagiar com o seu gosto e entusiasmo inigualável pela parasitologia. Obrigada pela disponibilidade e confiança com que me recebeu. Obrigada por todo o apoio, conselhos sábios e carinho com que me brindou ao longo dos últimos quase 10 anos.

Ao meu co-orientador, Professor Doutor Pedro Rodrigues, por ter aceite co-orientar o meu projeto de doutoramento. Obrigada pelas correções e sugestões que contribuíram para a elaboração desta tese.

Ao Professor Doutor Alexandre Lobo da Cunha e Professor Doutor Eduardo Rocha, que não sendo meus orientadores, me orientaram e apoiaram sempre que possível. Muito obrigada pela amabilidade com que me receberam, deixando que eu fizesse do Departamento de Microscopia uma segunda casa.

Ao Professor Doutor Carlos Antunes e restantes membros do Aquamuseu do Rio Minho, pelo esforço incansável na coleta de peixe e oligoquetas do Rio Minho, sem o qual não teria sido possível realizar a maior parte do trabalho integrante desta tese.

À Professora Doutora Maria João Santos, pela sua simpatia e boa-vontade em auxiliar-me sempre que necessário, auxiliar-mesmo em questões de última hora.

Ao meu colega, Doutor Luís Filipe Rangel, pelo auxílio prestado e pelas longas conversas telefónicas que, em várias ocasiões, me ajudaram a decidir aspetos cruciais da minha tese.

À Ângela, por todas as horas que dispensou comigo em viagens, amostragens, processamentos, e tantas mais tarefas que nem consigo enumerar. Digo, e não tenho dúvidas, que sem ti jamais teria conseguido terminar esta tese. Por isso, parabéns a nós duas!

À Elsa, por ser uma segunda mãe para mim, sempre disponível a ajudar-me e, mais importante, a dar-me conselhos que considero valiosos e que tenho a certeza me serão úteis por toda a vida. Obrigada pela preciosa ajuda nos processamentos e nos cortes infindáveis, os quais tantas vezes foram frustrados, mas que sempre me ajudou a percepcionar com positivismo.

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À Célia, por todo o apoio que me dá nas mais diversas “frentes” da minha vida, por seres uma excelente profissional e colega de trabalho, e acima de tudo por seres uma amiga inestimável. Obrigada por me ouvires e por estares sempre presente quando preciso.

À Fernanda, minha companheira de viagens, por ser essa luz brilhante cheia de vida que me animou em tantas ocasiões, dando-me força para continuar, mesmo quando a “coisa estava preta”.

À Cláudia, pelas sessões de fotos das tainhas e por todos os momentos de descontração em que nos rimos de tudo e de nada.

À Rute, pela pessoa generosa que é, tendo-se sempre prontificado a auxiliar no que fosse necessário, mesmo não tendo tempo para executar as suas próprias tarefas, e mesmo não tendo ido ainda de lua-de-mel.

À Ana Paula, pelas horas passadas à “cata da minhoca”, sempre com uma boa-disposição capaz de transformar horas verdadeiramente fastidiosas em momentos de diversão.

À Paula, por funcionar como um EndNote orgânico e pela sua infindável boa vontade em ajudar, mesmo nas tarefas mais ingratas.

À Susana, Ana Maria, Raquel, Tânia, Maria João, David e Tito, por todos os momentos em que, de uma forma ou de outra, me ajudaram e permitiram que desabafasse com eles as minhas frustrações, tendo recebido em resposta um positivismo revigorante.

A toda a minha família e amigos, pelo apoio incondicional que me dão em todos os aspetos da minha vida, e por aturarem todos os meus problemas, alguns mais objetivos, outros simplesmente existenciais.

À minha mãe, por ser uma força da vida e me ensinar que o caminho se faz sempre para a frente. Os meus sucessos são também o produto daquilo em que me moldaste. Obrigada pelas incontáveis vezes que tiveste que tomar conta do teu amado netinho para que eu pudesse ficar a trabalhar até mais tarde, ou pudesse estar “agarrada” horas a fio a um computador.

Ao Miguel, por ser o meu maior pilar em tudo. Obrigada pelas revisões, pelos esquemas, pelas medidas, pelas planches, pelas formatações, pelas horas, pela vida que dedicas a mim e à nossa pequena família. Sem ti eu não teria terminado esta tese, sem ti eu não reconheceria a minha vida, sem ti eu já não seria eu.

E ao meu Afonso José, que não me ajudou em nada na tese, só dificultou, mas a quem eu amo mais do que tudo na minha vida. És a minha luz e razão de fazer mais e ir mais além.

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Financial support

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

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Publications

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

Rocha, S., Casal, G., Rangel, L., Castro, R., Severino, R., Azevedo, C. and Santos, M.J.

(2015). Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae). Parasitology International 64, 305

‒

Rocha, S., Rangel, L.F., Castro, R., Severino, R., Azevedo, C., Santos, M.J. and Casal, G.

(2016). Ultrastructure and phylogeny of Ceratomyxa diplodae (Myxosporea: Ceratomyxidae), from gall bladder of European seabass Dicentrarchus labrax. Diseases of Aquatic Organisms 121, 117

‒

Rocha, S., Rangel, L.F., Castro, R., Severino, R., Azevedo, C., Santos, M.J. and Casal, G.

(2019). The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis. Journal of

Invertebrate Pathology 160,33

‒

Rocha, S., Azevedo, C., Oliveira, E., Alves, Â., Antunes, C., Rodrigues, P. and Casal, G.

(2019). Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular redescription of the cryptic species

Myxobolus exiguus. Parasitology 146, 479

‒

Rocha, S., Alves, Â., Fernandes, P., Antunes, C., Azevedo, C. and Casal, G. (2019).

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

Aquatic Organisms (In Press)

Rocha, S., Alves, Â., Antunes, C., Azevedo, C. and Casal, G. (2019). Molecular data infers

the involvement of a marine aurantiactinomyxon in the life cycle of the myxosporean parasite

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Rocha, S., Alves, Â., Antunes, C., Fernandes, P., Azevedo, C. and Casal, G. (2019).

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

Parasitologica (In Press)

Rocha, S., Casal, G., Alves, Â., Antunes, C., Rodrigues, P. and Azevedo, C. (2019). Myxozoan

(Cnidaria, Myxozoa) biodiversity in mullets (Teleostei, Mugilidae) unravels hyperdiversification of Myxobolus (Cnidaria, Myxosporea). Parasitology Research (In Press)

Rocha, S., Azevedo, C., Alves, Â., Antunes, C. and Casal, G. (2019). Morphological and

molecular characterization of myxobolids (Cnidaria, Myxozoa) infecting cypriniforms (Actinopterygii, Teleostei) endemic to the Iberian Peninsula. Parasite (Under review)

Rocha, S., Rangel, L.F., Casal, G., Azevedo, C., Rodrigues, P. and Santos, M.J. (2019).

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

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Abstract

Myxozoans are a highly diversified group of cnidarian endoparasites with a complex life cycle that alternates between two stages: a myxosporean stage that develops in a vertebrate intermediate host, mainly fish; and an actinosporean stage that develops in an invertebrate definitive host, namely an oligochaete or polychaete. For a long time, interest in myxozoans has been mostly associated with their recognized economic importance, given that some species are known to cause emerging diseases in wild and reared populations, with negative impact in fisheries and aquaculture industries. More recently, the unraveling of their extraordinary evolutionary history boosted interest in the group, given that their interactions probably represent important drivers of the evolution of parasitism in early diverging metazoans. Moreover, the increasing number of publications describing new species suggests that myxozoan biodiversity is greatly underestimated and that these parasites might be major ecological players in aquatic ecosystems.

Despite fishing and aquaculture constituting major economic activities in Portugal, a review of the available literature showed that few studies have targeted the myxozoan community inhabiting the extensive aquatic resources of this country. The present thesis aimed to tackle this issue by pursuing myxozoan surveys in three Portuguese estuaries, namely those of the Rivers Minho, Douro and Alvor, from which little or no information was previously available. Fishes and annelids were sampled and screened for the detection of myxozoan development in tissues and internal cavities. The results revealed a previously unknown rich diversity of this cnidarian group, with a total of 21 myxosporean species (17 new) and 15 actinosporean stages (13 new) being reported from fishes and oligochaete hosts, respectively. With the exception of Ceratomyxa auratae from the gall bladder of gilthead seabream Sparus

aurata Linnaeus, 1758 in the Alvor estuary, all other new species records refer to members of

the family Myxobolidae found infecting fish in the Minho estuary. Myxobolus arcasii n. sp., M.

duriensis n. sp. and Thelohanellus paludicus n. sp. are described from three distinct species

of Iberian endemic cypriniforms, respectively from the “bermejuela” Achondrostoma arcasii (Steindachner, 1866), the Northern straight-mouth nase Pseudochondrostoma duriense

(Coelho, 1985) and from the Southern Iberian spined-loach Cobitis paludica (de Buen, 1930).

The remaining 13 species, all belonging to the genus Myxobolus, were found to infect either the thinlip grey mullet Chelon ramada (Risso, 1827), the thicklip grey mullet Chelon labrosus (Risso, 1827) or the flathead grey mullet Mugil cephalus Linnaeus, 1758. The previously known species Ceratomyxa diplodae Lubat et al., 1989, Myxobolus pseudodispar Gorbunova, 1936,

M. exiguus Thélohan, 1895 and Ellipsomyxa mugilis (Sitjà-Bobadilla and Alvarez-Pellitero,

1993) were also observed among the fish specimens analysed.

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analysis of several criteria, including myxospore morphology, host specificity, site of infection and molecular data of the SSU rDNA gene. Comprehensive re-descriptions were also provided for C. diplodae Lubat et al., 1989 sensu Sitjà-Bobadilla and Alvarez-Pellitero, 1993 infecting European seabass Dicentrarchus labrax Linnaeus, 1758, and M. exiguus infecting C. ramada, given that the scarcity of data provided in their original descriptions led them to become potentially cryptic species complexes. Considering the enormous amount of Myxobolus spp. found in C. ramada and other mullet hosts, a detailed and critical review of all mugiliform-infecting myxobolids is further provided and will certainly prove to be extremely useful for establishing reliable taxonomic comparisons in future species descriptions of the group. Overall, the unreliability of using only morphological criteria for species differentiation, as well as for the distinction of actinosporean types and collective groups, was reinforced. Thirteen new types are described from the raabeia (1 type), aurantiactinomyxon 81 type) and sphaeractinomyxon (11 types) collective groups, but without the usage of molecular data could have been easily misidentified amongst themselves and in relation to previously known types. Moreover, the recognition of single types displaying overlap of morphological features that in the past have been used for the differentiation of distinct collective groups, lead to the demise of the echinactinomyxon, endocapsa and tetraspora collective groups. The first are united with raabeia and the two latter transferred to sphaeractinomyxon.

Phylogenetic analyses of the the SSU rDNA gene provided insight into the evolutionary patterns and drivers of the myxosporean groups analysed, either giving support or discrediting previously known assumptions. The phylogenetic analysis of C. auratae refuted the previously proposed common ancestry and geographically driven evolution of sparid-infecting

Ceratomyxa spp., while that of C. diplodae questioned the strict host specificity that it is

generally accepted for the members of the genus Ceratomyxa, namely by reinforcing the potentially cryptic nature of this species. Overall, the molecular data currently available for

Ceratomyxa was shown to be insufficient to unravel the evolutionary paths in place.

In turn, the phylogenetic analysis of cypriniform- and mugiliform-infecting myxobolids revealed clustering patterns that mostly reflected the evolutionary radiation of their hosts. While the formation of several leuciscid- and cyprinid-infecting subclades revealed that myxobolids entered different families of the order Cypriniformes multiple times during their evolution, the clustering of all mugiliform-infecting Myxobolus spp. within a single clade suggested a monophyletic origin of this group, establishing a clear parallelism to the monophyly of the order Mugiliformes. The positioning of all sphaeractinomyxon types with available molecular data (including former endocapsa and tetraspora) within this monophyletic clade of mugiliform-infecting Myxobolus, revealed a potential correlation between these morphotypes as counterparts of a common life cycle. This hypothesis was ultimately confirmed by the genetic matches obtained between Myxobolus spp. newly reported from the Minho estuary and three

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of the marine sphaeractinomyxon types found in the Douro estuary. The congruence of this

novel myxosporean/actinosporean association contradicts the overall reported lack of agreement between myxosporean genera and actinosporean morphotypes. The evolutionary success of this association is evidenced by the hyperdiversification of Myxobolus in mullet

hosts, which is suggested to correlate with the ecological plasticity of mullets, as well as with

the effectiveness of the Myxobolus and sphaeractinomyxon spore morphotypes in promoting dissemination and the invasion of their respective hosts.

A life cycle inference was further demonstrated between the aurantiactinomyxon type found infecting the marine oligochaete Tubificoides pseudogaster (Dahl, 1960) in the Minho estuary and the eel-infecting Paramyxidium giardi (Cépède, 1906) Freeman and Krist-mundsson, 2018.

In light of the associations established in these newly recognized life cycles, the

presence of a single actinospore type in both freshwater and marine oligochaete hosts could be clarified by their usage of a migratory fish as vertebrate host; thus strengthening the contention that the acquisition of fish as second hosts was crucial in enabling myxosporeans to cross environmental barriers and conquer new habitats.

Acknowledging the diversity of oligochaete species identified as hosts for the new actinosporean types in all three estuaries, as well as in previous studies, the family Naididae Ehrenberg, 1828 is suggested to have played a preponderant role in the settlement and evolution of myxosporeans in estuarine and marine habitats. This apparently successful parasite/host relationship is hypothesized to correlate primarily with the cosmopolitan nature and high availability of naidids in aquatic environments worldwide. Nonetheless, the body of knowledge currently available for myxosporean-annelid interactions is patchy and requires for future studies to recognize the abiotic and biotic factors shaping these relationships.

In conclusion, this work reinforces the importance of investing in the continuous research of the unexplored biodiversity of myxosporeans, not only in estuaries, but in aquatic ecosystems in general. This will certainly allow the identification of new life cycle associations and factors mediating myxosporean-host interactions, which are fundamental for recognizing the origin, diversification and evolutionary patterns of these ancient group of parasites – the oldest among metazoans and an example of evolutionary success.

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Resumo

As espécies do grupo Myxozoa são endoparasitas cnidários altamente diversificados e possuidores de um ciclo de vida complexo, durante o qual ocorre alternância entre dois estádios de vida: um estádio de mixosporídio que se desenvolve num hospedeiro intermediário vertebrado, comumente um peixe; e um estádio de actinosporídio que se desenvolve num hospedeiro definitivo invertebrado, nomeadamente um oligoqueta ou um poliqueta. Durante muito tempo, o interesse no estudo dos mixozoários esteve, principalmente, associado à sua reconhecida importância económica, uma vez que este grupo engloba várias espécies causadoras de parasitoses emergentes em populações de peixe selvagem e de cultivo, tendo, por isso, impacto negativo nas indústrias de pesca e de aquicultura. Mais recentemente, a descoberta da extraordinária história evolutiva deste grupo incrementou o interesse científico no seu estudo, por revelar as suas interações como sendo potencialmente representativas dos fatores que impulsionaram a evolução do parasitismo entre metazoários primitivos. Também, a publicação contínua de numerosos trabalhos que descrevem novas espécies, demonstra que a biodiversidade dos mixozoários se encontra muito subestimada, e que estes parasitas podem assumir grande importância na consciencialização de questões ecológicas existentes em ecossistemas aquáticos.

Não obstante a indústria pesqueira e de aquicultura constituírem atividades económicas importantes em Portugal, uma revisão da literatura disponível demonstrou que poucos estudos tiveram como alvo reconhecer a comunidade mixozoária que habita os extensos recursos aquáticos do nosso país. A presente tese pretendeu abordar esta questão por efetuar pesquisa de mixozoários em três estuários portugueses, nomeadamente, nos respeitantes aos rios Minho e Douro e ria de Alvor, dos quais pouca ou nenhuma informação estava previamente disponível. Neste contexto, peixes e anelídeos foram amostrados para a deteção de infeção por parasitas mixozoários nos tecidos e cavidades internas. Os resultados revelaram uma biodiversidade rica e previamente desconhecida deste grupo de cnidários, com um total de 21 espécies da classe Myxosporea (17 novas) e 15 estádios de actinosporídeos (13 novos) aqui reportados de peixes e oligoquetas, respetivamente. Excetuando a ocorrência de Ceratomyxa auratae na vesícula biliar da dourada Sparus aurata Linnaeus, 1758 proveniente do estuário da Ria de Alvor, todos os outros registos efetuados de novas espécies, referem-se a membros da família Myxobolidae encontrados em peixes amostrados do estuário do Rio Minho. Myxobolus arcasii n. sp., M. duriensis n. sp. e

Thelohanellus paludicus n. sp. são descritos a partir de três espécies distintas de cipriniformes

endémicos da Península Ibérica, respetivamente, do peixe ruivaco Achondrostoma arcasii

(Steindachner, 1866), da boga Pseudochondrostoma duriense (Coelho, 1985) e do verdemã

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género Myxobolus, são descritas a partir de infeções em diferentes espécies de tainhas, nomeadamente, Chelon ramada (Risso, 1827), Chelon labrosus (Risso, 1827) e Mugil

cephalus Linnaeus, 1758. As espécies Ceratomyxa diplodae Lubat et al., 1989, Myxobolus pseudodispar Gorbunova, 1936, M. exiguus Thélohan, 1895 e Ellipsomyxa mugilis

(Sitjà-Bobadilla e Alvarez-Pellitero, 1993) foram também registadas entre os espécimes de peixes analisados.

A descrição e posicionamento taxonómico das novas espécies foi realizada com base na análise combinada de vários critérios, incluindo morfologia dos mixosporos, especificidade do hospedeiro, sítio de infeção e informação molecular do gene da pequena subunidade ribossomal. Re-descrições detalhadas foram realizadas para as espécies C. diplodae Lubat et al., 1989 sensu Sitjà-Bobadilla e Alvarez-Pellitero, 1993 a partir de infeções no robalo

Dicentrarchus labrax Linnaeus, 1758 e M. exiguus a partir de infeções na tainha C. ramada,

dado que a escassez dos dados fornecidos nas suas descrições originais levou a que se tornassem complexos de espécies, potencialmente crípticas. Considerando a enorme biodiversidade de espécies do género Myxobolus encontrada nas espécies de tainha analisadas, e mais particularmente, em C. ramada, uma revisão detalhada e crítica de todos os mixobolídios parasitas de peixes mugiliformes é fornecida e certamente será extremamente útil para o estabelecimento de comparações taxonómicas em futuras descrições de espécies do grupo.

Em geral, as comparações taxonómicas efetuadas, reforçam a falibilidade do uso de critérios morfológicos para estabelecer diferenciação entre espécies, bem como para a distinção entre estádios de actinosporídeos e grupos coletivos. Treze novos tipos dos grupos coletivos raabeia (1 tipo), aurantiactinomyxon (1 tipo) e sphaeractinomyxon (11 tipos) são descritos de oligoquetas de água-doce e marinhas, mas é demonstrado que, sem o uso de técnicas moleculares, poderiam ter sido facilmente identificados erradamente entre si, e em relação a tipos previamente conhecidos. Também, o reconhecimento de tipos singulares que, simultaneamente, exibem características morfológicas que são comumente utilizadas para diferenciar grupos coletivos distintos, é dado como motivo para a invalidação dos grupos coletivos echinactinomyxon, endocapsa e tetraspora. Os primeiros são unidos aos raabeia e, os dois últimos, incluídos entre os membros do grupo coletivo sphaeractinomyxon.

Análises filogenéticas do gene da pequena subunidade ribossomal revelaram padrões evolutivos e condutores da evolução dos grupos de mixosporídeos analisados, apoiando ou desacreditando hipóteses anteriores. A análise filogenética da espécie C. auratae refutou a ancestralidade comum previamente proposta para as espécies do género Ceratomyxa que infetam esparídeos, bem como a suposta relevância da geografia como condutor da evolução deste grupo. Por sua vez, o posicionamento filogenético de C. diplodae, questiona a estrita

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Ceratomyxa, nomeadamente, por reforçar a natureza potencialmente críptica desta espécie.

Em geral, a informação molecular atualmente disponível para o género Ceratomyxa demonstra-se insuficiente para permitir o reconhecimento de trajetórias evolutivas e dos seus

elementos condutores.

Por sua vez, a análise filogenética dos mixobolídios parasitas de cipriniformes e de mugiliformes, revelou padrões filogenéticos que espelham a radiação evolutiva dos seus hospedeiros. A formação de várias subclades contendo separadamente espécies que parasitam leuciscídeos e ciprinídeos, revela que durante a sua evolução, os mixobolídios adquiriram múltiplas vezes os membros destas famílias e, potencialmente outras da ordem Cypriniformes, como hospedeiros vertebrados. Já o agrupamento de todos os Myxobolus que infetam tainhas numa única clade, sugere a origem monofilética deste grupo, estabelecendo um claro paralelismo à monofilia da ordem dos Mugiliformes. O posicionamento de todos os tipos de sphaeractinomyxon com dados moleculares disponíveis (incluindo antigos endocapsa e tetraspora) dentro desta clade monofilética de Myxobolus que infetam tainhas, revelou uma potencial correlação entre estes morfotipos, como contrapartes de um ciclo de vida comum. Esta hipótese foi confirmada pelo estabelecimento de correspondências genéticas entre as espécies do género Myxobolus descritas do estuário do Rio Minho, e três dos sphaeractinomyxon marinhos encontrados em oligoquetas do estuário do Douro. Reconhecida pela primeira vez neste trabalho, a congruência da associação entre estes morfotipos contradiz a falta de correspondência que é, geralmente, reportada entre os diferentes géneros de mixosporídeos e grupos coletivos de actinosporídeos. O sucesso evolutivo desta associação é evidenciado pela hiperdiversificação do género Myxobolus em hospedeiros mugilídeos, a qual se sugere correlacionar com a plasticidade ecológica desta família de peixes, bem como com a elevada eficácia dos morfotipos do género Myxobolus e do grupo coletivo sphaeractinomyxon em promover a disseminação e processos de invasão aos respetivos hospedeiros.

A descrição do tipo de aurantiactinomyxon encontrado no epitélio intestinal da oligoqueta marinha Tubificoides pseudogaster (Dahl, 1960) do estuário do Rio Minho permitiu ainda reconhecer o ciclo de vida do mixosporídio da enguia Paramyxidium giardi (Cépède, 1906) Freeman e Kristmundsson, 2018, por se verificar a existência de correspondência genética entre estes dois estádios de vida.

As associações estabelecidas pelos ciclos de vida demonstrados neste trabalho, permitiram compreender que a presença de um único tipo de actinosporídeo tanto em oligoquetas de água doce, como marinhas, é resultante do uso de um peixe migratório como hospedeiro vertebrado. Consequentemente, reforça-se a alegação de que a aquisição de peixes como hospedeiros secundários foi crucial para que os mixosporídeos pudessem ultrapassar barreiras ambientais e conquistar novos habitats.

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Com base na diversidade das espécies de oligoquetas identificadas como hospedeiras dos tipos de actinosporídeos encontrados nos três estuários analisados neste trabalho, bem como em estudos anteriores, sugere-se um papel preponderante da família Naididae Ehrenberg, 1828 na colonização e evolução dos mixosporídeos em ecossistemas estuarinos e marinhos. O sucesso desta relação parasita/hospedeiro parece correlacionar-se, primariamente, com a natureza cosmopolita e a elevada disponibilidade dos naidídeos em ambientes aquáticos de todo o mundo. No entanto, a informação presentemente disponível para as interações entre os mixosporídeos e os seus hospedeiros anelídeos é extremamente reduzida, e requer que estudos futuros se foquem em reconhecer os fatores abióticos e bióticos que as moldam.

Concluindo, este trabalho reforça a importância do investimento na investigação contínua da biodiversidade inexplorada destes organismos, não só nos estuários, mas nos ecossistemas aquáticos em geral. Isto seguramente permitirá identificar novas associações de ciclos de vida e fatores mediadores das interações dos mixosporídeos aos hospedeiros, considerados fundamentais para o reconhecimento da origem, diversificação e padrões evolutivos destes parasitas metazoários, que são os mais antigos à face da terra e um exemplo de sucesso evolutivo.

xix Table of Contents Agradecimentos………. v Financial support………..vii Publications………....ix Abstract………...xi Resumo………. xv

Chapter I - General Introduction………..1

Myxozoa……….3

Historical Overview………...4

Taxonomic system and main criteria used for classification………..6

Myxosporean diversity and morphotypes………..8

Myxosporean life cycles……….12

Myxosporean development………...15

Drivers of myxosporean evolution………18

Phylogenetic reconstruction………..21

Background in Portugal………..25

References……….. 29

Chapter II - Study aims and approach……….53

Study aims………55

Study approach………...55

Chapter III - Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae)……….. 57

Abstract……… 59

Introduction………..59

Materials and methods………...61

Results………..63

Discussion………67

Acknowledgments……….. 73

References……….. 74

Chapter IV - Ultrastructure and phylogeny of Ceratomyxa diplodae (Myxosporea: Ceratomyxidae), from gall bladder of European seabass Dicentrarchus labrax………79

Abstract……… 81

Introduction………..81

Materials and methods………...83

Page |xx

Discussion………88

Acknowledgments………...94

References………...94

Chapter V - The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis……….101

Abstract………..103

Introduction………103

Materials and methods……….105

Results………107

Discussion………. 114

Acknowledgments……… 119

References……… 120

Chapter VI - Phylogeny and comprehensive revision of mugiliform-infecting myxobolids (Myxozoa, Myxobolidae), with the morphological and molecular re-description of the cryptic species Myxobolus exiguus………...127

Abstract………..129

Introduction………129

Materials and methods……….131

Results………133

Discussion………. 138

Acknowledgments……… 151

References……… 151

Chapter VII - Description of a new actinosporean prompts union of the raabeia and echinactinomyxon collective groups (Cnidaria, Myxozoa), due to overlap in actinospore morphology……….163

Abstract………..165

Introduction………165

Materials and methods……….167

Results………170

Discussion………. 177

Acknowledgments……… 188

References……… 188

Chapter VIII - Molecular data infers the involvement of a marine aurantiactinomyxon in the life cycle of the myxosporean parasite Paramyxidium giardi (Cnidaria, Myxozoa)………195

Abstract………..197

xxi Materials and methods……….200 Results………201 Discussion………. 205 Acknowledgments……… 210 References……… 210

Chapter IX - Characterization of sphaeractinomyxon types (Cnidaria, Myxozoa) from marine

and freshwater oligochaetes in a Portuguese estuary, with the demise of the endocapsa collective group………..219 Abstract………..221 Introduction………221 Materials and methods……….223 Results………225 Discussion………. 234 Acknowledgments……… 239 References……… 240

Chapter X - Myxozoan (Cnidaria, Myxozoa) biodiversity in mullets (Teleostei, Mugilidae)

unravels hyperdiversification of Myxobolus (Cnidaria, Myxosporea)………245 Abstract………..247 Introduction………247 Materials and methods……….250 Results………253 Discussion………. 276 Acknowledgments……… 286 References……… 286

Chapter XI - Morphological and molecular characterization of myxobolids (Cnidaria, Myxozoa)

infecting cypriniforms (Actinopterygii, Teleostei) endemic to the Iberian Peninsula……….. 295 Abstract………..297 Introduction………297 Materials and methods……….299 Results………301 Discussion………. 311 Acknowledgments……… 318 References……… 318

Chapter XII - Molecular-based inferences confirm the involvement of sphaeractinomyxon in

the life cycle of mugiliform-infecting Myxobolus species (Cnidaria, Myxosporea)…………..327 Abstract………..329 Introduction………329

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Materials and methods……….332 Results………334 Discussion………. 340 Acknowledgments……… 345 References……… 345

Chapter XIII - General Discussion………..355

Myxosporean biodiversity………357 Taxonomic descriptions and species re-descriptions……….363 Phylogeny………..366 Life cycle inferences and novel myxosporean/actinosporean associations…………369

Myxosporean-annelid interactions………..371

References……… 373

Chapter XIV - Conclusion and Future Perspectives……….383

Conclusion and Future Perspectives……….385 References……… 389

C

hapter I

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Myxozoa

Myxozoans are cnidarian endoparasites that mainly infect aquatic invertebrates and vertebrates as definitive and intermediate hosts, respectively. Diverse and widely distributed, these parasites are important components of ecosystems, and have been previously estimated to represent ca. 18% of the presently known cnidarian diversity (Zhang, 2011). However, the increasing number of publications describing new species, clearly indicates that myxozoan diversity has been greatly underestimated and may surpass that of its free-living relatives (Okamura et al., 2015).

Interest in this group of parasites has been intrinsically linked to their considerable ecological, economic and even medical importance. Some species are known to cause emerging diseases that impact wild and reared populations (e.g. Ceratonova shasta,

Enteromyxum leei, Henneguya ictaluri, Myxobolus cerebralis, Kudoa thyrsites and Tetracapsuloides bryosalmonae), with significant economic losses having been reported in

aquaculture and fishery industries due to impaired growth, decreased body condition, loss of breeders, stock depletion and decreased marketability of fish carcasses, in addition to the costs associated with treatments and disinfection (e.g. Diamant et al., 1994; Kent et al., 1994b; Pote et al., 2000; Yanagida et al., 2004; Foott et al., 2007; Hallett and Bartholomew, 2012; Henning et al., 2013; Sarker et al., 2015; Marshall et al., 2016; Kotob et al., 2017). Human health may be challenged by these parasites when raw infected fish are consumed by immunocompromised individuals (McClelland et al., 1997; Boreham et al., 1998; Lebbad and Willcox, 1998; Moncada et al., 2001; Kawai et al., 2012; Iwashita et al., 2013).

The recent recognition of myxozoans as a radiation of endoparasitic cnidarians further increased interest in the group, as their interactions likely represent important drivers of the evolution of parasitism in early diverging metazoans. During their evolutionary history, myxozoans became miniaturized, incurred great morphological simplification and evolved complex life cycles by engaging in sophisticated interactions with their invertebrate and vertebrate hosts. The acquisition of vertebrates as intermediate hosts, in particular, was crucial for the successful diversification of myxozoans, as it facilitated alternative transmission and dispersion strategies that were decisive in the conquest of new habitats (Holzer et al., 2018). Fish are the most common intermediate hosts of myxozoans, but amphibians, reptiles, birds and mammals can also be infected by these parasites (Lom and Dyková, 2006). Some fish– parasitic myxozoans further evolved into hyperparasites of platyhelminth endo- and ectoparasites of fish (e.g. Overstreet, 1976; Siau, et al., 1981; Freeman and Shinn, 2011), and a muscle-dwelling Kudoa species was found in an octopus (Yokoyama and Masuda, 2001). Thus, the information currently available already demonstrates the distinct evolutionary success of this ancient parasitic group, which continued study may yet provide fundamental

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insight into metazoan endoparasitism.

Historical Overview

The first report of a myxozoan parasite was performed by Jurine (1825), who discovered cysts developing in the musculature of whitefish, Coregonus fera Jurine, 1825, in Lake Léman. Several descriptions followed without taxonomic allocation of these parasites (see Shulman, 1990 and references therein), which Müller named 'psorosperms' (Müller, 1841). It was only in the early 1880s that Otto Bütschli assigned myxozoans to Sporozoa, as the subclass Myxosporidia. The Sporozoa comprised a diverse group of spore-forming unicellular parasites of animals, further including the Microsporidia Sprague, 1977 [currently recognized as fungi associated with Cryptomycota (Weiss and Becnel, 2014)] and members of the protist phylum Apicomplexa Levine, 1970. Despite some structural characters giving support to their protist nature (see Marques, 1987; Lom and Dyková, 1997), recognition of their multicellularity prompted Štolc (1899) to propose the inclusion of myxozoans within Metazoa. Several subsequent studies gave additional support to this classification, namely by showing the presence of metazoan-related structures (e.g. septate and adherens-type cell junctions) in myxozoans (e.g. Emery, 1909; Ikeda, 1912; Weill, 1938). The study of Weill (1938) was particularly important since it demonstrated the similarity existent between myxozoan polar capsules and cnidarian nematocysts. This led the author to propose myxozoans as cnidarians, potentially related to the larval stages of Polypodium hydriforme Ussov, 1885, an enigmatic cnidarian parasite of the oocytes of sturgeon and paddlefish (Acipenseridae).

In 1970, Grassé attributed Myxozoa with the status of a phylum within the Metazoa, following what had previously been proposed by Lom (1969). Despite having been received with skepticism, this classification was ultimately confirmed in the 1990s, when sequencing of the SSU rDNA gene revealed myxozoans as highly modified metazoans that suffered extreme morphological simplification due to the acquisition of a endoparasitic life-style (Smoothers et al., 1994; Siddall et al., 1995; Schlegel et al., 1996). The recognition of their affinity to major metazoan groups, however, remained uncertain until very recently.

Early molecular studies, based mainly on analyses of the SSU rDNA gene, usually pointed Myxozoa as the sister taxon to P. hydriforme, whenever the latter was included in the analysis. Nonetheless, the position of this clade (named Endocnidozoa) was unreliable, appearing either placed as the sister clade to Bilateria or nested within Cnidaria, depending on taxon sampling, alignment, optimization method, and the characters considered (Smothers et al., 1994; Siddall et al., 1995; Kim et al., 1999; Siddall and Whiting, 1999; Zrzavý, 2001; Zrzavý and Hypša, 2003; Evans et al., 2008, 2010).

Page | 5 Medusozoa, received broad support from several studies showing morphological and functional homology between myxozoan polar capsules and cnidarian nematocysts (e.g. Weill et al.,1938; Lom, 1990; Siddall et al., 1995; Cannon and Wagner, 2003). However, it was argued that nematocyst-like structures could have evolved prior to the divergence of bilaterians and cnidarians or even have arisen independently within these groups (Jiménez-Guri et al., 2007).

The inclusion of the bizarre Buddenbrockia plumatellae Schröder, 1910 within Myxozoa fueled controversy, as its morphological features provided conflicting evolutionary signals. The classification of this rare vermiform-like endoparasite within Myxozoa was based on its morphological and biological similarities to the members of the then recently established class Malacosporea Canning et al., 2000 [specifically to the causative agent of Proliferative Kidney Disease Tetracapsuloides bryosalmonae (Monteiro et al., 2002)], and included the use of a freshwater bryozoan as host and the presence of “nematocyst-like” polar capsules in both the epidermis and infective spores, as well as of typical septate junctions. Its worm-like body and triploblastic organization, however, was more comparable to that of nematodes, despite B.

plumatellae lacking a recognizable nervous system, gut and external sense organs. Also, the

presence of four well-defined blocks of longitudinal muscles running its entire length, allowed this parasite to undergo nematode-like bending movements in the host's coelomic cavity, instead of the retractive and peristaltic movements commonly reported in cnidarians (Pickens, 1988). These mixed features led authors to suggest B. plumatellae as being representative of the missing link in the evolution of myxozoans from a bilaterian ancestor (Canning et al., 2002; Okamura and Canning, 2003). The characterization of bilaterian-like Hox genes in this species gave additional support to the bilaterian affinity of myxozoans (Anderson et al., 1998). Nonetheless, these results were later discredited by Jiménez-Guri et al. (2007), who further argued that the four blocks of muscle in B. plumatellae were radially distributed like in cnidarians, making it a tetraradial worm with a single axis of symmetry.

In more recent years, comprehensive multigene analyses (e.g. Jiménez-Guri et al., 2007; Nesnidal et al., 2013; Feng et al., 2014; Chang et al., 2015; Holzer et al., 2018) and the identification of synapomorphic genes between Myxozoa and Cnidaria (e.g. Feng et al., 2014; Shpirer et al., 2014, 2018; Holland et al., 2011) finally confirmed the myxozoan origin within Cnidaria. Holzer et al. (2018) further suggested that Myxozoa and P. hydriforme represent not one but two independent routes to endoparasitism in the Cnidaria, consequently invalidating the “Endocnidozoa”. Thus, after a long period of controversy, myxozoans are now widely accepted as highly diversified cnidarian parasites.

Another important breakthrough in myxozoan history was the unravelling of their two-stage life cycles, which involve alternation between a definitive invertebrate host and an intermediate vertebrate host. Prior to this groundbreaking discovery by Wolf and Markiw

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(1984), the different stages developing to produce spores in the fish hosts (myxospores) and in the annelid hosts (actinospores) were regarded as separate entities belonging to independent groups of parasites – the classes Myxosporea Bütschli, 1881 and Actinosporea Noble, 1980, respectively. This classification was mainly based on their presence in different host groups, but also in the distinctiveness of the morphological features found between myxospores and actinospores.

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

Taxonomic system and main criteria used for classification

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

Page | 7 evolutionary trajectory. Overall, myxosporeans are characterized by their derived features (e.g. lack of tissues and formation of complex spores). They utilize annelids (oligochaetes, poly-chaetes and sipunculids) as definitive hosts, and are able to use a broader array of vertebrate groups as intermediate hosts. Despite mainly infecting freshwater and marine fish, a few of these parasites have also been described from amphibians, reptiles, and even homeotherms (waterfowl and shrews) (Lom and Dyková, 2006).

The taxonomy of myxozoans is largely based on spore morphology and morphometry, following early classification systems of the group (Kudo, 1933; Tripathi, 1948; Shulman,

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1959). Other features traditionally used for classifying myxozoan taxa include identification of the host species and habitat, tissue of infection, and even the characterization of vegetative stages. The implementation of molecular tools to the study and classification of myxozoans, however, has shown that the reliable differentiation of taxa, especially at the species- and genus-level, must require the inclusion of molecular data due to the artificiality of morphological criteria. For instance, the genera Ellipsomyxa, Myxidium, Zschokkella and Sigmomyxa have convergent morphotypes that hinder correct identification of known and new species (see Fiala, 2006; Bartošová et al., 2009; Fiala and Bartošová, 2010; Liu et al., 2010; Rocha et al., 2013b; Fiala et al., 2015b).

Acknowledging that myxospore morphology-based taxonomy is mostly inconsistent with phylogenetic studies based on molecular markers (more commonly the SSU rDNA gene), numerous taxonomic revisions have been performed in the past few decades in order to resolve poly- or paraphyletic taxa. For instance, the families Pentacapsulidae, Hexacapsulidae and Septemcapsulidae were suppressed and their genera synonymized with Kudoa of the family Kudoidae, upon molecular evidence that the number of polar capsules constituted an artificial criterion for discriminating between these families (Whipps et al., 2004). The genus

Kudoa remained polyphyletic only due to the inclusion of two Sphaerospora sequences,

including S. dicentrarchi, which were ultimately synonymized with the genus, currently monophyletic (see Casal et al., 2019). Another example is that of the genus Polysporoplasma, suppressed due to clustering of its type species within the Sphaerospora sensu stricto lineage (Bartošová et al., 2013). The genus Leptotheca was also suppressed and its members synonymized with Ceratomyxa and Sphaerospora (Gunter and Adlard, 2010). In the other way around, for instance, the family Myxobilatidae was resurrected in order to encompass the phylogenetically related genera Acauda, Hoferellus and Myxobilatus, formerly ranked in different families (Whipps, 2011). Several new genera have also been erected to incorporate species that, whilst being morphologically similar to the morphotypes of other genera, can be differentiated on the basis of phylogenetic data and other biological data [e.g. Ceratonova and

Paramyxidium (Atkinson et al., 2014; Freeman and Kristmundsson, 2018)]. Despite these

advances, many myxozoan taxa remain poly- or paraphyletic; a situation that is expected to progressively be altered by the exponential increase of available molecular data.

Myxosporean diversity and morphotypes

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

Page | 9 latter are intracellular organelles, each containing an eversible polar tubule that, upon release, attaches to the host’s surface, allowing the sporoplasms (or their secondary cells – sporoplasm germ cells) to invade the host and begin infection. Despite sharing these main morphological features, myxospores and actinospores exhibit distinctive morphotypes (Lom and Dyková, 2006). Myxospores have a thick wall composed by hardened valve cells that unite along a conspicuous suture and, after release from the host's body, sink to the sediments, where they can remain infectious for annelids for months or even years due to their resistance (Hoffman and Putz, 1969; Hoffman and Markiw, 1977; El-Matbouli and Hoffmann, 1991; Lom and Dyková, 2006; Koel et al., 2010). Conversely, actinospores are short-lived and usually have inflatable valvular processes for increased floatability in the water column, so as to enhance contact with potential fish hosts (Yokoyama et al., 1993b; Xiao and Desser, 1998a; Lom and Dyková, 2006).

As previously mentioned, different myxospore morphotypes are classified into distinct myxosporean genera, distinguished based on morphological features such as the number and shape of valves, presence or absence of surface ornamentations and/or caudal processes, position of the suture line, and number and position of the polar capsules (Fig. 2; Lom and Dyková, 2006). Overall, morphotypes of the family Myxobolidae (specifically belonging to the genera Henneguya, Myxobolus and Thelohanellus) are the most commonly reported from freshwater habitats, comprising about 50% of the myxosporean diversity described to date. Together, members of this family account for more than 1,100 species, the majority of which is histozoic in fish hosts. Few species are coelozoic and even fewer have been reported to occur in amphibian hosts (see Eiras, 2002; Eiras et al., 2005, 2014; Lom and Dyková, 2006;

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

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Eiras and Adriano, 2012; Zhang et al., 2013). Other main myxospore morphotypes are those of the genera Ceratomyxa, Chloromyxum, Kudoa, Myxidium, Zschokkella, Sphaeromyxa and

Sphaerospora; the successful evolution of which is demonstrated by their multiple origin in

marine and freshwater lineages due to convergent events (Fiala and Bartošová, 2010). The remaining morphotypes represent solely about 10% of myxosporean biodiversity, being distributed among ca. 50 genera that have been rarely reported (Lom and Dyková, 2006; Fiala et al., 2015a).

In turn, actinospores are generally characterized by having triradiate symmetry and valves that inflate osmotically upon release into the environment, producing valvular processes that diverge in different directions to reduce sinking. Three polar capsules and numerous sporoplasms are located anteriorly to the valvular processes. Differentiation between morphotypes is, therefore, mainly based on the shape and size of the actinospores' body and valvular processes, shape and relative position of the polar capsules, and number of secondary cells in the sporoplasm (Fig. 3).

According to these criteria, actinosporean morphotypes are grouped into 21 collective groups (antonactinomyxon, aurantiactinomyxon, echinactinomyxon, endocapsa, guyenotia, helioactinomyxon, hexactinomyxon, hungactinomyxon, neoactinomyxum, ormieractinomyxon, pseudotriactinomyxon, raabeia, saccimyxon, seisactinomyxon, siedleckiella, sphaeractinomy-

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

Page | 11 xon, synactinomyxon, tretractinomyxon, tetraspora, triactinomyxon and unicapsulactinomy-xon), some of which were erected as genera of the former class Actinosporea (e.g. hexactinomyxon, raabeia, sphaeractinomyxon and triactinomyxon) (Lom and Dyková, 2006), while others were created in order to comprise distinct morphotypes described in more recent decades (e.g. endocapsa, saccimyxon, seisactinomyxon, tetraspora and unicapsulactinomy-xon) (Hallett et al., 1999; Hallett and Lester, 1999; Rangel et al., 2011; Milanin et al., 2017; Atkinson et al., 2019).

Despite sphaeractinomyxon and tetraspora sharing the same morphotype, the second was established based on the lower number of actinospores that it produces per pansporocyst: tetraspora forms groups of only four actinospores, while all others develop in groups of eight (Hallett and Lester, 1999). The validity of the usage of this criterion to differentiate between the sphaeractinomyxon and tetraspora collective groups was recently discussed by Rangel et al. (2016a).

An interesting feature that some morphotypes further evolved in order to increase flutuability and enable dispersion over longer distances is the formation of spore nets. Actinospores of the antonactinomyxon, hungactinomyxon, ormieractinomyxon, siedleckiella and synactinomyxon collective groups do no exit the hosts body as isolated units, but rather as nets of eight spores attaching to each other by the tips of their valvular processes (Lom and Dyková, 2006).

About 200 actinosporean types have been described to date (Lom and Dyková, 2006), but this number is expected to increase given its discrepancy to the number of known myxosporeans. Overall, the limited body of knowledge currently available for actinosporeans is mostly likely due to the little direct commercial and recreational value of their annelid hosts. In fact, research on this group only gathered momentum after Wolf and Markiw (1984) demonstrated actinosporeans as life cycle stages of potentially pathogenic myxosporeans. Moreover, their typically low prevalence of infection demands an enormous effort in collecting and examining potential hosts (McGeorge et al., 1997; Xiao and Desser, 1998a, b; Özer et al., 2002; Oumouna et al., 2003; Hallett et al., 2003; Eszterbauer et al., 2006; Marcucci et al., 2009; Rosser et al., 2014).

Thus far, aurantiactinomyxon, echinactinomyxon, neoactinomyxum, raabeia and triactinomyxon morphotypes are, undoubtedly, the most commonly reported from freshwater environments worldwide (Lom and Dyková, 2006). In turn, the few studies conducted in brackish/marine environments identify the sphaeractinomyxon and tetractinomyxon morphotypes as the most common in marine oligochaetes and polychaetes, respectively (see Marques, 1984; Hallett et al., 1997, 1998, 1999; Køie et al., 2004, 2007, 2008, 2013; Karlsbakk and Køie, 2012; Rangel et al., 2009, 2016a).

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Myxosporean life cycles

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

Since 1984, dozens of myxosporean life cycle have been elucidated, either through holistic transmission studies or DNA sequence match between myxosporean and actinosporean counterparts (see Eszterbauer et al., 2015). Holistic transmission experiments are extremely laborious and time-consuming, namely due to difficulties in acquiring and maintaining specific hosts under laboratory conditions. Moreover, while some success has been obtained in transmitting the parasitic infection from fish to annelids (e.g. El-Mansy and Molnár, 1997a,b; El-Mansy et al., 1998; Molnár et al., 1999; Székely et al., 1999, 2001, 2002; Eszterbauer et al., 2000; Rácz et al., 2004), attempts of infecting fish with viable actinospores

Page | 13 have mostly failed (e.g. Székely et al., 2001). The implementation of molecular tools to the study of myxosporean life cycles has further discredited the reliability of this methodology, having proved that erroneous associations may occur as a result of mixed infections (Holzer et al., 2004; Atkinson and Bartholomew, 2009; Marton and Eszterbauer, 2011; Székely et al., 2014). Consequently, life cycle studies now mostly rely on DNA match, specifically of the SSU rDNA gene, for inferring correspondence between myxosporean and actinosporean stages.

The vast majority of known myxosporean life cycles refer to species that parasitize potadromous, catadromous or anadromous fish in freshwater habitats; this includes mainly myxobolids of the genera Myxobolus, Henneguya and Thelohanellus, but also species of the genera Ceratonova, Chloromyxum, Hoferellus, Myxidium, Myxobilatus, Paramyxidium,

Parvicapsula, Sphaerospora and Zschokkella (e.g. Styer et al., 1991; Grossheider and Körting,

1992; Benajiba and Marques, 1993; Kent et al., 1993; Yokoyama et al., 1993a; Bartholomew et al., 1997, 2006; Lin et al., 1999; Eszterbauer et al., 2000, 2006; Holzer et al., 2004, 2006; Kallert et al., 2005; Atkinson and Bartholomew, 2009; Marton and Eszterbauer, 2011; Székely et al., 2014). Fewer studies provide information for myxosporeans that parasitize fish in brackish/marine habitats, with a total of 8 life cycles reported among species of the genera

Ceratomyxa, Ellipsomyxa, Gadimyxa, Kudoa, Ortholinea and Sigmomyxa(Køie et al., 2004,

2007, 2008, 2013; Rangel et al., 2009, 2015, 2016b, 2017; Karlsbakk and Køie, 2012). Overall, oligochaetes are the invertebrate hosts most commonly used by myxosporeans in freshwater habitats, while polychaetes appear to be the hosts of choice in brackish/marine habitats (Køie et al., 2004, 2007, 2008; Rangel et al., 2009, 2011, 2016b; Karlsbakk and Køie, 2012). Nonetheless, there are a few known exceptions: the triactinomyxon stages of Ortholinea

auratae Rangel et al., 2014 and O. labracis Rangel et al., 2017 infect the marine oligochaetes Limnodriloides agnes Hrabĕ, 1967 and a Capitella spp., respectively (Rangel et al., 2015,

2017), while the tetractinomyxon stages of Ceratonova shasta (Noble, 1950) and Parvicapsula

minibicornis Kent et al., 1977 use the freshwater polychaete Manayunkia speciosa Leidy, 1859

as invertebrate host (Bartholomew et al., 1997, 2006).

Even though only a small fraction of myxosporean life cycles have been resolved, the information so far acquired demonstrates that there is no obvious correlation between myxospore and actinospore morphotypes (Fig. 4). In fact, several different myxospore morphotypes have been shown to share the same actinospore morphotype. For instance, the species Ceratomyxa auerbachi, Ceratonova shasta, Ellipsomyxa gobii, E. mugilis, Gadimyxa

atlantica, Parvicapsula minibicornis, Sigmomyxa sphaerica and Sphaerospora dicentrarchi all

have tetractinomyxon counterparts developing in polychaete hosts (Bartholomew et al., 1997, 2006; Køie et al., 2004, 2007, 2008; Karlsbakk and Køie, 2012; Rangel et al., 2009; 2016b). In the same manner, species of the genera Chloromyxum, Henneguya, Hoferellus and

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developing in oligochaete hosts (e.g. Styer et al., 1991; Grossheider and Körting, 1992; Székely et al., 1998; Lin et al., 1999; Holzer et al., 2004; Eszterbauer et al., 2006; Zhao et al., 2016, 2017). These associations suggest that myxospores underwent greater morphological differentiation than actinospores during their evolution, probably as the result of adaptation to a broader array of tissues and hosts in different habitats. The production of distinct morphotypes (myxospores and actinospores) within the same life cycle evidences the considerable plasticity of myxosporean spore design and is hypothesized to correlate with optimizing transmission between vertebrate and invertebrate hosts (Fiala et al., 2015a). In fact, studies have shown that a single actinosporean genotype may produce more than one phenotype in the invertebrate host, probably representing designs that are intended for distinc fish hosts (Hallett et al., 2002; Holzer et al., 2004; Eszterbauer et al., 2006; Zhao et al., 2016).

Despite the growing interest in myxosporean life cycles, several actinospore morphotypes (e.g. hexactinomyxon, hungactinomyxon and sphaeractinomyxon), as well as many myxosporean genera (e.g. Ceratomyxa, Sphaeromyxa and Sphaerospora), have never been linked to any specific counterpart, neither by experimental transmission nor by DNA match. The acquisition of increased knowledge on this subject will almost certainly prove to be

Figure 4. Schematic drawing depicting the lack of congruence between myxosporean genera and actinosporean morphotypes: species of the genus Myxobolus have been paired with actinosporean types belonging to the aurantiactinomyxon, raabeia and triactinomyxon collective groups.