Anatomia Associada Ao Comportamento Reprodutivo De

Jimena García Rodríguez

Anatomia associada ao comportamento reprodutivo de

Cubozoa

Anatomy associated with the reproductive behavior of

Cubozoa

São Paulo

2015 Jimena García Rodríguez

Anatomia associada ao comportamento reprodutivo de Cubozoa

Anatomy associated with the reproductive behavior of Cubozoa

Dissertação apresentada ao Instituto de Biociências da Universidade de São Paulo para obtenção de Título de Mestre em Ciências, na Área de Zoologia

Orientador: Prof. Dr. Antonio Carlos Marques

São Paulo

2015

García Rodríguez, Jimena

Anatomia associada ao comportamento reprodutivo de Cubozoa 96 páginas

Dissertação (Mestrado) - Instituto de Biociências da

Universidade de São Paulo. Departamento de Zoologia.

1. Cubozoa; 2. Histologia; 3. Reprodução. I. Universidade de São Paulo. Instituto de Biociências. Departamento de Zoologia.

Comissão Julgadora

Prof(a) Dr(a) Prof(a) Dr(a)

Prof. Dr. Antonio Carlos Marques

A mis padres, hermana y en especial a mi abuelita

“Caminante, son tus huellas el camino y nada más; Caminante, no hay camino, se hace camino al andar. Al andar se hace el camino, y al volver la vista atrás se ve la senda que nunca se ha de volver a pisar. Caminante no hay camino sino estelas en la mar”

Antonio Machado, 1912

Agradecimentos

Em primeiro lugar, eu gostaria de agradecer ao meu orientador Antonio Carlos Marques, Tim, pela confiança desde o primeiro dia, pela ajuda tanto pessoal como profissional durante os dois anos de mestrado, pelas discussões de cada tema tratado e estudado e pelas orientações que tornaram possível a elaboração deste trabalho.

Agradeço também o apoio institucional do Instituto de Biociências e do Centro de Biologia Marinha da Universidade de São Paulo.

Obrigada ao CNPq, pela bolsa (Processo: 134663/2013-6) proporcionada durante o Mestrado.

Gostaria de agradecer também a todos os docentes que formaram parte deste estudo, em especial ao professor José Eduardo Marian, por me ensinar toda a histologia (que agora começo a entender) e pela paciência desde o começo. Obrigada também ao professor André C. Morandini por todo o material emprestado, por resolver muitas dúvidas e por me ajudar a compreender um pouco mais das Cubos. Obrigada também ao professor Alvaro Migotto pelas fantásticas imagens cedidas e aos doutores Allen Collins (National Museum of Natural History, Smithsonian Institution) e Sho Toshino pelos materiais fornecidos.

Infinitos agradecimentos à nossa colaboradora (e amiga) Cheryl Lewis Ames por sua estadia no Brasil, por me atender sempre que necessário e pelas grandes descobertas.

Obrigada aos técnicos Phillip Lenktaitis e Ênio Mattos pelo tempo que compartilhamos no grande laboratório de histologia e pelas dicas e risadas nos momentos mais frustrantes. Eu gostaria de agradecer também aos técnicos do CEBIMarpor todo seu apoio.

Muito obrigada aos colegas e, sobretudo, amigos de laboratório (LEM), María Mendoza, Amanda Cunha, Karla Paresque, Lucília Miranda, Thaís Miranda, Marina Fernández, Fernanda Miyamura, Adrián Jaimes e Adriana Morales. Obrigada pelo trabalho em equipe que tanto ajuda, pelas discussões, pelas dicas, pelos ânimos, por me escutar e por me fazer sentir em casa. Obrigada por ser a minha família aqui. Obrigada também a Melissa J. Acevedo pela estância na USP, pela paciência, por me transmitir as boas vibrações e por me ensinar tudo o que sabia.

Obrigada também a meus amigos fora do laboratório, em especial a Karina, Isabel e a Nilvea pelo apoio, muito obrigada por estar perto.

Obrigado aos meus amigos que não estão aqui, mas que mesmo à distância me apoiaram sempre e me fizeram sentir como se estivessem por perto. Obrigado à Lola, Olim, Ángel, Ser, Sara, Ainhoa, Vir e Laura.

Muito obrigada à toda a família Bressiani por cuidar de mim, por me fazer sentir como parte da família e por ser minha família aqui. Sem eles este trabalho não teria sido possível. Obrigado ao Paulo por ser meu melhor amigo e companheiro, pelo grande apoio, pelos conselhos e discussões, por toda confiança, por ajudar-me e dar-me forças para seguir com este trabalho e por haver estado sempre ao meu lado.

Finalmente, gostaria de agradecer a minha família, sempre presente me animando e aconselhando-me, especialmente aos meus pais por ter confiado em mim e ter me deixado voar.

Gracias a todos de corazón.

Índice

CAPÍTULO 1. Introdução ...... 1 A classe Cubozoa ...... 1 Reprodução sexuada dos Cubozoa ...... 2 Espécies estudadas ...... 2 Objetivos do trabalho ...... 4 Organização da Dissertação ...... 4 Referências ...... 5 Figuras ...... 10

CAPÍTULO 2. Histology of box jellyfish (Cnidaria: Cubozoa) gonads reveals variation among internal fertilizing species Alatina alata (Alatinidae) and Copula sivickisi (Tripedaliidae) (García-Rodríguez J.,C. Lewis, J. E. A. R. Marian, & A. C. Marques. A ser submetido) ...... 11 Abstract ...... 11 Resumo ...... 12 Introduction ...... 12 Material and methods ...... 14 Results ...... 16 Discussion ...... 19 References ...... 26 Figures ...... 30 Tables ...... 47

CAPÍTULO 3. Equivocal evolution in testes’ organization of cubozoans (Cnidaria) (García- Rodríguez J.,C. Lewis, J. E. A. R. Marian, & A. C. Marques. A ser submetido) ...... 49 Abstract ...... 49 Resumo ...... 50 Introduction ...... 51 Material and methods ...... 52 Results ...... 53 Discussion ...... 55 References ...... 58 Figures ...... 61 Tables ...... 68

CAPÍTULO 4. Internal fertilization and sperm storage in cnidarians: a response to Orr and Brennan (Marques, A. C., J. García, & C. Lewis Ames, 2015. Internal fertilization and sperm storage in cnidarians: a response to Orr and Brennan. Trends in Ecology & Evolution) ...... 72 References ...... 74 Figures ...... 76

CAPÍTULO 5. Considerações Finais ...... 77 Referências ...... 78

Resumo ...... 80 Abstract ...... 81

APÊNDICE I Protocolo para inclusão em historresina...... 82 APÊNDICE II Métodos De Coloração (Historresina) ...... 83 APÊNDICE III Preparação Dos Corantes (Fórmulas Para Preparar 300 ml) ...... 86 García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 1

CAPÍTULO 1. Introdução

A classe Cubozoa O filo Cnidaria é formado por dois clados, Anthozoa e Medusozoa (Werner, 1973a; Bridge et al., 1995; Marques & Collins, 2004), este diferenciado por ter DNA mitocondrial linear e fase de medusa no ciclo de vida. O subfilo Medusozoa, por sua vez, compreende quatro classes, Staurozoa, Hydrozoa, Scyphozoa e Cubozoa, distintas especialmente pelos ciclos de vida (Marques & Collins, 2004) (Figura 1).

Nosso modelo de estudo foi Cubozoa, com cerca de 50 espécies descritas (Bentlage et al., 2010) distribuídas por zonas costeiras ou neríticas, principalmente em regiões tropicais e subtropicais (Werner, 1973a). Os cubozoários, conhecidos em inglês como “box jellyfish”, eram considerados parte dos Scyphozoa até serem elevados à classe (Werner, 1973a; 1975) com base nas diferenças que existem no ciclo de vida e na morfologia de seu pólipo. Esta proposta foi corroborada também com base em estudos com nematocistos (Calder & Peters, 1975), microanatomia do pólipo (Chapman, 1978), comportamento (Hartwick, 1991; Stewart, 1996; Lewis & Long, 2005; Lewis et al., 2008; Garm et al., 2012) e inferências moleculares (Collins, 2002; Marques & Collins, 2004; Collins et al., 2006; Bentlage et al., 2010; Smith et al., 2012).

Algumas das características únicas de Cubozoa são a medusa em forma cúbica, metamorfose completa do pólipo em medusa, velário (uma dobra da subumbrela, semelhante em função ao véu das hidromedusas, com canais gastrovasculares) e pedálio (extensão da borda inferior da umbrela em cada aresta e que dá origem aos tentáculos) na medusa, quatro ropálios com estruturas sensoriais (estatocisto e olhos simples e complexos) em cada face da umbrela (Marques & Collins, 2004), além de toxinas potentes, como por exemplo, em Chironex fleckeri (Chirodropidae), considerada como “o animal mais venenoso da Terra” (Endean, 1988).

A classe Cubozoa é monofilética (Kim et al., 1999; Collins, 2002; Marques & Collins, 2004) e formada por duas ordens, também monofiléticas, Chirodropida e Carybdeida (Collins et al., 2006; Collins, 2009; Bentlage et al., 2010). Estas ordens são diferenciadas por características da medusa, como o número de tentáculos por pedálio (um em Carybdeida, mais de um em Chirodropida), morfologia do pedálio (simples em Carybdeida e geralmente ramificado em Chirodropida) e sáculos gástricos (estruturas subumbrelares digitiformes lisas presentes em Chirodropida) (Gershwin, 2005a). Atualmente se considera Carybdeida divididos

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 1 em cinco famílias, Carybdeidae, Tripedaliidae, Tamoyidae, Carukiidae e Alatinidae; e Chirodropida com três famílias, Chirodropidae, Chiropsalmidae e Chiropsellidae (Gershwin, 2006; Bentlage et al., 2010; Bentlage & Lewis, 2012; Toshino et al., 2015) (Figura 2).

Reprodução sexuada dos Cubozoa A literatura referente à reprodução sexuada em Cubozoa é escassa. Suas gônadas têm origem endodérmica (Hyman, 1940; Widersten, 1965; Campbell, 1974; Miller, 1983; Marques & Collins, 2004; Bentlage et al., 2010). A medusa adulta é gonocorística, em alguns casos com dimorfismo sexual da morfologia gonadal (e.g. Copula sivickisi em Lewis & Long, 2005; Lewis et al., 2008; Straehler-Pohl et al., 2014; Toshino et al., 2014; Garm et al., 2015; Tripedalia cystophora em Werner, 1973b; Straehler-Pohl et al., 2014).

A fertilização em Cubozoa pode ser externa ou interna. Fertilização externa é considerada distintiva de Chirodropida, a partir do conhecimento que existe para as espécies Chironex fleckeri e Chiropsella bronzie (Yamaguchi & Hartwick, 1980), sendo a fertilização interna considerada sinapomorfia de Carybdeida (Bentlage et al., 2010). Não há estudos sobre a reprodução para espécies de Tamoyidae, mas a fertilização externa para Carukiidae Morbakka virulenta põe em dúvida a fertilização interna como característica universal de Carybdeida. Tripedaliidae, por sua parte, apresenta características diferentes, em que os machos produzem espermatóforos que são transferidos para a fêmea por meio de um comportamento de corte, ocorrendo fertilização interna (Werner, 1973a, 1973b; Stewart, 1996; Lewis & Long, 2005; Lewis et al., 2008; Hartwick, 1991; Garm et al., 2015).

Espécies estudadas Neste estudo foram analisadas histologicamente as gônadas de seis espécies de famílias diferentes, cinco da ordem Carybdeida (Alatina alata, Carybdea marsupialis, Copula sivickisi, Morbakka virulenta e Tamoya haplonema) e uma Chirodropida (Chiropsalmus quadrumanus). Segue uma breve exposição das espécies:

Alatina alata (Reynaud 1830), Alatinidae: originalmente descrita como Carybdea alata (Arneson & Cutress, 1976; ver Gershwin, 2005b para lista de citações), tem umbrela até 80 mm de altura e 40 mm de largura. Mais comum em águas rasas do Atlântico Ocidental (incluindo Golfo do México e Mar do Caribe), possui também registros em águas mais profundas do Oceano Atlântico (Morandini, 2003; Lewis et al., 2013). Há registros de reprodução mensal em Bonaire (Holanda) em agregações noturnas cerca de 7-10 dias após luas cheias (Lewis et al., 2013). Possui fertilização interna, com blástula liberada após algumas horas pela fêmea

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 1

(Arneson & Cutress, 1976; Lewis et al., 2013). Os espécimes analisados são de Bonaire, depositados na coleção do National Museum of Natural History (NMNH), Washington DC, USA.

Carybdea marsupialis (Linnaeus, 1758), Carybdeidae: primeira espécie de Cubozoa descrita, com umbrela da medusa com até 40 mm de altura e 30 mm de largura. Atualmente tem registros no Mar Caribe, ao longo das costas africanas e portuguesas, e no Mar Mediterrâneo (Studebaker, 1972; Cutress, 1972; Corbelli et al., 2003; Di Camillo et al., 2006 Bordehore et al., 2011). Sua fertilização interna ocorre dentro da cavidade gastrovascular da fêmea (Studebaker, 1972). O material analisado prove do Mar Mediterrâneo, depositado na coleção do NMNH e do LIFE+ CUBOMED Project.

Copula sivickisi (Stiasny, 1926), Tripedaliidae: classificada anteriormente como Carybdea sivickisi, é a menor espécie de Cubozoa descrita, com uma umbrela até 12 mm de altura e 14 mm de diâmetro. Distribuída em algumas áreas tropicais dos oceanos Pacífico e Atlântico (Stiasny, 1922, 1926; Uchida, 1929; Hoverd, 1985; Hartwick, 1991; Matsumoto et al., 2002; Lewis & Long, 2005; Crow et al., 2006; Lewis et al., 2008; Bennett et al., 2013), apresenta dimorfismo sexual nas gônadas. Seus machos produzem espermatóforo que transferem para as fêmeas em um comportamento de corte. Sua fertilização é interna e a fêmea libera uma cadeia de embriões (“embryo strand”) na coluna de água (Hartwick, 1991; Lewis & Long, 2005; Lewis et al., 2008; Toshino et al., 2014; Garm et al., 2015). Os espécimes que estudamos provêm de diferentes localidades do Japão e estão depositados no NMNH.

Morbakka virulenta (Kishinouye, 1910), Carukiidae: umbrela pode alcançar 200 mm de altura. Ocorrem em Okayama (Japão) (Toshino et al., 2013), possui fertilização externa (cf. Toshino et al., 2013), embora esse mesmo estudo descreva a liberação de “ovos fertilizados” por parte da fêmea.

Tamoya haplonema F. Müller, 1859, Tamoyidae: umbrela chega até 150 mm de altura e 60 mm de largura. Distribui-se pela costa atlântica das Américas, com registros desde Argentina até o estado de New Jersey (EUA), bem como Congo, Guiné, África do sul e corrente de Benguela (Müller, 1859; Vannucci, 1957; Mianzan & Cornelius, 1999; Pastorino, 2001). Sua reprodução ainda não foi descrita. Os espécimes estudados provêm do Canal de São Sebastião (litoral de São Paulo) e do estado de New Jersey (EUA).

Chiropsalmus quadrumanus (F. Müller, 1859), Chiropsalmidae: única representante de Chirodropida neste estudo, sua umbrela alcança 100 mm de altura e 140 mm de largura. Tem distribuição desde o Sul do Brasil até a Carolina do Norte, EUA (Vannucci, 1954; Calder & Peters,

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 1

1975; Morandini et al., 2005; Gershwin, 2006). Sua reprodução não foi descrita, mas pode se esperar que sua fertilização seja externa se seguida a universalização do caráter sugerida para Chirodropida (Bentlage et al., 2010). Os espécimes estudados provêm do Canal de São Sebastião (litoral de São Paulo) e do estado de Carolina do Norte (EUA).

Objetivos do trabalho As análises morfológicas e histológicas, contrastadas aos dados comportamentais, foram usadas com o objetivo de (a) inferir um padrão geral na morfologia gonadal em relação ao tipo de fertilização; (b) estabelecer as relações da diversidade de modos de reprodução e morfologias associadas com a filogenia do grupo; (c) definir estruturas pós-espermáticas (os pacotes de esperma: espermatóforo e espermatozeugma) para Cubozoa Carybdeida; (d) inferir processos da utilização de estruturas de armazenamento de esperma (vesícula seminal em machos e espermateca em fêmeas), bem como dos sacos subgástricos presentes em Tripedaliidae.

Organização da Dissertação Esta tese está organizada em cinco capítulos, sendo o primeiro esta introdução, que expõe as características principais do estudo, seus objetivos e sua organização

O capítulo dois, “Histology of box jellyfish (Cnidaria: Cubozoa) gonads reveals variation among internal fertilizing species Alatina alata (Alatinidae) and Copula sivickisi (Tripedaliidae)”, tem como objetivo usar os dados comparados da histomorfologia das gônadas das espécies Alatina alata (Alatinidae) e Copula sivickisi (Tripedaliidae) para elucidar processos de fertilização interna com diferentes nuances de transferência de esperma em Cubozoa. Nele descrevemos as estruturas pós-espermáticas espermatóforo e espermatozeugma, presentes nos Carybdeida, conceituando o espermatozeugma em A. alata como um modo de transferência de esperma em pacotes desprovidos de membrana. Além disso, detalhamos diferentes estruturas de C. sivickisi importantes na reprodução, como os sacos subgástricos e as manchas velariais (presentes nas fêmeas).

O capítulo três, “Equivocal evolution in testes’ organization of cubozoans (Cnidaria)”, traz a primeira descrição histológica comparada da morfologia gonadal de machos e a espermatogênese de quatro espécies de Cubozoa. As espécies estudadas têm diferentes estratégias reprodutivas, de modo que o objetivo desse estudo foi inferir se a estrutura das gônadas masculinas possuem um padrão morfológico relacionado ao tipo de fertilização ou à filogenia.

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 1

O capítulo quatro, “Internal fertilization and sperm storage in cnidarians: a response to Orr and Brennan”, é um ensaio relacionado ao artigo “Sperm storage: distinguishing selective processes and evaluating criteria” (Orr & Brennan, 2015), com o objetivo de suplementar suas informações com organismos filogeneticamente basais no reino animal, e com hábito planctônico, como as cubomedusas, mas que também apresentam fertilização interna e estruturas de armazenamento de esperma.

O capítulo cinco apresenta as considerações finais do estudo e indica possíveis desdobramentos para essa área de estudo, como a inclusão de dados sobre mais espécies de Cubozoa e um aumento de conhecimento relacionado às fêmeas que, permitirá abordar questões levantadas ao final desse estudo.

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Stiasny, G., 1922. Papers from Dr. Th. Mortensen’s Pacific Expedition 1914-1916. XIL Die Scyphomedusen- Sammlung von Dr. Th. Mortensen nebst anderen Medusen aus dem Zoologischen Museum der Universität in Kopenhagen. Videnskabelige Meddelelser fra Dansk Naturhistorisk Forening i Kobenhavn Videnskabelige Meddelelser fra Dansk Naturhistorisk Forening i K0benhavn 73: 513–558.

Stiasny, G., 1926. Über einige Scyphomedusen von Puerto Galera, Mindoro (Philippinen). Zoologische Mededeelingen Deel. Leiden 9: 239–248.

Straehler-Pohl, I., A. Garm, & A. C. Morandini, 2014. Sexual dimorphism in Tripedaliidae (Conant 1897) (Cnidaria, Cubozoa, Carybdeida). Zootaxa 3785(4): 533–549.

Studebaker, J. P., 1972. Development of the Cubomedusa, Carybdea marsupialis. Department Marine Science. Mayaguez, University of Puerto Rico.

Toshino, S., M. Hiroshi, S. Ohtsuka, K. Okuizumi, A. Adachi, Y. Hamatsu, M. Urata, K. Nakaguchi, & S. Yamaguchi, 2013. Development and polyp formation of the giant box jellyfish Morbakka virulenta (Kishinouye, 1910) (Cnidaria : Cubozoa) collected from the Seto Inland Sea , western Japan. Plankton and Benthos Research 8(1): 1–8.

Toshino, S., H. Miyake, & H. Shibata, 2015. Meteorona kishinouyei, a new family, genus and species (Cnidaria, Cubozoa, Chirodropida) from Japanese Waters. ZooKeys 503: 121

Uchida, T., 1929. Studies on the Stauromedusae and Cubomedusae, with special reference to their metamorphosis. Japanese Journal of Morphology 2(2): 103–193.

Van Iten, H., A. C. Marques, J. D. M. Leme, M. L. Pacheco, & M. G. Simoes, 2014. Origin and early diversification of the phylum Cnidaria Verrill: major developments in the analysis of the taxon's Proterozoic–Cambrian history.Palaeontology, 57(4), 677-690.

Vannucci, M., 1954. Hydrozoa e Scyphozoa existentes no Instituto Oceanográfico II. Boletim do Instituto Oceanográfico 5(1-2): 95–149.

Vannucci, M., 1957. Distribuição de Scyphozoa nas Costas do Brasil. Anais da Academia Brasileira de Ciências 29: 593–598.

Werner, B., 1973a. New Investigations on systematics and evolution of the class Scyphozoa and the phylum Cnidaria. Publications of the Seto Marine Biological Laboratory 20: 35– 61.

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Werner, B., 1973b. Spermatozeugmen und Paarungsverhalten bei Tripedalia cystophora (Cubomedusae). Marine Biology 18(3): 212–217.

Werner, B., 1975. Bau und Lebensgeschichte des Polypen von Tripedalia cystophora (Cubozoa, class. no., Carybdeidae) und seine Bedeutung für die Evolution der Cnidaria. Helgoländer Wissenschaftliche Meeresuntersuchungen 27(4): 461–504.

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Yamaguchi, M., & R. Hartwick, 1980. Early life history of the sea wasp Chironex fleckeri (Class Cubozoa). Development and cellular biology of coelenterates 11-16.

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Figuras

Figura 1. Hipóteses filogenética entre os Cnidaria, segundo Van Iten et al. (2014).

Figura 2. Espécies analisadas neste trabalho inseridas na hipótese filogenética, simplificada no nível de famílias e ordens dos Cubozoa (Bentlage et al., 2010). As características, segundo o trabalho original, seriam: A- nerítico, B- modo de reprodução desconhecido, C- toxina(s) única(s), D- fertilização externa, E- ovoviviparidade, F- síndrome de Irukandji, G- pelágico, H- perda da síndrome de Irukandji, I- comportamento reprodutivo.

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CAPÍTULO 2. Histology of box jellyfish (Cnidaria: Cubozoa) gonads reveals variation among internal fertilizing species Alatina alata (Alatinidae) and Copula sivickisi (Tripedaliidae)

Jimena García-Rodríguez1, Cheryl Lewis Ames2,3, José Eduardo A. R. Marian1, Antonio Carlos Marques1,4

1Institute of Biosciences, University of São Paulo, R. Matão Tr. 14, 101, 05508-090, São Paulo, Brazil 2Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington DC, 20013-7012, USA 3BEES Concentration, Biological Sciences Graduate Program, University of Maryland, College Park, MD 20742, USA 4 Center for Marine Biology, University of São Paulo, Manoel Hypólito do Rego, km. 131,5, 11600-000, São Sebastião, Brazil

Abstract Alatina alata and Copula sivickisi are internal fertilizers with distinct reproductive strategies. We study the histological organization of the sperm packages of these species and we concluded that A. alata produce spermatozeugmata, i.e., non-encapsulated sperm clumps with motile sperm tails, while C. sivickisi produces spermatophores, i.e., encapsulated sperm packages. During spawning aggregations A. alata males release spermatozeugmata in the water column and the female take up the sperm into the gastrovascular system, whereas C. sivickisi males transfer the spermatophore to the female during mating with the aid of the tentacles. Nematocysts were found in the male gonad and within the embryo strand released by the female. The subgastric sacs of C. sivickisi males and females, previously believed to function as sperm storage structures, are filled with nematocysts but void of sperm. We found that the velarial spots in mature females are the site of sperm storage in C. sivickisi.

Keywords: Cubozoa, Alatinidae, Tripedaliidae, Internal fertilization, Gonads, Spermatophore, Spermatozeugmata, Subgastric sacs, Velarial spots, Nematocyst.

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Resumo Alatina alata e Copula sivickisi são fertilizadores internos com estratégias reprodutivas diferentes. Nós estudamos a estrutura histológica dos pacotes de esperma destas espécies e concluímos que A. alata produz spermatozeugmata, i.e., aglomerações de esperma não encapsuladas com flagelos do esperma dotados de mobilidade, enquanto C. sivickisi produz espermatóforos, i.e., pacotes de esperma encapsulados. Durante agregações reprodutivas, machos de A. alata liberam spermatozeugmata na coluna d’água e as fêmeas coletam o esperma para dentro de seu sistema gastrovascular, enquanto machos de C. sivickisi transferem os espermatóforos para as fêmeas com o auxílio de seus tentáculos. Foram encontrados nematocistos dentro da gônada de machos e no interior das cadeias de embriões liberados pelas fêmeas. Os sacos subgástricos de machos e fêmeas de C. sivickisi, cuja função acreditava-se que era o armazenamento de esperma, são ocupados por nematocistos e não contém esperma. Nós descobrimos que os pontos velariais em fêmeas maduras são o lugar de armazenamento de esperma em C. sivickisi.

Introduction Studies about the reproductive biology of the box-jellyfish are scattered along the last 50 years (Werner et al., 1971; Studebaker, 1972; Werner, 1973; Arneson & Cutress, 1976; Yamaguchi & Hartwick, 1980; Hartwick, 1991; Stewart, 1996; Corbelli et al., 2003; Lewis & Long, 2005; Lewis et al., 2008; Toshino et al., 2013; Garm et al., 2015). This dearth of histological studies on the gonads of sexually mature females and males leaves many unanswered questions about oogenesis and spermatogenesis in the cnidarian class Cubozoa. Conversely, gametogenesis, development and gonadal anatomy of the cnidarian classes Hydrozoa, Anthozoa and Scyphozoa are better known (e.g., Widersten, 1965; Hinsch & Clark, 1973; Campbell, 1974; Schmidt & Höltken, 1980; Miller, 1983; Eckelbarger & Larson, 1988; Avian & Sandrini, 1991; Eckelbarger & Larson, 1993; Eckelbarger, 1994; Franzén, 1996; Rouse & Pitt, 2000; Pitt & Kingsford, 2000; Morandini & Silveira, 2001; Ohtsu et al., 2007; Cartwright & Nawrocki, 2010; Tiemann & Jarms, 2010; Iguchi et al., 2010; Schiariti et al., 2012). Internal fertilization and accompanying ovoviviparity (i.e., temporary retention of zygotes inside eggs within the female body) has been documented in several cubozoan species of the order Carybdeida, viz. Carybdea marsupialis and Alatina alata (Arneson & Cutress, 1976; Lewis et al., 2013), Tripedalia cystophora (Werner, 1973; Stewart, 1996), and Copula sivickisi (Hartwick, 1991; Lewis & Long, 2005). However, this is not the only mode of reproduction in cubozoans, as there is evidence for external fertilization in the deadly box jellyfish Chironex fleckeri (order Chirodropida), in which gametes unite in the surrounding water after being

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 2 spawned by males and females (Yamaguchi & Hartwick, 1980), and possibly in the carybdeid Morbakka virulenta (Toshino et al., 2013). The first histological study looking into cubozoan gamete organization focused on Carybdea marsupialis (Avian et al., 1993). The same species had the spermatozoa ultrastructure published along with a short histological description of the gonads, although not including histological figures (Corbelli et al., 2003). The mating behavior and spermatophore transfer in the tiny copulating box jellyfish C. sivickisi (as Carybdea sivickisi) was subsequently described based on histological studies, in which it was characterized a developmental gradient within the female gonads (ovaries) (Lewis & Long, 2005). A recent publication on the histology of the gonads, spermatophore and gametes in Copula sivickisi (Garm et al., 2015) corroborates the finding of nematocysts associated with the embryo strand (Hartwick, 1991; Lewis & Long, 2005; Toshino et al., 2014), and described nematocysts associated with the ovaries and testis of mature females and males, respectively, as well as presented new findings of the ultrastructure of male testes, speculating about the function of its associated nematocysts. We have conducted a comparative study on two ovoviviparous carybdeids, Alatina alata (Alatinidae, see Lewis et al., 2013) and Copula sivickisi (Tripedaliidae, see Lewis & Long, 2005; Lewis et al., 2008), attempting to find a correlation between male gonad organization and mode of reproductive biology. Alatinidae was considered as the earliest diverging carybdeid clade, displaying internal fertilization monthly in near shore reproductive aggregations (Bentlage et al., 2010; Lewis et al., 2013), while Tripedaliidae is the apical most carybdeid clade and it exhibits sexual dimorphism of the gonads, spermatophore production and courtship behavior in seasonal copulation events (occurring during spring and summer months), usually found nocturnally near shore at the water surface (Matsumoto et al., 2002; Lewis & Long, 2005; Lewis et al., 2008; Bentlage et al., 2010; Bennett et al., 2013; Straehler- Pohl et al., 2014; Garm et al., 2015). This internal fertilization implies putative sperm storage structures in the females that likely evolved under selective pressures to streamline the species’ elaborate copulatory behavior as a highly efficient mode of fertilization (Marques et al., 2015). More specifically, Alatina alata (Reynaud, 1830) is a tropical and subtropical Atlantic jellyfish, both in neritic and oceanic domains, with bell height up to 100 mm (Fig. 1A). Near shore mature male and female A. alata medusae form massive nocturnal aggregations for sexual reproduction, approximately 7-10 days after the full moon on a monthly basis (Lewis et al., 2013). During these reproductive periods males release their entire stock of sperm into the surrounding water, leaving the bell completely void of gonadal material the following day

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(Lewis et al., 2013). Embryos are released as blastulae within hours of fertilization (Arneson, 1976; Lewis et al., 2013), the female bell also being completely void of gonadal material the following day (Lewis et al., 2013). On the other hand, Copula sivickisi (Stiasny, 1926) is a small (≤14 mm bell height) sexually dimorphic medusa (Fig. 2A-B) distributed in certain tropical areas of Pacific and Atlantic oceans. This species display a complex mating behavior, males producing spermatophore and transferring them to the female for internal fertilization (Hartwick, 1991; Lewis & Long, 2005; Lewis et al., 2008; Toshino et al., 2014; Garm et al., 2015; Straehler-Pohl et al., 2014). Here we describe the histological structure of the gonads in A. alata and C. sivickisi and its putative sperm storage structures in C. sivickisi hypothesizing how the organization of these structures might correspond to the respective sexual reproductive strategies. In this study new histological data are used to review and assess many previous hypotheses associated with the reproduction of some cubozoans. First, we discuss the history and inconsistent usage of the terms “spermatophore” and “spermatozeugma (plural: spermatazeugmata)” (Werner, 1973; Straehler-Pohl et al., 2014 for both terms in Tripedalia cystophora; and Hartwick, 1991; Lewis & Long, 2005; Lewis et al., 2008; Toshino et al., 2014 for spermatophore in C. sivickisi). Secondly, we clarify the mode of spermatozoa dispersal in A. alata, testing putative association and origin of nematocysts with gonads, sperm or embryos. Thirdly, we discuss the function of the subgastric sacs in C. sivickisi, traditionally associated to sperm storage sacs in both males and females (Hartwick, 1991; Lewis & Long, 2005; Straehler-Pohl et al., 2014), though no concrete evidence from dissections or histology has supported this hypothesis. Finally, we seek to verify the tissular structure and function of the species-specific velarial spots, conspicuous only in sexually active female C. sivickisi medusae, and currently supposed to be functional for mate recognition by males (Lewis & Long, 2005; Lewis et al., 2008).

Material and methods Material examined Alatina alata mature medusae (80 mm average bell height) were collected in Bonaire, The Netherlands, in June 2011 (see Lewis et al., 2013). Copula sivickisi mature medusae were collected in Shirahama, Japan, in July 2006 (5.5 mm average bell height; see Lewis et al., 2008) and in Okinawa, Japan in June 1996, 1997 (8 mm average bell height; see Lewis & Long 2005). Specimens were originally fixed in 8% formalin, and deposited into the collection of the National Museum of Natural History (NMNH), Washington, D.C., USA (Table 1 for supplemental data). The specimens analyzed for each species are: Alatina alata: two mature male gonads, as well as gametes and zygotes released into surrounding water during spawning

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 2 aggregation, from which squashes were prepared for the identification of sperm bundles, ova and zygotes under an optical microscope. Copula sivickisi (Shirahama): mature gonads and subgastric sacs of males (n=2) after several spermatophore releases, males (n=2) after one spermatophore release, aberrant “extra” male gonad (n=1) in mesoglea, female (n=1) velarial canals and female (n=1) gonads and subgastric sacs three hours after spermatophore ingestion. Copula sivickisi (Okinawa): mature gonads and subgastric sacs of males (n=2), and female (n=2) subgastric sacs. Additionally to histological analyses, squashes prepared from fixed tissues (gonads and subgastric sacs) were examined for nematocyst identification under an optical microscope.

Material dissection All dissections were made using scalpels under stereomicroscope Zeiss Stereo Discovery V8. During dissection, the medusae were placed on a glass slide in enough water to keep the specimen from drying out. Gonads sandwiched between the ectoderm and endoderm layers of the bell were excised from the medusa, therefore histological cuts include epithelia. Living and dissected specimens were photographed whenever possible.

For A. alata, male gonads were cut at three places along their length, namely at the top- closest to the apex, mid-way along the bell, and at the bottom closest to the bell margin. The material was placed into a vial with the original liquid they were preserved (i.e., 8% formalin).

For C. sivickisi, we dissected male and female gonads, subgastric sacs and female velarial spots. Medusae were cut longitudinally into halves, then the apex was cut off just below the sperm storage sacs. Stomach with three intact subgastric sacs of formalin-fixed female medusa from Shirahama, containing a recently ingested spermatophore, was cut out completely, so as not to disrupt the spermatophore inside. In males and females, each of the four subgastric sacs was separated from the four pairs of gonads. Velarial spots were cut from the velarium of mature female by removing a quadrant containing 8 velarial spots. A single aberrant “extra” male gonad found separate from the main gonads, in a corner inside the mesoglea of a male medusa was also dissected out. Subsamples were individualized into microcentrifuge tubes containing 4-8% formalin. The entire gonads of mature males from Shirahama (>5 mm) and Okinawa (8 mm) specimens were dissected from superior to inferior in cross-sections, and gonad morphology, spermatic follicles, nematocyst warts, interradial septum, epidermis, mesoglea and gastrodermis from the exumbrella and subumbrella were examined.

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Histological methods and light microscopy Dissected samples were dehydrated and embedded in glycol methacrylate following the instructions furnished with the kits (“Leica Historesin Embedding Kit’’, Leica Microsystems Nussloch GmbH, Germany). Thin-sections (3-5 µm) were cut with a Leica RM2255 microtome and were subjected to the following staining procedures (according to Humason, 1962; Behmer et al., 1976; Pearse, 1980; Bancroft & Stevens, 1982; Junqueira, 1995; Mendoza- Becerril et al., 2015): Hematoxylin-Eosin (HE) and Toluidine Blue (TB) for 3 µm sections (for general morphology), Periodic Acid-Schiff (PAS, identification of neutral polysaccharides), Gomori´s Trichrome (TG, identification of collagen) and Alcian Blue pH 2.5 (AB, identification of acidic polysaccharides such as glycosaminoglycans) for 5 µm sections (for histochemistry) (Table 2 for complementary staining methods). The sections were analyzed under a Zeiss Axio Imager.M2. Maps for the specimen’s collection sites were produced with ArcGis software.

Multimedia vouchers Using a pipette, contents were removed from the gastrovascular cavity of an ovulating A. alata female ingesting sperm from the surrounding water, and observed using light microscopy (S1).

Results

Description of gonadal organization and internal fertilization in Alatina alata (complementary to field observations made by Lewis et al., 2013). — Testes formed by simple cuboidal gastrodermal epithelium with apical nuclei and vacuoles, the latter showing no affinity for the applied stains (Fig. 3A-B). Gonads without spermatic follicles (i.e., no developmental gradient), only mature sperm present. Sperm tails overlap in parallel, heads (average 2.5 µ long) oriented in one direction protruding from either side of tight bundles (Fig. 3C). During spawning events, sperm released into gastrovascular cavity in clumps (Fig. 3D-E), male gonads became cloudy, and epithelium develops random lateral rupture sites along distal axis (Figs. 3F, 4). Within hours, sperm shed into surrounding water via the manubrium. As females ingested sperm from surrounding water via their manubria (either in ocean or lab buckets), female gonads became opaque (and yellowish in hue) as sperm entered into gastrovascular system destined to eight leaf-like interradial gonads (ovaries). Ovaries ruptured non-uniformly in multiple spots along their length, eggs ovulated into gastrovascular cavity (Fig. 3G). After several hours embryos seen circulating throughout gastrovascular system of female medusae, then released in clumps into surrounding water (either in aquaria or lab buckets).

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Description of release of sperm clumps by male Alatina alata.— Within 12 hours of A. alata male and female medusae spawning aggregation, zygotes shed by females were collected from buckets together with remained clumps of sperm expelled earlier by males (Fig. 5A). Squash preparation showed zygotes and individual spermatozoa intermingled (Fig. 5B-D). Histological cross-sections of sperm clumps showed multiple individual spermatozoa adhered to interspersed zygotes (possibly stuck into putative jelly coat fertilization envelope), amidst thousands of spermatozoa for every zygote (Fig. 5E-H). Eurytele nematocyst on sperm and zygote mixture (Fig. 5E), similar to those of embryos released into surrounding water by the female (Fig. 5I). Sperm clumps not covered by membrane or encapsulated (Fig. 5H).

Description of gonadal organization and spermatophore production in male Copula sivickisi (complementary to field observations by Lewis & Long, 2005; Lewis et al., 2008). — Juvenile medusae with bell height ~4 mm have evidence of gonadal development at region flanking interradial canals (Fig. 6A). Male medusae achieving bell height of 5 mm generally become sexually mature, developing two flattened hemispherical orange patches (fresh material) arranged into gastric pockets (ostia) at each interradius (i.e., eight hemigonads in total) (Fig. 6B). Gonads butterfly-shaped attached to uppermost part of interradial septum (Gershwin, 2005) (Fig. 6C-D). Fixed gonad (Fig. 6D) with transparent perimeter follicles, opaque white at central section of hemigonads, but deeply pigmented crimson or orange in life. Gonads bi-layered in cross-section: gastroderm (simple squamous gonadal epithelium) with gonadal mesoglea lying beneath. Follicles exhibit development gradient from perimeter towards center (Fig. 6E), whose density along with naturally occurring pigment render darkly pigmented center in life. Evidence of gametogenesis defined by spermatozoa within spermatic follicles (Pitt and Kingsford, 2000; Iguchi et al., 2010; Schiariti et al., 2012). Mature male with follicles at central region of hemigonad, oriented towards central “reservoir” functioning like seminal receptacle, filled with sperm and nematocysts (immature isorhizas), opening towards perradius by pore or sinus (Fig. 6F). Contrastingly, follicles around perimeter characterized by complete absence of sperm (Fig. 6G). Observations of copulating pairs in lab conditions indicate that sperm are released through each hemigonad pore as eight red strands, with male joining them into single bundle (= spermatophore) as they leave the manubrium. The spermatophore is then transferred to one of female’s tentacles. Subsequently, male released female, the latter inserting the spermatophore into her manubrium. Observations of a juvenile female (ca. 3. 5 mm bell height) after recent mating permitted to follow the pathway of the spermatophore within the gastrovascular cavity after ingestion: first within the gastric ostia

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(Fig. 7A-B) and then to the velarial canals (Fig. 7C). Analysis of an embryo strand released by a mature female (ca. 6 mm bell height) (Fig. 8A-C) revealed two kinds of nematocysts within the strand: euryteles (Fig. 8D) and isorhizas (Fig. 8E).

Description of sperm and nematocyst organization in Copula sivickisi male gonads.— Samples from Shirahama males after several spermatophore releases are similar to Shirahama specimens after single spermatophore release (lab observation one day after collection). Histological cross-sections along length of gonads from region nearest to apex to bottom region towards velarial turnover of males after several spermatophore releases (Fig. 9A-C) reveal spermatic follicles without sperm near top and bottom regions whereas central portion contains sperm and immature isorhiza nematocysts (Fig. 9D-E), and perimeter follicles are filled with unripe spermatozoa (Fig. 9F-G); developmental gradient also distinguished from oblique cuts of male gonads (Fig. 9H). Structurally, apical portion of bell different from lateral portions due to presence of apical pads glands on exumbrellar epithelium, these glands producing mucus-like sticky secretion (Fig. 9I). Nematocyst warts and other glandular secretions also present among pads (Fig. 9J). Histological cross-sections for male preserved after single spermatophore release (Fig. 10) with slightly different gonadal morphology, with more elongate gonad (cf. Fig. 9A with Fig. 10A) characteristically attached at interradial septum (Fig. 10B). Large spherical isorhizas also within exumbrellar warts (Fig. 10C). Spermatic follicles apparently connected by PAS-positive fibrous material (Fig. 10D-G). One mature male (average bell height 4.5 mm) with aberrant “extra” gonad (cf. Lewis et al. 2008) had two extra orange pigmented structures of unknown function situated towards middle of gastrovascular cavity (Fig. 11A). Histological section of extra orange pigmented structures resulted in same histological features of regular mature male gonads: spermatic follicles with developmental centripetal gradient filled with sperm and immature isorhizas (Fig. 11B-E). Samples from Okinawa are larger (average bell height 8 mm) than those of Shirahama (average bell height 5.5 mm), but with similar general morphology; medial region of gonads dark, with sperm and nematocysts (Fig. 12A-B); additional characters are subumbrellar gastrodermis with mucus gland cells (Fig. 12C), and greater amount of nematocysts within the gonads when compared with male specimens from Shirahama (Fig. 12D-E).

General description of a Copula sivickisi Shirahama female.— Female preserved three hours after spermatophore ingestion (Fig. 13) had gonad and subgastric sacs dissected. Remains of spermatophore still visible in gastrovascular cavity, between gonad and

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 2 subumbrellar epithelium (Fig. 13A-B) and between gastric cirri with sperm tails (Fig. 13C-D). Euryteles found within female gonads (Fig. 13E-F).

Subgastric sacs in Copula sivickisi males and females and cnidome information.— Mature males and females have flanking regions of gastric phacellae modified into subgastric sacs, i.e., asymmetrical dark-orange pigmented structures, apparently spherical to naked eye (Fig. 14A). Each subgastric sac appeared as widened version of one crumpled down gastric phacellae, creating many folds. As medusa grows a pair of hemispheric subgastric sacs develops flanking each of four gastric phacellae (made up of fine gastric cirri) (Fig. 14B-E), sacs being filled with nematocysts (up to tens of thousands of euryteles), nematoblasts near the gastrodermis (stained with hematoxylin), and adipocytes (unstained) (Fig. 14F-G). Subgastric sacs lack sperm in both male and females (Fig. 14F-G). Eurytele nematocysts also on gastric cirri (Fig. 14H). Subgastric sacs contiguous to base with centrally located gastric cirri (Fig. 15A) and easily removable at base from edge of gastric phacellae (Fig. 15B). Euryteles filled subgastric sacs in squash preparations (Fig. 15C-D). Juvenile medusae have subgastric sacs with less distinct spherical shape, containing only nematoblasts (Fig. 15E-F).

Sperm storage structures in Copula sivickisi females. — Quadrant of velarium containing velarial canals and spots (Fig. 16A-B) from Okinawa mature female (BH=8 mm) after recent embryo strand release was dissected. Darkly pigmented terminal spots filled with granular substance apparently leaking into to velarial canals with embryos (Fig. 16C-D). Under higher magnification velarial spots actually filled with sperm, with nuclei of sperm stained with hematoxylin (Fig. 16E-F).

Discussion Sperm packets in cubozoans: spermatophore vs. spermatozeugma Spermatophores are sperm packages that function in the transfer of sperm indirectly from the male to the female reproductive tract for internal fertilization (Werner 1973). They are commonly associated with reproduction in arachnids and insects where they consist of encapsulated and/or enveloped sperm packets (e.g., Kotrba, 1996). They occur more sporadically in marine invertebrates, varying broadly within and among taxa (Falese et al., 2011). They are best known in mollusks, e.g., compound structures secreted extracellularly containing eusperms in gastropods (Robertson, 2007), and complex structures with ejaculatory apparatus and spermatangium in cephalopods (Hoving & Laptikhovsky, 2007; Marian, 2012). There is a consensus that the tails of the sperm within spermatophores are not motile (cf. Ó

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Foighil, 1985; Hadfield & Hopper, 1980; Robertson, 2007). In cnidarians spermatophores have only been described from the tripedaliids Tripedalia cystophora and Copula sivickisi, as a simple non-capsulated structure (Werner, 1973; Lewis & Long 2005). On the other hand, spermatozeugmata (singular=spermatozeugma) are structures whose purpose would be the retention of viable sperm in high concentrations at the benthic and/or water column interface for prolonged periods following spawning (e.g., in oysters, see Ó Foighil, 1989) or spermcasting, i.e., sperm cast mating (e.g., in other bivalves, see Falese et al., 2011). Spermatophore and spermatozeugma are often used interchangeably when describing indirect fertilization in various animal taxa, and we know no single universally accepted definition to delineate the differences with respect to both form and function of the two, although some attempts have been made in this respect (Mann, 1984). In fact, spermatozeugma is defined as a sperm aggregate, and spermatophore as a sperm package contained within a capsule (Mann, 1984). Evidently, the use of the same descriptive term in different taxa (i.e., homonymy) does not guarantee the homology of the character, thus caution should always be used when interpreting reports in the absence of first-hand observation on sperm transfer or histological results. For instance, some studies differentiate the two structures by considering that spermatophores are of extracellular origin, while spermatozeugmata originate intracellularly (Robertson, 2007, for gastropods). Contrastingly, in some bivalves, for example, spermatozeugmata are composed of radically arrayed sperm cells connected by an extracellular matrix to a core of acellular membrane, with tails extending into the water column; sperm eventually disassociate from these so-called “sperm-balls”, and swimming independently fertilize species-specific eggs either internally or externally (Ó Foighil, 1989; Falese et al., 2011). Again, the high degree of morphological diversity suggests a lack of homology between the homonymic structures, and that the various types of sperm packages likely originated by homoplasy (Robertson, 2007). However, for reasons of consistency we recognize the need to establish a standard for the nomenclature used to describe sperm packages within the Cubozoa. The term spermatozeugma has only been referenced twice in the Cnidaria: 1) when both terms (spermatophore and spermatozeugma) were used to describe the mode of sperm transfer in T. cystophora: “Numerous spermatozeugmata form big globular bodies (spermatophores), which develop in small grooves on the inside surface of the stomach” (Werner, 1973, p. 212); and 2) “sperm bundle ‘spermatozeugma’ from the subgastral spermathecae [gastric phacellae] of the spawned female C. sivickisi” (Hartwick, 1991, p. 173). In both cases microscopy revealed numerous aggregates of active sperm with tails oriented

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 2 towards the water column (Hartwick, 1991). Based on their microscopic observations and no mention of “sperm package” encapsulated in either species, the term “spermatozeugma” would be the appropriate term in both instances, rather than spermatophore. However, the production and transfer of sperm packages was observed in C. sivickisi (as Carybdea sivickisi), which they referred to as “spermatophore production and transfer” (Lewis & Long, 2005, p. 479) propagating the use of the term in future studies (cf. Lewis et al., 2008; Straehler-Pohl et al., 2014). “Sperm packages” of C. sivickisi were described as having a monolayer epithelium (hence, contained within a capsule, a characteristic of spermatophores) that is digested when it passes through the stomach and comes in contact with the ovaries (Garm et al., 2015). However, Garm et al. (2015) refer to the structure as “spermatozeugmata”, further confounding the terminology. We also found “sperm packages” in the gastrovascular cavity of a mature C. sivickisi female that had been preserved within three hours of “spermatophore” ingestion, and within the gastric phacellae between the gastric cirri (corroborating Hartwick, 1991) (Fig. 13). These internal “sperm packages” (= spermatophores; Fig. 13B,D) also lacked a capsule, had several sperm tails on the surface, and lacked the associated nematocysts described in the sperm bundle removed from the stomach of C. sivickisi females (Hartwick, 1991; Garm et al., 2015). We conclude that Copula sivickisi transfer sperm through spermatophores, and that the use of “spermatozeugmata” is incorrect in this case and should be abandoned. We hypothesize likewise that the close relative T. cystophora also employs spermatophore transfer, and that future histological studies on this species sperm packages will allow testing this hypothesis. Our histological analysis of A. alata shows that the sperm clumps released by males into the surrounding water were not covered by any membrane (Fig. 5H), but were merely aggregates of sperm swimming together with their tail oriented towards the water column. This strategy of sperm transfer employed by Alatina alata could correspond to spermatozeugma. With monthly A. alata spermcasting events persisting for three to four days (i.e., 7-10 days after the full moon in Bonaire), spermatozeugmata provide a degree of protection for sperm cells and prolong viability, thus increasing the probability of encountering ova in egg-brooding A. alata females, as is the case in other animals (cf. Ó Foighil, 1989; Lynn, 1994). However, once the spermatozeugmata disassociate (observations made two hours after the sperm release) the female contracted her bell to get the individual spermatozoa into her gastrovascular system, where fertilization takes place. Evidence for this is seen in the videos we provide for supplemental material (S1) that show individual sperm in contact with ovulating eggs within the female gastrovascular cavity.

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Gonad structure and sperm release The histological data from gonads of A. alata males preserved during a spermcasting event revealed that the gonadal epithelium has multiple rupture sites (Fig. 3F and Fig. 4) where sperm is released in clumps (as spermatozeugmata) into the gastrovascular cavity (Fig. 3 D-E), and circulates throughout the gastrovascular system before being released into the water column. The gonads were filled entirely with ripe spermatozoa with long tails (Fig. 3C) suggesting a rapid development of the gonads. The few studies on the reproductive biology of A. alata suggested it is an iteroparous species (i.e., exhibiting multiple reproductive episodes during its lifetime), and may live for more than a year because gonads require a full year to mature (Arneson & Cutress, 1976). However, lab and field observations on A. alata showed that all sperm stock is exhausted in males within 24 hours, and that just a thin remnant remains along either side of the interradial canal in both males and females (that lacks gametes), rendering the sex of spent individuals undistinguishable at that point (Lewis et al., 2013; this study). The lack of any spermatogonia, or developmental gradient in the spermatic follicles of mature males, along with the fact that during the course of the monthly aggregations many spent medusae were found stranded and lifeless nearshore (Lewis et al., 2013), corroborates that A. alata is semelparous, i.e., exhibiting a single and exhaustive reproductive episode prior to death. Conversely, the gonads in C. sivickisi contain spermatogonia and a polarized developmental gradient of spermatic follicles with immature gonadal products found at the periphery of each of the eight “kidney shaped” hemigonad (Fig. 6E-G), developing towards the central portion which is darkly pigmented (orange or red) in live male individuals (Fig. 6B). Each spermatophore is synchronously exuded from a pore (Garm et al., 2015; this study) that opens into the perradius from the center of each hemigonad where mature sperm are held in reservoirs functioning as seminal receptacles (Fig. 6D-F). Fibrous material was observed connecting the spermatic follicles in cross-sections (Fig. 10D-G). We hypothesize that, as only the central section of the hemigonads contains mature sperm, perhaps developing sperm are shuttled from follicles in the periphery towards the central seminal receptacle via this fibrous material (i.e., a functional duct). This would be surprising though not new for medusozoans, as stauromedusae present follicles connected by actual ducts (Miranda et al., 2014). Additional histological studies are needed to test this hypothesis.

Structural variation between male gonads of different Copula sivickisi specimens The gonads from Shirahama males after one spermatophore release are longer than those after several spermatophores release (Fig. 9A-C and Fig. 10A) but this could have been

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 2 an artifact related with fixation or specimen size. The spermatic follicles on the centre of hemigonad, where there were ripe sperm, can be expected to remain empty after spawning (Schiariti et al., 2012). However, there were no differences between gonad structures in the specimens. This might indicate that gonads in C. sivickisi regenerate quickly. Moreover, regional differences might occur between Shirahama and Okinawa specimens: immature isorhiza nematocysts are abundant in males from Okinawa (Fig. 12), what could be explained by the larger dimensions of Okinawa’s specimens. In fact, it is possible that Okinawan specimens are the largest ever recorded for this species. Finally, regarding the aberrant “extra” male gonad in the mesoglea from one Shirahama’s male, histological data provides evidence that this structure is composed by spermatic follicles with sperm at different development stages and immature isorhizas as a gonad (Fig. 11C).

Subgastric sacs function as nematocyst nests in Copula sivickisi The subgastric sacs are hemispheric sacs below each of the gastric phacellae continuous to the gastric ostium (Hartwick, 1991; Lewis & Long, 2005; Lewis et al., 2008; Straehler-Pohl et al., 2014). They were long believed to contain sperm, mainly because dissected female sacs were described with “dense cluster of tailed sperm and numerous cnidocytes” (Hartwick, 1991, p. 173). For these reason, several authors (Hartwick, 1991; Lewis & Long, 2005; Lewis et al., 2008; Straehler-Pohl et al., 2014) called these structures as sperm- storage sacs, functioning as putative spermathecae in females and seminal vesicles in males. However, previously no histological studies were conducted to support or test this assumption. Our findings revealed that subgastric sacs are filled with nematocysts, but are void of sperm (Fig. 14F-G), thus rejecting the hypothesis they are used as sperm storage sacs (cf. Hartwick, 1991; Lewis & Long, 2005; Lewis et al., 2008; Straehler-Pohl et al., 2014). Instead, we conclude that C. sivickisi subgastric sacs function as reservoirs for nematocysts (i.e., nematocysts nests) in both males and females. Similar nematocysts nests (in which nematogenesis takes place prior to the migration of nematocysts to their final destination) are well-documented in Hydra (the fresh water solitary hydrozoan polyps) (David & Challoner, 1974, corroborated by Özbek, 2011), as well as suggested for stauromedusans (Miranda et al., 2014). Alternatively or complementarily, subgastric sacs could be just the pathway of sperm to the gonads (in females) or to the manubrium (in males). We hypothesize that nematocysts migrate from the reservoir (subgastric sacs) to the gastric cirri (also possessing nematocysts, Fig. 14H) where they could be added to the embryo strand in females, in addition to their regular digestive function.

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We compared also the subgastric sacs in males (after several and one spermatophore release) and females, from Shirahama and Okinawa, and concluded there are no differences between these specimens (Fig. 14). However, juveniles have a less distinct shape of their subgastric sacs, as well as they have just nematoblasts (Fig. 15E-F).

Copula sivickisi velarial spots function as female sperm storage structures (FSS) Some studies described that only sexually mature females (bell diameter of at least 5 mm) possessed conspicuous velarial spots, and males did not initiate courtship with females lacking velarial spots (as “velar spots” in Hartwick, 1991; Lewis & Long, 2005; Lewis et al., 2008). However, a female with faint velarial spots was reported to ingest four spermatophores during pair formation and copulation, but no viable embryo strand was generated (Lewis & Long, 2005). Our observations indicate that, after entering the female gastric ostium, the spermatophore is separated into several smaller pieces that circulate throughout the female’s gastrovascular cavity (Fig. 7). The following day, the female’s velarial spots became conspicuous, suggesting these structures appear after ingesting sperm (which have the same color as the velarial spots). Thus far, darkening of the velarial spots is the only known postcopulatory morphological change to occur in Cubozoa (Marques et al., 2015), but until now, neither the contents nor the function of these spots had been reported. Histological examination of velarial spots of mature females revealed their contents were entirely comprised of mature sperm (Fig. 16E-F), likely stored there by females from the onset of sexual maturity following the first copulation. Depending on how sexually mature a female’s gonads are at the time of the first copulation, not all copulation episodes necessarily result in fertilization and an ensuing embryo strand release (Lewis & Long, 2005; personal observations). It was noted however that velarial spots become darker with subsequent copulations, thus we conclude that C. sivickisi velarial spots function as female sperm storages structures. In C. sivickisi both polyandry and polygyny are pervasive, suggesting opportunities for sperm competition and postcopulatory female choice (Lewis & Long, 2005; Marques et al., 2015), and female medusae could use sperm from several males stored in their FSS to fertilize their eggs. Sperm is pigmented (orange or red) providing an observable cue when sperm disperses from the stomach into the gastrovascular cavity, internal fertilization occurring within hours to days. Histological sectioning of female ovaries in C. sivickisi reveals a development gradient (Lewis & Long, 2005; Garm et al., 2015; this study), and the fact that females accept sperm from males during gestation and after giving birth (i.e., embryo strand release) also suggests they might store sperm from multiple copulations for delayed fertilization, avoiding also sperm limitation if mating encounters are rare (e.g., Marian, 2012).

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Additionally, the distinct pattern formed by the 32 velarial spots around the velarium likely serves as a visual cue (as cubozoans have elaborated eyes; Coates et al., 2001) to males that the female is sexually mature and ready to copulate. Several specialized extra-gonadal areas have been documented in cnidarians functioning like seminal vesicle to store sperm following release from the male gonad, prior to release into the external environment, such as the genital sinus in Aurelia aurita (Widersten, 1965), and the subumbrellar space in pelagic Tubularia medusae (Miller, 1976). Nevertheless, the only documented case of specialized sperm storage structures cubozoans is found in cubozoan jellyfish of the family Tripedaliidae. These include the four gastral pockets (essentially grooves) in the manubrium of T. cystophora males and females (Werner, 1973; Straeher-Pohl et al., 2014), and the subgastric sacs in both sexes of C. sivickisi (Hartwick, 1991; Lewis & Long, 2005; Lewis et al., 2008; Straehler-Pohl et al., 2014), as discussed above. In this study, we identified velarial spots as putative spermathecae in females from which sperm appeared to be leaking into the gastrovascular system in association with embryos (Fig. 16D). According, “if sperm-storing features are apparent only when a female reaches sexual maturity and the sperm is positioned by the female in putative storage sites, this is supportive that such features play a role in female sperm storage” (Orr & Brennan, 2015, p. 270). Thus we conclude that velarial spots function as sperm storage structures, and future studies examining multiple fertilizations in C. sivickisi will further elucidate their effect on the reproductive success of C. sivickisi females.

Role of cnidocytes in reproduction Our histological results corroborate the finding of cnidocytes present in the gonads and their involvement in Cubozoa reproduction (Garm et al., 2015). Nematocysts seem to play a different role in male and female gonads. In male gonads there are immature isorhizas (Fig. 17A-B), in contrast with the euryteles found within the female gonads (Fig. 17C-F). The isorhizas from the male gonad are added to the spermatophore to presumably attach to the female. On the other hand, the euryteles found in the female gonads might be added to the embryo to possibly protect it (Garm et al., 2015). Furthermore, we reject the hypothesis that the subgastric sacs function like as sperm storage sites, and propose they function as nematocysts nests.

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Figures

Figure 1. Alatina alata (Reynaud, 1830). A. Mature female (Bonaire) (scale bar 4 mm) (Image courtesy of Allen Collins). feg=female gonad, p=pedalia, t=tentacle, v=velarium.

Figure 2. Copula sivickisi (Stiasny, 1926). A. Male specimen (scale bar 2mm). B. Female specimen (scale bar 2 mm) (Images courtesy of Alvaro Migotto). feg=female gonad, te=male gonad.

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Figure 3. Alatina alata (Reynaud, 1830) gonads. A. General view in cross-section with hematoxylin-eosin (HE) staining (scale bar 50 µm). B. Simple cuboidal vacuolated gastrodermal epithelium, stained with Toluidine Blue (TB) (scale bar 50 µm). C. Detail of sperm clumps within the gonad in cross-section, HE staining (scale bar 20 µm). D, E. Sperm release in clumps (from preserved material). White arrows indicate the spermatozeugma (scale bars 15 µm and 20 µm, respectively). F. Male gonad in cross-section with HE staining. The black arrows indicate lateral rupture sites (scale bar 50 µm). G. Female gonad (live). The black arrow indicates ovulation (scale bar 20 µm). nc=nucleus, vl=vacuole.

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Figure 4. Alatina alata (Reynaud, 1830) male gonad in cross-section, stained with hematoxylin-eosin (HE). A-L. Sequence of gastrodermal epithelium showing distal the rupture sites, possibly liberating sperm. (Scale bar 25 µm). Arrows indicate protuberances observed location.

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Figure 5. Alatina alata (Reynaud, 1830) sperm and zygotes released into surrounding water during spawning aggregation. A. General view of sperm aggregations (scale bar 200 µm). B-D. Squash preparations. Sperm is attached to zygotes (scale bars 25 µm). E-G. Histological sections of sperm clumps and zygotes with hematoxylin- eosin (HE) staining (scale bar 20 µm). H. Sectioned spermatozeugma (sperm clumps) stained with Toluidine blue (TB) (scale bar 20 µm). I. Nematocyst around the embryos in surrounding water (scale bar 50 µm). n=nematocyst, sp=sperm, zy=zygote.

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Figure 6. Copula sivickisi (Stiasny, 1926). A. Immature male medusa (Okinawa, Japan) (scale bar 1 mm). B. Live mature male medusa (Okinawa, Japan) (scale bar 2 mm) (Image courtesy of Alvaro Migotto). C. Lateral view of mature male medusa (Okinawa, Japan) with gonads and subgastric sacs (scale bar 1 mm). D. Mature male gonad excised from fixed material (Shirahama, Japan). White arrows indicate the centre of hemigonad with spermatic follicles filled with mature sperm (scale bar 1 mm), white in fixed material, but orange in life. E. Longitudinal-section of male gonad (Shirahama, Japan) stained with hematoxylin-eosin (HE) (scale bar 200 µm). F. Longitudinal-section of

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 2 male gonad (Shirahama, Japan) stained with Alcian Blue + Periodic Acid-Schiff + Hematoxylin (AB-PAS-H) . The centre of hemigonad is filled with ripe sperm and nematocysts. Arrow indicates the pore/sinus from which sperm is released (scale bar 100 µm). G. Detail of perimeter spermatic follicles filled with immature sperm. Toluidine Blue (TB) staining. Nematocysts visible in the gastrodermal epithelium. Specimen from Shirahama, Japan (scale bar 100 µm). g=gonad, n=nematocyst, nw=nematocyst warts, p=pedalia, r=rhopalia, sgs=subgastric sacs, t=tentacle, te=testes, sgs=subgastric sacs, *=septum.

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Figure 7. Juvenile of Copula sivickisi (Stiasny, 1926) female medusa (Okinawa, Japan). A-C. White arrows indicate the spermatophore circulating within the gastric ostia and tentacular canals (scale bars 1, 1 and 2 mm respectively); live specimen.

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Figure 8. Copula sivickisi (Stiasny, 1926) embryo strand (Okinawa, Japan). A. Mature female prepared to release the embryo strand (scale bar 2 mm). B. Embryo strand (scale bar 100 µm). C. Squash preparations of nematocysts within the embryo strand (scale bar 20 µm). D. Arrow indicates discharged eurytele (scale bar 5 µm). E. Isorhiza (scale bar 5 µm). r=rhopalia, sgs=subgastric sacs, t=tentacle, vs=velarial spots, zy=zygotes.

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Figure 9. Copula sivickisi (Stiasny, 1926) (Shirahama, Japan) male gonads after several spermatophore releases (cross-section). A-C. General view from superior to inferior section of the gonad with hematoxylin-eosin (HE) staining (scale bars 200 µm). D. Central region of the hemigonad filled with ripe sperm and immature isorhizas, stained with Toluidine Blue (TB). Detail form B (scale bar 20 µm). E. Squash preparation identifying immature

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 2 isorhizas within male gonads (scale bar 20 µm). F. Cross-section of the gonad near the septum with sperm follicles filled with unripe sperm, stained with TB. Detail from C (scale bar 50 µm). G. Distal region of the gonad with Periodic Acid-Schiff (PAS) staining, detail from C (scale bar 50 µm). H. Different stages of follicles development, detail from C, with Gomori’s Trichrome + Hematoxylin staining (TG-H) (scale bar 20 µm). I. Adhesive pads in the exumbrellar epithelium with mucus gland cells stained with TB, detail from A (scale bar 20 µm). J. Nematocyst warts in the exumbrellar epithelium with HE staining, detail from B (scale bar 25 µm). gt=gastrodermis, m=mesoglea, mgc=mucus gland cells, n=nematocyst, sp=sperm, *=septum.

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Figure 10. Cross-section of the male gonad Copula sivickisi (Stiasny, 1926) (Shirahama, Japan) after one spermatophore release. A. General view of hemigonad with proximal spermatic follicles with immature sperm, and the middle region with mature sperm and nematocysts; section stained with Toluidine Blue (TB) (scale bar 10 µm). B. Interradial septum region stained with TB (scale bar 20 µm) C. Hemigonad with sperm follicles and nematocyst warts in the subumbrellar epithelium stained with TB (scale bar 50 µm). D. Distal region of the gonad with a fibrous material connecting the spermatic follicles; hematoxylin-eosin (HE) staining (scale bar 50 µm). E-G. Middle region of the hemigonad stained with Periodic Acid-Schiff +Hematoxilin (PAS-H) (scale bars 50 µm, 50 µm, 20 µm, respectively). Black arrows indicate fibrous PAS-positive material. ep=epidermis, g=gonad, gt=gastrodermis, m=mesoglea, mc=musculature, n=nematocyst, nw=nematocyst wart, *=septum.

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Figure 11. Aberrant “extra” male gonad in Copula sivickisi (Stiasny, 1926) (Shirahama, Japan). A. Mature male with an extra gonad in the gastro-vascular cavity (scale bar 4 mm) (Image courtesy of Alvaro Migotto). B, C. Cross- sections stained with hematoxylin-eosin (HE) (scale bars 50 and 20 µm, respectively). D. Squash preparations showing immature isorhizas (scale bar 20 µm). E. Detail of immature isorhizas within the gonad; HE staining (scale bar 20 µm). n=nematocyst, sp=sperm.

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Fig. 12. Copula sivickisi (Stiasny, 1926) male gonad (Okinawa, Japan). Cross-sections with hematoxylin-eosin (HE) staining. A. General view of gonad (scale bar 200 µm). B. Hemigonad with nematocysts in the middle region (scale bar 100 µm). C. Subumbrellar wall (scale bar 50 µm). D. Detail of the center of the hemigonad with sperm and nematocysts (scale bar 20 µm). E. Abundant nematocysts in the gastrodermal epithelium and in the spermatic follicles (scale bar 20 µm). ep=epidermis, gt=gastrodermis, m=mesoglea, n=nematocyst, nw=nematocyst wart, *=septum.

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Figure 13. Copula sivickisi (Stiasny, 1926) (Shirahama, Japan). Female 3hrs after spermatophore ingestion. A, B. Female gonad in cross-section containing a spermatophore within the gastrovascular system; hematoxylin-eosin (HE) (scales bars 50, 20 µm, respectively). C-D. Female subgastric sacs with spermatophore between the gastric filaments; HE staining (scale bars 200 and 20 µm respectively). E-F. Euryteles within female gonads (scale bars 20 µm). E. Cross-section stained with HE. F. Squash preparation. ep=epidermis, gc=gastric cirri, gt=gastrodermis, mc=musculature, n=nematocyst, o=oocyte, sgs=subgastric sacs, st=spermatophore.

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Figure 14. Subgastric sacs of Copula sivickisi (Stiasny, 1926) male and female (Shirahama, Japan). A. Oral view of mature female (Okinawa, Japan) (scale bar 2 mm) (Image courtesy of Alvaro Migotto). B-E. Cross-section of male subgastric sacs stained with hematoxylin-eosin (HE), Toluidine blue (TB), HE and Alcian Blue + Periodic Acid-Schiff + Hematoxylin (AB-PAS-H), respectively (scale bars 100 µm). F-G. Euryteles within male (F) and female (G) subgastric sacs stained with HE (scale bar 50 and 20 µm, respectively). H. Euryteles in male gastric cirri with HE staining (scale bar 20 µm). ad=adipocytes, ap=adhesive pads, gc=gastric cirri, feg=female gonad, n=nematocyst, nb=nematoblasts, sgs=subgastric sacs, t=tentacle.

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Figure 15. Copula sivickisi (Stiasny, 1926) subgastric sacs morphology in mature and immature specimens (Shirahama, Japan). A-D. Subgastric sacs in mature male. A. Subgastric sacs and gastric cirra morphology (scale bar 500 µm). B. Subgastric sacs dissection (scale bar 250 µm). C,D. Euryteles within subgastric sacs in squash preparations (scale bar 10 µm). E, F. Subgastric sacs in immature female. E. Subgastric sacs and gastric cirra morphology (scale bar 250 µm). F. Nematoblasts within the immature subgastric sacs in squash preparations (scale bar 10 µm). gc=gastric cirra, n=nematocyst, nb=nematoblast, sgs=subgastric sacs.

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Figure 16. Female velarial spots of C. sivickisi (Stiasny, 1926) (Okinawa, Japan). A. Live mature female releasing embryo strand with velarial spots visible (scale bar 2 mm). B. Velarium with velarial spots in fixed material (scale bar 100 µm). C, D. Cross-section of velarium stained with hematoxylin-eosin (HE) (scale bars 300 and 100 µm, respectively) showing velarial spots and embryos within the gastro-vascular cavity. E. Cross -section of velarial spots stained with Toluidine blue (TB) (scale bar 50 µm). F. Detail of velarial spots filled with sperm stained with Alcian Blue + Periodic Acid-Schiff + Hematoxylin (AB-PAS-H) (scale bar 20 µm). e=embryo, r=rhopalia, vc=velarial canals, vs=velarial spots, *=septum.

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Tables Table 1. General data of specimens of Alatina alata (Reynaud, 1830) and Copula sivickisi (Stiasny, 1926) analyzed in this study. n/a: not analyzed; SP: São Paulo; *: most valuable and rare.

Number of Observations Total BH BW Species specimens/ Sex Body Part Notes Locality Date coll. Coll. Collection (field and lab Det. specimens (mm) (mm) samples codes and notes) analyzed

Top, middle and bottom of Bonaire (The USNM 1195805; AA_62511_TM_1 77 49 2 Males Gonads June/11 Collins et al. C. Lewis the gonads Netherlands) USNM 1195807 AA_62411_T+12 80 27

Sperm from male Sperm from male Bonaire (The USNM NAA1-7 1 Males June/11 A. Collins C. Lewis n/a n/a manubrium manubrium Netherlands) LEM AA_62411_sperm

NAA2-7 Alatina Bonaire (The USNM 1 - Zygotes in bowls/buckets Zygotes in bowls/buckets June/11 A. Collins AA_62411_buckets C. Lewis n/a n/a 3 alata Netherlands) LEM & bowls

Zygotes in buckets after Zygotes in buckets after 24 Bonaire (The USNM NAA3-7 1 - June/11 A. Collins C. Lewis n/a n/a 24 hrs hrs Netherlands) LEM AA_62411_24+hrs NAA4-7 Bonaire (The USNM 1 - Zygotes Zygotes June/11 A. Collins AA_62411_ZS, C. Lewis n/a n/a Netherlands) LEM from Ziploc Gonads and subgastric After several Shirahama, USNM 2 Males Aug/06 A. Collins AM1 & AM2 C. Lewis 5,5 5 sacs spermatophore releases (Japan) LEM Gonads and subgastric After 1 spermatophore Shirahama, USNM 2 Males Aug/06 A. Collins M1 & M2 C. Lewis 5,5 5 sacs release (Japan) LEM Gonads and subgastric Shirahama, USNM 1 Male 1 day after collected Aug/06 A. Collins M+1 C. Lewis 5,5 5 sacs (Japan) LEM

Gonads and subgastric Motobu, Okinawa USNM 2 Males - 1994 Tagawa OM1 & OM2 C. Lewis ~8 ~7 sacs (Japan) LEM Copula Aberrant “extra” male After several Shirahama, USNM 12 sivickisi 1 Male Aug/06 A. Collins AM2-8 C. Lewis 5,5 5 gonad in mesoglea spermatophore releases (Japan) LEM

Gonads and subgastric 3hrs after spermatophore Shirahama, USNM 1 Female Aug/06 A. Collins F1 C. Lewis 5 4,5 sacs ingestion * (Japan) LEM

Motobu, USNM 2 Females Subgastric sacs - May/95 C. Lewis OF1 & OF2 C. Lewis ~8 ~7 Okinawa,(Japan) LEM

Motobu, Okinawa USNM 1 Female Velarial canals - May/95 C. Lewis OF2-8 C. Lewis ~8 ~7 (Japan) LEM

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Table 2. Staining methods times in hystoresin for 3 µm section to morphology, and 5 µm sections to histochemistry.

Staining methods Reagents Minutes

TB 1,3 Toluidine Blue (TB) Distilled water Wash

H at 37°C 20

Tap water 3

Distilled water Wash

Hematoxylin-Eosin 3 µm sections µm 3 (HE) E at 37°C 5 Distilled water 2 min x 3 Alcohol 70% Differentiate Distilled water Wash

Periodic Acid 15

Distilled water 2 min x 3 Schiff 30 Periodic Acid-Schiff Tap water 5 (PAS) Distilled water Wash

H at 37°C (optional) 10 Tap water 3 Distilled water Wash

TG 15

Distilled water Wash Gomori´s Trichrome

(TG) Acetic acid 0,5 % Differentiate 5 µm sections µm 5 Distilled water Wash

AB 10

Tap water 3

Distilled water Wash Alcian Blue pH 2.5 (AB) H at 37°C (optional) 10

Tap water 3

Distilled water Wash

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CAPÍTULO 3. Equivocal evolution in testes’ organization of cubozoans (Cnidaria)

Jimena García-Rodríguez1, Cheryl Lewis Ames2,3, José Eduardo A. R. Marian1, Antonio Carlos Marques1,4

Abstract Reproductive behavior in the cnidarian class Cubozoa (box jellyfish) is highly diverse. Fertilization has been documented as both internal and external within the class. Complex sexual behavior (i.e., copulation) known in taxa of the family Tripedaliidae is unique among Cnidaria. Previous histological studies revealed major differences between testes structural organization in box jellyfish that copulate by spermatophore transfer and spermcasting males. We conducted histological analyses of the male gonads of four box jellyfish species from different cubozoan families, namely, Carybdea marsupialis (Carybdeidae), Chiropsalmus quadrumanus (Chiropsalmidae), Morbakka virulenta (Carukiidae) and Tamoya haplonema (Tamoyidae) to compare the testes with the corresponding putative reproductive mode (e.g., functional trends) and to determine if histological organization relates to phylogenetic placement (e.g., evolutionary trends) of the respective species. The discussion is enhanced by comparisons with recent findings on testes organization of Copula sivickisi and Alatina alata. Our results show spermatic follicles, in which spermatogenesis occurs within two layers, the gastrodermis (or simple columnar gonadal epithelium, in this case) and the gonadal mesoglea. Just one species analyzed, Tamoya cf. haplonema displayed rupture sites in the wall of the follicles indicating prior release of sperm, while other samples were of immature males or males which had not yet spawned. Results of this first interspecies comparative histological

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 3 study on cubozoans revealed that spermatogenesis occurs in a polarized manner, i.e., towards the lumen of the spermatic follicle, with mature spermatozoa in the center awaiting gonad rupture and release for both internal and external fertilizers. We conclude that there are insufficient discrepancies in testes organization among box jellyfish males to make broader inferences about reproductive modes or phylogenetic relationships.

Keywords: Reproduction, Fertilization, Histology, Spermatogenesis, Chiropsalmidae, Carukiidae, Tamoyidae, Carybdeidae.

Resumo O comportamento reprodutivo na classe de cnidários Cubozoa, conhecida em inglês como “box jellyfish”, é altamente diverso. Há registros de fertilização interna e externa dentre as espécies desta classe. Comportamento sexual complexo (i.e., copulação), conhecido em Tripedaliidae é único entre Cnidaria. Estudos histológicos anteriores revelaram diferenças significativas entre a estrutura gonadal masculina de Cubozoa que copulam através de transferência de espermatóforo e machos que liberam o esperma (“spermcasting”). Nós conduzimos análises histológicas de gônadas masculinas de quatro espécies de diferentes famílias de Cubozoa, a saber, Carybdea marsupialis (Carybdeidae), Chiropsalmus quadrumanus (Chiropsalmidae), Morbakka virulenta (Carukiidae) e Tamoya haplonema (Tamoyidae) para comparar as gônadas de machos com modo de reprodução putativo correspondente. (e.g., tendências funcionais) e para determinar se a organização histológica se relaciona com a filogenia (e.g., tendências evolutivas) das respectivas espécies. A discussão é aprofundada por comparações com descobertas recentes sobre a organização das gônadas de machos de Copula sivickisi e Alatina alata. Nossos resultados indicam que folículos espermáticos, nos quais ocorre a espermatogênese entre duas camadas, a gastroderme (ou epitélio gonadal columnar simples, neste caso) e a mesogleia gonadal. Apenas uma das espécies analisadas, Tamoya cf. haplonema apresentou pontos de ruptura na parede dos folículos indicando liberação de esperma, enquanto outras amostras eram de machos imaturos ou machos que ainda não tinham se reproduzido. Resultados deste primeiro estudo histológico comparativo entre espécies de Cubozoa revelaram que a espermatogênese ocorre de maneira polarizada, i.e., em direção ao lúmen do folículo espermático, com espermatozóides maduros no centro aguardando a ruptura e liberação em ambos tipos de fertilizadores, internos e externos. Nós concluímos que as discrepâncias entre gônadas de machos de Cubozoa são insuficientes para permitir inferências gerais sobre modos de reprodução ou relações filogenéticas.

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Introduction Cubozoa, commonly called box jellyfish, is a small class of the phylum Cnidaria with about 50 described species, distributed in two orders, namely Chirodropida and Carybdeida (Bentlage et al., 2010). Chirodropida comprises three families, Chiropsalmidae, Chirodropidae, and Chiropsellidae; and Carybdeida consists of five families, Alatinidae, Carukiidae, Tamoyidae, Tripedaliidae, and Carybdeidae (Bentlage et al., 2010). They are known best for their potent (and sometimes deadly) stings to unsuspecting beachgoers along tropical and subtropical coasts worldwide (Lewis et al., 2013; Brinkman et al., 2015). Many species are seasonal (Calder, 2009), sometimes occupying specific niches in mangroves (Studebaker, 1972; Stewart, 1996; Fenneret al., 2010) and at least one species has been found in the open ocean (Lewis et al., 2013). Overall, not much is known about their reproduction. Studies on box jellyfish reproduction are hampered by the difficulties of rearing medusae in the lab. The few studies addressing cubozoan reproduction indicate a sexual behavior repertoire not observed in other cnidarian classes. Among carybdeids, species of Tripedaliidae have sexual dimorphic gonads, indirect sperm transfer using spermatophores, and specialized courting behavior (Werner, 1973; Hartwick, 1991; Stewart, 1996; Lewis & Long, 2005; Lewis et al., 2008; Straehler-Pohl et al., 2014; Garm et al.,2015). In Tripedalia cystophora, more specifically, “the fertilized eggs [...] develop into planulae in the gastral pockets of the female medusa” (Werner et al., 1971, p. 582). Another tripedaliid, Copula sivickisi, releases an embryo strand (unique among cnidarians), following courtship behavior resulting in internal fertilization in the gastric ostium (Lewis & Long, 2005 as Carybdea sivickisi). Curiously, however, in T. cystophora males and females apparently release their “unused” gametes in the water 1–2 days after the ‘‘mating phase’’ (Werner, 1973), suggesting this species might use both strategies. Within the families Carybdeidae and Alatinidae ovoviviparity is documented following spermcasting, i.e., males release sperm into the water which is taken up by females and eggs are fertilized internally (Studebaker, 1972; Arneson, 1976; Lewis et al., 2013). The embryos are released within minutes to hours after fertilization (in Carybdea marsupialis see Studebaker, 1972; in Alatina alata, as Carybdea alata, see Arneson & Cutress, 1976). While the appearance of C. marsupialis in the water is seasonal (Studebaker, 1972), A. alata forms monthly aggregations for sexual reproduction (Lewis et al., 2013). The carukiid Morbakka virulenta is apparently the only known exception among the Carybdeida by having external fertilization (Toshino et al., 2013). However, this conclusion is dubious because, although external fertilization was inferred based on the absence of fertilized eggs in the females gonads, it was also contradictorily reported that adult medusae “spawn fertilized eggs” (Toshino et al., 2013, p. 6).

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Currently, no reports exist on reproduction in the cubozoan family Tamoyidae (Carybdeida). Likewise is the case for Chirodropida, but external fertilization was reported for Chironex fleckeri (Chirodropidae) and Chiropsella bronzie (Chiropsalmidae) (as Chiropsalmus quadrigatus) (Yamaguchi & Hartwick, 1980). There are few studies on the histology of the cnidarian gonads. The first histological observations of cubozoan gonads described male testes and female ovaries in Carybdea marsupialis (Avian et al., 1993), of which morphological studies briefly described male testes as “…formed by a simple row of follicles, the wall of which is formed by germinal cells at different stages of spermatogenesis” (Corbelli et al., 2003, p. 96). The histology of the female gonads in C. sivickisi was briefly described (Lewis & Long, 2005), and a more recent and detailed study described nematocysts within the gonads of this species (Garm et al., 2015), corroborating previous findings that suggested their role of defense in the embryo strand (Hartwick, 1991; Lewis & Long, 2005; Toshino et al., 2014). Both Garm et al. (2015) and García- Rodríguez et al. (2015) reported nematocysts in the male gonads and discussed their potential involvement in spermatophore attachment following copulation. Comparing aspects of reproductive strategy, ecology, and biology among different cnidarians facilitates a better understanding of evolutionarily adaptive strategies that might also be phylogenetically informative (Fautin, 2002). Herein we studied the histological and developmental structure of male testes across different box jellyfish taxa, namely Carybdea marsupialis, Chiropsalmus quadrumanus, Morbakka virulenta, and Tamoya haplonema, and compared our findings with those reported for Copula sivickisi and Alatina alata (García- Rodríguez et al., 2015). The aim of this study is to compare the morphology of cubozoan testes with the corresponding reproductive mode (e.g., functional trends) and to determine if histological organization is related to phylogeny (e.g., evolutionary trends). Herein we also provide histological data on testes development at different stages of reproductive maturity, providing additional insight into cubozoan ontogeny.

Material and methods Biological material

In total, we analyzed four specimens of Tamoya haplonema (one from São Sebastião and two from Ubatuba, São Paulo State, Brazil; one Tamoya cf. haplonema from New Jersey, USA, deposited in National Museum of Natural History, NMNH, Washington D.C., USA); three specimens of Chiropsalmus quadrumanus (two from São Sebastião, Brazil; one from North Carolina, USA, deposited in NMNH); two specimens of Carybdea marsupialis (one from

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Pescara, Italy, deposited in NMNH; one from Spain and accessioned through the LIFE+ CUBOMED project; LIFE08 NAT/ES/0064); and one specimen of Morbakka virulenta (from Okayama Prefecture, Japan, currently in S. Toshino collection; see Toshino et al., 2013). The specimens were fixed and preserved in 4-8% formalin until the dissection of gonads, except C. marsupialis from Italy that was originally fixed in 80% ethanol (Table 1 for supplemental data).

Material dissection (detailed protocol in García-Rodríguez et al., 2015) All dissections were made using a scalpel under a Zeiss Stereo Discovery V8 stereomicroscope. During dissection, the medusae were placed on a glass slide in enough water to keep the specimen from drying out. Gonads sandwiched between the ectoderm and endoderm layers of the bell were excised from the medusa, therefore histological cuts included epithelia. Living and dissected specimens were photographed whenever possible. Male gonads were cut at three places along their length: at the top-closest to the apex, mid- way along the bell, and at the bottom closest to the bell margin. The material was placed into a vial with the original liquid they were preserved (i.e., 4-8% formalin or 80% ethanol). Photographs of whole specimens were taken after incisions were made.

Histological methods and light microscopy (detailed protocol in García-Rodríguez et al., 2015) Dissected samples were dehydrated and embedded in glycol methacrylate following the instructions furnished with the kits (“Leica Historesin Embedding Kit’’, Leica Microsystems Nussloch GmbH, Germany). Thin-sections (3-5 µm) were cut with a Leica RM2255 microtome and were subjected to the following staining procedures (according to Humason, 1962; Behmer et al., 1976; Pearse, 1980; Bancroft & Stevens, 1982; Junqueira, 1995; Mendoza- Becerril et al., 2015): Hematoxylin-Eosin (HE) and Toluidine Blue (TB) for 3 µm sections (for general morphology), Periodic Acid-Schiff (PAS, identification of neutral polysaccharides), Gomori´s Trichrome (TG, identification of collagen) and Alcian Blue pH 2.5 (AB, identification of acidic polysaccharides such as glycosaminoglycans) for 5 µm sections (for histochemistry) (Table 2 for complementary staining methods). The samples were analyzed under a Carl Zeiss Axio Imager M2 Microscope.

Results Morphological and/or histological descriptions complemented by literature are presented below, organized by taxa. General patterns of the male gonads in Carybdeida.— Gonadal development begins on aboral part of region flanking each interradial septum; four gonad pairs (i.e., eight testes) gradually elongate towards oral region during medusa development (Uchida, 1970). Gonads

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 3 two-layered, with gastrodermis (or gonadal epithelium) and gonadal mesoglea. Male gonads (testes) formed by spermatic follicles undertaking spermatogenesis (García-Rodríguez et al., 2015). Follicle wall formed by germinal cells (gametes) at different spermatogenic stages, i.e., spermatogonia, spermatocytes or spermatids; follicular lumen with ripe spermatozoa in mature specimens. Follicles become enlarged when filled with mature spermatozoa, testes extends as eight wide leaf-like sheaths towards perradius, in some instances with adjacent gonads overlapping. Endodermal layer delimiting testes ruptures at various sites in irregular pattern (Corbelli et al., 2003; this study) in response to undetermined reproductive cues (e.g., environmental, molecular or chemotaxical), releasing spermatozoa into male gastrovascular cavity, then released into surrounding seawater (common for most carybdeids, but see García- Rodríguez et al., 2015 for discussion on differing modes of spermatogenesis in Copula sivickisi and Alatina alata). Particularities in Carybdea marsupialis (Carybdeidae).— Italian specimen (2.2 cm tall fixed in ethanol 80%) without clearly discernable patterns (Fig. 1B-C), apparently immature due to absence of sperm in gonads. Spanish specimen (2.1 cm tall fixed in formalin) with gonads with distinguishable germ cells (Fig. 1D-F); simple columnar gonadal epithelium with oval nucleus at apical position and large vacuole at basal position (Fig. 1E). Mature sperm absent, only spermatogonia and spermatocytes within spermatic follicles (Fig. 1F). Cells at different developmental stages along each follicular wall (Fig. 1F). Particularities in Morbakka virulenta (Carukiidae).— Four pairs (eight in total) of leaf- like gonads extending along both sides of interradial septa; mature medusae with whitish to whitish yellow gonads in males and females (Toshino et al., 2013). Okayama specimens (bell height 150 mm) (Fig. 2A) with spermatic follicles filled by spermatogonia and spermatocytes (Fig. 2B-C). Gonadal epithelium simple columnar, with oval nucleus at basal position, nucleolus visible in some nuclei (Fig. 2C). Gonadal mesoglea with PAS affinity. Specimen considered immature due to absence of mature sperm and small specimen size (M. virulenta reaches 200 mm at maximum bell height). Particularities in Tamoya haplonema (Tamoyidae).— We analyzed Tamoya haplonema from São Paulo State (Brazil, 8.5-11 cm tall, no sperm within gonads, Fig. 3) and Tamoya cf. haplonema from New Jersey State (USA, 10 cm tall, sperm within gonads, Fig. 4), both with 8 hemigonads, i.e., one half-gonad on each side of interradial septum (Tamoya pattern). Brazilian specimen with gonadal epithelium simple columnar, with apical hematoxylin-stained nucleus and huge unstained vacuole at basal position (Fig. 3B); follicles with spermatogonia and spermatocytes (Fig. 3C); gonadal mesoglea PAS-stained (Fig. 3D). USA specimen with gonadal epithelium not discernible, maybe lost during fixation or dehydration; follicles with

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 3 different developmental stages of gametes: spermatogonia and spermatocytes within follicle wall, sperm lying beneath wall (Fig. 4); irregular rupture sites along gonad periphery, from which sperm is released (Fig. 4B-F). General patterns of the male gonads in Chirodropida – Chiropsalmus quadrumanus (Chiropsalmidae).— General Chiropsalmus pattern with four pairs (eight in total) leaf-like gonads, attached along entire extension of interradial septa in mature specimens, extending out onto gastric saccules (Fig. 5A), genus up to ~80 mm in height (Gershwin, 2006). We analyzed Chiropsalmus quadrumanus from São Paulo State (Brazil, 40 mm tall, Fig. 5) and Chiropsalmus quadrumanus from North Carolina State (USA, 50 mm tall, Fig. 6), both displaying very few spermatozoa. Histological results showed no differences between male gonads from specimens from disparate localities (Figs. 5-6). Gonadal epithelium simple columnar, with hematoxylin-stained nucleus at apical position, and large basal vacuole (Fig. 5B). Gonadal mesoglea PAS-stained (Fig. 5C). Spermatic follicles with developmental gradient: spermatogonia on follicular wall developing into spermatocyte with condensed chromosomes; spermatid with elongated nucleus (suggesting chromosome packing), developing flagellum; center of lumen with motile sperm (Fig. 5C-D). USA specimens (more developed) with gonadal epithelium ciliated (Fig. 6B), lacking sperm within the follicles (Fig. 6B-C), suggesting immature developmental state.

Discussion External fertilization has been reported for the chirodropids Chironex fleckeri and Chiropsella bronzie (as Chiropsalmus quadrigatus) (Yamaguchi & Hartwick, 1980), and the strategy is hypothesized as distinctive and characteristic of Chirodropida (Bentlage et al., 2010), accordingly external fertilization is also assumed for Chiropsalmus quadrumanus, and likewise within the newly established family Chiropsellidae (Toshino et al. 2015). However, the universality of the trend “external fertilization” certainly surpasses Chirodropida, and minimally, from an evolutionary viewpoint, it would have to be assumed with multiple origins within Cubozoa. For instance, within Carukiidae reproductive behavior has only been observed for M. virulenta, but this shows fertilization occurring in the water, i.e., adult female and male medusae spawning eggs and sperm respectively (S. Toshino, in litteris). We consider that it is still necessary to test whether M. virulenta is an exclusive external fertilizer, as reports of sperm and eggs being released (spawned) due to stress or lack of sexual cues have occurred for Tripedalia cystophora (Werner, 1973), which is normally an internal fertilizer. Also to date, no studies dealing with fertilization in Tamoyidae have been reported. However, specimens of Tamoya haplonema cultivated under laboratory conditions released gametes that were

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 3 observed at the bottom of the aquarium, but fertilization itself was not documented despite the presence of females in the aquarium (A. C. Morandini pers. comm.). On the other hand, the phylogenetic corollary of the condition “external fertilization” of Chirodropida (Bentlage et al., 2010, Fig. 3 at p. 497) would be its counterpart condition, “internal fertilization” of Carybdeida. As the authors hypothesized (Bentlage et al., 2010, p. 497), “while information from Carukiidae and Tamoyidae are needed, it appears that internal fertilization is a synapomorphy of Carybdeida”– apparently rendering to both conditions the status of apormorphic. However, first, Morbakka virulenta has demonstrated that internal fertilization might not be universal for the taxon (see above). Second, when considering other medusozoans (cf. Marques & Collins, 2004; Collins et al., 2006; Van Iten et al., 2014), one can conclude that the ancestral condition for the cubozoans would be, actually, external fertilization. This reasoning is also supported by the evolution of planktonic reproduction in that reproductive efficiency would increase in those few groups presenting internal fertilization (Marques et al., 2015). Interestingly, there are many different types of internal fertilization in Carybdeida (Table 3). The carybdeid Carybdea marsupialis is reported to exhibit internal fertilization, in which spermatozoa released into the surrounding water by the male (i.e., spermcasting) enter the gastrovascular cavity of the female via the manubrium. The eggs are fertilized in the gastrovascular cavity (into which the ovaries ovulate mature ova), and zygotes remain there until blastulae are released (Studebaker, 1972; Arneson & Cutress, 1976). Conversely, tripedaliid species Tripedalia cystophora and Copula sivickisi, display a courtship behavior where males produce spermatophores that are transferred during copulation resulting in internal fertilization (Werner, 1973; Hartwick, 1991; Lewis & Long, 2005; Lewis et al., 2008; Straehler-Pohl et al., 2014). Furthermore, C. sivickisi females release an embryo strand into the water column (Hartwick, 1991; Lewis & Long, 2005; Lewis et al,. 2008; Toshino et al., 2014; Garm et al., 2015). It is surprising that testes histological organization lacks a correlation with both functional (e.g., modes of reproduction) and evolutionary (e.g., phylogeny) trends. For instance, the New Jersey specimen of T. cf. haplonema (evidently sharing similar gross morphology and gonadal structure with other individuals of the same lineage), surprisingly also shows similar rupture sites for sperm release to those observed in the testes of Carybdea marsupialis (Corbelli et al., 2003). On the other hand, C. marsupialis (Carybdeida: Carybdeidae) shows more similarities in gonad morphology (e.g., Figs. 1, 5 and 6; Table 4) to Chiropsalmus quadrumanus (Chirodropida: Chiropsalmidae), a phylogenetically distant and unrelated

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 3 species, than to Copula sivickisi (Carybdeida: Tripedaliidae), a less distant taxon in the phylogeny (Bentlage et al., 2010). The histological results are inconclusive regarding a putative correlation between gonad organization and reproductive mode. In fact, there are no major differences in the histological patterns of male gonads between those species with internal (e.g., C. marsupialis) and putative external (C. quadrumanus, M. virulenta) fertilization in this study. However, if we compare the histological results presented in this study with species with internal fertilization including different modes of sperm packaging (spermatophore in C. sivickisi, Tripedaliidae; or spermatozeugma in A. alata, Alatinidae) (García-Rodríguez et al., 2015) testes organization is completely different (Fig. 7 and Table 4). For example, in C. sivickisi, a development gradient is seen towards the center of each hemigonad, with mature sperm (spermatozoa) located exclusively in a central reservoir. This is in contrast to the testes analyzed in this study for species that do not produce intermittent sperm packages (spermatophores), and in which spermatic follicles have the same state of gamete development along the length of the gonads, with no evidence for a centrally polarized developmental gradient. Furthermore, Alatina alata release sperm in clumps, as spermatozeugmata, all at once during monthly spermcasting aggregations (García-Rodríguez et al., 2015); accordingly male gonads present yet a different pattern, in which the gonadal epithelium is simple cuboidal and testes lack a developmental gradient entirely. The limited number of mature male specimens examined in this study, combined with the almost complete lack of information on the reproductive behavior for these species naturally has compromised our ability to make broad conclusions about the functional and evolutionary ramifications of cubozoan testes organization. However, this study is the first to shed light on the gonadal organization of the earliest metazoans with sexual dimorphism, and provides a great deal of data suggesting the homology of cubozoan gonads with those of bilaterians – possessing “seminiferous tubules” (spermatic follicles in cubozoans) and the same mode of spermatogenesis. Our findings represent a preliminary study of reproductive biology in animals that have emerged over 600 million years ago (Van Iten et al., 2014) and that, despite lacking a brain, have evolved complex behavior for sexual reproduction. Additional taxon sampling, with an emphasis on examining mature male and female specimens, supplemented with studies on sexual behavior, fertilization and reproduction will provide insight into the evolution of different reproductive strategies in cubozoans.

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References

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Figures

Figure 1. Carybdea marsupialis (Linné, 1758) male gonads from two different localities (Italia and Spain). A. Live specimen from Spain (scale bar 2 cm) (Image courtesy of Eduardo Obis). B-C. Male gonads from Italian specimen in cross-section stained with hematoxylin-eosin (HE). Material fixed in ethanol (scale bars 100 µm and 20 µm respectively). D-F. Male gonads from Spanish specimen in cross-section stained with Toluidine blue (TB), HE and Gomori´s Trichrome + Hematoxylin (TG-H) respectively (scale bars 50 µm, 50 µm and 20 µm respectively). g=gonad, m=mesoglea, nc=nucleus, p=pedalia, ph=phacellae, sc=spermatocyte, sg=spermatogonia, t=tentacle, v=velarium, vl=vacuole.

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Figure 2. Morbakka virulenta (Kishinouye, 1910) from Okayama (Japan). A. Live specimen (scale bar 10 cm) (Image courtesy of Sho Toshino). B. Male gonad stained with Periodic Acid-Schiff + Hematoxylin (PAS-H) (scale bar 50 µm). C. Male gonad stained with Gomori´s Trichrome + Hematoxylin (TG-H) (scale bar 20 µm). m=mesoglea, nc=nucleus, ncl=nucleolus, sg=spermatogonia, st=spermatocyte, vl=vacuole.

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Figure 3. Tamoya haplonema F. Müller, 1859 male gonad from São Paulo State (Brazil). A. Live specimen (scale bar 2,5 cm) (Image courtesy of Alvaro E. Migotto). B. Male gonad in cross section stained with hematoxylin-eosin (HE) (scale bar 50 µm). C. Male gonad in cross section stained with Toluidine blue (TB) (scale bar 20 µm). D. Male gonad in cross section stained with Periodic Acid-Schiff (PAS) (scale bar 20 µm). g=gonad, m=mesoglea, mb=manubrium, nc=nucleus, p=pedalia, r=rhopalia, sc=spermatocyte, sg=spermatogonia, t=tentacle, v=velarium, vl=vacuole.

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Figure 4. Tamoya cf. haplonema from New Jersey State (USA). A-D. Male gonad in cross section stained with hematoxylin-eosin (HE) (scale bars 50 µm). E. Male gonad in cross-section stained with Gomori´s Trichrome + Hematoxylin (TG-H) (scale bar 50 µm). F. Male gonad in cross-section stained with Toluidine blue (TB) (scale bar 20 µm). G. Male gonad in cross-section stained with HE (scale bar 20 µm). Broad arrows indicate the sperm release. sc=spermatocyte, sg=spermatogonia, sp=sperm.

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Figure 5. Chiropsalmus quadrumanus (F. Müller, 1859) from São Paulo State (Brazil). A. Live specimen (scale bar 4 cm) (Image courtesy of Alvaro E. Migotto). B. Male gonad stained with hematoxylin-eosin (HE) (scale bar 50 µm). C. Male gonad stained with Periodic Acid-Schiff + Hematoxylin (PAS-H) (scale bar 20 µm). D. Male gonad in cross- section stained with Gomori´s Trichrome + Hematoxylin (TG-H) (scale bar 20 µm). g=gonad, gs=gastric saccules, m=mesoglea, nc=nucleus, p=pedalia, r=rhopalia, sc=spermatocyte, sd=spermatid, sg=spermatogonia, sp=sperm, t=tentacle, v=velarium.

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Figure 6. Chiropsalmus quadrumanus (F. Müller, 1859) (from North Carolina State (USA). A. Live specimen (scale bar 4 cm) (Image courtesy of Bastian Bentlage). B. Male gonad in cross-section stained with Toluidine Blue (TB) (scale bar 20 µm). C. Male gonad in cross-section stained with Periodic Acid-Schiff + Hematoxylin (PAS-H) (scale bar 50 µm). cl=cilia, m=mesoglea, p=pedalia, r=rhopalia, sc=spermatocyte, sg=spermatogonia, t=tentacle, v=velarium, vl=vacuole.

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Figure 7. Cubozoan phylogenetic relationships according to Bentlage et al., (2010), with optimization of different aspects of male gonads of species described herein and in García-Rodríguez et al., 2015 (scale bars 50 µm). The question mark indicates uncertainty about the reproductive mode..

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Tables Table 1. General data of specimens of Alatina alata (Reynaud, 1830) and Copula sivickisi (Stiasny, 1926) analyzed in this study. n/a: not analyzed; SP: São Paulo; *: most valuable and rare.

Number Observations Total of BH BW Species Sex Body Part Notes Locality Date coll. Coll. Collection (field and lab Det. specimens specimens (mm) (mm) codes and notes) analyzed / samples CM_1M; Top, middle and bottom Pescara 1 Male Gonads 2009 C. Camilo USNM USNM 1155726 B. Bentlage, 22,4 15 of the gonads (Italy) Carybdea 2 marsupialis LIFE+ LIFE+ Top, middle and bottom Denia 1 Male Gonads Aug/11 CUBOMED LEM CM_2M CUBOMED 21 14 of the gonads (Spain) project project

Top, middle and bottom São Sebastião, SP J. García- CH1, J. García- 2 Males Gonads June/14 LEM 40 40 of the gonads (Brazil) Rodríguez CH4 Rodríguez Chiropsalmus 3 quadrumanus Wrightsville Top, middle and bottom CQ_1M; 1 Male Gonads Beach, North 2007 B. Bentlage USNM B. Bentlage 50 43 of the gonads USNM 1124254 Carolina (USA) Uno Port, Morbakka MV_1M_T, Top, middle and bottom Okayama virulenta 1 Male Gonads Oct/09 S. Toshino LEM MV_1M_M, S. Toshino 150 140 1 of the gonads Prefecture MV_1M_B (Japan) Top, middle and bottom Ubatuba, SP TH1 85 35 2 Males Gonads July/12 A. Morandini LEM A. Morandini of the gonads (Brazil) TH4 110 45

Tamoya Top, middle and bottom São Sebastião, SP J. García- J. García- 1 Male Gonads June/14 LEM TH7 110 45 haplonema of the gonads (Brazil) Rodríguez Rodríguez 4

Barnegat Bay Top, middle and bottom Island, New J. Meany, A. A. Collins 1 Male Gonads Dec/14 USNM USNM 1110863 100,5 50 of the gonads Jersey X. Paul (USA)

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Table 2. Staining methods times used in hystoresin for 3 µm section to morphology and 5 µm sections to histochemistry.

Staining methods Dyes Minutes

TB 1,3 Toluidine Blue (TB) Distilled water Was

H at 37°C 20

Tap water 3

Distilled water Wash

Hematoxylin-Eosin 3 µm sections µm 3 (HE) E at 37°C 5 Distilled water 2 min x 3 Alcohol 70% Differentiate Distilled water Wash

Periodic Acid 15

Distilled water 2 min x 3 Schiff 30 Periodic Acid-Schiff Tap water 5 (PAS) Distilled water Wash

H at 37°C (optional) 10 Tap water 3 Distilled water Wash

TG 15

Distilled water Was Gomori´s Trichrome

(TG) Acetic acid 0,5 % Differentiate 5 µm sections µm 5 Distilled water Wash

AB 10

Tap water 3

Distilled water Wash Alcian Blue pH 2.5 (AB) H at 37°C (optional) 10

Tap water 3

Distilled water Wash

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Table 3. Comparing the reproductive characters in Carybdeida specimens analyzed in this study and in García-Rodríguez et al. 2015. n.d: no data

Gonads Mating behaviour Type of fertilization Embryo strand Sources

8 narrow leaf-like gonads extend in pairs Arneson & Cutress, Alatina alata within the gastric pockets, along either Internal (sperm released into the 1976; Gershwin, (Reynaud, 1830) side of interradial septa; filled with Spawning aggregation water and taken up by females). - 2005; Lewis et al., Alatinidae developing sperm or eggs in mature Females release blastulae 2013 females and males

Internal (within the gastrovascular spaces of the Studebaker, 1972; Carybdea marsupialis female). Within minutes fertilized Cutress, 1972; (Linné, 1758) 2 leaf-like gonads per quadrant Spawning aggregation - ova are released and in 24 hours Corbelli et al., 2003; Carydeidae they develop into swimming Gershwin, 2005

planulae

Hartwick, 1991; Lewis During courtship in spawning aggregation, Commences Sexual dimorphism within the gonads: & Long, 2005; males attach a tentacle to with transfer of the eggs females display two leaf-like gonads per Gershwin, 2005; Copula sivickisi the female and the female is brought in from the gastric pockets Carybdeida quadrant; Internal with spermatophore Lewis et al., 2008; (Stiasny, 1926) contact with the oral opening of the male through the gastric ostia, male gonads are in the form of two transfer Straehler-Pohl et al., Tripedaliidae bell; the male then passes over the openings of the orange-tinted sperm-filled hemigonads 2014; Toshino et al., a spermatophore to the female, which spermathecae and into per quadrant 2014; Garm et al., inserts it into her manubrium the stomach 2015

Morbakka virulenta 2 leaf-like Bentlage & Lewis, (Kishinouye, 1910) gonads in mature individuals per Spawning aggregation External - 2012; Toshino et al., Carukiidae quadrant 2013

Tamoya haplonema One half-gonad on each side of radial F. Müller, 1859 sinus; lamellar with frilled edges, when Spawning aggregation n.d. - Gershwin, 2005 Tamoyidae mature each extending to meet next

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Table 4. Comparing male gonad characters of specimens analyzed in this study and in García-Rodríguez et al. 2015.

Fibrous Spermatic Developmental connection Spermatic follicles follicles gradient of Gastrodermal Lumen Species Nematocyst Spermatozoa Sperm release between (Compartmentalized) development spermatogenesis Epithelium visible spermatic along the gonad within the gonad follicles

Multiple random Alatina alata No No Homogeneous Yes No Simple cuboidal and lateral rupture No No sites

Carybdea Immature Yes No Homogeneous Yes Simple columnar - Yes No marsupialis specimen

Chiropsalmus Yes No Homogeneous Yes Yes Simple columnar - Yes No quadrumanus

Towards the Simple One lateral rupture Copula sivickisi Yes Yes central region of Yes Yes Yes Yes squamous site the hemigonad Morbakka Immature Yes No Homogeneous Yes Simple columnar - Yes No virulenta specimen Multiple rupture sites, one per Tamoya Yes No Homogeneous Yes Yes Simple columnar spermatic follicle Yes No haplonema (in specimen from New Jersey)

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CAPÍTULO 4. Internal fertilization and sperm storage in cnidarians: a response to Orr and Brennan

1,2 1 3,4 Antonio Carlos Marques , Jimena García , and Cheryl Lewis Ames

1Institute of Biosciences, University of São Paulo, R. Matão Tr. 14, 101, 05508-090, São Paulo, Brazil 2Center for Marine Biology, University of São Paulo, Manoel Hypólito do Rego, km. 131,5, 11600-000, São Sebastião, Brazil 3Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington DC, 20013-7012, USA 4BEES Concentration, Biological Sciences Graduate Program, University of Maryland, College Park, MD 20742, USA

Orr and Brennan (2015) recently presented a synthesis of the diversity of sperm storage structures used in internal fertilization in animal taxa and speculated on the selective processes shaping their evolution. Orr and Brennan restrict their discussion of animals with internal fertilization and some type of sperm storage to terrestrial and marine or freshwater benthic and/or nektonic habitats, although they acknowledged that some ctenophores have internal fertilization (see Box 2 in Orr & Brennan, 2015). While we acknowledge that such restrictions are necessary in a short article, we would like to draw attention to a remarkable example that broadens the discussion of ecological and evolutionary variables connected to adaptation in an interesting and informative way. Here, we supplement Orr and Brennan’s (2015) work with information on internal fertilization and sperm storage structures in basal, planktonic cnidarians. Planktonic organisms exhibit low probability for random fertilization. The likelihood of success during external fertilization increases with higher numbers of gametes, synchronous spawning, and dense populations (generally the result of extensive asexual reproduction); all variables demand high energetic costs for the fertilization process. However, basal animals, such as medusozoan cnidarians (i.e., jellyfish) in which plesiomorphic external fertilization would be expected in adult medusae (Marques & Collins, 2004), offer several interesting examples of disparate jellyfish species exhibiting internal fertilization. The reduction of the sexual medusa to gonophores that remain attached to benthic polyps is a common strategy found in many phylogenetically distantly related hydrozoan clades (Cartwright & Nawrocki,

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 4

2010). Internal fertilization and larval brooding have also been documented in many scyphozoans (Schiariti et al., 2012). But no other early diverging lineage rivals the complexity of sexual reproductive behavior attained in certain cubozoans (i.e., venomous box jellyfish). While their plesiomorphic reproductive mode is external fertilization (e.g., in Chironex fleckeri) (Yamaguchi & Hartwick, 1980), there is evidence for an evolutionary transformation to the derived strategy of internal fertilization (Bentlage et al., 2010). Cubozoan species such as the carybdeid Carybdea marsupialis and the alatinid Alatina alata are ovoviviparous, a reproductive mode in which ova are internally fertilized by sperm taken up by the female medusae during spawning aggregations with males (Arneson & Cutress, 1976). At the climax of complex cubozoan sexual reproduction is the elaborate behavior seen only in species of Tripedaliidae – Copula sivickisi and Tripedalia cystophora. These tiny box jellyfish display sexual dimorphism and courtship behavior in which spermatophores are formed by coalescing sperm packets ejaculated from ‘seminal receptacles’ of the male testes (called hemigonads) (Werner, 1973; Hartwick, 1991; Stewart, 1996; Lewis & Long, 2005). Testes are depleted with successive ejaculations during breeding experiments, becoming visibly paler, but sperm content is replenished the next day for subsequent copulations. During pair formation the male uses his tentacles to control the female (Figure 1), bringing her in close to transfer the spermatophore that she invariably ingests (Figure 2). The sperm bundle is accommodated into her gastrovascular cavity (within several hours), which connects the stomach and specialized structures reported to function in sperm storage (Werner, 1973; Stewart, 1996; Lewis & Long, 2005). In C. sivickisi both polyandry and polygyny are pervasive (Lewis & Long, 2005), suggesting sperm competition and opportunities for postcopulatory female choice. Sperm is pigmented (orange or red) (Figure 1) providing an observable cue when sperm disperses from the stomach into the gastrovascular cavity, facilitating internal fertilization within hours to days, after which time the female deposits an embryo strand (unique among cnidarians) onto the substrate (Hartwick, 1991; Lewis & Long, 2005). One documented change induced by copulation in C. sivickisi is the presence of conspicuously dark pigmented velarial spots in sexually active females, which are absent in juvenile females and all males (Lewis & Long, 2005). Histological sectioning of female ovaries in C. sivickisi reveals a development gradient (Lewis & Long, 2005), and the fact that females accept sperm from males during gestation and after giving birth (i.e., embryo strand release) suggests they might store sperm from multiple copulations for delayed fertilization. The sperm storage index for C. sivickisi, following Orr and Brennan (2015), is estimated to be 3, exceeding estimates for sharks, earthworms, and even marsupial mammals (see Box 4

García Rodríguez, Jimena. Dissertação de Mestrado, 2015 Capítulo 4 in Orr & Brennan, 2015). It is clear that selective pressures to attain highly specialized internal fertilization (and corresponding sperm storage structures) resulted in several different and sometimes homoplastic strategies in animals. However, we believe that it is important to emphasize that in the case of tripedalid box jellyfish, selective pressures must have acted on both males and females (i.e., coevolution between the sexes in relation to sperm storage) to enhance fertilization efficiency. This is best put into perspective when recognizing that these morphologically rather simple basal animals inhabit the planktonic realm – a habitat in which organisms are driven by the currents, and, by definition, have incipient or no swimming capacity, drifting almost randomly with little control over mate choice. To increase our understanding of the evolutionary diversity of sperm storage adaptations, we must examine sexual selection in male and female medusozoans from a combined ecological, phylogenetic, and physiological perspective.

References Arneson, A. C., & C. E. Cutress, 1976. Life history of Carybdea Reynaud, 1830 (Cubomedusae). In Mackie, G. O. (Ed.), Coelenterate Ecology and Behavior. New York, NY: 227–236.