conformação gonadal, caracterização histoquímica e fileconformação gonadal, caracterização...

Conformação gonadal, caracterização histoquímica e

ultraestrutural da gônada masculina e

espermatozoides em espécies de águas-vivas

(Cubozoa e Scyphozoa, Medusozoa, Cnidaria)

Gonadal structure, histochemistry and ultrastructural

characterization of male gonad and sperm of

jellyfish species (Cubozoa and Scyphozoa,

Medusozoa, Cnidaria)

Gisele Rodrigues Tiseo

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. André Carrara Morandini

São Paulo

2016

Comissão Julgadora

Tiseo, Gisele Rodrigues

Conformação gonadal, caracterização histoquímica e

ultraestrutural da gônada masculina e espermatozoides em espécies de

águas-vivas (Cubozoa e Scyphozoa, Medusozoa, Cnidaria)

148 páginas

Dissertação (Mestrado) - Instituto de Biociências da

Universidade de São Paulo. Departamento de Zoologia.

1. Scyphozoa 2. Cubozoa 3. Espermatogênese

I. Universidade de São Paulo. Instituto de Biociências.

Departamento de Zoologia.

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

Prof. Dr. André Carrara Morandini

Agradecimentos

Agradeço a agência de fomento FAPESP (Fundação de Amparo a Pesquisa do

Estado de São Paulo Proc. MS 2014/08785-0, Proc. BEPE 2015/15527-0 e Proc.

Temático 2011/50242-5) pelo auxílio financeiro concedido para o desenvolvimento

desta pesquisa.

Ao Departamento de Zoologia, Instituto de Biociências, Instituto Oceanográfico,

CEBIMar e Universidade de São Paulo pela infra-estrutura utilizada.

Ao meu orientador Prof. Dr. André Carrara Morandini pela amizade, apoio,

sugestões e compreensão ao longo desses dois anos de trabalho.

Ao Prof. Dr. Peter L. Harrison, da Southern Cross University, Austrália, por ter

me recebido em seu laboratório e grupo de pesquisa e auxiliado no desenvolvimento do

projeto BEPE. Obrigada pela oportunidade de conhecer novas fronteiras e pelo

intercâmbio de conhecimento.

Agradeço de todo o coração aos técnicos Lea Taylor, Paul Kelly, Maxine Daves

e Barbara Harrison pela amizade e auxílio nas coletas, preparações das amostras e

infinitas conversas. Obrigada por serem minha pequena “família” no exterior.

Ao Prof. Jamie Seymour por ter me recebido em seu laboratório na James Cook

University em Cairns, Austrália. Agradeço pelo auxilio nas coletas e troca de

conhecimento.

Aos alunos de pós-graduação do Prof. Jamie Seymour – Robert Courtney e Sally

Browning pela ajuda nas coletas em Queensland e curta convivência.

Ao Prof. Dr. Alberto Ribeiro responsável pelo Laboratório de Microscopia

Eletrônica do Instituto de Biociências.

Aos integrantes do Laboratório de Microscopia Eletrônica do Departamento de

Genética – Camila e Alexandre – pelas disucssões produtivas e dicas de terminologias.

Aos técnicos Márcio Valentim Cruz e Sheila Carmo pelos cafés e amizade. E ao técnico

de microscopia eletrônica de transmissão Waldir Caldeira pela amizade e auxílio em

todas as preparações de ultraestrutura.

Aos técnicos do Laboratório de Histologia do Departamento de Zoologia, Enio

Mattos e Phillip Lenktaitis obrigada pela amizade e auxilio nas preparações de

histologia.

Aos integrantes do Laboratório de Cultivo e Estudos em Cnidaria – Júlia Beneti,

Renato Nagata, Henrique Alves, Clarissa Garbi Molinari, Priscila Cunha, Leandro

Santos, Max Maronna, Mayara Jordão, Edgar Gamero, Jonathan Lawley e Flávia

Murari – pela convivência, amizade, ajudas em coletas, discussões produtivas e apoio.

Muito obrigada por tudo!

Aos meus amigos (e respectivos) que sempre estão (e estarão) lá por mim –

Jaqueline Mariano, Bruna Gasbarra, Laianne Mendes, Roberto Radesca, Jefferson

Platini, Pedro Henrique, Renata Bannitz, Carla Peres de Paula, Luiza Saad, Bruna

Trevisan, Amanda Yano, Thalma e integrantes da República SF. Muito obrigada pela

amizade e suporte!!!

Aos amigos do Departamento de Zoologia e do Curso de Verão em Zoologia –

Rachel, Rafael Henrique, Adriana, Mariane, Carol, Guilherme, Alipio, Ana Bolatto,

Caio’s, Gabriel, Isabela, Jorge, Ligia, Marilia, Amanda, Pedro’s, Stefania, Thalles,

Victor, Loboda, Licia, Kleber, Karla Soares, Karla Paresque, Carolina Nisa, Anderson

Tama, Flávia Pentean e Juliana Bertacini – obrigada pela amizade, convivência e

risadas!

Agradeço ao meu namorado – Lucas Nastri – pelo apoio, cumplicidade e

compreensão (mesmo nas épocas malucas de entrega de relatório). Obrigada por estar

presente mesmo à distância e me fazer feliz.

Por fim, agradeço à minha família – Marcos, Marli e Jessica – sem vocês nada

em minha vida faria sentido e nada seria possivel. A realização de mais este trabalho so

foi possivel graças ao eterno apoio e suporte que vocês me proporcionam diariamante.

Amo vocês incondicionalmente e agradecerei por fazer parte desta familia hoje e

sempre!

Sumário

Introdução Geral ............................................................................................................................ 8

Ciclo de Vida, Reprodução sexuada e descrição do Sistema reprodutor masculino ............... 10

Espermiotaxonomia ................................................................................................................. 13

Referências .............................................................................................................................. 17

Capítulo 1 .................................................................................................................................... 27

Spermatogenesis in the cubozoans Tamoya haplonema Müller, 1859 and Chiropsalmus

quadrumanus (Müller, 1859), highlighting the gonadal cycle .................................................... 27

Abstract ................................................................................................................................... 28

Introduction ............................................................................................................................. 29

Material and Methods ............................................................................................................. 31

Results ..................................................................................................................................... 32

Discussion ............................................................................................................................... 35

Acknowledgements ................................................................................................................. 39

References ............................................................................................................................... 39

List of Figures ......................................................................................................................... 48

Capítulo 2 .................................................................................................................................... 54

Spermatogenesis and sperm morphology in cubozoans from SE Brazilian and Australian coasts

..................................................................................................................................................... 54

Abstract ................................................................................................................................... 55

Introduction ............................................................................................................................. 56


Results ..................................................................................................................................... 61

Discussion ............................................................................................................................... 67

Acknoledgements .................................................................................................................... 71

References ............................................................................................................................... 71

List of Figures ......................................................................................................................... 83

Capítulo 3 .................................................................................................................................... 94

Histochemistry and ultrastructure of spermatogenesis in Chrysaora lactea, Lychnorhiza lucerna

and Cassiopea sp. ........................................................................................................................ 94

Abstract ................................................................................................................................... 95

Introduction ............................................................................................................................. 96


Results ................................................................................................................................... 100

Discusssion ............................................................................................................................ 103

Acknowledgement ................................................................................................................. 106

References ............................................................................................................................. 106

List of Figures ....................................................................................................................... 116

Capítulo 4 .................................................................................................................................. 124

Considerações Finais ................................................................................................................. 124

Resumo ...................................................................................................................................... 129

Abstract ..................................................................................................................................... 131

Apêndice I - Protocolo de Fixador para Histologia ................................................................... 133

Apêndice II - Protocolo de Histologia em Historesina .............................................................. 135

Apêndice III - Técnicas Histológicas e Histoquímicas de Colorações ...................................... 138

Apêndice IV - Protocolos dos Fixadores para Microscopia Eletrônica de Transmissão........... 143

Apêndice V- Protocolo de Microscopia Eletrônica de Transmissão ......................................... 146

8

Introdução Geral

O filo Cnidaria é considerado um grupo monofilético composto por dois grandes

clados, Anthozoa e Medusozoa (Collins 2002; Collins et al. 2006), sendo esta divisão

bem suportada por caracteres morfológicos e moleculares (Schuchert 1993; Bridge et al.

1995; Odorico and Miller 1997; Collins 2002; Daly et al. 2007; Kayal et al. 2013;

Zapata et al. 2015). Seus representantes formam um grupo diverso de animais

relativamente simples caracterizados pela presença da cnida (Odorico and Miller 1997;

Collins 2002; Daly et al. 2007; Collins 2009; Reft and Daly 2012) e diversidade do

ciclo de vida com a presença ou ausência da alternância de gerações (Bridge et al.

1995).

A filogenia dos diferentes grupos de Cnidaria ainda permanece de certa forma

pouco resolvida, uma vez que não há um consenso de quais são as relações entre as

diferentes classes e ordens que compõem o filo. Os problemas classificatórios em

Cnidaria têm atraído o interesse de zoólogos (Werner 1973a) e são diversos os trabalhos

que apresentam teorias e hipóteses filogenéticas tentativas para classificar o grupo. As

principais divergências concernem na problemática sobre: a diversificação dos ciclos de

vida e qual dos dois estágios ou formas corporais (pólipo ou medusa) seria o mais basal

(Werner 1973a; Werner 1973b; Bridge et al. 1992; Schuchert 1993; Bridge et al. 1995;

Odorico and Miller 1997); na relação entre as quatro das atuais classes de Medusozoa

(Hydrozoa, Scyphozoa, Cubozoa e Staurozoa); e entre os subgrupos de Hydrozoa e

ordens de Scyphozoa (Marques and Collins 2004; Dawson 2004; Collins et al. 2006;

Daly et al. 2007; Kayal et al. 2012; Kayal et al. 2013; Zapata et al. 2015).

Kayal et al. (2013) ampliaram a amostragem dos genomas mitocondriais já

apresentados para Cnidaria a fim de reavaliar as relações filogenéticas dentro do filo.

Suas análises, baseadas em dados de mitogenômica apoiaram relações já propostas,

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como o monofiletismo do subfilo Medusozoa, das classes Cubozoa, Staurozoa e

Hydrozoa, da subclasse Discomedusae e das ordens Carybdeida e Chirodropida. Porém,

certos agrupamentos propostos anteriormente foram refutados como Scyphozoa uma

vez que Discomedusae se agrupa com Hydrozoa, e os demais grupos de Medusozoa que

não formam nenhum grupo observado em trabalhos anteriores. Embora os autores

apresentem a segunda filogenia mais atual de Cnidaria, esta ainda apresenta

inconsistências, levando os próprios a afirmarem que as relações mostradas entre os

diferentes táxons que compõem o filo são hipotéticas e sujeitas a testes. Deste modo,

estudos específicos nos diferentes grupos podem promover uma melhor compreensão

das relações entre os Cnidaria como um todo.

Existem aproximadamente 200 morfoespécies descritas de Scyphozoa ao redor

do mundo (Daly et al. 2007). Destas, 22 espécies são registradas no Brasil (Mianzan and

Cornelius 1999; Marques et al. 2003; Morandini et al. 2005) e 38 na Austrália

(Gershwin et al. 2010). A Classe Scyphozoa compreende dois grupos: as subclasses

Coronamedusae (ordem Coronatae) e Discomedusae (ordens Semaeostomeae e

Rhizostomeae) (Calder 2009). A classe distingue-se dos demais Medusozoa pela

presença da estrobilização, órgãos sensoriais marginais (ropálios) e o estágio de éfira

(Marques and Collins 2004; Collins et al. 2006; Daly et al. 2007).

Dentro de Cnidaria, a classe Cubozoa é a que possui menor número de espécies

descritas, 36 (Daly et al. 2007). As cubomedusas são nadadoras ágeis e extremamente

eficientes (Collins et al. 2011; Colin et al. 2013) caracterizadas por exibirem quatro

ropálios perradiais, contendo olhos complexos com ocelos, corpos vítreos, lentes,

córneas e retinas e, em cada um dos cantos da umbrela nota-se uma extensão espessa da

margem denominada pedálio (Pearse and Pearse 1978; Daly et al. 2007). A classe é

10

dividida em duas ordens Chirodropida e Carybdeida sendo que nesta última, cada

pedálio porta somente um único tentáculo (Bentlage and Lewis 2012).

No Brasil as espécies de Scyphozoa Lychnorhiza lucerna (Rhizostomeae) e,

Chrysaora lactea (Semaeostomeae) são abundantes (Morandini et al. 2005; Nogueira

Júnior et al. 2010). As cubomedusas Tamoya haplonema (Carybdeida) e Chiropsalmus

quandrumanus (Chirodropida) possuem abundância menor, mas são relativamente

comuns (Nogueira Júnior et al. 2010).

Na Austrália as espécies do gênero Cassiopea normalmente são encontrados

com a umbrella apoiada no substrato e a superficie oral para cima (Bigelow 1900;

Morandini et al. 2016b) e podem ser encontradas em Lizard Island lagoon e em

Vlashoff Cay (Cairns) (Templeman and Kingsford 2010; Templeman and Kingsford

2012). As cubozoas Carukia barnesi, Chironex fleckeri e Chiropselaa bronzie são

relativamente abundantes junto a Grande Barreira de Recifes de Coral em Queensland,

leste da Austrália (Williamson et al. 1996; Fenner and Hadok 2002; Tibballs 2006;

Gershwin and Kingsford 2008; Kingsford et al. 2012).

Ciclo de Vida, Reprodução sexuada e descrição do Sistema reprodutor masculino

O ciclo de vida metagenético ocorre em quatro das cinco classes de Cnidaria:

Hydrozoa, Scyphozoa, Cubozoa e Staurozoa (Bridge et al. 1995; Odorico and Miller

1997; Marques and Collins 2004; Morandini et al. 2016a). De um modo geral o ovo

fertilizado se desenvolve em uma larva plânula que ao assentar se diferencia em um

pólipo. O pólipo – estágio bentônico – se reproduz assexuadamente dando origem a

uma ou mais medusas livre-natantes dióicas, que quando adultas se reproduzem

sexuadamente (Morandini et al. 2014). Em Scyphozoa o pólipo é denominado cifístoma

e pode reproduzir-se assexuadamente originando outros pólipos (brotamento ou

11

formação de cistos) ou originar uma ou mais medusas jovens através de fissões

transversais (estrobilização) (Arai 1997; Adler and Jarms 2009). A estrobilização

tradicionalmente é caracterizada como sendo polidisco – com a formação de muitos

discos e várias medusas; ou monodisco – em que somente uma éfira é formada por vez

(Bigelow 1900; Morandini et al. 2004; Holst et al. 2007; Schiariti et al. 2008). As

ordens Coronatae e Semaeostomeae apresentam um padrão polidisco e Rhizostomeae

um padrão monodisco. No entanto, estudos sobre o ciclo de vida de algumas espécies

ressaltam que nem todos os membros de Rhizostomeae estrobilizam no padrão

monodisco e eram denominados polidisco (e.g. tabela 1 de Schiariti et al. 2008). Apesar

de o número de éfiras ser maior que um, ele pode ser considerado baixo (2-10). Com

isso alguns autores sugerem a utilização de um termo adicional para melhor definir esse

processo encontrado: oligodisco (Fuentes et al. 2011).

Dentro de Scyphozoa, das cerca de 200 espécies descritas, 49 tem seu ciclo de

vida conhecido (24,5%; Jarms 2010) e destes 47 são metagenéticos. O ciclo de vida de

L. lucerna, Cassiopea sp. e C. lactea seguem o padrão para Scyphozoa. A estrobilização

polidisco produz sempre três éfiras em L. lucerna e de 2 a 10 éfiras em C. lactea, e

sempre é monodisco em Cassiopea sp. (Bigelow 1900; Morandini et al. 2004; Schiariti

et al. 2008).

O ciclo de vida dos cubozoários T. haplonema e C. quadrumanus ainda não foi

descrito. Porém, para os cubozoários Chironex fleckeri e Carukia barnesi Southcott

(1956), Yamaguchi and Hartwick (1980) e Courtney et al. (2016) descrevem o ciclo de

vida enfatizando o padrão metagenético e evidenciando diferenças no processo

assexuado em que um pólipo se metamorfoseia em uma única cubomedusa (Werner

1973a; Werner 1973b; Werner 1975; Straehler-pohl and Jarms 2011).

12

O gonocorismo ocorre na maioria dos cifozoários (Berrill 1949) e Cubozoa é a

única classe exclusivamente gonocórica (Fautin 1992). No entanto, espécies de

cifomedusas como Chrysaora hysoscella e do gênero Cassiopea são conhecidas por

apresentarem hermafroditismo e gonocorismo (Berrill 1949; Hofmann and Hadfield

2002). Para a maioria dos cnidários dióicos, indivíduos de sexos diferentes são

macroscopicamente indistinguíveis, porém algumas características distintivas

relacionadas à diferenciação sexual podem estar presentes em indivíduos maduros: são

as próprias gônadas ou estruturas associadas a elas (Fautin 1992). Em alguns casos em

Scyphozoa e Cubozoa as gônadas podem ser diferenciadas quando maduras pela

coloração (Werner 1973a; Fautin 1992; Lucas and Reed 2010; Iguchi et al. 2010;

Schiariti et al. 2012). Na espécie L. lucerna os ovários normalmente possuem coloração

marrom claro ou escuro e os testículos coloração esbranquiçada ou esverdeada (Schiariti

et al. 2012). A espécie C. lactea apresenta variação na coloração gonadal podendo ser

esbranquiçada, marrom-amarelada ou rosa pálido (Morandini and Marques 2010), sem

evidenciar um padrão de cor por sexo. Para Cubozoa, Werner (1973b) afirma que as

colorações gonadais dos dois sexos diferem quando observadas através de finos tecidos

transparentes de algumas medusas.

O ‘órgão’ reprodutivo dos cnidários é descrito como um espaço preenchido com

células germinativas em desenvolvimento que migraram da endoderme ou ectoderme

para a mesogléia (Miller 1983). Para Scyphozoa, Anthozoa e Cubozoa o testículo é

originado de células intersticiais da gastoderme, as quais migram para o interior da

mesogléia originando os folículos testiculares. No interior dos folículos ocorrem os

processos de espermatogênese e espermiogênese, sendo os espermatozoides maduros

liberados (Conant 1898; Miller 1983; Harrison and Jamieson 1988). A classe

Scyphozoa apresenta duas estratégias de liberação dos espermatozoides: livres ou em

13

grupos e empacotados. Os espermatozoides livres são liberados na água via poro

localizado na gastroderme (Tiemann and Jarms 2010) ou por ruptura da parede do

folículo (Morandini and Silveira 2001; Ikeda et al. 2011). Os espermatozoides também

podem estar agrupados e serem liberados em pacotes (spermatozeugmata) (e.g.

Kikinger 1992). Em Cubozoa, são poucos os trabalhos abordando o processo de

espermatogênese e a estratégia de liberação do espermatozoide. Werner (1973b)

descreveu spermatozeugmata para a espécie T. cystophora e García-Rodriguez (2015)

para a espécie Alatina alata e alguns autores reportam a presença de espermatóforos em

Copula sivickisi (Hartwick 1991; Straehler-Pohl et al. 2014; Marques et al. 2015).

García-Rodriguez (2015) descreve o sistema reprodutor masculino de C. quadrumanus

e T. haplonema, caracterizando a estrutura gonadal macroscópica, a conformação geral

e o epitélio gonadal, porém não descreve o processo de espermatogênese para as

espécies do litoral brasileiro, uma vez que somente encontrou espécimes imaturos, não

sendo possível identificar todas as figuras celulares características do processo de

espermatogênese. Porém, reporta o tipo de estratégia de liberação para T. haplonema

(espécime de New Jersey) como sendo por ruptura do epitélio folicular.

Adicionalmente, a autora também descreve a morfologia gonadal para as cubomedusas

Morbakka virulenta, Alatina alata e Copula sivickisi, descrevendo a estratégia de

liberação dos espermatozoides e alguns aspectos reprodutivos para estas ultimas

espécies. Domo mesmo modo, Southcott (1956) descreve a organização gonadal para

Chironex fleckeri evidenciando certa sazonalidade graus de maturação gonadal.

Espermiotaxonomia

A espermiotaxonomia vem sendo utilizada como critério de suporte para

inferências filogenéticas por diversos autores em vários grupos dentro de Metazoa

14

(Franzén 1956) como Trematoda – Platyhelminthes (Jamieson and Daddow 1982),

Polychaeta e Oligochaeta – Annelida (Jamieson 1983; Rota and Lupetti 1997),

Decapoda – Crustacea (Jamieson 1994; Guinot et al. 1997; Benetti et al. 2008; Buranelli

and Mantelatto 2012) e Gastropoda e Bivalvia – Mollusca (Introíni et al. 2013; Giménez

2013). Em Cnidaria trabalhos como os de Miller (1983) e Harrison and Jamieson (1988)

apresentam, em sua revisão, a morfologia geral da ultraestrutura do espermatozoide de

todas as classes exceto Cubozoa.

Franzén (1956) propôs que há uma relação definida entre a morfologia do

espermatozoide e a biologia de fertilização. Porém o autor não faz uma descrição

ultraestrutural dos espermatozoides, sendo as comparações feitas com base em

observações em nível de microscopia de contraste de fase. De acordo com o autor foi

proposto que espermatozoides com cabeça arredondada, acrossomo esférico, peça

intermediária pequena, poucas (quatro) mitocôndrias, flagelos livres e longos, são

características “primitivas”. Este é geralmente associado com a ocorrência de

fertilização externa. Espermatozoides com morfologia da cabeça alongada e peça

intermediária com mitocôndrias em espiral são classificados como modificados, sendo

associados à fecundação interna. Porém, Rouse and Pitt (2000) citam que, em Cnidaria,

talvez esta relação esteja equivocada, uma vez que vários espermatozoides que se

enquadram na morfologia “primitiva” apresentam fecundação interna ou vice-versa.

No nível de microscopia de luz pode-se observar a morfologia geral do

espermatozoide de Cnidaria é composto por cabeça redonda-ovalada, cônica ou

alongada, peça intermediária visível com um flagelo ancorado à parte posterior (Franzén

1956; Harrison and Jamieson 1988). Já ao microscópio eletrônico de transmissão

observa-se certa simetria na cabeça do espermatozoide, com a presença do núcleo e de

pequenas vesículas situadas na região anterior deste, que podem ser precursoras da

15

vesícula acrossomal existente nos espermatozoides da maioria dos metazoários (Hinsch

and Clark Jr. 1973; Hinsch 1974; Miller 1983; Harrison and Jamieson 1988). A peça

intermediária é pequena geralmente com quatro ou mais mitocôndrias ao redor de um

par de centríolos, o proximal e o distal. É a partir do centríolo distal que se origina o

longo flagelo típico no espermatozoide da maioria dos metazoários, contendo o arranjo

usual de microtúbulos (9 + 2) e vários elementos acessórios, compondo o axonema

(Miller 1983; Harrison and Jamieson 1988; Corbelli et al. 2003).

De acordo com Harrison and Jamieson (1988) são poucas as informações

ultraestruturais do espermatozoide em Cnidaria sendo a maioria dos estudos realizados

nas classes Anthozoa (97 espécies) e Hydrozoa (31 espécies). Em Scyphozoa o autor

informa sobre a descrição ultraestrutural de espermatozoides de nove espécies,

incluindo um da ordem Coronatae, cinco da ordem Semaeostomeae e três

Rhizostomeae. Porém, atualmente sabe-se que esse número aumentou para pelo menos

12 espécies incluindo cinco Rhizostomeae, cinco Semaeostomeae e dois Coronatae

(Afzelius and Franzén 1971; Hinsch and Clark Jr. 1973; Hinsch 1974; Rouse and Pitt

2000). Para Cubozoa, Werner (1973a) faz uma descrição generalizada do

espermatozoide de Trypedalia cystophora e Corbelli et al. (2003) descrevem a

ultraestrutura do espermatozoide de Carybdea marsupialis, sendo somente estes dois

trabalhos referentes a classe.

Em Discomedusae, o espermatozoide é caracterizado por apresentar núcleo

cônico alongado, pequenas vesículas eletrondensas na cabeça e peça intermediária

sempre com quatro mitocôndrias, centríolos proximal e distal, sendo o último com

complexo pericentriolar formando um colar que envolve a porção proximal do flagelo.

A ordem Coronatae compartilha certos caracteres com Discomedusae como, por

16

exemplo, organelas idênticas na peça intermediária, mas destaca-se pela presença de um

núcleo cônico relativamente pequeno (Harrison and Jamieson 1988).

Ainda que sejam poucos os trabalhos em Cubozoa, Corbelli et al. (2003)

ressaltam possíveis autapomorfias do espermatozoide de Cubozoa como a presença de

processos intersecundários, a forma do apêndice “spur” que forma o aparato

pericentriolar, e a forma dos processos pericentriolares terciários. Porém, os próprios

autoes evidenciam que os espermatozoides de Cubozoa são parecidos com os de

Scyphozoa e Hydrozoa.

Apesar de a abordagem utilizada nesta dissertação ser a mesma para as

diferentes espécies de medusas estudadas, o detalhamento metodológico não pode ser o

mesmo para todas elas. Além disso optou-se para apresentar o texto como se fossem

artigos independentes e em formato para serem submetidos para publicação. Dessa

forma, a dissertação encontra-se dividida em 4 capítulos, a saber, Capítulo 1:

“Spermatogenesis in the cubozoans Tamoya haplonema Müller, 1859 and

Chiropsalmus quadrumanus (Müller, 1859), highlighting the gonadal cycle”,

Capítulo 2: “Spermatogenesis and sperm morphology in cubozoans from SE

Brazilian and Australian coasts”, Capítulo 3: “Histochemistry and ultrastructure

of spermatogesis in Lychnorhiza lucerna, Chrysaora lactea and Cassiopea sp.” e

Capítulo 4 “Considerações Finais”.

No Capítulo 1 descrevemos o processo de espermatogênese para os cubozoários

Tamoya haplonema e Chiropsalmus quadrumanus com base nos resultados obtidos

através da análise histológica e histoquímica. Adicionalmente, utilizando como base as

amostras gonadais em diferentes estágios de maturação gonadal, foi possível

descrevermos os diferentes estágios do ciclo gonadal dessas espécies, sendo esse o

primeiro registro detalhado nesse nível para a classe.

17

No Capítulo 2 é descrito o processo de espermatogênese utilizando técnicas

histológicas, histoquímicas e ultraestruturais para as espécies de Cubozoa Tamoya

haplonema e Chiropsalmus quadrumanus do Brasil, além das espécies Carukia barnesi,

Chironex fleckeri e Chiropsella bronzie coletadas na Austrália.

No Capítulo 3 descrevemos de modo comparativo o processo de

espermatogênese nas espécies Lychnorhiza lucerna, Cassiopea sp. e Chrysaora lactea

com base em análises histológicas, histoquímicas e ultraestruturais da gônada

masculina. Adicionalmente, também incluímos neste manuscrito a descrição

macroscópica da gônada masculina de Lychnohiza lucerna, bem como sua disposição

dentro da cavidade gastrovascular, complementando resultados previamente publicados

por Schiariti et al. (2012).

Ao final, o Capítulo 4 apresenta as considerações finais desta dissertação

comparando os dados da morfologia gonadal, histológicos e histoquímicos das espécies

de Cubozoa e Scyphozoa aqui estudadas (exemplares do Brasil e Austrália). Finalmente,

os dados descritivos do espermatozoide de todas as espécies trabalhadas forma

compilados e comparados ressaltando possíveis sinapomorfias.

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27

Capítulo 1

Spermatogenesis in the cubozoans Tamoya haplonema Müller, 1859 and

Chiropsalmus quadrumanus (Müller, 1859), highlighting the gonadal cycle

Gisele R. Tiseo1*

; Jimena Garcia-Rodriguez1; Fernando J. Zara

2; Cheryl Lewis

Ames3,4

; Antonio C. Marques1,5

& André C. Morandini1

1 Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, R.

Matão trav. 14, n.101, 05508-090, São Paulo, Brazil;

2 Departamento de Biologia Aplicada, Invertebrate Morphology Laboratory (IML),

Aquaculture Center (CAUNESP) and Instituto de Estudos Avançados do Mar

(IEAMar), Univ. Estadual Paulista (UNESP), Via de Acesso Prof. Donato Castellane,

S/N, Jaboticabal 14884-900, SP, Brazil;

3 Department of Invertebrate Zoology, National Museum of Natural History,

Smithsonian Institution, Washington DC, 20013-7012, USA;

4 BEES Concentration, Biological Sciences Graduate Program, University of Maryland,

College Park, MD 20742, USA;

5 Centro de Biologia Marinha, Universidade de São Paulo, Manoel Hypólito do Rego,

km. 131.5, 11600-000, São Sebastião, Brazil.

Running Title: Spermatogenesis of cubozoans from SE Brazil

*Corresponding author. Tel: +55 11 30917621

E-mail addresses: [email protected] (G.R. Tiseo), [email protected] (J.

García), [email protected] (F. J. Zara), [email protected] (C. Lewis),

[email protected] (A.C. Marques), [email protected] (A.C. Morandini)

mailto:[email protected]






28

Abstract

The spermatogenesis was little studied in cubozoans. Here we describe the

spermatogenesis of two Brazilian species Chiropsalmus quadrumanus and Tamoya

haplonema under light microscopy and histochemical techniques. Additionally, we

highlight aspects and stages of the gonadal cycle. Specimens were collected by hand at

the water surface or with shrimp trawls in three areas of São Paulo state (SE Brazil):

Cananéia lagoon estuarine system (south), Santos and São Vicente Bay (central), and

São Sebastião and Ubatuba (north). Samples of male gonad were excised, fixed in

paraformaldehyde 4% in sodium phosphate buffer 0,2M (pH 7, 2) by 24 hours and

posteriorly processed following protocol for historesin. The serial sections were

performed and the resulting slides were stained with hematoxylin and eosin and

techniques for proteins (mercuric bromophenol blue and ponceau xylidine), acids

materials (toluidine blue) and neutral polysaccharides (PAS). Male box jellyfish gonads

are located between the mesoglea and gastrodermis in a thin and independent layer,

organized in elongated follicles, macroscopically resembling a fingerprint pattern. The

follicle wall is composed of spermatogonia. The spermatocytes are found near the

follicle wall while spermatids and spermatozoa are located at the center. Sperm release

occurs through rupture of follicular wall in both species. We documented four gonadal

maturation stages for T. haplonema, viz. rudimentary, developing, mature, and spawned;

and three stages for C. quadrumanus, viz. rudimentary, developing, and mature. The

sperm of C. quadrumanus has a rounded head while T. haplonema has a conical shape

with an elongated projection. In both species there was no positive histochemical

reaction for the vesicles precursors of acrosome.

Key-words: Cnidaria, Cubozoa, histology, histochemistry, jellyfish.

29

Introduction

The class Cubozoa is the one with less number of described species among the

phylum Cnidaria, totaling approximately 50 species (Bentlage et al. 2010). The class is

divided in two monophyletic orders, Chirodropida and Carybdeida, whereas in the last

one, each umbranched pedalium have only one tentacle (Daly et al. 2007; Bentlage and

Lewis 2012). In Brazil there are four species of cubozoans (Mianzan and Cornelius

1999; Marques et al. 2003). The target species of this study – Tamoya haplonema

(Family Tamoyidae, Order Carybdeida) and Chiropsalmus quadrumanus (Family

Chiropsalmidae, Order Chirodropida) – are broadly distributed at the Western Atlantic

coast (Mianzan and Cornelius 1999; Migotto et al. 2002; Nagata et al. 2009).

There are some works addressing reproductive aspects of Cubozoa (Werner

1973a; Harrison and Jamieson 1988; García-Rodriguez 2015), being most of them

related to the description of life cycle, fertilization and strategies of release of gametes

(free in the water column, grouped or packed), mating behavior or gonadal macroscopic

description (Avian et al., 1993; Bentlage et al., 2010; Conant, 1898; Corbelli et al.,

2003; García-Rodriguez, 2015; Garm et al., 2015; Gershwin, 2006; Lewis and Long,

2005; Lewis et al., 2013, 2008; Marques et al., 2015; Müller, 1860; Southcott, 1956;

Straehler-Pohl et al., 2014; Thiel, 1936; Toshino et al., 2013; Werner, 1973a, 1973b).

Of all these works, Müller (1860), Thiel (1936) and Lewis and Long (2005) described

the histological structure of female gonad, and Thiel (1936), Southcott (1956), Avian et

al.(1993), Garm et al. (2015) and García-Rodriguez (2015) described the histological

structure of the male gonad (see Table 1 for details). Among Cubozoa, the

spermatogenesis was described only for few species given the difficulty in find males at

different stages of gonadal development as in Avian et al. (1993); Claus (1878) and

Southcott (1956). However a degree of gonadal maturity was already observed – as in

Chironex fleckeri Southcott (1956) and Carybdea xaymacana Conant (1898).

30

Cubozoa is a exclusively gonochoric class (Fautin 1992) and the gonads can be

differentiated by the color (Werner 1973b; Fautin 1992; Lewis and Long 2005;

Bentlage et al. 2010; Bentlage and Lewis 2012) and through the identification of a

pattern similar to a “fingerprint” (observed only in males) (Southcott 1956; Gershwin

2005). According to Gershwin (2005), all cubozoans have paired leaf-like lateral

gonads, that sometimes could overlap, and sometimes could be highly pleated. In most

carybdeids the gonads are attached along the length of the septum, having a butterfly

appearance in the tripedaliids (Gershwin 2005). In most chirodropids the lateral gonads

are broader and more developed in the upper half, and eventually the gonadal tissue

could envelop the gastric saccules – named superior gonads by Southcott (1956), and

perradial stomach pouches by Mayer (1910).

In a general way, the male gonad is originated of interstitial cells from the

gastrodermis, which migrate to the mesoglea differentiating and originating the male

follicles (Miller 1983). Inside these follicles occurs the spermatogenesis, and the sperm

release occurs in the water column by rupture of the follicle wall or transferred, in

packages (spermatozeugmata or spermatophores) to the female through mating behavior

(Conant 1898; Miller 1983; Harrison and Jamieson 1988; Lewis and Long 2005; Lewis

et al. 2008; Garm et al. 2015; Marques et al. 2015).

This work describes the spermatogenesis and the gross morphology of the sperm,

under light microscopy, for two species of different orders and families of box-

jellyfishes, viz. Chiropsalmus quadrumanus (Family Chiropsalmidae, Order

Chirodropida) and Tamoya haplonema (Family Tamoyidae, Order Carybdeida),

characterizing histochemically the sperm composition and gonadal tissues at different

degrees of gonadal maturation.

31

Material and Methods

Specimens of T. haplonema and C. quadrumanus were collected in four localities

of São Paulo state (SE Brazil) – viz. Cananéia lagoon estuarine system, Santos and São

Vicente Bay, São Sebastião and Ubatuba counties (Figure 1) – in the years 2009, 2010,

2012, 2014, and 2015 (Table 2). The exemplars were collected by hand at water surface

or with shrimp trawls, the specimens were transported alive to the laboratory in plastic

boxes and identified following Morandini et al. (2005). Samples of male gonad1 of six

individuals of each species were fixed in paraformaldehyde 4% in sodium phosphate

buffer 0,2M (pH 7,2) for 24hours. After the fixation, the samples were buffered for

more 24 hours, dehydrated in ethanol series (70-95%), embedded and included in

historesin glycolmethacrylate Leica®

. Serial cuts of 3 to 5 µm were carried in rotative

microtome and the slides were stained with hematoxylin and eosin (H&E) to traditional

histological description (Junqueira and Junqueira 1983), avoiding the ethanol and

xylene baths (Sant’Anna et al. 2010; Zara et al. 2012).The toluidine blue staining (AZT)

(Modificade de Audino et al. 2015) was used to describe spermatogenesis, the mercuric

bromophenol blue (AB) (Pearse 1960) and ponceau xylidine (Mello and Vidal 1980) to

demonstrate total proteins, and the Periodic acid of Schiff-Hematoxylin (PAS-H)

technique to demonstrate neutral polysaccharides with the groups 1-2-glycol (Junqueira

and Junqueira 1983).

To study and measure the germ cells at different spermatogenesis stages we used

preparations stained with H&E of 6 different males. In each preparation it was measured

30 nuclei of each cell type (spermatogonia, spermatocyte, spermatid and sperm) when

present. The nuclear measurements were made using the Leica® Microscope and the

software IM50 from the Laboratório de Morfologia de Invertebrados (UNESP-

1 Even the Cnidarians being the first animals with tissue organization, no exhibiting separate reproductive organs,

here we use the term “gonad” to refer to the places where the gametogenesis occurs, following Campbell (1974);

Marques and Collins (2004); Morandini and Marques (2010); Bentlage et al. (2010) and Straehler-Pohl et al. (2014).

32

Jaboticabal) and also the Nikon Eclipse 80i® Microscope from the Laboratório de

Cultivo e Estudos de Cnidaria (Instituto de Biociências, USP), using the software NIS-

Elements®, with the appropriate calibration to the lens used (Zara et al. 2012). Mean and

standard deviation of measurements were tested for normality by the Kolmogorov-

Smirnov test. Means were compared via the Dunn method (p ≤ 0.05) always when the

Kruskal-Wallis test, to heterocedastic data, indicated differences between the stages of

development and size of the germ cells.

Results

General histological morphology

The general morphology of the cubozoan male gonad is illustrated in Figure 2,

being this schematic draw done based in the histological micrographs presented below.

In both species the four male gonads are composed of a pair of hemigonads: a thin and

laminar tissue layer located between the mesoglea and gastrodermis, and is composed of

several follicles surrounded by an epithelium of gastrodermal origin. In longitudinal

section the follicles are elongated structures contiguous and juxtaposed one to another,

characterizing the pattern similar to a "fingerprint" (Fig. 2A). The gastrodermal

epithelium has cells with elongated nuclei and cytoplasm with large vesicles (Fig. 2B).

Spermatogenesis in Tamoya haplonema (Order Carybdeida, Family Tamoyidae)

The male gonad of T. haplonema is composed of several follicles surrounded by a

gastrodermal epithelium (Fig. 3A), and spermatogenesis occurs inside these male

follicles (Fig. 3A). The follicular epithelium is composed of spermatogonia organized in

just one layer of juxtaposed cells (Fig. 3B) or in more layers (Fig. 3C). The

spermatocytes have rounded nucleus with condensed chromatin (Fig. 3D, F). During

spermiogenesis, the spermatids reduce the cytoplasmic volume turning into sperm (Fig.

33

3E-F). The cells of the gastrodermal epithelium have an irregular shape containing

vesicles not stained by any of the histochemical techniques used (Fig. 3C-D, F).

During spermatogenesis process, it was observed differences between the nuclear

sizes of the different cellular types (Kruskal-Wallis test: H= 142.852; P < 0.0001, Fig.

4). The spermatogonia are cells with elongated nucleus (mean 6.1 ± 1.5µm), well

developed (Fig. 3B) and reactive to toluidine blue stain (Fig. 3D). The spermatocytes

have basophilic nucleus (Fig. 3F), are smaller than the spermatogonia (3.3 ± 1.4µm)

(Dunn test: z = 7.709; p <0.05) and intensely stained by toluidine blue (Fig. 3D).

Between spermatocytes and spermatids, there was significant reduction of the nuclear

diameter (Dunn test: z= 2.8638, p<0.05).

The spermiogenesis begins with the spermatids, which has basophilic, rounded

nucleus (2.6 ± 0.4 µm), showing a homogeneous chromatin stained (Fig. 3F). Among

spermatids and sperm, there was no reduction of the nuclear size (Dunn test: z = 1.4936;

p< 0.05). The mature sperm have an elongated conic head (Fig. 3E-G). Due to genetic

material compaction, the nucleus becomes slightly smaller (2.4 ± 0.5µm), being

strongly reactive to proteins (Fig. 3H). The anterior portion of the sperm head and the

flagellum of T. haplonema are negative to PAS-H stain (Fig. 3I). The mature sperm are

released by rupture of the follicle wall (Fig. 3H-I).

The gonadal cycle of T. haplonema can be divided in four stages of maturation

(Fig. 5): rudimentary, intermediate, mature and spawned. The rudimentary stage is

characterized by follicles with just the initial stages of spermatogenesis like

spermatogonia and spermatocytes (Fig. 5A). At the intermediate stage the follicles are

filled with few spermatids between the spermatocytes (Fig. 5B). The mature gonad is

characterized by the presence of spermatogonia and sperm inside the follicle (Fig. 5C-

D). Once the sperm is released, the male gonad starts a new cycle of sperm production,

34

characterizing the spawned stage (Fig. 5E and F). In this last stage, it can be noted the

presence of empty spaces and a large number of spermatogonia inside the follicle (Fig.

5F).

Spermatogenesis in Chiropsalmus quadrumanus (Order Chirodropida, Family

Chiropsalmidae)

Like in T. haplonema, the male gonad of C. quadrumanus is composed of several

follicles surrounded by a gastrodermal epithelium (Fig. 6A). Inside the follicles there is

only one type of germ cells (e.g. spermatogonia, spermatocytes or spermatids),

suggesting a synchronous process (Fig. 6B-C). The follicles have a centripetal

arrangement and can present, in the periphery, spermatogonia organized in one and

more layers (Fig. 6D-E). The spermatocytes have rounded nucleus and condensed

chromatin. Juxtaposed to the spermatogonial layer lays the thin spermatocytes layer,

detected through the meiotic signs in the chromatin (Fig. 6E). The spermatids showed

reduced cytoplasmic volume (Fig. 6F) giving rises to sperm (Fig. 6F-G). The cells of

the gastrodermal epithelium have an irregular shape containing vesicles not stained by

any of the histochemical stainings (Fig. 6C).

Differences were also detected between the nuclear size of the different types of

cells (Kruskal-Wallis test: H = 313.5568; p < 0. 001, Fig. 7) highlighting significant

reduction in nuclear size. The spermatogonia have a large and elongated nucleus (5.2 ±

0.9µm). The spermatocytes, when compared to the spermatogonia, present reduction in

the nucleus size (Dunn test: z = 7.7329; p < 0.05) with nucleus measuring 3.2 ± 0.9 µm.

This last cellular type can be in different phases of meiotic prophase (Fig. 6C, E).

The spermatids begin the spermiogenesis process and there is no difference in

nuclear size between spermatid and sperm (Dunn test: z = 2.082; p > 0.05). The

35

spermatids are characterized by the presence of rounded and basophilic nucleus (1.8 ±

0.4µm) (Fig. 6F). The sperm show rounded head (1.5 ± 0.3 µm) with also rounded and

strongly basophilic nucleus, which is reactive to proteins (Fig. 6G-I) as the acidophilic

flagellum (Fig. 6F-G). In C. quadrumanus, the sperm head is negative to PAS-H, as the

flagellum (Fig. 6J). When mature, the sperm is released through rupture of the follicle

wall (Fig. 6F).

The gonadal cycle of C. quadrumanus seems to be divided in the same four stages

presented in T. haplonema. The rudimentary stage has only spermatogonia and

spermatocytes (Fig. 6A). The intermediate stage contains spermatids between the

spermatocytes (Figure 6B) and the mature stage with all characteristic cellular stages of

spermatogenesis (Fig. 6C to J). The spawned stage was not observed in none of the

individuals.

Discussion

The male gonad of T. haplonema and C. quadrumanus have similar morphology

and the general pattern is also observed in all cubozoans already studied, like Carybdea

xaymacana (only description) (Conant 1898), Chironex fleckeri (Southcott 1956),

Carybdea marsupialis (Claus 1878; Avian et al. 1993; García-Rodriguez 2015), Copula

sivickisi (García-Rodriguez 2015; Garm et al. 2015), Alatina alata and Morbakka

virulenta (García-Rodriguez 2015). The organization of the male gonad in T.

haplonema and C. quadrumanus seems to be more similar to that observed in Ch.

fleckeri, Ca. marsupialis, A. alata and M. virulenta, in which the gonad is a thin,

laminar and independent tissue layer located between the mesoglea and gastrodermis.

The male gonad is composed of several follicles surrounded by a gastrodermal

epithelium and this structure was already observed in other cubozoans (Conant 1898;

Southcott 1956; Avian et al. 1993; García-Rodriguez 2015).

36

The gastrodermal epithelium is characterized by cells containing several vesicles

with negative reaction to all histological and histochemical staining used (Table 3). This

absence of reaction was also described by (Southcott 1956) for Ch. fleckeri and by

(García-Rodriguez 2015) for A. alata, Co. sivickisi, Ca. marsupialis, M. virulenta, T.

haplonema (specimen from New Jersey) and C. quadrumanus, but the authors called

them vacuoles. The composition of such secretion remains unknown, indicating the

need of further ultrastructural studies to solve this question.

The “fingerprint” pattern of the male gonad seems to be characteristic for all

cubozoans, thus including T. haplonema and C. quadrumanus. Such pattern was first

described for Ch. fleckeri (Southcott 1956) and later for other species (Gershwin 2005).

It is important to highlight that the use of the “fingerprint” to distinguish sexes in

Cubozoa is effective. However, this “fingerprint” pattern is not related with the gonadal

maturation.

For both studied species, the follicles have spermatogonia in the periphery

organized in one or more layers, just like (García-Rodriguez 2015) described to T.

haplonema (specimen from New Jersey). In T. haplonema and C. quadrumanus, the

spermatogonia are bulky, with the nucleus and nucleolus well developed. The

spermatocyte and the spermatid have round shape and the spermatids reduce the

cytoplasmic volume, turning into sperm. This description is very similar to that

observed in the micrographs of C. quadrumanus presented by García-Rodriguez (2015).

The sperm of T. haplonema undergo the modification of the head structure assuming the

conical morphology with elongated projection. However in C. quadramanus it is

observed the maintenance of the rounded morphology of the head.

For both species here studied, the sperm are accumulated in the mid region of the

follicle and the release occurs by rupture of the follicle wall as already described in the

37

cubozoans Ch. fleckeri (Southcott 1956; Yamaguchi and Hartwick 1980), and Ca.

xaymacana (Conant 1898). Yamaguchi and Hartwick (1980) and Toshino et al. (2013)

reported unfertilized eggs and external fertilization to Chiropsella bronzie (as

Chiropsalmus quadrigatus) and Morbakka virulenta, respectively, suggesting a release

of free sperm in the water column. This type of sperm release is different to that

described to the cubomedusae Tripedalia cystophora (Werner 1973a; Werner 1973b),

Alatina alata and Copula sivickisi (García-Rodriguez 2015; Garm et al. 2015). For T.

cystophora and A. alata the release of sperm is by spermatozeugmata. While, Copula

sivickisi presents copula behavior in which the male transfer the spermatophore from its

tentacle to the female's tentacles, that internalizes the spermatophore to gastric cavity,

followed by internal fertilization (Lewis and Long 2005; Bentlage et al. 2010; García-

Rodriguez 2015; Garm et al. 2015; Marques et al. 2015).

The sperm of cnidarians do not show a typical acrosome, but could present several

small vesicles in the anterior region of the head or near the mitochondria (Afzelius and

Franzén 1971; Hinsch and Clark Jr. 1973; Chapman 1974; Hinsch 1974; Miller 1983;

Harrison and Jamieson 1988; Rouse and Pitt 2000; Corbelli et al. 2003). These cells are

usually characterized by rounded head, small midpiece usually with four mitochondria

and a long flagellum (Franzén 1956; Harrison and Jamieson 1988; Corbelli et al. 2003).

To Cubozoa, only (García-Rodriguez 2015) describes the histochemical composition of

these cells and (Corbelli et al. 2003) the ultrastructure. The negative reaction of the

sperm head to the PAS staining in T. haplonema and C. quadrumanus could not be an

indicative of the presence of such little vesicles but, for both species, these small

vesicles can be seen in the ultrastrucutral cuts (Tiseo and Morandini, unpublished data).

There are few the works describing the gonadal cycle for cnidarians (Kessel 1968;

Chapman 1974; Rottini-Sandrini and Avian 1991; Macchi et al. 1995). Among them,

38

(Campbell 1974) presents general characteristics of the gametogenic cycle stating that it

exhibits, frequently, seasonal and circadian periodicity. For Cubozoa, even any of the

already published papers cite the cycle of gonadal maturation, there is some evidences

of this synchronous maturation of cells as presented by Conant (1898) and Southcott

(1956). Here we described the different stages of the gonadal cycle of both studied

species. In T. haplonema was observed four stages of maturation (rudimentary,

intermediate, mature and spawned) and for C. quadrumanus was recorded only three of

the four stages (rudimentary, intermediate, mature). Even the last stage not being

observed to C. quadrumanus, we believe that the spawned stage is also present, once

this species also presents synchrony in the spermatogenesis process and release of

sperm by rupture of the follicle wall. As we describe here, Macchi et al. (1995)

described the gonadal cycle of the male and female gonad of the hydromedusa Olindias

sambaquiensis. The authors divide the gonadal cycle in only four stages: immature

(individual that we cannot note sexual differentiation), in development, mature and

spent, but this division could not be used here once we noted differences in the gonadal

stages of the studied species and we also not observed any immature individual.

In conclusion, even both studied species belonging to different orders, the male

histological gonadal structure, the gonadal cycle and the spermatogenesis process are

similar. The general morphology of the sperm of C. quadrumanus (Family

Chiropsalmidae, Order Chirodropidae) seems to be more similar to the sperm found in

Scyphozoa, whiles the general sperm morphology of T. haplonema (Family

Carybdeidae, Order Carybdeida), seems to differ mainly in the head morphology. But

only an ultrastructural study could enumerate and confirm this hypothesis.

39

Acknowledgements

This work was supported by São Paulo Research Foundation (FAPESP) [GRT grants

2012/19080-1 and 2014/08785-1, FJZ grants BIOTA 2010/50188-8, ACM grants

2010/50174-7, 2011/50242-5, 2013/50484-4]; CAPES [ACM 23038.004309/2014-51];

and CNPq [ACMo (301039/2013-5, 486337/2013-8)]. Thanks are due to Enio Mattos,

Phillip Lenktaitis and Prof. Dr. José Eduardo Marian for help in the histology

procedures. We also thanks to anonymous reviewers for all comments and suggestions.

This is a contribution of NP-BioMar, USP. This study was conducted in accordance

with Brazilian laws (GRT SisBio license No. 611377-9; FJZ MMA SisBio permanent

license No. 34587-1; ACMo MMA SisBio permanent license No. 15031-2).

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45

Tables

Table 1: Previous studies describing the morphology of the gonad of cubozoans through histological approach.

Order Family Specie References

Chirodropidae

Chiropsalmidae Chiropsalmus quadrumanus García-Rodriguez (2015); This study

Chirodropidae Chironex fleckeri Southcott (1956)

Carybdeida

Tamoyidae

Tamoya haplonema (New

Jersey specimen)

García-Rodriguez (2015)

Tamoya haplonema This study

Tripedaliidae Copula sivickisi

Lewis and Long (2005); Garm et al. (2015); García-

Rodriguez (2015)

Carybdeidae Carybdea xaymacana Conant (1898)

Alatinidae

Carybdea marsupialis Claus (1878); Thiel (1936)

Alatina alata García-Rodriguez (2015)

Carukiidae Morbakka virulenta García-Rodriguez (2015)

46

Table 2: Details of sampling sites.

Sites of Collection Coordinates Species collected

Santos – São Vicente Bay (03/2009 and 04/2010) 23°59'33.97" S/ 46°22'12.64" O C. quadrumanus and T. haplonema

Municipality of Ubatuba (24 and 25/07/2012) 23⁰31'147''S/ 045⁰05'193''O T. haplonema

Municipality of São Sebastião (10/06/2014) 23⁰ 27' 5''S/ 45⁰1'47''O C. quadrumanus

Cananéia lagoon estuarine system (14 and

15/04/2015)

25⁰03'604''S/ 47⁰54'419''O C. quadrumanus and T. haplonema

47

Table 3: Summarized results of histochemistry techniques used to highlight the properties of secretions and sperm in Tamoya haplonema and

Chiropsalmus quadrumanus.

Histochemical Technique

Toluidine Blue

(Acids structures)

Bromophenol blue

(basic proteins)

Ponceau xylidine

(total proteins)

PAS (neutral

polysaccharides)

Tamoya

haplonema

Secretion of the vesicles of

gastrodermal epithelium - -

Not observed

-

Sperm head +++ +++ -

Flagellum ++ ++ +

Chiropsalmus

quadrumanus


gastrodermal epithelium - - - -

Sperm head +++ +++ +++ -

Flagellum + ++ ++ +

+++ = strongly positive; ++ = positive; + = weakly positive; − = negative.

48

List of Figures

Figure 1

Figure 1: Sampling sites along the coast of São Paulo state (SE Brazil). (A) Cananéia lagoon estuarine system. (B) Santos and São

Vicente Bay. (C) São Sebastião county. (D) Ubatuba county.

Figure 2

Figure 2: General morphology of the male cubozoan gonad. (A) Longitudinal section of male gonad, characterized

by the “fingerprint” pattern. (B) Cross section of a mature male gonad. (F) Follicle, (Ge) gastrodermal epithelium,

(Me) Mesoglea, (sg) spermatogonia, (sc) spermatocyte, (st) spermatid, (sz) sperm, (v) vesicle.

49

Figure 3

Figure 3: Spermatogenesis in Tamoya haplonema. (A) Hematoxylin and Eosin. Longitudinal section of the male gonad highlighting

the pattern similar to a “fingerprint”. (B) Hematoxylin and Eosin. Cross section of the male gonad. Follicle with spermatogonia with

an elongated nucleus and organized in only one layer. (C) Toluidine blue. Cross section of the male gonad. Note the spermatogonia

organized in more than one layer. (D) Toluidine Blue. Spermatocytes in different stages of meiosis. (E) Hematoxylin and Eosin.

Mature follicle with sperm. (F) Hematoxylin and Eosin. Follicle with spermatids between the spermatocytes. (G) Toluidine blue. Detail of the sperm inside the mature follicles. The sperm have an elongated head with an anterior conic projection and visible

flagellum. (H) Mercuric Bromophenol Blue. Sperm being released by rupture of follicle wall, note the nucleus strongly positive to

basic proteins. (I) PAS-Hematoxylin. The anterior portion of the sperm head had negative reaction to PAS. (F) Follicle, (Ge) Gastrodermal epithelium, (sg) Spermatogonia, (sc) Spermatocyte, (st) Spermatid, (sz) Sperm, (v) vesicle.

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Figure 4

Figure 4: Nuclear diameter of the different spermatic cells of Tamoya haplonema. The dots correspond to the average and the bars

to standard deviation.

51

Figure 5

Figure 5: Gonadal maturation in Tamoya haplonema. Hematoxylin and Eosin. (A) Rudimentary male gonad. Note only the presence of spermatogonia and spermatocytes. (B) Developing male gonad. Note spermatids between the spermatocytes. (C) Mature

male gonad with follicles full of sperm. (D) Detail of the mature gonad highlighting the sperm. (E) The spawned gonad is

characterized by the presence of empty spaces inside the follicles (black arrow) (F) Detail of the previous image highlighting the empty spaces (black arrow). (F) Follicle, (Ge) Gastrodermal epithelium, (sg) Spermatogonia, (sc) Spermatocyte, (st) Spermatid, (sz)

Sperm, (v) vesicle.

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Figure 6

Figure 6: Spermatogenesis in Chiropsalmus quadramanus. (A) Hematoxylin and eosin. General view of the rudimentary gonad with the follicles in a longitudinal section highlighting the pattern similar to a “fingerprint”. (B) Hematoxylin and eosin. Developing

gonad, with the presence of some spermatids between the spermatocytes. (C) Hematoxylin and eosin. Cross section of the mature

gonad. The spermatogonia have elongated basophilic nucleus. (D) Hematoxylin and eosin. Mature follicle with spermatogonia in the periphery and sperm in the center. (E) Toluidine blue. Spermatogonia with large nucleus and spermatocytes in different phases of

meiosis. (F) Hematoxylin and eosin. Final spermatids with rounded head. The sperm are released through rupture of the follicle

wall. (G) Hematoxylin and eosin. The sperm have rounded basophilic head, and acidophilic midpiece and flagellum. (H) Pounceau xylidine. Sperm with positive reaction to total proteins. (I) Mercuric bromophenol blue. Sperm with positive reaction to basic

proteins. (J) PAS. The sperm presented negative reaction to PAS. (F) Follicle, (Ge) Gastrodermal epithelium, (sg) Spermatogonia,

(sc) Spermatocyte, (st) Spermatid, (sz) Sperm, (v) vesicle.

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Figure 7

Figure 7: Nuclear diameter of the different spermatic cells of Chiropsalmus quadrumanus. The dots correspond to the average and the bars to standard deviation.

54

Capítulo 2

Spermatogenesis and sperm morphology in cubozoans from SE Brazilian and

Australian coasts

Gisele R. Tiseo1*

, Peter L. Harrison2, Jamie Seymour

3 and André C. Morandini

1

1 Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, R.

Matão trav. 14, n.101, 05508-090, São Paulo, Brazil;

2 Marine Ecology Research Centre, School of Environment, Science and Engineering,

Southern Cross University, PO Box 157, 2480, Lismore, NSW, Australia;

3Australian Institute for Tropical Health and Medicine, Centre for Biodiscovery &

Molecular Development of Therapeutics, Division of Tropical Health and Medicine,

James Cook University, Cairns Campus, Mcgregor Road, 4878, Cairns, Australia.

Running Title: Spermatogenesis in some species of Cubozoa

*Corresponding author. Tel: +55 11 30917621;

E-mail addresses: [email protected] (GRT); [email protected] (PLH);

[email protected] (JS); [email protected] (ACM).





55

Abstract

The spermatogenesis is poorly studied in members of the class Cubozoa. In this work

we describe the ultrastructure of spermatogenesis and sperm morphology of the

Brazilian species Tamoya haplonema and Chiropsalmus quadrumanus. And we also

describe the spermatogenesis and sperm morphology of three Australian species:

Carukia barnesi, Chironex fleckeri, and Chiropsella bronzie under light microscopy,

histochemistry and ultrastructure. Specimens of T. haplonema and C. quarumanus were

collected in two places along the São Paulo coast, Brazil. The Australian species were

collected near the Great Barrier Reef, Queensland using light traps to attract them. For

light microscopy, the male gonad was removed and fixed in paraformaldehyde 4% in

sodium phosphate buffer 0.2M (pH 7.2) for 24 hours and later processed following the

protocol for historesin. Serial sections were performed and the slides stained with

hematoxylin and eosin, and techniques for proteins (mercuric bromophenol blue and

ponceau xylidine), acid materials (toluidine blue) and neutral polysaccharides (PAS).

For transmission electronic microscopy (TEM) fragments of 1mm3 of the male gonad

were preserved in Karnovsky solution (2.5% glutaraldehyde with 2% paraformaldehyde

in 0.1 M sodium cacodylate buffer, pH 7.4, 2.5 mM of CaCl2 and saccharose) or in

Glutaraldehyde fixative solution (2.5% glutaraldehyde 0.1 M sodium cacodylate

buffered Millipore filtered seawater, pH 7.2-7.4) later processed for protocol in TEM.

The male gonad of all species is organized in elongated follicles, resembling a

fingerprint pattern. In a mature gonad, spermatogonia and spermatocytes are found near

the follicle wall while spermatids and spermatozoa are located at the center. The sperm

release occurs through rupture of follicular wall in T. haplonema, C. quadrumanus,

Carukia barnesi and Chiropsella bronzie. The main morphology of the sperm in the

studied species is very similar, with electrondense nucleus of granular chromatin,

56

electrondense vesicles above the nucleus, midpiece with 6 mitochondria, proximal and

distal centrioles and pericentriolar apparatus, and flagellum with the usual pattern of

9+2 microtubules.

Key-words: Spermatogenesis, box-jellyfish, sperm, Histology, TEM.

Introduction

The spermatogenesis is poorly studied in members of the class Cubozoa (Tiseo

et al. 2016 - chapter one of this dissertation). Under light microscopy there is only a few

works describing the histological structure of the male gonad or part of the

spermatogenesis (Thiel 1936; Southcott 1956; Avian et al. 1993; García-Rodriguez

2015; Garm et al. 2015). Under transmission electronic microscopy, were described

only aspects of the male gonad, spermatozeugmata and sperm of Copula sivickisi (Garm

et al. 2015) and the ultrastructure of the sperm of Carybdea marsupialis (Corbelli et al.

2003).

Box-jellyfish are gonochoric, with distinction between male and female gonads

done by color difference and recognition of the “fingerprint” pattern (Southcott 1956;

Werner 1973a; Gershwin 2005; Lewis and Long 2005; Bentlage et al. 2010; Bentlage

and Lewis 2012). The gonad has paired leaf-like hemigonads which are attached along

the length of the septum in most carybdeids and are broader and more developed in the

upper half, enveloping gastric saccules in most chiropdropids (Mayer 1910; Southcott

1956; Gershwin 2005).

For Scyphozoa, Anthozoa and Cubozoa the male gonad is derived from

interstitial cells of the gastrodermis, which migrate to the mesoglea differentiating the

male follicles (Miller 1983; Tiseo et al. 2016 - chapter one of this dissertation). The

spermatogenesis occurs inside these follicles and the mature sperm are released through

57

rupture of the follicle wall (Toshino et al. 2013; García-Rodriguez 2015; Tiseo et al.

2016 - chapter 1 of this dissertation) or transferred to the female through the mating

behavior (Conant 1898; Miller 1983; Harrison and Jamieson 1988; Lewis and Long

2005; Lewis et al. 2008; Marques et al. 2015). In Cubozoa there were reported two

different strategies of sperm release: free or packed. The free sperm are released in the

water by rupture of the follicle wall (García-Rodriguez 2015) and the packed strategy

consists of groups of sperm involved by an extracellular matrix (spermatozeugmata)

(e.g. Trypedalia cystophora – Werner, 1973b and Alatina alata – García-Rodriguez,

2015) or in spermatophores (e. g. Copula sivickisi – (Hartwick 1991; Straehler-Pohl et

al. 2014; García-Rodriguez 2015; Garm et al. 2015).

The general morphology of the cubozoans male gonad, viewed through

histological sections, is characterized by several follicles filled with spermatic cells

surrounded by a gastrodermal epithelium (Conant 1898; Southcott 1956; García-

Rodriguez 2015; Tiseo et al. 2016). The spermatogenesis is well known consisting of

pre-meiotic cellular stages (spermatogonia) with large nucleus and visible nucleolus,

and meiotic cellular stages (spermatocytes) with synaptonemal complex between

homologous chromosomes. The spermatocytes II divide into two spermatids that are

interconnected by cytoplasmic bridges. During the spermiogenesis, the spermatid

reduce the cytoplasmic volume turning into sperm (Miller 1983; Gaino et al. 2013).

There are few available information about cnidarians sperm ultrastructure and most

descriptions are for the Anthozoa and Hydrozoa classes (Harrison and Jamieson 1988).

For Cubozoa, Werner (1973a; 1973b) made a general description of the sperm of

Trypedalia cystophora and (Corbelli et al. 2003) described the ultrastructure

morphology of the sperm of Carybdea marsupialis.

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The general sperm morphology, under light microscopy, is composed of a

rounded, ovoid, conic or elongated head, a visible midpiece and a long flagellum

(Franzén 1956; Harrison and Jamieson 1988). Under transmission electronic

microscopy, it is observed the symmetry of the sperm head, the presence of small

vesicles (precursors of the acrosome) in the anterior region of the sperm, midpiece with

four or more mitochondria, Golgi complex, proximal and distal centrioles and the

pericentriolar apparatus of the flagellum (Hinsch and Clarck Jr. 1973; Hinsch 1974;

Miller 1983; Harrison and Jamieson 1988; Rouse and Pitt 2000; Corbelli et al. 2003).

The species Tamoya haplonema belongs to the family Tamoyidae (Carybdeida),

occurring in the Atlantic coast of America (Mianzan and Cornelius 1999; Morandini et

al. 2005), and is widely distributed along Brazil (Kramp 1961; Mianzan and Cornelius

1999; Morandini et al. 2005). Chiropsalmus quadrumanus (Chiropsalmidae,

Chirodropida) is one of the four species of the genus (Marques et al. 2003) being

distributed at the Atlantic coast of America (Kramp 1961; Mianzan and Cornelius 1999;

Morandini et al. 2005). Both species are found in São Paulo coast and are usually

related with stings caused by box-jellyfish in the country (Haddad et al. 2002;

Morandini et al. 2005).

The Cubozoa Carukia barnesi (Carukiidae, Carybdeida) is one of the most

smaller jellyfish in the world being distributed in the Exmouth county in Australia

Ocidental, across northern Australia and down the Queensland coast (Fenner and Hadok

2002; Kingsford et al. 2012). This species is commonly found during the jellyfish

season and also in the Northeastern Great Barrier Reef (GBR) in the inner and middle

cross-shelf transects at night (Kingsford et al. 2012). Chironex fleckeri (Chirodropidae,

Chirodropida) is one of the most common chirodropids in Australia coast (Williamson

et al. 1996) and is also found in Queensland during the danger period (October to May).

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The specie Chiropsella bronzie (Chiropsalmidae, Chirodropida) is an abundant specie

found from Port Douglas to Cairns during the summer months, being distributed from

Cooktown (in the North) to Magnetic Island (Townsville)(Gershwin 2006).

Here we describe the spermatogenesis and sperm morphology under light and

transmission electronic microscopy of five cubozoan species, two Carybdeida and three

Chirodropida. Additionally, we compare the sperm ultrastructure of the studied species

with the only cubozoan sperm already described in the literature, looking for common

features.


Sampling

For T. haplonema and C. quadrumanus, samplings occurred in Cananéia Estuary

(25ᵒ03'604''S 47ᵒ54'419''W) and Ubatuba county (23ᵒ31'147''S 045ᵒ05'193''W), São

Paulo State, SE Brazil (Figure 1), during April and May of 2015. The animals were

collected by hand in water surface or with shrimp trawls. The specimens were

transported alive to the laboratory and identified following Morandini et al. (2005).

The Australian species were collected along the eastern coast of Queensland.

Carukia barnesi was collected in Double Island Reef (16°43’25.55’’S 145°41’0.43’’W)

in November of 2013 and February of 2016. The specie Chironex fleckeri was found in

South Mission Beach (17°56’43.46’’ 146°6’13.08’’W) in 2015 and in Tully heads

(18°2’13.78’’S 146°3’5.08’’W) in April of 2016. Chiropsella bronzie was collected in

Pourt Douglas (16°29’6.10’’S 145°27’36.81’’W) in April of 2016 (Figure 2). The

collection occurred manually using a small bucket or hand net at the water surface,

during the night, using light traps to attract the jellyfish. The specimens were

transported alive to the Laboratory at James Cook University, Cairns, Queensland, and

60

were kept in plastics boxes until dissection. The animals were identified and measured

accordingly to Dawson (2005), Gershwin (2005), Tibballs (2006) and Gershwin and

Kingsford (2008).

Light microscopy

The animals were anesthetized by thermal shock (-20°C) for 5 minutes. Then,

samples of gonadal tissue were preserved in 4% formaldehyde solution buffered with a

saline solution of 0.2 M sodium phosphate (pH 7.2) for 24 hours. After fixation, the

samples were dehydrated in ethanol series (30-95%) and embedded in Leica®

methacrylate historesin. Serial sections 3-4 μm thick were made on a rotative

microtome. For histological description the tissue samples were stained with

hematoxylin and eosin (HE) (Modified from Junqueira and Junqueira 1983), without

baths in ethanol and xylene (Sant’Anna et al. 2010; Zara et al. 2012; Tiseo et al. 2014).

To analyze the chemical composition of the tissues, the slides were stained with

mercuric bromophenol blue and ponceau xylidine for proteins (Pearse 1960; Mello and

Vidal 1980). The PAS technique was used to visualize neutral polysaccharides

(Junqueira and Junqueira 1983) and the toluidine blue to visualize acid components

(Modified from Audino et al. 2015).

Transmission Electronic Microscopy (TEM)

For transmission electronic microscopy (TEM), fragments of 1mm3 of the male

gonad were preserved in Karnovsky solution (modified from Karnovsky 1965)

consisting of 2.5% glutaraldehyde with 2% paraformaldehyde in 0.1 M sodium

cacodylate buffer (pH 7.4) with 2.5 mM of CaCl2 and saccharose (Brazilian species)

and in Glutaraldehyde fixative solution consisting of 2.5% glutaraldehyde 0.1 M sodium

61

cacodylate buffered Millipore filtered seawater (pH 7.2-7.4) for 30 minutes at 4ºC

(Australian species), followed by post-fixation in 1% osmium tetroxide with the same

buffer, for one hour at the same temperature. Then, samples were dehydrated in ethanol

series (30 to 100%), and embedded in Spurr’s® resin. Semi-thin and ultrathin sections

were obtained with a Leica® ultramicrotome (gently provided by the Electronic

Microscopy Laboratory from the Department of Genetics, Institute of Biosciences of the

University of São Paulo). Grids with sections were contrasted with uranyl acetate and

lead citrate, and later examined and photographed in the Zeiss EM 900 Transmission

Electronic Microscope operated at 80kV.

Results

Ultrastructure of spermatogenesis in Tamoya haplonema (Tamoyidae)

The male gonad in T. haplonema is characterized by the presence of several

follicles with spermatic cells surrounded by a gastrodermal epithelium (Fig. 3A). The

cells that compose the gastrodermal epithelium have an acidophilic nucleus, located in

different positions of the basophilic cytoplasm which extends between the follicles.

Several large vesicles not reactive to hematoxylin and eosin are observed in the

cytoplasm (Fig. 3A). Under transmission electronic microscopy (TEM), these vesicles

are filled with granular electrondense material (Fig. 3B) that came from between the

follicles in smaller vesicles (Fig. 3C-F). The cells of gastrodermal epithelium, in TEM,

have cilia in the apical region (Fig. 3B), irregular format (Fig. 3B-C), several little

vesicles derived from Golgi complex in the cytoplasm (Fig. 3G-H) and electrondense

nucleus with euchromatin (Fig. 3I). The gastrodermal epithelium produces an

electrondense secretion that seems to give rise to the mesoglea found between and

inside the follicles (Fig. 3G-J). The follicular epithelium is composed of spermatogonia

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(Fig. 4A) characterized by elongated nucleus with chromatin of intermediate

electrondensity and a prominent nucleolus (Fig. 4B). The spermatocytes are

characterized by rounded nucleus with chromatin of intermediate electrondensity or

they can be in meiosis (Fig. 4C). The sperm are concentrated in the central portion of

the follicle immersed in the electrondense mesoglea (Fig. 4D) or in a granular substance

(Fig. 4E). The sperm have an ampuliform shape, elongated nucleus with ratio 1.5 ± 0.21

(n=18), midpiece with six mitochondria and a long flagellum (Fig. 4D-L). In the

anterior region of the nucleus can be observed several electrondense vesicles (Fig. 4E)

and several ribosomes (Fig. 4F). The midpiece is wider than longer with ratio 0.47 ±

0.11 (n=16). The pericentriolar apparatus is anchored in the distal centriole (Fig. 4G- I)

and this last one is connected to the proximal centriole (Fig. 4H). In the pericentriolar

apparatus can be noted part of the electrondense primary process (Fig. 4I). The

flagellum is immersed in the mesoglea and has an axoneme with the typical

microtubules pattern 9+2 (Fig. 4K-L).

Ultrastructure of spermatogenesis in Chiropsalmus quadrumanus (Chiropsalmidae)

The male gonad of C. quadrumanus is characterized by several follicles

surrounded by a gastrodermal epithelium (Fig. 5A). The gastrodermal epithelium has

cells with irregular shape with cilia in the apical portion (Fig. 5A-B), electrondense

nucleus with euchromatin (Fig.5C), and several vesicles of granular material in the

cytoplasm (Fig. 5A-C). The gastrodermal epithelium produces an electrondense

secretion that seems to give rise to the mesoglea found between the follicles (Fig. 5A,

C-E). Between the mesoglea can be seen a granular secretion that is transported to the

large vesicles (Fig. 5D-E). The follicular epithelium is characterized by the presence of

spermatogonia (Fig. 5D) with elongated nucleus and prominent nucleolus (Fig. 5F). The

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spermatocytes are located near the spermatogonia and are characterized by a rounded

nucleus (Fig. 5D) and can be in some stage of meiosis (Fig. 5G). The mature sperm is

released by rupture of the follicle wall (Fig. 5H). The sperm of C. quadrumanus is

immersed in a granular substance (Fig. A) and are longer than wider with ratio of 1.82 ±

0.18 (n=5). The sperm head has a bullet-shape with an elongated nucleus, midpiece with

six mitochondria and a long flagellum (Fig.6A-M). In the lateral region of the nucleus is

observed several electronlucid vesicles (Fig. 6C-D). In the anterior region of the nucleus

is observed several electrondense vesicles (Fig. 6E) and ribosome (Fig. 6E, H). The

midpiece is wider than longer with ratio 0.40 ± 0.10 (n= 8), and present a proximal and

distal centriole (Fig. 6G). The pericentriolar apparatus is anchored in the distal centriole

(Fig. 6G-J) and only the primary process could be observed (Fig. 6H). The flagellum

has an axoneme with the typical microtubules pattern 9+2 (Fig. 6L-M).

Histology and histochemistry of spermatogenesis in Carukia barnesi

(Carukiidae)

The male gonad of Carukia barnesi is a thin and laminar layer of tissue located

between the gastrodermis and the mesoglea (Fig. 7A). The male gonad present a

gastrodermal epithelium with elongated follicles (longitudinal section) adjacent and

juxtaposed one to another, characterizing the “fingerprint” pattern (Fig.7B-C). Between

the follicles lies the acidofilic mesoglea (Fig. 7B and C), strongly positive to acid

structures (Fig. 7D), positive to proteins (Fig. 7E), and strongly positive to neutral

polysaccharides (Fig. 7F). Inside these follicles spermatogenesis occurs, being a

synchronous process as we noted cells in the same stage of development (e.g. sperm in

Fig. 7B and spermatogonia in Fig. 7C). The gastrodermal epithelium present cells with

rounded basophilic nucleus and acidophilic cytoplasm. The epithelium has several

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vesicles with negative reaction to hematoxylin and eosin in the cytoplasm (Fig. 7B-C).

The follicular epithelium is composed by the spermatogonia (Fig. 7G). In light

microscopy, during the spermiogenesis process, the spermatids reduce the cytoplasmic

volume turning into sperm (Fig. 7G-J). The sperm is immersed in a substance with no

reaction to hematoxylin and eosin, is characterized by a rounded head with basophilic

nucleus, an acidophilic midpiece and flagellum (Fig. 7G). The sperm head was strongly

reactive to acid structures (Fig. 7H) and proteins (Fig. 7I), but present negative reaction

to neutral polysaccharides (Fig. 7J). The substance in which the sperm is immersed

presented negative reaction to all stainings used (Fig. 7H-J).

Histology, Histochemistry and ultrastructure of spermatogenesis in Chironex fleckeri

(Chirodropidae)

As in Carukia barnesi, Chironex fleckeri present the male gonad characterized

by a gastrodermal epithelium with elongated follicles surrounded by the mesoglea in

longitudinal section (Fig. 8A). In transversal section (Fig. 8B) we observe the

gastrodermal epithelium with several vesicles with negative reaction to hematoxylin and

eosin (Fig. 8B-C). The follicular epithelium is composed of spermatogonia with

rounded basophilic nucleus and acidophilic cytoplasm. Beneath these cells are the

sperm with rounded head with basophilic nucleus, acidophilic midpiece and a long

acidophilic flagellum (Fig. 8C). The sperm is immersed in a substance with no reaction

to hematoxylin and eosin (Fig.8C), positive reaction to acid structures (Fig. 8D),

negative reaction to proteins (Fig. 8E-F) and neutral polysaccharides (Fig. 8G). The

sperm head present strongly reaction to acid structures (Fig. 8D), to proteins (Fig. 8E-

F), but does not present reactivity to neutral polysaccharides (Fig. 8G).

65

Under transmission electronic microscopy the gastrodermal epithelium is

characterized by cells with rounded nucleus with euchromatin and cytoplasm with large

vesicles with granular material of intermediate electrondensity (Fig. 9A-B). These cells

produce an electrondense secretion present in the small vesicles derived from the Golgi

complex (Fig. 9D). The follicular epithelium is a thin layer of spermatogonia and

adjacent to them is the sperm (Fig. 9B). The spermatogonia has rounded nucleus with

prominent nucleolus (Fig. 9C). All the sperm are concentrated in the center of the

follicle immersed in the electrondense mesoglea (Fig.9E) and have a bullet-shape head

longer than wider (1.48 ± 0.21, n=4). They have an electrondense elongated nucleus

with granular chromatin and can present tiny electrondense vesicles in the anterior

region of the nucleus (Fig. 9F-G). The midpiece is wider than longer with ratio 0.36 ±

0.09 (n=6) and have six mitochondria with several crests (Fig. 9G-H). The anchorage

apparatus of the flagellum is present and the distal centriole can be noted (Fig. 9G). All

flagella are clusttered and immersed in the electrondense mesoglea (Fig. 9I) and present

simple axoneme with the typical microtubules pattern 9+2 (Fig. 9J).

Histology, Histochemistry and Ultrastructure of spermatogenesis in Chiropsella bronzie

(Chirodropidae)

Chiropsella bronzie present a male gonad also organized in several elongated

follicles (in a longitudinal section) adjacent one to another (Fig. 10A-B), characterizing

the “fingerprint” pattern, and an external gastrodermal epithelium containing several

vesicles with negative reaction to hematoxylin and eosin (Fig. 10A-C). The

spermatogenesis is synchronous [one individual only with spermatogonia and

spermatocytes (Fig. 10A), and another individual only with sperm and spermatogonia

(Fig. 10B-C)]. The follicular epithelium is composed of spermatogonia – cells with

66

large, rounded and basophilic nucleus (Fig. 10C-D). There is no substance surrounding

the mass of sperm being this last one released by rupture of the follicular wall (Fig.

10D). It is noted a substance with no reaction to any histochemical compound between

the follicular epithelium (Fig. 10F-H). The sperm have rounded head with basophilic

nucleus, visible and acidophilic midpiece and long and acidophilic flagellum (Fig. 10E).

The sperm head has positive reaction to acid structures (Fig. 10F), proteins (Fig. 10G-

H), but present negative reaction to neutral polysaccharides (Fig.10I).

In the transmission electronic microscopy it is noted the gastrodermal epithelium

characterized by cells with rounded nucleus with euchromatin and cytoplasm with

several vesicles. Inside these large vesicles there is a granular material of low

electrondensity (Fig. 11A). The follicular epithelium has spermatogonia organized in

centers and just below them there is a bunch of sperm immersed in the electrondense

mesoglea (Fig. 11A-B). All the sperm are accumulated in the center of the follicle (Fig.

11B). The sperm head has an ovoid shape, is longer than wider with a ratio of 1.19 ±

0.24 (n=5) and have an electrondense elongated nucleus with granular chromatin. No

acrosome is apparent but there are tiny electrondense vesicles in above the nucleus (Fig.

11C). The midpiece is wider than longer with ratio of 0.35 ± 0.05 (n=10), present six

mitochondria with several crests and the anchorage apparatus (Fig. 11D). In cross

section it is visible six mitochondria, and the proximal centriole clearly lies among them

(Fig. 11E). The flagellum is immersed in the electrondense mesoglea and presents an

axoneme with the typical microtubules pattern of 9+2 (Fig.11F). The midpiece presents

the Golgi complex with several vesicles (Fig. 11G).

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Discussion

For all studied species the gonadal structure is similar: the male gonad is a thin

and separate layer of tissue located near the mesoglea and gastrodermis. This

organization is also similar to that described for other cubozoan species like Carybdea

xaymacana (only description) (Conant 1898), Chironex fleckeri (Southcott 1956),

Carybdea marsupialis (Claus 1878; Avian et al. 1993; García-Rodriguez 2015), Alatina

alata and Morbakka virulenta (García-Rodriguez 2015), with the exception of Copula

sivickisi (García-Rodriguez 2015; Garm et al. 2015) in which the gonadal structure is

circular. With the exception of C. sivickisi (García-Rodriguez 2015; Garm et al. 2015)

all cubozoans already studied (including the five ones of this paper) present the male

gonad according to the general histological pattern presented by Tiseo et al. (2016) -

chapter one of this dissertation.

The male gonad present the “fingerprint” pattern in longitudinal section, in

which the follicles are surrounded by a gastrodermal epithelium with large vesicles with

negative reaction to all histochemical compounds tested (Table 1). These same vesicles,

in TEM, present a granular material (Table 2). In T. haplonema, C. quadrumanus, Ca.

barnesi and Ch. fleckeri a substance was observed inside the follicles between the sperm

that also presented negative reaction to the histochemical stainings and in TEM had a

granular aspect. In Chiropsella bronzie this substance is present only between the

follicles, and the sperm is immersed only in the electrondense mesoglea. Inside the

follicular epithelium of T. haplonema it was observed tiny vesicles transporting a

granular substance to the mesoglea and also some primary vesicles with granular

material fusing with the large vesicles found in the gastrodermal epithelium. We believe

that this transport of granular material from inside the follicle to the large vesicles in the

gastrodermal epithelium could be a residual of cytoplasmic material that is been

68

absorbed and stored until the end of the gonadal cycle. For all species here studied,

under TEM, the gastrodermal epithelium produces a homogeneous and electrondense

secretion that seems to give rise to the mesoglea. There is no published record of the

male gonad to produce the mesoglea, but this intermediate layer of tissue was already

described as an electrondense homogeneous matrix in the gonad of other cnidarians, as

Distichopora sp. (Hydrozoa, Sylasteridae) (Gaino et al. 2013).

Tiseo et al. (2016) - chapter one of this dissertation - described the different

stages of the gonadal maturation of T. haplonema and C. quadrumanus: Tamoya

haplonema have four stages of maturation and C. quadrumanus three. Southcott (1956)

and Avian et al. (1993) described a degree of gonadal maturity to Chironex fleckeri and

Carybdea marsupialis, respectively. For the Australian species here studied it was not

possible to describe the gonadal cycle because of the few number of male individuals

collected. Even we not describing the gonadal cycle of Carukia barnesi, Chironex

fleckeri and Chiropsella bronzie, like Tiseo et al. (2016) - chapter one of this

dissertation - stated to C. quadrumanus, it is supposed that a cycle of gonadal

maturation is present in all of them because of the observation of synchrony in sperm

production.

Here we report the release of sperm by rupture of the follicle wall for

Chiropsalmus quadrumanus (under TEM) and for Chiropsella bronzie (under light

microscopy). This strategy of sperm release was also reported for C. quadrumanus and

T. haplonema under light microscopy (García-Rodriguez 2015; Tiseo et al. 2016 -

chapter one of this dissertation), Chironex fleckeri (Southcott 1956; Yamaguchi and

Hartwick 1980), and Carybdea xaymacana (Conant 1898). Toshino et al. (2013)

reported unfertilized eggs and external fertilization to Morbakka virulenta suggesting

that the sperm is release in the water. García-Rodriguez (2015) described the sperm

69

release by spermatzeugmata to Alatina alata and in by a spermatophore for Copula

sivickisi. For Carukia barnesi and Chironex fleckeri it was not observed the sperm

release but the follicular structure with the sperm in the center and the flagellum

directed to one side, indicates the probable pole of follicles’ wall rupture.

The description of the spermatogenic process observed in the cubozoans studied

by us, is similar to the observed in scyphozoans (Morandini and Silveira 2001; Lucas

and Reed 2010; Ikeda et al. 2011b; Schiariti et al. 2012) and hydrozoans (Gaino et al.

2013). Gaino et al. (2013) also describes the spermatogonia as bulky cells, with nucleus

and nucleolus well developed and observed spermatocytes in meiosis with the

synaptonemal complex between homologous chromossomes visible. The spermatids

connected to each other forming cytoplasmic bridges was first described for the

scyphozoan Nemopilema nomurai (Ikeda et al. 2011a) and was also observed in

Distichopora sp (Gaino et al. 2013). Harrison and Jamieson (1988) noted evident

flagellum, and structures called collar in the spermatids posterior end of the midpiece

and this characteristics help us to differentiate the late spermatid to the sperm.

The knowledge about sperm morphology and their features can be linked to

systematics (spermiotaxonomy) and has been used as a support criterion for

phylogenetic inference in Cnidaria by some authors (Hinsch 1974; Miller 1983;

Harrison and Jamieson 1988; Corbelli et al. 2003). These studies can be used as a novel

approach and help further in resolving the relationships among the group. Several

authors (Miller 1983; Harrison and Jamieson 1988; Rouse and Pitt 2000; Corbelli et al.

2003) mentioned the presence of small vesicles, precursors of the acrosome, in the

anterior region of the sperm, in front of the nucleus (under TEM). Under light

microscopy, we could not observe such vesicles (using histochemistry) in the Australian

species, just like Tiseo et al. (2016) - chapter one of this dissertation - described for T.

70

haplonema and C. quadrumanus. But the presence of these vesicles in the TEM

micrographs, made us conclude that eventually their size was too small to express the

positive reaction to PAS.

Comparing the sperm morphology of the five Cubozoa studied with Carybdea

marsupialis (the only Cubozoa sperm available for comparison) some patterns and

slight differences could be noted (see Table 3 for details). With the exception of

Carukia barnesi, in which only histological samples were obtained, here we present the

main sperm morphology of all species. They all possess electrondense nucleus with

granular chromatin, electrondense vesicles in the anterior region of the nucleus, distal

centrioles, pericentriolar apparatus, six mitochondria in the midpiece and the typical

flagellum pattern 9+2 of microtubules. The main differences found were in the head

morphology (ampuliform in T. haplonema, ovoid in Carybdea marsupialis and

Chiropsella bronzie and bullet-shape in Chiropsalmus quadrumanus and Chironex

fleckeri), the presence or absence of the poli-ribosomes in the vestigial cytoplasm, the

presence or absence of Golgi complex and small vesicles in the midpiece. The proximal

centriole and the pericentriolar apparatus were not observed in Chironex fleckeri and

Chiropsella bronzie, but we strongly believe that these structures are present in both

species. They were not found in the cuts observed in the TEM, due to the difficulty to

separate only the sperm to analyze and in many different orientations. Corbelli et al.

(2003) described the existence of a “spurr” structure in the pericentriolar apparatus of

Carybdea marsupialis that was not observed in the species of this study. But, as already

explained we thought that maybe this could be due to an orientation resin block

problem. As no specific differences between the sperm of studied species were found,

maybe the sperm morphology, in a general way, is conserved at class level, at least for

cubozoans. Some interesting features are shared with representatives of other classes.

71

The presence of poli-ribosomes in the cytoplasm in Tamoya haplonema, Chiropsalmus

quadrumanus and Chironex fleckeri is shared with the hydroid Tubularia larynx

(Afzelius 1971) and the connection between the proximal and distal centriole observed

in T. haplonema is a characteristic of Anthozoa sperm (Harrison and Jamieson 1988).

Concluding, the male gonadal structure, the probable gonadal cycle and the

spermatogenesis process, in all species here studied, is similar. The sperm general

morphology of all cubozoans here described is very similar with some punctual

differences in the sperm head morphology and presence or absence of some organelles.

Acknoledgements

This work was supported by São Paulo Research Foundation (FAPESP) [GRT grants

2012/19080-1 and 2014/08785-1, ACM grants 2010/50174-7, 2011/50242-5,

2013/50484-4]; CAPES [ACM 23038.004309/2014-51]; and CNPq [ACM

(301039/2013-5, 486337/2013-8)]. Thanks are due to Lea Taylor, Maxine Daves and

Waldir Caldeira for all help in the transmission electronic microscopy and to Robert

Courtney and Sally Browning for help in the collects. Thanks are also due to

anonymous reviewers for all comments and suggestions. This is a contribution of NP-

BioMar, USP. This study was conducted in accordance with Brazilian laws (GRT

SisBio license No. 611377-9; ACM MMA SisBio permanent license No. 15031-2).

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Tables

Table 1: Secretions and sperm histochemistry of Tamoya haplonema, Chiropsalmus quadrumanus, Carukia barnesi, Chironex fleckeri

and Chiropsella bronzie.

Histochemical Technique References

Toluidine Blue

(Acids

structures)

Bromophenol

blue (basic

proteins)

Ponceau xylidine

(total proteins)

PAS (neutral

polysaccharides)

Tamoya

haplonema



Tiseo et al.

2016

(chapter 1)

Sperm head +++ +++ - -

Flagellum + ++ + +

Substance inside the follicles - - - -

Chiropsalmus

quadrumanus



Tiseo et al.

2016

(chapter 1)

Sperm head +++ +++ - -

Flagellum + ++ + +

Substanceinside the follicles - - - -

79

Carukia

barnesi



This study Sperm head +++ +++ - -

Flagellum ++ ++ + +

Substance inside the follicles - - - -

Chironex

fleckeri




Flagellum ++ ++ + +

Substance inside the follicles - + - -

Chiropsella

bronzie




Flagellum ++ ++ + +

Substance inside the follicles Absent Absent Absent Absent

+++ = strongly positive; ++ = positive; + = weakly positive; − = negative.

80

Table 2: Composition of secretions and substances found in Transmission Electronic Microscopy.

Transmission Electronic Microscopy

(TEM)

Tamoya haplonema

Secretion of the vesicles of gastrodermal

epithelium Granular

Substance inside the follicles Granular substance; Mesoglea (electrondense)

Chiropsalmus

quadrumanus


epithelium Granular


Carukia barnesi


epithelium Granular


Chironex fleckeri


epithelium Granular


Chiropsella bronzie


epithelium Granular

Substance inside the follicles Mesoglea (electrondense)

81

Table 3: Comparison between the sperm of Tamoya haplonema, Chiropsalmus quadrumanus, Carukia barnesi, Chironex fleckeri and

Chiropsella bronzie with other Cubozoa described in the literature.

Cubozoa Class

Carybdeida Chirodropida

Characteristics Tamoya

haplonema 1

Carukia

barnesi 1

Carybdea

marsupials 2

Chiropsalmus

quadrumanus 1

Chironex

fleckeri 1

Chiropsella

bronzie 1

Head length 3.7 ± 0.41 - 3.84 ± 0.34 2.98 ± 0.28 2.47 ± 0.17 2.73 ± 0.42

Head diameter 2.54 ± 0.24 - - 1.82 ± 0.16 1.95 ± 0.25 2.38 ± 0.38

Midpiece length 1.03 ± 0.23 - 1.1 0.73 ± 0.14 0.68 ± 0.14 0.78 ± 0.08

Midpiece diameter 2.23 ± 0.20 - 2.41 ±0.36 1.87 ± 0.25 1.99 ± 0.27 2.18 ± 0.30

Nucleus length 3.4 ± 0.48 - 2.83 ± 0.27 2.75 ± 0.29 2.23 ± 0.23 2.48 ± 0.4

Nucleus diameter 2.21 ± 0.19 - 1.84 ± 0.27 1.57 ± 0.14 1.59 ± 0.04 2.1 ± 0.32

Head morphology Ampuliform a Ovoid

b Ovoid

b? Bullet-shaped

c Bullet-shaped

c Ovoid

b

Nucleus ratio 1.5 ± 0.21 - 1.54 1.82 ± 0.18 1.48 ± 0.21 1.19 ± 0.24

Nuclear vesicle Absent a - Absent

a? Absent

a Absent

a Absent

a

Chromatin Granular a - Granular

a? Granular

a Granular

a Granular

a

Outer nuclear membrane Absent a - Absent

a? Absent

a Absent

a Absent

a

Number of Mitochondria 6 a - 6

a 6

a 6

a 6

a

Vesicles anteriorly to the

nucleus Present

a - Present

a Present

a Present

a Present

a

Vesicles diameter 0.09 ± 0.03 - - 0.09 ± 0.01 0.15 ± 0.009 0.12 ± 0.023

Proximal centriole Present a*

- Present a Present

a* Not observed

b* Not observed

b*

82

Distal centriole Present a - Present

a Present

a Present

a Present

a

Pericentriolar apparatus Present a - Present

a Present

a Present

a Present

a

Primary process Present a*

- Present a Present

a* Not observed

b* Not observed

b*

Interprimary process Present a - Present

a Present

a Not observed

b* Not observed

b*

Major striated bands Absent a*

- Present b Absent

c* Not observed

c* Not observed

c*

Secondary process Not observed a*

- Present b Not observed

a* Not observed

a* Not observed

a*

Tertiary Process Not observed a*


a* Not observed

a* Not observed

a*

Spurr Not observed a*


a* Not observed

a* Not observed

a*

Lamellas Absent a - Absent

a? Absent

a* Absent

a* Absent

a*

Poli-ribosomes Present a*

- Absent b?

Present a*

Present a*

Not observed b*

Golgi complex Absent a*

- Absent a?

Absent a*

Absent a*

Present b*

Vesicles in the midpiece Not observed a*


a* Not observed

a* Present

b*

Structure of the flagellum 9 + 2 - 9 + 2 9 + 2 9 + 2 9 + 2

1 This study;

2 Corbelli (2003).

Different letter indicate variation between the species; * = difference only between the species of the present study; ? = Not described

and observed in the published figure.

83

List of Figures

Figure 1

Figure 1: Sampling sites at the Brazilian coast. (A) Ubatuba (north) and Cananéia (south) counties in the coast of São Paulo state.

84

Figure 2

Figure 2: Sampling sites at the Australian coast. (A) All three points of collection in the north of Queensland State. (B) Pourt Douglas. (C) Double Island Reef. (D) South Mission Beach and Tully

Heads.

85

Figure 3

Figure 3: Ultrastructure of male gonad in Tamoya haplonema. (A) Hematoxylin and Eosin. Histology of the male gonad highlighting the germinal epithelium with several large vesicles and the follicles filled with sperm. B) Gastrodermal epithelium cells, under transmission electronic microscopy,

with several large vesicles containing granular secretion (C) Detail of the large vesicles in the cytoplasm of the gastrodermal epithelium that extends

to the follicles. (D) Detail of the vesicles (black arrowhead) originated in the follicular epithelium. (E) Detail of the granular substance immersed in the mesoglea, found between the follicles. (F) Small vesicles with granular substance funding with the large vesicles of granular material. (G) Golgi

complex producing small vesicles (white arrowhead) with the electrondese material that seems to give rise to the mesoglea. (H) Detail of the Golgi

complex with the electrondense vesicles. (I) Detail of the nucleus and thin portion of cytoplasm of gastrodermal epithelium with electrondense vesicles (arrowhead). (J) General view of the follicular epithelium surrounded by the mesoglea. Cillia (c); Follicular epithelium (ef); Follicle (F);

Gastrodermal epithelium (Ge); Golgi complex (gc); Mitochondria (M); Mesoglea (Me); Nucleus (N); Substance (Su); Spermatogonia (sg); sperm (sz);

gastrodermal epithelium; vesicle (v).

86

Figure 4

Figure 4: Spermatogenesis in Tamoya haplonema. (A) Detail of the gastrodermal epithelium with cilia in the apical region. At the follicular epithelium can be noted the spermatogonia and the sperm immersed in the mesoglea. (B) Detail of the spermatogonia. (C) Detail of the spermatocyte

in meiosis (D) General morphology of the sperm (longitudinal section). (E) Electrondense vesicles (arrowhead) in the anterior region of the sperm

head. (F) Detail of the poliribosomes (arrowhead) found in the anterior region of the sperm head. (G) Longitudinal section of the midpiece highlighting the pericentriolar apparatus (arrowhead). (H) Longitudinal section of the midpiece highlighting the connection (arrowhead) between the

proximal and distal centriole. (I) Cross section of the midpiece highlighting the pericentriolar apparatus (arrowhead). (J) Cross section of the midpiece

with six mitochondria. (K) Longitudinal section of the flagellum. (L) Cross section of the flagellum. The arrowhead indicates the microtubules duplete. Cillia (c); Follicle (F); flagellum (f); Gastrodermal epithelium (Ge); Golgi complex (gc); Mitochondria (M); Mesoglea (Me); Nucleus (N);

Nucleolus (nu); spermatogonia (sg); Spermatocyte (sc); sperm (sz); Substance (Su); vesicle (v).

87

Figure 5

Figure 5: Ultrastructure of male gonad in Chiropsalmus quadrumanus. (A) Cells of the gastrodermal epithelium, under transmission electronic

microscopy, with several large vesicles containing granular secretion. (B) Detail of the apical portion of the gastrodermal epithelium with cilia. (C)

Detail of the Golgi complex with the electrondense vesicles. (D) Two follicles immersed in the electrondense mesoglea. Between them can be noted the granular substance. (E) Detail of the granular substance (arrowhead) immersed in the mesoglea, found between the follicles. (F) Detail of the

spermatogonia. (G) Detail of the spermatocyte in telophase. (H) Release of the sperm by rupture of the follicle wall. Cillia (c); Follicle (F); Follicular

epithelium (Fe); Gastrodermal epithelium (Ge); Golgi complex (gc); Mitochondria (M); Mesoglea (Me); Nucleus (N); Nucleolus (nu); sperm (sz); vesicle (v).

88

Figure 6

Figure 6: Spermatogenesis in Chiropsalmus quadrumanus. (A) Several sperm inside the follicle. (B) General morphology of the sperm (longitudinal

section). (C) Detail of the small vesicles in the lateral of the nucleus. (D) Detail of the small vesicles near the midpiece. (E) Electrondense vesicle in the anterior region of the sperm head. (F) Longitudinal section of the midpiece highlighting the pericentriolar apparatus (arrowhead). (G) Longitudinal

section of the midpiece showing the proximal and distal centrioles. (H) Cross section of the midpiece highlighting the pericentriolar apparatus and the poli-ribosomes in the cytoplasm (arrowhead). (I) Detail of the pericentriolar apparatus. (J) Longitudinal section of the midpiece highlighting the

pericentriolar apparatus (arrowhead). (K) Cross section of the midpiece with six mitochondria. (L) Longitudinal section of the flagellum. (M) Cross

section of the flagellum. Primary process (1); Distal centriole (d); Flagellum (f); Mitochondria (M); Nucleus (N); Proximal centriole (p); Pericentriolar apparatus (pa); Sperm (sz); Substance (Su); vesicle (v).

89

Figure 7

Figure 7: Spermatogenesis in Carukia barnesi. (A) General gonad location. (B) Hematoxylin and Eosin. Longitudinal section of a rudimentary

gonad. The black arrow indicates the vesicles in the gastrodermal epithelium and the white arrow the substance inside the follicles. (C) Hematoxylin

and Eosin. Longitudinal section of a mature gonad, highlighting the “fingerprint” pattern. The black arrow indicates the vesicles in the gastrodermal epithelium and the white arrow the substance inside the follicles. (D) Toluidine Blue. Detail of gastrodermal epithelium with the follicles immersed in

the mesoglea positive to acid compounds. In the black arrow the gastrodermal vesicles with negative reaction to acid compounds. (E) Mercuric

Bromophenol Blue. In the gastrodermal epithelium is noted several vesicles that show negative reaction to the staining (black arrow). (F) PAS. The same mesoglea has positive reaction to neutral polysaccharides, but the vesicles in the gastrodermal epithelium does not. (G) The sperm present a

basophilic head and acidophilic midpiece and flagellum. The black arrow shows that the substance inside the follicle has negative reaction to the

staining. The sperm have positive reaction to Toluidine Blue (H), to basic proteins (I), not presenting reaction to PAS (J). In the black arrow note the substance with negative reaction to all staining. Follicle (F), Gastrodermal epithelium (Ge), Gonad (Go); Mesoglea (Me); Spermatogonia (sg), Sperm

(sz).

90

Figure 8

Figure 8: Spermatogenesis in Chironex fleckeri. (A) Hematoxylin and Eosin. Longitudinal section of the male gonad, highlighting the “fingerprint”

pattern. The black arrow indicates the substance inside the follicles with no reaction to the staining. B) Hematoxylin and Eosin. Cross section of the male gonad. The black arrow indicates the vesicles in the gastrodermal epithelium with no reaction to the staining. The white arrow indicates the

substance inside the follicles with no reaction to the staining (C) Hematoxylin and Eosin. Detail of the residual cytoplasmic substance inside the

follicle (black arrow) and the vesicles in the gastrodermal epithelium (white arrow), both with no reaction to the staining. (D) Toluidine Blue. Detail of the substance inside the follicle (black arrow) with positive reaction to acid compounds. (E) Mercuric Bromophenol Blue. The substance inside the

follicles has negative reaction to basic proteins and the sperm head positive. (F) Ponceau Xylidine. The substance inside the follicles has negative

reaction to total proteins and the sperm head positive. (G) PAS. The secretion inside the follicles and the sperm head have negative reaction to neutral polysaccharides. Follicle (F), Gastrodermal epithelium (Ge), Mesoglea (M); spermatogonia (sg), Sperm (sz).

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Figure 9

Figure 9: Ultrastructure of spermatogenesis in Chironex fleckeri. (A) General view of the gastrodermal and follicular epithelium. The arrow indicates the granular secretion inside the large vesicles of the gastrodermal epithelium. (B) Detail of the previous image indicating the large vesicles of the

gastrodermal epithelium (arrow). Between the gastrodermal epithelium and the follicular epithelium exists a substance of granular aspect. (C) Detail

of the follicular epithelium, with spermatogonia and the substance of granular aspect between the gastrodermal epithelium and the follicular epithelium. (D) Detail of the Golgi complex with vesicles of electrondense material. (E) Sperm clustered in the center of the follicle. (F)

Electrondense vesicles in the anterior region of the sperm head (arrow). (G) Detail of the pericentriollar apparatus and the distal centriole in

longitudinal section. (H) Cross section of the mitochondria. (I) Cross section of the flagellum. (J) Detail of the anterior micrograph highlighting the flagellum organization. Distal centriole (d); Follicle (F); Follicular epithelium (Fe); flagellum (f); Gastrodermal epithelium (Ge); Golgi complex (gc);

Mesoglea (Me); Mitochondria (M); Nucleus (N); Nucleolus (nu); Substance (Su); Spermatogonia (sg); sperm (sz).

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Figure 10

Figure 10: Spermatogenesis in Chiropsella bronzie. (A) Hematoxylin and Eosin. Longitudinal section of a rudimentary gonad highlighting the

“fingerprint” pattern. The arrow indicates the vesicles in the gastrodermal epithelium. (B) Hematoxylin and Eosin. Longitudinal section of a mature

gonad. The arrow indicates the vesicles in the gastrodermal epithelium. (C) Cross section of the male gonad. In the follicular epithelium note the spermatogonia with an elongated nucleus and organized in more than one layer. The arrow indicates the vesicles in the gastrodermal epithelium. (D)

Sperm being released by rupture of the follicle wall (arrow). (E) In the gastrodermal epithelium is noted several vesicles that shown negative reaction

to the staining (black arrow). (F) Toluidine blue. Detail of the substance (arrow) founded between the follicles and with no reaction to acid compounds. (G) Mercuric bromophenol blue. The sperm head present positive reaction to basic proteins (H) Ponceau Xylidine. The sperm head

present positive reaction to total proteins. The arrow indicates the substance with negative reaction to the staining. (I) PAS. The sperm head present

negative reaction to neutral polysaccharides. Follicle (F), Gastrodermal epithelium (Ge), Spermatogonia (sg), Spermatocyte (sc), Sperm (sz).

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Figure 11

Figure 11: Ultrastructure of spermatogenesis in Chiropsella bronzie. (A) General view of the gastrodermal and follicular epithelium. The black arrow indicates the large vesicles with granular material and the white arrow the mesoglea. (B) General view of the mature follicle. The white arrow

indicates the mesoglea. (C) Electrondense vesicles in the anterior region of the sperm head (arrow). (D) Detail of the pericentriolar apparatus (arrow)

in longitudinal section. In the arrowheads note the small vesicles in the midpiece. (E) Cross section of the mitochondria. The arrow indicates the pericentriolar apparatus between the mitochondria. (F) Cross section of the flagellum. (G) In the cytoplasm is observed the presence of the Golgi

complex. Distal centriole (d); Follicle (F); flagellum (f); Gastrodermal epithelium (Ge); Golgi complex (gc); Mitochondria (M); Nucleus (N); sperm

(sz).

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Capítulo 3

Histochemistry and ultrastructure of spermatogenesis in Chrysaora lactea,

Lychnorhiza lucerna and Cassiopea sp.

Gisele R. Tiseo1*

; Fernando J. Zara2; Peter L. Harrison

3, Jamie Seymour

4 and

André Carrara Morandini1

1 Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Rua

do Matão, travessa. 14, n. 101, Cidade Universitária, São Paulo CEP 05508-090, SP,

Brazil;

2 Departamento de Biologia Aplicada, Laboratório de Morfologia do Invertebrados

(IML), Centro de Aquicultura (CAUNESP) e IEAMar, Univ. Estadual Paulista

(UNESP), Prof. Donato Castellane, S/N, Jaboticabal 14884-900, SP, Brazil;

3 Marine Ecology Research Centre, School of Environment, Science and Engineering,

Southern Cross University, PO Box 157, 2480, Lismore, NSW, Australia;

4 Australian Institute for Tropical Health and Medicine, Centre for Biodiscovery &

Molecular Development of Therapeutics, Division of Tropical Health and Medicine,

James Cook University, Cairns Campus, Mcgregor Road, 4878, Cairns, Australia.

Running Title: Spermatogenesis of some Scyphozoa species

*Corresponding author. GRT, Tel: +55 11 30917621; +55 11 973084694

[email protected]

E-mail addresses: [email protected] (GRT); [email protected] (FJZ);

[email protected] (PLH); [email protected] (JS);

[email protected] (ACM).






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Abstract

Here is described the spermatogenesis of Chrysaora lactea, Lychnorhiza lucerna and

Cassiopea sp. under light microscopy, histochemistry and ultrastructure. Specimens of

C. lactea and L. lucerna were collected in four localities in São Paulo coast, SE Brazil.

Polyps from Cassiopea sp. were collected in Weipa County, Queensland, Australia, and

maintained in tanks until the jellyfishes develop gonads. For light microscopy, the male

gonad was removed and fixed in paraformaldehyde 4% in sodium phosphate buffer

0,2M (pH 7, 2) for 24 hours and posteriorly processed for protocol in historesin. Serial

sections were performed and were stained with hematoxylin and eosin and techniques

for proteins (mercuric bromophenol blue and ponceau xylidine), acid materials

(toluidine blue) and neutral polysaccharides (PAS). For transmission electronic

microscopy (TEM) fragments of 1mm3 of the male gonad were preserved in Karnovsky

solution (2.5% glutaraldehyde with 2% paraformaldehyde in 0.1 M sodium cacodylate

buffer, pH 7.4, 2.5 mM of CaCl2 and saccharose) or in Glutaraldehyde fixative solution

(2.5% glutaraldehyde 0.1 M sodium cacodylate buffered Millipore filtered seawater, pH

7.2-7.4) posteriorly processed for protocol in TEM. The male gonad of C. lactea, L.

lucerna and Cassiopea sp. is composed of a folded germinal epithelium divided in two

layers: the external and internal. The spermatogenesis is asynchronous in C. lactea and

L. lucerna and synchronous in Cassiopea sp. The spermatogonia are located in the

periphery of the follicle, and are bulky with prominent nucleolus. The spermatocytes

have rounded nucleus and meiotic figures could be seen. The spermatids are connected

by intercellular bridges and the sperm are located in the center of the follicles. In C.

lactea and L. lucerna the sperm are release by rupture of the follicle wall. On the

contrary, Cassiopea sp. releases sperm in spermatozeugmata. The sperm of all species

have electrondense nucleus, little electrondense vesicles above the nucleus and in the

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midpiece, have four mitochondria, proximal and distal centrioles and pericentriolar

apparatus. The flagellum has the typical microtubules pattern 9+2.

Key-words: Spermatogenesis, Scyphozoa, jellyfish, Histology, TEM.

Introduction

The phylum Cnidaria is composed of two monophyletic subphyla: Anthozoa and

Medusozoa. Among Medusozoa there are the classes Hydrozoa, Staurozoa, Cubozoa

and Scyphozoa, being this last one composed of the so-called true jellyfishes (Bridge et

al. 1995; Odorico and Miller 1997; Collins 2002; Collins et al. 2006; Daly et al. 2007;

Collins 2009; Kayal et al. 2013; Zapata et al. 2015). From the 200 scyphozoans

distributed around the world, only 22 are found in Brazil and 38 in Australia (Mianzan

and Cornelius 1999; Marques et al. 2003; Gershwin and Zeidler 2008). The

scyphozoans Lychnorhiza lucerna and Cassiopea sp. belongs to the order

Rhizostomeae, while the species Chrysaora lactea belongs to the order Semaeostomeae

(Kramp 1961; Marques et al. 2003; Morandini et al. 2005; Morandini and Marques

2010). Chrysaora lactea is one of the 16 species of the genus distributed in the Gulf of

Mexico, and from Caribbean to Argentina (Morandini and Marques 2010). Lychnorhiza

lucerna is one of the three species of the genus (Kramp 1961; Morandini et al. 2005;

Morandini 2009) being endemic to Western South Atlantic (Morandini et al. 2005;

Schiariti et al. 2012). Cassiopea sp. is a benthic jellyfish with wide distribution in

shallow tropical coastal waters exhibiting the behavior of rest upside down on the

substrate and possesses endosymbiotic dinoflagellates (zooxanthellae) (Templeman and

Kingsford 2010; Morandini et al. 2016).

Despite the several studies related to reproductive biology of Scyphozoa

(Afzelius and Franzén 1971; Lucas and Lawes 1998; Rouse and Pitt 2000; Morandini

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and Silveira 2001; Hofmann and Hadfield 2002; Lucas and Reed 2010), there are a few

works describing the spermatogenesis if considering the scyphozoan species diversity.

The spermatogenesis was already described for some species of the orders Coronatae

and Rhizostomeae (Kikinger 1992; Morandini and Silveira 2001; Lucas and Reed 2010;

Tiemann and Jarms 2010; Ikeda et al. 2011a; Schiariti et al. 2012).

Most scyphozoan are gonochoric but there are some cases of hermaphroditism

(Berrill 1949; Widersten 1965; Fautin 1992). The Semaeostomeae male gonad is

organized as a semicircle and can be reached through the subgenital ostia, they are great

folded rimming the gastric filaments (Morandini and Marques 2010). The gonad in most

Rhizostomeae is described as a cross-shaped structure in which each arm of the cross is

a band-like evagination of the gastrodermis forming several folds. The gonads are two-

layered in cross-section with a typical genital epithelium (Kikinger 1992; Schiariti et al.

2012). The male gonad is originated from the interstitial cells of the gastrodermis,

transported to the mesoglea and forming the male follicles (Miller 1983; Harrison and

Jamieson 1988). The spermatogenesis process is well known with the cellular types:

spermatogonia (pre-meiotic phase), primary spermatocytes (meiotic phase); secondary

spermatocytes, that divide into two spermatids that lost cytoplasmic volume turning into

sperm (Gaino et al. 2013).

The Cnidaria sperm consists of a head, midpiece and long flagellum. The shape

of the head is largely determined by the form of the nucleus, which may vary from

ovoid to conical or elongated. The cnidarian sperm lack the acrosome but present

several small electrondense vesicles in the apical region of head (Chapman 1974;

Harrison and Jamieson 1988). The midpiece has four or more mitochondria, could

present lipid and Golgi derivatives, the proximal and distal centrioles and the

pericentriolar apparatus anchoring the flagellum (Afzelius and Franzén 1971; Afzelius

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1971; Hinsch and Clarck Jr. 1973; Miller 1983; Harrison and Jamieson 1988; Rouse and

Pitt 2000; Corbelli et al. 2003).

The present work describe the gonadal structure, the spermatogenesis and the

sperm ultrastructure of two Brazilian scyphozoan species: Chrysaora lactea and

Lychnohiza lucerna and one Australian scyphozoan: Cassiopea sp. Additionally, we

compare the sperm ultrastructure of the studied species with the sperm of other

scyphozoans, looking for common characteristics.


Specimens of C. lactea and L lucerna were collected in four localities of São

Paulo coast (Figure 1) from March 2007 to June 2015. The collection was made by

hand at the water surface or with shrimp’s trawls. The animals was transported alive to

the laboratory and identified according to Morandini et al. (2005). Polyps of Cassiopea

sp. were collected at Weipa County, Queensland, Australia (Figure 2) and transported

alive to the Laboratory of Culture of the James Cook University in Cairns. The

individuals were kept inside the laboratory in circular aquariums until the ephyra was

released by strobilation. The mature jellyfishes were maintained in tanks with local

substrate of the collection site and were fed every day.

The animals were anesthetized by thermal shock (-20°C) for 5 minutes. The

male gonad of five individuals of C. lactea, L. lucerna and Cassiopea sp. was fixed in

4% formaldehyde buffered with a saline solution of 0.2 M sodium phosphate (pH 7.2)

for 24 hours. After fixation, the samples were buffered in sodium phosphate 0.2M (pH

7.2) for 24 hours. Later, the samples were dehydrated in ethanol series (30-95%) and

embedded in Leica® methacrylate historesin. Serial sections (3-4 μm thick) were made

on a rotative microtome. For traditional histological description the tissue samples were

99

stained with hematoxylin and eosin (HE) (modified from Junqueira and Junqueira

1983), avoiding baths in ethanol and xylene (Sant’Anna et al. 2010; Zara et al. 2012).

To analyze the chemical composition of the tissues, the slides were stained with

mercuric bromophenol blue and ponceau xylidine for proteins (Pearse 1960; Mello and

Vidal 1980), PAS-Hematoxylin technique to visualize neutral polysaccharides with the

groups 1-2-glycol (Junqueira and Junqueira 1983) and the toluidine blue to visualize

acid components (Modified from Audino et al. 2015).

For transmission electronic microscopy, fragments of 1mm3 of the male gonad

of C. lactea and L. lucerna were preserved in Karnovsky solution (modified from

Karnovsky, 1965) consisting of 2.5% glutaraldehyde with 2% paraformaldehyde in 0.1

M sodium cacodylate buffer (pH 7.4) with 2.5 mM of CaCl2 and saccharose for 24

hours; and samples of male gonad of Cassiopea sp. were fixed in Glutaraldehyde

fixative solution consisting of 2.5% glutaraldehyde 0.1 M sodium cacodylate buffered

Millipore filtered seawater (pH 7.2-7.4) for 30 minutes at 4ºC. The post-fixation was in

1% osmium tetroxide with the same buffer, for one hour at the same temperature. Then,

the samples were dehydrated in ethanol series 30 to 100%, and embedded in Spurr’s®

resin. Semi-thin and ultrathin sections were obtained with a Leica® UC-7

ultramicrotome from the Electronic Microscopy Laboratory of the Department of

Genetics, Institute of Biosciences of the University of São Paulo, Brazil. Grids with

sections were contrasted with uranyl acetate and lead citrate, and later examined at

80kV.

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Results

Spermatogenesis in Chrysaora lactea

The germinal epithelium of C. lactea is organized like in two layers and between

them is the mesoglea with the sperm follicles, with cells in different phases of

development, highlighting an asynchronous spermatogenesis (Fig. 3A-C). The external

layer of germinal epithelium is composed by cells of irregular format with nucleus in

different positions of cytoplasm (Fig. 3C). The mesoglea is acidophilic being slightly

positive to hematoxylin (Fig. 3B-C). The spermatogonia are located in the periphery of

the follicles (Fig. 3C-D). The spermatocytes reduce the nuclear diameter, have strongly

acidophilic nucleus (Fig. 3C), and can present nucleus in different meiotic phases (Fig.

3D). The spermatids reduce the cytoplasmic volume; have a basophilic flagellum, an

acidophilic nucleus (Fig. 3C), positive to acid structures (Fig. 3D). The sperm have

rounded basophilic head and acidophilic flagellum located in the central portion of the

follicle (Fig. 3E-G). The sperm head is strongly positive to acid structures (Fig. 3H),

positive to proteins (Fig. 3I-J), and negative to PAS (Fig. 3K). The follicles in more

advanced stages have ovoid shape being filled almost completely by sperm (Fig. 3E).

When mature the sperm is released through rupture in the follicle wall (Fig. 3F).

Under transmission electronic microscopy the external layer of germinal

epithelium is composed of cells with electrondense nucleus and several vesicles

containing a proteic material, Golgi complex and mitochondria (Fig. 4A). In the apical

portion can be seen cilia (Fig. 4A). The follicular epithelium is composed by

spermatogonia and the sperm are found in the center of follicle (Fig. 4B-C). The

spermatogonia have an elongated nucleus with euchromatin and proeminet nucleolus

(Fig. 4D). The spermatids reduce the cytoplasmic volume and are connected to each

other by intercellular bridges (Fig. 4E). The sperm of C. lactea have an ovoid head with

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electrondense nucleus (Fig.4F). Inside the nucleus can be noted a nuclear vesicle of

intermediate electrondensity (Fig.4F). Above the nucleus and in the midpiece are

observed little electrondenses vesicles (Fig. 4G-H). In the midpiece is observed 4

mitochondria with several crests (Fig. 4I), between them is the proximal centriole (Fig.

4J) and below the distal centriole (Fig. 4K). The pericentriolar apparatus is a complex

structure with primary, secondary and tertiary process (Fig. 4L-M). The flagellum is

anchored by the pericentriolar apparatus (Fig. 4M) and presents a simple axoneme with

the typical microtubules pattern of 9+2 (Fig. 4N-O).

Spermatogenesis in Lychnorhiza lucerna

The germinal epithelium is organized in two layers and between them is the

mesoglea with several follicles, in which, occurs the spermatogenesis (Fig. 5A). The

follicles have an ellipsoid format and inside them can be seen cells in all stages of

development, highlighting an asynchronous spermatogenesis (Fig.5B-D). Between and

inside the follicles can be seen the Sertoli cells (Fig. 5C-F). The spermatogenesis begins

in the periphery of the follicles (Fig. 5A-C). The spermatogonia are large cells with

well-developed rounded nucleus (Fig. 5C). The spermatocytes have a smaller and

basophilic nucleus (Fig. 5B-C), and is usual the presence of cells in different meiosis

phases (Fig. 5D-E). The spermiogenesis process begins with the spermatids,

characterized by rounded, homogeneous and basophilic nucleus (Fig. 5C). When stained

with toluidine blue, the nucleus presents α methacromasy (blue–greenwish) due the

DNA compaction (Fig. 5D). The sperm have rounded basophilic head and acidophilic

midpiece and flagellum (Fig. 5G-I). The rounded nucleus is positive to proteins (Fig.

5J-K), and negative to PAS-Hematoxylin (Fig. 5L). When mature, the sperm are

released by rupture of the follicle wall (Fig.5G-H, and M).

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Under transmission electronic microscopy, the germinal epithelium is

characterized by cells of irregular shape, with elongated nucleus and evident nucleolus

(Fig. 6A). The cytoplasm has many vesicles and in the apical portion is observed cillia

(Fig. 6A-B). Adjacent to the external germinal epithelium is the follicular epithelium,

characterized by the presence of cells of bulky and electrondense nucleus: the

spermatogonia (Fig. 6C). The sperm are clustered in the center of the follicle (Fig.6C-

D), have ovoid electrondense nucleus (Fig. 6E). Above the nucleus and in the midpiece

can be seen little electrondense vesicles (Fig. 6F-G). In the midpiece can be seen the

proximal and distal centrioles (Fig. 6H). From the distal centriole is the pericentriolar

apparatus (Fig. 6I-K). Above the pericentriolar apparatus are the Golgi complex (Fig.

6L) and four mitochondria (Fig. 6M). The flagellum presents a simple axoneme with the

typical microtubules pattern 9+2 (Fig.6N-O).

Spermatogenesis in Cassiopea sp.

The male gonad in Cassiopea sp. is a differentiation of the gastrodermis

epithelium and is composed by the external layer of germinal epithelium, which lies in

the mesoglea. It is inside the mesoglea that spermatogenesis takes place, with follicles –

in a general way – composed by cells in the same development stage (Fig. 7A). Below

the mesoglea is the internal germinal epithelium, followed by a space in which the

sperm – clustered by a secretion (spermatozeugmata) – are released (Fig. 7A-B).

Adjacent to this space is the gastrodermal epithelium composed by the external and

internal layers; between them is the cellular mesoglea with several zooxantellae

associations (Fig. 7A-B). The external germinal epithelium is a simple columnar

epithelium composed of cells juxtaposed to each other, with rounded basophilic nucleus

and acidophilic cytoplasm with large vesicles. These vesicles showing no reaction to

hematoxylin and eosin (Fig. 7C). The follicular epithelium is composed by

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spermatogonia organized in centers (Fig. 7C-E). The sperm is located in the center of

follicle and is surrounded by a secretion not stained by the hematoxylin and eosin (Fig.

7C). This secretion shows absence of reaction to acid compounds (Fig. 7D), proteins

(Fig. 7E) and neutral polysaccharides (Fig. 7F). However, the sperm present strong

reactivity to acid compounds (Fig. 7D), positive reaction to proteins (Fig. 7E) and

negative reaction to PAS (Fig. 7F). When mature, all sperm are clustered by this

secretion and the whole structure, called spermatozeugmata, is released in the space

between the germinal and gastrodermal epithelium (Fig. 7F). The secretion of the

spermatozeugmata is characterized by a secretion not reactive to any of the tested

histochemical compounds that maintain the sperm head attached and a free flagellum

(Fig. 7G-J).

Under transmission electronic microscopy the external germinal epithelium is

composed of cells with large and electrondense nucleus and several large vesicles

containing a granular material of intermediate electrondensity (Fig. 8A). The follicular

epithelium is composed by spermatogonia and in the center the sperm is immersed in a

secretion of granular material (Fig.8B-C). The sperm of Cassiopea sp. have an

elongated head with electrondense nucleus (Fig. 8D). In the midpiece is observed

mitochondria with several crests, between the distal centriole (Fig. 8E-H), proximal

centriole (Fig. 8F), little vesicles adjacent to the mitochondria (Fig. 8F-G) and lamellas

(Fig. 8G). The flagellum is anchored by the pericentriolar apparatus (Fig. 8G) and

presents a simple axoneme with the typical microtubules pattern of 9+2 (Fig. 8I-J).

Discusssion

In all studied species, the male gonad is composed of a germinal epithelium

divided in two layers with mesoglea in between them. In the mesoglea is found the

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follicles in which occurs the spermatogenesis. In the scyphozoans Atolla wyvillei,

Periphylla periphylla, Cotylorhiza tuberculata and Nausithoe aurea the male gonad is

described as an evagination of gastrodermis surrounded by the mesoglea (Kikinger

1992; Morandini and Silveira 2001; Lucas and Reed 2010). The germinal epithelium of

all species here studied is similar to the gastrodermal epithelium. We supposed that

because the germinal epithelium is derived from interstitial cells of gastrodermis.

The follicles have ellipsoid shape in L. lucerna and C. lactea as observed in

other Scyphozoa: for L. lucerna by Schiariti et al. (2012), Nemopilema nomurai (Ikeda

et al. 2011a), Atolla wyvillei and Periphylla periphylla (Lucas and Reed 2010). In

Cassiopea sp. the follicles have circular shape as already described to Cotylorhiza

tuberculata (Kikinger 1992) and other Cassiopea species (Smith 1936; Gohar and

Eisawy 1960). The follicles have spermatogonia in the follicular periphery organized in

one or more layers. In Cassiopea sp., L. lucerna and C. lactea the more developed

follicles present spermatogonia organized in germination centers and sperm in the

center like in Na. aurea (Morandini and Silveira 2001). The Sertoli like cells were

found only in Lychnorhiza lucerna and this is the first record of this cell type for

Scyphozoa. For all species in this study, the mature follicles have irregular format with

the sperm accumulating near the internal gastrodermal epithelium. In L. lucerna and C.

lactea, like in Na. aurea and Ne. nomurai (Morandini and Silveira 2001; Ikeda et al.

2011), the sperm release occurs through rupture of the follicular wall and this seems to

be the pattern in other Cnidaria (Miller 1983; Harrison and Jamieson 1988) as the

cubozoans Tamoya haplonema, Chiropsalmus quadrumanus, Chiropsella bronzie

(Tiseo et al. 2016, chapters 1 and 2 of this dissertation) and Morbakka virulenta

(García-Rodriguez 2015). However, there are other strategies of sperm released in

cnidarians: the spermartozeugmata and spermatophore. These are observed in

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Cassiopea species (Smith 1936; Gohar and Eisawy 1960) and Co. tuberculata (Kikinger

1992) and some cubozoan species as already presented in the previous chapters of this

dissertation. In Table 1 we compared all described strategies of sperm release in

scyphozoans and cubozoans.

In Cassiopea sp. all the sperm heads are immersed in a secretion forming the

structure called spermatozeugmata. This structure was already described for other

Cassiopea species and also for Cotylorhiza tuberculata (Kikinger 1992). A similar

structure was observed by Smith (1936) and Gohar and Eisawy (1960) for the species

Cassiopea frondosa and Cassiopea andromeda, but these authors did not call it

spermatozeugmata. The secretion found in the spermatozeugmata structure presented

negative reaction to all of histochemical staining used and if we had not observed, in the

TEM, the proteic composition of this secretion, we could argue that it could be

composed of lipids that could be lost in the dehydration. Here we also observed that the

spermatozeugmata secretion is produced by the follicular epithelium. Once all sperm are

mature, they are clustered in this secretion and then, released as a single structure.

In the TEM preparations, the spermatogonia are large cells, with well-developed

nucleus and nucleolus. The spermatocytes have rounded shape and the spermatids can

be united by intercellular bridges. This typical description of spermatogenic cells is

similar to that described for the scyphozoan Ne. nomurai (Ikeda et al. 2011) and for the

hydrozoan Distichopora sp. (Gaino et al. 2013). The cnidarian’s sperm do not have an

acrosome but, can present several electrondense small vesicles (Miller 1983; Harrison

and Jamieson 1988) as here observed in L. lucerna and C. lactea. Hinsch and Clarck Jr.

(1973) also described these electrondense vesicles in A. aurita and suggested that they

can be the precursor of the acrosome as Hinsch (1974) commented. Small vesicles were

described in the midpiece of Cassiopea sp. sperm as well as in the cubozoa Carybdea

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marsupialis (Corbelli et al. 2003). The sperm of all species here studied have an

electrondense nucleus, with granular chromatin, but only Chrysaora lactea have the

nuclear vesicle already described to Hydra caulinata. (Moore and Dixon 1972). The

sperm of all species here studied have four mitochondria in the midpiece and the typical

flagellum pattern (with 9+2 microtubules). Only half of pieces of the anchoring

apparatus are presented and in Cassiopea sp. could be observed the primary, secondary

and tertiary process. No significant differences between the main sperm morphology of

the different species were noted. But scyphozoan pattern of the sperm morphology

could be noted. See table 2 for the details of sperm in comparison. The main

morphology of the sperm is very similar to that described for A. aurita (Hinsch and

Clarck Jr. 1973) and Nausithoe sp. (Afzelius and Franzén 1971).

Acknowledgement

This work was supported by São Paulo Research Foundation (FAPESP)[GRT grants

2012/19080-1 and 2014/08785-1, FJZ grants BIOTA 2010/50188-8, ACM grants

2010/50174-7, 2011/50242-5, 2013/50484-4]; CAPES [ACM 23038.004309/2014-51];

and CNPq [ACM (301039/2013-5, 486337/2013-8)]. Thanks are due to Lea Taylor,

Maxine Daves and Waldir Caldeira for all help in the transmission electronic

microscopy and to anonymous reviewers for all comments and suggestions. This is a

contribution of NP-BioMar, USP. This study was conducted in accordance with

Brazilian laws (GRT SisBio license No. 611377-9; FJZ-MMA SisBio permanent license

No. 34587-1; ACM MMA SisBio permanent license No. 15031-2).

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Tables

Table 1: Reproductive strategies among members of classes Scyphozoa and Cubozoa.

Class Order Family Specie Sperm release Reference

Scyphozoa

Coronatae

Nausithoidae Nausithoe aurea Free sperm Morandini and Silveira, 2001

Periphylidae Periphylla periphylla Free sperm Lucas and Reed, 2010

Atollidae Atolla wyvillei Free sperm Lucas and Reed, 2010

Rhizostomeae

Lychnorhizidae Lychnorhiza lucerna Free sperm Schiariti et al., 2012; This study

Rhizostomatidae Rhizostoma pulmo Free sperm Paaspaleff, 1938; Widersten, 1965

Stomolophidae Nemopilema nomurai Free sperm Ikeda et al., 2011

Cassiopeidae

Cassiopea andromeda Spermatozeugmata Hoffmann and Hadfield, 2002

Cassiopea sp. Spermatozeugmata This study

Cepheidae Cotylorhiza tuberculata Spermatozeugmata Kikinger, 1999

Mastigiidae Phyllorhiza punctata Spermatozeugmata Rouse and Pitt, 2000

Catostylidae Catostylus mosaicus Spermatozeugmata Rouse and Pitt, 2000

Semaeostomeae Pelagiidae Chrysaora lactea Free sperm This study

Cubozoa Chirodropida Chiropsalmidae Chiropsalmus quadrumanus

Free sperm García-Rodríguez, 2015; Tiseo et al., 2016 -

chapter 1 and 2

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Chiropsella bronzie Free sperm Tiseo et al., 2016 - chapter 2

Chirodropidae Chironex fleckeri Free sperm Tiseo et al., 2016 - chapter 2

Carybdeida

Tamoyidae Tamoya haplonema Free sperm

García-Rodríguez, 2015; Tiseo et al., 2016 -

chapter 1

Carybdeidae Tripedalia cystophora Spermatozeugmata Werner, 1973

Tripedaliidae Copula sivickisi Spermatophore Garm et al., 2015; García-Rodríguez, 2015

Alatinidae Alatina alata Spermatozeugmata García-Rodríguez, 2015

Carukiidae

Morbakka virulenta Free sperm Toshino et al., 2013

Carukia barnesi Free sperm Tiseo et al., 2016 - chapter 2

Classification according to Kramp (1961) and Bentlage et al. (2010).

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Table 2: Comparison between the sperm morphology of the studied species (Lychnorhiza lucerna, Chrysaora lactea and Cassiopea sp.) and other

Scyphozoa.

Rhizostomeae Semeaostomeae Coronatae

Characteristics Lychnorhiza

lucerna 1

Rhizostoma

pulmo 2

Cassiopea sp. 1

Chrysaora

lactea 1

Chrysaora

hysoscella 2

Aurelia autita 2;3

Nausithoe sp. 4

Head length 2.01 ± 0.41 - 1.9 ± 0.15 2.17 ± 0.61 - - -

Head diameter 2.05 ± 0.61 - 1.59 ± 0.29 1.89 ± 0.57 - - -

Midpiece length 0.92 ± 0.26 - 0.75 ± 0.11 0.95 ± 0.25 - - -

Midpiece diameter 2.40 ± 0.54 - 1.35 ± 0.19 2.32 ± 0.75 - - -

Nucleus length 1.9 ± 0.35 - 1.76 ± 0.17 1.88 ± 0.59 - - -

Nucleus diameter 1.6 ± 0.49 - 1.31 ± 0.21 1.61 ± 0.43 - - -

Head morphology Ovoid a*

Elongated b?

Elongated b*

Ovoid c*

Ovoid c?

Elongated b?

Ovoid c?

Nucleus ratio 1.29 ± 0.52 - 1.4 ± 0.29 1.22 ± 0.45 - - -

Nuclear vesicle Absent a*

Not observed b?

Absent a*

Present c*

Not observed b?

Absent a?

Present c

Chromatin Granular a Granular

a? Granular

a Granular

a Granular

a? Granular

a? Granular

a?

Outer nuclear membrane Absent a*

Not observed b?

Present c*

Absent a*

Absent a?

Absent a?

Absent a?

Number of Mitochondria 4 a Not observed

b? 4

a 4

a 4

a? 4

a 4

a

Vesicles anteriorly to the

nucleus Present

a* Present

a? Not observed

b* Present

a* Not observed

b? Present

a Present

a

Vesicles diameter 0.16 ± 0.02 - - 0.12 ± 0.04 - - -

Electrondense vesicle

lateral to the nucleus Absent

a* Absent

a? Present

b* Absent

a* Absent

a? Absent

a? Absent

a?

Proximal centriole Present a Present

a? Present

a Present

a Present

a? Not observed

b? Present

a

Distal centriole Present a Present

a? Present

a Present

a Present

a? Present

a Present

a

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Pericentriolar apparatus Present a Present

a? Present

a Present

a Present

a? Present

a Present

a

Primary process Present a*

Not observed b?

Not observed b*

Present a*

Not observed b?

Present a Present

a

Interprimary process Present a*

Not observed b?

Not observed b*

Present a*

Not observed b?

Present a Present

a

Major striated bands Present a*

Not observed b?

Not observed b*

Present a*

Not observed b?

Present a Present

a

Secondary process Present a*

Not observed b?

Not observed b*

Present a*

Not observed b?

Not observed b?

Present a

Tertiary Process Present a*

Not observed b?

Not observed b*

Present a*

Not observed b?

Not observed b?

Present a

Spurr Not observed a Not observed

a? Not observed

a Not observed

a Absent

b? Not observed

a? Absent

b?

Lamellas Not observed a Not observed

a? Not observed

a Not observed

a Not observed

a? Absent

b Absent

b?

Poli-ribosomes Present a Not observed

b? Present

a Present

a Not observed

b? Absent

c? Absent

c?

Golgi complex Present a*

Not observed b?

Present a*

Absent c*

Not observed b?

Absent c?

Absent c?

Vesicles in the midpiece Present a Not observed

b? Present

a Present

a Not observed

b? Absent

c Present

a

Structure of the flagellum 9 + 2 a 9 + 2

a? 9 + 2

a 9 + 2

a 9+2

a? 9+2

a 9+2

a

1 This study;

2 Hedwig and Schafer (1986);

3 Hinsch (1974);

4 Afzelius and Franzen (1974).

Different letters indicate variation between the species; * = difference only between the species of the present study; ? = Not described and observed in

the published figure.

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List of Figures

Figure 1

Figure 1: Sampling sites in São Paulo coast, SE Brazil. (1) Cananéia (25°03'604''S/ 47°54'419''E); (2) Santos and São Vicente Bay (23°59'33.97"S/46°22'12.64"E); (3) São Sebastião county (23°27' 5''S/

45°1'47''E;) (4) Ubatuba county (23°31'147''S/ 045°05'193''E).

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Figure 2

Figure 2: Sampling site in Queensland coast, Australia. (A) Queensland coast; (B) Detail of the Northwestern coast of Queensland

indicating Weipa county (12°38’57.38’’S ̸ 141°50’48.66’’E).

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Figure 3

Figure 3: Spermatogenesis in Chrysaora lactea. (A) Hematoxylin and Eosin. General view of the male gonad. (B) Hematoxylin and Eosin. The germinal epithelium is divided in external and internal germinal epithelium. The follicles are immersed in the mesoglea.

(C) Hematoxylin and Eosin. Detail of the spermatogonia, spermatocytes and spermatids. (D) Toluidine blue. Follicle with cells in

different stages of spermatogenesis. (E) Hematoxylin and Eosin. More advanced follicle with the sperm organized in the center (arrow). (F) Hematoxylin and Eosin. Sperm released by rupture of follicular wall (arrow). (G) Hematoxylin and Eosin. Detail of

sperm. (H) Toluidine blue. Sperm head positive to acid structures. (I) Mercuric Bromophenol blue. Sperm head strongly positive to

neutral proteins. (J) Ponceau Xylidine. Sperm head positive to total proteins. (K) PAS. Sperm head negative to neutral polysaccharides. External germinal epithelium (Eg); Follicle (F); Gastroderm (Ga); Internal germinal epithelium (Ig); Mesoglea

(Me); Spermatogonia (sg), spermatocyte (sc), Spermatid (st), Sperm (sz).

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Figure 4

Figure 4: Ultrastructure of spermatogenesis in Chrysaora lactea. (A) General view of the external germinal epithelium with cells of irregular format. In the apical portion of the cell note the cilia. (B) Detail of the follicular epithelium with spermatogonia, the sperm

clustered in the center. (C) The sperm in the center of the follicle. (D) Detail of the spermatogonia and spermatocytes. (E) Detail of

the spermatids connected by the intercellular bridges (arrow). (F) Longitudinal section of the sperm. Note the nuclear vesicle. (G) Detail of the sperm head emphasizing the small vesicles precursor of the acrosome. (H) Detail of the sperm midpiece with the small

electrondense vesicles. (I) Cross section of the midpiece with 4 mitochondria. (J) Detail of the midpiece, highlighting the proximal

centriole. (K) Detail of the midpiece, highlighting the distal centriole and the pericentriolar apparatus. (L) Cross section of the pericentriolar apparatus. Note the primary, secondary and tertiary process. In the arrowhead note the interprimary pr ocess. (M)

Longitudinal section of the pericentriolar apparatus. (N) Longitudinal section of the flagellum. (O) Cross section of the flagellum.

1= primary process; 2= secondary process; 3= tertiary process; Cillia (c) Distal centriole (d); Follicular epithelium (fe); Flagellum (f); Germinal epithelium (Ge); Golgi complex (gc); Mesoglea (Me); Mitochondria (M); Nucleus (N); Nucleolus (Nu); Nuclear

vesicle (nv); Proximal centriole (p); spermatogonia (sg); Spermatocyte (sc); spermatid (st); sperm (sz); vesicles (v).

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Figure 5

Figure 5: Spermatogenesis in Lychinorhiza lucerna. (A). Hematoxylin and Eosin. General view of the male gonad near the

gastroderm. The germinal epithelium is divided in external and internal germinal epithelium. (B) Hematoxylin and Eosin. Detail of the follicle with spermatogonia in the periphery and sperm in the center. (C) Hematoxylin and Eosin. Detail of the spermatogonia,

spermatocytes and spermatids. (D) Toluidine blue. Spermatocytes with elongated nucleus in different stages of meiosis (arrow

head). (E) Toluidine Blue. Spermatids with rounded and homogeneous nucleus near the Sertoli cell. (F) Hematoxylin and eosin. Detail of the spermatogonias, spermatocytes and Sertoli cells. (G) Hematoxylin and Eosin. Sperm being released through the rupture

of follicle wall. (H) Hematoxylin and Eosin. Detail of the sperm being released (arrow). (I) Hematoxylin and Eosin. Sperm with

rounded basophilic head and midpiece and flagellum acidophilic. (J) Mercuric Bromophenol Blue. The sperm presents positive reaction to basic proteins. (K) Ponceau Xylidine. Sperm with positive reaction to total proteins. (L) PAS-Hemaotxylin. Sperm with

negative reaction in the anterior region of the sperm. (M) Hematoxyln and eosin. The sperm being released. External germinal

epithelium (Eg); Follicle (F), Gastroderm (Ga), Internal germinal epithelium (Ig); Mesoglea (Me), Sertoli cell (sec); Spermatogonia (sg), spermatocyte (sc), Spermatid (st), Sperm (sz).

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Figure 6

Figure 6: Ultrastructure of spermatogenesis in Lychnorhiza lucerna. (A) General view of the external germinal epithelium with cells of irregular format and elongated nucleus in different positions of the cytoplasm. (B) In the apical portion of the cell note the

microvilli. (C) Detail of the follicular epithelium with spermatogonia. (D) The sperm in the center of the follicle. (E) Longitudinal

section of the sperm. (F) Detail of the sperm head emphasizing the small vesicles precursor of the acrosome. (G) Detail of the sperm midpiece with the little electrondense vesicles. (H) Detail of the midpiece, highlighting the proximal and distal centrioles. (I)

Oblique section of the pericentriolar apparatus. (J) Cross section of the percentriolar apparatus. Note the primary, secondary and

tertiary process. In the arrowhead note the interprimary process. (K) Oblique section of the pericentriolar apparatus and detail of the proximal centriole. (L) Golgi complex in the midpiece. (M) Cross section of the midpiece with 4 mitochondria. (N) Longitudinal

view of the flagellum. (O) Cross section of the flagellum. 1= primary process; 2= secondary process; 3= tertiary process; Cillia (c)

Distal centriole (d); Flagellum (f); Germinal epithelium (Ge); Golgi complex (gc); Mesoglea (Me); Mitochondria (M); Nucleus (N); Nucleolus (Nu); Proximal centriole (p); spermatogonia (sg); spermatid (st); sperm (sz); vesicles (v).

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Figure 7

Figure 7: Spermatogenesis in Cassiopea sp. (A) Hematoxylin and Eosin. General view of the male gonad adjacent to the

gastroderm. The black arrow indicates the secretion with absence of reaction to the staining. (B) Hematoxylin an Eosin. Detail of the gastroderm and mesoglea with zooxantellae. (C) Hematoxylin and Eosin. Mature follicle inside the male gonad. The black arrow

indicates vesicle inside the external germinal epithelial cells, also with absence of reaction to the staining. In the white arrow note

the follicular secretion with negative reaction to the staining. (D) Toluidine blue. Mature follicle with sperm immerse in a secretion with no reaction (black arrow). The white arrow indicates the secretion in the epithelium with no reaction to the staining. (E)

Mercuric Bromophenol Blue. Secretion inside the mature follicle with negative reaction to proteins (black arrow). (F) PAS. Mature

follicle with sperm and secretion with absence of reaction to neutral polysaccharides. (G) to (J) Detail of the spermatozeugmata. (G) Hematoxylin and Eosin. (H) Toluidine Blue. (I) Mercuric Bromophenol Blue. (J) PAS. External germinal epithelium (Eg); Follicle

(F); Gastroderm (Ga); Internal germinal epithelium (Ig); Mesoglea (Me); Secretion (Se); Spermatogonia (sg); sperm (Sz);

spermatozeugmata (spgt); Zooxantellae (Zo).

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Figure 8

Figure 8: Sperm ultrastructure of Cassiopea sp. (A) General view of the germinal epithelium with the large vesicles. (B) Mature

follicle containing several sperm immersed in the proteic secretion. (C) Detail of the previous image highlighting the conncetion

between the sperm and the secretion. (D) Longitudinal section of the sperm. (E) Detail of the midpiece with mitochondria and the distal centriole. (F) Detail of the midpiece with mitochondria, the proximal centriole and the vesicles. (G) Detail of the midpiece

highlighting the lamella, vesicle and pericentriolar apparatus (arrowhead). (H) Cross section of the midpiece with 4 mitochondria. (I) Longitudinal section of the flagellum (J) Transversal section of the flagellum. 1= primary process; 2= secondary process; 3=

tertiary process; Distal centriole (d); Follicular epithelium (Fe); Flagellum (f); Germinal epithelium (Ge); Lamella (l); Mitochondria

(M); Nucleus (N); Proximal centriole (p); Secretion (Se); sperm (sz); vesicles (v).

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Capítulo 4

Considerações Finais

Nesta dissertação foi apresentada a estrutura gonadal, a espermatogênese e foi

descrito o espermatozoide de cinco espécies da classe Cubozoa e três espécies da classe

Scyphozoa. Estes resultados são de ampla contribuição para o entpendimento da

reprodução sexuada desses organismos.

A idéia inicial do trabalho era descrever a morfologia do espermatozoide de

forma comparativa e buscando esclarecer possiveis relações filogenéticas dos

reprentantes de ambas as classes. Como a espermiotaxonomia tem sido utilizada para

evidenciar diferenças entre os espermatozoides de espécies próximas, e para membros

de Scyphozoa e Cubozoa, quase não se tem dados da morfologia ultraestrutural dos

espermatozoides, o foco principal eram os detalhes ultraestruturais dos gametas

masculinos. Para tal, visando obter resultados ultraestruturais de qualidade, o esforço

amostral foi grande, utilizando a coleta de amostras de animais vivos e de rapida

preservação. Uma vez que o esforço amostral já era grande, foram coletadas amostras

tanto para ultraestrutura quanto para histologia e isso nos possibilitou descrever não

somente a morfologia dos espermatozoides, como também o processo de

espermatogênese a nivel histológico e ultraestrutural.

É importante ressaltar que o estudo de representantes das classes Cubozoa e

Scyphozoa apresenta dificuldades. Representantes de ambas as classes produzem grande

quantidade de muco que dificulta a separação dos tecidos alvos. A manutenção dos

animais vivos em laboratório apresenta dificuldade uma vez que, necessita-se de

aquários circulares não tão facilmente (e muitas vezes com custo inacessível)

encontrados, alimentação diária intensa dependente do tamanho do indivíduos

125

encontrados (indivíduos menores podem ser alocados em aquários menores e

demandam menos investimento na alimentação, indivíduos grandes precisam ser

alocados em aquários maiores se alimentando mais que o dobro do que os pequenos).

Dentro desta problemática, as cubomedusas são as mais difíceis de se manter em

laboratório, pois são nadadoras e predadoras ativas, não sobrevivendo muito mais do

que 48 horas em ambientes confinados.

O estudo da estrutura gonadal histológica em Cubozoa nos permitiu encontrar

padrões como a organização folicular, sendo estes circundados por um epitélio de

origem gastrodérmica e com características únicas descritas para os representantes da

classe, a saber: as vesículas não reativas a nenhum corante histoquímico testado e com

material granular ao MET. Através do estudo da espermatogênese sincronizada para das

espécies de cubozoários, também foi possível identificar um grau de maturação gonadal

cíclico que justifica o comportamento reprodutivo dos representantes desta classe e

também certa sazonalidade para se encontrar indivíduos maduros.

Do mesmo modo que encontramos padrões na organização gonadal histológica

de Cubozoa, para as três espécies de Scyphozoa, podemos dizer que a gônada masculina

também tem organização característica. Em L. lucerna, Cassiopea sp. e C. lactea a

gônada é composta por um epitélio germinal. Este, por sua vez, é dividido em camada

externa e interna, sendo interiorizado pela mesogléia. A espermatogênese não-

sincronizada de L. lucerna e C. lactea pode estar atrelada ao comportamento

reprodutivo desses organismos que apresentam fecundação externa. Do mesmo modo

que a espermatogênese sincronizada de Cassiopea sp. está associada com a produção e

possível transferência de spermatozeugmata.

Ao estudar a morfologia dos espermatozoides de quatro das cinco espécies de

Cubozoa, observamos que o número de mitocôndrias (seis) pode ser uma das

126

características do espermatozoide de Cubozoa, bem como a presença de vesículas na

região lateral ao núcleo (ver Tabela 1 para detalhes). Parece ser padrão para o

espermatozoide de Scyphozoa a presença de quatro mitocôndrias, a ausência de

vesículas na peça intermediária e também ausência do spurr no aparato pericentriolar.

Algumas das características encontradas como a ligação entre os centríolos proximal e

distal, em Tamoya haplonema, e a presença da vesícula nuclear em Chrysaora lactea,

são típicas de espermatozoides de representantes de outras classes. Estudos

ultraestruturais do espermatozoide de mais espécies de Cnidaria só acrescentariam às

analises comparativas e a delimitação de características únicas dos espermatozoides de

cada classe.

Algumas questões ainda permanecem indefinidas como a composição

histoquímica das vesículas encontradas no epitélio gastrodermal de Cubozoa e também

a composição da secreção em que os espermatozoides de Cassiopea sp. estão imersos,

formando a estrutura spermatozeugmata. Além disso, ainda são muitas as espécies que

não possuem a morfologia do espermatozoide descrita, bem como a estratégia de

liberação (se livres na coluna d’água, em spermatozeugmata ou espermatóforos), tipo de

produção (se sincronizada ou não) e o comportamento reprodutivo (fertilização interna e

externa).

127

Tabela 1: Comparação entre espermatozoides de Cubozoa e Scyphozoa espécies estudadas na presete dissertação com espermatozoide de outras

espéices já descrito na literatura.

Classe Scyphozoa Classe Cubozoa

Rhizostomeae Semeaostomeae Coronatae Carybdeida Chirodropida

Caracteristicas Lychnorhiza

lucerna 1

Rhizostoma

pulmo 2 Cassiopea sp. 1

Chrysaora

lactea 1

Chrysaora

hysoscella 2

Aurelia

autita 2;3 Nausithoe sp. 4

Tamoya

haplonema 1

Carukia

barnesi 1

Carybdea

marsupialis 5

Chiropsalmus

quadrumanus 1

Chironex

fleckeri 1

Chiropsella

bronzie 1

Comprimento da

cabeça 2,01 ± 0,41 - 1,9 ± 0,15 2,17 ± 0,61 - - - 3,7 ± 0,41 - 3.84 ± 0,34 2,98 ± 0,28 2,47 ± 0,17 2,73 ± 0,42

Diâmetro da

cabeça 2,05 ± 0,61 - 1,59 ± 0,29 1,89 ± 0,57 - - - 2,54 ± 0,24 - - 1,82 ± 0,16 1,95 ± 0,25 2,38 ± 0,38

Comprimento da

peça intermediária 0,92 ± 0,26 - 0,75 ± 0,11 0,95 ± 0,25 - - - 1,03 ± 0,23 - 1.1 0,73 ± 0,14 0,68 ± 0,14 0,78 ± 0,08

Diâmetro da peça

intermediária 2,40 ± 0,54 - 1,35 ± 0,19 2,32 ± 0,75 - - - 2,23 ± 0,20 - 2,41 ±0,36 1,87 ± 0,25 1,99 ± 0,27 2,18 ± 0,30

Comprimento do

núcleo 1,9 ± 0,35 - 1,76 ± 0,17 1,88 ± 0,59 - - - 3,4 ± 0,48 - 2,83 ± 0,27 2,75 ± 0,29 2,23 ± 0,23 2,48 ± 0,4

Diâmetro do

núcleo 1,6 ± 0,49 - 1,31 ± 0,21 1,61 ± 0,43 - - - 2,21 ± 0,19 - 1,84 ± 0,27 1,57 ± 0,14 1,59 ± 0,04 2,1 ± 0,32

Morfologia da

cabeça Ovoide a* Alongado b Alongado b* Ovoide c* Ovoide c Alongado b Ovoide c Ampuliforme a* Ovoide* Ovoide b? Forma de bala c*

Forma de

balac* Ovoide b*

Razão nuclear 1,29 ± 0,52 - 1,4 ± 0,29 1,22 ± 0,45 - - - 1,5 ± 0,21 - 1.54 1,82 ± 0,18 1,48 ± 0,21 1,19 ± 0,24

Vesicula nuclear Ausente a* Não

observado b Ausente a* Presente c*

Não

observado b Ausente a Presente c Ausente a* - Ausente a? Ausente a* Ausente a* Ausente a*

Cromatina Granular a Granular a Granular a Granular a Granular a Eletrondensa

b Granular a Granular a - Granular a? Granular a Granular a Granular a

Membrana

nuclear externa Ausente a*

Não

observado b Presente c* Ausente a* Ausente a Ausente a Ausente a Ausente a* - Ausente a? Ausente a* Ausente a* Ausente a*

Número de

mitocôndrias 4 a*

Não

observado b 4 a* 4 a* 4 a 4 a 4 a 6 b* - 6 b* 6 b* 6 b* 6 b*

Vesciulas acima

do núcleo Presente a* Presente a

Não observado b*

Presente a* Não

observado b Presente a Presente a Presente a* - Presente a Presente a* Presente a* Presente a*

Diâmetro das

vesiculas 0,16 ± 0,02 - - 0,12 ± 0,04 - - - 0,09 ± 0,03 - - 0,09 ± 0,01 0,15 ± 0,009 0,12 ± 0,023

Vesicular lateral

do núcleo Ausente a* Ausente a Presente b* Ausente a* Ausente a Ausente a Ausente a Presente a* - Presente a Presente a**

Não

observado b*

Não

observado b*

Centriolo

proximal Presente a* Presente a Presente a* Presente a* Presente a

Não

observado b Presente a Ausente a* - Ausente a Ausente a* Ausente a* Ausente a*

Centriolo distal Presente a Presente a Presente a Presente a Presente a Presente a Presente a Presente a - Presente a Presente a Presente a Presente a

Aparato

Pericentriolar Presente a Presente a Presente a Presente a Presente a Presente a Presente a Presente a - Presente a Presente a Presente a Presente a

Processo primário Presente a* Não

observado b

Não observado b*

Presente a* Não

observado b Presente a Presente a Presente a* - Presente a Presente a*

Não

observado b*

Não

observado b*

Processo

interprimario Presente a*

Não

observado b

Não observado b*

Presente a* Não

observado b Presente a Presente a Presente a - Presente a Presente a

Não

observado b*

Não

observado b*

128

Bandas estriadas

maiores Presente a*

Não

observado b

Não observado b*

Presente a* Não

observado b Presente a Presente a Ausente a* - Presente b Ausente c*

Não

observado c*

Não

observado c*

Processo

secundário Presente a*

Não

observado b

Não observado b*

Presente a* Não

observado b

Não

observado b Presente a

Não observado a*

- Presente b Não observado a* Não

observado a*

Não

observado a*

Processo terciário Presente a* Não

observado b

Não observado b*

Presente a* Não

observado b

Não

observado b Presente a

Não observado a*


observado a*

Não

observado a*

Spurr Não

observado a

Não

observado a

Não observado a

Não

observado a Ausente b

Não

observado a Ausente b

Não observado a

- Presente b Não observado a Não

observado a

Não

observado a

Lamelas Não

observado a*

Não

observado a

Não observado a*

Not observed a*

Não

observado a Ausente b Ausente b Ausente a* - Ausente a? Ausente a* Ausente a* Ausente a*

Poli-ribosomos Presente a* Não

observado b Presente a* Presente a*

Não

observado b Ausente c Ausente c Presente a* - Ausente b? Presente a* Presente a*

Não

observado b*

Complexo de

Golgi Presente a*

Não

observado b Presente a* Ausente c*

Não

observado b Ausente c Ausente c Ausente a* - Ausente a? Ausente a* Ausente a* Presente b*

Vesiculas na peça

intermediária Presente a

Não

observado b Presente a Presente a

Não

observado b Ausente c Presente a

Não observado a*


observado a* Presente b*

Estrutura do

flagelo 9 + 2 a 9 + 2 a 9 + 2 a 9 + 2 a 9+2 a 9+2 a 9+2 a 9 + 2 a - 9 + 2 a 9 + 2 a 9 + 2 a 9 + 2 a

1 Este estudo;

2 Hedwig e Schafer (1986);

3 Hinsch (1974);

4 Afzelius e Franzen (1974);

5 Corbelli et al. (2003).

Letras diferentes indicam variação entre as espécies; * = diferenças entre as espéices do presente estudo; ? = Não descrito e obsevado na figura

publicada.

129

Resumo

A filogenia dos diferentes grupos de Cnidaria permanece de certa forma pouco resolvida, uma vez

que não há um consenso de quais são as relações entre as diferentes classes e ordens que compõem

o filo. A espermiotaxonomia vem sendo utilizada como critério filogenético em diversos grupos de

Metazoa. Para Cnidaria são poucos os trabalhos descrevendo a morfologia do sistema reprodutor

masculino e do espermatozoide em nível de microscopia de luz e de microscopia eletrônica de

transmissão. Exemplares das espécies Tamoya haplonema e Chiropsalmus quadrumanus

(Cubozoa), Lychnorhiza lucerna e Chrysaora lactea (Scyphozoa), foram coletados próximos ao

Centro de Biologia Marinha da Universidade de São Paulo e junto às bases do Instituto

Oceanográfico da USP (Base de Cananéia e Base de Ubatuba) nos meses de agosto a outubro de

2014 e de abril a junho de 2015. Amostras das espécies Carukia barnesi, Chironex fleckeri e

Chiropsella bronzie (Cubozoa) e Cassiopea sp. (Scyphozoa) foram coletadas ao longo da costa leste

australiana de março a maio de 2016. Para a descrição histológica e histoquímica da gônada

masculina, amostras do tecido gonadal foram fixadas em paraformaldeído 4% preparado com água

do local da coleta e tampão fosfato de sódio 0,2M por 24 horas e as amostras foram processadas de

acordo com o protocolo para historesina. Fragmentos da gônada masculina foram fixados em

solução Karnovsky modificado (glutaraldeído 2,5% e 0,08% de paraformaldeído em tampão

cacodilato de sódio 0,1 M, pH 7,4) e solução de Glutaraldeído (2.5% glutaraldeido em tampão

cacodilato de sódio 0.1M em água do mar filtrada a vácuo, pH 7.2-7.4). Em seguida as amostras

foram processadas de acordo com protocolo de microscopia eletrônica transmissão. Na presente

dissertação é descrito de forma comparada o processo de espermatogênese dos cubozoários T.

haplonema e C. quadrumanus através da histologia e histoquímica evidenciando o ciclo gonadal de

ambos (capítulo 1). Descreve-se a espermatogênese e morfologia do espermatozoide, através da

microscopia de luz e ultraestrutura para as espécies de Cubozoa T. haplonema, C. quadrumanus,

Carukia barnesi, Chironex fleckeri e Chiropsella bronzie (Capítulo 2). Para as espécies de

Scyphozoa Lychnorhiza lucerna, Chrysaora lactea e Cassiopea sp. descreve-se o processo de

130

espermatogênese através da microscopia de luz e da microscopia eletrônica de transmissão, além do

aspecto macroscópico da gônada masculina (Capítulo 3). Adicionalmente, nas considerações finais,

é ressaltada a morfologia comparada dos espermatozoides de todas as espécies aqui estudadas

evidenciando possíveis características únicas de cada classe, enumerando eventuais caracteres

diagnósticos dos espermatozoides das espécies estudadas (Capítulo 4).

Palavras-chave: Cnidaria, medusas, reprodução, espermiotaxonomia, espermatozoide, MET.

131

Abstract

The phylogeny of the different groups of cnidarians remains elusive, since there is no consensus of

what are the relationships between the different classes and orders that comprise the phylum. The

spermiotaxonomy has been used as phylogenetic criteria in different groups of Metazoa. For

Cnidaria there are few studies describing the morphology of the male reproductive system and

sperm at the level of light microscopy and transmission electron microscopy. Specimens of the

species Tamoya haplonema and Chiropsalmus quadrumanus (Cubozoa), Lychnorhiza lucerna and

Chrysaora lactea (Scyphozoa), were collected near the Marine Biology Center of the University of

São Paulo and near the bases of the Oceanographic Institute of USP (Cananéia and Ubatuba Bases)

from August to October 2014 and from April to June 2015. Samples of the species Carukia barnesi,

Chironex fleckeri and Chiropsella bronzie (Cubozoa) and Cassiopea sp. (Scyphozoa), were

collected along the Australian east coast from March to May 2016. For histological and

histochemistry description of the male gonads, samples were fixed in 4% paraformaldehyde

prepared with water from the collection site and sodium phosphate buffer 0.2M for 24 hours. After

that, the samples were processed following the protocol for historesin. Fragments of male gonads

were fixed in modified Karnovsky solution (2.5% glutaraldehyde and 0.08% paraformaldehyde in

0.1 M sodium cacodylate buffer, pH 7.4) and glutaraldehyde (2.5% glutaraldehyde in cacodylate

buffer 0.1M sodium in seawater vacuum Millipore filtered, pH 7.2-7.4). Then the samples were

processed according to transmission electronic microscopy protocol. In this dissertation it is

described the comparative spermatogenesis of the cubozoans T. haplonema and C. quadrumanus,

under histology and histochemistry, showing the gonadal cycle (Chapter 1). We also describe the

spermatogenesis and sperm morphology, under light microscopy and ultrastructure, for the Cubozoa

species T. haplonema, C. quadrumanus, Carukia barnesi, Chironex fleckeri and Chiropsella

bronzie (Chapter 2). For the Scyphozoa species, Lychnorhiza lucerna, Chrysaora lactea and

Cassiopea sp., we describe the spermatogenesis process, under light transmission electronic

microscopy, describing the macroscopic structure of the male gonad (Chapter 3). Additionally, in

132

the final chapter, it is highlighted the comparative morphology of sperm of all species studied,

evidencing possible unique features of each class, enumerating possible sperm characters (Chapter

4).

Key-words: Cnidaria, jellyfish, reproduction, spermiotaxonomy, sperm, histology, MET.

133

Apêndice I - Protocolo de Fixador para Histologia

Paraformaldeído 4% em Tampão Fosfato de Sódio 0,2 M pH 7,2

1. Preparação de solução estoque de paraformaldeído 10%;

2. Preparação de solução estoque de Tampão Fosfato de Sódio 0,4M

3. Preparação do Fixador.

1. Preparação de solução estoque de paraformaldeído 10%

Água destilada ................................. 250 ml;

Paraformaldeído ...............................30 g.

a. Preparação:

- Aquecer a água destilada a 55ᵒC e separar em uma proveta 10 ml;

- Adicionar 240 ml em um bécker;

- Pesar 30 g de paraformaldeído;

- Adicionar o paraformaldeído no bécker com água destilada;

- Adicionar os 10 ml restantes e misturar em agitador magnético;

- Adicionar 4 pastilhas de Hidróxido de Sódio;

- Esperar até que solução fique transparente.

2. Preparação de Solução estoque de Tampão Fosfato de Sódio 0,4 M

Solução A:

Fosfato de Sódio Monobásico.................. 7,78g

Água destilada..........................................140ml

Solução B:

Fosfato de Sódio Bibásico.......................20,42g

Água destilada.........................................360ml

134

a. Preparação:

Para 500 ml de solução final:

- Misturar em um bécker Solução A e B;

- Corrigir o pH se necessário (pH 7,2 – 7,4).

3. Preparação do Fixador

Água destilada..........................................40 ml;

Tampão Fosfato de Sódio Na 0,4M..........200ml;

Paraformaldeído 10%................................160ml.

a. Preparação:

Para 400ml de solução final:

- Em um bécker adicionar 40ml de água destilada;

- Adicionar 200 ml de Tampão Fosfato de Sódio 0,4M;

- Adicionar 160 ml de Paraformaldeído 10%;

- Misturar em agitador magnético;

- Confirmar o pH (pH 7,2 – 7,4).

135

Apêndice II - Protocolo de Histologia em Historesina

1. Fixação

Colocar as pequenas amostras de tecido em solução de paraformaldeído a 4% em tampão

fosfato de sódio 0,2M por 24 horas. Sempre na proporção de 10:1 (10 partes de fixador para

1 de amostra).

2. Lavagem

Retirar o fixador e fazer 1 lavagem com o tampão fosfato de sódio por 24 horas em

geladeira.

3. Desidratação

- Retirar todo o tampão fosfato de sódio e colocar o material em séries de álcoois de 30% a

100%:

- Álcool 30%........... 30 min;

- Álcool 50%........... 30 min;

- Álcool 70%........... 30 min;

- Álcool 80%........... 30 min;

- Álcool 90%........... 30 min;

- Álcool 95%........... 30 min;

- Álcool 100%........... 1 hora.

4. Infiltração

- Retirar o álcool e colocar solução 1:1 de historesina + álcool. Deixar por 2 dias na

geladeira.

- Retirar a solução anterior e colocar em resina pura por 2 horas no vácuo.

- Trocar a resina pura por uma nova solução e deixar 48 horas na geladeira.

5. Inclusão

- Antes de começar a inclusão do material, organizar as amostras e anotar em um caderno

todas as informações necessárias para posterior identificação dos bloquinhos, como nome

da espécie, material, tipo de fixador, orientação da amostra (longitudinal ou transversal),

entre outras.

136

- Em um bécker adicionar resina líquida e o endurecedor (15ml de resina para 1ml de

endurecedor) e misturar em agitador magnético.

- Como a resina endurece em aproximadamente 30 minutos, colocar o bécker com a resina

(devidamente coberto com parafilm) em um pequeno vidro com gelo para que ela endureça

mais devagar.

- Após, com uma pipeta pasteur, adicionar um pouco de resina no molde desejado, orientar a

amostra o mais rápido possivel dentro do molde, e completar o restante do molde com

resina.

- Deixar polimerizar em estufa a 37ᵒC.

6. Fixação do bloco de resina em bloco de madeira

- Retirar os blocos de resina do molde pressionando o lado externo .

- Colar o bloco de resina no bloco de madeira utilizando cola especial* ou com cola

superbonder.

- Identificar a madeira com o nome do material.

* Araldite Profissional – Misturar partes iguais dos dois tubos até ficar uma mistura

homogênea de cor esbranquiçada, a cola seca em aproximadamente uma hora e

definitivamente em 24 horas

- Passar cola na base do bloco de resina e uma quantidade mais generosa no bloco de

madeira

- Pressionar o bloco de resina sobre o de madeira

- Cuidadosamente rearranjar a cola (com auxilio de um palito) pra que forme uma base

larga, que os lados estejam distribuídos uniformemente e que a altura da cola não alcance a

amostra.

7. Microtomia

- Limpar as lâminas com álcool e identificá-las.

- Cortar o material de modo contínuo a 10µm até chegar na amostra.

- Diminuir espessura dos cortes para 3 a 5µm.

137

- Depositar o corte na superfície da água destilada e esperar o corte esticar.

- Recolher com auxílio de um pincel posicionando na lâmina da forma desejada.

- Colocar a lâmina para secar na chapa aquecida;

- Após o término, deixar lâminas na estufa por pelo menos 15 min para secarem bem.

138

Apêndice III - Técnicas Histológicas e Histoquímicas de Colorações

Hematoxilina e Eosina (HE) (Behmer et al., 1976; Llewellyn, 2013)

1) Hematoxilina de Mayer (20min a 370C);

2) Água de torneira corrente (3min) – Lavar algumas vezes (2 ou 3) a cuba retirando o excesso

de corante e depois deixar apenas um fio de água não muito forte que elimine o resto do

excesso de corante;

3) Lavagem rápida em água destilada – Secar e analisar as lâminas sob microscópio para

verificar a intensidade da coloração;

4) Eosina (5min a 370C);

5) Lavar em água destilada (2min x 3) - Mergulhar para tirar o excesso;

6) Água destilada - analisar sob microscópio;

7) Secagem em chapa aquecedora (370C);

8) Secar em estufa (15-20 min);

9) Montagem final das lâminas com meio de montagem (Entellan ou Permount) e lamínula.

Preparação dos corantes:

Hematoxilina de Mayer (1891):

- Dissolver 1g de hematoxilina em 50ml de álcool a 95%;

- Dissolver 50g de sulfato de amônio e alumínio em 1000ml de água destilada;


- Deixar o corante maturar por meses (o frasco deve ser tampado com um chumaço de

algodão);

- Pode-se adicionar posteriormente 20ml de ácido acético glacial.

Eosina Y (solução aquosa com ácido acético) (Behmer et al., 1976):

- Dissolver 10g de eosina Y em 1000ml de água destilada;

- Adicionar 2ml de ácido acético glacial;

Azul de Toluidina com borax (Junqueira, 1995)

Para estruturas ácidas

1) Azul de Toluidina (1-2min) – Ajustar o tempo de acordo com a amostra aumentando

gradativamente o tempo até obter a coloração desejada;

139

2) Mergulhar a lâmina em outra cuba com água destilada (2 ou 3 vezes até o excesso de

corante ser removido);

3) Secar em chapa quente e observar a intensidade da coloração;

4) Secar em estufa (15-20 min);


Preparação do corante:

- Azul de toluidina 0,1 g.

- Borato de sódio 1 g;

- Água destilada 100 ml.

PAS – Ácido Periódico e Reativo de Schiff (McManus, 1946)

(Bancroft & Stevens, 1982; Kiernan, 1990)

Para polissacarídeos neutros

1) Ácido periódico (10-15min);

2) Lavar em água destilada (deixar por 2min x 3);

3) Reativo de Schiff (30min);

4) Água de torneira corrente (5min);

5) Água destilada (lavagem rápida);

6) Secar e observar sob microscópio se houve reação positiva;

Opcional:

7) Hematoxilina de Mayer (10-20min a 370C);

8) Água de torneira corrente (3min);




Preparação do corante e reagente:

Ácido Periódico:

- Ácido periódico 1g;

- Água destilada 200ml.

Reativo de Schiff:

- Dissolver 1g de fucsina básica em 200 ml de água fervente (um pouco antes da

fervura);

- Reduzir a temperatura da solução para 50°C;

140

- Adicionar 2g de metabissulfito de potássio e misturar em agitador magnético;

- Deixar a solução chegar à temperatura ambiente;

- Adicionar 2ml de ácido clorídrico concentrado;

- Adicionar 2g de carvão ativado, com agitação.

- Armazenar o frasco à temperatura ambiente e ao abrigo da luz.

- No dia seguinte, filtrar e manter o reagente em frasco âmbar e a 4°C.

Azul de Alcian (pH 2,5) – Alcian Blue (Bancrofft & Stevens, 1982)

Para polisacarídeos ácidos

1) Azul de Alcian a 1% em ácido acético a 3% (10-30 min) (melhor 60 min para resina);

2) Água de torneira corrente (3 min);


4) Secar e observar sob microscópio se houve reação positiva;

5) Hematoxilina de Mayer (10-20 min a 37°C);

6) Água de torneira corrente (3 min);




Preparação do corante:

Azul de Alcian (pH 2,5);

- Azul de Alcian - 2g;

- Ácido acético glacial a 3% (pH 2,5) - 200ml.

Azul de Bromofenol (Pearse, 1985)

Proteínas Básicas

1) Corar por 1 hora em temperatura ambiete;

2) Lavar em ácido acético 0,5% por 5 minutos;

3) Passar em Água corrente por 15 minutos;




141

Corante:

- Bicloreto de mercúrio 30 g;

- Azul de bromofenol 300 mg;

- Etanol (95%) 300 ml.

Ácido acético:

- 1 ml de Acido acético glacial;

- 200 ml de água destilada.

Xylidine Ponceau (Pearse, 1985)

Proteínas Totais

1) Corar por 30 minutos;

2) Passar no Tampão Acetato de Sódio por 1 minuto;

Observação: Medir o pH antes de usar o tampão (o pH 2,5 é o ideal);

3) Lavar em água destilada;




Xylidine

- 0,25 g de Xylidine;

- 250 ml de ácido acético a 2% (pH 2,5)

Ácido Acético 2%

- 5 ml de ácido acético glacial;

-250 ml de água destilada.

Tampão Acetato de Sódio

Solução A:

- 6,8 g de Acetato de Sódio

- 250 ml de água destilada.

Solução B:

- 15 ml de ácido acético;

142

- 250 ml de água destilada

- Adicionar solução B em A até atingir o pH desejado (2,5 a 3,5);

Observação: Fazer sempre a Solução B em maior quantidade.

143

Apêndice IV - Protocolos dos Fixadores para Microscopia Eletrônica de Transmissão

Fixador Karnovsky (utilizado para as espécies brasileiras)

OBS.: realizar todos os procedimentos em CAPELA e utilizando LUVAS e AVENTAL.

Para 200 ml de solução fixadora:

- Dissolver 4 g de paraformaldeído (pó) em 50 ml de água destilada aquecida a 60-70˚C (em

agitador magnético). Adicione algumas gotas de NaOH 1N até a solução ficar transparente.

CUIDADO, TÓXICO!

- Resfriar à temperatura ambiente e adicionar 20 ml de glutaraldeído a 25%. CUIDADO, TÓXICO!

- Adicionar 100 ml de tampão cacodilato de sódio 0.2M (4.28 g de cacodilato de sódio em 95 ml de

água destilada). CUIDADO, EXTREMAMENTE TÓXICO!

- Ajustar o pH para aprox. 7.4 com HCl 0.2N. CUIDADO, CORROSIVO!

- Adicionar 5 ml de CaCl2 0.1M (0.027g de CaCl2 em 5ml de água destilada)

- Adicionar 40 g de sacarose.

- Se necessário, adicionar água destilada para completar o volume de 200 ml (volume medido no

béquer graduado).

Obs.1: manter refrigerado. Retire somente o necessário para fixar (cobrir) suas amostras. A fixação

deve ser realizada em geladeira. Após o término do tempo de fixação, trocar a solução fixadora pela

solução tampão, descrita abaixo. Não descarte o fixador na pia (siga as normas para descarte).

Obs.2: concentrações finais do fixador: paraformaldeído a 2%, glutaraldeído a 2.5%, tampão

cacodilato de sódio (pH 7.4) a 0.1M e CaCl2 a 2.5mM.

Solução tampão cacodilato de sódio a 0.1M (para 200ml):

-Adicionar 100 ml de tampão cacodilato de sódio 0.2M (4.28 g de cacodilato de sódio em 95 ml de

água destilada). Utilize um agitador magnético. CUIDADO, EXTREMAMENTE TÓXICO!

- Ajustar o pH para aprox. 7.4 com HCl 0.2N.

- Adicionar 5 ml de CaCl2 0.1M (0.027 g de CaCl2 em 5 ml de água destilada)

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- Adicionar 40 g de sacarose.

- Completar o volume de 200 ml com água destilada (volume medido no béquer graduado).

Obs.: manter refrigerado. Retire somente o necessário para cobrir as amostras fixadas. Após trocar a

solução fixadora pela solução tampão, mantenha as amostras em geladeira. Não descarte o tampão

na pia (siga as normas para descarte).

Fixador Glutaraldeído 2,5% em água do mar filtrada a vácuo (utilizado para as espécies

australianas)

1. Filtrar Água do mar;

2. Fazer Tampão Cacodilato de Sódio 0,1M em água do mar;

3. Fazer o fixador;

1. Filtragem da água do mar

a. Filtrar 500 ml de água do mar a vácuo em filtro Millipore de 0,45 µm;

b. Armazenar em um frasco identificado.

2. Fazer Tampão Cacodilato de Sódio 0,1M em água do mar

Água do mar filtrada.................................... 200 ml;

Cacodilato de Sódio......................................3,831 g.

a. Preparo:

- Adicionar a água do mar em um bécker;

- Em uma balança analítica pesar 3,831 g de Cacodilato de Sódio;

- Adicionar o cacodilato de sódio no bécker com água e misturar em agitador

magnético;

- Verificar e corrigir pH (7,2).

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3. Fazer o Fixador

Tampão Cacodilato de Sódio 0,1M................100 ml

Glutaraldeído 25% (ampôla)...........................10 ml

a. Preparo:

- Separar 100 ml de Tampão Cacodilato de sódio 0,1M em um bécker;

- Adicionar os 10 ml de glutaraldeído 25%;


- Verificar e corrigir pH.

OBSERVAÇÃO: Todos esses procedimentos devem ser feitos em capela, com jaleco de algodão,

duas camadas de luvas de nitrilo e óculos.

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Apêndice V- Protocolo de Microscopia Eletrônica de Transmissão

1. Fixação I

Fixador Karnovsky: glutaraldeído a 2.5% + paraformaldeido 2% em tampão

cacodilato de sódio (pH 7.4) a 0.1M e CaCl2 a 2.5mM por 24 horas a 4oC (espécies do

Brasil).

Glutaraldeído 2,5% em Tampão Cacodilato 0,1M em água do mar filtrada a vácuo

Millipore (pH 7.2-7.4) – por 30 minutos a 4oC (espécies da Austrália).

2. Lavagem I

Mesmo Tampão Cacodilato utilizado para fazer o fixador 0,1M – 3 x 10 min a 4oC.

3. Fixação II (Pós-Fixação)

Ósmio 1% em Tampão Cacodilato 0,1M - 1 hora a 4oC

Receita para 10 ml:

_ Tetróxido de Ósmio a 2% aquoso ................................ 5 ml

_ Tampão Cacodilato 0,2M ............................................. 5 ml

4. Lavagem II

Opção 1 (com “En Bloc Staining” – amostras Brasil)

_ Água do mar filtrada ............................................................ 20 ml

Opção 2 (sem “En Bloc Staining” - amostras Austrália)

Tampão Cacodilato de Sódio 0,1M em água do mar – 3 x 10 min a 4oC

Receita:

_ Tampão Cacodilato 0,1M

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Obs: Nessa etapa, caso seja necessário, pode-se deixar o material na

geladeira por alguns dias.

5. “En Bloc Staining”

Acetato de Uranila 1% aquosa – 15 a 18 horas a 4oC

6. Desidratação (temperatura ambiente)

Álcool 50% ............... 10 min

Álcool 70% ............... 10 min

Álcool 95% ............... 15 min

Álcool 100% ............. 2 X 10 min

7. Embebição (temperatura ambiente)

Álcool 100% 2:1 Spurr .......................... 1 hora



Spurr puro .............................................. 2 x 2 horas

8. Emblocagem

Em molde de borracha ou cápsula de gelatina – 72 horas a 58oC (Amostras do Brasil);

Em molde de borracha ou cápsula de gelatina – 8 horas a 70oC (Amostras da

Austrália).

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____________________________________________________________

Receita:

1. Spurr tipo Standard

p / + 30 ml p / + 15 ml

_ ERL 4206 ........................ 10 g ............................... 5 g

_ DER 736 ............................ 6 g ................................ 3 g

_ NSA ................................. 26 g .............................. 13 g

_ S-1 (DMAE) ................... 0,4 g ............................. 0,2 g

2. Uranila 1% aquosa

_ Acetato de uranila .................................................... 1 g

_ água bi-destilada ................................................ 100 ml (q.s.p.)

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