UNIVERSIDADE FEDERAL DO RIO DE JANEIRO PROGRAMA DE PÓS-GRADUAÇÃO EM BIODIVERSIDADE E BIOLOGIA EVOLUTIVA INSTITUTO DE BIOLOGIA
Diversidade e genômica funcional de Symbiodinium spp. do coral Mussismilia do Banco de Abrolhos
Arthur Weiss da Silva Lima 2016 Arthur Weiss da Silva Lima
Diversidade e genômica funcional de Symbiodinium spp. do coral Mussismilia do Banco de Abrolhos
Tese de Doutorado apresentada ao Programa de Pós-Graduação em Biodiversidade e Biologia Evolutiva, Instituto de Biologia, Universidade Federal do Rio de Janeiro, como parte dos requisitos para a obtenção do título de doutor.
Orientadores: PhD. Fabiano Thompson PhD. Paulo Salomon
ii Silva-Lima, Arthur Weiss Diversidade e genômica funcional de Symbiodinium spp. do coral Mussismilila spp. do Banco de Abrolhos / Arthur Weiss da Silva Lima – Rio de Janeiro: Instituto de Biologia, 2016. x, 96 f. : il. ; 31 cm. Orientador: Fabiano Lopes Thompson. Co-orientador: Paulo S. Salomon Tese (doutorado) – UFRJ, IB, Programa de Pós-Graduação em Biodiversidade e Biologia Evolutiva, 2015. 1. Symbiodinium. 2. Mussimilia. 3. Diversidade. 4. Cultivo. 5. Branqueamento de corais. 6. Transcriptoma. 7. Estresse oxidativo. I. Thompson, Fabiano Lopes. II. Universidade Federal do Rio de Janeiro, IB, Programa de Pós- Graduação em Biodiversidade e Biologia Evolutiva. III. Título.
iii Folha de Aprovação
Arthur Weiss da Silva Lima Orientadores: Fabiano L. Thompson, Co-orientador: Paulo S. Salomon Diversidade e genômica funcional de Symbiodinium spp do coral Mussismilia spp. do Banco de Abrolhos
Aprovada em 28 de julho de 2016, por:
______Prof. Dr. Carlos Eduardo Guerra Schrago (UFRJ)
______Prof. Dr. Renato Crespo Pereira (UFF)
______Profa. Dra. Ana Carolina Paulo Vicente (Fiocruz/RJ)
______Profa. Dra. Claudia Mermelstein (UFRJ)
______Profa. Dra. Denise Carvalho (UFRJ)
______Prof. Dr. Paulo Cavalvanti Gomes Ferreira (UFRJ)
______Profa. Dra. Cassia Sakuragui (UFRJ)
iv
Junte um bico com dez unhas, quatro patas, trinta dentes E o valente dos valentes, ainda vai te respeitar Chico Buarque, Todos juntos
v Às minhas famílias
vi AGRADECIMENTOS
Várias pessoas foram importantes ao longo desses pouco mais de quatro anos, que aproveito para agradecê-las aqui. Inicialmente agradeço aos meus orientadores Fabiano Thompson e Paulo Salomon, pela orientação e suporte que tive para conduzir esse trabalho. Agradeço também pela confiança e autonomia que me foi dada, além de todas as discussões científicas. Agradeço também ao PPGBBE e seus professores pela oportunidade de desenvolver esse trabalho dentro do programa. Em especial agradeço às professoras Michelle Klautau e Daniela Takyia que, como cordenadoras do programa nos últimos anos, me receberam sempre que foi necessário e me ajudaram no que foi preciso. Foram tantas pessoas importantes durante meu estágio sanduíche que mereciam um capítulo à parte nessa tese. Agradeço, às professoras Janelle Thompson e Monica Medina por me receberem em seus laboratórios e pelas discussões científicas. Agradeço também a meus colegas dos laboratórios do MIT e da PSU, em especial Carolina Bastidas, Hanny, JP, Aki, Bishoy, Veridiana, Styles, Carlos, Ana, Joe, Megan e à Pepper. Alguns amigos, também fundamentais nesse processo, foram importados do Brasil: Adriana, Alessandra, Débora e Robin, Flavio, Rute e Camilinha, Fabio, Pauline e Juju, além dos meus primos Leo e Gabriel, das tias Norma e Sonia e dos cunhados Joana, Ken e Luma. À equipe das escolas Kennedy-Longfellow e Easterly Parkway. É impossível imaginar o estágio sanduíche sem essas pessoas. Aos meus amigos do laboratório de Microbiologia da UFRJ, pelo companheirismo e colaborações. Aos eternos amigos agostinianos, sempre presentes, apesar de todos os momentos em que eu não estive. Agradeço especialmente à minha família, pessoas que compartilharam grande parte desse processo e não mediram esforços para tornar isso posssível. Minha mãe, meu pai, minhas irmãs e meus sogros. Por fim, tenho orgulho de dividir todo esse processo com a Carolina, com o Lucas e, no último ano, com o Edu. Nossa roda cresceu e continuará a girar pelo mundo.
vii RESUMO
Dinoflagelados do gênero Symbiodinium são micro-organismos fotossintetizantes endossimbiontes de corais, entre outro hospedeiros. Eventos extremos de estresse podem desestabilizar a associação entre Symbiodinium e seu hospedeiro, levando ao branqueamento de corais. O estresse causado por aumentos na temperatura da água superficial pode causar o branqueamento de extensos bancos de corais, levando a mortalidade em massa de corais e a mudanças na estrutura do ecossistema. Apesar da grande importância ecológica, trabalhos abordando a interação Symbiodinium-corais na costa brasileira são escassos. Nessa tese foi desenvolvido o primeiro estudo de diversidade e o primeiro banco de cultivo de Symbiodinium associados a Mussismilia braziliensis, os principais construtores de recife do Atlântico Sudoeste. A diversidade molecular do espaçador interno ribossomal ITS2 indica que o gênero de corais Mussismilia é generalista, se associando à, pelo menos, três linhagens de Symbiodinium: C3, A4 e B19. Análise transcriptômica de Symbiodinium A4 em cultivo indica que o choque térmico causa variações na expressão gênica, em resposta à produção excessiva de espécies ativas de oxigênio (ROS). O consequente estresse oxidativo causa diversos impactos na célula: observa-se redução no potencial fotossintético do organismo, além de danos no enovelamento de proteínas. Repressão na expressão gênica indica a inibição de processos que geram ROS em diversos compartimentos celulares, como o cloroplasto, a mitocôndria e o retículo endoplasmático. Essa inibição é acompanhada por uma potencial resposta de aclimatação que envolve mudanças na composição de proteínas transmembranas, na homeostase dos íons Ca2+ e Fe2+ e na regulação do metabolismo do cloroplasto e da mitocôndria. Interessantemente, essa aclimatação é mediada pela atividade de proteínas que respondem ao estado oxidativo da célula, sugerindo um papel da sinalização redox na resposta celular. Apesar do deficit na produção de ATP, Symbiodinium A4 responde ativamente ao estresse oxidativo, investindo energia celular em uma resposta que possibilita sua sobrevivência, mas que pode desestabilizar a simbiose com corais.
Palavras-chave: Symbiodinium, Mussismilia, Diversidade, Cultivo, Branqueamento de corais, Transcriptoma, Estresse oxidativo.
viii ABSTRACT
Dinoflagelates from the Symbiodinium genera are photosynthettic micro-organisms, endosymbionts of corals, among other hosts. Extreme stress events can desestabilize the Symbiodinium-coral association, leading to coral bleaching. The stress caused by high superficial seawater temperature can trigger the bleaching of extensive reef banks, causing mass mortality of corals and changes in the structure of the reef environment. Despite this huge ecological importance, there are few studies on the Symbiodinium-coral interaction in the brazilian coastal waters. In this thesis, there is the first assessment of diversity and the first culture collection of Symbiodinium associated to the Mussismilia braziliensis coral, the main reef builder species in the Southwestern Atlantic Ocean. Molecular diversity of the ITS2 sequence revealed that Mussismilia is a generalist genera, wich associates to, at least, three Symbiodinium strains: C3, A4 and B19. Transcriptomic analysis of Symbiodinium A4 under culture conditions indicates that a heat shock causes variations on gene expression, in response to the increased reactive oxigen species (ROS) concentration in the cell. The consequent oxidative stress causes impatcs in the cell: reductions in the photosynthetic potential and damages to protein synthesis are observed. Repression of gene expression indicates the inhibittion of ROS generating processes in diverse cellular compartments, as chloroplast, mitochondria and the endoplasmatic reticulum. This inhibition is associated to a possible acclimatization reponse that involves modifications on composition of transmembrane proteins, Ca2+ and Fe2+ homeostasis and the regulation of chloroplast and mitochondrial metabolism. Interestingly, the activity of the proteins that mediates this acclimatization is regulated by the oxidative state of the cell, suggesting a role for redox signaling in the stress response. Despite the deficit in ATP production, Symbiodinium A4 responds actively to the oxidative stress, investing cellular energy in a response that enables survival, but that might still desestabilize the Symbiodinium-coral symbiosis.
Keywords: Symbiodinium, Mussismilia, Diversity, Culture, Coral Bleaching, Transcriptome, Oxidative stress.
ix SUMÁRIO
CAPÍTULO 1 13 Introdução 1.1 Recifes de Coral 13 1.1.1. Banco de recifes dos Abrolhos, Bahia 13 1.2 Interação Symbiodinium-coral 15 1.2.1 Diversidade taxonômica e funcional em Symbiodinium 16 1.3 Branqueamento de corais e o estresse oxidative 18 1.4 Genômica de Symbiodinium 21 1.4.1 Genoma mitocondrial e do cloroplasto 22
CAPÍTULO 2 24 Objetivos
CAPÍTULO 3 25 Multiple Symbiodinium strains are hosted by the Brazilian endemic corals Mussismilia spp. 3.1 Introdução 27 3.2 Métodos 29 3.3 Resultados 32 3.4 Discussão 35 3.5 Conclusão 39 3.6 Referencias 40 3.7 Material suplementar 47
CAPÍTULO 4 50 Heat stress induces a transcriptional response associated to diverse cellular compartments in the Mussismilia endosymbiont Symbiodinium 4.1 Introdução 51 4.2 Métodos 53 4.3 Resultados 57 4.4 Discussão 61 4.5 Conclusão 69 4.6 Referencias 69 4.7 Material suplementar 75 x CAPÍTULO 5 83 Discussão
CAPÍTULO 6 88 Conclusão
REFERÊNCIAS BIBLIOGRÁFICAS 89
APÊNDICE 96
LISTA DE TABELAS
Tabela 3.1. Amostras de Symbiodinium (isolados e tecido de coral) usados para a reconstrução filogenética...... 33 Tabela 3.2. Parâmetros morfológicos dos isolados de Symbiodinim...... 35 Tabela 3.S1. Alinhamento das sequências de ITS2 de linhagens de Symbiodinium do clado C, C1, C3, C15 and C90...... 47 Tabela 4.1. Estatística descritiva do transcriptoma de Symbiodinium………………………………...58 Tabela 4.2. Número de genes com domínios conservados de enzimas antioxidantes em Symbiodinium A4……………………………………………………………………………………………...59 Tabela 4.3. Genes diferencialmente expressos no choque térmico, compardo com o controle no escuro……………………………………………………………………………………………………...……75 Tabela 4.S1. Distribuição de fatores de transcrição no transcriptoma de Symbiodinium A4………77 Tabela 4.S2. Níveis de expressão (pseudo-counts) e log(fold-change) de genes associados à resposta antioxidante……………...………………………………………………………………………….78 Tabela 4.S3. Genes diferencialmente expressos na Luz, comparados com o controle no escuro…..79 Tabela 4.S4. Termos GOs enriquecidos de GOs, associados ao contraste Luz/Escuro...... 79 Tabela 4.S5. Termos GOs enriquecidos de GOs, associados ao contraste choque térmico/Escuro...... 80
xi LISTA DE FIGURAS
Figura 1.1. Localização do Banco de recifes dos Abrolhos, no sul do estado da Bahia...... 14 Figura 1.2. Filogenia molecular de Symbiodinium, ilustrando o relacionamento entre os 9 clados designados...... 16 Figura 1.3. Evolução do número de espécies formalmente descritas e da quantidade de estudos genômicos em Symbiodinium...... 18 Figura 1.4. Cadeia transportadora de elétrons da fotossíntese (Kegg:00195)...... 20 Figura 3.1. Reconstrução filogenética das sequências de ITS2 de Symbiodinium associadas a Mussismilia…………………………………………………………………………...………………………..34 Figura 3.2. Microscopia ótica revelando a morfologia de Symbiodinium A4 em cultivo...... 36 Figura 3.3. Curva de crescimento e potencial fotossintético de Symbiodinium A4 em cultivo...... 37 Figura 4.1. Efeito de um heat shock sobre a fisiologia de Symbiodinium A4 culture em cultvo…...57 Figura 4.S1. Comparação nas mudancas de expressao de genes no heat shock e na luz………..…81 Figura 4.S2. Média das temperaturas máximas da água do mar em Abrolhos, por mês nos últimos 15 anos………………………………………………………………………………………………………….82 Figura 5.1. Abundância relativa das sequências de ITS2 de Symbiodinium em amostras de tecido de M. braziliensis…………………………………………………………………..…………………………84
xii CAPÍTULO 1 INTRODUÇÃO
1.1 Recifes de Coral
Recifes de corais são encontrados em toda a faixa de mares tropicais, tipicamente em águas rasas e de temperatura amena. Esses ambientes abrigam uma grande diversidade de fauna associada, desde invertebrados a peixes e mamíferos marinhos (Muller-Parker & D'Elia 1997). A principal entrada de energia nesses ecossistemas de águas oligotróficas se dá através da associação entre corais da ordem Scleractinia e seus endossimbiontes fotossintetizantes, dinoflagelados do gênero
Symbiodinium (Muller-Parker & D'Elia 1997). Ocorrendo comumente em áreas costeiras, recifes de corais possuem uma grande importância econômica e social, devido à atividades de pesca e turismo, o que o torna mais exposto a impactos ambientais locais, como a sobrepesca e ao aporte de nutrientes e poluentes vindos dos continentes (Moura & Francini-Filho 2005). Essas ameaças locais se somam à mudanças climáticas globais causando uma degradação mundial dos ambientes de recifes de coral, observada nos últimos 40 anos (Hughes et al. 2003, Douglas 2003, Francini-Filho et al., 2008).
1.1.1 Banco de recifes dos Abrolhos, Bahia
Corais recifais são encontrados ao longo da quase toda a costa brasileira, desde a foz do rio
Amazonas (Moura et al 2016) ao estado de Santa Catarina (Capel et al 2012), em uma variação de mais de 25o latitudine. Entretanto, as maiores formação de recifes se concentram na costa nordeste, entre os estados do Rio Grande do Norte e Bahia (Leao & Kikuchi 2003). Observa-se comumente uma menor diversidade e um grande grau de endemismo de organismos recifais na região (Leao &
Kikuchi 2003, Robertson et al. 2006). Mesmo em espécies que ocorrem também no Atlântico norte, observam-se reduções na diversidade genética das populações no Atlântico Sul (Nunes et al. 2009,
Nunes et al 2011)
13 Acredita-se que esse endemismo seja uma consequência do isolamento causado pelas plumas dos rios Orinoco e Amazonas, isolando o Atlântico sul do Caribe (Robertson et al. 2006). Entretanto a recente descrição de um grande complexo recifal com ocorrência de corais sob a pluma do rio
Amazonas indicam uma via de conectividade entre esses domínios (Moura et al 2016).
Figura 1.1. Localização do Banco de recifes dos Abrolhos, no sul do estado da Bahia. Os polígonos vermelhos indicam a área sob proteção do Parque Nacional Marinho dos Abrolhos. Extraído de Bruce et al (2012).
O Banco dos Abrolhos é o maior recife de corais do Atlântico sul, localizado no alargamento da plataforma continental ao sul do estado da Bahia (Figura 1.1). Nesse banco está localizado o
Parque Nacional Marinho dos Abrolhos, com 88.000 hectares de área sob proteção (Bruce et al
2012). Das 23 espécies de corais descritas na costa brasileira, 20 estão presentes em Abrolhos, sendo as 6 mais abundantes (Mussismilia braziliensis, Mussismilia hispida, Mussismilia hartti,
Siderastrea stellata, Favia gravida e Favia leptophylla) endêmicas do Brasil (Leao & Kikuchi
2003, Bruce et al 2012). Os corais Mussismilia spp. são responsáveis por aproximadamente 70% da cobertura de coral do Banco dos Abrolhos, sendo os principais construtores, responsáveis pelas formações dos chapeirões característicos da bancada (Leão & Kikuchi, 2003). Entretanto, essa espécie está sob ameaça de espalhamento de patógenos e eventos de branqueamento (Leão &
Kikuchi 2003, Francini-Filho et al., 2008, Garcia et al 2013). Associada à pressão antrópica sobre o 14 ecossistema, isso explica porque esse bioma vem sofrendo degradação acelerada, com a perda de cobertura de corais formadores de recifes e diminuição da biomassa de peixes (Francini-Filho et al.,
2008).
1.2 Interação Symbiodinium-coral
Dinoflagelados do gênero Symbiodinium são organismos fotossintetizantes habitam a gastroderme de corais escleractíneos (Muller-Parker e D'Elia 1997). Há uma complexa sinalização celular que controla a entrada de células de Symbiodinium em corais e, posteriormente, a manutenção dessas células no tecido, sem que haja a digestão dos simbiontes (Davy et al 2012).
Estima-se que até 95% da energia consumida pelo coral seja produzida pelo endossimbionte e translocada para o coral hospedeiro, na forma de compostos derivados da fotossíntese, como glicose
(Venn et al 2008). Apesar de haver evidências da liberação de glicerol em cultivo (Muscatine 1967), existem dúvidas sobre a liberação de glicerol em Symbiodinium in hospite (Davy et al 2012, Lin et al. 2015). Em menores quantidades, carbono fixado em compostos como aminoácidos, lipídeos e
ácidos graxos também são transferidos para o hospedeiro (Venn et al 2008). Essa alta dependência energética torna a simbiose obrigatória em Scleractinia e o coral não sobrevive por longos períodos sem a presença do simbionte (Muller-Parker & D'Elia 1997).
Por sua vez, Symbiodinium depende de nitrogênio fixado, preferencialmente, em amônia para seu metabolismo (Miller & Yellowless 1989, Davy et al 2012). Alternativamente,
Symbiodinium pode absorver nitrato, reduzindo-o a amônia para a fixação em glutamato (Davy et al
2012). Enquanto nitrato e amônia podem ser absorvidos a partir da massa d'água, o principal aporte de amônia é derivado do metabolismo do coral (Davy et al 2012). A correlação entre a abundância de Symbiodinium e de bactérias diazotróficas no coral sugere que a fixação de nitrogênio (N2) por bactérias é também uma importante fonte de amônia para o simbionte (Silveira et al. submetido).
Assim, eventos de eutrofização e o consequente aporte de nitrato/amônia no ecossistema alteram a dinâmica de reciclagem de nitrogênio entre Symbiodinium, corais e bactérias, um importante
15 processo na ecologia dos corais (Radecker et al 2015).
Figura 1.2. Filogenia molecular de Symbiodinium, ilustrando o relacionamento entre os 9 clados designados (Pochon et al 2010). Reconstruções filogenéticas geradas com o gene nuclear 28s e com o gene 23s do cloroplasto geram árvores de topologias semelhantes. A única exceção é o clado E, representado nessas árvores por uma única sequência.
1.2.1 Diversidade taxonômica e funcional em Symbiodinium
Originalmente descrito como uma espécie única, Symbiodinium é reconhecido atualmente como um gênero bem diversificado (Freudenthal 1962, Pochon e Gates 2010). O gênero é atualmente dividido em 9 grandes clados (nomeados de A a I), de acordo com a filogenia do gene ribossomal 18s e reconstruções filogenéticas com genes mitocondriais (cox1) e do cloroplasto (23s rRNA) dão suporte a filogenia do gene nuclear 18s (figura 1.2). Linhagens dos clados A-D e G são encontradas em corais, enquanto que linhagens dos clados C, D, F, G, H e I são encontrados em diversos hospedeiros, como esponjas, moluscos, foraminíferos, ciliados, além de linhagens de vida livre (Carlos et al 1999, Pochon e Gates 2010, Mordret et al 2015). O clado E, pouco abundante, é representado por uma única espécie, S. voratum, de vida livre (Jeong et al 2014).
16 Observa-se uma grande variabilidade dentro dos clados ao utilizar marcadores moleculares com maior resolução filogenética, como, popularmente, o espaçador interno ribossomal ITS2
(LaJeunesse 2001). Estima-se que existam potencialmente centenas de espécies diferentes de
Symbiodinium, apesar do baixo número de espécies descritas (LaJeunesse 2005). Sobretudo nos clados B e C, extensas irradiações evolutivas ocorreram a partir dos últimos 6-9 milhões de anos
(LaJeunesse 2005) e estão associadas ao período de resfriamento global na transição entre o
Mioceno e o Plioceno (Pochon e Pawlowski 2006). O fechamento do Istmo do Panamá no período levou a eventos independentes nas comunidades do Caribe e do Pacífico. Assim, enquanto diferentes linhagens do clado C ocorrem em dominância em cada oceano, linhagens do clado B são raras no Pacífico, mas ocorrem em dominância no Caribe (LaJeunesse 2005).
Além da divisão entre oceanos, há evidências de zonação ecológica e divisão de nicho entre diferentes linhagens de Symbiodinium (Toller et al. 2001a, 2001b, LaJeunesse 2004) e essa zonação pode ocorrer até mesmo numa única colônia de coral (Rowan et al. 1997). Variações na linhagem de Symbiodinium dominante podem ocorrer também durante o desenvolvimento do coral (Little et al. 2004). A observação de eventos de branqueamento em campo (seção 1.3, Rowan et al. 1997,
Baker et al. 2004) e experimentos de transplantes (Baker 2001, Berkelmans & van Oppen 2006), indicam que, apesar de dominantes em condições amenas, linhagens dos clados C e B sejam mais vulnerável a fatores de estresse. Esse trade-off entre a eficiência como endossimbionte e a resistência a estresse foi confirmado em experimentos de laboratório, onde se observam mudanças na eficiência fotossíntese (Rowan 2004), na translocação de carbono para o hospedeiro (Stat et al
2008) e na assimilacão de nitrogênio (Baker et al 2013) de acordo com condições ambientais, em especial o aumento da temperatura. Esses resultados levaram a especulação de que Symbiodinium dos clados A e D poderiam, na verdade, ser parasitas de corais ou espécies oportunistas (Stat e
Gates 2010, Stat et al 2008).
Entretanto, com a utilização de marcadores moleculares com maior resolução filogenética, observa-se uma grande diversidade fisiológica também dentro dos clados (Sampayo et al 2008,
17 LaJeunesse et al 2010). Esses resultados indicam a ocorrência de espécies diferentes dentro de um
único clado (A-I). Recentemente, a combinação de variações fenotípicas, a distribuição ecológica das linhagens e marcadores moleculares menos conservados (ITS2, psbA e micro-satélites) tem sido utilizada para delimitar espécies no gênero (figura 1.2, LaJeunesse et al. 2014; LaJeunesse et al.
2015; Pettay et al. 2015). Assim, enquanto que S. necroappettens (ITS2 type A13) e S. trenchii
(D1a) podem de fato ser espécies oportunistas, S. microadriacticum (A1), S. “linuchae” (A4) e S. boreum (D15) são capazes de estabelecer simbioses saudáveis com corais (LaJeunesse et al. 2014;
LaJeunesse et al. 2015; Pettay et al. 2015). Em contraste, S. termophillum (C3), uma espécie que ocorre nas condições extremas de temperatura e salinidade do Golfo Pérsico/Arábico (Hume et al.
2015), é um membro do clado C, conhecido por ser sensível a estresse.
Figura 1.3. Evolução do número de espécies formalmente descritas e da quantidade de trabalhos utilizando sequenciamento em larga escala em Symbiodinium. Atualizado a partir de Shinzato et al (2014).
1.3 Branqueamento de corais e o estresse oxidativo
Eventos de branqueamento de corais, em que ocorre a dissociação da simbiose entre corais e
Symbiodinium, estão entre as principais ameaças aos recifes de corais no mundo (Hoegh-Guldberg
1999). A perda do endossimbionte pode levar a morte do coral, além de deixá-lo mais vulnerável ao ataque de patógenos (Silva-Lima 2010). Entre diversos fatores que podem levar ao branqueamento
18 de corais, a combinação de temperaturas altas e alta luminosidade é o mais evidente e, em condições de anomalias térmicas, pode ocorrer branqueamento em massa de corais, ao longo de todo recife (Hoegh-Guldberg 1999). Entretanto, diferentes linhagens de Symbiodinium apresentam variações na resistência ao branqueamento. O mesmo ocorre para espécies de coral, sendo assim, o limiar de branqueamento depende da associação Symbiodinium-coral específica (Abrego et al
2008).
Alternativamente, o branqueamento pode se dar também pela degradação de pigmentos de
Symbiodinium (Douglas 2003). Seja pela perda do simbionte ou pela perda de seus pigmentos, eventos de branqueamento causam uma redução na capacidade fotossintética do coral ou de
Symbiodinium em cultivo. Exitem ainda incertezas sobre o mecanismo primário de dano na célula de Symbiodinium, havendo evidências de danos na proteína D1 do fotossistema II (Warner et al
1996, Warner et al 1999, Takahashi et al 2008), redução na taxa de fixação de carbono (Jones et al
1998) e nas membranas tilacóides (Tchernov et al 2004). Em todos esses cenários, a redução na fotossíntese está associada a um estresse oxidativo, causado por uma maior produção de espécies reativas de oxigênio (ROS) na célula (Weis 2008). ROS são compostos altamente reativos, capazes de gerar danos a membranas biológicas, síntese de proteínas e à estrutura de ácidos nucleicos
(Lesser 2006). Se esses compostos não forem rapidamente metabolizados pelo sistema antioxidante de Symbiodinium, poderá haver danos à estrutura celular e vazamento de ROS para o coral. O vazamento de ROS para células do coral induz o branqueamento de diferentes formas: pela degradação de Symbiodinium dentro dos simbiossomos, pelo desligamento de células da gastroderme do coral ou pela morte dessas células, via apoptose ou necrose (Weis 2008, Bieri et al
2016).
O efeito do estresse luminoso se observa na geração de ROS na cadeia transportadora de elétrons da fotossíntese (Warner et al 1999, Tchernov et al 2004). O aumento na luminosidade gera um aumento na taxa de transporte de elétrons entre os fotossistemas, e uma maior taxa de redução de quinonas. O consequente aumento na pressão de excitação do fotossistema II causa o dano à
19 proteína D1 e a inativação do fotossistema II (Warner et al 1999). Um dos mecanismos de proteção
à fotoinibição, a reação de Mehler, é a principal via de dissipação de energia luminosa em
Symbiodinium (Jones et al 1998, Roberty et al 2014). Nessa reação, ocorre a geração de ROS, com a
- redução de oxigênio (O2) a peróxido (O2 ), que posteriormente será metabolizado por ação de enzimas antioxidantes, na via MAP (Mehler-Ascorbate-Peroxidase), regenerando água (Asada
1999).
Figura 1.4. Cadeia transportadora de elétrons da fotossíntese (Kegg:00195). Aumento na irradiância gera um aumento na taxa de redução do pool de quinonas e um consequente aumento na pressão de excitação do fotossistema II. Mecanismos de aclimatação envolvem a reação de Mehler no fotossistema I e o transporte cíclico de elétrons entre o fotossistema I e o pool de quinonas. Em ambos os casos ocorre a formação do gradiente de elétrons, síntese de ATP, mas não ocorre a redução de NADP+.
O efeito do estresse térmico tende a amplificar o efeito do estresse luminoso, gerando uma maior produção de ROS e uma maior redução do potencial fotossintético da célula. Entretanto, a geração de ROS pode ocorrer em diversas reações na célula e observa-se também que altas temperaturas podem levar ao branqueamento mesmo sem o estresse luminoso (Warner et al 1999,
Hill et al 2009, Tolleter et al 2013). Assim, o efeito do estresse térmico pode se dar também em outros compartimentos da célula. A cadeia respiratória mitocondrial é a principal via de formação de ROS em animais (Lesser 2006) e observa-se essa produção também em corais (Dunn et al 2012).
20 Assim, é importante considerar as diferentes vias de formação de ROS para entender os efeitos do estresse oxidativo em Symbiodinium e, consequentemente, no coral.
Existem variações na resistência e resiliência ao branqueamento entre diferentes linhagens de Symbiodinium. Entre os fatores atribuídos a essas variações estão o grau de saturação de lipídeos da membrana tilacóide (Tchernov et al 2004, Diaz-Almeyda at al 2011), o sistema de reparo do fotossistema II (Takahashi et al 2008, Takahashi et al 2014) e a produção de ROS e capacidade do sistema antioxidante da célula (McGinty et al 2012, Krueger et al 2014, Roberty et al 2016). Nesse sentido, o efeito do estresse oxidativo depende também do histórico de exposição ao stress do coral
(Grotolli et al 2014, Takahashi et al 2013).
1.4. Genômica de Symbiodinium
Dinoflagelados possuem uma série de características genômicas únicas, como a presença de cromossomos permanentemente condensados durante a intérfase, a baixa frequência de nucleossomos e a existência de uma proteína nuclear de origem viral (Gornik et al 2012). Os enormes genomas observados (de 1.2 a 200 Gb) são caracterizados por uma alta taxa de duplicação gênica e ocorrência de pseudogenes (LaJeunesse et al 2005, Hou e Lin 2009, Shoguchi et al 2013,
Lin et al 2015). Essas características limitavam a geração do conhecimento genômico sobre o grupo, mas, com o avanço recente nas tecnologias de sequenciamento, um número crescente de trabalhos tem sido publicados avaliando o transcriptoma e genoma de Symbiodinium (figura 1.3). O recente sequenciamento de dois genomas de Symbiodinium (S. minutum (B1), Shoguchi et al 2013 e
S. kawaguti (F), Lin et al 2015) e do genoma de Acropora digitifera (Shinzato et al 2011) possibilitou investigar a complementaridade entre o endossimbionte e o hospedeiro coral (Shinzato et al 2015). Interdependência entre os organismos foi sugerida pela ausência da síntese de cisteína em A. digitifera e pela sobreposição nos conjuntos de transportadores de membranas (envolvidos no metabolismo de nitrogênio, carbono, fosfato e metais (Lin et al 2015)
Especificamente em Symbiodinium, esses trabalhos permitiram identificar diversas
21 características genômicas do gênero, como a estrutura repetitiva do genoma nuclear (Shoguchi et al
2013), a existência de genes codificantes de histonas (Bayer et al 2012), a importância do metabolismo de nitrogênio e homeostase de Ca2+ (Rosic et al 2015) e a importância de mecanismos pós-transcricionais de controle da expressão gênica (Leggat et al 2007, Bayer et al 2012,
Baumgartem et al 2013, Shoguchi et al 2013, Rosic et al 2015). Curiosamente, poucos trabalhos avaliam diretamente o efeito do estresse térmico na resposta transcricional, observando em geral efeitos sobre a transcrição no cloroplasto (Baumgartem et al 2013) e em genes ligados ao ciclo celular (Levin et al 2016)
1.4.1. Genoma mitocondrial e do cloroplasto
Assim como o genoma nuclear, o genoma mitocondrial de Symbiodinium apresenta um enorme tamanho (300 kb), entretanto um conteúdo gênico reduzido a apenas três genes funcionais, todos codificando proteínas importantes para a cadeia respirtatória: cytB, cox1, cox3 (Shoguchi et al
2015). Já o genoma do cloroplasto, ao contrário dos genomas nuclear e mitocondrial, é extremamente reduzido, composto por 14 pequenos cromossomos circulares (1.8kb a 3.3 kb), cada um contendo um único gene (Barbrook et al 2013, Mungapkdee et al 2014). Os genomas das organelas de S. minutum (B1) apresentam também conteúdo GC reduzido em relação ao genoma nuclear e observam-se diferentes mecanismos específicos de edição de mRNA (Mungapkdee et al
2014, Shoguchi et al 2015). A redução do conteúdo gênico nas organelas se dá através de uma transferência massiva de genes para o genoma nuclear (Mungapkdee et al 2014), associados a uma alta taxa de duplicação de algumas dessas famílias gênicas (Maruyama et al 2015). Esses resultados implicam também na evolução de um sistema de transporte e importação de proteínas para o organelas.
Interessantemente, os genes mantidos nas organelas são fundamentais para a formação dos complexos proteicos dos fotossistemas e da cadeia respiratória (Mungpakdee et al 2014, Shoguchi et al 2015). A alta taxa de formação de ROS nas organelas e o consequente risco de danos ao DNA
22 causa uma pressão seletiva para a transferência de genes para o genoma nuclear (Allen et al 2011), mas a pressão seletiva para a manutenção de genes nas organelas é menos clara. A hipótese mais aceita, CoRR (Co-Location for Redox-Regulation), sugere que a transcrição de genes associados à cadeia transportadora de elétrons seja mediada pelo estado oxidativo da organela (Allen 2015).
Nessa hipótese, o estado oxidativo de proteínas carreadoras de elétrons serviria de sinal para a célula induzir a transcrição de proteínas específicas, mantendo o balanço necessário para o funcionamento das cadeias transportadoras de elétrons (Allen 2015). Assim, a co-localização do gene e do produto gênico permite uma resposta rápida ao estímulo ambiental.
23 CAPÍTULO 2
OBJETIVOS
Essa tese tem como objetivo geral estudar a interação entre Symbiodinium spp. e corais
Mussismilia spp. no Banco de Abrolhos, além de compreender a resposta de Symbiodinium à situações do estresse térmico simulado. Com isso pretende-se fornecer ferramentas para o manejo desse ambiente.
Entre os objetivos específicos, essa tese pretende:
- Verificar a diversidade molecular de Symbiodinium spp. em Mussismilia spp.;
- Estabelecer uma coleção de cepas de Symbiodinium spp. associados à Mussismilia spp.;
- Verificar a resposta funcional de Symbiodinium spp. ao estresse térmico através de estudos de transcriptoma;
24 CAPÍTULO 3
Multiple Symbiodinium strains are hosted by the Brazilian endemic corals
Mussismilia spp.
Arthur W. Silva-Lima1, Juline M. Walter1, Gizele D. Garcia1, Naiara Ramires1, Glaucia Ank1, Pedro M. Meirelles1, Alberto F. Nobrega3, Inacio D. Siva-Neto3, Rodrigo L. Moura1,2, Paulo S. Salomon1,2, Cristiane C. Thompson1,2 and Fabiano L. Thompson1,2* Publicado no periódico Microbial Ecology 70(2):301-10. doi: 10.1007/s00248-015-0573-z, Fevereiro/2015
25 Multiple Symbiodinium strains are hosted by the Brazilian endemic corals Mussismilia spp. Arthur W. Silva-Lima1, Juline M. Walter1, Gizele D. Garcia1, Naiara Ramires1, Glaucia Ank1, Pedro M. Meirelles1, Alberto F. Nobrega3, Inacio D. Siva-Neto3, Rodrigo L. Moura1,2, Paulo S. Salomon1,2, Cristiane C. Thompson1,2 and Fabiano L. Thompson1,2* 1Laboratório de Microbiologia, Instituto de Biologia, Universidade Federal do Rio de Janeiro (UFRJ), Brasil. 3SAGE-COPPE, UFRJ. 4Instituto de Microbiologia Prof Paulo de Goes, Universidade Federal do Rio de Janeiro. 5Laboratório de Protistologia, Instituto de Biologia, Universidade Federal do Rio de Janeiro. * corresponding author: Av. Carlos Chagas Fo. S/N - CCS - IB - Lab de Microbiologia - BLOCO A (Anexo) A3 - sl 102, Cidade Universitária, Rio de Janeiro, RJ – Brasil, CEP 21941-599, Phone/FAX: + 55 21 39386567. Centro de Gestão Tecnológica – CT2, Rua Moniz de Aragão, no.360 - Bloco 2. Ilha do Fundão - Cidade Universitária, Rio de Janeiro, RJ – Brasil. CEP 21.941-972. Phone: 3938- 7848 E-mail: [email protected]
Subject category: Microbial diversity, evolution, microbe-host interactions Abstract Corals of genus Mussismilia (Mussidae) are one of the oldest extant clades of scleractinians. These
Neogene relicts are endemic to the Brazilian coast and represent the main reef-building corals in the
Southwest Atlantic Ocean (SAO). The relatively low diversity/high endemism SAO coralline systems are under rapid decline from emerging diseases and other local and global stressors, but have not been severely affected by coral bleaching. Despite the biogeographic significance and importance for understanding coral resilience, there is scant information about the diversity of
Symbiodinium in this ocean basin. In this study we established the first culture collections of
Symbiodinium from Mussismilia hosts, comprising 11 isolates, four of them obtained by Fluorescent
Activated Cell sorting (FACS). We also analyzed Symbiodinium diversity directly from Mussismilia tissue samples (N=16) and characterized taxonomically the cultures and tissue samples by sequencing the dominant ITS2 region. Symbiodinium strains A4, B19 and C3 were detected.
Symbiodinium C3 was predominant in the larger SAO reef system (Abrolhos), while Symbiodinium
B19 was found only in deep samples from the oceanic Trindade Island. Symbiodinium strains A4 and C3 isolates were recovered from the same M. braziliensis coral colony. In face of increasing threats, these results indicate that Symbiodinium community dynamics shall have an important
26 contribution for the resilience of Mussismilia spp. corals.
Key-words: coral reefs, Symbiodinium, Mussismilia, ITS2, clonal cultures, Southwestern Atlantic
Ocean (SAO)
Introduction
The coral holobiont comprises the coral host, its microbiome (virus, prokaryotes, and eukaryotic microbes) and unicellular, photosynthetic endosymbiotic dinoflagellates of the genus
Symbiodinium, the so-called zooxanthellae [1]. Symbiodinium lives inside the coral tissues in extremely high densities, reaching more than 106 cells/cm2 [2]. In the intracellular compartment,
Symbiodinium cells receive protection and inorganic nutrients necessary for photosynthesis from the host coral, while providing organic carbon compounds and oxygen derived from photosynthesis [3-
7]. The coral-Symbiodinium symbiosis plays an important ecological role that is reflected by their geographic spread, with the occupation by modern coral reefs in tropical and subtropical waters of over 280,000 km2 [8], and by the great evolutionary diversification of both corals and zooxanthellae in the last 60 MYA [9]. In spite of this successful evolutionary history, the future of the coral holobiont is uncertain in face of the rapidly ongoing global climate changes [10-12]. Coralline reefs are currently challenged by unprecedented high rates of global warming, ocean acidification, and diseases [11]. Thermal stress, which leads to widespread episodes of coral bleaching, can be a foremost cause of coral mortality [13].
The life cycle of Symbiodinium comprises both a motile (flagellated) and a vegetative, coccoid phase [14]. Within the host cell, Symbiodinium is kept in the coccoid state, while the free- living forms might be motile or coccoid. Symbiodinium colonizes a vast array of hosts, including foraminiferans, sponges, jellyfishes, sea anemones, plathyhelminthes and molluscs [15-17]. The symbiosis is obligate for hermatypic scleractinian corals and the mode of transmission of
Symbiodinium among coral colonies depend mainly on the host’s reproduction type. In brooders, with larvae developing inside coral parents, transmission tends to be vertical, from the parent to the
27 offspring, while in corals that deliver gametes in the water column, the symbiont tends to be acquired from the environment [18, 19]. In this context, the availability of alternative hosts and the
Symbiodinium free living life stage is crucial for the maintenance of the symbiosis. This life stage was proposed to be transient [18], but recent work has been revealing a widespread occurrence of free-living forms [20-22].
Phylogenetic analysis of ribosomal RNA gene sequences (rRNA) revealed that the
Symbiodinium genus can be subdivided into nine (A – I) distinct clades [23-26]. Symbiodinium from clades A to D are the most commonly associated with corals, with clades B and C being dominant in central ecological niches [29, 30]. Clades A and D, although present in tropical seas, are dominant in stressed environments, such as high latitude locations, higher irradiance habitats, extreme temperature conditions and regions with higher coastal influence [31-37]. Furthermore, studies have applied molecular techniques to the rDNA internal transcribed spacer regions (ITS1 and ITS2) and revealed a great fine-scale diversity within these clades [27, 28],
Since clade H description in 2001, a new Symbiodinium clade was reported only in 2010, an endemic divergent lineage hosted by Hawaiian foraminiferans [17]. This decade novelty lag reinforces the importance of researching new habitats and hosts in order to unravel Symbiodinium diversity. The Southwestern Atlantic Ocean (SAO) is one major geographic gap, encompassing a low diversity/high endemism coral fauna dominated by one of the oldest extant genera of scleractinians, Mussismilia. These Neogene relicts, endemics to the Brazilian coast, are declining rapidly due to emerging diseases and other local and global stressors [38, 39]. In spite of such relevance, there is limited knowledge about the genetic and functional diversity of Symbiodinium in the SAO, particularly in the Abrolhos Bank [29, 40-44].
The Abrolhos Bank is the SAO's largest and richest coralline system, encompassing all scleractinian species recorded in the region [45-49]. Pan-Atlantic species show a remarkably low genetic variability in the region and eight of the 18 coral species commonly found in the Abrolhos
Bank are endemic to the SAO [50, 51]. The main reef-building coral species of the Abrolhos Bank
28 are the spawning corals of genus Mussismilia, which encompasses three described species: M. braziliensis, M. hartii and M. hispida [38, 52]. M. braziliensis has the narrowest distribution, restricted to the Bahia State, and may soon be listed as an endangered species due to its rapid decline caused by the infectious disease white plague [39, 40].
In this study we characterized the genetic diversity of Symbiodinium colonizing Mussismilia braziliensis and M. hispida by means of ITS2 sequences. We also established the first
Symbiodinium culture collection originated from M. braziliensis, and investigated it's morphology and physiology.
Materials and Methods
Sampling. Colonies of M. braziliensis were collected with SCUBA (5-20 m depths) using hammer and chisel in two locations (Sebastião Gomes - SG, 17°54′42.49″S,39°7′45.94W″; Parcel dos
Abrolhos - PAB, 17°57′32.7″ S, 38°30′20.3″ W) during the summer of 2012. Reefs SG (open access area) and PAB (inside the no-take Abrolhos National Marine Park) are 14 and 65 km off the coast, respectively [45, 46]. Five whole coral colonies (approx. 15 cm) were transported alive to the laboratory in separate coolers with seawater and kept in aquaria until Symbiodinium cell isolations for the establishment of cultures. Additionally, tissue samples of M. braziliensis were collected from healthy (n=7) and white plague infected (n=5) colonies during the summer of 2010 at PAB and SG reefs (Table 1, [40]). M. hispida tissue samples were collected in the summer of 2011 at the
Trindade Island, 1,600 km offshore (CVT20 - 20°31′33.6″ S, 29°18′37.3″ W) and at the Jaseur
(CVT13 - 20º 24,897´S; 36º 02,511´W) and Davis (CVT16 - 20º34,603´S; 34º48,387´W) seamounts
(Table 1). These samples were kept in liquid nitrogen until DNA extraction.
Isolation and establishment of Symbiodinium strains in cultures. After rinsing M. braziliensis colonies with filtered (0.45um) and autoclaved seawater, Symbiodinium cell suspensions were made by carefully scrapping tissue (one or two polyps) from coral specimens kept in the aquaria and re- suspending it in sterile sea water. Isolation of Symbiodinium cells from these suspensions was done
29 using two strategies: fluorescence-activated cell-sorting (FACS); and manual cell picking and transfers in an inverted microscope. Single-cell separation by FACS was done in a flow cytometer
(DakoCytomation® MoFlo) equipped with an electrostatic droplet deflection system and the
Cyclone sorting option for sorting single-cells. The flow cytometer was fitted with a 100 µm orifice nozzle tip and sheath pressure was kept at 12 PSI. Upon excitation with the blue laser line (488 nm,
100 mW), Symbiodinium cells were detected in two-parameter plots based on their chlorophyll content (red fluorescence) and size (forward scattered light). Using the sort-for-purity mode on the flow cytometer, single cells were individually deposited in each well of 96-well microtiter plates containing 150 µl of sterile F/2 medium [53]. Alternatively, Symbiodinium cells were isolated by manually picking them from freshly made coral tissue suspensions using a micropipette in an inverted microscope. Cells or small cell clumps were separated by successive transfers through F/2 medium in sterile Petri dishes or in the wells of 24-well sterile microtiter plates (on a 15 to 20-day interval basis). For manual isolation the medium was supplemented with a mix of antibiotics
(Gentamycin: 0.08mg/ml; Kanamycin: 0.02mg/ml; Nystatin: 0.015 mg/ml; Penicilin: 0.3 mg/ml;
Streptomycin: 0.08mg/ml; Germanium dioxide: 5 mg/L [54]). Microtiter plates with the isolated cells were kept in a culture chamber (24oC, photon flux of ca. 80 µE/m2/s, photoperiod of 14h light/10h dark) and monitored for growth on a weekly basis for ca. 10 weeks, using an inverted microscope. Cultures that grew during this period were transferred to larger volumes and incorporated into the collection, which is being kept by successive transfers since then.
Phylogenetic analysis. Molecular identification of Symbiodinium samples was done by direct sequencing the dominant nuclear ribosomal ITS2 region [55]. DNA extraction was performed for
Symbiodinium cultures using chloroform-ethanol washings [56]. To obtain total DNA of the coral holobiont, DNA extraction was done as described previously [40]. PCR amplification of partial 5.8s rDNA, complete ITS2 region and partial 28s rDNA was based on primers ITS2intfor (5’-
GAATTGCAGAACTCCGTG-3’) and ITS2reverse (5’-
GGGATCCATATGCTTAAGTTCAGCGGGT-3’) using a touch-down PCR strategy [57, 58]. PCR
30 products were purified with ExoSap-IT (USB Corporation, USA) and sequenced in both directions with the same primers above, using a capillary system (ABI3500).
The identity of the sequences obtained in this study was first identified by similarity (blastn algorithm [59]) and furthter checked by phylogenetic analysis. The best model of molecular evolution for the ITS2 sequences was chosen by ModelTest [60]. Maximum likelihood phylogenetic reconstructions were performed using a rooted Neighbor-joining guide tree, Kimura 2-parameter molecular evolution model and 2000 bootstrap replicates [61]. The final phylogenetic tree consisted of 27 ITS2 sequences generated in this study and 16 published Symbiodinium ITS2 sequences from clades A, B, and C. The closely related dinoflagellate Pelagodinium beii was used as the outgroup for the phylogenetic reconstructions. Gene sequences of cultures are deposited in Genbank under accession numbers KJ189553-KJ189564 and sequences of holobiont coral tissue under accession numbers KJ488961- KJ488977.
Symbiodinium cell morphology and physiology. Cell size and volume of seven representative
Symbiodinium cultures were measured with an automated inflow imaging system (FlowCAM®,
Fluid Imaging Technologies), which combines the capabilities of flow cytometry, microscopy and image analysis [62]. Before being analyzed in the FlowCam, each Symbiodinium culture was sonicated for 30 seconds (30 pulses of one second with three seconds intervals, 20% power, ultrasonic processor, Cole-Palmer) to disrupt cell clumps. The FlowCam was fitted with a 90 µm flow cell, and analysis was done at 100 µl/min sample flow for 10 min. Images were collected through a 10 X magnification objective in auto-image mode. A total of 1,500 cell images from each culture were selected for morphometric analysis of primary linear dimensions (cell length and width) and equivalent spherical diameter (ESD) using the software provided with the FlowCam.
Light micrographies of culture 043B7 living Symbiodinium cells were obtained with a differential interference contrast (DIC) equipped microscope (Axio Imager.A2, Zeiss, Germany).
In order to estimate growth parameters, Symbiodinium strain 043D10 (ITS2 type A4) was
31 grown as batch cultures in triplicate 500 milliliter glass flasks containing 200 ml F/2 medium, at 80
µE/m²/s; 14 light/10 dark and 24 +/- 1oC. Samples were taken at 3 to 4 days interval, sonicated as described above and fixed with acid Lugol's solution (1% final concentration). Cell counts were done in Palmer Maloney chambers using an inverted microscope (Nikon TS100) at 200x magnification. Based on cell densities, intrinsic growth rate, doubling time and maximum cell yield were then estimated for each replicate and averaged for each strain, as described previously [63,
64].
The photosynthetic potential of this strain (043D10) was determined by means of pulse amplitude modulated (PAM) fluorometry on cells collected at late exponential growth phase [65]. Culture was maintained in the same conditions as for the growth curve, except for an irradiance of ca. 60
µE/m²/s. Cell densities were adjusted to 106 cells/ml and twelve replicates were dark adapted for 20 minutes before measurements of maximum photosynthetic potential (Fv/Fm) with a blue light diving-PAM (Walz GmbH, Germany) under a saturation light pulse of 2500 uE/m²/s.
Results
Isolation and culturing of Symbiodinium. We established a culture collection of eleven
Symbiodinium strains originating from M. braziliensis hosts (Table 1). Both isolation methods produced actively growing cultures. Four Symbiodinium A4 clonal cultures originated from single cells sorted by FACS (042C5, 043B7, 043D10, 043G2), while the other seven isolates were obtained by manual cell picking and transfers.
32 Table 3.1. Symbiodinium samples (isolates and holobiont tissues) used in phylogenetic reconstruction, with strain designation, coral host, depth, health condition, year and sampling site. Blast results are summarized on columns “Best hit” and “Identity”. For holobiont coral tissue, each sample is from a different colony while for isolates the originating M. braziliensis colony is identified by numbers. SG: Sebastião Gomes; PAB: Parcel dos Abrolhos; CVT: Vitoria-Trindade seamount chain; WP: colonies diseased with White Plague.
Molecular diversity of Symbiodinium. Together, the culture dependent and independent analyses, revealed that Symbiodinium from three different clades (A, B and C) are associated with
Mussismilia (Table 1). Clade A sequences were all assigned to Symbiodinium A4, with at least 98% identity over 258 nucleotides. Clade B ITS2 sequences were most similar to Symbiodinium B19
(97% identity for sample CVT13), and sample CVT16 is a putative novel haplotype derived from
B19, with a 9 bp deletion and 20 base substitutions (88% identity). Due to the limited phylogenetic resolution of the ITS2 gene and the recent radiation event that occurred within this clade, identification of clade C Symbiodinium strains based on ITS2 sequences is difficult [44, 66]. C1 sequences clustered together on our phylogenetic analysis, but with a weak bootstrap support
33 (45%). Analysing the alignment of clade C ITS2 sequences (Table S1), C1 and C3 sequences were separated by a single base substitution on position 196 (C1: G , C3: A). As clade C sequences obtained in this study were placed outside the C1 clade and all had an A on position 196, they were assigned to the Symbiodinium C3 group (at least 98% identity).
The eleven cultures belonged to Symbiodinium strains A4 and C3 (Figure 1). C3 cultures ITS2 sequences grouped along with the sequences from M. braziliensis tissue, either healthy or white plague infected. Isolates identified as Symbiodinium A4 (103C2, 103C5) and from the C3 group
(103B3, 103C1, 103C6) originated from the same coral colony (Table 1), indicating that a single M. braziliensis colony can host multiple Symbiodinium strains.
Figure 3.1. Maximum likelihood phylogenetic reconstructions of Symbiodinium ITS2 sequences. All position containing gaps and missing data were eliminated, yielding a total of 151 positions in the final dataset. Bootstrap support (2000 replicates) are indicated above each node. Sequences obtained in this study for Mussismilia corals are marked by either filled triangles (culture collection) or circles (host tissue samples). Twenty seven Mussismilia associated Symbiodinium and sixteen formally and informally described Symbiodinium species ITS2 sequences were used in the final tree, with Pelagodinium beii as an outgroup. Symbiodinium clades A, B and C are marked accordingly. MB: Mussismilia braziliensis; MH: Mussismilia hispida. 34 Table 3.2. Morphological parameters of the isolated strains based on FlowCam data. Mean and standard deviations of 1,500 cells of each isolate sample. All parameters were measured at 40× magnifications. Units for length, width and Equivalent Spherical Diameter (ESD) are in micrometers.
Symbiodinium cell morphology and physiology. Symbiodinium cells of strains isolated from M. braziliensis displayed the characteristic brown color when observed in light microscopy due to photosynthetic pigments (chlorophylls and xanthophylls) typical of these dinoflagellates. During cultivation, cells were mostly found in their coccoid, nearly spherical, non-motile phase, and less frequently in their flagellated, gymnodinoid motile forms. Doublets, indicating the process of mitosis in the coccoid forms, were frequently observed throughout the growth curve, indicating healthy growth conditions (Figure 2). Average cell diameter (expressed as ESD) of the non-motile coccoid phase measured on seven cultured strains isolated from M. braziliensis ranged from 7.1 to
8.7 µm (Table 2).
The Symbiodinium culture 043D10 (Symbiodinium A4) reached stationary phase at ca. 20 days after inoculation, with cell concentrations remaining stable thereafter until the end of the experiment in day 40 (Figure 3). Growth rate during the exponential phase was estimated as 0.24 d-1, yielding doubling times of 2.9 days. Maximum cell density was as 4.4x105 cells/ml and mean photosynthetic potential (Fv/Fm) was 0.64.
Discussion
Symbiodinium strains isolation and physiology. Our isolation efforts using both FACS and manual cell transfers led to several established Symbiodinium cultures. The automated isolation
35 approach in the flow cytometer has the advantages of high cell-sorting throughput that allows processing many samples and isolating several hundred cells in short time intervals (<1hr), as well as the establishment of clonal cultures in one single step, directly from fresh cell suspensions [67,
68]. Remarkably, fewer dinoflagellate cultures have been established by FACS in comparison to other microalgal taxa [69, 70]. Dinoflagellates seem to be more sensitive to the process of cell sorting in the flow cytometer, showing lower success than other algal groups when single cells are deposited in the wells of microtiter plates [70]. Although flow cytometry has already been used to screen environmental Symbiodinium populations [20, 71], this is the first account of a successful use of FACS methodology for sorting viable Symbiodinium cells with the purpose of establishing clonal cultures. This results shows FACS has a great potential for retrieving coral-associated
Symbiodinium diversity and, with the rampant advances in single-cell genomics, for extending the knowledge on coral and Symbiodinium genomics [72].
Figure 3.2. Morphology of Symbiodinium A4 culture 043B7 (1000x DIC micrographs). Coccoid cells has spherical shape and great internal complexity. Dividing cells can be observed on the right and a newly encysted cell can be observed on the left image.
The specific growth rate of Symbiodinium A4 in our controlled cultivation experiments at 24oC are within the range typically observed for other Symbiodinium strains between 0.15 and 0.38 d⁻ ¹, yielding doubling times between 2 and 5 days, at temperatures from 20oC to 28oC [14, 64, 73-75].
Also, under controlled conditions, the photosynthetic potential of Symbiodinium A4 agrees with reported values for this genus [75,76]. Biogeographical and experimental studies have shown that
Symbiodinium C3 is more efficient on mild light and temperature conditions, while A4 symbionts
36 are typically associated to higher irradiance shallow waters [36, 75-79]. This diverse functional responses suggests a trade-off between physiological efficiency and stress resistance that plays a key role in Symbiodinium niche partitioning, reflecting the potential contribution of this symbionts for the coral holobiont Mussismilia spp. [12, 36, 78-83].
Figure 3.3. Growth curves of Symbiodinium A4, culture 043D10, cell densities expressed on base 10 logarithmic scale. On the inset the photosynthetic potential (Fv/Fm) of the same culture.
Symbiodinium biogeography in the Southwestern Atlantic ocean. Despite the isolation, relative small area (5% of Atlantic reefs) and the corresponding low coral host diversity in the SAO reefs
[84], three of the most commonly coral-associated clades (A, B and C) are present in the region.
Most of the Symbiodinium ITS2 types observed in the SAO (A4, B19, C1 and C3) are considered ancestral sequences, from which radiation events have occurred both in the Pacific and the
Caribbean [30, 44]. In fact, some of the samples on this study had less than 100% identity to known
ITS2 types and are probably derived from these ancestral sequences. Given the low number of hosts species surveyed and assuming that these ITS2 types are probably groups of species, the number of
Symbiodinium species reported in the SAO so far (~10) is likely an underestimation of its real richness [40-44].
Previous studies on deep reef environments showed the predominance of clade C and B
Symbiodinium strains [85-89]. The occurrence of Symbiodinium B19 at depths over 45 meters is
37 probably related to cold water tolerance of this lineage [30], but it is surprising the occurrence of M. hispida colonies harboring the shallow-water associated Symbiodinium A4 [36, 76-79], over twenty meters deep at the Trindade Island. Although it has already been reported at deep environments
[89], the dominance of this Symbiodinium type might reflect the higher light incidence and the impoverished conditions of Trindade Island reefs, compared to other mesophotic reefs of the region
[Meirelles PM, personal comunication].
Molecular clock reconstruction indicates that most Symbiodinium clades were established on a radiation event occurring between 25 and 50 MYA [9], prior to the formation of the Orinoco-
Amazon river plume (10 MYA) and the cooling of the Benguela current (2-3 MYA) [90], which are considered the main events that have limited the connectivity between the SAO and the Caribbean and tropical Pacific basins, respectively. From that perspective, the absence of clade D
Symbiodinium strains, specially S. trenchii (D1a) a widespread, generalist and stress-resistant species [37, 91] in our study is intriguing. Previous studies in different geographic regions of the
Brazilian coast, focused on the microbial diversity associated with different host species have reinforced the hypothesis that clade D is rare or absent in the SAO [40-44]. In this context,
Symbiodinium biogeography in the SAO is still challenging, including the distribution of the major clades and the recent fine-scale diversity radiations within clades (15MYA) [9, 30].
Multiple Symbiodinium strains inhabit simultaneously a single coral colony of Mussismilia braziliensis. As broadcast spawners, Mussismilia corals shall depend on environmental community composition in order to be re-colonized by Symbiodinium, a scenario that favors less specific interactions [18, 19, 92]. A significant finding of the present work was the demonstration that cells belonging to the Symbiodinium A4 and C3 may colonize the same coral host species, M. braziliensis. Regardless of sampling on shallow environments of the Abrolhos reef bank, we could not observe Symbiodinium A4 as the dominant symbiont in M. braziliensis tissue samples (Table 1).
Complementarly, Symbiodinium C3 strains were not obtained by FACS, suggesting it is more sensitive than A4 strains, either to the cell sorting process or to culture conditions [93, 94].
38 Combined, these facts suggests that M. braziliensis of the Abrolhos reefs harbors preferentially
Symbiodinium C3 with background populations of Symbiodinium A4.
Our observations that M. braziliensis can host at least two Symbiodinium strains has implications for the resilience of Mussismilia-dominated reefs, and might be partially connected to the relative resistance and resilience to bleaching of the SAO when compared to other biogeographic regions
[95]. Relevantly, the main coral mortality source reported for M. braziliensis is a white plague-like
(WPL) disease that has been recorded in all sampled sites in the Abrolhos Bank [39]. Symbiodinium
C3 was associated with healthy and diseased M. braziliensis, indicating that corals harboring these symbionts are susceptible to WPL infections, but it's not clear whether the occurrence of
Symbiodinium A4 is related to the current high WPL disease prevalence in M. braziliensis, or is indeed a baseline condition of SAO reefs. Subsequent studies shall test the hypothesis that the relative abundances of Symbiodinium strains in M. braziliensis might change under biotic or abiotic stress. In a severely threatened environment as the Abrolhos Bank [39], an understanding of the
Symbiodinium community responses to these factors is indispensable for the conservation of these reefs.
Conclusions
Our first survey of Symbiodinium in Mussismilia spp. corals reveals the occurrence of clades A, B and C, and the predominance of Symbiodinium ITS2 types A4 and C3 in the Abrolhos Bank, the largest and richest coralline reef in the SAO. Moreover, the endemic, relict and endangered coral genus Mussismilia spp. is a symbiont generalist group, associating to at least three different
Symbiodinium clades. Given the reported functional diversity observed within Symbiodinium, the fact that Mussismilia spp. corals can associate with multiple Symbiodinium strains, even within a single coral colony, might have profound implications for the resilience of Mussismilia holobionts and the dynamics of the SAO reef environments.
39 Acknowledgements The authors thank CNPq, CAPES, and FAPERJ for the core financial support to this work and Mr. Bruno Maia for technical assistance in the flow cytometer. The Abrolhos National Marine Park (ICMBio, Ministry of Environment), Brazilian Navy, Conservation International and the Rede Abrolhos (www.abrolhos.org) contributed with permits, logistics and field support in Abrolhos and Trindade Island.
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46 Table 3.S1. ClustalW alignment among Symbiodinium clade C sequences. C1, C3, C15 and C90 sequences from GenBank were used to support the analysis. All clade C sequences obtained in this study grouped together with C3 on phylogenetic analysis (Figure 1) and had an “A” on position 196, matching Symbiodinium C3 types.
47 48 49 CAPÍTULO 4
HEAT STRESS INDUCES A TRANSCRIPTIONAL RESPONSE
ASSOCIATED TO DIVERSE CELLULAR COMPARTMENTS IN THE
MUSSISMILIA ENDOSYMBIONT SYMBIODINIUM
ABSTRACT
Elevated temperature and irradiance are known to induce an accumulation of reactive oxygen species (ROS) in Symbiodinium, which might ultimately lead to the disruption of the coral-
Symbiodinium association. However, thermal-induced bleaching occurs independent of the photosynthetic activity and little is known about the possible ROS generation processes in
Symbiodinium, other than the photochemichal reactions. We performed a transcriptomic experiment to evaluate the light independent bleaching of cultured Symbiodinium A4 subjected to a 4oC heat shock. Compared to the control treatment at 24oC in the dark, the heat shock caused inhibition of expression of genes associated to ROS production processes located in diverse cellular compartments. Inhibition of genes associated to photochemichal reactions and respiratory electron transfer chains was observed and cytochrome p450 wast one of the most inhibited gene (log2FC=-
8), indicating a drastic reduction in the endoplasmatic reticulum monooxigenase activity. A possible acclimatization response was observed by the induction of genes controlling chloroplast protein import (tic32) and iron availability to the mitochondria and chloroplast (csd1). Noteworthy, the activity of proteins encoded tic32 and csd1 is regulated by the redox state of the cell. Modifications in the composition of transmembrane proteins was observed, indicating an active regulation of
Ca2+ and Fe2+ concentration in each cellular compartment. Overall, our results shows that
Symbiodinium A4 responds actively to heat-induced oxidative stress, despite the deficit in ATP synthesis. We argue that importance of ROS production in the endoplasmatic reticulum and
50 mitochondria has long been overshadowed by the generation in Symbiodinium chloroplast, under conditions of illumination. Moreover, our results suggests that oxidative stress response in
Symbiodinium is regulated by the redox state of the cell, in a negative feedback. The outcomes of this negative feedback and the utility of redox-sensitive genes as molecular markers for an acclimatization potential is Symbiodinium shall be investigated.
Keywords: Symbiodinium, oxidative stress, coral bleaching, transcriptome
INTRODUCTION
Photosynthetic dinoflagellates of the Symbiodinium genus lives in association with a wide array of metazoan hosts and also unicellular eukaryonts (Stat et al 2006). This association is obligatory for scleractinian hosts where the endosymbiont translocated photosynthetic derived carbon compounds can be responsible for up to 90% of the animal energy requirements (Muller-
Parker and D'elia 1997). Although this symbiosis is essential for the ecological and evolutionary success of reef-building corals over more than 50 MYA (Pochon and Pawlowski 2006), mass bleaching events have been causing a consistent decline on coral reef coverage over the last decades
(Hoegh-Guldberg 1999). Coral bleaching is an extreme stress event that culminates with the expulsion or degradation of the symbiotic dinoflagellate by the coral host. The resulting energetically impaired corals have lower growth rates, are more susceptible to the occurrence of diseases and mass mortality, favoring the shift to macro-algal dominated reefs (Dudgeon et al
2010).
Among other biotic and abiotic factors, thermal stress is the most well characterized trigger of coral bleaching (Hoegh-Guldberg 1999). World-wide mass bleaching events have been observed associated to seawater thermal anomalies, but the extent of bleaching depends on the coral and
Symbiodinium community composition (Rowan et al 1997). Thermal and high irradiance stress acts
51 in synergy compromising chloroplast functioning and leading to a higher production of reactive oxygen species and decreased photosynthetic potential (ROS, Hill et al 2009, Weston et al 2015).
The most well studied ROS generating process in Symbiodinium is Mehler reaction at photosystem
I, where electrons from the photosynthetic transfer chain are used to reduce dioxigen to superoxide
(Roberty et al 2014). However, bleaching also occurs in dark conditions, with no activity of photochemichal reactions (Hill et al 2009, Tolleter et al 2013) and it is likely that thermal stress affects ROS-generating processes in the cell in a non-specific manner (Lesser 2006). If the excess
ROS production is not scavenged by the Symbiodinium antioxidant system, ROS can cause damages to the membrane system and leak to the coral host, eliciting defensive responses in the coral host
(Weis 2008).
Different Symbiodinium strains have different susceptibility to thermal stress and bleaching.
Several factors have been attributed to this, including lipid composition of thylakoid membranes
(Tchernov et al 2004), repair system of thylakoid proteins (Takahashi et al 2008), production of
ROS and antioxidant capacity of the cell (Krueger et al 2014). Moreover, there is accumulating evidence that, at least some, Symbiodinium species may acclimatize to higher temperatures, either through changes in membrane composition (Diaz-Almeyda at al 2011), enhanced anti-oxidant activity (McGinty et al 2012) or denovo synthesis of chloroplastic proteins (Takahashi et al 2014).
In this sense, previous exposure to heat contribute to acclimatization of Symbiodinium (and coral holobionts) to higher seawater temperatures, pushing forward coral bleaching thresholds (Grotolli et al 2014, Takahashi et al 2013).
Although this scenario of oxidative damage is well documented in the literature, little is known about its genetic basis. It was only recently that comprehensive genomic resources have been developed for Symbiodinium, mostly due to its large genome size and complexity (LaJeunesse et al 2009, Shoguchi et al 2013). Dinoflagellates have several unique genomic characteristics such as a high level of methylation, chromosomes permanently condensed and the presence of a nuclear protein of viral origin, the dinoflagellate viral nuclear protein (Gornik et al 2012). The paucity of
52 transcription factors observed (Bayer et al 2012, Shoguchi et al 2013), as well as the high level of genes coding for post-translational modification and protein turnover suggests that most gene expression regulation might occur at the post-transcriptional level (Leggat et al 2007, Rosic et al
2015). Nevertheless, few studies have taken advantage of high throughput RNA-seq to analyze
Symbiodinium gene expression in different environmental factors in Symbiodinium, often finding small percentage of differentially expressed genes (~1% of the genes, Baumgartem et al 2013,
Xiang et al 2015, Levin et al 2016).
In this study, we evaluate the hypothesis of ROS production independent of photosynthetic activity observing the transcriptomic response of cultured Symbiodinium A4 to a heat shock experiment. Symbiodinium A4 is a widespread, generalist symbiont found from tidal pools to up to
20 meters deep reefs (Santos and LaJeunesse 2006, Silva-Lima et al 2015). It associates to a wide range of hosts and it was found to be resistant to thermal bleaching in association to Porites divaricata (LaJeunesse 2001, Grotolli et al 2014). It is found in association with Mussismillia spp. corals, the main reef builders of the Southwestern Atlantic Ocean, usually as a background symbiont in M. braziliensis (Silva-Lima et al 2015).
METHODS
CULTURE ORIGIN AND HEAT-STRESS EXPERIMENT
Symbiodinium A4 (043D10) was isolated from Mussismilia braziliensis corals, the main coral reef builders of the Southwest Atlantic Ocean. Isolation was done by single-cell sorting on a flow cytometer and the resulting clonal culture identified by ITS2 sequencing. Before the experiments, cultures were maintained on F/2 media at 24oC, and 70uE/m2/s, with 14/10 dark/light cycle (Silva-Lima et al 2015).
Algal cells were transferred to 30oC at the dark in four replicates, to assess the effect of an
53 acute heat stress over chloroplast functioning. Maximum photosynthetic potential (Fv/Fm) was measured 0.5, 1, 2, 21, 22 hours after the beginning of the heat shock, with a blue light diving-PAM
(Walz GmbH, Germany) under a saturation light pulse of 2500 uE/m²/s. Subsamples (200 ul) were taken at the onset and after 22 hours of the heat shock and fixed with acid Lugol's solution (1% final concentration) for estimating cell densities. Cell counts were done in Palmer Maloney chambers using an inverted microscope (Nikon TS100) at 200x magnification. Linear regression analysis and on the change of maximum photosynthetic potential over time and t-test on cell densities was done in R 3.2.4 (R Core Team 2016).
TRANSCRIPTOME EXPERIMENTAL SET-UP
Symbiodinium A4 culture was maintained on exponential growth and transcriptomic analysis were done with densities of 10e5 cells/ml over three different conditions, with two replicates per condition (table 1). 'Dark' treatment was done with 24oC after 24 hours without light and the 'Light' treatment was carried on standard culture conditions (24o C; 70 uE/m2/s) after 6 hours of illumination. Additionally, a heat-shock treatment was done where cells were subjected to 28oC at the dark for 2 hours, after 6 hours of illumination ('Heat'). A mix of antibiotics was used to control for bacterial growth two days before sampling cells, on all treatments (Polne-Fuller 1991, Silva-
Lima et al 2015).
Algal cells were collected by centrifugation (900g for 5 minutes), supernatant was discarded and pellet was instantly freezed in liquid nitrogen. Total RNA was extracted with TRIzol
(Invitrogen) and purified on columns (Qiagen RNEasy mini kit), according to manufacterer's instructions. cDNA was synthesized from poly-A mRNA with SMARTer PCR synthesis kit
(Clontech) and cDNA libraries were sequenced separately with 500 cycles paired-end NexteraXT on MiSeq (Illumina), generating pairs of 250-bases reads.
54 QUALITY CONTROL AND ASSEMBLY
Pairs of reads were merged with PEAR (Zhang et al 2014) and cutadapt was used to remove either SMARTer or Illumina adapter sequences (Martin 2011). Sequences with ambiguos bases and low mean quality (phred <25) were discarded while poli-A/T and low quality tails were trimmed with prinseq (Schmieder e Edwards 2011). The resulting high quality reads from the six replicates were combined in a single “cross-assemble” with Trinity (Haas et al 2013).
Ribossomal RNA reads were filtered with bowtie2 against the truncated SSU/LSU Silva database, version 119 (Quast et al 2013). Blastn analysis of the reads mapping the SILVA database indicated the occurrence of bacterial rRNA fragments, from the families Rhodospirillaceae
(Alphaproteobacteria), Flammeovirgacea (Cytophagales) and Flavobacteriales (Flavobacteria), indicating possible DNA contamination. To remove residual bacterial DNA fragments, assembled contigs were retained only if it presented less than 65% amino acid identity to bacterial sequences and more than 80% nucleotide identity to available Symbiodinium mRNA databases (Symbiodinium
C3 (Leegat et al 2007), Symbiodinium minutum – B1 (Bayer et al 2012, Shoguchi et al 2013),
Symbiodinium microadriacticum - A1 (Voolstra et al 2009, Bayer et al 2012)). High quality mRNA was mapped back to the final Symbiodinium transcriptome with bowtie2.
IDENTIFICATION OF TRANSCRIPTION FACTORS AND ANTIOXIDANT ENZYMATIC
SYSTEM
Identification of contigs with sequence specific transcription factor (TF) domains was based on pFam models compiled in (Ryu et al 2011). Nucleotide contigs from the transcriptome were translated in the 6 frames with transeq (EMBOSS) and HMMER searches were carried out, with an e-value threshold of 10e-6. We followed the approach of Bayer et al (2012) for quantification of TF domains: sequences were counted only once if multiple isoforms of the gene carries the same
55 domain or if a sequence contains repetitions of the same domain.
A similar approach was used to identify genes involved in the antioxidant response, with the list of pFam models described in Bayer et al (2012). Because of the potential role of DMSP- breakdown products as ROS scavengers (Sunda et al 2002), we further included the hmm model for bacterial DMSP-lyases (PF16867). Additionally, genes annotated (BLASTx at the SwissProt database) as the recently described algal DMSP-lyase gene were included in the comparison
(Alcolombri et al 2015).
DIFFERENTIAL EXPRESSION ANALYSIS
Analysis of differentially expressed (DE) genes was done for each pairwise contrast among treatments (Dark/Light, Dark/Heat, Light/Heat), on edgeR software (Ribinson et al 2010). To avoid possible influence of DNA sequences on DE results, reads mapping at bacterial contigs were discarded. Furthermore, reads with GC content lower than 45% were filtered out of the analysis.
This prevented the occurrence of unannotated bacterial sequences, but also of most sequences from the Symbiodinium chloroplast and mitochondrial genomes (Mungpakdee et al 2014, Shoguchi et al
2015). The different structures of organelle and nuclear genome and the varying number of mitochondria in each cell made this conservative approach necessary to avoid artifacts in DE genes calling. The final mRNA pool was used to quantify Symbiodinium contig expression level with
RSEM (Li and Dewey 2011) and contigs with a minimum support of 15 reads were selected for differential gene expression. Samples were compared after TMM normalization and DE genes were called at 0.05 significance level and a 10% false-discovery rate, with Benjamini-Hochberg correction.
The assembled transcriptome was annotated by BLASTx homology searches against the
SwissProt, TrEMBL and NCBI-nr databases, with an e-value threshold of 10e-5. Gene Ontology assignments were done by local mapping with the UniProt-GOA database
56 (ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/idmapping/). Analysis of GO enrichment on differentially expressed genes was checked by Fisher exact test on a 5% significance level and the 'weight01' algorithm to account for the GO topology, with the bioconductor package topGO (Alexa and Rahnenfuhrer 2010).
RESULTS
Figure 4.1. Effect of a heat-shock on a Symbiodinium A4 culture. Photosynthetic potential of cells transferred from 24oC to 30oC throughout 22 hours of exposure. In the insert results for cell counts (cells/ml) at the onset and 22 hours after to heat exposure.
The heat shock caused a consistent decrease on maximum photosynthetic capacity (Fv/Fm) along time, after the heat shock (slope=-0.005, p=3.74e-10; R2=0.89). After 22 hours,
Symbiodinium A4 cells presented a 20% decline in Fv/Fm, but this decline was not followed by a change in cell densities, which remained close to 6x10e5 cells/ml (t=-0.25; p=0.84).
57 Table 4.1. Annotation statistics of the Symbiodinium transcriptome
EVALUATION OF THE GENETIC REPERTOIRE OF SYMBIODINIUM ASSOCIATED TO
MUSSISMILIA spp..
The present study is the first transcriptome from Symbiodinium cultures isolated from
Mussismilia braziliensis and the first from Symbiodinium A4. The Symbiodinium A4 assembly is comparable with other assemblies in the genus. The GC content, the number of observed genes and the annotation ratio (table 1) is within the range observed for other lineages (Symbiodinium B1:
51%; Symbiodinium A1: 56,4%; Bayer et al 2012; Shoguchi et al 2013). Of the 36228 genes in the transcriptome, only 44 possessed TF domains (0.12%, TableS1). This proportion, lower than the observed for other eukaryotes, is in accordance with other Symbiodinium types (Bayer et al 2012,
Baumgartem et al 2013). Half of these TF were Cold Shock Domain, proteins present in most eukaryotic species, but abundant only in dinoflagellates (Bayer et al 2012).
We observed a diverse array of transcripts coding for antioxidant enzymes, most common are peroxidases, Glutaredoxin and the superfamily Thioredoxin. Despite there were no hits to the catalase domain, two genes were annotated as catalase-peroxidase (katG, table S2). Two of the four genes with the Sod_Ni domain were annotated as ubiquitinins, indicating the occurrence of genes
58 encoding Sod_Ni and ubiquitinin domains also in Symbiodinium A4 (table S2, Bayer et al 2012).
No genes with the bacterial type DMSP-lyase domain, but three genes homologous to the algal
DMSP-lyase were observed (table S2).
Table 4.2. Number of genes with antioxidant enzyme domains in Symbiodinium A4 (043D10) and comparison with Symbiodinium A1 (CassKB) and B1 (Mf1.05b).
DIFFERENTIAL EXPRESSION ANALYSIS
Considering all pairwise comparisons, few genes were found to be differentially expressed
(DE): 48 (0.1% of total) and 287 (0.7%) in the Light/Dark and the Heat/Dark contrasts, respectively. Of these, 25% (12/48) was annotated in the Light/Dark contrast and 32.4% in the
Heat/Dark contrasts (93/287). Surprisingly, no differentially expressed (DE) genes were observed when comparing the Light and Heat treatments. Of the 45 genes under-expressed in the light treatment, 39 were also under-expressed in the Heat-shock (86.8%). Similarly, two of the three over-expressed genes in the light were also over-expressed in the Heat-shock. These results suggests an influence of the period under illumination in the Heat-shock cellular response. The hypothesis that heat and light stress share a common cellular mechanism is re-forced by the observation that most DE genes in the Heat condition presented concordant log-fold changes of expression in the 59 light treatment, but with lower magnitude and not statically significant (Figure S1).
The paucity of DE genes expressed in the Light condition hampers a clear identification of the cellular response and few Gene Ontology (GO) terms were found enriched, mostly related endoplasmatic reticulum membrane and nuclear organization. A clearer response was observed in the Heat-shock experiment, where several enriched GO terms were associated to photosynthesis and membrane compartments, including chloroplasts, endoplasmatic reticulum and the Golgi apparatus.
Also of note were terms related to ribosomes, ionic balance and protein folding.
Enrichment of photosynthesis GO terms was caused by an overall under-expression of photosystem associated proteins. Noteworthy, membrane DE genes were not restricted to the chloroplast thylakoyd, indicating that re-arrangement might also be operating in other cellular membranes. Genes coding for 11 non-chloroplastic membrane related proteins were found under- expressed, including transmembrane proteins and an iron transporter. On the other way another 13 membrane related genes were over-expressed, including an ion transporter, potassium and calcium channels. Also associated to the membrane system were changes observed in endoplasmatic reticulum (ER) and Golgi apparatus functioning.
Seven genes coding for DNA and six for RNA binding proteins were also differentially expressed. While some DNA/RNA binding genes also points to a control of cell cycle (Cysteine- rich protein 2, Protein TBRG4), others points to mRNA modification and post-transcriptional control (Pentatricopeptide repeat-containing protein At1g12775, Nucleolar RNA helicase 2-B,
NFX1-type zinc finger-containing protein 1, Pentatricopeptide repeat-containing protein
At1g06710, mitochondrial).
60 DISCUSSION
DNA binding, transcription and post-transcriptional response
The unique characteristics of the dinoflagellate genome, associated to the paucity of sequence specific transcription factors observed have yielded the hypothesis that expression in dinoflagellates is mostly regulated by post-transcriptional control (Bayer et al 2012, Baumgartem et al 2013, Shoguchi et al 2013). This hypothesis is supported by the large occurrence of genes associated with mRNA processing in Symbiodinium (Leegat et al 2007, Rosic et al 2015), cis and trans-splicing in nuclear-encoded genes (Zhang et al 2007) and codon-specific mRNA editing in mithochondrial and chloroplast genes (Mungpakdee et al 2014, Shoguchi et al 2015). Our results are in agreement with this common notion, as only 0.12% of the genes had TF domains and less than 1% of the genes were differentially expressed in any condition.
Several DNA-binding genes were observed among the DE genes, most associated to modifications of chromatin organization, indicating gene regulation at the genome level (Bayer et al
2012). Variations on DNA-binding proteins observed could be related to DNA protection against
ROS damages (Lesser 2006) and possible implications of the heat stress for cell cycle evolution has been proposed (McLenon and DiTulio 2012, Levin et al 2016). A single site specific transcriptional response to the heat shock is evidenced by the reduced expression of Hsp70 and HSF1-binding protein, that leads to the release of the transcription promoter HSF1, a major regulator of heat-shock transcriptional response (Shamovsky and Nudler 2008).
Thermal shock and oxidative stress effects
Symbiodinium A4 is commonly found in shallow waters, likely to be affected by thermal stress. Our results shows that a 4oC heat shock can cause a significant impact on Symbiodinium A4 photosynthetic potential, although not necessarily lethal. Mean summer seawater temperature in
Abrolhos is around 28oC, sufficient to cause physiologic damages to Symbiodinium A4, and
61 seawater temperature of 29oC causes bleaching (Figure S3). Thermal stress causes an exacerbated production of reactive oxygen species (ROS) in the cell, not readily scavenged by the enzymatic antioxidant system (Weis 2008) and excess ROS has several defective effects, causing damages to membranes, proteins and nucleic acids DNA (Lesser 2006).
Despite the wide array of antioxidant enzymes observed in Symbiodinium A4 (table 2), none were statistically assigned as differentially expressed, neither in the Light nor in the Heat shock treatments. This could be attributed to the short duration of the Heat stress in our experiment, but this surprising result has also been observed in longer periods of stress (McGinty et al 2012,
Krueger et al 2015). Among the selected antioxidant genes, a DMSP-lyase is the most likely to be induced, what points out to the importance of non-enzymatic ROS scavenging in Symbiodinium
(Sunda et al 2002). It is intriguing that thioredoxins and glutaredoxins genes presented the largest changes in expression among the set of antioxidant genes. These proteins are indirectly involved in
ROS-scavenging, as part of the cellular redox-signalling system (Holmgren 1989).
Our results are coherent with a redox-signalling cascade and suggests a transcriptional response to heat stress in diverse cellular compartments. Oxidative stress can lead to inactivation of transcription factors, what may explain why most of the DE genes in thermal stress are down- regulated (table 3, Baumgarten et al 2013, Levin et al 2016). We observed under-expression of possibly ROS-generating processes located in the chloroplast, the endoplasmatic reticulum and the mitochondria (table 3), suggesting that thermal stress can affect ROS generating processes other than the photochemical reactions in the chloroplast.
Evidences of damages to protein synthesis
ROS attack to proteins can result in damages as specific aminoacids modifications, fragmentation of peptide chains and loss of function (Lesser 2006) and, indeed, we observed a series of modifications on ER and Golgi apparatus functioning (table 3). Genes coding for proteins involved in protein folding (FK506-binding protein 2, Peptidyl-prolyl cis-trans isomerase FKBP2),
62 post-translational modification (N-acetyltransferase), enzime-cytoskeleton binding (Ankyrin-1) and
ER to Golgi vesicle transport (ADP-ribosylation factor 3) were under-expressed. In opposition, protein OS-9, acting on misfolded protein degradation, was extremely stimulated (up to 60x increase) and retrograde vesicle transport from Golgi to ER was also increased (Coatomer subunit beta). These modifications are coherent with a scenario of defective protein synthesis, when newly synthesized proteins are transported back to the ER, instead of its actual cellular target. Accordingly, several ribosomal proteins were under-expressed and the activity of DnaJB8, an ATP-binding molecular chaperone that prevents misfolded protein aggregation (Hageman et al 2010), was induced.
Chloroplast
Decay of photosynthetic potential (Fv/Fm) was followed by an overall under-expression of genes associated to the thylakoid membrane. Reduced efficiency in photochemical reactions and
ATP synthesis is indicated by the under expression of genes coding for proteins in photosystem I and II (psaC, psbA, psbF, psbL, psbN), light harvesting complexes and ATP synthase (table 3).
Heat-induced erosion of transcription of chloroplast genes was observed in the heat sensitive S. microadriacticum (A1), Symbiodinium A13 and Symbiodinium C1b-c, but not in heat-tolerant strains
(McGinley et al 2012, Baumgartem et al 2013). In the Symbiodinium chloroplast, excess ROS can cause damages to photosynthetic proteins (Takahashi et al 2008) and the thylakoid membrane
(Tchernov et al 2004), but there still some controversy over the primary site of damage (Buxton et al 2012). Beyond the damage to functioning of the chloroplast thylakoid membrane and ATP synthesis, we observed an under-expression of Tbc2, a translation factor required for the initiation of psbC translation. This result re-enforces the hypothesis that biochemical conditions of stressed chloroplasts causes a decrease in the denovo synthesis of proteins, impairing repair processes
(Lesser 2006, Takahashi et al 2008, McGinley et al 2012). If left uncontrolled, this positive feedback between the stress over the membrane system and the inhibited synthesis of chloroplastic
63 proteins would, ultimately, lead to cell death.
Conversely, the over-expression of the gene TIC32, coding for an essential protein for chloroplast biosynthesis (Hormann et al 2004), is coherent with an acclimatization based on denovo synthesis of thylakoid proteins. Symbiodinium may acclimatise to higher temperatures and maximal acclimatisation in a heat-sensitive clade A Symbiodinium culture occurs in the time span of 6 hours
(Takahashi et al 2014). TIC32 attaches to the inner chloroplast membrane and is part of the chloroplast translocation protein complex. Protein import into the chloroplast depends on the binding of calmodulin to TIC32, which, itself, is dependent on the calcium concentration inside the organelle (Chigri et al 2006). Because binding of NADPH to TIC32 inhibits binding of calmodulin, activity of TIC32 is controlled by the redox state of the organelle (Chigri et al 2006). Redox control of TIC32 activity might represent an important chloroplast regeneration mechanism in cases of oxidative stress, where energy is dissipated by the water-water cycle with no reduction of NADP+
(Roberty et al 2014), favoring translocation of proteins into the chloroplast.
Mitochondrial fuctioning and Iron homeostasis
Mitochondrial electron transfer chain is an important processes of ROS generation in eukaryotic cells (Turrens 2003) and reduced mitochondrial activity in the heat shock is sugested by the inhibition of the phosphoglycerate kinase gene (pgk-1, table 3). As occurs with chloroplast protein import, oxidative state of the cell might regulate mitochondrial activity: mNT is an outer mitochondrial membrane protein binding a redox-sensitive FeS cluster, that might be reduced by glutathione reductase and further oxidized by H2O2 (Wiley et al 2007a, Landry et al
2015). Encoded by the Csd1 gene, mNT mediates mitochondrial iron import and regulates maximum respiratory rates (Wiley et al 2007a). Iron depletion itself limits the rate of mitochondrial electron transfer chains, as it is an important constituent of heme enzymes and FeS clusters (Wiley et al 2007b) and repression of Cisd1 gene will likely cause inhibition of iron import into the mitochondria (Wiley et al 2007b). A Csd1 homologous gene has been recently described in
64 Arabidopsis, where the protein might anchor also in the chloroplast (Nechushtai et al 2012).
Arabidopsis NEET-defficient mutants accumulated higher ROS concentration than wild types under control conditions, but presented similar levels under an induced burst of ROS (Nechushtai et al
2012), suggesting that repression of Csd1 in Symbiodinium shall be a response to the burst of ROS induced by the heat shock.
Lower mitochondrial activity and lower iron import is also supported by the strong inhibition of hydroxybutyrate dehydrogenase gene (Bdh2), responsible for the synthesis of 2,5-
DHBA, a siderophore that also mediates iron import into the mitochondria (Devirredy et al 2014).
This response might be a protective mechanism for the organelle, but accumulation of free iron in the cytoplasm might have adverse effects (Devirredy et al 2014). Increases in free iron content enhances ROS toxicity, through Fenton's reaction, fostering the rate of lipid peroxidation and cellular damage (Zangar et al 2004). The concomitant down regulation of iron transporter FTH1, a protein involved in remobilization of iron from the vacuole to the cytoplasm (Urbanowski et al
1999), adds further support for the hypothesis of an active control of iron content in each cellular compartment. However, our results points to a higher concentration of free iron in the cytosol and it is not clear what will be the effects of this regulation in terms of ROS toxicity for the cell.
Endoplasmatic reticulum
Despite the enhanced activity of protein repair (see above, damages to protein synthesis) the endoplasmatic reticulum (ER) itself is an important site of ROS generation in eukaryotic cells
(Lesser 2006). Cytochrome P4504F8 catalyzes the hydroxilation of fatty acids in the ER, a reaction that involves NAPH as an electron donnor and the reduction of dioxigen to water (Zangar et al
2004). Cytochrome p450 oxigenase activity is largely uncoupled, leading to the formation of ROS, even in the absence of substrates (Zangar et al 2004). Importance of ROS generation in the ER for
Symbiodinium has been long overshadowed by the ROS generation in the chloroplast, specially in cases of combined light and heat stress (Roberty et al 2014). However, it has been noted that
65 thermal stress can cause ROS production and bleaching even in the absence of light (Hill et al 2009,
Tolleter et al 2013). In our experiment, heat shock caused decay in Symbiodinium photosynthetic potential even two hours after incubation in the dark. Cytochrome P450 expression, and ROS production in the ER, can be transcriptionally repressed by oxidation of the transcription factor
NF1, in a negative stabilizing feedback (Barouk and Morel 2001). Reduced ER membrane electron transfer reactions is supported by the inhibition of cytochrome-b5 and a FAD-binding monooxigenase (table 3, Zangar et al 2004). Although it cannot be ruled out the possibility of ROS leakage from the chloroplast, it corroborates the negative feedback between ROS concentration and cytochrome P450 activity.
Cell signaling cascades
It is interesting that cytochrome p450 and Prostaglandin G/H synthase (PGTS2) presented large changes in expression compared to the Dark condition, even in the Light treatment (log2FC <
-8). This indicates a high sensitivity of the transcription factors to oxidative state of the cell, if the oxidative repression of P4504F8 transcription be confirmed in Symbiodinium. Both p4054F8 and
PGTS2 are part of the phosphatidylinositol signaling pathway, a conserved pathway in the
Symbiodinium genus (Rosic et al 2014), that might explain the dramatic changes in expression observed. Controlled activity of the phosphatidylinositol signaling pathway could be responsible for the extensive modifications observed on transmembrane proteins (table 3, Xiang et al 2015).
Pleckstrin-domain protein binds to specific membrane targets and are involved in lipid signal transduction and protein anchoring in membranes (Lenoir et al 2015). There is evidence of modifications both in the cellular membranes and in the internal membranes and the the involvement of the phosphatidylinositol signaling pathway in the calcium cellular homeostasis
(Berridge and Taylor 1988) is supported by the inhibition, in the same order of magnitude, of an extracellular calcium binding protein (Hemolysin-type).
Another important signaling pathway is calcium ion, involved in the regulation of
66 photosynthesis and respiration (Miller and Sanders 1987), cell signaling and response to stressors
(Schulz et al 2013). Accumulation of calcium in organelles has been linked to the establishment and maintenance of both chloroplasts and mitochondrial membrane potentials, required for ATP synthesis (DeLuca and Engstrom 1961, Miller and Sanders 1987), maintaining low levels of Ca 2+ in the cytosol. Increased Ca2+ concentration in the cytosol might lead to cell death, by either apoptosis or necrosis (Rizzuto et al 2012).
In our heat-shock experiment, we observed the induction of Calcium-dependent protein kinase (CDPK), a protein involved in Ca2+ homeostasis, cell signaling and response to stressors
(Schulz et al 2013). We hypothesize that CDPKs are involved in the formation of Ca2+ channels and ion transporters (Schulz et al 2013) in Symbiodinium internal membranes, because of the strong repression of the extracellular binding protein. Over-expression of diverse ion transporters was observed and enhanced activity of cyclic nucleotide-gated cation (Ca2+/K+) channels is corroborated by an over-expression of genes coding for a cyclic nucleotide-binding protein and Phosphoribosyl-
AMP cyclohydrolase, that catalyzes the formation of cAMP from AMP (Kaupp and Seifert 2002).
Excess of ROS impairs electron-transfer membranes dissipating the established H+ gradient, with
Ca2+ leakage to the cytosol (Tchernov et al 2004, Rizzuto et al 2012). Because high Ca2+ concentration in the cytosol are associated to apoptotic signaling (Rizzuto et al 2012) this mechanism shall represent an active Ca2+ sequestration back to the organelles. As it was observed in dark conditions in our experiment, this sequestration will likely occur in the mitochondria, but the enhanced activity of TIC32 suggests also a high Ca2+ concentration inside the chloroplast.
Fatty acid metabolism
Fatty acid metabolism is also affected in the stressed cell, with the down-regulation of 4'- phosphopantetheinyl transferase, involved in fatty acid biosynthesis, and the induction of fadB gene, coding for a desaturase acting on C18:3 fatty acids (Saito et al 2000). The over expression of fadB gene is unexpected because saturated fatty acids (SFA) conveys more stability to biological
67 membranes and are less susceptible to oxidation by ROS (Tchernov et al 2004). Bleaching resistant
Symbiodinium strains have higher relative content of saturated fatty acids (Tchernov et al 2004) and increases in the saturation level of C18 fatty acids have been observed at least one day after a thermal stress (Kneeland et al 2013, Revel et al 2016). Nevertheless, this response might be different for non-membrane fatty acids: throughout a heat stress experiment Symbiodinium A1 showed an increase in the whole cell content of the unsaturated C18:3 fatty acid, but this increased was not observed in the chloroplast membrane (Diaz-Almeyda et al 2011).
C18:3 and C18:4 polyunsaturated fatty acids (PUFA) are common in the Symbiodinium free pool of fatty acids (Zhukova and Titlyanov 2003) and increases in the abundance of PUFA have been observed in this pool (Diaz-Almeyda et al 2011, Hillyer et al 2016). Desaturation of fatty acids is an aerobic process and might help to alleviate oxidative stress, but this process must be transient, as unsaturated fatty acids are more susceptible to ROS (Hillyer et al 2016). Combined, these results suggests that activity of desaturases is limited in time and is specific to free fatty acids.
Interestingly, we found that fadB gene was over-expressed 2 hours after the heat shock. Whether activity of the fabB gene would decrease over the evolution of the heat stress and the resulting saturation level of the fatty acid pool remains to be investigated.
Repression of 4'-phosphopantetheinyl transferase activity is coherent with decreases in fatty acid biosynthesis observed for heat-stressed Symbiodinium, both in culture and in hospite (Diaz-
Almeyda et al 2011, Kneeland et al 2013, Revel et al 2016). With the observed reduced activity of electron-transfer chains and consequent deficit in ATP synthesis, energy consuming pathways should be down-regulated (Hillyer et al 2016). This inhibition, combined with consumption of fatty acids in storage lipids, could explain the observed decay in fatty acid abundance in stressed
Symbiodinium cells (Kneeland et al 2013).
Overall, our results shows that Symbiodinium A4 responds actively to oxidative stress, with investment of energy, despite the deficit in ATP synthesis. Although enabling Symbiodinium survival in culture, the effects of this cellular response for Symbiodinium in hospice is not
68 straightforward, introducing instability to the Symbiodinium-host interaction. If by one side, the control of ROS generating processes can reduce ROS leakage to the host and prevent bleaching, by the other, energy impoverished Symbiodinium cells presents a lower translocation of sugars and lipids to the host. Possible outcomes for this response remains to be investigated and shall likely depend on the Symbiodinium strain and the magnitude/duration of the thermal stress.
CONCLUSION
Our results indicates a stress response in Symbiodinium A4, a widespread and generalist zooxanthelae. Symbiodinium A4 can acclimate to thermal/oxidative stress, repressing ROS- generating processes and inducing expression of specific genes. The fact that ROS-generating processes located in different cellular compartments (chloroplast, the endoplasmatic reticulum and the mitochondria) might be down-regulated simultaneously is intriguing and leads to the speculation of a coordinated redox control of these processes. In this context, some of DE genes observed in this study could serve as molecular markers of an acclimatization response in Symbiodinium
(HSFB1/Hsp70, TIC32, Cisd1, p4504F8, fadB). Given the ecological importance of Symbiodinium response to oxidative stress, the activity of genes under redox control as TIC32 and Cisd1 deserves a deeper investigation.
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74 Table 4.3. Differentially expressed genes in the Heat shock treatment, compared to the dark condition. Gene description and accession number retrieved from the Swissprot database. Gene hypothetical function deducted from the Swissprot description, assigned GO term and/or literarure review.
75 Table 3 (con't). Differentially expressed genes in the Heat shock treatment, compared to the dark condition. Gene description and accession number retrieved from the Swissprot database. Gene hypothetical function deducted from the Swissprot description, assigned GO term and/or literarure review.
76 Table 4.S1. Distribution of transcription factors in the Symbiodinium A4 transcriptome.
Table 4.S2. Expression levels (pseudo-counts, counts adjusted to library size) and log(fold-change) of genes associated to antioxidant response. LogFC(DH) – ratio of expression between Heat and Dark treatments, p-value: associated p-value after BH adjustment for multiple tests. LogFC(DL) – ratio of expression between Light and Dark treatments. In each comparison, contigs supported by less than 15 reads were excluded from the DE analysis and there is no logFC for them.
77 78
Table 4.S3. Differentially expressed genes in the Light, compared to the dark condition.
Table 4.S4. Enriched GO terms associated to DE genes in the Dark/Light contrast. GO terms were retained if at least 2 genes were found among DE genes.
79 Table 4.S5. Enriched GO terms associated to DE genes in the Dark/Heat contrast. GO terms were retained if at least 2 genes were found among DE genes.
80 Figure 4.S1. Comparison of log2-fold change in the Heat/Dark and Light/Dark contrasts. No over- expressed gene in the Heat was found under-expressed in the Light treatment, and vice-versa. Changes in expression in the Light tend to have a lower magnitude (abs(log2FC)), than in the Heat treatment. Geneas that were also under-expressed in the light treatment are marked in red.
81 Figure 4.S2. Maximum monthly mean seawater surface temperature observed in the last 15 years in the Abrolhos reef bank, Brazil. Seawater temperatures over 29oC yield bleaching concerns. Period of april/2016 was associated to an extensive bleaching in Millepora sp. (Felipe Ribeiro, personal communication) Extracted from NOAA Coral Reef Watch.
NOAA Coral Reef Watch. 2013, updated daily. NOAA Coral Reef Watch Daily Global 5-km Satellite Virtual Station Time Series Data for Abrolhos Reef, Brazil. Jan. 1, 2001-May. 5, 2016. College Park, Maryland, USA: NOAA Coral Reef Watch. Data set accessed 2015-02-05 at http://coralreefwatch.noaa.gov/vs/index.php
82 CAPÍTULO 5
DISCUSSÃO
5.1. Diversidade de Symbiodinium em Mussismilia spp.
Nessa tese apresentamos a primeira coleção de cultivo de Symbiodinium estabelecida para corais do Atlântico sul. Mais especificamente, o estabelecimento de cultivos de Symbiodinium isolados de M. braziliensis permite maior manipulação experimental, possibilitando compreender a resposta desses simbiontes ao estresse térmico (capítulo 3), à acidificação dos oceanos (Hill 2016) e
à incidência de vírus patógenos (Benites 2016). Sendo M. braziliensis a principal espécie construtora de recifes no Banco dos Abrolhos, essa coleção é uma importante contribuição para um melhor entendimento e para a conservação desse ecossistema.
O estabelecimento de cultivos clonais por meio de citometria de fluxo (FACS) é também um importante marco desse trabalho, pois permite uma associação consistente entre a resposta biológica observada e a identificação taxonômica do simbionte. Esse é o primeiro registro da viabilidade de células de Symbiodinium após a separação por citometria de fluxo, um fator importante à medida que se avançam as tecnologias de sequenciamento com base em células únicas (single-cell genomics, Sandberg 2014). Entretanto, obteve-se uma maior taxa de sucesso no isolamento por meio de diluições (25% em contraste com 0,4% por meio de FACS). Assim, é necessário ponderar os objetivos específicos do trabalho para decidir a estratégia de isolamento de Symbiodinium.
O sequenciamento de amostras de tecido de corais associado a abordagem dependente de cultivo indicaram que M. braziliensis pode se associar a, ao menos, duas linhagens (ITS2) de
Symbiodinium: C3 e A4. Symbiodinium C3 foi a linhagem dominante de todas as amostras de tecido de M. braziliensis, enquanto que se observou uma dominância da linhagem Symbiodinium A4 nos cultivos. Esses resultados permitem inferir que Symbiodinium C3 é o simbionte dominante in hospite, enquanto que Symbiodinium A4 pode co-ocorrer nas mesmas colônias, porém com menor 83 abundância relativa. Essa conclusão pode ser verificada em um trabalho de meta-transcriptoma de
M. braziliensis, em andamento no laboratório de Microbiologia/IB-UFRJ (Figura 4.1, Froes et al, em preparação).
Figura 5.1. Abundância relativa das sequências de ITS2 de Symbiodinium em amostras de M.
braziliensis saudáveis (H) ou afetadas por praga branca (WP). Reads de rRNA foram comparados por similaridade (blastn) com o banco de ITS2 de Symbiodinium, extraído do NCBI, em 13/04/2015. Sequências com percentual de identidade superior a 95% em um alinhamento mínimo de 100 bp foram selecionadas.
5.2. Transcriptoma
Devido ao avanço das tecnologias de sequenciamento e diminuição dos custos, observa-se um grande aumento no número de trabalhos de genoma e transcriptoma de Symbiodinium nos
últimos cinco anos. Esses trabalhos trouxeram grandes avanços para a compreensão da história evolutiva do grupo, como a organização do genoma nuclear e presença de histonas (Bayer et al
2012, Shoguchi et al 2013), a transferência de genes das organelas para o núcleo (Barbrook et al
2013, Mungpakdee et al 2014, Shoguchi et al 2015) e diferentes mecanismos de transcrição e edição de mRNA (Zhang et al 2007, Barbrook et al 2013, Mungpakdee et al 2014, Shoguchi et al
2015). Entretanto, esses trabalhos adicionaram pouco a compreensão da resposta funcional de
Symbiodinium a a fatores ambientais. A escassez de fatores de transcrição sítio específicos associada a abundante maquinaria de edição de mRNA (Leggat et al 2007, Rosic et al 2015) levaram à 84 percepção de que “a transcrição em Symbiodinium é constante, estática, com poucos genes sob controle transcricional sítio específico” (Shoguchi et al 2013). Essa percepção é suportada pelo baixo número de genes diferencialmente expressos em condições ambientais de estresse (em geral menor que 1% do total de genes).
Entretanto, outros fatores relacionados a estrutura do genômica de Symbiodinium que podem interferir nas análises transcriptômicas tem sido negligenciados. Primeiro, devido ao tamanho do genoma nuclear (ao menos 1,2 Gb, Shoguchi et al 2013, Lin et al 2015), é necessário um enorme esforço de sequenciamento para se obter uma boa cobertura do transcriptoma. Apesar de o custo de sequenciamento ter diminuído, essa ainda é uma limitação, especialmente para estudos ecológicos, onde o desenho experimental tende a crescer de maneira fatorial. Tipicamente, essa maior cobertura no sequenciamento é alcançada às custas de um menor número de réplicas entre as amostras. Em segundo lugar, a alta incidência de genes duplicados e expansão de famílias gênicas (Shoguchi et al
2013, Maruyana et al 2015) dificultam a associação inequívoca das sequências de mRNA geradas
(reads) aos transcritos montados (contigs), introduzindo ruído à análise, que, por sua vez, demandaria ainda mais replicação. Em nossa análise, o número de genes diferencialmente expressos aumentou consideravelmente, após a remoção de contigs com baixo suporte de reads (veja tabela
4.S2, como exemplo). Esse aumento se deve a uma menor variabilidade entre as amostras.
Assim, enquanto a maior parte de estudos de RNA-seq em Symbiodinium tem usado um número limitado de réplicas (n<=2) e obtido uma baixa frequência de genes diferencialmente expressos (<1%, Baumgartem et al 2013, Xiang et al 2015, capítulo 3), um menor coeficiente de variação e maior frequência de genes diferencialmente expressos (3%) se observa com um número maior de réplicas (n=4, Levin et al 2016). Assim, a percepção de uma “transcrição constante” não é suportada por dados empíricos. Na prática, o que se observa é uma alta variabilidade entre réplicas
(tabela S2), que dificulta a detecção de diferenças entre os tratamentos. Apesar de haver poucas dúvidas em relação a importância do processamento de mRNA, essa percepção desestimula estudos de transcriptoma que busquem entender a resposta biológica de Symbiodinium a fatores ambientais.
85 5.3 – Estresse oxidativo em Symbiodinium spp. e em corais
O efeito do estresse térmico para a relação coral-zooxantela tem sido estudo extensivamente nos últimos 30 anos, apontando para um efeito no aumento na produção de espécies reativas de oxigênio (ROS) e uma complexa sinalização entre coral e hospedeiro que leva ao rompimento da simbiose (Weis 2008). Entretanto, essa resposta ao estresse varia entre diferentes linhagens de
Symbiodinium, podendo estar associada ao grau de saturação de lipídeos de membranas (Tchernov et al 2004, Diaz-Almeyda et al 2011), organização estrutural dos fotossistemas (Reynolds et al
2008, Slavov et al 2016) e síntese de proteínas da membrana thylakoide (Takahasi et al 2009).
Apesar de o mecanismo celular ser bem estudado e haverem diversos processos candidatos, até o momento não há marcadores genéticos descritos que permitam identificar um potencial de resistência ou aclimatação de Symbiodinium ao estresse térmico.
Nessa tese são identificados quatro genes de Symbiodinium A4 (tic32, csd1, p450-4F8, fadB), associados ao estresse térmico e, potencialmente, regulados pelo estado oxidativo da célula.
Estudos com corais tem reconhecido a importância do controle redox para a resistência e aclimatação do coral a altas temperaturas (DeSalvo et al 2008, Voolstra et al 2011, Dixon et al 2015,
Parkinson et al 2015, Maor-Landaw e Levy 2016), mas apenas recentemente essa hipótese foi levantada em Symbiodinium (Weston et al 2015). Além das dificuldades associadas ao estudo de transcriptomas em Symbiodinium, o sinergismo entre os efeitos de luminosidade e temperatura
(Roberty et al 2014, Weston et al 2015) dificultam a observação de mudanças transcricionais de processos geradores de ROS. De fato, no transcriptoma apresentado aqui não se observam diferenças significativas na expressão de genes na luz ou no estresse térmico (sem luz) e trabalhos anteriores compararam os efeitos em tratamentos de alta temperatura/com luz com controles com temperatura amena/com luz (Baumgartem et al 2013, Levin et al 2016). Assim, os resultados apresentados no capítulo 3 estão de acordo com um recente proteoma de coral submetido ao
86 estresse luminoso, onde genes de Symbiodinium associados ao estresse oxidativo e metabolismo de cálcio foram induzidos (Weston et al 2015).
A produção de oxigênio durante a fotossíntese faz com que organismos fotossintetizantes estejam mais sujeitos ao acúmulo de ROS (Asada 2006). Os resultados do transcriptoma apresentado indicam a inibição do citocromo p450 e sugerem que a transcrição de genes ligados ao funcionamento da mitocôndria e à homeostase de ferro também foi reprimida no tratamento com luz, apesar de não significativo estatisticamente (csd1: log2FC=-1.3, p-valor(aj)=0.68; bdh2: log2FC=-7.2, p-valor(aj)=0.26). A geração de energia a partir da atividade fotossintética é um processo primordial em Symbiodinium, porém que está associada à uma grande produção de oxigênio e ROS. Dessa forma, é possível que a inibição de processos geradores de ROS em períodos de iluminação seja uma estratégia evolutiva em resposta à alta produção de ROS nas reações fotoquímicas de Symbiodinium, amenizando assim o estresse oxidativo na célula.
87 CAPÍTULO 6
CONCLUSÕES
Essa tese permitiu alguns avanços para o conhecimento sobre Symbiodinium, em especial aos simbiontes associados ao coral brasileiro Mussismilia spp.. Entre as principais contribuições, destacam-se:
1. Foi demonstrada a viabilidade do uso de citometria de fluxo para o estabelecimento de cultivos de Symbiodinium;
2. O coral brasileiro Mussismilia spp. é generalista quanto ao seu endossimbionte, podendo se associar a linhagens divergentes em três diferentes clados de Symbiodinium;
3. O estresse térmico induz uma resposta celular em Symbiodinium associada a mecanismos de controle do estresse oxidativo;
4. A análise de expressão gênica permitiu identificar possíveis mecanismos de aclimatação ao estresse oxidativo em Symbiodinium.
Novas perspectivas se abrem com base nesses resultados e, futuramente, deve-se avaliar a dinâmica de colonização das diferentes linhagens de Symbiodinium em Mussismilia, de acordo com fatores ambientais e em resposta a infecções. Com relação ao estresse oxidativo, deve ser avaliado a amplitude e eficiência da resposta celular observada, assim como possíveis variações entre diferentes linhagens de Symbiodinium. O papel da sinalização redox na aclimatação da célula ao estresse oxidativo e o possível feedback negativo entre os processos geradores e a concentração de
ROS devem ser investigados. Por fim, deve-se verificar a utilidade dos genes observados nessa tese como marcadores genéticos do potencial de aclimatação de Symbiodinium ao estresse térmico/oxidativo em amostras de campo.
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95 APÊNDICE______
O capítulo 3 desta tese é a reproducão do trabalho “Multiple Symbiodinium Strains Are Hosted by the
Brazilian Endemic Corals Mussismilia spp.”, publicado em Microbial ecology, 70(2), 301-310. Fev/2015
96 Microb Ecol DOI 10.1007/s00248-015-0573-z
MICROBIOLOGY OF AQUATIC SYSTEMS
Multiple Symbiodinium Strains Are Hosted by the Brazilian Endemic Corals Mussismilia spp.
Arthur W. Silva-Lima & Juline M. Walter & Gizele D. Garcia & Naiara Ramires & Glaucia Ank & Pedro M. Meirelles & Alberto F. Nobrega & Inacio D. Siva-Neto & Rodrigo L. Moura & Paulo S. Salomon & Cristiane C. Thompson & Fabiano L. Thompson
Received: 11 July 2014 /Accepted: 21 January 2015 # Springer Science+Business Media New York 2015
Abstract Corals of genus Mussismilia (Mussidae) are one about the diversity of Symbiodinium in this ocean basin. of the oldest extant clades of scleractinians. These Neo- In this study, we established the first culture collections of gene relicts are endemic to the Brazilian coast and repre- Symbiodinium from Mussismilia hosts, comprising 11 iso- sent the main reef-building corals in the Southwest Atlan- lates, four of them obtained by fluorescent-activated cell tic Ocean (SAO). The relatively low-diversity/high-ende- sorting (FACS). We also analyzed Symbiodinium diversity mism SAO coralline systems are under rapid decline from directly from Mussismilia tissue samples (N=16) and char- emerging diseases and other local and global stressors, but acterized taxonomically the cultures and tissue samples by have not been severely affected by coral bleaching. De- sequencing the dominant ITS2 region. Symbiodinium spite the biogeographic significance and importance for strains A4, B19, and C3 were detected. Symbiodinium understanding coral resilience, there is scant information C3 was predominant in the larger SAO reef system (Abrolhos), while Symbiodinium B19 was found only in deep samples from the oceanic Trindade Island. Symbiodinium strains A4 and C3 isolates were recovered Subject category: Microbial diversity, evolution, microbe-host interactions from the same Mussismilia braziliensis coral colony. In face of increasing threats, these results indicate that Electronic supplementary material The online version of this article (doi:10.1007/s00248-015-0573-z) contains supplementary material, Symbiodinium community dynamics shall have an impor- which is available to authorized users. tant contribution for the resilience of Mussismilia spp. : : : : corals. A. W. Silva-Lima: J. M.: Walter G. D.: Garcia N. Ramires: G. Ank P. M. Meirelles: R. L. Moura P. S. Salomon C. C. Thompson F. L. Thompson . . . . Laboratório de Microbiologia, Instituto de Biologia, Keywords Coral reefs Symbiodinium Mussismilia ITS2 Universidade Federal do Rio de Janeiro (UFRJ), Av. Carlos Chagas Clonal cultures . Southwestern Atlantic Ocean (SAO) Fo. S/N - CCS - IB - Lab de Microbiologia - BLOCO A (Anexo) A3 - sl 102, Cidade Universitária, Rio de Janeiro, RJ, Brazil 21941-599
R. L. Moura : P. S. Salomon : F. L. Thompson (*) Sage/Coppe, Centro de Gestão Tecnológica–CT2, Rua Moniz de Aragão, no.360 - Bloco 2, Introduction Ilha do Fundão - Cidade Universitária, Rio de Janeiro, Brazil 21.941-972 The coral holobiont comprises the coral host, its microbiome e-mail: [email protected] (virus, prokaryotes, and eukaryotic microbes), and unicellular, A. F. Nobrega photosynthetic endosymbiotic dinoflagellates of the genus Instituto de Microbiologia Prof Paulo de Goes, Symbiodinium, the so-called zooxanthellae [1]. Symbiodinium Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil lives inside the coral tissues in extremely high densities, reaching more than 106 cells/cm2 [2]. In the intracellular com- I. D. Siva-Neto Laboratório de Protistologia, Instituto de Biologia, partment, Symbiodinium cells receive protection and inorgan- Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil ic nutrients necessary for photosynthesis from the host coral, A. W. Silva-Lima et al. while providing organic carbon compounds and oxygen de- new habitats and hosts in order to unravel Symbiodinium rived from photosynthesis [3–7]. The coral-Symbiodinium diversity. The Southwestern Atlantic Ocean (SAO) is one symbiosis plays an important ecological role that is reflected major geographic gap, encompassing a low-diversity/high- by their geographic spread, with the occupation by modern endemism coral fauna dominated by one of the oldest coral reefs in tropical and subtropical waters of over 280, extant genera of scleractinians, Mussismilia. These Neo- 000 km2 [8] and by the great evolutionary diversification of gene relicts, endemics to the Brazilian coast, are declining both corals and zooxanthellae in the last 60 MYA [9]. In spite rapidly due to emerging diseases and other local and glob- of this successful evolutionary history, the future of the coral al stressors [38, 39]. In spite of such relevance, there is holobiont is uncertain in face of the rapidly ongoing global limited knowledge about the genetic and functional diver- climate changes [10–12]. Coralline reefs are currently chal- sity of Symbiodinium in the SAO, particularly in the lenged by unprecedented high rates of global warming, ocean Abrolhos Bank [27, 40–44]. acidification, and diseases [11]. Thermal stress, which leads to The Abrolhos Bank is the SAO’s largest and richest widespread episodes of coral bleaching, can be a foremost coralline system, encompassing all scleractinian species cause of coral mortality [13]. recorded in the region [45–49]. Pan-Atlantic species The life cycle of Symbiodinium comprises both a motile show a remarkably low genetic variability in the region, (flagellated) and a coccoid phase [14]. Within the host cell, and eight of the 18 coral species commonly found in Symbiodinium is kept in the coccoid state, while the free- the Abrolhos Bank are endemic to the SAO [50, 51]. living forms might be motile or coccoid. Symbiodinium colo- The main reef-building coral species of the Abrolhos nizes a vast array of hosts, including foraminiferans, sponges, Bank are the spawning corals of genus Mussismilia, jellyfishes, sea anemones, plathyhelminthes, and molluscs which encompasses three described species: Mussismilia [15–17]. The symbiosis is obligate for hermatypic braziliensis, Mussismilia hartii,andMussismilia hispida scleractinian corals, and the mode of transmission of [38, 52]. M. braziliensis has the narrowest distribution, Symbiodinium among coral colonies depends mainly on the restricted to the Bahia State and may soon be listed as host’s reproduction type. In brooders, with larvae developing an endangered species due to its rapid decline caused inside coral parents, transmission tends to be vertical, from the by the infectious disease white plague [39, 40]. parent to the offspring, while in corals that deliver gametes in In this study, we characterized the genetic diversity of the water column, the symbiont tends to be acquired from the Symbiodinium colonizing M. braziliensis and M. hispida by environment [18, 19]. In this context, the availability of alter- means of ITS2 sequences. We also established the first native hosts and the Symbiodinium free living life stage is Symbiodinium culture collection originated from crucial for the maintenance of the symbiosis. This life stage M. braziliensis,and investigated its morphology and was proposed to be transient [18], but recent work has been physiology. revealing a widespread occurrence of free-living forms [20–22]. Phylogenetic analysis of ribosomal RNA (rRNA) gene Materials and Methods sequences revealed that the Symbiodinium genus can be subdivided into nine (A–I) distinct clades [23–26]. Sampling Symbiodinium from clades A to D are the most commonly associated with corals, with clades B and C being domi- Colonies of M. braziliensis were collected with SCUBA (5– nant in central ecological niches [27, 28]. Clades A and 20 m depths) using hammer and chisel in two locations D, although present in tropical seas, are dominant in (Sebastião Gomes SG, 17°54′42.49″S, 39°7′45.94 W″; Parcel stressed environments, such as high-latitude locations, dos Abrolhos PAB, 17°57′32.7″S, 38°30′20.3″W) during the higher irradiance habitats, extreme temperature conditions, summer of 2012. Reefs SG (open access area) and PAB (in- and regions with higher coastal influence [29–35]. Further- side the no-take Abrolhos National Marine Park) are 14 and more, studies have applied molecular techniques to the 65 km off the coast, respectively [45, 46]. Five whole coral rDNA internal transcribed spacer regions (ITS1 and colonies (approx. 15 cm) were transported alive to the labora- ITS2) and revealed a great fine-scale diversity within these tory in separate coolers with seawater and kept in aquaria until clades [36, 37]. Symbiodinium cell isolations for the establishment of cultures. Since clade H description in 2001, a new Symbiodinium Additionally, tissue samples of M. braziliensis were collected clade was reported only in 2010, an endemic divergent from healthy (n=7) and white plague infected (n=5) colonies lineage hosted by Hawaiian foraminiferans [17]. This de- during the summer of 2010 at PAB and SG reefs (Table 1 cade novelty lag reinforces the importance of researching [40]). M. hispida tissue samples were collected in the summer Multiple Symbiodinium Strains Are Hosted by Mussismilia spp. of 2011 at the Trindade Island, 1600 km offshore (CVT20, 20° coral specimens kept in the aquaria and re-suspending it in 31′33.6″S, 29°18′37.3″W) and at the Jaseur (CVT13, 20°24, sterile sea water. Isolation of Symbiodinium cells from these 897′S, 36°02,511′W) and Davis (CVT16, 20°34,603′S, 34° suspensions was done using two strategies: fluorescence- 48,387′W) seamounts (Table 1). These samples were kept in activated cell sorting (FACS) and manual cell picking and liquid nitrogen until DNA extraction. transfers in an inverted microscope. Single-cell separation by FACS was done in a flow cytometer (DakoCytomation® Isolation and Establishment of Symbiodinium Strains MoFlo) equipped with an electrostatic droplet deflection sys- in Cultures tem and the cyclone sorting option for sorting single cells. The flow cytometer was fitted with a 100-μm orifice nozzle tip and After rinsing M. braziliensis colonies with filtered (0.45 μm) sheath pressure was kept at 12 PSI. Upon excitation with the and autoclaved seawater, Symbiodinium cell suspensions were blue laser line (488 nm, 100 mW), Symbiodinium cells were made by carefully scrapping tissue (one or two polyps) from detected in two-parameter plots based on their chlorophyll
Table 1 Symbiodinium samples (isolates and holobiont tissues) used in phylogenetic reconstruction, with strain designation, coral host, depth, health condition, year, and sampling site
Sample ITS2-type Identity Best Hit Host species Health status Depth (m) Site Year
Culture 042C5 A4 100 EU449050.1 M. braziliensis (#5) Healthy 4–8 PAB 2012 043B7 A4 97 EU449050.1 M. braziliensis (#5) Healthy 4–8 PAB 2012 043D10 A4 100 EU449050.1 M. braziliensis (#5) Healthy 4–8 PAB 2012 043G2 A4 99 EU449050.1 M. braziliensis (#5) Healthy 4–8 PAB 2012 102A3 A4 99 EU449050.1 M. braziliensis (#1) Healthy 3–5SG2012 103B3 C3 98 HG515026.1 M. braziliensis (#5) Healthy 4–8 PAB 2012 103C1 C3 98 AJ311943.1 M. braziliensis (#5) Healthy 4–8 PAB 2012 103C2 A4 100 EU449050.1 M. braziliensis (#5) Healthy 4–8 PAB 2012 103C3 C3 99 HG515026.1 M. braziliensis (#5) Healthy 4–8 PAB 2012 103C5 A4 99 EU449050.1 M. braziliensis (#5) Healthy 4–8 PAB 2012 103C6 C3 99 HG515026.1 M. braziliensis (#5) Healthy 4–8 PAB 2012 Holobiont coral tissue SGS2 C3 99 HG515026.1 M. braziliensis Healthy 3–5SG2010 SGS3 C3 99 HG515026.1 M. braziliensis Healthy 3–5SG2010 SGS5 C3 98 HG515026.1 M. braziliensis Healthy 3–5SG2010 SGW2 C3 100 HG515026.1 M. braziliensis Diseased (WP) 3–5SG2010 SGW4 C3 100 HG515026.1 M. braziliensis Diseased (WP) 3–5SG2010 P5S1 C3 100 HG515026.1 M. braziliensis Healthy 4–8 PAB 2010 P5S2 C3 100 HG515026.1 M. braziliensis Healthy 4–8 PAB 2010 P5S4 C3 100 HG515026.1 M. braziliensis Healthy 4–8 PAB 2010 P5W2 C3 100 HG515026.1 M. braziliensis Diseased (WP) 4–8 PAB 2010 P5W5 C3 98 HG515026.1 M. braziliensis Diseased (WP) 4–8 PAB 2010 P5W6 C3 98 HG515026.1 M. braziliensis Diseased (WP) 4–8 PAB 2010 C1D C3 99 HG515026.1 M. braziliensis Healthy 4–8 PAB 2010 CVT20.1 A4 99 EU449050.1 M. hispida Healthy 22 CVT 2011 CVT20.3 A4 100 EU449050.1 M. hispida Healthy 22 CVT 2011 CVT13 B19 97 FJ823612.1 M. hispida Healthy 60 CVT 2011 CVT16 B19 88 FJ823612.1 M. hispida Healthy 45 CVT 2011
Blast results are summarized on columns BBest hit^ and BIdentity^. For holobiont coral tissue, each sample is from a different colony while for isolates, the originating M. braziliensis colony is identified by numbers SG Sebastião Gomes, PA B Parcel dos Abrolhos, CVT Vitoria-Trindade seamount chain, WP colonies diseased with white plague A. W. Silva-Lima et al. content (red fluorescence) and size (forward scattered light). Symbiodinium Cell Morphology and Physiology Using the sort-for-purity mode on the flow cytometer, single cells were individually deposited in each well of 96-well mi- Cell size and volume of seven representative Symbiodinium crotiter plates containing 150 μlofsterileF/2medium[53]. cultures were measured with an automated inflow imaging Alternatively, Symbiodinium cells were isolated by manually system (FlowCAM®, Fluid Imaging Technologies), which picking them from freshly made coral tissue suspensions using combines the capabilities of flow cytometry, microscopy, a micropipette in an inverted microscope. Cells or small cell and image analysis [62]. Before being analyzed in the clumps were separated by successive transfers through F/2 FlowCam, each Symbiodinium culture was sonicated for medium in sterile Petri dishes or in the wells of 24-well sterile 30 s (30 pulses of 1 s with 3 s intervals, 20 % power, ultrasonic microtiter plates (on a 15- to 20-day interval basis). For manual processor, Cole-Palmer) to disrupt cell clumps. The FlowCam isolation, the medium was supplemented with a mix of antibi- was fitted with a 90-μm flow cell, and analysis was done at otics (gentamycin 0.08 mg/ml; kanamycin 0.02 mg/ml; nystatin 100 μl/min sample flow for 10 min. Images were collected 0.015 mg/ml; penicillin 0.3 mg/ml; streptomycin 0.08 mg/ml; through a ×10 magnification objective in auto-image mode. A germanium dioxide 5 mg/L [54]). Microtiter plates with the iso- total of 1500 cell images from each culture were selected for lated cells were kept in a culture chamber (24 °C, photon flux of morphometric analysis of primary linear dimensions (cell ca. 80 μE/m2/s, photoperiod of 14-h light/10-h dark) and moni- length and width) and equivalent spherical diameter (ESD) tored for growth on a weekly basis for ca. 10 weeks, using an using the software provided with the FlowCam. Light inverted microscope. Cultures that grew during this period were micrographies of culture 043B7 living Symbiodinium cells transferred to larger volumes and incorporated into the collection, were obtained with a differential interference contrast (DIC) which is being kept by successive transfers since then. equipped microscope (Axio Imager.A2, Zeiss, Germany). In order to estimate growth parameters, Symbiodinium strain 043D10 (ITS2 type A4) was grown as batch cultures Phylogenetic Analysis in triplicate 500-ml glass flasks containing 200 ml F/2 medi- um, at 80 μE/m2/s; 14 light/10 dark and 24±1 °C. Samples Molecular identification of Symbiodinium samples was done by were taken at 3- to 4-day interval, sonicated as described direct sequencing the dominant nuclear ribosomal ITS2 region above and fixed with acid Lugol’s solution (1 % final concen- [55]. DNA extraction was performed for Symbiodinium cultures tration). Cell counts were done in Palmer Maloney chambers using chloroform-ethanol washings [56]. To obtain total DNA using an inverted microscope (Nikon TS100) at ×200 magni- of the coral holobiont, DNA extraction was done as described fication. Based on cell densities, intrinsic growth rate, dou- previously [40]. PCR amplification of partial 5.8 s rDNA, com- bling time, and maximum cell yield were then estimated plete ITS2 region, and partial 28 s rDNA was based on primers for each replicate and averaged for each strain, as de- ITS2intfor (5′-GAATTGCAGAACTCCGTG-3′)and scribed previously [63, 64]. ITS2reverse (5′-GGGATCCATATGCTTAAGTTCAGCGG The photosynthetic potential of this strain (043D10) was GT-3′) using a touch-down PCR strategy [57, 58]. PCR determined by means of pulse amplitude modulated (PAM) products were purified with ExoSap-IT (USB Corporation, fluorometry on cells collected at late exponential growth USA) and sequenced in both directions with the same primers phase [65]. Culture was maintained in the same conditions above, using a capillary system (ABI3500). as for the growth curve, except for an irradiance of ca. The identity of the sequences obtained in this study was 60 μE/m2/s. Cell densities were adjusted to 106 cells/ml, and first identified by similarity (blastn algorithm [59]) and 12 replicates were dark adapted for 20 min before measure- furthter checked by phylogenetic analysis. The best model of ments of maximum photosynthetic potential (Fv/Fm) with a molecular evolution for the ITS2 sequences was chosen by blue light diving-PAM (Walz GmbH, Germany) under a sat- ModelTest [60]. Maximum likelihood phylogenetic recon- uration light pulse of 2500 μE/m2/s. structions were performed using a rooted neighbor-joining guide tree, Kimura 2-parameter molecular evolution model, and 2000 bootstrap replicates [61]. The final phylogenetic tree Results consisted of 27 ITS2 sequences generated in this study and 16 published Symbiodinium ITS2 sequences from clades A, B, Isolation and Culturing of Symbiodinium and C. The closely related dinoflagellate Pelagodinium beii was used as the outgroup for the phylogenetic reconstructions. We established a culture collection of 11 Symbiodinium strains Gene sequences of cultures are deposited in GenBank under originating from M. braziliensis hosts (Table 1). Both isolation accession numbers KJ189553-KJ189564 and sequences of methods produced actively growing cultures. Four holobiont coral tissue under accession numbers Symbiodinium A4 clonal cultures originated from single KJ488961-KJ488977. cells sorted by FACS (042C5, 043B7, 043D10, 043G2), Multiple Symbiodinium Strains Are Hosted by Mussismilia spp. while the other seven isolates were obtained by manual this study were placed outside the C1 clade and all had an A cell picking and transfers. on position 196, they were assigned to the Symbiodinium C3 group (at least 98 % identity). Molecular Diversity of Symbiodinium The 11 cultures belonged to Symbiodinium strains A4 and C3 (Fig. 1). C3 cultures ITS2 sequences grouped along with Together, the culture dependent and independent analyses re- the sequences from M. braziliensis tissue, either healthy or vealed that Symbiodinium from three different clades (A, B, white plague infected. Isolates identified as Symbiodinium and C) are associated with Mussismilia (Table 1). Clade A A4 (103C2, 103C5) and from the C3 group (103B3, 103C1, sequences were all assigned to Symbiodinium A4, with at least 103C6) originated from the same coral colony (Table 1), in- 98 % identity over 258 nucleotides. Clade B ITS2 sequences dicating that a single M. braziliensis colony can host multiple were most similar to Symbiodinium B19 (97 % identity for Symbiodinium strains. sample CVT13), and sample CVT16 is a putative novel hap- lotype derived from B19, with a 9-bp deletion and 20 base Symbiodinium Cell Morphology and Physiology substitutions (88 % identity). Due to the limited phylogenetic resolution of the ITS2 gene and the recent radiation event that Symbiodinium cells of strains isolated from M. braziliensis occurred within this clade, identification of clade C displayed the characteristic brown color when observed in Symbiodinium strains based on ITS2 sequences is difficult light microscopy due to photosynthetic pigments (chloro- [44, 66]. C1 sequences clustered together on our phylogenetic phylls and xanthophylls) typical of these dinoflagellates. Dur- analysis, but with a weak bootstrap support (45 %). Analyzing ing cultivation, cells were mostly found in their coccoid, near- the alignment of clade C ITS2 sequences (Table S1), C1 and ly spherical, non-motile phase, and less frequently in their C3 sequences were separated by a single base substitution on flagellated, gymnodinoid motile forms. Doublets, indicating position 196 (C1: G, C3: A). As clade C sequences obtained in the process of mitosis in the coccoid forms, were frequently
Fig. 1 Maximum likelihood phylogenetic reconstructions of Symbiodinium ITS2 sequences. All position containing gaps and missing data were eliminated, yielding a total of 151 positions in the final dataset. Bootstrap supports (2000 replicates) are indicated above each node. Sequences obtained in this study for Mussismilia corals are marked by either filled triangles (culture collection) or circles (host tissue samples). Twenty-seven Mussismilia-associated Symbiodinium and 16 formally and informally described Symbiodinium species ITS2 sequences were used in the final tree, with Pelagodinium beii as an outgroup. Symbiodinium clades A, B, and C are marked accordingly. MB Mussismilia braziliensis, MH Mussismilia hispida A. W. Silva-Lima et al. observed throughout the growth curve, indicating healthy The specific growth rate of Symbiodinium A4 in our con- growth conditions (Fig. 2). Average cell diameter (expressed trolled cultivation experiments at 24 °C is within the range as ESD) of the non-motile coccoid phase measured on seven typically observed for other Symbiodinium strains between cultured strains isolated from M. braziliensis ranged from 7.1 0.15 and 0.38 days−1, yielding doubling times between 2 to 8.7 μm(Table2). and5days,attemperaturesfrom20to28°C[14, 64, The Symbiodinium culture 043D10 (Symbiodinium A4) 73–75]. Also, under controlled conditions, the photosynthetic reached stationary phase at ca. 20 days after inoculation, with potential of Symbiodinium A4 agrees with reported values for cell concentrations remaining stable thereafter until the end of this genus [75, 76]. Biogeographical and experimental studies the experiment in day 40 (Fig. 3). Growth rate during the have shown that Symbiodinium C3 is more efficient on mild exponential phase was estimated at 0.24 days−1,yieldingdou- light and temperature conditions, while A4 symbionts are typ- bling times of 2.9 days. Maximum cell density was as 4.4× ically associated to higher irradiance shallow waters [34, 105 cells/ml and mean photosynthetic potential (Fv/Fm) was 75–79]. This diverse functional responses suggests a tradeoff 0.64. between physiological efficiency and stress resistance that plays a key role in Symbiodinium niche partitioning, reflecting the potential contribution of this symbionts for the coral Discussion holobiont Mussismilia spp. [12, 34, 78–83].
Symbiodinium Strains Isolation and Physiology Symbiodinium Biogeography in the Southwestern Atlantic Our isolation efforts using both FACS and manual cell trans- Ocean fers led to several established Symbiodinium cultures. The automated isolation approach in the flow cytometer has the Despite the isolation, the relatively small area (5 % of Atlantic advantages of high cell-sorting throughput that allows pro- reefs), and the corresponding low coral host diversity in the cessing many samples and isolating several hundred cells in SAO reefs [84], three of the most commonly coral-associated short time intervals (<1 h), as well as the establishment of clades (A, B and C) are present in the region. Most of the clonal cultures in one single step, directly from fresh cell sus- Symbiodinium ITS2 types observed in the SAO (A4, B19, pensions [67, 68]. Remarkably, fewer dinoflagellate cultures C1, and C3) are considered ancestral sequences, from which have been established by FACS in comparison to other radiation events have occurred both in the Pacific and the microalgal taxa [69, 70]. Dinoflagellates seem to be more Caribbean [28, 44]. In fact, some of the samples on this study sensitive to the process of cell sorting in the flow cytometer, had less than 100 % identity to known ITS2 types and are showing lower success than other algal groups when single probably derived from these ancestral sequences. Given the cells are deposited in the wells of microtiter plates [70]. Al- low number of host species surveyed and assuming that these though flow cytometry has already been used to screen envi- ITS2 types are probably groups of species, the number of ronmental Symbiodinium populations [20, 71], this is the first Symbiodinium species reported in the SAO so far (~10) is account of a successful use of FACS methodology for sorting likely an underestimation of its real richness [40–44]. viable Symbiodinium cells with the purpose of establishing Previous studies on deep reef environments showed the clonal cultures. This results shows FACS has a great po- predominance of clade C and B Symbiodinium strains tential for retrieving coral-associated Symbiodinium di- [85–89]. The occurrence of Symbiodinium B19 at depths over versity and, with the rampant advances in single-cell ge- 45 m is probably related to cold water tolerance of this lineage nomics, for extending the knowledge on coral and [28], but it is surprising the occurrence of M. hispida colonies Symbiodinium genomics [72]. harboring the shallow-water associated Symbiodinium A4 [34,
Fig. 2 Morphology of Symbiodinium A4 culture 043B7 (×1000 DIC micrographs). Coccoid cells has spherical shape and great internal complexity. Dividing cells can be observed on the right and a newly encysted cell can be observed on the left image Multiple Symbiodinium Strains Are Hosted by Mussismilia spp.
Table 2 Morphological parameters of the isolated strains based on tissue samples (Table 1). Complementarly, Symbiodinium C3 FlowCam data strains were not obtained by FACS, suggesting it is more Strain ITS2-type Length Width ESD sensitive than A4 strains, either to the cell sorting process or to culture conditions [93, 94]. Combined, these facts suggest 042C5 A4 8.4 (1.04) 7.29 (1.00) 7.92 (0.99) that M. braziliensis of the Abrolhos reefs harbors preferential- 043B7 A4 8.11 (1.08) 6.97 (1.05) 7.62 (1.05) ly Symbiodinium C3 with background populations of 043D10 A4 7.98 (1.16) 6.74 (1.05) 7.43 (1.07) Symbiodinium A4. 103C2 A4 8.43 (0.91) 7.40 (0.88) 7.99 (0.88) Our observations that M. braziliensis can host at least two 103C3 C3 8.66 (1.16) 7.51 (1.14) 8.20 (1.05) Symbiodinium strains has implications for the resilience of 103C5 A4 9.09 (0.83) 8.10 (0.83) 8.67 (0.81) Mussismilia-dominated reefs and might be partially connected 103C6 C3 8.67 (0.98) 7.63 (0.96) 8.22 (0.95) to the relative resistance and resilience to bleaching of the SAO when compared to other biogeographic regions [95]. Mean and standard deviations of 1500 cells of each isolate sample. All parameters were measured at ×40 magnifications. Units for length, width Relevantly, the main coral mortality source reported for and equivalent spherical diameter (ESD) are in micrometers M. braziliensis is a white plague-like (WPL) disease that has been recorded in all sampled sites in the Abrolhos Bank [39]. 76–79], over 20 m deep at the Trindade Island. Although it has Symbiodinium C3 was associated with healthy and diseased already been reported at deep environments [89], the domi- M. braziliensis, indicating that corals harboring these symbi- nance of this Symbiodinium type might reflect the higher light onts are susceptible to WPL infections, but it’s not clear incidence and the impoverished conditions of Trindade Island whether the occurrence of Symbiodinium A4 is related to the reefs, compared to other mesophotic reefs of the region current high WPL disease prevalence in M. braziliensis,oris [Meirelles PM, personal communication]. indeed a baseline condition of SAO reefs. Subsequent studies Molecular clock reconstruction indicates that most shall test the hypothesis that the relative abundances of Symbiodinium clades were established on a radiation event Symbiodinium strains in M. braziliensis might change under occurring between 25 and 50 MYA [9], prior to the formation biotic or abiotic stress. In a severely threatened environment of the Orinoco-Amazon river plume (10 MYA) and the as the Abrolhos Bank [39], an understanding of the cooling of the Benguela current (2–3MYA)[90], which are Symbiodinium community responses to these factors is indis- considered the main events that have limited the connectivity pensable for the conservation of these reefs. between the SAO and the Caribbean and tropical Pacific ba- sins, respectively. From that perspective, the absence of clade D Symbiodinium strains, specially S. trenchii (D1a) a wide- Conclusions spread, generalist, and stress-resistant species [35, 91]inour study is intriguing. Previous studies in different geographic Our first survey of Symbiodinium in Mussismilia spp. corals regions of the Brazilian coast focused on the microbial diver- reveals the occurrence of clades A, B, and C and the predom- sity associated with different host species have reinforced the inance of Symbiodinium ITS2 types A4 and C3 in the hypothesis that clade D is rare or absent in the SAO [40–44]. Abrolhos Bank, the largest and richest coralline reef in the In this context, Symbiodinium biogeography in the SAO is still challenging, including the distribution of the major clades and the recent fine-scale diversity radiations within clades (15 MYA) [9, 28].
Multiple Symbiodinium Strains Inhabit Simultaneously a Single Coral Colony of M. braziliensis
As broadcast spawners, Mussismilia corals shall depend on environmental community composition in order to be re- colonized by Symbiodinium, a scenario that favors less specif- ic interactions [18, 19, 92]. A significant finding of the present work was the demonstration that cells belonging to the Symbiodinium A4 and C3 may colonize the same coral host species, M. braziliensis. Regardless of sampling on shallow Fig. 3 Growth curves of Symbiodinium A4, culture 043D10, cell environments of the Abrolhos reef bank, we could not observe densities expressed on base 10 logarithmic scale. On the inset the Symbiodinium A4 as the dominant symbiont in M. braziliensis photosynthetic potential (Fv/Fm) of the same culture A. W. Silva-Lima et al.
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