INSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA – INPA PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA, CONSERVAÇÃO E BIOLOGIA EVOLUTIVA – Gcbev

INSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA – INPA PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA, CONSERVAÇÃO E BIOLOGIA EVOLUTIVA – GCBEv

Taxa metabólica e expressão do gene hif-1α em juvenis e adultos do ciclídeo amazônico Astronotus ocellatus em condição de hipóxia aguda

VANESSA LEIKO OIKAWA CARDOSO

Manaus – AM Agosto de 2018

VANESSA LEIKO OIKAWA CARDOSO

Taxa metabólica e expressão do gene hif-1α em juvenis e adultos do ciclídeo amazônico Astronotus ocellatus em condição de hipóxia aguda

ORIENTADOR: Dra. VERA MARIA FONSECA DE ALMEIDA E VAL

Dissertação apresentada ao Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva do Instituto Nacional de Pesquisas da Amazônia, como requisito para a obtenção do título de Mestre em Genética, Conservação e Biologia Evolutiva.

Manaus - AM Agosto de 2018 iii

VANESSA LEIKO OIKAWA CARDOSO

Taxa metabólica e expressão do gene hif-1α em juvenis e adultos do ciclídeo amazônico Astronotus ocellatus em condição de hipóxia aguda

Aprovada em 07/08/2018

BANCA EXAMINADORA:

Dr. Adalberto Luís Val, Instituto Nacional de Pesquisas da Amazônia

Dra. Gislene Almeida Carvalho-Zilse, Instituto Nacional de Pesquisas da Amazônia

Dra. Adriana Regina Chippari-Gomes, Universidade Vila Velha

C268 Cardoso, Vanessa Leiko Oikawa

Taxa metabólica e expressão do gene hif-1α em juvenis e adultos do ciclídeo amazônico Astronotus ocellatus em condição de hipóxia aguda / Vanessa Leiko Oikawa Cardoso. - Manaus: [sem editor], 2018. ix, 42 f.: il. color.

Dissertação (Mestrado) - INPA, Manaus, 2018. Orientadora: Vera Maria Fonseca de Almeida e Val. Programa: Genética, Conservação e Biologia Evolutiva.

1. Astronotus ocellatus. 2. Expressão gênica. I. Título

CDD 597.74

Sinopse

Estudou-se os ajustes fisiológicos e moleculares de juvenis e adultos do ciclídeo Astronotus ocellatus em condição de hipóxia aguda. Aspectos como a taxa metabólica massa-específica, nível crítico de oxigênio, metabólitos plasmáticos e níveis de transcritos do gene hif-1α foram analisados.

À minha mãe, Tereza, que sempre acreditou em mim.

Agradecimentos

À minha mãe, Tereza, que sempre me apoiou e incentivou a batalhar por meus objetivos e é por ela que eu acordo todos os dias e busco ser uma pessoa melhor.

À minha orientadora, Dra. Vera Val, minha “mãe científica”, que com muito carinho e atenção me acolheu e me deu a oportunidade de desenvolver minha carreira científica. A senhora é um exemplo de orientadora e pesquisadora.

Ao Dr. Adalberto Val, que sempre me inspirou a perseguir com determinação a carreira científica e fazer ciência de qualidade e para todos.

Ao Luiz, que acompanhou e me ajudou durante toda minha trajetória e nunca me deixou desistir nos momentos mais difíceis e duvidosos.

À Naza, que sempre esteve disposta a ajudar durante os contratempos e é uma pessoa fundamental na execução dos projetos do laboratório.

À Dra. Carol e ao Dr. Márcio, pela ajuda na biologia molecular e análises bioquímicas, respectivamente.

À Samara, Luciana e Yasmin, pela ajuda nas análises de biologia molecular e bioquímicas.

Ao Dinho, Derek e Arlan, pela ajuda nas análises estatísticas e contribuições para a melhoria do projeto.

Aos amigos que fiz em Manaus, William, Alber, Junielson, Ricardo, Priscylla, Jéssica, Danilo e Jhonatan, pelos momentos de alegrias e tristezas compartilhados.

Aos meus melhores amigos, Danielli e Édipo, que mesmo distantes, acompanharam meus momentos de frustrações e alegrias.

À minha família.

A toda equipe do LEEM, dona Rai, Claudinha, Raquel, Rogério, Reginaldo, Tiago, dona Val e dona Zizi.

A todos os demais estudantes do LEEM que me ajudaram direta ou indiretamente.

À secretaria e à coordenação do programa de pós-graduação GCBEv, pelo apoio e assistência.

Ao Portal de Periódicos da CAPES, por oferecer o acesso aos artigos científicos.

À CAPES, CNPq e FAPEAM, que custeiam o projeto ADAPTA.

Ao CNPq pela concessão de minha bolsa de estudo.

vii

“Magia é apenas ciência que ainda não entendemos” Arthur C. Clarke. viii

Resumo

Os lagos de várzea da bacia Amazônica passam por flutuações diárias nos níveis de oxigênio dissolvido, variando desde supersaturação durante o dia a quase total falta de oxigênio durante a noite e, além disso, a maior parte do oxigênio está localizada nos primeiros centímetros da coluna d’água. As espécies de peixes desses ambientes desenvolveram diferentes ajustes para lidar com os baixos níveis de oxigênio, desde estratégias comportamentais a ajustes fisiológicos, bioquímicos e moleculares, sendo que esses mecanismos podem ser diferentes ao longo do desenvolvimento do indivíduo. O objetivo deste estudo foi investigar, em nível fisiológico e molecular, como juvenis e adultos do ciclídeo Astronotus ocellatus lidam com a exposição à hipóxia aguda. Para isso, foram analisados o consumo de oxigênio massa-específico, o nível crítico de oxigênio (Pcrit), glicose e lactato plasmáticos, glicogênio hepático e o nível de transcritos do gene hif-1α de juvenis pesando 10g, 50g e 100g e adultos pesando 500g. Nossos resultados mostraram uma relação hipoalométrica entre o consumo de oxigênio massa-específico e a massa corporal dos peixes, com um expoente alométrico igual a -0,26 (ou 0,74). Quando expostos a cinco horas de hipóxia aguda, juvenis e adultos reduziram o seu metabolismo aeróbico, entretanto, apenas os juvenis ativaram a via fermentativa para manter o fornecimento de energia, uma vez que os níveis de glicose e lactato plasmáticos aumentaram nesse grupo, enquanto que a resposta dos adultos foi apenas a supressão metabólica como mecanismo para manter o equilíbrio entre a demanda e a oferta de energia, uma vez que não apresentaram aumento nos níveis da glicose e lactato plasmáticos. Juvenis e adultos expostos à normóxia e hipóxia aguda apresentaram os mesmos níveis de transcritos do hif-1α no fígado, exceto nos juvenis de 100g, que tiveram um aumento no nível dos transcritos quando expostos à hipóxia aguda. Este resultado pode ser uma consequência de esse grupo ter mostrado menor capacidade de extrair o oxigênio em situação de hipóxia, a qual foi evidenciada pelo maior valor de Pcrit e, dessa forma, a exposição aguda à hipóxia foi mais severa para esses animais. Os níveis de transcritos do hif-1α nas brânquias foram os mesmos entre os grupos e entre tratamentos. Como as brânquias de A. ocellatus não têm sua extração de oxigênio prejudicada pela exposição aguda à hipóxia, um aumento nos níveis de transcritos do hif-1α não foi necessário. Este estudo foi um dos poucos estudos a analisar os níveis de transcritos do hif-1α ao longo do desenvolvimento e análises da proteína HIF-1α poderá fornecer mais informações sobre os mecanismos moleculares dessa espécie, que é altamente tolerante à hipóxia.

Abstract

The Amazonian floodplain lakes face daily fluctuations in oxygen concentrations, ranging from supersaturation during the day to almost total lack of oxygen during the night and, moreover, the greatest amount of oxygen is located at the first centimeter of the water column. The ichthyofauna of these environments developed different adjustments to deal with low oxygen levels, like behavioral, physiological, biochemical and molecular mechanisms, and these strategies may be different over the development of the individual. The objective of this study was to investigate, at a physiological and molecular level, how juveniles and adults of the cichlid Astronotus ocellatus deal with exposure to acute hypoxia. For this, the mass-specific oxygen consumption, critical oxygen level (Pcrit), plasma glucose and lactate, liver glycogen and hif-1α gene transcription of juveniles weighing 10g, 50g and 100g, and adults weighing 500g were analyzed. Our results showed a hypoallometric relationship between mass-specific oxygen consumption and fish body mass, with an allometric exponent equal -0.26 (or 0.74). When exposed to five hours acute hypoxia, juveniles and adults reduced their aerobic metabolism, however, juveniles needed to activate the fermentative pathway for energy supply, once plasma glucose and lactate increased in this group, while adults relied only on metabolic suppression as a mechanism to maintain in equilibrium the demand and supply of energy, since they did not show any change on plasma glucose and lactate. Juveniles and adults exposed to normoxia and acute hypoxia had the same hif-1α transcripts levels in liver, except 100g juveniles, that showed an increase on hif-1α transcripts level when exposed to acute hypoxia. This result may be a consequence of their lower capacity to extract the oxygen under hypoxia, evidenced by their higher Pcrit value, and thus acute hypoxia exposure was stronger for these animals. The transcripts levels of hif-1α in the gills were the same between groups and treatments. Since the gills of A. ocellatus do not have their oxygen extraction impaired by acute hypoxia exposure, an increase on hif-1α transcripts levels was not necessary. This study was one of the few studies to address the hif-1α transcripts levels along the developmental stages and further analysis of the HIF-1α protein will provide more information about the molecular mechanisms of this unique hypoxia and anoxia-tolerant Amazonian cichlid.

Sumário

1. INTRODUÇÃO ...... 1 2. OBJETIVO GERAL...... 4 3. CAPÍTULO 1 ...... 5 Abstract ...... 6 Introduction ...... 7 Material and methods ...... 9 Animals ...... 9 Mass-specific metabolic rate ...... 10 Critical oxygen level determination ...... 11 Acute hypoxia exposure experiment ...... 11 Sample collection ...... 12 Glucose and lactate...... 12 Liver glycogen ...... 13 RNA extraction and cDNA synthesis ...... 13 Statistical analysis ...... 14 Results ...... 15 Mass-specific metabolic rate ...... 15 Critical oxygen level ...... 15 Glucose and lactate...... 15 Liver glycogen ...... 16 hif-1α transcript levels ...... 16 Discussion ...... 16 Conclusion ...... 21 Acknowledgements ...... 22 Figures ...... 23 Table ...... 30 References ...... 31 4. CONCLUSÃO ...... 38 5. REFERÊNCIAS ...... 39

1. INTRODUÇÃO

A bacia amazônica possui a maior riqueza de espécies de peixes de água doce do mundo, com 2411 espécies, sendo que dessas, 1089 espécies (45%) são endêmicas da bacia (Reis et al., 2016). Esse ecossistema é caracterizado por pulsos anuais de inundação, os quais são a principal força responsável pela existência, produtividade e interações da biota em sistemas de rio-planície de inundação (Junk et al., 1989). A grande diversidade de espécies de peixes da bacia amazônica pode ser explicada pela dinâmica e tamanho da bacia hidrográfica, assim como pela heterogeneidade de habitats que a região possui (Junk et al., 1983; Val e Almeida- Val, 1995; Val et al., 1998). Os lagos de várzea têm como característica as bruscas variações diárias dos níveis de oxigênio dissolvido, variando desde condições de supersaturação de oxigênio nas horas mais quentes do dia, até concentrações que chegam a quase zero durante a noite, além da variação espacial, na qual a maior parte do oxigênio se encontra nos primeiros centímetros da coluna d’água (Junk et al., 1983; Val et al., 2005). Essas características são devidas às combinações de fatores biológicos, físicos e químicos como, por exemplo, a taxa fotossintética do fitoplâncton, a decomposição da matéria orgânica, a estratificação térmica e química da coluna d’água e a intrínseca baixa capacidade de dissolução do oxigênio na água, sendo que a razão da quantidade de oxigênio no ar para a água destilada é de 30:1, à 20ºC, e a taxa de difusão do oxigênio no ar é 10.000 vezes maior do que na água (Val, 1996; Rabalais et al., 1999). Além disso, a maior parte do oxigênio dissolvido é encontrada nos primeiros centímetros da coluna d´água (Junk et al., 1983; Val et al., 2005). Essas acentuadas variações diárias e espaciais nos níveis de oxigênio nos lagos de várzea amazônicos possibilitaram que os organismos desses ambientes desenvolvessem respostas adaptativas importantes na evolução das espécies de peixes amazônicos (Val et al., 1998; Scott et al., 2008; Almeida-Val et al., 2011). A disponibilidade de oxigênio tem papel importante na evolução das adaptações fisiológicas dos organismos, uma vez que o metabolismo aeróbico é o principal meio de obtenção de energia (Webster, 2003). Algumas das respostas à hipóxia em ambientes aquáticos envolvem mecanismos comportamentais, fisiológicos, anatômicos e moleculares. A respiração na superfície aquática (RSA) é um exemplo de mecanismo comportamental em que o peixe nada até a camada mais 2

superficial da coluna d’água, onde a maior parte do oxigênio dissolvido está concentrada (Kramer e Mehegan, 1981; Braum e Junk, 1982; Junk et al., 1983). A diminuição da tomada de oxigênio e, consequentemente, a depressão do metabolismo aeróbico, são exemplos de mecanismo fisiológico para lidar com a hipóxia (Val, 1996; Almeida-Val et al., 2000). A expansão do lábio inferior em algumas espécies de Characiformes tem como função direcionar a camada de água mais superficial e rica em oxigênio para as brânquias, sendo um exemplo de ajuste anatômico que ocorre em situações de hipóxia (Saint-Paul, 1984; Val e Almeida-Val, 1995). Como exemplo de ajustes moleculares em situações de baixo nível de oxigênio na água, podemos citar o aumento da expressão de fatores de transcrição relacionados à homeostase do oxigênio, como o fator de transcrição induzido por hipóxia (HIF), o qual é o principal regulador da expressão gênica em células expostas à hipóxia e evolutivamente conservado nos vertebrados (Wenger, 2000; Rissanen et al., 2006; Kodama et al., 2012). O fator induzido por hipóxia é um fator de transcrição heterodimérico constituído pelas subunidades HIF-α e HIF-β. A sensibilidade do HIF ao oxigênio é conferida pela subunidade HIF-α e, nos mamíferos, a subunidade HIF-α é constitutivamente sintetizada, porém rapidamente degradada na presença de oxigênio. Em situação de hipóxia, a subunidade HIF-α é estabilizada, acumulada e translocada do citoplasma para o núcleo, onde irá se ligar à subunidade HIF-β. O fator de transcrição formado irá se ligar a sequências conservadas nas regiões promotoras ou potencializadoras dos seus genes alvo, regulando suas taxas de transcrição (Wenger, 2000; Nikinmaa e Rees, 2005; Shen et al., 2010; Rytkönen et al., 2011; Tariq et al., 2016). A primeira sequência do gene hif-1α de descrita para peixes foi a da truta arco- íris (Soitamo et al. 2001) e, a partir de então, tal sequência foi caracterizada para outras espécies de peixes. Em mamíferos, a transcrição de pelo menos 100 genes está sob controle do HIF-1, dentre eles genes envolvidos na angiogênese, transporte de íons, transporte de glicose na célula e no sangue, eritropoiese e glicólise (Semenza, 2002; Rytkönen et al., 2007). O gênero Astronotus, pertencente aos ciclídeos neotropicais, engloba espécies de peixes extremamente tolerantes a ambientes hipóxicos. A espécie Astronotus ocellatus Agassiz 1831, popularmente conhecida como acará-açu ou oscar, é uma espécie sedentária, territorialista, agressiva e habita ambientes lênticos e hipóxicos, típico de lagos de várzea e margens de rios da bacia Amazônica. Em relação às 3

demais espécies de ciclídeos, Astronotus ocellatus é considerada de porte médio, alcançando até 25cm de comprimento padrão e 1,5kg (Soares et al., 2008; Colatreli et al., 2012). Sabe-se que a tolerância à hipóxia nessa espécie está relacionada com o tamanho do animal, sendo que os animais maiores possuem maior tolerância à hipóxia do que os indivíduos menores (Almeida-Val et al., 2000; Sloman et al., 2006). Os mecanismos fisiológicos que contribuem com a alta tolerância à hipóxia dessa espécie vêm sendo amplamente estudados (Muusze et al., 1998; Almeida-Val et al., 1999; Almeida-Val et al., 2000; Sloman et al., 2006, Richards et al., 2007; Scott et al., 2008), entretanto, pouco se sabe sobre os mecanismos genéticos e moleculares envolvidos na homeostase do oxigênio em situação de hipóxia (Baptista et al., 2016; Cassidy et al., 2017). Investigar o efeito da massa corporal na tolerância à hipóxia em A. ocellatus utilizando ferramentas moleculares, como a expressão do gene hif-1α, permitirá melhor compreensão dos mecanismos que contribuem para tal tolerância em ambientes tropicais, onde altas temperaturas impõem um aumento do metabolismo aeróbico para a maioria dos animais (Pörtner, 2001; Campos et al., 2016). Com as previsões do Painel Intergovernamental sobre Mudanças Climáticas (IPCC 2014) para o aumento nos níveis de emissão de gases de efeito estufa, como o gás carbônico, e da temperatura média da superfície terrestre até o ano de 2100, é esperado que os rios da bacia amazônica passem por episódios mais longos e mais severos de hipóxia devido às alterações nos períodos de chuvas e, consequentemente, alterações na dinâmica dos pulsos anuais de inundação, sendo importante entender como e se esses animais ao longo do desenvolvimento conseguirão suportar as novas condições de hipóxia em seus habitats (Almeida-Val et al., 2006; Röpke et al., 2017). Considerando a plasticidade fenotípica dos ciclídeos e a tolerância de muitas de suas espécies às situações de hipóxia, e mesmo anóxia, em temperaturas acima de 28°C e considerando que tal tolerância varia conforme o tamanho do organismo, o presente estudo teve como objetivo verificar se juvenis e adultos do ciclídeo amazônico Astronotus ocellatus possuem estratégias fisiológicas e moleculares diferentes para lidar com situações de hipóxia.

2. OBJETIVO GERAL

Verificar se juvenis e adultos do ciclídeos amazônico Astronotus ocellatus possuem estratégias fisiológicas e moleculares diferentes quando expostos à hipóxia aguda.

2.2 OBJETIVOS ESPECÍFICOS

a) Verificar se o consumo de oxigênio está relacionado com a massa corporal;

b) Verificar se o ponto em que os indivíduos não mais conseguem manter a tomada de oxigênio constante conforme o nível de oxigênio diminui no ambiente está relacionada com a massa corporal;

c) Verificar se a ativação do metabolismo anaeróbico dos animais em hipóxia aguda tem relação com a massa corporal;

d) Verificar se a expressão do gene hif-1α em hipóxia aguda está relacionada com a massa corporal.

3. CAPÍTULO 1

Title: How juveniles and adults of an Amazonian cichlid deal with acute hypoxia exposure? A metabolic and molecular approach.

Running title: Hypoxia adjustments on A. ocellatus

Authors: Vanessa Leiko Oikawa Cardoso1*, José Luiz de Vasconcelos Lima1, Vera Maria Fonseca de Almeida-Val1

1Laboratory of Ecophysiology and Molecular Evolution, National Institute of Amazon Researches, Manaus, Brazil.

*Corresponding author: Email address: [email protected]

Keywords: Astronotus ocellatus, acute hypoxia, metabolism, hif-1α, body mass, scaling.

Abstract

The Amazonian floodplain lakes face daily fluctuations in oxygen concentrations, ranging from supersaturation during the day to almost total lack of oxygen during the night and, moreover, the greatest amount of oxygen is located at the first centimeters of the water column. The ichthyofauna of these environments developed different adjustments to deal with low oxygen levels, like behavioral, physiological, biochemical and molecular mechanisms, and these strategies may be different over the development of the individual. The objective of this study was to investigate, at a physiological and molecular level, how juveniles and adults of the cichlid Astronotus ocellatus deal with exposure to acute hypoxia. For this, the mass-specific oxygen consumption, critical oxygen level (Pcrit), plasma glucose and lactate, liver glycogen and hif-1α gene transcription of juveniles weighing 10 g, 50 g and 100 g, and adults weighing 500 g were analyzed. Our results showed a hypoallometric relationship between mass-specific oxygen consumption and fish body mass, with an allometric exponent equal -0.26 (or 0.74). When exposed to five hours acute hypoxia, juveniles and adults reduced their aerobic metabolism, however, juveniles needed to activate the fermentative pathway for energy supply, once plasma glucose and lactate increased in this group, while adults relied only on metabolic suppression as a mechanism to maintain in equilibrium the demand and supply of energy, since they did not show any change on plasma glucose and lactate. Juveniles and adults exposed to normoxia and acute hypoxia had the same hif-1α transcripts levels in liver, except 100 g juveniles, that showed an increase on hif-1α transcripts level when exposed to acute hypoxia. This result may be a consequence of their lower capacity to extract the oxygen under hypoxia, evidenced by their higher Pcrit value, and thus acute hypoxia exposure was stronger for these animals. The transcripts levels of hif-1α in the gills were the same between groups and treatments. Since the gills of A. ocellatus do not have their oxygen extraction impaired by acute hypoxia exposure, an increase on hif-1α transcripts levels was not necessary. This study was one of the few studies to address the hif-1α transcripts levels along the developmental stages and further analysis of the HIF-1α protein will provide more information about the molecular mechanisms of this unique hypoxia and anoxia-tolerant Amazonian cichlid.

Introduction

Studies about animal metabolism and body size are common since early 1800s and, in a general way, there is an allometric relationship between this two variables, that is known as the quarter power law, and the general allometric exponent for vertebrates is around 0.75, but this value is constantly debated (Kleiber, 1932; Hochachka et al., 2003; Witting, 2017). Allometry has been seen as a consequence of a physiological optimization of metabolism in relation to the resource transport network (West et al., 1997; Banavar et al., 1999). Researchers report that animals with greater capacity of oxygen diffusion and greater respiratory surface area show higher levels of aerobic activity, and are commonly named “athletes” (Pauly, 1981; Witting, 2017). The mass-specific metabolism determines the energy needed based on oxygen consumption per gram of body mass (Kleiber, 1932). The relationship between the mass-specific oxygen consumption rate (MO2) in fishes and body mass (W) can be described as

b mass-specific MO2 = aW (1)

where a is the allometric coefficient, which varies among taxa, and b is the allometric exponent, that in fishes can vary from 0.7 to 0.85 (Kleiber, 1932; Killen et al., 2010; Gillooly et al., 2016). The Amazon basin has the most diverse fish fauna, with 2411 species, of which 1089 are endemic species (Reis et al., 2016). Fishes live in environments that are subject to great variation of water dissolved oxygen (Nilsson and Östlund-Nilsson, 2008). In the Amazonian water bodies, normal or even supersaturated oxygen levels can be found in the rivers, while hypoxia and anoxia are common features in the floodplain lakes (Junk et al., 1983; Val and Almeida-Val, 1998). Daily and local oxygen fluctuations occurs in the Amazonian floodplain lakes, ranging from saturated during the day to almost zero oxygen during the night, with the greatest amount of oxygen found at the first centimeters of the water column (Junk et al., 1983; Val et al., 2005). The main driving force in the Amazon basin is the annual flood pulse and it is responsible for the existence, productivity and interactions between the biota and the river-floodplain system (Junk et al., 1989). Along with the great fish diversity comes a 8

range of adjustment to deal with hypoxia, such as behavioral, physiological, morphological, biochemical and molecular adjustments (Val and Almeida-Val, 1998; Almeida-Val et al., 1999a). An Amazonian cichlid species known for its high hypoxia tolerance is Astronotus ocellatus, and one of the strategies employed by this species to deal with decreasing oxygen levels is the depression of its aerobic metabolism (Muusze et al., 1998; Almeida-Val et al., 1999b). Besides, larger individuals of A. ocellatus have higher anaerobic power to cope with hypoxia, so the increase in body mass is an important aspect for the increase of the hypoxia tolerance along the development (Almeida-Val et al., 1999b; Almeida-Val et al., 2000; Sloman et al., 2006). Another important approach is the cellular response to hypoxia. The hypoxia- inducible factor-1 (HIF-1) is known as the master switch of genes related to the oxygen homeostasis, playing an important role in adaptive response to hypoxia (Semenza, 1998). Hypoxia-inducible factor-1 is a heterodimeric transcription factor comprised by two subunits, HIF-1α and HIF-1β. The stability of the subunit HIF-1α is regulated by molecular oxygen and, under normoxia, it is tagged by the proline-hydroxylase enzyme (PHD), which uses the oxygen as substrate, and directs HIF-1α to degradation via the ubiquitin-proteasome pathway. When the cell is in hypoxia, the activity of the PHD decreases, and consequently, HIF-1α is stabilized and goes from the cytoplasm to the nucleus to form the heterodimer with HIF-1β. The transcription factor formed, HIF-1, binds to the promotor or enhancer regions of its target genes and, regulating their expression (Semenza, 1998; Nikinmaa and Rees, 2005) In mammalian, several HIF-1 target genes related to oxygen homeostasis are known and their functions are related to erythropoiesis, angiogenesis, glycolysis, glucose uptake, cell differentiation and cell apoptosis (Wenger, 2000; Liu et al., 2012). In fishes, HIF-1 has similar function as those in mammals. Rashid et al. (2017) developed a database of hypoxia responsive genes in fish and reported 35 genes, like genes related to glucose transporter, erythropoietin and angiogenesis. The first reported hif-1α gene was in rainbow trout (Soitamo et al., 2001) and studies relating hif-1α expression with hypoxia tolerance during the development of fishes are scarce (Vuori et al., 2004; Robertson et al., 2014). In addition, the Intergovernmental Panel on Climate Change (IPCC 2014) forecasts the increase of greenhouse gas emission levels, such as carbonic dioxide, and the increase of the average temperature of the Earth's surface (approximately 9

4.8°C) by the year 2100. The effects of climate change, combined with the deforestation, foresee that the aquatic environments of the Amazon basin will experience longer and more severe episodes of hypoxia due to changes in the rainy season and, consequently, changes in the dynamics of the annual flood pulses (Röpke et al., 2017). It is important to understand the mechanisms of how the hypoxia tolerant Astronotus ocellatus deals with hypoxia situation throughout development. Thus, considering the phenotypic plasticity of cichlids and the tolerance of many of its species to situations of hypoxia, and even anoxia at tropical temperatures, and considering that such tolerance varies according to the developmental stage, the present study aimed to verify if juveniles and adults of the Amazonian cichlid Astronotus ocellatus have different physiological and molecular strategies to deal with hypoxic situations

Material and methods

Animals

Specimens of Astronotus ocellatus, weighing approximately 5 g, were obtained from a local fish farm (Santo Antonio Fish Farm, AM-010 Km-113, Rio Preto da Eva, Amazonas, Brazil - 2.6982° S, 59.6994° W) and transferred to the Laboratory of Ecophysiology and Molecular Evolution at the National Institute of Amazonian Research (INPA) (Manaus, Amazonas, Brazil - 3.1190° S, 60.0217° W), where the experiments were carried out. The animals were kept outdoor in a tank of 2000 L, with -1 natural light exposure, aerated water (5.5 ± 0.5 mg O2 L ) and water temperature at 27°C ± 1. The animals were fed once a day with commercial pellet food (36% protein) until satiety. A low flow-through water was maintained to prevent ammonia accumulation. The animals were reared under these conditions until the desired experimental body masses were reached, which were 10 g, 50 g, 100 g and 500 g. In this way, the experiments were performed in different times. Feeding was suspended 24 hours before the experiments and all the experiments were performed indoor. All the procedures were in accordance with the guidelines of the National Council for Control of Animal Experimentation and were approved by INPA’s Ethics Committee on Animal Use (protocol #058/2016).

Mass-specific metabolic rate

To verify how oxygen consumption is related with body mass of Astronotus ocellatus, respirometry experiments were performed. Randomly, the animal was removed from the outdoors tanks, weighted and placed inside a respirometry chamber. The animal was maintained inside the chambers overnight to acclimate and to recover from manipulation stress. The proportion of the respirometry chamber volume and fish volume always respected the ratio between 20 and 30 (Svendsen et al., 2016). The respirometry chamber was placed inside 100 L tank with aerated and temperature- controlled water (28°C ± 0.5). At 28°C, 1010 hPa and 0% salinity, 100% air-saturated -1 water contains 7.8 mg O2 L .

To determine the mass-specific oxygen consumption rate (MO2), the oxygen uptake was measured with an automated intermittent-flow respirometry system. Witrox 4 instrument and oxygen optode sensor (Loligo Systems, Viborg, Denmark) was used to measure the oxygen inside the chamber. DAQ-M instrument (Loligo Systems, Viborg, Denmark) was used for data acquisition in combination with the commercial software AutoResp (Loligo Systems, Viborg, Denmark), providing a real-time analysis. The intermittent-flow respirometry setup consisted of three periods: a measurement time, in which the chamber was sealed and the oxygen consumption inside the chamber, by the animal, was monitored and data were collected; a flush time, in which submersible pumps were turned on to wash out waste products and renew the water and the oxygen level inside the chamber; and a wait time, in which the submersible pumps were turned off and the water inside the chamber was allowed to mix completely before the measurement time. The measurement time was determined by the time that the oxygen saturation decreased from 100% to 80% saturation, and the flush time was enough to return the oxygen level to 100% saturation. The oxygen consumption measurements of each animal were performed during five hours. Calculation of the oxygen consumption was automatically performed by the software, resulting in one MO2 value per animal per measurement time and it -1 -1 was given in function of body mass and time (mg O2 g h ) (Rosewarne et al., 2016;

-1 -1 Svendsen et al., 2016). Log mass-specific MO2 (mg O2 g h ) was regressed against log body mass (g) and this relationship was expressed according to the power law equation (1). For this experiment, 24 fishes (n=24) weighing from 9.7 g to 635 g were used. 11

Critical oxygen level determination

To verify the effect of the progressive oxygen declining on oxygen consumption rate of animals with different body mass, the critical oxygen level (Pcrit) was determined from animals weighing 10.9 g ± 0.4 (juvenile 1; n=6), 53.6 g ± 4.8 (juvenile 2; n=6), 98.8 g ± 5.9 (juvenile 3; n=6) and 582.5 g ± 22.3 (adult; n=6), with a total of 24 animals. For each body mass group, the data of the six animals were plotted and analyzed together. The experimental setup and equipment used for Pcrit determination were the same above mentioned for the mass-specific metabolic rate but excluding the flush time. The respirometry chamber was kept sealed during the whole experiment until the oxygen level reached 1% saturation by the animal’s own consumption (Campos et al., 2016). This way, the duration of the experiment varied according to the oxygen consumption rate of each animal. For Pcrit calculation, log MO2 values were regressed -1 against their respective oxygen level (mg O2 L ) using SegReg software (www.waterlog.info/segreg.htm) and it was determined as the breakpoint of the segmented linear regression (Ultsch et al., 1978; De Boeck et al., 2013).

Acute hypoxia exposure experiment

To verify how fishes with different body mass deal, in a physiological and molecular way, with acute hypoxia exposure, two treatments were performed: one with

-1 animals exposed to five hours normoxia (6 mg O2 L ), and the other with animals -1 exposed to five hours acute hypoxia (0.7 mg O2 L ). The oxygen consumption rate was measured during the experiment, following the previous described procedures. The oxygen level for the acute hypoxia treatment was set to be beneath the lowest Pcrit value found in the critical oxygen level experiment to guarantee that all fishes were under hypoxia condition. Hypoxia was achieved by turning off the aeration and diffusing nitrogen gas into the water with air-stones, a procedure that took approximately 30 minutes. The water temperature was controlled and maintained at 28°C ± 0.5. For this experiment, a total of 22 animals for normoxia treatment and 22 for acute hypoxia treatment were used and fish weight was 10.3 g ± 0.4 (juvenile 1; n=6), 50.7 g ± 2.6 (juvenile 2; n=6), 103 g ± 3.5 (juvenile 3; n=6), and 569 g ± 23.0 (adult; 12

n=4). The group names (juvenile and adult) were based on the first gonadal maturation of the Astronotus ocellatus, that occurs at approximately 13.7 cm of standard length (Sánchez-Botero and Araújo-Lima, 2001) and the fishes belonging the juvenile groups had standard length below this value and the fishes of the adult group had standard length above this value.

Sample collection

After five hours of the acute hypoxia exposure experiment, the animals were removed from the respirometry chambers and blood was sampled from the caudal vein with heparinized syringes. The animals were euthanized by head concussion followed by rupturing the spinal cord. Liver and gills were collected, frozen in liquid nitrogen and stored at -80°C for further analysis.

Glucose and lactate

To analyze plasma metabolites, blood sample was immediately centrifuged at 3000 rpm during 10 minutes for plasma separation. For plasma lactate quantification, an aliquot of the total plasma was acidified with 8% perchloric acid and centrifuged at 3000 rpm during 10 minutes. The supernatant was removed and neutralized with 6 M potassium hydroxide and centrifuged at 3000 rpm during 3 minutes. The supernatant was pipetted in a microplate with glycine buffer solution (G5418, Sigma-Aldrich, MO, USA), β-nicotinamide adenine dinucleotide hydrate (N6522, Sigma-Aldrich, MO, USA) and L-lactic dehydrogenase (L2500, Sigma-Aldrich, MO, USA). The microplate was incubated during eight minutes at 37°C. The assay was conducted using a microplate reader (SpectraMax M, Molecular Devices, CA, USA) at the wavelength of 340 nm and the final plasma lactate concentration was given in mmol L-1. For plasma glucose quantification, a colorimetric enzymatic detection kit was used (InVitro, MG, Brazil). An aliquot of total plasma was mixed with the enzymatic reagent and incubated at 25°C during 10 minutes. The product was read using a microplate reader (SpectraMax M, Molecular Devices, CA, USA) at the wavelength of 500 nm and final plasma glucose concentration was given at mmol L-1.

Liver glycogen

For liver glycogen quantification, 0.03 g of liver sample was weighed and transferred to a microtube containing 6 N potassium hydroxide and the microtube was kept in a dry heat bath at 98°C until complete liver dissolution. An aliquot of the liver homogenate was added to 96% ethanol, mixed and 10% potassium sulphate was added to it and the solution was centrifuged during 3 minutes at 3000 rpm. The supernatant was discarded and pure water was added to the white pellet (Bidinotto et al., 1997). An aliquot was taken and 3% phenol and sulfuric acid were added to it. The solution was mixed and incubated during 10 minutes at 25°C (Dubois et al., 1956). The final solution was read using a microplate reader (SpectraMax M, Molecular Devices, CA, USA) at a wavelength of 480 nm and final liver glycogen amount was given in µmol glucosyl-glucose gwt-1.

RNA extraction and cDNA synthesis

To analyze the liver and gills transcripts levels of hif-1α gene, total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following manufacturer’s instructions. Total RNA concentration and its purity were verified using Nanodrop 2000 Spectrophotometer (Thermo Scientific, MA, USA), where a good quality RNA sample was the one with 260/280 and 260/230 ratios approximately 2.0, following the manufacturer’s guidelines. Total RNA integrity was checked by 1% agarose gel electrophoresis, where 28S and 18S rRNAs bands were intact. Total RNA was diluted with nuclease-free water to a final concentration of 500 ng µL-1, followed by DNase treatment with DNase I, Amplification Grade (Invitrogen, CA, USA). For this, 2 µL of total RNA, 1 µL 10X Reaction Buffer, 1 µL DNase 1U µL-1 and 7 µL DEPC treated water were added in a microtube and incubated during 15 minutes at room temperature, then 1 µL 25 mM EDTA was added to it and the microtube was heated at 65°C during 10 minutes, using Veriti Thermal Cycler (Applied Biosystems, CA, USA). The synthesis of cDNA was performed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA), following the manufacturer’s protocol.

Real Time qPCR

The levels of transcripts of the target gene hif-1α, and reference genes 18S and β-actin, were analyzed by real time qPCR (RT qPCR) with Fast SYBR Green Master Mix (Applied Biosystems, Ca, USA) on ViiA 7 Real Time PCR System (Applied Biosystems, CA, USA). RT qPCR mix contained 1 µL cDNA, 5 µL Fast SYBR Green Master Mix, 1 µL forward primer (2.5 pmol), 1 µL reverse primer (2.5 pmol) and 2 µL nuclease-free water. Each sample was analyzed in triplicate. The primers sequences (Baptista et al., 2016) were:

hif-1α forward: 51-GGAGAGCACCAACGGACAA-3’ hif-1α reverse: 5’-GGGTCACAGATCAAAACCAGGTA-3’ 18S forward:5’-GGGAGGTTCGAGACGATCAG-3’ 18S reverse: 5’-TCGCTAGTTGGCATCGTTTATG-3’ β-actin forward:5’-CCTTGATGTCACGCACGATT-3’ β-actin reverse: 5’-CAGAGCGTGGCTATTCCTTCA-3’

The relative hif-1α transcript level was calculated using the efficiency corrected calculation model described by Pfaffl (2006) and its expression was normalized with 18S and β-actin reference genes, which exhibited stable expression during the experiment.

Statistical analysis

Pearson correlation coefficient and simple linear regression analyses were performed to verify if oxygen consumption and critical oxygen level were related to body mass, with log10-transformed data. Student’s t-test or Mann-Whitney U test was performed to test for differences between treatments, and ANOVA one-way or Kruskal-Wallis test was performed to test for differences between groups. Statistical analyses were performed using SigmaStat 3.5 and SigmaPlot 11.0 software. Data were presented as mean ± standard error of the mean, and significance level determined as 0.05.

Results

Mass-specific metabolic rate

There was a significant correlation between mass-specific oxygen consumption rate and body mass and a negative relationship between these two variables (r=-0.847, p<0.001) (Fig. 1). Therefore, as the animal’s body mass increases, its mass-specific oxygen consumption decreases. The relationship between mass-specific oxygen consumption rate and body mass scales in an allometric way, with the allometric exponent b=-0.26 (or b=0.74, when the whole-body oxygen consumption is considered, since both values refer to the same ratio). Mass-specific oxygen consumption rates of the animals exposed to five hours normoxia and acute hypoxia are expressed in Figure 2. In normoxia, mass-specific oxygen consumption rate between groups were different (p<0.001). Juveniles 1 and juvenile 2 had similar mass-specific oxygen consumption rates (p=0.123) and they were higher (p<0.001) than the rates of juvenile 3 and adult group, that also had similar rates (p=0.212). In relation to the acute hypoxia treatment, there were no differences between the mass-specific oxygen consumption rates (p=0.360) between groups. However, all groups exposed to acute hypoxia reduced their oxygen consumption rate when comparing to the relative normoxia group (juvenile 1, p<0.001; juvenile 2, p<0.001; juvenile 3, p=0.024; adult, p=0.004).

Critical oxygen level

Figure 3 shows the Pcrit values obtained from the groups of fishes with different -1 body mass. The lowest Pcrit (mg O2 L ) value was from adult fishes (Pcrit=1.02) while the highest Pcrit value occurred in juvenile 3 (Pcrit=2.47). Juvenile 1 and juvenile 2 Pcrit values was 1.41 and 1.70, respectively. No significant correlation between Pcrit and body mass was found (r=-0.274; p=0.726).

Glucose and lactate

When in normoxia, all groups had the same plasma glucose level (p>0.052) and the same happened for the fishes exposed to acute hypoxia (p=0.186) (Fig. 4). 16

Between treatments, plasma glucose was higher when juveniles were exposed to acute hypoxia (juvenile 1, p=0.024; juvenile 2, p=0.002; juvenile 3, p=0.037) and no change was observed on adult fishes, which had the same plasma glucose level when exposed to normoxia and acute hypoxia (p=0.140) (Fig. 4). Plasma lactate presented similar pattern as plasma glucose, with higher levels on juveniles exposed to acute hypoxia (juvenile 1, p=0.029 juvenile 2, p=0.024; juvenile 3, p=0.010), but no difference was seen on adult fishes (p=0.345) (Fig. 5).

Liver glycogen

As regard to liver glycogen, no differences between groups exposed to normoxia (p=0.099) neither exposed to hypoxia (p=0.079) were found. When comparing normoxia with acute hypoxia within each group, liver glycogen also remained the same (p>0.05) (Table 1).

hif-1α transcript levels

Liver hif-1α relative transcripts levels did not change among fishes with different body mass exposed to five hours normoxia (p=0.252). When exposed to acute hypoxia, juvenile 3 was the only group that had an increase of its transcripts level (p=0.001) (Fig. 6). For the gills, there was no difference among the groups submitted to normoxia (p=0.996) neither to the groups submitted to acute hypoxia (p=0.609). When comparing the transcripts level between normoxia and acute hypoxia within the group, acute hypoxia had no effect on hif-1α transcripts levels (p=0.113, p=0.485, p=0.485, p=0.421, for juvenile 1, 2, 3 and adult, respectively) (Fig. 7).

Discussion

In our study, for fishes with body mass ranging from 9.7 g to 635 g (n=24), the allometric scaling exponent value found was b=-0.26 when mass-specific oxygen consumption is considered, and b=0.74 when whole-body animal oxygen consumption is being analyzed (Fig. 1). When b<|1|, it indicates a hypoallometry relationship (Shingleton et al., 2008) between oxygen consumption and body mass, meaning that 17

the oxygen consumption of the animal increases more slowly than the animal’s body, so oxygen consumption is proportionally lower in adults than in juveniles of Astronotus ocellatus. Almeida-Val et al. (1999b) studying the same species with body mass ranging from 7.5 g to 260 g (n=15), found an allometric exponent value of b=0.52. Sloman et al. (2006) also studying the same species with individuals ranging from 8.8 g to 308 g (n=35), obtained an allometric exponent value of b=0.78. This intraspecific variation of the allometric exponent value can be attributed to the differences among the body mass ranges analyzed by the authors (White and Seymour, 2011). In a review of published studies associating oxygen consumption, body mass and temperature, Clarke and Johnston (1999) analyzed 69 species of post-larval teleost fishes and determined that the mean value of the allometric exponent of the metabolic rate was b=0.79 ± 0.11. This way, our result agrees with the literature and even though fish’s species differ in their metabolic rate, this variation seems to be managed by a general allometric exponent. The critical oxygen level values for A. ocellatus described by the literature are varied and these data are shown in Table 2 along with the values found in our study. Different intraspecific Pcrit values are describer for many fish species (Robertson et al., 2015) and this intraspecific variation can be caused by feeding (De Boeck et al., 2013), physiological condition, life history of the individual (Johannsson et al., 2018) or even caused by the methodology utilized by the investigator (Robertson et al., 2015; Johannsson et al., 2018). Although in our study no correlation between Pcrit and body mass was found, we can verify that adults have lower Pcrit value than juveniles and this pattern was also verified by Sloman et al. (2006) and Scott et al. (2008), where larger individuals of A. ocellatus had lower Pcrit values. Since the critical oxygen level is a measure of the oxygen extraction capacity of the animal (Ultsch et al., 1978), adults of A. ocellatus can extract better the oxygen from water in hypoxia conditions and, together with their higher survivorship in hypoxic environments and their higher anaerobic potential (Almeida-Val et al., 1999b; Almeida-Val et al., 2000), our result contributes to the knowledge that hypoxia tolerance in A. ocellatus increases with body mass. The lower Pcrit values found for juveniles 1 and juvenile 2, when comparing to juvenile 3, indicates that smaller individuals would also have a good capacity to extract oxygen from hypoxic environments. Oxygen concentration is important for the spatial distribution and interactions of the fishes from the Amazonian floodplain lakes, with smaller amount of piscivorous fishes inhabiting hypoxic areas (Anjos et al., 2008) and 18

greater amount of juveniles fishes in these areas (Junk et al., 1983). So, hypoxic areas would serve as a physiological refuge for prey species (Chapman et al., 2002; Anjos et al., 2008). Yet, preference or avoidance for hypoxic areas is not necessarily related with hypoxia tolerance (Nilsson and Östlund-Nilsson, 2008) and this was shown by Sloman et al. (2006). These authors observed that small individuals of A. ocellatus spent less time under a shelter, localized in hypoxic area, when they were prior exposed to hypoxia and, when the small individuals were exposed to decreasing oxygen levels, the animals increased their vertical activity in order to search for more oxygenated water. In contrast, large individuals reduced their horizontal activity, contributing to their metabolic depression. In our study, juveniles and adults decreased their metabolism when exposed to five hours acute hypoxia. This metabolic depression of A. ocellatus when submitted to hypoxia is a well-known pattern and characterize the species as a high-hypoxia tolerant, with individual tolerating even four hours of anoxia at 28°C (Muusze et al., 1998; Almeida-Val et al., 1999b; Sloman et al., 2006; Lewis et al., 2007; Scott et al., 2008; De Boeck et al., 2013). Juvenile 3 group (103 g ± 3.5) reduced their metabolism by 60.2% and a similar reduction was found by Muusze et al. (1998) and Lewis et al. (2007), where individuals of A. ocellatus, with approximately 100 g, reduced their metabolism by 59% and 70%, respectively. Plasma glucose and lactate increased when fishes were exposed to five hours acute hypoxia, except for adult group, that did not present any change. The increase of plasma glucose when juveniles of A. ocellatus is exposed to acute hypoxia aims to supply the energy demand during hypoxia by activation of the glycolytic fermentation pathway, which is evidenced by increased plasma lactate during hypoxia (Richards et al., 2007; Scott et al., 2008; Baptista et al., 2016; Cassidy et al., 2018). In a study performed by Chippari-Gomes et al. (2005) with juveniles (43 – 67 g) of the congeneric Astronotus crassipinnis, the authors verified that, under gradual hypoxia exposure, lactate dehydrogenase (LDH-A), an enzyme that under anaerobic condition converts pyruvate to lactate, increased its activity in white muscle. Almeida-Val et al. (2011) verified an increase on ldh-a transcripts levels on white muscle of juveniles of A. crassipinnis exposed to acute hypoxia and anoxia, while in adult, no change on ldh-a transcripts levels was observed. We have some hypothesis to explain the absence of change on plasma glucose and lactate of adult fishes when exposed to acute hypoxia. Since adult fishes have an intrinsic low metabolic rate, the activation of the glycolytic 19

fermentation was not required under acute hypoxia situation. The expression of ldh-a is regulated by HIF-1 (Semenza et al., 1996; Goda and Kanai, 2012) and acute hypoxia exposure is not a stressor for adult fishes, so an increase of hif-1a expression was not required and this may explain the normal levels of plasma glucose and lactate during acute hypoxia exposure. Almeida-Val et al. (2011) also suggest that adults of A. ocellatus have other mechanisms to deal with hypoxia. An alternative explanation is that in adult fishes, acute hypoxia induces Cori Cycle in liver, which recycles glucose and lactate between liver and muscle, enhancing the lactate uptake for gluconeogenesis, preventing lactic acidosis (Suhara et al., 2015) and maintaining the plasma glucose and lactate levels constant. Hypoxia-inducible factor-1 has an important role in the adaptive regulation of the energy metabolism (Goda and Kanai, 2012), since it has as targets genes related to glucose transporters and glycolytic enzymes (Semenza, 2012; Suhara et al., 2015; Rashid et al., 2017). Juvenile 1 and juvenile 2 had an increase on plasma glucose and lactate when exposed to acute hypoxia, with no change on hif-1α transcripts levels, but we cannot affirm the same at protein HIF-1 level. Robertson et al. (2014) found a higher HIF-1α protein level expressed during hypoxia with no change of hif-1α mRNA transcripts. Liver glycogen did not change in any groups, neither when fishes were exposed to acute hypoxia. The same result was observed by De Boeck et al. (2013) and Cassidy et al. (2018), in which hypoxia did not affect liver glycogen levels. Glycogen stores in liver seem to be related to a more severe stress, like long-term food deprivation and prolonged hypoxia, than to anaerobic metabolism during acute hypoxia exposure (Hochachka, 1986; Liew et al., 2012). Liver lipids and protein stores appear to be the major energy sources during acute hypoxia exposure to support the anaerobic metabolism in A. ocellatus, conserving this way the large reserves of liver glycogen, a common feature in hypoxia tolerant fishes (Liew et al., 2012; De Boeck et al., 2013). Liver hif-1α relative expression was higher only in the juvenile 3 group exposed to five hours acute hypoxia. For the adult group, the lack of change on hif-1α gene transcription can be explained due to acute hypoxia did not affect these fishes, once their plasma and glucose levels did not change. However, the absence of increase on hif-1α gene transcription was an outcome unexpected for juvenile 1 and juvenile 2, since Baptista et al. (2016) and Heinrichs-Caldas (unpublished data) found an increase on liver hif-1α transcripts levels of wild juveniles of A. ocellatus and A. crassipinnis, 20

respectively, exposed to acute hypoxia. The higher transcripts levels in the juvenile 3 can be explained based on its higher Pcrit value, meaning that when oxygen levels -1 reached 2.47 mg O2 L , fishes were already in hypoxia situation, and that juvenile 3 was less capable to extract the oxygen from water, when comparing to the other -1 groups. Acute hypoxia was set as 0.7 mg O2 L and this oxygen level was a stronger stressor for juvenile 3. This does not mean that juvenile 1 and juvenile 2 groups did not feel hypoxia. As observed by Yang et al. (2017), the liver transcripts of hif-1α only increased after 24h of hypoxia exposure in largemouth bass. The lack of change on hif-1α expression during hypoxia was also found in many studies (Soitamo et al., 2001; Rahman and Thomas, 2007; Shen et al., 2010; Rimoldi et al., 2012; Kwasek et al., 2017). Liver hif-1α transcripts levels also remained the same in Oreochromis niloticus along 6h, 12h and 24h of hypoxia exposure (Li et al., 2017). In other hand, Robertson et al. (2014) found an increase on HIF-1α protein level expressed during hypoxia, with no change of hif-1α transcripts. The same authors also described that the cellular response to hypoxia is influenced both by the developmental stage and the degree of hypoxia for the organism. In a study carried out by Rytkonen et al. (2012), fishes preconditioned to hypoxia had different hif-1α expression than fishes exposed only once to hypoxia. The group that faced a single hypoxia exposure did not present a transcriptional response of hif-1α (Rytkonen et al., 2012). Thus, the increased hif-1α transcription level of juvenile 3 can be attributed to the degree of hypoxia felt by the fish (Zhang et al., 2017). We also hypothesize that when Astronotus ocellatus reaches approximately 100 g, the individual metabolic depression becomes more effective than molecular responses to deal with hypoxia. Gills hif-1α transcription was the same in all groups and hypoxia did not affect the transcripts levels. Li et al. (2017) also did not find difference on gills hif-1α transcripts levels in Oreochromis niloticus even after 6h, 12h and 24h of hypoxia exposure. The same was also found by Yang et al. (2017) in largemouth bass exposed to various durations of acute hypoxia. Scott et al. (2008) observed that respiration of isolated gills cells of A. ocellatus, exposed to gradual hypoxia, declined only when oxygen levels almost reached anoxia and with oxygen uptake not been affected. A study performed by Wood et al. (2007) observed that the active ion exchange of the gills decreased during hypoxia in A. ocellatus, with reduction on Na+/K+ ATPase activity and a reduction on general nitrogen-waste production (Richards et al., 2007; Wood et al., 2007), contributing to the whole animal metabolism depression and maintenance 21

of the equilibrium between ATP suppression and ATP demand, with larger animals having a more effective ionoregulatory response. This way, the gills of the hypoxia tolerant A. ocellatus are able to reduce their costs of ion regulation without compromising oxygen exchange during hypoxia, a feature that is a common trade-off between the gas exchange and ion regulation for hypoxia non-tolerant fishes (Wood et al., 2007; Wood et al., 2009; Matey et al., 2011). The stability and activation of HIF- 1α protein depends on the degree and duration of hypoxia exposure (Yang et al., 2017) and once the gills of A. ocellatus can keep their oxygen uptake during short-term acute hypoxia (Scott et al., 2008), an increase on hif-1α expression in gills would not be necessary, since HIF-1α proteins are degraded in presence of oxygen.

Conclusion

The mass-specific variation of the metabolic rate depends on the size of Astronotus ocellatus and as larger the fish, lesser is the dependence on the aerobic metabolism. The intrinsic low metabolism of the adults contributes with the metabolic depression to deal with acute hypoxia exposure, while the juveniles need to activate the anaerobic metabolism for energy production. The lower critical oxygen level for adults indicates their higher capacity of oxygen uptake in hypoxia condition, contributing to their greater hypoxia tolerance. Juveniles 1 (10 g) and juveniles 2 (50 g) groups also presented low Pcrit values, but this cannot be associated with great hypoxia tolerance, since they needed to activate the anaerobic metabolism to energy supply and metabolic by-products of the anaerobic metabolism cannot be sustained for a long time. There was no relationship between transcript levels of hif-1α gene and the body mass and hypoxia did not affect hif-1α transcription in liver and gills of A. ocellatus, except for the 100 g juveniles, which had an increase on liver hif-1α transcripts level when exposed to acute hypoxia. Together with their higher Pcrit value, this outcome suggest that this stage of development may be the critical point between anaerobic metabolism and metabolic depression as the main strategy for dealing with hypoxia, since it is about this phase (100 g) that the first gonadal maturation and physiological changes and energy trades-off may be influencing their capacity to deal with hypoxia. 22

Analysis of the HIF-1 protein may provide further information about the molecular mechanisms involved in hypoxia tolerance throughout the development of Astronotus ocellatus.

Acknowledgements

This study was funded and supported by the INCT/ADAPTA II and the fellowship conceded by the Brazilian National Research Council (CNPq). We would like to thank Dr. Adalberto Luis Val, Msc. Maria de Nazare Paula da Silva, Dr. Derek Felipe Campos and Msc. Waldir Heinrichs Caldas for their suggestions and contributions to this work.

Figures

Table

Table 1. Liver glycogen content under five hours normoxia and acute hypoxia from fishes with different body mass. There was no difference between groups submitted to normoxia (same lower-case letter, Kruskal-Wallis: p=0.099) nor acute hypoxia (same capital letter, one-way ANOVA: p=0,079). When comparing normoxia with acute hypoxia within the group, no difference was found (t-test: p>0.05).

-1 Liver glycogen (µmol glucosyl-glucose gwt )

Normoxia Acute hypoxia Juvenile 1 583,09 ± 40.40a 552,87 ± 37.28A n = 6

Juvenile 2 771.40 ± 47.70a 813.55 ± 111.60A n = 6 Juvenile 3 758.69 ± 56.69a 612.93 ± 43.99A n = 6 Adult 760.48 ± 70.71a 791.19 ± 114.20A n = 4

Table 2. A review of Pcrit values from the literature for Astronotus ocellatus with different body mass.

-1 Pcrit (mg O2 L ) Body mass (g) Reference 1.41 10.9 ± 0.4 Present study

3.81 16.2 ± 1,9 Sloman et al., 2006

1.70 53.6 ± 4.8 Present study

2.73 98 ± 3 De Boeck et al., 2013

2.47 98.8 ± 5.9 Present study

3.81 129 ± 11 Scott et al., 2008

2.56 230 ± 11 Sloman et al., 2006

1.70 388 ± 20 Scott et al., 2008

1.02 582.5 ± 22.3 Present study

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4. CONCLUSÃO

A variação massa-específica da taxa metabólica depende da massa corporal de Astronotus ocellatus e, quanto maior a massa, menor é a dependência do individual em relação ao metabolismo aeróbico. O intrínseco baixo metabolismo aeróbico dos adultos contribui com os mecanismos para lidar com a exposição à hipóxia aguda, enquanto que, para os juvenis, é necessário a ativação do metabolismo anaeróbico para a produção de energia. O menor nível de oxigênio crítico dos adultos indica a maior capacidade dos animais maiores em extrair o oxigênio da água, contribuindo para sua maior tolerância à hipóxia. Os juvenis 1 (10g) e juvenis 2 (50g) também apresentaram baixos valores de Pcrit, mas isso não pode ser associado à maior tolerância à hipóxia, uma vez que esses animais precisaram ativar o metabolismo anaeróbico para manter o suprimento de energia e os produtos do metabolismo anaeróbico não podem ser mantidos pelo organismo por muito tempo. Não houve relação entre os níveis de transcritos do gene hif-1α e a massa corporal e a hipóxia não afetou a transcrição do hif-1α no fígado e nas brânquias, exceto nos juvenis 3 (100g), que tiveram um aumento no nível de transcritos do hif-1α no fígado quando expostos à hipóxia acuda e, juntamente com o maior valor de Pcrit, esse resultado sugere que esse estágio do desenvolvimento possa ser o ponto crítico entre o uso do metabolismo anaeróbico e a depressão metabólica como principal estratégia para lidar com a hipóxia. Análises da proteína HIF-1 podem fornecer informações adicionais sobre os mecanismos moleculares envolvidos na tolerância à hipóxia durante o desenvolvimento de Astronotus ocellatus.

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