UNIVERSIDADE ESTADUAL PAULISTA – UNESP CENTRO DE AQUICULTURA DA UNESP β-Glucano na piscicultura: efeitos na incubação dos ovos, sistema imune inato e microbiota intestinal Raphael Barbetta de Jesus Jaboticabal, São Paulo 2019 UNIVERSIDADE ESTADUAL PAULISTA – UNESP CENTRO DE AQUICULTURA DA UNESP β-Glucano na piscicultura: efeitos na incubação dos ovos, sistema imune inato e microbiota intestinal Raphael Barbetta de Jesus Orientadora: Dra. Fabiana Pilarski Coorientador: Dr. Geert Frits Wiegertjes Tese apresentada ao Programa de Pós-graduação em Aquicultura do Centro de Aquicultura da UNESP - CAUNESP, como parte dos requisitos para obtenção do título de Doutor. Jaboticabal, São Paulo 2019 Jesus, Raphael Barbetta de J58b β-Glucano na piscicultura : efeitos na incubação dos ovos, sistema imune inato e microbiota intestinal / Raphael Barbetta de Jesus. – – Jaboticabal, 2019 vi, 80 p. : il. ; 29 cm Tese (doutorado) - Universidade Estadual Paulista, Centro de Aquicultura, 2019 Orientadora: Fabiana Pilarski Coorientador: Geert Frits Wiegertjes Banca examinadora: Fábio Sabbadin Zanuzzo, Geovana Dotta Tamashiro, Leonardo Susumu Takahashi, Marianna Vaz Rodrigues Bibliografia 1. Imunoestimulantes. 2. Microbiota intestinal. 3. Ovos. 4. Pacu. 5. Prebióticos. 6. Tilápia do Nilo. 7. β-glucano. I. Título. II. Jaboticabal-Centro de Aquicultura. CDU 639.3.034 Ficha Catalográfica elaborada pela STATI - Biblioteca da UNESP Campus de Jaboticabal/SP - Karina Gimenes Fernandes - CRB 8/7418 Sumário DEDICATÓRIA ....................................................................................................... iii APOIO FINANCEIRO .............................................................................................. v RESUMO ................................................................................................................ 1 ABSTRACT ............................................................................................................. 2 INTRODUÇÃO GERAL ........................................................................................... 3 MANUSCRITO I ...................................................................................................... 8 1. Introdução ....................................................................................................... 10 2. Material e métodos ......................................................................................... 10 3. Resultados ...................................................................................................... 12 4. Discussão ....................................................................................................... 13 5. Referências .................................................................................................... 15 MANUSCRITO II ................................................................................................... 18 1. Introdução ....................................................................................................... 20 2. Material e métodos ......................................................................................... 21 3. Resultados ...................................................................................................... 26 4. Discussão ....................................................................................................... 29 5. Referências .................................................................................................... 35 MANUSCRITO III .................................................................................................. 44 1. Introdução ....................................................................................................... 46 2. Material e métodos ......................................................................................... 48 3. Resultados ...................................................................................................... 52 4. Discussão ....................................................................................................... 58 5. Referências .................................................................................................... 64 DISCUSSÃO GERAL ............................................................................................ 74 REFERÊNCIAS COMPLEMENTARES ................................................................. 75 i ii EPÍGRAFE "Quem come o fruto da árvore do conhecimento é sempre expulso de algum paraíso.” (William Ralph Inge) iii DEDICATÓRIA Dedico esta obra ao meu pai Valdemar de Jesus Sobrinho (in memoriam) e a minha mãe Rosana Celeste Barbetta de Jesus. iv AGRADECIMENTOS Esta tese seja talvez o resultado mais visível do processo de construção em meio a uma conjuração de afetos e amizades. Dessa forma, agradeço àqueles que dela fizeram parte direta ou indiretamente ou, ainda, pelo simples fato de existirem. À minha querida amiga e orientadora Profa. Dra. Fabiana Pilarski, agradeço o apoio, a partilha do saber e as valiosas contribuições para o trabalho. Acima de tudo, sou grato por estimular o meu interesse pelo conhecimento e pela pesquisa. Ao meu coorientador Prof. Dr. Geert F. Wiegertjes e ao amigo Dr. Jules Petit por me receberem de forma afetuosa em seu país e pelas inúmeras e valiosas trocas de conhecimento. A todos os amigos que fizeram parte do Laboratório de Microbiologia e Parasitologia de Organismos Aquáticos (LAPOA) e que durante todos estes anos souberam fazer do ambiente de trabalho um lugar acolhedor, construtivo e agradável de se conviver. Aos queridos Prof. Dr. Roque Takahashi, Dr. Danísio P. Munari, Dra. Maria Imaculada Fonseca e Dra. Maria Inez E. G. Martins pela inspiração profissional e pessoal que são em minha vida. Aos meus ex-orientadores Prof. Dr. Jackson A. M. de Souza, Dra. Telma T. Berchielli e Dra. Eliana G. de M. Lemos por todas as oportunidades e lições aprendidas. Ao meu pai Valdemar de Jesus Sobrinho (in memoriam) pela grandeza do seu amor. Pai, sua morte precoce é marca viva. Sua memória é exemplo de dignidade e honestidade. Agradeço a você por ter sido meu pai. À minha mãe Rosana C. Barbetta de Jesus pela sabedoria em me educar, pela sua espiritualidade, pelo amor e carinho de mãe que soube me proteger e me ensinar os limites da vida. A todos aqueles que, embora não nomeados, me presentearam com seus inestimáveis apoios em distintos momentos, o meu sincero e reconhecido agradecimento. v APOIO FINANCEIRO Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Auxílio Financeiro (Processo nº 13/50418-1). O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. 1 RESUMO: O β-glucano é uma molécula estudada como aditivo alimentar na piscicultura há vários anos, sendo descritos na literatura como imunoestimulante e atuando positivamente de diversas formas, como no crescimento, resistência aos patógenos, expressão dos genes do sistema imune inato, entre outros parâmetros. No presente estudo, diferentes abordagens de utilização com diferentes moléculas de β-glucanos foram avaliadas. No primeiro estudo, avaliamos os efeitos do banho de solução de β-glucano (100 mg L-1) durante 8 dias, após o período de exposição ao β-glucano foi avaliado o desempenho (massa e comprimento) das larvas de tilápia do Nilo. O segundo estudo nos possibilitou avaliar o potencial de dois β-glucanos de leveduras residuais provenientes da fermentação alcoólica. Os peixes foram alimentados durante 21 dias com 1 g kg-1 de β-glucano e desafiados com Aeromonas hydrophila inativada. No terceiro estudo avaliamos a composição intestinal bacteriana de pacus alimentados com dois diferentes β-glucanos de leveduras provenientes do processo de fermentação alcoólica. Para avaliar a diversidade bacteriana intestinal, os peixes foram alimentados por 21 dias com 1 g kg-1 de β-glucano, o DNA da microbiota intestinal extraído e realizado o sequenciamento (16S rDNA) utilizando a plataforma Illumina MiSeq. Os resultados do primeiro experimento demonstraram que as larvas incubadas com β-glucano apresentaram melhor desempenho, sendo maiores e mais pesadas que às do tratamento controle. Os resultados do nosso segundo estudo indicaram que um dos β-glucanos obtidos de leveduras residuais da produção de álcool avaliado foi eficaz como imunoestimulante, apresentando efeito positivo na ativação da atividade respiratória dos leucócitos. Os resultados observados no terceiro experimento demonstraram grande prevalência em todos os tratamentos dos gêneros Cetobacterium, Plesiomonas e Epulopiscium, os quais representam aproximadamente 60% de todos os gêneros identificados. Pesquisas utilizando diferentes moléculas de β-glucano são de grande importância e necessárias para averiguar a eficácia e fundamentar a utilização comercial dos mesmos. PALAVRAS-CHAVE: imunoestimulantes, microbiota intestinal, ovos, pacu, prebióticos, tilápia do Nilo, β-glucano 2 ABSTRACT: β-glucan is a molecule studied as a feed additive in fish farming for several years and is described in literature as immunostimulating, and acting positively in several ways, such as growth, resistance to pathogens, expression of innate immune system genes, among other parameters. In the present study, different approaches of use with different β-glucan molecules were evaluated. In the first study, we evaluated the effects of β-glucan bath solution (100 mg L-1) for 8 days, after the exposure period to β-glucan we evaluated the performance (mass and length) of Nile tilapia larvae. The second study allowed us to evaluate the potential of two yeast β-glucans from alcoholic fermentation. Fish were fed for 21 days with 1 g kg -1 of β-glucan and challenged with inactivated Aeromonas hydrophila. In the third study we evaluated the bacterial intestinal composition of pacu fed with two different yeast β-glucans from the alcoholic fermentation process. To evaluate intestinal bacterial diversity, the fish were fed during 21 days with 1 g kg -1 of β- glucan, the DNA of intestinal microbiota was extracted and sequencing (16S rDNA) was performed using the Illumina MiSeq platform. The results of the first experiment demonstrated that the larvae incubated with β-glucan presented better performance, being bigger and heavier than those of the control treatment. The results of our second study indicated that one of the β-glucans obtained from residual yeasts from the alcohol production evaluated was effective as an immunostimulant, with a positive effect on the activation of the leukocyte respiratory activity. The results observed in the third experiment showed a high prevalence in all treatments of the genera Cetobacterium, Plesiomonas and Epulopiscium, which represent approximately 60% of all genera identified. Research using different β-glucan molecules is of great importance and necessary to ascertain the efficacy and substantiate the commercial use of them. KEYWORDS: immunostimulants, intestinal microbiota, eggs, pacu, prebiotics, Nile tilapia, β-glucan 3 INTRODUÇÃO GERAL A produção mundial de peixes tem crescido constantemente nas últimas cinco décadas, com aumento médio anual de 3,2%, ultrapassando o crescimento populacional mundial em 1,6%. O consumo global per capita de peixes aumentou de 9,0 kg em 1961 para 20,2 kg em 2015, a uma taxa média de crescimento de 1,5% ao ano. Estimativas preliminares para 2016 e 2017 apontam para um crescimento de 20,3 e 20,5 kg per capita, respectivamente. Este impressionante desenvolvimento tem sido impulsionado por uma combinação do crescimento populacional, aumento da renda, urbanização e forte expansão da produção de peixes (FAO, 2018). Neste contexto, o Brasil apresenta vantagens para o desenvolvimento da piscicultura. Com cerca de 5,5 milhões de hectares em reservatórios naturais e artificiais de água doce, clima favorável, terras disponíveis e mão de obra relativamente barata, bem como pacotes tecnológicos já finalizados algumas espécies potencialmente produtivas. A produção brasileira de pescado continental atingiu, em 2017, 691.700 mil toneladas, com um incremento de aproximadamente 8% em relação a 2016, demonstrando um crescimento consistente desse setor no Brasil (Baptista et al., 2018). A tilápia do Nilo (Oreochromis niloticus) é o peixe mais produzido no Brasil, e sua criação fez com que o país aumentasse sua produção de pouco mais de 12 mil toneladas em 1995 para 219 mil toneladas em 2015 (FAO, 2016). Em termos de produção regional, a região Sul é responsável por 42% da produção, seguida pelas regiões Sudeste (26%), Nordeste (24%) e Centro-Oeste (8%). O crescimento observado nas últimas décadas na tilapicultura alavancou o país ao posto de quarto maior produtor mundial de tilápia, atrás somente da China, Egito e Indonésia (Schulter e Vieira-Filho, 2017). A tilápia é uma das espécies mais indicadas para a criação intensiva por ser onívora, de rápido crescimento, aceitar vários tipos de alimento, dócil em várias fases da criação e por apresentar rusticidade, com fácil domínio da sua reprodução e bom rendimento de carcaça, além de adaptabilidade em diversas condições de criação. A espécie apresenta ainda requisitos exigidos pelo mercado consumidor, tais como, carne branca de textura firme, sabor delicado, de 4 fácil filetagem, ausência de espinhas em Y, características estas, que a colocam entre as principais espécies comerciais do mundo (Jory et al., 2000). Dentre os peixes nativos produzidos no Brasil, o pacu (Piaractus mesopotamicus) é um dos que mais se destaca, ocupando a segunda posição no ranking brasileiro. Nativo das bacias dos rios Paraná, Uruguai e Paraguai, ressalta-se entre as espécies por apresentar características zootécnicas desejáveis, como carne saborosa e de alto valor comercial, hábito alimentar onívoro e em sistema produtivo aceita bem dietas artificiais, possui rápido crescimento e rendimento no processamento de 46,73% de filé sem pele; 16,57% de cabeça e 88,98% de rendimento de carcaça (Vaz et al., 2000; Jomori et al., 2003; Faria et al., 2003; Bittencourt et al., 2010; Silva et al., 2012; Valladão et al., 2018). Frente ao exposto, a espécie é amplamente produzida em todas as regiões do Brasil. Segundo dados do último Boletim Estatístico da Pesca e Aquicultura, a produção brasileira de pacu e patinga no Brasil em 2016 foi de 13.065 toneladas (IBGE, 2016). Todavia, a intensificação da criação tem levado ao aparecimento de diversos organismos patogênicos, causando grandes prejuízos econômicos, uma vez que podem resultar em altas taxas de mortalidade e lesões que tornam a comercialização do peixe inviável, além do impacto negativo na conversão alimentar que ocasiona redução do crescimento e desempenho dos peixes (Luque, 2004; Takemoto et al., 2004; Pavanelli et al., 2008; Tavares-Dias e Martins, 2017). No Brasil, a aquicultura, apesar do constante crescimento, vem enfrentando entraves na produção e comercialização do pescado por problemas relacionados às enfermidades, as quais provocam danos, muitas vezes irreversíveis, aos tecidos dos peixes e uma elevada taxa de mortalidade, com consequentes prejuízos econômicos (Sebastião et al., 2015). Neste contexto, é necessário estabelecer formas adequadas para controlar e prevenir os surtos de doenças na produção. O conceito de alimento funcional é um paradigma emergente na aquicultura, o qual objetiva a formulação de dietas equilibradas, complementadas com aditivos que melhorem a saúde e resistência à doenças dos peixes (Gatlin e Li, 2004). Dentre os aditivos mais promissores que vêm sendo utilizado nas dietas de peixes, encontram-se os imunoestimulantes, substâncias químicas, sintéticas ou biológicas, capazes de aumentar a resistência 5 do animal às doenças infecciosas, atuando no sistema imune inato, através do aumento da atividade fagocítica e bactericida das células de defesa. O uso de imunoestimulantes é um meio efetivo de aumentar a imunocompetência e a resistência às infecções causadas por vírus, fungos, bactérias e parasitos (Mulero et al., 1998; Sakai, 1999; Bricknell e Dalmo, 2005). Entre os imunoestimulantes que vêm apresentando efeitos benéficos em peixes, encontra-se o β-glucano, formado por uma cadeia linear de poliglicoses, derivado de leveduras, também podendo ser encontrado em alguns cereais, principalmente aveia e cevada (Robertsen, 1999; Meena et al., 2013). Os β- glucanos podem ser diferenciados pela forma como as moléculas de glicose estão ligadas entre si. Na maior parte dos polissacarídeos, as moléculas de glicose estão unidas por ligações α-1,4, conferindo-lhes uma estrutura linear, já nos β- glucanos estas moléculas unem-se por ligações β-1,3 e β-1,6 o que lhes confere uma estrutura helicoidal. Esta conformação estrutural é reconhecida por componentes do sistema imune e resulta na ativação deste sistema (Robertsen et al., 1990). Muitas pesquisas têm sido desenvolvidas utilizando o β-glucano como um potente imunoestimulante. Os resultados destas demonstram que o uso do β- glucano atua positivamente de diversas formas, como no crescimento (Kühlwein et al., 2014; Pilarski et al., 2017), sobrevivência e resistência à patógenos (Das et al., 2009; Sang e Fotedar, 2010; Pilarski et al., 2017), na produção de anticorpos (Kamilya et al. 2006), na alteração da microbiota intestinal (Petit e Wiegertjes, 2016; Miest et al., 2016; Jung-Schroers et al., 2016), na expressão dos genes relacionados ao sistema imune inato (Lokesh et al., 2012; Pietretti et al., 2013; Miest et al., 2016; Salah et al., 2017) e na melhora da cicatrização (Przybylska- Diaz et al., 2013; Schmidt et al., 2015) em várias espécies de peixes. O β-glucano tem sua atuação principal na imunidade inata, por meio da estimulação da atividade fagocitária, que ocorre pela ligação do imunoestimulante com receptores presentes em macrófagos e outras células de defesa (Engstad al., 1992; Gantner et al., 2003; Herre et al., 2004). Estudos apontam vários tipos de receptores para o β-glucano presentes em macrófagos (receptores scavengers, receptores complemento CR3, lactosilceramidas, receptores dectin-1, receptores Toll-like), porém, os mecanismos exatos de ação ainda não estão completamente elucidados (Meena et al., 2013). Detalhes de como os imunoestimulantes, como o 6 β-glucano, podem ativar os receptores de macrófagos em pacus são desconhecidos, devido à falta de pesquisas sobre as interações ligante-receptor nesta espécie. O produto comercial Macrogard® é fonte de 1,3/1,6 β-glucano altamente purificado, exposto e preservado, produzido a partir de uma cepa especialmente selecionada da levedura Saccharomyces cerevisiae, sendo considerado o β- glucano mais estudado no mundo. Inúmeros trabalhos científicos comprovam sua capacidade de promover resistência e saúde aos animais. A célula intacta da levedura tem pouca ou nenhuma capacidade de ativar o sistema de defesa, uma vez que os β-glucanos biologicamente ativos estão contidos no interior da parede celular, cobertos por componentes (manoproteínas), que não são removidos pelo processo natural de digestão do trato gastrointestinal dos animais. Portanto, para criar um produto biologicamente ativo, esses componentes de superfície têm de ser removidos em um processo tecnológico exclusivo e complexo em que, através de diversas etapas de extração consegue-se liberar e expor a estrutura preservada dos 1,3/1,6 β-glucanos em sua forma ativa (Biorigin, 2018). Porém, o tempo do processo e custo de produção do Macrogard® ainda é um fator limitante à ampla disseminação e aproveitamento dos benefícios do uso deste imunoestimulante pela maioria das pisciculturas. Criar fontes alternativas mais acessíveis para este tipo de tecnologia é desejável para o desenvolvimento de uma piscicultura nacional mais competitiva e saudável. Uma alternativa para a produção de 1,3/1,6 β-glucanos com custo reduzido seria o uso de leveduras residuais responsáveis pela fermentação na produção de álcool nas usinas. A levedura (Saccharomyces cerevisiae) é usada como fermento para a obtenção de álcool a partir da cana-de-açúcar e pode ser considerada um resíduo da produção de álcool. De cada litro produzido, sobram cerca de 30 gramas de levedura seca (Costa, 2004). A produção brasileira anual de álcool na safra 2016/2017, segundo dados da Companhia Nacional de Abastecimento (CONAB, 2017) foi de aproximadamente de 27,8 bilhões de litros, sendo o resíduo de biomassa de leveduras está perto de 834 mil toneladas. Neste contexto, a empresa nacional “Biorigin” está desenvolvendo um produto, ainda não comercial e não testado, baseado na produção de 1,3/1,6 β- glucano extraído de leveduras residuais provenientes da produção de álcool. A utilização de leveduras residuais, além de possibilitar a redução do custo de 7 produção e, como consequência um produto final mais barato, está alinhada às novas demandas do mercado para o desenvolvimento de produtos ecologicamente corretos, uma vez que utiliza material residual. Pesquisas utilizando este novo produto serão de grande importância e necessárias para averiguar sua eficácia como imunoestimulante e fundamentar a utilização comercial do mesmo. 8 MANUSCRITO I SHORT COMMUNICATION An early β-glucan bath during embryo development increases larval size of Nile tilapia Raphael B. de Jesus a, Jules Petit b, Fabiana Pilarski a, Geert F. Wiegertjes b,c, João Fernando A. Koch d, Carlos A. F. de Oliveira d and Fábio S. Zanuzzo a* a Centro de Aquicultura da Unesp, UNESP - Univ Estadual Paulista. Via de Acesso Prof. Paulo Donato Castelane, 14.884-900, Jaboticabal, SP, Brazil b Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH Wageningen, The Netherlands c Aquaculture and Fisheries Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH Wageningen, The Netherlands d Department of Research and Development, Biorigin Company, Fazenda São José s/n, 17290-000 Macatuba, São Paulo, Brazil *corresponding author: Fábio Sabbadin Zanuzzo E-mail address: fabioszanuzzo@gmail.com Current address: Department of Ocean Sciences and Biology, Memorial University of Newfoundland, St. John’s, Canada A1C5S7. Paper was resubmitted with the requested corrections to Aquaculture Research 30/01/2019 9 Abstract Larvae production is considered a ‘bottle neck’ for many aquaculture species and early life history plays a key role in this process. Although considerable research has been conducted in different fields to improve fish larvae productivity, very few reports have investigated methods to improve embryo development and consequently larvae quality/production. Here, we show that β-glucan bathing during the embryo development and yolk sac larvae absorption phase enhances the larvae growth (mass and length) of Nile tilapia and this is the first report of this nature on fish larvae culture. We discuss the possibility that microbiota may be the key to understanding the action of β-glucan on the size of Nile tilapia larvae. However, further studies should be designed to address this finding. In summary, it is an innovative approach and a novel use of β-glucan in the aquaculture industry, and could potentially represent a new research field to improve fish embryo/larvae culture. Keywords Fish larvae; hatching; immunostimulant; Nile tilapia; prebiotics 10 1. Introduction The demand to improve fish farming has increased exponentially in the last several decades and larvae production is crucial in this process. High mortality of embryos and larvae is a common phenomenon in fish, and causes a significant financial loss annually in the aquaculture industry (Jiang et al., 2016). Thus, methods/procedures to improve embryo development, and fish larvae culture are necessary. Several studies have shown that the use of β-glucan has positive effects in fish production such as promoting growth (Misra et al., 2006; Kuhlwein et al., 2014; Pilarski et al., 2017), improving wound healing (Przybylska-Diaz et al., 2013) and increasing survival and resistance to pathogens (Das et al., 2009; Meena et al., 2013; Sang and Fotedar, 2010; Petit and Wiegertjes, 2016). Studies using β- glucan during the first larval feeding have shown positive results on parameters such as survival, the expression of genes involved in metabolism and immunity, and the beneficial modulation of intestinal microbiota (Skjermo et al., 2006; Miest et al., 2016). In this study, we investigated the effects of β-glucan bathing during the embryo development and larvae phase until absorption of the yolk sac, on larvae performance post-absorption of the yolk sac. Although β-glucan has been widely studied in fish, the present work represents one of the few studies investigating the use of β-glucan bathing during fish larvae production and the first to evaluate the performance of larvae from embryos incubated with β-glucan. 2. Material and methods 2.2. Broodstock husbandry and embryo collection The embryos were obtained from the Laboratory of Tilapiculture of Aquaculture Center of Unesp (Jaboticabal, SP, Brazil). The broodstock (GIFT tilapia) were 1.5 years old and were fed with commercial feed (crude protein 28%, crude lipid 4%, crude fiber 10%, ash 14%), (Presence, Nutripiscis SI, Brazil), three times per day (08h00, 11h30 and 17h00) at a ratio of 3% body mass/daily. The broodstock were held in 7000 L circular flexible PVC laminate tanks connected to the water recirculation system with mechanical and biological filters, continuous aeration. The photoperiod was 12h00 light : 12h00 dark. The ratio of females to males was 3:1. The eggs were naturally fertilized and collected by the counterflow 11 of water with the aid of a spruce from maternal oropharyngeal cavities of 5 different females. 2.2. Experimental procedure A pool containing 3,300 embryos at a similar stage of ontogenic maturation (uniformly yellow eggs) were collected, as described above, and redistributed into 6 individual incubators (550 embryos in each incubator) supplied with a recirculation water flow system and photoperiod of 12h00 light, 12h00 dark. Each incubator (2.5 L) had an individual pump (62 L h-1; AT-201, Chuangxing Electrical Appliances, Hong Kong, China) placed on a reservatory tank (70 L) equipped with a thermostat (H-606, Hopar®, Dantu Zhenjiang, China) and the water from incubators returned to the reservatory tank by gravity. Two systems completely independent with 3 incubators each were used. One system received the MacroGard® (source of β-glucan, concentration > 60%, Biorigin, SP, Brazil) at a dose of 100 mg L-1 (added in water) and the other system was used as a control. The experimental period with exposure to β-glucan lasted 5 days for embryo development and more 3 days after hatching when the larvae completed the absorption of the yolk sac. Immediately after this period, larvae were collected from each experimental replicate (n = 30), anesthetized with benzocaine (0.1 g L- 1), excess water was removed using paper towels and biomass was obtained on a precision scale (Bel Engineering, São Paulo, Brazil). For the measurement of length, photos of the larvae (n = 24) were used and measured individually with Image-Pro Plus software (Media Cybernetics, Silver Springs, Maryland, USA). During the experimental period, temperature (control 28.13 ± 1.15°C, and β- glucan 27.65 ± 1.12°C), pH (control 8.07 ± 0.06, and β-glucan 8.03 ± 0.15), oxygen (control 5.27 ± 0.32 mg L-1, and β-glucan 5.51 ± 0.20 mg L-1) and conductivity (control 268.50 ± 23.39 μS/cm, and β-glucan 282.75 ± 20.50 μS/cm) were monitored daily with a multiparameter probe (W23-XD, Horiba, Japan). No significant (P > 0.05) differences were observed in water parameters between treatments (temperature, P = 0.579; pH, P = 0.488; oxygen, P = 0.327; conductivity, P = 0.432). 12 2.3. Statistical analyses All data were analyzed for normality (Cramer-von Mises) and homoscedasticity (Brown-Forsythe). The larvae length and mass were analyzed using t-tests and P < 0.05 was used as the level of statistical significance in all analyses. Values in the text and figures are presented as the means ± 1 standard error (S.E.) of the mean. 2.4. Animal welfare statement The experiment was approved by the Ethics Committee on the Use of Animals (CEUA) of the School of Agricultural Sciences and Veterinary Medicine of Universidade Estadual Paulista (UNESP), Jaboticabal, SP, Brazil, under protocol number 016057/17. The approval was consistent with the ethical principles adopted by the Brazilian College of Animal Experimentation. 3. Results Larvae from embryos incubated with β-glucan significantly increased in mass (Fig. 1 A) and length (Fig. 1 B) compared with larvae from the control group (P < 0.05). Larvae that received the β-glucan treatment were ~20% heavier (10.2 mg - Control; 12.3 mg - β-glucan) and ~8.5% longer (0.82 cm - Control; 0.89 cm - β- glucan) compared to the control larvae. Both treatments had a similar embryo viability rate of ~ 90%. 13 Figure 1. Larvae mass (A, n = 30) and length (B, n = 24) from embryos incubated with β-glucan at a dose of 100 mg/L and control. Values are presented as the means ± 1 standard error (S.E.) of the mean. 4. Discussion Recently, extensive studies in the field of feeding efficiency and health have been addressed to improve fish production, but comparatively few reports have addressed the early life stages such as embryo development and larvae production. Here, we evaluated the effects of β-glucan bathing during the embryo development phase on larvae performance. The results showed that larvae incubated with β-glucan were larger and heavier than the control. These findings are relevant to the aquaculture industry and suggests a novel method for improving fish larvae production. Strand and Dalmo (1997) bathing larvae of Atlantic halibut (Hippoglossus hippoglossus) with 25 mg L-1 of radioactive and fluorescein-labelled Laminaran (source of β-glucan from brown algae) observed the presence of labelled Laminaran in the yolk sac and also in the posterior intestinal epithelial layer. This result confirmed the translocation of β-glucan through water to the yolk sac and into the intestine of the larvae. The prebiotic effect of β-glucan on fish intestinal microbiota is already well known. β-Glucans promote the improvement/diversity of beneficial intestinal microbes as lactic acid bacteria and reduce the frequency of disease/inflammation resulting from pathogenic bacteria (Kuhlwein et al., 2013; Jung-Schroers et al., 2016; Miest et al., 2016). Several studies have demonstrated the positive effect of lactic acid bacteria on the bone development of fish larvae 14 (Aubin et al., 2005; Avella et al., 2010; Lamari et al., 2013). Moreover, Miest et al. (2016) observed in turbot larvae fed with MacroGard® a higher expression of the osteocalcin gene, in which the transcription of protein is responsible for bone mineralization. As β-glucan is not considered a nutritional feed, the authors suggested that the increased expression of the osteocalcin gene is a secondary effect of changing the intestinal microbiota caused by β-glucan. Herein, we suggest that the best performance observed in larvae from embryos incubated with β-glucan may be related to the entry of this compound through the yolk sac and consequent alteration of the intestinal microbiota modulating the expression of the osteocalcin gene. Kiseleva et al. (2014) applied β-glucan bathing at a concentration of 0.5 mg m L-1 for 3 min during eggs fertilization of chum salmon (Oncorhynchus keta). The authors observed a weight gain of up to 40-55% in juveniles 3.5 months after hatching compared with the control group. They suggested that the increase in the survival rate and decrease in susceptibility to Saprolegnia infection by up to 2.5- fold led to this weight gain. Herein, β-glucan bathing was applied with a different approach and method, but even using different methods and species, the use of β- glucan bathing during embryo development or the fertilization of eggs corroborates in enhancing of larvae and juvenile performance. The design of our experiment does not allow for distinguishing if the application of β-glucan bathing during the embryo development phase or post- hatching or both were responsible for the increase in larvae performance. However, additional experiments are being designed to specify this as well the mechanisms behind the finding, the minimum dose required to obtain significant results and the further development of the larvae. Although β-glucan is a product with a relatively high cost, the low concentration used and low water volume required during fish larvae production demonstrates an economic viability due to the significant positive results on larvae weight and size. Moreover, we highlight the innovative application for β-glucan and the novel method to improve larvae culture. Finally, it is important to emphasize that β-glucan is a natural compound with an environment-friendly approach in modern aquaculture. 15 Acknowledgments The authors would like to thank Dra. Teresa Cristina R. D. Koberstein and the technician Luiz Fernando de Souza for the technical support and management of the fish breeding, Biorigin for the MacroGard® donation, intellectual and financial support. This research was part of the Joint Research Projects BioBased Economy NWO-FAPESP Programme (Project number 729.004.002) funded by the Netherlands Organization for Scientific Research (NWO) and São Paulo Research Foundation, Brazil (FAPESP, grant 2013/50418-1). 5. References Aubin, J., Gatesoupe, F.J., Labbe, L., Lebrun, L., 2005. Trial of probiotics to prevent the vertebral column compression syndrome in rainbow trout (Oncorhynchus mykiss Walbaum). Aquac. Res. 36, 758-767. Avella, M.A., Olivotto, I., Silvi, S., Place, A.R., Carnevali, O., 2010. Effect of dietary probiotics on clownfish: A molecular approach to define how lactic acid bacteria modulate development in a marine fish. Am. J. Physiol. 298, R359-R371. Das, B.K., Debnath, C., Patnaik, P., Swain, D.K., Kumar, K., Misrhra, B.K., 2009. Effect of beta-glucan on immunity and survival of early stage of Anabas testudineus (Bloch). Fish Shellfish Immun. 27, 678-683. Jiang, C., Wang, P., Li, M., Liu, S., Zhang, S., 2016. Dietary beta-glucan enhances the contents of complement component 3 and factor B in eggs of zebrafish. Dev. Comp. Immunol. 65, 107-113. Jung-Schroers, V., Adamek, M., Jung, A., Harris, S., Dóza, Ö.S., Baumer, A., Steinhagen, D., 2016. Feeding of β-1,3/1,6-glucan increases the diversity of the intestinal microflora of carp (Cyprinus carpio). Aquac. Nutr. 22, 1026-1039. Kiseleva, M., Balabanova, L., Elyakova, L., Rasskazov, V., Zvyagintseva, T., 2014. Effect of treatment of chum salmon Oncorhynchus keta (Walbaum) eggs with 16 1,3;1,6-beta-D-glucans on their development and susceptibility to Saprolegnia infection. J. Fish Dis. 37, 3-10. Kuhlwein, H., Merrifield, D.L., Rawling, M.D., Foey, A.D., Davies, S.J., 2014. Effects of dietary beta-(1,3)(1,6)-D-glucan supplementation on growth performance, intestinal morphology and haemato-immunological profile of mirror carp (Cyprinus carpio L.). J. Anim. Physiol. An. N. 98, 279-289. Kuhlwein, H., Emery, M.J., Rawling, M.D., Harper, G.M., Merrifield, D.L., Davies, S.J., 2013. Effects of a dietary beta-(1,3)(1,6)-D-glucan supplementation on intestinal microbial communities and intestinal ultrastructure of mirror carp (Cyprinus carpio L.). J. Appl. Microbiol. 115, 1091-1106. Lamari, F., Castex, M., Larcher, T., Ledevin, M., Mazurais, D., Bakhrouf, A., Gatesoupe, F.-J., 2013. Comparison of the effects of the dietary addition of two lactic acid bacteria on the development and conformation of sea bass larvae, Dicentrarchus labrax, and the influence on associated microbiota. Aquaculture. 376-379, 137-145. Meena, D.K., Das, P., Kumar, S., Mandal, S.C., Prusty, A.K., Singh, S.K., Akhtar, M.S., Behera, B.K., Kumar, K., Pal, A.K., Mukherjee, S.C., 2013. Beta-glucan: an ideal immunostimulant in aquaculture (a review). Fish Physiol. Biochem. 39, 431- 457. Miest, J.J., Arndt, C., Adamek, M., Steinhagen, D., Reusch, T.B., 2016. Dietary beta-glucan (MacroGard®) enhances survival of first feeding turbot (Scophthalmus maximus) larvae by altering immunity, metabolism and microbiota. Fish Shellfish Immun. 48, 94-104. Misra, C.K., Das, B.K., Mukherjee, S.C., Pattnaik, P., 2006. Effect of long term administration of dietary beta-glucan on immunity, growth and survival of Labeo rohita fingerlings. Aquaculture. 255, 82-94. 17 Petit, J., Wiegertjes, G.F., 2016. Long-lived effects of administering β-glucans: Indications for trained immunity in fish. Dev. Comp. Immunol. 64, 93–102. Pilarski, F., Ferreira de Oliveira, C.A., Darpossolo de Souza, F.P.B., Zanuzzo, F.S., 2017. Different beta-glucans improve the growth performance and bacterial resistance in Nile tilapia. Fish Shellfish Immun. 70, 25-29. Przybylska-Diaz, D.A., Schmidt, J.G., Vera-Jiménez, N.I., Steinhagen, D., Nielsen, M.E., 2013. β-glucan enriched bath directly stimulates the wound healing process in common carp (Cyprinus carpio L.). Fish Shellfish Immun. 35, 998–1006. Sang, H.M., Fotedar, R., 2010. Effects of dietary beta-1,3-glucan on the growth, survival, physiological and immune response of marron, Cherax tenuimanus (smith, 1912). Fish Shellfish Immun. 28, 957-960. Skjermo, J., Storseth, T.R., Hansen, K., Handa, A., Oie, G., 2006. Evaluation of beta-(1 -> 3, 1 -> 6)-glucans and high-M alginate used as immunostimulatory dietary supplement during first feeding and weaning of Atlantic cod (Gadus morhua L.). Aquaculture. 261, 1088-1101. Strand, H.K., Dalmo, R.A., 1997. Absorption of immunomodulating β(1,3)-glucan in yolk sac larvae of Atlantic halibut, Hippoglossus hippoglossus (L.). J. Fish Dis. 20, 41-49. 18 MANUSCRITO II β-Glucan isolated from alcohol yeast waste enhances the leukocyte respiratory burst of pacu (Piaractus mesopotamicus) after Aeromonas hydrophila inoculation Raphael Barbetta de Jesus a, Fábio Sabbadin Zanuzzo a, Jules Petit b, João Fernando Albers Koch d, Carlos Alberto Ferreira de Oliveira d, Elisabeth Criscuolo Urbinati a, Geert Frits Wiegertjes b,c and Fabiana Pilarski a* a São Paulo State University (UNESP), Aquaculture Center of Unesp (CAUNESP). Via de Acesso Prof. Paulo Donato Castelane, 14.884-900, Jaboticabal, SP, Brazil b Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH Wageningen, The Netherlands c Aquaculture and Fisheries Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH Wageningen, The Netherlands d Department of Research and Development, Biorigin Company, Fazenda São José s/n, 17290-000 Macatuba, São Paulo, Brazil *corresponding author: Fabiana Pilarski E-mail address: fabiana.pilarski@unesp.br Current address: Via de Acesso Prof. Paulo Donato Castellane, s/n, 14884-900 Jaboticabal, São Paulo, Brazil. Paper will be submitted to Aquaculture 19 Abstract The most common sources of β-glucans are derived from the cell wall of yeast Saccharomyces cerevisiae baker yeast, and they have been widely used as feed additive to improve immunological status in fish farming. However, the cost of baker yeast-derived immunostimulants is quite high. Exploring more affordable alternatives to produce β-glucans is desirable. An alternative for the production at reduced cost would be the use of residual yeasts used in the production of alcohol and brewer. Another promising subject to considered in this context is the technological process, cell wall of Saccharomyces cerevisiae has different polysaccharides, and then the process of purification can result in different glucans functionalities. Taking together the potential benefits of these two concepts and given the potential benefits of β-glucan in aquaculture. Thus, the purpose of this study was to evaluate whether feeding fish with β-glucans of different levels of purification would affect their immune and stress response to heat killed Aeromonas hydrophila inoculation. The fish were fed with three experimental diets for 21 days, one control diet, and two diets to which 1 g kg-1 of different β-glucan products were added. On the 22nd day, the fish had the blood drawn by caudal puncture prior and 6 and 24 hours post inoculation with Aeromonas hydrophila. We measured glucose and cortisol plasma levels, leukocyte respiratory burst, serum lysozyme concentration and hemolytic activity of alternative pathway of the complement system. Our study indicates that β-glucans obtained from residual yeasts of the production of alcohol is effective as immunostimulant and can be an alternative to reduce cost of fish production, besides to be aligned with the new demands of the market for the development of ecologically correct products. Keywords Fish, immunostimulant, innate immunity, prebiotics, industry waste 20 1. Introduction The most common sources of β-glucans are derived from the cell wall of yeast Saccharomyces cerevisiae baker yeast, and they have been widely used as feed additive to improve immunological status and control diseases in fish farming (Miest et al., 2016; Petit and Wiegertjes, 2016; Pilarski et al., 2017; Dawood et al., 2017). In vivo administration of β-glucan showed increased protection against parasitic (Bridle et al., 2005; Guselle et al., 2007), bacterial (Yoo et al., 2007; Gopalakannan and Arul, 2010, Sirimanapong et al., 2015b) and viral pathogens (Lapatra et al., 1998; Sealey et al., 2007). In addition to the immunostimulant effect, β-glucan has shown a prebiotic effect, acting as a modulator of the intestinal microbiota of fish (Jung-Schroers et al., 2016; Miest et al., 2016; Petit and Wiegertjes, 2016). β-Glucans contain pathogen-associated molecular patterns (PAMPs) and may be recognized by the host's cell pattern recognition receptors (PRR) eliciting an inflammatory cascade that leads to the enhancement of innate immune responses (Volman et al., 2008; Dalmo and Bøgwald, 2008). Glucans may activate leukocytes, by stimulating their phagocytic, cytotoxic, and antimicrobial activities, and the production of reactive oxygen species (ROS) (Paredes et al., 2013; Pilarski et al., 2017). In addition, β-glucans increase the production of lytic proteins such as lysozyme (Ai et al., 2007; Gopalakannan and Arul, 2009; Chang et al., 2013) and complement system proteins (Chang et al., 2013; Pionnier et al., 2014; Franco Montoya et al., 2018). However, the cost of baker yeast-derived immunostimulants is quite high (De Oliva-Neto et al., 2016) and is still a limiting factor to the wide dissemination and exploitation of their benefits for the production of fish species with lower market or farming costs. Exploring more affordable alternatives to produce β- glucans is desirable. An alternative for the production at reduced cost would be the use of residual yeasts used in the production of alcohol and brewer. When fermentation ceases, in both cases, yeast cells are separated by centrifugation, resulting in a concentrated yeast cell suspension with 60 to 70% (wet weight basis/v) of cells (Basso et al., 2011). The use of residual yeasts, besides allowing the reduction of the cost of production and as a consequence a cheaper final product, is aligned with the new demands of the market for the development of 21 ecologically correct products. Once glucan amounts an average of 30% of yeast cell wall, the possibility of exploitation of residual yeast extract to animal nutrition should be carefully considered in order to prevent another kind of residual material. Another promising subject to considered in this context is the technological process. Cell wall of Saccharomyces cerevisiae has different polysaccharides as glucan, mannans and chitin, as well as different combinations between them with themselves and with other compounds, like proteins, forming mainly chitin-glucan, mannan protein and glucomannan (Orlean, 2012). Then, the process of purification can result in different glucans functionalities. Taking together the potential benefits of these two concepts (residual raw material and process) and given the potential benefits of β-glucan in aquaculture, the Research Center of Biorigin developed two waste β-glucans with different purities. Thus, the purpose of this study was to evaluate whether feeding fish with β-glucans of different levels of purification would affect their immune and stress response to heat killed Aeromonas hydrophila inoculation. 2. Material and methods The protocol used in this experiment is in accordance with the Brazilian College of Animal Experimentation guidelines and was approved by the Ethics Committee on the use of animals (CEUA) of the FCAV – UNESP – Jaboticabal Campus (protocol number 11336/15). 2.1 Experimental animals A total of 162 pacu (163.0 ± 23.07 g, 16.0 ± 0.77 cm) were distributed in 9 tanks (450 L, 18 fish per tank) with aeration and open water flow. During the experimental period, the water temperature (30.3 ± 0.2 °C), pH (7.5 ± 0.06), dissolved oxygen (6.06 ± 0.90 mg L-1), conductivity (0.180 ± 0.00 mS cm-1), total solids (0.117 ± 0.0 g L-1) and salinity (0.01 ± 0.0%) were monitored. No differences (P > 0.05) were observed between fish weight and length, and water parameters among the 9 tanks. The light-dark cycle was fixed at 12h00 light and 12h00 dark. 22 2.2 Experimental design The experimental design was completely randomized with three treatments and three replicates (tanks). After 20 days of acclimation, the fish were fed with three experimental diets for 21 days: one control diet (no β-glucan), and two diets to which 1 g kg-1 (0.1%) of different β-glucan products (see diet preparation below; BG01 and BG02) were added. On the 22nd day, the fish had the blood drawn by caudal puncture prior (n = 6) and 6 and 24 hours post inoculation (hpi) with Aeromonas hydrophila (n = 12). We measured glucose and cortisol plasma levels, leukocyte respiratory burst, serum lysozyme concentration and hemolytic activity of alternative pathway of the complement system. 2.3 Experimental diet Two β-glucans isolated from yeast (Saccharomyces cerevisiae) from the alcohol-producing industry were used in this experiment: BG01 (glucan concentration = 69%, batch number = Q515244) and BG02 (glucan concentration = 56.4%, batch number = BT13_260), not yet commercially available, both developed by Biorigin (SP, Brazil). Three diets were formulated, two with β- glucans and a control diet free of β-glucan. The proportions of the ingredients and the chemical analysis of the diets are presented in Table 1. Additional information about the β-glucan samples are in supplementary Table 1 (Appendix). The diets were extruded in a single-screw extruder (Mex-250, Manzoni Industry Ltda, Brazil) at 136°C and the extruded speed regulated for pellet size of 6.71 ± 0.24 mm in diameter and 7.76 ± 0.37 mm in length. The diets were analyzed to determine dry matter, mineral matter and ether extract according to AOAC (2016). Nitrogen was determined using an LECO FP-528 nitrogen analyzer (LECO Corp., St. Joseph, MI, USA). The feed was offered twice a day (9:00 a.m. and 5:00 p.m.), totaling 3.0% of the live weight. No differences (P > 0.05) were observed between chemical composition among the 3 experimental diets. 23 Table 1. Ingredients and nutrient composition of the experimental diets. Control BG01 BG02 Ingredients (g/kg DM a) Fish meal 42.6 42.6 42.6 Poultry viscera meal 74.6 74.6 74.6 Soybean bran 312.0 312.0 312.0 Corn 283.1 283.1 283.1 Wheat bran 190.0 190.0 190.0 Broken rice 50.0 50.0 50.0 Soybean oil 13.0 13.0 13.0 Antioxidant (BHT) 0.2 0.2 0.2 Dicalcium phosphate 10.0 10.0 10.0 Calcitic limestone 10.2 10.2 10.2 Vitamin-mineral supplement b 1.9 1.9 1.9 Choline choride 1.3 1.3 1.3 Kaolin 11.1 10.1 10.1 β-glucan 0.0 1.0 1.0 Chemical composition Dry matter [g/kg DM] 938.9 947.9 941.4 Crude protein [g/kg DM] 286.6 278.7 277.1 Ether extract [g/kg DM] 31.1 30.0 33.1 Crude fiber [g/kg DM] 78.6 87.1 82.6 Mineral matter [g/kg DM] 91.0 102.4 99.6 Nitrogen free extract [g/kg DM] 451.7 449.8 449.0 Crude energy [MJ/kg DM] 17.3 17.2 17.2 a Dry matter (DM) b Mineral mix (Premix Nutrifish Guabi ® , Campinas, SP, Brazil) per kg of product: folic acid (1,250 mg); calcium pantothenate (12,000 mg); cupper (125 mg); iron (15,000 mg); iodine (375 mg;) manganese (12,500 mg); selenium (87.5 mg); zinc (12,500 mg); cobalt (125 mg); vitamin A (2,500,000 IU); vitamin B12 (4,000 mg); vitamin B1 (4,000 mg); vitamin B2 (4,000 mg); vitamin B6 (4,000 mg); vitamin C (50,000 mg); vitamin D3 (600,000 IU); vitamin E (37,500); vitamin K3 (3,750 mg); niacin (22,500 mg); biotin (15 mg). 2.4 Sampling Fish were euthanized by deepening the anesthetic plane with benzocaine (0.1 g L-1) before blood collection. For cortisol, lysozyme and complement analyzes the whole blood was allowed to clot at room temperature for 3 h, centrifuged for 10 min at 3,000 × g and the obtained serum was stored at -80°C. Whole blood was immediately used to measure glucose concentration and heparinized blood used to measure the respiratory activity of leukocytes. 24 2.5 Cortisol levels and plasma glucose Cortisol levels were measured by spectrophotometry using the DRG® Cortisol ELISA kit (EIA-1887; DRG International, Inc USA, http://www.drginternational.com) and concentration of glucose was measured by amperometric system using the G-TECH Free lite (Accumed-Glicomed, RJ, Brazil, https://www.accumed.com.br). 2.6 Leukocyte respiratory burst (LRB) The production of reactive oxygen species was measured using NBT (nitrotetrazolium blue chloride, Sigma-Aldrich, São Paulo, Brazil; #N6876), following protocol (Anderson and Siwicki, 1995), modified (Biller-Takahashi et al., 2013). An aliquot of 100 μL of heparinized blood was incubated with an equal volume of NBT buffer (0.2%) at room temperature for 30 min. Thereafter, 1 mL of dimethylformamide (DMF, Sigma-Aldrich, São Paulo, Brazil; #227056) was added to the samples, that were read in a spectrophotometer (Model Genesys 10S, Thermo Scientific Inc., Madison, WI, USA) at 540 nm, at room temperature. 2.7 Serum lysozyme concentration (SLC) Serum lysozyme concentration was determined according to Demers and Bayne (1997) with modifications by Zanuzzo et al. (2015). Briefly, standard solutions of hen egg white lysozyme (Sigma-Aldrich, São Paulo, SP, Brazil; #L6876) and serum samples were placed into a 96-well plate in triplicate with a suspension of Micrococcus lysodeikticus (Sigma-Aldrich, São Paulo, Brazil; #M3770). After mixing, absorbance was measured at 450 nm over 10 min using a microplate reader (Model Multiskan Ascent, Thermo Fisher Scientific Inc., Madison, WI, USA) at room temperature. The rate of decrease in absorbance for each sample was then compared to that obtained with the standard curve (ng µL- 1). 25 2.8 Hemolytic activity of the alternative pathway of the complement system (ACH50) Serum hemolytic activity of the complement (alternative pathway) was measured according to Zanuzzo et al. (2017) with modifications for pacu blood. Initially, a sample of rabbit blood was collected and the erythrocytes (RaRBCs) were washed and isolated. To optimize the assay, a series of dilutions (1:2, 1:3 and 1:4 in a final volume of 200 μL) was made by mixing a pool of aliquots of all serum samples with TEA-EGTA-Mg2+ buffer (triethanolamine ethylene glycol tetraacetic acid; 8 mM, with 2 mM of Mg2+ and 0.1% gelatin, pH 7.4) and the RaRBC suspension, and measuring absorbance at 700 nm using a spectrophotometer (Model Genesys 10S, Thermo Scientific Inc., Madison, WI, USA). Based on this preliminary work, the assay was carried out with a 1:2 dilution at 25°C. Hemolytic complement activity (ACH50) of each sample was measured as the time (seconds) required for the initial optical density to be reduced by one-half (50% of RaRBC hemolysis by the alternative pathway). 2.9 Preparation of A. hydrophila and inoculation A. hydrophila strains isolated from Nile tilapia Oreochromis niloticus (strain T3R), and identified by partial sequencing of the 16S rRNA gene (GenBank accession no. MH591949.1), were grown in TBS medium (Tripic Soy Broth, Sigma-Aldrich, MO, USA) and incubated at 28°C for 24 h, after this time the medium was centrifuged at 4°C for 10 min at 3.000 × g, the bacterial cells were washed twice with PBS buffer (0.01 M) under the same conditions and re- suspended in the same buffer. The fish were immunologically challenged by intraperitoneal inoculation. Their mass was recorded and they were given 10 μL g-1 of intraperitoneal injection of A. hydrophila inactivated in a water bath at 40°C for 30 min (Bozzo et al., 2007) at the concentration of 7 × 107 CFU mL-1. Inactivation of bacteria was performed to stimulate the immune system of fish, and to avoid mortality. 26 2.10 Statistical analysis Values were identified as outliers if they were >3 or <-3 of the studentized x predicted y, and only 7 values were removed from the analyses. Further, some of the data were transformed prior to statistical analysis as they failed normality (Cramer Von Mises) and/or homoscedasticity tests (Brown–Forsythe). Further, the experiment was analyzed as a two-way ANOVA [3 (treatments) × 3 (initial, 6 and 24 hpi)], followed by Duncan's new multiple range test post-hoc tests. P < 0.05 was used as the level of statistical significance in all analyses. Values in the figures are means ± 1 standard error (S.E.) of the mean. 3. Results No fish mortality was observed during the experimental period and fish from all treatments were fed adequately, ingesting 3.0% of the daily live weight. There were neither treatment effect (P = 0.69; F = 0.36, Fig. 1A) nor interaction between treatment and sampling points for cortisol levels (P = 0.17; F = 1.63, Fig. 1A). We only observed elevation in plasma cortisol levels (P < 0.05, Fig. 1A) at 24 hpi. Similar to cortisol, there were neither treatment effect (P = 0.90; F = 0.23, Fig. 1B) nor interaction between treatment and sampling points for glucose levels. 27 Figure 1. Plasma cortisol (A) and glucose (B) levels in pacu after 21 days feeding a control diet and diets supplemented with 0.1 g kg-1 BG01 (69% concentration) or BG02 (56.4% concentration), before (initial, N = 6) and 6 and 24 hours post inoculation (hpi, N = 12) with Aeromonas hydrophila. Different capital letters indicate differences between sampling points (P < 0.05). Regarding LRB values, we observed interaction between treatment and sampling points (P = 0.02; F = 3.24, Fig. 2A). BG02 values increased 6 hpi, and differed positively from control and BG01, up to 24 hpi. There was difference (P < 0.05, 24 hpi) between sampling points; LRB increased in relation to the initial and 6 hpi samplings. No interaction between treatment and sampling points was observed for SLC (P = 0.68; F = 0.57, Fig. 2B), and no difference was seen between treatments 28 (P = 0.97; F < 0.0001, Fig. 2B). However, at 6 hpi (independent on the treatment), fish had higher values of SLC (P < 0.05, Fig. 2B) compared to the initial point, which reduced at 24 hpi, returning close to initial values. Regarding the ACH50, we did not observe neither interaction between treatment and samplings (P = 0.36; F = 1.10, Fig. 2C) nor effect of treatments (P = 0.62; F = 0.03, Fig. 2C), however ACH50 improved (P < 0.05) at 24 hpi compared to initial sampling. Figure 2. Leukocyte respiratory burst (A), serum lysozyme concentration (B) and hemolytic activity of the alternative pathway of the complement system (ACH50) in pacu after 21 days feeding a control diet and diets supplemented with 0.1 g kg-1 29 BG01 (69% concentration) or BG02 (56.4% concentration), before (initial, N = 6) and 6 and 24 hours post inoculation (hpi, N = 12) with Aeromonas hydrophila. Different capital letters indicate differences between diets at a given time point, whereas different lower case letters indicate a difference between sampling points within a group (P < 0.05). Different capital letters indicate differences among sampling points diets (P < 0.05) for serum lysozyme concentration and ACH50 since that no significant (P < 0.05) effect was observed for treatments. Values are means + 1 standard error (S.E.). 4. Discussion Many studies have demonstrated the positive effects of dietary β-1.3/1.6- glucan on fish, but few studies have compared and exploited different molecules and their activity (Vetvicka and Vetvickova, 2014; Pilarski et al., 2017; Franco Montoya et al., 2018). In our study we tested two β-glucan molecules with different degrees of concentration, BG01 and BG02, and, in general, both showed similar activities, except by the BG02 (less concentrated) which improved the LRB, 6 hpi, in comparison to BG01 and the control treatment. In addition, we detected an activation of the innate immune system by the A. hydrophila inoculation. The inoculum increased cortisol levels 24 hpi, when LBR and ACH50 were activated. This activation was reflected in the higher levels of blood glucose observed at this sampling point. The lysozyme concentration increased earlier, 6 hpi. During the phagocytosis of pathogens, leukocytes increase oxygen uptake through NADPH oxidase and generate several reactive oxygen species (ROS), such as superoxide radical (O2), hydrogen peroxide (H2O2), singlet oxygen (1O2) and the hydroxyl radical (OH-) in a process called leukocyte respiratory burst. Superoxide and hydrogen peroxide are very toxic ROS and form a potent antibacterial system (Klebanoff, 1999). Several studies have shown that the dietary administration of 1.3/1.6 β-glucan stimulated the LRB in Oreochromis niloticus (Pilarski et al., 2017), Pangasianodon hypophthalmus (Sirimanapong et al., 2015a), Lutjanus guttatus (Del Rio -Zaragoza, Fajer-Ávila and Almazán- Rueda, 2011), Acipenser persicus (Aramli et al., 2015). In our study, BG02 had a positive effect on LRB (6 hpi). 30 One fact to be considered is that BG02, which presented less concentrated, presented 5.09 times more mannans than BG01 (Supplementary Table 1), what can have contributed for the positive effect on LRB. Mannan oligosaccharide (MOS) is a glucomannoprotein complex derived from the cell walls of yeast (Saccharomyces cerevisiae) (Sang and Fotedar, 2010). The immunostimulant effect of MOS in fish is already described (Carbone and Faggio, 2016). Some studies have demonstrated the effect of MOS on parameters such as macrophage phagocytic activity (Torrecillas et al., 2011), leukocyte counts (Ebrahimi et al., 2012), phagocytic activity of head kidney leukocytes (Torrecillas et al., 2007; Torrecillas et al., 2011), all related to the LRB, what reinforces the result of BG02 that we found in pacu. The effect of MOS as immune modulator is probably based in the activation of pattern recognition receptors (PRR) and proteins (PRP), as the mannose receptor (MR) that is an endocytic receptor expressed by macrophages and by endothelial cells that recognize both self-glycoproteins and microbial glycan ligands, triggering the innate immune system in response to a non-self- substance (Torrecillas et al., 2014; Carbone and Faggio, 2016). Serum lysozyme is an important molecule of the innate immune system in fish (Saurabh and Sahoo, 2008). It hydrolyzes chemical bonds between N- acetylmuramic acid and N-acetylglucosamine present in the peptidoglycan of bacterial cell walls. It is capable of cleaving some Gram-positive bacteria and, along with the complement system, even some Gram-negative bacteria (Paulsen et al., 2001; Gómez and Balcázar, 2008). The SLC was not affected by the β- glucan treatments, but there was a significant increase in SLC (6 hpi). Similarly, in pacu fed with 0.1% β-glucan for 7 days, Biller-Takahashi et al. (2014) did not observe difference in SLC levels, but only after experimental infection with A. hydrophila. Absence of effect of β-glucan (0.0, 0.2, 0.6 and 1.8%) was also described by Kunttu et al. (2009) after feeding rainbow trout (Oncorhynchus mykiss) during 21 days. Although some studies have reported elevation in SLC by effect of β-glucan (Sirimanapong et al., 2015a; Aramli et al., 2015; Franco Montoya et al., 2017), modulation of lysozyme by fungi or components of fungal structures may be limited. Comparing, in vivo, SLC between A. hydrophila infection (Das et al. 2009) and the Saprolegnia parasitica fungus (Das et al., 2013) in Anabas testudineus, the authors suggested that lysosomal enzymes produced 31 by the leukocyte cell suspension may be spared to counteract the fungal challenge (Das et al., 2013). The complement system comprises more than 35 soluble plasma proteins that are critical for the innate and adaptive immunity of fish. Activation of the complement system initiates a cascade of biochemical reactions followed by the generation of biologically active mediators that act against the antigens via lysis of its cellular membrane and on the activation of nonspecific mediators of the inflammation. Additionally, the complement system has an important role between innate and adaptive immune responses (Holland and Lambris, 2002; Gómez and Balcázar, 2008). Although BG01 and BG02 did not activate ACH50, the inoculation with A. hydrophila did it 24 hpi. Franco Montoya et al. (2017) fed matrinxã (Brycon amazonicus) with β-glucan (0.1%) during 15 days and did not observe changes in ACH50 compared to control group before the fish exposure to A. hydrophila. Similarly, Biller-Takahashi et al. (2012) did not observe modulatory effect of β- glucan (0.0%, 0.1% and 1.0%) on ACH50 in pacu fed for 7 days. Using the same experimental protocol and same fish, Biller-Takahashi et al. (2014) found activation of the ACH50 after experimental infection with A. hydrophila but not by effect of β-glucan. Different from our study, other authors related increase in ACH50 caused by β-glucan (Chang et al., 2013; Pionnier et al., 2014; Domenico et al., 2017; Lee et al., 2018). However, the lack of responsiveness could be related to a number of factors. Although we cannot state, some factors must be taken into account to explain the lack of β-glucan effect in our experiment. The β-glucan concentration or time of administration may have been insufficient to activate the receptors that trigger the mechanism of the complement system. Regarding the molecules tested, it is remarkable the difference between the mean size of BG01 (35.20 μm) and BG02 (23.40 μm) (Supplementary Table 1). However, particles with size larger than 50 μm present a difference of only 16.6% of BG01 in relation to BG02. Small particles (> 10 μm and > 20 μm) present the largest difference between molecules (33.9% and 41.8% respectively), thus increasing the mean, so in fact the percentage of larger particles between β- glucans is not so high. The size or molecular weight of β-glucans has been considered fundamental to their activity (Akramiene et al., 2007), however, particle size is not necessarily directly related to its activity, so generalization of this nature 32 is risky (Chen and Seviour, 2007). Hong et al. (2010) analyzed the degradation of fluorescence-labeled yeast glucan particles in mice macrophages and showed that macrophages were able to partially degrade the large molecules of β-glucan into biologically active smaller fragments that were taken up by granulocytes via the CR3 receptor. Complete macrophage degradation required 13 days. Typically ingested particles remained intact for 3 - 5 days, appeared to fragment into smaller particles and soluble material (5 - 10 days), and the intracellular fluorescence disappeared after 14 - 21 days. Although we did not observe effects of both β-glucans molecules on stress indicators, they affected differently the innate immunity of pacu. Similarly, other studies have shown that different processes to obtain β-glucans may affect immune fish responses. Pilarski et al. (2017) observed that different β-glucans showed distinct effects on Oreochromis niloticus, one of the molecules affected the fish immune response and the other the growth performance. Similarly, Franco Montoya et al. (2018) tested two distinct molecules of β-glucan and showed that one of them was more efficient on the induction of the cell-mediate immunity in matrinxã (Brycon amazonicus). Although most studies use these periods of β-glucan administration, there is no consensus on the optimal period. We should take into consideration that this period may vary depending on the structure of the β-glucan molecule, concentration, particle size, species of fish, among other factors. Our study indicates that β-glucan obtained from residual yeasts of the production of alcohol (BG02) is effective as immunostimulant and can be an alternative to reduce cost of fish production, besides to be aligned with the new demands of the market for the development of ecologically correct products. However further studies with different protocols using these molecules are needed to confirm their effectiveness. Acknowledgments The authors would like to thank the Aquaculture Center of Unesp (CAUNESP) for the fish supply, the Biorigin for the β-glucans donation and intellectual support, and would also like to thank Joint Research Project Biobased Economy: São Paulo Research Foundation (FAPESP, grant 2013/50418-1) and 33 Netherlands Organization for Scientific Research (NWO, number 729.004.002). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. 34 Appendix Supplementary Table 1. Compositions of BG01 and BG02 included in the diets. Parameters BG01 BG02 Reference pH (sol. 2%) 6.29 3.01 (1) AOAC 981.12 Moisture (%) 6.66 8.68 (1) AOAC 934.01 Protein (%) 5.99 13.60 (1) AOAC 990.03 Fat (%) 3.53 4.19 (1) AOAC 922.06 Ash (%) 2.40 0.50 (1) AOAC 942.05 Glucan (%) 69.00 56.40 (2) Mannan (%) 1.10 5.60 (2) Others carbohydrates (%) 11.32 11.03 (3) Ca (%) 0.11 0.04 P (%) 0.15 0.24 K (%) 0.16 0.04 Na (%) 0.93 0.03 Mg (%) 0.09 0.01 Cu (mg/kg) 0.93 3.82 Fe (mg/kg) 143.66 77.93 Mn (mg/kg) 6.99 < 0.50 Zn (mg/kg) 548.14 56.20 Co (mg/kg) ˂ 0.50 ˂ 0.50 (4) Mo (mg/kg) ˂ 0.50 ˂ 0.50 Ni (mg/kg) 0.62 ˂ 0.50 Pb (mg/kg) ˂ 0.50 ˂ 0.50 Cr (mg/kg) 2.20 ˂ 0.50 Ba (mg/kg) 3.90 1.22 Al (mg/kg) 125.06 7.03 Cd (mg/kg) ˂ 0.50 ˂ 0.50 S (%) 0.06 0.08 Particle size – Mean (µm) 35.20 23.40 Particle size ˃ 10 µm (%) 91.60 60.50 Particle size ˃ 20 µm (%) 72.10 41.90 Particle size ˃ 50 µm (%) 18.60 15.50 Particle size ˃ 100 µm (%) 3.80 00.00 Particle size ˃ 200 µm (%) 0.00 00.00 (5) Particle size ˃ 500 µm (%) 0.00 00.00 Particle size ˃ 1000 µm (%) 0.00 00.00 Particle size < 150 µm (%) 99.80 100.00 [1] A.O.A.C, Official Methods of Analysis, 14a. 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Prof. Dr. Antônio Celso Wagner Zanin, 18618-689, Botucatu, SP, Brasil. *autor correspondente: Fabiana Pilarski Endereço de e-mail: fabiana.pilarski@unesp.br Endereço atual: Centro de Aquicultura da Unesp, UNESP - Univ Estadual Paulista. Via de Acesso Prof. Paulo Donato Castelane, 14.884-900, Jaboticabal, SP, Brasil 45 Resumo Poucos são os estudos que enfatizam a compreensão da microbiota intestinal em peixes. A diversidade de habitats e a variação da natureza das dietas faz com que a simbiose entre peixes e microrganismos intestinais seja diversa. Os processos fisiológicos dos animais, incluindo o metabolismo e seu sistema imunológico, são intensamente influenciados pela microbiota. Estudos recentes tem relatado que imunoestimulantes são capazes de modular a microbiota intestinal dos peixes. Dentre os imunoestimulantes mais utilizados e estudados na produção animal está o β-glucano, que possuí estrutura química muito semelhante aos prebióticos. Assim, este trabalho teve como objetivo avaliar a composição intestinal bacteriana de pacus alimentados com dois diferentes β-glucanos residuais provenientes da fermentação alcoólica. Para avaliar a diversidade bacteriana intestinal, foram utilizados seis pacus por tratamento alimentados por 21 dias com 1 g kg-1 de β- glucano. Para a colheita do intestino, os peixes foram anestesiados, lavados em água corrente e pulverizados com álcool. Após a higienização, procedeu-se a necropsia e o intestino foi totalmente retirado. Os intestinos foram suspensos em tampão PBS contendo Tween 80, metanol e terc-butanol, acrescidos com 20 microesferas de vidro e vortexadas por 1 minuto. As suspensões foram centrifugadas e os sobrenadantes recuperados, transferidos para tubos estéreis, seguidos de nova centrifugação. O material precipitado foi utilizado para extração de DNA metagenômico. Em seguida, foi realizada a PCR (16S rRNA) e em sequência procedeu-se a construção da biblioteca e sequenciamento utilizando- se a plataforma Illumina MiSeq. Não foram observadas grandes diferenças entre os tratamentos nos principais gêneros bacterianos identificados. Observou-se grande prevalência em todos os tratamentos dos gêneros Cetobacterium, Plesiomonas e Epulopiscium os quais representam aproximadamente 60% de todos os gêneros identificados na microbiota intestinal do pacu. Os outros 40% distribuem-se em gêneros de menor expressão percentual. Palavras chave: Cetobacterium, Epulopiscium, intestino, Plesiomonas, microbiota, 16S rRNA 46 1. Introdução A maior parte dos estudos de comunidades microbianas intestinais de vertebrados tem se concentrado em mamíferos, os quais compreendem menos de 10% da diversidade total dos vertebrados. Poucos são os estudos que investigam a microbiota intestinal de peixes, que se originaram há mais de 600 milhões de anos e abrangem aproximadamente 28.000 espécies existentes (Nelson, 2016), o que representa quase metade do número total de espécies de vertebrados no mundo. A diversidade de habitats e a variação da natureza das dietas faz com que a simbiose entre peixes e microrganismos intestinais seja extremamente diversa. Quantificar, qualificar e desvendar como se dão essas relações são de profunda importância para o desenvolvimento de dietas mais específicas para cada espécie de peixe. Os processos fisiológicos dos animais, incluindo o metabolismo e seu estado imunológico e até mesmo a anatomia do intestino, são profundamente influenciados pela microbiota intestinal (Rawls et al., 2004; Cerf-Bensussan e Gaboriau-Routhiau, 2010). As bactérias, componentes desta microbiota são geralmente benéficas e contribuem para processos nutricionais importantes, como na degradação de moléculas complexas, produção de algumas vitaminas (Sugita et al., 1991) e também estão envolvidas na proteção do hospedeiro contra potenciais patógenos, pela produção de compostos inibitórios e competição por espaço e nutrientes (Rawls et al., 2004). Rawls et al. (2004), utilizando Danio rerio genobióticos, demonstraram que a microbiota pode regular a expressão de 212 genes, nos quais alguns estavam relacionados à estimulação da proliferação epitelial, promoção do metabolismo de nutrientes e resposta imune inata. Em decorrência destes fatos, estudos da comunidade microbiana gastrointestinal vêm sendo realizados com várias espécies de peixes, como Salmo salar (Zarkasi et al. 2014), Cyprinus carpio (Jung-Schroers et al., 2016); Acipenser baerii (Geraylou et al., 2013); Peocilia reticulata (Sullam et al., 2015); Salmo trutta trutta (Skrodenyte- Arbaciauskiene et al., 2008); Salmo trutta fario (Skrodenyte-Arbaciauskiene et al., 2006); Oreochromis niloticus (Hu et al., 2007); Danio rerio (Russo et al., 2015), entre outros. Estudos recentes tem relatado que imunoestimulantes são capazes de modular a microbiota intestinal dos peixes (Kühlwein et al., 2013; Carda-Diéguez, 47 Mira e Fouz, 2014; Jung-Schroers et al., 2016). A inclusão de imunoestimulantes na dieta de peixes é uma das principais estratégias para alcançar uma aquicultura sustentável. Estas dietas enriquecidas são administradas durante períodos curtos, quando o risco de contrair infecções por animais aquáticos é maior (Miguel Carda- Diéguez, Alex Mira e Bélen Fouz, 2014). Dentre os imunoestimulantes mais utilizados e estudados encontra-se o β- glucano. A estrutura química do β-glucano é muito semelhante aos prebióticos, consistindo em unidades monoméricas ligadas a β-glicosídicas. Os prebióticos, definidos como ingredientes alimentares não digeríveis, afetam beneficamente o hospedeiro, estimulando seletivamente o crescimento e/ou a atividade de bactérias intestinais associadas à saúde e ao bem-estar (Gibson et al., 2004). Diversas pesquisas têm sido desenvolvidas utilizando o β-glucano como um potente imunoestimulante. Estes estudos tem demostrado que uso do β- glucano atua positivamente em diversas variáveis, como crescimento (Kühlwein et al., 2014; Pilarski et al., 2017), sobrevivência e resistência à patógenos (Das et al., 2009; Sang e Fotedar, 2010; Petit e Wiegertjes, 2016), produção de anticorpos (Kamilya et al. 2006), expressão dos genes relacionados ao sistema imune inato (Lokesh et al., 2012; Pietretti et al., 2013) e alteração da microbiota intestinal (Kühlwein et al., 2013; Jung-Schroers et al., 2016; Miest et al., 2016) em várias espécies de peixes. Porém, o custo de produção do β-glucano ainda é um fator limitante para a ampla disseminação e aproveitamento dos benefícios do uso deste imunoestimulante. Criar fontes alternativas mais acessíveis para este tipo de tecnologia é desejável para o desenvolvimento de uma piscicultura mais competitiva. Neste contexto, o Centro de pesquisa da Biorigin desenvolveu um produto, ainda não comercial, baseado na produção de 1,3/1,6 β-glucano extraído de leveduras residuais (Saccharomyces cerevisiae) provenientes de fermentação alcoólica. A utilização de leveduras residuais, além de possibilitar a redução do custo de produção, está alinhada às novas demandas do mercado para o desenvolvimento de produtos ecologicamente corretos, uma vez que utiliza material residual. Assim, este estudo teve como objetivo alimentar durante 21 dias pacus com dietas contendo dois tipos de β-glucanos com diferentes níveis de purificação 48 e processos de extração, desenvolvidos pelo Centro de pesquisa da Biorigin e avaliar a diversidade bacteriana intestinal pelo sequenciamento do gene 16S rRNA através da plataforma Illumina MiSeq, a fim de observar possíveis efeitos modulatórios dos β-glucanos sobre a microbiota intestinal. 2. Material e métodos O protocolo utilizado neste experimento está de acordo com as diretrizes do Colégio Brasileiro de Experimentação Animal (COBEA) e foi aprovado pela Comissão de Ética no Uso de Animais (CEUA) da Faculdade de Ciências Agrárias e Veterinárias - UNESP (número do protocolo 11336/15). 2.1. Animais experimentais Foram utilizados 162 pacus (163,0 ± 23,07 g, 16,0 ± 0,77 cm) distribuídos em nove tanques (450 L, 18 peixes por tanque) com aeração e fluxo contínuo de água. Durante o período experimental, a temperatura da água (30,3 ± 0,2°C), pH (7,5 ± 0,06), oxigênio dissolvido (6,06 ± 0,90 mg