Transcriptomic insight into the hybridization mechanism of the Tambacu, a hybrid from Colossoma macropomum (Tambaqui) and Piaractus mesopotamicus (Pacu).

Interspecific hybrids are highly complex organisms, especially considering aspects related to the organization of genetic material. The diversity of possibilities created by the genetic combination between different species makes it difficult to establish a large-scale analysis methodology. An example of this complexity is Tambacu, an interspecific hybrid of Colossoma macropomum (Tambaqui) and Piaractus mesopotamicus (Pacu). Either genotype represents an essential role in South American aquaculture. However, despite this importance, the genetic information for these genotypes is still highly scarce in specialized databases. Using RNA-Seq analysis, we characterized the transcriptome of white muscle from Pacu, Tambaqui, and their interspecific hybrid (Tambacu). The sequencing process allowed us to obtain a significant number of reads (approximately 53 billion short reads). A total of annotated contigs were 37,285, 96,738, and 158,709 for Pacu, Tambaqui, and Tambacu. After that, we performed a comparative analysis of the transcriptome of the three genotypes, where we evaluated the differential expression (Tambacu vs Pacu = 11,156, and Tambacu vs Tambaqui = 876) profile of the transcript and the degree of similarity between the nucleotide sequences between the genotypes. We assessed the intensity and pattern of expression across genotypes using differential expression information. Clusterization analysis showed a closer relationship between Tambaqui and Tambacu. Furthermore, digital differential expression analysis selected some target genes related to essential cellular processes to evaluate and validate the expression through the RT-qPCR. The RT-qPCR analysis demonstrated significantly (p < 0.05) elevated expression of the mafbx, foxo1a, and rgcc genes in the hybrid compared to the parents. Likewise, we can observe genes significantly more expressed in Pacu (mtco1 and mylpfa) and mtco2 in Tambaqui. Our results showed that the phenotype presented by Tambacu might be associated with changes in the gene expression profile and not necessarily with an increase in gene variability. Thus, the molecular mechanisms underlying these "hybrid effects" may be related to additive and, in some cases, dominant regulatory interactions between parental alleles that act directly on gene regulation in the hybrid transcripts.

An insight on the impact of teleost whole genome duplication on the regulation of the molecular networks controlling skeletal muscle growth.

Fish muscle growth is a complex process regulated by multiple pathways, resulting on the net accumulation of proteins and the activation of myogenic progenitor cells. Around 350-320 million years ago, teleost fish went through a specific whole genome duplication (WGD) that expanded the existent gene repertoire. Duplicated genes can be retained by different molecular mechanisms such as subfunctionalization, neofunctionalization or redundancy, each one with different functional implications. While the great majority of ohnolog genes have been identified in the teleost genomes, the effect of gene duplication in the fish physiology is still not well characterized. In the present study we studied the effect of WGD on the transcription of the duplicated components controlling muscle growth. We compared the expression of lineage-specific ohnologs related to myogenesis and protein balance in the fast-skeletal muscle of pacus (Piaractus mesopotamicus-Ostariophysi) and Nile tilapias (Oreochromis niloticus-Acanthopterygii) fasted for 4 days and refed for 3 days. We studied the expression of 20 ohnologs and found that in the great majority of cases, duplicated genes had similar expression profiles in response to fasting and refeeding, indicating that their functions during growth have been conserved during the period after the WGD. Our results suggest that redundancy might play a more important role in the retention of ohnologs of regulatory pathways than initially thought. Also, comparison to non-duplicated orthologs showed that it might not be uncommon for the duplicated genes to gain or loss new regulatory elements simultaneously. Overall, several of duplicated ohnologs have similar transcription profiles in response to pro-growth signals suggesting that evolution tends to conserve ohnolog regulation during muscle development and that in the majority of ohnologs related to muscle growth their functions might be very similar.

The combination of resveratrol and exercise enhances muscle growth characteristics in pacu (Piaractus mesopotamicus).

Pacu is a tropical fish with important value to aquaculture. During cellular metabolism, reactive oxygen species (ROS) are produced, which can influence muscle growth. Resveratrol is an effective antioxidant that scavenges ROS and can modulate physical performance preventing oxidative stress. We investigated the effects of resveratrol and exercise on pacu muscle growth characteristics. Four groups were used: fish fed with control diet /without exercise (C); fish fed with control diet/subjected to exercise (CE); fish fed resveratrol-supplemented diet/without exercise (R); and fish fed resveratrol-supplemented diet/subjected to exercise (RE). At 30 days, the RE group presented a significant increase in body weight, fewer muscle fibers in the 20-40 µm and more fibers in the >60 µm diameter class compared to the C group. At day 7, catalase activity decreased in CE and RE groups. Superoxide dismutase activity decreased only in the CE group. Myod and mtor gene expression was higher in R and RE and igf-1 was up-regulated in the RE group. Murf1a level decreased in CE, R, and RE, while sdha expression was higher in the RE group. We suggest that resveratrol in combination with exercise was beneficial for muscle growth and metabolism, increasing the expression levels of genes related to muscle anabolism and oxidative metabolism, besides the decrease of catabolic gene expression. Notably, all of these changes occurred together with muscle hypertrophy and increased body weight. Our results show a positive application for resveratrol in association with exercise as a strategy to improve the growth performance of juvenile pacus.

Proteomic analysis of the fast-twitch muscle of pacu (Piaractus mesopotamicus) after prolonged fasting and compensatory growth.

Protocols that improve growth performance in fish while assuring product quality are important for aquaculture. Fasting followed by refeeding may promote compensatory growth, thus optimizing growth performance. During fasting and refeeding, fast-twitch muscle, which comprises most of fish fillet, undergoes intense plasticity. In this work, we studied the proteome of pacu (Piaractus mesopotamicus) fast-twitch muscle after 30 days of fasting (D30), 30 days of refeeding (D60) and 60 days of refeeding (D90) with two-dimensional electrophoresis, mass spectrometry and bioinformatics. Body mass, growth rate and muscle histology were also assessed. At D30, fish presented muscle catabolism and decreased growth. Proteomic analysis showed that metabolism proteins were the most affected, up and downregulated. Cytoskeleton and amino acid biosynthesis proteins were downregulated, while nuclear and regulatory proteins were upregulated. At D60, fish showed accelerated growth, despite the body mass not completely recovering. Metabolism proteins were still the most affected. Amino acid biosynthesis proteins became upregulated, while cytoskeleton proteins remained downregulated. At D90, the fish presented total compensatory growth. Many metabolic proteins were up or downregulated. Few cytoskeleton proteins remained differentially expressed. Amino acid biosynthesis proteins were mostly upregulated, but less than at D60. Prolonged fasting followed by refeeding also led to the regulation of possible meat quality biomarkers, such as antioxidant enzymes. This fact suggests possible consequences of this protocol on fish meat quality. Our work also enriches our knowledge on proteomic changes during muscle plasticity that occur during fasting and refeeding diet protocols.

Influence of temperature and exercise on growth performance, muscle, and adipose tissue in pacus (Piaractus mesopotamicus).

The aim of this study was to evaluate the effects of temperature and swimming exercise on fish growth in pacus (Piaractus mesopotamicus). Pacus weighing 0.9 - 1.9g and 2.7 - 4.2cm in standard length were cultivated at an initial density of 120 fish m-3 in 3 recirculation systems containing 6 water tanks at a volume of 0.5m3 each at temperatures of 24, 28 and 32°C. At each temperature, three tanks were modified to generate exercise activity in the specimens and force the fish to swim under a current speed of 27.5cms-1. At the end of the experiment, the following metrics were evaluated: fish performance, morphometry (length, width, height and perimeter in different body positions), and the diameter and density of muscle and subcutaneous ventral adipose tissues. At 28°C, pacus were both heavier and had greater weight gain after 240 days of cultivation. Additionally, exercise improved the feed conversion. An increase of 4°C (30°C) did not provide any improvement in the performance of the fish. However, swimming exercise improved the performance of pacus, providing increases of 38% and a 15% improvement in feed conversion. Both temperature and exercise influenced the body morphology (especially in the caudal region) and the cellularity of white and red muscle fibers and adipocytes.

Food restriction increase the expression of mTORC1 complex genes in the skeletal muscle of juvenile pacu (Piaractus mesopotamicus).

Skeletal muscle is capable of phenotypic adaptation to environmental factors, such as nutrient availability, by altering the balance between muscle catabolism and anabolism that in turn coordinates muscle growth. Small noncoding RNAs, known as microRNAs (miRNAs), repress the expression of target mRNAs, and many studies have demonstrated that miRNAs regulate the mRNAs of catabolic and anabolic genes. We evaluated muscle morphology, gene expression of components involved in catabolism, anabolism and energetic metabolism and miRNAs expression in both the fast and slow muscle of juvenile pacu (Piaractus mesopotamicus) during food restriction and refeeding. Our analysis revealed that short periods of food restriction followed by refeeding predominantly affected fast muscle, with changes in muscle fiber diameter and miRNAs expression. There was an increase in the mRNA levels of catabolic pathways components (FBXO25, ATG12, BCL2) and energetic metabolism-related genes (PGC1α and SDHA), together with a decrease in PPARß/δ mRNA levels. Interestingly, an increase in mRNA levels of anabolic genes (PI3K and mTORC1 complex: mTOR, mLST8 and RAPTOR) was also observed during food restriction. After refeeding, muscle morphology showed similar patterns of the control group; the majority of genes were slightly up- or down-regulated in fast and slow muscle, respectively; the levels of all miRNAs increased in fast muscle and some of them decreased in slow muscle. Our findings demonstrated that a short period of food restriction in juvenile pacu had a considerable impact on fast muscle, increasing the expression of anabolic (PI3K and mTORC1 complex: mTOR, mLST8 and RAPTOR) and energetic metabolism genes. The miRNAs (miR-1, miR-206, miR-199 and miR-23a) were more expressed during refeeding and while their target genes (IGF-1, mTOR, PGC1α and MAFbx), presented a decreased expression. The alterations in mTORC1 complex observed during fasting may have influenced the rates of protein synthesis by using amino acids from protein degradation as an alternative mechanism to preserve muscle phenotype and metabolic demand maintenance.

Differential microRNA Expression in Fast- and Slow-Twitch Skeletal Muscle of Piaractus mesopotamicus during Growth.

Pacu (Piaractus mesopotamicus) is a Brazilian fish with a high economic value in pisciculture due to its rusticity and fast growth. Postnatal growth of skeletal muscle in fish occurs by hyperplasia and/or hypertrophy, processes that are dependent on the proliferation and differentiation of myoblasts. A class of small noncoding RNAs, known as microRNAs (miRNAs), represses the expression of target mRNAs, and many studies have demonstrated that miR-1, miR-133, miR-206 and miR-499 regulate different processes in skeletal muscle through the mRNA silencing of hdac4 (histone deacetylase 4), srf (serum response factor), pax7 (paired box 7) and sox6 ((sex determining region Y)-box 6), respectively. The aim of our work was to evaluate the expression of these miRNAs and their putative target mRNAs in fast- and slow-twitch skeletal muscle of pacu during growth. We used pacus in three different development stages: larval (aged 30 days), juvenile (aged 90 days and 150 days) and adult (aged 2 years). To complement our study, we also performed a pacu myoblast cell culture, which allowed us to investigate miRNA expression in the progression from myoblast proliferation to differentiation. Our results revealed an inverse correlation between the expression of the miRNAs and their target mRNAs, and there was evidence that miR-1 and miR-206 may regulate the differentiation of myoblasts, whereas miR-133 may regulate the proliferation of these cells. miR-499 was highly expressed in slow-twitch muscle, which suggests its involvement in the specification of the slow phenotype in muscle fibers. The expression of these miRNAs exhibited variations between different development stages and between distinct muscle twitch phenotypes. This work provides the first identification of miRNA expression profiles in pacu skeletal muscle and suggests an important role of these molecules in muscle growth and in the maintenance of the muscle phenotype.

Rearing temperature induces changes in muscle growth and gene expression in juvenile pacu (Piaractus mesopotamicus).

Pacu (Piaractus mesopotamicus) is a fast-growing fish that is extensively used in Brazilian aquaculture programs and shows a wide range of thermal tolerance. Because temperature is an environmental factor that influences the growth rate of fish and is directly related to muscle plasticity and growth, we hypothesized that different rearing temperatures in juvenile pacu, which exhibits intense muscle growth by hyperplasia, can potentially alter the muscle growth patterns of this species. The aim of this study was to analyze the muscle growth characteristics together with the expression of the myogenic regulatory factors MyoD and myogenin and the growth factor myostatin in juvenile pacu that were submitted to different rearing temperatures. Juvenile fish (1.5 g weight) were distributed in tanks containing water and maintained at 24°C (G24), 28 °C (G28) and 32 °C (G32) (three replicates for each group) for 60 days. At days 30 and 60, the fish were anesthetized and euthanized, and muscle samples (n=12) were collected for morphological, morphometric and gene expression analyses. At day 30, the body weight and standard length were lower for G24 than for G28 and G32. Muscle fiber frequency in the <25 µm class was significantly higher in G24, and the >50 µm class was lower in G24. MyoD gene expression was higher in G24 compared with that in G28 and G32, and myogenin and myostatin mRNA levels were higher in G24 than G28. At day 60, the body weight and the standard length were higher in G32 but lower in G24. The frequency distribution of the <25 µm diameter muscle fibers was higher in G24, and that of the >50 µm class was lower in G24. MyoD mRNA levels were higher in G24 and G32, and myogenin mRNA levels were similar between G24 and G28 and between G24 and G32 but were higher in G28 compared to G32. The myostatin mRNA levels were similar between the studied temperatures. In light of our results, we conclude that low rearing temperature altered the expression of muscle growth-related genes and induced a delay in muscle growth in juvenile pacu (P. mesopotamicus). Our study provides a clear example of thermally induced phenotypic plasticity in pacu fish and shows that changing the rearing temperature during the juvenile stage can have a considerable effect on gene expression and muscle growth in this species.

Curvas de crescimento morfométrico de piracanjuba (Brycon orbignyanus) / Morphometric growth curves of piracanjuba (Brycon orbignyanus)

Conduziu-se um experimento no setor de piscicultura da Universidade Federal de Lavras M.G., com o objetivo de estabelecer curvas de crescimento morfométrico em função do peso corporal para a piracanjuba (Brycon orbignyanus). Alevinos de piracanjuba foram cultivados em viveiros de terra e alimentados com ração comercial até atingirem peso aproximado de 1 kg. Durante o período experimental realizaram-se amostragens aleatórias, onde foram avaliadas as medidas morfométricas comprimento da cabeça (CCAB), comprimento padrão (CP), altura (AND) e largura (LND) tomada no 1ºraio da nadadeira dorsal e as razões morfométricas (CCAB/CP, AND/CP, LND/CP, AND/LND) utilizando-se as funções de Brody, von Bertalanffy, Gompertz e Logística. As funções estudadas apresentaram bom ajuste para todas as medidas morfométricas e para a razão CCAB/CP. As funções de Brody, von Bertalanffy e Gompertz apresentaram qualidades de ajuste semelhantes e superiores à função Logística para as medidas morfométricas. Para a razão CCAB/CP a função Logística apresentou-se superior às demais. A taxa de crescimento da CCAB e LND foi superior às do CP e AND, indicando um crescimento mais rápido da cabeça e largura do que do comprimento padrão e altura. Conclui-se que todos os modelos estudados apresentaram bom ajuste, sendo o de Brody e o de von Bertalanffy os que descrevem melhor o crescimento morfométrico da piracanjuba.

An experiment was carried out at the fish farming sector of the Universidade Federal de Lavras M.G., with the objective of establishing morphometric growth curves as a function of the body weight of piracanjuba (Brycon orbignyanus). Fingerlings of piracanjuba were cultivated in earthen ponds and fed a commercial diet (ad libitum) until they reached weight of about 1 kg. During the experimental period, samplings were randomly selected to take morphometric measures of the head length (CCAB), standard length (CP), height (AND), and breadth (LND), taken at the first ray of the dorsal fin. Measures and the morphometric ratios CCAB/CP, AND/CP, LND/CP, AND/LND were evaluated by Brody, von Bertalanffy, Gompertz and Logistic functions. All models showed a good fit for morphometric measures and the CCAB/CP ratio. Brody, von Bertalanffy and Gompertz functions presented similar fit qualities when studying morphometric measures while the Logistic function provided poor fit. However, the Logistic function presented better fit quality for the CCAB/CP ratio than the other functions. Growth rate of the variables CCAB and LND was higher than CP and AND, indicating a faster growth of head and breadth in relation to standard length and height. All models studied presented good fit, but the Brody and von Bertalanffy functions better described the morphometric growth of the fish.

Avaliação de curvas de crescimento morfométrico de linhagens de tilápia do nilo (Oreochromis niloticus) / Evaluation of morhometric growth curves of tilapia of Nilo (Oreochromis niloticus) strains

Com o objetivo de avaliar curvas de crescimento morfométrico de linhagens de tilápias em função dos pesos de abate, um experimento foi conduzido no setor de piscicultura da Universidade Federal de Lavras, M.G.- Brasil. Foram avaliados 362 peixes da linhagem Chitralada e 345 da Supreme, pesando entre 1,7 e 800 g. Com o crescimento dos animais, amostras aleatórias foram tomadas, sendo os peixes abatidos, pesados e submetidos à avaliação morfométrica. Foram ajustados diferentes modelos de crescimento de medidas corporais em função do peso de abate. Pequenas diferenças entre os ajustes dos modelos foram encontradas para o valor assintótico (A) e grandes diferenças para a taxa de crescimento relativo à maturidade (K) para todas as medidas. Os modelos de Brody e Logístico apresentaram maiores diferenças, ficando os modelos de Gompertz e von Bertalanffy em posição intermediária. A linhagem Supreme obteve valores de "A" ligeiramente maiores, para o comprimento padrão (CP), e ligeiramente menores, para a altura do corpo, em relação aos valores obtidos pela linhagem Chitralada, e essa apresentou maior "K" para a relação comprimento da cabeça/comprimento padrão. Conclui-se que os modelos não lineares de Gompertz, Brody, von Bertalanffy e Logístico descrevem adequadamente o crescimento morfométrico de tilápias do Nilo em função do peso corporal, sendo mais indicados os modelos de Gompertz e von Bertalanffy, tanto para a linhagem Chitralada quanto para a Supreme, considerando que foram pequenas as diferenças existentes entre as duas linhagens.

The objective of this research was to evaluate the morphometric growth curves of tilapia strains in function of the body weight, an experiment was carried out at the Fish Culture of the Animal Sciences Department, Federal University of Lavras, M.G-Brazil. Three hundred sixty-two (362) fish of the Chitralada strain and three hundred forty-five (345) of the Supreme strain, weighting between 1,7 and 800 g were used. With the animals' growth, random samples were taken; the fish were slaughtered, weighted and submitted morphometric evaluation. Different models of growth of body measurements in function of the body weight were fitted. In general, the measurements were adequately adjusted. Shorts differences were found between the fit of the models for limiting values (A) and high differences for growth rate relative the maturity (K) for all the measurements. The Brody and Logistic models had higher differences than Gompertz and von Bertalanffy that stayed in intermediary position. The Supreme strain had "A" for standard length (SL) slightly higher and for body height slightly smaller than Chitralada strain. The Chitralada strain had higher "K" for head length/standard lenght relation than Supreme strain. The Gompertz, Brody, von Bertallanffy e Logistic models described adequately the morphometric growth of tilapia in function of body weight, being more indicate the models Gompertz and von Bertalanffy. The differences between Chitralada and Supreme tilapia strains were small.

Rendimento do processamento de linhagens de tilápias (Oreochromis niloticus) em função do peso corporal / Process yield of tilapias (Oreochromis niloticus) strains in function of body weight

Com o objetivo de avaliar os rendimentos do processamento de linhagens de tilápias em função dos pesos de abate, um experimento foi conduzido no setor de piscicultura da Universidade Federal de Lavras, M.G. - Brasil. Foram utilizados 93 peixes da linhagem Chitralada e 78 da Supreme, pesando entre 150 e 790 g. Os peixes foram cultivados em dois tanques de alvenaria de 40 m², sendo alimentados com ração comercial fornecida de acordo com a biomassa e a temperatura da água. A temperatura e o oxigênio da água foram tomados diariamente, no início do dia e no final da tarde, e o pH aferido semanalmente. Com o crescimento dos animais, amostras aleatórias foram tomadas. Nas amostragens, os peixes foram abatidos após terem passado por um jejum de 24 horas e insensibilização por choque térmico. Em seguida, foram pesados e dissecados. A análise de regressão indicou que em pesos mais elevados, a linhagem Chitralada apresenta maior porcentagem de cabeça e rendimento de pele. A linhagem Supreme apresentou maior peso de carcaça, filé e resíduos, sendo mais expressivos em pesos superiores a 500 g e menor valor à maturidade para a por cento de vísceras. Conclui-se que as duas linhagens possuem crescimento das partes componentes proporcional ao aumento do peso corporal, e este aumento não altera o rendimento desses constituintes. A linhagem Supreme apresentou maiores rendimentos de carcaça e filé do que a Chitralada, sendo a mais indicada para a produção e comercialização de filés mais pesados.

With the objective of evaluating the yield processing of tilapia strains in function of body weight, an experiment was carried out at the Fish Culture of the Animal Sciences Department, Federal University of Lavras, M.G. - Brazil. A total 93 fish of the Chitralada strain and 78 of the Supreme strain, weighting between 150 and 790 g were used. The fish were cultivated in two tanks of 40 m², fed with commercial ration supplied according to the biomass and water temperature. The water temperature and oxygen were evaluated every day, at the early morning and at the late afternoon, and the pH checked weekly. During the fish growth, random samples were taken. After 24 hours of fasting, the fish were insensibilized (thermal shock), slaughtered (anoxia), weighted and dissected. The regression analysis showed that in higher weight, the Chitralada strain presented higher head percentage and skin yield. The Supreme strain presented higher carcass, fillet and residue weights, being more expressive in body weights superior to 500 g and smaller limiting value for the percentage of viscera. It was concluded that the two strains have growth of body parts proportional to the increase of the body weight, and this increase does not modify the parts yield. The Supreme strain showed more carcass and fillet yield than Chitralada, and is should be indicated for production and commercialization of heavier fillet.