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.

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.

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.

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.

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.