Short communication: Differential expression of piwi1 and piwi2 genes in tissues of tambacu and zebrafish: A possible relationship with the indeterminate muscle growth.

Fish skeletal muscle is a component of the human diet, and understanding the mechanisms that control muscle growth can contribute to improving production in this sector and benefits the human health. In this sense, fish such as tambacu can represent a valuable source for exploring muscle growth regulators due to the indeterminate muscle growth pattern. In this context, the genes responsible for the indeterminate and determinate muscle growth pattern of fish are little explored, with piwi genes being possible candidates involved with these growth patterns. Piwi genes are associated with the proliferation and self-renewal of germ cells, and there are descriptions of these same functions in somatic cells from different tissues. However, little is known about the function of these genes in fish somatic cells. Considering this, our objective was to analyze the expression pattern of piwi 1 and 2 genes in cardiac muscle, skeletal muscle, liver, and gonad of zebrafish (species with determinate growth) and tambacu (species with indeterminate growth). We observed a distinct expression of piwi1 and piwi2 between tambacu and zebrafish, with both genes more expressed in tambacu in all tissues evaluated. Piwi genes can represent potential candidates involved with indeterminate muscle growth control.

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.

Degree of piRNA sharing and Piwi gene expression in the skeletal muscle of Piaractus mesopotamicus (pacu), Colossoma macropomum (tambaqui), and the hybrid tambacu.

PiRNAs are a class of small noncoding RNAs that, in their mature form, bind to Piwi proteins to repress transposable element activity. Besides their role in gametogenesis and genome integrity, recent evidence indicates their action in non-germinative tissues. We performed a global analysis of piRNA and Piwi gene expression in the skeletal muscle of juveniles pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum), and the hybrid tambacu to evaluate the degree of piRNA sharing among these three genotypes. Total RNA was sequenced and analyzed using specific parameters of piRNAs by bioinformatics tools. piRNA and Piwi gene expression was analyzed by RT-qPCR. We detected 24 piRNA clusters common to the three genotypes, with eight shared between pacu and tambacu, three between pacu and tambaqui, and five between tambaqui and tambacu; seven, five, and four clusters were unique to pacu, tambacu, and tambaqui, respectively. Genomic localization and fold change values showed two clusters and 100 piRNAs shared among the three genotypes. The gene expression of four piRNAs was evaluated to validate our bioinformatics results. piRNAs from cluster 17 were higher in tambacu than pacu and piRNAs from cluster 18 were more highly expressed in tambacu than tambaqui and pacu. In addition, the expression of Piwis 1 and 2 was higher in tambacu and tambaqui than pacu. Our results open an important window to investigate whether these small noncoding RNAs benefit the hybrid in terms of faster growth and offer a new perspective on the function of piRNAs and Piwis in fish skeletal muscle.

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.

Association of CAST2, HSP90AA1, DNAJA1 and HSPB1 genes with meat tenderness in Nellore cattle.

The objective of this study was to evaluate the association of expression of CAPN1, CAPN2, CAST, HSP90AA1, DNAJA1 and HSPB1 genes with meat tenderness in Nellore cattle. Three experimental groups were selected by shear force (SF): moderately tender (SF=34.3±5.8N), moderately tough (SF=56.8±7.8N), and very tough meat (SF=80.4±15N). Gene expression was evaluated by real-time PCR. Expression of the CAPN1, CAPN2, CAST and CAST1 genes did not differ between groups. Expression of the CAST2 was up-regulated (P<0.05) in the moderately tough and very tough meat groups. Down-regulation of the HSP90AA1, DNAJA1 and HSPB1 genes (P<0.05) was observed in the moderately tender meat group. The present results suggest that meat tenderness in Nellore cattle does not directly depend on the expression of the CAPN1 and CAPN2 genes, but is associated with the expression of other genes such as CAST2, HSP90AA1, DNAJA1 and HSPB1.

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.

High Final Energy of Low-Level Gallium Arsenide Laser Therapy Enhances Skeletal Muscle Recovery without a Positive Effect on Collagen Remodeling.

The aim of this study was to evaluate the effects of a Gallium Arsenide (GaAs) laser, using a high final energy of 4.8 J, during muscle regeneration after cryoinjury. Thirty Wistar rats were divided into three groups: Control (C, n = 10); Injured (I, n = 10) and Injured and laser treated (Injured/LLLT, n = 10). The cryoinjury was induced in the central region of the tibialis anterior muscle (TA). The applications of the laser (904 nm, 50 mW average power) were initiated 24 h after injury, at energy density of 69 J cm(-1) for 48 s, for 5 days, to two points of the lesion. Twenty-four hours after the final application, the TA muscle was removed and frozen in liquid nitrogen to assess the general muscle morphology and the gene expression of TNF-α, TGF-ß, MyoD, and Myogenin. The Injured/LLLT group presented a higher number of regenerating fibers and fewer degenerating fibers (P < 0.05) without changes in the collagen remodeling. In addition, the Injured/LLLT group presented a significant decrease in the expression of TNF-α and myogenin compared to the injured group (P < 0.05). The results suggest that the GaAs laser, using a high final energy after cryoinjury, promotes muscle recovery without changing the collagen remodeling in the muscle extracellular matrix.