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As an internal time-keeping mechanism, circadian rhythm plays crucial role in maintaining homoeostasis when in response to nutrition change; meanwhile, branched-chain amino acids (BCAA) in skeletal muscle play an important role in preserving energy homoeostasis during fasting. Previous results from our laboratory suggested that fasting can influence peripheral circadian rhythm and BCAA metabolism in fish, but the relationship between circadian rhythm and BCAA metabolism, and whether circadian rhythm regulates BCAA metabolism to maintain physiological homoeostasis during fasting remains unclear. This study shows that the expression of fifteen core clock genes as well as KLF15 and Bcat2 is highly responsive to short-term fasting in fast muscle of Siniperca chuatsi, and the correlation coefficient between Clock and KLF15 expression is enhanced after fasting treatment. Furthermore, we demonstrate that the transcriptional expression of KLF15 is regulated by Clock, and the transcriptional expression of Bcat2 is regulated by KLF15 by using dual-luciferase reporter gene assay and Vivo-morpholinos-mediated gene knockdown technique. Therefore, fasting imposes a dynamic coordination of transcription between the circadian rhythm and BCAA metabolic pathways. The findings highlight the interaction between circadian rhythm and BCAA metabolism and suggest that fasting induces a switch in KLF15 expression through affecting the rhythmic expression of Clock, and then KLF15 promotes the transcription of Bcat2 to enhance the metabolism of BCAA, thus maintaining energy homoeostasis and providing energy for skeletal muscle as well as other tissues.

In skeletal muscle, autophagy regulates the development and growth of muscle fibres and maintains the normal muscle metabolism. Under starvation and refeeding conditions, the effect of reactive oxygen species (ROS) levels on skeletal muscle autophagy is still unclear, although the excessive accumulation of ROS has been shown to increase autophagy in cells. The purpose of this study was to explore the effects of starvation and diet after starvation on the autophagy of adult Chinese perch muscle, and to determine the level of ROS in the muscle. We performed zero (Normal control), three and seven starvation treatments on adult Chinese perch, and returned to normal feeding for 3 days after starvation for 7 days. In the muscles of the adult Chinese perch muscle after 3 days of starvation, the autophagy marker protein LC3 and the number of autophagosomes remained basically the same as in the normal feeding situation. However, on starvation for 7 days, the mitochondrial autophagy was sensitive and the number of autophagosomes increased, but the antioxidant-related molecules (malondialdehyde, catalase, glutathione S-transferase, glutathione and anti-superoxide anion) decreased and the accumulation of ROS was obvious. In addition, the extended starvation time also increased the level of LC3 protein. However, by refeeding after starvation this nutritional stress resulted in a decrease in ROS levels and a partial restoration of antioxidant enzyme activity. Our data show that in the adult Chinese perch muscle, starvation could reduce the antioxidant activity through the accumulation of ROS, and that the number of autophagosomes continues to increase. Refeeding after starvation could effectively compensate for the level of ROS, and restore the mRNA abundance of antioxidant genes and the activity of antioxidant enzymes to reduce autophagy and improve feed efficiency. Further research should optimize starvation conditions to reduce autophagy in muscles and maintain normal muscle metabolism.

DNA methylation and demethylation are crucial epigenetic modification and regulation for animal development, and their dynamic changes may affect skeletal muscle development. The ten-eleven translocation (Tet) family proteins are demethylases which are involved in the dynamic changes of DNA methylation. However, the expression pattern of Tet family genes and their role in myogenesis in fish remains unclear. In this study, the temporal and spatial expression profiles of Tet1, Tet2 and Tet3 were assayed with RT-qPCR techniques in Chinese perch, Siniperca chuatsi. The obtained data showed that the expressions of three Tet family genes were differentially expressed at different development stages. Tet1 was expressed low at blastula stage, but highly expressed at gastrula stage, then remained low until hatching. The expressions of Tet2 and Tet3 were significantly increased at late gastrula and kept high expression before hatching stage. At the spatial level, the Tet1 expression was highest in gill tissue, moderate level in brain and slow muscle. Tet2 was similar to that of Tet1 except that it was expressed at a lower level in slow muscle, and Tet3 exhibited a higher expression level in gill and brain, a moderate level in fast muscle. Cosinor analysis turned out that the expression of Tet1 and Tet2 displayed a significant daily rhythm in fast muscle, but Tet3 did not show daily rhythmicity. Inhibiting the activity of Tet1/2 proteins by injecting Bobcat339 significantly reduced the expression of MyoD and MRF4, but not MyoG and Myf5, by which leads to the increase of the number of satellite cells and proliferating myoblasts. Together, the results suggest that Tet1/2 may target to MyoD and MRF4 resulted in DNA demethylation and promote their expression, and therefore stimulate myoblast differentiation.

The autophagic lysosomal protein degradation pathway is an evolutionarily conserved pathway, which utilizes lysosomes to degrade and to circulate cell components. Autophagy has been observed in many different types of cells, but its role in skeletal muscle protein degradation has not been thoroughly studied, especially in aquatic species. This study assessed the expression of antioxidant-related signaling genes and the effects of starvation on antioxidant capacity, reactive oxygen species (ROS) content, autophagy-related gene, and autophagosome formation in the skeletal muscle of juvenile Chinese perch after short-term starvation. The results indicated that after starvation for 2 days, the expression of antioxidant-related signaling genes, such as Nrf2 and S6K, was upregulated, while Keap1 was downregulated in the muscle of juvenile Chinese perch. The amounts of antioxidant enzymes ROS, MDA, AHRFR, and ASA and the activities of SOD, CAT, GPx, and GST were increased, and the mRNA levels of GPx, GSTA, GST4A, GSTT1, MnSOD, ZnSOD, and CAT were upregulated. Meanwhile, there was no significant change in the level of LC3-II protein. When starvation was prolonged to 5 days, Nrf2 and S6K1 continued to increase and mTOR and Keap1 significantly decreased; ROS and ASA content continued to be significantly increased, but the MDA and AHRFR content and the SOD, CAT GR, and GPx activities all decreased. The expression of MnSOD, ZnSOD, and GR decreased significantly, and GST4A, GSTT1, and CAT tended to decrease to levels consistent with normal feeding. The expression of all autophagy-related genes except Ulk1 significantly increased, the formation of autophagosomes and autolysosomes was enhanced in muscle, and LC3 protein levels in muscle increased significantly. Our data suggested that the autophagy that occurs in the skeletal muscle tissue of Chinese perch due to dietary deprivation is involved in a series of molecular and physiological responses, including changes in antioxidant signaling molecules, in antioxidant capacity and in autophagy and autophagy-related gene expression.

An eight-week feeding trial discusses how methionine affects intestinal barrier and lipid transport on rice field eel (Monopterus albus). Six isoenergetic and isonitrogenous feeds contained different levels of methionine (0, 2 g/kg, 4 g/kg, 6 g/kg, 8 g/kg, or 10 g/kg). Compared with M0 (0 g/kg), gastric amylase, lipase, and trypsin were remarkably increased as dietary methionine ( P < 0.05 ); intestinal amylase, lipase, and trypsin were remarkably increased in M8 (8 g/kg) ( P < 0.05 ). Compared with M0, gastric fovea was remarkably increased ( P < 0.001 ), gastric epithelium is neater in M8 than that in M0, intestinal villus height and muscular thickness are remarkably increased in M8 ( P < 0.001 ), and amounts of goblet cells per root in M8 were increased ( P > 0.05 ), while intestinal crypt depth was markedly decreased ( P < 0.001 ). Lipid droplets in the intestinal villus and mucosal layer in the M8 (8 g/kg) group were more than that in M0 (0 g/kg). Compared with M0 (0 g/kg), the intestinal gcn2 and eif2α were downregulated in M8 (8 g/kg) ( P < 0.01 and P < 0.05 , respectively), while occ, cl12, cl15, zo-1, zo-2, hdlbp, ldlrap, npc1l1, cd36, fatp1, fatp2, fatp6, apo, apoa, apob, apoc, apoe, mct1, mct2, mct8, lpl, mttp, moat2, and dgat2 were upregulated markedly in M8 (8 g/kg). Intestinal eif2α expression was positively correlated with gcn2, and intestinal zo-1, cl15, fatp6, ldlrap, mct2, mct8, apo, apob, mct1, apoc, fatp1, mttp, cd36, occ, npc1l1, hdlbp, fatp2, apoe, lpl, and moat2 gene expression was negatively correlated with gcn2. In conclusion, methionine deficiency affected the gastric and intestinal structures, damaged the intestinal barrier, and decreased lipid and fatty acid transport. Besides, gcn2 could be activated when M. albus was fed methionine-deficient feed.

Ginkgo biloba leaf is widely used in traditional medicine in China. The present study aimed to illustrate the effects of dietary Ginkgo biloba leaf extract (GBLE) on growth performance and immune responses in common carp infected by Aeromonas hydrophila. Six different diets either not treated (control) or treated with 0.5, 1, 2, 5 and 10 g/kg of GBLE were designed to feed the fishes for 8 weeks. The results indicated that, compared to the control groups, 10 g/kg dietary GBLE significantly increased body growth and feed utilization. In GBLE dietary groups, red blood cell levels, white blood cells, hematocrit, hemoglobin, total protein, albumin and globulin were significantly increased relative to the control groups. Dietary supplementation with 5 g/kg GBLE increased the phagocytic ratio, and phagocytic indexes increased in the 2, 5 and 10 g/kg groups relative to the control groups. Moreover, 2, 5 and 10 g/kg GBLE diets increased O2− production compared to the control groups. Additionally, GBLE diets stimulated lysozyme activity (in 10 g/kg group) and inhibited bactericidal activity (in 0.5, 2, 5 and 10 g/kg group). Quantitative real-time PCR showed that IL1β, IL8, TNF-α, IL10, TGFβ, and inducible enzyme genes were prone to decrease while SAA, hepcidin and GPX1 were increased due to the GBLE diet in the intestine. In the head-kidney, the GBLE treatment decreased IL1β, IL8, TNF-α, IL10, TGFβ, INOS and arginase gene expressions, whereas SOD upregulation was found in the GBLE condition. The mRNA expressions of IL1β, IL8, TNF-α, IL10 and INOS were decreased, but SAA, hepcidin, GPX1 and SOD mRNA levels were increased in the spleen in the GBLE diet compared to the control. Additionally, diet supplemented with GBLE improved the survival rate infected with A. hydrophila. Our observations suggest that GBLE effectively enhanced growth performance, modulated immune-related gene expression. It improved survival rate of common carp after A. hydrophila infection and the optimum concentration we recommend is 10 g/kg of GBLE.

Skeletal muscle exhibits significant dynamism and plasticity over the fish life cycle. However, the molecular mechanism of dynamic regulation of metabolic homeostasis in fish skeletal muscle remains unclear. PI3K/AKT signaling plays a central role in cellular physiology by mediating growth factor signals during organismal growth and critical cellular processes. In order to reveal the role of PI3K/AKT signaling pathway in metabolic regulation of Chinese perch muscle, Illumina sequencing technology was preformed to mine the alterations of muscle transcriptome under PI3K inhibitor (LY294002) treatment. Total 619 unigenes were identified as significantly differentially expressed genes (DEGs) between the control and LY294002 treatment groups. Functional enrichment analysis revealed that DEGs were mostly associated with metabolic homeostasis, such as Biosynthesis of amino acids, Glycolysis/gluconeogenesis, Glycerolipid metabolism process. Furthermore, the histological section analysis showed that the spaces between muscle fibers in LY294002 injection group were increased and the cytoplasmic contents were reduced, which was consistent with the characteristics of muscle atrophy. Together with the results, it was suggested that the PI3K/AKT signaling pathway plays a crucial role in maintaining the balance of metabolism in fish muscle, which in turn ensures normal muscle physiology and growth. In addition, this study could provide a direction for the future research of elucidating the molecular mechanisms of metabolic homeostasis of PI3K/AKT signaling in fish muscle.

Two smyd1 paralogues, smyd1a and smyd1b, have been identified in zebrafish. Although Smyd1b function has been reported in fast muscle, its function in slow muscle and the function of Smyd1a, in general, are uncertain. In this study, we generated 2 smyd1a mutant alleles and analyzed the muscle defects in smyd1a and smyd1b single and double mutants in zebrafish. We demonstrated that knockout of smyd1a alone had no visible effect on muscle development and fish survival. This was in contrast to the smyd1b mutant, which exhibited skeletal and cardiac muscle defects, leading to early embryonic lethality. The smyd1a and smyd1b double mutants, however, showed a stronger muscle defect compared with smyd1a or smyd1b mutation alone, namely, the complete disruption of sarcomere organization in slow and fast muscles. Immunostaining revealed that smyd1a; smyd1b double mutations had no effect on myosin gene expression but resulted in a dramatic reduction of myosin protein levels in muscle cells of zebrafish embryos. This was accompanied by the up-regulation of hsp40 and hsp90-α1 gene expression. Together, our studies indicate that both Smyd1a and Smyd1b partake in slow and fast muscle development although Smyd1b plays a dominant role compared with Smyd1a.-Cai, M., Han, L., Liu, L., He, F., Chu, W., Zhang, J., Tian, Z., Du, S. Defective sarcomere assembly in smyd1a and smyd1b zebrafish mutants.

Fish skeletal muscles are composed of spatially well-separated fiber types, namely, red and white muscles with different physiological functions and metabolism. To compare the DNA methylation profiles of the two types of muscle tissues and identify potential candidate genes for the muscle growth and development under epigenetic regulation, genome-wide DNA methylation of the red and white muscle in Chinese perch Siniperca chuatsi were comparatively analyzed using bisulfate sequencing methods. An average of 0.9 billion 150-bp paired-end reads were obtained, of which 86% were uniquely mapped to the genome. Methylation mostly occurred at CG sites at a ratio of 94.43% in the red muscle and 93.16% in the white muscle. The mean methylation levels at C-sites were 5.95% in red muscle and 5.83% in white muscle, whereas the mean methylation levels of CG, CHG, and CHH were 73.23%, 0.62%, and 0.67% in red muscle, and 71.01%, 0.62%, and 0.67% in white muscle, respectively. A total of 4192 differentially methylated genes (DMGs) were identified significantly enriched in cell signaling pathways related to skeletal muscle differentiation and growth. Various muscle-related genes, including myosin gene isoforms and regulatory factors, are differentially methylated in the promoter region between the red and white muscles. Further analysis of the transcriptional expression of these genes showed that the muscle regulatory factors (myf5, myog, pax3, pax7, and twitst2) and myosin genes (myh10, myh16, myo18a, myo7a, myo9a, and myl3) were differentially expressed between the two kinds of muscles, consistent with the DNA methylation analysis results. ELISA assays confirmed that the level of 5mC in red muscle was significantly higher than in white muscle (P 0.05). The RT-qPCR assays revealed that the expression levels of the three DNA methylation transferase (dnmt) subtypes, dnmt1, dnmt3ab, and dnmt3bb1, were significantly higher in red muscle than in white muscle. The higher DNA methylation levels in the red muscle may result from higher DNA methylation transferase expression in the red muscles. Thus, this study might provide a theoretical foundation to better understand epigenetic regulation in the growth and development of red and white muscles in animals, at least in Chinese perch fish.

Skeletal muscle metabolism is highly adaptive in response to nutritional changes, as occur during food deprivation. Kruppel-like factor 15 (KLF15), as a critical regulator of branched-chain amino acids (BCAAs) metabolism, which plays an important role in energy metabolism. But the role for KLF15 in adaptation and physiology of fish skeletal muscle remains unclear. In this study, we obtained the complete cDNA of Chinese perch (Siniperca chuatsi) KLF15 and investigated its expression in different tissues as well as its expression characteristics during short-term (3 or 7 days) fasting and glucocorticoid (prednisolone) treatment in Chinese perch. Sequence analysis indicated that ScKLF15 showed a highly conservative Zinc finger C2H2 superfamily domain at its C terminal with those of other vertebrates. Tissue specificity analysis showed that ScKLF15 was highly expressed in fast muscle. During the short-term fasting, the expression of KLF15 was significantly increased after 3 days fasting and decreased after 7 days fasting as well as the expression of branched-chain aminotransferase 2 (BCAT2) and the activity of alanine aminotransferase (ALT). Under the prednisolone stimulation, the expression of KLF15 and BCAT2 were significantly increased at 5 days post injection. Furthermore, the activity of ALT and aspartate aminotransferase (AST) showed the same tendency after the injection. Thus, this work revealed that the catabolism of BCAAs was enhanced to maintain the physiological homeostasis in the fish when faced with food deprivation and glucocorticoids stress. More importantly, the study suggested that KLF15 plays a role in adaptive regulation of BCAAs metabolism in skeletal muscle of Chinese perch.

Background: Soy isoflavone and soy saponin are heat-stable glycosides present in soybean meal, which is the major factor restrict soy protein using in aquatic feed. This study is aimed to conduct that dietary soy isoflavone and soy saponin on growth performance, intestinal structure, intestinal immunity and gut microbiota community on rice field eel (Monopterus albus), and revealed its mechanism.Results: In current study, dietary 1g/kg soy isoflavone, 2g/kg soy saponin and their mixed feed decreased growth of M. albus. Dietary 1g/kg soy isoflavone significantly increased serum alkaline phosphatase (P < 0.05), dietary 2g/kg soy saponin remarkably declined intestinal immunoglobulin M (P < 0.05). Dietary 1g/kg soy isoflavone and 2g/kg soy saponin mixed feed significantly decreased intestinal complement 3, complement 4 and immunoglobulin M, amounts of intestinal goblet cells per root and shannon of gut microbiota (P < 0.05). Dietary 1g/kg soy isoflavone, 2g/kg soy saponin and their mixed feed down regulated intestinal tight junction protein (Interleukin-12, Interleukin-15, Tight junction protein ZO-2-like) and anti-inflammatory (Interleukin-10, Transforming growth factor beta 2) mRNA expression, up regulated pri-inflammatory factors (Interleukin-1 β, Interleukin-6, Interleukin-8, Interleukin-12 and Interleukin-15) mRNA expression.Conclusion: Based on the current results, dietary soy isoflavone and soy saponin could damage the intestinal structure and changes gut bacteria community of M. albus, and destroyed the intestinal barrier, eventually induced intestinal inflammatory occurring, soy isoflavone and soy saponin mixed feed has more serious effect than they single supplemented.

The intestine is a main organ responsible for energy and nutrition absorption and digestion and is an important immune barrier in fish. In recent years, the intestinal health of fish has gained increasing attention in the aquaculture industry. The intestine is a direct target organ of starvation and is highly sensitive to nutritional changes. However, the adaptive responses of the fish intestine to starvation are not well studied. The present study aimed to reveal the effects of starvation on antioxidant systems, autophagy, and mitochondrial function in the intestine of Chinese perch. Therefore, Chinese perch (250 ± 22 g) were starved for 0, 7, and 14 days. Our results indicate that the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) were significantly increased in the intestine of Chinese perch after 7 days and 14 days of starvation, indicating that starvation could induce oxidative stress in the intestine. Meanwhile, the activities and mRNA expression of antioxidative enzymes, including superoxide dismutases (SOD), glutathione peroxidases (GPx), and catalases (CAT), were upregulated in the intestine after the starvation treatment, which might be a compensatory response to oxidative stress. Starvation also increased the expression of nrf2 in the intestine, thereby resulting in the upregulation of antioxidative gene expression. However, the glutathione content and its generation in the intestine were decreased under starvation stress. The ultrastructural analysis of the intestine demonstrated that starvation induced autophagy in the intestine of Chinese perch. The RT–qPCR analysis showed that starvation induced the mRNA expression of genes involved in autophagosome membrane initiation, autophagosome membrane expansion, vesicle recycling, and cargo recruitment, including beclin1, ulk1, beclin1, lc3a, atg4, atg5, atg7, p62, and atg9. Mitochondria are the main ATP-generating organelles and target organelles for ROS. The present study indicates that starvation induced a decrease in the ATP content in the intestine of Chinese perch. In addition, starvation downregulated the expression of mitochondrial oxidative phosphorylation-related and mitochondrial ATP generation-related genes (cox1, atp5f1, atp5a1, and mt-cyb) in the intestine of Chinese perch, suggesting that starvation could suppress mitochondrial energy synthesis. Taken together, our data demonstrate that starvation can induce oxidative stress, disrupt antioxidant systems, and cause autophagy and mitochondrial dysfunction in the intestine of Chinese perch. These results provide insight into the mechanisms by which starvation disrupts intestinal homeostasis in fish.

The branched-chain amino acids (BCAAs) play a key role in the energy metabolism of the muscle tissue and the Krüppel-like factor 15 (KLF15) as a transcription factor, which is a key regulator of BCAA metabolism in the skeletal muscle. This study assessed the effect of starvation for 0, 3, 7, and 15 days on BCAA metabolism in the skeletal muscle of Nile tilapia. The results showed that the expression of KLF15 showed a trend of increasing first and then decreasing during starvation, as well as the expression and activity of branched-chain aminotransferase 2 (BCAT2) and alanine aminotransferase (ALT). On the other hand, the content of BCAA was at first decreased and then upregulated, and it reached the lowest level after starvation for 3 days. In addition, through dual-luciferase reporter assay and injection experiments, it was found that KLF15 is the target gene of miR-125a-3p, which further verified that miR-125a-3p can regulate the BCAA metabolism by targeting KLF15 in the skeletal muscle. Thus, our work investigated the possible mechanisms of BCAA metabolism adapting to nutritional deficiency in the skeletal muscle of Nile tilapia and illustrated the regulation of BCAA metabolism through the miR-125a-3p-KLF15-BCAA pathway in the skeletal muscle.

Spinibarbus caldwelli is an omnivorous cyprinid fish that is distributed widely in China. To investigate the adaptive evolution of S. caldwelli, the muscle transcriptome was sequenced by Illumina HiSeq 4000 platform. A total of 80,447,367 reads were generated by next-generation sequencing. Also, 211,386 unigenes were obtained by de novo assembly. Additionally, we calculated that the divergence time between S. caldwelli and Sinocyclocheilus grahami is 23.14 million years ago (Mya). And both of them diverged from Ctenopharyngodon idellus 46.95 Mya. Furthermore, 38 positive genes were identified by calculating Ka/Ks ratios from 9225 orthologs. Among them, several immune-related genes were identified as positively selected, such as POLR3B, PIK3C3, TOPORS, FASTKD3, CYPLP1A1, and UACA. Our results throw light on the nature of the natural selection of S. caldwelli and contribute to future immunological and transcriptome studies.

Nrf2 is an important transcription factor involved in the antioxidant response and is widely expressed in animal tissues. The function of Nrf2 is regulated by its negative regulator Keap1 by inducing its cytoplasmic degradation. Recent studies have suggested that Nrf2 is also regulated post-transcriptionally via miRNAs. However, to date, how miRNAs regulate Nrf2 in fish skeletal muscles is unknown. In this study, the full-length cDNAs with 2398 bp of the Nrf2 was firstly cloned by SMART RACE amplification tools from Chinese perch. The Nrf2 gene structure and its 3’-UTR region for possible miRNA binding sites, as well as its spatial expression profile were assayed. Then, we employed TargetScan Fish tool MiRNAnome to predict putative sites for five miRNAs including miR-181a-5p, MiR-194a, MiR-216a, miR-459-5p, and miR-724. Using qRT-PCR assay, we found that Nrf2 mRNA levels have negative correlation with all five miRNAs expression in muscle of nutritionally deprived fish, and that ectopic expression of miR-181a-5p alone reduces Nrf2 mRNA levels. Luciferase reporter assay in a heterologous cell system revealed that each of the five miRNAs reduced Nrf2 expression, suggesting a direct regulatory mechanism. Moreover, the miR-181a-5p suppression using specific antagomir led to a significant increase in Nrf2 expression in vivo. At the same time, the expression levels of the antioxidant enzymes CAT, ZnSOD, GPx, GSTA, and GSTA genes increased significantly after injecting miR-181a-5p antagomir. Taken together, these findings provide evidence that miRNAs are involved in the Nrf2 signaling networks in regulation of oxidative stress in fish, at least in Chinese perch muscle.

Muscle is an important structural tissue in aquatic animals and it is susceptible to bacterial and fungal infection, which could affect flesh quality and health. In this study, Chinese soft-shelled turtles were artificially infected with two pathogens, Proteus vulgaris and Elizabethkingia meningoseptica and the effects on muscle nutritional characteristics, oxidative stress and autophagy were assayed. Upon infection, the muscle nutritional composition and muscle fiber structure were notably influenced. Meanwhile, the mRNA expression of Nrf2 was down-regulated and Keap1 up-regulated, thus resulting in a decrease in antioxidant capacity and oxidative stress. However, with N-acetylcysteine treatment, the level of oxidative stress was decreased, accompanied by significant increases in antioxidant enzyme activities and the mRNA levels of SOD, CAT, GSTCD, and GSTO1. Interestingly, there was a significant increase in autophagy in the muscle tissue after the pathogen infection, but this increase could be reduced by N-acetylcysteine treatment. Our findings suggest that muscle nutritional characteristics were dramatically changed after pathogen infection, and oxidative stress and autophagy were induced by pathogen infection. However, N-acetylcysteine treatment could compromise the process perhaps by decreasing the ROS level and regulating Nrf2-antioxidant signaling pathways.

The branched-chain amino acids (BCAA) play an important role in muscle energy metabolism, and Kruppel-like factor 15 (KLF15) is an essential regulator of BCAA metabolism in muscle under nutritional deficiency. In this study, we analyzed the effect of normal feeding (starvation for 0 day), starvation for 3, 7, 10, 15 days, and refeeding for 7 days after 15 days of starvation on the expression of KLF15 and BCAA metabolism in muscle of Chinese soft-shelled turtles by a fasting-refeeding trial. The results showed that the level of KLF15 transcription was increased first and then decreased in muscle during short-term starvation, and the protein level was gradually increased. Both the mRNA and protein level of the KLF15 returned to normal feeding level after refeeding for 7 days. The changing trend of the activities of branched-chain aminotransferase (BCAT) and alanine aminotransferase (ALT) was consistent to that of KLF15 mRNA, but at the transcription level, the expression of BCAT mRNA was consistent with the change of enzyme activity as well as ALT continued to increase in muscle under starvation. In addition, BCAA content showed a trend that decreased first and then increased under starvation, while the alanine (Ala) was the contrary. The above results indicated that the regulatory role of KLF15 in BCAA catabolism of muscle in Chinese soft-shelled turtles under nutritional deficiency, which might be activated the catabolism of BCAA in muscle to provide energy and maintain the homeostasis by KLF15-BACC signaling axis.

An eight-week feeding trial was performed to determine how methionine regulates muscle growth in the rice field eel Monopterus albus (initial weight 25.08 ± 0.31 g). Six isonitrogenous and isoenergetic experimental feeds were formulated, and different levels of methionine (0, 2 g/kg, 4 g/kg, 6 g/kg, 8 g/kg, or 10 g/kg) were added to the basic diet (110 g/kg fish meal, 400 g/kg soy concentrate meal). The weight gain rate (WGR) of M. albus markedly increased as methionine concentrations increased to 6 g/kg (P

Circadian clock genes and myogenic factors are tightly integrated to influence muscle growth upon dietary deprivation in animals. In this study, we reported that upon short-term fasting of Nile tilapia juveniles for 7 and 15 days, the growth of the fish stagnated and the size of muscle fibers decreased. To reveal the molecular mechanisms of how starvation affects fish muscle growth, we analyzed the rhythmic expression of circadian clock genes and myogenic factors. After 7 and 15 days of fasting treatment, the muscle tissues were collected for 24 h (at zeitgeber times ZT0, ZT3, ZT6, ZT9, ZT12, ZT18, ZT21, and ZT24) from tilapia juveniles. Among the 27 clock genes, the expression of cyr1b, nr1d1, per1, clocka, clockb, ciarta, and aanat2 displayed a daily rhythmicity in normal daily cycle, while arntl2, cry1a, cry1b, npas2, nr1d2b, per2, per3, rorαb, clocka, clockb, nfil3, cipca, and cipcb exhibited daily rhythmicity in the fasting fish muscles. The transcript levels of clockb showed moderate positive correlation with the aanat2, ciarta, cry1b, and nr1d1 in the muscle tissue of normally fed Nile tilapia juvenile. In comparison of the two treatment modes, the expression levels of clocka, clockb, and cry1b showed the rhythmicity, but clockb expression was significantly decreased and the acrophase had shifted. The transcript levels of fbxo32 and pdk4 had either moderate or strong positive correlations with other daily expression of clock genes except arntl2 in the muscle after 7-day fasting. The expressions of myogenic regulatory factors were also either upregulated or downregulated. These observations demonstrated that dietary starvation might affect fish muscle growth by modulating the differential expression of circadian clock genes and myogenic factors. Thus, our work provides a better understanding of the molecular mechanism of dietary starvation on fish growth and may provide dietary administration in aquiculture.

The red swamp crayfish (Procambarus clarkii) is widely cultivated and consumed for its tender meat. The quality of P. clarkii is increasingly deteriorating due to a possible shortage of feed nutrition, posing a significant threat to its sustainability. To avoid the phenomenon mentioned above and enrich the culture technology of P. clarkii, the aim of this study was to assess the effect of re-feeding on alleviating oxidative damage caused by short-term starvation, as well as to investigate the relationship between oxidative stress and meat quality and its molecular regulation mechanism. A total of 600 crayfish weighing about 20.11 g were randomly divided into 4 groups: S0F0 (the control group), S0F3 (0 days of starvation and 3 days of re-feeding), S3F0 (3 days of starvation and 0 days of re-feeding) and S3F3 (3 days of starvation and 3 days of re-feeding). The results showed that starvation slowed down the growth of P. clarkii and significantly decreased the saturated fatty acids (SFA) contents in the muscle (P

In the present study, we sought to investigate the influence of high fat diet on the core clock genes and the muscle functional genes daily expression in the skeletal muscle of Chinese soft-shelled turtle. The turtles were fed by two diets including a control fat diet (the CON treatment, 7.98% lipid) and a high fat diet (the HFD treatment, 13.86% lipid) for six weeks and administrated by the photophase regimen of 24h light/dark (12L:12D) cycle. After the feeding trial experiment, we measured the daily expression levels of 17 core clock genes (Clock, Bmal1/2, NPAS2, Tim, Cry1/2, Per1/2, DBP, AANAT, NIFL3, BHLHE40, NR1D2, RORA, RORB, RORC) and 12 muscle functional genes (FBXO32, MBNL1, MSTN, Myf5, Myf6, MyoD, MyoG, MyoM1, PPARa, PDK4, Trim63, UCP3) in the skeletal muscle of the two treatments. The results showed that except for Bmal1, NPAS2, Per2 and RORB, the expression of the other 13 core clock genes exhibited circadian oscillation in the CON treatment. Among the 12 muscle functional genes, MBNL1, PDK4 and MyoM1 did not exhibit circadian oscillation in the CON treatment. In the HFD treatment, the circadian rhythms expressional patterns of the 8 core clock genes (Clock, Bmal2, Cry2, Per1, DBP, NFIL3, BHLHE40 and RORA) and 6 muscle functional genes (MSTN, Myf5, MyoD, MyoG, PPARa and Trim63) were disrupted. In addition, compared with the CON treatment, the circadian expression of the 5 core clock genes (Tim, Cry1, AANAT, NR1D2, RORC) and the 3 muscle functional genes (FBXO32, Myf6, UCP3) showed the advanced or delayed expression peaks in the HFD treatment. In CON treatment, the circadian expression of the MyoG, MyoD, Myf6, FBXO32 and PPARa showed positive or negative correlation with the transcription pattern of Clock, Bmal2, Cry1/2, Per1/2. However, only the FBXO32 and Myf6 presented positive or negative correlation with the circadian expression of Cry1, RORB, AANAT and Tim in HFD treatment. In summary, these results demonstrate that the disruption of the circadian rhythm of the core clock genes and muscle functional genes in the skeletal muscle is closely associated with feeding high fat diet to Chinese soft-shelled turtle.