The circadian rhythm regulates branched-chain amino acids metabolism in fast muscle of Chinese perch (Siniperca chuatsi) during short-term fasting by Clock-KLF15-Bcat2 pathway.

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.

Genome-wide DNA methylation profiles provide insight into epigenetic regulation of red and white muscle development in Chinese perch Siniperca chuatsi.

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.

The miRNA expression profile directly reflects the energy metabolic differences between slow and fast muscle with nutritional regulation of the Chinese perch (Siniperca chuatsi).

Fish skeletal muscles are composed of two distinct types, slow and fast muscles, and they play important roles in maintaining the body's movement and energy metabolism. The two types of muscle are easy to separate, so they are often used as the model system for studies on their physiological and functional characteristics. In this study, we revealed that the carbohydrate and lipid metabolic KEGG pathways are different between slow and fast muscles of Chinese perch with transcriptome analysis. In fast muscle, glucose metabolism was catabolic with higher glycolysis capacity, while in slow muscle, glucose metabolism was anabolic with more glycogen synthesis. In addition, oxidative metabolism in slow muscle was stronger than that in fast muscle. By analyzing the expression levels of 40 miRNAs involved in metabolism in the muscles of Chinese perch, 18 miRNAs were significantly upregulated and 7 were significantly downregulated in slow muscle compared with fast muscle. Based on functional enrichment analysis of their target genes, the differential expression levels of 17 miRNAs in slow and fast muscles were reflected in their carbohydrate and lipid metabolism. Among these, 15 miRNAs were associated with carbohydrate metabolism, and 6 miRNAs were associated with lipid metabolism. After 3 days of starvation, the expression levels of 15 miRNAs involved in glucose metabolism in fast and slow muscles increased. However, after 7 days of starvation, the mRNA levels of miR-22a, miR-23a, miR-133a-3p, miR-139, miR-143, miR-144, miR-181a and miR-206 decreased to basal levels. Our data suggest that the possible reason for the difference in glucose and lipid metabolism is that more miRNAs inhibit the expression of target genes in slow muscle.

Investigation of Proteus vulgaris and Elizabethkingia meningoseptica invasion on muscle oxidative stress and autophagy in Chinese soft-shelled turtle (Pelodiscus sinensis).

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.

Analysis of Natural Selection of Immune Genes in Spinibarbus caldwelli by Transcriptome Sequencing.

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.

Exposure to waterborne cadmium induce oxidative stress, autophagy and mitochondrial dysfunction in the liver of Procypris merus.

The present study was performed to determine the effect of waterborne cadmium (Cd) exposure on oxidative stress, autophagy and mitochondrial dysfunction, and to explore the mechanism of Cd-induced liver damage in freshwater teleost Procypris merus. To this end, P. merus were exposed to waterborne 0, 0.25 and 0.5 mg/L Cd for 30 days (equal to 0, 2.22 and 4.45 µmol Cd/l). The waterborne Cd exposure significantly increased hepatic Cd accumulation and impaired histological structure of the liver of P. merus. both low and high-dose waterborne Cd exposure induced oxidative stress in the liver of P. merus, through increases Malondialdehyde (MDA) and reactive oxide species (ROS) accumulation in the liver. The Cd-induced oxidative stress in liver may result from reduction of enzyme activities (superoxide dismutases (SOD), catalases (CAT), GSH-S-transferases (GST)) and transcriptional expression of antioxidant related genes (gpx1, gpx2, cata, gsta1, sod1). Furthermore, the present study showed that waterborne Cd exposure decreased the transcriptional factor (nrf2) expression, which might lead to the down-regulation of antioxidant gene expression. Transmission electron microscopy (TEM) observations demonstrated that waterborne Cd exposure induced autophagy in the liver of P. merus. Gene expression analysis showed that waterborne Cd exposure also induced mRNA expression of a set of genes (beclin1, ulk1, atg5, lc3a, atg4b, atg9a, and p62) involved in the autophagy process, indicating that the influence of Cd on autophagy involved transcription regulation of autophagy gene expression. Waterborne Cd exposure induced a sharp decrease in ATP content in the liver of P. merus. In addition, the expression of mitochondrial function genes (sdha, cox4i1, cox1, atp5f1, and mt-cyb) are significantly decreased in the liver of P. merus in Cd treated groups, manifesting the suppression of Cd on mitochondrial energy metabolism. Taken together, our experiments demonstrate that waterborne Cd exposure induced oxidative stress, autophagy and mitochondrial dysfunction in the liver of P. merus. These results may contribute to the understanding of mechanisms that hepatotoxicity of Cd in teleost.

MiR-125a-3p-KLF15-BCAA Regulates the Skeletal Muscle Branched-Chain Amino Acid Metabolism in Nile Tilapia (Oreochromis niloticus) During Starvation.

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.

Impact of short-term starvation and refeeding on the expression of KLF15 and regulatory mechanism of branched-chain amino acids metabolism in muscle of Chinese soft-shelled turtle (Pelodiscus sinensis).

The branched-chain amino acids (BCAA) play an important role in muscle energy metabolism, and Krüppel-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.

Expression analysis of the heat shock protein genes and cellular reaction in dojo loach (Misgurnus anguillicaudatus) under the different pathogenic invasion.

As molecular chaperones, heat shock proteins (HSPs) play essential roles in cells in response to stress conditions. Recent studies about immune functions of HSPs in fish have also been reported. In this study, based on the reported cDNA sequences of the four HSP genes, HSP70, HSC70, HSP90α and HSP90ß, the temporal expression patterns of the four genes during embryonic development of dojo loach(Misgurnus anguillicaudatus) was assayed with qRT-PCR. All of the four genes were ubiquitously expressed in all detected embryonic developmental stages. Among of them, HSP70, HSC70 and HSP90ß were highly expressed in the organ formation stage, while HSP90α was the highest expressed in myotome formation stage. Further, the immune responses of the four HSP genes were assayed when loach were infected with three different pathogens, bacterium (Flavobacterium cloumnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia). All of the four genes were differentially expressed in four tissues such as skin, gills, spleen and kidney in response to the pathogenic invasion, but both HSP70 and HSP90α expressions were dramatically up-regulated. Further, the cellular responses of the loach skinand gill tissues were observed, in which the number of the skin goblet cells were significantly increased, and the gill lamellae became shorter and wider after infected. Thus, our work indicated that the HSPs may directly or indirectly involved in immune defense in fish, at least in the loach.

Dietary Ginkgo biloba leaf extract alters immune-related gene expression and disease resistance to Aeromonas hydrophila in common carp Cyprinus carpio.

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 IL1ß, IL8, TNF-α, IL10, TGFß, 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 IL1ß, IL8, TNF-α, IL10, TGFß, INOS and arginase gene expressions, whereas SOD upregulation was found in the GBLE condition. The mRNA expressions of IL1ß, 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.

Impact of Short-Term Fasting on The Rhythmic Expression of the Core Circadian Clock and Clock-Controlled Genes in Skeletal Muscle of Crucian Carp (Carassius auratus).

The peripheral tissue pacemaker is responsive to light and other zeitgebers, especially food availability. Generally, the pacemaker can be reset and entrained independently of the central circadian structures. Studies involving clock-gene expressional patterns in fish peripheral tissues have attracted considerable attention. However, the rhythmic expression of clock genes in skeletal muscle has only scarcely been investigated. The present study was designed to investigate the core clock and functional gene expression rhythms in crucian carp. Meanwhile, the synchronized effect of food restrictions (short-term fasting) on these rhythms in skeletal muscle was carefully examined. In fed crucian carp, three core clock genes (Clock, Bmal1a, and Per1) and five functional genes (Epo, Fas, IGF1R2, Jnk1, and MyoG) showed circadian rhythms. By comparison, four core clock genes (Clock, Bmal1a, Cry3, and Per2) and six functional genes (Epo, GH, IGF2, Mstn, Pnp5a, and Ucp1) showed circadian rhythms in crucian carp muscle after 7-day fasting. In addition, three core clock genes (Clock, Per1, and Per3) and six functional genes (Ampk1a, Lpl, MyoG, Pnp5a, PPARα, and Ucp1) showed circadian rhythms in crucian carp muscle after 15-day fasting. However, all gene rhythmic expression patterns differed from each other. Furthermore, it was found that the circadian genes could be altered by feed deprivation in crucian carp muscle through the rhythms correlation analysis of the circadian genes and functional genes. Hence, food-anticipatory activity of fish could be adjusted through the food delivery restriction under a light⁻dark cycle. These results provide a potential application in promoting fish growth by adjusting feeding conditions and nutritional state.

Effects of short-term fasting on the rhythmic expression of core circadian clock and functional genes in skeletal muscle of goldfish (Carassius auratus).

Molecular oscillators exist in peripheral tissues like pacemaker cells. Food intake is a dominant zeitgeber for peripheral clocks in vertebrates. Fasting is a physiological stress that elicits well-known metabolic adaptations, however, little is known about the effects of the rhythmic expression of clock components in skeletal muscle following short-term fasting in goldfish. Here, we characterized the molecular clock components and their daily transcription in COSINOR, and assessed the effect of 7-day fasting on the circadian patterns of the candidate genes expression in goldfish skeletal muscle. For the core clock genes, clock, bmal1a, cry1, cry2, cry3, per1, per2 and per3 showed circadian rhythmicity in fed goldfish, but not for bmal1a, cry2 and per1 in the fasted state. Of the 8 candidate functional genes analyzed, igf1, igf2 and igfbp2 showed circadian rhythmicity in the fed state, but circadian pattern was only observed for mRNA of myog, igfbp2 and mstn in fasted goldfish. Additionally, Spelman's correlation analysis showed the circadian expression of the myog and mstn presented positive and negative correlation with the transcription pattern of clock and per2 genes in fasted goldfish, respectively. Our results demonstrated that the peripheral clocks might be reset to respond rapidly to withholding of food in teleost skeletal muscle.

Global analysis of transcriptome sequences highlights accelerated evolution of immune genes in Danio choprae and Danio albolineatus.

Danio fishes, a small type animal with short sexual cycles, are model vertebrate species. To investigate the genic evolution of this genus, the transcriptomes from Danio choprae and Danio albolineatus were sequenced by Illumina HiSeq 4000 platform. A total of 128,427,304 sequence reads from two Danio fishes were generated by Next Generation Sequencing. The resulting in two assemblies contained 88,682 and 88,029 unigenes in the Danio choprae and Danio albolineatus. Analysis of the orthologs from the Danio choprae and Danio albolineatus provided consistent evidence for the accelerated genic evolution in the Danio fishes. Several genes referring to immune functions under positive selection were identified by branch site model analysis, such as REL, GTF2E1, STAT6, MPG in Danio choprae and CYP17A1, ADORA2A, MYCN in Danio albolineatus. Our data provide novel insights into the adaptation in Danio fishes and is useful for understanding the genetic basis of adaptation in zebrafish.