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This study aimed to investigate the impact of dietary protein levels (DPLs) on the growth performance, plasma parameters, nutritional metabolism, and the mTOR pathway in Chinese perch. Three replicates of Chinese perch (34.39 ± 0.16 g) were provided with five diets containing varying protein levels (39.99 %, 43.09 %, 46.04 %, 49.14 %, and 52.12 %) over an 8-week period. The results revealed that, as DPLs increased, the final weight (FW), daily weight gain (DWG)), specific growth rate (SGR) and protein efficiency ratio (PER) initially rose and then declined, reaching their peak at 49.14 % DPL group. The feed conversion ratio (FCR) decreased first and then increased. In addition, compared to 46.04 % DPL group, the 39.99 % DPL group significantly suppressed whole body crude protein, liver crude protein, and liver crude lipid contents. The 52.12 % DPL group significantly increased liver crude lipid content and aspartate aminotransferase (AST) activity, and increased plasma total cholesterol (TC), total triglyceride (TG) and total lipase (TL) concentrations. However, in Chinese perch fed with 46.04 % DPL, the expression of genes associated with liver amino acid catabolism (ast, gdh and ampd), lipid hydrolysis, and oxidation (hsl, lpl and cs), as well as glycolysis (pk), was significantly increased. And the expression levels of mTOR pathway-related genes (s6k and mtor) and the phosphorylation level of p-S6 were significantly elevated in the liver of fish fed with 46.04 % DPL. In summary, a low DPL (39.99 %) significantly impedes the growth performance of Chinese perch, resulting in an elevated feed conversion ratio. And a high DPL (52.12 %) can induce lipid deposition in the liver of Chinese perch, increase metabolic stress, and lead to liver damage. Additionally, piecewise regression analysis and second-degree polynomial analysis revealed that optimal protein level in Chinese perch feed is suggested to range between 46.10 % and 48.71 %. This study provides partial theoretical basis for the design of commercial practical feed formulas for Chinese perch.

The objective of the study was to investigate the effects of dietary carbohydrate on growth performance, feed utilization, proximate compositions, and hepatic glucolipid metabolism in Chinese perch. Triplicate groups of Chinese perch (29.51 ± 0.24 g) were fed five isonitrogenous and isolipidic diets containing graded levels of carbohydrate (2.4%, 9.7%, 16.5%, 23.9%, and 30.1%) for 8 weeks. The results based on second-order polynomial regression analysis of specific growth rate (SGR) proved that Chinese perch obtained the best growth performance at the 10.93% dietary carbohydrate level. Compared to 2.4%-carbohydrate group, 9.7%-carbohydrate group exhibited the increase in daily weight gain (DWG) and protein retention ratio (PER) and the decrease in feed conversion efficiency (FCR). Moreover, 9.7%-carbohydrate group showed the increase in the mRNA levels of pfk and cs involved in aerobic oxidation pathway, the content of liver glycogen increased significantly, and no change in glucose level. Above results suggested that a part of carbohydrates may be converted into energy for growth by aerobic oxidation pathway, or converted into glycogen for storage to maintain glucose homeostasis in Chinese perch fed with moderate-carbohydrate diet. Differently, 23.9%-carbohydrate and 30.1%-carbohydrate groups showed the decrease in DWG and PER, and the increase in FCR, compared to 2.4%-carbohydrate group. Additionally, 23.9%-carbohydrate and 30.1%-carbohydrate group presented the increase in the mRNA levels of g6pca and pc involved in gluconeogenesis, which caused the increase in plasma glucose level. However, other plasma indices related to TG-metabolite and its transport (total cholesterol, and low-density lipoprotein) did not show difference, which combined with elevated fatty acid synthesis-related gene accα mRNA level resulted in excessive liver lipid deposition. Above results indicated that unregulated gluconeogenesis and invalid outward transportation of liver TG-rich metabolites may account for the glucose intolerance of Chinese perch fed with carbohydrate-rich diet. Furthermore, compared to the results from the 4th week, the expression of gk, pfk, pc, cs, and accα genes significantly increased in the 30.1%-carbohydrate group at the 8th week. Both liver and muscle exhibited a significant elevation in crude fat content, while Daily DWG experienced a noticeable decrease. These results indicated that the glucose metabolism and growth of Chinese perch in the high-carbohydrate group are influenced by the interaction of feeding duration and carbohydrate levels. And within a certain time frame, Chinese perch can tolerate a diet rich in carbohydrates.

Metabotropic glutamate receptor 4 (mGluR4) is widely regarded as an umami receptor activated by L-glutamate to exert essential functions. Numerous studies have shown that umami receptors participate in food intake regulation. However, little is known about mGluR4’s role in mediating food ingestion and its possible molecular mechanism. Mandarin fish, a typical carnivorous fish, is sensitive to umami substances and is a promising vertebrate model organism for studying the umami receptor. In this study, we identified the mGluR4 gene and conducted evolutionary analyses from diverse fish species with different feeding habits. mGluR4 of mandarin fish was cloned and functionally expressed to investigate the effects of L-glutamate on mGluR4. We further explored whether the signal pathway mGluR4-Ca2+-ERK1/2 participates in the process in mandarin fish brain cells. The results suggest that L-glutamate could regulate Neuropeptide Y (Npy) via the mGluR4-Ca2+-ERK1/2 signaling pathway in mandarin fish. Our findings unveil the role of mGluR4 in feeding decisions and its possible molecular mechanisms in carnivorous fishes.

Neuropeptide Y receptor Y2 (npy2r) is an important receptor gene involved in anxiety and feeding regulation in mammals. Since NPY receptors have different receptor gene deletions in mammals and teleost fish, it is not clear whether npy2r has the similar function in fish as in mammals. In this study, we used the CRISPR/Cas9 system to establish npy2r-deficient medaka (Oryzias latipes). Unexpectedly, the deletion of npy2r resulted in the npy2r+/− medaka were all-male, therefore, npy2r homozygous mutant lines could not be established. The deletion of npy2r increased the food intake in medaka, and the expression levels of appetite stimulating genes (agrp, npy) increased significantly, while the expression levels of anorexia factors (cck, pomc) decreased significantly. Moreover, the absence of npy2r significantly increased the total length and body weight of medaka. The mirror test and open field test showed that npy2r+/− medaka improved sociability and reduced anxiety-like behavior, qRT-PCR analysis showed that the expression levels of anxiety related genes (th1, th2, gr1, gr2, and mr) in npy2r+/− medaka were significantly decreased. So far, this is the first npy2r gene knockout model established in fish and demonstrates that npy2r plays an important role in the regulation of reproduction, feeding and anxiety in fish.

Visual perception plays a crucial role in foraging, avoiding predators, mate selection, and communication. The regulation of color vision is largely dependent on opsin, which is the first step in the formation of the visual transduction cascade in photoreceptor cells. Short-wave-sensitive 1 (sws1) is a visual pigment that mediates short-wavelength light transduction in vertebrates. The depletion of sws1 resulted in increased M-opsin in mice. However, there is still no report on the visual function of sws1 in teleost fish. Here, we constructed the sws1 knockout medaka using CRISPR/Cas9 technology. The 6 dph (days post-hatching) medaka sws1−/− larvae exhibited significantly decreased food intake and total length at the first feeding stage, and the mRNA levels of orexigenic genes (npy and agrp) were significantly upregulated after feeding. The swimming speed was significantly reduced during the period of dark-light transition stimulation in the sws1-mutant larvae. Histological analysis showed that the thickness of the lens was reduced, whereas the thickness of the ganglion cell layer (GCL) was significantly increased in sws1−/− medaka larvae. Additionally, the deletion of sws1 decreased the mRNA levels of genes involved in phototransduction (gnb3b, grk7a, grk7b, and pde6c). We also observed increased retinal cell apoptosis and oxidative stress in sws1 knockout medaka larvae. Collectively, these results suggest that sws1 deficiency in medaka larvae may impair visual function and cause retinal cell apoptosis, which is associated with the downregulation of photoconduction expression and oxidative stress.

Mandarin fish (Siniperca chuatsi) is one of the most economically important fish in China. However, it has the peculiar feeding habit that it feeds solely on live prey fish since first-feeding, while refuses dead prey fish or artificial diets. After the specific training procedure, partial individuals could accept dead prey fish and artificial diets. The genetic basis of individual difference in artificial diet feeding habit is still unknown. In the present study, the resequencing was performed between 10 individuals which could be domesticated to accept artificial diets and 10 individuals which could not. Through the selective sweep analysis based on heterozygosity (Hp) and population differentiation coefficient (Fst), 57 candidate windows were identified as the putative selected regions for feeding habit domestication of mandarin fish, involved in 149 genes. These genes were related to memory, vision and olfaction function, which could be potential targets of molecular marker assistant breeding of artificial diet feeding trait. Beside of the DNA sequence, we also explored the potential role of DNA methylation in feeding habit domestication in mandarin fish. Whole-genome bisulfite sequencing was performed between the individuals which could be domesticated to accept artificial diets and those could not. 5,976 differentially methylated regions were identified, referring to 3,522 genes, such as the genes involved in cAMP signaling pathway. The DNA methylation changes of these genes might contribute to the adaption of artificial diets in mandarin fish. In conclusion, the putative selected regions and the differentially methylated regions were identified in the whole genome, providing new insights into the feeding habit domestication from live prey fish to artificial diets in mandarin fish. And the involved genes were identified as the candidate genes for molecular breeding of artificial diet utilization in mandarin fish.

Vertebrates need suitable lipases to digest lipids for the requirement of energy and essential nutrients; however, the main digestive lipase genes of fishes have certain controversies. In this study, two types of digestive lipase genes (pancreatic lipase (pl) and bile salt-activated lipase (bsal)) were identified in mammals and fishes. The neighborhood genes and key active sites of the two lipase genes were conserved in mammals and fishes. Three copies of PL genes were found in mammals, but only one copy of the pl gene was found in most of the fish species, and the pl gene was even completely absent in some fish species (e.g., zebrafish, medaka, and common carp). Additionally, the hydrophobic amino acid residues (Ile and Leu) which are important to pancreatic lipase activity were also absent in most of the fish species. The PL was the main digestive lipase gene in mammals, but the pl gene seemed not to be the main digestive lipase gene in fish due to the absence of the pl gene sequence and the important amino acid residues. In contrast, the bsal gene existed in all fish species, even two to five copies of bsal genes were found in most of the fishes, but only one copy of the BSAL gene was found in mammals. The amino acid residues of bile salt-binding sites and the three-dimensional (3D) structure modeling of Bsal proteins were conserved in most of the fish species, so bsal might be the main digestive lipase gene in fish. The phylogenetic analysis also indicated that pl or bsal showed an independent evolution between mammals and fishes. Therefore, we inferred that the evolutionary selection of the main digestive lipase genes diverged into two types between mammals and fishes. These findings will provide valuable evidence for the study of lipid digestion in fish.

The purpose of this study is to compare the effects of three live feeds for the larvae of the domesticated strain of mandarin fish (Siniperca chuatsi). Larvae of this strain of mandarin fish with salinity activation ingested Artemia nauplii was firstly observed. The larvae at 3 days post hatch (dph) were reared from first feeding to 9 dph and fed on starving (no food supplied), zebrafish (Danio rerio) (newly hatched larvae), Artemia nauplii and Daphnia magna, respectively. The results showed that the highest survival was found in the newly hatched larvae group (41.7 ± 1.5 %), and the lowest growth was found in the Daphnia group, respectively. A high food intake was found in the Artemia nauplii group, and the npy expression between the Artemia nauplii and newly hatched larvae groups had no significant difference (p＞0.05), whereas they were significantly lower than that of the starving group (p＜0.05). At the first feeding day, the activities of amylase, lipase and trypsin and the target of rapamycin (tor) expression in the Artemia nauplii and newly hatched larvae groups were significantly higher than those of the starving group. These results suggested that larvae of this strain of mandarin fish could feed on the newly hatched larvae and Artemia nauplii, and both of them could be digested, but the digestion might be insufficient in the Artemia nauplii group. Together, this study indicates that larvae of this strain of mandarin fish can ingest and digest Artemia nauplii during first-feeding.

The medaka (Oryzias latipes) is an excellent vertebrate model for studying the development of the retina. Its genome database is complete, and the number of opsin genes is relatively small compared to zebrafish. Short wavelength sensitive 2 (sws2), a G-protein-coupled receptor expressed in the retina, has been lost in mammals, but its role in eye development in fish is still poorly understood. In this study, we established a sws2a and sws2b knockout medaka model by CRISPR/Cas9 technology. We discovered that medaka sws2a and sws2b are mainly expressed in the eyes and may be regulated by growth differentiation factor 6a (gdf6a). Compared with the WT, sws2a−/− and sws2b−/− mutant larvae displayed an increase in swimming speed during the changes from light to dark. We also observed that sws2a−/− and sws2b−/− larvae both swam faster than WT in the first 10 s of the 2 min light period. The enhanced vision-guided behavior in sws2a−/− and sws2b−/− medaka larvae may be related to the upregulation of phototransduction-related genes. Additionally, we also found that sws2b affects the expression of eye development genes, while sws2a is unaffected. Together, these findings indicate that sws2a and sws2b knockouts increase vision-guided behavior and phototransduction, but on the other hand, sws2b plays an important role in regulating eye development genes. This study provides data for further understanding of the role of sws2a and sws2b in medaka retina development.

In mammals, lipoprotein lipase (LPL) has been found to play an important role in lipid mentalism and deposition. LPL deficiency in humans (Homo sapiens) and mice (Mus musculus) tends to cause hypertriglyceridemia. The lpl gene is not expressed in adult mammalian liver, but is in adult fish liver. The functions provided by the lpl gene are diverse in vertebrates. Here, we knocked out the lpl gene in Japanese medaka (Oryzias latipes) with the CRISPR/Cas9 system. The lpl-knockout (KO) homozygous individuals showed severe developmental defects with an extremely emaciated and deformed body and only accounted for about 5% of the F2 fish. This is consistent with the findings in mice but disaccords with the results for zebrafish (Danio rerio). Compared with wild-type (WT) madaka, the mRNA level of lpl in lpl-KO heterozygous mutant was significantly higher in the muscle, showed no significant difference in the liver, and was significantly lower in the heart. Under lpl heterozygous deficiency, the relative area of Oil Red O and triglycerides (TG) level in the liver, heart and muscle tissue covaried with levels of lpl mRNA in medaka. The lpl heterozygous deficiency did not affect the levels of TG, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and total cholesterol (TC) in the plasma of medaka, which is inconsistent with the findings in mammals. In general, the lpl gene plays an important role in the growth and development and is closely related to lipid deposition of medaka.

Abstract Background As economical traits, food habits domestication can reduce production cost in aquaculture. However, the molecular mechanism underlying food habits domestication has remained elusive. Mandarin fish (Siniperca chuatsi) only feed on live prey fish and refuse artificial diets. In the present study, we domesticated mandarin fish to feed on artificial diets. The two groups were obtained, the fish did not eat artificial diets or ate artificial diets during all of the three domestication processes, named Group W or X, respectively. Results Using transcriptome and metabolome analysis, we investigated the differentially expressed genes and metabolites between the two groups, and found three common pathways related to food habit domestication, including retinol metabolism, glycerolipid metabolism, and biosynthesis of unsaturated fatty acids pathways. Furthermore, the western blotting and bisulfite sequencing PCR analysis were performed. The gene expression of TFIIF and histone methyltransferase ezh1 were significantly increased and decreased in the fish of Group X, respectively. The total DNA methylation levels of TFIIF gene and tri-methylation of histone H3 at lysine 27 (H3K27me3) were significantly higher and lower in the fish of Group X, respectively. Conclusion It was speculated that mandarin fish which could feed on artificial diets, might be attributed to the lower expression of ezh1, resulting in the decreased level of H3K27me3 and increased level of DNA methylation of TFIIF gene. The high expression of TFIIF gene might up-regulate the expression of genes in retinol metabolism, glycerolipid metabolism and glycerophosphoric metabolism pathways. Our study indicated the relationship between the methylation of DNA and histone and food habits domestication, which might be a novel molecular mechanism of food habits domestication in animals.

Low occurrence of swimbladder inflation often leads to vertebral skeletal malformations and poor swimming ability, which exert numerous effects on growth, feeding, buoyancy regulation and survival rate of fish larvae. In the present study, 713 mandarin fish (Siniperca chuatsi) larvae samples ranging from 5 to 11 days post hatch (DPH) were collected to detect the rate of swimbladder inflation and calculate the body density of each sample. Swimbladder inflated fish larvae were increased with the development of DPH except 7 DPH, with 86.21% in 11 DPH whereas 27.59% in 5 DPH. Our results indicate that 5–11 DPH is the main stage for swimbladder inflation in mandarin fish larvae, following the onset of exogenous feeding. The body density of swimbladder inflated (SBI) fish larvae (0.983 ± 0.016 g/cm3) was lower than the swimbladder non-inflated (SBN) ones (1.023 ± 0.018 g/cm3), while SBI fish larvae were significant heavier (p

This study aims to investigate the effects of three feed attractants on the growth performance, body composition, biochemical indicators, liver histology, lipid metabolism and appetite of Chinese perch (Siniperca chuatsi). An eight-week feeding trial was conducted using four diets, including a basal diet of 51% fish meal without attractant (control), with 5% stickwater (SW), 5% squid paste (SP) and a 5% mixture of SW and SP a ratio of 1:2 (Mix), respectively. The results demonstrated that SW diet significantly increased the weight gain rate and feed intake (P

Abstract Background Early nutritional programming affects a series of metabolism, growth and development in mammals. Fish also exhibit the developmental plasticity by early nutritional programming. However, little is known about the effect of early amino acid programming on growth and metabolism. Methods In the present study, zebrafish (Danio rerio) was used as the experimental animal to study whether early leucine stimulation can programmatically affect the mechanistic target of rapamycin (mTOR) signaling pathway, growth and metabolism in the later life, and to undercover the mechanism of epigenetic regulation. Zebrafish larvas at 3 days post hatching (dph) were raised with 1.0% leucine from 3 to 13 dph during the critical developmental stage, then back to normal water for 70 days (83 dph). Results The growth performance and crude protein content of zebrafish in the early leucine programming group were increased, and consistent with the activation of the mTOR signaling pathway and the high expression of genes involved in the metabolism of amino acid and glycolipid. Furthermore, we compared the DNA methylation profiles between the control and leucine-stimulated zebrafish, and found that the methylation levels of CG-differentially methylated regions (DMGs) and CHH-DMGs of genes involved in mTOR signaling pathway were different between the two groups. With quantitative PCR analysis, the decreased methylation levels of CG type of Growth factor receptor-bound protein 10 (Grb10), eukaryotic translation initiation factor 4E (eIF4E) and mTOR genes of mTOR signaling pathway in the leucine programming group, might contribute to the enhanced gene expression. Conclusions The early leucine programming could improve the protein synthesis and growth, which might be attributed to the methylation of genes in mTOR pathway and the expression of genes involved in protein synthesis and glycolipid metabolism in zebrafish. These results could be beneficial for better understanding of the epigenetic regulatory mechanism of early nutritional programming.

Abstract Background Taste is fundamental to diet selection in vertebrates. Genetic basis of sweet taste receptor in the shaping of food habits has been extensively studied in mammals and birds, but scarcely studied in fishes. Grass carp is an excellent model for studying vegetarian adaptation, as it exhibits food habit transition from carnivory to herbivory. Results We identified six sweet taste receptors (gcT1R2A-F) in grass carp. The four gcT1R2s (gcT1R2C-F) have been suggested to be evolved from and paralogous to the two original gcT1R2s (gcT1R2A and gcT1R2B). All gcT1R2s were expressed in taste organs and mediated glucose-, fructose- or arginine-induced intracellular calcium signaling, revealing they were functional. In addition, grass carp was performed to prefer fructose to glucose under a behavioral experiment. Parallelly, compared with gcT1R2A-F/gcT1R3 co-transfected cells, gcT1R2C-F/gcT1R3 co-transfected cells showed a higher response to plant-specific fructose. Moreover, food habit transition from carnivory to herbivory in grass carp was accompanied by increased gene expression of certain gcT1R2s. Conclusions We suggested that the gene expansion of T1R2s in grass carp was an adaptive strategy to accommodate the change in food environment. Moreover, the selected gene expression of gcT1R2s might drive the food habit transition from carnivory to herbivory in grass carp. This study provided some evolutional and physiological clues for the formation of herbivory in grass carp.

The effective utilization of carbohydrate feed sources is important to save protein feed resources and reduce formula feed costs in fish aquaculture. The present study investigated whether high-carbohydrate diet stimulus in the early larval stage of the grass carp (Ctenopharyngodon idellus) could affect glucose metabolism in subsequent life stages (i.e., the juvenile). A 73-day feeding trial was divided to five groups (four treatments and a control). The trial was conducted from the first feeding of larval fish. During the first 66 days, the control was continuously fed a commercial grass carp diet (CD), the FF-3 group was initially fed with 60% malty dextrin diet (MD) for 3 days and then with the CD for 63 days, the YE-7 group was initially fed with 60% MD after yolk-sac exhaustion for 7 days and with the CD for 56 days, the FF-10 group was initially fed with 60% MD for 10 days and then with CD for 56 days, and the FF-66 group was continuously fed to 60% MD. During the next 7 days, all the treatment groups and the control were fed with 35% MD as challenge test. The results showed that short-term stimulation with high-carbohydrate resulted in significantly increased body weights. After 73 days of feeding, the final body weights of the FF-3 and YE-7 groups were significantly higher than that of the control. Early high-carbohydrate manipulation changed the normal structure of the gut tissues of juvenile fish. After a short challenge test (7 days) performed with a 35% carbohydrate diet in juvenile grass carp, the results showed that high-carbohydrate dietary histories affected plasma metabolites and liver (muscle) glycogen storage in the juvenile fish. The mRNA levels of glucokinase and fatty acid synthetase genes increased significantly at the juvenile stage in the first-feeding groups. The phosphoenolpyruvate carboxykinase gene expression in the FF-3 and FF-10 groups decreased by varying degrees. The glycogen synthase gene expression levels in all groups were not affected. Long-term intake of high-carbohydrate feed (FF-66 group) led to the nutritional deficiency and disorder of the glucose metabolism system. In conclusion, our results demonstrated that a high-carbohydrate diet stimulus at first-feeding stage can affect the growth performance, gut morphology, and the homeostasis of glucose metabolism in juvenile of grass carp.

The mandarin fish (Siniperca chuatsi) is an important economic fish species in China. Many carnivorous fish larvae feed on zooplankton or microdiets. However, the mandarin fish larvae feed on live prey fish exclusively, while refusing zooplankton or microdiets. A stable supply of palatable live prey fish results in high costs. Moreover, the application of live prey fish might bring the risk of pathogens. However, little is known about the genes underlying the food preference of mandarin fish larvae. Partial offspring of the domesticated strain could feed brine shrimp from three days post-hatching (dph), the open mouth day. In the present study, the mandarin fish larvae were randomly divided into three groups, then treated differently at 3 dph: (1) unfed; (2) fed with live prey fish; (3) fed with brine shrimp (Artemia). Differentially expressed genes were identified by RNA-seq. The differential expression of the transcription factors involved in retinal photoreceptor development and differentiation might contribute to the intake of brine shrimp in mandarin fish larvae. Meanwhile, the digestive enzyme genes involved in protein, fat, and carbohydrate digestion have been expressed in mandarin fish larvae at 3 dph, contributing to the digestion of ingested food. Our study provides an overview of genes and biological processes involved in the peculiar food preference at the first-feeding stage in mandarin fish larvae and has critical importance to the future application of non-fish live feeds in the culture of mandarin fish larvae.

The disease outbreak of Chinese perch poses a severe threat to aquaculture industry. To explore the relationship between microbial communities in farming water and those in the intestine, and to further reveal the relationship between fish disease and environmental microbes (internal and external environments), 16S rRNA were sequenced. Although the composition and abundance of bacterial phyla varied with month in water samples, Actinobacteria and Proteobacteria were always the top 2 dominant phyla. Furthermore, most dominant genera in water were not significantly affected by environmental factors and seasonal changes, but there were specific dominant genera for each month. Besides, most dominant genera in water were not pathogenic bacteria. The Chinese perch ponds often breakout disease in September. 16S rRNA of the gut samples from the diseased and healthy Chinese perch were sequenced. In September, high water temperature and high contents of nitrite and ammonia-N might be environmental factors causing the disease outbreak of Chinese perch. The gut samples presented similarity in the phylum categories between the diseased and healthy Chinese perch, but they exhibited different abundances of microbiota at phylum level. Fusobacteria (44 %) exhibited the highest abundance in diseased Chinese perch, whereas Firmicutes (58 %) displayed the highest abundance in healthy Chinese perch. In addition, Lactococcus were dominant genus in September water samples and gut samples from diseased fish, which might be the pathogenic bacteria to infect the Chinese perch. Our findings provide novel information for improving microbial communities in aquaculture environments.

Regulation of food intake is associated with nutrient-sensing systems and the expression of appetite neuropeptides. Nutrient-sensing systems generate the capacity to sense nutrient availability to maintain energy and metabolism homeostasis. Appetite neuropeptides are prominent factors that are essential for regulating the appetite to adapt energy status. However, the link between the expression of appetite neuropeptides and nutrient-sensing systems remains debatable in carnivorous fish. Here, with intracerebroventricular (ICV) administration of six essential amino acids (lysine, methionine, tryptophan, arginine, phenylalanine, or threonine) performed in mandarin fish (Siniperca chuatsi), we found that lysine and methionine are the feeding-stimulating amino acids other than the reported valine, and found a key appetite neuropeptide, neuropeptide Y (NPY), mainly contributes to the regulatory role of the essential amino acids on food intake. With the brain cells of mandarin fish cultured in essential amino acid deleted medium (lysine, methionine, histidine, valine, or leucine), we showed that only lysine deprivation activated the general control nonderepressible 2 (GCN2) signaling pathway, elevated α subunit of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation, increased activating transcription factor 4 (ATF4) protein expression, and finally induced transcription of npy. Furthermore, pharmacological inhibition of GCN2 and eIF2α phosphorylation signaling by GCN2iB or ISRIB, effectively blocked the transcriptional induction of npy in lysine deprivation. Overall, these findings could provide a better understanding of the GCN2 signaling pathway involved in food intake control by amino acids.

Abstract Background Previous studies in teleost have demonstrated the adaptive strategy to maintain hepatic lipid homeostasis within certain limit. The excess of fat-intake could induce abnormal lipid deposition in liver but not adipose tissue. However, the molecular mechanism between the impaired lipid homeostasis and the aggravated lipid deposition in liver has not been elucidated well in fish. Methods Four isonitrogenous diets with different fat levels (2, 7, 12 and 17%) were formulated, named L2, L7, L12 and L17 respectively, and fed Chinese perch (44.50 ± 0.25 g) to apparent satiation for five weeks. Growth index, triglyceride concentrations and expression of genes involved in lipid metabolism were measured. Results The maximal growth performance and food intake were observed in L12 group. The lipid content in liver and serum were comparable in L2, L7 and L12 groups, while they were increased significantly in L17 group. Histology analysis also demonstrated that mass lipid droplets emerged in hepatocyte and then induced hepatic steatosis in L17 group. Compared to L2 group, the lipolytic genes related to fatty acids (FAs) transport (lpl & hl) and FAs β-oxidation (cpt1 & cs) were increased in L7 and L12 group. Relative mRNA levels of the gluconeogenesis (pc, pepck & g6pase) were also increased, in contrast, the lipogenic genes (srebp1, accα & fas) were decreased. Compared to L12 group, L17 group had higher mRNA levels of the FAs transport and the lipogenesis. But the lipolytic genes related to FAs β-oxidation were steady and the mRNA levels of gluconeogenesis were down-regulated instead. Conclusions Within certain limit, the increase of dietary fat in L7 and L12 group was propitious to reduce the consumption of protein and improve growth performance in Chinese perch. It was due to the homeostasis of hepatic triglyceride (TG) pool and serum glucose through promoting the FAs β-oxidation and gluconeogenesis respectively. Both the increase of lipogenesis and the absence of FAs β-oxidation in L17 group could trigger the esterification of FAs, indeed, the inhibition of gluconeogenesis could also aggravate triglyceride accumulation in liver and induce hepatic steatosis.

Glucose-6-phosphatase (g6p) plays a crucial role in glucose homeostasis by completing the final step of gluconeogenesis in mammals. Multiple g6p homologous genes were identified in fish due to whole genome replication. However, the g6p homologous genes closely related to glucose metabolism have not been systematically screened and the DNA methylation of g6p homologous genes induced by dietary carbohydrates is also poorly explored in fish. In this study, g6p homologous genes from different species were compared and analyzed. The tissue expression profiles of g6p homologous genes were identified. Then, two diets with different starch levels (0%, 20 %) were fed to Chinese perch for 8 weeks to evaluate the relative mRNA levels and DNA methylation levels of g6p homologous genes in liver. Results showed that g6pcb2 was lost in genome of Chinese perch. g6pca and g6pcb1 were mainly expressed in liver. g6pc2 and g6pc3 showed extremely low expression levels in liver. Moreover, compared with 0% starch group, 20 % starch group exhibited the significant decrease in relative mRNA levels of g6pca and g6pcb1, no difference in relative mRNA levels of g6pc2 and g6pc3, and the significant increase in DNA methylation of g6pca. Our results suggested that g6pca and g6pcb1 might coordinate to regulate the gluconeogenesis pathway and high-carbohydrate diet might up-regulate the DNA methylation level of g6pca to inhibit the mRNA expression level of g6pca, thereby down-regulating the gluconeogenesis pathway. g6pc2 and g6pc3 might not be related to the post meal regulation of glucose metabolism.

As a carnivorous fish, Chinese perch have a poor ability to utilize carbohydrate. To explore the effect of glucose exposure on glucose and lipid metabolism in larval Chinese perch, as well as on the domestication with high-starch artificial diet in juvenile fish. After a short-term glucose immersion, the growth performance, glucose and lipid metabolism, and DNA methylation level of glucose metabolism relative genes were measured. After a 3-month normal feeding trial, the success rate of domestication and the feed intake were examined. Success rate of feed intake and growth performance (the total length, body length and weight) in larval and juvenile fish in GLU group were all significantly higher than CON group (P

High carbohydrate diet-induced damage in gut is linked to changes in gut permeability and microbiota. However, the mechanisms of action are not clear, especially in non-mammals. We performed the gut microbiota profiling in Chinese perch fed with different content of starch diets (0, 10, and 20%) by 16S rRNA sequencing. The gut permeability, metabolites, histological analysis, and inflammatory infiltration were evaluated. We found that gut microbial diversity, beneficial bacteria quantity, and lactic acid content were higher in C10 group than in the other groups. The lower level of gut microbial diversity was observed in C20 group, and mycoplasma was the overwhelmingly dominant species, but the butyric acid-producing bacteria and butyric acid level were significantly reduced. The gut permeability in C20 group was also increased due to the decreased mRNA expression levels of tight junction proteins caused by the butyric acid deficiency and gut lipid droplets accumulation. Then a large amount of LPS penetrated into the plasma, resulting in inflammation. These results suggested that high carbohydrate diet-induced damage in gut could be attributed to the endotoxemia, permeability, and gut microbiota, especially the role of mycoplasma and butyric acid-producing bacteria. In addition, predictive functional profiling of microbial communities by PICRUSt showed that C10 group enriched pathway related to membrane transport and down-regulated the pathways related to energy, coenzyme factor and vitamin metabolism, while C20 group exhibited reversed results. These data showed that the high-carbohydrate diet reversed the beneficial changes in gut microbial metabolism resulted from the medium-carbohydrate diet, and further demonstrated that microbiota played a key role in the gut damage caused by the high-carbohydrate diet. Our findings provide a reference for the targeted regulation of gut microbiota to mitigate the damage caused by the increase in starch content in fish feed (cost saving).

Leptin affects food intake regulation and energy homeostasis in mammals, as opposed to mammals who have a single leptin gene, fish have duplicated leptin gene paralogues. Until now, most functional studies on fish focused on the first reported paralogue without much explanation on specific gene paralogue. This study successfully expressed two homologous recombinant mandarin fish leptin genes (LepA and LepB) for the first time. To explore the differential roles of these two gene paralogues involved in food intake and energy homeostasis, mandarin fish were treated with homologous recombinant LepA and LepB proteins by acute IP administration. The results showed that LepB inhibited the food intake of mandarin fish after acute IP administration through modifying the expressions of hypothalamic orexigenic genes, while LepA had no significant effect on its food intake. In addition, LepB administration decreased the hepatic glycogen level through regulating the gene expressions of glycogen synthase and glycogen phosphorylase in mandarin fish until 4 d, while LepA did not change the hepatic glycogen level as it failed to change the expressions of these regulatory genes. Moreover, LepA and LepB downregulated the expressions of key gluconeogenic genes (phosphofructokinase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase), indicating both mandarin fish leptins could regulate the rate of glucose production. However, these two gene paralogues presented secondary effects on lipid metabolism as they only enhanced the triglyceride level by modifying the gene expressions of adipose triglyceride lipase or acetyl CoA carboxylase just for 1 d after IP. Therefore, LepB played an important role in food intake and glucose homeostasis regulation, while LepA showed a limited role in gluconeogenesis and lipid metabolism.

Although studies have determined that epigenetics plays an essential role in regulating metabolism in mammals, research on nutrition-related DNA methylation remains to be lacking in teleosts. In the present study, we provided a hepatic whole-genome DNA methylation analysis in grass carp fed with moderate- or excessive-carbohydrate-level diet. Although a high-carbohydrate (HC) diet significantly changed the mRNA expression levels of metabolic genes, it did not affect the global genomic DNA methylation levels in grass carp liver. However, compared with the control group, 3,972 genes were hyper-methylated and 2,904 genes were hypo-methylated in the promoter region. Meanwhile, 10,711 genes were hyper-methylated and 6,764 genes were hypo-methylated in the gene body region in the HC group. These differentially methylated genes (DMGs) were enriched in multiple pathways, including carbohydrate metabolism, insulin pathway, lipid metabolism, and adipocytokine signaling pathway. In addition, the variations in DNA methylation significantly regulated the transcription levels of key genes of metabolism, which could affect the glucose concentrations and the lipid deposition of grass carp. Furthermore, we compared the DNA methylation alterations of genes in glucose metabolism and obesity pathways of grass carp with those of mammalian models in different nutritional states. The results showed that most of the DMGs in grass carp were also regulated by DNA methylation in mammals when the nutritional state changed. The findings revealed more differentially methylated regions and candidate genes for glucose metabolism and broken species boundaries.

Food intake of carnivorous fish decreases after feeding on a carbohydrate-rich diet. However, the molecular mechanism underlying the anorexia caused by high-carbohydrate diets has remained elusive. We domesticated the mandarin fish to feed on carbohydrate-rich (8%) diets. After 61 days of feeding, several fish (Group A) fed well on artificial diets during the whole feeding period; the other fish (Group B) fed well on artificial diets at the beginning of the feeding period, with their food intake then decreasing to half (anorexia) and then to zero for 5 days; and, finally, a negative control (Group C) fed on live prey fish throughout the experimental process. The plasma glucose was significantly higher in the mandarin fish of Group B than in those of Group A, whereas levels of hepatic glycogen and plasma triglyceride were significantly lower. Using transcriptome sequencing, we investigated the differentially expressed genes between Groups A and B and excluded the genes that were not differentially expressed between Groups A and C. The activation of mTOR and Jak/STAT pathways were found in the mandarin fish with anorexia, which was consistent with the higher expression levels of pepck and pomc genes. We found a higher expression of histone methyltransferase setd1b gene and an increased histone H3 tri-methylated at lysine 4 (H3K4me3) in the fish of Group B. Furthermore, using ChIP assay and inhibitor treatment, we found that the up-regulated H3K4me3 could activate pepck expression, which might have contributed to the hyperglycemia and anorexia in the mandarin fish that fed on carbohydrate-rich diets. Our study initially indicated a link between histone methylation and pepck expression, which might be a novel regulatory mechanism of fish who are fed a carbohydrate-rich diet.

Chinese perch (Siniperca chuatsi) is an important economic freshwater carnivorous fish. Commonly feeds on other live fish or bait fish, and accept an artificial diet only after standard training. The feeding habit of Chinese perch caused high aquaculture costs and consumer prices. However, only a few studies are conducted on Chinese perch live feed (bait fish) or live feed (mori) feeds. Therefore, the present study was conducted to investigate the effect of live feed (mori) and live feed (mori) feeds protein to energy ratio on Chinese perch. Three Iso-energetic diets (16 kJ/g) with graded levels of P/E ratios (19.79, 22.19 and 24.46 mg protein/kJ) each with a protein level of 28.18 %, 31.36 %, and 33.91 % (named A, B, and C) were formulated. Two separate experimental tanks systems were used. In one tanks system, a total of 90 kg live bait fish C. mrigala were distributed into three groups (triplicate tanks (1000 L) for each group) and fed with respective P/E ratio diets. In another tanks system, a total of 270 Chinese perch (30 fish/tank) were divided into three groups (triplicate tanks for each group), distributed into 9 (1000-L) fiberglass tanks and fed with assigned live bait fish at 4 % fish wet body weight, twice a day for 8 weeks. The results showed that P/E ratio in bait fish diets significantly affected bait fish and Chinese perch growth performance, feed utilization, body composition, nitrogen metabolism, protein synthesis, and energy homeostasis. Bait fish in Group A showed significantly lower weight gain (WG), specific growth rate (SGR), and higher Viscerosomatic index (VSI) and Hepatosomatic index (HSI) as compared to other groups. Correspondingly, bait fish in Group A showed significantly low whole-body crude protein and high lipid contents as compared to other groups. Similarly, Chinese perch in Group A, showed significantly lower WG, SGR, protein efficiency ratio (PER) and highest feed intake (FI), feed conversion ratio (FCR), VSI, HSI, and Intraperitoneal fat (IPF), as compared to other groups. Furthermore, Chinese perch in Group A, showed significantly lower whole body and muscle crude protein, and higher lipid contents as compared to other groups. Similarly, the nitrogen metabolism enzyme activities (ALT, AST, GDH, AMPD) and gene expression of GDH and AMPD in Group A of Chinese perch, were significantly lower than other groups. Moreover, the AMPK and mTOR pathways gene expression showed a low energy status and protein catabolism in Group A of Chinese perch as compared to other groups. From our results, we conclude that the P/E ratio in bait fish diet significantly affected bait fish and Chinese perch growth performance and nutrients metabolism. Keywords: Chinese perch, Bait fish, Protein/energy ratio, Enzyme activity, Gene expression, AMPK, mTOR

Abstract Although previous studies have indicated importance of taste receptors in food habits formation in mammals, little is known about those in fish. Grass carp is an excellent model for studying vegetarian adaptation, as it shows food habit transition from carnivore to herbivore. In the present study, pseudogenization or frameshift mutations of the umami receptors that hypothesized related to dietary switch in vertebrates, were not found in grass carp, suggesting other mechanisms for vegetarian adaptation in grass carp. T1R1 and T1R3 strongly responded to L-Arg and L-Lys, differing from those of zebrafish and medaka, contributing to high species specificity in amino acid preferences and diet selection of grass carp. After food habit transition of grass carp, DNA methylation levels were higher in CPG1 and CPG3 islands of upstream control region of T1R1 gene. Luciferase activity assay of upstream regulatory region of T1R1 (−2500-0 bp) without CPG1 or CPG3 indicated that CPG1 and CPG3 might be involved in transcriptional regulation of T1R1 gene. Subsequently, high DNA methylation decreased expression of T1R1 in intestinal tract. It could be a new mechanism to explain, at least partially, the vegetarian adaptation of grass carp by regulation of expression of umami receptor via epigenetic modification.

Social learning plays important roles in gaining new foraging skills and food preferences. However, the potential role and molecular mechanism of social learning in acquiring new feeding habits is less clear in fish. In the present study, we examined the success rate of feeding habit domestication from live prey fish to dead prey fish, as well as the food intake of dead prey fish in mandarin fish with or without feeders of dead prey fish as demonstrators. Here, we found that mandarin fish can learn from each other how to solve novel foraging tasks, feeding on dead prey fish. In addition, the analysis of gene expressions and signaling pathways of learning through Western blotting and transcriptome sequencing shows that the expression of the c-fos, fra2, zif268, c/ebpd and sytIV genes were significantly increased, and the anorexigenic pomc and leptin a expressions were decreased in fish of the learning group. The phosphorylation levels of protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the learning group were significantly higher than those of the control group, while the phosphorylation level of S6 ribosomal protein (S6) was lower. With the inhibitors of PKA and CaMKII signaling and the chromatin immunoprecipitation (ChIP) assay, we further found that the social learning of new feeding habits in mandarin fish could be attributed to the activation of the CaMKII signaling pathway and then the stimulation of the expression of the c-fos gene, which might be an important transcriptional factor to inhibit the expression of the anorexigenic gene pomc, resulting in the food intake of dead prey fish in mandarin fish. Altogether, our results support the hypothesis that social learning could facilitate the acquisition of novel feeding habits in fish, and it considerably increases the rate of subsequent individual food intake and domestication through the interaction between the learning gene c-fos and the appetite control gene pomc.

Mandarin fish refuse dead prey fish or artificial diets and can be trained to transform their inborn feeding habit. To investigate the effect of memory on feeding habit transformation, we compared the reaction time to dead prey fish and the success rate of feeding habit transformation to dead prey fish with training of mandarin fish in the 1st experimental group (trained once) and the 2nd experimental group (trained twice). The mandarin fish in the 2nd group had higher success rate of feeding habit transformation (100%) than those in the 1st group (67%), and shorter reaction time to dead prey fish (1 s). Gene expression of cAMP responsive element binding protein I (Creb I), brain-derived neurotrophic factor (Bdnf), CCAAT enhancer binding protein delta (C/EBPD), fos-related antigen 2 (Fra2), and proto-oncogenes c-fos (c-fos) involved in long-term memory formation were significantly increased in the 2nd group after repeated training, and taste 1 receptor member 1 (T1R1), involved in feeding habit formation, was significantly increased in brains of the 2nd group after repeated training. DNA methylation levels at five candidate CpG (cytosine–guanine) sites contained in the predicted CpG island in the 5′-flanking region of T1R1 were significantly decreased in brains of the 2nd group compared with that of the 1st group. These results indicated that the repeated training can improve the feeding habit transformation through the memory formation of accepting dead prey fish. DNA methylation of the T1R1 might be a regulatory factor for feeding habit transformation from live prey fish to dead prey fish in mandarin fish.