Integrated mRNA and miRNA analysis reveals the regulatory network of oxidative stress and inflammation in Coilia nasus brains during air exposure and salinity mitigation.

BACKGROUND: Air exposure is an inevitable source of stress that leads to significant mortality in Coilia nasus. Our previous research demonstrated that adding 10‰ NaCl to aquatic water could enhance survival rates, albeit the molecular mechanisms involved in air exposure and salinity mitigation remained unclear. Conversely, salinity mitigation resulted in decreased plasma glucose levels and improved antioxidative activity. To shed light on this phenomenon, we characterized the transcriptomic changes in the C. nasus brain upon air exposure and salinity mitigation by integrated miRNA-mRNA analysis. RESULTS: The plasma glucose level was elevated during air exposure, whereas it decreased during salinity mitigation. Antioxidant activity was suppressed during air exposure, but was enhanced during salinity mitigation. A total of 629 differentially expressed miRNAs (DEMs) and 791 differentially expressed genes (DEGs) were detected during air exposure, while 429 DEMs and 1016 DEGs were identified during salinity mitigation. GO analysis revealed that the target genes of DEMs and DEGs were enriched in biological process and cellular component during air exposure and salinity mitigation. KEGG analysis revealed that the target genes of DEMs and DEGs were enriched in metabolism. Integrated analysis showed that 24 and 36 predicted miRNA-mRNA regulatory pairs participating in regulating glucose metabolism, Ca2+ transport, inflammation, and oxidative stress. Interestingly, most of these miRNAs were novel miRNAs. CONCLUSION: In this study, substantial miRNA-mRNA regulation pairs were predicted via integrated analysis of small RNA sequencing and RNA-Seq. Based on predicted miRNA-mRNA regulation and potential function of DEGs, miRNA-mRNA regulatory network involved in glucose metabolism and Ca2+ transport, inflammation, and oxidative stress in C. nasus brain during air exposure and salinity mitigation. They regulated the increased/decreased plasma glucose and inhibited/promoted antioxidant activity during air exposure and salinity mitigation. Our findings would propose novel insights to the mechanisms underlying fish responses to air exposure and salinity mitigation.

Effects of effective microorganisms on the physiological status, intestinal microbiome, and serum metabolites of Eriocheir sinensis.

The compound known as effective microorganisms (EMs) is widely used in aquaculture to improve water quality, but how they affect the health of Chinese mitten crab (Eriocheir sinensis) is unclear, especially in terms of intestinal microbiota and serum metabolites. In this study, we fed juvenile crabs with an EM-containing diet to explore the effects of EM on the physiological status, intestinal microbiome, and metabolites of E. sinensis. The activities of alanine aminotransferase and alkaline phosphatase were significantly enhanced by EM, indicating that EM supplementation effectively enhanced the antioxidant capacity of E. sinensis. Proteobacteria, Tenericutes, Firmicutes, Bacteroidetes, and Actinobacteria were the main intestinal microbes in both the control and EM groups. Linear discriminant effect size analysis showed that Fusobacteriaceae, Desulfovibrio, and Morganella were biomarkers in the control group, and Exiguobacterium and Rhodobacteraceae were biomarkers in the EM group. Metabolomics analysis revealed that EM supplementation increased cellular energy sources and decreased protein consumption, and oxidative stress. Together, these results indicate that EM can optimize the intestinal microbiome and serum metabolites, thereby benefiting the health of E. sinensis.

Promotion of improved intestinal barrier health by soybean-derived bioactive peptides in Chinese mitten crab (Eriocheir sinensis) fed a low fishmeal diet.

To alleviate the growth inhibition, and intestinal damage of Chinese mitten crab (Eriocheir sinensis) induced by low fishmeal diets (LF), an 8-week feeding trial was conducted to evaluate the addition of dietary soybean-derived bioactive peptides (SBP) in LF diets on the regulation of growth, digestion and intestinal health. The crabs were fed isonitrogenous and isoenergetic conventional diet and LF diets (10 % fishmeal replaced by soybean meal, LF) supplemented with 0, 1 %, 2 %, 4 % and 6 % SBP, respectively. The results showed that LF diet inhibited growth while inclusion of SBP quadratically remitted the growth inhibition induced by LF. For digestive function, increasing addition level of SBP quadratically improved the α-amylase and trypsin activities. For antioxidant function, LF group significantly increased the malondialdehyde content, while SBP linearly decreased the malondialdehyde level and cubically increased the anti-superoxide anion activity and total antioxidant capacity level. For intestinal health, the peritrophic membrane (PM) almost completely separated from the inner wall of the intestinal lumen, the epithelial cells reduced, the muscularis became thinner and the apoptotic signals increased in LF group; with SBP addition, the intestinal morphology was improved, with the PM adhering to the inner wall of the intestinal lumen, an increase in the number of epithelial cells and an increase in the thickness of the muscularis. Additionally, there was a decrease in apoptotic signals. Dietary SBP also increased the expression of PT and Crustin1 quadratically and decreased the expression of ALF1 linearly, ALF3 and ILF2 quadratically.

Resveratrol ameliorates intestinal lipid metabolism through the PPAR signaling pathway in high-fat diet-fed red tilapia (Oreochromis niloticus).

Feeding high-fat (HF) diets has been shown to cause hepatic and intestinal impairment in fish species, but the mode of action, especially the pathways involved in the intestine, has not been determined yet. In this study, the effects of resveratrol (RES) supplementation on the intestinal structure, microbial flora, and fat metabolism in red tilapia (Oreochromis niloticus) were determined. The results showed RES maintained the structural integrity of the intestine and significantly increased the number of goblet cells in the midgut. RES significantly induced interferon (IL)-1ß, IL-6, IL-10, and tumor necrosis factor (TNF)-α, serumal and fecal trimetlylamine oxide (TMAO) and lipopolysaccharides (LPS), intestinal acetic acid levels. However, the concentrations of bound bile acids increased in HF-fed red tilapia. Atp5fa1 and Pafah1b3 significantly increased, Pmt and Acss2 significantly decreased, respectively, with RES supplementation, which was alleviated and retained at the same level in the selisistat (EX527) group. While for transcriptome and proteomics results, RES was found to promote fatty acid ß-oxidation and arachidonic acid metabolism associated with the peroxisome proliferator-activated receptor (PPAR) signaling pathway. The next validation experiment showed some genes related to apoptosis and fatty acid metabolism pathways were altered by RES supplementation. Namely, sn6, loc100702698, new_14481, and prkaa1 were upregulated, while ffrs1, ap3s1, and loc100705861 were downregulated. RES significantly increased Planctomycetes and Verrucomicrobia while decreased Moonvirus, Citrobacter, and Pseudomonas. Akkermansia and Fusobacterium significantly increased and Aeromonas significantly decreased. Thus, unsaturated fatty acid biosynthesis significantly increased and carbohydrate/energy metabolism decreased. To conclude, RES enabled the body to complete fatty acid ß-oxidation and arachidonic acid metabolism, whereas the addition of inhibitors increased the expression of the phagosome transcriptome and reduced fatty acid ß-oxidative metabolism.

Transcriptome analysis of the response of four immune related organs of tilapia (Oreochromis niloticus) to the addition of resveratrol in feed.

Resveratrol (RES) has been found to have immunological enhancement effects on Oreochromis niloticus. In O. nilocticus, the liver, spleen and kidney act as immune target tissues, while intestine works for nutrition sensing organ. In the present study, we determined RES administration on these immune tissues transcriptomic response in genetically improved farmed tilapia (GIFT), and further analyzed the relationship between transcriptomic response and intestinal microbiota. As results, hepatic hemosiderin and intestinal goblet cells significantly increased with RES addition. Kyoto encyclopedia of genes and genomes (KEGG) pathways associated with herpes simplex virus 1 infection, calcium signaling pathway, cell adhesion molecules, apoptosis, and mitogen-activated protein kinase (MAPK)/peroxisome proliferators-activated receptors (PPAR) signaling pathways were enriched. In particular, the differentially enriched genes (DEGs) associated pathways were present in different sampling tissues, times, and comparisons, interestingly, the PPAR signaling pathway was enriched with increasing time of RES addition. The assembled DEGs presented verified expression in the kidney, liver, spleen, and intestine tissues, and fabp6 was highly expressed in the intestine. Serial DEGs of fatty acid-binding proteins (fabp7, fabp7a, fabp10a) decreased in the liver and kidney, and fabp6 significantly increased in the spleen. With time, the pathways of energy metabolism, glycan biosynthesis, and metabolism decreased and increased in the intestinal metagenome. Some Candidatus branches significantly increased (C. cerribacteria and C. harrisonbacteria) and while others decreased (C. glodbacteria, etc.), whereas C. verstraetearchaeota fluctuated with RES addition. slc27a6 and dbi were negatively correlated with bacteria involved in the lipid, energy, and carbohydrate metabolism pathways. The present study suggests that RES supplementation affected lipid metabolism in immune-related organs may be related to the PPAR signaling pathway.

Integrative analysis of microbiome and metabolome reveals the linkage between gut microbiota and carp growth.

Gut microbiota and their metabolites are increasingly recognized for their crucial role in regulating the health and growth of the host. The mechanism by which the gut microbiome affects the growth rate of fish (Cyprinus carpio) in the rice-fish coculture system, however, remains unclear. In this study, the gut contents of the fast-growing and slow-growing (FG and SG) carp were collected from the rice-fish coculture system for both the fish gut microbiome and metabolome analyses. High throughput 16 S rRNA gene sequencing showed that the overall gut microbiota of FG group was distinct from that of SG group. For example, the cyanobacteria were highly enriched in the guts of SG carp (18.61%), in contrast, they only represented a minor fraction of gut microbiota for FG group (<0.20%). The liquid chromatography-mass spectrometry (LC-MS)-based metabolomics analysis revealed that 191 identified metabolites mostly located in 18 KEGG pathways were differentially present between the two groups, of which more than 50% of these metabolites were involved in lipid and amino acids metabolism. Compared with the FG group, the gut microbiota of SG group significantly enriched the metabolic pathways involved in the steroid (hormone) biosynthesis, whereas reducing those associated with beta-alanine metabolism, biosynthesis of unsaturated fatty acids and bile secretion. The enrichment and depletion of these metabolic pathways resulted in an increase in steroid metabolites and a decrease in the concentration of spermidine, which may have a major impact on the growth rate of carp. The metabolome results were further supported by the predicated KEGG functions of the gut microbiomes of the two groups, pointing out that the gut microbiota could substantially affect the growth of fish via their unique metabolic functions. Together, our integrated fish gut microbiome and metabolome analysis has substantial implications for the development of engineered microbiome technologies in aquaculture.

Microcystin-LR-induced autophagy via miR-282-5p/PIK3R1 pathway in Eriocheir sinensis hepatopancreas.

With the intensifying climate warming, blue-green algae blooms have become more frequent and severe, releasing environmental hazards such as microcystin that pose potential threats to human and animal health. Autophagy has been shown to play a crucial role in regulating immune responses induced by environmental hazards, enabling cells to adapt to stress and protect against damage. Although microcystin-LR (MC-LR) has been identified to affect autophagy in mammalian, its impact on aquatic animals has been poorly studied. To investigate the toxicological effects of MC-LR in aquatic ecosystems, we constructed a microRNA profile of acute MC-LR stress in the hepatopancreas of the Chinese mitten crab. Interestingly, we found the MC-LR exposure activated autophagy in the hepatopancreas based on the following evidence. Specifically, mRNA expression level of ATG7, Beclin1 and Gabarap was significantly up-regulated, autophagy regulatory pathways were significantly enriched, and numerous autolysosomes and autophagosomes were observed. Additionally, we found that miR-282-5p and its target gene PIK3R1 played important regulatory roles in autophagy by in vivo and in vitro experiments. Overexpression of miR-282-5p mimicked MC-LR-induced autophagy by inhibiting PIK3R1 expression, while miR-282-5p silencing inhibited autophagy by promoting PIK3R1 expression. Altogether, our findings suggest that MC-LR increases miR-282-5p, which then targets inhibition of PIK3R1 to stimulate autophagy. This study focused on the stress response regulatory mechanisms of juvenile crabs to toxic pollutants in water, offering a potential target for alleviating the toxicity of MC-LR. These findings lay a foundation for reducing the toxicity of MC-LR and environmental hazards in organisms.

Effects of prometryn on oxidative stress, immune response and apoptosis in the hepatopancreas of Eriocheir sinensis (Crustacea: Decapoda).

Prometryn, a triazine pesticide product used to control weed growth, poses a high risk to aquatic organisms in the environment. Several toxicological evaluations have been performed on bony fish and shrimp exposed to prometryn. However, there have been no reports conducted on the toxic mechanism of prometryn with regard to Eriocheir sinensis. In this study, our research evaluated the toxic effects of prometryn via in vitro and in vivo toxicity tests on E. sinensis. Firstly, we estimated the exposure toxicity of prometryn to E. sinensis, and then we constructed a 6 h transcriptional profile and conducted an enrichment analysis. To further reveal the toxicity of prometryn, the hepatopancreas (hepatopancreatic cells) was analyzed for antioxidant, immune and lipid-metabolism-related enzymes, antioxidant- and apoptosis-related gene expression, histopathology and TUNEL. From the results, we determined that the 96 h-LD50 was 70.059 mg/kg, and using RNA-seq, we identified 933 differentially expressed genes (DEGs), which were mainly enriched in the amino and fatty acid metabolism and the cell-fate-determination-related signaling pathway. The results of the biochemical assays showed that prometryn could significantly decrease the activities/levels of CAT, SOD, GSH, AKP and ACP, reduce the levels of T-AOC, TG, TCH, C3 and C4, and increase the MDA content. In addition, the expression levels of Nrf2, GSTs and HO-1 were first upregulated and then downregulated with increasing time. Histopathology showed that prometryn damaged the structure of the hepatopancreas cells and induced apoptosis, suggesting that the PI3K-Akt signaling pathway may be involved in the damage process of hepatopancreas cells (PI3K, PDK and Akt were downregulated whereas Bax was upregulated), leading to their apoptosis. The above results indicated that prometryn could cause injury of the hepatopancreas through oxidative stress, induce cell apoptosis, disrupt the lipid metabolism and cause immune damage. This study provided useful data for understanding and evaluating the toxicity of prometryn to aquatic crustacea.

Integrated Analysis of Transcriptomics and Metabolomics Unveil the Novel Insight of One-Year-Old Precocious Mechanism in the Chinese Mitten Crab, Eriocheir sinensis.

Eriocheir sinensis is traditionally a native high-value crab that is widely distributed in eastern Asia, and the precocity is considered the bottleneck problem affecting the development of the industry. The precocious E. sinensis is defined as a crab that reaches complete sexual maturation during the first year of its lifespan rather than as normally in the second year. However, the exact regulatory mechanisms underlying the precocity are still unclear to date. This study is the first to explore the mechanism of precocity with transcriptome-metabolome association analysis between the precocious and normal sexually mature E. sinensis. Our results indicated that the phenylalanine metabolism (map00360) and neuroactive ligand-receptor interaction (map04080) pathways play an important role in the precocity in the ovary of E. sinensis. In map00360, the predicted aromatic-L-amino-acid decarboxylase and 4-hydroxyphenylpyruvate dioxygenase isoform X1 genes and the phenethylamine, phenylethyl alcohol, trans-2-hydroxycinnamate, and L-tyrosine metabolites were all down-regulated in the ovary of the precocious E. sinensis. The map04080 was the common KEGG pathway in the ovary and hepatopancreas between the precocious and normal crab. In the ovary, the predicted growth hormone secretagogue receptor type 1 gene was up-regulated, and the L-glutamate metabolite was down-regulated in the precocious E. sinensis. In the hepatopancreas, the predicted forkhead box protein I2 gene and taurine metabolite were up-regulated and the the L-glutamate metabolite was down-regulated in the precocious crab. There was no common pathway in the testis. Numerous common pathways in the hepatopancreas between male precocious and normal crab were identified. The specific amino acids, fatty acids and flavorful nucleotide (inosine monophosphate (MP), cytidine MP, adenosine MP, uridine MP, and guanosine MP) contents in the hepatopancreas and gonads further confirmed the above omics results. Our results suggest that the phenylalanine metabolism may affect the ovarian development by changing the contents of the neurotransmitter and tyrosine. The neuroactive ligand-receptor interaction pathway may affect the growth by changing the expressions of related genes and affect the umami taste of the gonads and hepatopancreas through the differences of L-glutamate metabolite in the precocious E. sinensis. The results provided valuable and novel insights on the precocious mechanism and may have a significant impact on the development of the E. sinensis aquaculture industry.

Genome-wide identification of the NHE gene family in Coilia nasus and its response to salinity challenge and ammonia stress.

BACKGROUND: In aquatic environments, pH, salinity, and ammonia concentration are extremely important for aquatic animals. NHE is a two-way ion exchange carrier protein, which can transport Na+ into cells and exchange out H+, and also plays key roles in regulating intracellular pH, osmotic pressure, and ammonia concentration. RESULTS: In the present study, ten NHEs, the entire NHE gene family, were identified from Coilia nasus genome and systemically analyzed via phylogenetic, structural, and synteny analysis. Different expression patterns of C. nasus NHEs in multiple tissues indicated that expression profiles of NHE genes displayed tissue-specific. Expression patterns of C. nasus NHEs were related to ammonia excretion during multiple embryonic development stages. To explore the potential functions on salinity challenge and ammonia stress, expression levels of ten NHEs were detected in C. nasus gills under hypotonic stress, hypertonic stress, and ammonia stress. Expression levels of all NHEs were upregulated during hypotonic stress, while they were downregulated during hypertonic stress. NHE2 and NHE3 displayed higher expression levels in C. nasus larvae and juvenile gills under ammonia stress. CONCLUSIONS: Our study revealed that NHE genes played distinct roles in embryonic development, salinity stress, and ammonia exposure. Syntenic analysis showed significant difference between stenohaline fish and euryhaline fishes. Our findings will provide insight into effects of C. nasus NHE gene family on ion transport and ammonia tolerance and be beneficial for healthy aquaculture of C. nasus.

Microcystin-LR induces apoptosis in Juvenile Eriocheir sinensis via the mitochondrial pathway.

Microcystin-LR (MC-LR), the toxic substance of cyanobacteria secondary metabolism, widely exists in water environments and poses great risks to living organisms. Some toxicological assessments of MC-LR have performed at physiological and biochemical levels. However, plenty of blanks about the potential mechanism in aquatic crustacean remains. In this study, we firstly assessed the exposure toxicity of MC-LR to juvenile E. sinensis and clarified that the 96 h LD50 of MC-LR was 73.23 µg/kg. Then, hepatopancreas transcriptome profiles of MC-LR stressed crabs were constructed at 6 h post-injection and 37 differential expressed genes (DEGs) were identified. These DEGs were enriched in cytoskeleton, peroxisome and apoptosis pathways. To further reveal the toxicity of MC-LR, oxidative stress parameters (SOD, CAT, GSH-px and MDA), apoptosis genes (caspase 3, bcl-2 and bax) and apoptotic cells were detected. Significant accumulated MDA and rise-fall enzyme activities verified the oxidative stress caused by MC-LR. It is noteworthy that quantitative real-time PCR and TUNEL assay indicated that MC-LR stress-induced apoptosis via the mitochondrial pathway. Interestingly, activator protein-1 may play a crucial role in mediating the hepatotoxicity of MC-LR by regulating apoptosis and oxidative stress. Taken together, our study investigated the toxic effects and the potential molecular mechanisms of MC-LR on juvenile E. sinensis. It provided useful data for exploring the toxicity of MC-LR to aquatic crustaceans at molecular levels.

Effects of Submerged Macrophytes on the Growth, Morphology, Nutritional Value, and Flavor of Cultured Largemouth Bass (Micropterus salmoides).

Aquaculture environment plays important roles in regulating the growth, morphology, nutrition, and flavor of aquatic products. The present study investigated growth, morphology, nutrition, and flavor formation in largemouth bass (Micropterus salmoides) cultured in the ponds with (EM group) and without (M group) the submerged macrophytes (Elodea nuttallii). Fish in the EM group showed a significantly greater body length, higher growth rate, and lower hepatosomatic index than those in the M group (p< 0.05). Moreover, compared with fish in the M group, those in the EM group showed improved muscle quality with significantly elevated levels of crude protein, total free and hydrolysable amino acids, and polyunsaturated fatty acids (p < 0.05). Specifically, certain amino acids related to flavor (Glu, Asp, Ala, and Arg) and valuable fatty acids (C18:2, C18:3n3, C20:3n3, and C22:6) were more abundant in the EM group (p < 0.05). In addition, the levels of 19 volatile (p < 0.05) were significantly higher in the EM group than in the M group. Therefore, E. nuttallii significantly improved growth, morphological traits, nutritional components, and characteristic flavor in largemouth bass, indicating the superior nutritional value and palatability of fish cultured with submerged macrophytes.

Dietary Histidine Supplementation Maintained Amino Acid Homeostasis and Reduced Hepatic Lipid Accumulation of Juvenile Largemouth Bass, Micropterus Salmoides.

This 56-day research aimed to evaluate the recommended histidine requirement and the influence of dietary histidine levels on the protein and lipid metabolism of juvenile largemouth bass (Mieropterus salmoides). The initial weight of the largemouth bass was 12.33 ± 0.01 g, which was fed with six graded levels of histidine. The results showed that appropriate dietary histidine had a positive effect on growth, with a higher specific growth rate, final weight, weight gain rate, protein efficiency rate, and a lower feed conversion rate and feed intake rate being observed in 1.08-1.48% dietary histidine groups. Furthermore, the mRNA levels of GH, IGF-1, TOR, and S6 showed an increasing trend first and then declined, similar to the trend of the growth and protein content of the whole body composition. Meanwhile, dietary histidine levels could be sensed by the AAR signaling pathway, representing as downregulation of core genes of AAR signaling pathway with the increased dietary histidine levels, including GCN2, eIF2α, CHOP, ATF4, and REDD1. In addition, increased dietary histidine levels decreased the lipid content of the whole body and the liver by upregulating the mRNA levels of core genes of the PPARα signaling pathways, including PPARα, CPT1, L-FABP, and PGC1α. However, increased dietary histidine levels downregulated the mRNA levels of core genes of the PPARγ signaling pathways such as PPARγ, FAS, ACC, SREBP1, and ELOVL2. These findings were also supported by the positive area ratio of hepatic oil red O staining and the TC content of plasma. According to the specific growth rate and feed conversion rate, the recommended histidine requirement of juvenile largemouth bass was 1.26% of the diet (2.68% of dietary protein) by regression lines calculated using a quadratic model. In general, histidine supplementation promoted protein synthesis and lipid decomposition and reduced lipid synthesis by activating the TOR, AAR, PPARα, and PPARγ signaling pathways, which provided a new perspective to solve the fatty liver problem of largemouth bass by nutritional means.

Dietary leucine supplementation improves growth performance, metabolic responses of liver via GCN2/ATF4, and insulin signaling pathways in largemouth bass (Micropterus salmoides).

An 8-week growth experiment was conducted to investigate the effects of dietary leucine on growth performance, body composition, and gene expression of hepatic nutrient metabolism in the largemouth bass (Micropterus salmoides). Six isonitrogenous (49.87%) diets with graded leucine levels (2.62, 3.07, 3.60, 3.87, 4.20, 4.71% of dry diet) were fed to triplicate groups with 20 juvenile fish (20.00 ± 0.13 g). The results revealed that the specific growth rate (SGR) and weight gain (WG) increased significantly with increasing dietary leucine levels, reached their maximal value in the Leu-4.20% groups, and then decreased slightly. Although the feed conversion ratio (FCR) showed decreasing trends, no significant difference was detected. Leucine supplementation significantly improved the content of body protein and total plasma protein (TP). Additionally, a higher expression level of target of rapamycin (TOR) and ribosomal protein S6 (S6) mRNA was observed in the Leu-3.87% and Leu-4.20% diets, whereas the GCN2 (general control nonderepressible2 kinase) and AFT4 (activating transcription factor 4) mRNA expression levels were suppressed. The lipid content of the body was not influenced by leucine levels, whereas the content of total triglyceride (TG) first decreased significantly with increasing dietary leucine levels from 2.62 to 3.87% and then increased with increasing leucine levels (4.20% to 4.71%). The total cholesterol (TC) and low-density lipoproteins (LDL) trended in a similar direction but did not achieve statistical significance (P > 0.05). The expression of insulin receptor substrate 1 (IRS-1) was significantly elevated by dietary leucine levels, while protein kinase B (AKT) and phosphatidylinositol 3-kinase (PI3K) expression was inconsistently upregulated. Furthermore, leucine supplementation decreased plasma glucose and hepatic glycogen contents, and the expression levels of glucokinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6pase) were significantly inhibited at 4.20% and 4.71% leucine diets. Analyses of the change in SGR and FCR using the quadratic regression model estimated that the optimum dietary leucine requirement of juvenile largemouth bass was 4.42% and 4.63% of the dry diet (8.86% and 9.28% of dietary protein), respectively.

Appropriate dietary phenylalanine improved growth, protein metabolism and lipid metabolism, and glycolysis in largemouth bass (Micropterus salmoides).

The purpose of this study was aimed to determine the appropriate level of dietary phenylalanine and explored the influences of phenylalanine on target rapamycin (TOR) signaling and glucose and lipid metabolism in largemouth bass. Six isonitrogenous/isoenergetic diets with graded phenylalanine levels (1.45% (control group), 1.69%, 1.98%, 2.21%, 2.48%, and 2.76%) were designed. Experimental feed was used to feed juvenile largemouth bass (initial body weight 19.5 ± 0.98 g) for 8 weeks. The final body weight, specific growth rate (SGR), feed efficiency ratio (FER), and weight gain (WG) reached their highest values in the 1.98% dietary phenylalanine group and then declined with increasing phenylalanine addition. No significant difference was found in the whole-body composition of largemouth bass between different dietary phenylalanine groups. Compared with the control group, 1.69% dietary phenylalanine significantly reduced the contents of plasma glucose (GLU) and total protein (TP), and total cholesterol (TC) contents increased significantly in the 1.98% dietary phenylalanine group (P < 0.05). The key gene expressions of TOR signaling pathway and lipid metabolism was significantly inhibited by 2.21% dietary phenylalanine (P < 0.05). The 1.98% dietary phenylalanine group showed significantly increased expression of genes related to insulin signaling pathway and factors involved in fatty acid synthesis (P < 0.05). Furthermore, 2.76% dietary phenylalanine group inhibited glucose metabolism by lowering the key gene expressions of glucose metabolism (P < 0.05). According to quadratic regression analyses based on the WG and FER, the appropriate level of dietary phenylalanine for largemouth bass were 2.00% and 2.02% of the diet (4.23% and 4.27% dietary protein), respectively, with a constant amount of tyrosine (1.33%). Hence, the total aromatic amino acid requirements were 3.33% and 3.35% of the diet (equivalent to 7.03% and 7.09% of the protein content), which may provide a theoretical basis for the development of largemouth bass feed formulas. Therefore, the growth and metabolism of largemouth bass could be promoted by controlling the content of phenylalanine in the diet, or the imbalance of phenylalanine can form a specific pathological model.

Whole-genome resequencing of three Coilia nasus population reveals genetic variations in genes related to immune, vision, migration, and osmoregulation.

BACKGROUND: Coilia nasus is an important anadromous fish, widely distributed in China, Japan, and Korea. Based on morphological and ecological researches of C. nasus, two ecotypes were identified. One is the anadromous population (AP). The sexually mature fish run thousands of kilometers from marine to river for spawning. Another one is the resident population which cannot migrate. Based on their different habitats, they were classified into landlocked population (LP) and sea population (SP) which were resident in the freshwater lake and marine during the entire lifetime, respectively. However, they have never been systematically studied. Moreover, C. nasus is declining sharply due to overfishing and pollution recently. Therefore, further understandings of C. nasus populations are needed for germplasm protection. RESULTS: Whole-genome resequencing of AP, LP, and SP were performed to enrich the understanding of different populations of C. nasus. At the genome level, 3,176,204, 3,307,069, and 3,207,906 single nucleotide polymorphisms (SNPs) and 1,892,068, 2,002,912, and 1,922,168 insertion/deletion polymorphisms (InDels) were generated in AP, LP, and SP, respectively. Selective sweeping analysis showed that 1022 genes were selected in AP vs LP; 983 genes were selected in LP vs SP; 116 genes were selected in AP vs SP. Among them, selected genes related to immune, vision, migration, and osmoregulation were identified. Furthermore, their expression profiles were detected by quantitative real-time PCR. Expression levels of selected genes related to immune, and vision in LP were significantly lower than AP and SP. Selected genes related to migration in AP were expressed significantly more highly than LP. Expression levels of selected genes related to osmoregulation were also detected. The expression of NKAα and NKCC1 in LP were significantly lower than SP, while expression of NCC, SLC4A4, NHE3, and V-ATPase in LP was significantly higher than SP. CONCLUSIONS: Combined to life history of C. nasus populations, our results revealed that the molecular mechanisms of their differences of immune, vision, migration, and osmoregulation. Our findings will provide a further understanding of different populations of C. nasus and will be beneficial for wild C. nasus protection.

Gills full-length transcriptomic analysis of osmoregulatory adaptive responses to salinity stress in Coilia nasus.

Salinity changes will threaten the survival of aquatic animals. However, osmoregulatory mechanism of Coilia nasus has not been explored. Oxford Nanopore Technologies (ONT) sequencing was performed in C. nasus gills during hypotonic and hyperosmotic stress. 23.8 G clean reads and 27,659 full-length non-redundant sequences were generated via ONT sequencing. Alternative splicing, alternative polyadenylation, transcript factors, and long noncoding RNA were identified. During hypotonic stress, 58 up-regulated differentially expressed genes (DEGs) and 36 down-regulated DEGs were identified. During hypertonic stress, 429 up-regulated DEGs and 480 down-regulated DEGs were identified. These DEGs were associated with metabolism, cell cycle, and transport. The analysis of these DEGs indicated that carbohydrate and fatty acid metabolism were activated to provide energy for cell cycle and transport during hypotonic and hypertonic stress. Cell cycle was also promoted during hypotonic and hypertonic stress. To resist hypotonic stress, polyamines metabolism, ion absorption and water transport from extra-cellular to intra-cellular were promoted, while ion secretion was inhibited. During hypotonic stress, glutamine, alanine, proline, and inositol metabolism were activated. Ion absorption and water transport from intra-cellular to extra-cellular were inhibited. Moreover, different transcript isoforms generated from the same gene performed different expression patterns during hypotonic and hypertonic stress. These findings will be beneficial to understand osmoregulatory mechanism of Coilia nasus.

Transcriptome analysis of the brain provides insights into the regulatory mechanism for Coilia nasus migration.

BACKGROUND: Coilia nasus (C. nasus) is an important anadromous fish species that resides in the Yangtze River of China, and has high ecological and economical value. However, wild resources have suffered from a serious reduction in population, attributed to the over-construction of water conservancy projects, overfishing, and environmental pollution. The Ministry of Agriculture and Rural Affairs of the People's Republic of China has issued a notice banning the commercial fishing of wild C. nasus in the Yangtze River. Wild C. nasus populations urgently need to recover. A better understanding of C. nasus migration patterns is necessary to maximize the efficiency of conservation efforts. Juvenile C. nasus experience a simultaneous effect of increasing salinity and cold stress during seaward migration, and the brain plays a comprehensive regulatory role during this process. Therefore, to explore the early seaward migration regulation mechanism of juvenile C. nasus, we performed a comparative transcriptome analysis on the brain of juvenile C. nasus under salinity and cold stress simultaneously. RESULTS: Relevant neurotransmitters, receptors, and regulatory proteins from three categories of regulatory pathway play synergistic regulatory roles during the migration process: neuronal signaling, the sensory system, and environmental adaptation. The significant differential expression of growth-related hormones, thyroid receptors, haptoglobin, and prolactin receptors was similar to the results of relevant research on salmonids and steelhead trout. CONCLUSIONS: This study revealed a regulatory network that the brain of juvenile C. nasus constructs during migration, thereby providing basic knowledge on further studies could build on. This study also revealed key regulatory genes similar to salmonids and steelhead trout, thus, this study will lay a theoretical foundation for further study on migration regulation mechanism of anadromous fish species.

Optimum feeding frequency of juvenile largemouth bass (Micropterus salmoides) reared in in-pond raceway recirculating culture system.

This study was conducted to determine the effects of feeding frequency on the growth, serum biochemical parameters, antioxidant status and hepatic growth hormone (GH), insulin-like growth factor I (IGF-I), lipoprotein lipase (LPL) and hepatic lipase (HL) gene expression levels of juvenile largemouth bass (Micropterus salmoides) reared in an in-pond raceway recirculating culture system (IPRS). Fish (initial body weight 5.0 ± 0.4 g) were hand-fed with a commercial diet under one of three different feeding frequency treatments (2, 3 or 4 meals/day) for 120 days. The results indicated that no significant differences were observed in the final body weight, weight gain and specific growth rate of fish fed different feeding frequencies on 30 days and 60 days (P > 0.05). Fish fed 2 times/day had higher growth than that fed 4 times/day on 90 days but had higher growth than those fed 3 and 4 times/day on 120 days. No significant differences were found in serum alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) activities, total protein (TP), lysozyme and triglyceride (TG) content, hepatic total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX) activities and malondialdehyde (MDA) content among fish fed different feeding frequency (P > 0.05). Serum glucose (Glu) content and catalase (CAT) activity decreased, while total cholesterol (TC) content increased with increasing feeding frequency. Fish fed 2 times/day had higher hepatic total superoxide dismutase (T-SOD) than that fed 4 times/day on 60 days, 90 days and 120 days (P < 0.05). Fish fed 2 times/day had higher IGF-1 gene mRNA expression on 30 days, 60 days and 120 days (P < 0.05), while no significant difference on 90 days. No significant difference was found in GH gene mRNA expression on 30 days and 60 days, while fish fed 4 times/day had lower values than that fed 2 times/day on 90 days and 120 days (P < 0.05). Fish fed 2 times/day had significantly higher LPL mRNA expression level than that fed 4 times/day on 60 days and 90 days and had significantly higher HL mRNA expression level on 60 days, 90 days and 120 days (P < 0.05). Based on growth, physiology, hepatic gene expression levels, labour costs and intensity, the optimal feeding frequency of largemouth bass (average body weight 5.0 ± 0.4 g) reared in IPRS is 2 times/day. These data are very necessary for the optimizing of culture conditions and feeding management strategy in IPRS culture operations.

The effects of crowding stress on the growth, physiological response, and gene expression of the Nrf2-Keap1 signaling pathway in blunt snout bream (Megalobrama amblycephala) reared under in-pond raceway conditions.

We evaluated the effects of high density stress on growth performance, antioxidant parameters, and Nrf2 pathway signaling molecules after different lengths of exposure (30, 60, or 90 days) of Megalobrama amblycephala to in-pond raceway aquaculture systems (IPRS). M. amblycephala (average initial weight 2.33 ±â€¯0.15 g) were reared at two different initial densities (low density group [LD] had 534 fish/m3 and high density group [HD] had 1073 fish/m3) for 90 days. The growth performance was adversely influenced by the high stocking density. The HD group had elevated white blood cell counts, hemoglobin content, and hematocrit on days 60 and 90. The mRNA levels of NOX2 on days 60 and 90, Nrf2 on days 30, 60, and 90, Keap1 on day 30, Bach1 on days 30 and 60, SOD on day 30, and CAT on day 30 were significantly higher in the HD group than in the LD group. Similarly, higher trends were observed in the enzymatic activities of SOD on day 60, CAT on days 60 and 90, and GPx on day 60 in the HD group, compared to the LD group. Furthermore, HD bream showed an increased MDA content on days 60 and 90 compared to that of the LD group. This study demonstrates that high density-induced antioxidant defenses were involved in modifications to the enzymatic and transcriptional regulation of Nrf2-Keap1 signaling molecules and that M. amblycephala growth was reduced in a crowded IPRS.