Comprehensive mRNA and microRNA analysis revealed the effect and response strategy of freshwater fish, grass carp (Ctenopharyngodon idella) under geosmin exposure.

Geosmin is an environmental pollutant that causes off-flavor in water and aquatic products. The high occurrence of geosmin contamination in aquatic systems and aquaculture raises public awareness, however, few studies have investigated the response pathways of geosmin stress on freshwater fish. In this research, grass carp were exposed to 50 µg/L geosmin for 96 h, liver tissue was sequenced and validated using real-time qPCR. In total of 528 up-regulated genes and 488 down-regulated genes were observed, includes cytochrome P450 and uridine diphosphate (UDP)-glucuronosyltransferase related genes. KEGG analysis showed that chemical carcinogenesis-DNA adducts, metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450 pathway was enriched. Common genes from the target genes of microRNAs and differential expression genes are enriched in metabolism of xenobiotics cytochrome P450 pathway. Two miRNAs (dre-miR-146a and miR-212-3p) down regulated their target genes (LOC127510138 and adh5, respectively) which are enriched cytochrome P450 related pathway. The results present that geosmin is genetoxic to grass carp and indicate that cytochrome P450 system and UDP-glucuronosyltransferase play essential roles in biotransformation of geosmin. MicroRNAs regulate the biotransformation of geosmin by targeting specific genes, which contributes to the development of strategies to manage its negative impacts in both natural and artificial environments.

Berberine regulates glucose metabolism in largemouth bass by modulating intestinal microbiota.

This study examined the role of intestinal microbiota in berberine (BBR)-mediated glucose (GLU) metabolism regulation in largemouth bass. Four groups of largemouth bass (133.7 ± 1.43 g) were fed with control diet, BBR (1 g/kg feed) supplemented diet, antibiotic (ATB, 0.9 g/kg feed) supplemented diet and BBR + ATB (1g/kg feed +0.9 g/kg feed) supplemented diet for 50 days. BBR improved growth, decreased the hepatosomatic and visceral weight indices, significantly downregulated the serum total cholesterol and GLU levels, and significantly upregulated the serum total bile acid (TBA) levels. The hepatic hexokinase, pyruvate kinase, GLU-6-phosphatase and glutamic oxalacetic transaminase activities in the largemouth bass were significantly upregulated when compared with those in the control group. The ATB group exhibited significantly decreased final bodyweight, weight gain, specific growth rates and serum TBA levels, and significantly increased hepatosomatic and viscera weight indices, hepatic phosphoenolpyruvate carboxykinase, phosphofructokinase, and pyruvate carboxylase activities, and serum GLU levels. Meanwhile, the BBR + ATB group exhibited significantly decreased final weight, weight gain and specific growth rates, and TBA levels and significantly increased hepatosomatic and viscera weight indices and GLU levels. High-throughput sequencing revealed that compared with those in the control group, the Chao one index and Bacteroidota contents were significantly upregulated and the Firmicutes contents were downregulated in the BBR group. Additionally, the Shannon and Simpson indices and Bacteroidota levels were significantly downregulated, whereas the Firmicutes levels were significantly upregulated in ATB and BBR + ATB groups. The results of in-vitro culture of intestinal microbiota revealed that BBR significantly increased the number of culturable bacteria. The characteristic bacterium in the BBR group was Enterobacter cloacae. Biochemical identification analysis revealed that E. cloacae metabolizes carbohydrates. The size and degree of vacuolation of the hepatocytes in the control, ATB, and ATB + BBR groups were higher than those in the BBR group. Additionally, BBR decreased the number of nuclei at the edges and the distribution of lipids in the liver tissue. Collectively, BBR reduced the blood GLU level and improved GLU metabolism in largemouth bass. Comparative analysis of experiments with ATB and BBR supplementation revealed that BBR regulated GLU metabolism in largemouth bass by modulating intestinal microbiota.

New Insights into the Relationships between Bacterial Dynamics and Water Quality of Aquaculture Systems Supplemented with Carbon Source and Biofilm Substratum.

Aquaculture is crucial for achieving the FAO's goal of a world without hunger and malnutrition. Recently, biofilm substratum has been proposed as an effective means to control waste pollution caused by excessive nutrient inputs from aquaculture, but key bacterial communities involved in the remediation remain unclear. Here we reported a freshwater mesocosm study where the addition of biofilm substrata with external carbon effectively controlled the total ammonia nitrogen and improved fish growth. 16S rRNA study and Weighted UniFrac analysis revealed that bacterial compositions were significantly different (999 permutations, p-value < 0.01) between the biofilm-substrata-added and biofilm-substrata-free systems. Planctomycetes were found, as key bacteria benefited from the biofilm substrata addition and exerted the major function of ammonia nitrogen control. Our study demonstrated that the addition of biofilm substrata and an external carbon source favored fish growth and improved the aquaculture environment by the formation of a unique bacteria community.

The complex evolution of the metazoan HSP70 gene family.

The metazoan 70-kDa heat shock protein (HSP70) family contains several members localized in different subcellular compartments. The cytosolic members have been classified into inducible HSP70s and constitutive heat shock cognates (HSC70s), but their distinction and evolutionary relationship remain unclear because of occasional reports of "constitutive HSP70s" and the lack of cross-phylum comparisons. Here we provide novel insights into the evolution of these important molecular chaperones. Phylogenetic analyses of 125 full-length HSP70s from a broad range of phyla revealed an ancient duplication that gave rise to two lineages from which all metazoan cytosolic HSP70s descend. One lineage (A) contains a relatively small number of genes from many invertebrate phyla, none of which have been shown to be constitutively expressed (i.e., either inducible or unknown). The other lineage (B) included both inducible and constitutive genes from diverse phyla. Species-specific duplications are present in both lineages, and Lineage B contains well-supported phylum-specific clades for Platyhelminthes, Rotifera, Nematoda, Porifera/Cnidaria, and Chordata. Some genes in Lineage B have likely independently acquired inducibility, which may explain the sporadic distribution of "HSP70" or "HSC70" in previous phylogenetic analyses. Consistent with the diversification history within each group, inducible members show lower purifying selection pressure compared to constitutive members. These results illustrate the evolutionary history of the HSP70 family, encouraging us to propose a new nomenclature: "HSP70 + subcellular localization + linage + copy number in the organism + inducible or constitutive, if known." e.g., HSP70cA1i for cytosolic Lineage A, copy 1, inducible.

Identification and analysis of lipid droplet-related proteome in the adipose tissue of grass carp (Ctenopharyngodon idella) under fed and starved conditions.

Fat accumulation in the mesenteric adipose tissue is a serious problem in grass carp (Ctenopharyngodon idella) culture. Lipid droplet-related proteins (LDRPs) are involved in the formation, degradation, and biological functions of lipid droplets. In this study, we aimed to provide reference proteomics data to study lipid droplet regulation in fish. We isolated LDRPs from the mesenteric adipose tissue of grass carp (1-year-old) after normal feeding and 7 days of starvation, and identified and analysed them using isobaric tags for relative and absolute quantitation (iTRAQ) technology. Short-term starvation had no significant effect on the body weight, condition factor, visceral index, hepatopancreas index, intraperitoneal fat index, adipose tissue triglyceride content, and adipocyte size of grass carp. Nine hundred and fifty proteins were identified and annotated using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; they are involved in a variety of metabolic and signalling pathways, including amino acid, lipid, and carbohydrate metabolism, and the PI3K-Akt signalling pathway. There were 296 differentially expressed proteins (DEPs), with 143 up-regulated and 153 down-regulated proteins. Three proteins involved in triglyceride and fatty acid syntheses and two proteins involved in autophagy were up-regulated, and six proteins involved in lipid catabolism were down-regulated. These results indicate that under short-term starvation, lipid droplets in the adipose tissue of grass carp may maintain their shape by promoting fat production and inhibiting lipolysis, and autophagy may be one of the main strategies for coping with short-term energy deprivation.

Feeding Faba Beans (Vicia faba L.) Reduces Myocyte Metabolic Activity in Grass Carp (Ctenopharyngodon idellus).

In this study, we aimed to explore the effects of faba bean (Vicia faba L.) on the energy metabolism of grass carp (Ctenopharyngodon idellus). A total of 180 fish (∼2900 g) were randomly assigned to six tanks (2.5 × 2.5 × 1.2 m; 30 individuals per tank) and fed either faba bean (Vicia faba L.) or a commercial diet for 120 days (3% body weight, twice per day). The results showed that faba bean-fed grass carp (FBFG) had significantly lower growth and higher fat accumulation in the mesenteric adipose tissue and hepatopancreas than commercial diet-fed grass carp (CDFG). Compared with CDFG, FBFG exhibited no significant difference in proximate composition of the muscle; however, an obvious decrease in muscle fiber size and significantly higher hardness, chewiness, and gumminess were observed. Transcriptome results showed that a total of 197 genes were differentially regulated in the dorsal muscle. Down-regulated genes included four genes annotated with myocyte development and 12 transcripts annotated with components of myofibrils. In addition, the FBFG group exhibited significantly lower expression of genes associated with oxygen transport, the mitochondrial respiratory chain, and creatine metabolism, suggesting reduced energy availability in the muscle of the FBFG. Moreover, using western-blotting and enzyme assays, we found decreased protein levels in the mitochondrial electron transport respiratory chain and creatine metabolism activities, as well as increased expression of autophagy marker protein levels, in the muscle of FBFG. Overall, our results suggest that an abnormal energy distribution may exist in grass carps after feeding with faba bean, which is reflected by a mass of fat deposition in the adipose tissue and hepatopancreas and subdued metabolic activity in the muscle.

Lipid droplets participate in modulating innate immune genes in Ctenopharyngodon idella kidney cells.

Lipid droplets (LDs) are increasingly being recognized as important immune modulators in mammals, in additional to their function of lipid ester deposition. However, the role of LDs in fish immunity remains poorly understood. In this study, the function of LDs in the innate immune response of Ctenopharyngodon idella kidney (CIK) cells, which are the equivalent of myeloid cells in vertebrates, was investigated. LD number and TG content significantly increased in the CIK cells following exposure to lipopolysaccharide (LPS), peptidoglycan (PGN), and polyriboinosinic-polyribocytidylic acid (Poly [I: C]) for 24 h, accompanied by increases in the relative expression of several innate immune genes. However, fatty acid compositions of the triglycerides were not changed after treatment with these three pathogenic mimics. LPS, PGN, and Poly (I: C) did not alter the relative expressions of lipogenic (FAS, SCD, and DGAT) and lipid catabolic (PPARα, ATGL, and CPT-1) genes. However, these treatments did increase the mRNA levels of lipid transportation genes (FATP/CD36, ACSL1, and ACSL4), and also decreased the non-esterified fatty acid level in the medium. To further explore the role of LDs in the immune response, CIK cells were incubated with different concentrations (0, 100, 200, 300, 400, 500 µM) of exogenous lipid mix (LM; oleic acid [OA]:linoleic acid [LA]:linolenic acid [LNA] = 2:1:1), and were then transferred to a lipid-free medium and incubated for 24 h. LD size and number increased with the increase in lipid levels, and this was accompanied by increased expression of innate immune genes, including MyD88, IRF3, and IL-1ß, which were expressed at their highest levels in 300 µM exogenous lipid mix. Interestingly, after incubating with different fatty acids (LM, OA, LA, LNA, arachidonic acid [ARA], and docosahexaenoic acid [DHA]; 300 µM), ARA and DHA were more potent in inducing LD formation and innate immune gene expression in the CIK cells. Finally, atglistatin, an ATGL inhibitor, effectively attenuated the expression of most genes upregulated by ARA or DHA, suggesting that lipolysis may be involved in the regulation of immune genes at the transcriptional level. Overall, the findings of this study demonstrate that LDs are functional organelles that could act as modulators in the innate immune response of CIK cells. Additionally, long-chain polyunsaturated fatty acid enriched LDs play a unique role in regulating this process.

Dietary arachidonic acid decreases the expression of transcripts related to adipocyte development and chronic inflammation in the adipose tissue of juvenile grass carp, Ctenopharyngodon idella.

Overdevelopment of adipose tissue in cultured fish is one of the biggest issues plaguing current aquaculture industry, leading to unhealthy status of fishes and production losses. Diet supplemented with 0.30% arachidonic acid (ARA) has been found to reduce adipogenesis and inflammation in grass carp, but the potential mechanism is not comprehensively understood. To fully reveal the effects of dietary ARA on the mRNA profiles of adipose tissue, transcriptome techniques were applied in this study. A 10-weeks feeding experiment was performed using two isonitrogenous and isoenergetic purified diets, namely ARA-free (control) and 0.30% ARA (ARA group). Results showed increased ARA content and decreased intraperitoneal fat index and adipocyte size in the adipose tissue of fish fed ARA (P < 0.05). A total of 611 and 973 genes of the adipose tissue were significantly up-regulated and down-regulated, respectively, in fish fed ARA (P < 0.05). Dietary ARA upregulated LOX pathway but downregulated CYP450 pathway annotated genes expression. A total of 65 cell development annotated genes including 30 adipocyte proliferation, 21 adipocyte differentiation, and 14 cell apoptosis annotated genes were down-regulated in the ARA group. In addition, 19 lipid catabolism annotated genes were increased. The mRNA expression levels of 5 chemokines, 10 cytokines, 26 cytokine and chemokine receptors, 15 cell adhesion, 6 oxidative stress, and 6 angiogenesis annotated genes were all down-regulated in fish fed ARA. Finally, dietary ARA also decreased the expression of transcripts annotated with glucose transportation, glycolysis and gluconeogenesis. Overall, our results demonstrate that dietary ARA has a fat reducing role, and tends to retard adipocyte development and attenuate chronic inflammation based on these adipose transcript expression results in grass carp.

Smad4-dependent regulation of type I collagen expression in the muscle of grass carp fed with faba bean.

Smad4 is the key regulator in the transforming growth factor ß1 (TGF-ß1)/Smads signal pathway, and is also the crux of the regulation of type I collagen expression in mammals. In fish, however, the relationship between Smad4 and type I collagen is still unknown. Given the widely accepted importance of type I collagen in fish muscle hardness, we seek to explore this issue by analyzing the expressions of the TGF-ß1/Smads pathway molecules and type I collagen in the muscle of crisp grass carp fed with faba bean, which shows increased muscle hardness. The study found that (1) in the process of feeding the grass carp with faba bean, the mRNA and protein expressions of TGF-ß1, Smad2 and Smad4 all increased along with the increase of type I collagen expression (Col1α1 and Col1α2); (2) one day after the injection of Smad4 over-expression vector, both mRNA and protein expressions of Col1α1 and Col1α2 significantly increased, reaching the maximum on the 2nd and 5th day, respectively; (3) one day after the injection of Smad4 RNAi interference vector, the mRNA and protein expressions of Col1α1 and Col1α2 decreased, reaching the minimum on the 5th day. These results revealed that Smad4 is the major regulator of type I collagen in the muscle of grass carp fed with faba bean. This study would provide an important mechanistic basis for nutritional regulation of type I collagen in the muscle of fish.

Lipid accumulation in grass carp (Ctenopharyngodon idellus) fed faba beans (Vicia faba L.).

Feeding faba beans (Vicia faba L.) to grass carp (Ctenopharyngodon idellus) increases muscle compactness but decreases growth and motility. The lipid metabolism of grass carp was examined to assess potential effects of feeding faba beans on physiological properties using a total of 180 fish. The treatment group was fed faba beans for 120 days and a commercial diet for another 30 days. The control group received a commercial diet for 150 days. Fish were sampled every month. Weight gain was significantly lower in the treatment group than in the control. Hardness, springiness, chewiness, cohesiveness, and gumminess of the dorsal muscle increased significantly with the feeding faba beans from 30 to 120 days, which was not reversed by the subsequent feeding of commercial diet. Fat accumulation increased significantly in the treatment group as suggested by the condition factor, viscera index, hepatopancreatic index, and intraperitoneal fat index (IPFI), hepatopancreas, and muscle fat content but was not affected by subsequent feeding with the commercial diet. Serum triglyceride and total cholesterol levels were significantly reduced in the experimental diet group. In the hepatopancreas and intraperitoneal fat IPF, monounsaturated fatty acids showed significantly higher content in faba bean feeding fish, whereas polyunsaturated fatty acid content showed the reversed pattern. In the hepatopancreas, the activities of the lipogenic enzymes malate dehydrogenase and glucose 6-phosphate dehydrogenase were higher in the treatment than in the control group. Moreover, the treatment group showed lower mRNA levels of carnitine palmitoyltransferase-1. Overall, our results clearly demonstrate increasing lipid accumulation in the viscera of faba bean-fed grass carp.

The Wnt/ß-catenin pathway contributes to the regulation of adipocyte development induced by docosahexaenoic acid in grass carp, Ctenopharyngodon idellus.

Docosahexaenoic acid (DHA) has been suggested to inhibit adipogenesis in fish; the mechanism behind this observation remains largely unknown. The present study was to investigate the effects of DHA on the adipocyte differentiation of grass carp by using transcriptomic technology. Confluent primary cultured differentiating adipocytes were co-incubated with either 0µM or 100µM DHA for 2days (early phase of differentiation) and 7days (late phase). The results showed that 100µM DHA promoted adipogenesis at 2days but decreased lipid accumulation at 7days of treatment compared to that of the control. Cells treated with DHA for 2days showed up-regulation of adipogenic genes, such as PPARγ, C/EBPs, and FAS; 7day treatment showed down-regulation of these genes. Interestingly, ß-catenin, which plays important roles in suppressing adipogenesis in mammals, was also up-regulated after 7days of treatment. To address this, administration of ß-catenin inhibitor FH535 was used and abolished the inhibition of the adipogenesis induced by DHA for 7days. However, FH535 did not rescue the inhibition of adipogenic gene expression (including that of PPARγ, C/EBPß, and C/EBPγ) in the presence of 100µM DHA, but it attenuated the up-regulation of ß-catenin downstream target genes, e.g. c-myc and cyc D1. This suggests that DHA might regulate the adipogenic junction through the ß-catenin pathway. Overall, our results demonstrate that continuous DHA treatment could promote the early step of adipocyte differentiation and suppress late phase adipogenesis in grass carp. Moreover, Wnt/ß-catenin might be a new potential signaling pathway by which DHA suppresses adipocyte development.

Comparative analysis of effects of dietary arachidonic acid and EPA on growth, tissue fatty acid composition, antioxidant response and lipid metabolism in juvenile grass carp, Ctenopharyngodon idellus.

Four isonitrogenous and isoenergetic purified diets containing free arachidonic acid (ARA) or EPA (control group), 0·30 % ARA, 0·30 % EPA and 0·30 % ARA+EPA (equivalent) were designed to feed juvenile grass carp (10·21 (sd 0·10) g) for 10 weeks. Only the EPA group presented better growth performance compared with the control group (P<0·05). Dietary ARA and EPA were incorporated into polar lipids more than non-polar lipids in hepatopancreas but not intraperitoneal fat (IPF) tissue. Fish fed ARA and EPA showed an increase of serum superoxide dismutase and catalase activities, and decrease of glutathione peroxidase activity and malondialdehyde contents (P<0·05). The hepatopancreatic TAG levels decreased both in ARA and EPA groups (P<0·05), accompanied by the decrease of lipoprotein lipase (LPL) activity in the ARA group (P<0·05). Fatty acid synthase (FAS), diacylglycerol O-acyltransferase and apoE gene expression in the hepatopancreas decreased in fish fed ARA and EPA, but only the ARA group exhibited increased mRNA level of adipose TAG lipase (ATGL) (P<0·05). Decreased IPF index and adipocyte sizes were found in the ARA group (P<0·05). Meanwhile, the ARA group showed decreased expression levels of adipogenic genes CCAAT enhancer-binding protein α, LPL and FAS, and increased levels of the lipid catabolic genes PPAR α, ATGL, hormone-sensitive lipase and carnitine palmitoyltransferase 1 (CPT-1) in IPF, whereas the EPA group only increased PPAR α and CPT-1 mRNA expression and showed less levels than the ARA group. Overall, dietary EPA is beneficial to the growth performance, whereas ARA is more potent in inducing lipolysis and inhibiting adipogenesis, especially in IPF. Meanwhile, dietary ARA and EPA showed the similar preference in esterification and the improvement in antioxidant response.

Proteomic signature of muscle fibre hyperplasia in response to faba bean intake in grass carp.

Fish muscle growth is important for the rapidly developing global aquaculture industry, particularly with respect to production and quality. Changes in muscle fibre size are accomplished by altering the balance between protein synthesis and proteolysis. However, our understanding regarding the effects of different protein sources on fish muscle proteins is still limited. Here we report on the proteomic profile of muscle fibre hyperplasia in grass carp fed only with whole faba bean. From the results, a total of 99 significantly changed proteins after muscle hyperplasia increase were identified (p < 0.05, ratio <0.5 or >2). Protein-protein interaction analysis demonstrated the presence of a network containing 56 differentially expressed proteins, and muscle fibre hyperplasia was closely related to a protein-protein network of 12 muscle component proteins. Muscle fibre hyperplasia was also accompanied by decreased abundance in the fatty acid degradation and calcium signalling pathways. In addition, metabolism via the pentose phosphate pathway decreased in grass carp after ingestion of faba bean, leading to haemolysis. These findings could provide a reference for the prevention and treatment of human glucose-6-phosphate dehydrogenase deficiency ("favism").

Isolation of Tanichthys albonubes beta actin gene and production of transgenic Tanichthys albonubes.

A beta actin cDNA of Tanichthys albonubes was isolated through the RT-PCR and RACE approach. The cDNA was 1,787-bp in length, including a 1,128-bp CDS, a 95-bp 5'UTR and a 564-bp 3'UTR. Genomic DNA containing the transcription region and 5'-flanking region was cloned based on the beta actin cDNA by Genome walker. A 3,000-bp beta actin gene promoter was then produced by PCR according to the sequences of the 5'-flanking region and the first intron. This promoter consisted of a 1,800-bp 5'-flanking region, and a 1,200-bp 5'-UTR. 3 transcription elements, CAAT box, CArG motif and TATA box were found in the 5'-flanking region. This promoter was inserted into the vector pDsRed2-1 and microinjected into fertilized eggs of Tanichthys albonubes to prove its transcription activity. The beta actin promoter and GH CDS of Tanichthys albonubes were then fused to construct an expression vector pTLA-GH. GH-transgenic Tanichthys albonubes was obtained by microinjection of the pTLA-GH into the fertilized eggs. Fast-growth individuals were observed in the transgenic group and the body weight of the largest individual was 2.1-fold that of the maximum in its non-transgenic siblings in 100 dph. In addition, a co-injection strategy was employed with pTLA-DsRed and pTLA-GH vector and proven to enhance the efficiency of GH-transgenic fish detection.