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.

Proteomic and metabolomic basis for improved textural quality in crisp grass carp (Ctenopharyngodon idellus C.et V) fed with a natural dietary pro-oxidant.

Reactive oxygen species (ROS) regulate metabolism and chemical composition of various tissues. To understand how ROS affect the textural quality of fish muscle, we performed a multi-omics analysis on an established crisp grass carp model fed with a natural pro-oxidant faba bean. ROS levels were systemically and significantly increased up to three-fold in crisp grass carp, improving the muscle texture. Lipid metabolism was significantly enhanced up to five-fold in muscle and liver possibly to compensate the impaired carbohydrate metabolism of these tissues, but this caused further local ROS production. Mitochondrial damage associated with autophagy was evident in crisp grass carp. Proteomics revealed that elevated ROS likely disturbed the actin-myosin interaction and collagen turnover inducing fragmentation of myofibrillar proteins, all of which could have positively impacted the textural quality. The systemic metabolic changes that lead to the partial collapse of redox regulation likely underlie the ROS-induced improvement of textural quality.

Quantitative phosphoproteomic analysis of soft and firm grass carp muscle.

Fish muscle firmness is an important quality trait for consumer acceptance. Phosphorylation is known to change chemical and physical properties of proteins and is thus expected to affect muscle firmness, but only few such phosphoproteins have been identified. To explore phosphoproteins that affect fish muscle firmness, firm muscle (crisp grass carp) and soft muscle (ordinary grass carp) were analyzed by quantitative phosphoproteomics. We identified 27 up-regulated and 22 down-regulated phosphopeptides in crisp grass carp (ratio ≥1.5 or ≤0.667, and P-value < 0.05) and their potential upstream kinases. Protein-protein interaction analysis clustered these phosphoproteins into four groups, many of which have been suggested to impact muscle firmness and its postmortem changes: muscle fiber, connective tissue, carbohydrate metabolism and signal regulation. These results provide novel insights into the role of protein phosphorylation in fish muscle firmness and will contribute to the quality improvement of fish products.

Artificial substrata increase pond farming density of grass carp (Ctenopharyngodon idella) by increasing the bacteria that participate in nitrogen and phosphorus cycles in pond water.

Aquaculture has become a primary method to produce various aquatic products, and intensive aquaculture technologies have become commercially important. To improve the efficiency of intensive aquaculture per unit area without reducing the growth rate of cultured fish, the present study explored the potential of artificial substrata in ponds. Our results showed that the concentrations of total nitrogen (TN) and total phosphorous (TP) in the ponds with different stocking densities of grass carp were lower than those in the control group in most cases. Further, the feed conversion rate of grass carp was significantly reduced by introducing these artificial substrata, and the culture density could be significantly increased without reducing the growth rates of these fish. Artificial substrata also significantly enriched specific bacteria and changed the structure of the microbiota in pond water. The relative abundance of Proteobacteria was significantly increased, and bacteria closely related to N and P cycles, such as Hyphomicrobium, Chitinimonas, Legionella, Shewanella, Roseiflexus, and Planktothrix were significantly enhanced. These results showed that the artificial substratum could increase TN and TP removal in aquaculture pond water by enriching N and P cycle-related bacteria, thus significantly increasing the specific growth rate of grass carp and significantly reducing their feed conversion rate. Finally, the stocking density of grass carp and the yield per unit area of pond could be increased without reducing the growth rate.

Hydrodynamics of an in-pond raceway system with an aeration plug-flow device for application in aquaculture: an experimental study.

An in-pond raceway system (IPRS) is an effective intensive aquaculture practice for regions with high water consumption and limited land resources. Water flow and dissolved oxygen (DO) are important for sustainable aquaculture. Several innovations have been made in IPRS design and operation to increase water exchange and DO concentration; one of these is the aeration plug-flow device (APFD). The APFD is commonly used in China as the only power source for water recirculation in aquaculture ponds. Understanding of the hydrodynamics of the system is necessary to improve the design of the IPRS with APFD. To this end, we performed experimental studies on a model system. We measured three-dimensional velocity at various locations using an Acoustic Doppler Velocimeter. Velocity distribution and turbulence characteristics were assessed, and plug-flow characteristics were analysed. Two patterns of velocity and turbulence in horizontal sections were observed: near the APFD, the water flow was intensively pushed downstream and simultaneously recirculated; farther away, the reflux area gradually decreased and the velocity and turbulence distribution trended towards uniform. Secondary flows occurred in different directions, which improved the diffusion of materials and DO retention. The system is effectively self-circulating, and the plug-flow capability may be scaled up for commercial application.

Berberine Influences Blood Glucose via Modulating the Gut Microbiome in Grass Carp.

Berberine (BBR), an isoquinoline alkaloid, is a major pharmacological component of the Chinese herb Coptis chinensis, which has been listed in the Chinese Fisheries Pharmacopeia as a common drug for the control of bacterial fish diseases. However, BBR is poorly absorbed into the systemic circulation but is significantly accumulated in the intestine. It is difficult to explain the mechanism of clinical effects of BBR based on systemic genes and pathways; it has been proved that the function of BBR in mammals is associated with the host metabolic phenotypes mediated by the structural modulation of gut microbiota. The mechanism of pharmacological effects of BBR in fish remains unclear. Here, we fed grass carp (Ctenopharyngodon idellus) a diet supplemented with BBR at a dose of 30 mg/Kg body weight daily and compared them with grass carp fed a regular fish feed diet. Biochemical analysis revealed that fish fed BBR had significantly reduced serum glucose, total cholesterol (TC), and triglyceride (TG) levels, and increased TC (p < 0.05) and TG (p < 0.01) levels in the liver. Deep amplicon sequencing of the V4 region of 16S rRNA genes of the gut microbiota revealed: (i) the composition of gut microbiota after BBR feeding was more diverse than that in the control group; (ii)before fish were fed BBR, the enriched operational taxonomic units (OTUs) mainly belonged to Firmicutes while most enriched OTUs came from Proteobacteria, Planctomycetes, Bacteroidetes, and Firmicutes during BBR feeding and after BBR feeding stopped; (iii) the ratio of Firmicutes to Bacteroidetes was significantly decreased in fish fed BBR. Spearman's rank correlation showed that 32 berberine-OTUs were significantly negative correlated with glucose (p < 0.05). It indicates that BBR may affect the levels of serum glucose by the structural modulation of gut microbiota. Our results provide insight into the effect of BBR on fish metabolism and gut microbiomes, which would be beneficial for the fish welfare.

Epizootic ulcerative syndrome causes cutaneous dysbacteriosis in hybrid snakehead (Channa maculata♀ × Channa argus♂).

Cutaneous microbiota play an important role in protecting fish against pathogens. Aphanomyces infection causes epizootic ulcerative syndrome (EUS) in fish, and by perturbing the integrity of the cutaneous microbiota, increases the potential for infection by pathogenic bacteria. However, whether the composition of the cutaneous microbiota is altered in fish with EUS, and if so, which species are changed and how this might influence infected fish, is still largely unclear. Considering the importance of cutaneous microbiota in maintaining host health, we hypothesized that Aphanomyces infection significantly enhances the presence of certain bacterial pathogens in the cutaneous microbiota and causes cutaneous dysbacteriosis. To test this hypothesis, we compared the cutaneous microbiota compositions of hybrid snakehead (Channa maculata♀ × Channa argus♂) with and without Aphanomyces infection using Illumina Miseq sequencing of the 16S rRNA gene. Our results showed that the cutaneous microbiota of hybrid snakehead were significantly altered subsequent to EUS infection and that the numbers of potentially pathogenic bacteria classified into the genera Anaerosinus, Anaerovorax, Dorea, and Clostridium were significantly enhanced in the cutaneous microbiota of hybrid snakehead with EUS, whereas bacteria classified into the genera Arthrobacter, Dysgonomonas, Anoxybacillus, Bacillus, Solibacillus, Carnobacterium, Lactococcus, Streptococcus, Achromobacter, Polynucleobacter, Vogesella, and Pseudomonas were significantly reduced. These results imply that treatment for EUS should not only take into consideration the control of Aphanomyces reproduction but should also focus on regulating the cutaneous microbiota of infected fish.

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.

Identification and expression analysis of miRNA in hybrid snakehead by deep sequencing approach and their targets prediction.

MicroRNAs (miRNAs) play important regulatory roles in numerous biological processes, but there is no report on miRNAs of hybrid snakehead. In this study, four independent small RNA libraries were constructed from the spleen, liver kidney and muscle of hybrid snakehead. These libraries were sequenced using deep sequencing technology, as result, a total of 1,067,172, 1,152,002, 1,653,941 and 970,866 clean reads from these four libraries were obtained. 252 known miRNAs and 63 putative novel miRNAs in these small RNA dataset were identified. The stem-loop RT-qPCR analysis indicated that eight known miRNAs and two novel miRNAs show different expression in eight different kinds of tissues. For better understanding the functions of miRNAs, 95,947 predicated target genes were analyzed by GO and their pathways, the results indicated that these targets of the identified miRNAs are involved in a broad range of physiological functions.

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.

Identification and comparative analysis of the miRNA expression profiles from four tissues of Micropterus salmoides using deep sequencing.

In the present study, four small RNA libraries were constructed from an M. salmoides population and sequenced using deep sequencing technology. A total of 9,888,822; 8,519,365; 20,566,198; and 15,762,254 raw reads representing 666,097; 755,711; 978,923; and 840,175 unique sequences were obtained from the spleen, liver, kidney, and muscle libraries, respectively. As a result, 509 known miRNAs belonging to 143 families and 1157 novel miRNAs were identified. The miRNAs displayed diverse expression levels among the four libraries, among which most of the known miRNAs were expressed at higher levels than the novel miRNAs. Furthermore, stem-loop qRT-PCR was applied to validate and profile the expression of the differentially expressed miRNAs in the four different tissues, which revealed that some miRNAs showed tissue specific expression. The identification of miRNAs in M. salmoides will provide new information and enhance our understanding of the functions of miRNAs in regulating biological processes.

Broad Bean (Vicia faba L.) Induces Intestinal Inflammation in Grass Carp (Ctenopharyngodon idellus C. et V) by Increasing Relative Abundances of Intestinal Gram-Negative and Flagellated Bacteria.

Constant consumption of broad bean (Vicia faba L.) induces intestinal inflammation and reduces growth rate in grass carp (Ctenopharyngodon idellus C. et V). However, the mechanisms underlying these effects are unclear. In mammalian models of inflammatory bowel disease (IBD), endotoxin and flagellin cause intestinal inflammation through upregulation of tumor necrosis factor (TNF)-α expression. We therefore speculated that broad bean consumption alters intestinal microbiota composition, thereby increasing the relative abundance of endotoxin-producing Gram-negative and flagellated bacteria and resulting in upregulation of TNF-α and intestinal inflammation in grass carp. We tested this hypothesis by comparing intestinal microbiota compositions of grass carp fed broad bean (GCBB), hybrid giant napier (Pennisetum sinese Roxb, GCHG), or formula feed (GCFF) by 16S rRNA gene sequencing. We also performed a histological analysis of the intestinal inner wall by scanning electron microscopy and measured intestinal wall and serum concentrations of TNF-α. Our results revealed epithelial cell damage including microvillus effacement and synechia along with increased TNF-α levels in the intestinal wall in the GCBB group as compared to the GCHG and GCFF groups. The relative abundances of Gram-negative and flagellated bacteria were also higher in the GCBB group than in the GCHG and GCFF groups; this was accompanied by upregulation of genes expressing endotoxin and flagellin in intestinal microbiota. Thus, broad bean-induced intestinal inflammation in grass carp shares features with IBD. Our findings demonstrate that the microbiome in fish is directly influenced by diet and provide a reference for deconstructing host-intestinal microbiota interactions.

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").

Genome-wide identification and characterization of conserved and novel microRNAs in grass carp (Ctenopharyngodon idella) by deep sequencing.

MicroRNAs (miRNAs) are post-transcriptional regulators which bind to target to regulate protein expression by repressing translation or promoting degradation of the target mRNA. Studies have shown that deep sequencing is a powerful tool for the identification of miRNAs, and it is believed that may more miRNAs remain to be discovered in grass carp. In the present study, a pool of equal amounts of RNA obtained from 8 different adult grass carp tissues (spleen, liver, muscle, kidney, skin, testis, gut and heart) was sequenced using deep sequencing technology. A total of 16.579.334 raw reads were yielded from the pooled small RNA library. Using bioinformatics analysis, we identified 160 conserved miRNAs and 18 novel miRNAs in grass carp. Randomly selected 6 conserved and 2 novel miRNAs were confirmed their expression by stem-loop qRT-PCR assay. Furthermore, the 1212 potential targets of these miRNAs were predicted using miRNA target prediction tool. GO and KEGG pathway enrichment analyses indicated relevant biological processes. Our study provides the first genome-wide investigation of miRNAs from 8 mixed tissues of grass carp, and the data obtained expand the known grass carp miRNA inventory and provide a basis for further understanding functions of grass carp miRNAs.

Microbial succession in biofilms growing on artificial substratum in subtropical freshwater aquaculture ponds.

Biofilms can be used to improve the water quality in aquaculture ponds, and elucidating the process of microbial succession in biofilms would allow the characterization of metabolic processes and permit optimization. In the present study, microbial succession of a biofilm growing on artificial substrata in a subtropical freshwater pond was investigated by high-throughput sequencing. Providing artificial substrata effectively reduced the concentrations of total nitrogen and total phosphorus in the pond. Relatively stable microbiota were formed after approximately 1 week. The dominant phyla in the mature biofilm were Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. The relative abundances of denitrifiers and phosphorus-removing bacteria, such as those in Comamonadaceae and Neisseriaceae, were significantly increased. The use of avermectin B1 changed the community structure of the microbiota; the microbiota were more similar to those at Week 0 than to those at Week 3. However, the microbial community structure recovered after approximately 1 week. Our results indicate that using artificial substrata can create a habitat for denitrifiers and phosphorus-removing bacteria, and thereby improve pond water quality. This study provided insight into how the use of artificial substrata could improve water quality and elucidated the environment-biofilm relationship in a subtropical freshwater pond.

Gene Expression Profiling of Grass Carp (Ctenopharyngodon idellus) and Crisp Grass Carp.

Grass carp (Ctenopharyngodon idellus) is one of the most important freshwater fish that is native to China, and crisp grass carp is a kind of high value-added fishes which have higher muscle firmness. To investigate biological functions and possible signal transduction pathways that address muscle firmness increase of crisp grass carp, microarray analysis of 14,900 transcripts was performed. Compared with grass carp, 127 genes were upregulated and 114 genes were downregulated in crisp grass carp. Gene ontology (GO) analysis revealed 30 GOs of differentially expressed genes in crisp grass carp. And strong correlation with muscle firmness increase of crisp grass carp was found for these genes from differentiation of muscle fibers and deposition of ECM, and also glycolysis/gluconeogenesis pathway and calcium metabolism may contribute to muscle firmness increase. In addition, a number of genes with unknown functions may be related to muscle firmness, and these genes are still further explored. Overall, these results had been demonstrated to play important roles in clarifying the molecular mechanism of muscle firmness increase in crisp grass carp.

[Induction of pond-cultivated Anguilla japonica gonadial development by controlling temperature in high temperature season].

In this study, the pond-cultivated Anguilla japonica was induced to mature by artificially controlling temperature at 18 +/- 2 degrees C in high temperature season (from June to September), with injection of hCG and CPE. The results showed that both the male and the female eel could be induced to mature, and the artificial maturation induction rate was 73.33% and 67.77%, while under non-controlling temperature 25 approximately 32 degrees C, the maturation induction rate was 0 and 33.33%, respectively. In an ovulation induction test, the matured female eels were induced, with a spawning inducement rate of 70%, and a fertilization rate of 32%, which approached or reached the levels in low temperature season (with an average of 62.1% and 34.2%, respectively), and the zygote developed normally. At the temperature 22 and 24 degrees C, the zygote hatched to fry in about 39 h 15 min and 34 h 9 min. The experiment proved that it was possible to induce the eel to mature by controlling temperature in high temperature season, which provided some theoretical foundations for the techniques of artificially breeding eel in whole year.