Evaluation of four novel non-grain protein sources completely replacing soybean meal on growth performance, serum biochemistry, amino acid transport and intestinal health of grass carp (Ctenopharyngodon idella) at different water temperatures.

To investigate the effects of non-grain protein source and water temperature on growth and feed utilization differences of grass carp, the effects of different protein sources on the growth performance, serum biochemistry, digestive enzymes, amino acid transport and intestinal health of grass carp were studied at 24°C, 28°C and 32°C. In this study, a total of 1350 grass carp (5.00 ± 0.02 g) were selected, and Clostridium autoethanogenum protein (CAP), Tenebrio molitor meal (TMM), cottonseed protein concentrate (CPC) and Chlorella powder (CHP) were used as a single protein source to completely replace soybean meal for 56 days. The results showed that the final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR) and protein efficiency ratio (PER) of grass carp increased significantly with the increasing temperature (P < 0.001). The CHP and SBM groups showed no significant differences in FBW, WGR, SGR and PER (P > 0.05), which were higher than the CAP, TMM and CPC groups (P < 0.001). The alanine transaminase (ALT), aspartate aminotransferase (AST), total protein (TP) and triglyceride (TG) concentrations of grass carp at 32°C were significantly lower than those at 24°C and 28°C (P < 0.001). The acid phosphatase (ACP) activity decreased significantly with the increase of temperature (P = 0.001). The amylase (AMS) activity of the TMM, CPC and CHP groups was significantly lower than that of the SBM and CAP groups (P < 0.001), and the ACP and lipase (LPS) activities in the TMM group were significantly lower than those in the SBM group (P < 0.001). In addition, the interaction between temperatures and protein sources significantly affected the gene expression levels of amino acid transport including solute carrier family 1 member 3 (SLC1A3), solute carrier family 7 member 1 (SLC7A1), solute carrier family 7 member 5 (SLC7A5), solute carrier family 15 member 1b (SLC15A1b), solute carrier family 7 member 7 (SLC7A7), target of rapamycin (TOR), 4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1), intestinal inflammatory including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-8 (IL-8), interleukin-10 (IL-10) and tight junction proteins (occludin, claudin1, claudin3, claudin7 and claudin11) (P ≤ 0.001). Collectively, our results indicated that CHP could be a potential protein source in the case of complete replacement of soybean meal in grass carp.

Analysis of the genetic structure of the introduced clearhead icefish (Protosalanx chinensis) populations in northern China.

When species are introduced to a new environment, they can quickly adapt to the environment and may differ from the indigenous species. The indigenous population of Protosalanx chinensis has a high level of genetic diversity, but it is unclear on the genetic diversity of the introduced populations in northeast China, which supports the major production of P. chinensis in the world. A total of 556 individuals of P. chinensis were collected during 2016-2021, from Lianhuan Lake (LHL), Xingkai Lake (XK), and Shuifeng Reservoir (SF), and one population was collected from the indigenous Taihu Lake (TH). Overall, 36 haplotypes were detected, and the genetic differences in P. chinensis populations within and between river basins were investigated. The nucleotide diversity (π) of the populations ranged from 0.0005 to 0.0032, and the haplotype diversity (Hd) ranged from 0.455 to 0.890, with the highest genetic diversity in the TH population, followed by the SF population, and lower genetic diversity in the XK and LHL populations. The analysis of the genetic differentiation index (Fst) and the genetic distance between populations showed that there was significant genetic differentiation between the TH population and the other populations. More sampling points have been set up in LHL for further analysis; the Dalong Lake (DL) and the Xiaolong Lake (XL) populations were far from the other populations within the LHL population. In this study, we didn't find a correlation between population size, stability, and genetic diversity, and the ecological measures of management should be decisive to the population dynamics. These results provide a basis for the rational utilization and effective management of P. chinensis.

Momordica charantia saponins administration in low-protein-high-carbohydrate diet improves growth, blood biochemical, intestinal health and microflora composition of juvenile common carp (Cyprinus carpio).

An 8-week feeding trial was conducted to explore the feasibility of Momordica charantia saponins (MCS) administration to facilitate the protein-sparing action of high carbohydrate in diets for juvenile common carp (Cyprinus carpio) with initial mass of 5.41 ± 0.02 g. Based on our previous study, four diets with different the ratio of protein and carbohydrate (P/C ratio) were designed: 32%P/40%C, 30%P/43%C, 28%P/46%C, 28%P/46%C supplemented with 0.16% MCS (28%P/46%C + MCS). Each diet treatment was divided into 3 replicates. Results revealed that 30%P/43%C group increased growth performance and intestinal digestion, decreased intestinal inflammation, and optimized the intestinal microbiota compared to 32%P/40%C group, which presented the stronger protein-sparing action of high carbohydrate. But if the P/C ratio reduced to 28%P/46%C or less, the saving action would be restrained. However, compared to the 30%P/43%C and 28%P/46%C groups, 28%P/46%C + MCS group significantly elevated growth performance and activities of digestive enzymes and antioxidative enzymes, whilst the opposite trend occurred in the contents of glucose, triglyceride, total cholesterol, low density lipoprotein cholesterol, blood urea nitrogen, glutamic oxalacetic transaminase, glutamic-pyruvic transaminase and malondialdehyde. In addition, 28%P/46%C + MCS group markedly upregulated the expressions of GH/IGF axis genes, genes involved in protein synthesis, antioxidant genes and anti-inflammatory cytokine, whilst the opposite trend occurred in the expressions of pro-inflammatory cytokines. Moreover, 28%P/46%C + MCS group obtained the remarkably higher Enterococcus proportion and lower Lactococcus proportion compared to the 30%P/43%C and 28%P/46%C groups, whereas the opposite occurred in 30%P/43%C group, which indicated that there existed differences in the improvement mechanism on intestinal microflora composition between MCS and appropriate P/C ratio. Combined with the above mentioned changes in our research, we concluded that 0.16% MCS administration in a 28%P/46%C diet could facilitate the protein-sparing action of high carbohydrate in diets for common carp, which could decrease the 5% dosage of soybean meal and synchronously reduce the 4% crude protein of diets without affecting the growth and immune ability for common carp.

Genetic Diversity of Four Populations of Silver Carp (Hypophthalmichthys molitrix) Based on Mitochondrial Sequences.

Three mitochondrial DNA sequences (COI, ATP 8&6, and D-loop) were employed to assess the genetic diversity of four populations of silver carp from three main drainages in China, including the Yangtze River, the Amur River, and the Pearl River. As a result, 98 haplotypes were identified in combined sequences of COI, ATP8&6, and D-loop. A total of 196 variable sites and 116 parsimony-informative sites were observed. AMOVA based on assembled sequences indicated that 12.12% of the variation was among populations, while 87.88% of the variation was within populations. Additionally, the phylogenetic relationships of populations were depicted in a phylogenetic tree based on assembled sequences. Mismatch distribution analysis and the negative significant Fu's Fs values supported population expansion in all populations. Despite the high level of genetic diversity, the establishment of a state-level original breeding farm in the Amur River basin and the Pearl River basin may be an effective conservation strategy for the protection of local unique haplotypes.

Comprehensive assessment of detoxification mechanisms of hydrolysis fish peptides in largemouth bass (Micropterus salmoides) under copper exposure: Tracing from bioaccumulation, oxidative stress, lipid deposition to metabolomics.

As a heavy metal, copper is toxic to aquatic organisms in water, causing oxidative stress and lipid deposition. However, there is currently no effective dietary strategy to prevent damage caused by copper exposure. Here, copper bioaccumulation, antioxidant enzymes, lipogenic enzymes, lipid metabolism-related gene expression levels and metabolic pathways were synthesized and evaluated in copper-exposed largemouth bass (Micropterus salmoides) after hydrolysis fish peptides (HFP) pretreatment. The results showed that supplementation with 1% (P < 0.05), 3% (P < 0.01) and 5% (P < 0.05) HFP significantly reduced the copper bioaccumulation in largemouth bass. Hydrolysis fish peptides supplementation significantly reduced the activities of total antioxidant capacity (P < 0.01) and catalase (P < 0.01) and the contents of glutathione (P < 0.01) and malondialdehyde (P < 0.05). Fatty acid synthetase concentration was significantly reduced in fish supplemented with 3% (P < 0.05) and 5% HFP (P < 0.05). Similarly, fish fed 3% (P < 0.05) and 5% (P < 0.01) HFP significantly reduced the glucose-6-phosphate dehydrogenase concentration. Serum metabolomics revealed that 85, 144 and 207 differential metabolites were obtained in fish supplemented with 1%, 3% and 5% HFP, respectively. The differential metabolites were mainly lipids and lipid-like molecules, which were associated with the lipid metabolism pathways. The expression levels of fatty acid synthase (P < 0.01), sterol regulatory element binding protein-1c (P < 0.05), liver X receptor (P < 0.001), peroxisome proliferator activated γ (P < 0.01), apolipoprotein B (P < 0.001) and fatty acid-binding protein 1 (P < 0.01) were significantly down-regulated and the expression levels of carnitine palmitoyltransferase 1α (P < 0.01), hormone-sensitive lipase (P < 0.001), apolipoprotein A 1 (P < 0.05) were significantly up-regulated in fish fed with 3% HFP. Additionally, supplementation with 3% (P < 0.01) and 5% (P < 0.001) HFP significantly up-regulated the expression level of B-cell lymphoma-2 with a dose-dependent effect. In conclusion, our study confirmed that HFP supplementation was closely associated with oxidative stress, enzymatic activities and related pathways of lipid metabolism, and apoptosis, and in general alleviated lipid deposition caused by copper exposure in largemouth bass.

Molecular characterization and expression patterns of two hormone-sensitive lipase genes in common carp Cyprinus carpio.

The hormone-sensitive lipase (HSL) gene plays an important role in mammals' lipid metabolism. Therefore, its function in fish is capturing increasing attention. In this study, two distinct cDNAs, designated HSL1 and HSL2, are firstly identified from common carp Cyprinus carpio. The full-length cDNA of HSL1 and HSL2 consists of 3379 bp and 2732 bp, encoding polypeptide of 693 and 847 amino acids, respectively, and shares 60.6% amino acid identity. Phylogenetic analysis suggests that HSL1 and HSL2 are derived from paralogous genes, which might have arisen during a teleost-specific genome duplication event. The two HSL mRNAs are differentially expressed, both in terms of distribution among tissues and in terms of abundance during embryogenesis. Moreover, both HSL mRNAs are expressed in various tissues, the highest in abdominal fat. Meanwhile, the two HSLs are detected at all stages of embryonic development, suggesting that they could be functional and involved in embryogenesis. In addition, the results show that the mRNA expression level of HSL2 in the high group of intramuscular fat content is significantly higher than that in the low group (P < 0.01). The research provides basic data for developing a further understanding of the function of HSL as well as molecular regulation mechanism in fat metabolism of common carp.

QTL variations for growth-related traits in eight distinct families of common carp (Cyprinus carpio).

BACKGROUND: Comparing QTL analyses of multiple pair-mating families can provide a better understanding of important allelic variations and distributions. However, most QTL mapping studies in common carp have been based on analyses of individual families. In order to improve our understanding of heredity and variation of QTLs in different families and identify important QTLs, we performed QTL analysis of growth-related traits in multiple segregating families. RESULTS: We completed a genome scan for QTLs that affect body weight (BW), total length (TL), and body thickness (BT) of 522 individuals from eight full-sib families using 250 microsatellites evenly distributed across 50 chromosomes. Sib-pair and half-sib model mapping identified 165 QTLs on 30 linkage groups. Among them, 10 (genome-wide P <0.01 or P < 0.05) and 28 (chromosome-wide P < 0.01) QTLs exhibited significant evidence of linkage, while the remaining 127 exhibited a suggestive effect on the above three traits at a chromosome-wide (P < 0.05) level. Multiple QTLs obtained from different families affect BW, TL, and BT and locate at close or identical positions. It suggests that same genetic factors may control variability in these traits. Furthermore, the results of the comparative QTL analysis of multiple families showed that one QTL was common in four of the eight families, nine QTLs were detected in three of the eight families, and 26 QTLs were found common to two of the eight families. These common QTLs are valuable candidates in marker-assisted selection. CONCLUSION: A large number of QTLs were detected in the common carp genome and associated with growth-related traits. Some of the QTLs of different growth-related traits were identified at similar chromosomal regions, suggesting a role for pleiotropy and/or tight linkage and demonstrating a common genetic basis of growth trait variations. The results have set up an example for comparing QTLs in common carp and provided insights into variations in the identified QTLs affecting body growth. Discovery of these common QTLs between families and growth-related traits represents an important step towards understanding of quantitative genetic variation in common carp.

Development and evaluation of the first high-throughput SNP array for common carp (Cyprinus carpio).

BACKGROUND: A large number of single nucleotide polymorphisms (SNPs) have been identified in common carp (Cyprinus carpio) but, as yet, no high-throughput genotyping platform is available for this species. C. carpio is an important aquaculture species that accounts for nearly 14% of freshwater aquaculture production worldwide. We have developed an array for C. carpio with 250,000 SNPs and evaluated its performance using samples from various strains of C. carpio. RESULTS: The SNPs used on the array were selected from two resources: the transcribed sequences from RNA-seq data of four strains of C. carpio, and the genome re-sequencing data of five strains of C. carpio. The 250,000 SNPs on the resulting array are distributed evenly across the reference C.carpio genome with an average spacing of 6.6 kb. To evaluate the SNP array, 1,072 C. carpio samples were collected and tested. Of the 250,000 SNPs on the array, 185,150 (74.06%) were found to be polymorphic sites. Genotyping accuracy was checked using genotyping data from a group of full-siblings and their parents, and over 99.8% of the qualified SNPs were found to be reliable. Analysis of the linkage disequilibrium on all samples and on three domestic C.carpio strains revealed that the latter had the longer haplotype blocks. We also evaluated our SNP array on 80 samples from eight species related to C. carpio, with from 53,526 to 71,984 polymorphic SNPs. An identity by state analysis divided all the samples into three clusters; most of the C. carpio strains formed the largest cluster. CONCLUSIONS: The Carp SNP array described here is the first high-throughput genotyping platform for C. carpio. Our evaluation of this array indicates that it will be valuable for farmed carp and for genetic and population biology studies in C. carpio and related species.

Dietary Lycium barbarum Polysaccharide Modulates Growth Performance, Antioxidant Capacity, and Lipid Metabolism in Common Carp (Cyprinus carpio) Fed with High-Fat Diet.

To investigate the ameliorative effects and mechanism of Lycium barbarum polysaccharide (LBP) on growth performance, oxidative stress, and lipid deposition in common carp (Cyprinus carpio) fed with high-fat diets, fish with an initial weight of 5.29 ± 0.12 g were divided into five experimental groups-including normal-fat diets, high-fat diets, and high-fat diets-supplemented with LBP (0.5, 1.0, and 2.0 g/kg) for 8 weeks. The results showed that high-fat diets resulted in significant decreases in final body weight, weight gain rate, and specific growth rate of fish, as well as causing a significant decrease in hepatic total antioxidant capacity, catalase, and glutathione peroxidase activities. These changes were accompanied by a significant decrease in lipase activity and ATP level and a significant increase in malondialdehyde content. The expression levels of lipid metabolism-related genes (acetyl coenzyme A carboxylase 1, stearoyl coenzyme A desaturase 1, fat synthase, peroxisome proliferator-activated receptor-γ, fructofuranose bisphosphatase, and glucose-6-phosphatase) were also markedly elevated by high-fat diets. Supplementation with 0.5-2.0 g/kg LBP in high-fat diets improved the reduced growth performance, increased hepatic total antioxidant enzymes, catalase, and glutathione peroxidase activities, and lowered malondialdehyde level in fish fed with high-fat diets. Additionally, dietary supplementation with LBP significantly downregulated hepatic gene expression levels of acetyl coenzyme A carboxylase 1, stearoyl coenzyme A desaturase 1, fat synthase, sterol regulatory element-binding protein 1, peroxisome proliferator-activated receptor-γ, fructofuranose bisphosphatase, and glucose-6-phosphatase. In conclusion, fish fed with high-fat diets demonstrated impaired growth performance, antioxidant capacity, and lipid metabolism, and dietary supplementation with 0.5-2.0 g/kg LBP ameliorated the impairments induced by high-fat diets.

[Microsatellite markers for parentage identification in Jian Carp (Cyprinus carpio var. Jian)].

Using 16 microsatellite loci we estimated the parentage of 647 progeny in 10 Jian Carp full-sib families. Cervus 3.0 analysis showed that mean PIC value of 16 microsatellites, mean number of allele, and mean expected heterozygosity were 0.7025, 6.63, and 0.7405, respectively. The combined probability of exclusion was 0.99922456 when both parents were unknown and the combined probability of exclusion was 0.99999557 when only one of the parental genotype was known, with the confidence level of 95%. Further simulations based on allele frequencies suggested that to achieve the requirements of paternity test usually took 8 to 12 microsatellite loci when both parents were unknown and 5 to 8 microsatellite loci when one parent was known. Out of 647 progenies, 94.6% were assigned to their parental pairs without the information of both parents in parentage analysis, which were lower than the theoretical assignment rates predicted by the Cervus simulations. This could be explained by the relationship between the candidate parents or existence of null and by typing errors. The identification of 9 families was useful for linkage analysis of Jian Carp and QTL location, also for marker assisted selection for economical traits.

Resveratrol ameliorates oxidative stress, inflammatory response and lipid metabolism in common carp (Cyprinus carpio) fed with high-fat diet.

High-fat diet is regarded as crucial inducers of oxidative stress, inflammation, and metabolic imbalance. In order to investigate the ameliorative potential of resveratrol against the progression of liver injury towards steatohepatitis, common carp (Cyprinus carpio) were distributed into six experimental groups and were fed with a normal-fat diet, a high-fat diet, and supplemented with resveratrol (0.8, 1.6, 2.4, and 3.2 g/kg diet) for 8 weeks. The high-fat diet decreased the antioxidant capacities, as well as causing the inflammatory response and lipid deposition of common carp. Resveratrol induced a marked elevation in the final body weight, weight gain rate, condition factor and significant decrease in the feed conversion ratio. Moreover, dietary resveratrol showed a significant decrease in the alanine aminotransferase, aspartate aminotransferase, triglyceride and low-density lipoprotein levels, which was accompanied by an increase in high-density lipoprotein concentration in serum. A significant elevation in total superoxide dismutase, catalase, glutathione peroxidase and a decreased malondialdehyde content were observed, along with a substantial elevation in antioxidant activities were found. Additionally, fish fed with resveratrol had an up-regulation of hepatic catalase, copper, zinc superoxide dismutase, glutathione peroxidase 1a, and glutathione peroxidase 1b gene expression via Nrf2 signaling pathway. Expectedly, our results also demonstrated that resveratrol regulates hepatic lipid metabolism in fish by inhibiting the expression of hepatic lipogenesis genes (acetyl-CoA carboxylase 1, fatty acid synthase, and sterol regulatory element binding protein 1), fatty acid uptake-related genes of lipoprotein lipase, and ß-oxidation-related genes via PPAR-γ signaling pathway. Furthermore, dietary resveratrol reduced inflammation, as evident by down-regulating the interleukin-1ß, interleukin-6, interleukin-8, and tumor necrosis factor-α expression levels and upregulating the interleukin-10 and transforming growth factor-ß2 expression levels via NF-κB signaling pathway. As a whole, our results demonstrated that resveratrol defensed the impacts against high-fat diet on the serum biochemical, hepatic antioxidants, inflammation, and lipid metabolism.

Phosphorus Nutrition in Songpu Mirror Carp (Cyprinus carpio Songpu) During Chronic Carbonate Alkalinity Stress: Effects on Growth, Intestinal Immunity, Physical Barrier Function, and Intestinal Microflora.

Carbonate alkalinity is a major environmental stress factor affecting aquatic feed configuration, which easily causes oxidative stress and hypoimmunity for fish. Hence, the purpose of the study is to assess the potential effect of phosphorus on growth, intestinal oxidation resistance, physical barrier function, and microflora for Songpu mirror carp (Cyprinus carpio Songpu) (initial average weight of 2.95 ± 0.21 g) reared at the high-concentration carbonate alkalinity environment. A two-factor, three-level (2 × 3) design was applied, in which diets with three different phosphorus levels (3.6, 7.0, and 10.5 g/kg dry matter) were randomly assigned to 0 and 15 mmol/L carbonate alkalinity groups with three replicate aquariums. After the 8-week trial, we found that weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), and lipase and amylase activities in the intestine significantly (p < 0.05) declined with increasing carbonate alkalinity. Carbonate alkalinity of 15 mmol/L significantly reduced glutathione peroxidase (GSHPx) activities in the intestine (p < 0.05). The relative expressions of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), glutathione peroxidase 1a (GPX1a), Clautin3, Clautin11, and tumor necrosis factor ß (TNF-ß) in the intestine were markedly downregulated by increasing carbonate alkalinity levels (p < 0.05), whilst the relative expressions of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in the intestine were markedly upregulated (p < 0.05). At the 15 mmol/L carbonate alkalinity treatment, Songpu mirror carp suffer from hypoimmunity status with failed digestion, antioxidant, inflammation, and immune response, thereby inducing impaired growth. Additionally, significant increments in the abundance of Proteobacteria and a significant decrease in the abundance of Fusobacteria and the Firmicutes/Bacteroidetes ratio were caused due to excessively high carbonate alkalinity (15 mmol/L) and excessively low dietary phosphorus supply (3.6 g/kg). Collectively, 7.0 g/kg dietary phosphorus supplementation was effective in promoting intestinal antioxidant enzyme activities and the corresponding gene expression via the Keap1-Nrf2 signaling pathway and in enhancing intestinal immunity by upregulating anti-inflammatory and downregulating pro-inflammatory genes. Appropriate dietary phosphorus supply could promote the formation of beneficial microflora in freshwater, and it has the potential ability to transfer the adverse effect of carbonate alkalinity stress to the structural composition of intestinal microflora. Hence, consideration should be given to suitable phosphorus supply for fish under the chronic carbonate alkalinity stress.

In Silico Screening and Development of Microsatellite Markers for Genetic Analysis in Perca fluviatilis.

Perca fluviatilis is an economically important species of freshwater fish. To understand the genetic structure of P. fluviatilis in China, 268 samples were collected from Wulungu Lake (WL), Jili Lake (JL), the Wulungu River (WR), and the Kalaeerqisi River (KR). These samples were then analyzed using microsatellite markers. A total of 98,425 microsatellite markers were developed based on the genomic data, and 29 polymorphic microsatellite markers were selected to analyze genetic diversity in this study. The number of alleles (Na) and observed heterozygosity (Ho) per population ranged from 4.621 (KR) to 11.172 (WL) and from 0.510 (KR) to 0.716 (JL), respectively. The results of the polymorphic information content (PIC) showed that the WL, JL, and WR populations were highly polymorphic (PIC≥ 0.5) and that the KR population was moderately polymorphic (0.25 ≤ PIC < 0.5). The genetic differentiation coefficient (Fst) among the four P. fluviatilis populations was 0.074, indicating moderate genetic differentiation among the populations in Xinjiang. The reason for the significant difference between the rivers and lakes could be the presence of a dam blocking the flow of P. fluviatilis. The development of microsatellite markers provides support for population genetics in the future. The evaluation of the genetic structure of P. fluviatilis in Xinjiang provides a reference for the reproduction and conservation of P. fluviatilis.

Assessment of Fish Protein Hydrolysates in Juvenile Largemouth Bass (Micropterus salmoides) Diets: Effect on Growth, Intestinal Antioxidant Status, Immunity, and Microflora.

Varying dietary inclusion levels of fish protein hydrolysates (FPH) were applied in a feeding experiment with juvenile largemouth bass (Micropterus salmoides) to assess their effects on growth, intestinal antioxidant status, immunity, and microflora. FPH were added in 4 dietary levels: 0 g/kg (control group, FPH-0), 10 g/kg (FPH-10), 30 g/kg (FPH-30), and 50 g/kg (FPH-50) dry matter, respectively substituting 0, 5.3, 16.3, and 27.3% of fish meal with dietary fish meal. Quadruplicate groups of 25 juvenile largemouth bass with initial body weight 9.51 ± 0.03 g were fed during the 56-day feeding experiment. Experimental results showed that fish fed FPH-30 obtained a significantly higher weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), and significant feed conversion rate (FCR) compared to the other three groups (P < 0.05). FPH-30 group also promoted protein synthesis and deposition, as evidenced by the higher whole-body crude protein contents, the higher expressions of GH1, IGF-1, TOR, and S6K in the liver, and SLC7A5, SLC7A8, SLC38A2, and SLC15A2 in the intestine than the other three groups. FPH-30 group could also enhance intestinal health status by increasing the activities of SOD, POD, CAT, GSH-Px, and T-AOC activities by upregulating the expressions of SOD, GSH-Px, IL1ß, and TNFß, and by reducing the MDA contents and the expressions of IL15, Caspase 3, Caspase 9, and Caspase 10 than other groups. Compared to the control group, the Actinobacteriota abundance markedly decreased in FPH treatments, while the variation tendency of the phylum Proteobacteria was opposite. The peak value of Firmicutes:Bacteroidetes ratio and the lowest of Bacteroidetes abundance were seen in largemouth bass fed FPH-30 (P < 0.05). Fish in three FPH treatments had lower abundances of opportunistic pathogens Staphylococcus and Plesiomonas than fish in the control group. In conclusion, FPH is a nutritious feed ingredient for juvenile largemouth bass, and can be added to a dietary level of 30 g/kg dry matter replacing fish meal without any negative effect on growth and feed utilization. FPH supplements could also strengthen the intestinal immune mechanisms of largemouth bass to tackle the immunodeficiency produced by fish meal replacement.

Transcriptomic Analysis Provides Insights to Reveal the bmp6 Function Related to the Development of Intermuscular Bones in Zebrafish.

Intermuscular bones (IBs) are small, hard-boned spicules located in the muscle tissue that mainly exist in the myosepta of lower teleosts, which hurt the edibleness and economic value of fish. The study of the development of IBs is very important for freshwater aquaculture fish, but the molecular mechanism of its formation and the key regulatory genes remain unclear. In this study, we first constructed two types of zebrafish mutants (the mutants losing IBs and the mutants with partial deletion of IBs) by knocking out bmp6. We then carried out a transcriptomic analysis to reveal the role of bmp6 in the developmental mechanism of IBs; we used the caudal musculoskeletal tissues of these mutants and wild-type zebrafish at three development stages (20, 45, and 60 dph) to perform transcriptomic analysis. The results showed that the deficiency of bmp6 upregulated sik1 and activated the TNF-A signaling via the NF-KB pathway, which inhibited the development of osteoblasts and promoted osteoclast formation, thereby inhibiting the formation of IBs. These results provided insights to understand the role of bmp6 in the development of IBs in zebrafish and are useful for selective breeding of IBs in cyprinids.

A genetic linkage map and comparative genome analysis of common carp (Cyprinus carpio L.) using microsatellites and SNPs.

A genetic linkage map is a powerful research tool for mapping traits of interest and is essential to understanding genome evolution. The aim of this study is to provide an expanded genetic linkage map of common carp to effectively carry out quantitative trait loci analysis and conduct comparative mapping analysis between lineages. Here, we constructed a genetic linkage map of common carp (Cyprinus carpio L.) using microsatellite and single-nucleotide polymorphism (SNP) markers in a 159 sibling family. A total of 246 microsatellites and 306 SNP polymorphic markers were genotyped in this family. Linkage analysis using JoinMap 4.0 organized 427 markers (186 microsatellites and 241 SNPs) to 50 linkage groups, ranging in size from 1.4 to 130.1 cM. Each group contained 2-30 markers. The linkage map covered a genetic distance of 2,039.2 cM and the average interval for markers within the linkage groups was approximately 6.4 cM. In addition, comparative genome analysis within five model teleost fish revealed a high percentage (74.7%) of conserved loci corresponding to zebrafish chromosomes. In most cases, each zebrafish chromosome comprised two common carp linkage groups. The comparative analysis also revealed independent chromosome rearrangements in common carp and zebrafish. The linkage map will be of great assistance in mapping genes of interest and serve as a reference to approach comparative mapping and enable further insights into the comprehensive investigations of genome evolution of common carp.

[Quantitative trait locus analysis of standard length, body depth and body thickness in mirror carp (Cyprinus carpio L.)].

Based on a full-sib family, the genetic linkage map was constructed with 246 microsatellite and 306 SNP markers, which was used to detect the QTLs for standard length (SL), body depth (H), body thickness (BT), and the ratio of standard length and body depth (SLH) in mirror carp by GridQTL software. The results indicated that a total of 14 related QTLs distributed on the 7 linkage groups were obtained. Seven QTLs were related to standard length, of which the linkage groups of LG6, LG17, LG21, LG23, and LG35 were at 5% significant level, and linkage group LG1 and LG28 were at 1% significant level, which explained 6.6%-12.6% of the phenotypic variance. Three QTLs were identified for body depth on the linkage groups of LG17, LG23 and LG28 (P amp; 0.01), accounting for 11.6%, 12.7%, and 15.6% of the phenotypic variance, respectively. Two QTLs were associated with body thickness on the linkage of LG23 and LG28 (P amp; 0.05), which explained 8.6% and 7.2% of the phenotypic variation, respectively. Two QTLs were responsible for the ratio of standard length and body depth on the linkage of LG21 and LG35 (P amp; 0.05), both of which explained 8.2% of the phenotypic variance. The results provide a useful reference for further candidate gene research and molecular marker assisted selection in mirror carp.

Excessive Dietary Lipid Affecting Growth Performance, Feed Utilization, Lipid Deposition, and Hepatopancreas Lipometabolism of Large-Sized Common Carp (Cyprinus carpio).

An 82-day study was conducted to assess the effect of the dietary lipid levels on growth performance, feed utilization, lipid deposition, and hepatopancreas lipometabolism of large-sized common carp (Cyprinus carpio). Six isonitrogenous (300 g/kg protein) pelletized diets with different dietary lipid levels (30, 60, 90, 120, 150, and 180 g/kg) were fed in triplicate to fish groups with 75 individuals (with an initial mean weight of 247.00 ± 16.67 g). The results showed that there was a significant increase in weight gain (WG) rate (WGR), specific growth rate (SGR), and protein efficiency ratio (PER) as dietary lipid levels increased from 30 to 60 g/kg (p < 0.05) and then there was a decline. Feed conversion rate (FCR) was observed to be significantly lower in 60 g/kg lipid treatments (p < 0.05). Muscle crude protein contents were obtained to be significantly higher in 60 and 90 g/kg treatments (p < 0.05). The crude lipid content in the hepatopancreas increased significantly with an increase in dietary lipid levels (p < 0.05). The expression of lipoprotein lipase (LPL) and carnitine palmitoyltransferase-1 (CPT1) in the hepatopancreas was significantly downregulated with an increase in dietary lipid levels while the expression of growth hormone (GH), insulin-like growth factor-1 (IGF-1), fatty acid synthase (FAS), acetyl-CoA carboxylase-1 (ACC-1), and sterol regulatory element binding protein (SREBP) was upregulated first in 30 and 60 g/kg lipid treatments and then downregulated significantly in other treatments. The results revealed that excess dietary lipid supplements (more than 60 g/kg) would inhibit WG and would aggravate the lipid decomposition in the hepatopancreas. Based on WGR and FCR, the dietary lipid levels of 59.5 and 70.4 g/kg were optimal for growth performance and feed utilization of large-sized common carp.

Genome-wide identification of interleukin-17 (IL17) in common carp (Cyprinus carpio) and its expression following Aeromonas hydrophila infection.

Interleukin-17 (IL17) family cytokines are well known for having pro-inflammatory actions as important mediators of mucosal immune responses and are tightly regulated by various kinds of signals. However, most studies of IL17 genes have focused on mammals, and much less is known about IL17 genes in fish species. To better understand the scope and actions of the IL17 gene family in common carp, we characterized seven IL17 gene homologs from genomic and transcriptomic databases that could be classified into three subclasses according to different comparative genomic analyses. Phylogenetic analysis revealed that most IL17s are highly conserved, though recent gene duplication and gene loss events do exist. Through observation, we found that IL17D has undergone gene duplication in common carp and that all the IL17E genes were lost in vertebrates except mammals. The expression patterns of IL17 genes in common carp were examined during early developmental stages and in various healthy tissues, and the results indicated that most IL17 genes are ubiquitously expressed during early development and show particular tissue-specific expression in various healthy tissues, with relatively high levels in the spleen, liver, and kidney. To gain insights into the mucosal actions of inflammatory processes, the expression profiles of IL17 genes in gills from common carp were investigated after experimental challenge with Aeromonas hydrophila. After A. hydrophila infection, most IL17 genes were upregulated at 4 h postinfection in the gill and then gradually declined, while IL17A/F2 and IL17N were generally upregulated at 12 h postinfection, and IL17D2 maintained an increasing tendency. In contrast, IL17D showed the third phenomenon, rising expression, suggesting that immunogenes have different response strategies to bacterial invasion. Overall, the expression of IL17 in unstimulated tissues and toxicity attack test results demonstrated that these genes play critical roles under normal conditions and during bacterial infection. Moreover, this common carp IL17 gene family research provides a genomic resource for future studies on IL17 gene evolution, fish disease management and immune regulation.

Quantitative trait loci for morphometric traits in multiple families of common carp (Cyprinus carpio).

Quantitative trait locus (QTL) mapping is frequently used to understand the genetic architecture of quantitative traits. Herein, we performed a genome scan for QTL affecting the morphometric characters in eight full-sib families containing 522 individuals using different statistical methods (Sib-pair and half-sib model). A total of 194 QTLs were detected in 25 different regions on 10 linkage groups (LGs). Among them, 37 QTLs on five LGs (eight, 13, 24, 40 and 45) were significant (5% genome-wide level), while the remaining 40 (1% chromosome-wide level) and 117 (5% chromosome-wide level) indicated suggestive effect on those traits. Heritabilities for most morphometric traits were moderate to high, ranging from 0.21 to 0.66, with generally strong phenotypic and genetic correlations between the traits. A large number of QTLs for morphometric traits were co-located, consistent with their high correlations, and may reflect pleiotropic effect on the same genes. Biological pathways were mapped for possible candidate genes on QTL regions. One significantly enriched pathway was identified on LG45, which had a P-value of 0.04 and corresponded to the "regulation of actin cytoskeleton pathway". The results are expected to be useful in marker-assisted selection (MAS) and provide valuable information for the study of gene pathway for morphometric and growth traits of the common carp.