Integrated microbiome and metabolome analyses reveal the effects of low pH on intestinal health and homeostasis of crayfish (Procambarus clarkii).

Low pH (LpH) poses a significant challenge to the health, immune response, and growth of aquatic animals worldwide. Crayfish (Procambarus clarkii) is a globally farmed freshwater species with a remarkable adaptability to various environmental stressors. However, the effects of LpH stress on the microbiota and host metabolism in crayfish intestines remain poorly understood. In this study, integrated analyses of antioxidant enzyme activity, histopathological damage, 16S rRNA gene sequencing, and liquid chromatography-mass spectrometry (LC-MS) were performed to investigate the physiology, histopathology, microbiota, and metabolite changes in crayfish intestines exposed to LpH treatment. The results showed that LpH stress induced obvious changes in superoxide dismutase and catalase activities and histopathological alterations in crayfish intestines. Furthermore, 16S rRNA gene sequencing analysis revealed that exposure to LpH caused significant alterations in the diversity and composition of the crayfish intestinal microbiota at the phylum and genus levels. At the genus level, 14 genera including Bacilloplasma, Citrobacter, Shewanella, Vibrio, RsaHf231, Erysipelatoclostridium, Anaerorhabdus, Dysgonomonas, Flavobacterium, Tyzzerella, Brachymonas, Muribaculaceae, Propionivibrio, and Comamonas, exhibited significant differences in their relative abundances. The LC-MS analysis revealed 859 differentially expressed metabolites in crayfish intestines in response to LpH, including 363 and 496 upregulated and downregulated metabolites, respectively. These identified metabolites exhibited significant enrichment in 24 Kyoto Encyclopedia of Genes and Genomes pathways (p < 0.05), including seven and 17 upregulated and downregulated pathways, respectively. These pathways are mainly associated with energy and amino acid metabolism. Correlation analysis revealed a strong correlation between the metabolites and intestinal microbiota of crayfish during LpH treatment. These findings suggest that LpH may induce significant oxidative stress, intestinal tissue damage, disruption of intestinal microbiota homeostasis, and alterations in the metabolism in crayfish. These findings provide valuable insights into how the microbial and metabolic processes of crayfish intestines respond to LpH stress.

A chromosome-level genome assembly of East Asia endemic minnow Zacco platypus.

Zacco platypus is an endemic colorful freshwater minnow that is intensively distributed in East Asia. In this study, two adult female individuals collected from Haihe River basin were used for karyotypic study and genome sequencing, respectively. The karyotype formula of Z. platypus is 2N = 48 = 18 M + 24SM/ST + 6 T. We used PacBio long-read sequencing and Hi-C technology to assemble a chromosome-level genome of Z. platypus. As a result, an 814.87 Mb genome was assembled with the PacBio long reads. Subsequently, 98.64% assembled sequences were anchored into 24 chromosomes based on the Hi-C data. The chromosome-level assembly contained 54 scaffolds with a N50 length of 32.32 Mb. Repeat elements accounted for 52.35% in genome, and 24,779 protein-coding genes were predicted, with 92.11% were functionally annotated with the public databases. BUSCO analysis yielded a completeness score of 96.5%. This high-quality genome assembly provides valuable resources for future functional genomic research, comparative genomics, and evolutionary studies of genus Zacco.

Molecular Cloning, Tissue Distribution, and Pharmacological Characterization of GPR84 in Grass Carp (Ctenopharyngodon Idella).

The G-protein-coupled receptor GPR84, activated by medium-chain fatty acids, primarily expressed in macrophages and microglia, is involved in inflammatory responses and retinal development in mammals and amphibians. However, our understanding of its structure, function, tissue expression, and signaling pathways in fish is limited. In this study, we cloned and characterized the coding sequence of GPR84 (ciGPR84) in grass carp. A phylogenetic analysis revealed its close relationship with bony fishes. High expression levels of GPR84 were observed in the liver and spleen. The transfection of HEK293T cells with ciGPR84 demonstrated its responsiveness to medium-chain fatty acids and diindolylmethane (DIM). Capric acid, undecanoic acid, and lauric acid activated ERK and inhibited cAMP signaling. Lauric acid showed the highest efficiency in activating the ERK pathway, while capric acid was the most effective in inhibiting cAMP signaling. Notably, DIM did not activate GPR84 in grass carp, unlike in mammals. These findings provide valuable insights for mitigating chronic inflammation in grass carp farming and warrant further exploration of the role of medium-chain fatty acids in inflammation regulation in this species.

Histopathological observation and comparative transcriptome analysis reveal immune response mechanisms to Aeromonas dhakensis infection in Macrobrachium rosenbergii.

The Macrobrachium rosenbergii industry is threatened by various Aeromonas, resulting in high mortality of adult prawns. However, there are few studies on the immune response of M. rosenbergii infected with Aeromonas dhakensis. In this study, we observed the hepatopancreas and gills histopathologically, performed a comparative transcriptome analysis of the hepatopancreas, and analyzed the candidate gene expression of immune-related genes in the hemolymph, hepatopancreas, and gills of M. rosenbergii that had been infected with A. dhakensis. Histopathology revealed the hepatopancreas was successively inflamed, followed by cellular vacuolation, lumen deformation, and finally tissue erosion; partial and severe inflammation of the gills occurred successively, and eventually the gill tissue atrophy and the gill filaments detached from the gill arch. Transcriptome analysis showed that a total of 77,742 unigenes and 8664 differentially expressed genes (DEGs), and the immune-related DEGs were mainly enriched in lysosome and phagosome pathways. In addition, 4 immune-related candidate genes (RhoA, CASP9, PKC, and DSCIGN) based on KEGG and PPI analysis were monitored at 6, 12, and 24h post injection (hpi) in hepatopancreas, hemolymph and gills. Their spatio-temporal expression results indicated that A. dhakensis have activated the immune system of M. rosenbergii. The present study may provide new information on the complex immune mechanism of M. rosenbergii.

Analysis of mRNA and MicroRNA Expression Profiles of Nervous Tissues and Reproductive Tissues in Male Procambarus clarkii After Silencing IAG.

The insulin-like androgenic gland hormone gene (IAG), primarily expressed in the androgenic gland (AG), plays a crucial role in controlling male sex differentiation and maintaining male secondary sexual characteristics in decapods. In this study, we investigated the mRNA and microRNA expression profiles of male Procambarus clarkii to understand the transcriptomic regulatory mechanism of IAG after the injection of an efficient siRNA (GsiRNA) designed based on IAG. The results revealed that several differentially expressed genes were enriched in reproduction-related pathways, such as the wnt signaling pathway, MAPK signaling pathway, and GnRH signaling pathway. In the testis (Te), the injection of GsiRNA led to the up-regulation of many ovary-related genes and down-regulation of testis-related genes. Moreover, the brain (Br) and abdominal nerve cord (AN) appeared to be involved in the regulation of IAG, with numerous differentially expressed genes found in Br and AN. Notably, the expression of five neuropeptide genes, Crustacean hyperglycemic hormone, pigment-dispersing hormone, red pigment concentrating hormone precursor, corazonin, and gonadotropin-releasing hormone II receptor isoform X1 in Br/AN, was significantly changed. Additionally, three ovary-related miRNAs (miR-263a, miR-263b, miR-133) highly expressed in Te/AG showed significant up-regulation after GsiRNA injection. Furthermore, the long-term interference of GsiRNA was found to inhibit the development of male external sexual characteristics during the juvenile stage and delay it during the adult stage. This research provides valuable insights into the molecular regulatory mechanism and function of IAG in P. clarkii.

The Dynamics of Gene Expression Unraveling the Immune Response of Macrobrachium rosenbergii Infected by Aeromonas veronii.

To further investigate the immune response of Macrobrachium rosenbergii against Aeromonas veronii, comparative transcriptomic analyses of the M. rosenbergii hepatopancreas were conducted on challenge and control groups at 6, 12, and 24 h post-infection (hpi), independently. A total of 51,707 high-quality unigenes were collected from the RNA-seq data, and 8060 differentially expressed genes (DEGs) were discovered through paired comparisons. Among the three comparison groups, a KEGG pathway enrichment analysis showed that 173 immune-related DEGs were considerably clustered into 28 immune-related pathways, including the lysosome, the phagosome, etc. Moreover, the expression levels of the four key immune-related genes (TOLL, PAK1, GSK3ß, and IKKα) were evaluated at various stages following post-infection in the hepatopancreas, hemolymph, and gills. Both PAK1 and GSK3ß genes were highly up-regulated in all three tissues at 6 hpi with A. veronii; TOLL was up-regulated in the hepatopancreas and hemolymph but down-regulated in the gill at 6 hpi, and IKKα was up-regulated in hemolymph and gill, but down-regulated in the hepatopancreas at 6 hpi. These findings lay the groundwork for understanding the immune mechanism of M. rosenbergii after contracting A. veronii.

Alterations of the Gut Microbiota and Metabolomics Associated with the Different Growth Performances of Macrobrachium rosenbergii Families.

To investigate the key gut microbiota and metabolites associated with the growth performance of Macrobrachium rosenbergii families, 16S rRNA sequencing and LC-MS metabolomic methods were used. In this study, 90 M. rosenbergii families were bred to evaluate growth performance. After 92 days of culture, high (H), medium (M), and low (L) experimental groups representing three levels of growth performance, respectively, were collected according to the weight gain and specific growth rate of families. The composition of gut microbiota showed that the relative abundance of Firmicutes, Lachnospiraceae, Lactobacillus, and Blautia were much higher in Group H than those in M and L groups. Meanwhile, compared to the M and L groups, Group H had significantly higher levels of spermidine, adenosine, and creatinine, and lower levels of L-citrulline. Correlation analysis showed that the abundances of Lactobacillus and Blautia were positively correlated with the levels of alpha-ketoglutaric acid and L-arginine. The abundance of Blautia was also positively correlated with the levels of adenosine, taurine, and spermidine. Notably, lots of metabolites related to the metabolism and biosynthesis of arginine, taurine, hypotaurine, and fatty acid were upregulated in Group H. This study contributes to figuring out the landscape of the gut microbiota and metabolites associated with prawn growth performance and provides a basis for selective breeding.

Integrated Transcriptomic and Metabolomic Analyses Reveal Low-Temperature Tolerance Mechanism in Giant Freshwater Prawn Macrobrachium rosenbergii.

Water temperature, as an important environmental factor, affects the growth and metabolism of aquatic animals and even their survival. The giant freshwater prawn (GFP) Macrobrachium rosenbergii is a kind of warm-water species, and its survival temperature ranges from 18 °C to 34 °C. In this study, we performed transcriptomic and metabolomic analyses to clarify the potential molecular mechanism of responding to low-temperature stress in adult GFP. The treatments with low-temperature stress showed that the lowest lethal temperature of the GFP was 12.3 °C. KEGG enrichment analyses revealed that the differentially expressed genes and metabolites were both enriched in lipid and energy metabolism pathways. Some key genes, such as phosphoenolpyruvate carboxykinase and fatty acid synthase, as well as the content of the metabolites dodecanoic acid and alpha-linolenic acid, were altered under low-temperature stress. Importantly, the levels of unsaturated fatty acids were decreased in LS (low-temperature sensitive group) vs. Con (control group). In LT (low-temperature tolerant group) vs. Con, the genes related to fatty acid synthesis and degradation were upregulated to cope with low-temperature stress. It suggested that the genes and metabolites associated with lipid metabolism and energy metabolism play vital roles in responding to low-temperature stress. This study provided a molecular basis for the selection of a low-temperature tolerant strain.

Identification and Molecular Characterization of a Novel Carlavirus Infecting Chrysanthemum morifolium in China.

Chrysanthemum (Chrysanthemum morifolium) is an important ornamental and medicinal plant suffering from many viruses and viroids worldwide. In this study, a new carlavirus, tentatively named Chinese isolate of Carya illinoinensis carlavirus 1 (CiCV1-CN), was identified from chrysanthemum plants in Zhejiang Province, China. The genome sequence of CiCV1-CN was 8795 nucleotides (nt) in length, with a 68-nt 5'-untranslated region (UTR) and a 76-nt 3'-UTR, which contained six predicted open reading frames (ORFs) that encode six corresponding proteins of various sizes. Phylogenetic analyses based on full-length genome and coat protein sequences revealed that CiCV1-CN is in an evolutionary branch with chrysanthemum virus R (CVR) in the Carlavirus genus. Pairwise sequence identity analysis showed that, except for CiCV1, CiCV1-CN has the highest whole-genome sequence identity of 71.3% to CVR-X6. At the amino acid level, the highest identities of predicted proteins encoded by the ORF1, ORF2, ORF3, ORF4, ORF5, and ORF6 of CiCV1-CN were 77.1% in the CVR-X21 ORF1, 80.3% in the CVR-X13 ORF2, 74.8% in the CVR-X21 ORF3, 60.9% in the CVR-BJ ORF4, 90.2% in the CVR-X6 and CVR-TX ORF5s, and 79.4% in the CVR-X21 ORF6. Furthermore, we also found a transient expression of the cysteine-rich protein (CRP) encoded by the ORF6 of CiCV1-CN in Nicotiana benthamiana plants using a potato virus X-based vector, which can result in a downward leaf curl and hypersensitive cell death over the time course. These results demonstrated that CiCV1-CN is a pathogenic virus and C. morifolium is a natural host of CiCV1.

Integrated comparative transcriptome and weighted gene co-expression network analysis provide valuable insights into the response mechanisms of crayfish (Procambarus clarkii) to copper stress.

One of the significant limitations of aquaculture worldwide is the prevalence of divalent copper (Cu). Crayfish (Procambarus clarkii) are economically important freshwater species adapted to a variety of environmental stimuli, including heavy metal stresses; however, large-scale transcriptomic data of the hepatopancreas of crayfish in response to Cu stress are still scarce. Here, integrated comparative transcriptome and weighted gene co-expression network analyses were initially applied to investigate gene expression profiles of the hepatopancreas of crayfish subjected to Cu stress for different periods. As a result, 4662 significant differentially expressed genes (DEGs) were identified following Cu stress. Bioinformatics analyses revealed that the "focal adhesion" pathway was one of the most significantly upregulated response pathways following Cu stress, and seven DEGs mapped to this pathway were identified as hub genes. Furthermore, the seven hub genes were examined by quantitative PCR, and each was found to have a substantial increase in transcript abundance, suggesting a critical role of the "focal adhesion" pathway in the response of crayfish to Cu stress. Our transcriptomic data can be a good resource for the functional transcriptomics of crayfish, and these results may provide valuable insights into the molecular response mechanisms underlying crayfish to Cu stress.

DNA methylation mediates gonadal development via regulating the expression levels of cyp19a1a in loach Misgurnus anguillicaudatus.

DNA methylation-mediated transcriptional regulation has been considered to significantly impact some steroidogenic enzyme genes expression. To uncover the roles of DNA methylation on the regulation of aromatase gene expression during gametogenesis in Misgurnus anguillicaudatus, the expression profiles and cellular localization of cyp19a1a and cyp19a1b were analyzed, and the landscape of DNA methylation dynamics was investigated. We found that cyp19a1a was predominantly expressed in granulosa cells of oocytes, while cyp19a1b expression was enriched in radial glial cells of the forebrain. In ovary, cyp19a1a was highly expressed until the vitellogenesis stage. The average methylation levels, especially for two CpG sites within the cAMP response element, were negatively correlated with cyp19a1a expression levels, indicating that methylation could regulate cyp19a1a transcriptional activity by modulating the binding efficiency of cAMP to its response elements. Compared with in ovary, cyp19a1a showed lower expression in testis but was hypermethylated. Cyp19a1b in female brain weakly expressed before the vitellogenesis stage, but significantly elevated at the maturation stage. In both sexes, it maintained high methylation levels in brain despite the obvious fluctuation of the cyp19a1b expression. This study revealed that DNA methylation plays a key role in establishing cyp19a1a spatiotemporal expression patterns and thus mediates gonadal development in teleosts.

Comparative transcriptome analysis of brain and gonad reveals reproduction-related miRNAs in the giant prawn, Macrobrachium rosenbergii.

Macrobrachium rosenbergii (M. rosenbergii), as a species of common prawn, is a delicacy that is consumed all over the world. By interacting with the target gene 3'-untranslated region (3'-UTR), microRNAs (miRNAs) regulate its expression and ultimately participate in the regulation of reproductive development. However, research focusing on miRNA regulation during gonadal development in M. rosenbergii received very little attention. To explore the association between miRNA and reproduction, we performed RNA sequencing (RNA-seq) on brain and gonad organs in male and female M. rosenbergii. A total of 494 miRNAs were obtained in RNA-seq, including 31 and 59 differentially expressed (DE) miRNAs in the brain and gonads, respectively. Furthermore, 9 DE miRNAs were randomly selected from the brain and gonads, and qRT-PCR was conducted to validate the results of RNA-seq. Interestingly, dpu-miR-133 was found to be substantially expressed in the male brain and testis but poorly expressed in the female brain, ovary, and other organs. Analysis of dpu-miR-133 by Targetscan and MiRanda predicted to target 5-HT1. Furthermore, the dual-luciferase reporter assay manifested that dpu-miR-133 can combine with 5-HT1. Overall, our research work provides basic data for further study on the miRNA-mediated regulation of brain, gonad, and reproductive development of study M. rosenbergii.

The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification.

Vibrio anguillarum, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for V. anguillarum infection. Herein, we investigated the effects of flagellins (flaA, flaB, flaC, flaD and flaE) on cell apoptosis, TLR5 expression, and production of IL-8 and TNF-α. FlaB exhibited the strongest immunostimulation effects. To explore the functions of flaB in infection, we constructed a flaB deletion mutant using a two-step recombination method, and in vitro experiments showed a significant decrease in the expression of TLR5 and inflammatory cytokines compared with wild-type cells. However in the in vivo study, expression of inflammatory cytokines and intestinal mucosal structure showed no significant differences between groups. Additionally, flaB induced a significant increase in TLR5 expression based on microscopy analysis of fluorescently labelled TLR5, indicating interactions between the two proteins, which was confirmed by native PAGE and yeast two-hybrid assay. Molecular simulation of interactions between flaB and TLR5 was performed to identify the residues involved in binding, revealing two binding sites. Then, based on molecular dynamics simulations, we carried out thirteen site-directed mutations occurring at the amino acid sites of Q57, N83, N87, R91, D94, E122, D152, N312, R313, N320, L97, H316, I324 in binding regions of flaB protein by TLR5, respectively. Surface plasmon resonance (SPR) was employed to compare the affinities of flaB mutants for TLR5, and D152, D94, I324, N87, R313, N320 and H316 were found to mediate interactions between flaB and TLR5. Our comprehensive and systematic analysis of V. anguillarum flagellins establishes the groundwork for future design of flagellin-based vaccines.

Genetic Diversity and Population Differentiation of Kashgarian Loach (Triplophysa yarkandensis) in Xinjiang Tarim River Basin.

The distribution of Triplophysa yarkandensis is restricted to Xinjiang's Tarim River basin. We collected 119 T. yarkandensis samples from nine geographic populations in the Tarim River basin and utilized the RAD-seq method for SNP genotyping. In this study, a total of 164.81 Gb bases were generated with the Illumina platform, and 129,873 candidate SNPs were obtained with the Stacks pipeline for population genetic analyses. High levels of genetic diversity were detected among nine populations. The AMOVA results showed that the majority of genetic variations originated from among populations (FST = 0.67), and the pairwise FST values ranged from 0.4579 to 0.8736, indicating high levels of genetic differentiation among these populations. The discriminate analysis of principal components (DAPCs) and neighbor joining (NJ) tree revealed that the nine populations could be separated into two clusters (i.e., south and north populations), and modest genetic differentiation between south and north populations was observed, while the individuals from several populations were not clustered together by geographical location. The evidence of two genetic boundaries between south and north populations (except TTM) was supported by barrier analysis. The Bayesian skyline plotting indicated that T. yarkandensis populations in the Tarim River basin had not experienced genetic bottlenecks, and the effective population size remained stable. This study first clarified the genetic diversity and differentiation of T. yarkandensis populations in the Tarim River basin, and it provided valuable molecular data for conservation and management of natural populations.

Molecular identification and functional analysis of MyD88 in giant freshwater prawn (Macrobrachium rosenbergii) and expression changes in response to bacterial challenge.

Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein for Toll-like receptor (TLR)-mediated signaling pathways and plays an important role in immune response. In this study, the full-length cDNA of MyD88 from Macrobrachium rosenbergii (MRMyD88) was cloned. The MRMyD88 cDNA is 1758 bp long and contains a 1398-bp open reading frame. Multiple sequence alignment and phylogenetic analysis revealed that the amino acid sequence of MRMyD88 shared high identity with the known MyD88 proteins. The MRMyD88 mRNA was widely expressed in all examined tissues, with highest level in intestine, followed by gonad and pleopod. Furthermore, the MRMyD88 promoter region, spanning 1622 bp, contains several transcription factor-binding sites, including nine GATA-1 box motifs. Electrophoretic mobility shift assay showed that Gfi-1, SRF, and Oct-1 bind to the upstream region of MRMyD88. Additionally, the results showed that the expression levels of TLR1, TLR2 and TLR3 were different in response to Vibrio anguillarum, Lactobacillus plantarum and Aeromonas hydrophila infections. However, these bacteria significantly increased the expression levels of MyD88 and prophenoloxidase. These data suggest that the TLR-mediated signaling pathway is MyD88-dependent in response to pathogenic and probiotic bacteria in M. rosenbergii.

Construction of a Vibrio anguillarum flagellin B mutant and analysis of its immuno-stimulation effects on Macrobrachium rosenbergii.

Vibrio anguillarum is a globally distributed aquatic pathogen, and its flagellin B (FlaB) protein can evoke innate immune responses in hosts. In order to explore the role of FlaB in V. anguillarum infection, we constructed a FlaB-deficient mutant using overlapping PCR and two-step homologous recombination, and gene sequencing confirmed successful knockout of the FlaB gene. Scanning electron microscopy showed no significant differences in the morphological structure of the flagellum between wild-type and FlaB-deficient strains. The mutant was subsequently injected into the freshwater prawn (Macrobrachium rosenbergii) to explore its pathogenicity in the host, and expression of myeloid differentiation factor 88, prophenoloxidase, catalase, superoxide dismutase and glutathione peroxidase was investigated by real-time PCR. The results showed that deletion of FlaB had little effect on V. anguillarum-induced expression of these immune-related genes (p > 0.05). In general, the FlaB mutant displayed similar flagella morphology and immune characteristics to the wild-type strain, hence we speculated that knockout of FlaB might promote the expression and function of other flagellin proteins. Furthermore, this study provides a rapid and simple method for obtaining stable mutants of V. anguillarum free from foreign plasmid DNA.

Screening of Biomarkers Related to Ovarian Maturation and Spawning in Blunt Snout Bream (Megalobrama amblycephala) Based on Metabolomics and Transcriptomics.

In fish breeding practices, gamete maturity of females is vital to reproductive success. For some species, it is possible to estimate the female maturation status based on abdomen observation, but quite difficult for some species which mature at big size. To screen out the potential biomarker in fish blood relating to female maturation, we employed the approach integrating the UPLC-MS/MS and RNA-seq techniques to investigate the metabolites and genes reflecting the sexual maturation and spawning of female blunt snout bream Megalobrama amblycephala. The study included four groups, 1-year-old immature female individuals, 2-year-old immature female individuals, 2-year-old sexually mature female individuals, and 2-year-old sexually mature female individuals after 24 h of successful spawning. The upregulated metabolites in mature females were involved in "steroid hormone biosynthesis," "metabolic pathways," "glycerophospholipid metabolism," etc. compared with those of immature individuals. As the key intermediate of steroid hormone biosynthesis, 17α-hydroxypregnenolone exhibited the highest level in 2-year-old mature females than in the immature females. Meanwhile, the metabolites (i.e., dodecanoic acid and myristic acid) participating in fatty acid synthesis exhibited much lower levels in the females after spawning than those before spawning. In addition to the metabolites, the genes involved in ovarian steroidogenesis were significantly upregulated in the 2-year-old immature females compared to the 1-year-old immature females, indicating that the ovarian steroidogenesis plays important roles in ovarian development of M. amblycephala at the early stages. The significant upregulation of genes (i.e., itpr1, camk2, and mekk2) involved in the "GnRH signaling pathway" was observed in the mature females compared with the immature females, which indicated that the estrogen levels increased after female maturation in M. amblycephala. Moreover, many genes (e.g., gck, creb1, tf2-9, ryr2, asgr1, and creb1) regulating insulin secretion and thyroid hormone synthesis were significantly downregulated after female spawning. The dynamics of gene expression and metabolites observed in this study provide novel cues for guiding fish practical artificial reproduction.