TLR21 is involved in the NF-κB and IFN-ß pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor.

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-ß (IFN-ß) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-ß (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Molecular characterization, expression and functional analysis of TAK1, TAB1 and TAB2 in Nile tilapia (Oreochromis niloticus).

The MAPK pathway is the common intersection of signal transduction pathways such as inflammation, differentiation and proliferation and plays an important role in the process of antiviral immunity. Streptococcus agalactiae will have a great impact on tilapia aquaculture, so it is necessary to study the immune response mechanism of tilapia to S. agalactiae. In this study, we isolated the cDNA sequences of TAK1, TAB1 and TAB2 from Nile tilapia (Oreochromis niloticus). The TAK1 gene was 3492 bp in length, contained an open reading frame (ORF) of 1809 bp and encoded a polypeptide of 602 amino acids. The cDNA sequence of the TAB1 gene was 4001 bp, and its ORF was 1491 bp, which encoded 497 amino acids. The cDNA sequence of the TAB2 gene was 4792 bp, and its ORF was 2217 bp, encoding 738 amino acids. TAK1 has an S_TKc domain and a coiled coil structure; the TAB1 protein structure contains a PP2C_SIG domain and a conserved PYVDXA/TXF sequence model; and TAB2 contains a CUE domain, a coiled coil domain and a Znf_RBZ domain. Homology analysis showed that TAK1 and TAB1 had the highest homology with Neolamprologus brichardi, and TAB2 had the highest homology with Simochromis diagramma (98.28 %). In the phylogenetic tree, TAK1, TAB1 and TAB2 formed a large branch with other scleractinian fishes. The tissue expression analysis showed that the expression of TAK1, TAB1 and TAB2 was highest in the muscle. The expression of TAK1, TAB1 and TAB2 was significantly induced in most of the tested tissues after stimulation with LPS, Poly I:C and S. agalactiae. The subcellular localization results showed that TAK1 was located in the cytoplasm, and TAB1 and TAB2 had certain distributions in the cytoplasm and nucleus. Coimmunoprecipitation (Co-IP) results showed that TRAF6 did not interact with the TAK1 protein but interacted with TAB2, while TAB1 did not interact with P38γ but interacted with TAK1. There was also an interaction between TAK1 and TAB2.

Nile tilapia TBK1 interacts with STING and TRAF3 and is involved in the IFN-ß pathway in the immune response.

Nile tilapia (Oreochromis niloticus) occupies an important position in the culture of economic fish in China. However, the high mortality caused by streptococcal disease has had a significant impact on the tilapia farming industry. Therefore, it is necessary to clarify the immune mechanism of tilapia in response to Streptococcus agalactiae. As a hub in the natural immune signaling pathway, the junction molecule can help the organism defend against and clear pathogens and is crucial in the signaling pathway. In this study, the cDNA sequence of Nile tilapia TBK1 was cloned, and the expression profile was examined in normal fish and challenged fish. The cDNA sequence of the TBK1 gene was 3378 bp, and its open reading frame (ORF) was 2172 bp, encoding 723 amino acids. The deduced TBK1 protein contained an S_TKc domain, a coiled coil domain and a ubiquitin-like domain (ULD). TBK1 had the highest homology with zebra mbuna (Maylandia zebra) and Lake Malawi cichlid fish (Astatotilapia calliptera), both at 97.59%. In the phylogenetic tree, TBK1 forms a large branch with other scleractinian fish. TBK1 expression was highest in the brain and lowest in the liver. LPS, Poly I:C, and S. agalactiae challenge resulted in significant changes in TBK1 expression in the tissues examined. The subcellular localization showed that TBK1-GFP was distributed in the cytoplasm and could significantly increase IFN-ß activation. Pull-down results showed that there was an interaction between TBK1 and TRAF3 and an interaction between STING protein and TBK1 protein. The above results provide a basis for further investigation into the mechanism of TBK1 involvement in the signaling pathway.

Asymmetric expression of homoeologous genes contributes to dietary adaption of an allodiploid hybrid fish derived from Megalobrama amblycephala (♀) × Culter alburnus (♂).

BACKGROUND: Hybridization, which can quickly merge two or more divergent genomes and form new allopolyploids, is an important technique in fish genetic breeding. However, the merged subgenomes must adjust and coexist with one another in a single nucleus, which may cause subgenome interaction and dominance at the gene expression level and has been observed in some allopolyploid plants. In our previous studies, newly formed allodiploid hybrid fish derived from herbivorous Megalobrama amblycephala (♀) × carnivorous Culter alburnus (♂) had herbivorous characteristic. It is thus interesting to further characterize whether the subgenome interaction and dominance derive dietary adaptation of this hybrid fish. RESULTS: Differential expression, homoeolog expression silencing and bias were investigated in the hybrid fish after 70 days of adaptation to carnivorous and herbivorous diets. A total of 2.65 × 108 clean reads (74.06 Gb) from the liver and intestinal transcriptomes were mapped to the two parent genomes based on specific SNPs. A total of 2538 and 4385 differentially expressed homoeologous genes (DEHs) were identified in the liver and intestinal tissues between the two groups of fish, respectively, and these DEHs were highly enriched in fat digestion and carbon metabolism, amino acid metabolism and steroid biosynthesis. Furthermore, subgenome dominance were observed in tissues, with paternal subgenome was more dominant than maternal subgenome. Moreover, subgenome expression dominance controlled functional pathways in metabolism, disease, cellular processes, environment and genetic information processing during the two dietary adaptation processes. In addition, few but sturdy villi in the intestine, significant fat accumulation and a higher concentration of malondialdehyde in the liver were observed in fish fed carnivorous diet compared with fish fed herbivorous diet. CONCLUSIONS: Our results indicated that diet drives phenotypic and genetic variation, and the asymmetric expression of homoeologous genes (including differential expression, expression silencing and bias) may play key roles in dietary adaptation of hybrid fish. Subgenome expression dominance may contribute to uncovering the mechanistic basis of heterosis and also provide perspectives for fish genetic breeding and application.

Construction of a high-density linkage map and mapping of sex determination and growth-related loci in the mandarin fish (Siniperca chuatsi).

BACKGROUND: The mandarin fish (Siniperca chuatsi) is an important and widely cultured fish in China. However, the lack of selective breeding of mandarin fish in previous decades has resulted in a decline in the growth rate of pond-cultured fish, a shortened period of sexual maturity, and reduced disease resistance; these issues seriously affect the quality and safety of the fish products. Therefore, it is necessary to establish a selective breeding program for the mandarin fish to improve the economical traits of the fish and to sustain the development of the mandarin fish industry. RESULTS: We constructed a high-density linkage map for it based on double digest restriction site associated DNA sequencing (ddRAD-Sequencing). This map contained 3283 dimorphic single nucleotide polymorphism markers and 24 linkage groups (LGs). The total map-length was 1972.01 cM, with an average interlocus distance of 0.61 cM. One significant quantitative trait locus (QTL) for sex determination trait was detected on LG23, which was supported by five markers, clustered between 60.27 and 68.71 cM. The highest logarithm of odds value (17.73) was located at 60.27 cM, near the marker r1_73194, accounting for 53.3% of the phenotypic variance. Genotypes of all the male fish on r1_33008 were homozygous, whereas those of all females were heterozygous. Thus, LG23 was considered a sex-related linkage group. Eleven significant QTLs, for three growth traits, at two growth stages and the increased values were distributed on four LGs; their contributions to the phenotypic variation were quite low (12.4-17.2%), suggesting that multiple genes affected the growth traits. CONCLUSION: This high-resolution genetic map provides a valuable resource for fine-mapping of important traits and for identification of sex-related markers that should facilitate breeding of all-female mandarin fish for aquaculture and mechanistic studies on sex determination.

Grass carp reovirus-GD108 fiber protein is involved in cell attachment.

Viral attachment to specific host receptors is the first step in viral infection and serves an essential function in the selection of target cells. In this study, structure analysis, neutralization assays, and cell attachment assays were carried out to evaluate the cell attachment functions of the outer capsid fiber protein of grass carp reovirus GD108 strain (GCRV-GD108). The GCRV-GD108 fiber protein contained 512 amino acids encoded by S7 segment and shared sequence similarities with mammalian reovirus cell attachment protein σ1 and adenovirus fiber. Structural analyses predicted the presence of a coiled-coil tail domain, three adenoviral shafts in the body domain, and a globular head domain, similar to other fiber proteins. Neutralization assays showed that polyclonal antibodies against the fiber protein could prevent viral infection in both fish and grass carp snout fibroblast cells (PSF), suggesting that the recombinant fiber protein could induce neutralized antibodies against GCRV-GD108. Cell attachment assays showed that recombinant fiber protein could bind to PSF cells, demonstrating that the fiber protein functioned as the cell attachment protein in GCRV-GD108. These results provided the basis for further studies of the pathogenesis of grass carp reovirus.

Two myostatin genes exhibit divergent and conserved functions in grass carp (Ctenopharyngodon idellus).

Myostatin (MSTN) is an important negative regulator of myogenesis, which inhibits myoblast proliferation and differentiation. Here, we report the isolation and characterization of two mstn genes in grass carp (Ctenopharyngodon idellus). Grass carp mstn-1 and mstn-2 cDNAs are highly divergent, sharing a relatively low amino acid sequence identity of 66%. In adult fish, both orthologs are expressed in numerous tissues and they are differentially regulated during a fasting/refeeding treatments. During embryogenesis, the mRNA levels of both mstn-1 and -2 were upregulated significantly at the beginning of somitogenesis, and maintained at high levels until hatching. Using in situ hybridization, grass carp mstn-1 mRNA was found to ubiquitously express at 12hpf, with strong signals in the notochord, and in the eyes, brain and tailbud at 24hpf, and in brain and notochord at 36hpf. In comparison, the mstn-2 mRNA can be detected in the eyes, brain and notochord at 24hpf, and in the notochord and hindbrain at 36hpf. Further overexpression of mstn-1 mRNA caused a strongly ventralized phenotype by inhibiting dorsal tissue development, while injection of mstn-2 mRNA resulted in obvious embryonic abnormalities in grass carp. These results provide some new insights into the functional conservation and divergence of mstn genes in teleost species.

Characterization of four heat-shock protein genes from Nile tilapia (Oreochromis niloticus) and demonstration of the inducible transcriptional activity of Hsp70 promoter.

Heat-shock proteins (Hsps), known as stress proteins and extrinsic chaperones, play important roles in the folding, translocation, and refolding/degradation of proteins. In this study, we identified four Hsps in Nile tilapia (Oreochromis niloticus), which display conserved Hsp characteristics in their predicted amino acid sequences. Further analyses on the structures, homology, and phylogenetics revealed that the four Hsps belong to Hsp70 family. One of them does not contain introns and is named Hsp70, while all the other three contain introns and are named Hsc70-1, Hsc70-2, and Hsc70-3. Expressions of the four Hsp proteins were observed in all examined tissues. Six hours after infection of Streptococcus agalactiae in Nile tilapia, the expression of Hsp70 was significantly increased in the liver, head kidney, spleen and gill, while Hsc70s' expression was unchanged in all examined tissues except the head kidney that showed significantly reduced expression of both Hsc70-2 and Hsc70-3. These results suggest that Hsp70 may participate in the defense against S. agalactiae infection. We then isolated the promoter of Hsp70 gene and inserted it into the donor plasmid of Tgf2 transposon system containing green fluorescent protein (GFP) gene. The plasmid was microinjected into zebrafish embryos, where the expression of GFP was induced by heat shock, S. agalactiae immersion challenge, indicating that the isolated Hsp70 promoter has transcriptional activity and is inducible by both heat shock and bacterial challenge. This promoter may facilitate the future construction of disease-resistant transgenic fish. The work also contributes to the further study of immune response of tilapia after bacterial infection.

Genome-Wide Identification, Sequence Alignment, and Transcription of Five Sex-Related Genes in Largemouth Bass (Micropterus Salmoides).

BACKGROUND: Largemouth bass (Micropterus Salmoides) is an economically important fish species in China. Most research has focused on its growth, disease resistance, and nutrition improvement. However, the sex-determining genes in largemouth bass are still unclear. The transforming growth factor-beta (TGF-ß) gene family, including amh, amhr2 and gsdf, plays an important role in the sex determination and differentiation of various fishes. These genes are potentially involved in sex determination in largemouth bass. METHODS: We performed a systematic analysis of 5 sex-related genes (amh, amhr2, gsdf, cyp19a1, foxl2) in largemouth bass using sequence alignment, collinearity analysis, transcriptome, and quantitative real-time polymerase chain reaction (qRT-PCR). This included a detailed assessment of their sequences, gene structures, evolutionary traits, and gene transcription patterns in various tissues including gonads, and at different developmental stages. RESULTS: Comparative genomics revealed that the 5 sex-related genes were highly conserved in various fish genomes. These genes did not replicate, mutate or lose in largemouth bass. However, some were duplicated (amh, amhr2 and gsdf), mutated (gsdf) or lost (amhr2) in other fishes. Some genes (e.g., gsdf) showed significant differences in genomic sequence between males and females, which may contribute to sex determination and sex differentiation in these fishes. qRT-PCR was applied to quantify transcription profiling of the 5 genes during gonadal development and in the adult largemouth bass. Interestingly, amh, amhr2 and gsdf were predominantly expressed in the testis, while cyp19a1 and foxl2 were mainly transcribed in the ovary. All 5 sex-related genes were differentially expressed in the testes and ovaries from the 56th day post-fertilization (dpf). We therefore speculate that male/female differentiation in the largemouth bass may begin at this critical time-point. Examination of the transcriptome data also allowed us to screen out several more sex-related candidate genes. CONCLUSIONS: Our results provide a valuable genetic resource for investigating the physiological functions of these 5 sex-related genes in sex determination and gonadal differentiation, as well as in the control of gonad stability in adult largemouth bass.

Goldfish transposase Tgf2 presumably from recent horizontal transfer is active.

Hobo/Activator/Tam3 (hAT) superfamily transposons occur in plants and animals and play a role in genomic evolution. Certain hAT transposons are active and have been developed as incisive genetic tools. Active vertebrate elements are rarely discovered; however, Tgf2 transposon was recently discovered in goldfish (Carassius auratus). Here, we found that the endogenous Tgf2 element can transpose in goldfish genome. Seven different goldfish mRNA transcripts, encoding three lengths of Tgf2 transposase, were identified. Tgf2 transposase mRNA was detected in goldfish embryos, mainly in epithelial cells; levels were high in ovaries and mature eggs and in all adult tissues tested. Endogenous Tgf2 transposase mRNA is active in mature eggs and can mediate high rates of transposition (>30%) when injected with donor plasmids harboring a Tgf2 cis-element. When donor plasmid was coinjected with capped Tgf2 transposase mRNA, the insertion rate reached >90% at 1 yr. Nonautonomous copies of the Tgf2 transposon with large-fragment deletions and low levels of point mutations were also detected in common goldfish. Phylogenetic analysis indicates the taxonomic distribution of Tgf2 in goldfish is not due to vertical inheritance. We propose that the goldfish Tgf2 transposon originated by recent horizontal transfer and maintains a highly native activity.

Transcriptome profiling and digital gene expression analysis of Nile tilapia (Oreochromis niloticus) infected by Streptococcus agalactiae.

Tilapia is an important freshwater aquaculture species worldwide. In recent years, streptococcal diseases have severely threatened development of tilapia aquaculture, while effective prevention and control methods have not yet been established. In order to understand the immunological response of tilapia to infection by Streptococcus agalactiae (S. agalactiae), this study employed Solexa/Illumina RNA-seq and digital gene expression (DGE) technology to investigate changes in the tilapia transcriptome before and after S. agalactiae infection. We obtained 82,799 unigenes (mean size: 618 bp) using de novo assembly. Unigenes were annotated by comparing against databases including Nr, Swissprot, cluster of orthologous groups of proteins, Kyoto encyclopedia of genes and genomes, and gene ontology. Combined with DGE technology, transcriptomic changes in tilapia before and after bacteria challenging were examined. A total of 774 significantly up-regulated and 625 significantly down-regulated unigenes were identified, among which 293 were mapped to 181 signaling pathways including 17 immune-related pathways involving 65 differentially expressed genes. We observed a change in the expression of six genes in the Toll-like receptor signaling pathway, and this was subsequently confirmed via quantitative real-time PCR. This comparative study of the tilapia transcriptome before and after S. agalactiae infection identified important differentially-expressed immune-related genes and signaling pathways that will provide useful insights for further analysis of the mechanisms of tilapia defense against S. agalactiae infection.

Identification of a second follistatin gene in grass carp (Ctenopharyngodon idellus) and its regulatory function in myogenesis during embryogenesis.

Follistatin can antagonize the function of myostatin as a competitive binding protein and promote muscle growth in vivo. Here, we report the isolation and characterization of a second follistatin gene fst2 in grass carp (Ctenopharyngodon idellus). The grass carp fst2 cDNA was 1,376 bp in length, with an open reading frame (ORF) encoding 350 amino acid residues. A relatively low sequence identity of 78% was found between grass carp Fst2 and its paralog Fst1. Sequence and phylogenetic analyses suggest that the grass carp fst2 originated from fish-specific gene duplication. In adult fish, fst2 mRNA expression was observed in most tissues but was strongly expressed in the eyes, muscles, skin and ovary. Grass carp fst2 mRNA could be detected as early as 16 h post-fertilization (hpf), while fst1 mRNA was detected throughout embryogenesis. Using in situ hybridization, fst2 transcripts were detected in the anterior somites at 24 hpf and in the brain and posterior somites at 36 hpf. Meanwhile, fst1 mRNA was transcribed mainly in the optic vesicle and at the cephalic mesoderm at 12 hpf, in the eyes, cephalic mesoderm and at the lateral edge of most somites at 24 hpf, and mainly in the brain at 36 hpf. Furthermore, overexpression of fst2 mRNA markedly affected the formation of the embryonic midline and somite structures. Based on comparisons with fst1, our findings suggest that fst2 retained the ancestral functions of regulating muscle development and growth during embryogenesis in grass carp.

Genomic organization and gene evolution of two warm temperature acclimation proteins (Wap65s) of Micropterus salmoides and their responses to temperature and bacterial/viral infections.

Warm temperature acclimation-related 65-kDa proteins (Wap65s) are fish plasma acute-phase glycoproteins homologous to hemopexin with high affinity and clearance for heme. The study characterized Mswap65-1 and Mswap65-2 genes in Micropterus salmoides. Structural analysis showed MsWap65s contained conserved heme-binding sites. MsWap65-1 had a chloride-binding site similar to hemopexin, while MsWap65-2 had an additional calcium-binding site. Phylogenetic and Ka/Ks analysis showed that fish Wap65s were evolutionarily conserved and underwent strong purifying selection. Functional divergence analysis indicated that fish Wap65-2 retained the putative function of ancestral Wap65, while Wap65-1 underwent neofunctional differentiation. QPCR showed Mswap65s were predominantly expressed in liver, but prolonged hyperthermy inhibited Mswap65-2 expression. Mswap65-2 expression was up-regulated in liver and spleen after Nocardia seriolae infection, while Mswap65-1 was down-regulated. MsWap65-2 may be associated with pathogenesis and play potential role in pathogen resistance. LMBV infection resulted in both significant downregulation of Mswap65s were both significantly down-regulated, with differences observed between sexes. We speculated the immune system might suppress expression after viral infection. Exogenous rMsWap65s were prepared, and injection of rMsWap65s alleviated phenylhydrazine-induced hemolysis and inhibited increases in heme, complement C3 and inflammatory symptoms. Our results contribute to an advanced understanding of the functions and mechanisms of MsWap65s in stress resistance.

Development of a Highly Permissive Mandarin Fish (Siniperca chuatsi) Kidney Cell Line for Mandarin Fish Ranavirus Using a Single-Cell Cloning Method.

Mandarin fish ranavirus (MRV) infection poses a substantial challenge to the mandarin fish culture industry as no effective preventive or therapeutic measures currently exist. The creation of a highly permissive cell line from a natural host is crucial for developing a vaccine for MRV and understanding its pathogenic mechanisms. In this research, the mandarin fish (Siniperca chuatsi) kidney cell line (SCK) was isolated from mandarin fish kidneys. Subsequently, SCK-a to SCK-g monoclonal cell lines were derived from the SCK cell population, distinguished by morphological variations. Notably, MRV infection induced an advanced cytopathic effect (CPE) in almost all cells of the SCK-f clone. Further tests showed that MRV achieved a peak viral titer of 1010.7 50% tissue culture infectious dose (TCID50)/mL and consistently exceeded 1010 TCID50/mL across nine passages in SCK-f cells. Electron microscopy verified the MRV virion integrity within SCK-f. In vivo experiments revealed that MRV infections led to cumulative mortality rates of 86.9% in mandarin fish and 88.9% in largemouth bass (Micropterus salmoides). Such results suggest that SCK-f is highly permissive to MRV. This study underscores the importance of cellular diversity in developing viral permissive cell lines. The SCK monoclonal cell line pool may offer potential for generating highly permissive cell lines for other mandarin fish viruses.

Whole-genome resequencing reveals recent signatures of selection in five populations of largemouth bass ( Micropterus salmoides).

Largemouth bass ( Micropterus salmoides) is an economically important fish species in North America, Europe, and China. Various genetic improvement programs and domestication processes have modified its genome sequence through selective pressure, leaving nucleotide signals that can be detected at the genomic level. In this study, we sequenced 149 largemouth bass fish, including protospecies (imported from the US) and improved breeds (four domestic breeding populations from China). We detected genomic regions harboring certain genes associated with improved traits, which may be useful molecular markers for practical domestication, breeding, and selection. Subsequent analyses of genetic diversity and population structure revealed that the improved breeds have undergone more rigorous genetic changes. Through selective signal analysis, we identified hundreds of putative selective sweep regions in each largemouth bass line. Interestingly, we predicted 103 putative candidate genes potentially subjected to selection, including several associated with growth (p sst1 and grb10), early development ( klf9, sp4, and sp8), and immune traits ( pkn2, sept2, bcl6, and ripk2). These candidate genes represent potential genomic landmarks that could be used to improve important traits of biological and commercial interest. In summary, this study provides a genome-wide map of genetic variations and selection footprints in largemouth bass, which may benefit genetic studies and accelerate genetic improvement of this economically important fish.

Comparative Analysis of the Gut Microbiota of Mandarin Fish (Siniperca chuatsi) Feeding on Compound Diets and Live Baits.

Siniperca chuatsi feeds on live fry throughout their life. The sustainable development of its farming industry has urgently necessitated the development of artificial diets to substitute live baits. It has been demonstrated that gut microbiota assists in feed adaptation and improves the feed conversion rate in fish. Therefore, this study aimed to understand the potential role of intestinal microorganisms in the domestication of S. chuatsi with a compound diet. Accordingly, we performed 16S rRNA sequencing of the gut microbial communities in S. chuatsi groups that were fed a compound diet (including large and small individuals) and live baits. A total of 2,471 OTUs were identified, and the large individual group possessed the highest number of unique OTUs. The α-diversity index of the gut microbiota in groups that were fed a compound diet was significantly higher (p < 0.05) than that in the live bait group. There were no significant differences in the α-diversity between the large and small individual groups. However, relatively higher numbers of Lactococcus, Klebsiella, and Woeseia were observed in the intestines of the large individual group. Prediction of the metabolic function of the microbiota among these three fish groups by Tax4Fun revealed that most metabolic pathways, such as glycan metabolism and amino acid metabolism, were typically more enriched for the larger individuals. The results indicated that certain taxa mentioned above exist in large individuals and may be closely related to the digestion and absorption of compound diets. The present study provides a basis for understanding the utilization mechanism of artificial feed by S. chuatsi.

Exposure to heat stress causes downregulation of immune response genes and weakens the disease resistance of Micropterus salmoides.

In order to understand the molecular mechanism of response to heat stress in largemouth bass (LMB) Micropterus salmoides, we performed transcriptome analysis of spleen tissue of LMB subjected to heat stress and challenged with A. veronii under heat stress. A total of 2162 DEGs were identified between the heat stressed (32 °C) and control groups (24 °C) after 7 d treatment. Gene Ontology (GO) annotation analysis revealed that these differentially expressed genes (DEGs) were mainly enriched on GO terms of biological regulation, membrane part, and binding. ELISA validation indicated that except major histocompatibility complex II (Mhc II), the protein levels of t-Sod, caspase 3 (Casp3), tumor necrosis factor-α (Tnf-α), and complement component 3 (C3) were consistent with RNA-seq results. In the experiment of A. veronii challenged under heat stress (32 °C), 2899 and 2663 DEGs were obtained from the heat stress-challenged group (H6 vs H0, H12 vs H0), while 1485 and 3501 DEGs from the control-challenged group (C6 vs C0, C12 vs C0). GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that immune-related categories and pathways were significantly enriched, such as immune system process, immune response and positive regulation of immune response in GO enrichment analysis, and cytokine-cytokine receptor interaction, human cytomegalovirus infection in KEGG signaling pathways. The expressions of f11, c1q and c3 in complement and coagulation pathway, as well as that of proinflammatory genes tnf-α and il-8, were deeply inhibited. Real-time quantitative PCR validation for nine DEGs showed that most of them had consistent expression trends with RNA-seq results. Our results indicated that heat stress affects the immunity and metabolism of LMB. In particular, it aggravates the inhibitory effects of A. veronii on the complement and coagulation systems while downregulating proinflammatory cytokine expression, thereby weakening the resistance of LMB to pathogen infection. Our results contribute to the elucidation of A. veronii infection pathogenic mechanisms in LMB under heat stress.

Characterization of kiss2/kissr2 system in largemouth bass (Micropterus salmoides) and Kiss2-10 peptide regulation of the hypothalamic-pituitary-gonadal axis.

The kisspeptin system, which lies upstream of the hypothalamic-pituitary-gonadal (HPG) axis, is believed to function as a regulator of reproduction in teleosts. In this study, we isolated and characterized kiss2 and its receptor kissr2 in largemouth bass (Micropterus salmoides). The complete coding sequences of kiss2 and kissr2 were 375 and 1134 bp long and encoded precursor proteins 124 and 377 amino acid long, respectively. Real-time PCR showed that kiss2 and kissr2 were primarily expressed in the HPG axis. The expression profile of kiss2 and kissr2 varied with gonadal development, with the highest and lowest expression levels being detected during the immature and final maturation stages, respectively. Intraperitoneal injection of exogenous Kiss2-10 peptide increased the transcript levels of gnrh3, kissr2, fshß, lhß, ar, and er2 within 24 h (p < 0.05), as well as plasma levels of 17ß-estradiol and testosterone. Histological analysis indicated that chronic administration of exogenous Kiss2-10 peptide accelerated vitellogenesis in females and spermatogenesis in males. Further, in situ hybridization revealed that kiss2 is expressed in the ooplasm and vitelline envelope of oocytes and the spermatocytes of testes. In addition, experiments using gonad tissue primary cell cultures indicated that exogenous Kiss2-10 peptide stimulates the expression of reproduction-related genes. Collectively, our findings indicate that the kiss2/kissr2 system in largemouth bass is involved in regulating gonadal development through the HPG axis.

IGF binding protein 1 is correlated with hypoxia-induced growth reduce and developmental defects in grass carp (Ctenopharyngodon idellus) embryos.

The effects of hypoxia on embryonic development and the underlying cellular and molecular mechanisms are poorly understood in teleost fish, although the hypoxic effects on embryonic growth and development have been reported in the zebrafish model. Here, we found that hypoxia caused significant developmental delay and growth retardation during grass carp embryogenesis. Placing the embryos in hypoxic conditions at different developmental stages strongly induced the mRNA expression of insulin-like growth factor-binding protein 1 (IGFBP1), an inhibitory protein that binds to IGF and inhibits its subsequent actions in vivo. Further gain-of-function analysis results provided strong evidence to support the hypothesis that IGFBP1 plays an important role in mediating hypoxic-induced growth and developmental defects. Overexpression of IGFBP1 mRNA reduced the growth rate to a degree that was similar to hypoxia. Additionally, overexpression of IGFBP1 caused significant developmental defects in the formation of midline, somite and hindbrain structures during grass carp embryogenesis. Taken together, our studies suggest that IGFBP1 plays a key role in mediating these hypoxia-induced embryonic growth retardation and developmental delay in grass carp.

Chromosome-level genome assembly for the largemouth bass Micropterus salmoides provides insights into adaptation to fresh and brackish water.

Largemouth bass (LMB; Micropterus salmoides) has been an economically important fish in North America, Europe, and China. This study obtained a chromosome-level genome assembly of LMB using PacBio and Hi-C sequencing. The final assembled genome is 964 Mb, with contig N50 and scaffold N50 values of 1.23 Mb and 36.48 Mb, respectively. Combining with RNA sequencing data, we annotated a total of 23,701 genes. Chromosomal assembly and syntenic analysis proved that, unlike most Perciformes with the popular haploid chromosome number of 24, LMB has only 23 chromosomes (Chr), among which the Chr1 seems to be resulted from a chromosomal fusion event. LMB is phylogenetically closely related to European seabass and spotted seabass, diverging 64.1 million years ago (mya) from the two seabass species. Eight gene families comprising 294 genes associated with ionic regulation were identified through positive selection, transcriptome and genome comparisons. These genes involved in iron facilitated diffusion (such as claudin, aquaporins, sodium channel protein and so on) and others related to ion active transport (such as sodium/potassium-transporting ATPase and sodium/calcium exchanger). The claudin gene family, which is critical for regulating cell tight junctions and osmotic homeostasis, showed a significant expansion in LMB with 27 family members and 68 copies for salinity adaptation. In summary, we reported the first high-quality LMB genome, and provided insights into the molecular mechanisms of LMB adaptation to fresh and brackish water. The chromosome-level LMB genome will also be a valuable genomic resource for in-depth biological and evolutionary studies, germplasm conservation and genetic breeding of LMB.