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.

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.

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.

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.

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.

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.

Isolation, identification, and pathogenic characteristics of Nocardia seriolae in largemouth bass Micropterus salmoides.

The largemouth bass Micropterus salmoides is an important freshwater aquaculture fish in China. Recently, largemouth bass at a fish farm in Guangdong province experienced an outbreak of a serious ulcer disease. As part of the investigations conducted to identify the aetiology and identify potentially effective control measures, we isolated a pathogenic bacterium (NK-1 strain) from the diseased fish. It was identified as Nocardia seriolae through morphological observation, physiological and biochemical analysis, and molecular identification, and its pathogenicity was verified by experimental infection. Pathological changes in the diseased fish included granulomatous lesions in the liver and spleen, destruction of renal tubules, necrosis of intestinal epithelial cells, infiltration of inflammatory cells in the brain, vacuolation of cells, and swelling and cracking of the mitochondria and endoplasmic reticulum. Bacterial detection using qPCR showed that the spleen and intestine were the main organs targeted by N. seriolae. The mortality of largemouth bass experimentally infected with N. seriolae at 21°C was significantly lower than that in fish infected at higher temperatures between 24 and 33°C; there were no significant differences in the levels of mortality at these higher temperatures. The level of mortality of largemouth bass infected with N. seriolae was lowest at a neutral water pH of 7 but increased significantly at higher and lower pH. Of the tested Chinese herbal medicines, Chinese sumac Galla chinensis and Chinese skullcap Scutellaria baicalensis exhibited the best antibacterial effects. This study lays a foundation for the clinical diagnosis and scientific control of ulcer disease in largemouth bass.

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.

A Chromosome-Level Genome Assembly of Mozambique Tilapia (Oreochromis mossambicus) Reveals the Structure of Sex Determining Regions.

The Mozambique tilapia (Oreochromis mossambicus) is a fascinating taxon for evolutionary and ecological research. It is an important food fish and one of the most widely distributed tilapias. Because males grow faster than females, genetically male tilapia are preferred in aquaculture. However, studies of sex determination and sex control in O. mossambicus have been hindered by the limited characterization of the genome. To address this gap, we assembled a high-quality genome of O. mossambicus, using a combination of high coverage of Illumina and Nanopore reads, coupled with Hi-C and RNA-Seq data. Our genome assembly spans 1,007 Mb with a scaffold N50 of 11.38 Mb. We successfully anchored and oriented 98.6% of the genome on 22 linkage groups (LGs). Based on re-sequencing data for male and female fishes from three families, O. mossambicus segregates both an XY system on LG14 and a ZW system on LG3. The sex-patterned SNPs shared by two XY families narrowed the sex determining regions to ∼3 Mb on LG14. The shared sex-patterned SNPs included two deleterious missense mutations in ahnak and rhbdd1, indicating the possible roles of these two genes in sex determination. This annotated chromosome-level genome assembly and identification of sex determining regions represents a valuable resource to help understand the evolution of genetic sex determination in tilapias.

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.

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.

Comparative Genomics Studies on the dmrt Gene Family in Fish.

Doublesex and mab-3-related transcription factor (dmrt) genes are widely distributed across various biological groups and play critical roles in sex determination and neural development. Here, we applied bioinformatics methods to exam cross-species changes in the dmrt family members and evolutionary relationships of the dmrt genes based on genomes of 17 fish species. All the examined fish species have dmrt1-5 while only five species contained dmrt6. Most fish harbored two dmrt2 paralogs (dmrt2a and dmrt2b), with dmrt2b being unique to fish. In the phylogenetic tree, 147 DMRT are categorized into eight groups (DMRT1-DMRT8) and then clustered in three main groups. Selective evolutionary pressure analysis indicated purifying selections on dmrt1-3 genes and the dmrt1-3-2(2a) gene cluster. Similar genomic conservation patterns of the dmrt1-dmrt3-dmrt2(2a) gene cluster with 20-kb upstream/downstream regions in fish with various sex-determination systems were observed except for three regions with remarkable diversity. Synteny analysis revealed that dmrt1, dmrt2a, dmrt2b, and dmrt3-5 were relatively conserved in fish during the evolutionary process. While dmrt6 was lost in most species during evolution. The high conservation of the dmrt1-dmrt3-dmrt2(2a) gene cluster in various fish genomes suggests their crucial biological functions while various dmrt family members and sequences across fish species suggest different biological roles during evolution. This study provides a molecular basis for fish dmrt functional analysis and may serve as a reference for in-depth phylogenomics.

Correlation analysis of mandarin fish (Siniperca chuatsi) growth hormone gene polymorphisms and growth traits.

Screening of trait-associated molecular markers can be used to enhance the efficiency of selective breeding. Previously, we produced the first high-density genetic linkage map for the mandarin fish (Siniperca chuatsi) and identified 11 quantitative-trait loci significantly associated with growth, of which one is located within the growth hormone (GH) gene. To investigate the GH gene polymorphisms and their correlation with growth, the complete sequence was cloned and 32 single-nucleotide polymorphisms (SNPs) and one simple-sequence repeat (SSR) were identified. Of which, eight SNPs (G1-G8) and the SSR (GH-AG)were selected for genotyping and correlation analysis with growth traits in a random population. The results showed that the four novel polymorphicloci (G1, G2, G3 and GH-AG) were significantly correlated with growth traits of mandarin fish (P < 0.05). Of these, G1, G3 and GH-AG showed highly significant correlations with multiple growth traits (P < 0.01) and the combined SNP analysis showed that G1-G3 formed four effective diplotypes (D1-D4), among which D1 was highly significantly greater than D4 (P < 0.01) for some important growth traits. In conclusion, our results show that the four polymorphic loci G1-G3 and GH-AG within the mandarin fish GH gene are significantly correlated with growth traits and could be used as candidate molecular markers for selective breedingof superior varieties of mandarin fish.

Genomic organization and sexually dimorphic expression of the Dmrt1 gene in largemouth bass (Micropterus salmoides).

Doublesex and Mab-3 related transcription factor (Dmrt) genes play important roles in the process of sex determination and differentiation. In this study, a Dmrt1 gene open reading frame sequence was obtained from the gonadal transcriptome data of largemouth bass (Micropterus salmoides), and identified by cloning and sequencing. The ORF of Dmrt1 is 900 bp long, encodes 298 amino acids, and contains the DM region which is characteristic of Dmrt1. Full gDNA sequence of Dmrt1 was composed of five exons and four introns. RT-PCR and Q-PCR analysis of Dmrt1 were conducted in eight tissues and three developmental stages of mature male and female individuals. In situ hybridization was used to locate the expression of Dmrt1 in the testis and ovary of largemouth bass. The results showed that Dmrt1 was highly expressed in the testis of mature fish, but only weakly expressed in other tissues such as heart, liver, and brain, and exhibited gender dimorphism in the gonads of male and female fish at different stages. Furthermore, the expression level in female fish was very low and decreased gradually with ovary maturation. In situ hybridization indicated positive signals were found in early oocytes, but not in mature oocytes, while strong positive signals were found in all types of mature testis cells. The study showed that the sequence and structure of Dmrt1 were highly conserved and exhibited significant gender dimorphism in largemouth bass, as in other fish species. It is suggested that Dmrt1 plays an important role in sex determination and differentiation in largemouth bass.

Phylogenetic analysis of sooty grunter and other major freshwater fishes in the suborder Percoidei based on mitochondrial DNA.

Perciformes is the largest order of fishes and vertebrates. Sooty grunter (Hephaestus fuliginosus) is an economic fish species in the Terapontidae family of Percoidei, a suborder within Perciformes. To conduct molecular-level analysis of the phylogenetic relationships between sooty grunter and major freshwater fishes in Percoidei, we analysed the entire sooty grunter mitochondrial genome sequence and obtained the mitochondrial genome information of 19 fishes from Terapontidae, Serranidae, and Centrarchidae families in Percoidei from GenBank. The complete length of the sooty grunter mitochondrial genome was 16,770 bp; it encoded 13 proteins, 2 rRNAs, 22 tRNAs, and a displacement loop (D-loop). Other than ND6 and eight tRNA genes that are encoded by the light strand, the majority of genes are encoded by the heavy strand. The sequence and distribution of sooty grunter mitochondrial-encoded genes and non-coding segment were similar to those of most vertebrates. The results of neighbour joining, maximum parsimony, and Bayesian inference analyses of the complete mitochondrial genome and six genes, including cytochrome oxidase I, cytochrome B, 12S rRNA, ND2, ND4, and ND5, were consistent. In the phylogenetic trees, fishes in Terapontidae and Centrarchidae formed monophyletic clades, whereas those in Serranidae were divided into two clades, each containing Lateolabrax and Siniperca species. Among the three freshwater fish species in Terapontidae, the freshwater Terapontidae were more closely related to jade perch than with silver perch, suggesting that freshwater Terapontidae fishes originate from marine fishes. In addition, the phylogenetic results indicated that Micropterus salmoides salmoides and Micropterus salmoides floridanus in Centrarchidae should be designated as two independent species, and Siniperca in Serranidae should be considered an independent family. The sooty grunter mitochondrial genome sequence obtained in this study could be used to conduct population genetic diversity and germplasm resource studies. Furthermore, the phylogenetic analysis results of freshwater fishes in Percoidei could provide a molecular basis for cross-breeding.

Cloning, SNP detection, and growth correlation analysis of the 5' flanking regions of two myosin heavy chain-7 genes in Mandarin fish (Siniperca chuatsi).

Myosin heavy chains (MYHs) play important roles in muscle growth and contraction. In fish, MYHs contribute to hyperplasia and hypertrophy of muscle fibers, which can continue into adult life and thus result in indeterminate growth in some species. We previously identified two MYH genes, MYH-7a and MYH-7b, that are differentially expressed in Mandarin fish (Siniperca chuatsi) and appear to function in early growth. However, the regulatory role of their 5' flanking regions is unknown. To examine the effects of single nucleotide polymorphisms (SNPs) in these regions, we used genome walking to amplify their flanking sequences and analyzed the regulatory elements and binding sites. A single SNP locus was found in the flanking sequence of each gene. These SNP loci are located in the conserved glucocorticoid receptor binding region (MYH-7a: G-614A; Allele frequency: G:A = 94.9:5.1; GG (89.76) and AG (10.24) genotypes) and the LIM homeobox domain transcription factor binding sequence (MYH-7b: C-1933A; Allele frequency: C:A = 54.8:45.2; AA (20.82), AC (48.81), and CC (30.37) genotypes). At the G-614A loci, the GG genotype exhibited more superior growth traits (total length, body length, body height, etc.) than the AG genotype, with the exception of caudal peduncle length. Alternatively, at the C-1933A loci, the AC and AA genotypes showed significant differences in all growth traits, except for head length, with AC exhibiting superior traits. The AA and CC genotypes showed significant differences in caudal peduncle length and height, while no differences were observed between the AC and CC genotypes. Thus, these SNPs in the 5' flanking regions of MYH-7a and MYH-7b are correlated with superior growth and can be used for selecting Mandarin fish during breeding.

Interaction of Group B Streptococcus sialylated capsular polysaccharides with host Siglec-like molecules dampens the inflammatory response in tilapia.

Group B Streptococcus (GBS, S. agalactiae) infection in tilapia (Oreochromis niloticus) causes widespread death of this species and is a significant issue for the aquaculture industry. The major virulence factor for GBS is its sialylated capsular polysaccharides (CPs). These CPs interact with sialic acid-binding immunoglobulin-like lectins (Siglecs) on the host immune cells to regulate the downstream inflammatory response and evade detection. Previously, we cloned multiple Siglec-like molecules from an O. niloticus cDNA library, all of which were shown to interact with the sialylated CPs of GBS. In the present study, we investigated the effects of GBS infection on the expression of pro- and anti-inflammatory cytokines in O. niloticus as well as OnSiglec-like-transfected macrophage cells. Eukaryotic expression vectors containing full-length OnSiglec-1-like, -4b-like, -14-like were constructed and used to transfect RAW264 macrophages in vitro as well as live tilapia in vivo prior to GBS infection. The expression of the anti-inflammatory cytokine interleukin (IL)-10 and the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, IL-6, and interferon (INF)-ß were then analyzed by qPCR. Our results indicate that as infection progressed, IL-10 expression was significantly upregulated, while that of TNF-α and IL-6 were significantly downregulated in the OnSiglec-like-transfected cells. INF-ß expression was also downregulated in cells transfected with OnSiglec-1-like and -4b-like, but was not significantly effected in OnSiglec-14-like-transfected cells. Notably, the magnitude of these cytokine expression changes was greatly decreased when a ΔneuA GBS mutant was used to infect the OnSiglec-1-like-transfected cells. In GBS-infected tilapia, IL-10 expression was significantly upregulated in all tissues, whereas INF-ß expression in the spleen, kidney, and gills was significantly downregulated at 12 hpi. While the expression of TNF-α was slightly upregulated, this change was not significant. In GBS ΔneuA mutant-infected O. niloticus, IL-10 expression in all of the tissues was significantly lower than that observed for the wild-type GBS group, while TNF-α expression was higher in the mutant infected group. There was no significant difference in INF-ß expression between the two groups. Taken together, sialylated CPs on GBS appear to interact with host OnSiglec-like molecules to transmit negative regulatory signals via enhanced anti-inflammatory cytokine IL-10 production and reduced pro-inflammatory cytokine production, ultimately leading to dampening of the host immune response. The results of this study further elucidate the molecular mechanism underlying GBS infection in tilapia and also provide candidate drug target molecules.

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.