Complete Genome Sequence of Pseudomonas plecoglossicida XSDHY-P, a Strain That Is Pathogenic for the Marine Fish Larimichthys crocea.
ABSTRACT: Pseudomonas plecoglossicida is a Gram-negative fish pathogen responsible for visceral granular disease in large yellow croaker, Larimichthys crocea. Here, we report the complete genome sequence of a verified virulent strain, XSDHY-P, that was isolated from spleen tissue of a diseased large yellow croaker.
Project description:In the present study, Larimichthys crocea and Pseudomonas plecoglossicida were selected as a host-pathogen interaction model for teleosts and prokaryotic pathogens. Five shRNAs were designed and synthesized to silence the fliA gene, all of which resulted in pronounced reductions in fliA mRNA; the mutant strain with the best silencing efficiency of 92.16% was chosen for subsequent analysis. A significant decrease in motility, intracellular survival and escape was observed for the fliA-RNAi strain of P. plecoglossicida, whereby silencing of the fliA gene led to a 30% decrease in mortality and a four-day delay in the onset of infection in L. crocea. Moreover, silencing of P. plecoglossicida fliA resulted in a significant change in both the pathogen and host transcriptome in the spleens of infected L. crocea. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of pathogen transcriptome data showed that silencing fliA resulted in downregulation of 18 flagellum-related genes; KEGG analysis of host transcriptome data revealed that infection with the fliA-RNAi strain caused upregulation of 47 and downregulation of 106 immune-related genes. These pathogen-host interactions might facilitate clearance of P. plecoglossicida by L. crocea, with a significant decrease in fliA-RNAi P. plecoglossicida strain virulence in L. crocea.
Project description:Host-pathogen interactions are highly complex, involving large dynamic changes in gene expression during infection. These interactions are fundamental to understanding anti-infection immunity of hosts, as well as the pathogenesis of pathogens. For bacterial pathogens interacting with animal hosts, time-resolved dual RNA-seq of infected tissue is difficult to perform due to low pathogen load in infected tissue. In this study, an acute infection model of Larimichthys crocea infected by Pseudomonas plecoglossicida was established. The spleens of infected fish exhibited typical symptoms, with a maximum bacterial load at two days post-injection (dpi). Time-resolved dual RNA-seq of infected spleens was successfully applied to study host-pathogen interactions between L. crocea and P. plecoglossicida. The spleens of infected L. crocea were subjected to dual RNA-seq, and transcriptome data were compared with those of noninfected spleens or in vitro cultured bacteria. Results showed that pathogen-host interactions were highly dynamically regulated, with corresponding fluctuations in host and pathogen transcriptomes during infection. The expression levels of many immunogenes involved in cytokine-cytokine receptor, Toll-like receptor signaling, and other immune-related pathways were significantly up-regulated during the infection period. Furthermore, metabolic processes and the use of oxygen in L. crocea were strongly affected by P. plecoglossicida infection. The WGCNA results showed that the metabolic process was strongly related to the entire immune process. For P. plecoglossicida, the expression levels of motility-related genes and flagellum assembly-related genes were significantly up-regulated. The results of this study may help to elucidate the interactions between L. crocea and P. plecoglossicida.
Project description:Interleukin 1? (IL-1?), the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the cDNA and genomic DNA sequences of the IL-1? gene from the large yellow croaker, Larimichthys crocea. Phylogenetic analysis indicated that the IL-1? (LcIL-1?) gene was most closely related to that of European seabass (Dicentrarchus labrax), sharing 67.8% amino acid identity. In healthy large yellow croaker, LcIL-1? transcription was detected in all tested tissues, with the highest level found in the head kidney. Upon Vibrio alginolyticus infection, LcIL-1? transcription in all tested tissues was significantly upregulated. Intraperitoneal injection of recombinant LcIL-1? (rLcIL-1?) improved the survival rate and reduced the tissue bacterial load after V. alginolyticus infection. In addition, rLcIL-1? induced monocytes/macrophages (MO/M?) chemotaxis and increased phagocytosis and bactericidal activity in vitro. These results suggest that LcIL-1? plays an important role in the large yellow croaker immune response against V. alginolyticus.
Project description:High-density genetic maps are essential for genome assembly, comparative genomic analysis and fine mapping of complex traits. In this study, 31,191 single nucleotide polymorphisms (SNPs) evenly distributed across the large yellow croaker (Larimichthys crocea) genome were identified using restriction-site associated DNA sequencing (RAD-seq). Among them, 10,150 high-confidence SNPs were assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 5451.3 cM with an average distance of 0.54 cM between loci. This represents the densest genetic map currently reported for large yellow croaker. Using 2889 SNPs to target specific scaffolds, we assigned 533 scaffolds, comprising 421.44 Mb (62.04%) of the large yellow croaker assembled sequence, to the 24 linkage groups. The mapped assembly scaffolds in large yellow croaker were used for genome synteny analyses against the stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes). Greater synteny was observed between large yellow croaker and stickleback. This supports the hypothesis that large yellow croaker is more closely related to stickleback than to medaka. Moreover, 1274 immunity-related genes and 195 hypoxia-related genes were mapped to the 24 chromosomes of large yellow croaker. The integration of the high-resolution genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits in large yellow croaker.
Project description:<i>Pseudomonas plecoglossicida</i> is a rod-shaped, gram-negative bacterium with flagella. It causes visceral white spot disease and high mortality in <i>Larimichthys crocea</i> during culture, resulting in serious economic loss. Analysis of transcriptome and quantitative real-time polymerase chain reaction (PCR) data showed that <i>dksA</i> gene expression was significantly up-regulated after 48 h of infection with <i>Epinephelus coioides</i> (log <sub>2</sub>FC=3.12, <i>P</i><0.001). RNAi of five shRNAs significantly reduced the expression of <i>dksA</i> in <i>P. plecoglossicida</i>, and the optimal silencing efficiency was 96.23%. Compared with wild-type strains, the symptoms of visceral white spot disease in <i>L. crocea</i> infected with RNAi strains were reduced, with time of death delayed by 48 h and mortality reduced by 25%. The <i>dksA</i> silencing led to a substantial down-regulation in cellular component-, flagellum-, and ribosome assembly-related genes in <i>P. plecoglossicida</i>, and the significant up-regulation of <i>fliC</i> may be a way in which virulence is maintained in <i>P. plecoglossicida</i>. The GO and KEGG results showed that RNAi strain infection in <i>L. crocea</i> led to the down-regulation of inflammatory factor genes in immune-related pathways, which were associated with multiple immune response processes. Results also showed that <i>dksA</i> was a virulence gene in <i>P. plecoglossicida</i>. Compared with the wild-type strains, RNAi strain infection induced a weaker immune response in <i>L. crocea</i>.
Project description:The large yellow croaker Larimichthys crocea (L. crocea) is one of the most economically important marine fish in China and East Asian countries. It also exhibits peculiar behavioral and physiological characteristics, especially sensitive to various environmental stresses, such as hypoxia and air exposure. These traits may render L. crocea a good model for investigating the response mechanisms to environmental stress. To understand the molecular and genetic mechanisms underlying the adaptation and response of L. crocea to environmental stress, we sequenced and assembled the genome of L. crocea using a bacterial artificial chromosome and whole-genome shotgun hierarchical strategy. The final genome assembly was 679 Mb, with a contig N50 of 63.11 kb and a scaffold N50 of 1.03 Mb, containing 25,401 protein-coding genes. Gene families underlying adaptive behaviours, such as vision-related crystallins, olfactory receptors, and auditory sense-related genes, were significantly expanded in the genome of L. crocea relative to those of other vertebrates. Transcriptome analyses of the hypoxia-exposed L. crocea brain revealed new aspects of neuro-endocrine-immune/metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia. Proteomics data demonstrate that skin mucus of the air-exposed L. crocea had a complex composition, with an unexpectedly high number of proteins (3,209), suggesting its multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. Our results reveal the molecular and genetic basis of fish adaptation and response to hypoxia and air exposure. The data generated by this study will provide valuable resources for the genetic improvement of stress resistance and yield potential in L. crocea.
Project description:<i>Larimichthys crocea</i> (large yellow croaker) is a type of perciform fish well known for its peculiar physiological properties and economic value. Here, we constructed an improved version of the <i>L. crocea</i> genome assembly, which contained 26,100 protein-coding genes. Twenty-four pseudo-chromosomes of <i>L. crocea</i> were also reconstructed, comprising 90% of the genome assembly. This improved assembly revealed several expansions in gene families associated with olfactory detection, detoxification, and innate immunity. Specifically, six hepcidin genes (LcHamps) were identified in <i>L. crocea</i>, possibly resulting from lineage-specific gene duplication. All LcHamps possessed similar genomic structures and functional domains, but varied substantially with respect to expression pattern, transcriptional regulation, and biological function. LcHamp1 was associated specifically with iron metabolism, while LcHamp2s were functionally diverse, involving in antibacterial activity, antiviral activity, and regulation of intracellular iron metabolism. This functional diversity among gene copies may have allowed <i>L. crocea</i> to adapt to diverse environmental conditions.
Project description:High-density single-nucleotide polymorphism (SNP) genotyping array is an essential tool for genetic analyses of animals and plants. Large yellow croaker (Larimichthys crocea) is one of the most commercially important marine fish species in China. Although plenty of SNPs have been identified in large yellow croaker, no high-throughput genotyping array is available. In this study, a high-throughput SNP array named NingXin-I with 600K SNPs was developed and evaluated. A set of 82 large yellow croakers were collected from different locations of China and re-sequenced. A total of 9.34M SNPs were identified by mapping sequence reads to the large yellow croaker reference genome. About 1.98M candidate SNPs were selected for further analyses by using criteria such as SNP quality score and conversion performance in the final array. Finally, 579.5K SNPs evenly distributed across the large yellow croaker genome with an average spacing of 1.19 kb were proceeded to array production. The performance of NingXin-I array was evaluated in 96 large yellow croaker individuals from five populations, and 83.38% SNPs on the array were polymorphic sites. A further test of the NingXin-I array in five closely related species in Sciaenidae identified 26.68-56.23% polymorphic SNP rate across species. A phylogenetic tree inferred by using the genotype data generated by NingXin-I confirmed the phylogenetic distance of the species in Sciaenidae. The performance of NingXin-I in large yellow croaker and the other species in Sciaenidae suggested high accuracy and broad application. The NingXin-I array should be valuable for quantitative genetic studies, such as genome-wide association studies (GWASs), high-density linkage map construction, haplotype analysis, and genome-based selection.
Project description:Active coating could improve the fish quality and extend the shelf life. This study investigates the effect of locust bean gum (LBG) and sodium alginate (SA) active coatings containing lemon verbena (<i>Lippa citriodora</i> Kunth.) essential oil (LVEO) emulsions on microbiological, physicochemical and organoleptic evaluation of large yellow croaker (<i>Larimichthys crocea</i>) samples during refrigerated storage at 4°C. Results showed that LBG-SA coatings incorporated with 0.30 or 0.60% LVEO emulsions significantly inhibited the growth of mesophile bacteria, <i>Pseudomonas</i> spp., H<sub>2</sub>S-producing bacteria, lactic acid bacteria (LAB) and psychrophilic bacteria, and reduce the productions of trimethylamine (TMA), total volatile basic nitrogen (TVB-N) and ATP-related compounds. Further, the LVEO treatments also retarded the water migration and maintained the organoleptic evaluation results of large yellow croaker during storage at 4°C. In conclusion, the LBG-SA active coatings incorporated with LVEO emulsions maintained the quality and extended the shelf life of large yellow croaker during refrigerated storage.
Project description:Large-scale transcription studies have revealed numerous lncRNAs (long non-coding RNAs). lncRNAs have been proposed to participate in the regulation of a diverse range of biological processes, including transcriptional regulation. Although lncRNAs have attracted increasing attention, the studies in large yellow croaker (<i>Larimichthys crocea</i>) are still rare, and they lack systematic analysis. In this study, 101 RNA-seq datasets varied in ages, sexes, and tissues were retrieved from the NCBI database to generate a comprehensive catalog of large yellow croaker transcriptome database. A set of 14,599 high-confidence lncRNAs from 13,673 loci were identified and characterized. Furthermore, RNA-seq datasets obtained from the infection of <i>C. irritans</i> were employed to investigate the differential expression pattern of lncRNAs and analyze potential biological functions. A total of 77 differentially expressed lncRNAs targeting to 567 protein-coding genes were identified by using expression analysis. Several immune genes, including TLR5, CD2AP, and MMP9, were highlighted. With GO enrichment and KEGG pathway analysis, the immune-related terms or pathways were enriched. This study created a comprehensive dataset of lncRNAs for large yellow croaker, which would be helpful for the researches of functional roles of lncRNAs in large yellow croaker.