Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.

The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of this pathogen is type III secretion system 2 (T3SS2), which is encoded on the 80-kb V. parahaemolyticus pathogenicity island (Vp-PAI), the gene expression of which is governed by the OmpR-family transcriptional regulator VtrB. Here, we found a positive autoregulatory feature of vtrB transcription, which is often observed with transcriptional regulators of bacteria, but the regulation was not canonically dependent on its own promoter. Instead, this autoactivation was induced by heterogeneous transcripts derived from the VtrB-regulated operon upstream of vtrB. VtrB-activated transcription overcame the intrinsic terminator downstream of the operon, resulting in transcription read-through with read-in transcription of the vtrB gene and thus completing the autoregulatory loop for vtrB gene expression. The dampening of read-through transcription with an exogenous strong terminator reduced vtrB gene expression. Furthermore, a V. parahaemolyticus mutant with defects in the vtrB autoregulatory loop also showed compromises in T3SS2 expression and T3SS2-dependent cytotoxicity in vitro and enterotoxicity in vivo, indicating that this autoregulatory loop is essential for sustained vtrB activation and the consequent robust expression of T3SS2 genes for pathogenicity. Taken together, these findings demonstrate that the regulatory loop for vtrB gene expression based on read-through transcription from the upstream operon is a crucial pathway in T3SS2 gene regulatory network to ensure T3SS2-mediated virulence of V. parahaemolyticus.

Characterization and genomic analysis of a broad-spectrum lytic phage PG288: A potential natural therapy candidate for Vibrio infections.

Vibrio parahaemolyticus, an important zoonotic pathogen, can cause severe diseases and even death in aquatic animals and humans. As the widespread use of antibiotics gradually diminishes their effectiveness, phages, which can selectively lyse bacteria, are garnering increased attention as a valuable alternative antibacterial strategy. This study characterized PG288, a lytic phage utilizing V. parahaemolyticus strain G855 as its host. Morphologically, the phage features a polyhedral head and a long, non-retractable tail. Bactericidal assays revealed that phage PG288 exhibited a strong lytic ability against V. parahaemolyticus strain G855 and demonstrated a broad host range, as evidenced by the ability to infect several distinct Vibrio species. The one-step growth curve indicated a latent period of approximately 50 min for phage PG288, with a burst size of roughly 92 PFU per cell. Additionally, phage PG288 exhibited remarkable stability within a temperature range of 20-50°C and a pH range of 4-10. Genomic analysis unveiled 105 ORFs within phage PG288, notably devoid of genes associated with antibiotic resistance, virulence, and lysogenic activity. Phylogenetic analysis conclusively identified it as a new member of the genus Mardecavirus within the class Caudoviricetes. In summary, this study contributes valuable insights to the phage database, presenting phage PG288 as a promising candidate for phage therapies against Vibrio infections.

Characterization of caspase gene family in Sebastes schlegelii and their expression profiles under Aeromonas salmonicida and Vibrio anguillarum infection.

The caspase, functioning as a proteinase, plays a crucial role in eukaryotic cell apoptosis, regulation of apoptosis, cellular growth, differentiation, and immunity. The identification of caspase gene family in Sebastes schlegelii is of great help to understand its antimicrobial research. In S. schlegelii, we totally identified nine caspase genes, including four apoptosis initiator caspases (caspase 2, caspase 8, caspase 9 and caspase 10), four apoptosis executioners (caspase 3a, caspase 3b, caspase 6, and caspase 7) and one inflammatory executioner (caspase 1). The duplication of caspase 3 genes on chr3 and chr8 may have been facilitated by whole genome duplication (WGD) events or other complex evolutionary processes. In general, the number of caspase genes relatively conserved in high vertebrates, while exhibiting variation in teleosts. Furthermore, syntenic analysis and phylogenetic relationships analysis supported the correct classification of these caspase gene family in S. schlegelii, especially for genes with duplicated copies. Additionally, the expression patterns of these caspase genes in different tissues of S. schlegelii under healthy conditions were assessed. The results revealed that the expression levels of most caspase genes were significantly elevated in the intestine, spleen, and liver. To further investigate the potential immune functions of these caspase genes in S. schlegelii, we challenged individuals with A. salmonicida and V. anguillarum, respectively. After infection with A. salmonicida, the expression levels of caspase 1 in the liver and spleen of S. schlegelii remained consistently elevated throughout the infection time points. The expression levels of most caspase family members in the intestine exhibited significant divergence following V. anguillarum infection. This study provides a comprehensive understanding of the caspase gene families in S. schlegelii, thereby establishing a solid foundation for further investigations into the functional roles of these caspase genes.

Characterization of a Vibriophage Infecting Pathogenic Vibrio harveyi.

Bacterial diseases caused by Vibrio spp. are prevalent in aquaculture and can lead to high mortality rates among aquatic species and significant economic losses. With the increasing emergence of multidrug-resistant Vibrio strains, phage therapy is being explored as a potential alternative to antibiotics for biocontrol of infectious diseases. Here, a new lytic phage named vB_VhaS_R21Y (R21Y) was isolated against Vibrio harveyi BVH1 obtained from seawater from a scallop-farming area in Rongcheng, China. Its morphology, infection cycle, lytic profile, phage stability, and genetic features were characterized. Transmission electronic microscopy indicated that R21Y is siphovirus-like, comprising an icosahedral head (diameter 73.31 ± 2.09 nm) and long noncontractile tail (205.55 ± 0.75 nm). In a one-step growth experiment, R21Y had a 40-min latent period and a burst size of 35 phage particles per infected cell. R21Y was highly species-specific in the host range test and was relatively stable at pH 4-10 and 4-55 °C. Genomic analysis showed that R21Y is a double-stranded DNA virus with a genome size of 82,795 bp and GC content of 47.48%. Its high tolerance and lytic activity indicated that R21Y may be a candidate for phage therapy in controlling vibriosis in aquacultural systems.

A circRNA therapy based on Rnf103 to inhibit Vibrio anguillarum infection.

The losses caused by Vibrio infections in the aquaculture industry are challenging to quantify. In the face of antibiotic resistance, a natural and environmentally friendly alternative is urgently needed. In this study, we identify E3 ubiquitin-protein ligase RNF103 (rnf103) as a crucial target involved in immune evasion by Vibrio anguillarum. Our research demonstrates that Rnf103 promotes immune escape by inhibiting Traf6. Interestingly, we discover a circular RNA (circRNA), circRnf103, formed by reverse splicing of the Rnf103 gene. Predictive analysis and experimentation reveal that circRnf103 encodes Rnf103-177aa, a protein that competes with Rnf103 and binds to Traf6, preventing its degradation. Notably, circRnf103 therapy induces Rnf103-177aa protein production in zebrafish. In zebrafish models, circRnf103 exhibits significant effectiveness in treating V. anguillarum infections, reducing organ burden. These findings highlight the potential of circRNA therapy as a natural and innovative approach to combat infectious diseases sustainably, particularly in aquaculture and environmental management.

Diploid and triploid Chinook salmon (Oncorhynchus tshawytscha) have altered microRNA responses in immune tissues after infection with Vibrio anguillarum.

Production of sterile fishes through artificial retention of a third set of chromosomes (triploidy) is a sustainable alternative for aquaculture since it reduces escapee pressure on wild populations. However, these fishes have reduced survival in stressful conditions and in response to infection. In this study, the impact of Vibrio anguillarum infection on diploid and triploid Chinook salmon (Oncorhynchus tshawytscha) was investigated to identify if there was any significant immune regulation by microRNAs (miRNA). Small RNAs from hindgut, head kidney, and spleen were sequenced to determine if miRNA transcript abundance was altered due to ploidy and infection in nine-month old full-sibling diploids and triploids. All three tissues had differentially expressed miRNA prior to infection, indicating subtle changes in epigenetic regulation due to increased ploidy. Additionally, miRNA were altered by infection, but there was only a difference in spleen miRNA expression between diploids and triploids at three days of infection. Furthermore, one miRNA (ssa-miR-2188-3p) was confirmed as having an altered response to infection in triploids compared to diploids, implicating potential immune dysregulation due to increased ploidy. The miRNAs identified in this study are predicted to target immune pathways, providing evidence for their importance in regulating responses to pathogens. This study is the first to investigate how increased ploidy alters miRNA expression in response to infection. Additionally, it provides evidence for epigenetic dysregulation in triploid fishes, which may contribute to their poor performance in response to stress.

The TRAF gene family in turbot (Scophthalmus maximus): Identification, characterization, molecular evolution and expression patterns analysis.

Tumor necrosis factor receptor-associated factor (TRAF) is an important structural protein, which can bind to TNF receptors and participate in the regulation of TNF signaling pathway. Nonetheless, few studies have been conducted to investigate the systematic identification of TRAF gene family in teleost and role in innate immunity of turbot (Scophthalmus maximus). In this study, eight TRAF genes, namely SmTRAF2aa, SmTRAF2ab, SmTRAF2b, SmTRAF3, SmTRAF4a, SmTRAF5, SmTRAF6 and SmTRAF7, were identified and annotated in turbot by using bioinformatics methods. Analysis of the phylogenetic, syntenic and molecular evolution demonstrated that all SmTRAF members were evolutionarily conserved in teleost. Domain analysis showed all SmTRAF proteins contained a typical conserved N-terminal RING finger domain. Most SmTRAF proteins contained a MATH domain at the C-terminal, while SmTRAF7 contains seven duplicate WD40 domains. In addition, quantitative real-time PCR was performed to detect the expression patterns of SmTRAFs in tissues from healthy and Vibrio anguillarum infected turbots. The results indicated SmTRAFs had diverse tissue expression patterns and the expression of TRAF gene changed significantly after V. anguillarum infection. This study provided a basis for understanding the roles of TRAFs in the innate immune response of turbot.

Genome-wide characterization of integrin (ITG) gene family and their expression profiling in half-smooth tongue sole (Cynoglossus semilaevis) upon Vibrio anguillarum infection.

Integrins (ITGs) are transmembrane heterodimer receptors with ITGα subunit and ITGß subunit, participating in various physiological processes, including immunity. At present, systematic research on ITGs in teleost is scarce, especially in half-smooth tongue sole (Cynoglossus semilaevis). In this study, a set of 28 ITG genes in half-smooth tongue sole have been identified and characterized. The phylogenetic analysis showed that ITGα and ITGß subunits were respectively classified into five and two clusters, consistent with previous studies. The selection pressure analysis indicated that most of ITG genes were under purifying selection, except for ITGα11b and ITGαL with positive selection. The expression profiles of eight selected ITG genes, including ITGα1, ITGα5, ITGα8, ITGα11, ITGß1, ITGß2, ITGß3, and ITGß8, were analyzed in healthy tissues and after infection with Vibrio anguillarum, revealed their implications in immune response. The study provided a comprehensive characterization and expression analysis of ITG genes in half-smooth tongue sole, setting a solid foundation for further functional studies and promising potential in disease control.

Validation of a QTL associated with resistance to Vibrio anguillarum in rainbow trout (Oncorhynchus mykiss).

Vibriosis is a bacterial disease in fish caused by the Gram negative bacterium Vibrio anguillarum with severe impact on rainbow trout (Oncorhynchus mykiss) farming. Sustainable control methods should be developed and we here show that marker assisted selective breeding of fish naturally resistant to the disease is feasible. We have validated the use of a single nucleotide polymorphism (SNP) marker SNP AX-89,945,921 (QTL on chromosome 21). The QTL was previously found associated with resistance to vibriosis and described following a genome wide association analysis (GWAS) of trout exposed to the bacterium. For this validation spawners were genotyped by use of the 57 K Axiom®Trout Microarray (Affymetrix) and homozygous male fish carrying the allele with the SNP AX-89,945,921 were then selected and used to fertilize eggs from outbred female trout resulting in fish all carrying the SNP (QTL-fish). Control fish (non-QTL fish) were produced by fertilizing the same batch of eggs by use of male parents negative for the SNP. The fish were exposed in freshwater to V. anguillarum (water bath infection) at 19 C°. A total of 900 fish were challenged in a common garden set-up in triplicate. A bacterial solution of V. anguillarum (serotype O1) was added to each of three freshwater fish tanks, each with 150 QTL and 150 non-QTL fish. Fish were tagged by tail fin cut (upper/lower) to discern the two groups, whereafter fish were monitored around the clock to detect disease signs and remove moribund fish. Clinical vibriosis developed within two days in non-QTL-fish (overall morbidity of 70%). QTL fish developed clinical signs later and the morbidity was significantly lower and did not reach 50%. Rainbow trout farming may benefit from using the QTL associated with higher resistance towards vibriosis. The effect may be optimized in the future by use of both male and female parents homozygous for the marker allele.

Identification and functional characterization of caspases in turbot (Scophthalmus maximus) in response to bacterial infection.

Apoptosis is the autonomous and orderly death of cells under genetic control to maintain the stability of the internal environment, and is a programmed cell death process with unique morphological and biochemical properties that is regulated by a variety of factors. Caspase gene family has a significant function in the process of apoptosis. However, the knowledge of caspases in turbot remains largely unknown. In present study, a total of nine turbot caspase genes were identified. The mRNA length of these caspase genes was ranged from 1225 bp (caspase-7) to 3216 bp (caspase-2), and the protein length was ranged from 281 aa (caspase-3a) to 507 aa (caspase-10). Phylogenetic analysis showed these caspase genes were divided into three subfamilies. The qRT-PCR results showed that turbot caspase genes were expressed in all the examined organs, especially the intestine, kidney, blood and gills. Meanwhile, we explored the expression patterns of caspase genes in the intestine, skin and gills after Vibrio anguillarum and Aeromonas salmonids infections. The results showed that caspase genes showed different expression patterns in mucosal tissues after bacterial infection, demonstrating the critical role of caspase genes in mucosal immune responses. In addition, protein-protein interaction analysis showed that caspase proteins interacted with immune molecules such as NLR, IL-1ß, and birc. The results of interference and overexpression experiments showed that caspase-1 might play key roles in the regulation of the IL-1ß production, but the detailed mechanism needs to be further studied. The results of this study provide valuable information for further study the roles of caspase genes in turbot, which could help us to further understand the inflammatory pathways in teleost.

Genomics and transcriptomics reveal new molecular mechanism of vibriosis resistance in fish.

Infectious diseases have caused dramatic production decline and economic loss for fish aquaculture. However, the poor understanding of fish disease resistance severely hampered disease prevention. Chinese tongue sole (Cynoglossus semilaevis) is an important economic flatfish suffering from vibriosis. Here we used genomic, transcriptomic and experimental approaches to investigate the molecular genetic mechanisms underlying fish vibriosis resistance. A genome-wide comparison revealed that the genes under selective sweeps were enriched for glycosaminoglycan (GAG) chondroitin sulfate (CS)/dermatan sulfate (DS) metabolism. Transcriptomic analyses prioritized synergic gene expression patterns in this pathway, which may lead to an increased CS/DS content in the resistant family. Further experimental evidence showed that carbohydrate sulfotransferases 12 (Chst12), a key enzyme for CS/DS biosynthesis, has a direct antibacterial activity. To the best of our knowledge, this is the first report that the chst12 gene has a bactericidal effect. In addition, CS/DS is a major component of the extracellular matrix (ECM) and the selection signatures and fine-tuned gene expressions of ECM-receptor interaction genes indicated a modification in the ECM structure with an enhancement of the barrier function. Furthermore, functional studies conducted on Col6a2, encoding a collagen gene which constitutes the ECM, pointed to that it may act as a cellular receptor for Vibrio pathogens, thus plays an important role for the Vibrio invasion. Taken together, these findings provide new insights into the molecular protective mechanism underlying vibriosis resistance in fish, which offers crucial genomic resources for the resistant germplasm breeding and infectious disease control in fish culturing.

RNA-seq analysis revealing the immune response of Neocaridina denticulata sinensis gill to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus causes serious economic losses to the shrimp farming industry. There is still a lack of adequate understanding of the changes in the overall response of N. denticulata sinensis caused by V. parahaemolyticus, particularly with respect to gill tissue, which is severely damaged by the pathogen. In this study, a total of 1358 differentially expressed genes were identified between the PBS control and Vibrio stimulation groups using transcriptome sequencing techniques. After further screening and analysis, many immune-related genes were obtained, involving lysosome pathway, metabolic process, chitin-binding protein, and serine protease family members. In addition, we randomly selected six DEGs in the lysosome pathway for qRT-PCR verification, and the results showed that their expression patterns were consistent with the RNA-seq. The results demonstrate the molecular regulation of the gill tissue response to V. parahaemolyticus infection in N. denticulata sinensis, contributing to the understand of the complex and efficient innate immune system and defense mechanisms in crustaceans.

Integrative analyses of mRNA and microRNA expression profiles reveal the innate immune mechanism for the resistance to Vibrio parahaemolyticus infection in Epinephelus coioides.

Vibrio parahaemolyticus, as one of the main pathogens of marine vibriosis, has brought huge losses to aquaculture. However, the interaction mechanism between V. parahaemolyticus and Epinephelus coioides remains unclear. Moreover, there is a lack of comprehensive multi-omics analysis of the immune response of grouper spleen to V. parahaemolyticus. Herein, E. coioides was artificially injected with V. parahaemolyticus, and it was found that the mortality was 16.7% in the early stage of infection, and accompanied by obvious histopathological lesions in the spleen. Furthermore, 1586 differentially expressed genes were screened by mRNA-seq. KEGG analysis showed that genes were significantly enriched in immune-related pathways, Acute-phase immune response, Apoptosis, Complement system and Cytokine-cytokine receptor interaction. As for miRNA-seq analysis, a total of 55 significantly different miRNAs were identified. Further functional annotation analysis indicated that the target genes of differentially expressed miRNAs were enriched in three important pathways (Phosphatidylinositol signaling system, Lysosome and Focal adhesions). Through mRNA-miRNA integrated analysis, 1427 significant miRNA-mRNA pairs were obtained and "p53 signaling pathway", "Intestinal immune network for IgA production" were considered as two crucial pathways. Finally, miR-144-y, miR-497-x, novel-m0459-5p, miR-7133-y, miR-378-y, novel-m0440-5p and novel-m0084-3p may be as key miRNAs to regulate immune signaling pathways via the miRNA-mRNA interaction network. The above results suggest that the mRNA-miRNA integrated analysis not only sheds new light on the molecular mechanisms underlying the interaction between host and V. parahaemolyticus but also provides valuable and new insights into resistance to vibrio infection.

Characterization, evolution and expression analysis of Toll-like receptor 7 (TLR7) in turbot (Scophthalmus maximus L.).

The pattern recognition receptors (PRRs) can recognize the conserved molecular structures of pathogens to active the innate immune responses, and subsequently induce the antigen-specific adaptive immune responses for the clearance of infected pathogen. Among the PRRs, Toll-like receptors (TLRs) are the first and best characterized PRRs across all the species. Among the TLR members, TLR7 showed significant conservation across the vertebrates, with the lowest rate of evolution for its LRR domains from primates to fishes. In the current study, one TLR7 (SmTLR7) gene was captured in turbot, with a 3144 bp open reading frame (ORF), that encoding 1047 amino acid residues. Following multiple sequence comparison, SmTLR7 was found to have the highest similarity and identity both to Paralichthys olivaceus with 91.9% and 85.9%, respectively. In phylogenetic analysis, SmTLR7 was firstly clustered with Japanese flounder, and then clustered with fugu, rainbow trout, and zebrafish. In addition, SmTLR7 was widely expressed in all the examined tissues with the highest expression level in spleen, followed by skin, while the lowest expression level was detected in blood. Following both Edwardsiella tarda and Vibrio anguillarum challenge, SmTLR7 was significantly down-regulated in gill and intestine, and up-regulated in skin. Moreover, SmTLR7 was significantly up-regulated in head kidney macrophages following LPS, LTA, PGN and polyI:C stimulation, as well as showed the strongest binding ability to LPS, followed by PGN, LTA, and polyI:C in a dose-dependent manner. Finally, following RNAi of SmTLR7, MyD88 and IL-1ß were slightly up-regulated, while TRAF6 and IL-8 were significantly down-regulated. The characterization of TLR7 can expand our understanding of the PRRs in teleost fishes, and eventually aid the exploration of interactions between host and pathogen.

Antimicrobial and immunoregulatory activities of the derived peptide of a natural killer lysin from black rockfish (Sebastes schlegelii).

Natural killer lysin (NK-lysin) is a small molecule antimicrobial peptide secreted by natural killer cells and T lymphocytes. In this study, we characterized a cDNA sequence encoding an NK-lysin homologue (SsNKL1) from black rockfish, Sebastes schlegelii. The open reading frame (ORF) of SsNKL1 encodes a putative protein of 149 amino acids and shares 44%-87% overall sequence identities with other teleost NK-lysins. SsNKL1 possesses conserved NK-lysin family features, including a signal sequence and a surfactant-associated protein B (SapB) domain, sequence analysis revealed that SsNKL1 is most closely related to false kelpfish (Sebastiscus marmoratus) NK-lysin (with 87% sequence identity). SsNKL1 transcripts were detected in all the tested tissues, with the highest level in the kidney, followed by the spleen and gills. Upon Listonella anguillarum infection, the mRNA expression of SsNKL1 in the black rockfish was significantly up-regulated in the liver and kidney. The derived peptide SsNKLP27 from SsNKL1 was synthesized, and its biological function was studied. SsNKLP27 showed direct antibacterial activity against Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, L. anguillarum, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio vulnificus. SsNKLP27 treatment facilitated the bactericidal process of erythromycin by enhancing the permeability of the outer membrane. In the process of interaction with the target bacterial cells, SsNKLP27 changed the permeability and retained the morphological integrity of the cell membrane, then penetrated into the cytoplasm, and induced the degradation of genomic DNA and total RNA. In vivo studies showed that administration of SsNKLP27 before bacterial and viral infection significantly reduced the transmission and replication of pathogens in tissues. In vitro analysis showed that SsNKLP27 could enhance the respiratory burst ability and regulate the expression of some immune-related genes of macrophages. In summary, these results provided new insights into the function of NK-lysins in teleost fish and support that SsNKLP27 is a new broad-spectrum antimicrobial peptide that has a potential application prospect in aquaculture against pathogenic infection.

Two ACTH analogs exert differential effects on monocytes/macrophages function regulation in ayu (Plecoglossus altivelis).

Adrenocorticotropic hormone (ACTH), a bioactive peptide of the family of melanocortins, is generated from pro-opiomelanocortin (POMC). So far, the research on the specific functions of ACTH in the immune system of teleosts is limited. We determined two complementary DNA (cDNA) sequences of POMC in ayu (Plecoglossus altivelis), termed PaPOMC-A and PaPOMC-B. PaPOMCs transcripts occurred in all examined tissues, and their expression in immune tissues changed following experimental infection with Vibrio anguillarum. PaACTH-B, but not PaACTH-A, suppressed the phagocytosis of monocytes/macrophages (MO/MФ). Two isoforms of PaACTH increased the bactericidal capacity of MO/MФ. PaACTH-A increased anti-inflammatory cytokine expression, while PaACTH-B decreased pro-inflammatory cytokine expression in MO/MФ. Compared with PaACTH-B treatment, the PaACTH-A treatment improved survival rate and reduced the bacterial load in V. anguillarum-infected ayu through interleukin (IL)-10. Our results indicate that the two PaACTH isoforms exert different effects in the host defense against bacterial infection.

Characterization of Host lncRNAs in Response to Vibrio splendidus Infection and Function as Efficient miRNA Sponges in Sea Cucumber.

Long non-coding RNAs (lncRNAs) have been reported to play critical roles during pathogen infection and innate immune response in mammals. Such observation inspired us to explore the expression profiles and functions of lncRNAs in invertebrates upon bacterial infection. Here, the lncRNAs of sea cucumber (Apostichopus japonicus) involved in Vibrio splendidus infection were characterized. RNA-seq obtained 2897 differentially expressed lncRNAs from Vibrio splendidus infected coelomocytes of sea cucumbers. The potential functions of the significant differentially expressed lncRNAs were related to immunity and metabolic process based on the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Moreover, we identify a lncRNA (XLOC_028509), which is downregulated with Vibrio splendidus challenged, further study indicated that XLOC_028509 adsorb miR-2008 and miR-31 as competing endogenous RNAs (ceRNAs) through base complementarity, which in turn decreased the amount of miRNAs (microRNAs) bound to the 3'UTRs (untranslated regions) of mRNAs to reduce their inhibition of target gene translation. These data demonstrated that the lncRNAs of invertebrates might be important regulators in pathogen-host interactions by sponging miRNAs.

Immune correlates of NF-κB and TNFα promoter DNA methylation in Japanese flounder (Paralichthys olivaceus) muscle and immune parameters change response to vibrio anguillarum infection.

Vibrio anguillarum infection can activate NF-κB/TNFα pathway in the immune organs of fish. Fish muscle is also an important immune organ, but the research on its immune function is few. Our aim was to study regulating mechanism of NF-κB and TNFα gene expressions in the muscle of Japanese flounder (Paralichthys olivaceus) which was under Vibrio anguillarum infection (0, 24, 48, 72 and 96 h). The results showed that the expressions of NF-κB and TNFα increased significantly at 48 h, and there was a significant positive correlation between them. In situ hybridization confirmed the co-existence of NF-κB and TNFα genes in Japanese flounder muscle. Interestingly, the expression of the TNFα gene was regulated by the DNA methylation and its methylation level was negatively correlated with the expression. The lowest methylation level of TNFα occurred at 48 h under Vibrio anguillarum infection (P < 0.05). And more, when the fragment (-2122 âˆ¼ -730) was deleted on TNFα gene promoter, double luciferase activity was the highest, indicating that fragment (-730-0) was the transcription factor binding region. The site (-78 ~ -69) on the fragment (-730-0) binding NF-κB was mutated, and double luciferase activity decreased significantly. The results confirmed that the site (-78 ~ -69) was indeed an important binding site for NF-κB. In addition, the activity of TNFα in the serum of Japanese flounder changed with the prolongation of vibrio anguillarum infection, and the concentration of other immune factors such as ALP, ALT, AST and LDH also changed in the muscle under vibrio anguillarum infection. They all showed a trend of first increasing and then decreasing. Above studies implied that Japanese flounder responded to Vibrio anguillarum infection at the immune level with the change of its methylation status and the activation of transcription factor. By studying the mechanism of immune pathways, understanding the response to immune stress is great significant to the research of fish breeding for disease resistance.

Genome-wide identification and analysis of chemokine receptor superfamily in miiuy croaker, Miichthys miiuy.

The chemokine receptor (ChemR) superfamily, which is divided into 4 subfamilies (CXCR, CCR, XCR, and CX3CR), is the main receptors of chemokines in innate immune responses. In the current study, we have identified 27 ChemRs in miiuy croaker: 13 CCR genes, 11 CXCR genes, and 3 XCR genes. Multiple characteristics of these genes, including phylogeny, gene structures, conserved motifs, chromosome locations, evolutionary mechanism, and expression levels upon the bacterial challenge were analyzed. Gene structure and location analysis showed that all ChemR genes contain fewer introns (≤4) and they are unevenly distributed on the 12 chromosomes. And the XCR subfamily of miiuy croaker don't have the DRY motif of ChemR. Phylogenetic and synteny analysis showed that these genes experienced tandem and segmental duplication event in several species, and tandem duplication might be the main expansion way in miiuy croaker. The major ChemRs of each orthologous group in vertebrates were selected for molecular evolution analysis, the results of which indicated that compared with vertebrates, ChemRs of teleost fishes may have a relatively high evolutionary dynamic. In addition, a total of 21 positively selected codons were detected in vertebrate ChemRs under Model 8. RNA-Seq analysis and qRT-PCR verification demonstrated that CXCR3.2, CXCR5, and XCR1 genes were up-regulated significantly upon the Vibrio harveyi infection. These results provide valuable information for investigating the evolutionary relationships of chemokine receptor superfamily in miiuy croaker and laid the basis for further functional analysis.