Characterization of the ferric uptake regulator VaFur regulon and its role in Vibrio anguillarum pathogenesis.

The Gram-negative marine bacterium Vibrio anguillarum is able to cause vibriosis with hemorrhagic septicemia in many fish species, and iron acquisition is a critical step for virulence. Despite the fact that genes specific to certain processes of iron transport have been studied, the iron-regulated circuits of the V. anguillarum strains remain poorly understood. In this study, we showed that in V. anguillarum strain 775, iron could affect the expression of a number of critical metabolic pathways and virulence factors. The global iron uptake regulator VaFur is the major actor to control these processes for the bacterium to respond to different iron conditions. A VaFur binding motif was identified to distinguish directly and indirectly regulated targets. The absence of VaFur resulted in the aberrant expression of most iron acquisition determinants under rich-iron conditions. A similar regulation pattern was also observed in the transcription of genes coding for the type VI secretion system. The expression of peroxidase genes is positively controlled by VaFur to prevent iron toxicity, and the deletion of Vafur caused impaired growth in the presence of iron and H2O2. VaFur also upregulates some virulence factors under limited-iron conditions, including metalloprotease EmpA and motility, which are likely critical for the high virulence of V. anguillarum 775. The deletion of VaFur led to reduced swimming motility and decreased extracellular protease activity under limited-iron conditions, thereby leading to attenuated pathogenicity. Our study provides more evidence to better understand the VaFur regulon and its role in the pathogenesis of V. anguillarum.IMPORTANCEVibriosis, the most common disease caused by marine bacteria belonging to the genus Vibrio, leads to massive mortality of economical aquatic organisms in Asia. Iron is one of the most important trace elements, and its acquisition is a critical battle occurring between the host and the pathogen. However, excess iron is harmful to cells, so iron utilization needs to be strictly controlled to adapt to different conditions. This process is mediated by the global iron uptake regulator Fur, which acts as a repressor when iron is replete. On the other hand, free iron in the host is limited, so the reduced virulence of the Δfur mutant should not be directly caused by abnormally regulated iron uptake. The significance of this work lies in uncovering the mechanism by which the deletion of Fur causes reduced virulence in Vibrio anguillarum and identifying the critical virulence factors that function under limited-iron conditions.

Integrated microRNA study and pathological analysis provides new insights into the immune response of Ruditapes philippinarum under Vibrio anguillarum challenge.

Manila clam (Ruditapes philippenarum) is an important shellfish aquaculture product. The large-scale breeding of clams is often affected by V. anguillarum and causes large-scale death. However, the pathogenesis, immune response and metabolic pathway of V. anguillarum are still unclear. In this study, we found that the bacterial load in the hepatopancreas of R. philippinarum peaked at 48 h after V. anguillarum infection, and then gradually decreased, while the activity of lysozyme reached the peak at 12 h. Tissue section observation reveals that the infected hepatopancreas cells lost normal structure or necrosis. Additionally, six small RNA libraries were constructed using hepatopancreas of clams. A total of 15 differentially expressed (DE) microRNA (miRNA) were identified at 48 h after V. anguillarum infection, including 8 upregulated and 7 downregulated miRNAs. GO and KEGG enrichment results indicated the prediction of 48 known miRNAs and 127 new miRNAs, with functional annotation suggests that endocytosis pathway and bacterial recognition proteins may play key roles in immune response. The sequencing results were basically consistent with the qRT-PCR validation, indicating the accuracy of the data. This study provides a new idea to explore the immune regulation mechanism of shellfish after V. anguillarum infection, which brings important reference significance for modern immunological research.

Characterization of Myxovirus resistance (Mx) gene from Chinese seabass Lateolabrax maculatus: Insights into the evolution and function of Mx genes.

Chinese seabass (Lateolabrax maculatus) stands out as one of the most sought-after and economically significant species in aquaculture within China. Diseases of L. maculatus occur frequently due to the degradation of the germplasm, the aggravation of environmental pollution of water, and the reproduction of pathogenic microorganisms, inflicting considerable economic losses on the Chinese seabass industry. The Myxovirus resistance (Mx) gene plays pivotal roles in the antiviral immune response ranging from mammals to fish. However, the function of the Mx gene in L. maculatus is still unknown. Firstly, the origin and evolutionary history of Mx proteins was elucidated in this study. Subsequently, an Mx gene from L. maculatus (designed as LmMxA gene) was identified, and its functions in combating antiviral and antibacterial threats were investigated. Remarkably, our findings suggested that while Mx group genes were present in chordates, DYN group genes were present in everything from single-celled animals to humans. Furthermore, our investigation revealed that the LmMxA mRNA level increased in the kidney, spleen and liver subsequent to Vibrio anguillarum and poly(I:C) challenged. Immunofluorescence analysis indicated that LmMxA is predominantly localization in the nucleus and the cytoplasm. Notably, the expression of MAVS, IFN1 and Mx1 increased when LmMxA was overexpression within the EPC cells. Moreover, through assessment via cytopathic effect (CPE), virus titer, and antibacterial activity, it becomes evident that LmMxA exerts a dual role in bolstering both antiviral and antibacterial immune responses. These compelling findings laid the foundation for further exploring the mechanism of LmMxA in response to innate immunity of L. maculatus.

Manipulated C5aR1 over/down-expression associates with IL-6 expression during bacterial inflammation in half-smooth tongue sole (Cynoglossus semilaevis).

The complement component 5a/complement component 5 receptor 1 (C5a/C5aR1) pathway plays a crucial role in the onset and development of inflammation, but relevant studies in fish are lacking. In this study, we successfully characterized the relationship between half-smooth tongue sole (Cynoglossus semilaevis) C5aR1 (CsC5aR1) and bacterial inflammation. First, we showed that the overexpression of CsC5aR1 significantly increased bacterial pathological damage in the liver and intestine, whereas inhibition attenuated the damage. The in vitro experiments suggested that CsC5aR1 was able to positively regulate the phagocytic activity and respiratory burst of tongue sole macrophages. In terms of both transcriptional and translational levels, overexpression/inhibition of CsC5aR1 was followed by a highly consistent up-regulation/decrease of its downstream canonical inflammatory factor interleukin-6 (CsIL-6). Furthermore, we stimulated macrophages by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and found a broad-spectrum response to bacterial infections by the C5a/C5aR1 complement pathway together with the downstream inflammatory factor CsIL-6. Subsequently, we directly elucidated that CsIL-6 is an indicator of C5a/C5aR1-mediated inflammation at different infection concentrations, different infectious bacteria (Vibrio anguillarum and Mycobacterium marinum), and different detection levels. These results might provide a new inflammation bio-marker for early warning of bacteria-induced hyperinflammation leading to fish mortality and a promising target for the treatment of bacterial inflammation in teleost.

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.

Valp1, a Newly Identified Temperate Phage Facilitating Coexistence of Lysogenic and Non-Lysogenic Populations of Vibrio anguillarum.

Vibrio anguillarum is a pathogen for several fish and shellfish species. Its ecology is influenced by diverse factors, including bacteriophages. Here, we identify and characterize a new temperate bacteriophage (Valp1) of V. anguillarum. Valp1 is a myovirus with a 60 nm head and a 90 nm contractile tail. Its double-stranded DNA genome of 42,988 bp contains 68 genes, including a protelomerase gene, typical of telomeric phages. Valp1 inhibits the growth of the virulent strain of V. anguillarum PF4, while the derived lysogenic strain P1.1 presents a slight reduction in its growth but is not affected by the presence of Valp1. Both strains present similar virulence in a larval zebrafish (Danio rerio) model, and only slight differences have been observed in their biochemical profile. Co-culture assays reveal that PF4 and P1.1 can coexist for 10 h in the presence of naturally induced Valp1, with the proportion of PF4 ranging between 28% and 1.6%. By the end of the assay, the phage reached a concentration of ~108 PFU/mL, and all the non-lysogenic PF4 strains were resistant to Valp1. This equilibrium was maintained even after five successive subcultures, suggesting the existence of a coexistence mechanism between the lysogenic and non-lysogenic populations of V. anguillarum in conjunction with the phage Valp1.

Identification, characterization and complete genome analysis of a Vibrio anguillarum isolated from Sebastes schlegelii.

Vibrio anguillarum is an important fish pathogen in mariculture, which can infect fish with great economic losses. In this study, a Vibrio anguillarum isolated from Sebastes schlegelii was named VA1 and was identified and characterized from aspects of morphology, physiological and biochemical characteristics, 16SRNA, virulence genes, drug sensitivity, and extracellular enzyme activity. At the same time, The VA1 was investigated at the genomic level. The results showed that a Gram-negative was isolated from the diseased fish. The VA1 was characterized with uneven surface and visible flagella wrapped in a sheath and microbubble structures. The VA1 was identified as Vibrio anguillarum based on the 16S RNA sequence and physiological and biochemical characteristics. The VA1 carried most of the virulence genes (24/29) and was resistant to penicillin, oxacillin, ampicillin, cefradine, neomycin, pipemidic acid, ofloxacin, and norfloxacin. The pathogenicity of the isolated strain was confirmed by an experimental analysis, and its LD50 was 6.43 × 106 CFU/ml. The VA1 had the ability to secrete gelatinase, protease, and amylase, and it had α-hemolysis. The whole genome size of the VA1 was 4232328bp and the G + C content was 44.95 %, consisting of two circular chromosomes, Chromosome1 and Chromosome2, with no plasmid. There were 1006 predicted protein coding sequences (CDSs). A total of 526 genes were predicted as virulence-related genes which could be classified as type IV pili, flagella, hemolysin, siderophore, and type VI secretion system. Virulence genes and correlation data were supported with the histopathological examination of the affected organs and tissues. 194 genes were predicted as antibiotic resistance genes, including fluoroquinolone antibiotic, aminoglycoside antibiotic, and beta-lactam resistant genes, which agreed with the results of the above drug sensitivity, indicating VA1 to be a multidrug-resistant bacterium. This study provided a theoretical basis for a better understanding of pathogenicity and antibiotic resistance, which might contribute to the prevention of V. anguillarum in the future.

Deletion of speA and aroC genes impacts the pathogenicity of Vibrio anguillarum in spotted sea bass.

Vibrio anguillarum is one of the major pathogens responsible for bacterial infections in marine environments, causing significant impacts on the aquaculture industry. The misuse of antibiotics leads to bacteria developing multiple drug resistances, which is detrimental to the development of the fisheries industry. In contrast, live attenuated vaccines are gradually gaining acceptance and widespread recognition. In this study, we constructed a double-knockout attenuated strain, V. anguillarum ΔspeA-aroC, to assess its potential for preparing a live attenuated vaccine. The research results indicate a significant downregulation of virulence-related genes, including Type VI secretion system, Type II secretion system, biofilm synthesis, iron uptake system, and other related genes, in the mutant strain. Furthermore, the strain lacking the genes exhibited a 67.47% reduction in biofilm formation ability and increased sensitivity to antibiotics. The mutant strain exhibited significantly reduced capability in evading host immune system defenses and causing in vivo infections in spotted sea bass (Lateolabrax maculatus), with an LD50 that was 13.93 times higher than that of the wild-type V. anguillarum. Additionally, RT-qPCR analysis of immune-related gene expression in spotted sea bass head kidney and spleen showed a weakened immune response triggered by the knockout strain. Compared to the wild-type V. anguillarum, the mutant strain caused reduced levels of tissue damage. The results demonstrate that the deletion of speA and aroC significantly reduces the biosynthesis of biofilms in V. anguillarum, leading to a decrease in its pathogenicity. This suggests a crucial role of biofilms in the survival and invasive capabilities of V. anguillarum.

Unravelling the main immune repertoire of Paracentrotus lividus following Vibrio anguillarum bath challenge.

Paracentrotus lividus is the most abundant echinoid species in the North East Atlantic Ocean and Mediterranean Sea. Although there is abundant genomic information of the species, there is no deep characterisation of the genes involved in the immune response. Here, a reference transcriptome of male and female coelomocytes was produced. The generated P. lividus transcriptome assembly has 203,511 transcripts, N50 transcript length of 1079 bp, and more than 90% estimated gene completeness in Eukaryota and Metazoa BUSCO databases, respectively. Differential gene expression analyses showed 54 and 55 up-regulated genes in P. lividus female and male coelomocyte tissues, respectively. These results suggest a similar immune gene repertoire between sexes. To examine the immune response, P. lividus was challenged with Vibrio anguillarum, one of the candidate pathogens for bald disease. Immune parameters were evaluated at cell and humoral levels, as well as the expression analysis of immune related genes at an early response stage. No differences were found at cellular and humoral levels with the exception of the increase of nitric oxide in perivisceral fluid of challenged animals. At the gene expression level, a total of 2721 genes were upregulated in challenged animals, 13.6 times higher expression than control group. Our analysis revealed that four major KEGG pathways were enriched in challenged animals: Autophagy (KEGG:04140), Endocytosis (KEGG:04144), Phagosome (KEGG:04145) and Protein processing in endoplasmic reticulum (KEGG:04141). Several toll-like receptors (TLR), scavenger receptors cysteine-rich (SRCR) or nucleotide-binding oligomerisation domain like receptors (NLR) were identified as major family genes for pathogen recognition and immune defence. This study provides a valuable transcriptomic resource and unfolds the molecular basis of immune response to V. anguillarum exposure. Overall, our findings contribute to the conservation effort of the P. lividus populations, as well as its sustainable exploitation in an aquaculture context.

Remodulation of bacterial transcriptome after acquisition of foreign DNA: the case of irp-HPI high-pathogenicity island in Vibrio anguillarum.

The high-pathogenicity island irp-HPI is widespread in Vibrionaceae and encodes the siderophore piscibactin, as well as the regulator PbtA that is essential for its expression. In this work, we aim to study whether PbtA directly interacts with irp-HPI promoters. Furthermore, we hypothesize that PbtA, and thereby the acquisition of irp-HPI island, may also influence the expression of other genes elsewhere in the bacterial genome. To address this question, an RNAseq analysis was conducted to identify differentially expressed genes after pbtA deletion in Vibrio anguillarum RV22 genetic background. The results showed that PbtA not only modulates the irp-HPI genes but also modulates the expression of a plethora of V. anguillarum core genome genes, inducing nitrate, arginine, and sulfate metabolism, T6SS1, and quorum sensing, while repressing lipopolysaccharide (LPS) production, MARTX toxin, and major porins such as OmpV and ChiP. The direct binding of the C-terminal domain of PbtA to piscibactin promoters (PfrpA and PfrpC), quorum sensing (vanT), LPS transporter wza, and T6SS structure- and effector-encoding genes was demonstrated by electrophoretic mobility shift assay (EMSA). The results provide valuable insights into the regulatory mechanisms underlying the expression of irp-HPI island and its impact on Vibrios transcriptome, with implications in pathogenesis.IMPORTANCEHorizontal gene transfer enables bacteria to acquire traits, such as virulence factors, thereby increasing the risk of the emergence of new pathogens. irp-HPI genomic island has a broad dissemination in Vibrionaceae and is present in numerous potentially pathogenic marine bacteria, some of which can infect humans. Previous works showed that certain V. anguillarum strains exhibit an expanded host range plasticity and heightened virulence, a phenomenon linked to the acquisition of the irp-HPI genomic island. The present work shows that this adaptive capability is likely achieved through comprehensive changes in the transcriptome of the bacteria and that these changes are mediated by the master regulator PbtA encoded within the irp-HPI element. Our results shed light on the broad implications of horizontal gene transfer in bacterial evolution, showing that the acquired DNA can directly mediate changes in the expression of the core genome, with profounds implications in pathogenesis.

Effects of miR-722 on gene expression and alternative splicing in the liver of half-smooth tongue sole after infection with Vibrio anguillarum.

MicroRNAs play crucial roles in various biological processes, including but not limited to differentiation, development, disease, and immunity. However, their immunoregulatory roles in half-smooth tongue sole are lacking. Our previous studies indicated that miR-722 could target C5aR1 to modulate the complement pathway to alleviate inflammatory response and even affect the mortality after the bacterial infection with Vibrio anguillarum. Driven by the purpose of revealing the underlying mechanisms, in this study, we investigated the effects of miR-722 on the gene expression and alternative splicing (AS) in the liver of half-smooth tongue sole after Vibrio anguillarum infection, with the approach of miR-722 overexpression/silencing and subsequent RNA-seq. Among the different comparisons, the I group (miR-722 inhibitor and V. anguillarum) versus blank control (PBS) exhibited the highest number of differentially expressed genes (DEGs), suggesting that the immune response was overactivated after inhibiting the miR-722. In addition, enrichment analyses were performed to reveal the functions of DEGs and differential AS (DAS) genes, reflecting the enrichment of RNA splicing and immune-related pathways including NF-κB and T cell receptor signaling pathway. Comparing the M group (miR-722 mimic and V. anguillarum) with the negative control (random sequence and V. anguillarum), two immune-related genes, cd48 and mapk8, were differentially expressed, of which mapk8 was also differentially spliced, indicating their importance in the immune response. Furthermore, representative gene analysis was performed, suggesting their corresponding functional changes due to AS. To verify the RNA-seq data, quantitative real-time PCR was employed with twenty pairs of primers for DEGs and DAS events. Overall, our results demonstrated that miR-722 could mediate the transcriptome-wide changes of gene expression and AS in half-smooth tongue sole, and provided insights into the regulatory role of miR-722 in immune responses, laying the foundation for further functional analyses and practical applications in aquaculture.

Noctiluca scintillans bloom alters the composition and carbohydrate utilization of associated bacterial community and enriches potential pathogenic bacterium Vibrio anguillarum.

Noctiluca scintillans (red) is a widely distributed heterotrophic dinoflagellate and a prominent red tide forming species. This study investigated the effects of Noctiluca blooms on marine microbial diversity and functionality using multi-omics approaches. Our findings revealed significant differences in the community composition of Noctiluca-associated bacteria compared to those associated with autotrophic plankton and free-living bacteria in the surrounding seawater. The dominant bacterial groups within the Noctiluca-associated community shifted at various bloom stages, which could be attributed to changes in prey composition of Noctiluca. During the non-bloom stage, Burkholderiaceae, Carnobacteriaceae, and Pseudomonadaceae dominated the community, while Vibrionaceae became dominant during the bloom stage, and Saprospiraceae, Crocinitomicaceae, and Pirellulaceae thrived during the post-bloom stage. Compared to the non-bloom stage, Noctiluca-associated bacterial community at the bloom stage exhibited significant down-regulation of genes related to complex carbohydrate metabolism, while up-regulation of genes related to glucose transportation and utilization. Furthermore, we identified Vibrio anguillarum, a potential pathogenic bacterium to marine fish, as a major component of the Vibrionaceae family during the bloom stage. The occurrence of V. anguillarum associated with Noctiluca blooms may be attributed to the increased availability of its preferred carbon sources and its high capabilities in glucose transportation, motility and chemotaxis. Moreover, the presence of Vibrio infection genes (hap, hlyA, rtxA) encoding vibriolysin, hemolysin, and RTX (Repeats-in-toxin) toxin in the V. anguillarum genome, with the hap gene showing high expression levels during Noctiluca blooms, indicates an elevated risk of infection. This study underscores the unique composition of the bacterial community associated with red tide forming heterotrophic dinoflagellates and suggests that Noctiluca cells may serve as reservoirs and vectors for pathogenic bacteria, potentially posing a threat to fish-farming and the health of other marine organisms.

Evolutionary, comparative, and functional analyses of STATs and regulation of the JAK-STAT pathway in lumpfish upon bacterial and poly(I:C) exposure.

Background: The Janus kinase/signal transducers and activators of transcription (JAK-STAT) system regulates several biological processes by affecting transcription of genes as a response to cytokines and growth factors. In the present study, we have characterized the STAT genes in lumpfish (Cyclopterus lumpus L.), belonging to the order Perciformes, and investigated regulation of the JAK-STAT signaling pathway upon exposure to bacteria (Vibrio anguillarum) and poly(I:C), the latter mimicking antiviral responses. Methods: Characterization and evolutionary analyses of the STATs were performed by phylogeny, protein domain, homology similarity and synteny analyses. Antibacterial and antiviral responses were investigated by performing KEGG pathway analysis. Results: We observed that lumpfish have stat1a, 2, 3, 4, 5a, 5b, and 6. Transcriptome-wide analyses showed that most components of the JAK-STAT pathway were present in lumpfish. il-6, il-10, il-21, iκBα and stat3 were upregulated 6 hours post exposure (hpe) against bacteria while type I interferons (IFNs), irf1, irf3, irf10, stat1 and 2 were upregulated 24 hpe against poly(I:C). Conclusions: Our findings shed light on the diversity and evolution of the STATs and the data show that the STAT genes are highly conserved among fish, including lumpfish. The transcriptome-wide analyses lay the groundwork for future research into the functional significance of these genes in regulating critical biological processes and make an important basis for development of prophylactic measure such as vaccination, which is highly needed for lumpfish since it is vulnerable for both bacterial and viral diseases.

Inflammatory reaction and immune response of half-smooth tongue sole (Cynoglossus semilaevis) after infection with Vibrio anguillarum.

Frequently occurred bacterial diseases have seriously affected the aquaculture industry of half-smooth tongue sole (Cynoglossus semilaevis). Notably, vibriosis, with Vibrio anguillarum as one of the causative pathogens, is the most severe bacterial disease with severe inflammatory response of the host, leading to high mortality rates. In the present study, we explored the relationship between bacterial concentrations and host mortality, inflammatory reaction, and immune response in half-smooth tongue sole after infection with V. anguillarum at different concentrations (Treatment 1, 6.4 × 105 CFU/mL; Treatment 2, 6.4 × 106 CFU/mL). The mortality of Treatment 2 (77.5%) was significantly higher than that of Treatment 1 (10%), corresponding with bacterial concentrations. Although the number of deaths varies, intensive deaths were observed within 24 h post infection (hpi) in both bacterial concentration groups. Histopathological analyses revealed that fish tissues were most severely damaged at 24 or 48 hpi, and Treatment 2 was more severe than Treatment 1. A qRT-PCR-based detection method with virulence factor gene empA was established to quantify the bacterial loads in various tissues, and the bacterial loads were the highest at 24 hpi in Treatment 2, and at 48 hpi in Treatment 1. Additionally, the expression levels of complement genes (C5a, C3, C5, and C6), inflammatory factors (IL-1ß, TNF-α, and IL-10), and other immune-related genes (jak2, NF-κB1, stat3, and tlr3) were increased in various tissues after infection in both treatment groups, with most genes being most expressed at 24 or 48 hpi, and expression levels of inflammatory factors in Treatment 2 were higher than those in Treatment 1. Moreover, the expression of C5a was positively correlated with that of proinflammatory cytokines in both bacterial concentration groups. According to the results of this study, 24-48 hpi was a key node for early vibriosis detection and intervention. Compared with the low mortality of Treatment 1, the mass death of fish in Treatment 2 was suggested to be caused by uncontrolled excessive inflammatory reaction induced by the overactivation of complement system, especially C5a. We believe these results could provide theoretical basis for prevention, evaluation, and treatment of vibrio disease in tongue sole aquaculture, and lay a solid foundation for future functional analyses.

Regulatory mechanism of miR-722 on C5aR1 and its functions against bacterial inflammation in half-smooth tongue sole (Cynoglossus semilaevis).

MicroRNAs (miRNAs) are small non-coding RNAs involved in various biological processes, including immunity. Previously, we investigated the miRNAs of half-smooth tongue sole (Cynoglossus semilaevis) and found that miR-722 (designated Cse-miR-722) was significantly differentially expressed after infection with Vibrio anguillarum, reflecting its importance in immune response. Our preliminary bioinformatic analysis suggested that Cse-miR-722 could target C5aR1 (designated CsC5aR1), which was known to play crucial roles in complement activation and inflammatory response, as a receptor of C5a. However, the underlying mechanisms of their interactions and specific functions in inflammatory and immune response are still enigmas. In this study, we successfully cloned the precursor sequence of Cse-miR-722 (94 bp) and the full length of CsC5aR1 (1541 bp, protein molecular weight 39 kDa). The target gene of Cse-miR-722 was verified as CsC5aR1 by a dual luciferase reporter assay, and Cse-miR-722 was confirmed to regulate CsC5aR1 at the protein level using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The expression of CsC5aR1 and Cse-miR-722 in liver cells and four immune tissues of half-smooth tongue sole changed significantly after LPS stimulation and infection with V. anguillarum. To explore the functional role of Cse-miR-722 in half-smooth tongue sole, we performed both in vitro and in vivo experiments. Cse-miR-722 was observed to affect phagocytosis and respiratory burst activity of macrophages by regulating CsC5aR1 in half-smooth tongue sole. Furthermore, we found that Cse-miR-722 regulated the expression of CsC5aR1, CsC5a, and the inflammatory factors CsIL1-ß, CsIL6, CsIL8, and CsTNF-α both in vitro and in vivo. In addition, Cse-miR-722 reduced mortality and pathological damage. This study clarified the regulatory mechanism of Cse-miR-722 on CsC5aR1 and provided insight into the regulatory roles of Cse-miR-722 in immune responses, laying a theoretical foundation for the feasibility of using miR-722 to prevent and control bacterial diseases in teleost.

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.

Genome-wide identification, evolution and expression analysis of tight junction gene family and the immune roles of claudin5 gene in turbot (Scophthalmus maximus L.).

Tight junction proteins (TJs) are important component proteins that maintaining the structure and function of TJs, connecting to each other to form a TJ complex between cells, maintaining the biological homeostasis of the internal environment. In this study, a total of 103 TJ genes were identified in turbot according to our whole-transcriptome database. Transmembrane TJs were divided into seven subfamilies, including claudin (CLDN), occludin (OCLD), tricellulin (MARVELD2), MARVEL domain containing 3 (MARVELD3), junctional adhesion molecules (JAM), immunoglobulin superfamily member 5 (IGSF5/JAM4), blood vessel epicardial substance (BVEs). Moreover, the majority of homologous pairs of TJ genes showed highly conserved alongside length, exon/intron number and motifs. As for phylogenetic analysis for 103 TJ genes, eight of them have undergone a positive selection and JAMB-like has undergone the most neutral evolution. The expression patterns of several TJ genes showed the lowest expression levels in blood, while the highest expression levels were detected in intestine, gill and skin, which all belong to mucosal tissues. Meanwhile, most examined TJ genes showed down-regulated expression patterns during bacterial infection, while several TJ genes exhibited up-regulated expression patterns at a later stage (24 h). At the same time, several potential candidate genes (such as CLDN-15, CLDN-3, CLDN-12, CLDN-5 and OCLD) were significantly down-regulated, which may indicate their important functions that involved in the regulation of bacterial infection. Currently, there is little research on CLDN5 in the intestine, but it is highly expressed in the intestine and has significant changes in intestinal expression after bacterial infection. Thus, we knocked down CLDN5 by the method of lentiviral infection. The result showed CLDN5 was related to cell migration (wound healing) and apoptosis, and the method of dualluciferasereporterassay showed that the functions of CLDN5 could be regulated by miR-24. The study of TJs may lead to a better understanding of the function of TJs in teleost.

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.

Screening of Suitable Reference Genes for Immune Gene Expression Analysis Stimulated by Vibrio anguillarum and Copper Ions in Chinese Mitten Crab (Eriocheir sinensis).

The reference gene expression is not always stable under different experimental conditions, and screening of suitable reference genes is a prerequisite in quantitative real-time polymerase chain reaction (qRT-PCR). In this study, we investigated gene selection, and the most stable reference gene for the Chinese mitten crab (Eriocheir sinensis) was screened under the stimulation of Vibrio anguillarum and copper ions, respectively. Ten candidate reference genes were selected, including arginine kinase (AK), ubiquitin-conjugating enzyme E2b (UBE), glutathione S-transferase (GST), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF-1α), α-tubulin (α-TUB), heat shock protein 90 (HSP90), ß-actin (ß-ACTIN), elongation factor 2 (EF-2) and phosphoglucomutase 2 (PGM2). Expression levels of these reference genes were detected under the stimulation of V. anguillarum at different times (0 h, 6 h, 12 h, 24 h, 48 h and 72 h) and copper ions in different concentrations (11.08 mg/L, 2.77 mg/L, 0.69 mg/L and 0.17 mg/L). Four types of analytical software, namely geNorm, BestKeeper, NormFinder and Ref-Finder, were applied to evaluate the reference gene stability. The results showed that the stability of the 10 candidate reference genes was in the following order: AK > EF-1α > α-TUB > GAPDH > UBE > ß-ACTIN > EF-2 > PGM2 > GST > HSP90 under V. anguillarum stimulation. It was GAPDH > ß-ACTIN > α-TUB > PGM2 > EF-1α > EF-2 > AK > GST > UBE > HSP90 under copper ion stimulation. The expression of E. sinensis Peroxiredoxin4 (EsPrx4) was detected when the most stable and least stable internal reference genes were selected, respectively. The results showed that reference genes with different stability had great influence on the accurate results of the target gene expression. In the Chinese mitten crab (E. sinensis), AK and EF-1α were the most suitable reference genes under the stimulation of V. anguillarum. Under the stimulation of copper ions, GAPDH and ß-ACTIN were the most suitable reference genes. This study provided important information for further research on immune genes in V. anguillarum or copper ion stimulation.

Comparative proteomics study of exosomes in Vibrio harveyi and Vibrio anguillarum.

Exosomes are a class of extracellular vesicles released by bacteria and contain diverse biomolecules. In this study, we isolated exosomes from Vibrio harveyi and Vibrio anguillarum, which are both serious pathogens in mariculture, using a supercentrifugation method, and the proteins in the exosomes of these two vibrios were analyzed by LC-MS/MS proteomics. Exosome proteins released by V. harveyi and V. anguillarum were different; they not only contained virulence factors (such as lipase and phospholipase in V. harveyi, metalloprotease and hemolysin in V. anguillarum), but also participated in the important life activities of bacteria (such as fatty acid biosynthesis, biosynthesis of antibiotics, carbon metabolism). Subsequently, to verify whether the exosomes participated in bacterial toxicity, after Ruditapes philippinarum was challenged with V. harveyi and V. anguillarum, the corresponding genes of virulence factors from exosomes screened by proteomics were tested by quantitative real-time PCR. All the genes detected were upregulated which suggested that exosomes were involved in vibrio toxicity. The results could provide an effective proteome database for decoding the pathogenic mechanism of vibrios from the exosome perspective.