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.

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.

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.

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.

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.

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.

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.

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.

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.

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.
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.
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).
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.