Nuclear factor-κB (NF-κB)/p65, a vital signaling molecule in the NF-κB pathway, participates in diverse physiological functions and host-virus interactions. However, the involvement of NF-κB/p65 in fish virus infection remains poorly understood. In this study, we explored the role of the p65 in virus infection and its impact on IL-18 regulation in largemouth bass (Micropterus salmoides). Bioinformatics analysis showed that the ORF sequence of Msp65 spanned 1941 bp, encoding 646 amino acids with two conserved functional domains, including RHD and IPT domain. Msp65 mRNA was presented in various tissues, with higher levels detected in the liver and gill. After exposure to largemouth bass virus (LMBV), red grouper nervous necrosis virus, lipopolysaccharide and poly (I:C), Msp65 expression was activated in vivo. In addition, the antiviral role of Msp65 were explored. In vitro, Msp65 overexpression hindered LMBV replication and formation of viral assembly site. In vivo, we found that disruption of Msp65 by using maslinic acid (MA) notably promoted the infectivity of LMBV, indicating its antiviral capabilities in largemouth bass. Besides, the downregulation of Msp65 suppressed the expression of inflammatory and interferon signaling molecules. Conversely, Msp65 overexpression boosted the activities of IFN-I, IFN-III and ISRE promoters, suggesting the positive regulation of Msp65 on interferon immune pathway. Furthermore, to unveil the regulatory role of Msp65 on MsIL-18, a promoter investigation was conducted. The luciferase reporter assay demonstrated that Msp65 positively influenced the expression of MsIL-18. Subsequent analysis suggested that the putative binding sites for MsIL-18 could potentially reside within the -228 to -203 bp of the MsIL-18 promoter. These findings illustrated that Msp65 involved in LMBV infection by modulating immune responses, presenting a novel insight into the antiviral mechanisms of p65 in bony fish.

The large-scale mining and utilization of rare earth elements have significantly increased their concentration in the environment, especially in regions surrounding mining areas. These environmentally-enriched rare earth elements accumulate in agricultural products and organisms through soil and water, potentially impacting in human health through the food chain. Erbium (Er), a rare earth element of the lanthanide series (Group IIIB), plays a crucial role in various modern technological applications. It is primarily utilized in ceramics, glass coloring, optical fibers, laser technology, and the nuclear industry, among others. However, a paucity of information on the health effects and ecotoxicity of erbium is currently available. In this study, we used the zebrafish as experimental animal to investigate the potential impact of the rare earth element erbium on the immune system. We exposed fertilized zebrafish embryos to different concentrations of erbium (0, 4, 8 and 16 mg/L) from 6 hours post-fertilization (hpf) until 72 hpf. We found that with increasing concentrations of erbium exposure, there was an increasing and dispersing trend in the number of zebrafish neutrophils; a decreasing trend in the number of macrophages. Exposure to erbium was demonstrated to impair the phagocytic capability of macrophages, reduce the recruitment of neutrophils to the wound site, and lower the resistance of zebrafish to Escherichia coli infection. Erbium exposure led to macrophage apoptosis and upregulation of oxidative stress in the zebrafish. The individual application of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine, the IKBKB inhibitor resveratrol and the NF-κB inhibitor andrographolide were demonstrated to alleviate erbium-induced immune toxicity, as confirmed by assays including acridine orange staining, neutrophils enumeration and recruitment, and real-time quantitative PCR. Therefore, the rare earth element erbium induced immune toxicity in zebrafish through the ROS/NF-κB pathway. The findings of this study provide information for assessing the impact of rare earth elements on human health and ecosystems.

The Mediterranean mussel (Mytilus galloprovincialis) is a key species in European aquaculture, known for its economic and societal importance, particularly as a primary source of income for local fisheries in European coastal areas. While historically resilient to the mass mortality events that have affected other bivalve species, M. galloprovincialis may face increasing threats from emerging pathogens, including bacteria, viruses, and eukaryotic parasites. These microorganisms, often opportunistic, pose heightened risks in the current climate change scenario, where heatwaves are becoming increasingly frequent and the persistent presence of pollutants is suspected to impair the functional response of hemocytes. Over the past decade, significant advancements in immunological research have provided deeper insights into the cellular and molecular mechanisms underlying the robust defense system of M. galloprovincialis, which allows this species to efficiently cope with a broad range of infections. By analyzing the scientific literature published on mussel immunology over the past ten years, this review consolidates current knowledge on the immune system of the Mediterranean mussel. We place a particular focus on the cellular and molecular components involved in the recognition and elimination of microbial pathogens and discuss how the most recent discoveries may inform improved management and disease mitigation strategies for Mediterranean mussel farming in the in the years to come.

Mycobacterium marinum is a major pathogen in aquaculture, posing a substantial threat to the health and sustainability of tongue sole (Cynoglossus semilaevis) farming. This study investigated the genetic basis of immune response in tongue sole by comparing transcriptome profiles of liver and spleen tissues from symptomatic (susceptible) and healthy (resistant) individuals during a natural M. marinum outbreak. Transcriptomic analyses identified differentially expressed genes and enriched pathways related to immune responses. Key genes, including atp6ap1, gpi, and idh3a, were found to be crucial in immune response to M. marinum infection, involved in immune processes such as signal transduction, antigen processing, and metabolic pathways. Protein-protein interaction networks highlighted central hub genes such as nedd8, jun and junb, which play pivotal roles in immune regulation. These findings provide insights into the orchestrated immune responses to mycobacteriosis, which can inform selective breeding strategies for disease-resistant tongue sole strains. This is the first comprehensive transcriptome analysis of M. marinum natural infection in tongue sole, offering valuable data for future research and disease management in aquaculture.

Lipopolysaccharide (LPS) destroys intestinal mechanical barrier and causes apoptosis by triggering oxidative stress and inflammatory responses. Glutamine (Gln) can maintain normal intestinal function under various stressed or pathological conditions. Thereby, this study aims to evaluate the protection of glutamine on intestinal health of snakehead (Channa argus), specifically regarding the NF-κB/MLCK/MLC2 signaling pathway mediated by tight junction affecting oxidative stress, inflammation and apoptosis. In this work, a model of intestinal tight junction injury in intestine of snakehead was constructed by injecting 4 mg/mL LPS into anus for 96 h. Before constructing the model, fish were treated with different levels of alanyl-glutamine (Ala-Gln) (0 %, 0.3 %, 0.6 %, 0.9 %, 1.2 % and 1.5 %) for 56 days. Microstructure and ultra microstructure showed that LPS-induced obvious intestinal damage and tight connection destruction, while Gln effectively alleviated these phenomena. In addition, results also showed that Gln can effectively inhibit LPS-induced damage to intestinal tight junction (zo-1, occludin, claudin5, claudin1, nf-κb p65, mlck and mlc2), alleviate oxidative stress (nrf2, sod, gsh, gpx and cat), ameliorate intestinal inflammation (tnf-α, il-1β, il-8, tlr5 and tlr2), thereby reduce apoptosis (p38mapk, caspase9, caspase8, caspase3 and bax). Crucially, the above results were related to NF-κB/MLCK/MLC2 signaling pathway mediated by tight junction. In conclusion, Gln has a good protective effect on LPS-induced intestinal injury in northern snakehead, providing a new perspective for regulating fish intestinal health.

MicroRNAs (miRNAs) are highly conserved endogenous non-coding RNAs that play a crucial role in fish immune response by regulating gene expression at the post-transcriptional level. In recent years, the viral diseases caused by infectious hematopoietic necrosis virus (IHNV) have caused significant economic losses in rainbow trout (Oncorhynchus mykiss) aquaculture, whereas the immune regulatory mechanisms of miRNAs involved in rainbow trout resistance to IHNV infection remains largely undefined. In this study, we analyzed the structural characteristics of Oncorhynchus mykiss tumor necrosis factor receptor-associated factor 3 (OmTRAF3) by bioinformatics software and explored the molecular mechanism of miR-203-3p in rainbow trout resistance to IHNV by regulating OmTRAF3 in vivo and in vitro. The open reading frame (ORF) of OmTRAF3 gene was 1 731 bp and encoded 576 amino acids including an N-terminal RING finger domain, two zinc finger domains, a coiled-coil domain, and a C-terminal MATH domain. The expression pattern analysis showed that the expression of miR-203-3p and OmTRAF3 in immune-related tissues (head kidney, spleen, and liver) and liver cells of rainbow trout infected with IHNV varied with some regularity and had opposite trends at key time points, and a targeting relationship between miR-203-3p and OmTRAF3 was confirmed using a dual luciferase reporter gene assay. Further, we found that in vivo and in vitro overexpression of miR-203-3p significantly reduced the expression of OmTRAF3, downstream immune-related genes (OmTANK, OmIKKε, OmIFN1, and OmISG15) and promoted IHNV copy number replication, while silencing of miR-203-3p yielded opposite results. More importantly, OmTRAF3 and downstream genes as well as IHNV copy number changed accordingly with the silencing of OmTRAF3. The above results revealed that miR-203-3p participates in the immune response against IHNV by targeting OmTRAF3, and provide a theoretical basis for the screening of antiviral drugs in rainbow trout.

Sexual dimorphism is well-documented in aquaculture, particularly regarding growth differences, wherein one sex often grows faster than the other. However, despite the phenomenon being so widely documented, its underlying molecular mechanisms remain poorly understood. As an important digestive and immune organ, the gut plays key roles in the regulation of fish growth. In this study, we conducted RNA-seq and metabolomic analysis on the gut of female and male common carp. We discovered that growth-related pathways, such as "Glycolysis/Gluconeogenesis" and "Riboflavin metabolism" are significantly enriched in the gut of female carp. Conversely, pathways linked to disease resistance, such as "Th17 cell differentiation" and "Autophagy-animal" are predominantly enriched in male carp. Following intraperitoneal injection of spring viraemia of carp virus (SVCV) into both male and female carp, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis and histopathological staining confirmed that male carp exhibit greater disease resistance compared to females. This study identified the disease resistance dimorphism in common carp and specific mechanisms underlying growth differences. Our findings offer valuable insights for the application of growth dimorphism and disease-resistant breeding in fish.

Single-cycle viruses hold great promise as fish viral vaccines due to their high protective efficacy. Although the efficacy of the vaccine in olive flounder and rainbow trout has been proven through previous research, safety must be additionally proven considering the environment of use for commercialization. This study comprehensively assesses the safety of rVHSV-GΔTM and its impact on both the host and the surrounding environment, including the coastal habitat of nearby species and seawater. We constructed an experimental tank geographically mimicking coastal conditions and evaluated its effects on co-reared olive flounder and other organisms. Results showed no detectable viral presence in co-reared organisms or concentrated culturing seawater, and the virus demonstrated limited proliferation under varying salinity conditions. The survival rate of olive flounder immunized with rVHSV-GΔTM was significantly higher (up to 90% protection against wild-type VHSV) compared to non-immunized fish. Additionally, we developed a prototype vaccine of rVHSV-GΔTM through freeze-drying. Immunization of olive flounder with the prototype vaccine conferred significantly higher protection against wild-type VHSV. These findings underscore the safety of rVHSV-GΔTM as a commercial vaccine, unaffected by the host or surrounding environment, and highlight the efficacy of freeze-drying in delivering single-cycle viruses.

Finding effective alternatives to antibiotics is crucial for sustainable aquaculture. Host-derived probiotics have great potential as a promising alternative to antibiotics for immune regulation and disease control in fish farming. However, limited research exists regarding the application of native probiotics in largemouth bass (Micropterus salmoides). This study aims to evaluate the potential of the endogenous strain Pediococcus acidilactici B49 as a probiotic in modulating host immunity and disease control through in vitro and in vivo experiments. The results demonstrated that P. acidilactici B49 exhibited no hemolytic activity and displayed susceptibility to most tested antibiotics. It successfully survived and colonized in the intestinal tract of the largemouth bass. Furthermore, this strain showed remarkable antibacterial activity against common aquatic pathogens, including gram-positive and gram-negative bacteria, and also exhibited resistance against Aeromonas hydrophila on the head kidney leukocytes of largemouth bass in vitro. Following an 8-week feeding trial, P. acidilactici B49 improved host immunity by increasing intestinal lysozyme activity, enhancing IL-8 expression, reducing TGF-β expression, and enhancing IgM levels in both serum and intestinal mucus. It also potentiated the phagocytic activity of peripheral blood lymphocytes. In addition, the B49 feeding group showed a significant increase in intestinal villus height. The challenge test with A. hydrophila demonstrated that the administration of P. acidilactici B49 effectively maintained intestinal barrier integrity, reduced gut inflammation, decreased pathogen load in the spleen, and improved survival rates in largemouth bass. In conclusion, the host-derived strain P. acidilactici B49 exhibited broad-spectrum antibacterial ability, biosafety, and intestinal colonization in largemouth bass. It effectively improved immune function, intestinal health, and resistance against A. hydrophila in the host. Therefore, P. acidilactici B49 is a promising probiotic for immunomodulation and disease control in largemouth bass aquaculture.

Mytilus are sessile filter feeders that live in close contact with numerous marine microorganisms. Hemocytes, the immunocompetent cells of Mytilus, participate in the immune response in a very efficient manner. Lipopolysaccharide (LPS) and peptidoglycan (PGN) follow specific microbe/pathogen-associated molecular patterns (MAMPs or PAMPs) and are involved in immune stimulation in host cells. This study evaluated the molecular profiles and reactions at protein level of Mytilus hemocytes after stimulation with LPS and PGN. Mytilus coruscus was challenged in vivo with LPS and PGN. The hemocytes were collected after 48 h and analyzed for quantitative proteomics, cell subpopulations, and the free amino acid composition. 4D-DIA technology-based proteomic analysis revealed different protein profiles, as well as different responses at protein level, under either the LPS or PGN challenge. C-type lectins, collagens, and CD151 protein were highly upregulated in LPS-challenged mussels, while phospholipase A2 and dCMP deaminase were highly upregulated in PGN-challenged mussels. Moreover, LPS challenge disrupted dsRNA-mediated translation and stimulated energy-related metabolism, while PGN challenge stimulated proteins involved in the inflammatory response and suppressed amino acid metabolism. In addition, the LPS and PGN challenges differed in their effects on the free amino acid composition and granulocytes ratio of the hemocytes. These findings highlight the different strategies employed by mussel hemocytes in response to different MAMPs, providing insights into the effects of LPS and PGN on Mytilus.

Dendritic cells (DCs) play a pivotal role in activating naïve T-cells and bridging innate and adaptive immunity. This study aimed to identify and characterize CD83 and CD276 as potential markers for DCs in olive flounder (Paralichthys olivaceus). Specific antibodies against these markers were developed and used to analyze their distribution in peripheral blood leukocytes (PBLs) and intestinal tissues. Flow cytometry and immunohistochemistry revealed that CD83 and CD276 are expressed on DCs, with peak expression observed one week after oral administration of an inactivated viral hemorrhagic septicemia virus (VHSV) vaccine. Gene expression analysis further demonstrated significant activation of immune-related genes, including CD3, IgM, and TLRs, indicating that oral vaccine administration effectively activates the intestinal mucosal immune system. These findings provide valuable insights into fish immune responses and establish CD83 and CD276 as promising DC markers, contributing to the development of mucosal vaccine strategies in aquaculture.

Frog virus 3-like ranaviruses (FV3-like viruses), particularly FV3 (Frog virus 3), represent typical species within the genus Ranavirus, primarily infecting amphibians and reptiles, thereby posing serious threats to aquaculture and biodiversity conservation. We designed a pair of universal primers and a probe targeting the conserved region of the major capsid protein (MCP) genes of FV3-like viruses. By integrating recombinase-aided amplification (RAA) with lateral flow dipstick (LFD) technology and real-time fluorescence (RF) modification, we established RAA-LFD and RF-RAA assays. The limit of detection (LoD) of RAA-LFD was determined to be 35.4 copies/μL under optimized amplification conditions at 35°C for 15 minutes. Similarly, the RF-RAA assay exhibited high specificity with a satisfactory LoD of 3.54 × 10 copies/μL at 39°C over a duration of 17-20 minutes. In diagnosing 53 clinical samples infected by an isolated strain of FV3-like viruses, both assays demonstrated consistency with results obtained from real-time fluorescence PCR assay. These experiments indicate that both methods serve as promising alternatives for point-of care testing (POST), adaptable to various scenarios. This study represents the first establishment of RAA-LFD and RF-RAA assays for FV3-likes viruses, enabling rapid and intuitive assessment of detection results while fulfilling on-site testing requirements, thus contributing positively to swift diagnosis and long-term monitoring in aquaculture.

Rahnella aquatilis is an emerging opportunistic pathogen that usually causes septicaemia in fish and poses a potential threat to human health. VgrG gene is an important virulence factor of type VI secretion system in R. aquatilis, but its regulatory mechanism underlying PANoptosis is still unknown. Here, VgrG deletion mutant strain of R. aquatilis (ΔVgrG-RA) and recombinant plasmid pET32a-VgrG were respectively constructed, and immunohistochemistry for VgrG as well as PANoptosis features were evaluated. Moreover, the interaction transcriptome of ΔVgrG-mediated pathogen and host was determined by dual RNA-seq using an in vitro model of the primary macrophage cells from crucian carp Carassius auratus, and a total of 889 and 3765 differentially expressed genes (DEGs) were identified in pathogen-host interaction genes, respectively. Notably, GO and KEGG enrichment analysis were significantly involved in the PANoptosis pathways in ΔVgrG mutant-infected macrophages. The regulatory relationship of potential PANoptosis-related genes (PRGs) were analysed comprehensively, and their binding interaction of several hub proteins (eg., YcgR, Bcl2a, Calr3a, IL-1β) were determined by molecular docking analysis. To our best knowledge, this is first report of R. aquatilis VgrG-mediated interactions between pathogen and host macrophage cells, which will provide a new reference for understanding of molecular mechanism underlying PANoptosis in fish.

Aeromonas veronii is a zoonotic pathogen that is commonly found in various aquatic environments and causes serious damage to the aquaculture industry. Anti-virulence strategies based on mutating the virulence factors are important antibiotic alternative methods against A. veronii infection. The type Ⅳ pili, polar flagella, and aerolysin are considered to be the major virulence factors. In this study, we constructed a triple-gene deletion mutant strain (cheZ/mshK/aerA, ΔCMA-AV) and evaluated its potential as a live attenuated vaccine (LAV) candidate. Pathogenicity test showed that the LD of ΔCMA-AV in crucian carp (Carassius auratus) was 5.34 times higher than that of WT-AV. Further research found that the decline of pathogenicity was associated with the decrease of hemolysis, biofilm formation ability and expression levels of virulence-related genes (type IV pili and flagella). Our preliminary results on ΔCMA-AV as a LAV showed that crucian carp immunized with ΔCMA-AV at a concentration of 3 × 10 CFU/mL did not show any evident pathological alterations or clinical symptoms. Non-specific immune indicators, including serum immune-related enzyme activities and the expression of immune-related genes in immune organs, showed an up-regulation trend in the ΔCMA-AV group. A. veronii specific antibody levels increased significantly from 2 - 4 weeks. The relative percent survival in ΔCMA-AV groups was 73.97% ± 3.57 %, significantly higher than that of the inactivated A. veronii groups (53.73% ± 10.87 %). These results indicate that the three genes (cheZ/mshK/aerA) play an important role in the pathogenicity of A. veronii, in additionΔCMA-AV could induce an effective immune response and provide strong protection against A. veronii in crucian carp without adverse effects.

During viral infection, RIG-I-like receptors (RLRs) are cytoplasmic pattern recognition receptors that recognize and bind to viral RNA components, initiating the transcription of interferon-related genes, inflammatory cytokines and other factors, thereby triggering the cellular production of an antiviral innate immune response. The protein inhibitor of activated signal transducer and activator of transcription (STAT) (PIAS) protein family has become a hot research topic due to its extensive involvement in the regulation of cytokines, inflammatory factors and innate immune signaling pathways. In the present study, we investigated the role of fish PIASy in Singapore grouper iridovirus (SGIV) and red spotted grouper nervous necrosis virus (RGNNV) infections. The homologous sequence of orange-spotted grouper (Epinephelus coioides) PIASy gene (EcPIASy) was cloned and characterized, which encoded a 498-amino acid protein with 99.20% homology to Plectropomus leopardus. EcPIASy is expressed mainly in gills, blood, and liver. Subcellular localization showed that EcPIASy was uniformly distributed in the nucleus. Overexpression of EcPIASy promoted SGIV and RGNNV replication, and inhibited the expression of interferon related genes and pro-inflammatory factors induced by viruses. In addition, EcPIASy interacts with RLR signaling pathway-related genes EcMDA5, EcIRF3 and EcIRF7, whereas the interaction between EcPIASy and EcIRF3 does not depend on any specific structural domain of EcPIASy.. The results provide a better understanding of the relationship between PIASy and viral infection in fish.

Infectious hematopoietic necrosis virus (IHNV) is a serious pathogen in the salmonid aquaculture industry and leads to economic losses in the world. This study aimed to develop a new oral DNA vaccine designed to protect rainbow trout against infection by IHNV. Fish were administered via the oral route by the attenuated Salmonella enterica serovar Typhimurium as a carrier of pcDNA3.1-IHNG (glycoprotein (G)) plasmid for 7 days and finally, fish were challenged by 10 pfu ml IHNV. The results revealed that the antigen gene was identified in different tissues of rainbow trout at 15-, 30-, and 45 days post-vaccination (dpv). Also, the recombinant vaccine elicited both an innate and specific immune response, resulting in a significant upregulation of the expression levels of ifn-1, mx-1, vig-1, igm, and igt. In addition, serum levels of neutralizing antibodies were observed to be elevated in the vaccinated fish, in contrast to the unvaccinated fish, following 30 dpv. Compared to trout that received empty S. Typhimurium, notable differences in cumulative percentage mortality were evident among the vaccinated fish. The relative percent survival (RPS) was recorded at 58.2 % for the group that received oral vaccine, while the group that received empty S. Typhimurium exhibited an RPS of 18.2 %. Therefore, our results showed that this bacterial vector can be a candidate carrier for pcDNA3.1-IHNG plasmid that may be employed to confer protection to rainbow trout against IHNV.

Holothuria scabra is one of the most valuable species of sea cucumber owing to its exploitation as a seafood product. This study aims to describe the main molecular and cellular actors in the immunology of the holothuroid H. scabra. First, a detailed description of the immune cells - the cœlomocytes - is provided, highlighting five main cell types including phagocytes, small round cells (SRCs), spherulocytes, fusiform cells, and crystal cells, with a further five subtypes identified using transmission electron microscopy. Cœlomocyte aggregates were also described morphologically, yielding two main types, one comprising three successive maturation stages. A comparison of the concentration and proportion of cell populations was carried out between the two main body fluids, namely the hydrovascular fluid of the Polian vesicle (HF) and the perivisceral fluid of the general cavity (PF), and no clear relation could be revealed. Next, the cœlomocyte immune response was studied 24 hours after lipopolysaccharide (LPS) injection in the two body fluids. Firstly, the fluctuation in cell populations was assessed, and despite a high inter-individual variability, it shows a decrease in the phagocyte proportion and an increase in the SRC proportion. Secondly, the differential gene expression of PF cœlomocytes was studied by de novo RNA-sequencing between LPS-injected and control-injected individuals: 945 genes were differentially expressed, including 673 up-regulated and 272 down-regulated in the LPS-injected individuals. Among these genes, 80 had a presumed function in immunity based on their annotation, covering a wide range of immune mechanisms. Overall, this study reveals a complex immune system at both molecular and cellular levels and constitutes a baseline reference on H. scabra immunity, which may be useful for the development of sustainable aquaculture and provides valuable data for comparative immunology.

Ocean acidification and warming are significant stressors impacting marine ecosystems, exerting profound effects on the physiological ecology of marine organisms. We investigated the impact of ocean acidification and warming on the immune system of mussels, focusing on the regulatory mechanisms of intrinsic and extrinsic apoptosis. The study explored the effects on the immune response ability of mussels (Mytilus coruscus) after 14 and 21 days under combined conditions of different temperatures (20 °C and 30 °C) and pH (8.1 and 7.7), as expected for the year 2100. The experimental results indicated that ocean acidification and warming have significant interactive effects on various immune parameters of M. coruscus. Specifically, ocean acidification and warming lead to an increase in ROS (Reactive Oxygen Species), apoptosis, TNF-α (Tumor Necrosis Factor-alpha), TGF-β (Transforming Growth Factor-beta), Caspase-8, and a decrease in IL-17 (Interleukin 17). These findings suggest that ocean acidification and warming trigger an immune inflammatory response in mussels. Regulating immune functions through apoptosis pathways may be a crucial coping mechanism in response to environmental variations, but its long-term impact on population health and sustainability remains uncertain. Our findings offer important insights into the complex interactions between bivalve immune responses and environmental stressors. This also underscores the need for further research into the adaptive capabilities of marine organisms facing the compounded challenges of ocean acidification and warming.

Tumor necrosis factor receptor-associated factors (TRAFs) act as signal transducers and are critical in many biological processes. However, in contrast to mammals, the function of TRAFs in teleost is still largely unknown. In this study, we identified and cloned eight TRAF genes in snakehead (Channa argus), namely CaTRAF2aa, CaTRAF2ab, CaTRAF2b, CaTRAF3, CaTRAF4a, CaTRAF5, CaTRAF6, and CaTRAF7. Bioinformatics analyses exhibited CaTRAF genes were evolutionarily conserved among teleost. Subcellular localization results demonstrated that eight CaTRAFs were all localized in cytoplasm. These CaTRAFs showed widespread but different expression profiles in various organs/tissues of snakehead and their expression could be induced by IHSV infection. Furthermore, almost all CaTRAFs can be impacted by poly (I:C) stimulation in HKLs. To sum up, this study provides a valuable foundation for further functional research on teleost TRAF genes.

Piwi-interacting RNAs (piRNAs) are small non-coding RNAs that play a crucial role in gene regulation and immune defense. This study investigates their function in Penaeus vannamei shrimp during White Spot Syndrome Virus (WSSV) infection. Analysis of small RNA libraries from WSSV-infected shrimp hemocytes identified 82,788 piRNA homologs, with 138 showing altered expression during infection. Putative piRNAs were mapped to both the P. vannamei nuclear and mitochondrial genomes, highlighting their diverse origins. Interestingly, some piRNA sequences from uninfected shrimp mapped to both the shrimp and WSSV genomes, suggesting potential subversion or integration of viral fragments into the host genome. We focused on piR-pva-926938, a downregulated piRNA targeting the WSSV186 gene. Introducing piR-pva-926938 into WSSV-infected shrimp suppressed WSSV186 expression, but paradoxically increased viral load by downregulating host immune genes like calcineurin B and dynamin-binding protein. This study is the first to report WSSV-responsive piRNAs in shrimp and reveals the complex interplay between piRNAs, viral genes, and host immunity during WSSV infection.

The Chinese mitten crab (Eriocheir sinensis) is an important component in Chinese aquaculture. Due to its lacking adaptive immune system as a crustacean, it exhibits poor tolerance to environmental stresses, particularly the deleterious impact of lipopolysaccharide (LPS) from pathogenic bacteria during E. sinensis culture. In a previous study, we isolated LGSPDVIVIR (cmP4) peptide from cottonseed meal hydrolysate, having excellent antioxidant and immune-enhancing properties in vitro. Expressing this peptide abundantly as a tandem (a tandem of five cmP4 peptides, cmP4') using the Bacillus subtilis expression system, we aimed to investigate the effects of incorporating recombinant B. subtilis into diets on growth performance, acute oxidative stress, and hepatopancreatic injury induced by LPS injection in E. sinensis. Crabs were cultured for a period of 12 weeks on three diets: basal diet, basal diet supplemented with 10 CFU/kg of unmodified B. subtilis, and recombinant B. subtilis, respectively. Results indicated that both B. subtilis species improved the growth performance of E. sinensis. Subsequent challenge with LPS at 400 μg/kg body weight for 6 h revealed that both B. subtilis groups exhibited improved antioxidant capacity, decreased oxidative stress indexes in hemolymph, enhanced mitochondrial membrane potential, and reduced hepatopancreatic damage compared to the single LPS-treated group. Notably, the recombinant B. subtilis had better performance, demonstrating superior effects. Specifically, compared with the single LPS-treated group, the oxidative stress indexes, mitochondrial membrane potential, and apoptosis-related gene expression in both B. subtilis groups followed a similar trend. However, the recombinant B. subtilis group displayed greater absolute changes in these indexes, a finding further supported by histopathological observations of the hepatopancreas. In conclusion, this study provides useful information for promoting the application of plant protein by-products in aquafeeds, promoting antimicrobial-free aquaculture practices for E. sinensis.