The Collectin family, an important member of the C-type lectin superfamily, participates in innate immune defense through Ca[2+]-dependent recognition of glycan ligands on pathogen surfaces. In this study, five Collectin family members were identified in the silver pomfret (Pampus argenteus) genome, and their gene structures, conserved motifs, and chromosomal localizations were systematically characterized. Notably, a novel member, collectin-11b (PaCL-11b) was found to encode a truncated isoform containing only the neck region and the carbohydrate recognition domain (CRD). It exhibited the highest expression level in the liver of healthy silver pomfret and was significantly downregulated in the liver, kidney, and gills following Vibrio parahaemolyticus challenge. Functional characterization of the recombinant protein rPaCL-11b demonstrated Ca[2+]-dependent binding, agglutination, direct antimicrobial, and antibiofilm activities against both Gram-negative (V. parahaemolyticus and Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria, with the most potent inhibitory effects observed against V. parahaemolyticus. This study presents a systematic identification of the Collectin family in silver pomfret and reveals the multifunctional immune roles of PaCL-11b in defense against V. parahaemolyticus infection, providing a theoretical basis for the prevention and control of bacterial diseases in silver pomfret aquaculture.

Summer mortality events continue to threaten Pacific oyster (Magallana gigas) production worldwide, having consequences for local economies and food security, yet the physiological mechanisms underlying these losses remain poorly resolved, particularly for emerging pathogens. The disease nocardiosis, caused by the bacterium Nocardia crassostreae, has been increasingly associated with oyster mass mortality, with evidence suggesting a role of increased temperature in these losses. We experimentally examined how exposure to elevated temperature modulates the immune and energetic responses of M. gigas exposed to N. crassostreae. Oysters were challenged for 42 days, after which energetic and immunological parameters were quantified. Infection resulted in reduced condition index and mantle glycogen stores, accompanied by elevated glucose, ADP and AMP concentrations, and increased haemocyte concentrations. These changes indicate mobilisation of energetic reserves to support immune defence. Elevated temperature independently reduced glycogen, ATP concentration, haemocyte viability, and gill Na[+]/K[+]-ATPase activity, demonstrating thermally driven compromised immunity, energetic strain and impaired cellular integrity. No statistically significant interaction between temperature and infection was detected under the experimental conditions and timescales tested, indicating that the physiological impacts of temperature and N. crassostreae exposure operated largely independently rather than synergistically. Our results show that M. gigas mounts an energetically costly immune response to N. crassostreae, while elevated temperature compromises both energy balance and immunity. These findings suggest that, rather than amplifying one another, these stressors impose parallel constraints on oyster physiology that may still combine to increase vulnerability under more complex or prolonged environmental scenarios.

In mammals, IFN-γ acts as a critical cytokine that triggers intracellular signaling by binding to specific IFNGR1 and IFNGR2 receptors. Unlike mammals, teleosts possess two members of type II IFN genes (IFN-γ and IFN-γrel), two IFNGR1 paralogs (CRFB13 and CRFB17) and a single IFNGR2 homolog (CRFB6). Despite the identification of type II IFN receptors in multiple fish species, the molecular features, expression profiles and signaling-associated function remain limited. In this study, three type II interferon receptor genes, AjCRFB6, AjCRFB13 and AjCRFB17 were identified in Japanese eel, Anguilla japonica. All three receptors possess typical conserved structural domains of class II cytokine receptors. Phylogenetic analysis assigned them to respective teleost CRFB subfamilies. These three genes displayed broad tissue expression, with predominant abundance in immune-related spleen, trunk kidney and liver tissues. Following Poly(I:C) stimulation, AjCRFB6 and AjCRFB13 generally showed significant upregulation from 6 hpi in the liver, trunk kidney and spleen, maintaining elevated expression at most subsequent time points, while AjCRFB17 exhibited a distinct expression pattern from AjCRFB6 and AjCRFB13, with a generally downregulated or unchanged expression trend across most tested time points. It was revealed that AjIFN-γ initiates downstream ISGs transcription and STAT1 phosphorylation through AjCRFB6+AjCRFB13 or AjCRFB6+AjCRFB13+AjCRFB17, whereas all AjCRFB17-containing complexes confer responsiveness to AjIFN-γrel. Furthermore, co-immunoprecipitation assays revealed that AjSTAT1 specifically interacts with AjCRFB13 and AjCRFB17, but not with AjCRFB6. These results provide a reference for further exploring the potential roles of type II IFN receptors in teleost immunity and signaling.

Interferon regulatory factor 7 (IRF7) functions as a central orchestrator of type I interferon (IFN) responses in mammals, yet the functional mechanisms of its homologue in teleost remains incompletely understood. Here, we characterized an IRF7 homologue (LcIRF7) in large yellow croaker (Larimichthys crocea). The expression of LcIRF7 was strongly induced by poly (I:C), lipopolysaccharide (LPS), and recombinant type I IFN (rLcIFNi). These inductions were dependent on the JAK-STAT signaling pathway, suggesting that LcIRF7 is an interferon-stimulated gene (ISG). Overexpression of LcIRF7 significantly enhanced the activity of two type I IFNs (LcIFNi and LcIFNd) promoters and promoted antiviral responses in cells infected with spring viremia of carp virus (SVCV). Protein pull-down assays confirmed that LcIRF7 interacts with LcTBK1 and undergoes LcTBK1-mediated phosphorylation. In addition to forming homodimers, LcIRF7 can also heterodimerize with LcIRF11, an IRF family member absent in tetrapods, suggesting a potential lineage-specific mechanism of IRF7-mediated antiviral responses. Collectively, these findings provide insights into the antiviral signaling mechanisms mediated by teleost IRF7.

Fish cell lines serve as valuable tools in aquaculture research, particularly in immunology, pathology, and toxicology. In this study, we successfully established a novel cell line derived from brain tissue of grass carp, designated CIB. This cell line has been subcultured over 100 times and exhibits a fibroblast-like morphology. Chromosomal analysis revealed that the diploid chromosome number of CIB cells is 2n = 48, while sequencing of the 18S rRNA gene confirmed the cell line's origin. Notably, CIB cells demonstrated a transfection efficiency of 62.4% with pEGFP-N3, highlighting their potential for studies involving exogenous gene expression. Furthermore, CIB cells were susceptible to grass carp reovirus genotype I (GCRV-I), as evidenced by cytopathic effects (CPE), increased synthesis of viral proteins, and accumulation of viral particles within the cells. Both viral infection and poly(I:C) stimulation significantly increased the expression of intracellular interferon-related signaling molecules. Additionally, electroporation of a gRNA-Cas9 ribonucleoprotein (RNP) complex into CIB cells achieved the first successful large-fragment gene knockout in cultured grass carp cells and generated a homozygous clonal line, thereby enhancing the antiviral response. In summary, this novel cell line represents a significant advancement for studying gene functions, host-virus interactions, and genetic engineering in teleost fish.

In recent years, the viral disease caused by largemouth bass virus (LMBV) has inflicted severe economic losses on China's largemouth bass (Micropterus salmoides) aquaculture, significantly restricting its development. As targeted antiviral drugs and commercial vaccines remain in the investigational stage, the discovery of novel antiviral agents to control LMBV infections is urgently needed. Consequently, this study systematically evaluated the antiviral efficacy and underlying immunopharmacological mechanisms of cepharanthine (CE), a natural bisbenzylisoquinoline alkaloid, against LMBV infection using integrated in vitro and in vivo models. In vitro, CE exhibited potent anti-LMBV activity in epithelioma papulosum cyprinid (EPC) cells, with a half-maximal inhibitory concentration (IC50) of 0.2136 μM and negligible cytotoxicity within the effective concentration range. Furthermore, CE treatment induced a significant, dose-dependent reduction in progeny viral titers (TCID50). Additionally, CE suppressed intracellular viral replication by inducing G0/G1 phase arrest and apoptosis, effectively blocking viral DNA synthesis and prematurely eliminating infected cells. In vivo, CE administration (40 mg/kg) significantly protected LMBV-infected largemouth bass, reducing cumulative mortality from 73.33% to 46.67%. Concurrently, CE alleviated histopathological damage, diminished viral loads in primary target organs (liver, spleen, and kidney), and dynamically modulated the mRNA expression levels of key immune-related genes (IL-1β, IL-6, TNF-α, and IFN-γ). Furthermore, transcriptomic profiling revealed that CE globally reversed LMBV-induced gene expression patterns, down-regulating core replication, cell cycle, and apoptotic pathways.Collectively, our findings demonstrate that CE is a promising multi-functional antiviral agent for the clinical management of ranavirus infections in aquaculture.

High-carbohydrate diet (HCD) is widely used in aquaculture to reduce protein costs, but it often induces pathological adipose tissue hypertrophy, chronic inflammation, and increased susceptibility to bacterial infections. This study investigated whether dietary supplementation with caffeic acid phenethyl ester (CAPE) could alleviate HCD-induced pathological adipose tissue hypertrophy and enhance resistance against Aeromonas hydrophila in largemouth bass (Micropterus salmoides), and explored the underlying mechanisms. Fish were fed a control diet, an HCD, or HCD supplemented with CAPE (400, 800, or 1200 mg/kg) for eight weeks, followed by bacterial challenge. HCD impaired growth, promoted adipocyte hypertrophy and visceral fat accumulation, and triggered inflammation and oxidative stress. CAPE reversed these effects, shifting adipose expansion toward hyperplasia, improving lipid and glucose metabolism, reducing inflammatory markers and ROS, and markedly improving survival after infection. The three CAPE doses exhibited comparable efficacy across all measured parameters, with the lowest dose (400 mg/kg) already achieving maximal benefits. Mechanistically, CAPE suppressed HCD-induced NF-κB p65 activity via PPARγ activation, and we identified the antimicrobial peptide dicentracin as a direct transcriptional target repressed by p65. CAPE relieved this repression, restoring dicentracin expression and antibacterial defense. Collectively, CAPE decouples metabolic stress from immune suppression through PPARγ-mediated relief of p65 repression. These findings reveal a novel p65-dicentracin axis linking adipose tissue expansion mode to antibacterial immunity, and highlight CAPE as a promising immunometabolic feed additive to address both metabolic stress and infectious diseases in aquaculture.

Autophagy is a highly conserved cellular process that plays a dual role during viral infection, contributing to antiviral defense while also being exploited by viruses to support their replication. However, the molecular mechanisms by which white spot syndrome virus (WSSV) influences host autophagy remain poorly understood. In this study, we identified LvTM9SF4, a phylogenetically conserved nine-transmembrane protein with predominantly expressed in the shrimp intestine, as a host factor that physically interacts with the WSSV structural protein VP24. Functional assays showed that RNAi-mediated knockdown of LvTM9SF4 significantly suppressed WSSV infection and enhanced shrimp survival after WSSV challenge in Pacific white shrimp (Litopenaeus vannamei). Subcellular localization analyses revealed substantial colocalization of LvTM9SF4 with the autophagy marker LC3B. Transcriptomic analysis further suggested that LvTM9SF4 knockdown affected autophagy-related pathways and genes, among which LvAtg2 exhibited the most substantial downregulation. Meanwhile, silencing LvAtg2 inhibited WSSV infection and improved shrimp survival, producing phenotypes similar to those observed following LvTM9SF4 knockdown. All the results indicated that LvTM9SF4 may participate in autophagy-related processes during WSSV infection, and that LvAtg2 may represent a potential downstream effector associated with this host-virus interaction. This study provides new insights into antiviral immunity in crustaceans and highlights potential host-targeted strategies against WSSV.

Ctenopharyngodon idella (grass carp) is the dominant species in freshwater aquaculture, but its farming industry is severely threatened by grass carp haemorrhagic disease (GCHD) caused by grass carp reovirus (GCRV). Autophagy plays a crucial role in viral infection, and ATG13 is a core factor for autophagy initiation. However, the functional mechanism of grass carp ATG13 (CiATG13) during GCRV-I/II infection remains unclear. In this study, the CiATG13 gene was cloned and characterized by bioinformatics analysis. The results showed that CiATG13 sequence is highly conserved in evolution, sharing the highest homology and closest evolutionary relationship with Chanodichthys erythropterus. The expression and function of CiATG13 were investigated using RT-qPCR, Western blotting, fluorescence microscopy, and CRISPR-Cas13d knockdown techniques at both cellular and individual levels. The key findings are summarized below: tissue distribution analysis revealed that CiATG13 is widely expressed in various tissues of healthy grass carp, with the highest expression in the liver, brain, and heart, and it responds actively to stimulation by pathogen-associated molecular patterns (PAMPs), such as poly (I:C) and lipopolysaccharide (LPS). GCRV-I/II infection induces the expression of CiATG13. Overexpression of CiATG13 significantly promotes GCRV-I replication, whereas knockdown of CiATG13 inhibits GCRV-I replication. Further mechanistic studies indicated that CiATG13 can induce autophagy and upregulate the expression of heat shock protein 70 (CiHSP70) through this pathway. CiHSP70 promotes GCRV-I replication, and quercetin (Qu) can block its pro-viral effect on GCRV-I/II replication by inhibiting CiHSP70. Moreover, treatment with the autophagy inhibitors chloroquine (CQ) and Spautin-1 suppressed GCRV-I replication and the associated cytopathic effect (CPE), accompanied by reduced CiHSP70 expression, overexpression of CiATG13 partially rescued these inhibitory effects.. This study reveals the molecular mechanism that CiATG13 mediates autophagy to regulate CiHSP70 expression and promote GCRV-I/II replication, enriches the understanding of the interaction between fish viruses and autophagic molecules, and provides the potential strategy targeting CiATG13 for the prevention and control of GCHD.

Streptococcus agalactiae is an important zoonotic pathogen with an expanding host range and increasing antibiotic resistance, presenting a significant risk to the aquaculture in China. Therefore, developing effective treatment approaches and reducing the risk of antibiotics is crucial. Chinese medicine compounds have shown great potential in the prevention and control of aquatic animal diseases. In this study, we investigated the antibacterial activity and mechanism of magnolol against S. agalactiae (SICAU SA001) derived from Schizothorax prenanti. The in vitro experiments showed that the minimum inhibitory concentration (MIC) of magnolol against S. agalactiae was 16 μg/mL, and the minimum bactericidal concentration (MBC) was 32 μg/mL. Under the treatment with magnolol at 1× MIC, the biofilm formation was reduced by 61.98%, the conductivity increased by 30.59%, and the extracellular DNA (eDNA) release increased by 2.86-fold. The integrated omics analysis and molecular docking found that magnolol can interfere with the expression of ribosomal proteins in S. agalactiae, and inhibit the gene and protein expression of the phosphotransferase system (PTS). The in vivo experiments showed that magnolol conferred a relative protection rate of 54.55% against S. agalactiae infection in S. prenanti, significantly reducing bacterial loads in the liver, spleen, and kidney while alleviating S. agalactiae-induced histopathological damage. Furthermore, magnolol can significantly increase the activities of non-specific immune enzymes (acid phosphatase, catalase, and superoxide dismutase), and suppress the overexpression of pro-inflammatory cytokines (IL-6, IL-1β, and IL-8). In summary, magnolol exhibits favorable inhibitory effect on S. agalactiae, mainly by disrupting the cell membrane function, interfering with the ribosome and the PTS, and enhancing the immunity of the host to exert its antibacterial effect. Our study provides a promising strategy for the antibiotic-free control of S. agalactiae and supports the sustainable development of Schizothorax aquaculture.

Hemopexin (Hpx) is a conserved acute-phase protein that participates in the immune regulation and anti-bacterial response of higher vertebrates. However, the immunological function of Hpx in bony fish remains largely unclear. In this study, we found that Paralichthys olivaceus Hpx-a (PoHpx-a) contains a signal peptide and four hemopexin-like domains, with double β-propeller structure. PoHpx-a was found to be broadly occurred at 9 tissues, with the most abundant expression in liver, and its transcription was significantly influence in immune-related tissues upon bacterial challenge. Recombinant PoHpx-a (rPoHpx-a) exhibited binding activity to different types of bacteria. Furthermore, rPoHpx-a notably regulated serum complement activity. rPoHpx-a could bind to peripheral blood leukocytes (PBLs), inhibit cells LDH release, promote cells proliferation, and enhance cells phagocytic activity. In vivo, rPoHpx-a prominently reduced bacterial loads in the liver, spleen, and kidney, downregulated the expression of related pro-inflammatory cytokines, alleviated pathological damage of tissues, and improved the survival rate of hosts infected by Edwardsiella piscicida. These results indicate that PoHpx-a acts as a key immunoregulatory factor in antibacterial defense by regulating complement activity, enhancing leukocyte function, suppressing excessive inflammation, and inhibiting bacterial spread. This study provides new insights into the immune function of Hpx in teleost fish.

2,6-Dibromophenol (2,6-DBP), a typical brominated phenolic disinfection byproduct, is frequently detected in drinking water after disinfection. However, its effect on tissue regeneration has not been previously reported. In this study, using the zebrafish larvae caudal fin regeneration model, we investigated the impact of acute exposure to a high concentration of 2,6-DBP on fin regeneration. The findings revealed that exposure to 2,6-DBP impaired tailfin regeneration and reduced locomotor activity, accompanied by increased apoptosis and decreased cell proliferation in the regenerating tailfin region. Furthermore, 2,6-DBP hindered the recruitment of neutrophils and macrophages, suppressed Wnt signaling pathway activity, and downregulated the expression of immune-related genes and genes associated with the Wnt signaling pathway. Pharmacological activation with either LPS (Lipopolysaccharide) or BML-284 partially rescued the regenerative defects in the tailfin region. Notably, pharmacological activation with BML-284 not only enhanced the recruitment of neutrophils and macrophages to the injury site but also restored the expression of immune-related genes. Collectively, these results indicate that 2,6-DBP inhibits fin regeneration in zebrafish larvae via Wnt-mediated recruitment of immune cells. This study provides novel insights into the ecological risks posed by disinfection byproducts to tissue repair and population recovery in aquatic vertebrates.

RAR-related orphan receptor alpha (RORα), a core member of the NR1F subgroup within the nuclear receptor superfamily, critically regulates lipid metabolism and immune responses. This study identified the Pm-RORα (Pm-RORα1 and Pm-RORα2) gene and its alternative splicing isoforms in Pinctada fucata martensii and investigated their functions through bioinformatic analysis, quantitative PCR (qPCR), and bimolecular fluorescence complementation (BiFC). Phylogenetic analysis revealed high homology between Pm-RORα and the Pacific oyster Crassostrea gigas ROR. Pm-RORα1_1 and Pm-RORα1_2 were generated by exon skipping of exons 2 and 6 in Pm-RORα1, respectively, while intron retention in exon 7 of Pm-RORα2 produced Pm-RORα2_1. Domain analysis showed that Pm-RORα1_1 and Pm-RORα1_2 lacked the ZnF_C4 and HOLI domains, respectively, while Pm-RORα2_1 was deficient in the HOLI domain. qPCR analysis demonstrated the expression patterns following LPS stimulation, Pm-RORα1_1 and Pm-RORα2_1 were significantly upregulated at 12 h, whereas Pm-RORα1_2 peaked at 6 h. Following nucleus transplantation, stimulation induced significant upregulation of Pm-RORα1, Pm-RORα1_1 and Pm-RORα1_2 at 6 h, while both of Pm-RORα2 and Pm-RORα2_1 was significantly downregulated from 6 to 24 h. Bimolecular fluorescence complementation assays confirmed protein interactions between Pm-RORα1 and the three isoforms (Pm-RORα1_1, Pm-RORα1_2, and Pm-RORα2_1). These findings indicate that Pm-RORα and its splicing isoforms potentially form protein interactions to modulate dynamic immune regulation in P. f. martensii, providing molecular insights into molluscan innate immunity and pearl cultivation optimization.

Toll-like receptor 2 (TLR2) is a pivotal pattern-recognition receptor in innate immunity. It is well characterized in vertebrates but remains poorly understood in mollusks. In this study, we identified a TLR2 homolog (RpTLR2) in Ruditapes philippinarum encoding a 575-amino acid protein with a canonical Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis placed RpTLR2 within the molluscan TLR2 clade, confirming its evolutionary conservation. RpTLR2 was constitutively expressed in all examined tissues, with the highest transcript levels in the gill and mantle. After Vibrio parahaemolyticus challenge, RpTLR2 mRNA and protein levels were significantly upregulated. Functional assays showed that RpTLR2 overexpression intensified inflammatory responses and increased the expression of key inflammation-related genes, whereas RNA interference-mediated RpTLR2 knockdown markedly reduced V. parahaemolyticus-induced inflammation in gill cells. Overall, our findings indicate that RpTLR2 acts as a positive regulator of V. parahaemolyticus-induced inflammatory responses. These results improve understanding of TLR2-mediated innate immunity in mollusks and provide a molecular basis for studying host-pathogen interactions in bivalves.

Mitogen-activated protein kinase (MAPK) pathways are evolutionarily conserved signalling cascades that regulate fundamental cellular processes such as proliferation, differentiation, and stress responses. In the colonial ascidian Botryllus schlosseri, MAPK activity is required for efficient phagocytosis of non-self cells or particles. In the present study, we identified two novel MAPK transcripts in B. schlosseri and analysed their modulation during the colonial blastogenetic cycle under various experimental conditions. Since phagocytic activity is essential for the cyclical renewal of the colony throughout the blastogenetic cycle, we also investigated the effects of colony exposure to MAPK inhibitors, PD98059 and SP600125, targeting the ERK and JNK signalling pathways, respectively. Colonies were microinjected with the inhibitors, and changes in morphology and transcriptional profiles were analysed. The inhibition of MAPK activity altered colony development and modulated the expression of genes associated with oxidative stress protection (sod, gpx-5) and stress granule dynamics (tiar, ttp), processes involved in post-transcriptional regulation. These findings suggest that MAPK signalling is indispensable for the proper progression of the blastogenetic cycle in B. schlosseri, integrating oxidative stress defence and post-transcriptional control to maintain colony homeostasis.