White spot syndrome virus (WSSV) remains one of the most destructive viral pathogens in shrimp aquaculture. Reactive oxygen species (ROS) are required for innate immune signaling, but uncontrolled ROS accumulation can damage host tissues and compromise antiviral defense. Catalase (CAT) decomposes hydrogen peroxide and is therefore positioned to connect redox homeostasis with immune resilience during viral infection. Here, we cloned and characterized a CAT gene (Pv-CAT) from Penaeus vannamei and examined its role in the anti-WSSV response. Pv-CAT contained a 1563-bp open reading frame encoding a 520-amino-acid CAT-like protein with a predicted molecular mass of 58.83 kDa. Bioinformatic analyses indicated that Pv-CAT lacks a signal peptide and transmembrane region and retains typical features of antioxidant enzymes. Pv-CAT was expressed in all tested tissues, with relatively high levels in the hepatopancreas and hemocytes. Its expression changed markedly after WSSV infection, especially in barrier and immune-related tissues. Silencing Pv-CAT decreased intestinal CAT activity, promoted hydrogen peroxide (H2O2) accumulation and lipid peroxidation, disturbed the expression of antimicrobial peptide genes and JAK/STAT pathway-related genes, and reduced shrimp survival after WSSV challenge. These findings suggest that Pv-CAT contributes to antiviral resilience in P. vannamei by limiting oxidative injury and helping maintain immune-signaling balance during WSSV infection. The study provides evidence that antioxidant enzymes form an important part of the crustacean antiviral-defense network.

The intestinal tract maintains animal health directly (barrier integrity, nutrient absorption, etc.) and indirectly (microenvironment for commensal organisms). Due to its close interactions with the internal and external environment, the intestinal tract is the site of interactions between resident stromal cells, commensal and pathogenic organisms, and immune cells. As a result of its complex functions, physiology, and structural morphology, in vivo studies have been the most reliable method for understanding intestinal biology, until recently. The development of microphysiological and 3D cell culture based in vitro models are powerful tools to help address questions of drug and heavy metal cytotoxicity, barrier function, effects of nutritional supplements on local immune responses, and pathogenic immunomodulatory effects, while also helping to minimize live animal use (3Rs). While these physiologically-relevant in vitro systems have historically been used in mammalian species, fish researchers have begun to adopt these models to study fish intestinal physiology, pathogen defenses, and mucosal immunity, though to a more limited extent. Compared to the thousands of fish species and over 900 fish cell lines, only 10 fish intestinal cell lines exist. Here, we discuss the various intestinal in vitro models, their current and future applications for studying fish mucosal immunity and overall health, as well as proposed future directions for studying intestinal pathogens and diseases in aquatic invertebrate species of economic and environmental importance.

Septins are conserved GTP-binding cytoskeletal proteins that regulate cytokinesis, membrane dynamics, and innate immunity. Here, we report the first molecular and functional characterization of septin 8a in rohu (Labeo rohita), an economically important Indian major carp. The full-length septin 8a cDNA encodes a 420-amino acid protein containing conserved GTP-binding motifs (G1-G5) belonging to the SEPT6 subgroup with a predicted molecular weight of ∼48.98 kDa, hydrophilic character and intracellular localization consistent with cytoskeletal scaffolding. Developmental expression analysis revealed peak transcript abundance at fertilization, suggesting maternal deposition and an early embryogenic role. Tissue profiling showed the highest constitutive expression in gills, kidney, and spleen. The challenge experiments with Aeromonas hydrophila, Argulus siamensis, and the viral mimic poly I:C demonstrated a broad pathogen-induced upregulation, particularly in immune tissues. Administration of generated recombinant septin 8a in vivo activated pro-inflammatory cytokines, antimicrobial enzymes, antioxidant genes, and the MX gene, and conferred 55% relative percent survival against A. hydrophila challenge. Indirect ELISA revealed progressive increase in protein levels within liver and kidney tissues during infection. CRISPR/Cas9-mediated F0 mutagenesis in zebrafish caused developmental delay, morphological abnormalities, and a survival rate of 26%, emphasizing its role during early development. Successful mutagenesis in rohu embryos validated cross-species sgRNA efficacy. Following bacterial challenge, exon 2-edited F0 crispants showed the highest mortality (80%) relative to wild-type controls (53.33%), whereas the mortality in exon 4-edited rohu did not differ from controls. These findings establish septin 8a as a multifunctional immune regulator and developmental factor with potential applications in aquaculture disease management.

Chlorine dioxide (ClO2) is a potent oxidative disinfectant with potential use in bacterial disease control; however, its antibacterial mechanism and its influence on host redox-immune responses during Aeromonas salmonicida infection in rainbow trout (Oncorhynchus mykiss) remain unclear. Here, we evaluated the antibacterial activity of ClO2 against common bacterial pathogens, examined its thiol-targeting antibacterial mechanism, and characterized host toxicity, innate immune responses, oxidative stress, and hepatic transcriptomic regulation. ClO2 markedly inhibited Aeromonas hydrophila and A. salmonicida and reduced bacterial thiol contents in a concentration-dependent manner, indicating that thiol-associated oxidative damage contributes to its antibacterial action. Acute exposure revealed dose-dependent host responses: low-to-moderate ClO2 exposure activated antioxidant and innate immune responses, whereas higher concentrations caused mortality, liver and gill injury, glutathione depletion, lipid peroxidation, and immune disturbance. RNA-seq analysis showed that ClO2 exposure and A. salmonicida infection both affected xenobiotic metabolism, glutathione metabolism, lipid metabolism, and immune-related pathways. In the AsClO2 vs As comparison, 1,877 differentially expressed genes were identified and were mainly enriched in drug metabolism-cytochrome P450, cytochrome P450-mediated xenobiotic metabolism, glutathione metabolism, PPAR signaling, C-type lectin receptor signaling, and cytokine-cytokine receptor interactions. qRT-PCR confirmed the regulation of representative immune-metabolic genes, including mmp9, fmo5, cyp3a27, mgst1, fabp1, apoa-i-2, and bnip3. These findings indicate that ClO2 suppresses Aeromonas through thiol-associated oxidative damage while modulating hepatic redox-immune and metabolic networks during bacterial infection, providing new insight into host-pathogen-disinfectant interactions in rainbow trout.

A primer-driven reverse-screening strategy was used to identify an RPA-Cas12a target suitable for the rapid preliminary screening of megalocytivirus-related viruses. The ISKNV reference genome NC_003494.1 was used as the initial template, and candidate amplification units were designed according to RPA primer-design requirements, primer physicochemical properties, and the availability of Cas12a protospacer-adjacent motif (PAM) sites and crRNA target sequences. Following preliminary amplification assessment, the retained candidate primers were aligned individually against 75 complete genome sequences of megalocytivirus-related viruses. Of these, 67 sequences met the predefined criteria for target-region integrity, primer-binding-site compatibility, and Cas12a recognition. Retrospective mapping to the reference genome located the candidate amplification region within ORF057L. Based on the resulting candidate detection unit, a one-pot RPA-Cas12a assay incorporating a commercially available lyophilized RPA amplification module was developed. Optimization showed that 400 nM reporter and 80 nM crRNA-1 provided relatively stable fluorescence output. A cut-off value of 1281.6 relative fluorescence units (RFU) was established as the mean plus three standard deviations of the endpoint fluorescence values obtained from 20 qPCR-negative samples. In analytical sensitivity testing, the assay generated fluorescence signals above the negative control at low plasmid copy numbers. However, because only a limited number of replicates were tested at these low template concentrations, these findings were not used to define a formal limit of detection. ISKNV, RSIV, and TRBIV samples tested positive, whereas the MRV sample produced an endpoint fluorescence value below the cut-off. Repeatability analysis of the same sample in six independent reactions yielded a coefficient of variation of 8.03%. Among the 39 samples examined, no discordant qualitative results were observed between the RPA-Cas12a assay and qPCR. These findings support the use of the ORF057L-targeted one-pot RPA-Cas12a assay as a rapid preliminary screening tool for megalocytivirus-related viruses. Nevertheless, its formal limit of detection, inter-batch stability, cross-reactivity with additional non-target pathogens, and clinical diagnostic performance require further evaluation.

Type I interferons (IFNs) serve as pivotal functional molecules in the antiviral immune response of fish, and their signaling pathways require precise negative regulation to avoid tissue damage caused by excessive immune activation. In the present study, DEAD-box RNA helicase 3b (DDX3b), an ATP-dependent RNA helicase involved in cellular RNA metabolism and host-pathogen interaction, was identified in black carp (Mylopharyngodon piceus). Sequence and structural analyses showed that bcDDX3b was evolutionarily conserved and contained the characteristic DEXDc and HELICc domains of DEAD-box RNA helicases. Subcellular localization analysis showed that bcDDX3b was predominantly distributed in the cytoplasm and formed distinct cytoplasmic aggregates. Functional assays verified that black carp DDX3b (bcDDX3b) acted as a negative regulator of type I IFN signaling. Overexpression of bcDDX3b markedly suppressed both basal and virus-induced IFN promoter activities and decreased the expression levels of interferon-stimulated genes (ISGs), thereby blocking the establishment of cellular antiviral status. Mechanistically, bcDDX3b directly interacted with bcIRF7, the core transcription factor of IFN signaling pathway, and the DNA-binding domain (DBD) of bcIRF7 was indispensable for the interaction. The binding of bcDDX3b to bcIRF7 significantly impaired bcIRF7-mediated antiviral defenses against spring viraemia of carp virus (SVCV) and grass carp reovirus (GCRV). Further mechanistic investigations revealed that bcDDX3b inhibited the phosphorylation and ubiquitination of bcIRF7 and facilitated bcIRF7 degradation via a lysosome-dependent pathway. Collectively, bcDDX3b functions as a critical negative modulator of RLR-triggered type I IFN signaling in black carp. This study provides novel insights into the regulatory mechanism of IRF7 and the immune homeostasis underlying antiviral responses in teleost fish.

Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that catalyzes the desaturation of saturated fatty acids to monounsaturated fatty acids. Although lipid metabolism is increasingly recognized to influence antiviral innate immunity, the role of SCD1 in host defense, particularly in teleost fish, has not been fully explored. Here, we show that viral infection markedly suppresses SCD1 expression in zebrafish. Overexpression of zebrafish SCD1 significantly suppresses antiviral gene expression and enhances viral replication. Pharmacological inhibition of SCD1 desaturase activity enhances antiviral gene expression, reduces viral replication, and protects zebrafish larvae from viral lethality, indicating that SCD1 suppresses antiviral immunity in an enzymatic activity-dependent manner. Mechanistically, palmitic acid (PA), the saturated fatty acid substrate of SCD1, acts as a potent enhancer of antiviral innate immunity. PA treatment upregulates antiviral gene expression in vivo and in vitro, and restricts viral replication in ZFL cells. We demonstrate that PA exerts its immunostimulatory effects by inhibiting the two zebrafish isoforms of peroxisome proliferator-activated receptor α, PPARαa and PPARαb, as overexpression of either isoform attenuates PA-induced antiviral responses while a PPARα antagonist mimics the effects of PA. Furthermore, we show that PPARαa and PPARαb physically interact with interferon regulatory factor 3 (IRF3) and suppress IRF3-induced antiviral gene expression, thereby inhibiting type I interferon signaling. Collectively, our findings establish an SCD1-PA-PPARα-IRF3 regulatory axis in which SCD1 consumes its substrate PA to sustain PPARα-mediated negative regulation of IRF3-dependent antiviral gene expression, thereby restraining antiviral innate immunity. This study provides new insights into the metabolic control of host defense in teleost fish and highlights potential targets for antiviral strategies in aquaculture.

The present study evaluated the effect of short-term feeding with graded dietary protein levels on skin wound healing in largemouth bass (Micropterus salmoides). Three isolipidic diets containing 48% (P48), 52% (P52), and 56% (P56) crude protein were fed to fish (initial mean weight 13.00 g) for 5 weeks, followed by a standardized mechanical skin-wounding challenge. Wound repair was assessed at 1, 3, and 7 days post-wounding (dpw). The P52 diet supported better growth performance than the P48 diet. However, P48 accelerated wound closure and promoted more advanced histological repair, characterized by earlier re-epithelialization, more efficient necrotic tissue clearance, reduced wound bed area, and a thinner, more mature neo-epidermis. At the molecular level, P48 induced early upregulation of re-epithelialization-related genes, including mmp2, mmp9, and egf, accompanied by stronger MMP9 immunohistochemical signals. P48 was also associated with rapid inflammatory activation, shown by early increases in MPO, NAG, and LZM activities and il1b and tnfa expression, followed by faster inflammatory resolution and sustained il10 upregulation. In addition, P48 showed higher antimicrobial-defense-related markers and stronger extracellular-matrix-, angiogenesis-, and collagen-associated responses, as evidenced by higher expression of hamp, prf, fn1, lamb2, vegf, shh, and col1a1, stronger CD31-positive immunofluorescence signals, and greater collagen fiber accumulation. Western blotting further showed that FN1, SHH, and CCN1 protein responses were broadly consistent with their corresponding transcript-level patterns. Temporal response analysis further indicated a coordinated transition from early epithelial and inflammatory responses to later repair and remodeling responses. Correlation analysis showed that reduced wound area was significantly and negatively associated with both ccn1 transcript abundance and CCN1 protein abundance. These findings indicate that short-term low-protein feeding enhances skin wound healing and may represent a stage-specific nutritional strategy for farmed largemouth bass.

This study utilized single-cell transcriptomics to reveal the cellular heterogeneity and cell-cell communication networks in free gossypol-induced enteritis in juvenile largemouth bass (Micropterus salmoides). Four isonitrogenous and isolipidic diets were formulated: a fishmeal-based control (FM) and three diets supplemented with 150, 300, or 600 mg/kg gossypol acetate (FG150, FG300, or FG600). Healthy fish (14.80 ± 0.10 g) were randomly assigned to four groups (four replicates of 25 fish each) and fed for eight weeks. Results showed that increasing dietary FG progressively impaired growth performance and intestinal health. Compared to FM group, FG600 group exhibited lowest growth performance; reduced plica height/width, muscular layer thickness, and goblet cell numbers; elevated serum lipopolysaccharide and D-lactic acid; decreased serum phosphatase activities; lowest intestinal total antioxidant capacity; downregulated tight-junction genes (such as zo-1, zo-2, occludin, and claudin15); upregulated pro-inflammatory cytokines (tnf-α, il-1β, il-8, and nf-κb1); and increased abundance of potentially pathogenic bacteria (Aeromonas and Vogesella). Single-cell transcriptomic identified nine intestinal cell types, including epithelial cells, B cells, macrophages, and others. In FG600 group, proportions of epithelial cells, macrophages, and B cells in the G1 phase decreased, along with reduced communication strength/probability among neuroendocrine cells, M1 macrophages and mature B cells. Pseudotime analysis identified branch point 1 as a critical immune-related node in epithelial differentiation. In conclusion, FG impairs growth performance and intestinal health in juvenile largemouth bass, likely by increasing abundance of potential pathogens, disrupting neuroendocrine-immune cell communication; activating macrophages/B cells, inducing inflammation, inhibiting epithelial repair, and compromising intestinal barrier function.

The Gasdermin (GSDM) family drives pyroptosis via membrane pore formation, regulating immunity and disease. In teleosts, Gasdermin E (GSDME) is the sole pyroptosis executor but diverged into paralogs (GSDMEa/b) in cyprinids. We identified four GSDME paralogs (CaGSDMEa1/a2/b1/b2) in Carassius auratus gibelio and characterized functional divergence between CaGSDMEb1/b2. Both paralogs show broad tissue expression and are upregulated during Aeromonas hydrophila infection or LPS stimulation, indicating the involvement of antibacterial immune responses. However, only CaGSDMEb2 executes pyroptosis by being cleaved by Cacaspase-19 (caspase-4/5 homolog) upon cytosolic LPS detection, although CaGSDMEb1-NT and CaGSDMEb2-NT form pores. Evolutionary analysis identified a key divergence that CaGSDMEb1 lacks the conserved aspartic acid (ASP) in the caspase cleavage motif, rendering it resistant to proteolytic activation. Notably, full-length CaGSDMEb1 undergoes subfunctionalization by binding the N-terminal domain of Cacaspase-19, enhancing caspase activity and amplifying pyroptosis. Thus, CaGSDMEb1 may regulate pyroptosis through a full-length protein-dependent mechanism, bypassing its NT domain. These findings suggest functional divergence between duplicated GSDME genes-CaGSDMEb2 retains ancestral executioner activity, while CaGSDMEb1 may have evolved a regulatory role-and suggest a unique amplification loop for pyroptosis. This study provides insights into the evolutionary plasticity of the ancient GSDME family.

Gene associated with retinoid-interferon-induced mortality 19 (GRIM19) can affect viral infection by regulating lipid metabolism, and modulate inflammatory factors via the mitochondrial respiratory chain. However, the function of fish GRIM19s remains unclear. In this study, the GRIM19 gene (Ec-GRIM19) was identified from orange-spotted grouper (Epinephelus coioides). The amino acid sequence of Ec-GRIM19 shared highest identities with Epinephelus lanceolatus GRIM19. The in vivo analysis indicated that Ec-GRIM19 has the highest expression levels in the liver and kidney. The expression of Ec-GRIM19 was significantly down-regulated in GS cells after Red-spotted Grouper Nervous Necrosis Virus (RGNNV) infection. In vitro, overexpression of Ec-GRIM19 downregulated the expressions of lipid metabolism-related genes (SREBP1, FASN) and Capsid Protein (CP) and RNA-dependent RNA polymerase (RdRp) gene of RGNNV, induced the expressions of interferon-related factors (NLRP3, IL-1β and IL-18). And the knockdown of Ec-GRIM19 in vitro have the opposite regulatory effects. Besides, the production of ATP was suppressed, and the ROS level was increased, and the mitochondrial membrane potential was decreased, indicating inhibition of the mitochondrial respiratory chain. These data provide new insights to further investigate the interaction between fish and viral infections.

This study examined the effects of using synthetic vs a natural source of astaxanthin (AXT) on the histomorphometry of mucosal organs (intestine, skin, and gills), erythrocyte morphology, liver and trunk kidney health, and the expression of mucosal immune-related mucin genes (muc2, muc5ac1, muc5ac2, muc5b) and antimicrobial peptide (AMP) genes (def1, def2, def3, cath1) in Atlantic salmon (Salmo salar). Three experimental diets were formulated based on AXT sources: a control diet (CO), synthetic astaxanthin, and two additional diets containing an emulsion of wall-broken whole-cell algal astaxanthin (AW) and extracted algal astaxanthin (AE), both obtained from the microalga Haematococcus pluvialis. Each dietary group had six replicates, and the feeding trial lasted for 105 days. Afterward, a subset of fish from each group underwent a 14-day challenge test to assess stress tolerance. The results showed that the histoarchitecture of the intestine, skin, and gills was affected in both the CO and AW groups during the feeding and challenge phases. Mucosal mapping revealed increases in volumetric density, mucous cell (MC) area, and mucosal barrier integrity in these groups. The frequency of erythrocyte abnormalities was lower in the CO and AW groups compared to the AE group. Conversely, the AE diet was associated with significantly larger vacuole area (%) and mean vacuole size (μm) in hepatocytes. Similarly, the AE group showed a higher percentage and larger neutral lipid droplets in liver tissue. Structural anomalies in the trunk kidneys, assessed via semi-quantitative scoring, were more severe in the AE group than in the CO and AW groups. The expression of mucosal immune-related mucins and AMP genes was upregulated in both the CO and AW groups compared to the AE group. Notably, muc2, muc5b, and def3 in the distal mid-intestine, and muc2 in the skin showed significantly higher expression in the CO group than in the AE group. Overall, the findings suggest that supplementing the diet with synthetic and emulsified wall-broken whole-algal cell astaxanthin (57.0 and 43.0 mg/kg, respectively) influenced mucosal mapping and histomorphometry, which are linked to a supportive mucosal immune status and improved health in Atlantic salmon through a combination of structural and physiological changes.

Piscidins are cationic α-helical antimicrobial peptides (AMPs) that constitute a key component of the innate immune defense of teleost fish, yet the relationship between their genomic organization, structural properties, and functional specialization remains incompletely understood. In this study, six piscidin peptides from Epinephelus akaara, Seriola dumerili, Thunnus maccoyii, Argyrosomus regius, Dicentrarchus labrax, and Epinephelus coioides were characterized through an integrated sequence-to-function approach combining comparative genomics, structural modeling, physicochemical analysis, and in vitro validation, with the aim of identifying candidates with potential for biomedical and biotechnological applications. All genes studied exhibited the conserved four-exon, three-intron architecture characteristic of teleost piscidins. Structural modeling and circular dichroism confirmed α-helical conformations under membrane-mimetic conditions, despite measurable differences in hydrophobicity, charge distribution, and predicted membrane insertion parameters. Antimicrobial assays revealed distinct functional profiles: Sd_FI25 and Epinecidin_1 displayed broad antibacterial activity against Gram-positive and Gram-negative pathogens, whereas Dl_FI22 showed selective activity with reduced temporal persistence associated with lower peptide stability. Ea_FF25 exhibited comparatively weak antibacterial potency. Antibiofilm activity varied among peptides and did not uniformly parallel planktonic MIC values. Computational predictions further suggested antiviral and antitumoral potential for several sequences, extending their prospective relevance beyond classical antibacterial roles. Conserved genomic architecture and α-helical structure coexist with pronounced functional diversification among teleost piscidins. These findings demonstrate that integrating structural prediction with experimental validation is an effective strategy for identifying fish-derived innate immune peptides as candidates for biomedical applications.

Hemoglobin-containing bivalves are a unique object for studying the interaction between respiratory pigments and the immune response, but the sources for reactive oxygen species (ROS) production during immune activation in these species remain poorly understood. In this work, we investigated for the first time the mitochondrial source of ROS production in A. kagoshimensis hemocytes under non-specific immune system activation conditions by lipopolysaccharide (LPS). Stimulation by LPS for 1.5 h led to a significant twofold increase in the intracellular ROS level and hyperpolarization of the mitochondrial membrane. The use of specific electron-transport chain inhibitors (rotenone, S3QEL, azide) supports a contribution of the ubiquinone site (Qo) of mitochondrial complex III to ROS production in both unstimulated and during immune activation. Inhibition of complexes I and IV reduced the membrane potential, did not significantly alter basal or LPS-induced ROS production level. Despite a significant oxidative burst, short-term stimulation provided no evidence of DNA damage or impaired hemocyte adhesion over the period examined. These findings suggest that mitochondrial complex III contributes to ROS production in regulating the oxidative burst during immune responses in hemoglobin-containing mollusks.

Cyclic GMP-AMP synthase (cGAS) could recognize double-stranded DNA in the cytoplasm and catalyze the synthesis of cyclic GMP-AMP (cGAMP). This molecule activates the endoplasmic reticulum-resident protein stimulator of interferon genes (STING), thereby initiating the TBK1 (TANK-binding kinase 1)-IRF3 (interferon regulatory factor 3) signal cascade and promoting the expression of type I interferons and various inflammatory factors. The cGAS-STING pathway serves as a fundamental signal axis for DNA recognition in the cytoplasm of vertebrates and is crucial for innate immune defense. Recent research on the cGAS-STING pathway in fish has gradually advanced, with gene cloning and analyses completed across various fish species, indicating that this pathway plays a significant role in antiviral responses in fish. However, compared to mammals, the cGAS-STING pathway exhibits relative deficiencies in molecular regulatory mechanisms, structural analyses, and in vivo functional validations in different aquatic animals. Teleost fish occupy a critical stage in vertebrate evolution. The cGAS-STING pathway in teleost fish is systematically summarized to enhance our comprehensive understanding for the innate immune regulatory network, and the potential molecular targets is to be identified for disease-resistant breeding, immune enhancement, and sustainable aquaculture practices.