Hepcidin is an important antimicrobial peptide (AMP), yet the structural based classification of AMPs can be debated due to the additional role of hepcidin in iron homeostasis. Sequence analyses showed that the pro-peptide region of fish hepcidin exhibits substantial diversification, and the mature peptide remains comparatively conserved in fish, characterized by eight cysteine residues forming four intramolecular disulfide bonds. These conserved cysteines are essential for stabilizing a hairpin structure composed of two antiparallel β-sheets, an amphipathic configuration that is believed to facilitate interaction with microbial membranes. However, this structural conserveness is also observed in isoforms dedicated for iron homeostasis, which has become a primary role in higher vertebrates predicted through evolution because the mature peptide also showed substantial difference in amino acid sequence in higher vertebrates compared to fish. Positive Darwinian selection observed in fish hepcidins, likely due to pathogen-driven evolutionary pressure in diverse aquatic environments. Although hepcidin is predominantly expressed in the liver, induced expression was also noticed in spleen, gills, intestine, and skin. Recent studies have demonstrated that after cleavage from the propeptide, the mature peptide predominantly localizes to mucosal surfaces, including the skin and gills, indicating its role as a first line of defense. Nevertheless, fish hepcidin exhibits broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria, viruses, and parasites. It interacts with the specific microbial membrane, resulting in membrane disruption and ultimately death of the microorganism. Beyond direct antimicrobial action, evidence of interaction of hepcidin with various immune pathways, including inflammatory responses, complement, Toll-like receptors, and lysozyme and involvement in iron homeostasis gives evidence of physiological networks of hepcidin. Therefore, instead of categorizing AMPs on the basis of function or structure, we need to open up our understanding of physiological systems. Although fish hepcidin is well established as a potent antimicrobial peptide, and its involvement in complex physiological networks highlights the sophistication of living systems still it's a perfect example how genes evolve to perform different function.
Climate change has been linked to increased disease outbreaks in aquatic ectotherms, threatening species persistence. Fish rely primarily on the innate immune system, yet its thermal sensitivity remains poorly understood. We investigated temperature effects on innate immune cell behavior in zebrafish (Danio rerio) larvae by quantifying neutrophil and macrophage recruitment to a wound site and measuring tail regeneration across five ecologically relevant temperatures (22 °C, 28 °C, 31 °C, 33 °C, and 35 °C). Using transgenic zebrafish with fluorescently labelled immune cells, we performed in vivo imaging for 12 h post-tail amputation to track cell recruitment dynamics. Tail regeneration was assessed over three days. Integrating these traits into the thermal performance curve framework enabled comparisons of temperature sensitivities, optima and performance breadth. Neutrophil recruitment rates increased consistently with warming, while macrophage recruitment and tissue regeneration peaked more narrowly around 30-31 °C. This indicates that these processes exhibit distinct thermal sensitivities. By providing real-time, in vivo data on immune cell behavior, this study contributes to improving our understanding of how temperature influences immune responses in fish. Overall, moderate warming (30-31 °C) may enhance the cellular immune response in zebrafish, while higher temperatures disrupt immune resolution, highlighting the need to integrate immune performance into climate change vulnerability assessments of aquatic ectotherms.
As an economically important freshwater fish, catfish (Silurus asotus) has been cultivated on an increasingly large scale in recent years. However, disease outbreaks caused by bacterial pathogens have grown more severe. In this study, a pathogenic strain of Aeromonas hydrophila was isolated from diseased catfish fry. An acute toxicity assay showed that the 96-h LC50 of A. hydrophila for catfish was 1.9 × 107 CFU/mL. Experimental challenge confirmed that reinfection with this strain caused pathological damage in the intenstins, spleen, and kidney tissues. Both in vitro and in vivo experiments demonstrated that overexpression of SaMyd88 enhanced the expression of multiple immune factors, including IL6 and IFNφ1, and subsequently reduced the bacterial load of A. hydrophila. Consistently, knockdown of SaMyD88 not only suppressed inflammatory factor expression but also markedly inhibited IFN expression. Furthermore, pharmacological inhibition of IFNφ1 with GSK8612 diminished the ability of MyD88 to control A. hydrophila replication in the spleen of catfish. Together, these results indicate that MyD88 bridges IL and IFN signaling pathways to coordinate a synergistic defense against A. hydrophila infection.
This study aimed to explore whether Lactobacillus amylovorus (LA) mitigates acute ammonia-induced intestinal damage in yellow catfish (Pelteobagrus fulvidraco). A 14-day experiment used 360 healthy fish (initial body weight: 6.26 ± 0.05 g), randomly assigned to 4 groups (3 replicates, 30 fish/replicate): control (CON, 0 mg/L T-AN), ammonia exposure (AM, 125 mg/L T-AN), LA supplementation (LA, 0 mg/L T-AN + LA), and LA + AM (125 mg/L T-AN + LA). LA was sprayed onto commercial feed at 1 × 108 CFU/g. The results showed that the LA + AM had a significantly higher survival rate (78.33 ± 12.58% vs. 40.00 ± 5.00% in AM, P < 0.05), reduced serum/intestinal ammonia levels, alleviated intestinal villi breakage/shedding, and mitigated oxidative stress (lower ROS, MDA; higher SOD, CAT, GSH, T-AOC, P < 0.05). LA regulated inflammation (downregulated il-1β/il-6/il-8/tnf-α/nf-κb mRNA and IL-1/IL-6/p-NF-κB p65 protein; upregulated IL-10 protein, P < 0.05), enhanced intestinal barrier function by upregulating tight/adherens junction (zo-1, claudin-1, occludin) gene/protein (ZO-1, E-cadherin) expression (P < 0.05), and modulated gut microbiota (increased Lactobacillus/Cetobacterium abundance, decreased Plesiomonas/Proteobacteria, elevated α-diversity, P < 0.05). In conclusion, LA protects yellow catfish from ammonia-induced intestinal damage via antioxidant, anti-inflammatory, barrier-enhancing and microbiota-modulating effects, promising as a nutritional strategy to alleviate ammonia stress in intensive aquaculture.
Vaccination is widely regarded as the most effective strategy for controlling infectious diseases that compromise the sustainability and productivity of global aquaculture. Although inactivated and subunit vaccines are commonly applied, their protective efficacy often depends on potent adjuvants to induce robust and durable immune responses. In aquatic settings, environmental exposure and physiological barriers in fish-including enzymatic degradation, pH fluctuations, osmotic stress, and mucosal and integumentary defenses-pose substantial challenges to antigen stability and delivery efficiency. To address these constraints, advanced delivery systems have been developed to enhance antigen protection, facilitate transport to lymphoid tissues, and improve antigen presentation to immune cells. Among these, biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA)-based microparticles (MPs) and nanoparticles (NPs) have emerged as versatile and promising platforms. PLGA has been extensively utilized in human pharmaceutical applications and is increasingly being investigated for veterinary purposes, including fish vaccines. This review summarizes the physicochemical properties of PLGA, elucidates its controlled degradation behavior and antigen release kinetics, and examines PLGA particle-mediated antigen uptake, processing, and presentation mechanisms that contribute to immune activation in teleost fish. Recent advances in PLGA-based vaccines targeting major aquatic pathogens are also discussed. By enhancing antigen stability, prolonging immune stimulation, and enabling non-invasive administration routes such as oral and immersion vaccination, PLGA-based delivery systems represent a promising strategy for aquatic immunoprophylaxis. Their broader implementation may further reduce antibiotic reliance in aquaculture, thereby mitigating antimicrobial resistance and environmental impacts while supporting sustainable industry development.
Reducing dependence on marine-derived ingredients like fishmeal, while maintaining the robustness of carnivorous species, like European seabass (Dicentrarchus labrax), remains a challenge for sustainable aquaculture. Photobacterium damselae subsp. piscicida (Phdp) outbreaks are still responsible for significant economic losses, but functional ingredients capable of modulating immune responses may enhance resilience to a Phdp challenge. In this study, fish were fed four balanced diets for 89 days: a plant-based control (CTRL) and three diets including three protein hydrolysates (SHARK, FISH and SWINE) at the expense of fishmeal protein. After the feeding trial, fish were intraperitoneally injected with Phdp and sampled at 0, 4, and 24 h post-infection for hepatic oxidative stress, plasma biochemical profile and gene expression related to intestinal integrity and immune response. All diets were well accepted and no differences in survival could be detected. Hydrolysate inclusion induced distinct baseline physiological states, likely shaping subsequent responses to infection. SWINE and SHARK diets increased hepatic glutathione reductase activity, while SHARK-fed fish consistently showed lower lipid peroxidation (LPO). In contrast, FISH-fed fish exhibited a higher LPO and lower circulating proteins. Upon infection, a metabolic reorganization was triggered across all groups, including a reduction of plasma glucose except for SHARK. In this group, stable glycemia occurred alongside reduced expression of intestinal tight junction markers, suggesting a potential trade-off between energy homeostasis and barrier integrity. Overall, replacing fishmeal protein with animal-derived hydrolysates in high plant-based diets modulated host responses through primarily metabolic and redox pathways, without clear improvements in resistance to Phdp challenge.
Sex differences are a basic but still underexplored feature of mucosal immune regulation. Forkhead box P3 (FOXP3) is a key regulator of regulatory T cell (Treg) function, but how its activity differs between sexes and across organs remains unclear. Here, we identify foxp3a as the functional zebrafish homolog of mammalian FOXP3 and use a foxp3a-deficient zebrafish model to examine how immune and metabolic processes are coordinated along the gut-liver-gonad axis. Loss of foxp3a was associated with clear sex- and age-dependent changes in intestinal structure and immune composition, including epithelial barrier damage, altered goblet cell patterns, and increased immune cell infiltration. Transcriptomic analyses showed sustained activation of innate immune and inflammatory pathways in males, while females showed a more transient immune response together with metabolic pathway enrichment, suggesting compensatory regulation. Similar sex-biased patterns were observed in extraintestinal organs. The liver showed ongoing metabolic disruption, while the gonads displayed delayed ovarian development and inflammatory and stress-associated signatures in the testis. Single-cell RNA sequencing of intestinal mucosal cells revealed sex-specific immune remodeling, including reduced Treg populations and expansion of Th1 and Th17 cells, alongside distinct immune-metabolic gene programs across cell types. Deficiency of foxp3a was also linked to progressive, sex-dependent changes in gut microbiota composition and predicted microbial functions. Integrated multi-omics analyses support a model in which foxp3a contributes to sex-biased coordination of immune, metabolic, and reproductive processes across organs. Together, these findings position foxp3a as a key factor in sexually dimorphic mucosal immune regulation and highlight zebrafish as a useful system for studying sex-specific immune dysfunction.
Antimicrobial peptides (AMPs) are effective against pathogens; however, their application in aquaculture remains limited due to low stability and poor delivery efficiency. In this study, we designed a xylose-induced-engineered probiotic to enable tightly controlled production and efficient targeted delivery of AMPs. Specifically, the tilapia-derived piscidin-1 and hepcidin were separately cloned into the Bacillus subtilis 168 (BS168) expression vector, resulting in the successful construction of two recombinant engineered strains-BS168-sfGFP-piscidin and BS168-sfGFP-hepcidin. These two engineered strains could significantly inhibit the growth of Aeromonas hydrophila compared with BS168 transformed with an empty vector (pSTOP1622) in vitro. To investigate the additive bacteriostatic activity, piscidin-1 and hepcidin were fused in tandem within the same expression vector, generating BS168-sfGFP-PH. These three engineered strains were orally administered to zebrafish to evaluate their antimicrobial efficacy in vivo. The results showed that zebrafish fed with BS168-sfGFP-PH exhibited the highest survival rate following A. hydrophila challenge. This protection was attributed to the strain's ability to reduce intestinal pathogen load, thereby suppressing inflammatory responses and improving intestinal integrity. Collectively, this work established an orally deliverable probiotic for secreting AMPs, serving as a promising strategy against bacterial infections in aquaculture.
Feeding low fishmeal diets (LF) in aquaculture can induce oxidative stress and impair intestinal and immune homeostasis of shrimp. This study explored the impact of dietary phytosterol on intestinal health, antioxidant capacity, immune-related responses, sterol metabolism, and microbiota composition in juvenile Litopenaeus vannamei fed an LF diet. A total of 800 shrimp (0.29 ± 0.03 g) were randomly allocated to 20 tanks and were fed one of five diets for 7 weeks: a high fishmeal diet (HF), a LF diet, or the LF diet enrinched with phytosterol at 0.02% (LP1), 0.04% (LP2), or 0.08% (LP3). Growth performance did not differ significantly among treatments. However, dietary phytosterol (0.02-0.08%) improved antioxidant status by increasing superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) and decreasing malondialdehyde (MDA), while also enhancing the hemolymph lipid profile via reduced triglycerides (TG) and total cholesterol (T-CHO) and increased high-density lipoprotein (HDL-C). Histologically, 0.08% phytosterol enhanced hepatopancreatic tubule integrity and intestinal microvillus structure. Phytosterol supplementation modulated the expression of genes involved in fatty acid oxidation, sterol metabolism, immune responses, and apoptosis regulation. Phytosterol supplementation enriched beneficial gut microbiota (Bacillus) while reducing potential pathogens. In conclusion, the results indicate that dietary phytosterol provides protective effect against oxidative and intestinal stress induced by LF diet and enhances immune-related physiological homeostasis for juvenile L. vannamei, thereby supporting its use as a functional immunonutritional additive in LF feeds.
Precise post-transcriptional regulation is essential for balancing antibacterial defense and inflammatory homeostasis in teleost innate immunity. Here, we identified a competing endogenous RNA (ceRNA) axis involving lncRNA MSTRG.14394.1, miR-144-3p, and the nuclear receptor NR1H3 in Cynoglossus semilaevis. miR-144-3p was markedly induced by bacterial pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN), and promoted hepatocyte injury and pro-inflammatory cytokine mRNA expression. Mechanistically, miR-144-3p directly targeted NR1H3 mRNA, whereas MSTRG.14394.1 acted as a ceRNA to sequester miR-144-3p and partially restore NR1H3 mRNA expression. These interactions were supported by luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. In vivo experiments in Danio rerio further showed that modulation of this axis significantly affected host survival following Vibrio anguillarum infection. Collectively, these findings identify a ceRNA-mediated regulatory network that modulates inflammatory responses in teleost fish and provide potential molecular targets for improving disease resistance in aquaculture.
The paracaspase MALT1 plays a central role in regulating NF-κB and MAPK signalling pathways that, in mammals, control lymphocyte differentiation and survival. Rainbow trout possess three MALT paralogues that show distinct tissue- and cell-specific expression patterns. Malt1 transcripts predominated in the head kidney, malt2 was most highly expressed in the spleen and trunk kidney and malt3 was dominant in the liver and intestine. Integration with publicly available bulk and single-cell RNA-seq datasets supported these patterns and indicated broad expression of malt3 across tissues. At the structural level, Malt3 differs from Malt1 and Malt2 by the absence of a death domain. We explored potential functional differences using CHSE-214 cells as a heterologous system for overexpression studies. Luciferase reporter assays revealed promoter-specific activation of NF-κB signalling, where Malt1 and Malt3 activated the human ELAM promoter and Malt2 and Malt3 induced the teleostean saa promoter, whereas none of the three Malt proteins activated the cxcl8 promoter. Overexpression of individual Malt proteins resulted in gene- and stimulus-dependent modulation that generally attenuated the stimulus-induced expression of several pro-inflammatory genes. A notable finding, however, was the strong upregulation of mx1 transcripts in Malt-overexpressing CHSE-214 cells upon stimulation with poly(I:C). Co-expression experiments further suggested combinatorial effects, including enhanced suppression following zymosan stimulation and selective enhancement of il1b expression in response to flagellin, when all three paralogues were expressed together. Overall, these results provide an initial indication that Malt paralogues may contribute to context-dependent modulation of innate immune signalling; however, further studies in physiologically relevant systems are required.
Edwardsiella piscicida is a potent intracellular pathogen that infects a wide variety of freshwater and marine fish, causing systemic hemorrhagic sepsis that results in lethality. Although vaccines against E. piscicida have been developed for many fish species, a direct comparison between innate and adaptive immunity in protecting fish from E. piscicida infection has not been performed. We established an il2rb mutant lacking natural killer (NK) cells, innate type lymphocytes, showing that NK cells are required for protection against E. piscicida infection in adult medaka. In addition, we elucidated the role of the TLR signaling pathway transmitted by Myd88 in resistance to E. piscicida infection in larvae and adults. These results indicate the requirement of innate immunity in resistance to this intracellular pathogen, providing insights into vaccination strategies for fish.
Although CD4+ T cells are known to play a crucial helper role in mammalian humoral immunity, the function of early-evolved CD4-1+ T cells in teleost fish for assisting B cell-mediated antibody production remain to be elucidated. Using grass carp (Ctenopharyngodon idella) as a model, this study demonstrated that CD4-1+ T cells play a critical role in vaccine-induced antibody production and antibacterial immunity. We confirmed that inactivated Aeromonas hydrophila vaccine significantly induced interleukin-21 (IL-21) expression in grass carp head kidney leukocytes (HKLs), and identified CD4-1+ T cells as the primary cellular source of IL-21. In vitro experiments showed that IL-21 effectively promoted B cell differentiation into plasma cells and enhanced IgM secretion, which synergized with A. hydrophila to exert a more pronounced effect. Meanwhile, our in vivo studies demonstrated that IL-21 possess molecular adjuvant function when co-immunized with A. hydrophila. Depletion of CD4-1+ T cells reduced A. hydrophila-elicited specific IgM levels and antibacterial immune protection, confirming their essential helper function. Injection of IL-21 into CD4-1+ T cell depletion fish can partially improve the survival rate but could not restore antibody levels, indicating that while IL-21 has direct immunoenhancing effects, CD4-1+ T cells are essential for a full antibody response. In conclusion, these results demonstrated the critical roles of CD4-1+ T cells and their secreted IL-21 in vaccine-induced humoral immunity in grass carp, providing a foundation for identifying Tfh-like cells in teleost fish and supporting the potential application of IL-21 as molecular adjuvant in fish vaccines.
In this study, the native IgM of half-smooth tongue sole (Cynoglossus semilaevis), an economically important marine flatfish, were purified, and used as the immunogen for preparation of monoclonal antibody (mAb). Based on the mAbs, double-antibody sandwich ELISA was constructed for the detection of IgM in C. semilaevis serum and capture ELISA were established for vaccine efficacy evaluation. Results showed that two monoclonal antibodies (mAbs) specifically targeting IgM of half-smooth tongue sole (Cynoglossus semilaevis) were successfully obtained (designated as 3A12 and 5F39). The mAbs exhibited high specificity to native C. semilaevis IgM, without cross-reactivity with IgM from other teleost species. Furthermore, a double-antibody sandwich ELISA was further established for the detection of serum IgM in C. semilaevis with the mAbs pair, which showed a linear detection range of 0.156-10 μg/mL for purified IgM, with a limit of detection (LOD) of 0.313 μg/mL. Using the developed sandwich ELISA, it was found that the IgM level in large-sized fish was significantly higher than that in small-sized fish, and immunostimulants could remarkably enhance the IgM level in fish. Moreover, the specific IgM antibody levels in the serum of C. semilaevis after immunization with inactivated Vibrio vulnificus were analyzed by capture ELISA using mAb 5F39 as the detection antibody, a time-dependent variation trend was found in the specific IgM antibody titer among the immunized group. In conclusion, the mAbs targeting C. semilaevis IgM could specifically and efficiently recognize the native IgM protein, and served as a reliable tool for the detection of serum IgM, providing essential technical support for immune status assessment and vaccine development in C. semilaevis aquaculture.
Aeromonas salmonicida subspecies salmonicida is a Gram-negative fish pathogen that synthesizes and secretes melanin, a dark pigment with a complex molecular structure. Melanin is formed by polymerized phenolic and/or indole monomers, which possess antioxidant, radical scavenging, photo-protective, and even semiconducting properties. The role of melanin in A. salmonicida pathogenesis is unknown. In this study, we evaluated the effect of A. salmonicida melanin on Atlantic salmon (Salmo salar) head kidney and spleen. Atlantic salmon were intraperitoneally injected with A. salmonicida melanin (100 μg/100 g of fish). Fish injected with Tris-OH 20 mM pH 8.0 buffer were used as a control. Head kidney tissue samples were taken at innate immune response (6, 24, 48 h post-injection (hpi)), adaptive immune activation (168 hpi), and metabolic adjustment phase (336 hpi) for transcriptomics and histological analysis. Transcriptomic analysis followed an innate immune response kinetics with a peak of dysregulated genes at 24 hpi, followed by a decrease at 48 hpi. At 7 days post-injection (168 hpi), a second peak of dysregulated genes was observed, and at 14 day post-infection (336 hpi) the gene expression decreased. These results suggests that A. salmonicida melanin is not toxic to Atlantic salmon, remains in the animal, and induction of innate and adaptive immunity. Gene ontology analysis, indicate that A. salmonicida melanin induced an anti-viral innate and T-cytotoxic cell adaptive immune response. Immunohistochemistry showed that melanin promote the synthesis of CD10 and IgM in spleen and head kidney, suggesting that A. salmonicida melanin induce lymphocyte division. In summary, melanin produced by A. salmonicida appears to promote the division of antigen-presenting cells, potentially supporting the formation of cellular niches for intracellular infection and growth.
Streptococcus agalactiae serotype Ia is a major bacterial pathogen in Nile tilapia (Oreochromis niloticus), and vaccines for juvenile fish, especially immersion vaccines, remain unavailable. We evaluated live attenuated vaccine candidates based on deletion of capsule alone (Δcps) or capsule plus hemolysin (Δcps/Δcyl), and compared them with an oil-adjuvanted inactivated vaccine. Nile tilapia were vaccinated by intraperitoneal injection with an inactivated bacterin, live Δcps, or live Δcps/Δcyl, or by immersion with live Δcps/Δcyl. Mock-vaccinated fish served as controls. After approximately 1000° days, fish were challenged intraperitoneally with a virulent S. agalactiae Ia at high, medium, and low challenge doses. ELISA measured antibody responses, and group means ELISA values were plotted against mortality for each challenge dose. Bacterial DNA in immune organs, post-challenge vaccination, and in brain tissue post-challenge. Intraperitoneal vaccination induced significant systemic antibody responses, whereas immersion vaccination induced only a modest, non-significant increase. Bacterial DNA persisted transiently in the spleen and kidney after intraperitoneal vaccination but was not significantly elevated after immersion at 1000°-days. Protection varied with vaccine type and challenge pressure. The oil-adjuvanted inactivated vaccine showed the highest efficacy, with relative percent survival of 95-100%. The live Δcps mutant given intraperitoneally yielded 71 to 100% relative percent survival, the live Δcps/Δcyl mutant given intraperitoneally yielded 38 to 89%, and immersion with Δcps/Δcyl yielded 33 to 61%. Only the inactivated oil-adjuvanted vaccine markedly reduced bacterial DNA in the brain after challenge. Across groups, higher mean ELISA antibody levels were generally associated with lower mortality at the high-, medium-, and low-challenge doses. Intraperitoneal delivery of both inactivated and live attenuated vaccines induced the strongest systemic responses and conferred the greatest protection. However, immersion with the live attenuated Δcps/Δcyl strain still conferred moderate protection and remains a promising approach for juvenile tilapia. The findings support the further development of live attenuated immersion vaccines, including possible prime-boost strategies, and suggest that capsule expression is not required to induce protective immunity against S. agalactiae Ia.
This study aimed to evaluate the effects of dietary supplementation with Bacillus velezensis FL9 on growth performance, immune response, intestinal health, and disease resistance of Cyprinus carpio. A total of 420 fish (initial weight of 1.86 ± 0.25 g) were randomly assigned into four groups: a control group (C) and three treatment groups fed diets supplemented with B. velezensis FL9 at 1 × 105 CFU/g (SC), 1 × 107 CFU/g (MC), and 1 × 109 CFU/g (LC), respectively. After 56 days of feeding, ten fish per group were intraperitoneally challenged with Aeromonas hydrophila (2 × 107 CFU/mL) for 48 h. The results showed that dietary B. velezensis FL9, especially at 1 × 109 CFU/g, significantly improved growth performance, evidenced by increased weight gain rate (WGR) and a reduced feed conversion rate (FCR) (P < 0.05). The LC group enhanced liver antioxidant and immune-related enzyme activities (ACP, AKP, SOD, GST), reduced MDA content, improved intestinal digestive enzyme activities (lipase, pepsin, and α-amylase), and upregulated liver expression of immune-related genes (C3, LYZ, TLR1, TLR2) (P < 0.05). Moreover, the experimental treatment significantly improved the intestinal morphology, including villus height and muscular thickness. It also improved intestinal morphology and increased microbial diversity, with Cetobacterium becoming dominant in the MC group. After A. hydrophila challenge, the experimental groups showed significantly lower mortality, elevated ACP activity, and a modulated liver expression of immune-related genes (C3, LYZ, TLR1, TLR2, IL-8) (P < 0.05). Microbiota analysis revealed that, alongside Aeromonas, the relative abundance of beneficial bacteria in the experimental groups. In summary, dietary B. velezensis FL9 can improve growth performance, immunity, intestinal health, and disease resistance in C. carpio. Furthermore, our study found that the optimal concentration of B. velezensis FL9 in the diet was 1 × 109 CFU/g.
FinTRIM (FTR) proteins are a lineage-specific subfamily of TRIM E3 ubiquitin ligases in teleost fish and play important roles in antiviral innate immunity; however, the functions and underlying mechanisms of many FTR members remain poorly understood. In this study, we characterized a previously unappreciated FTR protein, CcFtr82, in common carp (Cyprinus carpio) and investigated its function in antiviral responses against spring viremia of carp virus (SVCV). We found that Ccftr82 expression was markedly induced by SVCV infection both in vivo and in vitro. Functional analyses revealed that overexpression of CcFtr82 significantly inhibited SVCV replication, whereas knockdown of endogenous ftr82 enhanced viral proliferation. Mechanistically, CcFtr82 positively regulated type I interferon (IFN-I) signaling by interacting with interferon regulatory factor 3 (Irf3) and promoting its K63-linked polyubiquitination. This modification facilitated Tbk1-Irf3 complex formation, enhanced Irf3-mediated antiviral signaling, and led to increased expression of ifnφ1 and interferon-stimulated genes (ISGs). Collectively, our findings identify CcFtr82 as a novel positive regulator of Irf3-dependent antiviral innate immunity and expand the functional understanding of finTrim proteins in teleost fish, providing new insights into host defense mechanisms against viral infection.
Obesity-related chronic inflammation in adipose tissue is closely associated with macrophage infiltration and M1 polarization. Adipose tissue macrophages (ATMs) aggravate metabolic disorders and inflammation in adipocytes. However, the molecular mechanisms underlying this process in fish remain unclear. In this study, we established an in vitro co-culture system comprising M1 macrophages and adipocytes derived from large yellow croaker (Larimichthys crocea). The results demonstrated that M1 macrophages significantly upregulated the mRNA expression of pro-inflammatory genes in co-cultured adipocytes, while downregulating the anti-inflammatory gene expression. M1 macrophages significantly increased the phosphorylation levels of p38 and JNK MAPK signaling pathway. Inhibition of p38 and JNK significantly attenuated the M1-induced upregulation of pro-inflammatory genes in co-cultured adipocytes. Moreover, M1 macrophages significantly reduced insulin signaling-related genes expression and AKT phosphorylation level, suggesting impaired insulin signaling. Inhibition of AKT further aggravated the M1-induced inflammatory response in co-cultured adipocytes. Furthermore, M1 macrophages significantly downregulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) in co-cultured adipocytes. Activation of PPARγ with troglitazone significantly alleviated the M1-induced inflammation in co-cultured adipocytes, whereas its inhibition with GW9662 exacerbated the inflammatory response. The M1-induced changes in PPARγ expression were significantly modulated by inhibition of p38, JNK or AKT, indicating that PPARγ acts a downstream of MAPK and AKT pathways. Notably, PPARγ directly bound to the promoters of TNF-α and IL-1β, thereby suppressing their transcription. Collectively, these findings demonstrated that M1 macrophages induced inflammatory responses in co-cultured adipocytes via the MAPK/AKT-PPARγ signaling axis. This study provides novel insights into the molecular regulation of adipose tissue inflammation in fish and may offer potential targets for addressing obesity-related metabolic disorders from an evolutionary perspective.
Streptococcus iniae represents an important bacterial agent responsible for streptococcosis in marine fish, leading to substantial economic impacts in aquaculture worldwide. The development of effective vaccines is therefore a critical priority. EsxA, a conserved early-secreted and homolog of antigenic target six (ESAT-6), is involved in bacterial virulence and mediates interactions between the pathogen and its host via the type VII secretion system. In this work, EsxA was examined as a prospective vaccine antigen in golden pompano (Trachinotus anak) using both subunit and DNA vaccination strategies. Recombinant EsxA protein was expressed in Escherichia coli BL21(DE3) and administered intraperitoneally as a subunit vaccine, either alone or formulated with the oil-based adjuvant Montanide ISA 763A. Concurrently, a DNA vaccine was developed by cloning the complete esxA gene into the pVAX1 vector. Vaccinated fish were subjected to challenge with S. iniae at 8 weeks post-immunization to evaluate protective efficacy and assess the host's innate and adaptive immune responses. A high level of protection against S. iniae challenge was achieved with the EsxA-based subunit vaccine, particularly when formulated with adjuvant ISA 763A, whereas the DNA vaccine elicited moderate yet statistically significant protection. Immunological profiling revealed robust antigen-specific IgM production following subunit vaccination, while DNA vaccination significantly upregulated transcription of key immune-related genes associated with antigen presentation and cellular immunity, including MHC class I and CD8α. Furthermore, nonspecific immune parameters, including catalase, lysozyme, acid phosphatase, alkaline phosphatase activity and superoxide dismutase, were significantly elevated following vaccination, indicating potent activation of innate immune defense. Collectively, EsxA is a promising vaccine candidate against S. iniae in T. anak, and different vaccine platforms elicit distinct immune response profiles that may inform future vaccine optimization in marine aquaculture.