Edwardsiella tarda is a significant pathogen in aquaculture, causing severe systemic infections and considerable economic losses worldwide. The limited efficacy of current treatments and the emergence of multidrug-resistant strains underscore the urgent need for novel antimicrobial strategies. Antimicrobial peptides have garnered increasing interest due to their broad-spectrum activity and low risk of resistance development. In this study, we identified a novel immune-related gene from the mudskipper Boleophthalmus pectinirostris, named Pecbloodin, which encodes a mature peptide of 67 amino acids. A truncated peptide derived from this sequence, Pecbloodin(18-37), exhibited broad-spectrum antibacterial activity, high thermal stability, and tolerance to sodium ions. Functional assays demonstrated that Pecbloodin(18-37) exerts rapid bactericidal effects by compromising bacterial membrane integrity, inducing cellular content leakage, and triggering endogenous reactive oxygen species accumulation. Furthermore, it effectively inhibited bacterial biofilm formation and did not promote resistance under prolonged exposure. Importantly, in vivo experiments using an E. tarda-infected mudskipper model revealed that Pecbloodin(18-37) significantly improved host survival and modulated the immune response. Overall, Pecbloodin18-37 shows great potential as a promising alternative to conventional antibiotics for the control of E. tarda infections in aquaculture, addressing the pressing issue of antibiotic resistance. IMPORTANCE Edwardsiella tarda is an urgent threat to global aquaculture. We mined the mudskipper Boleophthalmus pectinirostris genome for antimicrobial peptide and identified Pecbloodin(18-37), a 20-aa thermostable peptide that rapidly permeabilizes bacterial membranes, elicits intracellular reactive oxygen species, blocks biofilm formation, and does not select for resistance. In E. tarda-challenged fish, a single dose reduced mortality by 25% and restored immune homeostasis. The peptide is readily synthesized and feed-compatible, providing an immediate, resistance-proof substitute for conventional antibiotics in fish farming.

Edwardsiella tarda poses a substantial threat to fish populations, inducing severe systemic infections, especially pronounced in economically valuable species such as the Japanese eel (Anguilla japonica). Current therapeutic options are limited, and the emergence of multidrug-resistant strains further underscores the urgent need to develop novel antimicrobial alternatives. Antimicrobial peptides present promising alternatives because of their extensive activity range and minimal potential for resistance development. In this study, we identified a novel functional gene from A. japonica, named Anguinin, which encodes a mature peptide comprising 99 amino acids. The Anguinin gene was broadly expressed in various tissues of healthy A. japonica and showed significant induction in immune-related tissues following E. tarda infection. A truncated peptide, Anguinin55-72, derived from this gene, exhibited potent antimicrobial properties against a wide range of pathogens. Mechanistic studies revealed that Anguinin55-72 disrupted bacterial membranes, increased membrane permeability, and triggered the accumulation of reactive oxygen species, ultimately causing bacterial death. The peptide also demonstrated anti-biofilm activity and maintained stability at high temperatures without cytotoxicity. The in vivo animal models were constructed using zebrafish and Japanese eels infected with E. tarda. The results showed that Anguinin55-72 significantly increased survival rates of Danio rerio and A. japonica. Additionally, in the Japanese eel model, Anguinin55-72 significantly reduced disease severity, decreased bacterial loads, downregulated the expression of pro-inflammatory cytokines and promoted the recovery of tissue lesions. Taken together, Anguinin55-72 holds promise as an alternative to conventional antibiotics for controlling E. tarda infections in aquaculture to address the challenge of escalating antibiotic resistance.

Secondary infection by Vibrio parahaemolyticus, triggered by Cryptocaryon irritans, has become one of the most difficult diseases to control in Larimichthys crocea aquaculture due to its complex pathogens and diverse infection mechanisms. Traditional antibiotic therapies often face challenges such as resistance and low efficacy. Previous studies have shown that the antimicrobial peptide (AMP) Scy-hepc has potential as an immune-enhancing alternative to antibiotics. This study identified typical features of a natural outbreak of V. parahaemolyticus secondary infection triggered by C. irritans in a marine aquaculture farm. Under these practical farming conditions, the overall protective efficacy and potential mechanisms of Scy-hepc were further evaluated. Compared to the same dosage of Vector, Florfenicol, Bacillus subtilis, and a basal diet (Control), Scy-hepc significantly improved the survival rate and increased the total body weight of L. crocea. qPCR results showed that Scy-hepc downregulated pro-inflammatory genes (IL-1β, TNF-α) and upregulated anti-inflammatory genes (TGF-β, IL-10) as well as the AMP genes (Hepcidin, NK-lysin). 16S rRNA sequencing revealed that although all groups experienced gut microbiota disruption due to infection, the gut microbiota in the Scy-hepc group was enriched with more specific microorganisms, characterized by an increased abundance of potential probiotics (Tenericutes, Bacillaceae, Bacteroidales) and a decreased relative abundance of opportunistic pathogens (Clostridiales, Fusobacteriaceae, Spirochaetes, and Vibrionaceae). Scy-hepc also enhanced microbial functions related to metabolism and immunity. Network analysis showed higher complexity and stability in the Scy-hepc group than in the Vector and Control groups. Mantel test analysis revealed that gut microbiota composition was significantly correlated with health-related indicators (survival rate and body weight) as well as immune-related indicators (NK-lysin, Hepcidin, GPx-1a, and CAT) in the Scy-hepc group. Overall, this study provides the potential evidence of Scy-hepc’s protective effects and potential mechanisms against secondary infection under practical farming conditions. These findings support its potential as a promising antibiotic alternative to promote the sustainable and healthy aquaculture of L. crocea.

Benzo[a]pyrene (BaP), a high-molecular-weight polycyclic aromatic hydrocarbon, is a persistent contaminant with well-documented developmental and endocrine-disrupting effects in aquatic organisms. This study examined how reproductive timing after exposure cessation influences transgenerational toxicity in marine medaka (Oryzias melastigma). Fish were exposed to environmentally relevant BaP concentrations (1, 4, and 8 μg/L) for 120 days, and F1 offspring were obtained from parents spawning 1, 30, and 60 days post-exposure. Offspring from 1-day post-exposure spawns showed up to 40 % reduced hatching, elevated mortality, malformations, and shorter body length, accompanied by downregulation of antioxidant (sod, cat, gpx) and steroidogenic (cyp17a, 17βhsd, cyp11b) genes and elevated thyroglobulin (tg) and vitellogenin (vtg1, vtg2). Partial recovery occurred in oxidative and apoptotic pathways at 30 days, while endocrine and growth disruptions persisted. By 60 days, most parameters normalized except thyroid- and growth-axis markers. Hormonal assays revealed increased adrenocorticotropic hormone, cortisol, elevated thyroxine, and suppressed growth hormone, indicating slow endocrine and growth recovery.

Vibrio parahaemolyticus is a halophilic Gram-negative bacterium widely distributed in marine and estuarine environments, and is an important pathogen associated with human and animal infections. The increasing prevalence of antibiotic resistance has prompted the development of alternative antimicrobial strategies. Antimicrobial peptides (AMPs), especially those derived from marine organisms, have attracted increasing attention due to their broad-spectrum activity and low tendency to induce resistance. In this study, a novel AMP, Laricrocin92-116, was identified from the large yellow croaker Larimichthys crocea. Laricrocin92-116 showed broad-spectrum antimicrobial activity, rapid bactericidal kinetics, and excellent stability. In addition, Laricrocin92-116 showed negligible cytotoxicity and hemolytic activity, indicating favorable biosafety. In infected fish models, Laricrocin92-116 significantly improved survival rates. In juvenile large yellow croaker, pretreatment with Laricrocin92-116 markedly reduced bacterial burden and alleviated pathological damage in the intestine and spleen. Furthermore, Laricrocin92-116 modulated the expression of immune-related genes and enhanced antioxidant defenses during infection. Mechanistic studies showed that it disrupted bacterial membrane integrity, induced intracellular oxidative imbalance, inhibited biofilm formation and bacterial motility, thereby impairing bacterial virulence. Collectively, these findings demonstrate that Laricrocin92-116 effectively protects L. crocea against V. parahaemolyticus infection and represents a promising alternative antimicrobial agent for the prevention of vibriosis in aquaculture.

Antibiotic resistance, driven by multidrug-resistant (MDR) pathogens, poses a major global health threat. Antimicrobial peptides (AMPs) have garnered significant attention as promising therapeutic agents for combating MDR bacteria. Here, we demonstrate the potent antimicrobial activity of scyreprocin against MDR strains, including Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Notably, scyreprocin eradicated the tested strains without inducing drug resistance, inhibited biofilm formation, and eliminated persister cells. In terms of in vivo efficacy, scyreprocin displayed excellent activity against S. aureus and K. pneumoniae in a full-thickness wound infection model. Furthermore, administration of scyreprocin reduced bacterial burden in a K. pneumoniae lung infection model, thereby attenuating the pulmonary inflammatory response. Crucially, under cumulative administration of 25 mg/kg for 5 days, scyreprocin treatment via intravenous injection exerted no in vivo toxicity to mice. Mechanistic studies indicated that scyreprocin triggered membrane damage by causing rapid membrane permeability and leakage. Taken together, the marine-sourced scyreprocin demonstrates effective therapeutic potential for treating infections caused by drug-resistant pathogens.

Pathogenic "non-cholera" Vibrio species of Vibrio parahaemolyticus (V. parahaemolyticus) and Vibrio vulnificus (V. vulnificus) frequently pose a serious threat to aquaculture security and public health by causing infectious diseases. In this study, we reported the discovery of a marine-sourced antimicrobial peptide (AMP) called Ajapocin, which identified through a sequence optimization strategy. Ajapocin exhibited potent activity against V. parahaemolyticus and V. vulnificus pathogens, with minimum inhibitory concentrations (MICs) of 6-12 μM-comparable to the clinical agent Polymyxin B (PMB). In vivo, a single administration of Ajapocin (1 mg/mL) displayed therapeutic efficacy in a zebrafish-Vibrio infection model. Multiple doses reduced bacterial burden and accelerated wound healing in a mouse model of V. vulnificus-infected skin wounds. Ajapocin showed no cytotoxicity in ZF4 cells and HaCaT cells at concentrations up to 32 μM. Notably, after intraperitoneal injection for 1 week, Ajapocin did not induce cumulative hepatic or renal toxicity, as confirmed by histopathology analysis and chemistry profiles. Mechanistically, membrane-interacting Ajapocin targeted negative cellular components, enhancing membrane permeation, inducing membrane depolarization, and ultimately causing membrane damage and bacterial dysfunction. Taken together, these results position Ajapocin as an appealing anti-Vibrio agent for combating vibriosis in both aquaculture and clinical settings.

Frequent outbreaks of eel “mucus sloughing and hemorrhagic septicemia disease” caused by Anguillid herpesvirus 1 (AngHV) are a major epidemic in both wild and farmed eels. This virus has garnered global attention due to heavy losses on eel farms and the lack of protective vaccines or effective drugs, highlighting the urgent need for potent antiviral agents. In this study, we revealed a hepcidin homolog LJ-hep2 from Japanese seabass (Lateolabrax japonicus) can bind to AngHV and impede viral entry into cells. LJ-hep2 could directly destroy the viral envelope and showed a higher anti-AngHV activity than AA-hep (a hepcidin homolog cloned from Anguilla anguilla). It was found that the destruction of viral structure by LJ-hep2 was related to the binding of the peptide to AngHV envelope protein ORF51, and the two amino acid residues at the N-terminus of the peptide (lysine and phenylalanine) might play a key role. Comparative antiviral experiments with mutated LJ-hep2 (LJ-hep2A4A5) and multi-species hepcidin further confirmed this finding and demonstrated that these two amino acids were indispensable in the inhibition of AngHV infection by LJ-hep2. In an established eel immersion infection model, LJ-hep2 treatment reduced viral accumulation in tissues, inhibited horizontal transmission, alleviated skin lesions, and improved eel survival. Taken together, this study suggests that LJ-hep2 could inhibit AngHV infection in vitro and in vivo, and identify ORF51 as a potential target for the development of anti-AngHV drugs.

Vibrio species are ubiquitous in aquatic environments and represent significant pathogens in marine aquaculture, with Vibrio alginolyticus being a primary threat to farmed aquatic animals. The limited availability of therapeutic drugs in aquaculture, compounded by widespread antibiotic resistance, necessitates the development of new antibacterial strategies. In this study, a previously uncharacterized functional gene, designated as Spamprin, was identified in Scylla paramamosain. Its transcripts exhibited tissue-specific distribution and were markedly upregulated following LPS stimulation. Through bioinformatics analysis and prediction using antimicrobial peptide databases, a truncated peptide, Spamprin4-23 derived from Spamprin was screened. The peptide demonstrated potent antimicrobial activity against a wide range of microorganisms in vitro. Mechanistically, Spamprin4-23 significantly increased bacterial membrane permeability and induced morphological changes in target microorganisms. Importantly, it exhibited no cytotoxic effects against crab hemocytes or mammalian HEK293T cells. Although Spamprin4-23 showed no direct bactericidal activity against V. alginolyticus in vitro, it reduced bacterial burden in host tissues and conferred significant protection, leading to improved survival of V. alginolyticus-challenged mud crabs. This effect was most likely associated with the immunomodulatory activity of the peptide, as evidenced by the modulation of immune-related gene expression. Collectively, these findings suggest that Spamprin4-23 may serve as a promising immunomodulatory agent for disease control in aquaculture, offering a viable alternative to conventional antibiotic-based strategies.

The escalating threat of antimicrobial resistance (AMR) has created an urgent need for alternative therapeutic strategies beyond traditional antibiotics. In this study, we identified a new antimicrobial peptide, Boleokidin39-61, encoded by the immune-regulated gene Boleokidin in the amphibious fish Boleophthalmus pectinirostris. Notably, the expression of this gene was significantly upregulated in the liver and spleen following Edwardsiella tarda infection. Boleokidin39-61 exhibited broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. Mechanistic analyses showed that the peptide kills bacteria by disrupting membrane integrity, inhibits biofilm formation, and does not induce resistance under prolonged exposure. Cytotoxicity assays revealed no significant toxicity toward mammalian cells or erythrocytes at antimicrobial concentrations. Importantly, in vivo infection studies using both zebrafish and mice models confirmed the peptide's therapeutic efficacy, demonstrating its ability to reduce bacterial burden and modulate host inflammatory responses. Collectively, these findings highlight Boleokidin39-61 as a promising AMP candidate with potent antimicrobial and immunomodulatory properties, offering the potential for application in aquaculture and biomedical fields.

Fusarium graminearum, the causal agent of Fusarium head blight, poses a serious threat to global cereal production and food safety due to severe mycotoxin contamination. Here, we report that Spampcin56-86, a marine antimicrobial peptide from Scylla paramamosain, exhibits potent antifungal activity by rapidly inhibiting spore germination and mycelial growth of F. graminearum. Mechanistically, the peptide disrupts fungal cell membrane integrity, leading to extensive leakage of intracellular contents. Concurrently, it induces lethal reactive oxygen species accumulation and membrane lipid peroxidation, triggering a compensatory chitin stress response. Importantly, Spampcin56-86 markedly suppresses deoxynivalenol (DON) biosynthesis through transcriptional interference with the TRI gene cluster. In planta assays further demonstrate that the peptide delivers robust therapeutic protection against foliar infections and effectively prevents wheat grain spoilage during storage, without causing phytotoxicity. Collectively, these findings highlight Spampcin56-86 as a promising, safe, and dual-action natural biocontrol agent for managing F. graminearum and mitigating mycotoxin risks.

White spot syndrome virus (WSSV) poses a critical threat to crustacean aquaculture, particularly shrimp, causing widespread pandemics. In crustaceans, hemocytes function as a key component of the innate immune system and play a pivotal role in both cellular and humoral immune responses by producing various immune factors, such as antimicrobial peptides (AMPs), to defend against pathogenic microorganisms. In this study, an uncharacterized functional gene named Litopeidin was identified in Pacific white shrimp (Litopenaeus vannamei). It exhibited heightened expression in hemocytes and demonstrated a significant response to WSSV infection. Further, a truncated peptide, Litopeidin28-51, derived from this gene, was characterized and identified as a novel AMP with robust antibacterial and antifungal properties, especially against common aquatic pathogens, including Vibrio spp. Moreover, Litopeidin28-51 significantly suppressed the expression of viral genes (IE1 and VP28, WSSV replication-related genes) and the VP28 protein, as well as reduced viral copy numbers in hematopoietic tissue (Hpt) cells following WSSV infection. Mechanistic studies revealed that Litopeidin28-51 exhibited a direct virucidal effect on WSSV and significantly upregulated immune-related gene expression (including Relish, ALF, Crustin, and LYZ1) in Hpt cells. Notably, in Cherax quadricarinatus and L. vannamei, either co- or pre-treatment with Litopeidin28-51 markedly reduced animal mortality and viral replication in tissues. Collectively, the findings suggest that Litopeidin28-51, a newly identified AMP with potent antibacterial activity, effectively inhibits WSSV replication by disrupting the viral envelope and regulating the cellular antiviral responses, making it a promising candidate for developing anti-infective agents or immunostimulants in aquaculture.

Benzo[a]pyrene (BaP), a widespread environmental pollutant, has been extensively studied; however, knowledge gaps remain regarding its sex-specific reproductive toxicity and the persistence of its transgenerational effects. Marine medaka (Oryzias melastigma) were exposed to environmentally relevant BaP concentrations (1, 4, and 8 μg/L) throughout the F0 generation, with transgenerational effects assessed in F1, F2, and F3 generations reared in clean seawater. BaP exposure significantly affected biometric responses and reproductive parameters, including impaired gametogenesis, reduced fecundity, and decreased fertilization rates. Males were more sensitive to oxidative stress and hormonal imbalances in the gonads and showed delayed recovery during depuration. Genes in the hypothalamus-pituitary-gonad-liver (HPGL) axis were disrupted in a sex-specific manner. A persistent feminization and poor egg quality were observed up to the F2 generation, indicating transgenerational endocrine disruption. Despite recovery initiation in F3, the results reveal persistent sex-specific reproductive toxicity, emphasizing the need to assess sex-specific and transgenerational effects in ecotoxicology.

Methicillin-resistant Staphylococcus aureus (MRSA) is a predominant pathogen causing skin and soft tissue infections, which significantly hinders the wound healing process and contributes to high mortality rates. The rise of multidrug-resistant bacteria, coupled with the limited availability of new antibiotics, underscores the pressing need for the development of innovative antimicrobial substances. Antimicrobial peptides (AMPs), with their multitargeted and rapid antimicrobial activity, are promising candidates to address this crisis. In this study, we identified a novel AMP, Scymicrosin7-26, derived from Scylla paramamosain, which demonstrated potent antimicrobial activity against a variety of MDR strains, particularly MRSA. Confocal microscopy and transmission electron microscopy observations showed that Scymicrosin7-26 bound to MRSA, and had a disruptive effect on cell walls and cell membranes, rapidly penetrating and killing MRSA. Notably, Scymicrosin7-26 exhibited good stability under various ionic conditions, thermal stresses and certain serum concentration, had no obvious toxic effects on HaCaT cells, and its ability to penetrate HaCaT cells indicated its potential for intracellular targeted therapy. In vitro, Scymicrosin7-26 significantly reduced the number of MRSA in HaCaT cells and inhibited intracellular MRSA proliferation. After verifying the low toxicity of Scymicrosin7-26 in vivo in the Marine model organism─marine medaka (Oryzias melastigma), a wound model of MRSA-infected mice was made, and topical administration of Scymicrosin7-26 in hypromellose gels could significantly reduce bacterial burden and promote wound closure. Histological analysis confirmed that Scymicrosin7-26 alleviated tissue damage and was comparable to the effect of vancomycin treatment. Collectively, Scymicrosin7-26 is promising for the treatment of MRSA wound infections and could be a valuable addition to the arsenal against antibiotic-resistant bacteria.

Antimicrobial peptides (AMPs) are considered a key component of innate immunity, playing a vital role in host defense. In the study, a novel functional gene, named Larimicin, was identified from large yellow croaker Larimichthys crocea. The Larimicin gene was widely distributed in multiple tissues of healthy L. crocea and was significantly induced in the liver after Vibrio alginolyticus or Vibrio parahaemolyticus infection. Larimicin78-102, a truncated peptide derived from Larimicin, showed broad-spectrum antimicrobial activity and a binding affinity with LPS. It exhibited effective bactericidal activity against the common aquatic pathogens Vibrio fluvialis, Pseudomonas fluorescens, and Pseudomonas putida. It also showed anti-biofilm activity against three aquatic pathogens. Moreover, Larimicin78-102 disrupted the integrity of the outer and inner membranes, resulting in ATP leakage and intracellular ROS accumulation, which ultimately led to bacterial cell death. Larimicin78-102 exhibited good thermal stability and cation tolerance, with no obvious cytotoxicity or hemolytic activity. Notably, Larimicin78-102 significantly improved the survival rate of L. crocea infected with V. fluvialis, raising it to 95 %, indicating its anti-infective role in vivo. In addition, Larimicin78-102 significantly reduced the expression of the pro-inflammatory cytokines TNF-α and IL-1β, while up-regulating the anti-inflammatory factor IL-4 mRNA level. It also elevated the expression levels of piscidin, hepcidin, and lysozyme, as well as enhanced the enzymatic activity of lysozyme. Taken together, Larimicin78-102 is a potential antibacterial agent for use in aquaculture to combat V. fluvialis infection diseases in the future.

Pseudomonas putida was identified in the testis of Scylla paramamosain, where it induced elevated expression of two interacting antimicrobial peptides (AMPs), SCY2 and Scyreprocin, in the gonad, indicating their potential role in reproductive immunity. In this study, we demonstrate that both peptides exerted systemic immune protection in males against this pathogen, which colonizes multiple host tissues and causes substantial mortality. Furthermore, they also mediate reproductive-specific immune functions in females. Administration of recombinant SCY2 or Scyreprocin (8 μg per crab, average weight 250 g ± 10 g) in vivo increased the survival of P. putida-infected males by 40-50 %. In addition, both peptides promoted bacterial clearance in key immune-related tissues, including the hepatopancreas, gills, hemocytes, testis, and ejaculatory duct, and modulated the expression of immune-related genes. Subsequently, the peptides' function in mated females was examined through in vivo blockade, with the efficacy validated by immunofluorescence and flow cytometry. As expected, the in vivo blockade of SCY2 and Scyreprocin in female crabs resulted in increased bacterial colonization and a significant reduction in survival rates, which declined to only 5-10 % within 96 h post-reinfection. Taken together, this study reveals a dual function for the male-derived AMPs SCY2 and Scyreprocin: they not only mediate systemic innate immunity in males but are also transferred to females to establish reproductive immunity. Consequently, this establishes a direct correlation between male immune competence and the reproductive success of the species.

The escalating misuse of antibiotics has precipitated a worldwide crisis of bacterial resistance, greatly complicating the clinical management of multidrug-resistant bacterial infections, which now present a profound threat and a growing burden on public health systems. This situation necessitates the development of innovative anti-infective therapeutics. This work focuses on Scymicrosin7–26, a newly identified antimicrobial peptide (AMP) sourced from the crustacean Scylla paramamosain. AMPs typically derived from crustaceans are often characterized by suboptimal potency, instability, potential toxicity, and a narrow spectrum of activity, whereas Scymicrosin7–26 exhibits certain improvements in these regards. It exhibited antibacterial activity against five types of common clinically isolated multidrug-resistant organisms (MDROs). It inhibited both the formation and maturation of biofilms in carbapenem-resistant Pseudomonas aeruginosa (CR-PA) as well as methicillin-resistant Staphylococcus aureus (MRSA) without readily inducing resistance. Scymicrosin7–26 retained stable antimicrobial activity under physiological salt conditions and showed no significant antagonism when combined with several conventional antibiotics. It also demonstrated low toxicity toward RAW264.7, HEK293T, and Beas-2B cell lines, as well as human erythrocytes. Using fluorescence and electron microscopy, we observed disruption of bacterial surface structures. DNA binding assays further indicated the peptide’s capacity to interact with bacterial genomic DNA. Moreover, Scymicrosin7–26 alleviated lipopolysaccharide (LPS)-triggered inflammatory responses via concurrent blockade of MAPK and NF-κB pathway activation. With its antibacterial activity against multidrug-resistant pathogens, anti-inflammatory property, and safety profile, Scymicrosin7–26 exhibits therapeutic potential for managing infections caused by multidrug-resistant bacteria.

The Pacific white shrimp (Litopenaeus vannamei) aquaculture faces emerging threats from novel pathogens and escalating antibiotic resistance. This study successfully isolated and identified the pathogenic bacterium Acinetobacter ursingii strain 31C2 from diseased L. vannamei using an integrated approach combining microbiological, biochemical, and molecular techniques. The pathogenicity of this strain was confirmed in L. vannamei and marine medaka (Oryzias melastigma) infection models, exhibiting a strong dose-dependent mortality, with median lethal doses (LD₅₀) of 2.83 × 10⁴ CFU/g shrimp and 2.58 × 10⁶ CFU/fish, respectively. Infection caused severe hepatopancreatic necrosis (tubular deformation and epithelial vacuolation) and intestinal villi destruction. Antimicrobial susceptibility testing revealed that the 31C2 strain was resistant to tetracycline and azithromycin. To identify effective agents targeting this strain, the antimicrobial peptide Scymicrosin7-26 (derived from Scylla paramamosain) was evaluated. The peptide had potent antibacterial activity against A. ursingii 31C2 in vitro (MIC: 3–6 µM). In vivo application significantly enhanced survival of L. vannamei and O. melastigma infected with 31C2 by 30

Skin ulceration syndrome (SUS) is a major threat to the aquaculture of Apostichopus japonicus, particularly in southern China, where it has shown high mortality rates and infectious potential. Traditional antibiotic treatments often lead to challenges such as antibiotic resistance. Antimicrobial peptides (AMPs), which are vital elements of innate immunity, represent a promising alternative for treating SUS. In the study, a novel AMP named Lvvibriocin-GK identified in Litopenaeus vannamei was found to have a strong antibacterial activity against multiple Vibrio species that possibly cause SUS. Through constructing a Vibrio harveyi-induced SUS model, we evaluated the efficacy of a 7-day Lvvibriocin-GK immersion treatment to SUS. Compared to doxycycline hydrochloride at the same concentration, Lvvibriocin-GK treatments could have ulcer area and numbers reduced, mortality decreased, the DAI index significantly lowered, as well as intestinal inflammatory cell infiltration decreased but no significant effect on body weight. The therapeutic effects of Lvvibriocin-GK were accompanied by significantly enhancing the activities of trypsin, lysozyme, T-NOS, and T-SOD and reducing Vibrio harveyi load in tissues. qPCR results indicated that Lvvibriocin-GK upregulated the expression of intestinal barrier proteins ZO-1 and Occludin, and downregulated pro-inflammatory factors such as IL17, p105, NLRP3, Rel, and Stat5. Furthermore, 16S rRNA sequencing revealed that the beneficial effects of Lvvibriocin-GK might be linked to favorable changes in A. japonicus 's gut microbiota, including increased microbial diversity, enhanced abundance of potential probiotics (Rhodobacteraceae, Bacillus, Serratia liquefaciens), and reduced the abundance of opportunistic pathogens (Acinetobacter and Bacteroides vulgatus). These changes resulted in a more complex microbial network and improved immune-associated functions, particularly through pathways such as NF-κB signaling. Mantel tests indicated stronger correlations between Lvvibriocin-GK-treated gut microbiota and disease phenotypes (gut pathology), enzymatic activities (lipase, lysozyme, T-NOS, T-SOD), intestinal barrier markers (Occludin), and immune-related genes (Stat5, Rel, FoxP, VEGF). Taken together, this study proposes a novel, environmentally friendly AMP immersion treatment for severe cases of SUS. The therapeutic effects are closely to effectively eliminate pathogens, modulate the gut microbiota and enhance host immunity. A comprehensive evaluation of the efficacy and mechanisms of AMP treatment in A. japonicus SUS will contribute to assessing its advantages and potential applications as an antibiotic alternative, promoting A. japonicus health and improving aquaculture practices.

Bacterial foodborne contamination poses a dual challenge of chemical preservative risks and antibiotic resistance, drives the need for green production of natural antimicrobial alternatives. The reported cationic antimicrobial peptide (AMP) Spgillcin177−189 derived from the Scylla paramamosain, has strong antimicrobial activity against Staphylococcus aureus and clinical isolation strains. To meet industry demand in future, large-scale production of Spgillcin177− 189 is essential. In the study, Pichia pastoris expression system was established for production of the recombinant Spgillcin177− 189 (rSpgillcin177−189). Then, multicopy strategy was selectively designed by employing the Golden Gate assembly technology to efficiently construct multi-copy plasmids, which significantly enhanced the expression level of Spgillcin177− 189. A yield of 126.1 mg/L was harvested with 2.75-fold higher that of the single-copy strain. In addition, the recombinant Spgillcin177 − 189 exhibited potent antibacterial activity against multiple foodborne pathogens within a MIC range of 5.25–84 µg/mL. It also showed effective bactericidal activity and anti-biofilm activity against Staphylococcus aureus and Vibrio parahaemolyticus. rSpgillcin177 − 189 exhibited good thermostability, with no obvious cytotoxicity and hemolytic activity. rSpgillcin177 − 189 may interact with microbial surface components via hydrogen bonding, which were vital for peptide activity in combating bacteria. The rSpgillcin177 − 189 specifically targeting the cell membrane, disrupted bacterial membrane integrity and leading to cell death. This study provided a very feasible genetic engineering strategy for large-scale production of rSpgillcin177 − 189, which will be applied at a lower cost in agricultural and food industries in future.