Emerging infectious diseases threaten shrimp aquaculture, causing economic losses and challenging disease preparedness. Translucent Post-larvae Disease (TPD) is associated with Vibrio parahaemolyticus strains harboring a virulence plasmid encoding Vibrio high virulent proteins (VHVP). TPD causes translucent or pale body coloration, and high mortality. In this study, shrimp exhibiting TPD-like signs were obtained from local farms. A pathogenic V. parahaemolyticus strain (P40) was isolated from cephalothorax homogenates and screened for TPDassociated virulence genes (vhvp1, vhvp2-1, and vhvp2-2) by colony PCR. Immersion challenge assays confirmed the pathogenicity of strain P40, reproduced TPD signs and high mortality. A single-colony isolate designated P40.49, was used for subsequent analyses. Based on quantitative PCR (qPCR) analysis, stomach, hepatopancreas, and intestine were primary tissues for early detection of TPD-associated bacterial genome copies. Histopathological examination demonstrated epithelial sloughing, necrosis and hemocytic infiltration in both hepatopancreas and intestine. TPD infection significantly modulated host responses, including upregulation of innate immune genes (proPO, PPAE1, Toll-like, Serpin7, and Pen3a) and antioxidative response genes (MnSOD2 and ROS modulator). Dietary supplementation with perilla powder attenuated TPD-associated clinical manifestations and reduced detection of vhvp genes in stomach tissue. Whole-genome sequencing confirmed a TPD-associated virulence plasmid, and phylogenetic analysis clustered the strain with reported TPD isolates from China and Southeast Asia. Comparative genomic analysis further identified four unique plasmid-associated genes specific to TPD-causing strains and infected samples, suggesting their potential as additional molecular markers for TPD diagnosis. These findings provided mechanistic insights into TPD pathogenesis and support improved diagnostics, nutritional and biosecurity strategies for sustainable shrimp aquaculture.

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease impacting shrimp farming worldwide. The disease was originally found to be caused by a specific strain of Vibrio parahaemolyticus harboring a unique ∼70-kbp plasmid encoding the binary PirAvp and PirBvp toxins. We have previously shown that AphBvp regulates the expression of these toxins. In this study, we investigated the role of AphBvp in regulating key genes associated with V. parahaemolyticus virulence and survival by using next-generation sequencing (NGS) of wild-type (3HP) and aphBvp-deleted mutant (ΔaphBvp) strains. Our RT-qPCR validation of NGS data showed that 6 genes, hnsvp, dgcvp, t6ssvp, tssBvp, ifpvp and DNA mtasevp were significantly downregulated in the ΔaphBvp compared to the 3HP, while vcrHvp was upregulated. Electrophoretic mobility shift assays (EMSA) confirmed that AphBvp directly binds to the promoter regions of hnsvp, dgcvp and t6ssvp. Notably, AlphaFold structural prediction indicates that DGCvp is a diguanylate cyclase involved in producing cyclic-di-GMP (c-di-GMP), a key second messenger for bacterial biofilm formation and persistence. An intracellular c-di-GMP assay further showed that the concentration of c-di-GMP in the ΔaphBvp was significantly lower than in the 3HP, while crystal violet staining and scanning electron microscopy revealed significant changes in both the biomass and structure of the biofilm formed by the ΔaphBvp. Collectively, our findings suggest that AphBvp could be a key regulator for V. parahaemolyticus virulence and biofilm formation. This study of the integrated AphBvp regulatory network deepens our understanding of the pathogenesis of AHPND and offers potential targets for disease control strategies.

Acute hepatopancreatic necrosis disease (AHPND) is a disease that has caused significant losses to shrimp farming since 2009. The primary mechanism of this disease involves the binary toxins PirAvp and PirBvp, which are produced by specific strains of Vibrio parahaemolyticus, and which lead to significant damage to the hepatopancreatic cells of shrimps. Recent studies on the pathology of AHPND have also highlighted the role of the Vibrio quorum sensing (QS) system, which affects growth, virulence, and biofilm regulation in Vibrio species. For example, deletion of the qseC gene reduces the virulence of the AHPND-causative V. parahaemolyticus. Most importantly, the QS regulators LuxOvp and AphBvp have been implicated as they control the growth-phase-dependent expression of the pirAvp/pirBvp genes. Additionally, given the growing problem of antibiotic resistance, this article reviews several alternative control strategies targeting the QS system, including QS inhibition using natural products, biofloc technology, and the development of small-molecule inhibitors against AphBvp. Finally, we also discussed the potential of using probiotics to enhance shrimp disease resistance through QS inhibition, highlighting the feasibility of targeting the QS system for AHPND control.

Shrimp aquaculture is part of global seafood production, but recurring disease outbreaks threaten its sustainability and productivity. Acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus strains harboring PirABvp toxins, remains one of the most devastating bacterial diseases in shrimp farming. In this study, we evaluated the potential of Perilla frutescens powder (PP), a phytogenic feed additive enriched in rosmarinic acid and flavonoids, as a functional dietary intervention against AHPND. Black tiger shrimp (Penaeus monodon) were fed diets supplemented with 0.5 or 1% PP for 14 days, followed by challenge with an AHPND-causing V. parahaemolyticus strain. Dietary PP significantly reduced mortality, bacterial loads, and PirABvp toxins in infected shrimp. The 1% PP group had enhanced immune and antioxidant responses, including activation of the proPO system, increased superoxide dismutase activity, and improved regulation of reactive oxygen species. Histopathological analyses showed reduced tissue damage following infection. In addition, stomach microbiota profiling revealed community shifts characterized by a reduction in Vibrionaceae (including Vibrio) and enrichment of beneficial taxa such as Flavobacteriaceae and Pseudoalteromonadaceae. These findings demonstrated that dietary PP supplementation enhanced immune competence, modulated stomach microbiota, and mitigated AHPND-associated pathology, supporting its potential as a sustainable functional feed strategy in shrimp aquaculture.

BACKGROUND:Viruses rely on host metabolism to complete their replication cycle. White spot syndrome virus (WSSV), a major pathogen in shrimp aquaculture, hijacks host metabolic pathways to fulfill its biosynthetic and energetic needs. Previous studies have demonstrated that WSSV promotes aerobic glycolysis (Warburg effect) and glutaminolysis during its replication stage (12 hpi). Therefore, glucose and glutamine serve as crucial metabolites for viral replication. Additionally, de novo nucleotide synthesis, including the pentose phosphate pathway and purine/pyrimidine synthesis, is significantly activated during WSSV infection. However, the precise association between WSSV and host glucose and glutamine metabolism in driving de novo nucleotide synthesis remains unclear. This study aimed to investigate the involvement of glucose and glutamine in nucleotide metabolism during WSSV replication and to elucidate how WSSV reprograms these pathways to facilitate its pathogenesis. METHODS:To assess changes in metabolic flux during WSSV replication, LC-ESI-MS-based isotopically labeled glucose ([U-13C] glucose) and glutamine ([A-15N] glutamine) were used as metabolic tracers in in vivo experiments with white shrimp (Litopenaeus vannamei). The in vivo experiments were also conducted to measure the expression and enzymatic activity of genes involved in nucleotide metabolism. Additionally, in vivo dsRNA-mediated gene silencing was employed to evaluate the roles of these genes in WSSV replication. Pharmacological inhibitors targeting the Ras-PI3K-Akt-mTOR pathway were also applied to investigate its regulatory role in WSSV-induced nucleotide metabolic reprogramming. RESULTS:The metabolite tracking analysis confirmed that de novo nucleotide synthesis was significantly activated at the WSSV replication stage (12 hpi). Glucose metabolism is preferentially reprogrammed to support purine synthesis, while glutamine uptake is significantly increased and contributes to both purine and pyrimidine synthesis. Consistently, gene expression and enzymatic activity analyses, along with gene silencing experiments, indicated the critical role of de novo nucleotide synthesis in supporting viral replication. However, while the inhibition of the Ras-PI3K-Akt-mTOR pathway suggested its involvement in regulating nucleotide metabolism, no consistent effect on WSSV replication was observed, suggesting the presence of alternative regulatory mechanisms. CONCLUSION:This study demonstrates that WSSV infection induces specific metabolic reprogramming of glucose and glutamine utilization to facilitate de novo nucleotide synthesis in shrimp. These metabolic changes provide the necessary precursors for nucleotide synthesis, supporting WSSV replication and pathogenesis. The findings offer novel insights into the metabolic strategies employed by WSSV and suggest potential targets for controlling WSSV outbreaks in shrimp aquaculture.

Although shrimp are a valuable protein source, shrimp aquaculture has numerous challenges from various infectious diseases and understanding molecular mechanisms of disease pathogenesis is crucial for disease management. In this study, a gene-to-gene correlation network generated from a transcriptomic database of the stomach of shrimp infected with acute hepatopancreatic necrosis disease (AHPND) was used to identify a new α-arrestin, termed arrestin domain containing-3 gene (LvARRDC3), with crucial roles in development of both AHPND and white spot disease (WSD). Double stranded RNA-mediated silencing or plasmid-mediated overexpression of LvARRDC3 gene significantly decreased expression of WSSV genes (IE1, VP28, and ICP11) and viral genome copy numbers. Nevertheless, in AHPND, silencing the LvARRDC3 gene increased the AHPND-associated plasmid and Pir toxins copy numbers, whereas overexpression of LvARRDC3 had the opposite effect. An in vitro pathogen binding assay with recombinant LvARRDC3 protein produced robust binding to WSSV virions and AHPND-causing V. parahaemolyticus. Moreover, based on immunofluorescence, LvARRDC3 was localized in the cytoplasm of Spodoptera frugiperda (Sf9) insect cells. Therefore, we inferred that LvARRDC3 has a role in pathogen internalization, making it a valuable target for addressing AHPND and WSD and also a biomarker for marker-associated shrimp breeding.

Acute hepatopancreatic necrosis disease (AHPND), a prevalent shrimp disease in Southeast Asia, is caused by a specific strain of Vibrio parahaemolyticus (VP) carrying a virulent pVA1 plasmid with PirABvp toxin genes. Several host and pathogenic factors affect AHPND pathogenesis. However, quorum sensing (QS), a cell density-based bacterial cell-to-cell communication that regulates virulence factors in Vibrios, mediated mechanisms of AHPND-causing VP are yet to be fully elucidated. Here, we used wild type V. parahaemolyticus, AHPND-causing strain 3HP, along with the 3HP LuxO and/or OpaR QS mutants (Delta opaR Delta luxO, Delta opaR, Delta luxO, and luxOD47E) to investigate the effects of bile acids and taurocholate, dietary supplements given to shrimp to improve growth, on the pathogenicity of AHPND-causing VP. Our results suggest that biofilm formation and PirABvp toxin release in AHPND-causing wild type and QS mutants VP strains were induced by bile acids and taurocholate. Furthermore, these dietary supplements affected genes related to flagellar apparatus (MotY, FlhB, FliK, flagellin), bacterial pilus (PilW), biofilm formation (VpsN), bile resistance (OmpT), quorum sensing (LuxR4425, LuxR2865, OpaR, LuxO) and PirABvp toxins (PirA, PirB). This suggests that bile acids or taurocholate may affect virulence mechanism of AHPND-causing VP in a LuxO-OpaR QS-independent manner. This study provided insights into host-pathogen interactions and molecular mechanisms of QS in biofilm formation and PirABvp toxin production in AHPNDcausing VP.

Background Extreme precipitation events often cause sudden drops in salinity, leading to disease outbreaks in shrimp aquaculture. Evidence suggests that environmental stress increases animal host susceptibility to pathogens. However, the mechanisms of how low salinity stress induces disease susceptibility remain poorly understood. Methods We investigated the acute response of shrimp gut microbiota exposed to pathogens under low salinity stress. For comparison, shrimp were exposed to Vibrio infection under two salinity conditions: optimal salinity (Control group) and low salinity stress (Stress group). High throughput 16S rRNA sequencing and real-time PCR were employed to characterize the shrimp gut microbiota and quantify the severity level of Vibrio infection. Results The results showed that low salinity stress increased Vibrio infection levels, reduced gut microbiota species richness, and perturbed microbial functions in the shrimp gut, leading to significant changes in lipopolysaccharide biosynthesis that promoted the growth of pathogens. Gut microbiota of the bacterial genera Candidatus Bacilliplasma, Cellvibrio, and Photobacterium were identified as biomarkers of the Stress group. The functions of the gut microbiota in the Stress group were primarily associated with cellular processes and the metabolism of lipid-related compounds. Conclusions Our findings reveal how environmental stress, particularly low salinity, increases shrimp susceptibility to Vibrio infection by affecting the gut microbiota. This highlights the importance of avoiding low salinity stress and promoting gut microbiota resilience to maintain the health of shrimp.

White spot syndrome virus (WSSV) is a very large dsDNA virus. The accepted shape of the WSSV virion has been as ellipsoidal, with a tail-like extension. However, due to the scarcity of reliable references, the pathogenesis and morphogenesis of WSSV are not well understood. Here, we used transmission electron microscopy (TEM) and cryogenic electron microscopy (Cryo-EM) to address some knowledge gaps. We concluded that mature WSSV virions with a stout oval-like shape do not have tail-like extensions. Furthermore, there were two distinct ends in WSSV nucleocapsids: a portal cap and a closed base. A C14 symmetric structure of the WSSV nucleocapsid was also proposed, according to our Cryo-EM map. Immunoelectron microscopy (IEM) revealed that VP664 proteins, the main components of the 14 assembly units, form a ring-like architecture. Moreover, WSSV nucleocapsids were also observed to undergo unique helical dissociation. Based on these new results, we propose a novel morphogenetic pathway of WSSV.

Acute hepatopancreatic necrosis disease (AHPND), a high-mortality-rate shrimp disease, is caused by specific Vibrio parahaemolyticus (Vp) strains with a plasmid encoding the PirABVp toxins. As a bacterial pathogen, the invasion of AHPND-causing Vp might impose pressure on commensal microbiota in the shrimp gut, while the relationship between the pathogenesis of AHPND and the dysbiosis of gut bacterial communities remains unclear. Here we explored the temporal changes of shrimp gut microbiota in response to AHPND-causing and non-AHPND-causing Vp strains, with the non-infected controls as a baseline of the shrimp gut microbiota. The diversity and composition of bacterial communities from 168 gut samples (covering three treatments at seven time points with eight individuals per set) were investigated using 16S rRNA gene metabarcoding with high-throughput sequencing. The results showed that (i) species diversity of gut bacterial communities declined in Vp-infected shrimp, independent of the strain pathogenicity; (ii) taxonomic compositions of gut bacterial communities were significantly different between shrimp infected by AHPND-causing and non-AHPND-causing Vp strains; (iii) short-term (within 6 hours) compositional shifts in the gut microbiota were found only in AHPND-causing Vp-infected shrimp; (iv) the gut microbiota of AHPND-causing Vp-infected shrimp was enriched with genera Photobacterium and Vibrio, with a decline in Candidatus Bacilliplasma; and (v) functional predictions suggested the loss of normal metabolism due to compositional shifts in the gut microbiota. Our work reveals distinct features of community dynamics in shrimp gut microbiota, associated with pathogenic versus non-pathogenic Vibrio infections, providing a new perspective of the pathogenesis of AHPND. IMPORTANCE Shrimp production is continually threatened by newly emerging diseases, such as AHPND, which is caused by specific Vp strains. Previous studies on the pathogenesis of AHPND have mainly focused on the histopathology and immune responses of the host. However, more attention needs to be paid to the gut microbiota, which acts as the first barrier to pathogen colonization. In this study, we revealed that shrimp gut microbiota responded differently to pathogenic and non-pathogenic Vp strains, with bacterial genera Photobacterium and Vibrio enriched in pathogenic Vp-infected shrimp, and Candidatus Bacilliplasma enriched in non-pathogenic Vp-infected shrimp. Moreover, functional predictions suggested that changes in taxonomic compositions would further affect normal metabolic functions, emphasizing the importance of sustaining an equilibrium in the gut microbiota. Several biomarkers associated with specific microbial taxa and functional pathways were identified in our data sets, which help predict the incidence of disease outcomes.

BACKGROUND:Wound healing is one of the major challenges in chronic diseases; the current treatment options are less effective with undesirable side effects and are expensive. Extensive research is carried out to develop cost-effective, natural, biodegradable wound dressings that can reduce oxidative stress and inflammation and prevent bacterial infections. Curcumin has a plethora of therapeutic applications; however, its low solubility limits its clinical use.OBJECTIVE:In this study, curcumin nanoparticles (Cur NP) and curcumin-chitosan nanoparticles (CCNP) were incorporated into the chitosan collagen vanillin scaffold, characterized, and investigated their potential wound healing properties.METHODS:The nano-scaffolds were prepared by freeze-drying method and were characterized using Fourier transform infrared spectroscopy, X-ray diffraction, nanoparticle tracking analysis, and scanning electron microscopy. The drug release, antioxidant, antibacterial, and wound healing properties were assessed by in vitro assays.RESULTS:Cur nano-scaffolds showed particle sizes of 195.9 nm and 110.6 nm for Cur NP+VC and CCNP+VC, respectively. The curcumin encapsulated in the Cur NP+VC and CC+VC nano-scaffolds showed a release profile of > 60% and an improved antioxidant activity of greater than 80%. The nanoscaffolds were antagonistic against Escherichia coli and Staphylococcus aureus and enhanced wound healing capacity of 85.62 % and 77.05% in the murine cell line.CONCLUSION:The curcumin nano-scaffold is a biodegradable and effective drug delivery system for topical use that can act as an antioxidant, facilitate wound healing, as well as prevent bacterial infections.

In WSSV pathogenesis, the molecular mechanisms and the key host factors that regulate the viral replication and morphogenesis remain unclear. However, like most viruses, WSSV is known to induce metabolic reprogramming in several metabolic pathways including the host glutamine metabolism, and several recent reports have suggested that the sirtuins SIRT3, SIRT4, and SIRT5, which belong to a family of NAD+-dependent deacetylases, play an important role in this regulation. Here we focus on characterizing LvSIRT4 from Litopenaeus vannamei and investigate its role in regulating glutamine dehydrogenase (GDH), an important enzyme that promotes glutaminolysis and viral replication. We found that LvSIRT4 silencing led to significant decreases in both WSSV gene expression and the number of viral genome copies. Conversely, overexpression of LvSIRT4 led to significant increases in the expression of WSSV genes and the WSSV genome copy number. Immunostaining in Sf9 insect cells confirmed the presence of LvSIRT4 in the mitochondria and the co-localization of LvSIRT4 and LvGDH in the same cellular locations. In vivo gene silencing of LvSIRT4 significantly reduced the gene expression of LvGDH whereas LvSIRT4 overexpression had no effect. However, neither silencing nor overexpression had any effect on the protein expression levels of LvGDH. Lastly, although GDH activity in uninfected shrimp was unchanged, the GDH enzyme activity in WSSV-infected shrimp was significantly increased after both LvSIRT4 silencing and overexpression. This suggests that although there may be no direct regulation, LvSIRT4 might still be able to indirectly regulate LvGDH via the mediation of one or more WSSV proteins that have yet to be identified.

Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.

To counter the recurrent outbreaks of bacterial (acute hepatopancreatic necrosis disease; AHPND) and viral (white spot disease; WSD) shrimp diseases, which still remain a threat to the global industry, shrimp gut microbiota research has been gaining more attention in recent years, and the use of probiotics in aquaculture has had promising results in improving shrimp gut health and immunity. In this review based on our studies on AHPND and WSD, we summarize our current understanding of the shrimp gastrointestinal tract and the role of the microbiota in disease, as well as effects of probiotics. We focus particularly on the concept of microbiota resilience, and consider strategies that can be used to restore shrimp gut health by probiotic intervention at a crucial time during gut microbiota dysbiosis. Based on the available scientific evidence, we argue that the use of probiotics potentially has an important role in controlling disease in shrimp aquaculture.

In addition to the Warburg effect, which increases the availability of energy and biosynthetic building blocks in WSSV-infected shrimp, WSSV also induces both lipolysis at the viral genome replication stage (12 hpi) to provide material and energy for the virus replication, and lipogenesis at the viral late stage (24 hpi) to complete virus morphogenesis by supplying particular species of long-chain fatty acids (LCFAs). Here, we further show that WSSV causes a reduction in lipid droplets (LDs) in hemocytes at the viral genome replication stage, and an increase in LDs in the nuclei of WSSV-infected hemocytes at the viral late stage. In the hepatopancreas, lipolysis is triggered by WSSV infection, and this leads to fatty acids being released into the hemolymph. β-oxidation inhibition experiment reveals that the fatty acids generated by WSSV-induced lipolysis can be diverted into β-oxidation for energy production. At the viral late stage, WSSV infection leads to lipogenesis in both the stomach and hepatopancreas, suggesting that fatty acids are in high demand at this stage for virion morphogenesis. Our results demonstrate that WSSV modulates lipid metabolism specifically at different stages to facilitate its replication.

Peritrophins are peritrophic membrane (PM) proteins that can interact with chitin fibers via chitin-binding domains. Peritrophins have essential roles in providing porosity and strength to the PM that lines the shrimp midgut. Acute hepatopancreatic necrosis disease (AHPND), caused by strains of V. parahaemolyticus, is known to initially colonize the shrimp stomach and simultaneously disrupt its structural barriers (e.g., cuticle or epithelial tissues) to reach the hepatopancreas. Although stomach and hepatopancreas were identified as target tissues involved in AHPND pathogenesis, our results indicated that peritrophin in peritrophic membrane has a crucial role in determining not only colonization of AHPND-causing bacteria but also their tissue distribution. As the interaction between LvPeritrophin (LvPT) and WSSV (white spot syndrome virus) is not well understood, we noted that LvPT expression was upregulated in shrimp stomach challenged with either WSSV or AHPND. In an in vitro pathogen binding assay, there was strong binding of recombinant LvPT WSSV and AHPND-causing V. parahaemolyticus, and various bacteria. Furthermore, dsRNA-mediated LvPT silencing inhibited WSSV gene expression and viral genome replication. However, downregulation of LvPT gene expression increased copies of AHPND-causing bacteria in shrimp digestive tract, and facilitated bacterial colonization in stomach. In conclusion, we speculated that LvPT might regulate bacterial colonization during AHPND, whereas in WSSV infection, LvPT silencing favored the host. Although recombinant LvPT had strong binding with WSSV, the precise role of LvPT in WSSV infection needs further investigation. These findings increased our understanding of host-pathogen interactions in AHPND and WSSV infection that can be applied in shrimp aquaculture for developing effective antibacterial and antiviral strategies.