Glycosaminoglycan lyase: A new competition between bacteria and the pacific white shrimp Litopenaeus vannamei.

Horizontal gene transfer (HGT) is an important evolutionary force in the formation of prokaryotic and eukaryotic genomes. In recent years, many HGT genes horizontally transferred from prokaryotes to eukaryotes have been reported, and most of them are present in arthropods. The Pacific white shrimp Litopenaeus vannamei, an important economic species of arthropod, has close relationships with bacteria, providing a platform for horizontal gene transfer (HGT). In this study, we analyzed bacteria-derived HGT based on a high-quality genome of L. vannamei via a homology search and phylogenetic analysis, and six HGT genes were identified. Among these six horizontally transferred genes, we found one gene (LOC113799989) that contains a bacterial chondroitinase AC structural domain and encodes an unknown glycosaminoglycan (GAG) lyase in L. vannamei. The real-time quantitative PCR results showed that the mRNA expression level of LOC113799989 was highest in the hepatopancreas and heart, and after stimulation by Vibrio parahaemolyticus, its mRNA expression level was rapidly up-regulated within 12 h. Furthermore, after injecting si-RNA and stimulation by V. parahaemolyticus, we found that the experimental group had a higher cumulative mortality rate in 48 h than the control group, indicating that the bacteria-derived GAG lyase can reduce the mortality of shrimp with respect to infection by V. parahaemolyticus and might be related to the resistance of shrimp to bacterial diseases. Our findings contribute to the study of the function of GAGs and provide new insights into GAG-related microbial pathogenesis and host defense mechanisms in arthropods.

Synergistic effects of combined probiotics Bacillus pumilis D5 and Leuconostoc mesenteroide B4 on immune enhancement and disease resistance in Litopenaeus vannamei.

This study investigated the effects of two distinct probiotics, Leuconostoc mesenteroides B4 (B4) and Bacillus pumilus D5 (D5), along with their combination, on the diet of white shrimp (Litopenaeus vannamei) during an eight-week feeding trial. The diets tested included B4 + dextran at 107 CFU/g feed (the B4 group), D5 alone at 107 CFU/g feed (the D5 group), and a combination of B4 + dextran and D5 at 5 × 106 CFU/g feed each (the B4+dextran + D5 group). Relative to the control group, those administered probiotics exhibited moderate enhancements in growth. By the eighth week, the weight gain for the B4, D5, and B4+D5 groups was 696.50 ± 78.15%, 718.53 ± 130.73%, and 693.05 ± 93.79%, respectively, outperforming the control group's 691.66 ± 31.10% gain. The feed conversion ratio was most efficient in the B4 group (2.16 ± 0.06), closely followed by B4+D5 (2.21 ± 0.03) and D5 (2.22 ± 0.06), with the control group having the highest ratio (2.27 ± 0.03). While phenoloxidase activity was somewhat elevated in the B4 and D5 groups, no significant differences were noted in respiratory burst activity or total hemocyte count across all groups. Challenge tests at weeks 4 and 8 showed that the B4 + D5 combination offered superior protection against AHPND-causing Vibrio parahaemolyticus. The 4-week cumulative survival rate was highest in shrimp treated with B4 + dextran + D5 (56.25%), followed by B4 + dextran (31.25%), control (18.75%), and lowest in D5 (12.5%). By week 8, the B4 + dextran + D5 (43.75%) and B4 + dextran (37.5%) groups significantly outperformed the control group (6.25%, p < 0.05), with no significant difference observed between the D5 group (37.5%) and the control group at day 56. Analysis of the shrimp's foregut microbiota revealed an increase in unique OTUs in the B4 and B4 + D5 groups. Compared to the control, Proteobacteria abundance was reduced in all probiotic groups. Potential pathogens like Vibrio, Bacteroides, Neisseria, Botrytis, Clostridioides, and Deltaentomopoxvirus were detected in the control but were reduced or absent in probiotic groups. Beneficial microbes such as Methanobrevibacter and Dictyostelium in the B4+D5 group, and Sugiyamaella in the B4 group, showed significant increases. Probiotics also led to higher transcript levels of nitric oxide synthase in the hemocytes, and lysozyme and transglutaminase in the midgut, along with lysozyme and α2-macroglobulin in the foregut. Notably, the combined B4 + D5 probiotics synergistically enhanced the expression of superoxide dismutase and prophenoloxidase in the foregut, indicating an improved immune response. In summary, this study demonstrates that the probiotics evaluated, especially when used in combination, significantly boost the expression of specific immune-related genes, enhance the bacterial diversity and richness of the intestine, and thus prevent the colonization and proliferation of Vibrio spp. in L. vannamei.

Cellular and transcriptomic response to pathogenic and non-pathogenic Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei.

The shrimp industry has historically been affected by viral and bacterial diseases. One of the most recent emerging diseases is Acute Hepatopancreatic Necrosis Disease (AHPND), which causes severe mortality. Despite its significance to sanitation and economics, little is known about the molecular response of shrimp to this disease. Here, we present the cellular and transcriptomic responses of Litopenaeus vannamei exposed to two Vibrio parahaemolyticus strains for 98 h, wherein one is non-pathogenic (VpN) and the other causes AHPND (VpP). Exposure to the VpN strain resulted in minor alterations in hepatopancreas morphology, including reductions in the size of R and B cells and detachments of small epithelial cells from 72 h onwards. On the other hand, exposure to the VpP strain is characterized by acute detachment of epithelial cells from the hepatopancreatic tubules and infiltration of hemocytes in the inter-tubular spaces. At the end of exposure, RNA-Seq analysis revealed functional enrichment in biological processes, such as the toll3 receptor signaling pathway, apoptotic processes, and production of molecular mediators involved in the inflammatory response of shrimp exposed to VpN treatment. The biological processes identified in the VpP treatment include superoxide anion metabolism, innate immune response, antimicrobial humoral response, and toll3 receptor signaling pathway. Furthermore, KEGG enrichment analysis revealed metabolic pathways associated with survival, cell adhesion, and reactive oxygen species, among others, for shrimp exposed to VpP. Our study proves the differential immune responses to two strains of V. parahaemolyticus, one pathogenic and the other nonpathogenic, enlarges our knowledge on the evolution of AHPND in L. vannamei, and uncovers unique perspectives on establishing genomic resources that may function as a groundwork for detecting probable molecular markers linked to the immune system in shrimp.

Unveiling the positive impact of biofloc culture on Vibrio parahaemolyticus infection of Pacific white shrimp by reducing quorum sensing and virulence gene expression and enhancing immunity.

This study aimed to evaluate and unveil the positive impact of biofloc culture on Vibrio parahaemolyticus infection of Pacific white shrimp by reducing quorum sensing (QS) and virulence gene expression and enhancing shrimp's immunity. The shrimp with an average body weight of 0.50 ± 0.09 g were reared in containers with a volume of 2.5 L, 21 units, and a density of 20 shrimp L-1. The shrimp were cultured for 5 days, with each treatment including biofloc system maintenance with a C/N ratio of 10 and a control treatment without biofloc, followed by a challenge test through immersion using V. parahaemolyticus at densities of 103, 105, and 107 CFU mL-1 initially. The results of the in vitro experiment showed that biofloc suspension can inhibit and disperse biofilm formation, as well as reduce the exo-enzyme activity (amylase, protease, and chitinase) of V. parahaemolyticus. Furthermore, the biofloc treatment significantly reduced the expression of the QS regulatory gene OpaR, the PirB toxin gene, and the virulence factor genes T6SS1 and T6SS2 in both in vitro and in vivo. The biofloc system also increased the expression of shrimp immunity-related genes (LGBP, proPO, SP, and PE) and the survival rate of white shrimp challenged with V. parahaemolyticus.

Identification of a potential antigen stimulating immune response against Vibrio parahaemolyticus infection in hybrid tilapia (Oreochromis aureus♂ × Oreochromis niloticus♀).

Vibrio parahaemolyticus (V. parahaemolyticus) is a major pathogen that causes substantial losses in the marine fishery. With the emergence of antibiotic resistance, vaccines have become the most effective approach against V. parahaemolyticus infection. Adhesion factors on the cell surface are pivotal in the colonization and pathogenesis of V. parahaemolyticus within the host, highlighting their potential as vaccine candidates. This study aims to assess the immunogenicity and potential of recombinant V. parahaemolyticus MAM7 (rMAM7) as a vaccine candidate. Initially, we cloned and purified the MAM7 protein of V. parahaemolyticus. Moreover, after 4 weeks of vaccination, the fish were challenged with V. parahaemolyticus. rMAM7 demonstrated a certain protective effect. Immunological analysis revealed that rMAM7 immunization-induced antibody production and significantly increased acid phosphatase (ACP) and alkaline phosphatase (AKP) activity in hybrid tilapia. Furthermore, serum bactericidal tests demonstrated a lower bacterial survival rate in the rMAM7 group compared to PBS and rTrxa. qRT-PCR results indicated that rMAM7 significantly upregulated CD4, CD8 and IgM gene expression, suggesting the induction of Th1 and Th2 responses in hybrid tilapia. Overall, these findings highlight the potential application of MAM7 from V. parahaemolyticus in the development of protein vaccines.

Identifying the function of the PI3K-AKT pathway during the pathogenic infection of Macrobrachium rosenbergii.

The phosphoinositide-3-kinase/protein kinase b (PI3K-Akt) pathway is a signalling pathway based on protein phosphorylation and can be activated by a wide range of factors. To investigate the function of the PI3K-AKT signalling pathway in antibacterial immunity, we analysed the gene expression level of three key factors (PI3K, AKT and FoxO) and innate immune factors in immune tissues at different time points after Vibrio parahaemolyticus and Staphylococcus aureus infection. Tissues analysis showed that PI3K, AKT, and FoxO were expressed at high levels in the intestinal, hemocytes and hepatopancreas. Moreover, the expression levels of PI3K, AKT and FoxO can be regulated postinfection by different pathogens. In hemocytes and the intestine, V. parahaemolyticus infection was found to regulate the levels of PI3K, AKT, and FoxO more rapidly; however, an S. aureus infection regulated the levels of these factors more rapidly in the hepatopancreas and gills. Analysis showed that V. parahaemolyticus and S. aureus infection caused changes in the gene expression level of crustin, caspase 3 and NF-κB. Therefore, PI3K-AKT regulates the downstream immune pathway differentially in different immune tissues and participates in the regulation of cell apoptosis and the inflammatory response by activating caspase and NF-κB, respectively, following infection with V. parahaemolyticus and S. aureus.

Dietary administration of yeast (Saccharomyces cerevisiae) hydrolysate from sugar byproducts promotes the growth, survival, immunity, microbial community and disease resistance to VP (AHPND) in Pacific white shrimp (Litopenaeus vannamei).

This study investigated the effects of yeast hydrolysate (YH) from sugar byproducts on various parameters in Pacific white shrimp (Litopenaeus vannamei). The study found no significant differences in water quality parameters across all treatment tanks, ensuring that the observed effects were not due to environmental variations. There were no significant differences in growth parameters between the control group and groups receiving YH at different dosages. However, the group given YH at 10.0 g/kg feed exhibited a notably higher survival rate and higher expression of growth-related genes (IGF-2 and RAP-2A) in various shrimp tissues. YH was associated with enhanced immune responses, including lysozyme activity, NBT dye reduction, bactericidal activity, and phagocytic activity. Notably, the 10.0 g/kg feed group displayed the highest phagocytic index, indicating a dose-dependent immune response. Expression of immune-related genes (ALF, LYZ, ProPO, and SOD) was upregulated in various shrimp tissues. This upregulation was particularly significant in the gills, hepatopancreas, intestine, and hemocytes. While total Vibrio counts remained consistent, a reduction in green Vibrio colonies was observed in the intestine of shrimp treated with YH. YH, especially at 5.0 and 10.0 g/kg feed dosages, significantly increased survival rates and RPS values in response to AHPND infection. The findings of this study suggest that incorporating additives derived from yeast byproducts with possible prebiotic properties obtained from sugar byproducts can lead to positive results in terms of enhancing growth performance, immunity, histological improvements, and resistance to V. parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND).

Impact of rising seawater temperature on a phagocytic cell population during V. parahaemolyticus infection in the sea anemone E. pallida.

Climate change is increasing ocean temperatures and consequently impacts marine life (e.g., bacterial communities). In this context, studying host-pathogen interactions in marine organisms is becoming increasingly important, not only for ecological conservation, but also to reduce economic loss due to mass mortalities in cultured species. In this study, we used Exaiptasia pallida (E. pallida), an anemone, as an emerging marine model to better understand the effect of rising temperatures on the infection induced by the pathogenic marine bacterium Vibrio parahaemolyticus. The effect of temperature on E. pallida was examined at 6, 24, or 30 h after bath inoculation with 108 CFU of V. parahaemolyticus expressing GFP (Vp-GFP) at 27°C (husbandry temperature) or 31°C (heat stress). Morphological observations of E. pallida and their Hsps expression demonstrated heat stress induced increasing damage to anemones. The kinetics of the infections revealed that Vp-GFP were localized on the surface of the ectoderm and in the mucus during the first hours of infection and in the mesenterial filaments thereafter. To better identify the E. pallida cells targeted by Vp-GFP infection, we used spectral flow cytometry. E. pallida cell types were identified based on their autofluorescent properties. corresponding to different cell types (algae and cnidocytes). We identified an AF10 population whose autofluorescent spectrum was identical to that of human monocytes/macrophage, suggesting that this spectral print could be the hallmark of phagocytic cells called "amebocytes''. AF10 autofluorescent cells had a high capacity to phagocytize Vp-GFP, suggesting their possible role in fighting infection. This was confirmed by microscopy using sorted AF10 and GFP-positive cells (AF10+/GFP+). The number of AF10+/GFP+ cells were reduced at 31°C, demonstrating that increased temperature not only damages tissue but also affects the immune response of E. pallida. In conclusion, our study provides a springboard for more comprehensive studies of immune defense in marine organisms and paves the way for future studies of the dynamics, activation patterns, and functional responses of immune cells when encountering pathogens.

Effects of ß-1,3-glucan on growth, immune responses, and intestinal microflora of the river prawn (Macrobrachium nipponense) and its resistance against Vibrio parahaemolyticus.

In this study, we investigated the impact of ß-1,3-glucan on the immune responses and gut microbiota of the river prawn (Macrobrachium nipponense) in the presence of Vibrio parahaemolyticus stress. Shrimps were fed one of the following diets: control (G1), 0.2% curdlan (G2), 0.1% ß-1,3-glucan (G3), 0.2% ß-1,3-glucan (G4), or 1.0% ß-1,3-glucan (G5) for 6 weeks and then challenged with V. parahaemolyticus for 96 h. Under Vibrio stress, shrimps in G4 exhibited the highest length gain rate, weight gain rate, and survival rate. They also showed increased intestinal muscle thickness and villus thickness compared to the control and 0.2% curdlan groups. The apoptosis rate was lower in G4 than in the control group, and the digestive enzyme activities (pepsin, trypsin, amylase, and lipase), immune enzyme activities (acid phosphatase, alkaline phosphatase, lysozyme, and phenoxidase), and energy metabolism (triglyceride, cholesterol, glycogen, and lactate dehydrogenase) were enhanced. Expression levels of growth-related genes (ecdysone receptor, calmodulin-dependent protein kinase I, chitin synthase, and retinoid X receptor) and immune-related genes (toll-like receptor 3, myeloid differentiation primary response 88, mitogen-activated protein kinase 7, and mitogen-activated protein kinase 14) were higher in G4 than in the control. Microbiota analysis indicated higher bacterial abundance in shrimps fed ß-1,3-glucan, as evidenced by Sob, Chao1, and ACE indices. Moreover, 0.2% ß-1,3-glucan increased the relative abundances of Bacteroidota and Firmicutes while reducing those of Corynebacteriales and Lactobacillales. In summary, ß-1,3-glucan enhances immune enzyme activities, alters immune-related gene expression, and impacts gut microbial diversity in shrimp. These findings provide valuable insights into the mechanisms underlying ß-1,3 glucan's immune-enhancing effects.

Gut bacterial consortium enriched in a biofloc system protects shrimp against Vibrio parahaemolyticus infection.

BACKGROUND: Shrimp cultured in a biofloc system (BFS) have a lower disease incidence than those farmed in a water exchange system (WES). Although a number of studies have reported that the gut bacterial community induced by BFS is highly associated with shrimp disease resistance, the causal relationship remains unknown. Here, the promotive roles of gut bacterial community induced by BFS in pathogenic Vibrio infection resistance and its potential micro-ecological and physiological mechanisms were investigated by gut bacterial consortium transplantation and synthetic community (SynCom) construction. RESULTS: The BFS induced a more stable and resistant gut bacterial community, and significantly enriched some beneficial bacterial taxa, such as Paracoccus, Ruegeria, Microbacterium, Demequina, and Tenacibaculum. Transplantation of a gut bacterial consortium from BFS shrimp (EnrichBFS) greatly enhanced the stability of the bacterial community and resistance against pathogenic V. parahaemolyticus infection in WES shrimp, while transplantation of a gut bacterial consortium from WES shrimp significantly disrupted the bacterial community and increased pathogen susceptibility in both WES and BFS shrimp. The addition of EnrichBFS in shrimp postlarvae also improved the pathogen resistance through increasing the relative abundances of beneficial bacterial taxa and stability of bacterial community. The corresponding strains of five beneficial bacterial taxa enriched in BFS shrimp were isolated to construct a SynComBFS. The addition of SynComBFS could not only suppress disease development, but also improve shrimp growth, boost the digestive and immune activities, and restore health in diseased shrimp. Furthermore, the strains of SynComBFS well colonized shrimp gut to maintain a high stability of bacterial community. CONCLUSIONS: Our study reveals an important role for native microbiota in protecting shrimp from bacterial pathogens and provides a micro-ecological regulation strategy towards the development of probiotics to ameliorate aquatic animal diseases. Video Abstract.

Efficacy of sodium chlorite in inactivating Vibrio parahaemolyticus attached to polyethylene terephthalate surfaces.

The inactivation of Vibrio parahaemolyticus cells attached to a polyethylene terephthalate (PET) disc in a sodium chlorite (NaClO2) solution was kinetically studied in a weakly acidic pH range of 4.0 - 6.5. The logarithmic reduction in the survival ratio depended on the concentration-time product. All inactivation curves showed a linear reduction phase, and the reduction in viable cells was greater than 4-log. No significant desorption of attached cells was observed during the inactivation treatment. The first-order inactivation rate constant (k) increased by approximately 4.5-fold for every 1.0 unit fall in pH. At all pH values, the k values calculated for the attached cells were approximately half of those for the unattached cells. These findings indicate that a weakly acidic NaClO2 solution is effective in inactivating bacteria attached to hard surfaces.

Basigin binds bacteria and activates Dorsal signaling to promote antibacterial defense in Penaeus vannamei.

The NF-κB pathway plays an important role in immune regulation. Basigin, an immunoglobulin superfamily membrane protein, is involved in the activation of NF-κB. However, its role in NF-κB signaling in response to pathogen infection remains unclear. In this study, we identified the Basigin gene from Pacific white shrimp, Penaeus vannamei, a representative species for studying the innate immune system of invertebrates. Basigin promoted the degradation of the IκB homolog Cactus, facilitated the nuclear translocation of the NF-κB family member Dorsal, and positively regulated the expression of Dorsal pathway downstream antimicrobial peptide genes. Interestingly, recombinant Basigin protein could bind a variety of Gram-positive and Gram-negative bacteria. Silencing of Basigin inhibited the Dorsal signaling activated by V. parahaemolyticus infection and significantly decreased the survival rate of V. parahaemolyticus-infected shrimp. The expression levels of the antimicrobial peptides ALF1 and ALF2 were downregulated, and the phagocytosis of hemocytes was attenuated in Basigin-silenced shrimp. Similar results were observed in shrimp treated with a recombinant extracellular region of the Basigin protein that was able to compete with endogenous Basigin. Therefore, to the best of our knowledge, this study is the first to demonstrate the function of Basigin as a pathogen recognition receptor that activates NF-κB signaling for antibacterial immunity in shrimp.

Insights into molecular aspects of pathogenesis and disease management in acute hepatopancreatic necrosis disease (AHPND): An updated review.

Shrimp aquaculture is a rapidly growing sector that makes a significant economic contribution. However, the aquaculture industry is confronted with significant challenges, and infectious diseases, notably Acute Hepatopancreatic Necrosis Disease (AHPND), have emerged as severe threat. AHPND is caused by pathogens carrying the pVA-1 plasmid, which expresses the PirAB toxin, and it has wreaked havoc in shrimp aquaculture, imposing substantial economic burdens. To address this issue, it is crucial to delve into shrimp's immune responses. Therefore, this comprehensive review offers an in-depth examination of AHPND outbreaks, encompassing various facets such as environmental factors, host susceptibility, and the mechanisms employed by the pathogens. Traditional approaches to combat AHPND, primarily relying on chemicals and antibiotics, have raised concerns related to antibiotic resistance and have demonstrated limited success in disease control. Hence this review spotlights recent advancements in molecular diagnostics, therapeutic agents, and research related to shrimp immunity. Understanding these developments is crucial in the ongoing battle against AHPND. In conclusion, this review underscores the pressing need to comprehend the underlying mechanisms of AHPND pathogenesis and emphasizes the importance of developing comprehensive and effective solutions to combat this devastating disease, which continues to threaten the sustainability of shrimp farming.

Essential role of the HSC70 in the mud crab Scylla paramamosain in response to Vibrio parahaemolyticus infection.

Heat shock proteins play an important role in host defense, and modulate immune responses against pathogen infection. In this study, a novel HSC70 from the mud crab (designated as SpHSC70) was cloned and characterized. The full length of SpHSC70 contained a 58 bp 5'untranslated region (UTR), an open reading frame (ORF) of 2,046 bp and a 3'UTR of 341 bp. The SpHSC70 protein included the conserved DnaK motif. The mRNA of SpHSC70 was highly expressed in the hemocytes, heart and hepatopancreas, and lowly expressed in the intestine. The subcellular localization results indicated that SpHSC70 was localized in both the cytoplasm and the nucleus. Moreover, SpHSC70 was significantly responsive to bacterial challenge. RNA interference experiment was designed to investigate the roles of SpHSC70 in response to bacterial challenge. V. parahaemolyticus infection induced the expression levels of SpPO, SpHSP70, SpSOD and SpCAT. Knocking down SpHSC70 in vivo can decrease the expression of these genes after V. parahaemolyticus infection. These results suggested that SpHSC70 could play a vital role in defense against V. parahaemolyticus infection via activating the immune response and antioxidant defense signaling pathways in the mud crab.

Effects of high stocking density on growth performance and expression of MyD88, and its temporal expression under the challenge of Vibrio parahaemolyticus in the noble scallop Chlamys nobilis.

High stocking density has been regarded as an adverse factor in bivalve aquaculture. However, its subsequent molecular response to pathogenic bacteria has been little studied. In order to study the question, a novel MyD88 was first cloned using adult noble scallops Chlamys nobilis (CnMyD88), and its tissue distribution was investigated. Then, 1860 juvenile scallops were divided into two groups with two initial densities of high density (200 individuals/layer, HD) and normal density (110 individuals/layer, ND) and in-situ cultured for three months, in which their growth, survival, and the differential expression of CnMyD88 were examined, respectively. Finally, scallops were injected with the Vibrio parahaemolyticus to assess the temporal expression of CnMyD88. As the results show, CnMyD88 cDNA has a full length of 2241 bp and contains an 1107 bp ORF that encodes a 368-derived protein. It was widely expressed in examined tissues with a significantly higher level in hemolymph, intestine, mantle, and gonad than others. Besides, the HD group showed lower growth (0.39 ± 0.05 mm/day) and survival (37.00 ± 8.49%) than the ND group (0.55 ± 0.02 mm/day and 76.82 ± 5.78%). More importantly, the HD group exhibited significantly lower expression levels of CnMyD88 in their examined tissues than the ND group. After V. parahaemolyticus challenging, CnMyD88 had significantly lower expression levels in the scallops from the HD group than that of the scallops from the ND group at 6th, 24th, and 36th. The present results indicated that high stocking density not only made adverse impacts on growth and survival but also may induce immunosuppression in the noble scallop. Therefore, appropriate low stocking density may be worth considering to adopt in scallop aquaculture.

Effect of fed dietary yeast (Rhodotorula paludigena CM33) on shrimp growth, gene expression, intestinal microbial, disease resistance, and meat composition of Litopenaeus vannamei.

Yeast is a health-promoting and bio-therapeutic probiotic that is commonly used in aquaculture. Rhodotorula paludigena CM33 can accumulate amounts of intracellular carotenoids and lipid, which are regarded as nutritionally beneficial compounds in various aspects. The aim of this study was to evaluate the impact of different levels of R. paludigena CM33 (RD) incorporated in a dietary composition at 0% (control), 1% (1% RD), 2% (2% RD), and 5% (5% RD) on the growth of shrimp (Litopenaeus vannamei), their immune-related gene expression, intestinal health, resistance to Vibrio parahaemolyticus (VPAHPND) infection, and meat composition. The results showed significant improvements in the specific growth rate, weight gain, and survival of shrimp fed with 1% RD, 2% RD, and 5% RD, which were higher than the control group after 4 weeks of administration. The administration of 5% RD group resulted in a decrease in cumulative mortality upon VPAHPND challenge when compared to the control group. Furthermore, the expression levels of immune-responsive genes, including proPO system (prophenoloxidase-2: PO2), antioxidant enzyme (superoxide dismutase: SOD, glutathione peroxidase: GPX, and catalase: CAT), JAK/STAT pathway (signal transducer and activator of transcription: STAT, gamma interferon inducible lysosomal thiol reductase: GILT), IMD pathway (inhibitor of nuclear factor kappa-B kinase subunit beta and epsilon: IKKb and IKKe), and Toll pathway (Lysozyme) genes, were up-regulated in the 5% RD group. In the context of microbiota, microbiome analysis revealed that the main phyla in shrimp intestines were Proteobacteria, Firmicutes, Bacteroidota, Campilobacterota, Actinobacteriota, and Verrucomicrobiota. At the genus level, Vibrio was found to be reduced in the 5% RD group, whereas the abundance of potentially beneficial bacteria Bifidobacterium was increased. The 5% RD group showed a significant increase in the levels of crude protein and crude lipid, both of which are essential nutritious components. Our results show the capability of R. paludigena CM33 as a probiotic supplement in shrimp feed in improving growth, antimicrobial responses against VPAHPND, and meat quality by increasing protein and lipid content in shrimp.

Characterization of a crustin-like peptide involved in shrimp immune response to bacteria and Enterocytozoon hepatopenaei (EHP) infection in Palaemon carinicauda.

Crustins represent one type of antimicrobial peptides (AMPs) that are key components of the innate immune process of crustaceans. This study successfully identified a novel crustin-like peptide, EcCrustin2, in ridgetail white prawn, Palaemon carinicauda (formerly Exopalaemon carinicauda). EcCrustin2 was found to be 1082 bp in length, with a 378 bp open reading frame (ORF) encoding 125 amino acids. The deduced amino acid sequence of EcCrustin2 exhibited characteristics of crustins in crustacean, including a Cys-rich region at the N-terminus as well as a whey acidic protein domain at the C-terminus. Phylogenetic analysis revealed that the EcCrustin2 was first clustered with Type I crustins, then with other crustins. Expression of EcCrustin2 was mainly detected in immune tissues, including hemocytes, gill and stomach. The expression level of EcCrustin2 was also significantly up-regulated after being exposed to lipopolysaccharide (LPS), lipoteichoic acid (LTA), Vibrio parahaemolyticus and Staphylococcus aureus. EHP infection could also induce EcCrustin2 expression in P. carinicauda. Knockdown of EcCrustin2 with siRNA increased the mortality of V. parahaemolyticus challenged shrimp. Finally, the recombinant EcCrustin2 protein was obtained and demonstrated a wide spectrum of antibacterial activity in vitro. These results indicated that EcCrustin2 takes part in the immune response against bacteria and EHP infection.

Peroxiredoxin-4 supplementation modulates the immune response, shapes the intestinal microbiome, and enhances AHPND resistance in Penaeus vannamei.

Peroxiredoxin-4 from Penaeus vannamei (LvPrx4) is considered a damage-associated molecular pattern (DAMP) that can activate the expression of immune-related genes through the Toll pathway. We previously demonstrated that the recombinant LvPrx4 (rLvPrx4) can enhance shrimp resistance against Vibrio parahaemolyticus, causing acute hepatopancreatic necrosis disease (VPAHPND), which causes great production losses in shrimp farming. Herein, we showed that the rLvPrx4 had a thermal tolerance of around 60 °C and that the ionic strength had no noticeable effect on its activity. We discovered that feeding a diet containing rLvPrx4 to shrimp for three weeks increased the expression of the immune-related genes LvPEN4 and LvVago5. Furthermore, pre-treatment with rLvPrx4 feeding could significantly prolong shrimp survival following the VPAHPND challenge. The shrimp intestinal microbiome was then characterized using PCR amplification of the 16S rRNA gene and Illumina sequencing. Three weeks of rLvPrx4 supplementation altered the bacterial community structure (beta diversity) and revealed the induction of differentially abundant families, including Cryomorphaceae, Flavobacteriaceae, Pirellulaceae, Rhodobacteraceae, and Verrucomicrobiaceae, in the rLvPrx4 group. Metagenomic predictions indicated that some amino acid metabolism pathways, such as arginine and proline metabolism, and genetic information processing were significantly elevated in the rLvPrx4 group compared to the control group. This study is the first to describe the potential use of rLvPrx4 supplementation to enhance shrimp resistance to VPAHPND and alter the composition of a beneficial bacterial community in shrimp, making rLvPrx4 a promising feed supplement as an alternative to antibiotics for controlling VPAHPND infection in shrimp aquaculture.

The Hippo-Yki pathway downstream transcription factor Scalloped negatively regulates immune defense against Vibrio parahaemolyticus infection in shrimp.

The Hippo-Yki signaling pathway plays a crucial role in numerous biological processes. Previous studies have demonstrated the significance of signal transduction components of the Hippo pathway in the immune response of shrimp. In this study, the downstream transcription factor of Hippo signaling, Scalloped, was analyzed in the context of Vibrio parahaemolyticus infection in Pacific white shrimp, Penaeus vannamei. Upon bacterial and fungal infections, the expression of Scalloped was upregulated in hemocytes. Scalloped was found to localize in the nucleus and interact with the Hippo pathway downstream transcriptional co-activator Yki. With the assistance of Yki, Scalloped activated the promoter of Cactus, a cytoplasmic inhibitor of the NF-κB pathway, leading to the inhibition of the nuclear translocation of the NF-κB family member Dorsal in shrimp. By inhibiting the Dorsal pathway, Scalloped reduced the expression of immune functional proteins and negatively regulated the immune response against bacterial infection in shrimp. RNAi-mediated silencing of Scalloped significantly enhanced the survival rate of V. parahaemolyticus-infected shrimp and reduced the bacterial load in tissues. These findings demonstrate the potential of Scalloped as a therapeutic target for vibriosis in crustaceans and contribute to our understanding of the shrimp's antibacterial defense and the functional roles of Hippo signaling in animal immunity.

Quorum sensing signal synthases enhance Vibrio parahaemolyticus swarming motility.

Vibrio parahaemolyticus is a significant food-borne pathogen that is found in diverse aquatic habitats. Quorum sensing (QS), a signaling system for cell-cell communication, plays an important role in V. parahaemolyticus persistence. We characterized the function of three V. parahaemolyticus QS signal synthases, CqsAvp , LuxMvp , and LuxSvp , and show that they are essential to activate QS and regulate swarming. We found that CqsAvp , LuxMvp , and LuxSvp activate a QS bioluminescence reporter through OpaR. However, V. parahaemolyticus exhibits swarming defects in the absence of CqsAvp , LuxMvp , and LuxSvp , but not OpaR. The swarming defect of this synthase mutant (termed Δ3AI) was recovered by overexpressing either LuxOvp D47A , a mimic of dephosphorylated LuxOvp mutant, or the scrABC operon. CqsAvp , LuxMvp , and LuxSvp inhibit lateral flagellar (laf) gene expression by inhibiting the phosphorylation of LuxOvp and the expression of scrABC. Phosphorylated LuxOvp enhances laf gene expression in a mechanism that involves modulating c-di-GMP levels. However, enhancing swarming requires phosphorylated and dephosphorylated LuxOvp which is regulated by the QS signals that are synthesized by CqsAvp , LuxMvp , and LuxSvp . The data presented here suggest an important strategy of swarming regulation by the integration of QS and c-di-GMP signaling pathways in V. parahaemolyticus.