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Shrimp aquaculture has experienced a significant global growth in the past twenty years, making a huge contribution to economic revenue. However, the emergence of Acute Hepatopancreatic Necrosis Disease (AHPND) imposed a huge economic, welfare, and environmental burdens to aquaculture sector globally. In this review, we emphasized on two key applications on AHPND treatment and AHPND diagnosis through the lens of metallic nanoparticles (MNPs) interventions. We review the potentialities and implications of MNPs through accumulated evidence and focussed investigations specifically targeting the AHPND causative agent, Vibrio parahaemolyticus, to improve the understanding of MNPs application from shrimp aquaculture perspective. MNPs treatment offers a novel antibacterial application, providing a new alternative to antibiotic therapy in combating AHPND, but also the implication from toxicological perspective challenges the integration of this technology into aquaculture disease management and treatment applications. We examine how the toxicological considerations have impacted the direction of MNPs synthesis in aquaculture research, aiming to produce MNPs that are biocompatible and suitable for aquaculture disease and treatment program. The lack of practicality in the diagnostic approach for AHPND hindered easy practise of disease detection among shrimp farmers. Thus, we also explore how the incorporation of MNPs into the existing AHPND diagnostic approach has been improved, leading to the development of a practical and user-friendly diagnostic tool for detecting V. parahaemolyticus. Application of nanotechnology through MNPs integration into AHPND treatment and diagnosis has big potentials to improve the disease management strategy, potentially revolutionizing shrimp aquaculture on a global scale. [ABSTRACT FROM AUTHOR]

Environmental stress can disrupt the intestinal microbiota of aquatic animals, thereby impacting their health status. In this study, we aimed to elucidate the dynamic changes in the bacterial community within shrimp intestines under short-term ammonia nitrogen stress and investigate the subsequent ecological risks associated with it. Our findings revealed a significant reduction in the Chao1 diversity index within shrimp intestines of the ammonia nitrogen stress group compared to the Control group. Furthermore, distinct disparities in the bacterial community structures were observed between the two groups at 72 h and 120 h post-stress. Additionally, ammonia nitrogen stress significantly altered the abundances of specific bacterial taxa within shrimp intestines. Specifically, the abundance of Bacillus subtilis increased while that of Lactococcus garvieae decreased in the intestine under stress at 72 h. Additionally, Roseivivax jejudonensis exhibited an increase, whereas uncultured_o_Saccharimonadales and Hyphomicrobium sp. showed a decrease at 120 h. Notably, opportunistic pathogens such as Vibrio parahaemolyticus experienced a significant increase at 72 h post-stress, potentially due to a substantial contribution from stochastic processes in shaping the assembly of the intestinal community. Additionally, the intestinal bacterial community of shrimp displayed a noticeable decrease in complexity of bacterial co-association networks under stress conditions. Thus, short-term exposure to ammonia nitrogen disrupts the integrity of shrimp's intestinal community, indicating potential ecological risks within their microhabitat. Confirmatively, we conducted the immersion challenge experiment of V. parahaemolyticus on shrimp post community disturbance under stress, and observed a significantly higher copy numbers of V. parahaemolyticus in host intestines of stressed compared to control shrimp. These findings can provide novel insights into the interactions among ammonia nitrogen stress, intestinal microbiota and host health of aquatic animals, and guide the practices for shrimp healthy culture. [Display omitted] • The impact of short-term ammonia nitrogen stress on the intestinal bacterial community and health of shrimp is investigated. • Short-time ammonia nitrogen stress induces dysbiosis of the intestinal bacterial community of shrimp. • Short-time ammonia nitrogen stress facilitates the enrichment of Vibrio parahaemolyticus in intestines of shrimp. [ABSTRACT FROM AUTHOR]

Pathogenic infections are one of the main causes of molt failure and high mortality during molting on mud crab (Scylla paramamosain) farms. However, the molecular mechanisms associated with the immune defense during crab molting have not been clearly elucidated. Peritrophin, a classical chitin-binding protein, plays a key role in molting development and immunity of insects and crustaceans. In the present study, a new peritrophin homologous gene, SpPT1, was identified in S. paramamosain , and its full-length cDNA sequence was containing a single type II chitin-binding domain. During mud crab development, the SpPT1 gene was highly expressed at the zoea V stage, and its transcripts were significantly upregulated after molting compared to the pre-molt stage of juvenile crabs, particularly in the presence of bacterial exposure. A similar pattern was observed in the gills and subcuticular epidermis during molting in subadult crabs. Interestingly, this gene is regulated by 20E, a key steroid hormone during molting. In adult crabs, the highest expression of SpPT1 was detected in the midgut, followed by the stomach and hepatopancreas. This gene responded significantly to bacterial challenge in the hepatopancreas. Recombinant SpPT1 (rSpPT1) expressed in prokaryotes exhibited potent binding activity to chitin and several common aquatic pathogens, such as Vibrio alginolyticus , Vibrio parahaemolyticus , and Aeromonas hydrophila , as well as to four pathogen-associated molecular patterns (PAMPs). Notably, rSpPT1 promoted the phagocytosis of hemocytes in a dose-dependent manner in vitro , enhanced the clearance of V. alginolyticus from the hepatopancreas and gills, and significantly increased the survival of subadult S. paramamosain after bacterial infection in vivo. In addition, rSpPT1 markedly upregulated the expression of immune-related genes and enhanced the enzymatic activities of acid phosphatase and peroxidase in the hepatopancreas. Taken together, this study demonstrates that the peritrophin homolog SpPT1 might play a role in immune defense during molting, which provides new perspectives for the development of strategies to effectively control molt failure on crab farms. • SpPT1 gene was upregulated after molting and more significantly in response to bacterial challenge. • rSpPT1 exhibited potent binding activity to aquatic pathogens and PAMPs. • rSpPT1 promoted hemocyte phagocytosis in a dose-dependent manner. • rSpPT1 enhanced the bacterial clearance and crab survival. [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) and acute hepatopancreatic necrosis disease caused by virulence gene-containing Vibrio bacteria (AHPND) are two important infectious diseases that affect shrimp, including the economically important species Penaeus vannamei. Because these pathogens can spread rapidly in shrimp aquaculture facilities and because effective therapies are presently lacking, WSSV and AHPND have resulted in significant economic losses in the shrimp farming industry. Thus, the early detection of WSSV and AHPND is essential for shrimp farming. This study combines recombinase polymerase amplification (RPA) technology with the nuclease Argonaute from Pyrococcus furiosus (Pf Ago) to develop a duplex in-one-tube detection method with color indication (DIDCi) for WSSV and AHPND utilizing a combination of two fluorescence probes. The method successfully achieves a satisfactory sensitivity of a single gene copy per reaction for WSSV and 102 gene copies per reaction for AHPND. The detection exhibits good specificity and convenience, with the detection results indicated by different colors that can be read with the naked eye, thereby providing an effective tool for the early detection of WSSV and AHPND. This study marks the first application of Pf Ago for multiplex nucleic acid detection of aquacultural pathogens and explores the strategy of using combinations of fluorescence probes for the identification of multiple targets in one tube. • A duplex in-one-tube method for early detection of WSSV and AHPND. • Detection results conveniently indicated by different colors. • Combination of fluorescence probes for color distinction of multiple targets. [ABSTRACT FROM AUTHOR]

We evaluated the effects of resveratrol (RES) supplementation on the growth performance, physiological health, transcriptomic response, and intestinal health of Litopenaeus vannamei. L. vannamei (initial body weight: 0.40 ± 0.02 g) were fed using six types of experimental diets (R0, R50, R100, R200, R400, and R800, supplemented with 0, 50, 100, 200, 400 and 800 mg/kg of RES, respectively) for 56 days. The results showed that dietary RES had no significant effect on the growth of L. vannamei. The survival rate (SR) of shrimp in the R400 group was significantly higher than that in the R0 group after exposure to Vibrio parahaemolyticus. RES supplementation dramatically enhanced the immunity of L. vannamei , as indicated by the increased serum levels of important indicators of immune function. The activities of intestinal digestive enzymes increased dramatically with increasing RES supplementation. Transcriptome analysis revealed that adding RES had a considerable effect on various metabolic and immune-related pathways in L. vannamei , including beta-alanine metabolism, ascorbate and aldarate metabolism, and drug metabolism-other enzymes. 16S rDNA analysis indicated that RES supplementation improved the intestinal microflora composition of L. vannamei by enhancing the abundance of beneficial bacteria and reducing the abundance of harmful bacteria. In conclusion, this study indicated that RES supplementation at an appropriate dose (400 mg/kg) could improve the physiological health, regulate the response at the transcriptional level, and ameliorate the intestinal microflora structure of L. vannamei. These results offer novel ideas and theoretical support for using RES in L. vannamei cultures as an immune-boosting supplement. • Dietary resveratrol (RES) improved the intestinal digestive capacity of Litopenaeus vannamei. • Dietary RES improved the resistance to Vibrio parahaemolyticus of L. vannamei. • Dietary RES affected the expression of genes related to metabolic pathway. • Dietary RES optimized the bacteria structure in the intestine of L. vannamei. [ABSTRACT FROM AUTHOR]

Aquaculture is a rapidly growing industry, accounting for more than half of the worldwide fish production. However, this sector is often affected by bacterial infections, threatening farmed animals and consumer safety. This study explored the potential of Photodynamic Inactivation (PDI) as an innovative antimicrobial approach for the inactivation of Gram-negative bacteria relevant to the aquaculture sector: Vibrio parahaemolyticus , Vibrio anguillarum , and Escherichia coli. The experiments were conducted in the presence of the cationic photosensitizers (PSs) 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP) and methylene blue (MB). For V. parahaemolyticus, the protocol was extended to the neutral and anionic PSs curcumin (CUR), chlorin e 6 trimethyl ester (Ce 6 Me), and 5,10,15,20-tetrakis(4-sulfophenyl)porphyrin (TPPS 4). The assays were performed in the absence or presence of potassium iodide, well-known to improve PDI effect, while its potential for aquaculture application is yet to be studied. These evaluations were performed in phosphate-buffered saline (PBS) and in artificial seawater (ASW) to simulate aquaculture water disinfection. The cationic PSs TMPyP and MB were the most efficient in the bacterial inactivation. Although higher concentrations of PS were required to achieve effective bacterial inactivation in ASW compared to PBS, the application of KI enhanced the effectiveness of all PSs, reducing the treatment time at least by threefold. The bacterial inactivation profiles revealed higher susceptibility of Vibrionaceae bacteria which may be linked to the differential PDI impact on the outer cell membranes of these bacteria. These findings indicate that treatments with PSs, particularly TMPyP and MB , in combination with KI constitute a potential approach for bacteria control in the aquaculture sector. • The study explores Photodynamic Inactivation (PDI) of bacteria affecting aquaculture. • Experiments were conducted using cationic, neutral and anionic photosensitizers (PSs). • The application of potassium iodide (KI) boosted the success of PDI. • The Vibrionaceae bacteria, with negative impact on aquaculture, was highly susceptible to PDI. • PDI in combination with KI is a potential approach for bacteria control in aquaculture. [ABSTRACT FROM AUTHOR]

Acute hepatopancreatic necrosis disease (AHPND) is a major bacterial disease caused by Vibrio parahaemolyticus (VP AHPND) that results in significant economic losses to Pacific white shrimp Litopenaeus vannamei aquaculture. Breeding of disease-resistant broodstocks is regarded as a fundamental strategy for solving the disease problem. This study evaluated the feasibility of genomic selection (GS) for enhancing the AHPND resistance of L.vannamei. Following the VP AHPND challenge, the survival time was utilized as a phenotypic measure to assess the AHPND resistance. The reference population consisted of 447 genotyped and 288 no-genotyped individuals with phenotypes. The candidate population consisted of 206 uninfected and genotyped individuals. The liquid chip "Yellow Sea Chip No.1" with 40 K single nucleotide polymorphism (SNP) generated genotypes for the reference and candidate populations. Heritabilities of survival time obtained using the average information REML method with a relationship matrix (A or H) and an animal model were 0.16 ± 0.06 and 0.22 ± 0.07, respectively. Predictive accuracies of pedigree-based best linear unbiased prediction (PBLUP), single-step genomic BLUP (ssGBLUP), and single-step Bayesian regression (ssBR) were evaluated using random, full-sib and between-family cross-validation. Compared to PBLUP, the predictive accuracies of ssGBLUP and ssBR for the survival time increased by 15.00 % and 67.50 % in random cross-validation, and 13.33 % and 53.33 % in full-sib cross-validation, respectively. All methods exhibited a reduction in predictive accuracy when moving from the random cross-validation or full-sib cross-validation to the between-family cross-validation. The predictive accuracies of Genomic BLUP (GBLUP) and Bayesian A (BayesA) were evaluated using full-sib cross-validation. Compared to GBLUP, the predictive accuracy of BayesA increased by 33.33 %. ssBR was used to predict the candidates' genomic estimated breeding values (GEBVs). 80 candidates with high GEBVs, 86 with medium GEBVs, and 40 with low GEBVs were selected to breed offspring with a spectrum of resistance levels. In the VP AHPND challenge test, the high resistance offspring had mean survival times that were 10.15 % longer than the moderate resistance offspring and 102.01 % longer than the low resistance offspring. This study validates the practicality and success of GS in improving resistance to AHPND in L.vannamei. • ssGBLUP and ssBR improved AHPND resistance prediction by 15.00 % and 67.50 % over PBLUP. • High resistance offspring outlived moderate and low resistance ones by 10.15 % and 102.01 %. • Study proved genomic selection's effectiveness for AHPND resistance via progeny testing. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs) are essential for maintaining cellular homeostasis and regulating immune responses across diverse species. In aquaculture animals, the HSP70 family plays a crucial role in regulating innate immunity against pathogenic infections, although the mechanisms remain unclear. In this study, we investigate the role of HSP70 from Litopenaeus vannamei (LvHSP70) in regulating immune responses and intestinal microbiota composition during Vibrio parahaemolyticus infection. Using RNA interference (RNAi) to knock down LvHSP70 in shrimp, we observed significant impacts on homeostasis, including reduced activity, loss of feed intake, hepatopancreas damage, and individual death. During V. parahaemolyticus infection, shrimp with silenced LvHSP70 exhibited increased bacterial loads and elevated tissue damage. Transcriptomic analysis revealed that LvHSP70 knockdown significantly downregulated the expression of a broad range of immune-related genes, including antimicrobial peptides, cytokines, and components of the prophenoloxidase (ProPO) system. Additionally, 16S rDNA sequencing demonstrated a marked increase in the relative abundance of Vibrio in the intestines of LvHSP70-deficient shrimp. These findings underscore the essential role of LvHSP70 beyond its classical heat shock response in maintaining shrimp homeostasis, regulating immune responses, and controlling intestinal microbiota. • HSP70 is essential for maintaining cellular homeostasis and regulating immune responses in shrimp. • Silencing of HSP70 leads to reduced activity, loss of feed intake, hepatopancreas damage and increased mortality. • HSP70 knockdown results in downregulation of immune-related genes and an increased abundance of Vibrio in shrimp intestines. [ABSTRACT FROM AUTHOR]

Exosomes play an important role in the innate immune system. Many types of cells can secrete exosomes in both normal and abnormal status. However, the mechanism of exosome secretion in invertebrates has not been reported. In this study, the mechanism of the secretion of exosomes regulated by the Ca2+ signaling pathway in the hemocytes of mud crabs (Scylla paramamosain) under infection with Vibrio parahaemolyticus was investigated. The results showed that the increase of exosome secretion in mud crabs infected with V. parahaemolyticus was caused by the increase in intracellular Ca2+ level. In addition, transcriptome sequencing and differential expression analysis revealed that Sp VAMP2, a vesicle-associated membrane protein 2, was found to be involved in the exosome secretion regulated by Ca2+ signaling. Further research revealed that Sp VAMP2 can inhibit MVB from entering the lysosome for degradation and promote the release of exosomes. Additionally, the secretion of exosomes upregulated the expression of anti-lipopolysaccharide factors and enhanced the survival rate of mud crabs after V. parahaemolyticus infection. This study clarified the exosome secretion regulated by the Ca2+- Sp VAMP2 pathway, which, in turn, increased the innate immune response in mud crabs to resist the invasion of V. parahaemolyticus. • The level of Ca2+ in the hemocytes regulates the secretion of exosomes, which was mediated by Sp VAMP2 in mud crabs. • Sp VAMP2 can regulate the secretion of exosomes through inhibiting autophagy. • Sp VAMP2 resists Vibrio parahaemolyticus infection by promoting exosome secretion of hemocytes in mud crabs. [ABSTRACT FROM AUTHOR]

Biofloc technology is a promising strategy for the super-intensive cultivation of Pacific white shrimp (Litopenaeus vannamei). However, conventional bioflocs originating from pre-existing systems have shown to be inferior to bioflocs with constant probiotic supplementation regarding the stability in yield. Hence we compared bioflocs supplemented with Rhodospirillum rubrum (RR), Bacillus subtilis (BS), and Providencia rettgeri (PR) to the clear water system (CT), in terms of growth performances, immune status, nitrogen substances, and Vibrio populations in cultures of L. vannamei together with microbial profiles in both the midgut and bioflocs. Our findings exhibited increased average daily gain (ADG) and protein efficiency ratio (PER) among shrimp reared in BS and PR while BS also reduced feed conversion ratio (FCR). Gene expression analyses of growth-related markers revealed an upregulation of amylase and peptide transporter 1 in shrimp of PR treatment, whilst the changes did not outperform the BS supplement in growth promotion. Furthermore, PR treatment was found to enhance the expression of immune-related genes toll-like receptor , prophenoloxidase (proPO), and heat shock protein 70 (hsp70). All biofloc treatments effectively reduced NO 2 -N level and mitigated Vibrio parahaemolyticus proliferation, particularly in the BS and PR groups. The redundant analysis illustrated shrimp growth closely associated with improved water quality instead of the expression of genes participating in digestion, absorption, or immunity. Moreover, microflora sequencing exhibited the supplemental probiotics altered microbial communities in bioflocs and gut, whereas they did not dominate in either environment. In spite of the discrepancies in microbial composition between bioflocs and midgut of shrimp, BS and PR treatments enriched Rhodobacteraceae populations in both environments. In summary, B. subtilis and P. rettgeri supplementary bioflocs promote L. vannamei growth through nutritional supplementation and water environment optimization. Supplementation of P. rettgeri will simultaneously enhance immune responses and stress resistance of L. vannamei via activating proPO cascade and upregulating hsp70. Given the apparent benefits of heterotrophic probiotic supplementary bioflocs, it is imperative to investigate more microbial species or their combinations that can stimulate bioflocs to produce more nutrients. • B. subtilis and P. rettgeri supplementary bioflocs boost shrimp growth performance. • Growth-promoting effects of biofloc rely on nutrient supply and water improvement. • P. rettgeri raises shrimp immunity and stress tolerance by proPO cascade and hsp70. • Probiotics supplemented bioflocs favor beneficial bacteria and suppress pathogens. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the antioxidant and immune responses of crayfish Procambarus clarkii to V. parahaemolyticus infection through a comparative analysis of the immune index, antioxidant capacity, histomorphology, and transcriptome data of V. parahaemolyticus -infected and PBS-treated P. clarkii. The findings indicated that V. parahaemolyticus infection resulted in intestinal cell necrosis, a reduction in the number and height of intestinal villi, and an increased absorption rate of the intestinal jejunum. The prolonged infection time of V. parahaemolyticus resulted in an initial increase in intestinal immunity and antioxidant enzyme activity, followed by a subsequent decrease, ultimately returning to levels similar to those in the control group. The intestinal transcriptional response to V. parahaemolyticus infection was assessed using RNA sequencing (RNA-seq). A total of 27,867 genes were significantly assembled, and 2124 genes showed differential expression, including 849 upregulated and 1275 downregulated genes. These genes were primarily enriched in KEGG pathways such as Ribosome, Oxidative phosphorylation, Phagosome, Inositol phosphate metabolism, and adrenergic signaling in cardiomyocytes. Simultaneously, ATP synthesis-coupled electron and proton transport, mitochondrial electron transport, and other energy metabolism pathways were significantly enriched in the Gene Ontology (GO) database. qRT-PCR analysis confirmed the reliability of the RNA-seq results for five randomly selected differentially expressed genes (DEGs). Our experimental data contributes to the transcriptome database of P. clarkii , enhances our understanding of the immune and antioxidant defense mechanisms of P. clarkii , and provides a foundation for future research on disease control in crayfish aquaculture. • The intestine transcriptomes of Procambarus clarkii were constructed using high-throughput sequencing. • Infection of crayfish with V. parahaemolyticus will cause changes in intestinal structure and antioxidant indexes. • GO terms and KEGG pathways were significantly enriched after V. parahaemolyticus treatment. • Phagosome pathway plays an important role in the immune defense of crayfish against V. parahaemolyticus infection. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is a severe pathogenic bacterium which causes significant mortality and economical loss in shrimp farming. To control vibriosis in whiteleg shrimps (Litopenaeus vannamei), we developed synbiotic beads by microencapsulating probiotic of Lactobacillus plantarum and prebiotic of xanthan gum in agarose matrix through emulsion technique. Coculture method indicated that synbiotic beads reduce viability of Vibrio parahaemolyticus by 3 orders of magnitude. The high inhibitory activity of synbiotic beads against pathogen could result from the antibacterial substances secreted from encapsulated probiotics, and access blocking of pathogens to prebiotics as food source so that the encapsulated prebiotics solely stimulate nearby probiotic growth without boosting pathogen growth. Bacterial challenge test indicated that exposure to synbiotic beads in advance can delay mortality and increase shrimp survival from 10% to 57%. As a smart encapsulation platform, our design can be extended to various probiotics, prebiotics, and pathogens, thus providing an alternative strategy to the use of antibiotics for disease prevention and sustainable aquaculture. [Display omitted] • Prebiotic xanthan gum supports the growth of probiotic Lactobacillus plantarum. • Probiotic Lactobacillus plantarum inhibits growth of pathogens in aquaculture. • Synbiotic encapsulation enhances probiotic potency against pathogens. • Synbiotic beads increase shrimp survival in bacterial infections. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the adaptability of commercial probiotics to the biofloc system, as well as the influence on bacterial community, water quality and aquatic animal growth. Two biofloc systems nursing Litopenaeus vannamei postlarvae for 28 days were designed, the control (cBFT) and pBFT with addition of commercial probiotics (containing Bacillus , Clostridium , Enterococcus , Lactobacillus and Rhodopseudomonas). Results showed that at the end of the experiment, only the absolute abundance and the proportion of Bacillus contained in commercial probiotics (2.1 ± 0.8 × 105 genome copies mL−1 and 0.12 ± 0.03%) significantly increased in pBFT compared to those in cBFT (1.7 ± 0.8 × 104 genome copies mL−1 and 0.03 ± 0.02%), respectively (P < 0.05). Absolute abundances and proportions of several autochthonal bacterial genera (i.g., Acinetobacter , Anaerospora , Arthrobacter , Devosia , Luteolibacter , Marivita and Mycobacterium) also significantly elevated in pBFT (P < 0.05). Total suspended solids (TSS) and turbidity (415.2 ± 79.6 mg L−1 and 379.0 ± 86.4 NTU) were significantly higher in pBFT than both parameters in cBFT (118.1 ± 23.9 mg L−1 and 109.4 ± 44.3 NTU), respectively (P < 0.05). Whereas, zootechnical indices (except survival rate) were significantly lower in pBFT than those in cBFT, respectively (P < 0.05). The integrated impact model of commercial probiotics explained 57.9%–77.1% variations of shrimp growth performance, and showed that commercial probiotics as well as some autochthonal bacteria positively correlated with turbidity which in turn negatively correlated with zootechnical indices (P < 0.05). The findings suggested that added probiotics (especially Bacillus) induced changes on the autochthonal bacterial community, leading to changes of water quality which consequently resulted in the depression of growth performance of shrimp. [Display omitted] • Bacillus in the commercial probiotics mixture well adapted to biofloc system. • Commercial probiotics did not show benefit on shrimp growth unexpectedly. • Changes on autochthonal bacterial community alleviated probiotics' effects. • Probiotics alone contributed 8.0%–14.7% of variations on growth performance. • The integrated model explained 57.9%–77.1% of variations on shrimp growth. [ABSTRACT FROM AUTHOR]

The present study investigated on the potential use of marine benthic diatom Halamphora sp. and harpacticoid copepod Amphiascoides neglectus enriched with Halamphora sp. on the growth, biochemical composition, water quality performance and disease tolerance of black tiger shrimp Penaeus monodon postlarvae culture. The introduction of Halamphora sp. and enriched harpacticoid copepod (EHC) as feed to the postlarvae increased the final weight gain (38.10 ± 1.50 to 40.43 ± 1.17 mg/individual), weight gain (1267.97 ± 24.59 to 1321.09 ± 22.53%) and specific growth rate (9.34 ± 0.22 to 9.47 ± 0.26% day−1) significantly (p < 0.05) compared to the commercial feed. The protein and lipid contents of the postlarvae fed with EHC (protein: 15.26 ± 0.40% wet weight (WW); lipid: 1.91 ± 0.07% WW) were significantly higher (p < 0.05) compared to Halamphora sp. (protein: 13.66 ± 0.51%WW; lipid: 1.70 ± 0.01% WW) and the commercial feed. Moreover, the inclusion of Halamphora sp. and EHC into the postlarvae zero exchange water culture yielded lower nitrite (0.141 to 0.181 mg L−1), nitrate (3.15 to 8.50 mg L−1), orthophosphate (1.38 to 1.90 mg L−1), and total ammonia nitrogen (0.01 to 0.77 mg L−1) levels compared to the commercial feed. Finally, the incorporation of EHC into the diet also protected the postlarvae from the acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus by demonstrating the highest survival rate (63.33%) of the postlarvae compared to Halamphora sp. (mortality rate: 63.33%) and commercial feed (mortality rate: 100%) treatments after 72 h of the challenge test. The current findings highlighted the great potential of using EHC as live food for better growth performance of P. monodon postlarvae as well as the protection against virulent shrimp pathogens. • The diatom Halamphora sp. enhanced the nutritional values of harpacticoid copepod and black tiger shrimp postlarvae. • Harpacticoid copepod enriched with diatom Halamphora sp. promoted the growth of black tiger shrimp postlarvae. • The black tiger shrimp postlarvae fed with enriched harpacticoid copepod demonstrated a protective effect towards Vibrio parahaemolyticus responsible for Acute Hepatopancreatic Necrosis Disease. • Halamphora sp. and harpacticoid copepod showed bioremediation properties through efficient water nutrients removal. [ABSTRACT FROM AUTHOR]

Probiotic properties are highly species- or even strain- specific. Strain-specific responses of growth performance, non-specific immunity, disease resistance, and intestinal microbiota in Penaeus vannamei to two Enterococcus faecium strains supplementation were investigated in this study. Basal diet (control, CON) was supplemented with E. faecium HK and LYB at the dose of 1 × 1010 CFU / kg (HK group) and 1 × 1011 CFU / kg (LYB group), respectively. The results indicated that the HK and LYB groups exhibited significant enhancements in growth performance, non-specific immunity, and disease resistance of shrimp against Vibrio parahaemolyticus compared to the control (P < 0.05). However, the specific growth rate of shrimp in the LYB group was significantly better than that in the HK group (P < 0.05). Furthermore, the HK group showed significant increases in phenoloxidase, total nitric oxide synthase activities, lysozyme content in the serum, as well as the relative expression levels of proPO , LGBP , Imd , Relish , and eIF4E2 genes in the hepatopancreas compared to the LYB group (P < 0.05). These results suggested the strain HK exhibited better non-specific immunity efficiency while the strain LYB exerted better growth enhancement in shrimp. Dietary both of strain HK and LYB significantly altered microbial community structure (P < 0.05), and the strain HK significantly improved intestinal microbial diversity in shrimp. The relative abundance of Rubritaleaceae and Rhodobacteraceae were significantly increased, whereas those of facultative anaerobes and potential pathogens (i.e., Vibrionaceae , Flavobacteriaceae and Erysipelotrichaceae , etc.) were reduced in response to dietary supplementation of E. faecium HK and LYB. Furthermore, dietary both of strain HK and LYB could promote intestinal microbial community stability by increasing average connectivity, cooperation interactions, the number of generalists within the network. When growth performance and intestinal microbial homeostasis of shrimp are considered, E. faecium LYB might be better than the strain HK in shrimp farming. • Enterococcus faecium HK and LYB enhanced growth, non-specific immunity and disease resistance of shrimp. • Strain HK and LYB altered intestinal microbial diversity and community structure in shrimp. • Strain HK and LYB decreased the abundance of potentially pathogens in shrimp intestine. • Strain HK and LYB promoted intestinal microbial homeostasis of shrimp. • Strain HK exhibited better non-specific immunity efficiency while the strain LYB exerted better growth enhancement in shrimp. [ABSTRACT FROM AUTHOR]

Bacterial infections caused high mortality of Manila clam (Ruditapes philippinarum) after the sudden drop in salinity of seawater, which caused serious economic losses. Therefore, we explored the molecular mechanism of V. parahaemolyticus -mediated immunity of R. philippinarum under low-salinity condition. In this study, Manila clams treated with low-salinity (CD), V. parahaemolyticus -treated under low-salinity condition (HCD), and a blank group (CC) were sequenced through the Illumina HiSeq platform, and we verified 15 immune-related DEGs (12 up-regulated genes and 3 down-regulated genes) through quantitative real-time PCR (qRT-PCR), the results were consistent with those detected from the transcriptome. In addition, the results of KEGG pathway enrichment analysis showed that the different expression genes (DEGs) were mostly enriched in the lysosome and phagosome pathway exhibited upregulated expression throughout the V. parahaemolyticus infection. Among them, lysosome and phagosome-related genes were induced after R. philippinarum treated with V. parahaemolyticus. Thus, our results proved that the important role of regulatory mechanism of immune homeostasis mediated by the lysosome and phagosome pathway in R. philippinarum against V. parahaemolyticus infection. Altogether, this work provides a new information about the immune regulatory mechanisms of Manila clam response to V. parahaemolyticus challenge in a low-salinity environment. • Manila clam was subjected to V. parahaemolyticus infection under low-salinity condition. • The survival rate of clams in V. parahaemolyticus + low-salinity group was obviously lower than that in low-salinity group. • Lysosome and phagosome pathways were activated after clam treated with V. parahaemolyticus under low-salinity conditions. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the effect of fermentation (F; a phase without aeration) and respiration (R; a phase with aeration) processes, as well as varying processing times of the synbiotic system fertilizer, on the nitrification process, plankton composition, and the growth of Penaeus vannamei during the nursery phase comparing it with a biofloc system. A trial with a stocking density of 2400 shrimp m−3 was conducted over 48 days comprising the following treatments (with three repetitions each): CW: clear water (control); BFT: biofloc system; F12: fermentation for 12 h; F12 + R12: fermentation for 12 h + respiration for 12 h; F24: fermentation for 24 h; F24 + R24: fermentation for 24 h + respiration for 24 h; R12: respiration for 12 h; R24: respiration for 24 h. Rice bran, a commercial powder probiotic consisting of Bacillus subtilis and Bacillus licheniformis , sodium bicarbonate as a buffer, molasses, and water were utilized in the fertilizer. Treatments F12 + R12 and R24 demonstrated a more rapid control of TAN. A dominance of amoeba was noted in BFT, F12, F12 + R12, and F24 treatments. Treatments F12 + R12, F12, and F24 + R24 exhibited a higher abundance of ciliates compared to CW, BFT, F24, and R12 at the end of the experimental period. The abundance of vibrio at the end of the experiment was higher in the F24 + R24 treatment than in the other treatments. By the end of the trial, treatments F12, F12 + R12, F24, and R12 showed a lower FCR than the CW treatment. Yield was higher in F12, F12 + R12, F24, R12, and R24 compared to the CW treatment. Our findings suggest that reducing fertilizer processing time is viable for synbiotic system fertilization, optimizing the system management. Both fermentation and respiration phases are equally crucial, and therefore, the F12 + R12 treatment emerged as the most effective fertilization strategy, exhibiting significant effects on shrimp FCR, yield, and plankton composition of the system. • Synbiotic fertilizer with fermentation for 12 h and respiration for 12 h controls ammonia in Penaeus vannamei nurseries. • F12 + R12 treatment provides the growth of a high load of microorganisms in P. vannamei intensive nurseries. • F12 + R12 treatment reduces FCR and increases yield in P. vannamei nurseries. [ABSTRACT FROM AUTHOR]

Infectious myonecrosis virus (IMNV) causes 40–80% cumulative mortality in Penaeid shrimp aquaculture, with severity being dependant on pathogen variant and culture conditions. With an increasing presence of IMNV in shrimp aquaculture and devastating impacts in recent years on commercial production, there is increased demand for cost effective surveillance, early detection, and early mitigation strategies for this pathogen in combination with other devastating pathogens including White Spot Syndrome virus (WSSV), virulent strains of Vibrio parahaemolyticus (which cause acute hepatopancreatic necrosis disease (AHPND)) and Enterocytozoon hepatopenaei (EHP) that are also highly prevalent around the world. This study undertook assay validation of the IMNV test within the commercially available Shrimp MultiPath™ (SMP) technology in comparison to an existing quantitative PCR recommended by the World Organisation for Animal Health (WOAH). A total of 254 samples from three Penaeus vannamei populations from farms in the Indo-Pacific region that had reduced feed intake were used for this study. Key performance metrics for analytical and diagnostic performance of the IMNV assay in Shrimp MultiPath™ were assessed in addition to reproducibility and repeatability following the WOAH validation pathway. Establishing these metrics is important to provide the global industry with a cost-effective reliable tool that can be used to look at complete pathogen profiles to mitigate disease risk in shrimp farming. Limit of detection was determined to be 6.6 copies per microlitre of total nucleic acid sample. The diagnostic sensitivity of the IMNV SMP was 99.2% and diagnostic specificity 98.0%, whereas the diagnostic sensitivity and specificity of qPCR were estimated to be 86.9% and 98.2%, respectively. Importantly, the SMP IMNV assay detects both Indo-Pacific and Eastern Latin American variants of IMNV. Overall, the IMNV SMP assay outperforms currently published assays for early detection, and early risk mitigation of this pathogen in shrimp culture, whilst having the added benefit of a full pathogen profile due to its multiplexed nature. • Novel IMNV detection method using highly specific and sensitive multiplex PCR-based Shrimp MultiPath™ (SMP) technology. • Cost-effective high throughput method suitable for surveillance, translocation testing and confirmation of clinical disease. • SMP IMNV assay shows comparable analytical and diagnostic performance compared to WOAH qRT-PCR assay (Andrade et al., 2007). • Bayesian Latent Class Model approach used for determining Diagnostic metrics. • Updated performance, analytical and diagnostic validation metrics for Andrade qRT-PCR assay. [ABSTRACT FROM AUTHOR]

In early 2020, the shrimp farming in China suffered a severe setback due to an outbreak of the translucent post-larvae disease (TPD) in Penaeus vannamei. The emerging disease was caused by a hypervirulent strain of Vibrio parahaemolyticus (Vp TPD). Although some progress has been made in understanding virulence factors of Vp TPD , the diversity and prevalence of TPD pathogen are still unclear. Therefore, this study was undertaken to systematically investigate the epidemiology of TPD in China. In this study, potentially pathogenic bacteria present in naturally infected shrimp samples were isolated, identified, and preserved firstly. The further investigation showed that different Vibrio species carrying the key virulence genes of Vp TPD can infect post-larvae of P. vannamei and cause TPD, revealing the diversity of TPD pathogens, and the pathogenic Vibrio strains has been temporarily named as Vibrio causing TPD (V TPD). In addition, epidemiological surveillance based on TaqMan qPCR detection and histopathology techniques analysis showed that V TPD positive samples were detected in most shrimp aquaculture ponds in different shrimp aquaculture provinces of China, with prevalence rate exceeding 50% in some areas. And seawater cultured species such as P. vannamei and P. japonicus were more sensitive to V TPD and were more likely to be infected or carry it. Freshwater cultured shrimp such as Macrobrachium rosenbergii and Procambarus clarkii may not be affected by V TPD. However, shrimp bait organisms are still at risk of carrying or being infected with V TPD. The result suggests that V TPD is still prevalent and spreading in coastal shrimp farming areas in China, with a high risk of transmission. This effort of present study provided a basic epidemiological understanding as well as important technical insights for prevention and management of emerging shrimp diseases. • Translucent post-larvae disease (TPD) caused by V TPD threatens shrimp aquaculture. • Different Vibrio species carrying the key virulence genes of V p TPD can cause TPD. • Shrimp bait organisms have the risk of carrying or being infected with V TPD. • V TPD is prevalent and spreading in coastal shrimp farming areas in China. [ABSTRACT FROM AUTHOR]

The innate immunity is widely regulated by microRNAs (miRNAs) and that miRNA-125 (miR-125) is involved in many diseases and host immune response. This research was aimed to explore how miR-125 regulates innate immunity in Scylla paramamosain when response to pathogen infection. AMO-miR-125 (anti-miRNA-125 oligonucleotide) was applied to specifically inhibit miR-125 in mud crabs. We found that miR-125 induced hemocyte proliferation, enhanced total hemocyte count in the crabs and protected crabs from challenged with white spot syndrome virus (WSSV) and Vibrio parahaemolyticus. Astakine regulates hemocyte proliferation, and the expression of Astakine increased after miR-125 knockdown, the results indicated that miR-125 may regulate hemocyte proliferation and total hemocyte count by regulating the expression of Astakine. Moreover, miR-125 knockdown also reduced phagocytosis and apoptosis, showing that miR-125 could affect the two processes in crab hemocytes. And the cumulative mortality of WSSV or V. parahaemolyticus infected crabs increased after AMO-miR-125 treatment, which indicated that miR-125 could reduce the death of crab caused by the pathogens and significantly affect the crab's immune response to pathogen. In summary, our data confirmed that miR-125 participates in hemocyte proliferation, the innate immunity, and the resistance to microbial disease in Scylla paramamosain. • miR-125 induced hemocyte proliferation, total hemocyte count, host phagocytosis. • miR-125 can regulate hemocyte proliferation and total hemocyte count by regulating the expression of Astakine. • miR-125 plays an important role in the crab's immune response to pathogen infection. [ABSTRACT FROM AUTHOR]

Accumulating evidence has indicated that Vibrio parahaemolyticus stains resistant to multiple drugs may pose a serious threat to public health and could lead to global economic. Phage therapy is an alternative and eco-friendly biocontrol strategy for the prevention and control of diseases in the aquatic systems. Therefore, this study was oriented to isolate and characterize the applicability of phages for V. parahaemolyticus (VP) lysis in aquaculture. Here, twenty-two lytic bacteriophages (BP1–22) were isolated from sewage collected in a local shrimp farming area and from the tissue of AHPND-infected shrimp and oysters collected in Taiwan. Among these, phage BP14 could effectively lyse V. parahaemolyticus , this bacterium causes acute hepatopancreatic necrosis disease (AHPND), resulting in severe mortality in Litopenaeus vannamei and Penaeus monodon , two marine shrimp species that are commonly grown in aquaculture farms. Characterization of the morphology of the virion particle via electron microscopy revealed that the virus belonged to the family Inoviridae , as suggested by its filamentous shape. The genome of phage BP14 was composed of 9071 bp with a 43.85% GC content and was inserted into a chromosome depending on the occurrence of Xer site-specific recombination. Furthermore, phage BP14 established a chronic productive infection, thus continuously releasing virions from infected cells. A viral load of approximately 12 plaque-forming units (PFUs) per cell was determined for phage BP14 under laboratory growth conditions. Western blotting and ELISA analyses revealed that phage BP14 decreased the levels of toxin-PirB and PirA protein by approximately 40%–60% compared to the wild-type VP. Collectively, our findings demonstrated the potential applicability of phage BP14 for the biological control of VP AHPND. • A total of 22 phages lysis the V. parahaemolyticus (VP) were collected in Taiwan. • Phage BP14 can infect a broad range of VPs including VP AHPND PD2 in Taiwan and 3HP in Thailand. • Phage BP14 belongs to the family Inoviridae. • Phage BP14 establishes a chronic productive infection, thus continuously releasing virions from infected cells. • Phage BP14 decreased the production of toxin-proteins, demonstrating its potential as a biological agent against VP AHPND. [ABSTRACT FROM AUTHOR]

Crassostrea hongkongensis is a main oyster species farmed in South China Sea offshore area, and commonly suffers from vibriosis. Vibrio parahaemolyticus is one of the principal pathogens that causes the oyster vibriosis. The integrin from C. hongkongensis was studied for its function against V. parahaemolyticus in the current work. The full-length cDNA sequence was 3274 bp with an open reading frame (ORF) of 2397 bp that encoded a polypeptide of 798 amino acids. The polypeptide included typical domains of β integrin (such as INB domain, three EGF-like domains, and highly conserved NPX[Y/F] motif), and phylogenetically clustered with β3 integrins of other invertebrates, thereby, designated as Ch Intβ3. The mRNA transcripts of ChIntβ3 were significantly higher in the heart, hemocytes, adductor muscle and hepatopancreas than those in gills and mantle of C. hongkongensis. Following stimulation with V. parahaemolyticus , the mRNA expression level of ChIntβ3 was significantly up-regulated in both hemocytes and hepatopancreas. The recombinant Ch Intβ3 had significantly agglutinating activity on Escherichia coli , V. parahaemolyticus , Bacillus amyloliquefaciens , Pichia pastoris and the red blood cells of rabbit. In the one hand, the survival rate (83.33 ± 5.77%) of the oyster after V. parahaemolyticus infection at 30 days was significantly higher in the Ch Intβ3 overexpression group than those in the physiological saline control group (26.67 ± 20.82%) (p < 0.01) and the overexpressed GFP group (26.67 ± 5.77%) (p < 0.01); in the other hand, the cumulative survival duration of C. hongkongensis was significantly shorter in the RNAi treatment group (197.6 ± 2.77 h) than that in the control group (247.2 ± 36.24 h) (p < 0.01). The results indicated that Ch Intβ3 was involved in the recognition and clearance of V. parahaemolyticus through cell adhesion, and contributed to the further understanding of the function of Ch Intβ3 against infection of pathogens. • The sequence of ChIntβ3 was firstly cloned from Crassostrea hongkongensis. • The mRNA level of ChIntβ3 showed positive response post Vibrio stimulation. • Ch Intβ3 agglutinated Gram-negative bacterium, Gram-positive bacterium, yeast and the red blood cells of rabbit. • Ch Intβ3 was involved in the recognition and clearance of V. parahaemolyticus through cell adhesion. [ABSTRACT FROM AUTHOR]

The proliferation of opportunistic pathogens Vibrio parahaemolyticus , Vibrio alginolyticus , and Vibrio owensii have impeded the rapid development of aquaculture farming. The study aimed to analyze the whole genome of pathogenic Vibrio strains associated with hepatopancreatic necrosis in Penaeus vannamei. The analysis included genomic characterization, virulence genes profiling, antibiotic resistance genes (ARGs) identification, antimicrobial resistance phenotypes, clustered regularly interspaced short palindromic repeats (CRISPR) and prophage prediction, multi-locus sequence typing, and pan-genome analysis of Vibrio isolates from Guangdong and Jiangsu provinces, China. Sequence analysis of the specimens revealed 29 V. parahaemolyticus isolates , three V. alginolyticus isolates, and one V. owensii isolate. Their genomes are 4.9–5.9 Mbp in size, with a GC content of 44.4–45.4%. Each Vibrio isolate harbored >120 virulence genes. Out of the 33 isolates, 13 (39%) demonstrated multidrug-resistant (MDR). Notably, at least 14 isolates exhibited heightened resistance to sulfamethoxazole, erythromycin, and amoxicillin, with a cumulative detection of 20 ARGs. Nevertheless, no significant correlation was found between the composition of ARGs and the observed antimicrobial resistance phenotypes. All Vibrio isolates differed in the presence of prophage and CRISPR sequences; and two of these prophages harbored virulence genes but not ARGs. The study identified 17 distinct sequence types (STs) in V. parahaemolyticus isolates, primarily ST415 (n = 5), while two Vibrio isolates were classified as ST3. Using the 117 V. parahaemolyticus strains available in the National Center for Biotechnology Information database as a reference, pan-genomic analysis and phylogenetic analysis of the core genome revealed that clinical isolates of the same STs clustered into the same clade, irrespective of isolation date and geographic origins. In summary, these data enhance our understanding of the molecular characteristics and epidemiological risk of Vibrio strains in southern China, especially regarding shrimp aquaculture. • Comprehensive whole genome analysis of 33 pathogenic Vibrio isolates. • Discrepancy observed between ARG composition and phenotypic drug resistance. • Prophage sequences were found in all Vibrio isolates, and two of these prophages harbored virulence genes. • Superior resolution of core genome analysis compared to MLST. [ABSTRACT FROM AUTHOR]

The judicious application of antibiotics in shrimp aquaculture is vital for addressing bacterial infections, yet monitoring bacterial resistance during distinct aquaculture phases remains deficient. This study focused on tracking antibiotic resistance dynamics in Vibrio strains sourced from both shrimp and their breeding settings. Over a standard aquaculture period (day 0, 20, 40, 60, and 80), we isolated eighty-eight Vibrio cholerae and ninety Vibrio parahaemolyticus strains from water, sediment, and shrimp samples. All isolates underwent assessment for antibiotic resistance phenotype and genotype. Both Vibrio species demonstrated resistance to nine antimicrobials. Notably, Vibrio cholerae isolates exhibited resistance rates of up to 46.6% for cephazolin, 30.7% for streptomycin, and 11.4% for ampicillin. Conversely, V. parahaemolyticus isolates displayed pronounced resistance to ampicillin (100%), cephazolin (62.2%), and cefoxitin (36.7%). Moreover, we identified 38 antibiotic resistance genes (ARGs) across all samples, with a higher prevalence observed in the water. Genetic diversity and clonal origins of the Vibrio isolates were quantified using Multilocus Sequence Typing (MLST). New Sequence Types (STs) were predominant among V. cholerae (95.4% of 43 isolates) and V. parahaemolyticus (92.9% of 28 isolates). Predominantly, V. parahaemolyticus isolates formed two extensive clonal groups: CC1420 and CC744. As the shrimp cultivation period progressed, both the resistance development rate among isolates and the average ARG count within the same STs of Vibrios steadily increased. This study underscores the potential role of ARGs transmission in the aquaculture environment as a crucial conduit for Vibrio antibiotic resistance development. Insights gained contribute to understanding the dissemination and transmission of antibiotic resistance in aquaculture, aiding in devising strategies to curb antibiotic resistance in aquatic environments. [Display omitted] • Vibrio spp. were isolated from both shrimp and their culture environment. • Vibrio isolates are highly resistant to antibiotics and carry multiple resistance genes. • Antibiotic resistance in Vibrio spp. increases with shrimp cultivation period. • ARGs in aquatic environment might promote antibiotic resistance of Vibrio spp. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is known to cause disease and infection in humans. This study was conducted to understand the prevalence of V. parahaemolyticus and the presence of virulence factors in environmental isolates of V. parahaemolyticus , collected from three major shrimp producing states viz , West Bengal, Andhra Pradesh and Gujarat of India during February 2014 to June 2017. Total 183 V. parahaemolyticus were isolated from moribund shrimp and identified using toxR and 16S rRNA gene. Polymerase Chain Reaction (PCR) was performed to detect virulence genes under T3SS1 and T3SS2 along with AHPND. All the isolates showed presence of vcrD1 , vp1680 and vopD1 gene under T3SS1 but negative for AHPND. Only 3% of isolates showed presence of all the virulence genes (vcrD2 , vopD2 , vopB2 and vopC) used in the present study under T3SS2. The isolates were also tested for the presence of tdh , trh or both the genes; 8% of the isolates had trh gene and 3% of the isolates had tdh gene. The genetic diversity within and between V. parahaemolyticus isolated from three different geographical locations, was analyzed by using 16S rRNA gene. Though the biotype diversity (h) was high for all three populations, phylogenetic study of V. phaemolyticus revealed an admixture of biotypes. Tajima's D (−2.59470; p <.001) test of selective neutrality and Fu's Fs (−32.422; p <.001) test were negative and significant, suggesting a sudden population expansion with limited time for population differentiation. • Vibrio parahaemolyticus were isolated from shellfish (L. vannamei) samples and identified based on molecular diagnostics. • Virulence genes vcrD1 , vp1680 , vopD1 under T3SS1 and vcrD2 , vopD2 , vopB2 , vopC under T3SS2 have been identified. • The phylogenetic and phylogeographic relationship among different isolates was revealed by using 16S rRNA. • The mortalities observed in shrimp in absence of pir vp AB genes may be due to presence of some unknown virulence gene(S) [ABSTRACT FROM AUTHOR]

Biofloc systems are microbial mature environments that are potentially less conducive disease outbreaks. We hypothesized that the way in which biofloc microbial communities are managed determines the level of disease protection. To investigate such hypothesis, Litopenaeus vannamei post-larvae were cultured for 21 days in biofloc environments created by different water management procedures. Five different types of bioflocs were created: autotrophic bioflocs without probiotics, autotrophic bioflocs with probiotics, heterotrophic bioflocs without probiotics, heterotrophic bioflocs with probiotics, and a flow-through system as a control. Heterotrophic bioflocs were obtained by daily addition of carbon (glucose) at an estimated C/N ratio of 18 throughout the experiment. For autotrophic bioflocs this input of carbon was applied only to start up the system and upon appearance of bioflocs (TSS > 100 mg L−1) and a drop in total ammonium nitrogen concentration below 0.05 mg L−1, carbon dosing was stopped. Bioflocs cultured with addition of probiotics received a 0.5 ppm dose every 48 hours. After 21-d culture period, a 96 h challenge test was performed with a Vibrio parahaemolyticus strain known to cause AHPND. For each biofloc type, this challenge was performed in three different approaches: 1- Shrimp were taken out of their biofloc tanks and challenged by applying new seawater; 2-Shrimp from biofloc tanks were challenged in their respective biofloc suspensions; and 3- Non-experimental shrimp, randomly selected from a recirculation (RAS) system were challenged in the types of biofloc suspensions. Mortality was high when shrimp were challenged in new seawater, independent of treatment. When challenged in their respective biofloc suspensions shrimp survival was the highest in heterotrophic bioflocs with and without probiotic supplementation and the autotrophic bioflocs with probiotics, whereas shrimp survival in autotrophic bioflocs without probiotics was 50%. These results were similar when non-experimental shrimp originating from a RAS system were challenged in these biofloc suspensions. Taken together, results suggest that bioflocs as such can decrease the impact of a Vibrio parahaemolyticus challenge and that this protection depends on the operational parameters of the biofloc system. Moreover, probiotics can be used to complement the protective effect of bioflocs. This information reinforces the importance of microbial community management as a tool to reduce the risk of disease and establish highly biosecure systems. • Two operational parameters of biofloc system were evaluated based on carbon input and probiotic application. • Litopenaeus vannamei post-larvae previously cultured in the two types of bioflocs were challenged with Vibrio parahaemolyticus. • Heterotrophic bioflocs showed high survival with and without probiotic supplementation. • The use of probiotics increased the survival in bioflocs operated in an autotrophic way. [ABSTRACT FROM AUTHOR]

Off-bottom cultivation of oysters, Crassostrea virginica , is increasing in the Gulf of Mexico. The warm ambient air and water temperatures found in the Gulf of Mexico, coupled with the target market for off-bottom cultivated oysters for live raw consumption, raise concerns about the potential infections by human health pathogens, Vibrio parahaemolyticus and V. vulnificus. Regular practices associated with off-bottom cultivation, such as desiccation, expose oysters to ambient air to eliminate bio-fouling and are also known to increase these Vibrio spp. levels in oysters. Along with cultivation methods being introduced in the Gulf of Mexico, the use of triploid oysters is becoming increasingly popular. Triploid oysters are used a majority of the time in off-bottom cultivation due to their sterility, which results in rapid growth and high summer meat quality. Research also suggests that the lack of gonad tissue may correlate with lower Vibrio spp. levels in oysters. In this study, triploid and diploid oysters were cultured in Australian long line systems and subjected to two typical desiccation practices, air dried and freshwater dipped/air dried, and then evaluated for V. parahaemolyticus and V. vulnificus abundances over time. Three two-week long studies determined that Vibrio spp. levels in oysters that underwent either desiccation treatment returned to levels similar to those of submersed oysters by day three, referred to as returning to background levels. However, the Vibrio spp. levels in the treated oysters remained not significantly different from the elevated levels seen immediately following the desiccation treatment until seven days after re-submersion. There was no significant difference in Vibrio spp. levels between triploid and diploid oysters, nor a difference in the time of re-submersion needed to return levels to background. These results suggest that oysters that have been desiccated should be re-submersed for at least seven days prior to harvest to mitigate any human health risk contributed by desiccation practices, regardless of oyster ploidy. • Suggests an absence of correlation between ploidy and Vibrio spp. levels in cultured oysters that undergo desiccation. • Suggests that ploidy has no effect on the time needed for Vibrio spp. levels return to background levels post-desiccation. • Identifies the length of time necessary to reduce the increased risk of Vibrio spp. infection from desiccated oysters. [ABSTRACT FROM AUTHOR]

Abstract Vibrio parahaemolyticus (V. parahaemolyticus) carrying the toxic plasmid of pirA and pirB has been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND), which has caused serious economic loss to the aquaculture industry of penaeid shrimp Litopenaeus vannamei (L. vannamei) in recent years. To effectively control the outbreak of this disease, breeding of Vibrio resistant broodstocks of L. vannamei was regarded as an important approach. Due to the advantages in selection accuracy and efficiency, genomic selection (GS) was expected to be a feasible alternative to accelerate the genetic improvement of disease resistance traits. In the present study, the heritability of shrimp resistance to V. parahaemolyticus was estimated and the feasibility of GS was evaluated in L. vannamei based on the real and simulation data. The heritability of shrimp resistance against V. parahaemolyticus was around 0.15–0.24, which indicated that the genetic improvement can be achieved by selective breeding. Subsequent analysis for GS based on real data showed that the genomic best linear unbiased prediction (GBLUP) can result in more accurate prediction than the traditional pedigree-based best linear unbiased prediction (PBLUP), with a 6.8% increase in the prediction accuracy for the survival time, and a 3.5% increase for binary survival. Similarly, for the simulated data, a relative increase (3.0% and 5.0%) in the prediction accuracy was obtained for survival time and binary survival when comparing the PBLUP to GBLUP. Overall results suggest that GS could be an alternative approach to improve the genetic gains in L. vannamei for the resistance to V. parahaemolyticus. Highlights • Near-to-moderate heritability has been detected for the resistance of Litopenaeus vannamei against Vibrio parahaemolyticus • Genomic selection can be a powerful tool to improve the resistance of L. vannamei against V. parahaemolyticus. • The GBLUP can be a promising model for the application of genomic selection in L. vannamei. [ABSTRACT FROM AUTHOR]

Abstract Shrimp cultivation has been faced with pathogenic bacterial infections, such as luminous vibriosis and acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio harveyi and Vibrio parahaemolyticus , respectively. Biodegradable compounds that act to prevent both serious shrimp diseases are to be explored for producing safe shrimp. The probiotic purple nonsulfur bacteria (PNSB) are safe for use in producing antivibrio compounds against shrimp pathogenic vibrios. The probiotic PNSB, Rhodobacter sphaeroides (SS15, TKW17) and Afifella marina STW181, released antivibrio compounds inhibiting various shrimp pathogenic Vibrio spp. in the optimum conditions that were similar to the conditions of shrimp cultivation. The antivibrio compounds by these PNSB were stable over the wide pH range from 3 to 10, and at elevated temperatures up to 121 °C for 20 min; they were also quite sensitive to lipase, pronase and α-chymotrypsin. All the antivibrio compounds had bactericidal activity as evidenced by damaged cells with many holes, and they also showed bacteriolytic activity with the highest found for the strain STW181. One of the purified antivibrio compounds produced by the strain SS15, a low molecular weight (<3000 Da) cationic compound containing NH 2 group(s), was the most effective in inhibiting AHPND-causing V. parahaemolyticus SR2. Regarding toxicity to brine shrimp, the extract from water layer in ethyl acetate extraction of strain SS15 (extract-SS15) containing antivibrio peptides had LC50 at 6.79 ± 0.63 mg mL−1. In challenge tests the extract-SS15 at 2.68 mg mL−1 adversely affected water quality; however, it could significantly decrease the mortality of white shrimp infected with the virulent AHPND strain SR2. All the tested probiotic PNSB produced antivibrio compounds that are biodegradable with great potential to control serious shrimp pathogenic vibrios for sustainable shrimp cultivation. Highlights • R. sphaeroides (SS15, TKW17) and A. marina STW181 released antivibrio compounds under the conditions of shrimp cultivation. • Antivibrio compounds of all tested probiotic strains were biodegradable but stable under wide ranges of pH and temperature. • The purified cationic compound with amino group(s) from strain SS15 strongly inhibited AHPND-causing V. parahaemolyticus SR2. • Effective dose of the extract-SS15 to control the virulent strain SR2 adversely affected water quality for rearing shrimp. [ABSTRACT FROM AUTHOR]

Abstract Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease caused by virulent strain of Vibrio parahaemolyticus (VP AHPND) that results in significant economic losses to shrimp aquaculture. The V. parahaemolyticus encodes deadly toxins (VP AHPND toxins) that are responsible for shrimp mortality during AHPND. Therefore, to better understand the toxicity mechanism of VP AHPND toxins, we have used the gnotobiotic Artemia franciscana model to determine the toxicity of recombinant PirAVP and PirBVP toxins in vivo. Subsequently, the study was validated by VP AHPND ECP30, ECP10 and ECP3 concentrated with 30, 10 and 3 kDa amicon filters to establish the toxicity towards brine shrimp larvae. It was found that recombinant PirBVP is more toxic to brine shrimp larvae as compared to PirAVP. Moreover, the survival of brine shrimp larvae challenged with a mixture of PirAVP and PirBVP toxins decreased ~2-fold as compared to PirBVP toxin and ~3-fold as compared to PirAVP toxin, as anticipated, as these 2 toxins seem to form an active complex. The study also confirms that VP AHPND ECP10 and ECP3, comprising PirAVP and PirBVP toxins, exhibits toxic effect to brine shrimp larvae. The ECP30, lacking PirAVP, showed reduced toxicity. The VP AHPND ECP10 and ECP3 (containing PirAVP and PirBVP toxins, along with other proteins) were more toxic than their respective equivalent amounts of pure toxins, suggesting that VP AHPND ECP10 and ECP3 contains additional, but uncharacterized, toxins. Hence, our study provides substantial evidence that toxicity of V. parahaemolyticus AHPND strains is mediated by VP AHPND ECP comprised of PirAVP and PirBVP (mostly) and other ECP or toxins produced by the bacterium. Highlights • PirBVP is more toxic to brine shrimp larvae as compared to PirAVP, moreover, the survival of larvae decreased when challenged with PirAVP and PirBVP toxins mixture, as anticipated these 2 toxins seem to form an active complex. • VP AHPND ECP10 and ECP3, comprising PirAVP and PirBVP toxins, exhibits toxic effect to brine shrimp larvae and ECP30, lacking PirAVP, showed reduced toxicity. • The VP AHPND ECP10 and ECP3 were more toxic than their respective equivalent amounts of pure toxins, suggesting that VP AHPND ECP10 and ECP3 contains additional, but uncharacterized toxins contribute in the toxicity. • Toxicity of V. parahaemolyticus AHPND strains is mediated by VP AHPND ECP comprised of PirAVP and PirBVP (mostly) and other ECP or toxins produced by the bacterium. [ABSTRACT FROM AUTHOR]

Abstract Vibrio parahaemolyticus is a common zoonotic bacterium that causes infections and disease in both sea cucumbers (Apostichopus japonicus) and humans, potentially greatly impacting both aquaculture and human health. In this study, two phages with the ability to control V. parahaemolyticus were isolated and designated as vB_VpaP_VP-ABTNL-1 (PVP1) and vB_VpaS_VP-ABTNL-2 (PVP2). Morphological analyses showed that PVP1 belonged to C3 phage group Podoviridae while PVP2 belonged to the family Siphoviridae. In vitro phage-treated cultures showed significant (P < 0.05) inhibition of growth for V. parahaemolyticus VP-ABTNL when compared to untreated groups. Subsequently, we determined that skim milk might be the best protective agent, as the dissolution time and moisture content were both significantly (P < 0.05) higher than other agents tested. We also explored the utility of using feedstuff mixed with freeze-dried phage powder as a form of protection for sea cucumbers against V. parahaemolyticus infection. The antibiotic group had a significant(P < 0.05)difference with MOI = 1, but not with the two phage treatments (MOI = 10, MOI = 100), indicating an increase of phage protection with MOI = 10 and MOI = 100 against V. parahaemolyticus VP-ABTNL. There were no significant differences in weight gain, wall-to-wall ratio, feeding rate or food conversion efficiency among sea cucumbers fed the two phage treatments compared with those fed the non-supplemented diet (P > 0.05). Hence, feeding phage cocktails might be another optimal therapeutic agents to treat V. parahaemolyticus infections in sea cucumber aquaculture. Highlights • Two bacteriophages lytic to Vibrio parahaemolyticus were isolated. • Skim milk was considered to be a better lyophilized powder protectant during phage feeding experiment. • This study monitored the efficiency of feeding phages against Vibrio parahaemolyticus infection. [ABSTRACT FROM AUTHOR]

Abstract Acute hepatopancreatic necrosis disease (AHPND) is a shrimp disease caused by a unique strain of Vibrio parahaemolyticus (Vp AHPND). This strain contains a highly pathogenic ~69 kbp (pVA1) plasmid encoding PirA vp and/or PirB vp deadly toxins. The pVA1-like plasmid is also reported to be found in Vibrio harveyi, Vibrio campbellii and Vibrio owensii. Therefore, prompt detection of the bacterial species carrying the PirA vp and/or PirB vp toxins producing genes is essential to control the spread of the disease. In this study, a new 5-plex PCR detection method was developed and optimised to detect Vp AHPND or tetracycline resistant Vp AHPND strain. This technique is designed to include five pairs of primers, which allow a comprehensive PCR diagnostic method for AHPND. The primers flanked five regions which are the Photorhabdus insect-related (Pir) toxin-like genes; pir A vp and pir B vp , thermolabile hemolysin (tlh), housekeeping (tox R) or tetracycline resistance (tet B) genes and also 16S ribosomal RNA (16s rRNA) gene as an internal amplification control (IAC). The detection limit of the 5-plex PCR method is at 10 pg/μl of extracted genomic DNA. Whereas, the specificity of the method analysed using nine Vp AHPND strains, three non-AHPND Vibrio species and ten other bacterial species revealed that the method does not give unspecific reactions with non-AHPND bacteria. The 5-plex PCR method evaluated using three locally isolated Vp AHPND strains and 41 isolates of Vibrio spp. obtained from field samples confirmed that the method developed allows rapid, sensitive and reliable detection of AHPND-causing bacterial strains. The Vp AHPND strains that were detected positive for the tet B gene also showed resistant to tetracycline by disk diffusion antibiotic susceptibility test. The presence of IAC and the primers targeting the tet B gene helps to indicate false-negative results and to detect tetracycline resistant AHPND strains respectively thus differentiating this method from previously reported AHPND PCR methods. Highlights • This manuscript presents a new 5-plex PCR method for Acute hepatopancreatic necrosis disease (AHPND). • The method enables the detection of Vp AHPND or tetracycline resistant Vp AHPND strain in a single PCR reaction. • This method can indicate other bacterial species that carry the pir A vp , pir B vp and tetracycline resistance genes. • The 5-plex PCR for AHPND is proven specific and sensitive to detect at 10 pg/µl of extracted genomic DNA. • The 5-plex PCR method also has been successfully evaluated on field samples. [ABSTRACT FROM AUTHOR]

Abstract Acute hepatopancreatic necrosis disease (AHPND) is an emerging disease in shrimp aquaculture which directly caused by binary photorhabdus insect-related (Pir) toxins PirABvp from Vibrio parahaemolyticus. To delineating the evolutionary origin and structure variation of PirAB, the protein sequence of PirABvp resembles from bacteria and insects were retrieved from the public database. The evolutionary relationship of PirABvp and resembles were analyzed phylogenetically. I-TASSER server was used to predict the protein-structure of PirA/B in investigated species. The cell-free supernatant obtained from the broth of V. parahaemolyticus and crude and heterologously expressed PirABvp proteins were respectively administrated to shrimp (Litopenaeus vannamei), crab (Eriocheir sinensis), European seabass (Dicentrarchus labrax), bloodworm (Nereis succinea) and mosquito (Aedes albopictus) to confirm the host range of toxin. Results showed that majority of PirAB located in a genomic island with low GC content. Phylogenetic analysis of PirB from bacteria and insects divided them into three lineages, namely Marine, Soli and Insect lineage respectively, with the majority belonging to Soli lineage. Overall, resembles of PirABvp were structurally similar to the PirABvp, but harbored three highly variable regions in receptor binding region and ligand binding sites, respectively. Shrimps bioassayed with cell-free supernatant of V. parahaemolyticus , crude and heterologously expressed PirABvp proteins resulted in 76.5%, 68.7% and 86.6% mortality, respectively. However, almost no mortality was found in crab, bloodworm, mosquito and European seabass. In summary, results indicated that PirBvp and its homologs have significant sequence divergence and remarkable variability in the receptor binding sites, which might explain why PirABvp are shrimp-specific toxins. Homology analysis of proteins revealed that there were three evolutionary directions for PirAB and the emergence of PirABvp was an ancient event that not associated with the aquaculture practices of shrimp. This study enhanced our understanding of the evolution of pirAB , which provided new insights into the origins of AHPND. However, why only shrimp is highly susceptible to PirABvp remains mysteries and need to solve in the future research. Highlights • The emergence of PirABvp was an ancient event that not associated with the aquaculture practices of shrimp. • PirB has species-specific receptor binding sites, which might affect its pathogenicity against the different host. • PirABvp has no adverse effects on other three animals and seems to be shrimp-specific toxins. [ABSTRACT FROM AUTHOR]

Crude ethanolic extract from Terminalia catappa L. leaves was investigated for its ability to interfere with biofilm formation of acute hepatopancreatic necrosis disease (AHPND)-causing and a reference Vibrio parahaemolyticus strains and modulate the immune response of Penaeus vannamei shrimp postlarvae. Results showed that biofilms formed by both AHPND-causing and reference strains of V. parahaemolyticus were rendered susceptible to dispersal upon treatment with the sublethal concentration of the extract. Furthermore, results of the qPCR analyses showed upregulation of six immune-related genes after 7 and 14 d of feeding. Pre-infection survival rates equivalent to the shrimp fed with un-supplemented feed suggest that T. catappa extract is safe for shrimp consumption. Upon infection with the AHPND strain, V. parahaemolyticus R1, the survival rate of the treatment group was 77% - significantly greater than those fed with un-supplemented feed. Similarly, those fed with T. catappa -supplemented feed and subsequently infected with V. parahaemolyticus ATCC17802 had a 93% survival rate, which is as high as the uninfected control group. These results ultimately indicate that feed supplementation with T. catappa increases the survival rate of the shrimp, possibly via attenuation of V. parahaemolyticus pathogenesis and stimulation of the shrimp's immune response against infections. • Terminalia catappa L. extract rendered the pre-formed biofilms of Vibrio parahaemolyticus more susceptible to dispersal. • Immune-related genes of Penaeus vannamei were differentially-expressed after being feed supplementation with T. catappa. • T. catappa supplementation does not negatively affect the growth indices and pre-infection survival rate of P. vannamei. • T. catappa extract increased the survival rate of P. vannamei postlarvae against the two V. parahaemolyticus strains. • T. catappa is suitable as shrimp feed supplement. [ABSTRACT FROM AUTHOR]

Host gut bacterial community has strong resistance to the invasion of pathogens. However, the specific gut taxa that could confer the resistance against pathogens in shrimp are largely unknown. Here, we demonstrated that the shrimp in the same tank showed different resistance after pathogenic Vibrio parahaemolyticus challenge. Subsequently, the gut bacterial communities of healthy, diseased, and moribund shrimp were analyzed. The results indicated that the gut bacterial community in the healthy shrimp still kept a high stability after challenge, while they were gradually disordered with the severity of disease in the diseased, and moribund shrimp. Certain specific taxa belonging to Rhodobacteraceae (e.g., Ruegeria , Paracoccus , Sulfitobacter , and Phaeobacter), Flavobacteriaceae (e.g., Tenacibaculum) and Demequinaceae (e.g., Demequina) were mainly depleted in the moribund shrimp, compared to that in healthy, and diseased shrimp. Seven corresponding strains of above specific taxa including five Rhodobacteraceae, one Tenacibaculum and one Demequina were isolated from the shrimp gut. Then, four different synthetic communities (SynComs) were constructed by using these isolates: SynComA, all five strains from Rhodobacteraceae; SynComB, SynComA + T. lutimaris HBG23; SynComC, SynComA + D. globuliformis TCG4; and SynComD, SynComA + T. lutimaris HBG23 + D. globuliformis TCG4. The addition of these SynComs could not only improve the fitness of shrimp, but also protect them against subsequent V. parahaemolyticus challenge, especially the addition of SynComD. Our study revealed that specific gut bacteria inhabited in healthy shrimp could improve shrimp resistance against pathogenic microbes. • Shrimp in the same box showed different resistance after Vibrio challenge. • Gut bacterial communities of healthy, diseased and moribund shrimp differed. • Specific taxa were almost depleted in moribund shrimp but not in diseased shrimp. • SynComs were constructed by using the corresponding isolates of above specific taxa. • Addition of SynComs protected the shrimp against Vibrio challenge. [ABSTRACT FROM AUTHOR]

One of the major constraints on aquaculture is the susceptibility of farmed fish to diseases or slow growing. Antimicrobial peptide I20, as a novel feed additive, is used to cope with these limiting factors. In the current study, the Pichia pastoris expression system was used to produce I20 in large quantities, thereby reducing its application cost. In terms of molecular strategy, the I20 expression codon was optimized according to the preference of P. pastoris codon. Through traditional strategy optimization (expression strain, time, methanol concentration, temperature, and pH), the expression level of I20 in shaking bottles was 21.09 ± 1.10 mg/L. Surprisingly, the expression level of I20 in a 20 L high-cell-density cultivation was 514.78 ± 22.07 mg/L after methanol induction of 72 h. Comprehensive analyses using a series of fluorescence and microscopy revealed that I20 killed bacteria by increasing bacterial membrane permeability and binding bacterial DNA. I20 effectively killed a variety of pathogenic bacteria (Aeromonas hydrophila , Aeromonas veronii , Elizabethkingia miricola , Flavobacterium columnare , Edwardsiella ictaluri , Vibrio parahaemolyticus , Streptococcus agalactiae , Nocardia seriolae , Escherichia coli , and Staphylococcus aureus). I20 was found to have excellent biocompatibility after both feeding and injection studies. Grass carp were fed I20 diet supplemented with 200 mg/Kg I20 for 8 weeks. I20 can effectively promote intestinal villus length, secretion of digestive enzymes, and expression of growth genes to improve growth performance. I20 significantly promotes immune response to reduce tissue damage and bacterial load, enhancing survival after Aeromonas hydrophila infection. In conclusion, we systematically optimized the expression of antimicrobial peptide I20 in P. pastoris , and achieved a satisfactory efficiency for producing antimicrobial peptide I20 by integrating several strategies. The results herein showed that I20-feed promoted growth performance and disease resistance in fish, suggesting a feed supplement with great potential applications for aquaculture. • A combinational strategy was developed for high production of I20 in P. pastoris. • The production of I20 in fermentation tank is about 25 folds of that in shake flask. • Fermentative peptide I20 has broad-spectrum and efficient antibacterial activity. • I20 can significantly promote growth and disease resistance of grass carp. [ABSTRACT FROM AUTHOR]

The frequency of gastroenteritis outbreaks caused by Vibrio parahaemolyticus has been increasing in many parts of the world including New Zealand. Some outbreaks in New Zealand have occurred at times when V. parahaemolyticus concentrations in shellfish in growing waters are low. V. parahaemolyticus is mesophilic, so is not expected to grow under refrigeration. Therefore, one possible explanation for the outbreaks is that the organism was able to grow to hazardous concentrations (e. g. >10,000/g) because of inadequate post-harvest refrigeration. To model the response of growth rates to temperature, we collected Pacific oysters (Crassostrea gigas) containing natural populations of V. parahaemolyticus over three consecutive summers , stored them for various times at 5, 10, 15, 20, 25 and 30 °C and determined V. parahaemolyticus concentrations. A simple log-linear fit was best to determine the growth rates at each temperature for each season. Applying a square root model (√growth rate (MPN/h) = b x (Temperature (°C) - T 0) to all the growth rates gave a good fit (R2 = 0.84, RMSE = 0.0178) with b = 0.0096 and T 0 = 8.44 °C. Fitted growth rates were considerably lower than previously reported, but the hypothetical minimal growth temperature was also lower. Thus, concentrations of V. parahaemolyticus are likely to increase more slowly in New Zealand oysters than was previously thought, but the organism may also be able to grow at lower temperatures. Both models suggest that the recommended minimum storage temperature of 7 °C in current New Zealand and US regulatory schemes is adequate. This information will be useful for evaluating food safety risks from storing Pacific oysters at different temperatures for different times. It will also assist in formulating time-temperature recommendations for safe handling of Pacific oysters. • We modelled temperature effects on Vibrio parahaemolyticus growth in Pacific oysters. • Square root modelling gave lower growth rates than previously reported. • Oyster farmers can now select safe post-harvest storage temperatures and times. • Public health risk management will be improved, reducing vibriosis illness rates. [ABSTRACT FROM AUTHOR]

Aspergillus niger is a unique fungus with benefits that can help improve the performance of terrestrial and aquatic animals. However, studies on the use of A. niger on aquatic animal species are limited. The present study investigated the effects of A. niger preparation as a dietary supplement on growth, gut microbiota, and immune response of white shrimp, Penaeus vannamei. The experiment was conducted over a 56-day trial period, with shrimps fed a control diet, and three diets containing three doses of A. niger preparation 0.5, 1 and 1.5 g (kg diet)−1) designated as P500, P1000 and P1500, respectively. The results showed that increasing concentrations of A. niger preparation led to higher weight gain and final weight in shrimp. Moreover, increasing levels of A. niger preparation of P1000 and P1500 reduced the mortality rate of shrimp challenged with Vibrio parahaemolyticus. In A. niger groups, PA, RBs, PO and LYS activities of shrimp significantly increased compared to the control group. Microbial analysis revealed significantly higher Vibrio -like counts in the gut of shrimp fed the control diet than in all A. niger- supplemented diets. Shewanella belonging to the dominant phylum Proteobacteria was highly expressed in the microbiota of shrimp fed P1000 diet. Meanwhile, Vibrio was relatively abundant in the control group. Unique genera, such as JGI0000069P2 and Marinicella, promoted bioremediation activities, while Halobacteriovorax aided in the elimination of V. parahaemolyticus infection in shrimp. Notably, a symbiotic relationship between Shewanella and Halobacteriovorax bacteria in the gut jointly boosted the immune responses of shrimp in defense against V. parahaemolyticus infection. These findings suggest that the A. niger preparation, due to its beneficial effects, could potentially be included in the diet to promote growth and a healthy intestinal microbiota, while also serving as a prophylactic treatment and immunological booster for shrimp. • This is the first study to report on the beneficial effects of Aspergillus niger -probiotic on the performance of white shrimp. • A. niger enhanced immunity and gut microbiota, which reduced the mortality of white shrimp after V. parahaemolyticus infection. • The altered microbiota in A. niger -fed shrimps consisted of ammonia and nitrite oxidizing bacteria [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of selenium nanoparticle (Nano-Se) supplementation on the growth performance, physiological parameters, antioxidant system, and immune response of seabream (Sparus aurata) under normal conditions, as well as after low salinity stress and challenge with Vibrio parahaemolyticus. The fish were divided into five experimental groups that received experimental diets supplemented with different levels of Nano-Se (0, 0.125, 0.25, 0.5, and 0.75 mg/kg Nano-Se) for 14 weeks. The results revealed that Nano-Se supplementation had a positive impact on growth performance, including final weight, weight gain, daily weight gain, specific growth rate, and feed conversion ratio. Nano-Se significantly increased the expression of insulin-like growth factors 1 and 2. The findings suggest that Nano-Se promotes growth in seabream under both normal and stressful conditions. Low salinity stress and Vibrio parahaemolyticus challenge were found to increase the levels of serum cortisol and the heterophil and lymphocyte (H/L) ratio while decreasing the population of lymphocytes, white blood cells, and immunoglobulin M. However, Nano-Se supplementation mitigated these effects. Nano-Se exhibited a protective effect by restoring the H/L ratio to normal levels and enhancing the immune response. Our expression analyses indicated a significant interaction between Nano-Se and stress. Nano-Se reduced the upregulated levels of heat shock protein 70 and interleukin 1β caused by low salinity and bacterial challenge, bringing them closer to normal values, compared to the groups without Nano-Se supplementation. Nano-Se slightly increased the mRNA levels of interleukin 1β under healthy conditions, particularly at concentrations of 0.5 and 0.75 mg/kg. Additionally, Nano-Se improved the serum biochemical profiles and carcass composition of seabream. Notably, the concentrations of 0.5 and 0.75 mg/kg increased the survivability of seabream exposed to low salinity stress and V. parahaemolyticus challenge. These findings suggest that Nano-Se can be effectively utilized to enhance the health and performance of seabream in aquafarms. • Nano-Se supplementation enhanced the growth, physiological parameters, and immune response of seabreams. • Nano-Se supplementation increased the survivability of seabream under both healthy and stressful conditions. • The most effective concentration of Nano-Se for seabream was found to be 0.5 mg/kg of diet. [ABSTRACT FROM AUTHOR]

Shrimp farming production is affected severely by the presence of toxigenic strains of Vibrio parahaemolyticus , the leading etiological agent of acute hepatopancreatic necrosis disease that causes as much as 100% mortality. To date, conventional methods have not effectively controlled AHPND, and only a few studies have reported bioassays testing phage therapy against this shrimp disease, none with a metagenomic approach. Here, Penaeus vannamei juveniles infected with AHPND-causing Vibrio parahaemolyticus were treated with phage to assess shrimp survival, tissue damage, as well as water microbiota dynamics. Phage therapy reduced by 4% shrimp mortality compared to the positive control. Histopathological analysis showed a 3 h delay in mortality of shrimp in the acute stage treated with phage therapy concerning the positive control, as well as a shortening of the terminal phase. Further, surviving shrimp in the terminal stage were only found in the positive control treatment. No significant differences were observed in the diversity of water microbiota between these treatments. Interestingly, V. parahaemolyticus caused up to 65% mortality, although its density decreased rapidly, and its relative abundance in the water microbiota ranged from 0.1% to 0.4%, while the marine bacteria that predominated in the microbiota was Donghicola eburneus (25.2%–54.6%). In addition, two copies of the pVA1 plasmid per V. parahaemolyticus genome were detected during the first 12 hpi in the positive control and phage therapy treatments. Phage therapy using a single phage could not effectively control AHPND in this investigation. Therefore, methods combining different approaches to prevent or treat the AHPND, such as a phage cocktail and a toxin binder, should be considered to increase the success rate in mitigating the disease. • Phage therapy decreased AHPND-causing Vibrio parahaemolyticus mortality by 4%, although it was not statistically significant. • Phage-treated shrimp displayed 3-h delay in acute phase mortality vs. positive control, along with shortened terminal phase. • Vp caused 65% mortality despite low relative abundance in tank water microbiota (0.1-0.4%) and no increase in pVA1 copies. • Donghicola eburneus dominated tank water microbiota (25%–54% relative abundance) across all treatments and sampling times. [ABSTRACT FROM AUTHOR]

Highly lethal Vibrio disease (HLVD) is a newly emerging disease caused by virulent strains of Vibrio parahaemolyticus (Vp HLVD), which leads to acute and mass mortality in shrimp post-larvae. Previous research suggests that the pathogenicity of Vp HLVD may be associated with an unknown toxin(s). To screen for the HLVD-related bacterial toxin(s), we compared the protein profiles of Vp HLVD and non-HLVD-causing Vibrio strains and identified two Vp HLVD -specific protein bands of ∼300 kDa and ∼ 200 kDa. They were co-purified from the bacterial lysate with a protein of ∼100 kDa by ion-exchange chromatography. Immersion challenge using this protein fraction produced the HLVD pathology in Penaeus vannamei. LC-MS/MS analysis indicated that the three major bands in this toxic fraction are Tc toxin subunits, TcA, TcB, and TcC. The genome of Vp HLVD strain vp-HL-202005 carries two sets of plasmid-encoded Tc toxins, which contain three distinct TcC subunits. The specific expression of these Tc toxin subunits in Vp HLVD strains was confirmed by RT-PCR and Western blotting. Removal of the toxin-encoding plasmids completely abolished the HLVD-causing ability of Vp HLVD. These data demonstrate that HLVD is caused by the Tc toxins produced by Vp HLVD. • Tc toxins are exclusively expressed in HLVD-causing Vibrio parahaemolyticus (Vp HLVD). • Purified Tc toxins cause HLVD-related syndrome in Penaeus vannamei. • Vp HLVD contains two sets of plasmid-encoded Tc toxin genes. • Elimination of Tc toxin-encoded plasmids abolishes the HLVD-causing ability of Vp HLVD. • The Tc toxins produced by vibrios are the primary causes of HLVD. [ABSTRACT FROM AUTHOR]

Toll-like receptor 5 (TLR5) is an important pattern recognition receptor (PRR) for bacterial flagellin. There are two kinds of subtypes in fish, the membrane-associated Toll-like receptor 5 (TLR5M) and the soluble form of TLR5 (TLR5S). Among them, TLR5M is a homolog of mammalian TLR5, and TLR5S is fish-specific. TLR5M in orange-spotted grouper (Epinephelus coioides) was involved in recognizing flagellin and activating the nuclear factor (NF)-κB signalling pathway while Ec TLR5S negatively regulated the NF-κB pathway. In mammals, the NF-κB pathway is critical to the expression of inflammatory cytokines and effectors. To better understand the activity of the TLR5M gene, we characterized the TLR5M 5′-flanking sequence region from E. coioides and assayed the TLR5M promoter luciferase reporter activity in HEK 293 T cells and grouper spleen cells (GS). The 5′-flanking region of TLR5M included three NF-κB binding sites. To elucidate the molecular basis of TLR5M gene expression, the activity of the TLR5M gene promoter was characterized and the deleted or site-directed mutants were generated to explore the functional significance of these kinds of binding sites. The luciferase activity of the TLR5M promoter in HEK 293 T cells was detected after flagellin was treated. The overexpression of NF-κB/p65 greatly increased the wild-type TLR5M promoter luciferase activity higher than the mutants. TLR5M promoter luciferase activity was increased more in the presence of p65 overexpression and Vibrio parahaemolyticus Flagellin C (vp FlaC) stimulation. These results suggest that NF-κB may be the important transcription factor and vp FlaC may act as a ligand, they could maximally increase the TLR5M promoter luciferase activity. • NF-κB would be the important transcription factor for activating the transcriptional activation of Ec TLR5M. • The luciferase activity of Ec TLR5M promoter could be induced by NF-κB/p65 through binding with the NF-κB cis-acting element. • vp FlaC may act as a ligand to enhance the luciferase activity of Ec TLR5M promoter. [ABSTRACT FROM AUTHOR]

Abstract Acute hepatopancreatic necrosis disease (AHPND) is an emerging disease that has caused mass mortality in shrimp in many countries in Asia and South America. A new strain of Vibrio parahaemolyticus (Vp AHPND), carrying one or more extrachromosomal ~70 kbp plasmids that encode homologues of the Photorhabdus insect-related (Pir) binary toxins (PirA vp and PirB vp) is the causative agent of AHPND. Bioassay challenges using immersion, reverse gavage and per os (feeding) treatments have been shown to induce the pathology characteristics of AHPND in experimentally infected shrimp. Moreover, a number of polymerase chain reaction (PCR) methods have been found to facilitate early detection of the disease. However, non- V. parahaemolyticus species close to Vibrio harveyi , Vibrio owensii and Vibrio campbellii have recently been identified as carrying pVA1-like plasmids, signifying that these plasmids are capable of being transmitted to different Vibrio species. AHPND is thus a threat and a serious concern for the aquaculture industry. Against this background, this paper presents an overview of experimental methodologies and diagnostic procedures for AHPND. It also includes fundamental findings related to the disease as guidelines which may be useful for further experimental work on AHPND and other similar shrimp diseases. Highlights • Experimental methodologies and diagnostic procedures for acute hepatopancreatic necrosis disease (AHPND) are presented. • Bioassay models for pathogenicity study of AHPND using immersion, reverse gavage, per os and cohabitation. • Ethical principles and biorisk management procedures to conduct research using live shrimp and pathogenic microorganism. [ABSTRACT FROM AUTHOR]

Abstract Isolates of Vibrio parahaemolyticus (Vp AHPND) that carry a plasmid encoding two Pir -like toxins cause acute hepatopancreatic necrosis disease (AHPND), a disease that has caused devastating economic losses to the shrimp industry, particularly in Asia. However, lower prevalence of AHPND infection has been associated with farms that operate with biofloc or lower salinity culture water. Therefore, the aim of this present study was to investigate the effects of biofloc and Nile tilapia (Oreochromis niloticus)-conditioned water prepared at different culture water salinities on survival of whiteleg shrimp (Penaeus vannamei) bath-challenged experimentally with Vp AHPND. First, groups of shrimp were bath-challenged with Vp AHPND in clear 15 ppt seawater (CW) or in the presence of a pre-cultured biofloc at 25%, 50% and 100% (v /v). Survival during 96 h post-challenge was significantly greater in groups cultured in 50% and 100% biofloc (p < 0.05). In a second trial, the effect of shrimp stocking density on biofloc protection against bath challenge with Vp AHPND was determined and shrimp challenged in 100% biofloc again had significantly greater survival (p < 0.05) compared to the CW group, whilst under our experimental conditions stocking density had no significant influence on survival post-challenge. In a third trial, shrimp were challenged with Vp AHPND in three different salinities of CW or Nile tilapia-conditioned (NTC) water (5 ppt, 10 ppt and 15 ppt). Survival in this final trial was 33% at 96 h in 5 ppt CW compared to just 7% in the 10 ppt and the 15 ppt CW groups, though these differences were not statistically significant. Moreover, shrimp survival in the 5 ppt and 10 ppt NTC water groups was significantly greater than in the 15 ppt NTC water group (p < 0.05), while significantly greater survival was observed in 10 ppt NTC water compared to 10 ppt CW (p < 0.05). The results indicate that biofloc and NTC water may provide some protection against AHPND, whilst low salinity culture water may also offer a degree of protection against this bacterium. These findings may allow for the implementation of inexpensive strategies in the shrimp industry to assist in minimising the impact of Vp AHPND as part of pond management practices. Highlights • Shrimp bath challenged with V. parahaemolyticus (Vp AHPND) had significantly greater survival in biofloc than clear seawater. • Stocking density did not influence survival when bath challenged with Vp AHPND in either biofloc or clear seawater conditions. • Survival was sig. greater in Nile tilapia-conditioned water prepared at 5 ppt and 10 ppt than in the other water conditions. • Biofloc and Nile tilapia-conditioned water, as inexpensive disease management control strategies, may protect against AHPND. [ABSTRACT FROM AUTHOR]

The present study was carried out to investigate the effects of dietary fermented vegetable product (FVP) on the innate immune response, immune-related genes expression and resistance of kuruma shrimp Marsupenaeus japonicus against Vibrio parahaemolyticus . Four hundred and eighty healthy kuruma shrimp (15.3 ± 1.15 g) were distributed into four treatment groups and fed with diets containing 0 (control), 0.55, 5.5, and 55 g FVP (kg diet) −1 for 11 days. The results revealed that administration of FVP significantly elevated the innate immune parameters of kuruma shrimp including total hemocyte count (THC), phagocytosis, phenoloxidase activity, reactive oxygen species (ROS) production and bactericidal activity (P < 0.05). Furthermore, dietary supplementation of 5.5, and 55 g FVP (kg diet) −1 remarkably up-regulated the expression of antimicrobial peptides (AMPs) and toll-like receptors (TLR) in various tissues, particularly in the intestine. Shrimp groups supplemented with FVP diets showed significantly lower cumulative mortality percentage after being challenged with Vibrio parahaemolyticus (P < 0.05). The results suggest that FVP can be recommended as a feed supplement to elevate innate immune response, upregulate immune related genes expression and increase disease resistance in kuruma shrimp. [ABSTRACT FROM AUTHOR]

Green synthesis of silver nanoparticles (AgNPs) was undertaken using seagrass Cymodocea serrulata, and their characteristics were confirmed by indispensible instrumental analysis. UV spectroscopy analysis (UV) showed a strong peak in the wavelength of 431 nm, resembling the surface resonance peak (SPR) specific for AgNPs. Zeta potential analysis confirmed the stability (−28.4 ± 0.35 mV) of AgNPs. Transmission electron microscopy (TEM) result shows that the particles were in the size range of 40.49–66.44 nm. X-ray diffraction analysis (XRD) confirms the crystalline nature of AgNPs. Fourier transform infra-red spectroscopy (FTIR) analysis indicated the possible presence of polyphenol and protein encapsulates on AgNPs. Antibacterial efficacy of the AgNPs was tested against Vibrio parahaemolyticus induced vibriosis in the juvenile shrimps Penaeus monodon. Shrimps were fed on normal formulated feeds supplemented either with an aqueous extract of seagrass or AgNPs. Half of the experimental groups were infected with Vibrio parahaemolyticus (MTCC 451) (100 μl-LD 50 dose) after 14 days of scheduled feeding. After two weeks of experiment about 65% mortality could be observed in Vibrio challenged control prawns. 25.1% mortality was observed in prawns supplemented with seaweed aqueous extract, whereas the mortality was 15.2% in seaweed SNP supplemented group. Hepatopancreas samples of all the groups were subjected to biochemical and histological studies at the culmination of the feeding experiment (28 days). In vivo analysis of biochemical parameters (GOT, GPT, LDH, ALP and ACP) and the histological architecture provided evidence for the antibacterial effects of AgNPs. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus and Vibrio harveyi are commonly associated with brine shrimp, where they proliferate at the time of hatching and colonize nauplius surfaces, becoming a potential risk to organisms feeding on them. Among the strategies to prevent their occurrence, the use of chemotherapeutic agents and disinfectants could be considered; however, these strategies have side effects on the quality of brine shrimp and the environment. As a consequence, the use of bacteriophages (phages) is gaining interest in aquaculture. In this study, we investigated the effect of phage therapy (single phages and cocktails) on hatching and survival of brine shrimp ( Artemia franciscana ) cysts and nauplii exposed to pathogenic strains of V. parahaemolyticus and V. harveyi. The results revealed that application of single phages enhanced significantly ( p  < 0.05) the hatching success and survival rate, even at low multiplicity of infection (MOI). Subsequently, these promising phages were mixed (cocktails) and administered to brine shrimp cysts and nauplii exposed to both pathogenic Vibrio species. The results also revealed an enhanced hatching success (100% in both cases) and survival rate (from 85 to 89%) in groups treated with phage cocktails, whereas control groups had a hatching success of around 50% and survival rate from 40 to 50%. Given this, phage cocktails offer an alternative to chemotherapeutic agents routinely used in brine shrimp production. [ABSTRACT FROM AUTHOR]

The study was aimed to assess the growth and immunomodulatory potential of Syzygium cumini leaf powder against virulent strain of Vibrio parahaemolyticus on the juveniles of Litopenaeus vannamei . Two diets such as control diet (without immunostimulant) and immunostimulant diet (containing 1% S. cumini leaf powder) were prepared. The juveniles were distributed in four treatments namely; daily feeding of control diet (CD), daily feeding of immunostimulant diet (ID), alternate day feeding of control and immunostimulant diet (CD/ID) and two days control diet and one day immunostimulant diet (2CD/ID) feeding. After feeding trial, shrimps from each treatment group were challenged with 0.1 mL of virulent strain of V. parahaemolyticus suspension at a concentration of 10 7  CFU mL −1 . The post-challenge sampling was performed after 5 days of injection. The haemolymph prophenol oxidase, superoxide dismutase, catalase and respiratory burst activity of different treatments were significantly different (P < 0.05) in both pre- and post-challenge conditions. The haemolymph total protein, globulin, lysozyme and phagocytic activities of L. vannamei fed with Syzygium cumini leaf powder improved than the control group in pre- as well as post-challenge phase. The survival rate of shrimps fed with immunostimulant diet was significantly (P < 0.05) higher as compared to control group. Therefore, Syzygium cumini leaf powder added diet protects the host effectively against virulent strains of V. parahaemolyticus through both continuous and alternate day feeding. [ABSTRACT FROM AUTHOR]

In order to reduce disease outbreaks in shrimp aquaculture, biosafe farming practices have been created, such as the biofloc system and the use of feed additives to improve shrimp immune response and growth performance. Many studies with feed additives have tested probiotics in biofloc rearing systems; yet to date, no studies have reported on the use of prebiotics in the feed of farmed shrimp in biofloc systems. Moreover, the effects of mannoprotein (MP) prebiotics on shrimp and shrimp farming are also unknown. Therefore, this study aimed to evaluate the effects of dietary supplementation with mannoproteins (MP) on the performance, immune response, and midgut intestinal villi of L. vannamei raised in a biofloc system. Three diets with different MP concentrations were formulated (0.02%; 0.08%; 0.12%) and one control group without MP additive. The experiment utilized 12 experimental units (800 L) stocked with 400 shrimp/m 3 , with 3.64 ± 0.07 g of initial weight, in triplicate. Shrimp were fed four times per day, and both histology of the midgut and growth indices were assessed after 65 days. Intestinal villi presented a larger internal surface area in animals that were fed dietary supplements of 0.08% and 0.12% MP (p < .05). Shrimp fed with MP showed an approximate 10% increase in survival compared to animals in the control treatment (p < .05). No statistical differences were found in the growth parameters (final weight, weekly weight gain, final biomass, feed efficiency) or survival after challenge with Vibrio parahaemolyticus . Prior to infection, no differences were found in any of the immunological parameters analyzed. However, after infection, the production of superoxide anion after feeding with 0.12% MP showed higher ROI values. Therefore, dietary supplementation with different concentrations of mannoproteins resulted in an increase in survival and average surface area of shrimp midgut, as well as higher superoxide anion production in shrimp fed with 0.12% MP after infection with V. parahaemolyticus . [ABSTRACT FROM AUTHOR]