Acute Hepatopancreatic Necrosis Disease Infection Research Articles

Species Richness and Diversity of Gut Microbiota may Reduce AHPND in the Whiteleg Shrimp, Litopenaeus vannamei

Acute hepatopancreatic necrosis disease (AHPND) causes massive economic loss in shrimp culture sector. Eco-friendly feed additives, which can reduce antibiotic resistance in bacteria and antibiotic residues present in aquaculture products, have become popular in shrimp farming. These additives have proven to maintain a balance host gut microbiota in preventing pathogen infection. In the present study, gut microbiota of Litopenaeus vannamei, which had been administrated with different diets (probiotic: Bacillus B2; prebiotic: Gracilaria changii, G; and a combination of Bacillus B2 and G. changii, B2+G) and post-challenged with AHPND-causing Vibrio parahaemolyticus strain 3HP, was determined using the PCR-DGGE method. The results showed that the gut microbiota of L. vannamei was dominated by the phylum Proteobacteria. The abundance of Proteobacteria was higher in the B2+G and G treatment groups. High abundance of native gut microbiota, such as Proteobacteria might prevent the adhesion of virulent bacteria by competitive exclusion, and thus, reducing the chance of AHPND infection. Moreover, the shrimp from the B2+G treatment group with higher survival in the challenge tests possessed a higher diversity and species richness of gut microbiota. This suggests that higher diversity and species richness of beneficial gut microbiota probably interfering bacterial communication among the pathogens by either destructing or neutralising their quorum sensing signals. Consequently, this will prevent the colonisation of pathogens in the gut and resulted in a higher survival of the shrimp challenged with V. parahaemolyticus.

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

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

Insights into the fatty acid binding protein of Pacific white shrimp (Litopenaeus vannamei) suffering acute hepatopancreatic necrosis disease infection

Acute hepatopancreatic necrosis disease (AHPND) has a significant economic impact on aquaculture worldwide. However, the molecular mechanism responsible for AHPND remains unknown. Our previous study demonstrated that the Litopenaeus vannamei fatty acid binding protein (LvFABP) interacted with the Vibrio parahaemolyticus toxin PirB, and that silencing LvFABP using RNA interference improved AHPND resistance in shrimp. Polyclonal antibodies against recombinant LvFABP and PirBvp toxin were produced to further elucidate the role of LvFABP in AHPND resistance. Immunofluorescence localization showed that LvFABP was abundant throughout the cytoplasm of the hepatopancreas cells, and a co-localization assay revealed that PirBvp directly interacted with LvFABP. Antibodies against LvFABP were able to partially block the binding of LvFABP to PirBvpin vitro and inhibited AHPND infection in vivo. Exogenous recombinant LvFABP had a partially neutralizing effect against AHPND and gave L. vannamei significant protection against infection. In addition, recombinant LvFABP treatment of L. vannamei significantly reduced the morphological damage to the shrimp following pathogen challenge. The transcriptomes of the L. vannamei hepatopancreas with and without V. parahaemolyticus infection after silencing of LvFABP by RNA interference were also explored. The results showed that AHPND infection could affect the health of L. vannamei via functional changes in carbohydrate metabolism, lipid metabolism, and transport and catabolism. Furthermore, silencing LvFABP could effectively protect metabolism, transport and catabolic homeostasis, thus enabling adaptation to further or prolonged AHPND infections. These findings provided a unique and important dataset that deepened our understanding of the pathological mechanism of AHPND and may help in the development of strategies to prevent and ameliorate the effects of bacterial diseases.

Dietary montmorillonite clay improved Penaeus vannamei survival from acute hepatopancreatic necrosis disease and modulated stomach microbiota

Acute hepatopancreatic necrosis disease (AHPND) is causing a bacterial pandemic in shrimp farming. AHPND is caused by pathogenic strains of Vibrio parahaemolyticus (VPAHPND) that produces the bacterial toxins, PirA and PirB. Shrimp exposure to AHPND-causing bacteria is hypothesized to be through the oral route, ingested into the gut where the bacteria initially colonized the stomach and then releases the binary toxins to damage the hepatopancreas. In view of changing global regulatory controls on the use of antibiotics, there is much interest in the development of functional aquafeeds. One innovative method to mitigate AHPND is to bind the bacterial toxins by adding adsorbent clay minerals into shrimp feeds. A feeding trial using triplicate groups of Penaeus vannamei post-larvae (PL30) was conducted to evaluate the efficacy of a montmorillonite (MMT) clay [Calibrin®-Z (CL)] on growth, gut health and disease resistance to AHPND. The addition of 0.25% or 0.50% CL did not significantly (P > 0.05) impact growth and feed utilization efficiency compared to control groups fed diets with 0% CL. When challenged with VPAHPND, survival of shrimp fed 0.25% or 0.50% CL were 83.3 ± 5.5% and 93.8 ± 0.1%, respectively, and were significantly higher (P < 0.05) compared to the VPAHPND-challenged positive control group (39.6 ± 10.4%) but not significantly different compared to the unchallenged negative control group (95.8 ± 2.1%). Vibrio and total cultivable bacteria counts in the hepatopancreas of shrimp fed CL-added diets were significantly lower compared to positive control. Hepatopancreas histopathology of infected shrimp fed CL-added diets showed less damage. During AHPND infection, bacterial diversity was depressed but dietary CL tended to restore stomach bacterial richness and α-diversity index. Dietary CL modulated the stomach bacterial community possibly with beneficial impact to shrimp survival. At the phylum level, Proteobacteria and Bacteroidetes were the main groups but the relative abundance of Verrucomicrobia, Acidobacteria and Firmicutes was more prevalent in the microbiota of shrimp fed CL-added diets. Bacteria of the genus Pseudoalteromonas, Tenacibaculum, and Marinimicrobium were identified as members of the healthy microbiota and Lysobacter was observed to be relatively enriched in AHPND-infected shrimp. Demequina was identified as a potential biomarker and was significantly enriched in the stomach of the CL-added groups. This is the first report on the effectiveness of dietary clay mineral in AHPND mitigation. At least 0.25% CL is suggested to be included in commercial shrimp feeds to mitigate against production and economic losses from AHPND outbreaks in shrimp farms.

Antibacterial activity and immunomodulatory role of a proline-rich antimicrobial peptide SpPR-AMP1 against Vibrio campbellii infection in shrimp Litopenaeus vannamei

Antimicrobial peptides (AMPs) constitute one of the most promising sources of natural molecules used for the design of effective antimicrobial agents alternative to antibiotics. Previously, we have showed that a crab proline-rich AMP designated as SpPR-AMP1 is a potent AMP that exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Here, we demonstrated the importance of SpPR-AMP1 peptide in treating a virulent acute hepatopancreatic necrosis disease (AHPND) Vibrio campbellii VH-639 isolate and eliciting the innate immune response to counter the AHPND infection in shrimp Litopenaeus vannamei. SpPR-AMP1 exhibited a strong antimicrobial activity against V. campbellii VH-639 at MIC value of 0.195–0.39 μM. Scanning electron microscopy (SEM) revealed the membrane disruption potential of SpPR-AMP1 against the V. campbellii VH-639 cells. The in vivo effect of SpPR-AMP1 in shrimp L.vannamei was investigated and the results showed that SpPR-AMP1 was capable of modulating the innate immune response by stimulating the expression levels of AMP transcripts in shrimp hemocytes. Moreover, treatments with SpPR-AMP1 could promote the resistance of shrimp against V. campbellii VH-639 infection as demonstrated by a significant increase in shrimp survival rate and decrease in both the bacterial load and the expression levels of bacterial PirA and PirB toxin gene transcripts in the infected shrimp. These results suggest the potential of SpPR-AMP1 peptide with the combined antimicrobial and immunoenhancing capabilities as promising antimicrobial agent to treat V. campbellii VH-639 causing AHPND infection in shrimp aquaculture.

Litopenaeus vannamei peritrophin interacts with WSSV and AHPND-causing V. parahaemolyticus to regulate disease pathogenesis

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

Supplementation of ex situ produced bioflocs improves immune response against AHPND in Pacific whiteleg shrimp (Litopenaeus vannamei) postlarvae.

The emergence of Vibrio diseases, including acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio spp., had resulted in heavy losses in global shrimp production. Biofloc technology is a closed aquaculture system developed as one of the sustainable solutions to increase system resilience in the shrimp industry. In this study, biofloc was formed externally (ex situ biofloc) with probiotics Bacillus sp. strain BME and Bacillus sp. strain BCE, diatom microalgae Chaetoceros calcitrans, and a consortium of nitrifying bacteria, in the ratio of 1:1:6:6 as a starter. The study showed that the ex situ biofloc supplementation in Pacific whiteleg shrimp (L. vannamei) postlarvae culture can increase the shrimp culture performance (shrimp survival and growth), reduce Vibrio counts in the water and shrimp body, and provide stimulation of the shrimp immune response through humoral immune responses, such as pattern recognition protein (C-type lectin) and melanization process (proPO). Overall, the results indicate that the supplementation of ex situ biofloc provided protection to shrimp under Vibrio infection, regardless of the timing of addition (before, simultaneously, or after addition of Vibrio sp. strain VPA). This suggests that the ex situ biofloc can be effective as a preventive and a supportive treatment against potential AHPND infection in L. vannamei postlarvae culture. Taken together, the ability of the ex situ biofloc to modulate immune-related gene expression and resistance of L. vannamei against potentially AHPND-causing Vibrio sp. strain makes it an effective aquaculture technology for infectious disease control in shrimp production with high-density and minimal water exchange culture. KEY POINTS: • Supplementation of ex situ produced biofloc in shrimp postlarvae culture. • Ex situ biofloc reduces Vibrio counts in the water and shrimp body. • Ex situ biofloc stimulates shrimp humoral immune responses and survival.

Biochemical indexes and gut microbiota testing as diagnostic methods for Penaeus monodon health and physiological changes during AHPND infection with food safety concerns

Severe shrimp disease outbreaks have a destructive impact on shrimp aquaculture and its associated downstream food processing industries. Thus, it is essential to develop proper methods for shrimp disease control, which emphasizes the importance of food safety. In this study, we performed biochemical tests and gut microbiome analysis using uninfected control and Vp AHPND‐infected Penaeus monodon samples. Biochemical tests were performed to assess the phenoloxidase (PO) activity, respiratory Burst (RB) activity, nitrite concentration, superoxide dismutase (SOD) activity, total hemocyte count (THC), and total protein concentrations. Overall, upregulations were detected in these biochemical tests, which showed the activation of the immune response in P. monodon during acute hepatopancreatic necrosis disease (AHPND) infection, especially at 6 hpi and 12 hpi. Besides that, shrimp gut samples were collected and pooled (n = 3), followed by DNA extraction, PCR amplification targeting the V3/V4 16S ribosomal RNA (rRNA) region, next‐generation sequencing (NGS), and bioinformatics analysis. Proteobacteria was the most abundant phylum in both samples. The Rhodobacteraceae family and Maritimibacter genus were proposed to be vital forshrimp health maintenance. Vp AHPND bacterial colonization and secondary Vibrio infections were postulated to have occurred based on the higher abundances of Vibrionaceae family and Vibrio genus in the Vp AHPND‐infected sample. Firmicutes phylum together with Photobacterium and Aliiroseovarius genera were inferred to be pathogenic or related factors of AHPND infections. In conclusion, physiology (immune response activation) and gut microbiome changes of disease tolerant P. monodon during AHPND infection were identified. Both biochemical tests and 16S rRNA analysis are proposed as a combined strategy for shrimp health diagnosis for ensuring shrimp health maintenance, disease control, and food safety.

Metabolic Alterations in Shrimp Stomach During Acute Hepatopancreatic Necrosis Disease and Effects of Taurocholate on Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), a recently emerged bacterial shrimp disease, has increased shrimp mortality and caused huge economic losses in many Asian countries. However, molecular factors underlying pathogenesis of this disease remain largely unknown. Our objective was to characterize metabolic alterations in shrimp stomach during AHPND and determine effects of taurocholate on AHPND-causing Vibrio parahaemolyticus. Based on metabolomics, pathways for lipid metabolism and for primary bile acid (BA) synthesis were majorly affected following AHPND infection. Bile acid metabolites, namely taurocholate, were downregulated in the metabolomics database. This prompted us to study effects of taurocholate on biofilm formation, PirABvp toxin release and biofilm detachment capabilities in AHPND-causing V. parahaemolyticus. Treatment of this bacterium with high concentration of taurocholate, a primary bile acid, induced biofilm formation, PirABvp toxin release and facilitated the dispersion of bacterial cells. Taken together, our findings suggest that AHPND infection can affect the lipid metabolites in shrimp stomach, and further suggest that the primary bile acid taurocholate is important for the virulence of AHPND-causing V. parahaemolyticus.

Dysbiosis of the shrimp ( Penaeus monodon ) gut microbiome with AHPND outbreaks revealed by 16S rRNA metagenomics analysis

Acute hepatopancreatic necrosis disease (AHPND) or early mortality syndrome (EMS) is a severe hepatopancreatic disease of shrimp, caused mainly by Vibrio parahaemolyticus. Growing evidence has shown that gut bacterial imbalance is linked to bacterial pathogenesis. AHPND disease also imposes selective pressure on the gut microbiota, which was revealed by metagenomic analysis of 16S rRNA gene. Firstly, 46 bacterial strains and 8 V. parahaemolyticus were isolated from the intestine of AHPND-infected Penaeus monodon. Secondly, PCR detection of 4 major virulence genes was carried out in 8 V. parahaemolyticus. The results showed that pirAVp could be detected in 5 strains. Thirdly, 16S rRNA gene was used to sequence the intestinal microbiota of asymptomatic and AHPND-infected shrimp. There were significant differences in gut bacteria structure between the two groups, because pirAVp toxin alone is sufficient to change the core microbiota of infected shrimp. The microbial communities of infected shrimp were enriched with Firmicutes and Bacteroidetes, but the number of Proteobacteria was reduced compared with the healthy shrimps. However, in healthy shrimp, the core microbial abundance was opposite to that of infected shrimp. AHPND infection was positively correlated with the levels of SCFAs (butyrate and propionate) producing species such as Bacteroides, Bifidobacterium, Lactobacillus and Staphylococcus. Significant differences were found in the functional gene profile category between healthy and infected shrimp gut. These findings improve the current understanding of AHPND disease's impact on the assembly of shrimp gut microbiota.

QseC Inhibition as a Novel Antivirulence Strategy for the Prevention of Acute Hepatopancreatic Necrosis Disease (AHPND)-Causing Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus resulted in great economic losses in global shrimp aquaculture. There is an urgent need for development of novel strategies to combat AHPND-causing V. parahaemolyticus (Vp AHPND), given that one of the greatest challenges currently is the widespread use of antibiotics and subsequent emergence of multidrug-resistant bacteria. Here, we proposed a broad-spectrum antivirulence approach targeting a conserved histidine kinase, QseC, which has been demonstrated to activate virulence expression in several Gram-negative pathogens. Our results showed that QseC mediated the catecholamine stimulated effects on growth and flagellar motility of Vp AHPND. Transcriptome analysis revealed that QseC was involved in the global regulation of the virulence of Vp AHPND as the ΔqseC mutant exhibited a decreased expression of genes related to type IV pilin, flagellar motility, and biofilm formation, while an overexpression of type VI secretion system and cell wall biosynthesis. Subsequently, the bacterial catecholamine receptor antagonist LED209 not only neutralized the stimulatory effects of host catecholamines on the growth and motility of Vp AHPND in vitro, but also attenuated the virulence of Vp AHPND towards brine shrimp larvae and white shrimp in vivo. Additionally, LED209 presented no interference with pathogen growth, nor the toxicity to the experimental animals. These results suggest that QseC can be an attractive antivirulence therapy target, and LED209 is a promising candidate for development of broad-spectrum antivirulence agents. This is the first study that demonstrated the role of QseC in the global regulation of Vp AHPND infection and demonstrated the antivirulence potential of LED209, which provides insight into the use of an antivirulence approach for targeting not only Vp AHPND, but also a much larger collection of pathogenic bacteria.

In Litopenaeus vannamei, the cuticular chitin-binding proteins LvDD9A and LvDD9B retard AHPND pathogenesis but facilitate WSSV infection

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease caused by V. parahaemolyticus strains which contain a virulent plasmid that encodes a binary pore-forming Pir toxin. Typically, these AHPND-causing bacteria first colonize in the shrimp stomach and then later cross to the hepatopancreas. To do this, they must pass through structural barriers which include the pliant cuticular lining of the stomach lumen. A previous transcriptomic study of shrimp challenged with the virulent 5HP strain of V. parahaemolyticus found significant upregulation of a contig associated with the cuticular proteins LvDD9A and LvDD9B. Here, we confirmed that the mRNA levels of these two genes were significantly upregulated not only in 5HP-infected shrimp, but also in the stomach of shrimp challenged with the white spot syndrome virus (WSSV). Using dsRNA-mediated gene silencing, we found that AHPND-causing bacteria migrated to the hepatopancreas within 3 h of AHPND infection in LvDD9A/B-silenced shrimp. Shrimp shell hardness of LvDD9A/B-silenced shrimp was also significantly decreased. Conversely, we found that silencing of LvDD9A/B significantly inhibited both WSSV gene expression and genome replication. Taken together, our data suggests that LvDD9A and LvDD9B are involved in both AHPND and WSSV infection. However, in AHPND, these cuticular proteins help to prevent bacterial migration from the stomach to the hepatopancreas, whereas in WSSV infection, they facilitate viral gene expression and genome replication.

Potential muscle activity disturbance in Penaeus monodon during Acute Hepatopancreatic Necrosis Disease (AHPND) infection: Inference through gene expression, calcium concentration, and MicroRNA

Global shrimp aquaculture farmers have suffered major economic losses due to disease outbreaks. A notable shrimp disease is Acute Hepatopancreatic Necrosis Disease (AHPND), which is caused by a new strain of Vibrio parahaemolyticus bacteria (VpAHPND) that mainly inhabits the shrimp gut and damages the hepatopancreas. Fewer studies have investigated whether this disease will affect shrimp muscle functioning or cause any muscle damage. We challenged Penaeus monodon shrimp with VpAHPND bacteria using an immersion method. Expression of Dystrophin gene, an important regulatory gene for maintenance of muscle integrity, was quantified from muscle samples using qRT-PCR. Additional verification was conducted by determining calcium concentration and bta-miR-4286 and dre-miR-107b miRNAs expression. P. monodon dystrophin gene demonstrated the highest expression level during AHPND infection when muscle calcium concentration was detected at its lowest level at 6 h post-infection (hpi). The highest muscle calcium concentration, determined at 36 hpi, was supported by higher bta-miR-4286 miRNA expression and lower dre-miR-107b miRNA expression in VpAHPND-infected samples compared to uninfected samples at the same time point. We deduced an interactive relationship between dystrophin gene expression, calcium concentration, and miRNA expression in P. monodon muscle tissues triggered by the invading VpAHPND bacterium.

Acute hepatopancreatic necrosis disease in penaeid shrimp

AbstractAsian countries are the major producers of cultured Penaeid shrimps such as Penaeus monodon and Penaeus (Litopenaeus) vannamei. In recent years, shrimp production has declined due to the emergence of a bacterial disease called acute hepatopancreatic necrosis disease (AHPND). This disease is mainly caused by Vibrio parahaemolyticus, but other Vibrio species are also known to cause AHPND in shrimps. Here, in addition to reviewing recent developments in the field of AHPND diagnosis, host responses to AHPND, the role of microbiota during AHPND infection and the current treatment options for AHPND, we also describe a model of AHPND pathogenesis.

Transcriptomic analysis of Pacific white shrimp (Litopenaeus vannamei, Boone 1931) in response to acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), caused by marine bacteria Vibrio Parahaemolyticus, is a huge problem in shrimp farms. The V. parahaemolyticus infecting material is contained in a plasmid which encodes for the lethal toxins PirABVp, whose primary target tissue is the hepatopancreas, causing sloughing of epithelial cells, necrosis, and massive hemocyte infiltration. To get a better understanding of the hepatopancreas response during AHPND, juvenile shrimp Litopenaeus vannamei were infected by immersion with V. parahaemolyticus. We performed transcriptomic mRNA sequencing of infected shrimp hepatopancreas, at 24 hours post-infection, to identify novel differentially expressed genes a total of 174,098 transcripts were examined of which 915 transcripts were found differentially expressed after comparative transcriptomic analysis: 442 up-regulated and 473 down-regulated transcripts. Gene Ontology term enrichment analysis for up-regulated transcripts includes metabolic process, regulation of programmed cell death, carbohydrate metabolic process, and biological adhesion, whereas for down-regulated transcripts include, microtubule-based process, cell activation, and chitin metabolic process. The analysis of protein- protein network between up and down-regulated genes indicates that the first gene interactions are connected to oxidation-processes and sarcomere organization. Additionally, protein-protein networks analysis identified 20-top highly connected hub nodes. Based on their immunological or metabolic function, ten candidate transcripts were selected to measure their mRNA relative expression levels in AHPND infected shrimp hepatopancreas by RT-qPCR. Our results indicate a close connection between the immune and metabolism systems during AHPND infection. Our RNA-Seq and RT-qPCR data provide the possible immunological and physiological scenario as well as the molecular pathways that take place in the shrimp hepatopancreas in response to an infectious disease.

Differential transcriptome analysis of the disease tolerant Madagascar\u2013Malaysia crossbred black tiger shrimp, Penaeus monodon hepatopancreas in response to acute hepatopancreatic necrosis disease (AHPND) infection: inference on immune gene response and interaction

BackgroundPenaeus monodon is the second most widely cultured marine shrimp species in the global shrimp aquaculture industry. However, the growth of P. monodon production has been constantly impaired by disease outbreaks. Recently, there is a lethal bacterial infection, known as acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus AHPND strain (VpAHPND), which led to mass mortalities in P. monodon. Unfortunately, there is still insufficient knowledge about the underlying immune response of P. monodon upon AHPND infection. The present study aims to provide an insight into the antibacterial immune response elicited by P. monodon hepatopancreas towards AHPND infection.MethodsWe have employed high-throughput RNA-Seq technology to uncover the transcriptome changes of P. monodon hepatopancreas when challenged with VpAHPND. The shrimps were challenged with VpAHPND through immersion method with dissected hepatopancreas samples for the control group (APm-CTL) and treatment group at 3 (APm-T3), 6 (APm-T6), and 24 (APm-T24) hours post-AHPND infection sent for RNA-Seq. The transcriptome de novo assembly and Unigene expression determination were conducted using Trinity, Tgicl, Bowtie2, and RSEM software. The differentially expressed transcripts were functionally annotated mainly through COG, GO, and KEGG databases.ResultsThe sequencing reads generated were filtered to obtain 312.77 Mb clean reads and assembled into 48662 Unigenes. Based on the DEGs pattern identified, it is inferred that the PAMPs carried by VpAHPND or associated toxins are capable of activating PRRs, which leads to subsequent pathway activation, transcriptional modification, and antibacterial responses (Phagocytosis, AMPs, proPO system). DAMPs are released in response to cell stress or damage to further activate the sequential immune responses. The comprehensive interactions between VpAHPND, chitin, GbpA, mucin, chitinase, and chitin deacetylase were postulated to be involved in bacterial colonization or antibacterial response.ConclusionsThe outcomes of this research correlate the different stages of P. monodon immune response to different time points of AHPND infection. This finding supports the development of biomarkers for the detection of early stages of VpAHPND colonization in P. monodon through host immune expression changes. The potential genes to be utilized as biomarkers include but not limited to C-type lectin, HMGB1, IMD, ALF, serine proteinase, and DSCAM.

Bioactivity-guided identification of anti-AHPND (acute hepatopancreatic necrosis disease) metabolites of Ecklonia arborea

Acute hepatopancreatic necrosis disease (AHPND) of shrimps is a disease caused by Vibrio parahaemolyticus which has caused a dramatic drop in shrimp production worldwide. Seaweeds of the genus Ecklonia have been reported as natural sources of antibacterial compounds. The present study aims to develop a bio-guided fractionation using the Fast-Centrifugal Partition Chromatography (FCPC) to isolate and identify bactericidal compounds against V. parahaemolyticus AHPND+ from Ecklonia arborea extract. The enriched-phlorotannins extract (EPE) showed a bactericidal activity 4.6-fold higher than crude extract of E. arborea and this activity was increased to 10-fold in pool 8 with a minimum bactericidal concentration (MBC) of 350 μg mL−1 after FCPC fractionation. Eckol and dieckol were the major compounds in pool 8 with 5.23 mg g−1 and 1.67 mg g−1, respectively. The isolated eckol showed weak antibacterial activity compared to pool 8, whereas a dieckol-rich extract showed bactericidal activity at 750 μg mL−1. Data suggested that the bactericidal activity of pool 8 could be attributed to the content of phlorotannins, mainly eckol, and dieckol. EPE and its purified fraction pool 8 from E. arborea collected from Mexico may be useful to develop antibiotic agents for medicated shrimp feed additive to combat AHPND infection.

Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

Prophylactic properties of biofloc- or Nile tilapia-conditioned water against Vibrio parahaemolyticus infection of whiteleg shrimp (Penaeus vannamei)

Isolates of Vibrio parahaemolyticus (VpAHPND) 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 VpAHPND. First, groups of shrimp were bath-challenged with VpAHPND 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 VpAHPND 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 VpAHPND 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 VpAHPND as part of pond management practices.

The Rho signalling pathway mediates the pathogenicity of AHPND-causing V.parahaemolyticus in shrimp.

An emerging bacterial disease, acute hepatopancreatic necrosis disease (AHPND), is caused by strains of Vibrio parahaemolyticus with an additional AHPND-associated plasmid pVA1 encoding a virulent toxin (Pirvp ) that damages the shrimp's hepatopancreas. Like other species of Vibrio, these virulent strains initially colonise the shrimp's stomach, but it is not yet understood how the bacteria or toxins are subsequently able to cross the epithelial barrier and reach the hepatopancreas. Here, by using transcriptomics and system biology methods, we investigate AHPND-induced changes in the stomach of AHPND-causing V.parahaemolyticus (5HP)-infected shrimp and identify host molecular mechanisms that might explain how the integrity of the stomach barrier is compromised. We found that the expression of 376 unique genes was differentially regulated by AHPND infection. Gene ontology, protein interaction, and gene-to-gene correlation expression interaction analyses indicated that in addition to the immune system, a number of these genes were involved in cytoskeleton regulation by Rho GTPase. The involvement of Rho pathway regulation during AHPND pathogenesis was further supported by experiments showing that while Rho inhibitor pretreatment delayed the infection, pretreatment with Rho activator enhanced the pathogenicity of 5HP, and both the bacteria and toxin were detected sooner in the hepatopancreas. Further, disruption of the stomach epithelial structure was found in both Rho preactivated shrimp and in 5HP-infected shrimp. Taken together, we interpret our results to mean that Rho signalling helps to mediate AHPND pathogenesis in shrimp.