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The global aquaculture industry has suffered significant losses due to the outbreak of Acute Hepatopancreatic Necrosis Disease (AHPND) caused by Vibrio parahaemolyticus. Since the use of antibiotics as control agents has not been shown to be effective, an alternative anti-infective regimen, such as phage therapy, has been proposed. Here, we employed high-throughput screening for potential phages from 98 seawater samples and obtained 14 phages exhibiting diverse host specificity patterns against pathogenic VP AHPND strains. Among others, two Chimallinviridae phages, designated Eric and Ariel, exhibited the widest host spectrum against vibrios. In vitro and in vivo studies revealed that a cocktail derived from these two nucleus-forming vibriophages prolonged the bacterial regrowth of various pathogenic VP AHPND strains and reduced shrimp mortality from VP AHPND infection. This research highlights the use of high-throughput phage screening that leads to the formulation of a nucleus-forming phage cocktail applicable for bacterial infection treatment in aquaculture., (© 2023. Springer Nature Limited.)

Peroxiredoxin-4 from Penaeus vannamei (LvPrx4) is considered a damage-associated molecular pattern (DAMP) that can activate the expression of immune-related genes through the Toll pathway. We previously demonstrated that the recombinant LvPrx4 (rLvPrx4) can enhance shrimp resistance against Vibrio parahaemolyticus, causing acute hepatopancreatic necrosis disease (VP AHPND ), which causes great production losses in shrimp farming. Herein, we showed that the rLvPrx4 had a thermal tolerance of around 60 °C and that the ionic strength had no noticeable effect on its activity. We discovered that feeding a diet containing rLvPrx4 to shrimp for three weeks increased the expression of the immune-related genes LvPEN4 and LvVago5. Furthermore, pre-treatment with rLvPrx4 feeding could significantly prolong shrimp survival following the VP AHPND challenge. The shrimp intestinal microbiome was then characterized using PCR amplification of the 16S rRNA gene and Illumina sequencing. Three weeks of rLvPrx4 supplementation altered the bacterial community structure (beta diversity) and revealed the induction of differentially abundant families, including Cryomorphaceae, Flavobacteriaceae, Pirellulaceae, Rhodobacteraceae, and Verrucomicrobiaceae, in the rLvPrx4 group. Metagenomic predictions indicated that some amino acid metabolism pathways, such as arginine and proline metabolism, and genetic information processing were significantly elevated in the rLvPrx4 group compared to the control group. This study is the first to describe the potential use of rLvPrx4 supplementation to enhance shrimp resistance to VP AHPND and alter the composition of a beneficial bacterial community in shrimp, making rLvPrx4 a promising feed supplement as an alternative to antibiotics for controlling VP AHPND infection in shrimp aquaculture., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific Vibrio parahaemolyticus (VP AHPND ) strain, results in a great loss of global shrimp production. This study performed suppression subtractive hybridization (SSH) to identify differentially expressed genes from white shrimp Penaeus vannamei hemocyte upon VP AHPND infection. Among the immune-related genes identified, Vago5, kunitz, secretory leukocyte proteinase inhibitor, and profilin are the most abundant genes classified as the up-regulated genes in the SSH library. The qRT-PCR results show that only Vago5 was highly up-regulated at 3 and 6 h post-VP AHPND challenge, whereas kunitz, secretory leukocyte proteinase inhibitor, and profilin were highly up-regulated at 48 h post-VP AHPND challenge. As an early VP AHPND infection-responsive gene, Vago5 was further functional characterized by RNA interference. Knockdown of Vago5 gene resulted in the significantly rapid increase of shrimp mortality and the number of bacteria in the stomach and hepatopancreas upon VP AHPND infection. Moreover, downstream genes of Toll, IMD, and JAK/STAT pathways and phenoloxidase system were analyzed for the expression in the VP AHPND -infected shrimp hemocyte after dsVago5 treatment. Vago5 gene knockdown resulted in a significant decrease in transcript levels of PEN4, TNF, and PO2 genes as well as PO activity in the hemolymph, suggesting that Vago5 might modulate antibacterial infection through activation of the genes in the NF-κB mediated pathways, JAK/STAT pathway, and phenoloxidase system., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Acute hepatopancreatic necrosis disease (AHPND) caused by PirABVP-producing strain of Vibrio parahaemolyticus, VPAHPND, has seriously impacted the shrimp production. Although the VPAHPND toxin is known as the VPAHPND virulence factor, a receptor that mediates its action has not been identified. An in-house transcriptome of Litopenaeus vannamei hemocytes allows us to identify two proteins from the aminopeptidase N family, LvAPN1 and LvAPN2, the proteins of which in insect are known to be receptors for Cry toxin. The membrane-bound APN, LvAPN1, was characterized to determine if it was a VPAHPND toxin receptor. The increased expression of LvAPN1 was found in hemocytes, stomach, and hepatopancreas after the shrimp were challenged with either VPAHPND or the partially purified VPAHPND toxin. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the stomach after VPAHPND toxin challenge. In addition, LvAPN1 silencing prevented the toxin from causing severe damage to the hemocytes and sustained both the total hemocyte count (THC) and the percentage of living hemocytes. We found that the rLvAPN1 directly bound to both rPirAVP and rPirBVP toxins, supporting the notion that silencing of LvAPN1 prevented the VPAHPND toxin from passing through the cell membrane of hemocytes. We concluded that the LvAPN1 was involved in AHPND pathogenesis and acted as a VPAHPND toxin receptor mediating the toxin penetration into hemocytes. Besides, this was the first report on the toxic effect of VPAHPND toxin on hemocytes other than the known target tissues, hepatopancreas and stomach., Competing Interests: The authors have declared that no competing interests exist.

While Vibrio parahaemolyticus (VP AHPND ) has been identified as the cause of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, mechanisms of host response remain unknown. Understanding these processes is important to improve farming practices because this understanding will help to develop methods to enhance shrimp immunity. Pre-treatment of shrimp with 5-minute chronic non-lethal heat stress (NLHS) for 7 days was found to significantly increase Litopenaeus vannamei survival against VP AHPND infection. To elucidate the mechanism involved, mRNA and miRNA expression profiles from the hemocyte of L. vannamei challenged with VP AHPND after NLHS with corresponding control conditions were determined by RNA-Seq. A total of 2,664 mRNAs and 41 miRNAs were differentially expressed after the NLHS treatment and VP AHPND challenge. A miRNA-mRNA regulatory network of differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) was subsequently constructed and the interactions of DEMs in regulating the NLHS-induced immune-related pathways were identified. Transcriptomic data revealed that miRNA and mRNA interactions contribute to the modulation of NLHS-induced immune responses, such as the prophenoloxidase-activating system, hemocyte homeostasis, and antimicrobial peptide production, and these responses enhance VP AHPND resistance in L. vannamei.

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific strain of Vibrio parahaemolyticus (VP AHPND ), results in great loss of global shrimp production. Despite this, studies on shrimp defense mechanisms protecting against AHPND are few. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes from white shrimp Litopenaeus vannamei hepatopancreas upon VP AHPND infection at the early stages: 3 and 6 h post challenge and in the late stage at 48 h post challenge. Hemocyanin (HMC) is the most abundant gene identified as the up-regulated gene in the SSH library. Various hemocyanin subunits such as hemocyanin (HMC), hemocyanin subunit L1 (HMCL1), L2 (HMCL2), L3 (HMCL3), and L4 (HMCL4) were analyzed for their expression levels upon VP AHPND infection and in response to challenge with partially purified toxin of VP AHPND by qRT-PCR. Only HMC was highly up-regulated at 3 and 6 h post challenge in response to VP AHPND challenge. Two HMC subunits, HMCL3 and HMCL4, were up-regulated in the early phase of VP AHPND toxin injection. Furthermore, all subunits were down-regulated in the late phase of VP AHPND and toxin challenges. The native hemocyanin protein purified from shrimp hemolymph, identified as mixture of HMC and HMCL1, exhibited agglutination activity on VP AHPND . Injecting the purified native hemocyanin along with VP AHPND into shrimp decreased the number of bacteria in the hemolymph as compared to the VP AHPND challenged control. Moreover, pre-incubation of the purified native hemocyanin and VP AHPND toxin prior to injection into shrimp resulted in the decrease of cumulative mortality of shrimp when compared to the control. In addition, protein-protein interaction analysis carried out by ELISA technique indicated that hemocyanin exhibited VP AHPND toxin-neutralizing activity through direct interaction with PirA subunit with a dissociation constant of 6.83 × 10 -6  M. Our results indicated that upon VP AHPND infection the expression of hemocyanin was induced and hemocyanin functions might involve agglutination of invading VP AHPND and also neutralization of VP AHPND secreted toxin via direct interacting with the PirA protein., (Copyright © 2018 Elsevier Ltd. All rights reserved.)