Roles of bacterial toxin receptor and piwi-interacting RNA of pacific white shrimp penaeus vannamei in response to vibrio parahaemolyticus-causing acute hepatopancreatic necrosis disease infection

Abstract

Acute hepatopancreatic necrosis disease (AHPND) or Early mortality syndrome (EMS) caused by virulent strains of Vibrio parahaemolyticus AHPND (VPAHPND) has been a leading cause of significant losses of shrimp production. In this study, we aim to explore the response and the role of immune-related gene/protein and small RNA in the VPAHPND-infected shrimp. This research was divided into 2 parts. In the first part, we identified candidate genes of VPAHPND toxin receptor and further confirmed its function as a VPAHPND toxin receptor. APN has been reported as a Cry toxin from Bacillus thuringiensis, whose structure is similar to VPAHPND toxin. Therefore, we identified aminopeptidase N (APN) from the transcriptomic data of VPAHPND-infected Litopenaeus vannamei hemocyte. According to LvAPN1 and LvAPN2 gene expression analysis, only LvAPN1 were highly upregulated in stomach, hepatopancreas and hemocyte after VPAHPND infection and VPAHPND toxin injection. Silencing of LvAPN1 gene reduced mortality, the clinical signs of AHPND in the hepatopancreas and the number of virulent VPAHPND bacteria in the stomach. In addition, observation of hemocyte morphology by scanning electron microscope showed that the LvAPN1 silencing prevented severe damage of hemocyte morphology causing by VPAHPND toxin like what clearly observed in the control group. At the protein level, rLvAPN1 directly bind to the recombinant protein of PirA and PirB toxins. Our results indicated that not only stomach and hepatopancreas, but also hemocytes are the target tissues of VPAHPND toxin and act as the VPAHPND toxin receptor. The second part aims to study on piRNAs that are expressed in response to VPAHPND infection. Firstly, we identified piRNAs from small RNA-Seq data of VPAHPND-infected L. vannamei. Totally 150 types of piRNA homologs were identified. Target gene identification of those piRNA identified 53 target genes involving in gene expression and protein synthesis/degradation. Six differentially expressed piRNAs (DEPs) were discovered. Two DEPs were upregulated whereas another 4 DEPs were downregulated after VPAHPND infection. Expression analysis of DEPs and the target genes showed that only 2 piRNAs such as piR-lva-29948104 and piR-lva-26449194 had the negative expression correlation with their mRNA targets which are E3 ubiquitin-protein ligase RNF26-like (RNF26) and circadian locomoter output cycles protein kaput-like (Clock), respectively. According to target gene’s function, these 2 piRNAs might play the role in gene expression and protein synthesis/degradation processes in VPAHPND-infected shrimp. Collectively, our study provides the new insight into the role of VPAHPND toxin receptor, LvAPN1, and the expression of piRNA in response to VPAHPND infection. This knowledge will provide alternative strategies for fighting against VPAHPND infection in shrimp farming.