Differentially expressed genes in hemocytes of Litopenaeus vannamei challenged with Vibrio parahaemolyticus AHPND (VPAHPND) and VPAHPND toxin

While toxin-harboring Vibrio parahaemolyticus has been previously established as the causative agent of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, information on the mechanistic processes that happen in the host during infection is still lacking. Here, we examined the expression responses of the shrimp hemocyte transcriptome to V. parahaemolyticus AHPND (VPAHPND) by RNA sequencing (RNA-seq). Using libraries (SRA accession number SRP137285) prepared from shrimp hemocytes under experimental conditions, a reference library was de novo assembled for gene expression analysis of VPAHPND-challenged samples at 0, 3/6, and 48 h post infection (hpi). Using the library from 0-hpi as the control, 359 transcripts were found to be differentially expressed in the 3/6-hpi library, while 429 were differentially expressed in the 48-hpi library. The expression patterns reported in the RNA-seq of 9 representative genes such as anti-lipopolysaccharide factor (LvALF), crustin p (CRU), serpin 3 (SER), C-type lectin 3 (CTL), clottable protein 2 (CLO), mitogen-activated protein kinase kinase 4 (MKK4), P38 mitogen-activated protein kinase (P38), protein kinase A regulatory subunit 1 (PICA) and DNAJ homolog subfamily C member 1-like (DNJ) were validated by qRT-PCR. The expression of these genes was also analyzed in shrimp that were injected with the partially purified VPAHPND toxin. A VPAHPND toxin-responsive gene, LvALF was identified, and its function was characterized by RNA interference. LvALF knockdown resulted in significantly rapid increase of shrimp mortality caused by toxin injection. Protein-protein interaction analysis by molecular docking suggested that LvALF possibly neutralizes VPAHPND toxin through its LPS-binding domain. The data generated in this study provide preliminary insights into the differences in the immune response of shrimp to the bacterial and toxic aspect of VPAHPND as a disease.