Identification and functional characterization of genes and microRNAs from Penaeus vannamei in response to pathogens and heat stress

In the present day, high-throughput (HT) sequencing techniques provide in-depth data on how the transcripts are expressed and regulated and in this case shed light on how innate immunity responds to different stimuli. Acute Hepatopancreatic Necrosis Disease (AHPND) caused by Vibrio parahaemolyticusproducing Pir toxin (VPAHPND) is a current severe bacterial disease in shrimp. Suppression subtractive hybridization (SSH) was used to identify differentially expressed genes (DEGs) in hepatopancreas and hemocyte of VPAHPND-challengedPenaeus vannamei. The hemocyaninand Vago5 were found as up-regulated genes in hepatopancreas and hemocyte of VPAHPND-infectedP. vannamei, respectively. Their functions in shrimp immunity against VPAHPNDinfection were then characterized. The native hemocyanin protein purified from hemolymph was able to agglutinate VPAHPNDin vitroand neutralize of VPAHPNDsecreted toxin via direct interaction with the PirA protein. The Vago5, the early VPAHPND-responsive gene in P. vannameihemocyte, was functionally characterized by RNA interference. Vago5knockdown in VPAHPND-infectedP. vannameiresulted in the increase in shrimp mortality and the number of bacteria in stomach and hepatopancreas. Moreover, Vago5knockdown caused a significant decrease in PEN4, PO2and TNFexpression suggesting its role in modulating antibacterial responses. As reported previously, non-lethal heat shock (NLHS) could enhance the resistance of P. vannameito VPAHPNDinfection and induce expression of immune-related genes. Herein, HT-sequencing of total RNAs and small RNAs from P. vannameihemocyte challenged with VPAHPNDunder NLHS and non-heat stress (NH) conditions brought about the data on NLHS-induced DEGs and differentially expressed miRNAs (DEMs) and the miRNA-mRNA regulatory network. Furthermore, pathway analysis of the NLHS-induced DEMs and their target DEGs which have immune-related functions, demonstrated that NLHS induces changes in expression of genes involved in prophenoloxidase system, hemocyte homeostasis and antimicrobial peptide production of VPAHPND-infected P. vannamei. Focusing on VPAHPND-responsive miRNAs targeting immune genes, lva-miR-4850 was studied for its function in regulating prophenoloxidase 2 (PO2)geneexpression. Introducing the lva-miR-4850 mimic into the VPAHPND-infected shrimp caused the reduction of the PO2transcript and the PO activity but significantly increased the number of bacteria in the VPAHPNDtargeted shrimp tissues. White spot disease caused by white spot syndrome virus (WSSV), is highly contagious and lethal in shrimp. The method called, Psoralen Analysis of RNA Interactions and Structures (PARIS) was performed to determine the transcriptome-wide base pairing interactions in hemocyte of WSSV-infected P. vannamei. Although the sequencing data have not yet obtained, we demonstrated for the first time the success of psoralen-induced RNA cross-linking in invertebrates tissues. In conclusions, the P. vannameitransciptome-wide interactions during pathogen infections and NLHS elucidated by HT sequencing based technologies and protein function analysis, revealed the complexity of the shrimp innate immune responses to promote effective host defense.