In innate immunity, antimicrobial peptides (AMPs) play a vital role in combating microbial pathogens. Among the AMPs identified in Penaeus monodon, only anti-lipopolysaccharide factor isoform 3 (ALFPm3) has been reported to exhibit activity against white spot syndrome virus (WSSV). However, the mechanism(s) involved are still not clear. In the present study, ALFPm3-interacting proteins were screened for from a WSSV library using the yeast two-hybrid screening system, revealing the five potential ALFPm3-interacting proteins of WSSV186, WSSV189, WSSV395, WSSV458 and WSSV471. Temporal transcriptional analysis in WSSV-infected P. monodon revealed that all five of these WSSV gene transcripts were expressed in the late phase of infection (24h and 48h post-infection). Of these, WSSV189 that was previously identified as a structural protein, was selected for further analysis and was shown to be an enveloped protein by Western blot and immunoelectron microscopy analyses. The in vitro pull-down assay using recombinant WSSV189 (rWSSV189) protein as bait confirmed the interaction between ALFPm3 and WSSV189 proteins. Moreover, pre-incubation of rWSSV189 protein with rALFPm3 protein interfered with the latter's neutralization effect on WSSV in vivo, as shown by the increased cumulative mortality of shrimp injected with WSSV following prior treatment with pre-incubated rWSSV189 and rALFPm3 proteins compared to that in shrimp pre-treated with rALFPm3 protein. Thus, ALFPm3 likely performs its anti-WSSV action by binding to the envelope protein WSSV189 and possibly other WSSV structural proteins.

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 NLHSinduced 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. During its first outbreak, the devastating effect of early mortality syndrome (EMS) on the global shrimp industry was primarily caused by the lack of information regarding the disease and its causative agent 1. Later studies that were focused on mitigating this disease eventually identified the cause to be a toxin-harboring Vibrio parahaemolyticus, which causes Acute hepatopancreatic necrosis disease (AHPND) (VP AHPND) 2. This highlights how understanding disease etiology and a subsequent elucidation of host response can help to mitigate the effects of an outbreak. Understanding the functions of the shrimp immune system during disease progression is thus expected to create opportunities for the development of effective and efficient management strategies for VP AHPND infection. Likewise, information regarding the molecular mechanisms of AHPND tolerance can lead to platforms for the development of AHPND-resistant shrimp through selective breeding using markers mined from transcriptome analyses at different rearing conditions.

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