MicroRNA and mRNA interactions coordinate the immune response in non-lethal heat stressed Litopenaeus vannamei against AHPND-causing Vibrio parahaemolyticus

Abstract

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.

Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor

“…In the present study, we retrieved Lv APN1 and Lv APN2 sequences from our transcriptomic database of VP AHPND -challenged Litopenaeus vannamei hemocytes [ 18 ]. Gene expression of Lv APN1 was analyzed after challenge with AHPND-causing bacteria and partially purified VP AHPND toxins.…”

Regulation of shrimp prophenoloxidase activating system by lva-miR-4850 during bacterial infection

“…In combination with the previous transcriptomic data of VP AHPND -infected P. vannamei hemocytes 22 , we analyzed the data based on the negative correlation in gene and miRNA expression and the complementary miRNA/mRNA target prediction to further define the miRNA/mRNA interaction involved in antibacterial responses. According to Zheng et al (2018) 27 , 12 miRNAs and their predicted target genes are possibly involved in modulating several immune-related processes in the pathogenesis of AHPND.…”

“…The indexed libraries were normalized, pooled, and then, sequenced with a PhiX control spiked at 7.5% using MiSeq Reagent Kits v2 (Illumina) in a MiSeq sequencer (Illumina). The sRNA-Seq analysis was performed as previously described by Boonchuen et al (2020) 22 . Briefly, the Galaxy instance (https ://usega laxy.org/) was used for 5′-and 3′-adapter trimming and for quality control of raw reads 40 .…”

“…Therefore, identification of the target mRNA could provide clues regarding the role of miRNA in the shrimp's immune response against VP AHPND infection. The transcriptome database of VP AHPND -infected P. vannamei 22 was used for mRNA target prediction using CU-Mir (https ://cumir .shrim p-irn.org/), an in-house developed miRNA target prediction program. Although several target genes were predicted, this study emphasized the immune-related genes by targeting VP AHPND -responsive miRNAs.…”

“…Prediction of miRNA targets. The miRNA targets were identified by searching for the miRNA complementary sequence in the transcriptome data of VP AHPND -challenged P. vannamei hemocytes 22 using the CU-Mir software (http://shrim p-irn.org/mirta rget/index .php) that was developed in-house 42 . The CU-Mir software was used to search for locations on mRNA targets that seed sequences (2-8 nucleotides from the 5′ end) of miRNA that can bind perfect complementary or one mismatch at any different region; an open reading frame (ORF); 3′-untranslated region (UTR) and 5′-UTR.…”