Discovery and characterization of Vibrio parahaemolyticus AHPND-responsive piRNAs in modulating shrimp immune response

Piwi-interacting RNAs (piRNAs) represent the largest class of small non-coding RNAs, typically ranging from 24 to 31 nucleotides in length. When complexed with PIWI protein, piRNAs suppress expression of transposons and protein-coding genes, acting at both the transcriptional or post-transcriptional levels. While a recent study suggest that piRNAs play a regulatory role in shrimp during viral infections, their involvement in antibacterial responses remains unexplored. This study aims to identify piRNAs in the hemocyte of Penaeus vannamei infected with Vibrio parahaemolyticus AHPND (VP_AHPND). By re-analyzing our previous small RNA data generated from the shrimp hemocytes of heat-shock-treated challenged with VP_AHPND, we identified a total of 150 piRNA homologs across all libraries, with six piRNAs showing significant dysregulation in response to VP_AHPND infection. The target genes of piRNAs were identified from our in-house P. vannamei transcriptome database. Expression profiling revealed a negative correlation between piRNAs and their predicted targets, suggesting a potential regulatory role. Among them, we further characterized piR-pva-29948104 which targets E3 ubiquitin-protein ligase RNF26-like (PvRNF26) —a STING ortholog involved in modulating shrimp immune responses via the STING-IKKβ-Relish pathway. Introducing of piR-pva-29948104 mimic suppressed PvRNF26 expression resulting in the activation of downstream genes PvVago5 and PvPEN4 of the STING-IKKβ-Relish pathway. Correspondingly, PvRNF26 knockdown enhanced immune gene activation, significantly reducing both shrimp mortality and bacterial load during VP_AHPND infection. In summary, the piRNA homologs were discovered in shrimp, P. vannamei and their potential roles were highlighted in regulating immune gene expression during VP_AHPND infection, offering new insights into shrimp immune response mechanisms.