Identification and Functional Analysis of V-ATPaseA and C Genes in .

Vacuolar (H)-ATPases (V-ATPases) are ATP-driven proton pumps that play multifaceted roles across various organisms. Despite their widespread significance, the functional implications of genes in , an invasive forest pest with a global presence, have yet to be elucidated. In this study, two specific genes from were identified and analyzed, namely (accession number: OR217451) and (accession number: OR217452). Phylogenetic analysis and multiple sequence alignment reveal that HcV-ATPase A shares the highest amino acid sequence similarity with SfV-ATPase A, while HcV-ATPase C is most similar to HaV-ATPase C. Spatiotemporal expression profiles, determined via RT-qPCR, demonstrate that both and are expressed throughout all larval developmental stages, with predominantly expressed in the midgut and showing high expression in the epidermis. RNA interference (RNAi) targeting of these genes significantly suppressed their expression by 62.7% and 71.0% 120 h post-injection, leading to halted larval growth and increased mortality rates of 61.7% and 46.7%, respectively. Further investigations using immunohistochemistry, hematoxylin and eosin (HE) staining, and transmission electron microscopy (TEM) revealed that gene silencing induced vesiculation and subsequent losses or sloughing of intestinal parietal cells, alongside an increase in the number of autophagic cells. Additionally, the silencing of and genes resulted in a reduced gut epidermal cell layer thickness and further increases in goblet cell numbers. Importantly, RNAi of and did not affect the expression levels of one another, suggesting independent functional pathways. This study provides foundational insights into the role of in and identifies potential targets for the biocontrol of its larvae, contributing to the understanding of mechanisms and their application in pest management strategies.