Unveiling role of HSP70 genes for development and survival of Indian malaria vector Anopheles culicifacies

Heat shock proteins (HSPs) play a pivotal role in maintaining cellular homeostasis and mediating stress responses across diverse organisms. Among them, the HSP70 family is crucial for protein folding and stress regulation. However, its functions remain underexplored in mosquito species, particularly in major Indian malaria vectors such as Anopheles culicifacies (Ac). This study aims to contribute to mosquito control by investigating the role of HSP70 in An. culicifacies. Given the persistent global challenge posed by malaria, understanding the regulatory mechanisms of HSP70 is essential for developing effective control strategies. In this study, we identified seven HSP70 genes in An. culicifacies and analyzed their expression profiles across different life stages. Six of these HSP70 genes (1, 2, 3, 5, 6, and 7) exhibited significant upregulation during the third instar larval stage, emphasizing their critical role in larval development. Using specific HSP70 inhibitors, quercetin and KNK437, we observed that KNK437 displayed potent larvicidal activity, comparable to the widely used insecticide temephos. Additionally, we successfully purified and characterized recombinant AcHSP70-1, which demonstrated unique interactions with adenosine triphosphate (ATP) and its co-chaperone AcHSP40, distinguishing it from other HSP70 systems. Through a combination of confocal microscopy, qRT-PCR analysis, and inhibitor assays, we further established the essential role of HSP70 in both larval development and adult female mosquitoes during blood meal acquisition. These findings provide novel insights into the functional diversification and regulatory mechanisms of HSP70 genes in An. culicifacies. This study not only enhances our understanding of their developmental roles but also highlights innovative targets for the development of mosquito control strategies.