Abstract

Azadirachtin, a major botanical insecticide, is widely recognized as a sustainable alternative to synthetic pesticides, yet the molecular mechanisms underlying its effects on insect hosts remain poorly understood. Transcriptomic analysis, RT-PCR, and qPCR revealed significant upregulation of Sirt5 in the midgut following azadirachtin exposure. In the present study, we investigated the role of Sirt5 , a key regulator of metabolic and stress responses, in mediating midgut damage in Spodoptera litura under azadirachtin stress. Functional assays showed that overexpression of Sirt5 in SpLi-221 cells suppressed lysosomal activity under stress, whereas CRISPR/Cas9-mediated knockout (KO) of Sirt5 led to elevated expression of autophagy-related genes ( Atg6, Atg16, Atg101 ) and the apoptosis marker caspase-1 . Compared with the wild-type control group, the mortality of the larvae lacking Sirt5 increased from 49% to 95% when fed with the median lethal dose of azadirachtin. These findings indicate that Sirt5 mitigates azadirachtin-induced midgut damage by modulating autophagy and apoptosis pathways. The present study provides the first experimental evidence linking Sirt5 to host responses against azadirachtin, offering new mechanistic insights into botanical insecticide action and identifying a potential target for enhancing sustainable pest management strategies.