Azadirachtin Induces Fat Body Apoptosis by Suppressing Caspase-8 in the Fall Armyworm, Spodoptera frugiperda.

Azadirachtin is a widely used botanical pesticide for agricultural pest control worldwide. However, the molecular mechanisms of azadirachtin in insects are not fully understood. In this study, histological analysis and RNA sequencing were conducted to investigate the impact of azadirachtin on the larval development of Spodoptera frugiperda. Under azadirachtin exposure, the development was completely inhibited, and the major internal tissues, fat body, and midgut were strongly damaged under histological analysis. Differential gene expression analysis demonstrated that nutrient absorption and detoxification metabolism-related genes are differentially expressed. Interestingly, the expression of the apoptosis-related gene, caspase-8, was significantly inhibited under exposure to azadirachtin. In addition, after knocking down the expression of the caspase-8 gene, the fat body displayed a similar apoptotic phenotype as azadirachtin treatment; the distribution of chromatin and lipid droplets was uneven in the fat body cells. Thus, the results in this study demonstrated that exposure to azadirachtin rapidly activates apoptosis, resulting in innate tissue disruption, ultimately arresting larval development in S. frugiperda.

Azadirachtin Inhibits Nuclear Receptor HR3 in the Prothoracic Gland to Block Larval Ecdysis in the Fall Armyworm, Spodoptera frugiperda.

Azadirachtin has been used to control agricultural pests for a long time; however, the molecular mechanism of azadirachtin on lepidopterans is still not clear. In this study, the fourth instar larvae of fall armyworm were fed with azadirachtin, and then the ecdysis was blocked in the fourth instar larval stage (L4). The prothoracic glands (PGs) of the treated larvae were dissected for RNA sequencing to determine the effect of azadirachtin on ecdysis inhibition. Interestingly, one of the PG-enriched genes, the nuclear hormone receptor 3 (HR3), was decreased after azadirachtin treatment, which plays a critical role in the 20-hydroxyecdysone action during ecdysis. To deepen the understanding of azadirachtin on ecdysis, the HR3 was knocked out by using the CRISPR/Cas9 system, while the HR3 mutants displayed embryonic lethal phenotype; thus, the stage-specific function of HR3 during larval molting was not enabled to unfold. Hence, the siRNA was injected into the 24 h L4 larvae to knock down HR3. After 96 h, the injected larvae were blocked in the old cuticle during ecdysis which is consistent with the azadirachtin-treated larvae. Taken together, we envisioned that the inhibition of ecdysis in the fall armyworm after the azadirachtin treatment is due to an interference with the expression of HR3 in PG, resulting in larval mortality. The results in this study specified the understanding of azadirachtin on insect ecdysis and the function of HR3 in lepidopteran in vivo.