Azadirachtin A inhibits the growth and development of Bactrocera dorsalis larvae by releasing cathepsin in the midgut

Azadirachtin, a botanical insecticide with high potential, has been widely used in pest control. Azadirachtin has shown strong biological activity against Bactrocera dorsalis in toxicological reports, but its mechanism remains unclear. This study finds that azadirachtin A inhibits the growth and development of Bactrocera dorsalis larvae. The larval weights and body sizes of the azadirachtin-treated group were significantly less than those of the control group in a concentration-dependent manner. Further, pathological sections revealed that azadirachtin destroyed the midgut cell structure and intestinal walls, while TUNEL staining showed that azadirachtin could induce apoptosis of midgut cells, and Western blot analysis indicated that Bcl-XL expression was inhibited and cytochrome c (CytC) released into the cytoplasm. The results also imply azadirachtin-induced structural alterations in the Bactrocera dorsalis larvae midgut by activation of apoptosis. RNA-seq analysis of midgut cells found that 482 and 708 unique genes were upregulated and downregulated, respectively. These differentially expressed genes (DEGs) were enriched in apoptotic and lysosomal signaling pathways and included 26 genes of the cathepsin family. qRT-PCR verified the expression patterns of some DEGs, indicating that Cathepsin F was upregulated by 278.475-fold and that Cathepsin L and Cathepsin D were upregulated by 28.06- and 8.97-fold, respectively. Finally, association analysis between DEGs and DEMs (differentially expressed metabolites) revealed that azadirachtin significantly reduced the digestion and absorption of carbohydrates, proteins, fats, vitamins and minerals in the midgut. In conclusion, azadirachtin induces the release of cathepsin from lysosomes, causing apoptosis in the midgut. Ultimately, this leads to reduced digestion and absorption of nutrient metabolites in the midgut and inhibition of the growth and development of Bactrocera dorsalis larvae.