Azadirachtin‑A from Azadirachta indica Impacts Multiple Biological Targets in Cotton Bollworm Helicoverpa armigera

Azadirachtin-A
(AzaA) from the Indian neem tree (Azadirachta indica) has insecticidal properties;
however, its molecular mechanism
remains elusive. The “targeted and nontargeted proteomic profiling”,
metabolomics, matrix-assisted laser desorption/ionization time of
flight (MALDI-TOF) imaging, gene expression, and in silico analysis
provided clues about its action on Helicoverpa armigera. Fourth instar H. armigera larvae
fed on AzaA-based diet (AzaD) suffered from significant mortality,
growth retardation, reduced larval mass, complications in molting,
and prolonged development. Furthermore, death of AzaD-fed larvae was
observed with various phenotypes like bursting, blackening, and half-molting.
Liquid chromatography–mass spectrometry (LC–MS) data
showed limited catabolic processing of ingested AzaA and dramatic
alternations of primary metabolism in H. armigera. MALDI-TOF imaging indicated the presence of AzaA in midgut of H. armigera. In the gut, out of 79 proteins identified,
34 were upregulated, which were related to digestion, immunity, energy
production, and apoptosis mechanism. On the other hand, 45 proteins
were downregulated, including those from carbohydrate metabolism,
lipid metabolism, and energy transfer. In the hemolymph, 21 upregulated
proteins were reported to be involved in immunity, RNA processing,
and mRNA-directed protein synthesis, while 7 downregulated proteins
were implicated in energy transfer, hydrolysis, lipid metabolism,
defense mechanisms, and amino acid storage-related functions. Subsequently,
six target proteins were identified using labeled AzaA that interacted
with whole insect proteins. In silico analysis suggests that AzaA
could be efficiently accommodated in the hydrophobic pocket of juvenile
hormone esterase and showed strong interaction with active site residues,
indicating plausible targets of AzaA in H. armigera. Quantitative polymerase chain reaction analysis suggested differential
gene expression patterns and partly corroborated the proteomic results.
Overall, data suggest that AzaA generally targets more than one protein
in H. armigera and hence could be a
potent biopesticide.

印楝素-A（Azadirachtin-A, AzaA）源自印度印楝树（Azadirachta indica），具备杀虫活性，但其分子作用机制至今仍未阐明。本研究通过靶向与非靶向蛋白质组学分析、代谢组学、基质辅助激光解吸电离飞行时间（matrix-assisted laser desorption/ionization time of flight, MALDI-TOF）成像、基因表达分析以及计算机辅助分析（in silico analysis），为解析其对棉铃虫（Helicoverpa armigera）的作用机制提供了重要线索。

取食含AzaA饲料（AzaD）的四龄棉铃虫幼虫，出现显著死亡率升高、生长迟滞、幼虫体重下降、蜕皮异常以及发育周期延长等表型。此外，取食AzaD饲料的幼虫死亡时呈现多种异常表型，包括体壁破裂、体色黑化以及半蜕皮。

液相色谱-质谱联用（liquid chromatography–mass spectrometry, LC-MS）数据显示，取食的AzaA仅发生有限的分解代谢，且棉铃虫体内的初级代谢发生显著紊乱。MALDI-TOF成像结果证实，AzaA可在棉铃虫中肠内检出。在中肠中，共鉴定出79种蛋白质，其中34种表达上调，这些蛋白质参与消化、免疫、能量产生及细胞凋亡（apoptosis）通路；另有45种蛋白质表达下调，其功能涵盖碳水化合物代谢、脂质代谢及能量转运过程。

在血淋巴中，21种上调蛋白质参与免疫应答、RNA加工及mRNA指导的蛋白质合成过程；另有7种下调蛋白质则涉及能量转运、水解反应、脂质代谢、防御机制及氨基酸储存等功能。后续通过标记AzaA与全虫蛋白质进行亲和富集，成功鉴定出6种靶标蛋白质。

计算机辅助分析结果显示，AzaA可高效结合保幼激素酯酶（juvenile hormone esterase）的疏水口袋，并与其活性位点残基产生强相互作用，提示AzaA在棉铃虫体内存在多类潜在作用靶标。定量聚合酶链式反应（quantitative polymerase chain reaction）分析显示，基因表达谱存在显著差异，且部分验证了蛋白质组学实验结果。

综上，本研究数据表明，AzaA在棉铃虫体内可靶向多种蛋白质，具备开发为高效生物农药的潜力。