Table1_Cry1Ac toxin binding in the velvetbean caterpillar Anticarsia gemmatalis: study of midgut aminopeptidases N.DOCX

The velvetbean caterpillar Anticarsia gemmatalis is one of the main soybean defoliators in Brazil. Currently, the main biopesticide used to control insect pests worldwide is the bacteria Bacillus thuringiensis (Bt), which produces entomopathogenic Crystal toxins (Cry) that act in the midgut of susceptible insects, leading them to death. The mode of action of Cry toxins in the midgut involves binding to specific receptors present on the brush border of epithelial cells such as aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, and others. Mutations in these receptors, among other factors, may be involved in the development of resistance; identification of functional Cry receptors in the midgut of A. gemmatalis is crucial to develop effective strategies to overcome this possible scenario. This study’s goal is to characterize APNs of A. gemmatalis and identify a receptor for Cry1Ac in the midgut. The interaction of Bt spores with the midgut epithelium was observed in situ by immunohistochemistry and total aminopeptidase activity was estimated in brush border membrane vesicle (BBMV) samples, presenting higher activity in challenged individuals than in control ones. Ten APN sequences were found in a A. gemmatalis’ transcriptome and subjected to different in silico analysis, such as phylogenetic tree, multiple sequence alignment and identification of signal peptide, activity domains and GPI-anchor signal. BBMV proteins from 5th instar larvae were submitted to a ligand blotting using activated Cry1Ac toxin and a commercial anti-Cry polyclonal antibody; corresponding bands of proteins that showed binding to Cry toxin were excised from the SDS-PAGE gel and subjected to mass spectrometry analysis, which resulted in the identification of seven of those APNs. Quantitative PCR was realized to compare expression levels between individuals subjected to sublethal infection with Bt spores and control ones, presenting up- and downregulations upon Bt infection. From these results, we can infer that aminopeptidases N in A. gemmatalis could be involved in the mode of action of Cry toxins in its larval stage.

绒毛豆毛虫 Anticarsia gemmatalis 是巴西大豆主要落叶害虫之一。目前，全球范围内用于控制害虫的主要生物杀虫剂为苏云金芽孢杆菌（Bacillus thuringiensis，简称Bt），该菌株能产生对害虫具有病原性的晶体毒素（Cry），这些毒素作用于敏感昆虫的中肠，导致其死亡。Cry毒素在中肠中的作用机制涉及与上皮细胞刷状缘上特定的受体结合，如氨基肽酶N（APN）、碱性磷酸酶（ALP）、钙粘蛋白等。这些受体的突变以及其他因素可能参与抗性发展的过程；在 A. gemmatalis 中肠中鉴定功能性的Cry受体对于开发克服这一潜在挑战的有效策略至关重要。本研究的目标是对 A. gemmatalis 的 APN 进行表征并鉴定中肠中的 Cry1Ac 受体。通过免疫组化方法在原位观察了 Bt 芽孢与中肠上皮的相互作用，并通过分析刷状缘膜囊泡（BBMV）样本中的总氨基肽酶活性，发现受挑战的个体中活性高于对照组。在 A. gemmatalis 的转录组中发现了十个 APN 序列，并对其进行了多种计算机模拟分析，包括系统发育树、多序列比对以及信号肽、活性域和GPI锚定位点的识别。第五龄幼虫的 BBMV 蛋白通过使用活化的 Cry1Ac 毒素和商业抗 Cry 多克隆抗体进行配体印迹分析；从 SDS-PAGE 凝胶中切取与 Cry 毒素结合的蛋白质条带，并进行质谱分析，最终鉴定出七个 APN。通过定量PCR比较了受到 Bt 芽孢亚致死感染个体与对照个体的表达水平，结果显示在 Bt 感染后出现上调和下调。据此，我们可以推断 A. gemmatalis 中的氨基肽酶N可能在其幼虫阶段参与了 Cry 毒素的作用机制。