Table2_Cry1Ac toxin binding in the velvetbean caterpillar Anticarsia gemmatalis: study of midgut aminopeptidases N.XLSX

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.

巴西大豆的重要食叶害虫之一为绒毛豆毛虫Anticarsia gemmatalis。当前，全球用于控制害虫的主要生物农药为苏云金杆菌（Bacillus thuringiensis，简称Bt），该细菌可产生对害虫有致病作用的晶体毒素（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，可能参与抗药性的发展；在A. gemmatalis中肠中鉴定功能性的Cry受体对于开发克服这种潜在情况的有效策略至关重要。本研究的目标是对A. gemmatalis的APNs进行特征化，并识别中肠中的Cry1Ac受体。通过免疫组化和刷状缘膜囊（BBMV）样品中的总氨基肽酶活性测定，在原位观察了Bt孢子与中肠上皮的相互作用，结果显示受挑战个体中的活性高于对照组。在A. gemmatalis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，如系统发育树、多序列比对以及信号肽、活性域和GPI-锚定信号的识别。第五龄幼虫的BBMV蛋白被用于结合激活的Cry1Ac毒素和商业抗Cry多克隆抗体的配体印迹分析；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，从而鉴定出七个APN。通过定量PCR比较了接受Bt孢子亚致死感染个体与对照个体之间的表达水平，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可能在幼虫阶段Cry毒素的作用机制中发挥作用。