Table1_Four MicroRNAs, miR-13b-3p, miR-278-5p, miR-10483-5p, and miR-10485-5p, Mediate Insecticide Tolerance in Spodoptera frugiperda.XLSX

Spodoptera frugiperda is the world’s major agricultural pest and has the distinctive features of high fecundity, strong migratory capacity, and high resistance to most insecticides. At present, the control of S. frugiperda in China relies mainly on the spraying of chemical insecticides. MicroRNAs (miRNAs) are a class of small, single-stranded, non-coding RNAs and play crucial regulatory roles in various physiological processes, including the insecticide resistance in insects. However, little is known about the regulatory roles of miRNAs on the resistance of S. frugiperda to insecticides. In the present research, the miRNAs that were differentially expressed after cyantraniliprole, spinetoram, and emamectin benzoate treatment were analyzed by RNA-Seq. A total of 504 miRNAs were systematically identified from S. frugiperda, and 24, 22, and 31 miRNAs were differentially expressed after treatments of cyantraniliprole, spinetoram, and emamectin benzoate. GO and KEGG enrichment analyses were used to predict the function of differentially expressed target genes of miRNAs. Importantly, ten miRNAs were significantly differentially expressed among the treatments of three insecticides. miR-278-5p, miR-13b-3p, miR-10485-5p, and miR-10483-5p were significantly downregulated among the treatments of three insecticides by RT-qPCR. Furthermore, the overexpression of miR-278-5p, miR-13b-3p, miR-10485-5p, and miR-10483-5p significantly increased the mortality of S. frugiperda to cyantraniliprole and emamectin benzoate. The mortality was significantly increased with spinetoram treatment after the overexpression of miR-13b-3p, miR-10485-5p, and miR-10483-5p. These results suggest that miRNAs, which are differentially expressed in response to insecticides, may play a key regulatory role in the insecticide tolerance in S. frugiperda.

草地贪夜蛾（Spodoptera frugiperda）是全球范围内的重大农业害虫，其典型特征为繁殖力强、迁飞能力突出，且对多数杀虫剂具备高水平抗性。目前，我国针对草地贪夜蛾的防治主要依赖化学杀虫剂喷施。微小RNA（microRNAs, miRNAs）是一类小型单链非编码RNA，在昆虫的多种生理过程中发挥关键调控作用，其中包括对昆虫杀虫剂抗性的调控。然而，目前关于miRNAs对草地贪夜蛾杀虫剂抗性的调控作用，相关研究仍较为匮乏。本研究利用RNA测序（RNA-Seq）技术，分析了草地贪夜蛾经氰氟虫胺（cyantraniliprole）、多杀霉素（spinetoram）及甲氨基阿维菌素苯甲酸盐（emamectin benzoate）处理后，体内差异表达的miRNAs。研究共从草地贪夜蛾中系统鉴定出504个miRNAs，经氰氟虫胺、多杀霉素、甲氨基阿维菌素苯甲酸盐处理后，分别有24、22及31个miRNAs呈现显著差异表达。通过基因本体（Gene Ontology, GO）富集分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析，对差异表达miRNAs的靶基因功能进行了预测。尤为关键的是，在三种杀虫剂的处理组间，共有10个miRNAs呈现显著差异表达。经实时荧光定量PCR（Reverse Transcription Quantitative Polymerase Chain Reaction, RT-qPCR）验证，miR-278-5p、miR-13b-3p、miR-10485-5p及miR-10483-5p在三种杀虫剂处理后均显著下调。进一步功能验证结果显示，过表达上述4个miRNAs可显著提升草地贪夜蛾对氰氟虫胺与甲氨基阿维菌素苯甲酸盐的死亡率；而过表达miR-13b-3p、miR-10485-5p及miR-10483-5p后，草地贪夜蛾经多杀霉素处理后的死亡率亦显著升高。上述研究结果表明，响应杀虫剂胁迫而发生差异表达的miRNAs，可能在草地贪夜蛾的杀虫剂耐受性调控中发挥关键作用。