关键词: 中华蜜蜂, 亚致死剂量, 吡虫啉, RNA-seq, 实时荧光定量PCR

Abstract:

【Background】The target of neonicotinoid insecticides is the acetylcholine receptor in the nervous system of insects. Due to its good systemicity and low toxicity to humans and animals, it has been widely used in agricultural production. Thus there is still a low residue in plants, and this sublethal dose residue can still cause adverse effects on the behavior and nervous system of flower-visiting insects like bees.【Objective】The objective of this study is to clarify the effect of sublethal dose imidacloprid (a kind of neonicotinoid insecticides) on the nervous physiological and metabolism system of Apis cerana cerana.【Method】In this study, the worker bees were treated with two sublethal concentration gradient doses of 5 and 10 μg·L^-1 imidacloprid for 10 days (three biological replicates). After total RNA was extracted, the resulting library was analyzed by RNA-seq. Throughput sequencing, and the bioinformatics technology was used to assemble and annotate the sequence de novo, and the differentially expressed genes (DEGs) after sublethal doses imidacloprid treatment were analyzed by clustering and enrichment. Finally, real-time fluorescence quantitative PCR (RT-qPCR) technology was used to verify the DEGs related to the nervous and metabolic systems.【Result】A total of 9 sequencing libraries were obtained, the ratio of effective sequencing data exceeded 94.45%. From the obtained 37 364 unigenes, 571 DEGs were identified. The enrichment analysis of GO and KEGG found that the DEGs were mainly related to multiple pathways such as protein translation, redox, oxidative phosphorylation, and ribosome, indicating that sublethal doses of imidacloprid had an impact on multiple physiological processes and metabolic pathways of A. c. cerana. Nine DEGs (e.g. neuropeptide F (NPF), neuropeptide SIFamide receptor (SIFaR), phosphoinositide 3-dependent kinases (PDK1), A-kinase anchoring protein 1 (AKAP1), carbonic anhydrase 3 (CA3), superoxide dismutase 2 (SOD2), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10 (ND42), cuticle protein 12 (CP12), and odorant-binding protein (OBP17)) related to nerve signal transmission and metabolic function were selected for RT-qPCR verification. Their expression patterns were completely consistent with the transcriptome results.【Conclusion】The sublethal dose of imidacloprid can affect lots of aspects of A. c. cerana such as nerve signal transduction, cellular respiration, immune response, maintenance of homeostasis, and olfactory perception.

Key words: Apis cerana cerana, sublethal dose, imidacloprid, RNA-seq, RT-qPCR