Transcriptomic And Metabolomic Analysis Of Honeybees (Apis Mellifera Ligustica) Response To Neonicotinoid Insecticides

Honeybees have a high social and economic value since they not only produce various bee products,but also play important roles in pollination services of crops and wild plants and maintaining ecological diversity.In recent years,the constant decline of bee populations has attracted the attention of the world.Despite the specific reasons for the current decline in bee populations still being unclear,many studies have shown that the heavy use of insecticides in agriculture may be one of the most significant contributing factors causing this phenomenon.Neonicotinoid insecticides is one of the most widely used insecticides for pest control in nectariferous plant.Nowdays numerous studies have assessed and explored the harm of neonicotinoids on honeybee survival,physiology,and behaviour development.Despite this,few studies have been carried out on the molecular effects of neonicotinoids.In this study,two commonly used neonicotinoids,thiamethoxam and thiacloprid,were used as the insecticides against the Apis mellifera ligustica.Based on the optimal sublethal doses of thiamethoxam and thiacloprid,the response of honeybees to neonicotinoids were explored at transcription and metabolic levels,which can expand the understanding of the molecular basis of the complex interactions between neonicotinoids and honeybees.The results were obtained as follows:?1?Using high-throughput RNA-seq,the effects of thiamethoxam?0.01 mg/L?onhoneybee transcriptome profiles were studied.In addition,ds RNA-mediated RNA interference?RNAi?technology was used to assess the susceptibility of honeybees to thiamethoxam after silencing the expression of cytochrome P450 gene Cyp6as5.RNA-seq results showed that 609 genes were significantly differentially expressed in honeybees upon exposure to thiamethoxam?Q<0.05?,of which 225 were up-regulated and 384 were down-regulated.Ten selected genes were further investigated by q RT-PCR,and the results were consistent with those of RNA-seq.GO enrichment analysis showed that the enriched DEGs were mainly linked to metabolism,biosynthesis and translation.KEGG pathway analysis showed that thiamethoxam affected biological processes including ribosomes,the oxidative phosphorylation pathway,tyrosine metabolism pathway,pentose and glucuronate interconversions?Q<0.05?.Furthermore,results of RNAi combination with bioassay showed that inhibition of Cyp6as5 significantly increased the mortality rate of honeybees upon thiamethoxam treatment,which suggest the significant role of Cyp6as5 in detoxification of thiamethoxam.?2?Influence of thiamethoxam?0.01 mg/L?on mi RNAs expression in the honeybees was explored by s RNA-seq.The results showed that exposure to thiamethoxam significantly affected the expression of seven mature known mi RNAs?Q<0.01,|log₂?fold change?|>1?.Among these,ame-mi R-124,ame-mi R-971,amemi R-981,and ame-mi R-6038 were up-regulated,while ame-mi R-279b,ame-mi R-3791,and ame-mi R-6051 were inhibited.Expression of these seven mi RNAs was further investigated by q RT-PCR,and the results were almost the same as RNA-seq.Some predicted target genes of the differential mi RNAs were related to behavior,immunity,and neural function.GO enrichment analysis showed that the predicted targets were mainly linked to cellular structure and regulation of cellular process,while 111 KEGG pathways were obtained,endocytosis pathway was the most highly enriched.?3?Using LC-MS-based combined with GC-MS-based non-target metabolomic approaches,the metabolic changes that occur in the heads of honeybee after exposure to thiacloprid?2 mg/L?were profiled.The results showed that there were 115 metabolites significantly changed upon exposure to thiacloprid?VIP>1,P<0.05?.Among these,more than 70%?83?of differential metabolites were up-regulated.Functional analysis of differential metabolites suggests that thiacloprid may hinder the brain neural activity implicated in learning and behaviour development,while honeybees might protect against thiacloprid stress by speeding up their metabolism and activating glutathione metabolism.