Study On Molecular Mechanism Of Effects Of A Sublethal Dose Of Imidacloprid On Learning Behavior In Apis Mellifera L.

Honeybees not only promote the development of agricultural production through pollination services,but also play an important role in maintaining the balance of ecosystems.In recent years,however,the number of honeybees has decreased under the stress of biological and abiotic factors,and it has attracted global attention.Among them,the widespread use of neonicotinoid insecticides is considered to be one of the factors contributing to the decrease in the number of honeybees.Imidaclorprid is the most widely used pesticide among neonicotinoid pesticides and acts on the brain's nicotinic acetylcholine receptor of honeybees.It can cause olfactory learning deficits in honeybees.In this study,the relevant functional genes were screened by the transcriptome analysis,and the temporal and spatial expression of functional genes was analyzed by qRT-PCR.At the same time,aiming at the regulation of mrjp1 on learning and memory behavior of honeybees,RNAi technique was used to verify the gene function.The effect of sublethal dose of imidacloprid on learning behavior of bees was analyzed for the first time from the point of view of molecular mechanism.1.In the present study,we first selected the dose of imidacloprid(20ppb)that honeybees may be exposed to in the realistic foraging environment and studied the effects of sublethal dose of imidacloprid onhoneybees? learning behavior at the same age.After exposure to 20 ppb imidacloprid for 11 days,honeybees were used for the olfactory associative learning experiments with learning equipment.The results showed that although 20 ppb imidacloprid had no lethal toxicity to honeybees,it significantly decreased the proboscis extension reflex rate in the second and third learning(P< 0.01),that is,the learning ability of honeybees was decreased after being stressed by imidacloprid.2.The brains of imidacloprid-treated and control bees were used for comparative transcriptome analysis by RNA-Seq to elucidate the effects of imidacloprid on honey bee learning capacity.Gene expression profiles between imidacloprid treatment and the control revealed that 131 genes were differentially expressed(padj<0.05),of which 130 were downregulated in imidacloprid–treated honeybees.Downregulation of brain genes involved in immune,detoxification and antioxidant response genes.Gene ontology annotation indicated that the oxidation-reduction imbalance might exist in the brain of honey bees due to oxidative stress induced by long-term exposure to imidacloprid.KEGG revealed that transient receptor potential and Arrestin 2 in the phototransduction pathway were significantly downregulated in imidacloprid-treated honeybees,and ingenuity pathway analysis(IPA)revealed that five downregulated genes may also be involved in the regulation of honeybees olfactory learning behavior.3.Using qRT-PCR to analyze the temporal and spatial expression of functional genes,it was found that the results of qRT-PCR and RNA-Seq had high consistency,and the results of sequencing were proved to be reliable.Among them,20 genes were significantly down-regulated in the treatment group(P < 0.05 or P< 0.01).In this chapter,the experimental results are further suggest that the sublethal dose of imidacloprid decrease the expression of genes related to immune detoxification and chemosensors in the brain of honeybees and result in poor olfactory learning ability of honeybees under imidacloprid stress.4.The RNA-Seq data indicated that major royal jelly protein1(mrjp1)was significantly down-regulated in the honeybee's brain.mrjp1 was distributed in the antennal leaf,visual leaf and mushroom body of the honeybees.The mrjp1 gene specific silence by the RNAi technology to further verify that mrjp1 plays a key role in the olfactory learning behavior of honeybees.There was significant difference between ds-mrjp1 group and ds-egfp group of learning behavior(P< 0.05).The learning ability of ds-mrjp1 group was lower than that in ds-egfp group.However,there was no significant difference in 2 h memory between the two groups(P> 0.05).The results of qRT-PCR also showed that the expression level of mrjp1 in the experiment group with decreased learning ability was also decreased(P< 0.05),which further indicated that mrjp1 gene regulated the learning behavior of honeybees.