亚致死剂量吡虫啉胁迫对意大利蜜蜂内勤蜂与外勤蜂免疫解毒相关基因表达及免疫解毒酶系活力的影响

Effects of sublethal doses of imidacloprid on the expression of immune and detoxification related genes and the activities of immune and detoxification related enzymes in house and field bees of Apis mellifera ligustica

【目的】吡虫啉(imidaclorprid)是广泛使用的新烟碱类杀虫剂之一。大量研究表明亚致死剂量吡虫啉影响意大利蜜蜂Apis mellifera ligustica(简称“意蜂”)幼虫的发育和成年蜜蜂的采集、学习等行为。本实验旨在探究亚致死剂量吡虫啉对意大利蜜蜂内勤蜂(1日龄成年工蜂)与外勤蜂(21日龄成年工蜂)免疫解毒相关基因表达及免疫解毒酶系活力的影响,进而为蜜蜂健康的维护提供科学依据。【方法】测定饲喂含0.1 ng/μL吡虫啉的50%蔗糖溶液不同时间后意蜂成年工蜂的存活率;利用荧光定量PCR检测饲喂含0.1 ng/μL吡虫啉的50%蔗糖溶液6 d后其体内免疫基因多酚氧化酶基因(PPOA3,GenBank登录号:GB43738),Abaecin类抗菌肽基因(ABA,GenBank登录号:GB18323),葡萄糖脱氢酶基因(GLD,GenBank登录号:GB43007)和解毒基因细胞色素P450基因(CYP4506a2,GenBank登录号:GB49876)的表达,并采用双抗体一步夹心法酶联免疫吸附试验测定其体内细胞色素P450酶(cytochrome P450,CYP450)含量和多酚氧化酶(polyphenol oxidase,PPO)活力。【结果】1日龄和21日龄意蜂成年工蜂连续饲喂6 d含0.1 ng/μL吡虫啉的50%蔗糖溶液后,其存活率与对照组(饲喂含0.1 ng/μL丙酮的50%蔗糖溶液)无显著差异;连续饲喂9 d含0.1 ng/μL吡虫啉的50%蔗糖溶液后,1日龄意蜂成年工蜂存活率与对照组无显著差异,而21日龄意蜂成年工蜂存活率与对照组有显著差异。1日龄意蜂成年工蜂自由取食含0.1 ng/μL吡虫啉的蔗糖溶液6 d后,PPOA3,CYP4506a2,ABA和GLD表达水平,细胞色素P450含量以及多酚氧化酶活力与对照组相比均有显著下调趋势;而21日龄意蜂成年工蜂取食该药液6 d后,CYP4506a2,ABA和GLD表达水平及多酚氧化酶活力与对照组相比均有显著下调趋势,PPOA3表达水平和细胞色素P450含量有显著上调趋势。【结论】亚致死剂量吡虫啉影响意大利蜜蜂内勤蜂与外勤蜂免疫解毒相关基因的表达及免疫解毒酶系活力;吡虫啉短期胁迫对意大利蜜蜂内勤蜂与外勤蜂的存活无显著影响,长期胁迫则会影响意大利蜜蜂内勤蜂与外勤蜂的存活。

【Aim】Imidacloprid is one of the widely used neonicotinoid insecticides.Studies indicated that sublethal doses of imidacloprid affect the larval development of honeybees and the foraging and learning behaviors of adult honeybees.This study aims to investigate the effects of sublethal doses of imidacloprid on the expression of immune and detoxification related genes and the activities of immune and detoxification related enzymes in house bees(1 day-old adults)and field bees(21 day-old adults)of Apis mellifera ligustica,so as to provide a scientific basis for the maintenance of honeybee health.【Methods】The 1 and 21 day-old adults of A.m.ligustica were fed with 50%(w/v)sucrose solution containing 0.1 ng/μL of imidacloprid ad libitum,the survival rates at different time after treatment were determined.The expression levels of PPOA3(GenBank accession no.:GB43738),CYP4506a2(GenBank accession no.:GB49876),ABA(GenBank accession no.:GB18323),GLD(GenBank accession no.:GB43007)in the adult bees at 6 d after treatment were detected by real-time quantitative PCR,and the content of cytochrome P450(CYP450)and the activity of polyphenol oxidase(PPO)in them were determined by double antibody one-step sandwich enzyme-linked immunosorbent assay.【Results】After the 1 day-old and 21 day-old adult bees of A.m.ligustica were continuously fed with 50%sucrose solution containing 0.1 ng/μL imidacloprid for 6 d,their survival rates showed no significant difference from the control[fed with 50%(w/v)sucrose containing 0.1 ng/μL of acetone](P>0.05).After the 1 and 21 day-old adult bees of A.m.ligustica were continuously fed with 50%sucrose solution containing 0.1 ng/μL imidacloprid for 9 d,the survival rate of the 1 day-old adult bees showed no significant difference from the control,while the survival rate of the 21 day-old adult bees showed significant difference from the control.The expression levels of PPOA3,CYP4506a2,ABA and GLD,the CYP450 content and the PPO activity in 1 day-old adult bees exposed to 0.1 ng/μL imidacloprid for 6 d were significantly downregulated as compared to those in the control.The expression levels of CYP4506a2,ABA,and GLD and the PPO activity in 21 day-old adult bees exposed to 0.1 ng/μL imidacloprid for 6 d were significantly downregulated,while the expression levels of PPOA3 and the CYP450 content were significantly elevated as compared to those in the control group.【Conclusion】Sublethal doses of imidacloprid affect the expression of immune and detoxification related genes and the activities of immune and detoxification related enzymes in both house and field bees of A.m.ligustica.The short-term stress of imidacloprid has no significant effect on the survival of the house and field bees of A.m.ligustica,but long-term stress may affect their survival.