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【目的】蜜蜂是重要的授粉昆虫,但一直受到新烟碱类杀虫剂(如吡虫啉)的危害,该类杀虫剂主要作用于蜜蜂脑神经细胞。本研究旨在明确吡虫啉对意大利蜜蜂Apis mellifera ligustica脑神经细胞致凋亡作用。【方法】利用亚致死剂量吡虫啉饲喂成年意大利蜜蜂,通过原位末端标记法(TUNEL)检测脑神经细胞的凋亡情况,利用免疫荧光法检测Caspase-1激活情况,通过透射电镜观察脑神经细胞的超微结构。【结果】在对选取的脑部7个部位进行总体观测后,摄入亚致死剂量的吡虫啉(9.90 ng/蜜蜂)诱导成年意蜂工蜂脑神经细胞凋亡率随时间递增而增加,在处理9 d和12 d时,处理组与空白对照组差异极显著(P<0.01);Caspase-1阳性细胞率也随时间递增而增加,在处理3,6,9和12 d时,处理组与空白对照组差异极显著(P<0.01)。凋亡率和Caspase-1阳性细胞率均表现出时间效应,且两者成正相关性。超微结构表明凋亡的神经细胞呈现凋亡和自噬双重特征,包括:细胞固缩、染色质浓缩、凋亡小体和自噬体出现;线粒体肿胀,有的被自噬泡包裹后进行线粒体自噬。【结论】本研究从细胞水平证实了亚致死剂量吡虫啉对成年意蜂工蜂脑神经细胞具有致凋亡作用,并且其凋亡途径与Caspase-1和细胞自噬有关,这为利用细胞凋亡法评价杀虫剂对非靶标生物的神经毒性提供一定的理论基础。
【Aim】 Bees are important pollinators, but they are always endangered by neonicotinoid insecticides such as imidacloprid, which mainly act on bee brain neurons. The aim of this study was to determine the effect of imidacloprid on the apoptosis of neurons in Apis mellifera ligustica. 【Method】 Adult Italian bees were fed on a sublethal dose of imidacloprid. The apoptosis of neurons was detected by TUNEL. The activation of Caspase-1 was detected by immunofluorescence. Ultrastructure. 【Results】 The results showed that the apoptotic rate of bee berry neurons increased with the increase of time after ingesting sublethal doses of 9.90 ng / At 12 d and 12 d, the difference between the treatment group and the blank control group was significant (P <0.01), and the positive rate of Caspase-1 positive cells increased with time. At 3, 6, 9 and 12 d, The difference between the control group was significant (P <0.01). Apoptosis rate and Caspase-1 positive cell rate showed time effect, and the two were positively correlated. The ultrastructure shows that apoptotic neurons present dual characteristics of apoptosis and autophagy, including cell shrinkage, chromatin condensation, appearance of apoptotic bodies and autophagosomes, swelling of mitochondria, and some of them are carried by autophagic vesicles Mitochondrial autophagy. 【Conclusion】 The present study demonstrated that sub-lethal doses of imidacloprid have apoptosis-inducing effect on adult bee-cultured bee neurons at the cellular level, and its apoptotic pathway is related to the expression of Caspase-1 and autophagy. Evaluation of insecticides on non-target organisms neurotoxicity provides a theoretical basis.