【作者】 王强；

【导师】 蔡青年；

【作者基本信息】 中国农业大学 ， 农业昆虫与害虫防治， 2016， 博士

【摘要】 蜜蜂是一种重要的经济昆虫,在农业中通过帮助植物传花授粉,从而大大提高农产品的产量和品质。但随着设施农业的发展,农业生态环境中杀虫剂等的大量施用对蜜蜂造成的毒害越来越引起人们的重视。为更好的保护蜜蜂为农作物及自然界植物授粉,有必要研究环境有害物质对蜜蜂的毒性作用机理并建立一套更为灵敏准确的毒性风险评价技术。本项研究从几种主要杀虫剂在亚致死剂量下对蜜蜂生物学的影响入手,研究了其对蜂群繁殖的影响。同时以溴氰菊酯(DM)为素材研究了对意大利蜜蜂工蜂脑神经细胞内游离钙离子浓度([Ca2+]i)的影响规律以及对钙通道的作用进行了研究,通过实验得到了如下结果：1、亚致死剂量下,联苯菊酯、溴氰菊酯及氟氨氰菊酯均极限著地降低了蜂王产卵量；联苯菊酯与溴氰菊酯极显著地降低了意大利蜜蜂封盖率和发育成功率并可极显著的延长工蜂由卵发育至成蜂的历期。上述研究结果表明这三种杀虫剂在亚致死剂量下仍会显著影响蜜蜂蜂群的正常繁殖。2、建立了针对蜜蜂脑神经细胞体外培养及钙离子浓度测量技术的一套较完整的方法。该项测量技术的建立将有助于形成一套以细胞[Ca2+]i变化为衡量指标的细胞水平的环境有毒物质对蜜蜂风险的更为迅速而准确的评价系统,从而改变目前常用的利用蜜蜂生物学方法进行毒性评价的手段。3、实验明确了DM对蜜蜂脑神经细胞[Ca2+]i的影响规律：(1)即使极低浓度的DM仍会迅速并极显著地影响细胞内[Ca2+]i;(2)伴随着时间推移,不同浓度DM处理组间出现显著差异：当DM浓度>0.25 mg/L时,即使后期去除DM的影响,[Ca2+]i仍维持在高位,说明由于DM浓度过高已造成细胞Ca2+的过载而死亡；当DM浓度<0.25 mg/L时,去除DM的影响,[Ca2+]i能够逐渐恢复,说明细胞仍处于存活状态,具有一定的恢复功能；(3)将DM浓度降低至0.03 mg/L,神经细胞恢复后与正常细胞的[Ca2+]i的值相比仍存在极显著差异,这说明低浓度的DM虽不能造成细胞的中毒致死,但仍对细胞造成了损害并进而可能对蜜蜂神经及机体的正常生理活动造成影响。4、通过实验明确了DM刺激造成蜜蜂神经细胞[Ca2+]i升高的主要靶位是位于细胞膜上的电压依赖型T-型钙离子通道蛋白,DM将特异性的开启T-型钙离子通道,使得胞外Ca2+大量进入胞内,从而造成胞内[Ca2+]i的迅速升高。5、通过亚致死剂量下DM饲喂意大利蜜蜂工蜂不同时间后脑神经细胞T-型钙离子通道蛋白α-1H亚基mRNA表达差异的研究表明,亚致死剂量的溴氰菊酯会显著降低蜜蜂脑神经细胞T_型钙离子通道a-1H亚基基因的表达。更多还原

【Abstract】 Honeybee is an important economic insect that can help agricultural plant in pollinating, so that yield and quality of agricultural products can be significantly increased. However, along with development of facility agriculture, people pay more and more attention to the harm on honeybee caused by large-scale use of pesticide etc in agro- ecological environment. In order to protect bees to pollinate for crops and natural plants, it’s necessary to research toxicity mechanism of harmful environmental substances on bees and establish a set of flexible and accurate risk assessment technology. The research starts from influences of several main pesticides on biology of bee with dosage of sub-lethal damage and research on the influence on reproduction of bee group. At the meanwhile, influence rule as well as influence mechanism of sub-lethal dosage of DM on free calcium ion concentrate ([Ca2+]i) inside brain nerve cell of honeybee (Apis mellifera ligustica Spinola) developed in vitro, and following results were achieved through the test:1. With sub-lethal dosage, bifenthrin, deltamethrin and Tau-fluvalinate shall obviously reduce egg amount laid by queen bee; bifenthrin and deltamethrin shall obviously reduce capping rate and successful developmental rate of honeybee(Apis mellifera ligustica Spinola) and obviously extend the period for worker bee to grow from egg to adult bee. Above research result shows that the three kinds of pesticides shall obviously influence normal reproduction of bee group under sub-lethal dosage.2. A complete set of method is established for culture honeybee brain nerve cell in vitro and for calcium ion measuring technique. Establishment of the measuring technique shall be good for creating a set of fast and accurate system to evaluate risks of environmental toxic substances on honeybee by taking cell [Ca2+]i as measurement index, so as to change present method to evaluate toxicity by biological significance of honeybee.3. The test has clarified influence rule of DM on brain nerve cell of bee [Ca2+]i:(1) Even DM with low concentration shall rapidly and obviously impact [Ca2+]i inside cell; (2) As time goes by, there are obvious differences among different tests with different DM concentration:when DM concentration>0.25 mg/L, [Ca2+]i shall still maintain high though influence of DM is removed then. It shows that high DM concentration cause death of cell due to overload of Ca2+; when DM concentration is<0.25 mg/L, [Ca2+]i shall gradually recover without influence of DM. It shows that cell is still alive and has recovery function; (3) Compared with normal cell, final recovery rate of cell [Ca2+]i has obvious difference when DM concentration is reduced to 0.03125 mg/L, it shows that DM with lowest concentration shall cause irreversible influences on cell and exert influences on normal physiological activity of bee’s nerve and organism.4. The test has clarified that major target position for nerve cell [Ca2+]i increasing of bee(Apis mellifera ligustica Spinola) caused by DM stimulation is the voltage-dependent defect T-type of calcium ion passage protein, and DM can specifically open T-type of calcium ion passage, so that Ca2+ outside the cell shall enter into the cell and cause fast increase of [Ca2+]i inside cell.5. After feed worker bee (Apis mellifera ligustica Spinola) with sub-lethal dosage of DM in different duration, to find whether mRNA expression of T-type of calcium ion passage protein a-1H subunit are different. The research shows that deltamethrin with sub-lethal dosage shall obviously cause different for mRNA expression of T-type of calcium iron passage protein.更多还原