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Introduction
Protocol
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생화학

评估农药对孤独蜜蜂幼虫的影响

Published: October 15, 2021
doi:
10.3791/62946
, , , ,
1Institute of Plant Protection,Shandong Academy of Agricultural Sciences, 2College of Plant Protection,Shandong Agricultural University, 3State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology,Chinese Academy of Sciences

Summary

本议定书解释了一种将受农药污染的食品喂给孤独蜜蜂 Osmia excavata的方法。该程序检查杀虫剂对孤独蜜蜂幼虫的生态毒性。

Abstract

目前对授粉媒介杀虫剂的生态风险评估主要只考虑了实验室条件。对于独居蜜蜂的幼虫，从人口学角度来看，摄入受杀虫剂污染的幼虫可能会增加幼虫的死亡率，降低收集率和下一年成年独居蜜蜂的数量。但是关于杀虫剂对孤独蜜蜂幼虫的影响的研究有限。因此，了解农药如何影响独居蜂的幼虫应被视为农药生态风险评估的一个组成部分。本研究提出了一种将独生蜜蜂 Osmia excavata 的幼虫暴露于致死或亚致死剂量的杀虫剂的方法，跟踪幼虫体重增加，发育持续时间，闭合能力和摄入食物的食物消耗效率转换。为了证明该方法的有效性，向 挖掘螈虫 幼虫喂食含有急性致死性和亚致死剂量的毒死蜱。然后，研究了处理过的幼虫的上述指标。该技术有助于预测和减轻杀虫剂对授粉媒介的风险。

Introduction

传粉媒介在现代全球农业的生态系统服务中发挥着关键作用。而蜜蜂（Apis mellifera;膜翅目：Apidae）传统上被认为是作物的基本经济授粉媒介，最近的研究表明， Osmia （膜翅目：Megachilidae）在改善某些作物的授粉，增加果实大小和种子数量以及减少世界不同地区商业果园中不对称果实的比例方面也非常重要1。 Osmia excavata 被认为是苹果授粉的理想物种，主要在亚洲，如中国北部和西北部以及日本2，3，4。它可以为某些具有相似或有时效率更高的作物提供授粉服务。在这方面，它们已被证明可以取代蜜蜂或与蜜蜂4，5，6协同作用。

与群居蜜蜂相比， 挖掘狸藻 的生物学特征是独一无二的。其单伏，孤独和筑巢活动主要发生在春季和初夏。 挖掘 草的巢通常存在于预先存在的孔中，通常在自然条件下的枯木，空心植物，稻草管和竹茎中3。成年 的挖掘鸟 从茧中出来交配，收集花粉，并筑巢产卵，一周后开始孵化。受精卵发育成雌性，而未受精卵发育成雄性3。雌性分布在蜂管的底部，相应的规定更为显著。相比之下，雄性在管出口附近，次要规定7，所以雄性先出来，雌性出来后。雌性将花粉与少量花蜜混合成一个湿润的斑点，这是细胞8中每个幼虫的唯一食物来源。

几项研究报告说，授粉昆虫的数量减少了9，10。农药的广泛使用已被确定为减少授粉媒介丰度和多样性的主要因素之一，也可能危及授粉服务11，12。为了减少和减轻农药的不利影响，有必要对授粉媒介进行农药风险评估。一些国家已经建立了监管框架，以确保蜜蜂安全免受农药使用的13，14。最近的研究表明， Osmia 比蜜蜂更容易受到杀虫剂的影响1，15。

有趣的是，大多数风险评估都集中在成年蜜蜂11，12;对 挖掘狸藻，特别是幼虫的研究很少。此外，由杀虫剂直接引起的 Osmia 的死亡率最常被认为是16。尽管如此，诸如幼虫体重增加，发育持续时间，进食模式，诱发能力，随后的成年行为和繁殖力等慢性毒性可能与急性致死毒性具有相同的危害，并且由于缺乏针对孤独蜜蜂的有效实验方法而经常被忽视17。

到目前为止，采用两种方法评估农药对独居蜂幼虫的影响：（1）在规定的局部部位施用适量农药而不去除独居蜂卵1，18，19，20;（2）用含有特定量农药的人工花粉-花蜜混合物代替规定21。但是，上述两种方法存在一些限制。前者只能测量急性毒性，但不能测量慢性毒性，因为幼虫在短时间内摄入了整个剂量;后者将导致高死亡率，因为人为操作1。本文通过模拟真实环境中以残留农药为食的幼虫行为，描述了浸泡法，研究了在高度受控的研究条件下农药对漏斗草的生态毒性。本研究的方法解决了上述两种方法的缺点，适用于测量有害物质对急性和慢性毒性的影响。

Protocol

1. 喂料管的准备 使用电绕组铁在 2 mL 离心管的盖子上打孔（直径约 0.3 mm）（参见 材料表）。使用这种离心管来维持 挖掘芽孢 杆菌幼虫及其供应质量。 2. 农药的制备 将工业级农药（见 材料表）溶解在丙酮中，以获得1 x 104μg 的储备溶液。然后，对溶液进行梯度稀释至五种以上的浓…

Representative Results

规定中常用农药、毒死蜱、吡虫啉、芬地非脲、磷、阿维菌素含量均小于对照组定量限量（0.01~0.02 mg kg-1）;这些结果排除了农药残留对每次处理的影响。评估对照组48 h后有或未从供应中去除幼虫的死亡率;结果显示没有显着差异（表1），表明存在轻微的人为错误。 在急性致死毒性试验（表2）中，规定浸泡在七种稀释的农药溶液（0.1、0.2、0.4?…

Discussion

对于成年传粉媒介，有两种主要方法可以测量农药的生态毒性。一种是接触法，将农药施用于成虫的原胸;另一种是胃毒性法，其中用含有农药25，26的蜂蜜水喂养成年传粉者。近年来，人们发现挖掘蜉的授粉效果和宸死率相对较低27. 据推测，施用农药对幼虫生长发育的影响是主要原因之一。然而，关于农药对挖掘芽孢杆?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

本研究由国家重点研发计划（2017YFD0200400）、重大科技创新项目（2017CXGC0214）、山东省蜜蜂产业创新团队、山东省农业科学院农业科技创新项目（CXGC2019G01）和山东省农业科学院农业科技创新项目（CXGC2021B13）资助。

Materials

Abamectin Jinan Lvba Pesticide Co. Ltd
Black-light lamps Kanghua Medical Device Co., Ltd
Centrifugal tube box with 100 Wells Shanghai Rebus Network Technology Co., Ltd
Centrifuge tube Shanghai Rebus Network Technology Co., Ltd 2 mL;  Serve as bee tube
Electric soldering iron Kunshan Kaipai Hardware Electromechanical Co., Ltd
Electronic scale Sartorius Scientific Instruments (Beijing) Co., Ltd 3137510295
Graduated cylinder Anhui Weiss Experimental Equipment Co. Ltd
Petri dishes (60 mm diameter) Qingdao jindian biochemical equipment co., LTD
Pollen provision Yantai Bifeng Agricultural Science and Technology Co. Ltd
Soft brush Wengang Wenhai painting material factory
Solitary bees Yantai Bifeng Agricultural Science and Technology Co. Ltd
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Tags

Keywords: PesticidesSolitary BeesOsmia ExcavataLarvaFeeding TubePesticide TreatmentAcute Lethal ToxicitySublethal ToxicityMass RearingProvisions Consumption
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Song, Y., Li, R., Li, L., Ouyang, F., Men, X. Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees. J. Vis. Exp. (176), e62946, doi:10.3791/62946 (2021).
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