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Biologie

评估交配蜜蜂女王的农用化学品风险

Published: March 03, 2021
doi:
10.3791/62316
, , ,
1Invasive Species and Pollinator Health Research Unit,USDA-ARS, 2Carl R. Woese Institute for Genomic Biology,University of Illinois at Urbana-Champaign, 3Neuroscience Program,University of Illinois at Urbana-Champaign, 4Department of Entomology,University of Illinois at Urbana-Champaign

Summary

该协议的制定是为了加强对农用化学品如何影响蜜蜂（Apis mellifera）繁殖的理解，方法是建立方法，在受控的实验室环境中将蜜蜂及其工人看护人暴露于农用化学品中，并仔细监测它们的相关反应。

Abstract

目前对蜜蜂的风险评估策略在很大程度上依赖于对成年或未成熟的工蜂进行的实验室测试，但这些方法可能无法准确捕获农用化学品暴露对蜂王的影响。作为蜜蜂群落内受精卵的唯一生产者，蜂王可以说是一个正常运作的蜂群中最重要的单一成员。因此，了解农用化学品如何影响蜂王健康和生产力应被视为农药风险评估的一个关键方面。在这里，提出了一种适应性的方法，使蜜蜂蜂王和工蜂王服务员暴露于通过工人饮食施用的农用化学品应激源，然后在实验室中跟踪产卵量，并使用专门的笼子（称为蜂王监测笼）评估第一龄的关闭情况。为了说明该方法的预期用途，描述了一项实验的结果，其中工人女王服务员被喂食含有亚致死剂量的吡虫啉的饮食，并监测了对女王的影响。

Introduction

由于全球对农产品的需求增加，现代农业实践通常需要使用农用化学品来控制已知会降低或损害作物产量的众多害虫1。同时，许多水果，蔬菜和坚果作物的种植者依靠商业蜜蜂群落提供的授粉服务来确保丰收2。这些做法可能导致传粉媒介，包括蜜蜂（Apis mellifera），暴露于有害水平的农药残留3。同时，蜜蜂群落中广泛存在的寄生Varroa破坏螨虫侵扰经常需要养蜂人用杀螨剂处理其蜂巢，这也可能对蜂群的健康和寿命产生负面影响4，5，6。为了减少和减轻农用化学品的有害影响，有必要在实施之前全面评估其对蜜蜂的安全性，以便提出使用建议以保护有益昆虫。

目前，环境保护署（EPA）依靠对蜜蜂农药暴露的分层风险评估策略，其中包括对成年蜜蜂和有时对蜜蜂幼虫7进行实验室测试。如果较低级别的实验室检查无法减轻对毒性的担忧，则可能建议进行高级别的现场和半现场测试。虽然这些实验室测试为农用化学品对工人寿命的潜在影响提供了有价值的见解，但它们并不一定预测它们对女王的影响，这与工人生物学8和行为9显着不同。此外，农用化学品对昆虫有许多潜在的影响，除了死亡之外，这可能对依靠协调行为作为殖民地单位10，11的社会昆虫产生相当大的影响。

虽然死亡率是农用化学品最普遍考虑的影响12，但这些产品可以对目标和非目标节肢动物产生广泛的影响，包括改变行为13，14，15，16，排斥性或吸引性17，18，19，进食模式的变化20，21，22和增加或减少的生育能力20，21，22，23，24，25。对于群居昆虫来说，这些影响会系统性地破坏群体的相互作用和功能11。在这些功能中，严重依赖由殖民地单元9的其余部分支持的单个产卵女王的繁殖可能特别容易受到农药暴露的干扰。

对未成熟的蚁后进行的研究表明，发育暴露于杀螨剂会影响成年蚁后的行为，生理学，存活率26，27。类似地，使用全尺寸或缩小尺寸的菌落的研究表明，农用化学品可以通过减少交配成功率28，减少产卵29和降低产生的卵的生存能力来影响成年蜂王25，30，31。以前，如果不使用整个菌落，这些现象很难观察到，这主要是由于缺乏可用的实验室方法。然而，最近已经采用了一种使用女王监测笼（QMC）32在严格控制的实验室条件下研究女王产卵的方法，以检查农用化学品对女王生育能力的影响33。在这里，详细介绍了这些技术以及测量和跟踪QMC中工人饮食消耗的其他方法。

这些方法比需要全尺寸菌落的实验更有利，因为它们允许向大大减少的工人数量施用精确剂量的农用化学品，相对于通常存在于蚁群内的数万人，然后蚁后提供蚁后。这种曝光技术反映了女王在现实世界中会经历的二手曝光，因为在蚁群中，蚁后不会自己进食，而是依靠工人为它们提供饮食9。同样，蚁后通常不会离开蜂巢，除非在蜂群繁殖（蜂群）期间交配飞行35。交配的蜂王可以从商业蜂王种购买并在一夜之间发货。通常，女王育种者在确认它们已经开始产卵后直接出售女王，这被视为成功交配的标志。如果需要有关女王年龄或亲缘关系的更精确信息，研究人员可以在下订单之前咨询女王育种者。

QMC可以精确观察和量化蜂王的产卵和卵孵化率32，33，产生与农用化学品暴露对蜂王繁殖力的影响相关的宝贵数据。这里提出的代表性结果描述了在长期暴露于全身性神经毒性剂新烟碱类杀虫剂吡虫啉36的现场相关浓度下量化QMC的产卵，饮食消耗和胚胎活力的实验。一旦施用，吡虫啉会转移到植物组织37，并且已经检测到许多蜜蜂授粉植物的花粉和花蜜38，39，40的残留物。暴露于吡虫啉会对蜜蜂产生广泛的不利影响，包括觅食性能受损16，免疫功能受损41，以及群体扩张和存活率下降42，43。在这里，选择吡虫啉作为测试物质，因为田间实验表明它可以影响蜂王产卵29

Protocol

1. QMC组装 从部件（图1A）组装QMC，插入一个产卵板（ELP），如图 1B所示。在将工人添加到笼子之前，不要添加喂料管。用实验室级胶带暂时覆盖 4 个进纸器孔。 将蜂王排除器和喂食室门插入喂养室上方，以防止蜂王进入喂养室并接触经过处理的饮食。有关更多组装详细信息，请参见 Fine 等人32。 在成年休养前24?…

Representative Results

在如上所述组装和维护的QMC中监测鸡蛋的产量，每天观察一次鸡蛋产量和每个处理组15个网箱。新交配的主要是卡尼奥兰种的蜂王从女王饲养员那里购买并连夜运输，并且蜜蜂工人是从伊利诺伊大学厄巴纳 – 香槟分校的蜜蜂研究设施中按照标准商业方法维护的3个蜂群中获得的。这里，使用4个饮食治疗组：1）蔗糖溶液和花粉补充剂中的50 ppb（g / g）吡虫啉（50 ppb – p + s），2）蔗糖溶液和花粉补充剂…

Discussion

雌性独居昆虫以及真社会昆虫群落中的蚁后的繁殖力可能受到非生物应激源的影响，例如农用化学品25，28，29，30，33。在蜜蜂中，农用化学品对蜂王的影响可能是间接的，因为它们可以通过工蜂的护理和喂养的变化而发生。我们的代表性结果与基于现场的研究2…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

感谢Amy Cash-Ahmed博士，Nathanael J. Beach和Alison L. Sankey在开展这项工作时提供的帮助。本出版物中提及商品名称或商业产品仅用于提供特定信息，并不意味着美国农业部的推荐或认可。美国农业部是一个机会均等的提供者和雇主。这项研究得到了国防高级研究计划局#HR0011-16-2-0019对Gene E. Robinson和Huimin Zhao，USDA项目2030-21000-001-00-D以及伊利诺伊大学厄巴纳香槟分校社区学院学生表型可塑性研究经验的支持。

Materials

Fluon BioQuip, Rancho Dominguez, CA 2871A
Honey bee queens Olivarez Honey Bees, Orland, CA
Imidacloprid Sigma-Aldritch, St. Louis, MO 37894
MegaBee Powder MegaBee, San Dieago, CA
Microcentrifuge tubes 2 mL ThermoFisher Scientific, Waltham, MA 02-682-004
Needles 20 gauge W. W. Grainger, Lake Forest, IL 5FVK4
Potassium Sulfate Sigma-Aldritch, St. Louis, MO P0772
Queen Monitoring Cages University of Illinois Urbana-Champaign Patent application number: 20190350175
Sucrose Sigma-Aldritch, St. Louis, MO S8501
Universal Microplate Lids ThermoFisher Scientific, Waltham, MA 5500
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Tags

Keywords: Agrochemical RiskHoneybee QueensQueen Monitoring CagesWorker BeesQueen ExposureQueen Egg-layingCarbon DioxideControlled ConditionsInvasive Species And Pollinator Health Research Unit
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Fine, J. D., Torres, K. M., Martin, J., Robinson, G. E. Assessing Agrochemical Risk to Mated Honey Bee Queens. J. Vis. Exp. (169), e62316, doi:10.3791/62316 (2021).
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