作者 (s) 感谢威斯康星大学生物技术中心 DNA 测序设施, 提供放大和测序设施和服务, 凯特琳卡尔森, 珍妮弗诀窍, 杰克奥托, 和最大 Haase 提供技术援助分子分析。这项工作得到了农业部-农业研究处拨款资助 (目前的研究信息系统 #3655-21220-001)。国家科学基金会提供进一步支助 (赠款 No。DEB-1442148), 美国能源部大湖生物能源研究中心 (能源部 DE-FC02-07ER64494), 和美国农业部国家食品和农业研究所 (孵化项目 1003258)。C.T.H. 是一个皮尤学者在生物医学科学和阿尔弗雷德克劳斯·特普费尔教员研究员, 支持的皮尤慈善信托基金和亚历山大·冯洪堡基金会分别。

1. 使用田间网箱试验检查杀菌剂照射对蜂群成功的影响 <ol> <li> 在燕麦种植场中设置了十网笼。在每个笼子周围挖一条沟, 把网笼的四条边挖到地上, 以确保蜜蜂不能逃脱。将笼子与盆栽, 开花的植物, 是已知的吸引蜜蜂 ( 如 荞麦, 琉璃, alyssum, 宇宙和向日葵) ( 图 2 )。
	<li> 用一个单托盘 (36 厘米 x 42 厘米) 的盛开三叶草补充笼子。集群花卉资源在笼子的一个角落里, 占用一个空间大约 2.5 m x 1 m. 植物剩余的笼子区域由燕麦. </li>
	<li> 随机分配大黄蜂蜂群, 每个包含工人和一个女王, 治疗 (杀菌剂存在/缺席, N = 5 殖民地每治疗, 10 殖民地共计), 然后安置他们在一个领域笼子 ( n = 1/笼子) 29 天 (6月23日-2014年7月21日). </li>
	<li> 将菌落盒定向到菌落和 #39; s 开口指向南方, 为蜜蜂提供最佳的航行条件。用糖水囊来补贴蜂群, 放在蜂巢盒子里, 以补充花蜜的可用性. </li>
	<li> 在研究期间 (0 和 13), 使用手持杀虫剂喷雾器, 在五个杀菌剂处理笼中应用 chlorothalonil-based 杀菌剂在田间相关水平 (20 克/升) 到开花植物。将花朵均匀地涂上, 这样就不会有进一步的液体附着在花表面 ( 图 3 ). </li>
	<li> 在字段保持架研究结束时, 将 B. 凤仙花 从笼子中手工取出, 通过放置-20 和 #176 冷却蜂房; C 型冷冻机20分钟 </li>
	<li> 使用无菌钳清除蜜蜂并记录幼虫、蛹、成年雌性的数量 ( i . e . 觅食) 和成年男性。使用分析天平记录母亲皇后、幼虫、蛹、成年雌性 ( 即 觅食) 和成年雄性的干重. </li>
</ol> 2。使用实验室 在体内 试验中检测杀菌剂暴露对花粉中微生物群落的影响 <ol> <li> 用标准的方法将商业购买的花粉粉碎为细粉实验室球磨机。通过浸泡70% 乙醇, 使花粉在紫外线照射下一夜之间蒸发, 从而杀菌。在 general-purpose 琼脂培养基上通过电镀0.5 毫克来验证花粉的不育性. 
	<ol> <li> 对干燥、灭菌的花粉, 使用灭菌管, 添加田间相关剂量的杀菌剂: 环在 14.3%; 嘧22.9% 用于治疗 (0.74 和 #181; l 和0.65 和 #181; 分别/天/蜂房)。用消毒的木棍拌匀. </li>
	</ol> </li> <li> 在室温下保持的干净、卫生的实验室中放置6个实验性荨麻疹 (n = 3 个用于控制和治疗). 台式。每一天体重4.27 克的花粉 34 , 35 与杀菌剂混合 (用于治疗) 或无菌水 (用于控制) 在一个罩内使用标准的无菌技术. 
	<ol> <li> 使用包含蜂巢的纸板盒侧面提供的陷阱门, 介绍蜂房内的花粉。每周用消毒的糖溶液补充蜂房。继续喂养制度四周. </li>
	</ol> </li> <li> 在实验室研究的结论中, 通过放置-20 和 #176 来冷却荨麻疹; C 型冷冻机20分钟, 用灭菌钳和铲子在不育的储藏管中, 用消毒的镊子将花粉的规定刮掉。存储在-80 和 #176; c. 在实验开始和结束时清点和记录工人和王后母亲的体重 (步骤 1.7). </li>
	<li> 使用商业上可用的 dna 隔离套件从花粉提供样品中分离出 dna (详见材料表). 
	<ol> <li> 添加0.25 克花粉-提供给萃取管, 短暂的漩涡混合. </li>
		<li> 在去离子蒸馏水中制作200毫克/毫升溶菌酶溶液, 足够50和 #181; L/样品。用力摇动, 完全形成解决方案. </li>
		<li> 添加50和 #181; 溶菌酶溶液中的萃取管与样品在其中, 并混合好几次。在水浴中孵育试管10分钟, 在37和 #176; C。如果沉淀形成, 热溶液到60和 #176; C 直到溶解在用途之前. </li>
		<li> 添加70和 #181; L 对萃取管进行水裂解处理, 在平板涡垫上用胶带将其水平固定, 在室温下为三十年代 1万 x g 离心管10分钟. </li>
		<li> 将上清液转移到干净的2毫升收集管。添加250和 #181, L 蛋白质沉淀溶液, 和涡流 5 s. 在4和 #176; C 在冰浴中孵育5分钟. 
		注: 400 至 #181; 升至 500 #181; 上清。上清液可能还含有一些微粒. </li>
		<li> 在室温下离心管的温度为1分钟, 在 1万 x g, 并转移到600和 #181; 升上清到一个干净的2毫升收集管。添加200和 #181; 水抑制剂去除溶液, 涡旋, 并孵育在4和 #176; C 为5分钟离心管在室温下为1分钟, 在 1万 x g. 转移到750和 #181; 升上清成干净的2毫升收集管. </li>
		<li> 添加1200和 #181; 升水溶液到上清, 和涡流 5 s. 加载约675和 #181; l 上清到一个旋转过滤器, 离心在 1万 x g, 在室温下1分钟。放弃流. </li>
		<li> 重复2.4.7 两次. 
		注: 所处理的每个样品总共需要三负载. </li>
		<li> 添加500和 #181; L 乙醇, 在室温下离心三十年代在 1万 x g. 放弃流动, 并再次离心在室温下1分钟, 在 1万 x g. 在干净的2毫升收集管中放置旋转滤镜. </li>
		<li> 添加100和 #181; 洗脱缓冲液到滤膜中心。离心机在室温下为三十年代在 1万 x g. 丢弃旋转滤镜。在-20 和 #176 之间存储收集的 dna; c 和-80 和 #176; c </li> </ol> </li> <li> 使用分离的 dna 进行测序. 
	<ol> <li> 通过荧光分析对 2 ng/和 #181 的分离 DNA 进行量化和规范化;
		<ol> <li> 为每个提取的样品准备一式三份的反应, 比较 28S (植物) 的相对数量, 分析其 (真菌) 和每个花粉的 16S (细菌) 成分--提供样本 36 。确保每一个反应包含 10 ng 的总 DNA, 2x 的不对称的菁染料为主混合, 和2.5 和 #181; L 每个向前和反向底漆对28焦/28 b 37 用于植物和 ITS1/ITS5.8R 真菌 DNA 的 38.</li>
		</ol> </li> <li> 使用下列参数放大 DNA: 2 分钟 pre-denaturation 在50和 #176; c, 2 分钟初变性在95和 #176; c, 40 周期 (十五年代在98和 #176; c, 十五年代在58和 #176; c, 六十年代在72和 #176; c), 后跟一个熔体曲线. </li>
		<li> 使用面向 16S rRNA V3/V4 变量区域和其/5.8s rRNA 间隔区域的下一代测序库准备2步嵌套的 PCR 协议。
		<ol> <li> 将12.5 和 #181 添加到每个底漆和2x 主组合的 5 pmol。对每个区域分别作出反应 (16S 或其; 请参见 表 1 )。修改区域特定的引物, 如前面所描述的 39 , 40 , 以向特定于基因的序列添加排序器特定的适配器悬垂核苷酸序列. </li>
			<li> 使用以下参数执行初始放大: 3 分钟初变性在95和 #176; c, 25 周期 (三十年代在95和 #176; c, 三十年代在55和 #176; c, 三十年代在72和 #176; c), 5 min 最后的延长在72和 #176; c. </li>
			<li> 在初始放大后, 通过电泳移动验证库大小和数量, 并使用1x 体积固相可逆 imm 进行清洗obilization 珠去除残留底漆和反应试剂。池16S 和它的增定量地为每个示例创建一个增池. </li>
			<li> 使用以下引物添加排序器特定的适配器和示例特定的双索引 (请参见 表 1 )。增加2.5 和 #181; L 放大 DNA 到 5 pmol 的每个底漆和2x 主混合。使用以下参数执行库放大: 3 分钟初变性在95和 #176; c, 8 周期 (三十年代在95和 #176; c, 三十年代在55和 #176; c, 三十年代在72和 #176; c), 5 min 最后的引伸在72和 #176; c. </li>
		</ol> </li> <li> 遵循 PCR 方法, 使用 1 x 体积的固相可逆固定化微珠清洁成品库。使用电泳移动和荧光分别评估成品库的质量和数量。在排序前将库标准化到2和 #181; M 和池. </li>
		<li> 在与次级 PCR 中添加的适配器类似的平台上执行下一代测序, 其长度适当以覆盖整个增 41 . </li>
	</ol> </li> <li> 序列注释和微生物成分分析。
	<ol> <li> 将对所有顺序库的对端排序数据 (R1 和 R2) 合并为单个重叠。在合并 R1 和 R2 文件之后, 将生成每个库的单个 fasta 文件. 
		注意: 此步骤和下面描述的过程 (除非另有说明) 在 Mothur 版本 1.38.0 38 中执行. </li>
		<li> 筛选每个 fasta 文件以删除不明确的基、意外的长序列和长物. 
		注: 筛选的参数和序列去除 maxambig = 0, maxlength = 600, 和 maxhomop = 8 为两个基因。删除相同的序列, 但为所有库分别保留一个 contingence 表 (又称 Mothur 中的计数表). </li>
		<li> 将基因16S 库的唯一序列与席尔瓦数据库版本123对齐, 并将其库与其数据库进行联合, 以使其不被合并为 42 . 
		注意: 序列必须从王国水平到属层的注释。
		<ol> <li> 随后, 使用 diff=5 对具有函数前的群集的群集对齐序列, 并删除合体. </li>
		</ol> </li> <li> 根据席尔瓦 (用于基因 16S) 的方法对序列进行分类, 并将其 (用于基因的) 分类文件 (截止值为 80%) 联合使用. 
		<li> 加载到 R 44 在 Mothur 的分类过程中生成的 contingence 表 (每个基因一个)。在每个分类学层面上, 获取每个图书馆的相对丰度, 进一步分析微生物群落的变化. 
		注意: 在每个分类学层面上, 当相对丰度小于2% 时, 所有的实验重复都将分类组合并在一起. </li>
	</ol>
	</li>
</ol>

作者没有什么可透露的。

对杀菌剂对蜜蜂健康的影响的调查仍然是害虫管理战略的一个理解方面。我们的研究目的是通过一套互补的技术来弥合这一知识鸿沟, 这一方法明确地隔离了推动蜜蜂衰落的潜在因素。下面详细介绍这些实验的规划、基本原理和渲染。
重要的是要确保不允许蜜蜂逃脱笼实验的网格, 因为这将损害人口学分析。同样重要的是, 人工巢有足够的绝缘, 以防止雨水和阳光直射。应注意...

管理和野生蜜蜂物种正经历着广泛的下降, 对自然和农业系统的主要影响1。尽管共同努力, 以了解这一问题的原因, 驱动蜂蜜蜂下降的因素仍然没有很好地理解2,3,4。对于某些种类的野生, 本地蜜蜂, 情况已经变得可怕5,6。如果蜜蜂种群在与工业农业相交时不能持续, 其种群数量将继续下降, 而需要传粉者的作物 (全球总产量的 35%7) 将会减少收成。
虽然许多潜在的因素, 如农药暴露, 疾病和栖息地损失1,4,8,9,10都牵连到蜜蜂的衰落,在农业系统内部或附近, 这些压力源对本地蜜蜂健康的交互作用的了解相对较少。许多目前的研究工作继续侧重于杀虫剂, (例如, 烟碱11,12), 尽管过去的研究表明, 杀菌剂也可能通过损害记忆形成而在蜜蜂减少中发挥作用,嗅觉接收13, 巢识别14, 酶活性和代谢功能15,16,17。在全球范围内, 杀菌剂继续应用于开花的农作物。最近的研究表明, 蜜蜂通常会将杀菌剂残留物带回蜂巢18, 事实上, 研究显示, 有很大比例的被测试的蜂箱含有杀菌剂残留物19,20。进一步的工作表明, 杀菌剂残留与蜜蜂幼虫死亡率的高比率21,22,23和在殖民地内存在 "被埋葬的花粉", 虽然无毒,缺乏微生物活性, 营养受损24。尽管杀菌剂长期以来被认为是 "蜜蜂安全" 的事实, 但现在有证据表明, 单独接触杀菌剂可能会在本地大黄蜂物种中造成严重的蜂群损失,熊凤仙花25。
为了建立杀菌剂暴露与群体死亡率之间的因果关系, 需要确定这些化学品的作案手法。如土壤26, 沉积物27和水生环境28, 通过瞄准真菌, 杀菌剂最有可能改变真菌丰度和花粉中的多样性-规定, 从而调用一个主要的社区转移,可能强烈偏爱细菌。没有真菌竞争者或拮抗剂, 某些致病细菌可以增殖相对不受控制, 促进花粉变质的规定。过去的研究表明, 微生物, 特别是酵母菌和丝状真菌, 作为营养共生的蜜蜂29,30,31, 防止寄生虫和病原体32 ,33, 并提供花粉存储的长期保存。因此, 杀菌剂可能会通过破坏微生物群落来间接地伤害幼蜂, 因为它们需要提供这些服务和/或增加对机会性病原体和寄生虫的易感性12。随着对粮食生产的需求日益增加, 世界各地的农作物每年都在开花时喷洒杀菌剂, 这突出了需要了解这种杀菌剂引起的影响的严重性。
到目前为止, 与本地蜜蜂微生物生态学有关的主要知识鸿沟可以由以下问题来表示: 在何种程度上杀菌剂改变蜂花粉中的微生物群落-规定？什么是消耗花粉与一个深刻改变的微生物群落的下游影响？与这些生态学上相关的问题, 实验开发了以显露的主要目标 1) 仅杀菌剂残余可能导致严重殖民地衰落在本地蜂种;2) 花粉中微生物群落的程度--规定被杀菌剂改变, 3) 蜜蜂的健康受到严重改变的微生物群落的影响。实验目标的定义是通过结合实验室和实地实验来解决上述问题。本研究利用先进的基因和分子技术和传统的野外观察方法, 将杀菌剂对蜜蜂健康的潜在影响结合起来。
这项研究的第一个目的是证明单独的杀菌剂暴露会在本地蜜蜂物种中造成巨大的群体损失。一项涉及大型田间网箱的研究被用来调查杀菌剂暴露对熊凤仙花群体生长的影响, 这是一个无处不在的、丰富的美国本土蜜蜂 (图 1、图 2、图 3)。据推测, 与医护蜂房相比, 杀菌剂治疗的蜂箱会呈现较低的适应性和不典型的人口学。从这个实验中获得的数据证实了这一假说, 证明花粉中的杀菌剂残留物可能是造成本地大黄蜂物种严重群体损失的唯一原因25。本研究的第二个目的是探讨花粉微生物对杀菌剂暴露的反应。据推测, 花粉中微生物的群落组成--暴露在杀菌剂中的成分会与未经处理的花粉不同。虽然真菌的丰度和多样性预计会显著下降, 但细菌和/或单一的优势真菌物种在没有其他竞争真菌的情况下可能会无节制地生长。通过一系列的体内试验, 将利用基因分析微生物群落组成的这些变化。

<table><tbody><tr><td>Natupol Beehive</td><td>Koppert</td><td>USRESM1</td><td>16 hives</td></tr><tr><td>Propiconazole 14.3</td><td>Quali-Ppro</td><td>60207-90-1</td><td>Propiconazole 14.3%</td></tr><tr><td>Abound</td><td>Syngenta</td><td>4033540</td><td>Azoxystrobin 22.9%</td></tr><tr><td>Chlorothalonil</td><td>Syngenta</td><td>3452</td><td>Fungicide used for trials</td></tr><tr><td>Pollen granules</td><td>Bee rescued</td><td>B004D5650C</td><td>3X 16oz bottles, pollen for trials</td></tr><tr><td>Bacterial strains for inoculation</td><td>Currie Lab</td><td></td><td></td></tr><tr><td>Yeast strains for inoculation</td><td>Hittinger lab</td><td></td><td></td></tr><tr><td>Primer pairs</td><td>UW Biotech Center</td><td></td><td></td></tr><tr><td>DNA Isolation Kit</td><td>Mo Bio</td><td>12830-50</td><td>Commercial DNA isolation kit</td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher</td><td>Q32851</td><td>DNA quantification tool</td></tr><tr><td>Select Master Mix for CFX</td><td>Thermo Fisher</td><td>4472952</td><td>Used to perform real-time PCR using SYBR GreenER dye.</td></tr><tr><td>Real-Time PCR Detection System</td><td>Bio Rad</td><td>1855196</td><td>Instrument used for PCR amplification</td></tr><tr><td>PCR Clean-Up Kit,</td><td>Axygen</td><td>10159-696</td><td>Used for efficient removal of unincorporated dNTPs, salts and enzymes</td></tr><tr><td>DNA 1000 Kit</td><td>Agilent</td><td>5067-1504</td><td>Used for sizing and analysis of DNA fragments</td></tr><tr><td>MiSeq Sequencer</td><td>Illumina</td><td></td><td>Used for next-generation sequencing</td></tr><tr><td>Assorted glassware (beaker, flasks, pipettes, test tubes, repietters)</td><td>VWR</td><td></td><td></td></tr></tbody></table>

empirical, metagenomic, and computational techniques illuminate the mechanisms by which fungicides compromise bee health

种植者经常在开花时使用杀菌剂来保护农作物免受疾病的危害, 从而使蜜蜂暴露于杀菌剂残留物中。虽然被认为是 "蜜蜂安全", 但有越来越多的证据表明花粉中的杀菌剂残留物与蜜蜂的下降有关 (蜂蜜和蜜蜂的种类)。虽然这些机制仍然不为人知, 但研究人员推测, 蜜蜂微生物共生的参与。微生物在保存和/或处理花粉方面起着举足轻重的作用, 它们作为幼虫的营养。通过改变微生物群落, 杀菌剂可能破坏这些微生物介导的服务, 从而危害蜜蜂的健康。这份手稿描述了用于研究杀菌剂可能导致蜂群衰退的间接机制的协议。笼子试验揭露蜜蜂对杀菌剂处理过的花朵已经提供了第一个证据, 即杀菌剂造成了本地大黄蜂 (熊凤仙花) 的严重蜂群损失。利用田间相关剂量的杀菌剂, 进行了一系列的实验, 以提供更精细的描述微生物群落动态的杀菌剂暴露花粉。通过下一代测序和基因分析, 研究了花粉微生物群中真菌和细菌组合结构组成的变化。本文所开发的实验, 旨在提供一个机械的了解如何杀菌剂影响花粉的微生物-规定。最终, 这些发现应该揭示出杀菌剂可能导致蜂群衰退的间接途径。

野外网箱研究:
从笼型实验中获得的数据表明, 大黄蜂的蜂群对杀菌剂暴露有明显的反应。杀菌剂处理的蜂箱比控制单元 (43.2 ± 11.2, F19= 6.8, p = 0.03) (图 4) 产生的工人数量显著减少 (12.2 ± 3.8, 平均± SE)。此外, 杀菌剂处理的蜂箱的蜂生物量 (0.91 克± 0.15) 明显低于对照蜂房 (2.36 克± ...

Watch this Scientific Journal Video about Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health at JoVE.com

经验、宏基因组和计算技术阐明了杀菌剂损害蜜蜂健康的机制环境 |视频

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

黄蜂蜂巢内的微生物联合体丰富和保存蜂幼虫的花粉。使用下一代测序, 连同实验室和实地实验, 这份手稿描述的协议用于测试的假说, 杀菌剂残留改变花粉微生物群, 和群体人口统计, 最终导致殖民地损失.

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

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9.6K 次观看.  USDA-ARS. 黄蜂蜂巢内的微生物联合体丰富和保存蜂幼虫的花粉。使用下一代测序, 连同实验室和实地实验, 这份手稿描述的协议用于测试的假说, 杀菌剂残留改变花粉微生物群, 和群体人口统计, 最终导致殖民地损失.

视频: Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

从个体到群体度评价环境化学物质对蜜蜂发展的影响

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

科研

环境科学

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

用于蜂蜜蜜蜂筛选化学成分的视频跟踪协议

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

体外孤蜂饲养: 一种评估幼虫危险因素的工具

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

Quantification of the Potential Impact of Glyphosate-Based Products on Microbiomes

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway, which is virtually universal in plants. The inhibition of the enzyme stops the production of three essential amino acids: phenylalanine, tyrosine, and tryptophan. EPSPS is also present in fungi and prokaryotes, such as archaea and bacteria; thus, the use of GBP may have an impact on the microbiome composition of soils, plants, herbivores, and secondary consumers. This article aims to present general guidelines to assess the effect of GBP on microbiomes from field experiments to bioinformatics analyses and provide a few testable hypotheses. Two field experiments are presented to test the GBP on non-target organisms. First, plant-associated microbes from 10 replicated control and GBP treatment plots simulating no-till cropping are sampled and analyzed. In the second experiment, samples from experimental plots fertilized by either poultry manure containing glyphosate residues or non-treated control manure were obtained. Bioinformatics analysis of EPSPS protein sequences is utilized to determine the potential sensitivity of microbes to glyphosate. The first step in estimating the effect of GBP on microbiomes is to determine their potential sensitivity to the target enzyme (EPSPS). Microbial sequences can be obtained either from public repositories or by means of PCR amplification. However, in the majority of field studies, microbiome composition has been determined based on universal DNA markers such as the 16S rRNA and the internal transcribed spacer (ITS). In these cases, sensitivity to glyphosate can only be estimated through a probabilistic analysis of EPSPS sequences using closely related species. The quantification of the potential sensitivity of organisms to glyphosate, based on the EPSPS enzyme, provides a robust approach for further experiments to study target and non-target resistant mechanisms.

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. In this article, we introduce general guidelines to quantify the effect of GBP on microbiomes, from field experiments to bioinformatics analyses.

量化草甘膦产品对微生物组的潜在影响

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme ...

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ingestion of provisions contaminated with pesticides may increase the mortality rate of the larvae, decrease the collection rate and the population of adult solitary bees in the next year from a demographic perspective. But there are limited studies on the effects of pesticides on the larvae of solitary bees. Therefore, understanding how pesticides influence the larvae of solitary bees should be considered an integral part of pesticide ecological risk assessment. This study presents a method to expose the larvae of solitary bee, Osmia excavata, to lethal or sublethal doses of pesticide, tracking larval weight gain, developmental duration, eclosion ability, and food consumption efficiency conversion of ingested food. To demonstrate the effectiveness of this method, the larvae of O. excavata were fed with provisions containing acute lethal and sublethal doses of chlorpyrifos. Then, the above indexes of the treated larvae were investigated. This technique helps to predict and mitigate the risk of pesticides to pollinators.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

评估农药对孤独蜜蜂幼虫的影响

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

生物化学

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

约束和自由飞行，蜜蜂的神经药理学操纵，<em&gt;蜜蜂</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

神经科学

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

蜜蜂致死农药暴露对蜂群水平的影响

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

生物学

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis mellifera)

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee health, including exposure to viral pathogens. Such viruses can cause a wide variety of devastating effects and can often be challenging to study due to multiple factors that make it difficult to separate the effects of experimental treatments from preexisting background infection. Here we present a method to mass produce large quantities of virus particles along with a high throughput bioassay to test viral infection and effects. Necessitated by the current lack of a continuous, virus-free honey bee cell line, viral particles are amplified in vivo using honey bee pupae, which are extracted from the hive in large volumes using minimally stressful methodology. These virus particles can then be used in honey bee cage bioassays to test inocula viability, as well as various other virus infection dynamics, including interactions with nutrition, pesticides, and other pathogens. A major advantage of using such particles is that it greatly reduces the chances of introducing unknown variables in subsequent experimentation when compared to current alternatives, such as infection via infected bee hemolymph or homogenate, though care should still be taken when sourcing the bees, to minimize background virus contamination. The cage assays are not a substitute for large-scale, field-realistic experiments testing virus infection effects at a colony level, but instead function as a method to establish baseline viral responses that, in combination with the semi-pure virus particles, can serve as important tools to examine various dimensions of honey bee-virus physiological interactions.

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera

Here we describe two protocols: first to propagate, extract, purify, and quantify large quantities of honey bee non-enveloped virus particles, including a method for removing honey bee pupae and second to test the effects of viral infection using a highly repeatable, high-throughput cage bioassay.

一种富含病毒的接种物用于蜜蜂口腔感染（Apis mellifera）的制备

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee ...

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

评估交配蜜蜂女王的农用化学品风险

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

Cost-Effective Method for Safely Handling and Sampling Field-Collected Bees

A Rapid Method to Confine and Safely Handle Bees in the Field

Improving understanding of the basic biology and ecology of many insect pollinators, particularly specialist or rare taxa, is a priority for many researchers. As such, there is often a need to temporarily confine field-collected organisms in a non-injurious manner in order to gain information or support additional studies. This protocol represents a thoroughly tested, quick, and inexpensive field method for safely handling bees of conservation concern that can easily be tailored toward specific project needs, including organism identification, pollen removal, marking, and/or collection of non-lethal tissue samples for genetic analysis. This methodology can serve as an additional option in the researcher's toolbox to use when certain scenarios arise. It is anticipated that this methodology can be adapted for use with other insect species as well as used by individuals of varying experience and skill levels. It can be of great value to researchers studying specialist bees or conducting host-specific studies. The data collection made possible by this protocol will be invaluable to help researchers address critical data gaps for many pollinator species, plant-pollinator network structures, and pollinator conservation and management initiatives.

We demonstrate a tested method for safely handling field-collected bees. This method allows swift manipulation, identification, genetic sampling, and confirmation of plant-insect interactions via pollen collected while sampling. Easily adaptable, this approach offers a cost-effective, non-lethal means to study rare insect groups.

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

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