Name: evaluating the effect of pesticides on the larvae of the solitary bees
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Description: Watch this Scientific Journal Video about Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees at JoVE.com

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。

1. 喂料管的准备
<ol><li>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。</li>
</ol>2. 农药的制备
<ol><li>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...

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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Abamectin</td>
			<td>Jinan Lvba Pesticide Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Black-light lamps</td>
			<td>Kanghua Medical Device Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal tube box with 100 Wells</td>
			<td>Shanghai Rebus Network Technology Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Shanghai Rebus Network Technology Co., Ltd</td>
			<td></td>
			<td>2 mL; &#160;Serve as bee tube</td>
		</tr>
		<tr>
			<td>Electric soldering iron</td>
			<td>Kunshan Kaipai Hardware Electromechanical Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic scale</td>
			<td>Sartorius Scientific Instruments (Beijing) Co., Ltd</td>
			<td>3137510295</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Anhui Weiss Experimental Equipment Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (60 mm diameter)</td>
			<td>Qingdao jindian biochemical equipment co., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pollen provision</td>
			<td>Yantai Bifeng Agricultural Science and Technology Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Soft brush</td>
			<td>Wengang Wenhai painting material factory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solitary bees</td>
			<td>Yantai Bifeng Agricultural Science and Technology Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating the effect of pesticides on the larvae of the solitary bees

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。

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?...

Watch this Scientific Journal Video about Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees at JoVE.com

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

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。

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, ...

该协议可用于预测和减轻农药对孤独蜜蜂的不利影响。该技术的主要优点是模拟幼虫以残留农药为食。这种方法的视觉演示至关重要，因为与群居蜜蜂相比，Osmia excavata的生物学特征是独特的，并且对O.excavata，特别是幼虫的研究很少。为了准备进料管，使用电动绕组铁将一个直径为0.3毫米的孔打入两毫升离心管的盖子中。为了制备农药，将工业级农药溶解在丙酮中以获得每毫升10毫克的储备溶液，然后将溶液进行梯度稀释至五个浓度以上。接下来，从大规模饲养计划中获取含有供应品和新孵化的Osmia excavata幼虫的塑料蜂管。使用软刷，轻轻地将幼虫与幼虫分开，根据幼虫的大小和巢内的细胞位置选择雌性幼虫，然后将与所选雌性幼虫一起均匀大小的幼虫放置在直径为60毫米的培养皿中，并将它们放在一边使用。在进行农药处理之前，请权衡规定。要用农药处理规定，首先要测量农药溶液的体积。然后将所选均匀大小的铼剂浸泡在稀释的农药溶液中10秒钟。用农药处理规定后，再次测量农药溶液的体积。在无菌工作台上对规定进行空气干燥后，将它们添加到准备好的带孔的离心管中。接下来，称量60只雌性幼虫并使用软刷，将幼虫转移到自然干燥的准备物的表面，每管添加一个幼虫。将幼虫在25升或负2摄氏度，相对湿度为65%至75%的黑暗中将幼虫饲养在生长室中。对于急性致死毒性试验，测量幼虫在放置到治疗和对照规定48小时后的死亡率。接下来，称取昆虫饲养48小时后剩余的配料量，确定每只幼虫消耗的配料量，然后利用该公式，根据供给量的百分比和每只讧中农药含量，计算出每只幼虫在每种浓度下消耗的农药剂量。对于亚致死毒性试验，在黑光灯下茧化期间，每天观察O.excavata，以测量幼虫发育的持续时间。然后在治疗14天后称量幼虫，以确定幼虫体重增加。接下来，称量喂食14天后剩余的食物，以计算摄入食物的消耗量和转化效率。当对照蜜蜂长成成虫时，用小剪刀狙击茧，检查茧的次数。急性致死毒性试验表明，挖掘蜱幼虫毒死蜱的LD50值为每只蜜蜂0.001 μg。在亚致死毒性试验中，随着剂量的增加，幼虫体重增加、消耗量和摄入食物转化效率的指标值在治疗中降低，相对于对照值的最低值在每只蜜蜂0.013微克毒死蜱中观察到。相反，与对照处理相比，每只蜜蜂0.016微克毒死蜱观察到最延长的幼虫发育持续时间。对毒死蜱对eclosion速率的影响的评价表明，摄入剂量的毒死蜱与O.excavata的eclosion速率之间存在显着的负线性关系。当摄入的剂量超过每只蜜蜂0.02微克时，eclosion率比对照治疗中的剂量低得多。在该协议中，筛选出具有统一尺寸的规定对于最大限度地减少测试误差至关重要。该技术有助于探索农药对独居蜜蜂成虫飞行和繁殖力的影响，从而解决回收率低和种群数量下降的问题。

Research

JoVE Journal

Biochemistry

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

蛋白质的表征(按尺寸-排除色谱与多角度光散射相结合(SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

在氧化应激期间细胞粘附和纤维蛋白扩散动力学

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

使用随机微种子基质筛选与 I3C 的蛋白质晶体的衍生

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...