Name: in vitro rearing of solitary bees: a tool for assessing larval risk factors
Uploaded: 2018-07-16T14:00:00.000+00:00
Duration: 8 min 50 s
Description: Watch this Scientific Journal Video about In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors at JoVE.com

作者感谢克拉克和蒂姆克罗提供蜂筑巢芦苇, 梅瑞狄斯 Nesbitt 和莫莉比德韦尔协助在实验室, Drs. 卡梅伦·柯里, Christelle Guédot, 特里格里斯沃尔德, 迈克尔 Branstetter 和三匿名评论家为他们有用的评论, 改进了手稿。这项工作得到了农业部-农业研究服务划拨资金 (目前研究信息系统 #3655-21220-001)、威斯康星农业、贸易和消费者保护部 (#197199)、国家科学基金会 (根据格兰特号DEB-1442148), 能源部大湖生物能源研究中心 (能源部的科学 DE-FC02-07ER64494)。

1. 为杀菌剂暴露实验准备丙环唑解决方案
<ol><li>通过在无菌水中溶解适当数量的商业购买的丙环唑 14.3%, 准备0.1x 杀菌剂解决方案的一天的实验。确保只有新配制的杀菌剂溶液用于所有治疗。</li>
	<li>添加23µL 的0.1x 杀菌剂溶液每克花粉规定, 以获得丙环唑的最大浓度以前报告的蜂收集花粉24 (0.361 PPM 或µg 活性成分 g-1的花粉)。</li>
</ol>2. 从蜂芦苇中收获卵和寄主花粉的规定
<ol><li>使用消毒手术刀, 解剖新鲜堵塞的蜂筑巢芦苇, 分裂成两个部分沿芦苇的长度, 以暴露各自的房间。 注: 每个巢可能包含在8到14个房间和一个单独蛋在房间之内。</li>
	<li>根据先前公布的准则77, 目视检查芦苇以识别含有雄性卵的腔室。使用灭菌弯曲针去除每个花粉供应连同伴生的蛋从巢芦苇, 并且安置在干净的重量小船。</li>
	<li>用干净的细漆刷将鸡蛋从规定中轻轻地分开, 用标准的实验室平衡记录花粉供应和卵子的新鲜重量。计算男性花粉规定的平均重量。</li>
	<li>执行随后的步骤, 以最小的延迟, 以减少伤害的机会, 从暴露在过量的温度和脱水。</li>
</ol>3. 准备寄主植物花粉的规定
<ol><li>目视检查母系收集的寄主植物花粉从巢室挖掘, 以确保没有寄生虫存在78。为了减少任何潜在的产妇偏见, 将花粉的规定组合成一个不育的培养皿中的单一质量, 并使用消毒针混合良好。</li>
	<li>将组合质量分为新的花粉规定, 确保每一个重组的规定的重量大约等于一个自然分配的男性提供的平均重量 (平均值, 0.35, 0.01 克, N = 42)。 注: 由于蜂 sp。将较小的花粉规定分配给雄性后代, 这会导致雄性幼虫的体重降低77。为了避免因性别差异而引起的任何这种偏见, 在实验中只使用雄性卵子。</li>
</ol>4. 准备非寄主植物花粉供应
<ol><li>粉碎商业购买蜂蜜蜂收集花粉使用一个标准的实验室球磨机的细粉。</li>
	<li>根据母系花粉的含水量 (~ 20%), 利用适当体积的40% 种灭菌糖溶液79 , 将花粉粉水合, 并将其彻底混合, 形成面团状的稠度。</li>
	<li>分成单独的花粉质量, 每一个体重约相当于一个自然分配的男性规定的平均重量。 注: 母系收集的花粉的含水量可以通过比较30随机选择的男性腔80中花粉的新鲜和干重来进行标准化。为了获得干重, 花粉的规定应冷冻干燥的冻干机 (1.5 Pa 为72小时)。</li>
</ol>5. 制备多井细胞培养板
<ol><li>用蒸压锡杯 (5 x 9 厘米) 的无菌48井培养板的线个体井。使用无菌钳, 轻轻地把胶囊的顶端边缘, 以便它可以容纳花粉供应。</li>
	<li>使用基于治疗组的无菌工具, 在锡杯内放置单一质量的寄主或非宿主花粉供应。 注意: 为了避免交叉污染, 使用单独的钢板治疗和控制组。</li>
</ol>6. 添加杀菌剂
<ol><li>用不育的木棍在花粉质量中放置一个中央凹陷。使用新的棍子为每个花粉供应。</li>
	<li>添加适当数量的杀菌剂溶液 (用于治疗), 或无菌水 (用于控制) 进入抑郁症。用无菌钳捏住抑郁症的开口, 以尽量减少杀菌剂/无菌水与卵子之间的表面接触。</li>
	<li>确保实验设计的阶乘设置与示意图表示形式 (图 4) 中所描述的对齐。</li>
</ol><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57876/57876fig4.jpg"> 图4。实验装置的示意图表示法.该实验表示一个完全交叉的 2 x 2 阶乘设置。因子1代表杀菌剂暴露, 包括2级: (i) 无杀菌剂 (n = 10) 和 (ii) 杀菌剂 (n = 10)。因子2代表花粉源, 包括2层: (i) 寄主花粉 (n = 8) 和 (ii) 非寄主花粉 (n = 8)。<a href="/files/ftp_upload/57876/57876fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
7. 后方和观察幼虫
<ol><li>使用干净的细漆刷将随机选择的雄性卵放在花粉供应的顶端表面。一旦鸡蛋被放置在所有的规定, 更换细胞培养板的盖子, 确保它与标签胶带在角落。</li>
	<li>把井板放在干净的托盘上, 用深色的布盖住, 以阻挡直接光线的接触。在托盘内放置6个含30毫升不育水的井板, 以防止脱水。在室温下将孵化盘放在孵化器内不受干扰。</li>
	<li>每天在解剖显微镜下观察井板, 而不去除井板盖。通过检查运动来确保幼虫活着。如果检测不到运动, 丢弃含有死幼虫和剩余花粉的锡杯。允许所有幸存的幼虫在井板内不受干扰地发育, 直到到达 prepupal 阶段。</li>
	<li>从锡杯中取出幼虫, 一旦到达 prepupal 阶段41。用刷子清洁丝绸茧里的大便。用解剖显微镜仔细切开丝茧, 用橡皮钳提取预蛹。</li>
	<li>轻轻地处理预蛹, 确保工具不会刺穿柔软的身体。记录预蛹 (prepupal 生物量) 的新鲜重量和从卵到 prepupal 阶段 (幼虫发育时间) 的发育时间。 注: 任何死幼虫应立即丢弃, 以防止不需要的微生物生长在尸体和剩余的花粉供应。这减少了感染的风险, 其余的健康幼虫。</li>
</ol>

作者没有什么可透露的。

在实验室条件下, 在其天然筑巢芦苇外饲养蜜蜂, 可以测试与幼虫体质有关的多个假说。如果不明因素继续导致蜜蜂死亡, 使用体外实验进行的风险评估研究可以帮助确定潜在的威胁, 并告知该物种丰富的野生传粉者群的管理做法12 ,38,49,76,81,82?...

鉴于它们作为昆虫传粉者的主导群体1, 全球蜜蜂种群的损失对粮食安全和生态系统稳定性构成威胁2、3、4、5、6 ,7。管理和野生蜜蜂种群的下降趋势归因于几个共同的危险因素, 包括生境分裂、新出现的寄生虫和病原体、遗传多样性的丧失以及入侵物种的引进3 ,4,7,8,9,10,11,12。特别是, 杀虫剂的使用急剧增加 (例如, neonicotinoids) 与蜜蜂13、14、15之间的有害影响直接相关。一些研究表明, neonicotinoids 和麦角甾醇生物合成抑制 (EBI) 杀菌剂之间的协同作用可能导致高死亡率横跨多蜂种类16,17,18,19,20,21,22. 然而, 长期以来被认为是 "蜜蜂安全" 的杀菌剂继续喷洒在开花作物上, 但没有对23进行过多的审查。觅食蜜蜂已被记录下来, 例行带回花粉负荷污染的杀菌剂残留物24,25,26。这种杀菌剂 ladenpollen 的消费量会导致幼虫蜂27、28、29、30之间的高死亡率, 以及成年蜜蜂中的一组亚致死效应16,31,32,33,34. 最近的一项研究表明, 杀菌剂可能会通过改变蜂巢储存的花粉中的微生物群落来造成蜜蜂的损失, 从而扰乱蜜蜂和花粉传播的微生物之间的关键共生35。
虽然孤蜂对几种野生和农业植物的授粉至关重要,36、37、38, 但这种不同的传粉群体在农药监测研究中受到的关注较少。一个成年的独居女性的巢包含5-10 个封闭的巢室, 每个都有一个有限质量的母系收集的花粉和花蜜, 和一个单一的鸡蛋39。孵化后, 幼虫依赖于分配的花粉提供, 和相关的花粉传播的微生物群获得足够的营养40,41。因为他们缺乏社会生活方式的好处, 孤独的蜜蜂可能更容易受到杀虫剂的照射42。例如, 虽然在喷洒后的社会蜜蜂的赤字可能会得到工人和新出现的巢的一些扩大, 一个单一的成年单身女性的死亡结束所有的生殖活动43。这种易感性的差异突出表明需要在生态研究中纳入不同的蜜蜂分类, 以确保对被管理和野生蜜蜂有足够的保护。然而, 除了少数研究, 对杀菌剂暴露的影响的调查主要集中在社会蜜蜂18,23,32,44,45 ,46,47,48,49。
属于蜂属的孤蜂 (图 1) 已被全世界用作多种重要水果和坚果作物的有效传粉者, 39,50,51,53,53. 与其他被管理的授粉小组 24,54,55,56,57,58, 成年蜂蜜蜂通常暴露于杀菌剂喷洒在开花作物44。在最近喷洒的农作物上觅食的成年雌性可能会收集和储存他们的巢室与杀菌剂的花粉, 后来形成了唯一的饮食发育幼虫。食用受污染的花粉的规定随后可将幼虫暴露于杀菌剂残留物42。在 oligolectic 物种中, 接触的风险可能较高, 仅在少数几个密切相关的寄主植物上觅食59、60、61。例如, 某些 megachilid 蜜蜂似乎优先饲料低品质的菊科花粉, 作为减少寄生62的一种手段。然而, 杀菌剂对 oligolectic 孤蜂幼虫适应性的影响程度尚未得到实证量化。本研究的目的是制定一个协议, 以检验杀菌剂暴露和花粉源对体外饲养孤蜂的适应性的主要和交互作用。为了调查, ribifloris lato (广义) 的卵可以在商业上获得 (材料表)。这个人口是理想的, 因为它作为一个土生土长的授粉的重要性, 并且它强烈偏爱花蜜丰富的十大功劳 aquifolium (俄勒冈葡萄) 发现在区域53,63,64(图 2)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57876/57876fig1.jpg"> 图1。一张成人蜂 ribifloris的高分辨率照片。照片信贷, 美国农业部研究昆虫学家吉姆. 甘蔗,<a href="/files/ftp_upload/57876/Figure_1.pdf" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57876/57876fig2.jpg"> 图2。芦苇蜂 ribifloris (广义)的嵌套芦苇, 在前景中嵌套雌性. 芦苇的室隔断和端子插头是由咀嚼叶构造的。照片信用 NativeBees.com 先生,<a href="/files/ftp_upload/57876/57876fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
本研究的第一个目的是评估食用杀菌剂处理花粉对幼虫体质的影响 (以发育时间和 prepupal 生物量计算)。虽然接触到普遍应用的杀菌剂丙环唑已与成年蜜蜂之间的死亡率增加23,24,32,44,45, 54、55、56、57、58、65、66、67, 对蜜蜂幼虫的影响较小知道。本研究的第二个目的是评估食用非寄主花粉对幼虫体质的影响。以往的研究表明, oligolectic 蜜蜂的幼虫在被迫食用非寄主花粉68时未能发育。这种结果可能归因于蜜蜂生理学69、花粉生物化学70和与天然花粉有关的有益菌群的变化 (71)。本研究的第三个目的是评价杀菌剂处理和日粮花粉对幼体健康的交互作用。
众所周知, 许多生物学特性包括产妇体型、调配率、觅食策略和花粉量72、73、74、75都影响了孤蜂幼虫的适应性。这些因素可以在芦苇之间引入显著的变异性, 这在评估幼虫的健康状况时, 对发展防御性实验设计构成了挑战。此外, 鉴于幼虫的发育发生在密封的筑巢芦苇内, 这种变异对后代的影响很难想象和量化而不使用非致命技术 (图 3)。为了克服这一挑战, 本研究中的所有假说都是使用饲养在巢状芦苇外的幼虫来测试的。实验设计代表一个完全交叉的 2 x 2 阶乘设置, 每个因素由2级组成;因子 1: 杀菌剂暴露 (杀菌剂;无杀菌剂);因子 2: 花粉源 (寄主花粉, 非寄主花粉)。在受控实验室条件下, 蜜蜂在无菌多井细胞培养板中由卵向 prepupal 阶段培养。每个井都有一个标准的花粉供应量和一个鸡蛋。孵化后, 幼虫在井内分配的花粉上进食, 完成幼虫发育, 并启动蛹。过去的研究表明, 在这种人工饲养环境中所饲养的蜜蜂中, 无法解释的死亡率低于野外49、76所遇到的。在体外饲养的蜜蜂的使用比基于田间的研究有几个优点: 1) 它最大限度地减少自然变异性和不受控制因素的混杂效应, 通常与实地研究相关;2) 它允许在治疗组中同时测试每个感兴趣因素的多层次操作;3) 复制的次数可以预先确定, 每个复制的实验因素可以单独操作;4) 幼虫响应变量可在不干扰相邻幼虫的情况下, 独立地进行可视化和记录;5) 可以修改该协议, 以适应涉及多个因素和响应变量的更复杂的实验设计。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57876/57876fig3.jpg"> 图3。蜂 ribifloris (广义)天然嵌套芦苇中的含量.关闭(a)解剖的芦苇, 显示个别的房间, 花粉的规定和分区, (B)新鲜收获的花粉规定, 和相关的鸡蛋 (用黑色圆圈表示)。<a href="/files/ftp_upload/57876/57876fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table><tbody><tr><td>eggs of &lt;em&gt;O. ribifloris&lt;/em&gt; sensu lato (s.l.)</td><td></td><td></td><td>Kaysville, Davis County, Utah, USA</td></tr><tr><td>Osmia reeds</td><td>Nativebees.com</td><td>NA</td><td>Freshly plugged reeds</td></tr><tr><td>Dissection set</td><td>VWR</td><td>89259-964</td><td>Sterilize before use</td></tr><tr><td>Long Nose Pliers</td><td>Husky</td><td>1006</td><td></td></tr><tr><td>6 well culture plates</td><td>VWR</td><td>10062-892</td><td>Sterile sealed</td></tr><tr><td>48 well culture plates</td><td>VWR</td><td>10062-898</td><td>Sterile sealed</td></tr><tr><td>Petri dishes</td><td>VWR</td><td>25373100</td><td>Sterile sealed</td></tr><tr><td>Square Weighing Boats</td><td>VWR</td><td>10770-448</td><td></td></tr><tr><td>Camel Hair Brush</td><td>Bioquip</td><td>1153A</td><td></td></tr><tr><td>Tin capsules</td><td>EA Consumables</td><td>D1021</td><td>Sterilize before use</td></tr><tr><td>Sucrose</td><td>VWR</td><td>470302-808</td><td></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>Honey bee pollen</td><td>Bee energised</td><td>897098001244</td><td>Untreated, natural, raw pollen</td></tr><tr><td>Microbalance</td><td>VWR</td><td>10204-990</td><td></td></tr><tr><td>Pulverisette</td><td>LAB SYNERGY INC.</td><td>30334913</td><td></td></tr><tr><td>Wooden sticks</td><td>VWR</td><td>470146908</td><td>Sterilize before use</td></tr><tr><td>Sealing tape</td><td>VWR</td><td>89097-912</td><td></td></tr><tr><td>Microscope</td><td>VWR</td><td>89403-384</td><td></td></tr><tr><td>Planting tray</td><td>VWR</td><td>470150-632</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>BDH1158-4LP</td><td></td></tr><tr><td>Centrifuge tube</td><td>VWR</td><td>21008936</td><td></td></tr><tr><td>Microsyringe</td><td>Cole-Palmer</td><td>UX-07940-07</td><td></td></tr><tr><td>Rubber tweezer</td><td>Amazon</td><td>B0135HWPN4</td><td></td></tr><tr><td>Syringe needles</td><td>VWR</td><td>89219-334</td><td></td></tr><tr><td>Freeze drier</td><td>Labcono</td><td>LFZ-1L</td><td></td></tr><tr><td>Statistical software</td><td>SPSS</td><td>Version 21.0</td><td></td></tr></tbody></table>

in vitro rearing of solitary bees: a tool for assessing larval risk factors

虽然孤蜂为野生和被管理的农作物提供重要的授粉服务, 但在农药管制研究中, 这种富含物种的群体已基本被忽视。如果喷雾发生在或靠近寄主植物的情况下, 当蜜蜂收集花粉来供应巢时, 接触杀菌剂残留物的风险可能会特别高。对于从一组植物中摄取花粉的蜂物种 (oligolecty), 不能利用非寄主植物的花粉可以增加其与杀菌剂相关毒性的危险因素。本手稿描述了用于成功后方 oligolectic 梅森蜜蜂,蜂 ribifloris严格 lato, 从鸡蛋到 prepupal 阶段在细胞培养板的标准化实验室条件下的协议。在体外饲养的蜜蜂随后被用来研究杀菌剂暴露和花粉来源对蜜蜂健康的影响。以 2 x 2 全交叉因子设计为基础, 研究了杀菌剂暴露和花粉源对幼虫体质的主要影响和交互作用, prepupal 生物量、幼虫发育时间和存活率进行量化。这种技术的一个主要优点是, 使用体外饲养的蜜蜂可以减少自然背景的变异性, 同时也能同时操作多个实验参数。描述的协议提供了一个通用的工具, 假设测试涉及一系列影响蜜蜂健康的因素。为了使保护工作取得重大的、持久的成功, 对导致蜜蜂下降的生理和环境因素的复杂相互作用的这种洞察将被证明是至关重要的。

幼虫的体质是使用三指标 (i) 幼虫发育时间, (ii) prepupal 生物量, (iii) 存活率。利用杀菌剂暴露 (两个层次: 无杀菌剂、杀菌剂) 和花粉源 (两个层次: 寄主花粉、非寄主花粉) 作为独立变量, 以幼虫发育时间为相关变量, 进行了双向方差分析。杀菌剂暴露的主要效果 (F128 = 1.24, P = 0.28) 在杀菌剂治疗 (平均 SE) (28.14 ±1.98 d, N = 14) 和未处理 (25.39 @ 1.65 d, <e...

Watch this Scientific Journal Video about In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors at JoVE.com

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

体外孤蜂饲养: 一种评估幼虫危险因素的工具

在开花植物上喷洒杀菌剂可以使孤蜂接触到高浓度的花粉传播杀菌剂残留物。本研究以实验为基础, 结合体外饲养蜂幼虫, 探讨了寄主和非寄主植物对杀菌剂处理花粉的影响。

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

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Research

JoVE Journal

Environment

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

8.1K 次观看.  University of Wisconsin-Madison. 在开花植物上喷洒杀菌剂可以使孤蜂接触到高浓度的花粉传播杀菌剂残留物。本研究以实验为基础, 结合体外饲养蜂幼虫, 探讨了寄主和非寄主植物对杀菌剂处理花粉的影响。

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

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

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are also a useful scientific model to study motion vision and flight dynamics in a controlled laboratory setting. As the larvae develop in organically polluted water, they are useful models for investigating investment in microbial immunity. While large scale commercial breeding for agriculture already occurs, there are no standardized protocols for maintaining captive populations for scientific studies. This is important as commercial captive breeding programs focusing on mass output during peak pollination periods may fail to provide a population that is consistent, stable and robust throughout the year, as is often needed for other research purposes. Therefore, a method to establish, maintain and refresh a captive research population is required. Here, we describe the utilization of an artificial hibernation cycle, in addition to the nutritional and housing requirements, for long term maintenance of Eristalis tenax. Using these methods, we have significantly increased the health and longevity of captive populations of E. tenax compared to previous reports. We furthermore discuss small scale rearing methods and options for optimizing yields and manipulating population demographics.

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

The overall goal of these procedures is to establish, maintain and refresh a captive population of Eristalis tenax in a research setting.

研究性学习的Eristalis 管Hoverflies 的饲养和长期维护

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

长鼻扩展响应协议为侦查行为可塑性的昆虫：应用基础，生物医学和农业研究

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

神经科学

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

Exploring Life History Choices: Using Temperature and Substrate Type as Interacting Factors for Blowfly Larval and Female Preferences

Blowflies (Diptera: Calliphoridae) present a wide range of larval lifestyles, typically classified as obligate parasitism, facultative parasitism, and complete sapro-necrophagy. Several parasitic species, both obligate and facultative, are considered to be of sanitary and economic importance, as their larvae can cause myiasis (maggot infestation in live tissue). However, it is noteworthy that the adult female plays a decisive role as she chooses the oviposition site, and, therefore, largely determines the feeding habit and developmental conditions of the larvae. In this study, two protocols are proposed to test larval feeding preference and female oviposition site preference considering two interacting factors: meat substrate type and temperature. The setups presented here allowed to test Lucilia cuprina larvae and gravid females in a four-choice assay with two temperatures (33 &plusmn; 2 &#176;C and 25 &plusmn; 2 &#176;C) and two types of meat substrates (fresh meat supplemented with blood and 5-day-old rotten meat). Larvae or gravid females can choose to burrow or lay their eggs, respectively, in either of the following: rotten meat at 25 &#176;C (simulating a necrophagous species condition), fresh meat supplemented with blood at 33 &#176;C (simulating a parasitic species condition), and two controls, rotten meat at 33 &#176;C, or fresh meat supplemented with blood at 25 &#176;C. The preference is assessed by counting the number of larvae or eggs laid in each option for each replicate. Comparing the observed results to a random distribution allowed for the estimation of the statistical significance of the preference. The results indicated that L. cuprina larvae have a strong preference for the rotten substrate at 25 &#176;C. Conversely, oviposition-site preference by females was more varied for the meat type. This methodology can be adapted to test the preference of other insect species of similar size. Other questions can also be explored by using alternative conditions.

Herein, two protocols for assessing food source and oviposition preferences in larvae and females of blowflies are detailed. These comprise four choices with two interacting factors: substrate type and temperature. The assays enable the determination of the food source preference of the larvae and the oviposition site preference for the females.

探索生活史选择:使用温度和基质类型作为吹蝇幼虫和雌性偏好的相互作用因素

Blowflies (Diptera: Calliphoridae) present a wide range of larval lifestyles, typically classified as obligate parasitism, facultative parasitism, and ...

行为学

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

获取标本减慢，加速和反老化的蜜蜂型号

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

生物学

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 ...

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs.

风险蝴蝶捕获繁殖计划，以提高生命历史知识和有效的异地保护技术

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

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 ...