我们感谢奥斯曼Kaftanoglu的拍摄过程中有益的建议和帮助。我们要感谢匿名审稿人有见地的意见。这项工作是由挪威研究理事会（补助180504，191699，213976和），玛丽Curie/FP7（项目编号。238665），国家老化研究所（NIA授予P01 AG22500）和皮尤慈善信托基金的支持。

1。从实际年龄衰老脱钩本节介绍的双队列的殖民地，它包括确定个人共享同一个实足年龄（"单年龄组"）和巢蜜蜂的队列的队列的设置。单年龄组的同年龄人最终会分成不同的工人类型不同老化动力学 - 这些都是护士与蜜蜂放缓，采集蜜蜂与加速功能下降。所有的程序都描述一个实验殖民地。然而，我们建议，进行实验用于至少两个菌落复制，使细胞集落的影响可以被控制为（2 - 复制 - 设计）。 <ol><li> 准备蜂巢箱为双队列殖民地:准备一台普通的蜂巢框，接收两个食品梳子蜂蜜，花粉另一种食物梳子，和两个空的梳子。一定要找到一个交配女王，以及一个供体的殖民地超过3,000窝蜜蜂。双方将在后面介绍（1.3）。 </li><li> 获取和标记相似的实足年龄人士:收集梳子与密封育雏，即将涌现。对于一个复制预期收回梳子总3,000-5,000上限育雏细胞。对于每个复制使用育雏的平衡量从至少三个不同的蜂巢来源，以避免母体基因型（蜂巢原点）的偏斜分布。 <ol><li>将育雏梳子在孵化器设定为34℃，60％-70％的相对湿度。确保存储梳子以这样一种方式，新兴育雏无法逃脱。 </li><li>让蜜蜂出现两天并与胸廓（ 如统一POSCA，三菱铅笔有限公司）小漆标签标记这些蜜蜂。油漆标记将允许识别单个年龄组（出苗天）的蜜蜂，并从其他重复的菌落区别开来。 </li></ol></li><li> 设置一个双队列殖民地，包括i的队列dentified，单身年龄蜜蜂:在一天年轻的蜜蜂已经被标记，收集从捐赠者的殖民地约2500-3000巢蜜蜂（比较第1节中讨论。），并添加这些标记的蜜蜂来之前准备了蜂巢箱（见步骤1.1）。后者个人将构成不明巢蜂队列。 <ol><li>添加王后，这将是最初仅限于女王笼（市售）。密封笼食用糖果（ 如 Apifonda，Südzucker公司，曼海姆/ Ochsenfurt，德国），使工蜂慢慢松开女王。 </li><li>添加新出现的和显着的蜜蜂，这将构成一个年龄组。这些蜜蜂是唯一显着的个体，并为以下所有步骤的焦点小组。 </li></ol></li><li> 监测发病觅食和标记征粮:为了评估发病和护士对觅食的单年龄组的过渡动态，监测觅食活动辰人口统计发展很一天每个殖民地。开始计数5天后菌落进行设置（ 图1）。 <ol><li>计数蜜蜂从内按固定时间3×20分钟观察期间觅食航班（入口计数）返回的总数。请务必在定向航班期间不计蜜蜂（见讨论）。 </li><li>当入口计数表明相当觅食活动开始（&gt; 100入口数/天），开始标记觅食。要做到这一点，单年龄组（单标记个体）的觅食后，收到来自他们的第一觅食航班返回第二个油漆标记。这个标志漆将指定觅食发病的当天，并且将允许以后识别觅食年龄每觅食。 </li><li>每日重复标记直到蜜蜂足够数量的已标记。用于估计标记觅食足够数量的，期望的不超过5％-10％的检索率这些蜜蜂已经老化后，通常经过14天觅食。 </li></ol></li><li> 取样:由于所有的初始标记蜜蜂具有类似的实足年龄，护士和老觅食的年龄匹配的基团，可以同时采集，当觅食觅食已为≥14天。 <ol><li>单标记的护士蜜蜂蜂房内收集，并通过护理行为（喂食和清洗幼虫在开放育雏细胞放下头）确定。 </li><li>双标征粮也收集蜂巢内每天觅食活动开始之前。 </li><li>收集蜜蜂提供充足的通风笼（管，盒），并保持黑暗，直到进一步的处理。另外，对于转录组，表观遗传或蛋白质组学研究，直接扣在液氮中冷冻蜜蜂。收集个人的均衡数量从所有测试组和复制的殖民地。 </li></ol></li></ol> 2。与快速与缓慢老化工人工人通过改变蜂房的人口&lt;逆转/ P&gt; 本节详细介绍如何逆转，从工人的加速老化（觅食）工人与减缓老化（护士蜜蜂）执行。这种行为逆转诱导，觅食时遇到一个缺乏护士的蜜蜂，通常从事育雏舍。在回归过程将分离的单个菌落复制成两个蜂箱:一个配置单元的护士蜂分数（"护士衍生"），另一个与觅食分数（"觅食来源"）。后成功逆转，塑料可能出现的症状和逆转衰老可以研究在单一年龄组与恢复工人，继续征粮，持续护士蜜蜂和新招募的觅食。和以前一样，确定了单年龄组，而不是不明巢蜜蜂队列的蜜蜂，构成了实验焦点小组。 <ol><li> 制备方法:复制荨麻疹护士（单标）和征粮（双标）是在上一节中所述提供。确保不复归开始，每个重复菌落小于500标觅食，以确保有足够的检索复归完成后。 <ol><li>逆转后的安全鉴定测试组的关键是整个觅食人口在原来的蜂巢已经回归之前被标记为关闭。下面的步骤描述为一个复制。 </li><li>回归前一天，准备一个额外的蜂巢箱为觅食衍生蜂房（见步骤1.1）。找到两个皇后和两个育雏梳子捐助荨麻疹。转移到实验前殖民地刷了所有的成年蜜蜂从这些梳子。一笼女王（见步骤1.3），以及一个育雏梳将在原有的蜂巢箱代替女王和育雏梳子。另一组的女王和育雏梳将要使用的第二天新蜂巢框。新的育雏梳子和外来皇后两个，原来和新蜂巢建议，以确保类似的分配分开觅食和护士s将会同样经历改变了蜂房线索（"蜂巢闻"）。 </li></ol></li><li> 逆转:早晨，就在回归前，笼女王和育雏梳添加到新的方块，将接收觅食衍生部分。等到觅食高峰时开始。然后将原来的殖民地注明来源为觅食和护士的蜜蜂至少有100米的距离原来的位置。 <ol><li>在原来的位置上，建立了觅食来源的蜂房与育雏，只有女王的新盒子。 </li><li>征粮将离开原脱臼蜂巢框，头回原来的位置。允许觅食返回到2小时的原始位置，以达到下一个完成觅食人口从巢蜂分离。 </li><li>然后，终止分离，关闭掉原来的，现在的"护士衍生的"配置单元，它至少有3公里远转移到一个养蜂场。 </li></ol></li><li> 蜂巢维护和监控对于成功的社会任务反转:定期检查实验荨麻疹健康，开放的育雏。 <ol><li>在殖民地操纵后的第一天更换无人值守和死育雏，以减少潜在的病原体负荷。 </li><li>为了验证成功逆转的觅食来源的蜂房内，采取育雏梳子的图片介绍这些之前，再次当梳子被替换或者当复归试验完成（ 图2）。与以前不封顶的育雏和开放，现场育雏区域是护理活动中觅食的集落可靠标记。 </li></ol></li><li> 采样:觅食和护士后3-8天可以检测伴随社会反转生理效应分离。 <ol><li>我们建议所有取样试验组， 即恢复工人和持续觅食（觅食衍生蜂巢），以及持续的护士和新招聘的觅食（护士衍生蜂巢）8日内开始逆转后。 </li><li>关口如步骤1.5中所述课样品。 </li></ol></li></ol> 3。由脂褐质的定量分析工型特异性细胞衰老模式脂褐质是细胞衰老的一个普遍的生物标志物。作为一个内在的累积产物，脂褐质的特定的自体荧光（发射最大 = 530-650纳米），可用于检测18。 <ol><li> 解剖和固定:冰上冷藏蜜蜂，直到一动不动，删除并剖析出所需的组织样本。 <ol><li>在4℃下过夜孵育;转移到固定剂（pH 7.2的4％多聚甲醛的磷酸盐缓冲盐水，PBS）中</li><li>在PBS中洗涤样品3次。 </li></ol></li><li> 组织处理和安装:通过使用振动切片机刀片， 例如莱卡VT 1000S（徕卡生物系统公司，旅游Nussloch，德国）切割的组织样品与不超过40微米厚的切片，例如。 <ol><li&gt;对于结算，过夜孵育组织切片在30％甘油中（PBS），和另外2小时，在50％甘油中（PBS）中。 </li><li>安装在显微镜玻片切片在50％甘油（PBS）中。对于长期储存密封盖玻片指甲油。 </li></ol></li><li> 图像采集:为了检测脂褐素，我们建议使用激光扫描共聚焦显微镜，提供激光线，λ= 514，561nm或类似的激励，并与探测器的带宽设置为570-650纳米。 <ol><li>为了更好地识别脂褐素，包括第二通道，并做了同步扫描在更短的波长光谱（激发= 405 nm处;排放量= 410-450纳米）。较长波长信道将揭示两者，脂褐素颗粒，但也非特异性的"背景"，由于自身荧光气管等非粒状结构。第二，较短的波长信道将只显示非特异性自体荧光，但不脂褐素。因此，脂褐素标识可以是facili<html>通过比较两个通道的信号，只有其中一人流露颗粒与脂褐素特异性荧光tated。 </li><li>对采集的高分辨率图像使用目的与40X放大率或更高，以及1.25或更高优选的数值孔径。扫描图像叠加约100×100×10μm的3个维度。每一个个人和组织样本必须由多个图像堆栈来表示。 </li><li>为了减少因技术变化个体内和个体间变异，始终保持激光功率和探测器灵敏度恒定。 </li><li>由一天到一天的技术变化减少偏见扫描每个数扫描会话的所有测试组的相等样本数。 </li></ol></li><li>图像处理:使用软件包的模块，允许对显微图像堆栈先进的加工，例如ImageJ的（健康，马里兰州贝塞斯达，美国美国国家研究院​​，gej.nih.gov / IJ /"目标="_blank"&gt; http://imagej.nih.gov/ij/）。 <ol><li>生成一个二维最大投影为每个3D图像堆栈。 </li><li>为了衰减高频噪声，并保持结构与脂褐素颗粒的尺寸适用于一个高斯滤波器具有适中的籽粒大小。 </li><li>合并两个颜色通道，以方便识别脂褐素（见步骤3.2）。 </li></ol></li><li> 图片分析:确保执行量化步骤的主题将是盲目的测试群体认同。 <ol><li>对于所有的图像，先选择感兴趣区（ROI），涵盖了相关结构，并具有类似的尺寸从其他图像中感兴趣区域的区域。 </li><li>然后选择代表每个ROI脂褐素颗粒的期望数量。当选择一个ROI内脂褐素颗粒，下列规则的应用将减少主观偏见。 <ol><li>选择一致的位置，用于选择所述第一麻粒乐。这可以是，例如，一个ROI的最左边，并且该颗粒是最接近的边缘将始终是第一选择。 </li><li>此起彼伏，颗粒选择是最接近先前的选择（下一个邻居）。 </li><li>当选择一个邻居，只移动到一个方向，例如只能从先前的选择搜索的权利。这条规则可以防止该评选是由密密麻麻的颗粒偶尔集群为主。 </li><li>当选择完成后，通过概述和测量各颗粒面积评估每个脂褐素颗粒的大小。使用适当的统计测试，以比较不同的测试组的个体。 </li></ol></li></ol></li></ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+learning+deficits+can+be+reversible+in+honeybees+Apis+mellifera.">Age-related learning deficits can be reversible in honeybees Apis mellifera.</a> Exp Gerontol. 47, 764-772 (2012).</li><li>Capaldi, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+orientation+flight+in+the+honeybee+revealed+by+harmonic+radar.">Ontogeny of orientation flight in the honeybee revealed by harmonic radar.</a> Nature. 403, 537-540 (2000).</li><li>Marco Antonio, D. S., Guidugli-Lazzarini, K. R., do Nascimento, A. M., Simoes, Z. L., Hartfelder, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-mediated+silencing+of+vitellogenin+gene+function+turns+honeybee+(Apis+mellifera)+workers+into+extremely+precocious+foragers.">RNAi-mediated silencing of vitellogenin gene function turns honeybee (Apis mellifera) workers into extremely precocious foragers.</a> Naturwissenschaften. 95, 953-961 (2008).</li><li>Whitfield, C. W., Cziko, A. -. M., Robinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+in+the+brain+predict+behavior+in+individual+honey+bees.">Gene expression profiles in the brain predict behavior in individual honey bees.</a> Science. 302, 296-299 (2003).</li><li>Schmidt, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attraction+of+reproductive+honey+bee+swarms+to+artificial+nests+by+Nasonov+pheromone.">Attraction of reproductive honey bee swarms to artificial nests by Nasonov pheromone.</a> Journal of Chemical Ecology. 20, 1053-1056 (1994).</li><li>De Moraes, R., Bowen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+cell+death+in+the+hypopharyngeal+gland+of+the+honey+bee+(Apis+mellifera+L).">Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera L).</a> Cell Biol Internat. 24, 737-743 (2000).</li><li>Sheehy, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow-cytometric+method+for+quantification+of+neurolipofuscin+and+comparison+with+existing+histological+and+biochemical+approaches.">A flow-cytometric method for quantification of neurolipofuscin and comparison with existing histological and biochemical approaches.</a> Arch Gerontol Geriatr. 34, 233-248 (2002).</li><li>Hsieh, Y. S., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honeybee+trophocytes+and+fat+cells+as+target+cells+for+cellular+senescence+studies.">Honeybee trophocytes and fat cells as target cells for cellular senescence studies.</a> Exp Gerontol. 46, 233-240 (2011).</li><li>Wolschin, F., Munch, D., Amdam, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+proteomic+analyses+reveal+regional+brain+differences+during+honeybee+aging.">Structural and proteomic analyses reveal regional brain differences during honeybee aging.</a> J Exp Biol. 212, 4027-4032 (2009).</li></ol>

我们什么都没有透露。

在这里，我们采用前面所述的方法8,16,17,19,20，并将它们集成到一个单一的工作流程，这将有利于灵活学习老龄化蜜蜂。我们的目标是为科学家是新手，这个领域与标准化的工具集，以取得相关样本材料，并改善不同的研究小组之间的实验重现性。虽然我们的程序简化，不需要特殊设备，在前面的描述（比较例如8），预防的一些措施，建议及以下收集。 从...

高度社会化的动物的复杂的菌落结构通过生殖种性的相互作用维持，并且通常是非再现工人的辅助阶级不同社会任务的行为。在不同的工人，具体的生理适应性使不同的同胞关心的行为，也与极端的寿命差异。蜜蜂和鼹鼠代表了最发达的动物模型，研究如何社会性链接到的加速，可以忽略不计或逆转老化的1-3模式。 在蜜蜂的殖民地，一个产蛋女王是由成千上万的工人，往往育雏，饲料的粮食援助，并参与防守，体温调节或卫生行为4。在这些工人中是极其短暂的征粮，护士蜜蜂与中间，和冬季（diutinus）与蜜蜂的寿命最长。个人，但是，不会永久绑定到一个特定的窝rker型，但会显示一条灵活的行为个体发育:他们从一个社会任务的行为更改为另一种（"时间种姓"）。乳臭未干的蜜蜂可以换到育雏抚育护士蜜蜂，最终可能会改变外界觅食。然而，乳臭未干巢蜜蜂也可以变身为最长寿的冬季蜜蜂和短命觅食，甚至可以恢复到通常寿命更长护士。极端（冬季蜜蜂）和中级（保姆蜂）寿命工人有发达的食品生产和贮藏器官与丰富的资源-而不是短暂的觅食（1,5审查）。然而，个人寿命的调节超越于个人的资源平衡简单的修改建议通过在卵黄蛋白，它具有不同的适应功能，在非再现工人等级，比如果冻生产6，激素控制7，免疫研究8，抗氧化剂防御9。 的f模式unctional下降（衰老）工人镜的寿命差距，为建立嗅觉，同时还为其他脑或运动功能10-13。具体地，仅在两个星期的觅食之后的显著下降学习函数相匹配类似的死亡率级数中觅食14，相对于缺乏可检测的下降（可忽略衰老）的长寿命的冬季蜜蜂15。 为了确定我们适应建立实验范式，使监测和处理老化型转变8,16,17灵活衰老的分子指纹。实验1的详细信息如何获取样品中的实足年龄和老龄工作者类型的具体的社会行为的影响可以分开。实验2描述了征粮与加速进入护士蜜蜂与减缓老化动力学的逆转。实验3提供了解剖学探测细胞衰老的影响的方法已建立的生物标志物细胞衰老（脂褐素）18升量化。

<table><tbody><tr><td>Apifonda</td><td>S&amp;#252;dzucker AG, Mannheim/Ochsenfurt, Germany</td><td></td><td></td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>phosphate-buffered saline</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>Glycerol</td><td>Merck</td><td>1.04094.1000</td><td></td></tr></tbody></table>

obtaining specimens with slowed, accelerated and reversed aging in the honey bee model

高度社会化的动物的社会功能密切相关的个体之间的广袤的寿命差异。其中社会性昆虫，蜜蜂是最好建立的模型，以研究如何可塑性寿命和衰老是由社会因素来解释。 蜜蜂的工人等级包括护士蜜蜂，这往往育雏，并采集蜜蜂，负责收集花蜜和花粉。以前的工作表明，大脑功能和飞行性能的征粮衰老的速度比护士。然而，大脑的功能可以恢复，觅食时恢复到哺乳的任务。加快并逆转衰老的功能，例如模式都与代谢改变资源水平，在蛋白丰度的改变和免疫功能。卵黄，卵黄蛋白与激素控制和细胞防御适应功能，可作为中，控制工人的不同老化动力学网络的主要调控元件。在这里，我们描述了如何护士和觅食的出现可以被监控，和操作，包括逆转，从典型的短命觅食成更长寿命的护士。我们的代表结果表明，与同类实足年龄个体如何分化成蜂和护士蜜蜂实验条件下。我们举例说明如何行为逆转，从觅食回到护士可以进行验证。最后，我们显示如何不同细胞衰老可通过测定脂褐质的积聚，衰老的普遍的生物标记物进行评估。 为了研究可能连结社会影响的机制和老化的可塑性，该协议提供了一个标准化的工具集，以获取有关样品材料，并提高未来的研究中数据的可比性。

协议第1和2详细测试组如何能够获得学习的加速属性，减缓和扭转与一个单一的年龄组菌落老化。为了监视工人型分化，伴随着个体发育正常，我们评估觅食计数（"高考计数"）的6个菌落（ 图1，比较第1节）。该图显示，通常观察不到从护士相当大的变化的觅食状态前的人更超过10天。在觅食计数标记变异观察与问候觅食不同群体之间发生的时机，作为一个显着的一天到一天的每一个?...

Watch this Scientific Journal Video about Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model at JoVE.com

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

在蜜蜂的工人，老化依赖于社会行为，而不是实际年龄。在这里，我们将展示如何工人种类具有非常不同的老化模式可以得到和细胞衰老分析。

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 ...

obtaining-specimens-with-slowed-accelerated-reversed-aging-honey-bee

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.2K 次观看.  Norwegian University of Life Sciences. 在蜜蜂的工人，老化依赖于社会行为，而不是实际年龄。在这里，我们将展示如何工人种类具有非常不同的老化模式可以得到和细胞衰老分析。 

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

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

在蜜蜂的长鼻扩展响应的经典条件反射行为药理学（蜜蜂）

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

科研

神经科学

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 ...

环境科学

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 ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi介导的双基因敲除和味觉感知测量蜜蜂（<em&gt;蜜蜂</em&gt;）

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

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). ...

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies&#160;&#8211; a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

单队列殖民地和工蜂的激素治疗的准备来分析生理学与关联作用和/或内分泌系统

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

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 ...

Histology Basics and Cell Death Detection in Honeybee Tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

蜜蜂组织中的组织学基础和细胞死亡检测

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...