This work was supported by a Grant-in-Aid for Scientific Research (B) and a Grant-in Aid for Scientific Research on Innovative Areas 'Systems Molecular Ethology' from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. T.U. was the recipient of a Grant-Aid from the Japan Society for the Promotion of Science for Young Scientists.

1.单队列殖民地的制备<ol><li>准备三蜂群创建两个单队列菌落并获得足够数量的新出现的工人。 <ol><li>检查在梳皑皑的外周细胞的一些蛹有棕色的眼睛和开放使用镊子封顶梳子色素角质层。如果外围梳状细胞中存在这些蛹，在整个梳子最蛹会出现在大约1-3天。 </li><li>随后，收集所有含有成人贴壁蜂已经用刷子去除后这些蛹的梳子，混合来自三个殖民地梳子，以减少菌落中的变异。 </li></ol></li><li>放置收集梳子在一个空蜂巢框和在33℃潮湿条件下用C（&gt; 60％相对湿度）孵育。收集大约6000新出现过工人从收集的梳子3天（每殖民地3000名工人）。 </li><li>确定借机的量基于五个新兴工人的重量RS随机抽取未经处理的殖民地收集。 <ol><li>重5使用称重机未经处理的殖民地随机抽取新出现的工人。 注:五个新出现的工人的重量一般500-600毫克的。 </li><li>通过将所收集的工作人员的重量由平均重量五个新出现工人估计收集工人的数量。 </li></ol></li><li>涂漆标志使用水性漆的海报标志约1,800工人（每900菌落工人）的thoraces。 注意:可洗的标记被用于施加涂料中列出的材料的表 。 </li><li>介绍一个皇后和大约0-2天的工人3000到包含两个梳子，蜂蜜含有梳1和花粉如腌制食品和产蛋女王另一个空梳一个新的蜂巢框。收集含有蜂蜜和花粉FR梳OM所有贴蜜蜂陆续未经处理的殖民地被删除。 注:建议使用核蜂巢箱（小型蜂巢盒）的。 </li><li>收集护士蜜蜂觅食早熟用油漆标记6〜8天创造的单队列殖民地之后。 <ol><li>为了收集护士蜜蜂，拿起那把他们的头梳进入细胞采取育雏保健工作者。使用镊子从包含许多育雏和成年工人收集梳理护士蜜蜂。 注意:如果收集工人HPGS如步骤1.7中所述解剖时被开发，这些工人被定义为护士蜜蜂。拿住镊子thoraces建议从梳子拿起工人。 </li><li>为了收集征粮，使用防虫网，以捕获返回自己的蜂巢对他们的后腿花粉负载工人。 注意:如果在步骤1.7中所述解剖时拍摄的工人HPGS出现缩水，这些工人被定义为觅食。 </li></ol></li><li> CLASSIFY的HPG分成三组，"开发"，"中间"和"皱缩"通过在立体显微镜下解剖。 <ol><li>麻醉在昆虫笼10-15蜜蜂在冰箱10-15分钟，直到蜜蜂无法飞行或步行。然后，移动蜜蜂在冰上，并完成在冰上麻醉5分钟。 注意:大约需要总15-20分钟达到麻醉。如果麻醉时间需要缩短，蜜蜂在笼子的数目应减少到每笼1-3蜜蜂。 </li><li>用剪刀和镊子解剖从主体的头部。固定放置在培养皿销牙科蜡头。将两个引脚到天线的基地，以固定头部。浸泡固定磁头在昆虫盐水（130毫米氯化钠，5毫米氯化钾，1毫米氯化钙2）。 </li><li>双目显微镜下除去用细镊子头部和手术刀的角质层的前方面。 注:HPGS可以在头部很容易找到去除的C后uticle。该HPG的是，在头部左右脑存在葡萄状的器官。 </li><li>取出气管组织是表皮下的白色膜状组织。然后，用小镊子缓缓地提拉他们解剖从头部HPGS。 </li><li>分类HPGS大和循环腺泡为"发达"。分类HPGS小和歪曲腺泡为"皱缩"。相应的分类既不"发达"，也没有"皱缩"HPGS为"中级"。 注意:代表的照片，显示这三个类HPG状态在图2中表示。 </li><li>主题"发达"和"皱缩"HPGS以RNA提取为"护士蜂HPGS&#39;和&#39;觅食HPGS"，分别和护士蜜蜂觅食（第4节）之间的HPGS比较基因表达。 </li></ol></li></ol> 2. 20E注射<ol><li>从收集典型菌落为DESCR护士蜜蜂IBED在过程1.6.1。 注:HPGS不能在这些蜜蜂进行审查，作为蜜蜂会被饲养。 </li><li>麻醉在4℃蜜蜂在冰箱（未在冰上）。 注意:这需要大约30分钟，使麻醉。如果麻醉时间被缩短，在笼蜜蜂的数目应减少到每笼1-3蜜蜂。 </li><li>使用镊子，固定放置在培养皿上的牙模的蜜蜂。 </li><li>注入1微升20E溶液成使用来自用玻璃针拔出器校准的毛细管微量制成的注射尖端的头部的前方面。 <ol><li>溶解20E在乙醇-昆虫盐水（130毫摩尔NaCl，5mM的氯化钾，1mM的氯化钙2）的混合物（1:4）以2.5微克/微升。 </li><li>喷射末梢连接到橡胶管中，和一个黄色枪头连接到管的相反的一侧。 </li><li>使用微量上放置封口膜1微升20E溶液，保持黄色微量移液器尖端在口中，一个d拉伸溶液进入注射小费。 </li><li>插入喷射尖端进入天线的基础上，并通过经由黄色微量移液器尖端吹注入该溶液。 注意:在此步骤中显示的协议是代表注射方法，但建议在必要时20安全使用自动注射机。 </li></ol></li><li>后在33-36℃的昆虫笼注入蜜蜂（理想温度为35℃）进行1或3天阴暗潮湿条件下（&gt; 60％相对湿度）下。供应蜂蜜 - 水混合物（1:1），以蜜蜂。如果需要的话，算上注射后幸存的蜜蜂。 </li></ol> 3.烯虫酯中的应用<ol><li>收集包含从典型菌落蛹所有附着的成年蜜蜂用刷子去除后，梳子。孵育梳子在33-36℃（理想温度为35℃）在潮湿的条件下为1天（&gt; 60％相对湿度）。 </li><l我&gt;收集从收集的梳新兴蜜蜂和应用的标志漆用海报涂料作为在协议中用于制备单队列菌落描述其thoraces。返回标记的蜜蜂他们的殖民地。 </li><li> 6天后，收集来自克隆彩绘工人（6日龄）。通过举办他们的thoraces用镊子拿起从梳子彩绘工人。 </li><li>麻醉在4℃蜜蜂在冰箱（未在冰上），为在步骤2.2。使用镊子，固定放置在培养皿上的牙模蜜蜂和应用1-5微升溶于丙酮中使用微量头甲氧普林溶液（50-250微克/微升）。使用过滤嘴是向丙酮抗性。 注意:丙酮可能会对蜜蜂导致死亡的有害影响。如果观察蜜蜂的死亡率高，建议丙酮在最小的体积为1微升的降低。 </li><li>后在昆虫笼蜜蜂在33-36°C（理想Ťemperature是35℃）下阴暗潮湿的条件下7天（&gt; 60％相对湿度）。供应蜂蜜 - 水混合物（1:1），以蜜蜂。如果需要的话，算上局部应用后幸存的蜜蜂。 注:在本文中，幸存的蜜蜂申请（ 表3）后7天计数。 </li></ol> 4. RNA提取和定量RT-PCR <ol><li>麻醉蜜蜂和步骤1.7.1说明1.7.4解剖HPGS。 </li><li>收集在干冰上和存储微量​​离心管的解剖HPGS在-80℃直到使用。 </li><li>添加400微升试剂为蛋白质变性。 注:此试剂是市售的（见材料表 ）。 </li><li>使用均质与离心管内适合一个同质化的杵举行电动搅拌机一只手HPG组织。均质化在约300rpm下的旋转速度在冰上打破和裂解组织细胞1分钟。 材料表 ）。 </li><li>加入80微升氯仿拌匀。在室温下孵育5分钟。 </li><li>离心机离心管在14170 XG在4℃下15分钟。将水相转移至新管中。添加异丙醇和1μl糖原（5毫克/毫升）的等体积。孵育在-20℃至少20分钟。 </li><li>离心微量离心管在14170×g离心10分钟，在4℃下，去除上清。 </li><li>加入400微升75％乙醇的颗粒拌匀，在4℃，14170 XG离心离心管15分钟，然后取出上清。重复这些步骤一次。 </li><li>空气干燥沉淀5-10分钟，然后将其溶解于10μl无RNA酶的水。 注:沉淀完全干燥可能导致RNA沉淀的不溶化于水中。因此，这是微湿的沉淀是适当的溶解。 </li><li>使用分光光度计等的NanoDrop或类似测量RNA浓度。 </li><li>通过在37℃温育30分钟以降解任何污染的基因组DNA的治疗500ng的总RNA用DNase I的2.0ü。 </li><li>反向转录的200ng DNA酶I处理过的RNA，并使用市售的试剂盒进行实时PCR（见材料与试剂的表的列表）和基因特异性引物（MRJP2 5&#39;-AAATGGTCGCTCAAAATGACAGA-3&#39;和5 &#39;-ATTCATCCTTTACAGGTTTGTTGC-3&#39;，Hbg3 5&#39;-TACCTGGCTTCGTGTCAAC-3&#39;和5&#39;-ATCTTCGGTTTCCCTAGAGAATG-3&#39;）。执行的PCR如下:[95℃×30秒+（95℃×5秒+ 60℃×15秒+ 72℃×20秒）×45-55个循环]。 </li><li>规范化成绩单量与延伸因子1α-F2（EF1α˗F2）或核糖体蛋白49（RP49）9,16,21,22的。这些基因是可靠的控制功能g烯类在使用QRT-PCR的HPGS分析基因表达。 </li><li>执行双尾t检验来检测使用统计两个软件实验组（护士对蜜蜂觅食早熟，20E治疗蜜蜂与控制蜜蜂和烯虫酯处理与蜜蜂蜜蜂控制）之间的显著差异。 注意:如果F-检验假设方差的同质性，学生t检验可以使用。如果不是，可以使用韦尔奇氏t检验。 </li></ol>

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<li>Pandey, A., Bloch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Juvenile+hormone+and+ecdysteroids+as+major+regulators+of+brain+and+behavior+in+bees%5BTitle%5D)+AND+%22Current+Opinion+in+Insect+Science%22%5BJournal%5D)">Juvenile hormone and ecdysteroids as major regulators of brain and behavior in bees.</a> Current Opinion in Insect Science. 12, 26-37 (2015).</li>
</ol>

The authors have nothing to disclose.

单队列殖民地的制备在这里，我们描述了我们先前的研究16用于为与工蜂的角色转变相关的生理HPG分析准备单队列殖民地的协议。在单队列菌落（ 表1）中观察到护士蜜蜂和早熟觅食出满足在程序1.6-1.7和图2中所描述的标准。在HPG分类为基准规定的图2中的照片是有用的。基于这两个工人行为和HPG发展的标准是有效的，用于定义典型菌落护士蜜蜂觅食或。从护士角色的转变，逐步进行觅食这些殖民地:在护理行为花费的时间逐渐减少，同时，在觅食行为的作用变速2时逐渐增加。因此，这将是很难收集工人该专只有行为准则执行哺乳或觅食。 ，在HPGS MRJP2和Hbg3的表达与工人的角色相关的结果清楚地表明，这里所描述的协议可以采用审查护士蜜蜂觅食早熟从单队列菌落（ 图3）HPG生理学。基于这两个工人行为和HPG发展的标准也可以适用于从单队列菌落界定护士蜜蜂觅食早熟。除了HPGS，在头部其他外分泌腺经受在与角色移关联的生理变化。例如，从脂肪酸的合成为育雏食物报警信息素2-庚酮2的合成下颌腺的变化的功能。这里描述的协议因此可以适应审查除其他HPGS外分泌腺的相关角色的生理。 n个成功采集制备菌落时URSE蜜蜂和早熟觅食从单队列菌落取决于足够数量的新出现的工人的集合。如果收集新出现的工人数量较少，这将是很难收集到足够的护士蜜蜂觅食早熟。此外，女王的条件是成功的样本收集的另一个关键点。用大腹皇后是良好的，因为这些通常皇后开发卵巢产卵。因此，工人的劳动被认为是因为育雏不断被女王提供有效的，即使在单队列殖民地划分。如果没有育雏提供，这将是很难从单一的队列菌落收集护士蜜蜂。最后，使用标线漆海报工人必须确保工人从单队列殖民地收集，因为征粮偶尔会返回错误殖民地。为了有效地收集早熟征粮，油漆痕迹应该是应用到尽可能多的工人可能（超过900每单队列菌落新出现的工人）。可以建议使用用于标记水性油墨的，因为油基墨，其通常含有挥发性有机化合物，可能会破坏蜜蜂之间的化学连通。水性墨痕在实验的过程中并没有褪色非常多。 激素治疗我们还描述的协议注入到20E工人的首长审查蜕皮激素对HPG生理（MRJP2和Hbg3表达）的影响。用昆虫盐水或20E溶液的溶剂注入蜜蜂的存活率为足以用于随后的分析，表明协议此处所述的注射用的注射尖端和溶剂20E溶液可以为20E注射实验（ 表2中可以通过）。此外，在HPGS基因表达的比较20E处理蜜蜂和控制蜜蜂之间表明注射20E改变MRJP2和Hbg3，这两者都与HPG功能（ 图5）的表达。这一结果表明，这里描述的方案将是用于检查在HPG生理学蜕皮激素的作用是有用的，并可以用于检查在头部其他外分泌腺，如下颌腺通过。此外，该喷射方法可用于注射其他化学品通过。例如，这里描述的方案将是合适的用于注射的双链RNA解成用于基因敲除实验磁头。 除了20E中，我们描述一个协议用于施加烯虫酯，这是一个JH类似物。烯虫酯通常适用于腹部。另一方面，烯虫酯施加到工人的头上，在这里。几乎烯虫酯或丙酮的局部应用所有的蜜蜂成活治疗后7天，这表明5微升甲氧普林或丙酮溶液的该应用程序到磁头对工人的存活率（ 表3）的影响不大。此外，在methoprene-和丙酮处理蜜蜂之间HPGS MRJP2和Hbg3表达的比较表明，甲氧保幼素应用抑制MRJP2表达并增强Hbg3表达（ 图6）。这些结果表明，这里所描述的协议是适当的，用于分析在外分泌腺的生理学烯虫酯，如在头部HPGS的影响。 成功的激素治疗取决于处理蜜蜂的存活率。在冰箱中的低温曝光可能是适当的麻醉蜜蜂。不建议在冰上麻醉因为蜜蜂会得到在冰上湿，导致低的存活率。此外，注射法可能是饲养蜜蜂生存的另一个关键点。它是到m重要阿克注射尖端长和薄，并且使用切割机，以减少对蜜蜂通过注射破坏它的形状，以一个尖锐的端部。然而，注射方法可能不适合用于检查上HPG生理学甲氧普林的影响，因为丙酮溶剂为烯虫酯负面影响蜂生存（ 表4）。此外，这里所描述的注射方法可能不适合于作为注射蜜蜂会从它们的菌落被排除当它们被注射后重新引入菌落行为分析。有轻伤的工人可能会被排除在殖民地。其它方法，如激素的应用或口服给药可能是适当的行为分析。 单队列菌落已被用来检查工蜂10,11的分工的调节。此外，与保幼激素治疗已经用于行为和脑泛函的检查对相关劳动11分工。然而，对于单队列菌落的制备和JH的治疗都详细方案先前尚未在这样的细节提供。近日，在分工蜕皮激素的重要性正日益认可，且由JH劳动和蜕皮激素分工的规定，建议23。单队列殖民地和JH激素治疗等，并在这篇文章中描述蜕皮激素的成功编制了详细的协议可能有助于推进在工人生理学研究。

欧洲蜜蜂， 蜜蜂 ，是一种完全社会性昆虫具有高度组织性的社会1。工蜂（劳动等级）从事各种工作，以保持集落的活性，并且这些任务根据羽化后工蜂的年龄，这被称为劳动2-4的年龄相关的分割发生变化。青年工人（&lt;13日龄）照顾在蜂巢的育雏通过分泌蜂王浆（蜂护士），而年纪较大的工人（&gt; 15天）采集花蜜和花粉蜂巢（征粮）2-4之外。工人的生理与这个角色转变的关联变化。例如，下咽腺（HPGS）的功能，配对位于头外分泌腺，改变与护理的作用转变为觅食2,5关联。护士蜂HPGS主要合成王浆主蛋白，是蜂王浆主要成分。另一方面，觅食HPGS主要合成碳水化合物代谢酶，如α葡糖苷酶III，通过将蔗糖转化为葡萄糖和果糖处理花蜜到蜂蜜。我们以前的研究显示，MRJP2的表达，其编码的一个主要蜂王浆蛋白质和Hbg3，它的作用移6-9中编码α葡糖苷酶III，变更。 要确定是否在工人组织中的生理变化，包括HPGS，与角色的转变或与工人的年龄，这将是操纵蜜蜂殖民地的人口组成，如准备单队列有用菌落在几乎相同年龄的工人执行不同的任务10,11。 Robinson 等人。（1989）描述了用于建立单一的队列菌落10的方法。单队列殖民地最初包括女王和0-2日龄工人。建立殖民地几天后，ALM工人OST同龄承担不同的任务。有些工人进行护理任务，如在典型菌落，而其他工作人员执行任务觅食比往年提前，并因此被称为早熟觅食。护士蜜蜂觅食早熟之间的基因表达的比较将提供有关工作的组织12-16的相关角色的生理有用的信息。这里，我们描述了制备单队列菌落HPGS 16的角色和/或与年龄相关的生理分析的详细协议。我们还简要介绍了如何通过定量逆转录-聚合酶链反应（RT-PCR）检查MRJP2和Hbg3的基因的表达来评价HPG生理学。 除了工人生理学单队列菌落的分析，内分泌系统的检查是用于分析相关联的角色的工人生理学的调节机制重要。幼激素（JH），其硝酸钾WN在昆虫幼虫的&#39;现状&#39;的激素，加速从护理的作用，在工蜂11觅食的转变。此外，蜕皮激素，这是变态过程称为蜕皮激素，可能参与的作用移作为编码蘑菇体被表达，工人脑17-19的较高中心蜕皮激素信号传导分子的基因。因此，我们还描述了在我们以前的研究16用于与20E治疗工人的详细协议，它是蜕皮激素的活性形式，和烯虫酯，一JH类似物，为的内分泌系统的上HPG生理效应分析（表达MRJP2和Hbg3）。

<table><tbody><tr><td>UNIPOSCA</td><td>Mitsubishi pencil</td><td>PC-5M</td><td>Marker pen for the application of marks to bees&amp;nbsp;&amp;nbsp;</td></tr><tr><td>20-hydroxyecdysone</td><td>Sigma Aldrich</td><td>H5142</td><td></td></tr><tr><td>Methoprene</td><td>Sigma Aldrich</td><td>33375</td><td></td></tr><tr><td>Breeding case insect</td><td>IRIS OHYAMA</td><td>CP-SS</td><td></td></tr><tr><td>Electromotion mixier&amp;nbsp;</td><td>ISO</td><td>23M-R25</td><td>homogenization of tissue</td></tr><tr><td>TRIZol Reagent</td><td>Invitrogen</td><td>15596-026</td><td>the reagent for total RNA extraction</td></tr><tr><td>DNase I&amp;nbsp;</td><td>Takara</td><td>2270A</td><td></td></tr><tr><td>PrimeScript RT reagent kit</td><td>Takara</td><td>RR037A</td><td>the reagent for reverse transcription</td></tr><tr><td>SYBR Premix ExTaq II</td><td>Takara</td><td>RR820A</td><td>the reagent for real-time PCR</td></tr><tr><td>LightCycle 1.2 Instrument</td><td>Roche</td><td>12011468001</td><td>the instrument for real-time PCR</td></tr><tr><td>LightCycle Capillaries (20&amp;mu;l)</td><td>Roche</td><td>4929292001</td><td>the material for real-time PCR</td></tr></tbody></table>

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.

该协议用 ​​于制备单队列菌落的概述图1A中示出。从制备单队列菌落样品收集实验时间过程示于图1B。该纳护理行为或取食行为的行为准则工人从单队列菌落收集，并且HPG发展这些工人估计表1示出的HPG发展的分类为三组。 "开发"，"中间"和"皱缩"根据大小。示出这三个类HPGS的代表性照片在图2中所示，结果表明，这两个护士蜜蜂和早熟觅食，根据在协议中所述的标准，也以单队列菌落观察。在HPGS MRJP2和Hbg3的表达是恩富硒分别护士蜜蜂和早熟征粮，表明HPG功能与工人的角色（ 图3）16有关。 用于注射20E到工人头上的程序在图4中示出。存活蜜蜂计数1，注射以检查存活后5至7天 用昆虫盐水或溶剂为20E实验前20E溶液注入蜜蜂速率。在注射后7天，注射蜜蜂约60-80％存活，而用溶剂为20E溶液注入蜜蜂存活率比得上用昆虫盐水（ 表2）。的20E喷射的效果降低两者MRJP2和Hgb3在HPGS的表达（ 图5）16。除了​​20E，局部应用烯虫酯对MRJP2和Hbg3效果</eM&gt;进行了检查在HPGS表达。通过局部施用与methoprene-或丙酮处理蜜蜂约70-100％的应用（ 表3）后存活7天。另一方面，只蜜蜂的约20％至30％的注射丙酮/蜂盐水（1:4）溶液存活注射后7天，当存活蜜蜂计数1，3，注射后5和7天（ 表4） 。因此，对于烯虫酯治疗中的应用方法获得通过。抑制甲氧普林的应用MRJP2的表达和增强Hbg3的表达（ 图6）16。 <img alt="图1" src="/files/ftp_upload/54240/54240fig1.jpg" /> 图1. 概述单队列殖民地的实验。（A）计划准备单队列殖民地。 （B）试验的时间过程从preparin摹单队列殖民地收集护士蜜蜂觅食早熟。水平箭头表示在单队列殖民地工人的一生中。与垂直线的数字表示工人的年龄。不仅早熟觅食而且过时效护士蜜蜂（28-32天龄）报告于单队列菌落15被发现。因此，超龄护士蜜蜂觅食和之间的生理比较可以在单队列菌落进行，尽管这不是在这项研究中完成的。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54240/54240fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54240/54240fig2.jpg" /> 图2. 照片显示三类HPG状态。开发（A），中级（B）和皱缩（C </str翁&gt;）HPGS在步骤中描述的协议的1.7.5示于该图中。每班HPG的三种典型照片示。在每个子图下图表示的虚线正方形区域在上面板的放大视图。比例尺= 500微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54240/54240fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54240/54240fig3.jpg" /> 图3. 角色相关 的工人从单队列殖民地HPGS MRJP2 和 Hbg3 的表达 。该图显示护士蜜蜂（N）和早熟觅食（PF）之间的HPGS MRJP2（A）和Hbg3（B）的mRNA水平的两个单队列合作的比较lonies。每个基因的mRNA水平与核糖体蛋白49（RP49）的归一化。在护士蜂HPGS各基因的相对mRNA水平被定义为1 MRJP2在一些早熟觅食的mRNA水平不能因为信号强度低于检测阈值的量化。因此，采样的数目的基因是不同的。相对mRNA水平以表示标准错误。星号表示护士蜜蜂觅食早熟之间显著的差异（* P &lt;0.05; t检验）。这个数字已经从上野等人转载，（2015）16。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54240/54240fig3large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54240/54240fig4.jpg" /> 图4 的协议20E injec方案化，程序2.3，并在该协议第2.4节在本图所示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54240/54240fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54240/54240fig5.jpg" /> 图 20E注射液对 MRJP2 和 Hbg3 表达 5.图形 效果 在HPGS 16。该图显示MRJP2的mRNA水平的比较 在20E处理蜜蜂（20E +）和控制蜜蜂（20E-）之间的HPGS（A）和Hbg3（B）。在图表中的数字表示20E治疗后的第二天。每个基因的mRNA水平与延伸因子1α-F2（EF1α-F2）的归一化。相对mRNA水平在控制蜂HPGS各基因被定义为1的相对mRNA表达水平与标准误差表示。星号表示20E处理蜜蜂和蜜蜂控制之间的差异显著（*，P &lt;0.05; t检验）。这个数字已经从上野等人转载，（2015）16。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54240/54240fig5large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54240/54240fig6.jpg" /> 图 6. MRJP2 和 Hbg3 表达 烯虫酯应用的影响 在HPGS 16。该图显示MRJP2（A）和Hbg3（B）的mRNA水平的保幼激素治疗的蜜蜂和蜜蜂控制之间的HPGS的比较。该每个基因的mRNA水平与EF1α-F2的标准化。被定义在控制蜜蜂HPGS每个基因的相对mRNA水平为1的相对mRNA水平与标准误差表示。星号表示烯虫酯治疗蜜蜂和蜜蜂控制之间的差异显著（*，P &lt;0.05; t检验）。这个数字已经从上野等人转载，（2015）16。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54240/54240fig6large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2">殖民地号</td><td rowspan="2">观察到的行为</td><td colspan="3">个人在每个HPG类Number </td><td></td></tr><tr><td>发达</td><td>中间</td><td>瘪</td><td>总</td></tr><tr><td rowspan="2"> 1 </td><td>看护</td><td> 8 </td><td> 1 </td><td> 1 </td><td> 10 </td></tr><tr><td>觅食</td><td> 0 </td><td> 4 </td><td> 7 </td><td> 11 </td></tr><tr><td rowspan="2"> 2 </td><td>看护</td><td> 4 </td><td> 2 </td><td> 0 </td><td> 6 </td></tr><tr><td>觅食</td><td> 0 </td><td> 0 </td><td>五</td><td>五</td></tr></tbody></table> 表1:工人的护理或觅食行为在单队列菌落观察HPGS分类 。 HPG大小在从两个单队列菌落工人（菌落号1和2）被分为三类; "发达"，"中级"和"皱缩"按大小。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td>天注射后</td><td>没有注入</td><td>昆虫生理食盐水</td><td>溶剂20E SOLN </td></tr><tr><td rowspan="4">试验1 </td><td> Day0 </td><td> 15/15 </td><td> 15/15 </td><td> 14/15 </td></tr><tr><td>第一天</td><td> 15/15 </td><td> 14/15 </td><td> 13/15 </td></tr><tr><td>第五天</td><td> 14/15 </td><td> 13/15 </td><td> 11/15 </td></tr><tr><td>第七天</td><td> 14/15 </td><td> 12/15 </td><td> 9/15 </td></tr><tr><td rowspan="4">试验2 </td><td> Day0 </td><td> 15/15 </td><td> 15/15 </td><td> 15/15 </td></tr><tr><td>第一天</td><td> 15/15 </td><td> 13/15 </td><td> 15/15 </td></tr><tr><td>第五天</td><td> 15/15 </td><td> 11/15 </td><td> 12/15 </td></tr><tr><td>第七天</td><td> 14/15 </td><td> 10/15 </td><td> 12/15 </td></tr></tbody></table> 表2:用溶剂注射液20E蜜蜂成活率 。 在每个实验组中制备十五工人。 <table border="1"FO:保together.within页="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td></td><td>几天后申请</td><td>丙酮</td><td>烯虫酯</td></tr><tr><td>试验1 </td><td> 0天</td><td> 7/7 </td><td> 8/8 </td></tr><tr><td></td><td> 7天</td><td> 7/7 </td><td> 6/8 </td></tr><tr><td>试验2 </td><td> 0天</td><td> 7/7 </td><td> 8/8 </td></tr><tr><td></td><td> 7天</td><td> 7/7 </td><td> 8/8 </td></tr></tbody></table> 表3:与各实验组制备七至八个工人烯虫酯治疗蜜蜂的成活率 。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>天注射后</td><td>没有注入</td><td>丙酮/蜂盐水（1:4） </td></tr><tr><td> 0天</td><td> 15/15 </td><td> 15/1五</td></tr><tr><td> 1天</td><td> 15/15 </td><td> 6/15 </td></tr><tr><td>第3天</td><td> 15/15 </td><td> 5/15 </td></tr><tr><td>第5天</td><td> 15/15 </td><td> 4/15 </td></tr><tr><td> 7天</td><td> 15/15 </td><td> 4/15 </td></tr></tbody></table> 表4:（4 1）解决方案 ，各实验组制备十五工人用丙酮/只注射生理盐水蜜蜂成活率 。丙酮/蜂盐水（1:4）溶液注射到工人的腹部。蜂盐水的组合物，是130毫摩尔NaCl，6毫氯化钾，4mM的MgCl 2的，2.5mM的氯化钙 ，160毫蔗糖，25mM的葡萄糖和10mM HEPES（pH 7.0）中。

Watch this Scientific Journal Video about Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System at JoVE.com

工蜂单群菌落的制备和激素处理以分析与角色和/或内分泌系统相关的生理学|神经科学 |视频

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

在这里，我们描述详细的协议单队列蜂群的准备 - 分析相关角色工人生理的有用工具。我们还描述了用于治疗工人幼激素和蜕皮激素评估这些激素在工作者行为和/或生理调控的参与详细协议。

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 ...

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15.8K 次观看.  The University of Tokyo. 在这里，我们描述详细的协议单队列蜂群的准备 - 分析相关角色工人生理的有用工具。我们还描述了用于治疗工人幼激素和蜕皮激素评估这些激素在工作者行为和/或生理调控的参与详细协议。 

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

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 ...

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神经科学

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

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

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 ...

环境科学

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 ...

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 ...

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired glands that are located in the head of the bee are highly sensitive to the quantity and quality of pollen and pollen substitutes that the nurse bee consumes. The glands get smaller when nurses are fed deficient diets and are large when they are fed complete diets. Because nurse hypopharyngeal gland size is a robust indicator of nurse nutrition, it is essential that those studying honey bee nutrition know how to measure these glands. Here, we provide detailed methods for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal glands. We present comparisons of unstained and stained tissue and data that were used to study the impact of pollen on gland size. This method has been used to test how diet impacts hypopharyngeal gland size but has further use for understanding the role of these glands in hive health.

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers

Hypopharyngeal gland acinus size is a robust measure of nurse honey bee nutrition. Here, we provide a detailed protocol for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal gland acini.

蜂蜜蜂 (api 蜜蜂) 中下咽腺性腺泡大小的测定

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired ...

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). ...

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 ...

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 ...

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 ...