Name: preparation of single-cohort colonies and hormone treatment of worker honeybees to analyze physiology associated with role and/or endocrine system
Uploaded: 2016-09-06T13:00:00
Description: 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

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>...

<ol>
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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...

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 border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">UNIPOSCA</td>
			<td style="width:120px;">Mitsubishi pencil</td>
			<td style="width:91px;">PC-5M</td>
			<td style="width:327px;">Marker pen for the application of marks to bees&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20-hydroxyecdysone</td>
			<td>Sigma Aldrich</td>
			<td>H5142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methoprene</td>
			<td>Sigma Aldrich</td>
			<td>33375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Breeding case insect</td>
			<td>IRIS OHYAMA</td>
			<td>CP-SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electromotion mixier&#160;</td>
			<td>ISO</td>
			<td>23M-R25</td>
			<td>homogenization of tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIZol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>the reagent for total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I&#160;</td>
			<td>Takara</td>
			<td>2270A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PrimeScript RT reagent kit</td>
			<td>Takara</td>
			<td>RR037A</td>
			<td>the reagent for reverse transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR Premix ExTaq II</td>
			<td>Takara</td>
			<td>RR820A</td>
			<td>the reagent for real-time PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycle 1.2 Instrument</td>
			<td>Roche</td>
			<td>12011468001</td>
			<td>the instrument for real-time PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycle Capillaries (20&#956;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?...

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

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 ...

The overall goal of these procedures is to examine the physiology of worker honeybees in association with the workers'role and the status of their endocrine system. It's a method which has been widely used for the analysis of worker behavior, can also help answer key questions about the relationship between physiology and the role of worker honeybees. The main advantage of this technique is that it is relatively easy to perform.To create two single-cohort colonies, begin with three standard colonies and collect newly-emerged worker bees from each of the three. Using tweezers, regularly inspect the colonies for the appearance of puby in the capped peripheral cells with brown eyes and pigmented cuticles. Once found, most of the puby will emerge in the next few days.Then brush the adults off the combs and remove the combs from the colony. Combine one such comb from each of the three colonies into an empty nuclear hive box. Establish two such hive boxes and incubate them at 33 degrees Celsius with a relative humidity of at least 60%Over the next three days, collect about 3, 000 newly-emerged workers from each new hive box.To approximate the number of bees in each collection, weigh five randomly selected newly-emerged workers and use their average mass compared to the mass of the collected workers to estimate the number of bees. Prepare new hive boxes with a comb that is loaded with honey and pollen from an unmanipulated colony and another comb that is empty. Next, label the thoraxes of approximately 900 workers in each new colony using water-based poster paint markers.Then transfer the collection of worker bees into these new hive boxes and add one queen. Six to eight days after setting up the single-cohort colonies, collect some of their nurse bees and precocious foragers for analysis. To collect the nurse bees, use tweezers to select bees that put their heads into comb cells to care for the brood.Pick them up by the thorax. To collect foragers, use an insect net to capture workers that return to their hives with pollen loads on their hind legs. To classify the HPGs of honeybees, transfer a collection of 10 to 15 bees to an insect cage and anesthetize them at four degrees Celsius for 10 to 15 minutes until the bees cannot fly or walk.Then transfer the bees onto ice to cool them into a completely anesthetized state after about five minutes. Then leave the bees on ice until needed. Now, dissect their heads from their bodies with scissors and tweezers.In a Petri dish, fix the heads into dental wax using insect pins. To do this, use two pins inserted into the base of the antennae. Now, soak the fixed heads in insect saline and transfer the dish to a stereoscope.Using fine tweezers and a surgical knife, remove the anterior cuticle from the head to access the areas around the brain. Next, remove the white membranous tracheal tissue under the cuticle to expose the HPGs. Then dissect the HPGs from the head by slowing pulling them out using tweezers.Classify the HPGs with large and circular aseni developed and consider them to be from nurse bees. Classify HPGs with small and distorted aseni as shrunken and consider these to come from forager bees. Classify HPGs corresponding to neither developed nor shrunken as intermediate.Use the developed and shrunken HPGs for RNA extraction to compare their gene expression. Collect nurse bees from typical colonies as previously described. Anesthetize the nurse bees at four degrees Celsius for about 30 minutes.Then using tweezers, immobilize the bees on dental wax on a Petri dish. Once immobilized, inject the heads of the nurse bees with one microliter of 20E solution. To do this, use a calibrated capillary micropipette.Connect the injection tip to a rubber tube and connect the 200 microliter micropipette tip to the opposite end of the tube. Next, load one microliter of 20E solution onto a piece of parafilm. Then use mouth aspiration to draw the solution into the injection tip.Next, insert the injection tip into the base of the antennae and inject the solution by exhaling on the tube. Alternatively, use an automated injection machine if available. Rear the injected bees in insect cages at 35 degrees Celsius under dark and humid conditions.Include honey water for nourishment and track survivorship as needed. Collect combs containing puby from a colony and incubate them at 35 degrees Celsius with 60%or more relative humidity for up to one day. Collect the newly-emerged bees from the combs and apply paint marks to their thoraxes using poster paint.Then return these bees to their colonies. After six days, collect the painted workers from the colonies and anesthetize them at four degrees Celsius for 30 minutes. Once anesthetized, immobilize the bees using tweezers and dental wax as previously described.Then apply one to five microliters of methoprene and acetone onto their heads using a micropipette resistant to acetone. Rear the bees in cages as previously described. If a high mortality is observed, reduce the volume of acetone to one microliter.Workers that satisfy the behavioral criteria for nursing behavior or foraging behavior were collected from single-cohort colonies and their HPG development was estimated. Based on the HPG RNA extractions and quantitative RTPCR, expression of MRJP2 was enriched in nurse bees and Hbg3 was enriched in precocious foragers. Selected workers were injected with 20E and surviving bees were counted.Seven days after injection, approximately 60%to 80%of injected bees were alive. The control injection had similar results. Quantitative RTPCR analysis show that the 20E injection decreased expression of MRJP2 in the HPGs.The same occurred with Hbg3 in the HPGs. In addition to 20E, the effect of topical methoprene application on MRJP2 and Hbg3 expression in the HPGs was examined. After topical application, most bees survived to seven days.RNA harvested from these survivors show that application of methoprene inhibited the expression of MRJP2 and enhanced the expression of Hbg3. After watching this video, you should have a good understanding of how to prepare single-cohort colonies and treat the bees with hormone in order to analyze the worker physiology associated with roles and endocrine system. Once mastered, the and the classification of HPG can be done in five minutes if it is performed properly.While attempting the injection for hormone solution, it's important to remember to minimize the damage of bees by anesthesia and injection in order to keep bees alive when reared. Don't forget that working with honeybees can be hazardous and precautions such as wearing bee suits and gloves should always be taken while performing collection of bees and manipulation of colonies.

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SCIENTISTS IN ACTION

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

科研

神经科学

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

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During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

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The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

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Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition

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We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) ...

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

在在线 MRI 图像的指导下,对流增强将视遗传阿德诺相关病毒载体输送到 Rhesus Macaque 的皮质

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Role of Diffusion MRI Tractography in Endoscopic Endonasal Skull Base Surgery

扩散MRI造影术在内窥镜内窥镜颅骨基础外科中的作用

Endoscopic endonasal surgery has gained a prominent role in the management of complex skull base tumors. It allows the resection of a large group of ...

超声引导下穿刺松解术联合皮质类固醇注射

Ultrasound-Guided Needle Release Combined with Corticosteroid Injection for the Treatment of Supinator Syndrome

作者聚焦： 超声引导下松针联合皮质类固醇注射治疗旋后综合征  

超声引导下穿刺释放联合皮质类固醇注射治疗后导管综合征

The two main branches of the radial nerve (RN) are the deep branch (DBRN) and the superficial branch (SBRN). The RN splits into two main branches at the ...