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. Preparation of Single-cohort Colonies
<ol>
	<li>Prepare three honeybee colonies to create two single-cohort colonies and to obtain a sufficient number of newly emerged workers.
	<ol>
		<li>Check that some pupae in the capped peripheral cells in the combs have brown eyes and a pigmented cuticle by opening the capped combs using tweezers. If these pupae exist in peripheral comb cells, most of pupae in the whole combs will emerge in approximately 1-3 days.</li>
		<li>Subsequently, collect the combs containing these pup...

<ol>
	<li>Wilson, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The insect societies. , (1971).</li><li>Winston, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The biology of the honey bee. , (1987).</li><li>Lindauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ein+Beitrag+zur+Frage+der+Arbeitsteilung+im+Bienenstaat.">Ein Beitrag zur Frage der Arbeitsteilung im Bienenstaat.</a> Zeitschrift f&#252;r vergleichende Physiologie. 34 (4), 299-345 (1952).</li><li>Sakagami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untersuchungen+uber+die+Arbeitsteilung+in+einen+Zwergvolk+der+Honigbienen.+Beitrage+zur+Biologie+des+Bienenvolkes,+Apis+mellifera+L.">Untersuchungen uber die Arbeitsteilung in einen Zwergvolk der Honigbienen. Beitrage zur Biologie des Bienenvolkes, Apis mellifera L.</a> I. Jap J Zool. 11, 117-185 (1953).</li><li>Kubo, T., 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=Change+in+the+expression+of+hypopharyngeal-gland+proteins+of+the+worker+honeybees+(Apis+mellifera+L.)+with+age+and/or+role.">Change in the expression of hypopharyngeal-gland proteins of the worker honeybees (Apis mellifera L.) with age and/or role.</a> J Biochem. 119 (2), 291-295 (1996).</li><li>Ohashi, K., Natori, S., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Change+in+the+mode+of+gene+expression+of+the+hypopharyngeal+gland+cells+with+an+age-dependent+role+change+of+the+worker+honeybee+Apis+mellifera+L.">Change in the mode of gene expression of the hypopharyngeal gland cells with an age-dependent role change of the worker honeybee Apis mellifera L.</a> Eur J Biochem. 249 (3), 797-802 (1997).</li><li>Ohashi, K., Natori, S., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+amylase+and+glucose+oxidase+in+the+hypopharyngeal+gland+with+an+age-dependent+role+change+of+the+worker+honeybee+(Apis+mellifera+L).">Expression of amylase and glucose oxidase in the hypopharyngeal gland with an age-dependent role change of the worker honeybee (Apis mellifera L).</a> Eur J Biochem. 265 (1), 127-133 (1999).</li><li>Ohashi, K., Sawata, M., Takeuchi, H., Natori, S., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+cDNA+and+analysis+of+expression+of+the+gene+for+alpha-glucosidase+from+the+hypopharyngeal+gland+of+the+honeybee+Apis+mellifera+L.">Molecular cloning of cDNA and analysis of expression of the gene for alpha-glucosidase from the hypopharyngeal gland of the honeybee Apis mellifera L.</a> Biochem Biophys Res Commun. 221 (2), 380-385 (1996).</li><li>Ueno, T., Nakaoka, T., Takeuchi, H., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+expression+in+the+hypopharyngeal+glands+of+worker+honeybees+(Apis+mellifera+L.)+associated+with+an+age-dependent+role+change.">Differential gene expression in the hypopharyngeal glands of worker honeybees (Apis mellifera L.) associated with an age-dependent role change.</a> Zoolog Sci. 26 (8), 557-563 (2009).</li><li>Robinson, G. E., Page, R. E., Strambi, C., Strambi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+and+genetic+control+of+behavioral+integration+in+honey+bee+colonies.">Hormonal and genetic control of behavioral integration in honey bee colonies.</a> Science. 246 (4926), 109-112 (1989).</li><li>Sullivan, J. P., Fahrbach, S. E., 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=Juvenile+hormone+paces+behavioral+development+in+the+adult+worker+honey+bee.">Juvenile hormone paces behavioral development in the adult worker honey bee.</a> Horm Behav. 37 (1), 1-14 (2000).</li><li>Ben-Shahar, Y., Robichon, A., Sokolowski, M. B., 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=Influence+of+gene+action+across+different+time+scales+on+behavior.">Influence of gene action across different time scales on behavior.</a> Science. 296 (5568), 741-744 (2002).</li><li>Lehman, H. K., 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=Division+of+labor+in+the+honey+bee+(Apis+mellifera):+the+role+of+tyramine+beta-hydroxylase.">Division of labor in the honey bee (Apis mellifera): the role of tyramine beta-hydroxylase.</a> J Exp Biol. 209 (Pt 14), 2774-2784 (2006).</li><li>Mutti, N. S., Wang, Y., Kaftanoglu, O., 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=Honey+bee+PTEN--description,+developmental+knockdown,+and+tissue-specific+expression+of+splice-variants+correlated+with+alternative+social+phenotypes).">Honey bee PTEN--description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes).</a> PLoS One. 6 (7), e22195 (2011).</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 (5643), 296-299 (2003).</li><li>Ueno, T., Takeuchi, H., Kawasaki, K., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+Gene+Expression+Profiles+of+the+Hypopharyngeal+Gland+of+Worker+Honeybees+in+Association+with+Worker+Behavior+and+Hormonal+Factors.">Changes in the Gene Expression Profiles of the Hypopharyngeal Gland of Worker Honeybees in Association with Worker Behavior and Hormonal Factors.</a> PLoS One. 10 (6), e0130206 (2015).</li><li>Paul, R. K., Takeuchi, H., Matsuo, Y., Kubo, T. <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+of+ecdysteroid-regulated+gene+E74+of+the+honeybee+in+ovary+and+brain.">Gene expression of ecdysteroid-regulated gene E74 of the honeybee in ovary and brain.</a> Insect Mol Biol. 14 (1), 9-15 (2005).</li><li>Takeuchi, H., 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=Identification+of+a+novel+gene,+Mblk-1,+that+encodes+a+putative+transcription+factor+expressed+preferentially+in+the+large-type+Kenyon+cells+of+the+honeybee+brain.">Identification of a novel gene, Mblk-1, that encodes a putative transcription factor expressed preferentially in the large-type Kenyon cells of the honeybee brain.</a> Insect Mol Biol. 10 (5), 487-494 (2001).</li><li>Takeuchi, H., Paul, R. K., Matsuzaka, E., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EcR-A+expression+in+the+brain+and+ovary+of+the+honeybee+(Apis+mellifera+L).">EcR-A expression in the brain and ovary of the honeybee (Apis mellifera L).</a> Zoolog Sci. 24 (6), 596-603 (2007).</li><li>Yamane, T., Miyatake, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+female+mating+receptivity+and+activation+of+oviposition+in+two+Callosobruchus+species+due+to+injection+of+biogenic+amines.">Reduced female mating receptivity and activation of oviposition in two Callosobruchus species due to injection of biogenic amines.</a> Journal of Insect Physiology. 56 (3), 271-276 (2010).</li><li>Ben-Shahar, Y., Leung, H. T., Pak, W. L., Sokolowski, M. B., 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=cGMP-dependent+changes+in+phototaxis:+a+possible+role+for+the+foraging+gene+in+honey+bee+division+of+labor.">cGMP-dependent changes in phototaxis: a possible role for the foraging gene in honey bee division of labor.</a> J Exp Biol. 206 (Pt 14), 2507-2515 (2003).</li><li>Danforth, B. N., Ji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elongation+factor-1+alpha+occurs+as+two+copies+in+bees:+implications+for+phylogenetic+analysis+of+EF-1+alpha+sequences+in+insects.">Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects.</a> Mol Biol Evol. 15 (3), 225-235 (1998).</li><li>Pandey, A., Bloch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juvenile+hormone+and+ecdysteroids+as+major+regulators+of+brain+and+behavior+in+bees.">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>

Preparation of single-cohort colonies
Here we described the protocol used in our previous study16 to prepare single-cohort colonies for analysis of HPG physiology associated with the shift in the role of worker bees. Nurse bees and precocious foragers that satisfied the criteria described in procedures 1.6-1.7 and Figure 2 were observed in the single-cohort colonies (Table 1). The photographs in Figure 2 would be useful in classific...

The European honeybee, Apis mellifera, is a eusocial insect with a highly organized society1. Worker honeybees (labor caste) are engaged in various tasks to maintain colony activity, and these tasks change according to the worker honeybee&#39;s age after eclosion, which is referred to as age-related division of labor2-4. Young workers (&#60;13 days old) take care of the brood in the hive by secreting royal jelly (nurse bees), while older workers (&#62;15 days old) collect nectar and pollen outside of the hive (foragers)2-4. The physiology of the workers changes in association with this role shift. For example, the function of the hypopharyngeal glands (HPGs), paired exocrine glands located in the head, changes in association with the role shift from nursing to foraging2,5. Nurse bee HPGs mainly synthesize major royal jelly proteins, which are major components of bee milk. On the other hand, forager HPGs mainly synthesize carbohydrate-metabolizing enzymes, such as &#945;-glucosidase III, to process nectar into honey by converting sucrose into glucose and fructose. Our previous studies revealed that the expression of mrjp2, which encodes a major royal jelly protein, and Hbg3, which encodes &#945;-glucosidase III, changes during the role shift6-9.
To determine whether the physiological changes in the worker tissues, including the HPGs, is associated with the role shift or with the age of the workers, it would be useful to manipulate the population composition of a honeybee colony, such as to prepare single-cohort colonies in which workers of almost the same age perform different tasks10,11. Robinson et al. (1989) described a method for establishing a single-cohort colony10. Single-cohort colonies initially comprise a queen and 0-2 day old workers. Several days after establishing the colonies, workers of almost the same age assume different tasks. Some workers perform nursing tasks as in typical colonies, whereas other workers perform foraging tasks earlier than usual and are thus called precocious foragers. Gene expression comparisons between nurse bees and precocious foragers would provide useful information about the role-associated physiology of worker tissues12-16. Here, we describe a detailed protocol for preparing single-cohort colonies for analysis of the role- and/or age-associated physiology of HPGs16. We also briefly describe how to examine the gene expression of mrjp2 and Hbg3 by quantitative reverse transcription-polymerase chain reaction (RT-PCR) to evaluate HPG physiology.
In addition to the analysis of worker physiology in single-cohort colonies, examination of the endocrine system is important for analyzing the regulatory mechanisms of role-associated worker physiology. Juvenile hormone (JH), which is known as the &#39;status quo&#39; hormone in insect larvae, accelerates the shift in the role from nursing to foraging in worker honeybees11. Furthermore, ecdysone, which is known as the molting hormone during metamorphosis, might be involved in the role shift as genes encoding ecdysone signaling molecules are expressed in the mushroom bodies, a higher center of the worker brain17-19. Therefore, we also describe the detailed protocol used in our previous study16 to treat workers with 20E, which is an active form of ecdysone, and methoprene, a JH analogue, for analysis of the effect of the endocrine system on HPG physiology (expression of mrjp2 and 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.

An overview of the protocol for preparing single-cohort colonies is illustrated in Figure 1A. Time-course of experiments from preparing single-cohort colonies to sample collection is shown in Figure 1B. Workers that satisfied the behavioral criteria for nursing behavior or foraging behavior were collected from single-cohort colonies, and HPG development was estimated in these workers. Table 1 shows the classification of HPG development in...

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

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.

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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15.7K Aufrufe.  The University of Tokyo. 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 sta...

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