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. 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...

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

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 ...

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