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. Fremstilling af Single-kohorte Kolonier <ol><li> Forbered tre honningbikolonier at oprette to single-kohorte kolonier og at opnå et tilstrækkeligt antal nyligt dukket arbejdstagere. <ol><li> Kontroller, at nogle pupper i udjævningen perifere celler i kamme har brune øjne og en pigmenteret neglebånd ved at åbne udjævnede kamme med en pincet. Hvis der disse pupper i perifere kam celler, vil de fleste af pupper i hele kamme dukke op i cirka 1-3 dage. </li><li> Efterfølgende indsamle kamme indeholder disse pupp...

<ol>
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Udarbejdelse af single-kohorte kolonier Her beskrev vi den anvendte protokol i vores tidligere undersøgelse 16 til fremstilling single-kohorte kolonier til analyse af HPG fysiologi forbundet med skiftet i rollen som bier. Sygeplejerske bier og fremmelige finsnittere der opfyldte kriterierne i procedurerne 1,6-1,7 og figur 2 blev observeret i de enkelt-kohorte kolonier (tabel 1). Fotografierne i figur 2 ville være nyttig i klassi...

Den europæiske honningbi, Apis mellifera, er en eusocial insekt med en velorganiseret samfund 1. Worker honningbier (arbejdskraft kaste) er engageret i forskellige opgaver for at sikre koloni aktivitet, og disse opgaver ændres i henhold til arbejdstageren honningbien alder efter eclosion, der omtales som aldersrelateret arbejdsdeling 2-4. Unge arbejdstagere (&lt;13 dage gamle) tager sig af yngel i bikuben ved at udskille gelé royal (sygeplejerske bier), mens ældre arbejdstagere (&gt; 15 dage gamle) indsamle nektar og pollen uden for bikuben (finsnittere) 2-4. Fysiologi arbejderne ændringer i forbindelse med denne rolle skift. For eksempel funktionen af de hypopharyngeal kirtler (HPGs), parret eksokrine kirtler placeret i hovedet, ændringer i forbindelse med rollen skift fra sygepleje til fouragering 2,5. Sygeplejerske bi HPGs hovedsageligt syntetisere store Gelée royale proteiner, som er vigtige bestanddele af bi mælk. På den anden side, finsnittere HPGs hovedsageligtsyntetisere kulhydrat-metaboliserende enzymer, såsom α-glucosidase III, til at behandle nektar til honning ved at omdanne saccharose til glucose og fructose. Vores tidligere undersøgelser viste, at ekspressionen af mrjp2, som koder for et større gelée royale protein og Hbg3, som koder α-glucosidase III, ændringer i den rolle skift 6-9. For at bestemme om de fysiologiske ændringer i worker væv, herunder HPGs, er forbundet med den rolle skift eller med en alder af arbejderne, ville det være nyttigt at manipulere befolkningen ifølge en honningbi koloni, såsom at fremstille enkelt-kohorten kolonier, hvor arbejdstagerne på næsten samme alder udføre forskellige opgaver 10,11. Robinson et al. (1989) beskrevet en metode til indførelse af et enkelt-kohorte koloni 10. Single-kohorte kolonier omfatter indledningsvis en dronning og 0-2 dage gamle arbejdere. Flere dage efter oprettelse kolonierne, arbejderne i almost på samme alder antager forskellige opgaver. Nogle arbejdere udfører sygepleje opgaver som i typiske kolonier, mens andre arbejdstagere udfører fouragering opgaver tidligere end normalt og derfor kaldes fremmelige finsnittere. Genekspression sammenligninger mellem sygeplejerske bier og fremmelige finsnittere vil give nyttige oplysninger om den rolle-associerede fysiologi arbejdstager væv 12-16. Her beskriver vi en detaljeret protokol til at forberede enkelt kohorte kolonier til analyse af rolle- og / eller alder-associeret fysiologi HPGs 16. Vi har også kort beskrive, hvordan at undersøge genekspression af mrjp2 og Hbg3 ved kvantitativ revers transkription-polymerasekædereaktion (RT-PCR) for at evaluere HPG fysiologi. Ud over analysen af ​​arbejdstager fysiologi i single-kohorte kolonier, undersøgelse af det endokrine system er vigtigt for at analysere de regulatoriske mekanismer rolle-associeret arbejdstager fysiologi. Juvenil hormon (JH), som er known som &quot;status quo&quot; hormon i insektlarver, accelererer skiftet i rollen fra sygepleje til fouragere i arbejdsbier 11. Endvidere ecdyson, der er kendt som den molting hormon under metamorfose, kan være involveret i rollen skift som gener, der koder ecdyson signalmolekyler udtrykkes i svampen organer, et højere center af arbejderen hjerne 17-19. Derfor beskriver vi også den detaljerede protokol, der anvendes i vores tidligere undersøgelse 16 til behandling af arbejdere med 20E, som er en aktiv form for ecdyson og methopren, en JH-analog, til analyse af virkningen af det endokrine system på HPG fysiologi (ekspression af mrjp2 og 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.

En oversigt over protokollen for at forberede enkelt kohorte kolonier er illustreret i figur 1A. Tidsforløbet for eksperimenter fra fremstilling single-kohorte kolonier prøve samling er vist i figur 1B. Arbejdstagere, der opfyldte de adfærdsmæssige kriterier for sygepleje adfærd eller fourageringsadfærd blev indsamlet fra single-kohorte kolonier, og HPG udvikling blev anslået i disse arbejdere Tabel 1 viser klassifikationen af HPG...

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

Her beskriver vi vores detaljerede protokol for forberedelse af single-kohorte honningbikolonier - et nyttigt redskab til at analysere den rolle-associerede arbejdstager fysiologi. Vi beskriver også detaljerede protokoller til behandling af arbejdstagere med juvenile hormon og ecdyson at vurdere inddragelsen af ​​disse hormoner i reguleringen af ​​arbejdstager adfærd og / eller fysiologi.

Udarbejdelse af Single-kohorte Kolonier og Hormone Behandling af Worker Honningbier at analysere Fysiologi associeret med Rolle og / eller endokrine 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 ...

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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