We thank Osman Kaftanoglu for helpful advice and assistance during filming. We would like to thank the anonymous reviewers for insightful comments. This work was supported by the Research Council of Norway (grants 180504, 191699, and 213976), Marie Curie/FP7 (project ref. 238665), the National Institute on Aging (grant NIA P01 AG22500), and the Pew Charitable Trusts.

1. Decoupling Senescence from Chronological Age
This section describes the setup of double cohort colonies, which consist of a cohort of identified individuals that share the same chronological age ("single age cohort") and a cohort of nest bees. Same aged individuals of the single age cohort will eventually separate into different worker-types with different aging dynamics - these are nurse bees with slowed and forager bees with accelerated functional decline. All procedures are described for one experimental colony. We advise, however, to perform experiments for at least two colony replicates so that colony effects can be controlled for (two-replicate-design).
<ol>
	<li>Preparing hive boxes for the double cohort colonies: Prepare one regular hive box that receives two food combs with honey, another food comb with pollen, and two empty combs. Make sure to locate a mated queen as well as a donor colony with more than 3,000 nest bees. Both will be introduced later (1.3).</li>
	<li>Obtaining and marking individuals with similar chronological age: Collect combs with sealed brood that is about to emerge. For one replicate expect to collect combs with a total of 3,000-5,000 capped brood cells. For each replicate use a balanced amount of brood from at least three different hive sources to avoid skewed distributions of maternal genotypes (hive origin).
	<ol>
		<li>Place brood combs in an incubator set to 34 &deg;C with 60-70% relative humidity. Make sure to store combs in such a way that emerging brood cannot escape.</li>
		<li>Let bees emerge for two days and mark these bees with a small paint tag on the thorax (e.g. Uni POSCA, Mitsubishi Pencil Co. Ltd.). The paint mark will allow identifying the bees of the single age cohort (day of emergence), and to distinguish them from other replicate colonies.</li>
	</ol>
	</li>
	<li>Setting up a double cohort colony, which includes the cohort of identified, single age bees: On the day young bees have been marked, collect about 2,500-3,000 nest bees from a donor colony (compare section 1. in Discussion), and add these unmarked bees to the hive box that was prepared before (see step 1.1). The latter individuals will constitute the unidentified nest bee cohort.
	<ol>
		<li>Add the queen, which will be initially confined to a queen cage (commercially available). Seal the cage with edible candy (e.g. Apifonda, S&uuml;dzucker AG, Mannheim/Ochsenfurt, Germany) to make worker bees slowly release the queen.</li>
		<li>Add the newly emerged and marked bees, which will constitute the single age cohort. These bees are the only marked individuals, and are the focus group for all the following steps.</li>
	</ol>
	</li>
	<li>Monitoring foraging onset and marking foragers: To assess onset and dynamics of the nurse-to-forager transition in the single age cohort, monitor the demographical development of foraging activity every other day for each colony. Begin counting five days after colonies were set up (Figure 1).
	<ol>
		<li>Count the total number of bees returning from foraging flights (entrance counts) within 3 x 20 min observation periods at fixed times. Make sure to not count bees during periods of orientation flights (see Discussion).</li>
		<li>When entrance counts indicate considerable foraging activity to commence (&gt;100 entrance counts/day), begin marking foragers. To do so, foragers of the single age cohort (single marked individuals) receive a second paint mark upon returning from their first foraging flights. This paint mark will specify the day of foraging onset, and will allow to later identify the foraging age for each forager.</li>
		<li>Repeat daily markings until a sufficient number of bees has been marked. For estimating a sufficient number of marked foragers, expect a retrieval rate of no more than 5-10% after these bees had been aged, typically after 14 days of foraging.</li>
	</ol>
	</li>
	<li>Sampling: Since all initially marked bees have a similar chronological age, age-matched groups of nurses and old foragers can be collected simultaneously, when foragers have foraged for &ge;14 days.
	<ol>
		<li>Single marked nurse bees are collected within the hive, and are identified by nursing behavior (feeding and cleaning of larvae with heads put down in open brood cells).</li>
		<li>Double marked foragers are also collected within the hive before daily foraging activity begins.</li>
		<li>Collect bees in cages (tubes, boxes) that provide sufficient ventilation, and keep dark until further processing. Alternatively, for transcriptomic, epigenetic or proteomic studies, directly snap freeze bees in liquid nitrogen. Collect balanced numbers of individuals from all test groups and replicate colonies.</li>
	</ol>
	</li>
</ol>
2. Reversal of Workers with Rapid to Workers with Slowed Aging by Changing the Hive's Demography
This section details how the reversal from workers with accelerated aging (foragers) to workers with slowed aging (nurse bees) is performed. Such behavioral reversal is induced, when foragers experience a lack of nurse bees, which normally engage in brood care. The reversion procedure will separate a single colony replicate into two hives: one hive with the nurse bee fraction ("nurse-derived"), and another one with the forager fraction ("forager-derived"). After successful reversal, possible symptoms of plastic and reversed aging can be studied in the single age cohort with reverted workers, continuing foragers, continuing nurse bees and newly recruited foragers. As before, identified bees of the single age cohort, not the cohort of unidentified nest bees, constitute the experimental focus group.
<ol>
	<li>Preparation: Replicate hives with nurses (single marked) and foragers (double marked) are made available as described in the previous section. Make sure to not begin reversion with less than 500 marked foragers per replicate colony to ensure sufficient retrieval after the reversion has been completed.
	<ol>
		<li>For safe identification of test groups after reversal it is crucial that the entire forager population in the original hive has been marked off before reversion. The following procedure is described for one replicate.</li>
		<li>The day before reversion, prepare one additional hive box for the forager-derived hive (see step 1.1). Locate two queens and two brood combs from donor hives. Before transfer to the experimental colonies brush away all adult bees from these combs. One caged queen (see step 1.3) and one brood comb will replace queen and brood combs in the original hive box. The other set of queen and brood comb will be used the next day for the new hive box. Similar allocation of new brood combs and alien queens for both, the original and the new hive is advised to make sure that separated foragers and nurses will equally experience changed hive cues ("hive smell").</li>
	</ol>
	</li>
	<li>Reversion: In the morning, just before the reversion, add the caged queen and the brood comb to the new box that will receive the forager-derived fraction. Wait until the peak foraging time begins. Then move the original colony with marked foragers and nurse bees at least 100 m away from the original location.
	<ol>
		<li>At the original location, set up the new box for the forager-derived hive with brood and queen only.</li>
		<li>Foragers will leave the dislocated original hive box, and head back to the original location. Allow foragers to return to the original location for 2 hr in order to achieve next to complete separation of the forager population from nest bees.</li>
		<li>Then, to terminate separation, close off the original, now "nurse-derived" hive, and transfer it to an apiary at least 3 km away.</li>
	</ol>
	</li>
	<li>Hive maintenance and monitoring for successful social task reversal: Check the experimental hives regularly for healthy, open brood.
	<ol>
		<li>During the first days after colony manipulation replace unattended and dead brood to reduce potential pathogen load.</li>
		<li>To validate successful reversal within the forager-derived hive, take pictures of brood combs before introducing these, and again when combs are replaced or when the reversion experiment is completed (Figure 2). Areas with previously uncapped brood and with open, live brood are reliable markers of nursing activity in forager-derived colonies.</li>
	</ol>
	</li>
	<li>Sampling: Physiological effects that accompany social reversal can be detected 3-8 days after foragers and nurses were separated.
	<ol>
		<li>We advise sampling all test groups, i.e. reverted workers and continuing foragers (forager-derived hive), as well as continuing nurses and newly-recruited foragers (nurse-derived hive) 8 days after reversal is initiated.</li>
		<li>Collect samples as described in step 1.5.</li>
	</ol>
	</li>
</ol>
3. Analyzing Worker-type Specific Cellular Senescence Patterns by Quantification of Lipofuscin
Lipofuscin is a universal biomarker of cellular senescence. As an intrinsic accumulation product, lipofuscin's specific autofluorescence (emissionmax = 530-650 nm) can be used for detection18.
<ol>
	<li>Dissection and fixation: Chill bees on ice until motionless; remove and dissect out the desired tissue sample.
	<ol>
		<li>Transfer into fixative (4% paraformaldehyde in phosphate-buffered saline, PBS; pH 7.2) for overnight incubation at 4 &deg;C.</li>
		<li>Wash samples 3 times in PBS.</li>
	</ol>
	</li>
	<li>Tissue processing and mounting: Cut tissue samples into sections with no more than 40 &mu;m thickness, for example by using a vibrating blade microtome, e.g. Leica VT 1000S (Leica Biosystems, Nussloch, Germany).
	<ol>
		<li>For clearing, incubate tissue sections overnight in 30% glycerol (PBS), and another 2 hr in 50% glycerol (PBS).</li>
		<li>Mount sections on microscopic slides in 50% glycerol (PBS). For long-term storage seal cover slips with nail polish.</li>
	</ol>
	</li>
	<li>Image acquisition: To detect lipofuscin, we suggest using a laser scanning confocal microscope that provides laser lines with &lambda; = 514, 561nm or similar for excitation, and with the detector bandwidth set to 570-650 nm.
	<ol>
		<li>For better identification of lipofuscin, include a second channel, and do a simultaneous scan at shorter wavelength spectra (excitation = 405 nm; emission = 410-450 nm). The longer wavelength channel will reveal both, granular lipofuscin, but also unspecific "background" due to autofluorescent trachea and other non-granular structures. The second, shorter wavelength channel will only reveal unspecific autofluorescence, but not lipofuscin. Thus, lipofuscin identification can be facilitated by comparing the signals in both channels, with only one of them revealing the granules with lipofuscin specific fluorescence.</li>
		<li>For acquisition of high-resolution images use an objective with 40X magnification or higher, and preferably a numerical aperture of 1.25 or higher. Scan image stacks with dimensions of about 100 x 100 x 10 &mu;m3. Every individual and tissue sample has to be represented by multiple image stacks.</li>
		<li>To reduce intra-individual and inter-individual variation caused by technical variation, always keep laser power and detector sensitivity constant.</li>
		<li>To reduce bias by day-to day technical variations scan equal sample numbers of all test groups in each of the several scanning sessions.</li>
	</ol>
	</li>
	<li>Image processing: Use software packages with modules that allow for advanced processing of microscopic image stacks, for example ImageJ (U.S. National Institutes of Health, Bethesda, Maryland, USA, <a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a>).
	<ol>
		<li>Generate a 2D maximum projection for each of the 3D image stacks.</li>
		<li>Apply a Gauss filter with a modest kernel size in order to attenuate high frequency noise, and to preserve structures with dimensions of lipofuscin granules.</li>
		<li>Merge both color channels to facilitate identification of lipofuscin (see step 3.2).</li>
	</ol>
	</li>
	<li>Image analyses: Make sure that the subject performing the quantification steps will be blind to test group identity.
	<ol>
		<li>For all images, first choose a region of interest (ROI) that covers the relevant structures, and has similar dimensions as the ROIs from other images.</li>
		<li>Then select the desired number of lipofuscin granules that represent each ROI. When selecting lipofuscin granules within a ROI, the application of the following rules will reduce subjective bias.
		<ol>
			<li>Choose a consistent location for selecting the first granule. This can be, for example, the leftmost edge of a ROI, and the granule that is closest to the edge will always be the first selection.</li>
			<li>One after another, granules are chosen that are closest to the previous selection (next neighbor).</li>
			<li>When choosing the next neighbor, move only into one direction, for example only search right from the previous selection. This rule prevents that the selection is dominated by occasional clusters of densely packed granules.</li>
			<li>When selection is completed, assess the size of each lipofuscin particle by outlining and measuring the respective granule area. Use appropriate statistical tests to compare individuals of the different test groups.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Munch, D., 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=The+curious+case+of+aging+plasticity+in+honey+bees.">The curious case of aging plasticity in honey bees.</a> FEBS Lett. 584, 2496-2503 (2010).</li><li>Buffenstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negligible+senescence+in+the+longest+living+rodent,+the+naked+mole-rat:+insights+from+a+successfully+aging+species.">Negligible senescence in the longest living rodent, the naked mole-rat: insights from a successfully aging species.</a> J Comp Physiol B. 178, 439-445 (2008).</li><li>Parker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+social+insects+telling+us+about+aging?.">What are social insects telling us about aging?.</a> Myrmecological News. 13, 103-110 (2010).</li><li>Seeley, T. D. <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 Wisdom of the Hive. , (1995).</li><li>Amdam, G. V., Omholt, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulatory+anatomy+of+honeybee+lifespan.">The regulatory anatomy of honeybee lifespan.</a> J Theor Biol. 216, 209-228 (2002).</li><li>Amdam, G. V., Norberg, K., Hagen, A., Omholt, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+exploitation+of+vitellogenin.">Social exploitation of vitellogenin.</a> Proc Natl Acad Sci U S A. 100, 1799-1802 (2003).</li><li>Guidugli, K. R., 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=Vitellogenin+regulates+hormonal+dynamics+in+the+worker+caste+of+a+eusocial+insect.">Vitellogenin regulates hormonal dynamics in the worker caste of a eusocial insect.</a> FEBS Lett. 579, 4961-4965 (2005).</li><li>Amdam, G. V., 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=Social+reversal+of+immunosenescence+in+honey+bee+workers.">Social reversal of immunosenescence in honey bee workers.</a> Exp Gerontol. 40, 939-947 (2005).</li><li>Seehuus, S. C., Norberg, K., Gimsa, U., Krekling, T., 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=Reproductive+protein+protects+functionally+sterile+honey+bee+workers+from+oxidative+stress.">Reproductive protein protects functionally sterile honey bee workers from oxidative stress.</a> Proc Natl Acad Sci U S A. 103, 962-967 (2006).</li><li>Scheiner, R., 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=Impaired+tactile+learning+is+related+to+social+role+in+honeybees.">Impaired tactile learning is related to social role in honeybees.</a> J Exp Biol. 212, 994-1002 (2009).</li><li>Behrends, A., Scheiner, R., Baker, N., 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=Cognitive+aging+is+linked+to+social+role+in+honey+bees+(Apis+mellifera.">Cognitive aging is linked to social role in honey bees (Apis mellifera.</a> Exp Gerontol. 42, 1146-1153 (2007).</li><li>M&#252;nch, D., Baker, N., Kreibich, C. D., Braten, A. T., 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=In+the+laboratory+and+during+free-flight:+old+honey+bees+reveal+learning+and+extinction+deficits+that+mirror+mammalian+functional+decline.">In the laboratory and during free-flight: old honey bees reveal learning and extinction deficits that mirror mammalian functional decline.</a> PLoS One. 5, e13504 (2010).</li><li>Vance, J. T., Williams, J. B., Elekonich, M. M., Roberts, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+age+and+behavioral+development+on+honey+bee+(Apis+mellifera)+flight+performance.">The effects of age and behavioral development on honey bee (Apis mellifera) flight performance.</a> J Exp Biol. 212, 2604-2611 (2009).</li><li>Dukas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+rates+of+honey+bees+in+the+wild.">Mortality rates of honey bees in the wild.</a> Insect Soc. 55, (2008).</li><li>Behrends, A., Scheiner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+at+old+age:+a+study+on+winter+bees.">Learning at old age: a study on winter bees.</a> Front Behav Neurosci. 4, 15 (2010).</li><li>Huang, Z. -. Y., 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=Honeybee+colony+integration:+Worker-worker+interactions+mediate+hormonally+regulated+plasticity+in+division+of+labor.">Honeybee colony integration: Worker-worker interactions mediate hormonally regulated plasticity in division of labor.</a> Proc Natl Acad Sci USA. 89, 11726-11729 (1992).</li><li>Huang, Z. Y., 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=Regulation+of+honey+bee+division+of+labor+by+colony+age+demography.">Regulation of honey bee division of labor by colony age demography.</a> Behavioral Ecology and Sociobiology. 39, 147-158 (1996).</li><li>Double, K. L., 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=The+comparative+biology+of+neuromelanin+and+lipofuscin+in+the+human+brain.">The comparative biology of neuromelanin and lipofuscin in the human brain.</a> Cell Mol Life Sci. 65, 1669-1682 (2008).</li><li>Fonseca, D. B., Brancato, C. L., Prior, A. E., Shelton, P. M., Sheehy, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+rates+reflect+accumulating+brain+damage+in+arthropods.">Death rates reflect accumulating brain damage in arthropods.</a> Proc Biol Sci. 272, 1941-1947 (2005).</li><li>Baker, N., Wolschin, F., 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=Age-related+learning+deficits+can+be+reversible+in+honeybees+Apis+mellifera.">Age-related learning deficits can be reversible in honeybees Apis mellifera.</a> Exp Gerontol. 47, 764-772 (2012).</li><li>Capaldi, E. A., 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=Ontogeny+of+orientation+flight+in+the+honeybee+revealed+by+harmonic+radar.">Ontogeny of orientation flight in the honeybee revealed by harmonic radar.</a> Nature. 403, 537-540 (2000).</li><li>Marco Antonio, D. S., Guidugli-Lazzarini, K. R., do Nascimento, A. M., Simoes, Z. L., Hartfelder, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-mediated+silencing+of+vitellogenin+gene+function+turns+honeybee+(Apis+mellifera)+workers+into+extremely+precocious+foragers.">RNAi-mediated silencing of vitellogenin gene function turns honeybee (Apis mellifera) workers into extremely precocious foragers.</a> Naturwissenschaften. 95, 953-961 (2008).</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, 296-299 (2003).</li><li>Schmidt, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attraction+of+reproductive+honey+bee+swarms+to+artificial+nests+by+Nasonov+pheromone.">Attraction of reproductive honey bee swarms to artificial nests by Nasonov pheromone.</a> Journal of Chemical Ecology. 20, 1053-1056 (1994).</li><li>De Moraes, R., Bowen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+cell+death+in+the+hypopharyngeal+gland+of+the+honey+bee+(Apis+mellifera+L).">Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera L).</a> Cell Biol Internat. 24, 737-743 (2000).</li><li>Sheehy, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow-cytometric+method+for+quantification+of+neurolipofuscin+and+comparison+with+existing+histological+and+biochemical+approaches.">A flow-cytometric method for quantification of neurolipofuscin and comparison with existing histological and biochemical approaches.</a> Arch Gerontol Geriatr. 34, 233-248 (2002).</li><li>Hsieh, Y. S., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honeybee+trophocytes+and+fat+cells+as+target+cells+for+cellular+senescence+studies.">Honeybee trophocytes and fat cells as target cells for cellular senescence studies.</a> Exp Gerontol. 46, 233-240 (2011).</li><li>Wolschin, F., Munch, D., 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=Structural+and+proteomic+analyses+reveal+regional+brain+differences+during+honeybee+aging.">Structural and proteomic analyses reveal regional brain differences during honeybee aging.</a> J Exp Biol. 212, 4027-4032 (2009).</li></ol>

We have nothing to disclose.

We here adopt previously described approaches8,16,17,19,20, and integrate them into a single workflow that will facilitate studying flexible aging in honey bees. Our aim is to provide scientists that are novice to this field with a standardized tool set to obtain relevant sample material, and to improve experimental reproducibility among different research teams. While our procedures are simplified and do not require special equipment as in earlier descriptions (compare for example8), some measures ...

The complex colony structures of highly social animals are maintained through the interaction of a reproductive caste, and a helper caste of typically non-reproducing workers with different social task behaviors. In the different workers, specific physiological adaptations enable distinct sib care behaviors, and are also linked to extreme lifespan differences. Honey bees and mole rats represent the best-developed animal models to study how sociality is linked to patterns of accelerated, negligible or reversed aging1-3.
In honey bee colonies, a single egg-laying queen is assisted by thousands of workers that tend the brood, forage for food, and engage in guarding, thermoregulation or hygienic behaviors4. Among these workers are the extremely short-lived foragers, nurse bees with intermediate, and winter (diutinus) bees with longest lifespans. Individuals, however, are not permanently bound to a certain worker-type, but display a flexible behavioral ontogeny: they change from one social task behavior to another ("temporal castes"). Callow bees can change to brood tending nurse bees, which eventually may change to outside foraging. However, callow nest bees can also transform into longest-lived winter bees, and short-lived foragers can even revert into typically longer-lived nurses. Workers with extreme (winter bees) and intermediate (nurse bees) lifespan have well-developed food production and storage organs with copious resources - as opposed to short-lived foragers (reviewed in1,5). However, that the regulation of individual lifespan goes beyond simple changes in an individual's resource balance is suggested by research on a yolk protein, which has diverse adapted functions in the non-reproducing worker caste, such as jelly production6, hormonal control7, immune8 and anti-oxidant defense9.
Patterns of functional decline (senescence) mirror lifespan disparities among workers, as established for olfactory, and also for other brain or motor functions10-13. Specifically, the significant decline in learning function after only two weeks of foraging matches a similar mortality progression in foragers14, as opposed to the lack of detectable decline (negligible senescence) in long-lived winter bees15.
To identify the molecular fingerprints of flexible aging we adapted established experimental paradigms that allow for monitoring and manipulating aging-type transitions8,16,17. Experiment 1 details how to obtain samples in which the effects of chronological age and worker-type specific social behaviors on aging can be separated. Experiment 2 describes the reversal of foragers with accelerated into nurse bees with slowed aging dynamics. Experiment 3 provides an approach for probing effects of cellular senescence by anatomical quantification of an established biomarker for cellular aging (lipofuscin)18.

<table><tbody><tr><td>Apifonda</td><td>S&amp;#252;dzucker AG, Mannheim/Ochsenfurt, Germany</td><td></td><td></td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>phosphate-buffered saline</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>Glycerol</td><td>Merck</td><td>1.04094.1000</td><td></td></tr></tbody></table>

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.

Protocol sections 1 and 2 detail how test groups can be obtained to study attributes of accelerated, slowed and reversed aging in colonies with a single age cohort. To monitor worker-type differentiation that accompanies the normal ontogeny we assessed forager counts ("entrance counts") for 6 colonies (Figure 1, compare section 1). The graphs show that considerable change from nurse to the forager state is typically not observed before individuals are more than 10 days old. Marked variability in forager ...

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Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

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.

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

obtaining-specimens-with-slowed-accelerated-reversed-aging-honey-bee

Research

JoVE Journal

Biology

11.2K Views.  Norwegian University of Life Sciences.  In honingbij werknemers, ouder worden, hangt af van sociaal gedrag in plaats van op chronologische leeftijd. Hier laten we zien hoe de werknemer-typen met zeer verschillende veroudering patronen kan worden verkregen en voor cellulaire veroudering geanalyseerd. 

Video: Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

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.

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

Neuroscience

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.

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 ...

Environment

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.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

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.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

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.

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 ...

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.

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.

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 ...

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

Histology Basics and Cell Death Detection in Honeybee Tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

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