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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 aging^1-3.

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 in^1,5). 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 functions^10-13. 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 transitions^8,16,17. 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.

Protokół

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).

1. 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).
2. 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).
   1. Place brood combs in an incubator set to 34 °C with 60-70% relative humidity. Make sure to store combs in such a way that emerging brood cannot escape.
   2. 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.
3. 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.
   1. Add the queen, which will be initially confined to a queen cage (commercially available). Seal the cage with edible candy (e.g. Apifonda, Südzucker AG, Mannheim/Ochsenfurt, Germany) to make worker bees slowly release the queen.
   2. 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.
4. 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).
   1. 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).
   2. When entrance counts indicate considerable foraging activity to commence (>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.
   3. 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.
5. 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 ≥14 days.
   1. 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).
   2. Double marked foragers are also collected within the hive before daily foraging activity begins.
   3. 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.

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.

1. 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.
   1. 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.
   2. 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").
2. 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.
   1. At the original location, set up the new box for the forager-derived hive with brood and queen only.
   2. 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.
   3. Then, to terminate separation, close off the original, now "nurse-derived" hive, and transfer it to an apiary at least 3 km away.
3. Hive maintenance and monitoring for successful social task reversal: Check the experimental hives regularly for healthy, open brood.
   1. During the first days after colony manipulation replace unattended and dead brood to reduce potential pathogen load.
   2. 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.
4. Sampling: Physiological effects that accompany social reversal can be detected 3-8 days after foragers and nurses were separated.
   1. 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.
   2. Collect samples as described in step 1.5.

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 (emission_max = 530-650 nm) can be used for detection¹⁸.

1. Dissection and fixation: Chill bees on ice until motionless; remove and dissect out the desired tissue sample.
   1. Transfer into fixative (4% paraformaldehyde in phosphate-buffered saline, PBS; pH 7.2) for overnight incubation at 4 °C.
   2. Wash samples 3 times in PBS.
2. Tissue processing and mounting: Cut tissue samples into sections with no more than 40 μm thickness, for example by using a vibrating blade microtome, e.g. Leica VT 1000S (Leica Biosystems, Nussloch, Germany).
   1. For clearing, incubate tissue sections overnight in 30% glycerol (PBS), and another 2 hr in 50% glycerol (PBS).
   2. Mount sections on microscopic slides in 50% glycerol (PBS). For long-term storage seal cover slips with nail polish.
3. Image acquisition: To detect lipofuscin, we suggest using a laser scanning confocal microscope that provides laser lines with λ = 514, 561nm or similar for excitation, and with the detector bandwidth set to 570-650 nm.
   1. 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.
   2. 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 μm³. Every individual and tissue sample has to be represented by multiple image stacks.
   3. To reduce intra-individual and inter-individual variation caused by technical variation, always keep laser power and detector sensitivity constant.
   4. To reduce bias by day-to day technical variations scan equal sample numbers of all test groups in each of the several scanning sessions.
4. 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, http://imagej.nih.gov/ij/).
   1. Generate a 2D maximum projection for each of the 3D image stacks.
   2. 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.
   3. Merge both color channels to facilitate identification of lipofuscin (see step 3.2).
5. Image analyses: Make sure that the subject performing the quantification steps will be blind to test group identity.
   1. 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.
   2. 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.
      1. 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.
      2. One after another, granules are chosen that are closest to the previous selection (next neighbor).
      3. 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.
      4. 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.

Wyniki

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 ...

Dyskusje

We here adopt previously described approaches^{8,16,17,19,20}, 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 ...

Materiały

Name	Company	Catalog Number	Comments
Apifonda	Südzucker AG, Mannheim/Ochsenfurt, Germany
paraformaldehyde	Sigma-Aldrich	158127
phosphate-buffered saline	Sigma-Aldrich	P4417
Glycerol	Merck	1.04094.1000