Name: obtaining specimens with slowed, accelerated and reversed aging in the honey bee model
Uploaded: 2013-08-29T16:10:00
Description: Watch this Scientific Journal Video about Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model at JoVE.com

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 o...

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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 border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Apifonda</td>
 <td>S&uuml;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 ...

Watch this Scientific Journal Video about Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model at JoVE.com

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 ...

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