The overall goal of the following experiment is to obtain honeybee worker types in which mechanisms have slowed rapid and reversed. Aging can be studied. This is achieved by first decoupling senescence from chronological age by assembling colonies that receive newborn bees of the same age.
These bees eventually engage in different social tasks, either nursing or foraging while having the same chronological age. The nurse bees can show slowed aging compared with more rapid forager aging. Next, a reversal of aging dynamics from foraging to nursing is induced.
First forger are separated from nurse bees, then foragers that experience a lack of nurses revert to nursing tasks. Results are obtained by comparing symptoms of cellular aging in reverted workers, regular forger and nurse bees. This method can help answer key questions in the aging field, such as what mechanisms can contribute to slowing functional decline during aging.
A key advantage to this procedure is that it does not require special equipment. Thus, it should be easy to reproduce experiments using these methods. Demonstrating some of the procedures will be Osman Craft ulu project manager at the honeybee research lab here at the Arizona State University.
This section describes the setup of a two cohort colony containing an age-matched cohort of tagged bees and a cohort of random nest bees. The tagged bees separate into either nurse bees or forager bees, which have different aging dynamics. Experiments should consist of two or more such colonies.
First, prepare a regular hive box with two food combs with honey, another food comb with pollen and two empty combs. Then locate a mated queen as well as a donor colony with more than 3000 nest bees. Next, collect combs with sealed brood about to emerge between three and 5, 000 capped brood cells are needed for one colony.
These combs should be collected from at least three hive sources to avoid skewed distributions of maternal genotypes. Now incubate the collected brood combs at 34 degrees Celsius with 60 to 70%relative humidity. Let the bees emerge for two days and make sure the combs are stored so the emerging brood cannot escape.
Next, mark the emerged bees with a small paint tag on the thorax to identify the bees of the single age cohort and to distinguish them from other replicate colonies. On the day, young bees have been marked. Collect between 2, 503, 000 nest bees from a donor colony and add these unmarked bees to the prepared hive box.
Then add a queen bee confined to a cage that has been sealed with edible candy. The robust adult worker bees will slowly release the queen. Finally, add the newly emerged marked bees.
The focus of the study to assess the onset and dynamics of the nurse to forage your transition. Monitor the demographic development of foraging activity every other day for each colony. Begin counting five days after colonies were set up.
Monitoring is accomplished by counting the total number of bees returning from foraging flights via entrance counts. This is done in three 20 minute periods at fixed times. Do not count bees during periods of orientation.
Flights when the entrance counts exceed 100 per day. Begin marking forages. Use soft forceps to collect single marked bees when they return from their flights to facilitate catching bees.
After landing, narrow the hive entrance with foam or mesh. Apply a second paint mark that specifies the day, thus allowing later scoring of the age of onset of foraging activity. Use different colors or color combinations to mark the bees on each day.
When considering how many bees need to be marked for the study, expect to retrieve five to 10%of the marked foraging bees After at least two weeks of foraging activity, collect the marked nurses and forger simultaneously collect the marked nurse bees from the hive. These bees are feeding and cleaning the larvae, putting their heads in open brood cells. Also collect the double marked foragers.
Collect balanced numbers from all the test groups and replicate colonies. Transfer the bees to ventilated cages and keep them in the dark until they're further processed. For molecular studies directly snap, freeze the collected bees in liquid nitrogen.
This procedure separates a single colony into two hives, one with the nurse, B fraction, and the other with the forger fraction. This requires a hive as prepared in the previous section that retains at least 500 marked foragers and a completely marked foraging population. The day before the reversion prepare a second new hive box as previously described.
Next, collect two brood combs from donor hives. Brush away all the adult bees from the selected combs. Also collect two donor hive queens, one queen in a candy sealed cage and one brood comb.
Replace the queen and brood combs in the original hive box. Another queen and brood comb will be used the next day for the other hive box. This is a control to later provide both the nurse and forager derived colonies with a novel hive smell.
On the morning of the reversion day, photograph the state of the new brood combs. Then add one new queen in a candy sealed cage and one brood comb to the new box. During that day, at the peak foraging time, move the original colony with marked forger and nurse bees at least 100 meters away from the original location back at the original location.
Set up the new box for the forger derived hive. Allow the forger to return to the original location for two hours. This achieves a nearly complete separation of the forger population from the nest bees.
To terminate the separation close off the original hive and transfer it to an apiary that is at least three kilometers away. Check the experimental hives regularly for healthy open brood. During the first few days, replace any unattended and dead brooded to reduce the potential pathogen load areas with previously uncapped brood and with open live brood are reliable markers of nursing activity in forager derived colonies eight days after the reversal was initiated.
Using the previously described method for sampling sample reverted workers and continuing foragers as well as continuing nurse bees. When the experiment is completed, take another photo of the introduced combs to monitor worker type differentiation that accompanies the normal for forger counts were assessed for six colonies. A considerable increase in foraging activity was observed after 10 days.
Some variability marked by the red time points is due to changing weather conditions. The reversed ontogeny from foraging back to nursing tasks was validated by inspecting brood combs introduced into the forager derived colonies. On top is the comb before it was introduced.
Below is the same comb eight days after it was introduced. Patches of newly ca brooded healthy larvae and increased pollen storage around brooded cells all indicate that former foragers successfully performed typical nest building and nursing tasks. Accumulation of lipofuscin, a highly conserved symptom of cellular senescence was assessed in various bee tissues in age-matched nurse and forager bees.
17 days of foraging resulted in a significant increase in lipofuscin granule size. This clear increase in lipofuscin accumulation and granule size was linked to long forging durations as depicted with arrows in D.While attempting these procedures, it's extremely important to directly identify worker types. For example, avoid faults identification of forage of bees by discontinuing marking bees during daily periods with orientation flights, when also pre foraging stage.
Bees will depart from or enter the hive following our procedures. Other methods like proteomics and transcriptomics can be performed in order to identify molecular features of flexible aging.
