The overall goal of the following experiment is to measure longevity in drosophila while minimizing artifacts and bias. This is achieved by first preparing synchronized larvae to provide age matched adults for analysis. This next, the newly hatched males and females are allowed to mate for two days before they're separated into vials at a constant density, so gender specific effects can be measured throughout their lives.
The food supplies replenished no less than every two to three days, and their deaths are recorded in order to maintain their health and document the time course of mortality. Ultimately, results are obtained that show an effective experimental intervention on longevity based on comparison of survivorship and mortality between groups. Measuring longevity in any organism is technically straightforward but highly susceptible to bias.
We present this technique to demonstrate important aspects of the experiment that are essential, but may not be immediately obvious. This technique can be used to identify interventions that increases the robustness of the whole organism, reducing the risk of diseases that contribute to mortality over time. Though this method can provide insight into longevity and drosophila, it can also be applied to other measurements such as stress resistance.
Likewise, the principles contained in this protocol can be applied to measurement of longevity in a wide variety of organisms. Generally, individuals new to this method will struggle because lack of attention in the early steps can lead to major ambiguities. Later on.
The field is full of experimental artifacts. It can be easily avoided through the steps provided in this protocol, which have been developed over many years of experimental effort in multiple laboratories. Lifespan analysis requires stringent quality control of the food supply and use of healthy, genetically controlled fly stocks.
All stored foods should be refrigerated, used within one to two months and inspected for mold and dryness before their use. Likewise, environmental conditions are also standardized. The incubator where the experimental animals live should be set to 25 degrees Celsius with 60%relative humidity and a 12 to 12 light cycle prior to beginning this procedure equilibrate the following foods to room temperature, the yeast based grape auger plates and CT food bottles.
First, prepare a grape auger plate by spreading a dab of yeast paste at its center. Next anesthetize 150 to 200 pairs of flies in a large egg collection cage on a CO2 pad mesh side down. Using a funnel, transfer the flies into the egg collection cage.
Next, place the grape auger plate on the open end of the cage and secure with an end cap. Lay the cage on its side until flies wake up. Then place the cage with the auger plate side down in the incubator overnight.
On the next day, swap the plate with a freshly yeasted grape auger plate. However, only apply a one centimeter diameter swirl of yeast base to the surface of this plate. Discard the used plate.
Then return the cage to the incubator for 16 to 22 hours. The next day, discard the parent flies and wash the surface of the grape auger plate with one XPBS. To collect the embryos, be sure to remove the remaining yeast base before performing this wash.
Step eggs can be mobilized by gently scraping with a cotton swab. Take care nut to scratch damage or scrape thin pieces of auger off the surface of the plate. Now with the eight of a funnel, pour the washed eggs into a 15 milliliter conical tube.
Let the egg settle to the bottom of the tube. Then carefully pour off the supernat, leaving two to three milliliters of embryos in PBS. Now using additions of PBS, repeat the decanting.
Step two to three more times until the sate runs clear. Wash the eggs two to three times more by adding PBS to the tube and repeating the decanting. Step after the eggs settle down with the eggs.
In a small volume of clean PBS deeply submerge a wide boar pipette into the eggs and collect a 32 microliter Eloqua using a quick release of the plunger, the eggs in the pipette tip should be compact with little or no liquid aspirated. Deposit the eggs in a CT larva growth bottle and repeat this process to seed at least two to four CT bottles. For each experimental group, a larger number of larva bottles is better to mitigate potential artifacts associated with a single anomalous bottle.
Place the seated CT bottles back in the incubator throughout fly development at around day nine, discard the flies that were first to emerge and place the bottles back in the incubator overnight. This avoids inadvertent selection for early emergent flies. The next day, transfer the day old adult flies into room temperature.
10%SY food bottles record this as their first day of adulthood if needed, another batch can be collected. The next day return flies back into the incubator and allow them to reach sexual maturity and mate for two days. At two days, anesthetize small groups and sort the sexes using a paintbrush.
When using CO2, it is critical to minimize the anesthesia time to minimize possible long lasting health issues that can compromise the integrity of the experiment. Trans transfer 30 flies of the same gender into individual room temperature, 10%SY food vials. Typically, each bottle should produce around three to four vials of each sex.
This step should take no more than three to four minutes. Continue collecting until between eight and 10 vial replicates for each gender and experimental treatment have been established. Once all the vials for each experimental condition have been collected, use the D Life experiment setup wizard or a similar program to assign a randomized numerical ID to each vial.
Then arrange the vials and trays by ID number. This process eliminates bias associated with the vial location in the incubator and obscures the vial identity to the experimenter vials containing fresh food should always be at room temperature prior to their use. During the experiment, transfer the flies every two days for the first three weeks to keep females apart from larva.
After the first three weeks, flies can be transferred to new vials. Every two to three days, all the vials in a single experiment should be flipped together. This transfer should be completed by tapping flies into a fresh vial without anesthesia, which can induce acute mortality, particularly in older flies.
During each vial transfer record the age of the vial and the number of discarded dead and carried dead flies. The dead flies stuck to the old vial must be counted as well as those carried into the new vial. The number of new deaths is a total of newly discarded and carried dead flies minus the number of previously carried dead flies.
Sometimes a fly leaves the experiment during transfer by escaping or by accidental death. These flies are right censored and must be counted separately from deaths. Inappropriate censoring is probably the number one source of bias in longevity.Assessment.
Censoring should only be applied when escape or accidental death is observed by the experimenter. Otherwise, prevention is the best policy for avoiding ambiguous data. Several bile conditions can lead to anomalous deaths.
In general, any vial with small crevices where flies may get stuck and die should be avoided. Likewise, bacterial growth on the surface of the food can increase mortality. Bacteria usually appear transparent and shiny, but can also form white colonies.
Vials exhibiting any of these conditions should be noted and further considered in the analysis, but not removed from the experiment or bias would be introduced. Continue the transferring process until the last fly dies, then proceed with the analysis using the outlined protocol. A survivorship analysis of wild type flies demonstrates that adult males usually live shorter lives.
Both populations achieve a mean and median longevity of greater than 50 days. Survivorship remains high in the early part of the experiment and then declines exponentially. Drosophila lifespan is affected by temperature.
Adults typically live markedly shorter as temperature is increased, these data are derived from male flies. Likewise, the effective diet on lifespan of female flies shows that they live longer on a dilute diet than a more concentrated diet. The density of the cohorts during development can influence adult lifespan and alter developmental timing.
If there are too few eggs, adult fly yield is poor and the food surface is susceptible to drying. On the other hand, larval development is retarded in overcrowded bottles. The survivorship curve of the cohorts as a whole can be influenced significantly by anomalous vial effects.
An anomalous survival curve for an individual vial may have several causes, including bacterial, fungal, accumulation, and infection. Once mastered, this technique can allow one investigator to easily monitor over 200 vial in parallel spending approximately two hours three times a week While attempting this procedure, it's important to remember to randomize, avoid censoring, and maintain a large sample size As a follow up to this procedure. Measurement of age-related changes in functional and structural parameters can provide additional insight into the effects of a candidate intervention After its development.
This technique paved the way for researchers in the field of aging to explore the genes underlying longevity regulation.
