Measurement of Lifespan in Drosophila melanogaster

Summary

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Abstract

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.

The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts^1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (http://sitemaker.umich.edu/pletcherlab/software). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Protocol

We recommend storing experimental foods, yeast paste, and grape agar plates that appear in the protocol at 4 °C and using them within 1-2 months as long as mold and dryness have not set in. Standard environmental conditions for both the larval and adult stage involve maintenance of flies in an incubator at 25 °C with a 12:12 hr light dark cycle and 60% relative humidity.

1. Preparation of Experimental Food

1. For larval growth, we use a modified Caltech Medium⁵, which is abbreviated in this protocol as CT.
2. We recommend a diet for adult Drosophila (SY) that consists of sugar (sucrose) and yeast (lyophilized whole brewer's yeast) in a 2% agar base, that has been boiled, supplemented with antibiotics and anti-fungal agents, and distributed (10 ml per vial)⁶. Food should be allowed to solidify and evaporate for 12-24 hr prior to storage. Because the nutrient environment can substantially impact longevity, consistency in cooking processes is essential both within an experiment and for comparison between experiments.
3. If a pharmacological agent is to be added to the adult food, this drug can be mixed into a small batch of food and layered (2 ml) on the food surface, with control vials receiving layers containing vehicle alone.

2. Preparation of a Live Yeast Paste

Combine 5-6 ml of water with 3 g of active dry yeast and mix well. The consistency of the yeast paste should be that of a smooth peanut butter.

3. Preparation of a Grape Agar Plate

1. Add a packet of a grape agar premix in 500 ml of distilled water in a 1,000 ml flask and follow the instructions on the packet to dissolve the grape agar mix.
2. Carefully pour a thick layer of the mixture into 100 mm Petri dishes while avoiding bubble formation. One packet of premix produces approximately 14 grape agar plates.
3. Let the media cool and solidify in room temperature with lids on for 15 min. Plates can be kept at 4 °C, wrapped in plastic wrap, or used immediately.

4. Collection of Synchronized Eggs

All media used in this protocol, i.e. yeast, grape agar plates, CT and 10% SY food, should be at room temperature.

1. Spread a 2-3 cm diameter layer of yeast paste on a grape agar plate and set aside.
2. Place a large egg collection cage on a CO₂ pad with the mesh-side down to anesthetize the flies. Nitrogen-based anesthesia is an alternative to CO₂, used by some laboratories, that can produce high quality results⁷.
3. Using a funnel, transfer 150-200 pairs of flies into the egg collection cage.
4. Place the grape agar plate to cover the open end of the cage and secure with an end cap.
5. Lay the cage on its side until flies wake up. Then, keep the cage, grape agar plate side down, in the incubator overnight.
6. On the next day, swap the grape plate with a newly yeasted grape plate. Only put about 1 cm diameter of yeast paste onto the surface of the plate. Discard the 1^st day grape plate.
7. Allow embryos to collect on the surface of the grape agar plate for 16-22 hr. Once done, collect the grape agar plate and discard the parent flies.
8. Wash the surface of the grape agar plates with 1x Phosphate Buffered Saline (PBS). Eggs can be mobilized by gently scraping with a cotton swab. Take care not to scratch, damage, or scrape thin pieces of agar off the surface of the plate. With the aid of a funnel, pour the washed eggs into a 15 ml conical tube.
9. Let the eggs settle to the bottom of the tube and pour off the supernatant, being careful not to lose any eggs. The remaining volume should be around 2-3 ml.
10. Add 8-10 ml of PBS to the tube and repeat the above step 2-3 more times to thoroughly wash the eggs until the supernatant is clear. Getting rid of residual yeast is the key in this step.
11. After the wash, drain all supernatant until remaining volume is 2 ml. Aliquot 32 μl of eggs into CT bottles using a wide-bore pipette tip. Eggs in the pipette tip should be compact, with little to no liquid aspirated. This can be achieved by inserting the pipette tip deep into the egg settlement and quickly releasing the plunger to aspirate.
12. Place seeded CT bottles back in the incubator throughout fly development.

5. Collection of Age-matched Adult Flies

1. Adults will typically eclose from day 9 onward. Discard the flies that emerged on the first day and place bottles back in the incubator overnight. This practice will avoid inadvertent selection for early emergents and allow for the collection of a maximum number of synchronized flies.
2. 16-22 hr later, transfer the day-old adult flies into 10% SY food bottles. If needed, another batch can be collected the next day.
3. Return flies back into the incubator and allow flies to reach sexual maturity and mate for two days. Record the day of transfer to 10% SY bottles as the first day of adulthood.

6. Sorting Flies and Setting up Longevity Experiment

1. Anesthetize small groups of flies on the anesthetic pad, then sort males and females into two groups using a paintbrush. If using CO₂, it is critical to minimize exposure to prevent possible long-lasting health issues that can compromise the integrity of the longevity experiment.
2. Place 30 flies of the same gender into individual vials. Assuming no balancer chromosomes in the population and healthy progeny, each bottle should produce around 3-4 vials of each sex. Aliquoting flies into individual vials should take no more than 3-4 min, so that, in total, flies are only exposed to anesthesia for a maximum of 9-10 min.
3. Repeat steps 6.1-6.2 until there are 8-10 vial replicates for each gender and experimental treatment.

7. Setting Up the Excel spreadsheet to Track the Longevity Experiment

1. We recommend randomizing vial position rather than grouping vials by experimental condition in order to avoid bias associated with the vial location in the incubator and to obscure the vial identity to the experimenter. To do this, first assign a randomized numerical ID to each vial in a spreadsheet program, then arrange the vials in trays by ID number. If you are using the dLife experiment management software, follow the tutorial on experiment setup to generate the ID number for each vial.
2. (Optional) If you are using an RFID reader or barcode reader in association with dLife, attach an RFID or barcode tag to each vial and associate the tag with the vial's numerical ID in dLife. The program will recognize each vial when scanned with a reader and guide one to record the data in the correct location in a spreadsheet. Usage of a tag reader in conjunction with dLife significantly reduces data collection time and recording error.

8. Maintaining the Longevity Experiment

The vials containing fresh food should be at room temperature for each transfer.

1. During the experimental period, transfer flies onto new vials containing fresh food every 2 days (young females), or 3 times a week (males or females >3 weeks of age). This step will ensure that the feeding environment for young females is not disrupted by the presence of larvae. This transfer should be completed without anesthesia, which can induce acute mortality, particularly in older flies (Pletcher, personal observations).
2. During each vial transfer, record the age, count the dead flies in the old vial, and the dead flies that are carried to the new vial. Record this information separately in two columns in a spreadsheet (either dLife or your own spreadsheet). This will ensure that the carried flies are not double-counted. The total number of deaths (dead + carried) should at least equal the number of carried flies from the previous transfer. Subtract the number of previously carried flies from the total number of deaths to determine the number of new deaths.
3. A fly is considered right-censored if it left the experiment prior to natural death through escape or accidental death. Animals exiting the experiment in this way should be entered into a separate column on the day that the fly exited the experiment. Censored flies are not recorded as dead (see below).
4. Continue to repeat steps 8.1-8.2 until the last survivor is dead. Be aware that as the flies age, some flies may lie on their back and appear dead due to their inactiveness. Therefore when counting carried (dead) flies, tap on the side of the vials to determine if there are leg movements. If so, these flies are still alive. In the case where flies remain stuck to the food in the old vial but alive, they should not be counted as dead and should be rescued by further tapping of the vial to dislodge the fly. Censoring such flies should be used with caution as it may result in experimental bias.

9. Data Analysis

1. The survivorship curve displays the probability that an individual survives to a given age and is typically calculated using a Kaplan-Meier approach (Figure 1)⁸. In the absence of right-censored data, the formula can be simplified such that age-specific survivorship at age x (S_x) is determined by dividing the number of individuals alive at the start of a census time at age x (N_x) by the total number of flies in the experiment (N₀); S_x=N_x/N₀. 9.2) Survivorship curves are the most common form of data presentation, and they can be tested for equality between groups using a log-rank test. Reasonable inference can be drawn from as few as 50-100 individuals in a cohort. Survivorship is a cumulative measure, however, and therefore deaths that are not aging-related, such as those early in life, will depress survivorship throughout the lifespan making it difficult to ascertain effects specific to aging.
2. A second data visualization method is the age-specific mortality function, which displays the risk of dying through each age interval and presents a more nuanced description of survival data^9,10. Mortality measures are independent from one age to another, and the shape of the mortality curve is useful for inference about the dynamics of aging, particularly when treatments are adjusted during adult life. Estimates of age-specific mortality, however, lack both precision and accuracy with small sample sizes, and often require several-to-many hundreds of individuals per cohort for a reliable estimate¹¹.
3. Other methods for estimating longevity differences include parametric (e.g. Gompertz) and semi-parametric (e.g. Cox regression) models. These models can be powerful but should be applied with caution because of the assumptions made about the shape of the mortality curves and the nature of treatment effects, which could lead to incorrect inference^11,12.

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Results

A simplified scheme of the protocol is presented in Figure 1, where key steps are outlined. The synchronization part of the protocol can be used for various assays that require age-matched adult flies.

Typical survivorship curves of wild-type flies are shown in Figure 2a, using the dLife experiment management software (Figure 2b,c). Adult males usually live shorter, with both populations achieving a mean and median longevity of >50 days on ...

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Discussion

The protocol presented here describes a method for producing reproducible measurements of adult longevity in Drosophila that is adaptable for assessment of genetic, pharmacological, and environmental interventions. Crucial aspects of the protocol include carefully controlling the larval development environment, minimizing adult stress, and minimizing bias across experimental groups and controls. We also present the use of the dLife lifespan experiment management software. By simply attaching a bar code or RFID t...

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Materials

Name	Company	Catalog Number	Comments
Active Dry Yeast	Fleishmann’s Yeast	2192
Grape Agar Powder Premix	Genesee Scientific	47-102
Large Embryo Collection Cages	Genesee Scientific	59-101
Large Replacement End Caps	Genesee Scientific	59-103
6 oz Square Bottom Bottles, polypropylene	Genesee Scientific	32-130
Flugs Closures for Stock Bottles	Genesee Scientific	49-100
Drosophila Vials, Wide, Polystrene	Genesee Scientific	32-117
Flugs Closures for Wide Vials	Genesee Scientific	49-101
Wide Orifice Aardvark Pipet Tips, 200 ul	Denville Scientific	P1105-CP
Flystuff Flypad, Standard Size	Genesee Scientific	59-114
BD Falcon 15 ml Conical Centrifuge Tubes	Fisher Scientific	14-959-70C
Fisherbrand Petri Dishes with Clear Lids, Raised Ridge; 100 O.D. x 15 mm H;	Fisher Scientific	08-757-12
Kimax* Colorware Flasks 1,000 ml yellow	Fisher Scientific	10-200-47
PBS pH 7.4 10x	Invitrogen	70011044
Gelidium Agar	Mooragar	n/a
Brewer's Yeast	MP Biomedicals	0290331280
Granulated Sugar	Kroger	n/a
Tegosept	Genesee Scientific	20-266	Fly Food Preservative
Propionic Acid, 99%	Acros Organics	149300025	Fly Food Preservative
Kanamycin Sulfate	ISC BioExpress	0408-10G
Tetracycline HCl	VWR	80058-724