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 ....

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 .......

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.......

Materials

List of materials used in this article

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