Protocols to study ageing in Drosophila

Summary

The fruit fly Drosophila melanogaster offers a host of advantages for studying the biology of ageing: a well understood biology, a wide range of genetic reagents, well defined dietary requirements and a relatively short lifespan, with a median of ~80 days and maximum ~100 days. Several phenotypes can be used to assess the ageing process, but the simplest and most widely used metric is length of life. Here we describe a standard lifespan assay for Drosophila housed on a simple sugar/yeast diet.

1. Introduction

As populations around the world age, increasing effort is being devoted to the development of new approaches to improve the health of older people. Remarkably, experimental work on worms, flies and mice over the last 20 years has provided a positive outlook on this prospect (Kenyon, 2010; Partridge et al., 2011). For these model organisms, genetic, environmental and pharmacological interventions have been described that extend healthy lifespan (Niccoli and Partridge, 2012). Even more remarkably given the very different lifespans of these model organisms, these interventions often act on common mechanisms to extend lifespan, implying some degree of evolutionary conservation of mechanisms of ageing. Thus there is great promise that studies of ageing in laboratory model organisms will yield insights into ageing that will ultimately benefit humans.

The challenges of experimental gerontology are enormous. Experiments require long time-scales, genetic manipulations, large populations and well-controlled animal stocks and conditions. These factors make the work perfectly suited to the small, short-lived and well-characterized model organisms such as the fruit fly Drosophila melanogaster.

Lifespan experiments have been conducted on Drosophila for the last 100 years (Loeb and Northrop, 1916) and over time the conditions have been refined (Piper and Partridge, 2007). In general, the protocol can be simple, but small and seemingly insignificant modifications to experimental protocols can have large effects on outcomes. For example, by not controlling for diet quality, genetic background or the interactions between mating frequency and diet, the experiment may report the effects on lifespan of an uncontrolled, trivial experimental procedure, rather than the focal intervention of the study. (Partridge and Gems, 2007).

Here we outline the basic procedure for rearing, isolating and maintaining flies for lifespan experiments, highlighting a number of the known pitfalls that have misled researchers in the past. We provide a basic protocol for wild type flies housed under our standard laboratory conditions and then we provide modified protocols for studying the effects on lifespan of diet, drugs or genetic interventions.

2. Materials

2.1. Media

All media are prepared using reverse osmosis water. Cooking can be done on a gas hob using a standard saucepan and stirring with a heavy-duty whisk (see note ¹)

1. Egg collection medium (volume sufficient for ~10 x 15cm petri dishes): to 250ml cold water add 12.5g agar and stir to mix. Bring to boil while stirring and maintain boiling for ~2min to ensure agar is completely melted. Add 150ml red grape juice (see note ²) and stir until the mixture returns to the boil. Remove from heat. Add 25ml cold water and stir until temperature drops to ~65ºC. Mix in10.5ml 10% nipagin (methyl 4-hydroxybenzoate in 95% ethanol) and pour solution into petri dishes. Allow to cool at room temperature, allowing steam to escape. Ensure to protect the plates from any flies at this stage to avoid contamination. Cover and store at 4ºC.

2. Fly food for rearing and maintenance (makes 1L of 1SY (Bass et al, Exp Gerontol., 2007; see note ³): add 15g agar to 700ml cold water and stir. Heat until boiling. While continuing to stir, add 50g table sugar (sucrose) and 100g yeast (whole yeast autolysate and not water soluble yeast extract). After returned to boil, remove from heat and add cold water to make up to final volume of 1L. Stir and allow to cool to ~65ºC. Mix in 30ml 10% nipagin and 3ml propionic acid to act as preservatives. This is also the point at which to mix in any small volume additions of drugs/ transgene inducers/ vehicle control. For larger volume additions, reduce the cold water addition after cooking to ensure final total volume remains at 1L. Using a peristaltic pump with clean, sterilized tubing, dispense into clean vials or bottles. Allow to cool at room temperature for several hours (see note ⁴). To avoid contamination, ensure to protect cooling food from flies (see note ⁵). Plug individual vials with cotton balls (see note ^{6 7}). Store at 4ºC.

3. Live yeast paste for stimulating egg laying: Mix dried baker’s yeast granules with cold water at a ratio of approx. 1:1 by weight to make a stiff paste (ice cream consistency). Best when used immediately, but can be stored covered at 4ºC for 2 weeks.

2.2. Plastic / glassware for housing and handling eggs and flies⁸

1. 15cm diameter plastic petri dishes (see note ⁹)

2. fly “cage” for housing parental flies: an ~15cm long plastic cylinder that fits a petri dish snugly at one end, and is covered with mesh at the other

3. Bottles: ~250ml (polypropylene or glass ; ~60mm OD x 130mm H)

4. Vials / tubes: ~15ml (polystyrene, polypropylene or glass; ~ 25mm O.D. x 95mm H)

5. Cotton wool balls or high density synthetic bungs to close tubes

6. squeeze bottle

7. pipette (20 – 100μl)

8. wide bore pipette tips (note ¹⁰)

9. CO₂ stream – supplied via a water bubbler and low-static porous diffusion pad

10. fine paint brush (size 000 - 0000)

11. handle-mounted metal pick

2.3. Solutions for handling eggs and flies

1. Phosphate buffered saline. Mix pre-formulated tablets (Sigma, P4417) with water according to instructions on container. This yields 0.01M phosphate buffer, 0.0027M potassium chloride, 0.137M sodium chloride, pH 7.4.

3. Methods

3.1. Parental generation

Preparing stocks for egg collection: most laboratory stocks are kept in small numbers and under crowded conditions, both of which alter adult lifespan (Partridge and Gems, 2007; Zwaan et al., 1991)

1. ). It is important to implement procedures to control these factors so that they do not confound interpretations of alterations in fly lifespan.

   1. “wild types”: to escape the trans-generational effects of stock crowding on lifespan, we passage stock-derived flies through two generations of our standard density procedure before use in lifespan experiments.

   2. Genetic crosses: it is extremely important to standardize the genetic background of all mutant lines to be compared in a lifespan experiment. Failure to do so is common and leads to incorrect conclusions about the effects of genetic interventions to extend life. Most experimental transgenic flies are generated by crossing two inbred lines, with one containing the transgene to be activated and the other containing a genetic construct that drives the expression of the first. This cross also produces a hybrid genetic background, and this will generally increase lifespan when compared with that of the inbred controls, as a consequence of heterosis and irrespective of any effect of the transgenes (Pearl, 1921). To avoid this problem, all transgenes and mutants should first be back-crossed into a standardised genetic background for at least 6 generations. To maintain the lines an additional 2-3 backcrosses should be repeated every 6-12 months (see note ¹¹). Furthermore, each of the transgenic lines used to construct the experimental line should be included as a control in the lifespan experiment, because transgenes can cause insertional mutagenesis and which can in turn modify longevity.

2. To collect staged embryos:

   • a.house parental flies in “cages”. Provide a generous smear of live yeast paste (~1 tsp) at the centre of the egg laying plate
   • b.after 48h, replace egg laying surface (see note ¹²) with a fresh plate harbouring a fresh aliquot of live yeast paste (egg laying peaks ~72h after introduction to rich food) (see note ¹³)
   • c.leave overnight (see note ¹⁴)
   • d.Collect embryos for development at standard density. To achieve this, we either use a pipette to allocate a fixed volume of a dense embryo suspension into new media for development, or use a mounted metal pick to collect and transfer individual larvae to development media (see note ¹⁵):

     Pipetting method (ideal for robust genotypes, to yield large numbers of experimental flies):

     1. anaesthetise flies in cage and remove egg laying plate on which fertilized eggs lie (see note ¹⁶)

     2. using a squeeze bottle containing PBS, cover the plate with a thin layer of buffer

     3. dislodge eggs by “brushing” the egg laying surface with a fine paint brush

     4. pour egg / PBS suspension into a 15ml falcon tube and allow eggs to settle

     5. pour off most of the PBS and add more fresh PBS to wash the eggs

     6. allow eggs to settle and pour off most of the PBS, leaving only sufficient to just cover the settled egg mass

     7. allow eggs to settle

     8. using 100μl pipette with wide bore tip (see note¹⁷), set volume to 18-20ul and insert tip into the solution so the tip is level with the top of settled egg mass; quickly release plunger while dropping tip into the mass of eggs

     9. inspect tip for a dense, even, mass of eggs (see note ¹⁸)

     10. dispense egg mass on to surface of ~70ml SY medium in a 250ml bottle

   • Picking method (more labour intensive than pipetting, but more fragile genotypes tend to fare better using this method):.

     1. Incubate egg laying plate with staged embryos for 24h at 25ºC (see note¹⁹)

     2. using a dissecting microscope, locate 1^st instar larvae (see note ²⁰) on plate and touch with the metal pick. They will stick.

     3. With practice, up to ~20 larvae can be collected on one pick

     4. Gently transfer picked larvae into a fresh vial with food for development by wiping larvae off the needle on to the surface of the food (see note ^{21 22})

   • e.plug vial / bottle tightly with cotton wool ball(s) (see note ²³)
   • f.incubate at 25ºC with 65% humidity and 12:12 h light:dark

3.2. Experimental generation

3.2.1. measuring lifespan of one batch of mated wild type flies on one food type

1. after 10 days, transfer freshly emerged flies to fresh bottles or vials containing SY medium (see note ^{24 25})

2. return bottles of flies to controlled environment (25ºC, 65% humidity and 12:12 h light:dark) for 48h to allow all flies to mate

3. anaesthetise flies with CO₂ and manipulate carefully using a soft brush (see note ²⁶)

4. separate males from females and allocate target individuals to experimental containers (refer to notes ^{27 28 29 30 31}). Various aspects of courtship and mating modify lifespan of the different sexes to different extents (Partridge and Andrews, 1985; Partridge and Farquhar, 1981; Partridge et al., 1986, 1987; Sgrò and Partridge, 1999). Also, genotype and food quality interact with courtship and mating frequency (Chapman and Partridge, 1996; Wigby et al., 2011). Housing experimental flies as a single sex population avoids the confounding effects of sex X treatment interactions that modify lifespan.

5. store vials at 25ºC, 65% humidity, 12:12h light:dark (refer to note ³²)

6. transfer flies to fresh food every 2 -3 days (refer to notes ^{33 34 35 36})

   1. if recording female egg laying of experimental flies, a good and simple summary can be generated by counting all eggs in all vials once or twice a week for the first 4 – 5 weeks

      1. count the number of eggs on the surface of the food ~18h after transferring in flies (see note ^{37 38 39})

      2. data for a vial are expressed as the number of eggs per fly per day. For each vial, sum the egg lay for the average female across all count days to generate an index of lifetime egg laying.

7. for each transfer, score deaths and censors until all flies are dead (see Figure 1 for survival data examples).

Figure 1.

Examples of good and poor quality survival data. The survival characteristics of a healthy population of flies is demonstrated in blue. There are relatively few deaths up until day 60, from which point there is rapid loss of life. By contrast, the population illustrated by the red line suffers substantial numbers of deaths beginning at day 20. Thus many flies are dying at young and middle ages, rather than predominantly at old age. This is a sign of poor housing conditions or a genetically fragile stock.

3.2.2. protocol modifications for measuring the effects of dietary interventions

• 1.repeat steps 1-3 from 3.2
• 4.separate males from females and allocate target individuals to experimental containers (refer to note ⁴⁰)
• 5.store vials at 25ºC, 65% humidity, 12:12h light:dark
• 6.transfer to fresh food every 2-3 days.

  1. conduct egg counts as described in section 3.2

• 7.at each transfer score deaths and censors until all flies are dead

3.2.3. protocol modifications for measuring effects of genetic interventions to modify lifespan

• 1.repeat steps 1-3 from 3.2 (see note ⁴¹)
• 4.separate males from females and allocate target individuals to experimental containers (refer to notes ⁴²)
• 5.store vials at 25ºC, 65% humidity, 12:12h light:dark
• 6.transfer to fresh food every other day

  1. conduct egg counts as described in section 3.2

• 7.For each transfer, score deaths and censors until all flies are dead

3.2.4. modifications for measuring the effects of pharmacological interventions to alter lifespan

• 1.repeat steps 1-3 from 3.2
• 4.separate males from females and allocate target individuals to experimental containers (refer to notes ⁴³)
• 5.store vials at 25ºC, 65% humidity, 12:12h light:dark
• 6.transfer to fresh food every other day

  1. conduct egg counts as described in section 3.2

• 7.For each transfer, score deaths and censors until all flies are dead

3.3. Data handling

1. For each lifespan, record the date on which the experiment started, the genotype and conditions used in the experimental setup as well as any notes about the experimental setup that will modify or help interpretation of the outcomes. For consensus guidelines on what constitutes the minimal information to be recorded for lifespan experiments, see (Ziehm et al., 2015).

2. Throughout the experiment, record deaths and censors for each vial for each day on which they were observed (see note ⁴⁴)

3. These data can be used to generate lifespan curves for comparison using standard life table analyses (Lee and Wang, 2003)

4. An important recent advance has been the publication of an openly available database for storing lifespan data, called SurvCurv (Ziehm and Thornton, 2013; Ziehm et al., 2013, 2015). Users can upload data for secure storage as well as use an array of statistical tools to analyse the experimental outcomes. Additional tools available on the site allow the lifespans to be compared to others in the database and so can be used to aid further biological discoveries.