Obra civil

Stress responses have been connected with healthy ageing. Many lifespan extending interventions, including longevity selection, can also increase stress resistance (Kampkötter et al., 2008;

Deepashree, Shivanandappa and Ramesh, 2017). These observations are in accordance with the stress theory of ageing, a development of the rate of living theory, positing that inherited stress resistances are responsible for differences in maximum lifespan (Parsons, 1995).

21.1

Heat Stress

A very easy measure of the stress response in Drosophila is their reaction to severe heat. This is usually measured as the time to immobilisation or death, but can be measured with a lower fatality risk by measuring time to knockdown using specialised apparatus (Huey et al., 1992). Heat stress resistance is associated with ageing in two ways. Firstly, it shows an age-related decline, with older flies more susceptible to extreme heat (Grotewiel et al., 2005) and secondly, changes in heat resistance are often correlated with long-life. These correlations can either be positive (Lin, Seroude and Benzer, 1998; Scannapieco, Sambucetti and Norry, 2009) or negative (Kuether and Arking, 1999; Broughton et al., 2005), suggesting a complex relationship between the heat stress response

mechanism and ageing.

The heat stress response is not unique to heat stress. Heat shock proteins are involved in a wide variety of stress responses, acting as chaperones for protein folding and transport, monitoring protein production in non-stressful conditions and have a role in presentation of antigens to the immune system (Lindquist and Craig, 1988). The change in function of heat shock proteins between stressful and normal environments may explain why they behave differently across long-lived organisms. It appears that overexpressing heat shock proteins constitutively is not enough to improve longevity, although they are clearly involved in many longevity phenotypes.

21.2

Oxidative Stress

Oxidative stress resistance is frequently correlated with longevity, which would be expected according to the FRTOA. Increased oxidative stress resistance has been found in longevity selected lines, with the UC Irvine lines showing increased resistance to paraquat, a superoxide generator (Harshman and Haberer, 2000). The Wayne State University lines also exhibited an increase in oxidative stress resistance, which could be significantly reduced by treating both short and long-lived lines with a catalase inhibitor. This shows that at least part of the increased oxidative stress

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resistance was due to increased catalase activity in the long-lived lines (Dudas and Arking, 1995). Other lifespan extending phenotypes can also have an effect, for instance long-lived methuselah mutants show an increase in oxidative stress resistance (Lin, Seroude and Benzer, 1998).

21.3

Starvation and Desiccation

Selecting on starvation and desiccation has been shown to extend lifespan as a correlated response (Rose et al., 1992). Both of these stress resistances are also commonly increased when selecting for longevity, for instance desiccation resistance was increased in the Rose lines due to reduced rates of water loss under desiccation conditions (Nghiem et al., 2000) and starvation was increased in D. buzzatii females selected for delayed fecundity (Scannapieco, Sambucetti and Norry, 2009). Interestingly, selecting directly on lifespan has not previously shown a change in starvation resistance, at least relative to the controls. Rather, flies directly selected for lower longevity saw a decrease in starvation resistance, but there was no difference between controls and long-life selected lines (Zwaan, Bijlsma and Hoekstra, 1995). The lines selected by Bubliy and Loeschke (2005) had mixed results with longevity selected flies seeing a significant increase in starvation resistance, but not in desiccation.

22

Conclusion

A huge range of functions are affected by ageing, and as such are potentially affected by longevity selection and other lifespan extending effects. The challenge in studying correlated responses to longevity selection is in narrowing down the options to those phenotypes which may not only be affected by an altered ageing phenotype, but which may also be genetically linked to the lifespan extension mechanism itself. Thus, choosing the correct measurements is partly reliant on precedent (to compare a selection experiment to others it is helpful to have overlap) and partly on observation, because during the selection procedure itself there are many casual observations which can be made that may inform the choice of phenotypic analysis. Finally, it is important to think carefully about the questions being asked of a selection experiment to narrow down phenotypic analysis experiments to those which will be most useful or interesting.

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Materials and Methods

23

Fly Maintenance

23.1

Rearing Conditions and Larval Density Control

All flies were reared in standard conditions (25°C, 70% humidity and on a 12:12 light:dark cycle). To control for larval density and parental effects, flies were generated for assay according to the following procedure.

Approximately 100 age-matched males and 100 age-matched females were placed into a breeding cage (Genesee Scientific, Cat# 59-101) fitted with a petri dish half-filled with laying medium. Yeast paste was prepared by mixing live yeast with water to a smooth consistency and painted onto the center of each dish before fitting them to the cages. Flies were mated and allowed to lay any unfertilized eggs for 1 day, before fresh plates were put on and the flies were allowed to lay for 12- 16 hours. The plates were then removed, and the eggs washed off into a 15ml tube using PBS and a paintbrush. Eggs were washed with more PBS to remove residual yeast paste, and then pipetted into fresh bottles containing cornmeal medium at a controlled amount of 26ul eggs per bottle. To

achieve consistency, eggs were pipetted using a widened pipette tip and abruptly lifting the plunger to collect the eggs, using this method the eggs are densely packed into the pipette tip, and numbers pipetted are consistent (Clancy and Kennington, 2001).

Bottles seeded with eggs were then incubated in standard conditions until the flies eclosed, at which point all flies eclosing each day were collected (for experiments requiring virgins the flies were sorted at this stage) and allowed to mate for two days before being sorted into pre-experimental conditions at 3 days old. Unless otherwise stated, all flies tested were mated males.

23.2

Generation Specifications

There were slight differences in these protocols, depending on which generation they were carried out and for what purpose.

23.2.1 Generation Three

From the breeding stocks at generation three, 5 males and 5 females were collected from the three longest lived families of the S lines, and three randomly selected families of the C lines. These flies were kept in their family groups and allowed to mate in bottles (two per family) which had small plates of laying medium inserted into the lid. The flies were allowed to mate for 24 hours and the

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eggs collected as in Section 23. After this first generation eclosed, another generation was generated using the method outlined in Section 23. For all phenotyping experiments, these flies were kept in their distinct families. Due to low fertility in the initial generation, only two S2 families were carried through to the phenotyping experiments.

23.2.2 Generation Five

Two generations of controlled larval density were allowed after generation five of the selection. Breeding stocks from the selection were large enough that the standard larval density control method was used for both generations. For this generation the three longest lived families from each S line and three random families from each C line, were crossed within their line.

23.3

Media Recipes

Media Recipe

Standard Cornmeal 13.2% sucrose, 5.7% cornmeal, 3.3% autolysed yeast, 1% agar, 0.2%

nipagin, 0.5% propionic acid

Laying 8% autolysed yeast, 8% sucrose, 2% agar, 0.3% nipagin, 0.5% propionic

acid

Stock 8% autolysed yeast, 8% sucrose, 1.6% agar, 0.3% nipagin, 0.5%

propionic acid

Charcoal As standard cornmeal spiked with 2% ground activated charcoal

Starvation 1% agar, 0.16% KCl, 0.045% KH2PO4, 0.06% CaCl2, 0.259% MgSO4, 0.21%

NaCl, 0.07% Na2HPO4, 0.04% NaHCO3, 0.18% L-glutamine Table 16. Recipes for the various media used throughout the selection and phenotypic analyses. Ingredients were mixed into water at the given % (W/V).