Statistical Analysis

All analyses were carried out in the R statistical software package (R Core Team, 2018) using RStudio (RStudio Team, 2016). For each analysis that was sufficiently normally distributed, a mixed effects linear model of was created using the ‘lme4’ package of R (Bates et al., 2015). In the case of experiments which had been carried out only once a linear model was fitted with line as a random effect, while in the case of experiments which had been carried out at both generations 3 and 5, generation was included as a blocking factor. Finally, in the case of the lipid data, technician was

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included as a blocking factor to account for the large discrepancy between the plates caused by different technicians producing them. After modelling, the selection regimes were compared by Type III ANOVA.

For non-normal data, regimes were compared by the Kruskal-Wallis test, and for the stress

resistance tests survival was compared using log-rank test to compare the selection regimes. Again, where an assay was performed at generation 3 and generation 5 these data have been grouped for statistical testing.

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Results

To investigate phenotypic changes that may have occurred because of the selection process or that may have been causative of the observed long-lived phenotype, several physiological measures were made at generations 3 and 5 of the selection.

These were based on two broad categories typically associated with lifespan extension and long- lived organisms: life history characteristics and resistance to various stresses.

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Life History Characteristics

Characteristics concerned with development and fitness are frequent targets of both selection and manipulation for lifespan extension. For instance, selection on delayed reproduction is the most common method of longevity selection (Rose, 1984; Luckinbill and Clare, 1985; Partridge and Fowler, 1992), while selection on development time has been carried out in the context of ageing research. Likewise, lifespan extending interventions such as dietary restriction can have a large effect on these characteristics; DR slows development time, reduces adult body weight and reduces fecundity (Tu and Tatar, 2003) to list a few examples.

Since the lifespan results from within the selection experiment relied on small sample sizes by necessity, larger experiments (n=200) were carried out at 25°C to provide more robust lifespan comparisons between the selection regimes (Figure 12A+B). These results are discussed at length in Chapter 2 Section 4.5 but are presented again here for reference during the discussion of the correlated responses.

To determine if the selection was influencing early fecundity, a common response in longevity selection experiments (Rose, 1984; Luckinbill and Clare, 1985; Zwaan, Bijlsma and Hoekstra, 1995), the 3-day average fecundity of 4-day old flies was tested after 5 generations of selection.

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Figure 12. Life history characteristics of the selection lines. (A) Lifespan at 25°C after 3 generations of selection. (B) Lifespan at 25°C after 5 generations of selection. (C) Average fecundity over a 3-day period starting at age 4.

Median lifespan was significantly longer in the S regime at both generation 3 and generation 5 by 17% and 26% respectively (Figure 12A+B), however there was no change in early life fecunduty at generation 5 (Figure 12C).

According to developmental run-on theories of ageing, early life development and maturation may be intrinsic to ageing, as the continuance of these developemtal programs past their usefulness may be the primary driver of ageing (Blagosklonny, 2012; de la Guardia et al., 2016). To investigate this stage of development in the selection lines, egg-to-adult development time was measured at generations 3 and 5. At generation 5 the proportion of larvae that survived to adulthood, and then the sex ratio of those flies that successfully eclosed was also recorded.

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Figure 13. Larval development life-history characteristics of the selection lines. (A) Egg-to-adult development time. (B) Percent of larvae successfully developing to adults. (C) Ratio of males to females of successfully eclosing flies.

There were no changes in any of the developmental life-history characteristics, although there was a highly significant increase (p < 2x10-16_{) in development time for all lines between generations 3 and} 5.

Related to, and typically corellated with, larval development is adult body weight (Nunney, 1996). Flies were weighed whilst still alive in the first instance to calculate wet body weight without the risk of water loss, then placed in an oven at 45°C for 24 hours to dry them out before being weighed again. Finally the water fraction could be caluclated from these weight measurements.

Figure 14. Weights and composition measures of the selection lines. (A) Weight of live flies. (B) Dry body weight of flies. (C) Percent of body weight accounted for by water.

There were no changes in wither wet or dry weight, or water content (Figure 14).

Finally, to complement the water content results, other body composition measures were taken, total protein and lipid content. Protein content declines with age and is correlated with protein synthesis rates (Webster and Webster, 1983) while lipid content is associated with starvation

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resistance (Chippindale, Chu and Rose, 1996) and has been correlated with longevity in females (Service, 1987).

Figure 15. Body composition measures of selection lines. (A) Total protein content of flies. (B) Percent of dry body weight accounted for by lipids.

An increase in mean protein content of 12.3% approached significance in the S regime (p = 0.065) (Figure 15A), although there was no change in lipid content (Figure 15B).

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Stress Responses

Numerous stress responses were also studied as possible correlated responses to selection. Stress responses are closely associated with ageing and longevity, being altered in numerous longevity phenotypes (Service et al., 1985; Broughton et al., 2005; Bubliy and Loeschcke, 2005) and are downstream of genetic mechanisms known to affect ageing such as TOR (Chou et al., 2012) and JNK (Wang, Bohmann and Jasper, 2003) signalling pathways.

Heat stress resistance was a correlated response to longevity selection in the UC Irvine lines (Service et al., 1985), while mild heat stress can extend lifespan in Drosophila (Hercus, Loeschcke and Rattan, 2003) and heat stress response mimetic drugs can extend lifespan in C. elegans (Benedetti et al., 2008). To test heat stress resistance, survival of the selection lines was measured at 38°C at both generations 3 and 5.

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Figure 16. Resistance of the selection lines to heat stress (38°C). (A) The distribution of survival times at 38°C. (B) Kaplain-Meier survival curves showing survival at 38°C.

A 15% reduction in mean heat stress resistance of the S regime approached significance (p=0.086) and comparing the survival curves by log-rank showed a highly significant reduction in heat stress resistance of the S regime (p=1.42x10-7_{) (Figure 16).}

Desiccation and starvation resistance were measured at both generations 3 and 5. These traits are common correlated responses to longevity selection (Service et al., 1985; Bubliy and Loeschcke, 2005). Both desiccation and starvation resistance were measured at generations 3 and 5.

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Figure 17. Resistance of the selection lines to desiccation. (A) The distribution of survival times under desiccation conditions. (B) Kaplain-Meier survival curves showing survival under desiccation conditions. There was no significant change in mean desiccation resistance, although there was a significant decline from generation 3 to generation 5 (p=2x10-16_{), and comparing survival by log-rank showed a} significant increase in desiccation resistance in the S regime (p=0.016).

Figure 18. Resistance of the selection lines to starvation. (A) The distribution of survival times of under starvation conditions. (B) Kaplain-Meier survival curves showing survival of flies under starvation conditions. Mean starvation resistance did not change between the selection regimes, however there was a significant (p=1.04x10-14_{) increase between generations 4 and 5. Survival of the S regime was highly} significantly increased when compared by log-rank 4 (p < 2x10-16_).

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Finally, oxidative stress resistance and measures of oxidative damage can help determine the possible involvement of molecular damage responses in the longevity phenotype. To study this, resistance to paraquat (a producer of superoxide radicals), and adult levels of protein carbonyl levels were measured after 5 generations of selection.

Figure 19. Resistance of the selection lines to oxidative stress (20mM paraquat). (A) The distribution of survival times of flies fed 20mM paraquat. (B) Kaplain-Meier survival curves showing survival of flies fed 20mM paraquat.

There was no difference in either mean oxidative stress resistance, or the survival curves of the selection regimes (Figure 19).

Figure 20. Carbonyl content of individual flies from the selection line measured by dot blot, expressed as nmols carbonyl per mg total protein.

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As with the oxidative stress resistance assay, there was no difference in carbonyl accumulation between the selection lines (Figure 20).

Discussion

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Introduction

Correlated responses are changes in phenotype that are a result of a selection regime but were not the subject of direct selection. They are studied in order to determine potential genetic and

mechanistic links between the studied phenotype and other phenotypes, however they are typically far more variable than direct responses (Gromko, 1995). Between, and sometimes within, selection experiments correlated responses can be heterogeneous, making comparisons with other studies difficult, however some traits for instance stress responses and numerous life history characteristics have broad representation in longevity selection experiments.

One flaw of using correlated responses to determine possible genetic mechanisms is that, much like the genetic changes themselves, correlated responses can be the result of drift, genetic fixation or inadvertent direct selection brought about by an imperfect method.

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Life History

Life history characteristics are perhaps the most variable responses commonly measured in longevity selection lines. Even small changes in environment, handling or parental conditions can produce changes in life history (Crill, Huey and Gilchrist, 1996; Vijendravarma, Narasimha and Kawecki, 2010; Nystrand and Dowling, 2014). Nonetheless, they are still a useful measure and can provide useful insight into the mechanisms of ageing, due to how closely linked longevity is to the various stages of development and reproduction.

Development time has been altered in longevity selected lines. The UCL lines showed significantly reduced development time relative to the controls (Partridge, Prowse and Pignatelli, 1999) although in this case Drosophila selected for early reproduction also showed this change. In our study, there was no difference in development time between the regimes, however there was a highly significant increase (p < 2x10-16_{) in development time across both regimes from generation 3 to 5. This was} likely due to declining health of the lines due to inbreeding.

Drosophila weight has been shown to be both positively and negatively correlated with longevity in Drosophila, much like development time. The Wayne State longevity selected lines have a lower

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body weight than the controls (Buck et al., 2000) possibly due to an altered larval development phenotype ultimately reducing the effects of developmental overrun in later life. In our study there was no change in body weight between the lines, which coupled with the lack of change in

development time suggest that the lifespan extension observed was unlikely to have been caused by an alteration of larval developmental programs.

Viability and sex ratio can be indicative of parental biological age, with viability declining with maternal age (Kern et al., 2001) and the sex ratio of offspring being skewed depending on either the paternal or maternal age (Yanders, 1965). We observed no change in either viability or sex ratio across the selection lines. However, S1 tended towards a tighter grouping at a lower viability than the other lines potentially expected due to the higher incidence of inbreeding in the S lines

compared to the controls, however this was not significant. Likewise, C2 tended to a wider grouping with more males to females (Appendix 1).

Finally, there was no change in early fecundity, disagreeing with the results of Zwaan, et al. (1995). However, the period we measured fecundity over, ages 3-5 days, is in the period at which Zwaan, et al. detected the smallest difference between the lines in early life period. There are also plenty of examples whereby selecting to extend lifespan did not affect early fecundity (Partridge, Prowse and Pignatelli, 1999).

To evaluate functional ageing, three molecular measures were taken, total protein content, protein carbonylation levels in adult flies and total lipid content. Due to technical issues, only a small subset of the lipid data was usable.

Total protein was increased 12.3% in the S lines, approaching significance (𝑝𝑝= 0.065). This was mostly due to high levels in S2. The Wayne State lines saw no change in protein in long-lived adults, despite their larvae having roughly half the total protein of their short-lived counterparts. This was considered to be due to altered feeding behaviour in the larvae of the selected lines, however after the flies reached adulthood the disparity in protein disappeared, possibly as a result of the different levels of fecundity between the selection lines (Riha and Luckinbill, 1996). In the case of our lines this explanation seems unlikely since they were selected in a way that should not affect early fecundity, and indeed fecundity was not altered in the S lines (Figure 12C). However, it is still possible that early life metabolic tradeoffs allowed a greater amount of protein synthesis in the S lines.

There was no significant change in lipid content between the selection regimes (Figure 15B). The UC Irvine lines showed an increase in lipid content in the long-lived lines at young ages, possibly due to their decreased fecundity allowing increased lipid storage (Djawdan et al., 1996). As such it is

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unsurprising that our lines did not show a change in lipid content, since they also did not show altered fecundity, and thus this result contributes to the conclusion that the lifespan extension was not achieved by a reduction in reproduction or reproductive development.

The lack of change in most life history characteristics across the selection lines is interesting because it suggests that the lifespan extension was not achieved by simply slowing developmental processes or modulating reproduction. Further study could confirm this by covering a wider range of life history characteristics, for instance male competitive ability, and lifetime female fecundity.

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Stress Resistance

A reduction in mean heat stress resistance in the S lines approached significance (p = 0.086) while the survival analysis showed the lifespan curves to be highly significantly different between the control and selected regimes (p = 1.42x10-7_{). Whether this is because there is a tradeoff between} heat stress resistance and longevity or simply that heat stress resistance suffered inbreeding

depression is unclear without further study. Crossing the lines within each selection regime and then comparing heat stress resistances could help determine if inbreeding depression was at play.

Reduced heat stress resistance has been associated with extended lifespan in previous studies. Ablating insulin-like peptide producing cells extends lifespan and lowers heat tolerance (Broughton et al., 2005), while at least one other line selected for longevity has expressed a sensitivity to heat stress (Kuether and Arking, 1999). This sensitivity could be indicative of a frailty in the flies, although the lack of differences in life history characteristics suggests this is not the case. Another explanation could be that the heat stress response in the long-lived lines is constitutively more active, but less adaptive to the extreme stress of the assay.

Like heat stress, oxidative stress resistance relies on a network of molecular chaperone proteins, as well as antioxidant complexes to tackle ROS directly, and these processes are associated with ageing (Zou et al., 2000). Because of the overlap in function, it is somewhat surprising that while we saw a possible decline in heat stress resistance, there was no observed change in oxidative stress

resistance (Figure 19A). This may be due to the changes in heat stress resistance being such that they do not overlap with oxidative resistance, or possibly there were changes in other oxidative defences that counteracted a decline in the general stress response. A final possibility is that, at 20mM diluted in solid food medium, the oxidative stress caused by the paraquat was not sufficient to highlight any differences in the lines, where a higher concentration or different delivery, for instance in sucrose solution, may have an effect, however high sucrose diets can also be stressful so this may introduce a confounding factor (Rzezniczak et al., 2011).

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Protein carbonyl content is one possible measure of the oxidative damage that accumulates with ageing. High protein carbonyl is associated with advanced age as well as numerous ageing related diseases such as Alzheimer's disease, rheumatoid arthritis and diabetes (Dalle-Donne et al., 2003). We saw no change in carbonyl content at age 25, suggesting that if oxidative stress responses are altered in the S lines it does not influence carbonyl accumulation up to this age. An interesting future study would be to examine carbonyl levels in the selection lines at a more advanced age, possibly with other measures of oxidative damage such as advanced glycation end-product (AGE)

accumulation. These are protein modifications that accumulate with age, are associated with diabetes, ageing and other disorders and have links to carbonylation (Singh et al., 2001).

Desiccation resistance is associated with longevity and a decreased water loss rate, characteristics that have been observed in flies selected for longevity (Nghiem et al., 2000). Water loss rates in Drosophila are tied chiefly to three causes, respiratory loss, excretion and cuticular loss and is thus modulated by metabolism, spiracle activity and surface lipid content (Gibbs, Fukuzato and Matzkin, 2003). While there was no mean change in desiccation resistance, when comparing the survival curves there was a significant increase in desiccation resistance of the S regime (𝑝𝑝= 0.02). Looking at the results for desiccation at generations 3 and 5 separately (Appendix 1) it appears that there was a highly significant increase in desiccation in the S lines at generation 3, driven by S1, but no change at generation 5. This could indicate that the desiccation response was merely a result of fixation of extreme resistance alleles within S1 which were since lost by generation 5. This would mean that desiccation resistance was unlikely to be involved in the longevity phenotype observed in the S regime at generation 5.

Unsurprisingly, starvation resistance mimicked the results of the desiccation assay. Starvation and desiccation resistance are very closely connected in Drosophila, both being highly dependent on activity and lipid content (Hoffmann and Parsons, 1993; Hoffmann and Harshman, 1999). Selecting Drosophila for increased starvation resistance can extend longevity in a background previously selected for longer life (Rose et al., 1992). Starvation resistance is usually accompanied by increases in lipid content, much like desiccation resistance (Djawdan et al., 1998; Ballard, Melvin and Simpson, 2008). In this case, lipids were only measured at generation 5, and there were no significant

differences between the lines Figure 15B, which agrees with the lack of differences in the stress assays. These results suggest that the mechanism behind the longevity phenotype may at least in part overlap with starvation and desiccation resistance mechanisms, although in a lipid content independent manner.

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Longevity Strategies

It is apparent that the longevity phenotype achieved by our selection experiment was, at least in part, due to changes in stress responses rather than in life-history and developmental

characteristics. Heat stress was noticeably affected the S regime, suggesting that modulation of chaperones or repair proteins may be responsible for extending healthy lifespan.

The decline in heat stress resistance in the S lines, coupled with the lack of improvement in oxidative stress resistance, suggests that the selected flies may be frailer, perhaps expressing stress resistance mechanisms at a higher level constitutively, but being less able to increase them in response to severe stress. This would explain the longevity phenotype, repair mechanisms would be higher in the S lines under normal conditions, as well as the lowered stress resistance as the flies would be less adaptable.

As mentioned previously, the loss of desiccation resistance by generation 5 suggests that desiccation resistance mechanisms did not contribute to the lifespan extension. A possible explanation for this is