The final lifespan experiment was performed to test if the lifespan differences were retained after 3 generations of relaxed selection following generation 5 of selection. The siblings of these flies, reared in the same conditions and at the same time, were collected for RNA-Seq analysis at 25 days old.
Figure 10. Kaplain-Meier survival curve of the siblings of the flies analysed by RNA-Seq. Lifespan was measured at 25°C (n=200).
Line n Onset of Ageing (day) Median Lifespan (days) LCI UCI
Control 1 199 55 73 69 77
Control 2 200 51 75 71 77
Selected 1 199 59 83* 79 85
Selected 2 201 57 85* 83 87
Table 13. Sample size, median lifespan and confidence intervals for each line when tested at 25°C, of the generation used for RNA-Seq analysis. * indicates significance relative to both controls of at least p < 0.05 as tested by pairwise log-rank and corrected for multiplicity using the Benjamini-Hochberg procedure.
This lifespan experiment gave similar results to the investigative lifespan at generation 5 (Figure 5B) although with a smaller effect. There was an increase in median lifespan of 12% and 15% for S1 and S2 respectively (Table 13).
Effect Hazard Ratio (hr) LCI (hr) UCI (hr) p-value
Treatment 0.48 0.39 0.59 1.25x10-12
Control(Line) 1.04 0.85 1.26 0.72
Selected(Line) 0.79 0.65 0.97 0.02
Table 14. Cox proportional hazard models of the selection when tested at 25°C, of the generation used for RNA-Seq analysis. Table shows the effect of treatment, line nested within each treatment, as well as confidence intervals for the hazard ratio and its significance.
0.00 0.25 0.50 0.75 1.00 0 30 60 90 Age (days) S ur v iv al E s ti m at e Line C1 C2 S1 S2
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A significant reduction in hazard accompanied the lifespan extension of the S lines, and as with the investigative lifespan at generation 3, there was no significant difference in hazard between the controls (Table 14).
Line Age Independent Mortality (a) (x10-5) (x10LCI(a) -5) UCI(a) (x10-5) Age Dependent Mortality (b) LCI (b) UCI (b)
Control 1 17.3 9.14 32.8 0.08 0.07 0.09
Control 2 7.47 3.59 15.5 0.09 0.08 0.10
Selected 1 11.6 5.81 23 0.08 0.07 0.08
Selected 2 2.46 1.08 5.61 0.09 0.08 0.10
Table 15. Gompertz parameter estimates for each line when tested at 25°C, of the generation used for RNA- Seq analysis. Table includes the confidence intervals for each parameter.
The onset of ageing was delayed by about 7% in S1 relative to C1, the healthiest control. There was no apparent change in the survival curve shape between the lines with the exception that S2 was the only line to not suffer an incidence of early mortality at around age 32, resulting in a lower age- independent mortality than the other lines (Table 15).
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Discussion
11
Introduction
The aim of this selection was to create a suitable model organism for studying possible evolutionary routes for longevity, with the hope of identifying longevity mechanisms which could become targets for intervention. Drosophila were used because they are convenient to select, having a predictable reproductive cycle, obvious sexual dimorphism and well understood genetics (Ashburner, 1989; Adams et al., 2000). Other advantages of Drosophila are that they are relatively short lived
compared to other model organisms, facilitating easy lifespan experiments, as well as being cheap to culture and being easy to genetically modify, resulting in a wide range of pre-existing transgenic constructs which could be used for further investigation after gene targets are identified. Finally, there is precedent for using Drosophila in selections relating to ageing, making comparisons of relative successes, limitations and pitfalls simple.
We carried out a five-generation familial selection on wild-caught D. melanogaster, selecting directly on male virgin median lifespan. Four lines were established: two lines selected for longevity (S1 and S2) and two randomly selected controls (C1 and C2). S1 showed a consistent median lifespan increase relative to the controls throughout the experiment, peaking at 18.2% extension at
generation 4 (Figure 6). S2 did not respond as obviously, showing no significant lifespan extension at 27°C, although a non-significant increase of 7.3% was observed at generation 4. After the selection, S2 had responded, showing significant median lifespan extension of 21% when measured at 25°C (Figure 9B).
12
Experimental Design
Our selection was largely based on the familial selection method of Zwaan, et al. (1995), but with some improvements and limitations. This method was selected since it offers the best chance at both extending lifespan (whereas the only other attempt at a direct selection method did not achieve this, see Lints, et al. (1979)) whilst avoiding selection on factors related to ageing such as fecundity or stress responses. Indirect methods of extending lifespan by selection bias the lifespan extension mechanism, making them ideal for studying the connections between life-history or stress and longevity, but less useful for the identification of novel drug targets.
Another benefit of the familial design is that the lifespan metric selected on can be tightly
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a hazardous activity, and although its accompanying increase in mortality does not appear to be due to accelerated ageing (Partridge and Andrews, 1985), the low sample sizes of each family during the lifespan experiments necessitated removing as much age-independent mortality as possible. Additionally, since virgin males and females were required to be collected to be reared as potential breeding pairs, it was convenient to collect virgin males for the lifespan assays as well. Median lifespan was chosen as the selected measure because it better reflects healthy lifespan than the mean, is more easily measured than other possible metrics (for instance direct measures of functional ageing) and could be determined before the entire cohort had died, allowing the next generation to be established before the breeding pairs were too old. A criticism of median lifespan as a measure of ageing effects is that extension of median lifespan without extension of maximum lifespan could be interpreted as the removal of disease and frailty in middle age, rather than a slowing of the ageing process itself (Wang et al., 2004). This causes problems when attempting to study the mechanisms of ageing, however when searching for gene targets to extend healthy lifespan an extension in median lifespan is very welcome. Obviously, the ideal would be a strong extension in both median and maximum lifespan, representing a slowing of the ageing process coupled with better general health.
The main departure from Zwaan, et al. in our study was the use of wild-caught Drosophila, where they used an outbred lab strain which had been kept in bottle culture for approximately 10 years at the time of the experiment. Lab adaptation of wild Drosophila has a demonstrated effect on many life-history traits including development time, reproductive schedule and fecundity with a clear selection pressure in favour of rapid reproduction and competitive larvae, with little regard for healthy adulthood. As such, lifespan tends to decline in lab strains relative to wild Drosophila, with bottle culture having the most pronounced effect in this regard (Sgrò and Partridge, 2000). Thus, we used recently wild-caught Drosophila to ensure the base population and the ensuing controls of the selection had the longest lifespan possible. This was effective, as the starting population gave a robustly healthy survival curve with little early mortality (Figure 5A). This healthy initial population means that any lifespan extension in later generations was less likely to be due to the purging of unhealthy alleles and more due to the selection for healthy alleles.
Using wild-caught flies also meant that genetic variation in the starting population would be higher than if we had used a lab strain (Sgrò and Partridge, 2001). Increasing variation was also the reason we generated the base population by crossing two wild-caught strains, caught in different
environments within Lancaster. High initial variation is important in selection experiments, because it increases the chance that alleles of interest will be in the population, and barring mutation, selection can only increase or decrease the prevalence of an allele that is already present. Indeed,
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within the base population there was a high degree of variation in lifespan (Figure 5B), giving good stock from which to select longer lived flies.
A final change is that we measured lifespan within the selection at 27°C rather than 29°C. This was intended to provide a compromise between rapidly assessing lifespan and not subjecting the flies to an overly stressful environment. The flies used in this experiment were sourced from a warmed butterfly house and a domestic glasshouse in October, a typically cool month, and so represent a population with mixed tolerance to temperature. A temperature increase of just 1°C above the standard living conditions of Drosophila begins to have significant effects on fertility (Rohmer et al., 2004), which may influence age-independent mortality. We found that flies reared at 27°C
demonstrated healthy survival, while still being short-lived enough to quickly ascertain median lifespan. Reducing the temperature at which the selection metric was measured was also intended to reduce the effect of inadvertently selecting for heat resistance.
The food used for this experiment is a high sugar, cornmeal-yeast medium. The high sugar content is palatable to flies, mimicking the taste of fruit juice, while the cornmeal-yeast mixture is a preferred source of protein relative to either one of these ingredients alone (Hutner et al., 1937). This food medium gives good, healthy lifespan curves in our lab, reducing the chance that the lifespan extension seen was caused by selection against overeating behaviours.
13
Selection on Lifespan
Based on the selection of Zwaan, et al. it is expected that lifespan would be modestly, but
significantly, increased in the S lines. After 5 generations they saw an increase in median lifespan of about 8 days (roughly 25% increase) when measured at 29°C. This closely reflects our own results at the same point, with a few key differences. After 5 generations of selection, S1, the most responsive line, had a significant median lifespan extension of 14.5%. This is less than the extension seen by Zwaan, et al. although this can be explained to a certain extent by the heterogeneity of lifespan data in general, the fluctuations commonly seen in selection experiments and the slight differences in methods. At generation 4 of both experiments for instance, the lifespan extension observed were very similar, around 16% for Zwaan, et al. and 18.2% for our S1 line. Additionally, the effect may be partially temperature dependent, and so the different assay conditions may be responsible for these differences in response. Minor differences aside, our results are remarkably similar to those of Zwaan, et al. establishing a typical expectation of the lifespan extension from this kind of selection. The similarities of this selection with that of Zwaan, et al. relative to the results of indirect selection can be clearly seen. Drosophila selected for delayed fecundity typically show a large, highly
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significant increase in both median and maximum lifespan (Figure 11A), while a direct familial selection method sees a less pronounced increase in median lifespan, and does not necessarily see an increase in maximum lifespan at all, which may indeed be higher in the controls (Figure 11B). This pattern was reflected in our study, with multiple generations showing this pattern (Figure 6). As previously mentioned, an increase in median without an increase in maximum lifespan could be indicative of only removing disease alleles from the population, rather than slowing the ageing process. In our experiment we appear to have achieved both. While some generations show only median lifespan extension, others (Figure 7B) and especially the lifespan results at 25°C (Figure 9), show a clear maximum lifespan extension in either one or both selected lines.
Figure 11. Comparison of lifespan extension achieved by selecting for delayed fecundity (A) or by selecting directly on familial lifespan (B). Adapted from Wit, et al. (2013).
The relatively low increases in lifespan caused by familial selection can be readily explained. Familial selection acts only on lifespan, with a minimal amount of selection against causes of early mortality. It is thus possible for disease alleles that act during late life to slip through the selection, since the breeding stock are considerably biologically younger at the point of reproduction than their siblings in which lifespan was measured. Compare this to selection on delayed reproduction, where the age at reproduction is pushed further and further forward, with age at egg collection getting as high as 70 days old or higher in some cases (Partridge and Fowler, 1992). Because only flies which have survived to this advanced age are able to reproduce, late acting disease alleles are strongly selected against. The result of this is that familial selection without alterations to select against late life age- independent mortality (for instance selecting on a statistic generated by combining the median and maximum lifespan) will necessarily be limited in the degree of lifespan extension it can achieve, unless over many generations of selection.
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Another reason for the smaller increase in lifespan by the familial method is the quality of the controls. In a familial selection, the controls are almost identical in treatment to the selected lines; they undergo the same bottlenecks, the same number of generations and reproduce at the same age. When selecting on delayed fecundity, the selected lines will be treated quite differently from the controls. The longer-lived lines will undergo far fewer generations of selection, since each generation takes considerably longer, and they will be propagated from much older individuals. The time of reproduction is important, for since in these experiments the controls are typically allowed to reproduce at an early age, there will be a strong selective pressure for early fecundity in the control lines and non-existent selection against late acting disease alleles (as with all lines in the familial method). As such, it is possible that the control lines are getting selected for a shorter lifespan, while the selected lines get selected for a longer lifespan, thus exaggerating the final difference between the two regimes. Finally, the differences in number of generations within the selection could mean that the controls will be far more inbred by the point of comparison, which could lead to inbreeding depression of their lifespan, again exaggerating the difference.
A noticeable improvement in this regard can be seen in our selection. Our controls, when compared to those of Zwaan, et al. (Figure 11B) do not show anywhere close to the same levels of early mortality. Indeed, the survival curves for all four of our lines were healthy, showing low age- independent mortality and a rapid rate of age-dependent mortality. These characteristics can be seen in the base population (Figure 5) and throughout the selection, with only occasional examples of early mortality. This is indicative of a healthy population (Eakin and Witten, 1995), with low incidences of genetic disease or other frailties, and further support the lifespan extension as being a modulation of the ageing process, rather than the purging of disease alleles.
14
Investigations
The purpose of this selection was to generate a model organism for further investigation into the ageing process. We carried out numerous investigations, phenotypic characterisation of flies derived from generations 3 and 5, and an RNA-Seq transcriptomics study, also on flies derived from
generation 5.
Figure 9A shows the lifespan at 25°C of flies derived after two generations of relaxed selection after generation 3. The siblings and offspring of these flies (up to two generations after this lifespan measurement) were used for the phenotypic evaluation at generation 3. The curve in Figure 9A is healthy, and S1 shows a significant increase in median lifespan of 21%, accompanied by a delayed onset of ageing, while S2 shows a 13% increase in median lifespan, also accompanied by a delayed onset of ageing. It is interesting that both S1 and S2 show lifespan extension at this point, because
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S2 did not show a consistently longer life within the selection experiment up to this point, and S1 looked like it was starting to drop off as well. Since this experiment took place after two generations of relaxation, it is possible that the observed longevity in S1 and especially S2 is the result of
alleviation of inbreeding depression.
Figure 9B likewise shows the lifespan at 25°C of flies derived after two generations of relaxed selection after generation 5. This time, a strong lifespan extension was seen in both S1 and S2 of 30% and 21% respectively. This was accompanied by a delay in the onset of ageing in both lines and a large increase in maximum lifespan in S1. This change in response for S2 allowed for more solid conclusions on what correlated responses were associated with longevity, and which ones may be due to drift or inadvertent selection. The strong response in S1, as well as the increased maximum lifespan, also show this generation to be fit for purpose as a longevity model.
Finally, the RNA-Seq was carried out on 25-day old siblings from C1 and S1 reared in the same conditions at the same time as the flies represented in Figure 21. Compared to most lifespan experiments within the selection, there is noticeable early mortality across all four lines. The cause of this is not clear, although it may have been due to inbreeding, undetected bacterial infection or some other unforeseen environmental variable. Since all four lines were affected it is still valid to compare them, and since the mortality starts at around day 27, the flies collected for RNA-Seq may not have been affected. This issue aside, there is still a strong lifespan extension in S1 relative to C1, the two lines compared by RNA-seq.