3.1
Selection on Fitness Components
By far the most frequently used method for the selection of longevity in Drosophila is the selection of some sort of fitness characteristic, typically achieved by collecting eggs only from flies that live to a certain age, or by selecting old flies that have retained the highest fecundity in a population. These experiments were initially concerned with unpicking the evolutionary theory of ageing, aiming to determine whether mutation accumulation (Medawar, 1952) or antagonistic pleiotropy (Williams, 1957) were primarily responsible for the ageing process.
The first successful attempt to select on fitness characteristics in Drosophila with a view to extending lifespan began in 1981 with a small pilot experiment by Michael Rose and Brian Charlesworth, accompanied by a sib analysis of Drosophila females investigating the link between fecundity and lifespan (Rose & Charlesworth 1981a; Rose & Charlesworth 1981b). The means of selection was based on egg laying ability. Egg laying was measured over a five-day period for each line and the flies
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laying the most eggs were selected to continue the line. A control line, selecting the best layers from days 1-5 post-eclosion, and a selected line, selecting the best layers from days 21-25, were carried through three generations. This method achieved a modest increase in lifespan, despite the lack of replicate lines, accompanied by a decline in early fecundity and egg laying rate, corroborating with the sib analysis to suggest that ageing evolved in Drosophila primarily due to antagonistic pleiotropy. This pilot was followed up with a larger, more rigorous selection experiment (Rose, 1984). Five control lines and five selected lines were created (although due to accidental loss of two selected lines, only three lines of each treatment were reported), with the control lines being reared in discrete generations of 14 days, while the selected lines were reared in discrete generations of progressively longer time periods, starting at 28 days and finishing at 70. The selection was successful in extending lifespan, with selected males living 13% and selected females living 22% longer than the controls. Again, this was coupled with a decline in early fecundity for the selected lines, although no change in total lifetime fecundity was seen.
Concurrently, Luckinbill et al. carried out a similar experiment, selecting on both early and late fecundity (Luckinbill et al., 1984; Luckinbill and Clare, 1985). Early selected lines reproduced at 2-6 days post-eclosion for 26-29 generations, while the late selected lines reproduced at 22 days in the first instance which eventually progressed to 70 days by the 21st generation. This selection was very successful in extending lifespan with a more than 2-fold increase in the late selected lines at its highest point at generation 13. Subsequent generations also saw in increase in absolute lifespan for the late selected lines, however there was also a substantial increase in lifespan for the early selected lines over these generations, reducing the gap between them. Furthermore, an additional selection experiment was reported in these papers, showing that controlling larval density in the selection led to a much lesser response in the late selected lines.
3.2
Criticisms and Developments
These experiments were criticized for jumping to conclusions about the evolution of ageing, despite methodological flaws. It was noted that the control and selected lines were not necessarily assayed at the same time and certainly not at the same generation since the start of the experiment (Baret and Lints, 1993). This is an important point due to the well-known and studied fluctuation of lifespan in laboratory Drosophila populations, presumably due to non-random but uncontrollable
environmental factors (Lints et al., 1989). This particular criticism was answered with a large scale and more rigorous lifespan experiment on the selected lines of Luckinbill and Clare (1985), using a larger sample size and initiating the lifespans concurrently for both selected and control lines (Fukui, Pletcher and Curtsinger, 1995). A strong lifespan extension was shown in both males and females,
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selected males showing a 46% increase in mean lifespan while selected females saw a 21% increase. Additionally, continuation of the selection experiments long after the original publications showed the lifespan extension to be robust, as well as correlated responses to selection (Arking and Buck, 1995).
Another key criticism was that these selection experiments were carried out with uncontrolled larval density, and that when larval density was controlled the response to selection disappeared. This criticism was also extended to the life-history assays carried out in both experiments, since larval density and competition were known at the time to contribute to variation in many life-history traits, a view since supported by further research (Miller & Thomas 1958; C. Roper et al. 1993a; Leips & Mackay 2000). The lack of response to selection when controlling for larval density was conceded, with the conclusion that the lifespan extension achieved by the delayed fecundity method, in this case, was dependent on a minimum threshold larval density (Luckinbill and Clare, 1986).
In order to tighten up the design and provide firm answers on the effect of longevity selection on life history traits, a new selection was carried out and larval density controlled during the life history assays (Partridge & Fowler 1992; Roper et al. 1993b). Two selections were set up using different base stocks, both outbred laboratory strains although one was far more recently collected. Each strain produced three control and three selected lines, and the method of selection was similar to that of previous experiments. Lifespan was assayed after controlling for larval density, along with a host of other life-history characteristics. Lifespan was successfully extended in both males and females, although males did not respond in one of the two strains. Interestingly, there was no decrease in early fecundity observed in the selected females. This is opposed to the results of Rose and Luckinbill, suggesting that altered fecundity is not inextricably linked to long-life.
Further improvements were made when another selection was carried out, controlling larval density throughout the experiment (Partridge, Prowse and Pignatelli, 1999; Sgrò et al., 2000). Again,
controls could reproduce in early life, while selected flies reproduced 3-4 weeks later, larvae were collected and transferred to vials to develop at a standard, low density. The selection was successful, extending female mean lifespan by about 23%, and differed from previous studies in that there was no increase in late-life fecundity in the selected flies. This corroborated Partridge and Fowler (1992), again suggesting that the lifespan extension from this method of selection is not necessarily due to a delay in fecundity, but could be due to a decrease in overall female fertility, or potentially the 'purging' of deleterious mutations in the selected lines as predicted by the mutation accumulation theory.
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3.3
Other Approaches and New Questions
Another slightly different selection method was employed by Promislow and Bugbee (2000). Instead of selecting on late reproduction, they selected on time to physiological maturity i.e. the time until the flies were able to lay fertile eggs. Using Drosophila simulans, flies were either selected to be the earliest in the cohort to start laying, or the latest in the cohort to start laying. Although this method is less direct than those previously used, it still led to a modest lifespan increase in one of the female lines, although this may have been in part due to genetic drift.
Modern technologies have also allowed further investigation into longevity selection. For instance, Drosophila selected for late female fecundity were generated, extending virgin lifespan in both males and females, and then subjected to genomic sequencing and microarray expression analysis (Remolina et al., 2012). This experiment was able to identify numerous genetic variants associated with microbial defences, as well as in genes related to proteolysis. Significant increases in expression associated with these processes was also detected in numerous genes. As new techniques develop, ever deeper investigation into long-lived selected organisms will be possible.
Selecting for delayed fecundity has been used when investigating the link between stress responses and longevity. Norry and Loeschke (2003) used flies selected for delayed fecundity and flies selected for improved heat stress resistance to examine the effects on the molecular chaperone Hsp70. They found that selecting for heat stress resistance increased both male lifespan and Hsp70 expression, whereas the long-lived delayed fecundity selected flies had a decreased Hsp70 expression, but also saw lifespan extension in females. These experiments were further developed by selecting for a range of traits, including delayed fecundity, heat stress, heat knockdown, cold stress, starvation and desiccation resistances (Bubliy and Loeschcke, 2005). Finally, delayed fecundity selection in
Drosophila buzzatii has been used to study the connection between lifespan, life-history and stress resistance (Scannapieco, Sambucetti and Norry, 2009). Flies were successfully selected for increased lifespan in both males and females (although this was at least partly due to a reduction in frailty, rather than onset or rate of rate). Tradeoffs with longevity were found in fecundity and development time, while stress resistances generally increased, including heat and cold stress.