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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:1246-1252 (2006)
© 2006 The Gerontological Society of America

Effect of Deleterious Mutations on Life Span in Drosophila melanogaster

Yi Gong, James N. Thompson, Jr.2 and R. C. Woodruff

1 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio.
2 Department of Zoology, University of Oklahoma, Norman.

Address correspondence to R. C. Woodruff, PhD, Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403. E-mail: rwoodru{at}bgnet.bgsu.edu


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
Evolutionary theories of aging assume that the accumulation of deleterious mutations will reduce life span. We tested this assumption in Drosophila melanogaster by a newly designed mating scheme, in which mutations accumulate on the Binscy balancer X chromosome in heterozygous females in the absence of selection and recombination. We found that the life span of Binscy/RYL males from this cross decreased faster than the life span of their sibling controls over time in two of three runs, and that there was an age-specific increase in mortality in the Binscy/RYL males with time in one of three runs. Therefore, the accumulation of deleterious mutations can decrease life span by increasing fragility and can cause age-specific changes in mortality. These results support the evolutionary theory of aging.

AGING, or senescence, is a late-life decline in fertility and probability of survival (1). With all else being equal, aging reduces individual fitness. To explain why aging occurs despite selection that should presumably reduce or eliminate it, the evolutionary theory of aging has provided two related mechanisms, the mutation accumulation hypothesis and the antagonistic pleiotropy hypothesis (1–12). Medawar (2), in his mutation accumulation theory of aging, pointed out that deleterious mutations the effects of which occur only in late life after reproduction are less strongly selected against than are mutations the effects of which occur early. Because mutations that are selected only weakly can persist in mutation-selection balance, those with effects in late life can accumulate and cause senescence. The antagonistic pleiotropy hypothesis also defines aging as the accumulation of late-acting deleterious mutations (3,13). However, in the antagonistic pleiotropy theory of aging, mutations that are detrimental late in life may have beneficial effects early in life. For example, those mutations that increase reproduction are favored by natural selection, even if they are deleterious at a later stage of life. The disposable soma theory, which claims that organisms have a selective advantage when allocating more of their resources to reproduction and fewer to somatic maintenance and repair (9,14–16), is an example of the antagonistic pleiotropy theory of aging.

Both the mutation accumulation hypothesis and the antagonistic pleiotropy hypothesis assume that at least some deleterious mutations have aging effects. To verify this assumption, experiments have been performed to measure the decline in fitness of individuals in populations that are not subjected to selection and therefore accumulate new deleterious mutations. For example, Shabalina and colleagues (17) found that the average number of surviving offspring declined by 0.2%–2% every generation in a Drosophila melanogaster population in which deleterious mutations were allowed to accumulate, but no significant mutational effect on longevity was detected in their study. In contrast, other mutation accumulation experiments have reported that the accumulation of mutations does increase mortality (for example, 18–22). Yet, there has been little evidence for the occurrence of mutations with only late-age effects (12,18,23,24).

In this study, we have designed experiments in which deleterious mutations both do and do not accumulate in morphologically distinct siblings that have similar genetic backgrounds and are raised in identical environments (24–27). Then by comparing the life span of flies that carry new deleterious mutations with the life span of sibling control flies that do not accumulate new deleterious mutations, we are able to investigate the aging effect of new deleterious mutations.

Both evolutionary hypotheses of aging rely on the assumption that some mutations that influence survival and reproductive ability have effects only at late ages. Until recently, however, few experiments have tested this assumption directly (18,22,28,29). Our study was also planned to detect whether there are any age-specific mutational effects on mortality.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
To design a mutation accumulation experiment with better controls, we synthesized the Binscy assay (see Figure 1) by modifying a mating scheme used by Muller and Oster (30). Binscy is a balancer X chromosome with the B (Bar eyes, dominant) and y (yellow body, recessive) mutations, plus multiple inversions that eliminate recombination of the X chromosome in females; C(1;YS)oc ptg is a compound X and short arm of the Y chromosome, with the oc (ocelliless, missing ocelli, homozygous female sterile) and ptg (pentagon, thoracic trident dark) recessive mutations; RYL is the long arm of the Y chromosome in the shape of a ring (31). The Binscy/RYL males are sterile because of the missing male fertility factors in the short arm of the Y chromosome, and C(1;YS)oc ptg/C(1;YS)oc ptg females are sterile because of the homozygous oc mutation (31). In setting up the G0 crosses, Binscy/Y males, which were free of X-linked lethals, were mated with Binscy/C(1;YS)oc ptg females. Each generation, one Binscy/C(1;YS)oc ptg female was mated with one C(1;YS)oc ptg/RYL male per vial at 25°C on standard cornmeal and agar medium.


Figure 01
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  		Figure 1. Mating scheme for the accumulation of deleterious mutations in the Binscy X chromosome of Drosophila melanogaster. In every generation (G0, G1, G2, ... etc.), one single female and one single male were randomly selected to mate for each line. Life span of the Binscy/RYL and C(1;YS)oc ptg/RYL males were observed at generations 1, 9, and 17 for Run A, and at 1, 6, 11, 16, 21, and 26 for Runs B and C

 
This mating scheme is designed to allow new deleterious mutations to accumulate over generations on the Binscy balancer X chromosome in Binscy/C(1;YS)oc ptg females. Because C(1;YS)oc ptg homozygous females and Binscy/RYL males from this mating scheme are sterile, new deleterious mutations that occur on the X chromosome of C(1;YS)oc ptg/RYL males are in a hemizygous state and are eliminated by selection. Conversely, deleterious mutations that occur on the Binscy X chromosome in Binscy/C(1;YS)oc ptg females are buffered from selection because mutations are maintained as heterozygotes against wild-type alleles on the C(1;YS)oc ptg chromosome. Hence, with time, deleterious mutations will accumulate on the Binscy chromosome, but not on the C(1;YS)oc ptg chromosome. To the extent that deleterious mutations might temporarily be present on C(1;YS)oc ptg chromosomes, some reduction in life span of the C(1;YS)oc ptg/RYL control males may also occur. The difference in accumulation of mutations on these two chromosomes can be revealed by measuring the life span of the sibling C(1;YS)oc ptg/RYL males and the Binscy/RYL males every few generations. If some deleterious mutations accumulating on the Binscy X chromosome have effects on aging, the life span of Binscy/RYL males will decrease over time. We have previously used this mating scheme to measure the deleterious genomic mutation rate in D. melanogaster (26).

Three experimental runs were performed, in which the life span of males was measured at G1 (generation 1), G9, and G17 for Run A and at G1, G6, G11, G16, G21, and G26 for Runs B and C. For each generation, approximately 400 male flies of both genotypes were collected at the first day when they eclosed from pupae. These flies were kept in plastic vials with 12 flies per vial, and the vials were placed in an incubator at 25°C. The food in the vials was a dextrose medium of cornmeal, yeast, agar, dextrose, lexgard, and benzyl benzoate. Dextrose was used instead of sucrose to reduce the growth of a lactobacillus that produces mucus on the surface of standard cornmeal–molasses medium that may trap flies (32). The flies were transferred into new vials every 7 days to keep the medium fresh. The number of living flies in each vial was counted every day to measure life span. This technique has previously been used to study the influence of transposable DNA elements and DNA repair on aging in D. melanogaster (33–36).

Besides studying the changes in life span of the two kinds of male flies, it is also important to examine the age-specific death rates to verify whether any mutations have aging effects only at late ages. Several mathematical models have been proposed in survival analysis or longevity studies, such as the Weibull model, the Gompertz model, the Gompertz–Makeham model, and the logistic model (7,13,37–40). Among them, the Gompertz model and the Gompertz–Makeham model are the most widely used by biologists and demographers to characterize age-specific mortality parameters (38). The Gompertz model assumes a hazard increasing exponentially with age. In the Gompertz model, the hazard function is expressed as


Formula

where h(t) is the hazard function that describes the conditional death rate over a very short period of time, t is age, a is the initial mortality parameter, and b is the rate parameter. When parameter a (or lna) is greater, the death rate is higher at all ages. When parameter b is greater, the death rate increases faster with an increase in age. The Gompertz–Makeham model adds one more parameter (c) to the Gompertz model to take care of age-independent cause of death (38,40), so that the hazard function becomes:


Formula

These parameters were estimated from the life-span data using maximum likelihood method in the software package WinModest (38). If some deleterious mutations increase mortality, either parameter (a or b) should increase over time. Furthermore, if some aging effects are only at late ages, only parameter b should increase over time.


   RESULTS
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 Methods
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Discussion
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Mean and median life span are recorded in Table 1, and survival curves are shown in Figures 2–4GoGo. To check whether life span of the C(1;YS)oc ptg/RYL males and the Binscy/RYL males had decreased over time, the log-rank test for trend (22,41–42) was used for both genotypes in each of the three experimental runs. The statistical tests were performed using the software PRISM (GraphPad, San Diego, CA), and the outcomes are given in Table 2. Results indicated that both C(1;YS)oc ptg/RYL and Binscy/RYL males had a significantly decreasing trend in life span in Run A and Run C, but not in Run B (Table 2).


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  		Table 1. Mean and Median Life Span for Drosophila melanogaster Sibling Males.

 

Figure 02
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  		Figure 2. Survival curves of Drosophila melanogaster sibling males in Run A. Life spans of both genotypes were measured at generations 1, 9, and 17

 

Figure 03
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  		Figure 3. Survival curves of Drosophila melanogaster sibling males in Run B. Life spans of both genotypes were measured at generations 1, 6, 11, 16, 21, and 26

 

Figure 04
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  		Figure 4. Survival curves of Drosophila melanogaster sibling males in Run C. Life spans of both genotypes were measured at generations 1, 6, 11, 16, 21, and 26

 

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  		Table 2. Results of Log- Rank Test for Trend (One-Tail Test) for Drosophila melanogaster Sibling Males in Three Experimental Runs.

 
Although the life span of both kinds of sibling males declined in two of the three experimental runs, the decreasing rates are different. The rates of reduction in median life span over time were investigated using a logistic regression model


Formula

where Y is the median life span, X is generation, {alpha} is the intercept, ß is the slope, and {varepsilon} is the error term. From the formula above, the rate of decrease in median life span per generation is 1 – eß, which is close to ß if ß is small. The parameter ß was estimated by the survival analysis function in MINITAB (Minitab Inc., State College, PA), and then the rates of decline in median life span were calculated (Table 3). In Run A, the median life span of Binscy/RYL males decreased by 2.68% per generation and the median life span of the C(1;YS)oc ptg/RYL sibling males decreased by 1.85% per generation. In Run C, the median life span of Binscy/RYL males decreased by 1.12% per generation and the median life span of the C(1;YS)oc ptg/RYL sibling males decreased half as fast (0.69% per generation). In both Runs A and C, interval estimations indicated that the differences between rates are statistically significant (see Table 3). That is, the decline in median life span was significantly faster in the Binscy/RYL males than in the C(1;YS)oc ptg/RYL males in both Run A and Run C. However, neither genotype showed a significant reduction in median life span in Run B.


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  		Table 3. Decreases in Median Life Span Every Generation with 95% Confidence Intervals, Calculated by the Logistic Regression Model.

 
Parameters of the Gompertz and Gompertz–Makeham models were estimated for each fly population (Tables 4 and 5). For the Gompertz model, parameter a (or lna) increased significantly for both males in Run A and Run C, whereas no significant changes occurred in Run B. No significant increase in parameter b (slope) was observed in either genotype in any run of this experiment (Figure 5). Therefore, deleterious mutations can increase mortality at all ages, and there is no evidence that the change in mortality occurs only at late ages. In contrast, for the Gompertz–Makeham model, the rate parameter b significantly increased in the Binscy/RYL males but not in the C(1;YS)oc ptg/RYL males in Run B. This result suggests that there might be an increase in mortality rate with age in the presence of new mutations.


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  		Table 4. Estimated Parameters of the Gompertz Model: h(t) = aebt.

 

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  		Table 5. Estimated Parameters of the Gompertz–Makeham Model: h(t) = aebt + c.

 

Figure 05
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  		Figure 5. Ln mortality curves for life span of Drosophila melanogaster sibling males in the first and last generations of Runs A, B, and C

 

   DISCUSSION
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 Methods
 Results
 Discussion
References
 
In two of the three experimental runs (Run A and C), both the Binscy/RYL males and the C(1;YS)oc ptg/RYL males showed a decline in life span over time. Part of these declines can be attributed to inbreeding depression (43,44), as sibling matings were performed throughout the experiment. However, the life span of the Binscy/RYL males, where deleterious mutations were allowed to accumulate, decreased significantly faster than the life span of the C(1;YS)oc ptg/RYL control males. Therefore, deleterious mutations can cause a decline in longevity, which is consistent with the results of mutation accumulation experiments in D. melanogaster by Pletcher and colleagues (18) and Yampolsky and colleagues (22).

Because there is no selection on the Binscy X chromosome in our experiments, mutations will accumulate and lead to senescence as the evolutionary theory of aging predicts (2). However, the Binscy/RYL males are sterile, and the Binscy X chromosomes are not under selection in these males. Therefore, not only mutations having late-age effect, but also mutations with an effect at any stage will accumulate. This is probably why mortality did not increase only at late stages in this study. Heterogeneity of novel mutation load, caused by negative epistasis among aging-related mutations, is another possible reason for an age-specific effect not being observed.

The antagonistic pleiotropy theory of aging claims that mutations causing senescence are fixed because they have some beneficial effects such as increased fecundity (13,45). In our experiments, the fecundity of the Binscy/RYL males is impossible to measure because the flies are sterile. However, no increase in the fecundity of the C(1;YS)oc ptg/Binscy females was observed in any run.

The rate of decrease in life span of the Binscy/RYL males is influenced by the mutation rate of the Binscy X chromosome. We applied the Bateman–Mukai method (46,47), and estimated mutation rates in the three experimental runs. The estimated mutation rates of Runs A, B, and C are 0.054, 0.017, and 0.063 per X chromosome per generation, respectively, giving an average deleterious mutation rate of 0.025 per X chromosome per generation, or 0.31 per diploid genome per generation (26). The mutation rate in Run B is much lower than that in Run A or Run C, which is one possible explanation for the decline in life span being significant only in Run A and C.

Summary
Our results show that the accumulation of new deleterious mutations can reduce life span. This study, therefore, supports the evolutionary theory of aging. The results from this study, however, do not differentiate between the mutation accumulation or antagonistic pleiotropy mechanisms.


   Acknowledgments
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 Results
 Discussion
 References
 
This study was sponsored by NASA grants NAG 2-1427 and NCC 2-1355.

We thank Robert Arking, Priti Azad, Sheng Gu, and Elizabeth Morgan for their valuable advice.


   Footnotes
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Decision Editor: James R. Smith, PhD

Received October 6, 2005

Accepted June 8, 2006


   References
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