Functional significance of allelic variation at methuselah, a candidate gene for aging Annalise Paaby and Paul Schmidt, University of Pennsylvania
Introduction
In Drosophila melanogaster, disruption of the G­protein­coupled receptor methuselah (mth) produces atypically long lifespan and increased stress resistance1. Along the latitudinal gradient of the U.S. east coast, wild populations of D. melanogaster show changing patterns of longevity and stress resistance2 and a cline in the frequency of the most common mth haplotype3. We hypothesize that shifting selection pressures across this heterogeneous environment are driving alternative life histories and that mth may be an important target in this regime.
Fecundity
Methods. Eggs laid on disposable collection plates in the lifespan bottle cages were counted every other day.
In order to establish whether allelic diversity at mth produces functional differences in phenotype, we tested 8 wild lines for lifespan, fecundity and oxidative stress resistance. To control for background variation, we looked for changes in the magnitude of difference between each line assayed over a mth mutant (we used both a deletion, mth­del, and a P­element insertion, mth­insert) and over a precise P­
element excision that restored the wild type mth phenotype (mth­wt).
Figure 3. Average lifetime fecundity per line for all genotype combinations. Error bars show 95% confidence. ANOVA shows significant effects by line, mth allele, and line × allele interaction (p<0.02 for all).
Lifespan
Methods. Bottle cages were established with 40 males and 40 females for each of the 24 possible genotypes (8 wild lines × 3 mth alleles); each genotype was replicated 3 times. Dead flies were scored every other day.
Figure 4. The difference in eggs laid per female per day between each line over one of the mth mutants (mth­del, left; mth­insert, right) minus the line over mth­wt. ANCOVA shows significant effects by line, mth allele, and line × allele interaction (p<0.02 for all).
Stress resistance
Methods. Resistance to oxidative stress was evaluated by exposure to paraquat. For each of the 24 possible genotypes (8 wild lines × 3 mth alleles), 5 males and 5 females were placed in a media­free vial with cotton saturated with 30 mM methyl viologen; each genotype was replicated 4 times. After 19 hours of exposure, dead flies were scored.
Figure 1. Survivorship curves (left) for a sample of line pairs showing varying differences between the line over mth­del (top) or over mth­insert (bottom) and the line over mth­wt; the magnitude of those differences in survivorship (right).
Figure 5. Odds ratios for each line over one of the mth mutants (mth­del, left; mth­insert, right) relative to the line over mth­wt. Positive ratios indicate greater risk of death. Error bars show 95% confidence. Nominal logistic regression shows significant effects by line, mth allele, and line × allele interaction (p<0.001 for all). There was no effect by sex.
Conclusions
The allelic diversity at mth in wild populations of D. melanogaster contributes significantly to functional differences in lifespan, fecundity and stress resistance. These data suggest that mth genetic variation is visible to selection, and that selection at mth may contribute to natural variation in longevity and other life history traits.
Figure 2. Risk ratios for each line over one of the mth mutants (mth­del, left; mth­insert, right) relative to the line over mth­wt. Females are above, males below. Positive ratios indicate greater risk of death. Error bars show 95% confidence. A proportional hazards model shows significant effects by line, mth allele, sex, and all interactions (p<0.01 for all). Literature cited
1. Lin, Y­J, L Seroude and S Benzer. 1998. Extended life­span and stress resistance in the Drosophila mutant methuselah. Science 282: 943­946.
2. Schmidt, PS, L Matzkin, M Ippolito and WF Eanes. 2005. Geographic variation in diapause incidence, life­history traits and climatic adaptation in Drosophila melanogaster. Evolution 59(8): 1721­1732.
3. Schmidt, PS, DD Duvernell and WF Eanes. Adaptive evolution of a candidate gene for aging in Drosophila. PNAS 97(20): 10861­10865. Acknowledgments
We thank Ravi Ranjan for the mutant allelic series and the Binns­Williams Fund for financial support.