Locomotor activity as a function of age and life span in Drosophila melanogaster overexpressing hsp70.

Heat shock protein induction might be responsible for the longevity increase conferred by exposure to non-lethal stresses. To test this hypothesis, we studied in transgenic Drosophila melanogaster overexpressing hsp70 and controls, two behavioral variables (spontaneous locomotor activity and climbing activity) to evaluate the rate of aging, and life span. The results showed that in flies kept in groups, life span was decreased in transgenic flies compared to the parental line, but the contrary was observed in individually kept flies. Hsp70 overexpression had no dramatic effect on life span. Furthermore, we did not detect any advantage of Drosophila overexpressing hsp70 on the two measurements of locomotor activity. These results indicate that the rate of aging in transgenic flies is not different than in non-transgenic lines and that they are not more able to cope with the effects of aging on locomotor activity.

Why do life spans differ? Partitioning mean longevity differences in terms of age-specific mortality parameters.

Populations typically differ in mean life spans because of genetic, environmental, or experimental factors. In this paper methods are presented that clarify the relationship between differences in the longevity of two populations and differences in their underlying age-specific patterns of mortality. Data are examined from rodent and fruit fly (Drosophila melanogaster) experiments that investigated the longevity effects of a variety of environmental and genetic manipulations, including temperature, dietary restriction, laboratory selection for increased longevity, and severe inbreeding. Analyses suggest that longevity differences mediated by temperature and dietary restriction result predominantly from differences in the rate of increase in mortality with age. Increases in longevity through laboratory selection result primarily from a reduction in baseline mortality and not a slowing of the rate of aging. Although the methods are applied primarily in the context of simple mathematical models of mortality (e.g., the Gompertz model), they are quite general and can be applied to mortality models of arbitrary complexity. Mathematica protocols ("notebooks") and computer software have been developed to perform all the analyses discussed and are available from the first author.

Genetic analysis of extended lifespan in Drosophila melanogaster III. On the relationship between artificially selected and wild stocks.

Adult lifespans, age-specific survival, age-specific mortality, survival times on paraquat, and survival times on DDT were assayed in seven lines of Drosophila melanogaster, including two genetically heterogeneous wild lines recently collected from nature, and three inbred and recombinant inbred lines derived from an artificial selection experiment for increased lifespan. Survival on paraquat is positively correlated with adult lifespan. DDT resistance is uncorrelated with either paraquat resistance or lifespan. The wild lines are unexceptional with respect to average lifespan, paraquat resistance, age-specific survivorship, and leveling off of mortality rates at advanced ages, but have high levels of DDT resistance. Cluster analysis groups the wild lines with three unselected laboratory stocks in one cluster, while two long-lived elite recombinant inbred lines form a second cluster. Long-lived laboratory-adapted lines are quantitatively differentiated from the wild stocks, both with respect to average adult lifespans and resistance to an oxidizing agent. We reject the 'recovery' hypothesis, which proposes that Drosophila artificially selected for long life have phenotypes that merely recover the wild state.

The fractionation experiment: reducing heterogeneity to investigate age-specific mortality in Drosophila.

Age-specific mortality rates decelerate at older ages in both genetically homogenous and heterogeneous populations of Drosophila. One explanation proposed for deceleration is population heterogeneity. This hypothesis suggests that a population consists of sub-populations that differ in mortality characteristics and that the deceleration is the result of selective survival of stronger individuals. Here we describe an experiment that fractionates populations into several sub-populations without changing the physiological characteristics of the post-fractionated populations. Through a careful process of selection of Drosophila eggs, larvae, pupae and adults, we attempt to reduce as much as possible the degree of pre-adult, environmentally induced heterogeneity among individuals of a genetically identical cohort. We then ask whether such cohorts, when compared to non-fractionated populations, exhibit a lesser degree of mortality deceleration at advanced ages. From a total of 106 fractionated and control populations, consisting of 51331 individuals, 101 populations (93% of the fractionated populations and 100% of the control populations) exhibit a significant amount of mortality deceleration late in life. These observations suggest that environmental heterogeneity accrued during larval development is not a major factor contributing to mortality deceleration at older ages.

Biodemographic trajectories of longevity.

Old-age survival has increased substantially since 1950. Death rates decelerate with age for insects, worms, and yeast, as well as humans. This evidence of extended postreproductive survival is puzzling. Three biodemographic insights--concerning the correlation of death rates across age, individual differences in survival chances, and induced alterations in age patterns of fertility and mortality--offer clues and suggest research on the failure of complicated systems, on new demographic equations for evolutionary theory, and on fertility-longevity interactions. Nongenetic changes account for increases in human life-spans to date. Explication of these causes and the genetic license for extended survival, as well as discovery of genes and other survival attributes affecting longevity, will lead to even longer lives.

Genetic analysis of extended life span in Drosophila melanogaster. I. RAPD screen for genetic divergence between selected and control lines.

Using lines selected for long life by Luckinbil and his co-workers, we screened two selected and two control lines for allelic frequency differences at 1200 randomly chosen RAPD marker loci. Twenty-three marker loci showed frequency differences in excess of 80%, and five were greater than 90%. Age-specific effects of the five most differentiated loci were estimated by collecting complete survival data in segregating backcross populations. Alleles at four of the five marker loci were associated with significant extension of life span in males, while two marker loci had significant effects in females. Eighty percent of the total selection response in males can be explained by the identified QTL's, under the assumption of additivity. The N14+ marker allele accounted for a 12-day life span extension in males, but had little effect in females. Both sex-limited and sex-shared effects were observed. Analysis of age-specific mortality rates suggests that life span extension occurs by a combination of genetic factors that moderate both the level of mortality and the rate at which mortality increases with age.

Genetic analysis of extended life span in Drosophila melanogaster. II. Replication of the backcross test and molecular characterization of the N14 locus.

We are interested in localizing chromosomal regions that extend life span in Drosophila. Using stocks artificially selected for long life by Luckinbill and his colleagues, we have identified marker loci that are highly divergent in allelic frequencies between replicated long-lived lines and controls (Curtsinger et al., 1998). Several of the most divergent loci have been found to be associated with effects on life span in segregating backcross populations. Here we report an independent replication of the backcross test for the N14 marker locus, previously reported to extend male life spans by 12 days. The life span effect successfully replicates in males. N14 accounts for 30% of the total selection response in males. Life span extension occurs by a decrease in age-specific mortality rates at all ages, and is not attributable to modification of the slope of the age-specific mortality curve. The effect in females is small or nonexistent. Sequencing of the N14 locus shows that it is non-coding and not obviously regulatory, suggesting that the phenotypic effect arises from linkage disequilibrium with another locus or loci that directly affect life span. N14 DNA hybridizes to 63F/64A on the left arm of chromosome 3. The location is consistent with previous whole-chromosome substitution studies, and suggests new candidate genes for life span extension in Drosophila, including ras2.

Heat-induced longevity extension in Drosophila. I. Heat treatment, mortality, and thermotolerance.

Survival data were collected on a total of 28,000 Drosophila melanogaster adults in order to investigate mortality patterns and induced physiological responses after a mild thermal stress. A brief, nonlethal heat treatment extends adult life span at normal temperatures by an average of 2 days (64), compared to nontreated controls of the same genotypes. Life expectancy is extended as a demographic consequence of reduced age-specific mortality over a period of up to several weeks after the heat treatment. Heat treatment also increases tolerance to subsequent, more severe thermal stress. Observations on single-sex populations suggest that heat-induced longevity extension is independent of the suppression of reproductive activity.

Effect of density on age-specific mortality in Drosophila: a density supplementation experiment.

Age-specific mortality rates were studied at two adult density levels in four inbred lines of Drosophila melanogaster. In experimental populations, adult densities were maintained at constant levels throughout the experiment by replacing dead flies with live, marked mutants. In control populations, densities declined naturally as the cohorts aged. For all experimental populations the best mortality model is the two-stage Gompertz model, with slower mortality acceleration at older ages. Flies in the experimental populations generally lived longer than flies in control populations, regardless of sex, genotype, or initial density level. The data demonstrate that deceleration of age-specific mortality rates at older ages is not caused by declining cohort densities. Mortality deceleration is a real phenomenon that raises serious questions about the evolution of senescence.

Age-specific patterns of genetic variance in Drosophila melanogaster. I. Mortality.

PETER MEDAWAR proposed that senescence arises from an age-related decline in the force of selection, which allows late-acting deleterious mutations to accumulate. Subsequent workers have suggested that mutation accumulation could produce an age-related increase in additive genetic variance (VA) for fitness traits, as recently found in Drosophila melanogaster. Here we report results from a genetic analysis of mortality in 65,134 D. melanogaster. Additive genetic variance for female mortality rates increases from 0.007 in the first week of life to 0.325 by the third week, and then declines to 0.002 by the seventh week. Males show a similar pattern, though total variance is lower than in females. In contrast to a predicted divergence in mortality curves, mortality curves of different genotypes are roughly parallel. Using a three-parameter model, we find significant VA for the slope and constant term of the curve describing age-specific mortality rates, and also for the rate at which mortality decelerates late in life. These results fail to support a prediction derived from MEDAWAR's "mutation accumulation" theory for the evolution of senescence. However, our results could be consistent with alternative interpretations of evolutionary models of aging.

Age-specific patterns of genetic variance in Drosophila melanogaster. II. Fecundity and its genetic covariance with age-specific mortality.

Under the mutation accumulation model of senescence, it was predicted that the additive genetic variance (VA) for fitness traits will increase with age. We measured age-specific mortality and fecundity from 65,134 Drosophila melanogaster and estimated genetic variance components, based on reciprocal crosses of extracted second chromosome lines. Elsewhere we report the results for mortality. Here, for fecundity, we report a bimodal pattern for VA with peaks at 3 days and at 17-31 days. Under the antagonistic pleiotropy model of senescence, it was predicted that negative correlations will exist between early and late life history traits. For fecundity itself we find positive genetic correlations among age classes > 3 days but negative nonsignificant correlations between fecundity at 3 days and at older age classes. For fecundity vs. age-specific mortality, we find positive fitness correlations (negative genetic correlations) among the traits at all ages > 3 days but a negative fitness correlation between fecundity at 3 days and mortality at the oldest ages (positive genetic correlations). For age-specific mortality itself we find overwhelmingly positive genetic correlations among all age classes. The data suggest that mutation accumulation may be a major source of standing genetic variance for senescence.

Effect of adult cohort density on age-specific mortality in Drosophila melanogaster.

Mortality rates decelerate at older ages in experimental populations of Drosophila. It is unclear whether this reflects a real slow-down in the aging process, or an artifact of declining density. Mortality was studied in age-synchronized cohorts of four inbred lines at three initial densities that varied 10-fold. A total of 70,000 flies of both sexes were studied. There were large line x density, line, and sex effects, but no systematic relationship between density and life span was detected. Mortality curves level off at older ages in 23 out of 24 sex-genotype combinations, irrespective of initial cohort density. Density has only second-order effects on the pattern of oldest-old mortality over the range of densities studied here. The dramatic departure from Gompertz-type mortality dynamics at older ages is not an artifact of declining density in Drosophila.

Stress experiments as a means of investigating age-specific mortality in Drosophila melanogaster.

Age-specific mortality rates level off at older ages in genetically homogeneous experimental populations of Drosophila. Here we describe an experiment that is informative about the causes of mortality rate changes. By applying a brief, nondebilitating stress that increases mortality early in life and then observing subsequent mortality trajectories, it is possible to determine whether populations are heterogeneous for factors influencing mortality. We show that 24-h exposure to a desiccating air flow causes a spike and then a decrease in mortality rates in experimental populations compared to controls. If there is no stress-induced enhancement of vitality, then the results demonstrate the existence of heterogeneity for mortality rates in genetically homogeneous populations.

Genetic variation and aging.

Life span is subject to genetic modification in yeasts, nematodes, fruit flies, mice, humans, and other vertebrates and invertebrates. There are a few single-gene mutants known that extend life span in yeast and nematodes; in other experimental systems the character is treated quantitatively, and generally has a low to moderate heritability. Life span responds to artificial selection in Drosophila and Caenorhabditis. There are many candidate genes presently under investigation, including the anti-oxidizing enzymes and heat-shock proteins. The main evolutionary models of senescence are antagonistic pleiotropy and mutation accumulation, neither of which has substantial experimental support. The incorporation of analytical techniques from demography is playing an increasing role in research on aging.