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Keywords:

  • body size;
  • diet restriction;
  • fecundity;
  • insulin;
  • juvenile hormone;
  • ovariole

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Mutations of the insulin signal pathway in Drosophila melanogaster produce long-lived adults with many correlated phenotypes. Homozygotes of insulin-like receptor (InR) and insulin-like receptor substrate (chico) delay time to eclosion, reduce body size, decrease reproduction and increase life span. Because these mutations are expressed through all life stages it is unclear when insulin signals must be reduced to increase life span. As a first analysis of this problem in D. melanogaster we have manipulated the larval diet to determine if changes in metabolic regulation at this stage are sufficient to slow aging. We controlled the dietary yeast fed to third instar larvae and studied the size, mortality, fecundity and hormones of the resulting adults, which were fed a normal, yeast-replete diet. Adults from yeast-deprived larvae phenocopied many traits of InR and chico mutants: small body size, delayed eclosion, reduced ovariole number and reduced age-specific fecundity. But unlike constitutive mutants of the insulin/IGF system, adults from yeast-deprived larvae had normal patterns of demographic senescence, and this was accompanied by normal insulin-like peptide and juvenile hormone syntheses. Surprisingly, the normal aging in these adults was also associated with greatly reduced fecundity. Although nutritional conditions of the larvae can affect the subsequent body size and fecundity of adults, these are not sufficient to slow aging.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Drosophila melanogaster homozygous for mutants of insulin-like receptor (InR) or for the insulin-like receptor substrate (chico) are long-lived (Clancy et al., 2001; Tatar et al., 2001a; Tu et al., 2002). They are also variously small, slow to develop and infertile (Chen et al., 1996; Bohni et al., 1999). As InR and chico mutations are expressed throughout the larval and adult stages, these genotypes may age slowly because they alter development to produce adults predisposed to exceptional longevity regardless of changes in metabolic regulation of the adult. Conversely, independent of effects upon development, reduced insulin-like signalling specifically within the adult may retard aging. Here, to provide an initial understanding of how stage-specific consequences of metabolism affect aging in D. melanogaster we manipulate the nutrition of third instar larvae and examine patterns of age-specific mortality and reproduction in fully fed adults.

Beadle et al. (1938) observed that D. melanogaster larvae died if starved before 72 h of growth but grew into small adults if starved after this time. We follow this approach as described by Robertson (1960) and Sang (1962) where third instar larvae are transferred from a full diet (yeast, sugar, cornmeal, agar) to diet without yeast (sugar, cornmeal, agar), which delays eclosion and produces small adults. Third instar larvae reared under conditions of limited yeast are also known to produce adults with few ovarioles (Hodin & Riddiford, 2000). Each of these traits – reduced size, delayed eclosion and, as we report here, reduced ovariole number – are traits observed in mutants of InR and chico. In many ways, the larval diet-restriction phenocopies insulin/IGF mutations.

We study the demographic and hormonal profiles of fully fed adults following their development as yeast-deprived or yeast-fed larvae. Subsequent differences in adults therefore must originate in the nutritional conditions experienced exclusively during development. Contrary to the many morphological similarities between mutants of InR or chico and adults that developed under conditions of larval yeast-deprivation, diet manipulation in larvae did not alter aging measured by mortality. Surprisingly, this normal demographic aging was accompanied by a 64% decrease in reproduction. We conclude that nutritional conditions experienced during development can profoundly affect adult size and reproduction but need not influence life span.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Body size

Yeast-deprived larvae exhibited delayed time to eclosion by about 2 days and produced small adults. Heads of adult females and males from yeast-deprived larvae were 0.66 ± 0.04 mm and 0.61 ± 0.04 mm, respectively; heads of females and males from yeast-fed larvae were 0.83 ± 0.03 mm and 0.77 ± 0.03 mm, respectively (each sample, n = 30). Wing length was reduced: 1.19 ± 0.09 mm in yeast-deprived vs. 1.55 ± 0.05 mm in yeast-fed (females, each n = 30).

Age-specific mortality

Mortality rate µx is an informative measure of senescence (Sacher, 1977; Finch, 1990; Promislow et al., 1999). Mortality rate is the continuous-time form of age-specific mortality, the likelihood of death at a specific age x conditional on survival to x. Because mortality is age-specific its trend across ages estimates senescence without biases caused by transient differences in death rate. This characteristic contrasts with inferences based on survivorship, lx, which represents the proportion remaining alive in a cohort as a function of age. Survivorship of necessity decreases with age and always carries forward all previous history of mortality, including the effects of accident and age-independent risk.

These comments are important because although we sometimes observed transient differences in mortality at young adult ages, we found that larval yeast-restriction did not improve the overall pattern of mortality rate as it increased with age in females (Fig. 1) or in males (Table 1). Once mortality began to increase with age, typically at around 15 days, the trajectory for adults from yeast-deprived larvae did not differ in any important way from the pattern from adults of yeast-fed larvae, regardless of differences in life expectancy at eclosion (Table 1). Life expectancy at eclosion was reduced for adults of yeast-deprived larvae in some trials because their mortality was transiently elevated at young ages.

image

Figure 1. Mortality rate in yeast-fed females developed from either yeast-deprived larvae (solid) or yeast-fed larvae (open). Mortality rate µx was estimated as –ln(1 − qx) where qx (age-specific mortality) is dx/Nx, dx is the number observed deaths in the interval x − 1 to x, and Nx is the number alive at the beginning of interval x. (A) Canton-S, (B) w1118, (C) yw, (D) Oregon R.

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Table 1.  Summary statistics of complete life tables for adults in four genetic backgrounds. Adults were fed yeast-diet after they developed as third instar larvae with yeast (fed) or without (deprived). N is initial cohort size at eclosion. Life expectancy is estimated from eclosion and from age 15 days. Proportional hazard survival analysis is based on data left censored at age 15 days and estimates the mortality risk (exp β) of adults from yeast-deprived larvae relative to yeast-fed controls (probability by Wald-test). Male data were not collected for w1118.
StrainLarval treatmentFemalesMales
NLife expectancy atProportional hazard statistics from age 15 dNLife expectancy atProportional hazard statistics from age 15 d
eclosion15 dexp βProb.eclosion15 dexp βProb.
Canton-Sfed31231.616.71.260.09028938.422.91.200.051
deprived67225.616.4  26632.321.2  
ywfed45535.7420.80.910.14123339.726.41.140.14
deprived56037.724.6  23838.525.7  
w1118fed46553.038.20.960.614     
deprived23749.734.7       
Oregon Rfed58740.526.21.140.04058556.742.61.31< 0.001
deprived62832.222.0  57643.935.3  

Ovariole number and fecundity

Reproduction was altered in adults that developed from yeast-deprived larvae. Previous work has shown that females of yeast-fed larvae produce many advanced egg chambers by 2 days post-eclosion even if they are yeast-deprived as adults (Good & Tatar, 2001). Here we found that adults of yeast-deprived larvae uniformly arrested egg development near stage 7 (mean egg stage: 7.3 ± 0.8, n = 25) and remain in this state until they were fed yeast (mean egg stage after fed yeast: 13 ± 0.7, n = 26). The ability to produce mature eggs upon eclosion appears to be a function of larval nutrient status.

Hodin & Riddiford (2000) showed that limiting yeast during the third instar modestly reduced ovariole number. Here we found that complete yeast-deprivation during the third instar reduced ovariole number by 50% (yeast-deprived: 6.5 ± 2 per ovary, n = 16; yeast-fed: 13.5 ± 3.5 per ovary, n = 26). The ovarioles of females from yeast-deprived larvae were morphologically normal and as measured by fusome number possessed the usual complement of two or three germline stem cells (data not shown).

Ovariole number also varied as a function of insulin signalling. Wild-type chico+/chico+ females had ∼15 ovarioles per ovary (14.7 ± 1.5, n = 53). Homozygote chico1/chico1 females, which are dwarf and long-lived (Clancy et al., 2001; Tu et al., 2002), had 50% fewer ovarioles (6.9 ± 1.7, n = 49). However, chico+/chico1 females, which are also long-lived and normal sized, had a full complement of ovarioles (13.9 ± 1.7, n = 48). Thus, as a function of larval nutrient deprivation and of chico, ovariole number does not correlate with longevity.

Fecundity was reduced in females from yeast-deprived larvae, despite their feeding upon yeast as adults. Females of yeast-deprived larvae laid fewer eggs at every age (Fig. 2) and produced a total of 273 ± 14 eggs (n = 37) compared with 769 ± 16 eggs (n = 35) from females of yeast-fed larvae. Because females from yeast-deprived larvae laid small eggs of normal proportion we also estimated reproductive effort (total investment in eggs/unit volume of adult). We measured egg length and assume all eggs have a proportionally similar cylindrical shape; then, eggs laid by females of yeast-deprived larvae have 0.753 the volume of eggs from yeast-fed females. If we represent adults as proportional cylinders, based on the measures of head diameter, adult females from yeast-deprived larvae are 0.51 the volume of control females, which is similar to the change in mass caused by InR mutations (Tatar et al., 2001a). Thus, reproductive effort was (273 eggs) × (0.753)/0.512 = 401.5 for females of yeast-deprived larvae relative to (769) × (1)/1 = 769 for females of yeast-fed females. Measured either as absolute fecundity or as relative effort, reproduction was reduced in females from yeast-deprived larvae.

image

Figure 2. Fecundity of yeast-fed females developed from either yeast-deprived larvae (solid) or yeast-fed larvae (open). Per capita fecundity of females (w1118) from yeast-deprived larvae and from yeast-fed larvae; daily mean of eggs per live female with standard error. Inset: cumulative survivorship through age 35 days.

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Insulin and Juvenile Hormone (JH)

Although adult body size and reproduction were reduced by larval nutrient conditions, adult insulin and JH activity were regulated by the adult nutritional state (Figs 3 and 4). Females of yeast-fed larvae increased JH synthesis soon after eclosion when fed yeast as adults, but they suppressed JH when they were deprived of adult nutrient yeast. Surprisingly, females from yeast-deprived larvae synthesized small quantities of JH at eclosion independent of adult yeast feeding; this level increased when these adults were fed yeast and decreased when they were not (Fig. 3). Thus, juvenile nutrition affects the level of JH synthesis at eclosion but nutrition in the adult determines hormone synthesis of the imago.

image

Figure 3. Juvenile hormone synthesis of adult corpora allata at four ages measured by incorporation of 3H-methionine. Females from yeast-deprived larvae were maintained as adults with standard yeast-diet (solid square) or on agar-sugar-cornmeal diet lacking yeast (open circle). Females from yeast-fed larvae were maintained as adults with standard yeast-diet (hash-mark) or on agar–sugar–cornmeal diet lacking yeast (closed circle). Larval Yeast-Fed: LYF; Larval Yeast-Deprived: LYD; Adult Yeast-Fed: AYF; Adult Yeast-Deprived: AYD.

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image

Figure 4. Insulin-like peptide producing cells (IPC) and their size in adult females derived from yeast-deprived larvae. Neurosecretory cells in the pars intercebralis were located with antiserum against Insulin-like peptide in adult females derived from yeast-deprived larvae. (A) Adults held since eclosion (2 days) without dietary yeast (yeast-deprived). (B) Adults at 2 days, fed yeast since eclosion. (C) Estimated volume of the pars intercebralis cell body (IPC) from yeast-deprived and yeast-fed adult females.

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To assess insulin activity in response to dietary yeast in adults, we examined the distribution of insulin-like protein in adult pars intercebralis (Fig. 4). At eclosion females from yeast-deprived larvae had compact immunostaining of the pars intercebralis and few insulin-positive vesicles within or about the cells. Once these adults were fed yeast, the pars intercebralis rapidly attained normal insulin-like protein distributions: the cells enlarged and many insulin-positive vesicles were produced.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

The nutritional condition of third instar D. melanogaster has strong effects on adult fitness-related traits. Previous studies have shown that larval dietary yeast influences adult body size, fecundity and ovariole number (Beadle et al., 1938; Robertson, 1960; Hodin & Riddiford, 2000). We find that these nutrient-controlled aspects of adult morphology and reproduction are not associated with changes in adult mortality as a function of age. By contrast, diet restriction imposed at the adult stage is well documented to retard aging in D. melanogaster (Chippindale et al., 1993; Chapman & Partridge, 1996; Clancy et al., 2002; Mair et al., 2003). Thus, although nutrient deprivation of larvae influences many adult traits, nutrition appears directly to affect aging through processes acting in the adult.

From our data we may begin to understand when insulin/IGF signalling itself must act to influence aging in D. melanogaster. Stage-specific effects of insulin/IGF were demonstrated for the nematode Caenorhabditis elegans. RNAi designed to suppress the insulin/IGF receptor locus daf-2 increase life span when applied to adults but not when exclusively directed at larvae (Dillin et al., 2002). Reproduction is unaffected by the adult directed daf-2 RNAi but the same construct given to larvae strongly reduces adult fecundity. In D. melanogaster, metabolic changes induced by yeast deprivation in larvae do not impact adult aging. To the extent that larval yeast-deprivation reduces insulin/IGF, as demonstrated to occur by Ikeya et al. (2002), or affects other aspects of physiology, these larval metabolic factors do not affect senescence in the adult. Because insulin/IGF and JH can vary in adults as a function of diet, and diet restriction at that stage efficiently slows aging, we suggest that insulin/IGF signals must act specifically within the adult fly to affect senescence.

Because larval nutrition does not influence aging-related mortality, senescence can occur independently of body size and, remarkably, reproduction. Across taxonomic groups life span correlates positively with body size, but within many species it is negatively associated with individual stature (Li et al., 1996; Miller et al., 2000; Bartke et al., 2001; Speakman et al., 2003). In mammals, small stature and extended life may be associated through the common effects of reduced IGF (Bartke et al., 2001). Within D. melanogaster the relationship of body size to longevity is less clear. Many studies report a correlation between large size and long life, and these observations have been argued to support the ‘developmental theory of aging’ in which slow growth and large size confer superior longevity (Lints, 1978; Mayer & Baker, 1985). The well-controlled studies of Zwaan et al. (1991) demonstrated that the development of large size does not favour slow senescence in D. melanogaster. When egg density was varied to manipulate development rate and body size, life span was inversely correlated with adult body size. When larval nutrition was limited in order to slow development and reduce adult body size, stature again was inversely associated with life span, at least among females. In further work, size was manipulated by rearing larvae at three temperatures while adult longevity was assessed in common thermal conditions (Zwaan et al., 1992). Among groups, larvae from the lowest temperature had the largest adult body size but the shortest life span. In each case, slow growth and large size did not delay senescence, and if anything small size favoured longevity. Our current study now clarifies that small size is not sufficient to extend longevity in D. melanogaster.

Unlike body size, manipulations that suppress reproduction have been consistently observed to increase life span (Partridge, 1989; Partridge & Barton, 1993; Partridge & Mangel, 1999; but see Carey et al., 2002). We found that ovariole number and fecundity were reduced by 65% in females that developed from yeast-deprived larvae. Unexpectedly, these females had normal demographic aging. We also found that ovariole number was reduced by mutations of the insulin-signal pathway. Long-lived hypomorphic chico1/chico1 have about 50% the wild-type number of ovarioles (as do InRp5545/E19; data not shown). By contrast, long-lived heterozygote chico1/chico+ (Clancy et al., 2001; Tu et al., 2002) have a normal complement of ovarioles. Thus, as a function of nutrition or genotype, ovariole number and fecundity need not correlate with adult longevity.

We might interpret these unexpected relationships between reproduction and aging of the fly with insights based on how somatic and germline tissues regulate aging in C. elegans. In the worm, ablation of the whole gonad does not alter life span but longevity is increased when germline cells are eliminated (Hsin & Kenyon, 1999). Kenyon and co-workers propose that somatic and germline gonadal tissue produce counterbalancing regulatory signals that, respectively, favour and attenuate adult survival (Hsin & Kenyon, 1999). We find D. melanogaster females that develop from larval diet-restriction have fewer ovarioles, but each ovariole retains a full complement of germline stem cells, and presumably of somatic cells. Thus, when ovariole number is reduced fecundity will decline but the ratio of cell types within each ovariole is constant. Under these conditions females might retain the normal balance of gonad-derived longevity signals and not become long lived despite their reduced fecundity. This interpretation does not contradict the many manipulative studies in which reduced egg production increases longevity. If egg production is suppressed in the adult after the somatic gonad is mature, signals that depend on active germline tissue could decrease relative to the mass of the already formed somatic gonad, and thus slow aging.

Our discussion assumes that demographic aging is normal in adults that develop from diet-restricted larvae. It is possible that low fecundity in these adults improves their survival but at the same time they are developmentally compromised and carry a juvenile-derived mortality risk into adulthood that cancels the mortality advantage of reduced reproduction. This case seems unlikely, however, because it requires the historically derived mortality risk to mirror exactly the dynamic mortality associated with reproduction. By contrast, we find that elevated mortality, which could be attributed to developmental experience, is relatively constant and limited to young adults.

We have manipulated nutrition during development and found that body size reduced by larval nutrition had no impact on demographic aging. Small size itself is not sufficient to slow senescence and it seems unlikely that dwarf chico or InR genotypes can be long lived because they are small. Ovariole number and egg production are strongly affected by larval nutrition. Ovariole number is also reduced by some mutations of the insulin signal pathway. As with body size, adult survival is independent of these developmentally regulated traits. These influences of larval nutrition on growth and adult reproduction but not on aging are similar to the stage-specific effects of daf-2 upon reproduction and aging in C. elegans. Whether these stage-specific effects of metabolic regulation are a unique property of moulting animals or extend to mammals is a future problem for integrative research on development, aging and evolution.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Culture and diet

We investigated D. melanogaster strains w1118, yellow-white (yw), Oregon-R and Canton-S. Stocks were reared in uncrowded conditions on standard sucrose–cornmeal–agar–yeast diet (components, respectively, 10.5%, 5.0%, 0.7%, 2.0% with the remainder water) with live yeast supplemented to the surface. Eggs were collected over 8 h. Larvae were reared on standard diet until early third instar (92–96 h after egg deposition) when they were separated from the media by suspension in a 20% sucrose solution and washed with distilled water. Clean larvae were returned to standard diet (yeast-fed) or to diet lacking yeast but containing sucrose, cornmeal and agar (yeast-deprived). At emergence, all adults were placed on standard diet supplemented with grains of live yeast, unless otherwise noted. Adults were simultaneously collected from all control and yeast-deprived treatments; because of differences in development rate, in some trials fewer adults of the yeast-deprived group were available during the collection period.

We also measured ovariole number in genotypes of chico. In lines derived from 18 generations of backcross to a cn;ry background, the chico1 and chico+ allele segregate among highly isogenic sibs (Tu et al., 2002). Females and males with chico+/chico+, chico+/chico1 and chico1/chico1 genotypes are progressively longer lived. Here we measured the ovariole number of these genotypes in two independent backcross lines (de-2 and de-5).

Demography and survival statistics

We compared the mortality rates of adults from yeast-fed larvae to those of yeast-deprived larvae. Mortality rate was estimated from data pooled across 3–5 replicate 1-litre demography cages (cage design in Tatar et al., 2001b). Each cage was initiated with ∼300 adult flies, of mixed sex, collected within 48 h of eclosion. Food vials were changed and dead flies were removed and counted every other day. Life tables for each sex were calculated by the extinct cohort method (Chaing, 1984). Proportional hazard analysis was used to assess differences in mortality rates among yeast-fed and yeast-deprived groups (Lee, 1992).

Daily egg production was estimated from 37 w1118 females individually held in shell vials with standard yeast-supplemented diet and one male of the same strain and yeast treatment. All eggs were counted daily when females were transferred to fresh vials.

Morphology

We measured head capsule width and wing length of adults from yeast-fed and yeast-deprived larvae. Head capsules at the widest transect were measured by ocular graticule under 40× magnification. Wings were fixed in DPX Mount and the length measured from anterior to apical cross-vein along the radial vein. Ovariole number and the stage of the most advanced egg chamber (stage 1–14 of King, 1970) was scored from fixed tissue of 2-day-old females. Immunocytochemistry (see Ohlstein & McKearin, 1997) with monoclonal supernatant 1B1 (anti-1B1 Ag) was used to detect fusomes, which uniquely mark germline stem cells, in ovaries from 2- to 4-day-old females.

Hormone assays

JH synthesis was measured from incubated corpora allata (CA) by radiochemical assay (methods in Tatar et al., 2001a) from adults aged 1–4 days. For each datum, H3-labelled products synthesized by pooled CA of three females were separated by thin-layer chromatography, standardized against JHB3, JH III and methyl-farnesoate (MF). With immunocytochemistry (see Cao & Brown, 2001) polyclonal antibody against the A-chain of D. melanogaster insulin-like peptide (provided by M. Brown, University of Georgia) was used to define brain tissue containing insulin-like proteins. The size of insulin-positive cells was estimated from a compressed Z-axis series of confocal images.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

M.-P.T. received support for this project from the American Federation for Aging Research. Support to M.T. was provided by NIH AG16632 and from the Ellison Medical Foundation.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References