Increased transsulfuration mediates longevity and dietary restriction in Drosophila.

The mechanisms through which dietary restriction enhances health and longevity in diverse species are unclear. The transsulfuration pathway (TSP) is a highly conserved mechanism for metabolizing the sulfur-containing amino acids, methionine and cysteine. Here we show that Drosophila cystathionine ß-synthase (dCBS), which catalyzes the rate-determining step in the TSP, is a positive regulator of lifespan in Drosophila and that the pathway is required for the effects of diet restriction on animal physiology and lifespan. dCBS activity was up-regulated in flies exposed to reduced nutrient conditions, and ubiquitous or neuron-specific transgenic overexpression of dCBS enhanced longevity in fully fed animals. Inhibition of the TSP abrogated the changes in lifespan, adiposity, and protein content that normally accompany diet restriction. RNAi-mediated knockdown of dCBS also limited lifespan extension by diet. Diet restriction reduced levels of protein translation in Drosophila, and we show that this is largely caused by increased metabolic commitment of methionine cycle intermediates to transsulfuration. However, dietary supplementation of methionine restored normal levels of protein synthesis to restricted animals without affecting lifespan, indicating that global reductions in translation alone are not required for diet-restriction longevity. Our results indicate a mechanism by which dietary restriction influences physiology and aging.

Lipidomic Profiling Reveals Concerted Temporal Patterns of Functionally Related Lipids in Aedes aegypti Females Following Blood Feeding.

We conducted a lipidomic analysis of the whole body of female Aedes aegypti mosquitoes at different time points over the course of feeding and reproduction. There were temporal biphasic increases of more than 80% of lipids identified at the time of feeding and from 16 h to 30 h post blood meal (PBM). During these two increases, the abundance of many lipids dropped while body weight remained stable, probably reflecting blood lipid digestion and the synthesis of vitellogenin in this period. A concerted temporal pattern was particularly strong at the second peak for membrane and signalling lipids such as phosphatidylethanolamine (PE), phosphatidylinositol (PI), cardiolipin (CL), hexosylceramide (HexCer) and lyso-phosphatidic acid (LPA). Lyso-glycerophospholipids showed three distinct change patterns that are functionally related: Lyso-PE and Lyso-phosphatidylcholine (LPC), which are membrane lipids, showed little change; LPA, a signalling lipid, showed a significant increase from 16 to 30 h PBM; Lyso-PI, a bioactive lipid, and both lyso-phosphatidylglycerol (LPG) and lyso-phosphatidylserine (LPS), which are bacterial membrane lipids, showed one significant increase from the time of feeding to 16 h post blood meal. The result of our study on the anautogenous insect Ae. aegypti point to specific lipids likely to be important in the reproductive process with a role in the formation and growth of ovarian follicles.

Analyses of fruit flies that do not express selenoproteins or express the mouse selenoprotein, methionine sulfoxide reductase B1, reveal a role of selenoproteins in stress resistance.

Selenoproteins are essential in vertebrates because of their crucial role in cellular redox homeostasis, but some invertebrates that lack selenoproteins have recently been identified. Genetic disruption of selenoprotein biosynthesis had no effect on lifespan and oxidative stress resistance of Drosophila melanogaster. In the current study, fruit flies with knock-out of the selenocysteine-specific elongation factor were metabolically labeled with (75)Se; they did not incorporate selenium into proteins and had the same lifespan on a chemically defined diet with or without selenium supplementation. These flies were, however, more susceptible to starvation than controls, and this effect could be ascribed to the function of selenoprotein K. We further expressed mouse methionine sulfoxide reductase B1 (MsrB1), a selenoenzyme that catalyzes the reduction of oxidized methionine residues and has protein repair function, in the whole body or the nervous system of fruit flies. This exogenous selenoprotein could only be expressed when the Drosophila selenocysteine insertion sequence element was used, whereas the corresponding mouse element did not support selenoprotein synthesis. Ectopic expression of MsrB1 in the nervous system led to an increase in the resistance against oxidative stress and starvation, but did not affect lifespan and reproduction, whereas ubiquitous MsrB1 expression had no effect. Dietary selenium did not influence lifespan of MsrB1-expressing flies. Thus, in contrast to vertebrates, fruit flies preserve only three selenoproteins, which are not essential and play a role only under certain stress conditions, thereby limiting the use of the micronutrient selenium by these organisms.

Environmental and genetic perturbations reveal different networks of metabolic regulation.

Progress in systems biology depends on accurate descriptions of biological networks. Connections in a regulatory network are identified as correlations of gene expression across a set of environmental or genetic perturbations. To use this information to predict system behavior, we must test how the nature of perturbations affects topologies of networks they reveal. To probe this question, we focused on metabolism of Drosophila melanogaster. Our source of perturbations is a set of crosses among 92 wild-derived lines from five populations, replicated in a manner permitting separate assessment of the effects of genetic variation and environmental fluctuation. We directly assayed activities of enzymes and levels of metabolites. Using a multivariate Bayesian model, we estimated covariance among metabolic parameters and built fine-grained probabilistic models of network topology. The environmental and genetic co-regulation networks are substantially the same among five populations. However, genetic and environmental perturbations reveal qualitative differences in metabolic regulation, suggesting that environmental shifts, such as diet modifications, produce different systemic effects than genetic changes, even if the primary targets are the same.

Natural genetic variation in transcriptome reflects network structure inferred with major effect mutations: insulin/TOR and associated phenotypes in Drosophila melanogaster.

BACKGROUND: A molecular process based genotype-to-phenotype map will ultimately enable us to predict how genetic variation among individuals results in phenotypic alterations. Building such a map is, however, far from straightforward. It requires understanding how molecular variation re-shapes developmental and metabolic networks, and how the functional state of these networks modifies phenotypes in genotype specific way. We focus on the latter problem by describing genetic variation in transcript levels of genes in the InR/TOR pathway among 72 Drosophila melanogaster genotypes. RESULTS: We observe tight co-variance in transcript levels of genes not known to influence each other through direct transcriptional control. We summarize transcriptome variation with factor analyses, and observe strong co-variance of gene expression within the dFOXO-branch and within the TOR-branch of the pathway. Finally, we investigate whether major axes of transcriptome variation shape phenotypes expected to be influenced through the InR/TOR pathway. We find limited evidence that transcript levels of individual upstream genes in the InR/TOR pathway predict fly phenotypes in expected ways. However, there is no evidence that these effects are mediated through the major axes of downstream transcriptome variation. CONCLUSION: In summary, our results question the assertion of the 'sparse' nature of genetic networks, while validating and extending candidate gene approaches in the analyses of complex traits.

Pyrethroid Susceptibility Has Been Maintained in the Dengue Vector, Aedes aegypti (Diptera: Culicidae), in Queensland, Australia.

Although pesticide resistance is common in insect vectors of human diseases, the evolution of resistance might be delayed if management practices are adopted that limit selection of resistance alleles. Outbreaks of dengue fever have occurred in Queensland, Australia, since the late 1800s, leading to ongoing attempts to control the mosquito vector, Aedes aegypti (L.). Since the 1990s, pyrethroid insecticides have been used for this purpose, but have been applied in a strategic manner with a variety of delivery methods including indoor residual spraying, lethal ovitraps, and use of insect growth regulators as larvicides. Separate selection experiments on mosquitoes from Queensland using Type I and Type II pyrethroids did not produce resistant lines of Ae. aegypti, and bioassays of field material from Queensland showed only weak tolerance in comparison with a susceptible line. There was no evidence of knockdown resistance (kdr) mutations in Ae. aegypti from Queensland, in stark contrast to the situation in nearby southeast Asia. We suspect that careful management of pyrethroid insecticide use combined with surveillance and interception of exotic incursions has helped to maintain pyrethroid (and particularly kdr-based) susceptibility in Ae. aegypti in Australia.

Environmental stress and reproduction in Drosophila melanogaster: starvation resistance, ovariole numbers and early age egg production.

BACKGROUND: The Y model of resource allocation predicts a tradeoff between reproduction and survival. Environmental stress could affect a tradeoff between reproduction and survival, but the physiological mechanisms underlying environmental mediation of the tradeoff are largely unknown. One example is the tradeoff between starvation resistance and early fecundity. One goal of the present study was to determine if reduced early age fecundity was indeed a robust indirect response to selection for starvation resistance, by investigation of a set of D. melanogaster starvation selected lines which had not previously been characterized for age specific egg production. Another goal of the present study was to investigate a possible relationship between ovariole number and starvation resistance. Ovariole number is correlated with maximum daily fecundity in outbred D. melanogaster. Thus, one might expect that a negative genetic correlation between starvation resistance and early fecundity would be accompanied by a decrease in ovariole number. RESULTS: Selection for early age female starvation resistance favored survival under food deprivation conditions apparently at the expense of early age egg production. The total number of eggs produced by females from selected and control lines was approximately the same for the first 26 days of life, but the timing of egg production differed such that selected females produced fewer eggs early in adult life. Females from lines selected for female starvation resistance exhibited a greater number of ovarioles than did unselected lines. Moreover, maternal starvation resulted in progeny with a greater number of ovarioles in both selected and unselected lines. CONCLUSION: Reduced early age egg production is a robust response to laboratory selection for starvation survival. Ovariole numbers increased in response to selection for female starvation resistance indicating that ovariole number does not account for reduced early age egg production. Further, ovariole number increased in a parallel response to maternal starvation, suggesting an evolutionary association between maternal environment and the reproductive system of female progeny.

A dataset for assessing temporal changes in gene expression during the aging process of adult Drosophila melanogaster.

A Drosophila melanogaster genome-wide transcriptome dataset is available for studies on temporal patterns of gene expression. Gene expression was measured using two-dye color oligonucleotide arrays derived from Version 2 of the Drosophila Genomics Resource Center. A total of 15,158 oligonucleotide probes corresponded to a high proportion of the coding genes in the genome. The source of the flies was a highly genetically heterogeneous population maintained in an overlapping generation population regime. This regime was designed to maintain life history traits so that they were similar to those found in natural populations. Flies collected for the cohorts were obtained in a short period of time in a carefully controlled manner before virgin females and males were allowed to mate. Mated females were introduced into two large population cages in unusually high numbers (approximately 12,000 per cage) for a Drosophila laboratory longevity study. Samples were taken weekly from each cohort for 11 weeks; only a small proportion of surviving flies were present at the last two collection time points and thus they were exceptionally old compared to those collected in early-to-midlife samples. The data set is useful for studies of temporal patterns of gene expression as flies age. The very large size of each cohort, and relatively frequent incidence of temporal samples, allows for a fine-scale study of gene expression from young to very old flies.

Environment-dependent survival of Drosophila melanogaster: a quantitative genetic analysis.

Summary Survival under starvation conditions was investigated in relationship to survival when food was present because these traits could be linked by evolutionary history. Recombinant inbred lines derived from natural populations of Drosophila melanogaster were used to test genetic correlations and architecture of these survival traits. Sexes were genetically correlated within traits and there was significant correlation between survival traits. A number of quantitative trait loci (QTLs) were present for starvation survival and/or survival on food. In general, the QTL effects were consistent for sexes and environments. QTL effects were found on each major chromosome, but the major effects were largely localized on the second chromosome. Importantly, the 'four-allele' progenitor of the recombinant inbred lines used in the present study allowed the sign and magnitude of effects to be assigned to linkage groups. One such linkage group on the second chromosome conferred starvation resistance and longevity, supporting the hypothesis of an association between starvation resistance and lifespan.

The symmetry of longevity.

We tested the hypothesis that relatively symmetrical flies live longer. Vein measurements on the left and right wings from the same individual were used to characterize bilateral symmetry in relationship to age-specific survival in defined cohorts. A longitudinal cohort study supported the hypothesis of a correlation between bilateral symmetry and longevity. For another type of experiment, wings were removed from females and males at approximately the beginning of adult life. Thus, there would be no effect of wings per se on adult survival. These wings were measured to characterize bilateral asymmetry, and the day of death of each dewinged individual was determined. Wing symmetry of females and males proved to be a statistically significant predictor of life span, especially for males.

Insulin signaling is necessary for vitellogenesis in Drosophila melanogaster independent of the roles of juvenile hormone and ecdysteroids: female sterility of the chico1 insulin signaling mutation is autonomous to the ovary.

It has been suggested that insulin signaling mutations of Drosophila melanogaster are sterile and long-lived because of juvenile hormone (JH) and ecdysteroid deficiency. However, female sterility of an insulin/IGF-like signaling mutant (chico(1)) of D. melanogaster is not mediated by downstream systemic signaling in terms of major alterations in JH or ecdysteroid levels. chico(1) is a null mutation in the insulin substrate protein (CHICO) gene of D. melanogaster. Homozygous chico(1) females are sterile and their oocytes do not mature beyond the last previtellogenic stage. Homozygous chico(1) females exhibit approximately wild-type rates of JH biosynthesis, ovarian release of ecdysteroids and haemolymph ecdysteroid levels, suggesting that these two major hormone systems play no role in producing the sterility. Previtellogenic wild-type ovaries transplanted into homozygous chico(1) females underwent vitellogenesis, showing that systemic factors present in mutant females are sufficient to support normal vitellogenesis. chico(1) ovaries transplanted into wild-type females did not undergo vitellogenesis indicating that CHICO is necessary in the ovary for vitellogenic maturation. The ovary transplant experiments corroborate the endocrine results and demonstrate that insulin/insulin-like signaling (IIS) is necessary for vitellogenesis even when sufficient levels of JH, ecdysteroids or other factors are present.

Genome-Wide Gene Expression in relation to Age in Large Laboratory Cohorts of Drosophila melanogaster.

Aging is a complex process characterized by a steady decline in an organism's ability to perform life-sustaining tasks. In the present study, two cages of approximately 12,000 mated Drosophila melanogaster females were used as a source of RNA from individuals sampled frequently as a function of age. A linear model for microarray data method was used for the microarray analysis to adjust for the box effect; it identified 1,581 candidate aging genes. Cluster analyses using a self-organizing map algorithm on the 1,581 significant genes identified gene expression patterns across different ages. Genes involved in immune system function and regulation, chorion assembly and function, and metabolism were all significantly differentially expressed as a function of age. The temporal pattern of data indicated that gene expression related to aging is affected relatively early in life span. In addition, the temporal variance in gene expression in immune function genes was compared to a random set of genes. There was an increase in the variance of gene expression within each cohort, which was not observed in the set of random genes. This observation is compatible with the hypothesis that D. melanogaster immune function genes lose control of gene expression as flies age.

Biotin deficiency decreases life span and fertility but increases stress resistance in Drosophila melanogaster.

Biotin deficiency is associated with fetal malformations and activation of cell survival pathways in mammals. In this study we determined whether biotin status affects life span, stress resistance, and fertility in the fruit fly Drosophila melanogaster. Male and female flies of the Canton-S strain had free access to diets containing 6.0 (control), 4.8, 2.5, or 0 pmol biotin/100 mg. Biotin concentrations in diets correlated with activities of biotin-dependent propionyl-CoA carboxylase and biotin concentrations in fly homogenates, but not with biotinylation of histones (DNA-binding proteins). Propionyl-CoA carboxylase activities and biotin concentrations were lower in males than in females fed diets low in biotin. The life span of biotin-deficient males and females was up to 30% shorter compared to biotin-sufficient controls. Exposure to oxidative stress reversed the effects of biotin status on survival in male flies: survival times increased by 40% in biotin-deficient males compared to biotin-sufficient controls. Biotin status did not affect survival of females exposed to oxidative stress. Exposure of flies to cold, heat, and oxidative stress was associated with mobilization of biotin from yet unknown sources. Biotin deficiency decreased fertility of flies. When biotin-deficient males and females were mated, the hatching rate (larvae hatched per egg) decreased by about 28% compared to biotin-sufficient controls. These findings are consistent with the hypothesis that biotin affects life span, stress resistance, and fertility in fruit flies.

Methionine restriction extends lifespan of Drosophila melanogaster under conditions of low amino-acid status.

Reduced methionine (Met) intake can extend lifespan of rodents; however, whether this regimen represents a general strategy for regulating aging has been controversial. Here we report that Met restriction extends lifespan in both fruit flies and yeast, and that this effect requires low amino-acid status. Met restriction in Drosophila mimicks the effect of dietary restriction and is associated with decreased reproduction. However, under conditions of high amino-acid status, Met restriction is ineffective and the trade-off between longevity and reproduction is not observed. Overexpression of InRDN or Tsc2 inhibits lifespan extension by Met restriction, suggesting the role of TOR signalling in the Met control of longevity. Overall, this study defines the specific roles of Met and amino-acid imbalance in aging and suggests that Met restiction is a general strategy for lifespan extension.

Drosophila melanogaster Selection for Survival after Infection with Bacillus cereus Spores: Evolutionary Genetic and Phenotypic Investigations of Respiration and Movement.

Laboratory populations of D. melanogaster have been subjected to selection for survival after live spores of B. cereus were introduced as a pathogenic agent. The present study was designed to investigate correlated traits: respiration as a metabolic trait and movement as a behavioral trait. An underlying hypothesis was that the evolution of increased survival after B. cereus infection exerts a metabolic cost associated with elevated immunity and this would be detected by increased respiration rates. There was support for this hypothesis in the male response to selection, but not for selected-line females. Two phenotypic effects were also observed in the study. Females especially showed a marked increase in respiration after mating compared to the other assay stages regardless of whether respiration was measured per fly or adjusted by lean mass or dry weight. Given that mating stimulates egg production, it is feasible that elevated metabolism was needed to provision oocytes with yolk. Females also moved less than males, perhaps due to behaviors related to oviposition whereas elevated male activity might be due to behaviors associated with seeking females and courtship. Relatively low movement of females indicated that their elevated respiration after mating was not due to a change in locomotion.

Heritability and inter-population differences in lipid profiles of Drosophila melanogaster.

Characterizing and understanding the complex spectrum of lipids in higher organisms lags far behind our analysis of genome and transcriptome sequences. Here we generate and evaluate comprehensive lipid profiles (>200 lipids) of 92 inbred lines from five different Drosophila melanogaster populations. We find that the majority of lipid species are highly heritable, and even lipids with odd-chain fatty acids, which cannot be generated by the fly itself, also have high heritabilities. Abundance of the endosymbiont Wolbachia, a potential provider of odd-chained lipids, was positively correlated with this group of lipids. Additionally, we show that despite years of laboratory rearing on the same medium, the lipid profiles of the five geographic populations are sufficiently distinct for population discrimination. Our data predicts a strikingly different membrane fluidity for flies from the Netherlands, which is supported by their increased ethanol tolerance. We find that 18% of lipids show strong concentration differences between males and females. Through an analysis of the correlation structure of the lipid classes, we find modules of co-regulated lipids and begin to associate these with metabolic constraints. Our data provide a foundation for developing associations between variation in lipid composition with variation in other metabolic attributes, with genome-wide variation, and with metrics of health and overall reproductive fitness.

Drosophila melanogaster Selection for Survival of Bacillus cereus Infection: Life History Trait Indirect Responses.

To study evolved resistance/tolerance in an insect model, we carried out an experimental evolution study using D. melanogaster and the opportunistic pathogen B. cereus as the agent of selection. The selected lines evolved a 3.0- to 3.3-log increase in the concentration of spores required for 50% mortality after 18-24 generations of selection. In the absence of any treatment, selected lines evolved an increase in egg production and delayed development time. The latter response could be interpreted as a cost of evolution. Alternatively, delayed development might have been a target of selection resulting in increased adult fat body function including production of antimicrobial peptides, and, incidentally, yolk production for oocytes and eggs. When treated with autoclaved spores, the egg production difference between selected and control lines was abolished, and this response was consistent with the hypothesis of a cost of an induced immune response. Treatment with autoclaved spores also reduced life span in some cases and elicited early-age mortality in the selected and wound-control lines both of which were consistent with the hypothesis of a cost associated with induction of immune responses. In general, assays on egg production yielded key outcomes including the negative effect of autoclaved spores on egg production.

A hierarchical Bayesian model for a novel sparse partial diallel crossing design.

Partial diallel crossing designs are in common use among evolutionary geneticists, as well as among plant and animal breeders. When the goal is to make statements about populations represented by a given set of lines, it is desirable to maximize the number of lines sampled given a set number of crosses among them. We propose an augmented round-robin design that accomplishes this. We develop a hierarchical Bayesian model to estimate quantitative genetic parameters from our scheme. For example, we show how to partition genetic effects into specific and general combining abilities, and the method provides estimates of heritability, dominance, and genetic correlations in the face of complex and unbalanced designs. We test our approach with simulated and real data. We show that although the models slightly overestimate genetic variances, main effects are assessed accurately and precisely. We also illustrate how our approach allows the construction of posterior distributions of combinations of parameters by calculating narrow-sense heritability and a genetic correlation between activities of two enzymes.

Bioassays of compounds with potential juvenoid activity on Drosophila melanogaster: juvenile hormone III, bisepoxide juvenile hormone III and methyl farnesoates.

Metabolites of the 6,7,10,11 bisepoxide juvenile hormone III (JHB(3)), and other potential juvenoids, were tested for juvenile hormone activity using early instar or early stage pupae of Drosophila melanogaster. Importantly, methyl farnesoates were tested as they might have JH-like activity on Dipteran juveniles. Larvae were exposed to compounds in medium, or the compounds were applied to white puparia. In the assays employed in the present study, there was no indication for JH activity associated with the metabolites of JHB(3). The activity of methyl farnesoate (MF) was higher than that of JH III and far greater than bisepoxide JH III. As opposed to the two endogenous juvenile hormones, methyl farnesoate has weak activity in the white puparial bioassay. When fluorinated forms of methyl farnesoate, which is unlikely to be converted to JH, were applied to Drosophila medium to which fly eggs were introduced, there was a high degree of larval mortality, but no evidence of subsequent mortality at the pupal stage. One possible explanation for the results is that methyl farnesoate is active as a hormone in larval stages, but has little activity at the pupal stage where only juvenile hormone has a major effect.

Overexpression of methionine-R-sulfoxide reductases has no influence on fruit fly aging.

Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine residues in proteins. This function implicated Msrs in antioxidant defense and the regulation of aging. There are two known Msr types in animals: MsrA specific for the reduction of methionine-S-sulfoxide, and MsrB that catalyzes the reduction of methionine-R-sulfoxide. In a previous study, overexpression of MsrA in the nervous system of Drosophila was found to extend lifespan by 70%. Overexpression of MsrA in yeast also extended lifespan, whereas MsrB overexpression did so only under calorie restriction conditions. The effect of MsrB overexpression on lifespan has not yet been characterized in animal model systems. Here, the GAL4-UAS binary system was used to drive overexpression of cytosolic Drosophila MsrB and mitochondrial mouse MsrB2 in whole body, fatbody, and the nervous system of flies. In contrast to MsrA, MsrB overexpression had no consistent effect on the lifespan of fruit flies on either corn meal or sugar yeast diets. Physical activity, fecundity, and stress resistance were also similar in MsrB-overexpressing and control flies. Thus, MsrA and MsrB, the two proteins with similar function in antioxidant protein repair, have different effects on aging in fruit flies.