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Articles by Linda Partridge in JoVE

 JoVE Neuroscience

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

1Institute of Healthy Ageing, and GEE, University College London - UCL, 2School of Biosciences, University of Kent

JoVE 2412

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

Other articles by Linda Partridge on PubMed

A Mortality Cost of Virginity at Older Ages in Female Mediterranean Fruit Flies

Mortality rates were measured over the lifetime of 65,000 female Mediterranean fruit flies, Ceratitis capitata, maintained in either all-female (virgin) cages or cages with equal initial numbers of males, to determine the effect of sexual activity and mating on the mortality trajectory of females at older ages. Although a greater fraction of females maintained in all-female (virgin) cages survived to older ages, the life expectancy of the surviving virgins was less than the life expectancy of surviving non-virgins at older ages. This was due to a mortality crossover where virgin flies experience lower mortality than mated flies from eclosion to Day 20 but higher mortality thereafter. These results suggest that there are two consequences of mating--a short-term mortality increase (cost) and a longer term mortality decrease (benefit).

Dietary Restriction in Long-lived Dwarf Flies

Mechanisms of Ageing: Public or Private?

Ageing--the decline in survival and fecundity with advancing age is caused by damage to macromolecules and tissues. Ageing is not a programmed process, in the sense that no genes are known to have evolved specifically to cause damage and ageing. Mechanisms of ageing might therefore not be expected to be as highly conserved between distantly related organisms as are mechanisms of development and metabolism. However, evidence is mounting that modulators of the rate of ageing are conserved over large evolutionary distances. As we discuss in this review, this conservation might stem from mechanisms that match reproductive rate to nutrient supply.

Genome-wide Transcript Profiles in Aging and Calorically Restricted Drosophila Melanogaster

We characterized RNA transcript levels for the whole Drosophila genome during normal aging. We compared age-dependent profiles from animals aged under full-nutrient conditions with profiles obtained from animals maintained on a low-calorie medium to determine if caloric restriction slows the aging process. Specific biological functions impacted by caloric restriction were identified using the Gene Ontology annotation. We used the global patterns of expression profiles to test if particular genomic regions contribute differentially to changes in transcript profiles with age and if global disregulation of gene expression occurs during aging.

The Evolution of Longevity

A Lethal Side-effect

Life History Response of Mediterranean Fruit Flies to Dietary Restriction

The purpose of this study was to investigate medfly longevity and reproduction across a broad spectrum of diet restriction using a protocol similar to those applied in most rodent studies. Age-specific reproduction and age of death were monitored for 1200 adult males and 1200 females, each individually maintained on one of 12 diets from ad libitum to 30% of ad libitum. Diet was provided in a fixed volume of solution that was fully consumed each day, ensuring control of total nutrient consumption for every fly. Contrary to expectation and precedence, increased longevity was not observed at any level of diet restriction. Among females, reproduction continued across all diet levels despite the cost in terms of increased mortality. Among males, life expectancy exceeded that of females at most diet levels. However, in both sexes, mortality increased more sharply and the pattern of survival changed abruptly once the diet level fell to 50% of ad libitum or below, even though the energetic demands of egg production has no obvious counterpart in males. We believe that a more complete picture of the life table response to dietary restriction will emerge when studies are conducted on a wider range of species and include both sexes, more levels of diet, and the opportunity for mating and reproduction.

Interpreting Interactions Between Treatments That Slow Aging

A major challenge in current research into aging using model organisms is to establish whether different treatments resulting in slowed aging involve common or distinct mechanisms. Such treatments include gene mutation, dietary restriction (DR), and manipulation of reproduction, gonadal signals and temperature. The principal method used to determine whether these treatments act through common mechanisms is to compare the magnitude of the effect on aging of each treatment separately with that when two are applied simultaneously. In this discussion we identify five types of methodological shortcomings that have marred such studies. These are (1) submaximal lifespan-extension by individual treatments, e.g. as a result of the use of hypomorphic rather than null alleles; (2) effects of a single treatment on survival through more than one mechanism, e.g. pleiotropic effects of lifespan mutants; (3) the difficulty of interpreting the magnitude of increases in lifespan in double treatments, and failure to measure and model age-specific mortality rates; (4) the non-specific effects of life extension suppressors; and (5) the possible occurrence of artefactual mutant interactions. When considered in the light of these problems, the conclusions of a number of recent lifespan interaction studies appear questionable. We suggest six rules for avoiding the pitfalls that can beset interaction studies.

Different Cell Size and Cell Number Contribution in Two Newly Established and One Ancient Body Size Cline of Drosophila Subobscura

Latitudinal genetic clines in body size occur in many ectotherms including Drosophila species. In the wing of D. melanogaster, these clines are generally based on latitudinal variation in cell number. In contrast, differences in wing area that evolve by thermal selection in the laboratory are in general based on cell size. To investigate possible reasons for the different cellular bases of these two types of evolutionary response, we compared the newly established North and South American wing size clines of Drosophila subobscura. The new clines are based on latitudinal variation in cell area in North America and cell number in South America. The ancestral European cline is also based on latitudinal variation in cell number. The difference in the cellular basis of wing size variation in the American clines, which are roughly the same age, together with the similar cellular basis of the new South American cline and the ancient European one, suggest that the antiquity of a cline does not explain its cellular basis. Furthermore, the results indicate that wing size as a whole, rather than its cellular basis, is under selection. The different cellular bases of different size clines are most likely explained either entirely by chance or by different patterns of genetic variance--or its expression--in founding populations.

Rapid Laboratory Evolution of Adult Wing Area in Drosophila Melanogaster in Response to Humidity

We examined the evolutionary response of wing area (a trait highly correlated with other measures of body size) to relative humidity (RH), temperature, and their interaction in Drosophila melanogaster, using replicated lines that had been allowed to evolve at low or high humidity at 18 degrees C or at 25 degrees C. We found that after 20 weeks of selection (5-10 generations), low RH lines had significantly greater wing areas than high RH lines in both sexes. This evolutionary response may have resulted from selection of larger flies with a smaller surface area for water loss relative to their weight, or as a correlated response to selection on some other unidentified trait. There were no evolutionary effects of temperature on wing area or cell density. This may have been due to the short duration of the selection experiment, and/or counteracting selection pressures on body size at warm temperature.

The Sex Peptide of Drosophila Melanogaster: Female Post-mating Responses Analyzed by Using RNA Interference

Mating induces profound changes in female insect behavior and physiology. In Drosophila melanogaster, mating causes a reduction in sexual receptivity and an elevation in egg production for at least 5 days. Injection of the seminal fluid sex peptide (SP) induces both responses in virgin females, but only for 1-2 days. The role of SP in eliciting the responses to mating remains to be elucidated. Functional redundancy between seminal fluid components may occur. In addition, mating with spermless males results in brief (1- to 2-day) post-mating responses, indicating either that there is a "sperm effect" or that sperm act as carriers for SP or other seminal fluid components. Here we used RNA interference to suppress SP expression, to determine whether SP is required to elicit full post-mating responses, the magnitude of responses due to other seminal fluid components, and whether SP accounts for the "sperm effect." Receptivity was higher and egg production lower in females mated to SP knock-down males than in controls. Comparison with virgins showed that the responses were brief. SP is therefore required for normal magnitude and persistence of postmating responses. Sperm transfer and use were normal in mates of SP knock-down males, yet their post-mating responses were briefer than after normal matings, and similar to those reported in mates of spermless son-of-tudor males. The prolonged "sperm effect" on female receptivity and egg production is therefore entirely attributable to SP, but sperm are necessary for its occurrence.

Demography of Dietary Restriction and Death in Drosophila

Dietary restriction (DR) increases life-span in organisms from yeast to mammals, presumably by slowing the accumulation of aging-related damage. Here we show that in Drosophila, DR extends life-span entirely by reducing the short-term risk of death. Two days after the application of DR at any age for the first time, previously fully fed flies are no more likely to die than flies of the same age that have been subjected to long-term DR. DR of mammals may also reduce short-term risk of death, and hence DR instigated at any age could generate a full reversal of mortality.

Superoxide and Hydrogen Peroxide Production by Drosophila Mitochondria

Drosophila melanogaster is a key model organism for genetic investigation of the role of free radicals in aging, but biochemical understanding is lacking. Superoxide production by Drosophila mitochondria was measured fluorometrically as hydrogen peroxide, using its dependence on substrates, inhibitors, and added superoxide dismutase to determine sites of production and their topology. Glycerol 3-phosphate dehydrogenase and center o of complex III in the presence of antimycin had the greatest maximum capacities to generate superoxide on the cytosolic side of the inner membrane. Complex I had significant capacity on the matrix side. Center i of complex III, cytochrome c, and complex IV produced no superoxide. Native superoxide generation by isolated mitochondria was also measured without added inhibitors. There was a high rate of superoxide production with sn-glycerol 3-phosphate as substrate; two-thirds mostly from glycerol 3-phosphate dehydrogenase on the cytosolic side and one-third on the matrix side from complex I following reverse electron transport. There was little superoxide production from any site with NADH-linked substrate. Superoxide production by complex I following reverse electron flow from glycerol 3-phosphate was particularly sensitive to membrane potential, decreasing 70% when potential decreased 10 mV, showing that mild uncoupling lowers superoxide production in the matrix very effectively.

Testing for Asymmetrical Gene Flow in a Drosophila Melanogaster Body-size Cline

Asymmetrical gene flow is an important, but rarely examined genetic parameter. Here, we develop a new method for detecting departures from symmetrical migration between two populations using microsatellite data that are based on the difference in the proportion of private alleles. Application of this approach to data collected from wild-caught Drosophila melanogaster along a latitudinal body-size cline in eastern Australia revealed that asymmetrical gene flow could be detected, but was uncommon, nonlocalized, and occurred in both directions. We also show that, in contrast to the findings of a previous study, there is good evidence to suggest that the cline experiences significant levels of gene flow between populations.

QTL Mapping Reveals a Striking Coincidence in the Positions of Genomic Regions Associated with Adaptive Variation in Body Size in Parallel Clines of Drosophila Melanogaster on Different Continents

Latitudinal genetic clines in body size are common in many ectotherm species and are attributed to climatic adaptation. Here, we use Quantitative Trait Loci (QTL) mapping to identify genomic regions associated with adaptive variation in body size in natural populations of Drosophila melanogaster from extreme ends of a cline in South America. Our results show that there is a significant association between the positions of QTL with strong effects on wing area in South America and those previously reported in a QTL mapping study of Australian cline end populations (P < 0.05). In both continents, the right arm of the third chromosome is associated with QTL with the strongest effect on wing area. We also show that QTL peaks for wing area and thorax length are associated with the same genomic regions, indicating that the clinal variation in the body size traits may have a similar genetic basis. The consistency of the results found for the South American and Australian cline end populations indicate that the genetic basis of the two clines may be similar and future efforts to identify the genes producing the response to selection should be focused on the genomic regions highlighted by the present work.

Sex Differences in the Effect of Dietary Restriction on Life Span and Mortality Rates in Female and Male Drosophila Melanogaster

Dietary restriction (DR) has been shown to increase life span in taxonomically diverse animal species. In this study we tested for sex differences in the response of life span to graded severity of DR in Drosophila melanogaster. In both sexes, life span peaked at an intermediate food concentration and declined on either side. However, the magnitude of the response and the food concentration that minimized adult mortality differed significantly between the sexes. Female life span peaked at a food concentration 60% of the standard laboratory diet compared to a concentration of 40% for males. Moreover, female flies subject to DR lived up to 60% longer than did starved or fully fed females, whereas males subjected to DR lived only up to 30% longer. Analysis of age-specific mortality rates showed that DR extended life span by decreasing baseline mortality rates in both sexes, and to a greater extent in females. The differences in the response to DR in female and male Drosophila may be due to previously documented sex differences in sensitivity of life span to insulin/insulin-like growth factor-1 signalling or in nutrient/energy demand and allocation/utilization.

Long-lived Drosophila with Overexpressed DFOXO in Adult Fat Body

Reduced activity of the insulin/insulin-like growth factor signaling (IIS) pathway increases life-span in diverse organisms. We investigated the timing of the effect of reduced IIS on life-span and the role of a potential target tissue, the fat body. We overexpressed dFOXO, a downstream effector of IIS, in the adult Drosophila fat body, which increased life-span and reduced fecundity of females but had no effect on male life-span. The role of FOXO transcription factors and the adipose tissue are therefore evolutionarily conserved in the regulation of aging, and reduction of IIS in the adult is sufficient to mediate its effects on life-span and fecundity.

Lifespan Extension by Dietary Restriction in Female Drosophila Melanogaster is Not Caused by a Reduction in Vitellogenesis or Ovarian Activity

Dietary restriction (DR) extends lifespan in a wide range of organisms. DR also reduces daily and lifetime fecundity. The latter may be an evolutionary adaptation to survive periods of food shortage. Reproductive rate is often negatively correlated with lifespan, and a reduced cost of reproduction could be the mechanism by which DR extends lifespan. We tested this hypothesis in Drosophila melanogaster females, by directly suppressing different aspects of reproduction and measuring the effect on the response of lifespan and age-specific mortality to DR. DR resulted in lifespan extension in females kept with males, in females kept without males, in females with vitellogenesis blocked by the mutant ovoD1 and in females with no germline as a result of X-irradiation. Moreover, rapid (48 h) changes in age-specific mortality, previously seen in fertile females switched between full feeding and DR, were also seen in ovoD1 females. Furthermore, these rapid changes in age-specific mortality in cohorts of fertile wild type females were not accompanied by concurrent changes in egg-production. These results indicate either that reduced reproduction is not necessary for lifespan extension by DR in Drosophila females, or that the relevant aspects of reproduction act upstream of our interventions and were therefore not blocked in our experiments.

Lack of Correlation Between Mitochondrial Reactive Oxygen Species Production and Life Span in Drosophila

The free radical theory of aging proposes that mitochondrial production of reactive oxygen species (ROS) determines the rate of aging. Supporting this hypothesis, longer-lived species produce fewer ROS than shorter-lived ones, and calorically restricted rodents live longer and produce fewer ROS than controls. We studied such correlation in Drosophila melanogaster in caloric restriction and in mutant flies overexpressing the mitochondrial adenine nucleotide translocase (ANT). Caloric restriction extended life span, but there was no significant difference in mitochondrial ROS production compared with controls. ANT overexpressers had significantly lower ROS production (because they had lower membrane potential), but their life span was not extended compared to wild type. Our results show two examples in which mitochondrial ROS production and life span are not correlated.

Metabolic Rate is Not Reduced by Dietary-restriction or by Lowered Insulin/IGF-1 Signalling and is Not Correlated with Individual Lifespan in Drosophila Melanogaster

The link between resting metabolic rate and aging, measured as adult lifespan, was investigated in Drosophila melanogaster by (i) comparing lifespan and metabolic rate of individual flies, (ii) examining the effect of dietary-restriction on the metabolic rate of adult flies, and (iii) comparing the metabolic rate of wild-type and insulin/IGF-1 signalling mutant chico1 flies. The resting oxygen consumption of 65 individually housed and fully fed Drosophila was measured weekly throughout their lifetime. There was no significant difference in the mass-specific rate of oxygen consumption between cohorts that differed in lifespan. Nor was there any statistical correlation between mass-specific oxygen consumption and lifespan of individual Drosophila. The average mass-specific rate of oxygen consumption at 25 degrees C was 3.52+/-0.07 microl O2 mg(-1) h(-1). Variation in mass-specific metabolic rate explained only 4% of variation in individual life span in these flies. Contrary to predictions from the 'rate of living' theory of aging lifetime oxygen consumption was not constant and the lifespan of individual flies accounted for 91% of their lifetime oxygen consumption. An average Drosophila consumes about 3 ml O2 during its adult life. Dietary-restriction had no effect on mass-specific resting metabolic rate both when measured as oxygen consumption by respirometry and when measured as heat production by microcalorimetry. The mass-specific resting heat production of fully fed adult flies at 25 degrees C averaged 17.3+/-0.3 microW mg(-1). Similarly there was no difference in mass-specific metabolic rate of wild-type flies and longliving chico1 insulin/IGF-1 signalling mutant flies, either when measured as oxygen consumption or heat production. Thus, individual variation in lifespan in wild-type flies, and life extension by dietary-restriction and reduced insulin/IGF-1 signalling is not attributable to differences in metabolic rate.

The Interaction Between FOXO and SIRT1: Tipping the Balance Towards Survival

When overexpressed, the NAD-dependent protein deacetylase Sir2 extends the lifespan of both budding yeast and the nematode worm Caenorhabditis elegans. In the worm, this extension of lifespan requires the FOXO transcription factor daf-16. Three recent articles focusing on mammalian homologues of Sir2 and FOXO have highlighted the mechanisms that generate this genetic interaction. Mammalian SIRT1 deacetylates FOXO3 and/or FOXO4, thus attenuating FOXO-induced apoptosis and potentiating FOXO-induced cell-cycle arrest. SIRT1 might increase longevity by shifting FOXO dependent responses away from cell death and towards cell survival.

Computational Analysis of Alpha-helical Membrane Protein Structure: Implications for the Prediction of 3D Structural Models

Relatively little has been known about the structure of alpha-helical membrane proteins, since until recently few structures had been crystallized. These limited data have restricted structural analyses to the prediction of secondary structure, rather than tertiary folds. In order to address this, this paper describes an analysis of the 23 available membrane protein structures. A number of findings are made that are of particular relevance to transmembrane helix packing: (1) on average lipid-tail-accessible transmembrane residues are significantly more hydrophobic, less conserved and contain different residue types to buried residues; (2) charged residues are not always buried and, when accessible to membrane lipid tails, few are paired with another charge and instead they often interact with phospholipid head-groups or with other residue types; (3) a significant proportion of lipid-tail-accessible charged and polar residues form hydrogen bonds only with residues one turn away in the same helix (intra-helix); (4) pore-lining residues are usually hydrophobic and it is difficult to distinguish them from buried residues in terms of either residue type or conservation; and (5) information was gained about the proportion of helices that tend to contribute to lining a pore and the resulting pore diameter. These findings are discussed with relevance to the prediction of membrane protein 3D structure.

Genetic Variation for Total Fitness in Drosophila Melanogaster: Complex Yet Replicable Patterns

The extent of genetic variation in fitness is a crucial issue in evolutionary biology and yet remains largely unresolved. In Drosophila melanogaster, we have devised a method that allows the net effects on fitness of heterozygous wild-type chromosomes to be measured, by competing them against two different "balancer" chromosomes. We have applied the method to a large sample of 40 wild-type third chromosomes and have measured fitnesses of nonlethal chromosomes as well as chromosomes bearing recessive lethals. The measurements were made in the environment to which the population was adapted and did not involve inbreeding. The results show an extraordinary similarity in the behavior of replicates of the same chromosome, indicating consistent genetic effects on total fitness. Some invading chromosomes increased rapidly and some slowly, and some rose to appreciable frequency after several months, but then declined again: in every case, the same pattern was seen in each replicate. We estimated relative fitnesses, rates of change of fitness, and relative viabilities, for each chromosome. There were significant fluctuations around the fitted model, which were also highly replicable. Wild-type chromosomes varied substantially in their effects on heterozygous fitness, and these effects vary through time, most likely as a result of genotype x environment interactions.

Dietary Restriction, Mortality Trajectories, Risk and Damage

Restriction of food intake extends lifespan in evolutionarily diverse organisms, including mammals. Dietary restriction (DR) also delays the appearance of ageing-related damage and pathology and keeps organisms in a youthful state for longer. DR has hence been suggested to lower the rate of ageing. Analysis of mortality rates can be used to test this idea. During ageing, mortality rates in general increase, approximately exponentially. Lifespan can be extended either by a reduction in the rate of increase in mortality rate with age or a lowering of the initial rate of mortality. A reduction in the slope of a mortality trajectory has generally been taken to indicate that the rate of ageing has been lowered. Data on the effects of temperature on mortality in Drosophila are in accordance with this idea. Lowered temperature extends lifespan solely by lowering the slope of the mortality trajectory and flies with a hotter thermal history have permanently elevated death rates. In contrast, lowering of the initial rate of mortality has been taken to leave the rate of ageing unaffected. In Drosophila and in mice, but not in rats, DR extends lifespan by lowering the initial mortality rate. In Drosophila, the effect of DR is acute, and mortality rate switches rapidly between DR and control values with the corresponding changes in nutritional regime. DR in Drosophila therefore has no impact upon the rate of ageing. Possible mechanisms by which DR can both delay damage and pathology and yet act acutely to determine mortality rates are discussed. In rodents, some phenotypes associated with DR, including microarray profiles, show rapid switching with changed nutritional regime, pointing to potentially acute effects of DR in mammals.

Longer Lifespan, Altered Metabolism, and Stress Resistance in Drosophila from Ablation of Cells Making Insulin-like Ligands

The insulin/insulin-like growth factor-like signaling pathway, present in all multicellular organisms, regulates diverse functions including growth, development, fecundity, metabolic homeostasis, and lifespan. In flies, ligands of the insulin/insulin-like growth factor-like signaling pathway, the Drosophila insulin-like peptides, regulate growth and hemolymph carbohydrate homeostasis during development and are expressed in a stage- and tissue-specific manner. Here, we show that ablation of Drosophila insulin-like peptide-producing median neurosecretory cells in the brain leads to increased fasting glucose levels in the hemolymph of adults similar to that found in diabetic mammals. They also exhibit increased storage of lipid and carbohydrate, reduced fecundity, and reduced tolerance of heat and cold. However, the ablated flies show an extension of median and maximal lifespan and increased resistance to oxidative stress and starvation.

Sex and Death: What is the Connection?

A cost of reproduction, where lifespan and fecundity are negatively correlated, is of widespread occurrence. Mutations in insulin/IGF signaling (IIS) pathways and dietary restriction (DR) can extend lifespan in model organisms but do not always reduce fecundity, suggesting that the link between lifespan and fecundity is not inevitable. Understanding the molecular basis of the cost of reproduction will be informed by elucidation of the mechanisms by which DR and IIS affect these two traits.

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.

Dietary Restriction in Drosophila

The fruit fly Drosophila is a useful organism for the investigation of the mechanisms by which dietary restriction (DR) extends lifespan. Its relatively short generation time, well-characterised molecular biology, genetics and physiology and ease of handling for demographic analysis are all major strengths. Lifespan has been extended by DR applied to adult Drosophila, by restriction of the availability of live yeast or by co-ordinate dilution of the whole food medium. Lifespan increases to a maximum through DR with a progressive dilution of the food and then decreases through starvation as the food is diluted further. Daily and lifetime fecundities of females are reduced by food dilution throughout the DR and starvation range. Standard Drosophila food ingredients differ greatly between laboratories and fly stocks can differ in their responses to food dilution, and a full range of food concentrations should therefore be investigated when examining the response to DR. Flies do not alter the time that they spend feeding in response to DR. Both mean and maximum lifespan are extended by DR. The nutrients critical for the response to DR in Drosophila require definition. The extension of lifespan in response to DR is very much greater in females than in males. Two nutrient-sensing pathways, the insulin/IGF-like and TOR pathways, have been implicated in mediating this response of lifespan to DR in Drosophila, as have two protein deacetylases, dSir2 and Rpd3, although the precise nature of this interaction remain to be characterised. Although female fecundity is reduced by DR, the response of lifespan to DR appears normal in sterile females, possibly implying that reduced fecundity is not necessary for extension of lifespan by DR. There is no reduction in metabolic rate or in the rate of generation of superoxide and hydrogen peroxide from isolated mitochondria in response to DR. DR acts acutely and rapidly (within 48 h) to reduce the mortality of flies that are fully fed to the level found in animals exposed to DR throughout life. This rapid mortality rate recovery provides a powerful framework within which to further investigate the mechanisms by which DR extends lifespan.

Counting the Calories: the Role of Specific Nutrients in Extension of Life Span by Food Restriction

Reduction of food intake without malnourishment extends life span in many different organisms. The majority of work in this field has been performed in rodents where it has been shown that both restricting access to the entire diet and restricting individual dietary components can cause life-span extension. Thus, for insights into the mode of action of this intervention, it is of great interest to investigate the aspects of diet that are critical for life span extension. Further studies on the mechanisms of how food components modify life span are well suited to the model organism Drosophila melanogaster because of its short life span and ease of handling and containment. Therefore, we summarize practical aspects of implementing dietary restriction in this organism, as well as highlight the major advances already made. Delineation of the nutritional components that are critical for life-span extension will help to reveal the mechanisms by which it operates.

Calories Do Not Explain Extension of Life Span by Dietary Restriction in Drosophila

Dietary restriction (DR) extends life span in diverse organisms, including mammals, and common mechanisms may be at work. DR is often known as calorie restriction, because it has been suggested that reduction of calories, rather than of particular nutrients in the diet, mediates extension of life span in rodents. We here demonstrate that extension of life span by DR in Drosophila is not attributable to the reduction in calorie intake. Reduction of either dietary yeast or sugar can reduce mortality and extend life span, but by an amount that is unrelated to the calorie content of the food, and with yeast having a much greater effect per calorie than does sugar. Calorie intake is therefore not the key factor in the reduction of mortality rate by DR in this species.

Isogenic Autosomes to Be Applied in Optimal Screening for Novel Mutants with Viable Phenotypes in Drosophila Melanogaster

Most insertional mutagenesis screens of Drosophila performed to date have not used target chromosomes that have been checked for their suitability for phenotypic screens for viable phenotypes. To address this, we have generated a selection of stocks carrying either isogenized second chromosomes or isogenized third chromosomes, in a genetic background derived from a Canton-S wild-type strain. We have tested these stocks for a range of behavioral and other viable phenotypes. As expected, most lines are statistically indistinguishable from Canton-S in most phenotypes tested. The lines generated are now being used as target chromosomes in mutagenesis screens, and the characterization reported here will facilitate their use in screens of these lines for behavioral and other viable phenotypes.

Diet, Metabolism and Lifespan in Drosophila

Dietary restriction (DR) by dilution of the food medium can extend lifespan in Drosophila. DR results in a state that is characterized by reduced fecundity, increased starvation resistance and higher total lipid levels. In the past, each of these correlated phenotypes has been proposed to play a causal role in the lifespan-extending effects of food reduction. However, more recent data show that each phenotype can be uncoupled from the long-lived state to varying extents. In this mini-review, we summarize the principal findings of the effects of DR on Drosophila in order to address what these phenotypes can tell us about the physiological remodeling required for Drosophila to be long-lived. Current data indicate lifespan-extension by DR is likely to involve both enhancement of various defense and detoxification mechanisms and a complex range of metabolic alterations that make energy available for these processes.

Science Fact and the SENS Agenda. What Can We Reasonably Expect from Ageing Research?

Genome-wide Gene Expression in Response to Parasitoid Attack in Drosophila

Parasitoids are insect parasites whose larvae develop in the bodies of other insects. The main immune defense against parasitoids is encapsulation of the foreign body by blood cells, which subsequently often melanize. The capsule sequesters and kills the parasite. The molecular processes involved are still poorly understood, especially compared with insect humoral immunity.

Patterns of Diversity and Linkage Disequilibrium Within the Cosmopolitan Inversion In(3R)Payne in Drosophila Melanogaster Are Indicative of Coadaptation

The cosmopolitan inversion In(3R)Payne in Drosophila melanogaster decreases in frequency with increasing distance from the equator on three continents, indicating it is subject to strong natural selection. We investigated patterns of genetic variation and linkage disequilibrium (LD) in 24 molecular markers located within and near In(3R)Payne to determine if different parts of the inversion responded to selection the same way. We found reduced variation in the markers we used compared to others distributed throughout the genome, consistent with the inversion having a relatively recent origin (

The Effects of Exogenous Antioxidants on Lifespan and Oxidative Stress Resistance in Drosophila Melanogaster

We used the fruit fly Drosophila melanogaster to test the effects of feeding the superoxide dismutase (SOD) mimetic drugs Euk-8 and -134 and the mitochondria-targeted mitoquinone (MitoQ) on lifespan and oxidative stress resistance of wild type and SOD-deficient flies. Our results reaffirm the findings by other workers that exogenous antioxidant can rescue pathology associated with compromised defences to oxidative stress, but fail to extend the lifespan of normal, wild type animals. All three drugs showed a dose-dependent increase in toxicity in wild type flies, an effect that was exacerbated in the presence of the redox-cycling drug paraquat. However, important findings from this study were that in SOD-deficient flies, where the antioxidant drugs increased lifespan, the effects were sex-specific and, for either sex, the effects were also variable depending on (1) the stage of development from which the drugs were given, and (2) the magnitude of the dose. These findings place significant constraints on the role of oxidative stress in normal ageing.

The Effect of Dietary Restriction on Mitochondrial Protein Density and Flight Muscle Mitochondrial Morphology in Drosophila

Dietary restriction (DR) extends life span in diverse organisms and may do so by attenuating production of mitochondrial reactive oxygen species (ROS). However, measurements of ROS production from isolated mitochondria of organisms subjected to DR have produced inconsistent results. In the fruit fly Drosophila, DR does not reduce production of ROS from isolated mitochondria. In this study, we used Drosophila to test whether DR lowered mitochondrial density. We assessed mitochondrial densities of flies on DR and Control diets using (a) the activities of mitochondrial enzymes and (b) electron microscopy. Both methods showed no overall effect of DR on mitochondrial density; however, mitochondrial enzyme activities and morphology differed significantly between DR and Control flies. We concluded that life-span extension by DR in Drosophila is not mediated through a reduction in mitochondrial density. If DR in Drosophila extends life span by reducing ROS production, then it does so through mechanisms that operate only in vivo.

Flight Activity, Mortality Rates, and Lipoxidative Damage in Drosophila

In this study, the effect of flight activity on mortality rates and lipoxidative damage in Drosophila was determined to identify mechanisms through which oxidative damage affects life span. The results showed that flies allowed flying throughout life had higher mortality rates and decreased median and maximum life spans compared to controls. The mortality rate of the flight activity group could be lowered, but not completely reversed by switching to control conditions; and the accrued oxidative damage could not be eliminated. The levels of reactive oxygen species produced by mitochondria isolated from high activity and control flies did not differ significantly. However, the high activity flies had altered membrane fatty acid compositions, which made them prone to increased lipid peroxidation. The effect of flight activity on insect life span differs considerably from the beneficial effects of exercise in mammals; these differences may be caused by physiological differences between the two taxa.

No Extension of Lifespan by Ablation of Germ Line in Drosophila

Increased reproduction is frequently associated with a reduction in longevity in a variety of organisms. Traditional explanations of this 'cost of reproduction' suggest that trade-offs between reproduction and longevity should be obligate. However, it is possible to uncouple the two traits in model organisms. Recently, it has been suggested that reproduction and longevity are linked by molecular signals produced by specific reproductive tissues. For example, in Caenorhabditis elegans, lifespan is extended in worms that lack a proliferating germ line, but which possess somatic gonad tissue, suggesting that these tissues are the sources of signals that mediate lifespan. In this study, we tested for evidence of such gonadal signals in Drosophila melanogaster. We ablated the germ line using two maternal effect mutations: germ cell-less and tudor. Both mutations result in flies that lack a proliferating germ line but that possess a somatic gonad. In contrast to the findings from C. elegans, we found that germ line ablated females had reduced longevity relative to controls and that the removal of the germ line led to an over-proliferation of the somatic stem cells in the germarium. Our results contrast with the widely held view that it is downstream reproductive processes such as the production and/or laying of eggs that are costly to females. In males, germ line ablation caused either no difference, or a slight extension, in longevity relative to controls. Our results indicate that early acting, upstream reproductive enabling processes are likely to be important in determining reproductive costs. In addition, we suggest that the specific roles and putative patterns of molecular signalling in the germ line and somatic tissues are not conserved between flies and worms.

Beyond the Evolutionary Theory of Ageing, from Functional Genomics to Evo-gero

By the mid 1970s, the mechanisms by which ageing can evolve had a secure theoretical basis in population genetics. Here, we discuss how subsequent evolutionary work has focussed on testing and extending this theory, and on attempting to integrate it with other emerging facets of the biology of ageing, such as genetic studies of long-lived mutants and of phenotypic plasticity in ageing, such as in response to nutritional status. We also describe how functional genomic studies are providing new insights into the evolutionary forces shaping genome evolution and lifespan control. Future challenges include understanding the biochemistry of longevity and how its failure generates ageing and associated diseases, and the determination of the genetic basis of lifespan evolution and the great plasticity that it displays.

Coordinated Multitissue Transcriptional and Plasma Metabonomic Profiles Following Acute Caloric Restriction in Mice

Caloric restriction (CR) increases healthy life span in a range of organisms. The underlying mechanisms are not understood but appear to include changes in gene expression, protein function, and metabolism. Recent studies demonstrate that acute CR alters mortality rates within days in flies. Multitissue transcriptional changes and concomitant metabolic responses to acute CR have not been described. We generated whole genome RNA transcript profiles in liver, skeletal muscle, colon, and hypothalamus and simultaneously measured plasma metabolites using proton nuclear magnetic resonance in mice subjected to acute CR. Liver and muscle showed increased gene expressions associated with fatty acid metabolism and a reduction in those involved in hepatic lipid biosynthesis. Glucogenic amino acids increased in plasma, and gene expression for hepatic gluconeogenesis was enhanced. Increased expression of genes for hormone-mediated signaling and decreased expression of genes involved in protein binding and development occurred in hypothalamus. Cell proliferation genes were decreased and cellular transport genes increased in colon. Acute CR captured many, but not all, hepatic transcriptional changes of long-term CR. Our findings demonstrate a clear transcriptional response across multiple tissues during acute CR, with congruent plasma metabolite changes. Liver and muscle switched gene expression away from energetically expensive biosynthetic processes toward energy conservation and utilization processes, including fatty acid metabolism and gluconeogenesis. Both muscle and colon switched gene expression away from cellular proliferation. Mice undergoing acute CR rapidly adopt many transcriptional and metabolic changes of long-term CR, suggesting that the beneficial effects of CR may require only a short-term reduction in caloric intake.

Superoxide Dismutase Activities in Long-lived Drosophila Melanogaster Females: Chico1 Genotypes and Dietary Dilution

Superoxide dismutase (SOD) activities were determined for dietary dilution conditions that extend the life span of Drosophila melanogaster. The hypothesis motivating this research was that elevated SOD activity is associated with increased life span resulting from flies being held on a restricted diet. SOD activities were also measured for chico (1) which is a mutation in the insulin receptor substrate protein gene associated with life span extension. This allowed us to confirm the results of (Clancy et al. 2001) and extend the results by measuring CuZn SOD and Mn SOD activities in addition to the previously determined overall SOD activity. If the same form of SOD activity (CuZn SOD or Mn SOD) was elevated on the dilute diet that extends life span and in the long lived chico (1) homozygotes, then it would suggest that life span extension by dietary restriction and by insulin signaling mutations has a similar underlying mechanism. However, overall SOD activity, and CuZn SOD or Mn SOD activities did not differ among the diets tested. As observed previously (Clancy et al. 2001), overall SOD activity was elevated in chico (1) homozygotes compared to the heterozygote or wild type. Results from the present study indicate that elevated CuZn SOD activity, not Mn SOD, is the basis for the relatively high level of SOD activity in the chico (1) homozygotes.

A Functioning Ovary is Not Required for Sex Peptide to Reduce Receptivity to Mating in D. Melanogaster

In many species of invertebrates that mate multiply, mating induces a temporary reduction in sexual receptivity and an increase in the rate of egg laying. These processes often appear to be co-ordinately regulated, and triggered by the passage of seminal fluid components. However, little is known about the mechanisms of the links between these processes. In Drosophila melanogaster females, post-mating sexual receptivity is decreased and egg laying increased by the actions of the male ejaculate-derived sex peptide (SP). Effects of SP on egg laying and receptivity have not been observed separately, which has led to the suggestion that the reduction in receptivity is at least partially dependent on the status of egg development or egg laying, with the presence of an egg in the uterus being a strong predictor of receptivity state. Here, we examine the response to SP of females in which egg development is arrested at an early, pre-vitellogenic stage. We find that females in which egg development is arrested mate normally and that normal receptivity responses to SP are independent of early egg arrest. Among fertile control females that laid eggs, a significant effect of SP on receptivity was also observed, independent of whether an egg was present in the uterus. The results show that the effects of SP on receptivity are not dependent upon a fully functional ovary, and hence that egg development or laying is not causal in the SP receptivity response.

Comment by Matthew Piper, William Mair, Linda Partridge on Min, K.J., Flatt, T., Kulaots, I., Tatar, M. (2006) "Counting Calories in Drosophila Dietary Restriction"Exp. Gerontology, Doi:10.1016/j.exger.2006.10.009

Role of Insulin-like Signalling in Drosophila Lifespan

Regulation of lifespan by the insulin/insulin-like growth factor-like signalling (IIS) pathway has been conserved during evolution from the nematode worm to the mouse. In the insect Drosophila, regulation of lifespan by the IIS pathway was established by data showing that many mutations in single genes encoding IIS components result in an increase in lifespan. Recently, however, the focus has shifted from studying the effects of single gene mutations with ubiquitous effects to finding interventions that alter IIS in specific tissues and at specific stages in the life history of the fruitfly, in order to elucidate the signalling pathways at work and the mechanisms by which alterations in the IIS pathway can extend lifespan.

Dietary Restriction in Drosophila: Delayed Aging or Experimental Artefact?

Lifespan can be extended by reduction of dietary intake. This practice is referred to as dietary restriction (DR), and extension of lifespan by DR is evolutionarily conserved in taxonomically diverse organisms including yeast, invertebrates, and mammals. Although these two often-stated facts carry the implication that the mechanisms of DR are also evolutionarily conserved, extension of lifespan could be a case of evolutionary convergence, with different underlying mechanisms in different taxa. Furthermore, extension of lifespan by different methods of DR in the same organism may operate through different mechanisms. These topics remain unresolved because of the very fact that the mechanisms of DR are unknown. Given these uncertainties, it is essential that work on the mechanisms of DR is not clouded by imprecise descriptions of methods or by technical problems. Here we review the recent literature on DR in Drosophila to point out some methodological issues that can obscure mechanistic interpretations. We also indicate some experiments that could be performed to determine if DR in Drosophila operates through similar mechanisms to the process in rodents.

Dynamics of the Action of DFOXO on Adult Mortality in Drosophila

The insulin/insulin growth factor (IGF)-like signaling (IIS) pathway has a conserved role in regulating lifespan in Caenorhabditis elegans, Drosophila and mice. Extension of lifespan by reduced IIS has been shown in C. elegans to require the key IIS target, forkhead box class O (FOXO) transcription factor, DAF-16. dFOXO, the Drosophila DAF-16 orthologue, is also an IIS target, and its overexpression in adult fat body increases lifespan. In C. elegans, IIS acts exclusively during adulthood to determine adult survival. We show here, using an inducible overexpression system, that in Drosophila continuous dFOXO overexpression in adult fat body reduces mortality rate throughout adulthood. We switched the IIS status of the flies at different adult ages and examined the effects of these switches on dFOXO expression and mortality rates. dFOXO protein levels were switched up or down by the inducible expression system at all ages examined. If IIS status is reversed early in adulthood, similar to the effects of another intervention that reduces adult mortality in Drosophila, dietary restriction (DR), there is a complete switch of subsequent mortality rate to that of flies chronically exposed to the new IIS regime. At this age, IIS thus acts acutely to determine risk of death. Mortality rates continued to respond to a switch in IIS status up to 4 weeks of adult age, but not thereafter. However, unlike DR, as IIS status was altered at progressively later ages, mortality rates showed incomplete switching and responded with progressively smaller changes. These findings indicate that alteration of expression levels of dFOXO may have declining effects on IIS status with age, that there could be some process that prevents or lessens the physiological response to a switch in IIS status or that, unlike DR, this pathway regulates aging-related damage. The decreased mortality and increased lifespan of dFOXO overexpressing flies was uncoupled from any effect on female fecundity and from expression levels of Drosophila insulin-like peptides in the brain.

No Influence of Indy on Lifespan in Drosophila After Correction for Genetic and Cytoplasmic Background Effects

To investigate whether alterations in mitochondrial metabolism affect longevity in Drosophila melanogaster, we studied lifespan in various single gene mutants, using inbred and outbred genetic backgrounds. As positive controls we included the two most intensively studied mutants of Indy, which encodes a Drosophila Krebs cycle intermediate transporter. It has been reported that flies heterozygous for these Indy mutations, which lie outside the coding region, show almost a doubling of lifespan. We report that only one of the two mutants lowers mRNA levels, implying that the lifespan extension observed is not attributable to the Indy mutations themselves. Moreover, neither Indy mutation extended lifespan in female flies in any genetic background tested. In the original genetic background, only the Indy mutation associated with altered RNA expression extended lifespan in male flies. However, this effect was abolished by backcrossing into standard outbred genetic backgrounds, and was associated with an unidentified locus on the X chromosome. The original Indy line with long-lived males is infected by the cytoplasmic symbiont Wolbachia, and the longevity of Indy males disappeared after tetracycline clearance of this endosymbiont. These findings underscore the critical importance of standardisation of genetic background and of cytoplasm in genetic studies of lifespan, and show that the lifespan extension previously claimed for Indy mutants was entirely attributable to confounding variation from these two sources. In addition, we saw no effects on lifespan of expression knockdown of the Indy orthologues nac-2 and nac-3 in the nematode Caenorhabditis elegans.

Correcting for Sequence Biases in Present/absent Calls

The probe sequence of short oligonucleotides in Affymetrix microarray experiments can have a significant influence on present/absent calls of probesets with absent target transcripts. Probesets enriched for central Ts and depleted of central As in the perfect-match probes tend to be falsely classified as having present transcripts. Correction of non-specific binding for both perfect-match and mismatch probes using probe-sequence models can partially remove the probe-sequence bias and result in better performance of the MAS 5.0 algorithm.

Estimation and Correction of Non-specific Binding in a Large-scale Spike-in Experiment

The availability of a recently published large-scale spike-in microarray dataset helps us to understand the influence of probe sequence in non-specific binding (NSB) signal and enables the benchmarking of several models for the estimation of NSB. In a typical microarray experiment using Affymetrix whole genome chips, 30% to 50% of the probes will apparently have absent target transcripts and show only NSB signal, and these probes can have significant repercussions for normalization and the statistical analysis of the data if NSB is not estimated correctly.

Mapping Regions Within Cosmopolitan Inversion In(3R)Payne Associated with Natural Variation in Body Size in Drosophila Melanogaster

Associations between genotypes for inversions and quantitative traits have been reported in several organisms, but little has been done to localize regions within inversions controlling variation in these traits. Here, we use an association mapping technique to identify genomic regions controlling variation in wing size within the cosmopolitan inversion In(3R)Payne in Drosophila melanogaster. Previous studies have shown that this inversion strongly influences variation in wing size, a trait highly correlated with body size. We found three alleles from two separate regions within In(3R)Payne with significant additive effects on wing size after the additional effect of the inversion itself had been taken into account. There were also several alleles with significant genotype-by-inversion interaction effects on wing size. None of the alleles tested had a significant additive effect on development time, suggesting different genes control these traits and that clinal patterns in them have therefore arisen independently. The presence of multiple regions within In(3R)Payne controlling size is consistent with the idea that inversions persist in populations because they contain multiple sets of locally adapted alleles, but more work needs to be done to test if they are indeed coadapted.

Evolutionary Conservation of Regulated Longevity Assurance Mechanisms

To what extent are the determinants of aging in animal species universal? Insulin/insulin-like growth factor (IGF)-1 signaling (IIS) is an evolutionarily conserved (public) regulator of longevity; yet it remains unclear whether the genes and biochemical processes through which IIS acts on aging are public or private (that is, lineage specific). To address this, we have applied a novel, multi-level cross-species comparative analysis to compare gene expression changes accompanying increased longevity in mutant nematodes, fruitflies and mice with reduced IIS.

Some Highlights of Research on Aging with Invertebrates, 2006-2007

The invertebrate model organisms continue to be engines of discovery in aging research. Recent work with Drosophila stem cells has thrown light on their human equivalents, and on the role of stem cells and their niches in the decline in fecundity with age. Inspired by observations of aging in bacteria and yeast, a new theoretical study has revealed evolutionary forces that could favour asymmetry in the distribution of damaged cell constituents at division, and hence pave the way for the evolution of aging and selective maintenance of integrity of the germ line. Mechanisms of nutrient sensing and cell signalling in the response of lifespan to dietary restriction have been elucidated. Powerful invertebrate models of human aging-related disease have been produced, and used to start to understand how the aging process acts as a risk factor for disease. In the near future, studies of invertebrate aging are likely to move away from an exclusive reliance on genetic manipulation towards a more biochemical and physiological understanding of these systems.

Antagonizing Methuselah to Extend Life Span

A recent report describes the identification through the use of in vitro selection of a peptide that antagonizes Methuselah signaling in Drosophila in vitro and extends fly life span in vivo.

Effects of Resveratrol on Lifespan in Drosophila Melanogaster and Caenorhabditis Elegans

It was recently reported that the plant polyphenol resveratrol, found, e.g., in grape berry skins, extended lifespan in the fruit fly Drosophila melanogaster and the nematode worm Caenorhabditis elegans. This lifespan extension was dependent on an NAD(+)-dependent histone deacetylase, Sir2 in Drosophila and SIR-2.1 in C. elegans. The extension of lifespan appeared to occur through a mechanism related to dietary restriction (DR), the reduction of available nutrients without causing malnutrition, an intervention that extends lifespan in diverse organisms from yeast to mammals. In Drosophila, lifespan extension by DR is associated with a reduction in fecundity. However, a slight increase in fecundity was reported upon treatment with resveratrol, suggesting a mode of action at least partially distinct from that of DR. To probe this mechanism further, we initiated a new study of the effects of resveratrol on Drosophila. We saw no significant effects on lifespan in seven independent trials. We analysed our resveratrol and found that its structure was normal, with no oxidative modifications. We therefore re-tested the effects of resveratrol in C. elegans, in both wild-type and sir-2.1 mutant worms. The results were variable, with resveratrol treatment resulting in slight increases in lifespan in some trials but not others, in both wild type and sir-2.1 mutant animals. We postulate that the effect of resveratrol upon lifespan in C. elegans could reflect induction of phase 2 drug detoxification or activation of AMP kinase.

Optimization of Dietary Restriction Protocols in Drosophila

Dietary restriction (DR) extends life span in many organisms, through unknown mechanisms that may or may not be evolutionarily conserved. Because different laboratories use different diets and techniques for implementing DR, the outcomes may not be strictly comparable. This complicates intra- and interspecific comparisons of the mechanisms of DR and is therefore central to the use of model organisms to research this topic. Drosophila melanogaster is an important model for the study of DR, but the nutritional content of its diet is typically poorly defined. We have compared fly diets composed of different yeasts for their effect on life span and fecundity. We found that only one diet was appropriate for DR experiments, indicating that much of the published work on fly "DR" may have included adverse effects of food composition. We propose procedures to ensure that diets are suitable for the study of DR in Drosophila.

Benchmarks for Ageing Studies

Brief Carbon Dioxide Exposure Blocks Heat Hardening but Not Cold Acclimation in Drosophila Melanogaster

Carbon dioxide is a commonly used anaesthetic in Drosophila research. While any detrimental effects of CO2 exposure on behaviour or traits are largely unknown, a recent study observed significant effects of CO2 exposure on rapid cold hardening and chill-coma recovery in Drosophila melanogaster. In this study we investigated the effect of a brief CO2 exposure on heat hardening and cold acclimation in D. melanogaster, measuring heat knockdown and chill-coma recovery times of flies exposed to CO2 for 1 min after hardening or acclimation. CO2 anaesthesia had a significant negative effect on heat hardening, with heat knockdown rates in hardened flies completely reduced to those of controls after CO2 exposure. Chill-coma recovery rates also significantly increased in acclimated flies that were exposed to CO2, although not to the same extent seen in the heat populations. CO2 exposure had no impact on heat knockdown rates of control flies, while there was a significant negative effect of the anaesthetic on chill-coma recovery rates of control flies. In light of these results, we suggest that CO2 should not be used after hardening in heat resistance assays due to the complete reversal of the heat hardening process upon exposure to CO2.

Evidence for Lifespan Extension and Delayed Age-related Biomarkers in Insulin Receptor Substrate 1 Null Mice

Recent evidence suggests that alterations in insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) can increase mammalian life span. For example, in several mouse mutants, impairment of the growth hormone (GH)/IGF1 axis increases life span and also insulin sensitivity. However, the intracellular signaling route to altered mammalian aging remains unclear. We therefore measured the life span of mice lacking either insulin receptor substrate (IRS) 1 or 2, the major intracellular effectors of the IIS receptors. Our provisional results indicate that female Irs1-/- mice are long-lived. Furthermore, they displayed resistance to a range of age-sensitive markers of aging including skin, bone, immune, and motor dysfunction. These improvements in health were seen despite mild, lifelong insulin resistance. Thus, enhanced insulin sensitivity is not a prerequisite for IIS mutant longevity. Irs1-/- female mice also displayed normal anterior pituitary function, distinguishing them from long-lived somatotrophic axis mutants. In contrast, Irs2-/- mice were short-lived, whereas Irs1+/- and Irs2+/- mice of both sexes showed normal life spans. Our results therefore suggest that IRS1 signaling is an evolutionarily conserved pathway regulating mammalian life span and may be a point of intervention for therapies with the potential to delay age-related processes.

Role of DFOXO in Lifespan Extension by Dietary Restriction in Drosophila Melanogaster: Not Required, but Its Activity Modulates the Response

Dietary restriction (DR) increases lifespan in diverse organisms. However, the mechanisms by which DR increases survival are unclear. The insulin/IGF-like signaling (IIS) pathway has been implicated in the response to DR in some studies, while in others it has appeared to play little or no role. We used the fruitfly Drosophila melanogaster to investigate the responses to DR of flies mutant for the transcription factor dFOXO, the main transcription factor target of IIS. We found that lifespan extension by DR does not require dFOXO. However, flies with dFOXO overexpressed in the adult fat body showed an altered response to DR and behaved as though partially dietarily restricted. These results suggest that, although DR extends lifespan of flies in the absence of dFOXO, the presence of active dFOXO modulates the response to DR, possibly by modifying expression of its target genes, and may therefore mediate the normal response to DR.

Pitfalls of Measuring Feeding Rate in the Fruit Fly Drosophila Melanogaster

Stress-response Hormesis and Aging: "that Which Does Not Kill Us Makes Us Stronger"

Hormesis refers to the beneficial effects of a treatment that at a higher intensity is harmful. In one form of hormesis, sublethal exposure to stressors induces a response that results in stress resistance. The principle of stress-response hormesis is increasingly finding application in studies of aging, where hormetic increases in life span have been seen in several animal models.

Toward a Control Theory Analysis of Aging

Aging is due to the accumulation of damage over time that affects the function and survival of the organism; however, it has proven difficult to infer the relative importance of the many processes that contribute to aging. To address this, here we outline an approach that may prove useful in analyzing aging. In this approach, the function of the organism is described as a set of interacting physiological systems. Degradation of their outputs leads to functional decline and death as a result of aging. In turn, degradation of the system outputs is attributable to changes at the next hierarchical level down, the cell, through changes in cell number or function, which are in turn a consequence of the metabolic history of the cell. Within this framework, we then adapt the methods of metabolic control analysis (MCA) to determine which modifications are important for aging. This combination of a hierarchical framework and the methodologies of MCA may prove useful both for thinking about aging and for analyzing it experimentally.

Feeding, Fecundity and Lifespan in Female Drosophila Melanogaster

Male seminal fluid proteins induce a profound remodelling of behavioural, physiological and gene signalling pathways in females of many taxa, and typically cause elevated egg production and decreased sexual receptivity. In Drosophila melanogaster, these effects can be mediated by an ejaculate 'sex peptide' (SP), which, in addition, contributes significantly to the cost of mating in females. Recent research has revealed that SP can stimulate female post-copulatory feeding, raising the possibility that the widespread female cost of mating could be due to over-feeding. In this study, we used D. melanogaster as a model to test this hypothesis. We first show that elevated post-mating feeding is dependent upon egg production and does not occur in sterile ovoD1 mutant females. This conclusion was also supported by the increase in feeding of virgin females whose egg production was experimentally elevated. We then demonstrated that sterile ovoD1 and fertile females experienced identical survival costs of mating, related to their frequency of mating and not to female feeding rate or to egg production. We conclude that female mating costs are not the result of over-feeding, but may be due to other, potentially more direct, effects of ejaculate molecules.

Comment on "Brain IRS2 Signaling Coordinates Life Span and Nutrient Homeostasis"

Taguchi et al. (Reports, 20 July 2007, p. 369) reported that mice heterozygous for a null mutation in insulin receptor substrate-2 (Irs2) display a 17% increase in median life span. However, using the same mouse model, we find no evidence for life-span extension and suggest that the findings of Taguchi et al. were due to atypical life-span profiles in their study animals.

New Model of Health Promotion and Disease Prevention for the 21st Century

Stage Debut for the Elusive Drosophila Insulin-like Growth Factor Binding Protein

Insulin-like growth factor (IGF) binding proteins provide a layer of complexity to the insulin/IGF signaling system in mammals, but only now, in a recent study in Journal of Biology, has one such protein been functionally characterized in Drosophila.

Some Highlights of Research on Aging with Invertebrates, 2008

This annual review focuses on invertebrate model organisms, which shed light on new mechanisms in aging and provide excellent systems for in-depth analysis. This year, the first quantitative estimate of evolutionary conservation of genetic effects on lifespan has pointed to the key importance of genes involved in protein synthesis, a finding confirmed and extended by experimental work. Work in Caenorhabditis elegans and Drosophila has highlighted the importance of phase 2 detoxification in extension of lifespan by reduced insulin/Igf-like signalling. Thorough characterization of systems for dietary restriction in C. elegans is starting to show differences in the mechanisms by which these interventions extend lifespan and has revealed a requirement for autophagy. The response to heat shock in C. elegans turns out to be systemic, and mediated by sensory neurons, with potentially interesting implications for the response of lifespan to temperature. Work in Escherichia coli and yeast has revealed a role for retention of aggregated proteins in the parent in the rejuvenation of offspring while, as in C. elegans, removal of the germ line in Drosophila turns out to extend lifespan. Aging research has suffered the loss of a great scientific leader, Seymour Benzer, and his trail-blazing work on aging and neurodegeneration is highlighted.

Reduction of DILP2 in Drosophila Triages a Metabolic Phenotype from Lifespan Revealing Redundancy and Compensation Among DILPs

The insulin/IGF-like signalling (IIS) pathway has diverse functions in all multicellular organisms, including determination of lifespan. The seven insulin-like peptides (DILPs) in Drosophila are expressed in a stage- and tissue-specific manner. Partial ablation of the median neurosecretory cells (mNSCs) in the brain, which produce three DILPs, extends lifespan, reduces fecundity, alters lipid and carbohydrate metabolism and increases oxidative stress resistance. To determine if reduced expression of DILPs is causal in these effects, and to investigate possible functional diversification and redundancy between DILPs, we used RNA interference to lower specifically the transcript and protein levels of dilp2, the most highly expressed of the mNSC-derived DILPs. We found that DILP2 was limiting only for the increased whole-body trehalose content associated with mNSC-ablation. We observed a compensatory increase in dilp3 and 5 mRNA upon dilp2 knock down. By manipulation of dfoxo and dInR, we showed that the increase in dilp3 is regulated via autocrine insulin signaling in the mNSCs. Our study demonstrates that, despite the correlation between reduced dilp2 mRNA levels and lifespan-extension often observed, DILP2 reduction is not sufficient to extend lifespan. Nor is the increased trehalose storage associated with reduced IIS sufficient to extend lifespan. To understand the normal regulation of expression of the dilps and any functional diversification between them will require independent control of the expression of different dilps.

Endocrine Regulation of Aging and Reproduction in Drosophila

Hormonal signals can modulate lifespan and reproductive capacity across the animal kingdom. The use of model organisms such as worms, flies and mice has been fundamentally important for aging research in the discovery of genetic alterations that can extend healthy lifespan. The effects of mutations in the insulin and insulin-like growth factor-like signaling (IIS) pathways are evolutionarily conserved in that they can increase lifespan in all three animal models. Additionally, steroids and other lipophilic signaling molecules modulate lifespan in diverse organisms. Here we shall review how major hormonal pathways in the fruit fly Drosophila melanogaster interact to influence reproductive capacity and aging.

Effect of a Standardised Dietary Restriction Protocol on Multiple Laboratory Strains of Drosophila Melanogaster

Outcomes of lifespan studies in model organisms are particularly susceptible to variations in technical procedures. This is especially true of dietary restriction, which is implemented in many different ways among laboratories.

Insulin/IGF-like Signalling, the Central Nervous System and Aging

Enormous strides in understanding aging have come from the discovery that mutations in single genes can extend healthy life-span in laboratory model organisms such as the yeast Saccharomyces, the fruit fly Drosophila melanogaster, the nematode worm Caenorhabditis elegans and the mouse. IIS [insulin/IGF (insulin-like growth factor)-like signalling] stands out as an important, evolutionarily conserved pathway involved in the determination of lifespan. The pathway has diverse functions in multicellular organisms, and mutations in IIS can affect growth, development, metabolic homoeostasis, fecundity and stress resistance, as well as lifespan. The pleiotropic nature of the pathway and the often negative effects of its disruption mean that the extent, tissue and timing of IIS manipulations are determinants of a positive effect on lifespan. One tissue of particular importance for lifespan extension in diverse organisms is the CNS (central nervous system). Although lowered IIS in the CNS can extend lifespan, IIS is also widely recognized as being neuroprotective and important for growth and survival of neurons. In the present review, we discuss our current understanding of the role of the nervous system in extension of lifespan by altered IIS, and the role of IIS in determination of neuronal function during aging. The nervous system can play both endocrine and cell-autonomous roles in extension of lifespan by IIS, and the effects of IIS on lifespan and neuronal function can be uncoupled to some extent. Tissue-specific manipulation of IIS and the cellular defence mechanisms that it regulates will better define the ways in which IIS affects neuronal and whole-organism function during aging.

Expression of Human Uncoupling Protein-3 in Drosophila Insulin-producing Cells Increases Insulin-like Peptide (DILP) Levels and Shortens Lifespan

Uncoupling proteins (UCPs) can dissipate mitochondrial protonmotive force by increasing the proton conductance of the inner membrane and through this effect could decrease ROS production, ameliorate oxidative stress and extend lifespan. We investigated whether ubiquitous, pan-neuronal or neurosecretory cell-specific expression of human UCP3 (hUCP3) in adult Drosophila melanogaster affected lifespan. Low, ubiquitous expression of hUCP3 at levels found in rodent skeletal muscle mitochondria did not affect proton conductance in mitochondria isolated from whole flies, but high pan-neuronal expression of hUCP3 increased the proton conductance of mitochondria isolated from fly heads. Expression of hUCP3 at moderate levels in adult neurons led to a marginal lifespan-extension in males. However, high expression of hUCP3 in neuronal tissue shortened lifespan. The life-shortening effect was replicated when hUCP3 was expressed specifically in median neurosecretory cells (mNSC), which express three of the Drosophila insulin-like peptides (DILPs). Expression of hUCP3 in the mNSC did not alter expression of dilp2, dilp3 or dilp5 mRNA, but led to increased amounts of DILP2 in fly heads. These data suggest that lowering mitochondrial coupling by high expression of hUCP3 alters mNSC function in a way that appears to increase DILP-levels in fly heads and lead to a concomitant decrease in lifespan.

Longevity of Indy Mutant Drosophila Not Attributable to Indy Mutation

Quantification of Food Intake in Drosophila

Measurement of food intake in the fruit fly Drosophila melanogaster is often necessary for studies of behaviour, nutrition and drug administration. There is no reliable and agreed method for measuring food intake of flies in undisturbed, steady state, and normal culture conditions. We report such a method, based on measurement of feeding frequency by proboscis-extension, validated by short-term measurements of food dye intake. We used the method to demonstrate that (a) female flies feed more frequently than males, (b) flies feed more often when housed in larger groups and (c) fly feeding varies at different times of the day. We also show that alterations in food intake are not induced by dietary restriction or by a null mutation of the fly insulin receptor substrate chico. In contrast, mutation of takeout increases food intake by increasing feeding frequency while mutation of ovo(D) increases food intake by increasing the volume of food consumed per proboscis-extension. This approach provides a practical and reliable method for quantification of food intake in Drosophila under normal, undisturbed culture conditions.

Some Highlights of Research on Aging with Invertebrates, 2009

This annual review focuses on invertebrate model organisms, which shed light on new mechanisms in aging and provide excellent systems for both genome-wide and in-depth analysis. This year, protein interaction networks have been used in a new bioinformatic approach to identify novel genes that extend replicative lifespan in yeast. In an extended approach, using a new, human protein interaction network, information from the invertebrates was used to identify new, candidate genes for lifespan extension and their orthologues were validated in the nematode Caenorhabditis elegans. Chemosensation of diffusible substances from bacteria has been shown to limit lifespan in C. elegans, while a systematic study of the different methods used to implement dietary restriction in the worm has shown that they involve mechanisms that are partially distinct and partially overlapping, providing important clarification for addressing whether or not they are conserved in other organisms. A new theoretical model for the evolution of rejuvenating cell division has shown that asymmetrical division for either cell size or for damaged cell constituents results in increased fitness for most realistic levels of cellular protein damage. Work on aging-related disease has both refined our understanding of the mechanisms underlying one route to the development of Parkinson's disease and has revealed that in worms, as in mice, dietary restriction is protective against cellular proteotoxicity. Two systematic studies genetically manipulating the superoxide dismutases of C. elegans support the idea that damage from superoxide plays little or no role in aging in this organism, and have prompted discussion of other kinds of damage and other kinds of mechanisms for producing aging-related decline in function.

Invertebrate Models of Age-related Muscle Degeneration

Functional and structural deterioration of muscles is an inevitable consequence of ageing in a wide variety of animal species. What underlies these changes is a complex network of interactions between the muscle-intrinsic and muscle-extrinsic factors, making it very difficult to distinguish between the cause and the consequence. Many of the genes, structures, and processes implicated in mammalian skeletal muscle ageing are preserved in invertebrate species Drosophila melanogaster and Caenorhabditis elegans. The absence in these organisms of mechanisms that promote muscle regeneration, and substantially different hormonal environment, warrant caution when extrapolating experimental data from studies conducted in invertebrates to mammalian species. The simplicity and accessibility of these models, however, offer ample opportunities for studying age-related myopathologies as well as investigating drugs and therapies to alleviate them.

Chemical Changes in Aging Drosophila Melanogaster

The “Green Theory” of aging proposes that organismal lifespan is limited by the failure to repair molecular damage generated by a broad range of metabolic processes. Two specific predictions arise from this: (1) that these processes will produce a wide variety of stable but dysfunctional compounds that increase in concentration with age, and (2) that organisms maintained under conditions that extend lifespan will display a reduced rate of accumulation of such “molecular rubbish”. To test these predictions, novel analytical techniques were developed to investigate the accumulation of damaged compounds in Drosophila melanogaster. Simple preparative techniques were developed to produce digests of whole D. melanogaster for use in three-dimensional (3D) fluorimetry and 1H NMR spectrometry. Cohorts of Drosophila maintained under normal conditions showed an age-related increase in signals consistent with damage whereas those maintained under conditions of low temperature and dietary restriction did not. 1H NMR revealed distinct age-associated spectral changes that will facilitate the identification of novel compounds that both increase and decrease during aging in this species. These findings are consistent with the predictions of the “Green Theory”.

The Endosymbiont Wolbachia Increases Insulin/IGF-like Signalling in Drosophila

Insulin/IGF-like signalling (IIS) is an evolutionarily conserved pathway that has diverse functions in multi-cellular organisms. Mutations that reduce IIS can have pleiotropic effects on growth, development, metabolic homeostasis, fecundity, stress resistance and lifespan. IIS is also modified by extrinsic factors. For instance, in the fruitfly Drosophila melanogaster, both nutrition and stress can alter the activity of the pathway. Here, we test experimentally the hypothesis that a widespread endosymbiont of arthropods, Wolbachia pipientis, can alter the degree to which mutations in genes encoding IIS components affect IIS and its resultant phenotypes. Wolbachia infection, which is widespread in D. melanogaster in nature and has been estimated to infect 30 per cent of strains in the Bloomington stock centre, can affect broad aspects of insect physiology, particularly traits associated with reproduction. We measured a range of IIS-related phenotypes in flies ubiquitously mutant for IIS in the presence and absence of Wolbachia. We show that removal of Wolbachia further reduces IIS and hence enhances the mutant phenotypes, suggesting that Wolbachia normally acts to increase insulin signalling. This effect of Wolbachia infection on IIS could have an evolutionary explanation, and has some implications for studies of IIS in Drosophila and other organisms that harbour endosymbionts.

Ribosomal Protein S6 Kinase 1 Signaling Regulates Mammalian Life Span

Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.

Amino-acid Imbalance Explains Extension of Lifespan by Dietary Restriction in Drosophila

Dietary restriction extends healthy lifespan in diverse organisms and reduces fecundity. It is widely assumed to induce adaptive reallocation of nutrients from reproduction to somatic maintenance, aiding survival of food shortages in nature. If this were the case, long life under dietary restriction and high fecundity under full feeding would be mutually exclusive, through competition for the same limiting nutrients. Here we report a test of this idea in which we identified the nutrients producing the responses of lifespan and fecundity to dietary restriction in Drosophila. Adding essential amino acids to the dietary restriction condition increased fecundity and decreased lifespan, similar to the effects of full feeding, with other nutrients having little or no effect. However, methionine alone was necessary and sufficient to increase fecundity as much as did full feeding, but without reducing lifespan. Reallocation of nutrients therefore does not explain the responses to dietary restriction. Lifespan was decreased by the addition of amino acids, with an interaction between methionine and other essential amino acids having a key role. Hence, an imbalance in dietary amino acids away from the ratio optimal for reproduction shortens lifespan during full feeding and limits fecundity during dietary restriction. Reduced activity of the insulin/insulin-like growth factor signalling pathway extends lifespan in diverse organisms, and we find that it also protects against the shortening of lifespan with full feeding. In other organisms, including mammals, it may be possible to obtain the benefits to lifespan of dietary restriction without incurring a reduction in fecundity, through a suitable balance of nutrients in the diet.

A Drosophila Insulin-like Peptide Promotes Growth During Nonfeeding States

In metazoans, tissue growth relies on the availability of nutrients--stored internally or obtained from the environment--and the resulting activation of insulin/IGF signaling (IIS). In Drosophila, growth is mediated by seven Drosophila insulin-like peptides (Dilps), acting through a canonical IIS pathway. During the larval period, animals feed and Dilps produced by the brain couple nutrient uptake with systemic growth. We show here that, during metamorphosis, when feeding stops, a specific DILP (Dilp6) is produced by the fat body and relays the growth signal. Expression of DILP6 during pupal development is controlled by the steroid hormone ecdysone. Remarkably, DILP6 expression is also induced upon starvation, and both its developmental and environmental expression require the Drosophila FoxO transcription factor. This study reveals a specific class of ILPs induced upon metabolic stress that promotes growth in conditions of nutritional deprivation or following developmentally induced cessation of feeding.

The New Biology of Ageing

Human life expectancy in developed countries has increased steadily for over 150 years, through improvements in public health and lifestyle. More people are hence living long enough to suffer age-related loss of function and disease, and there is a need to improve the health of older people. Ageing is a complex process of damage accumulation, and has been viewed as experimentally and medically intractable. This view has been reinforced by the realization that ageing is a disadvantageous trait that evolves as a side effect of mutation accumulation or a benefit to the young, because of the decline in the force of natural selection at later ages. However, important recent discoveries are that mutations in single genes can extend lifespan of laboratory model organisms and that the mechanisms involved are conserved across large evolutionary distances, including to mammals. These mutations keep the animals functional and pathology-free to later ages, and they can protect against specific ageing-related diseases, including neurodegenerative disease and cancer. Preliminary indications suggest that these new findings from the laboratory may well also apply to humans. Translating these discoveries into medical treatments poses new challenges, including changing clinical thinking towards broad-spectrum, preventative medicine and finding novel routes to drug development.

Mechanisms of Life Span Extension by Rapamycin in the Fruit Fly Drosophila Melanogaster

The target of rapamycin (TOR) pathway is a major nutrient-sensing pathway that, when genetically downregulated, increases life span in evolutionarily diverse organisms including mammals. The central component of this pathway, TOR kinase, is the target of the inhibitory drug rapamycin, a highly specific and well-described drug approved for human use. We show here that feeding rapamycin to adult Drosophila produces the life span extension seen in some TOR mutants. Increase in life span by rapamycin was associated with increased resistance to both starvation and paraquat. Analysis of the underlying mechanisms revealed that rapamycin increased longevity specifically through the TORC1 branch of the TOR pathway, through alterations to both autophagy and translation. Rapamycin could increase life span of weak insulin/Igf signaling (IIS) pathway mutants and of flies with life span maximized by dietary restriction, indicating additional mechanisms.

DILP-producing Median Neurosecretory Cells in the Drosophila Brain Mediate the Response of Lifespan to Nutrition

Dietary restriction extends lifespan in diverse organisms, but the gene regulatory mechanisms and tissues mediating the increased survival are still unclear. Studies in worms and flies have revealed a number of candidate mechanisms, including the target of rapamycin and insulin/IGF-like signalling (IIS) pathways and suggested a specific role for the nervous system in mediating the response. A pair of sensory neurons in Caenorhabditis elegans has been found to specifically mediate DR lifespan extension, but a neuronal focus in the Drosophila nervous system has not yet been identified. We have previously shown that reducing IIS via the partial ablation of median neurosecretory cells in the Drosophila adult brain, which produce three of the seven fly insulin-like peptides, extends lifespan. Here, we show that these cells are required to mediate the response of lifespan to full feeding in a yeast dilution DR regime and that they appear to do so by mechanisms that involve both altered IIS and other endocrine effects. We also present evidence of an interaction between these mNSCs, nutrition and sleep, further emphasising the functional homology between the DILP-producing neurosecretory cells in the Drosophila brain and the hypothalamus of mammals in their roles as integration sites of many inputs for the control of lifespan and behaviour.

Molecular Evolution and Functional Characterization of Drosophila Insulin-like Peptides

Multicellular animals match costly activities, such as growth and reproduction, to the environment through nutrient-sensing pathways. The insulin/IGF signaling (IIS) pathway plays key roles in growth, metabolism, stress resistance, reproduction, and longevity in diverse organisms including mammals. Invertebrate genomes often contain multiple genes encoding insulin-like ligands, including seven Drosophila insulin-like peptides (DILPs). We investigated the evolution, diversification, redundancy, and functions of the DILPs, combining evolutionary analysis, based on the completed genome sequences of 12 Drosophila species, and functional analysis, based on newly-generated knock-out mutations for all 7 dilp genes in D. melanogaster. Diversification of the 7 DILPs preceded diversification of Drosophila species, with stable gene diversification and family membership, suggesting stabilising selection for gene function. Gene knock-outs demonstrated both synergy and compensation of expression between different DILPs, notably with DILP3 required for normal expression of DILPs 2 and 5 in brain neurosecretory cells and expression of DILP6 in the fat body compensating for loss of brain DILPs. Loss of DILP2 increased lifespan and loss of DILP6 reduced growth, while loss of DILP7 did not affect fertility, contrary to its proposed role as a Drosophila relaxin. Importantly, loss of DILPs produced in the brain greatly extended lifespan but only in the presence of the endosymbiontic bacterium Wolbachia, demonstrating a specific interaction between IIS and Wolbachia in lifespan regulation. Furthermore, loss of brain DILPs blocked the responses of lifespan and fecundity to dietary restriction (DR) and the DR response of these mutants suggests that IIS extends lifespan through mechanisms that both overlap with those of DR and through additional mechanisms that are independent of those at work in DR. Evolutionary conservation has thus been accompanied by synergy, redundancy, and functional differentiation between DILPs, and these features may themselves be of evolutionary advantage.

Regulation of Lifespan, Metabolism, and Stress Responses by the Drosophila SH2B Protein, Lnk

Drosophila Lnk is the single ancestral orthologue of a highly conserved family of structurally-related intracellular adaptor proteins, the SH2B proteins. As adaptors, they lack catalytic activity but contain several protein-protein interaction domains, thus playing a critical role in signal transduction from receptor tyrosine kinases to form protein networks. Physiological studies of SH2B function in mammals have produced conflicting data. However, a recent study in Drosophila has shown that Lnk is an important regulator of the insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway during growth, functioning in parallel to the insulin receptor substrate, Chico. As this pathway also has an evolutionary conserved role in the determination of organism lifespan, we investigated whether Lnk is required for normal lifespan in Drosophila. Phenotypic analysis of mutants for Lnk revealed that loss of Lnk function results in increased lifespan and improved survival under conditions of oxidative stress and starvation. Starvation resistance was found to be associated with increased metabolic stores of carbohydrates and lipids indicative of impaired metabolism. Biochemical and genetic data suggest that Lnk functions in both the IIS and Ras/Mitogen activated protein Kinase (MapK) signaling pathways. Microarray studies support this model, showing transcriptional feedback onto genes in both pathways as well as indicating global changes in both lipid and carbohydrate metabolism. Finally, our data also suggest that Lnk itself may be a direct target of the IIS responsive transcription factor, dFoxo, and that dFoxo may repress Lnk expression. We therefore describe novel functions for a member of the SH2B protein family and provide the first evidence for potential mechanisms of SH2B regulation. Our findings suggest that IIS signaling in Drosophila may require the activity of a second intracellular adaptor, thereby yielding fundamental new insights into the functioning and role of the IIS pathway in ageing and metabolism.

Biomarkers of Aging in Drosophila

Low environmental temperature and dietary restriction (DR) extend lifespan in diverse organisms. In the fruit fly Drosophila, switching flies between temperatures alters the rate at which mortality subsequently increases with age but does not reverse mortality rate. In contrast, DR acts acutely to lower mortality risk; flies switched between control feeding and DR show a rapid reversal of mortality rate. Dietary restriction thus does not slow accumulation of aging-related damage. Molecular species that track the effects of temperatures on mortality but are unaltered with switches in diet are therefore potential biomarkers of aging-related damage. However, molecular species that switch upon instigation or withdrawal of DR are thus potential biomarkers of mechanisms underlying risk of mortality, but not of aging-related damage. Using this approach, we assessed several commonly used biomarkers of aging-related damage. Accumulation of fluorescent advanced glycation end products (AGEs) correlated strongly with mortality rate of flies at different temperatures but was independent of diet. Hence, fluorescent AGEs are biomarkers of aging-related damage in flies. In contrast, five oxidized and glycated protein adducts accumulated with age, but were reversible with both temperature and diet, and are therefore not markers either of acute risk of dying or of aging-related damage. Our approach provides a powerful method for identification of biomarkers of aging.

Extending Healthy Life Span--from Yeast to Humans

When the food intake of organisms such as yeast and rodents is reduced (dietary restriction), they live longer than organisms fed a normal diet. A similar effect is seen when the activity of nutrient-sensing pathways is reduced by mutations or chemical inhibitors. In rodents, both dietary restriction and decreased nutrient-sensing pathway activity can lower the incidence of age-related loss of function and disease, including tumors and neurodegeneration. Dietary restriction also increases life span and protects against diabetes, cancer, and cardiovascular disease in rhesus monkeys, and in humans it causes changes that protect against these age-related pathologies. Tumors and diabetes are also uncommon in humans with mutations in the growth hormone receptor, and natural genetic variants in nutrient-sensing pathways are associated with increased human life span. Dietary restriction and reduced activity of nutrient-sensing pathways may thus slow aging by similar mechanisms, which have been conserved during evolution. We discuss these findings and their potential application to prevention of age-related disease and promotion of healthy aging in humans, and the challenge of possible negative side effects.

Inhibition of GSK-3 Ameliorates Abeta Pathology in an Adult-onset Drosophila Model of Alzheimer's Disease

Abeta peptide accumulation is thought to be the primary event in the pathogenesis of Alzheimer's disease (AD), with downstream neurotoxic effects including the hyperphosphorylation of tau protein. Glycogen synthase kinase-3 (GSK-3) is increasingly implicated as playing a pivotal role in this amyloid cascade. We have developed an adult-onset Drosophila model of AD, using an inducible gene expression system to express Arctic mutant Abeta42 specifically in adult neurons, to avoid developmental effects. Abeta42 accumulated with age in these flies and they displayed increased mortality together with progressive neuronal dysfunction, but in the apparent absence of neuronal loss. This fly model can thus be used to examine the role of events during adulthood and early AD aetiology. Expression of Abeta42 in adult neurons increased GSK-3 activity, and inhibition of GSK-3 (either genetically or pharmacologically by lithium treatment) rescued Abeta42 toxicity. Abeta42 pathogenesis was also reduced by removal of endogenous fly tau; but, within the limits of detection of available methods, tau phosphorylation did not appear to be altered in flies expressing Abeta42. The GSK-3-mediated effects on Abeta42 toxicity appear to be at least in part mediated by tau-independent mechanisms, because the protective effect of lithium alone was greater than that of the removal of tau alone. Finally, Abeta42 levels were reduced upon GSK-3 inhibition, pointing to a direct role of GSK-3 in the regulation of Abeta42 peptide level, in the absence of APP processing. Our study points to the need both to identify the mechanisms by which GSK-3 modulates Abeta42 levels in the fly and to determine if similar mechanisms are present in mammals, and it supports the potential therapeutic use of GSK-3 inhibitors in AD.

Insulin Signalling Regulates Remating in Female Drosophila

Mating rate is a major determinant of female lifespan and fitness, and is predicted to optimize at an intermediate level, beyond which superfluous matings are costly. In female Drosophila melanogaster, nutrition is a key regulator of mating rate but the underlying mechanism is unknown. The evolutionarily conserved insulin/insulin-like growth factor-like signalling (IIS) pathway is responsive to nutrition, and regulates development, metabolism, stress resistance, fecundity and lifespan. Here we show that inhibition of IIS, by ablation of Drosophila insulin-like peptide (DILP)-producing median neurosecretory cells, knockout of dilp2, dilp3 or dilp5 genes, expression of a dominant-negative DILP-receptor (InR) transgene or knockout of Lnk, results in reduced female remating rates. IIS-mediated regulation of female remating can occur independent of virgin receptivity, developmental defects, reduced body size or fecundity, and the receipt of the female receptivity-inhibiting male sex peptide. Our results provide a likely mechanism by which females match remating rates to the perceived nutritional environment. The findings suggest that longevity-mediating genes could often have pleiotropic effects on remating rate. However, overexpression of the IIS-regulated transcription factor dFOXO in the fat body-which extends lifespan-does not affect remating rate. Thus, long life and reduced remating are not obligatorily coupled.

Ageing in Drosophila: the Role of the Insulin/Igf and TOR Signalling Network

A remarkable discovery of recent years is that, despite the complexity of ageing, simple genetic interventions can increase lifespan and improve health during ageing in laboratory animals. The pathways involved have often proved to sense nutrients and to match costly activities of organisms, such as growth, metabolism and reproduction, to nutrient status. For instance, the insulin/insulin-like growth factor and Target of Rapamycin signalling network has proved to play a function in ageing, from yeast to mammals, seemingly including humans. In the fruit fly Drosophila, altered activity of several components of this network can increase lifespan and improve locomotor and cardiac function during ageing. The fly brain, fat body (equivalent of mammalian liver and white adipose tissue) and the germ line are important in determination of lifespan, with considerable communication between different tissues. Cellular detoxification pathways, increased autophagy and altered protein synthesis have all been implicated in increased lifespan from reduced IIS/TOR activity, with the role of defence against oxidative stress unresolved. Reduced IIS/TOR signalling can alter or block the response of lifespan to dietary restriction. Reduced IIS can act acutely to lower death rate, implying that it may ameliorate the effects of ageing-related damage, rather than preventing it.

Some Highlights of Research on Aging with Invertebrates, 2010

This annual review focuses on invertebrate model organisms, which continue to yield fundamental new insights into mechanisms of aging. This year, the budding yeast has been used to understand how asymmetrical partitioning of cellular constituents at cell division can produce a rejuvenated offspring from an aging parent. Blocking of sensation of carbon dioxide is shown to extend fly lifespan and to mediate the lifespan-shortening effect of sensory exposure to fermenting yeast. A new study of daf-16, the key forkhead transcription factor that mediates extension of lifespan by mutants in the insulin-signalling pathway in Caenorhabditis elegans, demonstrates that expression of tissue-specific isoforms with different patterns of response to upstream signalling mediates the highly pleiotropic effects of the pathway on lifespan and other traits. A new approach to manipulating mitochondrial activity in Drosophila, by introducing the yeast NADH-ubiquinone oxidoreductase, shows promise for understanding the role of mitochondrial reactive oxygen species in aging. An exciting new study of yeast and mammalian cells implicates deterioration of the nuclear pore, and consequent leakage of cytoplasmic components into the nucleus, as an important cause of aging in postmitotic tissues. Loss of, or damage to, chromosome-associated histones is also implicated in the determination of lifespan in yeast, worms and fruit flies. The relationship between functional aging, susceptibility to aging-related disease and lifespan itself are explored in two studies in C. elegans, the first examining the role of dietary restriction and reduced insulin signalling in cognitive decline and the second profiling aggregation of the proteome during aging. The invertebrates continue to be a power house of discovery for future work in mammals.

Lifespan Extension by Increased Expression of the Drosophila Homologue of the IGFBP7 Tumour Suppressor

Mammals possess multiple insulin-like growth factor (IGF) binding proteins (IGFBPs), and related proteins, that modulate the activity of insulin/IGF signalling (IIS), a conserved neuroendocrine signalling pathway that affects animal lifespan. Here, we examine if increased levels of an IGFBP-like protein can extend lifespan, using Drosophila as the model organism. We demonstrate that Imaginal morphogenesis protein-Late 2 (IMP-L2), a secreted protein and the fly homologue of the human IGFBP7 tumour suppressor, is capable of binding at least two of the seven Drosophila insulin-like peptides (DILPs), namely native DILP2 and DILP5 as present in the adult fly. Increased expression of Imp-L2 results in phenotypic changes in the adult consistent with down-regulation of IIS, including accumulation of eIF-4E binding protein mRNA, increase in storage lipids, reduced fecundity and enhanced oxidative stress resistance. Increased Imp-L2 results in up-regulation of dilp2, dilp3 and dilp5 mRNA, revealing a feedback circuit that is mediated via the fly gut and/or fat body. Importantly, over-expression of Imp-L2, ubiquitous or restricted to DILP-producing cells or gut and fat body, extends lifespan. This enhanced longevity can also be observed upon adult-onset induction of Imp-L2, indicating it is not attributable to developmental changes. Our findings point to the possibility that an IGFBP or a related protein, such as IGFBP7, plays a role in mammalian aging.

The New Science of Ageing

Replication of Extended Lifespan Phenotype in Mice with Deletion of Insulin Receptor Substrate 1

We previously reported that global deletion of insulin receptor substrate protein 1 (Irs1) extends lifespan and increases resistance to several age-related pathologies in female mice. However, no effect on lifespan was observed in male Irs1 null mice. We suggested at the time that the lack of any effect in males might have been due to a sample size issue. While such lifespan studies are essential to our understanding of the aging process, they are generally based on survival curves derived from single experiments, primarily due to time and economic constraints. Consequently, the robustness of such findings as a basis for further investigation has been questioned. We have therefore measured lifespan in a second, separate cohort of Irs1 null female mice, and show that, consistent with our previous finding, global deletion of Irs1 significantly extends lifespan in female mice. In addition, an augmented and completed study demonstrates lifespan extension in male Irs1 null mice. Therefore, we show that reduced IRS1-dependent signalling is a robust mechanism through which mammalian lifespan can be modulated.

Measurement of H2O2 Within Living Drosophila During Aging Using a Ratiometric Mass Spectrometry Probe Targeted to the Mitochondrial Matrix

Hydrogen peroxide (H(2)O(2)) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H(2)O(2) in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H(2)O(2) levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H(2)O(2) to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H(2)O(2) that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H(2)O(2) with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H(2)O(2) correlates with aging, it may not be causative.

Molecular Basis of Adaptive Shift in Body Size in Drosophila Melanogaster: Functional and Sequence Analyses of the Dca Gene

Latitudinal body size clines in animals conforming to Bergmann's rule occur on many continents but isolating their underlying genetic basis remains a challenge. In Drosophila melanogaster, the gene Dca accounts for approximately 5-10% of the natural wing size variation (McKechnie SW, Blacket MJ, Song SV, Rako L, Carroll X, Johnson TK, Jensen LT, Lee SF, Wee CW, Hoffmann AA. 2010. A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila. Mol Ecol. 19:775-784). We present here functional evidence that Dca is a negative regulator of wing size. A significant negative latitudinal cline of Dca gene expression was detected in synchronized third instar larvae. In addition, we clarified the evolutionary history of the three most common Dca promoter alleles (Dca237-1, Dca237-2, and Dca247) and showed that the insertion allele (Dca247), whose frequency increases with latitude, is associated with larger wing centroid size and higher average cell number in male flies. Finally, we showed that the overall linkage disequilibrium (LD) was low in the Dca promoter and that the insertion/deletion polymorphism that defines the Dca alleles was in strong LD with two other upstream sites. Our results provide strong support that Dca is a candidate for climatic adaptation in D. melanogaster.

A Longer and Healthier Life with TOR Down-regulation: Genetics and Drugs

Genetic down-regulation of a major nutrient-sensing pathway, TOR (target of rapamycin) signalling, can improve health and extend lifespan in evolutionarily distant organisms such as yeast and mammals. Recently, it has been demonstrated that treatment with a pharmacological inhibitor of the TOR pathway, rapamycin, can replicate those findings and improve aging in a variety of model organisms. The proposed underlying anti-aging mechanisms are down-regulated translation, increased autophagy, altered metabolism and increased stress resistance.

DFOXO-independent Effects of Reduced Insulin-like Signaling in Drosophila

The insulin/insulin-like growth factor-like signaling (IIS) pathway in metazoans has evolutionarily conserved roles in growth control, metabolic homeostasis, stress responses, reproduction, and lifespan. Genetic manipulations that reduce IIS in the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the mouse have been shown not only to produce substantial increases in lifespan but also to ameliorate several age-related diseases. In C. elegans, the multitude of phenotypes produced by the reduction in IIS are all suppressed in the absence of the worm FOXO transcription factor, DAF-16, suggesting that they are all under common regulation. It is not yet clear in other animal models whether the activity of FOXOs mediate all of the physiological effects of reduced IIS, especially increased lifespan. We have addressed this issue by examining the effects of reduced IIS in the absence of dFOXO in Drosophila, using a newly generated null allele of dfoxo. We found that the removal of dFOXO almost completely blocks IIS-dependent lifespan extension. However, unlike in C. elegans, removal of dFOXO does not suppress the body size, fecundity, or oxidative stress resistance phenotypes of IIS-compromised flies. In contrast, IIS-dependent xenobiotic resistance is fully dependent on dFOXO activity. Our results therefore suggest that there is evolutionary divergence in the downstream mechanisms that mediate the effects of IIS. They also imply that in Drosophila, additional factors act alongside dFOXO to produce IIS-dependent responses in body size, fecundity, and oxidative stress resistance and that these phenotypes are not causal in IIS-mediated extension of lifespan.

Unraveling the Biological Roles of Reactive Oxygen Species

Reactive oxygen species are not only harmful agents that cause oxidative damage in pathologies, they also have important roles as regulatory agents in a range of biological phenomena. The relatively recent development of this more nuanced view presents a challenge to the biomedical research community on how best to assess the significance of reactive oxygen species and oxidative damage in biological systems. Considerable progress is being made in addressing these issues, and here we survey some recent developments for those contemplating research in this area.

Genome-wide DFOXO Targets and Topology of the Transcriptomic Response to Stress and Insulin Signalling

FoxO transcription factors, inhibited by insulin/insulin-like growth factor signalling (IIS), are crucial players in numerous organismal processes including lifespan. Using genomic tools, we uncover over 700 direct dFOXO targets in adult female Drosophila. dFOXO is directly required for transcription of several IIS components and interacting pathways, such as TOR, in the wild-type fly. The genomic locations occupied by dFOXO in adults are different from those observed in larvae or cultured cells. These locations remain unchanged upon activation by stresses or reduced IIS, but the binding is increased and additional targets activated upon genetic reduction in IIS. We identify the part of the IIS transcriptional response directly controlled by dFOXO and the indirect effects and show that parts of the transcriptional response to IIS reduction do not require dfoxo. Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the indirect and dfoxo-independent effects. We demonstrate genome-wide evolutionary conservation of dFOXO targets between the fly and the worm Caenorhabditis elegans, enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.

Dietary Restriction and Aging: a Unifying Perspective

Dietary restriction (DR) and mutations in nutrient signaling pathways can extend healthy life span in diverse organisms. Studying the interaction between these interventions should reveal mechanisms of aging, but has yielded some apparently contradictory results. A multidimensional representation of nutrition, called the geometric framework, can better describe the responses of life span and other traits, including metabolism, and can reconcile these apparent contradictions. We provide examples showing that it is more informative to analyze DR in terms of dietary balance and that dietary optimization for life span is critical for studies examining the biology of aging and other traits.

Death and Dessert: Nutrient Signalling Pathways and Ageing

Reduction in nutrient intake without malnutrition can delay ageing and extend healthy life in diverse organisms from yeast to primates. This effect can be recapitulated by genetic or pharmacological dampening of the signal through nutrient signalling pathways, making them a promising target for intervention into human ageing and age-related diseases. Here we review the current knowledge of the interactions between nutrient signalling pathways and ageing, focusing on the findings emerged in the past few years.

Absence of Effects of Sir2 Overexpression on Lifespan in C. Elegans and Drosophila

Overexpression of sirtuins (NAD(+)-dependent protein deacetylases) has been reported to increase lifespan in budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans and Drosophila melanogaster. Studies of the effects of genes on ageing are vulnerable to confounding effects of genetic background. Here we re-examined the reported effects of sirtuin overexpression on ageing and found that standardization of genetic background and the use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high-level sir-2.1 overexpression abrogated the longevity increase, but did not abrogate sir-2.1 overexpression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low-copy-number sir-2.1 overexpression also abrogated longevity. A Drosophila strain with ubiquitous overexpression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported, but was not long-lived relative to the appropriate transgenic controls, and nor was a new line with stronger overexpression of dSir2. These findings underscore the importance of controlling for genetic background and for the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life-extending effect of dietary restriction on ageing in Drosophila has also been reported to be dSir2 dependent. We found that dietary restriction increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects of sirtuins on lifespan in C. elegans and Drosophila.

The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial MRNAs in Drosophila Melanogaster

The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation.

Current Developments at Philosophical Transactions of the Royal Society B

A Double Whammy for Aging? Rapamycin Extends Lifespan and Inhibits Cancer in Inbred Female Mice

Comment on: Anisimov VN, et al. Cell Cycle 2011; 10:4230-6.

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