Variation in sleep and metabolic function is associated with latitude and average temperature in Drosophila melanogaster

Regulation of sleep and metabolic homeostasis is critical to an animal's survival and under stringent evolutionary pressure. Animals display remarkable diversity in sleep and metabolic phenotypes; however, an understanding of the ecological forces that select for, and maintain, these phenotypic differences remains poorly understood. The fruit fly, Drosophila melanogaster, is a powerful model for investigating the genetic regulation of sleep and metabolic function, and screening in inbred fly lines has led to the identification of novel genetic regulators of sleep. Nevertheless, little is known about the contributions of naturally occurring genetic differences to sleep, metabolic phenotypes, and their relationship with geographic or environmental gradients. Here, we quantified sleep and metabolic phenotypes in 24 D. melanogaster populations collected from diverse geographic localities. These studies reveal remarkable variation in sleep, starvation resistance, and energy stores. We found that increased sleep duration is associated with proximity to the equator and elevated average annual temperature, suggesting that environmental gradients strongly influence natural variation in sleep. Further, we found variation in metabolic regulation of sleep to be associated with free glucose levels, while starvation resistance associates with glycogen and triglyceride stores. Taken together, these findings reveal robust naturally occurring variation in sleep and metabolic traits in D. melanogaster, providing a model to investigate how evolutionary and ecological history modulate these complex traits.     

Energy Stores Are Not Altered by Long-Term Partial Sleep Deprivation in Drosophila melanogaster

Recent human studies reveal a widespread association between short sleep and obesity. Two hypotheses, which are not mutually exclusive, might explain this association. First, genetic factors that reduce endogenous sleep times might also impact energy stores, an assertion that we confirmed in a previous study. Second, metabolism may be altered by chronic partial sleep deprivation. Here we address the second assertion by measuring the impact of long-term partial sleep deprivation on energy stores using Drosophila as a model. We subjected flies to long-term partial sleep deprivation via two different methods: a mechanical stimulus and a light stimulus. We then measured whole-body triglycerides and glycogen, two important sources of energy for the fly, and compared them to un-stimulated controls. We also measured changes in energy stores in response to a random circadian clock shift. Sex and line-dependent alterations in glycogen and/or triglyceride levels occurred in response to the circadian clock shift and in flies subjected to a single night of sleep deprivation using light. Thus, consistent with previous studies, our findings suggest that acute sleep loss and changes to the circadian clock can alter metabolism. Significant changes in energy stores were also observed when flies were subjected to chronic sleep loss via the mechanical stimulus, although not the light stimulus. Interestingly, mechanical stimulation resulted in the same change in energy stores even when it was not associated with sleep deprivation, suggesting that the changes are caused by stress rather than sleep loss. These findings emphasize the importance of taking stress into account when evaluating the relationship between sleep loss and metabolism.     

The mate choice brain: comparing gene profiles between female choice and male coercive poeciliids

Genes that mediate mate preferences potentially play a key role in promoting and maintaining biological diversity. In this study, we compare mate preference behavior in two related poeciliid fishes with contrasting behavioral phenotypes and relate these behavioral differences to gene profiles in the brain. Results reveal that one poeciliid fish, the Northern swordtail, exhibits robust mate preference as compared to the Western mosquitofish, which utilizes a coercive mating system. Female swordtails display no significant difference in association time between male- and female-exposure trials, whereas female mosquitofish spend significantly less time associating with males relative to females. Furthermore, the preference strength for large males is significantly lower in female mosquitofish relative to swordtails. We then examine expression of three candidate genes previously shown to be associated with mate preference behavior in female swordtails and linked to neural plasticity in other vertebrates: neuroserpin (NS), neuroligin-3 (NLG-3) and N-methyl-d-aspartate receptor (NMDA-R). Whole brain gene expression patterns reveal that two genes (NS and NLG-3) are positively associated with mate preference behavior in female swordtails, a pattern opposing that of the mosquitofish. In mosquitofish females, these genes are downregulated when females express biases toward males yet are elevated in association with total motor activity patterns under asocial conditions, suggesting that the presence of males in mosquitofish species may inhibit expression of these genes. Both gene expression and female behavioral responses to males exhibit opposing patterns between these species, suggesting that this genetic pathway may potentially act as a substrate for the evolution of mate preference behavior.     

Quantitative Genetic Variation of Odor-Guided Behavior in a Natural Population of Drosophila Melanogaster

Quantitative genetic variation in behavioral response to the odorant, benzaldehyde, was assessed among a sample of 43 X and 35 third chromosomes extracted from a natural population and substituted into a common inbred background. Significant genetic variation among chromosome lines was detected. Heritability estimates for olfactory response, however, were low, as is typical for traits under natural selection. Furthermore, the loci affecting naturally occurring variation in olfactory response to benzaldehyde were not the same in males and females, since the genetic correlation between the sexes was low and not significantly different from zero for the chromosome 3 lines. Competitive fitness, viability and fertility of the chromosome 3 lines were estimated using the balancer equilibrium technique. Genetic correlations between fitness and odor-guided behavior were not significantly different from zero, suggesting the number of loci causing variation in olfactory response is small relative to the number of loci causing variation in fitness. Since different genes affect variation in olfactory response in males and females, genetic variation for olfactory response could be maintained by genotype X sex environment interaction. This unusual genetic architecture implies that divergent evolutionary trajectories for olfactory behavior may occur in males and females.     

A QTL analysis of female variation contributing to refractoriness and sperm competition in Drosophila melanogaster

Sperm competition is an important fitness component in many animal groups. Drosophila melanogaster males exhibit substantial genetic variation for sperm competitive ability and females show considerable genetic variation for first versus second male sperm use. Currently, the forces responsible for maintaining genetic variation in sperm competition related phenotypes are receiving much attention. While several candidate genes contributing to the variation seen in male competitive ability are known, genes involved in female sperm use remain largely undiscovered. Without knowledge of the underlying genes, it will be difficult to distinguish between different models of sexual selection such as cryptic female choice and sexual conflict. We used quantitative trait locus (QTL) mapping to identify regions of the genome contributing to female propensity to use first or second male sperm, female refractoriness to re-mating, and early-life fertility. The most well supported markers influencing the phenotypes include 33F/34A (P2), 57B (refractoriness) and 23F/24A (fertility). Between 10% and 15% of the phenotypic variance observed in these recombinant inbred lines was explained by these individual QTLs. More detailed investigation of the regions detected in this experiment may lead to the identification of genes responsible for the QTLs identified here.     

The Perilipin Homologue,Lipid Storage Droplet 2, Regulates Sleep Homeostasis and Prevents Learning Impairments Following Sleep Loss

Extended periods of waking result in physiological impairments in humans, rats, and flies. Sleep homeostasis, the increase in sleep observed following sleep loss, is believed to counter the negative effects of prolonged waking by restoring vital biological processes that are degraded during sleep deprivation. Sleep homeostasis, as with other behaviors, is influenced by both genes and environment. We report here that during periods of starvation, flies remain spontaneously awake but, in contrast to sleep deprivation, do not accrue any of the negative consequences of prolonged waking. Specifically, the homeostatic response and learning impairments that are a characteristic of sleep loss are not observed following prolonged waking induced by starvation. Recently, two genes, brummer (bmm) and Lipid storage droplet 2 (Lsd2), have been shown to modulate the response to starvation. bmm mutants have excess fat and are resistant to starvation, whereas Lsd2 mutants are lean and sensitive to starvation. Thus, we hypothesized that bmm and Lsd2 may play a role in sleep regulation. Indeed, bmm mutant flies display a large homeostatic response following sleep deprivation. In contrast, Lsd2 mutant flies, which phenocopy aspects of starvation as measured by low triglyceride stores, do not exhibit a homeostatic response following sleep loss. Importantly, Lsd2 mutant flies are not learning impaired after sleep deprivation. These results provide the first genetic evidence, to our knowledge, that lipid metabolism plays an important role in regulating the homeostatic response and can protect against neuronal impairments induced by prolonged waking.     

Signal trait sexual dimorphism and mutual sexual selection in Drosophila serrata

Abstract The evolution of sexual dimorphism may occur when natural and sexual selection result in different optimum trait values for males and females. Perhaps the most prominent examples of sexual dimorphism occur in sexually selected traits, for which males usually display exaggerated trait levels, while females may show reduced expression of the trait. In some species, females also exhibit secondary sexual traits that may either be a consequence of a correlated response to sexual selection on males or direct sexual selection for female secondary sexual traits. In this experiment, we simultaneously measure the intersex genetic correlations and the relative strength of sexual selection on males and females for a set of cuticular hydrocarbons in Drosophila serrata . There was significant directional sexual selection on both male and female cuticular hydrocarbons: the strength of sexual selection did not differ among the sexes but males and females preferred different cuticular hydrocarbons. In contrast with many previous studies of sexual dimorphism, intersex genetic correlations were low. The evolution of sexual dimorphism in D. serrata appears to have been achieved by sex-limited expression of traits controlled by genes on the X chromosome and is likely to be in its final stages.     

Major Gene Influence on the Propensity to Store Fat in Trunk Versus Extremity Depots: Evidence From the Québec Family Study

Regional fat distribution is related to higher risks of cardiovascular morbidity and mortality, independent of general obesity. In particular, a centralized pattern of fat deposition, characterized by greater abdominal stores relative to extremity stores, is associated with a higher propensity to metabolic complications. Motivated by these considerations, we have initiated a systematic investigation of several measures of regional fat distribution aimed at the identification of possible major gene effects. Two measures approximate the size of subcutaneous fat stores: the sum of six skinfold thicknesses (SF6 = abdominal + suprailiac + subscapular + calf + triceps-+ biceps), and the sum of three trunk skinfold thicknesses (TSF3 = abdominal + suprailiac + subscapular). Both of these phenotypes are highly correlated with total fat mass, 0. 83 and 0.78 for SF6 and TSF3, respectively. The trunk to extremity ratio [TER = TSF3 / (calf + triceps + biceps)] is perhaps the most important of these phenotypes insofar as it is an index of centralized obesity; it is modestly correlated with fat mass (r = 0.18). Each of these phenotypes was adjusted for total fat mass by regression prior to analysis so that we could examine genetic effects on these measures of regional fat distribution without the confounding influence of the determinants of fat mass itself. Segregation analysis of SF6 and TSF3 controlled for total fat mass suggests the presence of a major effect underlying the observed phenotypic distribution; however, tests on the transmission probabilities did not substantiate the segregation of a Mendelian gene. Despite the sexual dimorphism in the expression of the TER, the distribution of age-, generation-, sex-, and fat mass-adjusted TER was not significantly heterogeneous comparing males to females. Consistent evidence of a recessive major gene determinant was obtained, accounting for 37% of the phenotypic variance with the frequency of the gene leading to high values of the TER being 0.35. This finding suggests that further studies to investigate the role of specific candidate genes are warranted.     

Individual variation in sleep behaviour in blue tits Cyanistes caeruleus: assortative mating and associations with fitness‐related traits

Sleep is ubiquitous in animals, but there is great inter‐ and intraspecific variation in the daily amount of sleep that is needed. Chronic sleep curtailment or experimental sleep deprivation are known to impair health and performance of individuals, but not much is known about the fitness consequences of naturally occurring variation in sleep behaviour. Here we test for assortative mating in sleep behaviour and for correlations between sleep phenotypes and reproductive success and survival in a free‐living blue tit population. We found that partners of a social breeding pair were mated assortatively in regard to standardized awakening time, i.e. awakening time relative to that of other birds of the same sex in the population. We found no evidence for assortative mating for other sleep parameters. In female blue tits no sleep parameter that we measured was significantly correlated with lay date or clutch size. Females that had extra‐pair young in their brood did not differ in awakening time, or in any other sleep parameter, compared to females without extra‐pair young. In males, the probability of siring extra‐pair young was related to sleep onset and sleep duration, but not as predicted. Males that began to sleep earlier and slept longer were more likely to sire extra‐pair offspring. None of the sleep parameters were significantly correlated with local survival of first‐year birds. Our results suggest that there is no strong effect of variation in sleep behaviour on fitness in blue tits, at least under natural conditions. Such a relationship might only become evident when natural sleep patterns are experimentally disturbed, or when sleep quality is affected by anthropogenic noise or light pollution.     

Evolution of starvation resistance in Drosophila melanogaster: measurement of direct and correlated responses to artificial selection

Laboratory selection for resistance to starvation has been conducted under relatively controlled conditions to investigate direct and correlated responses to artificial selection. With regard to starvation resistance, there are three physiological routes by which the trait can evolve: resource accumulation, energy conservation and starvation tolerance. A majority of energetic compounds and macromolecules including triglycerides, trehalose and other sugars, and soluble protein increased in abundance as a result of selection. Movement was additionally investigated with selected males moving less than control males and selected females exhibiting a similar response to selection. Results obtained from this study supported two of the possible evolutionary mechanisms for adaptation to starvation: energy compound storage and conservation. If the response to selection is based on an evolutionarily conserved pattern of genetic correlations (elevated lipid, elevated sugars and reduced movement), then the response to selection is medically relevant and the genetic architecture should be investigated in depth.     

DETECTING CRYPTIC INDIRECT GENETIC EFFECTS

Indirect genetic effects (IGEs) occur when genes expressed in one individual alter the phenotype of an interacting partner. IGEs can dramatically affect the expression and evolution of social traits. However, the interacting phenotype(s) through which they are transmitted are often unknown, or cryptic, and their detection would enhance our ability to accurately predict evolutionary change. To illustrate this challenge and possible solutions to it, we assayed male leg‐tapping behavior using inbred lines of Drosophila melanogaster paired with a common focal male strain. The expression of tapping in focal males was dependent on the genotype of their interacting partner, but this strong IGE was cryptic. Using a multiple‐regression approach, we identified male startle response as a candidate interacting phenotype: the longer it took interacting males to settle after being startled, the less focal males tapped them. A genome‐wide association analysis identified approximately a dozen candidate protein‐coding genes potentially underlying the IGE, of which the most significant was slowpoke. Our methodological framework provides information about candidate phenotypes and candidate single‐nucleotide polymorphisms that underpin a strong yet cryptic IGE. We discuss how this approach can facilitate the detection of cryptic IGEs contributing to unusual evolutionary dynamics in other study systems.     

Conserved and differential effects of dietary energy intake on the hippocampal transcriptomes of females and males

The level of dietary energy intake influences metabolism, reproductive function, the development of age-related diseases, and even cognitive behavior. Because males and females typically play different roles in the acquisition and allocation of energy resources, we reasoned that dietary energy intake might differentially affect the brains of males and females at the molecular level. To test this hypothesis, we performed a gene array analysis of the hippocampus in male and female rats that had been maintained for 6 months on either ad libitum (control), 20% caloric restriction (CR), 40% CR, intermittent fasting (IF) or high fat/high glucose (HFG) diets. These diets resulted in expected changes in body weight, and circulating levels of glucose, insulin and leptin. However, the CR diets significantly increased the size of the hippocampus of females, but not males. Multiple genes were regulated coherently in response to energy restriction diets in females, but not in males. Functional physiological pathway analyses showed that the 20% CR diet down-regulated genes involved in glycolysis and mitochondrial ATP production in males, whereas these metabolic pathways were up-regulated in females. The 40% CR diet up-regulated genes involved in glycolysis, protein deacetylation, PGC-1alpha and mTor pathways in both sexes. IF down-regulated many genes in males including those involved in protein degradation and apoptosis, but up-regulated many genes in females including those involved in cellular energy metabolism, cell cycle regulation and protein deacetylation. Genes involved in energy metabolism, oxidative stress responses and cell death were affected by the HFG diet in both males and females. The gender-specific molecular genetic responses of hippocampal cells to variations in dietary energy intake identified in this study may mediate differential behavioral responses of males and females to differences in energy availability.     

Male and female secondary sexual traits show different patterns of quantitative genetic and environmental variation in the horned beetle Onthophagus sagittarius

The expression of secondary sexual traits in females has often been attributed to a correlated response to selection on male traits. In rare cases, females have secondary sexual traits that are not homologous structures to secondary sexual traits in males and are thus less likely to have evolved in females because of correlated selection. In this study, we used the dung beetle Onthophagus sagittarius, a species with sex‐specific horns, to examine the environmental and quantitative genetic control of horn expression in males and females. Offspring subjected to different brood mass manipulations (dung addition/removal) were found to differ significantly in body size. Brood mass manipulation also had a significant effect on the length of male horns; however, female horn length was found to be relatively impervious to the treatment, showing stronger patterns of additive genetic variance than males. We found no correlations between horn expression in males and females. We therefore conclude that the horns of O. sagittarius females are unlikely to result from genetic correlations between males and females. Rather, our data suggest that they may be under independent genetic control.     

Seasonal variations in the biochemical composition and lipoperoxidation of Hyalella curvispina (Crustacea, Amphipoda)

We assessed the effect of seasonal variations on the biochemical composition and lipoperoxidation of an amphipod crustacean, Hyalella curvispina, which inhabits the coastal plain of the state of Rio Grande do Sul, and correlated this with information on the biology of the species. The animals were collected monthly from September 2003 to August 2005, in Gentil Lagoon, Tramandaí, Rio Grande do Sul, Brazil. Glycogen levels, total proteins, total lipids, triglycerides, and lipoperoxidation were measured for each sex separately. The results suggest that these animals have seasonal storage and degradation of the energy substrates investigated. During summer, glycogen, total lipids, and triglycerides were stored, and were utilized especially during autumn and winter. Proteins were utilized differently in males and females. Females stored proteins especially in spring and used them in summer; whereas males stored proteins especially in spring and used them in winter. The levels of lipoperoxidation during the year differed between females and males. These variations may be related to biotic factors (e.g., reproduction period and others) and to abiotic factors (e.g., water temperature, salinity and others).     

The genetics of phenotypic plasticity. IX. Genetic architecture, temperature, and sex differences in Drosophila melanogaster

Abstract.— We examined the genetic architecture of plasticity of thorax and wing length in response to temperature in Drosophila melanogaster . Reaction norms as a function of growth temperature were analyzed in 20 isofemale lines in a natural population collected from Grande Ferrade near Bordeaux (southern France) in two different years. We found evidence for a complex genetic architecture underlying the reaction norms and differences between males and females. Reaction norms were negative quadratics. Genetic correlations were moderately high between traits within environments. Among characteristic values, the magnitudes of genetic correlations varied among traits and sexes. We hypothesized that genetic correlations among environments would decrease as temperatures became more different. This expectation was upheld for only one trait, female thorax length. For males for both traits, the correlations were large for both very similar and very different temperatures. These correlations may constrain the evolution of the shape of the reaction norms. Whether the extent of independence implies specific regulatory genes or only a specific allelic regulation of trait genes can not be decided from our results.     

The ARF tumor suppressor inhibits tumor cell colonization independent of p53 in a novel mouse model of pancreatic ductal adenocarcinoma metastasis

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.     

Correlated responses to selection on female egg size in male reproductive traits in a butterfly

Genetic correlations between male and female traits can act as evolutionary constraints and, if involving reproductive traits, potentially influence sexual selection. Artificial selection on egg size in the tropical butterfly Bicyclus anynana has yielded highly divergent lines. Here we report evidence for correlated evolution in male traits. Males from the large-egg selected lines produced significantly heavier spermatophores independent of body size and tended to have more fertile sperm stored in their reproductive tracts than those from the small-egg selected lines. This may be due to an underlying genetic correlation in reproductive effort between the sexes. However, non-fertile sperm number and testis size remained unaffected by selection on egg size. Phenotypic correlations within an unselected population revealed that spermatophore mass and fertile sperm number, but not testis size and non-fertile sperm number, were positively related to male body size, and that larger spermatophores contained more fertile, but not non-fertile sperm. In addition, males provided larger females with bigger spermatophores and more fertile sperm, indicating males may be exercising mate choice during copulation.     

Glycogen in the brain of Drosophila melanogaster: diurnal rhythm and the effect of rest deprivation

One function of sleep is thought to be the restoration of energy stores in the brain depleted during wakefulness. One such energy store found in mammalian brains is glycogen. Many of the genes involved in glycogen regulation in mammals have also been found in Drosophila melanogaster and rest behavior in Drosophila has recently been shown to have the characteristics of sleep. We therefore examined, in the fly, variation in the glycogen contents of the brain, the whole head and the body throughout the rest/activity cycle and after rest deprivation. Glycogen in the brain varies significantly throughout the day (p=0.001) and is highest during rest and lowest while flies are active. Glycogen levels in the whole head and body do not show diurnal variation. Brain glycogen drops significantly when flies are rest deprived for 3 h (p=0.034) but no significant differences are observed after 6 h of rest deprivation. In contrast, glycogen is significantly depleted in the body after both 3 and 6 h of rest deprivation (p<0.0001 and p<0.0001, respectively). Glycogen in the fly brain changes in relationship to rest and activity and demonstrates a biphasic response to rest deprivation similar to that observed in mammalian astrocytes in culture.     

Energetic costs of mate guarding behavior in male stream-dwelling isopods

In the stream-dwelling isopod Lirceus fontinalis, males and females engage in a precopulatory mate guarding phase prior to mating. We examined the energetic costs of mate guarding behavior in males by separately assaying glycogen and lipid content at different time increments following mating. We found that males that had recently mated possessed reduced glycogen reserves and that these reserves were fully replenished within 36 h. Conversely, we found that male lipid reserves were unaffected by time since mating. We concluded that precopulatory mate guarding behavior is energetically costly to males and that glycogen is the energy source utilized to pay that cost. We also examined whether food deprivation during the mate guarding phase affected male energy reserves (glycogen) at the end of that phase. We found that males that were held in the laboratory and starved during mate guarding possessed reduced glycogen at the termination of the phase when compared to fed males. This reduced quantity was equivalent to the glycogen reserves of recently mated males collected from the field. We propose that food deprivation during the mate guarding phase explains the reduction in glycogen reserves at the termination of that phase. We discuss these results with reference to patterns of refuge use behavior during the mate guarding phase.     

A genetic screen for zygotic embryonic lethal mutations affecting cuticular morphology in the wasp Nasonia vitripennis

We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera; Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F1 females. We describe 74 of >100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila.