Octopamine mediates starvation-induced hyperactivity in adult Drosophila

Starved animals often exhibit elevated locomotion, which has been speculated to partly resemble foraging behavior and facilitate food acquisition and energy intake. Despite its importance, the neural mechanism underlying this behavior remains unknown in any species. In this study we confirmed and extended previous findings that starvation induced locomotor activity in adult fruit flies Drosophila melanogaster. We also showed that starvation-induced hyperactivity was directed toward the localization and acquisition of food sources, because it could be suppressed upon the detection of food cues via both central nutrient-sensing and peripheral sweet-sensing mechanisms, via induction of food ingestion. We further found that octopamine, the insect counterpart of vertebrate norepinephrine, as well as the neurons expressing octopamine, were both necessary and sufficient for starvation-induced hyperactivity. Octopamine was not required for starvation-induced changes in feeding behaviors, suggesting independent regulations of energy intake behaviors upon starvation. Taken together, our results establish a quantitative behavioral paradigm to investigate the regulation of energy homeostasis by the CNS and identify a conserved neural substrate that links organismal metabolic state to a specific behavioral output.The CNS plays an essential role in energy homeostasis (1). It actively monitors changes in the internal energy state and modulates an array of physiological and behavioral responses to enable energy homeostasis. Foraging behavior is critical for the localization and acquisition of food supply and hence energy homeostasis. It has been extensively documented both in ethological settings (2, 3) and under well-controlled laboratory conditions (4). Laboratory rodents with limited food access exhibit stereotypic food anticipatory activity (FAA) several hours before the mealtime, which is characterized by a steady increase in locomotion and other appetitive behaviors (5). The neural substrate that drives FAA still remains elusive (5, 6). Notably, the regulation of FAA seems to be dissociable from that of feeding behavior (7, 8). These results hint at the presence of an independent and somewhat discrete regulatory mechanism of foraging behavior.Foraging behavior has also been extensively studied in invertebrate species such as the roundworm Caenorhabditis elegans (9) and fruit flies Drosophila melanogaster (10). Roundworm populations exhibit two naturally emerged foraging patterns: “solitary” worms disperse across the bacterial lawn, and “social” worms aggregate along the food edge and form clumps (9). This behavioral dimorphism is controlled by natural variations of the npr-1 (neuropeptide receptor resemblance) gene that encodes a receptor homologous to the receptor family of orexigenic neuropeptide Y in mammals (9). A comparable scenario has also been identified in larval fruit flies (10), with two distinct forms of foraging present in nature: “rover” and “sitter.” On food sources, sitter but not rover reduces moving speed for feeding (11). Natural variations of a single gene named foraging that encodes a cGMP-dependent protein kinase are responsible for this behavioral dimorphism (10). It remains unclear, however, whether the foraging strategies outlined above are driven by animals’ metabolic state, and if so whether npr-1 and foraging are involved (4). It is worth noting that both the roundworm and fruit fly larvae are continuous feeders. It is therefore difficult to disassociate the effect of the internal energy state on foraging behavior from that of acute change in food availability.In this present study, we sought to characterize foraging behavior in an intermittent feeder, the adult fruit fly. We confirmed that starved flies exhibited robust and sustained increase in their locomotor activity and provided evidence that it partly resembled foraging behavior. Furthermore, we found octopamine, a biological amine structurally related to vertebrate norepinephrine with similar physiological roles, both necessary and sufficient for starvation-induced hyperactivity. To summarize, our results reveal a highly conserved neural mechanism that promotes locomotion upon starvation, shedding important light on the regulation of foraging behavior by the CNS.     

Deletion of Drosophila insulin-like peptides causes growth defects and metabolic abnormalities

Insulin/Insulin-like growth factor signaling regulates homeostasis and growth in mammals, and is implicated in diseases from diabetes to cancer. In Drosophila melanogaster, as in other invertebrates, multiple Insulin-Like Peptides (DILPs) are encoded by a family of related genes. To assess DILPs'' physiological roles, we generated small deficiencies that uncover single or multiple dilps, generating genetic loss-of-function mutations. Deletion of dilps1–5 generated homozygotes that are small, severely growth-delayed, and poorly viable and fertile. These animals display reduced metabolic activity, decreased triglyceride levels and prematurely activate autophagy, indicative of “starvation in the midst of plenty,” a hallmark of Type I diabetes. Furthermore, circulating sugar levels are elevated in Df [dilp1–5] homozygotes during eating and fasting. In contrast, Df[dilp6] or Df[dilp7] animals showed no major metabolic defects. We discuss physiological differences between mammals and insects that may explain the unexpected survival of lean, ‘diabetic’ flies.     

Gustatory and metabolic perception of nutrient stress in Drosophila

Sleep loss is an adaptive response to nutrient deprivation that alters behavior to maximize the chances of feeding before imminent death. Organisms must maintain systems for detecting the quality of the food source to resume healthy levels of sleep when the stress is alleviated. We determined that gustatory perception of sweetness is both necessary and sufficient to suppress starvation-induced sleep loss when animals encounter nutrient-poor food sources. We further find that blocking specific dopaminergic neurons phenocopies the absence of gustatory stimulation, suggesting a specific role for these neurons in transducing taste information to sleep centers in the brain. Finally, we show that gustatory perception is required for survival, specifically in a low nutrient environment. Overall, these results demonstrate an important role for gustatory perception when environmental food availability approaches zero and illustrate the interplay between sensory and metabolic perception of nutrient availability in regulating behavioral state.Starvation is a condition of extreme nutrient stress that leads to rapid death. On detecting the absence of environmental nutrient sources, organisms use multiple strategies to adjust resource allocation to maximize the chances of finding a food source, including inducing longer foraging searches (1) and limiting sleep behavior (2, 3). Sleep loss in Drosophila melanogaster is a characteristic response to nutrient deprivation that appears ∼12 h after the removal of a food source; in males, it is followed by death in another 12 h (2). Sleep loss is thought to represent a cost to the organism (4–6), and mechanisms for evaluating the environment and terminating this behavioral response when food is available would likely confer an adaptive benefit. A deeper understanding of how organisms perceive and respond to environmental stress could offer substantial benefit to humans attempting to maintain maximal health in the face of food shortages and unstable environmental conditions. The strategies used by organisms to evaluate the sufficiency of a food source and to initiate or suppress sleep loss under very low nutrient conditions remain largely unknown and represent one path toward understanding global stress response.     

Distinct signaling of Drosophila chemoreceptors in olfactory sensory neurons

In Drosophila, olfactory sensory neurons (OSNs) rely primarily on two types of chemoreceptors, odorant receptors (Ors) and ionotropic receptors (Irs), to convert odor stimuli into neural activity. The cellular signaling of these receptors in their native OSNs remains unclear because of the difficulty of obtaining intracellular recordings from Drosophila OSNs. Here, we developed an antennal preparation that enabled the first recordings (to our knowledge) from targeted Drosophila OSNs through a patch-clamp technique. We found that brief odor pulses triggered graded inward receptor currents with distinct response kinetics and current–voltage relationships between Or- and Ir-driven responses. When stimulated with long-step odors, the receptor current of Ir-expressing OSNs did not adapt. In contrast, Or-expressing OSNs showed a strong Ca²⁺-dependent adaptation. The adaptation-induced changes in odor sensitivity obeyed the Weber–Fechner relation; however, surprisingly, the incremental sensitivity was reduced at low odor backgrounds but increased at high odor backgrounds. Our model for odor adaptation revealed two opposing effects of adaptation, desensitization and prevention of saturation, in dynamically adjusting odor sensitivity and extending the sensory operating range.From insects to mammals, the sense of smell begins with odor detection by olfactory sensory neurons (OSNs) (1–6). Recently, rapid advances have been made in understanding chemoreceptors in Drosophila OSNs (7–9). To date, Drosophila is the only model organism for which odor selectivity is known for most of its odorant receptors (Ors) (10, 11), and an Or expression pattern has been mapped to OSNs (12, 13). In addition, another family of chemoreceptors called ionotropic receptors (Irs) has been identified and characterized (14–16). These two types of chemoreceptors respond to different odors, thus endowing Drosophila OSNs with unique and complementary properties for odor detection (17). In contrast to the advanced molecular understanding of these two types of chemoreceptors, the mechanisms of their cellular signaling in native OSNs remain unclear, particularly hampered by the technical difficulty of carrying out patch-clamp recordings of Drosophila OSNs.Drosophila OSNs are encased in hair-like sensilla in the antennae and maxillary palps, with each sensillum containing the dendrites of one to four OSNs that are wrapped by sheath cells (18). The responses of native Drosophila OSNs to odors have traditionally been measured by electroantennogram (EAG) (19), which extracellularly measures the potentials across the entire antenna. In addition, single-sensillum recording (SSR) was developed to provide a higher spatial resolution by measuring the local field potentials (LFPs) from a single sensillum (20–24). These methods, especially SSR, have greatly advanced understanding of the odor selectivity of both Ors and Irs (10, 11, 14). However, because sheath cells and other OSNs also contribute to EAG and SSR signals (25), the response characteristics obtained by such measurements are often contaminated. Patch-clamp recordings of single OSNs could ideally overcome this issue while facilitating the experimental manipulations of a cell’s membrane potential; however, this standard method has unfortunately not yet been routinely applied to Drosophila OSNs.Here, we developed a Drosophila antennal preparation and succeeded in performing patch-clamp recordings of single identified OSNs. By using a fast solution change system to deliver liquid-phase odor stimuli, we investigated the response properties of odor-induced receptor currents of Drosophila OSNs. We found that OSNs expressing Ors exhibited slow response kinetics, outward receptor current rectification, and strong adaptation to odors. We further demonstrated that this adaptation was produced by a Ca²⁺ influx into OSNs because it could be eliminated by voltage clamping at positive holding potentials, by removing extracellular Ca²⁺, or by removing internal free Ca²⁺ with a Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Importantly, in contrast to the long-held view that adaptation simply increases sensitivity, we found that Or-mediated adaptation selectively reduced odor-signaling gain at low odor backgrounds but increased the gain at high odor backgrounds, thereby extending the dynamic odor-operating range. In contrast, odor-induced receptor currents in Ir-expressing OSNs showed fast response kinetics and, surprisingly, did not adapt.     

Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

Characterization of the proteome of organelles and subcellular domains is essential for understanding cellular organization and identifying protein complexes as well as networks of protein interactions. We established a proteomic mapping platform in live Drosophila tissues using an engineered ascorbate peroxidase (APEX). Upon activation, the APEX enzyme catalyzes the biotinylation of neighboring endogenous proteins that can then be isolated and identified by mass spectrometry. We demonstrate that APEX labeling functions effectively in multiple fly tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle to demonstrate the power of APEX for characterizing subcellular proteomes in live cells. Further, we generate “MitoMax,” a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation. Altogether, APEX labeling in live Drosophila tissues provides an opportunity to characterize the organelle proteome of specific cell types in different physiological conditions.Specialized biological processes are carried out in specific organelles and subcellular compartments. For example, mitochondria are the site of oxidative respiration, neurons pass electrical or chemical signals to others through synapses, and apical and basolateral domains of epithelial cells are critical for their polarized functions. Understanding how these structures underlie specialized functions requires the comprehensive identification of proteins within spatially defined cellular domains.A common strategy to study the localization of a particular protein is to generate green fluorescent protein (GFP) fusion proteins. However, it is time-consuming and labor-intensive to investigate protein localization at a large scale using GFP tagging, especially in vivo. Therefore, highly sensitive mass spectrometry (MS) approaches have been developed to systematically characterize the proteome of subcellular compartments. However, using MS approaches to characterize the proteome of subcellular domains has been limited by purification methods and is commonly associated with numerous false positives and false negatives due to contamination and loss of components during purification, respectively. For example, mitochondria are composed of an outer membrane and an inner membrane, generating two subcompartmental regions: the intermembrane space and the matrix located within the inner membrane. Because the ultrastructure of mitochondria is often disrupted during isolation processes, the isolation of specific subcompartmental regions of mitochondria is prone to contamination.Recently, a method based on an engineered ascorbate peroxidase (APEX) has been developed and shown to function in cultured mammalian cells for proteomic mapping (1). Upon activation, the APEX enzyme turns a biotin-phenol substrate into a highly reactive radical that covalently tags neighboring proteins on electron-rich amino acids such as tyrosine. Biotinylated endogenous proteins can then be isolated and identified by MS. Thus, APEX labeling can be applied to bypass organelle purification steps, offering an alternative approach for systematic proteomic characterization in live cells. Here we report that the approach can be applied to characterize the subcellular proteome in live tissues and map the mitochondrial matrix proteome of Drosophila muscle. In addition to characterizing a number of uncharacterized putative mitochondrial proteins, we establish MitoMax, a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation.     

Pheromones mediating copulation and attraction in Drosophila

Intraspecific olfactory signals known as pheromones play important roles in insect mating systems. In the model Drosophila melanogaster, a key part of the pheromone-detecting system has remained enigmatic through many years of research in terms of both its behavioral significance and its activating ligands. Here we show that Or47b-and Or88a-expressing olfactory sensory neurons (OSNs) detect the fly-produced odorants methyl laurate (ML), methyl myristate, and methyl palmitate. Fruitless (fru^M)-positive Or47b-expressing OSNs detect ML exclusively, and Or47b- and Or47b-expressing OSNs are required for optimal male copulation behavior. In addition, activation of Or47b-expressing OSNs in the male is sufficient to provide a competitive mating advantage. We further find that the vigorous male courtship displayed toward oenocyte-less flies is attributed to an oenocyte-independent sustained production of the Or47b ligand, ML. In addition, we reveal that Or88a-expressing OSNs respond to all three compounds, and that these neurons are necessary and sufficient for attraction behavior in both males and females. Beyond the OSN level, information regarding the three fly odorants is transferred from the antennal lobe to higher brain centers in two dedicated neural lines. Finally, we find that both Or47b- and Or88a-based systems and their ligands are remarkably conserved over a number of drosophilid species. Taken together, our results close a significant gap in the understanding of the olfactory background to Drosophila mating and attraction behavior; while reproductive isolation barriers between species are created mainly by species-specific signals, the mating enhancing signal in several Drosophila species is conserved.In the vinegar fly Drosophila melanogaster, cuticular hydrocarbons (CHCs) act as pheremones and play important roles in courtship and aggregation behaviors. These pheremones include the female-specific aphrodisiacs (Z,Z)-7,11-heptacosadiene (7,11-HD) and (Z,Z)-7,11-nonacosadiene (7,11-ND) and the male specific antiaphrodisiacs (Z)-7-tricosene (7-T) and 11-cis-vaccenyl acetate (cVA) (1). However, several lines of evidence suggest that other unidentified pheromones likely contribute to courtship and aggregation behaviors. Previous studies have demonstrated that an unidentified volatile sex pheromone produced by female flies stimulates male courtship (2–6). Flies anosmic to cVA exhibit residual attraction to live male flies, suggesting that other attractive cues are produced by flies that are independent of cVA and its neural circuit (7). Furthermore, no specific ligands other than cVA have been identified for the potential pheromone receptors expressed in OSNs of antennal trichoid sensilla (8). Moreover, OSNs expressing olfactory receptors Or47a and Or88a housed in trichoid sensilla respond to unidentified odors in male and female body wash extracts (9).Although the CHC profile of D. melanogaster has been characterized by several analytical techniques (10–14), it is not yet complete (3). In the present study, we used thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) to determine whether flies harbor so far unidentified CHCs. TD-GC-MS provides a highly sensitive and labor-saving alternative to solvent extraction, and allows analysis of a wider volatility range of components than all previously mentioned techniques. In addition, this method has been applied to confirm the composition of sex pheromones in other insect species (15, 16).Here we demonstrate the presence of a truly positive fly-produced signal mediating mating and dissect the neural mechanism underlying its detection. With our findings, the understanding of male olfactory-based sexual arousal is becoming more complete, with all fru-positive OSNs now with known ligands. We also report the presence of the first fly odorants that exclusively mediate attraction in both sexes via a pathway separated from that involved in sexual and social behaviors. Interestingly, both systems and their ligands are remarkably conserved over a number of drosophilid species.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Causes of natural variation in fitness: Evidence from studies of Drosophila populations

DNA sequencing has revealed high levels of variability within most species. Statistical methods based on population genetics theory have been applied to the resulting data and suggest that most mutations affecting functionally important sequences are deleterious but subject to very weak selection. Quantitative genetic studies have provided information on the extent of genetic variation within populations in traits related to fitness and the rate at which variability in these traits arises by mutation. This paper attempts to combine the available information from applications of the two approaches to populations of the fruitfly Drosophila in order to estimate some important parameters of genetic variation, using a simple population genetics model of mutational effects on fitness components. Analyses based on this model suggest the existence of a class of mutations with much larger fitness effects than those inferred from sequence variability and that contribute most of the standing variation in fitness within a population caused by the input of mildly deleterious mutations. However, deleterious mutations explain only part of this standing variation, and other processes such as balancing selection appear to make a large contribution to genetic variation in fitness components in Drosophila.Advances in DNA sequencing methods have enabled geneticists to measure the amount of genetic variability in natural populations at the most basic level: the frequencies of variants in nucleotide sequences. This achievement has ended one component of a debate on the extent and causes of genetic variability that was initiated in the 1950s by Hermann Muller and Theodosius Dobzhansky (1, 2); we now know that DNA sequences are highly variable within the populations of most species (3). It has, however, been much harder to provide a definitive answer to the other component of this debate, which concerns the nature and intensity of the evolutionary forces that influence the frequencies of genetic variants within populations (1, 2, 4, 5). Are these variants mostly selectively neutral (6), with the fates of new mutations determined by random fluctuations in their frequencies (genetic drift)? Is selection on variants that affect fitness mostly purifying, so that mutations with harmful effects are rapidly removed from the population (1)? Or do many loci have variants maintained by balancing selection (2)? What fraction of newly arisen variants cause higher fitness and are in the process of spreading through the population and replacing their alternatives? How strong is the selection acting on nonneutral variants, and how much variation in fitness among individuals within populations is contributed by such variants? Does the existence of wide variation in fitness among individuals imply a genetic load that threatens the survival of the species (1)?These questions are very broad, and this paper deals only with one aspect of them. It focuses on the question of how recent inferences concerning the strength of purifying selection, derived from genome-wide surveys of DNA sequence variability, can be connected with the results of statistical studies of genetic variation in components of Darwinian fitness such as viability and fertility. I will refer to these two approaches as population genomics and quantitative genetics, respectively. The first approach sheds light on the general nature of the fitness effects of the DNA sequence variants found in natural populations, but says little about how these fitness effects are caused. The second tells us how much genetic variability exists for fitness traits, the rate at which it arise by mutation and something about the type of selection involved, but is silent about the nature of the underlying sequence variants.Surprisingly little attention has been paid to integrating these two lines of inquiry, except for ref. 7. I largely confine myself to results from studies of the fruitfly Drosophila, because this has been the most useful model organism for investigating these problems, especially by quantitative genetics methods. Current information derived from population genomics studies will first be reviewed, followed by an analysis of the results of quantitative genetics experiments on both mutational and standing variation. I show that the quantitative genetics results can only be explained if there is a significant input of new mutations with much larger effects on fitness than those inferred from population genomics. There also appears to be too much genetic variation in fitness components in natural populations to be explained purely by mutation selection balance, so that additional processes such as balancing selection must make an important contribution.     

Boundaries of Dachsous Cadherin activity modulate the Hippo signaling pathway to induce cell proliferation

The conserved Hippo tumor suppressor pathway is a key signaling pathway that controls organ size in Drosophila. To date a signal transduction cascade from the Cadherin Fat at the plasma membrane into the nucleus has been discovered. However, how the Hippo pathway is regulated by extracellular signals is poorly understood. Fat not only regulates growth but also planar cell polarity, for which it interacts with the Dachsous (Ds) Cadherin, and Four-jointed (Fj), a transmembrane kinase that modulates the interaction between Ds and Fat. Ds and Fj are expressed in gradients and manipulation of their expression causes abnormal growth. However, how Ds and Fj regulate growth and whether they act through the Hippo pathway is not known. Here, we report that Ds and Fj regulate Hippo signaling to control growth. Interestingly, we found that Ds/Fj regulate the Hippo pathway through a remarkable logic. Induction of Hippo target genes is not proportional to the amount of Ds or Fj presented to a cell, as would be expected if Ds and Fj acted as traditional ligands. Rather, Hippo target genes are up-regulated when neighboring cells express different amounts of Ds or Fj. Consistent with a model that differences in Ds/Fj levels between cells regulate the Hippo pathway, we found that artificial Ds/Fj boundaries induce extra cell proliferation, whereas flattening the endogenous Ds and Fj gradients results in growth defects. The Ds/Fj signaling system thus defines a cell-to-cell signaling mechanism that regulates the Hippo pathway, thereby contributing to the control of organ size.     

The apical transmembrane protein Crumbs functions as a tumor suppressor that regulates Hippo signaling by binding to Expanded

The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. At the core of the Hippo pathway is a kinase cascade extending from the Hippo (Hpo) tumor suppressor to the Yorkie (Yki) oncoprotein. The Hippo kinase cascade, in turn, is regulated by apical membrane-associated proteins such as the FERM domain proteins Merlin and Expanded (Ex), and the WW- and C2-domain protein Kibra. How these apical proteins are themselves regulated remains poorly understood. Here, we identify the transmembrane protein Crumbs (Crb), a determinant of epithelial apical-basal polarity in Drosophila embryos, as an upstream component of the Hippo pathway in imaginal disk growth control. Loss of Crb leads to tissue overgrowth and target gene expression characteristic of defective Hippo signaling. Crb directly binds to Ex through its juxtamembrane FERM-binding motif (FBM). Loss of Crb or mutation of its FBM leads to mislocalization of Ex to basolateral domain of imaginal disk epithelial cells. These results shed light on the mechanism of Ex regulation and provide a molecular link between apical-basal polarity and tissue growth. Furthermore, our studies implicate Crb as a putative cell surface receptor for Hippo signaling by uncovering a transmembrane protein that directly binds to an apical component of the Hippo pathway.     

Long-term dietary exposure to low concentration of dichloroacetic acid promoted longevity and attenuated cellular and functional declines in aged Drosophila melanogaster

Dichloroacetic acid (DCA), a water disinfection by-product, has attained emphasis due to its prospect for clinical use against different diseases including cancer along with negative impact on organisms. However, these reports are based on the toxicological as well clinical data using comparatively higher concentrations of DCA without much of environmental relevance. Here, we evaluate cellular as well as organismal effects of DCA at environmentally and mild clinically relevant concentrations (0.02–20.0 μg/ml) using an established model organism, Drosophila melanogaster. Flies were fed on food mixed with test concentrations of DCA for 12–48 h to examine the induction of reactive oxygen species (ROS) generation, oxidative stress (OS), heat shock genes (hsps) and cell death along with organismal responses. We also examined locomotor performance, ROS generation, glutathione (GSH) depletion, expression of GSH-synthesizing genes (gclc and gclm), and hsps at different days (0, 10, 20, 30, 40, 50) of the age in flies after prolonged DCA exposure. We observed mild OS and induction of antioxidant defense system in 20.0 μg/ml DCA-exposed organism after 24 h. After prolonged exposure to DCA, exposed organism exhibited improved survival, elevated expression of hsp27, gclc, and gclm concomitant with lower ROS generation and GSH depletion and improved locomotor performance. Conversely, hsp27 knockdown flies exhibited reversal of the above end points. The study provides evidence for the attenuation of cellular and functional decline in aged Drosophila after prolonged DCA exposure and the effect of hsp27 modulation which further incites studies towards the therapeutic application of DCA.     

Improved functional abilities of the life-extended Drosophila mutant Methuselah are reversed at old age to below control levels

Methuselah (mth) is a chromosome 3 Drosophila mutant with an increased lifespan. A large number of studies have investigated the genetic, molecular, and biochemical mechanisms of the mth gene. Much less is known about the effects of mth on preservation of sensorimotor abilities throughout Drosophila’s lifespan, particularly in late life. The current study investigated functional senescence in mth and its parental-control line (w1118) in two experiments that measured age-dependent changes in flight functions and locomotor activity. In experiment 1, a total of 158 flies (81 mth and 77 controls) with an age range from 10 to 70 days were individually tethered under an infrared laser-sensor system that allowed monitoring of flight duration during phototaxic flight. We found that mth has a statistically significant advantage in maintaining continuous flight over control flies at age 10 days, but not during middle and late life. At age 70 days, the trend reversed and parental control flies had a small but significant advantage, suggesting an interaction between age and genotype in the ability to sustain flight. In experiment 2, a total of 173 different flies (97 mth and 76 controls) with an age range from 50 to 76 days were individually placed in a large well-lit arena (60 × 45 cm) and their locomotor activity quantified as the distance walked in a 1-min period. Results showed that mth flies had lower levels of locomotor activity relative to controls at ages 50 and 60 days. These levels converged for the two genotypes at the oldest ages tested. Findings show markedly different patterns of functional decline for the mth line relative to those previously reported for other life-extended genotypes, suggesting that different life-extending genes have dissimilar effects on preservation of sensory and motor abilities throughout an organism’s lifespan.     

Mutations in insulin signaling pathway alter juvenile hormone synthesis in Drosophila melanogaster

Juvenile hormone (JH) is a key endocrine regulator of insect metamorphosis, reproduction, and aging. The synthesis of JH is regulated by neuropeptides and biogenic amines, but the molecular and cellular basis of this control remains largely unknown. Genetic analysis of JH synthesis in Drosophila melanogaster mutant for insulin signaling may provide new and powerful insights. Mutants of the insulin receptor (InR) are slow to develop, small, infertile, and long-lived. We previously reported that mutants of InR had reduced JH synthesis as young adults, and that normal longevity and vitellogenesis were restored by topical application of a JH analog [Science 292 (2001) 107]. Here, we describe the 10-day adult age course of JH synthesis from isolated corpus allatum (CA) of InR and of chico, the insulin receptor substrate homolog. JH synthesis increased in wildtype flies to a maximum of 30fmol/gland/h at day 10. In contrast, homozygous InR mutants produced no more than 3 fmol/gland/h JH within the first 5 days, and only 7 fmol/gland/h at day 10. InR mutation disproportionately reduced the synthesis of JH III-bisepoxide, the major JH subtype of the fly. Mutation of chico also reduces body size and extends longevity [Science 292 (2001) 104; Aging Cell 1 (2002a) 75]. Both homozygous and heterozygous chico genotypes reduced JH synthesis, but only to 47 and 67%, respectively, of wildtype and without influencing the ratio of JH subtypes. Because JH synthetic rate does not correlate with the size of CA, it is not likely that insulin signaling mediates JH by impeding endocrine tissue development. Alternatively, we find allatotropin-positive axons to be abundant in the adult brain and in the corpora cardiaca-corpus allatum complex but these neurons are less immunoreactive in the InR mutant genotype, suggesting that insulin signaling may affect JH synthesis through control of JH regulatory neuropeptides.     

Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary

Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line–derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms.Programmed cell death (PCD) is the genetically controlled elimination of cells that occurs during organismal development and homeostasis. Cells are considered dead when they have undergone irreversible plasma membrane permeabilization or have become completely fragmented (1). Apoptosis is the most well-characterized form of PCD, however there are at least a dozen cell death modalities that are morphologically, biochemically, and genetically distinct (2, 3). Two examples of nonapoptotic cell death are autophagic cell death and necrosis, but there are several alternative cell death mechanisms that are less well understood.Nonapoptotic PCD occurs on a large scale in the Drosophila ovary. Drosophila females can produce hundreds of eggs during their lifetime, and for every egg that is formed, developmental PCD of supporting nurse cells (NCs) occurs. However, the mechanisms of developmental PCD in the Drosophila ovary are poorly understood. Each egg forms from a 16-cell germ-line cyst, comprised of the single oocyte and 15 NCs that support the oocyte throughout 14 stages of oogenesis (4, 5). Hundreds of somatically derived follicle cells (FCs) surround the germ-line cyst, forming an egg chamber. At stage 11 of oogenesis, NCs rapidly transfer (“dump”) their cytoplasm into the oocyte. Concurrently, the NCs asynchronously undergo developmental PCD, resulting in mature stage 14 egg chambers that no longer contain any NCs (4–6). Interestingly, caspases, proteases associated with apoptosis, play only a minor role in the death of the NCs in late oogenesis (7–9). Furthermore, combined inhibition of caspases and autophagy does not significantly block NC death during late oogenesis (10). To date, defining the major mechanism of developmental PCD in the Drosophila ovary has remained elusive.An intriguing possibility is that the somatic FCs non–cell-autonomously promote developmental PCD of the NCs during late oogenesis. Non–cell-autonomous regulation of PCD occurs when a cell or group of cells extrinsically initiates or promotes the death of another cell. This concept challenges the idea that PCD is largely a self-regulated, autonomous suicide program in which a cell controls its own demise. One well-characterized example of non–cell-autonomous control of PCD is apoptosis induced by the death ligands Fas or TNF (11, 12).Another type of non–cell-autonomous PCD is phagoptosis (or primary phagocytosis), in which engulfing cells directly cause the death of other cells via “murder” or “assisted suicide.” Phagoptosis is distinct from the engulfment of cell corpses, as the engulfing cell plays an active role in the death of a cell, rather than simply degrading a cell that died via another mechanism. The defining characteristic of phagoptosis is that inhibition of phagocytosis leads to a failure in cell death (13, 14). Phagoptosis has been demonstrated in activated microglia that phagocytose viable neurons, resulting in their destruction (13–15). Entosis is another example of non–cell-autonomous PCD, often referred to as “cell cannibalism,” in which a viable cell invades another cell, where it is degraded by lysosomes. Entosis is distinct from phagoptosis, as the inhibition of phagocytosis genes does not prevent entosis (16). Phagocytosis has also been shown to promote PCD in Caenorhabditis elegans, although this is an example of assisted suicide, as dying cells also require apoptotic machinery (17, 18).Genetic studies in C. elegans have identified two partially redundant signaling pathways that control phagocytosis: the cell death abnormality (CED)-1, 6, 7 and CED-2, 5, 12 pathways (19–21). The CED-1, 6, 7 and CED-2, 5, 12 pathways act in parallel to promote the activation of CED-10, a Rac GTPase responsible for cytoskeletal rearrangements that allow for internalization of the cell corpse. In Drosophila, the roles of the Ced-1, 6, 7 and Ced-2, 5, 12 pathways appear to be conserved. The CED-1 ortholog, Draper, is a transmembrane protein that localizes to the surface of the engulfing cell and acts as a receptor to recognize dying cells. Draper was first shown to be required for engulfment of apoptotic neurons in the embryonic central nervous system with mutants displaying lingering cell corpses (22). Additionally, Draper has been shown to be important in several other contexts including the engulfment of severed axons, bacteria, imaginal disc cells, hemocytes, and apoptotic NCs in midoogenesis (23–27). In addition to Draper, other Drosophila engulfment receptors include Croquemort (28) and integrins (29–31). Croquemort is related to CD36, a scavenger receptor involved in engulfment in mammals (32), and integrins also act as engulfment receptors in C. elegans and mammals (33, 34). The upstream activators of the Ced-2, 5, 12 pathway are largely unknown, although integrins may activate the pathway (34). As in C. elegans, it appears that Ced-12 and draper function in separate pathways in Drosophila. Ced-12 and draper have been shown to function in distinct steps in axon clearance (35). In macrophages, Ced-12 has been shown to function in a separate pathway from simu, a bridging molecule that acts upstream of draper (36). A number of other engulfment genes have been identified in Drosophila, and their molecular interactions are under active investigation (36–39).Given the minor role for apoptosis and autophagic cell death during developmental PCD in the Drosophila ovary, we investigated the possibility that the FCs non–cell-autonomously promote NC death. Previously we showed that FCs of the Drosophila ovary are capable of phagoptosis in midoogenesis when phagocytosis genes are overexpressed (27), and we questioned whether phagocytosis genes might normally function to control cell death in late oogenesis. Indeed, we found that the phagocytosis genes draper and Ced-12/ELMO are required in the FCs for NC removal in late oogenesis and that they function partly in parallel. We also show that the FCs non–cell-autonomously control events associated with the death of the NCs, including nuclear envelope permeabilization, acidification, and DNA fragmentation. Furthermore, the genetic ablation of stretch FCs disrupted all cellular changes associated with developmental PCD of the NCs. Therefore, PCD of the NCs is a unique model of a naturally occurring developmental cell death program that is nonapoptotic and non–cell-autonomously controlled.     

Multiple-stress analysis for isolation of Drosophila longevity genes

Long-lived organisms tend to be more resistant to various forms of environmental stress. An example is the Drosophila longevity mutant, methuselah, which has enhanced resistance to heat, oxidants, and starvation. To identify genes regulated by these three stresses, we made a cDNA library for each by subtraction of "unstressed" from "stressed" cDNA and used DNA hybridization to identify genes that are regulated by all three. This screen indeed identified 13 genes, some already known to be involved in longevity, plus candidate genes. Two of these, hsp26 and hsp27, were chosen to test for their effects on lifespan by generating transgenic lines and by using the upstream activating sequence/GAL4 system. Overexpression of either hsp26 or hsp27 extended the mean lifespan by 30%, and the flies also displayed increased stress resistance. The results demonstrate that multiple-stress screening can be used to identify new longevity genes.     

Organ renewal and cell divisions by differentiated cells in Drosophila

For many organs, the processes of renewal and regeneration recruit stem cells to replace differentiated, postmitotic cells, but the capacity of an organ's differentiated cells to divide and contribute is uncertain. Most cells of the Drosophila adult are the descendants of dedicated precursors that divide and replace larval cells that are histolyzed during metamorphosis. We investigated the provenance of cells that reconstitute the second thoracic metamere of the tracheal system (Tr2). These cells contribute the precursors for Branchless(FGF)-dependent growth of the dorsal air sacs, the major tracheal organs of the adult fly. We found that, in contrast to the cells in other tracheal metameres that proceed through many cycles of endoreplication, the cells that constitute the Tr2 branches in young larvae do not. Like the cells in other tracheal metameres, these cells arrest mitotic cycling in the embryo and form differentiated, air-filled tracheal branches of the larva. We report here that they reinitiate cell divisions during the third instar (L3) to increase the Tr2 population by ≈10-fold with multipotent cells.     

The Toll pathway is important for an antiviral response in Drosophila

The innate immune response of Drosophila melanogaster is governed by a complex set of signaling pathways that trigger antimicrobial peptide (AMP) production, phagocytosis, melanization, and encapsulation. Although immune responses against both bacteria and fungi have been demonstrated in Drosophila, identification of an antiviral response has yet to be found. To investigate what responses Drosophila mounts against a viral infection, we have developed an in vivo Drosophila X virus (DXV)-based screening system that identifies altered sensitivity to viral infection by using DXV's anoxia-induced death pathology. Using this system to screen flies with mutations in genes with known or suggested immune activity, we identified the Toll pathway as a vital part of the Drosophila antiviral response. Inactivation of this pathway instigated a rapid onset of anoxia induced death in infected flies and increases in viral titers compared to those in WT flies. Although constitutive activation of the pathway resulted in similar rapid onset of anoxia sensitivity, it also resulted in decreased viral titer. Additionally, AMP genes were induced in response to viral infection similar to levels observed during Escherichia coli infection. However, enhanced expression of single AMPs did not alter resistance to viral infection or viral titer levels, suggesting that the main antiviral response is cellular rather than humoral. Our results show that the Toll pathway is required for efficient inhibition of DXV replication in Drosophila. Additionally, our results demonstrate the validity of using a genetic approach to identify genes and pathways used in viral innate immune responses in Drosophila.     

A tumor-suppressing mechanism in Drosophila involving cell competition and the Hippo pathway

Mutant larvae for the Drosophila gene lethal giant larva (lgl) develop neoplastic tumors in imaginal discs. However, lgl mutant clones do not form tumors when surrounded by wild-type tissue, suggesting the existence of a tumor-suppressing mechanism. We have investigated the tumorigenic potential of lgl mutant cells by generating wing compartments that are entirely mutant for lgl and also inducing clones of various genetic combinations of lgl⁻ cells. We find that lgl⁻ compartments can grow indefinitely but lgl⁻ clones are eliminated by cell competition. lgl mutant cells may form tumors if they acquire constitutive activity of the Ras pathway (lgl⁻ UAS-ras^V12), which confers proliferation advantage through inhibition of the Hippo pathway. Yet, the majority of lgl⁻ UAS-ras^V12 clones are eliminated in spite of their high proliferation rate. The formation of a tumor requires in addition the formation of a microenvironment that allows mutant cells to evade cell competition.