Dietary Ethanol Mediates Selection on Aldehyde Dehydrogenase Activity in Drosophila melanogaster

Ethanol is an important environmental variable for fruit-breedingDrosophila species, serving as a resource at low levels anda toxin at high levels. The first step of ethanol metabolism,the conversion of ethanol to acetaldehyde, is catalyzed primarilyby the enzyme alcohol dehydrogenase (ADH). The second step,the oxidation of acetaldehyde to acetate, has been a sourceof controversy, with some authors arguing that it is carriedout primarily by ADH itself, rather than a separate aldehydedehydrogenase (ALDH) as in mammals. We review recent evidencethat ALDH plays an important role in ethanol metabolism in Drosophila.In support of this view, we report that D. melanogaster populationsmaintained on ethanol-supplemented media evolved higher activityof ALDH, as well as of ADH. We have also tentatively identifiedthe structural gene responsible for the majority of ALDH activityin D. melanogaster. We hypothesize that variation in ALDH activitymay make an important contribution to the observed wide variationin ethanol tolerance within and among Drosophila species.     

Evolution of insect eye development: first insights from fruit fly, grasshopper and flour beetle

The molecular genetic dissection of Drosophila eye developmentled to the exciting discovery of a surprisingly large panelof genes and gene activities, which are functionally conservedacross phyla. Little effort has yet been made towards pinpointingnon-conserved gene functions in the developing Drosophila eye.This neglects the fact that Drosophila visual system developmentis a highly derived process. The comparative analysis of Drosophilaeye development within insects can be expected to enhance resolutionand accuracy of between phyla comparisons of eye development,and to reveal molecular developmental changes that facilitatedthe evolutionary transition from hemimetabolous to holometabolousinsect development. Here we review aspects of early Drosophilaeye development, which are likely to have diverged from thesituation in more primitive insects, as indicated by resultsfrom work in the flour beetle Tribolium castaneum and the grasshopperSchistocerca americana.     

Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+-ATPase, alter heart rate and rhythmicity in Drosophila

To analyze the role of cytosolic calcium in regulating heart beat frequency and rhythm, we studied conditional mutations in Drosophila Sarco-endoplasmic reticulum Ca²⁺-ATPase, believed to be predominantly responsible for sequestering free cytosolic calcium. Abnormalities in the amount or structure of the SERCA protein have been linked to cardiac malfunction in mammals. Drosophila SERCA protein (dSERCA) is highly enriched in Drosophila larval heart with a distinct membrane distribution of SERCA at cardiac Z-lines, suggesting evolutionarily conserved zones for calcium uptake into the sarcoplasmic reticulum. Heart beat frequency is strikingly reduced in mutant animals following dSERCA inactivation, (achieved by a brief exposure of these conditional mutants to non-permissive temperature). Cardiac contractions also show abnormal rhythmicity and electrophysiological recordings from the heart muscle reveal dramatic alterations in electrical activity. Overall, these studies underscore the utility of the Drosophila heart to model SERCA dysfunction dependent cardiac disorders and constitute an initial step towards developing Drosophila as a viable genetic model system to study conserved molecular determinants of cardiac physiology.     

Chemical Interactions in the Cactus-Microorganism-Drosophila Model System of the Sonoran Desert

The Cactus-Microorganism-Drosophila Model System of the SonoranDesert represents an excellent paradigm of the role of chemistryin plant-animal interactions. In this system, four species ofendemic Drosophila feed and reproduce in necrotic tissue offive species of columnar cacti. Studies over the past 35 yrhave characterized a myriad of interactions between the threemajor components of the model system. The cacti contain a varietyof allelochemicals which are primarily responsible for the highlyspecific pattern of host plant utilization exhibited by thedesert Drosophila. Plant chemistry, through its effect on themicrobially produced volatile patterns, is further involvedin host specificity because the flies use the volatile patternto cue in on necroses in the appropriate species of cactus.The metabolic activities of microorganisms (bacteria and yeasts)living in the necrosis can affect the substrate chemistry inboth positive and negative ways (i.e., acting to increase orto decrease the toxicity of the substrate). Finally, cactuschemistry may affect drosophilid mating behavior since larvalrearing substrate has been shown to influence adult hydrocarbonepicuticular composition. In D. mojavensis, adult hydrocarbonprofile has been implicated as a determinant of mate choiceleading to premating isolation between geographically isolatedpopulations that use chemically different cactus substrates.Current research is focused on the evolution and regulationof genes whose products (cytochrome P450 enzymes) are involvedin the specific insect-host plant relationships which existbetween the Drosophila species and the cactus species. There are many reasons why investigators choose to focus theirresearch efforts on what are referred to as "model systems."Typically included among these would be the idea that modelsystems are easier to study because they are less complex thanother scientific situations. At the same time, model systemsshould be representative of more complex, natural systems sothat information that is obtained from their study is broadlyapplicable. For almost a century, the fruit fly, Drosophilamelanogaster, has served as a model organism for the study ofgenetics. As a genetic paradigm, Drosophila is more tractableto scientific investigation than most organisms and has providedimportant insights into a wide variety of human maladies fromalcohol abuse to neurological brain disorders (Bellen, 1998).Similarly, the interrelationships of the columnar cacti andthe cactophilic Drosophila species of the Sonoran Desert have,for the past 35 yr, provided an excellent model system withwhich to study relevant questions in evolution, ecological genetics,and chemical ecology. The intent of this article is to brieflyreview and characterize the chemical interactions between theplants (cacti) and animals (Drosophila) of this model system,and, in addition, provide some thoughts on possible future directionsfor integrative approaches in this research area.     

A Pair of Dopamine Neurons Target the D1-Like Dopamine Receptor DopR in the Central Complex to Promote Ethanol-Stimulated Locomotion in Drosophila

Dopamine is a mediator of the stimulant properties of drugs of abuse, including ethanol, in mammals and in the fruit fly Drosophila. The neural substrates for the stimulant actions of ethanol in flies are not known. We show that a subset of dopamine neurons and their targets, through the action of the D1-like dopamine receptor DopR, promote locomotor activation in response to acute ethanol exposure. A bilateral pair of dopaminergic neurons in the fly brain mediates the enhanced locomotor activity induced by ethanol exposure, and promotes locomotion when directly activated. These neurons project to the central complex ellipsoid body, a structure implicated in regulating motor behaviors. Ellipsoid body neurons are required for ethanol-induced locomotor activity and they express DopR. Elimination of DopR blunts the locomotor activating effects of ethanol, and this behavior can be restored by selective expression of DopR in the ellipsoid body. These data tie the activity of defined dopamine neurons to D1-like DopR-expressing neurons to form a neural circuit that governs acute responding to ethanol.     

Developmental expression and biophysical characterization of a Drosophila melanogaster aquaporin

Aquaporins (AQPs) accelerate the movement of water and other solutes across biological membranes, yet the molecular mechanisms of each AQP's transport function and the diverse physiological roles played by AQP family members are still being defined. We therefore have characterized an AQP in a model organism, Drosophila melanogaster, which is amenable to genetic manipulation and developmental analysis. To study the mechanism of Drosophila Malpighian tubule (MT)-facilitated water transport, we identified seven putative AQPs in the Drosophila genome and found that one of these, previously named DRIP, has the greatest sequence similarity to those vertebrate AQPs that exhibit the highest rates of water transport. In situ mRNA analyses showed that DRIP is expressed in both embryonic and adult MTs, as well as in other tissues in which fluid transport is essential. In addition, the pattern of DRIP expression was dynamic. To define DRIP-mediated water transport, the protein was expressed in Xenopus oocytes and in yeast secretory vesicles, and we found that significantly elevated rates of water transport correlated with DRIP expression. Moreover, the activation energy required for water transport in DRIP-expressing secretory vesicles was 4.9 kcal/mol. This low value is characteristic of AQP-mediated water transport, whereas the value in control vesicles was 16.4 kcal/mol. In contrast, glycerol, urea, ammonia, and proton transport were unaffected by DRIP expression, suggesting that DRIP is a highly selective water-specific channel. This result is consistent with the homology between DRIP and mammalian water-specific AQPs. Together, these data establish Drosophila as a new model system with which to investigate AQP function. fluid homeostasis; osmosis; channel; membrane     

Interactions of Oxygen at High Pressure and Radiation in Drosophila

Oxygen at high pressure (OHP) and X-irradiation can interact in the fruit fly Drosophila melanogaster to potentiate toxic actions characteristic of one agent alone. 1000 kvp X-irradiation in doses of 30, 60, and 75 kr accelerated the acute immobilization of young male Drosophila by oxygen at 7.8 atm, up to rates twice that observed with such oxygen pressure alone. X-irradiation alone in these dosages did not acutely immobilize the Drosophila. X-irradiation during exposure to 7.8 atm pO₂ was more effective and consistent in producing this potentiation than was X-irradiation that preceded exposure to OHP. Acute OHP toxicity in young female Drosophila was not potentiated by 75 kr of X-irradiation. On the other hand, shortening of the life span of young male Drosophila by the above doses of X-irradiation was augmented significantly by a concurrent 40 min exposure to OHP (which alone did not significantly decrease life span). This shows, for the first time, that oxygen can affect not only the acute effects of radiation, but also the residual irreversible effects indicated by the life span shortening.     

Neuronal signaling systems and ethanol dependence

In recent years there have been remarkable developments toward the understanding of the molecular and/or cellular changes in the neuronal second-messenger pathways during ethanol dependence. In general, it is believed that the cyclic adenosine 3′, 5′-monophosphate (cAMP) and the phosphoinositide (PI) signal-transduction pathways may be the intracellular targets that mediate the action of ethanol and ultimately contribute to the molecular events involved in the development of ethanol tolerance and dependence. Several laboratories have demonstrated that acute ethanol exposure increases, whereas protracted ethanol exposure decreases, agonist-stimulated adenylate cyclase activity in a variety of cell systems, including the rodent brain. Recent studies indicate that various postreceptor events of the cAMP signal transduction cascade (i.e., G_s protein, protein kinase A [PKA], and cAMP-responsive element binding protein [CREB]) in the rodent brain are also modulated by chronic ethanol exposure. The PI signal-transduction cascade represents another important second-messenger system that is modulated by both acute and chronic ethanol exposure in a variety of cell systems. It has been shown that protracted ethanol exposure significantly decreases phospholipase C (PLC) activity in the cerebral cortex of mice and rats. The decreased PLC activity during chronic ethanol exposure may be caused by a decrease in the protein levels of the PLC-Β₁ isozyme but not of PLC-δ₁ or PLC-γ₁ isozymes in the rat cerebral cortex. Protein kinase C (PKC), which is a key step in the Pi-signaling cascade, has been shown to be altered in a variety of cell systems by acute or chronic ethanol exposure. It appears from the literature that PKC plays an important role in the modulation of the function of various neurotransmitter receptors (e.g., γ-aminobutyrate type A [GABAa], N-methyl-D-aspartate [NMDA], serotonin2A [5-HT2a], and 5-HT2C, and muscarinic [m1] receptors) resulting from ethanol exposure. The findings described in this review article indicate that neuronal-signaling proteins represent a molecular locus for the action of ethanol and are possibly involved in the neuroadaptational mechanisms to protracted ethanol exposure. These findings support the notion that alterations in the cAMP and the PI-signaling cascades during chronic ethanol exposure could be the critical molecular events associated with the development of ethanol dependence.     

巨尾阿丽蝇在自由状态下的电击回避学习能力(英文)

蜜蜂和果蝇具有良好的学习记忆能力。利用自主改良的研究装置对另一种具有强大生存本能的双翅目昆虫——巨尾阿丽蝇(Aldrichina grahami)在自由状态下电击回避学习能力进行研究。结果表明,巨尾阿丽蝇具有良好的学习记忆能力,因为当刺激电压范围为5V到45V时,观察到巨尾阿丽蝇有显著的回避电刺激行为,而当电压达到60V时会受到明显伤害。由此推测,巨尾阿丽蝇适合作为神经系统研究的动物模型。本实验所采用的实验范例较以往有所改进,适合作为自由状态下研究昆虫的工具。     

Biochemical Conservation of Recombinant Drosophila Tyrosine Hydroxylase with its Mammalian Cognates

Dopamine modulates several behavioral and developmental events; in the fruit fly Drosophila melanogaster, dopamine is a neurotransmitter, a neuromodulator, and a developmental signal. Studies in mammals suggest that these diverse roles for dopamine have been evolutionarily conserved. Fundamental regulation of dopamine occurs via tyrosine hydroxylase (TH), the first and rate-limiting enzyme in the catecholamine biosynthetic pathway. Mammalian TH is acutely regulated via phosphorylation–dephosphorylation mechanisms, which occur as a direct consequence of nerve stimulation. We have shown that the Drosophila homolog of TH, DTH, shares over 50% sequence identity with mammalian TH, and the serine residue corresponding to the major site of phosphorylation is conserved. We demonstrate using recombinant DTH protein generated in E. coli that its regulatory biochemical mechanisms closely parallel those from mammals. Drosophila thus provides a highly conserved and tractable model system in which to test the functional consequences of perturbing TH activity by acute regulatory mechanisms.     

Alcohol resistance in Drosophila is modulated by the Toll innate immune pathway

A growing body of evidence has shown that alcohol alters the activity of the innate immune system and that changes in innate immune system activity can influence alcohol‐related behaviors. Here, we show that the Toll innate immune signaling pathway modulates the level of alcohol resistance in Drosophila. In humans, a low level of response to alcohol is correlated with increased risk of developing an alcohol use disorder. The Toll signaling pathway was originally discovered in, and has been extensively studied in Drosophila. The Toll pathway is a major regulator of innate immunity in Drosophila, and mammalian Toll‐like receptor signaling has been implicated in alcohol responses. Here, we use Drosophila‐specific genetic tools to test eight genes in the Toll signaling pathway for effects on the level of response to ethanol. We show that increasing the activity of the pathway increases ethanol resistance whereas decreasing the pathway activity reduces ethanol resistance. Furthermore, we show that gene products known to be outputs of innate immune signaling are rapidly induced following ethanol exposure. The interaction between the Toll signaling pathway and ethanol is rooted in the natural history of Drosophila melanogaster.     

Developmental nicotine exposure affects larval brain size and the adult dopaminergic system of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background

Pregnant women may be exposed to nicotine if they smoke or use tobacco products, nicotine replacement therapy, or via e-cigarettes. Prenatal nicotine exposure has been shown to have deleterious effects on the nervous system in mammals including changes in brain size and in the dopaminergic system. The genetic and molecular mechanisms for these changes are not well understood. A Drosophila melanogaster model for these effects of nicotine exposure could contribute to faster identification of genes and molecular pathways underlying these effects. The purpose of this study was to determine if developmental nicotine exposure affects the nervous system of Drosophila melanogaster, focusing on changes to brain size and the dopaminergic system at two developmental stages.

Results

We reared flies on control or nicotine food from egg to 3rd instar larvae or from egg to adult and determined effectiveness of the nicotine treatment. We used immunohistochemistry to visualize the whole brain and dopaminergic neurons, using tyrosine hydroxylase as the marker. We measured brain area, tyrosine hydroxylase fluorescence, and counted the number of dopaminergic neurons in brain clusters.We detected an increase in larval brain hemisphere area, a decrease in tyrosine hydroxylase fluorescence in adult central brains, and a decrease in the number of neurons in the PPM3 adult dopaminergic cluster. We tested involvement of Dα7, one of the nicotinic acetylcholine receptor subunits, and found it was involved in eclosion, as previously described, but not involved in brain size.

Conclusions

We conclude that developmental nicotine exposure in Drosophila melanogaster affects brain size and the dopaminergic system. Prenatal nicotine exposure in mammals has also been shown to have effects on brain size and in the dopaminergic system. This study further establishes Drosophila melanogaster as model organism to study the effects of developmental nicotine exposure. The genetic and molecular tools available for Drosophila research will allow elucidation of the mechanisms underlying the effects of nicotine exposure during development.

     

Drosophila melanogaster as a model system for the study of the function of calcium/calmodulin-dependent protein kinase II in synaptic plasticity

Drosophila melanogaster has been used as a biological model system for almost a century. In the last several decades,Drosophila has been used as a system to probe the molecular basis of behavior and discoveries in the fly have been at the forefront of the elucidation of important basic mechanisms. This review will outline the variety of approaches that makeDrosophila an excellent model system with which to study the function of the enzyme calcium/calmodulin-dependent protein kinase II (CaMKII) in synaptic plasticity. CaMKII has a well documented role in behavior and synaptic plasticity in both vertebrates and invertebrates. The behavioral and genetic richness ofDrosophila allow for a multi-level approach to understanding the physiological roles of this enzyme's function.     

Beyond the mouse model: Using Drosophila as a model for sperm interaction with the female reproductive tract

Although the fruit fly, Drosophila melanogaster, has emerged as a model system for human disease, its potential as a model for mammalian reproductive biology has not been fully exploited. Here we describe how Drosophila can be used to study the interactions between sperm and the female reproductive tract. Like many insects, Drosophila has two types of sperm storage organs, the spermatheca and seminal receptacle, whose ducts arise from the uterine wall. The spermatheca duct ends in a capsule-like structure surrounded by a layer of gland cells. In contrast, the seminal receptacle is a slender, blind-ended tubule. Recent studies suggest that the spermatheca is specialized for long-term storage, as well as sperm maturation, whereas the receptacle functions in short-term sperm storage. Here we discuss recent molecular and morphological analyses that highlight possible themes of gamete interaction with the female reproductive tract and draw comparison of sperm storage organ design in Drosophila and other animals, particularly mammals. Furthermore, we discuss how the study of multiple sperm storage organ types in Drosophila may help us identify factors essential for sperm viability and, moreover, factors that promote long-term sperm survivorship.     

Matrix Metalloproteinase 2 Is Required for Ovulation and Corpus Luteum Formation in Drosophila

Ovulation is critical for successful reproduction and correlates with ovarian cancer risk, yet genetic studies of ovulation have been limited. It has long been thought that the mechanism controlling ovulation is highly divergent due to speciation and fast evolution. Using genetic tools available in Drosophila, we now report that ovulation in Drosophila strongly resembles mammalian ovulation at both the cellular and molecular levels. Just one of up to 32 mature follicles per ovary pair loses posterior follicle cells (“trimming”) and protrudes into the oviduct, showing that a selection process prefigures ovulation. Follicle cells that remain after egg release form a “corpus luteum (CL)” at the end of the ovariole, develop yellowish pigmentation, and express genes encoding steroid hormone biosynthetic enzymes that are required for full fertility. Finally, matrix metalloproteinase 2 (Mmp2), a type of protease thought to facilitate mammalian ovulation, is expressed in mature follicle and CL cells. Mmp2 activity is genetically required for trimming, ovulation and CL formation. Our studies provide new insights into the regulation of Drosophila ovulation and establish Drosophila as a model for genetically investigating ovulation in diverse organisms, including mammals.     

Ethanol sensitivity of BK(Ca) channels from arterial smooth muscle does not require the presence of the beta 1-subunit

Ethanol inhibition of large-conductance,Ca²⁺-activated K⁺ (BK_Ca) channelsin aortic myocytes may contribute to the direct contraction of aorticsmooth muscle produced by acute alcohol exposure. In this tissue,BK_Ca channels consist of pore-forming (bslo) and modulatory () subunits. Here, modulation of aortic myocyteBK_Ca channels by acute alcohol was explored by expressingbslo subunits in Xenopus oocytes, in the absenceand presence of ₁-subunits, and studying channelresponses to clinically relevant concentrations of ethanol in excisedmembrane patches. Overall, average values of bslo channelactivity (NP_o, with N = no. ofchannels present in the patch; P_o = probability of a single channel being open) in response to ethanol(3-200 mM) mildly decrease when compared with pre-ethanol,isosmotic controls. However, channel responses show qualitativeheterogeneity at all ethanol concentrations. In the majority of patches(42/71 patches, i.e., 59%), a reversible reduction inNP_o is observed. In this subset, the maximaleffect is obtained with 100 mM ethanol, at whichNP_o reaches 46.2 ± 9% of control. Thepresence of ₁-subunits, which determines channel sensitivity to dihydrosoyaponin-I and 17-estradiol, fails to modifyethanol action on bslo channels. Ethanol inhibition of bslo channels results from a marked increase in the meanclosed time. Although the voltage dependence of gating remainsunaffected, the apparent effectiveness of Ca²⁺ to gate thechannel is decreased by ethanol. These changes occur withoutmodifications of channel conduction. In conclusion, a new molecularmechanism that may contribute to ethanol-induced aortic smooth musclecontraction has been identified and characterized: a functionalinteraction between ethanol and the bslo subunit and/or itslipid microenvironment, which leads to a decrease in BK_Cachannel activity.

     

Effects of Environmental Stressors and Immunosuppressants on Immunity in Fundulus heteroclitus

Environmental stressors have been shown to alter immunocompetencein mammals. Similar effects have been reported in fish. Thepurpose of this paper is to review the literature concerningthe white blood cells and immunity in Fundulus heteroclitus.Evidence for immunosuppression following prolonged captivity,handling, hypophysectomy, radiation, changes in temperatureand salinity, and exposure to chemicals including environmentalpollutants is discussed. New evidence for immunosuppressionfollowing exposure to benzo-a-pyrene, pentachlorophenol, andhexachlorobenzene is presented. Possible mechanisms of immunealteration and the consequences to F. heteroclitus are discussed.     

JNK Pathway Activation Is Controlled by Tao/TAOK3 to Modulate Ethanol Sensitivity

Neuronal signal transduction by the JNK MAP kinase pathway is altered by a broad array of stimuli including exposure to the widely abused drug ethanol, but the behavioral relevance and the regulation of JNK signaling is unclear. Here we demonstrate that JNK signaling functions downstream of the Sterile20 kinase family gene tao/Taok3 to regulate the behavioral effects of acute ethanol exposure in both the fruit fly Drosophila and mice. In flies tao is required in neurons to promote sensitivity to the locomotor stimulant effects of acute ethanol exposure and to establish specific brain structures. Reduced expression of key JNK pathway genes substantially rescued the structural and behavioral phenotypes of tao mutants. Decreasing and increasing JNK pathway activity resulted in increased and decreased sensitivity to the locomotor stimulant properties of acute ethanol exposure, respectively. Further, JNK expression in a limited pattern of neurons that included brain regions implicated in ethanol responses was sufficient to restore normal behavior. Mice heterozygous for a disrupted allele of the homologous Taok3 gene (Taok3Gt) were resistant to the acute sedative effects of ethanol. JNK activity was constitutively increased in brains of Taok3Gt/+ mice, and acute induction of phospho-JNK in brain tissue by ethanol was occluded in Taok3Gt/+ mice. Finally, acute administration of a JNK inhibitor conferred resistance to the sedative effects of ethanol in wild-type but not Taok3Gt/+ mice. Taken together, these data support a role of a TAO/TAOK3-JNK neuronal signaling pathway in regulating sensitivity to acute ethanol exposure in flies and in mice.     

KCNQ Channels Show Conserved Ethanol Block and Function in Ethanol Behaviour

In humans, KCNQ2/3 channels form an M-current that regulates neuronal excitability, with mutations in these channels causing benign neonatal familial convulsions. The M-current is important in mechanisms of neural plasticity underlying associative memory and in the response to ethanol, with KCNQ controlling the release of dopamine after ethanol exposure. We show that dKCNQ is broadly expressed in the nervous system, with targeted reduction in neuronal KCNQ increasing neural excitability and KCNQ overexpression decreasing excitability and calcium signalling, consistent with KCNQ regulating the resting membrane potential and neural release as in mammalian neurons. We show that the single KCNQ channel in Drosophila (dKCNQ) has similar electrophysiological properties to neuronal KCNQ2/3, including conserved acute sensitivity to ethanol block, with the fly channel (IC₅₀ = 19.8 mM) being more sensitive than its mammalian ortholog (IC₅₀ = 42.1 mM). This suggests that the role of KCNQ in alcohol behaviour can be determined for the first time by using Drosophila. We present evidence that loss of KCNQ function in Drosophila increased sensitivity and tolerance to the sedative effects of ethanol. Acute activation of dopaminergic neurons by heat-activated TRP channel or KCNQ-RNAi expression produced ethanol hypersensitivity, suggesting that both act via a common mechanism involving membrane depolarisation and increased dopamine signalling leading to ethanol sedation.     

Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila

A diverse range of organisms shows physiological and behavioural rhythms with various periods. Extensive studies have been performed to elucidate the molecular mechanisms of circadian rhythms with an approximately 24 h period in both Drosophila and mammals, while less attention has been paid to ultradian rhythms with shorter periods. We used a video-tracking method to monitor the movement of single flies, and clear ultradian rhythms were detected in the locomotor behaviour of wild type and clock mutant flies kept under constant dark conditions. In particular, the Pigment-dispersing factor mutant (Pdf ⁰¹ ) demonstrated a precise and robust ultradian rhythmicity, which was not temperature compensated. Our results suggest that Drosophila has an endogenous ultradian oscillator that is masked by circadian rhythmic behaviours.