Three mutants have been isolated in which the normal 24-hour rhythm is drastically changed.One mutant is arrhythmic; another has a period of 19 hr; a third has a period of 28 hr. Both the eclosion rhythm of a population and the locomotor activity of individual flies are affected. All these mutations appear to involve the same functional gene on the X chromosome.Rhythmic variations in behavior are displayed by many organisms, ranging from single cells to man (1). When the rhythm persists under constant conditions, and has a period of around one day, depending little on temperature, the rhythm is called circadian (2). Many experiments have attempted to probe the mechanism (3), but the nature of the underlying oscillation remains unknown (4). Perturbations by inhibitors of RNA or protein synthesis suggest that such molecules are involved (5-8). Biochemical systems that oscillate with much shorter periods have been demonstrated both in vivo and in vitro (9, 10), but their relation to circadian rhythms is not clear.An approach that has been successful in unravelling mechanisms in some systems is the use of genetic alterations. Since the expression of a rhythm requires an integrated system, mutation of the genes responsible for development and function of the system could lead to abnormal rhythms. Various aspects of circadian rhythms have indeed been shown to be sensitive to genetic makeup (11-18). For genetic dissection of circadian rhythms in an organism having a nervous system, Drosophila offers certain advantages. Much is already known about the rhythm of eclosion (emergence of the adult fly from the pupa), and genetic methodology is readily available. This paper describes the first result of such an analysis. MATERIALS AND METHODSIsolation of mutants D. melanogaster of the C-S (Canton-Special) wild strain was maintained on cornmeal medium. Mutagenesis by ethyl methane sulfonate was according to Lewis and Bacher (19), the treated males being mated to virgin attached-X females, so that each F1 progeny male carri&Va treated X chromosome received from his father. Each male was mated individually to attached-X females, producing a stock of males bearing identical X chromosomes, plus normal-rhythm attached-X females. The stocks were reared at constant temperature under LD 12:12 (12 hr of 50 foot-candles or more of white fluorescent light, 12 hr of darkness each day).To detect X-linked rhythm mutants, the stocks were examined for ones in which males emerged abnormally. The normal females in each bottle served as an internal control, at least twice as many emerging during the light as during the dark period. In a few bottles, males emerged in approximately equal numbers during day and night. Each mutant candidate was examined in more detail by raising pupae in LD 12:12, then monitoring the adult eclosion rhythm in constant darkness. From a total of about 2000 F1 males, three rhythm mutants were obtained. Determination of eclosion and locomotor activity rhythmsEclosion rhythms, free-running in constant darkness, w...

We describe a paradigm for nociception in Drosophila. In response to the touch of a probe heated above 38 degrees C, Drosophila larvae produce a stereotypical rolling behavior, unlike the response to an unheated probe. In a genetic screen for mutants defective in this noxious heat response, we identified the painless gene. Recordings from wild-type larval nerves identified neurons that initiated strong spiking above 38 degrees C, and this activity was absent in the painless mutant. The painless mRNA encodes a protein of the transient receptor potential ion channel family. Painless is required for both thermal and mechanical nociception, but not for sensing light touch. painless is expressed in peripheral neurons that extend multiple branched dendrites beneath the larval epidermis, similar to vertebrate pain receptors. An antibody to Painless binds to localized dendritic structures that we hypothesize are involved in nociceptive signaling.

In many species, reducing nutrient intake without causing malnutrition extends lifespan. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence we examined the role of TOR pathway genes in regulating lifespan by using Drosophila. We show that inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila, TSC1 and TSC2 (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth. We find that overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction.

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Studies of feeding behavior in genetically tractable invertebrate model systems have been limited by the lack of proper methodology. We introduce the Capillary Feeder (CAFE), a method allowing precise, real-time measurement of ingestion by individual or grouped fruit flies on the scale of minutes to days. Using this technique, we conducted the first quantitative analysis of prandial behavior in Drosophila melanogaster. Our results allow the dissection of feeding into discrete bouts of ingestion, defining two separate parameters, meal volume and frequency, that can be uncoupled and thus are likely to be independently regulated. In addition, our long-term measurements show that flies can ingest as much as 1.7؋ their body mass over 24 h. Besides the study of appetite, the CAFE can be used to monitor oral drug delivery. As an illustration, we used the CAFE to test the effects of dietary supplementation with two compounds, paraquat and ethanol, on food ingestion and preference. Paraquat, a prooxidant widely used in stress tests, had a strong anorexigenic effect. In contrast, in a feeding preference assay, ethanol-laced food, but not ethanol by itself, acted as an attractant.appetite ͉ feeding ͉ ingestion ͉ preference U nderstanding the physiology and regulation of appetite is an indispensable step in tackling biomedical problems such as obesity and feeding disorders. Invertebrate model systems have provided invaluable mechanistic insight into the genetic control of various biological and pathological processes, but have contributed relatively little to the understanding of the genetic underpinnings and neuronal circuitry of appetite regulation. This dearth is largely due to the limits of the available methodology. In both Caenorhabditis elegans and Drosophila melanogaster, feeding behavior is often inferred from qualitative parameters such as the amount of time spent on a given food source or the percentage of animals from a population seen eating or simply loitering on the medium at a given time (1-3). A more direct method, widely used in the nematode, is the pharyngeal pumping rate, which assumes a constant ingestion volume per pharyngeal contraction (4-6). In Drosophila, food can be labeled with nonabsorbable dyes (6, 7) or radioactive isotopes (8-12), but these techniques also have several limitations. Dyes progress rapidly through the digestive tract, precluding long-term measurements. Isotope labeling, on the other hand, permits longterm recordings but does not distinguish between ingestion and intestinal absorption, leading to permanent tissue incorporation. Most importantly, labeling methods require killing the flies for each measurement, making it impossible to continuously monitor the behavior of individual animals.We describe a method allowing unambiguous recording of food ingestion in individual or groups of flies on the scale of minutes to the entire lifespan. Monitoring ingestion at short, 10-min intervals permitted the delineation of single meals. By modulating nutrient composition, we show that the pa...