Fly Activity Research Articles

CG17192 is a Phospholipase That Regulates Signaling Lipids in the Drosophila Gut upon Infection.

The chemoproteomics technique, activity-based protein profiling (ABPP), has proven to be an invaluable tool in assigning functions to enzymes. The serine hydrolase (SH) enzyme superfamily, in particular, has served as an excellent example in displaying the versatility of various ABPP platforms and has resulted in a comprehensive cataloging of the biochemical activities associated within this superfamily. Besides SHs, in mammals, several other enzyme classes have been thoroughly investigated using ABPP platforms. However, the utility of ABPP platforms in fly models remains underexplored. Realizing this knowledge gap, leveraging complementary ABPP platforms, we reported the full array of SH activities during various developmental stages and adult tissues in the fruit fly (Drosophila melanogaster). Following up on this study, using ABPP, we mapped SH activities in adult fruit flies in an infection model and found that a gut-resident lipase CG17192 showed increased activity during infection. To assign a biological function to this uncharacterized lipase, we performed an untargeted lipidomics analysis and found that phosphatidylinositols were significantly elevated when CG17192 was depleted in the adult fruit fly gut. Next, we overexpressed this lipase in insect cells, and using biochemical assays, we show that CG17192 is a secreted enzyme that has phospholipase C (PLC) type activity, with phosphatidylinositol being a preferred substrate. Finally, we show during infection that heightened CG17192 regulates phosphatidylinositol levels and, by doing so, likely modulates signaling pathways in the adult fruit fly gut that might be involved in the resolution of this pathophysiological condition.

Adult dietary experience influences mortality of the pest melon fly, Zeugodacus cucurbitae (Diptera: Tephritidae), to an ingested toxin

AbstractIngested toxins formulated as baits can be effective in the management of insect pests, but their efficacy may be influenced by insect foraging alongside active ingredients. There are several components that might influence insect consumption of toxic baits, one of which being prior nutritional exposure. In our study, we investigated how protein deprivation following adult emergence influences lethal and sublethal effects of boric acid on the pest tephritid melon fly, Zeugodacus cucurbitae. We performed a series of experiments to address the impact of prior diet on mortality, diet consumption, enzymes involved in detoxification and antioxidation and fly activity. Newly emerged melon fly adults were provided either diet containing 3:1 sucrose/yeast hyrdrolysate or just sucrose for three days prior to bioassays. Overall, protein deprivation had a strong effect on fly interactions with boric acid. In two separate experiments (continuous exposure and short duration feeding), insects only pre‐fed sucrose had significantly higher mortality compared with those pre‐fed protein. Insects that were pre‐fed only sucrose removed more liquid diet over the same duration of the experiments, suggesting compensatory foraging behaviours. Adults pre‐fed sucrose had higher catalase (1.5×) and superoxide dismutase (1.7×) activities compared with adults pre‐fed sucrose‐yeast, but we found no effect of boric acid on their activity or that of glutathione‐S‐transferase or acetylcholinesterase activity. Boric acid consumption also significantly reduced the activity of melon flies. Overall, our results demonstrate that prior diet in fly adulthood can impact subsequent exposure to and efficacy of ingested toxicants.

Can Monitoring Data of Forensically Important Blow Flies Be Transferred Between Cities?

ABSTRACTThe use of monitoring data to build prediction models for the abundance and activity of necrophagous blow flies is common practice in forensic entomology, but its advantages and disadvantages are still debated. A frequently asked question is the transferability of such species prediction models. So far no study has examined whether the assumption of low transferability of these data between cities and seasons holds true. In the present study, we evaluate whether models calibrated with a specific training data set from a specific place and time can be transferred to other data sets for different time periods and locations. We developed models using five different algorithms to predict the activity and abundance of four forensically relevant blow fly species (Calliphora vicina Robineau‐Desvoidy, Lucilia ampullacea Villeneuve, Lucilia caesar (Linnaeus), Lucilia sericata (Meigen)). The training data set was obtained from a single city, and the transferability of the models was evaluated using monitoring data from this and three other cities. The geographic transferability of the models was confirmed for all algorithms, but only for two species, C. vicina and L. sericata, and for two of the four cities. Lucilia caesar and L. ampullacea were rare in the test data set, and their species‐specific adaptation to environmental parameters was not captured by the models. Cities that did not work differed from the training data set in terms of climate and habitat features. To build generalised predictive models of blow fly abundance and activity, we need training data sets based on monitoring data from different regions, seasons and years to cover a wide range of environmental conditions. This is essential for describing and predicting natural variability.

Kaempferol Extends Male Lifespan Under Blue Light Irradiation in Drosophila.

Short-wavelength blue light is ubiquitous in daily life and has a lasting destructive influence. Its potential harm to biological health is significant. This study used Drosophila as a model organism to investigate the protective effects of kaempferol, a flavonoid, against the toxicity of blue light. It also examined its physiological effects on Drosophila under blue light irradiation. In this experiment, fruit flies were fed with three different concentrations of kaempferol solutions (0.1, 0.01, and 0.001 mol/L) dissolved in food. The survival rate and physiological indexes of Drosophila were investigated under blue light irradiation of 2500 lux. The results showed that 0.1 mol/L kaempferol increased the activity of male flies during the day and significantly extended the male survival time under blue light irradiation. However, the study found that kaempferol did not significantly prolong the survival time of Drosophila in the oxidative stress experiment, and no significant difference was observed in the feeding experiment. In summary, our research found that kaempferol, at the concentration of 0.1 mol/L, has a protective effect on Drosophila under blue light irradiation, potentially achieved through alterations in circadian rhythm.

Nutritional Profile and Antibacterial Activity of the Black Soldier Fly (Hermetia illucens) Larvae Meal as Potential Protein Source for Aquafeeds

Nutritional Profile and Antibacterial Activity of the Black Soldier Fly (Hermetia illucens) Larvae Meal as Potential Protein Source for Aquafeeds

Cross-resistance patterns in field strains of the sheep blowfly following laboratory-based selection pressure with dicyclanil or imidacloprid

Control of the sheep blowfly relies largely on the use of insecticides applied prophylactically in advance of expected fly activity. However, the blowfly has shown an ability to develop resistance to some of these insecticides. Recent reports of the co-occurrence of resistance to both dicyclanil and imidacloprid in in vitro bioassays with field-collected fly strains has raised the possibility that the two resistances may represent cross-resistance linked by a common mechanism. We investigated this by imposing insecticide selection pressure on larvae of two insecticide-resistant field strains over a number of generations using either dicyclanil or imidacloprid and then measuring changes in sensitivity to both the selecting chemical and the alternate chemical. Larvae selected over six generations with each chemical showed significant increases in resistance to the selecting chemical: resistance ratios at the IC50 5.5 - 8.1-fold higher for dicyclanil, and 3.1 - 3.8-fold for imidacloprid. The larvae also showed significant increases in levels of resistance towards the alternate chemical: resistance ratios 2.6 - 3.1-fold higher towards dicyclanil following selection with imidacloprid, and 2.2 - 3.2-fold higher towards imidacloprid following selection with dicyclanil. The increases in resistance to both chemicals after exposure to either suggests a common mechanism of resistance, at least in our laboratory-selected populations. Assays with the cytochrome P450 inhibitor aminobenzotriazole showed that this synergist was able to remove the increased resistance to both compounds in strains selected with either compound, suggesting that cytochrome P450 is responsible for the resistance observed to both chemicals. Our results confirm that cross-resistance exists between dicyclanil and imidacloprid in the sheep blowfly, and hence the two compounds should be considered as related entities in insecticide rotation strategies for flystrike control.

Gut Microbiota Dynamics and Phenotypic Changes Induced by Tetracycline in Drosophila melanogaster

Gut microbiota plays a crucial role in both physiological and pathological processes in humans and animals. Antibiotics, designed to combat bacterial infections, can induce alterations in the composition and abundance of the gut microbiota over prolonged exposure. This study addresses the limited understanding of the connections between gut microbiota and phenotypic profiles of metazoan species. We investigated the impact of early-life exposure to tetracycline in wild-type D. melanogaster, which were fed a standard diet, comparing them to a control group not exposed to tetracycline. The primary objective was to examine the consequences of early-life tetracycline exposure on gut microbiota and its implications for phenotypic profiles, including survival, locomotor activity, and reproduction in adult flies. Results revealed a significant reduction in lactic acid bacteria in adult flies exposed to tetracycline. However, tetracycline exhibited no interference with fly development, allowing them to maintain a normal lifespan. In adult flies, tetracycline significantly decreased the lifespan on day 35 at a concentration of 1000 µg/mL and reduced locomotion on day 27 at concentrations of 10 µg/mL and 1000 µg/mL. Remarkably, tetracycline did not impact the reproductive capabilities of the flies. This study demonstrates that while tetracycline led to a decline in lactic acid bacteria, locomotion, and lifespan in adult flies, it did not disrupt their development or reproductive processes.

Nocturnal activity and forestry-urban dispersal of phlebotomine sand flies (Diptera: Psychodidae) from an ecotourism park in Amazonian Brazil.

Phlebotomine sand flies are insects of notorious importance in public health, mainly due to their involvement in the transmission of Leishmania protozoa. Their flight activity occurs predominantly in the twilight/night period, being stimulated mainly by the need to search for food and reproduction. Despite being naturally wild, some species are able to invade anthropized environments. Present work aimed to assess the nocturnal activity and forestry-urban dispersal of phlebotomine sand flies from an ecotourism park in Belém, Amazonian Brazil. The study area comprised a horizontal transect, extending from a forest park to the neighboring urban environment, in Belém. Sampling was conducted with night-operating light traps. Nocturnal activity was assessed through time-set captures with a collection bottle rotator in the forest environment. Dispersal was assessed through captures carried out along the transect, starting from the forest edge (0 m), extending to the urban environment (50-200 m), phlebotomine sand flies were identified. Abundance, richness, diversity, and sampling sufficiency were estimated. Fourteen species were recorded in the surveyed environments, with Nyssomyia antunesi, Trichophoromyia brachipyga, and Trichophoromyia ubiquitalis being the most abundant. Nocturnal activity behavior of Ny. antunesi occurred between 8 PM and 4 AM while that of Th. brachipyga and Th. ubiquitalis occurred between 2 AM and 6 AM. In the urban environment, the 150 m site presented the highest abundance. Bichromomyia flaviscutellata was sampled in all sites. Gravid females of Pressatia choti and Bi. flaviscutellata were sampled in the urban environment. Putative differential activity between the species herein assessed and their urban dispersal observed are worthy of note, adding data for supporting vector surveillance at a local scale.

Transgenic Drosophila melanogaster Carrying a Human Full-Length DISC1 Construct (UAS-hflDISC1) Showing Effects on Social Interaction Networks.

Disrupted in Schizophrenia 1 (DISC1) is a scaffold protein implicated in major mental illnesses including schizophrenia, with a significant negative impact on social life. To investigate if DISC1 affects social interactions in Drosophila melanogaster, we created transgenic flies with second or third chromosome insertions of the human full-length DISC1 (hflDISC1) gene fused to a UAS promotor (UAS-hflDISC1). Initial characterization of the insertion lines showed unexpected endogenous expression of the DISC1 protein that led to various behavioral and neurochemical phenotypes. Social interaction network (SIN) analysis showed altered social dynamics and organizational structures. This was in agreement with the altered levels of the locomotor activity of individual flies monitored for 24 h. Together with a decreased ability to climb vertical surfaces, the observed phenotypes indicate altered motor functions that could be due to a change in the function of the motor neurons and/or central brain. The changes in social behavior and motor function suggest that the inserted hflDISC1 gene influences nervous system functioning that parallels symptoms of DISC1-related mental diseases in humans. Furthermore, neurochemical analyses of transgenic lines revealed increased levels of hydrogen peroxide and decreased levels of glutathione, indicating an impact of DISC1 on the dynamics of redox regulation, similar to that reported in transgenic mammals. Future studies are needed to address the localization of DISC1 expression and to address how the redox parameter changes correlate with the observed behavioral changes.

SOME BIOLOGICAL AND ECOLOGICAL FEATURES OF HYPODERMA LATREILLE, 1818 IN TAVUSH REGION OF ARMENIA

Background. Cattle hypodermosis is a chronic parasitic disease (entomosis) that occurs as a result of parasitizing the larvae of warble flies in the body of animals. Disease is common in more than 55 countries of the world, causing great economic damage. It is considered as a parasitic disease with a certain zoonotic potential. Purpose. To conduct research on biological and ecological features of the warble flies that determines certain epidemiological traits of the caused pathology in Tavush Region of Armenia. Materials and methods. The observations were carried out in Tavush Region, and the experiments were carried out at the Research Center for Veterinary Medicine and Veterinary Sanitary Expertise at the Armenian National Agrarian University. The warble flies marked with stain, were released at a distance of 0.5 to 1.5 km from livestock farms with subsequent search and trapping of insects, located on pastures and on the skin of animals. Generally, 275 warble flies were released. Warble fly activity was evaluated visually counting the comparative number of flying insects at the fixed point per minute at different times of the day, as well as animal anxiety state caused by warble flies was identified. Statistical processing of experimental data was performed by means of “Vassart.net” online calculator. Results. Female warble flies Hypoderma bovis flew up to 12.1 km, while female warble flies H. lineatum did it up to 8.6 km. Females of H. bovis and H. lineatum demonstrated various spatial and circadian flying activity that depends on season, and altitude above the sea level. Spring generation of warble flies is more resistant to low air temperatures compared to the summer generation. Mild and humid summers, as well as an increase in the number of animals, contribute to an increase in the population of warble flies, while high temperature and high soil moisture contribute to the mass death of warble fly pupae developing in the ground. Conclusion. The research data cited in the article, detect the presence of a wide range of biocenotic factors that determine the epidemiology of cattle hypodermosis in the Tavush region of the Republic of Armenia. Cattle hypodermosis control plan was designed based on the results of the research, as well as natural and climatic conditions of Tavush region. Pasture prevention activities and periodic treatment of the cattle with insecticides were emphasized.

Effect of Malathion on Catalase Enzyme and Acetylcholinesterase Activity in Adult Flies and 3rd Instar Larvae of Drosophila melanogaster

Effect of Malathion on Catalase Enzyme and Acetylcholinesterase Activity in Adult Flies and 3rd Instar Larvae of Drosophila melanogaster

Hover fly (Diptera: Syrphidae) diversity and seasonality in North Georgia apple and peach orchards.

Crop pollination and natural biological control provided by beneficial insects have an economic worth of hundreds of billions of dollars annually. Apple and peach production in North Georgia are economically important industries that benefit from these ecological services. Hover flies are dual ecosystem service providers that have been relatively understudied in orchard ecosystems. We investigated the diversity and seasonal activity of hover flies in apple and peach orchards at 2 sites in North Georgia from March to October 2020 and 2021. Bowl traps were used to sample hover flies in orchard edge and interior habitats. The aphidophagous species Toxomerus geminatus (Say) (Diptera: Syrphidae) and Toxomerus marginatus (Say) (Diptera: Syrphidae) comprised 86.6% of the total hover flies collected. Apple orchards yielded the greatest hover fly presence, species richness, and Toxomerus spp. abundance. Hover fly richness and diversity were greatest during postbloom, but Toxomerus spp. abundance was greatest during the bloom period. No differences in presence, richness, diversity, or Toxomerus spp. abundance were found between edge and interior habitats. Toxomerus geminatus and T. marginatus were dominant from March through August, with T. geminatus being more abundant than T. marginatus in March, early April, and August. October sampling produced the greatest hover fly richness. Our results suggest that hover flies are abundant in North Georgia orchards and exhibit substantial spatial and temporal variation in richness and diversity. Expanded studies incorporating additional sampling efforts and methods are needed to further characterize the hover fly fauna and their impact on North Georgia apple and peach orchards.

Antibacterial activity of the black soldier fly (Hermetia illucens) larva protein hydrolysates against some pathogenic bacterial strains on freshwater fish

The study was carried out to determine the antibacterial activity of protein hydrolysates from black soldier fly (Hermetia illucens) larva (PHBSF) on some pathogenic bacteria including E. ictaluri, A. hydrophila, A. verroni, and S. agalactiae on freshwater fish. The results showed that the tested bacteria were highly sensitive with significant difference (P < 0.05) in diameter clear zone compared to the negative control at the tested concentrations of 20, 35, and 70 mg/100 µL. Particularlly, E. ictaluri was sensitive at all three test concentrations with the diameter of clear zones following 14.0 ± 1.0, 18.7 ± 0.7, and 20.7 ± 0.7 mm, respectively. A. veronii and S. agalactiae were sensitive at concentrations of 35 and 70 mg/100 µL, and A. hydrophila with a clear zone diameter of 14.3 ± 0.3 mm at the concentration of 70 mg/100 µL. The PHBSF was able to kill A. veronii and S. agalactiae with MIC (minimum inhibitory concentration) = MBC (minimum bactericidal concentration) = 44 mg/mL, while E. ictaluri and A. hydrophila were inhibited at MIC 44 mg/mL, and MBC was 88 mg/mL. These results indicated that PHBSF could be potentially used to prevent fish diseases caused by E. ictaluri, A. hydrophila, A. verroni, and S. agalactiae.

Feeding Drosophila gut microbiomes from young and old flies modifies the microbiome

It is becoming increasingly evident that the myriad of microbes in the gut, within cells and attached to body parts (or roots of plants), play crucial roles for the host. Although this has been known for decades, recent developments in molecular biology allow for expanded insight into the abundance and function of these microbes. Here we used the vinegar fly, Drosophila melanogaster, to investigate fitness measures across the lifetime of flies fed a suspension of gut microbes harvested from young or old flies, respectively. Our hypothesis was that flies constitutively enriched with a ‘Young microbiome’ would live longer and be more agile at old age (i.e. have increased healthspan) compared to flies enriched with an ‘Old microbiome’. Three major take home messages came out of our study: (1) the gut microbiomes of young and old flies differ markedly; (2) feeding flies with Young and Old microbiomes altered the microbiome of recipient flies and (3) the two different microbial diets did not have any effect on locomotor activity nor lifespan of the recipient flies, contradicting our working hypothesis. Combined, these results provide novel insight into the interplay between hosts and their microbiomes and clearly highlight that the phenotypic effects of gut transplants and probiotics can be complex and unpredictable.

The landscape of fear in cattle farms? How the presence of barn swallow influences the activity of pest flies

Abstract Insectivorous birds are key suppliers of crucial ecosystem services, especially important for pest control in agricultural systems, and are globally decreasing at a fast rate. Among them, the declining barn swallow Hirundo rustica is particularly linked to cattle farms, where it is impacted by changes in livestock husbandry practices. The presence of swallows in cattle sheds might contribute to control insect pests, but such an effect had never been explored. With this work, we evaluated the potential control effect exerted by barn swallow on economically relevant pest insects, filth flies. To assess whether swallows inside the cattle sheds influence the activity rate of pest flies (especially Musca domestica and Stomoxys calcitrans), we assessed the relative abundance of swallows and flies over 16 weeks in nine different cattle farms in Non valley (Trentino, North‐east Italy), either occupied or not by nesting swallows. From April 2022 to August 2022, we collected weekly data on the activity rate of flies by placing eight white spot cards inside each of nine cattle sheds and counting the deposition of faecal and regurgitation spots by flies. Simultaneously, we collected weekly data regarding swallow occurrence, brood size, number and age of chicks. We applied generalised linear mixed effect models on the activity rate of flies, with spot card and farm as nested random effects, to account for potential random variability between cards and farms. We then assessed through model selection the effect of number of cows, number of swallows, and temperature or week alternatively. Temperature and week were modelled also in interaction with the number of swallows. Fly activity rate followed a seasonal trend with a peak in July, which was markedly lower where swallows were present. Moreover, the positive relationship between flies and temperature was lessened when swallows were abundant. Synthesis and applications. Barn swallows are effective biological control agents for lowering pest fly activity in livestock farms. Farmers were actively involved in swallow conservation within their sheds, also with a shared memorandum‐of‐intent. Harnessing the ecosystem services supplied by farmland birds could bring positive outcomes for people and biodiversity.

The possible role of haematophagous flies in the incidence of bovine teat papillomatosis.

The relationship between the incidence of bovine teat papillomatosis and the activity of haematophagous flies was investigated in Japan. A total of 15,737 flies consisting of 33 species were collected by dry ice-baited mosquito net (DMN) trap and a sweep net from udders of cattle. Simulium aokii (Takahasi) of Simuliidae (black flies) was the predominant species, followed by S. tobetsuense Ono and S. iwatense (Shiraki). Simulium aokii had the highest peak in October, followed by September. Numbers of blood spots from the bites per teat in nulliparous cattle were significantly correlated with numbers of S. aokii collected by DMN trap. Numbers of teats with warts and spots of blood from the bites per teat were significantly more abundant in anterior teats than posterior teats. The average incidence of teat papillomatosis in nulliparous cattle was significantly higher than that in parous cattle, and the highest incidence by month was in May, followed by April. Although bovine papillomavirus (BPV) DNA was not detected in flies examined, the presence of black flies and blood spots from their bites were associated with subsequent high incidence of growing warts. In particular, it would pay to give attention to species such as S. aokii that severely attack udders in the present locality. Further investigations for the detection of BPV DNA from flies parasitizing on teats are needed.

Recording physiological history of cells with chemical labeling.

Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.

A neural pathway for social modulation of spontaneous locomotor activity (SoMo-SLA) in Drosophila

Social enrichment or social isolation affects a range of innate behaviors, such as sex, aggression, and sleep, but whether there is a shared mechanism is not clear. Here, we report a neural mechanism underlying social modulation of spontaneous locomotor activity (SoMo-SLA), an internal-driven behavior indicative of internal states. We find that social enrichment specifically reduces spontaneous locomotor activity in male flies. We identify neuropeptides Diuretic hormone 44 (DH44) and Tachykinin (TK) to be up- and down-regulated by social enrichment and necessary for SoMo-SLA. We further demonstrate a sexually dimorphic neural circuit, in which the male-specific P1 neurons encoding internal states form positive feedback with interneurons coexpressing doublesex (dsx) and Tk to promote locomotion, while P1 neurons also form negative feedback with interneurons coexpressing dsx and DH44 to inhibit locomotion. These two opposing neuromodulatory recurrent circuits represent a potentially common mechanism that underlies the social regulation of multiple innate behaviors.

Unlocking the Potential of Substrate Quality for the Enhanced Antibacterial Activity of Black Soldier Fly against Pathogens.

Globally, antibiotics are facing fierce resistance from multidrug-resistant bacterial strains. There is an urgent need for eco-friendly alternatives. Though insects are important targets for antimicrobial peptides, it has received limited research attention. This study investigated the impact of waste substrates on the production of antibacterial agents in black soldier fly (Hermetia illucens L.) larvae (HIL) and their implications in the suppression of pathogens [Bacillus subtilis (ATCC 6051), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Escherichia coli (ATCC 25922)]. The 20% acetic acid (AcOH) extract from market waste had the highest antibacterial activity with an inhibition zone of 17.00 mm, followed by potato waste (15.02 mm) against S. aureus. Hexane extract from HIL raised on market waste also showed a significant inhibitory zone (13.06 mm) against B. subtilis. .Minimum inhibitory concentration (MIC) values recorded were 25 mg/mL against all test pathogens. The fastest time-kill of 20% AcOH extract was 4 h againstB. subtilis, E. coli, ,andP. aeruginosa. Lauric acid was also identified as the dominant component of the various hexane extracts with concentrations of 602.76 and 318.17 μg/g in HIL reared on potato and market waste, respectively. Energy from the market waste substrate correlated significantly (r = 0.97) with antibacterial activities. This study highlights the key role of substrate quality and extraction methods for enhancing the production of antibacterial agents in HIL, thus providing new insights into the development of potential drugs to overcome the alarming concerns of antimicrobial resistance.

Neural Stimulation during Drosophila Activity Monitor (DAM)-Based Studies of Sleep and Circadian Rhythms in Drosophila melanogaster.

Sleep is a fundamental feature of life for virtually all multicellular animals, but many questions remain about how sleep is regulated by circadian rhythms, homeostatic sleep drive that builds up with wakefulness, and modifying factors such as hunger or social interactions, as well as about the biological functions of sleep. Substantial headway has been made in the study of both circadian rhythms and sleep in the fruit fly Drosophila melanogaster, much of it through studies of individual fly activity using Drosophila activity monitors (DAMs). Here, we describe approaches for the activation of specific neurons of interest using optogenetics (involving genetic modifications that allow for light-based neuronal activation) and thermogenetics (involving genetic modifications that allow for temperature-based neuronal activation) so that researchers can evaluate the roles of those neurons in controlling rest and activity behavior. In this protocol, we describe how to set up a rig for simultaneous optogenetic or thermogenetic stimulation and activity monitoring for analysis of sleep and circadian rhythms in Drosophila, how to raise appropriate flies, and how to perform the experiment. This protocol will allow researchers to assess the causative role in the regulation of sleep and activity rhythms of any genetically tractable subset of cells.