Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases¹. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials^2-4. The rapid iterative negative geotaxis (RING) assay⁵ has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.     

The effects of age on radiation resistance and oxidative stress in adult Drosophila melanogaster

Drosophila melanogaster (fruit fly) is a well-established model organism for genetic studies of development and aging. We examined the effects of lethal ionizing radiation on male and female adult Drosophila of different ages, using doses of radiation from 200 to 1500 Gy. Fifty percent lethality 2 days postirradiation (LD(50/2)) in wild-type 1-day-old adult fruit flies was approximately 1238 Gy for males and 1339 Gy for females. We observed a significant age-dependent decline in the radiation resistance of both males and females. Radiation damage is postulated to occur by the generation of oxygen radicals. An age-related decline in the ability of flies to resist an agent that induces oxygen radicals, paraquat, was observed when comparing 10- and 20-day adults. Female flies are more resistant to paraquat than male flies. Oxidative stress mediated by paraquat was additive with sublethal exposures to radiation in young adults. Therefore, the ability to repair the damage caused by oxygen radicals seems to decline with the age of the flies. Because Drosophila adults are largely post-mitotic, our data suggest that adult Drosophila melanogaster can serve as an excellent model to study the factors responsible for radiation resistance in post-mitotic tissue and age-dependent changes in this resistance.     

Attraction of Drosophila melanogaster males to food-related and fly odours

The fruit fly Drosophila melanogaster has become a model for olfaction and odour-mediated behaviour. In the wild, Drosophila flies aggregate on decaying fruit where they mate and oviposit and a strategy to find mates would be to locate fruit which has already been colonized by other flies. We therefore developed a bioassay to investigate attraction of males to food and fly odours. We showed that upwind flights are initiated by food odours. At shorter distances, males are attracted by volatiles produced by conspecifics. However, only odours produced by copulating flies attract males. This suggests either a synergistic effect of both male and female odours or changes in pheromone release during mating, that indicate the presence of sexually receptive females. Our findings demonstrate the essential role of food odours and pheromones for mate location in D. melanogaster.     

RNA-based immunity terminates viral infection in adult Drosophila in the absence of viral suppression of RNA interference: characterization of viral small interfering RNA populations in wild-type and mutant flies

Replication of viral RNA genomes in fruit flies and mosquitoes induces the production of virus-derived small interfering RNAs (siRNAs) to specifically reduce virus accumulation by RNA interference (RNAi). However, it is unknown whether the RNA-based antiviral immunity (RVI) is sufficiently potent to terminate infection in adult insects as occurs in cell culture. We show here that, in contrast to robust infection by Flock house virus (FHV), infection with an FHV mutant (FHVΔB2) unable to express its RNAi suppressor protein B2 was rapidly terminated in adult flies. FHVΔB2 replicated to high levels and induced high mortality rates in dicer-2 and argonaute-2 mutant flies that are RNAi defective, demonstrating that successful infection of adult Drosophila requires a virus-encoded activity to suppress RVI. Drosophila RVI may depend on the RNAi activity of viral siRNAs since efficient FHVΔB2 infection occurred in argonaute-2 and r2d2 mutant flies despite massive production of viral siRNAs. However, RVI appears to be insensitive to the relative abundance of viral siRNAs since FHVΔB2 infection was terminated in flies carrying a partial loss-of-function mutation in loquacious required for viral siRNA biogenesis. Deep sequencing revealed a low-abundance population of Dicer-2-dependent viral siRNAs accompanying FHVΔB2 infection arrest in RVI-competent flies that included an approximately equal ratio of positive and negative strands. Surprisingly, viral small RNAs became strongly biased for positive strands at later stages of infection in RVI-compromised flies due to genetic or viral suppression of RNAi. We propose that degradation of the asymmetrically produced viral positive-strand RNAs associated with abundant virus accumulation contributes to the positive-strand bias of viral small RNAs.     

Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster

The facultative intracellular bacterial pathogen Listeria monocytogenes is capable of replicating within a broad range of host cell types and host species. We report here the establishment of the fruit fly Drosophila melanogaster as a new model host for the exploration of L. monocytogenes pathogenesis and host response to infection. Listeria monocytogenes was capable of establishing lethal infections in adult fruit flies and larvae with extensive bacterial replication occurring before host death. Bacteria were found in the cytosol of insect phagocytic cells, and were capable of directing host cell actin polymerization. Bacterial gene products necessary for intracellular replication and cell-to-cell spread within mammalian cells were similarly found to be required within insect cells, and although previous work has suggested that L. monocytogenes virulence gene expression requires temperatures above 30 degrees C, bacteria within insect cells were found to express virulence determinants at 25 degrees C. Mutant strains of Drosophila that were compromised for innate immune responses demonstrated increased susceptibility to L. monocytogenes infection. These data indicate L. monocytogenes infection of fruit flies shares numerous features of mammalian infection, and thus that Drosophila has the potential to serve as a genetically tractable host system that will facilitate the analysis of host cellular responses to L. monocytogenes infection.     

Fruit flies for anti-pain drug discovery

Recent work has indicated that fruit flies (Drosophila melanogaster) can be used in nociception research. Genetic screening identified a gene, painless, that is required for thermal and mechanical nociception in Drosophila larvae. On the other hand, pharmacological techniques and noxious heat were used to assay antinocieceptive behavior in intact adult Drosophila. In general, animal models for pain research are bound by ethical concerns. Since no serious ethical controversies have been raised regarding experiments in insects, Drosophila may be, for the time being an ethically acceptable animal model for combined genetic and pharmacological analgesia research.     

Development of diet-induced insulin resistance in adult Drosophila melanogaster

The fruit fly Drosophila melanogaster is increasingly utilized as an alternative to costly rodent models to study human diseases. Fly models exist for a wide variety of human conditions, such as Alzheimer's and Parkinson's Disease, or cardiac function. Advantages of the fly system are its rapid generation time and its low cost. However, the greatest strength of the fly system are the powerful genetic tools that allow for rapid dissection of molecular disease mechanisms. Here, we describe the diet-dependent development of metabolic phenotypes in adult fruit flies. Depending on the specific type of nutrient, as well as its relative quantity in the diet, flies show weight gain and changes in the levels of storage macromolecules. Furthermore, the activity of insulin-signaling in the major metabolic organ of the fly, the fat body, decreases upon overfeeding. This decrease in insulin-signaling activity in overfed flies is moreover observed when flies are challenged with an acute food stimulus, suggesting that overfeeding leads to insulin resistance. Similar changes were observed in aging flies, with the development of the insulin resistance-like phenotype beginning at early middle ages. Taken together, these data demonstrate that imbalanced diet disrupts metabolic homeostasis in adult D. melanogaster and promotes insulin-resistant phenotypes. Therefore, the fly system may be a useful alternative tool in the investigation of molecular mechanisms of insulin resistance and the development of pharmacologic treatment options.     

Activation of AMPK by the Putative Dietary Restriction Mimetic Metformin Is Insufficient to Extend Lifespan in Drosophila

The biguanide drug, metformin, commonly used to treat type-2 diabetes, has been shown to extend lifespan and reduce fecundity in C. elegans through a dietary restriction-like mechanism via the AMP-activated protein kinase (AMPK) and the AMPK-activating kinase, LKB1. We have investigated whether the longevity-promoting effects of metformin are evolutionarily conserved using the fruit fly, Drosophila melanogaster. We show here that while feeding metformin to adult Drosophila resulted in a robust activation of AMPK and reduced lipid stores, it did not increase lifespan in either male or female flies. In fact, we found that when administered at high concentrations, metformin is toxic to flies. Furthermore, no decreases in female fecundity were observed except at the most toxic dose. Analysis of intestinal physiology after metformin treatment suggests that these deleterious effects may result from disruptions to intestinal fluid homeostasis. Thus, metformin appears to have evolutionarily conserved effects on metabolism but not on fecundity or lifespan.     

Chemosensory apparatus of Drosophila larvae

Many insects, including Drosophila melanogaster, have a rich repertoire of olfactory behavior. Combination of robust behavioral assays, physiological and molecular tools render D. melanogaster as highly suitable system for olfactory studies. The small number of neurons in the olfactory system of fruit flies, especially the number of sensory neurons in the larval stage, makes the exploration of sensory coding at all stages of its nervous system a potentially tractable goal, which is not possible in the foreseeable future in any mammalian preparation. Advances in physiological recordings, olfactory signaling and detailed analysis of behavior, can place larvae in a position to ask previously unanswerable questions.     

Transformation of sensory organs by mutations of the cut locus of D. melanogaster

The identities of two types of sensory organs in the body wall of Drosophila, namely the external sensory organs and the chordotonal organs, are under genetic control. Embryonic lethal mutations in the cut gene complex transform the external sensory organs into chordotonal organs. The neurons, as well as the support cells forming the external sensory structures, change their morphological and antigenic characteristics to those of chordotonal organs, providing genetic evidence that these two types of sensory organs are homologous. Similar transformations of external sensory organs are observed in adult mosaic flies. Analysis of mosaic larvae and flies suggests that the cut gene function is required either in or near external sensory organs in order for them to acquire their correct identity.     

Robustness of Circadian Timing Systems Evolves in the Fruit Fly Drosophila melanogaster as a Correlated Response to Selection for Adult Emergence in a Narrow Window of Time

Robustness is a fundamental property of biological timing systems that is likely to ensure their efficient functioning under a wide range of environmental conditions. Here we report the findings of our study aimed at examining robustness of circadian clocks in fruit fly Drosophila melanogaster populations selected to emerge as adults within a narrow window of time. Previously, we have reported that such flies display enhanced synchrony, accuracy, and precision in their adult emergence and activity/rest rhythms. Since it is expected that accurate and precise circadian clocks may confer enhanced stability in circadian time-keeping, we decided to examine robustness in circadian rhythms of flies from the selected populations by subjecting them to a variety of environmental conditions comprising of a range of photoperiods, light intensities, ambient temperatures, and constant darkness. The results revealed that adult emergence and activity/rest rhythms of flies from the selected stocks were more robust than controls, as they displayed enhanced stability under a wide variety of environmental conditions. These results suggest that selection for adult emergence within a narrow window of time results in the evolution of robustness in circadian timing systems of the fruit fly D. melanogaster. (Author correspondence: vsharma@jncasr.ac.in or vksharmas@gmail.com)     

Behavioral, physical, and demographic changes in Drosophila populations through dietary restriction

Dietary restriction (DR) is a valuable experimental tool for studying the aging process. Primary advancement of research in this area has relied on rodent models, but attention has recently turned toward Drosophila melanogaster. However, little is known about the baseline effects of DR on wild-type Drosophila and continued experimentation requires such information. The findings described here survey the effects of DR on inbred, wild-type populations of Canton-S fruit flies and demonstrate a robust effect of diet on longevity. Over a circumscribed range of dietary conditions, healthy lifespan varies by as much as 121% for wild-type Drosophila females. Significant differences are also observed for male flies, but the magnitude of the DR effect is less robust. Mortality analyses of the survivorship data reveal that this variation in lifespan can be attributed to a modulation of the rate parameter for the mortality function - a change in the demographic rate of aging. Since the feeding of fruit flies is less easily controlled than that of rodents, this research also addresses the validity of applying a DR model to Drosophila populations. Feeding and body weight data for flies given the various dietary conditions surveyed indicate that Drosophila on higher-calorie diets consume a similar volume of food to those on a low-calorie diet, resulting in different levels of calorie intake. Fertility and activity levels demonstrate that the diets surveyed are comparable, and that increasing the calorie content of laboratory food up to twice the normal concentration is not pathologic for experimental fly populations.     

Role of oxidative stress in Drosophila aging.

We review the role that oxidative damage plays in regulating the lifespan of the fruit fly, Drosophila melanogaster. Results from our laboratory show that the lifespan of Drosophila is inversely correlated to its metabolic rate. The consumption of oxygen by adult insects is related to the rate of damage induced by oxygen radicals, which are purported to be generated as by-products of respiration. Moreover, products of activated oxygen species such as hydrogen peroxide and lipofuscin are higher in animals kept under conditions of increased metabolic rate. In order to understand the in vivo relationship between oxidative damage and the production of the superoxide radical, we generated transgenic strains of Drosophila melanogaster that synthesize excess levels of enzymatically active superoxide dismutase. This was accomplished by P-element transformation of Drosophila melanogaster with the bovine cDNA for CuZn superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide radical to hydrogen peroxide and water. Adult flies that express the bovine SOD in addition to native Drosophila SOD are more resistant to oxidative stresses and have a slight but significant increase in their mean lifespan. Thus, resistance to oxidative stress and lifespan of Drosophila can be manipulated by molecular genetic intervention. In addition, we have examined the ability of adult flies to respond to various environmental stresses during senescence. Resistance to oxidative stress, such as that induced by heat shock, is drastically reduced in senescent flies. This loss of resistance is correlated with the increase in protein damage generated in old flies by thermal stress and by the insufficient protection from cellular defense systems which includes the heat shock proteins as well as the oxygen radical scavenging enzymes. Collectively, results from our laboratory demonstrate that oxidative damage plays a role in governing the lifespan of Drosophila during normal metabolism and under conditions of environmental stress.     

Drosophila melanogaster as a model to study drug addiction

Animal studies have been instrumental in providing knowledge about the molecular and neural mechanisms underlying drug addiction. Recently, the fruit fly Drosophila melanogaster has become a valuable system to model not only the acute stimulating and sedating effects of drugs but also their more complex rewarding properties. In this review, we describe the advantages of using the fly to study drug-related behavior, provide a brief overview of the behavioral assays used, and review the molecular mechanisms and neural circuits underlying drug-induced behavior in flies. Many of these mechanisms have been validated in mammals, suggesting that the fly is a useful model to understand the mechanisms underlying addiction.     

鱼藤酮对果蝇运动行为及果蝇头部多巴胺合成相关酶基因表达的影响

为了探讨鱼藤酮对黑腹果蝇Drosophila melanogaster运动行为的影响与其头部多巴胺水平之间的关系,我们测定了鱼藤酮对黒腹果蝇成虫运动行为、头部多巴胺水平及酪氨酸羟化酶和多巴脱羧酶基因表达的影响。结果表明：与取食未加入药剂饲料的果蝇相比雌成虫用0.2～0.8 mmol/L、雄成虫用0.1～0.8 mmol/L浓度药液配制的饲料连续饲养6 d后运动能力显著下降,在0.8 mmol/L浓度下雌、雄成虫的运动能力分别仅为对照的55.6%和49.1%。取食用0.8 mmol/L浓度药液配制饲料6,12和21 d的果蝇雌、雄成虫头部多巴胺水平均显著下降,雌成虫头部多巴胺水平分别为对照雌成虫的83.2%,72.3%和59.8%;雄成虫头部多巴胺水平分别为对照雄成虫的79.3%,66.8%和53.2%。用0.8 mmol/L浓度鱼藤酮处理6,12和21d,雌成虫头部酪氨酸羟化酶基因（pale）的表达水平分别为对照的76.3%,51.4%和37.3%,多巴脱羧酶基因（Ddc）的表达水平分别为对照的87.1%,78.2%和63.5%, 均显著下降。结果提示,鱼藤酮可干扰果蝇成虫头部酪氨酸羟化酶和多巴脱羧酶基因的表达,导致果蝇头部多巴胺水平下降,进而影响了果蝇的运动行为。     

苯甲醛对果蝇视觉联想记忆的阻断

采用飞行模拟系统,以视觉模式为线索、热惩罚为负强化因子,对于在不同发育时期经受苯甲醛处理过的果蝇的视觉飞行定向条件化进行了检验。苯甲醛气味分别作用于果蝇幼虫和成虫阶段,将阻断果蝇成虫建立视觉联想记忆的能力;雌性果蝇在处女期对苯甲醛气味的接触,会阻断其子代建立视觉联想记忆,这种视觉联想记忆的能力可以通过对其子代连续３代的正常饲养而逐渐得到恢复。     

Ecological genetics of abdominal pigmentation in Drosophila falleni: a pleiotropic link to nematode parasitism

Drosophila falleni belongs to the quinaria species group, whose species vary considerably in patterns of wing and abdominal pigmentation. Drosophila falleni itself exhibits substantial variation among wild flies in abdominal spotting patterns. A selection experiment revealed that natural populations of D. falleni harbor high levels of genetic variation for spot number: in 10 generations of selection modal spot number within populations declined from 18 (the modal number in wild-caught females) to as low as zero. Rearing flies at different temperatures shows that some of the variation among wild flies is likely to reflect variation in the environmental conditions under which they developed. Fitness assays did not reveal any cost of reduced spot number with respect to development time, adult survival, or female fecundity. However, spotless flies were almost twice as susceptible to infection by the nematode parasite Howardula aoronymphium. Thus, selection exerted by nematode parasites may influence pigmentation patterns and other, genetically correlated traits in natural populations D. falleni.     

Delayed behavioural aging and altered mortality in Drosophila beta integrin mutants

The genetic basis for aging is being intensely investigated in a variety of model systems. Much of the focus in Drosophila has been on the molecular-genetic determinants of lifespan, whereas the molecular-genetic basis for age-related functional declines has been less vigorously explored. We evaluated behavioural aging and lifespan in flies harbouring loss-of-function mutations in myospheroid, the gene that encodes betaPS, a beta integrin. Integrins are adhesion molecules that regulate a number of cellular processes and developmental events. Their role in aging, however, has received limited attention. We report here that age-related declines in locomotor activity are ameliorated and that mean lifespan is increased in myospheroid mutants. The delayed functional senescence and altered mortality in myospheroid flies are independent of changes in body size, reproduction or stress resistance. Our data indicate that functional senescence and age-dependent mortality are influenced by beta integrins in Drosophila.     

The making of a pest: the evolution of a fruit-penetrating ovipositor in Drosophila suzukii and related species

Evolutionary innovation can allow a species access to a new ecological niche, potentially reducing competition with closely related species. While the vast majority of Drosophila flies feed on rotting fruit and other decaying matter, and are harmless to human activity, Drosophila suzukii, which has a morphologically modified ovipositor, is capable of colonizing live fruit that is still in the process of ripening, causing massive agricultural damage. Here, we conducted the first comparative analysis of this species and its close relatives, analysing both ovipositor structure and fruit susceptibility. We found that the ovipositor of the species most closely related to D. suzukii, Drosophila subpulchrella, has a similar number of enlarged, evolutionarily derived bristles, but a notably different overall shape. Like D. suzukii, D. subpulchrella flies are capable of puncturing the skin of raspberries and cherries, but we found no evidence that they could penetrate the thicker skin of two varieties of grapes. More distantly related species, one of which has previously been mistaken for D. suzukii, have blunt ovipositors with small bristles. While they did not penetrate fruit skin in any of the assays, they readily colonized fruit interiors where the skin was broken. Our results suggest that considering evolutionary context may be beneficial to the management of invasive species.