Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.     

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.     

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.     

Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans

Wolbachia manipulate insect host biology through a variety of means that result in increased production of infected females, enhancing its own transmission. A Wolbachia strain (wInn) naturally infecting Drosophila innubila induces male killing, while native strains of D. melanogaster and D. simulans usually induce cytoplasmic incompatibility (CI). In this study, we transferred wInn to D. melanogaster and D. simulans by embryonic microinjection, expecting conservation of the male-killing phenotype to the novel hosts, which are more suitable for genetic analysis. In contrast to our expectations, there was no effect on offspring sex ratio. Furthermore, no CI was observed in the transinfected flies. Overall, transinfected D. melanogaster lines displayed lower transmission rate and lower densities of Wolbachia than transinfected D. simulans lines, in which established infections were transmitted with near-perfect fidelity. In D. simulans, strain wInn had no effect on fecundity and egg-to-adult development. Surprisingly, one of the two transinfected lines tested showed increased longevity. We discuss our results in the context of host-symbiont co-evolution and the potential of symbionts to invade novel host species.     

N‐β‐alanyldopamine metabolism,locomotor activity and sleep in Drosophila melanogaster ebony and tan mutants

Drosophila melanogaster Meigen mutants for N‐β‐alanyldopamine (NBAD) metabolism have altered levels of NBAD, dopamine and other neurotransmitters. The ebony¹ mutant strain has very low levels of NBAD and higher levels of dopamine, whereas the opposite situation is observed in the tan¹ mutant. Dopamine is implicated in the control of movement, memory and arousal, as well as in the regulation of sleep and wakefulness in D. melanogaster. N‐β‐alanyldopamine, which is best known as a cuticle cross‐linking agent, is also present in nervous tissue and has been proposed to promote locomotor activity in this fly. The daily locomotor activity and the sleep patterns of ebony¹ and tan¹ mutants are analyzed, and are compared with wild‐type flies. The tan¹ mutant shows reduced locomotor activity, whereas ebony¹ shows higher levels of activity than wild‐type flies, suggesting that NBAD does not promote locomotor activity. Both mutants spend less time asleep than wild‐type flies during night‐time; ebony shows more consolidated activity during night‐time and increased sleep latency, whereas tan is unable to consolidate locomotor activity and sleep in either phase of the day. The daily level of NBAD‐synthase activity is measured in vitro using wild‐type and tan¹ protein extracts, and the lowest NBAD synthesis is observed at the time of higher locomotor activity. The abnormalities in several parameters of the waking/sleep cycle indicate some dysfunction in the processes that regulates these behaviours in both mutants.     

The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis

The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans.     

Dissecting chill coma recovery as a measure of cold resistance: evidence for a biphasic response in Drosophila melanogaster

Cold resistance in insects has traditionally been measured in terms of survival following a stress, but alternative methods are increasingly being used because of their relevance to the ecology of organisms and their utility in characterizing variation among species, populations and individuals. One such method capable of discriminating among Drosophila species and conspecific Drosophila populations from different environments is adult chill coma recovery time, the time taken for adults to become active again after being knocked down by a cold stress. Here we characterized the chill coma response of D.melanogaster in detail. Adults were exposed to a range of temperatures and stressful periods prior to measuring recovery. Recovery from chill coma in D.melanogaster was biphasic; as flies were stressed under cooler temperatures, recovery times leveled off and then decreased before sharply increasing again as mortality starts to occur. This biphasic response has previously been observed in D.subobscura where it has a somewhat different shape. A second mechanism therefore acts at relatively lower temperatures to ameliorate the effects of the cold stress. When D.melanogaster were reared at 19 and 25 °C for two generations, the shape of the curve relating temperature to recovery time was similar, but flies from the warmer temperature had longer recovery times and showed responses that leveled off and then decreased at relatively higher temperatures. As exposure time to cold stress was increased, recovery times also increased except at mild stress levels. Chill coma recovery in D.melanogaster is a complex trait and likely to reflect multiple underlying components.     

Trade-off of energy metabolites as well as body color phenotypes for starvation and desiccation resistance in montane populations of Drosophila melanogaster

Storage of energy metabolites has been investigated in different sets of laboratory selected desiccation or starvation resistant lines but few studies have examined such changes in wild-caught populations of Drosophila melanogaster. In contrast to parallel selection of desiccation and starvation tolerance under laboratory selection experiments, opposite clines were observed in wild populations of D. melanogaster. If resistance to desiccation and starvation occurs in opposite directions under field conditions, we may expect a trade-off for energy metabolites but such correlated changes are largely unknown. We tested whether there is a trade-off for storage as well as actual utilization of carbohydrates (trehalose and glycogen), lipids and proteins in D. melanogaster populations collected from different altitudes (512-2500 m). For desiccation resistance, darker flies (> 50% body melanization) store more body water content and endure greater loss of water (higher dehydration tolerance) as compared to lighter flies (< 30% body melanization). Based on within population analysis, we found evidence for coadapted phenotypes i.e. darker flies store and actually utilize more carbohydrates to confer greater desiccation resistance. In contrast, higher starvation resistance in lighter flies is associated with storage and actual utilization of greater lipid amount. However, darker and lighter flies did not vary in the rate of utilization of carbohydrates under desiccation stress; and of lipids under starvation stress. Thus, we did not find support for the hypothesis that a lower rate of utilization of energy metabolites may contribute to greater stress resistance. Further, for increased desiccation resistance of darker flies, about two-third of total energy budget is provided by carbohydrates. By contrast, lighter flies derive about 66% of total energy content from lipids which sustain higher starvation tolerance. Our results support evolutionary trade-off for storage as well as utilization of energy metabolites for desiccation versus starvation resistance in D. melanogaster.     

Role of Bacopa monnieri in the temporal regulation of oxidative stress in clock mutant (cryb) of Drosophila melanogaster

Accruing evidences imply that circadian organization of biochemical, endocrinological, cellular and physiological processes contribute to wellness of organisms and in the development of pathologies such as malignancy, sleep and endocrine disorders. Oxidative stress is known to mediate a number of diseases and it is notable to comprehend the orchestration of circadian clock of a model organism of circadian biology, Drosophila melanogaster, under oxidative stress. We investigated the nexus between circadian clock and oxidative stress susceptibility by exposing D. melanogaster to hydrogen peroxide (H₂O₂) or rotenone; the reversibility of rhythms following exposure to Bacopa monnieri extract (ayurvedic medicine rich in antioxidants) was also investigated. Abolishment of 24 h rhythms in physiological response (negative geotaxis), oxidative stress markers (protein carbonyl and thiobarbituric acid reactive substances) and antioxidants (superoxide dismutase, catalase, glutathione-S-transferase and reduced glutathione) were observed under oxidative stress. Furthermore, abolishment of per mRNA rhythm in H₂O₂ treated wild type flies and augmented susceptibility to oxidative stress in clock mutant (cry^b) flies connotes the role of circadian clock in reactive oxygen species (ROS) homeostasis. Significant reversibility of rhythms was noted following B. monnieri treatment in wild type flies than cry^b flies. Our experimental approach revealed a relationship involving oxidative stress and circadian clock in fruit fly and the utility of Drosophila model in screening putative antioxidative phytomedicines prior to their use in mammalian systems.     

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see ¹). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing "loser" effects that likely involve learning and memory ^2-4. The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals ^5,6. Many experimental procedures are used in different laboratories to study aggression ^7-9, however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of "loser" effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.     

Possible role of eclosion rhythm in mediating the effects of light-dark environments on pre-adult development in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

In insects, circadian clocks have been implicated in affecting life history traits such as pre-adult development time and adult lifespan. Studies on the period (per) mutants of Drosophila melanogaster, and laboratory-selected lines of Bactrocera cucurbitae suggested a close link between circadian clocks and development time. There is a possibility of clock genes having pleiotropic effects on clock period and pre-adult development time. In order to avoid such pleiotropic effects we have used wild type flies of same genotype under environments of different periodicities, which phenotypically either speeded up or slowed down the eclosion clock of D. melanogaster.     

Maximum voluntary temperature of insect larvae reveals differences in their thermal biology

We investigate the thermoregulatory behaviors of larvae of four species of Drosophila (D. melanogaster, D. subobscura, D. pseudoobscura, and D. mojavensis), a thermotolerant strain of Drosophila melanogaster (T strain) known to differ in thermal biology, and two mutant stocks of D. melanogaster that have (as adults) defective thermoregulatory behavior. We describe and evaluate new techniques to measure two indices of maximum voluntary temperature of insect larvae. Both measures were highly repeatable within lines (species, strains, or mutants). One measure (temperature at which larvae stood upright) differed among lines consistent with expectations based on adult thermal ecology, suggesting that this measure will be useful measures of thermoregulatory set-points of larvae. The second measure (temperature of emergence from media) is less discriminatory.     

A Novel Inhibitor of Amyloid β (Aβ) Peptide Aggregation: FROM HIGH THROUGHPUT SCREENING TO EFFICACY IN AN ANIMAL MODEL OF ALZHEIMER DISEASE*

Compelling evidence indicates that aggregation of the amyloid β (Aβ) peptide is a major underlying molecular culprit in Alzheimer disease. Specifically, soluble oligomers of the 42-residue peptide (Aβ42) lead to a series of events that cause cellular dysfunction and neuronal death. Therefore, inhibiting Aβ42 aggregation may be an effective strategy for the prevention and/or treatment of disease. We describe the implementation of a high throughput screen for inhibitors of Aβ42 aggregation on a collection of 65,000 small molecules. Among several novel inhibitors isolated by the screen, compound D737 was most effective in inhibiting Aβ42 aggregation and reducing Aβ42-induced toxicity in cell culture. The protective activity of D737 was most significant in reducing the toxicity of high molecular weight oligomers of Aβ42. The ability of D737 to prevent Aβ42 aggregation protects against cellular dysfunction and reduces the production/accumulation of reactive oxygen species. Most importantly, treatment with D737 increases the life span and locomotive ability of flies in a Drosophila melanogaster model of Alzheimer disease.     

The Imd Pathway Is Involved in Antiviral Immune Responses in Drosophila

Cricket Paralysis virus (CrPV) is a member of the Dicistroviridae family of RNA viruses, which infect a broad range of insect hosts, including the fruit fly Drosophila melanogaster. Drosophila has emerged as an effective system for studying innate immunity because of its powerful genetic techniques and the high degree of gene and pathway conservation. Intra-abdominal injection of CrPV into adult flies causes a lethal infection that provides a robust assay for the identification of mutants with altered sensitivity to viral infection. To gain insight into the interactions between viruses and the innate immune system, we injected wild type flies with CrPV and observed that antimicrobial peptides (AMPs) were not induced and hemocytes were depleted in the course of infection. To investigate the contribution of conserved immune signaling pathways to antiviral innate immune responses, CrPV was injected into isogenic mutants of the Immune Deficiency (Imd) pathway, which resembles the mammalian Tumor Necrosis Factor Receptor (TNFR) pathway. Loss-of-function mutations in several Imd pathway genes displayed increased sensitivity to CrPV infection and higher CrPV loads. Our data show that antiviral innate immune responses in flies infected with CrPV depend upon hemocytes and signaling through the Imd pathway.     

Impaired Sense of Smell in a Drosophila Parkinson’s Model

Parkinson’s disease (PD) is one of the most common neurodegenerative disease characterized by the clinical triad: tremor, akinesia and rigidity. Several studies have suggested that PD patients show disturbances in olfaction at the earliest onset of the disease. The fruit fly Drosophila melanogaster is becoming a powerful model organism to study neurodegenerative diseases. We sought to use this system to explore olfactory dysfunction, if any, in PINK1 mutants, which is a model for PD. PINK1 mutants display many important diagnostic symptoms of the disease such as akinetic motor behavior. In the present study, we describe for the first time, to the best of our knowledge, neurophysiological and neuroanatomical results concerning the olfactory function in PINK1 mutant flies. Electroantennograms were recorded in response to synthetic and natural volatiles (essential oils) from groups of PINK1 mutant adults at three different time points in their life cycle: one from 3–5 day-old flies, from 15–20 and from 27–30 days. The results obtained were compared with the same age-groups of wild type flies. We found that mutant adults showed a decrease in the olfactory response to 1-hexanol, α-pinene and essential oil volatiles. This olfactory response in mutant adults decreased even more as the flies aged. Immunohistological analysis of the antennal lobes in these mutants revealed structural abnormalities, especially in the expression of Bruchpilot protein, a marker for synaptic active zones. The combination of electrophysiological and morphological results suggests that the altered synaptic organization may be due to a neurodegenerative process. Our results indicate that this model can be used as a tool for understanding PD pathogensis and pathophysiology. These results help to explore the potential of using olfaction as a means of monitoring PD progression and developing new treatments.     

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.     

Consumption of dietary sugar by gut bacteria determines Drosophila lipid content

Gut microorganisms are essential for the nutritional health of many animals, but the underlying mechanisms are poorly understood. This study investigated how lipid accumulation by adult Drosophila melanogaster is reduced in flies associated with the bacterium Acetobacter tropicalis which displays oral–faecal cycling between the gut and food. We demonstrate that the lower lipid content of A. tropicalis-colonized flies relative to bacteria-free flies is linked with a parallel bacterial-mediated reduction in food glucose content; and can be accounted for quantitatively by the amount of glucose acquired by the flies, as determined from the feeding rate and assimilation efficiency of bacteria-free and A. tropicalis-colonized flies. We recommend that nutritional studies on Drosophila include empirical quantification of food nutrient content, to account for likely microbial-mediated effects on diet composition. More broadly, this study demonstrates that selective consumption of dietary constituents by microorganisms can alter the nutritional balance of food and, thereby, influence the nutritional status of the animal host.