Joel D. Levine

Signal analysis of behavioral and molecular cycles.

BackgroundCircadian clocks are biological oscillators that regulate molecular, physiological, and behavioral rhythms in a wide variety of organisms. While behavioral rhythms are typically monitored over many cycles, a similar approach to molecular rhythms was not possible until recently; the advent of real-time analysis using transgenic reporters now permits the observations of molecular rhythms over many cycles as well. This development suggests that new details about the relationship between molecular and behavioral rhythms may be revealed. Even so, behavioral and molecular rhythmicity have been analyzed using different methods, making such comparisons difficult to achieve. To address this shortcoming, among others, we developed a set of integrated analytical tools to unify the analysis of biological rhythms across modalities.ResultsWe demonstrate an adaptation of digital signal analysis that allows similar treatment of both behavioral and molecular data from our studies of Drosophila. For both types of data, we apply digital filters to extract and clarify details of interest; we employ methods of autocorrelation and spectral analysis to assess rhythmicity and estimate the period; we evaluate phase shifts using crosscorrelation; and we use circular statistics to extract information about phase.ConclusionUsing data generated by our investigation of rhythms in Drosophila we demonstrate how a unique aggregation of analytical tools may be used to analyze and compare behavioral and molecular rhythms. These methods are shown to be versatile and will also be adaptable to further experiments, owing in part to the non-proprietary nature of the code we have developed.

Specialized cells tag sexual and species identity in Drosophila melanogaster.

Social interactions depend on individuals recognizing each other, and in this context many organisms use chemical signals to indicate species and sex. Cuticular hydrocarbon signals are used by insects, including Drosophila melanogaster, to distinguish conspecific individuals from others. These chemicals also contribute to intraspecific courtship and mating interactions. However, the possibility that sex and species identification are linked by common chemical signalling mechanisms has not been formally tested. Here we provide direct evidence that a single compound is used to communicate female identity among D. melanogaster, and to define a reproductive isolation barrier between D. melanogaster and sibling species. A transgenic manipulation eliminated cuticular hydrocarbons by ablating the oenocytes, specialized cells required for the expression of these chemical signals. The resulting oenocyte-less (oe-) females elicited the normal repertoire of courtship behaviours from males, but were actually preferred over wild-type females by courting males. In addition, wild-type males attempted to copulate with oe- males. Thus, flies lacking hydrocarbons are a sexual hyperstimulus. Treatment of virgin females with the aversive male pheromone cis-vaccenyl acetate (cVA) significantly delayed mating of oe- females compared to wild-type females. This difference was eliminated when oe- females were treated with a blend of cVA and the female aphrodisiac (7Z,11Z)-heptacosadiene (7,11-HD), showing that female aphrodisiac compounds can attenuate the effects of male aversive pheromones. 7,11-HD also was shown to have a crucial role in heterospecific encounters. Specifically, the species barrier was lost because males of other Drosophila species courted oe- D. melanogaster females, and D. simulans males consistently mated with them. Treatment of oe- females with 7,11-HD restored the species barrier, showing that a single compound can confer species identity. These results identify a common mechanism for sexual and species recognition regulated by cuticular hydrocarbons.

A new role for cryptochrome in a Drosophila circadian oscillator

Cryptochromes are flavin/pterin-containing proteins that are involved in circadian clock function in Drosophila and mice. In mice, the cryptochromes Cry1 and Cry2 are integral components of the circadian oscillator within the brain and contribute to circadian photoreception in the retina. In Drosophila, cryptochrome (CRY) acts as a photoreceptor that mediates light input to circadian oscillators in both brain and peripheral tissue. A Drosophila cry mutant, cryb, leaves circadian oscillator function intact in central circadian pacemaker neurons but renders peripheral circadian oscillators largely arrhythmic. Although this arrhythmicity could be caused by a loss of light entrainment, it is also consistent with a role for CRY in the oscillator. A peripheral oscillator drives circadian olfactory responses in Drosophila antennae. Here we show that CRY contributes to oscillator function and physiological output rhythms in the antenna during and after entrainment to light–dark cycles and after photic input is eliminated by entraining flies to temperature cycles. These results demonstrate a photoreceptor-independent role for CRY in the periphery and imply fundamental differences between central and peripheral oscillator mechanisms in Drosophila.

Generalization of Courtship Learning in Drosophila Is Mediated by cis-Vaccenyl Acetate

Reproductive behavior in Drosophila has both stereotyped and plastic components that are driven by age- and sex-specific chemical cues. Males who unsuccessfully court virgin females subsequently avoid females that are of the same age as the trainer. In contrast, males trained with mature mated females associate volatile appetitive and aversive pheromonal cues and learn to suppress courtship of all females. Here we show that the volatile aversive pheromone that leads to generalized learning with mated females is (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). cVA is a major component of the male cuticular hydrocarbon profile, but it is not found on virgin females. During copulation, cVA is transferred to the female in ejaculate along with sperm and peptides that decrease her sexual receptivity. When males sense cVA (either synthetic or from mated female or male extracts) in the context of female pheromone, they develop a generalized suppression of courtship. The effects of cVA on initial courtship of virgin females can be blocked by expression of tetanus toxin in Or65a, but not Or67d neurons, demonstrating that the aversive effects of this pheromone are mediated by a specific class of olfactory neuron. These findings suggest that transfer of cVA to females during mating may be part of the males strategy to suppress reproduction by competing males.

Social experience modifies pheromone expression and mating behavior in male Drosophila melanogaster

Summary Background The social life of animals depends on communication between individuals. Recent studies in Drosophila melanogaster demonstrate that various behaviors are influenced by social interactions. For example, courtship is a social interaction mediated by pheromonal signaling that occurs more frequently during certain times of the day than others. In adult flies, sex pheromones are synthesized in cells called oenocytes and displayed on the surface of the cuticle. Although the role of Drosophila pheromones in sexual behavior is well established, little is known about the timing of these signals or how their regulation is influenced by the presence of other flies. Results We report that oenocytes contain functional circadian clocks that appear to regulate the synthesis of pheromones by controlling the transcription of desaturase1 ( desat1 ), a gene required for production of male cuticular sex pheromones. Moreover, levels of these pheromones vary throughout the day in a pattern that depends on the clock genes and most likely also depends on the circadian control of desat1 in the oenocytes. To assess group dynamics, we manipulated the genotypic composition of social groups (single versus mixed genotypes). This manipulation significantly affects clock gene transcription both in the head and oenocytes, and it also affects the pattern of pheromonal accumulation on the cuticle. Remarkably, we found that flies in mixed social groups mate more frequently than do their counterparts in uniform groups. Conclusions These results demonstrate that social context exerts a regulatory influence on the expression of chemical signals, while modulating sexual behavior in the fruit fly.BACKGROUND The social life of animals depends on communication between individuals. Recent studies in Drosophila melanogaster demonstrate that various behaviors are influenced by social interactions. For example, courtship is a social interaction mediated by pheromonal signaling that occurs more frequently during certain times of the day than others. In adult flies, sex pheromones are synthesized in cells called oenocytes and displayed on the surface of the cuticle. Although the role of Drosophila pheromones in sexual behavior is well established, little is known about the timing of these signals or how their regulation is influenced by the presence of other flies. RESULTS We report that oenocytes contain functional circadian clocks that appear to regulate the synthesis of pheromones by controlling the transcription of desaturase1 (desat1), a gene required for production of male cuticular sex pheromones. Moreover, levels of these pheromones vary throughout the day in a pattern that depends on the clock genes and most likely also depends on the circadian control of desat1 in the oenocytes. To assess group dynamics, we manipulated the genotypic composition of social groups (single versus mixed genotypes). This manipulation significantly affects clock gene transcription both in the head and oenocytes, and it also affects the pattern of pheromonal accumulation on the cuticle. Remarkably, we found that flies in mixed social groups mate more frequently than do their counterparts in uniform groups. CONCLUSIONS These results demonstrate that social context exerts a regulatory influence on the expression of chemical signals, while modulating sexual behavior in the fruit fly.

Hierarchical chemosensory regulation of male-male social interactions in Drosophila

Pheromones regulate male social behaviors in Drosophila, but the identities and behavioral role(s) of these chemosensory signals, and how they interact, are incompletely understood. We found that (z)-7-tricosene, a male-enriched cuticular hydrocarbon that was previously shown to inhibit male-male courtship, was essential for normal levels of aggression. The mechanisms by which (z)-7-tricosene induced aggression and suppressed courtship were independent, but both required the gustatory receptor Gr32a. Sensitivity to (z)-7-tricosene was required for the aggression-promoting effect of 11-cis-vaccenyl acetate (cVA), an olfactory pheromone, but (z)-7-tricosene sensitivity was independent of cVA. (z)-7-tricosene and cVA therefore regulate aggression in a hierarchical manner. Furthermore, the increased courtship caused by depletion of male cuticular hydrocarbons was suppressed by a mutation in the olfactory receptor Or47b. Thus, male social behaviors are controlled by gustatory pheromones that promote aggression and suppress courtship, and whose influences are dominant to olfactory pheromones that enhance these behaviors.

Advanced analysis of a cryptochrome mutation's effects on the robustness and phase of molecular cycles in isolated peripheral tissues of Drosophila

BackgroundPreviously, we reported effects of the cryb mutation on circadian rhythms in period and timeless gene expression within isolated peripheral Drosophila tissues. We relied on luciferase activity driven by the respective regulatory genomic elements to provide real-time reporting of cycling gene expression. Subsequently, we developed a tool kit for the analysis of behavioral and molecular cycles. Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs.ResultsIsolated antennal pairs, heads, bodies, wings and forelegs were evaluated under light-dark cycles. In these conditions, the cryb mutation significantly decreases the number of rhythmic specimens in each case except the wing. Moreover, among those specimens with detectable rhythmicity, mutant rhythms are significantly weaker than cry+ controls. In addition, cryb alters the phase of period gene expression in these tissues. Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others. We also analyze rhythms produced by antennal pairs in constant conditions.ConclusionsThese analyses further show that circadian clock mechanisms in Drosophila may vary in a tissue-specific manner, including how the cry gene regulates circadian gene expression.

Pheromonal and Behavioral Cues Trigger Male-to-Female Aggression in Drosophila

By genetically manipulating both pheromonal profiles and behavioral patterns, we find that Drosophila males showed a complete reversal in their patterns of aggression towards other males and females

Flies by night: Effects of changing day length on Drosophila's circadian clock.

In Drosophila, two intersecting molecular loops constitute an autoregulatory mechanism that oscillates with a period close to 24 hr. These loops touch when proteins from one loop, PERIOD (PER) and TIMELESS (TIM), repress the transcription of their parent genes, period (per) and timeless (tim), by blocking positive transcription factors from the other loop. The arrival of PER and TIM into the nucleus of a clock cell marks the timing of this interaction between the two loops; thus, control of PER:TIM nuclear accumulation is a central component of the molecular model of clock function. If a light pulse occurs early in the night as the heterodimer accumulates in the nucleus of clock cells, TIM is degraded, PER is destabilized, and clock time is delayed. Alternatively, if TIM is degraded during the later part of the night, after peak accumulation, clock time advances. Current models state that the effect of a light pulse depends on the state of the PER:TIM oscillation, which turns on the changing levels of TIM. However, previous studies have shown that light:dark (LD) regimes mimicking seasonal changes cause behavioral adjustments while altering clock gene expression. This should be reflected in the adjustment of PER and TIM dynamics. We manipulated LD cycles to assess the effects of altered day length on PER and TIM dynamics in clock cells within the central brain as well as light-induced resetting of locomotor rhythms.

Social structures depend on innate determinants and chemosensory processing in Drosophila

Flies display transient social interactions in groups. However, whether fly–fly interactions are stochastic or structured remains unknown. We hypothesized that groups of flies exhibit patterns of social dynamics that would manifest as nonrandom social interaction networks. To test this, we applied a machine vision system to track the position and orientation of flies in an arena and designed a classifier to detect interactions between pairs of flies. We show that the vinegar fly, Drosophila melanogaster, forms nonrandom social interaction networks, distinct from virtual network controls (constructed from the intersections of individual locomotor trajectories). In addition, the formation of interaction networks depends on chemosensory cues. Gustatory mutants form networks that cannot be distinguished from their virtual network controls. Olfactory mutants form networks that are greatly disrupted compared with control flies. Different wild-type strains form social interaction networks with quantitatively different properties, suggesting that the genes that influence this network phenotype vary across and within wild-type populations. We have established a paradigm for studying social behaviors at a group level in Drosophila and expect that a genetic dissection of this phenomenon will identify conserved molecular mechanisms of social organization in other species.