Drosophila Courtship Research Articles

Developmental remodeling repurposes larval neurons for sexual behaviors in adult Drosophila

Most larval neurons in Drosophila are repurposed during metamorphosis for functions in adult life, but their contribution to the neural circuits for sexually dimorphic behaviors is unknown. Here, we identify two interneurons in the nerve cord of adult Drosophila females that control ovipositor extrusion, a courtship rejection behavior performed by mated females. We show that these two neurons are present in the nerve cord of larvae as mature, sexually monomorphic interneurons. During pupal development, they acquire the expression of the sexual differentiation gene, doublesex; undergo doublesex-dependent programmed cell death in males; and are remodeled in females for functions in female mating behavior. Our results demonstrate that the neural circuits for courtship in Drosophila are built in part using neurons that are sexually reprogrammed from former sex-shared activities in larval life.

Nested neural circuits generate distinct acoustic signals during Drosophila courtship.

Many motor control systems generate multiple movements using a common set of muscles. How are premotor circuits able to flexibly generate diverse movement patterns? Here, we characterize the neuronal circuits that drive the distinct courtship songs of Drosophila melanogaster. Male flies vibrate their wings toward females to produce two different song modes-pulse and sine song-which signal species identity and male quality. Using cell-type-specific genetic reagents and the connectome, we provide a cellular and synaptic map of the circuits in the male ventral nerve cord that generate these songs and examine how activating or inhibiting each cell type within these circuits affects the song. Our data reveal that the song circuit is organized into two nested feedforward pathways with extensive reciprocal and feedback connections. The larger network produces pulse song, the more complex and ancestral song form. A subset of this network produces sine song, the simpler and more recent form. Such nested organization may be a common feature of motor control circuits in which evolution has layered increasing flexibility onto a basic movement pattern.

Single-Pair Courtship and Competition Assays in Drosophila.

Courtship in Drosophila melanogaster involves a series of innate, complex behaviors that allow male and female flies to exchange sensory information and assess the quality of a potential mate. Although highly robust and stereotypical, courtship behaviors can be modulated by internal state and experience. This protocol describes methods for designing and carrying out experiments that measure courtship performance in single-pair assays, in which a male is paired with a female, or in competitive assays, in which a male is presented with a female and with a male competitor. It also includes approaches for measuring female sexual receptivity during courtship.

Dopaminergic neurons mediate male Drosophila courtship motivational state

Motivational states are important determinants of behavior. In Drosophila melanogaster, courtship behavior is robust and crucial for species continuation. However, the motivation of courtship behavior remains unexplored. We first find the phenomenon that courtship behavior is modulated by motivational state. A male fly courts another male fly when it first courts a decapitated female fly, however, male–male courtship behavior rarely occurs under normal conditions. Therefore, in this phenomenon, the male fly's courtship motivational state is induced by its exposure to female flies. Blocking dopaminergic neurons synaptic transmission by expressing Tetanus toxin light chain (TNTe) decreases motivational state induced male–male courtship behavior without affecting male–female courtship behavior. Vision cues are another key component in sexually driven Drosophila male–male courtship behavior. Here, we identify a base theory that the inner motivational state could eventually decide Drosophila behavior.

Regulation of Drosophila courtship behavior by the Tlx/tailless-like nuclear receptor, dissatisfaction

Sexually dimorphic courtship behaviors in Drosophila melanogaster develop from the activity of the sexual differentiation genes, doublesex (dsx) and fruitless (fru), functioning with other regulatory factors that have received little attention. The dissatisfaction (dsf) gene encodes an orphan nuclear receptor homologous to vertebrate Tlx and Drosophila tailless that is critical for the development of several aspects of female- and male-specific sexual behaviors. Here, we report the pattern of dsf expression in the central nervous system and show that the activity of sexually dimorphic abdominal interneurons that co-express dsf and dsx is necessary and sufficient for vaginal plate opening in virgin females, ovipositor extrusion in mated females, and abdominal curling in males during courtship. We find that dsf activity results in different neuroanatomical outcomes in females and males, promoting and suppressing, respectively, female development and function of these neurons depending upon the sexual state of dsx expression. We posit that dsf and dsx interact to specify sex differences in the neural circuitry for dimorphic abdominal behaviors.

Social behavior: Using visual cues to guide dancing on the fly

Quantitative behavioral analysis of Drosophila courtship reveals that visual cues of a female's body influence which actions a male performs during courtship. These actions in turn influence female actions, producing a mutual synchronization of courtship between male and female flies.

Behavioral signatures of structured feature detection during courtship in Drosophila

Many animals detect other individuals effortlessly. In Drosophila, previous studies have examined sensory processing during social interactions using simple blobs as visual stimulation; however, whether and how flies extract higher-order features from conspecifics to guide behavior remains elusive. Arguing that this should be reflected in sensorimotor relations, we developed unbiased machine learning tools for natural behavior quantification and applied these tools, which may prove broadly useful, to study interacting pairs. By transforming motor patterns with female-centered reference frames, we established circling, where heading and traveling directions intersect, as a unique pattern of social interaction during courtship. We found circling to be highly visual, with males exhibiting view-tuned motor patterns. Interestingly, males select specific wing and leg actions based on the positions and motions of the females' heads and tails. Using system identification, we derived visuomotor transformation functions indicating history-dependent action selection, with distance predicting action initiation and angular position predicting wing choices and locomotion directions. Integration of vision with somatosensation further boosts these sensorimotor relations. Essentially comprised of orchestrated wing and leg maneuvers that are more variable in the light, circling induces mutually synchronized conspecific responses stronger than wing extension alone. Finally, we found that actions depend on integrating spatiotemporally structured features with goals. Altogether, we identified a series of sensorimotor relations during circling, implying that during courtship, flies detect complex spatiotemporally structured features of conspecifics, laying the foundation for a mechanistic understanding of conspecific recognition in Drosophila.

Consolidation and maintenance of long-term memory involve dual functions of the developmental regulator Apterous in clock neurons and mushroom bodies in the Drosophila brain.

Memory is initially labile but can be consolidated into stable long-term memory (LTM) that is stored in the brain for extended periods. Despite recent progress, the molecular and cellular mechanisms underlying the intriguing neurobiological processes of LTM remain incompletely understood. Using the Drosophila courtship conditioning assay as a memory paradigm, here, we show that the LIM homeodomain (LIM-HD) transcription factor Apterous (Ap), which is known to regulate various developmental events, is required for both the consolidation and maintenance of LTM. Interestingly, Ap is involved in these 2 memory processes through distinct mechanisms in different neuronal subsets in the adult brain. Ap and its cofactor Chip (Chi) are indispensable for LTM maintenance in the Drosophila memory center, the mushroom bodies (MBs). On the other hand, Ap plays a crucial role in memory consolidation in a Chi-independent manner in pigment dispersing factor (Pdf)-containing large ventral–lateral clock neurons (l-LNvs) that modulate behavioral arousal and sleep. Since disrupted neurotransmission and electrical silencing in clock neurons impair memory consolidation, Ap is suggested to contribute to the stabilization of memory by ensuring the excitability of l-LNvs. Indeed, ex vivo imaging revealed that a reduced function of Ap, but not Chi, results in exaggerated Cl− responses to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in l-LNvs, indicating that wild-type (WT) Ap maintains high l-LNv excitability by suppressing the GABA response. Consistently, enhancing the excitability of l-LNvs by knocking down GABAA receptors compensates for the impaired memory consolidation in ap null mutants. Overall, our results revealed unique dual functions of the developmental regulator Ap for LTM consolidation in clock neurons and LTM maintenance in MBs.

Sexual arousal gates visual processing during Drosophila courtship

Long-lasting internal arousal states motivate and pattern ongoing behaviour, enabling the temporary emergence of innate behavioural programsthat serve the needs of an animal, such as fighting, feeding, and mating. However, how internal states shape sensory processing or behaviour remains unclear. In Drosophila, male flies perform a lengthy and elaborate courtship ritual that is triggered by the activation of sexually dimorphic P1 neurons1-5, during which they faithfully follow and sing to a female6,7. Here, by recording from males as they court a virtual 'female', we gain insight into how the salience of visual cues is transformed by a male's internal arousal state to give rise to persistent courtship pursuit. The gain of LC10a visual projection neurons is selectively increased during courtship, enhancing their sensitivity to moving targets. A concise network model indicates that visual signalling through the LC10a circuit, once amplified by P1-mediated arousal, almost fully specifies a male's tracking of a female. Furthermore, P1 neuron activity correlates with ongoing fluctuations in the intensity of a male's pursuit to continuously tune the gain of the LC10a pathway. Together, these results reveal how a male's internal state can dynamically modulate the propagation of visual signals through a high-fidelity visuomotor circuit to guide his moment-to-moment performance of courtship.

Different diets can affect attractiveness of Drosophila melanogaster males via changes in wing morphology

Courtship in Drosophila involves exchange of chemosensory, auditory, tactile and visual signals. Differences in courtship signals are strongly influenced by environmental factors, among which nutrition plays one of the more important roles. Through influence on development, morphology, physiology and behaviour, nutrition may affect both sexual selection and isolation. However, the impact of specific nutritive regimes on male mating success has not been extensively studied. Therefore, the aim of this study was to explore how diets affect male attractiveness via changes in wing morphology during long-term rearing of flies on different diets. Through a series of female choice tests, we investigated the mating success of males reared on five different diets for more than 350 generations. Further, we analysed wing size and wing shape of mated and unmated males to link wing morphology with mating success. The results demonstrate significant differences in male mating success between strains. We also found significant differences in wing size between the strains and established that female choice is positively correlated with male wing size, that is, females of all strains preferred males with larger wings. In addition, we noted a significant difference in wing shape between the strains, as well as between mated and unmated males. Our results indicated that among strains, males with elongated wings were more successful in mating than males with rounded wings. Within strains, mated males had more elongated wings than unmated ones, which had more rounded wings. Our results support the hypothesis that nutrition represents an important environmental factor that may affect male attractiveness via changes in wing morphology, thereby playing an important role in sexual selection of Drosophila melanogaster . • Male mating success of Drosophila is related to wing size and shape. • Wing morphology in D. melanogaster may depend on diet. • In this study, we used flies reared for 350 generations on different diets. • Larger males reared on a tomato diet had the highest mating success. • Males with elongated wings had higher mating success than those with rounded wings.

Distinct Roles and Synergistic Function of FruM Isoforms in Drosophila Olfactory Receptor Neurons

Sexual dimorphism in Drosophila courtship circuits requires the male-specific transcription factor fruM, which is alternatively spliced to encode the FruMA, FruMB, and FruMC isoforms. Most fruM-positive neurons express multiple variants; however, the functional significance of their co-expression remains undetermined. Do co-expressed isoforms each play unique roles to jointly regulate dimorphism? By focusing on fruM-positive olfactory receptor neurons (ORNs), here, we show that FruMB and FruMC are both required for males' age-dependent sensitization to aphrodisiac olfactory cues in a cell-autonomous manner. Interestingly, FruMB expression is upregulated with age in Or47b and Ir84a ORNs, and its overexpression mimics the effect of age in elevating olfactory responses. Mechanistically, FruMB and FruMC synergistically mediate response sensitization through cooperation of their respective downstream effectors, namely, PPK25 and PPK23, which are both required for forming a functional amplification channel in ORNs. Together, these results provide critical mechanistic insight into how co-expressed FruM isoforms jointly coordinate dimorphic neurophysiology.

A Computational Error and Restricted Use of Time-series Analyses Underlie the Failure to Replicate period-Dependent Song Rhythms in Drosophila.

From 1980 to 1991, Kyriacou, Hall, and collaborators (K&H) reported that the Drosophila melanogaster courtship song has a 1-min cycle in the length of mean interpulse intervals (IPIs) that is modulated by circadian rhythm period mutations. In 2014, Stern failed to replicate these results using a fully automated method for detecting song pulses. Manual annotation of Stern's song records exposed a ~50% error rate in detection of IPIs, but the corrected data revealed period-dependent IPI cycles using a variety of statistical methods. In 2017, Stern et al. dismissed the sine/cosine method originally used by K&H to detect significant cycles, claiming that randomized songs showed as many significant values as real data using cosinor analysis. We first identify a simple mathematical error in Stern et al.'s cosinor implementation that invalidates their critique of the method. Stern et al. also concluded that although the manually corrected wild-type and perL mutant songs show similar periods to those observed by K&H, each song is usually not significantly rhythmic by the Lomb-Scargle (L-S) periodogram, so any genotypic effect simply reflects "noise." Here, we observe that L-S is extremely conservative compared with 3 other time-series analyses in assessing the significance of rhythmicity, both for conventional locomotor activity data collected in equally spaced time bins and for unequally spaced song records. Using randomization of locomotor and song data to generate confidence limits for L-S instead of the theoretically derived values, we find that L-S is now consistent with the other methods in determining significant rhythmicity in locomotor and song records and that it confirms period-dependent song cycles. We conclude that Stern and colleagues' failure to identify song cycles stems from the limitations of automated methods in accurately reflecting song parameters, combined with the use of an overly stringent method to discriminate rhythmicity in courtship songs.

Environmental Light Is Required for Maintenance of Long-Term Memory in Drosophila.

Long-term memory (LTM) is stored as functional modifications of relevant neural circuits in the brain. A large body of evidence indicates that the initial establishment of such modifications through the process known as memory consolidation requires learning-dependent transcriptional activation and de novo protein synthesis. However, it remains poorly understood how the consolidated memory is maintained for a long period in the brain, despite constant turnover of molecular substrates. Using the Drosophila courtship conditioning assay of adult males as a memory paradigm, here, we show that in Drosophila, environmental light plays a critical role in LTM maintenance. LTM is impaired when flies are kept in constant darkness (DD) during the memory maintenance phase. Because light activates the brain neurons expressing the neuropeptide pigment-dispersing factor (Pdf), we examined the possible involvement of Pdf neurons in LTM maintenance. Temporal activation of Pdf neurons compensated for the DD-dependent LTM impairment, whereas temporal knockdown of Pdf during the memory maintenance phase impaired LTM in light/dark cycles. Furthermore, we demonstrated that the transcription factor cAMP response element-binding protein (CREB) is required in the memory center, namely, the mushroom bodies (MBs), for LTM maintenance, and Pdf signaling regulates light-dependent transcription via CREB. Our results demonstrate for the first time that universally available environmental light plays a critical role in LTM maintenance by activating the evolutionarily conserved memory modulator CREB in MBs via the Pdf signaling pathway.SIGNIFICANCE STATEMENT Temporary memory can be consolidated into long-term memory (LTM) through de novo protein synthesis and functional modifications of neuronal circuits in the brain. Once established, LTM requires continual maintenance so that it is kept for an extended period against molecular turnover and cellular reorganization that may disrupt memory traces. How is LTM maintained mechanistically? Despite the critical importance of LTM maintenance, its molecular and cellular underpinnings remain elusive. This study using Drosophila is significant because it revealed for the first time in any organism that universally available environmental light plays an essential role in LTM maintenance. Interestingly, light does so by activating the evolutionarily conserved transcription factor cAMP response element-binding protein via peptidergic signaling.

EFFECTS OF HIGH URACIL DIET-INDUCED GUT DYSBIOSIS ON COURTSHIP BEHAVIOR IN DROSOPHILA

AbstractPast research has established that Drosophila gut inflammation via the dual oxidase (DUOX) regulatory pathway is induced by exposure to bacterial-derived uracil, which can be secreted by allochthonous and autochthonous bacteria in the midgut. However, the effects of the inflammatory response and resulting gut dysbiosis on the courtship behavior of the Drosophila host have yet to be determined. This work studies the changes in Drosophila courtship behavior resulting from diet-based exposure to uracil, a ligand for DUOX-dependent reactive oxygen species (ROS) generation in the epithelia of the midgut. The effects of a high-uracil diet on courtship behavior were determined by comparing courtship index (CI) values of flies treated with 20 nM of uracil for 16-hr (short-term) or 10-d (long-term) exposure to age-matched control flies. Although short-term uracil treatment caused no change in courtship behavior, long-term uracil treatment resulted in a significant decrease in CI. This result suggests that the underlying association for the behavioral change may be influenced by gut inflammation initiated by long-term exposure to uracil. By measuring the effects of an innate immune response such as exposure to a ligand for DUOX-dependent ROS generation on the courtship index of Drosophila, the current understanding of the relationship between gut inflammation and behavioral changes in animals can be expanded. Given that prior research has also established that the DUOX-dependent response can be found in animals ranging from Drosophila to humans, the behavioral changes observed from induction of the pathway have the potential to expand this connection in the future.

Recurrent Circuitry Sustains Drosophila Courtship Drive While Priming Itself for Satiety.

Motivations intensify over hours or days, promoting goals that are achieved in minutes or hours, causing satiety that persists for hours or days. Here we develop Drosophila courtship as a system to study these long-timescale motivational dynamics. We identify two neuronal populations engaged in a recurrent excitation loop, the output of which elevates a dopamine signal that increases the propensity to court. Electrical activity within the recurrent loop accrues with abstinence and, through the activity-dependent transcription factor CREB2, drives the production of activity-suppressing potassium channels. Loop activity is decremented by each mating to reduce subsequent courtship drive, and the inhibitory loop environment established by CREB2 during high motivation slows the reaccumulation of activity for days. Computational modeling reproduces these behavioral and physiological dynamics, generating predictions that we validate experimentally and illustrating a causal link between the motivation that drives behavior and the satiety that endures after goal achievement.

Neuropeptide F regulates courtship in Drosophila through a male-specific neuronal circuit.

Male courtship is provoked by perception of a potential mate. In addition, the likelihood and intensity of courtship are influenced by recent mating experience, which affects sexual drive. Using Drosophila melanogaster, we found that the homolog of mammalian neuropeptide Y, neuropeptide F (NPF), and a cluster of male-specific NPF (NPFM) neurons, regulate courtship through affecting courtship drive. Disrupting NPF signaling produces sexually hyperactive males, which are resistant to sexual satiation, and whose courtship is triggered by sub-optimal stimuli. We found that NPFM neurons make synaptic connections with P1 neurons, which comprise the courtship decision center. Activation of P1 neurons elevates NPFM neuronal activity, which then act through NPF receptor neurons to suppress male courtship, and maintain the proper level of male courtship drive.

Gαi and GƔ30A act downstream of Tre1 in Drosophila courtship

The role of genes in morphological development is well understood for a variety of model organisms, but there remains a gap in our understanding of how genetics mediate behavior. Are there master genes that regulate behavior? Answering this question will lead to a better understanding of the development and function of the central nervous system, eventually allowing us to map out the pathways that regulate specific behaviors. We are using Drosophila melanogaster as a model organism and the male courtship ritual as the behavior of interest to study the relationships between genes, neural development, and behavior. Trapped in endoderm 1 (Tre1), a gene encoding an orphan G-protein coupled receptor (GPCR), is required for normal courtship behavior in fruit flies, but how this receptor regulates behavior is not yet understood. Here, we characterize the signaling cascade downstream of Tre1 by testing mutations in the Drosophila G-proteins for courtship defects similar to those seen in Tre1. Our results demonstrate that Gαi is a candidate downstream effector for Tre1, while also implicating Gγ30A in courtship behavior. Future goals include completing the characterization of the G-protein mutations and conducting experiments to explore the complex interaction between G-protein signaling and courtship initiation.

Sexual selection drives the evolution of male wing interference patterns.

The seemingly transparent wings of many insects have recently been found to display unexpected structural coloration. These structural colours (wing interference patterns: WIPs) may be involved in species recognition and mate choice, yet little is known about the evolutionary processes that shape them. Furthermore, to date investigations of WIPs have not fully considered how they are actually perceived by the viewers' colour vision. Here, we use multispectral digital imaging and a model of Drosophila vision to compare WIPs of male and female Drosophila simulans from replicate populations forced to evolve with or without sexual selection for 68 generations. We show that WIPs modelled in Drosophila vision evolve in response to sexual selection and provide evidence that WIPs correlate with male sexual attractiveness. These findings add a new element to the otherwise well-described Drosophila courtship display and confirm that wing colours evolve through sexual selection.

Drosophila Courtship: Neuronal Coordination of Behavioural Sequences and a 60-Year-Old Hypothesis

Drosophila courtship consists of a stereotypic sequence of behaviours, involving increasing levels of excitation. A pair of neurons coordinate some of these sequences using a spike-counting model that echoes an idea first outlined in 1959.

Threshold-Based Ordering of Sequential Actions during Drosophila Courtship

Goal-directed animal behaviors are typically composed of sequences of motor actions whose order and timing are critical for a successful outcome. Although numerous theoretical models for sequential action generation have been proposed, few have been supported by the identification of control neurons sufficient to elicit a sequence. Here, we identify a pair of descending neurons that coordinate a stereotyped sequence of engagement actions during Drosophila melanogaster male courtship behavior. These actions are initiated sequentially but persist cumulatively, a feature not explained by existing models of sequential behaviors. We find evidence consistent with a ramp-to-threshold mechanism, in which increasing neuronal activity elicits each action independently at successively higher activity thresholds.