Fly Courtship Research Articles

An automatic system for recognizing fly courtship patterns via an image processing method

Fruit fly courtship behaviors composed of a series of actions have always been an important model for behavioral research. While most related studies have focused only on total courtship behaviors, specific courtship elements have often been underestimated. Identifying these courtship element details is extremely labor intensive and would largely benefit from an automatic recognition system. To address this issue, in this study, we established a vision-based fly courtship behavior recognition system. The system based on the proposed image processing methods can precisely distinguish body parts such as the head, thorax, and abdomen and automatically recognize specific courtship elements, including orientation, singing, attempted copulation, copulation and tapping, which was not detectable in previous studies. This system, which has high identity tracking accuracy (99.99%) and high behavioral element recognition rates (> 97.35%), can ensure correct identification even when flies completely overlap. Using this newly developed system, we investigated the total courtship time, and proportion, and transition of courtship elements in flies across different ages and found that male flies adjusted their courtship strategy in response to their physical condition. We also identified differences in courtship patterns between males with and without successful copulation. Our study therefore demonstrated how image processing methods can be applied to automatically recognize complex animal behaviors. The newly developed system will largely help us investigate the details of fly courtship in future research.

Neural network organization for courtship-song feature detection in Drosophila

Animals communicate using sounds in a wide range of contexts, and auditory systems must encode behaviorally relevant acoustic features to drive appropriate reactions. How feature detection emerges along auditory pathways has been difficult to solve due to challenges in mapping the underlying circuits and characterizing responses to behaviorally relevant features. Here, we study auditory activity in the Drosophila melanogaster brain and investigate feature selectivity for the two main modes of fly courtship song, sinusoids and pulse trains. We identify 24 new cell types of the intermediate layers of the auditory pathway, and using a new connectomic resource, FlyWire, we map all synaptic connections between these cell types, in addition to connections to known early and higher-order auditory neurons-this represents the first circuit-level map of the auditory pathway. We additionally determine the sign (excitatory or inhibitory) of most synapses in this auditory connectome. We find that auditory neurons display a continuum of preferences for courtship song modes and that neurons with different song-mode preferences and response timescales are highly interconnected in a network that lacks hierarchical structure. Nonetheless, we find that the response properties of individual cell types within the connectome are predictable from their inputs. Our study thus provides new insights into the organization of auditory coding within the Drosophila brain.

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.

Drosophila functional screening of de novo variants in autism uncovers damaging variants and facilitates discovery of rare neurodevelopmental diseases.

SUMMARYIndividuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through “humanization” rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Sex Mysteries of the Fly Courtship Master Regulator Fruitless.

The fruitless (fru) gene of Drosophila melanogaster generates two groups of protein products, the male-specific FruM proteins and non-sex-specific FruCOM proteins. The FruM proteins have a 101 amino acids (a.a.)-long extension at the N-terminus which is absent from FruCOM. We suggest that this N-terminal extension might confer male-specific roles on FruM interaction partner proteins such as Lola, which otherwise operates as a transcription factor common to both sexes. FruM-expressing neurons are known to connect with other neurons to form a sexually dimorphic circuit for male mating behavior. We propose that FruM proteins expressed in two synaptic partners specify, at the transcriptional level, signaling pathways through which select pre- and post-synaptic partners communicate, and thereby pleiotropic ligand-receptor pairs for cell-cell interactions acquire the high specificity for mutual connections between two FruM-positive cells. We further discuss the possibility that synaptic connections made by FruM-positive neurons are regulated by neural activities, which in turn upregulate Fru expression in active cells, resulting in feedforward enhancement of courtship activities of the male fly.

Quantitative and Discrete Evolutionary Changes in the Egg-Laying Behavior of Single Drosophila Females.

How a nervous system assembles and coordinates a suite of elementary behavioral steps into a complex behavior is not well understood. While often presented as a stereotyped sequence of events, even extensively studied behaviors such as fly courtship are rarely a strict repetition of the same steps in a predetermined sequence in time. We are focusing on oviposition, the act of laying an egg, in flies of the genus Drosophila to describe the elementary behavioral steps or microbehaviors that a single female fly undertakes prior to and during egg laying. We have analyzed the hierarchy and relationships in time of these microbehaviors in three closely related Drosophila species with divergent egg-laying preferences and uncovered cryptic differences in their behavioral patterns. Using high-speed imaging, we quantified in depth the oviposition behavior of single females of Drosophila suzukii, Drosophila biarmipes and Drosophila melanogaster in a novel behavioral assay. By computing transitions between microbehaviors, we identified a common ethogram structure underlying oviposition of all three species. Quantifying parameters such as relative time spent on a microbehavior and its average duration, however, revealed clear differences between species. In addition, we examined the temporal dynamics and probability of transitions to different microbehaviors relative to a central event of oviposition, ovipositor contact. Although the quantitative analysis highlights behavioral variability across flies, it reveals some interesting trends for each species in the mode of substrate sampling, as well as possible evolutionary differences. Larger datasets derived from automated video annotation will overcome this paucity of data in the future, and use the same framework to reappraise these observed differences. Our study reveals a common architecture to the oviposition ethogram of three Drosophila species, indicating its ancestral state. It also indicates that Drosophila suzukii’s behavior departs quantitatively and qualitatively from that of the outgroup species, in line with its known divergent ethology. Together, our results illustrate how a global shift in ethology breaks down in the quantitative reorganization of the elementary steps underlying a complex behavior.

The Blow Fly Waltz: Field and Laboratory Observations of Novel and Complex Dipteran Courtship Behavior

Complex courtship has been well documented in the Diptera. However, studies have focused on a limited number of taxa and mostly using lab populations, where behavior can differ substantially compared to nature. To broaden our understanding of dipteran courtship, studies are required in a wider range of species, across both wild and captive populations. The blow flies (Diptera: Calliphoridae) include some of the most commonly encountered flies, yet courtship has been documented in less than 1% of species and is reported to be brief and simple throughout the family. To further investigate blow fly courtship, and to assess the effect of captivity on behavior, this study aimed to document the courtship of a habitat specialist that is endemic to Australasia, Chrysomya flavifrons. Video footage of wild and captive groups was recorded and analyzed using behavioral analysis software. The specific aims were 1) to quantify the behavioral sequences that constitute courtship and 2) to compare courtship between wild and captive flies. We found that the courtship behavior of Ch. flavifrons was complex and stereotyped, consisting of five discrete behaviors, which starkly contrasts with the simple courtship observed in most calliphorids. All of these stereotyped behaviors were observed in both wild and captive groups. However, the proportion of time males spent on these behaviors differed substantially. These findings highlight that blow fly courtship behavior may be more complex than currently perceived, and that careful consideration should be given to the influence of the laboratory environment in future studies of fly behavior.

Social effects on fruit fly courtship song.

Courtship behavior in Drosophila has often been described as a classic innate behavioral repertoire, but more recently extensive plasticity has been described. In particular, prior exposure to acoustic signals of con‐ or heterspecific males can change courtship traits in both sexes that are liable to be important in reproductive isolation. However, it is unknown whether male courtship song itself is socially plastic. We examined courtship song plasticity of two species in the Drosophila melanogaster subgroup. Sexual isolation between the species is influenced by two male song traits, the interpulse interval (IPI) and sinesong frequency (SSF). Neither of these showed plasticity when males had prior experience of con‐ and heterospecific social partners. However, males of both species produced longer bursts of song during courtship when they were exposed to social partners (either con‐ or heterospecific) than when they were reared in isolation. D. melanogaster carrying mutations affecting short‐ or medium‐term memory showed a similar response to the social environment, not supporting a role for learning. Our results demonstrate that the amount of song a male produces during courtship is plastic depending on the social environment, which might reflect the advantage of being able to respond to variation in intrasexual competition, but that song structure itself is relatively inflexible, perhaps due to strong selection against hybridization.

Prior information and social experience influence male reproductive decisions

Lay SummaryMate preferences vary among individuals. We show that male fruit fly courtship preferences are influenced by their previous experiences. Males that previously experienced a population of mostly large, high-quality females biased their subsequent courtship behavior toward large females more than males that had previously experienced mostly small females. Males that had experienced mostly small, low-quality females, and who had mated with a small female, tended to bias their subsequent courtship toward such low-quality females.

Apterous Brain Neurons Control Receptivity to Male Courtship in Drosophila Melanogaster Females

Courtship behaviours allow animals to interact and display their qualities before committing to reproduction. In fly courtship, the female decides whether or not to mate and is thought to display receptivity by slowing down to accept the male. Very little is known on the neuronal brain circuitry controlling female receptivity. Here we use genetic manipulation and behavioural studies to identify a novel set of neurons in the brain that controls sexual receptivity in the female without triggering the postmating response. We show that these neurons, defined by the expression of the transcription factor apterous, affect the modulation of female walking speed during courtship. Interestingly, we found that the apterous neurons required for female receptivity are neither doublesex nor fruitless positive suggesting that apterous neurons are not specified by the sex-determination cascade. Overall, these findings identify a neuronal substrate underlying female response to courtship and highlight the central role of walking speed in the receptivity behaviour.

An unsupervised method for quantifying the behavior of paired animals

Behaviors involving the interaction of multiple individuals are complex and frequently crucial for an animal’s survival. These interactions, ranging across sensory modalities, length scales, and time scales, are often subtle and difficult to characterize. Contextual effects on the frequency of behaviors become even more difficult to quantify when physical interaction between animals interferes with conventional data analysis, e.g. due to visual occlusion. We introduce a method for quantifying behavior in fruit fly interaction that combines high-throughput video acquisition and tracking of individuals with recent unsupervised methods for capturing an animal’s entire behavioral repertoire. We find behavioral differences between solitary flies and those paired with an individual of the opposite sex, identifying specific behaviors that are affected by social and spatial context. Our pipeline allows for a comprehensive description of the interaction between two individuals using unsupervised machine learning methods, and will be used to answer questions about the depth of complexity and variance in fruit fly courtship.

Fruit fly courtship: The female perspective.

Research on fruit fly courtship has mostly focused on males' behavioral repertoire whereas females have been assumed to respond by either rejecting or accepting males. In many fruit fly species including Drosophila melanogaster, however, mating typically follows an extended period of courtship, which provides ample opportunities for females to inform males about their likelihood of mating. Our experiments indeed revealed that sexually immature females in both D. melanogasterand D. simulans showed responses to conspecific males that were distinct from those of sexually mature females. Furthermore, females' responses to conspecific males were different from their responses to heterospecific males. Our data indicate that females' behavioral repertoire early in courtship can inform males about their probability of mating if they persist in courting. We hypothesize (i) that males can rely on behavioral feedback from females for optimally allocating their courtship efforts towards distinct female classes, (ii) that males may learn to modulate their courtship behavior based on specific feedback from females, and (iii) that females may learn to alter their behavior towards distinct types of males in order to elicit the desired male response. Overall, we suggest that, although little explored, female behavior determines the dynamics of courtship and mating and can thus influence sexual selection and incipient speciation.

Specificity of the receptor for the major sex pheromone component in Heliothis virescens.

In a previous study, the Drosophila melanogaster OR67dGAL4;UAS system was used to functionally characterize the receptor for the major component of the sex pheromone in the tobacco budworm, Heliothis virescens Fabricius (Lepidoptera: Noctuidae), HvOR13. Electrophysiological and behavioral assays showed that transgenic flies expressing HvOR13 responded to (Z)-11-hexadecenal (Z11-16:Ald). However, tests were not performed to determine whether these flies would also respond to secondary components of the H. virescens sex pheromone. Thus, in this study the response spectrum of HvOR13 expressed in this system was examined by performing single cell recordings from odor receptor neuron in trichoid T1 sensilla on antennae of two Or67dGAL4 [1]; UAS-HvOR13 lines stimulated with Z11-16:Ald and six H. virescens secondary pheromone components. Fly courtship assays were also performed to examine the behavioral response of the Or67dGAL4[1]; UAS-HvOR13 flies to Z11-16:Ald and the secondary component Z9-14:Ald. Our combined electrophysiological and behavioral studies indicated high specificity and sensitivity of HvOR13 to Z11-16:Ald. Interestingly, a mutation leading to truncation in the HvOR13 C-terminal region affected but did not abolish pheromone receptor response to Z11-16:Ald. The findings are assessed in relationship to other HvOR13 heterologous expression studies, and the role of the C-terminal domain in receptor function is discussed. A third line expressing HvOR15 was also tested but did not respond to any of the seven pheromone components.

Mutation of Drosophila dopamine receptor DopR leads to male–male courtship behavior

In Drosophila, dopamine plays important roles in many biological processes as a neuromodulator. Previous studies showed that dopamine level could affect fly courtship behaviors. Disturbed dopamine level leads to abnormal courtship behavior in two different ways. Dopamine up-regulation induces male–male courtship behavior, while down-regulation of dopamine level results in increased sexual attractiveness of males towards other male flies. Until now, the identity of the dopamine receptor involved in this abnormal male–male courtship behavior remains unknown. Here we used genetic approaches to investigate the role of dopamine receptors in fly courtship behavior. We found that a dopamine D1-like receptor, DopR, was involved in fly courtship behavior. DopR mutant male flies display male–male courtship behavior. This behavior is mainly due to the male’s increased propensity to court other males. Expression of functional DopR successfully rescued this mutant phenotype. Knock-down of D2-like receptor D2R and another D1-like receptor, DAMB, did not induce male–male courtship behavior, indicating the receptor-type specificity of this phenomenon. Our findings provide insight into a possible link between dopamine level disturbance and the induced male–male courtship behavior.

Invertebrate Neurobiology

Invertebrate Neurobiology G. North and R. J. Greenspan (eds) Cold Spring Harbor Laboratory Press, 2007. $135.- (hardcover), xii + 665 pp. ISBN 978-087969819-5 This is the second book published by Ralph Greenspan (together with Geoffrey North) in 2007 (see also preceding). It covers many different aspects of invertebrate neurobiology, although it will be clear that given the vastness of the subject, it can hardly be complete. Covering sensory and motor systems, learning and memory, evolution, and other behaviors such as foraging and courtship in flies, worms, and other organisms, the book succeeds in passing Greenspan’s message that researchers studying vertebrates can learn a lot from what is being discovered in invertebrates, despite the sometimes obvious lack of homology. Given how close this comes to the philosophy underlying Genes, Brain and Behavior’s editorial scope, it should not come as a surprise that I recommend this book warmly to all neurobiologist, regardless of their pet animal subject.

Scent of a Fly

Sexual courtship is a highly ritualized behavior in many animals. Recent work in the vinegar fly, Drosophila melanogaster, has illuminated how the pheromone cis-vaccenyl acetate modulates sexual behavior in the fly. Chemosensory receptors and a sexually dimorphic circuit activated by this pheromone have been identified. This minireview highlights recent advances in the field of fly courtship.

Increased Male–Male Courtship in Ecdysone Receptor Deficient Adult Flies

Male-male courtship is infrequent among mature adult Drosophila melanogaster. After pairs of mature adult males expressing a temperature-sensitive allele of the ecdysone receptor (EcR) gene were treated at a restrictive temperature, however, they engaged in elevated levels of male-male courtship. EcR-deficient males courted wildtype males and females, but were not courted by wildtype males. These results suggest that the ecdysone steroid hormone system may have a role in courtship initiation by adult male fruit flies.

The Role of the Flabellar and Ellipsoid Bodies of the Central Complex of the Brain of Drosophila Melanogaster in the Control of Courtship Behavior and Communicative Sound Production in Males

The role of flabellar and ellipsoid bodies of the central complex of the brain of Drosophila melanogaster males in controlling courtship behavior and the accompanying sound production was studied by comparative analysis of the characteristics of courtship and singing in wild-type flies and individuals of five mutant lines with different anatomical defects in these parts of the brain. Investigations were performed using the following fly lines: Canton S (wild-type, controls), ebo(KS263), with defects only in the ellipsoid bodies, and ebo(1041), ceb(849), ceb(892), and cba(KS96), with different levels of abnormality in both parts of the central complex. The data obtained here indicated that the flabellar and ellipsoid bodies are involved in: 1) maintaining a high level of courtship activity; 2) regulating the precision of male movements while following females; 3) in controlling the nature and stability of the sound elements from which communicative signals are constructed; 4) in regulating the rhythmic structure of signals dependent on the stability of pacemakers function; 5) establishing the relationship between behavior and the context in which it occurs.

Courtship in long-legged flies (Diptera: Dolichopodidae): function and evolution of signals

Analyzing the courtship behavior of long-legged flies (Diptera: Dolichopodidae), we focus on the evolutionary development of courtship signals. Long-legged flies may serve as a model for this evolutionary process, because males of some species present sexually dimorphic badges during courtship, whereas others do not exhibit such conspicuous signals but present lavish courtship behavior, including dynamic flight maneuvers. A comparison of these two groups within a single taxonomic family provides insight into the evolution of courtship signals and the corresponding behavior. Males of the closely related Empididae do not possess such badges. Within the super-family Empidoidea, we propose an evolutionary shift from dynamically courting and mating on the wing (in Empididae) to courting and mating on ground (in Dolichopodidae), accompanied by signaling through badge-waving. By comparing previously published data and observations on courtship behavior in Dolichopodidae, we present the hypothesis that the latter replaced the former energetically expensive behavior as a case of automimicry and sensory trap. Copyright 2003.

Accumulation of phenylpropanoids in the rectal glands of males of the Oriental fruit fly,Dacus dorsalis

Two phenylpropanoid compounds, 2-allyl-4,5-dimethoxyphenol(II) and coniferyl alcohol(III), were characterized from body tissue of wild males of the Oriental fruit fly,Dacus dorsalis. These compounds accumulated in the rectal glands only when laboratory-reared males were fed with methyl eugenol. Compound II was released into the air during dusk, which coincides with the fly courtship period. Pheromonal and allomonal effects of the phenylpropanoids were examined.