Acoustic communication between insects serves as an excellent model system for analyzing the neuronal mechanisms underlying auditory information processing. The detailed organization of auditory neural circuits in the brain has not yet been described. To understand the central auditory pathways, we used the brain of the fruit fly Drosophila melanogaster as a model and performed a large-scale analysis of the interneurons associated with the primary auditory center. By screening expression driver strains and performing single-cell labeling of these strains, we identified 44 types of interneurons innervating the primary auditory center. Five types were local interneurons whereas the other 39 types were projection interneurons connecting the primary auditory center with other brain regions. The projection neurons comprised three frequency-selective pathways and two frequency-embracive pathways. Mapping of their connection targets revealed that five neuropils in the brain-the wedge (WED), anterior ventrolateral protocerebrum, posterior ventrolateral protocerebrum (PVLP), saddle (SAD), and gnathal ganglia (GNG)-were intensively connected with the primary auditory center. In addition, several other neuropils, including visual and olfactory centers in the brain, were directly connected to the primary auditory center. The distribution patterns of the spines and boutons of the identified neurons suggest that auditory information is sent mainly from the primary auditory center to the PVLP, WED, SAD, GNG, and thoracico-abdominal ganglia. Based on these findings, we established the first comprehensive map of secondary auditory interneurons, which indicates the downstream information flow to parallel ascending pathways, multimodal pathways, and descending pathways.

The antennal ear of the fruit fly, called the Johnston's organ (JO), detects a wide variety of mechanosensory stimuli, including sound, wind, and gravity. Like many sensory cells in insect, JO neurons are compartmentalized in a sensory unit (i.e., scolopidium). To understand how different subgroups of JO neurons are organized in each scolopidial compartment, we visualized individual JO neurons by labeling various subgroups of JO neurons in different combinations. We found that vibration-sensitive (or deflection-sensitive) neurons rarely grouped together in a single scolopidial compartment. This finding suggests that JO neurons are grouped in stereotypical combinations each with a distinct response property in a scolopidium.

Male Aedes aegypti (Ae. aegypti) mosquitoes rely on hearing to identify conspecific females for mating, with the male attraction to the sound of flying females ("phonotaxis") an important behavior in the initial courtship stage. Hearing thus represents a promising target for novel methods of mosquito control, and hearing behaviors (such as male phonotaxis) can be targeted via the use of sound traps. These traps unfortunately have proven to be relatively ineffective during field deployment. Shifting the target from hearing behavior to hearing function could therefore offer a novel method of interfering with Ae. aegypti mating. Numerous neurotransmitters, including serotonin (5-hydroxytryptamine, or 5-HT) and octopamine, are expressed in the male ear, with modulation of the latter proven to influence the mechanical responses of the ear to sound. The effect of serotonin modulation however remains underexplored despite its significant role in determining many key behaviors and biological processes of animals. Here we investigated the influence of serotonin on the Ae. aegypti hearing function and behaviors. Using immunohistochemistry, we found significant expression of serotonin in the male and female Ae. aegypti ears. In the male ear, presynaptic sites identified via antibody labelling showed only partial overlap with serotonin. Next, we used RT-qPCR to identify and quantify the expression levels of three different serotonin receptor families (5-HT1, 5-HT2, and 5-HT7) in the mosquito heads and ears. Although all receptors were identified in the ears of both sexes, those from the 5-HT7 family were significantly more expressed in the ears relative to the heads. We then thoracically injected serotonin-related compounds into the mosquitoes and found a significant, reversible effect of serotonin exposure on the male ear mechanical tuning frequency. Finally, oral administration of a serotonin-synthesis inhibitor altered male phonotaxis. The mosquito serotonergic system and its receptors thus represent interesting targets for novel methods of mosquito, and thus disease, control.

Animal behavior is the final and integrated output of brain activity. Thus, recording and analyzing behavior is critical to understand the underlying brain function. While recording animal behavior has become easier than ever with the development of compact and inexpensive devices, detailed behavioral data analysis requires sufficient prior knowledge and/or high content data such as video images of animal postures, which makes it difficult for most of the animal behavioral data to be efficiently analyzed. Here, we report a versatile method using a hybrid supervised/unsupervised machine learning approach for behavioral state estimation and feature extraction (STEFTR) only from low-content animal trajectory data. To demonstrate the effectiveness of the proposed method, we analyzed trajectory data of worms, fruit flies, rats, and bats in the laboratories, and penguins and flying seabirds in the wild, which were recorded with various methods and span a wide range of spatiotemporal scales-from mm to 1,000 km in space and from sub-seconds to days in time. We successfully estimated several states during behavior and comprehensively extracted characteristic features from a behavioral state and/or a specific experimental condition. Physiological and genetic experiments in worms revealed that the extracted behavioral features reflected specific neural or gene activities. Thus, our method provides a versatile and unbiased way to extract behavioral features from simple trajectory data to understand brain function.

In birds and higher mammals, auditory experience during development is critical to discriminate sound patterns in adulthood. However, the neural and molecular nature of this acquired ability remains elusive. In fruit flies, acoustic perception has been thought to be innate. Here we report, surprisingly, that auditory experience of a species-specific courtship song in developing Drosophila shapes adult song perception and resultant sexual behavior. Preferences in the song-response behaviors of both males and females were tuned by social acoustic exposure during development. We examined the molecular and cellular determinants of this social acoustic learning and found that GABA signaling acting on the GABAA receptor Rdl in the pC1 neurons, the integration node for courtship stimuli, regulated auditory tuning and sexual behavior. These findings demonstrate that maturation of auditory perception in flies is unexpectedly plastic and is acquired socially, providing a model to investigate how song learning regulates mating preference in insects.

Many animal species form groups. Group characteristics differ between species, suggesting that the decision-making of individuals for grouping varies across species. However, the actual decision-making properties that lead to interspecific differences in group characteristics remain unclear. Here, we compared the group formation processes of two Drosophilinae fly species, Colocasiomyia alocasiae and Drosophila melanogaster, which form dense and sparse groups, respectively. A high-throughput tracking system revealed that C. alocasiae flies formed groups faster than D. melanogaster flies, and the probability of C. alocasiae remaining in groups was far higher than that of D. melanogaster. C. alocasiae flies joined groups even when the group size was small, whereas D. melanogaster flies joined groups only when the group size was sufficiently large. C. alocasiae flies attenuated their walking speed when the inter-individual distance between flies became small, whereas such behavioural properties were not clearly observed in D. melanogaster. Furthermore, depriving C. alocasiae flies of visual input affected grouping behaviours, resulting in a severe reduction in group formation. These findings show that C. alocasiae decision-making regarding grouping, which greatly depends on vision, is significantly different from D. melanogaster, leading to species-specific group formation properties.

During courtship, many animals, including insects, birds, fish, and mammals, utilize acoustic signals to transmit information about species identity. Although auditory communication is crucial across phyla, the neuronal and physiologic processes are poorly understood. Sound-evoked chaining behavior, a display of homosexual courtship behavior in Drosophila males, has long been used as an excellent model for analyzing auditory behavior responses, outcomes of acoustic perception and higher-order brain functions. Here we developed a new method, termed ChaIN (Chain Index Numerator), in which we use a computer-based auto detection system for chaining behavior. The ChaIN system can systematically detect the chaining behavior induced by a series of modified courtship song playbacks. Two evolutionarily related Drosophila species, Drosophila melanogaster and Drosophila simulans, exhibited dramatic selective increases in chaining behavior when exposed to specific auditory cues, suggesting that auditory discrimination processes are involved in the acceleration of chaining behavior. Prolonged monotonous pulse sounds containing courtship song components also induced high intense chaining behavior. Interestingly, the chaining behavior was gradually suppressed over time when song playback continued. This behavioral change is likely to be a plastic behavior and not a simple sensory adaptation or fatigue, because the suppression was released by applying a different pulse pattern. This behavioral plasticity is not a form of habituation because different modality stimuli did not recover the behavioral suppression. Intriguingly, this plastic behavior partially depended on the cAMP signaling pathway controlled by the rutabaga adenylyl cyclase gene that is important for learning and memory. Taken together, this study demonstrates the selectivity and behavioral kinetics of the sound-induced interacting behavior of Drosophila males, and provides a basis for the systematic analysis of genes and neural circuits underlying complex acoustic behavior.

The fruit fly Drosophila melanogaster responds behaviorally to sound, gravity, and wind. Johnston's organ (JO) at the antennal base serves as a sensory organ in the fruit fly to detect these mechanosensory stimuli. Among the five anatomically defined subgroups of sensory neurons in JO, subgroups A and B detect sound vibrations and subgroups C and E respond to static deflections, such as gravity and wind. The functions of subgroup-D JO neurons, however, remain unknown. In this study, we used molecular-genetic methods to explore the physiologic properties of subgroup-D JO neurons. Both vibrations and static deflection of the antennal receiver activated subgroup-D JO neurons. This finding clearly revealed that zone D in the antennal mechanosensory and motor center (AMMC), the projection target of subgroup-D JO neurons, is a primary center for antennal vibrations and deflection in the fly brain. We anatomically identified two types of interneurons downstream of subgroup-D JO neurons, AMMC local neurons (AMMC LNs), and AMMC D1 neurons. AMMC LNs are local neurons whose projections are confined within the AMMC, connecting zones B and D. On the other hand, AMMC D1 neurons have both local dendritic arborizations within the AMMC and descending projections to the thoracic ganglia, suggesting that AMMC D1 neurons are likely to relay information of the antennal movement detected by subgroup-D JO neurons from the AMMC directly to the thorax. Together, these findings provide a neural basis for how JO and its brain targets encode information of complex movements of the fruit fly antenna.

Diverse mechanosensory neurons detect different mechanical forces that can impact animal behavior. Yet our understanding of the anatomical and physiological diversity of these neurons and the behaviors that they influence is limited. We previously discovered that grooming of the Drosophila melanogaster antennae is elicited by an antennal mechanosensory chordotonal organ, the Johnston's organ (JO) (Hampel et al., 2015). Here, we describe anatomically and physiologically distinct JO mechanosensory neuron subpopulations that each elicit antennal grooming. We show that the subpopulations project to different, discrete zones in the brain and differ in their responses to mechanical stimulation of the antennae. Although activation of each subpopulation elicits antennal grooming, distinct subpopulations also elicit the additional behaviors of wing flapping or backward locomotion. Our results provide a comprehensive description of the diversity of mechanosensory neurons in the JO, and reveal that distinct JO subpopulations can elicit both common and distinct behavioral responses.

Commensal microbes influence various aspects of vertebrate and invertebrate brain function. We previously reported that Lactiplantibacillus plantarum SBT2227 promotes sleep in the fruit fly, Drosophila melanogaster. However, how widely the sleep-promoting effects are conserved in gut bacterial species remains unknown. In this study, we orally administered human intestinal and food-associated bacterial species (39 in total) to flies and investigated their effects on sleep. Six species of bacteria were found to have significant sleep-promoting effects. Of these, we further investigated Bifidobacterium adolescentis, which had the greatest sleep-promoting effect, and found that the strength of the sleep effect varied among strains of the same bacterial species. The B. adolescentis strains BA2786 and BA003 showed strong and weak effects on sleep, respectively. Transcriptome characteristics compared between the heads of flies treated with BA2786 or BA003 revealed that the gene expression of the insulin-like receptor (InR) was increased in BA2786-fed flies. Furthermore, a heterozygous mutation in InR suppressed the sleep-promoting effect of BA2786. These results suggest that orally administered sleep-promoting bacteria (at least BA2786), may act on insulin signaling to modulate brain function for sleep.

Many animals use acoustic signals to attract a potential mating partner. In fruit flies (Drosophila melanogaster), the courtship pulse song has a species-specific interpulse interval (IPI) that activates mating. Although a series of auditory neurons in the fly brain exhibit different tuning patterns to IPIs, it is unclear how the response of each neuron is tuned. Here, we studied the neural circuitry regulating the activity of antennal mechanosensory and motor center (AMMC)-B1 neurons, key secondary auditory neurons in the excitatory neural pathway that relay song information. By performing Ca2+ imaging in female flies, we found that the IPI selectivity observed in AMMC-B1 neurons differs from that of upstream auditory sensory neurons [Johnston's organ (JO)-B]. Selective knock-down of a GABAA receptor subunit in AMMC-B1 neurons increased their response to short IPIs, suggesting that GABA suppresses AMMC-B1 activity at these IPIs. Connection mapping identified two GABAergic local interneurons that synapse with AMMC-B1 and JO-B. Ca2+ imaging combined with neuronal silencing revealed that these local interneurons, AMMC-LN and AMMC-B2, shape the response pattern of AMMC-B1 neurons at a 15 ms IPI. Neuronal silencing studies further suggested that both GABAergic local interneurons suppress the behavioral response to artificial pulse songs in flies, particularly those with a 15 ms IPI. Altogether, we identified a circuit containing two GABAergic local interneurons that affects the temporal tuning of AMMC-B1 neurons in the song relay pathway and the behavioral response to the courtship song. Our findings suggest that feedforward inhibitory pathways adjust the behavioral response to courtship pulse songs in female flies.SIGNIFICANCE STATEMENT To understand how the brain detects time intervals between sound elements, we studied the neural pathway that relays species-specific courtship song information in female Drosophila melanogaster We demonstrate that the signal transmission from auditory sensory neurons to key secondary auditory neurons antennal mechanosensory and motor center (AMMC)-B1 is the first-step to generate time interval selectivity of neurons in the song relay pathway. Two GABAergic local interneurons are suggested to shape the interval selectivity of AMMC-B1 neurons by receiving auditory inputs and in turn providing feedforward inhibition onto AMMC-B1 neurons. Furthermore, these GABAergic local interneurons suppress the song response behavior in an interval-dependent manner. Our results provide new insights into the neural circuit basis to adjust neuronal and behavioral responses to a species-specific communication sound.

We established a comprehensive projection map of the auditory receptor cells (Johnston's organ neurons: JONs) from the antennae to the primary auditory center of the Drosophila brain. We found 477 +/- 24 cell bodies of JONs, which are arranged like a "bottomless bowl" within the auditory organ. The target of the JONs in the brain comprises five spatially segregated zones, each of which is contributed by bundles of JON axons that gradually branch out from the antennal nerve. Four zones are confined in the antennal mechanosensory and motor center, whereas one zone further extends over parts of the ventrolateral protocerebrum and the subesophageal ganglion. Single-cell labeling with the FLP-out technique revealed that most JONs innervate only a single zone, indicating that JONs can be categorized into five groups according to their target zones. Within each zone, JONs innervate various combinations of subareas. We classified these five zones into 19 subareas according to the branching patterns and terminal distributions of single JON axons. The groups of JONs that innervate particular zones or subareas of the primary auditory center have their cell bodies in characteristic locations of the Johnston's organ in the antenna, e.g., in concentric rings or in paired clusters. Such structural organization suggests that each JON group, and hence each zone of the primary auditory center, might sense different aspects of sensory signals.

Many animals utilize auditory signals to communicate with conspecific individuals. During courtship, males of the fruit fly Drosophila melanogaster and related species produce a courtship song comprised of sine and pulse songs by vibrating their wings. The pulse song increases female receptivity and male courtship activity, indicating that it functions as a sexual signal. One song parameter, interpulse interval (IPI), varies among closely related species. In D. melanogaster, a song with a conspecific IPI induces a stronger behavioral response than heterospecific songs, indicating the ability of the flies to discriminate conspecific IPI. Traditionally, the fly's response to the song is measured under grouped conditions, in which the effect of sensory modalities other than audition cannot be excluded. Here, to quantify the individual ability to discriminate a conspecific song, we systematically analyzed the auditory response of single male flies to sound with various parameters. Moreover, we applied this method, termed SMART (Single Male Auditory Response Test), to two sister species for potential application in a comparative approach. By quantifying the locomotor activity of single D. melanogaster males during sound exposure, we detected increased locomotor activity in response to pulse songs, but not to white noise or pure tone. The conspecific song evoked stronger response than the heterospecific songs, and ablation of their antennal receivers severely suppressed the locomotor increase. A pulse song with a small IPI variation evoked a continuous response, while the response to songs with highly variable IPIs tends to be rapidly decayed. This provides the first evidence that fruit flies discriminate IPI variations, which possibly inform the age and social contexts of the singer. Sister species, D. sechellia, exhibited a locomotor response to pulse song, while D. simulans exhibited no behavioral response. This suggests that auditory and other stimuli that elicit this behavioral response are diversified among Drosophila species.

In the early phase of courtship, female fruit flies exhibit an acute rejection response to avoid unfavorable mating. This pre-mating rejection response is evolutionarily paralleled across species, but the molecular and neuronal basis of that behavior is unclear. Here, we show that a putative incoherent feedforward circuit comprising ellipsoid body neurons, cholinergic R4d, and its repressor GABAergic R2/R4m neurons regulates the pre-mating rejection response in the virgin female Drosophila melanogaster. Both R4d and R2/R4m are positively regulated, via specific dopamine receptors, by a subset of neurons in the dopaminergic PPM3 cluster. Genetic deprivation of GABAergic signal via GABAA receptor RNA interference in this circuit induces a massive rejection response, whereas activation of GABAergic R2/R4m or suppression of cholinergic R4d increases receptivity. Moreover, glutamatergic signaling via N-methyl-d-aspartate receptors induces NO-mediated retrograde regulation potentially from R4d to R2/R4m, likely providing flexible control of the behavioral switching from rejection to acceptance. Our study elucidates the molecular and neural mechanisms regulating the behavioral selection process of the pre-mating female.

Increased levels of dysfunctional mitochondria within skeletal muscle are correlated with numerous age-related physiopathological conditions. Improving our understanding of the links between mitochondrial function and muscle proteostasis, and the role played by individual genes and regulatory networks, is essential to develop treatments for these conditions. One potential player is the mitochondrial outer membrane protein Fis1, a crucial fission factor heavily involved in mitochondrial dynamics in yeast but with an unknown role in higher-order organisms. By using Drosophila melanogaster as a model, we explored the effect of Fis1 mutations generated by transposon Minos-mediated integration. Mutants exhibited a higher ratio of damaged mitochondria with age as well as elevated reactive oxygen species levels compared with controls. This caused an increase in oxidative stress, resulting in large accumulations of ubiquitinated proteins, accelerated muscle function decline, and mitochondrial myopathies in young mutant flies. Ectopic expression of Fis1 isoforms was sufficient to suppress this phenotype. Loss of Fis1 led to unbalanced mitochondrial proteostasis within fly muscle, decreasing both flight capabilities and lifespan. Fis1 thus clearly plays a role in fly mitochondrial dynamics. Further investigations into the detailed function of Fis1 are necessary for exploring how mitochondrial function correlates with muscle health during aging.

In internal fertilization animals, maintaining a copulation posture facilitates the process of transporting gametes from male to female. Here, we present a protocol to investigate the neural basis for copulation posture of fruit flies using a closed-loop real-time optogenetic system. We describe steps for using deep learning analysis to enable optogenetic manipulation of neural activity only during copulation with high efficiency. This system can be applied to various animal behaviors other than copulation. For complete details on the use and execution of this protocol, please refer to Yamanouchi et al. (2023).1.

In internal fertilization animals, reproductive success depends on maintaining copulation until gametes are transported from male to female. In Drosophila melanogaster, mechanosensation in males likely contributes to copulation maintenance, but its molecular underpinning remains to be identified. Here we show that the mechanosensory gene piezo and its' expressing neurons are responsible for copulation maintenance. An RNA-seq database search and subsequent mutant analysis revealed the importance of piezo for maintaining male copulation posture. piezo-GAL4-positive signals were found in the sensory neurons of male genitalia bristles, and optogenetic inhibition of piezo-expressing neurons in the posterior side of the male body during copulation destabilized posture and terminated copulation. Our findings suggest that the mechanosensory system of male genitalia through Piezo channels plays a key role in copulation maintenance and indicate that Piezo may increase male fitness during copulation in flies.

Lactic acid bacteria (LAB) influence multiple aspects of host brain function via the production of active metabolites in the gut, which is known as the pre/probiotic action. However, little is known about the biogenic effects of LAB on host brain function. Here, we reported that the Lactobacillus plantarum SBT2227 promoted sleep in Drosophila melanogaster. Administration of SBT2227 primarily increased the amount of sleep and decreased sleep latency at the beginning of night-time. The sleep-promoting effects of SBT2227 were independent of the existing gut flora. Furthermore, heat treatment or mechanical crushing of SBT2227 did not suppress the sleep-promoting effects, indicative of biogenic action. Transcriptome analysis and RNAi mini-screening for gut-derived peptide hormones revealed the requirement of neuropeptide F, a homolog of the mammalian neuropeptide Y, for the action of SBT2227. These biogenic effects of SBT2227 on the host sleep provide new insights into the interaction between the brain and gut bacteria.

The antennal ear of the fruit fly detects acoustic signals in intraspecific communication, such as the courtship song and agonistic sounds. Among the five subgroups of mechanosensory neurons in the fly ear, subgroup-A neurons respond maximally to vibrations over a wide frequency range between 100 and 1,200 Hz. The functional organization of the neural circuit comprised of subgroup-A neurons, however, remains largely unknown. In the present study, we used 11 GAL4 strains that selectively label subgroup-A neurons and explored the diversity of subgroup-A neurons by combining single-cell anatomic analysis and Ca2+ imaging. Our findings indicate that the subgroup-A neurons that project into various combinations of subareas in the brain are more anatomically diverse than previously described. Subgroup-A neurons were also physiologically diverse, and some types were tuned to a narrow frequency range, suggesting that the response of subgroup-A neurons to sounds of a wide frequency range is due to the existence of several types of subgroup-A neurons. Further, we found that an auditory behavioral response to the courtship song of flies was attenuated when most subgroup-A neurons were silenced. Together, these findings characterize the heterogeneous functional organization of subgroup-A neurons, which might facilitate species-specific acoustic signal detection.

Many animals rely on acoustic cues to decide what action to take next. Unraveling the wiring patterns of the auditory neural pathways is prerequisite for understanding such information processing. Here, we reconstructed the first step of the auditory neural pathway in the fruit fly brain, from primary to secondary auditory neurons, at the resolution of transmission electron microscopy. By tracing axons of two major subgroups of auditory sensory neurons in fruit flies, low-frequency tuned Johnston's organ (JO)-B neurons and high-frequency tuned JO-A neurons, we observed extensive connections from JO-B neurons to the main second-order neurons in both the song-relay and escape pathways. In contrast, JO-A neurons connected strongly to a neuron in the escape pathway. Our findings suggest that heterogeneous JO neuronal populations could be recruited to modify escape behavior whereas only specific JO neurons contribute to courtship behavior. We also found that all JO neurons have postsynaptic sites at their axons. Presynaptic modulation at the output sites of JO neurons could affect information processing of the auditory neural pathway in flies.