Behavioral screening of sleep-promoting effects of human intestinal and food-associated bacteria on Drosophila melanogaster.

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.

Molecular and neural mechanisms regulating sexual motivation of virgin female Drosophila.

During courtship, multiple information sources are integrated in the brain to reach a final decision, i.e., whether or not to mate. The brain functions for this complex behavior can be investigated by genetically manipulating genes and neurons, and performing anatomical, physiological, and behavioral analyses. Drosophila is a powerful model experimental system for such studies, which need to be integrated from molecular and cellular levels to the behavioral level, and has enabled pioneering research to be conducted. In male flies, which exhibit a variety of characteristic sexual behaviors, we have accumulated knowledge of many genes and neural circuits that control sexual behaviors. On the other hand, despite the importance of the mechanisms of mating decision-making in females from an evolutionary perspective (such as sexual selection), research on the mechanisms that control sexual behavior in females has progressed somewhat slower. In this review, we focus on the pre-mating behavior of female Drosophila melanogaster, and introduce previous key findings on the neuronal and molecular mechanisms that integrate sensory information and selective expression of behaviors toward the courting male.

GABAergic Local Interneurons Shape Female Fruit Fly Response to Mating Songs.

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.

A single male auditory response test to quantify auditory behavioral responses in Drosophila melanogaster.

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.

Softness sensing and learning in Drosophila larvae.

Mechanosensation provides animals with important sensory information in addition to olfaction and gustation during feeding behavior. Here, we used Drosophila melanogaster larvae to investigate the role of softness sensing in behavior and learning. In the natural environment, larvae need to dig into soft foods for feeding. Finding foods that are soft enough to dig into is likely to be essential for their survival. We report that larvae can discriminate between different agar concentrations and prefer softer agar. Interestingly, we show that larvae on a harder surface search for a softer surface using memory associated with an odor, and that they evaluate foods by balancing softness and sweetness. These findings suggest that larvae integrate mechanosensory information with chemosensory input while foraging. Moreover, we found that the larval preference for softness is affected by genetic background.

The Nutrient-Responsive Hormone CCHamide-2 Controls Growth by Regulating Insulin-like Peptides in the Brain of Drosophila melanogaster.

The coordination of growth with nutritional status is essential for proper development and physiology. Nutritional information is mostly perceived by peripheral organs before being relayed to the brain, which modulates physiological responses. Hormonal signaling ensures this organ-to-organ communication, and the failure of endocrine regulation in humans can cause diseases including obesity and diabetes. In Drosophila melanogaster, the fat body (adipose tissue) has been suggested to play an important role in coupling growth with nutritional status. Here, we show that the peripheral tissue-derived peptide hormone CCHamide-2 (CCHa2) acts as a nutrient-dependent regulator of Drosophila insulin-like peptides (Dilps). A BAC-based transgenic reporter revealed strong expression of CCHa2 receptor (CCHa2-R) in insulin-producing cells (IPCs) in the brain. Calcium imaging of brain explants and IPC-specific CCHa2-R knockdown demonstrated that peripheral-tissue derived CCHa2 directly activates IPCs. Interestingly, genetic disruption of either CCHa2 or CCHa2-R caused almost identical defects in larval growth and developmental timing. Consistent with these phenotypes, the expression of dilp5, and the release of both Dilp2 and Dilp5, were severely reduced. Furthermore, transcription of CCHa2 is altered in response to nutritional levels, particularly of glucose. These findings demonstrate that CCHa2 and CCHa2-R form a direct link between peripheral tissues and the brain, and that this pathway is essential for the coordination of systemic growth with nutritional availability. A mammalian homologue of CCHa2-R, Bombesin receptor subtype-3 (Brs3), is an orphan receptor that is expressed in the islet ß-cells; however, the role of Brs3 in insulin regulation remains elusive. Our genetic approach in Drosophila melanogaster provides the first evidence, to our knowledge, that bombesin receptor signaling with its endogenous ligand promotes insulin production.

The neural basis of Drosophila gravity-sensing and hearing.

The neural substrates that the fruitfly Drosophila uses to sense smell, taste and light share marked structural and functional similarities with ours, providing attractive models to dissect sensory stimulus processing. Here we focus on two of the remaining and less understood prime sensory modalities: graviception and hearing. We show that the fly has implemented both sensory modalities into a single system, Johnston's organ, which houses specialized clusters of mechanosensory neurons, each of which monitors specific movements of the antenna. Gravity- and sound-sensitive neurons differ in their response characteristics, and only the latter express the candidate mechanotransducer channel NompC. The two neural subsets also differ in their central projections, feeding into neural pathways that are reminiscent of the vestibular and auditory pathways in our brain. By establishing the Drosophila counterparts of these sensory systems, our findings provide the basis for a systematic functional and molecular dissection of how different mechanosensory stimuli are detected and processed.

Distinct sensory representations of wind and near-field sound in the Drosophila brain.

Behavioural responses to wind are thought to have a critical role in controlling the dispersal and population genetics of wild Drosophila species, as well as their navigation in flight, but their underlying neurobiological basis is unknown. We show that Drosophila melanogaster, like wild-caught Drosophila strains, exhibits robust wind-induced suppression of locomotion in response to air currents delivered at speeds normally encountered in nature. Here we identify wind-sensitive neurons in Johnston's organ, an antennal mechanosensory structure previously implicated in near-field sound detection (reviewed in refs 5 and 6). Using enhancer trap lines targeted to different subsets of Johnston's organ neurons, and a genetically encoded calcium indicator, we show that wind and near-field sound (courtship song) activate distinct populations of Johnston's organ neurons, which project to different regions of the antennal and mechanosensory motor centre in the central brain. Selective genetic ablation of wind-sensitive Johnston's organ neurons in the antenna abolishes wind-induced suppression of locomotion behaviour, without impairing hearing. Moreover, different neuronal subsets within the wind-sensitive population respond to different directions of arista deflection caused by air flow and project to different regions of the antennal and mechanosensory motor centre, providing a rudimentary map of wind direction in the brain. Importantly, sound- and wind-sensitive Johnston's organ neurons exhibit different intrinsic response properties: the former are phasically activated by small, bi-directional, displacements of the aristae, whereas the latter are tonically activated by unidirectional, static deflections of larger magnitude. These different intrinsic properties are well suited to the detection of oscillatory pulses of near-field sound and laminar air flow, respectively. These data identify wind-sensitive neurons in Johnston's organ, a structure that has been primarily associated with hearing, and reveal how the brain can distinguish different types of air particle movements using a common sensory organ.

Neuronal encoding of sound, gravity, and wind in the fruit fly.

The fruit fly Drosophila melanogaster responds behaviorally to sound, gravity, and wind. Exposure to male courtship songs results in reduced locomotion in females, whereas males begin to chase each other. When agitated, fruit flies tend to move against gravity. When faced with air currents, they 'freeze' in place. Based on recent studies, Johnston's hearing organ, the antennal ear of the fruit fly, serves as a sensor for all of these mechanosensory stimuli. Compartmentalization of sense cells in Johnston's organ into vibration-sensitive and deflection-sensitive neural groups allows this single organ to mediate such varied functions. Sound and gravity/wind signals sensed by these two neuronal groups travel in parallel from the fly ear to the brain, feeding into neural pathways reminiscent of the auditory and vestibular pathways in the human brain. Studies of the similarities between mammals and flies will lead to a better understanding of the principles of how sound and gravity information is encoded in the brain. Here, we review recent advances in our understanding of these principles and discuss the advantages of the fruit fly as a model system to explore the fundamental principles of how neural circuits and their ensembles process and integrate sensory information in the brain.

Piezo-mediated mechanosensation contributes to stabilizing copulation posture and reproductive success in Drosophila males.

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.

Protocol to investigate the neural basis for copulation posture of Drosophila using a closed-loop real-time optogenetic system.

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.

Evolutionary conservation and diversification of auditory neural circuits that process courtship songs in Drosophila.

Acoustic communication signals diversify even on short evolutionary time scales. To understand how the auditory system underlying acoustic communication could evolve, we conducted a systematic comparison of the early stages of the auditory neural circuit involved in song information processing between closely-related fruit-fly species. Male Drosophila melanogaster and D. simulans produce different sound signals during mating rituals, known as courtship songs. Female flies from these species selectively increase their receptivity when they hear songs with conspecific temporal patterns. Here, we firstly confirmed interspecific differences in temporal pattern preferences; D. simulans preferred pulse songs with longer intervals than D. melanogaster. Primary and secondary song-relay neurons, JO neurons and AMMC-B1 neurons, shared similar morphology and neurotransmitters between species. The temporal pattern preferences of AMMC-B1 neurons were also relatively similar between species, with slight but significant differences in their band-pass properties. Although the shift direction of the response property matched that of the behavior, these differences are not large enough to explain behavioral differences in song preferences. This study enhances our understanding of the conservation and diversification of the architecture of the early-stage neural circuit which processes acoustic communication signals.

Biogenic action of Lactobacillus plantarum SBT2227 promotes sleep in Drosophila melanogaster.

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.

Distinct decision-making properties underlying the species specificity of group formation of flies.

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.

Serotonin modulation in the male Aedes aegypti ear influences hearing.

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.

Loss of Fis1 impairs proteostasis during skeletal muscle aging in Drosophila.

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.

Mechanical feedback amplification in Drosophila hearing is independent of synaptic transmission.

Vertebrate inner-ear hair cells use mechanical feedback to amplify sound-induced vibrations. The gain of this 'cochlear amplifier' is centrally controlled via efferent fibres that, making synaptic contacts with the hair cells, modulate the feedback gain. The sensory neurons of the Drosophila ear likewise employ mechanical feedback to assist sound-evoked vibrations, yet whether this neuron-based feedback is also subject to efferent control has remained uncertain. We show here that the function of Drosophila auditory neurons is independent of efferent modulation, and that no synaptic transmission is needed to control the gain of mechanical feedback amplification. Immunohistochemical, mechanical and electrophysiological analyses revealed that the Drosophila auditory organ lacks peripheral synapses and efferent innervations, and that blocking synaptic transmission in a pan-neural manner does not affect the afferent electrical activity of the sensory neurons or the mechanical feedback gain. Hence, unlike the cochlear amplifier of vertebrates, mechanical feedback amplification in Drosophila is not associated with an efferent control system but seems to be a purely local process that is solely controlled peripherally within the ear itself.

A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila.

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.

Distinct subpopulations of mechanosensory chordotonal organ neurons elicit grooming of the fruit fly antennae.

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.