Hayato M. Yamanouchi,Ryoya Tanaka,Azusa Kamikouchi
Piezo-mediated mechanosensation contributes to stabilizing copulation posture and reproductive success in Drosophila males
Mechanobiology,Molecular physiology,Behavioral neuroscience
Summary 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.
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.

In internally fertilizing animals, reproduction is dependent on prolonging copulation until gametes are transported from the male to the female reproductive organ. Copulation termination before gamete transport prevents females from becoming fertile., During copulation, the male keeps coupling his genitalia to the female’s genitalia by maintaining a specific copulation posture suggesting that maintaining copulation posture is important to maintain genital coupling., The genitalia and abdominal structures of both males and females are adaptively tuned in a species-specific manner, like a lock and key, to maintain copulation posture.,, Despite accumulating knowledge of morphological aspects of copulation posture maintenance, the neuronal and/or molecular mechanisms underlying it are largely unexplored. The fruit fly Drosophila melanogaster serves as a suitable model for such studies, as its mating behavior is easily quantified under laboratory conditions, together with the availability of various genetic tools to manipulate gene expression and neural activity., Before starting copulation, male D. melanogaster display courtship behaviors toward a target female, including orientation, tapping, following, singing, licking, and attempted copulation. When the male initiates courtship, females typically exhibit signatures of pre-mating rejection such as escaping and kicking. However, as the male continues courtship, females gradually decrease locomotion and eventually accept copulation. At the attempted copulation stage, a male bends his abdomen in an effort to couple his genitalia with that of the female, and if the female is sufficiently receptive, they will couple their genitalia to initiate copulation., Following their genital coupling, the male mounts on the female and maintains his position.,, The duration of copulation (i.e., genital coupling) is almost the same as that of the “male mounting on a female” period, during which the male maintains his copulation posture.,,, The male needs to continue this posture at least until he transfers sufficient sperm for fertile mating, which occurs about 8 min after copulation initiation. Regulation of copulation posture likely involves the interplay of the central and peripheral nervous systems, including the mechanosensory system of the male genitalia. The male genitalia are covered with sensory bristles, with sensory neurons projecting to the base. Among these sensory bristles, a single pair of long mechanosensilla is known for its importance in determining copulation posture, as ablation of a single one tends to tilt the posture of copulating males. In the male abdominal ganglion, mechanosensory neurons at the sensory bristles of male genitalia have synaptic and functional connections to a sexually dimorphic motor circuit that mediates the motor sequence of initiating and terminating copulation. The motor neurons in this circuit regulate the phallic and periphallic musculature, which regulates genital grasping of the female genitalia, depending on mechanosensory inputs. Inhibition of fruitless (fru) neurons in the male genitalia (including fru+ mechanosensory neurons) results in shorter copulation duration, raising the possibility that mechanosensory neurons of the male genitalia are involved in the regulation of copulation duration. These previous studies together suggest that males receive mechanosensory information from females in copula via mechanosensory bristles of the genitalia, and use that information to maintain copulation until gametes are transported to the female. Which mechanosensory channels are expressed in these bristles and how they contribute to the maintenance of copulation, however, remains underexplored. Several types of mechanosensory channels have been identified in D. melanogaster. Among them, piezo, nompC, nanchung (nan), and inactive (iav) are expressed in the sensory bristle neurons of Drosophila. Functionally, NompC, Nanchung (Nan), and Inactive (Iav) are known as hearing genes; these three transient receptor potential (TRP) channels are expressed in the mechanosensory neurons within the fly ear and are involved in the auditory mechanotransduction.,, In mechanosensory neurons of Johnston’s organ (the sensory organ of the fly ear), Nan and Iav form heteromeric channels and are required for electrical signaling, possibly at the step of auditory mechanotransduction., Mutations in nompC channels reduce the amplitude and sensitivity of sound-evoked nerve responses from the Johnston’s organ, suggesting that NompC acts as a mechanical pre-amplifier in the fly ear. In the larval body wall neurons, NompC senses gentle touch and regulates locomotion., Another mechanosensory channel, Piezo, is a Drosophila homolog of the mammalian Piezo channels. It induces a mechanically activated current as is the case for its mammalian counterparts.,, In adult D. melanogaster, Piezo channels are responsible for internal and external mechanosensation.,,, As part of its function as an internal sensor, Piezo channels detect the expansion and contraction of the crop, which contributes to the regulation of feeding behavior.,, As an example of its role as an external sensor, Piezo expressed in the sensory neurons of the mechanosensory bristles at female genitalia and/or in neurons innervating the reproductive tract contributes to female mating experience. In this study, we explored the mechanosensory mechanisms underlying male copulation maintenance. To select candidate channels involved in the copulation maintenance of males, we utilized the single-cell transcriptome database of the entire adult fly. We found piezo as a highly enriched mechanosensory gene in the neural clusters of the male body wall dataset, which includes the mechanosensory bristle cells of the male genitalia. We observed the copulation of piezo mutant (PiezoKO) males and found their body axis tended to tilt during copulation, suggesting piezo is involved in copulation posture maintenance. In addition, wild-type females that copulated with PiezoKO males produced fewer offspring than those copulated with control males, indicating reduced male fitness. Molecular-genetic analysis showed that piezo was expressed in the mechanosensory bristle neurons in the male genitalia, which play important roles to maintain copulation. When piezo-expressing neurons on the posterior side were optogenetically suppressed during copulation, the copulation was disrupted and terminated. Taken together, our findings revealed a significant role of Piezo-mediated mechanosensation in copulation maintenance and reproductive success.

Mechanosensory bristles on the male external genitalia are important for maintaining male copulation postures. Mechanoreception in the male genitalia during copulation, likely mediated by mechanosensory channels, thus possibly plays a significant role in male copulation posture. Previous studies have reported gene expression of mechanosensory channels piezo, nompC, nanchung (nan), and inactive (iav) in the sensory bristle neurons of Drosophila, making them candidates for the molecular component involved in keeping the male copulation posture. To narrow down candidate mechanosensory channels that contribute to this process, we used a large-scale single-cell RNA-seq analysis platform, SCope., Since no dataset of genital sensory neurons was available in SCope, we used the male’s body wall data that potentially include the sensory neurons in the external genitalia. In the SCope database, the body wall data is annotated into cell types, such as the peripheral or ventral nervous system, fat body, and hemocyte (Figure S1). Because previous studies suggested that males detect genital coupling via genital mechanosensory neurons during copulation,, we focused on neuronal clusters in this dataset which potentially include sensory neurons: the peripheral and ventral nervous system clusters. These two clusters abundantly expressed the neuronal Synaptobrevin (nSyb) gene, a marker of neuronal cells, verifying their neuronal properties (Figure 1). In these two neuronal clusters, we detected the expression of three mechanosensory channels, piezo, nompC, and iav (but not nan), among which piezo was the most abundant (Figure 1). A previous study using male mice identified Piezo1 expression in the ejaculatory ducts and surrounding tissues, indicating that Piezo1-mediated mechanosensation is possibly involved in male ejaculatory function. We thus focused on the Drosophila piezo gene in the following experiments. The piezo gene encodes a component of a mechanosensory channel36. In females, mechanosensation by Piezo reduces receptivity after copulation. However, the effect of Piezo on copulation in males remains underexplored, including its involvement in the copulation posture. In order to investigate the possible role of Piezo in copulation postures, we observed copulation behaviors of PiezoKO males paired with wild-type females. PiezoKO males actively courted females, and in most cases successfully started copulation, as judged by genital coupling between the male and female. However, although their genitalia kept coupled, the copulation postures of PiezoKO males were unstable; they often tilted during the entire copulation period when compared to the wild-type males (Figures 2A and 2B). This phenotype looked similar to the tilted copulation postures observed in wild-type males whose one of the genital long mechanosensilla was ablated. Using DeepLabCut, a machine-learning-based markerless animal pose estimation tool, we quantified the postures of males throughout copulation events. The ratio of time in which the male engaged in tilted copulation (i.e., copulation angle, the angle between the body axes of two flies engaged in copulation, was ≥ 25°) was significantly larger in PiezoKO homozygous males than in control flies (Figures 2C and S2A–S2K). Furthermore, the copulation angle of PiezoKO homozygous males was indeed unstable when compared to that in control flies, as copulations of PiezoKO homozygous males fluctuated between tilted and stereotypic angles more frequently than those of control flies, resulting in a significantly larger variance of copulation angle (Figures 2B, 2D, and 2E). The tilted copulation observed in PiezoKO homozygous males may be due to the rejection behavior of females in copula. Indeed, a prior study in D. melanogaster observed that the female kicks the male with her hind legs during copulation. We examined if females coupled with PiezoKO homozygous males showed more intense kicking behavior than those with wild-type males. Contrary to our speculation, females kicked wild-type males as frequently as PiezoKO homozygous males (Figure S3A). This finding suggests that piezo mutation in males has no significant effect on female kicking behaviors during copulation. Another possibility we speculated was that the piezo mutation might affect the ability of the male genitalia to capture the female’s genitalia, which is required for successful copulation. We addressed this possibility by measuring the frequency of male genital touching before copulation (i.e., the male bending his abdomen and touching female genitalia before copulation initiation). Again, no significant difference was detected in this touching frequency between PiezoKO homozygous males and wild-type males (Figure S3B). These results exclude the possibility that tilted copulation posture in PiezoKO males was due to females’ rejection behaviors in copula or by genitalia mis-coupling, and lend support to the hypothesis that piezo-mediated mechanosensation in males contributes to stabilizing their copulation postures. We next tested if unstable copulation posture affected male fertility. Wild-type females that copulated with PiezoKO homozygous males produced significantly fewer offspring than those that copulated with heterozygous (PiezoKO/+) control males (Figure 3A). At the step of copulation behavior, however, copulation duration and rate for PiezoKO homozygous males were virtually identical to those of control males (Figures 3B and 3C). This finding supports the hypothesis that general courtship activity is comparable between PiezoKO homozygous males and control males. We further tested for deficits in vitality or physical agility caused by piezo mutation. Prior studies reported that Piezo expressed in the digestive organs affects the control of feeding behaviors., Accordingly, we observed that locomotor activity and the body size in PiezoKO homozygous males were significantly increased compared to control flies (Figures S4A and S4B). These results suggest that general markers of health do not appear compromised in PiezoKO homozygous. Moreover, a previous study reported that the body size of males had no significant effect on male fertility, suggesting that the larger body size of PiezoKO homozygous males does not cause the reduction of the offspring number. Fitness reduction in PiezoKO homozygous males may be due to lower sperm production. The sizes of the testes, seminal vesicles, and accessory glands, however, appeared similar between PiezoKO homozygous males and control males (Figure S4C). Moreover, the seminal vesicles of PiezoKO homozygous males contained much sperm, and were indistinguishable from that of control males (Figure S4D). These results suggest that the decreased offspring number of PiezoKO homozygous males, who showed unstable copulation postures, is potentially due to inefficient sperm transport rather than spermatogenesis defects. Stabilizing copulation posture might be important not only in stable conditions but also under disturbances. Under natural conditions, various external stimuli can potentially disturb copulation, such as wind and substrate vibrations.,, We tested if the unstable posture of PiezoKO homozygous males affected copulation persistency under physical disturbances by applying mechanical vibrations in the middle of copulation using a vortex mixer. The disturbance was applied 5 min into copulation, when “copulation persistence” is at its peak. The ratio of pairs that got separated during the vibrations, however, seemed similar among PiezoKO homozygous males, heterozygous males, and wild-type males (Figure S5). This result suggests that the disruption of piezo has less impact on copulation persistency under disturbances, highlighting its role in stabilizing the copulation posture of males under undisturbed conditions.

Males interact with females through various body parts, including the genitalia, during copulation (Figure 4A). During copulation, the male genital mechanosensory neurons are suggested to detect genital coupling., We, therefore, speculated that unstable copulation in PiezoKO males might be due to the failure of Piezo-mediated mechanosensation in the male genitalia. It is reported that piezo is expressed in sensory neurons and non-neuronal tissues in both adults and larvae. We then evaluated the hypothesis that piezo-expressing sensory neurons are involved in the stability of the copulation postures in males. To test if piezo is expressed in the sensory neurons of genitalia, we visualized piezo-GAL4 expressing cells using the GAL4/UAS system. In the male genitalia, piezo-GAL4 positive signals were detected in the mechanosensory neurons that project to the base of sensory bristles in the surstylus (including long and short mechanosensilla), epandrial ventral lobe, cercus, and hypandrum (Figures 4B–4D). A previous study indicated that fruitless (fru) -expressing mechanosensory neurons in male genitalia are involved in the copulation duration. We further tested if fru-GAL4 expression overlapped with piezo-GAL4 expression in the male genital mechanosensory neurons. fru-GAL4 positive signals were observed in many mechanosensory neurons of the surstylus, epandrial ventral lobe, cercus, and hypandrum of the male genitalia (Figure 4E), suggesting that many fru-GAL4 positive signals overlapped with piezo-GAL4 positive signals in male genital mechanosensory neurons. To evaluate the co-expression of piezo-GAL4 and fru-GAL4 signals, we focused on 11 types of bristles on the male genitalia that were identifiable across animals (Figure 4F). For all of these bristles, neurons that projected to the base of each bristle were positive for both piezo-GAL4 and fru-GAL4 signals (Figure 4F). To directly confirm the co-expression of piezo and fru, we combined piezo-GAL4 and fru-LexA and observed overlaps in genitalia by expressing red fluorescent protein (RFP) and GFP markers respectively. This co-expression experiment however yielded only faint signals for both markers (Figures S6A and S6B). Despite these faint signals, we detected an overlap of piezo-GAL4 positive signals and fur-LexA positive signals in a part of mechanosensory neurons that project to the base of sensory bristles in the male genitalia, especially in the surstylus (Figure S6A). Notably, these piezo-GAL4 and fru-LexA double-positive bristles included the long mechanosensilla known to influence male copulation posture. These results strongly suggest that piezo and fru are expressed in male genitalia, presumably in the mechanosensory neurons of the same sensory bristles. To investigate the involvement of piezo-expressing neurons during copulation, we suppressed these neurons only during copulation. Automated photostimulation systems triggered by selected animal behaviors enable us to conduct optogenetic experiments efficiently with high reproducibility. To this end, we established a DeepLabCut-Live! -based system that recognizes copulation instantly and then automatically controls hardware to silence piezo-expressing neurons (Figure 5A). This system tracked body parts of both males and females and detected copulation events based on the distances between them (See Materials and Methods). In a copulation chamber, we set up a copulation stage to which a wild-type female was attached. A male expressing the green-light-gated anion-channel GtACR1 in piezo-expressing cells was then introduced to the chamber and video-recorded to allow DeepLabCut-Live! to detect potential copulation events in real-time (Figure 5B). It should be noted that muscle tissue and neuronal cells are potentially affected by GtACR1-mediated suppression. However, expression of the piezo gene in the muscle system was not anatomically observed in previous studies, suggesting that only nervous system cells are suppressed in this setup. Five min after DeepLabCut-Live! had detected the copulation event in each pair, piezo-expressing neurons of males were suppressed by green-light photostimulation that covered the entire chamber. During photostimulation, the male’s copulation posture was disrupted in the experimental group, in which most pairs terminated copulation, whereas the control pairs were not (Figure 5C, Video S1). This result suggests that the suppression of piezo-expressing neurons terminates copulation by disrupting mechanosensation. We also observed that during photostimulation, males typically showed head grooming behaviors with their forelegs while continuing the copulation (Video S1, Figure S7A), possibly due to the suppression of piezo-expressing neurons in the head. Video S1. Suppression of piezo-expressing neurons in the male whole body destabilized the male’s copulation posture and terminated copulation, related to Figure 5 The head grooming behaviors observed during photostimulation might be due to the suppression of piezo-expressing neurons around the head. In support of this speculation, we confirmed that piezo-GAL4 positive signals were widely distributed around the head, both in the brain and sensory neurons of Johnston’s organ (Figures 5D and S8). A previous study demonstrated that Johnston’s organ neurons are responsible for head grooming,, further supporting the speculation that the head grooming behaviors we observed were due to the manipulation of piezo-expressing neurons around the head. We then narrowed down the piezo-expressing neurons responsible for the maintenance of copulation posture by focusing the photostimulation area to gate GtACR1 in piezo-expressing neurons using laser illumination which starts 5 min after the copulation initiation. First, we evaluated the illumination area of our experimental setup, using a beam profiler (Figure S9). The intensity distribution data identified a beam diameter of 1.639 mm, which covers about half of a male fly body length (Figure S9B). The laser illumination intensity outside of the laser diameter was too low to induce neuronal inhibition by GtACR1. Using this laser illumination system, we targeted the photostimulation only on the posterior side (focusing on male genitalia) of copulating males to keep the piezo-expressing neurons around the head unaffected (Figures 5E and S9A). In the experimental group, this targeted photostimulation destabilized the male’s copulation posture significantly after the initiation of photostimulation (Figures 5F and 5G; Video S2) and terminated the copulation (Figure 5H). In contrast, no such tendency was observed in control males (Figures 5F and 5G). Video S2. Suppression of piezo-expressing neurons on the posterior side of the male body destabilized the male’s copulation posture and terminated copulation, related to Figure 5 This finding suggests that piezo-expressing neurons on the posterior side of the body are responsible for maintaining the copulation posture of males. Interestingly, this targeted photostimulation did not significantly induce head grooming (Video S2, Figure S7B). In contrast, when the male piezo-expressing neurons on the anterior side of the body were selectively illuminated, head grooming was observed as observed in the whole body illumination (Video S3, Table S1). In some cases, photostimulation on the anterior side of the male body disrupted copulation in the experimental group (Table S1). These results suggest that the piezo-expressing neurons of both the anterior and posterior sides of the body are important for maintaining copulation. In terms of anterior side inhibition, copulation disruption possibly occurred due to piezo-expressing neurons in the forelegs and head, the latter of which induces anterior side movements, especially head grooming. In terms of posterior side inhibition, piezo-expressing neurons near the genitalia are presumably involved in the maintenance of copulation. Video S3. Suppression of piezo-expressing neurons on the anterior side of the male body induced head grooming, related to Figure 5

Piezo is a mechanosensory channel involved in various Drosophila behaviors, such as regulation of feeding behavior and experience-dependent female mating behavior.,,, In addition to this, this study revealed its involvement in male copulation behavior, specifically copulation posture maintenance, for the first time. PiezoKO males tended to tilt during the entire copulation period, indicating the contribution of the Piezo channel in stabilizing copulation posture. We also observed that wild-type females that copulated with PiezoKO males produced significantly fewer offspring. This finding suggests that stabilization of copulation by Piezo is possibly required for increasing offspring number in Drosophila males in nature. Furthermore, inhibiting male piezo-expressing neurons on the posterior side of the body during copulation destabilized posture and terminated copulation, lending credence to the idea that these neurons are important for maintaining copulation, or possibly body postures in general. Previous research in mammals, flies, and nematodes has linked mechanosensation with mating behaviors.,,, In rats, the mechanosensory activity of the penis during mating is suggested to be involved in male ejaculation. In nematodes (Caenorhabditis elegans), the MEC-4/MEC-10 mechanoreceptor complex (DeG/ENaC channel) is involved in regulating turning behavior during mating. These findings suggest that mechanosensation is involved in the control of mating behavior, and it is likely conserved across taxa. However, how animals maintain copulation until the gametes are transported to the female reproductive organ remains underexplored. Notable exceptions are found in studies using fruit flies which showed that mechanosensation in male genitalia affected copulation duration and posture,, suggesting that mechanosensation is involved in copulation maintenance. The male needs to maintain copulation at least until he transfers sufficient sperm for fertile mating, which occurs about 8 min after copulation initiation. We here revealed for the first time the mechanosensory components (i.e., genes and neurons) underlying copulation maintenance of males. When PiezoKO males were engaged in copulation, the coupling of the male and female genitalia was normal in appearance, but the male copulation posture was unstable and tended to tilt during the entire copulation period (Figure 2). We also demonstrated that the piezo mutation in males does not affect the ratio of female kicking behaviors during copulation (Figure S3A). Therefore, female rejection behaviors do not explain the different copulation posture phenotypes observed between PiezoKO and wild-type males. No differences were found in genital touch frequencies prior to copulation initiation either (Figure S3B), suggesting that the piezo mutation in males has a particularly pronounced effect during copulation rather than contributing to copulation initiation. Previous studies suggested that some male genital regions (i.e., surstylus, hypandrium, cercus, and epandrial ventral lobe) are involved in grasping the female genitalia during copulation.,,, These regions are covered with sensory bristles that house mechanosensory neurons. Among them, a single pair of long mechanosensilla at the male genital surstylus is known for its importance in the regulation of the copulation posture, as ablation of one of them tends to tilt the copulating males. This phenotype resembles that observed in PiezoKO males. In accordance with this, this study detected the mechanosensory neurons at the sensory bristles of male genital regions as piezo-GAL4 positive. It can therefore be inferred that the male recognizes the physical coupling with the female genitalia through the male genital piezo-expressing mechanosensory neurons and then stabilizes posture during copulation. Notably, PiezoKO males showed no defects in copulation duration and success rate, whereas inhibition of piezo-expressing neurons during copulation induced a severe effect, i.e., copulation termination. A possible reason for this phenotypic difference would be the difference between a gene mutation and neuronal inhibition, as potentially other mechanosensory channels that complement the Piezo function are expressed orthotopically with Piezo. According to the Scope database,, genes for piezo and other mechanosensory channels (i.e., nompC, nan, and/or iav) are co-expressed in the sensory neuron class throughout the body (Figures S10A–S10C). This study finds nompC-GAL4 positive signals in the mechanosensory neurons that project to the base of sensory bristles in the surstylus of male genitalia, whereas no signal was observed for nan-GAL4 and iav-GAL4 (Figures S10D–S10F). This finding suggests that the nompC is also responsible for the regulation of copulation posture. Further experiments, which analyze the copulation behavior of males upon disruption of both piezo and nompC, will shed light on the potentially redundant roles of channel function in the copulation maintenance of male flies. Our results suggest that mechanosensation mediated by piezo-expressing neurons located at the posterior side of the body is necessary to regulate copulation posture in male flies. Moreover, only a few piezo-GAL4 positive signals were detected at the base of sensory bristles on the male ventral abdomen, which possibly touches the female body during copulation (Figure S11A). It supports the scenario that piezo-expressing mechanosensory neurons in male genital regions are part of the neural circuit that regulates the copulation posture of males. On the other hand, inhibition of piezo-expressing neurons in the entire body or its anterior side either elicited two major behaviors: copulation termination and head grooming. These observations suggest that terminating copulation primarily involves the piezo-expressing neurons on the posterior side, while head grooming is largely caused by the inhibition of piezo-expressing neurons on the anterior side of the fly. A previous study showed that manipulation of a subset of mechanosensory neurons in Johnston’s organ (JO neurons), located at the antennae, elicits antennal grooming., We found piezo-GAL4 positive signals in JO neurons (Figure S8), which also supports the possibility that the head grooming behavior we monitored largely depended on manipulation of piezo-expressing neurons on the head side. Moreover, piezo-GAL4 positive signals were also observed at the base of sensory bristles in the forelegs, which hold the female body during copulation (Figure S11B). It is thus likely that piezo-expressing neurons in the male forelegs also play a role in maintaining the copulation posture. Our results showed that the frequency of copulation termination via inhibiting piezo-expressing neurons was higher during suppression in the entire body than when restricted to the posterior body side. The release of the genital coupling under anterior side illumination is possibly due to the suppression of piezo-expressing neurons in the male forelegs and grooming-induced body movement. A previous study identified a copulation neural circuit of Drosophila that mediates the action sequence of copulation from initiation to termination, such as the male genital attachment, intromission, and uncoupling. This copulation neural circuit is comprised mechanosensory neurons, motor neurons, and some interneurons. In this circuit, mechanosensory neurons detect signals from the mechanosensory bristles in the male genitalia. A retrograde labeling experiment showed that they send their axons to the male abdominal ganglion and synapse to motor neurons, possibly transmitting the sensory information directly to the motor neurons there from the genitalia. The motor neurons in this circuit, in turn, regulate the phallic and periphallic musculature in the male genitalia, by which adjusts the genital grasping of the female genitalia depending on the mechanosensory inputs. A part of the mechanosensory neurons in this circuit was suggested to express fru gene. This previous research suggests that the fru-expressing sensory neurons in this circuit send signals to the motor neurons to adjust the appropriate positioning of the male throughout an entire copulation event by controlling the male genital grasping of the female genitalia. In this study, we found that cell bodies of piezo-GAL4 positive mechanosensory neurons innervating the genitalia are located in the abdominal ganglion and their peripheral ends projected to the genital mechanosensory bristles. These piezo-GAL4 positive sensory neurons mostly overlapped with the distribution of fru-positive sensory neurons in male genitalia (Figures 4D–4F and S6). Previous studies reported that the mechanosensory neurons in the copulation neural circuit express fru, but which mechanosensory genes were expressed in these neurons remained unclear., Our results suggest that piezo is expressed in the mechanosensory neurons of the copulation circuit for transmitting signals from the genital mechanosensory bristles (Figures 4B–4E)., In addition, the mechanosensory neurons that projected to the male genital long mechanosensilla, whose ablation induced the tilted copulation postures in wild-type males, were positive for both piezo-GAL4 and fru-GAL4 signals (Figure S6). Our findings together suggest that the piezo-GAL4 positive neurons in the male genitalia possibly receive mechanosensory input during copulation and then transmit signals to the copulation neural circuit that adjusts the phallic and periphallic musculature, resulting in copulation maintenance. PiezoKO males showed reduced fertility, indicating reduced fitness. Unexpectedly, we found that PiezoKO males have no obvious defect in copulation duration and copulation rate. We also showed that the reduction of male fertility resulting from piezo mutation was unlikely to be caused by general deficits in the fly’s health or decreased sperm levels in male reproductive organs. The reduced male fertility found in PiezoKO males is, therefore, possibly caused by reductions in sperm transport due to disrupted copulation posture. It is also possible that PiezoKO males fail to detect mechanosensory input during copulation, resulting in a lower amount of sperm being transported. Further research is needed to investigate these possibilities, e.g., by determining if the PiezoKO males reduce the amount of sperm delivered to the female’s internal reproductive organs. Furthermore, since we have not directly evaluated the fertilizing ability of PiezoKO male sperm, the reduced fertility observed in these males may also be attributable to impaired sperm function. In Drosophila, copulating males adjust the number of their sperm transferred based on the female mating status, fecundity, and age. It has been suggested that the male senses the female’s state and regulates the number of sperm to transfer, possibly by detecting pheromones from the female. The findings of this study propose another type of signal with which the male detects the female’s condition in copula, i.e., the mechanosensory signal mediated via piezo-expressing mechanosensory neurons, to regulate the number of their sperm. The transfer of sperm and seminal fluid depends on the activation of neurons in the abdominal ganglion that co-express fru and corazonin (crz). The mechanism that regulates the activity of fru+crz+ neurons remains unclear. An interesting possibility is that fru+crz+ neuronal activity is controlled by the mechanical senses via piezo-expressing neurons. To evaluate this possibility, the functional connection between sperm transport and mechanosensation is to be confirmed by observing the activity of fru+crz+ neurons in the abdominal ganglion during mechanical stimulation from the genitalia (or optogenetic activation of piezo-expressing neurons) using calcium imaging. This study shows for the first time that Piezo and piezo-expressing neurons are necessary for stabilizing copulation posture and maintenance, respectively, in male D. melanogaster. Although it remains to be tested whether reduced fertility in PiezoKO males could be rescued by forced expression of piezo, our findings suggest that copulation maintenance and stabilization of copulation by Piezo is possibly required for increasing offspring number for male flies. In adult D. melanogaster females, piezo is expressed in reproductive organs, and mechanosensation mediated by Piezo channels contributes to the female mating experience. Piezo-mediated mechanosensation via genitalia would be, therefore, important for both males and females. The piezo homologs are widely found among various species and homologous genes of piezo exist even in plants and protists, implying that Piezo channels regulate many physiological processes across organisms. Additionally, Piezo has been implicated in underpinning the mating behaviors of males. In mammals, two types of Piezo channels (Piezo1 and Piezo2) are responsible for detecting mechanical stimuli and converting them into electrical signals. In mice, Piezo1, a homolog of the piezo family, was found in some structures of male internal genitalia (i.e., prostate gland, seminal vesicles, and ejaculatory ducts), suggesting that Piezo1 serves a role in detecting the amounts of glandular content and possibly involved in the contraction and release of glandular secretions during ejaculation. In a wild-type rat subpopulation that shows premature ejaculation, the knockdown of Piezo2, another homolog in the piezo family, significantly improved the ejaculation latency, as well as mounting frequency and ejaculation frequency. This research implies the involvement of Piezo channels in male ejaculation and mounting in mammals. In C. elegans, mechanosensation is involved in the control of the mating behavior. pezo-1, a C. elegans homolog in the piezo family, is expressed in male-specific neurons with key roles in mating behavior; pezo-1 mutations compromise mating efficiency and multiple steps of mating behaviors in C. elegans males. These findings in mice, rats, and C. elegans together suggest that the mechanism to control mating behaviors through Piezo-mediated mechanosensation is widely shared across animal species that engage in mating behavior. Although prior studies have suggested the involvement of Piezo-mediated mechanosensation during mating, there has been little research on the mechanosensation underlying copulation maintenance. This study reveals for the first time that the mechanosensory system, mediated via Piezo channels, contributes to copulation maintenance and male fitness and opens a new avenue for revealing the copulation maintenance mechanism through mechanosensation.

Our study demonstrated that a mechanosensory gene piezo and its expressing neurons are responsible for copulation maintenance in Drosophila males. Our findings suggest that copulation maintenance mediated by Piezo possibly increases the fitness of males by stabilizing copulation. We attempted to limit the photostimulation area used to gate the GtACR1 in piezo-expressing neurons using a laser. Although we narrowed down the responsible neurons to those located in the posterior side of the male body, a lack of appropriate genetic tools prevented us from directly evaluating if piezo-expressing neurons in the sensory bristles on the male genitalia mediate copulation maintenance. This is due to the difficulty of labeling and manipulating specific peripheral piezo-expressing neurons. Previous research suggests that the male genital mechanosensory neurons are connected to the motor neurons that innervate male genital muscles and are involved in the copulation maintenance., Building on these previous studies, we hypothesize that piezo-expressing mechanosensory neurons that send their cilia to sensory bristles on the male genitalia detect the genital coupling, and by doing so contribute to copulation maintenance. We showed PiezoKO males produced significantly fewer offspring than control males. This phenotype is unlikely to be due to general effects such as reduced locomotor activity or nutritional deficits resulting from piezo mutation. We hypothesize that reduced fertility was due to PiezoKO males delivering less sperm to females than wild-type males. Due to technical limitations, however, we were not able to directly evaluate this idea by counting sperm in female reproductive organs.



Further information and requests for resources and reagents should be directed to, and will be fulfilled by, the lead contact: Azusa KAMIKOUCHI, kamikouchi.azusa.r4@f.mail.nagoya-u.ac.jp.

This study did not generate new unique reagents.

Fruit flies D. melanogaster were raised on standard yeast-based media at 25°C and 40% to 60% relative humidity on a 12 h light/12 h dark (12 h L/D) cycle. Canton-S (Hotta-lab strain, a gift from K. Ito) was used as a wild-type strain. w1118 was obtained from the Vienna Drosophila Resource Center. piezoKO, piezo-GAL4 (chr 3), piezo-GAL4 (chr 2), iav-GAL4, nan-GAL4 , nompC-GAL4, 20XUAS-IVS-mCD8::GFP (attP2), UAS-GtACR1.d.EYFP (attP2), 10XUAS-IVS-mCD8::RFP (attP18), 13XLexAop2-mCD8::GFP (attP8), UAS-piezo.GFP were obtained from the Bloomington Drosophila Stock Center. fru-GAL4 was a kind gift from Dr. B. Dickson. fru-LexA was a kind gift from Dr. B. S. Baker. For behavioral experiments, adult flies were maintained at 25°C under a 12 h L/D cycle. They were transferred to new tubes every 2 to 3 days, but not on the day of experiment. Wild-type adult females were used as mating partners. Both sexes of flies were collected within 8 h after eclosion to ensure their virgin status. Male flies were kept singly in a plastic tube (1.5 mL, Eppendorf) containing ∼200 μL fly food. Females were kept in groups of 10 to 30. Males and females 5 to 8 days after eclosion were used for experiments. Males and females were used only once. All experiments were performed between Zeitgeber Time 1-11 at 25 ± 1°C and 50 ± 10% relative humidity. For immunolabeling, males 5-10 days after eclosion were used.

Male genitalia were dissected in phosphate-buffered saline (PBS: Takara Bio Inc., #T900; pH 7.4 at 25°C), kept in 50% Vectashield mounting medium (Vector Laboratories, #H-1000; RRID: AB_2336789) in deionized water for ∼5 min, and mounted on glass slides (Matsunami Glass IND., LTD, Osaka, Japan) using Vectashield mounting medium. Male forelegs and heads were dissected in PBS (pH 7.4 at 25°C), kept in 50% glycerol in PBS for ∼5 min, and mounted with 80% glycerol in deionized water. Male internal genitalia were dissected in PBS (pH 7.4 at 25°C), fixed with 4% paraformaldehyde for 15 min at 25ºC, kept in 50% glycerol in PBS for ∼10 min, and mounted with 80% glycerol in deionized water containing 1% DAPI (Sigma-Aldrich, # D9542). Immunolabeling of the brains was performed as described previously with minor modifications. Briefly, brains were dissected in PBS (pH 7.4 at 25ºC), fixed with 4% paraformaldehyde for 60-90 min at 4ºC, and subjected to antibody labeling. Brains were kept in 50% glycerol in PBS for ∼1 h, 80% glycerol in deionized water for ∼30 min, and then mounted. Rabbit polyclonal anti-GFP (Invitrogen, #A11122; RRID: AB_221569; 1:1000 dilution) was used for detecting the mCD8::GFP. Mouse anti-Bruchpilot nc82 (Developmental Studies Hybridoma Bank, #nc82, RRID:AB_2314866; 1:20 dilution) was used to visualize neuropils in the brain. Secondary antibodies used in this study were as follows: Alexa Fluor 488-conjugated anti-rabbit IgG (Invitrogen, #A11034; RRID:AB_2576217; 1:300 dilution) and Alexa Fluor 647-conjugated anti-mouse IgG (Invitrogen, #A21236; RRID: AB_2535805; 1:300 dilution).

Serial optical sections were obtained at 0.84 μm intervals with a resolution of 512 × 512 or 1024 × 1024 pixels using an FV1200 or FV3000 laser-scanning confocal microscope (Olympus) equipped with a silicone-oil-immersion 30× (UPLSAPO30XSIR, NA = 1.05; Olympus) or 60× Super Apochromat objective lens (UPLSAPO60XS2, NA = 1.30; Olympus). The size, contrast, and brightness of the images were adjusted using Fiji software (version 2.3.0; RRID: SCR_002285).

We used CMOS cameras equipped with a 50 mm focal length lens (MVL50M23, Thorlabs, Inc. or VS-5026VM, VS Technology Corporation) for all experiments. The cameras used to record the fly behavior in each assay are as follows: DFK 33UP1300 (The Imaging Source Asia Co., Ltd) in the copulation assay; DMK33UX273 (The Imaging Source Asia Co., Ltd.) equipped with a light-absorbing and infrared transmitting filter (IR-82, FUJIFILM) in the optogenetic assays. To time-stamp photostimulations, we used additional cameras as follows: DFK 33UP1300 (The Imaging Source Asia Co., Ltd.) for optogenetics using light emitting diode (LED); DFK 33UP1300 (2 sets; The Imaging Source Asia Co., Ltd.) for optogenetics using a laser. All recordings were performed at a resolution of 640 by 480 pixels and 30 frames/sec.

Male homozygous piezoKO mutants, heterozygous piezoKO mutants, or wild-type flies were used. A round courtship chamber (10 mm diameter and 4 mm deep) enclosed with a slide glass and an acrylic plate as a lid and bottom, respectively, was used. A pair of male and female flies was introduced into the chamber by gentle aspiration without anesthesia. After the video recording, we collected copulated females. Each female was kept in a plastic vial with food for 7 days and then removed. The number of pupae in each vial was counted 10 or 11 days after copulation.

A pair of male and female flies were introduced into a 2 mL plastic tube by gentle aspiration without anesthesia. To facilitate copulation, we narrowed the space in the tube by inserting a 3D-printed plug into the tube until copulation (Figure S12). Tubes were monitored to identify the copulation initiation. When the copulation was initiated, we capped the tube and wrapped it with tape (1.5 mm width and ∼80 mm length). 5 min after copulation initiation, we shook the tube for 30 s using a microtube mixer (MT-360, TOMY SEIKO CO. LTD.) at a frequency of ∼38 Hz.

DeepLabCut, the network model pretrained on the ResNet50, was used to detect male copulation posture automatically. We created four DeepLabCut models as follows: male copulation posture, copulation detection for LED, copulation detection for laser, and male copulation poster movement (See Table S2 for each training condition). In each training condition, movies were processed by Adobe Premiere Pro 2022 (Adobe Inc.) or VideoProc Converter (Chengdu Digiarty Software, Inc.) to collect the video clips of target events. We extracted images randomly from video clips and manually labeled the “keypoints” in each image (Figures S13A–S13C). To evaluate the performance of the trained DeepLabCut, we calculated the Root Mean Squared Error (RMSE), a measure to verify the goodness of the model, and confirmed that the pose predictions were sufficiently correct (Table S2).

In all experiments, we defined copulation as a male mounting on a female for more than 1 min., A custom-made Python script (version 3.8.13) was used for this evaluation. We collected the copulation video clips from the movie files on copulation assays using Adobe Premiere Pro 2022 (Adobe Inc.) or VideoProc Converter (Chengdu Digiarty Software, Inc.). We used the “male copulation posture model” of DeepLabCut to detect the wing bases of fly pairs in these video clips. Using coordinates of the wing bases, we defined the vectors and , where A, B, C, and D correspond to the coordinates of the male-left, male-right, female-left, and female-right wing bases, respectively (Figure S14A). The vectors and are perpendicular to the body axes of the male and female, respectively, and thus were used to obtain the copulation angle, which represents the angle between the body axes (Figure S14A; note that the angle between these two vectors is equivalent to the copulation angle). In this analysis, we used the frames in which all the keypoints on the wing bases showed≥ 0.9 likelihood. We used movie files to compare the data when the total duration of the calculated frames was at least 5 min, to avoid large discrepancies between the actual value and the calculated value due to the short observation duration (Figures 2C and 2D). The total frame numbers that were analyzed in PiezoKO groups were not significantly different from that in heterozygous (PiezoKO/+) control groups (Figure S14B). The ratio of time in which the male engaged in tilted copulation was significantly larger in PiezoKO homozygous males than in control flies even when the threshold of copulation angle to define the tilted copulation was changed between 20° and 30° (Figures S2A–S2K). In optogenetic assays using a laser, the male copulation position was defined by the middle point of wing bases, , during copulation. The movement of the male copulation position during photostimulation was quantified as the difference of the copulation position coordinate from the previous frame (1 mm = 40.36 ± 0.17 pixels). The average displacement of the male copulation position per second was obtained by:where is the cumulative displacement of the male copulation position during the observation time (Time), which was defined as follows: (1) For pairs that continued copulation during photostimulation, we set their observation time as the duration of photostimulation. (2) For pairs that terminated copulation during photostimulation, we ended their observation time at the time of copulation termination. In optogenetic assays using LEDs, we did not assess the male copulation position because the movement of the anterior body (especially due to head grooming) during photostimulation induced a large fluctuation of the male copulation position.

Males of piezo>GtACR1 flies were maintained under a dark condition for 4 to 6 days after eclosion and then transferred to a plastic tube (1.5 mL, Eppendorf) containing ∼150μL of fly food. Plastic tubes that contain males were divided into two groups: experimental and control groups. For the experimental group, 2 μL of all trans-retinal (R2500, Sigma-Aldrich), 25mg/mL dissolved in 99.5% ethanol (14033-80, KANTO KAGAKU), was placed on the food surface. For the control group, 2 μL of 99.5% ethanol was placed instead. All trans-retinal is a light-sensitive pigment that enables the triggering of ion flux through green-light absorption. The male flies were kept on each food for 24 to 36 h before being used for the assays.

We used a piece of eraser (∼2 mm length, ∼1 mm width, and ∼0.5 mm height) as the copulation stage, which was fixed at the center of a slide glass using light-curing adhesives (1771E, ThreeBond Holdings Co., Ltd.). Each female was anesthetized on ice for ∼30 sec and glued to the eraser using the light-curing adhesives. Males were introduced after the recovery of females. In all optogenetic assays, we used glued females.

A courtship chamber (10-mm diameter and 4-mm height for LED photostimulation; 10 mm x 10 mm x 4 mm with an 1 mm fillet radius for laser photostimulation) made of an aluminum plate with an anodic oxide coating to prevent light reflection, was used. The chamber was illuminated from above by an infrared LED ring light (FRS5CS, 850 nm, OptoSupply) to enable recordings in dark conditions. A female attached to the copulation stage was placed in the center of the courtship chamber. After the recovery of females from anesthesia, a male fly was gently aspirated into the chamber without anesthesia and sealed with a cover glass (Matsunami Glass IND., LTD, Osaka, Japan). We used a custom script for Bonsai, a visual programing language, to detect the copulation initiation. Five min after the copulation was initiated, we started photostimulation using LED or laser. We set the intensity of the photostimulation in the same intensity range used in a previous study. For photostimulation with LEDs, a 525 nm LED (OSG5XNE3E1E, OptoSupply) was applied for 30 sec from the bottom of the chamber at the intensity of 5.8 mW/cm2 and 30 Hz with a duty ratio of 0.2. In the assay using laser, a 532 nm laser light (JPM-1-3, Lightvision Technologies, Corp.) was applied for 30 sec near male genitalia from the left or right side of the body at the intensity of 5.6 mW/cm2 and 30 Hz with a duty ratio of 0.5. The light intensity of photostimulation was calibrated with a photometer (PM100D, Thorlabs, Inc.). In optogenetic assays with a laser, we saved a snapshot of the illumination area from the side camera video, at the first frame after light intensity saturated (∼0.5 sec after the laser onset). We measured the laser focusing point (the center of the photostimulation area assuming that all photostimulation areas are of uniform value) using Fiji software (version 2.3.0; RRID: SCR_002285). We confirmed in all videos that the area of the laser diameters, defined by the laser profiling, from the center of gravity of the photostimulation did not include the thorax of a male fly.

We evaluated the laser beam diameter used in optogenetic assays to ascertain the area of photostimulation. A laser beam profiler (Beamage-3.0, Gentec-EO) was used to measure the laser light intensity distribution at 23°C (Figures S9A and S9B). From the intensity distribution data, we calculated the beam diameter as 1.639 mm. The laser diameter was defined as the distance between two points equal to 1/e2 times the maximum intensity. Outside of the laser diameter, the laser intensity was too low to activate GtACR1 channels. R (version 4.2.1) and Python (version 3.8.13) were used.

We used a desktop PC equipped with NVIDIA GeForce RTX 2080 as GPU (OS, Windows 10; CPU, Intel Core i7-9700K; RAM, 32 GB). The program scripts were written by Bonsai. We adopted the trained DeepLabCut models (copulation detection models for LED and laser, respectively) for the DeepLabCut-Live! to track the keypoints on flies online during video recordings. We then obtained the coordinate of the middle point of wing bases, , as well as that of eyes, , to detect the copulation event. To detect copulation events, two distance values were used: (1) the distance between the female and male , and (2) the distance between the male and the female scutellums (Figure S14C). Copulation was judged positive when both values were less than or equal to (; 1 mm = 40.73 0.25 pixel), which was determined by measuring the distances in the five video clips of the training dataset. With this threshold value, no false negatives were detected. False positives were detected, however, and thus we set an additional criterion that uses three checkpoints (2, 5, and 15 sec after the first copulation detection) to reduce the false positives; only when it judged the image as positive at all three checkpoints, the system accepted it as the copulation event. In this step, we used a custom Arduino script. We used Arduino IDE (version 1.8.19, Arduino) written on a microcomputer AE-ATMEGA-UNO-R3 (Base on Arduino UNO; AKIZUKI DENSHI TSUSHO CO., LTD.) to turn on the LED upon receiving a trigger signal from the custom Bonsai scripts that had detected the copulation event. See Table S3 for a list of false negatives and false positives that indicates the evaluation of each DeepLabCut-Live! Model.

For copulation rate analysis (Figure 3C), we calculated the ratio of pairs showing copulation success. We defined copulation success as maintenance of male and female genital coupling for longer than 30 sec. For female kicking analysis (Figure S3A), we divided each movie file into 1-sec bins and counted the number of bins that contained the event of female kicking the male with her hind legs during copulation. The ratio of event-positive bins per total bins that cover the entire copulation period was calculated. For genitalia touching analysis (Figure S3B), we measured the number of time male genitalia touched female genitalia when the male performed abdominal bending prior to copulation success. The number of genitalia touching was normalized by the latency to copulation initiation.

Male locomotor activity was quantified using the Drosophila activity monitoring system (Trikinetics Inc). Each male was introduced into a glass tube (5 65mm) with standard yeast-based media. After acclimation to the new environment overnight, locomotor activity was measured for 10h (ZT1-11). Locomotor activity data was collected in 1-min bins, and the number of the beam-breaks was analyzed.

Statistical analyses were conducted using R (version 4.2.1) and Python (version 3.8.13). Aligned rank transform one-way analysis of variance (ART one-way ANOVA) and Brunner–Munzel tests were used for comparisons between conditions after verifying the equality of variance (Bartlett’s test for three-groups comparisons; F-tests for two-groups comparisons) and normality of the values (Shapiro-Wilk test) (Table S4). For the ART one-way ANOVA, the ARTool package (version 0.11.1) was used (https://github.com/mjskay/ARTool/)., After ART one-way ANOVA, p values were adjusted using the Benjamini-Hochberg method in the post hoc test. For the Brunner-Munzel test, the brunnermunzel package (version 1.4.1) was used (https://github.com/toshi-ara/brunnermunzel). The statistical methods and values are summarized in Table S5. Statistical significance was set at p < 0.05. Boxplots were drawn using the R package ggplot2 (https://ggplot2.tidyverse.org/). Boxplots represent the median and interquartile range (the distance between the first and third quartiles), and whiskers denote 1.5 × the interquartile range. The statistical methods and results in this study are summarized in Table S5.