https://orcid.org/0000-0002-2295-4898
Figures
Abstract
Males have finite resources to spend on reproduction. Thus, males rely on a ‘time investment strategy’ to maximize their reproductive success. For example, male Drosophila melanogaster extends their mating duration when surrounded by conditions enriched with rivals. Here we report a different form of behavioral plasticity whereby male fruit flies exhibit a shortened duration of mating when they are sexually experienced; we refer to this plasticity as ‘shorter-mating-duration (SMD)’. SMD is a plastic behavior and requires sexually dimorphic taste neurons. We identified several neurons in the male foreleg and midleg that express specific sugar and pheromone receptors. Using a cost-benefit model and behavioral experiments, we further show that SMD behavior exhibits adaptive behavioral plasticity in male flies. Thus, our study delineates the molecular and cellular basis of the sensory inputs required for SMD; this represents a plastic interval timing behavior that could serve as a model system to study how multisensory inputs converge to modify interval timing behavior for improved adaptation.
Author summary
To maximize their return on investment, male flies utilize a wide variety of sensory inputs, including memories of past sexual encounters. Therefore, when males have sufficient sexual experiences, they shorten their mating duration. The term "Shorter-Mating-Duration" (SMD) was coined to describe this behavior. SMD must be triggered by sugar and pheromone, which is detected by cells and receptors in the male forelegs. We found that these cells include male-specific sensory neurons that are tuned to collect on a male’s sexual experiences and relay that information to the brain, where it is used to determine how long to spend mating the next available mate. We hypothesize that SMD can serve as a straightforward genetic model system through which we can investigate "interval timing", the capacity of animals to distinguish between periods ranging from minutes to hours in duration.
Citation: Lee SG, Sun D, Miao H, Wu Z, Kang C, Saad B, et al. (2023) Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster. PLoS Genet 19(5): e1010753. https://doi.org/10.1371/journal.pgen.1010753
Editor: John Ewer, Universidad de Valparaiso, CHILE
Received: November 29, 2022; Accepted: April 20, 2023; Published: May 22, 2023
Copyright: © 2023 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: No datasets were generated during the current study. snRNAseq dataset analyzed in this paper is published in Li et al., doi:10.1126/science.abk2432 [79] and available at the Nextflow pipelines (VSN, https://github.com/vib-singlecell-nf), the availability of raw and processed datasets for users to explore, and the development of a crowd-annotation platform with voting, comments, and references through SCope (https://flycellatlas.org/scope), linked to an online analysis platform in ASAP (https://asap.epfl.ch/fca). The mating duration and offspring data are available in the S1 and S2 Raw Data Files.
Funding: This research was supported a University of Ottawa Startup grant 602496 to WJK, Startup funds from HIT Center for Life Science to WJK, a University of Ottawa Interdisciplinary Research Group Funding Opportunity (IRGFO stream 1 and 2) grants 148101 and 148747 to WJK, a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant (reference: 211406) to WJK, a University of Ottawa Brain and Mind Research Institute/Center for Neural Dynamics Open call project grant 150950 to WJK, a Mitacs Globalink Research Internship Program grant 17268 to WJK. This research was also supported by the Brain Pool Program of the National Research Foundation in Korea grant ZYM5041911 to WJK, Burroughs Wellcome Fund Collaborative Research Travel Grants (reference: 1017486) to WJK and a NVIDIA Academic Hardware Grant Program to WJK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. SGL received salary from the ‘University of Ottawa Startup grant to WJK’ and HM from the ‘Startup funds from HIT Center for Life Science to WJK’.
Competing interests: The authors have declared that no competing interests exist.
Introduction
From basic behaviors to complicated decisions, all animals have to make choices throughout their life to maximize their utility function [1]. The reproductive success of a male animal depends predominantly on how many of its sperm are successful in fertilizing eggs [2]. Males have a finite resource to spend on reproduction [3] and must make choices throughout their life to optimize how their resources are utilized [4]. For example, males that invest a long period of time for mating might expose themselves to the action of predators or various environmental hazards, thereby losing their competitiveness. In this regard, the ‘time investment strategy’ (the optimum allocation of time spent on given activities to achieve maximal reproductive success)’ is crucial for males. Male Drosophila, for instance, respond to the presence of competitors by extending the mating duration in order to guard the female and pass on their genes. Hence, female guarding has typically evolved as a tactic for males to invest their time [5].
Recent studies have revealed that male D. melanogaster shows wide variation in terms of their level of interest in females, thus providing evidence that males have also evolved to mate selectively [6]. When mating opportunities are constrained, males that show a preference for more fecund females will benefit directly by increasing the number of offspring they produce [7]. The selective mating investment exhibited by male D. melanogaster may have evolved for several reasons. First, sexual activity reduces the lifespan of males [8] due to costs arising from vigorous courtship [9], the production of ejaculates [10] and possibly also due to immunosuppression [11]. Second, repeated mating by males within a 24 h period depletes limiting components of the ejaculate [12]. Third, the quality of potential female mates is highly variable [13].
Behavioral plasticity is advantageous when specific aspects of the environment (e.g., the intensity of socio-sexual encounters) are prone to rapid and unpredictable variation [14–20]. The best-studied example of plastic behavioral responses in males is ‘longer-mating-duration (LMD)’ in which exposure to rivals before mating increases investment through mating duration [14,15,17–31].
It has been reported that previous sexual experience with females influences the mating duration of male D. melanogaster [14,19,32]; however, the neural circuits and physiology underlying this behavior have not been deeply investigated. Here, we report the sensory integration mechanisms by which sexually experienced males exhibit plastic behavior by limiting their investment in copulation time; we refer to this behavior as "shorter mating duration (SMD)."
Results
Sexual experiences diminish male Drosophila’s mating duration via chemosensory cues from females
To investigate how sexual experience affects the mating duration of male D. melanogaster, we introduced virgin females to group-reared males one day before the assay (this condition is referred to as ‘experienced’ hereafter) and compared mating duration of experienced males with group-reared males that had never encountered sexual experience (this condition is referred to as ‘naïve’ hereafter) (Fig 1A). We found that the mating duration of Canton S, WT-Berlin, Oregon-R, and w1118 naïve males are significantly longer (wild type 15.7~15.8%, w1118 12.4%) than that of sexually experienced males (Figs 1B–1D and S1A). Despite the fact that our previously reported LMD behavior is dependent on the white mutant genetic background [21], these findings show that the effect of the white mutant genetic background was not obvious in SMD behavior.
(A) Naïve males were kept for 5 days after eclosion in groups of 4 males. Experienced males were kept for 4 days after eclosion in groups then experienced with 5 virgin females 1 day before assay; for detailed methods, see the EXPERIMENTAL PROCEDURES. (B) Mating duration (MD) assays of Canton-S (CS), (C) WT-Berlin, and (D) w1118 males. Light grey dots represent naïve males and pink dots represent experienced ones. (E) Courtship index of naïve and experienced males. See the EXPERIMENTAL PROCEDURES section for detailed methods. (F) Courtship latency of naïve and experienced males. See the EXPERIMENTAL PROCEDURES section for detailed methods. (G) Mating initiation time of naïve and experienced males. (H) The locomotion of naïve and experienced male flies was quantified as velocity by a climbing assay paradigm. (I-L) MD assays of CS males with different exposure time with females. Each group of males was reared with females for (I) 2 h, (J) 6 h, (K) 12 h or (L) 24 h. (M) A diagram showing the results of MD assays of CS males with different exposure times with females. (N) MD assays for son-of-tudor mutants. Genotypes are described as in a previous report [33]. Dot plots represent the MD of each male fly. The mean value and standard error are labeled within the dot plot (black lines). Asterisks represent significant differences, as revealed by the Student’s t test (* p<0.05, ** p<0.01, *** p<0.001). The same notations for statistical significance are used in other figures. Number signs represent significant differences, as revealed by Dunn’s Multiple Comparison Test (# p<0.05). The same symbols for statistical significance are used in all other figures. See the EXPERIMENTAL PROCEDURES for a detailed description of the statistical analysis used in this study.
To test whether fatigue causes SMD behavior, we examined other behavioral repertoires of naïve and experienced male flies, such as courtship index, courtship latency, copulation latency and locomotion; there was no significant difference between experienced and naïve males (Figs 1E–1H, S1B and S1C). Thus, we conclude that potential fatigue from repetitive sexual experiences is not a causative factor for SMD behavior.
To determine the time required by males to be exposed to females in order to induce SMD behavior, we varied the exposure time of males to females and found that males significantly reduced their mating duration when their exposure to females lasted for longer than 12 h but not for less than 6 h, thus suggesting that SMD requires chronic exposure to females for longer than 6 h (Fig 1I–1M). To determine whether SMD is a reversible behavior, we separated males from females after 24 h or 48 h of sexual experience and then tested these males in a mating duration assay. We found that separating experienced males from females for 24 h was sufficient to restore the MD to the level of naïve males (S1D–S1G Fig), thus suggesting that SMD is plastic and dependent on sexual experience with females but can change over time.
To confirm the lack of effect of sperm depletion on SMD behavior, we depleted sperm prior to MD assays and found that sperm depletion did not affect SMD behavior (S1H–S1L Fig). We also tested the son-of-tudor males that lack germ cells and are therefore devoid of sperm [33]; we found that the son-of-tudor males also exhibited SMD (Fig 1N). Consistent with a previous report [34], these data suggest that sperm depletion does not cause SMD behavior in male D. melanogaster.
Next, to identify the sensory modalities that modulate SMD behavior, we tested multiple mutants with defects in various sensory modalities [21,35]. By using constant dark conditions (Fig 2A) and several mutants with impaired vision (GMR-Hid in Fig 2B; ninaE17 in Fig 2C) [21,35], impaired olfaction (Orco1/Orco2 in Fig 2D and Orco-GAL4/UAS-KNCJ2 in S2A Fig) [36], impaired gustation (GustDx6 in Fig 2E and Poxn-GAL4/Poxn-RNAi in S2B Fig) [37,38] and impaired auditory ability and mechanosensation (iav1 in Fig 2F) [30,39–41], we concluded that gustatory, auditory and mechanosensory pathways are involved in generating SMD behavior but not visual or olfactory pathways. S1 Table summarizes the settings we controlled to determine the sensory modality for SMD.
(A) To test whether the vision is required for SMD, CS males were reared and sexually experienced in constant dark for 5 days (dark). (B) MD assays of GMR-Hid males, and blind animal. (C) MD assays of ninaE17 mutant males animal lacking the opsin R1-6 photoreceptors [35]. (D) MD assay of Orco1/Orco2 trans-heterozygote mutant males with defects in olfaction [87]. (E) MD assays of GustDx6 mutant males showing aberrant responses to sugar and NaCl [88]. (F) MD assays of iav1 males, the auditory and mechanosensory mutant [89]. (G) MD assays of CS males exposed to sexually experienced females 1 day before assay. To generate mated females, 4-day-old 10 CS virgin females were placed with 5-day-old 20 CS males for 6 hours and then transferred to an empty vial. These females were used for experienced females 1 day after separation. (H) MD assay of CS males experienced with D. pseudoobscura females. (I) MD assay of CS males experienced with Dfexel6234 females, a deficiency strain that lacks the expression of the sex-peptide receptor (SPR) [90]. (J) MD assays of CS males experienced with virgin females behaving as mated females. To make virgin females behave as mated females, flies expressing UAS-mSP (a membrane bound form of male sex-peptide) were crossed with flies expressing fru-GAL4 driver, as described previously [44,91]. (K) MD assays of CS males experienced with oenocyte-deleted females. To generate oenocyte-deleted females, virgin flies expressing UAS-Hid/ crossed with flies expressing tub-GAL80ts, oeno-GAL4 males; then the female progeny were kept in 22°C for 3 days. Flies were moved to 29°C for 2 days before assay to express UAS-Hid/rpr and kill the oenocytes in these females. The oeno-GAL4 (PromE(800)-GAL4) was described previously [92]. (L) MD assays of CS males exposed to oenocyte-masculinized females. To generate oenocytes-masculinized females, flies expressing UAS-tra-RNAi were crossed with oeno-GAL4 driver. (M) MD assays of CS males exposed to pan-neuronally masculinized females. To generate pan-neuronally masculinized females, flies expressing UAS-tra-RNAi were crossed with elavc155 driver [93]. (N) MD assays of CS males exposed to feminized females. To generate feminized males, flies expressing actin-GAL4 were crossed with flies expressing UAS-traF [46,93]. (O) CS male courting with a feminized male and showing licking behavior, leading to (P) successful mating.
Next, we attempted to identify the physiological cues from females that play important roles in the induction of SMD behavior in males. To do this, we used various genotypes of females as experienced sexual partners. Mated females and Drosophila pseudoobscura females did not induce SMD, thus suggesting that cues originate from virgin D. melanogaster females [42] (Fig 2G and 2H). In contrast, female D. simulans, a closely related species of D. melanogaster, can induce SMD, indicating that cues for SMD are also present in female D. simulans (S2C Fig). It is well known that D. melanogaster and D. simulans can create hybrid offspring [43]. Sexual experiences with sex peptide receptor (SPR) mutant females, who have a delayed post-mating reaction and consequently display multiple mating with males compared to wild type females [44], showed no additional influence on SMD (Fig 2I). Virgin females behave like mated females by expressing a membrane-bound version of male sex-peptide in fruitless-positive neurons, hence rejecting the male’s copulation attempt [44]. Males that were experienced with these females did not show SMD, thus suggesting that both cues from females and successful copulation are required for SMD (Fig 2J).
We produced odorless and tasteless females by killing female oenocytes (oenocyte(-)) [45] and females that produced a male odor via the masculinization of female oenocytes (oeno-GAL4/tra-RNAi) [46]. Males that had experience with these females did not show SMD, thus suggesting that female-specific pheromones produced by oenocytes are important cues for SMD (Fig 2K and 2L). However, males experienced with females which contained masculinized neurons showed intact SMD, thus suggesting that female forms of odor, and not female forms of neural circuits, are critical for inducing SMD behavior (Fig 2M). Interestingly, feminized males, created by overexpressing the female form of the tra2 protein driven by a broad GAL4 driver, can provide the cues required for SMD, thus suggesting that developmental phenotypes that are regulated by tra2 can provide both cues from females that are sufficient to induce SMD (Fig 2N). By tracking videos of the mating assay, we were able to confirm that males exhibited a full repertoire of courtship behavior and mated successfully with oenocyte-masculinized females (S2D–S2I Fig) and feminized males (Figs 2O, 2P and S2J–S2K), thus suggesting that these experienced partners can provide a mating drive for male D. melanogaster. We also found that SMD was completely normal even when an oenocyte-masculinized female (S2L Fig) was used for assay partners, thus suggesting that SMD is independent of the genotypes of the assay partners used for mating duration assays. Collectively, these data suggest that both sexual experience and female D. melanogaster-specific odor (produced in the oenocytes) are required to induce SMD behavior. The genotypes of experienced females used to define the sensory modality for SMD are summarized in S2 Table.
In flies, taste and touch signals are primarily conveyed to the brain by sensory neurons in the legs and mouthparts. To understand how sensory information for SMD is mediated via the legs or proboscis, we first tested the SMD behavior of males for which each pair of legs had been removed; we found that the foreleg is critical for generating SMD behavior (Fig 3A–3C). When we carefully watched the position of each pair of legs during mating, we found that the male’s foreleg touches the female body most of the time during mating; the midleg only partially touches the female body while the hind leg does not touch the female at all (Fig 3D–3G). The point at which the male’s leg touched the female body was mostly the tarsus, an area that is known to recognize taste [47] and pheromones [48] via chemoreception (S3A Fig). Although we cannot rule out the role of the proboscis, wings and other unidentified taste organs in the reception of stimuli for SMD behavior, our present results suggest that the male’s foreleg is the major sensor for SMD behavior.
(A) MD assays of CS males in which the foreleg, (B) midleg, or (C) hindleg were removed 1 day before assay. Forelegs, midlegs, or hindlegs of 4-day-old males were removed by surgery and then treated as naïve or experienced for 1 d. (D) Dorsal view of the mating posture of CS males and females. The touching point of the male foreleg is marked with a white arrow. (E) Lateral view of the mating posture of CS males and females. The touching points of the male foreleg and midleg are marked with a white arrow. (F) Lateral view of the mating posture of CS males and females. The touching point of the male midleg is marked with a white arrow. (G) Ventral view of the mating posture of CS males and females. The touching points of the male midleg and genitalia are marked with a white arrow.
Gr5a-expressing sweet cells are required for SMD behavior
Of the various gustatory receptors, Gr5a marks cells that recognize sugars and mediate taste acceptance, whereas Gr66a marks cells that recognizes bitter compounds and mediates avoidance [49,50]. Gr5a and Gr66a are expressed in different cells in a sensillum of the foreleg and exhibit different sensory projections into the central brain region (Fig 4A and 4B). We found that male flies with ablated Gr5a-positive neurons that mediate sweet-taste detection did not exhibit SMD behavior while male flies lacking Gr66a-positive neurons that mediate bitter-taste detection exhibited normal SMD (Fig 4C and 4D). SMD was also impaired when we inhibited synaptic transmission via the expression of TNT in Gr5a-positive neurons but not in Gr66a-positive ones in an adult-specific manner by shifting flies bearing tub-GAL80ts to restrictive temperature (29°C) after eclosion (Fig 4E and 4F). The inactivation or hyperexcitation of Gr5a-positive neurons, but not Gr66a-positive neurons, by expressing the KCNJ2 potassium channel or NachBac bacterial sodium channel in an adult-specific manner using tub-GAL80ts, also resulted in impaired SMD (Fig 4G–4J). These data and genetic background control data (S4A–S4D Fig) suggest the cell populations of gustatory cells that mediate acceptance signals are associated with SMD behavior and that these Gr5a-positive neuronal populations and their neuronal activities are required for SMD.
(A) 4T and 5T of the male foreleg of flies expressing Gr5a-lexA and Gr66a- GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under a fluorescent microscope. Red arrows indicate Gr5a-positive neurons and green arrows indicate Gr66a-positive neurons. Scale bars represent 50 μm. (B) Brains of flies expressing Gr5a-GAL4 or Gr66a-GAL4 together with UAS-mCD8GFP, UAS-RedStinger were immunostained with anti-GFP (green), anti-DsRed (red) and nc82 (blue) antibodies. Scale bars represent 100 μm. The right panels indicate magnified regions of the left panels that are presented as a grey scale to clearly show the axon projection patterns of gustatory neurons in the adult sub-esophageal ganglion (SOG) labeled by GAL4 drivers. (C-D) MD assays for GAL4 driven cell death which labelled (C) sweet cells (Gr5a) or (D) bitter cells (Gr66a) using UAS-Hid/rpr. (E-F) MD assays of (E) Gr5a- or (F) Gr66a-GAL4 drivers for the inactivation of synaptic transmission via the expression of UAS-TNT transgene together with the tub-GAL80ts, such that UAS-TNT expression could be triggered by temperature shifts were crossed with flies expressing tub-GAL80ts (G-J) electrical silencing or hyperexcitation of Gr5a-positive neurons abolished SMD behavior. Flies expressing (G-H) potassium channel UAS-KCNJ2 or (I-J) bacterial voltage-gated sodium channel UAS-NachBac together with the tub-GAL80ts, such that UAS-KCNJ2 or UAS-NachBac expression could be triggered by temperature shifts, were crossed with flies expressing (G and I) Gr5a- or (H and J) Gr66a-GAL4 drivers. Flies were reared at 29°C for the first 2 days to strongly induce UAS-KCNJ2 or UAS-NachBac expression and then transferred to 25°C for the last 3 days for the mild induction of UAS-KCNJ2 or UAS-NachBac transgenes.
In addition, we found that Gr5a-positive cells were abundantly localized in the tarsus from tarsomeres 2 (2T) to tarsomeres 5 (5T) (S4E Fig). We also found that males have more Gr5a-positive cells than females (S4F Fig). On average, males had 10 ± 1 neurons in the tarsus (4 cells in 5T, 2 ± 1 cells in 4T, 1 ± 1 cells in 3T, 2 cells in 2T and no cells in 1T) and 0 ± 1 cells in the tibia; however, females had 6 cells in the tarsus (4 cells in 5T and 2 cells in 4T) (S4E and S4–S4F Fig). These data suggest that Gr5a-positive cells show sexual dimorphism and might have a male-specific function to generate SMD.
The sexual dimorphism of sensory structure and function generates neural circuitries that are important for gender-specific behaviors. In Drosophila, fruitless (fru) is an essential neural sex determinant that is responsible for male-specific behavior [51]. To determine whether sexually dimorphic sensory neurons are involved in SMD, we used intersectional methods to genetically dissect approximately 1500 fru neurons into smaller subsets. We used a combination of the fruFLP allele that drives FLP-mediated recombination specifically in fru neurons with UAS>stop>X genotype (X represents various reporters or effector transgenes) to express a UAS transgene in only those cells that were labeled by the GAL4 driver and were also fru-positive; this was controlled by the FLP-mediated excision of the stop cassette (>stop>).
We found that the sensory projections of a subset of Gr5a-positive neurons, but not Gr66a-positive neurons, were positive for fruitless, an essential neural sex-determinant that is responsible for male-specific behaviors [51] (Figs 5A and S5A). To test whether the small subset of fru-positive Gr5a cells is involved in SMD, we expressed tetanus toxin light chain (UAS>stop>TNTactive) with Gr5a- or Gr66a-GAL4 drivers along with fruFLP to inhibit synaptic transmission in sexually dimorphic subsets of fru-positive cells. We found that SMD was abolished when UAS-TNT was expressed only in male-specific Gr5a-positive neurons (Fig 5B and 5C). As a control, we found that SMD was unaffected when we used each of these GAL4 drivers in combination with UAS>stop>TNTinactive to express an inactive form of the tetanus toxin light chain (Fig 5D and 5E). The systemic expression of a female form of tra cDNA (UAS-traF) in a male during development is known to lead to the expression of female characteristics [52]. We found that SMD was eliminated by the feminization of Gr5a-GAL4 labeled cells but not by the expression of UAS-traF in Gr66a-positive neuronal subsets (Fig 5F and 5G), thus suggesting that the feminization of Gr5a-positive neurons nullifies the male-specific sensory function of those cells to detect sensory inputs for SMD behavior. Together with genetic background control experiments (S5B–S5D Fig), these data suggest that SMD requires the male-specific role of a subset of Gr5a-positive neurons.
(A) Brains of male flies expressing Gr5a-GAL4 or Gr66a-GAL4 together with UAS>stop>mCD8GFP; fruFLP were immunostained with anti-GFP (green) and nc82 (magenta) antibodies. Scale bars represent 100 μm in the colored panels and 10 μm in the grey panels. White boxes indicate the magnified regions of interest presented in the right panels. The right panels are presented as a grey scale to clearly show the axon projection patterns of gustatory neurons in the adult sub-esophageal ganglion (SOG) labeled by GAL4 drivers. (B-C) MD assays of (B) Gr5a- and (C) Gr66a-GAL4 drivers for the inactivation of synaptic transmission of fru-specific neurons among each GAL4-labelled neuron via UAS>stop>TNTactive; fruFLP. (D-E) Control experiments of (B-C) with the inactive form of UAS-TNT using UAS>stop>TNTinactive; fruFLP. (F-G) MD assays for (F) Gr5a- and (G) Gr66a-GAL4 drivers for the feminization of neurons via UAS-traF. (H) Male foreleg, midleg and hindleg tarsus of flies expressing fru-lexA and Gr5a- GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under a fluorescent microscope. Red arrows indicate Gr5a-positive neurons and green arrows indicate Gr66a-positive neurons. Scale bars represent 50 μm. (I) Male foreleg of flies expressing Gr5a-Gal4 together with fru-GAL80. White arrows indicate Gr5a-positive and fru-negative neurons. Dotted white arrows indicate missing neurons by adding fru-GAL80, as shown in S4F Fig.
By using the genetic intersectional method [53], we found that the male foreleg contains 5–6 Gr5a- and fru-positive cells in the tarsus (1 in 4T, 2–3 in 3T and 2 in 2T) while the midleg contains 1 (1 in 4T) (Fig 5H). However, we could find one of these cells in the male proboscis (S5E Fig). We also confirmed the number and position of Gr5a-expressing fru-positive cells using fru-GAL80 combined with Gr5a-GAL4, as shown in Fig 5H (Fig 5I). Together with the data arising from leg removal experiments (Fig 3), these data suggest that Gr5a-expressing male-specific sensory cells in the male leg provide the major sensory input for SMD generation.
Specific sugar receptors are essential for SMD sensory information
Next, we asked whether sugar receptors in the sexually dimorphic sugar sensory neurons are involved in the generation of the sensory input pathways that generate SMD. Sugars are the main group of chemicals underlying sweet taste and provide essential nutritional value for many mammals and insects [54]. Sweet taste in D. melanogaster is mediated by eight, closely related gustatory genes: Gr5a, Gr61a, and Gr64a-Gr64f [55]. The Gr5alexA allele refers to the Gr5a gene replaced by the mini-white transgene [55] results in a lack of SMD, thus suggesting that Gr5a itself is an important receptor for generating SMD (Fig 6A). We knocked down all known sugar receptors in fru-positive cells using a fru-GAL4 driver and found that only Gr5a and Gr64f are important for the generation of SMD in male-specific fru-positive cells (Figs 6B–6D and S6A–S6G).
(A) MD assay of Gr5a-lexA homozygote males in which the Gr5a coding sequence was replaced with a sequence encoding a lexA::VP16 driver [94]. (B-D) MD assays of flies expressing the fru-GAL4 driver together with (B) Gr5a-RNAi (C) Gr43a-RNAi (D) Gr64f-RNAi. (E) Male foreleg (upper panels), midleg (middle panels) and hindleg (bottom panels) of flies expressing Gr5a-lexA and Gr64f-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under a fluorescent microscope. Yellow arrows indicate Gr5a-positive neurons and Gr64f-positive neurons. Scale bars represent 50 μm. (F) Male foreleg of flies expressing Gr5a-lexA and Gr43a-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under a fluorescent microscope. (G) Male proboscis of flies expressing Gr5a-lexA and Gr43a-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under a fluorescent microscope. Yellow arrows indicate Gr5a-positive neurons and Gr43a-positive neurons. Scale bars represent 50 μm. (H-I) MD assays of (H) Gr64f- and (I) Gr43a-GAL4 drivers for the inactivation of synaptic transmission of fru-specific neurons among each GAL4-labelled neuron via UAS>stop>TNTactive; fruFLP. Tested gustatory sugar receptors were selected based on a previous study [55].
By using the genetic intersectional method, we found that Gr5a is co-expressed with Gr64f in 5T - 1T of the male foreleg and 5T - 4T in midleg (Fig 6E). However, Gr5a is co-expressed with Gr64f in 5T - 4T in the female foreleg/midleg and 5T in the female hindleg (S6H Fig). In contrast, there are no Gr5a-positive cells expressing the fructose sensor Gr43a [56] in the male foreleg (Fig 6F). Although no cells co-expressed Gr5a and Gr43a in the leg, several cells co-expressed Gr5a and Gr43a in the male proboscis (Fig 6G). We were unable to detect any fru-positive cells expressing Gr64f in the male proboscis (S6I Fig). When we expressed UAS-TNT only in male-specific Gr64f-positive neurons, we found that SMD was abolished; however, SMD remained intact in Gr43a-positive neurons (Fig 6H and 6I). Gr proteins are known to function as heterodimeric or multimeric complexes [57–59]. In addition, Gr64f is required broadly as a co-receptor for the detection of sugars and works together with Gr5a protein to illicit behavioral responses to trehalose [60]. Collectively, these data suggest that co-expression of the sugar receptor Gr5a and its co-receptor Gr64f in male-specific leg sensory neurons is crucial for the sensory inputs underlying SMD behavior.
Pheromone-sensing molecules and receptors are involved in the processing of SMD sensory information
Next, we tested the role of pheromone processing molecules in male legs in the generation of SMD behavior [61]. The knockdown of LUSH, an odorant-binding protein [62] in Gr5a-positive neurons, but not in Gr66a-positive neurons, led to the abolishment of SMD behavior (Fig 7A and 7B). SNMP1 is a member of the CD36-related protein family and functions as an important player for the rapid kinetics of pheromonal response in insects [63,64]. We found that the expression of Snmp1 on the snmp1 mutant background via the Gr5a-GAL4 driver, but not the Gr66a-GAL4 driver, could rescue SMD behavior (Fig 7C–7H), thus suggesting that the expression of the pheromone sensing proteins LUSH and Snmp1 in Gr5a-positive gustatory neurons is critical for generating SMD behavior. By using the genetic intersectional method, we found that the male antenna contains an abundance of snmp1-positive cells but did not find any Gr5a-positive or snmp1-positive cells (Fig 7I). Surprisingly, we found one cell that was both snmp1-positive and Gr5a-positive in the 2T of the male tarsus (Fig 7J). SMD behavior is disrupted by snmp1 knockdown utilizing Gr5a-GAL4 but not Gr66a-GAL4 (S7G–S7H Fig). Collectively, these data suggest that the expression of LUSH and SNMP1 in the male leg is crucial for sensory inputs for SMD behavior.
(A-B) MD assays for GAL4 mediated knockdown of LUSH via UAS-lush-IR; UAS-dicer (lush-RNAi) using (A) Gr5a-GAL4 and (B) Gr66a-GAL4 drivers. (C-H) MD assays of snmp1 genetic rescue experiments. Genotypes are indicated below each graph. (I) Male antenna of flies expressing Gr5a-lexA and Snmp1-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under a fluorescent microscope. Scale bars represent 50 μm. (J) Male foreleg of flies expressing Gr5a-lexA and Snmp1-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under a fluorescent microscope. Yellow arrows indicate Gr5a-positive and Snmp1-positive neurons. Scale bars represent 50 μm.
Next, we tested the importance of degenerin/epithelia Na+ channels (DEG/ENaC), ppk23, ppk25 and ppk29 in the excitability of pheromone-sensing cells [65,66]. By using RNAi-mediated knockdown experiments, we found that ppk25 and ppk29, but not ppk23, are crucial for generating SMD behavior in Gr5a-positive cells but not Gr66a-positive cells (Figs 8A–8F and, S8A–S8C). By using the genetic intersectional method, we found that ppk25 was co-expressed with Gr5a in 5T of the male foreleg and 4T of the midleg (Fig 8G). We also found that ppk29 was co-expressed with Gr5a in 2T and 4T of the male foreleg (Fig 8H). However, we did not detect any cells that co-expressed ppk23 and Gr5a in the legs of males (S8D Fig). Of the Deg/ENaC sodium channel family, ppk28 is reported to be expressed in gustatory neurons and is known to mediate the detection of water taste [67]. By using RNAi-mediated knockdown experiments, we found that ppk28 is dispensable for SMD behavior in Gr5a-positive neurons (S8H–S8J Fig). These data suggest that ppk25/ppk29, but not ppk23/ppk28, are crucial for pheromonal detection in the induction of SMD behavior in Gr5a-positive leg neurons in males.
(A-B) MD assays for GAL4 mediated knockdown of PPK23 via ppk23-RNAi using (A) Gr5a-GAL4 and (B) Gr66a-GAL4 drivers. (C-D) MD assays for GAL4 mediated knockdown of PPK25 via ppk25-RNAi using (C) Gr5a-GAL4 and (D) Gr66a-GAL4 drivers. (E-F) MD assays for GAL4 mediated knockdown of PPK29 via ppk29-RNAi using (E) Gr5a-GAL4 and (F) Gr66a-GAL4 drivers. (G) Male foreleg (upper panels), midleg (middle panels) and hindleg (bottom panels) of flies expressing Gr5a-lexA and ppk25-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under a fluorescent microscope. Yellow arrows indicate Gr5a-positive and ppk25-positive neurons. Scale bars represent 50 μm. (H) Male foreleg (upper panels), midleg (middle panels) and hindleg (bottom panels) of flies expressing Gr5a-lexA and ppk29-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under a fluorescent microscope. Yellow arrows indicate Gr5a-positive neurons and Gr64f-positive neurons. Scale bars represent 50 μm.
Three ppk family members (ppk23, ppk25 and ppk29) can sense the female pheromone 7,11-heptacosadiene [65] and express fruitless, a factor that is crucial for mating behavior in males [66]. By using RNAi-mediated knockdown, we found that the expression of ppk25/ppk29 in fru-positive cells is crucial for SMD behavior, but not ppk23 expression (Fig 9A–9C). By using the genetic intersectional method, we identified that ppk23 was co-expressed with fru in 5T - 2T of the male foreleg and 2T of the hindleg (Fig 9D). We also found that ppk25 was co-expressed with fru in 5T - 2T of the male foreleg and 4T of the midleg (Fig 9E) and that ppk29 was co-expressed with fru in 5T - 2T of the male foreleg (Figs 9F and S9A). We also confirmed that ppk29-GAL4 labels cells only in males and not in females (S9B and S9C Fig). These data suggest that the expression of ppk25 and ppk29 in fru-positive male-specific cells is crucial for SMD behavior.
(A-C) MD assays for GAL4 mediated knockdown of (A) PPK23, (B) PPK25, and (C) PPK29 via ppk23-RNAi, ppk25-RNAi, and ppk29-RNAi using the fru-GAL4 driver. (D-F) Male foreleg (upper panels), midleg (middle panels) and hindleg (bottom panels) of flies expressing fru-lexA and (D) ppk23-GAL4, (E) ppk25-GAL4, and (F) ppk29-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under a fluorescent microscope. Yellow arrows indicate fru-positive and ppk23-, ppk25-, or ppk29-positive neurons. Scale bars represent 50 μm. (G-I) MD assays for GAL4 mediated knockdown of LUSH via lush-RNAi using (G) ppk23-GAL4, (H) ppk25-GAL4, (I) ppk29-GAL4 drivers. (J) MD assays for GAL4 mediated knockdown of SNMP1 via snmp1-RNAi using the ppk25-GAL4 driver.
Next, to decipher whether DEG/NaC channel-expressing pheromone sensing neurons require the function of OBP, we expressed lush-RNAi using ppk23-, ppk25- and ppk29-GAL4 drivers to knockdown LUSH in each channel-expressing neuron. The knockdown of LUSH in ppk25- and ppk29-GAL4 labeled cells, but not in ppk23-GAL4 labeled cells, led to a disturbance in SMD behavior, thus suggesting that LUSH functions in ppk25- and ppk29-positive neurons to detect pheromones and elicit SMD behavior (Fig 9G–9I). The knockdown of SNMP1 in ppk25- or ppk29-GAL4- labeled neurons inhibited SMD behavior (Fig 9J and S9I Fig), thus suggesting that SNMP1 also functions in ppk29-positive neurons to induce SMD behavior.
The Drosophila melanogaster genome bears two members of the SNMP/CD36 gene family; the proteins these genes encode are expressed in distinct cells [68,69]. SNMP2 is known to contribute to gender recognition during courtship; however, its precise functional role remains unknown [69,70]. To compare the function of SNMP2 with SNMP1, a factor that is crucial for SMD behavior, we reduced the gene expression of SNMP2 in ppk23-, ppk25-, ppk29-GAL4 expressing pheromone sensing neurons and found that SNMP2 is dispensable in these pheromone-sensing neurons for eliciting SMD behavior (S9D–S9F Fig). We also found that SNMP2 was not required for SMD behavior in Gr5a- and Gr64f-GAL4 labeled sugar sensing neurons (S9G–S9H Fig). Combining with genetic control experiments (S12 and S13 Figs), all these data suggest that SNMP1, but not SNMP2, is specifically involved in pheromone detection for SMD behavior in the male leg system.
Activation of Gr5a-positive cells is sufficient to shorten the mating duration, and this relates to calcium accumulation in these cells
To determine whether the temporal activation of Gr5a-positive neurons may generate SMD behavior in the absence of sexual experiences, we expressed the heat-sensitive Drosophila cation channel TrpA1 in Gr5a-positive cells and then transferred the experimental group only to the activation temperature (29°C). Surprisingly, the flies expressing TrpA1 in Gr5a-positive neurons at the activation temperature showed a shorter mating duration than those that remained at 22°C (Fig 10A). Neither the genetic control (Fig 10B and 10C) nor the flies expressing shits that could disrupt synaptic transmission in a temperature-sensitive fashion (Fig 10D) showed changes in their mating duration between 22°C and 29°C. These findings indicate that the stimulation of Gr5a-positive neurons is sufficient to generate SMD behavior.
(A-D) MD assay for the temporal temperature-shift of flies expressing UAS-TrpA1 or UAS-shits by Gr5a-GAL4. Genotypes are labelled below the graph. Blue groups were reared at 22°C for five days and red groups were reared at 22°C for four days and moved to 29°C overnight. (E-G) MD assay for the temporal temperature-shift of flies expressing UAS-shits by Gr5a-GAL4. Genotypes are labelled below the graph. Grey groups reared at 22°C for four days and moved to 29°C for overnight. Red groups reared at 22°C for four days and moved to 29°C overnight with sexual experiences. (H) Different levels of neural activity of the 4th and 5th sensory neurons as revealed by the CaLexA system in naive versus mated male flies. Male flies expressing Gr5a-GAL4 along with LexAop-CD2-GFP, UAS-mLexA-VP16-NFAT and LexAop-CD8-GFP-2A-CD8-GFP were dissected after at least 10 days of growth (mated male flies had 1-day of sexual experience with virgin females). GFP is pseudo-colored as “red hot”. Dashed boxes represent the magnified area of interest and show the right section of each condition. Dashed circles represent the location of Gr5a-positive cells. White colors represent the naïve condition while the yellow color represents the experienced condition. Scale bars represent 20 μm. (I and J) Quantification of GFP fluorescence. GFP fluorescence of the 4th (I) or 5th (J) tarsus was normalized to that in auto-fluorescence. The conditions of flies are described above: naïve, naïve male flies; exp., male flies with sexual experience. Bars represent the mean of the normalized GFP fluorescence level with error bars representing the SEM. Asterisks represent significant differences, as revealed by the Student’s t test and ns represents non-significant difference (*p < 0.05, **p < 0.01, ***p < 0.001).
By using the expression of shits with Gr5a-GAL4, we then attempted to inhibit the synaptic transmission of Gr5a-positive neurons during sexual experiences. We discovered that inhibiting Gr5a-positive neurons during sexual interactions by increasing the temperature to 29°C could impair SMD behavior (Fig 10E). The genetic control exhibited no such result (Fig 10F and 10G). These findings imply that the neural stimulation of Gr5a-positive neurons during the sexual experiences is a crucial trigger for SMD behavior.
To determine whether neuronal activities undergo alterations in neurons associated with SMD, we utilized the CaLexA (calcium-dependent nuclear import of LexA) system [71]. This system is based on the activity-dependent nuclear import 1of the transcription factor nuclear factor of activated T cells (NFAT). Because SMD needs at least 6–12 h of sexual interaction, repeated sensory inputs might theoretically lead to the buildup of the modified transcription factor within the nucleus of activated neurons in vivo. Indeed, sexual encounters affected the neural activity of some Gr5a-GAL4-labeled neurons. Male flies with sexual experience and carrying Gr5a-GAL4 and LexAop-CD2-GFP; UAS-mLexA-VP16-NFAT, LexAop-CD8- GFP-2A-CD8-GFP exhibited strong fluorescence in the 5th tarsus following an overnight sexual experience. In contrast, no similar signals were identified in males with no prior experience. In contrast to Gr5a-positive neurons in the 5th tarsus, cells in the 4th tarsus did not exhibit a significant increase in GFP fluorescence (Fig 10H–10J), thus indicating that sexual encounters change the neuronal activity of Gr5a cells in the 5th tarsus.
SMD is an evolutionary adaptive trait
To explore the adaptive value of SMD, we developed a theoretical model to test the adaptive value of SMD behavior based on the marginal value theorem [72,73] (S1 Box). This model assumes that (i) the differences in mating duration occur largely due to the variation in post-ejaculation period (mate guarding) [15,19] and (ii) both the benefits and costs of mate guarding accumulate over time, but with different aspects.
The benefit refers to the number of eggs fertilized by the guarding male while the costs refer to the guarding-associated potential costs such as increased predation risk or the loss of opportunities for other forms of mating or foraging activity [74]. The model suggests that shortened mating durations can be preferred in experienced males if (1) experienced males can fertilize a fewer number of eggs in total than naïve males (Fig 11A) and that the rate of fertilization is (2) faster (Fig 11B) or (3) slower (Fig 11C) for experienced males while the total number of eggs that can be fertilized remains the same as for naïve males, and/or 4) the costs accumulate faster in experienced males (Fig 11D). Next, we empirically tested which scenario(s) could explain the observed SMD behavior. We focused on testing scenarios 1–3 but not 4, firstly because it was hard to identify a rationale for how the costs of mate guarding differ between experienced and naïve males and secondly, to experimentally manipulate the costs.
(A)-(D) show the four different scenarios by which SMD can evolve (see S1 Box). SMD can evolve when α gets larger (A), β gets smaller (B), γ gets larger (assuming β/α < γ < e*β/α) (C) and/or γ gets smaller (assuming γ > e*β/α) (D). (Ε) Relative ratio of total egg number comparing the eggs produced by females mated with naïve males to the eggs produced by females that mated with experienced males. (naïve = control bar for comparing, exp. = eggs from naïve males/eggs from exp. males). (F) Relative ratio of total progeny number comparing the progeny produced by the females mated with naïve males to the progeny produced by females mated with experienced males. (naïve = control bar for comparing, exp. = progeny from naïve males/progeny from exp. males). (G) Percentage of progeny originated from sepia (se) male versus CS male. se male was introduced to se female as first mate then followed by CS males as the second mate. The eye color of progeny was counted and interpreted as the source of the father; for detailed methods, see the EXPERIMENTAL PROCEDURES. (H) Summary of this study showing the multisensory inputs modulating SMD behavior.
We found that the total number of eggs produced by females that mated with experienced males was comparable to those that mated with naïve males (Fig 11E); however, the number of progeny from the experienced males was significantly lower than those from naïve males (Fig 11F). When females that mated with an experienced or naïve male were subsequently introduced to another male after 24 hours, the number of progenies arising from the experienced males was also significantly fewer than those from the naïve males (Figs 11G and S11A). This suggests that (i) the number of sperm or seminal proteins from experienced males for fertilization in a given period of time was lower than that from naïve males [32,75] or ii) females reduced the use of sperm from experienced males for fertilization when they had a choice. These results support scenario 1 and potentially scenario 3 in that SMD has evolved because the reproductive payoffs of experienced males through mate-guarding are consistently lower than those of naïve males.
Discussion
Our study provides new lines of evidence that male flies invest less time for mating duration when they are sexually experienced. Males retain a memory of sexual experience for several hours and economize mating duration accordingly (Fig 1). This behavior relies primarily on gustatory input from the male forelegs, indicating that contact chemoreception is required for SMD induction (Figs 2 and 3). Sugar cells expressing Gr5a, but not bitter cells expressing Gr66a, were found to be involved in the induction of SMD (Fig 4). We also found that male-specific, fru-expressing Gr5a-positive sensory neurons are required to recognize the presence of females (Fig 5). Sugar receptors such as Gr5a/Gr64f, but not fructose sensor Gr43a, are important for the sensory inputs required for SMD behavior (Fig 6). Chemosensory proteins such as lush and SNMP1, as well as female pheromone receptors (DEG/ENaC channel ppk25 and ppk29) are important for generating SMD (Figs 7, 8 and 9). We discovered that temporal stimulation of Gr5a neurons reduces mating duration, which is related to calcium accumulation (Fig 10). Using both theoretical and empirical approaches, we further showed that SMD represents the adaptive behavioral plasticity of male flies (Fig 11).
Previous research by our group and others demonstrated that past exposure to rivals lengthens mating duration, a characteristic known as longer-mating-duration (LMD) [14–23,76]. The two behavioral circuits for LMD and SMD might have evolved independently since they use different sensory cues for detecting ‘rivals’ or ‘females’ for ‘sexual competition’ or ‘mating investment’, respectively. We propose that multisensory inputs from male forelegs detect the chemical signals from the female body and contribute to the determination of mating investment in male Drosophila melanogaster (S10A Fig and Fig 11H). The visual inputs from the male’s compound eye are the most crucial sensory cue to generate LMD [22]; however, multisensory inputs from the foreleg are required to induce SMD (Fig 11H). To confirm that LMD does not require female pheromone signaling, we reduced the expression of the female pheromone receptor ppk29 in all neuronal populations using RNAi-mediated knockdown experiments and found that the neuronal expression of ppk29 is only essential for SMD but not for LMD behavior (S11B–S11C Fig). Consistent with our previous report on the different neural circuitry for LMD and SMD [23], these data clearly show that male flies use different sensory modalities to generate LMD or SMD, respectively.
In our sugar receptor screening for SMD behavior, we found that only Gr5a and Gr64f were required for SMD behavior (Fig 6A, 6B, and 6D). The other known sugar receptors (Gr61a, Gr64a, Gr64b, Gr64c, Gr64d, and Gr64e) are not required for SMD behavior (S6A–S6G Fig). Fujii et al reported the expression code for specific sweet neurons in labial palp and tarsal sensilla [55]. In this code, Gr5a- and Gr64f-positive but Gr43a-negative neurons are referred to as “f4b”, “f4s”, “f5s”, and “f5b”. In the foreleg, the hair cells expressing Gr43a do not express Gr5a [55]. Gr43a is the fructose sensor and is co-expressed with Gr61a [56]. In summary, we suggest that the sugar receptors Gr5a and Gr64f in fruitless-positive cells provide crucial sensory information for SMD behavior.
It is known that the type of neurons expressing ppk23, ppk25, and ppk29 is referred to as a “female” cell (F cell) from its responses to female aphrodisiac pheromones; the other type of neurons, expressing ppk23 and ppk29 but not ppk25, is referred to as the “male” cell (“M” cell) from its response to male anti-aphrodisiac pheromones. M and F cells both express fruitless gene [66]. SMD requires female pheromonal inputs through the contact chemoreception pathway in males (Fig 2K–2M). We also identified that there are ppk25- and/or ppk29-positive neurons among Gr5a-positive sugar detecting neurons (Fig 8A–8H). Thus, we hypothesize that F cells, which can detect sugar taste, are responsible for SMD behavior. Several groups have reported that ppk23-expressing cells respond to pheromones but not to water, salt, or sugars; in addition, this response is abolished by the mutation of either ppk23 or ppk29 [48,77,78]. Genetic rescue studies revealed that although all three subunits are co-expressed and function in the gustatory cells required for the activation of courtship by pheromones, each has a non-redundant function within these cells [66]. Thus, we suggest that the ppk25 and ppk29 receptors expressed in fruitless-positive “F cells” are critical for detecting female body pheromones via contact chemoreception and generating SMD behavior.
One of the findings of this report is that Gr5a-positive taste neurons also express the female pheromone receptors ppk25 and ppk29. To further validate our experimental data, we made use of a scRNA sequencing dataset of fruit flies that is available on the SCope website [79]. We reviewed the expression levels of essential marker genes for SMD behavior in several sensory organs, including the leg, wing, proboscis, antenna, trachea, and oenocyte, and concluded that these genes are expressed comparably in the leg and wing, but not in other sensory organs (Fig 12A–12F). In addition, we discovered that Gr5a and Gr64f are expressed in gustatory receptor neurons and other sensory neurons in the leg, which are pheromone-sensing neurons in the wing, as we continued to divide cell types (Fig 12G and 12H). Comparable to the leg, the wing may be an organ that can receive signals from females. Recent research found that pheromone sensing ppk29 and ppk23 were significantly expressed in the wing [80], thus indicating that the wing is also an intriguing organ for pheromone sensing function and may contribute to the mating behavior of males. Future research will investigate the potential role of the wings in SMD behavior.
Dot plot of the 8 genes involved in SMD in each cell type in different tissues (A-H) The size of dots represents the percentagew of cells in one cell type expressing the gene of interest; the intensity of color reflects the average scaled expression. We used broad annotation for (A-F) and detailed one for (G) and (H).
In summary, we report a novel sensory pathway that controls mating investment related to sexual experiences in Drosophila. Since both LMD and SMD behaviors are involved in controlling male investment by varying the interval of mating, these two behavioral paradigms will provide a new avenue to study how the brain computes the ‘interval timing’ that allows an animal to subjectively experience the passage of physical time [81–86].
Materials and methods
Fly rearing and strains
Drosophila melanogaster were raised on cornmeal-yeast medium at similar densities to yield adults with similar body sizes. Flies were kept in 12 h light: 12 h dark cycles (LD) at 25°C (ZT 0 is the beginning of the light phase, ZT12 beginning of the dark phase) except for some experimental manipulation (experiments with the flies carrying tub-GAL80ts). Wild-type flies were Canton-S. To reduce the variation from genetic background, all flies were backcrossed for at least 3 generations to CS strain. All mutants and transgenic lines used here have been described previously.
We are very grateful to the colleagues who provided us with many of the lines used in this study. We obtained the following lines from Dr. Joel D. Levine and Joshua J. Krupp (University of Toronto, Canada): PromE(800)-GAL4 (oeno-GAL4 in this study); from Dr. Barry Dickson (HHMI Janelia Research Campus, USA): UAS[stop]mCD8GFP; fruFLP, UAS[stop]nsybGFP; fruFLP, UAS[stop]TNTactive; fruFLP, fru-GAL4; from Dr. Toshiro Aigaki (Tokyo Metropoitan University, Japan): UAS-mSP; from Dr. Martin Heisenberg (Universität Würzburg, Germany): WT Berlin, ninaE17; from Dr. Michael Gordon (University of British Columbia, Canada): Gr5a-lexA, Gr64f-lexA, ppk23-GAL4, ppk25-GAL4, ppk29-GAL4.
The following lines were obtained from Bloomington Stock Center (#stock number): Orco1 (#23129), Orco2 (#23130), UAS-tubGAL80ts (#7018), Df(1)Exel6234 (#7708), UAS-traF (#4590), GustDx6 (#8607), Gr66a-GAL4 (#28801), UAS-mCD8GFP (#5130), UAS-RedStinger (#8547), snmp11 (#25043), snmp12 (#25042), UAS-snmp1 (#25044), Snmp2-RNAi (#51432), UAS-TNT (#28997), UAS-dicer (#24650, #24651), tra-RNAi (#28512), lush-RNAi (#31657), tud1 (son-of-tudor males were the sons of Oregon R males and virgin tudor females: tud1 bw sp/tud1 bw sp) (#1786), lexAop -tdTomato.nls, UAS-Stinger (#66680), Gr5a-RNAi (#31282), Gr43a-RNAi (#64881), Gr61a-RNAi (#54030), Gr64c-RNAi (#36734), ppk23-RNAi (#28350), ppk25-RNAi (#27088), ppk29-RNAi (#27241), fru-lexA (#66698), Gr64f-GAL4 (#57668), se1 (sepia mutants for fecundity test, #1668), elavc155; UAS-Dcr-2 (#25750), CalexA (#66542), UAS-TrpA1 (#26264), UAS-shits (#44222), Poxn-GAL4 (#66685), Poxn-RNAi (#26238); from Vienna Drosophila Stock Center: Gr5a-RNAi (#v13730), Gr43a-RNAi (#v39518), Gr61a-RNAi (#v106007), Gr64a-RNAi (#v103342), Gr64b-RNAi (#v42517), Gr64d-RNAi (#v29422), Gr64e-RNAi (#v109176), Gr64f-RNAi (#v105084). Following transgenic stocks are available from Korea Drosophila Resource Center (KDRC): UAS[stop]TNTinactive; fruFLP (1124).
Mating duration assays
Mating duration assay was performed as previously described [21,22]. For naïve males, 4 males from the same strain were placed into a vial with food for 5 days. For experienced males, 4 males from the same strain were placed into a vial with food for 4 days then eight CS virgin females were introduced into vials for last 1 day before assay. Five CS females were collected from bottles and placed into a vial for 5 days. These females provide both sexually experienced partners and mating partners for mating duration assays. At the fifth day after eclosion, males of the appropriate strain and CS virgin females were mildly anaesthetized by CO2. After placing a single female in to the mating chamber, we inserted a transparent film then placed a single male to the other side of the film in each chamber. After allowing for 1 h of recovery in the mating chamber in a 25°C incubator, we removed the transparent film and recorded the mating activities. Only those males that succeeded to mate within 1 h were included for analyses. Initiation and completion of copulation were recorded with an accuracy of 10 sec, and total mating duration was calculated for each couple. All assays were performed from noon to 4 pm. We conducted blinded studies for every test.
Sperm depletion from males
To deplete sperm from males, 40 virgin Defexel6234 females which lacks SPR and shows multiple mating with males [44] were placed in a vial containing four CS males for indicated time (2 h, 4 h, 8 h, and 24 h).
Courtship assays
Courtship assay was performed as previously described [95], under normal light conditions in circular courtship arenas 11 mm in diameter, from noon to 4 pm. Courtship latency is the time between female introduction and the first obvious male courtship behavior such as orientation coupled with wing extensions. Once courtship began, courtship index was calculated as the fraction of time a male spent in any courtship-related activity during a 10 min period or until mating occurred. Mating initiation is the time after male flies successfully mounted on female.
Locomotion assays
For climbing assay, individual flies were placed in a 15 ml falcon tube (Fisher Scientific) and were gently tapped to the bottom of the tube. The time taken for the flies to climb 8 cm of the tube wall was recorded. Each fly was tested 5 times. Other than a single instance, all flies were seen to reach the target height within 2 min, which was the experimental cut-off time. Velocity was obtained by dividing the lines (mm) a fly crossed (distance walked) by time (sec) a fly reached the line of the tube. For horizontal (spontaneous) locomotor activities, a single fly was first brought to the middle of the column by gentle shaking and then the fly movement was constantly monitored for 5 min and recorded. Total fraction of time flies walked during 5 min was calculated and number of stops during 5 min was also counted then calculated [96].
Immunostaining and antibodies
As described before [22], brains dissected from adults 5 days after eclosion were fixed in 4% formaldehyde for 30 min at room temperature, washed with 1% PBT three times (30 min each) and blocked in 5% normal donkey serum for 30 min. The brains were then incubated with primary antibodies in 1% PBT at 4oC overnight followed with fluorophore-conjugated secondary antibodies for 1 hour at room temperature. Brains were mounted with anti-fade mounting solution (Invitrogen, catalog #S2828) on slides for imaging. Primary antibodies: chicken anti-GFP (Aves Labs, 1:1000), rabbit anti-DsRed express (Clontech, 1:250), mouse anti-Bruchpilot (nc82) (DSHB, 1:50), mouse anti-PDF (DSHB, 1:100). Fluorophore-conjugated secondary antibodies: Alexa Fluor 488-conjugated goat anti-chicken (Invitrogen, 1:100), Alexa Fluor 488-conjugated donkey anti-rabbit (Invitrogen, 1:100), RRX-conjugated donkey anti-rabbit (Jackson Lab, 1:100), RRX-conjugated donkey anti-mouse (Jackson Lab, 1:100), Dylight 649-conjugated donkey anti-mouse (Jackson Lab, 1:100).
Quantitative analysis of GFP fluorescence
To quantify the calcium level in leg sensory neurons, we measured fluorescence intensity using the measure tool of ImageJ (National Institutes of Health, http://rsb.info.nih.gov/ij).). Fluorescence was quantified in a manually set region of interest (ROI) of the 4th or 5th tarsus. To compensate for differences in fluorescence between different ROI, GFP fluorescence for CaLexA was normalized to autofluorescence, and then the fluorescence of ROI was quantified using the measure tool of ImageJ. All specimens were imaged under identical conditions.
Statistical analysis
Statistical analysis of mating duration assay was described previously [21,22]. More than 36 males (naïve or experienced) were used for mating duration assay. Our experience suggests that the relative mating duration differences between naïve and experienced condition are always consistent; however, both absolute values and the magnitude of the difference in each strain can vary. So, we always include internal controls for each treatment as suggested by previous studies [30]. Therefore, statistical comparisons were made between groups that were naively reared or sexually experienced within each experiment. As mating duration of males showed normal distribution (Kolmogorov-Smirnov tests, p > 0.05), we used two-sided Student’s t tests. We summarized the normality and lognormality test of mating duration in S1M–S1N Fig and S3 Table. Each figure shows the mean ± standard error (s.e.m) (*** = p < 0.001, ** = p < 0.01, * = p < 0.05). All analysis was done in GraphPad (Prism). Individual tests and significance are detailed in figure legends.
When we compare the difference of mating duration in experiments without internal control built in, we always performed control experiments of wild type for each independent experiment for internal comparison. And in this case, we analyzed data using ANOVA for statistically significant differences (at a 95.0% confidence interval) between the means of mating duration for all conditions. If a significant difference between the means was found by Kruskal-Wallis test, then the Dunn’s Multiple Comparison Test was used to compare the mean mating duration of each condition to determine which conditions were significantly different from condition of interest. (# = p < 0.05)
Besides traditional t-test for statistical analysis, we added estimation statistics for all MD assays and two group comparing graphs. In short, ‘estimation statistics’ is a simple framework that—while avoiding the pitfalls of significance testing—uses familiar statistical concepts: means, mean differences, and error bars. More importantly, it focuses on the effect size of one’s experiment/intervention, as opposed to significance testing [97]. In comparison to typical NHST plots, estimation graphics have the following five significant advantages such as (1) avoid false dichotomy, (2) display all observed values (3) visualize estimate precision (4) show mean difference distribution. And most importantly (5) by focusing attention on an effect size, the difference diagram encourages quantitative reasoning about the system under study [98]. Thus, we conducted a reanalysis of all of our two group data sets using both standard t-tests and estimate statistics. In 2019, the Society for Neuroscience journal eNeuro instituted a policy recommending the use of estimation graphics as the preferred method for data presentation [99].
Egg and progeny counting
We performed egg laying assay as previously described [44]. In short, wild type females mated with naïve or experienced males were transferred to a fresh new vial and allowed to lay eggs for 24 hr at 25°C. After 24 hr of egg laying, number of eggs were counted under the stereomicroscope. After we count the number of eggs, we kept vials in 25°C incubator and counted the total number of progenies ecolsed from them.
Fecundity test by introducing the second male
Basically, we followed the protocols previously described by other group [19]. In short, se1 or CS virgin females were introduced to se1 or CS males either as naïve or experienced condition for 24 hours to be confident of all females’ mating with the first males. Then we introduced the second males for 24 hours. After this treatment, we separated females from second males then counted the number of progenies from females. To confirm that the effect from this fecundity test was not originated from the genotype background, we performed the same experiments by reversing the genotypes of the first and second males (se1 then CS vs. CS vs. se1). We calculated the percentage of progeny either from the first male or the second male by counting the eye color of progeny.
Single-nucleus RNA-sequencing analyses—data and code availability
snRNAseq dataset analyzed in this paper is published in Li et al., doi:10.1126/science.abk2432 [79] and available at the Nextflow pipelines (VSN, https://github.com/vib-singlecell-nf), the availability of raw and processed datasets for users to explore, and the development of a crowd-annotation platform with voting, comments, and references through SCope (https://flycellatlas.org/scope), linked to an online analysis platform in ASAP (https://asap.epfl.ch/fca).
Gene expression pattern analyses in different tissues
For the gene expression pattern of the 10 genes involved in SMD in each cell type of leg and other tissues, we used the single-cell RNA-seq data from https://flycellatlas.org [79], and the 10x Genomics stringent loom files were downloaded. The cell types are split by FCA.
The digital expression matrices were analyzed with the Seurat 4.1.0 R package [100]. The dot plots of the 10 genes involved in SMD in each cell type of different tissues were then made using the ‘DotPlot’ function with broad annotation (broad cell types) and the annotation (detailed cell types), respectively.
Supporting information
S1 Fig. General characteristics of ‘Shorter-Mating-Duration (SMD)’ behaviour.
(A) Mating duration (MD) assays of Oregon-R males. (B) Locomotion of naïve and experienced male flies were quantified as velocity by locomotion activity by horizontal paradigm, and (C) stop frequency by horizontal paradigm. See EXPERIMENTAL PROCEDURES section for detailed methods. (D-F) MD assays of CS males after isolated from female experience. Males were reared with sufficient numbers of virgin females for 24 h to be assured having sexual experience then isolated. Assay times after isolation are below the boxes as (D) no recovery, (E) 24 h recovery, and (F) 48 recoveries. (G) The diagram of MD assays of CS males after different time of isolation after sexual experience with females. (H-K) MD assays of CS males after sperm deleted as shown in (L). (M) Histogram of SMD behavior shown in Fig 1B. (N) Normal QQ plot of SMD behavior shown in Fig 1B.
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S2 Fig. Sensory inputs required for inducing SMD behavior.
(A) MD assays of UAS-KCNJ2 crossed with Orco-GAL4. (B) MD assays of Poxn-RNAi crossed with Poxn-GAL4. (C) MD assay of CS males experienced with D. simulans females. (D) CS male court oenocytes-musicalized female and show orientation behavior, (E) chasing (F) licking (G) wing extension, (H) copulation attempt, and (I) can successfully mate with it. (J) CS male court feminized male and show wing extension behavior and (K) copulation attempt. (L) MD assays of CS males with oenocytes-masculinized female as a female partner to test whether genotypes of female partners affect MD.
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S3 Fig.
The foreleg of male Drosophila melanogaster (A) The anatomical structures of male foreleg are labeled. Ta1-Ta5 comprise fore tarsus and represents tarsomeres 1–5, respectively.
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S4 Fig.
Control experiments for MD assays in Fig 4 and the location of Gr5a-positive neurons in male foreleg (A-D) MD assays of (A) UAS-Hid/rpr (B) UAS-TNT, tub-GAL80ts (C)UAS-KCNJ2, tub-GAL80ts (D) UAS-NachBac, tub-GAL80ts crossed with CS. (E) Foreleg tarsus of male flies expressing Gr5a-lexA together with lexAOP-mCherry. White arrows indicate Gr5a-positive neurons and numbers represent the order from the distal part of the leg. (F) Foreleg tarsus (left panels) and tibia (right panels) of male (top panels) or female (bottom panels) flies expressing Gr5a-GAL4 together with UAS-RedStigner. White arrows indicate Gr5a-positive neurons. White arrows with dotted line indicate missing neurons in female leg compared to male leg.
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S5 Fig.
(A) Brains of male flies expressing Gr5a-GAL4 together with UAS>stop>nSybGFP; fruFLP were immunostained with anti-GFP (green) and nc82 (magenta) antibodies. Scale bars represent 100 μm in the colored panels and 10 μm in the grey panels. White boxes indicate the magnified regions of interest presented next right panels. The right panels are presented as grey scale for clearly showing the axon projection patterns of gustatory neurons in the adult subesophageal ganglion (SOG) labeled by GAL4 drivers. (B-D) Control experiments for MD assays in Fig 5. MD assays of (B) UAS>stop>TNTactive; fruFLP (C) UAS>stop>TNTinactive; fruFLP (D) UAS-traF crossed with CS. (E) Proboscis of male flies expressing fru-lexA; lexAOP-tdTomato.nls, UAS-Stinger with Gr5a-GAL4 were imaged in live. Scale bars represent 100 μm.
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S6 Fig.
(A) Control experiments for MD assays in Figs 6B–6D and S6B–S6G. (B-G) MD assays of flies expressing fru-GAL4 driver together with (B) Gr61a-RNAi (C) Gr64a-RNAi (D) Gr64b-RNAi (E) Gr64c-RNAi (F) Gr64d-RNAi (G) Gr64e-RNAi. (H) Female foreleg (upper panels), midleg (middle panels), and hindleg (bottom panels) of flies expressing Gr5a-lexA and Gr64f-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under the fluorescent microscope. Yellow arrows indicate Gr5a-positive and Gr64f-positive neurons. Scale bars represent 50 mm. (I) Male proboscis of flies expressing fru-lexA and Gr64f- GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under the fluorescent microscope. Tested gustatory sugar receptors were selected based on previous study [55].
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S7 Fig.
(A) Control experiments for MD assays in Fig 7A and 7B. (B-C) Control experiments for MD assays in Fig 7C–7H. (D) Female foreleg of flies expressing Gr5a-lexA and Snmp1-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under the fluorescent microscope. Scale bars represent 50 μm. (E-F) MD assays for GAL4 mediated knockdown of LUSH via different lush-RNAi using Gr5a-GAL4. The stock numbers are written at the bottom of each graph. (G-H) MD assays for GAL4 mediated knockdown of SNMP1 via different snmp1-RNAi using (G) Gr5a-GAL4 or (H) Gr66a-GAL4.
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S8 Fig.
(A-C) Control experiments for MD assays in Fig 8A–8F. (D) Male foreleg (upper panels), midleg (middle panels), and hindleg (bottom panels) of flies expressing Gr5a-lexA and ppk23-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under the fluorescent microscope. Scale bars represent 50 μm. (H) Control experiments for MD assays in S8I–S8J Fig. (I-J) MD assays for GAL4 mediated knockdown of PPK28 via ppk28-RNAi using (I) Gr5a-GAL4 and (J) Gr66a-GAL4 drivers.
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S9 Fig.
(A) Male foreleg of flies expressing ppk29-GAL4 together with UAS-RedStinger; UAS>stop>mCD8GFP; fruFLP were imaged live under the fluorescent microscope. Scale bars represent 50 μm. (B) Male foreleg of flies expressing ppk29-GAL4 together with UAS-RedStinger, UAS-mCD8GFP were imaged live under the fluorescent microscope. Scale bars represent 50 μm. (C) Female foreleg of flies expressing ppk29-GAL4 together with UAS-RedStinger, UAS-mCD8GFP were imaged live under the fluorescent microscope. Scale bars represent 50 μm. (D-H) MD assays for GAL4 mediated knockdown of SNMP2 via snmp2-RNAi using (D) ppk23-GAL4, (E) ppk25-GAL4, (F) ppk29-GAL4, (G) Gr5a-GAL4, and (H) Gr64f-GAL4 drivers. (I) MD assays for GAL4 mediated knockdown of SNMP1 via snmp1-RNAi using ppk25-GAL4.
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S10 Fig.
(A) A diagram of the cells in the male legs expressing genes involved in SMD behavior.
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S11 Fig.
(A) Percentage of progeny originated from sepia (se) male vs. CS male. CS male was introduced to se female as first mate then followed se males as second mate. The eye color of progeny was counted and interpreted as the source of farther; for detailed methods, see EXPERIMENTAL PROCEDURES. (B-C) MD assays for GAL4 mediated knockdown of PPK29 via ppk29-RNAi using elavc155 for (B) LMD and (C) SMD behavior.
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S12 Fig.
(A-N) MD assay for GAL4, lexA, and RNAi control experiments. Genotypes are labelled below the graph.
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S13 Fig.
(A-H) MD assay for GAL4, RNAi, and UAS-dicer control experiments. Genotypes are labelled below the graph.
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S1 Table. Summary of MD assay results with various conditions and sensory mutants to identify the sensory modality for SMD behavior.
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S2 Table. Summary of MD assay results with various genotypes of experienced females.
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S3 Table. Summary of normality and lognormality tests of the mating duration result of Canton S naïve and exp. condition.
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S1 Raw Data. All mating duration data generated in this study.
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S2 Raw Data. All offspring data shown in Fig 11 and S11 Fig.
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Acknowledgments
We thank Dr. Joshua Bagley (ahead bio AG, Austria), Dr. Kyeongjin Kang (Korea Brain Research Institute, KBRI), Seokjun Moon (Yonsei University, Korea), Youngjoon Kim (Gwangju Institute of Science and Technology, GIST, Korea), Dr. Kweon Yu (Korea Research Institute of Bioscience and Biotechnology, KRIBB, Korea), Ashley Kim (Mountain View Hospital, USA), Drs. Yuh Nung Jan and Lily Yeh Jan (UCSF, USA) for helpful comments and support on this paper. We are also very appreciative to the colleagues who supplied us with several fly strains; Drs. Joel D. Levine and Joshua J. Krupp (University of Toronto, Canada), Dr. Barry Dickson (HHMI Janelia Research Campus, USA), Dr. Toshiro Aigaki (Tokyo Metropoitan University, Japan), Dr. Martin Heisenberg (Universität Würzburg, Germany), Dr. Michael Gordon (University of British Columbia, Canada), Dr. Richard Benton (Université de Lausanne, Switzerland), and Dr. Ralf Stanewsky (University of Münster, Germany). The authors would like to express their gratitude to EditSprings (https://www.editsprings.cn) for the expert linguistic services provided and to NVIDIA Academic Hardware Grant Program for providing GPU for behavioral analysis.
References
- 1. Louâpre P, Alphen JJM van, Pierre J-S. Humans and Insects Decide in Similar Ways. Plos One. 2010;5: e14251. pmid:21170378
- 2. PARKER GA. Sperm Competition and the Evolution of Animal Mating Systems. 1984; 1–60.
- 3. Parker GA, Pizzari T. Sperm competition and ejaculate economics. Biol Rev. 2010;85: 897–934. pmid:20560928
- 4. Dewsbury DA. Ejaculate Cost and Male Choice. Am Nat. 1982;119: 601–610.
- 5. Parker GA. Courtship Persistence and Female-Guarding as Male Time Investment Strategies. Behaviour. 1974;48: 157–183.
- 6. Gowaty PA, Steinichen R, Anderson WW. INDISCRIMINATE FEMALES AND CHOOSY MALES: WITHIN- AND BETWEEN-SPECIES VARIATION IN DROSOPHILA. Evolution. 2003;57: 2037–2045. pmid:14575325
- 7. Byrne PG, Rice WR. Evidence for adaptive male mate choice in the fruit fly Drosophila melanogaster. Proc Royal Soc B Biological Sci. 2006;273: 917–922. pmid:16627276
- 8. Partridge L, Farquhar M. Sexual activity reduces lifespan of male fruitflies. Nature. 1981;294: 580–582.
- 9. CORDTS R, PARTRIDGE L. Courtship reduces longevity of maleDrosophila melanogaster. Anim Behav. 1996;52: 269–278.
- 10. LEFEVRE G JONSSON UB. Sperm transfer, storage, displacement, and utilization in Drosophila melanogaster. Genetics. 1962;47: 1719–36. pmid:13929245
- 11. McKean KA, Nunney L. Increased sexual activity reduces male immune function in Drosophila melanogaster. Proc National Acad Sci. 2001;98: 7904–7909. pmid:11416162
- 12. Demerec M, Kaufman BP. Time Required for Drosophila Males to Exhaust the Supply of Mature Sperm. Am Nat. 1941;75: 366–379.
- 13. Lefranc A, Bundgaard J. The Influence of Male and Female Body Size on Copulation Duration and Fecundity in Drosophila Melanogaster. Hereditas. 2000;132: 243–247. pmid:11075519
- 14. Bretman A, Westmancoat JD, Gage MJG, Chapman T. COSTS AND BENEFITS OF LIFETIME EXPOSURE TO MATING RIVALS IN MALE DROSOPHILA MELANOGASTER. Evolution. 2013;67: 2413–2422. pmid:23888861
- 15. Dore AA, Rostant WG, Bretman A, Chapman T. Plastic male mating behavior evolves in response to the competitive environment*. Evolution. 2021;75: 101–115. pmid:32844404
- 16. Bretman A, Rouse J, Westmancoat JD, Chapman T. The role of species-specific sensory cues in male responses to mating rivals in Drosophila melanogaster fruitflies. Ecol Evol. 2017;7: 9247–9256. pmid:29187965
- 17. Rouse J, Watkinson K, Bretman A. Flexible memory controls sperm competition responses in male Drosophila melanogaster. Proc Royal Soc B Biological Sci. 2018;285: 20180619. pmid:29848652
- 18. Bretman A, Westmancoat JD, Chapman T. Male control of mating duration following exposure to rivals in fruitflies. J Insect Physiol. 2013;59: 824–827. pmid:23727302
- 19. Bretman A, Fricke C, Chapman T. Plastic responses of male Drosophila melanogaster to the level of sperm competition increase male reproductive fitness. Proc Royal Soc B Biological Sci. 2009;276: 1705–1711. pmid:19324834
- 20. Bretman A, Gage MJG, Chapman T. Quick-change artists: male plastic behavioural responses to rivals. Trends Ecol Evol. 2011;26: 467–473. pmid:21680050
- 21. Kim WJ, Jan LY, Jan YN. Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals. Nat Neurosci. 2012;15: 876–883. pmid:22561453
- 22. Kim WJ, Jan LY, Jan YN. A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating. Neuron. 2013;80: 1190–1205. pmid:24314729
- 23. Wong K, Schweizer J, Nguyen K-NH, Atieh S, Kim WJ. Neuropeptide relay between SIFa signaling controls the experience-dependent mating duration of male Drosophila. Biorxiv. 2019; 819045.
- 24. Lizé A, Doff RJ, Smaller EA, Lewis Z, Hurst GDD. Perception of male–male competition influences Drosophila copulation behaviour even in species where females rarely remate. Biol Letters. 2012;8: 35–38. pmid:21752815
- 25. Rouse J, Bretman A. Exposure time to rivals and sensory cues affect how quickly males respond to changes in sperm competition threat. Anim Behav. 2016;122: 1–8.
- 26. Bretman A, Fricke C, Hetherington P, Stone R, Chapman T. Exposure to rivals and plastic responses to sperm competition in Drosophila melanogaster. Behav Ecol. 2010;21: 317–321.
- 27. Maguire CP, Lizé A, Price TAR. Assessment of Rival Males through the Use of Multiple Sensory Cues in the Fruitfly Drosophila pseudoobscura. Plos One. 2015;10: e0123058. pmid:25849643
- 28. Bretman A, Fricke C, Westmancoat JD, Chapman T. Effect of competitive cues on reproductive morphology and behavioral plasticity in male fruitflies. Behav Ecol. 2016;27: 452–461. pmid:27004011
- 29. Price TAR, Lizé A, Marcello M, Bretman A. Experience of mating rivals causes males to modulate sperm transfer in the fly Drosophila pseudoobscura. J Insect Physiol. 2012;58: 1669–1675. pmid:23085556
- 30. Bretman A, Westmancoat JD, Gage MJG, Chapman T. Males Use Multiple, Redundant Cues to Detect Mating Rivals. Curr Biol. 2011;21: 617–622. pmid:21439827
- 31. Fowler EK, Leigh S, Rostant WG, Thomas A, Bretman A, Chapman T. Memory of social experience affects female fecundity via perception of fly deposits. Bmc Biol. 2022;20: 244. pmid:36310170
- 32. Linklater JR, Wertheim B, Wigby S, Chapman T. EJACULATE DEPLETION PATTERNS EVOLVE IN RESPONSE TO EXPERIMENTAL MANIPULATION OF SEX RATIO IN DROSOPHILA MELANOGASTER. Evolution. 2007;61: 2027–2034. pmid:17683443
- 33. Xue L, Noll M. Drosophila female sexual behavior induced by sterile males showing copulation complementation. Proc National Acad Sci. 2000;97: 3272–3275. pmid:10725377
- 34. Crickmore MA, Vosshall LB. Opposing Dopaminergic and GABAergic Neurons Control the Duration and Persistence of Copulation in Drosophila. Cell. 2013;155: 881–893. pmid:24209625
- 35. Cook T, Pichaud F, Sonneville R, Papatsenko D, Desplan C. Distinction between Color Photoreceptor Cell Fates Is Controlled by Prospero in Drosophila. Dev Cell. 2003;4: 853–864. pmid:12791270
- 36. Benton R, Sachse S, Michnick SW, Vosshall LB. Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo. Plos Biol. 2006;4: e20. pmid:16402857
- 37. Rodrigues V, Sathe S, Pinto L, Balakrishnan R, Siddiqi O. Closely linked lesions in a region of the X chromosome affect central and peripheral steps in gustatory processing in Drosophila. Mol Gen Genetics Mgg. 1991;226–226: 265–276. pmid:2034219
- 38. Raad H, Ferveur J-F, Ledger N, Capovilla M, Robichon A. Functional Gustatory Role of Chemoreceptors in Drosophila Wings. Cell Reports. 2016;15: 1442–1454. pmid:27160896
- 39. Gong Z, Son W, Chung YD, Kim J, Shin DW, McClung CA, et al. Two Interdependent TRPV Channel Subunits, Inactive and Nanchung, Mediate Hearing in Drosophila. J Neurosci. 2004;24: 9059–9066. pmid:15483124
- 40. Kwon Y, Shen WL, Shim H-S, Montell C. Fine Thermotactic Discrimination between the Optimal and Slightly Cooler Temperatures via a TRPV Channel in Chordotonal Neurons. J Neurosci. 2010;30: 10465–10471. pmid:20685989
- 41. Agrawal S, Dickinson ES, Sustar A, Gurung P, Shepherd D, Truman JW, et al. Central processing of leg proprioception in Drosophila. Elife. 2020;9: e60299. pmid:33263281
- 42. Fan P, Manoli DS, Ahmed OM, Chen Y, Agarwal N, Kwong S, et al. Genetic and Neural Mechanisms that Inhibit Drosophila from Mating with Other Species. Cell. 2013;154: 89–102. pmid:23810192
- 43. Wood D, Ringo JM. Male Mating Discrimination in Drosophila melanogaster, D. simulans and Their Hybrids. Evolution. 1980;34: 320. pmid:28563423
- 44. Yang C, Rumpf S, Xiang Y, Gordon MD, Song W, Jan LY, et al. Control of the Postmating Behavioral Switch in Drosophila Females by Internal Sensory Neurons. Neuron. 2009;61: 519–526. pmid:19249273
- 45. Billeter J-C, Atallah J, Krupp JJ, Millar JG, Levine JD. Specialized cells tag sexual and species identity in Drosophila melanogaster. Nature. 2009;461: 987–991. pmid:19829381
- 46. Clough E, Jimenez E, Kim Y-A, Whitworth C, Neville MC, Hempel LU, et al. Sex- and Tissue-Specific Functions of Drosophila Doublesex Transcription Factor Target Genes. Dev Cell. 2014;31: 761–773. pmid:25535918
- 47. Stocker RF. The organization of the chemosensory system in Drosophila melanogaster: a rewiew. Cell Tissue Res. 1994;275: 3–26. pmid:8118845
- 48. Thistle R, Cameron P, Ghorayshi A, Dennison L, Scott K. Contact Chemoreceptors Mediate Male-Male Repulsion and Male-Female Attraction during Drosophila Courtship. Cell. 2012;149: 1140–1151. pmid:22632976
- 49. Wang Z, Singhvi A, Kong P, Scott K. Taste Representations in the Drosophila Brain. Cell. 2004;117: 981–991. pmid:15210117
- 50. Dweck HKM, Carlson JR. Molecular Logic and Evolution of Bitter Taste in Drosophila. Curr Biol. 2020;30: 17–30.e3. pmid:31839451
- 51. Ryner LC, Goodwin SF, Castrillon DH, Anand A, Villella A, Baker BS, et al. Control of Male Sexual Behavior and Sexual Orientation in Drosophila by the fruitless Gene. Cell. 1996;87: 1079–1089. pmid:8978612
- 52. Belote JM, Baker BS. Sexual behavior: its genetic control during development and adulthood in Drosophila melanogaster. Proc National Acad Sci. 1987;84: 8026–8030. pmid:3120181
- 53. Venken KJT, Simpson JH, Bellen HJ. Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit Fly. Neuron. 2011;72: 202–230. pmid:22017985
- 54. Scott K. Taste Recognition: Food for Thought. Neuron. 2005;48: 455–464. pmid:16269362
- 55. Fujii S, Yavuz A, Slone J, Jagge C, Song X, Amrein H. Drosophila Sugar Receptors in Sweet Taste Perception, Olfaction, and Internal Nutrient Sensing. Curr Biol. 2015;25: 621–627. pmid:25702577
- 56. Miyamoto T, Slone J, Song X, Amrein H. A Fructose Receptor Functions as a Nutrient Sensor in the Drosophila Brain. Cell. 2012;151: 1113–1125. pmid:23178127
- 57. Slone J, Daniels J, Amrein H. Sugar Receptors in Drosophila. Curr Biol. 2007;17: 1809–1816. pmid:17919910
- 58. Lee Y, Moon SJ, Montell C. Multiple gustatory receptors required for the caffeine response in Drosophila. Proc National Acad Sci. 2009;106: 4495–4500. pmid:19246397
- 59. Jiao Y, Moon SJ, Montell C. A Drosophila gustatory receptor required for the responses to sucrose, glucose, and maltose identified by mRNA tagging. Proc National Acad Sci. 2007;104: 14110–14115. pmid:17715294
- 60. Jiao Y, Moon SJ, Wang X, Ren Q, Montell C. Gr64f Is Required in Combination with Other Gustatory Receptors for Sugar Detection in Drosophila. Curr Biol. 2008;18: 1797–1801. pmid:19026541
- 61. Mucignat-Caretta C, Wyatt T. Neurobiology of Chemical Communication. Front Neurosci-switz. 2014;20140707: 1–22.
- 62. Kim M-S, Repp A, Smith DP. LUSH Odorant-Binding Protein Mediates Chemosensory Responses to Alcohols in Drosophila melanogaster. Genetics. 1998;150: 711–721. pmid:9755202
- 63. Benton R, Vannice KS, Vosshall LB. An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature. 2007;450: 289–293. pmid:17943085
- 64. Li Z, Ni JD, Huang J, Montell C. Requirement for Drosophila SNMP1 for Rapid Activation and Termination of Pheromone-Induced Activity. Plos Genet. 2014;10: e1004600. pmid:25255106
- 65. Liu T, Wang Y, Tian Y, Zhang J, Zhao J, Guo A. The receptor channel formed by ppk25, ppk29 and ppk23 can sense the Drosophila female pheromone 7,11-heptacosadiene. Genes Brain Behav. 2020;19. pmid:30345606
- 66. Pikielny CW. Sexy DEG/ENaC Channels Involved in Gustatory Detection of Fruit Fly Pheromones. Sci Signal. 2012;5: pe48–pe48. pmid:23131844
- 67. Cameron P, Hiroi M, Ngai J, Scott K. The molecular basis for water taste in Drosophila. Nature. 2010;465: 91–95. pmid:20364123
- 68. Nichols Z, Vogt RG. The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum. Insect Biochem Molec. 2007;38: 398–415. pmid:18342246
- 69. Ashourian KT. The Effects of SNMP-2 Gene expression on mating discrimination in male Drosophila melanogaster. 2014.
- 70. Vogt RG, Miller NE, Litvack R, Fandino RA, Sparks J, Staples J, et al. The insect SNMP gene family. Insect Biochem Molec. 2009;39: 448–456. pmid:19364529
- 71. Masuyama K, Zhang Y, Rao Y, Wang JW. Mapping Neural Circuits with Activity-Dependent Nuclear Import of a Transcription Factor. Journal of Neurogenetics. 2012;26: 89–102. pmid:22236090
- 72. Charnov EL. Optimal foraging, the marginal value theorem. Theor Popul Biol. 1976;9: 129–136. pmid:1273796
- 73. Parker GA, Simmons LW. Evolution of phenotypic optima and copula duration in dungflies. Nature. 1994;370: 53–56.
- 74. Alcock J. Postinsemination Associations Between Males and Females in Insects: The Mate-Guarding Hypothesis. Annu Rev Entomol. 1994;39: 1–21.
- 75. Hopkins BR, Sepil I, Thézénas M-L, Craig JF, Miller T, Charles PD, et al. Divergent allocation of sperm and the seminal proteome along a competition gradient in Drosophila melanogaster. P Natl Acad Sci Usa. 2019;116: 17925–17933. pmid:31431535
- 76. Lee SG, Kang C, Saad B, Nguyen K-NH, Guerra-Phalen A, Bui D, et al. Multisensory inputs control the regulation of time investment for mating by sexual experience in male Drosophila melanogaster. Biorxiv. 2022; 2022.09.23.509131.
- 77. Lu B, LaMora A, Sun Y, Welsh MJ, Ben-Shahar Y. ppk23-Dependent Chemosensory Functions Contribute to Courtship Behavior in Drosophila melanogaster. Plos Genet. 2012;8: e1002587. pmid:22438833
- 78. Toda H, Zhao X, Dickson BJ. The Drosophila Female Aphrodisiac Pheromone Activates ppk23+ Sensory Neurons to Elicit Male Courtship Behavior. Cell Reports. 2012;1: 599–607. pmid:22813735
- 79. Li H, Janssens J, Waegeneer MD, Kolluru SS, Davie K, Gardeux V, et al. Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly. Science. 2022;375: eabk2432. pmid:35239393
- 80. He Z, Luo Y, Shang X, Sun JS, Carlson JR. Chemosensory sensilla of the Drosophila wing express a candidate ionotropic pheromone receptor. Plos Biol. 2019;17: e2006619. pmid:31112532
- 81. Buhusi CV, Meck WH. What makes us tick? Functional and neural mechanisms of interval timing. Nat Rev Neurosci. 2005;6: 755–765. pmid:16163383
- 82. Merchant H, Harrington DL, Meck WH. Neural Basis of the Perception and Estimation of Time. Annu Rev Neurosci. 2012;36: 313–336. pmid:23725000
- 83. Allman MJ, Teki S, Griffiths TD, Meck WH. Properties of the Internal Clock: First- and Second-Order Principles of Subjective Time. Annu Rev Psychol. 2013;65: 743–771. pmid:24050187
- 84. Rammsayer TH, Troche SJ. Neurobiology of Interval Timing. Adv Exp Med Biol. 2014; 33–47.
- 85. Golombek DA, Bussi IL, Agostino PV. Minutes, days and years: molecular interactions among different scales of biological timing. Philosophical Transactions Royal Soc B Biological Sci. 2014;369: 20120465. pmid:24446499
- 86. Jazayeri M, Shadlen MN. A Neural Mechanism for Sensing and Reproducing a Time Interval. Curr Biol. 2015;25: 2599–2609. pmid:26455307
- 87. Larsson MC, Domingos AI, Jones WD, Chiappe ME, Amrein H, Vosshall LB. Or83b Encodes a Broadly Expressed Odorant Receptor Essential for Drosophila Olfaction. Neuron. 2004;43: 703–714. pmid:15339651
- 88. Rodrigues V, Siddiqi O. Genetic analysis of chemosensory pathway. Proc Indian Acad Sci—Sect B. 1978;87: 147–160.
- 89. Homyk T, Szidonya J, Suzuki DT. Behavioral mutants of Drosophila melanogaster. Mol Gen Genetics Mgg. 1980;177: 553–565. pmid:6770227
- 90. Yapici N, Kim Y-J, Ribeiro C, Dickson BJ. A receptor that mediates the post-mating switch in Drosophila reproductive behaviour. Nature. 2008;451: 33–37. pmid:18066048
- 91. Häsemeyer M, Yapici N, Heberlein U, Dickson BJ. Sensory Neurons in the Drosophila Genital Tract Regulate Female Reproductive Behavior. Neuron. 2009;61: 511–518. pmid:19249272
- 92. Wang L, Han X, Mehren J, Hiroi M, Billeter J-C, Miyamoto T, et al. Hierarchical chemosensory regulation of male-male social interactions in Drosophila. Nat Neurosci. 2011;14: 757–762. pmid:21516101
- 93. Yamamoto D, Fujitani K, Usui K, Ito H, Nakano Y. From behavior to development: genes for sexual behavior define the neuronal sexual switch in Drosophila. Mech Develop. 1998;73: 135–146. pmid:9622612
- 94. Mishra D, Miyamoto T, Rezenom YH, Broussard A, Yavuz A, Slone J, et al. The Molecular Basis of Sugar Sensing in Drosophila Larvae. Curr Biol. 2013;23: 1466–1471. pmid:23850280
- 95. Griffith LC, Ejima A. Courtship learning in Drosophila melanogaster: Diverse plasticity of a reproductive behavior. Learn Memory. 2009;16: 743–750. pmid:19926779
- 96. Mohammad F, Singh P, Sharma A. A Drosophila systems model of pentylenetetrazole induced locomotor plasticity responsive to antiepileptic drugs. Bmc Syst Biol. 2009;3: 11. pmid:19154620
- 97. Claridge-Chang A, Assam PN. Estimation statistics should replace significance testing. Nat Methods. 2016;13: 108–109. pmid:26820542
- 98. Ho J, Tumkaya T, Aryal S, Choi H, Claridge-Chang A. Moving beyond P values: data analysis with estimation graphics. Nat Methods. 2019;16: 565–566. pmid:31217592
- 99. Bernard C. Estimation Statistics, One Year Later. Eneuro. 2021;8: ENEURO.0091-21.2021. pmid:33795354
- 100. Satija R, Farrell JA, Gennert D, Schier AF, Regev A. Spatial reconstruction of single-cell gene expression data. Nat Biotechnol. 2015;33: 495–502. pmid:25867923