Copulation Song in Drosophila: Do Females Sing to Change Male Ejaculate Allocation and Incite Postcopulatory Mate Choice?

Drosophila males sing a courtship song to achieve copulations with females. Females were recently found to sing a distinct song during copulation, which depends on male seminal fluid transfer and delays female remating. Here, it is hypothesized that female copulation song is a signal directed at the copulating male and changes ejaculate allocation. This may alter female remating and sperm usage, and thereby affect postcopulatory mate choice. Mechanisms of how female copulation song is elicited, how males respond to copulation song, and how remating is modulated, are considered. The potential adaptive value of female signaling during copulation is discussed with reference to vertebrate copulation calls and their proposed function in eliciting mate guarding. Female copulation song may be widespread within the Drosophila genus. This newly discovered behavior opens many interesting avenues for future research, including investigation of how sexually dimorphic neuronal circuits mediate communication between nervous system and reproductive organs.


Introduction
The mating of flies might seem insignificant to an uninitiated eye, and hardly an expedient topic for scientific inquiry. The Danish expression "flueknepperi" (hairsplitting, nitpicking, a pejorative term for investigating and paying attention to minor details), literally "sexual intercourse of flies," poignantly expresses this preconception. Flueknepperi, however, has led to a long list of impactful studies in genetics, neuroscience, and evolutionary biology. In recent neuroscience research, sexual behavior in Drosophila helped uncover principles of sensory processing, [1][2][3] DOI: 10.1002/bies.202000109 motor patterning, [4,5] context-dependent action selection, [6,7] experience and memory, [8,9] and evolution of circuits in the nervous system. [10,11] Early descriptions of mating behavior within the Drosophila genus highlight that in many species, males vibrate their wings during courtship. [12,13] Wing vibrations were speculated to serve visual stimulation, to fan pheromones toward the female or to produce sounds. Such sounds or "love songs" were first recorded and shown to function as auditory stimuli in the 1960s. [14,15] Since then, a fascinating variety of over hundred species specific male precopulatory courtship songs has been reported. [16][17][18] In species of the Drosophila virilis group, females respond to male songs with their own precopulatory songs, which stimulate the male and lead to duetting behavior. [19][20][21][22] Precopulatory courtship songs serve both sex and species recognition and increase the likelihood of copulation. Once copulation is initiated, these aims are usually achieved and indeed males of most species stop singing. An exception is the Drosophila montium subgroup, where male copulatory courtship very widespread and many species only sing in copula. [17,23] Here, females prematurely end copulations with males that do not sing or sing a heterospecific copulation song. [24][25][26] More enigmatic and so far less investigated is female copulation song. Over 30 years ago, female D. sechellia and D. orena were found to produce sound bursts during copulation. The function of these songs was not apparent. [27] We revived this finding by a recent study documenting female copulation song not only in D. sechellia, but also in D. melanogaster and the two sibling species, D. simulans and D. mauritiana. [28] Exploring female copulation song in D. melanogaster allowed us to use the genetic toolbox of this widely studied model species and to gain more mechanistic and functional insight into the behavior. Females sing in copula by vibrating both wings at characteristic frequency, generating high pitched pulses that are distinct from male courtship song (Figure 1a). Song pulses tend to come in trains and occur throughout the 15-20 min D. melanogaster stays in copula. While male precopulatory courtship has little or no influence on how much females sing in copula, the composition of ejaculate strongly modulates female copulation song. [28] To shed more light on the function of female copulation song, Figure 1. Female copulation song delays remating and could be a signal in mate choice. a) Oscillograms (3 s) of male precopulatory courtship song and female copulation song in D. melanogaster. b) Copulation song delays female remating. Copulation song or silence is played back to a copulation with a mute female, and the female is thereafter allowed to remate with subsequent males. Latencies to remating for females from copulations receiving song playback (red triangles) or silence (blue circles). Each data point represents one female, error bars indicate median and interquartile range, **p = 0.0035, Mann-Whitney test (two-sided). Graph reproduced with permission. [28] Copyright 2010, Elsevier. c) Schematic illustrating the hypothesis that female copulation song is a signal, with the copulating male as receiver responding with changes in seminal fluid allocation. song was played back to mating pairs with muted females. Copulations that received playback left females more reluctant to remate with subsequent males than silent control copulations (Figure 1b). Since a delay in remating can increase the paternity share of the first male, female copulation song was proposed to have an effect on postcopulatory mate choice. [28] Here, we elaborate on tentative mechanisms and functions of female copulation song in Drosophila. In particular, we want to scrutinize the hypothesis that female copulation song acts as signal in postcopulatory mate choice (Figure 1c). Animal signals have been defined as specialized behaviors or traits of a sender (or signaler, transmitter) that influence the behavior of a receiver in such a way that the sender benefits. Both signaling and response to the signal have evolved for this function. In contrast to signals, cues are inadvertently released by the sender and their reception by an eavesdropping receiver might not necessarily be beneficial. Signaling behavior is often elicited by releasers such as visual, mechanical or chemical (pheromonal) stimuli. [29,30] When studying a potential signal, releasing factor(s) and sender, receiver, and response to the signal, as well as the adaptive value of the communication should be identified.
We first discuss if male ejaculate acts as releaser for female copulation song. Assuming copulation song is a signal sent by the female, we consider potential receivers, notably, the male copulation partner, and his response. Next, we speculate how copulation song can benefit the copulating female by serving as a mechanism to change the amount or composition of ejaculate she receives from the male. Changed ejaculate receipt could Female copulation song is modulated by ejaculate composition. a) Schematics of male and female reproductive organs of D. melanogaster and signaling routes for ejaculate components to the female central nervous system. The male accessory gland mainly consists of main cells, with around 43 secondary cells located at the distal tips of the lobes. Left part of the figure (male) reproduced with permission. [28] Copyright 2010, Elsevier. b) Amount of female song elicited in copulations of wild type females with wild type males (gray) males with no or altered seminal fluid (cyan, males depleted of ejaculate by multiple recent matings and iab-6 cocu(D5) mutant males with impaired secondary cells of the accessory gland) and with males without sperm (orange, Son of Tudor mutants and X0 chromosome aberrancy). Each data point represents one female, error bars indicate median and interquartile range, *p = 0.01, **p = 0.006. ****p < 0.0001, Kruskal-Wallis test with Dunn's multiple comparison (two-sided). Data replotted from Figure 3a,c in ref. [28]. c) Confocal image of the distal part of a male accessory gland with secondary cells (Gfp) in green, actin (Phalloidin stain) in red, and DNA (Dapi stain) in blue. Gfp is expressed under the control of the secondary cell specific iab6(D1)-GAL4 driver described by Gligorov et al. [39] d) Combined transmitted light and fluorescence image of bursa with sperm storage organs (ST, spermatheca, SR seminal receptacle) from a recently mated female. The mating plug (autofluorescence) is in blue, transferred ejaculate (containing Gfp-tagged Ovulin from an Acp26AaGFP male [33] ) is marked in green. Scale bars in (c) and (d) are 100 µm. explain the delay in female remating and the increased use of sperm from the first male, which is observed after female song playback. [28] Both female influence on male ejaculate allocation by signaling in copula and male modulation of ejaculate in response to this signaling may represent a form of postcopulatory mate choice. Here, we briefly compare Drosophila copulation song to female copulatory vocalizations in vertebrates regarding their proposed adaptive value. In the end of this article, we add some short evolutionary considerations.

Do Females Sense Seminal Fluid Transfer in Copula and Respond with Copulation Song?
Like most animals with internal fertilization, copulating male Drosophila transfer sperm together with a complex mixture of seminal fluid, containing hundreds of proteins, peptides and other bioactive molecules. Seminal fluid is produced in the male accessory gland, the ejaculatory bulb, and secretory tissues of the reproductive tract [31,32] (Figure 2a). During the 15-20 min of a D. melanogaster copulation, sperm is released in a relatively short bout, around 7-8 min after intromission. By contrast, seminal fluid is already transferred before sperm and detectable in the female 1-3 min after the beginning of copulation. [33][34][35] Females receive ejaculate in their uterus (also called bursa) and store sperm in specialized organs-the seminal receptacle and the paired spermatheca. [36] Some components of the seminal fluid, such as the accessory gland protein ovulin, pass the vaginal wall and rapidly enter the female circulatory system after transfer [33,37,38] (Figure 2a).
Experiments indicate that female copulation song critically depends on ejaculate. Females mating with males that are depleted of seminal fluid sing very little. Female song is also almost completely abolished in copulations with iab6 cocu mutant males. iab6 cocu mutants have dysfunctional secondary cells (specialized secretory cells in the accessory gland) and a defective seminal fluid composition, with abnormal abundance and glycosylation of several accessory gland proteins. [39,40] Males that lack sperm elicit unusual high levels of female singing (Figure 2b,c). [28] The reason for that is unclear. It should be tested if spermless males transfer more seminal fluid or seminal fluid molecules in a more accessible form (unbound to sperm). Alternatively, females might directly sense sperm and suppress singing in response. In summary, these observations suggest that females sense ejaculate while it is transferred and react to it with more or less intense copulation song.
Notably, the male initiates precopulatory singing when he touches the female abdomen during courtship and samples gustatory female pheromones. These pheromones thus act as releasers for male courtship song, i.e., external stimuli that trigger the behavior with high probability in a specific context. [1,41,42] Could molecules in the male seminal fluid act in a similar way as releasing stimuli for female singing, binding to specific receptors, activating sensory neurons in the female reproductive tract and eliciting an immediate female song motor response?
Seminal fluid modulates female physiology and behavior in many animal species, critically impacting fertility. [43][44][45] In Drosophila, the effect of a few of the hundreds of seminal fluid proteins on the female reproductive organs and nervous system are especially well studied. [32] Sex peptide, a product of the accessory gland main cells, binds to sensory neurons in the uterus, which relay information to the brain, mediating behavioral changes after mating including reduced receptivity and increased egg laying. [46][47][48][49][50] Ovulin, another accessory gland protein, mediates the release of eggs by the ovary and egg laying [51,52] by acting on neurons expressing the neuromodulator octopamine. [53] These prominent examples of seminal fluid molecules acting on the female nervous system make it tempting to speculate about a "song releasing seminal fluid factor," which acts as a pheromone, i.e., a secreted chemical factor that affects the behavior of conspecifics. Since impairment of the secondary cells of the male accessory gland abolishes female song, [28] secondary cell products are good candidates. Of note, recent work established that besides producing and processing specific seminal fluid components, [39,40] secondary cells also alter the metabolism and secretion of the main gland cell type and have a critical role in determining the overall composition of seminal fluid. [54] The female internal reproductive organs (Figure 2a,d) are innervated by efferent sensory, neuromodulatory, and motor neurons from the ventral nerve cord (the insect's equivalent of a spinal cord) as well as local afferent sensory neurons. [48,49,55,56] Gustatory receptors are expressed there and on the genitalia. [57,58] Since copulation song starts immediately after intromission, [28] sensing of a song releasing factor through local neurons in the reproductive tract might be more likely than an effect via molecules that have entered the circulatory system and thus gained access to the central nervous system. An alternative explanation is that mechanosensation of intromission and seminal fluid transfer elicits female copulation song. Even in the absence of seminal fluid, females detect copulation via peripheral neurons expressing the mechanoreceptor Piezo and connected ascending neurons in the central nervous system, which mediate a short-term reduction in receptivity. [59] Silencing this pathway does not affect female copulation song, [28] but other proprioceptive channels could still play a role. Chemo-and mechanosensation not necessarily exclude each other, as integration on the level of the circuit, the sensory neuron, and even the receptor has been demonstrated. [60] In summary, the molecules, receptors, and neuronal circuits that convey information from the female reproductive tract to the central nervous system and trigger female copulation have not yet identified. We propose that seminal fluid molecules could act as pheromones and release female singing when they are sensed by neurons in the female reproductive tract. With regard to the releasing stimulus, the control of female copulation singing could be thus analogous to the control of male courtship singing, which is initiated when the male tastes female contact pheromones. [1,41,42]

Do Males Respond to Copulation Song of Their Mates with Changes in Ejaculate Allocation?
Signals are directed at receivers who respond with specific behavior. If female copulation song is a signal, who is the receiver of the signal? Copulation song could be detected by the male mating partner or nearby "bystander" flies. When copulation song was played back to a copulating pair with a mute female, the female subsequently remated later than after a control copulation without playback. [28] Since no "bystander" flies were present in this assay, we first explore the hypothesis that the male mating partner is the receiver. We suggest that effect of female song on female remating behavior is mediated via a response of the mating male to copulation song.

Males Can Strategically Adjust Ejaculate Transfer in Response to Various Stimuli: Is Female Song One of Them?
If the copulating male is the receiver of the signal, how does he respond? Once in copula, males could modulate the duration of copulation and/or the allocation of sperm and seminal fluid. Experiments show that copulation song does not affect copulation duration. [28] Could song influence ejaculate allocation? In many animals, males practise so-called "ejaculate economics" and "strategic/plastic ejaculate allocation": they modulate the quantity or quality of the ejaculate they transfer depending on female attractiveness, their own status and/or the presence of rival males [31,[61][62][63][64][65][66][67][68][69][70][71][72] (Table 1). These strategies are thought to arise when sperm competition takes place, i.e., when females mate with more than one male in a reproductive cycle and ejaculates from different males compete in the female reproductive tract for fertilization of ova. [73,74] In such a situation, males can become depleted of ejaculate and/or have to spend considerable investments in ejaculate production, making them choosy and prudent about how to spend it on available mates. [61,75] For most documented cases of male plastic ejaculate allocation, the exact stimuli eliciting differential allocation as well as the physiological mechanism effecting differences in transfer are not entirely understood. Even when a characteristic of sperm, such as viability or motility, is modulated, the effect is often due to changes in seminal fluid composition. [76][77][78] In D. melanogaster, various forms of male strategic ejaculate allocation are described [79][80][81][82][83] ( Table 1). Both prolonged precopulatory exposure to competitive social environments and "ad hoc" stimuli (only present shortly before or during copulation) from rival males or the female partner have been shown to influence quantity of sperm and/or seminal fluid composition, demonstrating that Drosophila males have sophisticated control over ejaculate transfer.

Strategic Allocation of Factors Delaying Remating: What Are the Candidates?
So far, the indication that copulation song impacts male strategic ejaculate allocation is only indirect, suggested by the delay in  [31,62], and [63].

Species
Strategy Time scale, eliciting stimulus, and potential mechanism Meadow voles More sperm transferred per copulation in the presence of odors from other males [64] Ad hoc modulation of sperm quantity based on olfactory stimuli. Suggested mechanism: increased contractibility of cauda epididymis and/or vas deferens Horse Larger ejaculates and more motile and viable sperm transferred in response to oestrous than to diestrous females [65] Ad hoc modulation of ejaculate quality, supposedly mediated by changes in seminal fluid quantity and/or quality, unknown mechanism Human Ejaculation of a) higher percentage of motile sperm after viewing sperm competition images compared to non-sperm competition images [66] b) larger volume and more motile sperm after viewing images of novel females [67] Ad hoc modulation of sperm quality based on visual stimuli, unknown mechanism Fowl More sperm transferred per copulation a) in the presence of other males b) to new copulation partners c) to females with large combs [68] Ad hoc modulation of sperm quantity, supposedly based on visual stimuli, unknown mechanism More motile sperm transferred to females with large combs [76] Ad hoc modulation of sperm quality, supposedly based on visual stimuli, effect depends on seminal fluid. Suggested mechanism: modulation of ejaculatory duct contractions (bilateral vs unilateral duct usage) Chinook salmon Subdominate males spawn ejaculate with more motile sperm [78] Modulation within 48 h of change of social status. Effect depends on seminal fluid, supposedly changes in seminal fluid protein production/composition

Nematode (Rhabditis regina)
Transfer of a larger mating plug in the presence of rival males [69] Modulation within hours, unknown mechanism Broad-horned flour beetle More sperm transferred to females which are perfumed with male contact pheromones [70] Ad hoc modulation of sperm quantity based on chemical stimuli, unknown mechanism.

Australian field cricket
Viability of transferred sperm depends on female mating status, being highest for single mated and lower for virgin or multiple mated females [71] Modulation within hours, supposedly based on chemical stimuli, unknown mechanism More viable sperm transferred after rearing in presence of courtship song from other males [ 77,72] Long term modulation of sperm viability based on acoustic stimuli, mediated by increased expression of seminal fluid protein genes Fruit fly (Drosophila melanogaster) More key seminal fluid proteins (sex peptide and ovulin) transferred after precopulatory exposure to rival males [79] Modulation within days, modulation of seminal fluid protein quantity might depend on increased mating duration.
More sperm and differentially composed seminal fluid transferred after precopulatory exposure to rival males [80] Modulation within days, depends on complex changes in seminal fluid protein production and transfer patterns.
More sperm transferred when male rivals are introduced during copulation [81] Ad hoc modulation of sperm quantity, based on acoustic and chemical stimuli, unknown mechanism More sperm transferred to virgin versus mated, young versus old and large versus small females [82] Ad hoc modulation of sperm quantity, virgin and young female might be recognized based on chemical stimuli, large females based on visual stimuli, unknown mechanism Modulation of transferred seminal fluid proteins dependent on female mating status: ratio of fecundity stimulating ovulin: receptivity inhibiting sex peptide higher in copulations with virgin females [83] Ad hoc modulation of seminal fluid composition, unknown mechanism remating that it elicits when played back to a mating couple [28] ( Figure 1b). In many species, female remating probability is strongly influenced by seminal fluid components, which reduce either her receptivity or her attractiveness to subsequent males. [31,32,84] In D. melanogaster, various proteins and peptides act on different time scales in this type of "chemical mate guarding." Best studied among them are sex peptide with its interacting network and constituents forming the mating plug. We reason that mating males could increase the remating interval of their mates if they for example chose to transfer a greater amount of sex peptide or a greater amount of substances that affect the retention period of the mating plug in the female genital tract. Drosophila sex peptide is a product of the male accessory gland. It is transferred to the female as a part of seminal fluid and mediates a long-term reduction of female receptivity after mating. [46,85] Sex peptide binds to sperm, from where it is slowly released over days, thus serving as readout for presence of sperm in the storage organs. [86] Interaction of sex peptide with sperm is mediated via several other seminal fluid proteins, [87] which influence the action of sex peptide. Sex peptide plays a prominent role in how the female changes behavior after mating, but even in the absence of sex peptide, female receptivity is strongly reduced in the first few hours after mating. [46] This effect is at least in part mediated by the mating plug. Mating plugs (sometimes also called copulatory plugs) are structures at the entry site of female reproductive organs that form by male derived material (Figure 2d). They occur in many animal species from insects to mammals and have been compared to biological "chastity belts". [73] Although they differ between species, mating plugs may share common functions such as temporary reduction of remating and promotion of sperm storage. [88][89][90] Manipulation of the Drosophila mating plug protein composition increases remating during the first 4 h following a copulation. [91] Females eject the plug together with unstored sperm and large amounts of the male pheromone cisvaccenyl acetate (cVA) within 2-3 h after mating. [92][93][94] Ejection is thought to increase remating, because it removes both a physical barrier and the antiaphrodisiac action of cVA on courting males. The timing of female ejection behavior is modulated by a brain neuronal circuit [93] that might receive input from stimuli increasing early female remating such as food. [95] So far, it remains to be tested if the composition of the mating plug itself and/or the simultaneously transferred ejaculate influence ejection timing. [96] The mechanism that delays remating after female copulation song exposure is still unknown. Since the effect is seen within hours after copulation, [28] it is attractive to hypothesize that it depends on the timing of mating plug ejection, which could be in turn altered by strategic ejaculate allocation. Alternatively, the male might differentially transfer sex peptide or any other, mating plug independent component of the extensive "transferome," [97,98] which is functionally largely uncharacterized and likely to contain unexplored factors influencing female remating.
In summary, we propose the following model for how female song affects female remating: the mating male hears female copulation song and depending on song amount and/or intensity changes his seminal fluid allocation. These changes in allocation include factors that delay female remating (Figure 2d). Candidates for such "remating delay seminal fluid factors" include sex peptide and components that affect mating plug retention. Of note, the "song releasing seminal fluid factors" discussed in Section 2 are not necessarily identical with the "remating delay seminal fluid factors." Sex peptide, for example, has no effect on the amount of female copulation song. [28] This does not exclude the possibility that differential allocation of sex peptide is part of the male response to female song. A previous study has indeed shown that male can adjust the amount of sex peptide they transfer in a copulation depending on female quality. [83] Although it is a parsimonious hypothesis that the mating male is the receiver of female copulation song, the possibility that female song affects "bystander" flies should be explored in future experiments. Specifically, it could be tested if female copulation song increases courtship of noncopulating males or receptivity of females. Conceptually, however, there seems no immediate explanation at hand why females should profit from broadcasting the fact that they are mating to other, noninvolved flies. In contrast, signaling to the mating partner could directly benefit the female-how and why will be discussed in the following section.

What Is the Adaptive Value of Signaling during Copulation? Can It Result in Postcopulatory Mate Choice?
Stimuli that transmit information are considered to represent signals rather than mere cues if they benefit the sender. [29,30] How could females gain advantage from signaling in copulation? Can signaling in copulation influence reproductive success of females and result in a form of postcopulatory mate choice?

Why Do Females Signal in Copula? Examples from Other Species
While male copulation song that occurs in some Drosophila species has been studied and found to be important to maintain genital coupling, [23][24][25][26] we are not aware of any functional explanation for female copulation song in Drosophilids. Female copulation song does not affect the length of copulation or occurrence of insemination. [28] What is known about female copulation signals in other species? There are reports of possible female signaling in copula in insects and spiders, [99,100] but little is known about the adaptive value of such behaviors. The copulation stridulation of female short-bodied cellar spiders (Physocylus globulus) induces males to loosen squeezing with their genitalia, which increases their paternity. [101] A similar interaction seems to take place in tsetse flies (Glossina pallidipes), where female wing vibration in copula is correlated with male genitalic squeezing. [102] Female copulatory calls or vocalizations of some vertebrate species, especially primates, have received comparatively more attention [103][104][105][106][107][108][109][110][111][112] (for details, examples and references, see Table 2). It has been hypothesized that they are directed at bystander males to incite male-male competition, and thus give the signaling female access to higher ranking or more diverse males. When directed at the copulation partner, the calls are thought to influence ejaculate transfer or to encourage postcopulatory mate guarding. In this case, they could benefit the female by making copulations more effective and coordinated, facilitating insemination or fertilization. The selective solicitation of mate guarding, on the other hand, would give females the possibility for postcopulatory mate choice. [113] According to the last hypothesis, females preferentially call in copulations with dominant, high quality partners. The calls induce the partner to stay in close proximity after the end of copulation where he prevents matings with subsequent males (physical mate guarding), thus ensuring the success of his sperm.
Is it possible that Drosophila female copulation song functions in an analogous way, encouraging mate guarding not in a physical, but rather a chemical form via the increased allocation of seminal fluid products (including mating plug components) that reduce female remating propensity (Figure 1c)?

Females Benefit from Control over Remating
Clearly, it is in the interest of males to prevent females from remating. However, it might be costly for a male to do so, and therefore adaptive to assess female quality for a more strategic allocation instead of always investing the maximum of his  [ 104] Japanese macaque, [ 105] Barbary macaque, [ 106] Domestic fowl [107] Female gets access to more males at shorter interval: increased sperm competition Chacma baboon, [ 108] Barbary macaque [ 106] Paternity uncertainty of males: increase in infant care and decrease in aggression toward infants Chacma baboon [ 108] Mating male Coordinate/incite ejaculate transfer Facilitation of fertilization Humans, [ 109,110] Barabary macaque [ 111] Selectively encourage mate guarding Female postcopulatory choice of high quality male Guinea Baboon [ 112] chemical mate guarding arsenal. [61,75] For females, benefits versus costs of remating (polyandry) are more complex. [114][115][116] Remating protects against insufficient supply of sperm and seminal fluid, and generates genetically diverse, higher quality offspring.
On the other hand, it can be costly to females, since it takes time and exposes them to the risk of predation, infection, [117] injury, [118] and ejaculate components that reduce longevity. [119] In D. melanogaster, the timing between matings and between mating and mating plug ejection determines the relative usage of sperm from two different males. [92,120] For a female, indirect control over her remating rate by titrating male ejaculate allocation would thus be adaptive, since it could balance costs and benefits of remating and serve as a mechanism of mate choice.

Females Remating Rate as a Mechanism of Female Postcopulatory Mate Choice
Mate choice is occurring when members of one sex nonrandomly select members of the other sex for offspring production based on phenotypic traits, or nonrandomly invest resources in specific mates. Mate choice can lead to differences in reproductive success between individuals and is a mechanism of sexual selection with an important impact on evolution of physiology and behavior of animals. [121] Mate choice and sexual selection can continue to some extent after two individuals have committed to copulation. [32,[73][74][75]121] As discussed in Section 3, males may strategically allocate ejaculate to females depending on their attractiveness and thereby increase the reproductive success of specific partners. Females may exert postcopulatory choice by using sperm received from certain males more efficiently than sperm from others. [32,122] Female remating rate can be considered as a mechanism of postcopulatory choice: by deciding when and how often she mates with subsequent mates, the female can expose the sperm of the first male to different levels of sperm competition and impact his paternity share of her clutch. [92,120]

Strategic Ejaculate Allocation as a Mechanism of Male Mate Choice
In this context, it is important to consider the signal content of female copulation song: which information does the male potentially extract from the song of his partner (Figure 1c)? Is it pos-sible that males exert postcopulatory mate choice based on female copulation song and allocate costly ejaculate components preferentially to females with specific song characteristics? Since structured wing vibrations rely on the motor neurons powering flight, [28] female song pulses might display flight ability, neuromuscular coordination, and female fitness. They further could be a readout of female sensitivity to seminal fluid and receptivity. Together, these parameters could represent female quality and the value of additional ejaculate investment for the male.

Female Song as a Mechanism to Negotiate Optimal Ejaculate Transfer?
Alternatively, the amount of female copulation song might just reflect the relative composition of ejaculate, for example, the ratio between different seminal fluid proteins or seminal fluid and sperm. Lack of song or highly elevated song (as observed in copulations with spermless males) would thus inform the male about an insemination that is suboptimal for both partners. Continuous feedback about received ejaculate could help the male to fine tune and adjust transfer to individual females in a cooperation between the sexes. Female song might thus not have directly evolved to delay remating, but rather as a female mechanism to influence ejaculate allocation and receive a "perfect mix" to her own taste. One (but not necessarily the most important) aspect of this "perfect mix" could be an optimal remating interval, others optimal stimulation of sperm storage, ovulation and immunoresponse. Notably, it has been suggested before that females evolved mechanisms controlling exposure to male ejaculate and the impact of seminal fluid on their postmating physiology. [63,96] Female copulation song might be such a mechanism-an attempt of the female to change the ejaculate she receives by signaling during the transfer process, and an evolutionary response to male strategic ejaculate allocation.

How Conserved Is Drosophila Female Copulation Song?
In the recent study, female copulation song was also observed in three different species of the D. simulans clade, [28] which diverged from D. melanogaster around 3 million years ago [123] (Figure 3a). The female songs of D. melanogaster, D. simulans, and D. mauritiana are very similar in their acoustic parameters. In D. sechellia, Figure 3. Female copulation song is conserved within the Drosophila melanogaster subgroup. a) Phylogenetic relationship of D. melanogaster and its closest sibling species, tree drawn after. [123] Photographs of D. melanogaster (mel) and D. sechellia (sec) females. b) Exemplary copulation song pulses of D. melanogaster and D. sechellia with spectrograms, highlighting the differences in song patterning. The oscillogram and spectrogram for D. sechellia is adapted with permission. [28] Copyright 2010, Elsevier.
an endemic island species from the Seychelles, female song has a lower pulse fundamental frequency, longer and more variable pulses and shorter inter pulse intervals compared to the other species [28] (Figure 3b). It would be very interesting to compare the functional relevance of female song and the genes and neuronal circuits for song production in D. melanogaster and D. sechellia, an endeavour that is facilitated by the rapid process in establishing D. sechellia as a genetically accessible model species. [124] Further, the occurrence of female song in evolutionarily more distant species of the Drosophila genus remains unexplored. In D. melanogaster, a wing control muscle that is functionally relevant for male courtship song shows male specific enlargement. [125] A comprehensive analysis of 18 species revealed that dimorphic, male enlarged song control muscles are widespread. Interestingly, there are also three instances of female enlarged muscles, namely, in D. erecta, D. ananassae, and D. prostipennis. [126] Could enlargement of key wing control muscles in these species be linked with prevalence or special attributes of female song? D. sechellia was not included in the recent study of muscle evolution, so it is unknown if there is an anatomical correlate to its prominent female song.
If females sing in a wide range of species during copulation, it could be also of interest to study potential correlation between female song and the evolution of copulation duration, [18,127] female remating, [114] seminal fluid proteins, [128] and sperm storage organs. [36] As mentioned in the introduction, male courtship song shows large interspecific variation in patterning and the occurrence of different song types. [16,17] Male and female song might share in part the genetic and neuronal circuit basis for song generation. [28] So far, the study of male song neurons and circuits has mainly focused on explaining why males sing a courtship song and females do not (or only after artificial activation of circuit elements considered as "rudimentary" in females). [41,42,[129][130][131][132][133] In future studies of the neuronal circuits for action selection and patterning of song, consideration of a distinct female song could change the functional interpretation of sexual dimorphisms.
Can we speculate about a functional connection between male and female song? Males might have evolved plastic seminal fluid allocation strategies in response to cues from rival males, including auditory cues such as male courtship song. It is not known if playback of male courtship song from a rival during copulation has a similar effect on subsequent female remating as playback female copulation song. If so, female song might have arisen from females acoustically mimicking the presence of other males. Such an idea, however, does not account for the distinct acoustic properties of male and female song. Male, but not female song playback enhances female receptivity (Kerwin and von Philipsborn, unpublished), indicating that at least females are very well able to distinguish between the two song types.
Summing up, the phylogenetic distribution and diversity of female copulation song within the genus Drosophila remains to be studied, as well as the relationship of female song to mating traits and male precopulatory song within a species.

Conclusions and Outlook
Female Drosophila copulation song is still very little understood. To answer the question if female song acts as a signal and is implicated in postcopulatory mate choice, it is imperative to experimentally address the different aspects of the hypothetical signal. While it seems parsimonious that the copulating male is the receiver of female song, a possible effect on bystander flies should be more rigorously tested.
When we understand better which stimuli trigger female singing and how female song changes depending on male and female condition and social context, the hypotheses about signal content can be revised. The potential male response, i.e., changes in ejaculate transfer, should be directly tested by quantitative proteomics or ELISA assays of candidates. Experiments testing which mechanisms and molecules (mating plug ejection timing, differential transfer of sex peptide) mediate delayed remating will give further insight into the male response. Female copulation song is an excellent new model system for investigating the signaling between nervous system and reproductive organs in the two sexes, as well as the communication between the two sexes during mating. The complexity of insect mating systems [73,134,135] has since long inspired studies in behavioral ecology, evolution of behavior and behavioral neuroscience. With the full connectome of D. melanogaster within reach, [136,137] it is gratifying to realize that this well-studied model organism can still surprise with overlooked behaviors whose underlying neuronal substrates and circuit mechanisms await discovery.