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Sperm and sex peptide stimulate aggression in female Drosophila

Introductory paragraph Female aggression towards other females is associated with reproduction in many taxa, and traditionally thought to be related to the protection or provisioning of offspring, such as through increased resource acquisition. However, the underlying reproductive factors causing aggressive behaviour in females remain unknown. Here we show that female aggression in the fruit fly Drosophila melanogaster is strongly stimulated by the receipt of sperm at mating, and in part by an associated seminal fluid protein, the sex peptide. We further show that the post-mating increase in female aggression is decoupled from the costs of egg production and from post-mating decreases in sexual receptivity. Our results suggest that male ejaculates can have a surprisingly direct influence on aggression in recipient females. Male ejaculate traits thus influence the female social competitive environment with potentially far-reaching ecological and evolutionary consequences.


Introduction
Female aggression directed towards other females (hereafter "female aggression") can have important fitness consequences for females and their offspring1-4. In both vertebrate and invertebrate taxa, females act aggressively to acquire resources or mates, defend territories, and protect and provision eggs or offspring5-10. Females are expected to be more flexible than males in their use of aggression, with aggression heightened at particular stages of reproduction or life-history11, and aggression is generally associated with maximising the production or survival of their offspring10. One observed pattern is an elevation of female aggression after mating, whether it be during offspring development6 or after egg production12-14. These increases in aggression typically occur in parallel with other dramatic changes in female behaviour and physiology, including alterations in sexual receptivity, feeding rates, and hormone levels4, 15-17. In many species, male ejaculate components can induce striking changes in female postmating behaviour and physiology. For example, in insects such as the fruit fly, Drosophila melanogaster, male ejaculate components increase ovulation and egg laying, alter immunity, and decrease female receptivity to remating15,18. Thus, ejaculate components are potential candidates for stimulating post-mating increases in female aggression. Male ejaculates could act either as a cue for females to optimally modify their behaviour, indicating upcoming physiological changes that require females to increase their levels of aggression, or could alter female behaviour for the male's benefit, which could either be in the interests of females, or represent a source of conflict between the sexes19.
There are two main pathways through which male ejaculates could stimulate post-mating aggression in females. First, by stimulating increased egg production20, ejaculates increase the demand for resources which could in turn lead to aggressive behaviour (Figure 1.a). Females lay eggs at high rates after mating and require more, or different, nutrients relative to virgin females16,17,21-23, potentially increasing the motivation for females to compete aggressively over food. Another possibility is a more direct stimulation of female aggression by males during mating, with egg production as a separate, but simultaneous, pathway ( Figure 1.b). Disentangling these alternatives is required to understand the physiological and molecular pathways that coordinate female aggression with reproduction, and thus unravelling the processes that drive the evolution of female aggressive behaviour.
In this study, we tested whether mating induces changes in female aggression in D. melanogaster. In the wild, this species aggregates on rotting fruit24,25, and lab studies have shown that females will engage in fights when food and oviposition sites are limited26,27. Differential reproductive success may therefore result from variation in female ability to aggressively acquire and defend food and oviposition sites. Males and females both display aggressive behaviours in D. melanogaster in the laboratory, although there are significant sex differences in the duration of encounters and the types of behaviour observed27,28. There are also differences in aggressive behaviour between mated and virgin females, with mated females fighting for longer and potentially more likely to escalate to higher levels of intensity27. In addition, female D. melanogaster display other dramatic post-mating changes and reductions in sleep15. These identified post-mating responses are mediated by male seminal fluid proteins, some of which associate with sperm, such as the 'sex peptide '29,30, suggesting that female aggression could also be influenced by male ejaculate components.
We hypothesize that mating may stimulate aggression in females via two potential pathways, either indirectly through egg production or directly through ejaculate contents ( Figure 1). We tested for these pathways by assessing the levels of aggression in females with genetically reduced egg-laying or blocked vitellogenesis, and in females mated to males lacking specific ejaculate components, such as sperm and sex peptide.

Methods
We conducted five experiments to test our main hypotheses about the causes of female aggression:

1.
Effects of mating: we measured aggression in pairs of mated females, virgin females, and mated vs. virgin females to test for effects of mating on aggression.

2.
Effects of egg production: to examine whether any increase in female aggression after mating is mediated by the demands and costs of egg-production20,31, we used the ovo D1 mutation, which blocks oogenesis prior to vitellogenesis32. The presence of the ovo D1 mutation in females abolishes the mortality costs associated with egg production: if these same costs drive female aggression, any differences in post-mating aggression between mated and virgin females should be abolished in ovo D1 flies.

3.
Effects of sperm: to test whether sperm are necessary to stimulate female postmating aggression, we measured aggression in females mated to spermless sonof-Tudor and control males33.

4.
Effects of the male seminal fluid protein, 'sex peptide', and the female 'sexpeptide-receptor': to determine whether the seminal fluid protein 'sex peptide' (SP) is involved in stimulating increased aggression after mating, we compared aggression in females mated to SP-null males with that of control-mated and virgin females. We also used females that lacked the receptor to SP ('sex peptide receptor', SPR) to examine the downstream pathway through which SP could potentially stimulate female aggression.

Fly stocks and culture
All flies were kept and experiments conducted at 25°C on a 12:12 light: dark cycle. We used the Dahomey genetic background as our stock population, and genetic mutations were backcrossed into this background for >5 generations where appropriate.
Production of wild-type females-Wild-type Dahomey females34 were reared in bottles at standardized larval density35. Virgins were collected within 8hrs of eclosion, and sexes were housed separately (females individually) in vials containing standard fly food media36, with no live yeast. For the experiments using mated vs. virgin flies, ovo D1 , and SPR knockout females, Dahomey males were used as mates.
Production of sterile (ovo D1 ) females-To produce sterile ovo D1 females, we crossed males carrying the dominant ovo D1 mutation to Dahomey virgin females37. Sterile ovo D1 females have ovaries that degenerate prior to S5, meaning that vitellogenesis is blocked and females cannot produce eggs32. Dahomey females, from the stock into which the ovo D1 flies were crossed, were used as controls. We tested the efficacy of the mutation by counting offspring collected from overnight vials following mating. No ovo D1 females produced offspring, confirming their complete sterility. Previous research has found mixed evidence for the effects of the ovo D1 mutation on feeding behaviour37, so we tested the effect of the mutation on feeding behaviour in this study (see Results).
Production of spermless males-To produce males that did not produce sperm, we used tudor33. tudor is a maternal effect mutation which prevents germ plasm assembly. Sons of homozygous tudor females have no germline and so do not produce sperm33,38. We collected the male offspring of homozygous tudor females mated to wild-type Dahomey males as our spermless males. Males of the same genetic background, but with mothers that did not possess the tudor mutation, were used as controls.
Production of sex peptide-less males-To produce males that did not produce sex peptide (SP null) and their controls (SP+), we used stocks created by Liu & Kubli29. SP null males carry one non-functional SP gene, and a deletion (Δ130) which removes SP29. The SP + control males are genetically matched with one non-functional SP gene and one functional SP gene, and show wild-type SP expression. To verify the phenotype of SP null and SP+, we counted offspring from a subset of female overnight vials. Females mated to SP+ males produced more eggs and offspring than females mated to SP null males, confirming that our mutants were acting in the expected way (i.e. SP null males were not transferring SP; GLM with quasipoisson distribution: Block 1 -females mated to SP+ males produced more offspring than those mated to SP null males: X 2 1, 109 = 82.917, P = 0.009; Block 2 -females mated to SP+ males produced more eggs than those mated to SP null males or virgins: SP+ vs. SP null: q 2, 46 = 11.089, P < 0.001; SP+ vs. V: q 3, 46 = 9.711, P < 0.001; Supplementary   Figure 1). The SP experiment was carried out over two blocks.
Production of sex peptide receptor-less females-To produce females that lack the sex peptide receptor (hereon, SPR deficiency females), we used the genetic deficiency Df(1)Exel6234, which deletes the SPR gene, and four other genes of unknown function39,40. SPR deficiency females do not produce the SPR. As expected, SPR deficiency females did not display reduced receptivity after remating, nor did they show elevated levels of offspring production relative to virgin females (GLM on offspring production: X 2 1, 101 = 111.22, P < 0.001; Supplementary Figure 2)39. SPR deficiency females have also previously been shown to have slightly shorter copulation duration than wild-type females41.

Experimental Design
3d post-eclosion virgin females were marked with acrylic paint (red or yellow) on the thorax to allow individual identification27, and returned to individual vials. 24hrs later, females in the "mated" treatments were placed individually with one male, and a single copulation was

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Europe PMC Funders Author Manuscripts observed. After each female mated exactly once, males were discarded and all females (i.e. both mated and virgins) were individually transferred to fresh vials, again containing standard media and no live yeast.
The following morning (5d post-eclosion), females were individually placed in vials containing damp cotton wool and no food for 2hrs after which pairs of females were simultaneously aspirated from these vials into the contest arena. The arena was a single well of a twelve-well plate, containing an Eppendorf tube cap filled with standard fly food media and a ~2 µL drop of yeast paste, providing a limited resource to compete over27. Females were either both mated, both virgin, or -to test the relative competitive ability of mated vs virgin females -one of each. Females were allowed 5 minutes to acclimatise, and then behaviour was recorded for 30 mins using Toshiba Camileo X400 HD video cameras (short sample videos are available in Supplementary Information).

Behavioural analysis
Videos were scored blind with respect to treatment. Fighting behaviours (head-butt, leg "fencing" (later excluded due to difficulties accurately quantifying the behaviour), shove, retreat27) and feeding behaviours were recorded using the program JWatcher + Video42. Specifically, we quantified the number and duration of aggressive encounters, the identity of females initiating each encounter, and the outcome of each encounter (i.e. win, lose, draw).
An encounter began when one female head-butted or shoved the other female and continued until the females separated or stopped interacting (e.g. were still within touching distance but both resumed eating). We used total duration of aggressive encounters as our primary response variable, which was measured as the sum of time spent in all encounters. We used total duration as it offered the best indicator of overall aggression as it took into account both the number of encounters and the time spent in encounters, providing a more accurate indicator of aggression than number of encounters alone. We also quantified the time females spent in a feeding posture where females were standing on the food cap with their heads tilted down towards the food in a position consistent with feeding.

Statistical analysis
The response variables (i.e. duration of aggressive encounters and time spent in feeding posture) were continuous and most closely fitted a Gamma distribution. Thus, we used generalized linear models (GLMs) with a Gamma error distribution to test the effects of mating status, egg production status, and various ejaculate components on total aggressive contest duration and time spent in feeding posture. The models in R followed the following format, with the model from the ovo D1 experiment given as an example: glm(Fight duration + 1 ~ Mating status * Egg production, family=Gamma(link="log")) Fight duration + 1 was the response variable measured in seconds, while mating status and egg production were the explanatory variables. The explanatory variables differed depending on which experiment was being analysed.
For feeding posture analyses, we additionally included proportion of encounters won as an explanatory variable. The models then followed this format: glm(Feeding duration + 1 ~ Treatment * Proportion of encounters won, family=Gamma(link="log")) Because Gamma error distributions use a logistic function, we added 1 to all scores of total contest duration prior to transformation to include replicates with scores of 0. We tested for outliers using the Grubbs outlier test and excluded outliers below a threshold of P = 0.001. We excluded one outlier in the egg production experiment in the control mated treatment (G = 6.15, P < 0.0001). Winsorizing the data did not qualitatively alter the results (in the winsorized analysis, mating status had a significant effect on fighting duration (Dev 1, 127 = 13.413, P < 0.001), egg production capability was marginally non-significant (Dev 1, 126 = 1.364, P = 0.069), and there was no interaction between egg production capability and mating status (Dev 1, 125 = 0.124, P = 0.583). We excluded the outlier in our final analysis even though the results did not qualitatively change as it seemed that even when winsorized, the one outlier was still exerting undue leverage on the results. To allow for nonindependence of individuals, we used each dyad as the unit of replication in the GLMs, rather than individual flies. For treatments with one mated and one virgin female, we randomly chose one focal individual from each dyad and analysed the proportion of encounters won using a GLM with a quasibinomial distribution. For multiple comparisons within experiments, we used Tukey tests. All models were run in R version 3.0.143.

Mating elevates female aggression
We found that aggression was significantly elevated by mating: pairs with two mated females spent more time fighting over food (GLM: Dev 1, 240 = 26.01, P < 0.001), than pairs with two virgins (compare MM and VV in Figure 2.a). Moreover, there was no significant difference in total contest duration between pairs with two mated females and pairs with one mated and one virgin female (Tukey test: q 2, 20 = 0.81, P = 0.58), whereas pairs with at least one mated female spent more time fighting than pairs with two virgin females (MM vs VV: q 3, 24 = 4.81, P = 0.006; MV vs VV: q 2, 24 = 3.69, P = 0.015; overall model: Dev 2, 74 = 11.36, P = 0.004; Fig. 2.a). We next paired mated females with virgin females to test relative fighting ability. We found no evidence that mating status influenced who won or lost aggressive encounters (X 2 1, 26 = 2.59, P = 0.41; Fig. 2.b). Thus, the presence of one mated female is sufficient to elevate the overall aggression level of a dyad, but mated females do not then win significantly more aggressive encounters.

Effects of egg production and male ejaculate components on female aggression
We found that mating-induced increases in aggression were not significantly different between sterile (ovo D1 ) females and controls (mating status, Dev 1, 126 = 17.32, P < 0.001; sterile vs control, Dev 1, 125 = 1.27, P = 0.12; interaction, Dev 1, 124 = 0.25, P = 0.49; Fig.  3.a). Thus, post-mating increases in female aggression are not restricted to females capable of vitellogenesis and egg laying, and are thus not driven by the energetic demands of egg production post-vitellogenesis.
Sperm is therefore required for the mating-induced increase in female aggression.
Aggression in females mated to males that did not transfer the seminal fluid protein 'sex peptide' (SP null males) was significantly higher than in virgin females (q 2, 30 = 3.4, P = 0.023) but significantly lower than in mates of males that transferred SP (q 2, 30 = 3.05, P= 0.04; overall model Dev 2, 127 = 11.26, P < 0.001; Fig. 3.c). Replicate block had no significant effect on total contest duration (Dev 1, 126 = 1.225, P = 0.131). These results suggest that SP contributes partially to increased post-mating female aggression, but that SP cannot fully explain the sperm effect on aggression. SPR deficiency females did not significantly differ from wild-type females in their aggression response to mating (mating status, Dev 1, 101 = 11.53, P < 0.001; interaction mating status x SPR, Dev 1, 99 = 0.05, P = 0.78; Fig. 3.d). SPR deficiency females also spent more time fighting than wild-type females overall, irrespective of their mating status (Dev 1, 100 = 12.71, P < 0.001).

Feeding behaviour responses to mating, egg production
In the majority of our experiments, the time females spent in feeding posture was qualitatively similar to patterns of aggressive behaviour: for example, mated females generally spent more time in feeding posture than virgins (Supplementary Figure 3). In the egg production experiment, there was a marginally non-significant trend for more time spent in feeding position in mated females (Dev 1, 102 = 1.128, P = 0.056; Supp. Fig. 3.a) but no significant effect of the ovo D1 mutation (Dev 1, 101 = 0.15, P = 0.49) or interaction (Dev 1, 99 = 0.31, P = 0.97). Therefore, ovo D1 females did not differ from control females in time spent in feeding postures, despite spending less time participating in aggressive encounters.

Discussion
Our results show that mating strongly stimulates aggressive behaviour in female fruit flies. Mated females fought for more than twice as long as virgins, and the full increase in aggression requires the receipt of sperm and sex peptide (SP) in the male ejaculate, but not the ability of females to complete vitellogenesis or begin egg laying, nor the presence of the female sex peptide receptor (SPR)39. ovo D1 females displayed the same increase in aggression after mating as wild-type females, despite lacking the costs of egg production20, while females that lacked the SPR gene (SPR deficiency) spent more time fighting than wild-type females despite producing fewer eggs (Supplementary Results and Figure 2). These results suggest that the costs of egg production and the levels of aggression can be fully decoupled and are thus modulated by divergent pathways (Figure 1.b).
The receipt of SP was necessary for the full increase in post-mating aggression. It should be noted, however, that as we conducted tests of aggression 24 hours after mating, it is likely that only seminal fluid proteins that bind to sperm remained in the female, as other seminal fluid proteins will no longer be present in females at this time44,45. SP is known to bind to sperm, though it is unclear which other seminal fluid proteins also bind to sperm46. It is therefore possible that other seminal fluid proteins bound to sperm may also influence female aggression, or proteins not bound to sperm could influence female aggression on a shorter time scale than the 24 hours we measured.
Seminal fluid proteins (including SP) can, under certain conditions, lower female lifetime survival and reproductive output47-49, raising the possibility that ejaculate-stimulated female aggression could contribute to these costs and thus represent an arena of sexual conflict rather than cooperation. However, the net fitness costs and benefits of female aggression, and the role of males in determining this, remains an area ripe for exploration. Surprisingly, females lacking the SPR displayed full increases in post-mating aggression suggesting that SPR or one of the other deleted genes may affect aggressive behaviour, SP may act through alternative pathways to stimulate female aggression, or that SP may act indirectly through association with other seminal proteins50. In addition to acting through the SPR, SP stimulates juvenile hormone production in the corpora allata, although the mechanism of this stimulation is unknown51,52. Juvenile hormone facilitates vitellogenesis in D. melanogaster, playing a crucial role in reproduction53. The amount of juvenile hormone present in the haemolymph has also been linked to aggression in both sexes in other insect species, such as burying beetles, paper wasps, and cockroaches54-56. It is therefore possible that SP acts to increase female aggression by stimulating juvenile hormone production, though this has yet to be tested.
Male ejaculates could potentially stimulate post-mating aggression through inducing increased feeding, though our data are not fully consistent with this idea. Results from the sperm and SP experiments suggest an association between feeding and aggression, but results from the ovo D1

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Europe PMC Funders Author Manuscripts ovo D1 females do not suffer the lifespan costs of egg production)20,37, it is possible that eggs do still play a role in inducing female aggression. Further investigation using females that do not possess a germline may help to further clarify the role of egg production in stimulating female aggression.
It is possible that mating results in a reduction of general social tolerance in females towards other individuals, as females are both less receptive to remating with males and more aggressive towards other females. However, the behaviours involved in rejection of males are very different to those involved in incidents of female aggression27,57. In addition, rejection behaviours and aggressive behaviours appear to be activated through different pathways, as rejection behaviours are primarily stimulated through the SPR but our results show that female aggressive behaviours are not.
Our results fit into a broader trend, across taxa, of mating leading to increased female aggression. For example, gestating and lactating mammals and fish display higher levels of aggression towards conspecifics6,12,58,59. However, our results suggest that it is possible to decouple aggression from increased energetic demands of reproduction after mating in D. melanogaster. Instead, we have shown that female aggression in D. melanogaster is stimulated more directly by male ejaculates, with downstream indirect effects on other females, whereby post-mating aggression affects the wider female competitive environment. For example, the levels of aggression experienced by females may depend not only on the abundance of resources and rivals in their environment, but also upon the seminal characteristics of their mates and mates of their rivals.
These findings have potential implications for a wide range of sexually reproducing animals. In many insect species, females compete aggressively54,60 and male accessory gland products induce striking changes in female behaviour and physiology other than female aggression18,61,62. Females of some mammal species also display increased aggression after mating63, and in some mammal species ejaculate components have been shown to influence female physiology64,65. Thus, it is intriguing to speculate that ejaculate-induced female aggression may occur in mammals, analogous to what we have shown here for D. melanogaster. However, further studies are required to verify whether ejaculate-mediated effects on female aggression occur in other species. Further key areas of focus for future research include identifying the neuronal mechanisms producing increased post-mating aggression, and understanding the fitness implications for individual females, their competitors and their mates.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.