1 Drosophila melanogaster females change mating behaviour and
2 offspring production based on social context
3 4 Jean-Christophe Billeter1,2, Samyukta Jagadeesh1, Nancy Stepek1, Reza Azanchi1 and
5 Joel D. Levine1,*.
6 1
7 Mississauga, ON, L5L 1C6, Canada.
8 2
9 Groningen, Groningen, 9700 CC, Netherlands.
10 Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road,
Present address: Centre for Behaviour and Neurosciences, PO Box 11103, University of
*to whom correspondence should be addressed: [email protected]
11 12 Keywords: Drosophila melanogaster; social behaviour; reproduction; mate choice;
13 sperm competition; Bruce effect.
14 1 15 ABSTRACT
16 In Drosophila melanogaster, biological rhythms, aggression and mating are modulated
17 by group size and composition. However, the adaptive significance of this group effect is
18 unknown. By varying the composition of groups of males and females, we show that
19 social context affects reproductive behaviour and offspring genetic diversity. Firstly,
20 females mating with males from the same strain in the presence of males from a different
21 strain are infertile, analogous to the Bruce effect in rodents, suggesting a social context-
22 dependent inbreeding avoidance mechanism. Secondly, females mate more frequently in
23 groups composed of males from more than one strain; this mitigates last male sperm
24 precedence and increases offspring genetic diversity. However, smell-impaired Orco
25 mutant females do not increase mating frequency according to group composition; this
26 indicates that social context-dependent changes in reproductive behaviour depend on
27 female olfaction, rather than direct male-male interactions. Further, variation in mating
28 frequency in wild-type strains depends on females and not males. The data show that
29 group composition can affect variance in the reproductive success of its members, and
30 that females play a central role in this process. Social environment can thus influence the
31 evolutionary process.
32 2 33 1. INTRODUCTION
34 The adaptive value of group life has been illustrated in many species by studies from both
35 field and laboratory [1,2]. Group membership can facilitate biological functions including
36 reproduction, foraging and protection against predators [1]. Such traits are controlled by
37 the genotype of an individual as well its social partners, a phenomenon termed Indirect
38 Genetic Effects (IGE) [3,4]. IGE are predicted to affect the evolutionary process when
39 interactions with social partners affect variance in the individual’s fitness, known as
40 social selection [3-6].
41 Drosophila melanogaster swarm on food substrates, where they feed and breed
42 communally [7,8]. Members of these groups derive benefits from this social environment.
43 For instance, females lay eggs communally [9], which increases the chance of larval
44 survival [10]. Group composition is an important parameter influencing Drosophila
45 social life. Studies on wild populations and laboratory lines suggest that the genotype of a
46 minority can influence behaviour in the whole group [11-13]. For example, introduction
47 of arrhythmic period mutants in a group of wild-type flies resets the locomotor activity
48 rhythms of the wild types, changes the blend of pheromones they display, and increases
49 mating frequency [14-16]. These effects have molecular correlates as the presence of the
50 mutant flies modulates gene expression in the brain, and in the pheromone-producing
51 cells of the wild-type flies [15]. Social interactions within D. melanogaster groups are
52 thus clearly subjected to IGE [16], allowing for the phenotype of others to affect the
53 behaviour of an individual. However, little is known about the fitness consequences of
54 these group interactions.
3 55 Genotypic frequency in a population is affected by mate preference. In the laboratory,
56 mate choice is often tested by observing how a single male and female select one another.
57 However genetic monogamy is rare and both males and females of many species mate
58 with multiple individuals in their lifetime [17-19]. In species where females have large
59 broods, such as in D. melanogaster, multiple matings in one reproductive episode may
60 lead to multiple paternities but each male may not sire equal amount of offspring [20-21].
61 Mating success for a male thus does not guarantee reproductive success.
62 D. melanogaster males deploy several strategies to ensure paternity not only before but
63 also after copulation. For instance, when a female re-mates, the second male sires a
64 majority of her offspring by out-competing his predecessor, a phenomenon called last
65 male sperm precedence [20-21]. Because of male-imposed constraints, re-mating could
66 have little benefit to females and may even reduce female lifespan [22-25]. Yet, multiple
67 matings per female in a single reproductive episode and polyandry have been regularly
68 reported in D. melanogaster [15,26-29], so the adaptive significance of this behaviour
69 remains unclear. In other species, multiple matings and/or polyandry are reported to have
70 a positive effect on female fitness, including an increase in number of offspring produced
71 [17] and offspring genetic diversity [18].
72 We have previously reported high incidence of re-mating when flies are housed
73 continuously for 24hrs in groups of six males and six females in the laboratory [15]. This
74 high mating frequency is dramatically higher than what is observed in most laboratory
75 assays of re-mating in D. melanogaster, which include a single male and female [23,
76 30,31]. This discrepancy in re-mating frequencies between assays suggests that social
77 context affects mating frequency in D. melanogaster [32] and provides a possible
4 78 example of social selection - if group composition leads to variance in reproductive
79 success.
80 Here, we show that the social environment affects the reproductive success of individual
81 flies. Group assays with six males and six females demonstrate high frequencies of re-
82 mating. We show that housing males from different strains to increase social
83 heterogeneity within these groups further increases re-mating frequency and affects
84 female mate choice. Females are central to this effect of the social environment, assessing
85 group composition by olfactory mechanisms. We also report that females can block
86 fecundity with certain males or cause a breakdown in last male sperm precedence in a
87 social context-dependent manner, thereby directing genotypic distribution in her
88 offspring. Females appear to derive indirect benefit from re-mating by increasing genetic
89 diversity in their brood. This study shows that the social environment can affect variance
90 in reproductive success and thus impact the evolutionary process.
91 2. MATERIALS AND METHODS
92 (a) Fly rearing
93 Flies were reared on medium containing agar, glucose, sucrose, yeast, cornmeal, wheat
94 germ, soya flour, molasses, propionic acid and Tegosept in a 12:12 hr light/dark cycle
95 (LD 12:12) at 25°C. Virgin adults were collected using CO2 anaesthesia and aged in
96 same-sex groups of 20 in food vials for 5-6 days.
97 (b) Group mating assays
5 98 Group mating assays were performed in disposable 55 x 8 mm Petri dishes containing a
99 fly food slice (22 x 5 mm). Assays were set up by sequentially introducing six virgin
100 females followed by different types and numbers of virgin males using a mouth pipette.
101 We report four different variations on this assay.
102 Experiment 1 (figure1) tests the effect of group composition on individual mating and
103 reproductive success over 8-hrs. The mating behaviour of two common laboratory wild-
104 type strains, Canton-S and Oregon-R, was assayed in groups of six females housed with
105 six males. Five group compositions were tested side-by-side by changing the type and
106 ratio of males as follows: groups of six males comprising only Canton-S or Oregon-R
107 males (homogeneous groups) or heterogeneous groups comprising both Canton-S and
108 Oregon-R males at different ratios: 2:4, 3:3 and 4:2 Canton-S to Oregon-R male
109 respectively. Flies were marked with acrylic paint (pink, blue, yellow, gold, white or
110 black) dotted onto the thorax upon collection for identification and determination of mate
111 choice. To allow colour discrimination, assays ran from Zeitgeber Time (ZT) 1 (10 am)
112 to ZT8 (6pm) under bright illumination and were continuously recorded using a Sony
113 AVCHD camcorder. An observer, blind to the flies’ colour code, scored the time to
114 mating, and identity of mating partners using the Picture Motion Browser software (Sony,
115 Inc.). Females were extracted after the assays and placed individually in food vials for
116 egg laying at 25°C in a 12:12 LD cycle. Vials were changed daily for 5 days; females
117 were left in the last vial to lay for another 5 day. Freshly eclosed adult offspring were
118 counted daily to determine female fecundity, then aged in fresh food vials for 3-7 days
119 before paternity analysis (see Paternity analysis).
6 120 Experiment 2 (figure 2) tests the effect of group composition on mating frequency over
121 24hrs [15]. Frequency and time of mating of Canton-S and Oregon-R flies, as well as
122 Orco mutants (née Or83b) [33] in a Canton-S genetic background were assayed. Orco
123 encodes an odorant co-receptor expressed in the olfactory system and required for the
124 functioning of most olfactory receptor neurons [33]. The original w1118;+;Orco2 was
125 outcrossed to a Canton-S-isogenised white stock for five generations and the X and 2nd
126 chromosomes were replaced by those of Canton-S. Six females were housed with groups
127 of males composed either solely of Canton-S or Oregon-R males, or of four males of
128 these strains with two males mutant for the yellow and white genes (y,w males; unknown
129 genetic background). Assays were started at ZT 8 (5 pm) in an incubator set at 25°C and
130 LD 12:12. Northern eclipse software (v. 7.0) operating a Hitachi CCD camera, or the
131 ZoomBrowser EX software (Canon, Inc.) operating a Canon S10 digital camera, were
132 used to take images of the assays at 2-minute intervals for 24 hrs. Red light was used to
133 monitor mating during the dark phase. This 24hr period necessarily included flies
134 entering a night phase, preventing identification of colour tagged individuals. The lighter
135 body and eye colours of y,w mutants allowed discrimination from wild-type flies by the
136 observer scoring mating events. Images were surveyed for copulating pairs and scored if
137 a pair was observed for at least four consecutive frames.
138 Experiment 3 (figure 3) assesses the effect of multiple female matings on last male sperm
139 precedence in groups over 8-hrs. Six Canton-S females were housed with 3 GFP-marked
140 and 3 non-GFP-marked males. The GFP-marked males express the Green Fluorescent
141 Protein (GFP) ubiquitously due to an homozygous Ubiquitin::GFP transgene. The non-
142 GFP males do not contain this transgene, but are otherwise in the same foragings wild-
7 143 type background. These males were chosen because they had been shown to be
144 equivalent despite the GFP transgene [34]. This experiment was otherwise performed and
145 scored as in Experiment 1. Colour tags permitted identification of mate choice and mating
146 order. Females were extracted at the end of the assay as in Experiment 1 for fecundity and
147 offspring paternity analysis, but paternity was determined through presence or absence of
148 GFP using a Leica MZ10F fluorescence stereomicroscope.
149 Experiment 4 (figure 4) assays group mating frequency in different wild-type strains.
150 Groups were composed of six females and six males, either all from the same strain, or in
151 conditions where the sexes were from different strains. Experiments were set-up and
152 scored as in Experiment 2.
153 (c) Paternity analysis
154 Abdominal segments A6 and A7 of Canton-S females are more darkly pigmented than
155 Oregon-R females and daughters from an Oregon-R dam and a Canton-S sire have a
156 pigmentation pattern similar to Canton-S (figure S2). Paternity was determined based on
157 this pigmentation dimorphism. Daughters from Canton-S dams and Oregon-R sires
158 however have the same pigmentation as Canton-S females when raised at 25°C, but
159 displayed intermediate pigmentation when raised at 30°C. The eggs of Canton-S dams
160 were therefore transferred to a 30°C incubator after having been laid by the female at
161 25°C, allowing unambiguous paternity identification.
162 (d) Statistical analysis
163 Statistical analyses were performed with SPSS software (version 19.0, SPSS inc.).
164 ANOVAs were followed by the post-hoc Tukey-Kramer test for multiple comparisons
8 165 except when data did not fit a normal distribution or had unequal variances, in which case
166 Kruskal-Wallis ANOVA was used followed by Dunn’s post-hoc test for multiple
167 comparisons. Differences in temporal distribution of mating were tested using repeated
168 measurement ANOVA after degrees of freedom were corrected using Greenhouse-
169 Geisser estimates of sphericity.
170 171 3. RESULTS
172 (a) Experiment 1: social context modulates male mating success, reproductive success
173 and offspring genotype
174 We assayed mating behaviour of six females from Oregon-R or Canton-S wild-type
175 strains housed with groups of six males comprising different mixes of Oregon-R or
176 Canton-S to determine effects of the social environment on reproductive behaviour (see
177 Material and Methods: experiment 1). In 8hrs of continuous housing with males, Oregon-
178 R females mated on average twice and Canton-S females three times (figure S1a1-2).
179 Housing females with heterogeneous groups of such males did not affect female mating
180 frequency (figure S1a1-2), but did affect male mating success in some of the groups
181 (figure 1a). For males housed with Oregon-R females, different social contexts
182 influenced male mating success in a strain-specific way (figure 1a1), showing that a
183 male’s mating success is not only determined by his own strain identity, but also by the
184 identity of other male group members. Interaction between social context and male strain
185 accounted for approximately 21% of the total variance in mating success between all
186 groups housed with Oregon-R females (2-way ANOVA; F3,108=10.9, P<0.0001) (figure
187 1a1).
The social environment mainly affected Canton-S males, which gained more
9 188 mating when in a 2:4 minority than in any other social contexts (ANOVA: F3,54=9.761,
189 P<0.0001)(figure 1a1). Oregon-R male mating success was not significantly affected by
190 the social context (ANOVA: F3,54=2.567, P=0.064). The influence of the social context
191 on a male’s mating success depends on the females’ strain because male mating success
192 was not affected when similar groups of males were housed with Canton-S females
193 (Figure 1a2). Male mating success was similar for both strains (2-way ANOVA;
194 F1,82=0.04, P=0.84), and was unaffected by social context (2-way ANOVA; F2,82=0.66,
195 P=0.52) (Figure 1a2).
196 Changes in social context mildly affected mating success (Figure 1a), but had dramatic
197 effects on reproductive success. Oregon-R female fecundity dropped in heterogeneous
198 social groups, compared to groups composed only of one male strain (figure S1b1). This
199 drop was not statistically significant when considering females irrespective of what males
200 they mated with (Kruskal-Wallis test: H=5.78, P=0.22). However, comparison of females
201 who mated solely with one type of male (Canton-S or Oregon-R) in different social
202 contexts revealed that Oregon-R females who mated with Oregon-R males in the
203 presence of Canton-S males, were on average 70% less fecund than when they mated
204 with Oregon-R males in the absence of Canton-S males (figure 1b1) (Kruskal-Wallis test:
205 H=24.41, P<0.0001). Fecundity of those Oregon-R females who mated solely with
206 Canton-S males in the same conditions was not affected (ANOVA: F3,129= 0.60,
207 P=0.61)(figure 1b1). Social context and the strain of the males interact to determine
208 reproductive success (2-way ANOVA; F3,212= 2.94, P=0.034). The social context in
209 which individuals mate can be as important as mating itself because mating does not
210 ensure reproductive success in all social environments (figure 1b1). These effects are not
10 211 due to incompatibility between strains because Oregon-R females had similar numbers of
212 offspring with Canton-S than with Oregon-R males when mating in homogenous groups
213 (figure S1b1), indicating equal male fecundity and no reproductive incompatibility.
214 Interaction between social context and male strain affecting reproductive success was not
215 observed when similar groups of males were housed with Canton-S females (ANOVA:
216 F2,155= 1.96, P=0.14)(figure 1b2), indicating again that influence of the social context on
217 male reproductive success depends on the identity of the females. Decreased fecundity of
218 Oregon-R females in heterogeneous groups is not linked to a general fecundity problem
219 because they are normally more fecund than Canton-S females when housed with
220 homogeneous groups of males (t-test: P=0.027) (figure S1b1-2).
221 We determined paternity in the offspring of females that mated in different social context
222 to determine the fitness of different male strains (See Material and Methods). We observed
223 a striking pattern of offspring production by Oregon-R females across social contexts
224 despite changes in mating and reproductive success (figure 1a1 and b1): Canton-S males
225 consistently sired ~80% of offspring in heterogeneous social groups and Oregon-R males
226 20% (figure 1c1). This stable percentage is a group property and is not found in any one
227 female. This effect was not observed when similar groups of males were housed with
228 Canton-S females, and their offspring were sired equally by Canton-S and Oregon-R males
229 (figure 1c2). We conclude that social environment can influence mate choice and fecundity,
230 and notably genotypic diversity in the next generation.
231 (b) Experiment 2: Social heterogeneity affects mating frequency
232 Male strain diversity within a group strongly influences mating behaviour of Oregon-R
233 but not Canton-S females, leaving the generality of this phenomenon unclear. We
11 234 extended the assay from 8 to 24 hrs to determine a potential longer term effect of the
235 social environment on Canton-S female mating behaviour, and created socially
236 heterogeneous groups by housing mutant males with wild-type males and female Canton-
237 S or Oregon-R (see Material and Methods: experiment 2). Mutant males were yellow,
238 white (y,w), which are sluggish courters with low mating success [35], making them
239 unlikely to directly influence mating frequency. This allows testing the indirect effect of
240 their mutant genotype on the mating success of the wild-type males by comparing mating
241 patterns in homogeneous groups composed of six females and males from either the
242 Canton-S or the Oregon-R strain to that of socially heterogeneous groups comprising four
243 males (either all Canton-S or Oregon-R) and two y,w mutant males (figure 2).
244 The presence of y,w males indirectly increased Canton-S males mating success (figure
245 2a1). Canton-S males gained 38% more matings in socially heterogeneous groups than in
246 groups with no y,w males present (figure 2a1), indicating that social heterogeneity can
247 also affect Canton-S reproductive behaviour. We ruled out a confound of social density.
248 Because the mating success of the two y,w males is negligible (less then 3% of all
249 matings in Canton-S groups), we reasoned that the mating frequency of four Canton-S
250 males with whom they are housed is better compared with that of four Canton-S males
251 without y,w males, but that changes social density. Density was not an issue because
252 homogeneous groups of Canton-S flies with either four or six Canton- S males were not
253 different in terms of individual male mating frequency (figure 2a1).
254 No significant increase in mating frequency was observed when Oregon-R flies were
255 mixed with mutant males (P=0.11) (figure 2a2). Differences in response to social
12 256 heterogeneity between Canton-S and Oregon-R between Experiment 1 and 2 may be
257 partially caused by a temporal difference in behaviour. Graphing the temporal
258 distribution of mating over the 24hrs of Experiment 2 reveals that the effect of mixing y,w
259 males in Canton-S groups become apparent 10-12hrs after the start of the experiment
260 (after ZT18; figure 2b1). Further, Canton-S and Oregon-R have different temporal
261 distributions of mating (figure 2b1-2). These findings raise the possibility that additional
262 matings of Canton-S females were not observed in Experiment 1 (figure S1) because the
263 8-hr observation was too short.
264 Increased mating frequency in socially heterogeneous groups of Canton-S flies (figure
265 2a1) may be caused by Canton-S females perceiving male diversity and increasing
266 receptivity. Because flies use chemical communication during social interactions [36], we
267 hypothesized that introduction of smell-impaired Canton-S females would result in no
268 mating frequency increase in groups comprising y,w and Canton-S males, compared to
269 the group composed of only Canton-S males. We re-tested the mating behaviour of
270 Canton-S flies in the presence of y,w males, using Canton-S females carrying a null Orco
271 allele, impairing their olfaction. Unlike groups including wild-type Canton-S females
272 (figure 2a1), groups including Orco mutant females did not show increased mating
273 frequency (figure 2a3) indicating that social context perception is mediated by an
274 olfactory cue perceived by females. Orco females behaved like Canton-S females in
275 homogeneous groups in terms of mating frequency (Figures 2a1,3) and temporal
276 distribution of mating (Figures 2b1,3) indicating no basic reproductive defects caused by
277 the mutation. We conclude that effects of social heterogeneity on mating behaviour are
278 mediated by females.
13 279 (c) Experiment 3: female mating frequency affects last male sperm precedence
280 The adaptive value of Canton-S increased mating frequency in heterogeneous groups is
281 unclear (figure 2a1). Females produce large broods (~100), so increasing number of
282 mates could increase offspring genetic diversity. However, males possess mechanisms
283 ensuring sperm precedence [21] predicting that female mating will not achieve greater
284 offspring diversity because the last male will sire most offspring [25].
285 We tested this prediction by examining the percentage of offspring sired by the last male
286 when mating with Canton-S females who had already mated with one, two, three or four
287 males (See material and methods: Experiment 3). Groups were composed of 3 males
288 genetically marked with a GFP transgene and 3 unmarked housed with 6 Canton-S
289 females, allowing determination of paternity in offspring who inherited GFP. We
290 observed classical instances of last male sperm precedence in twice mated females, with
291 the last male siring a majority of offspring (figure 3a1-2). Last male precedence was
292 however not observed in females that mated more than twice, and the portion of offspring
293 sired by the last male was reduced to a third or less (figure 3a1-2). This rejects the
294 prediction that increased remating does not change offspring diversity. A one-way
295 ANOVA with the proportion of offspring sired by the last male as the dependent variable
296 showed that last male sperm precedence is significantly affected by the number of times a
297 female mated (For GFP male F2, 52=17.52, P<0.0001; for non-GFP males F2, 52=13.35,
298 P<0.0001). The higher percentage of offspring sired by non-GFP (figure 3a1) compared
299 to GFP (figure 3a2) last males is probably linked to an effect of the ubiquitous expression
300 of GFP, which may reduce the ability of sperm from GFP males to compete with sperm
301 from non-GFP males [21]. Female fecundity was not significantly affected by numbers of
14 302 mating or male types, indicating that multiple matings have no detrimental effect on
303 female fecundity (figure S3).
304 Males reportedly transfer fewer sperm in successive copulations, until no more sperm is
305 transferred after 4-5 copulations [27]. During this 8hr-assay, we observed on average 3.89
306 mating per female (s.e.m.=0.08; n=464), each spaced by an average 135 min (s.e.m.=3
307 min). GFP males mated 3.4 times (s.e.m.=0.12; n=217) and non-GFP males mated 4.9
308 times (s.e.m.=0.13; n=230). This high re-mating frequency may allow females to affect
309 sperm competition by depleting male sperm reserves. We indirectly checked sperm
310 depletion in males who had mated last with three times mated females by comparing
311 those males who had previously mated with no more than two females (“fresh” males)
312 and those who had mated with three or more (“exhausted” males). The ability of
313 “exhausted” non-GFP males to sire offspring was reduced compared to “fresh” ones,
314 suggesting that sperm depletion is a factor in last male precedence reduction (figure 3b).
315 However, it cannot be the only factor as the number of offspring sired by “fresh” non-
316 GFP males in twice-mated females (figure 3a) and three-time mated females (figure 3b)
317 was significantly reduced in three-times mated females (t-test P= 0.03). The sperm
318 depletion effect was not observed in GFP males (figure 3b). The effect of female mating
319 history must therefore involve an interaction between sperm depletion and additional
320 female cryptic effects.
321 (d) Experiment 4: variation in re-mating behaviour between wild-type strains
322 As discussed in the introduction, the high female re-mating frequencies observed in our
323 assays are at odds with several reports of low female re-mating frequency in D.
324 melanogaster. To better characterize re-mating behaviour in our assay, we tested six
15 325 laboratory wild-type strains to estimate between-strain variability in mating frequency.
326 The number of matings was quantified in groups of six males and six females housed
327 together for 24-hrs. In addition to Canton-S and Oregon-R, we tested the C0-3, TW-1,
328 Urbana-S and Amherst-3 wild-type strains randomly selected from the Bloomington
329 Drosophila stock centre.
330 We found that mating frequency is strain-specific and ranges on average from two to five
331 matings per individual (figure 4a). Re-mating is thus a standard behaviour. The
332 percentage of individuals that mated more than once is also strain-specific and ranged
333 from 50% to 100% (figure 4b). We also observed strain differences in the time interval
334 between the first and second mating (figure 4c). Canton-S mate for the second time (first
335 re-mating) 4 hours after the virginal mating, while other strains, such as Oregon-R, mate
336 for the second time 10 hours later (figure 4c). Temporal distributions of re-mating over
337 24hrs of the two wild-type strains with the highest re-mating frequencies (Oregon-R and
338 Canton-S) are clearly different (Repeated measure ANOVA: F1,7.5=19.08, P<0.001)
339 (figure 4d). Canton-S re-mated at the highest frequency in the first half of the night,
340 whilst Oregon-R peaked in the second half of the night and in the early morning (figure
341 4d).
342 We investigated the contribution of each sex to mating frequency. We selected three
343 strains with high, medium and low mating frequency (Canton-S, Oregon-R and CO-3
344 respectively) (figure 4a) and quantified mating in groups composed of the 9 possible
345 permutations of males and females (figure 4f). In this factorial design, Canton-S females
346 showed the highest mating frequency with males from any strain, whilst Oregon-R and
347 C0-3 showed medium and low frequency respectively (figure 4f). Oregon-R males mated
16 348 more than Canton-S or CO-3 males with females from a given strain (figure 4f). A 2-way
349 ANOVA revealed that the strain of females significantly affects mating frequency and
350 accounted for 47% of the variance between groups (F2,124=133.87, P<0.0001). Strain of
351 the male also significantly affects mating frequency but accounts for only 11% of the
352 variance (F2,124=30.33, P<0.0001). The male by female genotypic interaction was small
353 and accounted for only 3% of the variance (F4,124=4.77, P=0.001). Temporal distribution
354 of mating also tracks the distribution observed in the strain of the female and not that of
355 the male (figure 4e) and so does the time to first re-mating (figure 4g). We conclude that
356 re-mating is a common behaviour in groups, but both the frequency and temporal
357 distribution of mating vary between strains. Although males exert some influence on
358 mating frequency, females are the main determinant of when and how often re-mating
359 occurs.
360 361 4. DISCUSSION
362 363 We investigated the effect of group composition on mating behaviour and offspring
364 production to determine the connection between an individual’s social environment and
365 its reproductive success. In groups, male reproductive success was predicted not only by
366 his strain identity, but also by the strain of the other males in the group (figures 1,2).
367 Thus, the contribution of the strain of an individual male to his own reproductive success
368 can be small relative to the contribution of group composition: effects of group
369 composition on male reproductive success were dependent on the type of females they
370 were housed with (figure 1). We hypothesize that females assess all potential mates in
17 371 their social environment and adjust mate preference and re-mating frequency as well as
372 fecundity accordingly (figures 1,2). Sexual selection can thus be influenced by the social
373 environment.
374 Several studies have indicated that female re-mating may increase offspring fitness
375 reviewed [reviewed in 18-19]. A general result across our assays is that increase in social
376 heterogeneity is accompanied by changes in female re-mating behaviour resulting in an
377 increase in offspring genetic diversity. This happens either via disassortative mate choice
378 and selective fecundity (figure 1), or by increase in re-mating frequency diluting last male
379 sperm precedence (figure 2,3). Our data in D. melanogaster suggest that indirect fitness
380 benefits to females are linked to effects of the social environment.
381 Females from different strains differ in their reaction to the social environment, perhaps
382 reflecting adaptations to local social environments in the natural populations from which
383 they were originally derived, or could have been picked up in the lab due to different
384 levels of genetic heterogeneity amongst stocks [37]. For instance, Canton-S females
385 increase mating frequency with Canton-S males in the presence of y,w mutant males, but
386 Oregon-R females do not (figure 2). We surmise that y,w males may not be perceived as
387 being genetically different enough from Oregon-R males to warrant extra mating.
388 Females may assess the relatedness of potential mates in their social environment and
389 mate assortatively and/or dissassortatively accordingly. This would offer an interpretation
390 for Oregon-R females blocking offspring production when mating with males from their
391 own strain in the presence of unrelated males (figure 1b1). We do not know how this
392 fecundity block is achieved, but we can exclude indirect male-male interactions like
393 sperm competition because this phenomenon is observed in females who only mated with
18 394 one type of males (figure 1b2). It must therefore stem from cryptic female choice, such as
395 the active discarding of sperm [21,38], and has an adaptive function because it prevents
396 inbreeding. This phenomenon recalls the “Bruce effect” in which female rodents
397 terminate pregnancy if exposed to a male from a different strain than the original stud
398 male [39]. This similarity is intriguing because the Bruce effect is a pheromonal response
399 depending on female olfaction to identify the “other” male [39]. Smell-impaired
400 Drosophila females do not react to a mix of males in their group (figure 2), indicating
401 that recognizing males occurs via chemical cues as in rodents.
402 We studied the effect of the social context on reproductive success by measuring the
403 frequency of re-mating in groups. The observation of high re-mating frequency contrasts
404 with several studies reporting low levels of re-mating in D. melanogaster. These studies
405 employ assays where a female is mated with a single male, isolated for 24hrs, and
406 subsequently housed with a different male, typically resulting in very low re-mating
407 frequencies, [e.g. 23,30-32]. Our assay differs dramatically in the number of flies, length,
408 and presence of food, explaining discrepancies between reports. Our observation of high
409 female re-mating frequencies does not appear to be an experimental artefact because it
410 matches data on polyandry in the wild. Wild-caught D. melanogaster females commonly
411 carry sperm from several males and will have offspring from 4-5 different sires [26,28],
412 indicating that our assay may be more naturalistic than classical assays. Further, our study
413 shows that female re-mating is connected to genetic heterogeneity amongst potential
414 mates, which may reflect the social environment of wild females on food aggregates [7].
415 Females in some lab assays may have lower level of re-mating due to limited mate choice
416 and low genetic diversity within strains. Differences in mating frequency may also be
19 417 linked to genetic variation between populations, as implied by the strain-specificity of
418 this phenotype (figure 4). Strain differences also extend to temporal distribution of
419 mating (figure 4). Temporal preference in mating may create reproductive barriers
420 between species [40]. We may speculate that strain-specific temporal differences in re-
421 mating may also limit reproduction between populations, to the extent that patterns of
422 variance observed in lab strains reflects differences between wild populations [37].
423 424 D. melanogaster is a model for last male precedence, but this phenomenon has mainly
425 been explored in the context of females mating twice and with long intervals between
426 copulations (1-2 days) [e.g. 21,25]. In this context, females mating three times failed to
427 show an effect of increased mating on last male sperm precedence [25]. That high re-
428 mating frequency in our assay dilutes last male precedence (figure 3) is therefore at odds
429 with previous publications. This discrepancy may be partially due to ejaculate depletion
430 resulting from fast mating frequency, which would not be an issue in classical assays.
431 However, this factor is not sufficient to fully explain the breakdown in sperm precedence
432 we observe. Studies in other arthropods have shown that last male sperm precedence
433 breaks down when females mate more than twice or at high frequencies [41,42], showing
434 precedents for this effect.
435 Observations in this study support the evolutionary theory of indirect genetic effects,
436 which states that the reproductive success of an individual can be influenced by the
437 genotype of another, thereby affecting allelic distribution in the next generation and in
438 populations [3,4]. Evolutionary changes may occur whenever the breeding value of one
439 individual covaries with the phenotype of its social partners [5]. Here we show that the
20 440 reproductive success of a given male can depend more on the genotypes and abundance
441 of other males in the group than his own genotype. Females appear to mediate this social
442 effect creating a behavioural equivalent of other variation-generating mechanisms such as
443 meiotic recombination and sex itself.
444 445 Acknowledgement
446 We thank J. Schneider for help with Statistics and M. de la Paz Fernández, M.E. Maan,
447 F.H. Rodd, M.F. Wolfner and A.J. Moore for comments on the manuscript. The Orco 448 mutant strain was a gift from L. Vosshall (Rockefeller University). J.-C.B. was
449 supported by a fellowship for advanced researcher from the Swiss National Science
450 Foundation. This work was supported by grants from the Canadian Institutes of Health
451 Research, Canada Research Chair grants and NSERC awarded to J.D.L.
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549 550 24 551 FIGURE LEGENDS:
552 FIGURE 1: Social context of Drosophila melanogaster affects (a) male mating success,
553 (b) reproductive success and (c) the frequency of genotypes in the F1 offspring. A group
554 of 6 Canton-S males (black circles) and/or Oregon-R males (grey circles) were housed at
555 a range of ratios (indicated below the x-axis) with either 6 Oregon-R females (a1-c1) or 6
556 Canton-S females (a2-c2) to vary social contexts. The first digit (underlined) of each ratio
557 indicates the males for which the data are presented; the second digit represents the males
558 from the other strain. (a) Mean numbers of matings per male of the indicated strain were
559 determined in different social contexts. Replicates ranged from 10-18. (b) Number of
560 offspring of females who mated exclusively with Canton-S (black) or Oregon-R males
561 (grey) in different social contexts. Sample size: 6-54. The female offspring were
562 genotyped to determine the fraction (c) of offspring sired by each male type (Oregon-R:
563 grey; Canton-S: black) in different socials contexts. Sample sizes: 29-54. Error bars: ±
564 SEM.
25 565 FIGURE 2. Social heterogeneity affects (a) mating frequency and is perceived by female
566 olfaction. (a1-3) Mean number of matings per male in 24hrs, excluding y,w males, in
567 groups of six females whose genotype is indicated above each graph in either
568 homogenous groups of males (light grey bars) or in mixed groups comprising four wild-
569 type males and two y,w mutant males (black). The four males were from the same strain
570 as the females in (a1-2). In the Orco females experiment (a3), the males were Canton-S.
571 Number of matings per male was estimated by dividing total number of matings by the
572 number of wild-type males (four or six). Bars labelled by same letter (A or B) are not
573 statistically different (ANOVA, P>0.05). n.s. indicates no significant difference between
574 groups determined by a two-tailed Student’s t-test (P>0.05). (b) Temporal distribution of
575 mating in groups of the indicated genotypes and composition as in (a). Data points
576 indicate the mean number of matings observed per 2 hr time bins over 24hrs. Numbers of
577 replicates are between brackets. Error bars: ±SEM.
578 26 579 FIGURE 3: Female mating affects pattern of last male sperm precedence. (a) Mean
580 percentage of offspring sired by the last male in a mating series ranging from one to five
581 matings. Mating series are indicated below the graphs. Numbers between brackets
582 indicate females recorded with this particular mating series. Histograms labelled by
583 different letters (A, B or C) are statistically different (ANOVA, P>0.05). (b) Effect of
584 male mating history on last male precedence in three-time mated females. Offspring
585 percentages are those presented in (a) for three-times mated females. Data were separated
586 between offspring sired by males having mated with other females twice or less before
587 and those that had mated more, as indicated below the X-axis. Asterisks indicate
588 significant differences determined by a two-tailed Student’s t-test. Error bars: ±SEM.
589 Sample size is between brackets.
590 27 591 Figure 4: Re-Mating differs in (a,b) frequency and (c,d,e) temporal distribution between
592 wild-type strains because of strain differences in female behaviour (f,g). (a) Mean
593 number of matings over 24-hr per individual in groups of six males and six females.
594 Number of matings was estimated by dividing the sum of matings in each experiment by
595 six (the number of potential couples). Each replicate was taken as a single data point. (b)
596 Fraction of individuals that mated more than once. (c) Time to first re-mating. Values
597 derived from the mean time to first six re-mating. The horizontal bars in box plots
598 represent the median, boxes denote 25th -75th percentile range, and whiskers the min. and
599 max. data points. (d) Temporal distribution of mating. Fraction of the total mating
600 observed over 24 hrs binned per 2 hrs. (e) Temporal distribution of mating as in (d), but
601 with males and females from different strains. (f) Mean number of mating per individual
602 in groups of six males and six females from different strains. Asterisks indicate
603 significant differences determined by ANOVA followed by Tukey-Kramer post-hoc test
604 (**, P<0.001; ***, P<0.0001).
605 combinations of females and males of the indicated genotypes. Box plots are as in (c).
606 White or grey boxes indicate significant differences (ANOVA, P<0.0001). Sample size is
607 between brackets. Error bars: ± SEM except for (c,g).
(g) Time to first re-mating measured as in (c), in
28 Billeter et al.
Figure 1
(a1) Mating success
(b1)
1
6:0
5
4:2
3:3
2:4
Canton-S
4
3
2
1
0
6:0
4:2
Oregon-R sire
Canton-S sire
(b2)
2:4
120
100%
Oregon-R
F1 genotype
100
80
60
40
0
70
60
50
40
30
20
10
0
Oregon-R
80%
60%
40%
20%
20
6:0
4:2
3:3
2:4
Canton-S
(c2)
0%
6:0
4:2
3:3
Canton-S
100%
F1 genotype
# progeny per female
2
0
# matings per male
Oregon-R
3
(a2)
Offspring genotype frequency
Oregon-R
Canton-S
# progeny per female
# matings per male
Oregon-R
Canton-S
4
(c1)
Reproductive success
80%
60%
40%
20%
6:0
4:2
2:4
0%
6:0
4:2
2:4
2:4
Billeter et al.
# matings per male
(a1) Canton-S
9
8
7
6
5
4
3
2
1
0
(a2) Oregon-R
(25)
(16)
Figure 2
(14)
n.s.
4
3
(16)
9
8
7
6
5
4
3
2
1
0
(22)
2
A
A
B
1
0
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
ZT
n.s.
(9)
6 females X 4 males
6 females X 6 males
6 females X 4 males, 2 y,w males
(10)
6 females X 4 males
6 females X 6 males
6 females X 4 males, 2 y,w males
(b3) Orco (Canton-S)
(b2) Oregon-R
(b1) Canton-S
Mean # matings [2 hr bin]
(a3) Orco (Canton-S)
2
1.5
1
0.5
8
10 12 14 16 18 20 22 24
2
4
6
0
ZT
8
10 12 14 16 18 20 22 24
2
4
6
3.5
3
2.5
2
1.5
1
0.5
0
ZT 8
10 12 14 16 18 20 22 24
2
4
6
Billeter et al.
% offspring sired by last male
(a1)
Figure 3
(a2)
non-GFP male
GFP male
100
(9)
100
(28)
80
(15)
B
C
(2)
1
2
3
4
5
Position in mating series
20
0
(22)
A
B
non-GFP male
GFP male
(6)
80
(21)
40
A
Effect of prior male matings on
sperm competitive ability
*
100
60
(4)
40
0
(9)
80
60
20
(b)
(4)
(4)
C
1
2
3
4
5
Position in mating series
60
n.s.
40
20
0
≤2
(6)
(7)
>2
≤2
(9)
>2
Number of prior matings
Figure 4
Billeter et al.
** ***
7
(18)
6
5 (36)
**
***
(6)
4
(30)
(28)
3
(8)
2
1
0
Canton-S Oregon-R
4
Canton-S
Oregon-R
CO-3
**
(19)
(13)
(14)
CO-3
6
Ca
5
5
0
0
ZT 8 10 12 14 16 18 20 22 24 2
(g)
16
Females
Canton-S Oregon-R
12
8 (36) (11)
4
(26)
(18)
0
Males
4
6
(11)
18
(4)
16
(30)
(5)
14
(8)
(5)
12
10
8 (33)
6
4
2
0
n
O tonre S
go
nCO R
-3
Am TW
he -1
Fl rstor 3
U ida
rb -9
an
aS
Time to 1st re-mating [Hr]
on
re S
go
nR
CO
-3
TW
Am -1
he
r
Fl st-3
or
id
U a-9
rb
an
aS
nt
10
re S
go
nR
ZT 8 10 12 14 16 18 20 22 24 2
10
O
0
15
-R
5
15
Canton-S
on
10
20
Canton-S (36) 25
Oregon-R (18)
20
25
on
15
(e)
nt
Canton-S (34)
Oregon-R (30)
re
g
# matings per individual
(f)
20
0
Ca
% total matings [2 hr bin]
(d)
(9)
O
nt
o
O n-S
re
go
nR
CO
-3
TW
Am he 1
Fl rstor 3
id
U a-9
rb
an
aS
0
(10)
20
-S
1
(13)
40
on
(10)
(9)
nt
(13)
O
(24) (10)
Percent re-mating [%]
2
(33)
(10)
60
Ca
3
(24)
80
% total matings [2 hr bin]
4
(36)
(c)
(36) (33)
100
Time to 1st re-mating [Hr]
5
(b)
Ca
6
Ca
# matings per individual
(a)
Oregon-R
Oregon-R (30)
Canton-S (28)
ZT 8 10 12 14 16 18 20 22 24 2
6
4 6
Billeter et al.
Figure S1
Female mating frequency
6
0
2
4
3
3
4
2
(14)
(14) (15) (15)
2
Canton-S
Oregon-R
1
0
# progeny
(10) (16)
Canton-S
4
3
0
6
6
0
0
6
Female fecundity
Oregon-R
150
(16)
1
0
(b1)
(b2)
2
4
4
2
(9) (9) (11)
(10) (13)
100
50
0
Canton-S
Oregon-R
# progeny
# matings per female
(18)
2 (17)
Canton-S
Oregon-R
(a2)
Oregon-R
3
# matings per female
(a1)
6
0
0
6
4
2
3
3
2
4
Canton-S
150
100 (13) (14) (15)
(14)
50
Canton-S
Oregon-R
0
6
0
0
6
2
4
4
2
Figure S1. Social context does not affect (a) female mating frequency and overall (b) female fecundity. A group of 6 Oregon-R females (top row of graphs) or 6 Canton-S females (bottom row) were
housed for 8hrs with a group of 6 males composed of males from the Canton-S strain and/or Oregon-R strain at a range of ratios indicated below the x-axis to create different social contexts. (a) The
mean number of matings per (a1) Oregon-R or (a2) Canton-S female was determined in different
social contexts. These social contexts did affect the mating frequency of neither (a1) Oregon-R
(ANOVA: F4,73=1.80, P=0.14), nor (a2) Canton-S females (ANOVA: F3,54=1.22, P=0.47). Females
were isolated at the end of the experiment and the adult offspring they produced were counted. (b)
Mean number of offspring from individual females from the (b1) Oregon-R and (b2) Canton-S strain
that mated in different social contexts. Number of replicates is between brackets. Error bars indicate
± SEM.
Figure S2
Billeter et al.
(a)
(b)
Canton-S
A6
A7
(c)
Oregon-R
A6 A7
Canton-S
Oregon-R
A6 A7
Figure S2. Pattern of female abdominal pigmentation in different strains of D. melanogaster. Pictures show the dorsal segments of filleted abdomens in (a) Canton-S and (b)
Oregon-R females or (c) daughters from a cross between a Canton-S male and an Oregon-R female. The terminal posterior segments, termed A6 and A7, are indicated on the
pictures. A6 and A7 are darker in Canton-S than in Oregon-R females. The darker
pigmentation of Canton-S is dominant over the lighter pigmentation of Oregon-R, and (c)
daughters from an Oregon-R female crossed to a Canton-S male have a Canton-S pattern
of abdominal pigmentation.