Journal of Physiology 46 (2000) 1355–1363 www.elsevier.com/locate/jinsphys

Copula in yellow dung flies ( stercoraria): investigating competition models by histological observation D.J. Hosken *, P.I. Ward

Zoologisches Museum, Universita¨tZu¨rich-Irchel, Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland

Received 13 October 1999; received in revised form 21 February 2000; accepted 22 February 2000

Abstract

While has been extensively studied, the mechanisms involved are typically not well understood. Nevertheless, awareness of sperm competition mechanisms is currently recognised as being of fundamental importance for an understanding of many behavioural strategies. In the yellow dung fly, a model system for studies of sperm competition, second male sperm precedence appears to result from a combination of sperm displacement and sperm mixing. Displacement was until recently thought to be directly from the female’s sperm stores, the spermathecae (i.e. males were thought to ejaculate directly into these stores), and under male control. However, recent work indicates displacement is indirect (i.e. males do not ejaculate directly into the sperm stores) and that it is female-aided, although the evidence was not based on direct observation. Here, we used histological techniques to directly determine interactions during copula and sperm transfer. Our results are consistent with inference and clearly show that males ejaculate into the bursa copulatrix. Our data are also consistent with active female involvement in sperm displacement, which is indirect, and indicate the aedeagus may remove some spermatozoa from the bursa at the end of copula. In addition, evidence suggests females aid sperm transport to and from the spermathecae, possibly by muscular movement of a spermathecal invagination.  2000 Elsevier Science Ltd. All rights reserved.

Keywords: Scatophaga; Functional anatomy; Sperm precedence; Mechanism; Sperm removal

1. Introduction 1997). Nevertheless, sperm competition mechanisms are largely unknown in most taxa, although the importance Since Parker’s (1970a) ground-breaking conceptualis- of understanding them is becoming increasingly clear ation of sperm competition, the yellow dung fly since the adaptiveness of many reproductive processes, (Scathophaga stercoraria (L.)) has been extensively for example prolonged , cannot be appreciated investigated and has become a model system for sperm otherwise (Eberhard, 1996; Birkhead, 1998; Simmons competition studies (e.g. Parker, 1970b; Simmons and and Siva-Jothy, 1998). Parker, 1992; Ward and Simmons, 1991; Ward 1993, In yellow dung flies, males which copulate last typi- 1998; Simmons et al., 1996; reviewed in Hosken, 1999). cally fertilize about 80% of the subsequent clutch (i.e. Many aspects of sperm competition are difficult to exam- P2, the proportion of eggs fertilized by the second male ine directly since they take place within the female. to mate in twice mated females, Ϸ 0.8) (Parker, 1970b). Therefore, models have been constructed which assume The mechanism of second (last) male sperm precedence different mechanisms of sperm competition and generate involves sperm displacement, with second males displac- different paternity outcomes. These are then checked ing about 80% of previously stored sperm, since 1) the against empirical paternity data, and the mechanism(s) number of sperm stored by females does not appear to of sperm competition inferred (Birkhead and Parker, increase with successive copulations (Parker et al., 1990), and 2) P2 is strongly dependent on copula dur- ation (Parker, 1970b). Parker and Simmons (1991) mod- * Corresponding author. Tel.: +41-1-635-4974; fax: +41-1-635- elled sperm competition in yellow dung flies, treating 6818. the females’ three sperm storage organs, the sperma- E-mail address: [email protected] (D.J. Hosken). thecae, as a single unit and they assumed that males

0022-1910/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S0022-1910(00)00057-3 1356 D.J. Hosken, P.I. Ward / Journal of Insect Physiology 46 (2000) 1355–1363 directly displaced sperm from them (i.e. males ejaculate portion. Copulations proceeded for a set duration (5, 10, directly into the sperm stores). In their model, incoming 15, 20, 25, 30 min) or to completion, at which times sperm mix instantaneously with previously stored sperm pairs were either immersed in liquid nitrogen and/or within the spermathecae, and constant, random displace- transferred to Carnoy’s fixative, or separated and the ment of mixed sperm directly from these stores is females immediately placed with new males and allowed assumed (Parker and Simmons, 1991). The model also to copulate (again for set durations or to completion) for assumes displacement is under direct male control and a second time and treated as above. This protocol simu- also that stored sperm is displaced directly from the lated take-over situations; when males interrupt copu- female’s sperm stores at the same rate as incoming lations of rivals, and after a struggle, copulate with the ejaculate, with sperm therefore moving both up and female themselves (see Parker, 1978). In addition, some down the spermathecal ducts simultaneously. females copulated a second time one or two weeks later, While the average outcome of sperm competition in and some pairs were not immersed in liquid nitrogen dungflies is neatly explained by the direct displacement before fixing as this procedure may disrupt some tissue. model, it seems unlikely that it is an accurate description However, immersion in liquid nitrogen has the advan- of copulatory events, if only because it treats the three tage of stopping all movement virtually instantaneously, spermathecae as a single unit (Parker and Simmons, thus allowing genital interactions to be observed and 1991; Ward, 1993; Birkhead and Parker, 1997). More- sperm location within the female reproductive tract at over, sperm storage in yellow dung flies appears to be a particular time to be assessed (see e.g. Eberhard and influenced by male and female interactions (Otronen et Pereira, 1997). al., 1997; Hellriegel and Bernasconi, 2000), and recent After fixing overnight, flies’ abdomens were removed, work by Simmons et al. (1999) using radio-labelled washed in n–butanol and left overnight in a butanol and ejaculates provides strong evidence that direct displace- paraffin wax mixture. Specimens were then transferred ment does not occur from the spermathecae. Simmons to pure wax (60°C) and, after several wax changes, were et al.’s (1999) results indicate that sperm movement to wax embedded. Sections were cut at 6–8 µm, stained and from the sperm stores may be female mediated, as with haematoxylin (either Harris’s or Mayer’s), counter originally suggested by Ward (1993). However, they stained with eosin, and slide mounted (Bancroft and Ste- could only speculate on the mechanism involved and vens, 1990). Specimens whose internal anatomy was there have been no direct observations of sperm transfer measured were all oriented approximately the same way to confirm their findings. during embedding and we assumed this standardised Studies of sperm movement in other taxa (e.g. Waage, internal organ orientation. 1979; Radwan and Witalinski, 1991; Gack and Peschke, Slides were examined under a Wild M3C microscope 1994; Civetta, 1999) indicate that proposed mechanisms fitted with a Donpisha 3CCD camera that relayed images of sperm precedence can be either ruled out or more to a PC running Bioscan Optimas software, which was accurately inferred by direct observation. This in turn used to make all measurements. The hind tibia lengths enables us to better understand behavioural strategies of all males and females were measured, and in females employed during sperm competition (Birkhead, 1998). not immersed in liquid nitrogen the volume of sperma- The aim of the present study is to investigate sperm thecae was calculated (internal and external using the transfer in S. stercoraria using histological techniques formula for a prolate spheroid: 0.523×L×W2; Abbott and in an attempt to better understand genitalic interactions, Hearns, 1978), as was the length of the spermathecal sperm movement and precedence, and hence the mech- invagination, a finger-like projection into the spermathe- anism of sperm competition. At the same time, we test cal lumen. We used non-frozen females to avoid possible the validity of Simmons et al.’s (1999) recent con- tissue distortion caused by freezing. In addition to these clusions. measurements, fly anatomy, and, for pairs frozen in liquid nitrogen, genital interactions and the location of sperm within the female reproductive tract were noted. 2. Materials and methods Genitals were said to be fully engaged when the aede- agus was still seen to closely align with the spermathecal collected from dung pats in fields near Fehral- duct openings, i.e. there was no indication of aedeagus torf, Switzerland were brought to the laboratory and withdrawal. In our analysis, the spermathecal duct was allowed to lay eggs. From these clutches, virgin male divided into three sections: the portion at the junction and female flies were obtained (Ward and Simmons, with the bursa, the middle segment, and the section at the 1991). Virgin females were fixed in Carnoy’s solution junction with the spermathecae. Sperm were recorded as (Bancroft and Stevens, 1990) either without copulating present or absent from these segments. We used logistic (i.e. remained virgin), or were allowed to copulate (non- regression, a regression technique for dichotomous inde- virgins). For copulations, one male and one female fly pendent variables, to analyse these data. Sample sizes were placed into a small vial containing a small dung vary because of leg loss during freezing, damage to sec- D.J. Hosken, P.I. Ward / Journal of Insect Physiology 46 (2000) 1355–1363 1357 tions and because in some analyses only frozen or non- frozen specimens were used, as appropriate.

3. Results

The female reproductive tract is characterised by paired accessory glands, three spermathecae (a singlet and a doublet), each with its own narrow duct, and a large muscular bursa copulatrix which is met by the common oviduct anterio-ventrally (Ward, 1993). In addition to its substantial musculature, the copulatory bursa is also heavily cuticularised, presumably to afford females some protection from the numerous spikes and spines on the males aedeagus. A full anatomical descrip- tion is presented in Hosken et al. (1999). Here we con- centrate on features relevant to sperm competition mod- els. During copula the aedeagus is deeply inserted into the bursa copulatrix, with the gonopore facing and almost abutting directly against the spermathecal duct openings, and it was at this point that free sperm entered the female tract (Figs. 1 and 2). Close aedeagus/spermathecal duct alignment is typical of many Diptera (e.g. Lachmann, 1997), and this congruence indicates the genital align- ment described here is unlikely to have been altered by tissue-processing, and therefore accurately represents alignment during copula. However, some male abdomi- nal movement sporadically occurs during copula, but

Fig. 2. Longitudinal sections through copulating pairs of dung flies focusing on the area approximately from a to g in Fig. 1. (A) The phallomere orientation during copula (females posterior to left, and the spermathecal ducts, not yet in view will appear to the upper right of the gonopore; scale bar=30 µm); (B) A higher magnification view of the gonopore and its association with the spermathecal duct openings in another female (females posterior to right; scale bar=22 µm). Note the large amounts of sperm (arrows) around the “scraper” (s) in (A), and the movement of sperm away from the spermathecal ducts (arrowed) but sperm within the duct in (B). Key: a=aedeagus, bc=cuti- cle lining the bursa; bm=muscle of the bursa; c=male clasper; d=sper- mathecal duct; dt=female digestive tract; ed=ejaculatory duct (full of sperm); g=gonopore; o=common oviduct; s=aedeagus “scraper” a scoop-shaped aedeagus extension; and arrows point to sperm, the dark Fig. 1. Diagrammatic representation of a copulating pair of dung mass around the scraper and leaving the gonopore. flies. Dotted line shows the approximate plane of rotation of the scraper (s) when the male removes his aedeagus (see text and Fig. 5). Key: a=aedeagus; ab=female’s abdomen; c=male’s clasper; d=spermathecal there is no musculature that appears able to move the duct; dt=female’s digestive tract; f=male’s forcep, a bilobed sternital projection; g=gonopore; p=anterior pouch of the bursa; pa=male’s par- aedeagus alone (Figs. 2 and 5A). Interestingly, logistic amere; st=spermatheca, showing detail of the invagination and its asso- regression indicated that genitals were more likely to be ciated muscle (see text and Fig. 4). engaged after freezing and processing (i.e. as in Fig. 2) 1358 D.J. Hosken, P.I. Ward / Journal of Insect Physiology 46 (2000) 1355–1363 in females whose first copula was shorter (ß=Ϫ0.1521; Wald statistic=4.2; P=0.039; Fig. 3). This effect was independent of the duration of the second copula, or of male or female size, all of which had no significant effect on the likelihood of genitals remaining engaged (all PϾ0.27). It is noteworthy that the probability function is approximately linear and decreasing until just below a first copula duration of 40 minutes (i.e. a normal mean copulation duration, Parker, 1970b), and then flattens out. Spermatozoa were always found in the spermathecae within five minutes of the commencement of the first copula of virgin females (n=6). Such rapid arrival of spermatozoa has been interpreted as evidence of female involvement in sperm storage in other flies, as calculated swimming speeds and distances travelled indicate arrival time is too rapid to be accounted for by swimming alone (Linley and Simmons, 1981). Spermatozoa were also always present in the spermathecae of females which had copulated for longer (5–30 minutes) or had copulated twice (n=54). However, sperm did not always appear to be evenly distributed either among females with copu- Fig. 4. Sections through the spermathecae and spermathecal ducts of three different females, whose first copulations all went to completion, lations of similar durations (Fig. 4), or across a females’ after 20 minutes of their second copulation (A)(B)(C) and (D) shows spermathecae when copula was interrupted (see e.g. Fig. the invagination muscle attaching to the common oviduct. Key: ag=ac- 2; Hosken et al., 1999). In addition, spermatozoa were cessory gland, d=spermathecal ducts, i=spermathecal invagination, not always present throughout a female’s reproductive o=common oviduct, m=muscle from oviduct to invagination and tract (18 of 28 females frozen in copula, and 10 of 17 arrows mark the duct junction with the spermatheca. In (A), sperm are clearly visible within the spermatheca, but few-to-no sperm are visible females with genitals still engaged, lacked sperm at the duct entrance to the spermatheca or within the ducts (scale throughout their tracts. Sample sizes here indicate only bar=35 µm). In (B), the entrance to the spermatheca is almost totally specimens for which all sections could be examined). occluded by sperm (scale bar=15 µm). (C) shows a section of duct, Specifically, segments, or in extreme cases the whole approximately mid-way between the bursa and spermathecae, that is essentially totally occluded by spermatozoa (scale bar=30 µm). (D) shows a cut-away of the spermathecal invagination (i) revealing the inserted muscle (m) that connects to muscle surrounding the common oviduct (o) (scale bar=60 µm). Note that for (A)(B) and (C) males were still ejaculating.

length, of a spermathecal duct was often devoid of sperm in spite of the fact that males were still coupled and ejaculating and sperm were in the spermathecae (Fig. 3). Uneven sperm distribution was not due to tissue pro- cessing as sperm were absent from all of some duct por- tions but not others, even when they appeared on the same slide. It was also not due to observations of differ- ent portions of the female tract as all sections/female were examined. Occasionally sperm could even be seen to be leaving the gonopore and the orientation of the sperm (inferred from the staining: sperm heads stain darker than tails) indicated they were moving away from the ducts back down the bursa (Fig. 2B). Additionally, Fig. 3. The logistic regression results showing genital engagement sperm often occluded duct entrances to spermathecae during second copulations plotted against duration of first copula. during copula, although this was not always the case Genitals were said to be engaged (Yes) if the aedeagus was still aligned (Fig. 4: 30 spermatheca/duct junctions of females frozen with the spermathecal ducts (i.e. Figs. 1 and 2), and not engaged (=No) in copula (n=37 females) were judged as being totally when aedeagus withdrawal had begun (i.e. Fig. 5B). Included is a prob- ability plot (solid curve) of the likelihood of finding genitals engaged occluded, another 75 contained fewer or no sperm, six relative to the duration of first copulas, and note the line joining the were damaged), and there was no obvious pattern to the yes/no data is for visual purposes only. distribution of sperm. Neither male size, female size, D.J. Hosken, P.I. Ward / Journal of Insect Physiology 46 (2000) 1355–1363 1359 relative size of males to females, or copula duration or delay was significantly associated with sperm presence or absence from portions of the ducts or the occlusion of the spermathecal entrances (analysed as sperm present throughout or absent in portions of the singlet or doublet ducts within females) (Logistic regression; Ϫ16.1ϽßϽ13.1; all Wald statistic valuesϽ0.52; all P- valuesϾ0.47). There was strong evidence that males ejaculate in a continuous fashion: sperm were seen leav- ing the gonopore or within the ejaculatory duct of all frozen pairs that maintained genital contact, regardless of the copula duration (copula durations 5–30 mins; n=17). However, as stated above, in spite of obvious ejaculation and close aedeagus/spermathecal duct alignment, sperm were not always found in the spermathecal ducts. Inter- estingly, in the few cases where the direction of sperm movement/orientation within the spermathecal ducts could be inferred, there was no indication of bi-direc- tional movement: sperm were all apparently aligned the same way, either towards or away from the sperma- thecae, even in females during their second copulation (n=4). Moreover, in these (and other) instances, sperm virtually totally occluded the ducts (28 of 37 females frozen during copula had at least one duct occluded; Fig. 4C), making it highly unlikely that an equal volume of sperm, or any fluid for that matter, could also be moving in an opposite direction within the duct at the same time. Sperm were typically not found in the bursa copulatrix anteriorly of the spermathecal duct entrance (33 of 37 frozen pairs), but were often seen caudally to the sperm- athecal duct/bursa junction in the vestibular region of the bursa (n=36 of 37 frozen pairs) (Fig. 2). Additionally, in a number of frozen specimens large quantities of sperm were noted externally within the males genital pouch and around the females rectum (13 of 37). During copula, large quantities of sperm were also typically found around an aedeagus projection caudo-ventrally to the spermathecal ducts and gonopore (27 of 37 frozen pairs: Fig. 2), or within crypts or folds in the cuticular Fig. 5. (A) A whole mount of the aedeagus showing the phallosome (p) and the position of the “scraper” (s) (scale bar=20 µm). (B) The lining of the dorsal surface of the bursa, also caudally aedeagus during withdrawal (females posterior to left) (scale bar=18 of the spermathecal ducts. Interestingly, when the aede- µm). Note the change in position of the “scraper” (s) compared with agus is being withdrawn, the method of collapse drags Fig. 2 (and see Fig. 1 for diagrammatic representation of scraper the projection, around which large quantities of sperm movement). Key: bc=cuticle lining the bursa; c=male clasper; d=sper- = = = was noted (a scoop-shaped section of the aedeagus; Fig. mathecal duct; dt female digestive tract; ed ejaculatory duct; g gono- pore; p=base of phallosome; s=aedeagus “scraper”. 2), through the copulatory bursa (Figs. 1 and 5). This mode of aedeagus withdrawal apparently removes some sperm from the bursa. However, the location of these theca (Fig. 4A). This invagination is filled with muscle spermatozoa, especially those sequestered within cuticu- that attaches to the oviduct (Fig. 4D), and muscle con- lar folds, plus the folded nature of the cuticle lining the tractions could move it in and out in a piston-like man- female reproductive tract are likely to make mechanical ner, creating negative pressure in the spermatheca aiding removal difficult, and quantities of sperm were seen left sperm movement to storage. Supporting this hypothesis, behind by the scoop-like projection. In addition, varying the variation in the proportional depth to which this inva- amounts of sperm were noted in the bursa of females gination projected differed significantly between virgin whose copula had naturally ceased (10 of 11 specimens). and non-virgin females (two-tailed variance test ratio; = = 2 = 2 = Each spermatheca has a large finger-like invagination v1 v2 11; S non-virgins 0.005; S virgins 0.001; opposite the spermathecal duct entrance to the sperma- F=6.032; PϽ0.01). That is to say, there was more vari- 1360 D.J. Hosken, P.I. Ward / Journal of Insect Physiology 46 (2000) 1355–1363 ation in non-virgins than virgins, indicating there is and that female sperm stores have a fixed internal vol- (more) invagination movement in the former but not the ume, as we found. We also found a positive association latter. However, there was no significant difference in between female size and total internal store volume, the mean proportional depth (length of the which is arguably expected since organs generally scale invagination/internal diameter of the spermathecae) to with body size (Calder, 1984; and see Parker et al., 1999; which the invagination projected into the spermathecae Gage, 1998), but also has implications for how female of virgins and non-virgins (mean depth virgins 44.3%, variance in P2 is viewed: for example, can variance in non-virgins 47.5%: two tailed t–test comparison of P2 be attributed to cryptic female choice or not (Pitnick arcsin transformed %’s:–df=22; t=Ϫ1.422; P=0.17; and Brown, 2000). power of detecting mean differenceϾ0.8). Moreover, Sperm movement from the bursa to the spermathecae there were no other obvious anatomical differences appears to be intermittent and the anatomical data between virgin and non-virgin sperm stores. For strongly suggest female involvement in sperm move- example, while the total internal volume of a female’s ment (cf. Ward, 1993; see also Otronen et al., 1997; sperm stores was positively associated with body size Ward, 1998; Simmons et al., 1999; Hellriegel and Berna- = 2= = = (n 18; r 0.33; F1,16 7.39; P 0.01), there were no sig- sconi, 2000). In addition to the bursa being the ejacu- nificant differences in total spermathecal volume of vir- lation site, several other lines of evidence support these gins and non-virgins after correcting for body size conclusions. Firstly, the distribution of sperm within the (taking the residuals from the least squares regression) female’s spermathecal ducts and spermathecae during (df=14; t=0.295; P=0.77; power 0.8–0.9). This compari- copula was uneven. In many instances sperm were found son assumed the spermathecae were prolate spheroids in the spermathecae and could be seen leaving the aede- and ignored the spermathecal invaginations, and implies agus, but there was no sperm in the spermathecal ducts, that spermathecae are of fixed volume. and at times sperm could be seen to be moving out of the gonopore but away from the ducts in spite of the close aedeagus/duct alignment. This also indicates sperm 4. Discussion motility is unlikely to be solely responsible for sperm movement to storage as sperm distribution in the female Our examination of copula in the yellow dung fly tract would presumably be more even otherwise, clearly shows the site of ejaculation is the bursa cop- especially because males ejaculate continuously. Fur- ulatrix, at or very near the entrances of the spermathecal thermore, anaesthesia of female dung flies after copula ducts. Thus our anatomical data indicate that the direct clearly alters sperm distribution patterns (Ward, 1998; displacement model is not an accurate description of Hellriegel and Bernasconi, 2000). Moreover, Linley and events during copula in yellow dung flies, as previously Simmons (1981) provide compelling evidence that suggested (e.g. Parker and Simmons, 1991; Ward, 1993; sperm motility is unlikely to contribute to spermathecal Birkhead and Parker, 1997). Moreover, not only is there filling in a range of Diptera, since sperm arrive in storage no evidence for direct displacement from the sperma- faster than they could swim (see also Clement and Pot- thecae, there is also no indication that equal volumes of ter, 1967), and Arthur et al. (1998) clearly show that sperm move, or could move, both ways in the ducts. without full female nervous involvement, sperm storage Independently-derived data also indicate the bursa is the in melanogaster is severely hampered. We site of ejaculation (Simmons et al., 1999), further sup- also found sperm within the spermathecae soon after porting the notion of indirect sperm displacement: that copula begins. The rapid arrival of sperm to storage sites is, sperm are not deposited directly into the sperm stor- is in agreement with the findings of Parker et al. (1990) age organs as previously assumed. Additionally, a num- who reported relatively high female fertility with copu- ber of Simmons et al.’s (1999) assertions and assump- lations as short as five minutes. Finally, the nature of tions are justified by our data. Simmons et al. (1999) the duct/aedeagus alignment makes it difficult to imagine suggested there is no aedeagus extension which can how males could force fluid both up and down the reach the sperm stores, we found none, and that the aede- ducts simultaneously. agus and spermathecal ducts align during copula, as Other researchers have also suggested that female flies described in other flies (e.g. Lachmann, 1997), and as must aid sperm movement, and that bi-directional sperm we noted here. This general concordance with the typical movement is unlikely within narrow spermathecal ducts fly aedeagus/spermathecal duct alignment also indicates (e.g. Linley and Simmons, 1981; Ward, 1993; Birkhead that genital alignment in our flies is unlikely to have and Parker, 1997; Simmons et al., 1999). Linley and been disturbed by processing. Simmons et al. (1999) also Simmons (1981) suggested fluid is actively pumped out suggested that males do not introduce most of their of dipteran spermathecae creating negative pressure (due sperm into the bursa at the commencement of copula, in part to the thick-walled, fixed-volume sperm stores) which agrees with our data (e.g. spermatozoa were still which draws up sperm (and fluid) from the copulatory seen leaving the aedeagus after 30 minutes of copula), bursa, and Simmons et al. (1999) speculated that duct D.J. Hosken, P.I. Ward / Journal of Insect Physiology 46 (2000) 1355–1363 1361 contractions aid sperm movement. This second mech- indicating that after exceptionally long first copulations, anism has also been proposed for a mosquito based on full genital engagement is highly unlikely during second spermathecal duct morphology (Clement and Potter, copulas. Why this occurs is unclear, but will be the sub- 1967), and yellow dung flies have ducts containing ject of future investigation. essentially the same structures (Hosken et al., 1999). The discovery of a scoop-shaped section of the aede- However, while we have no direct evidence for either agus which appears to remove some sperm from the of these mechanisms, one female contrivance likely to bursa is of particular interest. Since females typically lay aid sperm movement in dung flies was found. The sper- immediately after copula and fertilization occurs in the mathecal invagination could potentially act like a bursa, such sperm removal may be an additional means hydraulic piston assisting sperm transit. This inva- of sperm precedence, and analogous devices have been gination is filled with muscle that attaches to the com- described in other (e.g. Waage, 1979; and see mon oviduct. By moving the invagination females could Simmons and Siva-Jothy, 1998). However, since fertiliz- create negative or positive pressure within the sperma- ation appears to occur more anteriorly than the locations thecae moving fluid (and sperm) in or out. Consistent of the displaced sperm the evolutionary significance of with the idea that females pump sperm up and down the direct removal remains unclear. Removal may be non- ducts is the finding that variation in the proportional selective, simply being a by-product of the method of depth to which the invagination projected into the sperm- aedeagus collapse (e.g. Siva-Jothy et al., 1996), and athecae was significantly greater in non-virgins than vir- sperm was found in the bursa of singly-mated females. gins. This mechanism seems especially plausable Nevertheless, partial sperm removal together with the because of the chitinized nature of the spermathecae and often cryptic location of sperm within the bursa may their fixed volumes. Moreover, deformations of this nat- explain why Otronen et al. (1997) detected few sperm ure are thought to aid sperm movement in a number of there. Similarly, partial sperm removal may partly other insects (Happ and Happ, 1975; also see Eady, explain why Simmons et al. (1999) found proportionally 1994), and spermathecal muscles have similarly been less second male ejaculate in the bursa than predicted. implicated in sperm storage in a number of taxa although Occasional loss of ejaculate from the female tract, as the mechanics of the systems differ markedly. For noted by us, would also help to explain this discrepancy. example, Rodriguez (1994) showed that cutting the sper- While a number of studies now unequivocally indicate mathecal muscle in a altered sperm distribution that sperm displacement, and other reproductive pro- and reduced fertility (and see e.g. King, 1962; Wilkes, cesses, in flies involve male and female interactions (e.g. 1965). We speculate that stretching of the female tract Arthur et al., 1998; Clark et al., 1999; Hosken and Ward, provides the stimulus for pumping since both copula and 1999; Otronen and Siva-Jothy, 1991; Rice, 1996; Ward, egg-laying require movement of sperm, and both stretch 1998), male yellow dung flies obviously influence the the female tract (Hosken et al., 1999), but note that other outcome of sperm competition: copulation duration gre- mechanisms may also be involved in sperm movement. atly influences P2, and when allowed to copulate freely, Interestingly, we occasionally noted what appeared to be uneven distributions of sperm across individuals’ males on average gain 80% of paternity in the sub- spermathecae and within the ducts when copulations sequent clutch. However, the variability in P2 (e.g. were interrupted (Ward, 1993; see also Hosken et al., Parker, 1970b; and see Simmons and Siva-Jothy, 1998) 1999; Hellriegel and Bernasconi, 2000). This may have also requires explanation (Lewis and Austad, 1990), and been due to filling order effects, the result of asynchro- active female involvement in sperm storage is one mech- nous female pumping to fill spermathecae, direct female anism which could conceivably generate this variation manipulation of ejaculates, aedeagus/duct alignment or (Hellriegel and Ward, 1998). Simmons et al. (1999) ask any combination of these factors. The assortative storage why females should assist sperm displacement? It may of sperm noted by Otronen et al. (1997) may also be be that males cause female responses via their seminal explained by these mechanisms as copulas were also fluids (as in and many other interrupted in their experiments. Importantly however, flies; Kubli, 1996; Chapman et al. 1995, 1998; Wolfner, females can selectively utilize sperm to a degree based 1997), and male dung flies have a large glandular on relative PGM genotype (Ward, 1998), suggesting an secretory surface in their ejaculatory duct (Hosken et al., even more active role in sperm storage/use than the ana- 1999). Additionally, male genital movement may stimu- tomical data. late egg-laying receptors causing females to begin sperm Our results also provide evidence that the strength of mobilization (for discussion of such mechanisms see genital coupling is influenced by a female’s previous Eberhard, 1996). Alternatively and/or additionally, it mating experience, with genitals staying fully engaged may pay females to increase the diversity of sperm in in females with shorter previous copulations. It thus storage since doing so may allow them to adaptively appears that uncoupling occurs more quickly after longer manipulate offspring genotype or avoid genetically first copulations, with the shape of the logistic curve incompatible males (e.g. Ward, 1998; Tregenza and 1362 D.J. Hosken, P.I. Ward / Journal of Insect Physiology 46 (2000) 1355–1363

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