Associations between female remating behavior, oogenesis and oviposition in Drosophila melanogaster and Drosophila pseudoobscura

Journal of Insect Physiology 46 (2000) 1489–1496 www.elsevier.com/locate/jinsphys

Associations between female remating behavior, oogenesis and oviposition in Drosophila melanogaster and Drosophila pseudoobscura Rhonda R. Snook *, Yee K. So Department of Biological Sciences, 4505 S. Maryland Parkway, University of Nevada Las Vegas, Las Vegas, NV 89154-4004, USA Received 10 January 2000; accepted 4 April 2000

Abstract An association between female remating behavior, oogenesis and oviposition was examined in Drosophila melanogaster and Drosophila pseudoobscura to investigate mechanisms that elicit remating. Females receptive to remating oviposited more eggs in both species; however, the species differed in the association between remating behavior and the number and distribution of oocyte stages. We found no differences in the number of either developing eggs of different stages or mature eggs between female D. pseudoobscura that were either receptive or nonreceptive to remating. In contrast, D. melanogaster females that are receptive to remating had significantly more mature eggs in the ovaries than nonreceptive females. Nonremating females had a significantly greater number of immature, vitellogenic oocytes. These results suggest that factors associated with oogenesis are related to female remating behavior in D. melanogaster but not in D. pseudoobscura. We discuss these results in conjunction with other evidence on the role male ejaculatory components play in mediating female remating behavior.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Remating; Oogenesis; Oviposition; Drosophila; Ejaculate

1. Introduction The study of insect mating systems has frequently served to link physiological and behavioral traits in an evolutionary context. One recurrent issue is the adaptive significance of female remating behavior on the reproductive success of both females and males (Thornhill and Alcock, 1983; Birkhead and Møller 1992, 1998; Chapman et al., 1995; Rice, 1996) and the physiological factors of both males and females that control female remating behavior (Manning 1962, 1967; Fuchs and Hiss, 1970; Leopold, 1970; Gromko et al., 1984; Scott, 1987; Young and Downe, 1987; Bergh et al., 1992; Gromko and Markow, 1993; Kalb et al., 1993; Kubli, 1996; Yuval et al., 1996; Wolfner, 1997; Miyatake et al., 1999). At least nine hypotheses have been suggested to explain the ultimate significance of female remating

* Corresponding author. Tel.: +1-702-895-1395; fax: +1-702-8953956. E-mail address: [email protected] (R.R. Snook).

behavior (Halliday and Arnold, 1987) and some proximate factors that mediate female remating behavior have been identified (Kubli, 1996; Wolfner, 1997). For example, females may benefit from remating by having higher fecundity and, in several Drosophila species, multiple mating by females does increase their reproductive success (Pruzan-Hotchkiss et al., 1981; Markow, 1996). This increase, however, is costly to Drosophila melanogaster females because it results in early death. Causes of increased mortality include exposure to males and increased egg production as a result of mating (Partridge et al., 1987), and male accessory gland proteins (Acps) transferred in the ejaculate (Chapman et al., 1995). Recent work by Wolfner, Kubli and their colleagues have shown that these Acps proximately mediate behavioral and physiological alterations in females related to remating and oviposition (Kubli, 1996; Wolfner, 1997). Female remating behavior is complex and influenced by several factors. Following mating, females in many insects exhibit two conspicuous behavioral alterations: decreased receptivity to mating and increased ovi-

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position (Chen, 1984; Gillot, 1988; Wolfner, 1997). These alterations have both short- and long-term physiological cues. The short-term effect, called the copulation effect in Drosophila (Manning, 1967), is related to decreased attractiveness to males through alterations in pheromones (Scott, 1986; Hall, 1994), decreased locomotor response to males (Tompkins et al., 1982), and decreased receptivity to remating through increased oviposition (Fuyama, 1995) and ejaculatory fluids (Kubli, 1996; Wolfner, 1997). In a variety of insects, molecules in the male’s accessory fluid have been shown to temporarily reduce female receptivity and induce oviposition (Chen, 1984; Gillot, 1988; Wolfner, 1997). For example, both sex peptide (SP) and Acp26Aa induce oviposition and SP also acts to decrease female receptivity to remating in D. melanogaster (Monsma and Wolfner, 1988; Aigaki et al., 1991; Schmidt et al., 1993a,b; Herndon and Wolfner, 1995; Soller et al. 1997, 1999). In D. melanogaster, but not in other insects (Chapman et al., 1998; Snook, 1998; Miyatake et al., 1999), longterm suppression of female remating has been indirectly linked to sperm load, and termed the sperm effect (Manning 1962, 1967; Gromko et al., 1984; Scott, 1987; Gromko and Markow, 1993; Kubli, 1996). Female receptivity is proximately controlled by the central nervous system (CNS) in D. melanogaster (Tompkins and Hall, 1983) and a mechanical stimulation of the CNS through sperm motility detected by innervation of the sperm storage organs has been suggested to mediate the long-term inhibition to female remating (Gromko et al., 1984). Positive correlations between female remating behavior and progeny production (an index of sperm use; Trevitt et al., 1988; Gromko and Markow, 1993) have been used to support the relationship between sperm load and female receptivity. For example, laboratory female D. melanogaster that oviposit more eggs or produce more progeny remate more quickly than females ovipositing fewer eggs or producing less progeny (Trevitt et al., 1988). In addition, field-caught D. melanogaster and Drosophila simulans females produced fewer progeny when they were interrupted during mating (prior to sperm transfer) compared with nonmating females randomly collected adjacent to the mating pair (Gromko and Markow, 1993). These results suggested that remating females had a smaller sperm load than nonremating females (Gromko and Markow, 1993). Further indirect evidence correlating female remating behavior with sperm load is the positive relationships between the number of sperm received per copulation and female remating interval in some Drosophila species (Pitnick and Markow, 1994a). However, no study has directly quantified sperm loads of receptive and nonreceptive D. melanogaster females. Female remating behavior in the Drosophila obscura species group also appears to be related to sperm load. Similar to D. melanogaster, females receptive to remat-

ing oviposit more eggs than nonreceptive females, and a positive relationship between either fecundity or productivity and female remating behavior exists (PruzanHotchkiss et al., 1981; Snook, 1998). In an effort to directly determine if female remating behavior and sperm load are linked, Snook (1998) quantified the number of sperm present in the sperm storage organs of Drosophila pseudoobscura females that were receptive to remating compared with nonremating females. No difference in sperm load was found between remating and nonremating females, although remating females did oviposit more eggs. Instead of a dependence on sperm, female receptivity to remating was most predictably related to the absence of an egg in the uterus (Snook, 1998). Thus, any long-term inhibition to remating in D. pseudoobscura has been shown to be unrelated to sperm and at least indirectly related to oviposition. Given that there was no association between female remating behavior and sperm load and that female remating was related to oviposition in D. pseudoobscura, Snook (1998) suggested that oviposition per se mediated female remating behavior in that species. In support of this notion, D. pseudoobscura exhibits rhythmic oviposition with regular alternation of periods of egg laying and quiescent periods (Shapiro, 1932; Dobzhansky, 1935; Donald and Lamy, 1937; Snook, personal observations). Additionally, across ovarioles, there is a parallel synchronization of egg development (Donald and Lamy, 1937). Donald and Lamy (1937) noted that the “alternate” egg-laying behavior by D. pseudoobscura depletes the ovarioles of mature eggs and that, during quiescent periods, mature eggs accumulate. Hence, a large number of eggs become ready for laying at one time. In contrast, D. melanogaster females oviposit fairly equal numbers of eggs each day (Shapiro, 1932; Dobzhansky, 1935; Donald and Lamy, 1937), and do not exhibit synchronization of ovariole development (Donald and Lamy, 1937). Thus, eggs are found at all stages of development in mated females and eggs do not ripen in batches (Donald and Lamy, 1937). The differences in oogenesis and oviposition between these two species (synchronous and thus rhythmic, versus asynchronous and constant) support testing the hypothesis that female remating behavior in D. pseudoobscura, but not D. melanogaster, may be mediated by oogenesis, and thus oviposition, per se. For example, female D. pseudoobscura may remate between deposition of successive synchronous egg clutches, similar to some birds (Birkhead and Møller, 1992), whereas receptivity to remating in D. melanogaster females would have no association with these factors. Here we tested the hypothesis that female remating behavior is at least indirectly mediated by the number and stage of developing oocytes and mature eggs in D. pseudoobscura but not in D. melanogaster. Given the different oogenesis and oviposition patterns, we pre-

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dicted that D. pseudoobscura females receptive to remating would have fewer mature eggs than nonremating females, whereas no difference would be found between remating and nonremating D. melanogaster females. We found the opposite: remating D. pseudoobscura females do not differ from nonremating females in the number of oocytes at any stage of development, whereas remating D. melanogaster females had significantly more mature eggs than nonremating females but significantly fewer vitellogenic immature eggs. We discuss these results with respect to the interaction between physiological mechanisms controlling receptivity, oogenesis and oviposition. 2. Materials and methods 2.1. Fly maintenance The Drosophila melanogaster culture was established in February 1999 from multiple females collected on fallen citrus in Tempe, Arizona (collected by T.A. Markow). Drosophila pseudoobscura was collected in the same manner from the same area in March 1999. All cultures were maintained on standard cornmeal–agar– molasses food with yeast and kept at room temperature, 22–25°C, and an approximate 12 h/12 h light/dark cycle. We collected virgin flies, separated males and females upon eclosion using CO2 anesthetization, and stored each sex at 10 flies per 8 dram yeasted food vials. All flies used were 5 days old and reproductively mature. 2.2. Remating tests Remating tests generally followed those described in Snook (1998). We paired two virgin males with a virgin female in a yeasted food vial in the morning. Upon mating and the completion of copulation, males were removed from the vial by aspiration. Forty-eight hours later, we paired two virgin males with a previously mated female in her vial for a maximum of 2 h. A female was considered receptive to remating if she remated. If females did not indicate a willingness to remate by the end of the 2 h receptivity test, they were considered nonreceptive. In these cases, males were removed from the vials. Remated and nonremated females were immediately dissected and processed for ovarian counts as described below. During dissections, we noted whether females had an egg in the uterus. We also counted the number of eggs oviposited in vials that held females during the time between the first mating and the test for receptivity to remating. 2.3. Scoring vitellogenic stages Ovarian development was scored by dissecting ovaries from females and processing the ovaries for epifluo-

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rescence microscopy. Ether-anesthetized females were dissected in phosphate buffered saline (PBS) and the ovaries from a single female were placed in a microcentrifuge tube containing heptane and devitillizing membrane buffer as previously described (Cooley et al., 1992). Ovaries were rinsed in PBS, stained with diaminophenylinidole (DAPI; Sigma) at 1 µl/ml PBST (PBS containing 0.1% Triton X-100) for 5 min, rinsed again in PBS, and placed on a microscope slide in mounting media containing 80/20 (v/v) glycerol and PBS, respectively containing 1% (w/v) n-propyl gallate (Giloh and Sedat, 1982). Ovarioles were then spread apart using insect pins to observe all egg stages and a coverslip placed over the sample. Oocyte stages were characterized as described in King (1970) using a Leica DMLB microsocpe equipped with epifluorescence. Images were visualized using the appropriate filter for DAPI fluorescence and a 40× Plan Fluotar lens (n.a.=0.70). As a single category, we scored pre-vitellogenic stages 1–7 and early vitellogenic stage 8. Immature vitellogenic oocytes of stages 9–13 and mature stage 14 eggs were individually enumerated. 2.4. Statistical analyses All statistical analyses were performed using Systat (Version 5). The number of eggs oviposited between the initial mating and the receptivity test and the total number of developing eggs found in remating and nonremating females were compared using a t-test. To analyze differences between remating and nonremating females (“treatment”) in the number of oocytes of different stages and mature eggs, we performed a repeated-measures analysis of variance (ANOVA). This statistical analysis was chosen because of the potential of the number of eggs of one stage influencing the number of eggs in a different developmental stage within individual females. Once again, stage 1–8 oocytes were analyzed together, whereas vitellogenic immature stages 9–13 and mature stage 14 eggs were considered separately.

3. Results Remating females of both species oviposited more eggs prior to remating than those females that were nonreceptive to remating (Fig. 1; D. pseudoobscura: df=58, t=3.196, P=0.002; D. melanogaster: df=64, t=2.445, P=0.017). Nonremating D. pseudoobscura females usually had an egg in the uterus whereas remating females did not (Table 1), as was found previously (Snook, 1998). This factor predicts ca. 70% of the remating response in D. pseudoobscura (Table 1). Nonremating D. melanogaster females also frequently had an egg in the uterus in contrast to remating females, and the

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Table 2 Results from a repeated-measures ANOVA on the relationship between oocyte stages and female remating behavior in D. pseudoobscura Source

MS

Between-subject effects (df=1, 56) Treatment 22.564 Error 139.446 Within-subject effects (df=6, 6, 336) Stage 19,600 Stage×Treatment 291.021 Error 197.603

Fig. 1. Total number of eggs oviposited between first remating and receptivity test (“Eggs laid”), the number of oocytes in various developmental stages, and the total number of eggs being produced (mean±standard error of the mean) by receptive and nonreceptive females for D. pseudoobscura and D. melanogaster. * indicates comparisons that are statistically significantly different (see Results).

Egg present in Egg absent in uterus uterus D. pseudoobscura Nonremating Remating c2=43.866, df=1, P⬍0.0001, R2=0.69 D. melanogaster Nonremating Remating c2=62.11, df=1, P⬍0.0001, R2=0.90

24 0

4 30

34 1

0 31

absence of an egg predicted ca. 90% of the remating response in D. melanogaster (Table 1). Remating and nonremating D. pseudoobscura females did not differ in the total number of eggs being produced

P

0.162

0.689

99.1 1.473

⬍0.001 0.187

(Fig. 1; Table 2, “between-subjects” effect). Within-subject effects were found to be significant, indicating that there was a relationship between the number of eggs of a particular developmental stage and the number of eggs in a different developmental stage. However, remating and nonremating females did not differ in this response (Table 2). Similar to D. pseudoobscura, remating and nonremating D. melanogaster females did not differ in the total number of eggs being produced (Fig. 1; Table 3). Within-subject effects were also found to be significant, indicating a dependence of the number of eggs in one stage on the number of eggs in another stage within an individual (Table 3). In contrast to D. pseudoobscura, however, this pattern differed between remating and nonremating females (Table 3). Subsequent univariate FTable 3 Results from a repeated-measures ANOVA on the relationship between oocyte stages and female remating behavior in D. melanogaster Source

Table 1 Chi-square analysis of the association between the presence or absence of an egg in the uterus and female remating behavior in D. pseudoobscura and D. melanogaster

F

MS

Between-subject effects (df=1, 64) Treatment 0.642 Error 98.422 Within-subject effects (df=6, 6, 384) Stage 15,900 Stage×Treatment 550.855 Error 58.216 Univariate F tests Stages 1–8 (df=1, 330.011 64) Error 206.127 Stage 9 41.776 Error 43.318 Stage 10 285.522 Error 49.386 Stage 11 0.243 Error 1.152 Stage 12 26.444 Error 5.611 Stage 13 8.001 Error 4.547 Stage 14 2613.776 Error 137.576

F

P

0.007

0.936

273.5 9.462

⬍0.001 ⬍0.001

1.601

0.210

0.964

0.330

5.781

0.019

0.211

0.648

4.713

0.034

1.76

0.189

18.999

⬍0.001

R.R. Snook, Y.K. So / Journal of Insect Physiology 46 (2000) 1489–1496

tests indicated that the difference between remating and nonremating females occurred at stage 10, stage 12 and stage 14 (Table 3; Fig. 1). Nonremating females produced significantly more vitellogenic, immature stage 10 and 12 oocytes, whereas remating females produced significantly more mature stage 14 eggs.

4. Discussion We found that remating females oviposited more eggs than nonremating females. These results are consistent with previous studies of D. melanogaster (Trevitt et al., 1988) and D. pseudoobscura (Snook, 1998) using different strains than those examined here. Formerly, the observation that remating females lay more eggs than nonremating females has been used to infer that sperm load mediated female remating behavior since females that oviposited more eggs had probably used more sperm and thus carried a smaller sperm load. However, Snook (1998) demonstrated that, while remating D. pseudoobscura females oviposited more eggs than nonremating females, no differences between females in sperm load were found. Instead, females receptive to remating tended to not have an egg in the uterus whereas nonreceptive females possessed an egg in the uterus (Snook, 1998). We therefore examined an alternative hypothesis to explain female receptivity to remating in D. pseudoobscura: female remating behavior is at least indirectly mediated by oogenesis and, thus, oviposition. We found that nonremating D. pseudoobscura females had an egg present in the uterus whereas remating females did not, as previously seen in a different D. pseudoobscura strain (Snook, 1998). Interestingly, D. melanogaster females also exhibited this phenomenon. This result has not previously been reported for this species. However, Fuyama (1995), using homozygous lozenge mutant females that have increased spontaneous ovulation, found virgin females more reluctant to mate than wild-type females. In contrast, Hihara (1981) reported that females genetically altered so as to not oviposit had the same remating interval as ovipositing females. Here we find that oviposition and female remating behavior in D. melanogaster are linked (through increased egg laying and the absence of an egg in the uterus). Whether the observation that remating females lack an egg in the uterus compared with nonremating females is general to Drosophila remains to be determined, but given the reproductive biology of this group, is intuitive. Males transfer sperm to females in the uterus and sperm then move into the female sperm storage organs. At the onset of oviposition, any sperm that remain in the uterus and have not been stored are evacuated by the act of egg deposition due to the fact that the egg occupies the entire space of the uterus (Scott, 1987). Males that transferred

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sperm to a female with an egg in the uterus would have his sperm posterior to the egg and may lose his investment when the egg is oviposited, because the sperm may be unable to maneuver around the egg in the female reproductive tract and thus would be evacuated when the female oviposits. Females may also not benefit from remating when an egg is in the uterus because Acps may still enter the hemolymph and cause decreased female life span (Chapman et al., 1995) with no corresponding benefit of additional mating (Gromko and Markow, 1993) since, as the egg is laid, recently transferred sperm would be removed by the act of oviposition (Scott, 1987). Therefore, selection would presumably act on males to discriminate between females with and without an egg in the uterus if the ejaculate (Pitnick and Markow, 1994b) and/or mating were costly or limited. Selection would act on females to avoid mating if mating is costly (Partridge et al., 1987; Chapman et al., 1995; Rice, 1996). An alternative explanation for the association between having an egg in the uterus and nonremating is that it may be a stochastic phenomenon, perhaps related to timing of courtship by males, rather than selective. Females may be unwilling to remate only during the time an egg is in the uterus. As soon as an egg is deposited, unless another egg moves immediately into the uterus, the female may be willing to remate. The lack of remating during the time when the uterus is empty may be a missed opportunity by the male, perhaps because he is not near the female and not actively courting. Thus, the association between having an egg in the uterus and a female not remating may be spurious. We think this is unlikely for several reasons. First, complete sperm storage takes several hours in D. melanogaster (Markow, 1996) and over 24 h in D. pseudoobscura (Snook et al., 1994). If females were constantly ovipositing, then males that mated with a female in which the uterus was only temporarily unoccupied would lose a large quantity of their investment since the next egg oviposited would evacuate unstored sperm. Second, dissections of young, healthy, mated D. pseudoobcura (Snook, personal observations) and D. melanogaster (S. Pitnick, personal observations) females frequently reveal eggs in their uterus. No study has examined either how long it takes an egg to become fertilized and subsequently oviposited, or what percentage of time during the day males are courting females in which the uterus is occupied by an egg. However, the observations that females typically have an egg in the uterus suggests that there is little opportunity of a vacant uterus and that when a female is ovipositing and producing mature eggs, an egg probably occupies the uterus a majority of the time. In toto, the time it takes for sperm storage and the observations that during bouts of oviposition the uterus is rarely empty suggest that timing (or mistiming) of

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male courtship does not explain the association between female nonreceptivity and the presence of an egg in the uterus. Instead, this association is likely a real phenomenon, the proximate basis of which needs to be elucidated. Perhaps stretch receptors in the uterus cue females as to their oviposition status and the number of mature eggs still to be oviposited. Another spurious relationship may be that because we examined only one line from each species, our results may reflect line-to-line variation as opposed to interspecies differences. We think this unlikely for several reasons. First, in two different lines of D. pseudoobscura, both strains exhibited a significant association between remating behavior and the absence of an egg in the uterus (Snook, 1998; this study). Second, females that were willing to remate had oviposited more eggs than females unwilling to remate in both these strains (Snook, 1998; this study). Third, we found that remating D. melanogaster females also had oviposited more eggs than females that did not remate, a result that has been reported several times in the literature using different strains (Trevitt et al., 1988, Chapman et al., 1994; Chapman and Partridge, 1996). Finally, in our D. melanogaster strain, we found that females, regardless of whether they were willing to remate or not, had equal amounts of stage 8 and 9 eggs, similar to previous reports (Soller et al., 1997). In toto, these results indicate that associations between remating behavior, oviposition and oogenesis are consistent across strains for both species, and thus our results are likely true reflections of interspecies differences. Oviposition and oogenesis are inextricably linked. We therefore examined the role oogenesis may play in mediating female remating behavior by determining whether receptivity could be predicted by the number of eggs of any particular oocyte stage. We anticipated that the synchronous oogenesis and, thus, rhythmic (i.e., clutch) oviposition found in D. pseudoobscura would result in females remating between successive clutches, and therefore remating females should have more immature eggs and fewer mature eggs compared with nonremating females. In contrast, we predicted that the asynchronous oogenesis (and thus constant oviposition) in D. melanogaster would not mediate female remating behavior. Therefore, there should be no difference between remating and nonremating females with respect to ovarian status. Our results did not support these predictions. No differences in ovarian status between remating and nonremating D. pseudoobscura females were found, whereas remating D. melanogaster females had more mature eggs and fewer stage 10 and 12 oocytes than nonremating females (moreover, combining vitellogenic stages 10–13 still results in significant differences between remating and nonremating females). Contrary to our original prediction, female remating in D. melanogaster is at least indirectly related to oogen-

esis and, thus, oviposition. Egg production and egg laying in D. melanogaster are tightly coupled to mating and sperm use (Kubli, 1996; Wolfner, 1997). Acps transferred by the male during copulation stimulates oogenesis, elevates oviposition, and influences sperm storage (Kubli, 1996; Wolfner, 1997; Neubaum and Wolfner, 1999; Tram and Wolfner, 1999). Kubli and colleagues have defined a “control point” during oogenesis in D. melanogaster related to mating that subsequently influences egg development (Soller et al. 1997, 1999). Mated females and those injected with physiological amounts of the male ejaculatory fluid, SP, had a four- to sixfold increase in the number of stage 10 eggs compared with unmated females and those injected with saline, but all treatments had equal amounts of stage 8 and 9 eggs (Soller et al., 1997). These results suggest that the progression of vitellogenic oocytes is stimulated after mating, through actions of SP on the females’ CNS that also controls receptivity and oviposition (Soller et al., 1999). Given these interactions, how might oviposition and oogenesis at least indirectly mediate female remating in D. melanogaster? One potential mechanism for the association between oogenesis and female remating is that SP, through the CNS, acts on nonreceptive females at the control point of oogenesis, resulting in increased numbers of stage 10 oocytes. The large number of stage 10 oocytes may then indicate to females that they recently mated and to start oviposition, and thus become nonreceptive. As the effect of SP declines (possibly through decreased titer in the hemolymph), females may return to a “virgin” state, that is few stage 10 eggs (Kubli, 1996), subsequently indicating to females to decrease oviposition and thus resulting in females becoming receptive to remating. This response could be either independent of or in conjunction with the sperm effect. Unlike D. melanogaster, oogenesis per se plays no role in influencing female remating in D. pseudoobscura, at least for the remating interval examined. No differences in ovarian status were found between remating and nonreceptive females. This result suggests that there may be different physiological influences of male ejaculatory components, such as SP, on females of the two species. Perhaps the half-life of SP and the cascade of female responses triggered by SP is different, or SP does not act on oogenesis at the same control point or in the same fashion. To our knowledge, the physiological effects of male ejaculatory components on female behavior, including receptivity, oviposition and oogenesis, have been investigated in species other than D. melanogaster (e.g., Schmidt et al., 1993a; Imamura et al., 1998) but not D. pseudoobscura. Drosophila female receptivity is a complex behavior that is influenced by several factors, including male ejaculatory fluids and sperm (Manning 1962, 1967; Gromko et al., 1984; Scott, 1987; Gromko and Markow,

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1993; Kalb et al., 1993; Kubli, 1996; Wolfner, 1997; Neubaum and Wolfner, 1999; Tram and Wolfner, 1999). We have presented evidence that suggests oogenesis and oviposition may also trigger female remating, either alone or in concert with male effects, in one species of Drosophila but not in another species. These results indicate new research directions to understand links between physiological and behavioral traits affecting male and female fitness and proximate mechanisms controlling these processes in different species.

Acknowledgements We thank Kathy Davidson and Cher Chang for technical support, Scott Pitnick and Steve de Belle for reviewing an earlier version of the manuscript, and T. Markow for reminding R.R.S. that oogenesis can be as interesting as spermatogenesis. This research was supported in part by a grant from the National Science Foundation to R.R.S. (DEB-9815962).

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