Cryptic female choice during spermatophore transfer in Tribolium castaneum (Coleoptera: Tenebrionidae)

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1 ARTICLE IN PRESS Journal of Insect Physiology 53 (2007) Cryptic female choice during spermatophore transfer in Tribolium castaneum (Coleoptera: Tenebrionidae) Tatyana Y. Fedina Department of Biology, Tufts University, Medford, MA 02155, USA Received 3 October 2006; received in revised form 15 October 2006; accepted 26 October 2006 Abstract Sexual selection in both males and females promotes traits and behaviors that allow control over paternity when female mates with multiple males. Nonetheless, mechanisms of cryptic female choice have been consistently overlooked, due to traditional focus on sperm competition as well as difficulty in distinguishing male vs. female influence over processes occurring during and after mating. The first part of this study describes morphology and transformation of Tribolium castaneum spermatophores inferred from dissecting females immediately after normal or interrupted copulations. T. castaneum males are found to transfer spermatophores as an invaginated tube that everts inside the female bursa and which is filled with sperm during copulation. This sequence of events makes it feasible for females to control the sperm quantity transferred in each spermatophore. Through manipulation of the male phenotypic quality (by starvation) and manipulation of female control over sperm transfer (by killing a subset of females), the second part of this study examines whether females use control over transferred sperm quantity as a cryptic choice mechanism. Fed males transferred significantly more sperm per spermatophore than starved males but only when mating with live females. These results suggest an active differentiation by live females against starved males and provide an evidence for the proposed cryptic female choice mechanism. r 2006 Elsevier Ltd. All rights reserved. Keywords: Sexual selection; Sperm number 1. Introduction It is now widely accepted that traditional pre-mating interactions between sexes, namely male male competition and female choice of mates, continue during and after mating in polyandrous species (reviewed by Eberhard, 1996; Simmons, 2001). Nonetheless, female-imposed paternity biasing mechanisms (cryptic female choice) have been consistently overlooked due to the traditional focus on sperm competition mechanisms, and due to some logistic difficulties in demonstrating cryptic female choice (Eberhard, 1996). In order to show that a particular female process or trait is, in fact, a mechanism of cryptic female choice, it is necessary to: (1) demonstrate the consistency of this choice, i.e., that through this process/trait females favor males with certain characteristics over others lacking these characteristics and (2) differentiate male vs. female Tel.: ; fax: address: tfedina@yahoo.com. control over the process since it is likely that both sexes have at least some influence. Several studies have differentiated the influence of male and female genotype over paternity using statistical partitioning after reciprocal double mating of related and unrelated mates (Wilson et al., 1997; Clark and Begun, 1998; Nilsson et al., 2003). While documenting significant effects of female genotype on paternity, these studies did not address the mechanisms of paternity biasing by females. Among potential cryptic female choice mechanisms, studies have demonstrated control over insemination during copulation (Tallamy et al., 2002, 2003; Fedina and Lewis, 2006), as well as spatial partitioning of stored sperm from different males and its differential use for fertilization (Otronen et al., 1997; Pitnick et al., 1999; Ward, 2000; Hellriegel and Bernasconi, 2000). Another potential cryptic female choice mechanism that has received less empirical attention is female influence over sperm quantity transferred during copulation. Experimental evidence suggests that sperm quantity transferred by a male during /$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi: /j.jinsphys

2 94 ARTICLE IN PRESS T.Y. Fedina / Journal of Insect Physiology 53 (2007) copulation affects his paternity relative to that of other males mated to the same female (reviewed by Eberhard, 1996; Simmons, 2001). One key aspect of sperm transfer that determines the possibility of female control over sperm quantity is whether the sperm is packaged into the spermatophore before or during its transfer to the female reproductive tract. Existing data suggest that in many Coleoptera and other advanced insect orders characterized by spermatophore transfer, the sperm is either injected into female bursa before spermatophore formation, or the sperm is injected into pre-spermatophore after it is transferred to or formed inside the female bursa (reviewed by Mann, 1984; Chapman, 1998). Consequently, in these insects ejaculate size is determined during copulation and thus can potentially be influenced by females (e.g. by interrupting copulation before all the sperm is transferred). On the opposite side of the spectrum, in Gryllidae and Tettigoniidae, a male manufactures the entire spermatophore before encountering a female (Mann, 1984; Chapman, 1998) and the number of sperm per spermatophore is determined solely by a male. In these insects, females control the number of sperm translocated into their reproductive tracts by allowing the externally attached sperm ampulla to remain attached for varying times (Sakaluk and Eggert, 1996; Simmons, 1986, 1987). In this study, I tested female control over sperm quantity transferred in the male spermatophore as a potential mechanism of cryptic female choice in the red flour beetle Tribolium castaneum. I first examined in detail the process of T. castaneum spermatophore transfer, since the specifics of this process can determine the potential for female control. In the second part of this study, I manipulated male phenotypic quality by starvation, as well as female ability to control sperm transfer by killing some females before mating. In order to distinguish male vs. female influence over sperm transfer, I compared quantities of sperm transferred by fed and starved males when they mated to dead or live females. 2. Methods 2.1. T. castaneum spermatophore morphology and transformation: dissection experiments Here, I summarize my observations on T. castaneum spermatophore structure and transformation recorded for more than 300 dissections (performed for different experiments in years ). Males and females used for matings were all wild type T. castaneum, and the females were either virgin or pre-mated and either alive or dead (see killing method below). For pre-mated females, pre-mating was conducted at least 24 h before their final mating. This was done because after 24 h all previous spermatophores are extruded from the female bursa copulatrix and only free sperm from previous matings is present in the female reproductive tract. All dissections were performed in Tribolium saline (10 mm Hepes buffer, 1% BSA, 0.85% NaCl, adjusted to ph 7.4) under X magnification. Females were dissected immediately after an observed copulation (within 30 s of pair separation) using fine forceps to pull the reproductive tract by the extruded ovipositor out of the female body; this procedure results in the separation of the whole female reproductive tract excluding the ovaries. The spermatophore was then either pulled out by its tail or carefully removed out of the female bursa by tearing the bursa open at its base. A subset of copulations was allowed to end naturally before dissection. However, to observe the rapid progression of events occurring during the early stages of spermatophore transfer I used two methods: (1) retrieval of spermatophores from females after interrupted copulations and (2) observing spermatophores dumped outside during unsuccessful copulation attempts. Copulations were interrupted by brushing a male off with a paintbrush during female quiescence stage, when spermatophore transfer is most likely to occur (Bloch Qazi, 2003). Spermatophore dumping sometimes occurs when males engage in copulations with resisting (usually pre-mated) females, with dead females, or with other males (Spratt, 1980). In these cases, the spermatophore gets deposited on the abdomen of either mounting or mounted beetle or on the surface of the mating arena (pers. obs.). Successful preparations were digitally recorded using Olympus DP11 camera attached to the Olympus SZ11 stereomicroscope. To outline the structure and transformation of T. castaneum spermatophores I employ the terminology used by Gadzama and Happ (1974) who described in great detail spermatophore structure and transformation in another tenebrionid beetle, Tenebrio molitor Female influence over sperm quantity transferred To determine whether T. castaneum females exercise cryptic female choice by controlling sperm quantity transferred per spermatophore, I compared the number of sperm transferred by starved (n ¼ 28) and fed (n ¼ 30) males to live (n ¼ 25) and recently killed (n ¼ 33) females. All beetles were 1 3-week-old wild-type virgins. Females in the dead treatment were killed by placing them in a jar with ethyl acetate vapors for h before mating. They were then left in the air for about 5 min before being used in mating observations. Male phenotypic quality (body condition) was manipulated by starvation. Males were kept individually for 7 days in scintillation vials with either: (1) 2 g King Arthur wheat flour and 30 cm of coiled nylon string on top (fed males treatment) or (2) string only (starved males treatment). The latter treatment resulted in 15% decrease in body weight (Fedina and Lewis, 2006). Behavioral interactions in male female pairs were observed on a C warming tray in plastic 15 mm mating arenas with a surface scratched to provide sufficient traction. Copulation was defined as at least one intromission by the male aedeagus lasting a minimum of 25 s.

3 ARTICLE IN PRESS T.Y. Fedina / Journal of Insect Physiology 53 (2007) Immediately after copulation, the female reproductive tract was dissected out and the number of sperm present was estimated following the methods in Fedina and Lewis (2006). Data were analyzed using ANOVA with male and female treatments as predictors and number of sperm per spermatophore as response variable (data distribution conformed to ANOVA assumptions). Non-parametric Mann Whitney U-tests were also performed to compare sperm quantity transferred by fed and starved males within each female treatment. Since I wanted to assess female control over sperm quantity transferred per spermatophore, all cases where a male transferred two spermatophores during copulation (n ¼ 4 fed males mated with dead females) were removed from analysis. On several occasions, after copulation the spermatophore was found partially protruding from the female s body. This resulted in sperm drying and forming clumps in saline, which would undermine the accuracy of sperm counts (see Fedina and Lewis, 2006). Hence, these cases were also removed from analysis (n ¼ 3 fed males mated with dead females, n ¼ 4 starved males mated with dead females, and n ¼ 1 fed male mated with live female). Finally, the occasional presence in the bursa copulatrix of an egg ready to be laid seems to interfere with normal sperm transfer, as it noticeably increases copulation duration, frequency of transfer failure and variability in number of sperm transferred (pers. obs.). Consequently, those cases were also excluded (n ¼ 1 fed male mated with dead female, n ¼ 2 fed males mated with live females, and n ¼ 2 starved males mated with live females). Screening the data as described above yielded final sample sizes of 10 live/fed, 10 live/starved, 10 dead/fed and 12 dead/starved female/male pairs, respectively. 3. Results 3.1. T. castaneum spermatophore morphology and transformation: dissection experiments T. castaneum spermatophores deposited outside the body ( dumped ) appeared as invaginated double-walled tube with a cap on the anterior tip (Fig. 1a), very similar to non-everted T. molitor spermatophores extracted after normal copulations (Fig. 1b; see also Fig. 18 in Gadzama and Happ, 1974). When placed in saline, these T. castaneum spermatophores underwent a two-stage elongation similar to that described for T. molitor spermatophores (Gadzama and Happ, 1974). In T. castaneum, however, this elongation process occurred within 5 10 s after contact with the saline (unlike 5 min for T. molitor) and proceeded so quickly that it was not possible to record intermediate stages. In addition, each elongation occurred along the same axis as previous one, and not at an angle as in T. molitor. Within approximately 1 min, the elongation was followed by the formation of a frothy disintegrating sac, very similar to Fig. 1. Morphological similarity of T. castaneum spermatophores dumped outside of the body with T. molitor spermatophores extracted from females after normal copulation: (a) non-everted T. castaneum spermatophore, inset shows a close-up of the anterior part in dark field view, (b) T. molitor spermatophore after 1st eversion (marked by bracket), and (c) T. castaneum spermatophore with anterior bulb divided into two visible compartments (see the text). The scale bars are equal to 100 mm. 1 Anterior part of spermatophore with a darker core (the gap in the normally continuous core in (a) is caused by forceps pinch); 2 posterior tail; 3 cap; 4 spermatophore bulb compartment containing dark material, 5 transparent bulb compartment. spermless spermatophores retrieved from females after interrupted copulation (described below). Dumped spermatophores contained no visible sperm, although free sperm was often found clumped around the tube. Some dumped spermatophores seem to have been proceeded through initial eversion stage, as they looked like small bulb with a tail (Fig. 1c). Two compartments were sometimes observed inside the bulb very similar to Fig. 25 and the corresponding description in Gadzama and Happ (1974). T. castaneum females that were dissected immediately after naturally terminated copulations always contained spermatophore (if transferred) that had already everted in the bursa copulatrix. This spermatophore appeared as a sperm-filled sac with a posterior tail (Fig. 2). When extracted at an earlier transformation stage, the sperm sac was divided into two visually distinct parts: a posterior part with a transparent content and a larger anterior part with darker material (Fig. 2a). Within minutes, the two parts had coalesced and sperm, now vigorously moving, expanded the sac even more (Fig. 2b). More frequently, when dissected out immediately after mating, the sperm sac was already expanded and ruptured at the anterior end (Fig. 2c), near the entrance to the spermathecal duct.

4 96 ARTICLE IN PRESS T.Y. Fedina / Journal of Insect Physiology 53 (2007) Fig. 2. Spermatophore transfer in T. castaneum: female dissections immediately following naturally terminated copulations: (a) spermatophore inside the female bursa copulatrix: two parts of the sperm sac are still distinct, (b) later stage of spermatophore transformation: two parts fused into an expanded uniform sperm sac, and (c) spermatophore dissected out of the female bursa. The scale bars are equal to 100 mm. 1 Sperm sac, 2 tail, 3 anterior part of the sperm sac, 4 posterior part of the sperm sac, 5 spermatheca, 6 spermathecal gland, 7 common oviduct Number of sperm transferred, x Fig. 3. Spermless T. castaneum spermatophore retrieved from a female after interrupted copulation. The scale bar is equal to 100 mm. 1 Disintegrating spermatophore sac, 2 spermatophore tail. Spermatophores that were retrieved from females after interrupted copulations appeared similar to normal everted spermatophores (Fig. 2c), but their sacs contained either no sperm, or visibly fewer sperm. These spermatophores often had tails with a distinct opening at their distal end; this opening led to a channel containing material moving into the sac. When retrieved from females even sooner after transfer, spermatophores appeared as tubes with a darker core (similar to dumped spermatophores after two-stage elongation described above); this tube, when placed in saline, immediately expanded at the anterior end into disintegrating spermless sac (Fig. 3). 0 dead females Fig. 4. Sperm quantity (mean7se) transferred per spermatophore by T. castaneum fed (filled bars) and starved (hatched bars) males mating with dead or live females. Sample sizes are shown inside the bars Female influence over sperm quantity transferred Starved males transferred significantly fewer sperm than fed males but only when they mated with live females (Fig. 4, Mann Whitney U ¼ 17.5, U 0 ¼ 82.5, P ¼ 0.014); there was no difference between fed and starved males in sperm quantity transferred to dead females (U ¼ 55.5, U 0 ¼ 64.5, P ¼ 0.767). When analyzed with ANOVA, the quantity of sperm transferred per spermatophore was affected by male treatment (F 1,38 ¼ 5.54, P ¼ 0.023), was live

5 ARTICLE IN PRESS T.Y. Fedina / Journal of Insect Physiology 53 (2007) not affected by female treatment (F 1,38 ¼ 0.09, P ¼ 0.762), but most importantly, the significant interaction was detected between male and female treatments (F 1,38 ¼ 4.60, P ¼ 0.038). 4. Discussion The main goal of this study was to demonstrate one of the mechanisms of cryptic female choice, namely the female influence over the quantity of sperm transferred in a male spermatophore. This mechanism was demonstrated through crossed factorial design, in which fed males transferred significantly more sperm than starved males when mating with live females but not when mating with dead females. Using novel experimental approach, this study presents one of the few explicit illustrations of cryptic female choice. As a first step, the study sheds light on the process of spermatophore transfer in T. castaneum flour beetles since this process determines the potential for female control. Given that T. castaneum is such an important pest and a model organism (reviewed in Sokoloff, 1974; Brown et al., 2003), it is surprising that thus far, there has been no consistent description of spermatophore morphology in this species. Previous record indicates a simple morphology of T. castaneum spermatophore consisting of a sperm sac with a posterior tail (Bloch Qazi et al., 1996). However, the present study found that the T. castaneum spermatophore represents an invaginated tube that everts into a sperm-containing sac inside the female bursa, and that it is similar in overall structure and transformation sequence to the T. molitor spermatophore (Gadzama and Happ, 1974). Unlike in T. molitor, in T. castaneum spermatophore eversion occurs within seconds after its transfer to the female. In fact, upon immediate dissection after naturally ending copulation, T. castaneum spermatophores transferred to the female bursa are always found already everted and with the sperm sac expanded. Such rapid spermatophore eversion followed by sperm release may reflect an adaptation to the high degree of sperm competition in T. castaneum created by frequent female remating (Pai and Yan, 2003; Lewis, 2004). In T. molitor, when a second male s spermatophore is transferred to a female within 5 min of a previously deposited spermatophore, it inhibits the eversion and sperm release from the first-male spermatophore, resulting in a complete loss of paternity by the first male (Drnevich et al., 2000). Rapid transformation of spermatophores transferred by T. castaneum males may therefore represent an adaptation to avoid similar spermatophore inhibition by rivals. Alternatively, the ephemeral nature of T. castaneum spermatophores may be a consequence of miniaturization: T. castaneum is about one quarter of the body length of T. molitor. An important result of this study is the finding of spermless spermatophores inside the female bursa after interrupted copulations. This suggests that T. castaneum males transfer a spermless prespermatophore that is filled with sperm while it is located inside the female s reproductive tract. This sequence of events offers a potential for T. castaneum females to influence ejaculate size during copulation. The experimental part of the study demonstrates that T. castaneum females do, in fact, exploit this potential, as they use control over sperm numbers transferred in the male spermatophore to exercise cryptic female choice. This conclusion follows from an observation that starved males transferred only about half the number of sperm compared to fed males, but only when mating with live females. No such difference between fed and starved males mating with dead females suggests that starvation itself does not limit sperm quantity transferred in a spermatophore, and that live females actively discriminate against starved males. The most plausible physiological mechanism of female control over sperm quantity in T. castaneum is contraction of bursal muscles, which may reduce sperm flow into the spermatophore either directly or by interrupting copulation. The contraction of bursal muscles was implicated previously in the translocation of sperm from the female bursa into her spermatheca (Bloch Qazi et al., 1998). This physiological process was suggested as the potential mechanism for cryptic female choice, but not demonstrated to be such. The demonstration of cryptic female choice requires experimental confirmation that females discriminate for males with certain characteristics and against males lacking these features. Another potential mechanism was proposed based on the positive association between duration of female quiescence (cessation of any motion during copulation) and numbers of sperm transferred (Bloch Qazi, 2003). Combined with the results of the present study, this association implies that longer female quiescence may reflect longer relaxation of the female bursal muscles. This may allow the male to inject more sperm into his spermatophore. From the male perspective, even though within 24 h about 86% of the transferred sperm is discarded by female (Bloch Qazi et al., 1996), the initial quantity of transferred sperm must be important when a female mates with two or more males in quick succession. In this scenario, which is typical for T. castaneum (Pai and Yan, 2003; Lewis, 2004), several complete ejaculates from different males overlap in the bursa, and a fair raffle (Parker, 1990) at least partially determines the relative representation of each male s sperm that is moved into female sperm storage organs. In species where it is more costly for a female to struggle against male copulation attempts and sperm transfer (e.g. due to elaborate male anchoring genitalia or due to sheer male persistence), females may have an option to expel sperm or spermatophore after transfer as it was shown for the chrysomelid beetle Chelymorpha alternans (Rodriguez, 1995), the cicindelid beetle Pseudoxychila tarsalis (Rodriguez, 1999), and for the fly Dryomiza anilis (Otronen, 1997). T. castaneum females also occasionally expel spermcontaining spermatophores soon after copulation (pers. obs.). Therefore, T. castaneum females may use several

6 98 ARTICLE IN PRESS T.Y. Fedina / Journal of Insect Physiology 53 (2007) cryptic female choice mechanisms during sequential stages of sperm transfer and storage. Specifically, females can selectively prevent spermatophore transfer during copulation (Fedina and Lewis, 2006) and regulate sperm quantity injected into the spermatophore (shown in this study). In addition, they can potentially control sperm quantity stored in spermatheca (Bloch Qazi et al., 1998; Fedina and Lewis, 2004) and expel the spermatophore soon after mating (pers. obs.). Such multi-level control is likely to be less error prone and may allow for fine adjustments of sperm representation from several mates. Overall, this study describes complex morphology and transformation of the T. castaneum male spermatophore suggesting that sperm is injected into the spermatophore after it is transferred into the female reproductive tract. This study also demonstrates that T. castaneum females use control over sperm quantity transferred in a male spermatophore as a cryptic mechanism for female choice based on male phenotypic quality. Accumulating evidence from T. castaneum and other polyandrous species suggests that the outcome of sexual interactions and resultant offspring paternity are influenced not only by the male traits but also in large by female control over cryptic processes occurring during and after copulation. Future studies focusing on these female processes might clarify widely reported variability in male paternity success in species with polyandrous females. Acknowledgments I would like to thank Dr. Sara Lewis, Dr. Nancy Milburn and Dr. Frances Chew as well as two anonymous reviewers for their helpful comments on the manuscript and Tufts University Graduate Student Research Award Fund for financial support. References Bloch Qazi, M.C., A potential mechanism for cryptic female choice in a flour beetle. 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