ARTICLE IN PRESS

Journal of Physiology 53 (2007) 93–98 www.elsevier.com/locate/jinsphys

Cryptic female choice during spermatophore transfer in Tribolium castaneum (Coleoptera: Tenebrionidae) Tatyana Y. FedinaÃ

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 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 . 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: ; Sperm number

1. Introduction control over the process since it is likely that both sexes have at least some influence. Several studies have It is now widely accepted that traditional pre-mating differentiated the influence of male and female genotype interactions between sexes, namely male–male competition over paternity using statistical partitioning after reciprocal and female choice of mates, continue during and after double mating of related and unrelated mates (Wilson mating in polyandrous species (reviewed by Eberhard, et al., 1997; Clark and Begun, 1998; Nilsson et al., 2003). 1996; Simmons, 2001). Nonetheless, female-imposed While documenting significant effects of female genotype paternity biasing mechanisms (cryptic female choice) have on paternity, these studies did not address the mechanisms been consistently overlooked due to the traditional focus of paternity biasing by females. on sperm competition mechanisms, and due to some Among potential cryptic female choice mechanisms, logistic difficulties in demonstrating cryptic female choice studies have demonstrated control over insemination (Eberhard, 1996). In order to show that a particular female during copulation (Tallamy et al., 2002, 2003; Fedina and process or trait is, in fact, a mechanism of cryptic female Lewis, 2006), as well as spatial partitioning of stored sperm choice, it is necessary to: (1) demonstrate the consistency of from different males and its differential use for fertilization this choice, i.e., that through this process/trait females (Otronen et al., 1997; Pitnick et al., 1999; Ward, 2000; favor males with certain characteristics over others lacking Hellriegel and Bernasconi, 2000). Another potential cryptic these characteristics and (2) differentiate male vs. female female choice mechanism that has received less empirical attention is female influence over sperm quantity ÃTel.: +1617 6273195; fax: +1617 6273805. transferred during copulation. Experimental evidence E-mail address: [email protected]. suggests that sperm quantity transferred by a male during

0022-1910/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.10.011 ARTICLE IN PRESS 94 T.Y. Fedina / Journal of Insect Physiology 53 (2007) 93–98 copulation affects his paternity relative to that of other 1% BSA, 0.85% NaCl, adjusted to pH 7.4) under 20–110 X males mated to the same female (reviewed by Eberhard, magnification. Females were dissected immediately after an 1996; Simmons, 2001). One key aspect of sperm transfer observed copulation (within 30 s of pair separation) using that determines the possibility of female control over sperm fine forceps to pull the reproductive tract by the extruded quantity is whether the sperm is packaged into the ovipositor out of the female body; this procedure results in spermatophore before or during its transfer to the female the separation of the whole female reproductive tract reproductive tract. Existing data suggest that in many excluding the ovaries. The spermatophore was then either Coleoptera and other advanced insect orders characterized pulled out by its tail or carefully removed out of the female by spermatophore transfer, the sperm is either injected into bursa by tearing the bursa open at its base. female bursa before spermatophore formation, or the A subset of copulations was allowed to end naturally sperm is injected into pre-spermatophore after it is before dissection. However, to observe the rapid progres- transferred to or formed inside the female bursa (reviewed sion of events occurring during the early stages of by Mann, 1984; Chapman, 1998). Consequently, in these spermatophore transfer I used two methods: (1) retrieval ejaculate size is determined during copulation and of spermatophores from females after interrupted copula- thus can potentially be influenced by females (e.g. by tions and (2) observing spermatophores ‘‘dumped’’ outside interrupting copulation before all the sperm is transferred). during unsuccessful copulation attempts. Copulations were On the opposite side of the spectrum, in Gryllidae and interrupted by brushing a male off with a paintbrush Tettigoniidae, a male manufactures the entire spermato- during female quiescence stage, when spermatophore phore before encountering a female (Mann, 1984; Chap- transfer is most likely to occur (Bloch Qazi, 2003). man, 1998) and the number of sperm per spermatophore is Spermatophore ‘‘dumping’’ sometimes occurs when males determined solely by a male. In these insects, females engage in copulations with resisting (usually pre-mated) control the number of sperm translocated into their females, with dead females, or with other males (Spratt, reproductive tracts by allowing the externally attached 1980). In these cases, the spermatophore gets deposited sperm ampulla to remain attached for varying times on the abdomen of either mounting or mounted beetle (Sakaluk and Eggert, 1996; Simmons, 1986, 1987). or on the surface of the mating arena (pers. obs.). In this study, I tested female control over sperm quantity Successful preparations were digitally recorded using transferred in the male spermatophore as a potential Olympus DP11 camera attached to the Olympus SZ11 mechanism of cryptic female choice in the red flour beetle stereomicroscope. To outline the structure and transforma- Tribolium castaneum. I first examined in detail the process tion of T. castaneum spermatophores I employ the of T. castaneum spermatophore transfer, since the specifics terminology used by Gadzama and Happ (1974) who of this process can determine the potential for female described in great detail spermatophore structure and control. In the second part of this study, I manipulated transformation in another tenebrionid beetle, Tenebrio male phenotypic quality by starvation, as well as female molitor. ability to control sperm transfer by killing some females before mating. In order to distinguish male vs. female 2.2. Female influence over sperm quantity transferred influence over sperm transfer, I compared quantities of sperm transferred by fed and starved males when they To determine whether T. castaneum females exercise mated to dead or live females. cryptic female choice by controlling sperm quantity transferred per spermatophore, I compared the number 2. Methods of sperm transferred by starved (n 28) and fed (n 30) males to live (n 25) and recently killed¼ (n 33) fema¼ les. 2.1. T. castaneum spermatophore morphology and All beetles were ¼1–3-week-old wild-type virgin¼s. Females in transformation: dissection experiments the ‘‘dead’’ treatment were killed by placing them in a jar with ethyl acetate vapors for 0.5–1 h before mating. They Here, I summarize my observations on T. castaneum were then left in the air for about 5 min before being used spermatophore structure and transformation recorded in mating observations. Male phenotypic quality (body for more than 300 dissections (performed for different condition) was manipulated by starvation. Males were kept experiments in years 2000–2006). Males and females used individually for 7 days in scintillation vials with either: (1) for matings were all wild type T. castaneum, and the 2 g King Arthur wheat flour and 30 cm of coiled nylon females were either virgin or pre-mated and either alive or string on top (fed males treatment) or (2) string only dead (see killing method below). For pre-mated females, (starved males treatment). The latter treatment resulted in pre-mating was conducted at least 24 h before their final 15% decrease in body weight (Fedina and Lewis, 2006). mating. This was done because after 24 h all previous  Behavioral interactions in male–female pairs were spermatophores are extruded from the female bursa observed on a 29–30 1C warming tray in plastic 15 mm copulatrix and only free sperm from previous matings is mating arenas with a surface scratched to provide sufficient present in the female reproductive tract. All dissections traction. Copulation was defined as at least one intromis- were performed in Tribolium saline (10 mM Hepes buffer, sion by the male aedeagus lasting a minimum of 25 s. ARTICLE IN PRESS T.Y. Fedina / Journal of Insect Physiology 53 (2007) 93–98 95

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 4 fed males mated with dead females) were removed from¼analysis. 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 3 fed males mated with dead females, n 4 ¼ ¼ Fig. 1. Morphological similarity of T. castaneum spermatophores starved males mated with dead females, and n 1 fed male ‘‘dumped’’ outside of the body with T. molitor spermatophores extracted mated with live female). Finally, the occasional¼presence in from females after normal copulation: (a) non-everted T. castaneum the bursa copulatrix of an egg ready to be laid seems to spermatophore, inset shows a close-up of the anterior part in dark field view, (b) T. molitor spermatophore after 1st eversion (marked by bracket), interfere with normal sperm transfer, as it noticeably and (c) T. castaneum spermatophore with anterior bulb divided into two increases copulation duration, frequency of transfer failure visible compartments (see the text). The scale bars are equal to 100 mm. 1— and variability in number of sperm transferred (pers. obs.). Anterior part of spermatophore with a darker core (the gap in the Consequently, those cases were also excluded (n 1 fed normally continuous core in (a) is caused by forceps pinch); 2—posterior male mated with dead female, n 2 fed males¼mated tail; 3—cap; 4—spermatophore bulb compartment containing dark material, 5—transparent bulb compartment. with live females, and n 2 starved¼ males mated with live females). Screening ¼the 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, spermless spermatophores retrieved from females after respectively. interrupted copulation (described below). ‘‘Dumped’’ spermatophores contained no visible sperm, although free 3. Results sperm was often found clumped around the tube. Some ‘‘dumped’’ spermatophores seem to have been proceeded 3.1. T. castaneum spermatophore morphology and through initial eversion stage, as they looked like small transformation: dissection experiments bulb with a tail (Fig. 1c). Two compartments were sometimes observed inside the bulb very similar to T. castaneum spermatophores deposited outside the Fig. 25 and the corresponding description in Gadzama and body (‘‘dumped’’) appeared as invaginated double-walled Happ (1974). tube with a cap on the anterior tip (Fig. 1a), very similar T. castaneum females that were dissected immediately to non-everted T. molitor spermatophores extracted after naturally terminated copulations always contained after normal copulations (Fig. 1b; see also Fig. 18 in spermatophore (if transferred) that had already everted Gadzama and Happ, 1974). When placed in saline, these in the bursa copulatrix. This spermatophore appeared T. castaneum spermatophores underwent a two-stage as a sperm-filled sac with a posterior tail (Fig. 2). elongation similar to that described for T. molitor When extracted at an earlier transformation stage, the spermatophores (Gadzama and Happ, 1974). In sperm sac was divided into two visually distinct parts: a T. castaneum, however, this elongation process occurred posterior part with a transparent content and a larger within 5–10 s after contact with the saline (unlike 5 min for anterior part with darker material (Fig. 2a). Within T. molitor) and proceeded so quickly that it was not minutes, the two parts had coalesced and sperm, now possible to record intermediate stages. In addition, vigorously moving, expanded the sac even more (Fig. 2b). each elongation occurred along the same axis as previous More frequently, when dissected out immediately after one, and not at an angle as in T. molitor. Within mating, the sperm sac was already expanded and ruptured approximately 1 min, the elongation was followed by the at the anterior end (Fig. 2c), near the entrance to the formation of a frothy disintegrating sac, very similar to spermathecal duct. ARTICLE IN PRESS 96 T.Y. Fedina / Journal of Insect Physiology 53 (2007) 93–98

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.

3.5

3.0 5 2.5

2.0

1.5

1.0 Number of sperm transferred, x10

0.5 12 10 10 10

0 Fig. 3. Spermless T. castaneum spermatophore retrieved from a female dead live after interrupted copulation. The scale bar is equal to 100 mm. 1— females Disintegrating spermatophore sac, 2—spermatophore tail. Fig. 4. Sperm quantity (mean7SE) transferred per spermatophore by T. castaneum fed (filled bars) and starved (hatched bars) males mating Spermatophores that were retrieved from females after with dead or live females. Sample sizes are shown inside the bars. interrupted copulations appeared similar to normal everted spermatophores (Fig. 2c), but their sacs contained either no 3.2. Female influence over sperm quantity transferred sperm, or visibly fewer sperm. These spermatophores often had tails with a distinct opening at their distal end; this Starved males transferred significantly fewer sperm than opening led to a channel containing material moving into fed males but only when they mated with live females the sac. When retrieved from females even sooner after (Fig. 4, Mann–Whitney U 17.5, U0 82.5, P 0.014); transfer, spermatophores appeared as tubes with a darker there was no difference betw¼een fed an¼d starved¼males in core (similar to ‘‘dumped’ spermatophores after two-stage sperm quantity transferred to dead females (U 55.5, ¼ elongation described above); this tube, when placed in U0 64.5, P 0.767). When analyzed with ANOVA, the saline, immediately expanded at the anterior end into quantity¼ of ¼sperm transferred per spermatophore was disintegrating spermless sac (Fig. 3). affected by male treatment (F 5.54, P 0.023), was 1,38 ¼ ¼ ARTICLE IN PRESS T.Y. Fedina / Journal of Insect Physiology 53 (2007) 93–98 97 not affected by female treatment (F1,38 0.09, P 0.762), with sperm while it is located inside the female’s but most importantly, the significant¼ interaction¼ was reproductive tract. This sequence of events offers a detected between male and female treatments potential for T. castaneum females to influence ejaculate (F1,38 4.60, P 0.038). size during copulation. The experimental part of the study ¼ ¼ demonstrates that T. castaneum females do, in fact, exploit 4. Discussion this potential, as they use control over sperm numbers transferred in the male spermatophore to exercise cryptic The main goal of this study was to demonstrate one of female choice. This conclusion follows from an observation the mechanisms of cryptic female choice, namely the female that starved males transferred only about half the number influence over the quantity of sperm transferred in a male of sperm compared to fed males, but only when mating spermatophore. This mechanism was demonstrated with live females. No such difference between fed and through crossed factorial design, in which fed males starved males mating with dead females suggests that transferred significantly more sperm than starved males starvation itself does not limit sperm quantity transferred when mating with live females but not when mating with in a spermatophore, and that live females actively dead females. Using novel experimental approach, this discriminate against starved males. study presents one of the few explicit illustrations of cryptic The most plausible physiological mechanism of female female choice. control over sperm quantity in T. castaneum is contraction As a first step, the study sheds light on the process of of bursal muscles, which may reduce sperm flow into the spermatophore transfer in T. castaneum flour beetles since spermatophore either directly or by interrupting copula- this process determines the potential for female control. tion. The contraction of bursal muscles was implicated Given that T. castaneum is such an important pest and a previously in the translocation of sperm from the female model organism (reviewed in Sokoloff, 1974; Brown et al., bursa into her spermatheca (Bloch Qazi et al., 1998). This 2003), it is surprising that thus far, there has been no physiological process was suggested as the potential consistent description of spermatophore morphology in mechanism for cryptic female choice, but not demonstrated this species. Previous record indicates a simple morphology to be such. The demonstration of cryptic female choice of T. castaneum spermatophore consisting of a sperm sac requires experimental confirmation that females discrimi- with a posterior tail (Bloch Qazi et al., 1996). However, the nate for males with certain characteristics and against present study found that the T. castaneum spermatophore males lacking these features. Another potential mechanism represents an invaginated tube that everts into a was proposed based on the positive association between sperm-containing sac inside the female bursa, and that it duration of female quiescence (cessation of any motion is similar in overall structure and transformation sequence during copulation) and numbers of sperm transferred to the T. molitor spermatophore (Gadzama and Happ, (Bloch Qazi, 2003). Combined with the results of the 1974). Unlike in T. molitor, in T. castaneum spermatophore present study, this association implies that longer female eversion occurs within seconds after its transfer to the quiescence may reflect longer relaxation of the female female. In fact, upon immediate dissection after naturally bursal muscles. This may allow the male to inject more ending copulation, T. castaneum spermatophores trans- sperm into his spermatophore. ferred to the female bursa are always found already everted From the male perspective, even though within 24 h and with the sperm sac expanded. Such rapid spermato- about 86% of the transferred sperm is discarded by female phore eversion followed by sperm release may reflect an (Bloch Qazi et al., 1996), the initial quantity of transferred adaptation to the high degree of sperm competition in sperm must be important when a female mates with two or T. castaneum created by frequent female remating (Pai and more males in quick succession. In this scenario, which is Yan, 2003; Lewis, 2004). In T. molitor, when a second typical for T. castaneum (Pai and Yan, 2003; Lewis, 2004), male’s spermatophore is transferred to a female within several complete ejaculates from different males overlap in 5 min of a previously deposited spermatophore, it inhibits the bursa, and a fair raffle (Parker, 1990) at least partially the eversion and sperm release from the first-male determines the relative representation of each male’s sperm spermatophore, resulting in a complete loss of paternity that is moved into organs. by the first male (Drnevich et al., 2000). Rapid transforma- In species where it is more costly for a female to struggle tion of spermatophores transferred by T. castaneum males against male copulation attempts and sperm transfer (e.g. may therefore represent an adaptation to avoid similar due to elaborate male anchoring genitalia or due to sheer spermatophore inhibition by rivals. Alternatively, the male persistence), females may have an option to expel ephemeral nature of T. castaneum spermatophores may sperm or spermatophore after transfer as it was shown for be a consequence of miniaturization: T. castaneum is about the chrysomelid beetle Chelymorpha alternans (Rodriguez, one quarter of the body length of T. molitor. 1995), the cicindelid beetle Pseudoxychila tarsalis (Rodri- An important result of this study is the finding of guez, 1999), and for the fly Dryomiza anilis (Otronen, spermless spermatophores inside the female bursa after 1997). T. castaneum females also occasionally expel sperm- interrupted copulations. This suggests that T. castaneum containing spermatophores soon after copulation (pers. males transfer a spermless prespermatophore that is filled obs.). Therefore, T. castaneum females may use several ARTICLE IN PRESS 98 T.Y. Fedina / Journal of Insect Physiology 53 (2007) 93–98 cryptic female choice mechanisms during sequential stages Fedina, T.Yu., Lewis, S.M., 2004. Female influence over offspring of sperm transfer and storage. Specifically, females can paternity in the red flour beetle Tribolium castaneum. Proceedings of selectively prevent spermatophore transfer during copula- the Royal Society of London B 271, 1393–1399. Fedina, T.Yu., Lewis, S.M., 2006. Proximal traits and mechanisms for tion (Fedina and Lewis, 2006) and regulate sperm quantity biasing paternity in the red flour beetle Tribolium castaneum injected into the spermatophore (shown in this study). In (Coleoptera: Tenebrionidae). Behavioral Ecology and Sociobiology addition, they can potentially control sperm quantity 60, 844–853. stored in spermatheca (Bloch Qazi et al., 1998; Fedina Gadzama, N.M., Happ, G.M., 1974. The structure and evacuation of the and Lewis, 2004) and expel the spermatophore soon after spermatophore of Tenebrio molitor L. (Coleoptera: Tenebrionidae). Tissue and Cell 6 (1), 95–108. mating (pers. obs.). Such multi-level control is likely to be Hellriegel, B., Bernasconi, G., 2000. Female-mediated differential sperm less error prone and may allow for fine adjustments of storage in a fly with complex spermathecae, Scathophaga stercoraria. sperm representation from several mates. Behaviour 59, 311–317. Overall, this study describes complex morphology and Lewis, S.M., 2004. Multiple mating and repeated copulations: effects on transformation of the T. castaneum male spermatophore male reproductive success in red flour beetles. Animal Behaviour 67, 799–804. suggesting that sperm is injected into the spermatophore Mann, T., 1984. Spermatophores: development, structure, biochemical after it is transferred into the female reproductive tract. attributes and role in the transfer of spermatozoa. Zoophysiology 15, This study also demonstrates that T. castaneum females use 1–217. control over sperm quantity transferred in a male Nilsson, T., Fricke, C., Arnqvist, G., 2003. The effects of male and female spermatophore as a cryptic mechanism for female choice genotype on variance in male fertilization success in the red flour beetle (Tribolium castaneum). Behavioral Ecology and Sociobiology 53, based on male phenotypic quality. Accumulating evidence 227–233. from T. castaneum and other polyandrous species suggests Otronen, M., 1997. Sperm numbers, their storage and usage in the fly that the outcome of sexual interactions and resultant anilis. 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