Behavioral Ecology doi:10.1093/beheco/arp033 Advance Access publication 25 March 2009 How important is for females of a haplodiploid species under local mate competition?

Sven Steiner and Joachim Ruther

Institut fu¨r Biologie, Freie Universita¨t Berlin, Haderslebener Strasse 9, 12163 Berlin, Germany Downloaded from https://academic.oup.com/beheco/article/20/3/570/185777 by guest on 25 September 2021

Virgin females of are still able to reproduce by laying unfertilized eggs that develop into male offspring (arrhe- notokous ). Therefore, these constrained females may face a conflict between searching hosts and obtaining immediate fitness or investing time and energy in finding a mate enabling them to produce offspring of either sex. We studied this conflict in Walker (Pteromalidae), a pupal parasitoid of numerous fly species, by investigating olfactory preferences of constrained females for the male sex pheromone and host odor, respectively. According to Godfray’s constrained model, females of haplodiploid species gain only little advantage from mating when population sex ratio is at equilibrium. However, in species with local mate competition like N. vitripennis, females are predicted to invest comparatively more time and energy in mating activities. In behavioral 2-choice experiments, virgin females were innately attracted to both male pheromone deposits and the odor of host puparia. When given the choice between these 2 stimuli, virgin females preferred the pheromone even when they were allowed to associate host odor with oviposition success prior to the bioassay. Immediately after mating, however, the olfactory preference of females switched to host odor. We conclude that constrained females of N. vitripennis are much more motivated to invest in mate finding than in host finding as predicted by the constrained model. The results are discussed with respect to the ecology of N. vitripennis. Key words: constrained model, host odor, male sex pheromone, mate finding, Nasonia vitripennis, parasitic . [Behav Ecol 20:570–574 (2009)]

exual reproduction in the Hymenoptera is characterized by to compensate for the increasing male-biased sex ratio of the Sarrhenotokous haplodiploidy, that is, fertilized eggs de- population by producing more females. For this reason, velop into diploid females and unfertilized eggs into haploid females of haplodiploid species with a balanced sex ratio are males (Quicke 1997; Normark 2003; Heimpel and de Boer predicted to invest fewer resources in mating activities 2008). Hence, mated females have a precise control over (Godfray 1990, 1994). However, sex ratios of haplodiploid the sex of each offspring by choosing whether they fertilize species are not invariably balanced in natural populations an egg or not (Charnov 1982; Cook 1993). One consequence (Queller 2006). In nonrandomly mating species, for example, of arrhenotoky is that virgin or sperm-depleted females are where only 1 or a few mothers colonize patchy resources and nevertheless able to reproduce but are constrained to pro- the offspring of these mate among themselves at the emer- duce exclusively male offspring (referred to as constrained gence site, natural selection favors strongly female-biased sex females, Godfray 1990). Therefore, virgin females of haplodi- ratios, because under these conditions mothers gain highest ploid species may face a trade-off between searching for ovi- fitness by producing as many daughters as possible and a min- position sites and obtaining immediate fitness by producing imum number of sons necessary to fertilize all sisters in the only male offspring or searching for mates and subsequently brood. Because males in those species commonly do not dis- having the option to lay eggs of either sex. A possible ap- perse, male–male competition for females is restricted to the proach to study this conflict is the investigation of female natal patch and has therefore been termed local mate com- olfactory preferences in species using food-associated chemi- petition (LMC) (Hamilton 1967). Females dispersing un- cal messengers (kairomones) to locate oviposition sites and mated from the site of emergence are likely to get no male-derived sex attractants for mate finding. further mating opportunities. Therefore, virginity of the fe- One prediction of Godfray’s constrained model (Godfray male leads to a reproductive dead end, when she is the only 1990), which deals with the influence of oviposition by con- foundress colonizing a new oviposition site (Green et al. 1982; strained females on population sex ratio of haplodiploid spe- Godfray and Hardy 1993). Thus, a higher selective pressure to cies, is that the motivation of constrained females to engage in engage in mate finding has been predicted for females of mate finding should depend on the mating structure of the species with LMC than for panmictic species, which may join species. In panmictic (randomly mating) species where pop- a populationwide mating pool (Godfray 1988, 1990, 1994). ulation sex ratio is balanced, the investment in sons and Although other hypotheses of the constrained model have daughters is of equal value for the mother (Fisher 1930). been tested in several species (e.g., Ode et al. 1997; King Therefore, it should be no disadvantage for constrained fe- 2002; Metzger et al. 2008), little attention has been paid to males to lay all-male broods, as long as mated females are able the importance of sex for females of haplodiploid species (but see Guertin et al. 1996). Nasonia vitripennis Walker (Pteromalidae), a parasitic wasp Address correspondence to J. Ruther. E-mail: ruther@zedat. developing in puparia of cyclorraphous fly species (Whiting fu-berlin.de. 1967), is a typical LMC species (Werren 1980, 1983; Shuker Received 14 August 2008; revised 14 January 2009; accepted 8 and West 2004; Shuker et al. 2005) characterized by 1) iso- February 2009. lated host patches (nests of hole-breeding birds), 2) a female- biased sex ratio, 3) protandrous males that typically compete The Author 2009. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. For permissions, please e-mail: [email protected] Steiner and Ruther • Importance of sex for haplodiploid females 571 for females emerging from the same host or host patch, re- spectively, 4) a high degree of sib mating, 5) females that disperse soon after emergence to search for new host patches if hosts are absent at the natal site, and 6) flightless males that are unable to disperse (King and Hopkins 1963; Werren 1980; van den Assem 1986; Molbo and Parker 1996). Hence, mat- ings at places other than the site of emergence are unlikely. Sexual communication in N. vitripennis involves a male-derived sex pheromone composed of a mixture of (4R,5R)- and (4R,5S)-5-hydroxy-4-decanolides and the trace component 4- Figure 1 methylquinazoline (Ruther et al. 2007, 2008). The phero- Linear still-air olfactometer used in the experiments (wa, walking arena; nz, neutral zone; tz1 and tz2, test zones 1 and 2; mt, mone is synthesized in the male rectal vesicle (Abdel-latief microcentrifuge tube (containing odor sources); gz, gauze; en, entry et al. 2008) and applied to the ground via the anal orifice for parasitoids). Downloaded from https://academic.oup.com/beheco/article/20/3/570/185777 by guest on 25 September 2021 by dabbing movements of the abdomen (Steiner S, Ruther J, unpublished results). These marked spots are highly attrac- tive for virgin females (Ruther et al. 2007). Unlike the sexual sheet of nontransparent fine-meshed gauze. Volatiles were al- communication, little is known about the infochemical use in lowed to diffuse from the microcentrifuge tubes into the olfac- N. vitripennis during host finding (Wylie 1958; Peters 2007; tometer tube for 5 min enabling the formation of an odor Shuker et al. 2007). However, from numerous studies on gradient. Subsequently, N. vitripennis females were released other parasitic , it is well established that the location individually in the center of the olfactometer tube through of the host habitat and the hosts within the habitat is com- a cylindrical entry (1 cm diameter), and the time parasitoids monly mediated by volatile compounds originating from the spent in the 2 test zones was recorded for 5 min using the host itself, its products (silk, feces) or host-associated organ- computer software The Observer 3.0 (Noldus, Wageningen, isms (bacteria, fungi, and mites) (reviewed by Vet and Dicke The Netherlands). The olfactometer was surrounded by 1992; Steidle and van Loon 2002). a white Box (20 cm length 3 20 cm width 3 9 cm height) Using an olfactory approach, the present study aimed at study- to minimize external visual influences. Furthermore, the ol- ing the possible conflict of constrained N. vitripennis females factometer was rotated by 180 after every observation to com- either to invest time and energy into the location of hosts or to pensate for any unforeseen asymmetry of the setup. The engage in mating activities and thereby testing Godfray’s pre- olfactometer was regularly cleaned with ethanol, and odor diction that haplodiploid species with LMC should be highly sources were renewed after 2 females had been tested. Pre- motivated to get mated before searching for hosts. For this liminary test had shown that both females responded equally purpose, we performed behavioral bioassays to study the re- to the odor sources, and thus, data were pooled for statistical sponses of N. vitripennis females to host volatiles and investi- analysis. gated their olfactory preferences when given the choice between infochemicals indicating the presence of hosts and Bioassays mates, respectively. Finally, we studied possible interactions between host volatiles and the male sex pheromone. To study the olfactory preferences of female parasitoids for vol- atile stimuli associated with mate and host finding, respectively, we tested 6 hypotheses using the following 2 types of odor sour- METHODS ces in different constellations: a) Male sex pheromone. The time window of pheromone release by N. vitripennis males can be highly variable, but Nasonia vitripennis was reared on pupae of the green bottle fly in a previous study, all males deposited the pheromone Lucilia caesar as described elsewhere (Ruther et al. 2007). within an observation time of 5 h (Ruther et al. 2007). Three types of females were tested in the olfactometer experi- Therefore, 2- to 3-day-old unmated males of equal size ments: 1) Virgin/naive females had neither oviposition expe- were kept individually for 5 h in a microcentrifuge tube rience nor contact with males. They were excised as pupae to deposit the pheromone. Parasitoids were then re- from host puparia and kept isolated in 1.5 ml microcentrifuge moved, and the tube was connected to the olfactometer tubes (Brand GmbH, Wertheim, Germany) until emergence. as an odor source. Male pheromone deposition was 2) Virgin/oviposition-experienced females had oviposition ex- detectable by the characteristic odor of the pheromone perience but no contact with males. They were allowed to lay component 4-methylquinazoline (Ruther et al. 2008). eggs on suitable host puparia for 24 h and tested subse- b) Host odor. One or 5 freeze-killed host puparia were quently, 3) Mated/naive females were inseminated but had thawed for 1 h and then placed in a microcentrifuge no oviposition experience. A single virgin female was kept tube of the olfactometer as an odor source. Those host togetherwithavirginmaleinanobservationchamber(0.5cm puparia are accepted as hosts by N. vitripennis females height, 7 cm diameter) until mating was observed under a ste- without any restriction (Steiner S, unpublished data. reomicroscope. These females were tested 5 min after copu- When increasing the number of host puparia in the ol- lation. All females were used only once in the bioassays at an factometer, we assumed that the concentration of vola- age between 2 and 4 days. tile stimuli would increase as well. Our first hypothesis predicted that both the male sex pher- omone and host odor are highly attractive for virgin females Linear still-air olfactometer when tested alone. To test this prediction, we studied their re- Experiments were performed in a linear still-air olfactometer sponse to microcentrifuge tubes containing male pheromone (Figure 1), consisting of a rectangular clear acrylic tube deposits or host odor (one host puparium) versus empty con- (14 cm 3 1cm31cm) that was divided into 2 4-cm test zones trol tubes (experiments a and b, Figure 2). Our second hy- at either end and a 6-cm neutral zone in the middle. Micro- pothesis predicted that virgin females prefer the sex centrifuge tubes with odor sources were connected to both pheromone over host odor irrespective of the concentration ends and separated from the main olfactometer area by a thin of the latter. We tested this hypothesis by giving them the 572 Behavioral Ecology Downloaded from https://academic.oup.com/beheco/article/20/3/570/185777 by guest on 25 September 2021

Figure 2 Residence time (mean 6 SE) of Nasonia vitripennis females in the test zones 1 and 2 of a linear still-air olfactometer during a 5-min observation period. Asterisks indicate significant differences (*P,0.05, **P,0.01, and ***P,0.001); ns, not significant (t-test for dependent data; numbers in the bars represent replicates). choice between 1 or 5 host puparia and male pheromone deposits of a single male and the odor emitted by a single deposits (experiments c and d). Hypotheses 3 and 4 predicted host puparium (Figure 2a and b). However, when given the that oviposition experience does not influence olfactory pref- choice between host odor and the sex pheromone, females erences of virgin females, whereas mating induces an olfactory clearly preferred the pheromone irrespective of the number switch from host odor to the pheromone. We tested these of host puparia offered (Figure 2c and d). Virgin females with predictions by repeating experiment d using oviposition ex- oviposition experience nevertheless preferred the sex phero- perienced virgins (experiment e) and mated females (exper- mone over host odor (Figure 2e). Immediately after mating, iment f). Finally, we performed experiments addressing the however, females changed their behavior and preferred host interaction between the 2 types of stimuli. We predicted that odor over the male sex pheromone (Figure 2f). the male sex pheromone enhances the attractiveness of host The combination of male pheromone deposits and host odor (hypothesis 5) and vice versa (hypothesis 6). To test odor was significantly more attractive to virgin N. vitripennis these hypotheses, we studied the response of virgin females females than host odor alone (Figure 2g). However, virgin to host odor (1 host puparium) versus host odor plus phero- females did not prefer the combination of the pheromone mone deposits (experiment g) and pheromone deposits ver- and host odor over the pheromone alone irrespective of sus pheromone deposits plus host odor (1 or 5 host puparia, whether 1 or 5 host puparia were offered (Figure 2h and i). experiments h and i). DISCUSSION Statistical analysis The present study clearly demonstrates that the male sex pher- Mean residence times of N. vitripennis females in the test zones omone rather than host odor is the superior olfactory cue for 1 and 2 of the olfactometer were compared by a t-test for constrained females of N. vitripennis although host odor is dependent data using Statistica 4.5 scientific software (Stat- attractive when offered alone. This preference is innate be- Soft, Hamburg, Germany). Parasitoids that walked for less cause the tested females were naive and never had the chance than 50% of the total observation time were assumed to be to experience male-derived chemicals before the experiments. unmotivated and not included in the statistical analysis. Constrained females that laid unfertilized eggs for 24 h and thus had the chance to associate host odor with oviposi- tion success still preferred chemicals indicating the presence RESULTS of mates over host-derived volatiles. This is so much more Virgin females of N. vitripennis without prior oviposition expe- remarkable because N. vitripennis is able to associatively learn rience were strongly attracted to both the sex pheromone both colors (Oliai and King 2000; Baeder and King 2004) and Steiner and Ruther • Importance of sex for haplodiploid females 573 odors (Schurmann et al. 2009). In fact, Nasonia females learn Hence, the olfactory switch of mated females from the sex associatively any odor they experience during oviposition, and pheromone to host odor is coherent and might initiate the even those chemicals that do not occur in natural habitats dispersal of the females from the natal site to search for new become strongly attractive when females are exposed to these host patches. This idea is supported by the finding that mat- stimuli during a single oviposition event (Schurmann et al. ing modifies not only olfactory preferences in N. vitripennis 2009). We therefore conclude that constrained females of N. females but also increases their flight activity drastically (King vitripennis are much more motivated to invest time and energy 1993). This raises the question of whether our experimental in finding mates rather than hosts. Thus, our data emphati- situation exposing females to host odor and male sex phero- cally support Godfray’s prediction that constrained females of mone simultaneously was a realistic one. One might argue haplodiploid species with LMC should be selected to remove that it is much more likely that virgin females encounter the the constraint rather than obtaining immediate fitness by male sex pheromone only at the natal site and host odors only searching for oviposition sites (Godfray 1990). Olfactory pref- after dispersal. However, bird’s nests may contain successive

erences of constrained N. vitripennis females match very well generations of ornithoparasitic (sucking the blood of living Downloaded from https://academic.oup.com/beheco/article/20/3/570/185777 by guest on 25 September 2021 the results of 2 previous studies investigating the structure of nestlings) and necrophagous (feeding on dead nestlings) hosts natural N. vitripennis populations using biochemical and mo- of N. vitripennis (Peters and Abraham 2004), and under these lecular techniques (Molbo and Parker 1996; Grillenberger circumstances, females may find suitable hosts already at the et al. 2008). Both studies demonstrated that colonization of natal site (Wylie 1958). host patches (nest boxes of hole-breeding birds) by a single Our experiments on the interaction between the male sex female is common in N. vitripennis. Forty-six percent (Molbo pheromone and host odor emphasized the importance of and Parker 1996) and 28% (Grillenberger et al. 2008) of the the male-derived chemicals for constrained N. vitripennis fe- nest boxes containing N. vitripennis were parasitized by a single males. The combination of host odor and pheromone was foundress. If these females had been constrained, the result- more attractive than host odor alone, whereas host odor did ing all-male broods would likely get no chance to reproduce not enhance the attractiveness of the pheromone. This is in because N. vitripennis males are flightless and therefore un- contrast to numerous other insects where food-related info- able to disperse (Whiting 1967). Hence, the danger of gaining chemicals may drastically enhance the attractiveness of sex no inclusive fitness from their sons is very high for con- pheromones (reviewed by Landolt and Phillips 1997; Reddy strained females leaving their natal patch unmated and and Guerrero 2004). explains their strong innate preference for the male sex pher- The present study on N. vitripennis is the first testing hy- omone. In fact, the level of virginity in natural N. vitripennis potheses on olfactory preferences of constrained females populations is very low (2–3%) (Beukeboom and Werren for pheromones and host odor, respectively. Further studies 2000; Grillenberger et al. 2008), supporting the importance including species without LMC that use male-derived sex of being inseminated for females of this parasitic wasp. pheromones are needed. However, compared with other in- Volatile sex pheromones often enable mate finding over sect taxa, chemoecological research on the pheromone com- long distances and thus are usually found in species where munication of parasitic wasps is still at an early stage, and males and females emerge at different parts of the habitat apart from N. vitripennis only few species are known to use (Godfray and Cook 1997). However, because in N. vitripennis male-derived sex pheromones (Gonzalez et al. 1985; Matthews both emerge in the near vicinity of one another and et al. 1985; Coˆnsoli et al. 2002). However, the recent exem- mating exclusively takes place at the site of emergence, the plary studies on the model organism N. vitripennis (Steiner male-derived pheromone might have a different function in et al. 2006; Ruther et al. 2007, 2008; Abdel-latief et al. 2008) this mating system. Nasonia vitripennis develop in groups of opened the door to better understand the role of infochem- 10–20 individuals, by majority females, inside the host pupar- icals in the sexual communication of parasitic Hymenoptera ium. Males emerge first by gnawing an exit hole in the wall and thus to enable a more complete picture on the relative and then compete aggressively for access to mates on the importance of mate and host finding for constrained females surface of the puparium (van den Assem et al. 1980). Single of haplodiploid species. dominant males defending territories around the exit holes might copulate with most of the emerging females. However, FUNDING because the females often leave the host puparium in rapid succession, males might not be able to mate with all of them Deutsche Forschungsgemeinschaft (RU 717/7, RU 717/8-1 to during this short time period. Thus, dominant N. vitripennis J.R.). males might use the sex pheromone to arrest emerging females within the patch and prevent virgins from dispersing. Inferior We thank David Shuker, Charles Godfray, and 2 anonymous reviewers males, on the other hand, might release the pheromone to lure for helpful comments and Sophia Bohlke for technical assistance. away females from the sphere of the dominant male. Within minutes after mating or in the absence of the male REFERENCES sex pheromone, N. vitripennis females are strongly attracted by Abdel-latief M, Garbe L-A, Koch M, Ruther J. 2008. An epoxide hy- host odor. This suggests that olfactory cues play a crucial role drolase involved in the biosynthesis of an sex attractant and also in host finding and demonstrates that the female mating its use to localize the production site. Proc Natl Acad Sci USA. status determines her olfactory preference. 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