Insect. Soc. (2012) 59:61–65 DOI 10.1007/s00040-011-0188-4 Insectes Sociaux RESEARCH ARTICLE Males, but not females, mate with multiple partners: a laboratory study of a primitively eusocial wasp Ropalidia marginata M. C. Shilpa • R. Sen • S. Samudre • R. Gadagkar Received: 15 November 2010 / Revised: 5 July 2011 / Accepted: 8 July 2011 / Published online: 29 July 2011 Ó International Union for the Study of Social Insects (IUSSI) 2011 Abstract The intense interest in social Hymenoptera, on R. marginata females to mate with multiple partners is account of their elaborate sociality and the paradox of consistent with the expectation of monogamy in primitively altruism, has often suffered from considerable gender eusocial species with totipotent females, although the imbalance. This is partly due to the fact that worker apparent discrepancy with a previous work with allozyme behaviour and altruism are restricted to the females and markers in natural colonies suggesting that females may partly because males often live off the nest. Yet, under- sometimes mate with two or three different males remains to standing the males, especially in the context of mating be resolved. biology is essential even for understanding the evolution of sociality. Mating patterns have a direct bearing on the levels Keywords Social wasps Á Ropalidia marginata Á of intra-colony genetic relatedness, which in turn, along Mating behaviour Á Multiple mating Á Single mating with the associated costs and benefits of worker behaviour, are central to our understanding of the evolution of sociality. Although mating takes place away from the nest in natural Introduction colonies of the primitively eusocial wasp Ropalidia mar- ginata, mating can be observed in the laboratory if a male Social hymenopteran females usually mate with one or and a female are placed in a transparent, aerated plastic more males early in their life and store the received sperm in container, and both wasps are in the range of 5–20 days of their spermatheca. This sperm is used in all future fertil- age. Here, we use this setup and show that males, but not izations required for laying female-destined, diploid eggs. females, mate serially with multiple partners. The multiple Females in this insect order also lay unfertilized haploid mating behaviour of the males is not surprising because in eggs, which develop parthenogenetically and give rise to nature males have to mate with a number of females, only a males. In female social Hymenoptera, both the number of few of whom will go on to lay eggs. The reluctance of mating partners and the effective number of matings vary and are associated with a variety of costs and benefits. Single mating increases the relatedness among the colony members, which may sometimes promote social harmony M. C. Shilpa Á R. Sen Á S. Samudre Á R. Gadagkar Centre for Ecological Sciences, Indian Institute of Science, (though not always, see Ratnieks et al., 2006), while mul- Bangalore 560012, India tiple mating is thought to bring the advantages of increased supply of sperm, genetic diversity among the brood & R. Sen ( ) (resulting in genetic polyethism and increased immunity) Department of Entomology, Purdue University, West Lafayette, IN 47907, USA and avoidance of the sex locus load, the costs of evolving e-mail: [email protected] specialized strategies for choosing or resisting mates, etc. (Strassmann, 2001; Crozier and Fjerdingstad, 2001). R. Gadagkar Female single mating is a common female reproductive Evolutionary and Organismal Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore strategy (effective paternity is often little over 1) for 560064, India stingless bees (Peters et al., 1999), bumble bees (Schmid- 123 62 M. C. Shilpa et al. Hempel and Schmid-Hempel, 2000), most ants and most show that under this setup, males often mate multiply but vespine and polistine wasps (reviewed in Strassmann, females do not mate with any more than one partner. 2001). However, multiple mating is abundantly found in honeybees (Estoup et al., 1995; Moritz et al., 1995; Oldroyd et al., 1997; Palmer and Oldroyd 2000), some fungus- Materials and methods growing ants (Bekkevold et al., 1999; Bekkevold et al., 1999; Murakami et al., 2000; Fjerdingstad and Boomsma, R. marginata nests with large number of pupae were col- 1998; 2000), harvester ants (Cole and Wiernasz, 1999) and lected in and around Bangalore (13°000N77°320E), India. is occasionally found in vespine wasps (Muralidharan et al., All adults, larvae and eggs were removed from the nests, 1986; Ross 1985, 1986). In all, high polyandry has evolved and the nests with pupae were maintained in ventilated on nine occasions in the social Hymenoptera and about two- plastic boxes and monitored daily for the presence of newly thirds of species are monandrous (Hughes et al., 2008a, b). eclosed wasps. All newly eclosed males and females were Males in social hymenopterans usually emerge from the removed from the nest and kept in isolation in transparent, pupa with mature sperm, and additional sperm production ventilated plastic bottles (22 cm 9 11 cm 9 11 cm) with does not take place in the adult stage (Dumser, 1980; the ad libitum food and water for 5–20 days until they were exception: ergatoid males of the ant genus Cardiocondyla, used for the experiments. Heinze and Ho¨lldobler, 1993). Hymenopteran males are To study multiple mating in males, we introduced one haploid (producing clonal sperm), short-lived and often male and one female simultaneously into a fresh plastic referred to as sperm packets, as they eclose to perform the bottle (as described above). The pair was observed for 1 h or sole function of mating. Single mating associated with till they performed long conjugation behaviour (Sen et al., leaving a mating plug or spermatophore in the female 2010), whichever was earlier. Every occurrence and dura- genital tract is a usual phenomenon for many social hyme- tion of all mating behaviours: unsuccessful attempt to nopteran males (reviewed in Boomsma et al., 2005), mount—UAM, mount—MO (male climbs on to the back of especially in stingless bees, honeybees and some ants. the female), short conjugation—SC (up to 5 s long con- Multiple mating is found in males of bumble bees, social nection of abdominal tips of the male and the female in the wasps and ants (reviewed in Boomsma et al., 2005). mounted condition without any sperm transfer) and long In the primitively eusocial, polistine wasp Ropalidia conjugation—LC (locking of abdominal tips for more than marginata (Gadagkar, 2001), females either become work- 20 s that usually includes sperm transfer) were recorded (for ers in their natal nests or leave to found a new nest of their a detailed description of each of these behaviours please see own. In the event of a loss of natural queen or experimental Sen et al., 2010). If the pair performed long conjugation queen removal in an established nest, one of the workers behaviour, then the female was removed from the bottle becomes aggressive and eventually becomes the next queen immediately and a new virgin female was introduced into of the colony (Gadagkar, 2001). Although many workers in a the bottle. Similar behavioural observation was carried out colony may mate, mating itself is not essential for females to for the second female for 1 h or until the pair performed obtain direct reproductive fitness; unmated females can long conjugation behaviour. We continued to remove the develop their ovaries, suppress the ovarian development of previous female and introduce a new female until the male other, even mated females and can even function as the sole stopped performing the long conjugation behaviour. egg layer of a colony, at least for a while (Chandrashekara Behavioural observations were conducted for every pair. and Gadagkar, 1991). R. marginata males, on the other hand, All females, who took part in long conjugation behaviour, live in their natal nest for about a week and then leave to lead were then dissected to check for the presence of sperm in the a nomadic life. Males appear to wait in the foraging areas and spermatheca. Multiple mating behaviour was studied for 19 mate with the foraging females (Gadagkar, 2001). males. Given the difficulty of studying mating behaviour in To study female multiple mating behaviour, we attemp- natural conditions, Sen et al. (2010) have recently described ted to follow a similar procedure but females who mating behaviour of R. marginata in a laboratory setup and performed the LC behaviour with one male did not take part showed that male and female wasps of 5–20 days of age in the same behaviour with another male in pilot studies. readily mate in transparent, aerated, closed plastic con- Therefore, we followed a different protocol to study if tainers. Using this setup, Sen et al. (2010) and Shilpa et al. females remate (Fig. 1). We first introduced a virgin male (2010) have shown that for either sex, the probability and and female into the plastic container. The pair was observed mate choice are not influenced by body size or nestmateship for 1 h or until the performance of the long conjugation of the partner. Here, we used the same setup to ascertain behaviour, whichever was earlier. Once a female performed whether, when given a chance, males and females would the LC behaviour with a male, we immediately removed the mate multiply (sequentially with multiple partners). We male and introduced the second male (this time was 123 Male multiple mating in the social wasp R. marginata 63 Fig. 1 Protocol to test multiple mating in females. The female and the first male were introduced simultaneously. Immediately after long conjugation (LC) behaviour, the first male was removed and the second male was introduced.