Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 Contents lists available at ScienceDirect Neuroscience and Biobehavioral Reviews journal homepage: www.elsevier.com/locate/neubiorev Review The trap of sex in social insects: From the female to the male perspective Laura Beani a,∗ , Francesco Dessì-Fulgheri a , Federico Cappa a , Amy Toth b a b Department of Biology, University of Florence, Florence, Italy Departments of Ecology, Evolution & Organismal Biology and Entomology, Iowa State University, USA a r t i c l e i n f o Article history: Received 10 February 2014 Received in revised form 14 September 2014 Accepted 22 September 2014 Available online 2 October 2014 Keywords: Sexual selection Male social Hymenoptera Sexual behavior Male neuroendocrine system Polistes dominula a b s t r a c t The phenotype of male Hymenoptera and the peculiar role of males has been neglected and greatly understudied, given the spectacular cooperative behavior of female social insects. In social insects there has been considerable progress in understanding the molecular mechanisms behind haplodiploid sex determination but, beyond that, very little is known concerning the neural, endocrine, and genetic correlates of sexual selection in males. An opportunity is being missed: the male phenotype in Hymenoptera is a natural experiment to compare the drives of natural versus sexual selection. In contrast to females, males do not work, they usually display far from the nest to gain mates, compete among rivals in nuptial flights or for a symbolic territory at leks, and engage in direct or ritualized conflicts. By comparing the available data on male paper wasps with studies on other social Hymenoptera, we summarize what we currently know about the physical, hormonal, neural and behavioral traits in a model system appropriate to examine current paradigms on sexual selection. Here we review male behavior in social Hymenoptera beyond sex stereotypes: the subtle role of “drones” in the colony, the lack of armaments and ornaments, the explosive mating crowds, the “endurance” race, the cognitive bases of the “choosy” male and his immune defense. Social insect males are not just simple-minded mating machines, they are shaped, constrained and perhaps trapped by sexual selection. © 2014 Elsevier Ltd. All rights reserved. Contents 1. 2. 3. 4. 5. 6. Are insects a good model to explore sex-dimorphism in behavior and brain? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The neglected drone: male social hymenopterans in the Darwinian scenario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mating syndromes of social Hymenoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Male insects in neuro-endocrine research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Organizational and activational effects of hormones on sexual behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Male hymenopterans: a brain for just one season . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A new model for cognitive and neuroendocrine adaptations under sexual selection: a focus on Polistes dominula males . . . . . . . . . . . . . . . . . . . . . . . 5.1. The reproductive apparatus and immune defense, from a male perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Studying lekking behavior: not just “child’s play” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. The smart male: from the field to the lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wider challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519 520 522 522 522 523 525 525 527 529 529 530 530 1. Are insects a good model to explore sex-dimorphism in behavior and brain? ∗ Corresponding author. Tel.: +39 055 22 881; fax: +39 055 222 565. E-mail address: laura.beani@unifi.it (L. Beani). http://dx.doi.org/10.1016/j.neubiorev.2014.09.014 0149-7634/© 2014 Elsevier Ltd. All rights reserved. The evolutionary tree is not a hierarchy. It is tempting for all of us to view animals with which we share a more recent common 520 L. Beani et al. / Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 ancestor as being just like us. Baboons and even bluebirds can look and act an awful lot like people. A good deal of my own research is done with insects, and one of the reasons I like working with them rather then with vertebrates is that is harder to see myself reflected in their behavior. Marlene Zuk, Sexual Selections. What We Can and Can’t Learn about Sex from Animals, 2002, Introduction, p. 3. The challenge of this review it is to describe what is known about the forces of sexual selection which have shaped mating behavior, morphology and neuro-endocrine system of males in social Hymenoptera: wasps, bees, ants; and also to provide a roadmap for future studies by highlighting key questions for future research. The male phenotype is like the dark side of the moon. However it represents a natural experiment that has been greatly undervalued, to compare the drives of natural versus sexual selection, parental versus mating efforts, and their associated neurogenomic mechanisms. In social Hymenoptera males and females represent two divergent morphs. The primary abode of females (queens and workers) is the colony, whereas mating – the focus and the final chapter of male life history – occurs mainly outside the nest. The male, devoid of the constraints imposed by caste specialization, may be viewed as the output of sexual selection. In The Descent of man, and Selection in relation to Sex (Darwin, 1871), the actors of sexual selection are “ardent males and choosy females”, i.e. male–male fighting and female choice. The “wonderful horns” of male rhinoceros beetles fit into Darwin’s armaments category. Other traits, both morphological and behavioral, evolved because the females preferred them, i.e. are ornaments: again, in certain Coleoptera, “the splendid metallic tints” and “stridulating organs” of males (Chapter XI, p. 422). While competition among males for the rights to mate with a female seemed reasonable enough to Darwin’s Victorian contemporaries, virtually none of them could swallow the idea that females–of any species, but especially the so-called dumb animals –could possibly do anything so complex as discriminating between males with slightly different plumage colors. [. . .] Largely because of the opposition to the idea of female choice, sexual selection as a theory lay dormant for several decades. Marlene Zuk, Sexual Selections. What We Can and Can’t Learn about Sex from Animals, 2002; Introduction, p.7. Modern behavioral ecology has moved beyond the paralyzing view of “dumb animals”, with an increasing appreciation for the behavioral complexity and cognitive capacities of insects. Thus, in addition to an easier evasion of the risk of anthropomorphism, studies on insects have played a lead role in developing new insights in sexual selection (Table 1). The brain is one of the most important sexual organs; indeed, most sexual selection mechanisms rely on sensory/neural/cognitive differences among potential partners or rivals. Neural plasticity and learning may be involved in mating tactics, from competition to mate choice, from advertisement displays to mate guarding and pair bonding, in birds (Keagy et al., 2012) as well as in insects (Dukas, 2006, 2008) and other taxa. Neural sex dimorphism – in human and non-human animals – is the obvious consequence of Darwin’s assumption: “sexual selection has apparently acted on both the male and the female side, causing the two sexes of man to differ in body and mind” (Chapter XXI, p. 402). Brain sex differences involve developmental, ecological and taxonomic differences. “Not all sexually selected traits are conspicuous. However, and when a sex difference consists of an enhancement of cognitive and perceptual ability, disentangling the separate actions of natural and sexual selection is difficult” (Jacobs, 1996). Nevertheless, some of these differences might be best understood within the framework of sexual selection and, in particular, in social Hymenoptera. While female castes are hot topics in neuroscience and genetic analysis, social hymenopteran males are seldom subjects of molecular and neural studies of behavior, being instead used to study sex determination, sperm competition, long-term sperm storage, with some exploratory studies on brain transcriptome expression (mostly in Apis: Collins et al., 2006; den Boer et al., 2009; Stürup et al., 2013; Zareie et al., 2013; Zayed et al., 2012). Excellent reviews on genetic and genomic analyses in insect societies do not consider the male role, as this is surely marginal in terms of colony division of labor (Smith et al., 2008). In contrast to males’ lack of social behavior, the mating biology of male social hymenopterans involves cognitive abilities, is flexible and open to alternative tactics. Thus, hymenopteran traits have been the result of diverse selective forces – individual selection, kin selection, group selection, and sexual selection – acting across species and between males and females. The main goals of this review are: (i) to identify the role of males in social Hymenoptera, in which a massive emergence of males turns into collective mating syndromes; (ii) to organize the scarce and scattered neuro-endocrine data on males in an updated theoretical scenario; and (iii) to provide an overview of behavior and physiology of male Polistes dominula, a suitable model organism to investigate the expression of sexually selected traits by means of modern neuro-endocrine and genomic approaches. 2. The neglected drone: male social hymenopterans in the Darwinian scenario Eusociality, evolved in ants, bees, wasps and a few other taxa, is a rare form of complex social behavior characterized by cooperative brood care, reproductive castes (queens/kings and workers), and overlapping generations (Michener, 1969). In the Hymenoptera, these impressive feats of cooperation are entirely limited to the female sex. Social insects are descended from solitary-nesting ancestors where only females care for young (Davies and Gardner, 2014). Thus, in social as well in solitary species, females are involved in nesting and brood care, due to sex-specific expression of genes for parental behavior (West-Eberhard, 2003). Sexuals (males and gynes, i.e. virgin potential future queens) typically emerge at the peak of colony development (with some exceptions, Strassmann, 1981). Long-lived queens leave the colony and, after mating, will start a new colony, whereas males die after the nuptial season (but see Shik et al., 2013; Kureck et al., 2013 about longevity in male ants). Males lack the anatomical and behavioral adaptations to be efficient workers, such as the sting (Starr, 1985) and hunting and foraging for brood provisions (West-Eberhard, 1975), with some exceptions (see the moderately developed pollen baskets in bumble bee males observed also by Darwin, a case of “cross-sexual transfer”, West-Eberhard, 2003). Not surprisingly, the sex ratio of the colony is usually female-biased, a fact that has been recognized as far back as Charles Butler’s 1609, The Feminine Monarchie, or the Historie of Bees, the first English book about beekeeping. In a bee hive, the males are less than 5% of the total number of females (Wilson, 2007). As a rule, in colonies of eusocial insects, the investment in males is one third of that in future queens (Trivers and Hare, 1976). This asymmetry is the output of the reproductive conflict between the queen and the workers, which are more related to sisters than to brothers or sons due to haplodiploidy (Hamilton, 1972). Sex is determined by multi-allelic sex-determining loci (SDL) which, depending on the species, can consist of a single locus (such as the gene complementary sex determination) or many loci (Beukeboom et al., 2007). Females arise from fertilized diploid eggs that are heterozygous at the SDL and males from unfertilized haploid eggs. Diploid males – homozygous at the SDL – are rare and L. Beani et al. / Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 521 Table 1 Recent studies on sexual selection mechanisms in insects. Book/review/study Sexual selection mechanism Animal model Focus Andersson (1994) Lloyd (1981, 1997) Lewis and Cratsley (2008) Thornhill and Alcok (1983) Beani (1996) Beani and Turillazzi (1999) Zuk et al. (1998) Contreras-Garduño et al. (2008) Endurance rivalry Comparative approach Bumblebees, Fireflies Mating success of males active during a prolonged breeding season Endurance as a measure of male quality, bioluminescent and pheromonal signals Persistent lekking and swarming behavior, “Marathoner hypothesis” and delayed mate choice West-Eberhard (1979) Nakano et al. (2010, 2013) Ryan (1990, 1998), Ryan and Cummings (2013) Kumaran et al. (2013) Bonduriansky (2001) Parker (2006) Gwynne (2008) Gershman et al. (2013) Stutt and Siva-Jothy (2001) Parker (1970) Parker and Pizzari (2010) Waage (1986) Fedorka et al. (2011) Gray and Simmons (2013) Worthington et al. (2013) den Boer et al. (2010) Birkhead (2000) Arnquist and Nilsson (2000) Torres-Vila (2013) Eberhard (1985, 1996, 2010) Social insects Pathogens, parasites and sexual signals Sensory trap Sensory exploitation Field crickets Call parameters may attract both females and parasites Rubyspot damselfly Comparative approach The red wing spot decreases under immune challenges Male traits are attractive resembling stimuli salient in other context (food detection, predation) Male courtship songs rendering females motionless as bat calls Males traits exploit pre-existing sensory biases of females Moths Comparative approach Male mate choice Sexual conflict Sperm competition Female promiscuity Genitalia divergent evolution, cryptic female choice Fruit flies Insects Comparative approach Insects Crickets Bedbug Damselflies Plant related chemicals as male lures Pre- and post-copulatory choice in relation to search cost and female quality An overview of sexual conflict over mating and fertilization Sexual conflict over nuptial gifts, including seminal contribution Food fight over free amino acids of spermatophylax Sexual conflict and traumatic insemination Sperm displacement of rivals, morphology of penis and bursa copulatrix Fruitflies Field crickets Seminal fluid proteins allocation, effects of perceived competition Strategic ejaculates in relation to acoustic cues Social insects Comparative approach Meta-analysis Moths Comparative approach Seminal fluid mediates ejaculate competition Promiscuity as an evolutionary history of sperm competition Direct effects of multiple mating on female fitness Variation among females in re-mating propensity Male and female genital co-evolution, post-copulatory female choice, sexually antagonistic co-evolution their fitness is reduced (but see Cowan and Stahlhut, 2004; Liebert et al., 2005; Cournault and Aron, 2009). Interestingly, haploidy appears to impose constraints on males, because in many species they have “reinvented diploidy” by doubling nuclear DNA content mainly in thoracic flight muscles and forelegs. This may serve to boost expression of genes necessary to meet the energetic demands of flight and may be driven by sexual selection, as the need to fly in search of females is probably more critical to fitness than predator avoidance and long distance dispersal (Aron et al., 2005). What is the male role in the colony? In social bees, wasps or ants, workers and soldiers are females. Males seem to represent no more than a cost to the colony, “intra-colony parasites”, “examples of absolute egocentrism” (Wilson, 1971). In the English Thesaurus, “drone” is translated as “idler, scrounger, parasite”, although “a more appropriate view is that of a frenetic male in desperate search of an elusive, receptive female” (Paxton, 2005). As expected, male altruism is rare (Table 2), independently of ploidy (for the evolution of helping due to ecological factors and sex-ratio, see Ross et al., 2013; Gardner and Ross, 2013). Overall, males of Hymenoptera have no or only minor tasks in the colony (Hölldobler and Wilson, 1990); there are scattered records of larval feeding in polistine wasps (Hunt and Noonan, 1979; Cameron, 1986; O’Donnell, 1999; Sen and Gadakar, 2006; Sinzato et al., 2009); nest construction and defense in apoid wasps (Brockmann, 1992; Lucas and Field, 2011); fanning on the nest to control brood temperature in bumble bees (Cameron, 1985); trophallaxis by males of Camponotus ants living for a long time in their nest (Hölldobler, 1966); silk production by male larvae in the weaver ants (Wilson and Hölldobler, 1980). Thus, in social Hymenoptera, males are typically of the “drone” type, while the “helper” type is well represented in termites (where males are diploid, and workers and soldiers are often immature and with little sex-dimorphism, see Noirot, 1989, 1990; Thorne, 1997), and male helpers appear also in other non-hymenopteran eusocial taxa (Choe and Crespi, 1997). Because male hymenopterans are lacking conspicuous armaments and ornaments (see Baer, 2014), it is not surprising that Darwin, in The Descent of Man, and Selection in Relation to Sex, devoted just two pages to Hymenoptera out of two chapters reserved to insects. Overall, male fighting appears to be an ancestral trait (Heinze et al., 2005) that was subsequently lost and now uncommon in these taxa. Fighting is often associated with a strong investment in size in males. Darwin noted that male social insects may be “pugnacious”, but in only a few cases they are “larger and stronger than the females. On the contrary, they are usually smaller, so that they may be developed within a shorter time, to be ready in Table 2 Male ‘types’ in social insects (H = haploid male; I = inbreeding; O = outbreeding). ’Drone’ type ’Helper’ type Hymenoptera: bees, wasps, ants (H,O) [Bartz (1982)] Homoptera (Pemphigidae, Hormaphididae): aphids with female sterile soldiers for housekeeping in galls and winged dispersing males (O) [Benton and Foster (1992), Stern and Foster (1997)] Coleoptera: Austroplatypus incompertus, an ambrosia beetle with female subfertile helpers and males leaving the gallery-nest (O) [Kent and Simpson (1992), Kent (2002)] Isoptera: soldiers and workers of both sexes (I/O) [Muller and Korb (2008)] Thysanoptera: gall-induced thrips, micropterous soldiers of both sexes, male guarding on egg mass (H,O) [Crespi (1992), Choe and Crespi (1997), Chapman et al. (2000, 2002)] Coleoptera (Platypodidae e Scolytidae): ambrosia and bark beetles with male helpers at the nest (I) [Kirkendall (1993), Kirkendall et al. (1997), Biedermann and Taborsky (2011)] From Beani and Turillazzi (2002). References have been updated. 522 L. Beani et al. / Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 large numbers for the emergence of the females” (p. 264). According to Darwin, sexual size dimorphism is the rule throughout solitary and social Hymenoptera (Stubblefield and Seger, 1994). Body size of males is rarely smaller than workers but usually smaller than that of queens (Boomsma et al., 2005). Aggressiveness among rivals, as well as a high mating frequency, are more common in relatively large males of social wasps and bumble bees, while small male ants form mass swarms and the proportion of male multiple mating is lower. But do ornaments matter for male social insects more than armaments? “The power to charm the female has been in some instances more important than the power to conquer other males in battle” (Darwin, 1871, Chapter VIII, p. 279). The only male ornaments so far described in social insects are the elliptical yellow abdominal spots in the paper wasp Polistes dominula (Izzo and Tibbetts, 2012), the black pigmentation on the head of P. simillimus (de Souza et al., 2014) and the sexually dimorphic white stripes displayed by male stenogastrine wasps by fully stretching their abdomens during their costly hovering and aerial duels (Beani and Turillazzi, 1999). Overall, Darwin (1871) rightly underlines that in Hymenoptera males are “inconspicuous, being defenceless. Slight differences in color, according to sex, are common, but conspicuous differences are rare except in the family of Bees (. . .), especially in the solitary species” (p. 237), which do exhibit some sex dimorphism (see also Stubblefield and Seger, 1994). 3. Mating syndromes of social Hymenoptera Social hymenopterans differ from solitary species for “the production for this single purpose of thousands of drones, which are utterly useless to the community for any other end, and which are ultimately slaughtered by their industrious and sterile sisters” (Darwin, 1859, Chapter VI, p. 202). But male hymenopterans are not “useless” at all in terms of colony fitness. Their ultimate goal is undoubtedly mating: they are driven by the “developmental trap due to sexual selection”, a bottleneck which screens for indicators of good genes in males and subtle discrimination for sexual signals in females (West-Eberhard, 2005). “Vigorous, and in other respects the most attractive males” – to reuse the words of Darwin (1871, Chapter VIII, 262) – are not directly represented in “sexy sons”, because males do not sire sons but only female offspring due to haplodiploidy. But a long-term paternal effect of mate choice is likely to act on “sexy grandsons” and on the fitness of all the colony members. Sexual selection has a profound effect on the evolution of complex societies (Rubenstein, 2012), because offspring relatedness is ultimately determined by mating systems. Monandry is the ancestral and more frequent state of social Hymenoptera (Boomsma and Ratnieks, 1996; Strassmann, 2001), but it is interesting to note that extreme polyandry has evolved as a derived trait in a few highly social lineages (honey bees, leafcutter, army and harvester ants, and vespine wasps (Hughes et al., 2008; Boomsma, 2009). If single paternity is widespread, the role of sexual selection is critical to determine the most competitive or most preferred male by females (Baer, 2014). Relatively few studies (see Alcock et al., 1978, the first comparative paper on this topic) are devoted to the evolution of male mating strategies in Hymenoptera for at least three good reasons (Boomsma et al., 2009). First, exclusion is attributable to the “spectacular” emphasis on the Hamiltonian predictions about relatedness and eusociality. Second, it can also be explained by the fact that “male ornaments and sperm displacement devices are absent and male fighting rare”. Third, practical considerations may be at fault, because hymenopteran mating behavior is “very hard to study in the field (and close to impossible in the laboratory)” (Boomsma et al., 2009). In fact, the two major mating syndromes, female calling and male aggregation, first proposed by Hölldobler and Bartz (1985) and then extended to social wasps and bees (Ayasse et al., 2001), are ephemeral pre-copulatory phenomena that are difficult to observe and study. Because social insect males are “elusive” (Shik et al., 2013), the knowledge gap about their behavior and physiology is not surprising (but see the special issue of Apidologie devoted to “the neglected gender: males in bees”, Koeniger, 2005; Hrassnigg and Crailsheim, 2005). The nuptial systems of social insects, unlike solitary species, have been classified as scramble competition at explosive mating assemblages and prolonged lek polygyny, due to their massive production, low mate monopolization potential and widespread outbreeding (Thornhill and Alcock, 1983; Alcock and Thornhill, 2014). Sex pheromones, released by both sexes, induce impressive aerial or substrate-based swarms (Sivinski and Petersson, 1997; Shelley and Whittier, 1997), spatially organized around speciesspecific landmarks or hilltops, receptive females, more rarely, within or close to natal nests (see lethal fights for a harem of queens among wingless males of Cardiocondyla ants, Heinze and Hölldobler, 1993). With regard to post-copulatory mechanisms, both sexes may influence the strategic allocation of ejaculates. Males of honey bees, stingless bees and army ants mate singly whereas multiple mating by males is common in bumble bees and social wasps (Boomsma et al., 2005). After eclosion, males produce a fixed amount of clonal sperm; nonetheless, sperm gene expression shows substantial variation that may affect sperm morphology and fertilization ability (Higginson and Pitnick, 2011). Due to the limited amount of spermstorage, we can expect male mate choice and some effect of seminal fluid on sperm viability. But males of social Hymenoptera have “no influence on the fate of ejaculates after mating” (den Boer et al., 2010): they just survive as stored sperm Males have been regarded as “simple and small mating machines” or “flying sperm containers” (Tsuji, 1996; Baer, 2003). Although they do not display spectacular sexually selected traits, males are not simple-minded organisms. While not strictly social, they can be highly gregarious in their collective nuptial parades, where they may perform impressive sexual displays. They are also capable of behavioral flexibility in the form of alternative mating tactics, possess sophisticated chemical – and perhaps visual – perception to find females, and in some cases exercise subtle mate choice. In his concluding remarks on insects, Darwin (1871) points out that the sexes “differ in their sense and means of locomotion, so that the males may quickly discover and reach the females”, as well as in devices “for retaining the females when found” (p. 264). Sexual selection, according to the drone role outside the colony, should act on sensory and physical skills to gain fast but lasting access to females, rather than on male attractiveness and fighting abilities. In a general sense, their life history is similar to that of workers: they spend the first days inside the nest and venture outside for orientation, lekking and mating flights (Stürup et al., 2013). Males are targets of both pre- and post-copulatory pressures and are thus excellent models to study the action of sexual selection, from a behavioral as well as from a neuro-endocrine perspective. 4. Male insects in neuro-endocrine research 4.1. Organizational and activational effects of hormones on sexual behavior Unlike vertebrates, where sexually selected traits are mostly controlled by androgens and estrogens produced by gonads and extra-gonadal tissues, insect sexual development has been regarded as strictly genetic, dependent on “cell-autonomous expression of sex specific genes”, independent of the hormonal milieu (Bear and Monteiro, 2013). Each cell is thought “to know its L. Beani et al. / Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 sexual identity” according to information on the sex chromosomes (Robinett et al., 2010). However, in the last decade, a comparative approach using both invertebrates and vertebrates suggests the existence of counterparts in insects for reproductive hormones and neuropeptides (De Loof, 2008; Van Wielendaele et al., 2013). As in vertebrates, the concepts of organizational and activational hormonal effects apply to insects, allowing for a continuum between variation in early hormone levels, neural substrates and behavior (Elekonich and Robinson, 2000). In insects the hormones that control molting and growth can partly or totally affect sexually selected traits, and thus act as sex hormones: the lipophilic ecdysteroids (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), produced in the prothoracic gland, ovaries, oenocytes and other tissues, and juvenile hormone (JH), produced in the corpora allata, a distinct pair of glands in the retrocerebral complex, and probably in accessory glands (De Loof et al., 2013). In these taxa, the masculinizing or feminizing action of hormones depends on the sex of the subject, its stage of development, the timing and the target tissue. The role of ecdysteroids as sex hormones and developmental factors in insects is probably underestimated (De Loof, 2006). Not surprisingly, ecdysteroids and JH play a role in male sexual maturation and the activity of the accessory gland (Happ, 1992; Gillot, 1996), a structure under strong sexual selection because its secretions, transferred to the female, are essential for mating success in terms of female physiological and behavioral manipulation, sperm survival and sperm competition (den Boer et al., 2013). Genes for the synthesis of ecdysone (E) are expressed in the accessory glands of beetles and fruit flies, while the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females (Hentze et al., 2013). It is likely that “E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol” (Hentze et al., 2013). However, an effect on male reproductive behavior of ecdysteroids has not been clearly demonstrated. On the other hand JH, the “status quo” factor in molting and metamorphosis and “a master regulator of the female reproduction syndrome” (Hartfelder, 2000), plays a multifaceted role in controlling many aspects of sexual behavior: dispersal and nuptial flights, calling behavior, courtship, post-copulatory changes in the female, and oviposition. “Quite different from the more conserved role of JH as a modulator of ecdysteroid action in larval-pupal development, the roles of JH in adult insects are, at a first sight, rather confusing.” (Hartfelder, 2000). Evidence of hormonal effects on male sexual behavior in insects is somewhat scattered. As regards armaments and ornaments, JH and ecdysteroid titers influence horn growth during the development of the dung beetle Onthophagus taurus, resulting in males with long horns, rudimentary horns or no horns at all (Emlen and Nijhout, 1999). In male cockroach fights, JH levels affect both rank establishment (Kou et al., 2009) and sex pheromone production (Sréng et al., 1999), i.e. both male-male competition and female choice, because “odor conveys status” (Moore et al., 1997). In the noctuid moth Agrotis ipsilon, JH increases the neural sensitivity of male antennal lobes to female sex pheromones (Duportets et al., 2013). Many of the effects induced by JH hormones are costly (Teal et al., 2013): in the tephritid flies Anastrepha and Bactrocera, the administration of methoprene (JH-analog) increases male sex pheromone production, lekking behavior, courtship, and mating success when males are supplemented with proteins (but see Shelly et al., 2009). From this perspective it not surprising that JH action (or JH directly) may have an immunosuppressive effect (Rantala et al., 2003), in agreement with the immunocompetence handicap hypothesis (Folstad and Karter, 1992; see Schmid-Hempel, 2011). In honey bees, the sexual maturation of drones, which terminates with nuptial flights, is modulated by the endocrine system: 523 the JH titer gradually rises during adulthood (Giray and Robinson, 1996) due to increased synthesis in the corpora allata (Tozetto et al., 1995), while the ecdysteroid titer decreases. Experimental manipulations of ecdysteroids have shown they are potent modulators of protein synthesis in the mucus glands during larval sexual maturation (Colonello and Hartfelder, 2003). On the other hand, JH does not affect maturation of reproductive organs such as testes, seminal vesicles, and mucus glands but regulates in-hive behaviors such as movements and diet transition (Harano, 2013) and the onset of mating flights (Giray and Robinson, 1996; Tozetto et al., 1997). Many of the effects of JH on male behavior are mediated by dopamine (DA). Harano et al. (2008) showed that DA system is downstream of JH by applying a JH analog onto drones. Akasaka et al. (2010) and Mezawa et al. (2013) showed that DA influenced flight activities. JH-elicited DA signals may influence flight initiation in honey bee drones as well as in carpenter bee males (Sasaki and Nagao, 2013). The regulation of reproductive behaviors by JHDA signaling in drones resembles that of more primitively eusocial insects such as female bumble bees and paper wasps (Sasaki et al., 2012), while in female honey bees ovarian development is DAregulated (reviewed by Sasaki and Harano, 2010) and JH is the major determinant of caste differentiation and worker-specific foraging (Robinson, 1987). A possible linkage between JH and DA might be conserved in honey bee males but lost in their females in the course of social evolution (Sasaki and Harano, 2010; Sasaki, 2013). 4.2. Male hymenopterans: a brain for just one season “Perhaps these insects are little machines in a deep sleep, but looking at their rigidly armored bodies, their staring eyes, and their mute performances, one cannot help at times wondering if there is anyone inside.” Vincent G. Dethier, To Know a Fly, 1962 The contributions of the entomologist and physiologist Vincent Dethier, in establishing a continuum between the “microscopic brains” (1964) of insects and the larger brains of vertebrates, was aptly highlighted by Marlene Zuk in the chapter of Sex on Six Legs devoted to The Inner Lives of Wasps (2011). Among hymenopterans, brain research has first focused on Apis mellifera (Kenyon, 1896), because brain dissection is easier in larger insects, and caste differentiation and communication provide great opportunities to evaluate neural plasticity and divergent pathways. Overall, insect brains are composed of sensory, motor and multimodal central components. The mushroom bodies bear a striking resemblance to the mammalian cortex due to their modular organization in cell subpopulations (Farris, 2005), the fact that they combine visual and olfactory inputs, and their involvement in learning, memory, initiation of motor activity, decision-making processes (Strausfeld et al., 1998) and sleep (Joiner et al., 2006). The central complex plays a role in organizing behavior, because it integrates sensory inputs (polarized light information) and motor commands (Strausfeld, 1999; Strauss, 2002). With regard to vision, hymenopterans are generally equipped with three (bees and wasps) or two (ants) photo-pigments for UV and color vision (Briscoe and Chittka, 2001), and are able to process information about motion, patterns, direction and landmarks (Strausfeld, 1989). Large eyes and large optic lobes (lamina, medulla, lobula), the primary vision centers, suggest complex visual processing. Antennal sensilla, with thousands of olfactory receptor neurons terminating in the glomeruli of the antennal lobe are the primary olfactory centers, and perceive mechanical stimuli, humidity, temperature, carbon dioxide concentration and an infinite number of chemical cues: “ordinary” odors, but also pheromones (see Korsching, 2001 for a review in insects and vertebrates). 524 L. Beani et al. / Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 Fig. 2. Scanning electron micrograph of the head of a gynandomorphic honey bee. Right antenna and compound eye worker-like, left antenna and compound eye drone-like. From Brockmann and Brückner (1999). Fig. 1. The relatively large brain of a female Polistes dominula (top) and the “miniaturized” brain of a worker ponerine ant Pachycondyla villosa (bottom). Microtome section (15 m) of osmium-stained material. Labels: antennal lobe (al), mushroom body caliyx (ca) and lobes (mbl), central body (cb), lamina (la), medulla (me), lobula (lo), protocerebral lobe (pc), median ocellus (oc). From Gronenberg et al. (2007). Very few studies have examined the brain of paper wasps (Ehmer and Hoy, 2000; Ehmer et al., 2001; O’Donnell et al., 2013, 2014). Brain structure (Fig. 1, Gronenberg et al., 2007) has been recently analyzed in Polistes species where females seem to use facial cues to discriminate conspecifics (Tibbetts, 2002; Tibbetts and Dale, 2004; Sheehan and Tibbetts, 2011; but see Cervo et al., 2008). This ability is related to subtle changes in the size of the mushroom bodies and antennal lobes. Again, females, not males have been the focus of studies on functional neuroanatomy, brain allometry and neuroplasticity (Gronenberg and Riveros, 2009). Unlike in bees and wasps, in ants there has been “a major emphasis on olfactory and pheromone processing and relatively less prominence of visual processing” (Gronenberg, 2008). This ability is strictly related to social tasks and communication inside the “female superorganism”, because in insects, unlike in vertebrates, there are no specific “social” brain components. But what about the male brain? Studies devoted to sexual dimorphism in the chemosensory system and the brain structure of social hymenopterans are very scarce, despite the fact that male sexual performance is costly and surely involves specialized motor and cognitive skills (Table 3). A common approach has been to assay neural development and changes by comparing males and workers. Overall, the number of glomeruli reflects the richness of antennal olfactory receptors, this input is then processed in the MBs and in the protocerebrum, together with other sensory information. Males usually have fewer glomeruli than females but do possess four macroglomerular complexes, i.e. sex-specific hypertrophied glomeruli (see for a definition, Streinzer et al., 2013b). This striking difference may reflect the multiplication of sex-pheromone-sensitive sensory neurons. Male eyes and/or antennae are enlarged because they are selectively tuned to detect receptive females or pheromones. In gynandromorphic honey bees, depending on the mosaic pattern of the head, one antenna – and its antennal lobe – can be dronelike and the other worker-like (Brockmann and Brückner, 1999, Fig. 2). In contrast to the increase in eye and antenna size, males have reduced communication within the colony as well as a simpler behavioral repertoire. In line with this, MBs are smaller in males compared to females in Hymenoptera compared to other insects (Streinzer et al., 2013b). Notably, the only study on paper wasp males (Molina and O’Donnell, 2008, see Table 3) is focused on “unusual” males with female-like behavior (O’Donnell, 1999). Mischocyttarus mastigophorus males remain on their natal nests and show dominance behavior toward females, taking food from incoming foragers. Like females, more aggressive males have an enlarged antenna-innervated MB component, suggesting these males receive olfactory and tactile information useful for nestmate recognition. Individual males who frequently leave the nest for mating opportunities (in this genus along patrol routes or at nuptial arenas see Litte, 1979, 1981) have larger MB than females in the calyx region, which receive optic information. Searching for females requires enhanced peripheral and central visual processing and flexible cognitive strategies. Age is a positive predictor of MB calyx volume in both sexes. The brains of honey bee drones have been better explored: the striking dimorphism between workers and drones is probably enhanced by their different lifestyles (Fig. 3). Drones are Fig. 3. Sexual dimorphism of the peripheral olfactory system and its representation in higher brain centers in Apis mellifera drone and worker. Labels: antennal lobe (AL), mushroom body (MB), central body (CB), lobula (Lo), remainder of supraesophageal ganglion (SEG). Modified from Streinzer et al. (2013b). L. Beani et al. / Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 525 Table 3 Sex differences in sensory and brain development of social hymenopterans. OL = optical lobes, AL = antennal lobes, MB = mushroom bodies, CB = central bodies, HC = cuticular hydrocarbons. Study Animal model Sensory centers Brain specialization Focus van Praagh et al. (1980) Arnold et al. (1985) Menzel et al. (1991) Brockmann et al. (1998) Nishino et al. (2009) Streinzer et al. (2013a,b) Alford (1975) Alcock and Alcock (1983) Ågren and Hallberg (1996) Streinzer and Spaethe (2014) Honeybee drones, respect to workers, have: Extended acute zone in the dorsal eye region Elongated antenna and flagellum, more sensilla placodea, Larger optic lobula AL with a less glomeruli and macroglomeruli Smaller MB Bumblebee drones (Bombus spp.), which may scent-mark patrol routes or wait for females at perches have: Larger eyes, higher number of ommatiia in case of waiting at perches Elongated antenna Flagellar sensilla on antennae Ocelli, larger compound eyes with 1000–1500 ommatidia Sex-specific acuity related to mating behavior Selective detection of sex-pheromone Low investment in learning and memory No conspicuous species-specific or behavior-related differences for flagella types and density Larger OL (medulla and lobula) AL with a less glomeruli and macroglomeruli Smaller MB Larger CB Similar volume of OL and AL Larger MB calyx collars Smaller MB calyx lips Acute vision, less plasticity, “hard-wired” behavior Gronenberg (2008) Mysore et al. (2009) Nakanishi et al. (2009, 2010) Ant males (Camponotus spp. and other) respect to workers, have: Molina and O’Donnell (2008) Males of paper wasp Myschocyttarus mastigophorus, respect to females, have: “embedded in the protective social network and leave the colony only for short, synchronized mating flights” (Streinzer et al., 2013b) to congregate at landmarks, waiting for a queen (guided by olfactory and visual cues, Winston, 1991). If unsuccessful, they can return to their colonies, so forays outside the nest are short and males are unable to forage and feed themselves (Ruttner, 1966; Koeniger et al., 2005). Further research linking male sexual behavior and neural organization is surely needed to understand how experience can modify brain development in many subtle ways (Jones et al., 2013). Changes in brain size and organization might reduce the “global cost” of neural tissue in the evolution of learning and memory (Snell-Rood et al., 2009). In male song birds, the neural system involved in song control changes in relation to courtship and vocal performance: “a brain for all seasons” (Nottebohm, 1981). Although the nuptial season is brief for insect males, neural plasticity may play a key role if alternative nuptial strategies occur. Behavior and brain development, including MB reorganization, are influenced by juvenile hormone (JH) titers in worker honey bees, which change as they switch tasks with age (Fahrbach and Robinson, 1996). Males could provide an excellent reference point, independent of sociality, to investigate the influence of sexual selection on both neural development and gene expression (Zayed et al., 2012). Males of solitary and some social hymenopterans, like bumble bees or paper wasps, abandon the nest and have to survive for days or weeks outside; thus, we would expect a lower degree of sex-dimorphism or other adaptations, such as sex-specific odor detection and locomotion/defensive abilities, when compared to the less self-supporting honey bee drones. 5. A new model for cognitive and neuroendocrine adaptations under sexual selection: a focus on Polistes dominula males In the primitively eusocial paper wasp Polistes, a model organism in evolutionary biology (Starks and Turillazzi, 2006), morphological castes are not clearly differentiated, but genetic relatedness is always fairly high (Strassmann et al., 1989). These wasps are not only manageable as laboratory animals for experimental manipulation, but also suitable to explore the brain gene expression underlying reproductive and brood provisioning status, i.e. the evolution of castes (Toth et al., 2007, 2009, 2010). Studies of brain gene expression in P. metricus, indicate that there are Greater visual capabilities Lower chemosensory capacity Gender- and age-related effects on brain volume some conserved genes and pathways associated with foraging and aggressive behavior in both bees and wasps, suggesting that there may be a “genetic toolkit” for certain forms of social behavior across social taxa (Toth et al., 2010, 2014). It is of interest to note that several genes associated with female dominance behavior in P. metricus were also associated with male territorial aggression in Drosophila (Edwards et al., 2006), suggesting that some genes retain deeply conserved functions in aggressive behavior across a wide variety of taxa and across sexes (Toth et al., 2014). An overlooked key factor is the diversity of lifestyles among males. Unlike honey bee drones, males of paper wasps lack colony support. P. dominula males – variable in body size but slightly smaller than gynes like other wasps – are barely tolerated (Starks and Poe, 1997) and leave their colonies shortly after emergence (at around one-two weeks of age). Like bumble bees, they have to survive for days far from the nest, face pathogens and parasites, compete with rivals, and search for receptive females. In most Hymenoptera, male lifespan is around two-three months, e.g. in temperate species, from mid-July to October, and they do not overwinter. From an evolutionary perspective, “Live hard, die young” is an apt description of Hymenoptera male reproductive strategies (Zuk, 1990; Zuk and Stoehr, 2010). Alexander et al. (1997) argued that, in insects, learning in the context of courtship and mating is negligible due to their short lifespan. Conversely, Reuven Dukas (2006) stressed that males have many opportunities to learn (e.g. to avoid sexually deceptive orchids, Ayasse et al., 2001), especially if they spend their lives outside the colony like paper wasp males do. In theory a male could embark on a mazy decision-making pathway of reproductive strategies (Edward & Chapman, 2011, Fig. 4), in which he may choose tactical changes and sometimes mate rejection. In all of these areas, P. dominula males are an ideal study system. Here, we review a relevant body of behavioral and morpho-functional features of male P. dominula, from classical studies to recent results (Table 4), and conclude that lekking males represent an ideal – not yet exploited – system to study how sexual selection shapes cognitive and perceptual skills. 5.1. The reproductive apparatus and immune defense, from a male perspective “The new bridge between sexual selection and reproductive physiology”, advocated by Eberhard and Cordero some years ago 526 L. Beani et al. / Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 Table 4 A list of potential sexually selected traits in Polistes dominula males. Behavioral repertoire Focus Morpho-functional analysis Focus Study Leks at traditional landmarks, field and lab study Rubbing abdomen and legs on the substrate Landmark- based system in 4 Polistes species Nuptial systems in paper wasps, comparative approach Sperm number in relation to single mated females Antennal courtship Large residents and small transients Body size overlap between residents and transients Condition dependent strategy, flexible tactic Beani and Turillazzi (1988) Resident males mark peculiar spots inside territories Temporal and spatial segregation Sternal and leg tegumental glands Hypothesis of a pheromonal release Beani and Calloni (1991a,b) Rare copulations late in the season, endurance rivalry, marathoner hypothesis Antennal vibrations and grasps are indicator of mating success Unmated females were attracted to male odor, but not the reverse Sexual trials in lab between nestmates Male competition and female choice in lab studies Sexual preference trials in lab No clear avoidance of both nestmates and inbreeding Small elliptical abdominal spots as quality signals Bimodal-size sample, lab study Beani (1996) Post-emergence maturation of reproductive apparatus Sex attraction by olfactomer bioassays Sexual selection in lab by parasitized males Beani et al. (1992) Males avoid workers and sexually interact with gynes, healthy and parasitized (castrated) Parasitized males maintain their sexual behavior and preference Territorial strategy, more than size, affects mating success Class 3 sex dimorphic glands in antennae with ampulla-like reservoires Male glands in gaster and legs One month old males store a growing number of sperm waiting for females Hypothesis of a pheromonal release Salerno and Turillazzi (2001) Orientation rather than copulatory behavior: interference from alarm pheromone venom? MacKenzie et al. (2008) Romani et al. (2005) Liebert et al. (2010) Izzo and Tibbetts (2012) HC profiles differ among workers and gynes, no parasite effect Caste chemical cues orient male choice, regardless of the reproductive value of females Cappa et al. (2013) Histological analysis of male apparatus The parasite Xenos vesparum does not heavily affect the apparatus Apparatus development unrelated to body size, larger AG are associated with more copulations The parasite Xenos vesparum does not heavily affect the apparatus neither the size of CA Honeybee male larvae have higher susceptibility to bacteria than wasp male larvae Cappa et al. (2014) Analysis of testes, seminal vesicles and accessory glands (AG) Age and parasite effects Structure/ultra-structure of male reproductive apparatus, corpora allata (CA) Haplodiploid susceptibility hypothesis Bacterial challenge to test the immune response of males and females (1995) emphasizes the importance of avoiding a focus on each sex separately. The male reproductive apparatus of Polistes (Fig. 6) was surely an object of less attention than the ovaries, which serve as “a kind of biography” (West-Eberhard, 1996) for female paper wasps, despite the fact that sexual structures are deeply co-evolved. Adult males emerge sexually immature, like females; when they leave the colony, at around 2 weeks of age, they are able to copulate (Beani, pers. obs.) and are equipped with a fixed amount of stored sperm because testes degenerate 10 days after emergence (Salerno and Turillazzi, 2001). The large discrepancy between the number of sperm found in the vesicles of mature virgin males (≈500,000 in unmated 30-days old males, (Salerno and Turillazzi, 2001) and in the spermatheca (≈6800 sperm), suggests that the male may be polygynous and the female monandrous, as in most polistine wasps (Strassmann, 2001, but see recent data on multiply paternity in Polistes, Seppä et al., 2011, and in bumble bees, Owen and Whidden, 2013). Thus, the capacity to mate and intrasexual competition for a receptive female is potentially high (Edward & Chapman, 2011, see Fig. 4) and the male reproductive strategy is influenced by age and sperm storage. Male multiple mating with the same or different females has been often recorded in Polistes (Beani, 1996; Izzo and Tibbetts, 2012; Beani and Zaccaroni, 2014) although it does not Beani and Zaccaroni (2014) Unpublish. data Cappa et al. (in press) necessarily imply multiple inseminations. This mismatch between single paternity and repeated mating has been highlighted by Baer (2014) in other monandrous social insects. The general absence in wasps of genital plugs or peculiar devices to displace sperm (Richards, 1978; Eberhard, 2004) could be balanced by the evolution of “non-genital contact structures” (Eberhard, 2010) beyond the sclerotized clasping parameres (Fig. 5), which are species-specific, as in other Hymenoptera (Eberhard, 1996). For example, the curled antennae of P. dominula grip and rub female antennae during the brief period of mating contact (West-Eberhard, 1969), and are covered by sensilla and secretory glands, and may be a possible source of sex-pheromones; thus antennation in males could be critical for social interactions, as in females (Romani et al., 2005). Inside a large cage we observed both frequent antennal copulatory courtship, combined with coupling and mate guarding (Beani and Zaccaroni, 2014). Postcopulatory female choice is likely happening in P. dominula. The long spermathecal duct of female P. dominula, with a large reservoir and glands opening directly into the duct, as in other Polistinae (Gotoh et al., 2008, 2009), might allow both sperm storage and selective sperm utilization (Eberhard, 1996). Sperm expulsion by females (Boomsma and Ratnieks, 1996) likely represents a form of L. Beani et al. / Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 Fig. 4. Selection for male mate choice is affected by mating and parental efforts, operational sex ratio (OSR), potential reproductive rate (PPR), but also by the ratio of available females/male mating capacity, a factor that is often undervalued, as well as the variation in female quality. From Edward and Chapman (2011). cryptic choice by females. In P. dominula, we observed a female that was grasped in flight by a male, the two wasps fell on the ground, and this was accompanied by the ejection of a dense liquid drop containing sperm (Beani and Zaccaroni, 2014). The role of male accessory glands (AG), which grow with age and are the source of a blend of secretions, would indeed be of special interest, due to the long sperm storage before reproduction. These glands, which are widespread in insects (Gillot, 1996; Gillott, 2003; Simmons, 2001), can influence female reproductive physiology and behavior through secretory proteins and antimicrobial peptides (Avila et al., 2011) transferred with sperm during copulation (Eberhard and Cordero, 1995). They are deeply involved in “the chemical warfare between sexes”, as described by Baer (2003), who stressed the necessity of a proteomic approch (Baer, 2014). In a sample of males interacting inside a large cage, the size of AG was significantly related to the number of copulations (Beani and Zaccaroni, 2014). In line with our data, in D. melanogaster, males with larger glands mated at a higher frequency than did males with smaller glands, regardless of body size (Bangham et al., 2002). Body size was not related to size of testes, vesicles, or AG; this development of AG, independent of the size, mirrors what is seen in Polistes females, where large size is not an absolute predictor of well-developed ovaries (Dapporto and Palagi, 2006). Morpho-functional studies on the male reproductive apparatus of P. dominula are still needed. To date, a natural experiment involving a parasite of these wasps suggests that neuro-endocrine mechanisms may differ between sexes. The parasitic castrator Xenos vesparum (Strepsiptera) produces dramatic phenotypic alterations in their P. dominula hosts. Parasitized females have 527 undeveloped ovaries and desert the colony without performing any social tasks (Hughes et al., 2004; Beani, 2006; Beani et al., 2011). In a pioneering neuroendocrine study (Strambi et al., 1982), parasitized females were found to have smaller JH-secreting organs (corpora allata) and lower JH-levels. In contrast, the volume of the corpora allata in males decreases with age, as expected, regardless of parasite’s presence (unpublished data). Moreover, neither the structure of the male reproductive apparatus (Fig. 5) nor the sexual performances were seriously compromised by the parasite (Cappa et al., 2014, Fig. 6), although subtle differences might occur in AG proteins of parasitized males (unpublished data). Both pathogens and parasites may affect sexual traits (Zuk, 1990, 2009; Zuk and Wedell, 2014). The controversial but highly heuristic “sicker sex hypothesis” predicts that males will have a lower immune defense compared to females (Poulin, 1996a,b). In social hymenopterans, there is a combined influence of genetic (“haploid susceptibility hypothesis”, O’Donnell and Beshers, 2004) and environmental factors (i.e. the investment in individual versus social immunity, depending on male lifestyle). Given that the sex of the host may have a strong influence on the impact of a parasite (Zuk and McKean, 1996; Cappa et al., 2014), we compared bacterial clearance in larvae of P. dominula and A. mellifera of both sexes (Cappa et al., in press). In agreement with predictions based on behavioral dimorphism, males of P. dominula, which spend a greater proportion of their lives away from the nest, showed higher bacterial clearance than workers. Honey bee drones, which benefit from the protection of the colony for most of their lives, had a higher susceptibility to bacteria compared to workers (in agreement with Laughton et al., 2011). However, further trials, testing humoral and cellular responses, are necessary to identify possible trade-offs in P. dominula males between two linked costly traits, mating effort and immune function. 5.2. Studying lekking behavior: not just “child’s play” The term lek in Swedish denotes nuptial arena of Galliform birds but also “child’s play”: in many ways, lekking resembles a form of behavioral play for adults. In P. dominula, hundreds of males can be found lekking around the tops of poles, trees and other landmarks, which may be conspicuous visually and also facilitate pheromonal diffusion into the wind (see Table 4). Scent-marked leaves and small perches along their patrol routes function as tiny exclusive territories within the lek. The same lek system may be used by sequential generations of males according to a speciesspecific temporal and spatial segregation at landmarks. There is some evidence for alternative mating tactics, as larger males tend to defend territories whereas smaller males spend more time searching for females in sub-optimal locations across territories, as in other Polistes (Post and Jeanne, 1983; Polak, 1993). However, body size is not strictly correlated with behavior, and there is a certain degree of flexibility (Beani and Turillazzi, 1988; Beani and Zaccaroni, 2014). Because male hymenopterans lack of ornaments and armaments, their behavioral performance becomes critical to securing a mate. The acrobatic repetitive movements among the same perches, following repetitive routes, may resemble for some features the dance of manakins (Baske et al., in this issue) and be attractive per se (Prum, 2012). Territorial males commonly mate on their scent-marked perches, which become the cross-points of this ‘dance’ (Beani and Zaccaroni, 2014). The lek paradox , i.e. the conservation of genetic variation among males, regardless of female preference for male extreme traits (recently reviewed by Alcock and Thornhill, 2014; see Zuk and Wedell, 2014), has probably overshadowed other components of lekking behavior, which is widespread in insects. Leks are full of potent visual and chemical signals and represent a changing social context. Lekking males must be equipped by a set of sensorial and 528 L. Beani et al. / Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 Fig. 5. Morphological comparison of the reproductive apparatus from unparasitized (A–D) and parasitized (E–H) sexually active males of P. dominula. Stereomicrographs (A and E) show no macroscopic difference in the apparatus: Ag, accessory gland; Sv, seminal vesicle; T, testis. Cross sections of testes (B and F) show the strong degenerative aspects (asterisk) of the mature gonads. Semithin sections of monolayered seminal vesicles (C and G) show developed epithelial layers (Ep) surrounding a vesicle lumen completely filled with sperm cells (Sp). Through cross sections of accessory glands (D and H), a double layer of muscle fibers (Ms) and a glandular epithelium (Ep) are visible. The glandular lumen is full of granular secretions (Se). From Cappa et al. (2014). cognitive skills: spatial learning to reach the same rendezvous sites, day after day; motor learning to patrol, mark, defend and memorize exclusive territories; the evaluation of sexual signals and quality of potential rivals and partners by visual/chemical cues; neural plasticity, by continually adjusting their performance in relation to male/female density; visual, olfactory, tactile assessments of females before mating and male mate choice. A crowded lek may also represent a “sensory trap” (see West-Eberhard, in this issue) Fig. 6. On the left: males significantly (***P < 0.001) directed their choosiness toward gynes (◦ = outliers). On the right: Stepwise discriminant analysis of CHCs of 20 healthy gynes (HG), 20 parasitized gynes (PG) and 20 workers (W) tested for male preference. From Cappa et al. (2013). L. Beani et al. / Neuroscience and Biobehavioral Reviews 46 (2014) 519–533 for highly gregarious future queens, which leave the colony to form extra-nidal clusters at the end of summer (Beani et al., 2011). In this way, males might exploit pre-existing sensory biases of females that are attracted by wasp aggregations and by major orientation landmarks (and thus these leks may not be “symbolic” at all, L. Pardi pers.obs.), located along female transit routes, in areas at high density of nests, hibernacula and foraging patches. Who is the winner in the lek play? With regard to mating success, long-term territorial males achieve more copulations, but records are scarce and occur mainly at the end of the nuptial season. Beani suggested the “marathoner hypothesis” (Beani, 1996) to explain this phenomenon, which occurs in 9 out of 19 Polistes species – specifically, that the longer a male remains at a lek, the higher his probability of mating success. This idea is a variant of the “endurance rivalry” hypothesis (see Table 1), an idea that has not yet been thoroughly investigated (Andersson and Iwasa, 1996), but recently that has received some support from studies in lekbreeding anurans (Castellano, 2009; Castellano et al., 2009). Lek endurance may be related to energy stores. Before and after the peak of the lek period, it is common to observe non-aggressive Polistes males on flowering patches, covered with pollen (WestEberhard, pers. obs.). In males of P. metricus, another temperate species, lipids and proteins significantly decrease from late summer emergence to fall (October), whereas there is a rise in carbohydrates due to foraging, which compensates for the loss of energy reserves (Judd et al., 2010). Females may use male lek persistence to select high quality males by delaying mate selection as long as possible, until stable leks have been established As Lloyd stressed in bumble bees and other insects, “by scoring endurance females have a good and reliable measure of male quality” (1981); the same is likely to be true in paper wasps. Noticeably, in the lek-like swarms of hover wasps in the subfamily Stenogastrinae, females approached aerial aggregations at the end of the flight period, when only vigorous males persisted in their costly displays of abdominal stripes (Beani and Turillazzi, 1999). Waiting for a persistent male could be a parsimonious mate choice providing females with an indicator of “good genes”. 5.3. The smart male: from the field to the lab To the human eye, a Polistes lek looks like a cloud of insects; this makes a difficult study subject. In addition, a bias is likely to occur in field studies centered on the core of lek and on “hot-shot” males (Höglund and Alatalo, 1995). An artificial lek, a large greenhouse with paper leaves as perches and scattered resources (see Beani and Zaccaroni, 2014), provides some real advantages, e.g. to observe rare behaviors such as antennation, coupling and mate guarding or the mating success of small males with well-developed AG (see 5.1, 5.2). Most importantly, observations are not biased toward the most conspicuous territorial males. In yet another experimental setting, observations of sexual interactions in tiny lab arenas can provide a more detailed view of other aspects of sexual selection, i.e. the role of male color, abdominal spots and relatedness (see Table 4). In this simplified environment using short-term trials, visual/chemical cues have been implicated as important traits for mate acquisition. The yellow face and the yellow abdominal surface of males in P. dominula, as well as the silvery hairs on the male clypeus of the polistine wasp Chartergellus (Chavarrìa-Pizarro and West-Eberhard, 2010) and the black pigmentation on the head of other male Polistes (de Souza et al., 2014) might be sex-dimorphic short-range signals, a subtle “ornament” ripe for further investigation. It is also important not to overlook the possibility that males exercise mate choice for females as well. In a review of sexual selection studies on a large spectrum of species (Andersson and 529 Iwasa, 1996), female mate choice was found in 167, while male mate choice only in 30 cases, especially when males prefer large and fecund females. In a review devoted to insects (Bonduriansky, 2001), male mate choice was observed or inferred in 58 species of insects, belonging to 37 families and 11 orders, although its evidence was “sketchy” in Hymenoptera. Beyond the Darwinian binomial “ardent males, choosy females”, in theory male mate choice could occur also in absence of male parental care, nuptial gifts and sex role reversal, when there is a “variation in female quality” (Edward and Chapman, 2011; see Fig. 4). Recent studies in P. dominula suggest males may indeed have the ability to show mate preference between workers and future queens (Cappa et al., 2013), i.e. castes differing in their reproductive potential (Fig. 6). The results indicated that “males do not like the working class” (Cappa et al., 2013); they preferred gynes, both healthy and parasitized (castrated) by X.vesparum, and this choice was probably made on the basis of chemical cues, i.e. cuticular hydrocarbons (CHCs). Healthy and parasitized gynes are very similar in their CHC profiles (Dapporto and Palagi, 2006; Cappa et al., 2013), and they both possess poorly developed ovaries, large body size and high lipid storage. These physiological similarities could explain the males’ confusion in mate choice, in which the parasitized females are “right” as far as their caste, but “wrong” according to their reduced reproductive value. However, the males’ fixed amount of sperm and the large variation in female quality should select for the evolution of male mate choice (see Fig. 4), although this may be cryptic and difficult to quantify. Lab trials in small arenas require some caveats and, for future studies, precautions must be made to ensure that the experimental design does not overestimate the opportunity for male mate choice with respect to the natural situation. 6. Wider challenges Despite a large body of literature available for Polistes male sexual behavior, we lack key information on how differences in body size, mating tactics and health actually relate to the evolution of polymorphic traits, at the levels of hormones, neurons and gene expression. As model organism, Polistes wasps offer several advantages (long-term studies on behavior and physiology, the ability to rear and mate them in laboratory, the availability of genomic tools, see Toth et al., 2010), which make them highly suitable to be used for pioneering work in this field. Future research will not only add further insights into the reproductive behavior of social hymenopterans, but also allow more general hypotheses about the interplay between natural and sexual selection. In wasps, bees, and ants sexually selected traits seem relatively “weak” (Boomsma et al., 2005) both in males, devoid of armaments and ornaments, and in females, where the choice of male traits is likely limited by ancestral monogamy and perhaps not appreciated because of overlooked post-copulatory behaviors. By combining quantitative behavioral data, experimental manipulation, and assessment of immune-competence in both sexes, within and across species, it will be possible to identify shared and novel genes involved in the expression of sexually selected traits, and to describe co-evolved pathways of male and female phenotypes. “Sex is an antisocial force in evolution” (Wilson, 1975). This bold statement, which Boomsma (2007) used to introduce a review on “kin selection versus sexual selection”, may require reconsideration. Perhaps the role of male mate choice is more important than has previously been realized; such a revelation could imply that male mating decisions can have a strong impact in colony life and long-term fitness. And perhaps, thus, the ‘Ant’ could finally meet the ‘Peacock’ (Cronin, 1991). 530 L. 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