Parental care is costly enough that species exist which exploit the parental care of other individuals to rear their own brood (i.e., brood parasites; Clutton-Brock, 1991; Royle et al., 2012). In social insects, where parental care is usually performed by workers, brood parasites exploit the social organization of host colonies (hence the name social parasites), and force the host worker caste to care for their brood, which is often composed of reproductive individuals only, as many species of social parasites produce no workers (Wilson, 1971; Hölldobler and Wilson, 1990). In this way, social parasites spare themselves not only the time and resources needed to build the nest and care for their young, but also those necessary to produce workers.

Studies on brood and social parasites have flourished in recent years, providing fascinating, novel examples of coevolution between parasites and hosts. These studies investigate reciprocal adaptations and counter-adaptations in the perspective of an arms race between parasites and hosts (Davies et al., 1989; Davies, 2011; Kilner and Langmore, 2011). For example, among bird brood parasites, cuckoos lay eggs which are nearly identical in shape, size and color to those of their hosts (Brooke and Davies, 1988; Davies, 2011; Spottiswoode et al., 2012). In host populations infested by cuckoos, hosts are often able to discriminate between their own and parasite eggs, and reject eggs dissimilar to their own; this ability declines when parasites become rarer (Thorogood and Davies, 2013) and does not exist in non-parasitized populations (Rothstein, 1990).

Whereas in birds the arms race between brood parasites and hosts mainly involves the visual mode, in social insects it is mainly based on olfactory cues (Kilner and Langmore, 2011). Social parasites and their offspring escape host detection and eventually coopt hosts into caring for them using chemical deception, which emerges through the selection pressures that hosts impose on social parasites by using chemical cues to discriminate against intruders (Lorenzi, 2006; Bagnères and Lorenzi, 2010). Nestmate/non-nestmate discrimination in social insects is mainly based on cuticular blends of hydrocarbons and is highly effective (Van Zweden and d'Ettorre, 2010), but social parasites circumvent it in different ways (Lenoir et al., 2001; Lorenzi, 2006; Bagnères and Lorenzi, 2010). For example, among primitively eusocial insects, such as Polistes social wasps, Polistes semenowi, and Polistes atrimandibularis parasites are poor in chemical recognition cues (Lorenzi and Bagnères, 2002; Lorenzi et al., 2004) and the same characteristic was found among eusocial insects, such as Polyergus slave making ant queens (Lenoir et al., 2001). It is likely that the scarcity of recognition cues makes it difficult for hosts to detect parasites, as the defensive responses of social insects are affected by the amount of recognition cues on the cuticle of intruders (the smaller the amount, the lower the aggressive response, Cini et al., 2009; Ichinose and Lenoir, 2010). Social parasites may also mimic host recognition cues as a way to bypass host detection, and again we have examples of such a deception trait in both eusocial and primitively eusocial insects. For example, both social wasp P. atrimandibularis and P. semenowi parasites, and the parasite queen of Acromyrmex insinuator ants match their host recognition cues (Bagnères et al., 1996; Lorenzi et al., 2004; Nehring et al., 2015), and this is likely to make it difficult for hosts to identify social parasites as intruders. Similar strategies have been reported in other inquiline ants (Guillem et al., 2014). Workers of Harpagoxenus sublaevis slave-making ants mimic the recognition cues of the different host species they enslave (Kaib et al., 1993; Bauer et al., 2010) and Solenopsis picea ants mimic their hosts, although to a limited extent (Emery and Tsutsui, 2016). In contrast, among the rare facultative slave-making ants (D'Ettorre and Heinze, 2001), Formica sanguinea ants integrate slaves in their colonies by contaminating them with the parasite odor (Wlodarczyk and Szczepaniak, 2014, 2017). Other social parasites use appeasement substances which reduce attacks by hosts. For example, a newly discovered special class of compounds, crematenones, enables Crematogaster ants to avoid aggression by Camponotus ants when they live in the same nest (parabiosis; Menzel et al., 2013).

Consistently with reports on how bird populations diverge locally in the reciprocal adaptations of hosts and parasites (Rothstein, 1990; Thorogood and Davies, 2013), the chemical strategies of integration into host nests may differ between populations of social parasites (Nash et al., 2008; Ruano et al., 2011). This is due to the uniqueness in the gene pools of both local parasites and local hosts, and to the uniqueness of the quality of the biological interactions which occur locally, both between hosts and parasites and with hosts, parasites and other interacting organisms (Thompson, 2005). In this perspective, studying multiple populations sheds light on the variety of evolutionary outcomes in the conflict of interests between hosts and parasites. However, reconstructing what pathways have led to the evolution of the specialized chemical integration strategies of social parasites has been challenging up to now. In this perspective, identifying intermediate steps along this route may increase our understanding of the evolution of deception mechanisms. Intraspecific social parasitism—where parasites use the nest and worker force of another individual of the same species to rear their own young—may represent the first step toward the evolution of obligate interspecific parasitism (Taylor, 1939; Cervo, 2006) and is widespread among insects (Field, 1992).

In birds, where intraspecific parasitism is a common female reproductive strategy, a recent review discusses the adaptive value of conspecific nest parasitism (Lyon and Eadie, 2008).

According to Lyon and Eadie (2008), intraspecific parasites may be females doing the best-of-a-bad-job when independent nesting is limited or restrained by environmental or phenotypic factors (e.g., when nesting sites are limited, or females emerge late from hibernation). Alternatively, intraspecific parasites may have lost their previous nests, e.g., to predators. Yet, they may be “lifelong specialist parasites,” i.e., females who rely exclusively on others to rear their own brood. The best-of-a-bad-job and the nest-loss hypotheses are conditional tactics and are maintained in the population even if they yield lower fitness payoffs than independent nesting (parasitizing conspecific nests is the only option to females who would not gain any fitness otherwise). In contrast, the lifelong-specialist-hypothesis implies that intraspecific parasitism is frequency-dependent, as it yields higher fitness payoffs than nesting when it is rare (Lyon and Eadie, 2008). Detailed studies are lacking that clarify which of these hypotheses explains intraspecific parasitism in social insects.

Whatever the adaptive benefits they gain, intraspecific parasites in social insects have been selected to find evolutionary solutions to the same proximate problems as those faced by obligate social parasites. Since they are usually unrelated to host workers (e.g., Seppä et al., 2011), intraspecific parasites have to conceal their identity to host workers, which have no fitness gains in rearing unrelated brood. However, the evolutionary outcomes of the conflict of interests between hosts and parasites may differ between intra and interspecific parasitism. Unlike interspecific social parasites, intraspecific parasites use exactly the same communication code as their hosts (i.e., cues, signals) which may facilitate enslaving hosts. Moreover, they have the same genes as their hosts (Lyon and Eadie, 2008). This is especially true if intraspecific parasitism is a conditional reproductive strategy, i.e., foundresses switch to invading conspecific nests after the loss of their nests. In this case, hosts and parasites share both the genes for founding nests and producing workers and those for parasitizing conspecific nests and enslaving host workers.

Intraspecific, social parasitism is common in Polistes wasps and it is often the only breeding option besides nest-founding in wasps with an annual colony cycle (Reeve, 1991; Cervo and Dani, 1996; Cervo, 2006).

Toward the end of the founding phase (that is, before workers emerge), fertile Polistes females can invade conspecific colonies and evict the foundresses (Reeve, 1991). Such females (often referred to as “usurpers” in social wasp literature) are unrelated with the foundress they evict, as documented by DNA microsatellite analyses (Seppä et al., 2011) and behave as social parasites (Cervo, 2006). In general, after violently entering the host colony and killing the legitimate foundress(es) (or inducing her—or them—to flee), intraspecific social parasites kill part of the foundresses' brood (eggs and young larvae) and begin laying their own eggs; the host workers accept the parasite and care for its brood, although the intraspecific parasites' reproductive success is usually poor (Cervo and Lorenzi, 1996a; Cervo, 2006; Seppä et al., 2011). Laboratory studies documented that foundresses forced to adopt alien colonies and to behave as intraspecific social parasites do not exhibit any chemical mimicry with host nests and mark them with their own signature (Lorenzi et al., 2007, 2011; Costanzi et al., 2013). However, we do not know whether these simulations of parasitism reliably describe the integration strategies used by intraspecific social parasites in the wild, nor whether such strategies are consistent among populations.

In the present study, we combine field work and laboratory analyses to investigate what chemical strategy paper wasps use when they behave as intraspecific social parasites as a strategy opposed to independent nest founding, and if this effect is similar in the different populations.

Our study model is Polistes biglumis, a social wasp with solitary nest foundation that inhabits open meadows in mountain areas (>1,000 m a.s.l.). This species is peculiar in many respects, including its social structure. P. biglumis populations are geographically separated, as the areas surrounding each population are comprised of mountain barriers and areas covered by forests or rocks, which are unsuitable for nesting (Lorenzi and Turillazzi, 1986). Previous work has shown that these populations have largely diverged, as they differ between each other in several life-history traits, including worker production (Fucini et al., 2009; Lorenzi and Thompson, 2011). Usually, workers are the first female offspring to emerge in Polistes colonies; they are indistinguishable from fertile females in their external morphology, but they have no fat bodies in their abdomen and forage actively. In general, they do not lay eggs. Fertile females emerge at the end of colony cycle; they have fat bodies, they spend most of the time resting at the natal colony, they mate, hibernate and found their own colony next year (Reeve, 1991). In P. biglumis, there are populations where even the very first female offspring have fat bodies in the abdomen, and thus cannot be classified as workers (measured in the first three females emerged, Fucini et al., 2009; Lorenzi and Thompson, 2011). However, in other populations, only one or two females per colony (on average) are workers (in some colony no workers are produced; Fucini et al., 2009; Lorenzi and Thompson, 2011). This was interpreted as the result of the combined effects of severe climatic conditions—which make the colony cycle very short and worker production less profitable—and high rates of interspecific social parasitism—which make worker production even less profitable (Fucini et al., 2009; Lorenzi and Thompson, 2011). The diversity in social structure between populations offers a unique opportunity to test whether the chemical signature of intraspecific social parasites differs from that of foundresses depending on whether host workers are, or are not, present. Indeed, we focused on one population where all colonies produced workers, and on two others where foundresses produced, on average, 1–2 workers only. Under the hypothesis that intraspecific social parasites are exposed to selection pressures imposed by host workers (namely, host workers' ability to discriminate between nestmates and non-nestmates), we expect that intraspecific social parasites are under stronger selection to exhibit chemical deception strategies in populations where host workers are more abundant.

We sampled foundresses and intraspecific social parasites in three geographically isolated populations. Obligate, interspecific parasites who parasitize the same host employ a double concealing strategy, composed of chemical insignificance and mimicry. They are poor in cuticular hydrocarbons and mimic their host signature, as they lose all parasite-specific hydrocarbons and become enriched in long-chained hydrocarbons, which are typical of their hosts (Bagnères et al., 1996; Lorenzi and Bagnères, 2002; Uboni et al., 2012). As a result, they are tolerated as nestmates by host workers (Lorenzi, 2003). Therefore, we asked whether intraspecific social parasites employ similar deception strategies.

P. biglumis intraspecific social parasites evict host foundresses and are the only adults on the host nests until host brood will emergence some weeks later (Lorenzi and Cervo, 1995). Therefore, chemically mimicking the hosts may be unlikely. However, other chemical changes might favor parasite integration in host colonies: compared to legitimate foundresses, the chemical signature of intraspecific social parasites might be poorer in recognition cues—so that they will go undetected—and/or might have enhanced proportions of branched or long-chained hydrocarbons, as occurs in the obligate social parasites which use the same host (Uboni et al., 2012).

These results show that the chemical integration strategies of intraspecific social parasite wasps—i.e., females who invade the colony of unrelated conspecific foundresses in the field—differ from those of obligate interspecific social parasites. Whereas the latter chemically mimic their hosts and/or are poor in recognition cues, intraspecific social parasites have hydrocarbon abundance similar to conspecific foundresses. However, we could chemically distinguish them from foundresses in one population, where intraspecific social parasites had hydrocarbon signatures characterized by significantly larger proportion of long-chained and branched hydrocarbons (in the other two populations such differences were not significant). Such signatures might have evolved in that population to facilitate the integration of intraspecific social parasites into host colonies and the exploitation and control of host workforce, because colonies consistently produce workers there. In contrast, we could not detect any specific signature in intraspecific social parasites in the two populations where colonies produce few workers, if any.

It may appear counterintuitive that intraspecific social parasites express changes in their cuticular signatures, which may consistently reveal themselves as parasites to host workers. However, social wasp workers learn their colony recognition odor at emergence from the paper nest (Gamboa, 2004), i.e., they generally use the recognition cues deposited by foundresses on the surface of the paper nest as a template for recognition processes. Intraspecific social parasites, which have recognition cues different from those of the unrelated foundresses they evict, will be unmasked by host workers if they do not interfere in such a process. Indeed, there is evidence that hosts unmask social parasites and their eggs, in P. biglumis (Cervo and Lorenzi, 1996a; Lorenzi and Filippone, 2000; Lorenzi, 2006) and in other social insects (e.g., Achenbach and Foitzik, 2009; Pamminger et al., 2013). We expect that this ability selects for deception strategies in parasites, which might include changes in the chemical signature such as higher proportion of long-chained and branched hydrocarbons.

Indeed, long-chained and branched hydrocarbons play special roles in social interactions in social insects. Menzel and Schmitt (2011) and Emery and Tsutsui (2013) showed that large amounts of long-chained and methyl-branched hydrocarbons (branched alkenes) are typical of parabiotic species of Camponotus ants—which nest within the nests of other species—when compared to free-living species of the same genus—that is, long-chained and branched hydrocarbons were relatively more abundant in species which were tolerated by other species. Of course, the conflict of interests between counterparts is much stronger in systems involving social parasites and their hosts than in those involving parabiotic ants, whose relationship is mutualistic. However, both systems imply overcoming aggressive responses to unrelated individuals, and might imply convergent chemical adaptations. There are also antagonistic interactions which involve social parasites shifting their chemical signature toward higher proportions of branched and long-chained hydrocarbons after settling in host colonies. Among paper wasps, the signature of the obligate social parasite P. atrimandibularis shift to a larger proportion of both branched and long-chained hydrocarbons after infiltrating host nests (Bagnères et al., 1996; Uboni et al., 2012). These parasites, initially violently attacked by hosts (Cervo et al., 1990), are accepted as nestmates after these changes have occurred (Lorenzi, 2003); in the meantime, foundresses regress their ovary development (Cervo and Lorenzi, 1996b). Similarly, among ants, the queens of Polyergus breviceps and P. rufescens exhibit a shift from linear to branched hydrocarbons (although not to long-chained hydrocarbons) as they enter host colonies (Johnson et al., 2001 and d'Ettorre and Errard, 1998, respectively). Therefore, several parasite and parabiotic species increase their proportions of long-chained and methyl-branched hydrocarbons when they enter their host colonies, which suggest that these compounds may play a role in deception mechanisms and/or in host control. However, these compounds might also increase for other functions, which might explain why they were relatively abundant in the signature of intraspecific social parasites. In the socially parasitic A. insinuator ants, for example, the abundance of unusually long-chained hydrocarbons (n-alkenes) “might help to hide the information from the shorter hydrocarbons” or might serve as appeasing substances (Lambardi et al., 2007). Intraspecific social parasites might hide their identity in similar ways.

In fact, an increase in branched hydrocarbons occurs at a cost, as it lowers the melting point of the hydrocarbon blends and diminishes the waterproofing properties of the cuticular layer, which are especially associated to linear hydrocarbons (Gibbs and Rajpurohit, 2010). Chain length appears to be less relevant to waterproofing, although longer chain-length hydrocarbons melt at relatively higher temperatures (Gibbs and Pomonis, 1995) and thus may contrast water loss caused by the increase proportions of branched hydrocarbons. In our study, the proportions of branched and long-chained hydrocarbons covaried in Carì and Ferrere (and were marginally correlated in Montgenèvre); such a correlation suggests that the increase in the proportion of branched hydrocarbons in intraspecific social parasites may drive a parallel increase in the proportion of longer chain-length hydrocarbons as a compensation for increased water loss. We do not know whether longer chained hydrocarbons are there for merely physiological reasons or whether they serve other functions.

Both branched and long-chained hydrocarbons may also act as fertility cues in social insects. For example, there is a shift to long-chained and branched cuticular hydrocarbons as females become fertile in the drywood termite Cryptotermes secundus (Weil et al., 2009) and in the ant Gnamptogenys striatula (Lommelen et al., 2006). A shift toward long-chained hydrocarbons also occurs in females of the ponerine ants Pachycondyla inversa (Heinze et al., 2002) and Harpegnathos saltator (Liebig et al., 2000) and it is positively associated with fertility. Recently, it has been shown that some hydrocarbons regulate worker reproduction (“queen pheromones,” Van Oystaeyen et al., 2014), and the rest of the blend may contribute to this effect (Smith et al., 2015). Indeed, obligate social parasites take over the dominant position in social wasp colonies and their chemical signatures match that of the most dominant host female in the colony (Dapporto et al., 2004), supporting the hypothesis that the changes in the chemical signature of parasites are driven by selection for regulating worker reproduction as well as for bypassing host detection.

We may hypothesize that when P. biglumis intraspecific social parasites take over the host colonies and begin laying eggs, they may keep host workers more efficiently under control if they signal both that they belong to the colony and that they are dominant, fertile females. We know that P. biglumis intraspecific social parasites exhibit an intense marking behavior when they enter host nests (Cervo and Lorenzi, 1996a) which results in overmarking the nest-foundress signature with their own hydrocarbon signature and in shifting the original nest odor toward their own signature (Lorenzi et al., 2011). Eventually, newly emerged (host) wasps will learn their colony odor as the odor of their intraspecific social parasites and will erroneously accept them as their mothers (Lorenzi et al., 2011). This is the first step to get access to the host colony, but intraspecific social parasites need also to be recognized as the dominant, fertile females by resident workers. P. biglumis intraspecific social parasites might have enhanced proportions of branched and long-chained hydrocarbons in their signature as a means to regulate host worker reproduction through dishonest signaling, as expected for social parasites (Heinze and d'Ettorre, 2009). Up to now, little is known about fertility signals in Polistes wasps (but see Dapporto et al., 2004; Oi, 2016), and we do not know whether single compounds, or blends (such as increased proportions of branched or long-chained hydrocarbons), act as fertility signals in P. biglumis. Although we might expect that the fertility of intraspecific social parasites is larger than that of foundresses (e.g., they might spare time and resources for nest construction), data on reproductive success depict a different scenario. P. biglumis intraspecific social parasites produced 25–50% the brood produced by foundresses, according to two separate studies (Lorenzi and Cervo, 1995; Seppä et al., 2011). However, these data described the success of intraspecific parasites in the populations of Montgenèvre and Ferrere, and we lack data for Carì.

Recent genetic analyses have confirmed that these wasp populations belong to the same species (Bonelli et al., 2015). However, P. biglumis populations are geographically separated and live at relatively high elevation in the Alps. Geographic distance, mountain barriers, and local biotic and abiotic factors have imposed distinct selective pressures on each population. We know that P. biglumis populations have diverged in several relevant traits, such as body size, behavioral, and life history traits (Fucini et al., 2004, 2009; Lorenzi and Thompson, 2011). For example, foundresses from different populations have quantitatively distinct cuticular chemical profiles (Bonelli et al., 2015), distinct behavioral profiles and different colony productivity (Fucini et al., 2014; Mignini and Lorenzi, 2015). More relevant to the scope of this study, foundresses produce workers in some populations, including Carì, but not in others (Fucini et al., 2009; Lorenzi and Thompson, 2011). Therefore, it does not come as a surprise that in the present research we found that intraspecific social parasites exhibit a special chemical signature in Carì, whereas their chemical signature is similar to that of foundresses in the other two populations (Ferrere and Montgenèvre), where foundresses produce almost no workers. We interpret these population differences as the result of the diversity of selection pressures acting on intraspecific social parasites in the different populations: where workers are present, as occurs in Carì, intraspecific social parasites have been selected—by host workers—to change their cuticular signals in ways that possibly conceal their identity, and/or advertise their fertility and manipulate workers. These pressures are not at play in Ferrere and Montgenèvre, where intraspecific social parasites rarely have host workers.

The divergence among phenotypic traits between populations may also be associated with the presence or absence of obligate social parasites. Obligate social parasites are absent in Carì, but intraspecific social parasites are relatively common there, whereas in the others two populations, the opposite occurs: obligate social parasites are relatively common and intraspecific intraspecific social parasites relatively rare. These differences have resulted in different selection gradients on foundress traits depending on the population of origin (Lorenzi and Thompson, 2011) and were associated with a divergence between populations in some features of the chemical signatures (Lorenzi et al., 2014). Intraspecific social parasites being more common in Carì, selection may be faster and traits can spread more rapidly; additionally, the concomitant selective pressures on chemical signatures imposed by obligate social parasites in Montgenèvre and Ferrere may have prevented the spread of intraspecific-parasite strategies. Indeed, foundresses from Montgenèvre themselves already have the highest proportions of branched hydrocarbons among the three populations (possibly as a consequence of the high prevalence of the obligate parasite P. atrimandibularis in that population, Lorenzi et al., 2014). It is reasonable that branched hydrocarbons do not further increase in intraspecific parasites because their increase would imply high fitness costs, at least in terms of waterproofing effects (Gibbs and Rajpurohit, 2010). Foundresses in Montgenèvre were special also in respect to the striking abundance of cuticular hydrocarbons—possibly another outcome of high, local, obligate social-parasite pressure—and intraspecific parasites appeared as chemical insignificant there relative to foundresses, as a byproduct of hydrocarbon abundance in foundresses.

It is not clear whether parasitizing colonies is a reproductive option conditional on nest loss or whether intraspecific social parasites are a genetically separated subset of the population, in which case even other hypotheses might be made to explain our results. Lorenzi et al. (2011) showed that foundresses behaved as intraspecific social parasites when experimentally deprived of their own nests and offered an alien one in the lab, which stands against the hypothesis that intraspecific parasites are genetically distinct from foundresses. They adopted the alien nest, marked it with their own signature and changed the host nest signature. The amount of branched hydrocarbons increased significantly in females forced to behave as intraspecific social parasites similarly to what the present data show about naturally occurring intraspecific parasites. There is both evidence that intraspecific social parasites invade host nest after nest loss (Lorenzi and Cervo, 1995) and that fertile females “sit and wait” for nest to invade without any prior nest founding attempt (Starks, 2001), so that we do not know whether intraspecific social parasites followed a conditional strategy or relied exclusively on conspecific nest usurpation to establish their colonies. In any case, the present results suggest that adaptations to social parasitism might emerge in free-living species and intraspecific social parasites display sophisticated chemical strategies which consist of adjusting their chemical signature likely in relation to the pressures imposed by host workers.

The collection of colonies and the experiments performed comply with the current laws in Italy and France. No specific permits were required for the collection neither for collection location. The species used in the experiments was not endangered or protected in these countries.

MCL conceived the study, MCL and LA designed the experiment, LA collected samples and field data and LA and AGB acquired chemical data. LA, AGB, and MCL analyzed the data, and MCL wrote the manuscript. All authors read, commented, and approved the manuscript.