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Facultative social parasites mark host nests with branched hydrocarbons
Animal Behaviour 82 (2011) 1143e1149
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Facultative social parasites mark host nests with branched hydrocarbons M. Cristina Lorenzi a, *, Rita Cervo b,1, Anne-Geneviève Bagnères c, 2 a
Dipartimento di Biologia Animale e dell’Uomo, Università di Torino Dipartimento di Biologia Evoluzionistica Leo Pardi, Università di Firenze c I.R.B.I., UMR CNRS 6035, Université de Tours b
a r t i c l e i n f o Article history: Received 26 May 2011 Initial acceptance 23 June 2011 Final acceptance 8 August 2011 Available online 13 September 2011 MS. number: 11-00433R Keywords: brood parasitism cuticular hydrocarbon nest usurpation Polistes biglumis scent marking social wasp
The chemical integration strategies of facultative social parasites of social insects have not received the scientific attention they deserve, even though there is considerable research being done on the strategies of obligate social parasites. We simulated intraspecific nest usurpations in the social paper wasp, Polistes biglumis, by dividing each nest into two parts and putting one half in the care of the original foundress and the other half in the care of a usurper. After 8 days, we removed and killed foundresses and usurpers, and later tested the responses of naïve, sister-offspring to them. In each half-colony, the offspring were more tolerant to the female that was last on the nest, regardless of whether she was the foundress or a usurper. This suggested that usurpers had the chemical means to be tolerated by the host offspring. Comparisons between the epicuticular hydrocarbon profiles of foundresses and usurpers showed that usurpers were neither chemically insignificant nor transparent, nor were they mimetic, as obligate parasites often are. Instead, usurpers had chemical profiles richer in methyl-branched hydrocarbons than those of the foundresses. Analyses of the hydrocarbon profiles of nest paper revealed that usurpers supplemented host nests with their own hydrocarbons, a sort of nest marking. As a result, the chemical profiles of the host nests became qualitatively more similar to those of the usurpers. These chemical strategies illustrate that branched hydrocarbons play a role as semiochemicals and that facultative parasites may not all be on the main pathway to obligate parasitism. Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Parasites exploit any resources that free-living organisms offer: from molecular engines to whole cells, from whole organisms to the structures they make. In their struggle to exploit hosts, parasites provide some of the best evidence of adaptation by natural selection. Brood parasites are species that do not target organisms but rather the resources that free-living organisms build or produce: their nests, their parental care and/or their social structures (Wilson 1971; Rothstein 1990). Brood parasites exhibit traits that give them an advantage in fooling and exploiting their hosts and these traits are the results of reciprocal hosteparasite interactions across evolutionary time (Brooke & Davies 1988). The recognition systems of birds and social insects protect nests from brood parasites. For example, the hosts of cuckoos discriminate between their own eggs and those of cuckoos visually, and so cuckoos lay visually mimetic eggs (Rothstein 1990; Rothstein & Robinson 1998). Furthermore, social insects distinguish nestmates * Correspondence: M. C. Lorenzi, Dipartimento di Biologia Animale e dell’Uomo, Università di Torino, Via Accademia Albertina 13, 10123 Torino, Italy. E-mail address: [email protected] (M. Cristina Lorenzi). 1 R. Cervo is at the Dipartimento di Biologia Evoluzionistica Leo Pardi, Università di Firenze, Via Romana 17, 50125 Firenze, Italy. 2 A.-G. Bagnères is at the I.R.B.I., UMR CNRS 6035, Université de Tours, Faculté des Sciences, Parc Grandmont, 37200 Tours, France.
from non-nestmates by means of chemicals, and social insect parasites (social parasites) trick their hosts about their own chemical identity (Bagnères & Lorenzi 2010). Doing so, social parasites enter host colonies, exploit host nests and use the host workforce for their own reproduction. The information about chemical identity in social insects is conveyed by epicuticular hydrocarbon blends (Howard & Blomquist 2005; Blomquist & Bagnères 2010). Generally, individuals from different species have epicuticular hydrocarbon blends that differ in composition (Bagnères & Wicker-Thomas 2010). Within species, individuals from different colonies have hydrocarbon blends that differ in the relative proportions of their compounds (BonavitaCougourdan et al. 1987; Bruschini et al. 2010; Van Zweden & d’Ettorre 2010). Within colonies, nestmates recognize each other because their chemical profiles are very similar. Social parasites escape detection by hosts in at least three ways: they mimic the chemical profiles of their hosts (chemical mimicry, sensu Dettner & Liepert 1994); they have chemical profiles that are poor in hydrocarbons (chemical insignificance, Lenoir et al. 2001); and/or they lack some of the hydrocarbons of their hosts (chemical transparency, Martin et al. 2008a). One illustrative example of chemical mimicry is that of the slavemaker Polyergus queens, which take on chemical profiles that
0003-3472/$38.00 Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2011.08.011
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match those of their Formica hosts (Habersetzer & BonavitaCougourdan 1993; D’Ettorre & Errard 1998; Johnson et al. 2001). Similarly, all three species of the obligate social parasite Polistes wasps perfectly mimic their congeneric host chemical profiles (Bagnères et al. 1996; Turillazzi et al. 2000; Lorenzi et al. 2004). The Polyergus parasite species and two of the Polistes species are also chemically insignificant when they invade host colonies (Lenoir et al. 2001; Lorenzi & Bagnères 2002, Lorenzi et al. 2004). These chemical strategies can only be explained as the result of hosteparasite coevolution (Lorenzi 2006; Bagnères & Lorenzi 2010). The original, basic function of insect hydrocarbons was probably physical: the limitation of desiccation. This function was probably coopted by a semeiotic function in a series of evolutionary steps that are mostly unknown (Le Conte & Hefetz 2008). Likewise, we know very little about the evolutionary steps that led social parasites to evolve chemical insignificance and chemical mimicry. In this respect, any research about the chemical mechanisms employed by nonspecialized, facultative, social parasites may contribute to identifying the steps towards the evolution of the chemical strategies that obligate parasites use to overcome host detection. Intraspecific facultative parasitism is common among social insects (Hölldobler & Wilson 1990; Cervo 2006; Beekman & Oldroyd 2008) and intraspecific parasites have been pointed out as potential obligate preparasites or incipient obligate parasites (Taylor 1939; Savolainen & Vepsalainen 2003; Cervo 2006; Buschinger 2009). None the less, we have not come across any published research investigating the chemical ecology of intraspecific social parasites through the analysis of their hydrocarbon blends. In Polistes, facultative social parasites (hereafter usurpers) invade host colonies during the founding phase, that is, the phase when the foundresses are the only adults in the colonies. Often, the targeted colonies are solitary foundations and hence a single foundress is the only adult defending her colony (Cervo & Dani 1996; Cervo 2006). Unlike most obligate parasites, usurpers attack, chase away or kill adult hosts. Then usurpers take over host colonies, comprising the host nests and the host immature brood, and ‘wait’ for the host brood to emerge. When the host brood emerge, usurpers dominate them and force them into rearing their own brood (Cervo & Dani 1996; Cervo 2006). In the social wasp Polistes biglumis Linnaeus, the singly founded colonies are often the targets of conspecific usurpers (Lorenzi & Cervo 1995). We investigated whether usurpers are chemically insignificant, mimetic or transparent by simulating intraspecific nest usurpation in the laboratory. Although limited to one species, we also hope this study broadens our knowledge of female scent marking in animal conflicts in species other than paper wasps. METHODS Model Species In the solitarily founding species P. biglumis (Lorenzi & Turillazzi 1986), up to 18% of the colonies are usurped by conspecific usurpers, possibly after they have lost their own nests (Lorenzi & Cervo 1995). Within a colony, the hydrocarbon profiles of adult wasps are similar to each other and to the profiles that cover the paper nest surfaces (Lorenzi et al. 1996). In contrast, hydrocarbon profiles differ between colonies (Lorenzi et al. 1997). General Procedure We split nests collected in the field into two parts: one half was reared by the original foundress (foundress half-colonies) and the other half was ‘put up for usurpation’ to an alien female (usurper; usurper half-colonies). Usurpers and ‘host’ colonies came from two
populations separated by mountain barriers. Therefore we expected that usurpers would not be related to the colony they usurped. We collected 24 colonies in Chalpe (Italian Western Alps: 44 560 N, 6 490 E) in mid-July 1997. At the same time, we also collected 24 P. biglumis colonies in Montgenèvre (French Hautes Alpes: 44 550 N, 6 430 E). The colonies were towards the end of the founding phase and single foundresses were on the nests. In the laboratory, we randomly chose 12 colonies from each collection site. We removed their foundresses and placed them separately in glass jars. We cut their paper combs in two with scissors so that the larger number of pupae was intact and the numbers of cells and old brood (large larvae and pupae) of the two parts were similar (foundress half-colonies: 17e28 cells; usurper half-colonies: 16e28 cells; Wilcoxon pairwise test: Z ¼ 0.420, P ¼ 0.674; foundress half-colonies: 5e8 brood; usurper half-colonies: 2e8 brood; Z ¼ 0.962, P ¼ 0.336). Each half comb was separately fastened to the wall of a plastic box (18 12 cm and 11 cm high) and either its original foundress (foundress half-colony) or a usurper (usurper half-colony) was put into the box. If the colony came from one population, its usurper came from the other. The half-colonies were kept under a 12:12 h light:dark photoperiod and supplied with honey, water and Tenebrio molitor larvae ad libitum. We kept foundresses and usurpers on their halfcolonies for 8 days, then we removed and froze them in individual glass vials (18 C). We used them later in behavioural tests and chemical analyses (see below). Because foundress and usurper half-colonies had immature offspring, we waited for their emergence. The first offspring emerging from the foundress halfcolonies were sisters to those emerging from the usurper halfcolonies and both were daughters of their original foundresses (usurpers usually keep the first offspring of the foundress when they usurp host colonies, Lorenzi & Cervo 1992; Cervo & Lorenzi 1996). The offspring emerged after we removed foundresses or usurpers. Consequently, the offspring never met their foundresses or usurpers during adulthood and were immature in the 8-day period that foundresses and usurpers spent on the half-colonies. Therefore the offspring were naïve to foundresses and usurpers when we performed the behavioural tests. Behavioural Tests We tested whether the offspring discriminated between their foundress (i.e. their mother) and usurper (i.e. the usurper of their own half-colony or of their sisters’ half-colony) by performing bioassays using the dead foundresses and usurpers. Bioassays using dead insects eliminate the confounding effects of behavioural or chemical actions by the insects introduced and are routinely used in recognition experiments in social insects (e.g. BonavitaCougourdan et al. 1987; Lorenzi et al. 1997; Ruther et al. 2002). The tests were performed at least 1 day after the offspring emerged. At room temperature, we introduced the foundress and the usurper separately into each pair of half-colonies in random order and at least 1 h apart. We used forceps to keep foundresses and usurpers 1 cm from the nest surfaces. The tests lasted 1 min from the first unambiguous reactions by the offspring. The observer did not know whether the dead female was the foundress or the usurper in the test half-colony. We noted the occurrence of the following behaviours: biting, fleeing from the nest by flying, attempting to sting and grasping. In the behavioural tests, we recorded the responses by 1.13 0.18 adult female offspring per foundress half-colony and by 1.33 0.44 adult female offspring per usurper half-colony. The offspring tested did not differ significantly in age between half-colonies (on average: 3.18 2.71 days old in foundress half-colonies; 6.38 2.72 days old in usurper halfcolonies; Wilcoxon pairwise test: Z ¼ 1.826, P ¼ 0.068).
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For our analysis of behavioural data, we computed the mean frequency of attacks as the sum of the behaviours recorded in 1 min in each half-colony divided by the number of adult offspring in that half-colony. Behavioural data came from pairs of sisters that emerged from foundress or from usurper half-colonies. Consequently, we analysed behavioural data as paired data by using a general linear model for repeated measures (GLM, repeated measures, with GreenhouseeGeisser correction), after 1/squareroot transformation to meet assumptions of normality and homoscedasticity. Chemical Analyses After the behavioural tests, we analysed the chemical profiles of 16 half-colonies, namely, eight foundresses, eight usurpers and a 1 cm2 piece of the paper from their half-colonies. We weighed the wasps and the pieces of nest paper using a precision balance (Precisa 125A). On average, wasps weighed 31.80 1.21 mg and the pieces of nest paper 18.36 2.40 mg. We extracted the hydrocarbons by dipping each wasp or piece of nest paper separately into 1 ml of pentane for 75 s. We added to each extract 800 ng of n-C20 as an internal standard to quantify the amount of hydrocarbons. For each extract, 2 ml samples were analysed by capillary gas chromatography with a Delsi Nermag DN200 gas chromatograph (GC). The GC was equipped with a flame ionization detector and a Chrompack CPSIL5 WCOT CB nonpolar capillary column (25 m, 0.25 mm, 0.12 mm). Helium was the carrier gas (1 bar). We used a 15 s splitless method of injection. Oven temperature increased from 70 C to 150 C at a rate of 30 /min. After an isotherm of 5 min, the temperature increased to 320 C at a rate of 5 /min. Data were registered with an Enica 31 integrator and compounds were identified by comparing their mass spectra and their retention times against those recorded earlier (Lorenzi et al. 1997). The large majority of peaks consisted of single hydrocarbons. A few peaks consisted of a mix of coeluted hydrocarbons. The data for each extract were analysed in two ways. (1) We calculated the overall amount of hydrocarbons (in ng per mg of wasp or piece of nest paper) as the overall sum of peak areas 800 ng divided by the area of the C20 peak in that extract; the resulting value was divided by the weight of the wasp or piece of nest paper. (2) We also calculated the relative proportion of branched to total hydrocarbons as the amount of branched hydrocarbons in ng divided by the total amount of hydrocarbons in that extract. Test for Chemical Insignificance We tested for differences in the overall amount of hydrocarbons between matched pairs of foundresses and usurpers and between matched pairs of foundress half-colonies and usurper half-colonies (GLM, repeated measures). Hydrocarbon amounts were ln transformed to meet assumptions of normality and homoscedasticity. We also measured the surplus (if any) in the amount of hydrocarbons in usurper half-nests as the difference in the overall amounts of hydrocarbons between matched pairs of usurper and foundress nest papers. Assuming that the surplus was caused by the usurpers, we checked whether the surplus in the amounts of hydrocarbons was correlated with the similarity of the chemical profiles of the usurpers and those of their respective half-colonies (as measured by BrayeCurtis similarities in the percentages of the methylbranched hydrocarbons).
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usurpers, and in the nest paper of their respective half-colonies. Because the hydrocarbons in P. biglumis are either linear or branched, they have binomial distributions and we analysed them by using a GLM with binomial errors and logit link function. Test for Chemical Mimicry Because our preliminary analyses showed that branched hydrocarbons played a role in the usurpers’ chemical strategies, we checked whether foundresses and usurpers had profiles of branched hydrocarbons similar to those of their respective half-colonies. Therefore we excluded linear alkanes from the analysis. We also excluded two branched-alkane peaks (peak 11a: 3-methylhexacosane; peak 42a: 5-methylhentriacontane) that were present in less than 25% of the samples. We divided the integration area of peak 46 þ 47 (n-docotriacontane and 3,9-þ3,11þ3,13-dimethylhentriacontane) and peak 58 þ 59 (n-tetratriacontane and 3,9-þ3,11-þ3,13-þ3,15-dimethyltritriacontane) by two, because both peaks contained mixtures of branched and linear alkanes (in unknown proportions). Therefore, for each extract, we had 51 branched-hydrocarbon peaks (chain length between 24 and 34 carbon atoms) and we computed their relative proportions. We handled these compositional data (percentage of compounds) by using the log-ratio transformation (Aitchison 1982). To this end, we computed the natural log of the proportion of each peak and divided it by the geometric mean of the proportions of the branched alkanes. We analysed log-ratio-transformed data by using a stepwise multiple discriminant analysis (DA). The DA used the colony as a grouping variable (within-group covariance matrix) and entered geomean-log-transformed data as independent variables (Mahalanobis distance method). We used half of the cases as a training subset and the remaining half as a test subset and we derived the discriminant functions on only a portion of the cases (foundresses and the nest paper of their half-colonies). We then assessed the performance of the discriminant functions on the remaining cases (usurpers and the nest paper of their half-colonies). We expected that DA would discriminate significantly between foundresses and the nest paper of their halfcolonies by colony. We also expected that DA would successfully classify usurpers and the nest paper of their half-colonies by colony only if the usurpers chemically mimicked their host colonies. If the DA misclassified usurpers and the nest paper of their half-colonies by colony, the usurpers would not chemically mimic their host colonies. We tested whether the attacks of offspring from usurper half-colonies against usurpers increased as the distances between the usurper and her half-nest in the DA space increased (Pearson correlation, after controlling for normality). We computed the distances between usurpers and their half-nests in the DA space as the differences between the values of the discriminant function scores for usurpers and those for the nest paper of their respective half-colonies. Statistical analyses were performed in SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, U.S.A.; see Garson 2008 for procedures). BrayeCurtis similarities were computed in PAST (Hammer et al. 2001). Descriptive statistics are given as means 1 SE, unless otherwise stated. RESULTS Behavioural Tests
Test for Chemical Transparency We tested how different from each other (if at all) the proportions were of branched to linear hydrocarbons in foundresses and
The aggressive responses of sisters to foundresses and usurpers depended on whether the sisters had emerged from the foundress or the usurper half-colonies as shown by a significant interaction
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term (GLM, repeated measures, half-colony*target of attacks: F1,30 ¼ 10.857, P ¼ 0.003; power was acceptable, that is, larger than 0.80 only for the two-way interaction). Foundresses tended to be attacked less by offspring from foundress half-colonies than by their sisters from usurper half-colonies (Fig. 1). Usurpers tended to be attacked less by offspring from usurper half-colonies than by their sisters from foundress half-colonies (Fig. 1). Chemical Analyses
The variation in the overall amount of hydrocarbons between usurper and foundress half-colonies differed between wasps and nest paper, as shown by a significant interaction term (GLM, repeated measures, half-colony*sample category: F1,14 ¼ 8.161, P ¼ 0.013; Fig. 2). Matched pairs of foundresses and usurpers had roughly equivalent amounts of hydrocarbons (F1,7 ¼ 2.224, P ¼ 0.179). In contrast, the nest paper of usurper half-colonies had significantly larger amounts of hydrocarbons than that of foundress half-colonies (F1,7 ¼ 6.190, P ¼ 0.042; Fig. 2). The larger the surplus of hydrocarbons on the nest paper of usurper half-colonies, the more similar the branched-hydrocarbon profiles of the usurpers were to those of their own half-colonies (Pearson correlation: r6 ¼ 0.898, P ¼ 0.015). Test for Chemical Transparency Usurpers had significantly higher proportions of branched hydrocarbons than foundresses and, similarly, the nest papers of usurper half-colonies had significantly larger proportions of branched hydrocarbons than those of foundress half-colonies. However, the slope of the variation was steeper for wasps than for nest paper (as shown by a significant interaction term in the c21 ¼ 2605:72, GLM, half-colony*sample category: Wald P < 0.0001; Fig. 3). In their epicuticular profiles, foundresses had on average 62% of branched hydrocarbons and usurpers 72%,
Usurper 6
5
2000
0 Foundress half-colony
Usurper half-colony
Figure 2. The total amount of hydrocarbons as a function of half-colony and sample category (half-colony categories: foundress and usurper half-colony; sample categories: wasp and nest paper). The total amount of hydrocarbons is expressed in ng per mg of wasp or of nest paper.
a significant difference (Wald c21 ¼ 4421:477, P < 0.0001). The nest paper of foundress half-colonies had on average 72% of branched hydrocarbons and that of usurper half-colonies 74% (Fig. 3). Although only 2%, these differences were highly significant (Wald c21 ¼ 397:549, P < 0.0001). Test for Chemical Mimicry Colonies composed of foundresses and pieces of their nests could be significantly distinguished from each other by the DA (Fig. 4). Despite large variation, the discriminant model as a whole was significant (step 1: Wilks’s l ¼ 0.222, F7,8 ¼ 4.002, P ¼ 0.035; step 2: Wilks’s l ¼ 0.019, F14,14 ¼ 6.332, P ¼ 0.001). Function 1, which accounted for 93.42% of the total variance, differed significantly in mean by colony (Wilks’s l42 ¼ 0.000002, P < 0.00001). Function 2, which accounted for only 5.9% of the total variance, differed significantly in mean by colony as well (Wilks’s l30 ¼ 0.001, P ¼ 0.006). This DA classification showed that 100% of foundresses and their nest paper were correctly assigned to their colonies. In contrast, only 12.5% of usurpers and their nest paper were correctly assigned to their original colonies. This suggested that usurpers and their nest paper had chemical profiles that did not match those of
Proportion of branched hydrocarbons
Attacks by workers
Foundress
3000
1000
Target of attacks
7
Nest Wasp
4000
The blend of epicuticular hydrocarbons of P. biglumis foundresses, usurpers and their nest surfaces consisted of 70 peaks representing homologous series (C24eC34) of linear and methylbranched alkanes and confirmed previous GCeMS data (Lorenzi et al. 1997). Test for Chemical Insignificance
Category of sample
5000
Total amount of hydrocarbons
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0.8
Category of sample
0.75
Nest Wasp
0.7 0.65 0.6 0
0 Foundress half-colony
Usurper half-colony
Figure 1. Mean frequency of attacks SE by Polistes biglumis sisters that emerged from the half-colonies towards their own foundresses, the usurpers of their sisters or their own usurpers.
Foundress half-colony
Usurper half-colony
Figure 3. The proportion of branched alkanes as a function of the half-colony in wasps and nest paper. The proportion of branched hydrocarbons was computed as the relative amounts of branched hydrocarbons in ng divided by the total amount of hydrocarbons.
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20
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(b)
10 0 −10 20
(c)
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(h)
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Function 2
0 −10 20 10 0 −10 20
Foundress Foundress half-nest
10
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0 −10
Usurper half-nest −40 −20
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Function 1 Figure 4. Discriminant analysis (discriminant scores) of 51 branched hydrocarbons of P. biglumis foundresses, usurpers and their half-nests for colonies aeh, showing how similar the half-nests were to their foundresses or usurpers. For nest f, the point representing the usurper half-nest had a function 1 value <40; therefore, it is not shown in the graph.
the original colonies. This further suggested that usurpers were not mimicking the chemical profiles of the colonies they had usurped. Finally, it suggested that usurpers had partially changed the chemical profiles of their nests. The dissimilarity between the chemical profile of usurpers and that of their host nests mediated aggression by the host offspring: the larger the dissimilarity, the stronger the aggression by the host offspring (Pearson correlation: r6 ¼ 0.839, P ¼ 0.037). DISCUSSION Facultative social parasites of P. biglumis employ a chemical strategy of integration in host colonies, but they were neither chemically insignificant nor transparent, relative to their hosts. They were not even chemical mimics of their hosts. Instead, usurpers had chemical profiles that were richer in methyl-branched hydrocarbons than those of the foundresses. In addition, usurpers deposited large quantities of hydrocarbons on the surfaces of their host nests, a type of scent overmarking that is common in mammals (Johnston 2005; Jordan et al. 2011). The deposition of hydrocarbons on host nest surfaces by usurpers changed the chemical profiles of the host nests, making them qualitatively more similar to the chemical profiles of usurpers themselves. These adjustments influenced the host colony signatures, which were the ones that the offspring that emerged from usurper nests were to learn. The nest paper is the place where newly emerged paper wasp offspring get their information about their colony’s chemical signature. During nest
foundation, foundresses mark the nest surfaces with their own signatures (e.g. Espelie et al. 1990; Dani et al. 1992; Layton & Espelie 1995; Lorenzi et al. 1996). The emerging offspring learn the signature of their colony from the nest surfaces soon after they emerge and then use the learned template to discriminate between nestmates and strangers (Gamboa 1996, 2004). Any wasp approaching the nest is accepted by the offspring if its chemical profile matches the profile they had learned from the nest and, if not, the approaching wasp is rejected. Usually, usurpers are unrelated to host foundresses (e.g. Seppä et al. 2011) and hence they exhibit chemical profiles that do not match those of host nests. Therefore, usurpers will be rejected unless they change the host nest surface chemistry in a way that biases it towards their own profiles. Our behavioural results show that the offspring from usurper half-colonies, which were naïve to usurpers, were relatively tolerant towards usurpers. Because these offspring had no contact with their usurpers before the bioassays, they must have learnt the usurper signatures from the usurper marks on the nest surfaces. Usurpers probably marked host nests through stroking, a behaviour peculiar to Polistes usurpers, which spend time rubbing their abdomen on nest surfaces (Cervo & Turillazzi 1989; Dani et al. 1992; Lorenzi & Cervo 1992; Cervo & Dani 1996; Cervo & Lorenzi 1996; Van Hooser et al. 2002;). Surprisingly, the proportion of branched hydrocarbons on the nest paper was higher than that on the respective wasps. This might be caused by overmarking: foundresses marked their nests by repeatedly placing their hydrocarbon marks on the top of their previous hydrocarbon marks. In turn, usurpers placed their
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hydrocarbon marks on top of the hydrocarbon marks of the foundresses. As a consequence, hydrocarbons increased in quantity on the nest paper. The different marks probably had different proportions of linear and branched hydrocarbons because the proportions of branched and linear hydrocarbons on the wasp cuticle varied. When we extracted the hydrocarbons from the nest paper, we analysed a mixture of past and recent marks, rather than only the mark on the top. This may explain the mismatch between the proportion of hydrocarbons of wasps and nests. In nature, usurpers are not a subset of the wasp population. In fact, foundresses shift to usurping conspecific nests as a conditional strategy when their colonies are killed by predators (Lorenzi & Cervo 1995). Similarly, the usurpers in this research project were a random subset of the collected foundresses. Therefore these results suggest that foundresses have a flexible chemical signature, which they adjust depending on the conditions they are facing. In this respect, they resemble obligate social parasites, whose chemical signature is highly flexible and changes with the phases of host colony invasion (e.g. Bagnères et al. 1996 in paper wasp parasites; reviewed in D’Ettorre & Errard 1998; Lenoir et al. 2001; Akino 2008; Bagnères & Lorenzi 2010). Apart from flexibility, however, the chemical strategies of usurpers are very different from those of most obligate social parasites. When obligate social parasites enter host nests and face their hosts for the first time, they are often chemically insignificant (but see Cini et al. 2011). What obligate parasites do next is to steal into the host social structures and mimic their hosts chemically. Our results show that the usurpers’ chemical strategies of integration are very different from those of obligate social parasites in that usurpers mark, rather than mimic. This suggests that P. biglumis usurpers are not likely to be incipient obligate parasites. This does not imply that obligate parasitism did not originate from conspecific usurpation in other species. For example, Polistes nimphus intraspecific usurpers seem to mimic host colonies (Lorenzi et al. 2007) and therefore exhibit a preadaptation to obligate parasitism that P. biglumis usurpers do not have. Most of the approximately 200 species of paper wasps behave as facultative parasites but only three monophyletic species behave as obligate parasites (Choudhary et al. 1994; Cervo & Dani 1996). In fact, the transition from facultative to obligate parasitism is rare. This implies that most facultative parasites, such as P. biglumis, do not have the preadaptations that allow them to shift from facultative to obligate parasitism. Usurpers and their host nests were particularly rich in methyl-branched hydrocarbons. We are far from deciphering the communication codes encrypted in the hydrocarbon profiles of social insects. We know that the different structural classes of hydrocarbons are likely to work in concert to convey recognition signals (Greene & Gordon 2007), but we also know that some classes of compounds, that is, methyl-branched hydrocarbons, may play key roles in recognition (Bonavita-Cougourdan et al. 1987; Jackson et al. 2007; Martin et al. 2008b). We found a significant increase in branched hydrocarbons in both usurpers and in usurper nest paper surfaces. Slavemaker Polyergus queens also exhibited a marked enrichment in branched hydrocarbons after invading the host nests (Johnson et al. 2001). Branched hydrocarbons seem to be more easily detectable and seem to convey more information than linear alkanes (Châline et al. 2005; Dani et al. 2001). However, they reduce the waterproofing effect of epicuticular hydrocarbon layers (Le Conte & Hefetz 2008; Gibbs & Rajpurohit 2010). Then, what advantage do usurpers gain by emphasizing their hydrocarbon profiles and enriching their branched-hydrocarbon components disproportionately? If branched hydrocarbons play an essential role in encoding information about a queen’s identity, then successful usurpers must cover the legitimate queen’s signature with their own. In effect, they virtually ‘bury’ the legitimate queen’s chemical
signature to make it easier for them to trick emerging host offspring about the queen’s identity. In conclusion, facultative parasites may use chemical strategies of integration into host colonies that are different from those of obligate parasites. These chemical strategies illustrate that branched hydrocarbons play a role as semiochemicals and that facultative parasites may not all be on the main pathway to obligate parasitism. These are findings that may very well be applicable to species other than P. biglumis.
Acknowledgments We thank Ilaria Cometto and Giuliana Marchisio for their support during the behavioural experiments. We also thank Laura Azzani and two anonymous referees for their helpful comments on the manuscript. Funding was provided by M.I.U.R. (ex 60%) to M.C.L.
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Facultative social parasites mark host nests with branched hydrocarbons M. Cristina Lorenzi a, *, Rita Cervo b,1, Anne-Geneviève Bagnères c, 2 a
Dipartimento di Biologia Animale e dell’Uomo, Università di Torino Dipartimento di Biologia Evoluzionistica Leo Pardi, Università di Firenze c I.R.B.I., UMR CNRS 6035, Université de Tours b
a r t i c l e i n f o Article history: Received 26 May 2011 Initial acceptance 23 June 2011 Final acceptance 8 August 2011 Available online 13 September 2011 MS. number: 11-00433R Keywords: brood parasitism cuticular hydrocarbon nest usurpation Polistes biglumis scent marking social wasp
The chemical integration strategies of facultative social parasites of social insects have not received the scientific attention they deserve, even though there is considerable research being done on the strategies of obligate social parasites. We simulated intraspecific nest usurpations in the social paper wasp, Polistes biglumis, by dividing each nest into two parts and putting one half in the care of the original foundress and the other half in the care of a usurper. After 8 days, we removed and killed foundresses and usurpers, and later tested the responses of naïve, sister-offspring to them. In each half-colony, the offspring were more tolerant to the female that was last on the nest, regardless of whether she was the foundress or a usurper. This suggested that usurpers had the chemical means to be tolerated by the host offspring. Comparisons between the epicuticular hydrocarbon profiles of foundresses and usurpers showed that usurpers were neither chemically insignificant nor transparent, nor were they mimetic, as obligate parasites often are. Instead, usurpers had chemical profiles richer in methyl-branched hydrocarbons than those of the foundresses. Analyses of the hydrocarbon profiles of nest paper revealed that usurpers supplemented host nests with their own hydrocarbons, a sort of nest marking. As a result, the chemical profiles of the host nests became qualitatively more similar to those of the usurpers. These chemical strategies illustrate that branched hydrocarbons play a role as semiochemicals and that facultative parasites may not all be on the main pathway to obligate parasitism. Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Parasites exploit any resources that free-living organisms offer: from molecular engines to whole cells, from whole organisms to the structures they make. In their struggle to exploit hosts, parasites provide some of the best evidence of adaptation by natural selection. Brood parasites are species that do not target organisms but rather the resources that free-living organisms build or produce: their nests, their parental care and/or their social structures (Wilson 1971; Rothstein 1990). Brood parasites exhibit traits that give them an advantage in fooling and exploiting their hosts and these traits are the results of reciprocal hosteparasite interactions across evolutionary time (Brooke & Davies 1988). The recognition systems of birds and social insects protect nests from brood parasites. For example, the hosts of cuckoos discriminate between their own eggs and those of cuckoos visually, and so cuckoos lay visually mimetic eggs (Rothstein 1990; Rothstein & Robinson 1998). Furthermore, social insects distinguish nestmates * Correspondence: M. C. Lorenzi, Dipartimento di Biologia Animale e dell’Uomo, Università di Torino, Via Accademia Albertina 13, 10123 Torino, Italy. E-mail address: [email protected] (M. Cristina Lorenzi). 1 R. Cervo is at the Dipartimento di Biologia Evoluzionistica Leo Pardi, Università di Firenze, Via Romana 17, 50125 Firenze, Italy. 2 A.-G. Bagnères is at the I.R.B.I., UMR CNRS 6035, Université de Tours, Faculté des Sciences, Parc Grandmont, 37200 Tours, France.
from non-nestmates by means of chemicals, and social insect parasites (social parasites) trick their hosts about their own chemical identity (Bagnères & Lorenzi 2010). Doing so, social parasites enter host colonies, exploit host nests and use the host workforce for their own reproduction. The information about chemical identity in social insects is conveyed by epicuticular hydrocarbon blends (Howard & Blomquist 2005; Blomquist & Bagnères 2010). Generally, individuals from different species have epicuticular hydrocarbon blends that differ in composition (Bagnères & Wicker-Thomas 2010). Within species, individuals from different colonies have hydrocarbon blends that differ in the relative proportions of their compounds (BonavitaCougourdan et al. 1987; Bruschini et al. 2010; Van Zweden & d’Ettorre 2010). Within colonies, nestmates recognize each other because their chemical profiles are very similar. Social parasites escape detection by hosts in at least three ways: they mimic the chemical profiles of their hosts (chemical mimicry, sensu Dettner & Liepert 1994); they have chemical profiles that are poor in hydrocarbons (chemical insignificance, Lenoir et al. 2001); and/or they lack some of the hydrocarbons of their hosts (chemical transparency, Martin et al. 2008a). One illustrative example of chemical mimicry is that of the slavemaker Polyergus queens, which take on chemical profiles that
0003-3472/$38.00 Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2011.08.011
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match those of their Formica hosts (Habersetzer & BonavitaCougourdan 1993; D’Ettorre & Errard 1998; Johnson et al. 2001). Similarly, all three species of the obligate social parasite Polistes wasps perfectly mimic their congeneric host chemical profiles (Bagnères et al. 1996; Turillazzi et al. 2000; Lorenzi et al. 2004). The Polyergus parasite species and two of the Polistes species are also chemically insignificant when they invade host colonies (Lenoir et al. 2001; Lorenzi & Bagnères 2002, Lorenzi et al. 2004). These chemical strategies can only be explained as the result of hosteparasite coevolution (Lorenzi 2006; Bagnères & Lorenzi 2010). The original, basic function of insect hydrocarbons was probably physical: the limitation of desiccation. This function was probably coopted by a semeiotic function in a series of evolutionary steps that are mostly unknown (Le Conte & Hefetz 2008). Likewise, we know very little about the evolutionary steps that led social parasites to evolve chemical insignificance and chemical mimicry. In this respect, any research about the chemical mechanisms employed by nonspecialized, facultative, social parasites may contribute to identifying the steps towards the evolution of the chemical strategies that obligate parasites use to overcome host detection. Intraspecific facultative parasitism is common among social insects (Hölldobler & Wilson 1990; Cervo 2006; Beekman & Oldroyd 2008) and intraspecific parasites have been pointed out as potential obligate preparasites or incipient obligate parasites (Taylor 1939; Savolainen & Vepsalainen 2003; Cervo 2006; Buschinger 2009). None the less, we have not come across any published research investigating the chemical ecology of intraspecific social parasites through the analysis of their hydrocarbon blends. In Polistes, facultative social parasites (hereafter usurpers) invade host colonies during the founding phase, that is, the phase when the foundresses are the only adults in the colonies. Often, the targeted colonies are solitary foundations and hence a single foundress is the only adult defending her colony (Cervo & Dani 1996; Cervo 2006). Unlike most obligate parasites, usurpers attack, chase away or kill adult hosts. Then usurpers take over host colonies, comprising the host nests and the host immature brood, and ‘wait’ for the host brood to emerge. When the host brood emerge, usurpers dominate them and force them into rearing their own brood (Cervo & Dani 1996; Cervo 2006). In the social wasp Polistes biglumis Linnaeus, the singly founded colonies are often the targets of conspecific usurpers (Lorenzi & Cervo 1995). We investigated whether usurpers are chemically insignificant, mimetic or transparent by simulating intraspecific nest usurpation in the laboratory. Although limited to one species, we also hope this study broadens our knowledge of female scent marking in animal conflicts in species other than paper wasps. METHODS Model Species In the solitarily founding species P. biglumis (Lorenzi & Turillazzi 1986), up to 18% of the colonies are usurped by conspecific usurpers, possibly after they have lost their own nests (Lorenzi & Cervo 1995). Within a colony, the hydrocarbon profiles of adult wasps are similar to each other and to the profiles that cover the paper nest surfaces (Lorenzi et al. 1996). In contrast, hydrocarbon profiles differ between colonies (Lorenzi et al. 1997). General Procedure We split nests collected in the field into two parts: one half was reared by the original foundress (foundress half-colonies) and the other half was ‘put up for usurpation’ to an alien female (usurper; usurper half-colonies). Usurpers and ‘host’ colonies came from two
populations separated by mountain barriers. Therefore we expected that usurpers would not be related to the colony they usurped. We collected 24 colonies in Chalpe (Italian Western Alps: 44 560 N, 6 490 E) in mid-July 1997. At the same time, we also collected 24 P. biglumis colonies in Montgenèvre (French Hautes Alpes: 44 550 N, 6 430 E). The colonies were towards the end of the founding phase and single foundresses were on the nests. In the laboratory, we randomly chose 12 colonies from each collection site. We removed their foundresses and placed them separately in glass jars. We cut their paper combs in two with scissors so that the larger number of pupae was intact and the numbers of cells and old brood (large larvae and pupae) of the two parts were similar (foundress half-colonies: 17e28 cells; usurper half-colonies: 16e28 cells; Wilcoxon pairwise test: Z ¼ 0.420, P ¼ 0.674; foundress half-colonies: 5e8 brood; usurper half-colonies: 2e8 brood; Z ¼ 0.962, P ¼ 0.336). Each half comb was separately fastened to the wall of a plastic box (18 12 cm and 11 cm high) and either its original foundress (foundress half-colony) or a usurper (usurper half-colony) was put into the box. If the colony came from one population, its usurper came from the other. The half-colonies were kept under a 12:12 h light:dark photoperiod and supplied with honey, water and Tenebrio molitor larvae ad libitum. We kept foundresses and usurpers on their halfcolonies for 8 days, then we removed and froze them in individual glass vials (18 C). We used them later in behavioural tests and chemical analyses (see below). Because foundress and usurper half-colonies had immature offspring, we waited for their emergence. The first offspring emerging from the foundress halfcolonies were sisters to those emerging from the usurper halfcolonies and both were daughters of their original foundresses (usurpers usually keep the first offspring of the foundress when they usurp host colonies, Lorenzi & Cervo 1992; Cervo & Lorenzi 1996). The offspring emerged after we removed foundresses or usurpers. Consequently, the offspring never met their foundresses or usurpers during adulthood and were immature in the 8-day period that foundresses and usurpers spent on the half-colonies. Therefore the offspring were naïve to foundresses and usurpers when we performed the behavioural tests. Behavioural Tests We tested whether the offspring discriminated between their foundress (i.e. their mother) and usurper (i.e. the usurper of their own half-colony or of their sisters’ half-colony) by performing bioassays using the dead foundresses and usurpers. Bioassays using dead insects eliminate the confounding effects of behavioural or chemical actions by the insects introduced and are routinely used in recognition experiments in social insects (e.g. BonavitaCougourdan et al. 1987; Lorenzi et al. 1997; Ruther et al. 2002). The tests were performed at least 1 day after the offspring emerged. At room temperature, we introduced the foundress and the usurper separately into each pair of half-colonies in random order and at least 1 h apart. We used forceps to keep foundresses and usurpers 1 cm from the nest surfaces. The tests lasted 1 min from the first unambiguous reactions by the offspring. The observer did not know whether the dead female was the foundress or the usurper in the test half-colony. We noted the occurrence of the following behaviours: biting, fleeing from the nest by flying, attempting to sting and grasping. In the behavioural tests, we recorded the responses by 1.13 0.18 adult female offspring per foundress half-colony and by 1.33 0.44 adult female offspring per usurper half-colony. The offspring tested did not differ significantly in age between half-colonies (on average: 3.18 2.71 days old in foundress half-colonies; 6.38 2.72 days old in usurper halfcolonies; Wilcoxon pairwise test: Z ¼ 1.826, P ¼ 0.068).
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For our analysis of behavioural data, we computed the mean frequency of attacks as the sum of the behaviours recorded in 1 min in each half-colony divided by the number of adult offspring in that half-colony. Behavioural data came from pairs of sisters that emerged from foundress or from usurper half-colonies. Consequently, we analysed behavioural data as paired data by using a general linear model for repeated measures (GLM, repeated measures, with GreenhouseeGeisser correction), after 1/squareroot transformation to meet assumptions of normality and homoscedasticity. Chemical Analyses After the behavioural tests, we analysed the chemical profiles of 16 half-colonies, namely, eight foundresses, eight usurpers and a 1 cm2 piece of the paper from their half-colonies. We weighed the wasps and the pieces of nest paper using a precision balance (Precisa 125A). On average, wasps weighed 31.80 1.21 mg and the pieces of nest paper 18.36 2.40 mg. We extracted the hydrocarbons by dipping each wasp or piece of nest paper separately into 1 ml of pentane for 75 s. We added to each extract 800 ng of n-C20 as an internal standard to quantify the amount of hydrocarbons. For each extract, 2 ml samples were analysed by capillary gas chromatography with a Delsi Nermag DN200 gas chromatograph (GC). The GC was equipped with a flame ionization detector and a Chrompack CPSIL5 WCOT CB nonpolar capillary column (25 m, 0.25 mm, 0.12 mm). Helium was the carrier gas (1 bar). We used a 15 s splitless method of injection. Oven temperature increased from 70 C to 150 C at a rate of 30 /min. After an isotherm of 5 min, the temperature increased to 320 C at a rate of 5 /min. Data were registered with an Enica 31 integrator and compounds were identified by comparing their mass spectra and their retention times against those recorded earlier (Lorenzi et al. 1997). The large majority of peaks consisted of single hydrocarbons. A few peaks consisted of a mix of coeluted hydrocarbons. The data for each extract were analysed in two ways. (1) We calculated the overall amount of hydrocarbons (in ng per mg of wasp or piece of nest paper) as the overall sum of peak areas 800 ng divided by the area of the C20 peak in that extract; the resulting value was divided by the weight of the wasp or piece of nest paper. (2) We also calculated the relative proportion of branched to total hydrocarbons as the amount of branched hydrocarbons in ng divided by the total amount of hydrocarbons in that extract. Test for Chemical Insignificance We tested for differences in the overall amount of hydrocarbons between matched pairs of foundresses and usurpers and between matched pairs of foundress half-colonies and usurper half-colonies (GLM, repeated measures). Hydrocarbon amounts were ln transformed to meet assumptions of normality and homoscedasticity. We also measured the surplus (if any) in the amount of hydrocarbons in usurper half-nests as the difference in the overall amounts of hydrocarbons between matched pairs of usurper and foundress nest papers. Assuming that the surplus was caused by the usurpers, we checked whether the surplus in the amounts of hydrocarbons was correlated with the similarity of the chemical profiles of the usurpers and those of their respective half-colonies (as measured by BrayeCurtis similarities in the percentages of the methylbranched hydrocarbons).
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usurpers, and in the nest paper of their respective half-colonies. Because the hydrocarbons in P. biglumis are either linear or branched, they have binomial distributions and we analysed them by using a GLM with binomial errors and logit link function. Test for Chemical Mimicry Because our preliminary analyses showed that branched hydrocarbons played a role in the usurpers’ chemical strategies, we checked whether foundresses and usurpers had profiles of branched hydrocarbons similar to those of their respective half-colonies. Therefore we excluded linear alkanes from the analysis. We also excluded two branched-alkane peaks (peak 11a: 3-methylhexacosane; peak 42a: 5-methylhentriacontane) that were present in less than 25% of the samples. We divided the integration area of peak 46 þ 47 (n-docotriacontane and 3,9-þ3,11þ3,13-dimethylhentriacontane) and peak 58 þ 59 (n-tetratriacontane and 3,9-þ3,11-þ3,13-þ3,15-dimethyltritriacontane) by two, because both peaks contained mixtures of branched and linear alkanes (in unknown proportions). Therefore, for each extract, we had 51 branched-hydrocarbon peaks (chain length between 24 and 34 carbon atoms) and we computed their relative proportions. We handled these compositional data (percentage of compounds) by using the log-ratio transformation (Aitchison 1982). To this end, we computed the natural log of the proportion of each peak and divided it by the geometric mean of the proportions of the branched alkanes. We analysed log-ratio-transformed data by using a stepwise multiple discriminant analysis (DA). The DA used the colony as a grouping variable (within-group covariance matrix) and entered geomean-log-transformed data as independent variables (Mahalanobis distance method). We used half of the cases as a training subset and the remaining half as a test subset and we derived the discriminant functions on only a portion of the cases (foundresses and the nest paper of their half-colonies). We then assessed the performance of the discriminant functions on the remaining cases (usurpers and the nest paper of their half-colonies). We expected that DA would discriminate significantly between foundresses and the nest paper of their halfcolonies by colony. We also expected that DA would successfully classify usurpers and the nest paper of their half-colonies by colony only if the usurpers chemically mimicked their host colonies. If the DA misclassified usurpers and the nest paper of their half-colonies by colony, the usurpers would not chemically mimic their host colonies. We tested whether the attacks of offspring from usurper half-colonies against usurpers increased as the distances between the usurper and her half-nest in the DA space increased (Pearson correlation, after controlling for normality). We computed the distances between usurpers and their half-nests in the DA space as the differences between the values of the discriminant function scores for usurpers and those for the nest paper of their respective half-colonies. Statistical analyses were performed in SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, U.S.A.; see Garson 2008 for procedures). BrayeCurtis similarities were computed in PAST (Hammer et al. 2001). Descriptive statistics are given as means 1 SE, unless otherwise stated. RESULTS Behavioural Tests
Test for Chemical Transparency We tested how different from each other (if at all) the proportions were of branched to linear hydrocarbons in foundresses and
The aggressive responses of sisters to foundresses and usurpers depended on whether the sisters had emerged from the foundress or the usurper half-colonies as shown by a significant interaction
M. Cristina Lorenzi et al. / Animal Behaviour 82 (2011) 1143e1149
term (GLM, repeated measures, half-colony*target of attacks: F1,30 ¼ 10.857, P ¼ 0.003; power was acceptable, that is, larger than 0.80 only for the two-way interaction). Foundresses tended to be attacked less by offspring from foundress half-colonies than by their sisters from usurper half-colonies (Fig. 1). Usurpers tended to be attacked less by offspring from usurper half-colonies than by their sisters from foundress half-colonies (Fig. 1). Chemical Analyses
The variation in the overall amount of hydrocarbons between usurper and foundress half-colonies differed between wasps and nest paper, as shown by a significant interaction term (GLM, repeated measures, half-colony*sample category: F1,14 ¼ 8.161, P ¼ 0.013; Fig. 2). Matched pairs of foundresses and usurpers had roughly equivalent amounts of hydrocarbons (F1,7 ¼ 2.224, P ¼ 0.179). In contrast, the nest paper of usurper half-colonies had significantly larger amounts of hydrocarbons than that of foundress half-colonies (F1,7 ¼ 6.190, P ¼ 0.042; Fig. 2). The larger the surplus of hydrocarbons on the nest paper of usurper half-colonies, the more similar the branched-hydrocarbon profiles of the usurpers were to those of their own half-colonies (Pearson correlation: r6 ¼ 0.898, P ¼ 0.015). Test for Chemical Transparency Usurpers had significantly higher proportions of branched hydrocarbons than foundresses and, similarly, the nest papers of usurper half-colonies had significantly larger proportions of branched hydrocarbons than those of foundress half-colonies. However, the slope of the variation was steeper for wasps than for nest paper (as shown by a significant interaction term in the c21 ¼ 2605:72, GLM, half-colony*sample category: Wald P < 0.0001; Fig. 3). In their epicuticular profiles, foundresses had on average 62% of branched hydrocarbons and usurpers 72%,
Usurper 6
5
2000
0 Foundress half-colony
Usurper half-colony
Figure 2. The total amount of hydrocarbons as a function of half-colony and sample category (half-colony categories: foundress and usurper half-colony; sample categories: wasp and nest paper). The total amount of hydrocarbons is expressed in ng per mg of wasp or of nest paper.
a significant difference (Wald c21 ¼ 4421:477, P < 0.0001). The nest paper of foundress half-colonies had on average 72% of branched hydrocarbons and that of usurper half-colonies 74% (Fig. 3). Although only 2%, these differences were highly significant (Wald c21 ¼ 397:549, P < 0.0001). Test for Chemical Mimicry Colonies composed of foundresses and pieces of their nests could be significantly distinguished from each other by the DA (Fig. 4). Despite large variation, the discriminant model as a whole was significant (step 1: Wilks’s l ¼ 0.222, F7,8 ¼ 4.002, P ¼ 0.035; step 2: Wilks’s l ¼ 0.019, F14,14 ¼ 6.332, P ¼ 0.001). Function 1, which accounted for 93.42% of the total variance, differed significantly in mean by colony (Wilks’s l42 ¼ 0.000002, P < 0.00001). Function 2, which accounted for only 5.9% of the total variance, differed significantly in mean by colony as well (Wilks’s l30 ¼ 0.001, P ¼ 0.006). This DA classification showed that 100% of foundresses and their nest paper were correctly assigned to their colonies. In contrast, only 12.5% of usurpers and their nest paper were correctly assigned to their original colonies. This suggested that usurpers and their nest paper had chemical profiles that did not match those of
Proportion of branched hydrocarbons
Attacks by workers
Foundress
3000
1000
Target of attacks
7
Nest Wasp
4000
The blend of epicuticular hydrocarbons of P. biglumis foundresses, usurpers and their nest surfaces consisted of 70 peaks representing homologous series (C24eC34) of linear and methylbranched alkanes and confirmed previous GCeMS data (Lorenzi et al. 1997). Test for Chemical Insignificance
Category of sample
5000
Total amount of hydrocarbons
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0.8
Category of sample
0.75
Nest Wasp
0.7 0.65 0.6 0
0 Foundress half-colony
Usurper half-colony
Figure 1. Mean frequency of attacks SE by Polistes biglumis sisters that emerged from the half-colonies towards their own foundresses, the usurpers of their sisters or their own usurpers.
Foundress half-colony
Usurper half-colony
Figure 3. The proportion of branched alkanes as a function of the half-colony in wasps and nest paper. The proportion of branched hydrocarbons was computed as the relative amounts of branched hydrocarbons in ng divided by the total amount of hydrocarbons.
M. Cristina Lorenzi et al. / Animal Behaviour 82 (2011) 1143e1149
20
(a)
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(b)
10 0 −10 20
(c)
(d)
(e)
(f)
(g)
(h)
10
Function 2
0 −10 20 10 0 −10 20
Foundress Foundress half-nest
10
Usurper
0 −10
Usurper half-nest −40 −20
0
20
40
−40 −20
0
20
40
Function 1 Figure 4. Discriminant analysis (discriminant scores) of 51 branched hydrocarbons of P. biglumis foundresses, usurpers and their half-nests for colonies aeh, showing how similar the half-nests were to their foundresses or usurpers. For nest f, the point representing the usurper half-nest had a function 1 value <40; therefore, it is not shown in the graph.
the original colonies. This further suggested that usurpers were not mimicking the chemical profiles of the colonies they had usurped. Finally, it suggested that usurpers had partially changed the chemical profiles of their nests. The dissimilarity between the chemical profile of usurpers and that of their host nests mediated aggression by the host offspring: the larger the dissimilarity, the stronger the aggression by the host offspring (Pearson correlation: r6 ¼ 0.839, P ¼ 0.037). DISCUSSION Facultative social parasites of P. biglumis employ a chemical strategy of integration in host colonies, but they were neither chemically insignificant nor transparent, relative to their hosts. They were not even chemical mimics of their hosts. Instead, usurpers had chemical profiles that were richer in methyl-branched hydrocarbons than those of the foundresses. In addition, usurpers deposited large quantities of hydrocarbons on the surfaces of their host nests, a type of scent overmarking that is common in mammals (Johnston 2005; Jordan et al. 2011). The deposition of hydrocarbons on host nest surfaces by usurpers changed the chemical profiles of the host nests, making them qualitatively more similar to the chemical profiles of usurpers themselves. These adjustments influenced the host colony signatures, which were the ones that the offspring that emerged from usurper nests were to learn. The nest paper is the place where newly emerged paper wasp offspring get their information about their colony’s chemical signature. During nest
foundation, foundresses mark the nest surfaces with their own signatures (e.g. Espelie et al. 1990; Dani et al. 1992; Layton & Espelie 1995; Lorenzi et al. 1996). The emerging offspring learn the signature of their colony from the nest surfaces soon after they emerge and then use the learned template to discriminate between nestmates and strangers (Gamboa 1996, 2004). Any wasp approaching the nest is accepted by the offspring if its chemical profile matches the profile they had learned from the nest and, if not, the approaching wasp is rejected. Usually, usurpers are unrelated to host foundresses (e.g. Seppä et al. 2011) and hence they exhibit chemical profiles that do not match those of host nests. Therefore, usurpers will be rejected unless they change the host nest surface chemistry in a way that biases it towards their own profiles. Our behavioural results show that the offspring from usurper half-colonies, which were naïve to usurpers, were relatively tolerant towards usurpers. Because these offspring had no contact with their usurpers before the bioassays, they must have learnt the usurper signatures from the usurper marks on the nest surfaces. Usurpers probably marked host nests through stroking, a behaviour peculiar to Polistes usurpers, which spend time rubbing their abdomen on nest surfaces (Cervo & Turillazzi 1989; Dani et al. 1992; Lorenzi & Cervo 1992; Cervo & Dani 1996; Cervo & Lorenzi 1996; Van Hooser et al. 2002;). Surprisingly, the proportion of branched hydrocarbons on the nest paper was higher than that on the respective wasps. This might be caused by overmarking: foundresses marked their nests by repeatedly placing their hydrocarbon marks on the top of their previous hydrocarbon marks. In turn, usurpers placed their
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hydrocarbon marks on top of the hydrocarbon marks of the foundresses. As a consequence, hydrocarbons increased in quantity on the nest paper. The different marks probably had different proportions of linear and branched hydrocarbons because the proportions of branched and linear hydrocarbons on the wasp cuticle varied. When we extracted the hydrocarbons from the nest paper, we analysed a mixture of past and recent marks, rather than only the mark on the top. This may explain the mismatch between the proportion of hydrocarbons of wasps and nests. In nature, usurpers are not a subset of the wasp population. In fact, foundresses shift to usurping conspecific nests as a conditional strategy when their colonies are killed by predators (Lorenzi & Cervo 1995). Similarly, the usurpers in this research project were a random subset of the collected foundresses. Therefore these results suggest that foundresses have a flexible chemical signature, which they adjust depending on the conditions they are facing. In this respect, they resemble obligate social parasites, whose chemical signature is highly flexible and changes with the phases of host colony invasion (e.g. Bagnères et al. 1996 in paper wasp parasites; reviewed in D’Ettorre & Errard 1998; Lenoir et al. 2001; Akino 2008; Bagnères & Lorenzi 2010). Apart from flexibility, however, the chemical strategies of usurpers are very different from those of most obligate social parasites. When obligate social parasites enter host nests and face their hosts for the first time, they are often chemically insignificant (but see Cini et al. 2011). What obligate parasites do next is to steal into the host social structures and mimic their hosts chemically. Our results show that the usurpers’ chemical strategies of integration are very different from those of obligate social parasites in that usurpers mark, rather than mimic. This suggests that P. biglumis usurpers are not likely to be incipient obligate parasites. This does not imply that obligate parasitism did not originate from conspecific usurpation in other species. For example, Polistes nimphus intraspecific usurpers seem to mimic host colonies (Lorenzi et al. 2007) and therefore exhibit a preadaptation to obligate parasitism that P. biglumis usurpers do not have. Most of the approximately 200 species of paper wasps behave as facultative parasites but only three monophyletic species behave as obligate parasites (Choudhary et al. 1994; Cervo & Dani 1996). In fact, the transition from facultative to obligate parasitism is rare. This implies that most facultative parasites, such as P. biglumis, do not have the preadaptations that allow them to shift from facultative to obligate parasitism. Usurpers and their host nests were particularly rich in methyl-branched hydrocarbons. We are far from deciphering the communication codes encrypted in the hydrocarbon profiles of social insects. We know that the different structural classes of hydrocarbons are likely to work in concert to convey recognition signals (Greene & Gordon 2007), but we also know that some classes of compounds, that is, methyl-branched hydrocarbons, may play key roles in recognition (Bonavita-Cougourdan et al. 1987; Jackson et al. 2007; Martin et al. 2008b). We found a significant increase in branched hydrocarbons in both usurpers and in usurper nest paper surfaces. Slavemaker Polyergus queens also exhibited a marked enrichment in branched hydrocarbons after invading the host nests (Johnson et al. 2001). Branched hydrocarbons seem to be more easily detectable and seem to convey more information than linear alkanes (Châline et al. 2005; Dani et al. 2001). However, they reduce the waterproofing effect of epicuticular hydrocarbon layers (Le Conte & Hefetz 2008; Gibbs & Rajpurohit 2010). Then, what advantage do usurpers gain by emphasizing their hydrocarbon profiles and enriching their branched-hydrocarbon components disproportionately? If branched hydrocarbons play an essential role in encoding information about a queen’s identity, then successful usurpers must cover the legitimate queen’s signature with their own. In effect, they virtually ‘bury’ the legitimate queen’s chemical
signature to make it easier for them to trick emerging host offspring about the queen’s identity. In conclusion, facultative parasites may use chemical strategies of integration into host colonies that are different from those of obligate parasites. These chemical strategies illustrate that branched hydrocarbons play a role as semiochemicals and that facultative parasites may not all be on the main pathway to obligate parasitism. These are findings that may very well be applicable to species other than P. biglumis.
Acknowledgments We thank Ilaria Cometto and Giuliana Marchisio for their support during the behavioural experiments. We also thank Laura Azzani and two anonymous referees for their helpful comments on the manuscript. Funding was provided by M.I.U.R. (ex 60%) to M.C.L.
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