Cristina Maria
email: cristina.lorenzi@unito.it

Laura Azzani

Anne-Geneviève Bagnères

Maria Cristina Lorenzi

Complexity and informational content of colony signature

Keywords: Polistes, Brood parasitism, Cipher, Nestmate recognition, Hydrocarbons, Geographic mosaic, Crozier's paradox

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Encrypted communication involves the transfer of simple information encoded in complex ways. This is the case, for example, of ciphers, i.e., secret and complex codes used by closed social units. Cipher complexity arises as a result of antagonistic interactions and ongoing coevolution between code makers and code breakers [START_REF] Singh | The Code Book: The Science of Secrecy from Ancient Egypt to Quantum Cryptography[END_REF]. Social insect colonies are closed social units, which defend their attractive resources, such as food stores, eggs, broods, and protected shelters from intruders [START_REF] Hölldobler | The Ants[END_REF]. Colony members protect their resources by discriminating unwanted intruders from nestmates, and rejecting intruders [START_REF] Fletcher | Kin Recognition in Animals[END_REF][START_REF] Guerrieri | Ants recognize foes and not friends[END_REF]. Nestmate/non-nestmate discrimination processes occur via recognition codes: social insects discriminate against individuals lacking their own colony signature [START_REF] Gamboa | Kin recognition in eusocial wasps[END_REF][START_REF] Howard | Ecological, behavioral and biochemical aspects of insect hydrocarbons[END_REF]. In this respect, social insects are code makers that use chemical codes to encrypt colony affiliation. Social insect colonies also include complex social structures, which involve both sophisticated social organizations and an efficient workforce. These resources are especially targeted by social parasites: insects which rely on the social organization and workforce of host species to rear their own offspring [START_REF] Wilson | The Insect Societies[END_REF]. Often, social parasites invade host colonies and integrate stably within their social structures. Usually, social parasites break the host colony recognition codes, either by concealing their true identity to hosts (chemical insignificance, [START_REF] Lenoir | Chemical ecology and social parasitism in ants[END_REF][START_REF] Uboni | Cleptoparasites, social parasites and a common host: Chemical insignificance for visiting host nests, chemical mimicry for living in[END_REF] or by breaking into their host recognition codes and "copying" them (chemical mimicry or camouflage, [START_REF] Bagnères | Chemical deception/mimicry using cuticular hydrocarbons[END_REF][START_REF] Nash | Communication between hosts and social parasites[END_REF]. However, host colony recognition codes are often very elaborate. In fact, the content of their messages is simple ("I am affiliated with colony x"), but the way information is encrypted is very complex [START_REF] Blomquist | Insect Hydrocarbons: Biology, Biochemistry and Chemical Ecology[END_REF][START_REF] Richard | Intracolony chemical communication in social insects[END_REF]. This complexity may have arisen as a result of antagonistic interactions with code breakers. The harder it is to counterfeit colony recognition codes, the lower the probability that parasites can threaten colony integrity. Therefore, as for ciphers, colony affiliation codes are continuously under attack by social parasites and are potentially the object of an ongoing coevolution, whereby parasites select hosts for complex codes and hosts select parasites for the ability to break codes.

As a result of this co-evolutionary process, social parasites and their hosts have commonly evolved complex suites of traits. The parasites, for example, often use many strategies to overcome host colony defenses and intercept host communication codes; these strategies have been widely investigated [START_REF] Lenoir | Chemical ecology and social parasitism in ants[END_REF][START_REF] Lorenzi | The result of an arms race: The chemical strategies of Polistes social parasites[END_REF][START_REF] Bagnères | Chemical deception/mimicry using cuticular hydrocarbons[END_REF]. The hosts often have complex nestmate recognition mechanisms that efficiently (albeit not completely) identify unwanted insects in the colony [START_REF] Gamboa | The evolution and ontogeny of nestmate recognition in social wasps[END_REF][START_REF] Gamboa | Kin recognition in eusocial wasps[END_REF][START_REF] Pickett | Nestmate recognition in the absence of a pheromone[END_REF][START_REF] Lorenzi | Social wasp parasites affect the nestmate recognition abilities of their hosts (Polistes atrimandibularis and P. biglumis, Hymenoptera,Vespidae)[END_REF]. Fewer studies have been undertaken on host defense, but these studies have suggested that hosts evolve morphological, behavioral, and chemical adaptations in the coevolutionary arms race with their social parasites (e.g. [START_REF] Foitzik | Locally adapted social parasite affects density, social structure, and life history of its ant hosts[END_REF][START_REF] Ruano | A geographical mosaic of coevolution in a slave-making host-parasite system[END_REF][START_REF] Lorenzi | The geographic structure of selection on a coevolving interaction between social parasitic wasps and their hosts hampers social evolution[END_REF]. Similar complexity of defense measures and counter-measures has been found in other kinds of parasitism, such as brood parasitism, where the focus of selection in those interactions has been more on traits such as egg polymorphism, nest defense and egg or chicks rejection (Lathi, 2005;[START_REF] Rothstein | Mechanisms of avian egg recognition: Which egg parameters elicit responses by rejecter species?[END_REF][START_REF] Soler | Brood-parasite interactions between great spotted cuckoos and magpies: A model system for studying coevolutionary relationships[END_REF][START_REF] Spottiswoode | How to evade a coevolving brood parasite: Egg discrimination versus egg variability as host defences[END_REF][START_REF] Colombelli-Négrel | Embryonic learning of vocal passwords in superb fairy-wrens reveals intruder cuckoo nestlings[END_REF].

In social insects, colony affiliation is advertized with chemical signatures composed mainly of cuticular hydrocarbons. These are lipid substances that primarily serve as anti-desiccation agents; they form a hydrophobic layer over the cuticle of insects, preventing water loss via transpiration through the cuticle [START_REF] Blomquist | Insect Hydrocarbons: Biology, Biochemistry and Chemical Ecology[END_REF]. The layer works best at preventing water loss at lower temperatures and when it is mainly composed of straight-chain alkanes, as insertion of double bonds and methyl-branching significantly lower its anti-desiccation properties [START_REF] Gibbs | Waterproofing properties of cuticular lipids[END_REF][START_REF] Gibbs | Lipid melting and cuticular permeability: New insights into an old problem[END_REF][START_REF] Gibbs | Cuticular lipids and water balance[END_REF][START_REF] Hefetz | The evolution of hydrocarbon pheromone parsimony in ants (Hymenoptera: Formicidae) -Interplay of colony odor uniformity and odor idiosyncrasy: A review[END_REF]. In contrast, the communicative function of cuticular hydrocarbon blends is likely to be enhanced by the presence of molecules with complex shapes, such as alkenes and methyl-branched compounds [START_REF] Dani | Deciphering the recognition signature within the cuticular chemical profile of paper wasps[END_REF]Lorenzi et al., 2011). Here we tested two competing hypotheses based on the dual role of cuticular hydrocarbons as agents of anti-desiccation and agents of communication. We reasoned that the communication role of hydrocarbons could assume even greater importance when social insects are under attack by social parasites. Social parasites employ different chemical strategies that facilitate their integration into host colonies. Often, social parasites mimic the colony recognition codes of their hosts (chemical mimicry, i.e., they exhibit recognition cues that match those of their hosts, [START_REF] Lenoir | Chemical ecology and social parasitism in ants[END_REF][START_REF] Bagnères | Chemical deception/mimicry using cuticular hydrocarbons[END_REF]. The better the parasite mimics the host's cuticular signature, the faster the adoption by host colonies [START_REF] Nash | A mosaic of chemical coevolution in a large blue butterfly[END_REF]. Parasites can also elude recognition (chemical insignificance, i.e., possess low concentration of recognition cues, [START_REF] Lenoir | Chemical ecology and social parasitism in ants[END_REF]. The lower the amount of recognition cues, the shorter the time hosts spend attacking [START_REF] Cini | A quantitative threshold for nestmate recognition in a paper social wasp[END_REF]. Host populations infested by such parasites would have to mount defenses on the same ground, endowing themselves with distinctive hydrocarbon signatures, in terms of absolute concentrations of hydrocarbons, proportions of branched hydrocarbons and/or profile variation. Indeed, increasing the concentrations of recognition cues is a simple way to enhance the efficacy of chemical communication [START_REF] Steiger | The origin and dynamic evolution of chemical information[END_REF]. There might not be such an advantage in less infested populations, as hydrocarbons, and especially branched hydrocarbons, are costly to produce and/or maintain and branched hydrocarbons deteriorate the anti-desiccation function (Hefetz, 2007, LeConte and[START_REF] Leconte | Primer pheromones in social Hymenoptera[END_REF].

Therefore, we tested whether differences in the hydrocarbon signatures between populations of social insects could be explained by parasite prevalence or whether they were solely an effect of local abiotic conditions such as temperature and humidity.

If social parasites impose selection on hosts for the evolution of distinctive hydrocarbon signatures, we predicted that social insects from populations with high parasite prevalence would display higher concentration of hydrocarbons, higher proportions of branched hydrocarbons and higher profile variation than less infested populations. Such variations would indicate that social insect populations under high parasite prevalence are under selection for enhancing the communicative role of their cuticular hydrocarbon blends. Alternatively, if selection on hydrocarbon signatures is mainly imposed by abiotic factors (such as air temperature or rainfall regime), we predicted that hydrocarbon signa-tures would vary in a consistent way with local climatic conditions, i.e., by enhancing the anti-desiccation role under higher temperature and/or lower rainfall regimes. Accordingly to the latter hypothesis, we expected populations under relatively higher temperature-regimes to have higher concentrations (total amount) of hydrocarbons and lower proportions of branched hydrocarbons. We also expected populations with relatively high rainfall-regimes to have lower concentrations of hydrocarbons and higher proportions of branched hydrocarbons. Finally, we expected that temperature and rainfall regimes did not affect within-population variation in profile (see Table 1 for a schematic presentation of predictions under the two scenarios).

Materials and Methods

Study species

Our model species was the social wasp Polistes biglumis. These wasps discriminate nestmate from nonnestmates using cuticular hydrocarbons [START_REF] Lorenzi | Polistes biglumis bimaculatus epicuticular hydrocarbons and nestmate recognition (Hymenoptera: Vespidae)[END_REF]. In this way they reject conspecific facultative social parasites attempting nest usurpation and defend their colonies from robbery by conspecifics and cleptoparasites [START_REF] Lorenzi | Usurpations and late associations in the solitary founding social wasp Polistes biglumis bimaculatus[END_REF][START_REF] Uboni | Poor odors, strength, and persistence give their rewards to Mutilla europaea visiting dangerous wasp nests[END_REF]. Populations of P. biglumis can also be infested by a congeneric, obligate social parasite P. a t r i m a ndibularis, which overcome host recognition using both chemical insignificance and chemical mimicry [START_REF] Bagnères | Chemical usurpation of a nest by paper wasp parasites[END_REF][START_REF] Lorenzi | Concealing identity and mimicking hosts: A dual chemical strategy for a single social parasite? (Polistes atrimandibularis, Hymenoptera: Vespidae)[END_REF][START_REF] Uboni | Cleptoparasites, social parasites and a common host: Chemical insignificance for visiting host nests, chemical mimicry for living in[END_REF].

Polistes biglumis is a free-living social wasp with annual colonies. Each colony is founded in late spring by a single foundress which builds the nest, lays eggs and rears her brood solitarily during a long founding phase [START_REF] Lorenzi | Behavioural and ecological adaptations to the high mountain environment of Polistes biglumis bimaculatus[END_REF]. Foundresses efficiently recognize their nestmates and their eggs [START_REF] Lorenzi | Species and colony components in the recognition odor of young wasps: Their expression and learning (Polistes biglumis and P. atrimandibularis, Hymenoptera Vespidae)[END_REF][START_REF] Lorenzi | Opportunistic discrimination of alien eggs by social wasps (Polistes biglumis, Hymenoptera Vespidae): A defense against social parasitism?[END_REF]. As other Polistes wasps, foundresses establish their colony signature by marking nests with their cuticular signatures, which is composed of > 60 hydrocarbons [START_REF] Lorenzi | Epicuticular hydrocarbons of Polistes biglumis bimaculatus (Hymenoptera: Vespidae): Preliminary results[END_REF][START_REF] Lorenzi | Polistes biglumis bimaculatus epicuticular hydrocarbons and nestmate recognition (Hymenoptera: Vespidae)[END_REF]Lorenzi et al., , 2011;;[START_REF] Costanzi | Nestmate recognition in social wasp is based on the relative proportions of cuticular hydrocarbons within species-specific ranges of hydrocarbon concentrations[END_REF]. During the founding phase, colonies can be invaded by the obligate, workerless, social parasite wasp Polistes atrimandibularis. A single parasite peacefully enters a host colony, subdues the host foundress and lives in the host colony until the end of the summer, inhibiting foundress reproduction and reducing her fitness [START_REF] Cervo | Nonaggressive usurpation of the nest of Polistes biglumis bimaculatus by the social parasite Sulcopolistes atrimandibularis (Hymenoptera Vespidae)[END_REF][START_REF] Lorenzi | Effects of social parasitism of Polistes atrimandibularis on the colony cycle and brood production of Polistes biglumis bimaculatus (Hymenoptera, Vespidae)[END_REF]Cervo and Lorenzi, 1996a). Parasites elude host recognition using a composite strategy that includes chemical insignificance and chemical mimicry. At host-nest invasion, these parasites have hydrocarbon signatures different from their hosts, but quantitatively poor (chemical insignificance) [START_REF] Lorenzi | Concealing identity and mimicking hosts: A dual chemical strategy for a single social parasite? (Polistes atrimandibularis, Hymenoptera: Vespidae)[END_REF]. As they live in host colonies, parasites change their hydrocarbon signatures; they lose parasite-specific compounds and enrich in host-specific compounds, and especially in branched-hydrocarbons (chemical mimicry) [START_REF] Bagnères | Chemical usurpation of a nest by paper wasp parasites[END_REF][START_REF] Lorenzi | Dynamics of chemical mimicry in the social parasite wasp Polistes semenovi (Hymenoptera Vespidae)[END_REF][START_REF] Uboni | Cleptoparasites, social parasites and a common host: Chemical insignificance for visiting host nests, chemical mimicry for living in[END_REF]. As a result, social parasites integrate into host colonies so well that hosts discriminate between their own parasites and alien parasites, and attack the latter only [START_REF] Lorenzi | Social wasp parasites affect the nestmate recognition abilities of their hosts (Polistes atrimandibularis and P. biglumis, Hymenoptera,Vespidae)[END_REF]. We do not know whether the acquisition of host-specific compounds occurs passively or whether it involves the expression of previously-silent biosynthetic pathways. Whatever the underlying mechanism, mimicry of host-specific compounds on the part of parasites may trigger chemical arms races with hosts. We expect that populations subject to obligate parasitism have enhanced the communication role in their signatures, but parasite-free populations will not lack it. Indeed, hydrocarbon signatures also allow the identification of conspecific cheaters, i.e., facultative social parasites which are common in this species [START_REF] Lorenzi | Usurpations and late associations in the solitary founding social wasp Polistes biglumis bimaculatus[END_REF]Cervo and Lorenzi, 1996b) and overmark hostnest signatures with their own signatures [START_REF] Lorenzi | The chemical strategies used by Polistes nimphus social wasp usurpers (Hymenoptera Vespidae)[END_REF](Lorenzi et al., , 2011)).

Study populations

P. biglumis populations have a patchy distribution in the Alps, due to geomorphology of their mountain habitat and to large areas unsuitable for nesting (rocky areas and forests). These geographic barriers contribute to separate populations together with foundress philopatry [START_REF] Reeve | Polistes[END_REF][START_REF] Hunt | The Evolution of Social Wasps[END_REF] and local matings (Seppä et , 2011). Indeed, populations differ in life-history traits, as a result of both local climate and social parasite pressures [START_REF] Fucini | Social wasps without workers: Geographic variation of caste expression in the paper wasp Polistes biglumis[END_REF][START_REF] Fucini | Geographic variation in air temperature leads to intraspecific variability in the behavior and productivity of a eusocial insect[END_REF][START_REF] Lorenzi | The geographic structure of selection on a coevolving interaction between social parasitic wasps and their hosts hampers social evolution[END_REF]. P. biglumis populations are small and scattered in the Alps above 1200 m a.s.l., where other host species are very rare. P. biglumis was the only free-living Polistes wasp nesting at the study sites. Therefore, where the social parasite P. atrimandibularis was present, there were no alternate hosts to P. biglumis.

Sampling on multiple mountain field-populations is time-consuming and longitudinal field-data on parasite prevalence are rarely available for social insects. Therefore, we had to restrict our analysis to three populations where the prevalence of the social parasite P. atrimandibularis across years was known. The sampled populations have different social-parasite prevalence: Montgenèvre, where social parasitism is high (23.5% parasitized colonies over 5 years), Ferrere, where it is intermediate (5.6% parasitized colonies over 5 years), and Carì, where parasites are absent (no parasite has been sampled since we discovered this population in 2006) [START_REF] Lorenzi | The geographic structure of selection on a coevolving interaction between social parasitic wasps and their hosts hampers social evolution[END_REF]. Populations were between 1600-1900 m a.s.l. and 70-250 km apart. At the moment, there is no information on genetic diversity in the study populations which might be useful to infer the demographic history of these populations and identify and date past population-size changes (expansions or bottlenecks).

Parasite prevalence

Parasite prevalence in each population was determined through field censuses. From the early founding phase (May-June) until the end of the nesting cycle, during each of 3-5 years, we monitored the breeding attempts of 1648 individually marked foundresses (Montgenèvre, years: 2004(Montgenèvre, years: -2008: n = 498;: n = 498;Ferrere, years: 2004Ferrere, years: -2008: n = 464;: n = 464;Carì, years 2006Carì, years -2008: n = 170 foundresses) (details in [START_REF] Lorenzi | The geographic structure of selection on a coevolving interaction between social parasitic wasps and their hosts hampers social evolution[END_REF]. Each population was visited every 2-10 days checking for foundress presence and whether the colony had been invaded by a P. atrimandibularis parasite female. When we found parasites, we individually marked them. Prevalence (percentage of colonies invaded by P. atrimandibularis parasite females) was roughly constant per population and year [START_REF] Lorenzi | The geographic structure of selection on a coevolving interaction between social parasitic wasps and their hosts hampers social evolution[END_REF].

Sample collection

We collected foundresses in the pre-emergence period, i.e., when the single foundress was the only adult wasp on the nest (Montgenèvre: n = 8, Ferrere: n = 10, Carì: n = 11 foundresses) (date: July 820, 2009; July 13-22, 2010). We were interested in differences between popu-lations in hydrocarbon signatures due to evolutionary changes. However, hydrocarbon signatures could be adjusted not only in evolutionary terms, where conditions encountered in subsequent generations select for optimal signatures, but also in terms of phenotypic plasticity, where enslaved foundresses make short-term adjustments in their signature as a response to enslavement. Therefore, none of the collected foundresses came from parasitized colonies or from colonies usurped by conspecific females, so that we could exclude short-term, parasite-mediated modifications of host hydrocarbon signatures.

Chemical analyses

Foundresses were killed by freezing and weighed (precision balance Precisa 125A). We extracted their cuticular compounds by separately suspending each foundress in 1-ml pentane for 60 sec. An internal standard (n-C20) was added to each extract to quantify compounds.

For quantitative analyses, we analysed samples using gas-chromatography with flame ionization detection (AGILENT 6850). Two μl of each extract (which included 800 ng of n-C20) were injected in the gaschromatograph equipped with a Chrompack CPSIL5 WCOT CB nonpolar-capillary-column (30 m length × 320 μm internal diameter × 0.25 μm thickness of stationary phase) (carrier gas: helium, 1 bar, 50 ml/min flux). Oven temperature increased from 70°C to 150°C at a 30°C/min rate and from 150°C to 320°C at a 5°C/min rate (10 min at 320°C final temperature). Results were registered in an Agilent-ChemStation. The area of each peak was converted to its proportional contribution to total peak area in that sample. The compounds in the profiles were determined via GC-MS as in previous work [START_REF] Bagnères | Chemical usurpation of a nest by paper wasp parasites[END_REF][START_REF] Lorenzi | Polistes biglumis bimaculatus epicuticular hydrocarbons and nestmate recognition (Hymenoptera: Vespidae)[END_REF][START_REF] Uboni | Cleptoparasites, social parasites and a common host: Chemical insignificance for visiting host nests, chemical mimicry for living in[END_REF] and checked for each population.

Meteorological data: Air temperature and precipitation

Meteorological data were obtained from weather stations (Montgenèvre and Ferrere: ARPA; Carì: ME-TEOMEDIA AG). The closest weather stations were at lower altitudes than the populations themselves, so a correction was applied to account for the temperature drop owing to altitude increase (lapse rate: 0.6°C each 100m) [START_REF] Körner | The use of 'altitude' in ecological research[END_REF]. For each population, we calculated temperature regime as the average among the maximum air-temperature (°C) recorded each day during the 30 days preceding foundress collection, and rainfall regime as the average precipitation (rain mm/day) during the same period. This was done for both years of collection (data averaged across years).

Statistical analyses 1.7.1 Differences in concentrations

Following procedures described elsewhere (e.g. [START_REF] Lenoir | Chemical ecology and social parasitism in ants[END_REF], Lorenzi et al., 2011[START_REF] Costanzi | Nestmate recognition in social wasp is based on the relative proportions of cuticular hydrocarbons within species-specific ranges of hydrocarbon concentrations[END_REF][START_REF] Quezada-Euán | Does sensory deception matter in eusocial obligate food robber systems? A study of Lestrimelitta and stingless bee hosts[END_REF], we measured the total concentration of hydrocarbons for each foundress as the sum of all peak areas relative to the area of n-C20. We divided these measures by the body weight of each foundress (ng of hydrocarbons/mg of body weight). This measure reflects the concentration of hydrocarbons on the cuticular surface. Differences in the total concentration of hydrocarbons among populations were analyzed using a Linear Mixed effects Model (LMM) (R package lme4, [START_REF] Bates | lme4: Linear mixed-effects models using Eigen and S4. R package version 1[END_REF], using identity link function, with population as the fixed factor and year a random factor. Concentration data (and model residuals) followed the assumptions of normality and homoscedasticity. As our model contained only one fixed factor, model comparison or simplification was not feasible; we tested the significance of the population effect using the function Anova from the package lmerTest (Kenward-Roger approximation for the denominator degrees of freedom) [START_REF] Kuznetsova | lmerTest: Tests for random and fixed effects for linear mixed effect models (lmer objects of lme4 package). R package version 2.0-3[END_REF].

Differences in proportions

The epicuticular hydrocarbons of P. biglumis are either linear alkanes or methyl-branched alkanes [START_REF] Lorenzi | Polistes biglumis bimaculatus epicuticular hydrocarbons and nestmate recognition (Hymenoptera: Vespidae)[END_REF](Lorenzi et al., , 2011)). Traces of alkenes may be present in negligible concentrations (< 0.5%) [START_REF] Uboni | Cleptoparasites, social parasites and a common host: Chemical insignificance for visiting host nests, chemical mimicry for living in[END_REF]. Therefore, differences between populations in the proportions of branched hydrocarbons (proportional contribution of all branched hydrocarbons to total peak area in each sample) were analyzed using a Generalized Linear Model (GZLM) for binomial distribution data (hydrocarbons were either linear or methyl-branched) and logit link function. We first fitted a Generalized Linear Mixed effects Model (fixed factor = population, random factor = year) in R (package glmmML, [START_REF] Broström | glmmML: Generalized linear models with clustering[END_REF], but as the deviance associated with the random factor was 0 (test of null hypothesis of no random effect, p = 0.5), we reverted to a GZLM (function glm from package stats, family = binomial, link = logit, fixed factor = population). Tukey's contrasts for multiple comparisons were used as post-hoc tests.

Associations between hydrocarbon signature traits and parasite prevalence, rainfall or air temperature

When testing for associations between hydrocarbon signature traits and biotic (or abiotic) variables, we used a directional test. Ordered Heterogeneity test (OH test) allows testing against simply ordered alternative hypotheses in the context of nondirectional tests [START_REF] Rice | The ordered-heterogeneity family of tests[END_REF]. This procedure tests for between-population differences while taking an a priori expectancy into account, i.e., it combines an existing k-sample heterogeneity test with the Spearman-rank-order-correlation coefficient between observation and expectancy. Here, it included the a priori expectancies that we summed up in Table 1. These expectancies were used to test the relationships between the two variables (concentrations of hydrocarbons and proportions of branched hydrocarbons) and the sources of selection (parasite prevalence, air temperature and precipitation). Therefore, we performed six OH tests, after ranking the three populations for 1) parasite prevalence; 2) temperature regime and 3) rainfall regime (Table 2). We first tested whether there were significant differences between populations in hydrocarbon concentrations and branched-hydrocarbon proportions using LMMs or GZLMs, and then tested whether population means were ordered in the expected direction using OH test. We computed the r s P c statistic values as the product r s (1 -P c ) where r s was the Spearman's-rank-correlation coefficient between each variable and the source of selection and P c was a P-value from the model. Critical values for the r s P c statistic are given in [START_REF] Rice | The ordered-heterogeneity family of tests[END_REF] for selected alpha values (exact P-values cannot be computed).

The two variables (total concentrations of hydrocarbons and proportions of branched hydrocarbons) were not significantly correlated with each other (Spearman's-correlation test, Montgenèvre: rho = 0.190, P = 0.651; Ferrere: rho = 0.384, P = 0.273; Carì: rho = 0.245, P = 0.467).

Multivariate dispersion of hydrocarbon profiles within populations

We tested the variation of hydrocarbon signature within and between populations using an analysis of multivariate homogeneity of group dispersion on the entire hydrocarbon signature (64 peaks), following Anderson's PERMDISP procedure [START_REF] Anderson | Distance-based tests for homogeneity of multivariate dispersions[END_REF]. This is a multivariate analogue of Levene's test for homogeneity of variances used to evaluate beta diversity among different species assemblages/areas [START_REF] Anderson | Distance-based tests for homogeneity of multivariate dispersions[END_REF]. Bray-Curtis dissimilarity measures on the relative proportions of hydrocarbons were calculated for each population, and submitted to betadisper, a function of the R package vegan [START_REF] Oksanen | vegan: Community Ecology Package. R package version 2.0-8[END_REF]) that implements PERMDISP2.

We visualized the relationships (and dispersion) between the chemical profiles of foundresses from the three populations by performing a two-dimensional Non-Metric Multidimensional Scaling (NMDS) (with 95% confidence ellipses) with the program PAST version 2.17 [START_REF] Hammer | PAST: Paleontological statistics software package for education and data analysis[END_REF].

Results

The chemical signatures of P. biglumis foundresses in the three populations were complex blends including more than 60 peaks representing homologous series (C23-C35) of linear and methylbranched alkanes (confirming previous data from a population, [START_REF] Lorenzi | Polistes biglumis bimaculatus epicuticular hydrocarbons and nestmate recognition (Hymenoptera: Vespidae)[END_REF][START_REF] Uboni | Cleptoparasites, social parasites and a common host: Chemical insignificance for visiting host nests, chemical mimicry for living in[END_REF] (Fig. 1).

Parasite prevalence was the highest in Montgenèvre, where on average 23.5% of colonies (1 every 5) were invaded by social parasites. It was intermediate in Ferrere, where 5.6% of the colonies (1 every 20) were parasitized. Parasites were absent in Carì (Table 2, Fig. 2A).

Differences in concentration and proportions 2.1.1 Concentration of hydrocarbons

The differences in the concentration of cuticular hydrocarbons between populations were significantly associated with parasite prevalence (OH test: r s P c = 0.9991, k=3, P < 0.01) and rainfall regime (OH test: r s P c = 0.9991, k=3, P < 0.01), but not with temperature (OH test: r s P c = 0.49955, k=3, P > 0.05) (Table 2).

Foundresses differed significantly in hydrocarbon concentration between populations (LMM fit via lme4 random effect: σ(year) = 2.851, σ(residual) = 46.301; fixed effect: population: F 2,25.37 = 12.538, P < 0.001). Foundresses from Montgenèvre, the most parasitized population, had on average higher hydrocarbon concentrations than those from Ferrere (Tukey's test, P < 0.001) and Carì, where parasites were absent (P < 0.001) (Fig. 2B).

Proportion of branched hydrocarbons

The differences in the proportion of branched hydrocarbons were significantly associated with parasite prevalence (OH test: r s P c = 0.94, k = 3, P = 0.01) but not with rainfall (OH test: r s P c = -0.94, k = 3, P = 0.99) or temperature regime (OH test: r s P c = -0.47, k = 3, P > 0.8) (Table 2). Among-populations differences in the proportions of branched hydrocarbons were marginally significant (GLM: population: Wald χ 2 = 5.618, df = 2, P = 0.060; Null deviance: 86.204 on 28 df, Residual deviance: 80.609 on 26 df) (Fig. 2C). Foundresses from Montgenèvre had marginally higher proportions of branched hydrocarbons than foundresses from Carì (Tukey's test, P = 0.060).

Multivariate dispersion of hydrocarbon profiles within populations

The within-population multivariate dispersions of the whole chemical profiles were significantly different (999 permutations, F 2,26 = 5.0236, P = 0.01), indicating that populations differed in the degree of among-colony variation in hydrocarbon profiles. The average distance to centroid for the three populations was lower in Carì (0.1120), than in Ferrere (0.1472) or Montgenèvre (0.1378) (Fig. 2D). Pairwise comparisons of group mean dispersion showed that Carì (where parasites were absent) differed significantly from both Ferrere (999 permutations, P = 0.004) and Montgenèvre (999 permutations, P = 0.042), whereas these two parasitized populations were not significantly between each other (999 permutations, P = 0.492) (Fig. 2D). The difference in within-population dispersion in chemical profiles of foundresses from the three populations is also visualized via NMDS (Fig. 3), where 95% ellipses are larger in parasite-infested populations (Montgenèvre and Ferrere) than in the parasite-free population (Carì).

Discussion

These results give empirical support for the hypothesis that social parasites impose directional selection on host hydrocarbon signatures and promote their variation. Mimetic social parasites selected hosts for increasing the concentration of cuticular hydrocarbons, the proportions of branched hydrocarbons and the within-population variation in chemical profiles. These three recognition signature traits had the largest values in the two parasitized population and the smallest values in the population where parasites were absent. They were therefore significantly and positively associated with parasite prevalence, suggesting that parasite pressure, and parasite chemical mimicry in particular, may act as a selective force that enhances the complexity of recognition codes and promote their communicative role.

The composition of cuticular hydrocarbon layers often reflects local climatic differences [START_REF] Toolson | Effects of rearing temperature on cuticle permeability and epicuticular lipid composition in Drosophila pseudoobscura[END_REF][START_REF] Gibbs | Genetic and acclimatory variation in biophysical properties of insect cuticle lipids[END_REF][START_REF] Wagner | Task-related environment alters the cuticular hydrocarbon composition of harvester ants[END_REF][START_REF] Noorman | Effects of relative humidity, temperature, and population density on production of cuticular hydrocarbons in housefly Musca domestica L[END_REF][START_REF] Parkash | Changes in cuticular lipids, water loss and desiccation resistance in a tropical drosophilid: Analysis of within population variation[END_REF]. Many insect species, though, have many internally-branched and unsaturated hydrocarbons on their cuticles. These hydrocarbons, owing to their high molecular complexity, have a high potential to encode information (LeConte and [START_REF] Leconte | Primer pheromones in social Hymenoptera[END_REF][START_REF] Blomquist | Insect Hydrocarbons: Biology, Biochemistry and Chemical Ecology[END_REF][START_REF] Richard | Intracolony chemical communication in social insects[END_REF] and indeed qualify as effective communication agents [START_REF] Dani | Nestmate recognition cues in the honey bee: Differential importance of cuticular alkanes and alkenes[END_REF]Lorenzi et al., 2011). The impact of abiotic factors (such as air temperature and rainfall regimes) on the cuticular blends of P. biglumis social wasps is not to be ruled out, but the patterns of hydrocarbon divergence between populations highlighted in this study are best explained by parasite prevalence rather than by local temperature or rainfall regimes. Abiotic factors were also poor predic- tors of the evolution of hydrocarbon signature in ants in phylogenetic analysis and comparative studies [START_REF] Van Wilgenburg | Evolution of cuticular hydrocarbon diversity in ants[END_REF][START_REF] Menzel | Tolerance requires the right smell: First evidence for interspecific selection on chemical recognition cues[END_REF][START_REF] Martin | Sources of variation in cuticular hydrocarbons in the ant Formica exsecta[END_REF].

Our results also suggest that social parasites may promote an overall increase in cuticular hydrocarbon concentration in hosts. Generally, we do not know whether social parasites attain chemical mimicry through passive acquisition and/or biosynthesis of new compounds [START_REF] Bagnères | Chemical deception/mimicry using cuticular hydrocarbons[END_REF][START_REF] Bauer | An ant social parasite in-between two chemical disparate host species[END_REF]. However, producing and/or acquiring hydrocarbons -and branched hydrocarbons in particular -is costly (LeConte and [START_REF] Leconte | Primer pheromones in social Hymenoptera[END_REF], leading to tradeoffs [START_REF] Stearns | Trade-offs in life-history evolution[END_REF][START_REF] Roff | The evolution of trade-offs: Where are we?[END_REF] in the expression of cuticular hydrocarbons. If social parasites do not exhibit enough of the appropriate hydrocarbons, they may fail to gain the membership status within host colonies [START_REF] Lenoir | Chemical ecology and social parasitism in ants[END_REF][START_REF] Bagnères | Chemical deception/mimicry using cuticular hydrocarbons[END_REF][START_REF] Uboni | Cleptoparasites, social parasites and a common host: Chemical insignificance for visiting host nests, chemical mimicry for living in[END_REF]. The need for parasites to gain membership may have selected for hosts that raise their concentration of recognition cues, advertising their identity with stronger cues, somehow exaggerating the concentration of cuticular hydrocarbons and especially their branched-hydrocarbon component. This, in turn, inadvertently raises the costs for co-evolving parasites: parasites that do not have enough recognition cues may have hard times in exploiting their hosts, as they may

Fig. 3 NMDS plot of the chemical signatures of Polistes biglumis

Each dot represents the signature of a foundress from one of three populations (Carì: yellow; Ferrere: orange; Montgenèvre: red). The 95% confidence ellipses visualize the difference in multivariate dispersion of chemical signatures, which was larger in the two parasitized populations (Ferrere and Montgenèvre) than where parasites were absent (Carì).

be "invisible" to their host workforce.

We also analyzed the level of variation in recognition cues within and between populations. Bearers of rare signatures have higher chances of being rejected (and incurring fitness costs) than bearers of common ones: this would lead to erosion of signature diversity and to the eventual lack of a level of label polymorphism adequate to maintain recognition (Crozier's paradox) [START_REF] Crozier | Genetic clonal recognition abilities in marine invertebrates must be maintained by selection for something else[END_REF][START_REF] Tsutsui | Scents of self: The expression component of self/nonself recognition systems[END_REF]. In this scenario, it is unclear how polymorphism of recognition cues is maintained. One possibility is that colonies with rare signatures discriminate intruders better than those with common signatures [START_REF] Ratnieks | The evolution of genetic odor-cue diversity in social Hymenoptera[END_REF]. Recently, a possible solution of Crozier's paradox has been suggested, involving the dual role of recognition cue in favoring both assortative cooperation and disassortative mating [START_REF] Holman | Crozier's paradox revisited: Maintenance of genetic recognition systems by disassortative mating[END_REF]. Finally, recognition cue polymorphism might be maintained by selection imposed by parasites against common recognition cues [START_REF] Martin | Is parasite pressure a driver of chemical cue diversity in ants?[END_REF].

Our results also suggest that parasites may promote the variation in the cuticular hydrocarbon profiles of their hosts. A larger cuticular hydrocarbon variation can be achieved qualitatively, by increasing the number of compounds in the cuticular blends, or quantitatively, by varying the relative proportions of compounds. For example, parasitized populations of Formica fusca ants had more dimethylalkane isomers than those that were parasite-free [START_REF] Martin | Is parasite pressure a driver of chemical cue diversity in ants?[END_REF]. Among Camponotus ants, the appearance of unusually long-chain unsaturated hydrocarbons might have evolved as a private communication channel within species [START_REF] Menzel | Tolerance requires the right smell: First evidence for interspecific selection on chemical recognition cues[END_REF], possibly later used in the heterospecific context [START_REF] Emery | Recognition in a social symbiosis: Chemical phenotipes and nestmate recognition behaviors of neotropical parabiotic ants[END_REF]. In our study, instead, we focused on quantitative variation in chemical profiles and found that the within-population variation in hydrocarbon proportions was associated with parasite prevalence. It could be argued that the lower level of within-population variation in the chemical profiles in Carì could be due to past bottlenecks through which this population might have passed. While it is true that we lack information to infer the demographic history of these populations, it is also true that past bottlenecks would explain the lower within-population variation in the chemical profiles in Carì, but would not make sense of the other trait values that we measured in the chemical signatures of the foundresses in this population. Instead, the relationship between within-population variation in hydrocarbon proportions and parasite prevalence may occur via negative, frequency-dependent selection, a form of balancing selection that promotes and maintains diversity [START_REF] Zimmer | Evolution: Making Sense of Life[END_REF]. Parasites may mimic hosts with common colony signatures more easily than those with rare colony signatures, in as much the same way as parasites infect common genotypes more easily than rare ones [START_REF] Lively | Parasite adaptation to locally common host genotypes[END_REF][START_REF] Kerstes | Antagonistic experimental coevolution with a parasite increases host recombination frequency[END_REF]. For example, hosts of avian brood parasites have evolved colour polymorphism in their eggs (i.e., between clutches in the same population) as an evolutionary response to egg mimicry by brood parasite pressure [START_REF] Kilner | Cuckoos versus hosts in insects and birds: Adaptations, counter-adaptations and outcomes[END_REF][START_REF] Spottiswoode | How to evade a coevolving brood parasite: Egg discrimination versus egg variability as host defences[END_REF]. Similarly, our data suggest that host populations infected by social parasites had larger hydrocarbon-signature polymorphism than the parasite-free population, possibly as an evolutionary response to chemical mimicry by social parasites. It is suggestive that a previous study found that, in Ferrere and Montgenèvre, the two populations showing the largest chemical signature polymorphism, foundresses mate with multiple males, unlike most Polistes wasps [START_REF] Seppä | Colony kin structure and breeding patterns in the social wasp Polistes biglumis[END_REF]. Multiple mating has been often explained as an evolutionary response to parasite pressure [START_REF] Sherman | Parasites, pathogens, and polyandry in social Hymenoptera[END_REF][START_REF] King | Does genetic diversity limit disease spread in natural host populations?[END_REF]. Multiple mating by P. biglumis foundresses might enhance brood genetic heterogeneity, including chemicalsignature heterogeneity. Indeed, selection by parasites may favour host foundresses with unusual proportions of hydrocarbons, as such foundresses are temporarily "out of reach" of coevolving social parasites. We know that social parasites exhibit host choice by preferentially targeting large colonies (e.g., [START_REF] Nash | A mosaic of chemical coevolution in a large blue butterfly[END_REF][START_REF] Lorenzi | The geographic structure of selection on a coevolving interaction between social parasitic wasps and their hosts hampers social evolution[END_REF] and less aggressive hosts [START_REF] Foitzik | Coevolution in host-parasite systems: Behavioural strategies of slavemaking ants and their hosts[END_REF]. It seems reasonable that they can also select hosts on the basis of their colony signature, since signature has a key role in parasite adaptation.

Nestmate recognition codes show remarkable chemical complexity, involving multiple biochemical pathways [START_REF] Tsutsui | Dissecting ant recognition systems in the age of genomics[END_REF]. This complexity provides the opportunity to evaluate the ecological and social conditions that favor the evolution of complex signaling in relation to parasite pressure through comparisons within species or between host and non-host species. It also provides an example of how parasites may act as sources of selection which promote the diversification of communication signal between populations, eventually leading to a geographic mosaic of recognition codes [START_REF] Thompson | The Geographic Mosaic of Coevolution[END_REF]. This is to our knowledge the first evidence in favour of the hypothesis that different trait in the host hydrocarbon signatures as a whole undergo evolutionary changes resulting from either directional or balancing selection imposed by social parasites. While the number of populations sampled is quite low, this is a very promising finding, which will hopefully be confirmed by the sampling of additional populations.

Fig. 1

 1 Fig. 1 The chemical signature of Polistes biglumis foundresses in the three populations (Carì, Ferrere, Montgenèvre)The graph shows the average proportion of each peak (± SE) (linear alkanes: grey; branched alkanes: red; mixed peaks (peak 46+47 and 58+59): red-grey). Peaks are as follows: peak 1: n-C23; peak 2: n-C24; peak 2b: 2-Me C24; peak 3: n-C25; peak 4: 11-+13-MeC25; peak 5: 7-MeC25; peak 6: 5-MeC25; peak7: 11,15-DiMeC25; peak 8: 3-MeC25; peak 9: n-C26; peak 10: 12-MeC26; peak 11: 2-MeC26; peak 11a: 3-MeC26; peak 12: n-C27; peak 13+13a: mix of monoMeC27; peak14: 7-MeC27; peak 15: 5-MeC27; peak 16: 11,15-diMeC27; peak 17: 3-MeC27; peak 18: 5,11-+5,13-DiMeC27; peak 19: n-C28; peak 20: mix of diMeC27; peak 21: mix of monoMeC28; peak 21a: 6-MeC28; peak 22: 4-MeC28; peak 23: 2-MeC28; peak 24: 3-MeC28; peak 26: n-C29; peak 27: mix of monoMeC29; peak 28: 7-MeC29; peak 29: 5-MeC29; peak 30: mix of diMeC29; peak 31: 3-MeC29; peak 32: 5,11-+ 5,13-diMeC29; peak 33: 5,9-diMeC29; peak 34: n-C30; peak 35: mix of diMeC29; peak 36: mix of monoMeC30; peak 36b: 8,14-diMeC30; peak 37+38: 2-MeC30+9,19diMeC30; peak 39: 4,12-diMeC30; peak 40: n-C31; peak 41+42: mix of monoMeC31+7-MeC31; peak 42a: 5-MeC31; peak 43+44: 11,15-diMeC31+ mix of diMeC31; peak 45: 5,11-+ 5,13-diMeC31; peak 46+47: n-C32+mix of diMeC31; peak 48: mix of monoMeC32; peak 49+50: mix of diMeC32 + 2-MeC32; peak 52: n-C33; peak 53: mix of monoMeC33; peak 54+55: mix of diMeC33; peak 56: mix of triMeC33; peak 58+59: n-C34+ mix of diMeC33; peak 60: mix of monoMeC34; peak 61: 2-MeC34; peak 61bis: n-C35; peak 62: mix of monoMeC35; peak 63: mix of diMeC35.

Fig. 2

 2 Fig. 2 Variation in parasite prevalence and chemical signatures among populations of Polistes biglumisVariation is measured in terms of A) parasite prevalence (% of colonies parasitized by congeneric obligate social parasites); B) overall concentration of hydrocarbons (CHC) (in ng/mg of wasp); C) proportion of branched hydrocarbons (to branched + linear hydrocarbons) and D) quantitative variation in the chemical profiles (as withinpopulation distance to centroid in a multidimensional Euclidean space). Carì: yellow; Ferrere: orange; Montgenèvre: red.

Table 1 Expectations based on the contrasting hypotheses that either biotic or abiotic factors affect traits that contribute to colony signature, in terms of concentration, composition or quantitative variation of the cuticular hydrocarbon blends

 1

	Colony signature traits

Table 2 Mean concentration of hydrocarbons (± SD), proportion of branched hydrocarbons, parasite prevalence, mean daily maximum air temperature (± SD) and mean rainfall (±SD) in the three population (and relative population ranking).

 2

	Population	Hydrocarbon concentration (ng/mg of wasp)	Hydrocarbon concentration (rank)	Proportion of branched hydrocarbons (range)	Proportion of branched hydrocarbons (rank)	Parasite prevalence*	Parasite prevalence (rank)
	Montgenèvre	317.88 ± 33.43	1	0.56 (0.480.63)	1	23.5%	1
	Ferrere	223.60 ± 47.10	2	0.53 (0.410.60)	2	5.6%	2
	Carì	219.90 ± 52.97	3	0.52 (0.470.58)	3	0%	3
	Population	Max Temperature (°C)	Temperature (rank)	Mean precipitation (rain mm/day)	Precipitation (rank)
	Montgenèvre	18.1±4.0	2	1.4±3.1	3
	Ferrere	19.5±2.9	1	2.5±5.0	2
	Carì	16.5±3.7	3	2.7±9.9	1

*measured as percentage of nests parasitized by P. atrimandibularis in 3-5 years,

[START_REF] Lorenzi | The geographic structure of selection on a coevolving interaction between social parasitic wasps and their hosts hampers social evolution[END_REF]

Acknowledgements

We are grateful to Augusto Vigna-Taglianti for discovering the population in Ferrere and to Jean-Philippe Christidès for help in chemical analyses. We appreciate the helpful comments of John N. Thompson and Jack Coggins on earlier versions of the manuscript. Funding for this work was obtained from the University of Turin (ex 60% to MCL).


Janine W Y Wong

Christophe Lucas

Mathias Ko ¨lliker
email: mathias.koelliker@unibas.ch

Cues of Maternal Condition Influence Offspring Selfishness

The evolution of parent-offspring communication was mostly studied from the perspective of parents responding to begging signals conveying information about offspring condition. Parents should respond to begging because of the differential fitness returns obtained from their investment in offspring that differ in condition. For analogous reasons, offspring should adjust their behavior to cues/signals of parental condition: parents that differ in condition pay differential costs of care and, hence, should provide different amounts of food. In this study, we experimentally tested in the European earwig (Forficula auricularia) if cues of maternal condition affect offspring behavior in terms of sibling cannibalism. We experimentally manipulated female condition by providing them with different amounts of food, kept nymph condition constant, allowed for nymph exposure to chemical maternal cues over extended time, quantified nymph survival (deaths being due to cannibalism) and extracted and analyzed the females' cuticular hydrocarbons (CHC). Nymph survival was significantly affected by chemical cues of maternal condition, and this effect depended on the timing of breeding. Cues of poor maternal condition enhanced nymph survival in early broods, but reduced nymph survival in late broods, and vice versa for cues of good condition. Furthermore, female condition affected the quantitative composition of their CHC profile which in turn predicted nymph survival patterns. Thus, earwig offspring are sensitive to chemical cues of maternal condition and nymphs from early and late broods show opposite reactions to the same chemical cues. Together with former evidence on maternal sensitivities to condition-dependent nymph chemical cues, our study shows context-dependent reciprocal information exchange about condition between earwig mothers and their offspring, potentially mediated by cuticular hydrocarbons.

Introduction

Parental care evolved due to its fitness benefits to offspring, and it often comes at a cost for parents [START_REF] Th | The evolution of parental care[END_REF][START_REF] Royle | The evolution of parental care[END_REF]. Offspring that vary in condition are expected to differ in the fitness gain per unit of provisioning obtained from their parents [START_REF] Godfray | Signalling of need by offspring to their parents[END_REF][START_REF] Johnstone | Begging signals and parent-offspring conflict: do parents always win?[END_REF][START_REF] Kilner | Parent-offspring conflict[END_REF][START_REF] Mock | Honest begging: expanding from signal of need[END_REF][START_REF] Royle | Begging for control: when are offspring solicitation behaviours honest?[END_REF], and parents that differ in condition should experience differential costs of provisioning [START_REF] Bateson | The dynamics of parent-offspring relationships in mammals[END_REF][START_REF] Hinde | Parent-offspring conflict and coadaptation[END_REF]. Because parents and offspring are closely related, there is an evolutionary (kin selected) incentive for parents to adjust their provisioning to offspring condition (i.e., need or quality [START_REF] Kilner | Parent-offspring conflict[END_REF][START_REF] Mock | Honest begging: expanding from signal of need[END_REF][START_REF] Godfray | Evolutionary theory of parent-offspring conflict[END_REF][START_REF] Godfray | Begging and bleating: the evolution of parent-offspring signalling[END_REF][START_REF] Mock | The evolution of sibling rivalry[END_REF]) in order to maximize their returns on investment. However, it should also pay off to offspring to adjust their demand to parental condition to moderate the cost of investment that offspring impose on their parents [START_REF] Bateson | The dynamics of parent-offspring relationships in mammals[END_REF]. As a consequence, it is in the overall interest of both parents and offspring to be sensitive to variation in each other's condition, and selection may favor the exchange of information about condition between parents and offspring through cues or signals (see [START_REF] Smith | Animal Signals[END_REF] for definitions of terms). The evolutionary conflict between parents and offspring over parental investment [START_REF] Kilner | Parent-offspring conflict[END_REF][START_REF] Godfray | Evolutionary theory of parent-offspring conflict[END_REF][START_REF] Mock | The evolution of sibling rivalry[END_REF][START_REF] Trivers | Parent-offspring conflict[END_REF] may have a modulating effect in the evolution of the signals, leading to ''information warfare'' [START_REF] Kilner | Information warfare and parent-offspring conflict[END_REF] between parents and offspring and the evolution of exaggerated and costly signals.

Previous research has focused on offspring begging signals conveying information about offspring condition as signals of need or quality [START_REF] Godfray | Signalling of need by offspring to their parents[END_REF][START_REF] Mock | Honest begging: expanding from signal of need[END_REF][START_REF] Godfray | Evolutionary theory of parent-offspring conflict[END_REF][START_REF] Grafen | Biological signals as handicaps[END_REF][START_REF] Johnstone | Models of begging as a signal of need[END_REF]. The reverse expectation that offspring should be sensitive to cues of parental condition [START_REF] Bateson | The dynamics of parent-offspring relationships in mammals[END_REF], or that parents even may have evolved signals to convey honest information about their condition to their offspring, has received less theoretical or empirical scrutiny. We may ultimately often expect a reciprocal form of parent-offspring communication where parents and offspring exchange information about their respective condition (and maybe even beyond, an information exchange among all family members in a communication network; [START_REF] Morales | Signals in family conflicts[END_REF]; see also [START_REF] Parker | Intrafamilial conflict and parental investment: a synthesis[END_REF][START_REF] Royle | The evolution of parental care: summary, conclusions and implications[END_REF]). Based on these arguments, one may expect offspring to adjust their demand or selfishness to cues or signals of parental condition. The question how selfish offspring should be, how much resources they should demand from their parents, and how competitive they should be against their siblings is at the heart of parent-offspring conflict theory [START_REF] Kilner | Parent-offspring conflict[END_REF][START_REF] Royle | Begging for control: when are offspring solicitation behaviours honest?[END_REF][START_REF] Godfray | Evolutionary theory of parent-offspring conflict[END_REF][START_REF] Mock | The evolution of sibling rivalry[END_REF][START_REF] Trivers | Parent-offspring conflict[END_REF]. In its most extreme form, offspring selfishness leads to siblicide, that is, the killing and possible consumption of a sibling offspring [START_REF] Mock | The evolution of sibling rivalry[END_REF][START_REF] Connor | Brood reduction in birds: selection for fratricide, infanticide and suicide?[END_REF][START_REF] Mock | Siblicidal aggression and resource monopolization in birds[END_REF][START_REF] Parker | How selfish should stronger sibs be?[END_REF]. So, if parents provide cues or signals about their condition to their offspring (either as inadvertently released information or as evolved signal of parental condition), and offspring are sensitive to these cues, how should offspring respond in terms of their selfishness? The prediction partly depends on the consequences of the parent's condition on the amount of obtained care, and on whether sibling interactions are purely competitive or if there is scope for cooperation among siblings (see [START_REF] Roulin | Sibling competition and cooperation over parental care[END_REF] for review of evidence of sibling cooperation). Under pure competition over limited resources, offspring perceiving that their parents are in poor condition, which therefore will provide low levels of care, should compete more intensely and maybe even attempt to kill their siblings earlier (or, alternatively, disperse). This is because the poor condition of the parents would indicate insufficient resources for all offspring, enhanced sibling competition and threat of mortality. In contrast, when cooperation between offspring can compensate partly for reduced care provided by parents in poor condition, offspring perceiving cues of poor parental condition may reduce their competitive drive due to the advantage of maintaining a larger number of siblings to cooperate with. Sibling cooperation may occur for example if larger groups/broods of young are better in predator defense, have enhanced foraging efficiency or directly cooperate for example by sharing food [START_REF] Biedermann | Larval helpers and age polyethism in ambrosia beetles[END_REF][START_REF] Yip | The influence of siblings on body condition in a social spider: is prey sharing cooperation or competition?[END_REF][START_REF] Falk | Sibling cooperation in earwig families provides insights into the early evolution of social life[END_REF].

Parental condition is often related to the timing of breeding, for example because individuals in good condition are able to breed early [START_REF]Reproductive success[END_REF]. Furthermore, early breeders may face quite different ecological conditions compared to late breeders in terms of population density, food availability, predation pressure, temperature, etc., which are all factors that may also contribute to variation in their condition, in the benefits/costs of parental care (e.g., [START_REF] Klug | Theoretical foundations of parental care[END_REF][START_REF] Wong | The evolution of parental care in insects: the roles of ecology, life-history and the social environment[END_REF]) and in the pay-off of sibling competition versus sibling cooperation. Correspondingly, parental cues/signals of condition and/or offspring sensitivities to these cues/signals may be expected to vary with the timing of breeding. Few studies investigated such context-dependent parent-offspring communication, but there is some evidence for different responses of parents to variation in offspring signals of quality by early and later breeders [START_REF] Mas | An offspring signal of quality affects the timing of future parental reproduction[END_REF][START_REF] Bize | A UV signal of offspring condition mediates context-dependent parental favouritism[END_REF].

The European earwig (Forficula auricularia) is an insect species with uniparental maternal care including egg-and offspring attendance and food provisioning [START_REF] Lamb | Parental behavior in the Dermaptera with special reference to Forficula auricularia (Dermaptera: Forficulidae)[END_REF][START_REF] Ko ¨lliker | Benefits and costs of earwig (Forficula auricularia) family life[END_REF][START_REF] Ko ¨lliker | Maternal attendance and the maintenance of family groups in common earwigs (Forficula auricularia): a field experiment[END_REF][START_REF] Staerkle | Maternal food regurgitation to nymphs in earwigs (Forficula auricularia)[END_REF][START_REF] Vancassel | Plasticity and adaptive radiation of dermapteran parental behavior: results and perspectives[END_REF][START_REF] Weyrauch | Experimentelle Analyse der Brutpflege des Ohrwurmes Forficula auricularia L[END_REF]. The offspring (nymphs) signal their condition by solicitation pheromones in the form of cuticular hydrocarbons (CHC) to which the females show two distinct responses: When exposed to CHC extracts from well-fed nymphs (as compared to poorly fed nymphs, or controls) females increase their food provisioning [START_REF] Mas | A chemical signal of offspring quality affects maternal care in a social insect[END_REF] and modify the timing of second clutch production [START_REF] Mas | An offspring signal of quality affects the timing of future parental reproduction[END_REF]. This latter response depends on the timing of breeding, with early females advancing and late females delaying second clutch production. Furthermore, females in poor condition provide food to fewer nymphs [START_REF] Wong | The Effect of Female Condition on Maternal Care in the European Earwig[END_REF] and they negatively affect their nymphs' survival under conditions of limited food availability, probably because of mother-offspring competition over the scarce food [START_REF] Meunier | When it is costly to have a caring mother: food limitation erases the benefits of parental care in earwigs[END_REF]. This is in contrast to the beneficial effects of maternal presence under conditions of plentiful food where female food provisioning enhances nymph survival [START_REF] Ko ¨lliker | Benefits and costs of earwig (Forficula auricularia) family life[END_REF]. Finally, siblicide and cannibalism are a primary cause of mortality throughout nymph development [START_REF] Dobler | Kin-selected siblicide and cannibalism in the European earwig[END_REF][START_REF] Dobler | Influence of weight asymmetry and kinship on siblicidal and cannibalistic beahviour in earwigs[END_REF], which makes F. auricularia an ideal model system to test the influence of maternal condition cues on offspring selfishness.

Materials and Methods

The animals used in this experiment originated from a laboratory population held according to our standard laboratory rearing protocol and based on a large founder population [START_REF] Meunier | Parental antagonism and parent-offspring coadaptation interact to shape family life[END_REF][START_REF] Meunier | One clutch or two clutches? Coexisting alternative female life-histories in the European earwig[END_REF]. In brief, groups of approximately 80 males and 80 females (randomly selected from the breeding stocks) were set up for mating in two plastic containers (37622625 cm) lined with Fluon to prevent the insects from escaping, humid sand as a substrate, and egg-cardboard and plastic tubes as shelters. The food consisted of an artificial diet [START_REF] Meunier | One clutch or two clutches? Coexisting alternative female life-histories in the European earwig[END_REF] and was changed twice a week. The containers were kept in a climatic chamber at 60% humidity and 14 h/10 h 20uC/20uC light/dark photoperiod cycle (''summer conditions''). Upon observation of the first oviposition on 21 January 2011, all females were set up individually in Petri-dishes (1062 cm) with humid sand as substrate and plastic shelters as nests and ad libitum food. All females were then transferred to ''winter conditions'', which consisted of one week at 10uC to trigger egg-production, and 15uC afterwards and 80% humidity (throughout without light). The females were held under these conditions until the eggs hatched ( = day 0). Food was changed twice a week from isolation to oviposition. No food was provided from oviposition to hatching [START_REF] Ko ¨lliker | Benefits and costs of earwig (Forficula auricularia) family life[END_REF]. One day after hatching the number of hatched nymphs was counted, and the clutches were standardized to a maximum of 25 nymphs in preparation for the experimental set up (see below). The female and five randomly selected nymphs were weighed to the nearest 0.001 mg using a Mettler-Toledo MT5 Micro-balance (Mettler, Roche, Basel), provided with ad libitum food and transferred to summer conditions (see above).

Experimental Design

The aim of the experimental design was to allow the earwig mother to release chemical cues in the substrate and to expose the nymphs to these cues over an extended time period, but preventing physical contact between mother and nymphs. We achieved this by keeping mothers and nymphs in separate Petri dishes and swapping them daily between the two Petri dishes. This treatment ensured that nymphs were exposed continuously to any chemical cues females released and left in the substrate, and that the maternal cues were renewed every other day.

The experiment was initiated on day 2 after hatching. The female and 20 nymphs (between 15 and 19 nymphs when brood size was smaller; 7 out of 37 cases) were separated and transferred to a pair of Petri-dishes (1062 cm) containing humid sand as substrate and plastic shelters, respectively. At this stage, the females were randomly assigned either to the high food (HF) or to the low food (LF) treatment. To obtain females in HF or in LF condition, while keeping nymph condition constant, we manipulated the degree of female food access (pollen pellets [START_REF] Staerkle | Maternal food regurgitation to nymphs in earwigs (Forficula auricularia)[END_REF]) and kept it constant for nymphs. HF females had daily access to large amounts of food (approx. 10 mg) for 3 hours. LF females had access only every second day to a smaller amount of food (,1 mg) for a period of 3 h (see also [START_REF] Wong | The Effect of Female Condition on Maternal Care in the European Earwig[END_REF]). The nymphs had daily access to ad libitum food (pollen pellets) during these 3 h of female treatment. In all samples, the remaining food was removed after the 3 h feeding period.

Because HF females had access to larger amounts of food for a longer total amount of time, we expected them to produce more frass, which would have biased nymph food intake through allocoprophagy and, hence, potentially nymph condition. To prevent such an effect, female frass was removed daily before swapping females and nymphs between Petri-dishes. The number of nymphs alive was counted daily. In this species, deaths due to siblicide and cannibalism cannot easily be directly observed because the attacked nymphs are consumed quickly and completely. The number of nymphs alive is therefore mostly a consequence of nymph cannibalism (only 33 dead bodies were observed over the course of the experiments; out of 721 nymphs set up in total). On day 40 after hatching, we counted the number of surviving nymphs, and we took again the weight of the female and of five randomly chosen nymphs (or fewer, depending on the number of survivors).

The sample size consisted of 37 replicates (Petri dish pairs), 18 females and their broods in the HF treatment and 19 females and their broods in the LF treatment. The experimental treatments were properly randomized as there were no significant differences between treatments in female egg-laying date (means 6 s.e.; HF:

Extraction and Quantification of Cuticular Hydrocarbons (CHC)

After termination of the experiment on day 40, all females were individually frozen at 230uC for later CHC extraction. For extraction, each female was immersed for 10 minutes in 800 ml of the extraction solution which consisted of n-Heptane (Rotisolv 99% pure, Carl Roth AG, Arlesheim, Switzerland) and 2.5 ng/ml n-Octadecane as an internal standard (C 18 H 38 ; Fluka Analytical, Sigma-Aldrich, Buchs, Switzerland). The female was then removed from the vial and the extract stored at 230uC. Chemical analysis was carried out using Gas-Chromatography/Mass-Spectrometry (Agilent GC 7890A/5975C MSD; electron impact: 70 eV). For analysis, 2 ml extract were injected in the GC (containing 262.5 ng = 5 ng of the internal standard) in splitless mode (splitless time = 2 min.) and a constant inlet temperature of 250uC. The GC-MS system was equipped with a HP-5MS fused silica capillary column (length: 30 m, inner diameter: 0.250 mm, film thickness: 0.25 mm; Agilent J & W GC columns, Agilent Technologies, USA). The GC temperature program started with a temperature of 70uC (held for 2 min), then increased at 15uC/min to 232uC (held for 11 min), and then at 5uC/min to 300uC (held for 7 min). The column helium flow rate was 1 ml/min, ion detection started after a five minute solvent delay, and the MSD was set to a scan range of 40-550 m/z. For quantification of the CHC profiles, we integrated 31 peaks (of which one was the internal standard octadecane; nC18) from the chemical chromatogram using Chemstation software (Agilent Technologies, Inc.). For quantification, we divided the area of each peak by the area of the internal standard in the same chromatogram and multiplied this ratio by 5 ng to obtain an estimate of the quantity for each peak in ng. We provide peak identifications based on comparison with previous unpublished CHC identification from earwigs (Wong et al. submitted) and using fragmentation analysis [START_REF] Mccarthy | Hydrogen atom transfer in mass spectrometric fragmentation patterns of saturated aliphatic hydrocarbons[END_REF][START_REF] Nelson | Long-chain methyl-branched hydrocarbons: occurrence, biosynthesis and function[END_REF][START_REF] Promonis | Insect hydrocarbons. 2. Mass spectra of dimethylalkanes and the effect of the number of methylene units between methyl groups on fragmentation[END_REF] with MassHunter B.06.00 software (Agilent Technologies, Inc.). Kovats retention indices were calculated according to [START_REF] Kovats | Gas-chromatographische Charakterisierung organischer Verbindungen. Teil 1: Retentionsindices aliphatischer halogenide, Alkohole, Aldehyde und Ketone[END_REF] based on a series of n-alkane standards (C8-C40, Fluka Analytical, Sigma-Aldrich, Buchs, Switzerland).

Statistical Analysis

We analyzed the effect of the female condition treatment on the proportion of nymphs alive using a generalized linear model with a logit link, a binomial error distribution (correcting for overdispersion), the number of nymphs alive as the dependent variable, the number of nymphs originally present at experimental set up as denominator, and the female condition treatment, hatching date and their interaction as fixed effects.

The measures of peak quantities were transformed using the power transformation y = x 0.2 which yielded approximately normal distributions. The values y of each peak were then standardized to a mean = 0 and standard deviation = 1 (z i = (y i -y -)/s y ) [as recommended in 46]. Given the large number of peaks in the CHC data (k = 30 peaks) relative to sample size (n = 37), and in order to take into account tight correlations among individual peak quantities, we used a variable clustering approach as implemented in JMPHPro 10.0.1 to reduce data dimensionality (for more information about variable clustering, see e.g. [START_REF] Varmuza | Introduction to multivariate statistical analysis in chemometrics[END_REF] or the SAS/ STAT User's Guide, SAS Institute Inc., Cary, NC). Variable clustering is analogous to principle component analysis, but joins highly correlated variables (pointing in a similar direction in multivariate space) in clusters [START_REF] Nelson | Long-chain methyl-branched hydrocarbons: occurrence, biosynthesis and function[END_REF], facilitating biological interpretation of the experimental results. After forming the clusters, the peak of each cluster that showed the strongest correlation with its own cluster as compared to the next closest cluster was used as the cluster representatives for further analysis [START_REF] Mccarthy | Hydrogen atom transfer in mass spectrometric fragmentation patterns of saturated aliphatic hydrocarbons[END_REF].

To analyze the effects of the female condition treatment and hatching date on the female's CHC profile we used a MANOVA with the cluster representatives as dependent variables (repeated measurements), and the treatment, hatching date and their interaction as fixed factors. To directly test for a quantitative relationship between the proportion of nymphs alive and maternal CHC we used a step-wise linear regression approach with hatching date dependent survival (see results for details on how this variable was calculated) as dependent variable and the cluster representatives as candidate explanatory variables. The model with the lowest value for the Bayesian Information Criterion (BIC) was chosen as the final model and confirmed using both forward and backward variable selection procedures. All statistical analyses were carried out using JMPHPro 10.0.1 statistical software (SAS Institute Inc.) and all reported p-values are two-tailed.

Results

As intended, females from the HF treatment gained significantly more weight over the course of the experiment (mean 6 s.e.; 12.879 mg 61.254) than females from the LF treatment (3.945 mg 61.225; t 35 = 25.088, p,0.0001), but the female food treatment did not affect nymph weight gain (from day 1 to day 40) (HF: mean 6 s.e.; 3.993 mg 60.331; LF: 4.258 mg 60.322; t 35 = 0.575, p = 0.569). Thus, our food manipulation successfully generated variation in female condition while keeping nymph condition unaffected.

The proportion of nymphs alive on day 40 was affected by the female condition treatment through an interaction with hatching date (GLM; LR-x 2 1 = 6.177, p = 0.013; Figure 1), while the main effects of the female condition treatment (LR-x 2 1 = 0.899, p = 0.343) and hatching date (LR-x 2 1 = 0.014, p = 0.907) were not significant. The interaction was due to a significantly higher proportion of nymphs alive in the LF treatment among early hatching broods (contrast; LR-x 2 1 = 7.016, p = 0.008) and the opposite, marginally non-significant, trend among late hatching broods (contrast; LR-x 2 1 = 3.456, p = 0.063) (see Figure 1). The statistical clustering of the 30 peaks resulted in six clusters of highly correlated peaks (summarized in Table 1) jointly explaining 80.2% of the total variance in compound quantities. Entering the representative chemical compounds for each cluster (see Table 1) as repeated measures in a MANOVA with female condition treatment, hatching date and their interaction as fixed terms revealed a significant effect of female condition (but not hatching date or their interaction) on the relative CHC quantities and, hence, the composition of the CHC profile (Table 2; within-subjects effects: compound 6 treatment interaction). Conversely, the total CHC quantity was not significantly affected by female treatment, but dependent on hatching date (Table 2; betweensubjects effects: hatching date).

In order to correlate nymph survival patterns (Figure 1) to maternal CHC, a new variable for hatching date dependent survival was computed as the product of the standardized residuals (with respect to treatment means) of the proportion of nymphs alive and of hatching date. Positive values for this variable contribute to a positive covariance, negative values to a negative covariance between survival and hatching date. Hatching date dependent survival was significantly different between the HF and LF treatment (t 35 = 22.151, p = 0.038; see Figure 2). To test if maternal CHC predict nymphs survival patterns, we used hatching date dependent survival as the dependent variable in a step-wise linear regression with the female condition treatment and the six compound cluster representatives as predictor variables. The final model included CHC clusters 3 and 6 (Table 1) as the sole significant linear predictor variables (positive and negative respectively; Table 3). The female condition treatment dropped from the model as its formerly significant effect was explained by these two predictors.

The quantity ratio of cluster 3 and cluster 6 CHC was affected by the maternal condition treatment (F 1,33 = 11.618, p = 0.002; Fig. 2), but it was not significantly related to hatching date (F 1,33 = 0.371, p = 0.546) or to an interaction between hatching date and treatment (F 1,33 = 0.026, p = 0.874). Thus, the relative quantity of cluster 3 CHC compared to cluster 6 CHC was a cue for female condition and significantly predicted the hatching date dependent nymph survival pattern, but it was not in itself significantly related to hatching date.

Discussion

Parents may transmit information about their condition or environmental conditions through pre-birth maternal effects, through their behavioral interactions with offspring, the provisioning of resources [START_REF] Royle | The evolution of parental care[END_REF][START_REF] Mousseau | Maternal effects as adaptations[END_REF][START_REF] Mu ¨ller | Manipulative Signals in Family Conflict? On the Function of Maternal Yolk Hormones in Birds[END_REF], but also through specific signals as part of a reciprocal exchange of information between parents and offspring. For example, treehopper (Umbilia crassicornis) nymphs signal predator threat to their tending mother through vibrational signals [START_REF] Cocroft | Offspring-parent communication in a subsocial treehopper (Hemiptera: Membracidae: Umbonia crassicornis)[END_REF], and the mothers produce vibrational signals to reduce the likelihood of falls alarms among her nymphs [START_REF] Hamel | Negative feedback from maternal signals reduces false alarms by collectively signalling offspring[END_REF]. In this study, we provided evidence in the European earwig Forficula auricularia that condition-dependent chemical cues/signals from the mother, as encoded in her CHC profile, predict offspring survival, and that the direction of this effect depended on the timing of breeding. The nymphs from both treatments had access to equal amounts of food throughout and did not differ in their weight, and cannibalism occurred in almost all cases of nymph death. Thus, the difference in survival between treatments was most likely due to variation in nymph siblicidal and cannibalistic drive, induced by cues of maternal condition.

The information transfer about female condition was not direct through a behavioral interaction from mothers to their offspring. We experimentally prevented any physical (visual, tactile, or other) contact between mothers and nymphs by keeping the mother and her nymphs in separate Petri dishes (and swapping them daily) to ensure that only chemical information about maternal condition, and not her behavior or the amount of maternal food provisioning, could mediate the observed effects on nymph siblicide and cannibalism. Thus, females must have released chemical cues in the form of non-volatile contact pheromones in the substrate, and the nymphs were exposed to these cues when subsequently placed in the same environment. Under natural conditions this indirect substrate-born signaling would occur in the breeding burrows during the period of maternal care. Female earwigs ''mark'' their breeding burrow with pheromone secretions (shown for the sand earwig Labidura riparia; [START_REF] Radl | Maternal behaviour and nest recognition in the subsocial earwig Labidura riparia Pallas (Dermaptera: Labiduridae)[END_REF]; pers. obs. for F. auricularia), to which the nymphs are then exposed while in the burrow.

The effect of maternal chemical cues of condition on nymph siblicide and cannibalism depended on the timing of breeding. Among early broods, nymphs exposed to maternal cues of poor condition showed a significantly and about two-fold higher survival rate than nymphs exposed to maternal cues of high condition. Interestingly, the effect was in the opposite direction among late broods. This effect could be either due to a quantitative or qualitative difference in the condition-dependent chemical cues among early and late breeding females or a difference in the response to the same condition-dependent cues among nymphs from early and late broods. Although our data does not allow us to fully disentangle the two possibilities, our further analyses indicate that the latter is the more likely explanation. Variation between females in CHC profiles was quantitative in nature. Early and late breeding females, and females in poor and good condition, had qualitatively the same CHC profiles. The composition of the female CHC profile (in particular the quantity ratio between cluster 3 and cluster 6 CHC) varied quantitatively with female condition, but not with timing of breeding, and it explained the treatment effect on nymph survival. We cannot fully rule out that other cues not measured by CHC extraction and GC-MS analysis (e.g., peptides or proteins) may be the causal agents underlying this effect, but any such cue would have had to be correlated with maternal CHC. Thus, our results indicate that nymphs born early and nymphs born late had opposite responses to maternal substrate-born cues of condition, expressing behavioral reaction norms [START_REF] Smiseth | Parent-offspring conflict and coadaptation: behavioural ecology meets quantitative genetics[END_REF] of opposite sign. The ratio of cluster 3 CHC quantities (mostly composed of nC25 alkanes with linear and methylated pentacosane; Table 1) to cluster 6 CHC quantities (composed of a mix of monoenes and dienes of C25, C27, C29 and C31; Table 1) was lower in females of poor condition, and was associated with lower cannibalism rates among early broods and higher cannibalism rates among late broods (and vice versa for higher ratios). This is evidence for context-dependence of offspring responses to maternal cues/ signals. If variation in hatching date has a genetic component, these results would show genotype 6 family environment interactions [START_REF] Ko ¨lliker | Parent-offspring coadaptation[END_REF] with the maternal chemical cues of condition being a component of the family environment to which the nymphs are sensitive. G 6 E is an important factor in the maintenance of heritable variation of phenotypic traits [START_REF] Lynch | Genetics and analysis of quantitative traits[END_REF][START_REF] Rutter | Genes and behavior[END_REF] and in the present case would contribute to maintained variation in cannibalistic tendencies.

We previously showed that the same manipulation of female food access affected the food provisioning rate of earwig mothers, with females in poor condition providing food to fewer nymphs The representative peak for each cluster is highlighted in bold 1 . Clusters, peaks within clusters and chemical compounds within clusters are numbered according to the order of their retention times. Clusters 3 and 6 (bold) were condition dependent and significant predictors of nymph survival patterns.

1

The compound with strongest correlation with its own cluster compared to the next closest cluster (i.e., compounds with lowest 1-r2 ratio) were chosen as cluster representatives.

than females in high condition [START_REF] Wong | The Effect of Female Condition on Maternal Care in the European Earwig[END_REF]. Furthermore, the presence of a mother can reduce nymph survival when the mother is in poor condition and food is scarce, because mothers in poor condition compete with offspring for access to the limited available resources [START_REF] Meunier | When it is costly to have a caring mother: food limitation erases the benefits of parental care in earwigs[END_REF]. As a consequence, nymphs should associate poor maternal condition with low expected food provisioning by their mother, and more costly interactions with her, and they should respond to the corresponding cues of maternal condition accordingly. Based on the predictions we formulated in the introduction, the higher cannibalism rate among late broods when exposed to chemical cues/signals of poor maternal condition fits a scenario of such enhanced competition when the mother is in poor condition.

Conversely, the lower cannibalism rate among early broods when exposed to cues of poor maternal condition would then suggest a differential benefit of living in larger sibships and/or of sibling cooperation when the mother is in poor condition. Recent experiments demonstrated that earwig nymphs not only compete (including siblicide) [START_REF] Dobler | Kin-selected siblicide and cannibalism in the European earwig[END_REF], but that they are also very gregarious over large parts of their juvenile development [START_REF] Wong | The Effect of Female Condition on Maternal Care in the European Earwig[END_REF][START_REF] Go ´mez | Maternal care, mother-offspring aggregation and age-dependent co-adaptation in the European earwig[END_REF], and that they cooperate by sharing food, a behavior particularly pronounced in the absence of physical interactions with their mother [START_REF] Falk | Sibling cooperation in earwig families provides insights into the early evolution of social life[END_REF]. Thus, there is scope for both sibling competition and cooperation in F. auricularia. But why should the benefits of cooperative versus competitive strategies vary with the timing of breeding? In earwigs, early broods are the first to emerge from their winter burrows and experience low densities, less cannibalism threat by other earwigs and more time for development before the next winter starts. The low density could imply that the costs of dispersing and self-foraging (to escape from a mother in poor condition with which nymphs would otherwise have to locally compete for food; [START_REF] Meunier | When it is costly to have a caring mother: food limitation erases the benefits of parental care in earwigs[END_REF]) may be lower for early brood nymphs. Concurrently, maintaining larger sib groups by keeping the level of siblicide low may be beneficial for self-foraging, for example because larger groups of nymphs are more efficient at foraging or provide a better protection against predators (see [START_REF] Krause | Living in groups[END_REF] for a review). However, further studies are required to test this hypothesis.

Our results showed that the maternal CHC profile contained reliable information about condition and was associated with timedependent behavioral responses in offspring (i.e., cannibalistic drive) that have immediate fitness consequences in terms of survival. Thus, there is selection on this cue, and it seems likely that variation in maternal CHC profiles may have evolved to some extent due to its signaling function. We do not know if the observed variation in CHC profiles carries strategic costs (i.e., is a signal of condition) or if it rather reflects a constraint of limited food intake (i.e., is an index of condition; [START_REF] Smith | Animal Signals[END_REF][START_REF] Holman | Costs and constraints conspire to produce honest signaling: insights from an ant queen pheromone[END_REF]). Given that CHC derive from the fat-metabolism (which necessarily partly depends on the quality and quantity of ingested food [START_REF] Blomqvist | Insect pheromone biochemistry and molecular biology[END_REF]), it is possible that limitation in food intake directly constrains the quantitative production of CHC influencing CHC profiles in turn. However, the female condition treatment did not affect the overall quantity of CHC, only its composition, implying that some CHC decreased (cluster 3 CHC -nC25 alkanes with linear and methylated pentacosane; Table 1) but others increased (cluster 6 CHC -monoenes and dienes of C25, C27, C29 and C31; Table 1) under food restriction. CHC are well known for their multitudes of functions in insect communication, especially their role as cues in insect (kin) recognition [START_REF]Insect hydrocarbons[END_REF][START_REF]Sociobiology of communication[END_REF][START_REF] Guerrieri | Ants recognize foes and not friends[END_REF]. A comparably well studied example in the context of parental care are burying beetles (Nicrophorus vespilloides), where adult CHC profiles display information about breeding status (breeding versus non-breeding), and to a lesser extent also about their sex and nutritional condition [START_REF] Steiger | The smell of parents: breeding status influences cuticular hydrocarbon pattern in the burying beetle Nicrophorus vespilloides[END_REF]. Male and female parents in this biparental beetle recognize each other based on these CHC [START_REF] Steiger | The smell of parents: breeding status influences cuticular hydrocarbon pattern in the burying beetle Nicrophorus vespilloides[END_REF][START_REF] Mu ¨ller | Nestmate recognition in burying beetles: the ''breeder's badge'' as a cue used by females to distinguish their mates from male intruders[END_REF], and CHC of adults in breeding status act as a trigger of begging behavior in the larvae [START_REF] Smiseth | Chemical stimuli from parents trigger larval begging in burying beetles[END_REF]. However, it is not known in burying beetles if larvae modulate their begging in response to condition-dependent variation in parental CHC. CHC have been invoked as signals of quality in other social contexts. For example, in black garden ants (Lasius niger) it was shown that ant queen CHC convey information about queen reproductive potential, and inhibits worker ovarian development and aggression [START_REF] Holman | Costs and constraints conspire to produce honest signaling: insights from an ant queen pheromone[END_REF]. While these studies previously showed that CHC can display information about various aspects of individual condition/quality, our study suggests that CHC act as maternal condition cues mediating offspring siblicide and cannibalism and, hence, their selfishness.

Conclusions

Taken together, our results on the effect of maternal conditiondependent cues on nymph siblicide and cannibalism reported here, and the former findings in F. auricularia showing that earwig nymphs express condition-dependent CHC profiles that affect maternal behavior [START_REF] Mas | A chemical signal of offspring quality affects maternal care in a social insect[END_REF] and reproductive physiology [START_REF] Mas | An offspring signal of quality affects the timing of future parental reproduction[END_REF], we provided to our knowledge the first evidence for CHC variation to be involved in a reciprocal information exchange about nutritional condition between parents and offspring in insects. The CHC exposure effects on nymph selfishness and maternal reproductive physiology both depend on the timing of breeding. Although the ultimate causes of this variation remain to be illuminated, our findings that behavioral consequences of information exchange depend on the timing of breeding suggest that adaptive responses in communication can be strongly context-dependent and include responses that are in opposite direction.

Figure 1 .

 1 Figure 1. Relationship between the proportion of nymphs alive and brood hatching date for the two female condition treatments. A) low-condition treatment, B) high-condition treatment. Female condition was manipulated by varying experimentally the quantity of food to which the females had access (see Materials and Methods). On the x-axis, a julian date is provided with 6.2.2011 corresponding to day 1. doi:10.1371/journal.pone.0087214.g001

  17.50063.607, LF: 19.68463.511; t 35 = 0.434, p = 0.667), clutch size (HF: 67.55662.501, LF: 63.52662.434; t 35 = 21.155, p = 0.256), hatching success (HF: 0.82660.042, LF: 0.81060.041; t 35 = 20.279, p = 0.782), female body weight at hatching (HF: 52.17861.805, LF: 49.82661.757; t 35 = 20.933, p = 0.357), or nymph body weight at hatching (HF: 1.58560.076, LF: 1.58060.074; t 35 = 20.045, p = 0.965).

Table 1 .

 1 Summary of peaks, chemical identity of maternal cuticular hydrocarbons and their statistical clustering.

			Kovats		r 2	own cluster /r 2 next closest cluster /	Prop. Var.
	Cluster	Ret. Time	Index 2	Peak-ID: Compounds	1-r 2 ratio	explained 3
	1	13.95	2098	CC1: nC21	0.865/0.434/0.238	0.805
		14.40	2146	CC2:5-MeC21	0.824/0.757/0.724
		14.61	2168	CC3:3-MeC21	0.914/0.606/0.219
		14.88	2198	CC4: nC22	0.903/0.663/0.289
		15.79	2275	CC5: X,X9-nC23:2+ X99-nC23:1	0.502/0.195/0.619
		16.05	2298	CC6: nC23	0.824/0.594/0.433
	2	16.59	2333	CC7:11-, 9-, 7-MeC23	0.939/0.698/0.201	0.884
		16.77	2345	CC8:5-MeC23	0.734/0.515/0.549
		17.11	2368	CC9:3-MeC23	0.941/0.757/0.241
		17.29	2380	CC10: X-nC24:1	0.865/0.451/0.246
		17.73	2407	CC12: unknown HC	0.939/0.698/0.201
	3	17.56	2397	CC11: nC24	0.833/0.557/0.377	0.845
		19.52	2498	CC14: nC25	0.704/0.338/0.447
		20.34	2529	CC15:13-, 11-, 9-MeC25	0.932/0.574/0.160
		20.56	2537	CC16:7-MeC25	0.860/0.573/0.329
		21.34	2567	CC17:3-MeC25	0.914/0.672/0.261
		22.26	2602	CC18: unknown HC	0.779/0.667/0.663
		23.33	2635	CC19:13-, 11-, 9-MeC26	0.892/0.625/0.289
	4	26.30	2734	CC22:13-, 11-, 9-MeC27	0.815/0.382/0.299	0.837
		26.51	2742	CC23:7-MeC27	0.789/0.569/0.491
		27.10	2764	CC24:7,15-; 7,19-; 11,15-; 11,17-; 11,19-diMeC27	0.874/0.308/0.183
		27.29	2772	CC25:2,17-; 2,19-; 2,21-; 2,23-diMeC27	0.871/0.269/0.176
	5	25.35	2698	CC21: nC27	0.199/0.011/0.809	0.704
		28.86	2838	CC26:13-, 11-, 9-, 7-MeC28	0.791/0.433/0.369
		30.94	2938	CC28:11-, 9-, 7-MeC29	0.946/0.140/0.062
		31.50	2964	CC29:7,19-; 9,19-; 11,17-; 11,19-diMeC29	0.878/0.144/0.142
	6	19.04	2473	CC13: X,X9-nC25:2+ X99-nC25:1	0.572/0.324/0.633	0.681
		24.60	2675	CC20: X,X9-nC27:2+ X99-nC27:1	0.775/0.508/0.458
		29.67	2875	CC27: X,X9-nC29:2	0.785/0.272/0.295
		33.38	3075	CC30: X-nC31:1	0.590/0.077/0.444

Table 2 .

 2 Effect of female nutritional condition on cuticular hydrocarbon profiles.Results from MANOVA with the six compound cluster representatives (see Table1) as dependent variables (i.e., within-subjects effect) and the female condition treatment and hatching date as between-subjects effects. doi:10.1371/journal.pone.0087214.t002 Figure 2. Hatching date dependent nymph survival in relation to the ratio of cluster 3/cluster 6 CHC. The hatching date dependent survival rate was computed as the product of the

	Between-subjects effects	F 1,33	p
	Condition treatment	0.254	0.617
	Hatching date	6.568	0.015
	Condition treatment 6 hatching date	0.131	0.720
	Within-subjects interactions	F 5,29	p
	Compound 6 condition treatment	5.222	0.002
	Compound 6 hatching date	1.643	0.180
	Compound 6 condition treatment 6	0.411	0.837
	hatching date

standardized residuals (with respect to treatment means) of the proportion of nymphs alive and of hatching date. Positive values imply lower than average survival in early hatching broods or higher than average survival in late hatching broods. Negative values imply higher than average survival in early hatching broods or lower than average survival in late hatching broods. The CHC clusters 3 and 6 were selected based on variable clustering and a step-wise linear regression (see Tables

1 & 3

). doi:10.1371/journal.pone.0087214.g002

Table 3 .

 3 Relationship between hatching date dependent nymph survival and female CHC.Results from step-wise linear regression with hatching date dependent nymph survival as dependent variable (see main text for definition) and the six compound cluster representatives (see Table1) and the female condition treatment as dependent variables. The final model (confirmed using both forward and backward model simplification) had BIC = 112.05, and r 2 = 0.229 (null-model BIC = 114.43).

		Regression
	Final model	coefficient (± s.e.) F 1,34	p
	CHC cluster 3	0.479 (0.165)	8.362	0.007
	CHC cluster 6	20.355 (0.165)	4.593	0.039
	Rejected terms		F
	Condition treatment	-	0.332	0.568
	CHC cluster 1	-	0.734	0.398
	CHC cluster 2	-	0.152	0.699
	CHC cluster 4	-	0.060	0.808
	CHC cluster 5	-	0.598	0.445

doi:10.1371/journal.pone.0087214.t003

PLOS ONE | www.plosone.org

January 2014 | Volume 9 | Issue 1 | e87214

Index computed according to[START_REF] Kovats | Gas-chromatographische Charakterisierung organischer Verbindungen. Teil 1: Retentionsindices aliphatischer halogenide, Alkohole, Aldehyde und Ketone[END_REF], and using a series of n-alkane standards (C8-C40).

Variance explained by the cluster divided by the total variance among the peaks of this cluster. doi:10.1371/journal.pone.0087214.t001

Acknowledgments

We thank Joe ¨l Meunier, Luke Holman and an anonymous referee for valuable comments on the manuscript. We are grateful to Anne-Genevie `ve Bagne `res and Michel Vancassel for offering access to unpublished results on identified CHC compounds of F. auricularia, which were helpful for their identification in this study, and to Alain Lenoir for his advices on chemical identification. Data used in this paper are archived at Dryad (http://www. datadryad.org): doi:10.5061/dryad.cb11d.

The study was financially supported by the Swiss National Science Foundation (grant-no. PP00P3-139188 to MK), the Fonds zur Fo ¨rderung von Lehre und Forschung (Freie Akademische Gesellschaft FAG Basel), the Nikolaus und Bertha Burckhardt-Bu ¨rgin-Stiftung (PhD support to JWYW), and the PRES Centre Val de Loire Universite ´(APR-IA2012 to CL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions

Conceived and designed the experiments: JWYW MK. Performed the experiments: JWYW. Analyzed the data: MK CL. Wrote the paper: MK JWYW.