Claudia Westhus

V Line

Edouard Ugelvig

Jürgen Tourdot

Claudie Heinze

Sylvia Doums

Cremer

D Naug

L V Ugelvig

S Cremer

J Heinze

E Tourdot

C Doums

Increased grooming after repeated brood care provides sanitary benefits in a clonal ant

Keywords: Host-parasite interactions, Sanitary brood care, Experience, Grooming, Platythyrea punctata, Metarhizium fungus

Repeated pathogen exposure is a common threat in colonies of social insects, posing selection pressures on colony members to respond with improved disease-defense performance. We here tested whether experience gained by repeated tending of low-level fungus-exposed (Metarhizium robertsii) larvae may alter the performance of sanitary brood care in the clonal ant, Platythyrea punctata. We trained ants individually over nine consecutive trials to either sham-treated or fungus-exposed larvae. We then compared the larval grooming behavior of naive and trained ants and measured how effectively they removed infectious fungal conidiospores from the fungus-exposed larvae. We found that the ants changed the duration of larval grooming in response to both, larval treatment and their level of experience: (1) sham-treated larvae received longer grooming than the fungus-exposed larvae and (2) trained ants performed less self-grooming but longer larval grooming than naive ants, which was true for both, ants trained to fungus-exposed and also to sham-treated larvae. Ants that groomed the fungus-exposed larvae for longer periods removed a higher number of fungal conidiospores from the surface of the fungus-exposed larvae. As experienced ants performed longer larval grooming, they were more effective in fungal removal, thus making them better caretakers under pathogen attack of the colony. By studying this clonal ant, we can thus conclude that even in the absence of genetic variation between colony members, differences in experience levels of brood care may affect performance of sanitary brood care in social insects.

5. CHAPTER 2: Necrophoresis is not everything: cadaver groomings and intranidal transports in the ant Cataglyphis velox.

ABSTRACT

Division of labor is a key characteristic of social insects and contributes to their ecological success. Especially in disease defense, the intra-colony partitioning of sanitary work can reduce disease transmission, keep nestmates available for other tasks and reduce costs associated with sanitary task performance (i.e. at the behavioral and physiological level).

Factors internal and external to the individual affecting sanitary task allocation are not well known and most studies investigated genetic differences between workers performing behavioral sanitary work. In the first two studies I addressed whether individual experience (through repeated exposure to a sanitary hazard or performance of the task) can generate interindividual differences in the performance of behavioral sanitary tasks. Repeated parasite exposure is a common threat in colonies of social insects, posing selection pressures on colony members to respond with improved disease-defense performance. In the clonal ant Platythyrea punctata, I tested whether experience gained by repeated tending of low-level fungus-exposed (Metarhizium robertsii) larvae alters the performance of sanitary brood care. I found that ants trained both with sham-and fungus-treated larvae groomed the brood longer than naive ants. Increased grooming of fungus-treated larvae resulted in more effective fungal removal, thus making trained ants better caretakers under parasite attack of the colony.

Decomposing cadavers pose a sanitary risk to social insect colonies, necessitating cadaver management. In the second study I investigated whether cadaver management (i.e. cadaver grooming and transports) is divided among workers and task allocation affected by recent individual experience or worker size in the polymorphic and polygynous ant Cataglyphis velox. Many individuals performed cadaver management infrequently and few individuals dominated task performance. Our results suggested low division of labor for cadaver grooming and transport and a reduced modulation of these behaviors by recurrent exposure to nestmate cadavers.

Polyandry increases the diversity of group members and thereby favours division of labor within the colony. Colonies with increased genetic diversity can also be more resistant towards disease, but the mechanisms underlying increased disease resistance are not well understood. I analyzed in C. velox whether patriline differences among workers could affect their investment into the immune enzyme phenoloxidase. I did neither find heritability for this trait, nor for worker size. Environmental determination of variation in worker size could be advantageous with respect to division of labor, by permitting colonies to adapt worker size ratios to changing conditions if worker size predicts task performance. Environmental determination of the level of phenoloxidase might confer increased disease defense only to individuals performing the riskiest tasks within the colony and thereby limiting costs of immune investment at the colony level. My work suggests that individual experience may influence the performance of sanitary tasks and that tasks may vary in their degree of plasticity. It further demonstrates the importance of future research effort to understand the underlying mechanisms of interindividual variability and the benefits and costs associated with increased nestmate diversity.

Key words: ecological immunology, division of labor, phenotypic plasticity, experience, brood care, parasite exposure, cadaver management, phenoloxidase, polyandry, heritability, Platythyrea punctata, Metarhizium robertsii, Cataglyphis velox

RÉSUMÉ

La division du travail est une caractéristique clé chez les insectes sociaux et contribue à leur succès écologique. En ce qui concerne les tâches sanitaires, la division du travail au sein d'une colonie peut permettre de réduire la transmission des maladies, de libérer certaines ouvrières pour d'autres tâches, permettant de diminuer les couts associés à l'exécution des tâches sanitaires (sur le plan comportementale et physiologique). Les facteurs externes et internes aux individus déterminant leur participation aux tâches sanitaires ne sont pas bien connus. La plupart des études portent sur l'importance des différences génétiques entre ouvrières. Dans les deux premières études, j'ai examiné le rôle de l'expérience des individus (par exposition répétée à des déchets sanitaires ou à l'exécution d'une tâche) sur la mise en place de différences interindividuelles dans l'exécution d'une tâche sanitaire comportementale. L'exposition à un parasite est une menace fréquente au sein de colonies d'insectes sociaux. En utilisant la fourmi clonale Platythyrea punctata, j'ai voulu savoir si une exposition répétée des individus à des larves portant une faible quantité de conidiospores du champignon Metarhizium robertsii affectait la performance des soins sanitaires portés au couvain. J'ai trouvé que la durée de nettoyage des larves était plus élevée chez des fourmis entrainées, aux larves exposées ou non exposées au champignon, que chez des fourmis inexpérimentées. Un temps de nettoyage plus élevé améliorait l'élimination des conidiospores. Ainsi les fourmis entrainées pourraient être plus efficaces pour éliminer les conidiospores lors d'une attaque parasitaire de la colonie.

La décomposition des cadavres représente un risque sanitaire dans les colonies d'insectes sociaux, nécessitant une gestion de cadavres. Dans la deuxième étude, j'ai étudié la possibilité d'une division du travail dans la gestion des cadavres (c'est-à-dire le nettoyage et le transport) chez les ouvrières de la fourmi polygyne et polymorphe Cataglyphis velox. J'ai plus spécifiquement testé si la propensité d'accomplir ces tâches était en rapport avec une récente expérience individuelle ou avec la taille des ouvrières. Nos observations ont montré que la majorité des individus de la colonie n'effectuait que rarement des tâches de gestion de cadavres même si quelques individus pouvaient être impliqués plus fréquemment, au moins sur un laps de temps cours, dans l'exécution de ces tâches. Les résultats suggèrent une faible division du travail dans le nettoyage et le transport des cadavres et une faible modulation de ces tâches par l'exposition répétée des ouvrières à des cadavres.

La polyandrie accroît la diversité entres les membres d'un groupe et par ce fait pourrait favoriser la division du travail au sein de la colonie. Les colonies ayant une plus grande diversité génétique pourraient être plus résistantes aux maladies, mais les mécanismes sousjacents permettant une meilleure résistance aux maladies restent mal connus. J'ai analysé chez C. velox si les différences génétiques entre ouvrières appartenant à des lignées paternelles différentes avaient un effet sur le niveau de l'enzyme immunitaire phénoloxydase dans l'hémolymphe et sur la taille des ouvrières. Aucune héritabilité n'a pas pu être prouvée, ni pour le niveau de l'enzyme phénoloxydase, ni pour la taille de ouvrières. Si des ouvrières de différentes tailles n'ont pas la même probabilité d'effectuer une tâche, l'effet de l'environnement sur la taille des ouvrières pourrait alors être avantageux pour la division du travail, en permettant aux colonies d'adapter la distribution de la taille de leurs ouvrières aux conditions environnementales fluctuantes. Un effet environnemental, en particulier de la taille, sur le niveau de la phénoloxydase pourrait contribuer à accroitre la défense aux maladies seulement chez les individus de la colonie effectuant les tâches les plus risquées; ce qui aurait pour conséquence de limiter l'investissement immunitaire au niveau de la colonie.

Cette thèse suggère que l'expérience individuelle pourrait moduler la propensité à exécuter des tâches sanitaires, et que les tâches peuvent différer dans leur niveau de plasticité. Mon travail démontre l'importance de développer de futures recherches afin de pouvoir comprendre les mécanismes entraînant une variabilité interindividuelle et les bénéfices et coûts associés à une variabilité accrue des ouvrières.

Mots-clés: immunologie écologique, division du travail, plasticité phénotypique, expérience, soin de couvée, exposition aux parasites, gestion des cadavres, phénoloxydase, polyandrie, héritabilité, Platythyrea punctata, Metarhizium robertsii, Cataglyphis velox

INTRODUCTION

Studies on division of labor in parasite defense combine exciting issues: the variability among nestmates of social insects to invest into physiological and behavioral disease defense mechanisms, both at individual and collective level. Ongoing research investigates how variability in immune defense arises and is maintained within social groups. This thesis aimed to investigate whether individual experience and genetic background, both sources of interindividual variability, affect the investment into immune defense of individual ants. These aspects were investigated for behavioral and physiological defenses at the individual and collective level. The following introduction presents a short overview on immune defense and interindividual variability in social insects.

Disease resistance in social insects

A major cost of social life is the increased threat imposed by macro-(e.g. helminth worms and arthropods) and microparasites (e.g. fungi, bacteria, viruses and some protozoa; [START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF][START_REF] Tella | The evolutionary transition to coloniality promotes higher blood parasitism in birds[END_REF][START_REF] Godfrey | Transmission mode and distribution of parasites among groups of the social lizard Egernia stokesii[END_REF]. Parasites can have various effects on their hosts, such as the suppression of the host's immune system [START_REF] Edlund | Evidence for two inhibitors from Bacillus thuringiensis interfering with the host humoral defence system of saturniid pupae[END_REF][START_REF] Dupas | Geographic variation and evolution of immunosuppressive genes in a Drosophila parasitoid[END_REF][START_REF] Richards | Venom from the endoparasitic wasp Pimpla hypochondriaca adversely affects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, Lacanobia oleracea[END_REF][START_REF] Wang | A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses[END_REF] or the manipulation of host behavior to improve parasitic spread (reviewed in e.g. [START_REF] Van Houte | Walking with insects: molecular mechanisms behind parasitic manipulation of host behaviour[END_REF], thereby often leading to disease and death. There are numerous ways in which social insect workers (i.e. ants, termites, social bees and wasps) can get in contact with infective microorganisms, for instance in their nest material, through contact with nestmates [START_REF] Rosengaus | Pathobiology and disease transmission in dampwood termites [Zootermopsis angusticollis (Isoptera: Termopsidae)] infected with the fungus Metarhizium anisopliae (Deuteromycotina: Hypomycetes)[END_REF]Hughes et al. 2002;Cremer et al. 2007;[START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF] or by sharing the same foraging grounds with infectious individuals of the same or other species [START_REF] Durrer | Shared use of flowers leads to horizontal pathogen transmission[END_REF]. A particularity of social insect colonies is that the transmission of infective agents is facilitated among a high density of locally confined, closely related and frequently interacting nestmates. Furthermore, colonies achieve homeostatic nest conditions (in temperature and humidity) and provide resources in form of brood or honey, which facilitate the establishment of parasites within them [START_REF] Shykoff | Parasites and the advantage of genetic variability within social insect colonies[END_REF][START_REF] Rosengaus | Pathobiology and disease transmission in dampwood termites [Zootermopsis angusticollis (Isoptera: Termopsidae)] infected with the fungus Metarhizium anisopliae (Deuteromycotina: Hypomycetes)[END_REF][START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF]Cremer et al. 2007;[START_REF] Fefferman | Disease prevention and resistance in social insects: modeling the survival consequences of immunity, hygienic behavior, and colony organization[END_REF]. In consequence of the increased selection pressure imposed by parasites, natural selection favored the evolution of sophisticated and collective defense strategies, which complement individual behavioral and physiological defense mechanisms [START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF]Cremer et al. 2007;[START_REF] Evans | Socialized medicine: individual and communal disease barriers in honey bees[END_REF]. In social insects, selection acts both at the individual and at the collective level and affects traits which significantly influence colony phenotype and thereby colony fitness [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF].

Understanding the kind of parasites which endanger social insect colonies and the evolution of natural defense mechanisms is of great importance for conservational, agricultural and economic reasons. Honeybees and bumblebees are important pollinators and their services used in agricultural settings but wild populations have declined during the last years, which is in part linked to emerging infectious diseases [START_REF] Fürst | Disease associations between honeybees and bumblebees as a threat to wild pollinators[END_REF] and the references therein). Research on the heredity of immune defense components will allow improved selection programs. Ants and termites are also important members of terrestrial ecosystems and comprise more than 50 % of the biomass in some tropical habitats (Hölldobler and Wilson 1990). Some species are considered as major pest insects and damage control requires integrated pest management strategies (e.g. with the help of entomopathogenic fungi [START_REF] Shah | Entomopathogenic fungi as biological control agents[END_REF].

Individual physiological immune defenses

The physiological immune system serves as an ultimate defense mechanism against parasites when higher levels (e.g. individual sanitary behaviors such as self-grooming or collective defense mechanisms (see 3.1.2)) do not prevent infection. Workers of most ant species are able to spread antimicrobial secretions of their metapleural glands onto the cuticle (reviewed in Yek and Muller 2010), thereby increasing boundary cuticle defense to hinder parasite entrance into the body. If the parasite nevertheless breaches the cuticle, host receptors recognise conserved microbial patterns such as peptidoglycans and lipopolysaccharids from bacteria and β-1,3-glucans from fungi [START_REF] Gillespie | Biological mediators of insect immunity[END_REF][START_REF] Söderhäll | Role of the prophenoloxidase-activating system in invertebrate immunity[END_REF][START_REF] Tzou | How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions[END_REF][START_REF] Siva-Jothy | Insect immunity: an evolutionary ecology perspective[END_REF]. Upon recognition, an individual can employ different defense mechanisms, which can be constitutively expressed (always present, unspecific and immediate) or induced at parasite presence (specific but more delayed; Schmid-Hempel 2005a;[START_REF] Hamilton | Two arms are better than one: parasite variation leads to combined inducible and constitutive innate immune responses[END_REF]. Such defense mechanisms comprise pathways leading to the secretion of antimicrobial factors like lysozyme, antimicrobial peptides and cytotoxic molecules, as well as responses including melanization, phagocytosis of small and encapsulation of larger microbes through hemocytes (reviewed in [START_REF] Gillespie | Biological mediators of insect immunity[END_REF][START_REF] Tzou | How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions[END_REF]Schmid-Hempel 2005a;[START_REF] Siva-Jothy | Insect immunity: an evolutionary ecology perspective[END_REF][START_REF] Lemaitre | The host defense of Drosophila melanogaster[END_REF]. Viruses are detected on the basis of their double-stranded RNA and can be defended with the help of the RNA interference pathway, producing small interfering RNAs which inhibit the replication of viral RNA (reviewed in [START_REF] Kingsolver | Insect antiviral innate immunity: pathways, effectors, and connections[END_REF]. The phenoloxidase cascade is presumably the main frontline defense against parasites and anti-microbial peptides are applied at later stages if parasites remain within the host [START_REF] Haine | Antimicrobial defense and persistent infection in insects[END_REF]. The phenoloxidase cascade leads to the production of melanin, which is used for cuticle hardening, wound healing and to protect against microbes [START_REF] Gillespie | Biological mediators of insect immunity[END_REF][START_REF] Söderhäll | Role of the prophenoloxidase-activating system in invertebrate immunity[END_REF][START_REF] Cerenius | The proPO-system: pros and cons for its role in invertebrate immunity[END_REF]. As melanin and intermediates of the melanin pathway are cytotoxic and can lead to autoimmune costs in form of self-harm (e.g. [START_REF] Sadd | Self-harm caused by an insect's innate immunity[END_REF], phenoloxidase is stored in its inactive form and only activated on demand [START_REF] Söderhäll | Role of the prophenoloxidase-activating system in invertebrate immunity[END_REF][START_REF] Cerenius | The proPO-system: pros and cons for its role in invertebrate immunity[END_REF].

Autoimmune reactions and the requirement of resources are examples for maintenance and usage costs of physiological immune defense mechanisms (e.g. [START_REF] Moret | Survival for immunity: the price of immune system activation for bumblebee workers[END_REF][START_REF] Sadd | Self-harm caused by an insect's innate immunity[END_REF]. Evolutionary costs comprise negative genetic covariance relationships, where the expression of an immune parameter negatively affects other fitnessrelevant traits (e.g. reproduction, growth) and leads to trade-offs among them (reviewed in [START_REF] Schmid-Hempel | Variation in immune defence as a question of evolutionary ecology[END_REF]. Evolutionary ecology assumes that an organism's resources are limited and must be distributed and traded off among immunocompetence and life-history traits [START_REF] Sheldon | Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology[END_REF][START_REF] Rolff | Invertebrate ecological immunology[END_REF][START_REF] Schmid-Hempel | Variation in immune defence as a question of evolutionary ecology[END_REF], 2005a), as well as among different immune mechanisms (e.g. phenoloxidase and antimicrobial peptides: [START_REF] Ruiz-González | Rapid induction of immune densitydependent prophylaxis in adult social insects[END_REF]. Given the trade-offs and fitness costs of immune defenses, individuals are expected to optimise physiological immune defenses and only invest if benefits outweigh the costs, which leads to a considerate plasticity in immune responses over individual states and parasitic pressures [START_REF] Sheldon | Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology[END_REF][START_REF] Schmid-Hempel | Variation in immune defence as a question of evolutionary ecology[END_REF].

Collective immune defenses

When comparing the highly social honeybees with non-social Drosophila melanogaster and Anopheles gambiae, individual honeybees possess fewer immune genes, which might reflect their strength of collective defense mechanisms [START_REF] Evans | Immune pathways and defence mechanisms in honey bees Apis mellifera[END_REF], but could also be driven by the fact that honeybees possess other immune genes than these dipteran species which were not detected in this study. The colony-level protection achieved by organisational, behavioral and physiological mechanisms performed by cooperating social insect nestmates is termed 'social immunity' (e.g. Cremer et al. 2007;[START_REF] Spivak | Genetic, individual, and group facilitation of disease resistance in insect societies[END_REF][START_REF] Evans | Socialized medicine: individual and communal disease barriers in honey bees[END_REF][START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]) and these mechanisms can both be prophylactic or activated on demand (reviewed in Cremer et al. 2007). Modelling demonstrated that disease threat within the colony is increased through frequent and homogenous contact among nestmates and reduced by colony structuring. Structuring arises through spatial nest complexity and heterogeneity among workers as well as in their social interactions [START_REF] Pie | Nest architecture, activity pattern, worker density and the dynamics of disease transmission in social insects[END_REF][START_REF] Naug | The role of colony organization on pathogen transmission in social insects[END_REF]'organizational immunity': Naug and Smith 2007;[START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF].

The behavioral defense mechanisms employed can vary greatly within and between species (Cremer et al. 2007), ranging from parasite avoidance (Epsky andCapinera 1988, Diehl-Fleig andLucchese 1991) and social exclusion of infected individuals and brood (Arathi et al. 2000;[START_REF] Renucci | Complex undertaking behavior in Temnothorax lichtensteini ant colonies: from corpse-burying behavior to necrophoric behavior[END_REF][START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF][START_REF] Baracchi | Evidence for antiseptic behaviour towards sick adult bees in honey bee colonies[END_REF] to intensified sanitary care [START_REF] Waddington | Behaviour associated with hairless-black syndrome of adult honeybees[END_REF]Rosengaus et al. 1998b;[START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF]. Nest sanitation is a prophylactic mechanism to avoid microbial growth within the nest and comprises the application of gland-or bacteria-derived antimicrobials (e.g. metapleural gland secretions: reviewed in [START_REF] Yek | The metapleural gland of ants[END_REF][START_REF] Fernández-Marín | Active use of the metapleural glands by ants in controlling fungal infection[END_REF]; secretions from a mutualistic bacterium: [START_REF] Currie | Fungus-growing ants use antibioticproducing bacteria to control garden parasites[END_REF]; salivary gland secretions: [START_REF] Lamberty | Insect immunity: constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect[END_REF]. The strength of immune defense mechanisms can be adjusted to parasitic pressure, as the level of hitchhiking in leaf-cutting ants is adjusted to parasite abundance (minor workers hitchhike on leaves to protect major workers against parasitic flies ;[START_REF] Feener | Defense against parasites by hitchhikers in leaf-cutting ants: a quantitative assessment[END_REF] and the quantity of antimicrobial secretions is positively correlated with parasite virulence (metapleural gland: [START_REF] Yek | Regulation and specificity of antifungal metapleural gland secretion in leaf-cutting ants[END_REF]. Nest sanitation is further achieved by incorporating faecal pellets (termites: Rosengaus et al. 1998a) or collected plant resin (ants: [START_REF] Christe | Evidence for collective medication in ants[END_REF]honeybees: Simone et al. 2009) into nest material, as well as by cadaver management (Howard and Tschinkel 1976[START_REF] Robinson | Genetic determination of guarding and undertaking in honey-bee colonies[END_REF][START_REF] Hart | Task partitioning, division of labour and nest compartmentalisation collectively isolate hazardous waste in the leafcutting ant Atta cephalotes[END_REF]. Behavioral processes directed towards the dead differ between social insect species, depending on nest ecology and feeding habits [START_REF] Neoh | Do termites avoid carcasses? Behavioral responses depend on the nature of the carcasses[END_REF] and comprise behaviors such as cadaver avoidance (e.g. [START_REF] Kramm | Termite pathogens: transfer of the entomopathogen Metarhizium anisopliae between Reticulitermes sp. Termites[END_REF][START_REF] Franks | Tomb evaders: house-hunting hygiene in ants[END_REF], grooming [START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF][START_REF] Neoh | Do termites avoid carcasses? Behavioral responses depend on the nature of the carcasses[END_REF], burial [START_REF] Renucci | Complex undertaking behavior in Temnothorax lichtensteini ant colonies: from corpse-burying behavior to necrophoric behavior[END_REF]Sun et al. 2013), cannibalism (especially common in termites: [START_REF] Neoh | Do termites avoid carcasses? Behavioral responses depend on the nature of the carcasses[END_REF]) and necrophoresis (also called 'undertaking', i.e. the removal of cadavers from the nest). Another way to keep parasites at bay is the performance of pro-active self-grooming [START_REF] Morelos-Juárez | Ant farmers practice proactive personal hygiene to protect their fungus crop[END_REF]. If the infective agent has already entered the nest, individuals can warn nestmates of its presence through alarm behavior (vibratory displays in termites: Rosengaus et al. 1999a;[START_REF] Myles | Alarm, aggregation, and defense by Reticulitermes flavipes in response to a naturally occurring isolate of Metarhizium anisopliae[END_REF]. Grooming is an effective behavior to remove infectious particles from the cuticle of brood [START_REF] Drees | Susceptibility and behavioral response of red imported fire ant (Hymenoptera: Formicidae) to selected entomogenous nematodes (Rhabditida: Steinernematidae & Heterorhabditidae)[END_REF]Tragust et al. 2013a), one-self (Hughes et al. 2002), nestmates (Rosengaus et al. 1998b;Hughes et al. 2002;[START_REF] Yanagawa | Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi[END_REF] or a mutualistic fungus (Currie and Stuart 2001) and thereby increases resistance of the individual (Rosengaus et al. 1998b, Hughes et al. 2002[START_REF] Yanagawa | Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi[END_REF]. It also serves to spread antimicrobial substances from exocrine glands over the insect cuticle (e.g. [START_REF] Fernández-Marín | Active use of the metapleural glands by ants in controlling fungal infection[END_REF]Tragust et al. 2013a).

Some of the behavioral defense mechanisms seem to be triggered by chemical cues (e.g. hygienic behavior: [START_REF] Martin | Potential mechanism for detection by Apis mellifera of the parasitic mite Varroa destructor inside sealed brood cells[END_REF][START_REF] Swanson | Odorants that induce hygienic behavior in honeybees: identification of volatile compounds in chalkbroodinfected honeybee larvae[END_REF]. Necrophoresis is elicited by accumulating decomposition products (e.g. oleic acid: [START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF][START_REF] Haskins | Notes on necrophoric behavior in the archaic ant Myrmecia vindex (Formicidae: Myrmeciinae)[END_REF][START_REF] Gordon | Dependence of necrophoric response to oleic acid on social context in the ant, Pogonomyrmex badius[END_REF]Lopez-Riquelme et al. 2006;Diez et al. 2013b) in some species or through the disappearance of cues showing the vitality of the individual (dolichodial and iridomyrmecin: [START_REF] Choe | Chemical signals associated with life inhibit necrophoresis in Argentine ants[END_REF]. Honeybee cuticular hydrocarbon profiles (essential in recognition processes and chemical communication of social insects: reviewed in [START_REF] Howard | Ecological, behavioral, and biochemical aspects of insect hydrocarbons[END_REF] are modified by immune system activation (using bacterial lipopolysaccharide injections: [START_REF] Richard | Modulation of social interactions by immune stimulation in honey bee, Apis mellifera, workers[END_REF] and might be a cue for differential treatment of infected individuals.

Social and individual immune defense are tightly linked. For instance, exocrine gland secretions serve both individual and collective sanitary defense (e.g. [START_REF] Nascimento | Chemistry of metapleural gland secretions of three attine ants, Atta sexdens rubropilosa, Atta cephalotes, and Acromyrmex octospinosus (Hymenoptera: Formicidae)[END_REF][START_REF] Bot | Variable sensitivity of fungi and bacteria to compounds produced by the metapleural glands of leaf-cutting ants[END_REF][START_REF] Fernández-Marín | Active use of the metapleural glands by ants in controlling fungal infection[END_REF]Tragust et al. 2013a) and the presence of nest resin requires lower individual immune investment [START_REF] Castella | The presence of conifer resin decreases the use of the immune system in wood ants[END_REF][START_REF] Simone | Resin collection and social immunity in honey bees[END_REF].

Division of labor in social insect colonies

How the behavior and interactions of up to several millions of individuals in social insect colonies (the so-called "superorganism") are integrated and form the colony phenotype is one of the central questions in insect sociobiology [START_REF] Wilson | The Insect Societies[END_REF][START_REF] Robinson | Regulation of division of labor in insect societies[END_REF]. Without central colony control ('self-organization': [START_REF] Bonabeau | Self-organization in social insects[END_REF], workers perform tasks in response to an array of different task-related stimuli in their local environment whose occurrence depends on the workers' occupied space, intranidal task needs and the behavior of nestmates (reviewed in Beshers and Fewell 2001). Performing a task, workers change the associated stimulus level and thus shape the environment of their nestmates and thereby collective behavior (e.g. Beshers and Fewell 2001). Division of labor, where individuals perform different tasks from the group repertoire, is a complex social phenotype and found across a diversity of social taxa [START_REF] Oster | Caste and ecology in the social insects[END_REF][START_REF] Bednarz | Cooperative hunting in Harris' hawks[END_REF][START_REF] Wilson | The Insect Societies[END_REF][START_REF] Stander | Cooperative hunting in lions: the role of the individual[END_REF][START_REF] Patterson | Division of labor among lesbian and heterosexual parenting couples: Correlates of specialized versus shared patterns[END_REF]. It is especially pronounced in social insect colonies (e.g. [START_REF] Oster | Caste and ecology in the social insects[END_REF][START_REF] Gerber | Polyethism in Macrotermes bellicosus (Isoptera)[END_REF]Hölldobler and Wilson 1990) and assumed to be a key factor responsible for their remarkable ecological success [START_REF] Oster | Caste and ecology in the social insects[END_REF][START_REF] Wilson | The Insect Societies[END_REF]. The primary division of labor is reproductive division of labor, where only one or a few individuals (usually the queen caste) monopolize reproduction and all other individuals (the worker caste) help to raise the offspring and perform daily colony tasks (reviewed in [START_REF] Wilson | The Insect Societies[END_REF][START_REF] Oster | Caste and ecology in the social insects[END_REF]Hölldobler and Wilson 1990). Labor can again be divided within the worker caste, with individuals showing different probabilities to perform certain tasks or sub-tasks, if a task is partitioned [START_REF] Wilson | The Insect Societies[END_REF][START_REF] Oster | Caste and ecology in the social insects[END_REF][START_REF] Robinson | Regulation of division of labor in insect societies[END_REF]. Some ant species and most termites possess distinct worker castes that can differ morphologically (e.g. in size; i.e. polymorphism) and show distinct behavioral profiles [START_REF] Wilson | The Insect Societies[END_REF][START_REF] Oster | Caste and ecology in the social insects[END_REF]Hölldobler and Wilson 1990). Soldiers of leaf-cutting ants possess bigger and stronger mandibles and specialize in colony defense (Hölldobler and Wilson 1990). Most species show temporal polyethism, where a worker sequentially performs different tasks within its life, starting with intra-colony duties when young and changing to outdoor activities with a higher mortality rate when older (ants: Hölldobler and Wilson 1990;[START_REF] Tripet | Foraging for work and age-based polyethism: The roles of age and previous experience on task choice in ants[END_REF][START_REF] Mersch | Tracking individuals shows spatial fidelity is a key regulator of ant social organization[END_REF]honeybees: Seeley and Kolmes 1991;termites: Gerber et al. 1988;bumblebees: Cameron 1989). Age-related transitions between tasks are coupled with physiological and neurological changes [START_REF] Robinson | Regulation of honey bee age polyethism by juvenile hormone[END_REF][START_REF] Robinson | Regulation of division of labor in insect societies[END_REF][START_REF] Withers | Selective neuroanatomical plasticity and division of labour in the honeybee[END_REF][START_REF] Schulz | Biogenic amines and division of labor in honey bee colonies: behaviorally related changes in the antennal lobes and age-related changes in the mushroom bodies[END_REF][START_REF] Fahrbach | Limits on volume changes in the mushroom bodies of the honey bee brain[END_REF][START_REF] Manfredini | Molecular and social regulation of worker division of labour in fire ants[END_REF]. Age polyethism can co-occur with worker polymorphism, within physical castes (Hölldobler and Wilson 1990). In addition to age and morphology, further factors can increase inter-individual differences between workers and 'fine-tune' division of labor within the colony. Physiological differences among workers contribute to age-related or morphological variance, but can by itself generate subgroups among workers of the same size and age [START_REF] Robinson | Physiology as a caste-defining feature[END_REF].

Response-threshold models (for review see Beshers and Fewell 2001) are the current paradigm to explain the emergence of division of labor in social insects. For any given colony task, workers are assumed to engage in task performance once their stimulus level (response threshold) for that task is met (e.g. [START_REF] Bonabeau | Quantitative study of the fixed threshold model for the regulation of division of labour in insect societies[END_REF]Beshers and Fewell 2001). Response thresholds for a particular task differ between nestmates and thereby determine task allocation within the colony [START_REF] Robinson | Genetic determination of nectar foraging, pollen foraging, and nest-site scouting in honey bee colonies[END_REF][START_REF] Bonabeau | Quantitative study of the fixed threshold model for the regulation of division of labour in insect societies[END_REF]Fewell andPage 1993, 2000;[START_REF] O'donnell | Thresholds of response in nest thermoregulation by worker bumble bees, Bombus bifarius nearcticus (Hymenoptera: Apidae)[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF]. Workers with the lowest response thresholds will be the first to engage in task performance and thereby lower the corresponding stimulus level within the colony (e.g. [START_REF] Bonabeau | Quantitative study of the fixed threshold model for the regulation of division of labour in insect societies[END_REF], thus decreasing the need for other workers to perform the same task. As their thresholds will be repeatedly met when the stimulus level increases, these workers will become task specialists (e.g. [START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF]. Task specialists (i.e. workers which perform tasks more frequently or longer than their nestmates) are more abundant in larger societies (Bourke 1999;[START_REF] Thomas | Colony size affects division of labour in the ponerine ant Rhytidoponera metallica[END_REF]Jeanson et al. 2007;[START_REF] Holbrook | Division of labor increases with colony size in the harvester ant Pogonomyrmex californicus[END_REF]) and also in societies with higher genetic diversity [START_REF] Oldroyd | Genetic diversity promotes homeostasis in insect colonies[END_REF]. They are expected to perform tasks more efficiently than their non-specialized nestmates (e.g. [START_REF] Oster | Caste and ecology in the social insects[END_REF][START_REF] Jeanne | The organization of work in Polybia occidentalis: costs and benefits of specialization in a social wasp[END_REF]Dukas and Visscher 1994;Trumbo and Robinson 1997;[START_REF] Julian | Undertaking specialization in the desert leaf-cutter ant Acromyrmex versicolor[END_REF], but literature is conflicting (e.g. see [START_REF] Dornhaus | Specialization does not predict individual efficiency in an ant[END_REF]. The diversity of response thresholds within the workforce can be achieved through interindividual differences in caste (e.g. [START_REF] Detrain | Caste differences in behavioral thresholds as a basis for polyethism during food recruitment in the ant Pheidole pallidula (Nyl.) (Hymenoptera: Myrmicinae)[END_REF][START_REF] Pankiw | The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.)[END_REF], sex [START_REF] Pankiw | The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.)[END_REF] and genotype (e.g. [START_REF] Pankiw | The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.)[END_REF][START_REF] Kryger | Genotypical variability for the tasks of water collecting and scenting in a honey bee colony[END_REF][START_REF] Masterman | Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)[END_REF][START_REF] Jones | Honey bee nest thermoregulation: diversity promotes stability[END_REF][START_REF] Scheiner | Effects of patriline on gustatory responsiveness and olfactory learning in honey bees[END_REF]. They can further underlie modulation with age [START_REF] Pankiw | The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.)[END_REF], nutrition [START_REF] Pankiw | Modulation of sucrose response thresholds in honey bees (Apis mellifera L.): Influence of genotype, feeding and foraging experience[END_REF], season [START_REF] Scheiner | Variation in water and sucrose responsiveness during the foraging season affects proboscis extension learning in honey bees[END_REF], hormone levels [START_REF] Scheiner | Behavioural pharmacology of octopamine, tyramine and dopamine in honey bees[END_REF][START_REF] Barron | Octopamine modulates responsiveness to foraging-related stimuli in honey bees (Apis mellifera)[END_REF], rearing conditions [START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF], recent experience of the individual [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF]) and depend on the rate of stimulus increase (ants: Yousif 2005; bumblebees: [START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF].

Plasticity is a key characteristic of the division of labor found in social insect colonies and has been linked to colony success [START_REF] Schmid-Hempel | The ergonomics of worker behavior in social Hymenoptera[END_REF], cited in Trumbo et al. 1997). Colonies are able to adjust the workforce engaged in different tasks to changing internal and external conditions (e.g. changing age demography: Huang andRobinson 1992, 1996;disturbances: Gordon 1989) because of the behavioral flexibility of individual workers [START_REF] Theraulaz | Social regulation of foraging activities in Polistes dominulus Christ: a systemic approach to behavioural organization[END_REF][START_REF] Robinson | Regulation of division of labor in insect societies[END_REF][START_REF] Gordon | The organization of work in social insect colonies[END_REF]. Workers are able to pass through age-typical tasks at different rates, performing tasks precociously [START_REF] Huang | Regulation of honey bee division of labor by colony age demography[END_REF] or reversing back to tasks they had performed when younger [START_REF] Robinson | Colony integration in honey bees: mechanisms of behavioral reversion[END_REF].

Division of labor in disease resistance and causes of interindividual variability in the workforce

Division of labor for sanitary tasks can be advantageous, as it increases behavioral and spatial compartmentalisation, as well as heterogeneity in the interaction network, thereby decreasing parasite transmission among workers [START_REF] Schmid-Hempel | Transmission of a pathogen in Bombus terrestris, with a note on division of labour in social insects[END_REF][START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF][START_REF] Naug | The role of colony organization on pathogen transmission in social insects[END_REF][START_REF] Pie | Nest architecture, activity pattern, worker density and the dynamics of disease transmission in social insects[END_REF][START_REF] Naug | Experimentally induced change in infectious period affects transmission dynamics in a social group[END_REF][START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]. Spatial and social isolation of sanitary task performers protects individuals of higher lifetime ergonomic value (the queen, brood and young workers; reviewed in [START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]) and can even be reinforced through aggressive interactions [START_REF] Hart | Task partitioning, division of labour and nest compartmentalisation collectively isolate hazardous waste in the leafcutting ant Atta cephalotes[END_REF][START_REF] Ballari | Waste management in the leaf-cutting ant Acromyrmex lobicornis: Division of labour, aggressive behaviour, and location of external refuse dumps[END_REF]. Without variability in sanitary task performance, workers might focus all efforts on some tasks (e.g. sanitary brood care) while not performing others (e.g. cleaning of the nest and removing cadavers) so that sanitary risks might increase without being attended to. Nestmates could further interfere in task performance (as has been demonstrated in necrophoric honeybees : Trumbo and Robinson 1997), reducing task efficiency [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Even though immune defense and division of labor in social insects have both been studied comprehensively, knowledge on the allocation of sanitary work loads within the worker caste is limited.

The probability of a worker to engage in a sanitary task may be affected by a variety of factors, which have been shown to affect allocation to other colony tasks. These factors can be of internal or external nature and include an individual's biotic and abiotic environment, its spatial location, age, morphology, genotype, physiology and individual experience (reviewed for non-sanitary tasks in Beshers and Fewell 2001;[START_REF] Robinson | Physiology as a caste-defining feature[END_REF]. Individual age, the genetic background and worker size are factors influencing sanitary task allocation in highly evolved honeybees and leaf-cutting ants [START_REF] Frumhoff | A genetic component to division of labour within honey bee colonies[END_REF]Robinson andPage 1988, 1995;Arathi et al. 2000;[START_REF] Breed | Demand for task performance and workforce replacement: Undertakers in honeybee, Apis mellifera, colonies[END_REF][START_REF] Ballari | Waste management in the leaf-cutting ant Acromyrmex lobicornis: Division of labour, aggressive behaviour, and location of external refuse dumps[END_REF][START_REF] Camargo | Age polyethism in the leaf-cutting ant Acromyrmex subterraneus brunneus Forel, 1911 (Hym., Formicidae)[END_REF][START_REF] Pérez-Sato | Multi-level selection for hygienic behaviour in honeybees[END_REF]Waddington and Hughes 2010;Waddington et al. 2010;Eyer et al. 2013a). Less research effort has been performed on sanitary-task allocation in simpler societies (sensu Bourke 2001), with reduced morphological differences among nestmates or among workers and reproductives, simpler nests and communication systems and smaller colony size (but see [START_REF] Turnbull | Antifungal activity in thrips soldiers suggests a dual role for this caste[END_REF]Diez et al. 2013a). It is also not well understood how experience affects the performance of sanitary behaviors in social insect colonies (see point 3.3.2).

When responding towards a sanitary hazard, workers can show variability in task performance itself (e.g. the behavioral speed, efficiency, duration, precision or location of the behavior) and in their responsiveness towards the sanitary stimulus [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]). An individual's responsiveness is characterised by several possibly independent parameters [START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF]): the time an individual takes to respond, a worker's response threshold (the level of the sanitary stimulus at which the response is initiated) and its response probability (the probability to respond once the individual's response threshold is exceeded by the stimulus ;[START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Task performers might further differ in their degree of behavioral plasticity [START_REF] Dingemanse | Between-individual differences in behavioural plasticity within populations: causes and consequences[END_REF]. Inter-individual variability in the before mentioned behavioral response parameters is of great importance for colonies as they underlie division of labor, increase colony productivity and allow colonies to flexibly and robustly respond to changing external and internal conditions [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. In the context of social immune defenses, research has mainly focussed on behavioral frequency, duration or efficiency (e.g. Trumbo and Robinson 1997;[START_REF] Julian | Undertaking specialization in the desert leaf-cutter ant Acromyrmex versicolor[END_REF][START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]Tragust et al. 2013b). Less information is available on individual responsiveness, but research demonstrated increased olfactory sensitivity for diseased brood in honeybee workers performing hygienic behavior [START_REF] Masterman | Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)[END_REF]Gramacho and Spivak 2003). Interindividual variability might be increased through intraindividual variability, where one worker changes its response or responsiveness over time, for instance due to acquired experience (see point 3.3.2).

Individuals with greater physiological immune investment and thus reduced susceptibility could constitute a subgroup among workers (division of labor arising through physiological differences among workers [START_REF] Robinson | Physiology as a caste-defining feature[END_REF] and serve as barriers against the spread of diseases, which require direct contact between nestmates (see e.g. Cremer et al. 2007;[START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]. One could thus consider physiological immune investment a worker task. In the ant Camponotus pennsylvanicus, individuals which mounted an immune response even transferred antimicrobial compounds in their trophallactic droplet to nestmates (Hamilton et al. 2011). The selection acting on the expression of immune parameters may differ among species depending on the relative importance of life history traits under certain conditions. In bumblebees, foraging activity decreased immune function (measured as the encapsulation response; [START_REF] König | Foraging activity and immunocompetence in workers of the bumble bee, Bombus terrestris L[END_REF][START_REF] Doums | Immunocompetence in workers of a social insect, Bombus terrestris L., in relation to foraging activity and parasitic infection[END_REF]. Higher immune investment of more exposed task groups is however suggested by the findings that Cataglyphis velox foragers possess higher levels of active phenoloxidase [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]) and waste workers of Atta sexdens rubropilosa bigger metapleural glands than workers engaged in less risky tasks [START_REF] Lacerda | Morphometry of the metapleural gland of workers engaged in different behavioral tasks in the ant Atta sexdens rubropilosa[END_REF]. But whether task performance results in higher immune levels (as suggested by [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF] or whether workers possessing higher levels are more likely to engage in task performance remains to be investigated. To understand the conditional expression of traits and thus phenotypic plasticity, it is important to investigate the relative importance of environmental and genetic factors, as well as their interplay.

Genetic variability

Even though multiple mating is rare in social insects, polyandry (a female mates with several males) occurs in some highly eusocial species such as honeybees and the herein studied Cataglyphis velox [START_REF] Boomsma | Paternity in Eusocial Hymenoptera[END_REF][START_REF] Strassmann | The rarity of multiple mating by females in the social Hymenoptera[END_REF]Eyer et al. 2013b). Polyandry, as well as polygyny (several functional queens present within one colony) and genetic recombination (intra-chromosomal recombination; e.g. [START_REF] Sirviö | High recombination frequency creates genotypic diversity in colonies of the leaf-cutting ant Acromyrmex echinatior[END_REF], increase the genetic diversity among the workforce. As multiple mating events and increased genetic diversity of the offspring can be disadvantageous for both the female reproductive and the collective unit, hypotheses concerning the evolution of polyandry are diverse (for review see [START_REF] Palmer | Evolution of multiple mating in the genus Apis[END_REF][START_REF] Crozier | Polyandry in social Hymenoptera -disunity in diversity?[END_REF]. Genetic diversity is believed to confer several advantages to social insect colonies, such as the reduction of parasite loads and improved disease resistance (e.g. [START_REF] Liersch | Genetic variation within social insect colonies reduces parasite load[END_REF][START_REF] Baer | Experimental variation in polyandry affects parasite loads and fitness in a bumble-bee[END_REF][START_REF] Schmid-Hempel | Polyandry Versus Polygyny Versus Parasites[END_REF][START_REF] Baer | Unexpected consequences of polyandry for parasitism and fitness in the bumblebee, Bombus terrestris[END_REF]Hughes and Boomsma 2004;[START_REF] Tarpy | Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queens[END_REF][START_REF] Seeley | Queen promiscuity lowers disease within honeybee colonies[END_REF][START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]; but see e.g. [START_REF] Tarpy | Within-and across-colony effects of hyperpolyandry on immune function and body condition in honey bees (Apis mellifera)[END_REF]. Mechanisms underlying these relationships remain to be investigated. Polyandry modulates division of behavioral sanitary defense in bees and ants, as patriline (the offspring of a single male) origin influences the probability of an individual to engage in guarding the nest entrance [START_REF] Robinson | Genetic determination of guarding and undertaking in honey-bee colonies[END_REF][START_REF] Oldroyd | Task specialization in a wild bee, Apis florea (Hymenoptera: Apidae), revealed by RFLP banding[END_REF], waste management (Waddington et al. 2010;Eyer et al. 2013a), necrophoresis (Robinson andPage 1988, 1995), allogrooming [START_REF] Frumhoff | A genetic component to division of labour within honey bee colonies[END_REF] and hygienic behavior [START_REF] Pérez-Sato | Multi-level selection for hygienic behaviour in honeybees[END_REF]. These task preferences of some patrilines potentially arise through lower response thresholds for sanitary stimuli. Higher sensitivity for diseased brood was determined for workers performing hygienic behavior [START_REF] Masterman | Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)[END_REF]Gramacho and Spivak 2003). Genetically based differences in task performance among nestmates benefits colonies by increasing colony homeostasis (e.g. [START_REF] Jones | Honey bee nest thermoregulation: diversity promotes stability[END_REF]) and generating a stable and resilient system of division of labor when faced with environmental perturbations (e.g. [START_REF] Oldroyd | Genetic diversity promotes homeostasis in insect colonies[END_REF].

Patriline origin further affected disease resistance (Hughes and Boomsma 2004) and investment into constitutive antibacterial activity and metapleural gland size in leaf-cutting ant workers [START_REF] Hughes | Caste-specific expression of genetic variation in the size of antibiotic-producing glands of leaf-cutting ants[END_REF][START_REF] Armitage | Immune defense in leafcutting ants: a cross-fostering approach[END_REF]. In honeybees however, subfamilies (offspring of different patrilines) did not differ in phenoloxidase investment or in their capacity to encapsulate a foreign body [START_REF] Tarpy | Within-and across-colony effects of hyperpolyandry on immune function and body condition in honey bees (Apis mellifera)[END_REF]. Determining the heritability of immune traits advances the understanding of how traits will adapt to natural selection and changing environmental conditions. In honeybees, heritability of hygienic behavior was determined to be high [START_REF] Padilha | Genetic parameters for five traits in Africanized honeybees using Bayesian inference[END_REF]) and candidate genes that affect an individual's propensity to perform this task are currently investigated [START_REF] Lapidge | Seven suggestive quantitative trait loci influence hygienic behavior of honey bees[END_REF][START_REF] Oxley | Six quantitative trait loci influence task thresholds for hygienic behaviour in honeybees (Apis mellifera)[END_REF]. Heritability estimates of honeybee immune traits can be used by breeders to select colonies with higher resistance towards disease-causing agents.

The sequencing of the honeybee genome (The Honeybee Genome Sequencing Consortium 2006) and subsequent sequencing of several ant [START_REF] Bonasio | Genomic comparison of the ants Camponotus floridanus and Harpegnathos saltator[END_REF][START_REF] Nygaard | The genome of the leaf-cutting ant Acromyrmex echinatior suggests key adaptations to advanced social life and fungus farming[END_REF]Smith et al 2011a;Smith et al. 2011b;[START_REF] Suen | The genome sequence of the leafcutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle[END_REF], Wurm et al. 2011) and one termite species [START_REF] Terrapon | Molecular traces of alternative social organization in a termite genome[END_REF]) was a major milestone in genetic and genomic analyses of social insects in recent years and will increase the understanding of division of labor and its underlying evolutionary and mechanistic molecular processes (Smith et al. 2008b). Social insects are excellent model organisms for sociogenomic studies and sequenced genomes together with advanced molecular tools (e.g. expressed sequence tags, reverse transcription PCR, DNA microarrays, RNA interference) have demonstrated differential gene expression in conserved pathways (associated with nutrition, foraging behavior, maternal care and reproduction) corresponding to division of labor between workers (mainly foraging versus intranidal; reviewed in Smith et al. 2008b). In honeybees for instance, the age-related transition from intranidal tasks to foraging correlated with an increase in the expression of the foraging gene [START_REF] Ben-Shahar | Influence of gene action across different time scales on behavior[END_REF]. Other epigenetic factors such as DNA methylation, histone modification and RNA editing, seem to be important for behavioral differences between eusocial castes and developmental stages, suggesting considerate genomic plasticity (e.g. Smith et al. 2008b;[START_REF] Bonasio | Genome-wide and caste-specific DNA methylomes of the ants Camponotus floridanus and Harpegnathos saltator[END_REF][START_REF] Simola | A chromatin link to caste identity in the carpenter ant Camponotus floridanus[END_REF][START_REF] Li | Caste-specific RNA editomes in the leafcutting ant Acromyrmex echinatior[END_REF]. Allelic variation and epigenetic factors could both modulate phenotypic plasticity and increase interindividual variation within the workforce.

Phenotypic variability

Eusocial insects are able to generate worker phenotypes with distinct morphological, physiological and behavioral phenotypes from the same genome through a variety of nonmutually exclusive mechanisms. In social insects, immune defense varies at the colony and at the individual level and worker immune investment has important fitness consequences as it may affect colony disease rate and productivity (e.g. [START_REF] Evans | Colony-level impacts of immune responsiveness in honey bees, Apis mellifera[END_REF][START_REF] Baer | Phenotypic variation in male and worker encapsulation response in the bumblebee Bombus terrestris[END_REF]. Several immune components underlie plasticity, depending on environmental conditions and factors such as task performance, age, diet or parasite exposure (Rosengaus et al. 1998b;Jaccoud et al. 1999;[START_REF] Doums | Immunocompetence in workers of a social insect, Bombus terrestris L., in relation to foraging activity and parasitic infection[END_REF][START_REF] Amdam | Social reversal of immunosenescence in honey bee workers[END_REF][START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF][START_REF] Castella | The presence of conifer resin decreases the use of the immune system in wood ants[END_REF][START_REF] Castella | Covariation between colony social structure and immune defences of workers in the ant Formica selysi[END_REF][START_REF] Simone | Resin collection and social immunity in honey bees[END_REF][START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF][START_REF] Kay | A carbohydraterich diet increases social immunity in ants[END_REF]). An individual's degree of plasticity in response to these factors is determined by the level of heritability of a trait (including a potential effect of plasticity-regulating loci which exert environmentally-dependent control over structural gene expression: [START_REF] Schlichting | Control of phenotypic plasticity via regulatory genes[END_REF]. Factors leading to phenotypic variability among the workforce are dynamic and enable colonies to more rapidly adjust to changing internal and external conditions, compared to solely genetic trait determination [START_REF] Passera | Increased soldier production in ant colonies exposed to intraspecific competition[END_REF][START_REF] Mcglynn | Food supplementation alters caste allocation in a natural population of Pheidole flavens, a dimorphic leaf-litter dwelling ant[END_REF][START_REF] Hughes | Worker caste polymorphism has a genetic basis in Acromyrmex leaf-cutting ants[END_REF][START_REF] Bargum | A genetic component to size in queens of the ant, Formica truncorum[END_REF][START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF].

Worker specialization on waste management, necrophoresis and hygienic behavior depends not only on genetic factors (Robinson andPage 1988, 1995;[START_REF] Pérez-Sato | Multi-level selection for hygienic behaviour in honeybees[END_REF]Waddington et al. 2010;Eyer et al. 2013a), but is further influenced by individual age as task performers are of middle age (older than nurses but younger than foragers; Arathi et al. 2000;[START_REF] Breed | Demand for task performance and workforce replacement: Undertakers in honeybee, Apis mellifera, colonies[END_REF][START_REF] Mersch | Tracking individuals shows spatial fidelity is a key regulator of ant social organization[END_REF][START_REF] Camargo | Age polyethism in the leaf-cutting ant Acromyrmex subterraneus brunneus Forel, 1911 (Hym., Formicidae)[END_REF]Waddington and Hughes 2010). Necrophoric individuals are presumably developmentally advanced, as they start foraging at an earlier age (Trumbo et al. 1997) and possess higher levels of juvenile hormone than bees of the same age performing other age-typical tasks [START_REF] Huang | Physiological correlates of division of labor among similarly aged honey bees[END_REF]. It has been demonstrated that physiological immune defenses of social insect workers change with age (immunosenescence; [START_REF] Doums | Senescence of immune defence in Bombus workers[END_REF][START_REF] Amdam | Hormonal control of the yolk precursor vitellogenin regulates immune function and longevity in honeybees[END_REF][START_REF] Amdam | Social reversal of immunosenescence in honey bee workers[END_REF][START_REF] Schmid | Adult honeybees (Apis mellifera L.) abandon hemocytic, but not phenoloxidase-based immunity[END_REF][START_REF] Moret | Immune responses of bumblebee workers as a function of individual and colony age: senescence versus plastic adjustment of the immune function[END_REF] and can be linked to the age-dependent performance of indoor or outdoor tasks [START_REF] Amdam | Social reversal of immunosenescence in honey bee workers[END_REF][START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]).

The social environment

The social environment creates opportunities for feedback loops between nestmates (direct interactions) and between individuals and their shared environment (indirect interactions; [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF], which can affect the investment into individual and social immune defenses of workers and amplify interindividual differences. The different spatial regions workers occupy depending on age and tasks performed lead to interindividual differences in connectivity to nestmates and to a differential exposure to environmental stimuli (e.g. [START_REF] Jeanson | Long-term dynamics in proximity networks in ants[END_REF][START_REF] Mersch | Tracking individuals shows spatial fidelity is a key regulator of ant social organization[END_REF][START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]. Individuals with fewer social contacts are less likely to both transmit (e.g. individuals performing sanitary tasks) and receive (e.g. valuable colony members) infectious agents to and from nestmates (reviewed in [START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]). An individual's position in the interaction network will thus reinforce asymmetry between nestmates (Jeanson and Weidenmüller 2014;[START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]. Agonistic interactions among nestmates could also reinforce interindividual differences and contribute to sanitary task allocation. Biting interactions affected an individual's propensity to forage in the wasp Polybia occidentalis (O 'Donnell 2003'Donnell , 2006) ) and were directed towards waste-contaminated workers of the ant Atta cephalotes, thereby reinforcing sanitary division of labor [START_REF] Hart | Task partitioning, division of labour and nest compartmentalisation collectively isolate hazardous waste in the leafcutting ant Atta cephalotes[END_REF]. Stress within social groups arising through reproduction-based dominance interactions led to immunosuppression in ants [START_REF] Bocher | Reproductive conflicts affect labor and immune defense in the queenless ant Diacamma sp. "nilgiri[END_REF]. Sanitary task performance by some workers will change the task-related stimulus level for nestmates, and might affect their probability to engage in the same task. In honeybees, an individual's performance of hygienic behavior and task partitioning depended on the proportion of hygienic bees (individuals with low response thresholds for diseased brood and high propensity of task performance) present in the colony (Arathi and Spivak 2001;[START_REF] Gempe | Mixing of honeybees with different genotypes affects individual worker behavior and transcription of genes in the neuronal substrate[END_REF].

Social interactions within colonies directly affect the physiological susceptibility of individual group members and can increase their resistance towards a parasite ('social immunisation': [START_REF] Traniello | The development of immunity in a social insect: evidence for the group facilitation of disease resistance[END_REF][START_REF] Ugelvig | Social prophylaxis: group interaction promotes collective immunity in colonies[END_REF][START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]Hamilton et al. 2011). Immunity can be socially transferred (reviewed in [START_REF] Masri | Individual and social immunisation in insects[END_REF], by passing antimicrobial molecules between workers (horizontal transfer, e.g. Hamilton et al. 2011) or to the offspring (vertical transfer, e.g. [START_REF] Sadd | Facultative but persistent trans-generational immunity via the mother's eggs in bumblebees[END_REF]. In leaf-cutter ants, mutualistic Pseudonocardiaceae bacteria (produce antibiotics controlling a fungal parasite of their symbiotic fungus) are not present on major Acromyrmex octospinosus workers directly after eclosion, but transferred from older to freshly eclosed workers [START_REF] Poulsen | Within-colony transmission and the cost of a mutualistic bacterium in the leaf-cutting ant Acromyrmex octospinosus[END_REF]. Trophallaxis, which can serve to transfer antimicrobial substances between nestmates (Hamilton et al. 2011), could also affect sanitary task allocation by permitting the transfer of behavior-activating or -inhibiting substances (as has been described in the context of foraging: [START_REF] Huang | Regulation of honey bee division of labor by colony age demography[END_REF][START_REF] Leoncini | Regulation of behavioral maturation by a primer pheromone produced by adult worker honey bees[END_REF]. Nestmates are able to increase survival rates of workers exposed to parasites by removing and killing the parasite through grooming and the application of antimicrobial secretions (e.g. Rosengaus et al. 1998b;Hughes et al. 2002;[START_REF] Traniello | The development of immunity in a social insect: evidence for the group facilitation of disease resistance[END_REF][START_REF] Yanagawa | Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi[END_REF]Tragust 2013a). During social contact, small quantities of a parasite can be transmitted, causing low-level infections in the recipient resulting in increased immune gene expression and a better protection at secondary exposure [START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF].

Theoretical (foraging context: [START_REF] Linksvayer | Honeybee social regulatory networks are shaped by colony-level selection[END_REF]) and empirical research (hygienic behavior: Arathi and Spivak 2001;[START_REF] Gempe | Mixing of honeybees with different genotypes affects individual worker behavior and transcription of genes in the neuronal substrate[END_REF] demonstrates that trait expression of individuals is modulated by the combination of surrounding phenotypes. The mere presence of nestmates affects the expression of immune genes (e.g. genes coding antimicrobial peptides) in bumblebees [START_REF] Richter | Social context-dependent immune gene expression in bumblebees (Bombus terrestris)[END_REF]. However, a short-term absence of nestmates did not affect immunocompetence (the level of phenoloxidase) in leaf-cutting ants [START_REF] Armitage | The effects of age and social interactions on innate immunity in a leaf-cutting ant[END_REF]. Social evolution is linked with increasing group size and potentially increasing individual density, which have been shown to affect individual immune status (e.g. [START_REF] Doums | Immunocompetence in workers of a social insect, Bombus terrestris L., in relation to foraging activity and parasitic infection[END_REF][START_REF] Ruiz-González | Rapid induction of immune densitydependent prophylaxis in adult social insects[END_REF][START_REF] Turnbull | Antimicrobial strength increases with group size: implications for social evolution[END_REF]) and both increased group size (e.g. Rosengaus et al. 1998b;Hughes et al. 2002) as well as demographic differences among nestmates [START_REF] Rosengaus | Disease susceptibility and the adaptive nature of colony demography in the dampwood termite Zootermopsis angusticollis[END_REF]) can be beneficial for individual survival.

Individual experience

Social insects gain experience in various life activities when responding to external stimuli, such as aggression, nursing, temperature control, predator avoidance, social interactions, sexual behavior, navigation, colony emigration and food acquisition (e.g. Dukas and Visscher 1994;[START_REF] Langridge | Improvement in collective performance with experience in ants[END_REF][START_REF] Langridge | How experienced individuals contribute to an improvement in collective performance in ants[END_REF][START_REF] Leadbeater | Social learning in insects -from miniature brains to consensus building[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF]Dukas 2008;[START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF][START_REF] Muscedere | Brood-care experience, nursing performance, and neural development in the ant Pheidole dentata[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF]. For certain tasks, experienced individuals have been shown to work more efficiently and thus benefit colony performance (but see e.g. [START_REF] Dornhaus | Specialization does not predict individual efficiency in an ant[END_REF]). With increasing experience, honeybee, wasp and bumblebee foragers showed higher foraging success (Dukas and Visscher 1994[START_REF] O'donnell | Forager success increases with experience in Polybia occidentalis (Hymenoptera: Vespidae)[END_REF][START_REF] Raine | The correlation of learning speed and natural foraging success in bumble-bees[END_REF]. Experienced ant workers were more efficient in colony emigration (Temnothorax albipennis: [START_REF] Langridge | Improvement in collective performance with experience in ants[END_REF][START_REF] Langridge | How experienced individuals contribute to an improvement in collective performance in ants[END_REF]) and thermal brood relocation (Camponotus rufipes: [START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF]) and thereby lead to a better output of the whole group. Recent experience is presumably one of the most important parameters modulating individual behavior and thereby increasing interindividual differences between nestmates. Workers can acquire experience either passively by being exposed to a sanitary stimulus or actively by performing the corresponding task [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Especially in longlived social insect colonies, individuals are likely repeatedly confronted with the same infectious microorganisms [START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF]Schmid-Hempel 2005b), by contracting them from nestmates [START_REF] Rosengaus | Pathobiology and disease transmission in dampwood termites [Zootermopsis angusticollis (Isoptera: Termopsidae)] infected with the fungus Metarhizium anisopliae (Deuteromycotina: Hypomycetes)[END_REF]Hughes et al. 2002;Cremer et al. 2007;[START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]Tragust et al. 2013b) or picking them up from their surroundings and bringing them back to the colony (e.g. [START_REF] Durrer | Shared use of flowers leads to horizontal pathogen transmission[END_REF]. How repeated exposure to infectious microorganisms affects the physiological and behavioral immune defense of individual workers is a relatively new research field which remains largely unexplored and empirical evidence that experience contributes to mitigate disease risk is still scarce. Recent advances demonstrate that the physiological immune system of social insects is capable of immune memory ('immune priming'), providing survival benefits upon secondary exposure to the same parasite (termites: Rosengaus et al. 1999b;bumblebees: Sadd and Schmid-Hempel 2006;ants: Konrad et al. 2012) through micro-infections and upregulation of physiological immune defense [START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]. The produced antimicrobial substances can be transferred to nestmates via trophallaxis ("passive social immunization"; Hamilton et al. 2011) and individuals can thus benefit from parasitic exposure of nestmates. Also the behavioral anti-parasite defenses of social insects were found to be upregulated after first parasite exposure, but studies were performed at the collective level. Ant colonies that had previous contact to the fungal parasite Metarhizium show increased expression of allogrooming towards sham-treated [START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF]) and infectious nestmates, resulting in their increased survival [START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF]). In the special case of social parasites, previous exposure to a slavemaker scout (Protomognathus americanus ant) lead to increased aggression in host colonies of Temnothorax longispinosus ants [START_REF] Pamminger | Increased host aggression as an induced defense against slave-making ants[END_REF]) and these colonies were able to rescue a higher proportion of brood during a later colony raid [START_REF] Kleeberg | Forewarned is forearmed: aggression and information use determine fitness costs of slave raids[END_REF].

Experience could improve the behavioral performance of sanitary tasks (e.g. [START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF][START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF]) by (i) reducing individual response thresholds and enabling individuals to react sooner towards a sanitary threat, (ii) increasing their response probability and making it more likely for them to perform the sanitary task once their response threshold is reached, (iii) leading to a faster transition from perception of the sanitary stimulus to the final response, (iv) increasing response duration or (v) improving motoric capacities (e.g. more efficient grooming of fungal spores or preening of nematodes). Individuals specialized on necrophoric behavior removed cadavers faster and more successfully than less experienced individuals (Apis mellifera: Trumbo and Robinson 1997;Acromyrmex versicolor: Julian and Cahan 1999), but an effect of experience was not demonstrated (Trumbo and Robinson 1997).

Theoretical models propose that response thresholds can be modulated via positive feedback ('self-reinforcement'), where successful task performance results in decreased responsethresholds, leading to an increased propensity to perform the task and thereby promoting task specialization. On the other hand, response thresholds are assumed to increase when the task is not performed, decreasing the propensity of task performance [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF]reviewed in Beshers and Fewell 2001). Empirically, it has been demonstrated that foraging success increased the probability of individual ants to repeat this task [START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF][START_REF] Robinson | Experience, corpulence and decision making in ant foraging[END_REF]) and thereby impacted colony organization [START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF]. Evidence concerning a modulation of response thresholds through recent experience is scarce and conflicting [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Whereas fanning response thresholds decreased with repeated task performance in thermoregulating workers of the bumblebee Bombus terrestris [START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF], a modulation which depends on the time interval between task performances [START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF], fanning response thresholds were not affected by experience in the bumblebee Bombus impatiens [START_REF] Duong | Ventilation response thresholds do not change with age or selfreinforcement in workers of the bumble bee Bombus impatiens[END_REF]. Both these results and the finding that spatial memory lasted only 7 minutes for necrophoric Myrmica rubra workers [START_REF] Diez | Orientation in corpse-carrying ants: memory or chemical cues?[END_REF], suggest short-term modulation for infrequent tasks. If the stimulus level increases and thereby the sanitary risk (e.g. through parasitic attack), either "all hands on deck" might be needed (e.g. [START_REF] Dornhaus | Specialization does not predict individual efficiency in an ant[END_REF][START_REF] Chittka | Learning, specialization, efficiency and task allocation in social insects[END_REF] to deal with the sanitary hazard or this situation might provide ample opportunity for experience effects to arise.

Aims of the thesis

Despite a lot of knowledge on both division of labor and immune defense in social insect colonies, both research fields have only been combined in recent years. Previous studies have mainly focused on differences between colonies or between castes in disease susceptibility and defense mechanisms (e.g. [START_REF] Spivak | Varroa destructor infestation in untreated honey bee (Hymenoptera: Apidae) colonies selected for hygienic behavior[END_REF][START_REF] Poulsen | Mutualistic bacteria and a possible trade-off between alternative defence mechanisms in Acromyrmex leaf-cutting ants[END_REF][START_REF] Poulsen | Differential resistance and the importance of antibiotic production in Acromyrmex echinatior leaf-cutting ant castes towards the entomopathogenic fungus Aspergillus nomius[END_REF]Gramacho and Spivak 2003; but see e.g. [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]. But even within morphological castes or age cohorts, interindividual differences between workers can modulate individual immune investment and sanitary-task allocation at the colony level. The allocation of workers to sanitary tasks is not well understood and previous research effort has mainly investigated direct and indirect genetic effects on the performance of hygienic and necrophoric behavior (e.g. [START_REF] Robinson | Genetic determination of guarding and undertaking in honey-bee colonies[END_REF]Arathi and Spivak 2001). As social insect colonies may repeatedly face the same parasites [START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF]Schmid-Hempel 2005b), individual experience (acquired through the exposure to a sanitary hazard or through the performance of sanitary work) could be similarly important for task performance and contribute to sanitary task allocation. In the first chapter I investigate whether experience shapes the expression of sanitary brood care, i.e. larval grooming and effectiveness of conidiospore (Metarhizium robertsii) removal. The ponerine and clonal ant P. punctata allowed studying an effect of individual experience in the absence of genetic differences between individuals (Schilder et al. 1999a). In the second chapter, I analyze whether experience affects sanitary task allocation and thus division of labor by determining if recent experience increases the propensity of an individual to perform cadaver management behaviors. I determine if the workload associated with cadaver management is equally distributed among workers and whether worker size affects task performance in the polymorphic ant Cataglyphis velox. The genetic diversity of group members is important for disease dynamics, providing increased resistance of the colony (e.g. [START_REF] Liersch | Genetic variation within social insect colonies reduces parasite load[END_REF]Baer andSchmid-Hempel 1999, 2001). The underlying mechanisms of this relationship remain to be investigated and could comprise differential immune investment of patrilines (as suggested by Hughes and Boomsma 2004;[START_REF] Armitage | The effects of age and social interactions on innate immunity in a leaf-cutting ant[END_REF]. Using individuals of known age and genotype, I analyze in the polyandrous and polymorphic ant C. velox whether there is a direct link between genotype and immunocompetence (the level of phenoloxidase) in the third chapter. Patriline-level comparisons allow separating genetic and environmental influences on traits as workers from different patrilines share maternal genes and environmental conditions.

The study systems

Platythyrea punctata

The genus Platythyrea belongs to the subfamily Ponerinae and consists of 37 species worldwide [START_REF] Bolton | A taxonomic and zoogeographical census of the extant ant taxa (Hymenoptera: Formicidae)[END_REF]. Platythyrea punctata Smith, 1958 (Fig. 1) occupies a wide geographical range from southern Texas to Costa Rica and from Florida to most islands in the West Indies and the Bahamas [START_REF] Seal | Phylogeography of the parthenogenic ant Platythyrea punctata: highly successful colonization of the West Indies by a poor disperser[END_REF]. Preferred habitats are relatively undisturbed, wooded areas and colonies nest in preformed cavities in the soil, in dead branches in trees or in rotten wood on the ground [START_REF] Heinze | Thelytokous parthenogenesis and dominance hierarchies in the ponerine ant, Platythyrea punctata[END_REF][START_REF] Kellner | When and where to have sex? Different modes of reproduction and life history traits in the facultative parthenogenetic ant Platythyrea punctata[END_REF]. Nest cavities contain organic material such as dead plant particles, prey remnants and empty cocoon cases [START_REF] Schilder | Safer without Sex? Thelytokous Parthenogenesis and Regulation of Reproduction in the Ant Platythyrea punctata[END_REF]. Occupying these nest environments might expose colonies to naturally occurring parasites, for instance to fungi and bacteria developing on decomposing organic material or to their resting forms in the soil. Solitary foragers hunt for small live and dead arthropods and feed larvae of their colony on these prey items [START_REF] Schilder | Safer without Sex? Thelytokous Parthenogenesis and Regulation of Reproduction in the Ant Platythyrea punctata[END_REF][START_REF] Torres | Niches and coexistence of ant communities in Puerto Rico: repeated patterns[END_REF]. Colonies are relatively small in size, comprising few to some hundred workers (mean size = 35 individuals/colony (range 2 -475) in 189 field colonies determined by Kellner et al. (as cited in Kellner and Heinze 2011a)). They presumably reproduce by splitting (fission or budding) and disperse by walking over land [START_REF] Seal | Phylogeography of the parthenogenic ant Platythyrea punctata: highly successful colonization of the West Indies by a poor disperser[END_REF]. Various alternative reproductive tactics and female phenotypes cooccur in P. punctata: sexually reproducing queens (alate and dealate) and gamergates (mated, egg-laying workers), parthenogenetically reproducing workers as well as intercastes (phenotypes between workers and queens; Schilder et al. 1999b). In most populations (including the populations used for this thesis), the queen caste is absent and reproduction is monopolized by usually one unmated worker, even though all workers are potentially able to reproduce [START_REF] Heinze | Thelytokous parthenogenesis and dominance hierarchies in the ponerine ant, Platythyrea punctata[END_REF]Hartmann and Heinze 2003;Schilder et al. 1999b). Reproductive monopolization probably evolved to increase colony efficiency and is achieved by worker policing [START_REF] Heinze | Thelytokous parthenogenesis and dominance hierarchies in the ponerine ant, Platythyrea punctata[END_REF]Hartmann et al. 2003). Unmated reproductive workers reproduce by thelytokous parthenogenesis (i.e. the production of clonal females from unfertilized eggs) and recombination events are extremely rare in this species, leading to genetically homogeneous colonies [START_REF] Heinze | Thelytokous parthenogenesis and dominance hierarchies in the ponerine ant, Platythyrea punctata[END_REF]Schilder et al. 1999a;[START_REF] Hartmann | Facultative sexual reproduction in the parthenogenetic ant Platythyrea punctata[END_REF]Kellner et al. 2010;Kellner and Heinze 2011b). A low level of division of labor among workers is based on age, with older non-reproductives switching from intranidal tasks to foraging (Hartmann and Heinze 2003).

Cataglyphis velox

The genus Cataglyphis (subfamily Formicinae) comprises more than 100 species occurring in arid habitats in Asia, Europe and Africa. Especially the Mediterranean C. bicolor, C. cursor and C. velox are valued model species to investigate the elaborate navigational abilities and thermal adaptations typical for this genus. One of the most polymorphic species is Cataglyphis velox, Santschi, 1929, with a continuous worker length range of 4.5 -12 mm [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF]. This species is endemic to the Iberian Peninsula and found in an elevation range between 0 and 2400 m [START_REF] Tinaut | Taxonomic situation of the genus Cataglyphis Förster, 1850 in the Iberian Peninsula II. New position for C. viatica (Fabricius, 1787) and the redescription of C. velox Santschi, 1929 stat. n. (Hymenoptera, Formicidae)[END_REF]) (Fig. 2). Colonies have small to medium size, containing from hundreds to a few thousands of workers and one to several queens (worker number = 1220 individuals/colony (range 230-3650); queen number = 5 queens/colony (range 1-14) in 25 field colonies).The colonies occupy an underground nest containing several chambers connected by tunnels (Fig. 2). Two colonies were observed to possess superficial chambers close to the soil surface, containing empty cocoon cases and cadavers. Waste dumps with cadavers were also found exterior to the colony, about 15 to 20 cm away from the nest exit (observation by C. Haussy). With high maximal activity temperature values (44 to 50°C), C. velox workers (Fig. 3) are able to forage at temperatures deadly for other insects, scavenging on fresh cadavers of small arthropods and rarely incorporating plant material into their diet [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF]. Thermal tolerance of C. velox is size related: the longer legs of large workers allow them to withstand higher temperatures, achieve higher running speeds and shorter foraging durations than smaller workers. In the field, large foragers are most abundant during the hottest hours of the day, whereas smaller and medium foragers scavenge for food in the early and late hours of the day. This division of labor based on worker size enables a longer daily activity period [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF][START_REF] Cerdá | Behavioural and physiological traits to thermal stress tolerance in two Spanish desert ants[END_REF]. Workers of Cataglyphis species have been described to follow the classical age-dependent polyethism (reviewed in [START_REF] Lenoir | Cataglyphis desert ants: a good model for evolutionary biology in Darwin's anniversary year: a review[END_REF]). In C. cursor however, only one third of the workers follow the classical age-dependent polyethism, with the remainder showing either fixed behavior or irregular deviations from the expected scheme [START_REF] Retana | Behavioural variability and development of Cataglyphis cursor ant workers (Hymenoptera, Formicidae)[END_REF]. Within a single colony, C. velox queens reproduce sexually to produce workers and use parthenogenesis to produce queens (thelytokous parthenogenesis) and males (arrhenotokous parthenogenesis). C. velox thus benefits from the advantages of both sexual and asexual reproduction (Pearcy et al. 2004a). Most queens within the colony are essentially clones, they mate with multiple males (generally with 2-5 males) from different genetic lineages (Eyer et al. 2013b), thus increasing genetic diversity in the workforce.

Metarhizium robertsii

Insect parasitic fungi play an important role in agriculture and public health. Especially the entomoparasitic fungi Metarhizium spp. (Fig. 4) and Beauveria bassiana have been applied as biological pesticides to control insects classified as agricultural pests (e.g. locusts in Africa) or malaria-causing parasites in mosquitoes (reviewed in [START_REF] Thomas | Can fungal biopesticides control malaria?[END_REF]. The general insect pathogenic fungus Metarhizium robertsii (Bischoff et al. 2009) is a natural pathogen of ants [START_REF] Rath | The influence of abiotic factors on the distribution and abundance of Metarhizium anisopliae in Tasmanian pasture soils[END_REF]Keller et al. 2003;[START_REF] Leger | New perspectives on insect pathogens[END_REF]) and occurs in soils worldwide (St. [START_REF] Leger | New perspectives on insect pathogens[END_REF]. Entomopathogenic fungi invade hosts by direct penetration of the cuticle. When conidiospores of Metarhizium adhere to the cuticle, they germinate under highhumidity conditions and penetration occurs by a combination of physical force and cuticluladegrading enzymes after approximately 24 hours [START_REF] Clarkson | New insights into the mechanisms of fungal pathogenesis in insects[END_REF][START_REF] Hajek | Interactions between fungal pathogens and insect hosts[END_REF][START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]. Inside the hemocoel, Metarhizium produces yeast-like blastospores to spread the infection within the insect body and evades insect immune responses by a collagenous protective coat [START_REF] Wang | A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses[END_REF]. The production of fungal toxins (e.g. destruxins) and nutrient depletion weaken the host and usually cause death within several days; the speed of killing depends on parasite dose, the host-parasite combination, environmental conditions [START_REF] Clarkson | New insights into the mechanisms of fungal pathogenesis in insects[END_REF][START_REF] Hänel | The life cycle of the insect pathogenic fungus Metarhizium anisopliae in the termite Nasutitermes exitiosus[END_REF][START_REF] Hajek | Interactions between fungal pathogens and insect hosts[END_REF]Thomas and Blanford 2003), secondary infections and host defense reactions. Upon the death of the host, the fungus penetrates host tissues and mycelia emerge from the intersegmental regions of the cadaver cuticle under humid conditions [START_REF] Arthurs | Effects of temperature and relative humidity on sporulation of Metarhizium anisopliae var. acridum in mycosed cadavers of Schistocerca gregaria[END_REF] optimal sporulation of M. anisopliae at relative humidity of >96% and at temperatures between 20 to 30°C). Conidiospore packages are asexually produced on the cadaver (Fig. 4) and passively dispersed in the environment. Under unfavourable conditions (e.g. harsh temperatures), Metarhizium can produce thick-walled resting spores (Chlamydospores) which persist in soil for long periods of time (reviewed in [START_REF] Shah | Entomopathogenic fungi as biological control agents[END_REF].

CHAPTER 1

Increased grooming after repeated brood care provides sanitary benefits in a clonal ant.

ClaudiaWesthus, Line V. Ugelvig, Edouard Tourdot, Jürgen Heinze, Claudie Doums, Sylvia Cremer

Behavioral Ecology and Sociobiology 68: 1701-1710

Introduction

Organisms benefit greatly from the ability to respond to repeated extrinsic stimuli by improved task performance. Insects are known to gather experience and to be able to learn in different contexts (Dukas 2008), such as foraging (Dukas and Visscher 1994;[START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF]) and thermal brood relocation [START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF]. Experience could for instance result in (i) a faster reaction time to the stimulus (e.g., [START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF]), (ii) a higher probability to perform the task (e.g., [START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF], and (iii) a more efficient task performance (e.g., O 'Donnell and Jeanne 1992;Dukas and Visscher 1994;Raine and Chittka 2007), or a combination of these.

Colonies of social insects (wasps, bees, ants, and termites) are a good model system to study the effects of experience in social groups, as they are characterized by a strong division of labor with different group members specializing on particular tasks [START_REF] Oster | Caste and ecology in the social insects[END_REF]. Task specialization, the longterm behavioral bias toward the performance of a task [START_REF] Oster | Caste and ecology in the social insects[END_REF], can be affected by factors such as age (age polyethism; reviewed, e.g., in Hölldobler and Wilson 1990;[START_REF] Robinson | Regulation of division of labor in insect societies[END_REF]Merschetal.2013) and genetic background (e.g., patriline: Robinson andPage 1988, 1989;Waddington et al. 2010;[START_REF] Schlüns | Genetic polyethism and nest building in the weaver ant Oecophylla smaragdina (FABRICIUS, 1775) (Hymenoptera: Formicidae)[END_REF]genetic colony composition: Arathi and Spivak 2001) and seems further modulated by experience [START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF];R a v a r ye ta l .2007). Response-threshold models propose that task specialization within groups arises from inter-individual variation in response thresholds for task-associated stimuli. Individuals with the lowest thresholds most likely become specialists, as they will be the first to respond and thereby decrease the stimulus level present in the nest, so that the higher thresholds of their nestmates will not be met (reviewed in Beshers and Fewell 2001). Furthermore, task performance of the acting individuals can further lower their future reaction threshold, increasing the propensity of future task performance and further increasing specialization (self-reinforcement: e.g., [START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF].

Whereas previous work studied the effect of experience on activities such as foraging, nursing, temperature control, and colony emigration in social insects (e.g., Dukas and Visscher 1994;[START_REF] Langridge | Improvement in collective performance with experience in ants[END_REF][START_REF] Langridge | How experienced individuals contribute to an improvement in collective performance in ants[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF];D u k a s2008; [START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF][START_REF] Muscedere | Brood-care experience, nursing performance, and neural development in the ant Pheidole dentata[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF], it is not well understood how experience in anti-pathogen defense affects the performance and efficiency of sanitary behaviors in the colonies of social insects. Yet, this is an important factor particularly in the long-lived colonies of ants and termites, where individuals likely are repeatedly confronted with the same pathogens within their lifespan of several months to years [START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF]Schmid-Hempel 2005). Repeated pathogen exposures can occur because individuals pick up the same infectious particles recurrently from their surroundings or by contraction from their sick nestmates inside the colony [START_REF] Rosengaus | Pathobiology and disease transmission in dampwood termites [Zootermopsis angusticollis (Isoptera: Termopsidae)] infected with the fungus Metarhizium anisopliae (Deuteromycotina: Hypomycetes)[END_REF];H u g h e se ta l .2002; Cremer et al. 2007;Konradetal.2012).

Repeated pathogen exposure may affect both the physiological immune system and the behavioral defense line, which in social insects can occur both at the individual and group level (e.g., [START_REF] Siva-Jothy | Insect immunity: an evolutionary ecology perspective[END_REF]Cremeretal.2007;[START_REF] Spivak | Genetic, individual, and group facilitation of disease resistance in insect societies[END_REF]). The physiological immune system of social insects (bumblebees: Sadd and Schmid-Hempel 2006;a n t s : [START_REF] Rosengaus | Immune-priming in ant larvae: social immunity does not undermine individual immunity[END_REF] shows lower susceptibility to a previously encountered pathogen upon secondary contact (referred to as immune priming in invertebrates; [START_REF] Kurtz | Alternative adaptive immunity in invertebrates[END_REF].Atlowexposuredoses,aninfectioncanevenleadtoa protective immune upregulation (termites: Rosengaus et al. 1999;ants: Konrad et al. 2012), and such low-level exposures may be contracted during social contact with a diseased nestmate ("social immunization";Konradetal.2012).

Also, the behavioral anti-pathogen defenses of social insects were found to be upregulated after first pathogen exposure. Ant colonies that had previous contact to the fungal pathogen Metarhizium show increased expression of collective sanitary behaviors, such as allogrooming [START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF][START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF]. During allogrooming, social insects efficiently remove infectious particles from the body surface of their nestmates (Rosengaus et al. 1998;Hughes et al. 2002;[START_REF] Yanagawa | Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi[END_REF]Tragust et al. 2013a). To our knowledge, it has not been studied, however, if repeated pathogen encounter of particular individuals changes their propensity to perform sanitary brood care of diseased brood. Brood care is an integral part of sociality in insect societies and is of particular importance in the context of disease defense, as the brood is, on one hand, particularly susceptible to disease [START_REF] Patterson | Potential of three biological control agents for suppression of Solenopsis invicta, the red imported fire ant[END_REF]and,onthe other hand, very valuable, as it represents the next generation of workers and sexuals in the colony.

Social insects react to pathogen-exposed brood either by intensive brood grooming (ants: [START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF], Tragust et al. 2013b), by cannibalism (termites: [START_REF] Rosengaus | Disease susceptibility and the adaptive nature of colony demography in the dampwood termite Zootermopsis angusticollis[END_REF], or by brood removal from the colony, the latter being termed "hygienic behavior" (bees: [START_REF] Rothenbuhler | Resistance to American foulbrood in honey bees. I. Differential survival of larvae of different genetic lines[END_REF][START_REF] Spivak | Genetic, individual, and group facilitation of disease resistance in insect societies[END_REF]ants: Ugelvig et al. 2010;Tragust et al. 2013b). Whereas little is known for other social insects, such hygienic behavior is performed by middle-aged individuals in honeybees (Arathi et al. 2000). Moreover, the propensity to perform the task depends on the genotype, the genetic colony composition, and is higher in individuals with increased olfactory sensitivity (Arathi and Spivak 2001;[START_REF] Masterman | Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)[END_REF]Gramacho and Spivak 2003;Arathi et al. 2006). In general, hygienic tasks do not seem to be equally performed by all individuals of a colony, but rather by a subset of specialized workers (Trumbo et al. 1997;A r a t h ie ta l .2000). This task specialization promotes behavioral and spatial nest compartmentalization, which is predicted to decrease the risk of pathogen transmission [START_REF] Schmid-Hempel | Transmission of a pathogen in Bombus terrestris, with a note on division of labour in social insects[END_REF]N a u ga n d Camazine 2002;Cremeretal.2007), yet the factors underlying hygienic task specialization and division of labor are not well understood.

The aim of the current study was to test whether individual experience in sanitary brood care-gained by repeated contact of workers to pathogen-exposed broodmay affect the performance or effectiveness of their hygienic actions. As a model system, we used the parthenogenetically reproducing (thelytokous) ant Platythyrea punctata (Schilder et al. 1999) and the general insect pathogenic fungus Metarhizium robertsii (Bischoff et al. 2009), a natural pathogen of ants [START_REF] Rath | The influence of abiotic factors on the distribution and abundance of Metarhizium anisopliae in Tasmanian pasture soils[END_REF]Keller et al. 2003;S t .L e g e re ta l .2011). We chose this ant species that forms small colonies by clonal reproduction to study the effect of experience on task performance in groups with a low level of division of labor (Hartmann and Heinze 2003) and in the absence of genetic variation among workers. In our experimental approach, we repeatedly confronted ant workers to Metarhizium-exposed or sham-treated larvae. We then compared ants before and after training, i.e., at first contact with the larvae (trial 1) and after nine consecutive contacts of the same type of larvae (all fungus-exposed or all controls; trial 9) to test whether trained as compared to naive ants may show (i) a faster response to the stimulus, (ii) increased grooming performance, and (iii) higher removal of fungal infectious particles, or a combination of these. This experimental design allowed us not only to determine whether experience may affect the performance or efficiency of sanitary brood care in the clonal ant P. punctata,b u ta l s ot o disentangle a general effect of repeated brood care (trial 1 vs. 9) from a specific effect of repeated pathogen exposure (interaction between trial and treatment).

Materials and methods

Ant colonies

Colonies of the thelytokous ant P. punctata were collected from two populations in Puerto Rico (2005, authorized by the Departamento de Recoursos Naturales y Ambientales DRNA), one from the Dominican Republic (2006, authorized by La Dirección General de Vida Sylvester y Biodiversidad) and one from Barbados (2007, authorized by the Ministry of Agriculture and Rural Development), as detailed in Kellner et al. 2013. Ants were reared in the laboratory under standard conditions (24±2 °C, 60-65 % humidity, 12:12 h light/dark cycle; see Hartmann and Heinze 2003). All ants used in the experiments had eclosed in the laboratory and were therefore naive to the fungal pathogen.

Eight colonies with confirmed clonal colony structure (microsatellite analysis; Kellner and Heinze 2011)w e r e used as source colonies to set up two subcolonies from each. All subcolonies consisted of 15 individually colormarked (enamel paint, PinRestore) adult ants with brood (ten larvae and pupae). The size of these subcolonies lies in the natural range of colony size of this ponerine ant species (Kellner and Heinze 2011). To ensure that only non-reproductive workers were used in the following experiment, the single reproductive individual that e s t a b l i s h e di ne a c hs u b c o l o n yw i t h i n2w e e k sa f t e rs e t up was determined by observation of egg-laying. It remained within its respective subcolony over the whole course of the experiment. Twenty additional colonies were used as "larval donors" to provide brood for the experiments, as colonies of P. punctata readily accept alien brood (Kellner et al. 2010). We excluded the smallest and largest larval stages, i.e., L1 and L5, respectively, and preferentially used L3 larvae (mean weight 5.01± 1.11 mg standard deviation as revealed from weighing of 116 larvae in a preliminary experiment).

Fungal pathogen exposure

As a pathogen, we used the entomopathogenic fungus M. robertsii (strain ARSEF 2575 (Fang et al. 2006), previously named Metarhizium anisopliae,b u tn o wr e c o g n i z e da sa sister species (Bischoff et al. 2009)). The non-sexually produced fungal transmission stage (conidiospores/conidia) was freshly harvested from malt agar plates 3 to 4 days before the start of each experiment [START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF], suspended in the sterile surfactant Triton X-100 (0.05 %; Sigma) at a concentration of 10 6 conidiospores/ml and the fungal germination rate determined (>97 % in all cases). In the fungus exposure treatment, ant larvae were exposed to 500 conidiospores of the pathogenic fungus by applying 0.5 μl of the conidiospore suspension, while the sham control was treated with the same amount of Triton X solution. Treated larvae were air-dried on sterile plastic for 1 h before start of each experiment. This exposure dose was chosen to (i) provide a stimulus that likely approximates a natural exposure dose from surrounding soil (Keller et al. 2003:10 4 colony forming units of M. anisopliae per gram soil) and to (ii) prevent tending ants from contracting the disease so we could study behavioral changes based on repeated hygienic brood care experience rather than sickness of the tending workers.

Experimental procedure

We randomly assigned one subcolony per source colony to the sham control and the other subcolony to fungus exposure. From each subcolony, eight intra-nest workers were taken from the brood chamber, isolated from the colony for 1 h, during which they were placed individually in an arena (petri dish, diameter 5.5 cm), where they encountered a larva (a Triton X-treated larva for ants from the control-subcolony and a fungus-exposed larva for ants from the fungus-subcolony). Despite their isolation from the colony, ant workers readily engaged in normal brood care behavior. We repeated this procedure with exactly the same eight ants per colony over five consecutive days, with two trials per day (morning and afternoon training session) on the first 4 days and one on the fifth day (morning session only), always using a new larva and arena, so that each ant was trained for nine consecutive trials (Fig. 1). Trials 1 and 9 could thus be compared without any potential confounding effect of time of day, which is a known factor influencing behavior (Blackmer and Byrne 2008). After each trial, the ants were placed back into their subcolony and left undisturbed until the next trial. We performed the experiments for the two subcolonies (sham control and fungus exposure) of each source colony at the same time and the eight experiments/ colonies in consecutive order within a period of 8 weeks.

Behavioral observations

"Naive ants" (trial 1) and "trained ants" (trial 9) were video-recorded immediately after the larvae were placed into the arena containing the ants (Logitech QuickCam Sphere AF). Videos were analyzed blindly with respect to larval treatment, using the software BioLogic (http:// sourceforge.net/projects/biologic/). We determined the time to first contact (mostly antennation behavior) of the ant to the larva, as well as the time to first performance of sanitary brood care (larval grooming; [START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF] after the start of the experiment. We also determined the frequency (number of events/hour) and duration of larval grooming and worker self-grooming during the complete hour of the trial. We did not detect any trophallaxis behavior between the adult ants and the larvae. In very rare cases, ants also showed mandible-opening behavior, which is generally classified as slightly aggressive behavior. Due to its rareness, it was not included in the analysis.

Conidiospore removal

To determine how many of the originally applied 500 conidiospores were removed by the ants during grooming, we washed off conidiospores from the surface of fungusexposed larvae (as in Hughes et al. 2002) after the 1-h contact to either naive (trial 1) or trained (trial 9) ants and counted them. To account for potential differences in fungal attachment to the larval cuticle [START_REF] Vestergaard | Light and electron microscopy studies of the infection of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae) by the entomopathogenic fungus Metarhizium anisopliae[END_REF], we determined the baseline number of conidiospores that could be washed off from additional fungus-exposed larvae for each source colony after 1 h in the absence of any ants, both at day 1 (trial 1) and day 5 (trial 9). Washes were performed by vortexing each larva for 2 min in 50-μl Triton X solution. After larval removal, the conidiospore suspension was conc e n t r a t e dt o1 0μl and the number of conidiospores determined by counting five droplets of 1 μl each per sample under a stereomicroscope (at×400 magnification).

Disease contraction by adult ants

At the end of the experiment, we determined whether adult ants had contracted the disease during contact with the larvae. To this end, all ants were killed by freezing for 6m i na t-20 °C, which does not affect conidiospore germination (M. Klatt and CW unpublished data). To check for internal fungal infection, each ant was surfacesterilized [START_REF] Lacey | Initial handling and diagnosis of diseased insects[END_REF] and monitored for outgrowth of M. robertsii hyphae and conidiospores over 3 weeks under humid conditions at 24 °C.

Data analysis

We used the program R (v. 2.12.1) for all statistical analyses.

Behavioral observations

We analyzed a mean of five (2-8) ants per colony and treatment, at both their first and ninth trial, to compare the same ant in its naive and trained state (total of 80 ants, 40 of which were trained to sham-treated larvae and 40 to fungus-exposed larvae; total 160 h of observations). Ninety-three percent (74/80) of the ants (37/40 in both groups) had contact to the larva in both trials 1 and 9 of the experiment, and only these ants were subjected to further statistical analysis. We compared the occurrence of larval grooming performed by naive and trained ants toward sham-treated and fungus-exposed larvae with Fisher exact tests and corrected the significance level α to 0.025 due to multiple testing (Bonferroni correction). We analyzed the time to first larval contact and the time delay between first contact and first grooming using survival analysis (Cox proportional regression, generating Wald statistics), as data were censored (R package "survival" (Therneau 2012)). Larval treatment (sham control vs. fungus exposure) and experience of the ants (trial 1 vs. 9) were included as fixed effects, with individual ants nested within their source colony as random effect using the frailty function in coxph. Non-significant interaction terms were removed from the final models. For behavioral frequencies and durations, we performed linear mixed-effects models (LMM) to analyze the effects of treatment (sham control vs. fungus exposure) and experience (trial 1 vs. 9) as fixed effects and their interaction, with individual ants nested within their source colony as random effect, thereby controlling for repeated measures of the same individual (R package "nlme" [START_REF] Pinheiro | nlme: Linear and Nonlinear Mixed Effects Models[END_REF]). Data either showed normal error distribution and homogeneity of variance or reached it by log(x+ 1)-transformation (applied for larval grooming frequency and frequency and duration of worker self-grooming). From the full model, the significance of the interaction and each fixed factor were tested by removing the factor of interest

Fungus exposure Sham control

Naive Conidiospore counts Trained Fig. 1 Experimental design. Platythyrea punctata ants were individually confronted with either one sham-treated control larva (blue group) or fungus-exposed larva (green group) in nine consecutive trials (each of 1 h). Behavioral observations were performed for naive ants at their first encounter with the respectively treated larva (trial 1; light colors) and for trained ants after repeated encounters (trial 9; dark colors). Conidiospore number of fungus-exposed larvae was counted after trial 1 and trial 9 to test for changes in conidiospore removal capacity of the ants due to experience (likelihood-ratio test) to obtain a minimal adequate model only containing significant terms.

Conidiospore removal

We tested for a potential difference in the basal fungal attachment rates in the absence of ants in trials 1 and 9 by performing a LMM with trial as fixed factor (trial 1: n=31, trial 9: n=37) and source colony as a random effect. Following, we determined whether the number of conidiospores washed off from fungus-exposed larvae depended on experience (trial 1 vs. 9) and grooming duration performed by the ants during the respective trial (n=24 ants, eachintrial1and9),byperformingaLMMincludingtrial as a fixed factor and grooming duration as covariate and individual ants and source colony as random effect. Conidiospore number was log-transformed to obtain a normally distributed error structure and homogeneity of variance. For display, we show the number of conidiospores removed by grooming (i.e., the applied 500 minus the count value of conidiospores still to be washed off after the experiment).

The data sets supporting the results of this article are available in the DRYAD repository (doi:10.5061/dryad. NNNNN).

Results

Performance of sanitary behavior

Occurrence of sanitary brood care Ninety-two percent of the ants that had larval contact performed larval grooming (trial 1: sham control: 36/37, fungus exposure: 32/37; trial 9: sham control: 36/37, fungus exposure: 33/37), with no difference between larval treatments (Fisher exact test with α-level of 0.025; sham control vs. fungus exposure: P=0.056) or experience level of the ants (trial 1 vs. trial 9: P=1.000).

Time to response Naive and trained ants (trials 1 and 9) of both treatment groups (sham control and fungus exposure) first approached larvae approximately 6 min after start of the experiment (Fig. 2a). Hence, there was no significant effect of treatment or experience in the time to first contact (Cox regression, treatment: Wald χ 2 =0.71, df=1, P= 0 . 4 0 0 ;t r i a l :W a l dχ 2 =0.33, df=1, P=0.560). Ants of all groups then started grooming approximately another 6 min after the first contact (Fig. 2b). There was thus also no effect of larval treatment or experience of the ant on the delay period between the first contact to the larva and the first grooming (treatment: Wald χ 2 =0.30, df=1, P= 0.580; trial: Wald χ 2 =0.60, df=1, P=0.440).

Frequency and duration of larval grooming The frequency of larval grooming did differ neither across treatments (sham control vs. fungus exposure) nor between naive and trained ants (trial 1 vs. 9; LMM: treatment: LR 1,4 =2.807, P=0.094; trial: LR 1,5 =0.118, P=0.731; interaction treatment*trial: LR 1,6 =0.0003, P=0.987; Fig. 3a). Grooming duration, however, was significantly affected by larval treatment (LMM: LR 1,5 =6.941, P =0.008) with sham-treated larvae being groomed longer than fungus-exposed larvae. Moreover, trained ants groomed the larvae longer than naive ants, such that ants in the sham control increased larval grooming duration from trial 1 to 9 by 14 % and ants in the fungus treatment even by 32 % (Fig. 3b;LR 1,5 =4.473, P=0.034). There was no significant interaction between treatment and trial (LR 1,6 = 0.253, P=0.615).

Frequency and duration of worker self-grooming The frequency of worker self-grooming was significantly affected by trial (LMM: trial: LR 1,4 =19.942, P<0.0001) with naive workers self-grooming more frequently than trained ants, irrespective of larval treatment (LMM: LR 1,5 =0.041, P= 0.840), with no significant interaction between treatment and trial (LR 1,6 =0.715, P =0.398; mean±SE in events/ hour; sham control: trial 1 56.6±3.5, trial 9 35.6±3.6; fungus exposure: trial 1 57.9±6.8, trial 9 34.2±2.9). Similarly, self-grooming duration was significantly affected by trial (LMM: LR 1,4 =4.683, P=0.031) with naive workers self-grooming longer than trained ants but remained unaffected by larval treatment (LMM: LR 1,5 =0.112, P = 0.738). Again, no significant interaction was found between treatment and trial (LR 1,6 =0.718, P=0.397; mean± SE in min; sham control: trial 1 5.9±0.7, trial 9 4.0±0.5; fungus exposure: trial 1 5.6±0.8, trial 9 4.6±0.7).

Conidiospore removal

We found that the basal attachment rate of the fungal conidiospores to the larvae, as measured in the absence of any workers, showed a non-significant trend of being higher in trial 9 vs. trial 1 (LMM: LR 1,4 =3.712, P=0.054). Despite this tendency toward stronger fungal attachment, trained ants removed a significantly higher number of conidiospores from the fungus-exposed larvae than naive ants (mean±SE of proportion conidiospores removed in trial 1 63.1±0.07 % and trial 9 76.2±0.05 %; LMM: LR 1,5 =4.243, P=0.039). Moreover, ants expressing longer larval grooming duration removed a higher number of conidiospores than ants that groomed the larvae shorter (Fig. 4;L M M :L R 1,5 = 20.937, P<0.0001). There was no significant interaction between the experience of the ants (trial 1 vs. 9) and their grooming duration (LR 1,6 =0.098, P =0.754), indicating that trained ants did not remove more conidiospores per unit time than naive ants.

Disease contraction by adult ants

None of the ants tending fungus-exposed larvae (n=40) showed any fungal outgrowth of M. robertsii after surface sterilization, and the same was true for the ants tending shamtreated larvae (n=40). We could therefore not detect any signs of internal infections of ants handling fungus-exposed brood.

Discussion

We tested whether repeated contact to either sham-treated or fungus-exposed larvae would affect the performance of sanitary brood care in the clonal ant P. punctata. The experience of individual ants did not affect their probability or time to respond. Independent of their experience and larval treatment, ants thus (i) were equally likely to contact and groom the larvae, (ii) showed no difference in time to detection (first contact; Fig. 2a) and reaction (first contact to first grooming; Fig. 2b), and (iii) performed larval grooming at equal frequencies (Fig. 3a). However, we found a plastic response for the duration of grooming behavior. Ants groomed sham-treated larvae longer than fungus-exposed ones and trained ants in their ninth trial performed longer grooming than naive ants in both treatments (Fig. 3b), thereby removing a larger number of infectious conidiospores from the brood (Fig. 4). Repeated brood care therefore led to a more effective anti-pathogen defense in P. punctata. Yet, this increased sanitary brood care came along with reduced individual worker hygiene, as trained ants performed less self-grooming.

Our results contrast to previous findings in Camponotus ants, where experience led to a faster response in brood Fig. 4 Conidiospore removal depending on larval grooming duration. Both naive (trial 1, light green) and trained ants (trial 9, dark green) removed significantly increasing numbers of conidiospores from the fungus-exposed larva with increasing larval grooming duration. Trained ants removed significantly more fungal conidiospores from the larvae than naive ants. Trend lines are shown for both groups (trial 1 and trial 9) in the respective color thermoregulation [START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF]). Yet, they are in line with previous studies on P. punctata and other ants thatwhilst not testing for experience but reaction to first pathogen contact-also found that ants did not differentiate between healthy and contaminated larvae during transport of larvae into their brood chamber [START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]Tragust et al. 2013b), even if the larval exposure dose used in the current experiment was thousandfold lower. Whereas it is known that social insects can contract Metarhizium infections from highdose-exposed nestmates [START_REF] Rosengaus | Pathobiology and disease transmission in dampwood termites [Zootermopsis angusticollis (Isoptera: Termopsidae)] infected with the fungus Metarhizium anisopliae (Deuteromycotina: Hypomycetes)[END_REF]Hughes et al. 2002;K o n r a de ta l .2012), none of the ants in our low-dose experiment showed any signs of infection with M. robertsii or died from the disease.

Many studies report an upregulation of grooming in response to pathogen exposure (adults: Rosengaus et al. 1998;Jaccoud et al. 1999;[START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF][START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF]brood: Ugelvig et al. 2010;Tragust et al. 2013b;fungus garden: Currie and Stuart 2001). The lower grooming of fungus-exposed brood was therefore unexpected, particularly since P. punctata ants do show increased grooming of Metarhizium-exposed brood when the applied exposure dose is thousandfold higher and the ants are in a colony context rather than in isolation (Tragust et al. 2013b). This suggests that the reduction in grooming toward fungus-exposed compared to sham-treated larvae in our experiment likely is a result of either the low exposure dose or the fact that ants have been removed from their colony context to be trained individually. Our experimental setting may thus have rather reflected the situation of an ant encountering the larvae outside of the nest, where avoidance of infectious items is common in social insects (Epsky and Capinera 1988;Diehl-Fleig and Lucchese 1991;[START_REF] Mehdiabadi | Colony-level impacts of parasitoid flies on fire ants[END_REF]Fouks and Lattorff 2011).

Despite these different basal grooming levels in the two treatments, brood grooming duration significantly increased by repeated brood care for both the sham-treated larvae (by 14 %) and the fungus-exposed larvae (by 32 %; Fig. 3b), whilst worker self-grooming was reduced. The absence of a significant interaction between trial and treatment for grooming duration indicates that both treatment groups reacted similarly to experience and that the presence of a pathogen neither caused nor interfered with prolonged brood grooming. It thus seems that experienced ants prophylactically upregulate their expression of sanitary brood care at the expense of individual hygiene. It was shown that ant colonies that have previously encountered Metarhizium fungi increase allogrooming toward adult nestmates that are either shamtreated [START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF] or fungus-exposed [START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF]) and fungus-growing ants whose fungus garden has experienced pathogen threat increase self-grooming [START_REF] Morelos-Juárez | Ant farmers practice proactive personal hygiene to protect their fungus crop[END_REF]. The novelty of our study is that P. p u n c t a t a ants intensify brood grooming after repeated brood care even in the absence of any previous pathogen encounter, thereby deviating from Pheidole ants, where nursing-experienced individuals did not perform more brood care than their naive nestmates [START_REF] Muscedere | Brood-care experience, nursing performance, and neural development in the ant Pheidole dentata[END_REF]).

While we can exclude that the presence of the fungal pathogen M. robertsii at the used low exposure level triggered the prolonged larval grooming by experienced ants, we cannot disentangle whether the intensified grooming may have been caused by repeated confrontation with the brood per se or with the detergent Triton X. The detergent is required to bring the hydrophobic fungal conidiospores into suspension and was thus the closest control to our fungus-exposure treatment. Some studies have reported that treatment with a detergent elicits the same grooming intensity as fungus exposure (Graystock and Hughes 2011;[START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF]Tragust et al. 2013b; but see, e.g., Rosengaus et al. 1998;U g e l v i g and Cremer 2007; [START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF][START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]Tragust et al. 2013b), suggesting a potential effect of the detergent on the performance of sanitary behaviors. Intensified grooming in our experiment could thus be the result of either repeated nursing itself or repeated contact to a potentially irritating compound that may even act as a "danger signal" [START_REF] Matzinger | Tolerance, danger, and the extended family[END_REF], causing potential damage to the host cells through its detergent properties. Our study moreover revealed that fungal attachment might be affected by the storage time of the conidiospore suspension, highlighting that this should be strictly controlled for in experimental work. The cause may be an increase in RNA and protein synthesis during soaking, leading to faster swelling and germination when later coming in contact with the insect cuticle (Dillon and Charnley 1985;Hassanetal.1989;Dillon and Charnley 1990).

As expected from previous work (Rosengaus et al. 1998;Hughes et al. 2002;[START_REF] Yanagawa | Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi[END_REF]Tragust et al. 2013a), ants removed higher conidiospore numbers when grooming the larvae longer (Fig. 4). Experienced ants, which increased larval grooming duration (Fig. 3b), thus removed the fungal pathogen more effectively than naive ants. Such improved performance through experience is not self-evident, given that, e.g., honeybee undertakers did not improve in cadaver removal with experience (Trumbo and Robinson 1997) and brood-care-experienced Pheidole dentata ants were not more efficient nurses [START_REF] Muscedere | Brood-care experience, nursing performance, and neural development in the ant Pheidole dentata[END_REF]). Higher conidiospore removal by brood-care-experienced individuals is likely key to reduce the infection probability of exposed larvae (as is known for adults; Walker and Hughes 2009), as well as the transmission of the pathogen within the colony. Furthermore, social immunity, i.e., sanitary care by nestmates, can be beneficial for the helper, as it can bestow a survival benefit upon future exposure to the same pathogen [START_REF] Traniello | The development of immunity in a social insect: evidence for the group facilitation of disease resistance[END_REF][START_REF] Ugelvig | Social prophylaxis: group interaction promotes collective immunity in colonies[END_REF]Hamiltonetal.2011;[START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF].

We can conclude that repeated brood care in P. punctata ants modulates their brood care activities, increasing their potential to fight fungal disease. As experience-dependent behavioral modulation is reversible and for instance depends on the time delay between task performances [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF], absence of stimuli could again lead to decreased intensity of brood care. Such experience-modulated expression of sanitary brood care likely allows colonies to respond flexibly to incoming pathogens. Response threshold models postulate that inter-individual variation in response thresholds for taskassociated stimuli can lead to division of labor in the colony and individuals with lower response thresholds are more likely to become specialists for the task (reviewed in Beshers and Fewell 2001). Task specialization (i.e., the tendency of some workers to perform tasks more frequently or longer than their nestmates) may be more pronounced in larger ant societies (Bourke 1999;[START_REF] Thomas | Colony size affects division of labour in the ponerine ant Rhytidoponera metallica[END_REF]Jeanson et al. 2007) and also in societies with higher genetic diversity (reviewed by [START_REF] Oldroyd | Genetic diversity promotes homeostasis in insect colonies[END_REF]). Yet, already in the absence of large colonies and genetic variation, as in the studied clonal ant P. punctata, repeatedly performed brood care makes ants better caretakers and may promote divergence between group members.

ABSTRACT

A major cost of social life is an increased exposure to parasites, leading to the evolution of sophisticated social immune defenses, which complement the physiological immune system of social insects. Decomposing cadavers pose a special danger to eusocial societies, as they exist in densely populated, enclosed and perennial nests with a high genetic proximity between individuals. To prevent parasite establishment within them, social insects evolved cadaver management behaviors, including cadaver avoidance, grooming, carrying, burial and necrophoresis (the removal of cadavers from the nest). Workers managing cadavers might be at higher risk of contracting disease due to potential parasite contact and leaving the confines of the nest. In this study I investigated whether there is division of labor for cadaver management and whether task allocation is affected by recent individual experience and worker size in the thermophilic ant Cataglyphis velox. Recurrent introduction of four nestmate cadavers of different size into nests of cadaver groups in ten trials demonstrated that nestmates were significantly skewed in the performance of cadaver management with few individuals dominating task performance. Many task performers performed the behaviors only once over all trials. Individuals performing one type of cadaver management were also more likely to perform the other and cadaver transporters showed a high propensity to also engage in foraging. Cadaver size did not modulate cadaver management behaviors. The repeated occurrence of cadavers did not affect the number and duration of groomings and transports a cadaver received, but lead to an increasing number of grooming individuals per cadaver. When reunited with less experienced individuals, the probability of experienced individuals to groom and transport a cadaver was increased in only one out of four colonies. Together, these results suggest low division of cadaver management between nestmates and a reduced modulation of these behaviors by recurrent cadaver exposure.

INTRODUCTION

Division of labor is a key feature of social insects and largely contributes to their ecological success [START_REF] Oster | Caste and ecology in the social insects[END_REF]Hölldobler and Wilson 1990). It occurs when individuals specialize on different colony tasks, such as foraging and brood care. A central question about division of labor is how individuals decide what task to execute [START_REF] Gordon | The organization of work in social insect colonies[END_REF]. The concept of self-organization assumes that division of labor is an emergent property of interacting individuals which obey simple behavioral rules [START_REF] Bonabeau | Self-organization in social insects[END_REF][START_REF] Jr | Self-organization and the evolution of division of labor[END_REF]; reviewed e.g. in [START_REF] Theraulaz | The formation of spatial patterns in social insects: from simple behaviours to complex structures[END_REF][START_REF] Duarte | An evolutionary perspective on self-organized division of labor in social insects[END_REF]. Social insect workers are assumed to engage in a task when the task-associated stimulus exceeds their internal response threshold (response threshold models reviewed in Beshers and Fewell 2001) and thresholds differ among nestmates (e.g. [START_REF] Bonabeau | Quantitative study of the fixed threshold model for the regulation of division of labour in insect societies[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Duong | Ventilation response thresholds do not change with age or selfreinforcement in workers of the bumble bee Bombus impatiens[END_REF]. Inter-individual variability in response thresholds can arise through several factors, such as genotype [START_REF] Pankiw | The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.)[END_REF][START_REF] Kryger | Genotypical variability for the tasks of water collecting and scenting in a honey bee colony[END_REF][START_REF] Masterman | Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)[END_REF][START_REF] Jones | Honey bee nest thermoregulation: diversity promotes stability[END_REF][START_REF] Scheiner | Effects of patriline on gustatory responsiveness and olfactory learning in honey bees[END_REF], age [START_REF] Pankiw | The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.)[END_REF], rearing conditions [START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF]) and experience (e.g. [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF]) and enables a graded and flexible colony response adjusted to current task needs. Individuals with the lowest response thresholds for a task are assumed to engage in task performance more frequently and thereby become task specialists (e.g. [START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF], reducing stimulus levels so that response thresholds of their nestmates will not be met. With increasing stimulus levels (due to more frequent or bigger task loads), task specialists become insufficient to reduce stimulus levels and individuals with higher response thresholds will also engage in task performance (Beshers and Fewell 2001). Task specialization presumably increases colony-level efficiency by reducing costs associated with task switching [START_REF] Wilson | Behavioral discretization and the number of castes in an ant species[END_REF][START_REF] Jeanne | The organization of work in Polybia occidentalis: costs and benefits of specialization in a social wasp[END_REF][START_REF] Goldsby | Task-switching costs promote the evolution of division of labor and shifts in individuality[END_REF] Experience is an additional factor which can lead to short-or long-term modulation of individual behavior and thereby affect division of labor within the colony (e.g. [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF][START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Workers can acquire experience either passively through repeated exposure to a stimulus, or actively through performance of the corresponding task. The acquired experience can then either modulate task performance itself (i.e. behavioral speed, efficiency, precision or location) and/or affect individual responsiveness towards a taskassociated stimulus [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Parameters of individual responsiveness comprise an individual's response threshold, its probability to respond once the threshold is reached, the time an individual takes to respond, the duration of response and the intensity (or efficiency) of the response, which can be independent processes [START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF][START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Successful task performance can provide a positive feedback factor, leading to selfreinforcement and increasing the probability of an individual to repeat the task, thereby promoting specialization (e.g. [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF]). In the ants Temnothorax albipennis and Cerapachys biroi, foraging success increased the probability of individuals to repeat this task [START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF][START_REF] Robinson | Experience, corpulence and decision making in ant foraging[END_REF]) and thereby impacted colony organization [START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF]. Theoretical models predict a modulation of response thresholds through recent experience ('threshold reinforcement'; [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF]), but empirical evidence is scarce and conflicting [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Whereas fanning response thresholds decreased with repeated task performance in thermoregulating workers of the bumblebee Bombus terrestris [START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF], a modulation which depends on the time interval between task performances [START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF], fanning response thresholds were not affected by experience in the bumblebee Bombus impatiens [START_REF] Duong | Ventilation response thresholds do not change with age or selfreinforcement in workers of the bumble bee Bombus impatiens[END_REF].

A major cost of social life is an increased exposure to parasites due to geographic and genetic proximity between frequently interacting individuals in enclosed and perennial nests under homeostatic conditions (e.g. [START_REF] Shykoff | Parasites and the advantage of genetic variability within social insect colonies[END_REF][START_REF] Rosengaus | Pathobiology and disease transmission in dampwood termites [Zootermopsis angusticollis (Isoptera: Termopsidae)] infected with the fungus Metarhizium anisopliae (Deuteromycotina: Hypomycetes)[END_REF][START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF]Cremer et al. 2007;[START_REF] Fefferman | Disease prevention and resistance in social insects: modeling the survival consequences of immunity, hygienic behavior, and colony organization[END_REF]). To counter the selection pressure imposed by parasites, social insects evolved sophisticated group-level defenses which comprise behavioral, spatial and physiological mechanisms, complementing the individual physiological immune system (i.e. 'social immunity': Cremer et al. 2007;[START_REF] Spivak | Genetic, individual, and group facilitation of disease resistance in insect societies[END_REF][START_REF] Evans | Socialized medicine: individual and communal disease barriers in honey bees[END_REF][START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]. Some defense mechanisms are prophylactic to avoid the establishment of parasites within the nest, such as the incorporation of antimicrobial plant resin into nests of ants and honeybees [START_REF] Christe | Evidence for collective medication in ants[END_REF][START_REF] Chapuisat | Wood ants use resin to protect themselves against pathogens[END_REF][START_REF] Simone | Resin collection and social immunity in honey bees[END_REF]) or cadaver management. Individuals which have died of age or from infection within the nest pose a sanitary risk to nestmates, as they might grow bacteria and fungi under the often warm and humid nest conditions. Waste material is harmful to leaf-cutting ants and their mutualistic fungus [START_REF] Fisher | Microfungi in the fungus gardens of the leaf-cutting ant Atta cephalotes: a preliminary study[END_REF][START_REF] Bot | Waste management in leaf-cutting ants[END_REF]Hughes et al. 2004;[START_REF] Brown | Mortality rates and division of labor in the leafcutting ant, Atta colombica[END_REF] and Myrmica rubra ant workers suffer increased mortality if they cannot remove their dead [START_REF] Diez | Keep the nest clean: survival advantages of corpse removal in ants[END_REF]. Social insects thus evolved cadaver management behaviors (reviewed in Sun and Zhou 2013), such as cadaver avoidance (e.g. [START_REF] Kramm | Termite pathogens: transfer of the entomopathogen Metarhizium anisopliae between Reticulitermes sp. Termites[END_REF][START_REF] Franks | Tomb evaders: house-hunting hygiene in ants[END_REF], grooming [START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF][START_REF] Visscher | The honey bee way of death: Necrophoric behaviour in Apis mellifera colonies[END_REF][START_REF] Neoh | Do termites avoid carcasses? Behavioral responses depend on the nature of the carcasses[END_REF], intranidal transport [START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF][START_REF] Visscher | The honey bee way of death: Necrophoric behaviour in Apis mellifera colonies[END_REF]Sun et al. 2013), burial [START_REF] Renucci | Complex undertaking behavior in Temnothorax lichtensteini ant colonies: from corpse-burying behavior to necrophoric behavior[END_REF]Sun et al. 2013), cannibalism (especially common in termites: [START_REF] Neoh | Do termites avoid carcasses? Behavioral responses depend on the nature of the carcasses[END_REF]) and necrophoresis (also called 'undertaking', i.e. the removal of cadavers from the nest). Necrophoresis, which is most prevalent in ants and honeybees, has been studied since over 50 years (e.g. in ants: [START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF][START_REF] Gordon | Dependence of necrophoric response to oleic acid on social context in the ant, Pogonomyrmex badius[END_REF][START_REF] Ataya | Le comportement nécrophorique chez la fourmi Lasius niger L[END_REF][START_REF] Julian | Undertaking specialization in the desert leaf-cutter ant Acromyrmex versicolor[END_REF]in honeybees: Visscher 1983;Trumbo et al. 1997;[START_REF] Breed | Demand for task performance and workforce replacement: Undertakers in honeybee, Apis mellifera, colonies[END_REF]. Research has focused on the description of the behavior, on death recognition cues and task allocation, mainly in highly evolved social insect species with large colony sizes, such as honeybees and leaf-cutting ants. Less information is available on cadaver-management behaviors which occur before cadavers are either rejected from or buried within the nest.

Behavioral processes directed towards the dead differ between social insect species, depending on nest ecology and feeding habits [START_REF] Neoh | Do termites avoid carcasses? Behavioral responses depend on the nature of the carcasses[END_REF]. But plasticity in cadaverdirected behaviors also exists within a single colony, depending on the quantity of the cadavers present [START_REF] Breed | Demand for task performance and workforce replacement: Undertakers in honeybee, Apis mellifera, colonies[END_REF] Diez et al. 2013a) and their motoric complexity (i.e. whether an individual is able to transfer motor skills from a non-sanitary task) or in the level of physiological adaptations required for task performance. We expect stronger division of labor between nestmates for tasks which occur frequently, involve a big workload (Diez et al. 2013a) and a high exposure risk or require physiological specializations of task performing individuals. Division of labor is further expected to increase with genetic diversity of the workforce (reviewed in [START_REF] Oldroyd | Genetic diversity promotes homeostasis in insect colonies[END_REF], colony size (Bourke 1999;[START_REF] Thomas | Colony size affects division of labour in the ponerine ant Rhytidoponera metallica[END_REF]Jeanson et al. 2007) and spatial complexity [START_REF] Tofts | Doing the right thing: Ants, honeybees and naked mole-rats[END_REF][START_REF] Bourke | Social Evolution in Ants[END_REF] which determines at what rate and in which intensity nestmates are exposed to stimuli. Some of these conditions are found in colonies of the highly evolved leaf-cutting ants and honeybees, for which worker specialization on necrophoresis and waste management has been reported and seems to be influenced by age, size and genotype of individuals (e.g. [START_REF] Visscher | The honey bee way of death: Necrophoric behaviour in Apis mellifera colonies[END_REF]; Robinson andPage 1988, 1995;Trumbo et al. 1997;[START_REF] Julian | Undertaking specialization in the desert leaf-cutter ant Acromyrmex versicolor[END_REF][START_REF] Julian | Genetic variation and task specialization in the desert leaf-cutter ant, Acromyrmex versicolor[END_REF][START_REF] Ballari | Waste management in the leaf-cutting ant Acromyrmex lobicornis: Division of labour, aggressive behaviour, and location of external refuse dumps[END_REF]. Waste workers of leaf-cutting ants are not only spatially segregated, but also socially isolated, which is reinforced through aggressive interactions [START_REF] Hart | Task partitioning, division of labour and nest compartmentalisation collectively isolate hazardous waste in the leafcutting ant Atta cephalotes[END_REF][START_REF] Ballari | Waste management in the leaf-cutting ant Acromyrmex lobicornis: Division of labour, aggressive behaviour, and location of external refuse dumps[END_REF]). To my knowledge, no reports on cadaver management specialization exist to date in termites (Sun and Zhou 2013) and how cadaver management is allocated among workers in "more basic" ant species remains to be investigated (but see Diez et al. 2013a). It is also not well understood how experience in sanitary tasks affects the performance of sanitary behaviors in social insect colonies.

Experience could improve sanitary task performance (e.g. [START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF][START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF][START_REF] Westhus | Increased grooming after repeated brood care provides sanitary benefits in a clonal ant[END_REF]) by (i) reducing individual response thresholds (enabling individuals to react sooner towards a sanitary threat), (ii) increasing their response probability (making it more likely for them to perform the sanitary task once their response threshold is reached), (iii) leading to a faster transition from perception of the necrophoric stimulus to the final response, (iv) increasing response duration [START_REF] Westhus | Increased grooming after repeated brood care provides sanitary benefits in a clonal ant[END_REF] or through (v) improved motoric capacities (e.g. more efficient grooming of fungal spores or preening of nematodes). In the leaf-cutting ant Acromyrmex echinatior, a previous encounter with a pathogenic fungus resulted in individuals being more likely to allogroom re-introduced fungus-exposed nestmates, thereby increasing their likelihood of survival, compared to controls in naïve nests [START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF]. Repeated brood care resulted in longer grooming duration and thereby higher removal of a pathogenic fungus in the ant Platythyrea punctata [START_REF] Westhus | Increased grooming after repeated brood care provides sanitary benefits in a clonal ant[END_REF]. The previously mentioned behavioral modifications by experience could increase the performance of cadaver management by experienced individuals, leading to individual specialization and division of labor for the management of cadavers. These individuals might also become more efficient in cadaver management through repeated task performance. Individuals specialized on necrophoric behavior removed cadavers faster and more successfully than less experienced individuals (Apis mellifera: Trumbo and Robinson 1997; Acromyrmex versicolor: Julian and Cahan 1999), but an effect of experience was not demonstrated (Trumbo and Robinson 1997). However, if the occurrence of intranidal cadavers is rare and infrequent, which is expected in healthy colonies (Diez et al. 2013a), only a short-term modulation of cadaver management is expected. Indeed, in the ant Myrmica rubra, short-term spatial memory of necrophoric individuals was demonstrated within a time frame of 7 minutes [START_REF] Diez | Orientation in corpse-carrying ants: memory or chemical cues?[END_REF]. As the modulation of response thresholds depends on the time interval between task performances [START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF], increased time intervals will presumably not lead to self-reinforcement when the task is successfully performed. Even though cadaver management presumably occurs with low frequency within a healthy colony, the sanitary risk imposed by cadavers (e.g. [START_REF] Diez | Keep the nest clean: survival advantages of corpse removal in ants[END_REF] might have a strong impact on colony fitness, especially under parasitic attack.

I studied cadaver management in the ant Cataglyphis velox, Santschi, 1929. Intranidal cadaver management could be of importance for these ants inhabiting extreme thermal environments (maximal activity temperature 46 °C: reviewed in Cerdá 2001; reviewed in [START_REF] Lenoir | Cataglyphis desert ants: a good model for evolutionary biology in Darwin's anniversary year: a review[END_REF] with increased extranidal mortality risk. Additionally, their diet consisting of fresh cadavers of small arthropods [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF]) might lead to increased cadaver management behaviors. This species further offers polygynous colonies, allowing the establishment of queenright subcolonies for empirical studies. Workers are highly polymorphic, enabling division of labor in foraging with the biggest workers foraging during the hottest hours of the day [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF][START_REF] Cerdá | Behavioural and physiological traits to thermal stress tolerance in two Spanish desert ants[END_REF]. Cataglyphis species generally show division of labor based on age, with workers passing from intranidal tasks when young to foraging when older (reviewed in [START_REF] Lenoir | Cataglyphis desert ants: a good model for evolutionary biology in Darwin's anniversary year: a review[END_REF]).

I studied necrophoresis (i.e. the removal of cadavers from the nest) but also cadaver grooming and intranidal transports of cadavers, which have rarely been addressed in current literature. Nestmate cadavers present a natural stimulus and stimulus quantity can easily be manipulated. My objectives were: (i) to determine whether division of labor exists for cadaver management within the colony by providing eight cadavers per day over a period of 5 days, (ii) to evaluate the relative influence of both worker and cadaver size on cadaver management and (iii) to test whether worker experience affects cadaver management by comparing workers from subcolonies that have been differentially exposed to cadavers. I predicted that if cadaver management were a result of stimulus exposure (i.e. opportunity) or recent task performance, individuals from subcolonies which were recently exposed to cadavers would be more likely to perform cadaver management than individuals from subcolonies without this exposure. Moreover, bigger cadavers might provide a stronger stimulus (e.g. because of an increased surface area), potentially eliciting stronger cadaver management behaviors and faster removal from the nest.

MATERIALS AND METHODS

Laboratory colonies

Four polygynous colonies of Cataglyphis velox were collected from a Sierra Nevada population near Grenada, Spain in May 2011 (37°08' N, 3°29' W, 1300 meters altitude).

Colonies occupied an underground nest containing from 700 to 1900 workers and six to 11 queens. They were reared in a climate chamber under LD 12 : 12 h photoperiod, at 35-50% relative humidity and at a mean temperature of 28°C (24.4-29.9°C) over a period of 2 months and fed 3 times/week with a mixed diet of frozen crickets, live meal worms, fruit (apple, orange, banana), and ad libitum sugar and water. Except cadaver obtention, experimental steps were performed in the same climate chamber.

Establishment of subcolonies

Eight weeks after collection, two subcolonies were established from each of four stock colonies (four treatment replicates). Subcolonies contained one queen, 100 workers and few pupae. In C. velox, workers exhibit strong and continuous size variation from 4.5 to 12 mm, but can be grouped into four size classes for experimental convenience [START_REF] Tinaut | Taxonomic situation of the genus Cataglyphis Förster, 1850 in the Iberian Peninsula II. New position for C. viatica (Fabricius, 1787) and the redescription of C. velox Santschi, 1929 stat. n. (Hymenoptera, Formicidae)[END_REF][START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF]. I kept the proportion of each size class constant in the eight subcolonies based on their mean proportion estimated from three different stock colonies (very small workers: 23%; small workers: 25%; medium workers: 32%; large workers: 20%). These proportions were similar to the ones detected by [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF]. At the time of subcolony establishment, workers of the two subcolonies were individually marked (Uni Paint Marker) with a unique color combination on the thorax and gaster. Very small workers (4.5 -6 mm body length) could not be color-marked due to practical limitations such as difficulties of color identification on computer screens and during scans as well as successful color removal by ants when color spots were very small. Even though the smallest workers were too small for individual observations but still comprised 23% of the colony, they were deliberately kept in the colony to not disturb worker size distribution and division of labor within the group.

The eight subcolonies were maintained in plastic boxes (37 x 27 x 11 cm) lined with plaster and coated with fluon, containing a plastered petri-dish nest (diameter 9 cm) allowing direct observations of intranidal activities. Four holes were drilled through the top of the petri dish nest to introduce cadavers in the nests with minimal nest disturbance during the experiment. Subcolonies of the same stock colony were randomly assigned to either control (without cadavers) or cadaver group.

Experimental procedure

The experiment was divided into two steps (Fig. 1). The first step of five days consisted of two trials per day, one in the morning and one in the afternoon (with at least 1.5 hours between the morning and afternoon trial). At the beginning of each trial, I placed four cadavers (one of each size class, originating from the same stock colony) into the nest of the cadaver groups. The cadavers were inserted in the nest centre through holes in the petri-dish cover using forceps. To control for potential stress effects due to forceps insertion, the same forceps were similarly inserted into the nest of the control groups but without the introduction of cadavers. Nest areas of cadaver groups were filmed from cadaver insertion until all cadavers had been taken out, or until a maximum filming duration of three hours (Sony HDR-XR520VE camera). After the last daily trial, cadavers were removed from the foraging arena of cadaver groups to avoid unobserved cadaver transport within the foraging arena. The subcolonies were observed after food was given for 10 minutes after the last trial of each experimental day and during one week after subcolony establishment (in total 9-12 times per group) to identify foragers. The second step was to reunite the control and cadaver groups of the same stock colony. Before reunification, I placed them into a refrigerator at 4°C for 1.5 hours to decrease aggression towards introduced ants. For two colonies (B and C), I transferred the control group into the box of the cadaver group. The reverse was done for the two other colonies (A and D). I preferred not to use a new box as colony installation into a previously unused nest takes time and would not have allowed us to perform the observation just after reunification. The reunited colonies were left undisturbed the following day, during which the ants present outside the nest were noted three times (morning, noon and afternoon) and foragers were observed during one 10 min interval to determine whether both splits groups were equally well established within the nest. On the following day, I introduced eight cadavers into the reunited nests in two trials in the same way as in the first step of the experiment. The nests were video-recorded and the videos later analyzed according to the first experimental step.

Up to five ants died in the subcolonies (cadaver groups A: 3, B: 3, C: 1, D: 2, control groups A: 0, B: 5, C: 3, D: 3) after marking and before the first experimental step (during 2.5 weeks) and a maximum of two dying ants (control groups A: 1, B: 0; C: 2, D: 2; cadaver groups A: 0, B: 0; C: 1, D: 2) were recorded during the first step of the experiment (5 days). These comprised marked ants and emerging callows and were soon after occurrence removed. The insertion of 40 nestmate cadavers during the first step of the experiment thus increased the daily occurrence of cadavers (0.147 cadavers naturally occurred in all subcolonies daily) by 5574 % in cadaver groups.

Cadaver obtention

To obtain cadavers, 100 workers (25 per size class) of each field colony were killed by freezing (10 min at -20 °C) at the time of colony splitting. They were deposited in an open plastic box in a climate chamber (22 -25 ºC, 50 -80 % humidity) for 13 days, until the start of the experiment. At this point, cadavers were transferred to a refrigerator (4 °C) to avoid stimulus change over the course of the experiment.

Size measurements

The body size of an individual was estimated using the tibia length of the right posterior leg as described in [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]. Tibias were photographed with a Sony XCD-SX910CR camera connected to a binocular microscope and tibia length was measured with the ImageJ software (version 1.40, Wayne Rasband, National Institutes of Health, USA).

Video analysis

During video analysis, the identity of worker ants and the duration of their behavior directed towards each cadaver were noted. As every cadaver belonged to a different worker size class (1-4), they could be followed individually. I noted cadaver grooming (licking a cadaver with mouthparts) and intranidal cadaver transport (lifting or dragging a cadaver with mandibles and displacing it) during a maximum of 30 min after cadaver insertion. Necrophoric events were noted during the whole observation time (180 min). Once the cadaver was transported out of the nest, it was no longer considered as the video recorded only the nest area.

Data organization and statistical analysis

The worker force was divided into four task groups: foragers, cadaver groomers, cadaver transporters and other workers. Foragers were defined as ants that had been observed feeding in the arena. Cadaver groomers or transporters were observed grooming or transporting (within and outside of the nest) a cadaver at least once during the 10 trials and are termed cadaver-managing individuals. Cadaver transporters contained both necrophoric workers (individuals which transported a cadaver outside the nest) and intranidal cadaver transporters because (i) intranidal chambers comprising cadavers were observed in the field, (ii) several cadavers were deposited onto an intranidal waste pile in colony D, (iii) 59% of necrophoric workers (cadaver group A: 42.9 %, B: 55.6 %, C: 87.5 %, D: 50 %) also performed intranidal transports and (iv) only few necrophoric workers were observed during the first experimental step (cadaver group A: 7, B: 9, C: 8, D: 4 workers), preventing separate statistical analyses.

Other ants which performed neither foraging nor cadaver management are termed "other workers". Each subcolony contained 77 marked workers and the percentages given are based on these workers.

Unmarked individuals performed 8.9 % necrophoric events during the first experimental step (cadaver group A: 32.4 %, B: 0 %, C: 3.4 %, D: 0 %). Whereas in colonies B and C nearly all cadaver transports (97.2 %) and groomings (98.7 %) in the first experimental step were performed by marked individuals, unmarked individuals performed 34.9 % cadaver transports (intranidal and necrophoric transports) and 11.9 % groomings in cadaver groups A and D.

Slightly aggressive behavior was directed towards the cadavers in that some individuals were observed standing in the vicinity of the cadavers showing mandible opening behavior during the whole observation time. Infrequently, cadaver biting occurred, especially during and right after cadaver insertion into the nests. Aggressive acts directed towards cadavers during and after insertion were probably elicited by the introduction of a foreign object and were not necessarily linked to the fact that this object was a cadaver. Two cadavers (1.4 %, one in each cadaver group A and B) were dismembered during the experiment.

Pattern of cadaver removal.

I analyzed the time to cadaver removal using survival analysis (parametric survival regression model), as data were censored (R package "survival" [START_REF] Therneau | A Package for Survival Analysis in S. R package version[END_REF]). The size of the cadaver (factorial), cadaver group (factorial) and trial were included as fixed effects. Nonsignificant interaction terms were removed from the final models.

Performance of cadaver management. I analyzed the performance of cadaver management from two different perspectives, from the perspective of a worker performing the tasks and from the perspective of a cadaver receiving the behavioral acts.

Worker level: To address whether nestmates differed in the total number of cadaver-related behaviors (i.e. an individual's absolute number of cadaver-related behaviors within the 30 min observation time, combined for all trials), I analyzed whether (i) all workers of the cadaver group and (ii) task-performers were skewed (i.e. unequal sharing of a task among individuals) in the number of cadaver groomings and transports (intranidal and necrophoric) they performed. I used the program Skew Calculator 2003 by Peter Nonacs (https://www.eeb.ucla.edu/Faculty/Nonacs/PI.html). Estimations were based on 1000 runs and the significance level of alpha was set to be 0.05% in two-tailed tests to determine the confidence interval (CI). I determined the factors associated with the performance of cadaver management using Generalized Mixed Models (LMER) to account for pseudoreplication based on different cadaver groups and binomial distributed errors (family = binomial). I tested whether the binary response variables grooming, transporting or foraging (individuals performed these tasks or not) depended on worker size and on the performance of the other two behaviors grooming, transporting or foraging (as binary covariates).

Cadaver level:

To analyze whether cadaver size and trial affected cadaver management by workers, I used Linear Mixed Effects Models (LMM) and analyzed whether the number or duration of cadaver groomings and transports a cadaver received or the number of task performers per cadaver depended on the covariates cadaver size (factorial), trial (numeric), duration the cadaver stayed in the nest (or until 30 min) and number of task performers. I controlled for pseudoreplication by including cadaver group as a random effect. Response variables were log-transformed and insignificant terms were subsequently removed from the full models.

Reunification: treatment effect.

After reunification, workers of the cadaver and of the control group were expected to be present in the colony boxes and forage with the same probability of 50%. To analyze whether the origin of the colony box (whether it previously housed the cadaver or control group) affected this ratio, a chi-squared test was performed per colony on the observed number of individuals, comparing the cadaver and control group. During the experiment, some workers (12 %) were not attributable to a subcolony due to partial color loss or marking errors. Partly unmarked individuals, which performed cadaver-directed behaviors during the reunited trials, could not be clearly assigned to a subcolony (even if already observed performing cadaverdirected behaviors in the cadaver group in the first experimental step) and were grouped separately in the analysis. I further analyzed in each colony whether the number of cadavergrooming and cadaver-transporting (intranidal and necrophoresis) individuals differed between the cadaver and the control group and between the cadaver group and workers which could not be assigned to either subcolony, using an exact binomial test.

All data gathered was analyzed using R (version 2.11.1).

RESULTS

Pattern of cadaver removal

In the first step of the experiment (Fig. 1), over all 10 trials, a total of 40 cadavers were inserted into the nest of a cadaver group. Due to technical difficulties (e.g. electricity shortings in the laboratory), video data could not be obtained for trial 3 in cadaver groups A, C and D (36 cadavers observed) and for trials 3 and 4 in cadaver group B (32 cadavers observed). Soon after cadaver insertions, cadavers were antennated very frequently by at least ten workers, which ceased after several minutes. Nearly all inserted cadavers (85 %) were removed from the nests within the observation period (180 min) except in one cadaver group (D) in which only 11 % of the cadavers were removed (Table 1). In this colony, cadavers were deposited next to a pile of food waste and pupal exuviae within the nest. On one occasion, a cadaver was brought back into the nest of cadaver group A. In the four cadaver groups, necrophoresis occurred between 32 sec to 30 min 32 sec (Table 1). The time elapsed to cadaver removal did not differ according to cadaver size (survival analysis, chi-two = -0.598, df = 3, p = 0.897) and did not change over the 10 trials (survival analysis, chi-two = -0.005, df = 1, p = 0.944). However, it significantly varied among colonies (survival analysis, chi-two = -93.289, df = 3, p < 0.0001). The significant effect of colony was still observed even after removing the peculiar colony D (survival analysis, chi-two = -10.589, df = 2, p = 0.005). Groomings and transports received by cadavers

The number of groomings cadavers received did neither change over trial, nor was it affected by cadaver size or by the duration a cadaver stayed in the nest (LMM, Table 2). Cadavers were groomed more often if they elicited grooming in a higher number of different grooming individuals (Table 2, Fig. 2, LMM, coefficient = 0.331). The number of transports also significantly increased with an increasing number of transporting individuals (Table 2, Fig. 2, LMM, coefficient = 0.134). Trial, cadaver size and the duration a cadaver stayed in the nest did not affect the number of transports received (Table 2).

The number of workers which groomed cadavers increased with trial ( The results for the duration of groomings and transports a cadaver received are qualitatively the same compared to the number of these behaviors (Table 2), except that the duration of cadaver transports was negatively affected by the duration a cadaver stayed in the nest (Table 2, LMM, coefficient: -0.001). Who is performing cadaver-related tasks?

At the colony level

The percentage of foragers was on average of 43.3 % in all subcolonies and did not differ between cadaver (40.6 %) and control groups (46.1 %; Pearson's chi-squared test with Yates' continuity correction, χ 2 = 1.692, df = 1, p = 0.193). Foragers were smaller than the other marked workers (LMER, z = -2.237, p = 0.025, coefficient = -0.811). In the four cadaver groups, I observed 31 % of workers grooming cadavers and 21% transporting them within and outside of the nest (Table 1). Of these cadaver-managing individuals, 42 % engaged in both cadaver grooming and transports (Table 1). Individuals differed in their propensity to perform cadaver-related behaviors (Fig. 4). The marked workers of the cadaver groups differed significantly in the number of groomings (cadaver group A: B = 0.102, p = 0, ci = 0.191, cadaver group B: B = 0.076, p = 0, ci = 0.183, cadaver group C: B = 0.045, p = 0, ci = 0.056, cadaver group D: B = 0.092, p = 0, ci = 0.114) and transports directed towards cadavers (cadaver group A: B = 0.118, p = 0, ci = 0.359, cadaver group B: B = 0.073, p = 0, ci = 0.173, cadaver group C: B = 0.082, p = 0, ci = 0.103, cadaver group D: B = 0.047, p = 0.006, ci = 0.107) . Among individuals transporting cadavers, I also found a significant skew for cadaver groups A (B = 0.088, p = 0, ci = 0.330), B (B = 0.035, p = 0.001, ci = 0.136) and C (B = 0.029, p = 0.001, ci = 0.057), but not among the transporters of cadaver group D (B = -0.043, p = 0.962, ci = 0.035). Among cadaver groomers, I found a significant skew in each cadaver group (A: B = 0.082, p = 0, ci = 0.170; B: B = 0.052, p = 0, ci = 0.159; C: B = 0.019, p = 0, ci = 0.029; D: B = 0.040, p = 0, ci = 0.057).

At the individual level

Of the cadaver groomers, 38.9% groomed cadavers only once (cadaver group A: 15, B: 8, C: 9, D: 6 individuals) and 52.6% of transporters performed only one transport (cadaver group A: 16, B: 9, C: 4, D: 6 individuals) over all trials. Only seven groomers (mean value; cadaver group A: 5, B: 10, C: 11, D: 2 individuals) and three transporters (mean value, cadaver group A: 1, B: 2, C: 7, D: 1 individuals) performed these tasks in at least three of the 10 trials, not permitting analyzing an effect of experience on individual responsiveness and task performance.

The propensity of a worker to groom cadavers was neither affected by worker size (LMER, z = -0.352, p = 0.725) nor by the performance of foraging (LMER, z = -1.218, p = 0.223). I observed 34.9 % of cadaver groomers (cadaver group A: 33.3 %, B: 37 %, C: 16 %, D: 53.3 %) feeding in the colony box. Cadaver groomers were however more likely to engage in cadaver transports (LMER, z = 6.909, p < 0.0001, coefficient = 2.699). Transporters were more likely to groom cadavers (LMER, z = 6.911, p < 0.0001, coefficient = 2.700) and to forage (LMER, z = 2.087, p = 0.037, coefficient = 0.814). Of the cadaver transporters, 47 % (cadaver group A: 39.1 %, B: 42.1 %, C: 40 %, D: 66.7 %) were observed feeding.

Transporters did not differ in size from the other marked workers (the three biggest worker groups; LMER, z = -0.245, p = 0.807).

Effect of experience after reunification

The day following reunification (Fig. 1), both queens of each colony were alive and resided within the nest and no workers were observed in the corners of the ant boxes. Some ants died directly after reunification of the subcolonies, likely due to increased aggression when introducing new ants, and they were immediately removed by the experimenter (col A: 3, col B: 1, col C: 5, col D: 0). In the four colonies, workers of both previous subcolonies were in equal proportion present outside the nest (Pearson's chi-squared test; col A: X 2 = 0.803, df = 1, p = 0.370; col B: X 2 = 3.462, df = 1, p = 0.063; col C: X 2 = 2.500, df = 1, p = 0.114; col D: X 2 = 0.397, df = 1, p = 0.529) and engaged in equal proportion in foraging (Pearson's chisquared test; col A: X 2 = 0.758, df = 1, p = 0.384; col B: X 2 = 3.270, df = 1, p = 0.071; col C: X 2 = 0.862, df = 1, p = 0.353; col D: X 2 = 2.314, df = 1, p = 0.128).

During the reunification trials (Fig. 1), a few (2.6 %) marked workers (2-7 workers/colony) which could not be clearly assigned to either control or cadaver group, were observed performing cadaver-related behaviors (Fig. 4). Among the assignable cadaver-managing workers, 62 % groomers (col A: 53 %, col B: 82 %, col C: 67 %, col D: 46 %) and 58 % transporters (col A: 50 %, col B: 83 %, col C: 67 %, col D: 33 %) originated from the cadaver groups (Fig. 4). In three out of four colonies (A, C and D), these workers did not engage in cadaver grooming (exact binomial test; col A p = 1, col C p= 0.152, col D p = 1) or cadaver transports (exact binomial test; col A p = 0.453, col C p= 0.180, col D p = 0.754) with a higher probability than workers from the control group. In colony B, individuals from the cadaver group had a higher likelihood to groom (exact binomial test, p = 0.013) and transport cadavers (exact binomial test, p = 0.021) than individuals from the control group. However, this significant difference did not hold true if I considered that all non-assignable workers originated from the control group (exact binomial test, grooming p = 0.189, transports p = 0.804), a situation which is however not very likely.

DISCUSSION

In this study I analyzed whether division of labor exists for cadaver management (groomings and transports) in the ant Cataglyphis velox. I investigated whether the performance of cadaver management would be affected by repeated cadaver occurrence and the size of the cadaver and whether task performance depended upon the size of the worker or the performance of foraging behavior. I found that nestmates were significantly skewed in the performance of cadaver management acts and few individuals dominated task performance. Many task performers performed the behaviors only once over all trials and could thus not acquire experience over several trials. Individuals performing one type of cadaver management were also more likely to perform the other and cadaver transporters showed a high propensity to also engage in foraging. Cadaver size did not modulate cadaver management behaviors. The repeated occurrence of cadavers did not affect the number and duration of groomings and transports a cadaver received, but lead to an increasing number of groomers per cadaver. When reunited with less experienced individuals, the probability of experienced individuals to groom and transport a cadaver was increased in one out of four colonies.

Cadaver management was previously investigated especially in termites, where they often serve nutritional purposes or are buried [START_REF] Neoh | Do termites avoid carcasses? Behavioral responses depend on the nature of the carcasses[END_REF]reviewed in Sun and Zhou 2013).

In ants and honeybees, research has focused on the performance of necrophoresis (e.g. [START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF][START_REF] Gordon | Dependence of necrophoric response to oleic acid on social context in the ant, Pogonomyrmex badius[END_REF][START_REF] Visscher | The honey bee way of death: Necrophoric behaviour in Apis mellifera colonies[END_REF]Trumbo et al. 1997 (Diez et al. 2013b). What happens until the occurrence of necrophoresis has rarely been empirically analyzed. In the present study I demonstrated that even old cadavers are groomed and transported within the nest before necrophoresis occurred. To my knowledge, this is also the first study investigating whether nestmate cadavers of various size are treated differently.

Grooming and transport of cadavers

Cadaver grooming was performed by 31 % of nestmates, a behavior which has been described in the ants Pogonomyrmex badius [START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF] and Lasius niger [START_REF] Ataya | Le comportement nécrophorique chez la fourmi Lasius niger L[END_REF], in some termite species [START_REF] Neoh | Do termites avoid carcasses? Behavioral responses depend on the nature of the carcasses[END_REF][START_REF] Myles | Alarm, aggregation, and defense by Reticulitermes flavipes in response to a naturally occurring isolate of Metarhizium anisopliae[END_REF][START_REF] Renucci | Complex undertaking behavior in Temnothorax lichtensteini ant colonies: from corpse-burying behavior to necrophoric behavior[END_REF]) and in honeybees [START_REF] Visscher | The honey bee way of death: Necrophoric behaviour in Apis mellifera colonies[END_REF]. Grooming is a sanitary behavior during which foreign particles such as fungal spores are removed from the insect cuticle (Rosengaus et al. 1998b;Hughes et al. 2002;[START_REF] Yanagawa | Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi[END_REF]Tragust et al. 2013a) and can then be deactivated in the groomer's alimentary tract [START_REF] Yanagawa | Resistance of the termite, Coptotermes formosanus Shiraki to Metarhizium anisopliae due to grooming[END_REF]. Grooming of nestmate cadavers before removal is not intuitive as it could potentially increase the transmission of infective agents, but it might (i) increase nest hygiene if cadavers are not removed but deposited in a remote location inside the nest, (ii) bestow a future survival benefit upon the groomer if pathogen contact results in micro-infections and priming of the immune system [START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]), (iii) be less costly than necrophoresis as individuals do not have to leave the safe confines of the nest, especially in the thermophilic C. velox. Applying different possible behaviors to solve a unique problem (increased sanitary risk due to intranidal cadavers) might be an adaptive advantage, even if the behaviors differ in terms of cost [START_REF] Renucci | Complex undertaking behavior in Temnothorax lichtensteini ant colonies: from corpse-burying behavior to necrophoric behavior[END_REF]. Additionally, cadaver grooming could be a prophylactic mechanism directed towards all intranidal cadavers, including foreign cadavers which are brought into the nest and serve as a food source [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF]) and might pose a sanitary risk to the colony. Prophylactic grooming has been reported in Acromyrmex echinatior ants which selfgroom before entering the nest chamber containing brood or their mutualistic fungus (Morelos-Juárez et al. 2010) and in Formica selysi ants which allogroom all nestmates reentering the nest [START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF].

Cadavers were transported within the nest, before necrophoresis or deposition onto internal waste dumps occurred. Intranidal transports have also been described in some ant species (e.g. [START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF][START_REF] Julian | Undertaking specialization in the desert leaf-cutter ant Acromyrmex versicolor[END_REF][START_REF] Renucci | Complex undertaking behavior in Temnothorax lichtensteini ant colonies: from corpse-burying behavior to necrophoric behavior[END_REF], in honey bees [START_REF] Visscher | The honey bee way of death: Necrophoric behaviour in Apis mellifera colonies[END_REF] and termites (e.g. Sun et al. 2013) and likely occur when cadavers are brought to more remote areas of the nest. In the field I observed a superficial chamber containing pupal exuviae and cadavers in two colonies (CW pers. obs.). Superficial chambers also exist in the species Cataglyphis cursor, in which an individual was also observed removing a C. cursor cadaver from the nest (CD pers. obs.). These observations suggest the natural occurrence of intranidal transports and a reduced likelihood that moribund C. velox workers leave the nest to die outside, as has been described in three different ant species [START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF][START_REF] Heinze | Moribund ants leave their nests to die in social isolation[END_REF][START_REF] Bos | Sick ants become unsociable[END_REF]) and for honeybees (Rueppell et al. 2010).

Cadaver size did not modulate cadaver management behaviors. The only difference observed among cadavers of different size was that the largest and second smallest cadavers were transported by fewer individuals. This differential treatment might result from heterogeneity of the presented cadavers in the quantity of microorganisms they harbored.

Distribution of cadaver-management workload among workers I found that cadaver transports and groomings were not randomly distributed among workers of the cadaver group (Figure 4), but nestmates were skewed in the performance of these cadaver-management behaviors. Furthermore, task performers differed in the number of cadaver groomings and transports they performed and only a few individuals performed the majority of the work directed towards cadaver during the first experimental step. Of the cadaver-managing workers, 38 % managed cadavers only once across all trials (one grooming only: 39 %; one transport only: 53 %). I conclude that cadaver management labor is divided among nestmates with some individuals having a heavier workload than others, but division of labor is not very strong, as still 37 % of the marked workers performed these tasks and most of them infrequently. Domination of necrophoresis by a few individuals has been demonstrated in ants (Myrmica rubra) and in honeybees, with an extreme specialist bee removing 114 cadavers on 13 days (Trumbo et al 1997;[START_REF] Diez | Orientation in corpse-carrying ants: memory or chemical cues?[END_REF]. It is generally assumed that strong division of labor exists for necrophoresis and waste management in honeybees and leaf-cutting ants (Trumbo et al. 1997;[START_REF] Julian | Undertaking specialization in the desert leaf-cutter ant Acromyrmex versicolor[END_REF]Hart andRatnieks 2001, 2002;[START_REF] Ballari | Waste management in the leaf-cutting ant Acromyrmex lobicornis: Division of labour, aggressive behaviour, and location of external refuse dumps[END_REF]). Even if I include the smallest unmarked workers and assume that none of them performed these behaviors, cadaver-management behaviors would still be performed by 29% of the complete workforce. Comparisons between studies are not straight-forward as the number of necrophoric individuals reported depends on the stimulus level and thereby on the experimental technique [START_REF] Breed | Demand for task performance and workforce replacement: Undertakers in honeybee, Apis mellifera, colonies[END_REF]. Up to 30% of Acromyrmex versicolor ant workers (Julian and Cahan 1999) and 21 -23 % of honeybees were specialized in necrophoresis and a significant proportion of these bees participated for only 1 day (Trumbo et al. 1997). It is currently unclear how a specialist should be defined and whether individuals performing a task only once should be included (Trumbo et al 1997).

Together with these studies on necrophoresis, I suggest that division of labor for cadavermanagement might be less pronounced than is currently assumed. Having numerous nestmates manipulate cadavers might expose them to a low pathogen dose, potentially resulting in immune priming and bestowing a survival benefit upon these individuals ('social immunization', [START_REF] Traniello | The development of immunity in a social insect: evidence for the group facilitation of disease resistance[END_REF][START_REF] Ugelvig | Social prophylaxis: group interaction promotes collective immunity in colonies[END_REF][START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]. These workers could serve as reserve labor or present an immune barrier in case of an accumulation of cadavers and epidemic within the nest. In the ant Myrmica rubra, workers suffer increased mortality if they were not able to remove their dead [START_REF] Diez | Keep the nest clean: survival advantages of corpse removal in ants[END_REF].

Who performs cadaver management?

Task allocation for cadaver-management is poorly understood (Sun and Zhou 2013) and has only been studied for waste management in leaf-cutting ants and necrophoresis. In honeybees, a genetic component for necrophoresis has been demonstrated (Robinson andPage 1988, 1995), which likely determines olfactoric sensitivity (response thresholds) of workers to necromones [START_REF] Julian | Undertaking specialization in the desert leaf-cutter ant Acromyrmex versicolor[END_REF]. The responsiveness to oleic acid has further found to be caste-specific, with unresponsiveness in the soldier caste in the ant Atta mexicana [START_REF] López-Riquelme | Antennal olfactory sensitivity in response to task-related odours of three castes of the ant Atta mexicana (Hymenoptera: Formicidae)[END_REF]). Cadaver-manipulating workers of the present study might also differ from non-responsive nestmates in a higher sensitivity towards necromones, which might be genetically determined in this polygynous and polyandrous species. Age is a further predictor of necrophoric and waste-management behavior and both tasks are performed by middle-aged individuals [START_REF] Breed | Demand for task performance and workforce replacement: Undertakers in honeybee, Apis mellifera, colonies[END_REF][START_REF] Mersch | Tracking individuals shows spatial fidelity is a key regulator of ant social organization[END_REF][START_REF] Camargo | Age polyethism in the leaf-cutting ant Acromyrmex subterraneus brunneus Forel, 1911 (Hym., Formicidae)[END_REF]Waddington and Hughes 2010) and necrophoric individuals are presumably developmentally advanced in that they start foraging at an earlier age (Trumbo et al. 1997) and possess higher levels of juvenile hormone than bees of the same age performing other age-typical tasks [START_REF] Huang | Physiological correlates of division of labor among similarly aged honey bees[END_REF]. In my study, transporters were more likely to forage than nontransporting individuals, a behavior which is typically performed by the oldest workers (Cataglyphis species reviewed in [START_REF] Lenoir | Cataglyphis desert ants: a good model for evolutionary biology in Darwin's anniversary year: a review[END_REF]). As C. velox foragers leave the confines of the nest, they presumably suffer the highest infection risk in the colony and possess higher levels of active phenoloxidase [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]), an important enzyme in individual immune defense (reviewed in [START_REF] Cerenius | The prophenoloxidase-activating system in invertebrates[END_REF]. Cadaver groomers and transporters did not differ in size from nestmates belonging to the three biggest size groups and not engaging in cadaver management. Not being able to include the smallest workers of the colony might have masked an effect of body size on cadaver management in this study. In another polymorphic ant with monophasic worker distribution, necrophoric labor was not divided by worker size (Solenopsis invicta, Howard and Tschinkel 1976). An effect of body size on waste management has been demonstrated in leafcutting ants, where waste managers are smaller than foragers [START_REF] Ballari | Waste management in the leaf-cutting ant Acromyrmex lobicornis: Division of labour, aggressive behaviour, and location of external refuse dumps[END_REF]Waddington and Hughes 2010). Foraging workers were found to be smaller compared to nonforaging workers, which could be explained by relatively low laboratory temperatures (24.4-29.9°C) compared to what can be found under field conditions (maximal activity temperature 46 °C in the field, reviewed in [START_REF] Cerdá | Behavioural and physiological traits to thermal stress tolerance in two Spanish desert ants[END_REF]). In the field, the mean size of foraging workers increased with temperature and smaller workers foraged preferentially at lower temperatures [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF]).

Individuals performing one type of cadaver-management behavior were also more likely to perform the other (42% of cadaver managers performed both groomings and transports). I further observed a significant overlap among cadaver transporting and foraging individuals (47 % of transporters also foraged). The reported overlap between cadaver-managing individuals and foragers might be increased in my study, given the high percentage of individuals observed feeding in the colony box. Having flexible workers which can switch between cadaver management and other tasks can be advantageous for the colony, if tasks are performed in the same spatial zone and inter-task travel time is thereby reduced or if individuals can transfer previously acquired improvements in one task to another which requires similar motor skills [START_REF] Wilson | Behavioral discretization and the number of castes in an ant species[END_REF]). Indeed, both foragers and individuals transporting nestmate cadavers perform cadaver transports in this species and presumably use the same motor patterns and are exposed to the same sanitary threats. Having the same workers perform both tasks could reduce parasite transmission within the colony.

Additionally, an overlap among task groups determines colony level patterns [START_REF] Wilson | Behavioral discretization and the number of castes in an ant species[END_REF][START_REF] Gordon | The organization of work in social insect colonies[END_REF]Beshers and Fewell 2001) and enables the group to adjust more rapidly to varying environmental conditions, such as the amount of sanitary risk imposed by cadavers [START_REF] Johnson | Organization of work in the honeybee: a compromise between division of labour and behavioural flexibility[END_REF]Diez et al. 2013a). Waste workers of Pogonomyrmex barbatus and necrophoric individuals of Myrmica rubra are also able to switch to foraging [START_REF] Gordon | Dynamics of task switching in harvester ants[END_REF]Diez et al. 2013a) and necrophoric honeybees were observed to further remove debris and perform hygienic behavior (i.e. the removal of diseased brood, [START_REF] Spivak | Genetic, individual, and group facilitation of disease resistance in insect societies[END_REF]) at low frequency (Trumbo et al. 1997). In leafcutting ants however, where foragers collect leaf material for their mutualistic fungus, contact with waste workers is avoided and they are even aggressed to avoid contamination [START_REF] Hart | Task partitioning, division of labour and nest compartmentalisation collectively isolate hazardous waste in the leafcutting ant Atta cephalotes[END_REF][START_REF] Ballari | Waste management in the leaf-cutting ant Acromyrmex lobicornis: Division of labour, aggressive behaviour, and location of external refuse dumps[END_REF]).

Experience as a factor of task allocation

Experience is an important factor which can improve individual and collective performance (e.g. Chittka and Thompson 1997;[START_REF] Langridge | Improvement in collective performance with experience in ants[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Langridge | How experienced individuals contribute to an improvement in collective performance in ants[END_REF][START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF]). However little is known about an effect of recent experience on collective sanitary behaviors (but see [START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF][START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF][START_REF] Westhus | Increased grooming after repeated brood care provides sanitary benefits in a clonal ant[END_REF]. A behavioral modulation through experience depends on the time interval between task performances [START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF]. Time intervals which last as long as a night can reduce previously acquired effects [START_REF] Keasar | Overnight memory retention of foraging skills by bumblebees is imperfect[END_REF][START_REF] Langridge | Improvement in collective performance with experience in ants[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF]. Recurrent task performance of individuals was low in my study with 39% of groomers and 53 % of transporters performing these behaviors only once and only 26% groomers and 15% transporters performing these tasks in two trials on the same day. I was thus unable to satisfactorily address an effect of individual experience on task performance of individual workers. In honeybees and ants, an effect of experience on necrophoresis was not demonstrated, even though specialized necrophoric individuals were more efficient (cadavers removed faster and dropped less) than non-specialists (Trumbo and Robinson 1997;[START_REF] Julian | Undertaking specialization in the desert leaf-cutter ant Acromyrmex versicolor[END_REF], likely due to a greater ability from the outset (Trumbo and Robinson 1997). In the ant Myrmica rubra, necrophoric workers demonstrated spatial memory within a time frame of 7 minutes [START_REF] Diez | Orientation in corpse-carrying ants: memory or chemical cues?[END_REF]. Colony phenotype can also be shaped by experience, through the collective actions of nestmates. Experienced ant workers have been shown to be more efficient in colony emigration (Temnothorax albipennis: [START_REF] Langridge | Improvement in collective performance with experience in ants[END_REF][START_REF] Langridge | How experienced individuals contribute to an improvement in collective performance in ants[END_REF]) and thermal brood relocation (Camponotus rufipes: [START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF]), leading to a better output of the whole group. Recurrent insertion of nestmate cadavers did neither affect the time delay to cadaver removal, nor the intensity of groomings and transports cadavers received. However, cadavers were groomed by a higher number of individuals with increasing trial, suggesting that the repeated occurrence of cadavers increased the probability of nestmates to perform cadaver grooming and thereby affected individual responsiveness. Rare tasks such as necrophoresis (or even cadaver management) might not provide ample opportunity to improve task performance, as the time interval between the appearances of the task-associated stimuli might be too large for selfreinforcement to last (Trumbo et al. 1997;Diez et al. 2013a). Furthermore, task groups differ in their ability to learn and honeybee pollen foragers demonstrated stronger learning, which is genetically determined and likely due to increased stimuli sensitivity [START_REF] Latshaw | Heritable variation in learning performance affects foraging preferences in the honey bee (Apis mellifera)[END_REF] and cue biases for learning (reviewed in [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. Some motor patterns are more easily modified by experience than others. The restriction of learning to some stimuli or motor patterns in complex and unpredictable situations is assumed to be adaptive, as the costs of learning (time and energy spent, errors) do not arise in more predictable situations where fixed responses are sufficient (reviewed in [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. Both cadaver transports and foraging involve transporting cadavers with the mandibles and grooming cadavers is further not expected to differ from self-or allogrooming. Motor skills acquired in these tasks are presumably transferable to cadaver management, so that these behaviors might be less modulated by experience than tasks where the nature of the substrate is not predictable on an evolutionary scale, such as handling live prey [START_REF] Chittka | Learning, specialization, efficiency and task allocation in social insects[END_REF].

Theoretical studies proposed that the experience gained from previous performances influences an individual's probability to engage in this task. The likelihood of task performance would increase if previous performance was successful, resulting in a positive feedback factor, and decrease in case of failure or the lack of opportunity (e.g. [START_REF] Oster | Caste and ecology in the social insects[END_REF][START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF][START_REF] Merkle | Dynamic polyethism and competition for task in threshold reinforcement models of social insects[END_REF]. In the context of foraging, where success or reward is easily quantified, experiencing success of task performance (or lack thereof) affected task allocation and thereby colony organization of the ant Cerapachys biroi [START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF]. Additionally, the social environment can affect the performance of a task. In constructed colonies of the ant Temnothorax albipennis comprising inexperienced and experienced workers, old experienced individuals were more likely to lead tandem runs than old inexperienced individuals during emigrations [START_REF] Franklin | Do ants need to be old and experienced to teach?[END_REF]. Another study examining the performance of hygienic behavior found that non-hygienic bees demonstrated a decreased rate, duration and probability of uncapping and removing dead brood in the presence of hygienic bees (Arathi et al. 2006). Reuniting individuals who had previously encountered 40 nestmate cadavers and nestmates without this experience did not lead to different proportions of individuals engaging in cadaver-management behaviors in three out of four colonies. In the fourth colony (colony B) however, experienced individuals had a higher likelihood to groom and transport cadavers compared to controls. Inter-colony variation is common in social insect studies and might in this case arise through stimulus differences. Did the cadavers of colony B pose a higher sanitary threat, thereby increasing positive feedback ants received when managing them (e.g. removal of microorganisms during grooming or necrophoresis of these cadavers)? Positive feedback is an important factor modulating an individual's propensity to again engage in a task [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF] and affected allocation of workers to foraging in the ant Cerapachys biroi [START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF][START_REF] Robinson | Experience, corpulence and decision making in ant foraging[END_REF]. The stimuli triggering cadaver management in my study or the mechanisms underlying increased cadaver management of experienced individuals in colony B remain elusive. Not detecting an effect of experience in colonies A, C and D might be due to a low sample size, the fact that control individuals were not completely naïve or could be explained by only weak modulation of cadaver-management behaviors through experience. It is experimentally difficult to achieve complete naivety in terms of cadavers, if individuals are kept in a typical social environment, where cadavers already occur when young workers eclose. But even though the control group was not completely naive, the cadaver group was exposed to 40 cadavers over a time period of 5 days only, increasing daily cadaver occurrence by 5574%. A previous study on honeybees suggests that learning may not be an important component of necrophoric specialization, given the short tenure of necrophoric bees of 2 -3 days (Trumbo et al. 1997).

Necrophoresis has been demonstrated to be genetically determined with individuals of certain honeybee patrilines having a higher likelihood of task performance (Robinson andPage 1988, 1995), which might constrain a colony's ability to flexibly adapt to changing conditions [START_REF] Robinson | Genotypic constraints on plasticity for corpse removal in honey bee colonies[END_REF]. However, the high percentage of nestmates performing cadaverrelated tasks and the overlap among them suggests that workers performing cadaver management can switch to and stay available for other tasks in the ant C. velox.

Experimental design and its limitations

The occurrence of several cadavers inside the nest, as in the present study, could occur under natural conditions in the case of pathogen transmission from returning foragers to nestmates, colony attack by predators or social parasites, nest damage, flooding and hibernal mortality. I introduced only four cadavers to avoid a sanitary emergency situation, where 'all hands on deck' would be needed and the high task thresholds of usually unresponsive individuals would also be met (Dornhaus 2008, Chittka and[START_REF] Chittka | Learning, specialization, efficiency and task allocation in social insects[END_REF]. The simple nest design (round petri dish) was chosen to maximize cadaver encounter by incoming, outgoing and intranidal workers and enable filming of the whole nest, but it reduced spatial nest complexity. It rather represented a colony in the early ontogenetic stages, where few workers inhabit a small and simply structured nest. More complexity and nest structure will presumably decrease cadaver encounter rate and thereby increase the division of labor found for cadaver management. The simple nest design could further explain the high number of foragers I observed, even though foragers were defined as ants feeding in the arena and not defined according to their presence in the arena (as has been done for C. velox in [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF] where lower foraging percentages were reported). When inserting cadavers through the nest cover, I observed several ants inspecting and biting them, which was likely due to colony disturbance and might have resulted in stimulus enhancement (i.e. the proximity of a nestmate to a cadaver attracted the attention of another individual towards it; [START_REF] Leadbeater | Social learning in insects -from miniature brains to consensus building[END_REF], increasing cadaver encounter rate by nestmates. Disturbed fire ant workers (Solenopsis invicta) have been reported to attack cadavers which would otherwise be removed (Howard and Tschinkel 1976). The behavioral differences I observed among colonies might be common intercolonial variation in behavior (e.g. [START_REF] Breed | Demand for task performance and workforce replacement: Undertakers in honeybee, Apis mellifera, colonies[END_REF][START_REF] Diez | Orientation in corpse-carrying ants: memory or chemical cues?[END_REF], for instance stemming from differential patriline distributions resulting in a different number of individuals with lower response thresholds for task, but it might also be due to heterogeneity of the cadaver stimuli.

The cadavers of some colonies could have harboured more microorganisms than those of others, which is expected to be natural situation.

Future research

It would be interesting to investigate whether cadaver-managing individuals show higher investment in physiological immunity as a protection against increased pathogen exposure during task performance or when exiting the nest ('exposure risk hypothesis': [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]. Increased immune investment of more exposed task groups is suggested by the findings that C. velox foragers possess higher levels of active phenoloxidase [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]) and waste workers of Atta sexdens rubropilosa bigger metapleural glands than workers engaged in less risky tasks [START_REF] Lacerda | Morphometry of the metapleural gland of workers engaged in different behavioral tasks in the ant Atta sexdens rubropilosa[END_REF]. On the other hand, individuals which already possess higher levels of individual immunity might be the ones engaging in cadaver-related tasks. It would further be interesting to know, whether the occurrence of cadaver grooming depends on the diet of the species and is prophylactically directed towards all cadavers, also nutritional ones. It would be interesting to investigate how stimulus quantity, quality (e.g. the virulence of a parasite) or temporal presentation (i.e. periodic or chronic exposure) modulates sanitary response behavior in social insects, a question which has not yet received enough empirical attention. Furthermore, more research is warranted on the actual perception of a disease stimulus by individuals and the transmission of this information within the colony.

CHAPTER 3

Are worker size and phenoloxidase activity of Cataglyphis velox workers genetically determined?

ClaudiaWesthus, Pierre Fédérici, Claudy Haussy,

Sylvia Cremer, Claudie Doums

In preparation

TITLE

Are worker size and phenoloxidase activity of Cataglyphis velox workers genetically determined?

ABSTRACT

Polyandry increases the diversity of group members and thereby favors division of labor within the colony. Colonies with increased genetic diversity can also be more resistant towards disease, but the mechanisms underlying increased disease resistance are not well understood. The effects of polyandry on division of labor and disease resistance could be linked if considering physiological disease defense a worker task. In the polymorphic and polyandrous ant Cataglyphis velox I analyzed whether there is a direct link between genotype, worker type (young laboratory-born versus old field-derived) and the phenotypic traits worker size and immune investment. As an indication of immune investment I analyzed the level of the enzyme phenoloxidase (PO). I did not find evidence for genetic determination of both worker size and the level of PO, neither for all workers, nor separately in both worker types (young laboratory-born workers versus old field-derived workers). Workers of different size did not differ in their levels of active PO. Interestingly, whereas bigger field-derived workers possessed higher levels of total PO, I could not detect this relationship in young laboratoryborn workers. The age (and potentially the environmental origin) of workers affected the size and immune investment of individuals: laboratory-born workers were smaller and had a reduced variance in size compared to field-derived workers. The older field-derived workers showed higher levels of total PO, but did not differ from the young laboratory-born workers in the variance for this trait and in the level of active PO. The variance in active PO was however lower in old field-derived workers.

INTRODUCTION

The evolution of multiple mating (polyandry, reviewed in e.g. [START_REF] Crozier | Polyandry in social Hymenoptera -disunity in diversity?[END_REF] resulted in increased genetic diversity within social insect colonies (e.g. Smith et al. 2008;Rueppell et al. 2012). Genetic diversity among nestmates can benefit colonies in (i) giving rise to division of labor; (ii) permitting flexibility and resiliency of the group; (iii) enhancing colony efficiency and productivity (e.g. [START_REF] Fewell | Genotypic variation in foraging responses to environmental stimuli by honey bees, Apis mellifera[END_REF]Beshers and Fewell 2001;[START_REF] Crozier | Polyandry in social Hymenoptera -disunity in diversity?[END_REF][START_REF] Brown | The evolution of female multiple mating in social hymenoptera[END_REF][START_REF] Jones | Honey bee nest thermoregulation: diversity promotes stability[END_REF][START_REF] Mattila | Genetic diversity in honey bee colonies enhances productivity and fitness[END_REF][START_REF] Oldroyd | Genetic diversity promotes homeostasis in insect colonies[END_REF][START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]) and (iv) enhancing disease resistance (e.g. Baer andSchmid-Hempel 1999, 2001;[START_REF] Tarpy | Genetic diversity within honeybee colonies prevents severe infections and promotes colony growth[END_REF][START_REF] Tarpy | Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queens[END_REF][START_REF] Seeley | Queen promiscuity lowers disease within honeybee colonies[END_REF][START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]. Division of labor, the performance of different tasks such as foraging and brood care by nestmates, is achieved through self-organization (i.e. without a central leader, [START_REF] Jr | Self-organization and the evolution of division of labor[END_REF][START_REF] Detrain | Self-organized structures in a superorganism: do ants "behave" like molecules?[END_REF]) and believed to be responsible for the high ecological success of social insects [START_REF] Oster | Caste and ecology in the social insects[END_REF]Hölldobler and Wilson 1990). A worker's genotype has been shown to affect its task preference (e.g. ants: [START_REF] Stuart | Genetic component to division of labor among workers of a leptothoracine ant[END_REF][START_REF] Snyder | The genetics of social behavior in a polygynous ant[END_REF][START_REF] Blatrix | Task allocation depends on matriline in the ponerine ant Gnamptogenys striatula Mayr[END_REF][START_REF] Julian | Genetic variation and task specialization in the desert leaf-cutter ant, Acromyrmex versicolor[END_REF]Waddington et al. 2010;Eyer et al. 2013a; honey bees: [START_REF] Frumhoff | A genetic component to division of labour within honey bee colonies[END_REF]Robinson andPage 1988, 1989;[START_REF] Oldroyd | Task specialization in a wild bee, Apis florea (Hymenoptera: Apidae), revealed by RFLP banding[END_REF][START_REF] Pérez-Sato | Multi-level selection for hygienic behaviour in honeybees[END_REF]social wasps: O'Donnell 1996social wasps: O'Donnell , 1998)), by affecting task-associated response thresholds. Nestmates are assumed to possess different response thresholds for a task (i.e. to respond at different stimulus intensities; response thresholds models reviewed in Beshers and Fewell 2001), which enables a graded colony response [START_REF] Robinson | Genetic determination of nectar foraging, pollen foraging, and nest-site scouting in honey bee colonies[END_REF]Beshers and Fewell 2001;[START_REF] Oldroyd | Genetic diversity promotes homeostasis in insect colonies[END_REF]. Even if genetically determined, response thresholds are not fixed, but can underlie short-term modulation by recent experience of the individual and environmental conditions individuals are exposed to (e.g. [START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF]. Genetic diversity can further affect disease dynamics, as it reduces parasite loads and improves disease resistance within the social insect colony (e.g. [START_REF] Liersch | Genetic variation within social insect colonies reduces parasite load[END_REF]Baer andSchmid-Hempel 1999, 2001;[START_REF] Schmid-Hempel | Polyandry Versus Polygyny Versus Parasites[END_REF][START_REF] Tarpy | Genetic diversity within honeybee colonies prevents severe infections and promotes colony growth[END_REF]Hughes and Boomsma 2004;[START_REF] Tarpy | Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queens[END_REF][START_REF] Seeley | Queen promiscuity lowers disease within honeybee colonies[END_REF]. Which proximate mechanisms result in increased disease resistance is not well understood. Compared to colonies with reduced genetic diversity (due to inbreeding), colonies with natural genetic diversity showed improved behavioral anti-parasite defense through higher allogrooming frequencies and earlier hygienic behavior (i.e. the removal of diseased brood from the nest; [START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]. Increased genetic diversity did however not lead to higher levels of physiological immune function such as the encapsulation response and the activity of the enzyme phenoloxidase when comparing honeybee larvae among colonies with natural mating numbers [START_REF] Tarpy | Within-and across-colony effects of hyperpolyandry on immune function and body condition in honey bees (Apis mellifera)[END_REF].

How tasks are allocated among nestmates is a central question about division of labor [START_REF] Gordon | The organization of work in social insect colonies[END_REF] and factors demonstrated to affect task allocation are for instance genotype, age, social interactions, rearing conditions, experience and worker size (e.g. [START_REF] Robinson | Genetic determination of guarding and undertaking in honey-bee colonies[END_REF]Hölldobler and Wilson 1990;[START_REF] Pankiw | The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.)[END_REF]Beshers and Fewell 2001;[START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF]Smith et al. 2008;[START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF]). Temporal polyethism, in which a sequential order of tasks is correlated with an age-dependent hormonal change, is widespread in social insects. With increasing worker age, individuals switch from intranidal activities such as brood care and cleaning to extranidal tasks such as foraging and nest defense with a higher extrinsic mortality risk [START_REF] Seeley | Adaptive significance of the age polyethism schedule in honeybee colonies[END_REF]reviewed in Robinson 1992;[START_REF] Mersch | Tracking individuals shows spatial fidelity is a key regulator of ant social organization[END_REF]. Another pattern of division of labor is morphological polyethism, in which different fixed body sizes or shapes of nestmates can be distinguished and predispose individuals to certain task repertoires [START_REF] Oster | Caste and ecology in the social insects[END_REF]. In extreme cases, where species have morphological worker castes, the largest workers commonly specialize in foraging and colony defense and the smallest ones in brood care (Hölldobler and Wilson 1990;[START_REF] Nowbahari | Polymorphism and polyethism in the formicinae ant Cataglyphis niger (Hymenoptera)[END_REF][START_REF] Mertl | Behavioral evolution in the major worker subcaste of twignesting Pheidole (Hymenoptera: Formicidae): does morphological specialization influence task plasticity ?[END_REF], but task-size matching can also occur in species with unimodal worker size distributions (e.g. [START_REF] Waser | Task-matching and short-term size shifts in foragers of the harvester ant, Messor pergandei (Hymenoptera: Formicidae)[END_REF]).

Larger workers of several ant species are better foragers (e.g. [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF][START_REF] Goulson | Can alloethism in workers of the bumblebee, Bombus terrestris, be explained in terms of foraging efficiency?[END_REF] and nest defenders [START_REF] Braendle | Size-correlated division of labour and spatial distribution of workers in the driver ant, Dorylus molestus[END_REF], show increased longevity [START_REF] Calabi | Worker longevity in the fire ant Solenopsis invicta: Ergonomic considerations of correlations between temperature, size and metabolic rates[END_REF][START_REF] Porter | Fire ant polymorphism: factors affecting worker size[END_REF]; but see: [START_REF] Camargo | Age polyethism in the leaf-cutting ant Acromyrmex subterraneus brunneus Forel, 1911 (Hym., Formicidae)[END_REF], for instance under starvation [START_REF] Heinze | The significance of latitudinal variation in body size in a holarctic ant, Leptothorax acervorum[END_REF], but are more expensive to produce compared to smaller workers [START_REF] Calabi | Worker longevity in the fire ant Solenopsis invicta: Ergonomic considerations of correlations between temperature, size and metabolic rates[END_REF]. Worker polymorphism increases intra-colony diversity, which is believed to result in higher colony efficiency (e.g. [START_REF] Oster | Caste and ecology in the social insects[END_REF][START_REF] Porter | Fire ant polymorphism: factors affecting worker size[END_REF][START_REF] Wilson | The sociogenesis of insect colonies[END_REF], for instance through better exploitation of external environmental conditions (e.g. [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF]. Colony heterogeneity through polymorphism might further be favored by selection as worker size differences can result in varying susceptibility to parasites [START_REF] Keller | Parasites, worker polymorphism, and queen number in social insects[END_REF][START_REF] Kermarrec | Comparison of the susceptibility of Acromyrmex octospinosus Reich (Attini, Formicidae) to two insect parasitic nematodes of the genera Heterorhabditis and Neoplectana (Rhabditina, Nematoda)[END_REF]).

Division of labor and individual disease resistance are often studied apart, but can be tightly linked if physiological immune investment is considered a worker task. To counter the selection pressures imposed by a variety of macro-and microparasites (such as helminth worms, fungi, bacteria, viruses and some protozoa, e.g. [START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF], social insects evolved anti-parasite defense strategies at the individual as well as at the group level, comprising physiological, behavioral and spatial mechanisms (e.g. Cremer et al. 2007;[START_REF] Spivak | Genetic, individual, and group facilitation of disease resistance in insect societies[END_REF][START_REF] Evans | Socialized medicine: individual and communal disease barriers in honey bees[END_REF][START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]. Behavioral sanitary tasks can be divided among nestmates and task allocation affected by genetic, age and size factors (e.g. [START_REF] Frumhoff | A genetic component to division of labour within honey bee colonies[END_REF][START_REF] Robinson | Genetic determination of guarding and undertaking in honey-bee colonies[END_REF][START_REF] Breed | Demand for task performance and workforce replacement: Undertakers in honeybee, Apis mellifera, colonies[END_REF][START_REF] Ballari | Waste management in the leaf-cutting ant Acromyrmex lobicornis: Division of labour, aggressive behaviour, and location of external refuse dumps[END_REF][START_REF] Camargo | Age polyethism in the leaf-cutting ant Acromyrmex subterraneus brunneus Forel, 1911 (Hym., Formicidae)[END_REF][START_REF] Pérez-Sato | Multi-level selection for hygienic behaviour in honeybees[END_REF][START_REF] Abramowski | Caste specialization in behavioral defenses against fungus garden parasites in Acromyrmex octospinosus leaf-cutting ants[END_REF]Waddington et al. 2010;[START_REF] Mersch | Tracking individuals shows spatial fidelity is a key regulator of ant social organization[END_REF], as well as by the social context (e.g. Arathi and Spivak 2001). Sanitary task performance by a subset of workers may limit the exposure of the entire colony to parasites, if few task performers have limited interactions with nestmates [START_REF] Hart | Task partitioning, division of labour and nest compartmentalisation collectively isolate hazardous waste in the leafcutting ant Atta cephalotes[END_REF][START_REF] Naug | The role of colony organization on pathogen transmission in social insects[END_REF]. Nestmates further differ in their investment into physiological defense mechanisms which serve collective parasite defense. The size of metapleural glands, which' secretions possess antimicrobial activities (reviewed in Yek and Mueller 2010), is positively correlated with worker size [START_REF] Hughes | Caste-specific expression of genetic variation in the size of antibiotic-producing glands of leaf-cutting ants[END_REF]) and bigger in task groups with higher infection risk [START_REF] Lacerda | Morphometry of the metapleural gland of workers engaged in different behavioral tasks in the ant Atta sexdens rubropilosa[END_REF]. Soldiers of eusocial thrips produce more antifungal secretions than dispersers [START_REF] Turnbull | Antifungal activity in thrips soldiers suggests a dual role for this caste[END_REF]. At the individual level, nestmates differ in the resources they allocate to individual physiological immune defense, for instance to their encapsulation response (e.g. [START_REF] Doums | Immunocompetence in workers of a social insect, Bombus terrestris L., in relation to foraging activity and parasitic infection[END_REF][START_REF] Vitikainen | Inbreeding and caste-specific variation in immune defence in the ant Formica exsecta[END_REF], constitutive antibacterial activity (e.g. [START_REF] Armitage | Immune defense in leafcutting ants: a cross-fostering approach[END_REF] or phenoloxidase activity (e.g. [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF][START_REF] Armitage | The effects of age and social interactions on innate immunity in a leaf-cutting ant[END_REF]. Spread of diseases which require direct contact between individuals occurs from infectious to susceptible neighbors. If these are neighbors with higher immune investment and increased immune resistance, they could reduce disease transmission within the nest (Cremer et al. 2007;[START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]). In the ant Camponotus pennsylvanicus, individuals which mounted an immune response could even confer a survival advantage to nestmates at a secondary immune challenge by transferring antimicrobial compounds in their trophallactic droplet (Hamilton et al. 2011). Increasing age is assumed to correlate with a decline in immune function ('immunosenescence': reviewed for vertebrates and invertebrates in [START_REF] Müller | Immunosenescence in vertebrates and invertebrates[END_REF], but empirical evidence is conflicting. Whereas some studies reported a decline in immunocompetence (e.g. encapsulation response and phenoloxidase activity) with age in insects (e.g. [START_REF] Rolff | Effects of age and gender on immune function of dragonflies (Odonata, Lestidae) from a wild population[END_REF][START_REF] Doums | Senescence of immune defence in Bombus workers[END_REF][START_REF] Amdam | Social reversal of immunosenescence in honey bee workers[END_REF][START_REF] Moret | Immune responses of bumblebee workers as a function of individual and colony age: senescence versus plastic adjustment of the immune function[END_REF], other studies found either no difference or even an increase in immunocompetence with age [START_REF] Schwarzenbach | Sex and immunity in the yellow dung fly Scathophaga stercoraria[END_REF]Wilson-Rich et al. 2008;[START_REF] Armitage | The effects of age and social interactions on innate immunity in a leaf-cutting ant[END_REF][START_REF] Roberts | Immunosenescence and resistance to parasite infection in the honey bee, Apis mellifera[END_REF].

The hypotheses on the advantage of genetic diversity for the emergence of division of labor and for increased collective disease resistance rely on the assumption that their components such as individual size and immune defense are in part genetically determined. Studies investigating a heritable component of worker size are conflicting, possibly due to differences in sampling (the year of the study, see [START_REF] Bargum | A genetic component to size in queens of the ant, Formica truncorum[END_REF]) and social context (e.g. [START_REF] Rüppell | Complex determination of queen body size in the queen size dimorphic ant Leptothorax rugatulus (Formicidae : Hymenoptera)[END_REF]. A genetic influence on worker size has been reported in several ant species [START_REF] Fraser | Genetic influence on caste in the ant Camponotus consobrinus[END_REF][START_REF] Hughes | Worker caste polymorphism has a genetic basis in Acromyrmex leaf-cutting ants[END_REF][START_REF] Rheindt | A genetic component in the determination of worker polymorphism in the Florida harvester ant Pogonomyrmex badius[END_REF][START_REF] Schwander | Division of labour and worker size polymorphism in ant colonies, the impact of social and genetic factors[END_REF][START_REF] Jaffé | Worker caste determination in the army ant Eciton burchellii[END_REF][START_REF] Evison | Genetic caste polymorphism and the evolution of polyandry in Atta leaf-cutting ants[END_REF][START_REF] Huang | Mating system evolution and worker caste diversity in Pheidole ants[END_REF], but not in Formica truncorum [START_REF] Bargum | A genetic component to size in queens of the ant, Formica truncorum[END_REF]). In the ant Cataglyphis cursor with continuous worker size distribution, individual size was also not influenced by patriline [START_REF] Fournier | Genetic diversity, worker size polymorphism and division of labour in the polyandrous ant Cataglyphis cursor[END_REF]Eyer et al. 2013a). Worker size determination has long been assumed to result from environmental cues (Hölldobler and Wilson 1990;[START_REF] Wheeler | Soldier determination in Pheidole bicarinata: inhibition by adult soldiers[END_REF], such as the amount of food available during larval development in holometabolous insects. Worker size has been demonstrated to increase with colony size (e.g. [START_REF] Wood | Quantification and modification of worker size variation in the fire ant Solenopsis invicta[END_REF][START_REF] Gibson | Soldier production in Camponotus novaeboracensis during colony growth[END_REF][START_REF] Tschinkel | Colony growth and the ontogeny of worker polymorphism in the fire ant, Solenopsis invicta[END_REF][START_REF] Tschinkel | Sociometry and sociogenesis of colonies of the fire ant Solenopsis invicta during one annual cycle[END_REF][START_REF] Tschinkel | Sociometry and sociogenesis of colonies of the harvester ant, Pogonomyrmex badius: I Worker characteristics in relation to colony size and season[END_REF]Hölldobler andWilson 1990, Clémencet andDoums 2007) and decrease with the presence of multiple queens within the colony (e.g. [START_REF] Schwander | Division of labour and worker size polymorphism in ant colonies, the impact of social and genetic factors[END_REF][START_REF] Rüppell | Complex determination of queen body size in the queen size dimorphic ant Leptothorax rugatulus (Formicidae : Hymenoptera)[END_REF]. The size of Cataglyphis cursor workers differed between natural habitats [START_REF] Clémencet | Habitat-related microgeographic variation of worker size and colony size in the ant Cataglyphis cursor[END_REF]. A genetic basis of disease resistance was demonstrated in several social insect species (e.g. Baer andSchmid-Hempel 1999, 2001;[START_REF] Tarpy | Genetic diversity within honeybee colonies prevents severe infections and promotes colony growth[END_REF][START_REF] Tarpy | Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queens[END_REF][START_REF] Seeley | Queen promiscuity lowers disease within honeybee colonies[END_REF]) even though the underlying mechanisms remain unclear in most cases. In leafcutting ant workers, disease resistance varied between patrilines (Hughes and Boomsma 2004), which could relate to a variation in immune response [START_REF] Armitage | The effects of age and social interactions on innate immunity in a leaf-cutting ant[END_REF]. Indeed, an effect of patriline was demonstrated on constitutive antibacterial activity [START_REF] Armitage | Immune defense in leafcutting ants: a cross-fostering approach[END_REF]) and on the size of the metapleural gland [START_REF] Hughes | Caste-specific expression of genetic variation in the size of antibiotic-producing glands of leaf-cutting ants[END_REF][START_REF] Armitage | Immune defense in leafcutting ants: a cross-fostering approach[END_REF]) in leaf-cutting ants. Heritability of hemolymph phenoloxidase and the ability to encapsulate a foreign body have been demonstrated in non-social insects (e.g. [START_REF] Ryder | Quantitative genetics of immune function and body size in the house cricket, Acheta domesticus[END_REF][START_REF] Cotter | Heritability of immune function in the caterpillar Spodoptera littoralis[END_REF][START_REF] Schwarzenbach | Sex and immunity in the yellow dung fly Scathophaga stercoraria[END_REF]. In honeybees however, larvae of different patrilines did not differ in these immune parameters [START_REF] Tarpy | Within-and across-colony effects of hyperpolyandry on immune function and body condition in honey bees (Apis mellifera)[END_REF]), but diseased larvae showed moderate heritability for the expression of the antimicrobial peptide abaecin [START_REF] Decanini | Variation and heritability in immune gene expression by diseased honeybees[END_REF]). The conditional expression of physiological immune parameters is influenced by many environmental factors, such as the presence of nestmates (e.g. [START_REF] Richter | Social context-dependent immune gene expression in bumblebees (Bombus terrestris)[END_REF]; but see: Armitage and Boomsma 2010) or antimicrobial compounds in the nest [START_REF] Castella | The presence of conifer resin decreases the use of the immune system in wood ants[END_REF][START_REF] Simone | Resin collection and social immunity in honey bees[END_REF].

We aimed to investigate whether interindividual variation in body size and immunocompetence are based on genetic differences between workers of the ant Cataglyphis velox, Santschi, 1929 [START_REF] Tinaut | Taxonomic situation of the genus Cataglyphis Förster, 1850 in the Iberian Peninsula II. New position for C. viatica (Fabricius, 1787) and the redescription of C. velox Santschi, 1929 stat. n. (Hymenoptera, Formicidae)[END_REF]). The underlying causes of phenotypic variability among workers are required to understand in what extent traits will adapt to natural selection and changing environmental conditions. I estimated immunocompetence of individuals by measuring the level of the immune enzyme phenoloxidase (both active and total PO). Within young laboratory-born workers of the same age, I was able to control for the confounding factor of individual age. I expected a patriline effect on worker size and immune function, whereby different sibling groups would vary from each other. I assumed lower heritability for the inducible active PO than for the constitutive form of the enzyme (total PO). Foragers of C.

velox possess higher levels of active PO [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]) and I thus expected higher levels of this enzyme in old field-derived workers. In field-collected leaf-cutting ant colonies (Acromyrmex octospinosus), the level of stored PO decreased over time in the laboratory [START_REF] Armitage | The effects of age and social interactions on innate immunity in a leaf-cutting ant[END_REF]. This finding leads to the assumption of either lower total PO values in young, laboratory-born workers or no difference among both worker types in this trait. Compared to young laboratory-born workers, I expected a higher variation in worker size in old field-derived workers, as they were presumably exposed to more diverse environmental conditions during larval development. A large environmental variation of size in this worker type could hide a genetic effect on worker size and I thus assumed higher worker size heritability values in the young, laboratory-born workers.

Experiments were performed on the polymorphic and polyandrous ant C. velox, which offers both genetic and morphological diversity among the workforce. The species reproduces by social hybridogenesis (Eyer et al. 2013b). Within single polygynous colonies, queens combine parthenogenetic reproduction to produce queens (thelytokous parthenogenesis) and males (arrhenotokous parthenogenesis) with sexual reproduction to produce hybrid workers. Nestmate queens are essentially clones and mate with multiple males (generally with 2-5 males) originating from different genetic lineages (Eyer et al. 2013b), thus increasing genetic diversity in the worker force. Colonies are therefore a mix of clones and siblings, ranging in relatedness.

METHODS

Colony collection and rearing

The thermophilic species C. velox occurs on the Iberian Peninsula in arid Mediterranean habitats with sparse vegetation [START_REF] Tinaut | Taxonomic situation of the genus Cataglyphis Förster, 1850 in the Iberian Peninsula II. New position for C. viatica (Fabricius, 1787) and the redescription of C. velox Santschi, 1929 stat. n. (Hymenoptera, Formicidae)[END_REF]). Colonies occupy an underground nest and have small to medium size, containing from hundreds to a few thousand workers. Workers of Cataglyphis species follow the classical age-dependent polyethism, passing from intranidal tasks when young to foraging when older (reviewed in [START_REF] Lenoir | Cataglyphis desert ants: a good model for evolutionary biology in Darwin's anniversary year: a review[END_REF]. C. velox workers are very polymorphic with a continuous worker length range of 4.5-12 mm [START_REF] Tinaut | Taxonomic situation of the genus Cataglyphis Förster, 1850 in the Iberian Peninsula II. New position for C. viatica (Fabricius, 1787) and the redescription of C. velox Santschi, 1929 stat. n. (Hymenoptera, Formicidae)[END_REF][START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF], which enables division of labor in foraging and thus a longer daily activity period. Larger workers withstand higher temperatures, achieve higher running speeds and shorter foraging durations than smaller workers and are able to forage at temperatures deadly for other insects. The species scavenges on fresh cadavers of small arthropods and rarely incorporates plant material into its diet [START_REF] Cerdá | Links between worker polymorphism and thermal biology in a thermophilic ant species[END_REF][START_REF] Cerdá | Behavioural and physiological traits to thermal stress tolerance in two Spanish desert ants[END_REF].

Twenty-five mature queenright colonies of Cataglyphis velox, Santschi, 1929, were collected from a population in the Sierra Nevada near Grenada, Spain (37°08' N, 3°29' W, 1300 meters altitude) in May 2011 (5 ± 4 queens and 1220 ± 820 workers; mean ± standard deviation). Of these colonies, four with the highest quantity of brood were chosen for this experiment. They were polygynous (six queens in colonies A, B, C and 13 in colony D) and contained 600 to 1900 workers and some brood in the egg and early larval stage at collection. I found no sexual offspring, which C. velox colonies typically produce in the Sierra Nevada in July (Eyer et al. 2013b). Complete colonies were brought to the laboratory and reared at a temperature of about 28°C with a 15/9 h day/night light cycle and fed 3 times/week with a mixed diet of live meal worms, frozen crickets, fruit (apple, orange, banana), artificial food (1 L distilled water, 10 g agar, two eggs, 124 g honey, 2 g vitamins (Nature'sPLUS), 2 g salt) and ad libitum sugar and water.

Establishment of young workers in the laboratory

To obtain laboratory-born workers of known age, two queenless subcolonies 1 and 2 were generated per colony, each containing 50 intra-and extranidal nestmates of all sizes. These workers originated from the field (field-derived workers). Three times a week, pupae were transferred from the stock colony to subcolony 1, which served as a nursery, until worker emergence. Subcolony 2 contained no brood and served to maintain newly emerged workers of known age (coming from subcolony 1) that were used in the experiment (laboratory-born workers). All field-derived workers of the two subcolonies were color-marked on the thorax (Uni Paint Marker) in order to be able to differentiate them from laboratory-born workers. The first week, newly emerged workers (lighter and unmarked) were transferred from subcolony 1 to 2 when they had achieved walking capacity. For the subsequent weeks, before the transfer was done from 1 to 2, the young workers of known age (13 ± 2 days) and the same number of field-derived workers from subcolony 2 were sampled for immune defense and size estimations (see below). To exclude an effect of task on immune investment [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]), I tested individuals at an age before they left the nest under laboratory conditions. In C. bicolor, the onset of foraging takes place at about 28 days in the field [START_REF] Schmid-Hempel | Life duration and turnover of foragers in the ant Cataglyphis bicolor (Hymenoptera, Formicidae)[END_REF]. In order to reduce environmental variation during the experiment, for each subcolony and each week, I performed manipulations to the same number of fieldderived workers and laboratory-born workers. The size of the subcolonies changed due to differing numbers of eclosing workers and the use of laboratory and field-derived workers for experiments. Field-derived workers were replenished by transferring them from the stock colonies into subcolony 1 and from subcolony 1 into subcolony 2. The transfer from subcolony 1 to subcolony 2 took place at least 1 week before immune measurements, comparable to eclosing laboratory workers.

Subcolonies were maintained in the same room under the same environmental conditions as the stock colonies and were kept in plastic boxes (27 x 17 x 11 cm) lined with plaster and coated with fluon. The colony boxes contained a plastered petri-dish nest (diameter 9 cm) darkened with cardboard paper, allowing direct access to hatched workers, and were moistened once a week.

Experiments were performed in July 2011 (2 months after collection, colonies B and C), November 2011 (6 months after collection, colony A) and in April 2012 (11 months after collection, colony D) and lasted about 1 month per colony. Colony D differs from the others in that experiments were performed after hibernation in the laboratory (3 months, temperature: 14.7 ± 1.8 °C, humidity: 58.1 ± 7.5 % humidity, fed about once a week with meal worms, orange, sugar and water). Given the seasonal cycle of C. velox (as has been described in C. iberica: [START_REF] Lenoir | Cataglyphis desert ants: a good model for evolutionary biology in Darwin's anniversary year: a review[END_REF], I infer that field-derived workers were at least 8 months old in colonies B and C, 11 months in colony A and 16 months in colony D. In colony A, no young workers eclosed before the experiment started and in colony D (the colony which overwintered), a few workers eclosed the previous summer which were marked with 3 color dots and colored ants were still distinguishable and not used for the experiments the following year. Due to rearing problems in the laboratory, I was not able to include old laboratory-born workers to analyze an effect of age on PO investment. However, since fieldderived workers stayed under controlled laboratory conditions for at least 2 months before the start of the experiment (colony A: 6 months, colony D: 11 months) and were at least 8 months old, they should mainly differ from young laboratory-born workers in their age (and the behavioral and physiological differences associated with it).

Phenoloxidase measurement

The phenoloxidase assay was adapted from [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF] and [START_REF] Helft | Effects of division of labour on immunity in workers of the ant Cataglyphis cursor[END_REF], with slight modifications. The level of the active (active PO) and total amount of phenoloxidase (total PO = active PO and stored PPO) was determined for each individual using a spectrophotometer (96-well Microplate reader 680; Bio-Rad, Hercules, CA, USA). After being anaesthetised on ice, workers were decapitated and hemolymph was removed from cutting sites at the head and thorax with a disposable graduated capillary tube (Hirschmann Laborgeräte ringcaps). All manipulations were conducted on ice-blocks to prevent enzyme activation. Hemolymph was diluted (1:100) in ice-cold sodium cacodilate/CaCl2 buffer (0.01 M Na-Cac, 0.005 M CaCl2, pH = 6.5) and stored at -80°C. Of each individual, 10 µL of diluted hemolymph was mixed with 5 µL of distilled water for active PO measurement and with 5 µL of alpha-chymotrypsin (2 mg mL -1 in distilled water; prepared freshly) for total PO measurement, both performed at the same time. Chymotrypsin proteolytically activates stored PO into active PO and thus allows measurement of the total enzymatic activity of the sample (total PO). Samples were incubated for 5 min at room temperature and 17.5 µL of L-DOPA (4 mg mL -1 in distilled water) was added in each well. The mixture absorbance at 490 nm was measured at 30°C every 10 sec for 50 min. The enzymatic activity was determined as the slope of the reaction curve during the linear phase of the reaction (V max) using Microplate Manager 5.2 software (Bio-Rad, Hercules, CA, USA). The enzyme level is proportional to the optical-density increase induced by the transformation of L-DOPA to dopachrome by phenoloxidase [START_REF] Söderhäll | Role of the prophenoloxidase-activating system in invertebrate immunity[END_REF]. Within-individual repeatability of enzyme measurements was obtained by analysing intra-well (active PO N = 22; total PO N = 23) and inter-well (active PO N = 36; total PO N = 39) replicates of the same individual. Both active PO (Pearson's product-moment correlation; intra-well: r 2 = 0.774; inter-well: r 2 = 0.899) and total PO data (Pearson's product-moment correlation; intra-well: r 2 = 0.683; inter-well: r 2 = 0.887) showed reasonable repeatability. Negative controls (wells containing only distilled water or only buffer) showed horizontal kinetic curves and minimal activity (0 -0.2 V max).

If the absorbance curve was too irregular, these data points were eliminated. The hemolymph volume collected from each worker varied from 0 to 3.00 µL. As active PO and total PO measures required 0.1 µL each, only active PO was assayed when less than 0.2 µL of hemolymph were collected and samples of less than 0.1 µL were excluded from the PO activity analyses. I favored active over total PO as a previous study found a higher variance for active PO among C. velox workers [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]). I obtained active PO values of 341 individuals (180 young laboratory-born workers and 161 old field-derived workers) and total PO values of 310 individuals (171 young laboratory workers and 139 old field-derived workers). Sample size was lower than the total of 425 workers sampled as for many individuals I did either not succeed in collecting enough hemolymph quantity or hemolymph of good quality.

Worker size measurements

The tibia length of the right posterior leg was used as an estimation of individual body size [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]). Tibias were photographed with a Sony XCD-SX910CR camera connected to a binocular microscope and tibia length was measured with the ImageJ software (version 1.40, Wayne Rasband, National Institutes of Health, USA).

Genetic analysis

After taking samples for size and immune measurements, individuals were stored in 90% alcohol with 10% Tris-EDTA. In total, 28 queens and 425 workers were genotyped from the four studied colonies, consisting of 229 laboratory-born and 196 field-derived workers (colony A: 65 laboratory-born and 60 field-derived workers; colony B: 57 laboratory-born and 53 field-derived workers; colony C: 78 laboratory-born and 50 field-derived workers; colony D: 29 laboratory-born and 33 field-derived workers; Table 1). Three queens of colony D could not be retrieved from the colony for genotypic analyses as they had presumably already died and were dismembered by the workers (we found not dead queens on the extranidal waste dumps). Ant DNA was extracted from half of the heads through digestion at 55 °C in a 10% Chelex buffer and 15 µL proteinase K at 10 mg/ml for 2 hours. After 3 min of centrifugation at 1400 g, the supernatant was taken and stored at 4 °C before being quickly used in a polymerase chain (PCR) reaction. Ants were genotyped with five microsatellite markers (CC11, CC26, CC58, CC63, CC99) previously developed by Pearcy et al. (2004b) for C. cursor. Each PCR reaction was carried out in a 10 µL volume, containing 1 µL Buffer 10x, 0.15 µL 0.4 mM dNTA, 0.5 units Taq polymerase (Quiagen) and 1µL DNA. The five loci were amplified in two multiplex PCR reactions, using 100 nM of CC26 and CC58 (first set of loci) or 100 nM of CC11, CC63 and CC89 (second set of loci). The reactions were run in a GeneAmp PCR system 9700 Thermal cycler (Applied Biosystems), starting for 10 min at 94°C to denaturate the DNA, followed by ten cycles of: 15 sec at 94 °C (DNA denaturation), 15 sec at 52 °C (primer annealing phase) and 30 sec at 72 °C (DNA elongation). These cycles were followed by 35 cycles of: 15 sec at 89 °C, 15 sec at 52°C and 30 sec at 72°C. The PCRs were completed with a DNA elongation phase at 72 °C for 10 min. The amplified fluorescent products were separated and visualized using an automated ABI Prism 310 sequencer (Applied Biosystems) with ROX 400 size markers and allele sizes estimated with the Genescan 3.2.1 software (Applied Biosystems).

Patriline determination

The absolute number of patrilines within each colony was estimated by comparing worker and mother genotypes inferred from microsatellite analyses. Even though there were multiple queens in each colony, the queens shared the same multilocus genotype within each colony as observed by Eyer et al. (2013b) except for two queens that were homozygote for a locus which was heterozygous for the other queens. This is probably due to the occurrence of recombination events that lead to homozygosity under meiotic thelytoky with central fusion. This mode of thelytoky has been demonstrated for the related species C. cursor [START_REF] Pearcy | Thelytokous parthenogenesis and its consequences on inbreeding in an ant[END_REF]. From a genetic perspective, colonies could therefore broadly be considered as "quasi"monogynous.

All workers examined, except two, are likely the offspring of the mother queens. These two non-natal workers were excluded from subsequent analysis. The fathers' genotypes were inferred from the queen and worker genotypes. When a patriline contained only a single worker and only differed by a single allele from the other fathers (occurred five times), I preferred to be conservative and considered that the different allele originated from a PCR error or mutation. A new patriline was therefore not created for a single worker and the worker assigned to the closest patriline.

In agreement with the mode of reproduction (Pearcy et al. 2004a;[START_REF] Pearcy | Thelytokous parthenogenesis and its consequences on inbreeding in an ant[END_REF]Eyer et al. 2013b), the level of worker heterozygosity was very high since all except four workers were heterozygotes at all loci. And in these four workers, only one locus was found in the homozygous state. Out of 29 alleles found over the five loci in the fathers, only one allele in one locus was common with the queens and was found at the heterozygous state in workers so that I could not determine the correct father genotype at this locus. I estimated the allelic frequency in the father gene pool without considering the undetermined genotype at a single locus in a single patriline.

Because of the unusual reproductive system of C. velox, with males and females having so different alleles that they could belong to different gene pools (Eyer et al 2013b), it would be incorrect to use the population allelic frequency based on workers to assess the probability of non-detection of a patriline. Given that I observed a total of 49 patrilines, I estimated the allelic frequency only in the male gene pool from the inferred father genotypes. I then estimated the probability that two fathers share the same genotype and would therefore lead to an undetected patriline using the following equation [START_REF] Boomsma | Paternity in Eusocial Hymenoptera[END_REF]:

∏ ∑ = - j i j i ection non f P 2 , det
with f i,j being the allelic frequency of the allele i at locus j in the fathers gene pool. Over the five loci, I obtained a probability that two fathers share the same genotype of 0.005, which suggests that the variability detected with the five microsatellites was sufficiently high for having only a low proportion of patrilines that could remain undetected.

Statistical analysis

To test whether patrilines were equally distributed among workers, I computed the reproductive skew among (i) all workers, (ii) laboratory-born workers and (ii) field-derived workers using the B index. This index is based on the observed variance in a group corrected by the expected variance from the binomial distribution [START_REF] Nonacs | Measuring and using skew in the study of social behavior and evolution[END_REF] and equals zero if patrilines are randomly distributed. I calculated B and tested the null hypothesis that B equals zero by using the program Skew Calculator 2003 (http:// www.eeb.ucla.edu/Faculty/Nonacs). Using two-sided Fisher exact tests, I further determined whether patriline distribution differed among laboratory-born and field-derived workers.

The broad sense heritability ((V A + V D ) / V T ; V A = additive genetic variance, V D = dominance genetic variance, V T = total phenotypic variance) [START_REF] Falconer | Introduction to Quantitative Genetics[END_REF] was estimated based on a half-sib design with sires nested within dam, that is patrilines nested within colonies separately for field-derived and laboratory-born workers. I decomposed the total phenotypic variation ) (

2 T T V σ =
into three variance components: inter-colony variation that included both environmental and genetic differences among colonies

) ( 2 C C V σ =
, interpatriline variation within colonies that included both additive and dominance genetic variance ) ( . The three variance components were estimated using a Linear Mixed-Effects Model (LMM) with patrilines embedded in colony as a random effect and a fixed intercept using Restricted Maximum Likelihood Method (REML) in the package nlme in R [START_REF] Pinheiro | Mixed-Effects Models in S and S-plus[END_REF]. The model was run separately for each worker type to estimate the level of broad sense heritability. Because of the haplodiploid sex determination, inter-patriline variation represents ½ of V A and ½ of V D [START_REF] Liu | Estimating quantitative genetic parameters in haplodiploid organisms[END_REF], hence The level of significance of the effect of patriline was tested by comparing the model with and without the effect of patriline using a log likelihood ratio test with a REML [START_REF] Pinheiro | Mixed-Effects Models in S and S-plus[END_REF]. To run the LMM I excluded the patrilines represented by single workers (eight patrilines for field-derived workers and seven patrilines for laboratory-born workers). For active PO and total PO analyses, data were log transformed to fulfill the assumption of residual normality and homoscedasticity. Moreover, I first tested an effect of the covariate size as a fixed factor in the LMM. If not significant, size was removed from the model to estimate the variance components.

We also ran a LMM on the full data set comprising both field-derived and laboratory-born workers in order to test for differences between both worker types. The full model included as the fixed effect the worker type (young laboratory-born or old field-derived) and as the random effect patriline embedded in colony. The significance level of the fixed effect was tested by comparing the models with and without the fixed effect using a likelihood ratio test.

For this test to be valid, I fitted the two models using the maximum likelihood method (ML) as advised by [START_REF] Pinheiro | Mixed-Effects Models in S and S-plus[END_REF]. The residuals demonstrated that the variance in worker size was higher in field than in laboratory workers. In order to fulfill the assumption of the model and to test whether the variance in laboratory workers was lower than in fieldderived workers, I also included in the model the possibility of having a different variance in laboratory and field-derived workers using the weight function (weights = varIdent(form=~1|AGE)).

To test for an interaction between the effect of worker type and colony, I removed the effect of patriline (which was not significant) in order to add the possibility that the effect of age randomly varied within colonies. I thus included age both as fixed effect and as a random effect within colonies.

RESULTS

Differences between young laboratory-born and old field-derived workers

Worker size:

Workers eclosed in the laboratory had a lower size variance than workers eclosed in the field (L-ratio = 24.295, p < 0.001) and were also smaller (L-ratio = 43.747, p < 0.001) (Figure 1). This was true in all colonies (no interaction between worker size and colony; L-ratio < 0.001; p = 0.999).

Active PO:

Both worker types did not differ in the level of active PO (L-ratio = 0.604; P = 0.437, interaction: L-ratio = 0.820; p = 0.365). Worker size neither affected the level of active PO in all workers (L-ratio = 0.697; p = 0.404), nor in each of the worker types (young laboratoryborn: L-ratio = 0.442; p = 0.506; old field-derived: L-ratio = 1.280; p = 0.258) (Figure 2). Young laboratory-born workers had a higher variance in active PO than old field-derived workers (L-ratio = 35.8; p < 0.001, coefficient 0.61 for laboratory workers). This is especially true for colonies B and C (Figure 2). Whereas I determined in young laboratory-born workers 0.962 ± 1.729 mOD min -1 and in old field-derived workers 0.705 ± 0.612 mOD min -1 active PO, I obtained in young laboratory-born workers 3.974 ± 4.395 mOD min -1 and in old field-derived workers 17.721 ± 11.932 mOD min -1 total PO. These estimates suggest that most of the enzyme was in the inactive (stored) form in both young laboratory-born and old fieldderived workers. The boxplots show mean, quartiles and range of the data.

Total PO:

The level of total PO was not affected by worker size in the laboratory (L-ratio = 0.790; p = 0.374) but was strongly affected by worker size in the field-derived workers (L-ratio = 13.206; p < 0.001). Thus, there was a significant interaction between worker size and type (Lratio = 5.531; p = 0.018). The level of total PO was higher for old field-derived workers than for young workers born in the laboratory (L-ratio = 104.120; p < 0.001) (Figure 3 and Figure 4). The variance in total PO was not different between old field-derived and young laboratory-born workers (L-ratio = 0.052, p = 0.820).

Number of patrilines and patriline distribution

Of the 49 different patrilines detected, 14 were present in colony A, 11 in colony B and 12 in colony C and D each (absolute and effective number of patrilines in Table 1). Patrilines present in a colony were not equitably represented, but some patrilines were present in a higher proportion of workers than others (skew calculation; B values in Table 1; colony A: p = 0; colony B: p = 0; colony C: p = 0; colony D: p = 0.001). The same was true for the Tibia size (mm) Log (total PO) patriline distribution among laboratory-born workers (skew calculation; B values in Table 1; colony A: p = 0; colony B: p = 0; colony C: p = 0) and among field-derived workers (skew calculation; B values in Table 1; colony A: p = 0.006; colony B: p = 0.007; colony C: p = 0.027; colony D: p = 0.011), with the exception of laboratory-born workers in colony D where patrilines were equally represented among workers (p = 0.601). I further found significant differences in patriline distribution comparing laboratory-born and field-derived workers (Fisher's exact tests, Table 1). In colony D, only one of the 12 patrilines was present in both young laboratory-born and old field-derived workers.

Table 1 Sample size and summary statistics of the genetic analysis of young laboratory-born and old field-derived workers and queens of four C. velox colonies. For each colony, results for the complete sample (Overall), the young laboratory-born and old field-derived workers are given. The number of queens analyzed (Nq) is followed by the number of workers analyzed (Nw) and the number of patrilines observed for each colony (Np). Those are followed by the effective paternity number (k e ) and the index of skewness (B). The results of an exact test of patriline differences between field and laboratory workers (T LF ) are further given per colony. Significance level of B and T LF : * p < 0.05; ** p < 0.01; *** p < 0.001; no asterisk: nonsignificant.

Overall

Laboratory-born workers Field-derived workers

Worker Size:

Most of the worker size variation (87%) was neither explained by colony nor by patriline effects (Table 2), both for field-derived and laboratory-born workers. Accordingly, the broad sense heritability for worker size was low (0.197) in the field-derived workers and nearly zero in the laboratory-born workers (Table 2). In agreement with this low worker size variation among patrilines, in both field-derived and laboratory-born workers, the effect of patriline was not significant even though the p value was much lower in the field-derived workers (Lratio = 2.851; p = 0.091) than in the laboratory-born workers (L-ratio < 0.001; p = 0.999), in accordance with a higher level of heritability in the field-derived workers (Table 2).

Table 2 Broad sense heritability (h 2 ) and variance components estimated for old field-derived and young laboratory-born workers for the three parameters worker size, active PO and total PO. The A circle (°) behind a value signifies that the variance could not be properly estimated in the mixed model as there was barely no patriline variation (the real value was < 0.0001 and the confidence intervals were big). The variance level was therefore set to zero.

Field-derived workers

Laboratory-born workers

h 2 σ 2 c σ 2 p σ 2 e h 2 σ

Active PO:

The effect of patriline on the level of active PO was not significant, both in the young laboratory-born workers (L-ratio = 1.467; p = 0.226) and in the old field-derived workers (Lratio = 0.335; p = 0.563). Accordingly, the levels of heritability were low (Table 2). The models explained not more than 10% of the variance in the old field-derived and in the young laboratory-born workers (Table2).

Total PO:

The effect of patriline was not significant, neither in the young laboratory-born (L-ratio < 0.001; p = 0.999) nor in the old field-derived workers (L-ratio < 0.01; p = 0.999). Accordingly, the level of heritability could not be estimated and was considered as zero (Table 2). The models explained no more than 10% of the variance in the field-derived and the laboratory-born workers (Table 2).

DISCUSSION

Phenotypic variation among individuals in a population underlies both genetic and environmental sources, and determination of their respective influence is important to assess the population's potential to adapt to and evolve in heterogeneous environments. In the ant C.

velox I investigated interindividual variation in body size and immune investment based on genetic differences between workers. Unexpectedly, the phenotypic traits worker size and level of active and total PO were not explained by genetic variation within both laboratoryborn and field-derived workers (i.e. heritability values close to zero), even though worker age and environmental conditions were controlled for in young laboratory-born workers (Table 2). Foragers of C. velox possess higher levels of active PO [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]) and I thus expected higher levels of this enzyme in old field-derived workers. However, both worker types did not differ in the level of active PO and the variance for this trait was lower in old field-derived workers. According to the assumption that the level of stored PO decreases over time in the laboratory (Armitage and Boomsma 2010), I detected higher levels of total PO in field-derived workers than in young workers born in the laboratory. The variance for this trait did not differ among worker types. Workers born in the laboratory were presumably exposed to reduced environmental variation during larval development and accordingly showed reduced variance in size compared to field-derived workers. Compared to field-derived workers, they were also smaller. Interestingly, individuals of different size did not differ in their levels of active PO, but total PO was affected by worker size only in old field-derived workers.

Social structure

As previously described by Eyer et al. (2013b), colonies were polygynous with queens sharing the same multilocus genotype and the heterozygous worker force belonged to several different patrilines (11-14 different patrilines per colony; Table 1). I further detected a significant skew in the distribution of patrilines between young laboratory-born and old fieldderived workers, suggesting that either (i) there is a queen turnover and not all queens are equally contributing to egg laying at least temporarily and/or (ii) that sperm are not mixed (sperm mixing seems to be common in some species: e.g. [START_REF] Laidlaw | Polyandry in honey bees (Apis mellifera L.): sperm utilization and intracolony genetic relationships[END_REF][START_REF] Holman | Random sperm use and genetic effects on worker caste fate in Atta colombica leaf-cutting ants[END_REF][START_REF] Stürup | Sperm mixing in the polyandrous leaf-cutting ant Acromyrmex echinatior[END_REF]but No effect of patriline on worker size Similar to other studies (e.g. [START_REF] Bargum | A genetic component to size in queens of the ant, Formica truncorum[END_REF][START_REF] Fournier | Genetic diversity, worker size polymorphism and division of labour in the polyandrous ant Cataglyphis cursor[END_REF][START_REF] Kovacs | Environmental and genetic influences on queen and worker body size in the social wasp Vespula maculifrons[END_REF]Eyer et al. 2013a), I found no heritability in the phenotypic trait worker size, indicating that most of the size variation between workers was due to environmental effects. Moderate heritability of worker size (heritability value = 0.26 ± 0.07) was reported in colonies of doubly-mated queens of the ant Formica selysi [START_REF] Schwander | Division of labour and worker size polymorphism in ant colonies, the impact of social and genetic factors[END_REF]. In Lasius niger and Leptothorax rugatulus, individual size heritability was demonstrated for sexuals [START_REF] Rüppell | Complex determination of queen body size in the queen size dimorphic ant Leptothorax rugatulus (Formicidae : Hymenoptera)[END_REF][START_REF] Fjerdingstad | Control of body size of Lasius niger ant sexuals -worker interests, genes and environment[END_REF]. Heritability estimates are typically context-dependent and independent estimates often do not agree. My relatively small sample size might not provide enough statistical power to detect stronger heritability of worker size in field-derived workers. Additionally, uncontrolled environmental variance could have masked a stronger influence on worker size on C. velox than was found in this study. If worker size is determined by a genotype-environment interaction, providing the same environment might not lead to the phenotypic expression of a patriline effect on size. For instance, if worker size is constrained by food and larval provisioning wasn't adequate, workers could all be of a certain size and no effect of patriline detected. In polygynous C. velox colonies, heritability estimates may have contained maternal effects also. However, these queens shared the same genotype (also described in Eyer et al. 2013b) and environment. Heritability estimates can vary between years (e.g. [START_REF] Bargum | A genetic component to size in queens of the ant, Formica truncorum[END_REF]) and underlie social effects, which can hardly be excluded even under controlled laboratory conditions [START_REF] Rüppell | Complex determination of queen body size in the queen size dimorphic ant Leptothorax rugatulus (Formicidae : Hymenoptera)[END_REF]. The environment of a social insect worker underlies genetic influences as it is shaped by the presence of nestmates (and their genotypes) and individual phenotypes are thus modulated by direct and indirect genetic effects. In honeybees, a colony's genotypic composition affected gene and trait expression of individual bees [START_REF] Linksvayer | Honeybee social regulatory networks are shaped by colony-level selection[END_REF][START_REF] Gempe | Mixing of honeybees with different genotypes affects individual worker behavior and transcription of genes in the neuronal substrate[END_REF]) and influenced the performance and partitioning of hygienic behavior (i.e. the removal of diseased brood; Arathi and Spivak 2001;[START_REF] Gempe | Mixing of honeybees with different genotypes affects individual worker behavior and transcription of genes in the neuronal substrate[END_REF]. Even though heredity of queen body size was demonstrated in the ant Leptothorax rugatulus, it was entirely superseded by social influences [START_REF] Rüppell | Complex determination of queen body size in the queen size dimorphic ant Leptothorax rugatulus (Formicidae : Hymenoptera)[END_REF]. The presence of multiple queens can result in smaller and less polymorphic offspring [START_REF] Rüppell | Complex determination of queen body size in the queen size dimorphic ant Leptothorax rugatulus (Formicidae : Hymenoptera)[END_REF][START_REF] Schwander | Division of labour and worker size polymorphism in ant colonies, the impact of social and genetic factors[END_REF]. Environmental determination of worker size can be advantageous with respect to division of labor, by permitting colonies to adapt worker size ratios to changing environmental conditions and needs (without being genetically fixed; [START_REF] Passera | Increased soldier production in ant colonies exposed to intraspecific competition[END_REF][START_REF] Mcglynn | Food supplementation alters caste allocation in a natural population of Pheidole flavens, a dimorphic leaf-litter dwelling ant[END_REF][START_REF] Hughes | Worker caste polymorphism has a genetic basis in Acromyrmex leaf-cutting ants[END_REF][START_REF] Bargum | A genetic component to size in queens of the ant, Formica truncorum[END_REF]) and by avoiding parasitic worker genotypes specializing in reproduction [START_REF] Hughes | Worker caste polymorphism has a genetic basis in Acromyrmex leaf-cutting ants[END_REF]. The growth of ant larvae is affected by nutrition and pheromones (e.g. Hölldobler and Wilson 1990;[START_REF] Wheeler | Soldier determination in Pheidole bicarinata: inhibition by adult soldiers[END_REF] and soldiers of Pheidole bicarinata ants can inhibit larval development into the same caste through a contact pheromone [START_REF] Wheeler | Soldier determination in Pheidole bicarinata: inhibition by adult soldiers[END_REF]. If a need for soldiers arises in the colony, pheromone levels will decrease and new soldiers develop, allowing colonies to adjust worker size distribution more flexibly and rapidly to current needs than by genetic determination only. Worker size in several species is thus likely determined through interplay of both environmental and genetic factors (Hölldobler and Wilson 1990;[START_REF] Wheeler | Soldier determination in Pheidole bicarinata: inhibition by adult soldiers[END_REF][START_REF] Hughes | Worker caste polymorphism has a genetic basis in Acromyrmex leaf-cutting ants[END_REF]). In the case of Pheidole bicarinata larvae, their response towards soldier pheromone is likely affected by genetically determined response thresholds [START_REF] Hughes | Worker caste polymorphism has a genetic basis in Acromyrmex leaf-cutting ants[END_REF], which differ between individuals and will favor certain genotypes to develop into soldier castes (genetic determination of worker morphology in ants: [START_REF] Hughes | Worker caste polymorphism has a genetic basis in Acromyrmex leaf-cutting ants[END_REF].

No effect of patriline on phenoloxidase Genetic diversity reduces parasite loads and increases disease resistance within social insect colonies (e.g. [START_REF] Liersch | Genetic variation within social insect colonies reduces parasite load[END_REF]Baer andSchmid-Hempel 1999, 2001;[START_REF] Schmid-Hempel | Polyandry Versus Polygyny Versus Parasites[END_REF][START_REF] Tarpy | Genetic diversity within honeybee colonies prevents severe infections and promotes colony growth[END_REF]Hughes and Boomsma 2004;[START_REF] Tarpy | Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queens[END_REF][START_REF] Seeley | Queen promiscuity lowers disease within honeybee colonies[END_REF] but the underlying mechanism remains undetermined [START_REF] Tarpy | Within-and across-colony effects of hyperpolyandry on immune function and body condition in honey bees (Apis mellifera)[END_REF]. In polyandrous honeybees (with naturally occurring mating numbers), genetic diversity did not increase levels of the physiological immune functions encapsulation response and phenoloxidase activity of larvae and these immune components were further not affected by patriline [START_REF] Tarpy | Within-and across-colony effects of hyperpolyandry on immune function and body condition in honey bees (Apis mellifera)[END_REF]. Accordingly, I did not find heritability in the level of phenoloxidase, neither for the active enzyme nor the total amount, in the ant C. velox at naturally occurring mating levels. Presumably, disease defense with the help of phenoloxidase is of too vital importance and thereby indispensable for individuals to vary among the worker force based on genetic differences [START_REF] Tarpy | Within-and across-colony effects of hyperpolyandry on immune function and body condition in honey bees (Apis mellifera)[END_REF]. Indeed, the innate phenoloxidase pathway is a central component of invertebrate disease defense in the hemolymph [START_REF] Söderhäll | Role of the prophenoloxidase-activating system in invertebrate immunity[END_REF]. PO activity has been linked for instance to reduced susceptibilty to fungal (e.g. [START_REF] Ochiai | Purification of a beta-1,3-glucan recognition protein in the prophenoloxidase activating system from hemolymph of the silkworm, Bombyx mori[END_REF], bacterial (e.g. [START_REF] Pye | Microbial activation of prophenoloxidase from immune insect larvae[END_REF]) and viral (e.g. [START_REF] Beck | A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway[END_REF] diseases. Unexpectedly, heritability of hemolymph phenoloxidase and the ability to encapsulate a foreign body have been demonstrated in nonsocial insects (e.g. [START_REF] Ryder | Quantitative genetics of immune function and body size in the house cricket, Acheta domesticus[END_REF][START_REF] Cotter | Heritability of immune function in the caterpillar Spodoptera littoralis[END_REF][START_REF] Schwarzenbach | Sex and immunity in the yellow dung fly Scathophaga stercoraria[END_REF]. Hypothetically, the observed genetic variation in these traits arises from antagonistic pleiotropy (i.e. a gene positively affects one trait and negatively another), which is the genetic basis of trade-offs between traits (Roff 1992, as cited in [START_REF] Cotter | Heritability of immune function in the caterpillar Spodoptera littoralis[END_REF][START_REF] Cotter | Costs of resistance: genetic correlations and potential trade-offs in an insect immune System[END_REF]. Presumably, different selection pressures of non-social and social insects lead to differences in the heritability of immune traits.

As PO activity involves costly immunopathology (e.g. [START_REF] Sadd | Self-harm caused by an insect's innate immunity[END_REF], conditional expression depending on the environment or performed task can be advantageous, with individuals performing the riskiest tasks (these are often the oldest workers in colonies with age polyethism) possessing higher PO levels and thus presumably better protection than their nestmates. Behaviors leading to increased parasite exposure (foraging, frequent nest relocation) correlate with higher levels of active PO (C. velox: Bocher et al. 2007) and stored PPO [START_REF] Scharf | Characterizing the collective personality of ant societies: aggressive colonies do not abandon their home[END_REF]. Different immune mechanisms such as behavioral defenses (e.g. hygienic behavior: [START_REF] Spivak | Varroa destructor infestation in untreated honey bee (Hymenoptera: Apidae) colonies selected for hygienic behavior[END_REF]Padhila et al. 2013;[START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]allogrooming: Ugelvig et al. 2010) or different physiological defense mechanisms (e.g. antimicrobial peptide abaecin: [START_REF] Decanini | Variation and heritability in immune gene expression by diseased honeybees[END_REF]; general antibacterial activity: [START_REF] Armitage | Immune defense in leafcutting ants: a cross-fostering approach[END_REF]metapleural gland size: Hughes et al. 2010;[START_REF] Armitage | Immune defense in leafcutting ants: a cross-fostering approach[END_REF]) seem to underlie stronger genetic modulation and could result in increased disease resistance in genetically diverse colonies. Indeed, Cardiocondyla obscurior colonies showed earlier performance of hygienic behavior and higher allogrooming frequencies than inbred colonies with reduced genetic diversity [START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]. Heritability of hygienic behavior in Africanized honeybees was determined to be high (heritability estimate = 0.52), providing potential for selection to act [START_REF] Padilha | Genetic parameters for five traits in Africanized honeybees using Bayesian inference[END_REF].

Difference between young laboratory-born and old field-derived workers I expected higher heritability estimates from laboratory-born workers than from field-derived workers due to less variable environmental conditions [START_REF] Falconer | Introduction to Quantitative Genetics[END_REF]. However, a comprehensive meta-analysis suggests that there is no significant difference between heritability estimates determined in the laboratory and from natural populations (Weigensberg and Roff 1997). Because of the uncontrolled age (and correlated task) and environmental conditions in old field-derived workers, I further expected a higher variation among colonies or a higher uncontrolled variation of the different traits in this worker type. However, both worker types did not differ in total PO heterogeneity. Unexpectedly, young laboratory-born workers showed higher variance in active PO than field-derived workers of higher age and with a higher uncontrolled variance in age.

According to previous studies, I expected lower levels of active PO in the laboratory-born workers, because (i) they had a lower risk of pathogen exposure and (ii) they were younger than field-derived workers. Behaviors leading to increased parasite exposure (foraging, frequent nest relocation) correlate with higher levels of active PO in C. velox [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]) and stored PO [START_REF] Scharf | Characterizing the collective personality of ant societies: aggressive colonies do not abandon their home[END_REF]) and increasing age led to higher levels of active PO in Acromyrmex octospinosus leaf-cutting ants [START_REF] Armitage | The effects of age and social interactions on innate immunity in a leaf-cutting ant[END_REF]. In bumblebees however, older individuals possessed lower levels of active PO [START_REF] Moret | Immune responses of bumblebee workers as a function of individual and colony age: senescence versus plastic adjustment of the immune function[END_REF], suggesting that an effect of age on active PO investment is not always clear and differs between species. I found no difference between both worker types in the level of active PO, which supports the assumption of [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF] that it is indeed task performance and not age which results in higher levels of active PO in C. velox in foraging ants. Because of the pathological effects of PO activation (e.g. toxic side products harming the host: [START_REF] Sadd | Self-harm caused by an insect's innate immunity[END_REF], the enzyme should only be activated when necessary, which might explain the same level of active PO in old field-derived and young laboratory-born workers when tested under laboratory conditions several months after collection (2-11 months). Neither task performance [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]) nor age [START_REF] Armitage | The effects of age and social interactions on innate immunity in a leaf-cutting ant[END_REF][START_REF] Roberts | Immunosenescence and resistance to parasite infection in the honey bee, Apis mellifera[END_REF] affected the level of total or stored PO in previous studies. To the contrary, I detected lower levels of total PO in young laboratory-born workers. This finding is in agreement with a study on Acromyrmex octospinosus leaf-cutting ants [START_REF] Armitage | The effects of age and social interactions on innate immunity in a leaf-cutting ant[END_REF], where the level of stored PO was negatively correlated with the duration colonies stayed in the laboratory, even though these colonies were not in a poorer condition. Both results suggest that controlled laboratory conditions decrease the need of individuals to invest into total or stored phenoloxidase, liberating resources for other life-history traits. Differences to previous studies on social insects might further arise from the fact that I tested older individuals (fieldderived workers) which overwintered in the field or both in the field and in the laboratory, thus comparing a bigger age span. Previously, social insect workers were either tested in annual colonies (e.g. [START_REF] Helft | Effects of division of labour on immunity in workers of the ant Cataglyphis cursor[END_REF][START_REF] Moret | Immune responses of bumblebee workers as a function of individual and colony age: senescence versus plastic adjustment of the immune function[END_REF] or up to an age of about 2-4 weeks (e.g. Wilson-Rich et al. 2008;[START_REF] Roberts | Immunosenescence and resistance to parasite infection in the honey bee, Apis mellifera[END_REF].

Worker size, a trait which can be environmentally determined (e.g. [START_REF] Wood | Quantification and modification of worker size variation in the fire ant Solenopsis invicta[END_REF][START_REF] Gibson | Soldier production in Camponotus novaeboracensis during colony growth[END_REF][START_REF] Tschinkel | Colony growth and the ontogeny of worker polymorphism in the fire ant, Solenopsis invicta[END_REF]) but does not vary with age in holometabolous insects (e.g. Hölldobler and Wilson 1990;[START_REF] Nylin | Plasticity in life history traits[END_REF], was observed to be lower in laboratory-raised workers and showed higher heterogeneity in field-derived workers. Accordingly, laboratory-born workers of the ant Temnothorax nylanderi were smaller than workers collected in the field [START_REF] Canovas | Effets de la température sur la variation de taille des ouvrières et la production d'intercastes chez Temnothorax nylanderi[END_REF]. Both findings might have resulted from colony disturbance by transferring them to the laboratory. In bumblebees, where worker size gradually increases in annual colonies, a transfer to the laboratory resulted in decreased size of consecutive age groups [START_REF] Knee | The seasonal size increase of bumblebee workers (Hymenoptera: Bombus)[END_REF]. [START_REF] Wetterer | The ecology and evolution of worker size-distribution in leaf-cutting ants (Hymenoptera: Formicidae)[END_REF] further observed laboratory Trachymyrmex colonies in declining health producing smaller workers. Even though food was diverse (live meal worms, frozen crickets, apple, orange, banana, a mix of proteins, minerals and vitamins) and provided three times per week in addition to sugar and water available ad libitum, the different diet might not have been sufficient for the production of bigger workers in the laboratory. Additionally, higher temperatures in the laboratory could have accelerated larval development (as has been demonstrated for the ant Solenopsis invicta: [START_REF] Porter | Impact of temperature on colony growth and developmental rates of the ant, Solenopsis invicta[END_REF]) and resulted in smaller eclosing workers.

Worker size and phenoloxidase

We observed a correlation between worker size and total PO in old field-derived workers, however not for active PO. This finding is in accordance with a previous study in this species, where the correlation between worker body size and total PO was much stronger than between size and active PO [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]). The larger field-derived workers presumably have lower maintenance costs of the same mass of tissue than smaller field-derived workers, leaving more resources for immune investment [START_REF] Peters | The ecological implications of body size[END_REF][START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]. Findings of different studies do not demonstrate this relationship, as bigger Cataglyphis cursor workers showed higher levels of active PO [START_REF] Helft | Effects of division of labour on immunity in workers of the ant Cataglyphis cursor[END_REF]) and larger Acromyrmex octospinosus workers did not vary in immune investment from smaller workers (the sampling did however exclude the smallest workers; [START_REF] Armitage | The effects of age and social interactions on innate immunity in a leaf-cutting ant[END_REF]. Research on different immune parameters agrees with my findings, as a correlation between worker size and metapleural gland size [START_REF] Hughes | Caste-specific expression of genetic variation in the size of antibiotic-producing glands of leaf-cutting ants[END_REF], as well as between individual size and the strength of the encapsulation response [START_REF] Vitikainen | Inbreeding and caste-specific variation in immune defence in the ant Formica exsecta[END_REF], have been described.

Interestingly, the link between size and total PO disappeared in the young laboratory-born workers. This suggests that the relationship does not strictly result from a physiological link between worker size and total PO. The mechanisms behind the link between body size and immune investment in old field-derived but not in young laboratory-born workers remain unclear and deserve further empirical attention. Furthermore, comprehensive studies are warranted which compare the heritability of several different immune parameters for a holistic view, using a large sample size and repeating measurements in different habitats and/or over several years. Especially the investigation of social immune defense mechanisms could be promising to disentangle how colony-level genetic diversity increases disease resistance.

CONCLUSIONS AND PERSPECTIVES

7.1 What are the benefits of interindividual variation in immune investment?

Behavioral performance of sanitary tasks

Division of labor is considered a key factor of the ecological success of social insects [START_REF] Oster | Caste and ecology in the social insects[END_REF][START_REF] Wilson | The Insect Societies[END_REF]) and it arises through variability among nestmates and a certain level of intraindividual consistency in task performance (reviewed in Beshers and Fewell 2001;[START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. In theoretical studies, interindividual variability benefited groups by permitting increased resilience when faced with external challenges, by decreasing fluctuations in task-associated stimulus levels and allowing more efficient allocation of workers to tasks (reviewed in [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Sanitary division of labor could increase colony survival not only by reducing the number of individuals in direct parasite contact, but also by increasing heterogeneity in nestmate connectivity and thereby limiting parasite transmission within the colony (reviewed in [START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]. Indeed, leaf-cutting ant workers performing waste management and potentially harbouring parasitic contaminations neither engaged in foraging nor entered chambers with their mutualistic fungus, and were socially avoided and aggressed, likely to minimize disease spread within the nest (Hart andRatnieks 2001, 2002;Hart et al. 2002;[START_REF] Ballari | Waste management in the leaf-cutting ant Acromyrmex lobicornis: Division of labour, aggressive behaviour, and location of external refuse dumps[END_REF]). The benefits and costs of division of labor and task specialization (i.e. an individual's preferential performance of one task over others) for a colony will strongly depend upon the level of both task specialization and division of labor existing for a specific trait. The performance of sanitary work by task specialists can reduce task switching costs, limit parasite exposure to these individuals and might improve task performance efficiency (e.g. [START_REF] Jeanne | The organization of work in Polybia occidentalis: costs and benefits of specialization in a social wasp[END_REF]Trumbo and Robinson 1997;Trumbo et al. 1997;[START_REF] Julian | Undertaking specialization in the desert leaf-cutter ant Acromyrmex versicolor[END_REF]; but see [START_REF] Dornhaus | Specialization does not predict individual efficiency in an ant[END_REF].

Spatial and temporal heterogeneity in the environment are assumed to be important factors in maintaining interindividual diversity [START_REF] Nettle | The evolution of personality variation in humans and other animals[END_REF][START_REF] Burns | Diversity of speed-accuracy strategies benefits social insects[END_REF]. Colony flexibility in the response towards environmental heterogeneity can be achieved through amongnestmate variation in the way a task is performed (i.e. 'behavioral types': [START_REF] Jandt | Behavioural syndromes and social insects: personality at multiple levels[END_REF], even in species with low genetic diversity (e.g. Bombus terrestris). Nestmate bees (B.

terrestris and the highly genetically variable Apis mellifera) differ in their foraging approaches (e.g. fast and imprecise or slow and precise) which allows colonies to respond more quickly to environmental fluctuations [START_REF] Chittka | Bees trade off foraging speed for accuracy[END_REF][START_REF] Burns | Diversity of speed-accuracy strategies benefits social insects[END_REF]Chittka et al. 2009). [START_REF] Burns | Diversity of speed-accuracy strategies benefits social insects[END_REF] suggest that this variability is maintained because it decreases the variation in resource acquisition in the field and they further assume that variation in resource availability will more likely negatively affect a single foraging approach than multiple approaches. Social insects come in contact with a diversity of parasites (e.g. Hughes et al. 2004), whose occurrence is expected to vary spatially and temporally. Such heterogeneity in parasite occurrence could favour the existence of different behavioral defense approaches within the same workforce.

Empirical work demonstrated benefits to maintaining genetic variation within colonies, as it confers behavioral variability and can thereby improve the colony phenotype (reviewed in Oldroyd andFewell 2007 and[START_REF] Jeanson | Long-term dynamics in proximity networks in ants[END_REF]Weidenmüller 2013). Increased behavioral variability in the workforce may increase the range of worker response behaviors, could improve task allocation and reduce costs associated with task switching [START_REF] Goldsby | Task-switching costs promote the evolution of division of labor and shifts in individuality[END_REF][START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Polyandry leads to the presence of several patrilines which can vary in task-associated response thresholds and this variation was demonstrated to improve intranidal thermal homeostasis in honeybees [START_REF] Jones | Honey bee nest thermoregulation: diversity promotes stability[END_REF]). According to the response threshold model, interindividual differences in response thresholds allow graded colony responses to increasing stimulus levels and thereby flexible responses towards changing internal and external conditions (reviewed in Beshers andFewell 2001 andin Oldroyd andFewell 2007). Both polyandry and polygyny modulate division of sanitary labor in bees and ants, as subfamily origin influences the probability of an individual to engage in a sanitary task [START_REF] Robinson | Genetic determination of guarding and undertaking in honey-bee colonies[END_REF][START_REF] Oldroyd | Task specialization in a wild bee, Apis florea (Hymenoptera: Apidae), revealed by RFLP banding[END_REF][START_REF] Donnell | Genetic effects on task performance, but not on age polyethism, in a swarm-founding eusocial wasp[END_REF]Waddington et al. 2010;Eyer et al. 2013a;Robinson andPage 1988, 1995;[START_REF] Frumhoff | A genetic component to division of labour within honey bee colonies[END_REF][START_REF] Pérez-Sato | Multi-level selection for hygienic behaviour in honeybees[END_REF]). These task preferences likely arise through lower response thresholds for sanitary stimuli, since workers performing hygienic behavior have reduced thresholds to take care of diseased brood [START_REF] Masterman | Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)[END_REF]Gramacho and Spivak 2003). The existence of these low-thresholds individuals is essential, as it permits colonies to eliminate sanitary hazards before parasites can establish within the nest. It has been observed that allogrooming generally occurs immediately upon the detection of a parasite-exposed individual or brood item, before infectious particles can enter the insect body and cause an infection (chapter one and e.g. [START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF][START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]. Individuals with high response thresholds will be unlikely to perform sanitary tasks in healthy colonies and thereby remain available to perform different tasks such as foraging or nest repair. These workers could serve as reserve labor during colony invasion by a parasite, when high levels of task-associated sanitary stimuli will be reached. It is generally assumed that individuals are totipotent and able to switch to other tasks if need arises [START_REF] Gordon | The organization of work in social insect colonies[END_REF]Beshers and Fewell 2001 and the references therein). Indeed, individuals can even switch between foraging and cadaver management, which has been observed in chapter two and demonstrated for waste workers of Pogonomyrmex barbatus [START_REF] Gordon | Dynamics of task switching in harvester ants[END_REF]) and necrophoric individuals of Myrmica rubra (Diez et al. 2013a). A switch to other tasks will depend on several factors, such as an individual's degree of behavioral plasticity (e.g. Dingemanse and Wolf 2013) and the number of individuals engaged in other tasks [START_REF] Gordon | The organization of work in social insect colonies[END_REF].

A highly variable workforce further possesses a higher probability to include rare outlier phenotypes, such as necrophoric individuals in honeybees [START_REF] Robinson | Genotypic constraints on plasticity for corpse removal in honey bee colonies[END_REF], which can benefit colonies under certain environmental conditions [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Demographic diversity of Zootermopsis dampwood termites reduced disease susceptibility of individuals compared to demographically homogeneous groups [START_REF] Rosengaus | Disease susceptibility and the adaptive nature of colony demography in the dampwood termite Zootermopsis angusticollis[END_REF]. Genetic diversity can also improve colony resistance to disease [START_REF] Liersch | Genetic variation within social insect colonies reduces parasite load[END_REF][START_REF] Baer | Experimental variation in polyandry affects parasite loads and fitness in a bumble-bee[END_REF][START_REF] Schmid-Hempel | Polyandry Versus Polygyny Versus Parasites[END_REF][START_REF] Baer | Unexpected consequences of polyandry for parasitism and fitness in the bumblebee, Bombus terrestris[END_REF]Hughes and Boomsma 2004;[START_REF] Tarpy | Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queens[END_REF][START_REF] Seeley | Queen promiscuity lowers disease within honeybee colonies[END_REF][START_REF] Reber | Experimentally increased group diversity improves disease resistance in an ant species[END_REF][START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]; but see e.g. [START_REF] Schmidt | No benefit in diversity? The effect of genetic variation on survival and disease resistance in a polygynous social insect[END_REF]), but the underlying mechanisms are not yet elucidated. Genetic diversity could increase the efficiency of sanitary task performance and intranidal communication of task needs, since it improved foraging-related signaling in honeybee colonies [START_REF] Mattila | Genetic diversity within honeybee colonies increases signal production by waggle-dancing foragers[END_REF][START_REF] Mattila | Does a polyandrous honeybee queen improve through patriline diversity the activity of her colony's scouting foragers?[END_REF]. Additionally, Cardiocondyla obscurior colonies showed earlier performance of hygienic behavior than inbred colonies with reduced genetic diversity [START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]). Genetic diversity had, however, no impact on short-term task efficiency for necrophoresis in the ant Linepithema humile [START_REF] Rosset | Experimental manipulation of colony genetic diversity had no effect on short-term task efficiency in the Argentine ant Linepithema humile[END_REF]). More research is necessary to understand the relationship between genetic diversity, individual behavioral differences and division of labor to answer questions such as whether the occurrence of different patrilines increases task specialization within the colony [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF] and could thereby improve disease resistance.

Physiological immune investment

Individual physiological immune investment typically differs among nestmates, but it is not always clear why variation within the workforce is maintained and why not all individuals show an optimal approach. Why do some individuals invest more in immune defense opposed to others, even though there are costs involved? And why do some individuals invest less even though social insects are assumed to be under high selection pressure imposed by parasites? Interindividual variability in how individuals respond to disease seems to reflect two different types of selection. First, life-history and resource tradeoffs in disease resistance could lead to varying investment among workers, with some individuals maintaining weak immune defenses (low cost but risky) while others invest more heavily (high cost but better protection; Evans and Spivak 2010). Indeed, individuals which perform tasks with a higher likelihood of parasite exposure seem to invest more into physiological defenses (but see: [START_REF] König | Foraging activity and immunocompetence in workers of the bumble bee, Bombus terrestris L[END_REF][START_REF] Doums | Immunocompetence in workers of a social insect, Bombus terrestris L., in relation to foraging activity and parasitic infection[END_REF]. Cataglyphis velox foragers possess higher levels of active phenoloxidase [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]) and waste workers of Atta sexdens rubropilosa bigger metapleural glands than workers engaged in less risky tasks [START_REF] Lacerda | Morphometry of the metapleural gland of workers engaged in different behavioral tasks in the ant Atta sexdens rubropilosa[END_REF]. A mixed strategy might be more adaptive as colonies whose members invest more strongly into individual immune defense are less productive [START_REF] Moret | Social life-history response to individual immune challenge of workers of Bombus terrestris L.: a possible new cooperative phenomenon[END_REF][START_REF] Evans | Colony-level impacts of immune responsiveness in honey bees, Apis mellifera[END_REF]. Second, interindividual variation in disease resistance (e.g. in resistance alleles of patrilines) could be an adaptation to host-parasite coevolution. An increased genetic diversity in offspring through sexual reproduction is believed to be advantageous to hosts under strong pressure from parasites adapted to the most common host genotype ('Red Queen Hypothesis': Ladle 1992 and the references therein). A more diverse workforce might be more likely to successfully recognize and fight different invading parasite strains or species than homogenous nestmates [START_REF] Evans | Socialized medicine: individual and communal disease barriers in honey bees[END_REF].

Potential costs of interindividual variation

Whereas the potential benefits of interindividual variation (especially linked to increased genetic diversity) have received research attention, the potential costs and limits of worker variability await further study. The mechanisms underlying worker variability presumably entail different costs at both the individual and the colony level [START_REF] Auld | Re-evaluating the costs and limits of adaptive phenotypic plasticity[END_REF][START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Experience-based differentiation requires increased resources for neuronal plasticity, especially in mushroom bodies (e.g. [START_REF] Laughlin | The metabolic cost of neural information[END_REF][START_REF] Dukas | Costs of memory: ideas and predictions[END_REF][START_REF] Farris | Experience-and age-related outgrowth of intrinsic neurons in the mushroom bodies of the adult worker honeybee[END_REF][START_REF] Riveros | Brain allometry and neural plasticity in the bumblebee Bombus occidentalis[END_REF]. Studies on fruit flies with artificially selected enhanced learning abilities demonstrated fitness costs (e.g. reduced larval competitive ability and decreased egg-laying rates) of learning under low food availability (Mery andKawecki 2003, 2004). Furthermore, gaining experience requires time and errors may occur occasionally [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. Additional genetic material, such as plasticity-regulating loci [START_REF] Schlichting | Control of phenotypic plasticity via regulatory genes[END_REF], is presumably necessary to encode not only a trait, but also its plasticity (Williams 1966, as cited in [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. In colony disease defense, interindividual variability in social interactions can lead to highly connected "super-spreaders", which are able to increase the transmission of disease within the colony [START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF].

To establish a genetically diverse workforce, a reproductive female either has to share the nest and reproduction with others (polygyny) or has to mate multiply, which often incurs costs (polyandry), such as the risk of injury and disease transmission, an increased risk of predation and increased investment of time and energy (Crozier and Fjerdingstand 2001). According to kin selection theory, increased genetic diversity will decrease relatedness among workers (e.g. [START_REF] Boomsma | Multiple paternity, relatedness and genetic diversity in Acromyrmex leaf-cutter ants[END_REF]) and thereby reduce the incentive for cooperation and increase the potential for intranidal conflicts (reviewed in [START_REF] Ratnieks | Conflict resolution in insect societies[END_REF]. Reproductive conflicts among workers led to immunosuppression in groups of the ant Diacamma sp. "nilgiri" [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF]) and could negatively impact non-reproductive division of labor as workers might instead invest into dominance interactions or reproduction. The occurrence of several different subfamilies within one nest could also lead to nepotism (i.e. favoring of one's own kin), where workers use hydrocarbon profile cues to preferentially rear larvae of their own patri-or matriline, if colony costs are not prohibitive. Although evidence for nepotism is generally weak in social insects, workers of the ant Formica fusca are able to detect kin relationships and favor their closest kin when rearing brood in polygynous colonies [START_REF] Hannonen | Worker nepotism among polygynous ants[END_REF]. Furthermore, higher genetic diversity might also make colonies more susceptible towards a larger suite of parasites, possibly increasing rates of acquiring infections [START_REF] Van Baalen | The costs and benefits of genetic heterogeneity in resistance against parasites in social insects[END_REF]. The potential costs linked to increased genetic diversity can be lower in polygynous colonies if reproductive queens are related and/or if there is a queen turnover (i.e. reduced reproductive lifespan of a single queen; van Baalen and Beekman 2006 and the references therein). In C. velox, nearly all queens are practically clones (chapter three and Eyer et al. 2013b) and multiply mated with males from another genetic lineage, leading to a high level of heterozygosity in the workforce (Eyer et al. 2013b). I further detected a significant skew in the distribution of patrilines between young laboratory-born and old field-derived workers, suggesting that either (i) not all queens are equally contributing to egg laying at least temporarily (queen turnover) and/or (ii) that sperm are not mixed (sperm mixing seems to be common in some species: e.g.

The modulation of sanitary division of labor and the costs and benefits of helping

Sanitary division of labor (i.e. individuals perform different sanitary tasks from the group repertoire) is considered a group-level trait and its expression (i.e. strength) will presumably depend on the investigated species, colony size, colony genetic and demographic diversity, on the focal task, individual experience and on the task-associated stimulus level (i.e. parasitic pressure). Increased division of labor is expected in bigger colonies (Bourke 1999;[START_REF] Thomas | Colony size affects division of labour in the ponerine ant Rhytidoponera metallica[END_REF]Jeanson et al. 2007;[START_REF] Holbrook | Division of labor increases with colony size in the harvester ant Pogonomyrmex californicus[END_REF]) and for more virulent parasites, which pose a higher sanitary risk. On the other hand, highly virulent parasites or high parasite abundance might lead to a weaker division of labor. "All hands on deck" could be needed to deal with the sanitary hazard, including task performance of individuals with high response thresholds for this task. The number of task-performing individuals (i.e. low or strong division of labor for this task) and their identity (e.g. high versus low social connectivity, high versus low activity level, naïve versus experienced) will affect intra-colony parasitic spread [START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]. Relatively weak division of labor seems to occur for cadaver management in Cataglyphis velox and a significant percentage of cadaver transporters were also observed foraging (chapter two). Presumably, the sanitary risk arising due to the management of nestmate cadavers is either limited or also occurs for foragers scavenging on fresh cadavers of other arthropods. The parallel performance of both tasks might thus not increase intranidal disease transmission in this species. At low exposure risk, a weak division of sanitary labor and thereby task performance by a higher percentage of nestmates might be expected if exposure results in immune priming and confers survival benefits upon a secondary parasite contact (e.g. [START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]. A strong division of sanitary labor would be expressed in a 'sanitary caste', whose existence has not yet been reported in social insects.

If collective sanitary defense is efficient against the establishment of parasites within the nest, the costs of colony-level physiological immune investment can be reduced [START_REF] Evans | Immune pathways and defence mechanisms in honey bees Apis mellifera[END_REF][START_REF] Simone | Resin collection and social immunity in honey bees[END_REF]).

The performance of sanitary tasks could affect an individual's cuticular hydrocarbon profile, either through direct parasite effects or through the activation of the immune system (e.g. [START_REF] Richard | Modulation of social interactions by immune stimulation in honey bee, Apis mellifera, workers[END_REF], which could increase agonistic interactions (e.g. [START_REF] Hart | Task partitioning, division of labour and nest compartmentalisation collectively isolate hazardous waste in the leafcutting ant Atta cephalotes[END_REF][START_REF] Richard | Modulation of social interactions by immune stimulation in honey bee, Apis mellifera, workers[END_REF] or decreasing social interactions and thereby reinforcing division of sanitary labor.

The performance of behavioral sanitary tasks can represent a double-edge sword both at the level of the task performer and the colony. Parasite-exposed and immune-stimulated individuals are groomed by nestmates which increases survival of the exposed individual through the removal of parasitic stages (e.g. [START_REF] Drees | Susceptibility and behavioral response of red imported fire ant (Hymenoptera: Formicidae) to selected entomogenous nematodes (Rhabditida: Steinernematidae & Heterorhabditidae)[END_REF]Rosengaus et al. 1998b;Hughes et al. 2002;[START_REF] Little | Defending against parasites: fungusgrowing ants combine specialized behaviours and microbial symbionts to protect their fungus gardens[END_REF][START_REF] Richard | Social management of LPS-induced inflammation in Formica polyctena ants[END_REF][START_REF] Yanagawa | Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi[END_REF][START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF][START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF]Tragust et al. 2013a). But at the same time, performing sanitary care increases the risk of disease uptake by the caregiver (e.g. [START_REF] Kramm | Termite pathogens: transfer of the entomopathogen Metarhizium anisopliae between Reticulitermes sp. Termites[END_REF]Cremer et al. 2007) and could facilitate parasitic spread within the colony [START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF]Hughes et al. 2002;[START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]reviewed in Stroeymeyt et al. 2014). In the leaf-cutting ant Atta sexdens rubropilosa, media workers who attempted to pile and cover Metarhizium anisopliae conidiospores had a higher mortality than nestmates with less contact (Jaccoud et al. 1999). Chapter one and other studies using Metarhizium anisopliae point however to a low risk of disease contraction among nestmates of different developmental stages (adults: Rosengaus et al. 1998;Hughes et al. 2002;brood: Ugelvig et al. 2010;Tragust et al. 2013a). A caregiver might further have to deal with costs associated with higher physiological immune investment to limit the increased risk of infection (e.g. [START_REF] Sadd | Self-harm caused by an insect's innate immunity[END_REF][START_REF] Lacerda | Morphometry of the metapleural gland of workers engaged in different behavioral tasks in the ant Atta sexdens rubropilosa[END_REF][START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]). These costs suggest that providing sanitary care requires altruistic acts of the task-performing nestmate. But performing sanitary tasks can increase inclusive fitness of the caregiver, if copies of its genes are passed on to the next generation (Hamilton's Kin selection theory [START_REF] Hamilton | The genetical evolution of social behaviour I, II[END_REF][START_REF] Foster | Kin selection is the key to altruism[END_REF]. Through priming of the physiological immune system, the caregiver can even receive survival benefits upon a future exposure with the same parasite (e.g. [START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]. A naïve individual might thus only limit its contact to parasites and exposed or infected nestmates at high parasite infectivity or quantity (Diehl-Fleig and Lucchese 1991;[START_REF] Milner | Biological control of termites: results and experiences within a CSIRO project in Australia[END_REF][START_REF] Mburu | Relationship between virulence and repellency of entomopathogenic isolates of Metarhizium anisopliae and Beauveria bassiana to the termite Macrotermes michaelseni[END_REF] or if the individual itself is highly susceptible or valuable [START_REF] Ugelvig | Social prophylaxis: group interaction promotes collective immunity in colonies[END_REF]. It is still unknown under which conditions an individual should either avoid a sanitary hazard or increase immune defense investment and research investigating the cost-benefit ratio of caregivers as well as parasite transmission within colonies, should receive more attention (Cremer et al. 2007;[START_REF] Ugelvig | Social prophylaxis: group interaction promotes collective immunity in colonies[END_REF][START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF].

Who is expected to invest more heavily into immune defense?

As the performance of parasite defense can be costly, individuals with a lower residual value for the colony, such as old workers, are generally expected to perform these tasks ('residual value theory' e.g. [START_REF] Kolmes | Stinging behavior and residual value of worker honey bees (Apis mellifera)[END_REF][START_REF] Bot | Waste management in leaf-cutting ants[END_REF]. Indeed, directionality in the mechanisms of task allocation has been detected with workers switching to more dangerous tasks with age [START_REF] Schmid-Hempel | Transmission of a pathogen in Bombus terrestris, with a note on division of labour in social insects[END_REF][START_REF] Tripet | Foraging for work and age-based polyethism: The roles of age and previous experience on task choice in ants[END_REF]). In chapter two, cadaver transporters showed significant overlap with foraging individuals, which might signify that cadaver transporters belong indeed to the oldest workers of the colony or that age polyethism is less pronounced in C. velox. Irregularities in the agepolyethism schedule have been described for the closely related Cataglyphis cursor (Retana andCerdá 1990, 1991). Honeybees performing hygienic behavior and necrophoresis, as well as leaf-cutting ants performing waste-management, are older than nurses but younger than foragers (Arathi et al. 2000;[START_REF] Breed | Demand for task performance and workforce replacement: Undertakers in honeybee, Apis mellifera, colonies[END_REF][START_REF] Mersch | Tracking individuals shows spatial fidelity is a key regulator of ant social organization[END_REF][START_REF] Camargo | Age polyethism in the leaf-cutting ant Acromyrmex subterraneus brunneus Forel, 1911 (Hym., Formicidae)[END_REF]Waddington and Hughes 2010). Having individuals of medium age perform sanitary tasks could be advantageous for the colony, as these individuals do not risk transmitting disease to the brood, but are young enough to be able to profit from an eventual immune protection acquired during task performance. In the case of sanitary brood care (chapter one), nurses seem most likely to perform the task within the nest as they will be the first to notice the sanitary threat and possess the highest probability to respond towards (non-sanitary) brood stimuli. Older worker cohorts typically have reduced contact with the brood. Rapid detection of diseased brood is especially important in ants, where brood is typically stacked facilitating cross-infection [START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF]Tragust et al. 2013b). If the physiological status of an individual interacts with its expression of sanitary tasks, one would expect workers performing sanitary tasks (and having the highest risk of parasite exposure) to show increased physiological immune investment [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF][START_REF] Lacerda | Morphometry of the metapleural gland of workers engaged in different behavioral tasks in the ant Atta sexdens rubropilosa[END_REF]; but see: [START_REF] König | Foraging activity and immunocompetence in workers of the bumble bee, Bombus terrestris L[END_REF][START_REF] Doums | Immunocompetence in workers of a social insect, Bombus terrestris L., in relation to foraging activity and parasitic infection[END_REF].

Morphology could be another factor of sanitary task allocation, as leaf-cutting ant waste managers were smaller than foragers (e.g. [START_REF] Ballari | Waste management in the leaf-cutting ant Acromyrmex lobicornis: Division of labour, aggressive behaviour, and location of external refuse dumps[END_REF]). Smaller individuals are less costly for the colony to produce (on an individual basis; e.g. [START_REF] Calabi | Worker longevity in the fire ant Solenopsis invicta: Ergonomic considerations of correlations between temperature, size and metabolic rates[END_REF]) and might thus be expected to have a higher probability of sanitary task performance, if task performance is not constrained by worker morphology. Social insects are associated with a variety of symbiotic microorganisms, which can for instance serve nutritional or disease defense purposes (e.g. [START_REF] Currie | Fungus-growing ants use antibioticproducing bacteria to control garden parasites[END_REF][START_REF] Hughes | Social insect symbionts: evolution in homeostatic fortresses[END_REF]. Nestmates may vary in the abundance of symbiotic partners on or within their bodies, which would lead to variability among nestmates in their contribution to colony disease defense. Major workers of the leafcutting ants Acromyrmex have a higher cuticular abundance of a mutualistic bacterium (an actinomycete which produces antibiotics and suppresses the growth of Escovopsis, a specialized fungal parasite of their mutualistic fungus garden) than minor workers. Major workers are also more present in parts of their fungus gardens where Escovopsis is most abundant [START_REF] Poulsen | Mutualistic bacteria and a possible trade-off between alternative defence mechanisms in Acromyrmex leaf-cutting ants[END_REF]. In social insect colonies, it is common to find inactive workers (e.g. [START_REF] Cole | The social behavior of Leptothorax allardycei (Hymenoptera, Formicidae): time budgets and the evolution of worker reproduction[END_REF][START_REF] Jandt | Spatial organization and division of labour in the bumblebee Bombus impatiens[END_REF] who might invest more resources into physiological immune defense since they spend less energy on the performance of behavioral tasks. They could thus serve as immune barriers against disease spread [START_REF] Bocher | Défenses immunitaires et division du travail chez les fourmis[END_REF][START_REF] Robinson | Physiology as a caste-defining feature[END_REF][START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]). In the ant C. velox however, inactive workers did not differ in the level of phenoloxidase from active workers [START_REF] Bocher | Défenses immunitaires et division du travail chez les fourmis[END_REF].

Having been previously infected by a parasite primes the physiological immune system and thereby confers a survival advantage upon a secondary challenge [START_REF] Konrad | Social transfer of pathogenic fungus promotes active immunisation in ant colonies[END_REF]reviewed in Masri and Cremer 2014). Either an infection or a previous exposure to a parasite could also affect sanitary response thresholds, a question which has to my knowledge not yet been investigated. If a previous infection would lower response thresholds for sanitary tasks, individuals with increased immune protection (immune-primed individuals) will be more likely to perform the riskiest tasks in the colony, conferring increased disease protection to the colony. Nestmates differ in their responsiveness towards sanitary stimuli (e.g. [START_REF] Masterman | Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)[END_REF]Gramacho and Spivak 2003;[START_REF] López-Riquelme | Antennal olfactory sensitivity in response to task-related odours of three castes of the ant Atta mexicana (Hymenoptera: Formicidae)[END_REF]) and studies on honeybees suggested that individuals with the lowest response thresholds for disease brood are indeed the ones who are more likely to perform hygienic behavior [START_REF] Masterman | Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)[END_REF]Gramacho and Spivak 2003). Furthermore, the proportion of foraging honeybees infected with Nosema apis is higher under harsher environmental conditions [START_REF] Woyciechowski | Division of labor by division of risk according to worker life expectancy in the honey bee (Apis mellifera L.)[END_REF].

Response thresholds can be genetically determined (e.g. [START_REF] Pankiw | The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.)[END_REF][START_REF] Kryger | Genotypical variability for the tasks of water collecting and scenting in a honey bee colony[END_REF][START_REF] Masterman | Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)[END_REF][START_REF] Jones | Honey bee nest thermoregulation: diversity promotes stability[END_REF][START_REF] Scheiner | Effects of patriline on gustatory responsiveness and olfactory learning in honey bees[END_REF] and further underlie plasticity due to factors such as age, nutrition, rearing conditions; recent experience of the individual and rate of stimulus increase [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF][START_REF] Pankiw | The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.)[END_REF][START_REF] Pankiw | Modulation of sucrose response thresholds in honey bees (Apis mellifera L.): Influence of genotype, feeding and foraging experience[END_REF][START_REF] Weidenmüller | The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response[END_REF][START_REF] Yousif | Die Thermoperiodizität und die Kontrolle der Bruttemperatur bei der Ameisenart Camponotus mus[END_REF][START_REF] Weidenmüller | Preimaginal and adult experience modulates the thermal response behavior of ants[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF]. Previous infection or exposure to parasites might be another source of response threshold variation for sanitary tasks.

Nature versus nurture: phenotypic plasticity in immune defense mechanisms

The relative contribution of genetic and environmental effects on traits (such as individual size) varies among species and ranges from mainly environmentally determined phenotypes to nearly complete genetic control (see for a review on genetic versus environmental determination of social insect caste: [START_REF] Schwander | Nature versus nurture in social insect caste differentiation[END_REF]. Individual phenotypes are determined by complex interactions of genotypic and environmental effects, which modulate gene expression patterns (Smith et al. 2008b). Especially during the last years, permitted by the development of new molecular genetic tools, differences in gene expression patterns among task groups have been detected in several species but most of the genes underlying heritable effects of social organization still need to be identified (Smith et al. 2008b). Smith et al. (2008b) even assume that the epigenetic differences detected among task groups could only be 'the tip of the iceberg' and that more discoveries of heritable effects on components of social organization are expected.

Disease resistance is an important life-history trait and its efficiency closely related to individual fitness [START_REF] Cotter | Costs of resistance: genetic correlations and potential trade-offs in an insect immune System[END_REF]. It is generally assumed that natural selection decreases additive genetic variation in traits determining individual fitness and that fitness-increasing alleles become fixed. Secondly, high environmental and non-additive genetic contributions to phenotypic variation may be responsible for a low heritability of fitness-related traits [START_REF] Merilä | Lifetime reproductive success and heritability in nature[END_REF] and the references therein). Unexpectedly, studies on non-social insects have demonstrated significant levels of additive genetic variation in immune components such phenoloxidase and antibacterial activity [START_REF] Kurtz | The immunocompetence handicap hypothesis: testing the genetic predictions[END_REF][START_REF] Ryder | Quantitative genetics of immune function and body size in the house cricket, Acheta domesticus[END_REF][START_REF] Cotter | Heritability of immune function in the caterpillar Spodoptera littoralis[END_REF][START_REF] Cotter | Costs of resistance: genetic correlations and potential trade-offs in an insect immune System[END_REF][START_REF] Schwarzenbach | Sex and immunity in the yellow dung fly Scathophaga stercoraria[END_REF]. Hypothetically, the observed genetic variation in these traits arises from antagonistic pleiotropy (i.e. a gene positively affects one trait and negatively another), which is the genetic basis of trade-offs between traits (Roff 1992, as cited in [START_REF] Cotter | Heritability of immune function in the caterpillar Spodoptera littoralis[END_REF][START_REF] Cotter | Costs of resistance: genetic correlations and potential trade-offs in an insect immune System[END_REF]. Increased physiological immune investment involves costs in form of resources and the risk of immunopathology (e.g. [START_REF] Moret | Survival for immunity: the price of immune system activation for bumblebee workers[END_REF][START_REF] Sadd | Self-harm caused by an insect's innate immunity[END_REF]. Trade-offs exist across several insect taxa both between immune defense and other life-history traits (e.g. reproduction, longevity, competitive ability: [START_REF] Sheldon | Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology[END_REF][START_REF] Kraaijeveld | Trade-off between parasitoid resistance and larval competitive ability in Drosophila melanogaster[END_REF][START_REF] Moret | Survival for immunity: the price of immune system activation for bumblebee workers[END_REF][START_REF] Armitage | Examining costs of induced and constitutive immune investment in Tenebrio molitor[END_REF]reviewed in Schmid-Hempel 2003) and between different components of physiological immune defense (e.g. phenoloxidase and antibacterial activity: Moret and Schmidt-Hempel 2001;[START_REF] Moret | Adaptive innate immunity? Responsive-mode prophylaxis in the mealworm beetle, Tenebrio molitor[END_REF][START_REF] Cotter | Costs of resistance: genetic correlations and potential trade-offs in an insect immune System[END_REF][START_REF] Ruiz-González | Rapid induction of immune densitydependent prophylaxis in adult social insects[END_REF]. Maximum immune defense is thus not optimal immune defense [START_REF] Zuk | Immune defense and host life history[END_REF] and can only be bought at the expense of other important traits, a possible reason why additive genetic variation in immune components could be maintained [START_REF] Cotter | Costs of resistance: genetic correlations and potential trade-offs in an insect immune System[END_REF].

Selection pressures vary across species and differ between non-social and social species, potentially leading to differences in the heritability of immune traits. Once workers were limited to serve mostly as non-reproductive helpers in social insects, natural selection could shape their traits to increase colony fitness (Smith et al. 2008b). In social insects, disease transmission is assumed to be increased due to the high density of closely related and interacting individuals and disease defense is typically performed by (mostly) sterile individuals (Cremer et al. 2007 and the references therein). For these workers, inclusive fitness gains are expected to outweigh individual defense [START_REF] Cotter | A direct physiological trade-off between personal and social immunity[END_REF]. Collective immune mechanisms might decrease the need to invest into physiological immune defense (e.g. [START_REF] Evans | Immune pathways and defence mechanisms in honey bees Apis mellifera[END_REF][START_REF] Castella | The presence of conifer resin decreases the use of the immune system in wood ants[END_REF][START_REF] Simone | Resin collection and social immunity in honey bees[END_REF]), which comprises not only costs at the individual but also at the colony level. Honey-and bumblebee colonies whose members invested more strongly into individual immune defense produced fewer offspring [START_REF] Moret | Social life-history response to individual immune challenge of workers of Bombus terrestris L.: a possible new cooperative phenomenon[END_REF][START_REF] Evans | Colony-level impacts of immune responsiveness in honey bees, Apis mellifera[END_REF]. In chapter 3, patrilines of the ant C. velox did not significantly vary in the level of phenoloxidase and thus showed no heritability for this trait. Accordingly, patriline differences in the level of phenoloxidase or in the capacity to encapsulate a foreign body were not detected among honeybee larvae [START_REF] Tarpy | Within-and across-colony effects of hyperpolyandry on immune function and body condition in honey bees (Apis mellifera)[END_REF]. Environmental determination and thus condition-dependence of immune investment might confer increased disease defense only to individuals performing the riskiest tasks within the colony and thereby limit costs of immune investment at the colony level. Furthermore, condition-dependent immune expression might provide colonies with a higher flexibility under rapidly changing environmental conditions. If for instance behavioral immune defense is modulated by individual experience or the rate of stimulus increase, as has been demonstrated for thermoregulating bumblebees [START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF]), colonies might be better able to limit the spread of a parasite within the nest.

According to expectations based on the trait individual size [START_REF] Bargum | A genetic component to size in queens of the ant, Formica truncorum[END_REF]), arguments both against (see above) and for a heritable component to immune investment exist. If interindividual variation in immune investment positively affects the efficiency of division of sanitary labor in that individuals with a higher protection perform the riskier tasks (as suggested by the findings of [START_REF] Bocher | Phenotypic plasticity of immune defence linked with foraging activity in the ant Cataglyphis velox[END_REF][START_REF] Lacerda | Morphometry of the metapleural gland of workers engaged in different behavioral tasks in the ant Atta sexdens rubropilosa[END_REF], but see: [START_REF] König | Foraging activity and immunocompetence in workers of the bumble bee, Bombus terrestris L[END_REF][START_REF] Doums | Immunocompetence in workers of a social insect, Bombus terrestris L., in relation to foraging activity and parasitic infection[END_REF] or efficiently hinder disease spread as immune barriers, then selection on worker immune investment should be diversifying. A diversifying selection would lead to heritability of immune investment and workers of genetically more diverse colonies would be expected to show a higher immune trait variance (which was not demonstrated in honeybee larvae: [START_REF] Tarpy | Within-and across-colony effects of hyperpolyandry on immune function and body condition in honey bees (Apis mellifera)[END_REF]). More research is needed in this field to determine the relative importance of both environmental and genetic effects on immune investment and to be able to address the question why colonies of higher genetic diversity are more resistant towards disease. Social immune defense behaviors seem a likely candidate (as in [START_REF] Ugelvig | Rapid anti-pathogen response in ant societies relies on high genetic diversity[END_REF], since behavior is expected to show stronger plasticity than other immune defense barriers [START_REF] Hughes | Plasticity in antiparasite behaviours and its suggested role in invasion biology[END_REF] and heritability for hygienic behavior has been demonstrated in africanized honeybees [START_REF] Padilha | Genetic parameters for five traits in Africanized honeybees using Bayesian inference[END_REF].

When is experience-modulated behavioral plasticity expected to occur?

Experience-dependent behavioral modulation is assumed to occur in all aspects of life and even short-lived fruit flies learn in feeding, predator avoidance, aggression and sexual behaviors (reviewed in Dukas 2008). The modification of behavior due to learning could in some cases increase fitness, as it increases foraging success in bees and wasps (e.g. Dukas and Visscher 1994;[START_REF] O'donnell | Forager success increases with experience in Polybia occidentalis (Hymenoptera: Vespidae)[END_REF]) and led to a higher growth rate in grasshoppers [START_REF] Dukas | Learning improves growth rate in grasshoppers[END_REF]. The estimation of fitness benefits is not trivial because it is not clear how time or energy (in which costs or benefits of learning are often measured) relate to fitness [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. To be able to satisfactorily answer whether insects should learn a certain task requires quantification of the fitness cost -benefit ratio of learning under natural conditions (Dukas 2008). Natural selection could alter learning, as variation in learning has a heritable component (e.g. [START_REF] Brandes | Estimation of hertability of learning behavior in honeybees (Apis mellifera capensis)[END_REF][START_REF] Brandes | Genetic differences in learning behavior in honeybees (Apis mellifera capensis)[END_REF] and because differences in learning ability could be associated with fitness differences (e.g. [START_REF] Dukas | Learning improves growth rate in grasshoppers[END_REF].

The neural economy hypothesis assumes that high environmental complexity does not allow programming all recognition and response behaviors into the DNA or nervous system, due to physiological constraints (reviewed in [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. If information storage is physiologically constrained, learning could be more advantageous than genetically fixed behavior if it is more economical in terms of the amount of useful information stored [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. Indeed, only a limited amount of information might be storable and interference could occur between acquired experiences (e.g. [START_REF] Lewis | Memory constraints and flower choice in Pieris rapae[END_REF][START_REF] Chittka | Learning, specialization, efficiency and task allocation in social insects[END_REF]. The environmental unpredictability hypothesis, another hypothesis to explain the evolution of learning, assumes that the high unpredictability of the environment within an individual's lifetime or over successive generations does not allow natural selection to program all appropriate recognition and response behaviors (reviewed in [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. It is expected that experience-dependent behavioral modulation more likely occurs in moderately unpredictable environments and less likely occurs in either extremely predictable or in extremely unpredictable environments than fixed behavior (reviewed in [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. Behavioral avoidance of parasites could be learned in response to selection from relatively predictable and high-cost parasites. Insects might rely on innate mechanisms to detect parasites but parasites could vary in spatial or temporal occurrence. If an abundance of parasites is positively correlated with a certain space or time, insects might learn these parameters to improve parasite avoidance. Task groups can differ in their capacity for learning which might result in a differential degree of experience-induced plasticity for different tasks. Honeybee pollen foragers for instance were better learners than foragers with nectar preference, which might result from a general higher sensitivity to environmental foraging-associated stimuli of these bees [START_REF] Latshaw | Heritable variation in learning performance affects foraging preferences in the honey bee (Apis mellifera)[END_REF]. Furthermore, individuals show cue biases for learning, which means that they learn certain stimuli faster than others. Honeybees learn odors similar to flower scents more rapidly than other odors (reviewed in [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. Additionally, some motor patterns are more susceptible to modification by experience than others [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF]. Restricting experience-dependent plasticity to certain stimuli or motor patterns is assumed to be adaptive, as it could confine learning to situations, which are too unpredictable or complex for pre-programmed responses [START_REF] Papaj | Ecological and evolutionary aspects of learning in phytophagous insects[END_REF].

Temporary limitation in acquired experience effects (or short-term memory limits [START_REF] Menzel | Neurobiology of learning and memory: The honeybee as a model system[END_REF] might be adaptive as it could allow higher flexibility towards changing environmental conditions and only allow the persistence of behavioral modulation, which is still advantageous under the current conditions. The persistence of acquired experience-effects depends on the time delay between task performances (e.g. [START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF]) and acquired experience may vanish over night (e.g. [START_REF] Keasar | Overnight memory retention of foraging skills by bumblebees is imperfect[END_REF][START_REF] Westhus | Behavioral plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase[END_REF]. Sanitary stimuli which occur only infrequently are presumably unlikely to lead to long-term behavioral modification, protecting against costs associated with learning in terms of neuronal resources (e.g. [START_REF] Laughlin | The metabolic cost of neural information[END_REF][START_REF] Farris | Experience-and age-related outgrowth of intrinsic neurons in the mushroom bodies of the adult worker honeybee[END_REF][START_REF] Riveros | Brain allometry and neural plasticity in the bumblebee Bombus occidentalis[END_REF], time required and occasional mistakes. For rare events, the infrequent benefits gained by experiencedependent behavioral modulation might not outweigh the costs. In the ant Myrmica rubra, short-term spatial memory of necrophoric ants lasted only 7 minutes, presumably leading to a dispersion of cadavers in the field [START_REF] Diez | Orientation in corpse-carrying ants: memory or chemical cues?[END_REF].

Research on how either repeated exposure to sanitary hazards or repeated performance of sanitary tasks affects individual response behavior was only lately initiated (e.g. Trumbo and Robinson 1997;[START_REF] Walker | Adaptive social immunity in leaf-cutting ants[END_REF][START_REF] Diez | Orientation in corpse-carrying ants: memory or chemical cues?[END_REF][START_REF] Reber | The expression and impact of antifungal grooming in ants[END_REF], even though social insects are presumably repeatedly exposed to sanitary threats. This research field might lead to exciting insights on an important mechanism of interindividual variation and deepen understanding of host-parasite interactions. Many questions await further study. It remains unexplored whether experience in sanitary task performance can result in task specialization and thus division of labor for these defenses. In chapter two, the repeated exposure to cadavers or performance of cadaver management increased the number of individuals engaging in cadaver grooming behavior and affected task allocation in only one out of four colonies. Inter-colony differences could arise through many factors, such as the sanitary stimulus presented. Of course this does not mean that other parameters of cadaver management are not affected by experience, simply that my experimental protocol was not able to reveal it. In chapter one, ant workers trained to both sham-and fungus-treated larvae increased larval grooming duration and thereby increased the removal of conidiospores by trained ants. If these experienced individuals were more likely to respond towards a sanitary threat within the colony, they could become task specialists. If experienced individuals specialize on the sanitary task, parasite spread might be reduced though behavioral compartmentalisation and higher task efficiency of experienced individuals. Increased efficiency of task performance could be achieved through lowered response thresholds (thus responding earlier towards a sanitary threat, at lower parasite abundance), a higher response probability, a reduced delay to response, an increasing response duration or improved motoric capacities (enabling the removal of more parasites in the same amount of time). Individuals specialized on necrophoric behavior removed cadavers faster and more successfully than less experienced individuals (Apis mellifera: Trumbo and Robinson 1997;Acromyrmex versicolor: Julian and Cahan 1999), but an effect of experience was not demonstrated (Trumbo and Robinson 1997). Feedback processes are important for self-reinforcement to occur [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF] and the successful performance of foraging resulted in lasting division of labor between successful and unsuccessful Cerapachys biroi workers [START_REF] Ravary | Individual experience alone can generate lasting division of labor in ants[END_REF]. It remains to be investigated what provides negative or positive feedback (What are the possible reinforcers?) in the context of disease defense. Do workers assess their own efficiency in performing the task, e.g. by sensing the decreasing number of fungal conidiospores on exposed nestmates? Does increased effectiveness in conidiospore removal affect future responses of these ants? Research effort is still necessary to address whether experience affects the allocation of sanitary labor and leads to sanitary task specialization. It further remains elusive whether specialists of different sanitary tasks are really more efficient. It would be interesting to analyze if and how stimulus quantity, quality (e.g. the virulence of a parasite) or temporal presentation (i.e. periodic or chronic exposure) affect experience-dependent modulation of sanitary response behaviors.

Empirical difficulties to analyze proximate mechanisms contributing to interindividual variation in immune defense

Interindividual differences among social insect nestmates have long been recognized and regained interest during the last years in studies on behavioral syndrome (reviewed in [START_REF] Jandt | Behavioural syndromes and social insects: personality at multiple levels[END_REF]). In social insects, studies on interindividual variability are complex in that selection acts both at the individual and at the colony level and worker variability can be caused by an interplay of non-mutually exclusive mechanisms [START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF]. Especially in species with temporal polyethism, an individual's age, physiology, the performed task and the level of acquired experience can strongly covary and their relative importance on individual response behavior and responsiveness are difficult to disentangle. Patriline and age of the individual might also be correlated, as some patrilines could be older than others (chapter 3), requiring studies on either age or patriline effects to also investigate the other factor. Furthermore, genetic factors can influence differential sensitivity towards environmental factors (e.g. by determining response thresholds; e.g. [START_REF] Masterman | Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)[END_REF]Smith et al. 2008a;[START_REF] Scheiner | Effects of patriline on gustatory responsiveness and olfactory learning in honey bees[END_REF] and influence an individual's rate of aging (e.g. [START_REF] Calderone | Genotypic variability in age polyethism and task specialization in the honey bee, Apis mellifera (Hymenoptera: Apidae)[END_REF] or its capacity to learn [START_REF] Brandes | Estimation of hertability of learning behavior in honeybees (Apis mellifera capensis)[END_REF][START_REF] Brandes | Genetic differences in learning behavior in honeybees (Apis mellifera capensis)[END_REF]. Many genes are pleiotropic (i.e. one gene influences multiple phenotypic traits) and traits polygenically influenced (West-Eberhard 1989 and the references therein; [START_REF] Butcher | Generalist genes and cognitive neuroscience[END_REF], adding further complexity. Studies investigating task allocation to sanitary work thus need meticulous protocols which try to disentangle the multitude of potential mechanisms.

Uncertainties about the definition of task specialists in the current literature could complicate comparisons and generalizations between tasks and among species. Whereas in some publications task specialization is described as a concept "in which a small fraction of sameage workers perform the majority of work of certain tasks" [START_REF] Julian | Undertaking specialization in the desert leaf-cutter ant Acromyrmex versicolor[END_REF], others describe specialization as a high concentration of individual "work effort in a single task" [START_REF] Dornhaus | Specialization does not predict individual efficiency in an ant[END_REF]. Task specialization seems to be rather a statistical concept, "reflecting an individual's tendency to perform particular tasks more often than others" [START_REF] Duarte | An evolutionary perspective on self-organized division of labor in social insects[END_REF], and ranges from short-term behavioral differences to fixed morphological variation in large insect societies [START_REF] Robinson | Regulation of division of labor in insect societies[END_REF][START_REF] Duarte | An evolutionary perspective on self-organized division of labor in social insects[END_REF]. The work by Trumbo et al. (1997) on necrophoric honeybees is often cited as an example of strong specialization on a sanitary behavior. But already these authors discuss the difficulties of classifying an individual as a task specialist due to an unclear definition, which needs to include an appropriate timescale of specialization and a minimal number of task performances (a certain performance threshold) (Trumbo et al. 1997). In chapter 2, I did not analyze the complete behavioral profile of workers because of low brood availability and thus do not know whether some individuals specialized on cadaver grooming and transports. The full behavioral profile of workers is necessary to be able to identify them as task specialists because frequent task performers could instead be highly active 'elite' individuals, which perform every task in high frequency [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF]Trumbo et al. 1997).

Especially in social insects, the social environment strongly influences individual response behavior and even gene expression (e.g. [START_REF] Calderone | Effects of interactions among genotypically diverse nestmates on task specialization by foraging honey bees (Apis mellifera)[END_REF]Arathi and Spivak 2001;Arathi et al. 2006;[START_REF] Gempe | Mixing of honeybees with different genotypes affects individual worker behavior and transcription of genes in the neuronal substrate[END_REF], complicating its study within the social context. A trait might not only underlie direct genetic effects (i.e. genetic origin), but trait differences among patrilines might also arise through indirect genetic effects (i.e. environmental effect), where the genes of nestmates modulate the phenotype of the focal individual. If nurses favour one patriline over others, larvae of the preferred patriline might grow faster [START_REF] Bargum | A genetic component to size in queens of the ant, Formica truncorum[END_REF] and the references therein). I have thus trained Platythyrea punctata workers individually (chapter one) to investigate an effect of individual experience on task performance. However, to analyze how colonies allocate workers to sanitary tasks, manipulations within the colony context are necessary and I have thus studied cadaver management within the nest. In this social environment however, the relative contribution of self-reinforcement might have been hindered by social interactions and their effect on division of labor [START_REF] Jeanson | Division of labour and socially induced changes in response thresholds in associations of solitary halictine bees[END_REF].

Investigating interindividual differences in disease defense is further complicated by a potential infection of the focal worker. Behavioral modifications of infected individuals increase distance to valuable colony members and reduce social interactions. Exposed or infected individuals have been shown to stay away from the brood chamber [START_REF] Ugelvig | Social prophylaxis: group interaction promotes collective immunity in colonies[END_REF][START_REF] Bos | Sick ants become unsociable[END_REF], to decrease queen attendance [START_REF] Wang | The division of labor and queen attendance behavior of Nosema-infected worker honey bees[END_REF], interact less with nestmates (e.g. [START_REF] Bos | Sick ants become unsociable[END_REF] or to precociously initiate foraging (e.g. [START_REF] Wang | The division of labor and queen attendance behavior of Nosema-infected worker honey bees[END_REF][START_REF] Goblirsch | Physiological and behavioral changes in honey bees (Apis mellifera) induced by Nosema ceranae infection[END_REF]. In some species, moribund individuals altogether leave the nest (e.g. [START_REF] Heinze | Moribund ants leave their nests to die in social isolation[END_REF][START_REF] Rüppell | Altruistic self-removal of health-compromised honey bee workers from their hive[END_REF][START_REF] Bos | Sick ants become unsociable[END_REF]. Behavioral modifications might stem from parasitic manipulation, disease side-effects and behavioral changes of the focal individual or its nestmates [START_REF] Ugelvig | Social prophylaxis: group interaction promotes collective immunity in colonies[END_REF][START_REF] Stroeymeyt | Organisational immunity in social insects[END_REF]. Not only infection, but already an immune response without parasitic exposure can lead to reduced learning abilities in honeybees (e.g. [START_REF] Mallon | Immune response inhibits associative learning in insects[END_REF][START_REF] Iqbal | Virus infection causes specific learning deficits in honeybee foragers[END_REF] and in bumblebees (e.g. [START_REF] Alghamdi | Immune response impairs learning in free-flying bumble-bees[END_REF]). An alternative for experimental work would be to use a disease proxy, which triggers a sanitary behavior in the same way as a real parasite, but does not result in infection. Finding a suitable proxy which triggers the same (so far unknown) feedback mechanisms, a prerequisite for selfreinforcement to occur [START_REF] Plowright | Elitism in social insects: a positive feedback model[END_REF][START_REF] Theraulaz | Response threshold reinforcement and division of labour in insect societies[END_REF], is not trivial. Methods estimating physiological immune investment further harbour weaknesses. If the ability of an individual to resist infection is qualified using a specific parasite, the obtained results might be specific to the parasite studied and the efficacy of the immune system will be only indirectly measured. Other factors such as behavioral mechanisms might confound the results [START_REF] Cotter | Costs of resistance: genetic correlations and potential trade-offs in an insect immune System[END_REF]). Due to the amount of work involved, studies examining immune investment tended to concentrate on just one or two components of the physiological immune system, rather than a comprehensive suite of immune traits. However, only a holistic approach will advance the field of ecological immunology.

Repeated exposure to a parasite can have multiple effects on the host, influencing parameters of individual responsiveness and sanitary task performance, as well as social interactions, spatial location or immune investment of the individual. Monitoring all of these parameters and manipulating one without the others seems a challenging task, potentially leading to the non-detection of weak effects. Increased research effort in the study of experience effects on immune defense and an increased support of publications containing negative results are required for a holistic view on if and how phenotypic plasticity in immune defense underlies individual experience. unnoticed, and to Simon Tragust for teaching us patiently everything he knew and established in the laboratory.

The team IST (S. Cremer workgroup), especially Miriam Stock, Matthias Konrad, Line Ugelvig and Christopher Pull. Line, thanks for your advice and help on stats and graphics and that you always take time to help and discuss with others and show interest in their work. Miriam and Matthias, thanks for the nice moments shared and your practical help in Regensburg as well as at the IST, it has been great being in a team with you. Chris, thank you for keeping me updated on the newest team literature.

The team Jussieu (C. Doums workgroup), especially Claudy Haussy, Romain Pérronet, Pierre Fédérici, Claire Tirard, Thibaud Monnin, David Sillam-Dussès, Mathieu Molet, Roberto Keller and Christian Peeters for their support and being open for discussion. Claudy, thank you for all the practical work and help with colony maintenance as well as the nice moments shared together. Romain, thank you for your practical support both in the laboratory and with colony maintenance, you were always there in times of need and your love for ants is wonderful. Pierre, thank you for the genetic work and your enthusiasm to teach. Romain, Claudy, Thibaud and David, thank you for colony collection, even under the hardest conditions in the Sierra Nevada. Roberto, thank you for teaching me about metapleural glands. Claire, thanks for your practical enthusiasm and the support for the phenoloxidase method and the different immune measurements tried.

My practical students Martina Klatt, Carlos Alberto Ortega Trujillo, Edouard Tourdot, Hélène Dudek for their motivation and help with practical work.

Céline Hauzy, Florence Helft, Sylvain Londe and many others from the research unit "Institut d'Écologie et des Sciences de l'Environnement" at Jussieu for the nice moments shared and the warm welcome.

Raphaёl Boulay, Yannick Moret, Mark Brown and Martine Maïbeche-Coisné for being in the thesis committee and Raphaёl as well as Yannick, thank you for the thesis reports.

Last but not least I would like to thank my husband Dima Missoh and my parents Sabine and Werner Westhus for their immense support. Your encouraging words, understanding, financial help and practical support in times of need were unvaluable. Levi, you brought so much joy into my life and finally the work-life balance that was needed.

3. 3

 3 Division of labor in disease resistance and causes of interindividual variability Increased grooming after repeated brood care provides sanitary benefits in a clonal ant.

Fig. 1

 1 Fig. 1 P. punctata worker photographed by Simon Tragust.

Fig. 2

 2 Fig. 2 Typical habitat of Cataglyphis velox at 1300 m altitude in the Sierra Nevada, close to Grenada, Spain (on the left). Individuals enter and exit the nest through a single tunnel and small exit hole (on the right; diameter ca. 1.5 cm).

Fig. 3

 3 Fig. 3 C. velox forager returning with collected bait (cookie crumb) to the colony. Thermophile foragers scavenge individually for food.

Fig. 4 A

 4 Fig. 4 A Lasius neglectus cadaver without fungal growth and with Metarhizium spp.. Hyphae (in white) and cylindrical green spore packages (picture on the right). Picture provided by Martina Klatt.

Fig. 2 Fig. 3

 23 Fig. 2 Time to response of brood care. Neither a the time to first contact of the ants to the larvae after start of the experiment nor b the delay between the first contact and performance of sanitary brood care (larval grooming) depended on larval treatment (sham control: blue; fungus

Figure 1 :

 1 Figure 1: Experimental design. Cataglyphis velox colonies were split into cadaver and control group of 100 individuals each. The first experimental step started 2.5 weeks after splitting and consisted of 5 days with two trials per day. In each trial, four cadavers were inserted into the nest of the cadaver group. At the same time, forceps were inserted into the nest of the control group to control for colony disturbance. During the second step, the subcolonies were reunited on day 6 of the experiment and ants present within the foraging arena recorded the next day. On day 8 of the experiment, eight cadavers were inserted into the nest of the reunited colonies in two trials (four in each trial).

Figure 2 :

 2 Figure 2: Relationship between the number of groomings (A) and transports (B) and the number of grooming (A) and transporting (B) workers for each of 140 cadavers over all trials. Black circles denote colony A, open circles colony B, black triangles colony C and open triangles colony D.

Figure 4 :

 4 Figure 4: The number of cadaver-management behaviors for every task-performing individual within each cadaver group (cadaver groups A to D displayed from top to bottom panel). Cadaver groomings of an individual are displayed in black and transports in white within each individual bar. The circles above bars display necrophoric individuals.

Figure 1 :

 1 Figure 1: Comparison of tibia length (mm) among laboratory-born (L) and field-derived (F) workers of colonies A, B, C and D (displayed in the four horizontal panels). The boxplots depict mean, quartiles and range of the data.

Figure 2 :

 2 Figure 2: Comparison of the level of active PO (log-transformed) among young laboratory-born (L) and old field-derived (F) workers of colonies A, B, C and D (displayed in the four horizontal panels).

Figure 3 :

 3 Figure 3: The level of total PO (log-transformed) is displayed among young laboratory-born (L) and old field-derived (F) workers of colonies A, B, C and D (in the four horizontal panels). The boxplots show mean, quartiles and range of the data.

Figure 4 :

 4 Figure 4: Relationship between total PO activity (data are log-transformed) and tibia length (in mm) of young laboratory-born workers (filled circles) and old field-derived workers (empty circles) for all workers of the four colonies.

  heritability was estimated as twice the variance components due to an effect of patriline in a LMM containing no fixed effect and patriline embedded in colony as a random effect. The number of individuals used for these heritability estimates are given between parentheses below heritability values. The values of the variance components inter-The proportion of variance of the three parameters worker size, active Po and total PO are given between parentheses for each variance component (below variance component estimates).

TABLE OF CONTENTS

 OF

	1. ABSTRACT	3
	2. RÉSUMÉ	5
	3. INTRODUCTION	7
	3.1 Disease resistance in social insects	7
	3.1.1 Individual physiological immune defenses	8
	3.1.2 Collective immune defenses	9
	3.2 Division of labor in social insect colonies

  Konrad M, Vyleta ML, Theis FJ, Stock M, Tragust S, Klatt M, Drescher V, Marr C, Ugelvig LV, Cremer S (2012) Social transfer of pathogenic fungus promotes active immunisation in ant colonies. PLoS Biol 10(4):e1001300. doi:10.1371/journal.pbio.1001300 Kurtz J, Armitage SAO (2006) Alternative adaptive immunity in invertebrates. Trends Immunol 27:493-496 Lacey LA, Brooks WM (1997) Initial handling and diagnosis of diseased insects. In: Lacey LA (ed) Manual of techniques in insect pathology.

	Trumbo ST, Huang Z-Y, Robinson GE (1997) Division of labor between	Robinson GE, Page REJ (1988) Genetic determination of guarding and Weidenmüller A (2004) The control of nest climate in bumblebee
	undertaker specialists and other middle-aged workers in honey bee	undertaking in honey-bee colonies. Nature 333:356-358 (Bombus terrestris) colonies: interindividual variability and
	colonies. Behav Ecol Sociobiol 41(3):151-163	Robinson GE, Page RE Jr (1989) Genetic determination of nectar forag-self reinforcement in fanning response. Behav Ecol 15(1):
	Ugelvig LV, Cremer S (2007) Social prophylaxis: group interaction pro-	ing, pollen foraging, and nest-site scouting in honey bee colonies. 120-128
	motes collective immunity in colonies. Curr Biol 17(22):1967-1971	Behav Ecol Sociobiol 24(5):317-323 Weidenmüller A, Mayr C, Kleineidam CJ, Roces F (2009) Preimaginal
	Ugelvig LV, Kronauer DJC, Schrempf A, Heinze J, Cremer S (2010)	Rosengaus RB, Traniello JFA (1997) Pathobiology and disease transmis-and adult experience modulates the thermal response behavior of
	Rapid anti-pathogen response in ant societies relies on high genetic	sion in dampwood termites [Zootermopsis angusticollis (Isoptera: ants. Curr Biol 19(22):1897-1902
	diversity. Proc R Soc B Biol Sci 277(1695):2821-2828	Termopsidae)] infected with the fungus Metarhizium anisopliae Westhus C, Kleineidam CJ, Roces F, Weidenmüller A (2013) Behavioral
	Academic Press, London, pp 1-16 Langridge EA, Franks NR, Sendova-Franks AB (2004) Improvement in collective performance with experience in ants. Behav Ecol Vestergaard S, Butt T, Bresciani J, Gillespie AT, Eilenberg J (1999) Light and electron microscopy studies of the infection of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae) CHAPTER 2 (Deuteromycotina: Hypomycetes). Sociobiology 30(2):185-195 Rosengaus RB, Traniello JFA (2001) Disease susceptibility and the adaptive nature of colony demography in the dampwood termite plasticity in the fanning response of bumblebees: the impact of experience and rate of temperature increase. Anim Behav 85(1): 27-34
	Sociobiol 56:523-529 by the entomopathogenic fungus Metarhizium anisopliae.J	Zootermopsis angusticollis. Behav Ecol Sociobiol 50:546-556 Wilson-Rich N, Spivak M, Fefferman NH, Starks PT (2009) Genetic,
	Langridge EA, Sendova-Franks AB, Franks NR (2008) How experienced Invertebr Pathol 73:25-33	Rosengaus RB, Maxmen A, Coates L, Traniello JFA (1998) Disease individual, and group facilitation of disease resistance in insect
	individuals contribute to an improvement in collective performance Waddington SJS, Santorelli LA, Ryan FRF, Hughes WOH (2010)	resistance: a benefit of sociality in the dampwood termite societies. Annu Rev Entomol 54:405-423
	in ants. Behav Ecol Sociobiol 62:447-456 Genetic polyethism in leaf-cutting ants. Behav Ecol 21:1165-1169	Zootermopsis angusticollis (Isoptera: Termopsidae). Behav Ecol Yanagawa A, Yokohari F, Shimizu S (2008) Defense mechanism of the
	Masterman R, Ross R, Mesce K, Spivak M (2001) Olfactory and behav-Walker TN, Hughes WOH (2009) Adaptive social immunity in leaf-	Sociobiol 44:125-134 termite, Coptotermes formosanus Shiraki, to entomopathogenic
	ioral response thresholds to odors of diseased brood differ between cutting ants. Biol Lett 5(4):446-448	Rosengaus RB, Traniello JFA, Chen T, Brown JJ (1999) Immunity in a fungi. J Invertebr Pathol 97:165-170
	hygienic and non-hygienic honey bees (Apis mellifera L.). J Comp	social insect. Naturwissenschaften 86:588-591
	Physiol A 187:441-452	Rosengaus RB, Malak T, MacKintosh C (2013) Immune-priming in ant
	Matzinger P (1994) Tolerance, danger, and the extended family. Annu Rev Immunol 12:991-1045 Mehdiabadi NJ, Gilbert LE (2002) Colony-level impacts of parasitoid Necrophoresis is not everything: cadaver groomings and larvae: social immunity does not undermine individual immunity. Biol Lett 9:20130563 Rothenbuhler WC, Thompson VC (1956) Resistance to American foul-
	flies on fire ants. Proc Biol Sci 269:1695-1699	brood in honey bees. I. Differential survival of larvae of different
	Mersch DP, Crespi A, Keller L (2013) Tracking individuals shows spatial fidelity is a key regulator of ant social organization. Science intranidal transports in the ant Cataglyphis velox. genetic lines. J Econ Entomol 49:470-475 Sadd BM, Schmid-Hempel P (2006) Insect immunity shows spec-
	340(6136):1090-1093	ificity upon secondary pathogen exposure. Curr Biol 16:
	Morelos-Juárez C, Walker TN, Lopes JFS, Hughes WOH (2010) Ant	1206-1210
	farmers practice proactive personal hygiene to protect their fungus	Schilder K, Heinze J, Gross R, Hölldobler B (1999) Microsatellites reveal
	crop. Curr Biol 20:R553-R554
	Muscedere ML, Djermoun A, Traniello JFA (2013) Brood-care experi-
	ence, nursing performance, and neural development in the ant
	Pheidole dentata. Behav Ecol Sociobiol 67(5):775-784
	Naug D, Camazine S (2002) The role of colony organization on
	pathogen transmission in social insects. J Theor Biol 215:	7-11
	427-439	Schmid-Hempel P (1998) Parasites in social insects. Princeton University
	O'Donnell S, Jeanne RL (1992) Forager success increases with experi-	Press, Princeton, New Jersey
	ence in Polybia occidentalis (Hymenoptera: Vespidae). Insect Soc	Schmid-Hempel P (2005) Natural insect host-parasite systems show
	39:451-454	immune priming and specificity: puzzles to be solved. Bio Essays
	Oldroyd BP, Fewell JH (2007) Genetic diversity promotes homeostasis in	27:1026-1034
	insect colonies. Trends Ecol Evol 22:408-413	Schmid-Hempel P, Schmid-Hempel R (1993) Transmission of a pathogen
	Oster GF, Wilson EO (1978) Caste and ecology in the social insects.	in Bombus terrestris, with a note on division of labour in social
	Princeton University Press, Princeton, NJ	insects. Behav Ecol Sociobiol 33:319-327
	Patterson R, Briano J (1993) Potential of three biological control agents	Siva-Jothy MT, Moret Y, Rolff J (2005) Insect immunity: an evolutionary
	for suppression of Solenopsis invicta, the red imported fire ant.	ecology perspective. Adv In Insect Phys 32:1-48
	Proceedings of the 1st International Conference on Insect Pests on	St. Leger RJ, Wang C, Fang W (2011) New perspectives on insect
	Urban Environments. Exeter, UK. pp. 35-43	pathogens. Fungal Biol Rev 25:84-88
	Pinheiro J, Bates D, DebRoy S, Sarkar D and the R Development Core	Theraulaz G, Bonabeau E, Deneubourg J-L (1998) Response threshold
	Team (2012) nlme: linear and nonlinear mixed effects models. R	reinforcement and division of labour in insect societies. Proc R Soc
	package version 3.1-110	Lond B Biol Sci 265:327-332
	Plowright RC, Plowright CMS (1988) Elitism in social insects: a positive	Therneau T (2012) A package for survival analysis in S. R package
	feedback model. In: Jeanne RL (ed) Interindividual behavioral var-	version 2.36-14
	iability in social insects. Westview Press, Boulder, Colorado, pp	Thomas ML, Elgar MA (2003) Colony size affects division of labour in
	419-432	the ponerine ant Rhytidoponera metallica. Naturwissenschaften 90:
	Raine NE, Chittka L (2007) Pollen foraging: learning a complex motor	88-92
	skill by bumblebees (Bombus terrestris). Naturwissenschaften 94:	Tragust S, Mitteregger B, Barone V, Konrad M, Ugelvig LV, Cremer S
	459-464	(2013a) Ants disinfect fungus-exposed brood by oral uptake and
	Rath AC, Koen TB, Yip HY (1992) The influence of abiotic factors on	spread of their poison. Curr Biol 23(1):76-82
	the distribution and abundance of Metarhizium anisopliae in	Tragust S, Ugelvig LV, Chapuisat M, Heinze J, Cremer S (2013b) Pupal
	Tasmanian pasture soils. Mycol Res 96(5):378-384	cocoons affect sanitary brood care and limit fungal infections in ant
	Ravary F, Lecoutey E, Kaminski G, Châline N, Jaisson P (2007)	colonies. BMC Evol Biol 13:225. doi:10.1186/1471-2148-13-225
	Individual experience alone can generate lasting division of labor	Traniello JFA, Rosengaus RB, Savoie K (2002) The development of
	in ants. Curr Biol 17:1308-1312	immunity in a social insect: evidence for the group facilitation of
	Reber A, Purcell J, Buechel S, Buri P, Chapuisat M (2011) The expression	disease resistance. PNAS 99(10):6838-6842
	and impact of antifungal grooming in ants. J Evol Biol 24:954-964	Trumbo ST, Robinson GE (1997) Learning and task interference by
	Robinson GE (1992) Regulation of division of labor in insect societies.	corpse-removal specialists in honey bee colonies. Ethology 103:
	Annu Rev Entomol 37:637-665	966-975

clonal structure of populations of the thelytokous ant Platythyrea punctata (F. Smith) (Hymenoptera; Formicidae). Mol Ecol 8:1497-1507 Schlüns EA, Wegener BJ, Robson SKA (2011) Genetic polyethism and nest building in the weaver ant Oecophylla smaragdina (FABRI-CIUS, 1775) (Hymenoptera: Formicidae). Myrmecol News 15:ClaudiaWesthus, Sylvia Cremer, Claudie Doums In preparation TITLE Necrophoresis is not everything: cadaver groomings and intranidal transports in the ant Cataglyphis velox.

  time, the presence of harmful microorganisms;Neoh et al. 2010;[START_REF] Renucci | Complex undertaking behavior in Temnothorax lichtensteini ant colonies: from corpse-burying behavior to necrophoric behavior[END_REF][START_REF] Fan | Pyrokinin β-neuropeptide affects necrophoretic behavior in fire ants (S. invicta), and expression of β-NP in a mycoinsecticide increases its virulence[END_REF] Sun and Zhou 2013). Recognition of dead individuals appears to be achieved through chemical cues (necromones), either accumulating decomposition products (e.g. oleic acid:[START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF][START_REF] Haskins | Notes on necrophoric behavior in the archaic ant Myrmecia vindex (Formicidae: Myrmeciinae)[END_REF][START_REF] Gordon | Dependence of necrophoric response to oleic acid on social context in the ant, Pogonomyrmex badius[END_REF] Lopez- Riquelme et al. 2006; Diez et al. 2013b) or disappearing cues showing the vitality of the individual (dolichodial and iridomyrmecin:[START_REF] Choe | Chemical signals associated with life inhibit necrophoresis in Argentine ants[END_REF].Division of labor for sanitary tasks can be advantageous, as it increases behavioral and spatial compartmentalisation and can, together with heterogeneity in the interaction network, decrease parasite transmission among workers[START_REF] Schmid-Hempel | Transmission of a pathogen in Bombus terrestris, with a note on division of labour in social insects[END_REF][START_REF] Schmid-Hempel | Parasites in Social Insects[END_REF][START_REF] Hart | Task partitioning, division of labour and nest compartmentalisation collectively isolate hazardous waste in the leafcutting ant Atta cephalotes[END_REF][START_REF] Naug | The role of colony organization on pathogen transmission in social insects[END_REF][START_REF] Pie | Nest architecture, activity pattern, worker density and the dynamics of disease transmission in social insects[END_REF] Cremer et al. 2007). But how colonies allocate risky sanitary tasks such as cadaver management is not well understood. Sanitary tasks vary in the pathogenic exposure risk of the task-performing individual, in the frequency with which they occur (i.e. the time interval between task performances;Diez et al. 2013a), in their workload (e.g. the quantity of cadavers which occur at the same time;

, the cadavers' origin (nestmate or foreign;

[START_REF] Ataya | Le comportement nécrophorique chez la fourmi Lasius niger L[END_REF][START_REF] Renucci | Complex undertaking behavior in Temnothorax lichtensteini ant colonies: from corpse-burying behavior to necrophoric behavior[END_REF] Diez et al. 2013b)

, and the associated sanitary risk (e.g. decomposition

Table 1 :

 1 For each cadaver group, the percentage of cadavers removed in the first experimental step is given, together with the minimum and maximum time of removal over all trials (not including unremoved cadavers). I further provide the percentage of cadaver groomers, transporters and the overlap between both, based on the number of marked workers per cadaver group.

							Overlap
	Cadaver group	Cadavers removed (%)	Minimal time until necrophoresis	Maximal time until necrophoresis	Groomers (%)	Transporters (%)	between groomers and transporters
							(%)
	A	94	59 sec	24 min 15 sec	39	30	42
	B	81	32 sec	30 min 32 sec	35	25	48
	C	81	2 min 22 sec	30 min 21 sec	32	19	38
	D	11	3 min 33 sec	19 min 41 sec	19	12	41

Table 2 ,

 2 LMM, coefficient = 0.051) and with the duration a cadaver stayed in the nest (Table2, LMM, coefficient = 0.001), but was not affected by cadaver size (Table2, LMM). The number of individuals which engaged in cadaver transports was neither affected by trial, nor by the duration a cadaver stayed in the nest, but depended on cadaver size (Table2, LMM). The largest and second smallest cadavers were transported by fewer individuals.

Table 2 :

 2 The statistical values of Linear Mixed Effects Models (LMM) testing an effect of trial, cadaver size, the number of task performers (groomers or transporters for grooming or transporting respectively) and the duration the cadaver stayed in the nest (rows) on the number and duration of cadaver groomings and transports and on the number of task performers (groomers or transporters) per cadaver (columns).

						Duration the
		Statistical values	Trial	Cadaver size	Number of task performers	cadaver stayed in the
						nest
	Number of	L	1.573	1.041	209.357	0.688
	groomings	P	0.209	0.791	< 0.0001	0.407
	Grooming duration	L P	1.113 0.292	2.041 0.564	128.031 < 0.001	0.513 0.474
	Number of	L	5.461	5.367		21.198
	groomers	P	0.019	0.147		< 0.0001
	Number of	L	0.002	4.936	32.139	2.967
	transports	P	0.968	0.177	< 0.0001	0.085
	Transport duration	L P	0.185 0.667	2.295 0.514	18.897 < 0.0001	8.912 0.003
	Number of	L	2.084	12.668		0.668
	transporters	P	0.149	0.005		0.414

  ; Julian and Cahan 1999; but see e.g.[START_REF] Ataya | Le comportement nécrophorique chez la fourmi Lasius niger L[END_REF][START_REF] Renucci | Complex undertaking behavior in Temnothorax lichtensteini ant colonies: from corpse-burying behavior to necrophoric behavior[END_REF]) and groomings and intranidal transports have, if observed, often only been described (e.g.[START_REF] Wilson | Chemical releasers of necrophoric behavior in ants[END_REF][START_REF] Visscher | The honey bee way of death: Necrophoric behaviour in Apis mellifera colonies[END_REF], but see Renucci et al. 2010 and Neoh et al 2012 which studied cadaver-management modulation due to corpses of different origin). A study on Myrmica rubra demonstrated that only a few fresh cadavers are removed immediately and 85% remain in the nest up to 6 days postmortem

  not in others: e.g.[START_REF] Sundström | Reproductive alliances and posthumous fitness enhancement in male ants[END_REF][START_REF] Wiernasz | Patriline shifting leads to apparent genetic caste determination in harvester ants[END_REF]) and workers of different patrilines are thus sequentially produced. Both mechanisms would increase genetic relatedness among nestmates of similar age and. The results highlight the need to consider age in genetic studies and hereditary influences in age studies as both can covary.

  [START_REF] Laidlaw | Polyandry in honey bees (Apis mellifera L.): sperm utilization and intracolony genetic relationships[END_REF][START_REF] Holman | Random sperm use and genetic effects on worker caste fate in Atta colombica leaf-cutting ants[END_REF][START_REF] Stürup | Sperm mixing in the polyandrous leaf-cutting ant Acromyrmex echinatior[END_REF]; but not in others: e.g.[START_REF] Sundström | Reproductive alliances and posthumous fitness enhancement in male ants[END_REF][START_REF] Wiernasz | Patriline shifting leads to apparent genetic caste determination in harvester ants[END_REF]) and workers of different patrilines are thus sequentially produced. Both mechanisms would increase genetic relatedness among nestmates of approximately the same age. Determining the benefits and costs of interindividual variation will be important to address the underlying mechanisms and help to advance questions such as if there is an optimal level of workforce variability, depending on colony size, tasks performed[START_REF] Jeanson | Interindividual variability in social insects -proximate causes and ultimate consequences[END_REF] or parasitic pressure. Increased research effort manipulating nestmate variability in immune investment (for instance the demography or the level of individual experience in a sanitary task) is warranted to address colony-level effects of nestmate diversity for disease defense.

Behav Ecol Sociobiol (2014) 68:1701-1710

Behav Ecol Sociobiol (2014) 68:1701-1710

Acknowledgments We thank Katrin Kellner for colony establishment and characterization, Mike Bidochka for the fungal strain, Meghan Vyleta for fungal strain characterization, Martina Klatt and Simon Tragust for help in the laboratory, Dimitri Missoh for developing the software Bio-Logic, and Mark Brown and Raphaël Jeanson for discussion and help with data analysis. The study was funded by the European Research Council (ERC Starting Grant to SC; Marie Curie IEF to LVU) and the German Research Foundation DFG (to SC and to JH), and CW received funding by the doctoral school Diversité du Vivant (Cotutelle project to CD and SC).

Ethical standards

The performed experiments comply with European laws.

Conflict of interest

The authors declare that they have no conflict of interest.

ACKNOWLEDGMENTS

I would like to thank everyone warmly who has supported me during my thesis work and the writing process (and if you are reading this, you very likely have). It has not been easy due to several difficulties such as experimental complications (seemingly endless construction work leading to the loss of brood in ant colonies and a delay of genetic work of nearly 8 months), administrative challenges or health problems. I was able to overcome them thanks to your support.

I thank especially:

Claudie Doums and Sylvia Cremer for the possibility to work on this project, for their support, understanding, encouraging words and patience. Thank you for giving me such scientific freedom and the possibility to shape this thesis with my own ideas. I would further like to thank you for your generous financial support, which enabled me to improve my statistical skills in an R course (and thank you Claudie for your big statistical help when I realized that the knowledge gained in such a course is still very basic), to collect the species Cataglyphis velox, travel between institutions and attend several conferences. I would like to thank Sylvia for being available even at 1 am in the morning and during maternity leave, for welcoming me in Regensburg and at the IST, for always being open to new ideas and providing a place to stay in Regensburg. Claudie, thank you for bringing 'reality' back into my huge project plans and at the same time being excited about new ideas and even the smallest results. There were many times during the thesis (especially during the last two years) when your encouraging words and practical help were invaluable.

Jürgen Heinze for the opportunity to study Platythyrea ants in his department, providing colonies and scientific stimulation in journal clubs and for meetings in Regensburg and Paris.

Mark JF Brown and Raphaёl Jeanson for being thesis mentors and for fruitful discussions during thesis committee meetings. Mark for the welcoming visit to his laboratory with a tasting of British beer afterwards and Raphaёl for statistical help, his understanding and support with applications for thesis prolongations as well as for his encouraging words and meetings in Banyuls and in Paris.