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 (Boomsma and Ratnieks 1996; Strassmann 2001; Eyer et al. 2013b). Polyandry, as well as polygyny (several functional queens present within one colony) and genetic recombination (intra-chromosomal recombination; e.g. Sirviö et al. 2006), 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 Palmer and Oldroyd 2000; Crozier and Fjerdingstad 2001). 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. Liersch and Schmid-Hempel 1998; Baer and Schmid-Hempel 1999; Schmid-Hempel and Crozier 1999; Baer and Schmid-Hempel 2001; Hughes and Boomsma 2004; Tarpy and Seeley 2006; Seeley and Tarpy 2007; Ugelvig et al. 2010; but see e.g. Wilson-Rich et al. 2012). 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 (Robinson and Page 1988; Oldroyd et al. 1994), waste management (Waddington et al. 2010; Eyer et al. 2013a), necrophoresis (Robinson and Page 1988, 1995), allogrooming (Frumhoff and Baker 1988) and hygienic behavior (Pérez-Sato et al. 2009). 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 (Masterman et al. 2001; Gramacho and Spivak 2003). Genetically based differences in task performance among nestmates benefits colonies by increasing colony homeostasis (e.g. Jones et al. 2004) and generating a stable and resilient system of division of labor when faced with environmental perturbations (e.g. Oldroyd and Fewell 2007).
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 (Hughes et al. 2010; Armitage et al. 2011). In honeybees however, subfamilies (offspring of different patrilines) did not differ in phenoloxidase investment or in their capacity to encapsulate a foreign body (Wilson-Rich et al. 2012). 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 (Padilha et al. 2013) and candidate genes that affect an individual’s propensity to perform this task are currently investigated (Lapidge et al. 2002; Oxley et al. 2010). 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 (Bonasio et al. 2010; Nygaard et al. 2011; Smith et al 2011a; Smith et al. 2011b; Suen et al. 2011, Wurm et al. 2011) and one termite species (Terrapon et al. 2014) 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 (Ben-Shahar et al. 2002). 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; Bonasio et al. 2012; Simola et al. 2013; Li et al. 2014). Allelic variation and epigenetic factors could both modulate phenotypic plasticity and increase interindividual variation within the workforce.
Eusocial insects are able to generate worker phenotypes with distinct morphological, physiological and behavioral phenotypes from the same genome through a variety of non-mutually 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. Evans and Pettis 2005; Baer and Schmid-Hempel 2006). 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; Doums and Schmid-Hempel 2000; Amdam et al. 2005; Bocher et al. 2007; Castella et al. 2008, 2010; Simone et al. 2009; Walker and Hughes 2009; Kay et al. 2014). 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: Schlichting and Pigliucci 1993). 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 (Passera et al. 1996; McGlynn and Owen 2002; Hughes et al. 2003; Bargum et al. 2004; Jeanson and Weidenmüller 2013). Worker specialization on waste management, necrophoresis and hygienic behavior depends not only on genetic factors (Robinson and Page 1988, 1995; Pérez-Sato et al. 2009; 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; Breed et al. 2002; Mersch et al. 2013; Camargo et al. 2007; 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 (Huang et al. 1994). It has been demonstrated that physiological immune defenses of social insect workers change with age (immunosenescence; Doums et al. 2002; Amdam et al. 2004, 2005; Schmid et al. 2008; Moret and Schmid-Hempel 2009) and can be linked to the age-dependent performance of indoor or outdoor tasks (Amdam et al. 2005; Bocher et al. 2007).
The social environment
The social environment creates opportunities for feedback loops between nestmates (direct interactions) and between individuals and their shared environment (indirect interactions; Jeanson and Weidenmüller 2013), 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. Jeanson 2012; Mersch et al. 2013; Stroeymeyt et al. 2014). 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 Stroeymeyt et al. 2014). An individual’s position in the interaction network will thus reinforce asymmetry between nestmates (Jeanson and Weidenmüller 2014; Stroeymeyt et al. 2014). 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, 2006) and were directed towards waste-contaminated workers of the ant Atta cephalotes, thereby reinforcing sanitary division of labor (Hart and Ratnieks 2001). Stress within social groups arising through reproduction-based dominance interactions led to immunosuppression in ants (Bocher et al. 2008). 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; Gempe et al. 2012).
Social interactions within colonies directly affect the physiological susceptibility of individual group members and can increase their resistance towards a parasite (‘social immunisation’: Traniello et al. 2002; Ugelvig and Cremer 2007; Konrad et al. 2012; Hamilton et al. 2011). Immunity can be socially transferred (reviewed in Masri and Cremer 2014), by passing antimicrobial molecules between workers (horizontal transfer, e.g. Hamilton et al. 2011) or to the offspring (vertical transfer, e.g. Sadd and Schmid-Hempel 2007). 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 (Poulsen et al. 2003). 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: Huang and Robinson 1996; Leoncini et al. 2004). 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; Traniello et al. 2002; Yanagawa et al. 2008; 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 (Konrad et al. 2012).
Theoretical (foraging context: Linksvayer et al. 2009) and empirical research (hygienic behavior: Arathi and Spivak 2001; Gempe et al. 2012) 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 (Richter et al. 2012). However, a short-term absence of nestmates did not affect immunocompetence (the level of phenoloxidase) in leaf-cutting ants (Armitage and Boomsma 2010). Social evolution is linked with increasing group size and potentially increasing individual density, which have been shown to affect individual immune status (e.g. Doums and Schmid-Hempel 2000; Ruiz-González et al. 2009; Turnbull et al. 2011) and both increased group size (e.g. Rosengaus et al. 1998b; Hughes et al. 2002) as well as demographic differences among nestmates (Rosengaus and Traniello 2001) can be beneficial for individual survival.
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 (Table 2, LMM, coefficient = 0.051) and with the duration a cadaver stayed in the nest (Table 2, LMM, coefficient = 0.001), but was not affected by cadaver size (Table 2, 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 (Table 2, LMM). The largest and second smallest cadavers were transported by fewer individuals.
Table 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).
Grooming and transport of cadavers
Cadaver grooming was performed by 31 % of nestmates, a behavior which has been described in the ants Pogonomyrmex badius (Wilson et al. 1958) and Lasius niger (Ataya and Lenoir 1984), in some termite species (Neoh et al. 2012; Myles 2002; Renucci et al. 2010) and in honeybees (Visscher et al. 1983). 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; Yanagawa et al. 2008; Tragust et al. 2013a) and can then be deactivated in the groomer’s alimentary tract (Yanagawa and Shimizu 2007). 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 (Konrad et al. 2012), (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 (Renucci et al. 2010). 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 (Cerdá and Retana 1997) and might pose a sanitary risk to the colony. Prophylactic grooming has been reported in Acromyrmex echinatior ants which self-groom 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 re-entering the nest (Reber et al. 2011).
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. Wilson et al. 1958; Julian and Cahan 1999; Renucci et al. 2010), in honey bees (Visscher 1983) 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 (Wilson et al. 1958; Heinze and Walter 2010; Bos et al. 2012) 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; Diez et al. 2011). 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; Julian and Cahan 1999; Hart and Ratnieks 2001, 2002; Ballari et al. 2007). 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 (Breed et al. 2002). 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 cadaver-management 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’, Traniello et al. 2002; Ugelvig and Cremer 2007; Konrad et al 2012). 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 (Diez et al. 2014).
Table of contents :
3.1 Disease resistance in social insects
3.1.1 Individual physiological immune defenses
3.1.2 Collective immune defenses
3.2 Division of labor in social insect colonies
3.3 Division of labor in disease resistance and causes of interindividual variability in the workforce
3.3.1 Genetic variability
3.3.2 Phenotypic variability
3.4 Aims of the thesis
3.5 The study systems
3.5.1 Platythyrea punctata
3.5.2 Cataglyphis velox
3.5.3 Metarhizium robertsii
4. CHAPTER 1: Increased grooming after repeated brood care provides sanitary benefits in a clonal ant.
5. CHAPTER 2: Necrophoresis is not everything: cadaver groomings and intranidal transports in the ant Cataglyphis velox.
6. CHAPTER 3: Are worker size and phenoloxidase activity of Cataglyphis velox workers genetically determined?
7. CONCLUSIONS AND PERSPECTIVES
7.1 What are the benefits of interindividual variation in immune investment?
7.1.1 Behavioral performance of sanitary tasks
3.1.2 Physiological immune investment
7.2 Potential costs of interindividual variation
7.3 The modulation of sanitary division of labor and the costs and benefits of helping
7.4 Who is expected to invest more heavily into immune defense?
7.5 Nature versus nurture: phenotypic plasticity in immune defense mechanisms
7.6 When is experience-modulated behavioral plasticity expected to occur?
7.7 Empirical difficulties to analyze proximate mechanisms contributing to interindividual variation in immune defense