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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).
The genus Platythyrea belongs to the subfamily Ponerinae and consists of 37 species worldwide (Bolton 1995). 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 (Seal et al. 2011). 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 (Heinze and Hölldobler 1995; Kellner 2009). Nest cavities contain organic material such as dead plant particles, prey remnants and empty cocoon cases (Schilder 1999). 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 (Schilder 1999; Torres 1984). 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 (Seal et al. 2011). Various alternative reproductive tactics and female phenotypes co-occur 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 (Heinze and Hölldobler 1995; Hartmann and Heinze 2003; Schilder et al. 1999b). Reproductive monopolization probably evolved to increase colony efficiency and is achieved by worker policing (Heinze and Hölldobler 1995; 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 (Heinze and Hölldobler 1995; Schilder et al. 1999a; Hartmann et al. 2005; 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).
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 (Cerdá and Retana 1997). This species is endemic to the Iberian Peninsula and found in an elevation range between 0 and 2400 m (Tinaut 1990) (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).
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 Thomas and Read 2007). The general insect pathogenic fungus Metarhizium robertsii (Bischoff et al. 2009) is a natural pathogen of ants (Rath et al. 1992; Keller et al. 2003; St. Leger et al. 2011) and occurs in soils worldwide (St. Leger et al. 2011). Entomopathogenic fungi invade hosts by direct penetration of the cuticle. When conidiospores of Metarhizium adhere to the cuticle, they germinate under high-humidity conditions and penetration occurs by a combination of physical force and cuticlula-degrading enzymes after approximately 24 hours (Clarkson and Charnley 1996; Hajek and St. Leger 1994; Ugelvig et al. 2010).
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 (Wang and St. Leger 2006). 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 (Clarkson and Charnley 1996; Hänel 1982; Hajek and St. Leger 1994; 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 (Arthurs and Thomas 2001: 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 Shah and Pell 2003).
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 (Tinaut 1990; Cerdá and Retana 1997). 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 Cerdá and Retana (1997). 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.
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.
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.
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