Regulation of Reproductive Division of Labor in the Ant Camponotus Floridanus : Behavioral Mechanisms and Pheromonal Effects

Regulation of reproductive division of labor in the ant Camponotus floridanus : Behavioral mechanisms and pheromonal effects

Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg

vorgelegt von:

Annett Endler aus Marienberg

Würzburg 2006

Eingereicht am: ......

Mitglieder der Promotionskommission:

Vorsitzender: ...... 1. Gutachter: Prof. Dr. Bert Hölldobler 2. Gutachter: Prof. Dr. Hans-Joachim Poethke

Tag des Promotionskolloquiums: ......

Doktorurkunde ausgehändigt am: ......

“In formica non modo sensus sed etiam mens, ratio, memoria.”

(Die Ameise hat nicht allein Sinnes-Empfindung, sondern auch Geist, Verstand und Gedächtnis.)

Cicero (De Nat. Deorum, Lib.III c.IX)

This thesis is based on following manuscripts:

Chapter II: Endler A, Liebig J, Schmitt T, Parker J, Jones G, Schreier P, Hölldobler B (2004). Surface hydrocarbons of queen eggs regulate worker reproduction in a social insect. Proceedings of the National Academy of Sciences USA 101:2945-2950

Chapter III: Endler A, Liebig J, Hölldobler B (2006). Queen fertility, egg marking and colony size in the ant Camponotus floridanus . Behavioral Ecology and Sociobiology 59:490-499

Chapter IV: Endler A, Hölldobler B., Liebig J. Lack of physical policing and fertility cues in egg-laying workers of the ant Camponotus floridanus . (subm.)

Table of Contents

I. General Introduction ...... 1 I.1 Maintenance of eusociality ...... 1 I.1.1. Kin-selected altruism ...... 1 I.1.2. Enforcement ...... 3 I.1.3. Mutual benefit and indirect reciprocity...... 6 I.2 Chemical Communication ...... 7 I.3 Reproductive division of labor in an evolutionary derived ant...... 8 I.3.1 The model system Camponotus ( Myrmothrix ) floridanus (Buckley) ...... 10 I.4 References...... 12 II. Surface hydrocarbons of queen eggs regulate worker reproduction ...... 20 II.1 Introduction...... 20 II.2 Materials and Methods...... 22 II.3 Results ...... 24 II.4 Discussion...... 29 II.5 Acknowledgements...... 32 II.6 References ...... 32 III. Queen fertility, egg marking and colony size in the ant ...... 35 III.1 Introduction ...... 36 III.2 Materials and Methods...... 39 III.3 Results...... 42 III.4 Discussion ...... 48 III.5 Acknowledgements...... 51 III.6 References ...... 51 IV. Lack of physical policing and fertility cues in egg laying workers ...... 56 IV.1 Introduction...... 57 IV.2 Materials and Methods...... 59 IV.3 Results...... 63 IV.4 Discussion ...... 69 IV.5 Acknowledgements ...... 72 IV.6 References...... 72

V. General Discussion ...... 78 V.1. References ...... 82 VI. Summary ...... 87 VII. Zusammenfassung ...... 90 VIII. Danksagung ...... 94 IX. Curriculum Vitae ...... 96

I. General Introduction 1

I. General Introduction

I.1 Maintenance of eusociality

Insect societies have long been perceived as being perfectly well organized groups of altruistic individuals engaged in peaceful cooperation (Keller & Chapuisat 1999). Indeed, the essential characteristic of the eusocial insects, which include all ants and termites as well as many bees and wasps, is the reproductive division of labor between one or few reproducing individuals (queens and males) and a majority of sterile conspecifics (workers) (Wilson 1971; Gadakar 1994; Sherman et al. 1995). To understand why insect societies are not destabilized by reproductive conflicts between reproductives and sterile conspecifics, we must investigate proximate mechanisms that maintain social cohesion. What makes infertile workers evolutionary successful and resolve reproductive conflicts in an insect society? Basically four processes – kin-selected altruism, enforcement, mutual benefit and indirect reciprocity – shape the preconditions for cooperation and maintain eusociality (Maynard Smith & Szathmáry 1995). These processes act complementary in varying degrees in all societies. This limits the effects of reproductive conflict and often leads to complete resolution. The present study will investigate the proximate mechanisms that expose the regulation of reproduction in an evolutionary derived ant species. This study discusses potential reproductive conflicts involved in the direct reproduction by individuals and manipulation of the reproduction of colony members. In contrast to recent work on more ancestral species, the system used in this study is characterized by high colony relatedness, large colony sizes and low reproductive potential of the workers, thus utilizing a class of insect societies that have been insufficiently investigated so far. The results presented here identify behavioral and pheromonal mechanisms by which reproductive conflicts are in fact resolved and therefore make a considerable contribution toward our understanding how social cohesion maintain insect societies.

I.1.1. Kin-selected altruism Altruism is defined by helpful behavior that raises the recipient’s direct fitness while lowering the donor’s direct fitness (Alcock 1998). Such altruistic behaviour, and in particularly extreme reproductive altruism involving sterility, seem to contradict Darwin’s theory of natural selection. This theory predicts that each individual is forced to maximize its number of offspring, the individual being defined as the “unit of self-interest”. He discussed the I. General Introduction 2 problems of altruism and sterility in social insects and postulated that workers could evolve if they are profitable to the community (Darwin 1892 p. 295). Thus, we might conclude that sterility might evolve if colonies that produce workers produce extra queens and males (reviewed in Bourke & Franks 1995). Sexuals of these colonies would have been favored by natural selection, since they transmit the profitable trait of worker-reproduction to their own offspring. One of the modern explanations of altruism evolved by natural selection is described by Hamilton’s kin-selection theory (1964). It indicates that individuals increase their personal fitness, thus propagation of their own genes, by helping a relative to rear offspring (inclusive or indirect fitness). However, the genetic perspective adopted in inclusive fitness theory also predicts within-group conflicts to arise whenever the co-operating individuals are not genetically identical; hence each individual gains most by favoring the offspring produced by closest relatives (Trivers & Hare 1976). Hamilton’s theory gains of importance in groups of social Hymenoptera, since offspring are asymmetrically related to each other due to their haplodiploid sex determination system. In most species, workers are not able to mate, and thus lack the ability to produce diploid female offspring, but retain functional ovaries and can lay male eggs (e.g. Ratnieks & Reeve 1992). If females have the same mother and father, they are related to their sisters by 0.75 but only by 0.25 to their brothers and 0.375 to their nephews. Their own male and female offspring is related by 0.5 to them. Under these conditions workers should prefer a female biased sib-rearing because sisters are more closely related than own offspring. However, a female-biased population sex ratio does not make worker behavior more profitable in female haplodiploids necessarily. If females are more abundant than males in a population, then females are reproductively less valuable than males (Maynard Smith & Szathmáry 1995). The advantage that workers gain by preferentially investing in their sisters disappears under these conditions. On the other hand, the asymmetrical relatedness results in a potential conflict over male production; thus females should prefer their own sons over their nephews (worker- worker) and both over their brothers (queen-worker) (Trivers & Hare 1976; Woyciechowski & Lomnicki 1987; Ratnieks 1988; Ratnieks & Reeve 1992; Wenseleers et al. 2004; Ratnieks et al. 2006). Worker reproduction, in the sense of the ability to lay unfertilized male-eggs (arrhenotoky) is common in ants (e.g. Brian 1979, 1980; Fletcher & Ross 1985; Bourke 1988; Choe 1988; Hölldobler & Wilson 1990). Total sterility in workers due to the absence of functioning ovaries is known from relatively few genera, including Solenopsis, Monomorium, Pheidole, Tetramorium , and Eciton (Bourke 1988). Workers may have retained their male- I. General Introduction 3 producing competence because of selection for worker reproduction under monogyny with singly mated queens. However, under other genetic and social circumstances, e.g. polygyny or even monogyny and single mating in some cases, worker reproduction is counter-selected (see below). In addition, if they are exposed to irreversibly queenless conditions it may be advantageous to workers to keep the ability to produce males (Ratnieks 1988). Costs affect the evolution of worker reproduction independent of kin structure. If worker reproduction decreases total colony productivity, worker should prevent nestmates from laying eggs successfully (worker policing) (Cole 1986; Ratnieks 1988; Pamilo 1991; Hammond & Keller 2004). Furthermore, if one individual forces another to cooperate, the latter can hardly be said to have acted altruistically.

I.1.2. Enforcement Alexander (1974) discussed the evolution of altruistic behavior in social insects. His parental manipulation hypothesis predicts that workers help rear the queen’s offspring because the queen manipulates them into doing so, for example, by physical dominance (Packer 1986). It seems that parental manipulation by definition involves social actions performed among kin (West-Eberhard 1975, Vehrencamp 1983, Bourke & Franks 1995), therefore standing in agreement with kin selection theory (Hamilton 1964). On the other hand, if parental manipulation occurs, offspring will be selected to resist and the strength of this selection will depend on relatedness, costs and benefits in terms of Hamilton (Trivers 1974, Crozier 1979, 1982, Metcalf 1980). In fact, dominance and relatedness both play a role in the evolutionary conflict of interest between relatives (Stubblefield & Charnov 1986; Trivers 1974, Trivers & Hare 1976). In this context, parental manipulation may well have been important in insect social evolution, but it is still a controversial topic. Other factors, e.g. ecological conditions, also affect whether individuals help to rear relatives or produce own offspring. If the ecology of the species is such that young dispersing adults have very little chance to reproduce successfully, then nondispersers benefit more from helping relatives than from personal reproduction (Alexander 1974, Queller 1989, Gadagkar 1990, Alcock 1998). For many social animals, critical resources are limited outside the natal nest. New nests are often very difficult to establish, but once constructed, they offer a safe structure. Under these circumstances, dispersing reproductives face the nearly hopeless task of building a new nest. A foundress female of a eusocial ambrosia beetle for example goes through quite an ordeal to establish her nest in a eucalyptus tree (Kent & Simpson 1992). She will need seven months of gnawing just to carve out the first five centimeters of what may I. General Introduction 4 become an extensive gallery in the heartwood of the tree. Most foundresses probably are killed before they get safely deep into the wood. High adult mortality rates and long periods of offspring dependence combine to provide a large selective advantage for helping behavior too. Polistine wasps that found colonies with helpers produce offspring faster than solitary individuals (Queller 1989). Many solitary reproducers die early and will fail to bring any young to independence, thus helpers that die at the same age may have made substantial fitness benefits by indirect offspring (Queller 1989, Gadagkar 1990). The theories described above discuss different constraints as factors for the origin of eusociality. These theories are just as fundamental as models where enforcement maintains social cohesion. The latter is especially important in the study of advanced eusocial species like ants, where solitary living is completely unknown, making it thus impossible to clarify the origin of eusociality (Bourke & Franks 1995). In these societies, maintenance of social cohesion is affected by reproductive conflicts among individuals involved in the direct reproduction of individuals and manipulation of the reproduction of colony members (e.g. worker policing see chapter I.1.1 Starr 1984; Woyciechowski & Lomnicki 1987; Ratnieks 1988; Frank 1995; Monnin & Ratnieks 2001; Ratnieks et al. 2006). Mutual control of reproduction (worker policing) may be positively selected by two parameters: the relative relatedness of workers to queen- and worker-produced males (relatedness hypothesis) and the colony-level costs of worker reproducing (efficiency hypothesis; Hammond & Keller, 2004). The relatedness hypothesis predicts that high mating frequencies (polyandry) as well as polygyny dilute worker relatedness with an average worker-produced male more than its relatedness with an average queen-produced male (Visscher 1996; Halling et al. 2001; Oldroyd et al. 2001; Foster & Ratnieks 2001 a,b; Whitfield 2002), thus workers should prevent fellow workers from laying eggs successfully. Otherwise, colony-level costs explain worker policing under monogynous and singly-mated conditions too (Foster & Ratnieks 2001b; Hammond & Keller 2004). In the presence of a fertile queen worker reproduction may divert resources from brood rearing, which imposes costs on colony productivity and reduces the indirect fitness gains of workers (Cole 1986; Pamilo 1991). Therefore, workers should rear brothers alone and police reproductive nestmates as well as their male eggs. Worker policing occur as selective removing of worker- laid eggs (oophagy) that otherwise develop into males (ants: Monnin & Peeters 1997; D’ Ettorre et al. 2004a; Helanterä & Sundstöm 2005; bees: Ratnieks & Visscher 1989; Foster & Ratnieks 2001a; Halling et al. 2001; Oldroyd et al. 2001; wasps: Foster & Ratnieks 2000; Tsuchida et al. 2003), or by directing aggression toward workers with developed ovaries I. General Introduction 5

(ants: Gobin et al. 1999; Kikuta & Tsuji 1999; Liebig et al. 1999; Monnin & Ratnieks 2001, Dietemann et al. 2003; Iwanishi et al. 2003; Kawabata & Tsuji 2005; wasps: Wenseleers et al. 2005). Another reproductive conflict, the conflict over the sex allocation ratio, can explain worker policing contrary to relatedness predictions (Foster & Ratnieks 2001b). The basic sex ratio theory by Trivers & Hare (1976) combine Fisher’s (1930) sex ratio theory and Hamilton’s kin selection theory (1964) to produce a sex ratio theory for Hymenoptera characterized by a haplodiploid sex determination system. Trivers & Hare (1976) pointed out that the queen will favor a 1:1 ratio of investment, and workers a 3:1 ratio of investment in females relative to males. Data on primitively eusocial bees support the latter ratio, and hence suggest that the workers control the sex ratio (Mueller 1991). Bourke and Franks (1995) arrive at the same conclusion that the stable investment ratio for workers is 3:1 (females : males). Sex allocation biasing favors worker policing because policing removes some males (sons of workers) at low cost at the egg stage rather than a higher cost on larval stage (sons of the queen) (Foster & Ratnieks 2001b, Ratnieks et al. 2006), because workers cannot determine the sex of queen-laid eggs (Nonacs & Carlin 1990). This is an important interaction between two reproductive conflicts, in which the presence of one conflict (sex allocation) favors the suppression of the other (male production by workers). Worker policing in terms of egg-removing or physical aggression towards ovary developed workers by other colony members assumes that infertile individuals are able to detect worker laid eggs and reproductive nestmates. Previous studies indicate that long- chained hydrocarbons present on the cuticle seem to play a major role in these identification mechanisms, e.g. they correlate with ovarian activity in various social insects (ants: Dahbi & Lenoir 1998; Monnin et al. 1998; Peeters et al. 1999; Liebig et al. 2000; Cuvillier-Hot et al. 2001; Hannonen et al. 2002; Heinze et al. 2002, Dietemann et al. 2003; D’Ettorre et al. 2004b; wasps: Bonavita-Cougourdan et al. 1991; Butts et al. 1995; Sledge et al. 2001; Dapporto et al. 2004; bumblebees: Ayasse et al. 1995). These suggest that cuticular hydrocarbons might reflect the presence and the physical condition of the reproductive colony members, thus honest fertility signals (Keller & Nonacs 1993). Strict manipulation by reproductive nestmates, as the parental manipulation theory predicts, (Alexander 1974, Ross & Matthews 1991) does not seem to have much support.

I. General Introduction 6

I.1.3. Mutual benefit and indirect reciprocit y Much evidence exists that a high relatedness is not an absolute precondition for sociality because some ant foundress associations, for example, are cooperations among non-kin (Hagen et al. 1988; Sasaki et al. 1996; Bernasconi & Strassmann 1999). In addition halictine bees, kingfishers, manakins and mongooses display a large amount of cooperation between non-relatives (Reyer 1984; Creel & Waser 1994; Kukuk & Sage 1994; McDonald & Potts 1994). Cooperation can evolve if individuals interact repeatedly. This mechanism is often referred as “direct reciprocity” or “reciprocal altruism” because individuals can reciprocate previous cooperative behavior of partners (Trivers 1971). Direct reciprocity can only be successful if individuals recognize their partners and remember the outcome of their previous encounter, or if individuals interact with only one partner for a long time (Dugatkin 2002). In the literature only few examples of reciprocity beside humans exist (Packer 1977, Wilkinson 1984), where individuals interacted repeatedly and donors were able to recognize recipients that failed to reciprocate. If the mentioned conditions are lacking, a population composed of reciprocal altruists would be vulnerable to invasion by individuals that accepted help but later do not return the favor. Such ‘cheaters’ would reduce the fitness of ‘noncheaters’, which would presumably make direct reciprocity unlikely to evolve in accordance with the model of game theory called “prisoner’s dilemma” (Axelrod & Hamilton 1981; Axelrod 1984; Nowak & Sigmund 1993). In complex social systems, especially human societies, other mechanisms than direct reciprocity are imaginable. Direct reciprocity is captured in the principle: “I help you and you help me over again”, but also it makes sense if we propose: “I help you and somebody else helps me”. The latter mechanism, called “indirect reciprocity”, appears when benevolence to one agent increases the chance to receiving help from others (Alexander 1987; Nowak & Sigmund 2005). The model assumes that two individuals interact at most once with each other, consequently repeated interactions with the same partner are not necessary. In contrast to direct reciprocity, it is impossible for a cheater to gain fitness benefits at the expense of a cooperative donor. Since partners of a social interaction often change, an individual should assess their partner by using social information such as reputation and make decisions whether to help him or not (Pollock & Dugatkin 1992; Dunbar 1996; Whiten & Byrne 1997; Nowak & Sigmund 1998; Ohtsuki 2004). To those who have ‘good’ social reputation, an individual reciprocates by giving aid, whereas those that have a ‘bad’ reputation, help will be refused. I. General Introduction 7

Cooperation based on similarity could be also applicable in situations where repeated interactions are rare, and reputations are not established (Riolo et al. 2001). In fact, when agents donate to others who are sufficiently similar to themselves in any observable trait for example, cooperation can arise. The basis for similarity can be completely arbitrary, such as for chemical markers or cultural attributes. Indirect reciprocity and other complex models of game theory offer many new approaches to understand the conditions for the maintenance of cooperation in evolving populations, especially for groups with non-related members (Reyer 1984; Creel & Waser 1994; Kukuk & Sage 1994; McDonald & Potts 1994). However, these models are suitable to only limited extent for most social insects with high colony relatedness.

I.2 Chemical Communication

Investigations on the proximate mechanisms such as chemical signaling contribute to our understanding of the reproductive division of labor, a fundamental characteristic of eusocial societies. In social Hymenoptera, chemical cues of reproductives should represent cooperative signals that inform workers how they can realize their fitness interests (see I.1.2; Seeley 1985; Keller & Nonacs 1993; West-Eberhard 2003). However, the importance of chemical communication in social insects is not restricted to regulation of reproduction. In fact, to live in animal societies require effective mechanisms of communication and the coordination of different behavioral tasks. Solitary individuals or species with parental care (subsocial) already possess numerous abilities to communicate with mates or offspring (Bradbury & Vehrencamp 1998). Social insects possess additional features such as foraging, defense of the nest area and division of labor. The required mechanisms differ depending on the senses employed; olfactory communication is generally the most important of all. Chemical cues are not only important for recruitment behavior but also in all other behavioral tasks such as kin- and nestmate recognition, food exchange, mating behavior, territoriality and alarm signaling (Wilson 1971; Hölldobler & Wilson 1990). A terminology to classify the functions of chemical substances was developed by (Nordlund 1981). A semiochemical is any substance used in communication, whether between species (as in symbioses) or between members of the same species (Law & Regnier 1971). A pheromone is a subclass of semiochemicals, usually a glandular secretion, that mediate communication between conspecifics. They are defined as “substances secreted to the I. General Introduction 8 outside by an individual and received by a second individual of the same species in which they release a specific behavior (releaser pheromone) or developmental process (primer pheromone)” (Karlson & Lüscher 1959). An allomone is a comparable substance employed in communication across species, adaptively favorable to the emitter but not to the receiver (Brown 1968, Brown et al. 1970). In contrast, the term kairomone cover chemicals emitted by an organism that elicit a response adaptively favorable to the receiver but not to the emitter (Brown et al. 1970). In the following chapters the terms ‘cue’ or ‘signal’ were used instead of ‘pheromone’ in cases where the exact activities of the chemical compounds in the investigated model system were unknown. In this context, a signal means the sequence of states of a communication channel that encodes information by a sender to a receiver (Bradbury & Vehrencamp 1998). A signal may include for example a chemical odor or an acoustical sign. The provision of information occurs deliberately because the sender benefits by altering the likelihood that the receiver will respond in one way instead of another (Bradbury & Vehrencamp 1998). On the other hand, animals can also produce stimuli whose perception by other animals is not beneficial to the emitter. These stimuli are usually called cues (Seeley 1989).

I.3 Reproductive division of labor in an evolutionary derived ant

In line with Hamilton’s kin selection theory (1964) social Hymenoptera are able to compensate the loss of direct fitness benefits by indirect fitness gains, when rearing closely related offspring (see I.1.1). Furthermore, it is in the interest of individuals to forgo personal reproduction if indirect fitness benefits exceed the loss of direct offspring. The theories discussed above (see I.1.2.) demonstrate that colony members can be manipulated by nestmates preventing own reproduction. Manipulation by physical aggression is known from species with small colony sizes (e.g. Pardi 1948; Breed & Gamboa 1977). In previous studies, it was postulated that the queen pheromone manipulates workers in Hymenoptera species with large colony sizes (e.g. Ross & Matthews 1991), whereas current studies show that queen pheromones are honest signals, which inform workers how they can realize their fitness interests (Ratnieks 1988, 2006; Nonacs & Keller 1993). If the queen is healthy, highly fertile and closely related to the workers, the latter should help to increase the colony production of reproductives. However, two requirements are necessary. The queen pheromone should contain information about health and fertility of the queen. On the other hand, worker reproduction should cause a decrease of colony productivity, thus imposing costs on colony I. General Introduction 9 level (Cole 1986, Bourke & Franks 1995). These costs can be created by the loss of work force or by interindividual aggression as a consequence of the reproductive attempt. Despite the existence of this signal, it seems difficult to understand how it should be effective in a large colony. Not every colony member can regularly contact the queen to perceive her signal directly. Thus, there must be alternative ways of indirect communication. One method is observed in the honeybee Apis mellifera, where messenger bees distribute the queen mandibular pheromone throughout the colony (Seeley 1979; Mohammedi et al. 1998; Oldroyd et al. 2001). A further possibility of indirect communication is the use of eggs as a vehicle to distribute a queen signal throughout the colony. However, no experimental proof exists so far for this hypothesis. Workers should be able to detect differences in the activity of ovaries, an assumption for fertility signaling. Examples are known from different Hymenopteran species (Gobin et al. 1999; Liebig et al. 1999; Ortius & Heinze 1999; Tsuji et al. 1999). However, the mentioned mechanism is poorly understood, despite indications for olfactory recognition. Cuticular hydrocarbons seem to play a major role by the information transfer. Previous studies show that workers are able to detect slight differences of the cuticular hydrocarbon profile, e.g. between nestmates and alien workers (Lahav et al. 1999; Thomas et al. 1999; Wagner et al. 2000). Furthermore, hydrocarbon profiles correlate with the rate of egg-laying in different species (ants: Dahbi & Lenoir 1998; Monnin et al. 1998; Peeters et al. 1999; Liebig et al. 2000; Cuvillier-Hot et al. 2001; Hannonen et al. 2002; Heinze et al. 2002, Dietemann et al. 2003; D’Ettorre et al. 2004b; wasps: Bonavita-Cougourdan et al. 1991; Butts et al. 1995; Sledge et al. 2001; Dapporto et al. 2004; bumblebees: Ayasse et al. 1995). It is also important for workers to control other infertile nestmates and prevent them from male-production (worker policing). Worker male production as well as aggressive policing behaviour produces costs on colony efficiency, which should finally induce workers to refrain from reproduction (self-restrain) (Ratnieks & Reeve 1992; Frank 1995; Wenseleers et al. 2004), even if workers are closely related in the colony. Function and kind of worker policing as well as potential costs of worker reproduction should be verified with adequate tests in this thesis. So far, the presence of a fertility signal and its compounds has been only shown in the honeybee and in more ancestral ant species, such as the Ponerinae (Butler 1957; Butler et al. 1962; Liebig et al. 2000; Heinze et al. 2002; Hoover et al. 2003; D’Ettorre et al. 2004b). Ancestral species are characterized by small colony sizes, only a slight caste dimorphism and I. General Introduction 10 a high reproductive potential of the workers. Only little is known about evolutionary derived species. The experiments reported in this thesis clarify the proximate mechanism of reproductive division of labor in the evolutionary derived ant Camponotus floridanus . In this species I tested the presence of queen signals on queen eggs and the function of cuticular hydrocarbons as fertility signal. This study also investigated whether workers from C. floridanus differentiate between eggs originating from workers, highly fertile queens from large colonies and from weakly fertile queens from small colonies. I furthermore investigated the profiles of the surface hydrocarbons whether differences exist among egg-layers as this would support the idea that hydrocarbons are responsible for egg recognition. Since I argue that the hydrocarbon profiles represent a fertility signal, I investigated the egg-laying rates of the queens and the respective colony size to see whether egg-laying rate is correlated with changes in the hydrocarbon profile. Furthermore, this study clarifies whether the pattern of reproductive regulation in C. floridanus differs from what is known about species with small colonies and with workers characterized by a high reproductive potential. In the latter, pronounced dominance interactions and physical policing of workers are typical in addition to the policing of eggs. I also looked at differences in the hydrocarbon profile of fertile and infertile workers because this is correlated with worker policing in many species (Gobin et al. 1999; Kikuta & Tsuji 1999; Liebig et al. 1999; Monnin & Ratnieks 2001; Iwanishi et al. 2003; Kawabata & Tsuji 2005). Finally, I tested whether individual worker behavior changes because of ovarian activation, causing a cost at the colony level by reducing their contribution to colony productivity. C. floridanus is the perfect model organism to investigate the proximate mechanisms mentioned above, as outlined below.

I.3.1 The model system Camponotus ( Myrmothrix ) floridanus (Buckley)

The carpenter ant Camponotus floridanus provides good conditions for bioassays and chemical analysis, because of the simple relatedness structure. This evolutionary derived species is characterized by large colonies, which contain only one singly mated queen (Gadau et al. 1996) and approximately 15000 workers, thus workers of a colony are closely related. Workers have a low reproductive potential, thus they lay only unfertilized male eggs. Furthermore, the colonies show a distinct dimorphism between the reproductive castes (Fig. 1.1A). I. General Introduction 11

This species is found widely distributed throughout Florida / USA and some neighboring states (Fig. 1.1.B) (Warner & Scheffrahn 2005), thus founding queens of the common species can be easily collected. They prefer nesting sites, which are old drywood termite galleries and wooden objects that have previous damage from other organisms including insects or fungi (Warner & Scheffrahn 2005). In addition, they use debris of almost any kind, coconuts left on the ground, under mulch, inside logs or wooden borders in gardens, railroad ties, under stones and so on. Queenless satellite nests are often founded within 5 to 30 meters of a mature nest. Winged reproductives fly in the evening or night during the rainy season (May through November), the exactly time of the mating flight is depending on weather and geographical position (e.g. differences between islands and mainland) (Warner & Scheffrahn 2005). Typically a monoandrous queen will start a new colony claustrally, caring for her first brood until they develop into workers that then begin to forage for food. When the colony is two to five years old, depending on environmental conditions, new winged reproductives will usually be produced.

A B

North America (USA)

Figure I.1 . A) Queen, workers and respective brood of C. floridanus (photo by A. Endler), B) Geographic distribution (green) of C.

floridanus (map by K.S. Hedlund, 2005)

Another advantage of this species is that it is very easy to maintain their colonies in the laboratory. In the first six months of laboratory rearing approximately 16% of the collected founding queens died, subsequently the mortality decreased to less than 5%. Incipient colonies grew to 1000-2000 individuals within the next one to two years. Ants were kept in plaster nests into which chambers had been formed and covered with glass plates to allow observation. C. floridanus is particularly suitable for behavioral tests as well as chemical analysis. In this study, SPME (Solid Phase MicroExtraction) was used to extract cuticular hydrocarbons from live individuals as well as surface hydrocarbons from eggs (Arthur & Pawliszyn 1990; Malosse et al. 1995; Monnin et al. 1998). The ants were immobilized by I. General Introduction 12 small doses of carbon dioxide during extraction to prevent defensive reactions such as biting and formic acid spraying, thus this procedure extracted only cuticular hydrocarbons. The ants survived repeated carbon dioxide treatments without any consequential damage. The extraction was performed on queens and workers for 3 min and on eggs for 2 min, enough time to get adequate number of hydrocarbons for gas chromatography. This non-destructive technique allowed further behavioral studies of the queens and their colonies therefore an optimal management of the ant keeping. Furthermore, this method is suited for collecting cuticular hydrocarbons without any contamination from internal tissues and glands.

I.4 References

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II. Surface hydrocarbons of queen eggs regulate worker reproduction in a social insect

A hitherto unresolved problem in behavioral biology is how workers are prevented from reproducing in large insect societies with high relatedness. Signals of the queen are assumed to inform the nestmates about her presence in the colony which leads to indirect fitness benefits for workers. In the ant Camponotus floridanus , we found such a signal located on queen-laid eggs. In groups of workers regularly provided with either queen eggs, larvae and cocoons, with larvae and cocoons or with no brood, only in the groups with queen eggs workers did not lay eggs. Thus, the eggs seem to inform the workers about queen presence that let them refrain from reproducing. The signal on queen eggs is presumably the same that enables workers to distinguish between queen and worker-laid eggs. Despite their viability the latter are destroyed by workers when given a choice between both types. Queen and worker- laid eggs differ in their surface hydrocarbons in a similar way as fertile queens differ from workers in the composition of their cuticular hydrocarbons. When we transferred hydrocarbons from the queen cuticle to worker eggs the eggs were no longer destroyed, indicating that they now carry the signal. These hydrocarbons thus represent a queen signal that regulates worker reproduction in this species.

II.1 Introduction

The fundamental difference between solitary and highly social insects is reproductive division of labor between one or a few breeders and their non-breeding helpers (1-3). It is assumed that in large insect societies reproduction is regulated by pheromones (4). One hypothesis suggests that these pheromones may be coercive tools of the breeder (the queen) to prevent its helpers (the workers) from reproducing against their own fitness interests (5, 6) as a form of parental manipulation (7). According to an alternative hypothesis, they may represent cooperative signals that inform workers how they can realize their fitness interests (8-10) in line with kin selection theory (11). In the presence of a fertile queen worker reproduction may impose costs on colony productivity which reduces the indirect fitness gains of workers (12-14). They should therefore either refrain from reproducing (self-policing) or control each other’s reproduction (worker policing) (13). So far, the presence of such a cooperative signal and its compounds has only been shown in the honeybee, Apis mellifera. Here, the queen mandibular pheromone with its main II. Surface hydrocarbons of queen eggs regulate worker reproduction 21 component 9 oxo-decenoic acid let workers refrain from reproducing (15-17). However, workers seem not always to respond to an artificial pheromone or to queen presence in A. mellifera (18, 19). In other species, there exists some evidence that such a signal also occurs (20, 21). Despite the existence of this signal it seems difficult to understand how it should be effective in a large colony, since not every colony member can regularly contact the queen to directly perceive her signal. Thus, there must be alternative ways of indirect communication. One way has become manifest in the honeybee A. mellifera in which messenger bees distribute the queen mandibular pheromone throughout the colony (22, 23). A further possibility of indirect communication is the use of eggs as a vehicle to distribute a queen signal throughout the colony. This has been suggested for the ant Myrmica rubra , where queen produced egg clusters had some inhibitory effect on worker ovarian development (24) and for large, monogynous and polydomous colonies of Aphaenogaster cockerelli or Oecophylla weaver ant, in which the queen remains in one restricted nest zone but her eggs are distributed by workers all over the large nest area (25). However, no experimental proof exists so far for this hypothesis. We tested the presence of queen signals on queen eggs in the ant Camponotus floridanus . In this species a single queen lays eggs while the majority of workers (presumably up to 10000 per colony) remain infertile. Even in subcolonies workers do not lay eggs although the queen is not present. However, brood items including eggs are usually carried into these subnests which suggest an indirect communication of a queen signal via eggs. In our experiments, we first demonstrated that the presence of queen eggs let workers refrain from reproducing. Then we showed that workers differentiate between queen- and worker-laid eggs. The pattern of discrimination corresponds to differences in the described composition of the egg surface hydrocarbons, which actually are qualitatively similar to the cuticular hydrocarbon profiles of the adults. Finally, a transfer of cuticular hydrocarbons of queens on worker eggs rescues the latter from being destroyed by workers, indicating that these hydrocarbons represent the hypothesized queen signal.

II. Surface hydrocarbons of queen eggs regulate worker reproduction 22

II.2 Materials and Methods

Animals. Queens of Camponotus floridanus (N = 75) were collected at the Florida Keys/USA after the mating flight in August 2001 and transferred to the laboratory. They were cultured at 25° Celsius (12h day, 12h night). Subsequently, the queens raised colonies with 1000 - 2000 individuals within the next year. Experimental worker groups were provided with honeywater and 1.5 cockroaches ( Nauphoetia sp.) twice a week.

Egg inhibition experiment. The brood composition of queenless worker groups (N = 19; for each treatment 19 worker groups out of 19 colonies) was varied in three different ways. Group (a) received 250 workers without any brood, group (b) 200 workers with 50 larvae and 50 pupae and group (c) 200 workers with 35 ± 5 queen eggs, 35 larvae and 35 pupae. The groups were controlled for the presence of eggs twice a week. Whenever the number of eggs present in group c had dropped below six another 35± 5 queen eggs was added. Brood from parental colonies was regularly added to approximately maintain the brood composition (Group b: 50 larvae, group c: 35 ± 5 eggs). The groups were regularly controlled for the presence of eggs. The beginning of worker egg-laying is very conspicuous, since up to 700 eggs are produced within a week. Sporadic egg-laying by workers in group c cannot be excluded. However, we have no evidence that this occurred. First, the number of eggs never increased before the conspicuous occurrence of a lot of egg piles, our defined onset of worker egg-laying. Second, no males were produced in these groups, which would be expected if workers perceive the absence of the queen.

Discrimination of queen- and worker-laid eggs. In the first experiment freshly orphaned groups each containing 150 workers were provided with eggs of different origin: they received either 30 to 35 eggs from sister workers, 30 to 35 eggs from their own queen, or 30 to 35 eggs from a foreign queen. Paired worker groups were used, i.e. nine queen colonies were used from which three worker groups were isolated each time. During the next five, days the surviving eggs were counted daily for five days. In the second experiment, four worker groups were isolated from queen colonies. In this case they received either 30 to 35 eggs from their mother queen or from sister workers (N = 9 queenright colonies for each treatment), or 30 to 35 eggs from sister workers treated either with the cuticular hydrocarbons of foreign queens (N = 9 queenright colonies) or with cuticular hydrocarbons from sister II. Surface hydrocarbons of queen eggs regulate worker reproduction 23 workers (N = 5). The sample size of the last group is smaller due to an insufficient number of eggs available at that time. Remaining eggs were counted 1h, 2h and 24 h after the transfer.

Extraction and transfer of compounds. Single queens or two workers, respectively, were extracted 15 min in 1 ml hexane for each experiment. The extracts were fractionated on conditioned SiOH columns (Macherey & Nagel, Chromabond 500mg, glass) with 4 ml hexane and the non-polar hydrocarbon fraction transferred onto clean glass slides and the solvent evaporated. Thirty worker eggs were then swiftly rolled on the extract for 5 min. A solid phase microextraction fiber (SUPELCO, fiber coated with a 7-µm polydimethylsiloxane film) was used to roll the eggs. This allowed to simultaneously sample the hydrocarbon profiles of the manipulated eggs. The extracted profiles were directly injected into the gas chromatograph. Programming of the gas chromatograph was the same as for the cuticular extraction (see below). The hexane used had been distilled to the highest possible purity.

Chemical Analysis. Cuticular hydrocarbons from queens and workers and from eggs were extracted with solid phase microextraction (see above). The fiber was swiftly rubbed on the tergites of queens and workers for 3 minutes and on eggs for 2 minutes. Then the fiber was directly injected into the injection port of a ThermoQuest Trace GC with a split/splitless injector. We used a non-polar capillary column (DB 1, J&W Scientific, Folsom, CA, 20 m x

0.18 mm, 0.18 µm film thickness) with H 2 as carrier gas. The temperature was kept at 60° for two minutes with the split closed for the same time. Then temperature was raised at 60° C/min to 200° C. Temperature subsequently increased at 4° C/min to 320° and then held constant. The injector port was kept at 260° C and the FID at 340° C. Peak areas were computed with Chrom-Card 1.19 (CE Instruments). One part of the GC/MS analysis was carried out with a Hewlett Packard 5890 GC directly coupled to a 5970B mass selective detector (quadrupole mass spectrometer with 70eV electron impact ionisation). The system was controlled by a Hewlett Packard series 300 computer with HP 5972/5971 Chem station. Chromatography was performed using a non- polar capillary column (Restek, RTX-5, 15m x 0.25mm, 0.25µm thickness), using Helium as the carrier gas at 1µl/min. Samples were injected in splitless mode, the split valve being closed before the sample was injected, and reopened 45 seconds later. The solvent delay was set at 3 minutes and the injector port at 250°C. The oven temperature was programmed to increase from 50°C (3 minutes) at 5°C/min to a final temperature of 300°C (10 minutes). II. Surface hydrocarbons of queen eggs regulate worker reproduction 24

Structures were determined by Equivalent Chain Length and the use of standard MS databases – NIST/EPA/NIH mass spectral library and J.Wiley and Sons. The other part of the GC/MS analysis was performed with a Fisons Instruments GC 8000 Series gas chromatograph (Fisons, Egelsbach, Germany) coupled to a Fisons Instruments MD 800 quadrupol mass detector. The GC was equipped either with a J & W DB-5 fused silica capillary column (30 m x 0.25 mm ID; df = 0.25 µm; J & W, Folsom, CA, USA; temperature program: from 60°C to 310°C at 5°C/min and held for 10 min at 310°C) or with a J & W DB-1 fused silica capillary column (30 m x 0.25 mm ID; df =0.25 µm; J & W; temperature program: from 60°C to150°C at 10°C/min, from 150°C to 310°C at 1.5°C/min and held for 10 min at 310°C). Helium was used as carrier gas at a constant pressure of 90 kPa. Injection was carried out at 250°C in the splitless mode for 60 sec. The electron impact mass spectra (EI-MS) were recorded with an ionisation voltage of 70 eV and a source temperature of 220°C. The software Xcalibur (ThermoFinnigan, Egelsbach, Germany) for windows was used for data acquisition. Methylalkanes were characterized by the use of standard MS databases, diagnostic ions and by determining Kovats indices by the method of Carlson et al. (26).

II.3 Results

When worker groups were isolated from the queen, some workers started laying eggs provided no brood or only larvae und pupae were present in the group (Fig. II.1). In the groups that started egg-laying we followed the production of males. On average 64 days (± 13.5 days SD) after the onset of worker egg-laying, new males regularly emerged in 21 of the 23 colonies with worker egg-laying. We did not wait for male emergence in the other two colonies, since they started very late with egg-laying (at day 139 and 158). However, one colony with no queen brood provided produced larvae and the other with queen brood produced male cocoons. In contrast to these groups when the isolated workers were exposed to queen laid eggs, they refrained from reproduction. This is confirmed by the absence of male production in these groups. The refraining from egg-laying suggests that workers can identify the origin of the eggs, thus that these eggs carry a queen signal.

II. Surface hydrocarbons of queen eggs regulate worker reproduction 25

18 larvae/pupae 16 14 without brood 12 eggs/larvae/pupae 10 8 6 4 2 0

layingof colonies -2 Cumulative number 0 20 40 60 80 100 120 140 160 Days Figure II.1 Inhibition of worker egg-laying by the presence of queen eggs. After 160 days of separation from the parental colonies the difference in worker egg-laying among the groups was significant (Overall comparison: Cochran Q test, N = 19, Q = 17.64, p < 0.0001; post hoc comparison one tailed Fisher’s exact test: group b – group c, p < 0.0001; group a – group c, p < 0.002, group a – group b, not significant).

If there is a specific queen signal on these eggs, worker eggs should not elicit this response and workers should be able to discriminate between worker- and queen-laid eggs. When eggs from a queen or sister workers were given to freshly orphaned worker groups, the eggs from sisters were destroyed whereas eggs from their mother or from a foreign queen were tolerated (Fig. II.2). Actually, it has been observed that workers always started destroying eggs immediately after egg transfer in groups with a final high loss of eggs. This indicates that queen eggs carry a specific signal that allows the workers to identify the origin of the eggs.

own queen foreign queen worker 100

80 60 40

Number of eggs (%) 0 0 h 24 h 48 h 72 h 96 h 120 h Tim e Figure II.2 Discrimination of untreated queen and worker laid eggs. Only medians are presented. Already after 24 hours more than 62% of the worker eggs disappeared on average while the queen eggs remained almost untouched. The differences between the queen eggs and the worker eggs were statistically significant (N groups = 9, Wilcoxon-Wilcox test for multiple comparisons, p < 0.01, own queen eggs versus worker eggs, p < 0.05, foreign queen eggs versus worker eggs, ns between queen eggs). II. Surface hydrocarbons of queen eggs regulate worker reproduction 26

But what makes the eggs different? Chemical analysis revealed that queen eggs differ from worker eggs in the composition of their surface hydrocarbons (Fig. II.3, II.4). We found qualitative as well as quantitative differences (Fig. II.4). The profiles of the egg surface compounds show qualitative similarities to the cuticular hydrocarbon profiles of either queens or workers (Fig. II.4). Therefore, we tested whether the surface hydrocarbons of the queen eggs may represent the hypothesized queen signal. Due to the similarity of the cuticular hydrocarbon profiles of adults and the surface profiles of their eggs (Fig. II.4) we simulated queen eggs by extracting and transferring hydrocarbon blends of the cuticle of foreign queens onto worker eggs. Successful manipulation was confirmed by gas chromatography of hydrocarbons extracted from the treated eggs (Fig. II.4).

120 7 65 15 queen queen egg

12 6 15 7 4 13

19 28 20 14 10 1 18 16 2 11 8 16 28 25 4 19 26 9 30 3 11 12 18 26 13 23 31 33 23 30 2 5 6 10 14 29 8 25 9 17 34 29 33 3 1 17 31 0 21 34

t 2122 24 27 32

l 25

o 0 V 15 30 20 22 24 27 32 19

m 18 worker egg 16 28 19 28 30 25 23 20 worker 16 26 12 29 33 26 1 30 18 25 23 17 31 31 14 4 11 14 29 33 21 34 17 15 11 12 13 7 8 13 34 0 0 21 22 24 27 32 6 10 15 20 22 24 27 20 32 25 6 1015 20 25 Time (min) Time (min)

Figure II.3 Chromatograms of the surface hydrocarbons of eggs and the cuticular hydrocarbons of fertile queens and workers. The compounds have been identified on the basis of retention times (in reference to GC/MS analysis) 1) n-pentacosane, 2) 3-methylpentacosane, 3) 10,14-dimethylhexacosane, 4) n-heptacosane, 5) 9- methyl-, 11-methyl-, and 13-methylheptacosane, 6) 11,15-dimethylheptacosane, 7) 3-methylheptacosane and 7,11-dimethylheptacosane, 8) n-octacosane, 9) 3,7-dimethyl-, 3,9-dimethyl-, 3,11-dimethyl-, and 3,13- dimethylheptacosane, 10) 10-methyl-, 12-methyl-, and 14-methyloctacosane, 11) 12,16-dimethyloctacosane, 12) n-nonacosane, 13) 9-methyl-, 11-methyl-, 13-methyl-, and 15-methylnonacosane, 14) 13,17-dimethyl-, 11,15- dimethyl-, and 9,13-dimethylnonacosane, 15) 3-methylnonacosane, 16) 3,7-dimethyl-, and 3,9- dimethylnonacosane, 17) 10-methyl-, 12-methyl-, and 14-methyltriacontane, 18) 4-methyltriacontane and 12,16- dimethyltriacontane, 19) 4,8-dimethyl-, 4,10-dimethyl-, 4,12-dimethyl-, and 4,14-dimethyltriacontane 20) n- hentriacontane, 21) 4,8,12-trimethyltriacontane, 22) 11-methyl-, 13-methyl-, and 15-methylhentriacontane, 23) II. Surface hydrocarbons of queen eggs regulate worker reproduction 27

7-methyl-, 9-methylhentriacontane and 13,17-dimethylhentriacontane, 24) 11,15-dimethylhentriacontane, 25) 3- methylhentriacontane, 26) 5,9-dimethyl-, 5,11-dimethyl-, and 5,13-dimethylhentriacontane, 27) 7,11,15- trimethylhentriacontane, 28) n-dotriacontane, 3,7-dimethyl-, and 3,9-dimethylhentriacontane, 29) 3,7,11- trimethylhentriacontane 30) 3,9,15,21-tetramethyl-, and 3,7,11,15-tetramethylhentriacontane, 31) 4,8-dimethyl-, 4,10-dimethyl-, 4,12-dimethyl-, and 4,14-dimethyldotriacontane, 32) n-tritriacontane 33) 4,8,12,16- tetramethyldotriacontane 34) 5,9,13,17-tetramethyltritriacontane.

linear alkanes A C25 C27 C28 C29 3, 7 DiMeC27 1 4 8 12 9

3 methylbranched alkanes central-branched alkanes 3 MeC25 3 MeC27 3 MeC29 9 MeC27 9 MeC29 2 7 15 5 13

odd - even central-branched dimethylalkanes 11, 15 DiMeC27 13, 17 DiMeC29 10, 14 DiMeC26 12, 16 DiMeC28 10 MeC28 R elative peak area (% ) 6 14 3 11 10

Queen Worker egg with B Queen egg queen profile

Worker Worker egg with Worker egg worker profile Median Quartile

Range Discriminant function

Figure II.4 Differences in surface hydrocarbons between adult queens and workers, between queen and worker eggs and between worker eggs treated with either queen or worker extracts of cuticular hydrocarbons (N = 28 (queens); 33 (workers); 16 (queen eggs); 13 (worker eggs); 9 (worker eggs treated with queen extracts); 5 (worker eggs treated with worker extracts)). Within a group all samples originated from different colonies to obtain independent data points. A) Major differences exist between queens and workers in 15 compounds. The differences in the medians of each compound between queens and workers and between queen eggs and worker eggs are significant (Wilcoxon test for paired samples, p < 0.001). The manipulation of the worker eggs either simulated queen origin or worker origin as before, since the direction of the differences in the medians between untreated eggs and between treated eggs were not different (Sign test, p > 0.6). Single variations are described by II. Surface hydrocarbons of queen eggs regulate worker reproduction 28 abbreviation of compound names. The numbers in the panels correspond to the compound names in figure 3, B) Differences in the profile of the remaining compounds of adult queens and workers were determined by a stepwise discriminant analysis. The resulting discriminant function was used to determine the similarity of the profiles of the untreated and treated eggs. Four compounds were excluded from the analysis. Compounds 20 and 34 were not normally distributed and compounds 16 and 17 did not show variance homogeneity according to Levene’s test. In both cases the significance levels were corrected for multiple comparison according to Bonferroni. The stepwise procedure selected the compounds 31, 24, 23, and 27 (see compound names in Fig. 3). Only one discriminant function was extracted. The differences between the queens and workers are statistically significant (Wilk’s lambda = 0.181, p < 0.001). The discriminant function correctly assigned queens and workers with the exception of three misclassifications of queens (leave-one-out criterion used). The plot of the egg hydrocarbon profiles employing this discriminant function shows, that the profiles of the selected compounds of the treated eggs are within the range of the natural profiles.

Subsequently, the reaction of worker ants towards eggs carrying a transferred queen hydrocarbon profile was compared with that exhibited towards unmanipulated eggs from queen and workers and worker eggs carrying a transferred worker hydrocarbon profile. The result was unequivocal: significantly fewer worker eggs carrying the transferred queen hydrocarbon profile were destroyed than worker eggs although the manipulated eggs did not have the full protection of queen-laid eggs (Fig. II.5).

queen queen extract worker worker extract 100

Numberof eggs (%) 0 0h 1h 2h 24h

Time

Figure II.5 The survival of eggs treated with cuticular hydrocarbons in comparison to untreated eggs. The difference between the untreated eggs from queens and workers and the eggs treated with queen profile is statistically significant after 24h (Friedman’s ANOVA, N groups = 9, p < 0.0005). The important difference is between the worker eggs treated with queen cuticular hydrocarbons and the untreated worker eggs (Wilcoxon test for paired samples, p < 0.02). The sample size of the last group is smaller due to an insufficient number of eggs available at that time. However, the difference among all groups remains statistically significant with only the five samples including the control with manipulated worker eggs (Friedman’s ANOVA, p < 0.005). The important differences between worker eggs treated with either queen extracts versus worker extracts are each II. Surface hydrocarbons of queen eggs regulate worker reproduction 29 significant (Wilcoxon test for paired samples, p < 0.05). The medians of eggs from workers and with worker profiles overlap.

II.4 Discussion

Our results show that queen eggs let workers refrain from reproducing in the ant Camponotus floridanus . This newly documented indirect way of queen signaling helps to understand the mechanisms of the regulation of reproduction in social insects, since the signaling way via eggs is of clearly cooperative character. Surface hydrocarbons of eggs seem to represent this signal, since workers use them to differentiate between queen- and worker-laid eggs as shown by our transfer experiment. These hydrocarbons reliably indicate the origin of the eggs, since they are closely connected to the differences in the cuticular hydrocarbon profiles between highly fertile queens and workers.

Egg inhibition. To our knowledge this study showed for the first time the inhibitory effect of queen eggs. Nevertheless, larvae have been shown to effect worker reproduction in at least two species. In the honeybee, Apis mellifera , larvae inhibit worker ovarian activation (19, 27- 30). Larvae have also been found to affect worker reproduction in queenless worker groups in the ant Pachycondyla apicalis (31). However, larvae do generally not directly signal queen presence and therefore, this regulation mechanism clearly differs from our results.

Egg identification. In our study, workers destroyed worker-laid eggs but let queen-laid eggs alive. We showed that workers identify egg origin via the differences in their surface hydrocarbons. Our manipulation experiment excludes two alternative explanations for the loss of worker eggs. First, worker eggs may posses a lower viability which triggers worker egg destruction. Second, workers may identify the sex/ploidy of the eggs and destroy haploid, male-destined eggs preferentially. However, the transfer of cuticular hydrocarbons of the queen on worker eggs prevented their destruction. Therefore, workers primarily destroy eggs on the basis of their hydrocarbon profiles and not as a consequence of different viability or male-determination. Actually, in the groups that were followed up for male production many males were produced indicating their viability. Furthermore, there is no evidence so far that workers can recognize the sex of eggs (32).

Surface hydrocarbons of eggs and reproductive physiology. Our data are further supported from a study in the queenless ant Dinoponera quadriceps (33). Here, the amount of one II. Surface hydrocarbons of queen eggs regulate worker reproduction 30 compound of the cuticle of reproductive workers correlates with the amount of this substance on their eggs. While in D. quadriceps the difference relates to one compound, eggs of workers in C. floridanus differ from queen eggs in many compounds specific to the composition of the queen’s cuticular hydrocarbons. The close linkage between cuticular hydrocarbons and surface hydrocarbons of eggs is based on specific transport mechanisms in the hemolymph (34). Hydrocarbons are transported by lipoproteins to different tissues in the insect body including the ovaries and the cuticle. In the ovaries they are incorporated in developing oocytes (34). Differences between the profiles of the eggs in and the cuticle in C. floridanus may either be due to a different transport mechanism of hydrocarbons or to changes after oviposition. In several ant species, the hydrocarbon profiles of adults correlate with the fertility of individuals which suggests that hydrocarbons represent a signal regulating reproduction (35- 45). In fact, workers can identify gradual differences in the fertility of nestmates in some of these species (42, 46) as well as in others (47-49). In Myrmecia gulosa , workers can differentiate between the hydrocarbon profiles of reproductives and infertile workers (44). However, it has previously not been shown that cuticular hydrocarbons regulate worker reproduction.

Egg marking. Our transfer of queen cuticular hydrocarbons on worker eggs protected them from being destroyed, indicating that they represent a queen signal. This kind of destruction of worker eggs is actually a case of worker policing, i.e. the mutual control of the workers’ reproduction (13, 32). Since C. floridanus is monogynous with a singly mated queen (50), workers should lay eggs even in the presence of their mother to maximize their inclusive fitness (13, 51). On the other hand, if worker reproduction reduces colony efficiency they should police each others reproduction despite their greater relatedness to their sons and nephews than to their brothers (13); this seems to be the case in C. floridanus . Egg marking is known from several other species. In the honey bee Apis mellifera , worker policing of eggs occurs as well (52). Queen eggs differ from worker eggs by several compounds that are also present in the queen’s Dufour’s gland (53). Egg-laying workers mimic the queen specific profiles of the eggs (54-56). Although, there is experimental evidence, that Dufour’s gland secrections let workers identify who laid the eggs (57), behavioral experiments failed so far to show which compounds are active (58-60). Egg marking also occurs in the fire ant Solenopsis invicta . Here, fertile queens apply Poison gland contents on their eggs (61). However, in this species workers do not lay eggs due to the lack II. Surface hydrocarbons of queen eggs regulate worker reproduction 31 of ovaries (4). Interestingly, the poison gland contents delay dealation of winged queens and consequently their ovarian activation in this species (62). In the queenless ant, Dinoponera quadriceps , the eggs of the reproductive workers are marked with a compound that is also found on their cuticle (33). Here, the marking is again not used for worker policing, but for queen policing, i.e. the dominant worker eats eggs from subordinates.

Regulation of reproduction. Our results in C. floridanus strongly suggest that components of the queens hydrocarbon profile serve as a signal that regulates reproduction in a dual way: i) it encourages workers to refrain from reproduction (self-policing (13)). (Fig. II.1) and ii) it enables workers to discriminate between queen and worker-laid eggs and to destroy the latter if necessary (worker policing (13)(Fig II.2)). Although it is not known if it is a single compound that is the active signal, we do know that it is not nest-specific but common to all C. floridanus queens and can be detected by all C. floridanus workers and hence is acting as a true queen signal present on the surface of the queen and her eggs. This also suggests that the workers are particularly sensitive to the signal as they are able to detect it within a mixture that contains a large number of other like molecules. This could be achieved by a specific pheromone binding protein which selectively transports the signal molecules from the surface of the antennae to the receptors on sensory neurons. Krieger and Ross (63) have recently reported such a pheromone binding protein in the fire ant S olenopsis invicta which in this case allows workers to distinguish between queens with different genotypes. On the other hand, the differences between queens and workers and their eggs in C. floridanus are largely linked to compound classes that are structurally different (Fig. II.3, II.4a). Therefore, high receptor specificity would not be required to detect these differences. In C. floridanus , hydrocarbons are reliable indicators of the presence of the queen and presumably also of her fertility due to the close linkage between hydrocarbon production and physiological processes. These processes are very basal and widespread as indicated by the close correlation of variations in the cuticular hydrocarbon profile and reproductive activity in many ant species (35, 37, 41, 42). Besides their function in protecting eggs and cuticle from desiccation (64-66) and contributing to nestmate recognition (67-69), hydrocarbon profiles additionally represent a queen signal that regulates reproduction in C. floridanus and maybe in many other social insects as well. Via the hydrocarbon profiles, workers could perceive the signal either directly from the queen or indirectly via her eggs. These two ways of signaling efficiently provide the workers with the information they rely on for their reproductive decisions. II. Surface hydrocarbons of queen eggs regulate worker reproduction 32

II.5 Acknowledgements

We thank Erhard Strohm and Tom Seeley for their useful comments on an earlier draft of this manuscript and the German Science Foundation (SFB 554 TP C3, B3) and the European Union (INSECTS, Integrated Studies of the Economy of Insect Societies, HPRN-CT-2000- 00052) for funding. The Social Insects Working Group of the Santa Fe Institute provided insightful discussions.

II.6 References

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III. Queen fertility, egg marking and colony size in the ant Camponotus floridanus

In ant societies workers do usually not reproduce but gain indirect fitness benefits from raising related offspring produced by the queen. One of the preconditions of this worker self- restraint is sufficient fertility of the queen. The queen is, therefore, expected to signal her fertility. In Camponotus floridanus , workers can recognize the presence of a highly fertile queen via her eggs, which are marked with the queen’s specific hydrocarbon profile. If information on fertility is encoded in the hydrocarbon profile of eggs, we expect workers to be able to differentiate between eggs from highly and weakly fertile queens. We found that workers discriminate between these eggs solely on the basis of their hydrocarbon profiles which differ both qualitatively and quantitatively. This pattern is further supported by the similarity of the egg profiles of workers and weakly fertile queens and the similar treatment of both kinds of eggs. Profiles of queen eggs correspond to the cuticular hydrocarbon profiles of the respective queens. Changes in the cuticular profiles are associated with the size of the colony the queen originates from, and her current egg-laying rate. However, partial correlation analysis indicates that only colony size predicts the cuticular profile. Colony size is a buffered indicator of queen fertility as it is a consequence of queen productivity within a certain period of time, whereas daily egg-laying rate varies due to cyclical oviposition. We conclude that surface hydrocarbons of eggs and the cuticular profiles of queens both signal queen fertility suggesting a major role of fertility signals in the regulation of reproduction in social insects.

Keywords : Queen signal, Honest signaling, Pheromone, Cuticular hydrocarbons, Worker policing, Conflict, Formicidae III. Queen fertility, egg marking and colony size 36

III.1 Introduction

The evolution of social insects is considered one of the major transitions in evolution (Maynard Smith and Szathmáry 1995). In insect societies only a minority of individuals reproduce directly whereas all others usually forego personal reproduction and instead help rearing the offspring of relatives. Understanding the ultimate and especially the proximate mechanisms of this kind of reproductive division of labor is still a challenge to evolutionary and behavioral biologists. Hamilton’s kin selection theory (Hamilton 1964) provides the framework for understanding division of reproductive labor in most insect societies (Bourke and Franks 1995; Pamilo and Crozier 1996). According to this theory a helper benefits when the condition r > c/b is true where r is the relatedness between the altruist and the beneficiary of the help, c the loss of direct fitness and b the gain of indirect fitness benefits of the altruist. It is immediately obvious that we have two parts in the term, relatedness and the cost-benefit ratio that determine whether helping is advantageous. Relatedness is especially important in social Hymenoptera, since offspring is asymmetrically related to each other due to their haplodiploid sex determination system. If females have the same mother and father they are related to their sisters by 0.75 but only by 0.25 to their brothers and by 0.375 to their nephews. On the other hand they are related by 0.5 to their male and female offspring. Under these conditions, females should prefer their own sons or nephews over their brothers (Ratnieks 1988). Since workers are restricted to male production in most hymenopteran societies, we expect them to produce sons in colonies with a single queen (monogynous) that is singly mated (monandreous) purely on relatedness grounds (Ratnieks 1988). However, a comparative analysis shows that relatedness is not sufficient to explain the pattern of male production in the social Hymenoptera (Hammond and Keller 2004). The common absence of worker produced males in monogynous and monandrous ant species may also be explained by colony efficiency. In the presence of a fertile queen worker reproduction may divert resources from brood rearing which imposes costs on colony productivity and reduces the indirect fitness gains of workers (Cole 1986; Pamilo 1991). Workers are expected to control each other’s reproduction (worker policing) (Ratnieks 1988; Monnin and Ratnieks 2001) which may finally induce them to refrain from reproduction (self-policing), if the reduction of their indirect fitness is sufficiently high (Ratnieks and Reeve 1992; Frank 1995; Wenseleers et al. 2004). III. Queen fertility, egg marking and colony size 37

Egg destruction and aggression towards fertile individuals are two efficient mechanisms, which either prevent worker-laid eggs from reaching adulthood or inhibit worker reproduction. In bees ( Apis sp.) and the common wasp Vespula vulgaris workers eliminate worker laid eggs via oophagy (egg-eating) in queenright colonies (Ratnieks and Visscher 1989; Ratnieks 1993; Visscher 1996; Foster and Ratnieks 2001; Halling et al. 2001; Oldroyd et al. 2001). In ants worker policing may either be employed by attacking workers with developed ovaries (Hölldobler and Carlin 1989; Gobin et al. 1999; Kikuta and Tsuji 1999; Liebig et al. 1999; Monnin and Peeters 1999; Hartmann et al. 2003; Iwanishi et al. 2003) or by selective destruction of worker-laid eggs as well, as in the ant Diacamma sp. (Kikuta and Tsuji 1999), Camponotus floridanus (Endler et al. 2004), Pachycondyla inversa (D’Ettorre et al. 2004a), and Formica fusca (Helanterä and Sundström 2005). Nevertheless, in several species individuals sometimes escape worker policing (Visscher 1989; Walin et al. 1998; Barron et al. 2001; Dietemann et al. 2003). Worker policing, however, is only advantageous in the presence of a fertile related queen. If the colony is orphaned, workers generally start laying eggs (Bourke 1988a; Choe 1988). We also expect that workers lay eggs if the productivity of the queen decreases and rearing capacities are unused (Seeley 1985; Keller and Nonacs 1993). In several species, pheromones are involved in the regulation of reproduction between queen and workers (Passera 1980; Hölldobler and Wilson 1983; Hoover et al. 2003; Endler et al. 2004) and between the queen and her gyne offspring (Vargo 1988; Vargo and Hulsey 2000). These pheromones both inform about the presence and the fertility of a queen (Ortius and Heinze 1999; Hannonen et al. 2002). In many species, this kind of queen pheromone seems to be represented by cuticular hydrocarbons that change their profile with fertility (Bonavita-Cougourdan et al. 1991; Ayasse et al. 1995; Monnin et al. 1998; Peeters et al. 1999; Liebig et al. 2000; Sledge et al. 2001; Cuvillier-Hot et al. 2002; Hannonen et al. 2002; Heinze et al. 2002; Tentschert et al. 2002; Cuvillier-Hot et al. 2004a; De Biseau et al. 2004). In large colonies not every colony member can monitor the queen’s condition directly suggesting indirect communication of her presence or fertility. In the honeybee ( Apis mellifera ) for example, messenger bees distribute the queen pheromone within the colony (Seeley 1979; Naumann et al. 1991). Alternatively, queen-laid eggs may be used as vehicle for the distribution of a queen signal among nestmates. Such a mechanism is present in the ant C. floridanus, where the queen indirectly signals her presence to workers via her eggs (Endler et al. 2004). The eggs are marked with a queen specific hydrocarbon profile that also occurs on the queen’s cuticle. Queen and worker III. Queen fertility, egg marking and colony size 38 profiles show qualitative differences in the composition of the compounds, approximately 46% of the total amount of CHCs are found only in highly fertile queens (10 of the major 35 compounds, see Endler et al. 2004). In C. floridanus workers refrain from own reproduction in the presence of queen eggs even though they are not in direct contact with the queen (self-policing). Worker eggs do not elicit such an effect, but instead they will be destroyed when presented to workers from a queenright colony (worker policing). Worker eggs lack the respective queen specific hydrocarbon components, but when they are coated with hydrocarbon extracts of the queen’s cuticle their rate of acceptance by workers increased by 34% (Endler et al. 2004). The results on C. floridanus indicate that workers differentiate between hydrocarbon profiles in the context of reproduction as suggested by behavioral and electrophysiological experiments in the ant species Myrmecia gulosa and Pachycondyla inversa (Dietemann et al. 2003; D’Ettorre et al. 2004b). However, it is unknown whether the eggs also transmit information about the fertility of the queen. In the study of Endler et al. (2004) only highly fertile Camponotus -queens from large colonies of more than 1000 workers were investigated. Nothing is known about queen signaling in smaller incipient colonies in which the egg-laying rate of the queen is relatively low. We hypothesize that in small colonies queens lack either single or all compounds of the cuticular hydrocarbon profile which characterizes queens in large colonies. As cuticular and egg profiles are similar in highly fertile queens, eggs from weakly fertile queens originating from incipient colonies should lack similar parts of the profile. If this is the case we expect workers from large colonies not to be able to distinguish the eggs from weakly fertile queens from worker eggs. They should accordingly destroy them like worker eggs due to lack of the appropriate queen signal (Endler et al. 2004). In this study we investigated whether workers from C. floridanus differentiate between eggs originating from highly fertile queens from large colonies and from weakly fertile queens from small colonies. We offered workers from large queenright colonies eggs from i) queens from large colonies, ii) queens from incipient nests and iii) workers as a control. We furthermore investigated the profiles of the surface hydrocarbons whether differences exist among egg-layers as this would support the idea that hydrocarbons are responsible for egg recognition. Since we argue that the hydrocarbon profiles represent a fertility signal, we investigated the egg-laying rates of the queens to see whether egg-laying rate is correlated with changes in the hydrocarbon profile. However, as egg-laying rates tend to fluctuate very much, a better indicator of fertility may be colony size as large colonies can only be III. Queen fertility, egg marking and colony size 39 maintained by highly fertile queens. Thus, colony size is included as another factor to explain variation in cuticular hydrocarbon profiles of queens as an indicator of overall ovarian activity.

III.2 Materials and Methods

Animals and egg discrimination experiment. Queens of Camponotus floridanus (N = 90) were collected at the Florida Keys/USA after the mating flight in August 2001, July 2002 as well as June 2003. The very common carpenter ant C. floridanus reaches colony sizes of over 10000 individuals. The colonies have only one single-mated queen (Gadau et al. 1996) but often several subnests. If the queen dies the colony will die as well, as requeening is unknown in strictly monogynous ant species (Hölldobler and Wilson 1990, Heinze and Keller 2000) except Nothomyrmecia macrops (Sanetra and Crozier 2002). Founding queens were transferred to the laboratory and were cultured in plaster nests at 25° Celsius and 50% humidity (12-h day, 12-h night). Twice a week they were fed with honeywater and cockroaches ( Nauphoeta cinerea ). Subsequently, approximately 80% raised colonies with 1000 – 2000 individuals within one to two years from which the experimental colonies originated. In our egg discrimination experiment we measured the differential survival of three groups of eggs originating either from queens from incipient colonies, from queens from large colonies from workers in order to establish whether queen eggs from incipient colonies can be identified by workers. Freshly orphaned worker groups (<2 hours isolated) each containing 130 individuals (30 foragers and 100 individuals from inside the nest) were provided with eggs of different origin: they received either 30-35 eggs from unrelated workers, 30-35 eggs from a foreign queen originating from a colony with more than 1000 workers or 30-35 eggs from a foreign queen originating from an incipient colony with less than 10 workers. Egg number varied, since we wanted to minimize the risk of unknowingly damaging an egg by singling them out from egg clumps. Only eggs without any conspicuously embryonic development were used (identifiable by the formation of the chorionic cavity). This indicates that the eggs have been laid relatively recently. Eggs from workers were laid in colonies that were orphaned for more than 60 days. For the discrimination experiment we used ten queen colonies. For one experimental trial we isolated three worker groups from one queen colony resulting in a paired experimental design. The survival of the eggs was calculated as the percentage of the eggs III. Queen fertility, egg marking and colony size 40 remaining from the original number added to the respective worker group. Survival was measured by counting the eggs 1h, 2h, and 24h after the transfer. During the experiment each worker group was provided with honeywater and 1 cockroach ( N. cinerea ).

Determination of fertility . In order to determine the relationship between fertility and queen signals we measured oviposition rates of differently fertile queens. We isolated queens of four categories from their colonies in small plastic boxes for 24 hours. Group A contained founding queens with less than 10 workers (N = 2), group B queens with 50 to 80 workers (N = 5), group C queens with 200 to 300 workers (N = 10) and group D queens from colonies over 1000 workers (N = 12). Each of them was provided with 10 workers and fed with honeywater and a half cockroach ( N. cinerea ). After this time the eggs were counted and the queens were returned to their colonies. All queens were only measured once and samples always originated from different colonies, thus each data point reflects an independent measurement. Sample sizes referring to eggs always represent different individuals that laid the eggs, but never different eggs laid by a single individual.

Chemical analysis. As the surface hydrocarbons of eggs have been shown to be a major determining factor of egg discrimination in workers (Endler et al. 2004), we investigated the profiles of eggs originating from workers and queens. We only used eggs less than 24h old, since eggs change their profile with time (Endler and Liebig unpublished). As we focus on differences in fertility among queens and fertility of queens is presumably associated with colony size, we analyzed the egg profiles of queens from differently sized colonies (A: N=20, B: N=16, C: N=14, D: N=19). In order to establish a connection of egg and cuticular profiles, we also investigated the cuticular hydrocarbon (CHC) profiles of queens. A high similarity between the two profiles supports the idea that CHC profiles directly act as a signal and, furthermore, allows directly using CHC profiles of the egg-layers instead of those of the eggs for further investigations. This avoids the time-consuming and disturbing isolation of the egg- layers which is necessary to obtain fresh eggs. The CHCs were extracted with solid phase microextraction (see e.g. Monnin et al. 1998; Liebig et al. 2000; Endler et al. 2004). The fiber was swiftly rubbed on the tergites of queens for 3 minutes and on eggs for 2 minutes. Afterwards the fiber was directly injected into the injection port of a ThermoQuest Trace GC with a split / splitless injector. Further details are described in Endler et al. (2004). Peak areas were computed with Chrom-Card 1.19 III. Queen fertility, egg marking and colony size 41

(CE Instruments). The compounds were identified by using Kovats indices on the basis of a GC/MS analysis (see Endler et al. 2004). Since the differences in the CHC profiles are mostly qualitative and thus represent absence-presence data, principle component or discriminant analyses are not applicable. As the transition from A to D queens is also accompanied by major changes in the presence of some hydrocarbon compounds we use the most parsimonious way to statistically describe the transition. We divided the cuticular CHC profiles of queens and the surface hydrocarbon profiles of eggs into two parts: 1) the shorter-chained queen compounds between retention time 7.0 min and 14.0 min (n-pentacosane to 10-methyl-, 12-methyl-, and 14- methyloctacosane) that are specific to queens of group D and 2) the longer-chained compounds present in both queens and workers (retention time >14.0 min, 12,16- dimethyloctacosane to 5, 9, 13, 17-tetramethyltritriacontane) (see Endler et al. 2004). Then we calculated the sum of the peak areas of the compounds of the respective parts. This allows describing changes with simple statistical correlations. In addition to the qualitative differences we also compared quantitative differences in egg profiles based on compounds present in eggs from A-queens, D-queens and workers from orphaned worker groups. These profiles are represented by the compounds or compound mixtures indicated by the numbers 11 (12,16-dimethyloctacosane) through 34 (5,9,13,17- tetramethyltritriacontane) described in Endler et al. (2004). In addition, we included the alkanes n-pentacosane, n- heptacosane, and n-octacosane, since they occur regularly on all eggs. The areas of the peaks were standardized to 100%. The resulting values were transformed according to Aitchison (1986) (see Dietemann et al. 2003). Six variables were excluded from the analysis as they either deviated significantly from the normal distribution (Kolmogorov-Smirnov test with Bonferroni correction, p < 0.01, p < 0.005, and three times p < 0.0001) or did not show homogenous variances (Levene’s test after Bonferroni correction, p < 0.05 for one variable). The remaining 20 variables were subject to a stepwise backward discriminant analysis. We identified five variables that significantly contributed to the separation. These are represented by three alkanes (n-pentacosane, n-heptacosane, n- hentriacontane) and a mixture of 4-methyltriacontane and 12,16-dimethyltriacontane and the compound 4,8,12,16-tetramethyldotriacontane. The compounds were identified in comparison to the GC/MS analysis from Endler et al. (2004) using Kovac indices. Statistical tests were performed with STATISTICA 6.1 (Statsoft) and SPSS 12.02 (SPSS Inc.).

III. Queen fertility, egg marking and colony size 42

III.3 Results

A greater fraction of eggs laid by D-queens was alive after 24h compared to eggs laid by A- queens and workers, whereas there was no difference between the latter two kind of eggs (Overall difference, Friedman’s ANOVA, p < 0.0001; Wilcoxon-Wilcox test for multiple comparisons: eggs from D-queens versus eggs from A-queens, p < 0.01, eggs from D-queens versus worker eggs, p < 0.01, eggs from A-queens versus worker eggs, p > 0.1, Fig. III.1). The differences in egg survival correspond to differences in the composition of their surface hydrocarbons. The median of the percentage of the shorter-chained compounds of the total profile is 47.4 in eggs from D-queens (range: 31.7 – 60.5%) and 2.2% in eggs from A- queens (range: 0.6 – 5.4%) and 1.9% in eggs from workers (range: 0.3 – 10.7%). High proportions of shorter-chained compounds in A-queens and workers are due to large amounts of linear alkanes. Differences are significant between D-queens and A-queens or workers, respectively but not between A-queens and workers (Kruskal-Wallis test: N of egg samples from D-queens = 19, from A-queens = 19 and from workers = 13, H = 35.15, p < 0.0001, posthoc comparison (Siegel and Castellan 1988), D-queens versus A-queens or workers, p < 0.0001, A-queens versus workers, p > 0.3).

Figure III.1 Discrimination of queen- and worker-laid eggs expressed as survival rate after transfer in worker groups. Presented are medians, quartiles, and range. Stars denote extreme values. N (egg samples) = 10 for each group. III. Queen fertility, egg marking and colony size 43

In the longer-chained part of the profile all focus compounds were present in all individuals allowing for a discriminant analysis (Fig. III.2). This part of the profile shows significant differences as well (N as before, Wilks-Lambda = 0.009, p < 0.0001). The profiles of eggs from D-queens are well separated from those of the other eggs by the first discriminant function (100% correct classification). However, the profiles of eggs laid by workers and by A-queens overlap completely on the first axis. The separation of the two groups is largely due to the second axis (91% correct classification) which, however, explains only 1.8% of the variance. The profiles of eggs laid by D-queens largely overlap with the profiles of the eggs from the other two groups on the second axis, which indicates that fertility, colony size, sex of the egg or caste of the egg-layer is not associated with the separation on the second axis. The compounds that were excluded from the analysis for statistical reasons show a pattern that supports the results of the discriminant analysis (Tab. III.1).

Figure III.2 Comparison of the profiles of eggs originating from workers, A-queens and D-queens. The percentage denotes variance explained by the discriminant function. Egg samples originate from 13 workers, from 19 A-queens, and from 19 D-queens.

III. Queen fertility, egg marking and colony size 44

Table III.1 : Compounds excluded from the discriminant analysis of egg profiles with their respective transformed values

Compound Worker A-queen D-queen

Median Range Median Range Median Range

9-methylnonacosane -1.14 -2.09 to -0.90 -1.44 -2.50 to -1.00 2.02 0.97 to 2.52

13,17-dimethyl-, 11,15- -2.27 -2.77 to -2.09 -2.76 -3.56 to -1.95 1.72 0.89 to 2.06 dimethyl- and 9,13- dimethylnonacosane 7-methyl-, 0.36 0.08 to 0.73 0.72 0.35 to 0.92 -1.3 -2.11 to 0.30 9-methylhentriacontane and 13,17-dimethylhentriacontane 11,15-dimethylhentriacontane -0.04 -0.64 to 0.42 -0.22 -1.17 to 0.77 -0.63 -1.04 to -0.04

N-dotriacontane 1.64 1.05 to 2.06 1.58 1.28 to 1.78 0.45 -0.07 to 1.22

N-tritriacontane -1.03 -1.59 to -0.66 -0.65 -1.18 to -0.23 -2.36 -2.78 to -1.75

The percentage of the amount of the shorter-chained part of the egg profile is positively correlated with the respective part of the CHC profiles of the egg-laying queens from groups A to D (Spearman rank correlation, NA-queens = 8, N B-queens = 7, N C-queens = 6, N D- queens = 19, r s = 0.93, p < 0.0001, Fig. III.3). Overall, the CHC profiles of egg-layers and the profiles of the surface hydrocarbons of their eggs are similar in the proportions of shorter- chained hydrocarbons and in the pattern of the profile (Fig. III.3, III.4).

III. Queen fertility, egg marking and colony size 45

As we were interested in the information content of the hydrocarbon signal we investigated the egg-laying rate of the queens of different groups and its relation to hydrocarbon profile changes. A-queens laid about 1 eggs / day on average (Median, N = 7, range: 0-9), B-queens 9.5 eggs / day (Median, N = 8, range: 3-12), and C queens 14.5 eggs / day (Median, N = 14, range: 4-41). The highest egg-laying rate with 28 eggs / day on average was observed in D-queens (Median, N = 17). Nevertheless, the daily egg-laying rate of queens from these large colonies varied strongly covering the whole range from 0 to 83. Although some queens produced none or only few eggs, their colonies contained much brood that developed into females. Furthermore, these queens lived for at least another year after counting egg-laying rate which suggests that these queens were healthy and produced female brood.

70 Spearman rank correlation 60 rs = 0.93 p < 0.0001 50

20 A) <10 workers B) 50-80 workers 10 C) 200-300 workers D) >1000 workers 0 Shorter-chained SHC's on eggs on (%) SHC'seggs Shorter-chained 0 10 20 30 40 50 60 70 Shorter-chained CHC's on queens (%)

Figure III.3 Comparison of the proportion of queen specific compounds on the cuticle (CHC) and the egg surface (SHC). Queens and their eggs originate from group A (N = 8); B (N = 7); C (N = 6) and D (N = 19).

The higher the average daily egg-laying rate the higher is the relative amount of shorter-chained hydrocarbons in the cuticular profile of the queens. A-queens with an egg- laying rate of 0 or 9 eggs/day have less than 0.6% shorter-chained hydrocarbons in their profile, whereas D-queens with egg-laying rates between 20 and 83 eggs/day (median: 37.5) have between 28.7 and 59.4% shorter-chained hydrocarbons in their profile (N = 12, Median: 50.3%, Fig. III.5). III. Queen fertility, egg marking and colony size 46

Figure III.4 Chromatograms of the cuticular hydrocarbons of typical representatives of their categories together with the surface hydrocarbons of their respective eggs. Only compounds between 6 and 25 minutes are shown, which represent the hydrocarbon profile. Minor not reproducible peaks between 0 and 6 minutes are not shown. For better comparison the elution times for n-alkanes with chain length from 25 to 33 are indicated. Colony sizes are indicated by letters (see methods!). III. Queen fertility, egg marking and colony size 47

20 A) <10 workers B) 50-80 workers 10 C) 200-300 workers D) >1000 workers 0 0 10 20 30 40 50 60 70 80 90 Shorter-chained CHC's on queens Shorter-chained CHC's(%) on Number of eggs in 24 hours

Figure III.5 The amount of shorter-chained hydrocarbons on the queen cuticle (CHC’s) versus the oviposition rate of the queens in 24 hours and colony size. The size of the colonies the queens originated from, are indicated by A to D. See text for details and statistics!

Besides egg-laying rate, the amount of shorter-chained hydrocarbons in the CHC- profile of queens is also associated with the size category of the colony. The larger the colony is from which the egg-laying queen originates the more shorter-chained hydrocarbons are present in her CHC-profile, whereas the strongest variation is present in C-queens (Fig. III.5). The amount of short-chained hydrocarbons increases with both egg-laying rate and colony size (Spearman rank correlation: N = 29, r s = 0.79, p < 0.0001 and r s = 0.88, p < 0.0001, respectively). As egg-laying rate and colony size are positively correlated (Spearman rank correlation, r s = 0.89, p < 0.0001) we used partial correlation analysis to factor out first the effect of colony size and then that of egg-laying rate. When controlling for colony size egg- laying rate had no effect on the amount of short-chained hydrocarbons (r s = 0.04, N = 29, p > 0.2), but when controlling for egg-laying rate, the amount of short-chained hydrocarbons increased significantly with colony size (r s = 0.63, N = 29, p < 0.001). This indicates that colony size is a better predictor of the CHC profile of egg-laying queens than egg-laying rate.

III. Queen fertility, egg marking and colony size 48

III.4 Discussion

Our results clearly show that workers of C. floridanus extract information about the fertility of queens from their eggs. The treatment of eggs corresponds to the hydrocarbon profiles of the egg surface and the cuticular profiles of the respective queens. A good predictor of the hydrocarbon profiles is the size of the colony the respective queen originates from. Since a large colony size can only be maintained by a certain productivity of a queen it may represent the moving average of the queen’s egg-laying rate whereas egg-laying rate itself is subject to daily or weakly variation. A strong correlation between colony size and hydrocarbon profiles thus supports the idea that hydrocarbon profiles represent a fertility signal that can be perceived via eggs and the cuticle of the respective egg-laying queen. The fertility signal on the eggs also seems to regulate the policing of worker-laid eggs by workers in C. floridanus (Endler et al. 2004) as well as in other ant species, e.g. differences in the hydrocarbon profiles of eggs are associated with worker policing in the ant Pachycondyla inversa (D’ Ettorre et al. 2004a) and “queen” policing in Dinoponera quadriceps (Monnin and Peeters 1997) although the differences between the eggs are not as pronounced as in C. floridanus . In our experiment, the destruction of eggs from A-queens by workers may be a consequence of the inability of the workers to differentiate between eggs from A-queen and those laid by workers, since the eggs of A-queens lack the fertility signal similar to worker eggs. We assume that the workers falsely perceived the A-queen eggs as worker-laid eggs what finally let them destroy the A-queen eggs. A further reason for the destruction of A-queen eggs could be their origin. Potentially, workers may recognize that neither A-queen eggs nor worker-laid eggs originated from their colony. However, D-queen eggs also originated from foreign colonies in this study as well as in the study of Endler et al. (2004) and they were accepted. In contrast, eggs laid by sister workers in the study of Endler et al (2004) were destroyed in worker groups. Thus foreign colony membership cannot generally be a cause of destruction. If information about colony membership is present, the fertility signal on D-queen eggs presumably prevents destruction. This would, however, not work for foreign eggs laid by A-queens and workers. Thus, destruction of eggs laid by workers or A-queens in this experiment could potentially be the consequence of the detection of foreign colony membership or of worker policing. A major cause for egg destruction as alternative to worker policing was put forward by Pirk et al. (2004). They claimed that worker-laid eggs have a high mortality per se in the honeybee, Apis mellifera . New data from honeybees, however, strongly oppose these results III. Queen fertility, egg marking and colony size 49

(Beekman and Oldroyd 2005). Our results do not support differential egg mortality as a factor either, as there was no difference in survival between eggs laid by A-queens and workers. In addition, the transfer of queen hydrocarbons increased the survival of worker-laid eggs in C. floridanus in another study (Endler et al. 2004). Thus we exclude increased mortality of worker-laid eggs as a factor explaining our results. Furthermore, the indiscriminate destruction of eggs laid by A-queens or workers excludes other potential causes for differential egg treatment, e.g. differences in morphology, physiology or other potentially sex- specific properties. Although workers use the differences in the hydrocarbon profiles to discriminate among eggs the question is what the differences indicate. If the hydrocarbons represent a fertility signal the most obvious measure is egg-laying rate. Although egg-laying rate differed among queens, this difference was also associated with differences in colony size. When we corrected for colony size, the partial correlation analysis shows no correlation between CHC patterns of the queens and their current egg-laying rate. On the other hand, colony size strongly correlates with the changes in the hydrocarbon profiles. Thus, colony size is a better predictor for the changes in the CHC profiles of the queens than current egg-laying rate. This may be the consequence of cyclical oviposition of the queens (Endler pers. obs.) which is known from other ants (Hölldobler and Wilson 1990; Dietemann et al. 2002, Cuvillier-Hot et al. 2004b). On the other hand colony size does not vary on a day to day basis. Since egg- laying rate of the queen determines colony size in combination with the mortality rate of workers, colony size is a buffered and delayed indicator of the fertility of the respective queen. Potentially, colony size could determine the fertility of the queen as a kind of feedback mechanism. This, however, seems only partly to be the case in C. floridanus , as this species is monogynous and founds colonies solitarily. A queen has to produce a surplus amount of brood which determines colony growth. On the other hand, successful raising of brood depends on a sufficient number of workers which leads to the close correlation of colony size and egg-laying rate in our analysis. The change in the hydrocarbon profiles of the queens is not only associated with an increase of colony size as a secondary effect but primarily with a change in physiology. Many studies showed a close link between CHC patterns and fertility (Bonavita-Cougourdan et al. 1991; Ayasse et al. 1995; Monnin et al. 1998; Peeters et al. 1999; Liebig et al. 2000; Sledge et al. 2001; Cuvillier-Hot et al. 2002; Hannonen et al. 2002; Heinze et al. 2002; Tentschert et al. 2002; Cuvillier-Hot et al. 2004a; De Biseau et al. 2004). These CHC patterns revert to those of unfertile individuals if ovarian activity stops in a ponerine ant (Liebig et al. 2000). In III. Queen fertility, egg marking and colony size 50 another ponerine ant, Streblognathus peetersi , the experimental manipulation of JH-patterns in fertile ants inhibited ovarian activity and this was preceded by changes in the CHC-patterns of the respective individuals (Cuvillier-Hot et al. 2004a). The underlying physiological mechanism also explains the close connection between the surface profiles of eggs and the CHC profiles of the respective queens. This could be a consequence of the same physiological pathway responsible for the transportation of hydrocarbons to target tissues. In the German cockroach Blattella germanica hydrocarbons are distributed by a hemolymph high-density lipophorin to different tissues in the insect body including the cuticle and the ovaries where they are incorporated in developing oocytes (Schal et al. 1998; Fan et al. 2003). A similar lipophorin transport pathway is known from the ant Pachycondyla villosa (Lucas et al. 2004). In C. floridanus , the surface hydrocarbons may thus be already present in the ovaries in similar proportions as on the cuticle and are presumably not transferred from the cuticle on the eggs during egg-laying. Eggs thus carry the same information as the cuticle of the egg-layer. Since the proportions of shorter-chained hydrocarbons in the surface profile of eggs and the cuticular profile of the respective queen increase with fertility, they both provide information about queen fertility. Although it is clear that workers use this fertility signal (Endler et al. 2004) for their reproductive decisions the finer mechanisms of their decision making is generally unclear. Is there any fertility threshold of the queen below which workers should start laying eggs? Should they start laying eggs at all if there is still an egg-laying queen in the colony? This probably depends on the potential benefits they receive from egg-laying and these vary depending on the situation (e.g. Foster 2004). One indication for a high threshold for egg- laying in this species is the relatively long delay between orphanage and first worker oviposition. In an isolation experiment of C. floridanus worker eggs appeared not before 60 days after orphanage and some worker groups did not yet oviposit after 158 days of isolation (Endler et al. 2004). In several ant species this delay is much shorter (earliest egg-laying after orphanage: 5 (mean 29.5) days ( Streblognathus peetersi ) Cuvillier-Hot et al. 2004b; 6 days (Harpagoxenus sublaevis ) Bourke 1988b, less than 13 days ( Diacamma ceylonense ) Cuvillier-Hot et al. 2002; 17 days ( Aphaenogaster cockerelli ) Hölldobler and Carlin 1989; less than 21 days (Harpegnathos saltator ) Liebig et al. 1999; 30 days (Gnamptogenys menadensis ) Gobin et al. 1999). This large variation in oviposition delay indicates that workers probably respond differently to variations in the fertility signal due to different potential benefits of own reproduction. One would expect therefore that workers invest more in monitoring the fertility of the queen if the potential benefits of own reproduction are III. Queen fertility, egg marking and colony size 51 higher. A better understanding of fertility signaling will therefore remain at the core for understanding of the mechanisms and evolution of reproductive regulation in social insects.

III.5 Acknowledgements

We thank Thibaud Monnin, Christian Peeters, Kazuki Tsuji, as referee, and especially Lotta Sundström for providing helpful comments on the manuscript; we also thank the German Science Foundation (SFB 554 TP C3), the European Union (INSECTS, Integrated Studies of the Economy of Insect Societies, HPRN-CT-2000-00052) and Aventis (travel grant to AE) for funding. The Social Insect Working Group of the Santa Fe Institute provided insightful discussions.

III.6 References

Aitchison J (1986) The statistical analysis of compositional data: Monographs in statistics and applied probability. Chapman & Hall, London Ayasse M, Maelovits T, Tengö J, Taghizadeh T, Francke W (1995) Are there pheromonal dominance signals in the bumblebee Bombus hypnorum L. (Hymenoptera, Apidae). Apidologie 26:163-180 Barron AB, Oldroyd BP and Ratnieks FLW (2001) Worker reproduction in honey-bees ( Apis ) and the anarchic syndrome: a review. Behav Ecol Sociobiol 50 :199-208 Beekman M, Oldroyd BP (2005) Honey bee workers use cues other than egg viability for policing. Biol. Letters 1:129-132 Bonavita-Cougourdan A, Théraulaz G, Bagnéres AG, Roux M, Pratte M, Provost E, Clément JL (1991) Cuticular hydrocarbons, social organisation and ovarian development in a polistine wasp: Polistes dominulus Christ. Comp Biochem Physiol B 100:667-680 Bourke AFG (1988a) Worker reproduction in the higher eusocial Hymenoptera. Q Rev Biol 63:291- 311 Bourke AFG (1988b) Dominance orders, worker reproduction, and queen-worker conflict in the slave- making ant Harpagoxenus sublaevis . Behav Ecol Sociobiol 23:323-333 Bourke AFG, Franks NR (1995) Social evolution in ants. Princeton University Press, Princeton Choe JC (1988) Worker reproduction and social evolution in ants (Hymenoptera: Formicidae). In: Advances in myrmecology (Trager JC, ed). Leiden: Britt, E. J.; 163-187 Cole BJ ( 1986) The social behavior of Leptothorax allardycei (Hymenoptera, Formicidae): time budgets and the evolution of worker reproduction. Behav Ecol Sociobiol 18 :165-173 Cuvillier-Hot V, Gadagkar R, Peeters C, Cobb M (2002) Regulation of reproduction in a queenless ant: aggression, pheromones and reduction in conflict. Proc R Soc Lond B 269 :1295-1300 III. Queen fertility, egg marking and colony size 52

Cuvillier-Hot V, Lenoir A, Peeters C (2004a) Reproductive monopoly enforced by sterile police workers in a queenless ant. Behav Ecol 15:970-975 Cuvillier-Hot V, Lenoir A, Crewe R, Malosse C, Peeters C (2004b) Fertility signalling and reproductive skew in queenless ants. Anim Behav 68:1209-1219 D’Ettorre P, Heinze J, Ratnieks FLW ( 2004a) Worker policing by egg eating in the ponerine ant Pachycondyla inversa . Proc R Soc Lond B 271:1427-1434 D’Ettorre P, Heinze J, Schulz C, Francke W, Ayasse M (2004b) Does she smell like a queen? Chemoreception of a cuticular hydrocarbon signal in the ant Pachycondyla inversa . J Exp Biol 207:1085-1091 De Biseau J-C, Passera L, Daloze D, Aron S (2004) Ovarian activity correlates with extreme changes in cuticular hydrocarbon profile in the highly polygynous ant, Linepithema humile . J Ins Physiol 50:585-593 Dietemann V, Hölldobler B, Peeters C ( 2002) Caste specialization and differentiation in reproductive potential in the phylogenetically primitive ant Myrmecia gulosa . Insectes Soc 49 :289-298 Dietemann V, Peeters C, Liebig J, Thivet V, Hölldobler B (2003) Cuticular hydrocarbons mediate discrimination of reproductives and nonreproductives in the ant Myrmecia gulosa . Proc Natl Acad Sci USA 100:10341-10346 Endler A, Liebig J, Schmitt T, Parker J, Jones G, Schreier P, Hölldobler B (2004) Surface hydrocarbons of queen eggs regulate worker reproduction in a social insect. Proc Natl Acad Sci USA 101:2945-2950 Fan Y, Zurek L, Dykstra MJ, Schal C (2003) Hydrocarbon synthesis by enzymatically dissociated oenocytes of the abdominal integument of the German cockroach, Blattella germanica . Naturwissenschaften 90 :121-126 Foster KR, Ratnieks FLW (2001) Convergent evolution of worker policing by egg eating in the honeybee and common wasp. Proc R Soc Lond B 268:169-174 Foster KR, 2004. Diminishing returns in social evolution: the not-so-tragic commons. J Evol Biol 17:1026-1034 Frank SA (1995) Mutual policing and repression of competition in the evolution of cooperative groups. Nature 377:520–522 Gadau J, Heinze J, Hölldobler B, Schmid M (1996) Population and colony structure of the carpenter ant Camponotus floridanus. Mol Ecol 5:785-792 Gobin B, Billen J, Peeters C (1999) Policing behaviour towards virgin egg layers in a polygynous ponerine ant. Anim Behav 58 :1117-1122 Halling LA, Oldroyd BP, Wattanachaiyingcharoen W, Baron AB, Nanork P, Wongsiri S (2001) Worker policing in the bee Apis florea . Behav Ecol Sociobiol 49:509-513 Hamilton WD (1964) The genetical evolution of social behavior I and II. J Theor Biol 7:1-52 III. Queen fertility, egg marking and colony size 53

Hammond RL, Keller L (2004) Conflict over male parentage in social insects. PLoS Biology 2:1472- 1482 Hannonen M, Sledge MF, Turillazzi S, Sundström L (2002) Queen reproduction, chemical signalling and worker bahaviour in polygyne colonies of the ant Formica fusca . Anim Behav 64 :477-485 Hartmann A, Wantia J, Torres JA, Heinze J (2003) Worker policing without genetic conflicts in a clonal ant. Proc Natl Acad Sci USA 100:12836-12840 Heinze J, Keller L (2000) Alternative reproductive strategies: a queen perspective in ants. Trends Ecol Evol 15:508-512 Heinze J, Stengl B, Sledge MF (2002) Worker rank, reproductive status and cuticular hydrocarbon signature in the ant, Pachycondyla cf . inversa . Behav Ecol Sociobiol 52:59-65 Helanterä H, Sundström L (2005) Worker reproduction in the ant Formica fusca . J Evol Biol 18:162- 171 Hölldobler B, Wilson EO (1983) Queen control in colonies of weaver ants (Hymenoptera: Formicidae). Ann Entomol Soc Am 76 :235-238 Hölldobler B, Carlin NF (1989) Colony founding, queen control and worker reproduction in the ant Aphaenogaster (= Novomessor ) cockerelli (Hymenoptera: Formicidae). Psyche 96:131-151 Hölldobler B, Wilson EO (1990) The ants. Harvard University Press, Cambridge Mass. Hoover SER, Keeling CI, Winston ML, Slessor KN (2003) The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften 90:477-480 Iwanishi S, Hasegawa E, Ohkawara K (2003) Worker oviposition and policing behaviour in the myrmecine ant Aphaenogaster smythiesi japonica Forel. Anim Behav 66 :513-519 Keller L, Nonacs P (1993) The role of queen pheromones in social insects: queen control or queen signal. Anim Behav 45:787-794 Kikuta N, Tsuji K (1999) Queen and worker policing in monogynous and monandrous ant, Diacamma sp. Behav Ecol Sociobiol 46:180-189 Liebig J, Peeters C, Hölldobler B (1999) Worker policing limits the number of reproductives in a ponerine ant. Proc R Soc Lond B 266 :1865-1870 Liebig J, Peeters C, Oldham NJ, Markstädter C, Hölldobler B (2000) Are variations in cuticular hydrocarbons of queens and workers a reliable signal of fertility in the ant Harpegnathos saltator ? Proc Natl Acad Sci USA 97 :4124-4131 Lucas C, Pho DB, Fresneau D, Jallon JM (2004) Hydrocarbon circulation and colonial signature in Pachycondyla villosa . J Insect Physiol 50:595-607 Maynard Smith J, Szathmary E (1995) The major transitions in evolution. W.H. Freeman and Company, New York Oxford Monnin T, Peeters C (1997) Cannibalism of subordinates' eggs in the monogynous queenless ant Dinoponera quadriceps . Naturwissenschaften 84:499-502 III. Queen fertility, egg marking and colony size 54

Monnin T, Malosse C, Peeters C (1998) Solid-phase microextraction and cuticular hydrocarbon differences related to reproductive activity in a queenless ant. J Chem Ecol 24 :473-490 Monnin T, Peeters C (1999) Dominance hierarchy and reproductive conflicts among subordinates in a monogynous queenless ant. Behav Ecol 10 :323-332 Monnin T, Ratnieks FLW ( 2001) Policing in queenless ants. Behav Ecol Sociobiol 50:97-108 Naumann K, Winston ML, Slessor KN, Prestwich GD, Webster FX (1991) Production and transmission of honey bee queen ( Apis mellifera L.) mandibular gland pheromone. Behav Ecol Sociobiol 29 :321-332 Oldroyd BP, Halling LA, Good G, Wattanachaiyingcharoen W, Barron AB, Nanork P, Wongsiri S, Ratnieks FLW ( 2001) Worker policing and worker reproduction in Apis cerana . Behav Ecol Sociobiol 50:371-377 Ortius D, Heinze J (1999) Fertility signaling in queens of a North American ant. Behav Ecol Sociobiol 45 :151-159 Pamilo P (1991) Evolution of colony characteristics in social insects. II. number of reproductive individuals. Am Nat 138:412-433 Pamilo P, Crozier RH (1996) Reproduktive skew simplified. Oikos 75:533-535 Passera L (1980) La fonction inhibitrice des reines de la fourmi Plagiolepis pygmaea Latr.: role des pheromones. Insectes Soc 27:212-225 Peeters C, Monnin T, Malosse C (1999) Cuticular hydrocarbons correlated with reproductive status in a queenless ant. Proc R Soc Lond B 266:1323-1327 Pirk CWW, Neumann P, Hepburn R, Moritz RFA, Tautz J (2004) Egg viability and worker policing in honey bees. Proc Natl Acad Sci USA 101:8649-8651 Ratnieks FLW (1988) Reproductive harmony via mutual policing by workers in eusocial Hymenoptera. Am Nat 132 :217-236 Ratnieks FLW, Visscher PK (1989) Worker policing in the honeybee. Nature 342:796-797 Ratnieks FLW, Reeve HK (1992) Conflict in single queen Hymenopteran societies: the structure of conflict and processes that reduce conflict in advanced eusocial species. J Theor Biol 158 :33-65 Ratnieks FLW (1993) Egg-laying, egg-removal, and ovary development by workers in queenright honey bee colonies . Behav Ecol Sociobiol 32:191-198 Schal C, Sevala VL, Young HP, Bachmann JA (1998) Sites of synthesis and transport pathways of insect hydrocarbons: Cuticle and ovary as target tissues. Am Zool 38:382-393 Sanetra M, Crozier RH (2002) Daughters inherit colonies from mothers in the ‘living-fossil’ ant Nothomyrmecia macrops . Naturwissenschaften 89:71-74 Seeley TD (1979) Queen substance dispersal by messenger workers in honeybee colonies. Behav Ecol Sociobiol 5:391-415 Siegel S, Castellan NJ Jr (1988) Nonparametric statistics for behavioral sciences. McGraw Hill Book Co., New York III. Queen fertility, egg marking and colony size 55

Sledge M, Boscaro F, Turillazzi S (2001) Cuticular hydrocarbons and reproductive status in the social wasp Polistes dominulus . Behav Ecol Sociobiol 49:401-409 Tentschert J, Bestmann HJ, Heinze J (2002) Cuticular compounds of workers and queens in two Leptothorax ant species - a comparison of results obtained by solvent extraction, solid sampling, and SPME. Chemoecology 12:15-21 Vargo EL (1988) A bioassay for a primer pheromone of queen fire ants ( Solenopsis invicta ) which inhibits the production of sexuals. Insectes Soc 35:382-392 Vargo EL, Hulsey CD (2000) Multiple glandular origins of queen pheromones in the fire ant Solenopsis invicta . J Insect Physiol 46:1151-1159 Visscher PK (1989) A quantitative study of worker reproduction in honey bee colonies. Behav Ecol Sociobiol 25:247-254 Visscher PK (1996) Reproductive conflict in honey bees: a stalemate of worker egg-laying and policing. Behav Ecol Sociobiol 39:237-244 Walin L, Sundström L, Seppä P, Rosengren R (1998) Worker reproduction in ants – a genetic analysis. Heredity 81:604-612 Wenseleers T, Helanterä H, Hart A, Ratnieks FLW (2004) Worker reproduction and policing in insect societies: an ESS analysis. J Evol Biol 17:1035-1047 IV. Lack of worker policing and fertility cues in egg-laying workers 56

IV. Lack of physical policing and fertility cues in egg-laying workers of the ant Camponotus floridanus

Conflict resolution is often a major factor in maintaining cooperation in insect societies. Workers usually do not reproduce but gain indirect fitness benefits from raising related offspring produced by the queen. Even though workers are expected to reproduce in queenright Hymenopteran societies with high intracolonial relatedness, they mutually control each other (worker policing) when worker reproduction sufficiently reduces colony productivity. Worker policing occurs either as aggression towards incipient egg-layers or as destruction of worker-laid eggs in ant societies with small colonies where workers retain a high reproductive potential. Beginning egg-layers as well as worker eggs can be recognized in many species by differences in their surface hydrocarbons. In this study, we investigated whether these mechanisms are also used in evolutionarily derived species with large colony sizes and with workers having low reproductive potential. We previously found that in the ant species Camponotus floridanus, worker policing of eggs is based on differences in the hydrocarbon profiles between worker- and queen-laid eggs. However, the current study shows that workers with developed ovaries are never attacked by nest-mates, which contrasts with most studies in species having workers with a higher reproductive potential. The pattern in C. floridanus is further supported by the absence of relevant differences in the cuticular hydrocarbon profile between egg-laying and infertile workers, although strong differences exist between highly fertile queens and workers. In C. floridanus, efficient mutual control by practically cost-free egg policing seem to have lead to worker self restraint without the need for additional policing mechanisms.

Keywords : Reproductive conflict, worker reproduction, worker policing, cuticular hydrocarbons, fertility signal, Formicidae

IV. Lack of worker policing and fertility cues in egg-laying workers 57

IV.1 Introduction

Reproductive division of labor is a fundamental characteristic of eusocial insects. In larger colonies of the Hymenoptera, one or several reproductively specialized queens reproduce while her helpers, reproductively degenerated workers, are functionally sterile and help rearing offspring of relatives. Nevertheless, most workers retained ovaries with which they can produce male destined fertile eggs due to the haplo-diploid sex determination system of the Hymenoptera (Bourke 1988a), which leads to a potential conflict between the queen and her workers over male production (reviewed in Ratnieks et al. 2006). Workers do not lay eggs in many queenright Hymenopteran societies, despite their capability to do so. However, in a monogynous colony with a singly mated queen, worker reproduction is expected on relatedness grounds (Hamilton 1964). In haplodiploid animals, females are related to their sister by 0.75 and with their brothers by 0.25. Workers in the social Hymenoptera are more related to their sons (0.5) and to their nephews (0.375) than to their brothers. They are, therefore, expected to replace brothers with their own male offspring (Hamilton 1964; Woyciechowski & Lomnicki 1987; Ratnieks 1988; Ratnieks & Reeve 1992; Wenseleers et al. 2004; Ratnieks et al. 2006). However, workers should prevent each other from reproduction when intracolonial relatedness among workers drops below 0.5 due to multiple mating of the queen, since their brothers are then more related to them than their nephews (<0.5). To maintain social cohesion, mechanisms have evolved that moderate individual selfishness and resolve within-group conflicts (Ratnieks et al. 2006). One such mechanism is mutual control, where members of a group collectively prevent individuals from acting in their own selfish interests (Frank 1995). The best examples of mutual control are found in social insects, where workers control each others’ reproduction (worker policing) by selectively removing worker-laid eggs (oophagy) that otherwise develop into males (ants: Monnin & Peeters 1997; D’ Ettorre et al. 2004a; Endler et al. 2004; Helanterä & Sundstöm 2005; bees: Ratnieks & Visscher 1989; Foster & Ratnieks 2001; Halling et al. 2001; Oldroyd et al. 2001; wasps: Foster & Ratnieks 2000; Tsuchida et al. 2003), or by directing aggression toward workers with developed ovaries (Hölldobler & Carlin 1989; Gobin et al. 1999; Kikuta & Tsuji 1999; Liebig et al. 1999; Monnin & Ratnieks 2001; Hartmann et al. 2003, 2005; Iwanishi et al. 2003; Dietemann et al. 2005; Kawabata & Tsuji 2005). The relative relatedness of workers to queen- and worker-produced males is only a partial explanation for worker policing (Hammond & Keller 2004, Ratnieks et al. 2006), IV. Lack of worker policing and fertility cues in egg-laying workers 58 although the levels of worker male production in colonies with low intracolonial relatedness fits the theoretical predictions (Ratnieks et al. 2006). If worker reproduction reduces colony productivity and thus the indirect fitness gains of workers in the presence of a fertile queen, workers should also police each other (Cole 1986; Ratnieks 1988, Pamilo 1991). In addition, worker policing is favored in colonies where workers manipulate sex ratio by killing male larvae as long as male recycling is sufficiently inefficient (Foster & Ratnieks 2001). Increasing costs of worker reproduction may finally induce workers to refrain from reproduction (self-policing) (Ratnieks & Reeve 1992; Frank 1995; Wenseleers et al. 2004). Worker policing in terms of physical aggression towards ovary developed workers by other colony members requires that infertile individuals are able to detect reproductive nestmates. Long-chained hydrocarbons present on the cuticle seem to play a major role in these identification mechanisms (Heinze 2004, Howard & Blomquist 2005). Cuticular hydrocarbons correlate with ovarian activity in various social insects (ants: Monnin et al. 1998; Peeters et al. 1999; Liebig et al. 2000; Cuvillier-Hot et al. 2001; Hannonen et al. 2002; Heinze et al. 2002, Dietemann et al. 2003; de Biseau et al. 2004; D’Ettorre et al. 2004b; wasps: Bonavita-Cougourdan et al. 1991; Butts et al. 1995; Sledge et al. 2001a; Dapporto et al. 2004; bumblebees: Ayasse et al. 1995). A change in cuticular hydrocarbon profiles after either the onset or a decline in egg-laying activity shows that they encode information about reproductive physiology (Monnin et al. 1998; Liebig et al. 2000; Cuvillier-Hot et al. 2001; Dietemann et al. 2003). Most of these studies focused on species with ancestral characters (e.g. small colony size, high reproductive potential of workers). In contrast, only little is known about physical aggression against reproductive workers and identification mechanisms in evolutionarily more derived species with large colonies and workers with low reproductive potential, such as the ant Camponotus floridanus . In this monogynous species usually only the singly mated queen lays eggs (Gadau et al. 1996). In large colonies, queens produce a highly distinct hydrocarbon signal that regulates reproduction in the colony (Endler et al. 2004). The signal is present on the cuticle and on the eggs (Endler et al. 2004, Endler et al. 2006). Workers living in large colonies with a highly fertile queen eat the eggs laid by workers or weakly fertile queens. These eggs lack the appropriate hydrocarbon signal which is represented by qualitative and quantitative differences. This queen signal is among the most pronounced known for social insects suggesting a highly derived state. We expected that the pattern of worker policing would match the derived pattern of queen signaling in this species. IV. Lack of worker policing and fertility cues in egg-laying workers 59

In our study, we investigated whether the pattern of reproductive regulation in C. floridanus is different from what is known from species with small colonies and with workers characterized by a high reproductive potential. In the latter, pronounced dominance interactions and physical policing of workers are typical besides the policing of eggs. We also looked at differences in the hydrocarbon profile of fertile and infertile workers as this is correlated with worker policing in many species (Gobin et al. 1999; Kikuta & Tsuji 1999; Liebig et al. 1999; Monnin & Ratnieks 2001; Iwanishi et al. 2003; Kawabata & Tsuji 2005). We performed two behavioral tests to see whether the differentiation of infertile workers into egg-layers releases aggressive responses by nestmates and how well this correlates with their respective cuticular patterns. First, we observed the change in the frequency of aggressive interactions in isolated worker groups until workers started egg-laying. An increase in frequency would suggest competition for reproduction. Second, we identified egg-laying workers in the isolated groups and observed whether they elicited any policing behavior after reintroduction into their parental queenright colony in comparison to controls. The lack of discrete cuticular hydrocarbon patterns of egg-laying workers would support the view of a derived situation as it is assumed that these hydrocarbons are mainly used to identify egg- layers as e.g. in the queens of this species. Finally, we investigated whether the individual worker behavior changes because of ovarian activation, causing a cost at the colony level by reducing their contribution to colony productivity.

IV.2 Materials and Methods

Animals. Queens of Camponotus floridanus (n = 70) were collected at the Florida Keys/USA after the mating flight in August 2001 and July 2002. The very common carpenter ant C. floridanus reaches colony sizes of more than 10000 individuals. The polydomous colonies have only one single-mated queen (Gadau et al. 1996). If this queen dies, the colony is doomed, as requeening is unknown in strictly monogynous ant species (Hölldobler & Wilson 1990). Founding queens were transferred to the laboratory and were cultured in plaster nests at 25° Celsius and 50% humidity (12-h day, 12-h night). Twice a week they were fed with honeywater and cockroaches ( Nauphoeta cinerea ). Subsequently, approximately 80% were raised to colonies with 1000-2000 individuals within one to two years from which the experimental colonies originated.

IV. Lack of worker policing and fertility cues in egg-laying workers 60

Dominance behavior within worker groups. In the absence of a fertile queen, workers start male production at the earliest 60 days after orphanage (Endler et al. 2004). Generally, this period is accompanied by an increase in the frequency of aggressive interactions among individuals as a consequence of reproductive competition where individuals identify nestmates with activated ovaries (Gobin et al. 1999; Kikuta & Tsuji 1999; Liebig et al. 1999; Monnin & Ratnieks 2001, Dietemann et al. 2003; Iwanishi et al. 2003; Kawabata & Tsuji 2005). If this is also the case in C. floridanus , we would expect respective changes in aggressive behavior. We accordingly investigated the occurrence of dominance behavior and / or aggression among isolated sister workers until they started egg-laying. Queenless worker groups (N = 9), each containing 280 individuals without any brood (80 foragers and 200 individuals from inside the nest), were provided with honeywater and 1 ½ cockroaches ( N. cinerea ) twice a week. We started observations two hours after separation and one hour after feeding to retain the behavior of the workers by 24-hours videotaping. Subsequently, the worker groups were recorded one time per week for 24h until the onset of worker egg-laying. The queenless groups were monitored with Panasonic NV-DS29EG Digital camcorders recording at six frames per second. We analyzed the videos of the first day and one to three days after they started egg-laying. The frequency of behavioral interactions between the sister workers was recorded every other hour for one minute and the observed behavior was classified in a 4- Level aggression scale (Table IV.1). The frequencies of behaviors were summed up and proportions of total behavior were calculated.

Table IV.1 Aggression scales used in scoring the behavioral interactions of sister workers as well as foreign workers (modified after Carlin & Hölldobler 1986).

4-Level Aggression Scale

0: Neutral antennal contacts, grooming, food exchange (trophallaxis)

1: Avoidance, open mandible threat

2: Weak attack, nipping, leg / antennae pulling

3: Full attack, locking together, biting, spraying acid

IV. Lack of worker policing and fertility cues in egg-laying workers 61

Discrimination between egg-laying and non egg-laying nestmates. A requirement for worker policing via physical aggression is the ability to identify individuals that activated their ovaries. We, therefore, tested whether workers of queenright colonies are able to discriminate between sisters with and without active ovaries. We used orphaned worker groups without brood from nine different colonies, each containing 150 individuals (100 from inside the nest and 50 marked foragers). The groups of 50 foragers were replaced with another 50 marked foragers from the respective queenright colony once a week for the first nine weeks of isolation and every two weeks thereafter to exclude isolation effects on aggression upon reintroduction of nestmates (see Boulay et al. 2003; 2004). Foragers were marked with color codes (Edding paint marker 780). We used this worker caste because foragers have only little contact to the queen and / or her eggs while younger workers that tend the queen may potentially carry the queen signal (see e.g. messenger workers in the honeybee (Seeley 1979)). The isolated worker groups were controlled for the presence of eggs twice a week until the workers started egg-laying. Experimental worker groups were provided with honey water and 1 ½ cockroaches ( N. cinerea ) twice a week. After the appearance of eggs six minor and six major workers from egg laying groups were marked with triple color codes for individual identification. We selected egg-laying and non egg-laying workers by the differences in their characteristically escape- and avoidance behavior in threatening situations. Correct selection was verified after the experiment by dissection of the ovaries (see below) . After 30 minutes when the color dried, the ants were united with 50 workers (from inside the nest) from the respective queenright colony. These groups were videotaped for 24 hours with Panasonic NV-DS29EG Digital camcorders recording at six frames per second. Interactions between isolated workers and sisters from the respective queenright colony were analyzed for the first hour after reunion and every four hours for three minutes. We differentiated frequency and kind of interactions according to the 4-level aggression scale (see “Dominance behavior within worker groups” Table IV.1).

Dissection. After the observations of the focus workers (N =108), they were frozen and dissected in water to check the reproductive status of their ovaries. Workers, which lacked oocytes in their ovarioles, were defined as “non egg-laying”. Workers with oocytes longer than 0.5 millimeters and clearly recognizable yellow bodies were classified as “egg-laying”.

Control of aggressive behavior between non nestmates. In order to exclude reduced or absent aggressiveness as a consequence of laboratory culture, we used nestmate recognition IV. Lack of worker policing and fertility cues in egg-laying workers 62 bioassays to quantify the level of aggressiveness still present. We isolated 50 workers from a queenright colony in small plaster arenas (10 cm x 10 cm x 5 cm). After 1hour of acclimatization, five major and five minor workers of a foreign colony were added to the group. They had been isolated and marked with color codes (Edding paint marker 780) one hour before the experiment. The behavior towards the foreign ants was observed for one hour and classified according to the previous aggression scale (see “Dominance behavior within worker groups” Table IV.1). The experiment was terminated when aggression level 3 or higher had been observed.

Chemical analysis. Recognition of individuals with active ovaries is often associated with a distinct cuticular hydrocarbon profile (see introduction). We, therefore, investigated whether fertile and infertile workers of C. floridanus differ in their cuticular hydrocarbon (CHC) profile as it would be expected from all other studies on this topic. The CHC profiles of eight major and eight minor workers (each with four egg-laying and four non egg-laying workers) of eight different worker groups were analyzed. The worker groups originated from different queenright colonies. The CHC’s were extracted with solid phase microextraction (SPME see e.g. Monnin et al. 1998; Liebig et al. 2000; Endler et al. 2004). The fiber was gently rubbed on the tergites of workers for 3 minutes. Afterwards the fiber was directly injected into the injection port of a ThermoQuest Trace gas chromatograph with a split / splitless injector. Further details are described in Endler et al. (2004). Peak areas were computed with Chrom-Card 1.19 (CE Instruments). CHC profiles of queens with various levels of fertility differed qualitatively and quantitatively (Endler et al. 2006). We assumed that differences among fertile and infertile workers are at least in the same range. Similar to the analysis of the queen profiles we divided the CHC profiles of egg-laying and non egg-laying workers into two parts: 1) the shorter- chained compounds (n-pentacosane to 10-methyl-, 12-methyl-, and 14-methyloctacosane) and 2) the longer-chained compounds (12,16-dimethyloctacosane to 5,9,13,17- tetramethyltritriacontane) (see Endler et al. 2004). Since the two parts of the profile primarily differ qualitatively, they basically represent absence-presence data that are not suited for standard principle component analysis or discriminant analysis. These methods are used for quantitative differences. We instead calculated the sum of the peak areas of the compounds of the respective parts which allowed describing changes with simple statistical comparisons. IV. Lack of worker policing and fertility cues in egg-laying workers 63

Quantitative differences between profiles of fertile and infertile workers may occur in the second part of the profile. Since the same compounds are compared, multivariate statistical methods are appropriate here. The areas of the peaks were standardized to 100%. The resulting values were transformed according to Aitchison (1986) (see Dietemann et al. 2003). The variables were subject to a linear discriminant function analysis after Fisher for multiple groups (Johnson & Wichern 1999). We identified three variables that significantly contributed to the separation. These are represented by the alkane N-hentriacontane, the compound 8,12-dimethyldotriacontane and 3,7,11-trimethylhentriacontane. Classification was achieved using the leave-one-out criterion. The compounds were identified in comparison to the GC/ mass spectrometry analysis from Endler et al. (2004) using Kovac indices. Statistical tests were performed with STATISTICA 6.1 (Statsoft) and SPSS 12.02 (SPSS Inc.).

Differences of activity patterns in fertile and infertile nestmates. We compared the behavior of fertile and infertile nestmates to investigate potential reduction in working performance in egg-layers. The recorded videotapes from the experimental setup “Discrimination between egg-laying and non egg-laying nestmates” (see above) were analyzed to quantify activity patterns of six minor- and six major workers (three egg-laying / non egg-laying workers respectively) from egg laying groups. Worker status was already determined in that experiment (see above). They were marked with triple color codes for individual identification. The behavior of the marked ants was observed every four hours for three minutes within 24 hours. After each observation session (3 minutes) we defined the activity patterns either as “resting behavior” (workers are completely inactive) or as “motion” (workers move away by a body-length within 3 minutes) which resulted in six data points per individual. The frequency of the activity patterns of the three individuals per group were summed up and the proportions of each activity were calculated.

IV.3 Results

Workers in orphaned groups changed their behavior anytime between 21 days after isolation and the beginning of egg-laying in this group. Workers of the observed groups started egg- laying on average 100.3 days after separation. The beginning varied highly among groups (SD ≤ 53.5 days). We found significant more aggressive interactions at level 2 (see Table IV.1) in egg-laying worker groups than in the same groups at the beginning of isolation (Fig. IV.1). Aggression did not depend on the time of isolation, since there is no correlation IV. Lack of worker policing and fertility cues in egg-laying workers 64

between the onset of egg-laying and aggression (Spearman rank correlation; N groups = 9, R = - 0.23, p > 0.54 n.s.). Aggression at level 1 and 3 occurred only infrequently or was totally absent, respectively. In the same period we also recorded a decreased number of tolerance / social interactions at level 0.

worker groups 2h after isolation 100 * egg-laying worker groups

Median; Box: 25%-75%; Whisker: Min-Max 95

90 10 * 5

Frequency of interactions (%) interactions of Frequency 0

0 1 2 3 Level of aggression

Figure IV.1 Frequency of aggressive interactions between queenless sister workers at the beginning of isolation and after the onset of egg-laying (N groups = 9). Pair wise comparison of the frequencies of aggression levels occurring in queenless worker groups shortly after isolation and after the beginning of egg-laying shows significant differences (Wilcoxon signed ranks test for paired samples with sequential Bonferroni correction (Sokal & Rohlf 2003): * p < 0.02, all other n.s. p > 0.5).

In a second test we investigated whether queenright workers were able to discriminate between egg-laying and non egg-laying sisters. We differentiated between the response to egg-laying minors and majors and between the interaction within the first hour of contact and within 24h. Different morphs may be treated differently and familiarity after 24h may contribute to this as well. We found no significant differences in the discrimination ability of queenright workers between egg-laying and non egg-laying major sister workers as well as related egg-laying and non egg-laying minor workers (Fig. IV.2).

IV. Lack of worker policing and fertility cues in egg-laying workers 65

A: major workers, 1h observation B: minor workers, 1h observation 100 100 non egg-laying non egg-laying egg-laying egg-laying 95 95

90 90 10 10

5 5

Frequency of interactions (%) of interactions Frequency 0 (%) of interactions Frequency 0 0 1 2 3 0 1 2 3 Level of aggression Level of aggression C: major workers, 24h observation D: minor workers, 24h observation 100 non egg-laying 100 non egg-laying egg-laying egg-laying 95 95

90 90 10 10

5 5 Frequencyof interactions (%) Frequency of interactions (%) of interactions Frequency 0 0 0 1 2 3 0 1 2 3 Level of aggression Level of aggression

Figure IV.2 A-D Discrimination between egg-laying and non egg-laying major and minor nestmates (N group = 9 for each block). Each data point reflects the average of three individuals. The percentage of interactions between reunited workers of a queenright colony and the respective egg-laying as well as non egg-laying sisters from isolated worker groups are presented. Interactions within the first observation hour: A) minor workers and B) major workers. Within 24h: C) minor workers and D) major workers. (Wilcoxon signed ranks test for paired samples, A) p = 1.0 n.s.; B) p > 0.28 n.s.; C) p > 0.1 n.s.; D) p > 0.28 n.s.; median, quartile (25%-75%) and range.

We controlled whether aggression generally may have decreased due to laboratory conditions by performing a nestmate recognition experiment (Fig. IV.3). Sister as well as foreign workers were introduced in a colony and observed for up to 1h. Sister workers from isolated groups were not attacked at level 3 after introduction into their parental colony whatever ovarian status they had. However, between 80% major and 100% minor workers on average were strongly attacked after introduction when they originated from a foreign colony (attack level 3). Aggression level 3 was reached after 25 sec on average (median).

IV. Lack of worker policing and fertility cues in egg-laying workers 66

A: major workers B: minor workers * * * * 100 100

80 80 Median Median 25%-75% 25%-75% 60 Min-Max 60 Min-Max

40 40

20 20 responseslevel at (%) 3 responses at level 3 (%) 3 level responsesat Proportionaggressive of Proportion of aggressive of Proportion 0 0 foreign colony with eggs foreign colony with eggs without eggs without eggs

Figure IV.3 Comparison of the aggression by queenright workers towards conspecifics from foreign colonies and non egg-laying as well as egg-laying sisters. The proportion of interactions at aggression level 3 (see Table

1) is shown. A: major workers (N group = 9; overall difference, Friedman’s ANOVA, p < 0.0001; Wilcoxon-

Wilcox test for multiple comparisons: * p < 0.01; all other n.s. p > 0.95) B: minor workers (N group = 9; overall difference, Friedman’s ANOVA, p < 0.0001; Wilcoxon-Wilcox test for multiple comparisons: * p < 0.01; all other n.s. p > 0.95). Each data point reflects the average of three individuals.

Figure IV.4 Comparison of the proportion of shorter-chained cuticular hydrocarbons (CHC) of egg-laying and non egg-laying major and minor workers. We tested paired categories (egg-laying / non egg-laying major workers and egg-laying / non egg-laying minor workers) each containing four workers of eight different colonies respectively (equals 32 workers per category, N total = 128). Wilcoxon signed ranks test for paired samples, *** p < 0.0001, comparison between minor workers n.s., p > 0.9. IV. Lack of worker policing and fertility cues in egg-laying workers 67

Our previous results show that workers of C. floridanus do not differentiate between fertile and non fertile sister workers in our experimental setup. This would suggest that their cuticular hydrocarbon patterns are similar, since these substances are supposed to provide the information for discrimination. We found that the shorter-chained part of the profile is significantly smaller in non egg-laying major workers than in egg-laying major workers (Fig. IV.4), whereas this part of the profile did not differ significantly between egg-laying and non egg-laying minor workers.

Figure IV.5 Comparison of cuticular hydrocarbons (CHC) compounds from the longer chained profile among egg-laying major workers (N = 29), non egg-laying major workers (N = 32), egg-laying minor workers (N = 27) and non egg-laying minor workers (N = 32). Differences in the profiles were determined by a linear discriminant function analysis after Fisher for multiple groups (Johnson & Wichern 1999). We identified three variables that significantly contributed to the separation. These are represented by the alkane N-hentriacontane, the compound 8,12-dimethyldotriacontane and 3,7,11-trimethylhentriacontane. Two discriminant functions were extracted. The difference between the worker categories are statistically significant (Wilk`s lambda = 0.425 and p < 0.0001). The percentage denotes variance explained by the discriminant function.

IV. Lack of worker policing and fertility cues in egg-laying workers 68

100 * *

Resting behavior (%) egg-laying 20 non egg-laying Median; Box: 25%-75%; Whisker: Min-Max 0 major workers minor workers

Figure IV.6 Comparison of the proportion of resting behavior of egg-laying and non egg-laying major and minor workers within 24h. We tested paired categories (egg-laying / non egg-laying major workers and egg- laying / non egg-laying minor workers). Wilcoxon signed ranks test for paired samples; N group = 8, * p < 0.02. Each data point reflects the average of three individuals.

In the longer-chained part of the profile all focus compounds were present in all individuals allowing for a discriminant analysis (Fig. IV.5). Although we found significant differences among the groups overall, this is not associated with fertility since the differences are mainly due to the non-laying major workers. They are 71.9 % correctly classified whereas the average correct classification of the other groups is only 42.5 %. We investigated whether fertile and infertile workers differ in their activity patterns (Fig. IV.6). We found that egg-laying major workers as well as egg-laying minor workers show significantly more “resting behavior” than non egg-laying nestmates.

IV. Lack of worker policing and fertility cues in egg-laying workers 69

IV.4 Discussion

Our results show that worker policing in the ant Camponotus floridanus is highly derived in comparison to other species where this has been investigated. Policing of fertile workers in queenright colonies is almost or totally absent. This pattern corresponds to the variations in their cuticular hydrocarbon profiles. Fertile workers do not have a distinct profile although significant differences among different worker castes with different reproductive status exist. Despite the absence of physical policing, worker reproduction may reduce overall productivity, since egg-laying workers are less active suggesting that by lowering their contribution to general tasks they decrease colony productivity. Although workers of C. floridanus activated their ovaries and laid eggs in orphaned groups, they were not physically policed when introduced into their parental colony besides the occurrence of some aggression towards transferred individuals independent of their ovarian activity. The lack of physical policing is unusual and has been described in only two species in which, however, workers can become a principal egg-layer (Blatrix & Jaisson 2000; Sledge et al. 2001b) in contrast to C. floridanus , where a queen is the major reproductive and workers are normally functionally sterile. On the other hand, worker policing by physical aggression is usually common in eusocial species with more ancestral characters, e.g. ponerine ants with small colonies as well as weak queen-worker dimorphism and workers with high reproductive potential (Kikuta & Tsuji 1999; Monnin & Ratnieks 2001). In these species, workers can mate and reproduce sexually, so there is a conflict over male production as well as over becoming the colony’s gamergate under queenless conditions (Peeters 1993; Liebig et al. 1999; Monnin & Ratnieks 2001). They reconcile their differences, amongst others, by worker policing in terms of physical aggression (Monnin & Peeters 1997; Gobin et al. 1999; Kikuta & Tsuji 1999; Liebig et al. 1999; Monnin & Ratnieks 2001; D’ Ettorre et al. 2004a; Kawabata & Tsuji 2005). Worker policing in terms of physical aggression is also known from non-ponerine ants (Hölldobler & Carlin 1989; Dietemann et al. 2003; Iwanishi et al. 2003). Many studies suggest that egg-laying workers are normally recognized by policing workers sensing the changed profiles of their cuticular hydrocarbons (Monnin & Peeters 1999; Liebig et al. 2000; Dietemann et al. 2003; Cuvllier-Hot et al. 2004). In C. floridanus however, we did not find consistent differences between the cuticular hydrocarbon profiles of egg-laying and infertile workers, which is in line with the absence of physical worker policing in this species. The absence of differences in the cuticular hydrocarbon profiles of egg-laying IV. Lack of worker policing and fertility cues in egg-laying workers 70 and infertile workers is surprising, since queens of this species show a well expressed fertility signal encoded in their cuticular hydrocarbon profile (Endler et al. 2004, Endler et al. 2006). They show qualitative and quantitative differences in their cuticular hydrocarbon compared to those of fertile and infertile workers. Between 28.7% and 59.4% of the queens’ total amount of hydrocarbons is comprised by shorter-chained hydrocarbons, whereas these compounds represent less than 1% of the total amount of hydrocarbons in the profiles of egg-laying and infertile workers. Queens develop this profile when colonies grow larger and their fertility increases (Endler et al. 2006). Workers respond to profile differences when they police worker eggs in queenright colonies in C. floridanus . The differences between the profiles of surface hydrocarbons of queen- and worker-laid eggs are similar to those found on the cuticle of adults (Endler et al. 2004). In this case, workers differentiate between the profiles by eating worker-laid eggs, which contrasts to the lack of discrimination behavior towards egg-laying workers. In other ant species, where differences in the hydrocarbon profiles of eggs are present and where eggs laid by subordinates are policed, workers normally attack the subordinate egg-laying workers (Monnin & Peeters 1997; Dietemann et al. 2003; D’Ettorre et al. 2004a). However, in the latter cases the cuticular hydrocarbon profile of egg-laying workers differs from that of infertile workers indicating that the policed workers had been identified based on their changed hydrocarbon profiles. Workers are generally able to respond to very small differences in the cuticular hydrocarbon profiles. In nestmate recognition, hydrocarbon profiles are a major factor for the accurate discrimination of foreign individuals (Lahav et al. 1999; Thomas et al. 1999; Wagner et al. 2000; Akino et al. 2004), although the differences among colony profiles are often minor compared to those between the hydrocarbon profiles of fertile and infertile individuals (e.g. Cuvillier-Hot et al. 2001). All these examples indicate that differences in hydrocarbon profiles are perceived by workers and serve as a basis for discrimination in different contexts. Thus, the lack of differences in the cuticular hydrocarbon profiles between egg-laying and infertile workers in C. floridanus very well fits to the absence of consistent physical policing behavior directed towards egg-layers in this species. Even within the isolated worker groups of our experiment aggression was weak. This contrasts with the strong dominance interactions known from many species where sometimes fatal fights occur (Gobin et al. 1999; Kikuta & Tsuji 1999; Liebig et al. 1999; Monnin & Ratnieks 2001, Dietemann et al. 2003; Iwanishi et al. 2003; Kawabata & Tsuji 2005). In C. floridanus , the weak aggression seems not to be related to dominance. The level of aggression IV. Lack of worker policing and fertility cues in egg-laying workers 71 did not correlate with the onset of egg-laying in these colonies, making it unlikely that this aggression is associated with ovarian activation in workers. The absence of distinct physical aggression against workers with active ovaries in C. floridanus is still in line with the policing hypothesis. Instead of policing workers directly, worker-laid eggs are efficiently destroyed by nestmates at almost no cost (Endler et al. 2004; 2006). This leads to low potential benefits of worker reproduction that contrast with the costs of worker reproduction at the colony level. An increasing number of egg-laying workers may significantly reduce colony efficiency, since these workers are less active and thus contribute less work as became evident in our last experiment. Such productivity effects alone support worker policing (Cole 1986; Ratnieks 1988; Pamilo 1991). The unfavorable cost-benefit ratio of worker reproduction may finally lead to self-restraint (Ratnieks 1988; Foster 2004). This view is supported by the strong effect of the queen signal. Eggs that carry the queen signal alone are sufficient to prevent workers from egg-laying (Endler et al. 2004). Furthermore, workers of C. floridanus delay unusually long the onset of egg-laying in orphaned worker groups. The earliest egg-laying occurred 60 days after orphanage and 26 percent of worker groups did not lay eggs after 158 days in another experiment (Endler et al. 2004) indicating a high threshold to start egg-laying. This threshold seems to be much lower in other ant species with a less derived regulation of reproductive division of labor (earliest egg-laying after orphanage: 5 (mean 29.5) days ( Streblognathus peetersi ) Cuvillier-Hot et al. 2004; 6 days ( Harpagoxenus sublaevis ) Bourke 1988b, less than 13 days ( Diacamma ceylonense ) Cuvillier-Hot et al. 2002; less than 21 days (Harpegnathos saltator ) Liebig et al. 1999; 30 days (Gnamptogenys menadensis ) Gobin et al. 1999). Two species with a derived regulation of reproduction also show a relatively short period of producing viable eggs after orphanage: Aphaenogaster cockerelli with 17 – 41 days (Hölldobler & Carlin 1989) and Oecophylla longinoda with 30 to 60 days (Hölldobler & Wilson 1983). In both cases, however, young workers produce trophic eggs which are fed to the queen larvae. Hence the ovaries are active and only have to switch to production of viable eggs after the queen signal has vanished. Why are then workers in C. floridanus not completely sterile? Most species of Camponotus are monogynous including C. floridanus , thus no other queen could continue the colony if the single queen dies. In addition no requeening exists, thus workers will gain some benefit by producing males when the colony is orphaned (Hölldobler & Wilson 1990). These males have a certain chance to mate with a queen, even though most of them will be produced at a time where no mating partners are present. IV. Lack of worker policing and fertility cues in egg-laying workers 72

This study indicates that potential conflicts between the queen and her workers over male production in the social Hymenoptera can be resolved. Presumably, the low benefits of worker reproduction lead to self-restraint that is reinforced by efficient and practically cost- free worker policing of eggs. The evolution of self-restraint is accompanied by the evolution of a strong queen signal represented by hydrocarbons profiles that informs the workers about queen presence and fertility. Worker policing by egg eating together with queen signaling are thus the basis for the resolution of the conflict over male production in this species.

IV.5 Acknowledgements

We thank Dr. Thomas Schmitt (Univ. Freiburg, Germany) for identification of 8,12- dimethyldotriacontane, Katja Tschirner for technical assistance, Prof. Dr. M. Falk and PD Dr. F. Marohn (Department of Applied Mathematics and Statistics, Univ. Würzburg, Germany) for expert advice, the German Science Foundation (SFB 554 TP C3) and the European Union (INSECTS, Integrated Studies of the Economy of Insect Societies, HPRN-CT-2000-00052) for funding. The Social Insect Working Group of the Santa Fe Institute provided insightful discussion.

IV.6 References

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Woyciechowski, M. & Lomnicki, A. 1987. Multiple mating of queens and the sterility of workers among eusocial Hymenoptera. Journal of Theoretical Biology , 128, 317-327. V. General Discussion 78

V. General Discussion

The present study contributes to the understanding of the maintenance of social cohesion and discusses proximate mechanisms of regulation of reproduction in an advanced insect society. These results identify behavioral and pheromonal mechanisms of reproductive division of labor in the evolutionary derived ant Camponotus floridanus . Colonies of this species contain one monoandrous queen, which lays eggs while a large number of workers remain infertile. However, if the queen is absent or became infertile, workers are able to produce males. The results of this study suggest that in large insect societies the reproductive conflict between the queen and their workers is resolved in line with kin-selection theory (Hamilton 1964) as well as other factors like costs on colony level and mutual control of colony members. In the large societies of C. floridanus reproduction is regulated by an honest queen signal, which informs workers how they can realize their fitness interests. In C. floridanus the queen signal is based on hydrocarbons regulating reproductive division of labor by preventing workers from egg-laying. More precisely, queen eggs inhibit ovary development and egg-laying in isolated workers, whereas isolated sister workers without brood produce males regularly. The sterility of workers under queenright conditions is not caused by parental manipulation but rather self-restraint of workers, which realize their fitness interests. The cuticular hydrocarbons inform about presence and fertility of the queen. In contrast to highly fertile queens, low fertile queens and infertile workers show a similar hydrocarbon profile, thus a strong correlation between egg-laying rate and presence of queen specific hydrocarbons. Similar differences were found on the surface of the respective eggs. On the basis of the queen signal workers of a colony with a highly fertile queen are able to discriminate between eggs of workers and different fertile queens by egg destruction. However, this study also shows that workers with developed ovaries were never physically attacked by nestmates, which contrasts with most studies in species having workers with a higher reproductive potential. The behavioral pattern in C. floridanus is further supported by the absence of relevant differences in the cuticular hydrocarbon profile between egg-laying and infertile workers. This suggests that efficient mutual control by practically cost-free egg policing seems to have lead to worker self-restraint without a need for additional policing mechanisms. Furthermore, since egg-laying workers were significantly less active than non egg-laying nestmates, resources may be diverted from brood rearing and thus reduce overall productivity by lowering their contribution to general tasks.

V. General Discussion 79

The present study shows that workers use the information of the honest queen signal located on the eggs and on the cuticle to optimize their fitness benefits, and thus restrain themselves from reproducing in the presence of a highly fertile queen. This occurs even though workers should be favored to lay eggs in high-related monogynous colonies with single-mated queens like C. floridanus (Hamilton 1964, Bourke 1988, Wenseleers et al. 2004). Recent articles discuss the influence of relatedness controversially (see e.g. Griffin & West 2002). Lower relatedness leads to more worker reproduction in two bumblebee species, for example (Birmingham et al. 2004; Lopez-Vaamonde et al. 2004). Low relatedness caused by multiple paternity can also help to resolve conflicts by favoring worker policing (Ratnieks 1988). In colonies with high relatedness such as C. floridanus , worker policing can also be favored if the killing of worker-laid eggs helps to increase the total colony productivity (Ratnieks 1988; Hammond & Keller 2004), if it results in a more female based sex-allocation ratio (Foster & Ratnieks 2001b) or if it gives policing workers a greater opportunity to reproduce directly (“selfish” policing) (Saigo & Tsuchida 2004; Wenseleers et al. 2005). There is evidence that in C. floridanus the first two explanations might play a role. Results of the present study suggest that workers of queenright colonies increase the total colony productivity by egg policing due to the hypothesis that egg-laying workers produce costs on colony level. This is attributed to the loss of work force, e.g. brood rearing, because of decreased activity in fertile workers. Therefore, under queenright conditions and mutual control, workers gain more fitness benefits by indirect as by direct reproduction. Distinct worker policing should also be suggest self-restraint (Ratnieks & Reeve 1992; Frank 1995; Wenseleers et al. 2004). Although egg-policing does not directly prevent workers from laying eggs, it greatly reduces the incentive for them to do so (Ratnieks 1988; Visscher 1989; Ratnieks & Reeve 1992). Similar results are known from the honeybee, where effective policing reduces the benefit of worker reproduction that only around 0.1 % of the honeybee workers activate their ovaries (Ratnieks 1993). In C. floridanus there are some evidence that worker policing or in particular self- restraint of workers help even to enforce a female-based sex ratio. Worker reproduction is restricted by egg-policing to queenless conditions, thus policing behavior enables workers to control the sex-allocation of the colony (Foster & Ratnieks 2001b). The worker optimum of sex-allocation ratio is female based which is consistent with an ongoing conflict between queen and workers. In some species, a sex allocation less extreme than 75% female, may also be compatible with full worker control whether the worker optimum is reduced by worker production of males (Trivers & Hare 1976; Pamilo 1991a), multiple mating by queens V. General Discussion 80

(Pamilo 1991a,b) or multiple related queens (Ratnieks 1991a,b). Workers have more influence on sex allocation as the queen, not only because they tend and feed the brood but because they outnumber the queen. However, only in the reproductive phase will single- mated queens of C. floridanus lay haploid eggs. The queen controls laying haploid eggs whereas workers presumably do not, because they obviously are not able to discriminate the sex of queen-laid eggs. There is some evidence that workers recognize the sex of the larvae not until a late developmental stage (Nonacs & Carlin 1990). The elimination of queen- produced males on larval stage produces significant costs on the colony level. Therefore, worker policing is favored because policing removes some males (sons of workers) at low cost at the egg stage rather than a higher cost on larval stage (sons of the queen) (Foster & Ratnieks 2001b, Ratnieks et al. 2006). Workers of C. floridanus realize their fitness interests by egg policing, but not by aggression against nestmates with developed ovaries. A recent study indicates that worker policing occurs in 29 species (Ratnieks et al. 2006) in terms of egg-destruction and/or physically policing. Physically policing is known from species with small colony size, where nestmates contact each other regularly (Gobin et al. 1999; Kikuta & Tsuji 1999; Liebig et al. 1999; Monnin & Ratnieks 2001, Dietemann et al. 2003; Iwanishi et al. 2003; Kawabata & Tsuji 2005; Wenseleers et al. 2005). In such colonies, workers reconcile their differences by acting aggressively toward workers with active ovaries. In contrast, workers with developed ovaries of C. floridanus are never attacked by nestmates. Furthermore, workers are able to discriminate very effectively between worker-laid eggs and eggs of a highly fertile queen. In C. floridanus the complete destruction of worker eggs by oophagy seems practically cost-free and replace physically policing which might be costly. There is also plenty of evidence from 36 species that the queen may police worker-laid eggs or attack egg-laying workers (e.g. Free et al. 1969; Michener & Brothers 1974; Saigo & Tsuchida 2004; Wenseleers et al. 2005, Ratnieks et al. 2006). In the large colonies of C. floridanus queen policing could not be observed so far. In addition, it seems unlikely, that the queen is able to control ovary development of 15000 workers on their own. A further queen strategy may be marking her own eggs with a pheromone in order to facilitate worker policing (Ratnieks 1988).

Workers of C. floridanus are able to recognize highly fertile queens and their eggs due to their cuticular hydrocarbon pattern. These hydrocarbons represent the basis for differentiation between worker-laid eggs and eggs of a highly fertile queen. The results of this study support V. General Discussion 81 also the idea that hydrocarbons function as honest signals representing a general feature in social Hymenoptera queen pheromones (Hölldobler & Wilson 1983; Ratnieks 1988). In C. floridanus the hydrocarbon signal is closely correlated with the fertility of the queen and therefore also with colony size. Workers of C. floridanus obviously use the honest signal, since worker reproduction is absent in colonies with a highly fertile queen. This study shows for the first time the inhibitory effect of queen-laid eggs. Nevertheless, larvae have been shown to affect worker reproduction in at least two species. In the honeybee, Apis mellifera , larvae inhibit worker ovarian activation (Jay 1970, 1972; Arnold et al. 1994; Oldroyd et al. 2001), and in the ant Pachycondyla villosa larvae affect worker reproduction in queenless groups (Heinze et al. 1996). However, in these cases larvae do not directly signal queen presence; therefore, this regulation mechanism clearly differs from the mechanism described in this study. Recent studies support a closely correlation between the activity of the ovaries and the occurrence of hydrocarbons (ants: Dahbi & Lenoir 1998; Monnin et al. 1998; Peeters et al. 1999; Liebig et al. 2000; Cuvillier-Hot et al. 2001; Hannonen et al. 2002; Heinze et al. 2002, Dietemann et al. 2003; D’Ettorre et al. 2004b; wasps: Bonavita-Cougourdan et al. 1991; Butts et al. 1995; Sledge et al. 2001; Dapporto et al. 2004; bumblebees: Ayasse et al. 1995). The close linkage between cuticular hydrocarbons and surface hydrocarbons of eggs is based on specific transport pathways in the hemolymph (Schal et al. 1998, Fan et al. 2002, Lucas et al. 2004). Hydrocarbons are transported by lipoproteins to different tissues in the insect body, including the ovaries and the cuticle, thus queens are able to offer information about their actual fertility status and health. Consequentely, parental manipulation by the queen can be ruled out. Workers can perceive the queen signal either directly from the queen or indirectly via their eggs. A comparable system is only known from the honeybee, where messenger bees distribute the queen mandibular pheromone (QMP) (Seeley 1979). The QMP causes workers to refrain from reproducing (Hoover et al. 2003) but the exact mechanism is still unknown. These two ways of signaling provide reliable information to the workers to adjust their reproductive activities. The model system C. floridanus is one of few examples (e.g. Passera 1980) where proximate mechanisms of regulation of reproduction in an evolutionary derived social species could be revealed. In colonies of C. floridanus with effective egg-policing, the potential conflict over male production does not disappear under queenright conditions, as there is selection on individuals to evade policing by self-restraint (Ratnieks & Reeve 1992; Frank 1995; Wenseleers et al. 2004; Ratnieks 2006). This is contrary to many ancestral species V. General Discussion 82 where workers have a high reproductive potential and reproductive conflicts are highly distinctive. In the investigated species, the fertility signal based on cuticular hydrocarbons is queen specific and offers information about health and fertility. The direct link to physiological pathways and the disability of workers to mimic the signal, eliminate any potential of manipulation. Such an honest fertility signal based on hydrocarbons enables workers to distinguish between eggs of different maternities very effective and to enforce their own interests. Furthermore, the occurrence of worker policing is forced by costs on the colony level and worker policing supports presumably conflict resolution over sex allocation in C. floridanus . The proximate mechanisms examined in this study contribute to the understanding of the maintenance of social cohesion and offer new perspectives in regulation of reproduction in evolutionary derived species by allowing to compare them across species.

V.1. References

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Heinze, J., Stengl, B. & Sledge, M. F. 2002. Worker rank, reproductive status and cuticular hydrocarbon signature in the ant, Pachycondyla cf. inversa . Behavioral Ecology and Sociobiology , 52, 59-65. Hölldobler, B. & Wilson, E. O. 1983. Queen control in colonies of weaver ants (Hymenoptera: Formicidae). Annals of the Entomological Society of America , 76, 235-238. Hoover, S. E. R., Keeling, C. I., Winston, M. L. & Slessor, K. N. 2003. The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften , 90, 477-480. Iwanishi, S., Hasegawa, E. & Ohkawara, K. 2003. Worker oviposition and policing behaviour in the myrmecine ant Aphaenogaster smythiesi japonica Forel. Animal Behaviour , 66, 513-519. Jay, S. C. 1970. The effect of various combinations of immature queen and worker bees on the ovary development of worker honeybees in colonies with and without queens. Canadian Journal of Zoology, 48, 169-173. Jay, S. C. 1972. Ovary development of worker honeybees when separated from worker brood by various methods. Canadian Journal of Zoology, 50, 661-664. Kawabata, S. & Tsuji, K. 2005. The policing behavior `immobilization´ towards ovary-developed workers in the ant, Diacamma sp. from Japan. Insectes Sociaux , 52, 89-95. Kikuta, N. & Tsuji, K. 1999. Queen and worker policing in monogynous and monandrous ant, Diacamma sp. Behavioral Ecology and Sociobiology , 46, 180-189. Liebig, J., Peeters, C. & Hölldobler, B. 1999. Worker policing limits the number of reproductives in a ponerine ant. Proceedings of the Royal Society of London Series B, 266, 1865-1870. Liebig, J., Peeters, C., Oldham, N. J., Markstädter, C. & Hölldobler, B. 2000. Are variations in cuticular hydrocarbons of queens and workers a reliable signal of fertility in the ant Harpegnathos saltator ? Proceedings of the National Academy of Sciences, U.S.A , 97, 4124- 4131. Lopez-Vaamonde, C., Koning, J. W., Jordan, W. C. & Bourke, A. 2004. A test of information use by reproductive bumblebee workers. Animal Behaviour , 68, 811-818. Lucas, C., Pho, D. B., Fresneau, D. & Jallon, J. M. 2004. Hydrocarbon circulation and colonial signature in Pachycondyla villosa . Journal of Insect Physiology , 50, 595-607. Michener, C. D. & Brothers, D. J. 1974. Were workers of eusocial Hymenoptera initially altruistic or oppressed? Proceedings of the National Academy of Sciences, U.S.A., 71, 671-674. Monnin, T., Malosse, C. & Peeters, C. 1998. Solid-phase microextraction and cuticular hydrocarbon differences related to reproductive activity in queenless ant. Journal of Chemical Ecology , 24, 473-490. Monnin, T. & Ratnieks, F. L. W. 2001. Policing in queenless ponerine ants. Behavioral Ecology and Sociobiology , 50, 97-108. V. General Discussion 85

Nonacs, P. & Carlin, N. F. 1990. When can ants discriminate the sex of brood? A new aspect of queen-worker conflict. Proceedings of the National Academy of Sciences, U.S.A., 87, 9670- 9673. Oldroyd, B. P., Halling, L. A., Good, G., Wattanachaiyingcharoen, W., Barron, A. B., Nanork, P., Wongsiri, S. & Ratnieks, F. L. W. 2001. Worker policing and worker reproduction in Apis cerana . Behavioral Ecology and Sociobiology , 50, 371-377. Pamilo, P. 1991. Evolution of colony characteristics in social insects. I. Sex allocation. American Naturalist , 137, 83-107. Pamilo, P. 1991. Evolution of colony characteristics in social insects. II. Number of reproductive individuals. Am. Nat. , 138, 412-433. Passera, L. 1980. La fonction inhibitrice des reines de la fourmi Plagiolepis pygmaea Latr.: role des pheromones. Insectes Sociaux , 27, 212-225. Peeters, C., Monnin, T. & Malosse, C. 1999. Cuticular hydrocarbons correlated with reproductive status in a queenless ant. Proceedings of the Royal Society of London Series B, 266, 1323- 1327. Ratnieks, F. L. W. 1988. Reproductive harmony via mutual policing by workers in eusocial Hymenoptera. American Naturalist , 132, 217-236. Ratnieks, F. L. W. 1991. Facultative sex allocation biasing by workers in social Hymenoptera. Evolution , 45, 218-292. Ratnieks, F. L. W. 1991. The evolution of genetic odor-cue diversity in social Hymenoptera. American Naturalist, 137, 202-226. Ratnieks, F. L. W. 1993. Egg-laying, egg-removal, and ovary development by workers in queenright honey bee colonies. Behavioral Ecology and Sociobiology , 32, 191-198. Ratnieks, F. L. W. & Reeve, H. K. 1992. Conflict in single queen Hymenopteran societies: the structure of conflict and processes that reduce conflict in advanced eusocial species. Journal of Theoretical Biology , 158, 33-65. Ratnieks, F. L. W., Foster, K. R. & Wenseleers, T. 2006. Conflict resolution in insect societies. Annual Review of Entomology , 51, 581-608. Saigo, T. & Tsuchida, K. 2004. Queen and worker policing in monogynous and monoandrous colonies of a primitively eusocial wasp. Proceedings of the Royal Society of London Series B, 271, S509-S512. Schal, C., Sevala, V. L., Young, H. P. & Bachmann, J. A. 1998. Sites of synthesis and transport pathways of insect hydrocarbons: Cuticle and ovary as target tissues. American Zoologist , 38, 382-393. Seeley, T. D. 1979. Queen substance dispersal by messenger workers in honeybee colonies. Behavioral Ecology and Sociobiology , 5, 391-415. V. General Discussion 86

Sledge, M. F., Boscaro, F. & Turillazzi, S. 2001. Cuticular hydrocarbons and reproductive status in the social wasp Polistes dominulus . Behavioral Ecology and Sociobiology , 49, 401-409. Trivers, R. L. & Hare, H. 1976. Haplodiploidy and the evolution of social insects. Science , 191, 249- 263. Visscher, P. K. 1989. A quantitative study of worker reproduction in honey bee colonies. Behavioral Ecology and Sociobiology , 25, 247-254. Wenseleers, T., Helanterä, H., Hart, A. & Ratnieks, F. L. W. 2004. Worker reproduction and policing in insect societies: an ESS analysis. Journal of Evolutionary Biology , 17, 1035-1047. Wenseleers, T., Tofilski, A. & Ratnieks, F. L. W. 2005. Queen and worker policing in the tree wasp Dolichovespula sylvestris . Behavioral Ecology and Sociobiology, 58, 80-86. VI. Summary 87

VI. Summary

The present study contributes to the understanding of the maintenance of social cohesion in insect societies. Proximate mechanisms of regulation of reproduction in the evolutionarily derived ant Camponotus floridanus are discussed. The presented results identify behavioral and pheromonal mechanisms of reproductive division of labor between one reproductive (queen) and a majority of infertile individuals (workers), which help rearing related offspring. It remains controversial, whether non reproductive colony members help in their own interest or due to manipulation by the reproductive caste. Involved proximate mechanisms are especially poorly understood in evolutionary derived species. Species of these systems are characterized by large colony sizes, a distinct caste dimorphism between reproductives and helping nestmates as well as a low reproductive potential of these workers. The latter are able to lay unfertilized male eggs or they forgo this ability completely. In evolutionary derived species with large colony sizes reproduction is regulated by pheromones, since not all colony members can regularly contact each other. Cuticular hydrocarbons seem to play a major role in controlling worker reproduction. The aim of this study was to determine the function of the hydrocarbons, whether they are a manipulative agent or an honest signal. The information contained in an honest signal can be used by workers to optimize their fitness benefits. If the queen is healthy, highly fertile, and closely related to the workers, the latter should help to increase offspring production. On the other hand worker reproduction may cause a decrease in colony productivity, thus impose a cost at the colony level. These costs are caused by the loss of work force or by interindividual aggression as a consequence of the reproductive attempt. It is not in the interest of the workers that other nestmates beside the queen reproduce, particularly with regard to cost of worker reproduction. Workers should control other infertile nestmates and prevent them from reproduction (worker policing). In this study, queen signaling and worker policing in terms of egg destruction and physical aggression toward fertile workers are investigated. The evolutionarily derived ant species Camponotus floridanus served as model organism due to several advantageous properties. The large colonies of this species contain only one singly mated queen and approximately 15000 workers. Thus workers of a colony are closely related and social structure of the colony is simple. In addition, queen-worker dimorphism is large and workers have a low reproductive potential, since they can only lay unfertilized male eggs in the absence of the queen. I provide conclusive evidence that cuticular hydrocarbons signal the presence of the queen in C. floridanus . The hydrocarbon profiles of highly fertile queens and workers show VI. Summary 88 qualitative and quantitative differences mainly in the shorter-chained hydrocarbons. Similar differences were found on the surface of the respective eggs. In a behavioral test, queenless workers were regularly provided with queen eggs. In this group, workers did not lay eggs whereas isolated sisters without queen-laid eggs started egg-laying after two months. This shows that the eggs carry the queen signal and distribute the signal throughout the colony. In addition, the results support the idea that the hydrocarbons are an honest signal providing information about queen fertility. There is a strong correlation between the rate of egg-laying and the presence of shorter-chained hydrocarbons. Hydrocarbon profiles of different fertile queens of different colonies were investigated. The results show that cuticular components typical for highly fertile queens increase with colony size and oviposition rate. However, there are no differences between the cuticular profiles of founding queens with a low fertility and infertile workers. Again, the surface compounds of the eggs possess a high qualitative similarity to the respective profiles of the different queens. These differences in the profiles are perceived by workers. A bioassay was used to show the ability of workers to discriminate between eggs of different origin. When eggs were added to newly orphaned worker groups originating from a colony with a highly fertile queen, workers destroyed worker-laid eggs as well as eggs laid by low fertile queens while eggs laid by highly fertile queens were accepted. The differences in the hydrocarbon profiles easily explain the pattern of discrimination. As a result of this study, C. floridanus was shown to be among the few species where workers control reproduction by selective egg eating. The hypothesis of worker policing also includes control of worker reproduction by physical aggression, which was shown by many ancestral ant species with a higher reproductive potential of workers. I therefore investigated whether egg-laying workers of C. floridanus police fertile nestmates by physical aggression. Queenright workers showed an indiscriminate behavior to egg-laying and infertile colony members. Workers with developed ovaries were never attacked by nest-mates. The behavioral pattern can be explained by the absence of relevant differences in the cuticular hydrocarbon profile between egg-laying and infertile workers, in contrast to the strong differences that exist between highly fertile queens and workers. In this study I could show that worker reproduction may reduce overall colony productivity, since workers change their behavior after egg-laying and decrease colony productivity by lowering their contribution to general tasks. It was found that egg-laying workers were significantly less active than non egg-laying nestmates, which may divert resources from brood rearing. Such productivity effects alone may suffice to facilitate worker VI. Summary 89 policing despite high colony relatedness. The high costs of worker reproduction may finally lead to self-restrain. The study shows for the first time that in C. floridanus hydrocarbons on cuticle and on egg surface of highly fertile queens regulate reproductive division of labor. The honest signal of the queen is strongly correlated with her fertility. The signal, as well as the relevant differences in the hydrocarbon profile, is absent in fertile workers and low fertile queens. Furthermore, the results offer an interesting outlook to the evolution of worker policing. Policing occurs only in terms of egg destruction, whereas fertile workers are never attacked physically. In addition, worker reproduction entails costs on the colony level due to lowered overall colony productivity. This is attributed to the loss of work force as a consequence of increased resting behavior in fertile workers. Improving our knowledge of the proximate mechanisms of the reproductive division of labor in evolutionary derived species like C. floridanus will help to understand the evolution of extreme reproductive altruism involving sterility as a characteristic feature of advanced eusocial systems.

VII. Zusammenfassung 90

VII. Zusammenfassung

Die vorliegende Studie erweitert unser Verständnis des sozialen Zusammenwirkens von Insektengemeinschaften durch die Diskussion proximater Mechanismen, insbesondere der Regulation von Reproduktion. Die dargestellten Untersuchungen bei der evolutionär abgeleiteten Ameisenart Camponotus floridanus bieten Einblick in die Verhaltens- sowie Pheromon gesteuerten Mechanismen der reproduktiven Arbeitsteilung zwischen einem reproduktiven Tier (Königin) und einer großen Anzahl infertiler Individuen (Arbeiterinnen), welche bei der Aufzucht verwandter Nachkommen helfen. Es wird nach wie vor widersprüchlich diskutiert, ob infertile Tiere aus eigenem Interesse helfen oder ob sie durch die reproduzierende Kaste manipuliert werden. Besonders in evolutionär abgeleiteten Arten sind die dabei beteiligten proximaten Mechanismen bisher kaum verstanden. Charakteristisch für Arten dieser Systeme sind eine große Anzahl an Koloniemitgliedern, ein ausgeprägter Kastendimophismus zwischen reproduktiven Tieren und helfenden Nestgenossen, sowie ein geringes reproduktives Potential dieser Arbeiterinnen. Letztere sind demnach nur in der Lage nicht befruchtete Eier zu legen aus denen Männchen schlüpfen, oder sie haben die Fähigkeit zur Eiablage ganz verloren. Es wird angenommen, dass bei abgeleiteten Arten mit einer großen Anzahl an Koloniemitgliedern die Reproduktion durch Pheromone geregelt wird, zumal bei großen Kolonien nicht jede Arbeiterin physischen Kontakt zu jedem Koloniemitglied halten kann. Es scheint, dass kutikuläre Kohlenwasserstoffe bei der Steuerung der Arbeiterinnenreproduktion eine Rolle spielen. Gegenstand der vorliegenden Studie war es, die Funktion der Kohlenwasserstoffe dahingehend zu untersuchen, ob sie einen manipulativen Charakter haben oder ob es sich hierbei um ehrliches Signal handelt. Arbeiterinnen könnten dieses Signal als Informationsquelle nutzen, um ihre Gesamtfitness zu optimieren. Ist die Königin gesund, hoch fertil und nah verwandt zu den Arbeiterinnen, so sollten letztere helfen, die Reproduktion von Nachkommen zu steigern. Zum anderen sollte Arbeiterinnenreproduktion eine Abnahme der Kolonieproduktivität und somit Kosten auf Kolonieebene verursachen. Solche Kosten können durch einen Verlust von Arbeitskraft oder durch interindividuelle Aggression als Folge von Reproduktionsversuchen entstehen. Es ist daher auch nicht im Interesse infertiler Arbeiterinnen, dass andere Nestgenossen außer der Königin reproduzieren. Sie sollten sich somit gegenseitig von der Reproduktion abhalten (worker policing). Das Königinsignal sowie „worker policing“ in Form von Eifrass bzw. physische Aggression gerichtet gegen fertile Arbeiterinnen, war ebenfalls Gegenstand der Untersuchung. VII. Zusammenfassung 91

Die evolutionär stark abgeleitete Ameisenart C. floridanus bietet eine Reihe von Vorteilen und ist somit als Modellorganismus gut geeignet. Jede der großen Kolonien dieser Art besitzt eine einfach verpaarte Königin und bis zu 15000 Arbeiterinnen. Zwischen den Arbeiterinnen einer Kolonie bestehen damit ein hoher Verwandtschaftsgrad und eine einfache Verwandtschaftstruktur. Darüber hinaus ist der Dimorphismus zwischen Königin und Arbeiterin stark ausgeprägt und die Arbeiterinnen besitzen nur ein eingeschränktes reproduktives Potential. Sie können demnach nur bei Verlust oder Infertilität der Königin Männchen produzieren. In dieser Arbeit wird der Nachweis erbracht, dass kutikuläre Kohlenwasserstoffe die Präsenz der Königin bei C. floridanus signalisieren. Die Profile hoch fertiler Königinnen und Arbeiterinnen unterscheiden sich qualitativ sowie auch quantitativ voneinander, besonders im kurzkettigen Bereich. Ähnliche Unterschiede sind auch bei den Oberflächenkohlen- wasserstoffen der jeweiligen Eier zu finden. In einem Verhaltensversuch wurden königinlos gehaltene Arbeiterinnen regelmäßig mit Eiern einer hoch fertilen Königin versorgt. Arbeiterinnen aus dieser Gruppe legten auch nach 2 Monaten keine Eier, während gleichzeitig isolierte Schwestern ohne Brut in diesem Zeitraum mit der Eiablage begannen. So konnte gezeigt werden, dass die Eier das Signal der Königin transportieren und in der gesamten Kolonie verbreiten. Die Ergebnisse der vorliegenden Studie unterstützen auch die Annahme, dass Kohlenwasserstoffe ein ehrliches Signal darstellen, welches über die Fertiliät der Königin informiert. Es besteht ein enger Zusammenhang zwischen Eiablagerate und dem Vorhandensein kurzkettiger kutikulärer Kohlenwasserstoffe. Dies wurde an den Kohlenwasserstoffprofilen von unterschiedlich fertilen Königinnen aus unterschiedlichen Kolonien untersucht. Die ermittelten Ergebnisse zeigen, dass die kutikulären Komponenten, welche typisch für hoch fertile Königinnen sind, mit steigender Koloniegröße und Eiablagerate zunahmen. Profile von Gründungsköniginnen mit einer geringen Eiablagerate zeigten hingegen keine Unterschiede zu den Profilen infertiler Arbeiterinnen. Die Oberflächenprofile der Eier wiesen wiederum eine starke Ähnlichkeit zu den jeweiligen kutikulären Profilen der unterschiedlich fertilen Königinnen auf. Die Unterschiede in den kutikulären Profilen können von Arbeiterinnen wahrgenommen werden. Um diese Fähigkeit der Arbeiterinnen zur Eiunterscheidung zu überprüfen, wurden Verhaltenstests durchgeführt. Es konnte nachgewiesen werden, dass wenn man isolierten Arbeiterinnen aus Kolonien mit einer hoch fertilen Königin Eier von anderen hoch fertilen Königinnen, niedrig fertilen Königinnen und Arbeiterinnen vorlegte, VII. Zusammenfassung 92 diese die Herkunft der Eier genau der jeweiligen eierlegenden Kaste zuordnen konnten. Eier von anderen Arbeiterinnen sowie von niedrig fertilen Königinnen wurden im Gegensatz zu Eiern von hoch fertilen Königinnen zerstört. Es liegt daher nahe, dass die Kohlenwasserstoffe die Grundlage zur Unterscheidung darstellen. C. floridanus ist damit eine der wenigen nachgewiesenen Arten, wo Arbeiterinnen durch selektiven Eifrass die Reproduktion kontrollieren. Die Hypothese des „worker policing“ beinhaltet auch, dass die Kontrolle der Reproduktion von Arbeiterinnen durch direkte physische Aggression erfolgen kann. Bei vielen untersuchten, allerdings oftmals sehr ursprünglichen Arten mit höherem reproduktivem Potential, konnte dies auch nachgewiesen werden. In der vorliegenden Studie wurde daher untersucht, inwiefern eierlegende Arbeiterinnen bei C. floridanus durch physische Aggression von der Reproduktion abgehalten werden. Arbeiterinnen zeigen in Anwesenheit einer Königin kein aggressives Verhalten gegenüber infertilen und eierlegenden Koloniemitgliedern, Arbeiterinnen mit aktiven Ovarien wurden nie von Nestgenossen angegriffen. Dieser Umstand wird auch unterstützt durch die Abwesenheit von relevanten Unterschieden im kutikulären Kohlenwasserstoffprofil zwischen eierlegenden und infertilen Arbeiterinnen, obwohl es massive Unterschiede im Profil zwischen hoch fertilen Königinnen und Arbeiterinnen gibt. Bei Arbeiterinnen von C. floridanus konnte außerdem gezeigt werden, dass Reproduktion von Arbeiterinnen die Gesamtproduktiviät der Kolonie reduziert, da sich mit der Eiablage das Verhalten so verändert, dass sie weniger zu den allgemeinen Aufgaben innerhalb einer Kolonie beitragen. Eierlegende Arbeiterinnen verbringen signifikant mehr Zeit mit Ruhen als ihre infertilen Nestgenossen. Dies bedeutet einen Verlust an Arbeitskraft, welcher sich z.B. negativ auf die Versorgung von Brut auswirken könnte. Solch Nebeneffekte von Arbeiterinnenreproduktion reichen aus, um „worker policing“ trotz hohem Verwandtschaftsgrad entstehen zu lassen. Das daraus resultierende ungünstige Kosten-Nutzen Verhältnis führt letztendlich zur Aufgabe der eignen Reproduktionsfähigkeit. Es konnte erstmalig nachgewiesen werden, dass die Kohlenwasserstoffe auf der Kutikula und der Oberfläche der Eier von C. floridanus Königinnen die Reproduktion regeln. Das abgegebene Signal korreliert eng mit der Fertilität und bestätigt die Vermutung eines ehrlichen Signals. Bei eierlegenden Arbeiterinnen und niedrig fertilen Königinnen fehlen dieses Signal und die relevanten Unterschiede im Kohlenwasserstoffprofil vollständig. Weiterhin zeigt diese Untersuchung interessante Perspektiven im Bezug auf die Evolution von „worker policing“, da „policing“ nur noch in Form von Eifrass nach der Ablage stattfindet VII. Zusammenfassung 93 und nicht mehr in Form von physischer Aggression gegenüber fertilen Nestgenossen. Außerdem konnte gezeigt werden, dass die Reproduktionsfähigkeit von Arbeiterinnen mit Kosten verbunden ist. Dies ist wahrscheinlich auf einen Verlust an Arbeitskraft zurückzuführen, welcher durch vermehrtes Ruhen fertiler Tiere verursacht wird. Die Aufklärung der Regulationsmechanismen der reproduktiven Arbeitsteilung bei stark abgeleiteten Arten wie C. floridanus liefert somit einen Beitrag zum Verständnis, wieso es im Laufe der Evolution zur reproduktiven Degeneration von Arbeiterinnen gekommen ist, einem Charakteristikum hoch entwickelter eusozialer Systeme.

VIII. Danksagung 94

VIII. Danksagung

Viele Menschen leisteten einen direkten oder indirekten Beitrag zu dieser Arbeit. Ihnen allen möchte ich, namentlich erwähnt oder unerwähnt, ganz herzlich danken.

Besonderer Dank gebührt Dr. Jürgen Liebig für sein Vertrauen und die uneingeschränkte Unterstützung in allen Phasen dieser Arbeit. Er ermöglichte mir den Einstieg in die Welt der Ameisen und half mir in vielen persönlichen Gesprächen die komplexen Zusammenhänge des sozialen Lebens zu verstehen. Von seinem großen Interesse und seinen Ideen hat die Arbeit durchgehend profitiert. „Natur: dies, was selbst den Gedanken manchmal keine Wahl läßt.“ – Danke für dieses Erlebnis!

Prof. Dr. Bert Hölldobler möchte ich danken, dass ich die Arbeit an seinem Lehrstuhl durchführen konnte, sein Interesse am Thema und seine stete Diskussionsbereitschaft.

Einzelne Kapitel dieser Arbeit (siehe auch entsprechende „Acknowledgements“) profitieren vom fachkundigen Beitrag folgender Kollegen und Freunde, denen nochmals herzlich gedankt sei: Dr. Thomas Schmitt, Dr. Graeme Jones und Jane Parker für die Identifizierung und Synthese der Substanzen des Königinsignals, Prof. Dr. Peter Schreier für die großzügige Versorgung mit destillierten Lösungsmitteln, Prof. Dr. Falk und PD Dr. Marohn für statistische Beratung, Annette Laudahn und Malu Obermaier teilten ihr reiches Wissen über Ameisen mit mir und Katja Tschirner danke ich für technische Hilfestellung im Labor. Bei den Sammelfahrten in den USA 2001-2003 hatte ich tatkräftige Unterstützung von Dr. Jürgen Liebig, Dr. Christoph Strehl, Claudia Bandulet und Sebastian Diedering, die alle heldenhaft Hitze und Moskitos Widerstand leisteten. Für die Unterstützung im Kampf mit der englischen Sprache gebührt Jon Seal und Dr. Heike Feldhaar ein besonderer Dank.

Ebenfalls danken möchte ich den vielen Hiwis, insbesondere Anastasios Saratsis, Kerstin Tüchert und Christine Betz für zum Teil jahrelanges unermüdliches Füttern und Putzen der bis zu 300 Ameisenkolonien.

Das Leben und Arbeiten am Lehrstuhl wurde durch zahlreiche Leute erleichtert und in vielfacher Weise bereichert. In dieser Hinsicht möchte ich vor allem Karl-Herbert Falk und Dr. Heike Feldhaar danken, ohne ihre Unterstützung wäre diese Arbeit so nicht zustande VIII. Danksagung 95 gekommen. Herzlichen Dank auch der „Mittagsrunde“, einem unerschöpflichen Quell an menschlicher Wärme, Humor, Information und zuweilen Kuchen. Für eine schöne Arbeitsatmosphäre und erbauliche Gespräche möchte ich mich bei Christoph, Christof, Oli, Sebbo, Claudia, Sebastian und Vincent bedanken. Allen Mitgliedern der Zoo II, die ich hier nicht alle namentlich aufzählen kann, danke ich für ihre mannigfaltige Unterstützung.

Zu Dank verpflichtet bin ich auch Franka, Julia, Martina und allen anderen Freunden, die trotz meiner Vernachlässigung stets da waren und es schafften, mich auch mal aus meinem Trott zu reißen.

Thomas - Verständnis, Geduld, konstruktive Kritik, unglaubliche Mengen an Kaffee und Kuchen, tausend tägliche Kleinigkeiten – DANKE!

Abschließend möchte ich meinen Eltern ganz herzlich danken, dass sie immer Verständnis aufbrachten, teilnahmen an meiner Freude an der Biologie und für ihre Unterstützung in meinem Studium, wann immer ich sie benötigte.

IX. Curriculum Vitae 96

IX. Curriculum Vitae

Name: Annett Endler Geburtsdatum / Geburtsort: 01. Oktober 1976 in Marienberg

Schulausbildung 1983 – 1992 Polytechnische Oberschule in Olbernhau 1992 - 1995 Naturwissenschaftliches Gymnasium in Olbernhau Abschluss Abitur

Hochschulstudium Okt. 1995 – Feb. 2001 Studium der Biologie an der Bayrischen Julius-Maximilians-Universität Würzburg Feb. 2001 Abschluss Diplom; Schwerpunkte: Tierökologie, Tropenbiologie, Mikrobiologie, Ökophysiologie und Vegetationsökologie Diplomarbeit: „Biologie und Ökologie aggregierender Baumwollwanzen (Heteroptera, Pyrrhocoridae) und deren Räuber in einem Savannen-Ökosystem Westafrikas“ bei Prof. Dr. K. Eduard Linsenmair seit März 2001 Promotionsstudium bei Prof. Dr. Bert Hölldobler, Betreuer: Dr. Jürgen Liebig, Lehrstuhl für Tierphysiologie und Soziobiologie, Bayrische Julius-Maximilians-Universität Würzburg

Auslandsaufenthalte März 1999 - Mai 1999 Forschungspraktikum Comoé Nationalpark, Côte d’Ivoire (Elfenbeinküste), Forschungsstation Universität Würzburg März 2000 – Juli 2000 Forschungsaufenthalt für Diplomarbeit, Comoé Nationalpark, Côte d’Ivoire (Elfenbeinküste), Forschungsstation Universität Würzburg

Würzburg, den 01.06.2006 Annett Endler

Ehrenwörtliche Erklärung

gemäß § 4 Abs. 3 Ziff. 3,5 und 8 der Promotionsverordnung der Fakultät für Biologie der Bayrischen Julius-Maximilians-Universität Würzburg

Hiermit erkläre ich ehrenwörtlich, dass ich die vorliegende Dissertation selbstständig angefertigt und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet habe.

Die Dissertation wurde bisher weder vollständig noch teilweise einer anderen Hochschule mit dem Ziel einen akademischen Grad zu erwerben, vorgelegt.

Am 21.02.2001 wurde mir von der Universität Würzburg der akademische Grad „Diplom- Biologin Univ.“ verliehen. Weitere akademische Grade habe ich weder erworben noch versucht zu erwerben.

Würzburg, den 01.06.2006

Annett Endler