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Abstract. Slave-making ants are social parasites which ex- ploit the workforce of heterospecific slaves for their own re- production, and to this end they have ...
Insect. Soc. 53 (2006) 291–299 0020-1812/06/030291-9 DOI 10.1007/s00040-006-0871-z © Birkhäuser Verlag, Basel, 2006

Insectes Sociaux

Research article

Convergent evolution of the Dufour’s gland secretion as a propaganda substance in the slave-making ant genera Protomognathus and Harpagoxenus M. Brandt1,2, J. Heinze1, T. Schmitt3 and S. Foitzik1,4,* 1

Department Biology I, University Regensburg, 93040 Regensburg, Germany Present address: Department Ecology & Evolutionary Biology, University of California, Irvine, Irvine CA 92697, USA 3 Institute of Biology I, University of Freiburg, 79104 Freiburg, Germany 4 Present address: Department Biology II, Ludwig Maximilians University Munich, 82152 Martinsried, Germany, e-mail: [email protected] 2

Received 24 February 2005; revised 28 July 2005 and 2 March 2006; accepted 6 March 2006. Published Online First 30 June 2006

Abstract. Slave-making ants are social parasites which exploit the workforce of heterospecific slaves for their own reproduction, and to this end they have developed a variety of morphological and behavioural adaptations. Furthermore, social parasites utilize the chemical communication system of their hosts by breaking their nestmate recognition code, and some slave-maker species additionally employ semiochemicals as weapons during colony foundation and slaveraiding. Here, we demonstrate the use of such a ‘propaganda allomone’ by the North American myrmicine slave-maker Protomognathus americanus. This substance is produced in the Dufour’s gland and may be employed during slave raids to elicit panic among defending host workers. Slave-maker Dufour’s gland secretions evoked agitation and heightened levels of activity among host workers when applied directly on a host nest, and strong aggressive responses of nestmates when applied onto a host worker. Although the hosts own Dufour’s gland secretion also elicits intra-colonial fights, no support for the hypothesis was found that the slave-maker propaganda substance mimics a fertility signal of the host, as the chemical profile of the gland secretions is highly divergent between the two species. Preliminary results on the chemical composition of the secretion obtained by gas chromatography indicate that the propaganda substance of P. americanus differs from that of the related European slavemaker Harpagoxenus sublaevis, and is thus likely to represent an independent evolutionary development. Keywords: Chemical warfare, Dufour’s gland, social parasites, fertility signal, propaganda substance. * Corresponding author

Introduction Social life requires complex interactions between group members, and social insects have therefore evolved highly sophisticated communication systems in which information is conveyed by chemical substances or pheromones. For example, insect societies are protected against the intrusion of alien individuals by a nestmate recognition system based on a shared hydrocarbon profile on the cuticle (Crozier and Dix, 1979; Howard, 1993; Lorenzi et al., 1996). Colony members learn the recognition cues shortly after eclosion, and individuals that do not match the internal template are usually rejected. In addition to the chemical cuticular signature, social insects have developed numerous exocrine glands responsible for the production of semiochemicals that function e.g. as alarm pheromones, territorial or trail markings, or signals of reproductive status (Ali and Morgan, 1990). The number and diversity of exocrine glands are particularly impressive in ants, and for this reason ant workers have been described as ‘walking glandular batteries’ (Billen, 1991). Social parasites that take advantage of the group behaviour of bee, wasp and ant colonies have evolved various adaptations to exploit the chemical communication system of their hosts. Among ant social parasites, chemical strategies are especially well-known for dulotic, or slave-making, species. A slave-maker colony is initiated when a mated parasite queen invades a host colony, kills the host queen and appropriates the brood. At this stage, some species (such as those in the genus Polyergus) are accepted by the resident host workers, whereas many myrmicine slave-makers kill or drive away all adults and take over only the larvae and pupae. Enslaved hosts form a social attachment to the slave-makers, perform their usual worker functions in the mixed-species

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colony, and also raise the slave-maker queen’s brood (Le Moli and Mori, 1985). Slave-maker workers are specialized on only a single task: they regularly conduct slave raids against neighbouring host colonies and pillage their larvae and pupae to replenish the work force in their own nest (D’Ettorre and Heinze, 2001; Mori et al., 2001). Especially slave-maker queens at the vulnerable founding stage have been shown to exhibit elaborate chemical strategies. For example, Polyergus queens are characterized by a reduced cuticular hydrocarbon profile before colony usurpation (“chemical insignificance”), but afterwards quickly acquire the host colony odour from the host queen (Topoff and Zimmerli, 1993; D’Ettorre and Errard, 1998; Johnson et al., 2001; D’Ettorre et al., 2002). Apart from breaking the nestmate recognition code of their hosts, some slave-maker species produce specific allomones, chemical weapons that trigger certain host behaviours at the advantage of the parasite. Polyergus queens, for example, possess a Dufour’s gland secretion that protects them from host aggression; this substance has been interpreted as an appeasement allomone (Topoff et al., 1988; Mori et al., 2000a, b) or a repellent substance (D’Ettorre et al., 2000). Similarly, compounds produced in the Dufour’s gland are used by slave-maker workers during slave raids as a means of ‘information warfare’ that diverts attacks by the usually much more numerous host defenders (Franks and Partridge, 1993). These ‘propaganda substances’ may either be directly applied onto host workers, which are subsequently attacked by their own nestmates (Harpagoxenus sublaevis; Buschinger, 1974; Allies et al., 1986; Foitzik et al., 2003), or they can be released in the host nest to cause panic and disorganisation among defending workers (Formica subintegra; Regnier and Wilson, 1971). A similar host alarm behaviour induced by the entrance of slave-maker workers into the nest has been described for the North American myrmicine slave-maker Protomognathus americanus (Wesson, 1939; Buschinger et al., 1980; Alloway and Del Rio Pesado, 1983) and has been interpreted as evidence for the existence of a propaganda substance in this species (Alloway, 1979; Buschinger et al., 1980). However, this hypothesis has never been empirically tested. Here we demonstrate the effect of the Dufour’s gland secretion of P. americanus on colonies of its Temnothorax (formerly Leptothorax; Bolton, 2003) host species. Furthermore, we present preliminary results on the chemical composition of P. americanus Dufour’s gland contents and compare them with the propaganda substance of the closely related slave-maker H. sublaevis. Since Dufour’s gland secretions of some social insects have been found to constitute egg-marking pheromones used e.g. by honey-bee queens to protect their eggs from worker policing (Ratnieks, 1995; Oldroyd and Ratnieks, 2000), a conceivable hypothesis is that substances from the slave-maker Dufour’s gland mimic fertility signals of their hosts, whose release in the nest causes confusion and misdirected aggression (‘worker policing’) among host workers. To test this hypothesis, we compared the effect of slave-maker Dufour’s glands with the impact of glands of host individuals of different caste and reproductive status.

Propaganda substances in slave-making ants

Materials and methods Study system, colony collection and ant maintenance P. americanus enslaves the three closely related host species Temnothorax longispinosus, T. curvispinosus, and T. ambiguus. These ants are widely distributed in deciduous forests in northeastern North America and nest in hollow acorns, nuts and rotting sticks on the forest floor. Slave-maker colonies are typically small, containing between two and eight slave-maker workers and 20–50 slaves. We collected ant colonies at the Huyck Preserve, Rensselaerville, Albany County, New York and in Harpersfield, Ashtabula County, Ohio, in summer 2003. Colonies were transported to the laboratory in their natural nesting sites, censused, and moved to artificial nest sites between two microscope slides for behavioural observations. Ants were housed under standard conditions (Heinze and Ortius, 1991) in an incubator (25 °C for 14 h light, 17 °C for 10 h dark).

Effect of P. americanus Dufour’s glands on host colonies Behavioural experiments were performed in fall 2003. In order to assess the effect of the Dufour’s gland contents of slave-maker workers on colonies of T. longispinosus, we individually marked five host workers with Edding paint dots on the gaster and reintroduced them to their colony one day prior to the experiment. The behaviour of the marked individuals was first observed for 5 min employing a scan sampling technique in 15 s intervals. Subsequently, a P. americanus worker was killed by freezing at –80 °C, dissected in distilled water under a stereomicroscope, and the intact Dufour’s gland was placed on the tip of an insect pin. The pin was then introduced into the host colony, and the gland crushed against the nest wall. A second observation period of 5 min was started immediately after the release of the Dufour’s gland contents. Again using scan sampling with 15 s intervals we recorded the following behaviours: resting, walking/running, self-grooming, allogrooming, trophallaxis, antennation (of nestmates), mandible opening and biting. As a control for colony disturbance caused by the introduction of the insect pin into the nest, the procedure described above was repeated with the same colony, except that the insect pin was dipped in water. Control and gland trials were performed with the same colonies in random order, with one day in between the two tests. Trials were conducted with five T. longispinosus and five T. curvispinosus colonies.

Comparison with Dufour’s glands of other species In the subsequent tests, the Dufour’s gland was not introduced into a host colony directly; instead, we applied its contents onto a host worker, reintroduced it into the colony, and observed its nestmates’ reactions. For this purpose, a host worker was marked with an Edding paint dot on the thorax approximately 1 h prior to the experiment. A Dufour’s gland was dissected, placed whole on the tip of an insect pin, and carefully smeared onto the host worker’s gaster. Immediately afterwards, the worker was placed back into its mother colony, and the nest entrance was sealed with cotton wool. Subsequently, all reactions of nestmates towards the focal worker were recorded with scan-sampling every 15 s for a period of 5 min. Behaviours recorded were antennating, mandible-opening, grooming, biting, dragging and stinging. An aggression score was calculated as the sum of mandible-opening, biting, dragging and stinging events, standardized for the number of antennal contacts to control for differential encounter rates with nestmates. These experiments were performed with glands of P. americanus workers and queens, T. longispinosus fertile workers, infertile workers, and queens, T. curvispinosus infertile workers, and infertile H. sublaevis workers. Conspecific glands always came from individuals belonging to a different colony than the tested one. Nestmate workers smeared with

Insect. Soc.   Vol. 53, 2006

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distilled water instead of gland contents were used as controls. We observed the reaction of ten host colonies that were confronted in random order with nestmates treated with water or a gland from one of the above categories, with at least one day in between individual tests. Ovary status of workers was determined in the course of dissection. Workers were counted as infertile if their ovaries were completely undeveloped and contained no developing oocytes, whereas workers whose ovaries contained at least one large egg were classified as fertile.

Chemical analyses In the chemical analysis of Dufour’s gland contents, most individual glands did not yield a strong enough signal. We therefore pooled glands of six T. longispinosus workers (N = 4 runs for fertile and infertile workers, respectively), three T. longispinosus queens (N = 3), and two P. americanus workers (N = 3), respectively, per GC run. Glands of the slightly larger P. americanus queens (N = 3) and H. sublaevis workers (N = 2) were analysed individually. After dissection of the ants, the Dufour’s gland was transferred into a glass vial filled with 20 µl of pentane with an insect pin. 2  µl of the glandular extracts were injected into an Agilent Technologies 6890N gas chromatograph equipped with an Rtx-5 capillary column (30 m × 0.25 mm × 0.50 µm, Restek, Bellefonte, USA). We used helium as carrier gas at 1 ml/min, the injector in splitless mode (1 min), and the temperature program of 3 min at 70 °C, to 150 °C at 30 °C/min, then held at 150 °C for 5 min, from 150 °C to 290 °C at 5 °C/min, and finally held constant at 290 °C for 16 min. Flame ionisation detector temperature was set at 300 °C. Since the GC profiles for the different categories of individuals contained markedly different sets of peaks, the obtained dataset contained

a) running

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many zeroes, and standard multivariate procedures such as Principal Components Analysis or Discriminant Analysis were not feasible. To visualize the relationship between the different categories, we therefore conducted a Multiple Correspondence Analysis (MCA) on the presenceabsence matrix of peaks, and plotted the resulting scores on the first two extracted factors. GC-MS analysis was done on an Agilent 5973 inert Mass Selective Detector with the GC machine, temperature program and parameters as for the GC analyses, but equipped with an RH-5ms+ fused silica capillary column (30 m × 0.25 mm × 0.25 µm). Electron impact mass spectra were obtained with an ionisation voltage of 70 eV and a source temperature of 230 °C. Agilent G1701DA MSD ChemStation was used for data acquisition. Compounds were characterized by use of standard MS databases, diagnostic ions and Kovats indices (Carlson et al., 1998).

Results Effect of P. americanus Dufour’ glands on host colonies Preliminary trials had indicated that the contents of a slavemaker Dufour’ glands only elicited a reaction if they were released within about 2–3 cm of the ants in the colony, thus the gland contents have only a short-distance effect. The general response to P. americanus Dufour’s gland secretions was agitation and increased activity in the host nest. This was evident in an increase in the time out of the 5 min the focal individuals spent running around (Fig. 1a, Friedman-ANO-

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Figure 1. Reaction of host colonies to Dufour’s gland contents of P. americanus workers, measured by the behaviours a) running, b) selfgrooming, c) antennating, and d) mandible-opening. Bars represent the difference in frequency of the behaviours between observations before and after exposure to water (controls, white bars) and gland contents (trials with glands, hatched bars). Tests were performed with colonies of T. longispinosus and T. curvispinosus. We show median and quartiles.

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VA, χ2 = 9.72, N = 5, p < 0.02 for both host species). The rise in running activity was more pronounced in T. longispinosus than in T. curvispinosus colonies (Mann-Whitney U tests, p > 0.2 for the ‘control before’, ‘control after’, and ‘trial before’ categories; ‘gland treatment’: U = 2.61, p < 0.01). T. curvispinosus workers groomed themselves significantly more often after the gland treatment (Fig. 1b, χ2 = 9.77, p < 0.02), which was not evident in T. longispinosus (χ2 = 3.48, ns). Workers of both host species more frequently antennated their nestmates in response to release of the propaganda substance (Fig. 1c, data pooled over both species: χ2 = 10.26, N = 10, p < 0.02), and showed a higher level of aggression indicated by more frequent mandible-opening (Fig. 1d, χ2 = 28.71, N = 10, p < 0.0001). However, fighting among nest mates, including biting or stinging as a reaction to the release of the parasite gland content in the colony was observed in only two of the 10 trials. Comparison with Dufour’s glands of other species The origin of the gland with which the nestmate was treated had a highly significant influence on the host colony’s response (Fig. 2, repeated measures ANOVA, F = 26.68, N = 10, p < 0.0001). T. longispinosus colonies showed virtually no aggression against nestmates treated with water (controls), as well as to glands of infertile T. longispinosus and T. curvispinosus workers (Fisher LSD test, control – T. l.: p = 0.93, control – T. c.: p = 0.92). In contrast, glands of fertile host females and of P. americanus workers and queens elicited more hostile reactions (Fisher LSD test, p < 0.001 in all comparisons against control). Host workers smeared with H. sublaevis glands received intermediate levels of aggression (Fig. 2, Fisher LSD test, H. s. – control: p < 0.01; H. s. – P. a. queens: p = 0.17; p < 0.001 in all other comparisons). Chemical analyses As emerges from Figure 3, the composition of Dufour’s gland extracts differed markedly between the seven species/ caste categories. Preliminary mass spectrometry could identify some of the peaks in the GC-profiles; the known substances are listed in Table 1. The most prominent compounds in the glands were alkanes, alkenes and dienes (range C15C33). In accordance with an earlier study (Ollett et al., 1987), GC-MS analysis of H. sublaevis Dufour’s glands additionally yielded farnesene. In the Multiple Correspondence Analysis (Fig. 4), glands of H. sublaevis workers are separated from all other samples by a large distance on the first axis, whereas P. americanus and its T. longispinosus hosts diverge mainly in their scores on the second axis. In this qualitative analysis of the peaks detected in the GC runs, P. americanus queens and workers could not be separated, but their chromatograms suggest that these two groups would be distinguishable by information on the relative amounts of the different compounds. T. longispinosus workers were characterized by a different gland

Propaganda substances in slave-making ants

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Figure 2. Response of T. longispinosus colonies to nestmates smeared with Dufour’s glands of different species and castes. The sum of aggressive acts (mandible opening, biting, stinging and dragging) is shown, standardized for the number of interactions with nestmates. Mean ± SD are given. The letters above the bars indicate significance groups.

composition than conspecific queens; however, there was no evident correlation between worker’s ovarian status and the composition of their Dufour’s gland secretion (Fig. 3), and fertile and infertile workers consequently cluster together in the MCA plot (Fig. 4). Discussion Our results demonstrate that the slave-maker Protomognathus americanus, as has been suggested from behavioural observations of slave raids (Wesson, 1939; Alloway, 1979; Alloway and Del Rio Pesado, 1983), indeed possesses a ‘propaganda substance’ that may aid slave-maker workers during the attack on a host colony. We found that the contents of P. americanus Dufour’s glands, when released directly in the nest, cause agitation and confusion in Temnothorax longispinosus and T. curvispinosus colonies. Furthermore, host workers treated with slave-maker Dufour’s gland extracts were heavily attacked by their nestmates. As Dufour’s glands of T. curvispinosus workers did not evoke a similarly aggressive response when applied to T. longispinosus, this finding cannot be attributed to a generally hostile reaction towards heterospecific compounds. It is thus likely that P. americanus – like several other slave-making species – has evolved a chemical weapon in the arms race with its host species. In contrast to the closely related European slave-maker H. sublaevis, which has been observed to smear host workers with its Dufour’s gland secretion (Buschinger, 1974), P. americanus does not directly apply its propaganda substance, but probably releases it into the host nest (Alloway, 1979; Buschinger et al., 1980; Alloway and Del Rio Pesado, 1983). In accordance with these different

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Retention time (min) Figure 3. Gas chromatograms of Dufour’s gland contents of T. longispinosus infertile and fertile workers and queens, of P. americanus workers and queens, and of H. sublaevis females. Peaks that could be identified by GC-MS are labelled with numbers; a list of these compounds is given in Table 1. Peaks labelled with x are unidentified substances that were specific for a particular profile.

usage modes of the allomones, our behavioural experiments indicate that the propaganda substance in P. americanus is not identical to that of H. sublaevis; otherwise the Dufour’s gland of the latter species should have provoked a much stronger reaction. This view is corroborated by the preliminary analysis of the gland content’s composition: Although based on low sample sizes and identification of only a few of the detected compounds, our data clearly suggests that the two slave-maker species indeed produce different substances. Considering that Dufour’s glands contents appear to be highly variable among ant species and exhibit an extreme diversity of chemical compounds (e.g., Ali et al., 1987, 1989; Ollett et al., 1987; Bagnères et al., 1991; Bestmann et al.,

1995; Visicchio et al., 2000; Morgan et al., 2003), glandular secretions are probably very versatile and can undergo rapid evolutionary changes. The propaganda substance of P. americanus thus likely represents a convergent phylogenetic development. The slave-making habit has apparently evolved independently more than ten times (D’Ettorre and Heinze, 2001), and this specialized lifestyle has produced a striking number of convergently developed traits in morphology and behaviour. Remarkably, it appears that chemical warfare has been evolved independently several times as well. Exactly which aspect of host social behaviour does the propaganda substance exploit? The answer is not straightforward, as the function of the Dufour’s gland varies greatly in

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Propaganda substances in slave-making ants

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Pentadecane Heptadecadiene Heptadecene Heptadecane m/z (%): 69 (100); 83 (46); 95 (39); 107 (28); 121 (28); 137 (23); 163 (23); 181 (8); 189 (4); 207 (18); 250 (1) m/z (%): 69 (100); 81 (33); 95 (36); 107 (27); 121 (26); 137 (22); 163 (21); 181 (4); 189 (4); 231 (3); 249 (2); 318 (7) m/z (%): 69 (100); 83 (34); 95 (52); 109 (38); 121 (29); 137 (28); 151 (25); 163 (18); 193 (6); 245 (7); 263 (7); 332 (