Cuticular hydrocarbons mediate discrimination of reproductives and nonreproductives in the gulosa

Vincent Dietemann*†, Christian Peeters‡,Ju¨ rgen Liebig*, Virginie Thivet*, and Bert Ho¨ lldobler*§

*Lehrstuhl Verhaltensphysiologie und Soziobiologie, Theodor-Boveri-Institut, am Hubland, D-97074 Wu¨rzburg, Germany; and ‡Laboratoire d’Ecologie, Centre National de la Recherche Scientifique Unite´Mixte de Recherche 7625, Universite´Pierre et Marie Curie, 7 Quai Saint Bernard, 75005 Paris, France

Contributed by Bert Ho¨lldobler, July 9, 2003 In many of social , the cuticular hydrocarbons of produce unfertilized, male-destined eggs in the presence of their adults vary with both colony identity and individual physiology queen, they are attacked by nestmates (25), which indicates that (oogenesis). Such variations have been shown in some and fertile workers of M. gulosa are recognized as already demon- social wasps to function in nestmate recognition, but as yet there strated in other ants (26–28). is no demonstration of their use by workers to recognize egg The ability of workers to discriminate between individuals of layers. We report that in the ant Myrmecia gulosa, workers can contrasting reproductive status enabled us to use bioassays to discriminate queens and fertile workers from infertile individuals investigate the underlying chemical mechanism. We first verified based on distinctive blends of long-chained hydrocarbons present the correlation between CHCs and ovarian activity in M. gulosa. both on the cuticle and in the postpharyngeal gland. The purified These HCs were extracted from the cuticle as well as the hydrocarbon fraction of cuticular extracts from queens elicited postpharyngeal gland (PPG), where CHCs accumulate during high interest in workers, unlike the nonhydrocarbon fraction. self-grooming (refs. 29–33 and R. Beard and V.D., unpublished However, both fractions were necessary to trigger a response of data). The extracts from fertile queens, workers laying repro- maximal intensity. In contrast, extracts of mandibular and Dufour ductive eggs, and infertile workers were transferred on glass glands from queens or infertile workers were not treated differ- slides that were presented to nestmate workers. If these extracts entially by workers. We suggest that cuticular hydrocarbons func- elicit different amounts of attention, then workers are able to tion as pheromones allowing for recognition of the queen as well recognize different blends of CHCs and use them to discriminate as egg-laying workers. reproductive from nonreproductive individuals.

n societies chemical communication plays essential Methods Iroles. At the group level, individuals must recognize colony Ant Collection and Laboratory Rearing. Complete nests of M. gulosa membership to avoid exploitation of resources by nonnestmates. were excavated in October 1999 and 2000 from a sandstone area At the individual level, the presence of a reproductive must be near Waterfall, , Australia. For practical detected by the infertile nestmates to maximize their reproduc- reasons, the number of individuals brought back for laboratory tive interests (e.g., ref. 1). In addition to their implication against rearing was reduced; together with the queen and all the eggs, desiccation (2–5), long-chained hydrocarbons (HCs) present on 500 workers and up to 250 larvae and 100 cocoons were selected the cuticle seem to play a major role in both these identification randomly. The ants were kept in plaster-of-Paris nests into which mechanisms. In solitary insects, cuticular HCs (CHCs) can be sex chambers had been molded. A glass plate covered each chamber attractants or used for species recognition (reviewed in refs. 2, to allow for observations and removal of individuals. The nests 3, and 6). In social insects, CHCs are known to be colony-specific were connected to foraging arenas where food (pieces of cock- (e.g., refs. 7 and 8) and in some ants were demonstrated to be roaches or entire crickets and honeywater) was deposited every the basis of nestmate recognition (9–11). In the latter studies, 1–2 days. The temperature was maintained at 24 Ϯ 1°C, the the purified HC fraction of cuticular extracts from workers photoperiod was set at 10:14-h (light͞darkness) cycles, and a was applied on live ants or artificial lures, and the response high humidity was maintained inside the nests by regularly of nestmates or nonnestmates was modified according to the moistening the plaster. origin of the extracts to which they were exposed. Nestmate and nonnestmate extracts elicited weak and strong aggression, Extraction of CHCs and GC. Solid-phase microextraction (34) was respectively. used to extract CHCs from live individuals (17). The ants were CHCs also correlate with ovarian activity in various solitary immobilized by blocking their postpetiole in a split cardboard, (12–14) and social (7, 8, 15–22) insects. It has been shown that CHC which reduced the mobility of the ant’s gaster and allowed the profiles shift in a predictable manner after either the onset or a rubbing of a solid-phase microextraction fiber (SUPELCO, decline in egg-laying activity. CHCs thus encode reliable informa- Bellefonte, PA, coated with a 7-␮m polydimethylsiloxane film) tion about reproductive physiology and have been suggested to on the tergites. The rubbing was performed for 30 sec in a function in the recognition of fertile and infertile nestmates (refs. standardized manner. The fiber then was inserted in the injection 18–23 and V. Cuvillier-Hot, A. Lenoir, R. Crewe, C. Malosse, and

port (260°C) of a Carlo Erba 8130 gas chromatograph equipped EVOLUTION C.P., unpublished data). However, bioassays are needed to dem- with a DB-1 nonpolar capillary column (30 m ϫ 0.32 mm ϫ 0.25 onstrate that ant workers can detect variations in CHC profiles. ␮m,J&WScientific, Folsom, CA). Helium was used as a carrier Myrmecia gulosa belongs to the phylogenetically primitive ant gas with a column head pressure of 95 kPa. Samples were run in subfamily . Queens are morphologically specialized relative to the workers. Their ovaries are bigger (44 ovarioles compared with 8–14 in workers), and thus they can lay more eggs Abbreviations: HC, hydrocarbon; CHC, cuticular HC; PPG, postpharyngeal gland. (24). A retinue of workers constantly surrounds the queen, and †Present address: Zoology and Entomology Department, Pretoria University, Pretoria 0002, in her presence, workers do not reproduce but lay unviable South Africa. trophic eggs (24). Queens thus monopolize reproduction and are §To whom correspondence should be addressed. E-mail: [email protected] clearly recognized. When workers are experimentally induced to wuerzburg.de.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.1834281100 PNAS ͉ September 2, 2003 ͉ vol. 100 ͉ no. 18 ͉ 10341–10346 Downloaded by guest on October 1, 2021 the splitless mode. The column temperature was maintained at tested against those of infertile individuals. These workers 60°C for 4 min and then raised to 250°C at a rate of 20°C⅐minϪ1 originated from groups of 100–200 individuals without the and from 250°Cto300°C at 2.5°C⅐minϪ1. Flame ionization queen. These groups were created by randomly choosing workers detector temperature was set at 310°C. from all chambers of the nests to avoid biasing the sample toward We extracted CHCs of 12 mated queens from 12 colonies as particular age or task classes. M. gulosa egg layers are difficult to well as CHCs of 12 virgin queens from 2 colonies. The queen- identify by direct observations. Some workers present near the right workers measured (n ϭ 12) originated from five colonies, egg pile had a protruding sting sheath and stood high on their and the workers laying reproductive eggs (n ϭ 12) originated legs, suggesting they were about to oviposit. To confirm this, they from five orphaned groups of workers. Because worker repro- were taken out of the nest and individually isolated overnight in duction only occurs in orphaned situations (24), we also ex- a plastic box with moist cotton. On the next day, the presence of tracted 10 trophic egg-laying workers from two orphaned colo- eggs in the box was checked. The workers that did not lay eggs nies to separate the effects of queen loss and of reproductive were not used in the experiment. The ovarian status of all the activity on the CHCs of workers. workers was verified later by dissection. The solid-phase microextraction͞GC analysis yielded 92 For the bioassays, we used one of the inhabited chambers peaks. The number of variables to be used in multivariate (90 ϫ 110 mm) of the rearing nests to provide a biologically statistical analysis was reduced (see ref. 18). We selected the relevant context. After blocking access to this chamber with a peaks that represented Ͼ0.3% relative peak area and that were cotton ball, it was emptied from its workers. Ten large and 10 present in all the individuals. The relative areas of the 17 selected small workers were selected randomly and left in the chamber peaks were restandardized to 100% and transformed following for 10 min to calm down. One-tenth of the gland extracts of Aitchison’s formula (35), either queen͞infertile worker or trophic͞reproductive egg layers was then applied on two 18 ϫ 18-mm glass cover slides ϭ ͓ ͞ ͑ ͔͒ Zij ln Yij g Yj , cleaned with pentane. After solvent evaporation, the cover slides were introduced in the middle of the chamber without where Zij is the standardized peak area i for individual j, Yij is the disturbing the ants. The slides were placed 4 cm apart from peak area i for individual j, and g(Yj) is the geometric mean of each other. The extracts were tested only with nestmates of the all peaks for individual j. The homogeneity of variance of these extracted individuals. The extracts were presented to 6–13 variables was tested with Levene’s test, and Bonferroni’s cor- different groups of 20 queenright nestmate workers in each of rection was applied. Only 13 variables had homogeneous vari- three colonies. In the queen vs. infertile worker bioassay, the ances and were considered. The transformed areas were used as same extracts were used in six repetitions (because only one variables in a principal component analysis. The four principal queen is available per nest), whereas in the reproductive vs. components extracted then were used as variables in a discrimi- infertile worker bioassay, a different extract was used for each nant analysis. This procedure does not allow the identification of repetition. The bioassays were done in the 48 h after the the discriminating compounds. A stepwise analysis was not extraction of queens and workers. Controls consisted of two performed, because the proportion of describing variables vs. cover slides on which 5 ␮l of hexane was applied. They were sample size was too large to fulfill the statistical requirements for presented to workers as described above. Gland extracts and this procedure. controls were presented to each group of workers in sequences In M. gulosa a large proportion of both large and small workers of randomized order. produce trophic eggs in the presence of their queen; these fragile The interest of workers was quantified as the cumulated yolk sacs without a rigid chorion are mostly given to larvae (24). number of antennal inspections received by the extracts or To determine whether the cuticular profiles varied with the rate controls during 5 min. An inspection started as an individual’s of trophic egg laying, two categories of workers were extracted: antennae touched the extract and was deemed to finish once the workers without oocytes or with only submature trophic oocytes antennae left the vertical projection area of the cover slides. The (n ϭ 9) and workers with mature trophic oocytes in most position of the two extracts tested was alternated for each ovarioles (n ϭ 9). Their cuticular profiles were compared with repetition by using a blind protocol. The cumulated numbers of those of the orphaned reproductive workers used in the previous antennal inspections were compared with a Wilcoxon test for analysis, but 18 compounds were selected and three principal paired samples for each colony. components were used in a discriminant analysis. Bioassays of Polar vs. Nonpolar Cuticular Compounds. HCs are the Bioassays of Glandular Extracts. Individuals were freeze-killed at major lipid constituents of insect epicuticle, but non-HC polar Ϫ70°C for 10 min, and PPGs, Dufour glands, and mandibular lipids are also present (cf. refs. 2 and 4). Non-HCs are found only glands were dissected out after thawing. The glands were excised in traces in M. gulosa and do not differ among individuals of in Ringer’s solution with pentane-cleaned forceps and stored in different morphological caste or reproductive status (R. Beard glass vials. The vials were frozen at Ϫ70°C for several hours, and and V.D., unpublished data). However, we tested the biological after1hofthawing, 100 ␮l of hexane was added. The vials then activity of polar and nonpolar constituents separately. The were placed in an ultrasonic bath for 5 min to release the procedure of Lahav et al. (9) to fractionate lipid extracts into HC chemicals they contained as completely as possible. The long- and non-HC compounds was adapted for M. gulosa. Total lipid chain HCs were extracted from either the cuticle or the PPGs. extracts (37) were obtained from the cuticle of single queens The PPGs store CHCs in several ant species (29–33), and large (after freeze-killing but before dissection, see above) and pools quantities of compounds can be collected from the reservoirs of five nestmate workers. Pools of several workers were used (31, 36). Poison glands of queens were always degenerated and because the queen produces more CHCs than individual workers empty (n ϭ 12; V.D., unpublished data) and thus were excluded (25). The extracts were evaporated and rediluted in 200 ␮lof from this study. hexane. These solutions were loaded onto a silica gel column We compared the interest triggered by queen vs. infertile (Chromabond 500 mg, Macherey & Nagel) and eluted with 10 ml worker extracts of each gland. In M. gulosa, most workers of hexane to obtain the HC fraction and then with 4 ml of produce unviable trophic eggs in the presence of their queen hexane͞chloroform (1:1), 5 ml of chloroform, and 5 ml of (24). For extraction, we selected workers that had just laid a methanol to obtain the more polar non-HC components. The trophic egg. fractions obtained were left to evaporate under a fume hood. Gland extracts from orphaned reproductive workers were also The HC fractions were rediluted in 200 ␮l of hexane, and all the

10342 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1834281100 Dietemann et al. Downloaded by guest on October 1, 2021 Fig. 2. Discriminant analysis of five categories of individuals based on the relative proportions of 13 cuticular compounds. Reproductive (filled symbols) and nonreproductive (open symbols) individuals are separated completely. The groups are encircled arbitrarily. Solid lines indicate individuals present in queenright colonies and dotted lines designate orphaned workers.

individuals. In addition to these conspicuous differences, a discriminant analysis with 13 other compounds (peaks num- bered in Fig. 1) revealed that each category of individuals has typical blends of these HCs [Wilk’s ␭ ϭ 0.03, F(16,153) ϭ 21.7, P Ͻ 0.01; Fig. 2]. All individuals able to lay reproductive eggs had similar CHC profiles regardless of morphological caste. Although all categories were statistically separated, virgin queens and infertile workers (both queenright and orphaned) clustered together, whereas fertile queens and workers laying reproductive eggs formed a second group (Fig. 2). These reproductive and nonreproductive poles were separated by the first discriminant function explaining 93.6% of the variance. Fig. 1. Chromatograms of CHCs of M. gulosa obtained by solid-phase Within these poles, misclassification occurred, but the model microextraction. (a) Fertile queen. (b) Worker laying reproductive eggs. (c) permitted the correct classification of 87.9% of the individuals. Infertile worker. Two compounds [9-pentacosene (p) and 3-methylpentaco- In M. gulosa, many of the infertile workers exhibit a limited sane (m)] clearly differentiate reproductives from nonreproductives. Used in degree of ovarian activity that allows them to lay trophic eggs. A the statistical analysis are pentacosane (peak 1), heptacosane (peak 2), discriminant analysis with 18 compounds [Wilk’s ␭ ϭ 0.09, 9,11,13,15-methylheptacosane (peak 3), 10,11,12,13,14-methyloctacosane F(6,50) ϭ 19.92, P Ͻ 0.01] showed that individuals with high and (peak 4), nonacosene (peak 5), nonacosane (peak 6), 9,11,13,15-methylnona- ϭ cosane (peak 7), 3-methylnonacosane (peak 8), hentriacontane (peak 9), low ovarian development could not be separated (P 0.36) but 9,11,13,15-methylhentriacontane (peak 10), 9,11,13,15-methylheptatriacon- that their cuticular profiles were very distinct from those of tane (peak 11), and unknown (peaks 12 and 13). workers laying reproductive eggs (P Ͻ 0.01 in both cases).

Gland Extracts and Queen Recognition. In all three colonies studied ␮ ͞ others were pooled in 200 l of chloroform methanol (1:2). The (I–K), the extracts of the queens’ PPGs triggered significantly ϭ quality and yield of the fractioning process were verified by GC more interest than those of the infertile workers (TI 0.0 and ϭ ϭ ϭ ϭ ϭ analysis. pI 0.03; TJ 0.0 and pJ 0.03; TK 1.0 and pK 0.046) (Fig. One hundredth of the queen extracts and a volume of worker 3). In contrast, the Dufour and mandibular gland extracts of extracts containing the equivalent quantity of HCs were used in queens were each preferred in only one colony (Wilcoxon test, ␮ ϭ ϭ ϭ ϭ the bioassays. Extracts in hexane (5 l) and hexane controls (5 n 6 in all cases, Dufour gland: TI 6.0 and pI 0.35; TJ ␮ ϭ ϭ ϭ l) were applied on glass cover slides, and the interest of 1.0 and pJ 0.046; TK 5.0 and pK 0.25; mandibular gland: ϭ ϭ ϭ ϭ ϭ nestmate workers was quantified and compared as described TI 10.0 and pI 0.92; TJ 8.5 and pJ 0.68; TK 1.0 and

ϭ EVOLUTION above. pK 0.046) (Fig. 3).

Results HC Fraction of Cuticle Extracts and Queen Recognition. Workers HC Profiles and Reproductive Status. The CHCs of M. gulosa are showed more interest in the total lipid extracts of queens’ cuticles a complex mixture of alkanes, alkenes, and methyl-branched than in those of the infertile workers (Wilcoxon test, n ϭ 6inall ϭ ϭ ϭ ϭ ϭ alkanes ranging from C23 to C39 (R. Beard and V.D., unpub- cases: TI 0.0 and pI 0.03; TJ 0.0 and pJ 0.03; TK 0.0 ϭ lished data). Two compounds (9-pentacosene and 3-methyl- and pK 0.03) (Fig. 4). The same was true for the purified HC pentacosane; Fig. 1) were present in high proportions (range fraction from these extracts (Wilcoxon test, n ϭ 6 in all cases: ϭ ϭ ϭ ϭ ϭ 12–43%) in reproductive individuals (both queens and work- TI 0.0 and pI 0.03; TJ 0.0 and pJ 0.04; TK 0.0 and ϭ ers) but were absent or in trace quantities in nonreproductive pK 0.03). No preference was shown when the non-HC fractions

Dietemann et al. PNAS ͉ September 2, 2003 ͉ vol. 100 ͉ no. 18 ͉ 10343 Downloaded by guest on October 1, 2021 Fig. 4. Bioassay of cuticular extracts from queens and infertile workers. Twenty workers were exposed to 1͞100th of cuticular extracts of nestmate queens and to the matching quantity of nestmate worker extracts. Total lipid extracts as well as their HC and non-HC fractions were tested. We monitored the preference of the workers for each extract in terms of cumulated number of antennal inspection in 5 min (* indicates significant Fig. 3. Bioassay of glandular extracts from queens and infertile workers. differences, Wilcoxon test, P Ͻ 0.05). Five microliters of hexane applied on Twenty workers were exposed to 1͞10th of mandibular, Dufour, and PPGs two cover slides constituted the solvent control. Because the attention extracts in each of three colonies. The cumulated number of antennal elicited was not significantly different for the two control slides (Wilcoxon inspections elicited in 5 min was compared with the Wilcoxon test (* test), results were pooled for clarity. The number of replications was six for indicates significant differences, P Ͻ 0.05). Five microliters of hexane each category. applied on two cover slides constituted the solvent control. Because the attention elicited was not significantly different for the two control slides (Wilcoxon test), results were pooled for clarity. The number of replications Gland Extracts and Recognition of Reproductive Workers. was six for each category. The com- parison of gland extracts from infertile workers and reproductive workers gives similar results to those for queen recognition. Only of the queen and infertile worker extracts were compared the PPG extracts allowed discrimination between the two cate- ϭ ϭ ϭ ϭ (Wilcoxon test, n 6 in all cases: TI 7.0 and pI 0.46; TJ gories. Reproductive worker PPG extracts elicited significantly ϭ ϭ ϭ 3.0 and pJ 0.47; TK 3.0 and pK 0.23). The intensity of more interest in nestmates than PPG extracts from infertile ϭ ϭ Ͻ antennation directed at the queen extracts was higher for the workers (Wilcoxon test, n 9–13, PPG: TI 0.0 and pI 0.01; ϭ ϭ ϭ ϭ total lipid extract compared with the HC fraction (Mann– TJ 4.5 and pJ 0.01; TK 5.5 and pK 0.04; Dufour gland: ϭ ϭ ϭ ϭ ϭ ϭ ϭ Whitney test, ntotal lipids 6 in all cases and nHC 6 in all cases: TI 12.0 and pI 0.74; TJ 28.5 and pJ 0.69; TK 7.5 and ϭ ϭ ϭ Ͻ ϭ ϭ ϭ ϭ ϭ UI 5.0 and pI 0.04; UJ 5.0 and pJ 0.01; UK 2.5 and pK 0.14; mandibular gland: TI 22.0 and pI 0.95; TJ 22.0 ϭ ϭ ϭ ϭ pK 0.01) (Fig. 4). and pJ 0.58; TK 12.5 and pK 0.23).

10344 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1834281100 Dietemann et al. Downloaded by guest on October 1, 2021 Discussion CHCs are important in the regulation of reproduction. None of CHCs Correlate with Reproductive Status. Variations in the CHCs the other gland products tested in this study (mandibular and of workers and queens in M. gulosa reflect individual repro- Dufour glands) unambiguously allowed for the recognition of ductive status. Distinct CHC profiles were always associated queens or reproductive workers. with reproductive activity in both morphological castes. Two compounds (both with 25 carbon atoms) were characteristic of CHCs as Queen Pheromone. Until recently, the origin of ant queen reproductives. The workers that shifted to the production of pheromones was only sought in exocrine glands, which often reproductive eggs also produced these molecules together with release their products on the cuticle. The excision of gland proportions of other CHCs typical of the fertile queens (Fig. reservoirs allows for collection of these products in large 2). Although the reproductive workers of M. gulosa cannot quantities for bioassays. However, only in fire ants and the mate, the resemblance of their cuticular profiles to those of the honey have queen pheromones been identified from queens is similar to that found for the gamergates and fertile glands (e.g., refs. 39–42). In contrast, numerous studies dem- queens in the ponerine ant Harpegnathos saltator (18). These onstrated the existence of pheromones on the cuticle of queens intracolonial variations are independent of age, because the but failed to locate their glandular source (e.g., refs. 43–47). age structure of orphaned and queenright groups was ran- This can be explained in the light of our findings. CHCs are domized in our experiments. Orphaned infertile workers kept produced by the oenocytes (48, 49), which are glandular cells a profile similar to that of queenright workers, showing that spread in the body cavity; the secretions are released in their changes in CHCs are not caused by our manipulations. Fur- hemolymph and transported to the cuticle (2, 48–54). The thermore, the profiles of fertile and virgin (infertile) queens absence of a structured reservoir storing CHCs has hindered did not overlap. Thus, as in other social (see the the investigation of this pheromonal source. Only two studies introduction), CHCs of M. gulosa encode reliable information envisaged the origin of queen pheromones to be epidermal about fertility. gland cells (45, 46). The possibility that CHCs could function Although fertility has a striking effect on the CHCs, workers as queen or gamergate (mated workers) recognition phero- laying trophic eggs had different cuticular profiles than those of mones was suggested only recently (18–23). reproductives. Our study of the variations of CHCs in an ant CHC profiles correlate with ovarian activity in a growing producing trophic eggs gives insight into which physiological number of species investigated (see the introduction). Their use traits affect the synthesis of CHCs. as queen-recognition pheromones thus could be widespread in the social Hymenoptera. Perception of the poorly volatile long- Recognition of Reproductive Status. In colonies of M. gulosa, chained CHCs necessitates direct contacts between emitters and queens and reproductive workers are recognized by their nest- receivers. Indeed in M. gulosa, direct contact is needed for the mates in which they elicit high interest; a retinue is formed workers to perceive the presence of the queen (25). With around queens, whereas workers that start reproducing are often increasing colony size, however, the probability of a worker surrounded by nestmates and immobilized (25). We bring evi- encountering the queen diminishes. In species with larger col- dence that these behaviors can be based on the detection of onies, queen pheromones can be transmitted efficiently in an specific CHC profiles. Indeed, cuticular compounds from the indirect manner via other workers or the substrate, as occurs in PPG of queens and reproductive workers elicited more interest the honey (55, 56). Alternatively, more volatile secretions in nestmates than those of infertile workers. In M. gulosa,asin might be needed in addition to the queen’s CHCs to attract other ants studied, the PPG content closely reflects the CHC workers to her immediate vicinity. profile of an individual, and PPG contents of queens and infertile and reproductive workers vary (R. Beard and V.D., unpublished Intracolonial Variation in CHC Profiles and Colonial Odor. The ability data). Furthermore, the HC fraction of the queen cuticular lipid of ant workers to use variations in CHC profiles to identify extracts played the major role in eliciting interest. The non-HC reproductives is in apparent contradiction to the Gestalt model fraction on its own lacked such effect, but its presence together for nestmate recognition. Indeed, it is claimed that all nestmates with the HC fraction increased the intensity of the nestmate share a colonial odor as a result of mixing their CHC profiles (29, workers’ response. Indeed, the total lipid extract (HC ϩ non-HC 30, 36, 57–59). Our results and others clearly show that this is not fractions) elicited more antennal inspections than the HC frac- the case because reproductives have a specific profile. Further- tion alone. Non-HC compounds represent only a small propor- more, the molecules characterizing reproductives are the same in tion of the cuticle constituents and are similar in all individuals all colonies. In most of the ants investigated, CHC profiles are irrespective of morphological caste or reproductive status a complex mixture of dozens of compounds. The presence or (R. Beard and V.D., unpublished data). They may therefore only absence of some compounds or the variations in their relative have made the glass slides smell more ant-like and thus more proportion allow for enough combinations to encode both kinds attractive in our experiment. Although the activity of both of information. It has already been demonstrated that nestmate fractions was not tested separately for the PPG extracts of queens or their extracts were preferred over nonnestmate queens reproductive workers and infertile workers, the HC fraction in several ant species (43, 59–63). Similarly, in M. gulosa, extracts presumably also plays the major role in their discrimination. of nestmate queens were preferred over those of foreign ones As hypothesized in various social Hymenoptera (see the (25). It is thus likely that CHCs are used as both reproductive and introduction), workers of M. gulosa detect changes in CHC colony labels. Determining the specific cuticular compounds profiles and use this information to discriminate reproductive involved in the recognition of either nestmates or reproductives EVOLUTION from nonreproductive nestmates. According to the signaling is the next step for future studies. hypothesis, fertile queens produce honest signals to which work- ers respond adaptively by refraining from reproduction (38). We We thank Richard Beard, Abraham Hefetz, and Graeme Jones for suggest that the CHCs represent such a signal by allowing the chemical analyses and helpful discussions, and Michael Schwarz, Katja Hogendoorn, Remko Leijs, Steve Shattuck, Archie McArthur, and workers to detect the presence of the queen. Further experi- Russell for hospitality and assistance during fieldwork. Thomas Seeley ments are needed to show that CHCs also have primer effects in and Stefano Turillazzi made helpful comments on the manuscript. This workers. However, the fact that changes in workers’ CHCs work was funded by the Deutsche Forschungsgemeinschaft SFB 554 correlate with the triggering of attacks as well as with the onset (C3) and the Graduiertenkolleg ‘‘Grundlagen des Arthropodenver- of reproductive activity in this species (25) supports the idea that haltens.’’

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