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Disentangling environmental and heritable nestmate recognition cues in a carpenter ant
Journal of Insect Physiology 55 (2009) 159–164
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Disentangling environmental and heritable nestmate recognition cues in a carpenter ant Jelle S. van Zweden 1,*, Stephanie Dreier 1, Patrizia d’Ettorre Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 September 2008 Received in revised form 3 November 2008 Accepted 3 November 2008
Discriminating between group members and strangers is a key feature of social life. Nestmate recognition is very effective in social insects and is manifested by aggression and rejection of alien individuals, which are prohibited to enter the nest. Nestmate recognition is based on the quantitative variation in cuticular hydrocarbons, which can include heritable cues from the workers, as well as acquired cues from the environment or queen-derived cues. We tracked the profile of six colonies of the ant Camponotus aethiops for a year under homogeneous laboratory conditions. We performed chemical and behavioral analyses. We show that nestmate recognition was not impaired by constant environment, even though cuticular hydrocarbon profiles changed over time and were slightly converging among colonies. Linear hydrocarbons increased over time, especially in queenless colonies, but appeared to have weak diagnostic power between colonies. The presence of a queen had little influence on nestmate discrimination abilities. Our results suggest that heritable cues of workers are the dominant factor influencing nestmate discrimination in these carpenter ants and highlight the importance of colony kin structure for the evolution of eusociality. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Ants Camponotus aethiops Cuticular hydrocarbons Kin recognition Multi-component signals
1. Introduction Nestmate recognition in social insects ensures that altruistic behavior is directed to the appropriate recipients; usually related individuals. By excluding non-nestmates from their nest, social insects avoid robbery and parasitism of their valuable resources (Wilson, 1971). When two individuals meet, they assess if the other is a nestmate or non-nestmate on the basis of chemical cues. In the case of non-nestmates they usually exhibit aggressive behavior; otherwise they may cooperate (Ho¨lldobler and Michener, 1980). In most social insects, and especially in ants, the recognition cues are primarily encoded in the cuticular hydrocarbon profile (Bonavita-Cougourdan et al., 1987; Lahav et al., 1999; reviewed by Lenoir et al., 1999). Individuals within one social insect species have approximately the same qualitative profile of hydrocarbons (the so-called colony odor), but these differ in their relative proportions depending on the colony of origin of the individual (e.g. Bonavita-Cougourdan et al., 1987; Espelie et al., 1990). With the exception of some ants, wasps and bees with small colony sizes (e.g. Buckle and Greenberg, 1981), the colony odor is thought to be
* Corresponding author. Tel.: +45 3532 1238; fax: +45 3532 1250. E-mail address: [email protected] (J.S. van Zweden). 1 These authors contributed equally to this work. 0022-1910/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2008.11.001
homogenized among nestmates by grooming and body contact alongside mixing of hydrocarbons in the postpharyngeal gland, which are then exchanged by trophallaxis together with regurgitated liquid food (Soroker et al., 1994; Meskali et al., 1995). This homogenization facilitates recognition by diminishing the possibility for errors (Reeve, 1989; Sherman et al., 1997). Among the different structural classes of cuticular hydrocarbons (linear alkanes, branched alkanes and alkenes), not all are thought to have equally important roles in nestmate recognition. It has been shown that linear alkanes are less important (Akino et al., 2004; Martin et al., 2008; but see Greene and Gordon, 2007), which has been attributed to their lack of discriminative features; linear alkanes can only be discriminated on their chain length, whereas branched and unsaturated hydrocarbons could also be discriminated on the position of the methyl group or the double bond, respectively. Multiple sources have been proposed to influence the colony odor, including genetically determined odors from all the individuals of a colony (e.g. Wallis, 1962; Crozier and Dix, 1979; Ho¨lldobler and Michener, 1980), environmental odors, like nest material or food (e.g. Kalmus and Ribbands, 1952; Crosland, 1989; Jutsum et al., 1979; but see Downs et al., 2001), queen odors (e.g. Carlin and Ho¨lldobler, 1986) or combinations of these (e.g. Obin and Vander Meer, 1988; Obin and Vander Meer, 1989; Provost, 1991). In the paper wasp Polistes fuscatus females do not discriminate between nestmates and non-nestmates under
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homogeneous laboratory conditions, whereas they do under field conditions (Gamboa et al., 1986). Diet can influence nestmate recognition cues and abilities, for example in Argentine ants (Liang and Silverman, 2000). Therefore, if environmental odors are the dominant factor, we would expect that colonies under homogeneous environmental conditions would slowly stop to discriminate non-nestmates from nestmates. We tested this in Camponotus aethiops by tracking the cuticular hydrocarbon profile of workers for a year after field collection and transfer to homogeneous laboratory conditions. We then investigated which hydrocarbons are most affected by uniform laboratory conditions, which hydrocarbons are most likely to act as nestmate recognition cues and if the homogeneous conditions would affect ants’ nestmate recognition abilities. We also asked whether the presence of the queen had an influence on any of these factors. 2. Materials and methods 2.1. Study organisms Five colonies of C. aethiops, each containing between 100 and 300 workers, were collected in April 2006 in Moraduccio (448100 3000 N, 118290 600 E), on the border of Emilia Romagna and Toscana, Italy, and transported to Copenhagen, Denmark. In two of the colonies (Cae1 and Cae12) the queen could not be found, so these may or may not have been queenless before. One of the queenright colonies (Cae2) was split into a queenright (Cae2a) and a queenless half (Cae2b) right after collection, each with approximately 150 workers. The last two colonies (Cae6 and Cae9) remained queenright, so that we had three queenright and three queenless colonies altogether. In the laboratory each colony was kept in a nest consisting of two plastic boxes (26.5 cm 17.5 cm 8.0 cm) connected with a short plastic tube, and both were coated with Fluon1 (De Monchy, The Netherlands). One half was darkened with a lid, had a moist plaster floor and contained cotton-stoppered glass tubes filled with water, while the other half was uncovered and served as foraging area. The colonies were fed diluted honey and mealworms, Tenebrio molitor, thrice a week and were kept at room temperature and humidity. All colonies were kept in a climate room at 23 2 8C during summer and at 15 2 8C during winter, under 12:12 LD photoperiod. 2.2. Timeline From each of the six colonies, we randomly collected five workers for chemical analysis right after field collection and subsequently each time after 2 months, until approximately 12 months had passed (2006: 25 April, 27 June, 22 August, 23 October, 13 December; 2007: 6 February, 13 April). For simplicity, we will present some of the results only for the start of the study (T0 = 25 April 2006), after 6 months (T1 = 23 October 2006) and after 12 months (T2 = 13 April 2007), but we will specify per analysis if all results or only these three points are presented. To evaluate nestmate recognition abilities, aggression tests were performed on T1 and T2. 2.3. Chemical analysis Workers were killed by freezing at 20 8C and cuticular hydrocarbons were extracted by immersing individual workers in 200 ml HPLC-grade pentane (Sigma–Aldrich, Denmark) for 10 min, of which the first and the last 15 s involved gentle vortexing. The solvent was then let to evaporate at room temperature in a laminar flow cupboard. The extract was
resuspended in 100 ml pentane, of which 2 ml were injected in an Agilent 6890N gas-chromatograph, equipped with a HP-5MS capillary column (30 m 250 mm 0.25 mm), a split-splitless injector and a 5975 Agilent Mass Spectrometer with 70 eV electron impact ionization. The carrier gas was helium at 1 ml min1. After an initial hold of 1 min at 70 8C, the temperature rose to 200 8C at a rate of 30 8C min1, and then to 300 8C at 3 8C min1. The areas of 36 hydrocarbons (or mixtures of few hydrocarbons, due to co-elution) found on the cuticles of all workers were integrated for further analysis. These were used as variables in Principal Component Analysis (PCA) in the program LatentiX 1.00 for Windows. On T0, T1 and T2 we performed PCAs including all colonies (five colonies on T0, as Cae2a and Cae2b were still a single colony at this point, and six colonies on both T1 and T2), after which the PC scores were used for Discriminant Analysis (DA) in the program STATISTICA 7.1 (StatSoft Inc., USA), in order to determine if colonies could be significantly discriminated according to their hydrocarbon profile on these three points in time. The Mahalanobis distance between colonies was calculated, which was normalized according to the maximum Mahalanobis distance observed in the DA (relative chemical distance, hereafter), in order to allow comparison between DA’s. To calculate the diagnostic power (DP) for each peak, the standard deviation of the standardized peak area over all colonies was divided by the pooled standard deviation within these colonies (Christensen et al., 2005). Hence, compounds with the highest DP are most variable between colonies, but comparatively most consistent within colony, and are therefore the most likely to act as nestmate recognition cues. To investigate the relative length of hydrocarbons in the profile, weighted retention time was calculated by multiplying the average retention time of each peak with the relative area of that peak (i.e. the weight), and taking the sum of these values over the entire profile. Mean weighted retention time was calculated separately for queenright and queenless colonies for each point in time, after which these were compared using a repeated measures ANOVA in STATISTICA 7.1. 2.4. Aggression bioassays To test whether homogeneous conditions, inducing a change in the hydrocarbon profile, would affect the nestmate recognition capabilities of the workers, we conducted aggression bioassays between a subset of the nests after 6 months (T1) and 12 months (T2). We made 11 combinations of colonies to test against one another, so that we covered the variables colony origin (nestmates or non-nestmates) and queen presence (both queenright (QR), both queenless (QL), or queenright vs. queenless), and replicated each combination six times. Non-nestmate combinations included three QR–QR pairs (Cae2a–Cae6, Cae2a–Cae9 and Cae6–Cae9), two QL–QL pairs (Cae12–Cae1 and Cae12–Cae2b) and three QR–QL pairs (Cae2a–Cae1, Cae6–Cae12 and Cae1–Cae9). Nestmate (control) combinations included one QR–QR pair (Cae1–Cae1), one QL– QL pair (Cae6–Cae6) and one QR–QL pair (Cae2a–Cae2b). The same combinations were tested twice, once on T1 and once on T2. No ant was used more than once, the observer was the same person for T1 and T2, and the observer was blind with respect to the colony origin of the ants. In each aggression bioassay a dyadic encounter was staged in a Petri dish (15.2 cm) lined with a clean filter paper. The ants were confined by a cylinder coated with Fluon1 and were separated from each other by a smaller Fluon1-coated cylinder until the bioassay started. The ants were allowed to interact with each other for 3 min, throughout which we used the software Etholog 2.25 (Ottoni, 2000) to record the duration of the following behaviors with their aggression indices in
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Fig. 1. Chromatogram of the cuticular hydrocarbon profile of Camponotus aethiops. Identification of the peaks is provided in Table 1.
duration of the ith behavior, and T is the sum of the durations of all interactions. Relative importance of colony origin, queen presence, time and chemical distance on the observed aggression was tested with a General Linear Model (GLM) in the program SAS 9.1 for Windows.
brackets: investigation with antennae (0), mandibles open (1), bite (2), curl gaster forward (3). We then calculated an overall aggression level using the from Errard and Pnformula (modified Hefetz, 1997): AIoverall ¼ AI t =T, in which AIoverall is the i i i¼1 overall aggression level, AIi and ti are the aggression index and
Table 1 Diagnostic power and average percentage for each compound in the hydrocarbon profile over time. The diagnostic power (DP) is the standard deviation over all individuals (for T0, T1 and T2), divided by the pooled standard deviation within colonies (for T0, T1 and T2). % (S.D.) = mean percentage over all colonies S.D. of each peak in the total hydrocarbon profile (for T0, T1 and T2). Peak #
Identification
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
2-MeC24 C25 13- + 11- + 9-MeC25 7-MeC25 5-MeC25 11,15- + 9,13-diMeC25 7,13- + 7,15-diMeC25 + 3-MeC25 5,9- + 5,13- + 5,17-diMeC25 C26 3,13- + 3,11- + 3,9- + 3,7-diMeC25 13- + 12- + 11-MeC26 8-MeC26 + x,y-diMeC26 6-MeC26 2-MeC26 x,y-diMeC26 4,8-diMeC26 C27 2,y-diMeC26 13- + 11-MeC27 9-MeC27 7-MeC27 5-MeC27 11,15- + 9,13-diMeC27 7,15- + 7,13-diMeC27 + 3-MeC27 5,9- + 5,13- + 5,15- + 5,17-diMeC27 C28 3,15- + 3,13- + 3,9- + 3,7-diMeC27 14- + 13- + 12- + 10- + 8-MeC28 12,16-diMeC28 + 4-MeC28 C29 15- + 13- + 11- + 9-MeC29 7-MeC29 5-MeC29 13,17- + 11,15- + 9,13-diMeC29 7,17-diMeC29 + 3-MeC29 5,17-diMeC29
Mean S.D.
T0
T1
T2
DP
% (S.D.)
DP
% (S.D.)
DP
% (S.D.)
2.40 1.46 4.12 2.24 1.96 4.93 2.33 6.69 1.34 2.59 2.50 1.17 1.14 3.16 4.60 4.87 1.07 4.71 3.27 1.44 2.23 5.46 6.22 1.59 4.32 1.36 4.69 3.04 1.48 1.27 2.82 1.48 2.51 6.33 2.56 6.92
0.6 1.3 3.0 1.6 0.4 1.8 1.6 1.0 0.9 3.5 1.0 3.4 0.4 6.4 0.4 0.8 4.7 0.5 8.0 5.3 6.0 2.4 12.9 3.5 2.7 0.5 7.9 3.8 1.1 0.9 3.4 2.1 0.4 1.7 1.8 2.1
(0.1) (0.4) (1.3) (0.3) (0.7) (0.8) (0.8) (0.8) (0.3) (1.4) (0.4) (1.1) (0.7) (1.4) (0.2) (0.3) (1.3) (0.3) (1.8) (0.6) (1.1) (1.4) (5.4) (0.3) (1.2) (0.2) (2.6) (0.7) (0.6) (0.4) (1.0) (0.4) (0.2) (2.0) (0.5) (1.4)
1.33 1.25 2.88 2.86 1.39 1.12 1.34 2.57 1.01 3.10 1.67 1.73 0.99 1.25 1.84 1.80 1.18 2.51 2.33 1.26 1.19 3.55 1.59 1.09 4.87 1.20 1.60 1.09 2.41 1.77 1.15 0.96 2.02 3.08 1.28 2.15
0.6 2.2 2.3 1.0 0.3 1.2 1.3 0.7 2.1 2.3 0.8 2.0 0.2 7.1 0.4 0.4 9.6 0.4 8.0 5.2 6.2 2.7 10.2 3.2 2.4 1.9 5.2 3.2 1.2 2.8 4.7 2.7 0.8 1.5 1.4 1.7
(0.2) (0.8) (1.1) (0.4) (0.2) (0.3) (0.7) (0.5) (0.8) (0.9) (0.3) (0.6) (0.1) (1.2) (0.2) (0.3) (3.6) (0.2) (2.1) (0.9) (1.3) (1.2) (2.8) (0.5) (0.9) (1.0) (1.2) (0.6) (0.5) (2.0) (1.0) (0.4) (0.4) (0.8) (0.4) (0.9)
1.75 1.98 2.58 2.62 3.09 1.49 4.07 4.00 1.48 2.38 2.63 1.34 1.02 1.18 2.26 2.33 1.18 2.96 2.33 1.26 1.64 2.51 1.97 2.15 3.14 1.07 2.11 1.97 2.78 1.71 2.11 1.17 2.27 5.23 2.50 4.16
0.5 2.2 2.1 0.7 0.1 1.3 0.7 0.5 2.2 1.7 0.7 1.7 0.1 7.0 0.2 0.4 9.5 0.3 8.2 4.6 5.2 2.2 11.2 3.1 2.4 2.1 4.9 3.5 1.5 3.2 6.0 3.1 0.8 2.2 2.0 1.8
2.8 (1.0)
1.85 0.88
2.8 (0.9)
2.29 0.96
3.12 1.76
(0.2) (1.1) (0.9) (0.3) (0.1) (0.3) (0.2) (0.4) (1.1) (0.5) (0.3) (0.4) (0.1) (1.6) (0.1) (0.1) (3.6) (0.2) (3.0) (1.0) (1.6) (0.9) (2.7) (0.3) (0.8) (1.0) (1.2) (0.6) (0.4) (1.6) (1.7) (0.5) (0.4) (1.7) (0.5) (0.7)
2.8 (0.9)
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We used PROC MIXED with the aggression index as dependent variable, colony origin (nestmates or non-nestmates), queen presence (both queenright, both queenless, or queenright vs. queenless) and time (T1 or T2) as categorical predictors and relative chemical distance as continuous predictor. 3. Results The cuticular hydrocarbon profile of C. aethiops of this Italian population consists of linear and branched alkanes of a chain length between 25 and 29 carbon atoms (Fig. 1, Table 1). We found a clear effect of homogeneous lab conditions on the hydrocarbon profiles of the workers. All colonies showed a similar change in the relative amounts of cuticular compounds (Fig. 2): the strongest effect was an increase in all the linear alkanes (C25, C26, C27, C28, and C29) and the branched alkane 5-MeC29. This made the colony profiles in a PCA converge slightly. Especially the queenless colonies were characterized by a strong increase in linear alkanes and 5-MeC29 (high on PC2), already after 4 months, and this became even stronger after 12 months. Queenright colonies did not change that fast and had less consistent patterns. In addition to the general increase in linear alkanes, queenright colonies were also found to have an increase in the mixtures of 11,15- + 9,13diMeC27 and 11,15- + 9,13-diMeC25. All colonies also had a relative increase in the mixture of 13,17- + 11,15- + 9,13-diMeC29 (high on PC1). Another trend that was to be found after prolonged exposure to the lab conditions, was a fluctuating and increasing relative amount of heavier compounds (Fig. 3; repeated measures ANOVA, F6,132 = 34.152, p < 0.0001). Generally, both linear and methylbranched hydrocarbons with a chain length of 29 carbon atoms increased in proportion to the rest of the hydrocarbon profile. However, the initial increase was followed by a sharp decline after 6 months, after which the average chain length started to increase again until the end of the year. Therefore, this may be attributed to seasonality. Relative chemical distances are based on all hydrocarbons found in the profile, but we have to take into account that not all compounds may have the same value in nestmate recognition. We found a subset of compounds at T0 to be >3.12 times (the mean at T0) more variable between colonies than within colonies (in bold in Table 1). These are most likely to play a role in nestmate
recognition. Some of these compounds (5,17-diMeC29, 5,9- + 5,13+ 5,17-diMeC25, 13,17- + 11,15- + 9,13-diMeC29, 5-MeC27, 2,ydiMeC26, 5,9- + 5,13- + 5,15- + 5,17diMeC27 and 13- + 11- + 9MeC25) remained high in their diagnostic power (DP) throughout T0, T1 and T2, whereas others became of less DP under laboratory conditions (11,15- + 9,13-diMeC27, 11,15- + 9,13-diMeC25, 4,8diMeC26 and 3,15- + 3,13- + 3,9- + 3,7-diMeC27). Generally, the DP of compounds declined over the year, as shown by the decline in the mean and standard deviation of all compounds across T0, T1 and T2 (Table 1). Nestmate recognition abilities were not impaired by the prolonged exposure to homogeneous laboratory conditions. Backward elimination of least significant variables in the model showed that the aggression between colonies was best explained by the relative chemical distance between their hydrocarbon profiles (relative chemical distance, F1,125 = 42.308, p < 0.0001). Colony origin and the interaction between relative chemical distance and time also had significant effects (colony origin, F1,125 = 3.966, p = 0.049; relative chemical distance time, F1,125 = 7.360, p = 0.008), whereas queen presence and time did not (queen
Fig. 2. PCA based on hydrocarbons of all colonies over 12 months under laboratory conditions. Sample points depict the centroid of five workers, sampled every 2 months. Filled symbols are queenright colonies, open symbols are queenless colonies. Star symbols are loadings of variables: the mixture 13,17- + 11,15- + 9,13diMeC29 scores highest on PC1, the average of linear alkanes and 5-MeC29 score high on PC2.
Fig. 4. The correlation between relative chemical distance and aggression displayed by Camponotus aethiops workers in aggression bioassays. Closed circles and complete line depict T1 (after 6 months), open circles and dashed line depict T2 (after 12 months).
Fig. 3. Mean (95% CI) weight of hydrocarbon profiles of queenless (QL) and queenright (QR) colonies over 12 months under laboratory conditions. Relative abundance of heavier compounds fluctuated and increased over time (repeated measures ANOVA, F6,132 = 34.152, p < 0.0001).
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Fig. 5. Aggression level observed between nestmates and non-nestmates, depending on queen presence. N is the number of combinations of colonies used for each category. Each combination was replicated 12 times, 6 times on T1 (after 6 months) and 6 times on T2 (after 12 months).
presence, F2,125 = 0.829, p = 0.439; time, F1,125 = 0.004, p = 0.947). The aggression towards non-nestmates was thus still significantly higher than towards nestmates after 1 year, although we find a slight but significant decrease in the aggression displayed with increasing relative chemical distances, as compared to after 6 months (Fig. 4). This is likely due to the absolute chemical distances between colonies becoming smaller. Despite not finding a significant interaction between colony origin and queen presence, we can see a slight trend of queenless colonies being less aggressive towards non-nestmates than queenright colonies (Fig. 5). 4. Discussion We have shown that the cuticular hydrocarbon profile of C. aethiops is affected by prolonged exposure to homogeneous laboratory conditions. Even though we found the nestmate recognition cues to be slightly converging between colonies, this change did not significantly impair nestmate recognition capabilities of the ants. Cuticular hydrocarbon profiles have repeatedly been shown to change over time under natural conditions (e.g. Vander Meer et al., 1989; Provost et al., 1993; Nielsen et al., 1999; Liu et al., 2001; Lahav et al., 2001; Steinmetz and Schmolz, 2005), which generally has been attributed to seasonal changes in for example food and nest material. However, the same may not apply to the change we observed in C. aethiops, since the lab environment provides them with a uniform food source and nest material. The most significant change in the workers’ hydrocarbon profiles was a strong relative increase in linear alkanes and 5MeC29. This can probably partly be attributed to the food the ants received. The cuticular hydrocarbon profile of mealworms, T. molitor, is characterized by three linear alkanes, C25, C27 and C29
(Dreier, unpublished data). However, this still does not explain the relative increase of 5-MeC29, C26 and C28. Linear alkanes have repeatedly been found to play little role in nestmate recognition in social insects (Dani et al., 2001, 2005; Martin et al., 2008; but see Greene and Gordon, 2007). In C. aethiops, too, linear alkanes do not seem to be important, according to their low diagnostic power between colonies. The variability of C25, C26, C27, C28 and C29 within colonies was almost just as high as between colonies (Table 1), which would make it difficult for an ant worker to discriminate between colonies. Therefore, since the major change in the cuticular profile concerned linear alkanes, our observation that nestmate recognition abilities between the colonies remained effective over time is in accordance with the current interpretation that linear alkanes do not represent relevant recognition cues at the inter-colony level. The other major change that we observed was an increase in heavier compounds over the year that we followed the hydrocarbon profiles. One hypothesis relating to this would be that increasing the average chain length reduces the volatility of the hydrocarbons, and hence increases the protection against desiccation (Tissot et al., 2001). We would therefore expect this change under higher temperature, higher aridity and/or lower atmospheric pressure. Average temperature in our climate room in Copenhagen, Denmark, may be slightly higher than the natural temperature in Moraduccio, Italy in April, and aridity in the boxes in which the ants were kept may have been higher as well. Our results show that nestmate recognition in C. aethiops predominantly depends on the genetic background of the workers of a colony. Environment had a considerable influence on the hydrocarbon profile, but not to an extend that significantly affected discrimination between nestmates and non-nestmates. However, aggression slightly decreased at a given chemical distance after 1 year under laboratory conditions, suggesting a shift in the
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acceptance threshold of ants (Reeve, 1989), which became more permissive possibly due to the abundance of food (Downs and Ratnieks, 2000). The presence of a queen also had some influence on the hydrocarbon profile, but again we could not find a significant influence on the aggression towards non-nestmates or nestmates. We therefore suggest that the hierarchy of nestmate discrimination sources is: worker > environment queen. This is different from the finding of Carlin and Ho¨lldobler (1986, 1988) in other Camponotus species, where the presence of the queen was the dominant factor influencing aggression towards other colonies. This may have been due to the size of the colonies, as these authors generally used small colonies of 10 or less workers, whereas we had at least a hundred workers in each colony. Altogether our data show that even though the multicomponent recognition signal represented by ants’ hydrocarbon profiles may change in some particular components, nestmate recognition is not necessarily affected. Some ant species appear to rely on genetically determined cues, while for others the environment might be more important, which emphasizes the complexity of the interaction between different factors involved in the formation of the nestmate recognition odor. Our results suggest that the heritable component is particularly important in carpenter ants and thus colony kin structure is a key factor for the stability of these advanced societies. Acknowledgements We thank Fernando J. Guerrieri for his help in setting up and carrying out the experiment, David Nash for comments on the manuscript and Bernhard Seifert and Alain Lenoir for identification of the ant species. We would also like to thank all members of the Copenhagen Centre for Social Evolution for providing a stimulating working environment. The work was supported by the EU Marie Curie Excellence Grant CODICES-EXT-CT-2004-014202 assigned to PdE. References Akino, T., Yamamura, K., Wakamura, S., Yamaoka, R., 2004. Direct behavioral evidence for hydrocarbons as nestmate recognition cues in Formica japonica (Hymenoptera: Formicidae). Applied Entomology and Zoology 39, 381–387. Bonavita-Cougourdan, A., Cle´ment, J.-L., Lange, C., 1987. Nestmate recognition: the role of cuticular hydrocarbons in the ant Camponotus vagus Scop. Journal of Entomological Science 22, 1–10. Buckle, G.R., Greenberg, L., 1981. Nestmate recognition in sweat bees (Lasioglossum zephyrum): does an individual recognize its own odour or only odours of its nestmates? Animal Behaviour 29, 802–809. Carlin, N., Ho¨lldobler, B., 1988. Influence of virgin queens on kin recognition in the carpenter ant Camponotus floridanus (Hymenoptera: Formicidae). Insectes Sociaux 35, 191–197. Carlin, N.F., Ho¨lldobler, B., 1986. The kin recognition system of carpenter ants (Camponotus spp.) I: hierarchical cues in small colonies. Behavioral Ecology and Sociobiology 19, 123–134. Christensen, J.H., Hansen, A.B., Karlson, U., Mortensen, J., Andersen, O., 2005. Multivariate statistical methods for evaluating biodegradation of mineral oil. Journal of Chromatography A 1090, 133–145. Crosland, M.W.J., 1989. Kin recognition in the ant Rhytidoponera confusa I. Environmental odour. Animal Behaviour 37, 912–919. Crozier, R.H., Dix, M.W., 1979. Analysis of two genetic models for the innate components of colony odor in social Hymenoptera. Behavioral Ecology and Sociobiology 4, 217–224. Dani, F.R., Jones, G.R., Corsi, S., Beard, R., Pradella, D., Turillazzi, S., 2005. Nestmate recognition cues in the honey bee: differential importance of cuticular alkanes and alkenes. Chemical Senses 30, 477–489. Dani, F.R., Jones, G.R., Destri, S., Spencer, S.H., Turillazzi, S., 2001. Deciphering the recognition signature within the cuticular chemical profile of paper wasps. Animal Behaviour 62, 165–171. Downs, S.G., Ratnieks, F.L.W., 2000. Adaptive shifts in honey bee (Apis mellifera L.) guarding behavior support predictions of the acceptance threshold model. Behavioral Ecology 11, 326–333.
Downs, S.G., Ratnieks, F.L.W., Badcock, N.S., Mynott, A., 2001. Honeybee guards do not use food-derived odors to recognize non-nest mates: a test of the odor convergence hypothesis. Behavioral Ecology 12, 47–50. Errard, C., Hefetz, A., 1997. Label familiarity and discriminatory ability of ants reared in mixed groups. Insectes Sociaux 44, 189–198. Espelie, K.E., Wenzel, J.W., Chang, G., 1990. Surface lipids of social wasp Polistes metricus Say and its nest and nest pedicel and their relation to nestmate recognition. Journal of Chemical Ecology 16, 2229–2241. Gamboa, G.J., Reeve, H.K., Ferguson, I.D., Wacker, T.L., 1986. Nestmate recognition in social wasps: the origin and acquisition of recognition odours. Animal Behaviour 34, 685–695. Greene, M.J., Gordon, D.M., 2007. Structural complexity of chemical recognition cues affects the perception of group membership in the ants Linepithema humile and Aphaenogaster cockerelli. Journal of Experimental Biology 210, 897–905. Ho¨lldobler, B., Michener, C.D., 1980. Mechanisms of identification and discrimination in social Hymenoptera. In: Markl, H. (Ed.), Evolution of Social Behavior: Hypotheses and Empirical Tests. Weinheim, VC, pp. 35–58. Jutsum, A.R., Saunders, T.S., Cherrett, J.M., 1979. Intraspecific aggression in the leafcutting ant Acromyrmex octospinosus. Animal Behaviour 27, 839–844. Kalmus, H., Ribbands, C.R., 1952. The origin of the odours by which honeybees distinguish their companions. Proceedings of the Royal Society B: Biological Sciences 140, 50–59. Lahav, S., Soroker, V., Hefetz, A., Vander Meer, R.K., 1999. Direct behavioral evidence for hydrocarbons as ant recognition discriminators. Naturwissenschaften 86, 246–249. Lahav, S., Soroker, V., Vander Meer, R.K., Hefetz, A., 2001. Segregation of colony odor in the desert ant Cataglyphis niger. Journal of Chemical Ecology 27, 927–943. Lenoir, A., Fresneau, D., Errard, C., Hefetz, A., 1999. Individuality and colonial identity in ants: the emergence of the social representation concept. In: Detrain, C., Deneubourg, J.-L., Pasteels, J.M. (Eds.), Information Processing in Social Insects. Birkha¨user Verlag, Basel, pp. 219–237. Liang, D., Silverman, J., 2000. ‘‘You are what you eat’’: diet modifies cuticular hydrocarbons and nestmate recognition in the Argentine ant, Linepithema humile. Naturwissenschaften 87, 412–416. Liu, Z.B., Bagne`res, A.G., Yamane, S., Wang, Q.C., Kojima, J., 2001. Intra-colony, intercolony and seasonal variations of cuticular hydrocarbon profiles in Formica japonica (Hymenoptera, Formicidae). Insectes Sociaux 48, 342–346. Martin, S.J., Vitikainen, E., Helantera¨, H., Drijfhout, F.P., 2008. Chemical basis of nestmate discrimination in the ant Formica exsecta. Proceedings of the Royal Society B: Biological Sciences 275, 1271–1278. Meskali, M., Bonavita-Cougourdan, A., Provost, E., Bagne`res, A.-G., Dusticier, G., Cle´ment, J.-L., 1995. 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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Disentangling environmental and heritable nestmate recognition cues in a carpenter ant Jelle S. van Zweden 1,*, Stephanie Dreier 1, Patrizia d’Ettorre Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 September 2008 Received in revised form 3 November 2008 Accepted 3 November 2008
Discriminating between group members and strangers is a key feature of social life. Nestmate recognition is very effective in social insects and is manifested by aggression and rejection of alien individuals, which are prohibited to enter the nest. Nestmate recognition is based on the quantitative variation in cuticular hydrocarbons, which can include heritable cues from the workers, as well as acquired cues from the environment or queen-derived cues. We tracked the profile of six colonies of the ant Camponotus aethiops for a year under homogeneous laboratory conditions. We performed chemical and behavioral analyses. We show that nestmate recognition was not impaired by constant environment, even though cuticular hydrocarbon profiles changed over time and were slightly converging among colonies. Linear hydrocarbons increased over time, especially in queenless colonies, but appeared to have weak diagnostic power between colonies. The presence of a queen had little influence on nestmate discrimination abilities. Our results suggest that heritable cues of workers are the dominant factor influencing nestmate discrimination in these carpenter ants and highlight the importance of colony kin structure for the evolution of eusociality. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Ants Camponotus aethiops Cuticular hydrocarbons Kin recognition Multi-component signals
1. Introduction Nestmate recognition in social insects ensures that altruistic behavior is directed to the appropriate recipients; usually related individuals. By excluding non-nestmates from their nest, social insects avoid robbery and parasitism of their valuable resources (Wilson, 1971). When two individuals meet, they assess if the other is a nestmate or non-nestmate on the basis of chemical cues. In the case of non-nestmates they usually exhibit aggressive behavior; otherwise they may cooperate (Ho¨lldobler and Michener, 1980). In most social insects, and especially in ants, the recognition cues are primarily encoded in the cuticular hydrocarbon profile (Bonavita-Cougourdan et al., 1987; Lahav et al., 1999; reviewed by Lenoir et al., 1999). Individuals within one social insect species have approximately the same qualitative profile of hydrocarbons (the so-called colony odor), but these differ in their relative proportions depending on the colony of origin of the individual (e.g. Bonavita-Cougourdan et al., 1987; Espelie et al., 1990). With the exception of some ants, wasps and bees with small colony sizes (e.g. Buckle and Greenberg, 1981), the colony odor is thought to be
* Corresponding author. Tel.: +45 3532 1238; fax: +45 3532 1250. E-mail address: [email protected] (J.S. van Zweden). 1 These authors contributed equally to this work. 0022-1910/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2008.11.001
homogenized among nestmates by grooming and body contact alongside mixing of hydrocarbons in the postpharyngeal gland, which are then exchanged by trophallaxis together with regurgitated liquid food (Soroker et al., 1994; Meskali et al., 1995). This homogenization facilitates recognition by diminishing the possibility for errors (Reeve, 1989; Sherman et al., 1997). Among the different structural classes of cuticular hydrocarbons (linear alkanes, branched alkanes and alkenes), not all are thought to have equally important roles in nestmate recognition. It has been shown that linear alkanes are less important (Akino et al., 2004; Martin et al., 2008; but see Greene and Gordon, 2007), which has been attributed to their lack of discriminative features; linear alkanes can only be discriminated on their chain length, whereas branched and unsaturated hydrocarbons could also be discriminated on the position of the methyl group or the double bond, respectively. Multiple sources have been proposed to influence the colony odor, including genetically determined odors from all the individuals of a colony (e.g. Wallis, 1962; Crozier and Dix, 1979; Ho¨lldobler and Michener, 1980), environmental odors, like nest material or food (e.g. Kalmus and Ribbands, 1952; Crosland, 1989; Jutsum et al., 1979; but see Downs et al., 2001), queen odors (e.g. Carlin and Ho¨lldobler, 1986) or combinations of these (e.g. Obin and Vander Meer, 1988; Obin and Vander Meer, 1989; Provost, 1991). In the paper wasp Polistes fuscatus females do not discriminate between nestmates and non-nestmates under
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homogeneous laboratory conditions, whereas they do under field conditions (Gamboa et al., 1986). Diet can influence nestmate recognition cues and abilities, for example in Argentine ants (Liang and Silverman, 2000). Therefore, if environmental odors are the dominant factor, we would expect that colonies under homogeneous environmental conditions would slowly stop to discriminate non-nestmates from nestmates. We tested this in Camponotus aethiops by tracking the cuticular hydrocarbon profile of workers for a year after field collection and transfer to homogeneous laboratory conditions. We then investigated which hydrocarbons are most affected by uniform laboratory conditions, which hydrocarbons are most likely to act as nestmate recognition cues and if the homogeneous conditions would affect ants’ nestmate recognition abilities. We also asked whether the presence of the queen had an influence on any of these factors. 2. Materials and methods 2.1. Study organisms Five colonies of C. aethiops, each containing between 100 and 300 workers, were collected in April 2006 in Moraduccio (448100 3000 N, 118290 600 E), on the border of Emilia Romagna and Toscana, Italy, and transported to Copenhagen, Denmark. In two of the colonies (Cae1 and Cae12) the queen could not be found, so these may or may not have been queenless before. One of the queenright colonies (Cae2) was split into a queenright (Cae2a) and a queenless half (Cae2b) right after collection, each with approximately 150 workers. The last two colonies (Cae6 and Cae9) remained queenright, so that we had three queenright and three queenless colonies altogether. In the laboratory each colony was kept in a nest consisting of two plastic boxes (26.5 cm 17.5 cm 8.0 cm) connected with a short plastic tube, and both were coated with Fluon1 (De Monchy, The Netherlands). One half was darkened with a lid, had a moist plaster floor and contained cotton-stoppered glass tubes filled with water, while the other half was uncovered and served as foraging area. The colonies were fed diluted honey and mealworms, Tenebrio molitor, thrice a week and were kept at room temperature and humidity. All colonies were kept in a climate room at 23 2 8C during summer and at 15 2 8C during winter, under 12:12 LD photoperiod. 2.2. Timeline From each of the six colonies, we randomly collected five workers for chemical analysis right after field collection and subsequently each time after 2 months, until approximately 12 months had passed (2006: 25 April, 27 June, 22 August, 23 October, 13 December; 2007: 6 February, 13 April). For simplicity, we will present some of the results only for the start of the study (T0 = 25 April 2006), after 6 months (T1 = 23 October 2006) and after 12 months (T2 = 13 April 2007), but we will specify per analysis if all results or only these three points are presented. To evaluate nestmate recognition abilities, aggression tests were performed on T1 and T2. 2.3. Chemical analysis Workers were killed by freezing at 20 8C and cuticular hydrocarbons were extracted by immersing individual workers in 200 ml HPLC-grade pentane (Sigma–Aldrich, Denmark) for 10 min, of which the first and the last 15 s involved gentle vortexing. The solvent was then let to evaporate at room temperature in a laminar flow cupboard. The extract was
resuspended in 100 ml pentane, of which 2 ml were injected in an Agilent 6890N gas-chromatograph, equipped with a HP-5MS capillary column (30 m 250 mm 0.25 mm), a split-splitless injector and a 5975 Agilent Mass Spectrometer with 70 eV electron impact ionization. The carrier gas was helium at 1 ml min1. After an initial hold of 1 min at 70 8C, the temperature rose to 200 8C at a rate of 30 8C min1, and then to 300 8C at 3 8C min1. The areas of 36 hydrocarbons (or mixtures of few hydrocarbons, due to co-elution) found on the cuticles of all workers were integrated for further analysis. These were used as variables in Principal Component Analysis (PCA) in the program LatentiX 1.00 for Windows. On T0, T1 and T2 we performed PCAs including all colonies (five colonies on T0, as Cae2a and Cae2b were still a single colony at this point, and six colonies on both T1 and T2), after which the PC scores were used for Discriminant Analysis (DA) in the program STATISTICA 7.1 (StatSoft Inc., USA), in order to determine if colonies could be significantly discriminated according to their hydrocarbon profile on these three points in time. The Mahalanobis distance between colonies was calculated, which was normalized according to the maximum Mahalanobis distance observed in the DA (relative chemical distance, hereafter), in order to allow comparison between DA’s. To calculate the diagnostic power (DP) for each peak, the standard deviation of the standardized peak area over all colonies was divided by the pooled standard deviation within these colonies (Christensen et al., 2005). Hence, compounds with the highest DP are most variable between colonies, but comparatively most consistent within colony, and are therefore the most likely to act as nestmate recognition cues. To investigate the relative length of hydrocarbons in the profile, weighted retention time was calculated by multiplying the average retention time of each peak with the relative area of that peak (i.e. the weight), and taking the sum of these values over the entire profile. Mean weighted retention time was calculated separately for queenright and queenless colonies for each point in time, after which these were compared using a repeated measures ANOVA in STATISTICA 7.1. 2.4. Aggression bioassays To test whether homogeneous conditions, inducing a change in the hydrocarbon profile, would affect the nestmate recognition capabilities of the workers, we conducted aggression bioassays between a subset of the nests after 6 months (T1) and 12 months (T2). We made 11 combinations of colonies to test against one another, so that we covered the variables colony origin (nestmates or non-nestmates) and queen presence (both queenright (QR), both queenless (QL), or queenright vs. queenless), and replicated each combination six times. Non-nestmate combinations included three QR–QR pairs (Cae2a–Cae6, Cae2a–Cae9 and Cae6–Cae9), two QL–QL pairs (Cae12–Cae1 and Cae12–Cae2b) and three QR–QL pairs (Cae2a–Cae1, Cae6–Cae12 and Cae1–Cae9). Nestmate (control) combinations included one QR–QR pair (Cae1–Cae1), one QL– QL pair (Cae6–Cae6) and one QR–QL pair (Cae2a–Cae2b). The same combinations were tested twice, once on T1 and once on T2. No ant was used more than once, the observer was the same person for T1 and T2, and the observer was blind with respect to the colony origin of the ants. In each aggression bioassay a dyadic encounter was staged in a Petri dish (15.2 cm) lined with a clean filter paper. The ants were confined by a cylinder coated with Fluon1 and were separated from each other by a smaller Fluon1-coated cylinder until the bioassay started. The ants were allowed to interact with each other for 3 min, throughout which we used the software Etholog 2.25 (Ottoni, 2000) to record the duration of the following behaviors with their aggression indices in
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161
Fig. 1. Chromatogram of the cuticular hydrocarbon profile of Camponotus aethiops. Identification of the peaks is provided in Table 1.
duration of the ith behavior, and T is the sum of the durations of all interactions. Relative importance of colony origin, queen presence, time and chemical distance on the observed aggression was tested with a General Linear Model (GLM) in the program SAS 9.1 for Windows.
brackets: investigation with antennae (0), mandibles open (1), bite (2), curl gaster forward (3). We then calculated an overall aggression level using the from Errard and Pnformula (modified Hefetz, 1997): AIoverall ¼ AI t =T, in which AIoverall is the i i i¼1 overall aggression level, AIi and ti are the aggression index and
Table 1 Diagnostic power and average percentage for each compound in the hydrocarbon profile over time. The diagnostic power (DP) is the standard deviation over all individuals (for T0, T1 and T2), divided by the pooled standard deviation within colonies (for T0, T1 and T2). % (S.D.) = mean percentage over all colonies S.D. of each peak in the total hydrocarbon profile (for T0, T1 and T2). Peak #
Identification
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
2-MeC24 C25 13- + 11- + 9-MeC25 7-MeC25 5-MeC25 11,15- + 9,13-diMeC25 7,13- + 7,15-diMeC25 + 3-MeC25 5,9- + 5,13- + 5,17-diMeC25 C26 3,13- + 3,11- + 3,9- + 3,7-diMeC25 13- + 12- + 11-MeC26 8-MeC26 + x,y-diMeC26 6-MeC26 2-MeC26 x,y-diMeC26 4,8-diMeC26 C27 2,y-diMeC26 13- + 11-MeC27 9-MeC27 7-MeC27 5-MeC27 11,15- + 9,13-diMeC27 7,15- + 7,13-diMeC27 + 3-MeC27 5,9- + 5,13- + 5,15- + 5,17-diMeC27 C28 3,15- + 3,13- + 3,9- + 3,7-diMeC27 14- + 13- + 12- + 10- + 8-MeC28 12,16-diMeC28 + 4-MeC28 C29 15- + 13- + 11- + 9-MeC29 7-MeC29 5-MeC29 13,17- + 11,15- + 9,13-diMeC29 7,17-diMeC29 + 3-MeC29 5,17-diMeC29
Mean S.D.
T0
T1
T2
DP
% (S.D.)
DP
% (S.D.)
DP
% (S.D.)
2.40 1.46 4.12 2.24 1.96 4.93 2.33 6.69 1.34 2.59 2.50 1.17 1.14 3.16 4.60 4.87 1.07 4.71 3.27 1.44 2.23 5.46 6.22 1.59 4.32 1.36 4.69 3.04 1.48 1.27 2.82 1.48 2.51 6.33 2.56 6.92
0.6 1.3 3.0 1.6 0.4 1.8 1.6 1.0 0.9 3.5 1.0 3.4 0.4 6.4 0.4 0.8 4.7 0.5 8.0 5.3 6.0 2.4 12.9 3.5 2.7 0.5 7.9 3.8 1.1 0.9 3.4 2.1 0.4 1.7 1.8 2.1
(0.1) (0.4) (1.3) (0.3) (0.7) (0.8) (0.8) (0.8) (0.3) (1.4) (0.4) (1.1) (0.7) (1.4) (0.2) (0.3) (1.3) (0.3) (1.8) (0.6) (1.1) (1.4) (5.4) (0.3) (1.2) (0.2) (2.6) (0.7) (0.6) (0.4) (1.0) (0.4) (0.2) (2.0) (0.5) (1.4)
1.33 1.25 2.88 2.86 1.39 1.12 1.34 2.57 1.01 3.10 1.67 1.73 0.99 1.25 1.84 1.80 1.18 2.51 2.33 1.26 1.19 3.55 1.59 1.09 4.87 1.20 1.60 1.09 2.41 1.77 1.15 0.96 2.02 3.08 1.28 2.15
0.6 2.2 2.3 1.0 0.3 1.2 1.3 0.7 2.1 2.3 0.8 2.0 0.2 7.1 0.4 0.4 9.6 0.4 8.0 5.2 6.2 2.7 10.2 3.2 2.4 1.9 5.2 3.2 1.2 2.8 4.7 2.7 0.8 1.5 1.4 1.7
(0.2) (0.8) (1.1) (0.4) (0.2) (0.3) (0.7) (0.5) (0.8) (0.9) (0.3) (0.6) (0.1) (1.2) (0.2) (0.3) (3.6) (0.2) (2.1) (0.9) (1.3) (1.2) (2.8) (0.5) (0.9) (1.0) (1.2) (0.6) (0.5) (2.0) (1.0) (0.4) (0.4) (0.8) (0.4) (0.9)
1.75 1.98 2.58 2.62 3.09 1.49 4.07 4.00 1.48 2.38 2.63 1.34 1.02 1.18 2.26 2.33 1.18 2.96 2.33 1.26 1.64 2.51 1.97 2.15 3.14 1.07 2.11 1.97 2.78 1.71 2.11 1.17 2.27 5.23 2.50 4.16
0.5 2.2 2.1 0.7 0.1 1.3 0.7 0.5 2.2 1.7 0.7 1.7 0.1 7.0 0.2 0.4 9.5 0.3 8.2 4.6 5.2 2.2 11.2 3.1 2.4 2.1 4.9 3.5 1.5 3.2 6.0 3.1 0.8 2.2 2.0 1.8
2.8 (1.0)
1.85 0.88
2.8 (0.9)
2.29 0.96
3.12 1.76
(0.2) (1.1) (0.9) (0.3) (0.1) (0.3) (0.2) (0.4) (1.1) (0.5) (0.3) (0.4) (0.1) (1.6) (0.1) (0.1) (3.6) (0.2) (3.0) (1.0) (1.6) (0.9) (2.7) (0.3) (0.8) (1.0) (1.2) (0.6) (0.4) (1.6) (1.7) (0.5) (0.4) (1.7) (0.5) (0.7)
2.8 (0.9)
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We used PROC MIXED with the aggression index as dependent variable, colony origin (nestmates or non-nestmates), queen presence (both queenright, both queenless, or queenright vs. queenless) and time (T1 or T2) as categorical predictors and relative chemical distance as continuous predictor. 3. Results The cuticular hydrocarbon profile of C. aethiops of this Italian population consists of linear and branched alkanes of a chain length between 25 and 29 carbon atoms (Fig. 1, Table 1). We found a clear effect of homogeneous lab conditions on the hydrocarbon profiles of the workers. All colonies showed a similar change in the relative amounts of cuticular compounds (Fig. 2): the strongest effect was an increase in all the linear alkanes (C25, C26, C27, C28, and C29) and the branched alkane 5-MeC29. This made the colony profiles in a PCA converge slightly. Especially the queenless colonies were characterized by a strong increase in linear alkanes and 5-MeC29 (high on PC2), already after 4 months, and this became even stronger after 12 months. Queenright colonies did not change that fast and had less consistent patterns. In addition to the general increase in linear alkanes, queenright colonies were also found to have an increase in the mixtures of 11,15- + 9,13diMeC27 and 11,15- + 9,13-diMeC25. All colonies also had a relative increase in the mixture of 13,17- + 11,15- + 9,13-diMeC29 (high on PC1). Another trend that was to be found after prolonged exposure to the lab conditions, was a fluctuating and increasing relative amount of heavier compounds (Fig. 3; repeated measures ANOVA, F6,132 = 34.152, p < 0.0001). Generally, both linear and methylbranched hydrocarbons with a chain length of 29 carbon atoms increased in proportion to the rest of the hydrocarbon profile. However, the initial increase was followed by a sharp decline after 6 months, after which the average chain length started to increase again until the end of the year. Therefore, this may be attributed to seasonality. Relative chemical distances are based on all hydrocarbons found in the profile, but we have to take into account that not all compounds may have the same value in nestmate recognition. We found a subset of compounds at T0 to be >3.12 times (the mean at T0) more variable between colonies than within colonies (in bold in Table 1). These are most likely to play a role in nestmate
recognition. Some of these compounds (5,17-diMeC29, 5,9- + 5,13+ 5,17-diMeC25, 13,17- + 11,15- + 9,13-diMeC29, 5-MeC27, 2,ydiMeC26, 5,9- + 5,13- + 5,15- + 5,17diMeC27 and 13- + 11- + 9MeC25) remained high in their diagnostic power (DP) throughout T0, T1 and T2, whereas others became of less DP under laboratory conditions (11,15- + 9,13-diMeC27, 11,15- + 9,13-diMeC25, 4,8diMeC26 and 3,15- + 3,13- + 3,9- + 3,7-diMeC27). Generally, the DP of compounds declined over the year, as shown by the decline in the mean and standard deviation of all compounds across T0, T1 and T2 (Table 1). Nestmate recognition abilities were not impaired by the prolonged exposure to homogeneous laboratory conditions. Backward elimination of least significant variables in the model showed that the aggression between colonies was best explained by the relative chemical distance between their hydrocarbon profiles (relative chemical distance, F1,125 = 42.308, p < 0.0001). Colony origin and the interaction between relative chemical distance and time also had significant effects (colony origin, F1,125 = 3.966, p = 0.049; relative chemical distance time, F1,125 = 7.360, p = 0.008), whereas queen presence and time did not (queen
Fig. 2. PCA based on hydrocarbons of all colonies over 12 months under laboratory conditions. Sample points depict the centroid of five workers, sampled every 2 months. Filled symbols are queenright colonies, open symbols are queenless colonies. Star symbols are loadings of variables: the mixture 13,17- + 11,15- + 9,13diMeC29 scores highest on PC1, the average of linear alkanes and 5-MeC29 score high on PC2.
Fig. 4. The correlation between relative chemical distance and aggression displayed by Camponotus aethiops workers in aggression bioassays. Closed circles and complete line depict T1 (after 6 months), open circles and dashed line depict T2 (after 12 months).
Fig. 3. Mean (95% CI) weight of hydrocarbon profiles of queenless (QL) and queenright (QR) colonies over 12 months under laboratory conditions. Relative abundance of heavier compounds fluctuated and increased over time (repeated measures ANOVA, F6,132 = 34.152, p < 0.0001).
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Fig. 5. Aggression level observed between nestmates and non-nestmates, depending on queen presence. N is the number of combinations of colonies used for each category. Each combination was replicated 12 times, 6 times on T1 (after 6 months) and 6 times on T2 (after 12 months).
presence, F2,125 = 0.829, p = 0.439; time, F1,125 = 0.004, p = 0.947). The aggression towards non-nestmates was thus still significantly higher than towards nestmates after 1 year, although we find a slight but significant decrease in the aggression displayed with increasing relative chemical distances, as compared to after 6 months (Fig. 4). This is likely due to the absolute chemical distances between colonies becoming smaller. Despite not finding a significant interaction between colony origin and queen presence, we can see a slight trend of queenless colonies being less aggressive towards non-nestmates than queenright colonies (Fig. 5). 4. Discussion We have shown that the cuticular hydrocarbon profile of C. aethiops is affected by prolonged exposure to homogeneous laboratory conditions. Even though we found the nestmate recognition cues to be slightly converging between colonies, this change did not significantly impair nestmate recognition capabilities of the ants. Cuticular hydrocarbon profiles have repeatedly been shown to change over time under natural conditions (e.g. Vander Meer et al., 1989; Provost et al., 1993; Nielsen et al., 1999; Liu et al., 2001; Lahav et al., 2001; Steinmetz and Schmolz, 2005), which generally has been attributed to seasonal changes in for example food and nest material. However, the same may not apply to the change we observed in C. aethiops, since the lab environment provides them with a uniform food source and nest material. The most significant change in the workers’ hydrocarbon profiles was a strong relative increase in linear alkanes and 5MeC29. This can probably partly be attributed to the food the ants received. The cuticular hydrocarbon profile of mealworms, T. molitor, is characterized by three linear alkanes, C25, C27 and C29
(Dreier, unpublished data). However, this still does not explain the relative increase of 5-MeC29, C26 and C28. Linear alkanes have repeatedly been found to play little role in nestmate recognition in social insects (Dani et al., 2001, 2005; Martin et al., 2008; but see Greene and Gordon, 2007). In C. aethiops, too, linear alkanes do not seem to be important, according to their low diagnostic power between colonies. The variability of C25, C26, C27, C28 and C29 within colonies was almost just as high as between colonies (Table 1), which would make it difficult for an ant worker to discriminate between colonies. Therefore, since the major change in the cuticular profile concerned linear alkanes, our observation that nestmate recognition abilities between the colonies remained effective over time is in accordance with the current interpretation that linear alkanes do not represent relevant recognition cues at the inter-colony level. The other major change that we observed was an increase in heavier compounds over the year that we followed the hydrocarbon profiles. One hypothesis relating to this would be that increasing the average chain length reduces the volatility of the hydrocarbons, and hence increases the protection against desiccation (Tissot et al., 2001). We would therefore expect this change under higher temperature, higher aridity and/or lower atmospheric pressure. Average temperature in our climate room in Copenhagen, Denmark, may be slightly higher than the natural temperature in Moraduccio, Italy in April, and aridity in the boxes in which the ants were kept may have been higher as well. Our results show that nestmate recognition in C. aethiops predominantly depends on the genetic background of the workers of a colony. Environment had a considerable influence on the hydrocarbon profile, but not to an extend that significantly affected discrimination between nestmates and non-nestmates. However, aggression slightly decreased at a given chemical distance after 1 year under laboratory conditions, suggesting a shift in the
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acceptance threshold of ants (Reeve, 1989), which became more permissive possibly due to the abundance of food (Downs and Ratnieks, 2000). The presence of a queen also had some influence on the hydrocarbon profile, but again we could not find a significant influence on the aggression towards non-nestmates or nestmates. We therefore suggest that the hierarchy of nestmate discrimination sources is: worker > environment queen. This is different from the finding of Carlin and Ho¨lldobler (1986, 1988) in other Camponotus species, where the presence of the queen was the dominant factor influencing aggression towards other colonies. This may have been due to the size of the colonies, as these authors generally used small colonies of 10 or less workers, whereas we had at least a hundred workers in each colony. Altogether our data show that even though the multicomponent recognition signal represented by ants’ hydrocarbon profiles may change in some particular components, nestmate recognition is not necessarily affected. Some ant species appear to rely on genetically determined cues, while for others the environment might be more important, which emphasizes the complexity of the interaction between different factors involved in the formation of the nestmate recognition odor. Our results suggest that the heritable component is particularly important in carpenter ants and thus colony kin structure is a key factor for the stability of these advanced societies. Acknowledgements We thank Fernando J. Guerrieri for his help in setting up and carrying out the experiment, David Nash for comments on the manuscript and Bernhard Seifert and Alain Lenoir for identification of the ant species. We would also like to thank all members of the Copenhagen Centre for Social Evolution for providing a stimulating working environment. The work was supported by the EU Marie Curie Excellence Grant CODICES-EXT-CT-2004-014202 assigned to PdE. References Akino, T., Yamamura, K., Wakamura, S., Yamaoka, R., 2004. Direct behavioral evidence for hydrocarbons as nestmate recognition cues in Formica japonica (Hymenoptera: Formicidae). Applied Entomology and Zoology 39, 381–387. Bonavita-Cougourdan, A., Cle´ment, J.-L., Lange, C., 1987. Nestmate recognition: the role of cuticular hydrocarbons in the ant Camponotus vagus Scop. Journal of Entomological Science 22, 1–10. Buckle, G.R., Greenberg, L., 1981. Nestmate recognition in sweat bees (Lasioglossum zephyrum): does an individual recognize its own odour or only odours of its nestmates? Animal Behaviour 29, 802–809. Carlin, N., Ho¨lldobler, B., 1988. Influence of virgin queens on kin recognition in the carpenter ant Camponotus floridanus (Hymenoptera: Formicidae). Insectes Sociaux 35, 191–197. Carlin, N.F., Ho¨lldobler, B., 1986. The kin recognition system of carpenter ants (Camponotus spp.) I: hierarchical cues in small colonies. Behavioral Ecology and Sociobiology 19, 123–134. Christensen, J.H., Hansen, A.B., Karlson, U., Mortensen, J., Andersen, O., 2005. Multivariate statistical methods for evaluating biodegradation of mineral oil. Journal of Chromatography A 1090, 133–145. Crosland, M.W.J., 1989. Kin recognition in the ant Rhytidoponera confusa I. Environmental odour. Animal Behaviour 37, 912–919. Crozier, R.H., Dix, M.W., 1979. Analysis of two genetic models for the innate components of colony odor in social Hymenoptera. Behavioral Ecology and Sociobiology 4, 217–224. Dani, F.R., Jones, G.R., Corsi, S., Beard, R., Pradella, D., Turillazzi, S., 2005. Nestmate recognition cues in the honey bee: differential importance of cuticular alkanes and alkenes. Chemical Senses 30, 477–489. Dani, F.R., Jones, G.R., Destri, S., Spencer, S.H., Turillazzi, S., 2001. Deciphering the recognition signature within the cuticular chemical profile of paper wasps. Animal Behaviour 62, 165–171. Downs, S.G., Ratnieks, F.L.W., 2000. Adaptive shifts in honey bee (Apis mellifera L.) guarding behavior support predictions of the acceptance threshold model. Behavioral Ecology 11, 326–333.
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