Tropical parabiotic ants: Highly unusual cuticular substances and low interspecific discrimination
© Menzel et al; licensee BioMed Central Ltd. 2008
Received: 30 May 2008
Accepted: 20 October 2008
Published: 20 October 2008
Associations between animal species require that at least one of the species recognizes its partner. Parabioses are associations of two ant species which co-inhabit the same nest. Ants usually possess an elaborate nestmate recognition system, which is based on cuticular hydrocarbons and allows them to distinguish nestmates from non-nestmates through quantitative or qualitative differences in the hydrocarbon composition. Hence, living in a parabiotic association probably necessitates changes of the nestmate recognition system in both species, since heterospecific ants have to be accepted as nestmates.
In the present study we report highly unusual cuticular profiles in the parabiotic species Crematogaster modiglianii and Camponotus rufifemur from the tropical rainforest of Borneo. The cuticle of both species is covered by a set of steroids, which are highly unusual surface compounds. They also occur in the Dufour gland of Crematogaster modiglianii in high quantities. The composition of these steroids differed between colonies but was highly similar among the two species of a parabiotic nest. In contrast, hydrocarbon composition of Cr. modiglianii and Ca. rufifemur differed strongly and only overlapped in three regularly occurring and three trace compounds. The hydrocarbon profile of Camponotus rufifemur consisted almost exclusively of methyl-branched alkenes of unusually high chain lengths (up to C49). This species occurred in two sympatric, chemically distinct varieties with almost no hydrocarbons in common. Cr. modiglianii discriminated between these two varieties. It only tolerated workers of the Ca. rufifemur variety it was associated with, but attacked the respective others. However, Cr. modiglianii did not distinguish its own Ca. rufifemur partner from allocolonial Ca. rufifemur workers of the same variety.
We conclude that there is a mutual substance transfer between Cr. modiglianii and Ca. rufifemur. Ca. rufifemur actively or passively acquires cuticular steroids from its Cr. modiglianii partner, while the latter acquires at least two cuticular hydrocarbons from Ca. rufifemur. The cuticular substances of both species are highly unusual regarding both substance classes and chain lengths, which may cause the apparent inability of Cr. modiglianii to discriminate Ca. rufifemur nestmates from allocolonial Ca. rufifemur workers of the same chemical variety.
Associations across different animal taxa require specific adaptations on one or both sides. In particular, recognizing the partner species is a crucial task to any form of association, albeit in host-parasite associations only the latter might need to recognize the partner . Nestmate recognition mechanisms in associating species must therefore go beyond the own species and include the partner species.
In ants, one of the closest and most intriguing interspecific associations is parabioses, where two ant species live together in a common nest. This phenomenon is found in several parts of the world, including Southeast Asia  and tropical South America . Parabiotic ants have nestmates not only from their own colony, but also from a completely different species. Their nestmate recognition system therefore needs to include allospecific nestmates. In ants and other social hymenoptera, recognition is based on colony-specific chemical cues on the body surface that are perceived through olfactory or contact chemoreception [4, 5]. Most of them are hydrocarbons [6–8]. Via allogrooming and trophallaxis, the individuals continually take up their nestmates' surface compounds into the postpharyngeal gland (PPG), where they are mixed and redistributed. Through this process, a colony-specific odour is created [5, 9–11]. This colony-specific odour is learned by the colony members and represented as a neuronal template in the nervous system . Nestmates are recognized by comparing the cuticular profile of the encountered individual to the neuronal template (phenotype matching), whereby a mismatch generally results in aggression .
Despite this complex nestmate recognition system, a considerable number of insect species manages to be accepted in Hymenoptera colonies, such as Lycaenid larvae, Staphylinidae, Ensifera, and Diptera [13–16] as well as social parasites, such as the parasitic bumblebee Psithyrus  and inquiline ant species [1, 6]. In many of these associations, the parasite chemically resembles the host (chemical mimicry) [1, 13–16, 18, 19]. Another possible mechanism to remain incognito is chemical insignificance . Several social parasite species are – like callows – chemically insignificant, i.e. they do not possess an individual surface profile and are hence not recognized as foreign by their hosts [1, 20, 21]. Hydrocarbon profiles of very long chain lengths are difficult to perceive and hence may also promote chemical insignificance [22, 23]. Still, numerous other social parasite species possess distinct profiles that do not resemble their hosts. Since these profiles neither show chemical mimicry nor insignificance, it has been supposed that the host species habituate to the parasites' profiles [24–26].
While the chemical mechanisms of tolerance between species have been studied in associations like social parasitism, little is known about parabiotic associations. It seems likely that parabiotic ants possess a nestmate recognition system that tolerates allospecific nestmates. In the present study we examined the relationship between interspecific tolerance and surface chemistry among the Southeast Asian parabiotic species Crematogaster modiglianii and Camponotus rufifemur. The two species tolerate ants from certain (but not all) foreign parabiotic nests but attack non-parabiotic ant species . We discovered that two morphological varieties of Ca. rufifemur (the 'red' and the 'black' variety, see Methods) also differ in their chemical profiles. This enabled us to study two different levels of chemical similarity – within and between the two varieties. Our research questions were:
(1) Do parabiotic species possess cuticular substances different from related, non-parabiotic species?
(2) Is there evidence for chemical mimicry, i.e., chemical overlap between parabiotic partners?
(3) Do chemical differences within species account for differences in interspecific allocolonial tolerance?
Cuticular substances: Hydrocarbons and other aliphatic components
Aliphatic cuticular substances found in Crematogaster modiglianii and the two varieties of Camponotus rufifemur
red Ca. rufifemur
black Ca. rufifemur
0.26 ± 0.01%
0.46 ± 0.03%
0.20 ± 0.01%
0.16 ± 0.01%
0.49 ± 0.06%
0.40 ± 0.09%
0.20 ± 0.16%
0.23 ± 0.06%
0.06 ± 0.05%
0.45 ± 0.02%
0.11 ± 0.09%
0.05 ± 0.02%
0.67 ± 0.17%
0.02 ± 0.01%
0.72 ± 0.17%
0.21 ± 0.03%
0.08 ± 0.02%
0.13 ± 0.11%
0.13 ± 0.01%
0.47 ± 0.39%
0.09 ± 0.04%
0.52 ± 0.08%
0.09 ± 0.06%
0.14 ± 0.01%
0.21 ± 0.17%
0.27 ± 0.02%
0.01 ± 0.01%
0.20 ± 0.03%
0.15 ± 0.11%
0.08 ± 0.03%
0.26 ± 0.09%
0.15 ± 0.04%
0.15 ± 0.06%*
0.76 ± 0.11%
17-MeC35, 15-MeC35, 13-MeC35
0.88 ± 0.25%
0.3 ± 0.13%
2.31 ± 0.28%
1.56 ± 0.12%
0.45 ± 0.07%
C37-13-ene, C37-14-ene, C37-15-ene, C37-16-ene
5.43 ± 0.49%
0.48 ± 0.15%
4.53 ± 0.4%
0.44 ± 0.07%
0.52 ± 0.1%
19-MeC37, 17-MeC37, 15-MeC37, 13-MeC37, 11-MeC37
11.47 ± 0.28%
0.18 ± 0.09%
6.02 ± 0.33%
0.63 ± 0.1%
1.99 ± 0.11%
1.12 ± 0.13%
1.46 ± 0.19%
15.23 ± 0.76%
13.7 ± 0.73%
3.66 ± 0.34%
0.55 ± 0.08%
7.62 ± 0.19%
1.7 ± 0.08%
88.66 ± 0.53%
3.15 ± 1.18%
19-MeC39, 17-MeC39, 15-MeC39, 13-MeC39, 11-MeC39
0.52 ± 0.24% (only 13-MeC39)
4.51 ± 0.2%
11,21-DiMeC39, 11,23-DiMeC39, 11,27-DiMeC39, 11,29-DimeC39
4.84 ± 0.52%
0.22 ± 0.01%
27-MeC40-14-ene, 27-MeC40-15-ene, 27-MeC40-16-ene++
3.41 ± 0.09%
1.04 ± 0.18%
0.36 ± 0.08%
3.39 ± 0.24%
3.01 ± 0.29%
3.35 ± 0.4%
0.65 ± 0.12%
0.45 ± 0.03%
3.34 ± 0.19%
3.01 ± 0.04%
4.17 ± 0.06%
1.09 ± 0.4%
2.10 ± 0.16%
0.98 ± 0.06%
1.07 ± 0.06%
15.11 ± 0.52%
8.72 ± 0.37%
4.43 ± 0.18%
22.95 ± 0.96%
4.10 ± 0.07%
9.20 ± 0.49%
1.49 ± 0.12%
1.29 ± 0.11%
1.91 ± 0.13%
0.54 ± 0.07%
2.55 ± 1.7%
2.40 ± 1.6%
1.25 ± 0.11%
Relative quantities of the different aliphatic substance classes in Cr. modiglianii and Ca. rufifemur.
red Ca. rufifemur
black Ca. rufifemur
0.64 ± 0.41%
1.25 ± 0.18%
1.29 ± 0.16%
0 ± 0%
37.44 ± 0.94%
23.06 ± 1.03%
0 ± 0%
28.77 ± 2.41%
39.83 ± 1.09%
0 ± 0%
0 ± 0%
2.8 ± 0.16%
0.58 ± 0.26%
0.45 ± 0.02%
28.07 ± 0.85%
98.1 ± 0.35%
13.37 ± 0.44%
3.15 ± 1.18%
0 ± 0%
1.9 ± 0.78%
0 ± 0%
0.91 ± 0.11%
16.82 ± 0.42%
1.77 ± 0.28%
Diagnostic ions of the methyl-branched alkenes in the red Ca. rufifemur variety.
diagnostic ions from hydration
diagnostic ions from DMDS derivatization
inferred double bond position
243*, 271, 355, 383*
14*, 16 or 21, 23*
25-MeC38-ene, 26-MeC37-ene, 27-MeC38-ene
182, 196, 210, 364, 379, 393
229, 243, 257, 271, 369, 383, 397, 411
13, 14, 15, 16 or 22, 23, 24, 25
243, 271, 383, 411
14**, 16** or 23, 25
243*, 257, 271, 397, 411, 425*
14*, 15, 16 or 24, 25, 26*
196, 224, 392, 421
243, 271*, 299, 383, 411*, 439
14, 16*, 18 or 23, 25*, 27
The profile of the black Ca. rufifemur variety consisted of even larger molecules, with 92.8% of the surface compounds between C44 and C49 (Table 1). At least 80% of the compounds were unsaturated (Table 2). Methyl-branched alkenes were also present, albeit not as abundant as in the red Ca. rufifemur variety. Minor compounds included n-alkanes, methyl-branched alkanes and aldehydes (Table 2). In both Ca. rufifemur varieties, PPG and surface extracts contained the same aliphatic compounds in similar relative quantities.
Cuticular substances: Steroids
Besides aliphatic compounds, the surface profile of both ant species contained up to 24 components with a basic steroid structure (as inferred from mass spectra and diagnostic ions). Their mass spectra indicate a close chemical interrelatedness of the compounds. Due to the high substance quantities necessary for NMR analysis, their spatial molecular structure has not yet been resolved but is under investigation. Crematogaster modiglianii possessed high amounts of steroids on the body surface (2.59 ± 0.58 μg/worker, n = 11 colonies, mean and SE) which by far exceeded the hydrocarbons (0.48 ± 0.05 μg/worker, n = 11 colonies, mean and SE). In contrast, postpharyngeal gland extracts only contained minor amounts of steroids but high quantities of hydrocarbons. High steroid amounts of the same quantitative composition were also found in the Dufour gland, in separate alitrunk and gaster cuticular extracts and, albeit in lower amounts, in head cuticular extracts. They also occurred in cuticular extractions of living ants with SPME fibres, thus confirming that their presence in hexane extracts was not an artefact of concomitantly extracted glands. Altogether, Cr. modiglianii extracts contained 24 different steroid components with an abundance higher than 0.1% in at least one colony (percent of total steroid abundance). Their retention indices ranged between 20.38 and 25.77. Six of the 24 steroids were found in all Cr. modiglianii colonies in similar relative compositions. An additional eleven steroids were abundant in certain colonies but absent in others. The remaining seven steroids were irregularly found and never occurred in relative abundances higher than 1% (percent of total steroid abundance). Camponotus rufifemur extracts (both varieties) contained up to eight different steroids, all of which also occurred in Cr. modiglianii. The absolute steroid quantities in Ca. rufifemur were lower than the hydrocarbon quantities (black variety: 0.66 ± 0.22 μg steroids/worker and 1.79 ± 0.29 μg hydrocarbons/worker, n = 3 colonies; red variety: 0.41 ± 0.14 μg steroids/worker and 9.71 ± 3.79 μg hydrocarbons/worker, n = 4 colonies, mean and SE given).
Chemical overlap among the parabiotic species
Eight of the steroids common in Cr. modiglianii were also frequently found in Ca. rufifemur (inclusion criterion: median abundance > 0% in 11 colonies of both species; Figure 1d). Their relative abundances varied between parabiotic nests but were significantly correlated among the two species within a nest (Mantel test: r = 0.49, p = 0.041, N = 11; Bray-Curtis distances: 0.13 ± 0.08 (Ca. rufifemur), 0.43 ± 0.31 (Cr. modiglianii); mean and s.d.). A second Mantel test considered only three steroids with very similar mass spectra, which were present in all extracts (retention indices: 21.92, 22.24, 24.47; marked with asterisks in Figure 1d). This test yielded a highly significant correlation of steroid abundance among the two species of each parabiotic nest (r = 0.620, p < 0.001, N = 11; Bray-Curtis distances: 0.06 ± 0.04 (Ca. rufifemur), 0.13 ± 0.07 (Cr. modiglianii)).
Differences in allocolonial tolerance
GLM for total aggression of Cr. modiglianii towards dead Ca. rufifemur workers from different colonies.
Ca. rufifemur variety
Ca. rufifemur colony
GLM for total aggression of Cr. modiglianii towards dead Ca. rufifemur from different colonies.
GLM for total aggression in interspecific live confrontations
Cr. modiglianii → Ca. rufifemur
Ca. rufifemur → Cr. modiglianii
In confrontations of Ca. rufifemur towards allocolonial Cr. modiglianii, Menzel et al.  had found low levels of aggression albeit they were higher than against intracolonial Cr. modiglianii. Similar to above, Ca. rufifemur workers were more aggressive towards Cr. modiglianii from the respective other variety (Figure 5b, Table 6).
Unusual features of the cuticular profiles in parabiotic ants
To our knowledge, steroids have not been found in surface extracts of other ant species up to now, and to our knowledge have been found on insect cuticles only in one halictid bee . However, various Crematogaster species are known to have highly efficient poisons [31, 32]. The genus Crematogaster has evolved a peculiar system of venom production which involves a cooperation of Dufour and poison gland. In several species the venom consists of precursors from the Dufour gland which are derivatized by enzymes from the poison gland [33, 34]. Crematogaster poisons – from Dufour and poison glands, but also from hypertrophied metapleural glands – belong to such different chemical classes as cyclohexan derivatives, crematofuranes (cembranoid diterpenes), coumarin derivatives, alkylphenols, alkylresorcinols, salicylic acids, resorcylic acids, and polyfunctionalized long-chain derivatives [33, 35–38]. Since extracts of Cr. modiglianii Dufour glands contained the same steroid composition as the body surface (but no other compounds), they are probably produced in this gland and then distributed onto the body surface. In Cr. modiglianii, steroid synthesis did not depend on biosynthetic precursors acquired from food. In two colonies kept in the laboratory for 15 and 6 months, respectively, the steroid profile did not change despite of an artificial diet of cockroaches, honey solution and Bhatkar diet (F.M. pers. obs.). Moreover, in one forest colony, the steroid profile remained relatively constant over three years, corroborating that the steroid composition is rather genetically determined than dependent on environmental factors.
It is notable that 98% of the entire hydrocarbon profile of the red Ca. rufifemur (and ≥ 13% of the black Ca. rufifemur hydrocarbon profile) were methyl-branched alkenes. This substance class seems to be generally very rare in insects and has been detected only in several Diptera and one Noctuid moth as pheromones [39–41]. Among ants, they have been found in traces in the ponerine ant Pachycondyla villosa and in two Leptothorax species [42, 43], but in higher abundances only in Nothomyrmecia macrops surface profiles, which is probably the most primitive existent ant species . That they make up almost the entire hydrocarbon profile is therefore highly unusual. Another unusual feature in both parabiotic species is the high hydrocarbon chain lengths. Although common in this study (Table 1), hydrocarbons beyond C37 have not been found in non-parabiotic Camponotus and Crematogaster species [9, 27, 28]; unpublished data. Other studies report small concentrations of heavier hydrocarbons in other ant genera, but always accompanied by high amounts of lighter ones [45, 46]. It is possible that extremely long-chain hydrocarbons are difficult to perceive by receptors and thus promote interspecific tolerance [23, 47]. In one case, we observed that a non-parabiotic Cr. modiglianii colony was initially very aggressive against (black) Ca. rufifemur workers but treated them amicably (and had hence become habituated) after less than 24 h of exposure. Unsaturation in these long-chain hydrocarbons might be necessary to maintain a minimum fluidity of the cuticular profile .
Chemical overlap among parabiotic partners
Given the high allocolonial tolerance between parabiotic partners, the hydrocarbon overlap of the two species is surprisingly small. While the red Ca. rufifemur variety shared two compounds with its partner, the black variety only shared three trace compounds with Cr. modiglianii but otherwise possessed a completely different hydrocarbon profile. We tentatively suppose that Cr. modiglianii acquires 27-MeC39-14-ene 27-MeC39-16-ene from its red Ca. rufifemur partner although Ca. rufifemur generally tolerates Cr. modiglianii workers, including those lacking these substances . In a Cr. modiglianii colony kept in the laboratory without its previous red Camponotus partner, the compound disappeared from the profile after eight months of separation (F.M. pers. obs.). It is possible that the other hydrocarbons of the red Ca. rufifemur are acquired by Cr. modiglianii as well but remain beyond detectability due to their low abundances. The hydrocarbons of the black Ca. rufifemur, in contrast, were never found on Cr. modiglianii surface extracts. This is probably due to their high chain lengths, which makes the cuticular profile more solid and do not allow chemical transfer . In the light of the low overall hydrocarbon overlap among the two parabiotic ant species, chemical camouflage, a mechanism often found in social parasites [13–15], must be dismissed as an explanation for mutual tolerance. However, the existence of only few substances common to both species might be a sufficient signal for tolerating the partner .
The steroid components, in contrast, showed high congruence among both species. We found that the relative composition of eight steroid compounds differs between colonies but is very similar among the two species of a parabiotic nest. Since it is highly improbable that Ca. rufifemur is able to synthetically copy the steroid profile of each respective partner colony, this result suggests that Ca. rufifemur acquires steroids from Cr. modiglianii. Notably, only a certain set of steroids is transferred to Camponotus, while others, despite of high abundance in Cr. modiglianii, were almost or completely absent from the Ca. rufifemur profile.
Possible transfer mechanisms
Two mechanisms seem possible for the observed transfer of chemical cues, namely trophallaxis and direct physical contact. Via trophallaxis, individual ants exchange not only food but also the PPG content, i.e. hydrocarbons relevant for nestmate recognition . The PPG of Cr. modiglianii indeed contained steroids, albeit in much lower concentrations than on the body surface, thus making trophallaxis a possible pathway for chemical transfer. Interspecific trophallaxis has been observed between the two parabiotic species (F.M. and A. Endler, pers. obs.) and also shown via stained food only fed to Cr. modiglianii (F.M., pers. obs.).
Another possible transfer mechanism is direct physical contact. We frequently observed that Cr. modiglianii climbed on living or dead Ca. rufifemur individuals (workers and alates). The latter sometimes tried to shake them off but did not show aggression. Though almost never observed in the field, this 'mounting behaviour' could be easily induced in the laboratory by keeping the two species separate for one or two days. Mounting may therefore represent another possible mechanism for transfer of surface chemicals.
Partner recognition is not colony-specific
The red and the black variety of Camponotus rufifemur are chemically distinct and – apart from trace compounds – do not share any hydrocarbons. The two dominant surface components of the red variety (substance #52, Table 1) are present in Crematogaster modiglianii colonies associated with this Ca. rufifemur variety but almost completely absent from those living with the black variety. Their abundance thus allows separating 'red' from 'black' Cr. modiglianii albeit the remaining surface profile is similar. The existence of two chemical Ca. rufifemur varieties accounts for most of the aggression variance in allocolonial encounters between the two species. Cr. modiglianii usually tolerated living or dead Ca. rufifemur workers of the same variety as their parabiotic partner but fiercely attacked those of the respective other variety (Figures 3, 4, 5, Tables 4, 5, 6). An analogous pattern was found in Ca. rufifemur. Despite of generally low aggression levels, black Ca. rufifemur workers were significantly more aggressive towards 'red' Cr. modiglianii workers than towards allocolonial 'black' Cr. modiglianii (Figure 5b). However, we did not detect a corresponding difference in the red Ca. rufifemur.
While much of the interspecific aggression can be explained by chemical differences, however, the low interspecific aggression within chemical varieties is still surprising. Rather than recognizing heterospecific nestmates, the two species seemingly recognize only the chemical variety of their partner and do not discriminate within these varieties. Nestmate recognition rather depends on volatile substances than on substances only perceivable through antennal contact . Due to their low volatility , very long-chain hydrocarbons are less detectable than short-chain molecules. Thus, olfactory receptors may additionally absorb traces of lighter hydrocarbons, thereby blurring inter-colony profile differences and hampering inter-colony discrimination . The role of the steroids in the nestmate discrimination process is still unclear and under investigation.
The high interspecific tolerance strongly contrasts with the South American parabioses of Crematogaster limata and the ponerine ant Odontomachus mayi, where the ants never tolerated heterospecific workers from foreign parabioses . In these associations, very low chemical overlap was found (no substance data given), suggesting that both species habituated to each other's colony-specific profiles. The associated Chilean species Camponotus morosus and Solenopsis gayi also showed distinct hydrocarbon profiles . In contrast to non-associated colonies, however, associated C. morosus had acquired small amounts of the S. gayi hydrocarbons. In both of these species, only individuals from associated colonies were tolerant towards allocolonial allospecifics , indicating that the acquisition of allospecific hydrocarbons promoted mutual tolerance.
In this study we document the cuticular chemistry of the parabiotically associated ant species Camponotus rufifemur and Crematogaster modiglianii. In contrast to neotropical parabioses, these ant species did not show heterospecific nestmate recognition. In our experiments, Cr. modiglianii did not discriminate its partner Ca. rufifemur colony from other Ca. rufifemur colonies of the same chemical variety (nor vice versa). Rather, Cr. modiglianii distinguished only between the two Ca. rufifemur varieties, accepting the familiar one but attacking the respective other. This reduced discrimination of heterospecific nestmates may be caused by two unusual properties of the cuticular surface: Transfer of Ca. rufifemur hydrocarbons to the Cr. modiglianii profile (in one of the Ca. rufifemur varieties only), and the generally high chain hydrocarbon lengths in the two parabiotic species. As hypothesized elsewhere , extremely long-chain hydrocarbons may be difficult to perceive by receptors and hence promote chemical insignificance (sensu ). It is currently investigated whether the cuticular steroids unique to these species play a role in nestmate or partner recognition.
Study site and ants
The studies were conducted at Danum Valley Conservation Area from September to November in the years 2004 and 2007. Danum Valley represents one of the major remaining patches of tropical lowland rainforest in Sabah (Malaysian Borneo). The site has a typical equatorial rainforest climate with a mean annual temperature of 26.9°C and a yearly rainfall of 2700 mm. We studied parabiotic associations of Camponotus (Myrmotarsus) rufifemur Emery 1900 and Crematogaster (Paracrema) modiglianii Emery 1900. Their nests are commonly found in hollow, living tree trunks in the rainforest. Extracts of one parabiotic nest from the Kuala Belalong Field Studies Center (Brunei) were analyzed in addition.
Camponotus rufifemur occurs in two sympatric morphological varieties that have not previously been described (although Emery  notes that specimen from Sarawak are darker in colour than those from Sumatra). While one variety (henceforth, 'red' variety) has a reddish alitrunk and light red-brown legs, the other one (henceforth, 'black' variety) possesses a black alitrunk and dark red-brown legs. The area between the frontal carinae of soldiers is dull in the red but shining in the black variety. Although the ratio head width/scape length (in frontal view) tends to be higher in the large soldier caste of the black variety than in that of the red variety, no significant morphometric differences were found. In the following, we will refer to the varieties as 'red' and 'black' Ca. rufifemur. In order to allow a differentiation, the respective associated Cr. modiglianii will be called 'red' and 'black' Cr. modiglianii although we did not find morphological distinctions within this species. Voucher specimen of Cr. modiglianii and both Ca. rufifemur varieties are deposited at the Department of Zoology III, University of Würzburg and at the Forest Resarch Center in Sepilok, Sabah (Malaysia).
Preparation of extracts
Extracts were prepared from both body surface and postpharyngeal glands (PPGs). For body rinses, 10 to 90 ants were killed by freezing and immersed in hexane for ten minutes. Extracts from single individuals contained quantities too low for reliable substance identification. Eleven parabiotic nests were sampled with one to eight (mean: 3.5) replicates per colony and species (ten from Danum Valley, one from Kuala Belalong). PPG extracts were obtained from three to four freshly dissected PPGs per sample dissolved in hexane. Octadecane (n-C18) was used as internal standard in most samples. Cuticular substances were additionally obtained from living Cr. modiglianii workers brought into the laboratory in Würzburg with solid-phase microextraction (SPME). A SPME fibre (Supelco) coated with a 100 μm polydimethylsiloxan film was rubbed on the ant for 3 min and then directly injected into a ThermoQuest Trace GC.
Substances were identified by coupled capillary gas chromatography-mass spectrometry (GC-MS) with a Hewlett Packard 6890 series gas chromatograph coupled to a HP 5973 Mass Selective Detector. The GC was equipped with a J&W Scientific DB-5 fused silica capillary column (30 m × 0.25 mm ID; df = 0.25 μm). Temperature was kept at 60°C for 2 min then increased by 60°C/min up to 200°C and subsequently by 4°C/min to 320°C, where it remained constant for 10 min. Helium was used as carrier gas with a constant flow of 1 ml/min. A split/splitless injector was installed at 250°C in the splitless mode for 30 s. The electron impact mass spectra (EI-MS) were recorded with an ionisation voltage of 70 eV, a source temperature of 230°C and an interface temperature of 325°C. For analysis of hydrocarbons beyond C41, we used a DB-1 HT column (30 m × 0.25 mm ID; df = 0,25 μm). Temperature was raised from 60°C by 5°C/min up to 350°C and then kept constant for 10 min. The interface had a temperature of 350°C. All other settings were as above. The software MSD ChemStation (Version A.03.00) for Windows was used for data acquisition. We restricted the analyses to substances with a retention time beyond that of C19 since compounds with shorter chain length are likely to be too volatile to be relevant for nestmate recognition [5, 6]. Substances present in less than 50% of the samples are given in Table 1 (marked with *) but were disregarded from further analysis.
For quantification of steroid-like compounds and aliphatics shorter than C33, we used ion counts from the GC-MS data and analysed both substance classes separately. Heavier hydrocarbons (beyond C33) were quantified using a high-resolution ThermoQuest Trace GC-FID with H2 as carrier gas in order to achieve a better separation of the substances. We used a nonpolar capillary column [DB1 (J&W Scientific, Folsom, CA), 20 m × 0.18 mm, 0.18 μm film thickness] and the first temperature program given above (split closed for 30 s for extracts and for 2 min when using SPME fibers). The split/splitless injector port was kept at 260°C and the flame ionization detector (FID) at 340°C. Peak areas were computed with Chrom-Card 1.19 (CE Instruments, Milan, Italy). Mean absolute substance quantities were estimated by comparing substance peak areas with that of the internal standard (acquired with GC-FID) and dividing by the number of extracted individuals.
Profile similarities between the two partner species were analyzed for eleven parabiotic nests (including one from Kuala Belalong Field Studies Center). The average proportions of the steroid components per colony and species were calculated. The distances between colonies were calculated for each species separately using Bray-Curtis index of similarity and then compared between species using a Mantel test (1000 permutations).
Identification of cuticular hydrocarbons
Alkanes, methyl-branched alkanes and alkenes were characterized using diagnostic ions and retention indices calculated using Kovats' method . Unsaturated methyl-branched hydrocarbons were hydrated under a H2 atmosphere using Palladium on activated carbon as catalyst to determine the position of the methyl group. The position of the double bond in methyl-branched and n-alkenes was determined using DMDS derivatization following . For methyl-branched alkenes, DMDS derivatization was insufficient for substance characterization since the position of the double bond relative to the methyl group remained unresolved and left two possible structures. Therefore, we cleaved the molecules in two parts at the position of the double bond via ozonisation. We diluted the sample in approx. 3 ml hexane, applied a constant flow of O3 (300 mg/h) for ten minutes from a glass pipette (EO3G Ozone Generator, Easelec Technology Inc.) and directly injected the sample into the GC-MS. Ozonisation succeeded for substance 52 but not for the substances 37, 43, 56, and 61 (surface compounds of the red Ca. rufifemur, Table 1). However, it is highly probable that all methyl-branched alkenes are produced via the same biosynthetic pathway. We therefore tentatively inferred the position of the double bond from the structure of substances 52 (Table 1) and possibilities left from the DMDS results, which had succeeded for all of the above substances. Double bond positions in alkenes with chain lengths higher than C41 as well as in dienes and trienes could not be determined due to their low abundance and/or their high chain length, which resulted in derivatives which could not be detected using GC-MS. Aldehydes were identified by comparing their mass spectra to a commercial library (Wiley 275) and therefore remain tentative.
For the substances of the black Ca. rufifemur profile beyond C44, retention indices were calculated based on the retention times of an n-alkane standard (C21 to C40), C47 and C49, and therefore remain preliminary. These substances were identified based on mass spectra and hydrated samples. Unsaturation was further confirmed via fractionation using a SiOH column treated with AgNO3. However, their characterization remains preliminary since the DMDS derivatized substances could not be detected using GC-MS.
We studied the reaction of Crematogaster modiglianii towards dead Ca. rufifemur workers from different colonies in Borneo. The reverse situation (Ca. rufifemur towards Cr. modiglianii) was not studied in this paper since Ca. rufifemur shows little discrimination between different Cr. modiglianii workers . A Cr. modiglianii colony (R0) had been collected in the forest circa one week prior to the experiments and was kept together with its red Ca. rufifemur partner in its original nest (a small tree trunk) in an open plastic box. The dead ants were placed onto the nest trunk with forceps such that several ants could interact with it simultaneously. During three minutes, each observed interaction was classified as peaceful (antennating), weakly (open mandibles) or strongly aggressive (biting or locking mandibles). An additional behaviour classified as peaceful was 'mounting', where the smaller Crematogaster (body length approx. 2–3 mm) climbed onto the Camponotus body (body length 5–13 mm). Continued interactions were recorded again after 10 s (the same behavioural classification as used in ).
The aggressiveness of two other Cr. modiglianii colonies was estimated in arena confrontations. The workers had been collected in the forest one day prior to the tests and were kept in a plastic box among nestmates (but separate from the partner species) over night. Five Cr. modiglianii individuals were placed into a fluon-covered plastic cylinder (Ø 7.5 cm, height 5 cm) on top of a paper sheet floor. After 1 min to calm down, a dead Camponotus specimen was introduced. For the following 100 s we recorded the behaviour of the ants as above. Each living or dead ant was used for one assay only. In all of the above assays, we performed ten replicates per treatment.
From each replicate we calculated the proportions of all aggressive versus all non-aggressive interactions. Both strong and total (including weak) aggression were analyzed using generalized linear models (GLM) with quasibinomial error distribution and logit link function. In order to determine whether confrontations within and across chemical varieties differ, we used the according explanatory variable 'within/across variety' with two factor levels (which collapsed to 'Camponotus variety' in the first dataset). The variable 'variety combination' (with the factor levels 'black→black', 'black→red', 'red→red', and 'red→black') was nested in the former one. Further explanatory variables were 'colony combination' (nested in 'variety combination'), which collapsed to 'Camponotus colony' in the first dataset, and 'intra-/allocolonial'. Due to their nested structure, no interactions between the variables were possible. The impact of each variable was determined by likelihood ratio tests (F tests). We also re-analyzed data from  in a similar way, where we included the number of workers present in the experimental arena as explanatory variables (see  for details on the experimental setup). Since the statistical results for total aggression and for strong aggression only were similar, only the former will be reported in the results section. All computations were performed in R Version 2.5.1 .
We thank Annett Endler and Heike Feldhaar for support regarding chemical analyses. Brigitte Fiala kindly provided samples from Brunei for further analysis. We are grateful to the Danum Valley Management Committee (DVMC) and Malaysian Economic Planning Unit (EPU) for research permission, the Royal Society's South East Asia Rainforest Research Programme (SEARRP) and the staff at Danum Valley Field Center, especially Bernadus 'Mike' Bala Ola for logistic support and help in the field. Dr. Arthur Chung (Forest Research Center, Sepilok) kindly supported our work as local collaborator. Finally, we would like to thank three anonymous reviewers for helpful comments on the manuscript. This research was supported by the Sonderforschungsbereich 'Mechanisms and Evolution of Arthropod Behaviour' (SFB-554) of the German Research Foundation (DFG) and complies with current laws of Malaysia and Germany. F.M. was supported by a doctoral fellowship provided by the German National Academic Foundation (Studienstiftung des deutschen Volkes) and the Bayerische Graduiertenförderung.
- Lenoir A, D'Ettorre P, Errard C, Hefetz A: Chemical Ecology and Social Parasitism in Ants. Annual Review of Entomology. 2001, 46: 573-599. 10.1146/annurev.ento.46.1.573.View ArticlePubMedGoogle Scholar
- Menzel F, Linsenmair KE, Blüthgen N: Selective interspecific tolerance in tropical Crematogaster-Camponotus associations. Animal Behaviour. 2008, 75: 837-846. 10.1016/j.anbehav.2007.07.005.View ArticleGoogle Scholar
- Orivel J, Errard C, Dejean A: Ant gardens: Interspecific recognition in parabiotic ant species. Behavioral Ecology and Sociobiology. 1997, 40: 87-93. 10.1007/s002650050319.View ArticleGoogle Scholar
- Hölldobler B, Wilson EO: The Ants. 1990, Berlin, Heidelberg: Springer VerlagView ArticleGoogle Scholar
- Vander Meer RK, Morel L: Nestmate Recognition in Ants. Pheromone communication in social insects. Ants, wasps, bees, and termites. Edited by: Vander Meer RK, Breed MD, Espelie KE, Winston ML. 1998, Boulder: Westview Press, 79-103.Google Scholar
- Lenoir A, Malosse C, Yamaoka R: Chemical mimicry between parasitic ants of the genus Formicoxenus and their host Myrmica (Hymenoptera, Formicidae). Biochemical Systematics and Ecology. 1997, 25: 379-389. 10.1016/S0305-1978(97)00025-2.View ArticleGoogle Scholar
- Lahav S, Soroker V, Hefetz A: Direct behavioral evidence for hydrocarbons as ant recognition discriminators. Naturwissenschaften. 1999, 86: 246-249. 10.1007/s001140050609.View ArticleGoogle Scholar
- Wagner D, Tissot M, Cuevas W, Gordon DM: Harvester ants utilize cuticular hydrocarbons in nestmate recognition. Journal of Chemical Ecology. 2000, 26: 2245-2257. 10.1023/A:1005529224856.View ArticleGoogle Scholar
- Boulay R, Hefetz A, Soroker V, Lenoir A: Camponotus fellah colony integration: worker individuality necessitates frequent hydrocarbon exchanges. Animal Behaviour. 2000, 59: 1127-1133. 10.1006/anbe.2000.1408.View ArticlePubMedGoogle Scholar
- Boulay R, Katzav-Gozansky T, Hefetz A, Lenoir A: Odour convergence and tolerance between nestmates through trophallaxis and grooming in the ant Camponotus fellah (Dalla Torre). Insectes Sociaux. 2004, 51: 55-61. 10.1007/s00040-003-0706-0.View ArticleGoogle Scholar
- Soroker V, Vienne C, Hefetz A, Nowbahari E: The Postpharyngeal Gland as a "Gestalt" Organ for Nestmate Recognition in the Ant Cataglyphis niger. Naturwissenschaften. 1994, 81: 510-513.Google Scholar
- Errard C, Hefetz A, Jaisson P: Social discrimination tuning in ants: template formation and chemical similarity. Behavioral Ecology and Sociobiology. 2006, 59: 353-363. 10.1007/s00265-005-0058-z.View ArticleGoogle Scholar
- Akino T: Chemical camouflage by myrmecophilous beetles Zyras comes (Coleoptera: Staphylinidae) and Diaritiger fossulatus (Coleoptera: Pselaphidae) to be integrated into the nest of Lasius fuliginosus (Hymenoptera: Formicidae). Chemoecology. 2002, 12: 83-89. 10.1007/s00049-002-8330-4.View ArticleGoogle Scholar
- Akino T, Knapp JJ, Thomas JA, Elmes GW: Chemical mimicry and host specificity in the butterfly Maculinea rebeli, a social parasite of Myrmica ant colonies. Proceedings of the Royal Society of London, Series B. 1999, 266: 1419-1426. 10.1098/rspb.1999.0796.View ArticleGoogle Scholar
- Akino T, Mochizuki R, Morimoto M, Yamaoka R: Chemical camouflage of myrmecophilous cricket Myrmecophilus. sp to be integrated with several ant species. Japanese Journal of Applied Entomology and Zoology. 1996, 40: 39-46.View ArticleGoogle Scholar
- Howard RW, Stanley-Samuelson DW, Akre RD: Biosynthesis and chemical mimicry of cuticular hydrocarbons from an obligate predator, Microdon albicomatus Novak (Diptera: Syrphidae) and its ant prey, Myrmica incompleta Provancheri (Hymenoptera: Formicidae). Journal of the Kansas Entomological Society. 1990, 63: 437-443.Google Scholar
- Dronnet S, Simon X, Verhaeghe J-C, Rasmont P, Errard C: Bumblebee inquilinism in Bombus (Fernaeldaepsithyrus) sylvestris (Hymenoptera, Apidae): behavioural and chemical analyses of host-parasite interactions. Apidologie. 2005, 36: 59-70. 10.1051/apido:2004070.View ArticleGoogle Scholar
- Elgar MA, Allan RA: Predatory spider mimics acquire colony-specific cuticular hydrocarbons from their ant model prey. Naturwissenschaften. 2004, 91: 143-147. 10.1007/s00114-004-0507-y.View ArticlePubMedGoogle Scholar
- Howard RW, Perez-Lachaud G, Lachaud JP: Cuticular hydrocarbons of Kapala sulcifacies (Hymenoptera: Eucharitidae) and its host, the ponerine ant Ectatomma ruidum (Hymenoptera: Formicidae). Annals of the Entomological Society of America. 2001, 94: 707-716. 10.1603/0013-8746(2001)094[0707:CHOKSH]2.0.CO;2.View ArticleGoogle Scholar
- D'Ettorre P, Mondy N, Lenoir A, Errard C: Blending in with the crowd: social parasites integrate into their host colonies using a flexible chemical signature. Proceedings of the Royal Society of London, Series B. 2002, 269: 1911-1918. 10.1098/rspb.2002.2110.View ArticleGoogle Scholar
- Jeral JM, Breed MD, Hibbard BE: Thief ants have reduced quantities of cuticular compounds in a ponerine ant, Ectatomma ruidum. Physiological Entomology. 1997, 22: 207-211. 10.1111/j.1365-3032.1997.tb01160.x.View ArticleGoogle Scholar
- Akino T: Cuticular hydrocarbons of Formica truncorum (Hymenoptera: Formicidae): Description of new very long chained hydrocarbon components. Applied Entomology and Zoology. 2006, 41: 667-677. 10.1303/aez.2006.667.View ArticleGoogle Scholar
- Lambardi D, Dani FR, Turillazzi S, Boomsma JJ: Chemical mimicry in an incipient leaf-cutting ant social parasite. Behavioral Ecology and Sociobiology. 2007, 61: 843-851. 10.1007/s00265-006-0313-y.View ArticleGoogle Scholar
- Errard C, Regla JI, Chambris A: Interspecific recognition in chilean parabiotic ant species. Presented at 3rd Jornadas Chile – Francia di Etologia e Sociobiologia. 1996, Universidad Mayor, Santiago, ChileGoogle Scholar
- Liu ZB, Yamane S, Yamamoto H, Wang QC: Nestmate discrimination and cuticular profiles of a temporary parasitic ant Lasius sp. and its host L. fuliginosus (Hymenoptera, Formicidae). Journal of Ethology. 2000, 18: 69-73. 10.1007/s101640070002.View ArticleGoogle Scholar
- Liu ZB, Bagneres AG, Yamane S, Wang QC, Kojima J: Cuticular hydrocarbons in workers of the slave-making ant Polyergus samurai and its slave, Formica japonica (Hymenoptera: Formicidae). Entomological Science. 2003, 6: 125-133. 10.1046/j.1343-8786.2003.00014.x.View ArticleGoogle Scholar
- Boulay R, Katzav-Gozansky T, Vander Meer RK, Hefetz A: Colony insularity through queen control on worker social motivation in ants. Proceedings of the Royal Society of London, Series B. 2003, 270: 971-977. 10.1098/rspb.2002.2325.View ArticleGoogle Scholar
- Endler A, Liebig J, Schmitt T, Parker JE, Jones GR, Schreier P, Hölldobler B: Surface hydrocarbons of queen eggs regulate worker reproduction in a social insect. Proceedings of the National Academy of Sciences of the United States of America. 2004, 101: 2945-2950. 10.1073/pnas.0308447101.PubMed CentralView ArticlePubMedGoogle Scholar
- Meskali M, Bonavitacougourdan A, Provost E, Bagneres AG, Dusticier G, Clement JL: Mechanism underlying cuticular hydrocarbon homogeneity in the ant Camponotus vagus (Scop) (Hymenopters, Formicidae) – role of postpharyngeal glands. Journal of Chemical Ecology. 1995, 21: 1127-1148. 10.1007/BF02228316.View ArticlePubMedGoogle Scholar
- Ayasse M, Engels W, Lübke G, Taghizadeh T, Francke W: Mating expenditures reduced via female sex pheromone modulation in the primitively eucosial halictine bee, Lasioglossum (Evylaeus) malachurum (Hymenoptera: Halictidae). Behavioral Ecology and Sociobiology. 1999, 45: 95-106. 10.1007/s002650050543.View ArticleGoogle Scholar
- Heredia A, De Bizeau JC, Quinet Y: Toxicity of the venom in three neotropical Crematogaster ants (Formicidae: Myrmicinae). Chemoecology. 2005, 15: 235-242. 10.1007/s00049-005-0317-5.View ArticleGoogle Scholar
- Marlier JF, Quinet Y, de Bizeau JC: Defensive behaviour and biological activities of the abdominal secretion in the ant Crematogaster scutellaris (Hymenoptera: Myrmicinae). Behavioural Processes. 2004, 67: 427-440. 10.1016/j.beproc.2004.07.003.View ArticlePubMedGoogle Scholar
- Leclerq S, Braekman JC, Kaisin M, Daloze D: Venom constituents of three species of Crematogaster ants from Papua New Guinea. Journal of natural products. 1997, 60: 1143-1147. 10.1021/np970202h.View ArticleGoogle Scholar
- Pasteels JM, Daloze D, Boeve JL: Aldehydic contact poisons and alarm pheromone of the ant Crematogaster scutellaris (Hymenoptera: Myrmicinae) – enzyme mediated production from acetate precursors. Journal of Chemical Ecology. 1989, 15: 1573-1561. 10.1007/BF01012379.View ArticleGoogle Scholar
- Daloze D, de Biseau JC, Leclerq S, Braekman J-C, Quinet Y, Pasteels JM: (13E,15E,18Z,20Z)-1-Hydroxypentacosa-13,15,18,20-tetraen-11-yn-4-one 1 acetate, from the venom of a Brazilian Crematogaster ant. Tetrahedron Letters. 1998, 39: 4671-4672. 10.1016/S0040-4039(98)00875-2.View ArticleGoogle Scholar
- Jones TH, Brunner SR, Edwards AA, Davidson DW, Snelling RR: 6-Alkylsalicylic acids and 6-alkylresorcylic acids from ants in the genus Crematogaster from Brunei. Journal of Chemical Ecology. 2005, 31: 407-417. 10.1007/s10886-005-1349-6.View ArticlePubMedGoogle Scholar
- Laurent P, Hamdani A, Braekman JC, Daloze D, Isbell LA, de Biseau JC, Pasteels JM: New 1-alk(en)yl-1,3,5-trihydroxycyclohexanes from the Dufour gland of the African ant Crematogaster nigriceps. Tetrahedron Letters. 2003, 44: 1383-1386. 10.1016/S0040-4039(02)02870-8.View ArticleGoogle Scholar
- Leclerq S, de Biseau JC, Braekman JC, Daloze D, Quinet Y, Luhmer M, Sundin A, Pasteels JM: Furanocembranoid diterpenes as defensive compounds in the dufour gland of the ant Crematogaster brevispinosa rochai. Tetrahedron. 2000, 56: 2037-2042. 10.1016/S0040-4020(00)00113-7.View ArticleGoogle Scholar
- Carlson DA, Mramba F, Sutton BD, Bernier UR, Geden CJ, Mori K: Sex pheromone of the tsetse species, Glossina austeni : isolation and identification of natural hydrocarbons, and bioassay of synthesized compounds. Medical and veterinary entomology. 2005, 19: 470-479. 10.1111/j.1365-2915.2005.00596.x.View ArticlePubMedGoogle Scholar
- Etges WJ, Jackson LL: Epicuticular hydrocarbon variation in Drosophila mojavensis cluster species. Journal of Chemical Ecology. 2001, 27: 2125-2149. 10.1023/A:1012203222876.View ArticlePubMedGoogle Scholar
- Francke W, Plass E, Zimmermann N, Tietgen H, Tolasch T, Franke S, Subchev M, Toshova T, Pickett JA, Wadhams LJ, Woodcock CM: Major sex pheromone component of female herald moth Scoliopteryx libatrix is the novel branched alkene (6Z, 13)-methylheneicosene. Journal of Chemical Ecology. 2000, 26: 1135-1149. 10.1023/A:1005475725742.View ArticleGoogle Scholar
- Tentschert J, Bestmann HJ, Heinze J: Cuticular compounds of workers and queens in two Leptothorax ant species – a comparison of results obtained by solvent extraction, solid sampling, and SPME. Chemoecology. 2002, 12: 15-21. 10.1007/s00049-002-8322-4.View ArticleGoogle Scholar
- Lucas C, Fresneau D, Kolmer K, Heinze J, Delabie JHC, Pho DB: A multidisciplinary approach to discriminating different taxa in the species complex Pachycondyla villosa (Formicidae). Biological Journal of the Linnean Society. 2002, 75: 249-259. 10.1111/j.1095-8312.2002.tb01425.x.View ArticleGoogle Scholar
- Brown WV, Jaisson P, Taylor RW, Lacey MJ: Novel internally branched, internal alkenes as major components of the cuticular hydrocarbons of the primitive Australian ant Nothomyrmecia macrops Clark (Hymenoptera: Formicidae). Journal of Chemical Ecology. 1990, 16: 2623-2635. 10.1007/BF00988074.View ArticlePubMedGoogle Scholar
- Dalecky A, Renucci M, Tirard A, Debout G, Roux M, Kjellberg F, Provost E: Changes in composition of cuticular biochemicals of the facultatively polygynous ant Petalomyrmex phylax during range expansion in Cameroon with respect to social, spatial and genetic variation. Molecular Ecology. 2007, 16: 3778-3791. 10.1111/j.1365-294X.2007.03429.x.View ArticlePubMedGoogle Scholar
- Lucas C, Pho DB, Fresneau D, Jallon JM: Hydrocarbon circulation and colonial signature in Pachycondyla villosa. Journal of Insect Physiology. 2004, 50: 595-607. 10.1016/j.jinsphys.2004.04.006.View ArticlePubMedGoogle Scholar
- Gibbs A, Pomonis JG: Physical properties of insect cuticular hydrocarbons: The effects of chain lengths, methyl branching and unsaturation. Comparative Biochemistry and Physiology. 1995, 112B: 243-249.View ArticleGoogle Scholar
- D'Ettorre P, Heinze J, Schulz C, Francke W, Ayasse M: Does she smell like a queen? Chemoreception of a cuticular hydrocarbon signal in the ant Pachycondyla inversa. Journal of Experimental Biology. 2004, 207: 1085-1091. 10.1242/jeb.00865.View ArticlePubMedGoogle Scholar
- Lenoir A, Fresneau D, Errard C, Hefetz A: Individuality and colonial identity in ants: the emergence of a social representation concept. Information Processing in Social Insects. Edited by: Detrain C, Pasteels JM, Deneubourg JL. 1999, Basel: Birkhäuser, 219-237.View ArticleGoogle Scholar
- Brandstaetter AS, Endler A, Kleineidam CJ: Nestmate recognition in ants is possible without tactile interaction. Naturwissenschaften. 2008, 95: 601-608. 10.1007/s00114-008-0360-5.View ArticlePubMedGoogle Scholar
- Errard C, Regla JI, Hefetz A: Interspecific recognition in Chilean parabiotic ant species. Insectes Sociaux. 2003, 50: 268-273. 10.1007/s00040-003-0677-1.View ArticleGoogle Scholar
- Emery C: Formiche raccolte da Elio Modigliani in Sumatra, Engano e Mentawei. Annali del Museo Civico di Storia Naturale di Genova, Serie 2. 1900, 20: 661-722.Google Scholar
- Carlson DA, Bernier UR, Sutton BD: Elution patterns from capillary GC for methyl-branched alkanes. Journal of Chemical Ecology. 1998, 24: 1845-1865. 10.1023/A:1022311701355.View ArticleGoogle Scholar
- Dunkelblum E, Tan SH, Silk PJ: Double-bond location in monounsaturated fatty acids by dimethyl disulfide derivatization and mass spectrometry: Application to analysis of fatty acids in pheromone glands of four Lepidoptera. Journal of Chemical Ecology. 1985, 11: 265-277. 10.1007/BF01411414.View ArticlePubMedGoogle Scholar
- R Development Core Team: A language and environment for statistical computing. 2007, Vienna, R Foundation for Statistical Computing, [http://www.R-project.org]Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.