Hydrocarbons ofNasutitermes acajutlae and comparison of methodologies for sampling cuticular hydrocarbons of caribbean termites for taxonomic and ecological studies

Using data from the arboreal nestingNasutitermes acajutlae (Holmgren), we propose standard collection and extraction methodology for characterization of cuticular hydrocarbons of termites under field conditions in the tropics. Specifically, we evaluated: (1) the effect of the duration and the number of extractions; (2) the effect of drying termites before extraction; (3) the effect of sample size; (4) the effect of solvents (ethanol versus hexane) on cuticular hydrocarbon profiles. Olefins comprise ca. 70% of the cuticular hydrocarbons ofN. acajutlae. Hydrocarbons consist of two distinct groups: early-eluting components, primarilyn-alkanes and methyl-branched alkanes, and late-eluting compounds, which consist almost exclusively of unsaturated components with one to six double bonds. Soldiers have more early-eluting compounds than workers or alates. Nests from the same island had qualitatively similar, but quantitatively dissimilar hydrocarbon mixtures. Brief extractions of 300 live workers in 10 ml of hexane for only 20 sec produced a hydrocarbon mixture equivalent to a 10-min extraction. Long-term extraction of 300 workers in hexane for two years resulted in different mixtures of hydrocarbons. Drying workers tended to enhance extraction of the less abundant unsaturated compounds such as C_41.4 and C_41.5. A single extraction of a minimum of 100 workers (live or dried), with hexane for 20 sec to 10 min is best; these extraction regimes resulted in mixtures of hydrocarbons that are quantitatively very similar. For quantitative comparisons, extracts from dried samples should not be compared to those from live samples. Storage in ethanol caused numerous unidentified, nonhydrocarbon compounds to be extracted either from the cuticle or from internal tissues.     

Alkadienes mediating courtship in the parasitoidCardiochiles nigriceps (Hymenoptera: Braconidae)

Courtship inCardiochiles nigriceps (Viereck) (Hymenoptera: Braconidae) involves male attraction to females and male antennation of females followed by mounting and copulation. Once the female is located, antennation and mounting of the female are mediated by the hydrocarbon fraction of female's Dufour's gland and cuticle. The identification of the cuticular hydrocarbons of males and females revealed a mixture of alkanes and alkenes, in addition to a series of alkadienes specific to females. These female-specific alkadienes reported from Braconidae are unusual among insect alkadienes in that the second double bond occurs in the middle of the molecule. Bioassays with three available alkadienes revealed that contact behaviors (antennation and mounting) are in part mediated by the (Z,Z)-7, 13-heptacosadiene and at least one other alkadiene in combination with other hydrocarbons found in males and females.     

A Non-lethal Water-based Removal-reapplication Technique for Behavioral Analysis of Cuticular Compounds of Ants

Interspecific relationships among insects are often mediated by chemical cues, including non-volatile cuticular compounds. Most of these compounds are hydrocarbons that necessitate the use of solvents for their extraction, identification, and manipulation during behavioral assays. The toxicity of these solvents often precludes the removal and reapplication of hydrocarbons from and to live insects. As a consequence, dummies often are used in behavioral assays, but their passivity can bias the behavior of the responding insects. To overcome these limitations, we propose a method where cuticular compounds are extracted from live ants by placing them into glass vials half-filled with tepid water (ca. 34°C) and vigorously shaking the vials to form an emulsion whose supernatant can be analyzed and/or reapplied to other ants. We demonstrate that cuticular compounds can be extracted from workers of the red fire ant, Solenopsis saevissima, and reapplied to the cuticle of workers from a sympatric species, Camponotus blandus (both Hymenoptera: Formicidae), while keeping the ants alive. Gas chromatographic-mass spectrometric analysis and behavioral assays were used to confirm the successful transfer of the behaviorally active compounds.     

Courtship Pheromones in Parasitic Wasps: Comparison of Bioactive and Inactive Hydrocarbon Profiles by Multivariate Statistical Methods

Cuticular hydrocarbons play a significant role in the regulation of cuticular permeability and also in the chemical communication of insects. In the parasitoid Lariophagus distinguendus (Hymenoptera: Pteromalidae), male courtship behavior is mediated by a female-produced sex pheromone. Previous studies have shown that the chemicals involved are already present in the pupal stage of both males and females. However, pheromonal activity in males decreases shortly after emergence. This pheromonal deactivation occurs only in living males, suggesting an active process rather than simple evaporation of bioactive compounds. Here, we present evidence that the sex pheromone of L. distinguendus is composed of a series of cuticular hydrocarbons. Filter paper disks treated with nonpolar fractions of cuticular extracts of freshly emerged males and females, 72-hr-old females, and yellowish pupae caused arrestment and stimulated key elements of courtship behavior in males, whereas fractions of 72-hr-old males did not. Sixty-four hydrocarbons with chain length between C₂₅ and C₃₇ were identified in the fractions by gas chromatography-mass spectrometry (GC-MS). Methyl-branched alkanes with one to four methyl groups were major components, along with traces of n-alkanes and monoalkenes. Principal component analysis, based on the relative amounts of the compounds, revealed that cuticular hydrocarbon composition differed among all five groups. By using partial least squares-discriminant analysis, we determined a series of components that differentiate bioactive and bioinactive hydrocarbon profiles, and may be responsible for pheromonal activity of hydrocarbon fractions in L. distinguendus.     

Cuticular hydrocarbon profiles of dominant versus subordinate male Nauphoeta cinerea cockroaches

Interactions between male Nauphoeta cinerea cockroaches are characterized by an elaborate ritual that leads to a stable dominant–subordinate hierarchy between two individuals. Chemical signals involving volatile sex pheromones and cuticular hydrocarbons play an important role in establishing and maintaining dominance status. The present study was performed to identify cuticular hydrocarbons in two- and three-times dominant or subordinate individuals obtained by forcing dyadic encounters. Two methods, i.e., solid-phase microextraction (SPME) and organic solvent extraction, were used to isolate cuticular hydrocarbons. A total of 23 peaks of cuticular hydrocarbons were identified. Analysis showed quantitative differences in hydrocarbon profiles of three-times dominant and subordinate individuals according to extraction method and dominance status. Dominant individuals were characterized by higher proportions of the monomethylalkanes 11- and 13-MeC₃₆, 13- and 15-MeC₃₈, and 11-, 13-, and 15-MeC₃₅, while subordinate individuals had higher proportions of the following monomethylalkanes: 7-, 9-, and 11-MeC₃₁, 5-MeC₃₁, 3- and 8-MeC₃₂, and 9-, 10- , 11-, and 12-MeC₃₂. Discussion focuses on the reliability of hydrocarbons as indicators of dominance status and on their correlation with physiological processes.     

Cuticular Hydrocarbon Composition Reflects Genetic Relationship Among Colonies of the Introduced Termite Reticulitermes santonensis Feytaud

Nestmate recognition plays a key role in kin selection to maintain colony integrity in social insects. Previous studies have demonstrated that nestmate recognition is dependent on detection of cuticular hydrocarbons. However, the absence of intraspecific aggression between some colonies of Isoptera and social Hymenoptera questions whether kin recognition must occur in social insects. The purpose of this study was to determine if cuticular hydrocarbon similarity and high genetic relatedness could explain the lack of intraspecific aggression among and within colonies of the introduced subterranean termite Reticulitermes santonensis. We performed both GC analysis of cuticular hydrocarbons and genotyping by using 10 DNA microsatellite loci on the same 10 workers from each of 14 parisian colonies. Multivariate analyses demonstrated correspondence between cuticular hydrocarbon patterns and genetic variation. By using a redundancy analysis combining chemical and genetic data, we found that a few hydrocarbons (mainly short vs. long chains; saturated vs. unsaturated alkanes) were associated with most genetic variation. We also found a strong positive correlation between chemical and genetic distances between colonies, thus providing evidence of a genetic basis for cuticular hydrocarbon variation. However, genetic distance did not account for all chemical variation, thus suggesting that some hydrocarbon variation was environmentally derived. Investigation at the intracolony level indicated that cuticular hydrocarbons did not depend on colony social structure. Based on our findings, we speculate that the absence of intraspecific aggression in R. santonensis may result from a loss of diversity in genetically derived recognition compounds in this species that presumably descended from R. flavipes populations imported from North America.     

Insect Adhesion Secretions: Similarities and Dissimilarities in Hydrocarbon Profiles of Tarsi and Corresponding Tibiae

Spatially controlled in vivo sampling by contact solid phase microextraction with a non-coated silica fiber combined with gas chromatography-mass spectrometry (GC-MS) was utilized for hydrocarbon profiling in tarsal adhesion secretions of four insect species (Nicrophorus vespilloides, Nicrophorus nepalensis, Sagra femorata, and Gromphadorhina portentosa) by using distinct adhesion systems, viz. hairy or smooth tarsi. For comparison, corresponding samples from tibiae, representing the general cuticular hydrocarbon profile, were analyzed to enable the statistical inference of active molecular adhesion principles in tarsal secretions possibly contributed by specific hydrocarbons. n-Alkanes, monomethyl and dimethyl alkanes, alkenes, alkadienes, and one aldehyde were detected. Multivariate statistical analysis (principal component and orthogonal partial least square discriminant analyses) gave insights into distinctive molecular features among the various insect species and between tarsus and tibia samples. In general, corresponding hydrocarbon profiles in tarsus and tibia samples largely resembled each other, both qualitatively and in relative abundances as well. However, several specific hydrocarbons showed significantly different relative abundances between corresponding tarsus and tibia samples, thus indicating that such differences of specific hydrocarbons in the complex mixtures might constitute a delicate mechanism for fine-tuning the reversible attachment performances in tarsal adhesive fluids that are composed of substances originating from the same pool as cuticular hydrocarbons. Caused by melting point depression, the multicomponent tarsal adhesion secretion, made up of straight chain alkanes, methyl alkanes, and alkenes will have a semi-solid, grease-like consistency, which might provide the basis for a good reversible attachment performance.     

Role of cuticular hydrocarbons of aphid parasitoids in their relationship to aphid-attending ants

Lysiphlebus cardui, the dominant aphidiid parasitoid of the black bean aphid,Aphis fabae cirsiiacanthoidis (Afc), on creeping thistle, is able to forage in ant-attended aphid colonies without being attacked by ants. Several behavioral observations and experimental studies led to the hypothesis thatL. cardui mimics the cuticular hydrocarbon profile of its host aphid. Chemical analysis of the cuticular extracts revealed that bothL. cardui and Afc exclusively possess saturated hydrocarbons:n-alkanes, monomethyl (MMA), dimethyl (DMA), and trimethyl alkanes (TMA). Comparison of the hydrocarbon profiles of parasitoid and aphid showed great qualitative resemblance between parasitoid and host:L. cardui possesses almost all host-specific compounds in addition to species-specific hydrocarbons of mainly higher molecular weight (>C₃₀). However, there is a lesser quantitative correspondence between parasitoid and host aphid. Furthermore, we analyzed the cuticular hydrocarbon profile of another parasitoid of Afc,Trioxys angelicae. This aphidiid species is vigorously attacked and finally killed by honeydewcollecting ants when encountered in aphid colonies. Its cuticular hydrocarbon profile is characterized by the presence of large amounts of (Z)-11-alkenes of chain lenghts C₂₇, C₂₉, C₃₁, and C₃₃, in addition to alkanes and presumably trienes. The role of the unsaturated hydrocarbons onT. angelicae as recognition cues for aphid-attending ants is discussed.     

How Reliable is the Analysis of Complex Cuticular Hydrocarbon Profiles by Multivariate Statistical Methods?

Numerous recent studies have correlated cuticular hydrocarbon profiles with a wide range of behaviors, particularly in social insects. These findings are wholly or partly based on multivariate statistical methods such as discriminate analysis (DA) or principal component analysis (PCA). However, these methods often provide limited insight into the biological processes that generate the small differences usually detected. This may be a consequence of variability in the system due to inadequate sample sizes and the assumption that all compounds are independent. A fundamental problem is that these methods combine rather than separate the effects of signal components. By using cuticular hydrocarbon data from previous social insect studies, we showed that: (1) in 13 species of Formica ants and seven species of Vespa hornets, at least one group of hydrocarbons in each species was highly (r ² > 0.8) correlated, indicating that all compounds are not independent; (2) DA was better at group separation that PCA; (3) the relationships between colonies (chemical distance) were unstable and sensitive to variability in the system; and (4) minor compounds had a disproportionately large effect on the analysis. All these factors, along with sample size, need to be considered in the future analysis of complex chemical profiles.     

Fine Tuning of Social Integration by Two Myrmecophiles of the Ponerine Army Ant, <Emphasis Type="Italic">Leptogenys distinguenda</Emphasis>

Myrmecophiles are animals that live in close association with ants and that frequently develop elaborate mechanisms to infiltrate their well-defended host societies. We compare the social integration strategies of two myrmecophilic species, the spider, Gamasomorpha maschwitzi, and the newly described silverfish, Malayatelura ponerophila gen. n. sp. n., into colonies of the ponerine army ant, Leptogenys distinguenda (Emery) (Hymenoptera: Formicidae). Both symbionts use chemical mimicry through adoption of host cuticular hydrocarbons. Exchange experiments between L. distinguenda and an undetermined Leptogenys species demonstrate that reduced aggression toward alien ants and increased social acceptance occurred with individuals of higher chemical similarity in their cuticular hydrocarbon profiles. We found striking differences in chemical and behavioral strategies between the two myrmecophiles. Spider cuticular hydrocarbon profiles were chemically less similar to the host than silverfish profiles were. Nevertheless, spiders received significantly fewer attacks from host ants and survived longer in laboratory colonies, whereas silverfish were treated with high aggression and were killed more frequently. When discovered and confronted by the host, silverfish tended to escape and were chased aggressively, whereas spiders remained in contact with the confronting host ant until aggression ceased. Thus, spiders relied less on chemical mimicry but were nevertheless accepted more frequently by the host on the basis of behavioral mechanisms. These findings give insights into the fine tuning of social integration mechanisms and show the significance of qualitative differences among strategies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cuticular Hydrocarbons of Buffalo Fly, <Emphasis Type="Italic">Haematobia exigua</Emphasis>, and Chemotaxonomic Differentiation from Horn Fly, <Emphasis Type="Italic">H. irritans</Emphasis>

We determined the quantity and chemical composition of cuticular hydrocarbons of different strains, sexes, and ages of buffalo flies, Haematobia exigua. The quantity of cuticular hydrocarbons increased from less than 1 μg/fly for newly emerged flies to over 11 μg/fly in 13-d-old flies. The hydrocarbon chain length varied from C₂₁ to C₂₉, with unbranched alkanes and monounsaturated alkenes the major components. Newly emerged flies contained almost exclusively C₂₇ hydrocarbons. Increasing age was accompanied by the appearance of hydrocarbons with shorter carbon chains and an increase in the proportion of alkenes. 11-Tricosene and 7-tricosene were the most abundant hydrocarbons in mature H. exigua. Cuticular hydrocarbons of H. exigua are distinctly different from those of horn flies, Haematobia irritans. The most noticeable differences were in the C₂₃ alkenes, with the major isomers 11- and 7-tricosene in H. exigua and (Z)-9- and (Z)-5-tricosene in H. irritans, respectively. Cuticular hydrocarbon analysis provides a reliable method to differentiate the two species, which are morphologically difficult to separate. The differences in cuticular hydrocarbons also support their recognition as separate species, H. exigua and H. irritans, rather than as subspecies.     

Cuticular hydrocarbons of the eastern subterranean termite,Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae)

Major cuticular hydrocarbon components in several castes ofReticulitermes flavipes (Kollar) have been identified and quantitated. Types of hydrocarbons present includen-alkanes, 2-methylalkanes, 3-methylalkanes, 5-methylalkanes, an alkene, and an alkadiene, with a range in carbon numbers from C₂₁ to C₂₆, This is the first report on insect cuticular hydrocarbons in which both 2- and 3-methylalkanes are present, as well as the first report of an insect with a conjugated alkadiene.     

Parasitoids Modify Their Oviposition Behavior According to the Sexual Origin of Conspecific Cuticular Hydrocarbon Traces

Hydrocarbons play a crucial role in insect behavior in general and in sexual recognition in particular. Parasitoids often modify their oviposition behavior according to hydrocarbons left by conspecifics on the reproductive patch, such as oviposition markers left by females after oviposition, or cuticular hydrocarbon (CHC) traces left by individuals by walking or rubbing. This study determined whether Eupelmus vuilleti females are able to distinguish CHCs left by male or female conspecifics on seeds. The results show that the cuticular profile of E. vuilleti differs according to its gender, and that females are able to detect the sexual origin of these CHCs. Moreover, they adjust their oviposition behavior according to the nature of these traces. Although females lay fewer eggs on hosts when confronted with female CHCs, they lay more daughters when confronted with male CHCs, thus changing the sex ratio.     

Task-Related Environment Alters the Cuticular Hydrocarbon Composition of Harvester Ants

Within a colony of harvester ants (Pogonomyrmex barbatus), workers in different task groups differ in the hydrocarbon composition of the cuticle. Foragers and patrollers, which spend extended periods of time outside the nest, have a higher proportion of saturated, unbranched hydrocarbons (n-alkanes) on the cuticle than nest maintenance workers, which spend only short periods of time outside the nest. We tested whether these task-related differences in ant cuticular chemistry arise from exposure to conditions outside the nest. Nest maintenance workers experiencing daily, short-term outside exposure developed a higher proportion of n-alkanes on the cuticle than workers kept inside the lab. Independent manipulations of ultraviolet radiation, relative humidity, and temperature revealed that only the combination of high temperature (ca. 38°C) and low relative humidity (ca. 8%) increased the proportion of cuticular n-alkanes. The results indicate that warm dry conditions, such as those encountered when an ant leaves the nest, trigger changes in cuticular chemistry.     

Cuticular Surface Hydrocarbons of Desert Locust Nymphs, Schistocerca gregaria, and Their Effect on Phase Behavior

The quantity of cuticular hydrocarbons is higher in solitarious nymphs of the desert locust, Schistocerca gregaria, compared to gregarious nymphs, but the total hydrocarbon fraction of solitarious nymphs does not significantly divert behavioral transition of isolated nymphs to the gregarious phase, while gregarious hydrocarbon extracts do. This suggests that qualitative differences in composition are responsible for the biological effect. The profile of cuticular hydrocarbon components is similar in the two phases, but some peak ratios differ. Crowding of solitarious nymphs leads to rapid changes in the profile of the hydrocarbon fraction, suggesting that specific hydrocarbons are produced and secreted as a consequence. Isolating previously crowded nymphs has an opposite effect. The composition of cuticular hydrocarbons from the migratory locust, Locusta migratoria, which differs considerably from that of S. gregaria, does not induce the gregarious behavioral phase in solitarious nymphs of the latter.     

Chemical Signals in the Stingless Bee, <Emphasis Type="Italic">Frieseomelitta varia</Emphasis>, Indicate Caste,Gender, Age,and Reproductive Status

Chemical compounds on the cuticle are a rich source of information used during interactions among social insects. Despite the multitude of studies on these substances and their function in ants, wasps, and honeybees, little is known about this subject in stingless bees (Hymenoptera: Apidae, Meliponini). We studied the chemical composition of the cuticle of the stingless bee, Frieseomelitta varia, by gas chromatography-mass spectrometry (GC-MS), to investigate potential chemical variation among castes, gender, age, and reproductive status. We found differences in the cuticular hydrocarbon composition among workers, males, and queens, recording both qualitative and quantitative differences among individuals of different ages and gender. The cuticle of physogastric queens presented a chemical profile that was distinct from all other groups in the analysis, with high relative abundances of alkenes and alkadienes with 27, 29, and 31 carbon atoms. We discuss the possibility that these compounds signal a queen’s presence to the colony, thereby initiating all vital worker-queen interactions.     

Identification of Termite Species by the Hydrocarbons in their Feces

Blends of abundant cuticular hydrocarbons are species-specific for termites (Isoptera) and can be used to identify a given taxon without the diagnostic castes, soldiers or adults. We demonstrate that hydrocarbon extracts of termite fecal pellets from damaged wood can also be characterized and used to identify termites responsible for damage, even though termites are no longer present or easily recovered. In structures infested by drywood termites, it is common to find fecal pellets, but difficult to extract termites from the finished wood in service. Nine species belonging to two families (Kalotermitidae and Termopsidae) were examined to compare the hydrocarbon composition of termites and their fecal pellets. Diversity was extensive: at least one half of the amount of the hydrocarbons from Neotermes connexus, Incisitermes immigrans, Cryptotermes brevis, Cryptotermes cynocephalus, Procryptotermes corniceps, and Zootermopsis nevadensis nuttingi was olefins. Incisitermes minor and Pterotermes occidentis incorporated only small amounts of olefins in cuticular hydrocarbons; Marginitermes hubbardi had no detectable olefins. Hydrocarbons extracted from fecal pellets were qualitatively and quantitatively similar to cuticular extracts and can be used to determine the termite species responsible without the termites present.     

Mechanism underlying cuticular hydrocarbon homogeneity in the antCamponotus vagus (SCOP.) (Hymenoptera: Formicidae): Role of postpharyngeal glands

The aim of the present study was to investigate the regulation and the homogeneity of the chemical signature between members ofCamponotus vagus after experimentally changing the cuticular chemical signature by topically applying hydrocarbons. Topical application of pentane (solvent) to the cuticle of isolated workers led to a significant decrease in the quantities of the cuticular hydrocarbons measured within 3 hr, followed by an increase within the following 3 hr and a period of relative stability from 9 hr to 14 days. On the other hand, after topical application to isolated workers ofn-tetracosane, a hydrocarbon existing only in trace quantity in this species, the quantity of this hydrocarbon measured over time in the epicuticular wax tended to level out at about 14 days after treatment. In contrast, topically applied (Z)-9-tricosene, an unsaturated hydrocarbon not normally synthesized by this species, decreased dramatically within a few hours and had completely disappeared within 14 days. (Z)-9-Tricosene applied to one member of a group was present in the postpharyngeal glands of the other members from 30 min to seven days of cohabitation. The highest levels were recorded in all six workers in each group after one day. GC-MS analyses showed that (Z)-9-tricosene was present in the cuticles of some untreated workers only after four and seven days of cohabitation with a treated worker. These data suggest: (1) that the deposited (Z)-9-tricosene decreased very quickly on the cuticle of the treated worker, although the total amount was spread over the cuticle and postpharyngeal gland and (2) that it was absorbed by the nontreated workers via the postpharyngeal glands during licking or grooming activities and reincorporated into the cuticle at four and seven days. When the treated worker was separated from the other ants by a wire mesh, (Z)-9-tricosene was detected neither in the cuticle nor in the postpharyngeal gland of nontreated workers.     

Characterization of a Female-Produced Courtship Pheromone in the Parasitoid Nasonia vitripennis

Males of the parasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) show a characteristic courtship behavior. We demonstrate that male arrestment and key behavioral elements of the courtship sequence are mediated by a female-derived contact sex pheromone. Males were arrested on paper disks treated with female extracts but not on those treated with male extracts. Male responsiveness was influenced by the surface to which female extracts were applied. Extracts applied to an extracted beetle elytron arrested males more strongly than those applied to filter paper of comparable size. However, more complex behavioral elements, such as head nodding and copulation attempts, were shown only when extracts were applied to extracted male cadavers, suggesting that tactile or visual cues synergize the male response. The chemicals involved are stable, of low volatility, and nonpolar. Dead females arrested males and elicited courtship behavior for at least 8 d. Males showed no sign of attraction to live females at a distance of 3 cm in an olfactometer. Fractionation of female extracts demonstrated that the activity was exclusively located in the nonpolar fraction. Analysis of the active fraction by gas chromatography–mass spectrometry revealed that cuticular hydrocarbons with chain lengths between 25 and 37 carbon units were present. Comparison of hydrocarbon profiles from males and females showed qualitative and quantitative differences. These results suggest that sex-specific cuticular hydrocarbons are the key signals mediating the male courtship behavior in N. vitripennis.     

Acceptance Threshold Hypothesis is Supported by Chemical Similarity of Cuticular Hydrocarbons in a Stingless Bee, Melipona asilvai

The ability to discriminate nestmates from non-nestmates in insect societies is essential to protect colonies from conspecific invaders. The acceptance threshold hypothesis predicts that organisms whose recognition systems classify recipients without errors should optimize the balance between acceptance and rejection. In this process, cuticular hydrocarbons play an important role as cues of recognition in social insects. The aims of this study were to determine whether guards exhibit a restrictive level of rejection towards chemically distinct individuals, becoming more permissive during the encounters with either nestmate or non-nestmate individuals bearing chemically similar profiles. The study demonstrates that Melipona asilvai (Hymenoptera: Apidae: Meliponini) guards exhibit a flexible system of nestmate recognition according to the degree of chemical similarity between the incoming forager and its own cuticular hydrocarbons profile. Guards became less restrictive in their acceptance rates when they encounter non-nestmates with highly similar chemical profiles, which they probably mistake for nestmates, hence broadening their acceptance level.