Genetic Distance and Age Affect the Cuticular Chemical Profiles of the Clonal Ant Cerapachys biroi

Although cuticular hydrocarbons (CHCs) have received much attention from biologists because of their important role in insect communication, few studies have addressed the chemical ecology of clonal species of eusocial insects. In this study we investigated whether and how differences in CHCs relate to the genetics and reproductive dynamics of the parthenogenetic ant Cerapachys biroi. We collected individuals of different ages and subcastes from several colonies belonging to four clonal lineages, and analyzed their cuticular chemical signature. CHCs varied according to colonies and clonal lineages in two independent data sets, and correlations were found between genetic and chemical distances between colonies. This supports the results of previous research showing that C. biroi workers discriminate between nestmates and non-nestmates, especially when they belong to different clonal lineages. In C. biroi, the production of individuals of a morphological subcaste specialized in reproduction is inversely proportional to colony-level fertility. As chemical signatures usually correlate with fertility and reproductive activity in social Hymenoptera, we asked whether CHCs could function as fertility-signaling primer pheromones determining larval subcaste fate in C. biroi. Interestingly, and contrary to findings for several other ant species, fertility and reproductive activity showed no correlation with chemical signatures, suggesting the absence of fertility related CHCs. This implies that other cues are responsible for subcaste differentiation in this species.     

Task-Related Differences in the Cuticular Hydrocarbon Composition of Harvester Ants, Pogonomyrmex barbatus

Colonies of the harvester ant, Pogonomyrmex barbatus, perform a variety of tasks. The behavior of an individual worker appears to depend on its recent history of brief contacts with ants of the same and other task groups. The purpose of this study was to determine whether task groups differ in cuticular hydrocarbon composition. We compared the cuticular hydrocarbon composition of ants collected under natural conditions as they performed one of three tasks: patrolling (locating food sources), foraging, or nest maintenance. Task groups differed significantly in the relative proportions of classes of hydrocarbon compounds, as well as in individual compounds. Relative to nest maintenance workers, foragers and patrollers had a higher proportion of straight-chain alkanes relative to monomethylalkanes, dimethylalkanes, and alkenes. There was no significant difference in the chain length of n-alkanes among the task groups. Foragers did not differ in hydrocarbon composition from patrollers. Colonies differed significantly from one another in hydrocarbon composition, but task groups differed in consistent ways from colony to colony, suggesting that the mechanism responsible for task-related hydrocarbon composition was the same in all colonies. P. barbatus workers switch tasks during their lifetimes, suggesting that cuticular hydrocarbon composition changes during adulthood as well. Nest maintenance workers are probably younger than foragers and patrollers and perform very little of their work outside of the nest. Task-related hydrocarbon differences detected here may be associated with worker age, and/or the abiotic characteristics (temperature, humidity, and ultraviolet light) of the interior and exterior work environments.     

Hydrocarbons on Harvester Ant (<Emphasis Type="Italic">Pogonomyrmex barbatus</Emphasis>) Middens Guide Foragers to the Nest

Colony-specific cuticular hydrocarbons are used by social insects in nestmate recognition. Here, we showed that hydrocarbons found on the mound of Pogonomyrmex barbatus nests facilitate the return of foragers to the nest. Colony-specific hydrocarbons, which ants use to distinguish nestmates from non-nestmates, are found on the midden pebbles placed on the nest mound. Midden hydrocarbons occur in a concentration gradient, growing stronger near the nest entrance, which is in the center of a 1–2 m diameter nest mound. Foraging behavior was disrupted when the gradient of hydrocarbons was altered experimentally. When midden material was diluted with artificial pebbles lacking the colony-specific hydrocarbons, the speed of returning foragers decreased significantly. The chemical environment of the nest mound contributes to the regulation of foraging behavior in harvester ants.     

Hydrocarbons in the Ant <Emphasis Type="Italic">Lasius niger</Emphasis>: From the Cuticle to the Nest and Home Range Marking

The cuticular hydrocarbons (CHCs) of the ant Lasius niger are described. We observe a high local colony specificity of the body cuticular profile as predicted for a monogynous and multicolonial species. The CHCs show a low geographical variation among different locations in France. The CHCs on the legs also are colony specific, but their relative quantities are slightly different from those on the main body. For the first time, we demonstrate that the inner walls of the ant nest are coated with the same hydrocarbons as those found on the cuticle but in different proportions. The high amount of inner-nest marking and its lack of colony-specificity may explain why alien ants are not rejected once they succeed in entering the nest. The cuticular hydrocarbons also are deposited in front of the nest entrance and on the foraging arena, with a progressive increase in n-alkanes relative amounts. Chemical marks laid over the substrate are colony specific only when we consider methyl-branched alkanes. Our data confirm that these “footprint hydrocarbons” are probably deposited passively by the contact of ant tarsae with the substrate. These results suggest that the CHCs chemical profiles used by ants in colony recognition are much more complex than a single template: ants have to learn and memorize odors that vary depending on their context of perception.     

Social closure,aggressive behavior,and cuticular hydrocarbon profiles in the polydomous antCataglyphis iberica (hymenoptera,Formicidae)

Nestmate recognition was studied in the polydomous antCataglyphis iberica (Formicinae) in the laboratory. The study examined six colonies collected from two different populations 600 km apart in the Iberian peninsula (Barcelona and Murcia). Introduction of an alien worker into an allocolonial arena always ended in death to the intruder, demonstrating that in this species societies are extremely closed. Dyadic encounters composed of individuals from different colonies in a neutral arena confirmed the existence of high aggression between allocolonial individuals. We also investigated variability in the composition of the major cuticular hydrocarbons between the colonies used in the behaviorial tests. There were marked quantitative differences between the profiles of ants from the two populations, suggesting that the populations are completely segregated. Cuticular profiles within a population tended to be more similar, but were nevertheless colony specific. The degree of colony closure inC. iberica seemed to be independent of geographic distance since aggression between the colonies was always at its maximum, irrespective of their population origin.     

Wax Lipids Signal Nest Identity in Bumblebee Colonies

The signalling functions of cuticular lipids, particularly cuticular hydrocarbons, have gained considerable attention in social insect communication. Information transfer between individuals by means of these substances has been examined extensively. However, communication with cuticular lipids is not limited to inter-individual recognition. Cuticular compounds can also have a signalling function in the nest environment. Workers of the bumblebee Bombus terrestris leave cuticular lipid traces, so-called footprints, that mark their nest entrance. In addition, there is evidence that bumblebees sense nesting material to identify their colony. In this study, we examined the signalling potential of bumblebee wax, and tested if bumblebee workers are able to identify their colony with the help of wax scent. Chemical analyses of wax extracts using coupled gas chromatography–mass spectrometry showed that wax from colonies of the bumblebee B. terrestris contained a complex blend of cuticular lipids, dominated by hydrocarbons and wax esters. Comparing the relative compound amounts of wax samples from different colonies, we found that wax scent patterns varied with nest identity. Olfactometer bioassays showed that bumblebees were able to discriminate between wax scents from their own and a foreign colony. Our findings suggest that wax emits characteristic olfactory profiles that are used by workers to recognize their colony.     

Colony Fusion in Argentine Ants is Guided by Worker and Queen Cuticular Hydrocarbon Profile Similarity

Introduced populations of the Argentine ant, Linepithema humile, have experienced moderate to severe losses of genetic diversity, which may have affected nestmate recognition to various degrees. We hypothesized that cuticular hydrocarbons (CHC) serve as nestmate recognition cues, and facilitate colony fusion of unrelated L. humile colonies that share similar CHC profiles. In this study, we paired six southeastern U.S. L. humile colonies in a 6-month laboratory fusion assay, and determined if worker and queen CHC profile similarity between colonies was associated with colony fusion and intercolony genetic similarity. We also compared worker and queen CHC profiles between fused colony pairs and unpaired controls to determine if worker and queen chemical profiles changed after fusion. We found that colony fusion correlated with the CHC similarity of workers and queens, with the frequency of fusion increasing with greater CHC profile similarity between colonies. Worker and queen CHC profile similarity between colonies also was associated with genetic similarity between colonies. Queen CHC profiles in fused colonies appeared to be a mix of the two colony phenotypes. In contrast, when only one of the paired colonies survived, the CHC profile of the surviving queens did not diverge from that of the colony of origin. Similarly, workers in non-fused colonies maintained their colony-specific CHC, whereas in fused colonies the worker CHC profiles were intermediate between those of the two colonies. These results suggest a role for CHC in regulating interactions among mutually aggressive L. humile colonies, and demonstrate that colony fusion correlates with both genetic and CHC similarities. Further, changes in worker and queen chemical profiles in fused colonies suggest that CHC plasticity may sustain the cohesion of unrelated L. humile colonies that had fused.     

Temporal changes in colony cuticular hydrocarbon patterns ofSolenopsis invicta

Heritable cuticular hydrocarbon patterns ofSolenopsis invicta workers are consistent within colonies for a given sampling time but vary sufficiently from colony to colony to distinguish the colonies from each other. In addition, cuticular hydrocarbon patterns change within colonies over time. Nestmate recognition cues found on the individual's cuticle, can be from heritable or environmental sources, and are a subset of colony odor. The cuticular hydrocarbons can be used as a model for heritable nestmate recognition cues. We propose that because potential nestmate recognition cues, both environmental and genetic, are dynamic in nature rather than static, during its lifetime a worker must continually update its perception (template) of colony odor and nestmate recognition cues.     

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.     

Surface lipids of social waspPolistes melricus say and its nest and nest pedicel and their relation to nestmate recognition

In eight replicate laboratory tests wherePolistes metricus adults were allowed to choose between their own nest, a second nest, and neither nest, they selected their own nest 66% of the observed time. When the surface hydrocarbons had been extracted from the nests, the wasps chose their own nest only 8% of the time, but after the hydrocarbons were reapplied to the surface of the respective nests, they selected their own nest 47% of the time. These changes are significant. The cuticular lipids were analyzed from individualP. metricus adult females collected from 13 colonies. Surface lipids were recovered from the paper and pedicels of their nests. Eighteen hydrocarbons were identified in these lipid fractions. The major components of the wasp cuticular lipids weren-heptacosane,n-nonacosane, methylhentriacontane, and methyltritriacontane. Factor analysis revealed that extracts of pedicels are all similar in composition, while cuticle and paper extracts vary, sometimes similarly according to colony identity.     

Cuticular Hydrocarbon Compounds in Worker Castes and Their Role in Nestmate Recognition in <Emphasis Type="Italic">Apis cerana indica</Emphasis>

Differences in cuticular hydrocarbons (CHCs) among worker castes and colonies were examined in Apis cerana indica. The roles of tetracosanoic acid, hexadecanoic acid, pentacosane, and (Z)-9-tricosene in nestmate recognition were studied. The CHC profiles of different castes, i.e., newly emerged bees, nurse bees, and forager bees, were found to differ among colonies. The CHC profiles of nurse bees were similar across different colonies, but forager bees in all colonies had significantly greater amounts of alkanes. In nestmate recognition experiments, guard bees reacted significantly more aggressively to foragers treated with tetracosanoic acid, hexadecanoic acid, and (Z)-9-tricosene. Pentacosane provoked no such effect.     

Induced mimicry of colony odors in ants

The cuticular hydrocarbons ofFormica selysi (Formicinae) andMonica rubida (Myrmicinae) reared in single species and in mixed species colonies were determined using gas chromatography (GC) and GC-mass spectrometry. In colonies containing both species, each species modified its species-specific recognition odor. This odor is composed, at least partly, of cuticular hydrocarbons. The cuticular hydrocarbons ofM. rubida consist only of saturated alkanes (n-alkanes and branched alkanes). InF. selysi the mixture also contains unsaturated compounds (monoenes and dienes). In hetero-specific colonies, a new chemical signature developed. This signature resulted from qualitative and quantitative changes in the spectrum of hydrocarbons produced by each species and permitted the two species to inhabit the same nest without displaying interspecific aggression. The readjustment seemed to be more an active synthesis or an active transfer than simply a passive transfer from one species to the other. This may imply that the ants synthesized some components of the hydrocarbon signature of the other species. These synthesizing processes may be activated under particular social environmental conditions.     

Venom Alkaloid and Cuticular Hydrocarbon Profiles Are Associated with Social Organization,Queen Fertility Status,and Queen Genotype in the Fire Ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Queens in social insect colonies advertise their presence in the colony to: a) attract workers’ attention and care; b) gain acceptance by workers as replacement or supplemental reproductives; c) prevent reproductive development in nestmates. We analyzed the chemical content of whole body surface extracts of adult queens of different developmental and reproductive stages, and of adult workers from monogyne (single colony queen) and polygyne (multiple colony queens) forms of the fire ant Solenopsis invicta. We found that the composition of the most abundant components, venom alkaloids, differed between queens and workers, as well as between reproductive and non-reproductive queens. Additionally, workers of the two forms could be distinguished by alkaloid composition. Finally, sexually mature, non-reproductive queens from polygyne colonies differed in their proportions of cis-piperidine alkaloids, depending on their Gp-9 genotype, although the difference disappeared once they became functional reproductives. Among the unsaturated cuticular hydrocarbons characteristic of queens, there were differences in amounts of alkenes/alkadienes between non-reproductive polygyne queens of different Gp-9 genotypes, between non-reproductive and reproductive queens, and between polygyne and monogyne reproductive queens, with the amounts increasing at a relatively higher rate through reproductive ontogeny in queens bearing the Gp-9 b allele. Given that the genotype-specific piperidine differences reflect differences in rates of reproductive maturation between queens, we speculate that these abundant and unique compounds have been co-opted to serve in fertility signaling, while the cuticular hydrocarbons now play a complementary role in regulation of social organization by signaling queen Gp-9 genotype.     

Cuticular hydrocarbons of dampwood termites,Zootermopsis: Intra- and intercolony variation and potential as taxonomic characters

Colonies ofZootermopsis were collected from the central Sierra Nevada and the Monterey Penninsula in California, and from southern Arizona. Cuticular hydrocarbons were identified by gas chromatography-mass spectrometry (GC-MS) and quantified by gas-liquid chromatography (GLC) for each caste of all colonies. Four consistent and distinct cuticular hydrocarbon patterns, or chemical phenotypes, were identified. Unique and abundant monomethyl- and dimethylalkanes, and ann-alkene provided easy separation of the various phenotypes. Significant differences in the proportions of the various components were found among castes within a colony and colonies within phenotypes from California. Differences in the hydrocarbon proportions for castes were not consistent between colonies. The current taxonomy of the genusZootermopsis recognizes three species. Our identification of four consistent, unique cuticular hydrocarbon phenotypes from the three described species should alert systematists and others to a major concern. If there are truly only three extant species, then the hypothesis that cuticular hydrocarbon profiles in this genus are species specific is not acceptable. Conversely, if cuticular hydrocarbon profiles are truly species specific, then there is at least one new, undescribed species ofZootermopsis.Isoptera: Termopsidae.     

Genetic Relatedness and Chemical Profiles in an Unusually Peaceful Eusocial Bee

Colonies of the stingless bee Tetragonilla collina frequently occur in unusually high densities and in direct neighborhood (nest aggregations), in rainforests of Southeast Asia. To investigate whether close relatedness and/or similar chemical profiles facilitate the co-occurrence of multiple T. collina colonies, we investigated aggressive behavior, genetic relatedness and cuticular hydrocarbon (CHC) profiles within and between colonies and nest aggregations. Although 17 out of 19 colonies within aggregations were largely unrelated, intraspecific aggression between different colonies was basically absent both within and among aggregations. This lack of aggression should favor social parasitism and hence the occurrence of unrelated individuals within a colony. However, low within-colony relatedness was found in only five out of 19 colonies where it may be explained by queen turnover or the occurrence of foreign workers. CHC profiles of colonies within and among aggregations were statistically different. However, many workers could chemically not be assigned to their maternal colony, indicating considerable overlap among colonies in odor profiles of workers. Moreover, odor profiles tended to be more similar within than among aggregations, although most colonies were unrelated. Thus, CHC profiles were a poor indicator of relatedness in T. collina. The lack of correlation between relatedness and chemical similarity in T. collina may be explained by the incorporation of resin-derived terpenes in their CHC profiles. The composition of these terpenes was highly similar among colonies, particularly within aggregations, hence potentially decreasing chemical distinctiveness and increasing behavioral tolerance.     

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 hydrocarbons and defensive compounds ofReticulitermes flavipes (Kollar) andR. santonensis (feytaud): Polymorphism and chemotaxonomy

Colonies ofReticulitermes flavipes andR. santonensis were collected from the southeastern United States (Georgia) and the southwest of France (Charente-maritime). Defensive compounds and cuticular hydrocarbons were identified by gas chromatography-mass spectrometry and quantified by gas chromatography using an internal standard for each caste and all colonies. These analyses show that although the cuticular hydrocarbons ofR. santonensis in Europe andR. flavipes in Georgia are identical, their relative proportions are different. However, the defensive compounds synthesized by their soldiers are different. A strong chemical polymorphism between sympatric colonies ofR. flavipes in the SW United States was detected in terms of both the hydrocarbons of the workers and soldiers and in the defensive secretions of the soldiers. The six defensive secretion phenotypes are based on the presence or absence of terpenes whereas the cuticular hydrocarbon phenotypes are based on significant differences in the proportions of the various components. A multivariate analysis (analysis of principal components) clearly permitted discrimination of four phenotypes (three inR. flavipes and one inR. santonensis) without intermediates. The hydrocarbons responsible for these variations were identified, and it was shown that the variations are neither seasonal nor geographic. The phenotypes of the cuticular hydrocarbons (workers and soldiers) and defensive compounds are linked in each colony, forming in three groups inR. flavipes Georgia, one subdivided into four subgroups according to the defensive secretion phenotypes. The role of these polymorphisms is discussed and ethological tests indicate that the chemical polymorphism do not determine aggressive behavior. The taxonomic significance of these results is considered and two hypothesis are formulated: (1) We only detected a strong genetic polymorphism in one unique species, and we believe thatR. santonensis was introduced into Europe in the last century from oneR. flavipes colony. (2) Chemical variability characterizes the sibling species that can be grouped into the same subspeciesR. flavipes. Unknown mechanisms of reproductive isolation separate them.     

Cuticular Hydrocarbons and Aggression in the Termite Macrotermes Subhyalinus

Cuticular hydrocarbons are among the prime candidates for nestmate recognition in social insects. We analyzed the variation of cuticular hydrocarbons in the termite species M. subhyalinus in West Africa (Comoë National Park) on a small spatial scale (<1 km). We found considerable variation in the composition of cuticular hydrocarbons among colonies, with four distinct chemical phenotypes. Different phenotypes occurred within each of the four habitats. The difference between these phenotypes is primarily due to unsaturated compounds. A clear correlation between the difference of the hydrocarbon composition and the aggression between colonies was found. This correlation also holds in a multivariate analysis of genetic similarity (measured by AFLPs), morphometric distances (measured by Mahalanobis-distances), as well as geographic distances between colonies. In a more detailed analysis of the correlation between the composition of cuticular hydrocarbons and aggression, we found that no single compound is sufficient to explain variation in aggression between pairings of colonies. Thus, termites seem to use a bouquet of compounds. Multiple regression analysis suggested that many of these compounds are unsaturated hydrocarbons and, thus, may play a key role in colony recognition.     

Colony-Specific Cuticular Hydrocarbon Profile in Formica argentea Ants

The cuticular hydrocarbons of the ant Formica argentea were identified by gas chromatography/mass spectrometry. Behavioral bioassays tested the role of each class of cuticular hydrocarbon in nestmate recognition, and statistical analyses looked for potential colony-specific signatures. The cuticular hydrocarbons of F. argentea consist of n-alkanes, alkenes, and methyl-branched alkanes. Behavioral bioassays demonstrated that changes in the alkene and methyl-branched alkane signature of F. argentea increased aggression, but changes in alkanes did not. Statistical analyses demonstrated that F. argentea workers present a colony-specific hydrocarbon profile based on their methyl-branched C₂₉ alkane signature. Using this signature alone, it is possible to group worker ants statistically by nest, suggesting that methyl-branched C₂₉ alkanes may be important in nestmate recognition for this species. These results support the idea that variation in positional isomers of cuticular hydrocarbons of the same carbon chain length may provide enough information for nestmate recognition. Although the addition of alkenes increased aggression in F. argentea, alkenes did not provide a colony-specific signature. This study reinforces the idea that investigators studying nestmate recognition should not examine cuticular hydrocarbon profiles as a whole but rather, should look for colony-specific signatures embedded in parts of the profile.     

Segregation of colony odor in the desert ant Cataglyphis niger

There are two separate, and presumably opposing, processes that affect colony odor in the desert ant Cataglyphis niger: (1) biosynthesis and turnover of these chemicals by individual ants, and (2) homogenization of colony odor through exchange of cues. The first increases signal variability; the latter decreases it. The impact of these factors was tested by splitting colonies and monitoring the profile changes occurring in the postpharyngeal glands (PPG) and cuticular hydrocarbons.From each of two polygynous nests four daughter colonies were formed, three monogynous and one queenless. Thereafter, 10 ants from each were randomly selected each month, for three successive months, for analyses of their PPG and cuticular hydrocarbons. From two colonies we also obtained ants from a known matriline. Over time, there was a shift in hydrocarbon profiles of both the PPG and cuticular washes in each of the tested colonies. Moreover, by subjecting selected hydrocarbon constituents to a discriminant analyses based on their relative proportions, all of the daughter colonies (queenright and queenless) were distinguishable from each other and from their respective mother colonies. In each of the queenright daughter colonies, the queen profile was indiscriminable from that of the workers and often was in the center of the group. Full sisters were clearly distinguishable from their nestmates, emphasizing the genetic versus environmental processes that govern colony odor. The effect of time was always superior to the separation effect in contributing to odor segregation. Comparison of the Mahalanobis distances indicated that the shift in hydrocarbon seems to proceed along parallel lines rather than in divergence. However, there was no overt aggression between ants that originated from the different subgroups in dyadic encounters. It appears that in this species a three-month separation period is not sufficient to change the hydrocarbon profile beyond the recognition threshold.