Cuticular Hydrocarbon Composition Research Articles

Deciphering the variation in cuticular hydrocarbon profiles of six European honey bee subspecies

The Western honey bee (Apis mellifera) subspecies exhibit local adaptive traits that evolved in response to the different environments that characterize their native distribution ranges. An important trait is the cuticular hydrocarbon (CHC) profile, which helps to prevent desiccation and mediate communication. We compared the CHC profiles of six European subspecies (A. m. mellifera, A. m. carnica, A. m. ligustica, A. m. macedonica, A. m. iberiensis, and A. m. ruttneri) and investigated potential factors shaping their composition. We did not find evidence of adaptation of the CHC profiles of the subspecies to the climatic conditions in their distribution range. Subspecies-specific differences in CHC composition might be explained by phylogenetic constraints or genetic drift. The CHC profiles of foragers were more subspecies-specific than those of nurse bees, while the latter showed more variation in their CHC profiles, likely due to the lower desiccation stress exerted by the controlled environment inside the hive. The strongest profile differences appeared between nurse bees and foragers among all subspecies, suggesting an adaptation to social task and a role in communication. Foragers also showed an increase in the relative amount of alkanes in their profiles compared to nurses, indicating adaptation to climatic conditions.

Conventional agriculture affects sex communication and impacts local population size in a wild bee

Man-made agricultural stressors have been identified to compromise the reproductive dynamics of bee populations within agricultural environments. With the aid of bee hotels, we explored the influence of conventional and organic farming systems on local population size and body traits of the mason bee, Osmia bicornis, in southern Germany. We further used a chemical ecology approach and bioassays to test whether farming management influence male pre-copulatory behaviors. We observed a positive relationship between the extent of organic agriculture in the landscape and both overall brood cell production and nesting frequency. Moreover, farming systems were found to influence body traits, with bees from organic sites being smaller in size and having a different cuticular hydrocarbon composition compared with those at conventional sites. Bioassays revealed that males were more sexually attracted to freeze-killed females from conventional sites compared with those from organic sites. Intriguingly, treating females from organic fields with synthetic semiochemicals enhanced their sexual attraction to levels comparable with females from conventional sites. Our findings shed light on the intricate interplay between farming practices and the reproductive behaviors of wild mason bees, emphasizing the need for a comprehensive understanding of these dynamics for effective conservation and management strategies.

Reproductive isolation arises during laboratory adaptation to a novel hot environment

BackgroundReproductive isolation can result from adaptive processes (e.g., ecological speciation and mutation-order speciation) or stochastic processes such as “system drift” model. Ecological speciation predicts barriers to gene flow between populations from different environments, but not among replicate populations from the same environment. In contrast, reproductive isolation among populations independently adapted to the same/similar environment can arise from both mutation-order speciation or system drift.ResultsIn experimentally evolved populations adapting to a hot environment for over 100 generations, we find evidence for pre- and postmating reproductive isolation. On one hand, an altered lipid metabolism and cuticular hydrocarbon composition pointed to possible premating barriers between the ancestral and replicate evolved populations. On the other hand, the pronounced gene expression differences in male reproductive genes may underlie the postmating isolation among replicate evolved populations adapting to the same environment with the same standing genetic variation.ConclusionOur study confirms that replicated evolution experiments provide valuable insights into the mechanisms of speciation. The rapid emergence of the premating reproductive isolation during temperature adaptation showcases incipient ecological speciation. The potential evidence of postmating reproductive isolation among replicates gave rise to two hypotheses: (1) mutation-order speciation through a common selection on early fecundity leading to an inherent inter-locus sexual conflict; (2) system drift with genetic drift along the neutral ridges.

Genetic Underpinnings of Cuticular Hydrocarbon Biosynthesis in the German Cockroach, Blattella germanica (L.): Progress and Perspectives.

Insect cuticular hydrocarbons (CHCs) serve as important waterproofing barriers and as signals and cues in chemical communication. Over the past 30 years, numerous studies on CHCs have been conducted in the German cockroach, Blattella germanica, leading to substantial progress in the field. However, there has not been a systematic review of CHC studies in this species in recent years. This review aims to provide a concise overview of the chemical composition, storage, transport, and physical properties of different CHCs in B. germanica. Additionally, we focus on the biosynthetic pathway and the genetic regulation of HC biosynthesis in this species. A considerable amount of biochemical evidence regarding the biosynthetic pathway of insect CHCs has been gathered from studies conducted in B. germanica. In recent years, there has also been an improved understanding of the molecular mechanisms that underlie CHC production in this insect. In this article, we summarize the biosynthesis of different classes of CHCs in B. germanica. Then, we review CHCs reaction to various environmental conditions and stressors and internal physiological states. Additionally, we review a body of work showing that in B. germanica, CHC profiles exhibit significant sexual dimorphism, specific CHCs act as essential precursors for female contact sex pheromone components, and we summarize the molecular regulatory mechanisms that underlie sexual dimorphism of CHC profiles. Finally, we highlight future directions and challenges in research on the biosynthesis and regulatory mechanisms of CHCs in B. germanica, and also identify potential applications of CHC studies in the pest control.

Life-cycle profiling of cuticular hydrocarbons in the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae)

Cuticular hydrocarbons (CHCs) are the main components of the wax layer present on insects’ surface, serving as protective agents against environmental stress and/or as communication signals. It has been documented that the chemical composition of CHCs varies with species and even with developmental stage or sex of the same species. However, studies involving in a life-cycle identification of CHCs are rare. In this work, we attempted to profile the components and relative abundance of CHCs in the cotton bollworm Helicoverpa armigera at all the four developmental stages and in both sexes of pupae and adults. A total of 29 CHCs were putatively identified by gas chromatography-mass spectrometer analysis, including 14 n-alkanes, 4 methyl-branched alkenes and 11 methyl-branched alkanes, with 15 of them being detected at all the four developmental stages. Of the 29 CHCs, the straight-chain alkanes with 27, 29 and 31 carbon atoms were the major CHCs. All three classes of CHCs (n-alkanes, methyl-branched alkanes and methyl-branched alkenes) were found in both larvae and pupae, while no unsaturated hydrocarbons were observed in eggs and adults. Methyl-branched alkanes were the main component found in eggs and adults, while the composition of n-alkanes and methyl-branched alkanes in larvae and pupae varied with ages. Principal component analysis (PCA) revealed that the CHC profiles in larvae and adults changed significantly with ages, while the CHC profiles in early pupae (1-day-old) showed distinct difference from those of late pupae. Furthermore, sexual dimorphism in the CHC profile was observed in mature adults (3-day-old and 5-day-old), with male adults possessing more n-C31 and n-C33, but less 3-MeC29 than females.

Colorimetric surface lipid quantification in Drosophila.

Insects are covered with free neutral cuticular hydrocarbons (CHC) that may be linear, branched, and unsaturated and vary in their chain length. The CHC composition is species-specific and contributes to the adaptation of the animal to its ecological niche. Commonly, CHCs contribute substantially to the inward and outward barrier function of the cuticle and serve pheromonal communication. They are generally determined by gas-chromatography, a time-consuming method requiring detailed expertize, but it is not available in many laboratories. Here, we report on the establishment of a colorimetric method allowing semi-quantitative determination of unsaturated CHCs in Drosophila flies. This method is based on the in vitro reaction of vanillin with double bounds in lipid molecules in an acidic solution to generate a reddish color. We found a robust correlation between gas chromatographic and vanillin-colorimetric data on unsaturated CHCs amounts in single flies. As the role of unsaturated CHCs in the performance of insects in their environment is only partly understood, we think that this novel method would allow fast and broad analyses of this type of CHCs in insects both in the field and in laboratories and thereby contribute to a substantial improvement in the investigation of this matter.

Discrimination of Diptera order insects based on their saturated cuticular hydrocarbon content using a new microextraction procedure and chromatographic analysis.

The nature and proportions of hydrocarbons in the cuticle of insects are characteristic of the species and age. Chemical analysis of cuticular hydrocarbons allows species discrimination, which is of great interest in the forensic field, where insects play a crucial role in estimating the minimum post-mortem interval. The objective of this work was the differentiation of Diptera order insects through their saturated cuticular hydrocarbon compositions (SCHCs). For this, specimens fixed in 70 : 30 ethanol : water, as recommended by the European Association for Forensic Entomology, were submitted to solid-liquid extraction followed by dispersive liquid-liquid microextraction, providing preconcentration factors up to 76 for the SCHCs. The final organic extract was analysed by gas chromatography coupled with flame ionization detection (GC-FID), and GC coupled with mass spectrometry was applied to confirm the identity of the SCHCs. The analysed samples contained linear alkanes with the number of carbon atoms in the C9-C15 and C18-C36 ranges with concentrations between 0.1 and 125 ng g-1. Chrysomya albiceps (in its larval stage) showed the highest number of analytes detected, with 21 compounds, while Lucilia sericata and Calliphora vicina the lowest, with only 3 alkanes. Non-supervised principal component analysis and supervised orthogonal partial least squares discriminant analysis were performed and an optimal model to differentiate specimens according to their species was obtained. In addition, statistically significant differences were observed in the concentrations of certain SCHCs within the same species depending on the stage of development or the growth pattern of the insect.

A supergene in seaweed flies modulates male traits and female perception.

Supergenes, tightly linked sets of alleles, offer some of the most spectacular examples of polymorphism persisting under long-term balancing selection. However, we still do not understand their evolution and persistence, especially in the face of accumulation of deleterious elements. Here, we show that an overdominant supergene in seaweed flies, Coelopa frigida, modulates male traits, potentially facilitating disassortative mating and promoting intraspecific polymorphism. Across two continents, the Cf-Inv(1) supergene strongly affected the composition of male cuticular hydrocarbons (CHCs) but only weakly affected CHC composition in females. Using gas chromatography-electroantennographic detection, we show that females can sense male CHCs and that there may be differential perception between genotypes. Combining our phenotypic results with RNA-seq data, we show that candidate genes for CHC biosynthesis primarily show differential expression for Cf-Inv(1) in males but not females. Conversely, candidate genes for odorant detection were differentially expressed in both sexes but showed high levels of divergence between supergene haplotypes. We suggest that the reduced recombination between supergene haplotypes may have led to rapid divergence in mate preferences as well as increasing linkage between male traits, and overdominant loci. Together this probably helped to maintain the polymorphism despite deleterious effects in homozygotes.

Effect of two larval diets on the reproductive parameters of the housefly, Musca domestica L. (Diptera, Muscidae)

Abstract Deep understanding of the optimal reproductive and biological parameters is necessary to obtain the maximum egg production in captive rearing of Musca domestica, the common housefly. The effect of larval diet is explored in this study, using two different media: poultry manure and an artificial medium based on cereals mixed with meat. The preimaginal mortality and development time, with other reproductive parameters that indirectly affect adult fecundity as: ovary and wing size, fluctuating asymmetry, and cuticular hydrocarbon composition, were analysed. The results show that poultry manure was related with better efficacy values, lower larval mortality rate, and a sex-dependent effect in fecundity. Asymmetry fluctuation and different hydrocarbon composition was detected in females. The females from larvae that grew on manure presented bigger ovaries and oocytes, but a smaller number of ovarioles per ovary. Their morphological asymmetry was also lower; and the cuticular hydrocarbon composition was distinct from the rest of the adults, with more alkanes and less alkenes than males or females related with the artificial diet. As conclusion, the effect of larval substrate on the survival and fecundity parameters is evident. The implications for the mass production of the house fly as animal feed are also indicated.

Degradation dynamics of cuticular hydrocarbons of empty puparia in forensically important blow fly Lucilia sericata in soil and under room conditions: Insights and machine learning applications

Empty puparia, remnants of blow fly larvae cuticles, aid in estimating long-term post-mortem intervals. Assessing alterations in cuticular hydrocarbons (CHCs) composition provides insights into environmental effects on their degradation, enabling age estimation methods for empty puparia found at the crime scene. The current study aimed to investigate the temporal variations of five predominant CHCs of empty puparia of Lucilia sericata (Diptera: Calliphoridae). The study was conducted over six months (180 days) under controlled conditions, comparing the storage of puparia in soil and non-soil pupation medium (room conditions). The five CHCs investigated are- n-pentacosane, n-hexacosane, n-heptacosane, n-octacosane, and n-nonacosane. Gas chromatography-mass spectrometry (GC–MS) was used to analyze the cuticular hydrocarbons after they were extracted in hexane. The findings indicated that the degradation rate of CHCs was influenced by the surrounding environment, with more rapid degradation observed in soil conditions compared to room conditions. This suggests that soil can impact the weathering process of empty puparial cases. To predict the age of the puparial cases, a Support Vector Machine (SVM) analysis was conducted based on the concentrations of hydrocarbons. The SVM models demonstrated promising performance in age prediction, exhibiting high R-squared values and low prediction errors. By highlighting the influence of environmental factors on CHC degradation and demonstrating the efficacy of SVM models in age prediction, this study lays the groundwork for further exploration and development of CHCs as valuable tools in forensic and ecological research.

Endosymbiosis allows Sitophilus oryzae to persist in dry conditions.

Insects frequently associate with intracellular microbial symbionts (endosymbionts) that enhance their ability to cope with challenging environmental conditions. Endosymbioses with cuticle-enhancing microbes have been reported in several beetle families. However, the ecological relevance of these associations has seldom been demonstrated, particularly in the context of dry environments where high cuticle quality can reduce water loss. Thus, we investigated how cuticle-enhancing symbionts of the rice-weevil, Sitophilus oryzae contribute to desiccation resistance. We exposed symbiotic and symbiont-free (aposymbiotic) beetles to long-term stressful (47% RH) or relaxed (60% RH) humidity conditions and measured population growth. We found that symbiont presence benefits host fitness especially under dry conditions, enabling symbiotic beetles to increase their population size by over 33-fold within 3 months, while aposymbiotic beetles fail to increase in numbers beyond the starting population in the same conditions. To understand the mechanisms underlying this drastic effect, we compared beetle size and body water content and found that endosymbionts confer bigger body size and higher body water content. While chemical analyses revealed no significant differences in composition and quantity of cuticular hydrocarbons after long-term exposure to desiccation stress, symbiotic beetles lost water at a proportionally slower rate than did their aposymbiotic counterparts. We posit that the desiccation resistance and higher fitness observed in symbiotic beetles under dry conditions is due to their symbiont-enhanced thicker cuticle, which provides protection against cuticular transpiration. Thus, we demonstrate that the cuticle enhancing symbiosis of Sitophilus oryzae confers a fitness benefit under drought stress, an ecologically relevant condition for grain pest beetles. This benefit likely extends to many other systems where symbiont-mediated cuticle synthesis has been identified, including taxa spanning beetles and ants that occupy different ecological niches.

Seasonal changes in photoperiod and temperature lead to changes in cuticular hydrocarbon profiles and affect mating success in Drosophila suzukii

Seasonal plasticity in insects is often triggered by temperature and photoperiod changes. When climatic conditions become sub-optimal, insects might undergo reproductive diapause, a form of seasonal plasticity delaying the development of reproductive organs and activities. During the reproductive diapause, the cuticular hydrocarbon (CHC) profile, which covers the insect body surface, might also change to protect insects from desiccation and cold temperature. However, CHCs are often important cues and signals for mate recognition and changes in CHC composition might affect mate recognition. In the present study, we investigated the CHC profile composition and the mating success of Drosophila suzukii in 1- and 5-day-old males and females of summer and winter morphs. CHC compositions differed with age and morphs. However, no significant differences were found between the sexes of the same age and morph. The results of the behavioral assays show that summer morph pairs start to mate earlier in their life, have a shorter mating duration, and have more offspring compared to winter morph pairs. We hypothesize that CHC profiles of winter morphs are adapted to survive winter conditions, potentially at the cost of reduced mate recognition cues.

Gene expression differences consistent with water loss reduction underlie desiccation tolerance of natural Drosophila populations

BackgroundClimate change is one of the main factors shaping the distribution and biodiversity of organisms, among others by greatly altering water availability, thus exposing species and ecosystems to harsh desiccation conditions. However, most of the studies so far have focused on the effects of increased temperature. Integrating transcriptomics and physiology is key to advancing our knowledge on how species cope with desiccation stress, and these studies are still best accomplished in model organisms.ResultsHere, we characterized the natural variation of European D. melanogaster populations across climate zones and found that strains from arid regions were similar or more tolerant to desiccation compared with strains from temperate regions. Tolerant and sensitive strains differed not only in their transcriptomic response to stress but also in their basal expression levels. We further showed that gene expression changes in tolerant strains correlated with their physiological response to desiccation stress and with their cuticular hydrocarbon composition, and functionally validated three of the candidate genes identified. Transposable elements, which are known to influence stress response across organisms, were not found to be enriched nearby differentially expressed genes. Finally, we identified several tRNA-derived small RNA fragments that differentially targeted genes in response to desiccation stress.ConclusionsOverall, our results showed that basal gene expression differences across individuals should be analyzed if we are to understand the genetic basis of differential stress survival. Moreover, tRNA-derived small RNA fragments appear to be relevant across stress responses and allow for the identification of stress-response genes not detected at the transcriptional level.

Cuticular hydrocarbons of alpine bumble bees (Hymenoptera: Bombus) are species-specific, but show little evidence of elevation-related climate adaptation

Alpine bumble bees are the most important pollinators in temperate mountain ecosystems. Although they are used to encounter small-scale successions of very different climates in the mountains, many species respond sensitively to climatic changes, reflected in spatial range shifts and declining populations worldwide. Cuticular hydrocarbons (CHCs) mediate climate adaptation in some insects. However, whether they predict the elevational niche of bumble bees or their responses to climatic changes remains poorly understood. Here, we used three different approaches to study the role of bumble bees’ CHCs in the context of climate adaptation: using a 1,300 m elevational gradient, we first investigated whether the overall composition of CHCs, and two potentially climate-associated chemical traits (proportion of saturated components, mean chain length) on the cuticle of six bumble bee species were linked to the species’ elevational niches. We then analyzed intraspecific variation in CHCs of Bombus pascuorum along the elevational gradient and tested whether these traits respond to temperature. Finally, we used a field translocation experiment to test whether CHCs of Bombus lucorum workers change, when translocated from the foothill of a cool and wet mountain region to (a) higher elevations, and (b) a warm and dry region. Overall, the six species showed distinctive, species-specific CHC profiles. We found inter- and intraspecific variation in the composition of CHCs and in chemical traits along the elevational gradient, but no link to the elevational distribution of species and individuals. According to our expectations, bumble bees translocated to a warm and dry region tended to express longer CHC chains than bumble bees translocated to cool and wet foothills, which could reflect an acclimatization to regional climate. However, chain lengths did not further decrease systematically along the elevational gradient, suggesting that other factors than temperature also shape chain lengths in CHC profiles. We conclude that in alpine bumble bees, CHC profiles and traits respond at best secondarily to the climate conditions tested in this study. While the functional role of species-specific CHC profiles in bumble bees remains elusive, limited plasticity in this trait could restrict species’ ability to adapt to climatic changes.

Immune Stimulation via Wounding Alters Chemical Profiles of Adult Tribolium castaneum

Group-living individuals experience immense risk of disease transmission and parasite infection. In social and in some non-social insects, disease control with immunomodulation arises not only via individual immune defenses, but also via infochemicals such as contact cues and (defensive) volatiles to mount a group-level immunity. However, little is known about whether activation of the immune system elicits changes in chemical phenotypes, which may mediate these responses. We here asked whether individual immune experience resulting from wounding or injection of heat-killed Bacillus thuringiensis (priming) leads to changes in the chemical profiles of female and male adult red flour beetles, Tribolium castaneum, which are non-social but gregarious. We analyzed insect extracts using GC-FID to study the chemical composition of (1) cuticular hydrocarbons (CHCs) as candidates for the transfer of immunity-related information between individuals via contact, and (2) stink gland secretions, with analysis of benzoquinones as main active compounds regulating ‘external immunity’. Despite a pronounced sexual dimorphism in CHC profiles, wounding stimulation led to similar profile changes in males and females with increases in the proportion of methyl-branched alkanes compared to naïve beetles. While changes in the overall secretion profiles were less pronounced, absolute amounts of benzoquinones were transiently elevated in wounded compared to naïve females. Responses to priming were insignificant in CHCs and secretions. We suggest that changes in different infochemicals after wounding may mediate immune status signaling in the context of both internal and external immune responses in groups of this non-social insect, thus showing parallels to social immunity.

Evidence for a chemical arms race between cuckoo wasps of the genus Hedychrum and their distantly related host apoid wasps

BackgroundBrood parasites can exert strong selection pressure on their hosts. Many brood parasites escape their detection by mimicking sensory cues of their hosts. However, there is little evidence whether or not the hosts are able to escape the parasites’ mimicry by changing these cues. We addressed this question by analyzing cuticular hydrocarbon (CHC) profiles of Cerceris and Philanthus wasps and their brood parasites, cuckoo wasps mimicking the CHC profiles of their hosts. Some of these hosts use hydrocarbons to preserve their prey against fungal infestation and thus, they cannot significantly change their CHC composition in response to chemical mimicry by Hedychrum brood parasites.ResultsWe found that the CHC overlap between brood parasites and their hosts was lower in case of host wasps not preserving their prey than in case of prey-preserving host wasps, whose CHC evolution is constrained. Furthermore, the CHC profiles in non-preserving host wasps is more strongly diversified in females than in males, thus in the sex that is chemically mimicked by brood parasites.ConclusionOur results provide evidence for a chemical arms race between those hosts that are liberated from stabilizing selection on their chemical template and their parasites.

Within and among population differences in cuticular hydrocarbons in the seabird tick Ixodes uriae

The hydrophobic layer of the arthropod cuticle acts to maintain water balance, but can also serve to transmit chemical signals via cuticular hydrocarbons (CHC), essential mediators of arthropod behavior. CHC signatures typically vary qualitatively among species, but also quantitatively among populations within a species, and have been used as taxonomic tools to differentiate species or populations in a variety of taxa. Most work in this area to date has focused on insects, with little known for other arthropod groups such as ticks. The worldwide distribution and extensive host-range of the seabird tick Ixodes uriae make it a good model to study the factors influencing CHC composition. Genetically differentiated host-races of I. uriae have evolved across the distribution of this species but the factors promoting sympatric population divergence are still unknown. To test for a potential role of host-associated CHC in population isolation, we collected I. uriae specimens from two of its seabird hosts, the Atlantic puffin (Fratercula arctica) and the common guillemot (Uria aalge) in different colonies in Iceland. Using gas-chromatography and mass-spectrometry, we detected a complex cuticular mixture of 22 hydrocarbons, including n-alkanes, methyl-alkanes and alkenes ranging from 17 to 33 carbons in length. We found that each population had a distinct CHC profile. The host group explained the greatest amount of population divergence, with long-chain hydrocarbons being more abundant in puffin tick populations compared to guillemot tick populations. Future work will now be required to test whether the different CHC signals reinforce assortative mating, thereby playing a role in generating I. uriae population divergence patterns, and to evaluate diverse hypotheses on the origin of distinct population signatures.

Why do ants differ in acclimatory ability? Biophysical mechanisms behind cuticular hydrocarbon acclimation across species.

Maintaining water balance is vital for terrestrial organisms. Insects protect themselves against desiccation via cuticular hydrocarbons (CHCs). CHC layers are complex mixtures of solid and liquid hydrocarbons, with a surprisingly diverse composition across species. This variation may translate into differential phase behaviour, and hence varying waterproofing capacity. This is especially relevant when temperatures change, which requires acclimatory CHC changes to maintain waterproofing. Nevertheless, the physical consequences of CHC variation are still little understood. We studied acclimatory responses and their consequences for CHC composition, phase behaviour and drought survival in three congeneric ant species. Colony sub-groups were kept under cool, warm and fluctuating temperature regimes. Lasius niger and Lasius platythorax, both of which are rich in methyl-branched alkanes, showed largely predictable acclimatory changes of the CHC profile. In both species, warm acclimation increased drought resistance. Warm acclimation increased the proportion of solid compounds in L. niger but not in L. platythorax. In both species, the CHC layer formed a liquid matrix of constantly low viscosity, which contained highly viscous and solid parts. This phase heterogeneity may be adaptive, increasing robustness to temperature fluctuations. In Lasius brunneus, which is rich in unsaturated hydrocarbons, acclimatory CHC changes were less predictable, and warm acclimation did not enhance drought survival. The CHC layer was more homogeneous, but matrix viscosity changed with acclimation. We showed that ant species use different physical mechanisms to enhance waterproofing during acclimation. Hence, the ability to acclimate, and thus climatic niche breadth, may strongly depend on species-specific CHC profile.

Increased complexity of worker CHC profiles in Apis dorsata correlates with nesting ecology.

Cuticular hydrocarbons (CHC) are known to serve as discrimination cues and will trigger defence behaviour in a plethora of eusocial insects. However, little is known how about nestmate recognition ability selects for CHC diversification. In this study we investigate differences in CHC composition of four major honey bee species with respect to the differences in their nesting behavior. In contrast to A. mellifera, A. cerana and A. florea, the giant honey bee A. dorsata prefers to build their nests in aggregations with very small spatial distances between nests, which increases the probability of intrusions. Thus, A. dorsata exhibits a particularly challenging nesting behavior which we hypothesize should be accompanied with an improved nestmate recognition system. Comparative analyses of the worker CHC profiles indicate that A. dorsata workers exhibit a unique and more complex CHC profile than the other three honey bee species. This increased complexity is likely based on a developmental process that retains the capability to synthesize methyl-branched hydrocarbons as adults. Furthermore, two sets of behavioral experiments provide evidence that A. dorsata shows an improved nestmate discrimination ability compared to the phylogenetically ancestral A. florea, which is also open-nesting but does not form nest aggregations. The results of our study suggest that ecological traits like nesting in aggregation might be able to drive CHC profile diversification even in closely related insect species.

Acclimation in ants: Interference of communication and waterproofing through cuticular hydrocarbons in a multifunctional trait

Abstract Organismal traits may experience conflicting selection pressures if they fulfil different functions simultaneously. This can require trade‐offs between functions or alternatively functional separation between elements of the trait. An important multifunctional trait in insects is the cuticular hydrocarbon (CHC) layer. CHCs cover the body of nearly all insects, protect against desiccation and serve as a communication signal. In social insects like ants, they provide cues for nestmate recognition. To maintain their waterproofing function, insects have to adjust CHC composition to current temperatures. These changes might affect information content and interfere with communication, which would be especially detrimental in social insects. Here, we studied how acclimation affects nestmate recognition in two sister species of the ant genus Lasius. Colony fragments were exposed to three climate regimes. We analysed behaviour towards same and differently acclimated conspecifics, and determined which CHCs were related to acclimatory changes, colony differences and inter‐individual aggression. Differential acclimation led to higher aggression and chemical distances among former nestmates. We identified small CHC subsets, which only differed among colonies or among acclimation treatments. Moreover, few compounds sufficed to explain inter‐individual aggression, suggesting that ants do not use the entire CHC profile for nestmate recognition and that colony identity is encoded in a redundant way. Across individual CHCs, their contribution to colony differences and to differences among acclimatory treatments was negatively correlated, indicating that there is some degree of functional separation. However, CHC classes could not be clearly assigned to one or another function, indicating that the role of each CHC is idiosyncratic and may differ among species. Acclimatory effects and colony differences were more independent from each other in L. platythorax than in L. niger, indicating that functional separation can differ even among sister species. Our results show that CHC functions are more intertwined than previously assumed, suggesting that insects cannot optimise all functions independently. The main constraint might be the need to maintain a certain phase behaviour of the CHC layer, which depends on CHC composition and affects functionality. The need to separate functions might depend on species‐specific ecological and life‐history parameters. Read the free Plain Language Summary for this article on the Journal blog.