Role of cuticular hydrocarbons in the chemical recognition between ant species in the Pachycondyla villosa species complex

Cuticular hydrocarbons (HCs) play important roles in insect communication but few studies clearly demonstrate the direct link between HCs and nestmate recognition. Therefore, cuticular lipids were extracted from ants, their HC and non-HC fractions as well as the three principal classes of HCs (n-alkanes, branched alkanes and alkenes) were purified and tested using an immobilizing "joust" device which allowed quantification of early pairwise behavioural responses, mandibular opening and antennal retraction, without occurrence of subsequent damages as in classic dyadic encounters. Chemical recognition of ants was studied at three levels of interactions (intra-colonial, intra-specific and inter-specific). Three closely related species already chemically characterized were used: Pachycondyla villosa (Pv), P. inversa (Pi) and P. subversa (Ps). Each species had its own behavioural responses. Moreover, responses of Pi and Ps towards Pv were significantly longer, than they were between themselves whereas Pv ants were equally aggressive towards Pi and Ps. These differences are in agreement with the results of the cluster analysis of the cuticular HCs profiles that place Pi closer to Ps. In support of the idea that components of cuticular lipids profiles are important for recognition, we found that only the HC fraction and its branched subfraction elicited a behavioural response of Ps workers. It is suggested that internally branched methyl- and dimethylalkanes are involved in recognition behaviour.     

A Drosophila male pheromone affects female sexual receptivity

Sex pheromones are chemical signals frequently required for mate choice, but their reciprocal role on mate preference has rarely been shown in both sexes. In Drosophila melanogaster flies, the predominant cuticular hydrocarbons (CHs) are sexually dimorphic: only females produce 7,11-dienes, whereas 7-tricosene (7-T) is the principal male CH. Males generally prefer females with 7,11-dienes, but the role of 7-T on female behaviour remains unclear. With perfumed males, control females mated faster and more often with males carrying increased levels of 7-T showing that this CH acts as a chemical stimulant for D. melanogaster females. Control females-but not antenna-less females-could detect small variation of 7-T. Finally, our finding that desat1 mutant female showed altered response towards 7-T provides an additional role for this gene which affects the production and the perception of pheromones involved in mate choice, in both sexes.     

Hydrocarbon circulation and colonial signature in Pachycondyla villosa

In ants, both cuticular and postpharyngeal gland (PPG) hydrocarbons (HCs) have been involved in nestmate recognition. However, no detailed comparison is available. A comparative study including also high density lipophorin (HDLp), an internal HC carrier, was therefore undertaken on Pachycondyla villosa. Purified HDLp is an 820 kDa lipoprotein with a density of 1.114 g/ml and two 245 and 80 kDa apo-proteins. Its hydrocarbon profile is very similar with the cuticular one, in agreement with its hydrocarbon carrier function. Conversely, n-alkanes and externally branched monomethylalkanes are markedly decreased in the PPG. According to their physical properties, this suggests that they are involved in waterproofing on the cuticle. The PPG actually contains only internally branched mono-, dimethylalkanes or monomethylalkenes; their greater fluidity is more adequate for chemical communication. The percentages of some of them are statistically not different between the cuticle and PPG. Their mixtures vary with colonies and they may thus be involved in colonial signature. A scheme for hydrocarbon circulation is discussed, involving lipophorin, cuticle, PPG and self-grooming in one individual, a pathway complementary or alternative to the selective delivery by lipophorin in some other insects. HCs are then distributed between nestmates' cuticles through allo-grooming and physical contacts.     

Sex- and age-related changes in the biophysical properties of cuticular lipids of the housefly,Musca domestica

We examined the biophysical properties of cuticular lipids isolated from the housefly, Musca domestica. Melting temperatures (T_m) of surface lipids isolated from female houseflies decreased from 39.3 °C to 35.3 °C as the females attained sexual maturity and produced sex pheromone, whereas those prepared from males did not change with age. Lipids melted over a 10–25 °C temperature range, and their physical properties were a complex function of the properties of the component lipids. The T_m of total cuticular lipids was slightly below that of cuticular hydrocarbons (HC), the predominant lipid fraction. Hydrocarbons were further fractionated into saturated, unsaturated, and methyl-branched components. The order of decreasing T_m was total alkanes > total HCs > methyl-branched alkanes > alkenes. For 1-day-old flies, measured T_ms of hydrocarbons were 1.3–5.5 °C lower than T_ms calculated from a weighted average of T_ms for saturated and unsaturated components. For 4-day-old flies, calculated T_ms underestimated T_m by 11–14 °C. © 1995 Wiley-Liss, Inc.     

Variation in the male pheromones and mating success of wild caught Drosophila melanogaster

Drosophila melanogaster males express two primary cuticular hydrocarbons (male-predominant hydrocarbons). These act as sex pheromones by influencing female receptivity to mating. The relative quantities of these hydrocarbons vary widely among natural populations and can contribute to variation in mating success. We tested four isofemale lines collected from a wild population to assess the effect of intrapopulation variation in male-predominant hydrocarbons on mating success. The receptivity of laboratory females to males of the four wild-caught lines varied significantly, but not consistently in the direction predicted by variation in male-predominant hydrocarbons. Receptivity of the wild-caught females to laboratory males also varied significantly, but females from lines with male-predominant hydrocarbon profiles closer to a more cosmopolitan one did not show a correspondingly strong mating bias toward a cosmopolitan male. Among wild-caught lines, the male-specific ejaculatory bulb lipid, cis-vaccenyl acetate, varied more than two-fold, but was not associated with variation in male mating success. We observed a strong inverse relationship between the receptivity of wild-caught females and the mating success of males from their own lines, when tested with laboratory flies of the opposite sex.     

Volatile Drosophila cuticular pheromones are affected by social but not sexual experience

Recognition of conspecifics and mates is based on a variety of sensory cues that are specific to the species, sex and social status of each individual. The courtship and mating activity of Drosophila melanogaster flies is thought to depend on the olfactory perception of a male-specific volatile pheromone, cis-vaccenyl acetate (cVA), and the gustatory perception of cuticular hydrocarbons (CHs), some of which are sexually dimorphic. Using two complementary sampling methods (headspace Solid Phase Micro-Extraction [SPME] and solvent extraction) coupled with GC-MS analysis, we measured the dispersion of pheromonal CHs in the air and on the substrate around the fly. We also followed the variations in CHs that were induced by social and sexual interactions. We found that all CHs present on the fly body were deposited as a thin layer on the substrate, whereas only a few of these molecules were also detected in the air. Moreover, social experience during early adult development and in mature flies strongly affected male volatile CHs but not cVA, whereas sexual interaction only had a moderate influence on dispersed CHs. Our study suggests that, in addition to their role as contact cues, CHs can influence fly behavior at a distance and that volatile, deposited and body pheromonal CHs participate in a three-step recognition of the chemical identity and social status of insects.     

Effect of the entomopathogenic fungus,Entomophthora muscae (Zygomycetes: Entomophthoraceae), on sex pheromone and other cuticular hydrocarbons of the house fly,Musca domestica

House fly (Musca domestica) males are highly attracted to dead female flies infected with the entomopathogenic fungus Entomophthora muscae. Because males orient to the larger abdomen of infected flies, both visual and chemical cues may be responsible for the heightened attraction to infected flies. Our behavioral assays demonstrated that the attraction is sex-specific-males were attracted more to infected females than to infected males, regardless of cadaver size. We examined the effect of E. muscae on the main component of the house fly sex pheromone, (Z)-9-tricosene, and other cuticular hydrocarbons including n-tricosane, n-pentacosane, (Z)-9-heptacosene, and total hydrocarbons of young (7 days old) and old (18 days old) virgin females. Young E. muscae-infected female flies accumulated significantly less sex pheromone and other hydrocarbons on their cuticular surface than uninfected females, whereas the cuticular hydrocarbons of older flies were unaffected by fungus infection. These results suggest that chemical cues other than (Z)-9-tricosene, visual cues other than abdomen size, or a combination of both sets of cues might be responsible for attraction of house fly males to E. muscae-infected females.     

Genetic divergence does not predict change in ornament expression among populations of stalk-eyed flies

Stalk-eyed flies (Diptera: Diopsidae) possess eyes at the ends of elongated peduncles, and exhibit dramatic variation in eye span, relative to body length, among species. In some sexually dimorphic species, evidence indicates that eye span is under both intra- and intersexual selection. Theory predicts that isolated populations should evolve differences in sexually selected traits due to drift. To determine if eye span changes as a function of divergence time, 1370 flies from 10 populations of the sexually dimorphic species, Cyrtodiopsis dalmanni and Cyrtodiopsis whitei, and one population of the sexually monomorphic congener, Cyrtodiopsis quinqueguttata, were collected from Southeast Asia and measured. Genetic differentiation was used to assess divergence time by comparing mitochondrial (cytochrome oxidase II and 16S ribosomal RNA gene fragments) and nuclear (wingless gene fragment) DNA sequences for c. five individuals per population. Phylogenetic analyses indicate that most populations cluster as monophyletic units with up to 9% nucleotide substitutions between populations within a species. Analyses of molecular variance suggest a high degree of genetic structure within and among the populations; > 97% of the genetic variance occurs between populations and species while < 3% is distributed within populations, indicating that most populations have been isolated for thousands of years. Nevertheless, significant change in the allometric slope of male eye span on body length was detected for only one population of either dimorphic species. These results are not consistent with genetic drift. Rather, relative eye span appears to be under net stabilizing selection in most populations of stalk-eyed flies. Given that one population exhibited dramatic evolutionary change, selection, rather than genetic variation, appears to constrain eye span evolution.     

A Drosophila protein family implicated in pheromone perception is related to Tay-Sachs GM2-activator protein

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.     

Male-limited evolution suggests no extant intralocus sexual conflict over the sexually dimorphic cuticular hydrocarbons of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Sexually dimorphic traits are likely to have evolved through sexually antagonistic selection. However, recent empirical data suggest that intralocus sexual conflict often persists, even when traits have diverged between males and females. This implies that evolved dimorphism is often incomplete in resolving intralocus conflict, providing a mechanism for the maintenance of genetic variance in fitness-related traits. We used experimental evolution in Drosophila melanogaster to directly test for ongoing conflict over a suite of sexually dimorphic cuticular hydrocarbons (CHCs) that are likely targets of sex-specific selection. Using a set of experimental populations in which the transmission of genetic material had been restricted to males for 82 generations, we show that CHCs did not evolve, providing experimental evidence for the absence of current intralocus sexual conflict over these traits. The absence of ongoing conflict could indicate that CHCs have never been the target of sexually antagonistic selection, although this would require the existing dimorphism to have evolved via completely sexlinked mutations or as a result of former, but now absent, pleiotropic effects of the underlying loci on another trait under sexually antagonistic selection. An alternative interpretation, and which we believe to be more likely, is that the extensive CHC sexual dimorphism is the result of past intralocus sexual conflict that has been fully resolved, implying that these traits have evolved genetic independence between the sexes and that genetic variation in them is therefore maintained by alternative mechanisms. This latter interpretation is consistent with the known roles of CHCs in sexual communication in this species and with previous studies suggesting the genetic independence of CHCs between males and females. Nevertheless, direct estimates of sexually antagonistic selection will be important to fully resolve these alternatives.     

Not only cuticular lipids: first evidence of differences between foundresses and their daughters in polar substances in the paper wasp Polistes dominulus

Several studies have shown that differences in the relative abundance of cuticular hydrocarbons occur between reproductives and non-reproductives in many social insects. These differences also exist between Polistes dominulus foundresses and their first emerged daughters (usually indicated as workers), but they gradually disappear when workers from orphaned colonies develop their ovaries and assume a reproductive role in the colony. However, hydrocarbons are not the exclusive components of cuticular layer of wasps. Mass-spectrometry analysis of cuticular methanol extracts from Polistes paper wasps showed a complex pattern of polar substances, partly or totally proteinaceous in nature. We found that these compounds, ranging from 918 to 2679 Da, showed a clear caste differentiation between foundresses and their first emerged daughters (usually indicated as "workers"), both in queen-right and in orphaned colonies. Conversely to hydrocarbons, workers from orphaned colonies maintain a significant difference from foundresses in the pattern of the medium molecular weight (MW) polar compounds obtained by MALDI-TOF. On the basis of such results we hypothesize that a reliable cue to identify foundresses from daughters, and not only their reproductive status, may exist in Polistes wasps. Although the great majority of previous work on social insect communication has focused on cuticular hydrocarbons, our findings suggest that the medium MW component of cuticular substances may be involved in recognition.     

白蚁表皮碳氢化合物研究进展

近年来, 固相微萃取等现代技术的使用显著促进了白蚁表皮碳氢化合物研究的开展。至今, 已有约29种白蚁的表皮碳氢化合物组分得到鉴定, 分属于木白蚁科、 鼻白蚁科、 原白蚁科和白蚁科, 其组分主要为正烷烃、 含有不同数量甲基的支链烷烃及少量烯烃。白蚁表皮碳氢化合物不仅具有一定的科、 属特异性, 大多数种类还具备特有组分, 表明其可作为种间识别的指标。表皮碳氢化合物组分在种内个体识别方面的作用, 在低等白蚁中多获得了支持性结果, 但也有研究认为在这些种类中表皮碳氢化合物不是种内个体识别（同巢个体识别）的唯一指标。发现其与品级分化的相关是近年来白蚁表皮碳氢化合物研究的重要进展。有些种类表皮碳氢化合物的年消长与生殖蚁的分化有关; 而另一些种类生殖蚁含有表皮碳氢化合物特有组分, 其含量与生殖蚁的生殖状态有关, 提示其可能在品级分化中发挥重要作用。作为研究白蚁品级分化和维持机理的新方向, 表皮碳氢化合物值得进一步研究探索。     

Purification of adult Drosophila melanogaster lipophorin and its role in hydrocarbon transport

Lipophorin was isolated from homogenized adult Drosophila melanogaster. It is stained by Sudan Black and has a native molecular mass of 640 kD and a density of 1.12 g/ml. It consists of two glycosylated apoproteins of 240 and 75 kDa. Gas chromatography and mass spectrometry showed that lipophorins isolated separately from virgin 3-day-old male and female flies were associated with specific hydrocarbons, and that these were the same hydrocarbons found in male and female cuticles, respectively. Moreover, a pool of internal hydrocarbons was demonstrated for the first time, with chain lengths similar to those of the cuticular pool. Studies on the fate of the hydrocarbons synthesized de novo after topical applications of radiolabelled fatty acid precursors showed a decrease of the internal pool of hydrocarbons with time, concomitant with an increase of the cuticular pool. These results suggest that hydrocarbons synthesized at an internal site, possibly in oenocytes, may be transported to the cuticle of the flies by lipophorin. © 1996 Wiley-Liss, Inc.     

Cuticular hydrocarbons and their role in copulatory behavior in Phormia regina (Meigen)

The cuticular hydrocarbons from adult Phormia regina (Meigen) were characterized by gas chromatography-mass spectrometry. Both sexes had similar components in nearly identical quantities, consisting of complex mixtures of saturated n-, monomethyl- and dimethylalkanes from 23 to 33 total carbons. Although no diet-, age-, or sex-specific differences were observed, cuticular hydrocarbons were shown to be involved in copulatory behavior. Hydrocarbon profiles of wild, compared to laboratory reared flies, showed no major differences. Behaviorally, males responded the same to dead decoys of either sex. Removal of the hydrocarbons, using hexane, from either male or female decoys, did not affect the number of mating strikes, but markedly reduced the number of copulatory attempts and the amount of time males spent mounted on either decoy. House fly, Musca domestica L., males when paired with a female M. domestica decoy produced copulatory attempts: whereas, when P. regina males were placed with M. domestica female decoys, there were no copulatory attempts. It is concluded that the cuticular hydrocarbons of P. regina function as species-specific but not sex-specific mating cues and elicit species-specific copulatory behavior in males.     

Levels of mate recognition within and between two Drosophila species and their hybrids

If sexual selection is to result in speciation, traits involved in mate choice within species need to be capable of producing sexual isolation between species. We investigated the association between mate choice and sexual isolation using interspecific hybrids between two sibling species, Drosophila serrata and Drosophila birchii. A perfuming experiment demonstrated that olfaction was involved in the sexual isolation between the two species. A quantitative genetic analysis using 30 populations of hybrids between the two species indicated that mating success in hybrid individuals was predominately determined by cuticular hydrocarbons; the average genetic correlation between mating success and cuticular hydrocarbon profile was 0.84, and in some instances exceeded 0.95. Multivariate analysis of the cuticular hydrocarbons of the two species revealed that there were three independent blends of cuticular hydrocarbons that separated three levels of organization: species, sex, and sex within species. The hydrocarbons used by hybrids in mate choice included those that separated the two species, demonstrating that species-specific characters may be used in mate choice within populations. The interspecific reciprocal cross had major effect on which cuticular hydrocarbons were associated with mating success, indicating that the expression of the cuticular hydrocarbons was strongly sex linked.     

Interaction between temperature and male pheromone in sexual isolation in Drosophila melanogaster

In Drosophila, female hydrocarbons are known to be involved in premating isolation between different species and pheromonal races. The role of male‐specific hydrocarbon polymorphism is not as well documented. The dominant cuticular hydrocarbon (CHC) in male D. melanogaster is usually 7‐tricosene (7‐T), with the exception of African populations, in which 7‐pentacosene (7‐P) is dominant. Here, we took advantage of a population from the Comoro Islands (Com), in which males fell on a continuum of low to high levels of 7‐T, to perform temperature selection and selection on CHCs' profiles. We conducted several experiments on the selected Com males to study the plasticity of their CHCs in response to temperature shift, their role in resistance to desiccation and in sexual selection. We then compared the results obtained for selected lines to those from three common laboratory strains with different and homogenous hydrocarbon profiles: CS, Cot and Tai. Temperature selection modified the CHC profiles of the Com males in few generations of selection. We showed that the 7‐P/7‐T ratio depends on temperature with generally more 7‐P at higher temperatures and observed a relationship between chain length and resistance to desiccation in both temperature‐ and phenotypically selected Com lines. There was partial sexual isolation between the flies with clear‐cut phenotypes within the phenotypically selected lines and the laboratory strains. These results indicate that the dominant male pheromones are under environmental selection and may have played a role in reproductive isolation.     

Water loss through the integument in the desiccation-sensitive mutant, Parched, of Drosophila melanogaster

Two desiccation-sensitive mutants of Drosophila melanogaster were isolated. Genetic analysis showed that the phenotype is controlled by a recessive gene parched located in 1A1-8 of the X-chromosome. In a desiccated environment without any water supply, the survival time of the mutant flies was considerably shorter than that of the wild-type flies. The rate of water loss in the mutant flies was significantly higher than that of the wild-type flies, whether dead or alive. The survival time of the mosaic flies, which have the mutant and wild-type cuticle, was prolonged in proportion to the amount of wild-type cuticle which they possessed. These results suggest that the mutant has a defect in some waterproofing mechanism of the integument. The mutant flies drank much more water than the wild-type flies, to compensate for the rapid water loss. The hydrocarbons, which are the predominant constituent of cuticular lipids, were analyzed by gas-liquid chromatography, but there were no significant quantitative nor qualitative differences between the wild-type and the mutant flies.