Does the Stereochemistry of Methylated Cuticular Hydrocarbons Contribute to Mate Recognition in the Egg Parasitoid Wasp Ooencyrtus kuvanae?

Close-range sexual communication of the egg parasitoid wasp Ooencyrtus kuvanae (Hymenoptera: Encyrtidae) takes place on host gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae), egg masses. We tested the hypothesis that mate recognition in O. kuvanae is mediated, in part, by low-volatility cuticular hydrocarbon (CHC) pheromone components. Gas chromatographic and GC-mass spectrometric analyses of body surface extracts of male and female wasps revealed no sex-specific components, but 5-methylheptacosane (5-me-27Hy) and 5,17-dimethylheptacosane (5,17-dime-27Hy) were consistently more abundant in extracts of males. The ratio of 5-me-27Hy and 5,17-dime-27Hy was similar in extracts of males and females, and quantitative differences alone seemed insufficient to impart sex-specific CHC profiles. Therefore, we further hypothesized that the absolute configuration of 5-me-27Hy and 5,17-dime-27Hy contributes to mate recognition or attraction. As the stereoisomers of 5-me-27Hy and 5,17-dime-27Hy cannot currently be separated chromatographically, we could not determine the stereochemistry of the insect-produced components. Instead, we synthesized all stereoisomers and bioassayed synthetic blends in laboratory experiments. Of eight 2-component blends, each blend containing one of the two enantiomers of 5-me-27Hy and one of the four stereoisomers of 5,17-dime-27Hy, the blend of (5S)-methylheptacosane and (5R,17S)-dimethylheptacosane attracted males, whereas the blend of (5R)-methylheptacosane and (5R,17R)-dimethylheptacosane repelled males. Apparent recognition of both pheromone components and pheromone antagonists by males supports the hypothesis that the stereochemistry of 5-me-27Hy and 5,17-dime-27Hy, and possibly other methylated CHCs, may differ between male and female O. kuvanae, and that these differences may serve in mate attraction and recognition.     

Cuticular lipids of insects: I. Hydrocarbons ofLeucophae maderae andBlatta orientalis

The major hydrocarbons found in the surface lipids of the cockroachesLeucophaea maderae andBlatta orientalis have been identified asn-heptacosane, 11-methylheptacosane, 13-methylheptacosane and 3-methylheptacosane. The hydrocarbons from the two genera of cockroaches are qualitatively identical and quantitatively similar.     

Nestmate Recognition and the Role of Cuticular Hydrocarbons in the African Termite Raiding Ant <Emphasis Type="Italic">Pachycondyla analis</Emphasis>

Cuticular hydrocarbons (CHCs) are used for chemical communication among nestmates in many ant species, and they may play a role in the discrimination of nestmates and non-nestmates. Using the mandible opening response (MOR) bioassay, we tested the response of the African termite raiding ant, Pachycondyla analis, to CHC extracts of nestmates and non-nestmates. The ants were able to distinguish control chemical cues, from nestmate CHCs, and from non-nestmate CHCs, and, based on a CHC recognition threshold, aggression was demonstrated toward non-nestmates. Gas chromatography (GC) and GC-mass spectrometric analyses showed that CHC components of different ant colonies had chain lengths ranging from C₈ to C₃₁, comprising mainly n-alkanes, alkenes, and methyl branched alkanes, with the n-alkanes occurring in the same proportions among all colonies. The ants were grouped successfully according to their colonies of origin by using discriminant analysis of CHCs. We demonstrate that nestmate recognition occurs in P. analis, and that some of the cues involved are evidently alkenes and methyl-branched alkanes.     

Soldier frontal glands of the termiteMacrotermes subhyalinus: Morphology,chemical composition,and use in defense

Soldiers of the East African fungus-growing termiteMacrotermes subhyalinus (Rambur) (Termitidae: Macrotermitinae) employ both mechanical and chemical defenses. Soldiers release a chemical secretion composed of long-chain saturated and monounsaturated hydrocarbons into wounds inflicted by their powerful mandibles. Chemical analysis of the secretion shows the paraffin fraction to consist primarily ofn-tricosane,n-pentacosane, 3- and 5-methylpentacosane, and 5-methylheptacosane. The major oleflns were identified as (Z)-9-heptacosene and (Z)-9-nonacosene. The secretion originates from the frontal glands of both major and minor soldiers; however, the hypertrophied gland of the major soldiers contains 500-fold more secretion than that of the minor soldiers. This secretion appears to impair the healing of wounds in test ants, and thus could represent a valuable supplement to the mechanical defense mechanism.     

Aphrodisiac pheromones of the sulfur butterfliesColias eurytheme andC. Philodice (Lepidoptera,Pieridae)

Male wing compounds involved in maintaining reproductive isolation between the sulfur butterfliesColias eurytheme andC. philodice have been identified. MaleC. philodice produce threen-hexyl esters; myristate, palmitate, and stearate, which are absent inC. eurytheme. A branched hydrocarbon, 13-methylheptacosane, is found on the wings of maleC. eurytheme, but notC. philodice. Several straight-chain hydrocarbons are on the wings of both species. The esters and 13-methylheptacosane have significant electrophysiological activity. Preliminary behavioral experiments indicate that the esters (especiallyn-hexyl myristate) function as species-recognition signals. The esters and 13-methylheptacosane also have low-to-moderate aphrodisiac activity.     

Chemical secretions of two sympatric harvester ants,Pogonomyrmex salinus andMessor lobognathus

Messor lobognathus, an apparent mimic ofPogonomyrmex salinus, shows little chemical resemblance to its exemplar. The mandibular glands ofM. lobognathus gave no volatile compounds. Those ofP. salinus contain chiefly 4-methyl-3-heptanone. Both species contain a mixture of straight-chain alkanes, alkenes, and methyl-branched alkanes in their Dufour glands. Tridecane (64%) is the major substance inM. lobognathus and dodecane (25%) and pentadecane (24%) are the major compounds ofP. salinus. No secretion induced trail-following in either species. A mixture of 9-, 11-, and 13-methylheptacosane formed the largest peak in the chromatograms obtained from the postpharyngeal glands of both species, but otherwise the hydrocarbon mixtures in this gland too were distinct.     

Surface lipids of the social waspPolistes annularis (L.) and its nest and nest pedicel

Cuticular lipids were recovered fromPolistes annularis workers and characterized by combined gas-chromatography-mass spectrometry. These lipids were found to be straight-chain and methyl-branched hydrocarbons with the major components being 13,17-dimethylhentriacontane (18%), 3-methyl-nonacosane (13%), and 3-methylheptacosane (11%). Cuticular lipids with a very similar composition were found on the surface of the larvae, eggs, and adult males ofPolistes annularis as well as on the surface of the nest and nest pedicel of the wasp. Hexadecanoic acid and octadecenoic acid were also found on the surface of the nest pedicel and these acids and/or the hydrocarbons may play a defensive role for the wasps.     

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.     

Sex Pheromone of Glossina tachinoides: Isolation, Identification, and Synthesis

Study of lipids from male and female laboratory-reared flies led to the demonstration of a potent contact sex stimulant in extracts and cuticular hydrocarbons of the female tsetse fly Glossina tachinoides (Westwood) against conspecific males. Thin-layer and column chromatography indicated that extracts contained hydrocarbons and saponifiable lipids. Biological activity was found in the alkanes from females, including prominent branched-chain alkanes that were detected by gas chromatography (GC). The alkanes were separated and collected by preparative gas chromatography (GC), and only the 37-carbon region showed biological activity. GC–mass spectrometry showed the major peak contained a mixture of isomeric 11,23-, 13,25- plus a minor amount of 11,21-dimethylheptatriacontane. Two racemic isomers were synthesized, and bioassays showed that the greatest activity was possessed by the 11,23- isomer with somewhat less activity in 13,25-dimethyl heptatria-contane. Dose–response data showed ED₅₀ at 5 g per decoy with solvent-washed males, nonspecific females, or corks as decoys. These alkanes released sexual activity in males that comprised most of the behaviors released by a female fly of the same species.     

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.     

Lack of intraspecific aggression in the ant Tetramorium bicarinatum: a chemical hypothesis

Tetramorium bicarinatum(Myrmicinae) is an ant species frequently found in tropical and subtropical areas, particularly in Africa, Southeast Asia (Japan), and South America (Brazil). The species is polygynous, reproduces by budding, and has sterile workers. Since the nests are widely distributed in a given area, the problem arises of territorial defense against conspecifics. Because not all ants defend territories, we assessed the defensive behavior of T. bicarinatumworkers through intraspecific and interspecific aggressiveness tests. A detailed behavioral study of the interactions between workers from several different colonies of T. bicarinatum(originating from Japan and Brazil) showed that workers do not discriminate against conspecific nonnestmate individuals, but they are highly aggressive towards allospecifics (Myrmica rubra, Myrmicinae). The results suggest that each colony from this ant species possesses a similar colonial odor. Chemical analyses of the cuticular hydrocarbons of these species were made with gas chromatography coupled to mass spectrometry. Results showed that the different colonies of T. bicarinatumpossess a common chemical profile mainly composed of straight-chain alkanes and alkenes, while M. rubrapossess more methyl-branched alkanes. We suggest that methyl alkane cues play a determining role in colonial recognition and that these results could explain the underlaying basis of the lack of intraspecific aggressiveness in T. bicarinatum.     

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.     

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.     

Species Recognition from Postpharyngeal Gland Contents of Ants of the Cataglyphis bicolor Group

The Cataglyphis bicolor group of species of desert-dwelling ants, difficult to identify from morphological features alone, can be readily recognized by the contents of their postpharyngeal glands. Analysis by linked gas chromatography–mass spectrometry of glands from colonies identified only by code numbers showed in all samples straight and branched-chain alkanes and linear alkenes. C. viaticus, C. bicolor, and C. savignyi, the three species most difficult to distinguish morphologically, each contained distinctly different patterns of hydrocarbons, as illustrated by cluster analysis. The 16 most abundant hydrocarbons in the whole group of samples were selected and plotted as windrose diagrams. The differences in the windroses have more visual impact than gas chromatograms of the same data. The only case where there was any similarity was that between C. bicolor and C. diehlii, and even there the resemblance was not close. C. bombycinus is a sympatric species but is recognized as not belonging to the bicolor group by its different mandibular gland substances. It also was easily distinguished by its postpharyngeal gland contents from the other species.     

Cuticular hydrocarbons of adults of the cowpea weevil,Callosobruchus maculatus

The composition of the cuticular hydrocarbons of the cowpea weevil,Callosobruchus maculatus (F.), was determined by using combined gas chromatography-mass spectrometry. The hydrocarbons constituted 88% of the cuticular lipids and were composed of four homologous series of alkanes. Mono- and dimethyl branched-chain alkanes made up 83% of the hydrocarbon fraction.Coleoptera: Bruchidae.Mention of a proprietary product or company name in this paper does not constitute a recommendation of this product by the U.S. Department of Agriculture.     

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.     

Diesel Oil Degradation Potential of a Bacterium Inhabiting Petroleum Hydrocarbon Contaminated Surface Waters and Characterization of Its Emulsification Ability

Degradation of poorly water soluble hydrocarbons, like n‐alkanes and polycyclic aromatic hydrocarbons are challenged by some bacteria through emulsification of hydrocarbons by producing biosurfactants. In diesel oil bioremediation, diesel oil degrading and surfactant producing bacteria are used to eliminate these pollutants from contaminated waters. Therefore, identifying and characterizing bacteria capable of producing surfactant and degrading diesel oil are pivotal. In this study, bacteria isolated from hydrocarbon contaminated river water were screened for their potential to degrade diesel oil. Primary selection was carried out by using conventional enrichment culture technique, emulsification index measurement, gravimetric and gas chromatographic analyses of diesel oil degradation. A bacterium with 60 % emulsification index and 92 % diesel oil degradation ability in 14 days was identified as Acinetobacter haemolyticus Zn01 by 16S rRNA sequencing. A. haemolyticus Zn01 was shown to harbor both catabolic genes alkB and C23O effective in diesel oil degradation. The biosurfactant of the bacterium was also characterized in terms of surface tension, zeta potential, Fourier transform infrared spectroscopy and scanning electron microscopy. Being able to emulsify and degrade diesel oil, A. haemolyticus Zn01 seems to have high potential for the elimination of diesel oil from polluted waters.     

Cuticular Hydrocarbons Provide Reliable Cues of Fertility in the Ant Gnamptogenys striatula

In ca. 150 species of queenless ants, a specialized queen caste is rare or absent, and mated workers take over the role of the queen in some or all of the colonies. Previously, it has been shown that reproduction in queenless ants is regulated by a combination of dominance behavior and chemical fertility signaling. It is unknown, however, whether chemical signals alone can sufficiently regulate reproduction. To investigate this possibility, we studied reproductive regulation in the facultatively queenless ant Gnamptogenys striatula, a species where dominance behavior is rare or absent. Active egg layers and infertile workers showed qualitative and quantitative differences in their cuticular hydrocarbon profile. Five long-chain methyl alkanes, 3,13- and 3,15-dimethyl pentriacontane, 3,13- and 3,15-dimethyl heptentriacontane, and 3,11,15-trimethyl heptentriacontane occurred only on the cuticles of virgin and mated egg layers. Pronounced quantitative differences were found in a further 27 alkenes; alkanes; and mono-, di-, and trimethyl alkanes. Workers that had recently stopped laying eggs had profiles similar to infertile workers, and mating status did not affect this chemical pattern. We conclude that the cuticular hydrocarbon profiles of G. striatula workers provide reliable information about their current fertility. In the interest of colony productivity, this allows reproduction to be regulated without the use of aggression.     

Hydrocarbon Signatures of Egg Maternity, Caste Membership and Reproductive Status in the Common Wasp

In most ants, bees, and wasps, the workers are capable of challenging the reproductive monopoly of the queen by laying unfertilized, male eggs. An important mechanism that can resolve this conflict is policing, whereby the queen or workers prevent successful worker reproduction by selectively eating worker-laid eggs or by attacking egg-laying workers. Egg policing by workers has been shown to occur in several social wasp species, but the information used by worker wasps to discriminate between queen-laid and worker-laid eggs has never been investigated. Our aim, therefore, was to investigate if hydrocarbons might be used in egg policing by workers in the common wasp, Vespula vulgaris, where worker policing previously has been shown to be effective. Our results show that 51 different hydrocarbons are present on the surface of newly-laid eggs, and that there are pronounced quantitative differences in the hydrocarbon profiles of queen-laid and worker-laid eggs, with longer-chained alkenes and methylated alkanes (C₂₈–C₃₁) in particular being more abundant on the surface of queen-laid eggs. We further show that the hydrocarbon profiles on the surface of queen-laid and worker-laid eggs resemble those found on the mother queen’s and workers’ cuticles. Interestingly, longer-chained methylated alkanes also were more abundant on the cuticle of both mother queens and reproductive workers, suggesting that these compounds are linked to fertility, as has also been found to be the case in several ant species.     

Sex pheromone of the stable fly: Evaluation of methyl- and 1,5-dimethylalkanes as mating stimulants

Each of 20 methyl-branched and 1,5-dimethyl-branched alkanes that comprise the active principle of saturated hydrocarbons of the female stable fly,Stomoxys calcitrans (L.), was synthesized and evaluated for mating stimulant activity. The compounds that showed the highest degree of activity in bioassays were 15-methyl- and 15,19-dimethyltritriacontanes.Diptera: Muscidae.Mention of a proprietary or commercial product in this paper does not constitute an endorsement by the U.S. Department of Agriculture.