Intercaste,intercolony, and temporal variation in cuticular hydrocarbons ofCopotermes formosanus shiraki (Isoptera: Rhinotermitidae)

We characterized the variation in cuticular hydrocarbon mixtures between seven colonies of the Formosan subterranean termite,Coptotermes formosanus Shiraki, from the same population. We report differences between castes, between colonies, and within the population over time to assess seasonality. Colonies ofC. formosanus from Oahu, Hawaii, were sampled for 25 months. Each month, one sample each of 200 workers, 50 soldiers, nymphs, or alates from each colony was subjected to gas chromatography-mass spectrometry (GC-MS) analysis of the cuticular hydrocarbons. We resolved 39 individual peaks and identified 52 individual or isomeric mixtures of hydrocarbons. Onlyn-alkanes and methyl-branched alkanes occur; no olefins were found. Internally branched monomethylalkanes were the most abundant class of hydrocarbons, representing 45% to 50% of the total 9-;11-;13-Methyl-heptacosane accounted for over 30% of the total hydrocarbon for all castes. 2-Methyl- and 3-methylalkanes comprise approximately 30% of the total. Internally branched dimethylalkanes constitute 15% to 20% of the total cuticular hydrocarbon. Only one trimethylalkane, 13,15,17-trimethylnonacosane, was found in small amounts. The hydrocarbon mixtures of all four castes were similar. Quantitative differences in hydrocarbon mixtures among the castes were easily displayed using canonical discriminant analysis. Soldiers and workers are significantly different from one another and from nymphs and alates. Nineteen peaks are statistically significant between workers and soldiers. Nymphs and alates were not statistically different. We detected statistically significant quantitative differences between colonies in 18 peaks for workers and 12 peaks for soldiers. Each of the colonies ofC. formosanus can be separated from the others by the proportions of their hydrocarbon components. We detected statistically significant differences between months of the year for 12 peaks for workers and four peaks for soldiers; two peaks each for workers and soldiers showed distinct, seasonal trends. This seasonal shift in proportions of hydrocarbons correlates with the production of alates.     

Cuticular lipids of the booklouse,Liposcelis bostrychophila: hydrocarbons,aldehydes, fatty acids,and fatty acid amides

The booklouse, Liposcelis bostrychophila, is an increasingly common pest of stored food products worldwide. We report here the cuticular lipid composition of this pest (the first report of the hydrocarbons of any member of the Order Psocoptera and the first report of fatty acid amides as cuticular components for any insect). No unsaturated hydrocarbons were present. A homologous series of n-alkanes (C₂₁–C₃₄), monomethyl alkanes (3-, 4-, 5-, 7-, 9-, 11-, 12-, 13- and 15-methyl-) with a carbon chain range of C₂₈–C₄₂, and dimethyl alkanes (3, 7-; 9, 13-; 11, 15-; 13, 17-; 9, 21-; 11, 19-; and 13, 21-); with a carbon number range of C₃₁–C₄₃ were identified. The relative abundances of these hydrocarbons were low, comprising approximately 0.0125% of total biomass. The amides were a homologous series (C₁₆–C₂₂ in chain length), with the major amide being stearoyl amide. In addition to the amides, free fatty acids (C_16:1, C_16:0, C_18:2, C_18:1, and C_18:0 in chain length) and three straight chain aldehydes (C₁₅, C₁₆, and C_17:1 in chain length) also occurred as cuticular components. These findings are discussed in terms of the chemical and physiological ecology of this species.     

Cuticular hydrocarbons ofAedes hendersoni cockerell andA. triseriatus (SAY)

Field-caught adult male and femaleAedes hendersoni are difficult to distinguish from the sibling speciesA. triseriatus. We found that mosquitoes from the same sex of the sibling species can not be readily separated either by unique cuticular hydrocarbon components or by differences in percent composition of those components. Multivariate analysis of the cuticular hydrocarbon data does not provide good separation. Cuticular hydrocarbons were identified using gas chromatography electron-impact mass spectrometry and gas chromatography chemical-ionization mass spectrometry. Flame-ionization capillary gas chromatography was used for quantitative analysis of individual mosquitoes. Sixty-four hydrocarbons with chain lengths from C₁₆ to greater than C₄₆ were common to both species. Identified hydrocarbon components weren-alkanes, monomethylalkanes, dimethylalkanes, trimethylalkanes, and alkenes.Diptera: Culicidae.     

Cuticular hydrocarbons ofReticulitermes virginicus (Banks) and their role as potential species- and caste-recognition cues

The cuticular hydrocarbon components of four castes ofReticulitermes virginicus (Banks) have been identified and quantitated. Components identified includen-alkanes; 2-, 3-, 11-, 13-, and 15-methyl-alkanes; 11,15-dimethylalkanes, (Z)-9-alkenes; (Z,Z)-7,9-dienes; and (E/Z)-6,9-dienes ranging in carbon number from C₂₁ to C₄₀. All caste forms ofR.virginicus contained the same components, but showed caste-specific proportions. Comparison of these hydrocarbons with those of the sympatric termiteR. flavipes (Kollar) suggest that cuticular hydrocarbons might serve as species- and caste-recognition cues. A bioassay was developed to test this species-recognition hypothesis, with the experimental results supporting the hypothesis.Isoptera: Rhinotermitidae.     

Cuticular lipids of insects: VIII. Alkanes of the mormon cricketAnabrus simplex

The cuticular hydrocarbons of the Mormon cricket,Anabrus simplex, are all saturated and consist of n-alkanes (29+%), 3-methylalkanes (12%), internally branched monomethylalkanes (26+%), and dimethylalkanes (28+%). The principal n-alkane is the C₂₉ component, with a range from C₂₃ to C₃₃. The 3-methylalkanes range from C₂₈ to C₃₂, and the internally branched monomethyl-and dimethylalkanes range from C₂₉ to C₃₉. When the branched alkanes ofA. simplex are compared to those from other insects in the order Orthoptera, interesting patterns of methyl branching are observed. Scientific Journal Series 631, Agricultural Experiment Station, University of Montana, Bozeman, Montana 59715.     

Mating stimulant pheromone and cuticular lipid constituents ofFannia femoralis (Stein) (Diptera: Muscidae)

The cuticular lipids of male and femaleFannia femoralis were similar for recently emerged insects but soon began to develop chromatographic patterns characteristic of each sex. Mature females contained more C₃₁ and C₃₃ monoolefin in the cuticular lipid than males. Also, the double bonds in the monoolefins of the female lipid were situated predominantly at the eleventh and thirteenth carbons, while most of those from the males were centrally located in the molecule or at the ninth carbon.The female C₃₁ monoolefin stimulated copulation by the males, but more mating activity occurred when the saturated hydrocarbons present in the female cuticular lipids were added. The synthetic monoolefin most active as a mating stimulant pheromone was (Z)-11-hentriacontene, but the addition of female alkanes or of syntheticn-alkanes to (Z)-11-hentriacontene increased the activity of the synthetic pheromone.A portion of a dissertation intended for submission by the first author to the Graduate School of the University of Maryland in partial fulfillment of the requirements for the Ph.D. degree.Mention of a proprietary or commercial product in this paper does not constitute an endorsement of this product by the U.S. Department of Agriculture or the University of Maryland.     

Biosynthesis of (Z,Z)-6,9-heptacosadiene in the American cockroach

The biosynthesis of (Z,Z)-6,9-heptacosadiene, the major cuticular hydrocarbon component of the American cockroach, was examined by radiotracer and¹³C-nuclear magnetic resonance (NMR) techniques. Sodium [1-¹⁴C] acetate was incorporated about equally into the saturated and diunsaturated hydrocarbons, whereas [1-¹⁴C] linoleate preferentially labeled the C₂₇ alkadiene and [9,10-³H] oleate labeled the C₂₇ alkadiene almost exclusively.¹³C-NMR demonstrated that [2-¹³C] acetate labeled carbons 25 and 27 but not carbon 3 of the C₂₇ alkadiene. In addition, ozonolysis of the diene labeled from [1-¹⁴C] acetate followed by radio-gas liquid chromatography showed that carbons 1–6 were not labeled, whereas the fragment containing carbons 10–27 was labeled. The data presented in this paper indicate that linoleate from the diet or synthesized de novo is elongated by the addition of acetate units and is then decarboxylated.     

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

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

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

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

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

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

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.     

Cuticular hydrocarbons of the screwworm,Cochliomyia hominivorox (Diptera: Calliphoridae)

A novel series of 2,X-dimethylalkanes were isolated and identified. The nonpolar fraction of the surface lipids secreted by the adult (5-day-old) screwworm,Cochliomyia hominivorax, contains over 130 different hydrocarbons comprising normal alkanes (32% of the total hydrocarbon), branched alkanes (53%), and monoalkenes (11%). Branched alkanes included monomethylalkanes with substitution in all possible positions except for 4-methylalkanes, internally branched dimethylalkanes, and 2,X- and 3,X-dimethylalkanes. At emergence, adults of both sexes of the 009 strain have nearly identical gas Chromatographic profiles, which diverge as the insect ages. Irradiation of pharate pupae does not affect the hydrocarbon produced.Mention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA.     

Cuticular hydrocarbons of gregarious and solitary locustsLocusta migratoria cinerascens

The cuticular hydrocarbons ofLocusta migratoria cinerascens—larvae and adults, males and females, gregarious and solitaries—have been investigated by combined gas chromatography-mass spectrometry. The hydrocarbons comprise 52–78% of the cuticular lipids and are divided inton-alkanes (28.7–47.3%), 3-, 4-, and 5-methylalkanes (11.3–15.8%), internally branched monomethylalkanes (13.7–19.9%), and internally branched dimethylalkanes (19.8–35.9%) with seven or nine methylenes between the two branch points. While the sexual dimorphism does not seem to be reflected in the cuticular hydrocarbon composition, clear quantitative variations favoring the longest chain alkanes have been observed between gregarious and solitary locusts, thus revealing a new phase character in these insects.     

Host-location kairomone fromPeriplaneta americana (L.) for parasitoidAprostocetus hagenowii (Ratzeburg)

Chemically mediated host location in the eulophid parasitoidAprostocetus hagenowii (Ratzeburg) was investigated. In Y-tube bioassays 77.6% of female parasitoids responded to aPeriplaneta americana (L.) ootheca; parasitoids did not respond to air with no volatile stimuli. Frass from adult cockroaches was as attractive as an ootheca. Bioassay of one ootheca equivalent of five lipid fractions (eluted with hexane and 1, 5, 10, and 30% ether in hexane) from silica gel column chromatography indicated that the active component was a hydrocarbon. Further separation and bioassay of oothecal hydrocarbons by AgNO₃-impregnated silica gel column chromatography indicated that the biological activity was in one fraction. Gas chromatographic (GC) analysis of this fraction revealed a single peak; this peak was identified by researchers in 1963, 1969, and 1972 as (Z,Z)-6,9-heptacosadiene. Qualitative and quantitative GC analyses of total hydrocarbons from oothecae, frass, and adult females were essentially identical; 6,9-heptacosadiene was the dominant hydrocarbon from each source. The alkadiene was 37 times more abundant in frass than on the ootheca. The volatilization of the alkadiene from oothecae was demonstrated by aeration and trapping on Super Q adsorbent. The current study is the first evidence for biological activity of (Z,Z)-6,9-heptacosadiene, a major hydrocarbon component on adult female American cockroaches, on their oothecae, and in their frass.This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the USDA or the University of Florida.     

Structural correlation between cuticular hydrocarbons and female contact sex pheromone of German cockroachBlattella germanica (L.)

The structural relationships between the cuticular hydrocarbons and the contact sex pheromone of the female German cockroach,Blattella germanica, were investigated. Cuticular hexane extracts were separated into hydrocarbon and ketone fractions by TLC or silicic acid column chromatography. The ketone fraction (which contains the major contact sex pheromone component) was analyzed by GC-MS before and after reduction to ydrocarbon. In addition to 3,11-dimethyl-2-nonacosanone, 3,11-dimethyl-2-heptacosanone was also identified. Females have the 3,11- and 3,9-dimethyl C₂₇ and C₂₉ alkanes, but only the 3,11- isomer of the dimethylketones. Inddition to the hydrocarbon components previously reported, a number of new components were characterized. Although the ratios of cuticular hydrocarbons differ among nymphs, adult males, and adult females, they have qualitatively identical hydrocarbon profiles, suggesting that the production of the contact sex pheromone results from the sex-specific oxidation of 3,11-imethylalkanes to pheromone components by the female.     

Tracing Pollinator Footprints on Natural Flowers

Many insects are known to leave lipid footprints while walking on smooth surfaces. Presumably, the deposited substances improve tarsal adhesion. In bumblebees, footprint hydrocarbons also function as scent marks that allow detection and avoidance of recently depleted flowers. I used GC-MS to detect hydrocarbons deposited by bumblebee (Bombus pascuorum) on flowers of Lamium maculatum. In addition to the plants' own cuticular lipids, extracts of corollas that had been visited by bumblebees contained odd-numbered alkenes. The amount of pentacosenes (C₂₅H₅₀) on corollas was linearly related to the number of bumblebee visits, with workers depositing approximately 16 ng per visit (extrapolated to a total of 65 ng of bumblebee cuticular hydrocarbons). Pentacosenes were retained on visited flowers without loss for 2 hr, and probably longer. This and results from flight cage experiments suggest that flower epicuticles retain a chemical record of pollinator visitation, including information on visiting bee species. Continuous footprint accumulation necessitates new explanations concerning the reversibility of “repellent scent marks” of bumblebees.     

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

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

Cuticular hydrocarbons of eight species of north american cone beetles,Conophthorus hopkins

A study to determine the degree of similarity and/or diversity among eight of the 15 described species ofConophthorus is reported. Cuticular hydrocarbons were evaluated forC. conicolens, C. ponderosae, C. cembroides, C. edulis, C. radiatae, C. coniperda, C. resinosae, andC. banksianae. Seventy-eight individual and isomeric mixtures of hydrocarbons were identified by gas chromatography-mass spectrometry, includingn-alkanes, alkenes, alkadienes, 2- or 4-methylalkanes, 3-methylalkanes, and single-component and isomeric mixtures of internally branched mono-, di-, and trimethylalkanes. Differences in alkenes and mono-, di-, and trimethylalkanes can be used easily to separate the eight species.Conophthorus conicolens andC. ponderosae contain the most complex blends. Hydrocarbon patterns in three geographically separated populations ofC. ponderosae, each from a different host, are qualitatively identical with the exception of a homologous series of 3,7-dimethylalkanes from adults collected fromPinus lambertiana cones. The latter could comprise a sibling species. Hydrocarbon mixtures of two eastern species,C. resinosae andC. banksianae, are qualitatively identical, supporting the suspicion thatC. banksianae may not be a valid species. Closely relatedC. cembroides andC. edulis have similar combinations of hydrocarbons except for a unique and abundant alkene (C₂₇₁) inC. edulis and two dimethyhexacosanes inC. cembriodes.Coleoptera: Scolytidae.     

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.     

A new component of the female sex pheromone ofBlattella germanica (L.) (Dictyoptera: Blattellidae) and interaction with other pheromone components

A fourth component, 3,11-dimethyi-2-heptacosanone, was identified as a cuticular contact sex pheromone of the female German cockroach,Blattella germanica. In behavioral assays, higher dosages of 3,11-dimethyl-2-heptacosanone were needed to elicit similar sexual responses in males to those elicited by the major pheromone component, 3,11-dimethyl-2-nonacosanone. A 1585 blend of the C₂₇ and C₂₉ methyl ketone homologs resulted in a dose-response curve intermediate between that of each of the components alone, indicating independence of activity of each component and lack of synergism. Moreover, the activity of 3,11-dimethyl-2-nonacosanone was not enhanced by female cuticular hydrocarbons. The relationship between sexual responses of males to females and to isolated female antennae, and the amount of cuticular pheromone on whole females was investigated. Cuticular sex pheromone found on females increased with the age of the female, as did the male response to whole females. However, a bimodal male response was elicited by isolated female antennae. Differences between behavioral and analytical assays of pheromone are discussed.