Comparison of the Chemical Compositions of the Cuticle and Dufour’s Gland of Two Solitary Bee Species from Laboratory and Field Conditions

Species-specific biochemistry, morphology, and function of the Dufour’s gland have been investigated for social bees and some non-social bee families. Most of the solitary bees previously examined are ground-nesting bees that use Dufour’s gland secretions to line brood chambers. This study examines the chemistry of the cuticle and Dufour’s gland of cavity-nesting Megachile rotundata and Osmia lignaria, which are species managed for crop pollination. Glandular and cuticular lipid compositions were characterized and compared to each other and according to the nesting experience of adult females. Major lipid classes found were hydrocarbons, free fatty acids, and wax esters. Many components were common to the cuticle and Dufour’s glands of each species, yet not identical in number or relative composition. Wax esters and fatty acids were more prevalent in Dufour’s glands of M. rotundata than on cuticles. Wax esters were more abundant on cuticles of O. lignaria than in Dufour’s glands. In both species, fatty acids were more prevalent in glands of field-collected females compared to any other sample type. Chemical profiles of cuticles and glands were distinct from each other, and, for O. lignaria, profiles of laboratory-maintained bees could be distinguished from those of field-collected bees. Comparison of percentiles of individual components of cuticular and glandular profiles of the same bee showed that the proportions of some cuticular components were predictive of the proportion of the same glandular components, especially for nesting females. Lastly, evidence suggested that Dufour’s gland is the major source of nest-marking substances in M. rotundata, but evidence for this role in O. lignaria was less conclusive.     

Kälteadaptation der Wachssynthese in der Harder'schen Drüse der Ratte

Adaptation to Coldness in the Synthesis of Wax Esters in the Harderian Gland of the Rat The Harderian gland of the rat has been shown to contain characteristic wax esters of [n-7] unsaturated alcohols and fatty acids. The change of the temperature in the surroundings from 25 to 4° C for six weeks does not change the pattern of exclusively [n-7] monoenoic fatty acids. The adaptation of this exocrine eye gland results in an increased synthesis of [n-5] monounsaturated alcohols and shortening of the chain length. Different pathways of unsaturation and/or chain modification and the regulation of lipid synthesis in this gland are discussed, as well as the biological role of these waxes.     

Temporal patterns of sex pheromone titers and release rates inHolomelina lamae (Lepidoptera: Arctiidae)

Six hydrocarbon components (2-methylhexadecane,n-heptadecane, 2-methylheptadecane, 2-methyloctadecane,n-nonadecane, and 2-methylnonadecane) were identified in sex-pheromone gland extracts and in airborne collections ofHolomelina lamae. Low variability in the ratio of components among individuals indicates tight regulation of blend composition. Minor changes were evident in the quantity and ratio of the blend as a function of time of day. Based on gland extracts, the total quantity of the six components increased from day 1 (X = 6299 ng) to day 4 (X = 7498 ng) and subsequently decreased. No significant correlations were found between total gland contents and wet or dry weights of moths. Emission rates ofH. lamae females were determined from pheromone adsorbed onto Porapak Q. Quantities released peaked shortly after the onset of calling and decreased rapidly as calling continued. Peak release rates ranged from 13 to 350 ng/10 min, and from 37 to 835 ng/60 min. Noncalling females did not emit detectable quantities of pheromone. Based on release rates and the rate of pulsation of the abdominal tip, the average amount released per pulse is not constant. The mean ratio of components released (0.787.45 84.802.842.591.53) was not very different from the ratio of components in gland extracts of 2-day-old females (0.704.1990.12 1.651.911.42). We propose that the blend is atomized rather than volatilized from the gland, thus retaining nearly the same ratios in the female's effluvium as in her gland.     

Liver lipids and fatty acids of the sting ray Dasyatis bleekeri (Blyth)

The sting ray, Dasyatis bleekeri (Blyth), has been studied for lipids and fatty acids of its liver. The neutral lipids identified were hydrocarbons, wax esters, steryl esters, 1-O-alkyl-2,3-diacylglycerols, triacylglycerols, and sterols. Neutral lipids were predominant (91.8%), major components being triacylglycerols (92.7%). Polyenoic fatty acids of n-3 series, viz. eicosapentaenoic acid and docosahexaenoic acid, were high in the phospholipid and neutral lipid fractions. Cholesterol was the major component (67.9%) in the steryl ester fraction. Glyceryl ethers, with chainlengths up to 30 carbons, were recorded with unsaturated, anteiso, iso, and normal chains. In wax ester alcohols, up to 32-carbon chains were recorded. Hydrocarbons were up to 36-carbon chains with anteiso, iso, and normal chains. Among branched chain hydrocarbons, pristane was the major component (6.7%) and squalene was present at the level of 3.5%. Chimyl and batyl alcohol backbones were the major components found in 1-O-alkyl-diacylglycerols.     

Tarsi of Male Heliothine Moths Contain Aldehydes and Butyrate Esters as Potential Pheromone Components

The Noctuidae are one of the most speciose moth families and include the genera Helicoverpa and Heliothis. Females use (Z)-11-hexadecenal as the major component of their sex pheromones except for Helicoverpa assulta and Helicoverpa gelotopoeon, both of which utilize (Z)-9-hexadecenal. The minor compounds found in heliothine sex pheromone glands vary with species, but hexadecanal has been found in the pheromone gland of almost all heliothine females so far investigated. In this study, we found a large amount (0.5–1.5 μg) of hexadecanal and octadecanal on the legs of males of four heliothine species, Helicoverpa zea, Helicoverpa armigera, H. assulta, and Heliothis virescens. The hexadecanal was found on and released from the tarsi, and was in much lower levels or not detected on the remaining parts of the leg (tibia, femur, trochanter, and coxa). Lower amounts (0.05–0.5 μg) of hexadecanal were found on female tarsi. This is the first known sex pheromone compound to be identified from the legs of nocturnal moths. Large amounts of butyrate esters (about 16 μg) also were found on tarsi of males with lower amounts on female tarsi. Males deposited the butyrate esters while walking on a glass surface. Decapitation did not reduce the levels of hexadecanal on the tarsi of H. zea males, indicating that hexadecanal production is not under the same neuroendocrine regulation system as the production of female sex pheromone. Based on electroantennogram studies, female antennae had a relatively high response to hexadecanal compared to male antennae. We consider the possible role of aldehydes and butyrate esters as courtship signals in heliothine moths.     

Are Insect-Synthesized Retronecine Esters (Creatonotines) the Precursors of the Male Courtship Pheromone in the Arctiid Moth Estigmene acrea?

The pyrrolizidine alkaloid (PA) profiles were determined for adults of the polyphagous arctiid Estigmene acrea, which as larvae had fed on artificial diet supplemented with Crotalaria-pumila powder with known concentrations of PAs. The larvae always had a free choice between alkaloid-containing and plain diets. The alkaloid profiles of adults revealed a striking sexual dimorphism. Both sexes contained macrocyclic PAs of the monocrotaline type sequestered from the diet and, in addition, a substantial proportion of supinidine and retronecine esters synthesized by the insects from necine bases derived from the dietary alkaloids and necic acids of insect origin. These insect alkaloids accounted for 35% and 55% of total PAs in males and females, respectively. The difference was that in females the retronecine esters (creatonotines) made up 58 g (43% of total PAs), while males contained a fivefold lower proportion, 12 g (13%). Four of the ten male individuals analyzed were found devoid of creatonotines. Based on the experimental data in combination with evidence from the literature, it is suggested that the creatonotines are direct pheromone precursors in E. acrea. It is hypothesized that this may represent a general mechanism of hydroxydanaidal formation from diverse macrocyclic PAs in arctiids.     

Chemistry of the Dufour's gland secretions of North American andrenid bees (Hymenoptera: Andrenidae)

The volatile components of the Dufour's gland secretions were examined in 22 Nearctic species of andrenid bees representing 10 subgenera. Farnesyl hexanoate was the dominant component in the secretion of 17 species. In others, various terpenoid esters such as geranyl octanoate, farnesyl octanoate and geranylgeranyl octanoate were the major components. Approximately 30 compounds have been identified in the secretions; many are newly identified in andrenid bees. Three different groups of secretions are discerned with exceptions. A discussion of the possible function of the Dufour's gland secretion in the biology of, and its use in, the systematics of these bees is presented.     

Role of semiochemicals in mate location by parasitic sea louse,Lepeophtheirus salmonis

The role of olfaction and diffusible pheromones in mate location behavior of sea lice, Lepeophtheirus salmonis, was assessed with Y-tube behavioral bioassays. The pheromone emitting animals were located in a chamber in one arm of a Y-tube arena, with artificial seawater flowing through both arms. Adult male sea lice displayed both activation and directional responses to seawater conditioned with preadult II virgin females, but were only activated by mated adult female conditioned water. Further, when males were given the choice of preadult II virgin females or mated adult females, a significant number of males chose the arm with the preadult II virgin females. Adult males showed activation responses when presented with water conditioned with adult males but were not attracted to them. When presented with adult males, preadult II virgin females showed only directional responses, but not activation responses. Preadult II virgin female conditioned water was extracted using solid-phase extraction (SPE) protocols pioneered for semiochemical isolation. Adult male sea lice showed significant directional responses to the preadult II virgin female SPE extract. Distillation under vacuum was performed on the extract to give a distillate comprising components with a molecular weight range and physical properties comparable to those of compounds utilized as volatile semiochemicals by terrestrial organisms and a residue comprising components with higher molecular weight range comparable to those utilized as involatile semiochemicals. Adult males were found to be both significantly activated and attracted to the distillate, but not to the residue. This research provides evidence that small, lipophilic organic molecules are used by sea lice as sex pheromone signals to locate a member of the opposite sex.     

Identification of the sex pheromone of the guernsey carpet beetle,Anthrenus sarnicus Mroczkowski (Coleoptera: Dermestidae)

It has been confirmed that adult virgin females ofAnthrenus sarnicus Mroczkowski exhibit a characteristic headstand posture that is associated with the release of a sex pheromone. Volatiles trapped on filter papers suspended above calling females were attractive to adult virgin males when tested in a two-choice target bioassay. Separate aeration extracts of males and females were analyzed by gas chromatography-mass spectrometry and showed that decanol and decyln-butyrate were released by females only. These components were present in approximately equal amounts and accounted for about 90% of the total area of the chromatogram. Decyl butyrate produced an electroantennogram response with a larger response from males than females. Behaviorally, a mixture of 10g of decanol and 10g of decyl butyrate attracted 88% of males and 10g of decyl butyrate alone attracted 82% of males in the bioassay. The role of decyl butyrate as a sex pheromone is convincing, but this is not the case for decanol.     

Defensive Chemistry of an Aposematic Bug, Pachycoris stallii Uhler and Volatile Compounds of Its Host Plant Croton californicus Muell.-Arg.

Volatile components of Pachycoris stallii scent gland secretions and the bug's host plant, Croton californicus, were identified by gas chromatography and mass spectroscopy. The predominant compounds isolated from C. californicus fruit and leaves were -myrcene and -caryophyllene. Metathoracic gland secretions of P. stallii contained mostly (E)-2-hexenal, (E)-4-oxo-2-hexenal, (E)-2-hexenyl acetate, and n-tridecane. In males, n-tridecane was present throughout the metathoracic gland, but in females this compound was found only in the median reservoir/accessory gland. (E)-2-Hexenal was present throughout the gland of female bugs, but in males was primarily present in the median reservoir/accessory gland. (E)-4-Oxo-2-hexenal and n-dodecane were isolated from the median reservoir/accessory gland of male and female bugs. Metathoracic glands were sexually monomorphic. Data from chemical analyses and anatomical observations suggest that dorsal abdominal glands of adults were apparently obsolescent. In nymphs, dorsal abdominal glands produced (E)-2-hexenal, (E)-4-oxo-2-hexenal, n-dodecane, n-tridecane, and tetradecanal. The proportion of the predominant constituent, (E)-4-oxo-2-hexenal, decreased from 72% in the first instar to 47% in the fourth instar. Proportions of tetradecanal and n-tridecane were greater in the fourth instar than in the first instar. Observations of dissected glands indicated that median and posterior dorsal abdominal glands of all nymphal instars were more developed than anterior dorsal abdominal glands. Scanning electron micrography revealed the presence of polygonal microsculpturing on the integument surrounding the ostioles of metathoracic and dorsal abdominal glands. Chemical, anatomical, and behavioral data indicated that P. stallii has a chemical defense system based on short-chain carbonyl compounds and that this system is directed against arthropods. The abundance of arthropod natural enemies apparently has forced P. stallii to maintain this defense system despite feeding on a toxic host plant.     

Evaluation of the synthetic major component of the sex pheromone of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

In wind-tunnel bioassays, dispensers loaded with 1 g of the synthetic major component (3E,8Z,11Z)-3,8,11-tetradecatrienyl acetate (TDTA) of the sex pheromone emitted by Tuta absoluta (Meyrick) females were found to be highly attractive to conspecific males. Field experiments were conducted to evaluate the efficacy of five trap designs. The best trap, baited with 100 g of the synthetic sex pheromone caught on average 1200 males per trap per night, while those baited with virgin females caught only 201 males. The male response to this pheromone is restricted to the same early-morning time window during which females exhibit calling behavior. The high biological activity of the synthetic pheromone suggests that it could be useful for pest monitoring and in mating disruption.     

Oligosaccharide composition and pheromonal activity of male tergal gland secretions of the German cockroach,Blattella gennanica (L.)

In the sequential courtship behavior of the German cockroach, Blattella germanica (Dictyoptera: Blattellidae), females feed on the tergal gland secretions from the male tergites. The phagostimulative components in the male secretions were composed of a complex mixture of oligosaccharides and phospholipids. Besides seven oligosaccharides previously identified, two new trisaccharides, O--D-glucopyranosyl-(16)--D-glucopyranosyl -D-glucopyranoside and O--glucopyranosyl-(14)-O--glucopyranosyl- (11)-myo-inositol, were characterized as the sugar components of the secretions. The activity of the sugar components was compared in combination with the lipid fraction. Individual oligosaccharides exhibited the activity either alone or synergistically with the phospholipids. A complex mixture of these primary substances in the tergal gland secretions, thus, serves as a pheromonal phagostimulant in the precopulatory behavior, strongly eliciting feeding response in the female cockroach.     

Individual variation in aggregation pheromone content of the bark beetle,Ips typographus

The total amounts of, and proportions among, components of the aggregation pheromone produced byIps typographus were found to vary considerably among individuals excised from attacks on standing spruce trees. Chemical analyses of 392 individual male beetles were made by GC-MS. Both unmated and mated males had log-normal frequency distributions in their content of the pheromone components 2-methyl-3-buten-2-ol (MB) andcis-verbenol (cV), since a large fraction of males had a low content. The amount of MB in male hindguts varied independently of cV and the other oxygenated monoterpenes, while the amount of cV covaried with the other pinene alcohols and showed a variation between beetles from different spruce trees. Mated males had, on average, lower amounts of MB than unmated, while the average content of cV in mated males varied with the resin content of their host trees. Ipsdienol and ipsenol were only found in mated males, but in less than 40% and 10%, respectively, of these mated males. Even-aged males exposed to -pinene in the laboratory showed slightly less variation in the amounts of verbenols, and the variations in ratio between cV and tV were similar to those among males attacking the same spruce tree.Coleoptera, Scolytidae.This study was made within the Swedish project Odour Signals for Control of Pest Insects.     

Intraspecific variation and interspecific differences in sex pheromones of sibling species inCtenopseustis obliquana complex

The specific status ofCtenopseustis obliquana pheromone-types I, II, and III has been more fully examined. Females of types I and III produce a mixture of (Z)-8- and (Z)-5-tetradecenyl acetates (Z8-14:Ac, Z5-14: Ac). The previously reported different ratios of these two components in females of the two types (type I=8020, III=9010) have been reinvestigated. The median ratios of each type differed significantly, although there was some overlap in the ranges of these ratios. A field cage trial showed that males of type III are attracted to females of type I, so the observed differences may be ascribed to intraspecific variation. In contrast to females of types I or III, females of type II produce Z5-14Ac but no Z8-14Ac. The electroantennogram (EAG) profile of antennae of type II males shows a maximum response to Z5-14Ac, while the EAG profiles of types I and III show a strong response to Z8-14Ac. In wind tunnel tests using mixtures of these two compounds, type II males prefer blends consisting of all or mostly Z5-14: Ac, while type I males showed a preference to a mix of 70% Z8-14Ac plus 30% Z5-14Ac. We found that type I males are attracted to type I females when offered a choice between type I and type II females in a field cage test and that type II males similarly prefer type II females. Males of types I and II have specialist cells for Z8-14Ac and Z5-14Ac but differ with respect to relative densities of these cells and to further cell types responsive to other alkenyl acetates. Type IIC. obliquana is considered therefore a sibling species of types I and III. In addition, the amount of Z5-14Ac produced by type II females varied geographically. Females from the North Island produced significantly less (median=1.2 ng) Z5-14Ac than females from the South Island (median=2.2 ng). Type II populations in the North Island morphologically resembled sympatric type I, rather than type II from the South Island and are designated as type II (North Island). Type II (North Island) populations have so far been found only at Rukuhia (near Hamilton) and from Kerikeri. In contrast, type II populations are sympatric with type III populations over much of the South Island. In a field cage trial, males of types II and II (North Island) were attracted to females of both II and II (North Island). We tentatively ascribe the differences between type II and type II (North Island) to intraspecific variation.     

Linalool in mandibular gland secretion ofColletes bees (Hymenoptera: Apoidea)

Uinalool was identified as the main component of the mandibular gland secretion in females and males of four species ofColletes bees, viz.,C. cunicularius, C. daviesanus, C. impunctatus andC. succinctus. It was also shown to be the dominant volatile compound in the same gland in maleC. floralis (the female of this species has not yet been investigated). Further, in another species,C. similis, linalool is present in the mandibular gland secretions of females and males, but the dominant volatile component in these secretions is geranial, together with neral. Females of the speciesC. fodiens seem to lack monoterpenes altogether; nonadecane is the dominant volatile compound of the cephalic secretion (based on analysis of a single individual). When linalool is put out in the area of nest aggregation ofC. cunicularius, where the males are patrolling, a distinct increase in flight activity is noted. The function of linalool is discussed on the basis of field observations.This report forms part XXI in the series Studies on Natural Odoriferous Compounds.     

Variation in a female sexual attractiveness pheromone controls male mate choice in garter snakes

Male red-sided garter snakes (Thamnophis sirtalis parietalis) display a courtship preference for larger females during the breeding season. Utilizing behavioral experiments and chemical analyses, we tested the hypothesis that males can discriminate among females of varying size solely by means of the sexual attractiveness pheromone, a previously characterized sex pheromone composed of a homologous series of long-chain saturated and -9 cis-unsaturated methyl ketones contained in the skin lipids of females. When presented with skin lipid extracts from large and small females, a greater proportion of males displayed courtship behaviors to large female extracts. This demonstrates that there is an intrinsic property of the female skin lipids that allows males to differentiate among large and small females. Analysis of the sexual attractiveness pheromone revealed that the necessary variation exists for this pheromone to function as a reliable indicator to males of female body size. Specifically, we observed a strong correlation between female snout–vent length and the relative concentration of saturated and -9 cis-unsaturated methyl ketones composing the pheromone; smaller females expressed pheromone profiles higher in saturated methyl ketones, while larger females expressed pheromone profiles dominated by unsaturated methyl ketones. The results of this study suggest that male red-sided garter snakes utilize compositional variation in the female sexual attractiveness pheromone to differentiate among potential mates of varying size.     

Cuticular Hydrocarbons and Novel Alkenediol Diacetates from Wheat Stem Sawfly (Cephus cinctus): Natural Oxidation to Pheromone Components

The cuticular lipids of the wheat stem sawfly Cephus cinctus (Hymenoptera: Cephidae) were investigated as part of a chemical ecology project with this species. The major cuticular lipids were n-alkenes and n-alkanes. Alkenes were the most abundant and exhibited dramatic sexual dimorphism. (Z)-9-Tricosene accounted for about half of the total hydrocarbon in males but was nearly absent from females. The dominant alkenes in females were (Z)-9-pentacosene and (Z)-9-heptacosene. The alkane profiles were similar in both sexes, with n-tricosane being the most abundant, followed by n-pentacosane and n-heptacosane. In both sexes, there were minor amounts of alkanes and alkenes with other chain lengths and n-alkadienes of 29 and 31 carbons. In males, about one tenth of the surface lipids consisted of (Z)-9-alkene-1, -diol diacetates with 22-, 24-, and 26-carbon chains. The same compounds were also detected from females but in much smaller amounts. The structures of these novel diacetates were proven by synthesis. By analogy to methyl oleate, a well-studied food lipid, the alkenes and diacetates were expected to undergo slow oxidation in air to release specific aldehydes and other volatile products, and these were generally detected in volatiles collected from living sawflies. Atmospheric oxidation of the diacetates was also demonstrated in the absence of sawflies. One product from the diacetates, 9-acetyloxynonanal, was shown in other research to be particularly active electrophysiologically and was also attractive in the field. Aldehydes from the alkenes also showed strong electrophysiological activity. The concept of volatile pheromones originating from heavy, unsaturated cuticular lipids is discussed.     

Morphology and chemistry of brown rat,Rattus norvegicus,preputial and clitoral glands

In contrast to previous results from laboratory rats, when sexual dimorphism occurred, clitoral glands from wild female rats were heavier and accounted for a greater proportion of body mass than preputial glands from wild males. Gland length and weight increased with body weight, but gland weight as a proportion of body weight did not. Preputial and clitoral glands showed seasonal differences in size: at one site glands were smaller in December than in April. Maturity, estrus, and pregnancy had no significant effect on clitoral gland size, but immature males had proportionally smaller preputial glands than mature males, and the absolute gland weight, but not gland weight as a proportion of body weight, increased with testes weight. There were no differences between the sexes in lipid production, which increased with preputial or clitoral gland weight. An homologous series of aliphatic acids and their methyl and ethyl esters, plus squalene, cholesterol and lanosterol, was revealed by GC-MS study of the trimethyl silyl derivatized lipid extract. Males and females could be distinguished by principal component analysis of chromatogram peaks.     

Volatile components in dorsal gland secretions of the collared peccary,Tayassu tajacu (Tayassuidae,mammalia)

Secretions of the dorsal gland of free-ranging adult male and female collared peccaries (Tayassu tajacu) were analyzed by gas chromatography-mass spectrometry. Both sexes contain (2E,6E,10E)-geranylgeraniol; squalene (allE isomer); and the following isomers of springene, a diterpene homolog of -farnesene: (3E,6E,10E)--springene, (3Z,6E,10E)--springene, and (6E,10E)--springene. A diterpene alcohol and an additional isomer each of squalene and springene also were observed. Straight- and branched-chain esters abound in the secretions of females, but they were not detected in males.     

Terminal steps in pheromone biosynthesis byHeliothis virescens andH. zea

In vivo application to the sex pheromone gland ofHeliothis Virescens andH. Zea of large quantities of alcohols normally present in small amounts resulted in the preferential conversion of the alcohols to the corresponding pheromonal aldehydes. Amounts of the minor component aldehydes were increased up to 15-fold by selectively applying large quantities of the alcohol precursors. Using this technique, we have inducedH. virescens to convert bombykol, the sex pheromone of the silkworm, to the corresponding aldehyde, bombykal, and have induced femaleH. zea to produce the same sex pheromone components used byH. virescens by applying tetradecanol and (Z)-9-tetradecenol to the surface of the gland. Further, treatedH. zea females were attractive toH. virescens males and caused males to attempt interspecific copulation repeatedly. We have also found that the enyzme involved in this conversion is dependent on the presence of molecular oxygen, indicating that a nonspecific alcohol oxidase is responsible for the terminal biosynthetic step in pheromone production by bothH. virescens andH. zea.Mention of a commercial product or proprietary does not constitute endorsement by either the University of Guelph or U.S.D.A.