Premating Behavior of Bombus Confusus Males and Analysis of Their Labial Gland Secretion

Premating behavior in the bumblebee Bombus confusus was studied. Visual searching for females is not the only premating strategy of this species, as was believed earlier. Males of B. confusus have a normally developed labial gland and its secretion is used to mark a perch from which they visually search for females. The labial gland secretion contains geranylcitronellol and (Z)-9-octadecenyl acetate as the main components.     

Comparison of Age-dependent Quantitative Changes in the Male Labial Gland Secretion of <Emphasis Type="Italic">Bombus Terrestris</Emphasis> and <Emphasis Type="Italic">Bombus Lucorum</Emphasis>

Age-related changes of antennal-active components of male labial gland extracts were studied in two closely related bumblebee species, Bombus terrestris and B. lucorum. In B. terrestris, compounds eliciting electroantennogram (EAG) responses of virgin queens were ethyl dodecanoate, 2,3-dihydrofarnesal, 2,3-dihydrofarnesol, hexadecan-1-ol, octadeca-9,12,15-trien-1-ol, and geranylcitronellol. Compounds that elicited EAG responses from queens of B. lucorum were ethyl dodecanoate, ethyl tetradec-7-enoate, ethyl tetradec-9-enoate, ethyl hexadec-9-enoate, hexadecan-1-ol, hexadec-7-enal, octadeca-9,12-dien-1-ol, octadeca-9,12,15-trien-1-ol, and octadecan-1-ol. Quantities of these compounds in the labial glands changed significantly over the lifetime of the respective males of the two species. In both species, concentrations of the respective compounds reached their maximum within seven days after eclosion. Subsequently, a rapid decrease in the amount of EAG-active compounds occurred in B. terrestris, whereas in B. lucorum the amount of active compounds stayed approximately constant or decreased at a slow rate. Microscopy showed that in B. terrestris secretory cells of the labial glands undergo apoptosis from the fifth to the tenth day of life, whilst in B. lucorum labial gland cells remain unchanged throughout the life of the males.     

Chemistry of the Cephalic Labial Gland Secretions of Male Bombus morrisoni and B. rufocinctus, Two North American Bumblebee Males with Perching Behavior

The labial gland secretions from males of the North American bumblebees Bombus morrisoni Cresson and B. rufocinctus Cresson were analyzed by gas chromatography/mass spectrometry. In both species, 3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl acetate was found as the major compound of a complex mixture of alkenols, acetates, hydrocarbons, and wax-type esters. In addition to a mixture of saturated and mono-unsaturated straight chain hydrocarbons, the labial gland of both species contained the isoprenoid hydrocarbons (6E, 10E)-3,7,15-trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene [β-springene], three isomers of 3,7,11,15-tetramethyl-hexadeca-1,3,6,10,14-pentaene [α-springene], and two further unidentified cyclic diterpenes. In B. morrisoni, 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol and 3,7,11,15-tetramethyl-6,10,14-hexadecatrien-1-ol were detected as characteristic alcohols, as well as small amounts of 9-hexadecenol and hexadecanol. Furthermore, a large peak of hexadecyl dodecanoate and minor amounts of 9-hexadecenyl, 9-octadecenyl, and eicosenyl 9-tetradecenoate were found as typical esters in this species. In B. rufocinctus, 9-hexadecenol, hexadecanol, and 9-octadecenol were present in considerable amounts, with their acetates and 9-tetradecenoic, tetradecanoic, hexadecanoic, and 11-octadecenoic acids. The chemical composition of cephalic labial glands in male bumblebees with perching behavior is discussed.     

Unusual Fatty Acids in the Fat Body of the Early Nesting Bumblebee, <Emphasis Type="Italic">Bombus pratorum</Emphasis>

Unusual fatty acids with 24, 26, and 28 carbon atoms were found in triacylglycerols (TAGs) isolated from fat body tissue of bumblebee Bombus pratorum. The most abundant one was (Z,Z)-9,19-hexacosadienoic acid. Its structure was determined by mass spectrometry after derivatization with dimethyl disulfide and by infrared spectroscopy. ECL (equivalent chain length) values of its methyl ester were determined on both DB-1 and DB-WAX capillary columns. (Z,Z)-9,19-Hexacosadienoic acid is quite rare in nature. So far it has been identified only in marine sponges, and this work is the first evidence of its occurrence in a terrestrial organism. HPLC/MS analysis of the bumblebee TAGs showed that (Z,Z)-9,19-hexacosadienoic acid is present in one third of all TAG molecular species. As it was found in all sn-TAG positions, it is likely that (Z,Z)-9,19-hexacosadienoic acid is transported to tissues. Interestingly, labial gland secretion of B. pratorum was found to contain (Z,Z)-7,17-pentacosadiene, a hydrocarbon with markedly similar double bond positions and geometry. Possible biosynthetic relationships between these two compounds are discussed.     

Chemistry of a Mating Plug in Bumblebees

In the bumblebee B. terrestris males transfer a mating plug into the queen's sexual tract shortly after sperm transfer. The plug is a sticky, opaque secretion of the male accessory gland. In order to clarify the meaning of the mating plug, we collected the plug substance directly from the male's accessory gland and identified the chemical substances present with gas chromatography. The main compounds found in the mating plug were four fatty acids (palmitic, linoleic, oleic, and stearic acids) and a cyclic peptide (cycloprolylproline). Mixing the four fatty acids resulted in a similar sticky, opaque mass as found in natural plugs, indicating that cycloprolylproline is not necessary for the physical attributes of the plug. The function of the fatty acids may therefore be to build up a physical barrier, optimizing sperm placement before the spermathecal duct or preventing sperm backflow. Cycloprolylproline, on the other hand, may influence female mating behavior so as to reduce her receptivity. In fact, peptides are known to reduce female receptivity in other insects. This would explain why queens of B. terrestris are only singly mated, although multiple mating is beneficial during the colony cycle with respect to parasitism and fitness.     

Hexyl Decanoate, the First Trail Pheromone Compound Identified in a Stingless Bee, Trigona recursa

Foragers of many species of stingless bees guide their nestmates to food sources by means of scent trails deposited on solid substrates between the food and the nest. The corresponding trail pheromones are generally believed to be produced in the mandibular glands, although definitive experimental proof has never been provided. We tested the trail following behavior of recruits of Trigona recursa in field experiments with artificial scent trails branching off from natural scent trails of this stingless bee. First-time recruits (newcomers) did not follow these trails when they were laid with pure solvent or mandibular gland extract. However, they did follow trails made with labial gland extract. Chemical analyses of labial gland secretions revealed that hexyl decanoate was the dominant component (72.4 ± 1.9% of all volatiles). Newcomers were significantly attracted to artificial trails made with synthetic hexyl decanoate, demonstrating its key function in eliciting scent-following behavior. According to our experiments with T. recursa, the trail pheromone is produced in the labial glands and not in the mandibular glands. Hexyl decanoate is the first component of a trail pheromone identified and proved to be behaviorally active in stingless bees.     

Attraction of pinyon pine bark beetle,Ips hoppingi,to conspecific andI. confusus pheromones (Coleoptera: Scolytidae)

Females of a pinyon pine bark beetle,Ips hoppingi Lanier, were less attracted by the aggregation pheromone produced by conspecific males than by the pheromone produced by the neighboring sibling species,I. confusus (LeConte). Cross-attraction was elicited by males infesting the regional pinyon pine hosts (P. discolor andP. edulis) of eitherIps species in south-eastern Arizona. Pheromonal specificity has not accompanied speciation in this species pair.     

Identity and Function of Scent Marks Deposited by Foraging Bumblebees

Foraging bumblebees can detect scents left on flowers by previous bumblebee visitors and hence avoid flowers that have been depleted of nectar. Tarsal secretions are probably responsible for this repellent effect. The chemical components of the tarsal glands were analyzed by combined gas chromatography–mass spectrometry for three species of bumblebee, Bombus terrestris, B. lapidarius, and B. pascuorum. The hydrocarbons identified were similar for each species, although there were interspecific differences in the relative amounts of each compound present. The tarsal extracts of all three species comprised complex mixtures of long-chain alkanes and alkenes with between 21 and 29 carbon atoms. When B. terrestris tarsal extracts were applied to flowers and offered to foraging bumblebees of the three species, each exhibited a similar response; concentrated solutions produced a repellent effect, which decreased as the concentration declined. We bioassayed synthetic tricosane (one of the compounds found in the tarsal extracts) at a range of doses to determine whether it gave a similar response. Doses 10^–12 ng/flower resulted in rejection by foraging B. lapidarius. Only when 10^–14 ng was applied did the repellent effect fade. We bioassayed four other synthetic compounds found in tarsal extracts and a mixture of all five compounds to determine which were important in inducing a repellent effect in B. lapidarius workers. All induced repellency but the strength of the response varied; heneicosane was most repellent while tricosene was least repellent. These findings are discussed in relation to previous studies that found that tarsal scent marks were attractive rather than repellent.     

Feeding Stimulating Signal in Labial Gland Secretion of the Subterranean Termite Reticulitermes santonensis

The paired labial glands of the French subterranean termite Reticulitermes santonensis Feytaud are located in the thorax. In the head, the glandular ducts join with those of the water sacs. In feeding choice tests with two semicircles of moist filter paper as food, workers of R. santonensis preferred the semicircle treated with labial gland extract compared to the semicircle treated with water (control). The labial gland secretion carries a signal that stimulates gnawing and feeding by termite workers during food exploitation. The extract of the labial gland even elicited feeding behavior when applied without food (on glass plates). The content of the water sacs was not effective as a feeding stimulus and neither were different body parts except for the fat-body. Water sacs are thus not a reservoir for the labial gland secretion, but their contents may serve as a solvent or carrier for the phagostimulant signal. The signal is highly polar, heat-resistant, nonvolatile, and thus very persistent. There is evidence that the signal from the labial gland is not species specific. Instead, it might be part of a general strategy by termites to exploit food sources.     

Cuticular Hydrocarbons as Chemotaxonomic Characters of Pine Engraver Beetles (Ips spp.) in the grandicollis Subgeneric Group

Cuticular hydrocarbons were extracted, identified, and evaluated as chemotaxonomic characters from all species of adult Ips pine engraver beetles in the grandicollis subgeneric group. The grandicollis group consists of Ips grandicollis (Eichhoff), I. cribricollis (Eichhoff), I. lecontei Swaine, I. montanus (Eichhoff), I. paraconfusus Lanier, I. confusus (LeConte), and I. hoppingi Lanier. In order to provide outgroups for a phylogenetic analysis, cuticular hydrocarbons were also analyzed from Orthotomicus caelatus (Eichhoff), I. latidens (LeConte) (latidens subgeneric group), and I. pini (Say) (pini subgeneric group). Two hundred forty-eight hydrocarbon components were identified by gas chromatography–mass spectrometry. The members of the grandicollis group provided 206 of these compounds. The components represented eight classes: n-alkanes, alkenes, alkadienes, terminally branched methylalkanes, internally branched methylalkanes, dimethylalkanes, trimethylalkanes, and tetramethylalkanes. Different populations of O. caelatus, I. grandicollis, I. lecontei, I. montanus, I. paraconfusus, I. confusus, and I. hoppingi provided no evidence for interpopulational variation in cuticular hydrocarbons. Single populations only were analyzed for I. latidens, I. pini, and I. cribricollis. Sexual dimorphism in cuticular hydrocarbons occurred only in I. lecontei where females produced eight unique components with a pentatriacontane parent chain. Several phylogenetic analyses based on hydrocarbon phenotypes agreed in general with the established morphologically based system of relatedness and with published phylogenies reconstructed from protein and nucleic acid characters. Nearly all hydrocarbon analyses suggested a close relationship between I. grandicollis and I. cribricollis; between I. lecontei and I. montanus; and among the sibling species I. paraconfusus, I. confusus, and I. hoppingi. The presence or absence of specific n-alkanes (n-docosane, n-triacontane); certain dimethylalkanes (terminally branched with octacosane and triacontane parent chains and internally branched with heptacosane, hentriacontane, and docotriacontane parent chains); and 3,7,11-; 3,7,15-trimethylheptacosane permit facile discrimination of I. paraconfusus, I. confusus, and I. hoppingi. These three sibling species are difficult to resolve by external morphology. These data support the species status of I. hoppingi rather than it being considered a host race of the I. confusus complex. They also support the species status of I. cribricollis rather than it being considered part of I. grandicollis. In contrast to other published phylogenies reconstructed from molecular data, phylogenies reconstructed from cuticular hydrocarbons repeatedly place I. lecontei as an integral part of the grandicollis subgeneric group. Thus, cuticular hydrocarbon and pheromone alcohol composition of I. lecontei support its inclusion in the grandicollis subgeneric group.     

Ancestral semiochemical attraction persists for adjoining populations of siblingIps bark beetles (Coleoptera: Scolytidae)

Three species ofIps pine bark beetles in Hopping's group IX (S.L. Wood'sgrandicollis group),Ips confusus, I. lecontei, andI. paraconfusus, are parapatrically distributed in the American Southwest. They share post-Pleistocene altitudinal ecotones with their host pines. Adjacent to these areas of host overlap, we tested the hypothesis that aggregation pheromones produced by male beetles and/or host volatiles are sufficient to elicit the speciesspecific colonization behaviors typical of these threeIps species in nature. A more distantly related species,I. pini (Hopping's group IV, S.L. Wood'spini group) was used for outgroup comparison. Under the influence of pheromone, males ofI. confusus andI. paraconfusus do not discern among unin-fested log bolts of host and nonhost pine prior to bark contact. Males responding to pheromones emanating from infested bolts are similarly undiscriminating. Females ofI. confusus andI. lecontei olfactorily discern the combination of conspecific males in host pine from other possible beetlepine combinations; females ofI. paraconfusus do not. FemaleI. pini discerned conspecific pheromone from that ofI. lecontei. The bark beetle predator,Enoclerus lecontei, is attracted by, but does not discriminate among, the male-produced volatiles of these Group IXIps species. These results support a hypothesis that divergence in pheromonal responses by these group IXIps species has evolved following their speciation, having been manifested first in the female sex. Evolutionarily, the derived pheromonal messages have preceded their behavioral discrimination by these beetles. Additional speciesspecific cues may operate between the sexes in the field that may preclude heterospecific pairings.     

Semiochemicals produced by western balsam bark beetle,Dryocoetes confusus Swaine (Coleoptera: Scolytidae)

The most prominent beetle-produced volatiles identified in the abdominal extracts of maleDryocoetes confusus Swaine after they had bored for 24 hr in logs of subalpine fir,Abies lasiocarpa (Hook.) Nutt. were:exo andendo-brevicomin,trans-verbenol, verbenone, myrtenol,trans-pinocarveol,cis- andtrans-p-menthen-7-ol, 3-caren-10-ol, and several monoterpenes and sesquiterpenes. Myrtenol was the only conspicuous compound in extracts from males that had been exposed toA.lasiocarpa resin volatiles for 24 hr. Laboratory bioassays indicated that both (–)- and (+)-exo-brevicomin were attractive to femaleD. confusus, and that the (–) enantiomer did not inhibit response to its antipode. Results from field trapping experiments indicated that bothero-brevicomin and myrtenol are aggregation pheromones forD. confusus.exo-Brevicomin baits were effective in causing attack byD. confusus on baited and surrounding trees, suggesting that this pheromone may have utility in manipulating populations of the beetle.Research supported by the Natural Sciences and Engineering Research Council of Canada, Operating Grants A3881 and A3706 and Strategic Grant G1039.     

Macrocyclic lactones and isopentenyl esters in the Dufour's gland secretion of halictine bees (Hymenoptera: Halictidae)

The volatile components of the Dufour's gland secretion of female halictid bees have been examined in 18 Nearctic species belonging toAgapostemon, Augochlora, Augochlorella, Augochloropsis, Dialictus, Evylaeus, Halictus, andLasioglossum. Nine saturated and unsaturated macrocyclic lactones ranging from C₁₈ to C₂₆ have been identified. Four of these compounds, the saturated C₂₆ and the unsaturated C₂₀, C₂₂, and C₂₄ lactones, are new natural products reported for halictine bees. A series of eight esters containing branched C₅-alkenols and fatty acids has been identified in several species. The cell linings and pollen ball inAugochlora pura pura contain the same major lactones as the Dufour's gland. A discussion of the significance of the Dufour's gland secretion for apoid systematics and its function in the Halictidae is presented.     

Behavioral correlates for minor volatile compounds from stink bugs (Heteroptera: Pentatomidae)

Many terrestrial Heteroptera have small, but functional, dorsal abdominal glands as adults. The chemistry, and associated intra- and inter-specific behavior, for dorsal abdominal gland secretions from 10 species representing four genera of Pentatomidae was investigated. Eighteen volatile compounds were identified in species-specific blends from the dorsal abdominal gland secretions ofEuschistus, Acrosternum, andEurydema adults, including aliphatic, aromatic, and terpenoid constituents. Evidence from bioassays is presented that parasitic Tachinidae (Diptera) and Scelionidae (Hymenoptera) use these secretions as kairomones. A field experiment was performed to test the hypothesis that minor volatiles increase the specificity of the main pheromone component from NearcticEuschistus species, methyl (2E,4Z)-decadienoate. However, significantly fewer individuals ofE. tristigmus were captured in traps baited with the complete blend for this species than in traps baited with methyl (2E,4Z)-decadienoate alone. Thus, at the concentrations tested, these kinds of dorsal abdominal gland secretions may be epideictic, promoting spacing in the natural habitat.     

Chemical constituents of the chest gland secretion of the thick-tailed galago (Galago crassicaudatus)

The naturally exuded chest gland secretions of adult male and female thick-tailed bush babies were collected directly in capillary tubes. The pure secretion was subjected to GC-MS analysis which revealed three major components. These were identified by comparison with the appropriate standard compounds: benzyl cyanide,p-hydroxybenzyl cyanide, and 2-(p-hydroxyphenyl)ethanol. Analysis of a male and a female secretion indicated that some sexual dimorphism in the relative concentrations of these components may exist.     

Mandibular Gland Secretion of <Emphasis Type="Italic">Melipona beecheii</Emphasis>: Chemistry and Behavior

The mandibular gland secretion of Melipona beecheii contains a rich mixture of terpenoid and oxygenated compounds and unsaturated and saturated hydrocarbons. However, it differs markedly from the 11 other Brazilian species examined in previous studies, both by the absence of 2-heptanol and the presence of rose oxides. The most abundant compound was geranyl hexanoate, whereas the most volatile compounds were cis- and trans-rose oxide and geraniol. The complete blend and five individual components found in the gland secretion were tested by electroantennography (EAG) and behavioral assays. The complete mandibular gland extract and geraniol elicited the strongest EAG responses, whereas these and farnesyl acetate induced the strongest attack response from workers. The role of the rose oxides remains to be elucidated, as they do not appear to play a major role as an alarm pheromone of this species.     

Geometrical and optical isomerism of pheromones in two sympatricDryocoetes species (Coleoptera: Scolytidae), mediates species specificity and response level

In a field-trapping experiment, western balsam bark beetles,Dryocoetes confusus Swaine, were highly attracted to a 51 mixture of (±)-exo-and (±)-endo-brevicomin. Beetles in the sympatric speciesD. affaber (Mann.), were best attracted to a 11 blend of these semiochemicals [either (±)(±) or (±)(±)], suggesting that both geometrical isomers are pheromone components in these species. In laboratory bioassays and further field experiments, attraction ofD. confusus was greatest when the (+) enantiomers of both geometrical isomers of brevicomin were presented in a 91 ratio. Responses by maleD. confusus to attractive mixtures were reduced in the presence of (–)-exo-brevicomin. Exploitation of the complete range of variability in pheromone structure (both geometrical and optical isomerism) would allow for optimization and regulation of response levels within a species and also could maintain reproductive isolation among sympatric congeneric species primarily through production and response to species-specific blends.During the course of the research, A.D. Camacho was on educational leave from the Escuela Nacional de Ciencias Biológicas. I.P.N. Depto. de Zoología. Prol. Carpio y Plan de Ayala, Mexico 11340D.F. México.     

Chemistry and functions of exocrine secretions of the antsTapinoma melanocephalum and T. erraticum

Volatile constituents produced by ant workers belonging to the speciesTapinoma melanocephalum andT. erraticum have been analyzed by gas chromatography-mass spectrometry. The pygidial (=anal) gland secretion ofT. melanocephalum is fortified with 6-methyl-5-hepten-2-one and actinidine (the mass spectrum of which is corrected in this paper). An unidentified compound was detected in cephalic extracts. The pygidial gland secretion ofT. erraticum was also dominated by 6-methyl-5-hepten-2-one, in addition to two isomers of iridodial, and iridomyrmecin. The sternal glands contained iridodial and C₁₅-C₂₀ hydrocarbons. Workers ofT. melanocephalum effectively utilize their pygidial gland secretions as an alarm-defense system during aggressive encounters with workers ofSolenopsis geminata. 6-Methyl-5-hepten-2-one is active as a releaser of alarm behavior, and actinidine is repellent to workers ofT. melanocephalum. Cephalic extracts possessed attractant and arrestant properties for workers of this species.     

Volatile compounds from the predatory insectPodisus maculiventris (Hemiptera: Pentatomidae)

In the predatory spined soldier bug,Podisus maculiventris, the dorsal abdominal glands are much smaller in adult females than males. Females produce a mixture of (E)-2-hexenal, (E)-2-octenal, (E)-2-hexenoic acid, benzaldehyde, and nonanal in these glands. The female dorsal abdominal gland secretion may be a close-range pheromone since the dorsal abdominal gland secretion from males has been shown to be a long-range aggregation pheromone in this species. The metathoracic scent gland secretions of male and female spined soldier bugs are apparently identical, and similar to that of other pentatomids, except for the presence of the monoterpene alcohol, linalool.Mention of a commercial product does not constitute an endorsement of this product by the USDA.     

Chemical Defense in Harvestmen (Arachnida,Opiliones): Do Benzoquinone Secretions Deter Invertebrate and Vertebrate Predators?

Two alkylated 1,4-benzoquinones were identified from the defensive secretion produced by the neotropical harvestman Goniosoma longipes (Gonyleptidae). They were characterized as 2,3-dimethyl-1,4-benzoquinone and 2-ethyl-3-methyl-1,4-benzoquinone. We tested the effectiveness of these benzoquinone secretions against several predator types, including invertebrates and vertebrates. Different predators were exposed to the harvestmen's gland secretion or to distilled water in laboratory bioassays. Our results indicate that secretions containing the 1,4-benzoquinones released by G. longipes can be an effective defense against predation, and that the effectiveness of the secretion is dependent on the predator type. The scent gland secretion repelled seven ant species, two species of large wandering spiders, and one frog species, but was not an effective defense against an opossum. Our study also demonstrates that the scent gland secretion of G. longipes can work as a chemical shield preventing the approach of three large predatory ants for at least 10 min. The chemical shield may protect the harvestman against successive attacks of the same ant worker and also allow the harvestman to flee before massive ant recruitment. Our data support the suggestion that chemical defenses may increase survival with some but not all potential predators. This variation in defense effectiveness may result from many interacting factors, including the attack strategy, size, learning ability, and physiology of the predators, as well as the chemical nature of the defensive compounds, type of emission, and amount of effluent released by the prey.