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.     

Characterisation of Acetyl‐CoA Thiolase: The First Enzyme in the Biosynthesis of Terpenic Sex Pheromone Components in the Labial Gland of Bombus terrestris

Buff‐tailed bumblebees, Bombus terrestris, use a male sex pheromone for premating communication. Its main component is a sesquiterpene, 2,3‐dihydrofarnesol. This paper reports the isolation of a thiolase (acetyl‐CoA thiolase, AACT_BT), the first enzyme involved in the biosynthetic pathway leading to formation of isoprenoids in the B. terrestris male sex pheromone. Characterisation of AACT_BT might contribute to a better understanding of pheromonogenesis in the labial gland of B. terrestris males. The protein was purified to apparent homogeneity by column chromatography with subsequent stepwise treatment. AACT_BT showed optimum acetyltransferase activity at pH 7.1 and was strongly inhibited by iodoacetamide. The enzyme migrated as a band with an apparent mass of 42.9 kDa on SDS‐PAGE. MS analysis of an AACT_BT tryptic digest revealed high homology to representatives of the thiolase family. AACT_BT has 96 % amino acid sequence identity with the previously reported Bombus impatiens thiolase.     

Changes in the Composition of Triacylglycerols in the Fat Bodies of Bumblebee Males During Their Lifetime

The age-dependent changes in the composition of triacylglycerols (TAG) in the fat bodies of bumblebee males were studied using HPLC/MS. Two related species (Bombus terrestris and B. lucorum) were compared, with the age of the males being 0–30 days. The total amount of TAG in B. lucorum was about 2.7 times higher than that in B. terrestris for all of the ages studied. One to three-day-old males had the highest content of TAG in their fat bodies (1.6–2.3 mg/individual in B. terrestris and 3.8–4.2 mg/individual in B. lucorum). The analytical data show different patterns in both species. The qualitative composition of fatty acids in TAG was similar, but the mean relative abundance between B. terrestris and B. lucorum differed: 14:0, 7 and 14%; 16:0, 20 and 44%; 18:3, 62 and 23%; 18:1, 3 and 8%, respectively (the data is based on a GC/MS integration). A statistical evaluation of the dynamic changes in the TAG composition revealed that in B. terrestris different age classes were well separated according to their TAG composition while in B. lucorum the TAG did not change substantially during the male’s life. The TAG analyses provide more precise information on the differences between the classes studied than the FA composition alone.     

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.     

Fatty acid metabolism of the calanoid copepodParacalanus parvus: 2. Palmitate,stearate, oleate and acetate

The de novo biosynthesis of fatty acids in the wild, calanoid copepodParacalanus parvus was studied. The incubation of labeled acetate proved the de novo biosynthesis of saturated and monounsaturated even fatty acids from 14 to 20 carbons and the 22∶1 acid. Saturated and monounsaturated uneven fatty acids from 15 to 21 carbons were also synthesized. The copepod could not synthesize linoleic and α-linolenic acids. By administration of [1-¹⁴C]palmitate, [1-¹⁴C] stearate and [1-¹⁴C]oleate, it was possible to elucidate the general pattern of the de novo biosynthesis of fatty acids in the wildP. parvus.     

Host recognition and the study of a chemical basis for attraction by cuckoo bumble bees (Hymenoptera: Apidae)

Species ofPsithyrus (Hymenoptera; Apidae) are obligate bumble bee social parasites. In this study, females ofP. vestalis andP. ashtoni were presented with pentane extracts prepared from different body parts of queens of their respective host species,Bombus terrestris andB. terricola. Parasites of both species were capable of distinguishing host bees from other bumble bee species using chemical cues contained within extracts. Among extracts of several body parts presented to parasites, the abdomen produced the greatest behavioral response, with Dufour's gland and terminal tergal segments eliciting the greatest response among abdominal regions. Extracts of these two body parts obtained fromB. terrestris queens shared a number of compounds, identified by GC-MS. Among the identified compounds are a number that have been reported to be of importance in bee sociochemistry.     

Sex pheromone biosynthesis in the red-banded leafroller moth,studied by mass-labeling with stable isotopes and analysis with mass spectrometry

A technique for mass-labeling was developed to study sex pheromone biosynthesis in the red-banded leafroller moth,Argyrotaenia velutinana. With this technique, the pheromone components and all fatty acyl groups in the pheromone gland were analyzed for incorporation of label in the same analytic ran with gas chromatography-mass spectrometry, using chemical ionization and selected ion monitoring (GC-SIM-CI-MS). Sex pheromone glands were incubated with fatty acids or triacylglycerols labeled with at least three deuterium atoms or carbon-13 atoms. The results of these incubations support an interpretation in which hexadecanoate is chain shortened to tetradecanoate, which is desaturated to produce (E)- and (Z)-11-tetradecenoate precursors for the sex pheromone components (E)- and (Z)-11-tetradecen-1-yl acetate. Labeled (E)- and (Z)-11-tetradecenoyl groups in synthetic triacylglycerols were not incorporated into the sex pheromone components, perhaps indicating that this lipid class is not a donor of the immediate fatty acyl precursors in sex pheromone biosynthesis.     

Identification of Queen Sex Pheromone Components of the Bumblebee Bombus terrestris

We investigated the origin and chemical composition of the queen sex pheromone of the primitively eusocial bumblebee, Bombus terrestris (Apidae). Physiologically and behaviorally active compounds were identified by coupled gas chromatography electroantennography (GC-EAD), gas chromatography-mass spectrometry (GC-MS), and laboratory behavioral tests. In the behavioral assays, virgin queens frozen previously at −20°C were highly attractive to males. Dummies impregnated with surface and cephalic extracts obtained from virgin queens that had been frozen at −50°C were more attractive to males than odorless dummies. Male mating behavior was stimulated by components of cephalic secretions that are smeared onto the cuticle surface by the queen. Overall, 21 compounds present in surface and cephalic extracts evoked electroantennographic responses in male antennae. These included saturated and unsaturated fatty acids, ethyl- and methyl esters of the fatty acids, heptacosene, 2-nonanone, and geranyl geraniol. A blend of synthetic versions of these compounds elicited typical male mating behavior. Since solvent-impregnated dummies were approached by the males, but did not release copulatory behavior, visual cues may be important in the initial step of stimulating male mating behavior. Close-range olfactory signals are more important for releasing male mating behavior as well as for species recognition. In further behavioral assays, the attractiveness of a frozen virgin queen decreased as the storage time at −20°C increased from 2 hr to 1 d. Therefore, the chemical composition of the sex pheromone may change during freezing as behaviorally active compounds may decompose.     

Age-Dependent Changes in the Chemistry of Exocrine Glands of Bombus terrestris Queens

Extracts of three different glands (mandibular, labial, and Dufour’s) of virgin Bombus terrestris queens at ten different ages (1–8, 12, and 18 days) were analyzed for chemical composition. One hundred and twenty-seven compounds were identified in the extracts. The mandibular and labial glands contained previously reported electroantennogram-active compounds (3-hydroxydecanoic acid, fatty acids of different chain lengths, their esters, and heptacosene). These compounds reached a maximum concentration in 3- to 7-d-old queens. Geranylcitronellol was found in both labial and Dufour’s glands. Its amount was inversely correlated to age of queens.     

Water-soluble organosulfur compounds of garlic inhibit fatty acid and triglyceride syntheses in cultured rat hepatocytes

The putative hypolipidemic effect of garlic remains controversial. To gain further insight into the effect of garlic on lipid metabolism, the present study determined the inhibitory effects of water-soluble organosulfur compounds present in garlic on triglyceride (TG) and fatty acid synthesis in cultured rat hepatocytes. When incubated at 0.05 to 4.0 mmol/L with cultured hepatocytes, S-allyl cysteine (SAC) and S-propyl cysteine (SPC) decreased [2-¹⁴C]acetate incorporation into triglyceride in a concentration-dependent fashion achieving a maximal inhibition at 4.0 mmol/L of 43 and 51%, respectively. The rate of [2-¹⁴C]acetate incorporation into phosphlipids was depressed to a similar extent by SAC and SPC. SPC, SAC, S-ethyl cysteine (SEC), and γ-glutamyl-S-methyl cysteine decreased [2-¹⁴C]acetate incorporation into fatty acid synthesis by 81, 59, 35, and 40%, respectively, at 2.0–4.0 mmol/L concentrations. Alliin, γ-glutamyl-S-allyl cysteine, γ-glutamyl-S-propyl cysteine S-allyl-N-acetyl cysteine, S-allylsulfonyl alanine, and S-methyl cysteine had no effect on fatty acid synthesis. The activities of lipogenic enzymes, fatty acid synthase (FAS), and glucose-6-phosphate dehydrogenase (G6PDH) were measured in cultured hepatocytes treated with the inhibitors. The activity of FAS in cells treated with 4.0 mmol/L SAC and SPC, respectively, was 32 and 27% lower than that of non-treated cells. Neither SAC nor SPC affected G6PDH activity. The results indicate that SAC, SEC, and SPC inhibit lipid biosynthesis in cultured rat hepatocytes, and further suggest that these S-alk(en)yl cysteines of garlic impair triglyceride synthesis in part due to decreased de novo fatty acid synthesis resulting from inhibition on FAS. Whether tissue concentrations of active garlic components can achieve levels required to inhibit TG synthesis in vivo warrants further investigation.     

Reevaluation of the Role of Mandibular Glands in Regulation of Reproduction in Bumblebee Colonies

Possible pheromonal control of worker reproduction was tested in Bombus terrestris. The mode of assay included exposure of callow workers to extracts originating from different queen parts and measuring the effect on the in vitro biosynthesis of juvenile hormone (JH), the apparent gonadotropin in this species. Both queen total body extracts applied to dummies consisting of oven-dried or Soxhlet-washed virgin queen bodies and cuticular washes applied to living virgin queens effectively inhibited the biosynthesis of JH in callow workers. None of the five exocrine glands (mandibular, hypopharyngeal, salivary, Dufour's, and tarsal) demonstrated inhibitory activity. Likewise, the use of synthetic 3-hydroxy acids, found in queen mandibular glands, were ineffective in blocking JH biosynthesis in queenless workers. The results suggest that the queen may use a primer pheromone spread on the epicuticle as a means to inhibit worker reproduction. However, our results are not consistent with the prevailing hypothesis that in B. terrestris the main source of the pheromone that inhibits worker reproduction is in the queen's mandibular glands.     

Biosynthesis of hydrocarbons by algae: Decarboxylation of stearic acid to N-heptadecane inAnacystis nidulans determined by13C- and2H-labeling and13C nuclear magnetic resonance

The distribution of isotopic labels inn-heptadecane enriched from [1,2-¹³C] and [2-¹³C, 2-²H₃) acetates byAnacystis nidulans has been determined by¹³C nuclear magnetic resonance (¹³C NMR). Labeling with [1,2-¹³C] acetate is consistent with assembly from¹³C−¹³C units derived from an acetate “starter” group and 8 malonate units, as in fatty acid biosynthesis, followed by production of a methyl group through bond cleavage of the terminal¹³C−¹³C unit. A comparison of the hydrocarbon with palmitic acid (the only fatty acid produced in sufficient amount for NMR analysis) enriched from [2-¹³C,2-²H₃]acetate by the same culture shows that they have retained the same fraction of²H at corresponding sites, and have therefore undergone identical biosynthetic and hydrogen-deuterium exchange processes, as would be expected ifn-heptadecane originates from de novo-synthesized stearic acid. NRCC No. 18251.     

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.     

A delta9 desaturase from Bombus lucorum males: investigation of the biosynthetic pathway of marking pheromones

The knowledge of the molecular basis of communication in bumblebee communities is limited. None of the enzymes that participate in pheromone production have been characterized. Here, we cloned the gene encoding the Delta(9) desaturase from cDNA prepared from the total RNA of the pheromone gland and fat bodies of Bombus lucorum male. Functional expression of BlucNPVE desaturase in Saccharomyces cerevisiae and GC-MS analyses revealed its preference for C(18) saturated fatty acids. This suggests that Delta(9) desaturase is involved in the desaturation of metabolic fatty acids stored in triacylglyceroles (TAGs), because oleic acid is the most abundant fatty acid bound in TAG in B. lucorum and it is present in low concentration in the pheromone blend. The incubation of pheromone precursors with a dissected labial gland as well as direct injection of labelled pheromone substrates into B. lucorum males revealed that esterification of pheromone products occurs in the labial gland. These results support both the biosynthesis of pheromones from common lipids and the de novo synthesis of unsaturated pheromones in the labial gland.     

Biosynthesis of fatty acids byTrypanosoma cruzi

The incorporation of [1-¹⁴C] acetate into fatty acids by cultured epimastigotes ofTrypanosoma cruzi was studied. After 8, 24, and 48 hr incubation with labeled precursor, up to 2.8% of the initial radioactivity added to the medium was found in theT. cruzi long chain fatty acids. Saturated (16∶0 and 18∶0), monounsaturated (18∶1ω9), and diunsaturated (18∶2ω6) fatty acids were synthesized. Both the pattern of incorporation of labeled acetate into the fatty acids and the decarboxylation ratios found suggest that de novo synthesis of fatty acids has taken place.     

Binding and hydrolysis of radiolabeled pheromone and several analogs by male-specific antennal proteins of the mothAntheraea polyphemus

Sensory hair proteins from antennae of males of the wild silk moth,Antheraea polyphemus (Lepidoptera, Saturniidae) were incubated with radiolabeled 6E,11Z-hexadecadienyl acetate in the presence of unlabeled pheromone analogs as competitive inhibitors. The two extracellular proteins of importance, a highly active sensillar esterase and an abundant 15,000 mol wt binding protein, interact to degrade labeled pheromone less efficiently in the presence of certain unsaturated acetate analogs of the natural pheromone.Enzymatic hydrolysis of the acetate (or diazoacetate) was also examined for three pheromone analogs: [11,12-³H₂]-6E,11Z-hexadecadienyl diazoacetate, [11,12-³H₂]-hexadecyl acetate, and [9,10-³H₂]-9Z-tetradecenyl acetate. The former two are poor substrates at concentrations over four orders of magnitude. The 9Z–14:Ac, however, is the best alternative substrate for this in vitro pheromone metabolism system. Unlabeled 9Z–14: Ac is also the best competitive inhibitor of the hydrolysis of labeled 6E, 11Z–16: Ac. Whereas the tritiated natural pheromone shows a flat response (ca. 40% conversion) to increasing concentrations from 3 × 10^–9 to 3 × 10^–6 M, tritiated 9Z–14: Ac is degraded more rapidly at higher concentrations.Fellow of the Alfred P. Sloan Foundation (1981–1985) and Camille and Henry Dreyfus Teacher-Scholar (1981–1986).     

Detection of Reactive Quinones in the Metabolism of Polycyclic Aromatic Hydrocarbons by the Formation of their Glutathione Conjugates

The biotransformation of polycyclic aromatic hydrocarbons to quinones by rat liver microsomes was investigated. The employment of an electrochemical detector allowed the specific detection of quinones separated by reverse phase HPLC with higher sensitivity as compared to UV detection. Microsomal incubations of benzo[a]pyrene (BP) resulted in the formation of 1,6-, 3,6- and 6,12-quinones, of naphthalene in the detection of naphthalene-1,4-quinone, whereas ortho-quinones could only be detected in trace amounts. Additional protein binding studies showed that only 9–22% of synthetic ortho-quinones could be recovered from microsomal incubations. In order to scavenge possible reactive quinone metabolites with glutathione (GSH) and to identify these metabolites, GSH-conjugates of naphthalene-1,2-quinone, naphthalene-1,4-quinone, chrysene-1,2-quinone, BP-7,8-quinone and BP-9,10-quinone were synthesized and spectroscopically characterized. After incubations of 1-naphthol or naphthalene with rat liver microsomes the GSH conjugate of naphthalene-1,2-quinone could be identified by cochromatography with the authentic reference compound.     

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.     

The Yeast ATF1 Acetyltransferase Efficiently Acetylates Insect Pheromone Alcohols: Implications for the Biological Production of Moth Pheromones

Many moth pheromones are composed of mixtures of acetates of long‐chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl‐CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non‐insect acetyltransferase alternative, we expressed a plant‐derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with EaDAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27‐ and 10‐fold higher in vivo and in vitro efficiency, respectively, compared to EaDAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.