Stereochemistry of two pheromonal components of the bumblebee wax moth, Aphomia sociella

The bumblebee wax moth, Aphomia sociella, is a parasite of bumblebees. In this species, males produce sexual pheromone to attract females, while females produce an aphrodisiac pheromone that initiates male courtship. Both pheromones contain 6,10,14-trimethylpentadecan-2-one (TMPD-one) and the corresponding alcohol, 6,10,14-trimethylpentadecan-2-ol (TMPD-ol) in sex specific quantities. Male sex pheromone consists of 7 components with TMPD-one as a minor one and traces of TMPD-ol. In female aphrodisiac pheromone, TMPD-ol is the major component, while TMPD-one is present in traces. Here we report on the absolute configuration of TMPD-one in male sex pheromone and TMPD-ol in female aphrodisiac pheromone of A. sociella. The configuration was determined from GC/MS of prepared (S)-acetoxypropionyl esters of TMPD-ol. TMPD-one was first reduced to the alcohol that was then derivatized with (S)-acetoxypropionyl chloride. The GC/MS data of obtained diastereoisomers were compared with synthetic standards. The absolute configuration of TMPD-one in the male pheromone was (6R,10R). The configuration of TMPD-ol in the female pheromone was (2R,6R,10R). Electrophysiological experiments showed that TMPD-one and TMPD-ol are perceived by both sexes. The synthetic standards of naturally produced stereoisomers elicited higher responses than mixtures of all stereoisomers.

gas chromatograph (GC) equipped with a polar Varian FactorFOUR VF-23ms column (30 m × 0.25 mm i.d., d f = 0.25 µm) and a HP 5973 MS-detector in electron ionization mode (EI). The carrier gas (1 mL min −1 ) was helium; the sample (1 µL) was injected in the splitless mode, the injector temperature was 250 °C, the transfer line was maintained at 250 °C and the MS source was set to 280 °C. For identification of the alcohol and ketone in crude extracts, the full scan mode was used and the column temperature increased from 50 °C by 10 °C min −1 up to 230 °C and held at 230 °C for 10 minutes. Mass spectra of main extract components are in the Supplementary material (Figs. S1-S3).
Purification of insect extracts. Extracts were purified using liquid chromatography (LC). Prior purification, female extracts were washed with NaHCO 3 (sat.aq.) to remove free carboxylic acids. Afterwards, male and female extracts were treated in the same way. In LC purification, pre-conditioned solid phase extraction column (500 mg, SPE, Chromabond) were used. SPE conditioning was done with EtOAc (1.5 mL), 10% EtOAc in pentane (1.5 mL) and pentane (1.5 mL). Then the columns were gradually eluted in steps of 1% increase of ethyl acetate in pentane (0-15%) with each fraction consisted of a volume of 750 µL.
TMPD-one was enriched in fractions 3-6 and TMPD-ol was enriched in fractions 9−11. In cases where the purity was not satisfactory, the combined fractions were subjected to the purification protocol again until no further improvement of purity was obtained. The combined TMPD-one fractions were then reduced to alcohols (described below). After reduction, the TMPD-ol fractions and those containing the reduced TMPD-one were derivatized and analyzed as described below (Fig. 1D).
Reduction of TMPD-one fraction. The collected ketone fractions were pooled and solvent evaporated under a stream of argon. The purified ketone was diluted in dry diethyl ether (1 mL) and 0.2 mL was reduced to alcohol with lithiumaluminumhydride in diethyl ether (1 M, 50 µL) at 0 °C for 1 hour (Fig. 1B). The reaction was quenched by addition of HCl (2 M, 100 µL) and H 2 O (100 µL). Phases were separated and the aqueous layer was extracted with Et 2 O (3 × 200 µL), the combined organic layers was washed with HCl (2 M, 100 µL) and brine (100 µL) and dried over Na 2 SO 4 (anhydr.) in a Pasteur pipette. The solvent was evaporated under a stream of argon. The sample was diluted with pentane (50 µL) and purified with the same SPE purification method as the crude extracts, prior to derivatization.

Derivatization.
To determine the stereochemistry of TMPD-ol enriched from female extract and TMPD-one enriched from male extract of A. sociella, both the pooled alcohol fraction and the reduced ketone fraction were derivatized with (S)−2-acetoxypropionyl chloride (SigmaAldrich) according to Bång 14 . Pooled extracted TMPD-ol and reduced TMPD-one were concentrated by means of evaporating solvent under a stream of argon. Dry cyclohexane (50 µL) was added to the vial containing the purified extract and subsequently was 1% pyridine in cyclohexane (65 µL) and 1% (S)-2-acetoxypropionyl chloride in dichloromethane (100 µL) added, the resulting mixture was heated at 80 °C for 30-60 minutes under argon atmosphere (Fig. 1D). A needle was inserted into the septum of the vial and the solvent was allowed to evaporate before allowing the vial to reach room temperature. www.nature.com/scientificreports www.nature.com/scientificreports/ HCl (1 M, 200 µL) and pentane or cyclohexane (1 mL) was added, the organic layer was subsequently dried over Na 2 SO 4 (anhydr.) in a Pasteur pipette. Solvent was either reduced to approximately 50 μL and the sample was analyzed as described below or the solvent was evaporated completely and sample dissolved in cyclohexane before analysis.

Electroantennography (EAG). Detailed description of EAG measurements on A. sociella is available in
Kalinová et al. 5 . Briefly, isolated antennae, glass Ag/AgCl electrodes and Syntech EAG recording and stimulus delivery system were used. Male and female antennal responses to stimulation by 10, 100, and 250 ng of racemic mixtures and pure enantiomers of TMPD-one and TMPD-ol were investigated. Tested compounds were diluted in n-hexane. Aliquots of 10 μL of respective solutions were loaded onto the filter paper discs within Pasteur pipette odor cartridges. During stimulation, 0.5 mL of humidized air was flown through loaded odor cartridge and injected into the air stream directed onto the antennal preparation. For each sex and stimulus type, 6 replicates were performed.
The antennal responses from each series of stimulations were normalized to the stimulations by 10 ng of 1-hexenol using the Syntech software and expressed as percents of 1-hexenol responses. For statistical evaluation, the data were subjected to logarithmic transformation to correct for the heteroscedasticity inherent to EAG data. The resulting datasets complied with the assumption of equal variances (tested using Levene's test) and the

Results and Discussion
Purification of extracts, derivatization, and analysis. GC/MS has been previously used to determine the stereoisomeric composition of TMPD-ol and TMPD-one in wing extracts of Bicyclus butterfly species 10 , stereoisomeric composition of TMPD-one in Euglossa bees 11 and of 3,7-dimethylpentadecan-2-ol in female extracts of pine sawflies 14 , where the stereoisomers of the chiral alcohols were separated as either (R)-trans-chrysanthemoyl or (S)-2-acetoxypropionyl esters. Here we used (S)-2-acetoxypropionyl derivatization method, because it is robust and more suitable for derivatization of compounds present at low concentrations 10,14 .
Male gland extracts gave stronger samples than female body extracts obtained from one specimen (Fig. S4). TMPD-one and TMPD-ol were found in both sexes, but their ratio in the sexes differed. In males, the ketone was present in substantially higher amount than the alcohol while in females, the alcohol dominated over the ketone. In the male wing glands, the trace amount of TMPD-ol was on the detection limit and the compound was only found after pre-cleaning the extract with SPE. A rough estimate of TMPD-ol quantity produced by females and TMPD-one produced by males was done using dodecanol as internal standard. In average, males produce 2−7 ng TMPD-one and females produce 1−5 ng TMPD-ol per individual.
Obtaining pure TMPD-ol from male glands was complicated due to its co-elution with mellein, which is a major component of the male-produced pheromone ( Fig. 2A, crude male extract, main peak). In the GC analysis of crude extracts TMPD-ol and TMPD-one co-elute, as a result of this a relatively large peak can be seen at the retention time of TMPD-ol/TMPD-one in the crude extract ( Fig. 2A, dotted line). After the first run of purification, mellein still dominated the fraction containing TMPD-ol (Fig. 2B, fraction containing TMPD-ol and mellein; Fig. 2C, fraction containing TMPD-ol and mellein, enlarged in Y-axis). Based on integration, the peak area of TMPD-ol in the fraction was 0.024% of the mellein peak area. The attempt to remove mellein by the reaction with KOH (1 M) was not successful and did not lead to the determination of the configuration of the minute amount of derivatized TMPD-ol.
Similar problems occurred during the purification of the female extract where TMPD-ol dominates over TMPD-one. Since the source of female pheromone is not known 3 , extract of whole female bodies was used. Purification of TMPD-one and TMPD-ol from the females was more difficult due to a higher contamination resulting from body extraction compared to gland extraction in males. TMPD-one from the extract of females was separated from TMPD-ol by means of SPE purification, but unfortunately the fractions were not pure. Several attempts of purification were performed. The ketone fraction was treated with KOH (1 M) in order to remove the excess of free fatty acids; this was however not successful. The ketone fraction was subjected to reduction and derivatization even though there were impurities present; it did not result in unambiguous determination of the stereochemistry. Thus, we were able to determine the stereochemistry of those pheromonal components present in higher amounts, i.e. of TMPD-one in the males and TMPD-ol in the females.
The synthetic mixture of eight stereoisomers and pure isomers of (2R,6R,10R)-and (2S,6R,10R)-TMPD-ol were utilized for comparisons on GC/MS. The pure isomers were synthesized by Hedenström 10 while working with Euglossa bees and Bicyclus butterflies 10,11 . The eight-stereoisomers mixture of TMPD-ol was synthesized according to Nieberding (supplemental material) 9 . All 8 derivatized stereoisomers separated well as (S)-2-acetoxypropionyl esters on the highly polar VF-23ms column (Fig. 3A). The male-extracted, reduced and derivatized pheromonal component, matched with the authentic standard of derivatized (2R,6R,10R)-TMPD-ol (Fig. 3B) leading to a conclusion that males produce (6R,10R)-TMPD-one (Fig. 3C). During reduction of TMPD-one a new stereocenter is introduced in position 2, that is the reason to why two peaks appear in the chromatogram after derivatization (Fig. 3C). Due to co-elution during SPE purification of the male-produced TMPD-ol with mellein ( Fig. 2) however, it was not possible to determine the stereochemistry of the native male TMPD-ol present in male wing glands in minute quantity. Female-produced TMPD-ol was determined as (2R,6R,10R)-TMPD-ol based on the match of the extracted and derivatized compound with the synthetic standard. The GC/MS analysis of female-produced and derivatized TMPD-ol showed one more peak at 397.5 min (Fig. 3D). However, retention time and mass spectral data did not match with any of the TMPD-ol isomers.
Schulz 16 showed that the Large Cabbage White, Pieris brassicae, sequester (R,R)-phytol from food sources and via oxidative processes transform phytol to (6R,10R)-TMPD-one. Eltz 11 reported on Euglossa bees carrying (6R,10R)-TMPD-one in their hind-leg pouch which is believed to be collected from a natural source, despite the source of TMPD-one has not been identified. This shows that some insects utilize the naturally derived (R,R)-phytol as a precursor. Since we kept A. sociella in the old bumblebee hives and not on an artificial diet of known composition, we cannot tell what the source of (2R,6R,10R)-TMD-ol was in this particular case.
The stereoisomers of TMPD-one found in males and TMPD-ol in females of A. sociella have the same configuration on chiral centres (2R,6R,10R) as 9 butterfly species of the genus Bicyclus studied earlier by Hedenström 10 and as male orchid bees of the genus Euglossa 11 . On the other hand, 3,7-dimethylpentadecan-2-ol, present in the female sex pheromone of the pine sawfly Neodiprion lecontei, has the configuration (2 S,3 S,7 S) 14 . The stereochemistry of TMPD-one and TMPD-ol in pheromones of related species of the subfamily Galleriinae (Galleria mellonella, Tirathaba mundella or Corcyra cephalonica) has not been studied so far. Thus, this paper is the first report on the configuration of the above pheromonal components within Galleriinae.

electroantennograhpy.
To determine the biological activity of native stereoisomers of TMPD-one and TMPD-ol, antennal responses of males and females of A. sociella were recorded and compared to those of a mixture of eight stereoisomers for TMPD-ol and/or a mixture of four stereoisomers for TMPD-one. Three concentrations for each treatment were tested to see the dose-response dependence (Fig. 4). The lowest selected concentration (10 ng) was approximately of the same order as 2 gland equivalents of males (wing glands are pair organs) or 1 body equivalent of females (the source of pheromone is not known, therefore the extract of the whole body was used). TMPD-one and TMPD-ol were perceived by both sexes. The lowest concentrations of both compounds elicited responses that were not signifficantly different from the solvent (n-hexane). Higher doses elicited higher antennal responses that were statistically different from the solvent at the levels 100 ng or 250 ng. Female antennal reactions were higher in response to native stereoisomer of TMPD-one (100 ng) in comparison with TMPD-one stereoisomeric mixture (Fig. 4A), while males responded in the same way on a higher concentration level (250 ng, Fig. 4C). On the other hand, both sexes responded significantly more strongly to the native stereoisomers of TMPD-ol than to the stereoisomeric mixture, both at the concentration level of 100 ng and 250 ng (Fig. 4B,D). www.nature.com/scientificreports www.nature.com/scientificreports/ conclusion Based on the purification and derivatization of the extract of male and female pheromone of A. sociella, we have determined the absolute configuration of TMPD-one in the male sex pheromone as (6R,10R)-6,10,14-trimethylpentadecan-2-one and of TMPD-ol in the female aphrodisiac pheromone as (2R,6R,10R)-6,10,14-trimethylpentadecan-2-ol. The pure stereoisomers elicit significantly stronger antennal responses than the tested stereoisomeric mixtures of both TMPD-ol and TMPD-one, respectively. We are aware that in the tested stereoisomeric mixtures only one forth (TMPD-one) or one eights (TMPD-ol) of the naturally produced isomer is present. We are showing here that the antennal responses increased with dose, for both the pure enantiomers as well as the mixtures of all stereoisomers. EAG experiments suggest that the antennal perception may be enantioselective. The significant difference in the perception of the pure isomer and the mixture might mean that none of the non-natural stereoisomers is antennaly active, has synergic affect, or influences the antennal response in any way. However, further experiments with pure stereoisomers are needed to support this hypothesis.