Materials and Methods

2.2.1 Specimens

Adult male beewolves were obtained from a laboratory population (see e.g. Strohm &

Linsenmair 1997). Freshly eclosed males were individually marked and kept as described before (Herzner et al. 2006). Since age has been shown to influence pheromone composition (Kaltenpoth & Strohm 2006) all males used for the analyses were the same age. Twelve to fourteen days after emergence, males were caught and stored individually in small polystyrene vials (35mm diameter, 82mm length, filled with 2 cm of moist sand) with rubber foam plugs for two days, so that they could replenish the reservoirs of their marking pheromone. They were then anaesthetized with CO2 and individually frozen at -18°C until chemical analyses were conducted.

2.2.2 Extracts

For the analyses males were thawed, decapitated, and the heads dissected under a stereo microscope. The PPG was carefully uncovered by removing the frontal part of the head capsule by cutting the cuticle between the eyes, toruli, and lateral ocelli very shallowly using newly broken pieces of razorblades in a blade holder. If the reservoir was not injured it then bulges out. A sample of the PPG content was taken by inserting the tip of a fine glass pipette directly into the gland reservoir. The content was automatically sucked into the pipette by capillary forces. The sample was then dissolved in re-distilled hexane.

For the complete inventory and characterization of the content of the PPG the samples of three males were combined and reduced in volume to approximately 100 µl by a stream of nitrogen at ambient temperature. An aliquot of 1 µl was analyzed by combined gas chromatography – mass spectrometry (GC-MS) (set-up 1, manual injection). To check for any unpolar substances hidden under the peaks of polar pheromone compounds, the extract was fractionated on a conditioned SiOH-glass-column (CHROMABOND, 500mg, Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions, and 1 µl of both the hexane and the dichloromethane fraction was analyzed by combined GC-MS (set-up 1, manual injection).

Some components that could be detected and characterized in the pooled samples could not be satisfactorily identified in samples of individual males due to the smaller amounts of secretion. Therefore, the number of peaks included in the respective analyses varies somewhat.

To compare samples directly taken from the PPG with those obtained from total head extracts, 15 males were dissected as described above. After a sample of the PPG content had been taken, the remaining head capsule still containing the PPG and most of its content was extracted in re-distilled hexane for three hours. The samples were reduced in volume to approximately 100 µl and an aliquot of 1 µl of each solution was analyzed by GC-MS (set-up 1, automatic injection).

To investigate a possible dimorphism in the composition of the PPG content, we analyzed a large sample of 45 males in order to be able to detect a low frequency of one morph. Males were obtained and treated as described above. For this second analysis we did not dissect the heads, however, but extracted whole heads that were incised to open the glands. The heads were individually extracted in re-distilled hexane for three hours, the sample volume reduced

to approximately 100 µl, and an aliquot of 1µl of each sample was analyzed by GC-MS (set-up 2, automatic injection).

2.2.3 Capillary Gas chromatography – mass spectrometry. Set-up 1

GC-MS analysis was performed with an Agilent 6890N Series gas chromatograph (Agilent Technologies, Böblingen, Germany) coupled to an Agilent 5973 inert mass selective detector.

The GC was equipped with an RH-5ms+ fused silica capillary column (30 m x 0.25 mm ID; df

= 0.25µm), and the temperature program ramped from 60°C to 300°C with 5°C/min. The temperature was held constant in the beginning at 60°C for 1 min and at the end at 300°C for 10 min. Helium was used as a carrier gas with a constant flow of 1 ml/min. A split/splitless injector was installed at 250°C and in the splitless mode for 60 sec. The electron impact mass spectra (EI-MS) were recorded with an ionisation voltage of 70 eV, a source temperature of 230°C and an interface temperature of 315°C. The software MSD ChemStation (Agilent Technologies, Palo Alto, CA, U.S.A.) for Windows was used for data acquisition.

2.2.4 Capillary Gas chromatography – mass spectrometry. Set-up 2

GC-MS analysis was performed with a Fisons Instruments (Fisons, Egelsbach, Germany) GC 8000 Series coupled to a Fisons Instruments MD800 quadrupol mass detector. The GC was equipped with a J&W DB-5 fused silica capillary column (30 m x 0.25 mm ID; df = 0.25µm) (J&W, Folsom, CA, USA), and the temperature program ramped from 60°C to 310°C with 5°C/min. The temperature was held constant at 310°C for 10 min. Helium was used as a carrier gas with a constant pressure of 90 mbar. A split/splitless injector was set at 240°C and was in the splitless mode for 60 sec. The electron impact mass spectra (EI-MS) were recorded with an ionisation voltage of 70 eV, a source temperature of 220°C and an interface temperature of 315°C. The software Xcalibur (ThermoFinnigan, Egelsbach, Germany) for Windows was used for data acquisition.

2.2.5 Chemicals and pheromone compound identification

Solvents (Fluka, Deisendorf, Germany) were distilled and checked for purity by GC-MS prior to use. (Z)-9-octadecen-1-ol and 1-eicosanol were identified by comparing retention times and mass spectra of the PPG extracts with those of the synthetic standard ((Z)-9-octadecen-1-ol: Merck Schuchard OHG (Hohenbrunn, Germany); 1-eicosan((Z)-9-octadecen-1-ol: Dr. Ehrensdorfer GmbH (Augsburg, Germany)). The n-alkanes were identified by comparing retention times and mass spectra of beewolf gland extracts with data from earlier analyses (Schmitt et al. 2003;

Strohm et al., unpublished data) and with data from a commercial MS library (NIST, Gathersburg, MD, U.S.A.). The corresponding alkenes were identified by their typical mass spectra and their retention times. The positions and geometries of the double bonds were inferred by comparison with earlier analyses on the chemistry of the pheromone (Schmitt et al. 2003) and of the PPG content or cuticular hydrocarbons of conspecific females that had been characterized by FTIR spectrometry and DMDS derivatization (Strohm et al., unpublished data); peaks with identical retention times were considered to be the same isomers. Some alkenes were present only in threshold amounts in all our earlier and the current analyses and could thus not be characterized completely. The alkadienes were characterized by their typical mass spectra and their retention times. Due to their very small amounts the position and geometry of the double bonds could not be determined. Methyl alkanes were identified by diagnostic ions, standard MS databases (see above), and by determining Kovats indices by the method of (Carlson et al. 1998). The ketone ∆-16-pentacosen-8-one was identified by comparing retention time and mass spectrum of the peak with the ∆-16-pentacosen-8-one present in the female PPG (Strohm et al., unpublished data).

2.2.6 Statistics. Head extracts vs. PPG content

Peak areas were obtained by manual integration (MSD ChemStation) and relative peak areas were transformed to logcontrasts (Aitchison 1986; Reyment 1989) prior to the analysis. Mean values of each individual peak were normalized by log-transformation. To test for a chemical congruency between the substances found in the PPGs and in the head extracts, we conducted a regression analysis between the proportions of components (Aitchison- and log-transformed) in the samples directly obtained from the PPG and in the head extracts (reduced major axis regression (RMA) (Legendre & Legendre 1998) using ‘RMA Software for Reduced Major Axis Regression v.1.17’ (A. J. Bohonak, San Diego University, U.S.A; freely

available at http://bio.sdsu.edu/pub/andy/RMA.html). To assess the chemical similarity between the PPG samples and the head extracts, we tested whether there was a direct proportionality: i.e. the slope of the resulting regression line should not deviate significantly from 1 and the y-intercept should not deviate significantly from 0.

2.2.7 Statistics. Chemical dimorphism

Peak areas were obtained by automatic integration (Xcalibur). The relative peak areas were transformed to longcontrasts (Aitchison 1986; Reyment 1989). Since in females the dimorphism is most striking for the compounds (Z)-9-pentacosene and (Z)-9-heptacosene (Strohm et al., unpublished data), we first focused on these peaks. The different isomers of the unsaturated hydrocarbons were not well separated in the chromatograms (see below).

Therefore, in the following ‘pentacosene’ and ‘heptacosene’ refer to the mixture of isomers.

However, in all cases the Z-9 isomers are most dominant.

Inspection of the chromatograms revealed that based on the proportions of pentacosene and heptacosene in their pheromone blends, males could be separated into two distinct types:

those with pentacosene as the by far most abundant hydrocarbon (C25–type males) and those with comparable proportions of pentacosene and heptacosene (C25/C27–type males). To test for a bimodal distribution we analyzed the frequency distribution of the proportion of heptacosene by use of a histogram plot (see results). The histogram revealed that the males could be assigned to two groups according to their proportion of heptacosene. We compared the proportions (relative peak areas Aitchison transformed) of all substances between these two groups with exact tests for two independent samples (using SPSS 13.0 for Windows, SPSS Inc., Chicago, IL, U.S.A.).