Enhanced chemstation

For Py–GC/MS analysis an HP-Plot/Q 30 m long column with a stationary phase of 0.32 mm thickness and 20 μm i.d. (Agilent Technologies, Santa Clara, CA, USA) was used. Small samples of pigment B15:3 were directly placed inside a glass tube which were then automatically inserted to the pyrolysis module at the thermal desorption unit (TDU) (both from Gerstel, Mühlheim, Germany) of the GC/MS inlet system. Pyrolysis was carried out at temperatures of 500–1,000 °C for 6 s. The temperature of the cold injection system (CIS), TDU and transfer line was maintained at 260 °C. The carrier gas flow rate was 1 ml/min using a split ratio of 1:30. The GC oven was kept at 50 °C for 2 min and afterwards ramped at 10 °C/min to 260 °C which was then held for 10 more minutes. The mass range was scanned in full scan mode from 10 to 550 m/z. Fragments were identified using a library of standards (US-NIST – National Institute of Standards and Technology – 2011 MS Library). Match and rematch for HCN, BCN and BDCN were above 900 on a scale to 999 being the best possible match. Data were analyzed using Enhanced ChemStation (E02.02.1431) from Agilent Technologies.

Each SPME fiber was desorbed in the injection port of an Agilent 7890B gas chromatograph (GC) coupled with an Agilent 5977A mass spectrometer (MS) (EI mode, 70 eV with a scanning range of 40.0–450.0 m/z), using a DB-5MS capillary column (Agilent, 30 m×0.25 mm i.d., 0.25 µm film thickness) in splitless mode, with helium carrier gas at constant flow rate of 1 ml/min. The injection port temperature was 280°C, and the oven temperature was held at 40°C for 1 min, ramped at 10°C/min to 300°C, then held for 25 min. Tentative identifications of the honeydew volatile components were made by comparing mass spectra with those in the mass spectral library database (Enhanced ChemStation, MSD Chemstation, Data Analysis software vF.01.00.1903, and NIST, v11, Agilent Technologies, Santa Clara, CA). Close matches were confirmed by obtaining and injecting authentic standards and comparing their Kovat’s indexes (KI), retention times, and mass spectra to ensure they matched. Compounds that were also present in controls are not reported. Peak areas representing the total ion abundance for each peak were used to calculate the percent (ratio) of each identified compound over all SPME volatile collections combined for each sex (4 Early, 2 Mid, and 1 Late). The sum of peak areas for each compound was divided by the total sum of all 13 compounds for males and for females to calculate ratios.