Phenotyping
Female sex pheromone can be extracted from mated and old females by injecting them with Pheromone Biosynthesis Activating Neuropeptide, i.e. PBAN (Groot et al., 2005). Females were injected with a 7.5 pmol (2 µL of a 0.0146 µg/µL) PBAN solution to activate pheromone production in the mated females. After a 90-minute incubation time, female glands were extruded by squeezing the abdomen then fixed by firmly holding the abdomen with forceps just anterior of the gland. The gland was excised with microdissection scissors. Excess abdominal tissue and eggs that remained in the ovipositor were removed, after which the glands were submerged in 50 μL hexane containing 200 ng pentadecane as an internal standard. After 30 minutes, the glands were removed and the extracts stored at -20°C until analysis.
Pheromone extracts were analyzed by injecting the concentrated samples into a splitless inlet of a 7890A GC (Agilent Technologies, Santa Clara, CA, USA). The area under the pheromone peaks was calculated using integration software implemented in Agilent ChemStation (version B.04.03). Pheromone peak areas were obtained for the 11 pheromone components: two 14-C aldehydes (14:Ald, Z7-14:Ald), four 16-C aldehydes (16:Ald, Z7-16:Ald, Z9-16:Ald, Z11-16:Ald), the three acetates (Z7-16:OAc, Z9-16:OAc, Z11-16:OAc), and two 16-C alcohols (Z9-16:OH, Z11-16:OH). Z7-16:Ald and Z9-16:Ald were difficult to separate by GC and were therefore integrated as one peak (referred to Z9-16:Ald). Absolute amounts (in ng) of each compound were calculated relative to a 200 ng pentadecane internal standard. All downstream analyses were done in R 3.6.1 (R Core Team, 2019). Samples containing < 20 ng were excluded, because the ratios of the components in such low titers cannot be reliably measured in the chromatogram. Relative amounts were calculated by dividing the absolute amounts by the total amount across all 11 components.