2.5 Sex pheromone production
Molecular data suggested that the Pacific coast parthenogenetic populations are genetically distinct from the Sea of Japan coast parthenogenetic populations which include p2 of which sex pheromone production was analyzed in Hoshino et al., (2019). Thus, the parthenogens in the Pacific coast were newly examined whether they produce sexual pheromone using a gas chromatography/mass spectrometry (GCMS). Females from parthenogenetic population p28 were used for GCMS. Cultured gametophytes were kept in 300 mL of sterilized seawater in a 500 mL flask at 15°C and gametes were released in the flask. Volatile secretions were trapped on Mono Trap RCC18 (GL science, Tokyo, Japan) using a closed-loop-stripping system at 15°C. After looping for 15 h, absorbed volatile compounds were eluted with 50 μL of CH2Cl2 and immediately analyzed by GCMS. [Zebron ZB-wax (phenomenex) 30 m × 0.25 μm; He as the carrier gas; program rate: 45-200°C at 5°C/min]. Compounds were identified by using the NIST MS library and similarity search program. A female gametophyte from sexual population p15 was also examined as positive control.