Molecular Sexing of Insect Larvae

All adults were sexed based on apparent sexually dimorphic features, specifically the presence/absence of androconia (the male-specific pheromone organ). Because larvae do not display any obvious sexual dimorphism, we extracted DNA from individuals using the KAPA Express Extract Kit (KAPA Biosystems; Wilmington, MA, USA) and amplified a female-specific W-chromosome microsatellite ([115 (link)]; Genbank accession no.: AY785080) using the primers from [116 (link)]. PCR products were then visualized by gel electrophoresis, and females were identified by the presence of a band at ~ 185 bp, while males lacked this band (Additional file 1: Fig. S9).