Mature males and virgin queens from Atta colombica ants were collected in panama, and their seminal fluid (SF) and spermathecal fluid (SpF), respectively, were extracted and analysed by shot-gun proteomics. For this, using a pooled SF sample of 25 males from five colonies, squeezing their abdomens to collect 2-4ul of ejaculate per male from the extended endophallus. The SF supernatant was collected from the pooled ejaculate after 2 centrifugations for10 min at 13,000xg. The Spf was obtained form 4 different colonies collecting 10 virgin queens per colony and “artificially inseminating them with 10ul of Hayes. All 40 queens were allowed to recover for 12 hours before retrieving the content by dissecting and rupturing the spermathecal in Hayes. The samples were vortexed for 3 min, followed by centrifugation for 3 min at 13,500 xg to separate SpF from tissue.