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https://avalanches.com/world_news/my/naturecom/natu_the_5225924_30_11_2022

In O. biroi colonies, larvae and pupae develop in discrete and synchronized cohorts26. Ten days after the first larvae had entered pupation in a large stock colony, the entire colony was anaesthetized using a CO2 pad, and white pupae were separated using a paintbrush. Pupae were individually placed in 0.2ml PCR tubes with open lid. These tubes were then placed inside 1.5ml Eppendorf tubes with 5µl sterile water at the bottom to provide 100% relative humidity. The outer tubes were closed and kept in a climate room at 25°C. The inner tube in this design prevents the pupa from drowning in the water reservoir. The outer tubes were kept closed throughout the experiment, except for once a day when the tubes were opened to remove pupal social fluid. Pulled glass capillaries were prepared as described elsewhere29, and used to remove and/or collect secretion droplets. We were careful to leave no remains of the secretion behind on the pupae or the inside of the tubes. To ensure that all secretion had been removed, pupae were taken out of the tube after fluid collection and briefly placed on a tissue paper to absorb any excess liquid. The inner tubes were replaced if neededfor example, if fluid traces were visible on the old tube after collection. Each pupa was checked daily for secretion (absent or present), onset of melanization and eclosion, and whether the pupa was alive (responding to touch). Control groups of 30 pupae and 30 adult ants from the same stock colony and cohort as the isolated pupae were placed in Petri dishes with a plaster of Paris floor, and the same parameters as for the isolated pupae were scored daily. Experiments ended when all pupae had either eclosed or died. Newly eclosed (callow) workers moved freely inside the tube and showed no abnormalities when put in a colony. A pupa was declared dead if it did not shed its pupal skin and did not respond to touch three days after all pupae in the control group had eclosed.

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