Electronic Supplementary Materials 1: Details of observation box setup and surgical methods
Observation box setup
Workers were collected with a hand-held, battery-powered insect vacuum (BioQuip Inc.).Collection took place during the day, and I waited for at least 30 minutes to collect nearly all returning foragers (initial observations suggest that 95% of foragers that return within 1 hour do so within the first 30 minutes of collection).The nest was then cut into a zip-close plastic bag, and the plastic chamber containing workers was placed on ice.
In the laboratory, the nest envelope was carefully removed by making an incision along one side.The combs typically consisted of a first comb of worker cells and the start of a second comb with no pupal caps; only occasionally did I use colonies that had begun construction of a third or fourth comb.The upper comb was glued into the top of a wooden frame observation box that was either 1.5 or 2 inches wide, depending on the size of the nest when collected (see Figure 1d).The envelope was trimmed on opposite sides to permit view of the comb, and then glued in place around the comb.
Workers and the queen were placed in a refrigerator until anesthetized.Queens were marked with a thin copper wire bent in a loop between abdomen and thorax, or with a large paint mark (Sharpie paint pen) on the thorax.This made identifying the queen easier in large colonies with post-emergence gynes.All individuals were placed inside the nest while still anesthetized.Cotton balls soaked in honey-water were placed in the bottom of the box, and then the glass walls were attached with duct tape.The colony was kept in the dark until after nightfall, when the box was installed in one of several sheds at the Liddell Field Station, and connected via a 1-inch-diameter Tygon tube to the outdoors.Foraging resumed the next morning.
Spermathecetomy
Prior to surgery, the queen was extracted from the nest either after collecting workers with a vacuum and net, or in 2013 after briefly anesthetizing the entire colony with CO2 (only enough to prevent workers from flying when the box was opened, typically no longer than 1 minute).The queen was placed abdomen-first into a modified 1500 μL Eppendorf tube (Figure 1c).This tube held the queen in place while CO2 was gently flowing from a Tygon tube connected to the mouth of the Eppendorf tube.The tube containing the queen was positioned using a movable jointed arm, and she was oriented with her ventral side upward, with the sting pointing toward me.Forceps on a micromanipulator grasped the ultimate sternite while a hook fashioned from the tip of an insect pin held the penultimate sternite.By retracting the ultimate sternite, the membrane between these two segments was exposed (Figure 1).I then made an incision in the membrane with a shard of sterilized razor blade, and immediately placed a drop of sterile Ringer’s solution into the aperture.Then, using two sets of fine forceps, the common oviduct was brought into view.Care was taken not to tear muscles that connect the two segments and run across this region, and the ventral nerve cord was also seen and avoided.Once the common oviduct was in view, it was a simple procedure to pull it to one side with one pair of forceps, and to grasp the spermatheca with the other pair.The spermatheca was then removed without rupturing it.Before closing the wound, a few crystals of phenylthiourea, penicillin G and streptomycin sulfate (ratio 2:1:1) were placed in the abdominal cavity (Schneiderman 1967).The wound was not sealed, but the overlapping segments closed the wound effectively.The entire process usually took around 3-4 minutes.This method was practiced on dozens of spring queens of various species before implementation on observation colony queens.
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