2.2 | Treatment with the tracer
We fed workers from each treated nest a nitrogen tracer mixed in sugar water. A solution with 102 mM of 15N-labeled glycine (98 atom%, Sigma-Aldrich, Inc., St. Louis, MO, USA) and 61.5 mM of unlabeled sucrose was created using distilled water. This concentration was determined based on a preliminary laboratory experiment with a small number of fire ant workers and an approximation of colony sizes in the field, which can exceed 250,000 workers within a single nest (Tschinkel, 2006). The solution was mixed in bulk at the beginning of the field experiment and frozen between uses to avoid mold growth. We filled 1-mL microtubes with 1 mL of the solution and stoppered each with cotton. Three of these vials were left on the surface of each treatment nest and replaced every other day for 14 days. Vials were placed directly on the mound surface to ensure that only the treated nest fed on the solution. Each treatment nest was fed a total of 160 mg of15N-labeled glycine in 21 mL of sugar solution over a 14-day period. Fire ant workers were observed feeding on the solution, and there was evidence of nest building over the vial opening, indicating worker foraging.
We collected workers from all treated and untreated nests once immediately before feeding the tracer to treated nests and once at 14 days after beginning the treatment. Workers were collected by disturbing a small section of each nest and aspirating 40-50 workers for isotopic and genetic analyses (see Genetic analyses section below). Workers were frozen at ‑10˚C. We then transferred 10-30 workers per nest to 95% EtOH for storage prior to DNA extraction and left the remaining workers for stable isotope analysis. Workers for stable isotope analysis were never stored in EtOH to avoid possible effects of EtOH on isotopic signatures (Tillberg, McCarthy, Dolezal, & Suarez, 2006).