2.6 | Nestmate discrimination within polygyne colonies
To evaluate polygyne nestmate discrimination, we conducted a laboratory
experiment testing worker tending behavior towards larvae from the same
nest and larvae from a different nest. To do this, we established six
single-lineage experimental colonies by collecting mated polygyne queens
following mating flights. All colonies were kept in standardized
laboratory conditions for at least two years to ensure they were large
enough to be divided into smaller experimental colonies (i.e., over 360
workers and 150 larvae). Colonies were confirmed as the polygyne social
form by screening workers for the presence of theGp-9b allele using the same methods as
described above (see Genetic analyses in the methods section).
Once incipient colonies were large enough, we used food dye to label the
brood of each colony. We dyed brood by giving workers two separate tubes
of 15 mL of water and 15 mL of artificial nectar each containing 0.9 ml
of food coloring (McCormick® Food Colors & Egg Dye, McCormick &
Company, Inc., 18 Loveton Circle, Sparks, MD). We gave three colonies
(colonies A, B, and C) yellow food coloring and three colonies (colonies
D, E, and F) green food coloring (Table 1). Colors were randomly
assigned. During this six-day period of brood dyeing, we did not give
the ants any proteinaceous food so that dye would be highly visible in
the guts of larvae.
Next, we created experimental colonies by combining 0.1g of workers
(~120 workers) with 50 larvae from the same natal colony
as the workers (i.e., nestmates) and 50 larvae from a different colony
(i.e., non-nestmates; see Table 1 for complete family combinations). Not
all permutations of families were logistically possible in this
experiment, so only larval combinations of different colors were
combined so that all possible two-color combinations were created (Table
1). In all, there were 18 experimental colonies.
To quantify the feeding of larvae by workers, experimental colonies were
given 7.5 ml artificial nectar containing 0.2 g of non-toxic,
fluorescent dye (DFDRY-C0 UV Dye from Risk Reactor, 2676 S. Grand Ave.,
Santa Ana, CA) for 18 hours. After 18 hours, we recorded the number of
larvae remaining from each family and used a black light to count the
number of larvae from each family fed the fluorescent dye. In order to
ensure accurate results for potentially variable behaviors, data for
experimental colonies were averaged across the three iterations of this
experiment. This allowed us to remove within colony temporal variation
and estimate the general behaviors of each experimental colony instead
of only looking at a single snapshot of their behavior.