Experiment 1: brood care
To determine the importance of adult worker antimicrobial
secretions for brood survival, we collected 120 leaf-cutting ant
workers and 160 weaver ant workers, split into two cohorts.
The leaf-cutting ant cohorts were each formed of 60 ants, with
20 ants from each of the three colonies, while the weaver ant
cohorts consisted of 80 ants, with 40 ants fromeach of the two
colonies used. Half the ants from each colony had their main
antimicrobial-producing glands (themetapleural gland in leafcutting
ants and the venom gland in weaver ants) blocked
using nail varnish, and the remaining workers had nail varnish
applied to the pronotum as a control (Poulsen et al. 2002;
Graystock and Hughes 2011). After 24 h,we collected 60 leafcutting
ant pupae and 80 weaver ant larvae, for each of the two
cohorts, and surface-treated half of them with the
Metarhizium parasite and the other half with 0.5 μl of a
0.05 % Triton X control solution using a micropipette. Each
pupa or larva was then placed in a pot (40 mm diameter) with
a single “nurse” worker ant from the same colony to give four
combinations of infected/uninfected brood and workers with
functional/nonfunctional glands, in a full factorial design, with
a total of 30 leaf-cutting ant and 40 weaver ant replicates of
each (Fig. S1). Ants were maintained in the pots with moistened
cotton wool to supply water and sucrose solution ad
libitum. Any workers which died during the experiment were
replaced with an identically treated worker. The survival of the
brood was monitored for 14 days. Dead brood were each
placed on moistened filter paper in a Petri dish at 26 °C and
80 % RH and checked daily for the appearance of fungal
conidia and conidiophores diagnostic of a Metarhizium infection.
To confirm that the blockage treatment did not affect
normal brood care behaviors, we also compared the behavior
of nurse workers for 20 ants of each species. Half the ants in
each species had their respective glands blocked and the other
half had the control treatment applied to the pronotum. The
ants were placed in a Petri dish with a single item of brood
(pupae for leaf-cutting ants and larvae for weaver ants) and (a)
the duration of any non-grooming interaction between nurse
and brood (e.g., carrying, antennation), (b) the frequency of
physical contact between nurse and brood, and (c) the frequency
of brood grooming by the nurse ant were recorded for
a 10-min period.