37v software. It was assumed that the darkest

gray received the highest encapsulation rate (total black). The background gray value was subtracted to correct the gray values of the implants. The colony was included see more as a random factor and treatments were analyzed by an ANOVA followed by an Unequal N HSD test at 5% probability. In this experiment, we used a fourth colony (colony D) to test the effects of removing bacteria on worker immunity. To kill the bacteria, we followed the methodology described by Poulsen et al. (2003a). We established six experimental treatments using workers with bacteria covering the whole body: (1) 22 without treatment, (2) 20 treated with a dry brush to remove their bacterial cover, (3) 20 treated with a wet (water only) brush, (4) 20 treated with a brush containing

a solution of penicillin G (622 mg/L), (5) 20 with a brush containing a solution of streptomycin sulfate (1230 mg/L) and (6) 20 treated with a brush containing a mixture of the two antibiotics. Ant workers were all about the same size (∼2.4 mm HW) and the brushing operation lasted approximately 10 s. Afterwards, all ants were marked with a dot of paint and placed in mini-colonies established in plastic pots containing 100 mL of fungus GKT137831 purchase garden and approximately 100 nestmate workers without visible bacteria coating. Ten days later, the marked workers were removed for an encapsulation assay, as described in Section 2.2. We verified that these marked workers did not show a visible white coating of bacteria in the integument, confirming that the treatments were effective. The groups

were compared by an ANOVA followed by an Unequal N HSD test at 5% probability. The aim of this study was to assess the metabolic rate and to infer ioxilan a possible energetic cost of maintaining ectosymbiotic bacteria. The production of carbon dioxide was measured in a carbon dioxide analyzer (TR 2; Sable System International, Las Vegas, Nevada, USA) using methods adapted from Hebling et al. (2000) and Guedes et al. (2006). A series of 25 mL flasks was used, each flask containing three workers (2.4 mm head capsule width) from each group (EXT, INB, and INØ) in a completely closed system. Carbon dioxide-free air was injected into the flasks for 2 min at 600 mL/min. An infrared reader was connected to the outlet of the system to quantify carbon dioxide (μmol). The test tubes were connected to the system for three hours before measurement of CO2 production from the workers, which was achieved by injection of CO2-free air into the vials for 2 min at a flow rate of 600 mL/min. This air flow directs CO2 to an infrared reader connected to the system and allows rapid quantification of the amount of CO2 produced on an hourly basis (in μmol). There were 14 replicates for each group, which were taken at the same proportion from three colonies (A, B, and C). In total, we took 42 workers from each colony.