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The original version of this article unfortunately contained some mistakes for the greek letter “Ɵ” in the proof manuscript.

Adult leaf-cutting ants of the subspecies Acromyrmex subterraneus subterraneus were fed with an Evans Blue dye solution, which allowed the investigation of subsequent exchange of liquids between ants by oral trophallaxis. Trophallactic behavior was filmed and the antennation patterns of donor and recipient ants were described. The ants’ crop capacity was measured following ad libitum feeding on dye solution. Ants previously fed on the dye solution (donors) were placed individually with unfed ants of the same caste (recipients) and the amount of dye solution passed from the donor to the recipient by oral trophallaxis was measured after one hour. The volume received was 0.26 ± 0.15μL (mean ± SD) and the residual volume of dye in the crop of the donors was 0.49 ± 0.23μL. There were 38 trophallactic events recorded for 50 pairs of ants. Trophallaxis was observed from 2.3 min to 21.5 min after initial exposure, with a mean latency of 8.4 ± 5.6 min. The mean duration of a trophallatic event was 2.3 ± 1.3 min. As far as we know, this is the first time that trophallaxis in leaf-cutting ants has been described and quantified.

Neotropical “Fungus-growing” leaf-cutting ants (tribe Attini) live in obligatory symbiosis with a fungus, which they grow on fresh leaves harvested by workers. Colonial recognition is likely based on chemical cues provided by cuticular hydrocarbons that have been found to be partly influenced by environmental odor sources. The diet breadth of Acromyrmex subterraneus subterraneus enabled us to test the impact of different plant diets on colonial recognition. The intermediary of the fungus in the ants’ feeding habit adds a special angle to the question. From a queenright (QR) mother colony of A. s. subterraneus we formed several groups of queenless (QL) workers with fungus (approx. 700 ants). The QR colony and two of the QL-groups were fed with the same diet of fresh bramble leaves. Two other QL-groups were fed with privet leaves and two with rose flowers. After 4 months, QR workers were significantly more aggressive towards the QL-group fed with rose flowers or privet leaves than towards workers of the QL-groups fed with fresh bramble leaves. Rose-fed QL workers were aggressive towards privet-fed QL workers and vice versa, but never towards workers of their counterpart group that fed on the same diet. These results suggest that the absence of the queen or the separation time between groups played a minor role in shaping nestmate recognition cues as compared to the diet. The behavioral studies were supplemented by chemical analyses of cuticles, postpharyngeal glands (PPG) and plant-food extracts revealing profiles variations that were correlated with the dietary changes. However, although the plant extract contained several hydrocarbons there was no congruency between the plant profile and the respective diet-group ants. These results support the hypothesis that the diet influences indirectly the chemical profiles and consequently the recognition cues in A. s. subterraneus.

The trail pheromone ofAcromyrmex subterraneus subterraneus consists simply of the heterocyclic ester methyl 4-methylpyrrole-2-carboxylate in the venom reservoir of workers at about 1.2 ng/ant. No pyrazines were detected, and no enhancement of trail-following was observed when pyrazines were added to the pyrrole compound.