In the course of the second project year, the two sampling expeditions were successfully executed and yielded material sufficient for analytical chemical and microbiological investigations. Activity profile, foraging and defence behaviour of COCY ants were documented by in situ and in vitro video materials. The morphology-based revision of the Colobopsis cylindrica species group proceeded by studying specimens from Southeast Asia. Samples from 2015 yielded the first identifiable males of COCY ants and led to the discovery of mermithism (parasitism by nematodes) and first description of COCY mermithogynes. As previous studies had revealed that COCY ant species have a kind of worker polymorphism that is unique within Camponotus/Colobopsis, we carried out additional comparative studies on two outgroup taxa with a distinct worker dimorphism. In 2015 we also studied types of relevant COCY species including the collection of Carlo Emery (Museum Genova, Italy) which led to the discovery of a new species from Indonesia.

The primary assessment of COCY nutrition has been summed up in a peer-reviewed publication (Davidson et al., 2016, Biotropica, in press). For the investigation of microorganisms in COCY-related habitats and for tests of COCY fungivory a library of ca 600 pure fungal and bacterial cultures was composed for ongoing molecular identification, ecophysiological profiling and in situ experiments. Multiloci molecular evolutionary analysis of 143 reference COCY and related ants confirmed monophyletic origin of exploding ants and revealed at least 10 genetically distinct lineages within them what is in line with morphological evaluations. With respect to mandibular gland (MG) secretion, more than 50 volatile substances were detected. Among those, 36 metabolites were successfully identified (19) or annotated (17) and found to belong to alkanes, alkenes, terpenoids, carboxylic acids, ketones, and phenols. 17 of the detected metabolites not been described for the investigated COCY species before. Interestingly, many of these substances have also been found in defensive secretions of other ants/insects, where they have been described as antimicrobials.

Results suggest that the MG content of ‘COCY’ ants may not only serve to directly kill opponents, but might also act against detrimental bacteria and fungi being present in their nests. A GC-MS protocol has been established for the determination of the ants’ cuticular hydcrocarbon (CHC) profiles with the aim to support taxonomic classification and to investigate the role of CHCs for intra- and inter-species communication. The initial behavioural and aggression tests demonstrate that COCY ants are capable of discriminating between non-nestmates and nestmates via CHC profiles.