Alarm signalling is of paramount importance to communication in all social insects. In termites, vibroacoustic and chemical alarm signalling are bound to operate synergistically but have never been studied simultaneously in a single species. Here, we inspected the functional significance of both communication channels in Constrictotermes cyphergaster (Termitidae: Nasutitermitinae), confirming the hypothesis that these are not exclusive, but rather complementary processes. In natural situations, the alarm predominantly attracts soldiers, which actively search for the source of a disturbance. Laboratory testing revealed that the frontal gland of soldiers produces a rich mixture of terpenoid compounds including an alarm pheromone. Extensive testing led to identification of the alarm pheromone being composed of abundant monoterpene hydrocarbons (1S)-α-pinene and myrcene, along with a minor component, (E)-β-ocimene. The vibratory alarm signalling consists of vibratory movements evidenced as bursts; a series of beats produced predominantly by soldiers. Exposing termite groups to various mixtures containing the alarm pheromone (crushed soldier heads, frontal gland extracts, mixture of all monoterpenes, and the alarm pheromone mixture made of standards) resulted in significantly higher activity in the tested groups and also increased intensity of the vibratory alarm communication, with the responses clearly dose-dependent. Lower doses of the pheromone provoked higher numbers of vibratory signals compared to higher doses. Higher doses induced long-term running of all termites without stops necessary to perform vibratory behaviour. Surprisingly, even crushed worker heads led to low (but significant) increases in the alarm responses, suggesting that other unknown compound in the worker's head is perceived and answered by termites. Our results demonstrate the existence of different alarm levels in termites, with lower levels being communicated through vibratory signals, and higher levels causing general alarm or retreat being communicated through the alarm pheromone.

Faculty of Electrical Engineering Czech Technical University Prague 166 27 Prague 6 Czech Republic

Faculty of Forestry and Wood Sciences Czech University of Life Sciences 165 21 Prague 6 Suchdol Czech Republic

Faculty of Forestry and Wood Sciences Czech University of Life Sciences 165 21 Prague 6 Suchdol Czech Republic Faculty of Science Charles University Prague 128 43 Prague 2 Czech Republic Institute of Organic Chemistry and Biochemistry Academic of Sciences of the Czech Republic 166 10 Prague Czech Republic

Laboratório de Interações Ecológicas Departamento de Ecologia Universidade Federal de Sergipe São Cristovão SE 49000 000 Brazil

Laboratório de Termitologia Departamento de Entomologia Universidade Federal de Viçosa Viçosa MG 36570 900 Brazil

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Alarm communication predates eusociality in termites

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