AbstractTreehoppers are known for their substrate-borne communication and some of them also for their subsocial behavior. Following a more general study of the natural history and substrate-borne signal repertoire of the treehopper Ennya chrysura, the objective of this paper was to explore in greater depth the signals and other behaviors associated specifically to egg-guarding. Theese were studied both in natural and laboratory conditions between July, 2000 and March, 2004. The spatial distribution of egg guarding females was studied in the natural population; recording equipment and playback experiments were used in the laboratory and then analyzed digitally. Under natural conditions (San Antonio de Escazú, Costa Rica), female E. chrysura guard their egg masses and egg-guarding was associated with lower parasitism of the eggs from the wasps Gonatocerus anomocerus and Schizophragma sp. (Mymaridae). Females tended to place their eggs close to other egg-guarding females and they produced substrate-borne vibrations when disturbed. An aggregated pattern under natural conditions was confirmed by calculating dispersion indices from egg-clutch data obtained from 66 leaves in the field. The disturbance signal was characterized from laboratory recordings of substrate-borne vibrations of 10 egg-guarding females. Experiments conducted in the laboratory with 18 egg-guarding females showed that those which were previously exposed to the disturbance signal of another female moved slightly or vibrated more during playbacks and that they reacted more quickly and exhibited more deffensive behaviors in response to a tactile stimulus. The signals produced while defending against egg parasites may therefore function as an alarm and favor aggregating behavior of egg-guarding females. Rev. Biol. Trop. 64 (3): 1209-1222. Epub 2016 September 01.