The behavioral mechanisms by which the larvae of sympatric Chilean populations of Drosophila melanogaster, D. simulans, D. hydei and D. busckii select pupation sites are described in terms of larval substrate preferences. These species share the same breeding sites in Central Valley of Chile. It is important to investigate larval substrate preferences to pupate in sympatric natural populations of species of Drosophila because such behavior could contribute to the coexistence of the species in the wild. D. busckii larvae preferred humid substrates with a smooth surface to pupate, whereas D. simulans larvae selected humid substrates with a rough surface. Larvae of D. melanogaster chose dry and humid substrates with a rough surface, whereas D. hydei larvae occupied dry substrates with a smooth surface to form puparia. D. melanogaster larvae dug deeper into dry than into humid sand, whereas D. simulans larvae dug more into humid sand. D. busckii larvae pupated in the upper layers of humid and dry sand, and D. hydei larvae dug more into humid than into dry sand. Pupae of the four Drosophila species showed aggregated distributions on the substrates. Larval prepupation behaviors of D. melanogaster, D. simulans, D. hydei and D. busckii could be important to their coexistence in the wild.
