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Tsetse flies significantly impact public health and economic development in sub-Saharan African countries by transmitting the fatal disease African trypanosomiasis. Unusually, instead of laying eggs, tsetse birth a single larva that immediately burrows into the soil to pupate. Where the female chooses to larviposit is, therefore, crucial for offspring survival. Previous laboratory studies suggested that a putative larval pheromone, n-pentadecane, attracts gravid female Glossina morsitans morsitans to appropriate larviposition sites. However, this attraction could not be reproduced in field experiments. Here, we resolve this disparity by designing naturalistic laboratory experiments that closely mimic the physical characteristics found in the wild. We show that gravid G. m. morsitans were neither attracted to the putative pheromone nor, interestingly, to pupae placed in the soil. By contrast, females appear to choose larviposition sites based on environmental substrate cues. We conclude that, among the many cues that likely contribute to larviposition choice in nature, substrate features are a main determinant, while we failed to find evidence for a role of pheromones. [ABSTRACT FROM AUTHOR]