Abstract Diathoneura tessellata Duda, 1925 (Diptera: Drosophilidae) uniquely and effectively uses the fleshy tepals of Anaxagorea crassipetala (Annonaceae), a small, understory tree of the primary lowland rain forest of Costa Rica, as a larval substrate and pupation site. This study is the first to document 1) the brood substrate for larvae of this species and the 2) use of flowers in the Annonaceae as a drosophilid larval substrate. Oviposition into the tough, immature flower buds is made possible by an enlarged oviscape. This relationship is unique in that these flowers support two sequential cohorts of larvae, one cohort mining the living tepals of immature and mature flowers and the second cohort consuming the fallen, postanthesis tepals. We refer to this phenomenon as sequential florivory/saproflorivory. The first cohort consists of fewer, larger larvae, whereas the second cohort consists of more numerous, smaller larvae. Both cohorts exhibit a female skewed sex ratio.