(A, B) Frequency of germaria containing 1, 2, and 3 GSCs (left axis) and the average number of GSCs per germarium (right axis) in virgin (v) and mated (m) female flies. Loss of NPFR (NPFRsk8/D(3R)BSC464) or ovarian knockdown of NPFR (tj-GAL4>NPFRRNAi) blocked the NPF-induced increase in GSC number after fly injection (panel A) or ex vivo ovary cultures (panel B). The number of germaria analyzed is shown inside the bars. For statistical analysis, a Wilcoxon rank sum test with Holm’s correction was used. ***P ≤ 0.001. Underlying data can be found in S1 Data. GSC, germline stem cell; NPF, neuropeptide F; NPFR, neuropeptide F receptor; NS, nonsignificant (P > 0.05); RNAi, RNA interference.