show Abstracthide AbstractPIWI-interacting RNAs (piRNAs) bind to PIWI proteins to assemble the piRISC, which represses germline transposons. Maelstrom (Mael) is necessary for piRISC biogenesis in germ cells, but its function remains unclear. Here, we show that Mael interconnects Spindle-E (Spn-E), a key piRISC biogenesis factor, with unloaded Siwi, one of two silkworm PIWI members. Mael also assembles a subset of nuage, a non-membranous organelle involved in piRISC biogenesis. Loss of Mael abrogated the Spn-E–Siwi interaction and Ago3-piRISC biogenesis, but Siwi-piRISC was produced. Bioinformatic analysis showed that Siwi-bound piRNAs in Mael-lacking cells were rich in transposon-targeting piRNAs as in normal cells but were biased toward transposons that are marginally controlled by Siwi-piRISC. This explains the impairment in Ago3-piRISC production because transposon mRNAs cleaved by Siwi are the origin of Ago3-loaded piRNAs. We argue that Mael plays a role in the production of primary Siwi-piRISC capable of regulating transposon expression in germ cells. Overall design: Six smRNA-seq libraries: PIWI-associated small RNA libraries were prepared from small RNAs associated with Flag-Siwi immunoprecipitates with size selection at 22-35nt. Six RNA-seq libraries: RNA libraries were prepared from total RNAs with poly-A selection.