LAUSR

Summary PIWI proteins and their guiding Piwi-interacting small RNAs (piRNAs) are crucial for fertility and transposon defense in the animal germline. In most species, the majority of piRNAs are produced from distinct large genomic loci, called piRNA clusters. It is assumed that germline-expressed piRNA clusters, particularly in Drosophila, act as principal regulators to control transposons dispersed across the genome. Here, using synteny analysis, we show that large clusters are evolutionarily labile, arise at loci characterized by recurrent chromosomal rearrangements, and are mostly species-specific across the Drosophila genus. By engineering chromosomal deletions in D. melanogaster, we demonstrate that the three largest germline clusters, which account for the accumulation of >40% of all transposon-targeting piRNAs in ovaries, are neither required for fertility nor for transposon regulation in trans. We provide further evidence that dispersed elements, rather than the regulatory action of large Drosophila germline clusters in trans, may be central for transposon defense.