show Abstracthide AbstractWe investigated a pool of small RNAs derived from transposon Penelope to probe the evolution of small RNA pathways in response to the transposon challenge in natural (D. virilis) and experimentally induced (D. melanogaster) colonization processes. In both species, Penelope was predominantly targeted by endo-siRNAs rather than by piRNAs. Although we observed correlations between Penelope transcription and dysgenesis, we could not correlate differences in maternally deposited Penelope piRNAs with the sterility of progeny. Instead, we found that strains, which produced dysgenic progeny, differed in their production of piRNAs from clusters in sub-telomeric regions, possibly indicating that changes in the overall piRNA repertoire underlie dysgenesis. Considered together, our data reveal unexpected plasticity in small RNA pathways in germ cells, both in the character of their responses to invading transposons and in the piRNA clusters that define their ability to respond to mobile elements. Overall design: Examination of total small RNA in 0-2h embryos, testes, ovaries and carcasses of several D. virilis strains and their hybrids; total small RNA and Ago2, Piwi, Aub and Ago3-associated small RNA in testes and ovaries of D. melanogaster.