show Abstracthide AbstractThe PIWI-interacting RNA (piRNA) pathway plays a crucial role in preventing endogenous genomic parasites, transposable elements (TEs), from damaging the genetic material of animal gonadal cells. Specific regions in the genome, called piRNA clusters, define each species' piRNA repertoire and therefore its capacity to recognize and silence transposons. In the somatic cells of the Drosophila melanogaster ovary, the flamenco (flam) unistrand cluster is the main source of piRNAs and primarily regulates Gypsy family TEs that are able to form virus-like particles and infect neighbouring germ cells. Disruption of the flam locus or failure to process flam precursor transcripts into piRNAs results in sterility, yet it remains unknown whether this silencing mechanism is employed widely across Drosophilidae. Here, using both synteny-based analyses and de novo TE annotation, we identify candidate loci sharing both their organisation and TE targeting repertoire with flam in widely divergent Drosophila species groups. Small RNA-sequencing validated these loci as bona-fide unistrand piRNA clusters and revealed their predominant expression in somatic cells of the ovary, likely to counter TE mobilisation in this tissue. This study provides compelling evidence of co-evolution between virus-like Gypsy family transposons and a host defence mechanism in form of soma-expressed, unistrand piRNA clusters. Overall design: Small RNA-seq from dissected ovaries (total or enriched for somatic follicle cells) from 15 Drosophila species, 1-3 replicates per species and library type.