GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE335nnn/GSE335290/suppl/GSE335290_SLX-19644_totalRNA_RNAseq_GeTMM_TE_sense.xlsxftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE335nnn/GSE335290/suppl/GSE335290_R18168_GeTMM_gene.xlsxftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE335nnn/GSE335290/suppl/GSE335290_R17585_TOsK_MZ-piwiaub_dKD_TE_GeTMM_sense.xlsxftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE335nnn/GSE335290/suppl/GSE335290_CutnTag_bam_Table_for_heatmap_log2CPMadd1_single_TE_insertions.xlsxftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE335nnn/GSE335290/suppl/GSE335290_CutnTag_single_TE_insertions_table.xlsxftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE335nnn/GSE335290/suppl/GSE335290_CutnTag_avg_GeTMM_TE_consensus_unistrand.xlsxftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE335nnn/GSE335290/suppl/GSE335290_SLX-17461_GeTMM_gene.xlsxftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE335nnn/GSE335290/suppl/GSE335290_R18168_GeTMM_TE_sense.xlsxftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE335nnn/GSE335290/suppl/GSE335290_R16775_1kb_tiles_RNA_mappers_sum-PlusMinus.xlsxftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE335nnn/GSE335290/primaryOK200GenomicsDrosophila melanogaster Genome binding/occupancy profiling by high throughput sequencingNon-coding RNA profiling by high throughput sequencing Expression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE335290GEOGSEfalseA developmental switch in the piRNA pathway ensures stage-specific transposon silencing in DrosophilaTransposable element (TE) mobilization poses a major threat to genome integrity and fertility. In animals, TEs are suppressed in the germline by the Piwi-interacting RNA (piRNA) pathway, which is well characterized during late-stage Drosophila oogenesis. However, it remains unclear how TEs and the piRNA pathway are regulated across the dynamic chromatin and transcriptional landscape of earlier germline development. Here, using stage-specific chromatin profiling, piRNA sequencing, and temporally controlled knockdown of piRNA pathway components, we characterize TE regulation throughout Drosophila female germline development and uncover two distinct phases of piRNA-mediated transposon defense. In the first phase, spanning primordial germ cells through mitotically dividing germline stem cells, only a subset of TE families is transcriptionally competent, driven by TE-intrinsic cis-regulatory elements. These TEs are targeted by a piRNA program that relies on promoter-driven piRNA clusters independently of Moonshiner, Kipferl, and H3K27me3. In the second phase, triggered at the transition to endocycling nurse cells, the coordinated upregulation of Moon, Kipferl, Nxf3, and Bootlegger combined with increased H3K27me3 deposition reconfigures the piRNA pathway for enhanced piRNA production from heterochromatic dual-strand clusters, targeting the additional TE families activated in the polyploid nurse cell environment. Stage-specific chromatin profiling reveals that Piwi-dependent H3K9me3 deposition at TE loci requires their transcriptional activation. Importantly, silencing established by the early piRNA program can be maintained independently of piRNA pathway activity in later development, consistent with an epigenetic lock mechanism. Our findings establish that the piRNA pathway is not merely maintained but actively remodeled across germline development to match changing TE silencing demands.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 melanogaster