Retrotransposon activation during spermatogenesis achieves massive ecDNA biogenesis but rare integration

Retrotransposon mobilization in germline cells enables the rewriting of genetic information to drive genome innovation, species evolution, and adaptation through the generation of de novo mutations. However, uncontrolled mobilization can cause DNA breaks and genome instability, often leading to sterility. How retrotransposon mobilization that can be retained for genome evolution persists despite negative outcomes of retrotransposon activity remains poorly understood. Here, we used Drosophila spermatogenesis as a model to investigate retrotransposon mobilization dynamics. Although many retrotransposon families are transcriptionally active, we found that the LTR retrotransposon nomad completes the full mobilization cascade (including mRNA export, protein translation, and reverse transcription) to produce double-stranded DNA (dsDNA) the most efficiently. Strikingly, despite successfully generating dsDNA, nomad rarely achieves genomic reintegration. Instead, its newly synthesized DNA predominantly forms extrachromosomal circular DNA (ecDNA). These findings show that retrotransposon-derived DNA largely remains as ecDNA. This could prevent widespread genomic integration during spermatogenesis, potentially preserving genome stability with the presence of limited retrotransposon activity.