Chandra, V.; Tseng, S. E.; Kann, A. P.; Bolanos, D. M.; Srivastava, M.

Acoel worms belong to an enigmatic and understudied animal lineage (Phylum Xenacoelomorpha). Sparse taxonomic and histological work suggests that these worms exhibit a diversity of reproductive anatomies and likely also a corresponding diversity in reproductive behavior. However, few acoels have been studied in detail. Here, we study the reproductive life history of the three-banded panther worm Hofstenia miamia, an acoel that is emerging as a new lab-tractable model system for regenerative biology. Using molecular labeling and histology, we describe H. miamia's reproductive organs, identifying structures previously unknown in acoels. Following a cohort of worms from zygote to adulthood, we quantify the developmental dynamics of their reproductive organs, and find that these organs are assembled step-wise, at increasing body sizes. Studying the dynamics of organ growth during regeneration and de-growth in starving adult worms, we show that reproductive organs follow similar growth rules in these contexts, suggesting that their development and maintenance is regulated by a size-dependent program. Finally, we study egg-laying behavior, finding that H. miamia lay their eggs through their mouths after loading them into their pharynges. Experimentally varying the worms' environment reveals that the worms make active decisions about when and where to lay their eggs. In summary, our work establishes foundational knowledge to enable the experimental study of reproductive anatomy, physiology, and behavior in acoels.