Comparative analysis of the survival and regeneration potential of juvenile and matured earthworm, Eudrilus eugeniae, upon in vivo and in vitro maintenance.

Eudrilus eugeniae is a clitellum-dependent earthworm that requires intact clitellum segments for its survival and regeneration. The present study aims to interconnect the survival and regeneration ability that varies between in vivo and in vitro maintenance upon different sites of amputation. The amputated portion of the worm that possesses intact clitellum (13th-18th segments) survived and had the potential to regenerate, whereas worms with partial or without clitellum segments only survived and were unable to regenerate. Besides segment length and clitellum segments, clitellum factors also determined the survival, blastemal initiation and differentiation potential. The survivability and regeneration potential of worms were augmented upon in vitro maintenance. Notably, the amputated segments (1st-10th segments) and posterior segments of similar length, which usually die within the 4th day in vivo, survived for more than 60 days in vitro but lacked the regeneration ability. On the other hand, the amputated posterior segments (30th to 37th segments) from juvenile worms, maintained in in vitro condition, survived and initiated blastema with multiple buds but lacked the ability to regenerate. Interestingly, the equal half of adult worm blastema that is maintained in in vitro conditions were able to form the blastema-like structure with the help of a unique stick. The anterior blastema failed to retain the regenerative structure but the posterior portion of the amputated blastema, which is also associated with a small portion of the body segment, showed the ability to retain the regenerative structure. Our results conclude that the survivability is enhanced upon in vitro maintenance and this condition favours the adult dedifferentiated blastemal and stem cell-enriched juvenile posterior segments to form a regenerative blastema.