Supplementary Material for: Understanding the Role of the Clitellum in the Regeneration Events of the Earthworm Eudrilus eugeniae

Regeneration is a complex mechanism to restore lost or damaged body parts. In earthworms, regeneration capability varies among different species, and it is important to explore the mechanism behind the regeneration process. Interestingly, regeneration in earthworms is either dependent or independent of clitellum segments. In the present study, juvenile earthworms (Eudrilus eugeniae) were amputated at 3 different sites, namely the head, clitellum, and tail segments (at segments 10, 15, and 30, respectively), and their regeneration ability was documented using a foldscope. The amputated segments having the intact clitellum were able to heal the wounds and form the regenerative blastema. The smaller portions of the amputated segments (segments 1–10 and 1–15) without intact clitellum were unable to heal the wound, and death occurs within 12–24 h. The larger portions of the amputated segments (segments 15 and 30 to anus) without intact clitellum were able to heal the wound but lacked the regeneration capability. In control worms, alkaline phosphatase (ALP) signals were observed at the anterior tip, clitellum, and gut epithelium tissues, whereas, upon amputation, the enriched signals from the clitellum diminished, but profound signals were observed at the amputation site and regenerative blastema. Interestingly, on days 3 and 4, blastemal tips lacked ALP signals due to initiation of the differentiation process in the regeneration blastema. In summary, using a foldscope microscope, the role of the clitellum in the regeneration mechanism was indicated by ALP activity.