Enchytraeus japonensis, a recently described terrestrial oligochaete, reproduces asexually by fragmentation and subsequent regeneration. Taking notice of its high potential as a new material for regeneration study, detailed studies were undertaken on the regeneration and reproduction of E. japonensis. The full-grown body divided into 6-13 fragments that regenerated into complete individuals in 4 days, grew to full length in 10 days, and then fragmented again. Regeneration of the head and tail was epimorphic, involving blastema formation, while old segments in the regenerating fragment morphallactically transformed into the appropriate segments to retain the proper body proportions, which could be visualized by histochemistry for alkaline phosphatase. Artificially cut fragments regenerated either normally or into dicephalic monsters with biaxial heads depending on the conditions. Fragmentation could be induced by decapitation, and sexual reproduction was also found inducible in the laboratory. These findings, together with its simple metameric morphology and ease of culture and handling, suggest that E. japonensis is an excellent material for studying animal regeneration.

Enchytraeus japonensis is a small terrestrial oligochaete which primarily reproduces asexually by fragmentation and regeneration. In order to introduce a molecular approach to the study of regeneration we developed a whole-mount immunostaining procedure for the worm. Using an antibody directed against acetylated tubulin in conjunction with confocal laser-scanning microscopy, we succeeded in clarifying the three- dimensional structure of the entire nervous system in the full-grown worm and its dynamics during the fragmentation and regeneration process. In addition, we examined the expression of neurotransmitters and neuropeptides in the worm using a fluorescently-labeled antagonist and various antibodies. In particular, we found two circumferential structures in the body wall muscle of each segment that react strongly with alpha-bungarotoxin, an antagonist of nicotinic acetylcholine receptors, and detected nerve fibers just underneath these structures. During the fragmentation process, the circular body wall muscles contract near one of these circumferential structures in the middle of the segment, which causes constriction and results in fission of the body. This alpha-bungarotoxin-positive structure was designated the neuromuscular junction of the circular muscle. During the regeneration process nerve fibers grow from the remaining ventral nerve cord and gradually form networks in both the anterior and posterior regeneration buds. The growing fibers extend to the prostomium (a sensory organ) at the anterior end prior to connecting to the presumptive brain rudiment. A neural network appears around the pygidium, and this is followed by growth of the body at the posterior end. The nervous system appears to play an important role in both anterior and posterior regeneration.

The fragmenting potworm Enchytraeus japonensis (Oligochaeta, Annelida) reproduces asexually by dividing the body into several fragments that then regenerate to complete individuals in 4 -5 days. Such large-scale regeneration, however, occurs only in some invertebrates. To better our understanding of why regeneration is so limited in many animals, despite their ability to undergo embryonic development from the single cell of a fertilized egg, comparisons were made between regeneration and embryonic development of E. japonensis by using two methods: histochemistry for alkaline phosphatase (ALP) and immunohistochemistry with an antibody against acetylated tubulin that visualizes nervous system development. The analyses revealed that both ALP expression patterns and central nervous system development differ between embryogenesis and the regeneration, suggesting that regeneration is not a simple reiteration of embryogenesis but involves different regulatory mechanisms. The study provides a basis for the elucidation of mechanisms that are unique and crucial to regeneration.

To identify genes specifically activated during annelid regeneration, suppression subtractive hybridization was performed with cDNAs from regenerating and intact Enchytraeus japonensis, a terrestrial oligochaete that can regenerate a complete organism from small body fragments within 4 -5 days. Filter array screening subsequently revealed that about 38% of the forward-subtracted cDNA clones contained genes that were upregulated during regeneration. Two hundred seventy-nine of these clones were sequenced and found to contain 165 different sequences (79 known and 86 unknown). Nine clones were fully sequenced and four of these sequences were matched to known genes for glutamine synthetase, glucosidase 1, retinal protein 4, and phosphoribosylaminoimidazole carboxylase, respectively. The remaining five clones encoded an unknown open-reading frame. The expression levels of these genes were highest during blastema formation. Our present results, therefore, demonstrate the great potential of annelids as a new experimental subject for the exploration of unknown genes that play critical roles in animal regeneration.

The term ‘neoblast’ was originally coined for a particular type of cell that had been observed during annelid regeneration, but is now used to describe the pluripotent/totipotent stem cells that are indispensable for planarian regeneration. Despite having the same name, however, planarian and annelid neoblasts are morphologically and functionally distinct, and many annelid species that lack neoblasts can nonetheless substantially regenerate. To further elucidate the functions of the annelid neoblasts, a comparison was made between the regeneration patterns of two enchytraeid oligochaetes, Enchytraeus japonensis and Enchytraeus buchholzi, which possess and lack neoblasts, respectively. In E. japonensis, which can reproduce asexually by fragmentation and subsequent regeneration, neoblasts are present in all segments except for the eight anterior-most segments including the seven head-specific segments, and all body fragments containing neoblasts can regenerate a complete head and a complete tail, irrespective of the region of the body from which they were originally derived. In E. japonensis, therefore, no antero-posterior gradient of regeneration ability exists in the trunk region. However, when amputation was carried out within the head region, where neoblasts are absent, the number of regenerated segments was found to be dependent on the level of amputation along the body axis. In E. buchholzi, which reproduces only sexually and lacks neoblasts in all segments, complete heads were never regenerated and incomplete (hypomeric) heads could be regenerated only from the anterior region of the body. Such an antero-posterior gradient of regeneration ability was observed for both the anterior and posterior regeneration in the whole body of E. buchholzi. These results indicate that the presence of neoblasts correlates with the absence of an antero-posterior gradient of regeneration ability along the body axis, and suggest that the annelid neoblasts are more essential for efficient asexual reproduction than for the regeneration of missing body parts.