Nervous system dynamics during fragmentation and regeneration in Enchytraeus japonensis (Oligochaeta, Annelida)

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.