Title Bipolar head regeneration induced by artificial amputation in Enchytraeus japonensis (Annelida, Oligochaeta) Author(s) Kawamoto, Shishin; Yoshida-Noro, Chikako; Tochinai, Shin Journal of Experimental Zoology Part A: Comparative Experimental Biology, 303A(8), 615-627 Citation https://doi.org/10.1002/jez.a.205 Issue Date 2005-07-12 Doc URL http://hdl.handle.net/2115/559 Rights Copyright (c) 2005 Wiley-Liss, Inc. Type article (author version) File Information 2005JEZa303-615.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP JOURNAL OF EXPERIMENTAL ZOOLOGY 303A: 615–627 (2005) Bipolar Head Regeneration Induced by Artificial Amputationin Enchytraeus japonensis (Annelida, Oligochaeta) 1* 2 1 SHISHIN KAWAMOTO , CHIKAKO YOSHIDA-NORO , AND SHIN TOCHINAI 1Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan 2Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan ABSTRACT The Enchytraeida Oligochaeta Enchytraeus japonensis propagates asexually by spontaneous autotomy. Normally, each of the 5–10 fragments derived from a single worm regenerates a head anteriorly and a tail posteriorly. Occasionally, however, a head is formed posteriorly in addition to the normal anterior head, resulting in a bipolar worm. This phenomenon prompted us to conduct a series of experiments to clarify how the head and the tail are determined during regeneration in this species. The results showed that (1) bipolar head regeneration occurred only after artificial amputation, and not by spontaneous autotomy, (2) anesthesia before amputation raised the frequency of bipolar head regeneration, and (3) an extraordinarily high proportion of artificially amputated head fragments regenerated posterior heads. Close microscopic observation of body segments showed that each trunk segment has one specific autotomic position, while the head segments anterior to the VIIth segment do not. Only the most posterior segment VII in the head has an autotomic position. Examination just after amputation found that the artificial cutting plane did not correspond to the normal autotomic position in most cases. As time passed, however, the proportion of worms whose cutting planes corresponded to the autotomic position increased. It was suspected that the fragments autotomized after the artificial amputation (corrective autotomy). This post-amputation autotomy was probably inhibited by anesthesia. The rate at which amputated fragments did not autotomize corresponded roughly to the rate of bipolar regeneration. It was hypothesized then that the head regenerated posteriorly if a fragment was not amputated at the precise autotomic position from which it regenerated without succeeding in corrective autotomy. J. Exp.Zool.303A: 615–627 ,2005. The Enchytraeida Oligochaeta Enchytraeus japonensis reproduces asexually only by spontaneous autotomy (fragmentation) in a mass culture condition. Among several hundred species of Enchytraeidae, only eight, including E. japonensis, have been reported to reproduce asexually by regeneration after fragmentation (Schmelt et al., 2000). When E. japonensis reaches 10–15mm in length, the worm spontaneously autotomizes into 5–10 fragments, each of which is comprised of 5–10 segments. Each fragment regenerates a head (prostomium to segment VII) anteriorly and a tail posteriorly, in about 4 days, at 24ºC (Myohara et al., ’99). Regeneration of the present material is possible even from a fragment comprising only two trunk segments (unpublished). Moreover, it is possible to induce sexual reproduction by changing the culture condition. This enables research into embryonic development complementary to the study of regeneration (Myohara, 2004). Compared to hydras and planarians, which have been widely used in research into the regeneration of invertebrates, Annelida Oligochaeta is a more advanced triploblastic animal, as it has a well-developed ladder-like central nervous system, a closed blood-vascular system, a highly developed endocrine system, a coelom, and segmentation. Although many classical regeneration studies in Annelidae were conducted using Polychaetae and Oligochaetae (Christensen, ’64; Herlant-Meewis, ’64), for a long time thereafter, the field was barely advanced. Recently, though, some pioneering molecular biological studies have been reported on asexual regeneration in the aquatic Oligochaeta Pristina leidyi, which reproduces through paratomy (Bely and Wray, 2001), and on the early development of Annelidae (Seaver and Shankland, 2001; Seaver et al., 2001; Prud’homme et al., 2003). To date, however, the information is still fragmentary. To understand the formation and regulation of an animal’s body plan, it is important to know whether or not the same mechanism determines the body axis in both embryogenesis and regeneration. How is an autotomized plane destined to form an anterior head vs. a posterior tail in E. japonensis? Although all spontaneously autotomic fragments regenerate a head anteriorly and a tail posteriorly without exception, artificially amputated fragments occasionally regenerate a head posteriorly, resulting in bipolar head regeneration (Myohara et al., ’99). The experiments in the present study are designed to clarify how the head is determined during the process of regeneration, by focusing on the bipolar head regeneration phenomenon. Our results suggest that the posterior head regenerates when regeneration occurs from a position other than the normal autotomy plane. Among regeneration studies of segmented animals, this is the first report to show that the position of the amputation within a segment is closely related to the type of structure formed. This phenomenon will serve as an interesting model for analyzing the axis determination in Annelidae. Grant sponsor: Hokkaido Foundation for the Promotion of Scientific and Industrial Technology, Japan; Grant number: H12-jounior scientist 059; Grant sponsor: Japan Space Forum, Ground- Based Research Announcement for Space Utilization Research; Grant number: Phase IB research for germinating 25; Grant sponsor: The Japanese Ministry of Education, Culture, Sports, Science and Technology, 21st Century Center of Excellence (COE) grant for the ‘‘Neo-Science of Natural History’’ Program (Leader: Hisatake Okada) at Hokkaido University; Grant number: H15-RA6; Grant sponsor: The Japanese Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research; Grant number: 09878168; Grant sponsor: The Japanese Ministry of Agriculture, Forestry and Fisheries; Grant number: trust research 15-3150. Correspondence to: Shishin Kawamoto, kita-10 nishi-8, Kita-ku, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. E-mail: [email protected] Received 29 March 2005; Accepted 20 May 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jez.a.205. MATERIALS AND METHODS Animals and cultures Throughout the experiment, we used roughly 400–500 E. japonensis, an Enchytraeida worm cultured in our laboratory since 1995 as a closed colony. The cultures were kept at 24ºC in 150-mm disposable Petri dishes (Falcon), each coated with 100 ml of 0.8% agar (Nacalai-Tesque) in Steinberg’s solution designed for anuran larvae (58 mM NaCl, 0.67 mM KCl, 0.34 mM Ca(NO3)2 4H2O, 0.85 mM MgSO47H2O, 4.6 mM Tris–HCl, pH 7.4) supplemented with 0.1% insoluble calcium carbonate salt. The worms had been fed powdered a rolled oats (Quaker Oats) every other day. The worms used for experimentation were placed in 60-mm disposable Petri dishes, each coated with 15 ml of 0.8% agar in Steinberg’s solution. Fragmentation Decapitation To induce autotomy, the head was removed from a worm placed on a glass slide by cutting at intersegment VII/VIII with a disposable surgical blade (No. 19, Futaba). Because autotomy usually occurred within 24 hr after this operation (Inomata et al., 2000), the fragments were collected 24 hr after decapitation, and this collection time marked the beginning of day 0. Electrical stimulus Autotomy by electrical stimulus (Christensen, ’64) was achieved by subjecting a worm to a direct current of 40 V (transformer Type RS-5A; Rikoh) for 0.5 sec several times on an agar plate dissolved with deionized water (Millipore). The fragments were transferred to experimental culture conditions within 10 min. Amputation The worms were amputated with a disposable surgical blade on a glass slide. The obtained fragments were placed on an agar plate or in deionized water. Some of the worms were anesthetized by a diluted L-menthol saturated solution (40%) for 10 min before amputation. The anesthetized worms transferred to agar plates or water awoke in 10–30 min. Histology Hematoxylin-eosin staining The worms and the fragments were fixed in Bouin’s fluid for 4 hr, embedded in paraffin, and sliced into 5-µm-thick sections. The sections were stained with hematoxylin and eosin using a routine method (Conn, ’77). Whole-mount paracarmine staining Worms were fixed in Bouin’s fluid for 4 hr, washed several times in 70% ethanol, and stained with paracarmine (1 g carminic acid, 0.5 g aluminum chloride, 4 g potassium chloride, 70% ethanol to 100 ml) for 15 min. After washing in 70% ethanol followed by dehydration in a graded series of alcohol, the worms were mounted in a mixture of benzylalcohol and benzylbenzoate (1:2). Whole-mount α-bungarotoxinstaining The worms and fragments were fixed in 10% formalin