Morphology Characterization of Anterior Regeneration in Diopatra claparedii

Summary

This protocol provides a focused framework to characterize the anterior regeneration process in Diopatra claparedii, serving as a critical step toward future regenerative studies.

Abstract

Polychaetes possess the ability to regenerate anterior and posterior segments following injury or self-amputation, offering a tractable system for studying wound repair and tissue patterning. To support comparative and mechanistic studies in annelid regeneration, this study presents a protocol for inducing and characterizing anterior regeneration in Diopatra claparedii (D. claparedii). The protocol outlines steps for specimen handling, immobilisation, and precise amputation, followed by scheduled observational time points to document morphological progression throughout regeneration. D. claparedii were gently removed from their tubes using a blunt-ended stick and immobilised in 4% magnesium chloride to induce relaxation. Anterior amputation was performed at the 4^th chaetiger under a stereomicroscope, and the regeneration process was monitored on days 1, 6, 15, 30, and 60 post-amputation. The procedure enables consistent induction of anterior regeneration and captures key morphological features across early wound closure, blastema formation, and segment re-establishment. By providing a reproducible workflow and morphological reference for D. claparedii, this protocol offers a practical foundation that supports future investigations that aim to relate morphological observations to underlying cellular or molecular processes in annelid regeneration.

Introduction

Polychaetes comprise nearly 65% of all described annelid species, making them one of the most diverse groups of marine invertebrates¹. A recent updated checklist of polychaete species (Annelida) recorded a total of 57 species belonging to 47 genera across 30 families from Malaysian coastal waters, with the majority of species (53 species) reported from Peninsular Malaysia². Among these, Diopatra claparedii (D. claparedii) Grube, 1878, or locally known as ruat sarung, is one of the commercially exploited polychaete species in Malaysia. It is predominantly found along mudflats on the west coast o....

Protocol

Ethics Statement

This study has been approved by the Universiti Malaysia Terengganu Research Ethics Committee under approval number UMT/JKEPH/2026/173

1. Collection and Habituation of D. claparedii

1. Locate D. claparedii in the mudflat area of the estuarine intertidal zone (medium tide level with medium fine sand), which serves as its natural habitat.
2. Excavate the upper portion of the polychaete tubes by gently digging into the sediment with a shovel to avoid damaging the tubes.
3. Confirm the presence of the worm inside each tube by gently pinching th....

Results

The regeneration process in D. claparedii was monitored continuously until complete head regeneration. The overall regeneration pattern was comparable to that reported for D. neapolitana (Figure 2). Wound closure at the amputation site was completed within 1 day post-amputation (p.a.). By day 6 p.a., a clearly defined blastema was observed in surviving individuals, indicating active tissue regeneration. At this stage, approximately 90% of wo.......

Discussion

Annelids, comprising segmented worms inhabiting marine, freshwater, and terrestrial environments, possess remarkable regenerative abilities across multiple levels of biological organization. These range from cellular repair and germ cell renewal to structural regrowth and, in some cases, complete body regeneration from small body fragments^8,10. The present protocol focuses on the anterior regeneration of D. claparedii.

In poly.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Artificial Seawater	Instant Ocean
Digital Handheld Salinity Hydrometer	Scionix
MgCl2·6H2O	Sigma	M2670
Scapel
Stereomicroscope	Leica Microsystem
TeraBits Complete Fish Food	Tetra
Tweezer