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 of Peninsular Malaysia, including sites such as Jeram Beach, Selangor^2,3.

D. claparedii belongs to the family Onuphidae and is a sedentary, tube-dwelling polychaete renowned for its ability to regenerate both anterior and posterior body segments. This includes components of the central and peripheral nervous systems⁴. Its well-defined segmental body plan and pronounced regenerative capacity make D. claparedii a promising and tractable invertebrate model for investigating tissue regeneration in relatively complex biological systems. The selection of D. claparedii as the model organism for regeneration studies is due to its ecological relevance, regional significance, and the current gaps in scientific knowledge surrounding its regenerative capacity. This species is commercially harvested and widely used as fishing bait in Malaysia, indicating that it is well known to local communities and not considered rare^2,3. Thus, investigating regeneration in D. claparedii holds practical and economical value, as the findings may help disseminate scientific knowledge to local stakeholders while potentially informing strategies to support population sustainability through improved understanding of regenerative mechanisms.

In addition, D. claparedii exhibits a specific geographic distribution across Southeast Asia, with particularly high representation in Malaysian coastal ecosystems^2,3. However, most regeneration studies on polychaetes and other annelids have focused on species from the Americas or other non-Asian regions^5,6. For example, the well-studied Diopatra neapolitana (D. neapolitana), frequently used as a regenerative biomarker species, has a broad distribution spanning the Red Sea, Indian Ocean, Mediterranean Sea, and Atlantic Ocean⁷. Consequently, regeneration research on annelids native to Southeast Asia, including D. claparedii, remains limited.

Previous studies on polychaetes have shown distinct and reproducible stages of regeneration, including wound closure, blastema formation, and segmental tissue differentiation^7,8. Although molecular-level studies on D. claparedii have been previously reported, investigations specifically focusing on regeneration are scarce. Moreover, existing work has largely emphasized molecular characterization rather than experimental regeneration protocols. Moreover, D. claparedii displays both dedifferentiation and redifferentiation processes, reflecting mechanisms that are more comparable to those in higher organisms⁹. This complexity in regenerative response highlights the growing recognition of polychaetes as valuable intermediate models, effectively bridging the gap between simple invertebrates and complex vertebrates in regeneration studies.

The present study addresses this gap by establishing and optimising a regeneration protocol for D. claparedii, providing a robust framework for future functional, developmental, and molecular investigations of regeneration in this regionally important polychaete species. Specifically, this protocol offers a reliable approach for examining the sequential stages and underlying mechanisms of regeneration in polychaetes. This enables systematic observation and characterization of key regenerative processes, including wound healing, blastema formation, and segmentation.

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 the tube and checking for movement or resistance.
4. Select only specimens with a minimum relaxed body length of 5 cm.
   NOTE: Choose polychaetes by body length to reduce handling damage and avoid fragmentation during weighing.
5. Collect sediment using a shovel from the same locations to maintain environmental consistency in the laboratory.
6. Transport the collected specimens to the laboratory in portable cooler containers at room temperature. Cover the tubes with a seawater-moistened towel to maintain humidity and reduce stress during transit.
7. In the laboratory, manually separate debris from the sediment, then air-dry the material under direct sunlight until it is completely dry and free of residual moisture.
8. Remove or clean out any other organisms (e.g., small crabs, seashells, or foreign substances), to ensure the sediment is free from contaminants.
9. Rinse the polychaete tubes with artificial seawater (ASW) to remove debris and any living organisms attached to the surface.
   NOTE: To make ASW, mix 1/4 cup of Instant Ocean Sea Salt in 1.9 L dechlorinated purified water.
10. Prepare aquaria using plastic aquarium, with sediment from the original site and fill with artificial ASW adjusted to 23-25 ppt salinity (Figure 1). Maintain the water temperature consistently at 28 °C throughout the habituation period.
    NOTE: The volume of ASW used was 58 L, determined based on the dimensions of the aquarium: 61.5 cm (L) x 32 cm (W) x 33.5 cm (H).
11. Transfer D. claparedii into cylindrical plastic containers (7 cm in diameter, 15 cm in height) filled halfway with sediment, at a density of one polychaete per container.
12. Submerge the containers fully in an aquarium with aerated ASW. Allow the polychaetes to burrow and re-establish themselves.
13. Feed the polychaetes with sinking-type fish food at a frequency of 2-3 times per week, to ensure continuous nourishment throughout the habituation period. Remove excess food regularly to prevent accumulation, fungal growth, and contamination.
14. Habituation was assessed through behavioural observations, with active exploration (body movement) and food-searching behaviour (response to stimuli) indicating a return to normal physiological condition.
    NOTE: Allow a habituation period of two weeks before commencing any experimental procedures, enabling the worms to recover from any stress or accidental amputation incurred during collection.

2. Regeneration

1. Select chaetiger 4 as the standard amputation site to ensure consistent and successful regeneration outcomes^7,9.
   NOTE: Selection of amputation site at 4^th is based on preliminary findings. Use segment 10 only if required, as it regenerates more slowly.
2. Prepare an anaesthetic solution with 4% (weight/volume) magnesium chloride hexahydrate (MgCl₂·6H₂O) in ASW (4 g MgCl₂·6H₂O in 1 L ASW).
3. Push the polychaete gently out of its tube using a blunt-ended stick inserted into the anterior opening.
   NOTE: Avoid damaging the body during removal.
4. Then immerse the polychaete in the anaesthetic solution until it is fully relaxed and immobilised (approximately 15 min).
5. Amputate at chaetiger 4 using a sterilized scalpel under a stereomicroscope, ensuring precision and minimal tissue damage (Figure 1).
   NOTE: Always wear appropriate personal protective equipment and care to avoid cuts while handling with a scalpel.
6. Return the posterior portion of the worm to its original tube using forceps, inserting the anterior end first to facilitate natural re-burrowing.
7. Following this procedure, monitor the worms daily for behavioural changes and physical condition, including signs of infection, tissue decay, or mortality.
   NOTE: Individuals exhibiting abnormal behaviour, visible necrosis, or progressive deterioration were considered unsuccessful in recovery and were excluded from further analysis.
8. Withhold food at this stage until regeneration progresses, as the mouth region is absent due to anterior amputation. Resume normal feeding behaviour once the mouth has developed, approximately 60 days post-amputation (p.a).
   NOTE: Maintain the same environmental conditions (water salinity range 23-25 ppt and the water temperature consistently at 28 °C) throughout the regeneration process.
9. Capture the images of successfully regenerated polychaetes using a stereomicroscope on certain days (1, 6, 15, 30 and 60) p.a.
10. Complete regeneration monitoring after 60 p.a.

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