This protocol provides a focused framework to characterize the anterior regeneration process in Diopatra claparedii, serving as a critical step toward future regenerative studies.
Method Article
This protocol provides a focused framework to characterize the anterior regeneration process in Diopatra claparedii, serving as a critical step toward future regenerative studies.
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 4th 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.
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, Selangor2,3.
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 rare2,3. 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 ecosystems2,3. However, most regeneration studies on polychaetes and other annelids have focused on species from the Americas or other non-Asian regions5,6. 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 differentiation7,8. 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.
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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
2. Regeneration
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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...
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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 fragments8,10. The present protocol focuses on the anterior regeneration of D. claparedii.
In poly...
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The authors declare no competing interests.
The research work was supported by the TAPE Talent and Publication Enhancement Research Grant (55269) awarded to SZA.
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| 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 |
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