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Investigating the regenerative potential of Tharyx sp. (Polychaeta: Cirratulidae) 


Harriet Lawrence 2015

Abstract
Regeneration has emerged and been lost repeatedly throughout the Animalia. Annelid polychaetes provide an ideal subject for the experimental investigation of regeneration but the capacity for regeneration in many families is yet to be described. This study describes the anterior and posterior regenerative stages of Tharyx sp. (Polychaeta: Cirratulidae) from the UQ aquaria in two treatments of amputation and draws comparisons with other cirratulids. Tharyx sp. successfully regenerated both anteriorly and posteriorly in individuals from both treatments. Tharyx sp. was found to have 4 stages of anterior regeneration and 2 stages of posterior regeneration. These results are supported by similar conclusions from other cirratulid studies. Other developmental variables which affect the regenerative success of the cirratulids are also discussed.

Introduction

Regeneration occurs in many animal phyla as both a response to injury and a method of asexual reproduction. Regeneration is thought to have arisen early in the Animalia, possibly in association with multicellularity (Bely 2010). Later specialisation of organ systems and increasingly adapted bauplans led to the secondary loss of regeneration as a result of increased cellular complexity (Zoran 2010). 

Annelids are an ideal study group for investigating the mechanisms and limits of regeneration as they are metameric; making regeneration of structurally identical segments very simple (Zoran 2010). Regeneration is thought to be an ancestral trait of the annelids which has been secondarily lost to a variable extent throughout the phyla (Zoran 2010). This variation in regenerative capacity can include variation in the biological structures which can be regenerated, the time taken for regeneration to occur, the number of segments which can be regenerated and the specie’s survival rate following amputation (Bely 2006).

Annelid polychaete worms are among the most abundant marine invertebrates; possibly as a result of highly successful asexual reproduction demonstrated in many polychaete families. Despite this, few studies have been undertaken to explore the capacity of regeneration potential within the Polycheata; which could provide useful ecological, developmental and evolutionary insights to aid in untangling the evolutionary history of both regeneration and the polychaete worms (Bely 2010) (Fauchald and Rouse 2005). 

The Cirratulidae are an understudied family of marine polychaetes which are thought to be highly abundant and diverse (Weidhase, Bleidorn and Helm 2014). Most species are infaunal and feed on sediment using grooved branchiae (Glasby 2000). The bipalpate Tharyx sp. (Polychaeta: Cirratulidae) (Figure 1) used in this study are observed soft-sediment deposit feeders (Figure 2).  Therefore they are expected to possess regenerative capabilities due to the risk associated with browsing benthic predators (Lindsay, Wethey and Woodin 1995). This study aims to investigate and compare the anterior and posterior regenerative potential of Tharyx sp. when individuals have been halved or quartered under experimental conditions.
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Figure 1
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Figure 2

Materials and Methods

Pilot Study
A pilot experiment was run in the first research week to investigate if Tharyx sp. would demonstrate any regenerative potential after 1 week. The pilot was conducted using 4 cirratulids taken from the abundant population within the UQ aquaria. Tharyx sp. was identified by the recognition of identical morphology, colouration and similar body size (Glasby 2000). All exterior sediment was removed from the worms using tweezers under a stereoscopic microscope to ensure that all individuals had no access to food. The body length of each Tharyx sp. was measured using 80mm and 10μm scales and averaged over 3 measurements before they were amputated. This measurement was used to guide even amputation along the body in order to minimize variation in regenerative capabilities caused by differing segment number among the amputated sections.   There were 3 separate treatments used in the pilot stage: halving, quartering and the amputation of all branchiae and palps to a length of <2mm using FST Vannas Spring micro-scissors with a 2.5mm cutting edge. A single individual underwent each treatment except in the case of halving where two individuals were used. Following treatment, the sections of each individual were kept together in petri dishes with 5ml of saltwater in each dish. Any evidence of regeneration was recorded a week later using regenerative morphology and total section length.

The pilot study showed distinct morphological evidence of regeneration in every amputated section but measuring the length of each section proved too inaccurate for comparative data due to the contractile nature of cirratulids. Some of the sections which had been kept together in the same petri dish had become entangled with one another and so separate storage of all sections was employed in the subsequent regeneration experiment. Individual P2, which had undergone palp/branchiae amputation, showed no visible morphological signs of regeneration and it was impossible to differentiate between moving filaments in order to measure any increase in length. As a result of this the investigation into palp/branchiae regeneration was abandoned.

Regeneration Experiment
Following the success of the pilot study, the experiment was repeated using 15 Tharyx sp. from the UQ aquaria which were chosen using a random timed search and identified using the same methodology as in the pilot study. The exterior sediment was removed from each individual and their body lengths recorded. 5 cirratulids underwent each of the 2 treatments. In treatment 1, individuals were halved to form 2 sections and in treatment 2, individuals were quartered to form 4 sections. A further 5 non-amputated individuals acted as a control. Each amputated cirratulid section and the 5 control individuals were kept separate within labelled 6-dish petri trays in order to avoid entanglement of individuals. Each petri dish contained 7.5ml of saltwater and no sediment. The posterior and anterior regeneration process was recorded through weekly photo-documentation of all of the cirratulids using a stereoscopic microscope and a Dino-Lite microscopic camera. Each week 5.1ml of seawater was changed in every petri dish to avoid suffocating the specimens. The experiment was run for 2 weeks alongside the pilot study.

Results

Throughout the results and discussion each amputated section of Tharyx sp. will be referred to as a single regenerating individual.
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Figure 3
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Figure 4
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Figure 5
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Figure 6

Discussion
Tharyx sp. were found to demonstrate the potential for asexual reproduction via architomy because of their ability to regenerate post-amputation (Weidhase, Bleidorn and Helm 2014). Individuals produced by treatment 1 displayed full regenerative potential of both the anterior and posterior within 2 weeks. The rate of anterior regeneration and the morphological stages which were seen during the study are supported the results of a similar study on Cirratulus cf. cirratus (Weidhase, Bleidorn and Helm 2014). In Weidhase’s study, though all of the cephalic features were present after 14 days, the head did not reach its original size until 1 month after decapitation and the anterior musculature and nervous systems were shown to only be fully developed after 22 days (Weidhase, Bleidorn and Helm 2014). Therefore it is possible that if the study were run over a month, further anterior regeneration would be seen than has been demonstrated in these two weeks.

Posterior regeneration is well documented in many polychaete families with only a few families being incapable of posterior regeneration (Bely, 2006). Posterior regeneration has been shown to be more successful than anterior regeneration in cirratulid Dorvillea spp. as a result of the higher energy requirement for anterior regeneration (Akesson and Rice 1992). Anterior regeneration may have evolved to be so successful in Tharyx sp. because of its feeding branchiae being located anteriorly and so it can be assumed that there is a greater risk and consequence associated with anterior injury than in posteriorly feeding families; like the sabellids (Murray et al. 2013).

Individuals produced by treatment 2 displayed regenerative potential up to stage 3 anteriorly (Figure 5) and up to stage 1 posteriorly (Figure 6) within 2 weeks. Complete regeneration of the anterior and posterior was seen within 3 weeks in the pilot group and so the limited reproductive potential seen within the second treatment group could be a result of the limited time scale of the experiment. It is therefore possible that the individuals from treatment 2 may have achieved full regeneration by week 3. When naturally autotomising as a means of asexual reproduction, only a few cirratulid species have demonstrated multiple fragmentation at once and instead equal binary fission has been shown to be more successful (Gibson 1976). This preference for binary fission may be due to the lower energy requirement for the regeneration of a single end and fewer segments which could be reflected in the greater rate of regeneration seen in the individuals from treatment 1 (Gibson 1976). There was a 60% survival rate in both treatment groups and an 80% survival rate within the control group. This suggests that there may be some mortality risk associated with amputation and regeneration. The regeneration success of cirratulids has been shown to be limited by starvation and so these results for Tharyx sp. must be interpreted in mind of a potential starvation limit (Akesson and Rice 1992). 

There were more distinct morphological regenerative stages seen during anterior regeneration than posterior regeneration during this study. This may be a result of the increased complexity of anterior tissue structures in comparison to posterior tissues (Zoran 2010) and the subsequent re-organisation of post-cephalic segments following anterior regeneration which is unnecessary in metameric posterior segments (Hyman 1940). 

Both anterior and posterior regeneration was seen to act unevenly, extending the tissue from the ventral side first (Figures 5C and 6B). This developmental pattern was also observed in cirratulids by Peterson (1999) and is thought to be due to the expansion of the ventrally located nerve cord during the first 12 days of regeneration which is followed by the expansion of the dorsal longitudinal muscle plate (Weidhase, Bleidorn and Helm 2014).
Unusual regeneration morphology was seen in individuals from both treatment groups (Figure 4: Table 2). Mutation during regeneration of cirratulids has been previously recorded and is an outcome of rapid replication of cells in deleterious individuals (Peterson 1999).

Anterior segmentation and regeneration potential have been shown by other cirratulid studies to be closely associated. Successful regeneration of the anterior segments has been shown to be subject to the position of amputation and the number of segments remaining in the post-amputated individual (Hyman 1940). In a study of Dodecaceria caulleryi, the regeneration of anterior segments was also limited by the number of regenerated segments lost, while the potential for posterior regeneration appeared limitless (Gibson and Clark, 1976). To explore this relationship in Tharyx sp. future experiments should observe the variation in regenerative capability of amputated individuals originating from different amputation sites along the axial length of the body as well as amputated individuals containing different numbers of segments.

References
Akesson, B. and Rice, S. A. 1992. Two new Dorvillea species (Polychaeta: Dorvilleidae) with obligate asexual reproduction. Zoologica Scripta 21:4, 351-362

Bely, A. E. 2006. Distribution of segment regeneration ability in the Annelida. Integrative and Comparative Biology 46:4, 508-518

Bely, A. E. 2010. Evolutionary loss of animal regeneration: pattern and process. Integrative and Comparative Biology 50:4, 515-527

Fauchald, K. and Rouse, G. 2005. Polychaete systematics: Past and present. Zoological Scripta 26:2, 71-138

Glasby, C.J. 2000. Family Cirratulidae: 208-211. In Beesley, P.L., Ross, G.J.B. and Glasby, C.J. (eds) Polychaetes & Allies: The Southern Synthesis. Fauna of Australia. ‘4A: Polychaeta, Myzostomida, Pogonophora, Echiura, Sipuncula’ CSIRO Publishing: Melbourne xii 465pp

Gibson, P. H. 1977. Reproduction in the cirratulid polychaetes Dodecaceria concharum and D. pulchra. Journal of Zoology 182:1, 89-102

Gibson, P. H. and Clark, R. B. 1976. Reproduction of Dodecaceria caulleryi (Polychaeta: Cirratulidae). Journal of the Marine Biology Association of the United Kingdom 56:3, 649-674

Hyman, L. H. 1940. Aspects of regeneration in annelids. The American Naturalist 74:755, 513-527

Lindsay, S. M., Wethey, D. S. and Woodin, S. A. 1995. Modelling interactions of browsing predation, infaunal activity, and recruitment in marine soft-sediment habitats. The American Naturalist 148:4, 684-699

Murray, J. M., Watson, G. J., Licciano, M. and Bentley, G. 2013. Regeneration as a novel method to culture marine ornamental sabellids. Aquaculture 410:129-137

Peterson, M. E. 1999. Reproduction and development in Cirratulidae (Annelida: Polychaeta). Hydrobiologia 402:107-128

Weidhase, M., Bleidorn, C. and Helm, C. 2014. Structure and anterior regeneration of musculature and nervous system in Cirratulus cf. cirratus (Cirratulidae, Annelida). Journal of Morphology 275:12, 1418-1430

Zoran, M. J. 2010. Regeneration in Annelids. eLS