Coelomic Transport and Clearance of Durable Foreign Bodies by Starfish (Asterias Rubens)

University of Southern Denmark

Coelomic transport and clearance of durable foreign bodies by starfish (Asterias rubens)

Olsen, Trine Bottos; Christensen, Frederik Ekholm Gaardsted; Lundgreen, K.; Dunn, P. H.; Levitis, D. A.

Published in: Biological Bulletin

DOI: 10.1086/BBLv228n2p156

Publication date: 2015

Document version: Final published version

Citation for pulished version (APA): Olsen, T. B., Christensen, F. E. G., Lundgreen, K., Dunn, P. H., & Levitis, D. A. (2015). Coelomic transport and clearance of durable foreign bodies by starfish (Asterias rubens). Biological Bulletin, 228(2), 156-162. https://doi.org/10.1086/BBLv228n2p156

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Coelomic Transport and Clearance of Durable Foreign Bodies by Starﬁsh (Asterias rubens)

TRINE BOTTOS OLSEN1, FREDERIK EKHOLM GAARDSTED CHRISTENSEN1, KIM LUNDGREEN1,2, PAUL H. DUNN1,2, AND DANIEL A. LEVITIS1,2* 1Department of Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark; and 2Max-Planck Odense Center on the Biodemography of Aging, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

Abstract. Echinoderms have excellent healing and re- through localized healing or the removal of foreign bodies. generation abilities, but little is known about how they deal Invertebrate immune responses have been shown to be with the related challenge of durable foreign bodies that dependent on the size of the foreign object (Leclerc, 1996). become lodged within their bodies. Here we report a novel Microorganisms are typically eliminated through cellular mechanism for foreign body elimination in starfish. When immune responses involving phagocytosis (Go¨tz, 1986). injected into the arm of a starfish, passive integrated tran- However, when larger objects become lodged in the tissues, sponder tags and magnets of similar dimensions are elimi- hollow organs, or body cavity of the animal, they are nated at a rate approximating 10% per day. These objects typically encapsulated by leukocytes (Go¨tz, 1986; Schmid- are forcefully ejected through the body wall at the distal tip Hempel, 1994) before being eliminated (a process called of an arm. Ultrasound images reveal that foreign bodies are “clearance”). The encapsulation of foreign bodies was first moved within the body cavity, and tracking of magnets investigated at the end of the nineteenth century using injected into starfish suggests that the movements are hap- starfish (Metchnikoff, 1883). The degree to which encapsu- hazard rather than directed. Constrictions of the body wall lation and subsequent elimination of the intruder succeeds near the foreign object are the likely mechanism for this in some species has been shown to depend on the physical transport process. Open questions include the ecological nature of the foreign object (e.g., wettability and charge, relevance of this behavior, why clearance occurs through Lackie, 1983) as well as its antigenic properties (Ghiradella, the distal tips of the arms, the neurological and muscular 1965). control of this behavior, what other animals use this mech- In human and veterinary medicine, problems involving em- anism, and the range of objects starfish can eliminate in this bedded (Cartee and Rumph, 1984) or ingested (Cheng and way. Tam, 1999) durable foreign objects (e.g., splinters, glass shards, thorns, spines) are common. If not eliminated, such Introduction foreign bodies can produce a wide variety of long-lasting The regenerative abilities of echinoderms (Hyman, 1955; problems, including internal injuries, chronic inflammation, Carneveli, 2006), and starfish in particular (Anderson, 1965; impeded movement, and increased risk of infection. Starfish, Mladenov et al., 1989; Fan et al., 2011), are well docu- as common benthic predators, are likely to similarly acquire mented, including regrowth of whole limbs and major or- potentially harmful biotic objects and rock shards. The ejection gans. Regeneration, however, is slow and expensive (Law- of those foreign bodies that cannot be broken down has been rence and Larrain, 1994; Ramsay et al., 2001; Maginnis, studied in vertebrates and can involve the intestines or the 2006); many breaks in the body wall can be better addressed bladder—organs that already actively move wastes out of the body (Chisholm and Hubert, 1985; Tracy et al., 2011). While encapsulation of foreign bodies has been studied widely, we Received 3 November 2014; accepted 23 January 2015. * To whom correspondence should be addressed. E-mail: levitis@ can find only passing reference to clearance of durable objects biology.sdu.dk in invertebrates (Porchet-Hennere´ et al., 1987).

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Our investigation began with a desire to mark the starﬁsh location on the animal where the tag came out. Demo-

Asterias rubens (Linnaeus), with passive integrated tran- graphic hazard rates (hx, a standard measure of mortality sponder tags (PIT-tags), which would have allowed auto- risk over age, deﬁned as minus the slope of the log number mated in situ identiﬁcation of individuals (Miller and Sadro, of surviving tags over time) were calculated for tag loss 2003). This technique is relatively successful in sea urchins each day using standard life-table methods (Preston et al.,

(Hagen, 1996; Duggan and Miller, 2001). Marking individ- 2001). We fit three linear models of hx over time (command ual starfish in a way that is long-lasting (external vital lm in statistical package R 3.0.2 [R Core Team, 2013]). staining, Feder, 1970; visible implant elastomer, Martinez et These assumed that hazard was constant, linear, or expo- al., 2013) or allows for remote data collection (archival nential over time. We compared these models using AICc, electronic tags tied to the animal, Lamare et al., 2009) is the small-sample Akaike information criterion (in R pack- feasible, but as with previous methods, PIT-tags fail to age AICcmodavg, Mazerolle, 2011). combine these features. Rather, we witnessed a previously undescribed mechanism of foreign body elimination: ejec- Experiment 2: Tracking. To understand how these foreign tion of PIT-tags through the distal ends of the arms. In this bodies were moving with the starfish, we next injected article, we analyze data from these and additional experi- magnetic stir rods of similar dimensions (length ϭ 8 mm, ments to better characterize this mechanism. diameter ϭ 2 mm, average weight ϭ 0.082 g), using the same procedure as for the PIT-tags. These magnets were not Materials and Methods individually identifying, but they allowed us to quickly determine where in the animal the object was. This was Experimental animals and maintenance done by hanging another magnet glued to a 20-cm-long hair Common starfish (Asterias rubens) were caught by local over the starfish and observing the interaction of the two fishermen (generally as bycatch) in the waters around Ker- magnets. When directly over the internal magnet, the hang- teminde, Funen, Denmark, and transported to the University ing magnet would twist and rotate vigorously. The hanging of Southern Denmark’s Marine Biological Research Centre magnet was kept at least 2 cm from the skin of the animal, in Kerteminde. Prior to experiments, animals were kept in which preliminary tests indicated would avoid moving the 1000-l tanks supplied with unfiltered running aerated sea- internal magnet. Preliminary testing revealed this method to water in a 10 °C temperature-controlled room with a 12: be repeatable across independent observers. Twenty indi- 12-h light/dark cycle. Salinity in the tank followed natural viduals were injected in this way and divided up among 10 fluctuations of incoming water from Kerteminde Fjord and buckets of aerated, frequently changed seawater at 10 °C, stayed around 20 psu during the experimental period. Live with each bucket having one starfish larger than 90 g and blue mussels (Mytilus edulis) were available in the tanks for one smaller than 90 g. This system allowed for repeated ad libitum feeding. Individuals that were injured or visibly identification of each individual based on its bucket and unhealthy at the start of the experiment were excluded. weight. We repeatedly recorded the location of each magnet within its starfish, whether it had come out, and (when possible) the location of the exit. These data were taken Experimental design and protocol three times during the first day after injection, and once Experiment 1: Retention times. To study PIT-tag retention, daily on days 2, 5, 6, and 10. we injected 53 starfish with PIT-tags (rod-shaped with To gain further insight into how this transport occurs, we rounded edges, length ϭ 12 mm, diameter ϭ 2 mm, used our data from Exps. 1 and 2 to compare retention times weight ϭ 0.095 g) using a syringe implanter. The tag was of PIT-tag versus magnets. Starfish in these two experi- injected through the aboral surface of the body wall of the ments differed both in object type and environment, such first arm counterclockwise from the madreporite, halfway that any differences in time to loss (tested for with Cox down the arm. A PIT-tag reader (Trovan LID 500) allowed Regression in R package survival) could be due to either daily individual identification of marked starfish and a de- factor. termination of when each tag was lost. This method did not allow us to determine where within the animal the tag was Experiment 3: Focused tracking and imaging. As internal located. The mass of each individual was tracked daily by movements of magnets were too rapid to be tracked in detail lightly patting it dry and then weighing it on a digital scale. using our Exp. 2 protocols, a small number of starfish were A control group of 25 starfish was exposed to sham injec- observed in greater detail. We followed the same procedure tions for comparison and transferred to additional 1000-l as described for Exp. 2 except that experiments were done tanks with identical culture conditions. As no pattern of at 16 °C, which we expected would shorten the time to limb loss, weight change, or mortality was observed in ejection, allowing for near-continuous observation. Place- either group, we present no further analysis of these con- ment of the magnets was recorded at least once per hour trols. We recorded the timing of tag loss, and when seen, the until loss. To better understand the anatomical details of this

Figure 1. Hazard rate (hx) of PIT-tag loss appears roughly constant over time. Error bars were calculated as 95% confidence interval: 1.96 ϫ sqrt(hx/lx). Only upper confidence limit is shown. Points without confidence limits are for ages with zero observed deaths, for which confidence limits are, by this formula, zero. Figure 2. Kaplan-Meier plot of retention times in Experiments 1 (PIT-tags, solid line) and 2 (magnets, dashed line). Magnets were lost more transport, ultrasound observations were taken at intervals of quickly than were PIT-tags, but differences between experiments (in con- 5 min or less on four of these individuals with a 2002 tainers used) leave open the possibility that object type is not the cause of Panther B&K medical ultrasound scanner (Analogic Ultra- this difference. sound, Herlev, Denmark). Simultaneously, constrictions of the body wall in the vicinity of the magnets were observed Experiment 2: Tracking for signs of motion along the arm’s major axis. All injected magnets moved repeatedly within the starfish, and in none was the movement solely distal. While Results most movement was in the injected arm, four magnets were Experiment 1: Retention times at some point observed in the central disc of the starfish, and two of these later moved into a different arm. One magnet Daily hazard rate (h , the risk of loss of each tag each day, x that was retained to the end of the experiment (Day 10) conditional on its having been retained to that day) of moved between three different arms. We witnessed four PIT-tags fluctuates around 0.09 (Fig. 1). As sample size magnets being ejected, three from the injected arm (Arm #1) decreases and the uncertainty in the hazard rate increases, and one from an adjacent arm (Arm #2). The inclusion of individual measurements wander further from 0.09. How- bucket-ID and individual size as random effects had no ever, using AICc, a model that assumes a constant hazard influence on preliminary statistical models of retention time rate is somewhat preferred (Table 1) to those that assume a and therefore were not further considered. linear or exponential dependence of hx on time. No time dependence is visually apparent. Comparing Experiments 1 and 2 All ejected tags but two were found, undamaged, in the bottom of the tank at the end of the experiment. The We observed a much earlier loss of magnets than of remaining two tags may have been lost in a drain hole. PIT-tags (Fig. 2). On the first day, 70% of the magnets were Following injection, glands of the pyloric caecum often lost, compared to a little more than 20% of the PIT-tags. briefly protruded from the entry wound, indicating that the Only one magnet lasted 10 days, while one PIT-tag was tag was entering the coelom. Newly ejected tags often had retained beyond 21 days. The hazard of ejection for magnets minute bits of soft tissue adhering to them; these were was more than twice that of PIT-tags (Cox regression, loosely attached and appeared randomly distributed. hazard ratio ϭ 2.11, z ϭ 2.7, P ϭ 0.007).

Table 1 Hazard functions for loss of tags in Exp. 1

Model Function AICc „AICc AICcWt

ϭ Ϫ Null model hx 0.086 43.14 0.00 0.55 ϭϪ ϩ Ϫ Linear time model hx 0.00089x 0.095 40.44 2.70 0.14 ϭ ϫ Ϫ2 ϫ Ϫ Ϫ10 x Ϫ Exponential time model hx 9.32 10 ( 4.97*10 ) 41.92 1.22 0.30

AICc calculations and Akaike weights. AICc: estimated information loss, AICcWt: Akaike weights, the relative probability that each model is best.

Figure 3. The movements of a magnet in a starﬁsh over time. Numer- als correspond to location at the following number of minutes post- injection: (1) 0, (2) 15, (3) 23, (4) 42, (5) 66, (6) 84, (7) 121, (8) 137, (9) 154, (10) 162, (11) 191. (M) is the madreporite. The magnet wandered, sometimes distally, sometimes proximally, and brieﬂy moved into the base of another arm. It was ejected from the end of the injected arm (12) 201 min after injection.

Experiment 3: Focused tracking and imaging Movements of magnets varied in both pace and direction (see Fig. 3 for an example). All seven magnets injected at 16 °C were lost within the ﬁrst 3 h. All such losses were via the tip of one of the arms (Fig. 4). The magnet was squeezed out through the skin after roughly 1 min of increasing stretching

Figure 5. Cross-sectional images through Arm #1 with magnet in coelom. (A) Ultrasound image. (B) Interpretive cartoon of same view. In all our observations, the magnet (black and white stripes) was parallel to the arm and is therefore seen end-on in the coelom (gray) between the body walls (white).

followed by a rupture and a few seconds of ejection, leaving a tiny tear barely detectable upon microscopic inspection. Cross- sectional ultrasound images of starﬁsh arms containing magnets (Fig. 5) show that the magnet is transported within the coelom, or body cavity, of the arm. In all of dozens of ultrasound views of four individuals, magnets were clearly visible within the coelomic space, and were generally well away from other spaces, such as the water vascular system. An externally visible constriction of the arm (Fig. 6) was often associated (roughly half the time, persisting for several minutes) with the location of the magnet, and ultrasound (Fig. 7) demonstrates Figure 4. Ejection of a magnet (white object in front of arrow) from the that this constriction generally narrows the coelomic space just aboral side of the distal end of an arm just above the sensory cluster. Across experiments, we directly observed 12 ejections of foreign bodies, all from this proximal or distal to the magnet. However, we observed no same location. movement of these constrictions, even when the associated

Figure 6. Constriction of Arm #1 (bottom left) seen from the outside of the starﬁsh. The magnet was located just distally of this constriction (arrow). magnet moved quickly. Again we observed a seemingly un- directed transport resulting in removal of magnets through the tips of arms in all four starﬁsh examined by ultrasound.

Comparing Experiments 2 and 3 Patterns of movement observed at 16 °C, as compared to 10 °C (Fig. 8) are quite similar, except that starﬁsh held at Figure 7. Longitudinal section of magnet inside starﬁsh arm. (A) Ultra- 16 °C lose their tags rapidly. The small sample size at 16 °C sound image and (B) interpretive cartoon of Arm #1 constricting just proxi- (n ϭ 7) and short retention of tags at the higher temperature mally (to the left) of a magnet (black and white stripes) that was observed to make Cox regression uninformative for this temperature be moving distally (to the right). The constriction is visible as a narrowing of contrast. the coelom (gray) between oral and aboral body walls (white).

Discussion face properties of the object can influence the immune re- Starfish have the ability to transport durable foreign bodies sponse to it, as previously observed in insects by Lackie through their coelom (Figs. 5 and 7) and eject them through the (1983). The degree to which starfish encapsulate different distal ends of their arms (Fig. 4). That the hazard of ejection is constant over time (Fig. 1) indicates both that the PIT-tag is not coming back out through the entrance wound, and that movement toward arm-tips involves a somewhat haphazard wander. A direct, coordinated distal movement would likely result in an increase in clearance rate with time as an increasing proportion of tags reached the tips of arms. Tracking of individual magnets (e.g., Fig. 3) confirms this wandering pattern. Despite this random aspect, the comparison of ejection times of PIT-tags versus magnets (Fig. 2) indicates that object type, the environment to which the animal is subjected (temperature, container, etc.), or their interaction can influence how fast an object is ejected. Further experiments will be needed to determine what controls rate of clearance. The magnets, in addition to being harder, denser, and 4-mm shorter than the PIT-tags (and therefore potentially easier to transport), may be more disturbing to Figure 8. Kaplan-Meier plot of magnet retention over time (h) com- starfish if they, like many other animals, can sense magnetic paring different water temperatures (10 °C, solid line, 16 °C dashed line). fields (Wiltschko and Wiltschko, 1995). Additionally, the sur- At 16 °C, loss occurs within 3 h.

This content downloaded from 130.226.087.174 on December 20, 2017 01:09:33 AM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). ELIMINATION OF DURABLE FOREIGN BODIES IN STARFISH 161 types of objects could perhaps affect the triggering and fre- the objects we injected were eliminated through the mouths quency of constrictions of the arms around the object, because of starfish, we saw no indication of this, and elimination muscle contractions have been shown to be involved in im- generally followed the magnet being close to the tip of an mune responses of other invertebrates (Leclerc, 1996). Simi- arm. Ghiradella (1965) observed that bits of foreign tissue larly, ejection of magnets at different temperatures (Fig. 8) injected into the coelom of two starfish species could some- suggests that temperature influences the time to ejection. Tem- times be ejected through the body wall by means of the perature likely speeds transport by increasing the physiological dermal brachiae—small projections of the coelom that com- and physical activity of the ectothermic echinoderms. The municate with the exterior seawater and function in waste hypothesis of active but loosely directed transport is confirmed removal and respiration. While this mechanism is likely by our ultrasound observations of magnets within starfish. related to what we report here, clearance of these small, soft These images revealed that the magnets were moving within bits of tissue was not through the arm tips and did not the coelom (Fig. 5). rupture the body wall as tags did; no constriction of the arm We frequently observed externally visible constrictions of was reported. The same author hypothesized that some of arms (Fig. 6) close to the location of magnets, and the magnets the foreign tissue could have been transported into the seemed to move most rapidly when pushing away from the stomach and either digested or ejected from there. It may be internal manifestations of these constrictions (Fig. 7). Constric- that while soft tissues could be eliminated in this way, no tions observed over several minutes of continuous observation mechanism exists for transporting hard, larger objects from did not appear to move along the length of an arm as would be the coelom across the stomach wall, a necessary step for expected in peristalsis, although our experiments were not elimination through the mouth. A breach of the barrier specifically designed to detect this. As we did not expect these between coelom and stomach could risk coelom infection contractions, we do not have good data on their prevalence, by digestive microbes. persistence, etc. Our impression based on ad hoc observation is How is this behavior controlled? The observed constric- that they persist for several minutes and occur in all injected tions used to move the foreign bodies through the coelom individuals roughly half the time. clearly involve the circular muscles of the body wall (Hy- This previously undescribed behavior functions to trans- man, 1955). The radial nerves within each arm have direct port and clear durable foreign bodies, raising several ques- control of that arm’s movements (Dale, 1999) with little tions. input from the central nerve ring (Migita et al., 2005). The What is the ecological relevance to Asterias rubens? The activity and coordination of these muscles remain poorly ability to clear durable foreign objects is useful to organisms understood in starfish, but it appears that they can be acti- only if such objects occur in the coelom frequently enough vated locally by sensing a foreign body within the coelom. to impose a significant selective cost on those lacking this What range of objects can be eliminated? We have in- ability. Endoparasites may grow in starfish’ coeloms (Stone, vestigated only two types of objects of similar shapes and 1987), and forcible expulsion could serve where encapsu- sizes, both plastic-coated. Whether this same mechanism lation fails. Durham (1887), who studied the clearance of would serve to eject soft-bodied endoparasites, small sand- microscopic particles from the coelom of this species, sug- grains, or a large and complex fragment such as a piece of gested that this ability was most needed when an arm is arthropod exoskeleton is unknown. shed. Extrapolating, it seems likely that most durable for- How wide a range of organisms beyond Asterias rubens eign bodies enter the coelom through injuries to the arms. A employs this mechanism? Although encapsulation has been compilation by Lindsay (2010) indicates that depending on studied in a wide range of organisms, clearance of durable species and environment, 0 to 69% of individuals bear such foreign bodies has not. Starfish and most other echinoderms injuries, with most populations in the 20% to 40% range. A inhabit and feed in benthic habitats where sharp detritus, survey of A. rubens reported that a mean of 26% of indi- prey-remains (e.g., molluscan shells), pieces of predators, viduals had arm damage (Marrs et al., 2000). Given the and potential endoparasites could end up inside their tissues efficiency with which objects are ejected from the coelom or cavities. How other echinoderms, let alone more dispa- and the fact that this aspect of organismal maintenance has rate taxa, clear durable foreign bodies is unknown. As sea rarely been investigated, it is not surprising that nonparasite urchins do not have this ability (Hagen, 1996) it may be durable intracoelomic foreign objects have not been re- unique to Asterozoa (starfish and brittle stars), or some ported in starfish. Ultimately, our results suggest that only subgroup thereof. internal examination of thousands of freshly caught wild We propose the following crude model to explain our ob- individuals from a variety of environments would reveal the servations: (1) An object gains entry to the coelom. (2) The frequency with which such objects occur. animal detects the object, perhaps through immune-mediated Why push an object out through the skin rather than the irritation. (3) The radial nerves cause a contraction of the mouth? Starfish can evert their stomachs and will eliminate circular muscles in the location of the irritation. (4) The result- digestive waste this way. While it is possible that some of ing pressure causes the object to move, either proximally or

This content downloaded from 130.226.087.174 on December 20, 2017 01:09:33 AM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). 162 T. B. OLSEN ET AL. distally. (5) This relieves the irritation at the initial location, Hagen, N. T. 1996. Tagging sea urchins: a new technique for individual allowing the constriction to relax, but moves the irritant to a identification. Aquaculture 139: 271–284. Hyman, L. H. 1955. The Invertebrates: Echinodermata, the Coelomate new location. (6) Steps 2 through 5 are repeated (accompanied Bilateria. McGraw-Hill, New York. by movement of the object caused by the normal behavioral Lackie, A. 1983. Effect of substratum wettability and charge on adhesion movements of the animal) until the object reaches an arm tip. in vitro and encapsulation in vivo by insect haemocytes. J. 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