Unique tagging of small echinoderms: a case study using the cushion star Parvulastra exigua

Authors


Correspondence author. E-mail: aline.martinez@sydney.edu.au

Summary

  1. Tagging animals is essential to evaluate animal population dynamics and behaviour. Using unique marks requires that tags must last for the duration of the study and not affect animal behaviour and health. Echinoderms, especially smaller species, are usually considered intractable for tagging because they readily discard both internal and external tags.
  2. We compared the utility of two tagging techniques for marking the small starfish Parvulastra exigua: (i) branding with a soldering iron, and (ii) injection of visible implant elastomer (VIE) of different colours. The efficiency of these techniques was evaluated by assessing (i) the effects of the tag on the mortality, growth and mobility of P. exigua; and (ii) the persistence of the tags over 30 days.
  3. VIE had no detectable effect on any variable tested, whereas brand marks caused a decrease in mobility. In addition, 95% of the starfish tagged with VIE were still tagged after 1 month.
  4. This technique will facilitate research on the ecology of this and other small asteroid species, and is also relevant for ecological studies of other small soft-bodied invertebrates. The combination of VIE colours plus the viability to track individuals offers a wide range of opportunities to investigate the ecology and behaviour of small invertebrates in the field.

Introduction

Much research on spatial or population ecology and especially animal behaviour requires the unique identification of individuals. For many animals, this is relatively easy; vertebrates can be marked with internal or PIT tags (Castro-Santos, Haro & Walk 1996; Gibbons & Andrews 2004) or external tags/labels such as fin tags(Tranquilli & Childers 1982; Oven & Lee Blankenship 1993), leg bands/rings (Ogilvie 1972; Blums, Mednis & Nichols 1994) and ear (Twigg 1975; Morley 2002). Some invertebrates, notably arthropods and molluscs are amenable to glued/painted tags (Southwood & Handerson 2000).

Many tagging techniques have been used to mark echinoderms, but these are mainly done on sea urchins. Tagging methods include plastic tubes over spines, labelled tags inserted into the sea urchin test or spine, coded wire, fluorochromes and PIT tags (see Ebert 2013, for review). Such methods have variable success with sea urchins, but fluorochromes have been shown to be efficient (Ebert 2013) and their use is increasing in studies on sea urchin and shelled invertebrates (e.g. Moran 2000; Moran & Marko 2005; Ellers & Johnson 2009). In contrast, soft-bodied echinoderms such as starfish and sea cucumbers are difficult to tag because they readily discard inserted or attached tags.

Several ecological studies have attempted to mark starfish, but found it difficult to apply an efficient technique that combined persistence of the tag with the capacity to identify individuals in the field. Early studies used Nile blue sulphate to tag starfish by staining vital organs by soaking individuals in a dye solution (Loosanoff 1937). Nile blue stain may persist for months, and it is possible to recognize individuals by staining different arms of the animal (Barahona & Navarrete 2010). For field-based studies, the major disadvantage of this method is that it is hard to find tagged individuals. Importantly, it is not possible to produce individual marks in small animals, which limits this technique to larger species and/or relatively small test populations. Finally, even with larger starfish, the number of treatment combinations which may be recorded is constrained. Marker pens and sharp pencils have been used to write numbers on the aboral surface of starfish, but such techniques are limited to much larger species (e.g. Asteria rubens, Oreaster reticulatus) (Kvalvagnaes 1972; Scheibling 1980). External tags such as plastic labels and electronic tags have been used to track individuals, and these methods are also applied to large asteroids (e.g. Marthasterias glacialis, Acanthaster planchi, Coscinasterias muricata, Protoreaster nodosus) (Savy 1987; Keesing & Lucas 1992; Lamare et al. 2009; Chim & Tan 2012a). Another option is to use natural marks of the starfish to identify individuals, but this technique can only be applied to species with a range of distinguishable natural body marks (Chim & Tan 2012b). So whilst the techniques to uniquely mark larger starfish are reasonably well determined, there are still major issues with marking smaller animals.

The ability to follow small individuals becomes important when the organisms concerned have the potential to influence ecosystem level outcomes, but whose behaviour in the field is less understood. Parvulastra (Patiriella) exigua is a small starfish (average radius 10 mm; Dartnall 1971), which occurs at great densities in and around rock pools on the mid-low intertidal shores of south-east Australia and South Africa. Grazing by P. exigua was suggested to have significant consequences for rocky shore assemblages (Jackson, Murphy & Underwood 2009). Grazing by small starfish such as P. exigua is much less studied compared with other small herbivores such as gastropod grazers (Poore et al. 2012), mainly because of the difficulties in tagging them.

The only method tested previously to mark small starfish was branding the aboral surface of Asterina pectinifera (radius size ca. 28 mm) with a soldering iron (Kurihara 1998). This technique identified specific individuals and marked groups for up to 60 days without reported effects on growth and mortality (Kurihara 1998). Branding is, however, very hard to use because it is difficult to ensure consistency in temperature, application pressure, application angle and duration of application of the soldering iron bit (A. S. Martinez, M. Byrne & R. A. Coleman, unpubl. data). More recently, researchers have used hypodermal marking to identify individual/groups of fish, amphibians, reptiles and, among invertebrates, earthworms, molluscs and crustaceans (e.g. Godin et al. 1996; Clark & Kershner 2006; Butt & Lowe 2007; Wallin & Latty 2008; Brewer & Norcross 2012). This technique consists of injecting visible implant elastomer (VIE) underneath the skin or deeper within the tissues without creating a permanent wound or lesion. The elastomer remains externally visible for a timescale of months in many species. VIE is a coloured nontoxic compound, which after mixing with a curing agent is injected as a liquid and then cures to a biocompatible solid (Northwest Marine Technology, Shaw Island, WA, USA).

Here, we compare the use of branding using a soldering iron (Kurihara 1998) and VIE tags on the starfish P. exigua. The challenge is to tag this small starfish without producing major injuries that may adversely affect growth, activity and/or mortality. Branding marks may represent a significant challenge to the health of the starfish because the soldering iron generates temperatures up to 370°C with a risk of overheating the individual. Injecting VIE is less of a challenge in terms of tissue damage, but is not easy as the starfish has a hard skin, and care is needed to avoid penetrating damage to organs or the circulatory system.

Effects of each tagging method on P. exigua were assessed by measuring mortality, growth and behavioural responses (i.e. righting and escape response) of the starfish. The righting response behaviour, commonly used to record activity levels in starfish (Lawrence & Cowell 1996), is the time taken for a starfish to correct its vertical orientation after being inverted. Escape responses from predator cues are useful to assess movements. P. exigua has a distinct escape response to touching by the predatory starfish Meridiastra calcar (Stevenson 1992); hence, it can test whether the tags modify rates of movement. We predicted that VIE would not affect mortality (H1), growth (H2), coordination (H3) and movement (H4) of P. exigua compared with unmarked individuals, whereas branding would. Also, we expected that the proportion of tagged starfish remaining at the end of the experiment would be greater for individuals tagged with VIE than with a soldering iron (H5).

Materials and methods

Tagging Parvulastra exigua

Individuals of P. exigua were collected from rock pools at Little Bay (33°58′44″S, 151°15′10″E) and Cape Banks (33°59′56″S, 151°14′53″E), NSW, Australia. The animals were taken to the laboratory and left in the container in which they were transported until the temperature of the seawater equilibrated with laboratory seawater. Subsequently, they were placed in two aquaria (25 L ea., 0·4 m × 0·3 m × 0·25 m) containing sandstone cobbles covered in biofilm/microalgae as a food resource. The first step of the tagging procedures was to place each animal in individual petri dishes covered with a thin layer (5 mm) of seawater. Then, each starfish was blotted dry and tagged.

Visible implant elastomer (VIE)

A pink fluorescent VIE (Northwest Marine Technology) was injected into the aboral surface between the arms near the edges of the aboral surface (Fig. 1a). A 0·3 mL insulin syringe with 30 G/12·7 mm needle was used to inject elastomer. The needle was introduced through the body wall parallel to the arm (Fig. 1b). After positioning the needle ≈2 mm deep, the syringe was slowly and gently depressed to inject elastomer (Fig. 1c). Since the elastomer is only visible through the puncture wound, application of VIE beyond the minima results in raised epidermis but no increase in visibility (Fig. 1d). We applied ca 4 μL, this was an average of the total volume used to inject VIE in 48 animals from a single syringe. The animals were then returned to their individual containers within the aquarium.

Figure 1.

Method of injecting visible implant elastomer (VIE) in P. exigua. (a) Aboral side of the starfish; (b–d) cross section of the starfish arm showing: (b) the needle being inserted, (c) VIE being injected and (d) the mark that is left after injecting the VIE and removing the needle.

Brand marks (BM)

The starfish were branded on the aboral surface following Kurihara (1998), but on the edge of their arms. We used a 40-W soldering iron with a pointed bit (1·2 mm diam.). The brand marks were made by gently placing the soldering iron on the starfish aboral surface for 3–4 s under slight pressure. Preliminary tests showed that excessive pressure or time (more than 5 s) resulted in increased mortality within a few days. After branding, the starfish was returned to their individual container within the aquarium.

Experiment 1: Testing the effects of tag techniques on mortality and growth of P. exigua and evaluating the duration of the tags

We attempted to use specimens of similar size (9–10 mm radius) and weight (0·4–0·6 g) in these experiments to avoid any potential confounding effects of size. The mean radius of each animal was determined from three haphazardly chosen arms measured from the centre of the oral disc to the arm tip with a digital calliper (nearest 0·1 mm). Then, the starfish was blotted dry (5 s) and the wet mass determined (to the nearest 10−3 g; model PB303, Mettler Toledo International Inc., Columbus, OH, USA). The radius size and wet weight of all individuals were measured at the first and last day of the experiment. Starfish were then randomly allocated to one of three treatments – individuals not tagged (NT – control), individuals tagged by injecting VIE and individuals branded with soldering iron (BM). Animals were kept in individual containers (70 mL – 54 mm high and 40 mm diameter) with two holes (40 mm diameter) covered with a mesh (1·4 mm). Sandstone cobbles covered with biofilm were put inside of each container as food supply during the whole experiment. The individual containers were then randomly allocated to one of three aquaria (0·6 m × 0·4 m × 0·2 m) filled with 42 L of seawater with individual filters until all aquaria contained equal numbers of individuals from each treatment. Water temperature and salinity varied between 15 and 17°C and 35–38, and between 16 and 18°C and 35–38, for the first and second experimental run, respectively. Compressed air bubbled through air stones maintained dissolved oxygen saturation at >80% and light cycle was 12 h:12 h.

The following experiments were repeated twice (August and November 2012), and each trial ran for 30 days, a time-scale representative of previous field experiments on movement and behaviour of invertebrate grazers (e.g. Underwood 1977; Levings & Garrity 1983; James 2000; Coleman, Underwood & Chapman 2004; Noel et al. 2009).

Mortality and growth of P. exigua

Each of the three aquaria started with 48 starfish containing 16 individuals of each treatment. To test the effects of tagging techniques on P. exigua mortality (H1), the percentages of dead starfish were measured at the end of the experiment for each treatment and data arcsin-transformed (Underwood 1997). The null hypothesis that the average percentage of dead individuals would not differ between treatments was tested by a two-factor anova (Experimental run: 2 levels – run 1, run 2, random; Treatment: 3 levels – NT, BM, VIE, fixed and orthogonal; n = 3) using WinGMAV5 software (EICC, University of Sydney) with the assumption of homogeneity of variance determined by Cochran's test (Underwood 1997). Mortality was determined for each aquarium as a replicate, thus ‘aquarium’ could not be included as a random factor.

Size and wet mass obtained at the first and last day from the experiment of mortality were used to test the effects of tagging on P. exigua growth (H2). For each animal, we calculated the slope of the line representing the change in radius and wet mass over the duration of the experiment and used this value as a response variable (Coleman et al. 1999). Data from nine surviving starfish of each treatment within each aquarium were randomly selected to allow comparisons with a balanced design (Underwood 1997). The null hypotheses that the average slopes of radius size and weight would not differ between treatments were tested through a three-factor anova as above (Experimental run: 2 levels – run 1, run 2, random; Aquarium: 3 levels – A, B, C, nested in time; and Treatment: 3 levels – NT, BM, VIE; n = 9).

Identification and duration of tags on P. exigua

The identification of tagged individuals and longevity of the tag was evaluated by recording the number of animals tagged every 3 or 4 days from the beginning to the end of the experiment (10 records). Branded starfish were checked by direct visual observations, and starfish tagged with elastomer were checked using an UV light. Two different lights were trialled: a regular UV light (400 nm 21 LED UV torch) used in the experiments at time 1 and 2 and a FL-1 LED light (NightSea, Bedford, MA, USA) used only for run 2. The number of tagged starfish observed with the regular light on the last day from experiment 1 and with the FL light from experiment 2 was compared through a G-test adjusted by Williams’ correction (Sokal & Rohlf 2011). Individual codes were used for both tagging techniques to evaluate the possibility of identifying individual tagged starfish. The codes were done by combining different number of spots and arm tagged using the madreporite as a reference (Fig. 2). The percentages of tagged starfish of each treatment were calculated for every interval of time to the initial time to evaluate the efficiency of each tagging technique (H5). The number of tagged animals observed on the last day of the experiment was compared between treatments for both identification of the tag and code through a G-test as above.

Figure 2.

Codes used to identify individual starfish: the number before and after the letter ‘M’ means, respectively, the number of spots tagged and the number of the arm where the code starts anticlockwise in relation to the starfish madreporite (black triangle symbol).

Experiment 2: Effects of tags on P. exigua mobility

Righting and speed response experiments were done to test potential effects of the tags on the coordination (H3) and movement (H4) of P. exigua. Starfish were collected from rock pools at Little Bay, and the animals were separated into three different treatments (16 in each) – individuals not tagged (NT - control), individuals tagged by injecting VIE and individuals branded with soldering iron (BM). Starfish were randomly reared in individual small containers (as in experiment 1) in three aquaria (0·4 m × 0·3 m × 0·25 m) containing 5–6 starfish of each treatment. These aquaria were placed within a single recirculated seawater system, and thus, aquarium could not be included as a factor in the analyses (see below). The radius and wet mass of each individual were measured before performing the experiment as described in experiment 1. These experiments were repeated twice (November 2012) and ran for 7 days to avoid possible confounding effects of weight and size losses.

The righting response was evaluated by placing the starfish on its aboral surface (i.e. oral surface faces up, and the tube feet are not in contact with the substratum) and measuring the time that the starfish completely returns the oral surface with the distal tips of each arm on the substrate (Lawrence & Cowell 1996). The movements of each starfish were recorded via a webcam connected to a computer, then observed later in the laboratory. The righting response of each starfish was recorded three times during two periods viz – 48 h before and after being tagged. The activity coefficient was calculated using the response time of each individual to evaluate potential levels of stress and well-being (AC = 1000/time), on which relatively lower activity coefficient is associated with higher levels of stress (Lawrence & Cowell 1996). The ‘before’ and ‘after’ activity coefficients are nonindependent and so direct comparison is not viable. Thus, the log response ratio of activity coefficient before and after tagging was calculated to allow comparisons using anova (Coleman 2012). The null hypothesis that the average of the activity coefficient ratio response would not differ between treatments was tested by a two-factor anova (Experimental run: 2 levels – run 1, run 2, random; Treatment: 3 levels – NT, BM, VIE, fixed and orthogonal; n = 16) using GMAV5 software as above. To evaluate the strength of the effect, the response ratio was tested against a null hypothesis of zero by a two-tailed t-test (Coleman 2012).

Specimens of M. calcar (ca. 3 cm radius), used to stimulate the escape response of P. exigua, were collected from Little Bay, kept in separate aquaria and fed ad libitum with P. exigua. During the experiment, each test P. exigua was placed in a plastic tray (30 cm × 40 cm) filled with a depth of 3 cm of running seawater. The base of the tray was marked with a 4-cm2 grid to facilitate tracking of movements. Each observation was recorded using a webcam connected to a computer. Data were extracted from the videos using the software Kinovea™ (www.kinovea.org) to minimize errors of measuring distance displaced as previous tests showed that many P. exigua do not move in a straight direction. First, the speed of undisturbed individual rate (i.e. normal speed) was recorded after the starfish started moving. Then, M. calcar was placed within the plastic tray with one arm over P. exigua, and the movement rate (i.e. escape speed) of P. exigua was recorded again. Their speed was obtained by the distance (cm) displaced during 1 min. Normal and escape speed did not correlate with body size, and thus data were not standardized. Starfish movements were recorded before and after being tagged as above. The log ratio of speed (normal and escape) before and after tagging was calculated to perform an anova (Coleman 2012). The null hypotheses that the average of normal and escape ratios response would not differ between treatments were tested by a two-factor anova (Experimental run: 2 levels – run 1, run 2, random; Treatment: 3 levels – NT, BM, VIE, fixed and orthogonal; n = 16) as above.

Results

Tagging effects on mortality and growth of P. exigua

There was no effect of tag techniques on the mortality of P. exigua, as the percentage of dead individuals at the last day of experiment did not differ (F(1, 12) = 0·02, P = 0·98; NT = 27·67 ± 1·89, VIE = 28. 68 ± 3·15, BM = 29·09 ± 4·02, mean percentage ± SE; Table S1). All specimens showed a slight decrease in radius (F(2, 2) = 0·40, P = 0·71; NT = −0·045 ± 0·004, VIE = −0·046 ± 0·004, BM = −0·050 ± 0·004, mean mm day−1 ± SE) and wet mass (F(2, 154) = 0·40, P = 0·36; NT = −4·196 ± 0·236, VIE = −4·078 ± 0·220, BM = −4·518 ± 0·247, mean mg day−1 ± SE; Table S2), but as for mortality, there were no differences between treatments. There were significant interactions between experimental runs for both experiments and also between aquaria for the growth experiment. The fact that the direction of the differences in treatments was consistent between experiments means (i) that we can interpret the main effect of treatment only and (ii) that these interactions arise from temporal variation between experiments and spatial variation between aquaria, which are random effects and so not interpreted further.

Tagging effects on coordination and mobility of P. exigua

There was no effect of tag techniques on the righting response of P. exigua since the ratio of activity coefficient did not differ between treatments (F(2, 90) = 0·81, P = 0·49; NT = −0·013 ± 0·039, VIE = 0·001 ± 0·043, BM =  −0·052 ± 0·049, mean Log (1000 s−1 after/before) ± SE; Table S3). Also, none of the ratio were significantly different from zero (t95 = 0·77, > 0·4). The mobility of P. exigua, however, was affected by tagging as differences in the ratio response were found between treatments for both normal (F(2, 90) = 5·78, P = 0·004; NT = −0·11 ± 0·03, VIE = −0·13 ± 0·02, BM = −0·24 ± 0·04, mean Log (cm min−1 after/before) ± SE) and escape speeds (F(2, 92) = 7·6, P =0·001; NT = −0·02 ± 0·02, VIE = 0·001 ± 0·02, BM = −0·09 ± 0·02, mean Log (cm min−1 after/before) ± SE; Fig. 3; Table S4). All individuals exhibited a small negative ratio response for normal and escape speeds, but only results for normal speed were significant different from zero (t65 = 0·64, < 0·001). The negative ratio response observed for the branded starfish, however, was greater than the other treatments for both speeds (Fig. 3), which indicates a decrease in the starfish movement due to the branding technique.

Figure 3.

Effects of treatments (NT, Not Tagged; BM, Brand Marked; VIE, Visible Implant Elastomer tag) on the ratio response (after/before) of activity coefficient (mean Log AC ± SE,= 32), normal speed (mean Log NS ± SE,= 32) and escape speed (mean Log ES ± SE, n = 32) in P. exigua. Post hoc SNK tests are shown for significant factors above the bars on graphs b and c.

Identification and duration of tags on P. exigua

The VIE was detectable over the duration of the experiment (Fig. 4). After 6 days, at least 90% of the starfish were still tagged and 50% by the end of experiment when using a basic UV torch to stimulate tag fluorescence. The use of a more powerful light (FL LED), however, showed that it is possible to improve identification of the tagged individuals (Fig. 5, Table 1). Using the FL light, we were able to identify 100% of the tagged starfish on day 5 and 95% after 30 days (Fig. 4a). The FL light enhanced successful identification of the codes on the starfish. More than 80% of the codes were identified during the 30 days of experiment (Fig. 4b). In contrast, with the basic UV torch, only ca. 20% of the codes were identified at the first day decreasing to 2% at the last day.

Table 1. Results from the G-test (adjusted by Williams’ correction) of the identification of tags on P. exigua. Comparisons were done between the proportions of tagged starfish observed with the regular light (RL) and the FL LED light (FL), and between tagging techniques (VIE – visible implant elastomer and BM – brand mark)
 Tag identificationCode identification
RL vs. FLVIE vs. BMRL vs. FLVIE vs. BM
df 1111
Gadj3·701·431·024·24
P >0·05>0·15<0·001<0·05
Figure 4.

Percentage of identified starfish tagged with visible implant elastomer for each day sampled in a period of 30 days: (a) identification of tagged starfish and (b) identification of the codes. The identification of tag and codes was recorded using two different lights viz the regular light used at experimental run 1 (circles) and experimental run 2 (inverted triangles), and the FL-1 LED light (squares) that was used only at the second repetition of the experiment.

Figure 5.

Photographs of a starfish tagged with an orange fluorescent elastomer (code 3 m3, as shown in the picture) under different lights at the sixth day of experiment: (a) white light, (b) regular UV light and (c) FL-1 LED under a yellow filter glass that comes with the light.

Identification of starfish branded with the soldering iron method was also successful, but less efficient than VIE with respect to identification of individuals (Fig. 6, Table 1). After the first day, 96% of tagged animals were identified declining to nearly 70% after 30 days. The identification of the codes started with nearly 80% identified and gradually decreased until the last day of the experiment, but this decline was not consistent due to differences in, probably due to healing of the wound. Initially, the wound was bright orange, contrasting with the background colour of the starfish body. As the wound healed, the colour of the marked areas became similar to the starfish body wall, making the identification more difficult. By day 15, the wound was dark in some starfish, making it better to identify. These colour changes in the branded area varied from individual to individual.

Figure 6.

Percentage of identified starfish marked with soldering iron for each day sampled in a period of 30 days for each replicated experiment (experimental run 1 – solid circles; experimental run 2 – open circles): (a) identification of tagged starfish; and (b) identification of the codes.

Discussion

The results indicated that VIE is an effective technique for tagging small starfish, whereas the branding method might not be as useful because of problems associated with inter-individual differences in wound healing. When comparing VIE with other methods used before to tag echinoderms (Table S5), even small animals can be identified as an individual, whereas the other techniques were developed with large species. Indeed, many techniques applied in other echinoderms for studies of a longer duration (i.e. months) cannot be applied to P. exigua because of the large size of the tags, which approximates to the size of individual P. exigua. Even though the effects of tags on echinoderms as well and their longevity are not well documented.

Over the duration of our experiment, mortality and growth of P. exigua were not affected by the two tagging techniques. The mortality noted across all treatments (≈ 20%) and an overall decrease in size and weight may have been caused by insufficient food during the experiment. We used small rocks covered with biofilm as food supply and did not change the rocks through time; thus, the growth of biofilm might have not been sufficient for the daily intake of the starfish.

Coordination was also not affected by tagging; however, speed of movements was altered. The escape response of controls and VIE tagged starfish did not differ, but normal speeds were slightly negatively different from zero, which is probably a consequence of husbandry during the experiment (e.g. food supply). In contrast, starfish tagged with the branding method were significantly slower than controls. Moreover, it was noted that some of the branded starfish lost part of their arms after a few days, despite efforts to minimize injury. The branding technique may be too aggressive for small starfish. Branding with a soldering iron was previously used by Kurihara (1998) on the starfish Asterina pectinifera, which is nearly four times bigger than P. exigua. The relationship between wound area from branding and body area would therefore be much smaller in A. pectinifera and thus did not cause a detectable effect on the starfish. It should be noted that mobility was not tested by Kurihara (1998). Moreover, identification of the brand-marked Pexigua was difficult due to the variation in the colour of the wound. This variation would increase the errors of identification in the field, meaning the method has limited applicability.

The small lesion caused by the needle used to inject elastomer did not appear to be detrimental to P. exigua. Indeed, the pore created by the needle is where the elastomer can be visible as the starfish surface is quite thick and not transparent. Kirshenbaum, Feindell and Chen (2006) excluded the possibility of using elastomer to tag sea cucumbers because it could only be visible on juveniles, which have a transparent body wall. Here, we showed that VIE can be applied to animals that do not have a transparent skin, although detection of the tag was facilitated by the thin body profile of P. exigua. The elastomer is likely to migrate in the coelom of a larger species, especially sea cucumbers. Some P. exigua released the elastomer from the injection pore before the elastomer cured. VIE, however, persisted at least in 80% of the tagged P. exigua after 30 days in experimental run 2. Moreover, the use of the FL-1 LED light (NightSea) improved the identification of the tagged starfish, specially the individual codes, which was not as efficiently detected with the regular UV light. The FL-1 light is used in coral reef studies to find microalgae in corals and coral recruits (e.g. Baird, Salih & Trevor-Jones 2006; D'Angelo et al. 2008), and thus can detect very small fluorescent spots. Moreover, this light facilitated location of tagged animals in the field even during bright, sunny days; however, it takes longer in this situation because starfish have to be handled and checked (A. S. Martinez unpublished data). The challenge now is to improve the fluorescence detection on bright days and a possible solution is to create shadows on the substrata when searching for fluorescence. Since these experiments were done under laboratory conditions, it is possible that the retention of VIE and the rates of mortality in the field may differ. Also, as we did not measure daily changes in size and weight, we cannot discard the possibility of a very short-term effect of the tag on growth.

Visible implant elastomer has been shown to be an efficient method to tag many invertebrates, including terrestrial animals such as slugs (Wallin & Latty 2008) and earthworms (Butt & Lowe 2007) and marine invertebrates such as shrimps (Godin et al. 1996), lobsters (Woods & James 2003), crabs (Davis et al. 2004), squid (Zeeh & Wood 2009) and octopus (Brewer & Norcross 2012). The animals from these studies were tagged in a soft and transparent part of the body, what makes identification easy to detect. Here, we showed that elastomer can also be applied to starfish, which have a rigid opaque surface and is hard to tag due to their capacity to easily eliminate tags from their body. Efficient tags to identify individual starfishes for a longer period (i.e. weeks or months) such as plastic labels and electronic tags were only possible for large species (e.g. Keesing & Lucas 1992; Lamare et al. 2009). The possibility of marking P. exigua will facilitate studies on the ecology of this starfish to address gaps in the knowledge in their role in marine communities (Branch & Branch 1980; Arrontes & Underwood 1991; Stevenson 1992; Jackson, Murphy & Underwood 2009; Pillay, Branch & Steyn 2010; Dawson & Pillay 2011).

Complex experimental designs for field-based tests of hypotheses may require many codes which could involve a greater frequency of injections to an individual animal. We suggest that researchers combine VIE methods for coding treatments and use gross whole-animal marking systems such as Nile blue (Loosanoff 1937) for factors such as ‘site’ or ‘time’. This will facilitate more informative marking whilst minimising disturbance of test animals.

Injecting VIE is a novel method to tag starfish and also has potential application for other small invertebrates that cannot be tagged externally (e.g. on shells). Using fluorescent elastomer, animals can be detected during the day and especially at night, which is helpful to track small invertebrates that have nocturnal behaviour. Furthermore, the availability of different colours of elastomer increases the possibility of developing more complex experimental designs by increasing the combination of codes in animals.

Acknowledgements

A.M. was funded by an Endeavour Scholarship. Additional funding was provided by the authors. We thank Paige Alderoty, Jessica Fine, Clarissa Fraser and Ana Bugnot for field and laboratory assistance. Animals were collected under NSW DPI Fisheries Permit number F96/146-7.2.

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