PREDATOR DEFENSE MECHANISMS IN SHALLOW WATER SEA CUCUMBERS (HOLOTHUROIDEA)

JESSICA A. CASTILLO

Environmental Science Policy and Management, University of California, Berkeley, California 94720 USA

Abstract. The various predator defense mechanisms possessed by shallow water sea cucumbers were surveyed in twelve different species and morphs. While many defense mechanisms such as the presence of Cuverian tubules, toxic secretions, and unpalatability have been identified in holothurians, I hypothesized that the possession of these traits as well as the degree to which they are utilized varies from species to species. The observed defense mechanisms were compared against a previously-derived phylogeny of the sea cucumbers of Moorea. Furthermore, I hypothesized that while the presence of such structures is most likely a result of the species’ placement on a phylogenetic tree, the degree to which they utilize such structures and their physical behavior are influenced by their individual ecologies. The presence of a red liquid secretion was restricted to individuals of the genus Holothuria (Linnaeus 1767) however not all members of the genus exhibited this trait. With the exception of H. leucospilota, which possessed both Cuverian tubules and a red secretion, Cuverian tubules were observed in members of the genus Bohadschia (Ostergren 1896). In accordance with the hypothesis, both the phylogenetics and individual ecology appear to influence predator defense mechanisms. However, even closely related species of similar ecology may differ considerably.

Key words: holothurians; defense; toxicity; Cuverian tubules; Moorea, French Polynesia

INTRODUCTION (Sakthivel et. Al, 1994). Approximately 20 species in two families and five genera, Sea cucumbers belong to the phylum including Holothuria, Bohadschia, and Thenelota, Echinodermata and the class Holothuroidea. are traded internationally (Anonymous, 2003). Echinoderms are distinguished by having a The topic of overhavesting and sustainable coelomic, water vascular system and aquaculture has become increasing important pentaradial symmetry. Holothurians in turn in recent years. are characterized by having feeding tentacles Studies have noted that predation plays a that extend from a circumoral ring. major role in influencing community structure Individuals can range in size from 10 to as well as spatial and temporal distribution of 2000mm in length (Lawrence, 1897). marine invertebrates (Bingham, 1986; Holothurians come in many shapes and sizes Francour, 1997). Predators of holothurians and inhabit a wide variety of habitats from the include fish, crustaceans, and most intertidal zone to the deep sea (Anonymous, importantly sea stars (Francour, 1997). 2003). Because of the shape and structure of sea Holothurians are important ecologically cucumbers, either a portion of the sea as well as economically and culturally. Sea cucumber must be bitten off by the predator, cucumbers have been collected and utilized as or the individual must be ingested whole a food item, especially in Southeast Asia, for (Lawrence, 1987). In response to predation generations. Food products include dried pressures, sea cucumbers possess a wide gonads, dried body meat and longitudinal variety of defense mechiansms. Such defenses muscles, and salted fermented intestine include toxicity, thickened body wall, evisceration, autotomy cryptic behavior, structure and texture, coloration, and unpalatability, and swimming or other active behavior. avoidance of the predator (Bingham, 1986). I expect that individuals will exhibit A toxic secretion has been identified in multiple defense mechanisms and that not all species belonging to the genus Holothuria, mechanisms will be expressed to the same particularly H. atra. This secretion is burgundy extent. Additionally, I expect to see some in color and is thought to be a toxin that was similarities among closely related species, but used by inhabitants of the Pacific to poison it is likely that the individuals’ life histories fish, thus it is most likely utilized as a defense will also influence the way they respond to mechanism by the sea cucumber (Delia, 1977). potential predators. Possibly the most studied defense mechanism is the expulsion of Cuverian METHODS tubules. Cuverian tubules are found in several species belonging exclusively to the family Collection and initial observations Holothuriidae (Flammang et. al, 2005). The tubules are white, intracoelomic caeca I surveyed shallow water sites on the attached to the basal part of the left northern and western sides of Moorea for sea respiratory tree (Van Den Spiegel, 1987). cucumbers. Sites were reached either by When physically stimulated, the sea swimming, snorkeling, or boat. The collection cucumbers expel a few tubules through the sites include Temae (1), Cook’s Bay directly off anus which become adhesive as they elongate of the Gump Station (2), Cook’s Bay near and stick to almost any surface (Flammang, Pihaena Point (3), a reef just offshore between 2003). The expulsion is directed toward the PK 14 and PK 15 (4), three sites at the Motu on point of stimulus and the number of tubules the northwest side of the island (5), and the expelled is proportional to the intensity of Mangrove Marsh near Haapiti (6) (Figure 1). I stimulation (Hamel, 2000). The tubules can also observed and collected sea cucumbers on elongate to up to 30 times their original Tetearoa; an atoll approximately 59km to the length, or approximately 60cm depending on North of Papeete, Tahiti. I picked up the the species, and can maintain both their individual sea cucumbers gently with rubber strength and adhesiveness for several days coated gloves and placed them in a plastic bin (Van Den Spiegel, 1995). while still underwater so as to disturb them as Cuverian tubules are particularly effective little as possible. They were kept in the bins against fish as the fish become entangled and until I returned to the Gump Station, where immobilized in the tubules (DeMoor, et. al, they were placed in a large concrete tank 2003). Furthermore, it is thought that the (8’x4’x2’) with flowing sea water. Ecological presence of Cuverian tubules is correlated factors such as depth, substrate, distance from with a higher degree of fish predation shore, and proximity to coral and other observed at lower latitudes (Lawrence, 1987). organisms were noted for each species This study includes a survey of the sea observed. cucumbers in shallow waters surrounding Individuals were identified to species Moorea, as well as an assessment of their whenever possible using Guille, 1986 and predator defense mechanisms. I investigated Clark and Rowe, 1971. When identifications the possible relationships between defense could not be made based on superficial mechanisms and habitat and life histories as characteristics alone, ossicles were extracted well as phylogeny and relatedness of species. from the body wall. This was done by soaking While an attempt was made to qualify all an approximately 2cm2 segment of body wall defense machanisms exhibited, I focused on in sodium hypochlorite for 48 hours at the following: presence of Cuverian tubules approximately 7 degrees celcius. The ossicles and or a liquid secretion, toxicity, body were collected from the bottom of the small jar, slide mounted, and photographed for identification. individuals that secreted a colored liquid when stimulated, I held them above a plastic cup and squeezed gently in a peristaltic motion to collect the liquid. Lastly, I placed the individuals in plastic freezer bags and placed them in the freezer for a minimum of six hours to sacrifice them before dissection.

Toxicity assessment

I removed the sea cucumbers from the freezer and allowed them to thaw for a few minutes before I dissected them using scissors FIG. 1. Study Sites. and a razor blade. I noted the presence or absence of Cuverian tubules within the body Individual and behavioral observations cavity. I then separated the external tegument and flesh from the internal organs, muscles, I observed the sea cucumbers individually and connective tissue. If a liquid secretion was in a small, glass aquarium (approximately present in the plastic bag, I collected it for the 25x50x25cm) with flowing sea water. Once toxicity assessment either by pouring it into a placed in the tank, I left the sea cucumbers graduated cylinder or by removing it from the alone for approximately 15 minutes to bag with a syringe. acclimate. At this time I took photographs for To test toxicity I performed a series of identification and voucher purposes, and I brine shrimp bioassays. I raised brine shrimp took physical measurement in an undisturbed using a salt water solution of 2 tablespoons sea state. I measured length and width of the sea rock salt and 1L of tap water. I measured cucumbers using a measuring tape and I 0.5mL of either Cuverian tubules, liquid measured volume using a large graduated secretion, external body, or internal body parts plastic container. by filling a graduated cylinder with 10mL salt I simulated a predator attack by water and adding the desired part until the mechanically stimulating the sea cucumbers water line read 10.5mL. The salt water using large, blunt-tipped, metal forceps to solution used was of the same concentration pinch the dorsal integument. I described and as the brine shrimp were raised in. I then recorded the response as well as the effort it poured the contents of the graduated cylinder took to elicit a response. If Cuverian tubules into a Petri dish and added 25 live brine were expelled, I removed the tubules and shrimp. I recorded the number of live brine placed them in a plastic cup using forceps and shrimp in each Petri dish after one hour and at while wearing inverted latex gloves (the multiple hours for a 24 hour period. adhesive tubules stuck to most surfaces, but I was able to remove them most easily from the Analysis smooth latex). I counted the number of times tubules were expelled as well as the The data collected from the bioassays approximate number of tubules expelled each were analyzed in a number of ways. First I time. If tubules were not expelled as a result of looked at mortality after one hour by pinching with forceps, I turned the sea subtracting the number of live brine shrimp in cucumbers over and poked their ventral side each test from the number of live brine shrimp either with forceps or my fingers. I then in the control to get the number of brine picked up the individuals and gently shrimp that died as a result of the test constricted along the body with my hands. substance. I then did three one-way Anova Lastly, I removed the cucumbers from the analyses comparing the various genera, water and held them momentarily. For the species, and sea cucumber parts to level of mortality. Additionally, I did a regression analysis comparing the volume of the black with soft spikes all over. They are thin individual ea cucumbers with mortality after and can reach a length of over 50cm. Every one hour. individual I observed was almost entirely To determine the estimated median lethal hidden under rock and when I tried to pick time (LT50), I extrapolated when mortality them up they became wedged under so tightly equals 50% for each assay. I repeated the same that it was impossible to collect them except three one-way Anova analyses for LT50 as when I was able to pick up the entire rock with mortality after one hour. itself. Pearse observed H. leucospilota at four Additional Anova analysis and regression locations around Moorea; always completely were done with Holothuria atra comparing or partially under either rubble or rock. volume and toxicity. Two individuals of Holothuria fuscogilva Finally, I used a phylogeny of Moorean were found; one light grayish and the other sea cucumbers created by K. Tremain, 2005 brownish purple. The lighter one was found and adapted it for the species included in this on coral sand between coral heads and the study. I overlaid the presence of Cuverian darker one was found partially under an tubules, a secretion, and various other overhang of coral. Both were difficult to see as physical characteristics onto the tree using they blended in with their surroundings. MacClade software. Pearse does not list this species, but another study conducted in the Opunohu Bay of RESULTS Moorea did note its presence (Parmentier, 2005). Collection and initial observations The second most abundant species encountered was Bohadschia argus (Jaeger I surveyed eight sites with varying 1833). I found three color morphs of this habitats and found 15 different species and species, but only included two in this study: a morphs of sea cucumbers in three different light purple with dark spots and a dark families (Appendix 1). Twelve species and burgundy with orange spots. The third color morphs are included in this study. The most morph was marbled brown with dark brown abundant species encountered was Holothuria spots. This species was found in all but two (Halodeima) atra Jaeger, 1833. I found H. atra at sites. It was also found in all but two habitats every site and in almost every habitat type. A which had shallow water less than 0.5m deep previous survey of the island conducted by J. with no live coral. Pearse observed the two Pearse in 1989 encountered this species and color morphs included in this study and noted noted that it was “probably the most that while both were abundant, the darker abundant and common echinoderm in French morph was encountered more often. During Polynesia” (Pearse, 1989). These small to collection I observed many individuals medium sized sea cucumbers are almost black expelling Cuverian tubules when disturbed in color and are often seen with sand stuck to either purposely or accidentally by me. About their bodies. When touched, most large one third to half of the total individuals individuals secreted a burgundy liquid observed were partially obscured either by through their skin at the place of contact. The sand or by coral. individuals ranged in size from seven cm to I also collected individuals of Bohadschia 27cm in length. I split the species into two marmorata?, Bohadschia vitiensis marmorata, and groups according to size in order to Bohadschia spp.. Pearse describes the species I investigate the possible relationship between am referring to as B. vitiensis marmorata as B. size and predator defence. vitiensis but notes that it is also referred to as Two other species of Holothuria were B. marmorata in Connon & Silver, 1986. It is observed: Holothuria leucospilota Clark, 1920 possible that these two species are in fact the and Holothuria fuscogilva Cherbonnier, 1980. I same species, but for the purposes of this only encountered Holothuria leucospilota in the study I consider them two different species. very shallow calcium carbonate composite of All species of Bohadschia were found in areas Temae. Individuals of this species are almost with sandy substrate and scattered coral heads. Some individuals were found in areas the paper became burgundy. After being of predominantly coral and coral rubble and a placed in a plastic bag and frozen, a small few were found in shallow water with sandy amount of burgundy liquid could be obtained substrate and with no nearby coral heads. from the plastic bag. Also, while collecting I observed individuals Like H. atra, H. leucospilota erected spikes from each species expell Cuverian tubules all over and pulled in their tentacles. They when provoked. All of the above species of became slightly shorter and more robust. One Holothuria and Bohadschia belong to the family individual was very active and tried to escape, Holothuriidae. but the other three showed little or no Two genera of the family Synaptidae were movement when being physically stimulated. encountered: Chiridota and Synapta. I collected A single individual expelled a few very thin Chiridota sp., tiny pink sea cucumbers 0.5 to Cuverian tubules when first disturbed. I five centimeters long, by sifting through the removed the tubules but could not get it to fine coral sand with my fingers. They were expel again. I did not observe a secretion buried two to six centimeters in the sand. during my observations, but after being frozen After collecting them my fingers were pink a secretion as observed in the plastic bag. where I had touched them. Pearse describes C. I did not observe any of the behaviors rigida from two locations around Moorea, exhibited by H. atra or H. leucospilota in H. however a study conducted in 2005 describes fuscogilva; except clinging to the substrate. The C. hawaiiensis from the same location I two individuals of H. fuscogilva were encountered them (Tremain, 2005). I believe extremely hard and I could not deform their that the same species, Chiridota spp., was shape in any way. During the dissection, I observed in the two previous studies. could not cut the tegument with scissors at all The second synaptid encountered is and it was very difficult to do so with a razor Synapta maculata. Thos species is long, blade. Their texture was also rough like coarse snakelike, and very active. Their integument sandpaper. feels and acts like Velcro hooks when touched; All the individuals belonging to the genus clinging even to latex gloves. In contrast to Bohadschia erected small spikes all over their Chiridota, this species was observed to reach bodies. These spikes were in the center of their lengths of over 130 centimeters. spots, except in B. vitiensis marmorata which Thenelota ananas is the largest of the does not have spots. Bohadschia sp. remained species I encountered and the only member of soft as it erected its spikes while all others the family Stichopodidae. They are grayish became more rigid. All but one individual beige in color with many forked spikes that, as expelled Cuverian tubules when physically the specific name suggests, resemble a stimulated. Bohadschia argus (dark), B. vitiensis pineapple. They also have a soft, bright marmorata, and B. marmorata? curved their pinkish-orange underside. anal end back in an upright “C” position and expelled Cuverian tubules toward the point Individual and behavioral observations where the physical stimulation was being exerted. One individual of B. argus (light) When physically disturbed, large made a slight motion and B. sp. made no individuals of H. atra pulled in their tentacles motion while expelling tubules. All used for feeding, erected spikes all over their individuals pushed water through their anal bodies, stuck to the glass of the aquarium, and opening as they were expelling tubules which secreted a burgundy liquid. Some individuals made the tubules move and appear greater in were active and attempted to escape, however number. most showed little or no movement. Small When disturbed with forceps in a Petri individuals of H. atra also erected spikes all dish full of water and no sand, individuals of over their bodies and stuck to the glass. They Chiridota sp. changed their shape slightly and were slightly more active than the larger move away from the disturbance, but did not individuals. I did not observe a liquid being appear to do anything else. In their natural secreted, but when wiped with tissue paper setting they were observed to burrow under A regression analysis showed that volume the sand. of the individual is correlated with mortality Synapta maculata were extremely active after one hour (p<0.0001). Additionally, a and move in a snake-like manner away from oneway Anova showed that individual the disturbance. When touched they clung to volume and species are highly correlated my gloves and often left tiny, hook-like (p<0.0001) as well. structures behind. I did not observe any other According to the calculated median defense mechanisms. lethal times (LT50), H. atra (large) displayed the highest mortality rate followed by T. KEY TO SPECIES NAMES ananas and B. marmorata, and the lowest was 1 Bohadschia argus (dark) displayed by H. atra (small). The oneway 2 Bohadschia argus (light) anova analyses showed a significant 3 Bohadschia marmorata? difference between LT50s at the species level 4 Bohadschia vitiensis marmorata (p<0.0001), a slight difference at the genus 5 Bohadschia spp. 6 Thenelota ananas 7a Holothuria atra (small) 20

7b Holothuria atra (large) 15 7 Holothuria leucospilota 8 Holothuria fuscogilva 10

9 Synapta maculata 5 Thenelota ananas do not appear to respond 0 to physical stimulation other than by clinging Number of Dead Brine Shrimp to the substrate and becoming slightly more 1 10 11 2 3 4 5 6 7 a 7 b 89 rigid. However, when the stimulation ceased Species they moved away from the location where it Figure 3: Comparing mortality after 1 hour took place relatively quickly. Fish, belonging between species. to the family Carapidae, were found in every individual of T. ananas dissected. One individual had two carapid fish inside it; one 20 of which was 22 cm long, or almost 40% of the 15 total body length. 10 Toxicity assessment 5

After one hour, the highest mortality was 0 Number of Dead Brine Shrimp observed in T. ananas, followed by B. Bohadschia Chiridota Synapta marmorata, and S. maculata and the least Holothuria Thene Genus mortality was observed in H. atra (small) (Figure 2). The highest mortality of all the Figure 2: Comparing mortality after 1 hour individual parts was observed in the external between genera. body parts of T. ananas and internal body level(p=0.049), and very little difference parts of S. maculata. among parts independent of taxa (p=0.25). The oneway anova analyses for variance Again, there was no significant difference showed that there is a significant difference in between the genera Bohadschia and Holothuria. mortality after one hour at both the genus and There is a significant difference in the species level (p is <0.0001 for both) (Figures 3 toxicity between large and small individuals and 4). However when comparing only the of Holothuria atra observed in both the genera Bohadschia and Holothuria, there is no mortality after 1 hour and LT50 (p<0.0001 for significant difference in mortality after one both) with larger individuals being much hour (p=0.32). more toxic. A regression analysis showed a 25

20

15 LT50 10

5

0 1 10 11 2 3 4 5 6 7 a 7 b 89 FIG. 5. Presence of Cuverian tubules.

Species Figure 4: Comparing LT50 between species. strong correlation (p<0.0001) between size and mortality after one hour. Anova analyses for large individuals showed no significant difference in mortality after one hour between secretions collected off of live individuals and from the bag after freezing (p=0.46). Thus being frozen did not have an effect on toxicity. No significant difference was observed in mortality after one hour between the secretions of individuals characterized as FIG. 6. Presence of a red, liquid secretion. either large or small (p=0.16).

Phylogeny

The presence of Cuverian tubules, a red liquid secretion, an active flight response, and a thickened tegument were mapped onto the phylogeny adapted from Tremain 2005. The phylogeny was originally created using 27 morphological and ecological characteristics (Tremain, 2005). The placement of Thenelota ananas between Bohadschia and Holothuria is FIG. 7. Presence of an active flight somewhat problematic because Thenelota belongs to the family Stichopodidae while the other two belong to the family Holothuriidae. However, for the purposes of this study the phylogeny is still very useful. Eight of the twelve species in Tremain’s study were present in this one. I omitted one species from the phylogeny and substituted a few of the species in this study for closely related species. For instance, I replaced Holothuria (Lessonothuria) with Holothuria fuscogilva, and Bohadschia unknown spp. K with Bohadschia spp. FIG. 8. Presence of a thickened tegument.

DISCUSSION but the regeneration of the intestinal tract is one of the most studied in regeration studies Sea cucumbers as a group have a wide of holothurians (Van Den Spiegel, 2000). variety of predator defense mechanisms and Immediately after evisceration, the sea most species utilize one or more (Bingham, cucumber rapidly crawled away from its 1986). I observed that individual species not intestines. This is an extremely effective only possess multiple mechanisms, but they defense behavior especially for predators that exhibit these mechanisms to varying degrees. prefer to only eat part of the cucumber, such The most striking of the defense mechanisms as fish or crustaceans (Francour, 1997). The observed was the expulsion of Cuverian predator is distracted by the intestines and the tubules. All members of the genus Bohasdchia sea cucumber has a chance to escape, however that I observed expelled tubules, but not all this is an extremely high energy cost to the the tubules produced the same mortality in cucumber and that is probably why I only the bioassays. For instance tubules from B. observed it after persistent agitation and argus (light) produced relatively high levels of removal of all Cuverian tubules. mortality after one hour while tubules from B. Holothuria leucospilota, the only species argus (dark) produced relatively low levels. It other than members of Bohadschia possessing is possible that the toxicity of the tubules Cuverian tubules, possessed multiple defense themselves may not be very important for mechanisms yet did not respond very much to their function in predator deterrence. It has agitation. Both Cuverian tubules and a liquid been noted that while the tubules do possess secretion were present in this species, but toxins (Parmentier, 2005), they function neither were utilized to a great extent during primarily by entangling and immobilizing this study. A previous study of H. leucospilota potential predators (DeMoor et. al, 2003). observed evidence of expulsion of Cuverian In addition to Cuverian tubules, the tubules in 2.3-6.1% of individuals in the field coloration of B. argus may also play a role in (Hamel, 2000), showing that they do in fact predator avoidance. Depending on the utilize this mechanism in the wild. Toxicity of substrate, the light or dark color morphs of this species is moderate compared to the other Bohadschia argus may be cryptic. The lighter species in this study. Holothuria leucospilota color morph blends in better with the calcium was observed to wedge itself under rocks to carbonate sand and light colored corals, while avoid predation. This is most likely a very the darker color morph blends in more with successful behavior for avoiding larger darker corals. However both morphs were predators and the moderate toxicity may be found out in the open, near all types of coral, enough to deter smaller predators. and partially buried in the sand. In addition to A significant difference in the toxicity color, the spots may serve to disrupt the between large and small individuals of overall shape of the cucumber and make it Holothuria atra was observed in both the even more cryptic; or the spots may be mortality after 1 hour and LT50. This suggests aposematic, warning potential predators to that there is an increase in toxicity as the stay away. The two are considered different individual ages. Individuals of all sizes were color morphs of the same species and a further observed out in the open and were usually look into how color is determined could covered in sand in the wild. Further study into provide more insight into the relationship the ontogeny of the species and any between color and predator defense. behavioral differences might provide more Bohadschia vitiensis marmorata did not insight into why larger individuals are more produce particularly high mortality rates in toxic. the toxicity assessment, however it expelled Holothuria fuscogilva is the most cryptically Cuverian tubules readily and in large colored of all the species observed in this quantity. One individual even eviscerated its study. I only encountered two, but it is likely entire digestive tract when I agitated it after that there were more because it was found in a having removed all the Cuverian tubules. I relatively common habitat type. The bioassays only observed this behavior in one individual, showed that it is only moderately toxic, however it is unlikely that many predators can were present in Holothuria and then were penetrate its extremely rough, thick subsequently lost by multiple species or integument. lineages. A previous phylogenetic study of the Thenelota ananas, the most toxic of the family Holothuriidae which included eight species studies, exhibited relatively passive species and five genera suggests that Cuverian forms of predator deterrence by becoming tubules were present before the initial slightly more rigid and clinging to the radiation of the family and then lost by substrate. It is possible that the spikey various members (Kerr et. al, 2005). The appearance suggests to predators that, results of this study support Kerr et. al’s although it is cryptically colored, T. ananas is findings. Kerr et. al also state that because a unpalatable. Another explanation for the basal lineage of the family possesses Cuverian spikey texture, in conjunction with a tubules which are small, not adhesive, and thickened integument, is to make it difficult rarely expelled, Cuverian tubules may have for potential predators to penetrate into the evolved for some other function than defense body. The bright underside may also serve in (Kerr et. al, 2005). predator defense by being aposematic, The red secretion was only observed in however I did not observe any behaviors that the species Holothuria atra and Holothuria might support this. leucospilota. According to the phylogeny Thenelota ananas is also the largest of the examined, this specific secretion probably species encountered and while a regression only evolved once. Thickened skin appears to analysis showed a strong positive correlation be characteristic of Bohadschia and has evolved between volume of the individual and multiple times in other species as well. Having toxicity, volume and species are also strongly an active flight response, on the other hand correlated. Therefore, it is uncertain whether appears to not necessarily be related to a individual size plays a role in toxicity, or if species’ phylogenetic history, but rather it both size and toxicity can be attributed to the varies from species to species and is most species independent of the other. likely a result of the particular ecology and life The four carapid fish found in three history of the animal. specimens of T. ananas is probably related to A total of 12 species and morphotypes the large size and absence of Cuverian were examined in this study and different tubules. A study of carapid fish in the defense mechanisms and levels of toxicity Opunohu Bay of Moorea found fish in 81% of were observed in each. Inheritance and T. ananas examined. The fish are generally phylogenetic relationships appear to play a found in the respiratory tree where they take large role in determining what mechanisms advantage of the water flow and if the are present. However the expression of the diameter of the respiratory tree is too small traits and utilization of certain mechanisms is the fish cannot penetrate into it. Thenelota also influenced by the individual’s ecology; ananas was observed in this study to have a thus there appears to be a degree of relatively high level of toxicity, however the phenotypic plasticity among the species. carapid fish are particularly resistant to sea Future studies including introducing cucumber toxins (Parmentier, 2005). Even potential predators such as asteroids, Cuverian tubules prove to be an ineffective crustaceans, and gastropods into a controlled defense mechanism against these well adapted setting may provide additional knowledge of fish as they are covered in mucus which keeps holothurian defense behavior. For example, a the tubules from adhering to them particular species may respond differently to (Parmentier, 2005). One small fish was found different predators and thus the presence of in a specimen of Holothuria leucospilota as well. multiple defense mechanisms within a single Comparing the presence and absence of species may be explained. Also, many of the Cuverian tubules to a phylogenetic study phylogenetic relationships within performed in 2005 suggests that Cuverian Holothuroidea remain unresolved. This study tubules either evolved separately in Bohadschia serves as a baseline for further study into sea and H. leucospilota, or more likely that they cucumber defense as well their ecology in Echinodermata. Springer-Verlag Berlin general. Heidelberg. 2005. Francour P. 1997. Predation on Holothurians: A Literature Review. Invertebrate Biology ACKNOWLEDGMENTS 116:52-60. Guille, A. Guide des etoiles de mer, oursins et I thank Jacques and Tony of the UC autres echinoderms du lagon de Nouvelle- Berkeley Gump station for taking me out on Caledonie. Collection Faune tropicale, no the boat to my study sites. I thank Zachary 25. Paris. 1986. Hanna for all his help finding cryptic sea Hamel J. -., A. Mercier. 2000. Cuvierian cucumbers that I would have otherwise tubules in tropical holothurians: missed and for diving down to get all the ones usefulness and efficiency as a defence I could not reach. Also, I thank Jaime mechanism. 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Maintaining the Line of Defense: Regeneration of Cuvierian

APPENDICES

APPENDIX A: Study sites and species encountered. West of Cook's Cook's Mangrove East Sheraton Bay at Bay Marsh North side of b/w Gump Reef near side of Motu PK14 & Genus Species Station Crest Haapiti Motu Tiahura Temae Tetearoa PK15 vitiensis Bohadschia marmorata x x

Holothuria leucospilota x Holothuria atra x x x x x x x x Thenelota ananas x x Synapta maculata x x Chiridota spp. x x

Bohadschia argus (dark) x x x x x x x

Bohadschia argus (light) x x x x x x x Bohadschia marmorata x x Bohadschia spp. x x Holothuria fuscogilva x

1 Temae: Composite CaCO3 near shore, predominantly coral with some sandy bottom further. 2 Cook's Bay at Gump Station: Sand bottom with coral rubble and scattered coral heads. 3 Cooks Bay near Pihaena Point: Sand bottom with coral rubble and scattered coral heads 4 West of Sheraton between PK14 & 15: Composite CaCO3 near shore, predominantly coral with some sandy bottom further. 5 a: East side of Motu Tiahura: Fine coral sand. b,c: North side of Motu: Sand and coral rubble bottom with scattered, large coral heads. Deep water ~1.5m 6 Mangrove marsh near Haapiti: Fine sand and sediment bottom. Scattered mangrove trees and occassional coral . 7 Tetearoa: Fine sand and sediment bottom. Scattered mangrove trees and occassional coral

APPENDIX B: Physical and behavioral characteristics observed by species.

Species Cuverian Secretion Thickened Active Change Retractable Burrowing/ Volume Volume Tubules Present body wall Escape in mouthparts Hiding >500mL >1000mL Expelled Behavior Shape/ under rock Texture behavior Bohadschia 1 0 1 1 1 1 1 1 1 argus (dark) Bohadschia 1 0 1 1 1 1 1 1 1 argus (light) Bohadschia 1 0 1 0 1 1 1 1 1 marmorata Bohadschia 1 0 1 0 1 1 1 1 0 spp. Bohadschia 1 0 1 1 1 1 1 1 0 vitiensis marmorata Chiridota spp. 0 1 0 1 0 1 1 0 0 Holothuria atra 0 1 0 0 1 1 1 1 0 (large) Holothuria atra 0 1 0 0 1 1 0 0 0 (small) Holothuria 0 0 1 0 0 1 0 1 1 fuscogilva Holothuria 1 1 0 1 1 1 1 0 0 leucospilota Synapta 0 0 0 1 1 1 0 0 0 maculata Thenelota 0 0 1 1 0 1 0 1 1 ananas

APPENDIX C: Characteristics of habitats inhabited by species of holothurians.

Species Shallow Shallow ~1m ~1m 1-2m 4-6 m sandy (<0.5m) (<0.5m) predominately coral sand/coral substrate w/ CaCO3 coral sand coral heads sand rubble w/ few coral Composite substrate and rubble w/ substrate scattered heads little sand w/ few coral coral heads heads Bohadschia argus 0 0 1 1 1 1 (dark) Bohadschia argus 0 0 1 1 1 1 (light) Bohadschia 0 0 0 1 1 0 marmorata Bohadschia spp. 0 0 0 1 1 0 Bohadschia vitiensis 0 0 0 1 1 0 marmorata Chiridota spp. 0 1 0 0 0 0 Holothuria atra 1 1 1 1 1 0 (large) Holothuria atra 1 1 1 1 1 0 (small) Holothuria 0 0 0 0 1 0 fuscogilva Holothuria 1 1 0 0 0 0 leucospilota Synapta maculata 1 0 1 1 0 0 Thenelota ananas 0 0 0 0 0 1

APPENDIX D: Species List and Photos

Bohadschia argus (dark) Bohadschia argus (light)

Bohadschia marmorata? Bohadschia vitiensis marmorata

Bohadschia spp. Chiridota spp.

Holothuria atra Holothuria fuscogilva

Holothuria fuscogilva Holothuria leucospilota

Synapta maculata Thenelota ananas