THE ROLE OF ADAPTIVE IMM1JNITY IN THE PREDATORY

BEHAVIOR OF DERHASTERIAS

A Thesis Presented to the Graduate Faculty

of

California State University, Hayward

In Partial Fulfillment

of the Requirements for the Degree

Master of Science in Biology

By

James M. Willard

December, 1981 ABSTRACT

Observations on the feeding behavior of Dermasterias

imbricata on sea anemones suggested a hypothesis that

these sea stars developed an immune response to counter­

act the toxic effects of the prey organisms. The hypothe­

sis was tested by attempting to demonstrate the existence

of an immune system involving 1) an inducible memory

response, 2) a cytotoxic or antagonistic response upon sen­

sitization, and 3) immunogenic specificity.

A memory response of three weeks duration was demon­

strated by re-expos1ng sea stars to sea anemones at ever

increasing intervals. With the onset of the fourth week

a drastic change in response time occurred, from a

response of 1.0 + 1.16 hr. to a response of 58.0 + 1.80 hr.

In vitro studies of sea anemone toxin neutralization using

coelomic fluid from immunized versus naive donors demon­

strated the existence of antagonism. Gastropod righting

response times, utilizing Mitra idae, were not signifi­

cantly affected by toxin incubated in whole coelomic

fluid from s~nsitized donors, (2.31 ! 0.37 minutes versus

1.65 + 0.38 minutes for the controls), while naive sea star whole coelomic fluid could only lessen, not eliminate

toxic effects (18.5 ~ 6.06 minutes versus 49.45 + 16.18

minutes for pure toxin injected snails).

ii iii

Antagonism was further demonstrated by the administra­ tion of the immunosuppressant drug, azathioprine, which prevented sea star feeding on sea anemones while not affecting feeding on squid. Coelomic fluid transfers from immunized donors accelerated sea star feeding rates by as much as 12 times (1.33 + 0.99 hr. versus 16.0 + 0 hr. in untreated sea stars).

Specificity experiments further suggest the existence of an immune response by the sea star. Dermasterias imbricata exposed to Corynactis californica were not able to feed on Anthopleura elegantissima in the time limits indicative of immunized behavior. Identical results were obtained in the reciprocal experiment with sea stars ini­ tially exposed to A. elegantissima, then exposed to

C. californica.

These results strongly suggest that Dermasterias imbricata utilizes its immune system to mediate its feeding response to sea anemones. Since sea anemones are a major part of D. imbricata's diet, this interac­ tion is of particular interest and significance in con­ sideration of the development of invertebrate immune systems and their relationships to the behavior of the animals. THE OF ADAPTIVE IMMD~I IN THE PREDATORY

BEHAVIOR OF ;...... ,;;;.;;;.,;;;;;.;;.;;..;;...;;....;;;...... ;;;...;__;.;;.._ _;...... ,....,...... ;~=

By

s M. 1!Jillard ACKNOWLEDGMENTS

In any published study, there are always many people

1n the background whose support the author found indis­ pensable in one way or another. This study is no different, and I would like to sincerely thank the following people, without whom this study would never have been done.

First, I would like to thank the members of my com­ mittee, Drs. Henry Hilgard, Edward B. Lyke, James

Nybakken, and Richard Tullis. Extra thanks to Dr. Lyke for advice, equipment, encouragement, and insightful comments. Drs. Hilgard and Tullis contributed critical

comments which greatly improved the quality of my study.

Special thanks to Dr. Ann Hurley who not only helped me

start this study, but whose classes taught me how to think

scientifically. Also, special thanks to Cynthia Annett, who helped fan the spark with her belief and advice.

Drs. Hildemann and Bigger of U.C.L.A. provided useful

suggestions and insights. Thanks also go out to Barbara

Baldwin and all the other patient people, especially

Sheila Baldridge, who have listened, encouraged, helped

in collecting, and many other ways, and are unfortunately

too numerous to mention.

v TABLE OF CONTENTS

Pa e

ABSTRACT . . . . ii

ACKNOWLEDGHENTS v

LIST OF TABLES AND FIGURES viii Chapter

1. INTRODUCTION ...... 1

2 • GENERAL METHODS AND MATERIALS 4

Experimental Animals . . . . . 4 Isolation of Corynactis californica Nematocyst Toxins 4

Marking Sea Stars 6

3. EXPERIMENTAL METHODS AND MATERIALS 8

Immunological memory 8

Immunosuppressant Effects 9

Transferability of Immunity 10

Toxin Bioassay and Coelomocyte Studies . 10

Bioassay 10

Coelomocyte Studies 11

Phagocytosis of Carmine Red 11

Neutralization of Toxin 12

Diversity and Specificity of the Immune response 13

4. RESULTS 15

Immunological Memory 15

Immunosuppressant Effects 16

vi vii

Chapter

Immunity Transfer Experiments . 20

Toxin Bioassay and Coelomocyte Studies. 20

Bioassay . 20

Coelomocyte Studies 20

Phagocytosis of Carmine Red 20

Neutralization of Toxin 23 Diversity and Specificity of the Immune Response 26

5. DISCUSSION 29

LITER.I\TURE CITED 35 LIST OF TABLES AND FIGURES

Table

1. Feeding response times of Dermasterias imbricata on Corynactis californica 18

2. Immunosuppressant drug effects on Dermastrias imbricata's feeding response to Co s californica 19

3. Effect of coelomic fluid transfers from immunized to non-immunized sea stars. 21

4. Righting time response of Mitra idae following toxin injection . 22

5. Effects of coelomic fluid on toxin activity as measured by Mitra idae righting time response . . . 25

6. Diversity of predatory responsiveness of rmasterias imbricata to toxic prey. 27

7. Specificity of s~nsitized Dermasterias imbricata predatory enhancement . 28

Figure

1. Feeding response times of Dermasterias imbricata to Corynactis californica . 17

2. Coelomocytes of Derrnasterias imbricata with phagocytized carmine red particles (x 400). 24

viii Chapter 1

INTRODUCTION

Interactions between species are very important ecologically. Those involving predators and their prey are of considerable interest, and investigations have taken several approaches, including theoretical (Ernlen,

1966; Slobodkin, 1968; Emlen and Ernlen, 1975); the role of predation in community structuring (Paine, 1966;

Connell, 1970); behavioral (Krebs, 1978); and physiological

(Vadas, 1978). The study reported on here involves a predator that utilizes its immune system to mediate its predatory behavior.

Dermasterias imbricata, the leather sea star, is commonly found in rocky areas on the West Coast of

North America (Morris, et al., 1980). In the Monterey

Bay region, where this study was carried out, D. imbricata is uncommon. It is primarily associated with a corallimorpharian sea anemone, Co ctis californica.

Corallimorpharians are known for possessing large holotrichous isorhiza nematocysts (Hand, 1954). These organelles contain toxins and are effective defense mechanisms for the cnidarians (Hyman, 1940; Blanquet,

1968; Mariscal, 1974). D. imbricata appears to be the sole predator of C. californica in the Monterey Bay region.

1 2

This study resulted from some unusual behavioral observations obtained in a class project for Behavior of

Marine Animals. On initial encounters with C. californica

D. imbricata was repulsed, and even occasionally paralyzed.

However, on future encounters, the sea stars were un- harmed by contacts with anemones. The behavioral se- quence involved in these encounters was investigated and led to the following hypothesis: Dermasterias imbricata utilizes an immunological response to mediate its pre datory encounters with toxic prey.

To test the hypothesis, a testable definition of an iiJUnune response -..-.ras required. Hildemann, et al.

(1979) provided the basis for such a definition ln invertebrate immune systems by suggesting an immune response must display: l) an inducible memory response,

2) a cytotoxic or antagonistic response upon sensiti­ zation, and 3) immunogenic specificity. These were the components chosen as criteria for demonstrating an adaptive immune system in D. imbricata.

Few studies have been performed on invertebrate

responsiveness to immunogenic proteins. Despite this,

it has been well demonstrated that invertebrates are able to respond effectively and specifically to allO­ grafts (Hi1demann, 1979), various bacterial antigens

(Bang, 1975; Cohen, 1975; Foley and Cheng, 1975), and 3

Tripp, 1975). Phillips and Yardley (1960) were able to

induce in the sea anemone elegantissima a factor

that could effect ly compete with rabbit anti-sera

for binding bovine globulins. However, they never

isolated the factor(s) responsible. Hilgard et al.

(1967) examined the uptake of bovine and human proteins

in the sea urchin purpuratus. They

demonstrated that the coelomocytes were the agents for clearing the prote from the coelomic fluid. However,

they did not demonstrate inducibility or memory after

sensitization to these proteins. Some specificity was

established through competition experiments, but the

authors could not clearly relate the clearance response

to an immunological response. In part, their failure to

demonstrate ecificity or memory may be due to the use

of vertebrate proteins as immunizing agents. Perhaps

the use of molecules the sea stars were exposed to in

nature would have been a better choice for immunizing

agents.

The present study, performed from January 1980 to

June 1981 at the Moss Landing Marine Laboratories,

attempted to explain some of the peculiarities of

invertebrate immune responses and to demonstrate con­

clusively that an immune system was operational in

interactions between the sea star Dermasterias icata

and the sea anemone Corynactis _c_a_l__ ~~--~ Chapter 2

~~TERIALS AND METHODS

The animals utilized for the experiments reported here were all collected from the Monterey Bay region of

California. Both Dermasterias irnbricata and Co act is c lifornica were collected in dives from the Monterey

Breakwater and the Stilhvater Cove areas off the ~'1onterey

Peninsula. Anth leura ele antissima used for experi­ mental purposes were collected from the Moss Landing

Jetty's intertidal zone at low tides. All other experi­ mental animals were collected by divers in Stillwater

Cove. Once animals were collected, they were maintained separately in 20 gallon aquaria with running seawater at the Moss Landing Marine Labs. D. imbricata utilized for experiments ranged in size from a radius of 4.5 em and a mass of 16 grams to a radius of 11 em and a mass of 200 grams. C. californica individuals were all approximately

2.5 em high and 1 em in diameter. A. elegantissima ranged in size from a radius of 1 em, with a mass of 3.5 grams, to a 3.5 ern radius, with a mass of 16 grams.

Isolation of Corynactis californica Nematocyst Toxins

The nematocyst toxin of Corynactis californica was isolated utilizing a modification of a method devised by

4 5

Phillips (1956) for isolating Metridium senile nematocyst toxins. This method entailed the homogenization of whole anemones in at least twice their volume of 1 M sucrose/ seawater solution in a tissue grinder. The purpose of the sucrose solution was to prevent the discharge of the nematocysts by osmotic stress (Phillips, 1956). The homogenate was then filtered through a graded series of polyethylene screens, with openings of 1.24 mm, 0.508 mm,

0.286 mm, and 0.157 mm, to remove most of the tissue fragments and large cellular materials. The remaining debris and intracellular materials were stored at 0°C without affecting the toxin's activity.

Since nematocysts are the largest intracellular structures (Mariscal, 1974), they are among the first to settle in a centrifugation. Therefore, several low speed-short time centrifugations were performed at approximately 150 x g alternating with short, 1 minute centrifugations to settle out sand and other heavier debris. The supernatant was collected after each centri­ fugation and saved for later assay of toxicity, and the pellet was resuspended and washed in the sucrose/seawater solution. Washing, resuspension, and centrifugations of the pellet were continued until microscopic examination of the pellet revealed only pure nematocysts.

Once the pure nematocysts were collected, they were discharged and used immediately, as a loss of total 6

activity occurs with 24 hours. Discharge was accom­ plished by adding distilled water to the pellet, thus causlng the nematocysts to rupture, thereby discharging the contents (Phillips, 1956). The supernatant containing the toxin was then utilizable for assays or immunological work.

Marking Sea Stars

It was useful to be able to identify individual animals in order to take individual differences into account through time. In the work performed here, indi- vidual sea stars were marked by using a nylon thread put through the integument of the aboral surface and attaching a small numbered square of underwater paper.

This method was quite satisfactory and caused no noticeable discomfort to the tagged individuals. Dermal branchiae were not interfered with, and the sea stars displayed no signs of stress. An attempt was made to mark Dermasterias imbricata with various dyes as sug­ gested by Feder (1955). Neutral red, aniline blue, and nile blue sulfate A all caused necrosis and sloughing of

the dermal layers on the aboral surface, the entir~

surface turned white, and dermal branchiae were no longer

extruded. Also, when dyed individuals were returned to

the holding tanks, unmarked stars would attack them, and

in one instance cannibalism occurred since the star was not rescued quickly enough. 7 Radii measurements and weights of all tagged sea stars were also recorded. Chapter 3

EXPERIMENTAL METHODS AND MATERIALS

Immunolo ical Memo

An important component of any immune response is memory. Short-term memory in invertebrates is char acterized by enhanced responsiveness to an immunological challenge that lasts usually not more than 40 days

(Hildemann et al., 1980). Long term memory lasts over 40 days, and may endure for years or perhaps even throughout the organism's lifetime (Hildemann et al., 1980).

Memory extent was determined by utilizing sea stars not feeding on anemones when collected. These sea stars could either be nonfeeding or feeding on other prey. Sea stars were then maintained in the laboratory for two months without contact with sea anemones. Thus, sea stars were starved throughout the maintenance interval until being exposed to sea anemones in an encounter chamber. The chambers used were 20 em long x 20 em wide x 15 em deep. They were open on top, and running sea water entered through an opening approximately 1 em above the bottom and 4 mm in diameter. Anemones were placed near the water intake and allowed to fully expand. Sea stars were then placed in the chambers upcurrent of the sea anemones, and the time until feeding began was recorded. The experiment was repeated with the same

8 9

sea star at increased weekly intervals until feeding

times equaled those of first exposure sea star response

times. For example, a sea star first exposed to sea

anemones on March l would be re-exposed on March 9, March

23, April 13, May 11, and June 8. The curve generated

from these observations manifests the length of memory.

Imrnunos ressant Effects

The possible influence of azathioprine, a commonly

utilized immunosuppressant drug, on Dermasterias imbricata's

predatory abilities was examined. The drug's immuno­

suppressive activity is derived from its prevention of

cell division through the inhibition of purine ring

biosynthesis (Calabresi and Parks, 1975). Dosages were

prepared equivalent to 3 mg/kg/day (Burroughs-Wellcome,

1978) and were administered with a 27 gauge needle on a

tuberculin syringe into the coelomic cavity of the sea

stars. Animals received the full dose in one injection.

For experiments lasing more than one day, the animals

received one injection each day of 3 mg/kg. Stock

solutions of 30 mg azathioprine (Sigma)/ 50 ml sterile

sea water were utilized. Controls received sterile sea water injections.

Experiments were carried out in encounter chambers.

Results were obtained by comparing predatory abilities of

treated sensitized sea stars to untreated sensitized sea

stars. Sea stars were exposed to prey one hour after

treatment. 10

Transferability of Immunity

One characteristic of an 1mmune response 1s that the components conferring immunity to one animal may be transferable to another animal (Cunningham, 1978). This would confer passive immunity on a naive sea star.

Transfer of immunity was attempted by withdrawing 0.25 ml of coelomic fluid from immunized sea stars and injecting it into a non-immunized sea star. Coelomic fluid was handled 1n a 1 ml tuberculin syringe with a 27 gauge needle. The aboral surface was swabbed with 70% ethanol before collecting the coelomic fluid. Immediately after collection, the coelomic fluid was injected into the naive sea stars. The coelomic fluid was collected from sea stars immediately after a successful predatory encounter with a sea anemone since it would be antici­ pated to be the time of the highest presence of the toxin neutralizing factor. The pre-treated sea stars were then immediately exposed to the appropriate prey.

Toxin Bioassay and Coelomocyte Studies

Bioassay. Bioassays must be developed so that they give clear results for the substances being tested. The gastropod righting response (Phillips and Abbott, 1957) was used to assay for the nernatocyst toxin isolated.

Mitra idae, a specialist predator on sipunculids that can be found near Moss Landing, which probably had not had previous exposure to, or been sensitized to, the toxins, 11 were used in this study. Another advantage of utilizing

M. idae is that it has no operculum, therefore the foot is readily accessible for injections.

Animals for the bioassay were maintained in glass jars 11 em ln diameter by 4 em deep. Average shell length forM. idae used was 3.2 em. For the assay,

animals received injections of the substances to be tested in their foot muscle. Each animal was used for

only one assay. The injections were always 0.5 ml.

Animals were then placed on their backs in the jars, and

their righting times were monitored. Controls received

injections of sterile sea water.

Cfrelomo e Studies.

Phagocytosis of Carmine Red. Carmine red particles were first suspended in sterile sea water. This suspen- sion was then injected aborally into Dermasterias imbricata

individuals. After one hour of incubation, a coelomic

fluid sample was withdrawn from the sea stars and smeared

on a slide, a coverslip was added, and the coelomocytes were examined microscopically at a magnification of 40Dx

for the presence of carmine red particles. An Olympus

Nomarski Interference Contrast binocular microscope was

utilized for photomicrographs and visual investigation of

the phagocytosis that occurred. The focal plane of the

microscope allowed for determining whether the carmine

red was internalized or bound to the outside of the

coelomocytes. 12

Neutralization 6f Toxin. The ability of the coelomic

fluid and coelomocytes to neutralize nematocyst toxins in

vitro was examined. A 0.5 ml demonstrably toxic fraction

of freshly isolated nematocyst toxin was incubated at

room temperature for one hour ln 0.5 ml of either cell

free or whole coelomic fluid. A 0.5 rnl dose of toxin was

also incubated for one hour as a control against loss of

activity.

Coelomic fluid for each set of experiments was

withdrawn from the same individual, thereby eliminating

one source of variability. Cellfree coelomic fluid was

obtained by centrifuging for 30 minutes at 150 x g. The

coelornocytes are very adherent to most surfaces (C.

·Bigger, personal communications, 1981), thus, when

decanted, the supernatant is virtually cell-free. This

was confirmed by microscopic examination of the super

natant.

In addition, the effectiveness of toxin neutrali­

zation by coelomic fluid from naive sea stars was corn­

pared to coelomic fluid from immunized sea stars. For

the experiments reported here, toxin was incubated in

coelomic fluid from naive as well as sensitized sea

stars.

After incubation for one hour, the coelomic fluid

suspension containing the toxin was injected into the

foot muscle of Mitra idae. Results were assayed for

toxic activity as in the regular toxin bioassays. This 13 allowed comparison of neutralized toxin to the standard toxin bioassays.

ecifici of the Immune Re onse

The specificity of the predatory response of Dermasteria5 imbricata was examined in two ways. First, the diversity of the response to various toxic prey was examined.

Next, the responsiveness of second contacts was explored.

For this experiment, D. imbricata were placed in an encounter chamber with one of the following potential prey: Anthopleura ele antissima, Epiactis prolifera, Tealia lofotensis, Metridium senile, Corynactis californica, Strongylocentrotus purpuratus, and

Strongylocentrotus franciscanus. Predators were mon­ itored until all had fed. Through this, the diversity of the sea stars' responsiveness to various toxins was assessable.

Specificity in immune reactions can only be examined on second contact. For this study, only sea stars sen~ sitized to Anthopleura elegantissima and Corynactis

alifornica were used, as they were the only sea anemones consistently available for research purposes.

Sea stars were placed in encounter chambers with either C. californica and allowed to become sensitized but not to feed. Sea stars were then placed in different encounter chambers with either the

same or different species of sea anemone. Sea stars were 14

allowed 3 hours to feed. This was the maximum value for feeding time obtained for first week sensitized sea stars. This value was obtained by the following formula;

Med + SMed = (1.253 x S) + Med where: S = the standard error of the mean

S d = standard error of the median me (Sakal and Rohlf, 1969). Chapter 4

RESULTS

Immunolo ical Memo

The response of Dermasterias imbricata to Corynactis californica has a definite memory component to it. On the initial encounter, D. imbricata requires a median time of 43.5 hr +a standard error (SE) of 31.42 hours

(Sakal and Rohlf, 1969). However, if the next encounter

1s a week later, D. imbricata can feed on C. californica in a median time of 1.46 +1.51 hour. The same order of magnitude for the feeding response times remains for two and three week separations of D. imbricata from C. californica. When a four week or more separation period occurs, D. imbricata once again requires a long period of sensitization in order to feed on Corynactis californica.

A time of 58 +1.8 hour is needed after 4 weeks, and a sensitization time of 58 +41.38 hour is needed after 6 weeks.

The sharp changes 1n reaction time between initial encounters and week #1 encounters and encounters of weeks

# 3 and 4 are quite apparent. During weeks #1, 2, and 3 after the initial encounters, D. imbricata is in the sensitized state and requires very little time to begin the feeding response. Initial encounters and encounters after four weeks of separation or longer require a

15 16 long period of time, approximately two days, before the immune system is fully capable of neutralizing anemone toxins. Figure 1 and Table 1 contain summaries of the data.

Immuno ressant Effects

The injection of azathioprine, an immunosuppressant drug, had a significant effect on the predatory behavior of Dermasterias imbricata. As demonstrated in Table 2, the drug prevents the feeding by D. imbricata on sea anemones for four days, while not affecting feeding on non-toxic prey. Dermasterias imbricata used for controls were able to feed on anemones in less than three hours on the average, and on non-toxic prey the longest time to feeding was 40 minutes.

Treatments had to be discontinued after four days due to prominent skin sloughing and blanching of the dermal layers in the sea stars. However, all sea stars looked normal three days after treatmments with azathio­ prine were discontinued. The drug never visibly affected the behavior of the sea stars except to prevent feeding on toxic sea anemones. Since the drug affects only cellular division and new DNA production, the immune system, and not the nervous system or and induced enzyme system, is at work here. 100

80 ::r:: ... 0c >'"'! Vl rt ... 0 60 ... .. '"Ij (]) (]) p.. f-1• ... ::s 40 q l)q

20 ...... _ __~j 0 2 3 4 5 6 (Initial Response) Weeks separated from prey Figure 1

Feeding response times of Dermasterias imbricata to Cor~nactis californica. Values are+ 1 standard error of Median Reaction Times (MRT). 18

Table

Feeding response times of Dermasterias imbricata on Corynactis californica 1-ffiT represents the time from initial contact to feeding

Weeks between Median Reaction Standard Encolinters· N Time (MRT) in hrs. Error

0 (Initial) 13 43.5 31 . 37

1 6 1.46 1. 51 2 5 4.o 3. 52 3 5 1 . 0 1 . 1 6 4 3 58.0 1 . 80

6 5 58.0 41 .38 Table 2 Immunosuppressant drug effects on Dermasterias imbricata's feeding response to Gorynactis californica. All times expressed in minutes

Experiment Control Immunosuppressant Control Immunosuppressant Number (Anemone Feeding) · (Anemone Feeding)· · (Sguid Feeding) ·~ Sguid Feeding) 1 25 5760* 15 15 2 45 5760* 10 30 3 55 5760* 28 30 4 120 5760* 40 55 5 378 5760* 40 55 6 420 5760* X 174 35 37 Standard 72.5 8.5 7.6 Error * Treatment discontinued due to condition of the sea stars. See text for further information. 20

Immunity Transfer Experiments

Upon injection of coelomic fluid from sensitized D. imbricata individuals into naive D. imbricata individuals, significant changes in the predatory abilities of the sea star were obtained. Table 3 demonstrates this enhance ment of the sea star's predatory abilities. Injection of coelomic fluid from sensitized donors allowed the naive sea stars to feed in 1.33 + .99 hour, whereas the naive sea stars receiving control injections took 16 + 0 hour to feed. This experiment clearly shows the factor res­ ponsible for the toxin neutralization is present in the coelomic fluid.

Toxin Bioass and Coel e Studies

Bioass The bioassays performed on the materials collected and hypothesized to be nematocyst toxins provided clear results. Table 4 demonstrates the large differences observed in gastropod righting times between the snails treated with nematocyst toxin and those receiving sterile seawater injections. The control group was able to right itself in 1.65 + .425 minutes, while the group rece1v1ng the toxin required 49.45 ! 18.25 minutes for righting.

Coelomo te Studies.

Phagocytosis of Carmine Red. Carmine red particles were readily phagocytized by Dermasterias imbricata 21

Table 3

Results of immunity transfer experiments utilizing coelomic fluid transfers from immunized to non-immunized sea stars

Experiment Non-Pretreated Pretreated Sea Number Sea Stars (Hrs.) Stars (Hrs.)

1 16 0.33 2 16 0.75 3 16 2.92 X 16 1.33 Standard 0 0.99 Error 22

Table 4-

Results of the toxin bioassays. Results are minutes of righting time by the gastropod Mi.tra idae.

Experiment · Nurriber Control · Toxin 0. 58 15

2 1 . 0 22

3 1. 58 25.25 4- 2.08 so 5 3.0 105 X 1.65 4-9.4-5 Standard 0.38 1 6. 1 8 Error 23 coelomocytes. Figure 2 is a photomicrograph depicting coelomocytes with phagocytized carmine red particles.

Though phagocytic capability has been demonstrated, it is not known which of the two types of coelomocytes par­ ticipates in the toxin neutralization response.

Neutralization of Toxin. Definite differences emerged in the ability of the coelomic fluid of different groups of sea stars to neutralize the nematocyst toxin.

The results can be seen in Table 5. Sensitized sea star whole coelomic fluid was able to lower toxin activity so that gastropod righting response took 2.3 + .42 minutes.

This value approaches the value for the control groups 1n the toxin bioassay (see Table 4) of 1.65 ±.425 minutes.

Sensitized sea star cell-free coelomic fluid and naive sea star whole coelomic fluid both had approximately equal abilities at lowering the toxin's activity, 23.3 ±

11.72 minutes and 18.5 ± 6.9 minutes, respectively for the snail righting times. However, neither of these two coelomic fluid preparations significantly lowered the toxin's activity as compared to the response given by the gastropods to toxin alone (see Table 4). Therefore, it is readily noticeable that a large reduction in toxin activity is achieved by incubation of the toxin with sensitized sea star whole coelomic fluid. 24 '

Figure 2

Coelomocytes of Dermasterias imbricata with phagocytized carm~ne red particles (x 400) Table 5

Results of investigations of coelomic fluid on toxin activity. Results are minutes of t to righting by the gastropod, Mitra idae..

Toxin incubated in Toxin incubated Toxin incubated in sensitized sea star in sensitized sea Experiment na sea star whole cell-free coelomic star.whole coelomic Number coelomic fluid fluid fluid 1 3 15 1.5 2 6 1 5 2.0 3 17.5 4-o 2.25 4- 26 3-5 5 4-0 18.5 23.3 2.31 Standard 6.06 6.8 0.37 Error 26

Diversity and Specificity of the Immune Response

rmasterias imbricata was able to feed on all the toxic prey offer to it. This is demonstrated in Table

6 where it is clear that the sea star was able to feed on all prey offered it. Therefore, the sea star is able to neutralize a large variety of toxins, but this diversity

responsiveness is only of use to the sea star if it continues feeding on the same prey species to which it is sensitized. This is seen in Table 7, where it can be seen that D. imbricata sensitized to one species is not able to feed on another toxic species within the period of time indicative of a response of a sensitized sea star. Therefore, the sensitization process does not apply to nematocysts or nematocyst tox s in general, but only to the nematocyst toxin of a species to which sensitization has occurred between that sea anemone species and the sea star. 27

Table 6

Diversity of predatory responsiveness of Dermasterias imbricata to toxic prey

· Dermasterias imbricata 1 s response (+ = feeding; Prey Species ·;.... = non-feeding) · · · ·

Anthopleura e gantiss +

Corynactis californica +

iactis prolifera + Metridium senile +

Strongylocentrotus franciscanus +

Strongylocentrotus purpuratus +

Tealia lofotensis + 28

Table 7

Specificity of sensitized Derrnasterias imbricata predatory enhancement.

Feeding Prey Prey Response in Sensitized Exposed 15 hours To To (fe~ding/non-feeding) Anthopleura A. egantissima 10/10 elegantissima A. elegantissima Corynactis 0/5 californica C. californica C. californica 6/6 C. californica A. elegantissima 0/5 Chapter 5

DISCUSSION

Predator-prey interactions have been equently

compared to an "arms race" (Ehrlich, 1968; Edmunds,

1974). Many of the recorded instances of predators

overcoming a potential prey's defenses involve an evolu­

tionarily derived mechanism. For example, several phytophagous insects are constantly e osed to either new mechanical hindrances evolved by plant hosts (Gilbert,

1971) or various secondary plant substances (Fraenkel,

1959; Whittaker and Feeny, 1971). Both require a res­ ponse at the genetic level, and therefore require time to spread through the population (Ehrlich and Raven, 1965).

In general, these responses are very ecific, and sometimes a repellent will become an attractant (Krieger et al., 1971). Also, many predators are able to store the secondary substances or de nse structures and utilize them for defense (Edmunds, 1974). Mammals have also evolved enzymatic mechanisms inducible by various secondary substances. However, these mechanisms are generally for dealing with certain chemical types, such as cardenolides, glycosides, or glucosides (Smith, 1968).

What would be the perfectly adapted predator? One aspect of a per ctly adapted predator would be the ability to adapt quickly to new defenses developed by

29 30 the prey. Dermasterias imbricata may be an optimal predator this re ect. A slow-moving predator of sessile prey, D. imbricata is readily able to adapt to the toxins contained in defensive structures of its prey, primarily sea anemones. This is accomplished by a system previously unknown to be involved in predatory behavior.

It involves a coelomocyte response to prey fenses, in this case nematocyst toxins, set in action by the sea stars' predatory activity. This system involves the immunologically competent cells of D. imbricata and is at present the only known instance of immune involvement in predatory sea star activities.

In order to ascertain if the immune system was involved, various aspects of D. imbricata's predatory behavior were tested for the presence of the three components involved Hildemann's (1979) de£ ition an immune system.

The present study has shown a three week memory component to be operational. The reason for the memory lasting three weeks is unknown. Unfortunately, little is known of the asteroid coelomocytes, the mediators of the immune response. Perhaps there is a three week turnover time for D. imbricata coelomocytes. The quick loss of memory between the third and fourth weeks is also a puzzling phenomenon. A much smoother transition would be anticipated. 31 The existence of an antagonistic reaction by the immunocytes is a more difficult question to investigate.

In order to answer this question, a knowledge of the immunocompetent factor is necessary. Fecent work has strongly indicated that the amoebic coelomocytes have an important immunological function. Kaneshiro and Karp

(1980), Hilgard et al. (1967), Karp and Hildemann (1976), and Bertheussen (1979) all have demonstrated that in echinoderm immune responses the phagocytic amoebocytes are intimately involved.

The present study confirmed the involvement of coelomocytes on the basis of the following results.

First, the immunosuppressant study demonstrated that prevention of cell division would inhibit D. imbricata from feeding on toxic sea anemones, but not on non-toxic prey. An active cellular response is indicated, involving reproduction of cells. This result is also inconsistent with the expected behavior if an induced enzyme system were involved in the toxin neutralization. Second, coelomic fluid transfers from sensitized donors to naive sea stars accelerated feeding times by as much as a factor of 12. This indicates the immunoreactive factor resides in the coelomic fluid. Lastly, in in vitro toxin neutralization studies, whole coelomic fluid from sensitized donors completely neutralized the toxin, while cell-free coelomic fluid had little or no effect on 32

the activity of the toxin. This seems to confirm that

coelomocytes are necessary to the immune response of

echinoderms, and in particular, D. imbricata's immune

response. Since naive sea star whole coelomic fluid had

little or no effect on sea anemone nematocyst toxin

activity, a definite antagonistic response upon sen­

sitization has been demonstrated to occur in immunocytes

activated by Q imbricata's predatory activity.

The specificity of the immune response was the last

component to be demonstrated. Since the sea anemones utilized for this test were from different orders, differences in the nematocyst toxin chemical structures would be anticipated. It was demonstrated that D.

irnbricata, once sensitized to a species of sea anemone, could only show the accelerated feeding times if its second encounter were with a member of the same species.

Exposure of a sea star to a spec1es to which it is not sensitized gives behavior undifferentiatable from that of a na1ve sea star. Therefore, sensitization to one spec1es is useless in encounters with another species of sea anemone.

It can now be conclusively stated that D. imbricata exhibits an adaptive immune response to the nematocyst toxins of many anthozoans. But, as in any scientific study, many new questions and future studies are indi­ cated. The first question seems to be, by what mechanism does this immune system function? What types of receptors 33 are involved? Could it involve precursors, homologues, or analogues to immunoglobul s? The immune system studies here may respresent a primitive T-type immuno­ logical system.

For me, the most rplexing a ect is how do the sea stars "realize" that er being stung, even into paralys , by a sea anemone they can later feed without receiving ill effects from nematocysts? This would seemingly involve some type of deeply ingrained search image (Krebs, 1978) at either a learned level, or on an

stinctual level. This would be an interesting question to investigate.

Another cinating study would be of prey choices.

In particular, a comparison of naive and sensitized sea stars could be quite informative. Considering ene expenditures to develop sensitization, the prudent immunologically mediated predator would be anticipated to continue feeding on the same prey. Does this really happen? And what would a naive sea star do? Would it choose the least toxic prey, or would choices be made on some other factor as yet unknown?

would also be of great interest to examine the behavior of other species associating with cnidarians.

Would these also utilize an immune response? Aeolid nudibranchs are able to feed on anemones and store nematocysts (Edmunds, 1974). The mesogastropod pelagic snail Janthina is able to feed on scyphozoans dmunds, 34

1974). Anemone fish seem to be able to withstand stings

while acclimating, and the mechanisms involved in the

specialist species of anemone fish versus the generalist

may yield interesting results (Dunn, 1981). The

Acanthaster/coral predator-prey system from the Pacific

could well be similar in nature to the Dermasterias

Corynactis interactions. LITERATURE CITED

Bang, F. B. 1975. Phagocytosis in invertebrates. In K. Maramorosch and R. E. Shope (Eds.). Invert rate immunity. Academic Press, N.Y. pp. 137-151. Bertheussen, K. 1979. The cytotoxic reaction in allo­ geneic mixtures of echinoid phagocytes. Exp. Cell Res. 120: 373-381.

Blanquet, R. 1968. Properties and composition of the nematocyst toxin of the sea anemone allida. Comp. Biochem. Physiol. 25: 893 90 .

Burroughs Wellcome. 1978. Manufacturer's product information on Azathioprine.

Calabresi, P. and R. E. Parks, Jr. 1975. Alkylating agents, antimetabolites, hormones, and other antiproliferative agents. In L. S. Goodman and A. Gilman (Eds.). The pharmacological basis of therapeutics. MacMillan Pub. Co., Inc. N.Y., N.Y.

Cohen, E. 1975. Immunological sifnificance of specifici­ ties of cellular agglutins of Limulus polyphemus. In W. H. Hildemann and A. A. Benedict (Eds.). Immunologic phylogeny. Plenum Press, N.Y. pp. 29 33.

Connell, J. H. 1970. A predator-prey system in the marine intertidal region. I. Balanus glandula and several predatory species Thais. Ecol. Mono. 40: 49-78.

Cunningham, A. J. 1978. Understanding immunology. Academic Press, N.Y. 254 pp.

Dunn, D. F. 1981. Clownfish sea anemones. Trans. Am. Phil. Soc. 71(1). Philadelphia, Penn.

Edmunds, M. 1974. Defence in animals. Longman Press, London. 288 pp.

Ehrlich, P. R. 1968. Biochemical coevolution. Oregon State Univ. Press. Corvallis, Ore.

Ehrlich, P. R. and P. H. Raven. 1965. Butterflies and plants: a study in coevolution. Evolution 18: 586 608.

35 36 Emlen, J. M. 1966. The role of time and energy in food preference. Amer. Nat. 100:611-617.

Emlen, J. M. and M. G. R. Emlen. 1975. Optimal choice in diet: test of a hypothesis. Amer. Nat. 109:427- 435.

Feder, H. M. 1955. The use of vital stains in marking Pacific Coast starfish. Calif. Fish and Game 41: 245-246.

Foley, D. A. and T. C. Cheng. 1975. Morphology, hematologic parameters, and behavior of hemolymph cells of the quahaug clam, Mercenaria mercenaria. Bio. Bull. 146: 335-356.

Fraenkel, G. S. ·1959. The raison d'etre of secondary plant substnaces. Science 129: 1466-1470.

Gilbert, L. E. 1971. Butterfly plant coevolution: has Passiflora adenopoda won the selectional race with Hellconilne butterflies? Science 172: 585-586. Hand, C. 1954. The sea anemones of Central California. Part I. The corallimorpharian and athenarian anemones. Wasmann J. Biol. 13: 345-375.

Hildemann, W. H. 1979. Immunocompetence and allogeneic polymorphism among invertebrates. Transplantation 27: 1-3.

Hildemann, W. H., I. S. Johnston, and P. L. Jokiel. 1979. Immunocompetence in the lowest metazoan phylum: transplantation immunity in sponges. Science 204: 420-422.

Hildemann, W. H., C. H. Bigger, P. L. Jokiel, and I. S. Johnston. 1980. Characteristics of immune memory in invertebrates. In M. J. Manning (Ed). Phylogeny of immunological memory. Elsevier/North Holland Biomedical Press, Amsterdam. pp. 9 15.

Hil rd, H. R., W. E. Hinds, and J, E. Phillips. 1967. The specificity of uptake of foreign proteins by coelomocytes of the purple sea urchin. Camp. Biochem. Physio. 23:815-824.

Hyman, L. H. 1940. The invertebrates. Volume 1: Protozoa through ctenophora. McGraw-Hill Book Co. N.Y. 726 pp. 37 Karp, R. D. and W. H. Hildemann. 1976. Specific allograft reactivity in the sea star Dermasterias imbricata. Transplantation 22: 434-43 .

Kaneshiro, E. S. and R. D. Karp. 1980. The ultrastructure of coelomocytes of the sea star Dermasterias imbricata. Bio. Bull. 159: 295-310.

Krebs, J. R. 1978. Optimal foraging: decision rules for predators. In J. R. Krebs and N. B. Davies (Eds.). Behavioral ecology. Blackwell Sci. Pub. London. pp. 23-63.

Krieger, R. I., P. P. Feeny, and C. F. Wilkinson. 1971. Detoxification enzymes in the guts of ceterpillars: an evolutionary answer to plant defenses? Science 172: 579 581.

Mariscal, R. N. 1974. Nematocysts. In L. Muscatine and H. M. Lenhoff (Eds.). Coelenterate biology. Academic Press, N.Y. pp. 129-178. McKay, D. and C. R. Jenkin. 1970. Immunity in invertebrates. The fate and distribution of bacteria in normal and irrununised crafish (Parachaeraps bicarinatus). Aust. J. Exp. Bio. Med. Sci. 48:599-607.

Morris, R. H., D. P. Abbott, and E. C. Haderlie. 1980. Intertidal invertebrates of California. Stanford Univ. Press, Stanford, Calif. 690 pp.

Paine, R. T. 1966. Food web complexity and species diversity. Amer. Nat. 100:65-76.

Phillips, J. H. 1956. Isolation of active nematocysts of Metridium senile and their chemical composition.

Phillips, J. H. and D. P. Abbott. 1957. Isolation and assay of the nematocyst toxin of Metridium senile fimbriatum. Bio. Bull. 113: 296-

Phillip~, J. H. and B. J. Yardley. 1960. Detection in invertebrates of inducible, reactive materials resembling antibody. Nature 188: 728-730. Slobodkin, L. B. 1968. How to be a predator. Amer. Zoo. 8: 43 51.

Sakal, R. R. and F. J. Rohlf. 1969. Biometry. W. H. Freeman and Co., San Francisco. 776 pp. 38

Smith, J. N. 1968. The comparative metabolism of xenobiotics. In 0. Lowenstein (Ed.). Advances in comparative-physiology and biochemistry. Academic Press, N.Y. pp. 173-232.

Trip tt, E. L., J. E. Cushing, and G. L. Durall. 1958. Observations on some immune reactions of the sipunculid worm Dendeostomum zostericolum. Amer. Nat. 92: 287-29 .

Tripp, M. R. 1975. Humoral factors and molluscan immunity. InK. Maramorosch and R. E. Shope (Eds.). Invertebrate immunity. Academic Press, N.Y. pp. 20]-224.

Vadas, R. L. 1978. Preferential eding: an optimization strategy in sea urchins. Ecol. Mono. 47: 337-371.

Whittaker, R. H. and P. P. Feeny. 1971. Allechemics: chemical interactions between species. Science 171: 757-770.