THE ACCLIMATION PROCESS BETWEEN FISH ANT ANEMONES IN MONTEREY BAY STEPHEN EMILLER Stanford University at Hopkins Marine Station Biology 175H, 1989 ABSTRACT Although there have been many studies on the relationships between certain fish and sea anemones in the tropics, very little is known about the fish-anemone interactions in the colder waters of the northeastern Pacific Ocean. Äfter a seven week investigation in Monterey Bay, I found that none of the fish observed will acclimate to the tentacles of either of the intertidal sea anemones Anthopleura xanthogrammica or Anthopleura elegantissima, although the wooly Sculpin (Clinocottus analis) will rest on the base or column of either. also found that the Painted Greenling (Oxylebius pictus), as well as other rock dwelling fish, will acclimate to the Strawberry Anemone (Corynactis californica). The acclimation process takes approximately 3-5 days, depending on the amount of contact between the acclimating fish and the anemones, and the amount of previous contacts with the anemones by other fish. There are two mechanisms involved in the acclimation process. First of all, through repeated contact, the fish picks up mucus from the anemones and adds it to its own mucus layer. This causes each anemone to recognize the acclimated fish as a part of itself and prevents the fish from stimulating the anemone's chemoreceptors for nematocyst discharge. Secondly, the anemones become habituated to contact by the fish. INTRODUCTION There are two fish that were concentrated on during this study. The first were the wooly Sculpins, Clinocottus analis. These fish inhabit the intertidal and shallow subtidal zones. They are found from Baja, California to Cape Medocino, California and are extremely abundant in the tidepools and shallows at Hopkins Marine Station in Monterey The other fish studied were Painted Greenlings (also called the convict fish), Oxylebius pictus. These fish live at depths ranging from just below the intertidal to 50 meters along the northeastern Pacific coast from Baja California to British Columbia. They live under the canopy forming brown kelp Macrocystis pyrifera Adult greenlings are residential, usually occupying areas less than 10 square meters (DeMartini), and feed from the rocks on a variety of invertebrates, especially crustaceans and worms (Burgess and Axelrod). Two intertidal species from the order Actiniaria were used in this study: Anthopleura xanthogrammica and Anthopleura elegantissima They each have strongly adherent bases and have columns covered with verrucae, which are evaginations with modified ectoderm and no nematocysts which are able to cling to such things as rocks and shells (Carlgren). They have large, strong tentacles and can therefore catch fairly large prey. Both practice dioecious reproduction, although A. elegantissima also has the ability to reproduce by longitudinal fission, forming aggregations of a single clone (Sebens). They are part ially supported by the symbiotic algae Zooxanthellea (Friese). The other anemone studied was the Strawberry Anemone, Corynactis californica. This anemone-like species is in the order Corallimorpharia It is morphologically similar to corals, but it lacks any skeleton. It has a smooth column and thin tentacles with acrospheres at the distal ends. Acrospheres are globular ends of tentacles containing numerous potent nematocysts (Carlgren). This anemone is very abundant in Monterey Bay and occurs in California from San Diego to Sonoma County. It is found in shaded areas from the low intertidal to 30 meters. Corynactis californica reproduces asexually by longitudinal fission and is found in clonal groups (Hand). In a recent publication, The Biology of Nematocysts (Thorington and Hessinger). Thorington and Hessinger described their study of nematocyst discharge and listed some of the factors affecting this discharge. Like most researchers of nematocysts, they found that concomitant mechanical and chemical stimulation of the cnidocytes is required to initiate or trigger the discharge of the cnidae. The cnidocytes are specialized secretory cells on the tentacles that are also considered independent effectors and directly perceive stimuli for discharge. The mechanism for discharge involves chemosensitization of the cnidocytes by the active chemicals, which then lower the mechanical threshold of cnida discharge. Chemical stimulation alone has occasionally been found to cause discharge, but only under more stringent conditions (using very harsh chemicals) than are found in the natural environments of the anemones. In some anemone tentacles mechanical stimulation alone can cause some discharge, but in every case adding appropriate chemical stimulation to mechanical stimulation causes much greater discharge. Some of the active chemicals have been isolated and include n-acetylated sugars and various amino compounds. N-acetylated sugars are common in the mucus layers of many organisms that are prey for anemones. These sugars cause immediate discharge on contact with the tentacles. Then, after nematocysts have pierced the prey, body fluids containing numerous amino compounds are released and stimulate further discharge. For years cnidocytes were considered independent effectors with direct control over the cnidae discharge and no intervention of the nervous system. Certain observations, however, show that the nervous system seems to have either a modulatory or an inhibitory influence on cnidae discharge. For example, a swimming anemone, Stomphia coccinea, will not capture any prey while its pedal disc is detached from the substratum. It was also found, using coverslips coated with food extract and the anemone Anemonia sargassensis, that tactile stimulation of the body column increases the amount of discharge. Even the ambient light intensity, though cnidocytes are not considered to be photosensitive, affects the responsivness of cnidocytes and the ability to capture prey. Other factors influencing the physiological state of the anemones, such as ions in the ambient medium and the amount of recent feeding, also seem to affect the amount of cnidae discharge (Thorington and Hessinger). The acclimation process between anemonefish and their hosts has been studied for years, but most of these studies have taken place in the tropics where the fish actually lives within the tentacles of a single host. There are a few different theories on how the acclimation takes place. The first set of theories assumes that the anemone makes certain changes and is responsible for the acclimation (Gohar). They feel that the anemone changes or inhibits its pattern of nematocyst discharge due to the behavioral, physical, or chemical stimuli of the acclimating fish. Contrary to these are the theories in which the fish is responsible for its protection from the anemones by in some way changing its mucus layer. Some feel that the mucus layer after acclimation contains no active agents or other factors which are known to stimulate the chemoreceptors, causing nematocyst discharge upon contact (Lubbock). Öthers feel that certain chemicals in the mucus layer inhibit nematocyst discharge (Davennport & Norris). This inhibition can occur in one of two ways. There is either direct inhibition of the cnidocyte-nematocyst complex on the tentacles, or this inhibitior is accomplished through the organism's nervous system. The final theories assume that the mucus of the anemone is transferred to the acclimating fish. One German researcher, Schlichter concluded in 1976 that substances which inhibit nematocyst discharge are produced by the anemones themselves (Mariscal). Through repeated contact, the acclimating fish coats its body with this inhibitor as to no longer induce nematocyst discharge. Another possible explaination is that the anemone identif ies the acclimated fish as a part of itself by recognizing its own mucus on the fish. MATERIALS AND METHODS All of the Clinocottus analis used were collected from tidepools during low tide at Hopkins Marine Station. All other fish tested (Oxylebius pictus, Coryphopterus nicholsii, and Orthonopias triacis) were captured with anet from depths of 5-10 meters at Hopkins Marine Station, the breakwater, or Lovers Point, all of which are located in Monterey Bay. For Tests With Anthopleura; In each experiment l used a tank with two A. xanthogrammica and one A. elegantissima, with approximate diameters of 15, 10. and 10 centimeters respectively. All three anemones were carefully removed from rocks in the intertidal zone of Hopkins Marine Station. They were left in isolation for one week before any experimentation was started, in which time they settled on rocks in the observation tank. During observational experiments (watching fish in tanks with anemones), all contacts and reactions were noted, described and timed. In the experiment with forced contact between anemones and Wooly Sculpins (Clinocottus analis), the sculpins were held by the jaw with forceps and placed tail first on one side of the ring of tentacles. Care was taken not to touch the fish with my hands For Tests With Corynactis californica: One rock of C californica was collected from a depth of 8 meters at the breakwater of Monterey Bay. All other rocks were collected from the Del Monte shalebeds of Monterey Bay at depths of 13-16 meters. For all observational experiments, tanks would be watched for a couple hours and then left for a few hours repeatedly over several days. All rocks with C californica were mapped such that individual anemones could be accounted for (See Maps 1-3). During a given observation period, all contacts were recorded by anemone number and type of contact. Three types were used: hit hard, hit, and brushed. One of four types of reactions by the anemone was then recorded: closed, closed 1/2, slight retraction, or none. When an anemone closed, the amount of time before opening was also marked for some experiments. For any observation period, a "closure value" could be calculated. Each of the reaction types was assigned a number: closed=3, closed 1/2=2, slight retraction=1, and none=O. For all contacts in a period, these numbers were added up and divided by the total number of contacts. This would result in a closure value between O and 3. One experiment using various parts of Painted Greenlings was also done. A fish was captured in a clean glass bowl to prevent contamination. The fish was then placed on its side in a clean pan, keeping its tail up with forceps. The spinal cord just behind the head was quickly severed and the tail and side facing up were left uncontaminated. The pectoral fin, ventral fin, and tail were then removed and tested on anemones. The anemones were put in just enough water to cover them such that the body parts could be touched to anemones without letting the areas cut touch the water. This prevented internal body fluids from entering the water and possibly triggering the chemoreceptors. The final experiment done with C californica involved an attempt to acclimate my finger to a clonal group of anemones from the shalebeds. Irubbed the end of my index finger against 172 of the anemones (which were closed) and then tested the reactions of open anemones to my touch. RESULTS: FIELD OBSERVATIONS Both Anthopleura xanthogrammica and Anthopleura elegantissimawere studied in the tide pools of Hopkins Marine Station. The only contact ever seen involved a wooly Sculpin and an A. Xanthogrammica The sculpin was sitting on the column of the anemone, approximately 1/2 way between the oral and pedal discs. Another observation involved an A. xanthogrammica, four Wooly Sculpins, and a Monkey Faced Eel. The anemone was attached to a vertical rock surface just above the bottom of the pool such that its tentacles reached the bottom. This formed a small shelter between the sand, rock, and tentacles (Diagram 1) saw a fish go behind the tentacles, but it was too dark to see where it went. I could see that nothing was touching the anemone's tentacles. By putting a net on one side of the anemone and sticking my hand under on the other side, I captured the four sculpins and the eel. Finally, a fellow student found a bead that she had used to mark a small Clinocottus analis near the mouth of an anemone in one of her study tide pools. All field observations with C californica were made using SCUBA. Painted Greenlings were almost always found on the same rocks as C californica, though contact was only observed once. A fairly large greenling was found at 8 meters on a field of anemones covering approximately one square meter. Using my hand, I caused him to move three times before he left. Each time he landed on another part of the anemone cluster, and none of the anemones closed. The only reactions observed were occasional slight retractions. Another interesting observation was the abundance of Snubnose Sculpins (Orthonopias triacis) and Blackeyed Gobies (Coryphopterus nicholsiD on the same rocks as the greenlings and C californica A Spubnose Sculpin was once seen with its pectoral fin on an anemone that was still open. Finally, a Northern Spearnose Poacher (Agonopsis emmelane) was found resting on four anemones. I pulled him off by the tail and released him. He immediately returned to the anemones, none of which showed any reaction. RESULTS: TANK OBSERVATIONS With Anthopleura: A Painted Greenling with bubble disease was placed in a small tank with the three Anthopleura In the first hour, the fish made contact with only the tentacles 5 times. In all 5 cases, the anemones responded with a feeding motion; the tentacles rolled in toward the mouth and then immediately back out. Then, as the fish died an hour later, it landed in one of the A. xanthogrammica twice. Both times the anemone attempted to eat the fish (exp. 1). The other greenling tested with Anthopleura was in a much larger tank. It stayed in the far left 1/3 of the tank, away from the anemones, for the entire week of observation (Map *4). Similar feeding responses were observed both times a Clinocottus analis touched an Anthopleura (exp. *4). But unlike the Painted Greenlings, Wooly Sculpins were often seen resting on the pedal discs and columns of these anemones. With Corynactis californica: All the Painted Greenlings that acclimated to C californica did so in approximately 3-5 days. For example, a greenling from Hopkins Marine Station was put in a tank with two different clones of C californica, one orange and one pink, neither of which had been previously in a tank with fish. As time passed, the closure values for the orange clone decreased, showing acclimation. These values are listed in Table *1 and graphed in Figure +1. In the first 75 hours, there were only 9 observed contacts with the pink anemones, so closure values were not calculated for individual observation periods. Of these 9 hits, 4 caused complete closure while 5 caused anemones to close 1/2 (a combined closure value of 2.4). However, in the 80 hour and 89 hour observation periods, there were more hits and closure values of 0.2 and 1.0 were found This demonstrates the greenling's ability to acclimate to more than one clone at a time. Interesting results were found when a greenling was acclimated to the orange anemones in one tank, moved to a new tank with pink anemones, and then returned to the original tank 11 days later. The original acclimation closure values with the orange anemones can be found in Table *2. Figure *2 shows the closure values when the fish is put in and taken out of the first tank, when the fish is put in the second tank, and when it is returned to the original tank. Note that after 11 days, the fish was still fairly acclimated (closure values for hit hard, hit, and brushed were 1.0, 0.81, and 0.6 respectively). It is also interesting to follow different situations for a particular group of C californica One at a time, four fish were introduced to the clonal groups of anemones in one of the tanks (exp. 6, 7 (Table *2), 8, & 10). Figure +3 shows the closure values calculated from the first observation périod for each of these four fish (2 greenlings, a Snubnose Sculpin, and then another greenling) Each greenling had been previously isolated and all were approximately the same size, yet the closure values upon introduction to the tank go down with each successive greenling. This implies some type of mechanical habituation by the anemones to repeated contact by fish. Another example of this mechanical habituation is the acclimation periods of the second and third greenlings mentioned above. The third greenling was entirely acclimated after 72 hours. The second greenling, however, was only partially acclimated: after 72 hours and was not fully acclimated until over 100 hours after introduction to the tank. This is also shown in Figure r3. Finally, I looked at all the "hit hard" contacts throughout my observations. For each contact, I counted how many times that particular anemone had been hit before. For anemones that closed, they had previously been hit an average of 1.6 times. Anemones that closed 172 had been hit an average of 4.0 times, while slight retraction anemones had been hit an average of 5.5 times. This shows the mechanical habituation by individual anemones. RESULTS: FORCED CONTACT EXPERIMENTS Using forceps to hold wooly Sculpins by the jaw, contact with the tentacles of Anthopleura was forced. One small A. xanthogrammica had its mouth already partly open. A small (3. cm.) sculpin was touched to the tentacles, and as soon as there was contact, the fish was engulfed. Within 1 second I could not even see the fish, let alone try to save it. Next I tested a similar fish (3.3 cm.) with A. elegantissima It was caught and disappeared as quickly as the fish above. Both anemones were fully opened around 8 minutes after catching the fish. Obviously, Anthopleura are willing to eat Clinocottus analis. Body parts from two acclimated greenlings were tested with both acclimated and non acclimated anemones. The results can be found in Table *3. For the pectoral fins of both fish and the ventral fin of the second fish, the acclimated anemones responded C less than the previously isolated anemones. But for the tails of both fish and the ventral fin of the first fish, there is little or no difference, possibly due to contamination by blood when the fish had its spinal cord cut. As a final experiment, l acclimated my index finger to a clone of C californica After ten minutes of rubbing my finger on closed anemones, I was able to lightly touch the tentacles of an opened anemone on the same rock without causing a reaction.f then touched this anemone with a different finger, the anemone would close. The acclimated finger was covered by a slimy substance that must have been anemone mucus. It is this mucus that protected my finger from nematocyst discharge. DISCUSSION AND CONCLUSIONS Observations with Anthopleura show that fish did not acclimate to the tentacles of either A. elegantissima or A. xanthogrammica. Painted Greenlings avoid these anemones completely. Wooly Sculpins, on the other hand, merely avoid the tentacles. They will rest on both the pedal disc and column of an anemone, and were even seen using an anemone for protection in the field. If contact with Anthopleura tentacles does occur, however, the anemone will attempt to eat the fish. It is also clear that Painted Greenlings (and probably Northern Spearnose Poachers, Snubnose Sculpins, and maybe Blackeyed Gobies) do acclimate to the Strawberry Anemone, Corynactis californica This acclimation period takes approximately 3-5 days. The acclimation process seems to occur through two main mechanisms. The first involves a change in the mucus layer of the fish. Though there have been claims that the fish secretes the protective substances, it seems more likely that the fish picks up anemone mucus through repeated contact. It is this mucus layer that prevents the fish from stimulating the chemoreceptors to cause nematocyst discharge. This is demonstrated by the acclimation of my finger to the anemones. Another observation that supports this theory was that even after acclimation, on the rare occasion that the face of a fish hit an anemone, the anemone always closed. If the fish was producing a protective mucus layer itself, the face would also be protected. Since the face rarely touched anemones, it did not accumulate enough anemone mucus for protection. The Painted Greenling seems able to maintain this protection through its mucus layer for a long time. One fish was removed from its acclimated anemones for 11 days and was still fairly acclimated upon return to the tank. This is quite different from the inability of Amphiprion bicinctus to remain acclimated to Radianthus koseirensis (Graefe, 1964). Graefe observed that if this fish was seperated from its host anemone for 1 hour or more, it would be stung upon its return. One main theory behind mucus protection is that the mucus does not contain any of the active chemicals needed to trigger the chemoreceptors. Another claims that certain chemicals inhibit nematocyst discharge, possibly by raising the threshold to mechanical stimuli. Finally, the anemone may recognize its own mucus on the fish and identify the fish as part of itself. It seems very unlikely that the mucus contains inhibitors to nematocyst discharge. If inhibitors were present in the anemone's mucus layer, they would interfere in the anemone's normal feeding and protective reactions. One experiment that may support the lack of excitatory factors was the testing of greenling body parts with anemones. While some fins caused a notable difference in reactions between acclimated and nonacclimated anemones, others did not. Those that did not may have been contaminated by blood when the fish was killed. If the fins were covered with something that inhibited nematocyst discharge, the contamination should not have caused all anemones to react the same. Even if the contamination contained chemicals that were active enough to override the inhibition (lower the threshold to mechanical stimult more than the inhibitors raised it), the reactions of acclimated anemones should still have been somewhat less than those that were not affected by the inhibitors at all. Inhibition must not be the mechanism. On the other hand, if the mucus layer merely contained no active chemicals, contamination would be expected to cause similar reactions by both acclimated and nonacclimated anemones. It is still possible, however, that there was merely a lack of mucus on the areas touched against the anemones. This would also cause all anemones, acclimated or not, to react the same. The most likely mechanism for protection is that the anemone recongnizes the fish as part of itself. All observations agree with this expanation. For example, consider that any particular greenling can acclimate to more than one clone of C; californica at a time. An acclimated fish put in a tank with a different clone of anemones will be entirely unacclimated. So even if there are no active chemicals to cause discharge of the one clone, another clone will react. A fish in a tank with two clones would pick up mucus from both clones. Assuming that the lack of active chemicals causes protection, this combined mucus should still contain chemicals from each clone that are active enough to cause discharge on the other clone. If, however, each clone gives the fish mucus that it can recognize, the fish will be recongnized by both and will not cause nematocyst discharge on either. This is what was observed, causing me to believe that this is the more probable mechanism. The other important aspect of acclimation involves habituation by the anemones to repeated contact by the fish. This was first observed in a three clone tank of C californica in which successive greenlings showed lower initial closure values and shorter acclimation periods. It was also seen in individual anemones in that stronger reactions were observed for anemones that had been previously hit less times. The mechanism for this habituation is unknown, though it probably involves modulation by the nervous system. Thorington and Hessinger (Thorington and Hessinger) showed that various types of modulation do occur for certain species of anemones. For example, one experiment showed that tactile stimulation of the body column of Anemonta sargassensis will increase the amount of discharge by the cnidae on the tentacles. No tests were done on habituation to repeated contact. Besides the mechanism of habituation to contact for the anemones, there are other aspects of acclimation that need to be studied further. A complete examination of anemone mucus would help to determine how the anemone recognizes its own mucus if this indeed is the mechanism behind the protection from nematocyst discharge. Possibly one could determine the chemicals detected by the chemoreceptors. It would also be interesting to see if an active chemical present on the fish could cause discharge despite recognition by the anemone of its own mucus. e LITERATURE CITED Burgess, W.E. and Axelrod, H.R.,1984, Fishes of California and Western Mexico, TFH Pub. Inc, New Jersey. Carlgren, O., 1949, A Survey of the Plychodactiacea, Corallimorpharia and Actiniaria, London. Davenport, D. and Norris, K., 1958, Observations on the Symbiosis of the Sea Anemone Stoichactis and the Pomacentrid Fish, Amphiprion Percula, University of Califonia. DeMartini, E.E., 1946, The Adaptive Significance of Territoriality and Egg Cannibalism In the Painted Greenling, Doctoral dissertation, University of Washington. Friese, U. Erich, 1972, Sea Anemones, TFH Publications, Hong Kong. Gohar, H.A.F., 1948, Commensalism Between Fish and Anemones, Pub. Marine Biology, Stn Ghardaga 6, pp 35-44. Hand, C., 1955, Sea Anemones of Central California, Wasmann journal of Biology. Lubbock, R., 1979, Why are Clownfish not Stung by Sea Anemones, University of Cambridge, G.B.. Mariscal, R.N., 1966, The Symbiosis Between Tropical Sea Anemones and Fishes: a Review. pp 157-171 in Bowman, R.I. ed., The Galapagos: Proceedings of the Symposia of the Galapagos International Scientific Project, University of CA press, Berkeley. Sebens, K., 1977, Habitat Suitability, Reproductive Ecology, and the Plasticity of Body Size in two Sea Anemone Populations, Doctoral Dissertation, University of Washington Thorington and Hessinger, 1988, Control of Discharge: Factors Affecting Discharge of Cnidae. pp 233-250 in Hessinger and Lenhoff ed.s, The Biology of Nematocysts, Academic Press, San Diego. APPENDIX Exp. *1: During the dying process (the last four minutes), the fish would shoot to the surface and then sink to the bottom. He landed with his tall in the small A. xanthogrammica and his tail was held by the tentacles. He swam out after 10 seconds and again went to the surface and sank. Again he landed with his tail in the small A. xanthogrammica and appeared to be dead. The anemone pulled the greenling in towards its mouth and after 90 seconds he was nearly 1/2 way in. Suddenly, the greenling started thrashing violently and after seven seconds was free. He landed on the sand and died. Exp. *4: A Wooly Sculpin was dropped into the tank. It was still somewhat dulled from CO» and just sank when 1 dropped it in. On the way down, it touched three tentacles of the large A xanthogrammica On contact, the fish immediately swam hard away from the anemone. The three touched tentacles rolled in and then immediately back out. The only other contact between anemone tentacles and fish occurred when a piece of squid was dropped to an anemone. A sculpin saw the squid just before it reached the anemone and swam for it. But before it got to the squid, the fish touched some tentacles and immediately changed direction and swam away quickly. The anemone got the squid and closed around it. A Painted Greenling was also in this tank for over a week. After the first hour, in which he sat on the right side of the tank where I had dropped him in, I never saw him as far right as the most left anemone (Map 4). He completely avoided them all. Exp. 46: A Painted Greenling was put in a tank with at least three different clones of C californica (Map 1). During observations in the first 90 minutes, closure values for hit hard, hit and brushed were 3, 3, and 2 respectively. The fish died of bubble disease before further observations could be made. Exp. *8: A Snubnose Sculpin was put in the same tank as (exp. 16). In the first 90 minutes, closure values for hit hard and hit were 3.0 and 1.13 respectively. For the next three days, the fish touched no anemones, and was then removed. Exp. +10: Another Painted Greenling was placed in the same tank (exp. *6) and observed for the first 1 hour. The closure value for hit during this period was 2.2. Observations were not made again until 72 hours after introduction to the tank. Closure values at this point were 1.0, 0.0, and 0.0 for hit hard, hit, and brushed. After 80 hours, hit and brushed still had closure values of 0.0. c TABLE *1 (PAINTED GREENLING WITH CORVNACTIS CALIFORNICA) CLOSURE VALUES THREE TYPES OF CONTACT HIT TIME (In Hours) HIT HARD BRUSHED 2.79 O- 1.33 — 1.5-3 1.0 Z.12 18-20 2.30 2.0 24-26 2.57 1.0 32-33 1.80 39-41 1.83 54-55 2.54 64-65 1.60 70-71 3.0 1.0 0.8 75-76 1.0 0.0 80-81 3.0 1.29 88-89 1.06 106-107 0.55 TABLE *2 (PAINTED GREENLING WITH CORWACTIS CALIFORNICA) CLOSURE VALUES THREE TYPES OF CONTACT TIME (In Hours) HIT HIT HARD BRUSHED O- 2.63 1.0 65-66 3.0 0.0 72-73 1.0 1.17 0.0 95-96 1.0 0.56 0.0 117-118 1.0 1.50 0 TABLE F3 (PAINTED GREENLING FINS TOUCHED TO BOTH ACCLIMATED AND NONACCLIMATED CORWACTIS CALIFORNICA) FISH FIN TYPE ANEMONE REACTION TIME BEFORE FULLY OPENED TAIL CLOSED 1/2 2.50 ACCL 410 TAIL ACCL CLOSED 1/2 420 TAIL NON CLOSED 5:15 TAIL NON CLOSED 410 VENTRAL ACCL CLOSED VENTRAL ACCL 5.30 CLOSED VENTRAL NON 5:15 CLOSED 5:10 VENTRAL NON CLOSED PECTORAL ACCL 2:40 CLOSED 3:40 PECTORAL ACCL CLOSED PECTORAL NON 8:50 CLOSED PECTORAL NON 9.30 CLOSED CLOSED 5:10 TAIL ACCL 6:40 TAIL ACCL CLOSED 6.30 TAIL CLOSED NON VENTRAL ACCL 0.30 CLOSED 1/2 450 VENTRAL ACCL CLOSED 1/2 VENTRAL NON 9.30 CLOSED 4.15 PECTORAL ACCL CLOSED 17/2 PECTORAL ACCL 3.10 CLOSED 1/2 PECTORAL ACCL 3.50 CLOSED 1/2 PECTORAL ACCL 6.20 CLOSED 173 PECTORAL NON CLOSED 12:00 PECTORAL NON CLOSED 8:10 ACCL= Anemone clone fish was acclimated to NON= isolated clont FIGURE LEGENDS FIGURE 1: This graph shows a Painted Greenling becoming acclimated to the C californica shown in Map 3. The line represents closure values for hits, the dark spots on top represent hit hard, and the plus signs represent brushes. The one high brush mark and the highest hit mark are from observations in which the greenling's face hit and closed anemones. Figure 2: This shows closure values for a greenling in a variety of different situations. First it is introduced to orange C californica and then is taken out after acclimation (140 hours). It is put in with a new clone (pink) and again is completely unacclimated. Finally, after 11 days, the fish is put back in with the orange anemones and is still fairly acclimated, but not completely. Figure 3: The first four bars represent the introduction of different fish to the same clone of C californica Notice the initial closure values go down for each successive greenling. The final two bars show that the second greenling introduced took longer to acclimate than the third greenling. This is due to mechanical habituation. 8 Fiqure + Closure value tataaaatataaaaaaaa- O ( Figore 2 CLOSURE VALUE 3 L testell A 2 O O 8 Figore 43 CLOSURE VALUE 2 - 4 o 2 2 O 8 Rock 3 17 - 8 Rock 2 22 580 283 38 64 . 4 8 6 5 a 2 95 e 3 — Miodle ak o Vier ao Rock E S a 15 16 1 5s 5 27 gd 8 Rock C 56 ttll, 200 flrrpt 33 3 55 34 53 59 A 135 10 3 130 e 827 3 45 46 97 85 627 Se 76 Rock 162 75 8 Not Shoun 65 63— ea 3 0 68 11 5 56 O 1 25 —. - L Lisel —— Mao Piak Corpnactis Tonk (Onse Right Tank) (once lesta, e 17 e 5 50 16 0 55 15 S26 -3 5 78 114 13 s0 153 45 83 ag 5 a 33 4 126 216 8 274 24 6 880 32 5 MapS Flat Orange Corpoactis Rock Leftak 0 la. Slat Lrck 852 — 62 3 — 559 — ae ges 568 D F 47 544 K 540 6a Dya 42 h 104 ee 138 1 37 Ant pa on 5 46 70 ta 2t 128 15 s 00 5 9 a3 4. 026 2 192 ) D 128 —2 15 61 15 188 a 120 105 Map Large Arthopleora Tank Top Vier osogoo0. Lorge osgesiee eg o De ertolizing ee Torer doked e Diagran Large Rock Ln Aad Tn this avea, betbeen Rock and lentacles dere foor Looh seolping ano a Nonkey Faced Eel. ACKNOWLEDGEMENT would like to thank the professors and staff (especially in the library) at Hopkins Marine Station for all their help. I would also like to thank the many graduate students who were willing to take the time to help when asked. Also, I thank Maggie Harrison for being my relfable dive partner. Finally, I want to give special thanks to Chuck Baxter for his time, insights, and guidance.