University of Southern California Dissertations and Theses

Feeding Behavior In Palythoa Townsleyi And Zoanthus Pacifica With Emphasis On The Chemical Control Of Their Feeding Reactions

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Feeding Behavior In Palythoa Townsleyi And Zoanthus Pacifica With Emphasis On The Chemical Control Of Their Feeding Reactions

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Content I I 71-12,412 REIMER, Amada Alvarez, 1941- FEEDING BEHAVIOR IN PALYTHOA TOWNSLEYI AND ZOANTHUS PACIFICA WITH EMPHASIS ON THE CHEMICAL CONTROL OF THEIR FEEDING REACTIONS. University of Southern California, Ph.D., 1970 Biology University Microfilms, A X ER O X Com pany, Ann Arbor, Michigan Copyright by Amada Alvarez Reimer 1971 t h t c nTSSERTATTON HAS BEEN MICROFILMED EXACTLY AS RECEIVED FEEDING BEHAVIOR IN PALYTHOA TOWNSLEYI AND ZOANTHUS PACIFICA WITH EMPHASIS ON THE CHEMICAL CONTROL OF THEIR FEEDING REACTIONS by Amada Alvarez Reimer A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biological Sciences) August 1970 UNIVERSITY O F SO U T H E R N CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES. CALIFORNIA 9 0 0 0 7 This dissertation, written by .......... AmadA. Mvax.&g. Rsim f i X..... under the direction of h $£.... Dissertation C o m mittee, and a p p ro ved by all its members, has been presented to and accepted by The G radu ate School, in partial fulfillment of require ments of the deyree of D O C T O R O F P H I L O S O P H Y Date... July.. X .3 , . . 1 . 9 7 0. DISSERTATION CO M M ITTEE ACKNOWLEDGMENTS It gives me great pleasure to acknowledge the gen erous advice, help and warm interest of the faculty, staff and of my fellow graduate students at the Department of Biological Sciences, University of Southern California, throughout the course of this study. I am especially grateful to Dr. Howard M. Lenhoff, University of California, Irvine; and to Dr. Leonard Musca tine, University of California, Los Angeles, who aroused my interest for feeding and nutrition in coelenterates, who taught me important techniques and who gave me counsel and encouragement throughout this study. I owe particular thanks to the members of my com mittee: Drs. Gerald J. Bakus, John S. Garth, Russel L. Zim mer, Walter E. Martin, John D. Soule, and Peter M. Shugar- man for their interest and helpful suggestions in the prep aration of this manuscript. I want to thank especially Drs. Gerald J. Bakus and John S. Garth for their invaluable advice and encouragement during the preparation of the manuscript. I would also like to express my gratitude to Dr. Maria W. Seraydarian, University of Southern California, for her valuable comments and suggestions; and to Dr. Ber- ii nard L. Strehler, University of Southern California, for the use of his laboratory and chromatography equipment. It is a pleasure to acknowledge the generous help of Mrs. June Siva, University of Southern California, who allowed me to use her reprint collection; Mr. Ralph L. Bowers, University of Hawaii, who collected and shipped the zoanthids from Hawaii to Los Angeles, and to Mr. William T. Petersen, University of Hawaii, for valuable information on the diversity and abundance of zooplankton in Kaneohe Bay, Hawaii. It gives me great pleasure to thank my husband, Mr. Roger D. Reimer, for the photographic work presented in this thesis and my brother, Mr. Mauricio L. Alvarez, for typing the first draft of the manuscript. My research was supported by a Fellowship from the American Association of University Women; a Biomedical Sciences Support Grant from the National Institutes of Health and a National Science Foundation grant to the Hawaii Institute of Marine Biology. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS..................................... ii LIST OF TABLES..................................... vii LIST OF ILLUSTRATIONS ............................. xi PREFACE.............................................. xvi INTRODUCTION ....................................... 1 GENERAL CONSIDERATIONS ON METHODOLOGY .............. 7 PART I. FEEDING BEHAVIOR IN PALYTHOA Chapter I. RESPONSE OF PALYTHOA TO A VARIETY OF FOODS. . 24 Material and Methods Results Discussion Conclusions II. GENERAL CHARACTERISTICS OF THE FEEDING REACTION ......................... 54 Material and Methods Results Discussion Conclusions III. CONTROL OF LIP FORMATION................... 84 Material and Methods Results Discussion Conclusions iv Chapter Page IV. CONTROL OP MOUTH OPENING.................. 95 Material and Methods Results Discussion Conclusions V. CONTROL OP THE INGESTION RESPONSE.......... 136 Material and Methods Results Discussion Conclusions PART II. FEEDING BEHAVIOR IN ZOANTHUS VI. RESPONSE OP ZOANTHUS TO A VARIETY OP POODS. . 200 Material and Methods Results Discussion Conclusions VII. GENERAL CHARACTERISTICS OP THE FEEDING REACTION ......................... 212 Material and MethodB Results Discussion Conclusions VIII. ABILITY OP ZOANTHUS TO TAKE UP GLYCINE PROM SOLUTION AND UTILIZATION OP THIS AMINO ACID........................... 236 Material and Methods Results Discussion Conclusions IX. IMPORTANCE OF ZOOXANTHELLAE FOR ZOANTHUS' WELL B E I N G ..................... 258 Material and Methods Results v Chapter Page Discussion Conclusions PART III. GENERAL CONSIDERATIONS X. DISCUSSION................................. 269 Feeding Methods Control of Feeding Response Evolutionary and Ecological Implications XI. SUMMARY AND CONCLUSIONS..................... 278 LITERATURE CITED ................................... 288 vi LIST OF TABLES Table Page 1. Effect of Starvation in the Response of Palythoa townBleyl to Filter Paper Imbibed by Proline 10-lM ................... 17 2. Influence of a Behavioral Response on the Ability of Palythoa townsleyl to Make Successive Captures of Artemia ............. 32 3. Percentage of Polyps of Palythoa townsleyl which Leave Tentacles Protruding after Capturing Artemia ......................... 33 4. Frequency of Escape of Artemia salina from Palythoa townsleyl ......................... 35 5. Frequency of Shrimp (Artemia salina) Capture by Palythoa townsleyl ............ 36 6 . Effects of Prey Density on the Ability of Palythoa townBleyl to Perform Con secutive Captures of Artemia Salina .... 40 7. Response of Palythoa townsleyl to Amino Acid from Artemla^Alcohollc Extract, on Filter P a p e r ........................... 45 8. Response of Palythoa townsleyl to Amino Acids and the tripeptide Glutathione on Filter Paper ............................... 63 9. Characteristics of the Response of Palythoa townsleyl to Different Concentrations of Proline ................ 65 10. Characteristics of the Response to Proline of Two Sized Categories of Palythoa townBleyl Polyps........................... 71 11. Characteristics of the Response of Palythoa townsleyl to Different Concentrations of Glutathione............................. 73 vii Table Page 12. Activation of the IngeBtion Response in Palythoa townsleyl by Proline and Glutathione in Solution ................... 74 13. Initial Responses of Palythoa townsleyl to Treated Filter Paper ................... 87 14. Initial Response of Palythoa townsleyl to Solutions of Several Amino Acids and the Tripeptide Glutathione ............ 92 15. Activation and Characteristics of Mouth- Opening Reaction in Palythoa townsleyl . . . 101 16. Activation and Characteristics of Mouth- Opening Reaction in Palythoa townsleyl Exposed to Different Solutions ............ 102 17. Activation of the Mouth-Opening Response in Palythoa townsleyl by Acid p H .......... 105 18. Interaction between Proline and Gluta thione in the Mouth-Opening Response of Palythoa townsleyl ..................... 106 19. Effect of the Interaction between Proline and Pipecolic Acid in the Mouth-Opening Reaction of Palythoa townsleyl ............ 118 20. Effect of the Interaction between Proline and l-Thiazolidine-4-Carboxylic Acid in the Mouth-Opening Reaction of Palythoa townsleyl................................. 126 21. Activation of the Ingestion Response in Palythoa townsleyl by Several Substances Imbibed by Filter Paper at Concentrations 10-lM..................................... 139 22. Response of Palythoa townBleyl to Untreated Filter Paper when Polyps Are Placed in a Solution of Glutathione or Proline at Different Concentrations .............. 144 2 3. Response of Palythoa townsleyl to Gluta thione 10-- 1 - M and S-methyl Glutathione 10-3-M Imbibed by Filter Pa p e r............ 148 viii Table Page 24. Effect of the Interaction between Prollne and Pipecolic Acid In the Ingestion Response of Palythoa townsleyl ............ 149 25. Effect of the Interaction between Prollne and l-Thlazolidine-4-Carboxylic Acid In the Ingestion Response of Palythoa townsleyl ......................... 153 26. Effect of the Interaction between Prollne and Glutathione In the Ingestion Response of Palythoa townsleyl ............ l6l 27. Effect of the Interaction between Prollne and Glutathione in the Ingestion Response of Palythoa townsleyl ............ 165 28. Effect of the Interaction between Prollne and Glutathione in the Ingestion Response of Palythoa townsleyl ............ 169 2 9. Response of Zoanthus paclflca to Amino Acids and a Tripeptide on Filter Paper . . . 205 3 0. Response of Zoanthus paclflca to a Tri- peptide and Several Amino Acids at 10"^M, Absorbed in Filter Papers 0.5mm2 in Size . . 208 31. Response of Zoanthus paclflca to Reduced Glutathione............................... 218 3 2. Response of Zoanthus paclflca to Glycine and the Glycine Analog a-Aminomethasul fonic Acid................................. 224 33. Effect of the Interaction between Glycine and its Analog a-Aminomethasulfonic Acid in the Response of Zoanthus paclflca. . 226 34. Response of Zoanthus paclflca to Feeding Activator, its Components' and a Glycine Analog, Absorbed in Filter Paper of 0 .5mm2 ................................. 228 _l4 35. Uptake of Glycine C from Solution by Zoanthus paclflca ......................... 240 ix Table Page 3 6. Radioactivity Lost In Controls of Glycine-l^C, without Animals .............. 243 _l4 37. Distribution of C in Alcohol-Soluble Fraction o-f Zoanthus paclflca as Determined by Radiochromatography ........ 247 14 3 8. Uptake of Glycine- C by Bacteria after 16 Hours' Incubation and Growth .......... 252 _l4 39. Distribution of C in Bacteria after 16 Hours' Growth and Incubation .......... 253 40. Changes in Size of Oral Disc in Starved Aposymbiotic Polyps of Zoanthus paclflca over a Period of 11 Days................... 264 x LIST OF ILLUSTRATIONS Figure Page 1. Lip Formation in Palythoa townsleyl upon Contact with Proline Paper ................ xviii 2. "Localized" Lip Securing and Pushing Proline-Paper toward Mouth ................ xviii 3. Lip Formation. Edge of DIbc Rising and Turning Inward ............................. xxi 4. Wide, Round-Mouth Opening and Ingestion of Pro line-Paper....................... xxi 5. Small, Round-Mouth Opening ................... xxiii 6 . Slit-Like Mouth Opening ..................... xxiii 7. Corner-of-the-Mouth Opening ................. xxvi 8 . Actinopharynx Exposure.............• • xxvi 9. Collection Sites for Palythoa and Zoanthus . . 12 10. Aquarium where Palythoa townsleyl (P) and Zoanthus pacifica (Z) Were Maintained (a-b) T ............................... 14 11. Palythoa townsleyl Fed and Unfed Polyps (a-b-c) ■ ..................................... 1 9, 22 12. Palythoa townsleyl Feeding on Artemia; a Generalized Lip Encloses Shrimp (a-b-c-d)...................................3 1, 38 1 3. Efficiency of Palythoa in Capturing Artemia as Compared to the Efficiency of a Sea Anemone Living in the Same Habitat (a-b) . . 42 14. Experimental Set Up for Studying Location of Proline-Receptors in Palythoa townsleyl . . 6l xi Figure Page 15. Activation and Characteristics of Mouth- Opening Reaction in Palythoa townBleyl Exposed to Different Solutions ............ 67 16. Correlation between Surface Area of Oral Disc in Palythoa townsleyl and the Dura tion of their Response to Proline 10 "3m Solution................................... 70 17- Response of Palythoa townsleyl to Untreated Filter Paper while in a Glutathione 10*" M Solution (a-b-c-d) ......................... 77,79 18. Lip Formation in Palythoa townsleyl ......... 89 19. Effect of Equimolar Concentrations of Proline and Glutathione in the Mouth- Opening Response of Palythoa townsleyl . . . 108 20. Effect of Glutathione in the Mouth-Open ing Reaction of Palythoa townsleyl to Proline 10 “5m ............................. Ill 21. Effect of Glutathione Concentration in the Mouth-Opening Response of Palythoa to Proline 10_4M 114 22. Effect of Glutathione Concentration in the Mouth-Opening Response of Palythoa town sleyl to Pro line 10-3m .................... 116 2 3. Effect of Pipecolic Acid Imbibed in Filter Paper (10-1M), on the Mouth-Opening Response of Palythoa townsleyl to Proline Solutions 7 ....................... 120 24. Effect of Proline Paper (P) (10-^M) in the Mouth-Opening Response of Palythoa town sleyl to Pipecolic Acid (P.A. J I 7 ........ 122 2 5. Effect of l-Thlazolidine-4-Carboxylic Acid (T) in the Mouth-Opening Response of Palythoa townsleyl to Proline Imbibed by Filter Paper at Concentration 10-1M . . . 125 xii Figure Page 26. Effect of l-Thiazolidine-4-Carboxylic Acid (T) Imbibed by Filter Paper (10-1M Con centration) on the Response of Palythoa townsleyl to Proline Solutions ............ 128 27. Chemical Structure of Glutathione ........... 132 28. Activation of the Ingestion Response in Palythoa townsleyl by Several Substances Imbibed by Filter Paper, at Concentration 10_1M ..................................... l4l 2 9. Activation of the Ingestion Response in Palythoa townsleyl by Proline and Glutathione in Solution ................... 146 30. Effect of Pipecolic Acid Imbibed in Filter Paper (lO-J-M) on the Ingestion Response of Palythoa townsleyl to Proline Solutions . 151 31. Effect of l-Thiazolidine-4-Carboxylic Acid Imbibed in Filter Paper (10"4m) on the Ingestion Response of Palythoa townsleyl to Proline Solutions ....................... 155 32. Effect of Proline-Paper (P) (lO'^M) in the Ingestion Response of Palythoa townsleyl to Pipecolic Acid (P.A. ' ) . ! 7 ............ 157 3 3. Effect of Proline-Paper (P) (lO^M) in the Ingestion Response of Palythoa townsleyl to l-Thiazolidine-4-Carboxylic Acid (T) . . 159 34. Effect of Reduced Glutathione in the Ingestion Response of Palythoa townsleyl to 10“^M Prollne Absorbed in Filter Paper ............................... 164 3 5. Effect of Proline in the Ingestion Response of Palythoa townsleyl to 10-1M Gluta thione Absorbed in Filter Paper .......... 167 3 6. Inhibition of the Ingestion Response in Palythoa townsleyl when Polys Are Placed in Equimolar Combination of Proline and Glutathione........................... 171 xiii Figure Page 37. Effect of Glutathione in the Ingestion Response of Palythoa townsleyl in Prollne 10_5m ............................... 174 3 8. Comparative Ranges of the Time Required by Palythoa townsleyl to Ingest Untreated Paper when the Polyps Are In Solutions of Glutathione or Combinations of Glutathione and Pro line..................... 176 39. Effect of Glutathione In the Ingestion Response of Palythoa townsleyl to Proline 10“4M ............................... 179 40. Comparative Ranges of Time Required by Palythoa townsleyl to Ingest Untreated Paper when the Polyps Are in Solutions of Glutathione or Combinations of Glutathione and Proline .................... 181 41. Effect of Glutathione Concentration in the Ingestion Response of Palythoa townsleyl to Prollne 10"3m Solution ................... 184 42. Comparative Ranges of Time Required by Palythoa townsleyl to Ingest Untreated Paper when the Polyps Are in Solutions of Glutathione or Combinations of Glutathione and Prollne .................... 186 4 3. Effect of Glutathione Dominance in Combina tions of Proline and Glutathione which Activate Ingestion Response in Palythoa townsleyl................................... 189 44. Enhancement of the Ingestion Response in Palythoa townsleyl under Certain Combina tions of Proline and Glutathione.......... 191 4 5. Response of Zoanthus paclflca In Glycine 1 0”1*! (a-b) . . ........................... 207 46. Position Assumed by Zoanthus paclflca after Exposure in 10-- * - or 1 0 Glutathione .... 217 47. Actinopharynx Exposure In Glutathione 10 . . 217 xiv Figure Page 48. Mouth-Opening Response in Glutathione 10 -4 to 10-DM............................. 217 49. Response of Zoanthus pacifica to Reduced Glutathione............................... 220 50. Response of Zoanthus pacifica to Glycine and the Glycine Analog a-Aminomethanesulfonic Acid....................................... 223 51. Response of Zoanthus pacifica to Feeding Activator, its Components and a Glycine Analog, Absorbed in Filter Paper, 0.5nun" in Size............................. 230 14 52. Uptake of Glycine- C from Solution by Zoanthus pacifica ......................... 242 53* Radiochromatogram of the Alcohol-Soluble Fraction of Zoanthus after Six Hours' Incubation with Glycine- C ............... 246 . _i4 54. Distribution of C in Alcohol-Soluble Fraction of Zoanthus as Determined by Radiochromatography ....................... 249 55* Response of Zoanthus pacifica in Glycine 10_1M (a-b) . . ......................... 261 xv PREFACE Clarification of the terminology used in this thesis is as follows: Feeding reaction is defined as a complex and or derly series of steps leading to the ingestion of food. In this study food represents any substance, liquid or solid, ingested by the zoanthids and kept in their coe- lenteron for a period of over 10 hrs. The feeding reaction of zoanthids consists of three major components: I. Lip Formation A group of tentacles seizes the food (Fig. l). The edge of the disc carrying these tentacles first con tracts, so that they group together around the food, then rises up and turns inward (Fig. 2), thereby folding tenta cles and food toward the mouth. The lip may be localized if it involves a re stricted number of tentacles (Fig. 2) or generalized if it involves all the tentacles (Fig. 3)• This clasping of food by the action of tentacles and disc has been described previously for sea anemones such as Anemonia sulcata (Pantln & Pantin, 19^3)• Torrey xvi Figure 1. Lip formation in Palythoa townsleyl upon contact with proline-paper. e, edge of oral disc Figure 2. "Localized" lip securing and pushing proline-paper toward mouth e, edge of the disc 1, lip xvii Figure 2 xviii (1904a.,b) described similar behavior in the Sagartia and in the hydroid Corymorpha. II. Mouth Opening When food reaches the mouth it opens; that is, the mouth borders separate to different degrees depending on the stimulus applied. 1. Wide, round-mouth opening when the borders of the mouth separate completely to give a maximum (Pig. 4) opening into the actinopharynx. 2. Small, round-mouth opening when the borders of the mouth separate partially (Pig. 5)• 3. Slit-like mouth opening when borders separate lengthwise only (Pig. 6). 4. Corner-of-the-mouth-opening when borders sepa rate at one end only (Pig. 7)• 5. Gaping-mouth opening when the borders separate completely, opening the mouth into the gastro- vascular cavity. Under certain circumstances the mouth-opening re sponse Is replaced by a step that I will call exposure of the actinopharynx. Mariscal & Lenhoff (1 9 6 8) described a general swelling or "Inflating" of the tissue immediately surrounding the mouth. The same phenomenon was reported by Lindstedt, Muscatine & Lenhoff (1 9 6 8) for the sea anemone xix Figure 3 . Lip formation. Edge of disc rising and turning inward. gl, generalized lip Figure 4. Wide, round-mouth opening and ingestion of proline-paper. b, border of mouth p, proline-paper mo, mouth opening xx Figure 4 xxi Figure 5 Figure 6 Small, round-mouth opening p, paper mo, mouth opening se, sand encrustations Slit-like mouth opening 1 1, localized lip mo, mouth opening xxii Figure 5 Figure 6 xxiii Boloceroides. The tissue surrounding the mouth corresponds to the glandular epithelium of the tube which leads from the mouth into the body cavity and which is called the ■actinopharynx. Under certain stimuli this tissue becomes inflated and may protrude as bladder-like lobes (Pig. 8). Ill. The Feeding Reaction Culminates with the Ingestion-Response --------- (Pig. 4)---------- A chemical substance found to stimulate the feeding reaction and carry it to completion is known as a feeding activator. Beck (1 9 6 5) reviewed the resistance of plants to insects., and classified the stimuli influencing different feeding-behavior responses. Those that apply to the zoan- thids studied are used in the context given by Beck; namely, "feeding incitant" is one that evokes biting or piercing reaction. Conversely, a Btimulus tending to prevent this response is designated as a "feeding suppressant." Stimuli tending to promote continuous feeding are termed "feeding stimulants" and those preventing the ter mination of feeding are designated "feeding deterrents." Rejection in the restricted sense used in this thesis describes the process by which zoanthids rid their oral disc of undesired material. Is is rather complex and Bimilar to that described by Jennings (1905a) for the sea anemone Stolchactls hellanthus. xxiv Figure 7 Figure 8 Corner-of-the-mouth opening p, paper mo, mouth opening Actinopharynx exposure p, paper al, actinopharynx lobes xxv Figure 7 Figure 8 XX vi The zoanthids remove debris from the oral disc by carrying it to the rim by ciliary currents. The edge with the waste sinks, the tentacles of the area collapse and the mass slides downward off the disc. Defecation designates the elimination of digestion products. Extrusion describes the elimination of symbiotic algae, zooxanthellae. xxvii INTRODUCTION The behavior of organisms is largely determined by the relation of their internal physiological processes to their environment. In no field is this so striking as in their behavior in obtaining materials for carrying on the processes of metabolism. General Considerations on the Species Used in This Thesis Palythoa townsley1 Walsh & Bowers (MS), Zoanthus danae, Verrill, 1896 and Zo an thus paciflca Walsh & Bowers (MS) belong to the family Zoanthidae, which includes Zoan- tharia with a mesogleal sphincter muscle and in which the sulcar element of the primitive sulco-lateral pair of mesenteries is imperfect (Walsh, 1 9 6 7). The manuscript by Walsh and Bowers is entitled "Zoantharia" and has been recom mended for publication. At the present time (June, 1970) it is being reviewed by Dr. Walsh. The names used in this thesis are those given by Walsh & Bowers in their manu script and are not valid until that manuscript is publish ed. The genus Palythoa is characterized by having a body wall heavily encrusted with sand and possessing a single mesogleal sphincter. The genus Zoanthus lacks en- 2 crustation of the body wall and has a double mesogleal sphincter (Walsh & Bowers, unpublished). Palythoa townsleyl is found in large numbers on the sand flats of Kaneohe Bay, Oahu, Hawaii. The polyps lie buried in fine sand to the level of the oral disc and are found solitary or in small groups (Walsh & Bowers, unpub lished) . The largest polyps measure up to 1.5 cm across the oral disc. Zoanthus paclflca is common in surge pools and rocky shores and on coral reefs of all Hawaiian islands. It is a colonial animal and is often found growing commen- sally with Palythoa vestltus (Walsh & Bowers, unpublished). Large polyps measure up to 0.5 cm across the oral disc. Zoanthus danae is commonly found in low intertidal rocky areas along the Gulf of California shores. It is colonial and often is found growing commensally with Eplzoanthus gabrlell (Carlgreen, 1951)• Previous Work Taxonomic work on the Hawaiian zoanthids has been reviewed by Walsh (1 9 6 7) and on the Eastern Pacific zoan thids by Cutress & Pequegnant (i9 6 0). Other work on zoan thids includes the discovery of a new amino acid in Zoanthus soclatuB (Kittredge & Hughes, 1964); some observations on the bioluminescence and reproduction of zoanthids from California (Cutress & Pequegnant, i9 6 0) and a detailed mor phological study on Eplzoanthus scotlnus (Wood, 1958). 3 The relationship of some zoanthids to their symbi otic algae has received considerable attention. Goreau (1964) observed loss of zooxanthellae by Palythoa carlbbea and Zoanthus soclatus after severe rains and floods in Eastern Jamaica. Muscatine (1 9 6 7) reported the ability of a subtropical subtidal zoanthid to excrete substantial quantities of glycerol when allowed to photosynthesize 14 Na2C 0^ in the presence of the host tissue homogenate. Von Holt & Von Holt (1968a) studied the transfer of photo synthetic products from zooxanthellae to its host Zoanthus flos marlnus. Feeding With respect to feeding there are, essentially, two groups of coelenterates: those that capture live prey— mostly zooplankton, and those that do not seem to take any exogenous food. Although they are equipped with nemato- cysts, the stinging cells that other ceolenterateB use to seize their prey, members of this second group do not cap ture any organisms even if theBe swim about over their oral disc and tentacles. Since Trembley published his famous Memoires in 1744, with his observations on the biology of hydra (sensu lato), a large number of papers have dealt with feeding in predatory coelenterates. Reviews covering this work are by Parker (1 8 9 6,1 9 1 7) Jennings (1905b), Boschma (1 9 2 5), Yonge (1930)j Pantin & Pantin (1943) and Lenhoff (1968a). The non-predatory group of coelenterates, however, has received considerably less attention. Gohar studied feeding in the Xeniidae (Gohar, 1940) and in the alcyonari- an Clavularia hamra (Gohar, 1948). He found no response to any exogenous food and concluded that the Xeniidae and Clavularia hamra depend almost entirely on the zooxanthel lae for the supply of nutritive substances. Goreau (1967) made observations on the lack of typical morphological di gestive structures in some species of tropical Zoanthus. The only work on the feeding biology of a zoanthid is that of Hadden (1 9 6 8), who found that Zoanthus soclatus could in gest pieces of frozen butterfly fish obtained from the same habitat where Zoanthus was found. Chemical Control of the Feeding Reaction During feeding, most coelenterates first capture and pierce the prey with their nematocysts. Next, a sub stance present in the fluids oozing from the wounds in flicted by the nematocysts on the prey, causes the tentacles to contract toward the mouth and the mouth to open. Lastly, on contact with the mouth, the food is ingested. Parker (1 8 9 6) first suggested that feeding of the Cnidaria might be under chemical control. Nagel (1 8 9 2), Abe (1938), Beutler (1924), Henschel (1935) and Pantin & Pantin (1943) found that food extracts alone cause many coelenterates to carry out part of the feeding reaction. In 1955, Loomis Identified the first feeding activator for the Cnidaria: the tripeptide glutathione, which induces the feeding reaction in Hydra llttoralls. Since then, numerous workers have studied feeding reaction in diverse coelente rates, using the techniques developed by Loomis (Loomis, 1 9 5 5)• A comprehensive review of the chemical control of feeding in coelenterates was presented by Lenhoff (1968a). No work has been published on the feeding reaction of the non-predatory coelenterates. Significance of Study This study is the first which deals with the con trol of feeding response in zoanthids. The hypothesis is that the two genera of subtropi cal zoanthids, Palythoa and Zoanthus, the first belonging to the predatory coelenterates and the second belonging to the non-predatory coelenterates, have feeding reactions which are controlled basically in similar ways, even though the feeding methods used by the animals to incorporate food into their systems and the relative importance of such exog enous food in the well-being of the polyps, may differ widely between the two genera. Description of the dynamic and changing patterns of animal behavior is a challenge, for the possible measure ments are almost infinite. Some are correlated, others vary independently. Some can be recorded directly by hu man senses, others require instruments to reveal them. GENERAL CONSIDERATIONS ON METHODOLOGY Early zoologists who studied the behavior of ani mals such as sea anemones were inclined to the view that all responses were directly evoked by external stimuli. The animal was believed to participate only as a passive recipient of environmental forces. But careful study has shown that movements in a sea anemone such as Metridlum occur constantly and apparently spontaneously (Bathan & Pantin, 1950). Jennings (1905a) placed strong emphasis upon the dynamic relationship between organisms and their surround ing environment. He pointed out that often the effect of an external stimulus upon an organism can be predicted only if the physiological state of the animal at the moment of stimulation Is known. The need to take account of the physiological state of an animal in predicting what the effect of a given ex ternal stimulus will be on its behavior relates in part to the fact that past stimulation may affect the response to a present stimulus. For example, Batham & Pantin (1950) found that a starved animal may show complete feeding re sponses to inappropriate objects such as grains of sand. Starvation lowers the threshold for the response in Metri- dium and satiation raises it. A partially satiated animal will wait longer before responding to food and may have to be presented with several pieces before it will show a com plete response. Still other factors affecting the feeding response have to be accounted for. Whether food stimuli will in fact evoke feeding depends on the state of the animal, which may be partly a function of its posture at the time, and the expansion or retraction of its tentacles. Furthermore, Allabach (1905) found that fatigue could affect the re sponsiveness of Metrldium to food. If the tentacles of a certain region of the disc of Metridium were given many pieces of food, one after the other, they became fatigued and after a time refused to take food. Analysis of the stimulus properties responsible for the environmental control of different action patterns is a central concern in the study of animal behavior. There is wide variation in the specificity of the effective ex ternal stimuli. Some behavior patterns are triggered by several types of stimuli, while in others the relationship is more specific. But in every case a given behavior pat tern is controlled by only a portion of the vast array of external stimuli that the animal can perceive. The animal, in the words of Jennings (1905a), responds to "representa tive stimuli." The external stimuli evoking feeding behavior in anemones have been analyzed In some detail. There are at least two distinct phases. First there Is the discharge of the nematocysts and secondly the actual grasping and in gestion of the food. Pantln (19^2) showed that In Anemonla sulcata a mechanical stimulus is usually not sufficient for nematocyst discharge. Normal discharge also requires sens itization by chemical stimuli which lowers the threshold of response to tactile stimuli without actually eliciting dis charge. Ewer (19^7) found that the two nematocyst types used by Hydra in feeding were sensitized by chemical stim uli from food and were triggered by mechanical contact. Not only is it necessary to know what kinds of stimuli to apply in order to elicit a feeding reaction but also it is important to know how to control the conditions under which these stimuli are applied. The importance of carefully regulating the condi tions of the experiment have been emphasized by Lenhoff (1965,1968a) and can be appreciated fully if the work of Forrest (1 9 6 2) is analyzed critically. She asserted that the feeding reaction in the ten North American hydra species [including the same species and strain used by Loomis (1955) and Lenhoff (1961a)] was independent of the presence of reduced glutathione, contrary to what had been reported by Loomis (1955) and Lenhoff (19 6la). She failed, however, to control the medium in which hydra were kept and in which the experiments were performed. She reported using "fil tered pond water" which of course could contain a number of substances, including glutathione, proceeding from the rup ture of small organisms upon filtration of the water. The most important factors to control in experi ments of feeding behavior are the condition of the medium and the degree of starvation or repletion in the experi mental animals. Collection of Specimens Palythoa townsley1 and Zoanthus paclflca were col lected on the North Reef of Coconut Island, Kaneohe Bay, (Pig. 9)» Oahu, Hawaii, on May 6, September 22, 1969* and January 2, 1970. The animals were transported to Los An geles by United Airlines and placed in 5-gallon aquaria (Pig. 10a,b) containing artificial sea water, within 15 to 24 hours after collection. Zoanthus danae was collected in Puerto Penasco, Mexico, on December 17, 1968, and brought to Los Angeles within one week after collection. Experimental Conditions All animals were held in well-aerated 5-gallon aquaria containing Instant Ocean (Aquarium Systems, Inc.) prepared according to the instructions of the manufacturer. Salinity was checked daily with a hydrometer and maintained at 33 o/oo. Temperature was held between 24 and 27° C by using a refrigeration system during the summer (Bakus, 1965) and aquarium thermostats during the remainder of the Figure 9. Collecting sites for Palythoa townsley1 (p) and Zoanthus paclflca (zj. 11 12 Figure 9 Figure 10. Aquarium where Palythoa townsleyi (p) and Zoanthus pacifica \z) were maintained. a, side view bj top view 13 14 Figure 10 b year. Illumination was provided by means of four cool, white fluorescent lamps About 200 polyps of Palythoa and as many as five colonies of Zoanthus were held in each aquarium. Water was changed every 15 to 30 days. Waste products of the polyps and other animals living commensally with them, such as some syllid polychaetes and some nemer- teans, accumulated in the aquaria and could affect the be havior of the animals. Therefore, experimental polyps were isolated in 250 ml finger-bowls containing fresh Instant Ocean for two hours preceding an experiment. Animals were used only once in a 24-hr. period and returned to the large aquarium after the experiments were done. To control the physiological state of the animal, in order to know its degree of starvation, previous workers have used animals that had faBted for one day [Lenhoff (1961b); Pulton (1 9 6 3); Lindstedt, Muscatine & Lenhoff (1 9 6 8) and Pardy & Lenhoff (1 9 6 8)]. This method could ap ply well to Polythoa, which could be fed Artemla without difficulty. Zoanthus, however, would not respond to food and therefore could not be fed. Consequently, and since my interest waB to compare both genera under similar condi tions, both animals were kept unfed, except for a group of Palythoa which was used in experiments to determine the characteristics of its response to Artemla and which was kept separated from the starved Palythoa and Zoanthus. 16 The degree of starvation, as discussed above, af fects the behavior of other animals; therefore, control ex periments had to be devised to determine if Palythoa or Zoanthus were affected by it. These experiments consisted in timing the speed with which the polyps responded to feeding activators and, in the case of Palythoa, to live Artemla. It was found that polyps starved for up to one month (Fig. 11a,b) showed no difference in their reactions to feeding activators, although the speed of reaction was slightly lower in starved animals (Table 1, fig. 11a). The first day in the laboratory after collection, the animals were very sensitive to mechanical stimulation and could not be used, for they would contract upon contact 2 with a 1 mm piece of filter paper. This sensitivity to a mechanical stimulus tapered off within 2 weeks. For the reasons considered above, Btarved polyps were used in all experiments except those with live Artemia and the period of experimentation on the animals was restricted to within 2 to 4 weeks after collection. Related experiments were all performed within 2 or 3 days of each other to limit the variations due to the physiological state of the animals. In most experiments with Palythoa, 20 polyps were used so that each polyp responding to a solution represents 5 percent of the total tested. The fact that no response was ever obtained In the controls run for each experiment TABLE 1 EFFECT OF STARVATION IN THE RESPONSE OF PALYTHOA TOWNSLEYI TO FILTER PAPER IMBIBED IN PROLINE 10-1M* Condition of Palythoa Condition of Paper Time to Form Lip (min.)** In Percent age Polyps Time to Ingest Paper (min.) In Percent age Polyps Reject Paper (min.) In Percent age Polyps Fed*** Imbibed in proline 10-1M 0.18 + 0.06 100 0.60 + O.57 100 - - Unfed**** Imbibed in proline 10-lM 0A3 + 0.53 100 1.08 + O.58 100 - - Fed Untreated - - - - 10.6 + 8.2^ 100 Unfed Untreated - - - - 12.0 + 11.90 100 *20 Polyps tested. v w Although the numbers are indicated as decimal fractions of minutes, they were collected with a stop watch measuring seconds and are only expressed as decimals to facilitate statistical handling of the data. ***Polyps fed Artemla regularly. xxx* ( polyps starved for 1 month. Figure 11a. Range of time required by Palythoa to initiate a feeding response to proline 10-lM imbibed in filter paper. Fed and unfed polyps are compared. 18 Fed polyps 1 " ! 0.5 Unfed polyps Figure 11a min. indicates that responses by 5 or 10 percent of the polyps are significant even though they represent only 1 or 2 polyps. Starvation of more than two weeks diminished the size of the Palythoa polyps and reduced their tentacles (Pig. llb,c) to a point where they could not capture live Artemla. For this reason the experiments with live brine shrimp were performed on Palythoa within one week of col lection . Figure llb,c. Palythoa townsleyl b. Colony starved for 1 month, m, mouth tj tentacles c. Colony recently collected, m, mouth t, tentacles od, oral disc per, peristome 21 Figure lib Figure 11 c PART I FEEDING BEHAVIOR IN PALYTHOA TOWNSLEYI. WALSH AND BOWERS 23 CHAPTER I RESPONSE OF PALYTHOA TO A VARIETY OF FOODS Palythoa townsley1 is a semicolonial animal found In large numbers on the sand flats of Kaneohe Bay, Oahu, Hawaii. The polyps lie burled In the fine sand to the level of the oral dlBc and are found solitary or In a small group. Palythoa belongs to the predatory coelenterates, a large group of animals that utilize complex Intracellular secretion products— the nematocysts, In the capture of prey. Because Palythoa Is sessile and has very limited movements of the column and oral disc, the animal depends largely on the natural water movements to carry food within the range of Its tentacleB. As Crisp (1 9 6 2) pointed out, the advantage to this system Is that food can be derived from a very wide area with the minimum effort on the part of the animal. The disadvantage, however, Is that the ani mal must be content with whatever food arrives by chance and cannot afford the luxury of a specialized diet. Hyman (1940) reported that sea anemones eat almost any live ani mals of suitable size. Yonge & Nichols (1931a) made the same observation for corals. 24 25 Anemones and zoanthids use their tentacles to seize food and naturally the size of their prey is restricted to that which can be captured by the tentacles. Other coelen- terates, such as many siphonophores, possess fishing fila ments (Mackie & Boag, 1 9 6 3). Some species, notably Phys- alla, use these structures to capture large prey, such as fish (Wilson, 1947). Other species, e.g., Nanomla, use their fishing filaments to catch small crustacean larvae (Vogt, 1854). Although very little is known about the natural diet of predatory coelenterates, most of them can be kept successfully in the laboratory on a diet of Artemla and the general consensus is that in nature they feed on crustacean microplankton. Errington (1 9 6 7) noted that most predatory species are adapted to do some fasting when necessary and are able to engorge themselves when they have access to an abundance of food. The rate of prey captured is usually proportional to the concentration of the food organisms (Crisp, 1 9 6 2) but there is a limit to how much the predator can ingest. Crisp (1 9 6 2) found that barnacles fed Artemla ingested only a limited amount of food, the excess material caught by the cirri being returned to the water. He concluded that the rate of Ingestion does not depend on the concentration of nauplii in the surrounding water, provided, of course, that it exceeds certain minimal requirements. 26 The experiments described in this chapter were de signed: 1. To obtain some information on the behavior of Palythoa as a predator on Artemla. 2. To determine whether the polyps have a diet re stricted by some controlling factor such as found by Loomis (1955) for Hydra; or if they feed in discriminately on any type of food or inert ob ject such as reported by Hyman (1940) for certain sea anemones. Materials and Methods Palythoa townsleyl polyps were collected in the North reef of Coconut Island, Oahu, Hawaii (Fig. 9), and shipped to Los Angeles, California by Mr. Ralph L. Bowers. Upon arrival they were placed in a well aerated 5-gallon aquaria (Fig. 10) at 25+1.5° C and 33 °/oo salinity. Preceding an experiment the polyps were removed from the large aquarium, placed in fresh Instant Ocean con tained in 2 5 0-ml finger bowls and two hours later offered the experimental food. Artemla nauplii were raised in the laboratory from eggs (Bay shrimp eggs, San FranciBco) in a cone-shaped plastic hatchery. Nauplii were rinsed in fresh Instant Ocean and offered to the zoanthids. Adult Artemla were purchased from Los Angeles Aquarium and offered to the zoanthids after rinsing in fresh Instant Ocean. 27 The reactions of the polyps were observed, timed (Cletimer Stop-watch) and recorded. Time of observation was completed only when food had been either Bwallowed or rejected. Once it was determined that Palythoa townsleyi responded to Artemla, an aqueous homogenate of brine shrimp was prepared as follows? a solid pack suspension of Artemla was homogenized in an electric grinder. The homogenate was either used directly or centrifuged and only the super natant tested. An extract of Artemla for chromatographic analysis was obtained by homogenization in distilled water, centri fugation at 1,500 rpm in an International Clinical Centri fuge, and the resulting aqueous supernatant mixed with an equal volume of 80 percent ethanol. The alcohol-soluble material was dried in a lyophilyzer, resuspended in 80 per cent ethanol and then recentrifuged. This procedure was re peated several times, first with 80 percent alcohol, then 95 percent alcohol and finally with absolute alqohol until no further precipitate was formed. The extract was spotted on Whatman paper #4, and one-dimensional descending chro matograms were run in butanol-propionic acid-water (4:2?3) solvents (Bassham & Calvin, 1957)• One strip was developed with one percent ninhydrin in acetone and its co-chromato gram was retained for testing. A blank paper was run through the same solvent system as the shrimp extract. It p was then offered to the polyps as small 1mm pieces, to 28 control for the possible effect of solvents in the response of the zoanthids. A second experimental control consisted 2 of 1mm pieces of clean filter paper to determine if this caused any feeding response in the polyps. When a strip that produced both good concentration 2 and separation of spots was detected, 1mm pieces were cut out of the undeveloped portion of the co-chromatogram and presented directly to the polyps. In this way the approxi mate region of the chromatogram which elicited mouth open ing could be determined. A piece of blank filter paper which had also been run through the solvent system and a piece of clean filter paper served as controls. To determine the identity of the compounds present in the chromatograms of Artemla extract which elicited a feeding response, the Rp value of each active spot was com pared to that of a known amino acid mixture containing glycine, serine, cysteine, proline, alanine, valine, leu cine and phenylalanine, that had been run through the same one-dimensional chromatography system. The Rp values were obtained by comparison of resulting spots with a chromato graphic map of amino acids (Bassham & Calvin, 1957)• Results Palythoa townsleyi shows the typical feeding reac tion that was described in the preface of this thesis. 29 a. Response to live material 1. Artemla sallna (adult shrimps) On contact with Artemla the tentacles of Palythoa writhe and discharge very few nematocysts to immobilize the shrimp. Examined prey seldom had more than two nemato- cyst's tubes piercing its lower abdomen. Once the prey has been secured by the tentacles, it is either pushed by these toward the peristome, or enclosed in a lip formed at the point of contact between the prey and the polyp’s ten tacles. The lip becomes generalized (Fig. 12a,b) involving all the tentacles. The mouth opens slightly and the oral disc closes over the shrimp (Fig. 12c,d), which is swallowed shortly afterwards. The entire reaction takes an average of 0.72 minutes. After ingestion of the shrimp a group of expanded tentacles protrudes above the rim of the contracted disc (Fig. 12b,c,d). This proved to be a behavioral re sponse intended to allow consecutive capture of prey. Table 2 shows that 73 percent of the polyps that maintain a bunch of tentacles protruding after capturing the first prey, do capture a second time. Of those that do not show the tentacle protrusion, only 25 percent achieves consecu tive captures. The results presented in Table 3 suggest that shrimp density may have an effect in producing the tentacle- protrusion response. Polyps exposed to either low (5 or 1 5 /2 5 0 ml) or high (9 0 /2 5 0 ml) densities of shrimp tend to Figure 12a,b. Palythoa townsleyi feeding on Artemla; a generalized lip encloses shrimp. a. side view gl, generalized lip mos, mouth opening small b. front view gt, group of tentacles el, enclosing lip s, shrimp 30 31 Figure 12.b TABLE 2 INFLUENCE OF A BEHAVIORAL RESPONSE* ON THE ABILITY OF PALYTHOA TOWNSLEYI TO MAKE SUCCESSIVE CAPTURES OF ARTEMIA Number of Percentage of Polyps Percentage of Polyps Polyps with Tentacles Pro Making Successive Tested truding after Catch Captures 69 66 73 50 0 25 *A group of tentacles protrude after animal closes the oral disc having captured live Artemla (Fig. 12c,d). TABLE 3 PERCENTAGE OF POLYPS OF PALYTHOA TOWNSLEYI WHICH LEAVE AF^TteR CAPTURING ARTEMIA TENTACLES PROTRUDING Number of Percentage of Polyps with Tentacles Pro Shrimp Density Polyps truding after per 250 ml Tested 1st Capture 5 5 100 16 16 100 30 20 50 50 20 46 70 20 30 90 20 78 uo CO show higher incidence of tentacle-protrusion than polyps exposed to intermediate (50 or 7 0 /2 5 0 ml) densities. Artemla density also seems to have an effect.on the fre quency with which the shrimp escapes after being captured by Palythoa. Table 4 indicates that only when the shrimp density is 15/250 ml or below, can Artemla escape after capture. This fact is also reflected in the somewhat longer time required by Palythoa to capture shrimp when they are at low densities (Table 5). It seems logical to assume that the density at which the largest number of shrimp is captured in the shortest time represents the op timum plankton density for Palythoa to prey upon. Such density was found to be 50 shrimp/2 5 0 ml. However, the experiments were discontinued after the second capture in densities of 70 shrimp/2 5 0 ml and after the first capture in densities 90 shrimp/2 5 0 ml, because the number of shrimp swimming about the polyps made extremely difficult to re cord accurately the number of shrimp being caught or the sequence of captures by a particular polyp. For this rea son it is not possible to say that 50 shrimp/2 5 0 ml repre sents the optimum concentration but only that this is the minimum optimum concentration of shrimp at which Palythoa can acquire food rapidly and efficiently. After 1.33 min utes the polyps have captured 74 percent of the shrimp present in the dish. In densities of 30 Artemla per 250 ml TABLE 4 FREQUENCY OF ESCAPE OF ARTEMIA SALINA FROM PALYTHOA TOWNSLEYI Shrimp Density per 250 ml Number of Polyps Tested Percentage of Shrimp Escap ing after Capture 5 20 50 16 20 22 30 20 0 50 20 0 70 20 0 90 20 0 oo VJI TABLE 5 FREQUENCY* OF SHRIMP (AKEEMIA SALINA) CAPTURE BY PALYTHOA TOWNSLEYI Shrimp Density per 250 ml Time of 1st Capture (min.) Time of 2nd Capture (min.) Time of 3rd Capture (min.) Time of 4th Capture (min.) 5 1.91 + 1.23 ( 5)** None None None 16 0.86 + 0.64 (16) None None None 50 0.42 + 0.19 (20) 2.75 + O.69 (6) 43.83 (3) 55.66 (1) 50 O.33 + 0.15 (20) 0.94 + 0.68 (6) 1.02 + 0.48 (6) 1-35 + 0.77 (5) 70 0.28+0.19 (20) 10.91 + 6.58 (6) Not observed Not observed 90 0.76 + O.65 (20) Not observed Not observed Not observed *Rates are expressed as mean values with standard deviation. **Number in parenthesis indicates number of polyps tested. tjO (Ti Figure 12c,d. Palythoa townsleyl feeding on Artemla; a generalized lip encloses shrimp. c. Shrimp being enclosed, as a group of tentacles protrude ed, enclosing disc gt, group of tentacles d. Oral disc closed over shrimp gt, group of tentacles protruding ode, oral disc closed s, shrimp 37 Figure 12c Figure i2d 39 it takes the polyps almost 1 hour to capture all the shrimp present in the dish. Another important feature of the prey-predator re lationship between Artemla and Palythoa is the number of Bhrimp that can be captured at any one time (Table 6). Here again the density of shrimp is very important in de termining the number of shrimp caught. The highest density gives the highest number of prey caught at any one time. The maximum observed was six shrimp captured by a single Palythoa polyp, simultaneously. This may not seem impres sive when compared to other coelenterates such as the Hawaiian sea anemone in Pig. 13a,b, which customarily cap tures five or more shrimp before beginning to ingest any of them, but Palythoa most often only captures one Artemla at a time and ingests this before capturing a second one. Only a few polyps capture more than three Artemla at any one time (Table 6). The shrimp captured is Ingested and digested, for, after 10-12 hrs., the clean exoskeletons of Artemla are extruded through the mouth, together with some brown-yellow, material and some granular, red material. 2. Syllid polychaetes, nemerteans and Tubifex worms Tentacles of Palythoa writhe, the mouth opens and food is ingested within one minute. TABLE 6 EFFECTS OF SHRIMP DENSITY ON THE ABILITY OF PALYTHOA TOWNSLEYI TO PERFORM CONSECUTIVE CAPTURE OF AEIEMIA SALINA Shrimp Density per 250 ml Number of Polyps Tested Percentage of Polyps Achieving Consecu tive Captures Percentage of Polyps Capturing 1 2 3 4 5 6 Shrimp, at any one time 5 5 0 100 16 16 0 100 30 20 20 80 20 50 20 20 80 10 10 TO 20 40 60 20 20 90 20 50 50 30 5 5 5 5 -t- o Figure 1 3. Efficiency of Palythoa in capturing Artemla as compared to the efficiency of a sea anemone living in the same habitat. a. Anemone has captured two shrimps p, Palythoa a, anemone b. After two minutes anemone has cap tured six shrimps pws., Palythoa with shrimp aws, anemone with shrimp 41 Figure 13 < a . Figure 13 b b. Response to dead material 1. Pishsticks (Certi-fresh breaded cod), frozen cod and freshly killed Gambusla (all cut in pieces 1x2 mm in size) All the polyps tested showed a characteristic feed ing reaction. When the food is placed on the peristome the first step of this reaction is mouth opening, then the area surrounding the mouth becomes depressed so that the oral disc of the animal resembles the upper portion of a funnel. At the same time the tentacles curl up and toward the mouth, enclosing the food, which is ingested within 1 to 3 minutes. When the food is placed on the margin of the oral disc, the first step of the reaction is the formation of a lip which encloses the food and literally pushes it toward the mouth; this opens when the food reaches the peristome and the food is ingested. After ingestion the polyps begin to relax and with in 8 to 10 minutes they are expanded and capable of re sponding to more food. 2. Freshly killed Artemla salina Regardless of where the shrimp is placed on the oral disc, a lip is formed (Pig. 12a). This encloses the food and carries it to the mouth, which opens within 30 seconds. The shrimp is ingested within 2 to 3 minutes. Water and alcohol extracts of Artemia cause lip formation 44 and mouth opening when squirted directly over the peristome. All food offered and ingested by Palythoa remained in the animal for 10 to 12 hours. After this period pel lets containing clean exoskeletons of Artemia mixed with a greenish-brown material and with red granules are elimi nated through the polyp's mouth. c. Response to treated filter paper 1. Imbibed in water- or alcohol-extracts of Artemla Water- and alcohol-extracts of Artemla spotted on filter paper are ingested by Palythoa within two to four minutes of being offered. 2. Filter paper proceeding from chromatograph of alcohol-extract of Artemla Amino acids separated by this method and present in 2 the paper, were offered to the polyps as pieces 1mm in size. Table 7 shows that only the yellow spot, correspond ing to proline, could always elicit ingestion of the paper. Alanine caused 30 percent of the polyps to ingest paper and lysine elicited ingestion in 10 percent. Controls of un treated paper and blank paper (chromatographed) served as controls and were rejected by Palythoa. Discussion Palythoa lies on the sand with the column buried to the level of the oral disc and shows no apparent movement. 45 TABLE 7 RESPONSE OP PALYTHOA TOWNSLEYI* TO AMINO ACID FROM ARTEMIA-ALCOHOLIC EXTRACT, ON FILTER PAPER Amino Acid Percentage of Polyps Ingesting Paper Blank (chromatographed) 0 Untreated paper 0 Alanine 30 Cysteine 0 Glycine 0 Leucine 0 Lysine 10 Phenylalanine 0 Proline 100 Serine 0 Valine 0 *10 polype tested In each experiment. The polyps., however, may have a full range of behavior pat terns, including postures for resting, defecating, being hungry or satiated. Perhaps these could be revealed by use of the time lapse photography and kymographlc record ings as was done for the sea anemone Metrldlum senile by Batham and Pantln (1950). The fact Is, though, that be cause such spontaneous and probably continuous movements cannot be detected visually and so are not distracting to the observer, he can study the response to an external stimulus, such as food, without having much difficulty In analyzing the different steps and details of feeding be havior. The seemingly Inactive polyps of Palythoa show quick and predictable responses to zooplankton. The steps of the feeding reaction, although somewhat modified, are essentially those described for most predatory coelenter- ates (Lenhoff, 1968a). Upon contact of Artemla with Paly thoa1 s tentacles, nematocysts are discharged and pierce the prey, a grasping lip secures and puBhes the food toward the mouth which opens and the prey is ingested. The function of nematocysts in capturing prey has been studied in detail for Hydra by Ewer (1947) and on Pen- narla by Pardy and Lenhoff (1 9 6 8). Both authors reported the penetration of the prey's exoskeleton with numerous nematocysts. Sea anemones living together with Palythoa in the same aquarium also discharged many (around 8 or 10) nematocysts per Artemla and were able to capture a consider able number of prey In very short time. Prey caught by Palythoa, however, consistently showed one or two nemato- cyst's tubes piercing the lower abdomen of the prey. This would struggle actively to liberate Itself but the grip of the lip formed by a group of tentacles and the contracted margin of the oral disc was always strong, and very seldom could the shrimp escape. Escape happened only when the prey was at very low densities, and very often the prey that escaped showed no signs of having been hurt. Palythoa captures prey with the tentacles by discharging few nemato cysts and relies more on the grasping action of a localized lip to secure and push the prey toward the mouth than on immobilizing the prey by toxins injected upon contact with it, such as occurs in Hydra (Ewer, 19^7) and in Pennarla (Pardy & Lenhoff, 1 9 6 8). Ab the prey comes closer to the mouth, the localized lip becomes generalized; that is, all the tentacles and the entire margin of the oral disc move upward and mouthward to enclose the prey. This method of capturing prey is slower than that used in Hydra or Pen narla, and Palythoa may compensate for this seemingly less efficient prey-capturing method by leaving tentacles pro truding from the closed oral disc immediately after enclos- ing Artemla. This group of protruding tentacles allows the polyps to catch new shrimp while ingesting the ones that have been just captured. As was reported in the results 48 section, if the density of shrimp is adequate, Palythoa can capture a minimum of 4 adult shrimp within 1.33 minutes by this system of consecutive captures. If Palythoa captures more than one shrimp at any one time, such as is the case in densities of 30 shrimp/2 5 0 ml or higher, then the number of prey caught can be much higher than 4. For example, if in the first capture Palythoa ingests three shrimp; in the second, two; in the third, one; and the fourth, one; the total prey caught could be Beven. By using Tables 2 and 5, a great number of possible shrimp captured and ingested can be calculated with the restriction, of course, that the number of shrimp ingested will be limited by the capacity of the tentacles to catch and hold prey and by the holding capacity of Palythoa1s gastral cavity. Regarding the first limitation, each tentacle contains a definite number of nematocysts and after all of them have been discharged new ones will have to be replaced before that tentacle can cap ture more prey. It is very unlikely, however, that the number of nematocysts per tentacle will limit the prey captured by Palythoa, for only one or two need to be dis charged to catch the shrimp and then the prey is secured by muscular activities of the tentacle. It is conceivable that after a certain number of captures the tentacles of Palythoa become fatigued and can no longer hold more prey. Allabach (1905), for example, 49 found that if the tentacles of a certain region of the disc of Metridlum were given many pieces of food, one after the other, they became fatigued and after a time refused to take food. Regarding the second limitation to the amount of prey caught by Palythoa, that is, the holding capacity of its gastral cavity, it is necessary to remember that, as Errington (1969) pointed out, predatory species are able to engorge when they have access to an abundance of food; consequently, Palythoa may be able to ingest unusual amounts of food. Because Palythoa is sessile, feeding in nature is undoubtedly a fortuitous and discontinous process, depend ent on the food available at particular times of the year. According to Peterson (personal communication), the waters near the reef where Palythoa was collected (Pig. 9) contain microzooplankton overwhelmingly dominated by one of two copepod genera and their respective naupliar and copepo- dite stages. The animals are Pseudocalanus sp. and Olthona sp. Peterson estimated that the copepodites of these crus taceans numbered 40,000/m^. If expressed as density per ml this could represent six copepodites/2 5 0 ml which compares to the lowest density of Artemla used in this study. Given the size of the copepodites, 0.18mm to 0 . 5mm for Pseudo calanus and 0.40mm for Oithona (cephalothorax length on both) and their density, they do not seem to represent much 50 food for Palythoa. The polyps probably capture not only microcrustaceans but also macrozooplankters such as barnacle nauplii, crab zoea larvae of Lucifer chacel (pelagic decapod shrimp) and Alpheus (snapping shrimp) mysls stages. Accord ing to Peterson’s estimates of the macrozooplankters named above, only 212 animals occur per cubic meter. The food available to Palythoa In the natural environment does not seem to amount to much but It Is Impossible to decide how significant It Is for Palythoa without knowing the meta bolic requirements of the polyps and the ways In which they might fulfill them. The number of zooplankters consumed by Palythoa will depend on the number of collisions of their tentacles with the plankters: It would seem that the higher the den sity of zooplankton the higher the possible number of col lisions. But when the density increases beyond a certain point the collisions will be so numerous that Palythoa would spend more time and capture less prey. There is an indication of this in the experiments where 90 shrimp/250 ml were offered, but the information gathered is very Bcant to place much importance on it. This observation agrees with the information reported by Cushing (1 9 6 8), who found that prey mortality decreases with prey density because the time spent capturing and eating increases. Cushing worked with grazing herbivorous copepods which obtain their food by tactile encounter as they move steadily through the 51 water. This method, according to Crisp (1 9 6 2), represents an inverted system with respect to that of sessile animals that are Btatic and spread out as a surface layer, while the food material is carried past them by the movement of the water. In both systems, however, the same rules seem to operate for both involve prey-predator relationships. Sessile animals such as Palythoa may have to util ize a very catholic diet, for, as discussed above, the quantity of live prey in their environment 1b very limited. Most coelenterates are observed to subsist only on live animal food (Lenhoff, 1968a). It was found that Palythoa does not respond to algal masses collected from its natural habitat. The polyps give positive responses to freshly killed and to frozen animal food, but not to inert material such as pieces of filter paper unless these are first soaked in Artemia extract (or certain components of this extract). Loomis (1955) found that Hydra responded to the tripeptide glutathione present in the extract and concluded that this was the mechanism by which an animal without brain nor sensory organs such as the eyes of higher animals could differentiate between living and dead prey. Since then, many coelenterates have been found to recognize dif ferent compounds in live prey. The chromatographic separa tion of Artemla extract showed that Palythoa responded with ingestion to a yellow spot in the chromatogram (Table 7)> corresponding to proline. The effect of this and other 52 amino acids in the feeding response of Palythoa are the subject of the next chapter. Conclusions 1. After Palythoa have captured live prey with their tentacles, they demonstrate a coordinated feeding re action . 2. The polyps compensate for their inability to inmobilize prey with nematocysts by using their tentacles and margin of the oral disc to hold it against the peris tome. 3. The behavioral response called "tentacle pro trusion" allows the polyps to capture additional prey while ingesting one just caught. 4. The density of zooplankton affects the speed with which Palythoa captures live prey. 5. Palythoa polyps can capture as many as six shrimp at any one time, when the Artemla density is 90 shrimp/250 ml. 6. The optimum density of zooplankton, which causes the fastest response and largest capture, was found to be 50 shrimp/2 5 0 ml. This is about ten times that cal culated for the natural environment of Palythoa. 7. The polyps digest Artemla within 10-12 hours and clean exoskeletons are egested through the mouth. 53 8. Palythoa Ingest fresh-killed or frozen animal food. 9. The polyps reject vegetable food. 10. The imino acid proline will elicit a feeding reaction in Palythoa. 11. A chemical stimulus is needed to provoke feed ing behavior because untreated paper is rejected by the polyps. CHAPTER II GENERAL CHARACTERISTICS OP THE FEEDING REACTION Trembley (1744) described the fact that hydra feed exclusively on living animals. Subsequent workers have confirmed and extended Trembley’s observations and have elicited feeding behavior in diverse coelenterates by of fering them constituents of their natural foods. The experiments of Henschel (1935) on the manubria of various medusae showed that feeding response could be obtained to proteins, peptones and various amino acids. Beutler (1924) observed that hydra would ingest small pieces of gelatine or fibrin soaked in crustacean Juice. Batham & Pantin (1950) studied the response of the sea anemone Anemonla sulcata and found that the active substances of natural foods causing feeding reactions in the anemone were soluble peptones and amino acids. They also found that the mouth of Anemonla responds to a greater variety of stimuli than the tentacles and has a higher chemical sensitivity. The chemical control of feeding be havior, however, could not be Btudied in detail until Loomis (1955) and Pulton (1963) developed the methods for rearing hydroids in the laboratory under rigorously con trolled conditions and established the procedures for 55 isolating the substances activating feeding reaction. Loomis (1955) found that the writhing of tentacles and mouth opening reactions of Hydra could be elicited by plac ing the polyps in a solution of glutathione 10~^M. Pulton (1 9 6 3) studied the chemical control of feeding in Cordylo- phora and found that this was activated by the amino acid proline. Numerous papers appeared since then, reporting studies on many diverse aspects of the feeding behavior in Hydra, such as quantification and characterization of the feeding reaction (Lenhoff, 1961a,b). Many workers studied the chemical control of feed ing in diverse coelenterates and found a varied number of amino acids to activate their feeding reactions. Lenhoff & Schneiderman (1959) found that glutathione provoked a feeding response in Campanularia flexulosa and in the si- phonophore Physalla physails. Another siphonophore, Nanomia cara, was also found to respond to glutathione (Mackie & Boag, 1 9 6 3). Wyman (1 9 6 5) reported Corymorpha palma to give positive responses to 10~^M concentrations of gluta thione, glycine, cysteine and Berine. Preliminary results (Siva, personal communication) indicate that the common sea anemone, Anthopleura elegantlssima also responds to gluta thione. A gymnoblastic hydroid, the marine Pennarla tlarel- — f i la, responded to proline in concentrations as low as 10“ M (Pardy & Lenhoff, 19 6 8). MariBcal & Lenhoff (1 9 6 8) reported that the coral, Cyphastrea ocellina responds to both proline and gluta thione. The scleractinian coral responded to proline at concentrations 10~ to 10’ ”' 5M, and to glutathione at concen- _2i trations 10 M. This represents the first well-documented case of a coelenterate responding to two different types of molecules. Recently the branched amino acid valine was shown to elicit ingestion response in the sea anemone Boloceroldes (Lindstedt, Muscatine & Lenhoff, 1 9 6 8). This anemone seems to need some sort of mechanical stimulation along with the chemical one, because the animal will not give a typical feeding response to a valine solution unless a solid parti cle is present on the oral disc. The fact that glutathione and proline are the most common activators found so far in coelenterates and that, at least in some scleractinian corals, both activate feed ing behavior is highly suggestive and deserves special at tention . Materials and Methods Two hours preceding an experiment, about 200 Paly thoa polyps were removed from the large aquarium and placed in 2 5 0-ml finger bowls containing fresh Instant Ocean. To find if Palythoa would respond to more than one amino acid or perhaps to the tripeptide glutathione, 10“^M solutions of all the naturally-occurring amino acids and 57 p the tripeptide were prepared and imbibed in 1mm pieces of filter paper. When one of these pieces of treated filter paper elicited a response, a series of dilutions of the Bame compound was prepared, placed in 50-ml Stender dishes and tested for its effect on the polyps by transferring 3 or 4 polyps at a time from the finger bowl to the 50-ml _2 dish. A stock solution 10 M concentration was prepared and diluted to the needed concentrations by adding the proper volumes of Instant Ocean. A control of 20 polyps transferred to fresh Instant Ocean was kept throughout each experiment. A negative response was one where no tentacular movements or mouth opening occurred for at least 15 minutes after the experiment had been initiated. A period of 15 minutes was considered adequate because all the positive response occurred within a maximum of 10 minutes after the initiation of an experiment. The time required for the polyps to open their mouths and the time when these were closed was recorded for each polyp. The ingestion response was studied by placing pol yps in dishes containing the test solution and offering 2 them clean 1mm pieces of filter paper. The time required for ingestion of this inert particle was recorded The duration of the response seemed to be corre lated with the size of the polyps. The size of those was estimated by measuring the diameter of the oral disc in 40 expanded polyps, and calculating the surface area for each 2 polyp using the formula nr . The polyps were placed in numbered dishes containing proline at 10-^M and the time for them to respond was recorded. The correlation coeffi cient between surface area of oral disc and time to respond waB calculated in a Friden calculator Model RQ 10 (Friden Statistical Methods, 1 9 6 3). The results were plotted on logarithmic paper. To study further the effect of oral disc size on the speed of the response to proline, 2 cate gories of polyps based on the plot were tested for the speed, duration and time of maximum mouth opening. To study the location of feeding receptors, two types of ex periments were performed. 1. Polyps were placed in glass dishes with a plas tic lid adjusted in such a way (Fig. 14) that two compart ments (upper and lower) were separated in the dish. The lid had two performations: one through which the mouth of a pipette was inserted and another large enough to have the contracted polyp fitting through. The animal was al lowed to relax (10 to 60 minutes) and when the oral disc was expanded, a seal between the lower and upper compart ments was accomplished. A measured amount (sufficient to produce a 10“^M solution) of reagent proline was added by pipette into the lower compartment of the dish so that the 59 proline solution could reach only the column of the animal. Behavior of the polyps was observed and recorded for 10 minutes. At the end of this period the pipette was removed and the proline solution was allowed to flow into the upper part of the dish. The response of the polyps was timed and recorded again. 2. Agar was mixed with a known concentration of the feeding activator and a small amount placed at the end of a fine entomological .needle. With this needle very re stricted sites around the mouth, peristome, tentacles and column were stimulated and the responses were timed and recorded. In all of these experiments salinity was 33 °/oo, temperature 24 to 27°C, illumination by fluorescent light and pH adjusted to that of sea water (8*15) by using 1 N solutions of HC1. To measure pH, a pH meter Beckman E 300 was used. The paper used in the experiments was Whatman #4 2 cut in 1mm pieces. Special care was taken in selecting papers of the same size and shape for all the experiments. Paper was handled with Inox #5 forceps which were washed with distilled water and dried before and after each ex periment. The small pieces of paper were dipped in fresh Instant Ocean or in the test solutions and used immediately, without drying. All reactions were timed with a Cletimer 3 0-minute Figure 14. Experimental set up for studying location of proline-receptors in Palythoa tovmsleyi. p, pipette wl, water level od, oral disc c, column gd, glass dish pi, plastic lid 60 Figure 14 62 stop watch. Standard deviations were calculated for all measurements, using a Friden calculator Model RQ 10. Results a. Response to naturally occurring amino acids and the tripeptide glutathione The results presented in Table 8 show that lO'^M concentrations of several amino acids and the tripeptide glutathione elicit feeding behavior in Palythoa. The re sponses to the paper consist of the formation of a lip, the closing of the oral disc over the paper and the ingestion of it. The two most effective substances to elicit the reaction are proline and glutathione, both of which stimu late lip formation and enclosure of the paper by all the polyps tested. Proline induces ingestion of paper in 100 percent of the polyps and glutathione in 60 percent of them. Alanine, serine, lysine and hydroxyproline stimu late one, two or all the steps of the reaction but in a significantly lesser proportion of the polyps. Controls of untreated paper and the rest of the naturally occurring amino acids gave no response during 15 minutes of observation. This period was considered adequate because all reactions to active substances occurred within 10 minutes or less. b. Response to proline The characteristics of the response to proline are 63 TABLE 8 RESPONSE OP PALYTHOA TOWNSLEYI TO AMINO ACIDS AND THE TRIPEPTIDE GLUTATHIONE ON FILTER PAPER Amino Acid* 10"^M Number of Percentage of Polyps Giving Imbibed by Polyps Positive Responses Filter Paper Tested + ++ +++ None 10 - - - Glycine 10 - - - Alanine 20 40 40 40 Valine 10 - - - Leucine 10 - - - Isoleucine 10 - - - Serine 20 25 - 25 Threonine 10 - - - Cysteine 10 - - - Cystine 10 - - - Methionine 10 - - - Glutamic acid 10 - - - Aspartic acid 10 - - - Lysine 10 40 6o 40 Hydroxylysine 10 - - - Arginine 10 - - - Histidine 10 - - - Phenylalanine 10 - - - Tyrosine 10 - - - Tryptophan 10 - - - Proline 20 100 100 100 Hydroxyproline 20 - 10 10 Tripeptide Glutathione 20 100 100 60 *Amino acid arranged by their chemical similarities. +Lip formation; ++oral disc closeB over paper; +++ingestion of paper. 64 shown in Table 9j Pig. 15* In the high concentrations, -2 -3 10 and 10 “W, almost all the polyps show a wide, round -4 mouth opening. In concentrations of 10 M only 32 percent of the animals give a small, slit-like mouth-opening re sponse. Lower concentrations cause no mouth opening. The time required for the reaction to start is shortest in the _2 10 M concentration, but the response lasts longest in 10~3m proline solution. The large size of the standard deviations calculated for the speed and duration of the response suggest a great of variability among the polyps. This could be a result of genetic differences, physiological stage, age, size of the animals The one factor that could be tested without dif ficulty was size. c. Effect of polyps' size in the duration of mouth-opening responses The correlation coefficient between surface area of the oral disc and time to respond to proline 1 0~^M was found to be O.6 3. When the points were plotted on logar ithmic paper (Pig. 16) it became clear that there were groups of polyps behaving differently. One group tended to fall in a line, the other was completely separated and so dispersed that no tendency to fall in a line was obvious. This last group was represented by individuals of less than 10mm oral disc diameter. In order to determine If size had TABLE 9 CHARACTERISTICS OF THE RESPONSE OF PALYTHOA TOWNSLEYI TO DIFFERENT CONCENTRATIONS OF PROLINE Proline Concentrat ion Number of Polyps Tested Percentage of Polyps Giving + Responses Type of Response Time for Mouth to Open (min.) Time for Mouth to Remain Open (min.) -2 10 M 49 94 Wide-round mouth opening 0.99 + 0.90 3.56 + 1.99 10-5M 90 99 Wide-round mouth, opening 1.21 + 1.10 5.01 + 4.26 -4 10 M 46 32 Small, slit-like mouth opening 2.46 + I.23 2.53 + 0.35 10“5M 13 8 Lip formation - - io-6m 10 0 No response - - Control sea vater 20 0 No response - - O '! Figure 1 5. Activation and characteristics of mouth opening reaction in Palythoa townsley1 exposed to different solutions. Control is Instant Ocean. 66 Figure 15 Control Percent of polyps opening mouth i\ j o - F - O (7 \ O 00 o o o Pro 10~2M Pro 10“3M Pro Pro 10 -'M GSH 10~2M GSH 10“^M o CT\ 68 an effect in the variability of the mouth opening duration, two groups of polyps were studied. The first one with Palythoa in which oral disc measured over 9mm in diameter; the other with polyps in which the oral disc measured less than 9mm in diameter, because these last ones formed a group apart from the large ones in Pig. 16. Table 10 shows that when the groups are separated the standard deviations for speed and duration of the re sponse become smaller within each group, and also that considerably lower proportion of small polyps than large oneB respond to proline at concentrations 10 J and 10 M. Certain proportions of the large polyps (80 percent in 10-2M; 30 percent in 10~^M and 12 percent in 10"^M) give a maximum mouth opening in which the mouth opens very widely and roundly. This consists of a very wide, round mouth opening that is reached and immediately begins to close down. The time required to reach this maximum open ing is shortest in concentration 10“^M. Small polyps show maximum mouth opening in a much smaller proportion of the polyps. d. Location of chemoreceptors The receptors for proline were found to be located on the oral disc, for only when this was stimulated did the animal show a response. When the column alone was put in contact with proline, the animal showed no reaction. The Figure l6. Correlation between surface area of oral disc in Palythoa townBleyl and time to respond to proline 10~3 solution. 69 Surface area of oral disc 70 400 T 300 f \ i 200 100. 90, 80 70- 60. 50. 40 30. 20j 10 ° o f 5 2 5 3 5 4 ( 5 55 5 5 70 5 5 96 l o ' o Time for mouth to open ( sec. ) Figure 16 TABLE 10 CHARACTERISTICS OF THE RESPONSE TO PROLINE OF TWO CATEGORIES OF PALYTHOA TOWNSLEYI POLYPS* Proline Concen tration Number of Polpys Tested Time for Mouth to Open (min.) In Percent age Polyps Time for Maximum Mouth Open ing (min.) In Percent age Polyps Time for Mouth to Remain Open (min.) -2 10 M 20 large 0.52 + 0.26 100 2.15 80 4.83 + 2.50 13 small 1.26 + O.85 100 0.91 38 2.20 + 0.12 io“5m 20 large 1.21 + O.93 100 3.05 30 5.08 + 2.04 23 small 1.30 + 1.20 50 1.16 12 2.70 + 1.66 -4 10 M 21 large 1.13 + O.93 29 0.75 8 2.70 + 1.30 10 small 4.00 10 - - 1.00 10_5M 10 large No mouth opening - - - - 10 small No mouth opening - - - - *Category 1 = (large) oral disc larger than Ston in diameter. Category 2 = (small) oral disc smaller than 9vm in diameter. 72 most sensitive area to proline was found to be the one sur rounding the mouth and the oral margin itself. The stimu lation of the oral margin in large polyps with proline _2 10 M mixed with agar provoked a response within 4-10 sec onds. Stimulation of the peristome provoked a reaction within 30 to 90 seconds and stimulation of the tentacles provoked lip formation to grasp the piece of agar and mouth opening within 9 0-100 seconds. e. Response to glutathione Table 11 shows that the only glutathione concentra- _2 tion of which Palythoa open their mouths is 10 M. The mouth opening is very similar to that elicited by proline -4 10 M. At the other concentrations glutathione provokes the exposure of the actinopharynx. The largest proportion of polyps giving this reaction iB 10_^M. The reaction iB _2 considerably faster in 10 M than in any of the other con- -4 centrations. The lowest effective concentration is 10 M, -5 -6 for 10 ^ and 10 cause a response in only 15 and 3 percent of the polyps respectively. f. Activation of the ingestion response — 2 — " 2 Proline at 10 M and 10 -'M solutions stimulate in gestion of untreated filter paper in 85 and 80 percent, respectively, of the polyps tested (Table 12). The inges tion takes over 10 minutes. TABLE 11 CHARACTERISTICS OF THE RESPONSE OF PAIYTHOA TOWNSLEYI TO DIFFERENT CONCENTRATIONS OF GLUTATHIONE Glutathione Concen tration Number of Polyps Tested Percentage of Polyps Giving + Response Type of Response Time for Response to Occur (min.) Duration of Response (min.) _2 10 M 52 68 Actinopharynx exposed + slit-like mouth opening 0.18 + 0.16 2.38 + 1.10 10-5M 58 91 Act inopharynx exposed 2.00 + 1.23 2.86 + O.76 10~ \ 49 52 Act inopharynx exposed 1.30 + O.58 2 M + 0.26 10"5M 59 15 Act inopharynx exposed 3.00 + 2.50 2.00 + 0.82 10~^M 20 5 Actinopharynx exposed 3.00 + 3.20 1.80 + 0.79 Control (sea water) 10 0 None - - (jo 74 TABLE 12 ACTIVATION OF THE INGESTION RESPONSE IN PALYTHOA TOWNSLEYI* BY PROLINE AND GLUTATHIONE IN SOLUTION Solution and Concentration Percentage of Polyps Ingesting Untreated Paper Time for Paper to be Ingested Proline 10"2M 85 10.64 + 2.64 Proline 10**^M 80 11.10 + 3-74 -4 Proline 10 M 0 - Glutathione 10~2M 25 5.00 + 2.44 Glutathione 10~3M 85 5.00 + 1.73 Glutathione 10-^M 35 4.57 ± 1.4l Glutathione 10~^M 15 5 .3 0 + 0 .0 0 Glutathione 10~6M 0 - Control** 0 - *20 polyps tested In each experiment. **Control 1b Instant Ocean. 75 Glutathione induces ingestion in 85 percent of the polyps when at concentration 10_^M. Higher or lower con centrations of the tripeptide elicit ingestion in much -4 fewer polyps. Glutathione at 10 M concentration is more effective than proline at the same concentration to elicit the reaction. Discussion The fact that proline and glutathione imbibed in filter paper cause respectively 100 percent and 68 percent of the polyps to ingest the paper suggests that these substances can activate the feeding reaction in Palythoa and in fact they do so when presented in solutions of con- -4 centrations above 10 M. If a piece of untreated paper is offered the animals form a lip to trap the paper and push it toward the mouth (Pig. 17t>), this opens (proline) or the actinopharynx becomes exposed (glutathione) (Pig. 17c), and the paper is ingested (Pig. 17d). In Hydra the ingestion response is independent of the presence of glutathione (Lenhoff, 1968a). As Kanev (1952) reported, Hydra will swallow an inert object such as pins if these are placed in their mouths. This is also the case with some sea anemones (Hyman, 1940). In Palythoat however, the Ingestion response is controlled chemically, for there is no ingestion when an inert object is placed on the mouth. Therefore the ingestion-response may be as im- Figure 1 7. Response of Palythoa townsleyl to untreated filter paper while in a glutathione 10-2M solution. a. Polyps begin to separate border of the mouth. smo, small mouth opening b. Lip formation 11, localized lip ae, actinopharynx exposed 76 77 ,smo Figure 17 f c Figure 17 Response of Palythoa townsley1 to untreated filter paper while in a glutathione 10”2M solution. c. Lip becomes generalized gl, generalized lip ae, actinopharynx exposure d. Ingestion of paper pi, ingested paper 78 Figure 17 c Figure Vt d 80 portant as the mouth-opening response when selecting some method to quantify the response activated chemically. Len- hoff (1961a,b) quantified the feeding response of Hydra llttoralis to glutathione by studying the duration of the response. Since then many workers (Fulton, 1 9 6 3; Mariseal & Lenhoff, 1968; Pardy & Lenhoff, 1 9 6 8) have used the degree of mouth opening as a measure of the activity of a certain molecule in a coelenterate. Lindstedt, Muscatine & Lenhoff (1 9 6 8) used the percentage of polyps ingesting treated fil ter paper as an indication of the activity of several amino acids in the feeding response of the swimming sea anemone Boloceroldes. In Palythoa both mouth opening and ingestion of treated filter paper were studied in order to characterize the reaction elicited by proline or glutathione. The duration of mouth-opening proved to be quite variable in Palythoa. One source for such variability was found in the size of the polyps. If more or less uniform reactions are to be obtained with Palythoa only polyps with an oral disc diameter larger than 9mm should be used. This of course does not give the uniformity of genetically iden tical animals, such as has been the case in experiments with Hydra (Lenhoff, 196la), but proved adequate because measurements of the times required to open the mouth and the duration of mouth opening had small standard deviations. 81 The receptors for proline are present in the oral disc. No columnar stimulation provoked a feeding reaction. The most sensitive area is the border of the mouth. This agrees with Batham and Pantin's (1950) report that the mouth has the highest chemical sensitivity of the whole body in Anemonia sulcata. Lenhoff (1961b) could not de termine the exact location of the chemoreceptors in Hydra, but found that isolated headB would give a maximum response to glutathione. The concentration of prollne and gluta thione found to activate feeding behavior in Palythoa are two orders of magnitude higher than those found to elicit feeding behavior in most other coelenterates (Lenhoff, 1968b), including the scleractinian Cyphastrea ocelllna which responds to both proline and glutathione (Mariseal & Lenhoff, 1968). Cyphastrea ocelllna shows a slight mouth opening in 73 percent of the polyps in proline solution of _7 concentrations as low as 10 'M. The same coral gives the same slight mouth opening response in presence of gluta thione, and 88 percent of the polyps respond in the tri- -5 peptide at concentrations of 10 ^M. In Palythoa concentra- _ji tions of 10 proline or glutathione elicit response in only 32 percent of the polyps. Mariscal and Lenhoff (1 9 6 8) reported that reduced glutathione has been shown to be abundant in Artemis nau- plii, Mariscal (personal communication) communicated that proline is also abundant in Artemla. However, the concen- trations of either substance that can be found over the oral disc of Palythoa when a prey has been captured and injured are unknown and it is very unlikely that they will ever reach concentrations similar to the ones found neces sary to stimulate feeding behavior in this study. Conse quently, the fact that proline and glutathione elicit a feeding reaction only at high concentrations places much doubt in whether they may activate feeding behavior in nat ural situations. To determine if glutathione and proline may have a synergistic effect and therefore be effective at naturally occurring concentrations, combinations of both activators were prepared and tested. The results of these experiments are the subject of the three following chapters. Conclusions 1. The feeding reaction of Palythoa townsleyl can be elicited by proline or glutathione at concentrations -4 above 10 M. 2. The mouth-opening and the ingestion response are chemically controlled. 3. The ingestion response is independent of the mouth-opening response and can be stimulated without pre vious mouth opening. 4. The duration of the mouth-opening response de pends in certain way on the size of the polyps. 5. The receptors for proline are located on the oral disc. 6. The month is the most sensitive area to stimu late mouth-opening response. 7. Although proline and glutathione elicited feed ing behavior, they cannot be considered as feeding activa tors for Palythoa because they only act at concentrations which are too high to be expected in the natural foods of the polyps. CHAPTER III CONTROL OP LIP FORMATION During feeding most coelenterates first capture and pierce the prey with their nematocysts, next a sub stance, present in the fluids oozing from the prey’s wounds, causes the tentacles to contract toward the mouth and the mouth to open. Lastly, on contact with the mouth, the food is ingested. In most of the coelenterates In which the chemical control of feeding has been studied, the initial response of the polyps is tentacle writhing and twisting toward the mouth (Ewer, 1957; Loomis, 1955; Lind- stedt, Muscatine & Lenhoff, 1 9 6 8). In several sea anemones, however, the initial response to pieces of food is an or derly contraction of a limited group of tentacles and of the edge of the disc carrying those tentacles in such a way that a clasping lip is formed around the food. The lip pushes the food toward the mouth, this opens and the food is ingested. This type of behavior was observed by Torrey (l904a,b) In Sagartia and in the hydroid Corymorpha and by Pantin & Pantin (1943) in Anemonla sulcata. The contraction of tentacle toward the mouth is under chemical control in Hydra llttoralls (Lenhoff, 1968a). 84 85 The sea anemone Sagartia and the hydroid Corymorpha, how ever, form a lip when they are stimulated in almost any possible way (Torrey, 1904a,b). Pantin & Pantin (19^-3) found that lip formation could be elicited by chemical, mechanical or electrical stimuli in Anemonia sulcata. Palythoa gives a very distinctive lip-formation re sponse to live andfbeshly killed prey, and represented a good experimental subject for the study of the control of lip formation. Material and Methods Materials and methods are the same as in Chapter II, pp. 5 6-6 2. Results Lip formation was observed very rarely to occur spontaneously and when it did occur in polyps that had not been stimulated experimentally it well might have been due to some stimulus present in the medium, but not detected by the observer. Polyps used as controls in clean Instant Ocean never showed lip or funnel formation. Lip formation is not to be confused with a reaction of closing the oral disc in response to mechanical action such as striking a group of tentacles or dropping a piece of filter paper on a group of tentacles or on the margin of the oral disc. Although this reaction may look some what similar to a generalized lip formation. It is not one 86 intended to trap food but rather a defensive action whereby the polyps close their oral disc completely. 2 Lip formation is elicited by 1mm pieces of filter paper only when paper is imbibed in lO'^M solutions of alanine, serine, lysine, proline, hydroxyproline or gluta thione (Table 13) but not with untreated paper. Table 12 showed that lip formation may be the first or second step in the feeding reaction depending on the chemical that is stimulating it. Alanine causes the formation of a local ized lip (Pig. 18a) in 40 percent of the polyps. Serine causes a very localized lip, involving just the few tentacles that have been excited by the paper (Pig. 18b). The reaction is present in 25 percent of the polyps. The lip is formed after 0.79 + 0.28 minutes of stimulation. Lysine elicits the formation of a loose lip (Pig. 18c) in 40 percent of the polyps within 1.87 + 0.09 minutes of stimulation and is preceded by the formation of a fun- nel-like structure. Proline causes the formation of a restricted or, more often, generalized lip (Pig. 18a) in all the polyps stimulated, within O .5 3 +0.43 minutes. Hydroxyproline does not elicit lip formation, but occasionally a funnel like structure forms and in 60 percent of the polyps the initial response to hydroxy-proline-paper is the exposure 87 TABLE 15 INITIAL RESPONSES OF PALYTHOA TOWNSLEYI* TO TREATED FILTER PAPER Paper Imbibed in 10-1M Solution ofs Type and Time for Initial Response (min.) In Percent age Polyps Type and Time for Second Response (min.) In Percent age Polyps Alanine Loose lip 1.87 + 0.00 1*0 Small, slit-like mouth opening 2.75 + 0.10 kO Serine Restricted lip O.79 + 0.28 25 Lysine Funnel formation O.73 + 0.43 100 Lip formation I.87 + 0.09 ho Glutathione Small,, slit-like mouth opening O.83 + 0.30 100 Lip formation 1.26 + 0.68 100 Proline Lip formation O.53 + 0.43 100 Mouth opening 1.83 + O.58 100 Hydroxyproline Act inopharynx exposure 2.50 60 Comer of mouth- opening 2.70 20 Pipecolic acid Small mouth- opening 0.18 + 0.04 100 Funnel formation 0.1*8 + 0.21 100 1-Thiazolidine- 4-carbox- ylic acid Weak lip formation 0.72 + 0.17 100 Lip becomes strong 1.25 + 0.26 100 Azetidine-2- Lip formation carboxylic acid O.kO + 0.30 100 Very small mouth opening 1.00 + 0.70 100 Prolylglycine -- -- Glycylproline Act inopharynx exposure 1.00 + 0.05 100 Funnel foims 1.58 + 0.58 100 None (control) -- -- *20 Polyps tested In each experiment. Figure 18. Lip formation in Palythoa townsleyi a, generalized lip b, localized lip c, loose lip 88 Figure 18 of the actinopharynx within 2 .5 0 minutes after stimulation. Other analogs of proline Buch as azetidine-2-carboxylic acid cause a generalized or localized lip to form in all the polyps tested within 0.40 + O.3O minutes. The six- membered ring analog of proline, pipecolic acid, does not cause the formation of a lip, but only the formation of a funnel-like structure. L-thiazolidine-4-carboxylic acid, another analog of proline, elicits lip formation in 100 percent of the polyps. This lip is weak and loose at the beginning ( 0 .2 2 + 0 .1 7 minutes) but becomes strong and tight after 1.25 +0.26 minutes of stimulation. Proline peptides such as glycylproline and prolylglycine do not provoke lip formation. In the presence of glycylproline, however, the initial step of the feeding reaction is the exposure of the actinopharynx in 100 percent of the polyps tested. The tripeptide glutathione induces lip formation in all the polyps tested after 1 .2 6 + 0 .6 8 minutes of stim ulation. This, however, comes after the mouth has opened and thus becomes the second step of the feeding reaction. The glutathione analog S-methyl glutathione does not elicit lip formation. In all the experiments described previously the chemical was used at concentrations lO^M and absorbed in 2 1mm pieces of filter paper. In order to separate the 91 chemical stimulus from the mechanical one represented by the piece of paper striking the oral disc, lip formation waB studied in polyps placed in solutions of alanine, pro line, serine, lysine, L-thiazolidine~4-carboxylic acid and glutathione (Table 14). It was found that only proline could induce the formation of a lip when no mechanical stimulus was pro vided, and then the number of polyps responding was rather —2 small. The higher concentrations of proline (10" M) induce lip formation in 50 percent of the polyps within a time which is almost twice the time required for lip formation when paper is offered. The lower concentration of proline (10-^M) induces lip formation in only 10 percent of the polyps. Discussion The function of the lip-formation response is to secure the food and push it toward the mouth (Pantin & Pantin, 1943)• For this reason it is logical to suspect that lip formation must require a mechanical stimulus, to be activated. This mechanical stimulus under natural con ditions will be represented by prey or particulate food hitting the tentacles or the oral disc. In the laboratory it can be represented by a piece of filter paper. Un treated filter paper does not elicit lip formation. This suggests that a chemical stimulus must also be supplied in 92 TABLE 14 INITIAL RESPONSE OP PALYTHOA TOWNSLEYI* TO SOLUTIONS OF SEVERAL AMINO ACIDS AfoD Tfcfe TRtPEPTIDfe GLUTATHIONE (GSH) Substance and Concentration Type and Time for Initial Response (min.) In Percent age Polyps Alanine 10“3-10-1M Serine 10"3-10“1M Lysine lO^-lO^M Proline 10“2M Proline 10~3M -4 Proline 10 M Proline 10~^M GSH 10"2M GSH 10“3M GSH 10"^M GSH 10"5M None None None Generalized lip formation 50 0.99 + 0.90 Generalized lip formation 30 0.28 Generalized lip formation 35 1.50 Generalized lip formation 10 0.30 Actinopharynx exposure 68 0.18 + 0.16 Actinopharynx exposure 32 2.00 + 1.23 Actinopharynx exposure 15 1.30 + 0.58 0 *20 polyps tested in each experiment. order to provoke lip formation. It was found that several amino acids, glutathione and several proline analogs can elicit the lip-formation response in Palythoa when the substances are imbibed in 2 1mm pieces of filter paper. If the chemical stimulus is _2i supplied by itself, only proline at concentrations 10 M and above can elicit formation of lip, and this in a limited proportion of polypB. The chemical and mechanical stimuli combined provoke a faster response in a higher pro portion of the polyps. However, the fact that a lip can be formed in response to a chemical stimulus alone suggests that this step may be very closely related, as in a chain reaction, to the following step: mouth opening. This sug gestion finds support also in the fact that in the presence of glutathione-paper the mouth opens first and then the lip forms. This seems a completely illogical sequence, for the animal would have to secure a prey (by forming a lip) be fore it is stimulated to open the mouth to swallow that prey. The fact that proline-paper elicits lip formation first, mouth opening second and glutathione provokes the same reactions but in Inverse order suggest that if both molecules activate the response, there must be some syner gistic effect whereby both influence the response of Paly thoa to food. Most proline analogs elicited lip formation In a high proportion of the animals. This suggests that the chemoreceptors for this reaction have affinity for the molecular structure of proline. The substitution on the imino group by the radical glycyl abolished the activity, but substitution for a S group did not. Substitutions in the carboxyl group did not destroy the activity of the molecule. This is contrary to what Pulton (1 9 6 3) found for the activation of feeding in Cordylophora. In this hydroid both the imino group and the carboxyl group must be intact to activate the feeding re sponse . Conclusions 1. Lip formation represents the first step in the feeding reaction of Palythoa. 2. The lip is initially very restricted ("local ized") involving the group of tentacles stimulated mechani cally by the prey. As the prey is pushed toward the mouth more and more tentacles participate in the lip formation and this becomes "generalized." 3. The lip grasps the food and pushes it toward the mouth. 4. Lip formation is elicited by chemical and me chanical stimuli combined. 5. Without a mechanical stimulus present, only proline can elicit lip formation, and this in very limited proportion of the polyps. 95 6. The receptors for lip formation are probably located on the tentacles and margin of the oral disc. 7. The chemoreceptors for lip formation recognize the proline molecule even with certain modifications of the size of the ring, substitutions on the carboxyl group and to certain extent substitutions on the imino group. 8. Glutathione can activate lip formation only after the mouth opening reaction has occurred. CHAPTER IV CONTROL OF MOUTH OPENINGS This phenomenon was carefully studied in Hydra llttoralls hy Lenhoff (I96lb). He found that the time re quired by Hydra to start opening their mouths depended (l) on the concentration of the feeding activator glutathione, (2) on the presence of a competitive inhibitor and (3) on the composition of the external medium in which Hydra were placed. His results showed that mouth opening is not an all-or-none response, and that graded responses could occur when conditions were not optimal. He observed that, in general, the duration of the response was shorter when the Hydra took longer to start responding. He also cautioned against confusing the normal feeding reflex with any ab normal mouth openings which occur in response to nonspecif ic compounds such as acids. The Hydra stimulated to open their mouth by such deleterious compounds cannot be made to ingest dead food and usually die within a few hours. Numerous workers after Lenhoff utilized different aspects of the mouth-opening reaction to express the degree of activation caused by certain molecules to the feeding behavior of diverse coelenterates. 96 97 Pulton (1 9 6 3) used the mouth-opening reaction of Cordylophora lacustrlB to Indicate completeness of the feeding response; Pardy & Lenhoff (1 9 6 8) did the same for the hydrold Pennarla tlarella which, as the hydrold Cordy lophora, also responds to proline. Mariscal & Lenhoff (19 6 8) used the degree of mouth opening and the proportion of polyps opening their mouths as an indication of the degree of activity of the feeding activators proline and glutathione and their analogs in the feeding reaction of Cyphastrea ocelllna and two other scleractinian corals. Palythoa townsley1 offered a good experimental sub ject in which the speed in responding, the duration of the mouth opening reaction, its degree and the proportion of polyps showing it could be studied without difficulty. Materials and Methods Materials and methods for these experiments are those of Chapter II and the ones described below. a. Effects of proline-glutathione interaction on the feeding reaction of Palythoa Two hours preceding an experiment a suitable number of Palythoa polyps was removed from the large aquarium and placed in fresh Instant Ocean. In order to prepare mixed solutions of proline and glutathione, a stock solution of -2 proline 10 M was prepared and maintained at room tempera- — 9 ture (22°C). A stock solution of glutathione 10"" M was prepared and its pH adjusted to that of sea water: 8.15 ty addition of 1 N HC1. This solution was kept in the refrig erator and used only to obtain dilutions to 10-^M concen- _Zi trations. Another stock solution was prepared for 10 M glutathione concentration and this was diluted to obtain -5 -6 10 and 10 M glutathione concentrations. Equal volumes (25 ml) of proline and glutathione were mixed Just prior to _2 experimentation. When 25 ml of proline 10 M were mixed _2 with 25 ml of glutathione 10 M the combination contained 5 x 10“^M proline + 5 x 10~^M glutathione. When 25 ml of _Q _2 proline 10 “T V ! were mixed with 25 ml of glutathione 10 M, -4 the combination contained 5 x 10 M proline and 5 x 10 JM glutathione, etc. The synergistic effects of the two ac tivators were analyzed by comparison with behavior of Paly thoa in either activator by itself at the proper concentra tion. The time required for the mouth to open and the pro portion of polyps opening their mouth were recorded. b. Specificity of mouth-opening chemoreceptors The time required for the mouth to open, the degree of mouth opening and the proportion of polyps giving the mouth-opening reaction were used as indicators of the ef fectiveness of diverse proline analogs in eliciting the feeding reaction. The selection of analogs was done in such a way that molecular shape and active groups of the molecule could be evaluated as to their relative abilities 99 in the recognition by the receptor sites. Twenty polyps of Palythoa were used for each ex periment. Temperature was 22°C, pH 8.15. Each polyp was used only once in a 24 hr. period. The experiments con- 2 sisted of offering Palythoa 1mm pieces of filter paper imbibed in lO'^M concentration of the analog or of proline to polyps placed in a dish containing 50 ml of proline at different concentrations or 50 ml of the analogs at differ ent concentrations. The time for mouth to open and the proportion of polyps responding in each case were recorded. c. Effect of pH on the mouth-opening reaction Because acid pH was found to stimulate mouth open ing, a series of solutions with pH's ranging from 1 to 14 were prepared by adding 1 N solutions of HC1 or NaOH to Instant Ocean. When the pH was 1.0 or 1.5 permanent damage was caused to the animals and they died. In pH's above 3.0 an apparent reaction could be detected. In pH 2.0, 2.5 and 3.0 the polyps showed different degrees of mouth opening and the time required for the reaction, as well as the proportion of polyps responding were recorded. The pH was measured with a Beckman pH meter, Model E 300. Results After Palythoa has secured food by the formation of an enclosing lip, the mouth opens. This reaction can be Induced by several chemicals imbibed in filter paper and presented at lCT^M (Table 15). The degree of mouth opening depends on the chemical activator used. Proline is the only substance to cause a wide, round mouth opening in all the polyps tested. The proline analogs azetidine-2-carboxylic acid and pipecolic acid also elicit response in 100 percent of the polyps, but the mouth opening is very small. Hy- droxyproline induces a very weak response in only 20 per cent of the polyps and the proline peptide prolylglycine causes a Bmall corner-of-the-mouth-opening in all polyps tested. In the presence of glutathione there is a small, slit-like mouth opening in 100 percent of the polyps, simi lar to one occurring in presence of alanine, where 40 per cent of the polyps respond. Lysine elicits corner-of-the- mouth-opening in only 15 percent of the polyps. The fast est reactions are obtained with glutathione and the proline analog pipecolic acid. If the chemicals are provided in solution, eliminating therefore the mechanical stimulus represented by the filter paper, only proline, hydroxypro- line, pipecolic acid, and glutathione can elicit the mouth opening response (Table 16). Proline is by far the most active molecule in inducing the reaction. Table 16 shows that the proportion of polyps responding to the activator —2 _ Q depends on its concentrations. At.10 and 10 JM concen- -h trations a large number of polyps respond, but at 10 M concentration only 32 percent of the polyps open their mouths. 101 TABLE 15 ACTIVATION AND CHARACTERISTICS OP MOUTH-OPENING REACTION IN PALYTHOA TOWNSLEYI* Activator of Concentration 10“% Imbibed in Filter Paper Type of Mouth Opening Percentage of Polyps Opening Mouth Time for Mouth to Open Alanine Small, slit-like 40 2.75 +0.10 Lysine Corner only 15 2.66 Glutathione Small, slit-like 100 0 .8 3 + 0 .3 0 Proline Wide, round 100 I.83 +0.58 Azetidine-2- carboxylic acid Very Small 100 1.00 + 0 .7 0 Hydroxyproline Corner only 20 2.50 +0.50 Pipecolic acid Very small 100 0 .1 8 +0.04 Prolylglycine Corner only 100 1.00 + 0 .0 5 *20 polyps tested in each experiment. TABLE 16 ACTIVATION AND CHARACTERISTICS OF MOUTH-OPENING REACTION IN PALYTHOA TOWNSLEYI EXPOSED TO DIFFERENT SOLUTIONS Number of Percentage of Activator and Polyps Polyps Time to Open Duration of the Concentration Tested Opening Mouth Mouth (min.) Response (min.) Proline 5 x 10~2M 1 * 9 94 0.99 + 0.90* 4.83 + 2.50 Pro line 5 x 10_3M 90 99 1.21 + 1.10 5-08 + 2.04 Proline 5 x 10_4M 46 32 2.1 * 6 + I.23 2.70 + 1.30 Proline 5 x lO^M 13 — Hydroxyproline 5 x lO^M 20 80 I.50 + O.63 2.36 + 1.06 Hydroxyproline 5 x 10 3M 20 15 O.85 1.6l + 1.6l Hydroxyproline 5 x 10~4M 20 — - - Pipecolic acid 5 x 10-2M 20 50 2.20 + O.73 4.55 + I.30 Pipecolic acid 5 x 10-3M 20 48 2.88 + O.73 4.21 + 1.21 Pipecolic acid 5 x 10-4M 20 — - - Glutathione 5 x lO^M 52 68 0.6l + 0.18 1.10 + O.38 Glutathione 5 x 10 ^ 58 — - Control** 20 — *Time is given as mean value + standard deviation. **Control was Instant Ocean. 102 103 A significant number of the polyps give a maximum response, that Is a gaping, round mouth opening, In the higher proline concentrations. Glutathione can Induce only a small, slit-like mouth opening In concentration —2 10 M (Table ll) and here only 68 percent of the polyps respond. The speed of reaction to glutathione has a much narrower range than the speed of reaction to proline (com pare 0.1 6 = 0 .1 8 minutes in glutathione to 0.99+0.90 minutes In proline) and the duration of the response Is reduced by over 75 percent. Lower concentrations of gluta thione elicit the exposure of the actinopharynx, a reac tion which can be just as Important as mouth opening In permitting Ingestion of food, as will be seen later. The optimal concentration of glutathione to Induce exposure of the actinopharynx Is 10-^M (Table ll), and below this con centration the number of polyps giving this response Is drastically reduced. _2 Hydroxyproline is effective at concentration 10 M (Table 16), Inducing a very weak reaction (only corner-of- the-mouth opens) in 80 percent of the polyps. The dura tion of the reaction is comparable with the duration of the -4 response to proline 10 M. At this concentration of pro line the mouth opening is also very small and compares well with the reaction to hydroxyproline. This indicates that hydroxyproline is 1/100 as effective as proline in provok- Ing a mouth opening reaction in Palythoa. The six-membered ring proline analog, pipecolic -2 -"3 acid is active at concentrations 10 and 10 JM in only 50 percent of the polyps (Table 16). The speed and dura tion of the response to this analog are comparable to those found in the response to proline. It was seen that the mouth-opening step of the feeding reaction in Palythoa is induced by acid pH (Table 17). A large, round, gaping mouth opening is observed in 95 percent of polyps tested when they are exposed to pH 2.0. The mouth opens very rapidly (10 times faster than in proline solution 10” M) and the response lasts 2.90 + 0 .3 2 minutes. A small, round, gaping mouth opens in all the polyps exposed to pH 2.5 and a large but slit-like mouth opening is observed in all polyps exposed to pH 3. 0 . Lower pH’s than 2.5 cause permanent damage to the animals and they die. Higher pH's than 3 .5 cause no apparent damage and do not elicit any visible reaction in the polyps. If solutions of proline and glutathione are mixed just before testing and the mouth opening reaction of the polyps is studied, a clear inhibition of the reaction is seen when the activators are in equimolar concentrations (Table 18, Pig. 19)- The strongest inhibition occurs when the activators are at concentrations 5 x 10-^M. Only 50 percent of the polyps open their mouths in this combination in circumstances under which 100 percent do so in proline 105 TABLE 17 ACTIVATION OP THE MOUTH-OPENING RESPONSE IN PALYTHOA TOWNSLEYI* BY ACID pH pH Time for Mouth to Open (min.) In Percent age Polyps Duration of Response (min.) In Percent age Polyps 2.0 0 .0 5 + 0 .0 6 95 2.40 + 0 .3 2 95 2.5 0.14 + 0 .0 9 100 1.10 + 0.10 100 3.0 0.09 + 0.05 100 1.50 + 0 .00 100 3.5 - 0 - 0 Con- tro1** - 0 - 0 *20 polyps were used in each experiment. **Control was Instant Ocean pH 8.15. TABLE 18 INTERACTION BETWEEN PROLINE (PRO) AND GLUTATHIONE* (GSH) IN THE MOUTH-OPENING RESPONSE OF PALYTHOA TOWNSLEYI (20 POLYPS) Solutions and Concentrations Percentage of Polyps Ingesting Untreated Filter Paper Time to Ingest Paper Pro 5 x 10 + GSH 5 x 10_®M GSH 5 x 10_ M GSH 5 x 10 5M GSH 5 x 10~®M 50 100 85 75 o.8o + o.i4 1.85 + 0.14 1.13 + 0.72 1.76 + 1.45 Pro 5 x 10 100 1.10 + 1.21 Pro 5 x 10 4M + GSH 5 x 10 GSH 5 x 10 M GSH 5 x 10~5M GSH 5 x 10_eM 100 40 50 55 1.10 + 0.82 0.85 + 0.24 3.66 + 0.00 3.75 + 2.25 Pro 5 x 10~ " 50 2.40 + 1.23 Pro 5 x 10 M + GSH 5 x 10_3M GSH 5 x 10_4M GSH 5 x 10 M GSH 5 x 10~®M 85 75 20 100 O.56 + 0.16 O.56 + 0.17 2.50 1.60 + i.4i Pro 5 x 10~SM Pro 5 x 10-eM GSH 5 x 10 ®M Control (instant Ocean) *25 ml of Pro 5 x 10 2M with 25 ml of GSH 5 x 10 2U, combination 5 x 10 TM Pro + 5 x 10~3M GSH. lar fashion. were mixed In order to obtain the Other combinations were prepared in simi- Figure 19. Effect of equimolar concentrations of pro line (P) and glutathione (G) in the mouth opening response of Palythoa townsleyl. 107 Percent of polyps opening mouth 108 '100 Proline P-G combination PlO^ P10"^P10"3 Iv .'.v .v X v mm XyX'XvX* mil ■ I P * Pl0"^P10_ZfP10_566 G10“^G10-ZfG10_3«6 GlO^C-io^G^Gio6 Figure 19 _"3 5 x 10 -I and no polyps open their mouths in glutathione 5 x 10"^M. The speed of the reaction Is not altered sig- _-2 nificantly when proline is at concentrations 5 x 10 JM and -4 -S 5 x 10 M but it is increased considerably when 5 x 10 JM -4 and 5 x 10 M concentrations of glutathione are mixed with proline 5 x 10*"^M. Proline 5 x 10~^M by itself does not elicit mouth opening, but when mixed with glutathione 5 x 10-^M 85 percent of the polyps respond. The increased pro portion of responsive polyps is due to some interaction be- tween both molecules because glutathione 5 x 10 J alone cannot elicit response. In combinations where both activa- tors are at concentration 5 x 10 only 20 percent of the polyps open their mouths. This would seem to contradict the statement about the inhibition to the response observed when the activators are equimolar, because 20 percent of polyps respond to the combination of proline and glutathione at 5 x 10”^M concentrations in circumstances under which no polyps respond to either activator at that concentration by itself. But response by 20 percent of polyps is very low if compared to the proportion of polyps (Pig. 20) respond- -5 ing to combinations where proline is kept at 5 x 10 and -■a the concentration of glutathione is varied from 5 x 10 J to 5 x 10~^M, all of which do not elicit mouth opening when offered to the polyps by themselves. The most effective combination proved to be Pro 5 x 10-5M GSH 5 x 10_6M (Fig. 20). In this combination Figure 20. Effect of glutathione (G) in the mouth opening reaction of Palythoa townsleyi to proline (P) 5 x 10“5m . 110 Figure 20 Percent of polyps opening mouth 111 112 all the polyps responded within 1.60 + 1.4l minutes. Neither prollne 5 x 10 J nor glutathione 5 x 10 M can elicit a mouth-opening reaction, thus the response is due to some interaction between both activators. When various concentrations of glutathione are used in conjunction with 5 x 10”^M proline, all concentra tions of glutathione except 10"^M inhibit the mouth opening induced by proline. The combination of 5 x 10"^M gluta thione and 5 x 10~^M proline gives a fast response (1.10 + 0.82 minutes) in all the polyps tested. When 5 x 10_\ -5 -6 5 x 10 and 5 x 10 M glutathione solutions are combined _2i with proline 5 x 10 M, there is a definite inhibition in the number of polyps opening their mouths and at least in the two laBt glutathione concentrations, an increment in the time needed for the reaction to occur. The lowest con centrations of glutathione have a slightly less inhibitory effect than the higher ones (Pig. 21), but increase the time needed for reaction more than the high concentrations. The effect of glutathione on proline 5 x 10~^M is inhibitory except when the tripeptide is at concentration 5 x 10~^M (Pig. 22). The combination Pro 5 x 10”^ GSH 5 x 10~^M elicits response in all the polyps within O .85 + 0.14 minutes. The inhibition of glutathione on the effect of proline 5 x 10"^M is strongest when both activators are at concentrations 5 x 10“^M. Here the number of polyps re sponding is reduced 50 percent. Glutathione 5 x 10-^ and Figure 21. Effect of glutathione concentration in the mouth-opening response of Palythoa to proline 5 x 10“^M. 113 Percent of polyps opening mouth Figure 22. Effect of glutathione (g) concen- ^ mo^th-°Pening response at p x jjy i ^ t0 proline 115 116 100 80 MfO 20 ■ $8$$$ iiSWi N W . w a r n S U M >>>:*$£ • •V •v .v i O P h fA I o c d • 4 - I o CD P^oline p-G combination fO i • • • 1 O f A | - 4 - \ J O 1 O A 1 VD CD CD O 1 O ’ CD CD ■A VO I I O c d o CD Figure 22 117 5 x 10”^M reduced the number of polyps responding 85 and 75 percent respectively. Pipecolic acid, an analog of prollne, was found to elicit a small mouth opening In 50 and 48 percent of the _ p polypB when offered In concentrations 5 x 10 and 5 x 10-^M respectively (Table 19). In the hope of finding more Information In the control of the speed of reaction, the Interaction between prollne and Its analog was studied by placing the polyps In prollne solutions of several con centrations and offering pipecolic acid 5 x lO'^M imbibed In filter paper and also by the reverse experiment: placing the polyps In pipecolic acid solution and offering prollne 5 x 10 filter paper (Table 19). It was found that the presence of pipecolic acid-paper did not affect signifi cantly the speed of the reaction or the proportion of polyps responding to the prollne solutions (Pig. 23) except -4 in the case of prollne 5 x 10 M, where it increased the proportion of polyps responding by about 50 percent. Pipecolic acid solution does not affect the time required to respond to proline-paper (Table 19) and only in concen- -2 tration 5 x 10 M lowers slightly the number of polyps re sponding from 100 to 85 percent (Pig. 24). l-Thiazolidine-4-carboxylic acid, another proline analog, was found to be completely inactive in eliciting mouth-opening response in Palythoa, either in solutions of 118 TABLE 19 EFFECT OF THE INTERACTION BETWEEN PROLINE (PRO) AND PIPECOLIC ACID* (P.A.) IN THE MOUTH-OPENING REACTION OF PALYTHOA TOWNSLEYI** Time for Paper Imbibed Percentage of Mouth by 10-1M Polyps to Open Solution of.. Opening Mouth (min.) Pro 5 x 10"2M P.A. - paper 100 0.43 o C O • o +1 Pro 5 x 10_3M P.A. - paper 85 1.05 1+ o VO o o Pro 5 x 10 "Sfl P.A. - paper 80 0.53 + 0.01 Control*** P.A. - paper 100 0.51 + 0 .6 1 P.A. 5 x 10"2M Pro - paper 85 2.01 ± 3.05 P.A. 5 x 10_3M Pro - paper 100 1.55 ± 0.73 P.A. 5 x 10_1*M Pro - paper 100 0.76 0 0 C O o +1 Control Pro - paper 100 1.08 0 0 LTV • o +1 P.A. 5 x 10_2M - 50 2.20 ± 0.73 P.A. 5 x 10_3M - 48 2.88 1+ o o ♦Activator and analog offered, one In solution, the other Imbibed In filter paper. **20 Polyps tested In each experiment. ***Control Is Instant Ocean. Solution and Concentration Figure 23. Effect of pipecolic acid (10- M), (P.A.) imbibed in filter paper, on the mouth- opening response of Palythoa townsleyl to proline solutions. CPA Control is Instant Ocean. 119 W m m Control P.A m >>>XvXv> •X'X'XvXv X.vl-Xv. m m m ■ t ■ 1 ■ m Figure 2k. Effect of proline paper (P) (10~ M) in the mouth opening response of Palythoa townsleyi to pipecolic acid TKA7). C. Control is Instant Ocean. 121 Percent of polyps opening their mouths V A V M W W W i W . iViV»V»V«V/iViViV*V PA10 Cj 1 0 - 0 1& 10-3 PA 10 PA 10 1 1 $ : II • 4 • > o > • M • H - CO 4 CO o CD o H I—1 ?“ * >4 4 < + P r i - H - ► d H - O CD O 4 4 4 SSI 100r 123 different concentrations or imbibed in filter paper. But this analog can inhibit considerably (Pig. 25) the response of polyps to proline paper. The inhibition is highest in the higher concentrations of thiazolidine. The time of the reaction is not affected significantly. If the con verse experiment is performed, that is, if thiazolidine-4- carboxylic acid-paper is offered to polyps placed in pro line solutions of different concentrations (Table 20, Pig. 26) there is no effect in the mouth-opening response. Discussion The only solutions capable of activating mouth- opening response in Palythoa were those of proline, hy- droxyproline, the proline analog pipecolic acid and gluta thione. All of them were only effective at concentrations which are too high (10_^M and above) to be expected in the natural foods of Palythoa. Since glutathione elicits ingestion-response with out the need for a previous mouth opening, it could be sug gested that mouth opening is not necessary, or, at least, not important for the completion of the feeding reaction in Palythoa. Some information about the properties of the chemoreceptors involved in the mouth-opening response can be obtained by the use of proline analogs. It waB found, for instance, that hydroxyproline, which has an extra OH group on the molecule is only 1 /1 0 0 as effective as proline. Figure 2 5. Effect of l-thiazolidine-4-carboxylic acid (T) in the mouth-opening response of Paly thoa townsleyi to proline (P; imbibed in filter paper at concentration 10~lM. 124 Percent of polyps opening mouth o r \ j o O C T s o O o o Control T10 SSI 100 126 TABLE 20 EFFECT OF THE INTERACTION BETWEEN PROLINE AND 1-THIAZOLIDINE-4-CARBOXYLIC acid* (t .) in THE MOUTH-OPENING REACTION OF PALYTHOA TOWNSLEYI** Solution and Concentration Paper Imbibed by 10-1M Solution of.. Percentage of Polyps Opening Mouth Time for Mouth to Open (min.) Pro 5 x 10-2M T. - paper 100 0 .2 6 + 0 .1 6 Pro 5 x 10 “3M T. - paper 100 0 .6 0 + 0 .2 6 Pro 5 x 10~4M T. - paper 50 1.66 + 1 .0 3 Control*** T. - paper - - T. 5 x 10“2M Pro - paper 60 1 .2 6 + 0 .9 1 T. 5 x 10-3M Pro - paper 60 I.2 3 + 0 .5 0 T. 5 x 10“4M Pro - paper 75 1 .2 6 + 0 .7 6 Control*** Pro - paper 100 1 .0 8 + 0 .5 8 T. 5 x 10"2M - - - T. 5 x 10-3M - - - *Activator and analog offered one In solution, the other Imbibed In filter paper. **20 Polyps tested In each experiment. ***Control Is Instant Ocean. Figure 26. Effect of l-thiazolidine-4-carboxylic acid (T) imbibed in filter paper (10_1M concentration) on the response of Palythoa tovmsleyl to pro line solutions. Control is Instant Ocean. 127 Percent of polyps opening mouth * ■ Control T T T Proline alone T.-paper offered Figure 26 128 129 Similar results were observed earlier in Cordylophora (Ful ton, 1963) and Cyphastrea (Mariscal & Lenhoff, 1968). Both coelenterates respond to proline and in both hydroxyproline is 1 /100 as effective as proline in eliciting mouth opening. The six-membered-ring analog of proline, pipecolic acid, is effective in a smaller proportion of the polyps, but the speed of the reaction and its duration are compar able to those observed for proline at the same concentra tions. Mariscal & Lenhoff (1 9 6 8) found pipecolic acid more effective than proline in activating mouth opening in CyphaBtrea and Fulton (1963) found that this analog was about 10 fold less active than proline. The fact that only proline analogs with the imino group intact, such as pipecolic acid, azetidine-2-carboxylic acid, hydroxyproline and the prolinepeptide proylglycine, can elicit mouth opening (Table 14) suggests that the re ceptors are specific for this imino group. Since azetidine- 2-carboxylic acid and pipecolic acid, both analogs where the size of the ring differs from the proline one, can elicit mouth opening in all the polyps tested, the size on the ring seems not to be important in the activation of the reaction. l-Thiazolidine-4-carboxylic acid does not elicit mouth opening when offered either as solution or imbibed in filter paper, but it inhibits the reaction to proline-paper. 130 It does not, however, Inhibit the reaction of the polyps to proline solution. The explanation of this contradictory behavior of the analog could be that when imbibed in paper the analog does not diffuse over the receptors fast enough to displace the proline molecules which are affecting the receptors for mouth opening. When the analog is in solu tion some of its molecules reach the receptors before the proline molecules, which in turn are diffusing from the piece of filter paper where proline is imbibed. The analog cannot elicit mouth opening, which indicates that the re ceptors for this reaction are very specific for the proline molecule, but it can inhibit the response to proline, prob ably by occupying the receptors before the proline does. Pipecolic acid elicits mouth opening in all the polyps when given imbibed in filter paper, but only in 50 and 48 percent of the polyps when presented in solutions of -2 -8 concentrations 5 x 10 and 5 x 10 JM. When offered to- -4 gether with proline, at 5 x 10 M concentration the propor tion of polyps responding is enhanced. If the receptors for mouth opening were specific for proline or certain proline analogs, how does one explain the response of polyps to high concentrations of glutathione (5 x lO”^ and 5 x 10“2M)? Isherwood (1959) hypothesized that glutathione may occur in ring structures (Pig. 27). If this is true the ring structures present in a solution of glutathione would be able to stimulate mouth opening Figure 27. Chemical structure of glutathione, according to Isherwood (1959). 131 SH I CH2 I HOOC- CH -CHr CH7- CO- N H-CH- CO - N H -C H2- C OOH NHj+ Glutathione ( straight-form chain ) CH,- CH7 XS-CHa f CH V ' * ' / \ y \ / c o o - N N H H Ilydroxyuyrrolidinc HO S — CH, O S------CH* 0 ,<k CH-C-NH- -CHl -C / \ n - < - c < N x v N / H Hydroxythiazolidine Thiazoline Possible rinr structure of glutathione -N»- 1 —1 Figure 27 Vo 133 because they have an intact imino group and a similar shape to the proline molecule. Since mouth opening occurs only _p at glutathione concentrations 10 , it is conceivable that enough molecules could have the ring structure. The analog S-methyl-glutathione, kindly supplied by Dr. H. M. Lenhoff, did not elicit mouth opening when offered imbibed in filter paper at lO'^M concentration. This suggests that the glutathione-sensitive receptors for mouth opening in Paly thoa may differ from those in Hydra, for in this fresh water hydroid S-methyl glutathione is effective in elicit ing mouth opening (Lenhoff, 1968a). It is very difficult to imagine proline present in -4 concentrations above 10 M or glutathione in concentrations _2 above 10 M in prey organisms used by Palythoa as food. Thus, the fact that the receptors for mouth opening seem to be specific to the imino group of proline is not very significant. What is very important is that mouth opening can be activated by the interaction between proline and _c glutathione when these are at concentrations 5 x 10 <M, and -6 5 x 10" M, respectively, both of which can reasonably be expected in the food of Palythoa. The chemical control of the mouth opening response seems very intricate. Although some important aspects of the chemical control of mouth opening in Palythoa can be determined from the information presented so far, many more details must be known before the control of the reaction 13^ can be fully understood. The fact that high concentration of H+ In the medium caused a mouth-opening reaction and the fact that the shape of this mouth opening seems to depend on such concentration suggests that some alteration of the receptors which control mouth opening has occurred In acid environment. However, It does not mean that acid pH can elicit a feeding reaction, for the polyps are not able to Ingest food under acid pH conditions. A similar phenomenon was found to occur In Hydra (Lenhoff, 1961b). The animals show a gaping mouth opening in the presence of many delete rious compounds such as acids, but they cannot be made to ingest dead food and usually die within a few hours. Fur thermore, the extreme acid pH’s tested in the experiments described, probably will never be encountered under natural conditions. Conclusions 1. Mouth opening is controlled chemically in Paly thoa townsleyl, and can be elicited only by chemical stimu li. 2. The mouth-opening receptors are located mainly on the margin of the mouth itself. 3. The receptors have high affinity for the imino group of proline. 4. The size of the proline ring is not a critical factor in the interaction of the activator and receptor in the mouth-opening response. 5. The receptors are altered in some way by pH’s 2.0 to 3-0. 6. Mouth opening is not required for ingestion to take place 7. Under natural conditions, the most probable -6 stimulus for mouth opening is glutathione at 5 x 10" M concentration and proline at 5 x 10 concentration acting synergistically. CHAPTER V CONTROL OP THE INGESTION RESPONSE Jennings, one of the giants of American biology during the first half of this century (Jensen, in JenningB 1905a), made some important contributions to the study of behavior in sea anemones. His description of the feeding reaction in the large sea anemone Stoichactlc helianthus (Jennings, 1905b) coincides in many ways with what has been observed in the feeding reaction of Palythoa, from the formation of a lip which secures the food and pushes it toward the mouth, to the interesting manner in which these coelenterates carry out the process of ingestion. After the lip has formed and the food is within close distance to the mouth, the peristome contracts . This causes the food and the mouth to approach each other. At this moment the lobes of the actinopharynx increase in size, extend to ward the food and reach. In Stoichactls the mouth may be transferred from the center of a disc 10 cm in diameter to within 1 cm of the edge (Jennings, 1905b). In Palythoa this twisting of the mouth is never as dramatic, but never theless, it is present. After this "the lobes of the ac tinopharynx extend over the food, while the tentacles pro- 136 137 gressively withdraw from it, till the food is lying on the contracted part of the disc, completely covered by the lobes of the actinopharynx which presses the food into the internal cavity." This is very much the way in which Paly thoa placed in a solution of glutathione respond to inert material. Many sea anemones have been reported to swallow inert material, such as pellets of paper, grains of sand, and the like, when they have not been fed for a long time. This has been observed in a number of Mediterranean sea anemones (Nagel, 1892), Sagartiaj (Torrey, 1904a), Metrldl- um (Allabach, 19 0 5), Alptasla (Jennings, 1905b), and Eplactls (Lenhoff & Schneiderman, 1959)* It can be con cluded then, that in various sea anemones, the ingestion response can be elicited by a pure mechanical stimulus. This is also the case in Hydra littoralls in which an ob ject that reaches the mouth is swallowed regardless of the presence or absence of the feeding activator glutathione (Lenhoff, 1968a). This tripeptide merely coordinates the movements of the tentacles to bring the food to the mouth and causes the mouth to open. The control of the ingestion response has been studied in much fewer coelenterates than the mouth opening response. Lenhoff& Schneiderman (1959) described the activa tion of a coordinated ingestion of food carried out by the gastrozooids of the siphonophore Physalia physails. Lin- 138 stedt, Muscatine & Lenhoff (1968) found that valine caused the sea anemone Boloceroides to ingest an inert object. The ingestion response in Palythoa represented a very interesting subject for investigation., particularly after determining how the mouth opening response was con trolled. Materials and Methods The materials and methods for this section are the same as those described in the preceding chapter with the 2 modification that an inert object, represented by 1mm pieces of filter paper, was offered to the polyps in each experiment. The purpose of this filter paper was to pro vide a solid object in order to be able to visualize in gestion . Results The feeding reaction in Palythoa culminates with the ingestion of food, represented by filter paper when this is treated with certain chemical substances (Table 21, Pig. 28). A general survey for activity of amino acids re vealed that alanine, serine and lysine could elicit inges tion response in Palythoa (Table 21), when they were imbibed in filter paper at concentrations 10-1M. Alanine causes ingestion response in 40 percent of 139 TABU 21 ACTIVATION OF THE INGESTION-HESPONSE IN PALYTHOA TOWNSIPYI (20 POLYPS) BY SEVERAL SUBSTANCES IMBIBED IN FILTER PAPER, AT CONCENTRATIONS 10_iM Substance Structure Percentage Polyps Ingesting Paper Time to Ingeet Paper (mln.) Proline ^ h^COOH H 100 2.97 ± l.**3 Hydroxyproline ” 1 1 S ^ ^ C O O H H 10 10 .0 0 + 0 .5 0 Azetldlne-2- carboxyllc eeld ">COOH N H 100 3 .5 0 + 1.0 0 Pipecolic aold ^sjj^COOH H 50 2.50 + 0.50 Prolylglycine CO-NH-CHa-COOH H 100 1 0 .1 0 + 2. 1 (0 Olutathlone aHN-^H—( CHj. )a-CO-NH-OI-COrE-CHa-COOH COOH CHgrSH 60 9. 1 ( 6 + 7 .2 8 S-methyl Olutathlone aHN-CH—(CHa)a-CO-NH-<jH-CO—NH-CHa-COOH COOH CHarS-CHa 20 7.50 + 1.73 Alanine NHa CHg-C-COOH > 1 0 3.00 + 0.1(0 Serine NHa HO-CHa-f-COOH H 25 3 .0 0 + 0 .5 0 Lysine HbN-<CH£) . 1 -C-COOH 100 5.75 + 1.73 Figure 28. Activation of the ingestion-response in Palythoa townsleyl by several sub stances imbibed in filter paper, at concentrations of 10“lM. 140 Figure 28 Proline Hydroxyproline Azetidine-2- carboxylic acid Pipecolic acid Prolylglycine Glutathione S-methyl glutathione Alanine Serine Lysine Percent of polyps ingesting paper ro - p - co o o o o o o ■ 1 142 the polyps after 3*00 + 0.40 min. of stimulation. Solu- _Q tlons of alanine in 5 x 10 or higher concentrations do not elicit Ingestion response. When filter paper Imbibed In a 5 x lO'^M solution of serine Is offered to the polyps several tentacles con tract and bend upward and toward the mouth, enclosing the paper and pushing It toward the mouth, where It Is In gested after 3.00 + 0 .5 0 minutes by 25 percent of the polyps. This reaction occurs only when the serlne-paper lands on the tentacles of the polpys. If the landing site Is the peristome or the margin of the oral disc, no re sponse Is observed. The fact that only 25 percent of the polyps respond to serine sheds doubt on Its significance as a possible activator of feeding in Palythoa. It is pos sible that the reagent amino acid used had some contamina tion and the response seen is not one due to serine but, rather, to the contaminant which could be lysine or proline. Since the contaminant would be in very small concentration the response would occur in very few polyps and be very weak. Serine solutions do not cause any response, even at _"2 concentrations higher than 5 x 10 lysine 10_1M imbibed in filter paper causes inges tion response in all the polyps after 5 -7 5 + 1*73 minutes. Ingestion is slow and takes an average of about 5 minutes from the moment when the polyps close over the paper, and the moment when this disappears in the coelenteron. No response to lysine is observed when the amino -1 -4 acid is in solution at concentration 5 x 10 to 5 x 10 M. If clean filter paper is offered to the polyps placed in any of these lysine solutions it is rejected within 30 minutes to 1 hour without any signs of acceptance by the polyps. Alanine, serine and lysine have a terminal struc ture (-CH2-(HNH2-(00H)) similar to that present at one end of the glutathione molecule (Table 21). This structure may represent an active portion of the tripeptide molecule, capable of eliciting an ingestion response in certain pro portions of the polyps. If untreated filter paper is of fered this is rejected unless the polyps are placed in a solution of proline or glutathione (Table 22, Pig. 29). The most effective solutions to induce ingestion in Paly- -2 -3 thoa are prollne at 5 x 10 and 5 x 10 JM concentrations both acting at comparable speed. The time required for the reaction is 3 .5 times that required when proline is offered absorbed in filter paper. Although glutathione solutions can induce ingestion response at concentrations as low as 5 x 10 ^M, the number of polyps responding Is significant _2 only at glutathione concentrations 5 x 10 M. Contrary to what was observed for proline, the speed of the reaction for glutathione paper is about 50 percent slower than the speed of the reaction to glutathione solution. TABLE 22 RESPONSE OF PALYTHOA TQWNSLEYI* TO UNTREATED FILTER PAPER WHEN POLYPS ARE PLACED IN A SOLUTION OF GLUTATHIONE** OR PROLINE*** AT DIFFERENT CONCENTRATIONS Activator Concentration Type of Initial Response In Percentage of Polyps Time for Paper to he Ingested (min.) In Percentage of Polyps GSH 10“2 Sllt-llke mouth opening 65 5.00 + 2.44 25 GSH 10“3 Actinopharynx exposed 100 5.00 + I.75 85 GSH 10 Actinopharynx exposed 55 4.57 + i.4i 35 GSH 10-3 Actinopharynx exposed 35 5.30 + 0.00 15 GSH 10“® None 0 - 0 Pro 10“2 Wide—round mouth opening 100 10.64 + 2.64 85 Pro 10“3 Wide-round mouth opening 100 11.10 + 3.74 80 Pro 10“4 Sllt-llke mouth opening 30 - - Pro 10“S None 0 - - *20 Polyps for each experiment. **GSH. u u U - ***Pro. 4=- 4 = - Figure 29 Activation of the ingestion response in Palythoa townsleyi by proline (Pro) and glutathione (GSH) in solution. Control is Instant Ocean. 145 Figure 29 o Percent of polyps ingesting clean paper o o Control o Pro 10~2M Pro 10“3M Pro lO”^ GSH 10“2M GSH 10"3M GSH 10 GSH 10"5H GSH 10 °M 146 147 The substitution of a methyl group for a hydrogen on the sulfhydryl group of the glutathione molecule sig nificantly reduces the activity of the tripeptide (Table 2 3). Only 20 percent of the polypB ingest filter paper imbibed in 10-1M solution of S-methyl glutathione, as com pared to 60 percent of the polyps that ingest paper imbibed in 10-1M glutathione. The affinities of the chemoreceptors for the in gestion response with proline were studied by determining the activity of several proline analogs and of two proline peptides. Hydroxyproline, which differs from proline in pos sessing one hydroxyl group, elicits ingestion in only 10 percent of the polyps, and this after very long time of stimulation (10 .0 0 + 0 .5 0 minutes). Pipecolic acid induced ingestion of clean filter paper in 45 to 50 percent of the polyps and had a clear inhibitory effect in the ingestion of proline-imbibed fil ter paper (Table 24, Pig. 3 0), when present in concentra- _2 tion 5 x 10 M. Here the number of polyps swallowing pro- line-paper is reduced from 100 percent in Instant Ocean to _2 30 percent in pipecolic acid at concentration 5 x 10 M. The analog is probably occupying the receptor sites, which can not be available to proline. The inhibition is much less marked in pipecolic acid 5 x 10 when the number of polyps ingesting proline-paper is reduced from 100 to 85 TABLE 23 RESPONSE OP PALYTHOA TOWNSLEYI TO GLUTATHIONE 10 “1M AND S-METHYL GLUTATHIONE 10-lMIMBIBED ON FILTER PAPER Substance Time to Ingest Paper (min.) In Percentage of Polyps Time to Reject Paper (min.) In Percentage of Polyps Glutathione 1.85 + 1.17 60 12.00 + 11.90 40 S-methyl Glutathione 7.50 + 1.73 20 1 3 .1 2 + 5.74 80 -pr 0 0 149 TABLE 24 EFFECT OF THE INTERACTION BETWEEN PROLINE AND PIPECOLIC ACID* IN THE INGESTION-RESPONSE OF PALYTHOA TOWNSLEYI** Solution and Concentration Paper Imbibed In 10~Im Solution on.. Percentage of Polyps Ingesting Pro 10 "2M P.A. - paper 35 Pro 10'3M P.A. - paper 10 Pro 10-^M P.A. - paper 45 Control P.A. - paper 50 P.A. 10“2M Pro - paper 30 P.A. 10_3M Pro - paper 85 P.A. 10~^M Pro - paper 100 Control*** Pro - paper 100 P.A. 10_2M Untreated paper 45 P.A. 10-3M Untreated paper 50 *Activator and analog offered, one In solution, the other Imbibed In filter paper. **20 Polyps tested In each experiment. ***Control Is Instant Ocean. Figure 3 0. Effect of pipecolic acid imbibed in filter paper (10“lM), on the ingestion response of Palythoa townsleyl to proline solutions. CPA Control is Instant Ocean. P.A. is pipecolic acid. 150 Percent of polyps giving ingestion response 100, 80 60- ZfO 20 '.yX vX v’ .y »»»:•: * • % v. v sill : < ■ > > > > > > : . 0 •v .v .v .v .v . v X*XvX*X * % • V h N v .v .v m m Control PA10"2PA10~2 PA10“3PA10~3 Figure 30 Prolinc paper Clean paper 152 percent (Table 24). Another analog of proline, thiazoli- dine-4-carboxylic acid does not induce the ingestion of _2 untreated filter paper even at concentrations 5 x 10 and 5 x 10_^M (Table 25). The shape of the thiazolidine ring is similar to that of proline but it has a S substituting for the imino group. Thiazolidine-4-carboxylic acid can inhibit the response of Palythoa to proline paper (Pig. 31) in a proportion that depends on the analog's concentration. When the analog is not present, 100 percent of the polyps _2 ingest proline paper. Concentrations 5 x 10 M of thia- zolidine-4-carboxylic acid reduce the proportion of polyps ingesting proline paper to 5 percent. When Palythoa is in a solution of l-thiazolidine-4-carboxylic acid at concen- _2i tration 5 x 10 J and 5 x 10 M, 55 percent and 75 percent of the polyps respectively swallow proline-paper. This suggests that the analog can occupy the receptor sites for proline and also that the imino group of proline must re main unchanged in order to elicit ingestion response. If the experiments are reversed, that is if paper imbibed in the analogs is presented to polyps placed in a proline solu tion, the results (Pigs. 32 and 3 3) corroborate the fact that proline competes with pipecolic acid and with 1-thia- zolidine-4-carboxylic acid for the receptors for the inges tion response. If proline Is not present or Is at concen trations 5 x 1 0"^M only 50 or 45 percent of the polyps 153 TABLE 25 EFFECT OF THE INTERACTION BETWEEN PROLINE (PRO) AND l-THIAZOLIDINE-4-CARBOXYLIC ACID* (T.) IN THE INGESTION-RESPONSE OF PALYTHOA TOWNSLEYI** Solution and Concentration Paper Imbibed in 10“lM Solution on.. Percentage of Polyps Ingesting Pro 10_2M T. - paper 10 Pro 10_3M T. - paper 30 Pro 10-^M T. - paper 0 Control*** T. - paper 0 T. 10"2M Pro - paper 5 T. 10~3M Pro - paper 55 T. 10~^M Pro - paper 75 Control*** Pro - paper 100 T. 10"2M Untreated paper None T. 10 “3M Untreated paper None *Activator and analog, one In solution, the other Imbibed In filter paper. **20 Polyps tested in each experiment. ***Control is Instant Ocean. Figure 31. Effect of l-thiazolidine-4-carboxylic acid (T) imbibed in filter paper on the ingestion-response of Palythoa townsleyl to proline (Pro) solutions. Control is Instant Ocean. 154 CONTROL P T10-2 T10 2 T10- - 5 TIO ^ P P Figure 31 J p r o l i n e p a p e r . . . . . . . . . . Untreated S g-jC lea n p a p e r Figure 32 Effect of proline-paper (P) (10 M) in the ingestion response to Palythoa towns ley 1 to pipecolic acid (P. A. ' ) Control (C) is Instant Ocean. 156 Percent of polyps giving ingestion response IQ i loot Figure 3 3. Effect of proline-paper (P) (10- M) in the ingestion response of Palythoa townBleyl to 1-thiazolidine-4-c arboxy11c acid. ( T ) Control ( c ) is Instant Ocean. 158 Percent of polyps giving ingestion response ■ : : : :*x*x* ; ! ; X*XvX. v’ •X vX vX v' : •x*x*x* mm C o n t r o l Figure 33 1 - t h i a z o l i d i n e Untreated c l e a n p a p e r 160 ingest pipecolic acid-paper (Pig. 32), when proline is at -2 concentrations of 5 x 10 M and 5 x 10 only 35 or 10 percent of the polyps ingest the pipecolic acid paper, while 85 or 80 percent of the polyps ingest clean paper (Pig 3 1). Thiazolidine-4-carboxylic acid-paper does not stimulate ingestion (Pig. 33) hut it does reduce the per centage of polyps swallowing untreated paper while in a —2 5 x 10 M proline solution from 85 to 10 percent; when _Q polyps are in a solution of proline at 5 x 10 -'M the reduc tion caused by 1-thiazolidine paper is from 80 to 30 per- -4 cent and in proline at 5 x 10 M concentrations 1-thiazoli dine paper has no effect. The study of the response of Palythoa to two pro line peptides (Table 3 1) showed again that the amino group 1b Important in the activation of the ingestion response. When this group is replaced, such as is the case in gly- cylproline, no ingestion occurs; however, if this group is kept intact, as in prolylglycine, 100 percent of the polyps ingest the paper. This indicates that substitutions in the carboxylic group do not alter the activity of the molecule. If either proline or glutathione are presented in concentration 10-1M imbibed in filter paper while the pol yps are placed in a solution of the other activator (Tables 26 and 2 7), the interaction between both causes a decreased proportion of ingestion response. Table 26 indicates that TABLE 26 EFFECT OF THE INTERACTION BETWEEN PROLINE (PRO) AND GLUTATHIONE (GSH)* IN THE INGESTION RESPONSE OF PLAYTHOA TOWNSLEYI** Solution and Concentration Condition of Paper Percentage of Polyps Ingest ing Paper Time to Ingest Paper GSH 10~2M Pro - paper 85 5 .2 8 + 4.04 GSH 10"3M Pro - paper 100 4.50 + 0 .8 0 GSH 10_2|M Pro - paper 75 5 .0 0 1+ 0 ro 4^ GSH 10"5M Pro - paper 75 4.10 + 2 .6 0 Contro1*** Pro - paper 100 2.97 ± 1.43 GSH 10"2M Untreated 25 5 .0 0 + 2.44 GSH 10 “3M Untreated 85 5 .0 0 ± 1-73 GSH 10 Untreated 35 4.57 + 1.41 GSH 10_5M Untreated 15 5.30 + 0 .0 0 GSH 10“6M Untreated 0 - Control*** Untreated 0 - *Glutathione offered in solution at the concentrations indicated, proline offered imbibed in filter paper at concentration 10“lM. **20 Polyps tested in each experiment. ***Control is Instant Ocean. 162 when proline is offered in 10~^M concentration absorbed in filter paper (Pig. 34) the interaction with glutathione caused a decreased proportion of ingestion responses, ex- _Q cept in solutions of 10 glutathione when almost all polyps respond just as it occurs in Instant Ocean. In the other solutions, glutathione seems to inhibit ingestion and the greatest inhibition is seen when the tripeptide is -4 -6 in concentrations 5 x 10 and 5 x 10 In all cases the reaction to proline paper is slower when glutathione is present in the medium. When glutathione is offered imbibed in filter paper while the polyps are placed in a solution of proline (Table 27, Pig. 35)> the effect of proline is to increase the number of polyps responding. The highest increase oc- -4 curs at proline 5 x 10 and 5 x 10 concentrations, both of which induce ingestion in all the polyps tested as com pared to only 60 percent polyps ingesting glutathione paper -4 -6 when no proline is present. Proline 5 x 10 or 5 x 10 cannot elicit second ingestion of untreated paper by them selves. Thus the stimulation of ingestion is due to some effect of proline and glutathione acting together. The times required for ingestion are very long and _2 variable. In 5 x 10 M prollne, the time of ingestion is increased when GSH paper is offered. In Pro 5 x 10""^M It is shortened slightly. Figure 3^* Effect of reduced glutathione (G) in the ingestion response of Palythoa townsley1 to lCT^M proline absorbed in filter paper. 163 Percent of polyps giving ingestion response * M w . v . v ■ Control 1.0 Figure 34 Proline-paper Untreated Clean-paper 165 TABLE 27 EFFECT OF THE INTERACTION BETWEEN PROLINE (PRO) AND GLUTATHIONE (GSH)* IN THE INGESTION RESPONSE OF PLAYTHOA TOWNSLEYI** Solution and Concentration Condition of Paper Percentage of Polyps Ingest ing Paper Time to Ingest Paper Pro 10_2M GSH - paper 80 GO OJ i —i + 2.25 Pro io_3m GSH - paper 80 8.70 + 1.23 Pro 10 GSH - paper 100 7.00 + 4.00 Pro 10 "5m GSH - paper 100 14.50 + 6.20 Control*** GSH - paper 60 9.46 + 7 .2 8 Pro io_2m Untreated 85 10.64 + 2.64 Pro io_3m Untreated 80 11.10 + 3-74 Pro io_4m Untreated - - Pro 10 “5m Untreated - - Control*** Untreated - - *Proline offered in solution at the concentration indi cated. Glutathione offered imbibed in filter paper at concentration 10“ **20 Polyps tested in each experiment. ***Control is Instant Ocean. Figure 3 5. Effect of proline (P) in the ingestion response of Palythoa townsleyl to 10-1M glutathione absorbed in filter paper (G-paper) and control paper moistened with Instant Ocean (U-paper). 166 Percent of polyps giving ingestion response l l l i i P R I I i f l l l l l l l l l i l l l - p a p e r 168 The time required for Ingestion of GSH-paper when the animals are placed In Instant Ocean has a large stand ard deviation and thus cannot be compared meaningfully with times required to ingest paper when the polyps are placed in a proline solution. If both activators are mixed in a series of combi nations (Table 28) an interaction between them becomes ap parent. As was seen in the study of the Pro-GSH combina tions on the mouth-opening response, the ingestion of fil ter paper is inhibited when the two activators are in equi- molar concentrations (Pig. 3 6) since a smaller proportion of polyps respond to the combinations than to either ac tivator by itself. Both glutathione and proline at con centrations of 5 x 10 induce ingestion reaction in 85 and 80 percent of the polyps when each one is in solution by itBelf. If they are combined, however, only 50 percent of the polyps respond. -h Glutathione at 5 x 10 M induces an ingestion re sponse In 35 percent of the polyps, but when combined with proline at the same molarity only 20 percent of the polyps respond. —B Glutathione 5 x 10 -'M Induces Ingestion of un treated paper in 15 percent of the polyps. When combined -5 with proline at 10 -'M it elicits the response in 30 percent of them. This may seem to contradict the previous state ment about equimolar concentrations of the activator causing TABLE 28 EFFECT OF THE INTERACTION BETWEEN PROLINE (PRO) AND GLUTATHIONE* (GSH) IN THE INGESTION RESPONSE OF PALYTHOA TOWNSLEYI (20 POLYPS) Solutions and Concentrations Percentage of Polyps Ingesting Untreated Filter Paper Time to Ingest Paper Pro 5 x 10 % + GSH 5 GSH 5 GSH 5 GSH 5 X X X X 10-3M 10-% io-5m 10-eM 50 55 85 10 1.17 + 1.18 5.81 + I.73 1.28 + 1.00 2.50 + 0.50 Pro 5 x 10"% 80 11.10 + 3.74 GSH 5 X 10-% 100 3.27 + 0.54 GSH 5 X 10-*M 20 4.00 + 0.01 Pro 5 x 10 M + GSH 5 X 10-SM 45 4.44 + l.4l GSH 5 X 10-SM 45 4.46 + 2.23 Pro 5 x 10~4M 0 - GSH 5 X 10-% 85 1.85 + 1.17 Pro 5 x lO^M + GSH 5 X 10“4M 75 2.73 + 2.00 GSH 5 X 10~5M 50 5.50 + 4.89 GSH 5 X 10-°M 100 2.60 + O.58 Pro 5 x 10-5M 0 - Pro 5 x 10-SJ4 GSH 5 X 10-QM 0 - Pro 5 x 10-«M 0 - GSH 5 x 10-®M 85 5.00 + I.73 GSH 5 x 10— 35 4.57 + l.4l GSH 5 x 10“5M 15 5.30 + 0.00 GSH 5 x 10“«M 0 - Control (Instant Ocean) 0 - *25 ml of Pro 5 x 10 2M with 25 ml of GSH 5 x 10 2M were mixed in order to obtain the combina tion 5 x 10“% Pro + 5 x 10~% GSH. Other combinations were prepared in similar fashion. Figure 3 6. Inhibition of the ingestion-response in Palythoa townsley1 when polyps are placed in equimolar combination of proline (P) and glutathione (G). 170 Percent of polyps giving an ingestion response _ -£■ cn O O t O_________O O_________ o__________o________ £ Q O c t - ( —1 CJ O H- cl- O 3 B CD H- o' O H- f c f 3 CD p ) e l s ' 1 2 Mm 2 o o » O ' M m Ui 100' 172 an Inhibition In the proportion of polyps giving the in gestion response. However, if 30 percent response when _c both proline and glutathione are at 10 molar concentra tions is compared (Pig. 37) to the percentages of polyps -S responding to combinations where proline is at 5 x 10 _ o _2i and glutathione at 5 x 10 , 5 x 10 , 5 x 10 M, 30 per cent becomes small enough to be considered to represent an inhibitory effect of the equimolar Pro-GSH combination. The most effective combination to elicit the in- gestion response proved to be proline 5 x 10 ^M, gluta- -6 thione at 5 x 10” M, in which all the polyps ingest clean paper within 2.60 + 0.58 minutes. Since neither activator alone elicits response at such concentration, the ingestion is stimulated by some combined action of the molecules. In combinations of proline 5 x 10 ^ with gluta- -•2 _il thione 5 x 10 J and 5 x 10 M, 85 and 75 percent of the polyps respond respectively. Furthermore the time required for the reaction to occur is shorter in the combination than in glutathione alone (Pig. 3 8). For instance, compare a response within a range of 0 .6 8 to 3 .0 2 minutes and a mean of I .8 5 + 1.17 minutes in the combination Pro 5 x 10 ”% + GSH 5 x 10”% with a response within 3.27 - 5*00 + 1 .7 3 - 6.43 minutes in glutathione 5 x 10”% by itself. Proline 5 x 10 by itself does not elicit ingestion re sponse. The effect of glutathione on proline at 5 x 10 ”% Figure 37. Effect of glutathione (G) in the ingestion response of Palythoa townsley! in proline (P) 10-bM. 173 P 10"5M Percent of polyps giving ingestion response o o ro XL -p- _Q_ CT n _e_ co _a_ o -P 3 01 c 4 ( D CO -J G 10“3M P 10"5M G 10"5m G 10"6M ••v .v .v v .v .v .v .v .v .v .v .v .v .v .v . V .W *W .V .V .V .V .V A V A V A V .V ,' > » : • : • : • .•.v .v .v .v : G 10"3M G 10-ZfM G 10"5M Figure 3 8. Comparative ranges of time required by Palythoa townsleyl to ingest untreated paper when the polyps are in solutions of glutathione (GSH), or combinations of glutathione and proline (P). 175 GSH 10 h i Pro 10“5M 10 YM 10~/ + I I GSH 10~6M Pro 10“5I I RL^uro 38 min. 177 (Pig. 39) is to increase the proportion of polyps respond ing with respect to the number of polyps responding in similar concentrations of glutathione alone. This phenome non occurB in all combinations except when both activators are in equimolar concentrations. The highest percentage of polyps to ingest paper -4 -4 _"5 was seen in combination Pro 5 x 10 x 10 GSH 5 x 10 -> M and the time range for the speed of reaction in the combi- nation is much narrower than for gluathione 5 x 10 JM alone (Pig. 40). The converse combination: Pro 5 x 10"^ GSH 5 x -4 10 M, however, does not increase the proportion of polyps responding, on the contrary it lowers it from 80 percent of polyps responding in proline 5 x 10 to 55 percent of polyps responding in the combination (Pig. 4l). Glutathione -4 5 x 10 by itself causes only 35 percent of the polyps to respond. A striking change in the time required for the reaction is apparent (Pig. 42). In proline at 5 x 10-^M, ingestion occurs within 7 .3 6 - (11.10 + 3*74) - 14.84 min- -4 utes; in glutathione at 10 M, ingestion occurs after 3.16 - (4.57 + 1.4l) - 5.98 minutes; and, in the combination, the time required for the reaction is 2 .0 8 - 3*81 + 1.73 - 4.54 minutes. The ranges for time required to ingest in gluta thione alone and in the combination, overlap, but the range for time required to ingest in glutathione alone is sepa rated from the range of time required to ingest in proline Figure 3 9. Effect of glutathione (G) in the ingestion response of Palythoa . townsley1 to proline (P) 5 x lb“^M. 178 Figure 39 10-ZfM Percent of polyps giving ingestion response o ro o - p - o CTs o co o o o P 10 “Si G IQ-^M G 10“S'I G 10"5M G 10“SM G 10“3M G 10“Sl G 10“5M M -< VO Figure 40. Comparative ranges of time required by Palythoa townsleyi to ingest untreated paper when the polyps are in solutions of glutathione (GSH), or combinations of glutathione and proline (Pro). 180 i i f".......T 5 GSH 1 0 “ 3 l P r o 1 0 - i 4 GSH 1 0 P r o 1 0 ^I'l Figure 40 GSH 10 - H - : j 7 ra in , oo i —■ 182 alone, and a still wider separation Is observed when the combination range Is compared to the proline range. This indicates that the ingestion response is considerably faster in glutathione than in proline, and suggests that the interaction between both feeding activators affects the speed of reaction, although not enough information was gathered to study such speed of reaction. Glutathione is less inhibiting of the proline at 5 x 10 activity when the tripeptide is at 10 ■'M concen trations (Pig. 4l) and in fact here it increases the number of polyps ingesting paper. Furthermore the time required to ingest is considerably reduced (Pig. 42) falling out side the ranges for either proline or glutathione alone. This is a particular effect of glutathione 5 x 10 pro- line 5 x 10 combination because it does not occur when proline concentration is changed. Glutathione 5 x 10~^M _3 combined with proline 5 x 10 does not cause the same effect but rather an opposite one, for only 10 percent of -8 the polyps respond. The combination proline 5 x 10 ^ and glutathione 5 x 10~^M (converse of proline 5 x 10~^ gluta thione 5 x 10~^M) also elicits response in 85 percent of the polyps and the time required for the ingestion is shorter (Pig. 37) in the combination than in glutathione -8 by itself Proline at 5 x 10 does not elicit ingestion reaction by itself. Figure 4l. Effect of glutathione (G) concentration in the ingestion response of Palythoa townsleyi to proline (P) at 5 x 10-3M. 183 Figure 4l P P 10' P 10' P 10' G G Percent of polyps giving ingestion response o ru o - F - O o CO .o o O 10"^M %v.v m m -ZfM 10 10“5M M oo Ft Figure 42. Comparative ranges of time required by Falythoa townsleyi to ingest untreated paper when the polyps are in solutions of glutathione (GSH) or combinations of glutathione and proline (Pro). 185 VO 00 tin:; ^71 XL 01 11 01 i'V-Ol 0J- c I K-o 3^7 oanOT^ i L , I ’ Ll_0 L USD m/_OL ojj i i uc 01 o*d H^_Ol USD & _0l c ] i t / ' ^" 01 I •id SO ,0 I --V Ot USD Hc„oi iisd 187 When glutathione dominates the combination (Fig. ^3) in general the response Is enhanced. The combination -4 proline 5 x 10 + glutathione 5 x 10 elicits ingestion -4 in all the polyps tested. Proline alone at 5 x 10 M con centration does not induce ingestion. Glutathione alone at concentration 5 x 10~^M induces ingestion in 85 percent of the polyps. The range of time (Fig. 40) required for re sponse in the combination is narrower and shifted toward less time than the range of time required to elicit the re- _"3 action in glutathione 5 x 10 alone. _"2 The interaction between glutathione 5 x 10 J and proline 5 x 10 does not affect the proportion of polyps responding but it shortens the time of reaction consider ably (Fig. 40). The interaction between glutathione 5 x -4 -*5 10 and proline 5 x 10 enhances the response, increas ing the number of polyps responding from 30 percent in -4 glutathione 5 x 10 M alone to 75 percent in the combina- tion. Pro line 5 x 10 -7M does not elicit ingestion. To summarize, the most effective concentrations of -4 proline and glutathione were found to be proline 5 x 10 M _c: + glutathione 5 x 10 JM and proline 5 x 10 M + glutathione -fl 5 x 10“ M (Fig. 44); at both combinations all the polyps re spond with ingestion of untreated filter paper. The in creased response is due to the interaction between both molecules rather than to a response to either one alone for Figure 43. Effect of glutathione (G) dominance in combinations of proline (P) and gluta thione which activate ingestion-response in Palythoa townsleyi. 188 gure 43 Percent of polyps giving ingestion response ro 0 - p - ■O C 7 \ . 0 Co J2_ o o. P 10_ZfM P 10“5M o o P 10 _ZfM G 10 "3 M P 1 0 G 10”^M P 10 M G 10 “4, Mmmmmmmrnm®®® G 1 0 “ 3 M G 10“Si 0 0 vo Figure 44. Enhancement of the ingestion-response in Palythoa townsleyi under certain combina tions of proline (Pro) and glutathione (GSH). 190 Figure 44 Percent of polyps giving ingestion response 8 5 g s 8 Pro line 10*"^ Proline 10~^M o WwvwwwOOC 191 -4 there Is no ingestion In proline at concentrations 5 x 10 -R -6 or 5 x 10 or glutathione 5 x 10 M concentration. Glutathione 5 x 10 elicits the ingestion re sponse in 85 percent of the polyps (Fig. 39), but the time has a wider range than the time required for reaction in _2i the combination of glutathione 5 x 10 J proline 5 x 10 M (Fig. 40). Although the ranges of time required to react in -4 — Q proline 5 x 10 glutathione 5 x 10 JM and in proline 5 x -R -6 10 ^ glutathione 5 x 10 M overlap, the time of reaction -R -6 in proline 5 x 10 glutathione 5 x 10 M can be considered shorter. This combination was also seen to be the most ef fective in eliciting mouth opening and, as it was discussed then, it is reasonable to imagine such combination being present in the prey organisms upon which Palythoa feeds. Discussion The fact that several amino acids can elicit in- gestlon-response when they are at high concentrations and imbibed in filter paper is not considered of much signifi cance for, although free amino acids are very abundant in Invertebrates (Awapara, 1 9 6 2), they probably do not reach the concentrations needed to elicit ingestion response in Palythoa. The response to such amino acids suggests that they affect the receptors for ingestion response in some way, but it does not prove that these amino acids cause 193 ingestion response in Palythoa under natural conditions. The activation of the ingestion response closely parallels the activation of the mouth-opening response and might be controlled by the same receptors. Ingestion rep resents the culmination of the feeding reaction and is not controlled chemically in most coelenterates studied so far (Lenhoff, 1968a). In Palythoa, however, ingestion oc curs only in the presence of certain chemicals. With the methods used in this study it is not possible to know if the ingestion response needs a mechanical stimulus or not, for, unless one is provided (in the form of visible parti cles of paper) the response cannot be visualized. This could be studied later by kymographic recordings such as used by Batham & Pantin (1950) in their work on the phases of activity and its relation to external stimuli in Metrld- lum senile. The same features of the activators* molecules dis cussed in the control of the mouth-opening response are valid for the control of the ingestion response. Concisely, these features are: 1. The methylation of the SH of glutathione re duces the activity of that activator. 2. The receptors recognize the a-imino group of proline and do not respond if this is altered such as is the case in l-thiazolidine-4-carboxylic acid and the peptide glycyproline. 194 3. The presence of OH group In hydroxyproline con siderably diminishes the activity of the molecule. 4. Pipecolic acid and l-thiazolidine-4-carboxylic acid effectively compete with proline for the receptor site and cause Inhibition In the Ingestion response. The similarities and differences of these facts with those reported In the literature for other coelenter- ates were analyzed In the discussion on control of mouth- opening response (Chapter IV, p. 123). The ingestion-response, as the mouth-opening re sponse are Induced In Palythoa by the synergistic action of glutathione in 5 x 10-^M concentration and proline in 5 x 10 concentration. So far, glutathione has been shown to activate the feeding reaction In some coelenter- ates (Loomis, 1955> Lenhoff, 1961a,b; Lenhoff & Schneider- man, 1959 and Mackie & Boag, 1 9 6 3)• Proline has been shown to activate the feeding reaction of yet other coelenterates (Pulton, 1 9 6 3; Pardy & Lenhoff, 1 9 6 8). Marlseal & Lenhoff (1 9 6 8) found that both feeding activators induced the feed ing reaction of some Hawaiian scl.eractinian corals. Cy- phastrea ocelllna responded to proline at concentrations 10-7 ^ - 0 io”^M and to pipecolic acid at concentrations 10”® -3 -4 to 10 JM, they also gave a response to 10 reduced gluta thione The results with this coral offer the first well- documented case of a coelenterate giving a feeding response 195 to two different molecules, proline low concentration and glutathione high concentration. The response of Palythoa to these two feeding ac tivators differs from that reported for Cyphastrea (Mariscal & Lenhoff, 1 9 6 8) in that much higher concentrations of both activators are needed in order to provoke feeding reaction in Palythoa and, most important of all, in that both acti vators are necessary to elicit feeding behavior in nature, because, even though they can elicit a complete feeding reaction by themselves, the concentrations required are too high to be expected under natural circumstances. There is an intricate interaction between the two activators which results in inhibitions or enhancement of the ingestion re sponse, depending on the concentrations of glutathione and proline that are combined. Considerable attention has been given to the evolu tion of receptor sites in coelenterates. An activator molecule must be widely present in prey organisms and have properties that distinguish it from closely related sub stances. There are many such molecules and thus many pos sible activators. Glutathione and proline have been found to induce feeding behavior in most of the coelenterates studied so far. After a receptor site for a specific com pound has been acquired during evolution, further modifica tion of the receptor site itself might occur. Pulton (1963) 196 suggested that the evolution of a receptor for glutathione into one for the a-imino acid proline may have proceeded by means of slight structural changes in the receptor. He postulated this because one of the possible cyclized forms of glutathione in solution is close in structure to an a- imino acid (isherwood, 1959)• Because proline is also present in the fluids released from prey organisms, the change in structure of the receptor site was advantageous to some coelenterates and it was selected for against the glutathione receptor. Perhaps the coral Cyphastrea which responds to both proline and glutathione (Mariscal & Len hoff, 1 9 6 8) may represent a form retaining both receptor types. The zoanthid Palythoa which was also shown to re spond to proline and glutathione, may represent still another stage in the evolution of one receptor type into another, for it was seen that both activators are necessary for the reaction to occur and also that there is some very complex interaction between both molecules. Conclusions 1. Ingestion-response is chemically controlled in Palythoa townsleyl and cannot be elicited by mechanical stimuli. 2. Several amino acids and the tripeptide gluta thione imbibed in filter paper at concentrations of lO'^M cause ingestion response in significant proportion of the 197 polyps; but in solutions only proline or glutathione can elicit ingestion of untreated paper. 3. Lower concentrations of glutathione than of proline can induce ingestion, but only in a small propor tion of the polyps. 4. The speed of ingestion of glutathione paper is about 50 percent slower than the speed of ingestion of un treated paper when glutathione is in the medium. 5. The speed of ingestion of proline-paper is about 66 percent higher than the speed of ingestion of un treated paper when proline is in the medium. 6. The alteration of the SH group in glutathione, by the addition of a CH^ group reduced the proportion of polyps giving an ingestion response. 7. Receptors for ingestion have affinity for the a-imino group of proline. 8. Compounds with a ring of similar size to that of proline do not elicit ingestion if the imino group is substituted, however, they probably combine with the recep tor site, for they effectively inhibit ingestion of proline- paper . 9. Substitutions in the carboxyl group do not af fect the activity of the proline molecule 10. The addition of an OH group effectively dimin ishes the affinity of the molecule for the ingestion-chemo- receptors. 11. Some very Intricate interaction occurs between the two feeding activators. They cancel each other out to certain degree when in equimolar concentrations. In some concentrations one activator inhibits the response of the other; in other concentrations, it enhances it. In most combinations, the combined action of the activators affect the time required for ingestion. 12. In natural situations, both proline and gluta thione are necessary for the stimulation of the ingestion response. They are most effective when 5 x 10~^M gluta- thione is combined with 5 x 10 of proline. PART II FEEDING BEHAVIOR IN ZOANTHUS PACIFICA WALCH AND BOWERS 199 CHAPTER VI RESPONSE OP ZOANTHUS TO A VARIETY OP FOODS Zoanthus paclflca is a colonial animal common in surge pools, rocky shores and coral reefs of all Hawaiian island. In the areas where It Is found it is strikingly abundant, covering large expanses of the reefs and shores. Zoanthus belongs to the group of non-predatory coelenter- ates and seems to survive without any Intake of exogenous food. Von Holt & Von Holt (1968a) reported unpublished observations made by Goreau (1 9 6 7) and Goreau & Neuman (1 9 6 7) regarding, respectively, the semi- or complete in dependence of exogenous food, and the lack of typical mor phological digestive structures in some tropical Zoanthus Bpecies. Gohar (19^0), in a comprehensive study of the Xeniidae at Ghardaqa and the Red Sea, and later (Gohar, 19^8), on the alcyonarian Clavularla hamra, reported that these coelenterates would not swallow, or even seize, food of any kind, plant or animal, alive or dead, whole or cut. He also noticed that Clavularla had very poorly developed organs of digestion. 200 201 The only report of a Zoanthus species Ingesting particulate food Is that of Hadden (1 9 6 8), who found that Zoanthus soclatus could swallow pieces of frozen butterfly fish obtained from the same habitat where Zoanthus was found. Long and patient observations on Zoanthus paclflca failed to give clues as to how these zoanthlds feed. Study ing their feeding behavior, therefore, represented a chal lenge that could not be Ignored. Materials and Methods Zoanthus paclflca colonieB of 100 to 200 polyps were collected In the North Reef of Coconut Island, Oahu, Hawaii (Fig. 9)j and shipped to Los Angeles, California by Mr. Ralph L. Bowers. Upon arrival they were placed In well- aerated 5-gallon aquaria at 25 + 1.5°C and 33 °/oo salinity. Two hourB preceding an experiment, colonies were removed from the large aquarium and placed In fresh Instant Ocean contained in 250-ml finger bowls and were offered several materials cut in pieceB small enough to fit into the mouth. The only food found to give positive responses was fish (Certi-fresh breaded cod fishsticks). This was homog- 2 enized in a small volume of water and spotted on 0 . 5mm pieces of filter paper. The fact that many polyps re sponded with Ingestion of the paper indicated that some 202 water-soluble substance present in the fishsticks could elicit an ingestion response in Zoanthus. Since most solu ble substances known to elicit feeding behavior in coelen- terates are amino acids or the tripeptide glutathione, and because it was not easy to separate chromatographically the components of fishstick extract, all the naturally occur ring amino acids were spotted on filter paper in concentra tions 10-^M. Since taurine occurs in most invertebrates in very high concentration this amino acid was also tested. The tripeptide glutathione was tested because it is found in most living systems and is known to be a feeding activa tor for several coelenterates. 2 The test consisted of offering 0.5mm pieces of filter paper spotted with the experimental solution, while the polyps were placed in fresh Instant Ocean. Results The feeding response of Zoanthus paclfica is simi lar to that of the predatory coelenterate Palythoa towns- leyl and was described in the preface of this thesis, a. Response to live material 1. Artemla nauplii and adults There was no reaction to shimp moving about the polyps or colliding with their tentacles. 2. Syllid polychaetes, nemerteans and Tublfex worms No reaction was observed. 203 b. Response to dead material 1. Fishsticks (Certi-fresh breaded cod, cut in 0.5 x 1mm pieces). All the polyps tested showed a definite reaction of accepting the food. The response consisted of curling down the tentacles, raiBing the mouth, exposing the actinophar- ynx and ingesting the food. The process of Ingesting a small piece of fishstick occurred after the food had been kept on the exposed lining of the actinopharynx for 1 to 3 minutes, and took about 30 minutes. During swallowing the column of the polyps contracted repeatedly and some what rhythmically. On several occasions, after 12-24 hrs. ingestion of a fishstick piece, a brown-greenish mass was eliminated through the polyp’s mouth. It contained red granules and is regarded as a product of digestion. 2. Freshly killed Artemia sallna (adult shrimps cut in 0.5mm long pieces) When deposited on the peristome or on the margin of the oral disc, dead Artemia caused a weak lip formation In 10 percent of the polyps tested. The rest of the animals rejected the food by creating a ciliary current which moved the material to the margin. Here, several tentacles col lapsed, the edge with the Artemia sank and the food slid downward off the oral diBc. 204 3. Filamentous green and blue green algal mass Rejected by all the polyps tested. c. Response to treated filter paper 1. Imbibed in fishstick extract Water extracts of fishstick imbibed in filter paper elicit responses similar to those described for fishstick pieceB, indicating that this food must contain some soluble substance able to provoke a feeding reaction in Zoanthus paclflca. Frozen cod and freshly killed Gambusla affinis caused responses similar to those observed for fishsticks. 2. Imbibed in amino acids or glutathione The results presented in Table 29 show that gluta thione provokes a characteristic feeding reaction with for mation of a lip, which is localized and limited to a few tentacles at the beginning but becomes generalized shortly afterwards and encloses the paper. Of the polyps tested, 55 percent ingest the paper within 9 to 15 minutes. The amino acid glycine is also very effective in eliciting a feeding reaction. Sixty percent of the polyps offered fil ter paper immersed in glycine 10_1M solution ingest the paper (Fig. 45a,b). There are diminished responses with several amino acids, the names and structures of which are presented in Table 30. 205 TABLE 29 RESPONSE OP ZOANTHUS PACIFICA TO AMINO ACIDS AND A TRIPEPTIDE ON FILTER PAPER Number of Percentage of Polyps Giving Polyps Positive Responses Amino Acid* Tested + ++ +++ None 10 - - - Glycine 23 5 13 60 Alanine 10 20 - - Valine 10 - - - Leucine 10 - - - Isoleucine 10 - - - Serine 15 33 - - Threonine 10 - - - Taurine 10 20 20 - Cysteine 10 10 - - Cystine 10 - - - Methionine 10 - - - Glutamic acid 10 10 - - Aspartic acid 10 30 - - Lysine 10 - - - Hydroxylysine 10 - - - Arginine 10 - - - Histidine 10 - - - Phenylalanine 10 - - - Tyrosine 10 - - - Tryptophan 10 - - - Pro line 10 - - - Hydroxyproline 10 - - - Tripeptide Glutathione 20 20 45 55 *Amino acids arranged according to their chemical similari ties . + -H- +++ Lip formation; oral disc closes over paper; ingestion of paper. Figure 45. Response of Zoanthus paclflca to glycine. a. Initial response gl, generalized lip me, mouth closed mo, mouth opening ae, actinopharynx exposure b. after 10 minutes ae, actinopharynx exposure ip, paper ingested 206 Figure 45 ^ TABLE 30 RESPONSE OF ZOANTHUS PACIFICA TO A TRIPEPTIDE AND SEVERAL AMINO ACIDS AT A CONCENTRATION OF 10-1M, ABSORBED IN FILTER PAPERS 0.5mm2 IN SIZE Substance Structure Number of Polyps Tested Percentage + Response Type of Response Glutathione NHg-CH—CH2-CB2—CO—NH—CH—CO—NH—CHe—COOH COOH CReSH 20 100 +, ++, +++ Glycine NH^-CHe-COOH 23 78 +, ++, +++ Alanine NHs 1 CH3—CH—COOH 10 20 + Serine M e OH—CH2—CH—COOH 15 33 + Taurine NRs—CHs-CHsSO 3H 10 ko +, ++ Cysteine NHs HS-CHs-CH-COOH 10 10 + Glutamic acid NHs NHg—CHs—CRs—CH—COOH 0 10 10 + +Lip formation. | | Oral disc closes over paper. | | Ingestion of paper. r o o CO 209 The tripeptide glutathione, which elicits a com plete feeding reaction in Zoanthus, is formed by glycine, cysteine and glutamine. Therefore the observation that glycine is effective in 78 percent of the polyps suggests that the active part of the glutathione molecule is that corresponding to the glycyl radical. The other components, cysteine and glutamine, only caused reaction in 10 percent of the polyps. Serine and alanine were also effective in eliciting feeding reaction to a certain degree. Taurine, which is very similar in structure to the glycine analog o-aminomethanesulfonic acid, caused response in 40 percent of the polyps. Discussion The facts that frozen butterfly fish is the only material reported to cause ingestion response in another species of Zoanthus (Hadden, 1 9 6 8) and that only fishsticks (frozen cod) were found to activate an ingestion response in Zoanthus paciflca suggest that these animals may feed on particulate matter originating from fish that are preyed upon by other organisms that lose part of their food while tearing the prey to consume it. Reduced glutathione is found only in living organ isms, for the tripeptide gradually oxidizes to a biologi cally inactive form (Loomis, 1955) when exposed to the at mosphere. If it is assumed that Zoanthus recognizes gluta thione and not some other activator* in the pieces of fish tested* then* since the animal has never been observed to eat living material* the material it does eat must come from freshly-killed animals which still contain reduced glutathione when they reach the polyps of Zoanthus. On the other hand, it may be that the reaction of Zoanthus to glutathione does not have significance for the animal and only represents remnant behavior from Zoanthus1 ancestors which were predatory and fed on live prey. This possibil ity* however, is remote and cannot be tested unless the metabolic requirements of Zoanthus are fully understood and all the possible ways of fulfilling those requirements are studied in detail. Furthermore* in order to accept the fact that the activation of feeding response in Zoanthus represents remnant behavior only and has no significance in the feeding behavior of the animal* extensive field obser vations would be needed to establish whether Zoanthus does or does not ingest particulate matter under natural condi tions . Conclusions 1. Zoanthus paciflca does not react to live Artem ia nauplii* syllid polychaetes* nemerteans or Tublfex worms. 2. Zoanthus paclflca ingests frozen breaded cod. 3. The feeding reaction consists of lip formation* mouth opening and ingestion. 4. The feeding response is elicited by glutathione and to a lesser degree by glycine and some related amino acids. CHAPTER VII GENERAL CHARACTERISTICS OF THE FEEDING REACTION The tripeptide glutathione has been found to acti vate the feeding behavior of numerous coelenterates. Loomis (1955) reported that his findings on the ac tivation of feeding behavior in Hydra llttoralls by gluta thione confirmed unpublished observations made earlier by H. Park, who, while testing glutathione as an antiradiation compound, saw hydras' mouths opening. Glutathione Is also known to activate feeding be havior In other coelenterates. Lenhoff & Schneiderman (1959) found that glutathione elicited feeding behavior In Physalia physails and Campanularla flexulosa. Mackie & Boag (1 9 6 3) found reduced glutathione to elicit "writhing" activities of the gastrozoids from the siphonophore Namonia cara. The study of the feeding reaction in several Hawai ian corals (Mariscal & Lenhoff, 1 9 6 8) proved that Cyphas- trea ocellina, Poeclllopora damlcornls and Fungia scutarla respond to reduced glutathione and also to another activa tor, the heterocyclic imino acid proline. The response to glutathione has been extensively studied by Lenhoff (1961a,b). He concluded that the major parameter of measurement was the "duration of the feeding 212 213 response," I.e., the length of time that the animal's mouth remained open In the presence of*reduced glutathione. The "backbone" of the glutathione molecule was found essential to elicit the feeding response In Hydra (Loomis, 1955). Lenhoff 8c Bovalrd (1 9 6 1) found that glu tamic acid, under certain conditions, acted as a competi tive inhibitor of glutathione and concluded that the gluta myl radical of the tripeptide is also important for the activation of the feeding response. Zoanthus paclflca gave positive responses to gluta thione and glycine. Glycine has not been found so far to activate the feeding reaction in any coelenterate, and the large concentrations needed to elicit response in Zoanthus preclude its acceptance as a feeding activator in Zoanthus. However, very interesting aspects of the feeding behavior could be determined by studying reactions of Zoanthus pa- ciflca to this amino acid. Materials and Methods Two hours preceding an experiment, four colonies of approximately 100 polyps each were placed in 2 5 0-ml finger bowls containing fresh Instant Ocean. Stock solutions (10~^M) of glutathione and glycine were prepared and diluted with Instant Ocean to the concen trations indicated for each experiment; pH was corrected to that of Instant Ocean (usually 8.15) by the addition of 1 N NaOH. 214 The experimental solutions were placed In 250-ml finger bowls and the Zoanthus colonies moved from the In stant Ocean to the test solutions. Because in this trans fer from one bowl to the other many polyps contracted, only a fraction of polyps In each colony showed a feeding reac tion that could be analyzed. This explains the disparity in the number of polyps tested for each experiment. In order to gain some Insight Into the specificity of the reaction for glycine, an analog of this amino acid, a-aminomethanesulfonlc acid, was used In solution at con- -1 -5 centratlons from 10 to 10 -^M, or Imbibed in filter paper at concentration 1 0~^M. The interaction between glycine and its analog was studied by placing the polyps in a solution of the analog -1 2 and offering them glycine 10 M imbibed in 0 . 5mm pieces of filter paper or pipetted directly over the mouth. The ingestion response of Zoanthus paclflca was studied by offering to the polyps several amino acids and _ 1 p glutathione 10 M each imbibed in 0 .5mm pieces of filter paper. The speeds with which the polyps gave a first re sponse to the solutions, opened their mouths or ingested filter paper were timed with a Cletimer stop-watch and re corded. Standard deviations were calculated for each ex periment. Since the responses were elicited only when the chemical stimulus was provided, the proportion of polyps 215 responding to each solution Is highly significant. Results a. Response to glutathione Solutions of the tripeptide at concentrations 10*"'1 ’ _2 and 10 M cause the polyps to assume and remain in a par ticular position (Pig. 46) for as long as two to three hours after they are removed from the glutathione solution and placed in fresh Instant Ocean. In glutathione at a _-a concentration of 10 -’ M the mouth rises in concurrence with a general swelling of the tissue immediately surrounding it. The actinopharynx bulges out as it becomes exposed (Pig. 47a,b). In concentrations 10“^ to 10~^M the mouth opens round and wide (Table 31) while the animal contracts (Pig. 48). The upper, short, thin-walled region of the column, called the capitulum, becomes clearly visible and separated from the scapus. The optimum responses are ob tained with glutathione concentrations ranging from 10“^ to 10""^M (Pig. 49). In the first concentration, however, the mouth remains closed as the actinopharynx becomes ex posed and protrudes as bladder-like lobes. b. Response to glycine At lO^M concentration glycine causes the polyps to give a wide, round mouth opening (Pig. 45a and Table —2 3 2). At 10" M concentration mouth opening becomes small Figure 46 Figure 47 Figure 48 . Position assumed by Zoanthus paclflca after exposure in 10-- * - or 10“2M glutathione gp, position in glutathione np, normal position m, mouth _Q Actinopharynx exposure in 10 glutathione alj actinopharynx lobes a, side view of oral disc b, top view of oral disc Mouth-opening response in glutathione at 10-4 to 10- % m, mouth c, capitulum s, scapus 216 217 £• P F i g u r e 46 a . i . a F i g u r e 47 ra c s Figure 48 218 TABLE 31 RESPONSE OF ZOANTHUS PACIFICA TO REDUCED GLUTATHIONE Concentration Number of Polyps Tested Percentage of + Responses Type of + Response 10_1M 56 22 Actinopharynx exposed 10 "2M 80 26 Actinopharynx exposed io-3m 90 98 Actinopharynx exposed -4 10 124 82 Mouth opening io_5m 130 98 Mouth opening io "6m 80 5 Mouth opening io_7m 50 28 Mouth opening 10 “8m 100 5 Actinopharynx showing 10 "9m 50 0 Control (instant Ocean) 50 0 Figure 49. Response of Zoanthus paclflca to reduced glutathione. Control Is Instant Ocean. 219 Percent of polyps responding to glutathione 220 V s s s I S 1 * - * s + > c \ J I T S NO CV CO ON c 1 1 1 1 1 1 1 1 1 o o o o o o O o o o O 1— Y— V— 1 — V— * -----------Glutathione concentration Figure 49 and slit-like. The optimum concentration to cause mouth —2 opening is 10" M (Pig. 50), for, although the degree of mouth opening is larger in 10"^M glycine, at this concen tration only 48 percent of the polyps respond, compared to _2 100 percent of those responding to concentrations 10 and 10-^M. Table 32 shows that as the concentration of glycine decreases, the time needed for the initial response of the polyps increases slightly. This difference in the speed of reaction is particularly strong when the time for mouth to open in glycine 10-1M (1.68 + O .3 3 min.) is compared with the time required for mouth to open in glycine 10 M (4 08 + 3*05 min.). At this last concentration the actino pharynx remains exposed for 1 0 -1 8 minutes after the mouth -4 closes. At 10 M glycine concentrations only 11 percent of the polyps show exposure of the actinopharynx and below this concentration glycine has no visible effect on the _Q polyps. Glycine 10 causes a number of after effects in the polyps, such as elongation of the column to reach twice the length of the polyps in normal position, rhythmic peri staltic waves to run through the column and writhing of the tentacles in a somewhat rhythmic fashion. c. Response to a-aminomethanesulfonic acid, a glycine analog The response of the polyps to a-aminomethanesul- fonic acid (Table 3 2) parallels the response to glycine, but the analog is more effective than glycine at concentra- Figure 50 Response to Zoanthus paciflca to glycine and the glycine analog a- aminomethanesulfonic acid. CIO is Control Instant Ocean. 222 Percent of polyps exposing actinopharynx P s > co o © o o o o CIO 1 ° 1 0 _ 1 M 1 0 “ 1M 10“2M 3 o q £ 4 CD vn o -M M M 10'*Z fM 10_ZfM I I I 1 0 '" 5 M 5 D 4 513 M O oq o I on H o H* 4 CD 10“5M £S2 RESPONSE OF TABIE 32 Z0ANTHUS PACIFICA TO GLYCINE AND THE GLYCINE ANALOG O-AMI NOMETHANE SULFONIC ACID Concentration Number of Polyps Tested Time for Actino- pharynx to Become Exposed (min.) In Percentage of Polyps Time for Mouth to Open (min.) In Percentage of Polyps Control sea vater 70 - - - - Glycine 10_1M ^5 1.16 + 0.18* 48 1.68 + O.33 100 Glycine 10 2M 129 4.00 + 0.35 100 4.08 + 3.05 27 Glycine 10 3M 110 5.36 + 0.00 100 - 0 Glycine 10_4M 56 24.00 11 - 0 Glycine lO^M 4o - 0 - 0 Analog 10 ^ 20 1.00 + 0.10 50 1.50 + 0.20 50 Analog 10_2M 50 0.71 + 0.50 100 1.73 + O.83 100 Analog 10-3M 40 0.72 + 0.55 100 10.00 5 Analog 10 4M 45 1.76 + 0.35 60 10.00 5 Analog 10 5M 60 4.00 5 3.00 5 *Times are expressed as mean values + standard deviation. ro r o - l i - 225 -4 tion 10 M and the response to the analog In concentrations -2 -3 -4 10 , 10 J and 10 M is considerably faster than that ob served In glycine at the same concentrations. The optimum _2 concentration of analog to cause mouth opening Is 10 M. At this concentration the actinopharynx remains exposed for 2 .5 to 6 minutes after the mouth has closed. The Interaction between glycine and its analog (Table 3 3) enhances the activation of the mouth-opening re sponse. The analog at 10~^M concentration induces mouth opening in only 5 percent of the polyps when presented by itself. With glycine, however, it causes 100 percent re sponse if glycine is squirted over the peristome directly and 50 percent response if glycine is offered imbibed in filter paper. If the time required for mouth to open when either the amino acid or its analog are by themselves (Table 3 2) is compared with the time required for mouth to open when both glycine and the analog are offered together (Table 3 3), it becomes obvious that the combined action of glycine and a-aminomethanesulfonic acid shortens the time required for mouth to open. An important aspect of the interaction is the pres ence of lip formation which 1b due not to the Interaction between glycine and its analog (Table 3 3), but rather to the effect of glycine being pipetted over the peristome or of glycine-imbibed filter paper being dropped over the peri stome. Glycine squirted over peristome while the polyps TABLE 33 EFFECT OF THE INTERACTION BETWEEN GLYCINE AND ITS ANALOG O-AMINOMETHANESULFONIC ACID IN THE RESPONSE OF 20ANTHUS PACIFICA Solution Where Polyps* Placed Glycine 10-1M Applied hy Time to Form Lip (min.) In Percent age Polyps Time to Open Mouth (min.) In Percent age Polyps Time of Mouth Opening Sea vater Squirted over peristome 0.43 + 0.36 100 O.58 + O.33 30 Small, slit-like Analog 10 Squirted over peristome O.33 + 0.25 100 0.68 + O.36 100 Large, round Analog 10 2M Imbibed in filter paper 1.15 + 0.88 100 O.75 + 0.20 100 Small, slit-like Analog 10 ^ Imbibed in filter paper - 80 2.36 + I.38 50 large, round Analog 10_3M Untreated paper as control - - - 5 Small, slit-like *20 Polyps tested in each experiment. **Times are expressed as mean values + standard deviation. 227 are In Instant Ocean causes lip formation. Lip formation requires both mechanical and chemical stimuli, for there is no lip formation without mechanical action (solution or paper hitting the peristome) and chemical action (untreated paper does not elicit lip formation). d. Ingestion response Glutathione causes ingestion of paper in 55 percent of the polyps after an average of 11.30 minutes. Glycine causes ingestion in 60 percent of the polyps at a much faster rates 0.45 minutes (Table 34). Glycine analog causes ingestion In only 3° percent of the polyps and after 14.50 minutes. The small or nil standard deviation calcu lated for the time required for ingestion In glutathione, glycine and a-aminomethanesulfonic acid Indicates that the ingestion response is fairly uniform among the polyps. Glutamine and cyBteine, the other components of the glutathione molecule, do not elicit ingestion in Zoanthus (Pig 51)• When the paper Is not ingested, ciliary currents move it to the rim of tentacles, several of these collapse and the paper slides downward, off the oral disc. Table 34 shows that the time required to reject paper is affected by the presence of glutathione. It takes 12.00 + 10.00 minutes to reject glutathione-paper in circumstances that clean paper is rejected within 0.16 +0.08 minutes. Rejection of TABLE 3k- RESPONSE OF ZOANTHUS PACIFICA* TO FEEDING ACTIVATOR, ITS COMPONENTS AND A GLYCINE ANALOG, ABSORBED IN FILTER PARER OF 0.5mm2 IN SIZE Condition of Paper Time for Oral Disc to Close over Paper (min.) Percentage of Polyps Closing Time for Ingestion of Paper (min.) Percentage of Polyps Ingesting Time for Rejec tion of Paper (min.) Untreated - - - - 0.08 + 0.16*** Glutathione 10_1M 0.75 + 0.20** 100 11.50 + 1.49 55 10.00 - 12.00 Glycine 10-1M 5.75 + 2.96 100 0.45 + 0.00 60 0.12 - 0.16 Glycine analog 10 1M 1.02 + 0.00 100 i4.8o + 0.00 50 0.05 - 0.25 Glutamine 10 "Si 2.00 10 - - 5.00 - 4.00 Cysteine 10 1.00 10 - - 5.00 - 4.00 *20 Polyps tested in each experiment. **Times are expressed as mean values + standard deviation. ***Times of rejection are expressed as the range. Figure 51. Response of Zoanthus paclfica to feeding activator, its components and a glycine 2 analog, absorbed in filter paper of 0.5mm in size. Control is Instant Ocean. 229 Figure 51 Control glutathione glycine glycine analog glutamine cysteine Percent of polyps ingesting paper ro .p- ca co o o o o o o o > -■" • — ■ ■ ■ — ■ I o ;IiI«IIIIt§It O O [V> uo o 231 paper Imbibed In glycine or Its analog takes times similar to those required for rejection of untreated paper. Discussion The response of Zoanthus paclflca to solutions of glycine at concentrations 10 or higher Is very similar to that of Palythoa to comparable concentrations of proline. The response of glutathione, however, differs in that In Zoanthus the higher concentrations of the tripeptide stimu late reaction in a very small proportion of the polyps and In Palythoa most polyps give positive responses. The opti mum concentrations of glutathione to elicit mouth opening -4 -5 in Zoanthus were 10 and 10 ^M. These concentrations were effective in Palythoa only in the presence of proline. Glutathione 10 was also found to be effective in eliciting a full response in Hydra llttoralls (Lenhoff, 1961a). Glutathione (10~^M) causes slight mouth opening in Cyphastrea ocellina, Fungia scutarla and Pocillopora dami- cornls (Mariscal & Lenhoff, 1 9 6 8). All of these corals re spond also to the amino acid proline. Wyman (1 9 6 5) re ported that the hydroid Corymorpha responds to glutathione, _-a glycine, serine and cysteine all in concentrations 10 and squirted directly over the distal tentacles of the hy droid. He failed, however, to describe what he called feed ing reaction beyond saying that the polyps gave a closing reaction. Unfortunately the high concentrations he used 232 and the lack of information as to how the experiments were controlled or how many experimental polyps were used* di minish the importance of his findings. Corymorpha may have a feeding reaction cc-ntrolled by glutathione and feeding receptors with high affinity for glycine and cysteine but more information is needed to establish these possibilities. It is conceivable that glycine and glutathione may act together in Zoanthus in a way similar to that described for proline and glutathione in the activation of feeding behavior in Palythoa. This, however, is not very likely for glutathione alone elicits a typical feeding reaction at concentrations which can be expected in natural conditions and would not need the reinforcement of another activator. Lenhoff & Bovaird (1 9 6 1) demonstrated that glutamine and glutamic acid are the only amino acids that compete with glutathione and cause an inhibition in the response of Hydra llttoralis to glutathione. They concluded that the recep tors of Hydra had a high affinity for the glutamyl part of the tripeptide molecule. The fact that Zoanthus responds to glycine could mean that the receptors for feeding response in this animal may have high affinity for the glycyl part of the gluta thione molecule. More information is needed to establish this possibility. The fact that glycine is effective in eliciting feeding behavior only at concentrations above 233 - • a 10 “’ M precludes Its consideration as a feeding activator by itself. Assuming that -Zo an thus cannot capture live prey, the fact that lip formation and ingestion responses are present suggests strongly that this animal has the ability to feed on particulate matter if the matter contains reduced gluta thione. Since glutathione oxidizes within 2 hours after exposure to the atmosphere (Loomis, 1955)» Zoanthus must feed on particles of food that proceed from recently killed prey which still contain enough glutathione to induce the feeding response. _Q Glutathione and glycine in concentrations of 10 and higher continue to affect the behavior of the polyps long after they have been replaced in fresh Instant Ocean. This kind of behavior is very similar to that described by Batham & Pantin (1950) in the sea anemone Metrldium, where the ingestion of food is followed by a sequence of phases including, among others, expansion of the disc and elonga tion of the column. Parker (1 8 9 6) reported that when Me trldium1 s tentacles are touched by food, the esophagus fre quently shows peristaltic contractions and the sphincter of the mouth closes. Peristaltic contractions were seen in Zoanthus when exposed to glycine 10-^M concentrations. These effects of glycine and glutathione on Zoanthus suggest that the animal may possess some mechanism to absorb small 234 molecules directly from solution. The examination of this hypothesis is the subject of the next chapter. Conclusions 1. Zoanthus paclflca shows a feeding reaction very similar to that described for the predatory coelenterate Palythoa. This reaction includes lip formation, mouth open ing and ingestion of solid material. 2. The feeding reaction of Zoanthus paclflca is initiated and controlled by glutathione. 3. The optimum concentrations of glutathione to activate feeding response are 10“^ to 10-^M. 4. Glutathione 10-^M elicits exposure of the ac- tinopharynx and important columnar movements. 3. Mouth opening response is elicited at gluta- -4 -6 thione concentrations 10 and 10 6. Glycine and a-aminomethanesulfonic acid, a gly cine analog, elicit mouth-opening response when in concen trations 10“1 or 10-2M. 7. The interaction between the amino acid and its analog does not Bhow competitive inhibition, but rather an enhancement of the reaction to the analog in concentration of 10 "2M. 8. Glycine may represent the active portion of the glutathione molecule. 235 9. Glutathione affects the speed with which the polyps reject extraneous materials from the oral disc. 10. Lip formation is controlled by mechanical and chenucal stimuli. 11. Mouth opening is controlled chemically. 12. High concentrations of glycine and glutathione cause important after effects in the polyps which last for 2 hours after these have been removed from the solutions. CHAPTER VIII ABILITY OP ZOANTHUS TO TAKE UP GLYCINE FROM SOLUTION AND UTILIZATION OP THIS AMINO ACID The effect of glycine on the behavior of Zoanthus paclfica raises the question of the possibility of this animal withdrawing the amino acid from solution in the me dium. In fact, the ability to remove amino acids and other small organic compounds from dilute solution is widespread among marine invertebrates. Stephens & Schinske (1 9 6 1) tested ten phyla of marine invertebrates and concluded that any soft bodied marine invertebrate exposed to an amino acid such as glycine or phenylalanine at concentrations be- tween 10 J and 10 M per liter showed the capacity to re move it quite rapidly from solution. If Zoanthus were capable of taking up significant amounts of glycine from the medium this would represent a possible way in which an animal that does not feed on zoo- plankton might incorporate nitrogen into its system. Materials and Methods _14 a. Incubation with glycine C _lli Glycine C was added to a beaker of 100 ml artifi cial sea water to give a final concentration of 2 .1 6 mg/ml 236 237 — P (2 87 x 10 M). A cluster of 5 to 10 zoanthid polyps was placed In the solution and Incubated at 28 + 0.5°C In a plexiglass water bath above four 40-watt Sylvania fluores cent lights. Incident Illumination was approximately 560 foot candles. After Incubation for times ranging from 2 to 13 hours, animals were removed, rinsed In 14 to 17 brief serial changes of Instant Ocean and analyzed as described below. b. Analysis of labelled zoanthlds Fractionation. Labelled zoanthlds were homogenized to a fine suspension In 95 percent ethanol using mortar and pestle. The suspension was washed Into a test tube and the volume adjusted to 10 ml. A 0.1 ml aliquot was removed for protein nitrogen determination (Lowry, Rosebrough, Farr & Randall, 1951)• The suspension was centrifuged (2 min. at 1,500 rpm, International Clinical Centrifuge) and the alco holic supernatant withdrawn. The Insoluble residue was further extracted with 0.5 ml portions of 80 percent and 50 percent ethanol and finally with hot distilled water, until a gray-white residue was obtained. Supernatant and extracts were combined and stored at -15°C. Insoluble residue was hydrolyzed In a sealed tube with 6n HC1 at 108°C for 24 hours. Chromatography. Ethanolic extracts and neutralized hydrolyzateB were analyzed by two-dimensional chromatography 238 on Whatman No. 4 paper (1 8 5 in x 22^ in) using phenoliwater (7 2:2 8; w/w) in the first dimension and butanols propionic acid:water (1246:620:884; v/v/v) in the second dimension. Papers were exposed to Kodak blue-sensitive "No-screen” medical x-ray film for 2-l4 days (Benson, Bassham, Calvin, Goodale, Hass & Stepka, 1950)• Assay of radioactivity. Fluid samples of known volume were dried on planchets and assayed with a thin end window Geiger tube of Nuclear-Chicago gas flow detector (Model 470), with correction for background. Radioactive spots on paper chromatograms were assayed with an end window probe. Radioactivity of each spot was expressed as a percentage of the total activity detected in the paper. Identification of radioactive unknowns. Tentative identifications were suggested (l) by comparison of un knowns with the chromatographic maps of Bassham & Calvin (1 9 5 7); (2) by elution and co-chromatography with authentic labelled and nonradioactive compounds; and (3), in the case of amino acids, by the appropriate use of ninhydrin (l per cent in acetone). c. Analysis of coelenteric bacteria Prior to each experiment, a sample of coelenteric fluid was removed and the number of bacteria in the sample determined with the aid of a hemocytometer. A sample of 239 coelenteric fluid was introduced to 100 ml of sterile In stant Ocean enriched with 0.5 gm Bactopeptone and 0.1 gm dextrose, and glycine“^C (l |ic/ml; approximately 0 .1 9 mg/ml). Growth was monitored daily by cell counts of sam ples. At the onset of stationary phase (16 to 20 hours), the suspension was centrifuged for 30 minutes at 1 2 ,0 0 0 rpm (Servall). The bacterial pellet obtained was washed in Instant Ocean and analyzed as described for zoanthlds. Results a. Animal Table 35 indicates the time course removal of gly- _l4 cine C from solution and its accumulation by the animals. ill Of the total C available, the animals acquired a maximum of 65 percent after 13 hours'incubation (Pig. 52). The relative specific activity of the animals also increased appreciably during thiB time, more than two-fold of its original value. Analysis of the sea water In which the animals were rinsed after two hours' Incubation revealed nearly 30 percent of the total glycine in the medium, and almost 25 percent after 6 hours. Whether or not this exoge nous glycine represents material from the surface of the animals or the coelenteric fluid, or efflux of Intracellular glycine, cannot be determined from the data acquired. Fur ther, It was noted In one control experiment, consisting of the Incubation mixture without the animals (Table 3 6), that TABLE 35 14 __ UPTAKE OF GLYCINE- C FROM SOLUTION BY ZOANTHUS PACIFICA Incubation Time (hrs.) In Medium Initial Final ( cpm) (cpm) In Animals (cpm) Percent In Wash (cpm) Percent Recovery Percent Specific Activity of Animals (cpm/mg protein N) 2 1,365,800 803,200 210,600 15.4 399,600 29.2 103.1 432 6 1,1+72,000 24- 9,400 614,100 41.5 368,100 25.0 83.5 787 10 1,148,700 450,500 544,900 47.5 49,700 4.3 91.0 865 13 1,861,000 182,900 1,214,700 65.O 86,900 4.6 79.4 1.084 ro o -14 Figure 52. Uptake of glycine C from solu tion of Zoanthus paclflca. 241 Percent of radioactivity in Zoanthus oo r t - OO v_n StTS 100 TABLE 36 RADIOACTIVITY LOST IN CONTROLS OP GLYCINE-^C, WITHOUT ANIMALS In Medium Incubation Time Initial Pinal Lost (hrs.) (cpm) (cpm) (cpm) % 10 1 7 8 ,9 0 0 164,500 14,400 7 .2 10 195,400 1 8 6 ,9 0 0 8 ,5 0 0 4.2 r o -!= • OO 244 i -14 4 to 7 percent of the initial glycine C could not be re covered after 10 hours. This suggests that a fraction of the glycine is removed from solution by some means other than accumulation by the animals. Once it was found that Zoanthus has the ability to take up glycine from solution, another question arose: Is the animal able to use this glycine? And, if so, how does the animal utilize this amino acid? It was found that, regardless of the length of in- 14 cubation, about 90 percent of the total C taken up by the zoanthlds was alcohol-soluble. Radiochromatographic analy sis of this fraction after 2, 6 and 10 hours revealed up to 8 different labelled products. A representative radiochro matogram of the compounds which were detected is shown in Pig* 53* The major intracellular radioactive compounds -14 / - were identified as glycine C and lipid. Of the other 6 labelled compounds, only one (compound # 3) exceeded 5 per- 14 cent of the total alcohol soluble C. The others were present in small amounts and are grouped together in Table 37 for convenience. This shows that the quantitative re lationships between these compounds varied with incubation time. The amount of glycine decreased from 81 percent to 48 percent of the total alcohol soluble material over 10 hours. There was a concomitant increase in the lipid frac tion from 1.6 percent to 42 percent during this time (Pig. 54). Compound #3 increased to 21 percent after 6 hours but Figure 5 3. Radiochromatogram of the alcohol-soluble fraction of Zoanthus after six hours’ in cubation with glycine_1^C. FW - Pheno1-water BPAW - Butanol propionic-acid water 245 246 “ , , d O ea 3 3 < o « = » 2 (— ~^) GLYCINE "I ORIGIN 1 6 <=» O FW Figure 53 247 TABLE 37 14 DISTRIBUTION OP C IN ALCOHOL-SOLUBLE FRACTION OF ZOANTHUS PACIFICA AS DETERMINED BY RADIOCHROMATOGRAPHY Radioactivity Percentage Incubation Time (hrs.) As Glycine As Lipid As Unknown #3 As Unknowns #1,2,4,3,6 2 8 1 .0 1.6 1.5 15.9 (#6 absent) 6 66.0 5.2 21.7 7.1 10 48.1 42.0 6.5 3.-4 (#1,2,4. absent) I 14 Figure 54. Distribution of C in alcohol-soluble fraction of Zo an thus as determined by- radiochromatography . 248 Percent radioactivit 249 100 30 Glycine 60 Lipid 20 Compound 0 8 2 6 10 0 Incubation time ( hrs ) Figure 5^ 250 then decreased to 6 .5 percent after 10 hours. The decrease of compound # 3 coincided with the sharp Increment of lipid. This suggests that compound # 3 may represent an intermediate in the synthesis of lipid. Co-chromatograms with glycerol and glycolic acid, two possible intermediates, revealed that compound # 3 is not glycerol nor glycolic acid. The fact that only a small amount of compound was present in the chromatograms prevented further tests to determine its nature. The alcohol-insoluble fraction, which represented 14 10 percent of the total C incorporated by the animal, was 14 hydrolyzed and chromatographed. It was found that C had been incorporated into 5 different compounds which occupy the conventional position of amino acids and gave a nin- hydrin-positive test. This indicated that 10 percent of 14 the C taken up by the animals was incorporated into pro teins . b. Coelenteric bacteria The coelenteric fluid of Zoanthus paciflca contains diverse unicellular organisms. The most numerous of these is an unidentified species of bacterium, usually occurring in l-2n motile rods, but occasionally forming chains. Di rect cell counts in a hemacytometer gave 9575 x 10^ cells/ml which is 2 -3 orders of magnitude higher than concentrations found in marine waters (Zobell & Peltham, 1938). To deter- 251 mine if this bacterial flora might be accumulating glycine from solution to a significant extent, In Inoculum of co elenteric fluid was cultured with glyclne-^^C for 16 to 20 hours. Results are shown In Table 3 8. In two trialB bac teria grew rapidly and removed 5 percent or less of the to tal labelled glycine In the medium. Table 39 shows distrib- 14 utlon of C In the bacterial fractions as determined by chromatographic analysis. Almost 90 percent of the total 14 C In the alcohol-soluble fraction was associated with the lipid fraction. The remainder was nearly all unincorporated _lli glycine C. The converse was observed In the hydrolyzed . alcohol-insoluble fraction. The major labelled constituent _l4 was glycine C while the labelled lipid accounted for only 15 percent of the total Insoluble activity. Since the con centration of bacteria was higher in the culture than in the coelenteric fluid and the uptake of glycine was only 5 percent or less, it was concluded that activities of the bacteria ln_ situ did not significantly affect the results of uptake of glycine by Zoanthus. This conclusion was based on the assumption that the coelenteric bacteria behaved similarly in the coelenteron and in culture. To determine whether bacteria present in Zoanthus were important in the uptake of glycine, an alternate ex periment had been planned. It consisted of studying glycine uptake in polyps without bacteria. To eliminate the bac teria the polyps were placed in an antibiotic mixture con- TABLE 38 UPTAKE OP GLYCINE-l4C BY BACTERIA AFTER 16 HOURS’ INCUBATION AND GROWTH Initial Number of Bacteria (X 104) per liter Final Number of Bacteria (X 104) Generation Time (hrs.) Initial ^C in Medium (cpm) 14C in (cpm) Bacteria % 375 34.250 4.5 170.800 6894 4 130 2.950 4.5 37.900 2268 5 r o V J l ro 253 TABLE 39 DISTRIBUTION OP l2|C IN BACTERIA AFTER 16 HOURS GROWTH AND INCUBATION B S B a s a s g a s c a j r 1 i i » Radioactivity Percentage Fraction As Glycine As Lipid As Unknown Alcohol-in soluble 9-7 88.2 2.1 Alcohol-insoluble 79.6 14.9 5.1 taining polymixin-B (20 fig), penicillin (100 units/ml) and tetracycline (20 ng). Such mixture was used successfully by Davis (personal communication) in Hydra, but it proved lethal to the zoanthids and the experiment could not be done. Discussion The experiments reported here, although preliminary, _l4 show that Zoanthus paclflca accumulates glycine C from l4 solution and incorporates a large proportion of the C into lipid. The organisms seem to harbor a large bacterial _lii flora which, in culture, can also accumulate glycine C from solution but to a lesser extent than the "host" zoan- thid. The significance of these observations may be related to the nutrition and metabolism of Zoanthus but much more investigation is required. Stephens (1963) demonstrated that the marine annelid Clymenella torquata can remove amino acids, including glycine, from solution and use these substrates in respiratory metabolism, pointing out that several criteria should be observed if such results are to be related meaningfully to the nutritional metabolism of the organism. These criteria include (l) the demonstration that the organism is physiologically capable of dealing with the organic material in dilute solution; (2) the pres ence of available organic substrate in the habitat of the organism; (3) some quantitative data on rate of uptake to 255 lend support to the significance of the process. In the present study these criteria were satisfied only in an in direct and tentative way. If it is assumed that no conventional overt feeding behavior is displayed under natural conditions during the day or night, there are two possibilities which might ex plain "feeding" by zoanthids. 1. The possibility that zoanthids can accumulate organic material from solution has been tested in a prelim inary way in this study. Although it was demonstrated that the organisms can take up glycine from dilute solution and incorporate it into lipid, there is no evidence yet that this is a significant nutritional process under natural con ditions. The potential contribution of accumulation of or ganic compounds to the metabolic requirements of Zoanthus could be realized only if the metabolic requirements of the animal were known and if the organic compounds investigated could be found in significant amounts in the habitat of Zoanthus. Unfortunately there are no measurements on the metabolic rate of Zoanthus and the amount of glycine present in the habitat of Zoanthus is unknown. However, some idea of the possible content of this amino acid in the environ ment might be drawn from reports in the literature. If the value found by Belser (1959) for inshore waters (777 mg of glycine/l) is combined with the value found by Stephens (1963) for mud flat interstitial waters (723 mg of glycine/ 256 l), they average 0.750 mg of glycine/l. This represents around one-third of the glycine concentration used in this study. However, the rate of uptake may compensate for a reduced amount of amino acid in solution and the process be significant to the animal's nutrition. 2. The possibility that zoanthids harbor a large bacterial flora within the coelenteron and perhaps feed on them has not been tested but certainly represents a source of nutrients which would preclude manifest feeding behavior. Although Zoanthus does not possess the very fine filtering mechanism required to catch individual suspended bacteria, it could catch them if the bacteria were clumped in aggre gates. According to Zobell (1 9 3 6), most bacteria are peri- phytes or epiphytes which grow attached to particles of organic matter, plankton or other surfaces, forming aggre gates that could be ingested by Zoanthus. Conclusions 1. Zoanthus paclflca can accumulate about 65 per- _l4 cent of the glycine C from a solution with an initial concentration of 2.l6 mg/ml (2 .8 7 x 10~^M). 2. After 13 hours, a large proportion of the gly- -14 14 cine C is metabolized and the C utilized in synthesis of protein and lipid. 3. The animals harbor a coelenteric bacterial flora which can be cultured in vitro. Cultured bacteria accumu- 257 _ nil late some glycine C and also utilize it in the synthesis of lipid and protein, but the quantity is too small as to suggest that the animal itself is capable of taking up glycine. 4. The possible importance of glycine uptake to nutrition of zoanthids is discussed and compared to an al ternative hypothesis: the role of bacteria as food. CHAPTER IX IMPORTANCE OP ZOOXANTHELLAE FOR ZOANTHUS1 WELL-BEING Intracellular symbiotic algae called zooxanthellae often have been thought to fulfill part of the nutritional requirements of their coelenterate hosts. Gohar (1948) concluded that as the Xeniidae (Gohar, 1940), Clavularla hamra depended almost entirely on their zooxanthellae for the supply of nutritive substances. Direct evidence for the role of algae on their coelenterate hosts appeared only 10 years later with the work of Muscatine (Muscatine & Hand, 1958 and Muscatine & Lenhoff, 19 6 3)• Through radioauto- 14 graphs he first observed that C-labeled material fixed by the photosynthesizing zooxanthellae of the sea anemone An- topleura elegantlsslma waB passed on to and incorporated by the host anemone cells (Muscatine & Hand, 1958). Muscatine & Lenhoff (1965a,b) determined that the growth, survival and excretion rates of Chlorohydra vlrldlsslma are affected by the symbiotic algae. Muscatine (1 9 6 7) reported that the zooxanthellae of a subtropical, subtidal zoanthid excrete substantial quan- 14 titles of glycerol when allowed to photo synthesize Na2C 0^ 258 259 in the presence of their host tissue homogenate. Evidence for assimilation of zooxanthellae photo- synthate by Zoanthus flos marlnus was given by Von Holt & Von Holt (1968a,b). Prom the preceding discussion it is clear that the symbiotic algae may have an important role in the nutrition of the coelenterate host. If this host does not feed on zooplankton, as is the case with Zoanthus, the animal may be affected drastically by any loss of zooxanthellae and may have developed mechanisms whereby it insures the main tenance of its symbionts. Indirect evidence of this was found by Goreau (1964), who observed that of all the coelen terate species affected by the Hurricane Flora, the only form that could retain its zooxanthellae was Zoanthus soclatus. Any study on the nutrition of Zoanthus therefore will need to investigate the relationships of the animal with its algal symbionts. This thesis deals only with feeding behavior in Zoanthus and touches aspects of nutri tion only wyen they are needed to corroborate some sugges tive information obtained while studying behavior. Materials and Methods Aposymbiotic polyps (Pig. 55*0 (animals without zooxanthellae) were obtained accidentally when the tempera- Figure 55- Response of Zoanthus paclflca in glycine 10-1M. a. Mouth-opening response gl, generalized lip me, mouth closed mo, mouth opening ae, actinopharynax exposure b. Ingestion-response ae, actinopharynx exposure ip, paper ingested 260 262 ture was raiBed to 30°C after power failure during the summer of 1969. Regression of the animals was studied by measuring size of zoanthids at different periods of time. Size was determined In contracted polyps as diameter of the oral disc. This direct measurement of the oral disc Is not a good Indication of size, especially for coelenterates that contract to different degrees. However, In colonial ani mals which cannot be separated from the subBtratum to which they are attached and which must be handled carefully If they are going to remain alive, there was no other practical method of measuring size. Results Zoanthus paclflca began losing its symbiotic algae upon sudden Increase In the temperature from 26.5°C to 30°C. Although this was corrected within 2 days, the polyps continued to extrude zooxanthellae until, at the end of three months, they were completely bleached. The extrusion of zooxanthellae occurred In uniform, polyp-like pellets approximately 0 .25mm In largest diameter which can be seen in Pig. 55b. Normal and aposymbiotic colonies were kept In the same aquarium and, while the former did not show any ap parent change In size, the aposymbiotic colonies regressed very rapidly. The tentacles disappeared, the polyps re 263 mained permanently contracted and lost the ability to rid the oral disc from extraneous materials such as sand grains and algal masses. The diameter of the oral disc was measured in 39 polyps belonging to 4 colonies (Table 40). It was found that in 11 days the oral-disc diameter of Zoanthus paclflca decreased between 1.1 and 1.7mm. Only 2 polyps showed no change in oral-disc diameter over an 11-day period. In view of the large symbiotic flora and the strik ing effect that its loss causes on Zoanthus, the question may be raised as to whether the zooxanthellae have any ef fect on the feeding behavior elicited by glutathione and glycine. Aposymbiotic polyps, placed in solutions of -5 -1 glutathione 10 -'M or glycine 10 M, gave mouth-opening re sponses similar to those described for normal polyps. Heat was the most effective method found for ridding Anthopleura elegantisslma of Its symbiotic algae (Buchsbaum, 1968). She found that the anemones would lose their zoo xanthellae when exposed to temperatures ranging from 30 to 32°C. Higher temperatures killed the anemones. The loss of zooxanthellae by Zoanthus paclflca In temperatures of 30°C agrees well with the occurrence of the same phenomenon in the other coelenterates mentioned above. One important observation made was that a colony of Zoanthus danae maintained under conditions similar to those of Zoan thus paclflca (they were In the same aquarium) did not lose TABLE 1+0 CHANGES IN SIZE* OF ORAL DISC IN STARVED-APOSYMBIOTIC POLYPS OF ZOANTHUS PACIFICA OVER A PERIOD OF 11 DAYS Colony Number Number of Polyps Tested Initial Measurement (mm) Final Measurement (mm) Change in Size (mm) 1 19 2.0 - 2.5 - 5.5 1.0 - 1.1 + - 2.5 0.5 - 1.1 + 0.0** - 2 12 1.0 - 2.2 - 3.0 0.3 - 0.9 - 2.0 0.0 - 1.3 + 0.0 - 2.0 3 5 o J r I r o v • K ' V 1 O • 1.0 - 1.1+ - 2.5 1.0 - 1.8 + 0.0 - 2.5 1 + 3 2.0 - 3.0 - k.O 1.0 - 1.5 - 2.0 0.0 - 1.5 + 0.0 - 2.5 l - k (combined) 39 1.0 - 2.5 - b.o 0.3 - 1.2 - 2.5 0.0 - 1.3 + 0.1+ - 2.5 *Sizes are expressed as mean values bounded by extremes. **Changes in size are expressed as mean values with standard deviations bounded by extremes. t795 265 the algal symbionts and remained unchanged during the pe riod In which Zoanthus paclflca was bleaching and diminish ing in size. This observation could indicate that the zoo xanthellae of Zoanthus danae resist high temperatures bet ter than the symbiotic algae of Zoanthus paclflca, or that the zoanthids themselves have a different ability in con trolling their symbiotic populations. If this second pos sibility is true, the assumption must be made that zoo xanthellae of different zoanthids are conspecific or at least have similar tolerances to stress conditions. The regulation of the zooxanthellar population will be shown not only by loss of the symbionts, but also by an increase of them beyond the normal population sizes found under natural conditions When Zoanthus paclflca is kept under strong illumination, its zooxanthellar population increases considerably, giving the polyps a much darker color than that which they show in their natural habitat (compare Figs. 45 and 55a). Since Zoanthus danae maintained a constant Bhade of color while exposed to the same condi tions under which Zoanthus paclflca showed striking changes, the former species is believed to have a better control of its algal symbiontB. Goreau (1964) suggested that Zoanthus soclatus presents a higher resistance to losing its zoo xanthellae than other species of coelenterates in the Jamai can reef where his studies were made. This differential power of zooxanthellar retention by diverse coelenterates 266 could also be Interpreted as a differential ability to control their population of algal symbionts. The most Important observation In relation to zoo xanthellae and Zoanthus paclflca is that when no exogenous food Is provided and the zooxanthellae are lost the polyps decrease rapidly In size and eventually become smothered with filamentous algae and sediment that settles over their oral disc and Is not removed. Colonies of Zoanthus that had not bleached and had been kept In the same aquarium with the aposymbiotic zoanthids while these were bleaching, showed no apparent changes In size. This Is Interpreted as indirect evidence that ZoanthuB paclflca may be nutrition ally dependent on the photosynthetic products of Its zoo xanthellae . Conclusions 1. Zoanthus paclflca was not able to maintain a constant zooxanthellar population under the condition kept in the laboratory. 2. Temperatures of 30°C cause Zoanthus paclflca to begin losing its zooxanthellae. 3. Once extrusion of zooxanthellae has begun it proceeds to termination even when the condition that caused it is connected. 4. Zooxanthellae are extruded in pellets of defi nite polyp-like shape. 267 5. Extrusion proceeds until the animal becomes completely white and no zooxanthellae can be found. 6. Loss of the algal symbiontB causes a marked regression in the polyps of Zoanthus paclflca and eventually leads to death of the animal. 7- The absence of zooxanthellae does not affect the activation of mouth opening by glutathione or glycine. PART III GENERAL CONSIDERATIONS 268 CHAPTER X DISCUSSION Most coelenterates are carnivores feeding on zoo- plankton which they capture with their nematocysts. Numer ous studies have been made on the ways in which predatory coelenterates capture their prey, notably those of Vogt (1854) in the syphonophore Nanomia; Ewer (19^7) on Hydra; Mackie & Boag (1 9 6 3) on Physalla physallB; and Pardy & Len- hoff (1 9 6 8) on Pennarla tlarella. Most of the studies have dealt with the use of the nematocysts in capturing the prey and all agree that numerous nematocysts1 tubes pierce the prey immobilizing it so that the tentacles may bring it to the mouth, which opens and the prey is ingested. Palythoa differs from these other predatory coelenterates in that it only discharges one or two nematocysts on the prey; it does not paralyze it, but rather creates a lip with part of the tentacles and oral disc. This lip secures the prey and pushes it toward the mouth. Variations in the way in which the coelenterates carry the food to the mouth after the prey has been cap tured include: coordinated movements of the tentacles to bring food to the mouth (Ewer, 19^7) in Hydra; bending of the oral cone toward the nauplii and spreading of the mouth 269 270 around the prey to swallow It in the hydroid Cordylophora (Pulton, 1 9 6 3) and the hydroid Pennarla (Pardy & Lenhoff, 1968). Palythoa shows a response similar to that of some sea anemones such as Sagartla, reported by Vogt (1854) to enclose the food in a lip and push it to the mouth, and, Anemonla sulcata, reported by Pantin & Pantin (19^3), to clasp dead food and push it toward the mouth in a similar way. It is interesting that coelenterates which are closer to each other phylogenetically show very similar re sponses which in turn differ from those present in groups phylogenetically more distantly related. This suggests that behavioral patterns are probably the product of evolu tion in the different coelenterate groups and are not in dependently acquired by each animal. The efficiency and speed with which Palythoa cap tures prey seems very low when compared to the activities of sea anemones living in the same habitat. However, Paly thoa compensates its Inability to capture more than one prey in the natural low densities of zooplankton by leaving a group of tentacles protruding immediately after closing over the prey. This group of tentacles insures the possi bility of consecutive prey captures. To a certain degree, this response can be compared to the behavioral modifica tion elicited in Hydra by tyrosine, called neck formation (Blanquet & Lenhoff, 1968). This reaction has the adaptive 271 value of allowing Hydra to capture new prey without losing some of the food particles being circulated In the coelen- teron and proceeding from digestion of prey captured pre viously. Since Palythoa cannot paralyze prey by the use of nematocysts It must rapidly close over the food caught without waiting for other prey to collide with its tenta cles. The closed oral disc could be compared to the neck that keeps food inside Hydra, and the group of tentacles protruding represent the adaptation to a need for capturing more prey. Other coelenterates do not capture live prey but have an entirely different feeding method. They are sus pension feeders which use the secretion of mucus to retain suspended particles as well as to carry them to the mouth, such aB occurs in the scleractinian coral Fungla (Abe, 1938), the Jelly fish Aurelia aurlta (Southward, 1955) and in the anthozoan Alcyonlum dlgltatum (Roushdy & Hansen, 1961). According to Jorgensen (1 9 6 6) the occurrence of sus pension feeding in coelenterates is erratic. The coelenter ates in which such feeding method has been reported ingest numbers of diatoms, ciliates, flagellates or detritus. Species of the genus Zoanthus have never been seen feeding in nature (Goreau, 1964 and personal observations). In the laboratory the only reports on feeding are those of Hadden, 1968, on Zoanthus soclatus ingesting small pieces of butterfly fish, and those of the present dissertation on Zoanthus paclflca ingesting freshly-killed Gambusla and frozen cod, cut in small pieces. The abundance of diatoms, bacteria and flagellates in the coelenteron of Zoanthus paclflca could suggest that the polyps might be feeding on such organisms suspended in the water, but those organisms collected from Zoanthus1 gastral cavity are never found to be affected in any way by digestive processes. Further more, algal masses with numerous diatoms do not elicit in gestion by the zoanthids. The fact that Zoanthus Bhows a feeding response similar to that of the predatory Palythoa and the fact that this reaction is controlled by the tri peptide glutathione, which is present only in live animals or freshly killed prey, point away from Zoanthus1 feeding on suspended small organisms and toward the possibility that the polyps feed on small particles of food, probably fish, proceeding from prey captured by large predators which lose particles of their meal as they feed. Yet another possible method of feeding is the up take of dissolved organic matter, such as amino acids or glucose, directly from solution in the medium. Fungla scu- torla was found to remove glucose and several small organic molecules from very dilute solution (Stephens, 1962). Zoan thus paclflca was reported in this dissertation to accumu late glycine directly from the medium. The significance of 273 such ability to take up glycine from solution, however, cannot be understood until the metabolic requirements of the animal are known. It is not likely that an animal may rely on direct absorption of small molecules alone to obtain its food, but rather it is probable that this represents a method of satisfying part of the needs of the animal. One very important aspect in the nutrition of many coelenterates is the role of their symbiotic zooxanthellae. Intracellular symbiotic algae have often been thought to fulfill part of the nutritional requirements of the coelen- terate (Yonge & Nicholls, 1931b; Gohar, 19^8). Such role has been demonstrated recently In a series of papers by Muscatine & Hand (1958); Muscatine & Lenhoff (19^3j 1965a*b). The role of symbiotic algae in members of the genus Zoanthus was studied by Yon Holt & Von Holt (1968a,b). They found that the algae produced and released into the medium a mix ture of organic materials Including amino acids, mono-, di-, and tri-carboxylic acids and a number of neutral molecules, presumably carbohydrates. They estimated that the uptake of photosynthate by the animal is of the order of 2 -3 ng of carbon or 0 .1 percent of the animal's carbon content per day, and concluded that the total carbon discharge of the zooxanthellae could contribute significantly toward satisfy ing the metabolic requirements of Borne of the zoanthids in vestigated. They based their conclusion on the assumption that the oxygen uptake of Zoanthus is comparable to that of 274 other Cnidaria 0.1-1 nmol/O^/g fresh weight per hour or 0 . 0 1 5-0 .1 5 per animal, and also on the assumption that the respiratory quotient 1b 1 or near to 1. The fact that polyps which lose their symbiotic algae degenerate and die very rapidly also demonstrates that the zoanthids may rely heavily on their zooxanthellae for the fulfillment of some important metabolic require ments. An important ecological observation which reinforces this point is that made by Goreau (1964) on the ability of Zoanthus soclatus to maintain its population of zooxanthel lae under stress conditions which cause extrusion of sym bionts in most other coelenterates in the reef. The im plication is that coelenterates, such as most corals and Palythoa, which feed on zooplankton may lose symbiotic al gae easily under stress conditions. But an animal that de pends strongly on its symbionts may have some mechanism to . retain them even under stress conditions. Although the feeding methods used by Palythoa and Zoanthus are very different, their feeding reactions are very similar and both are chemically controlled. Further more, the tripeptide glutathione is a feeding activator in both animals. It has been discussed throughout this disser tation that both glutathione and proline serve as inducers of feeding responses in a vast array of coelenterates. In some scleractinian corals both proline and glutathione were * 275 found to activate the response, either one acting sepa rately. In Palythoa, however, a very interesting and im portant phenomenon occurs. Both activators are needed in the proper concentrations in order for them to induce feed ing reaction. The interest of this observation resides in that this is the first report of such a phenomenon in coe lenterates, and the Importance of it lies in the possible evolutionary significance that such a requirement of two feeding activators may have. Most coelenterates have evolved receptor-effector Bystems that take care of their primary behavioral needs, most of which revolve around food and defense. At a basic level the behavior stimulated by the tripeptide glutathione in Hydra and many other coelenterates may be similar to such phenomenon as the stimulation of the uterus by the octapeptide hormone oxytocin (Lenhoff, 1 9 6 7). Lenhoff (1968b) further speculates on the possibility that hormone receptors perhaps evolved from behavioral chemore- ceptors. The molecules selected for as activators will be those ubiquitous in prey organisms, and probably have a number of carbons between 5 and 20 with a corresponding range in molecular weights from 80 to 300. The need for signal diversity cannot be met by less than 5 carbon atoms, since the number of possible molecular configurations would be too small (Wilson & Bossert, 1963). 276 As Wilson & Bossert (I9 6 3) further predicted, the molecular size of activators will determine to a certain degree the complexity of the signal and its specific dis tinctiveness. It is easy to imagine that internal peptide activators in the more complex organisms should be rela tively more complicated, because these organisms could not chance having one of their specialized control mechanisms activated by a simple, widely distributed compound present in the circulating fluids. Wilson (1968) points out that if mixtures and combi nations of different substances can be used in coding, elaborate systems of signals can be developed. The fact that Palythoa responds to such a specific mixture of two feeding activators can be interpreted as a complication of the simpler pattern known for other coelenterates, or could represent an intermediary stage in the acquisition of two receptor sites. The next step in such an evolutionary line would be represented by the scleractinian corals Cyphastrea, Poclllopora and Fungla in which two activators can elicit the feeding reaction independently of each other. Finally, the ecological position of Zoanthids in their natural habitat is one of highly successful animals which cover large expanses of the reef or beach. They rep resent terminal positions on food chains for, as Ross & Sut ton (1 9 6 7) pointed out, coelenterates rarely form the food 277 of other animals because their nematocysts render them both, unpalatable and dangerous to would-be predators. CHAPTER XI SUMMARY Palythoa townbley1 Walsh and BowerB and Zoanthus paclflca Walsh and Bowers are two subtropical, subtldal zoanthlds closely related phylogenetically but widely dif ferent In their feeding methods. Palythoa townsleyl Is a micropredator, while Zoanthus paclflca seems to be a par ticulate matter suspension feeder. In nature Palythoa feeds on microzooplankton, especially crustaceans. There Is an optimum zooplankton density which was calculated ex perimentally and found to be ten times the zooplankton den sity reported for Palythoa1s natural habitat. In the laboratory Zoanthus feeds on pieces of freshly-killed or frozen fish exclusively. In natural sit uations It has never been observed to feed. One possibility Is that the animal probably feedB on particulate matter pro ceeding from fish preyed upon by large predators which lose part of their meal while tearing pieces off the prey. After the food has been obtained, either by capture (Palythoa) or seizure of particles (Zoanthus), both animals show the same complex and orderly series of steps which Is called the feeding reaction and consist of: 278 279 1. Lip formation. A group of tentacles seizes the food; the edge of the disc carrying these tentacles first contracts so that they group together around the food, then rises up and turns inward, thereby folding tentacles and food toward the mouth. Lip formation is controlled by mechanical and chemical Btimuli and its function is to se cure food and push it to the mouth. 2. Mouth opening consists of separation of the borders of the mouth so that food can be ingested. It is chemically controlled. The stimulus necessary to elicit mouth opening in Palythoa townsleyl is a combination of the tripeptide glutathione in concentration 10 M and of the a-Imino acid proline in concentration 10~^M. Some very complex interaction occurs between gluta thione and proline. In equimolar concentrations they cancel each other’s effect on the activation of feeding. In cer tain concentrations glutathione inhibits the activating ac tion of proline, in others it enhances it. The combined action of both activators influences the speed of reaction as well as the proportion of polyps In which the reaction occurs. The stimulus necessary to elicit mouth opening in Zoanthus paclflca Is the tripeptide glutathione at concen- -4 -’ S trations 5 x 10 to 5 x 10 •'M. Glycine at concentration 10"^M and higher can induce mouth opening and peristaltic movements in the column of the polyps. 280 Mouth opening can be replaced by a response called exposure of the actinopharynx in which the walls become swollen and protrude as bladder-like lobes above the oral disc. 3. Ingestion response Is the culmination step of the feeding reaction and is chemically controlled in the same way as the mouth-opening reaction. It is believed that Palythoa townsleyl has two types of feeding receptors, some sensitive to proline and others sensitive to glutathione. The first ones have high affinity for the a-imino group. Substitutions in this group such as l-thIazolIdIne-4-carboxylic acid and glycyl proline Inhibit the activity of the proline molecule. The addition of an OH group such as in hydroxyproline diminishes the activity of the proline molecule 100-fold. Additions in the carboxyl group as In prolylglycine do not affect the activity of the molecule. The size of the ring can be al tered within certain limits such as in azetidine-2-carboxy- lic acid and pipecolic acid without affecting the activity of the molecule. The receptors for glutathione seem to have affinity for the glycyl terminal end of the molecule with . JIHg the structure -CH^-D-gOOH and also for the thiol group. Zoanthus paclflca has feeding receptors sensitive to glutathione. The active part of the tripeptide molecule is the glycyl radical. Glycine at concentrations above _-a 10 -'M can elicit wide mouth opening peristaltic movements 281 of the column and Ingestion of solid particles. The polyps can take up glycine directly from the medium and utilize it in the manufacture of lipids and proteins. Both Palythoa townsleyl and Zoanthus paclflca pos sess symbiotic algae called zooxanthellae. The importance of these for Zoanthus could be determined indirectly when aposymbiotic polyps (those that have lost their symbionts) rapidly regressed and died under starvation conditions. Since Zoanthus depends on the chance of getting very specif ic particles to utilize as exogenous food, it is believed that both the ability to take up and utilize glycine, and perhaps other amino acids, from solution, and also the de pendence on zooxanthellae represent adaptations of Zoanthus to compensate for a very restricted diet, which by itself cannot insure the animal with satisfaction on its metabolic requirements. Conclusions Palythoa townsleyl 1. Palythoa townsleyl belongs to the group of pred atory coelenterates which feed on microzooplankton and cap ture their food with specialized organelles called nemato- cysts. 2. Palythoa is a strict carnivore. 3. The density of zooplankton affects the rate of capture by Palythoa and also the number of prey caught at 282 any one time. There is an optimum zooplankton density at which the polyps capture most rapidly and efficiently. This optimum density calculated experimentally is 10 times the zooplankton density in Palythoa*b natural habitat. 4. Palythoa townsleyl stings the prey with very few nematocysts and does not paralyze it as is common for most predatory coelenterates. Instead, the animal seizes the prey with a group of tentacles which contract in con currence with the contraction of the corresponding area of the oral disc. This area rises and bends toward the center of the oral disc, pushing the food to the mouth. This proc ess Is called "lip formation." Its purpose Is to seize and push the food toward the mouth. The process is elicited by mechanical and chemical stimuli combined. 5. The mechanical stimulus that elicitB lip forma tion Is the collision of a prey with Palythoa tentacles or oral disc. 6. The chemical stimulus that elicits lip forma tion Is the Imino acid proline. The chemoreceptors for lip formation recognize the proline molecule even with cer tain modifications of the ring's size (azetidine-2-carboxy- lic acid causes lip formation but pipecolic acid does not) and certain substitutions on the imino group (l-thiazoli- dine-4-carboxylic acid causes lip formation but glycyl pro- line does not). 283 7. Lip formation is a response independent of mouth opening but so closely related that it can be con sidered as just one link in a chain reaction including lip formation, mouth opening and ingestion response in a normal sequence. The normal sequence can be altered by the pres ence of the tripeptide glutathione, which stimulates the formation of a lip immediately after the mouth-opening re sponse. 8. Mouth opening is the second step of Palythoa1s feeding reaction; it is chemically controlled. The recep tors ai?e located mainly in the mouth itself and have high affinity for the proline molecule with an intact imino group. l-Thiazolidine-4-carboxylic acid, in which the imino group is replaced by S, effectively competes with pro- line for the receptor sites but is not able to elicit mouth opening. 9. Mouth-opening response may be replaced by ac- tinopharynx exposure under certain stimuli, such as the presence of glutathione. The walls of the actinopharynx become swollen and protrude as bladder-like lobes above the level of the oral disc. The food is moved by ciliary currents over these lobes which close around it and push it down into the coelenteron, without an actual separation of the borders of the mouth as seen in the mouth-opening re sponse. The most effective stimulus to cause mouth opening 284 -S was found to be a combination of proline 5 x 10 and glutathione 5 x 10-^M. 10. Acid pH alters the chemoreceptors for mouth opening, causing certain degrees of response depending on the acidity of the medium. 11. The feeding reaction of Palythoa culminates with the ingestion response, which is chemically controlled. The most effective stimulus is represented by the combination -8 -6 of proline 5 x 10 and glutathione 5 x 10 M. Some very intricate interactions occur between the imino acid and the tripeptide. This interaction influences the speed with which ingestion occurs and the proportion of polyps in which the reaction is elicited. Either substance by itself can elicit ingestion but only effectively when present in concentrations too high to be expected under natural condi tions . 12. With respect to the proline molecule, the sta bility of the imino group is necessary in order for the response to occur. If this group is changed, such as by substitution as in l-thiazolidine-4-carboxylic acid or glycylproline, the reaction does not occur. Substitutions in the carboxylic group such as in prolylglycine do not af fect the activity of the proline molecule; addition of an OH"* group effectively reduces the activity of the proline molecule; and alterations of the proline ring's size such as in azetidine-2-carboxylic acid, pipecolic acid, affect 285 only the speed of the reaction but not Its Intensity as measured by the number of polyps responding to the solution. 13- With respect to the glutathione molecule the SH group seems to be very Important In the recognition of the molecule by the chemoreceptors. The addition of a CH^ group to the SH effectively reduces the proportion of polyps that are Induced to respond with Ingestion. The most Important area of the tripeptlde glutathione seems to be the glycyl terminal end of the molecule with the group -CHg-^-COOH but much more investigation with the use of glutathione analogs Is needed before the characteristics of the molecule responsible for the activation can be determined. Zoanthus paclflca 1. Zoanthus paclflca does not belong to the group of predatory coelenterates and at the present time cannot be placed in a definite feeding-type category, although the evidence points toward considering it as a suspension feeder. 2. Zoanthus paclflca is very selective of the kind of particulate matter that is ingested by the animal as food. This food probably consists of pieces of fish that are left from the meal of large predators and are carried by the water movements over the colonies of zoanthids. 3. Zoanthus paclflca show an orderly and well co ordinated feeding reaction similar to that observed in 286 Palythoa. It Involves the same steps with the exception that no live prey Is captured, but only particulate matter that is seized by a lip, carried to the mouth, which opens upon contact with the food, and ingested. 4. The feeding reaction of Zoanthus is under chem ical control. The activator for mouth opening and inges tion is the tripeptide glutathione, and the most effective -4 concentrations of the activator are 10 and 10 Higher concentrations are inhibitory of mouth opening and inges tion responses. 5. Glutathione probably affects the cilia on the oral disc of Zoanthus, for its presence increased consider ably the time required for elimination of extraneous mate rial from the oral disc. 6. The active part of the glutathione molecule is that corresponding to the glycyl radical. Glycine at con- _Q centrations above 10 can elicit wide-mouth opening in gestion responses. 7. Zoanthus can accumulate glycine from the medium and incorporate it into the metabolism of lipids and pro teins . 8. Zoanthus harbors a coelenteric bacterial flora which can be cultured ln_ vltro. Cultured bacteria accumu- _l4 late some glycine C and also utilize it in the synthesis of lipid and protein. 287 9. 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Creator Reimer, Amada Alvarez (author)

Core Title Feeding Behavior In Palythoa Townsleyi And Zoanthus Pacifica With Emphasis On The Chemical Control Of Their Feeding Reactions

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Biological Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, general,OAI-PMH Harvest

Language English

Advisor Bakus, Gerald J. (committee chair), Martin, Walter E. (committee member), Shugaram, Peter M. (committee member), Soule, John D. (committee member), Zimmer, Russel L. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-461146

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Rights Reimer, Amada Alvarez

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