THE ULTRASTRUCTURE AND FUNCTION OF THE GUT OF PATELLA VULGATA. by Maxwell Simon Bush, Department of Zoology, Bedford College, University of London. Thesis submitted for the degree of PhD. ProQuest Number: 10098528 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10098528 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 R.H Q N.( ifij-c'îAnYj CLASS S%:M No. 6 ^ 1 ACC No Dats ACO. Klov’ gt I dedicate this work to my parents, whose co-operation and financial support made it possible. ABSTRACT. The limpet gut consists of a long coiled tube lined by a ciliated columnar epithelium, into which open the ducts of the salivary and digestive glands. Six sections can be distinguished: the buccal cavity, oesophagus, stomach, style sac, intestine and rectum. The oesophagus can be subdivided into a dorsal food channel and a series of lateral pouches forming the oesophageal gland. The intestine is divisible into sections designated A, B, C, D and E. Ultrastructural and histochemical analyses revealed nine cell types, seven of them glandular. A single type of gland cell lining the tubules of the salivary gland produces a viscous secretion that lubricates the radula and entraps particles rasped from the substratum. Mucous cells occur in the buccal cavity, dorsal food channel and rectum; in the rectum, mucus aids defaecation, but elsewhere it entraps loose particles that are consequently transported to the stomach. The only extracellular enzyme, an amylase derived from the gland cells of the oesophageal gland, is mixed with the food in the dorsal food channel. Ciliated and unciliated columnar cells lining the ducts of the digestive gland, stomach, style sac and anterior intestine, release blebs of cytoplasm into the lumen to consolidate loose particles into a faecal rod that is rotated along the intestine. Clavate gland cells and possibly basal gland cells in the posterior intestine, cover the faecal rod with their secretion to form a durable rod. The vacuolated digestive cells of the digestive gland, digest food intracellularly releasing undigestible residues in spherules of cytoplasm, these are bound into a liver string by the proteinaceous secretion of the basophilic cells. Both these cell types and the amylase-secreting cells exhibit phases of activity, but only that of the latter is related to the tidal cycle. Tritiated D-glucose was absorbed by the oesophagus, intestine and digestive gland by a mechanism inhibited by 2,4-DNP and phloridzin. The mechanisms operating in the oesophagus and posterior intestine were sodium-dependent. Fluid movements from the intestinal lumen to the blood occurred. TABLE OF CONTENTS, PAGE. TITLE 1. DEDICATION 2 . ABSTRACT 3-4. TABLE OF CONTENTS 5-9. LIST OF PLATES . 10-11. LIST OF FIGURES AND TABLES 12-14. INTRODUCTION 15-31. MATERIALS AND METHODS 32-52. Collection site 32. Collection, transport and maintenance 32-33. Dissection techniques 33. Preparation of sections 33-35. 1.Wax .... 33-34. 2 .Thick resin . 34. 3.Frozen . 34-35. Preparation of tissue for TEM 35-36. Preparation of tissue for SEM 36-37. Histochemistry 37-38. Biochemical enzyme demonstrations 38-39. Cytochemistry 39-42. 1.Acid phosphatase 39. 2 .Alkaline phophatase 39. 3 .Carbohydrates . 39-41. A).PTA 39-40. B).PA-SM and PA-TSC-SP 40-41. 4.Tannic acid 41. 5.Ferritin tracing 41-42. pH of gut fluid 42. Ciliary currents 42. Phasic activity 43. Starvation 43. Radiotracer experiments 43-47. l.In vitro experiments 43-47. A).Glucose absorption 43-45. B).Glucose flux 45-46. C).Chromatography 46-47. D).Net water movement 47. 2 .In vivo experiments 47. 3.Statistical methods 48. Autoradiography 48-49. Photographic techniques 49. Word processing 49. RESULTS 53- THE CILIATED AND UNCILIATED EPITHELIUM 53-82. Ultrastructure . 56-66. Cyto- and histochemistry 66-72. 1 .General 66-67. 2 .Carbohydrates . 68-70. 3.Proteins 70. 4.Lipids 70. 5.Pigments 70. 6 .Enzymes 70-72. Plates 1-7 73-82. THE BUCCAL CAVITY 83-89. Plates 8-9 86-89. THE SALIVARY GLANDS . 90-103. l.The ducts 90-93. 2 .The tubules .... 93-98. A).Histology and ultrastructure 93-96. B).Histochemistry 96-98. Plates 10-12 99-103. THE OESOPHAGUS 104-133. 1 .Anatomy 104-108. 2 .Ciliary currents 108-109. 3.Epithelium of food channel 109-113. A).The ciliated cells 109. B).The mucous gland cells 109-113. 4.Epithelium of oesophageal gland 113-120. A).The ciliated cells 115. B).The amylase-secreting cells 115-120. Plates 13-22 121-133. THE STOMACH AND STYLE SAC . 134-151. l.SEM 134-140. 2 .Ultrastructure and histochemistry 140-141. 3.Ciliary currents 141-144. Plates 23-27 145-151. THE DIGESTIVE GLAND . 152-184. 1.The ducts 152-155. A).Ultrastructure 152-153. B).Histochemistry 153-155. 2 .The tubules 155-173. A) .Basophilic cell 155-163. i).Ultrastructure . 155-161. ii).Histochemistry . 161-163. B).Digestive cell 163-173. i).Ultrastructure . 163-168. ii).Histochemistry . 168-173. 8 Plates 28-35 174-184. THE INTESTINE . 185-210. 1.Section A ' 185. 2.Section B 185-187. 3.Section C 187-193. A).Basai gland cell 187-193. i).Ultrastructure 187-191. ii).Histochemistry 191-193 4.Section D 193-197. A).Clavate gland cell 193-197. i).Ultrastructure 193-195. ii).Histochemistry 195-197. 5.Section E 197. Plates 36-45 198-210. THE RECTUM 211-216. Plates 46-47 214-216. THE EFFECTS OF STARVATION ON THE GUT 217-219. Plate 48 . 220-221. PHASIC ACTIVITY 222-253. 1.The oesophageal gland 222-234. A).Ultrastructural characteristics 222-229. B).Synchrony of phases in gland 229-233. C).Synchrony of phases with tide 233-234. 2 .The digestive gland . 234-241. A) .Digestive cell 234-238. i).Holding phase 234-236. ii).Absorbing phase 236. iii).Digesting phase 236-237. iv).Fragmenting phase 237-238. v).Disintegrating phase . 238. B).Basophilic cell 238. C).Synchrony of phases with tide 238-241. Plates 49-57 242-253. RADIOTRACER EXPERIMENTS 254-288. l.In vivo experiments . 254-258. 2 .Glucose flux experiments 258-267. 3.Glucose absorption experiments 268-280. A).Absorption 258&268. B).Inhibitors 268-272. C).Sodium concentration 272. D).Effect of pH . 272-274. E).Kinetics 274-280. 4.Chromatographic experiments 280-285. 5.Net water movement experiments 285-288. AUTORADIOGRAPHY 289. DISCUSSION 290-339. SUMMARY 340-341. ACKNOWLEDGEMENTS 342. REFERENCES 343-367. APPENDICES 368-374. lA . 368-370. IB . 370-371. 2 371-373. • 373-374. 10 LIST OF p l a t e s . PLATES. PAGE. Abbreviations used in plates 1-7 • 73. 1-7 Ciliated and unciliated columnar cells. 74-82. 1-5 Transmission Electron Micrographs (TEM) of cells • 76-80. 6-7 Cytochemistry of cells • 81-82. Abbreviations used in plates 8-9 • 86. 8-9 Scanning Electron Micrographs (SEM) of buccal cavity 87-89. Abbreviations used in plates 10-12 . • 99. 10-12 Salivary glands. 100-103. 10 SEM of glands, TEM of the duct cells • 101. 11-12 TEM of the tubule cells • 102-103. Abbreviations used in plates 13-22 . • 121. 13-22 The oesophagus. 122-133. 13 Light micrograph (LM) and SE>1 of oesophagus • 124. 14 TEM of oesophageal epithelia • 125. 15 TEM of epithelium of food channel . • 126 16 LM post-oesophagus, SEM food channel epithelium • 127. 17 SEM and TEM of mucous cells . • 128. 18 SEM of oesophageal gland • 129. 19-21 TEM of amylase-secreting cell • 130-132. 22 Amoebocytic blood cell, starch-film method • 133. Abbreviations used in plates 23-27 . • 145. 23-27 The stomach and style sac. 146-151. 23 SEM of stomach .... •147. 24 SEM of digestive gland duct and style sac . • 148. 25 SEM of typhlosoles .... •149. 26 LM of typhlosoles and SEM of white secretion • 150. 27 TEM secretory tract cells, SEM liver string • 151. Abbreviations used in plates 28-35 . 174. 11 28-35 The digestive gland. 175-184. 28 TEM of duct cells, LM of tubule 177. 29 TEM of tubule epithelium . 178. 30-31 TEM of basophilic cell 179-180. 32 TEM digestive cells (absorbing & digesting) 181. 33-34 TEM digestive cell .... 182-183. 35 Starch-film and gelatin-film methods 184. Abbreviations used in plates 36-45 . 198. 36-45 The intestine. 199-210. 36 SEM and TEM of section A . 201. 37 SEM and TEM of section B 202. 38 SEM and TEM of section C 203. 39 TEM and SEM of basal gland cell (bgc) 204. 40-41 TEM and cytochemistry of bgc, TEM of section D 205-206. 42-44 TEM and SEM of clavate gland cell . 207-209. 45 TEM and SEM of section E 210. Abbreviations used in plates 46-47. 214. 46-47 SEM, LM and TEM of rectum 215-216. Abbreviations used in plate 48 220. 48 TEM of starved digestive gland cells 221. Abbreviations used in plates 49-57 . 242. 49-57 Phasic activity. 243-253. 49 LM of oesophageal gland during both phases 245. 50-51 TEM of amylase-secreting cell (secretory phase) 246-247. 52 TEM of amylase-secreting cell (heterophagic phase 248.