oa eT ee ee oi 3 3 H Marine Biological Laboratory Library } (| Woods Hole, Mass. it (] Aer Ml (]] Presented by (] ll sssociation of American M } University Presses (] fN Aug. 25, 1961 (] Mt N M N I 0 | 3 §3 ae Se ee E OS09100 TOED OG W000 IOHM/1981N iH) belie ey : orp Nit Fu THE AMERICAN ARBACIA AND OTHER SEA URGHINS am 4 7 Ave * ‘ 7 I ; ‘ Pak) ' cM c i : i) ie iy a ms AA an WG H&S c-3 THE AMERICAN ARBACIA AND OTHER SEA URCHINS Bue ETHEL’ BROWNE HARVEY 1956 PRINGETON UNIVERSITY PRESS PRINCETON, NEW JERSEY Published, 1956, by Princeton University Press London: Geoffrey Cumberlege, Oxford University Press ALL RIGHTS RESERVED No part of this book may be reproduced in any form without permission of the author Device on title page, Aristotle’s Lantern, from a wood- cut by Edward Forbes, A History of British Star- fishes, and other Animals of the Class Echinodermata (London, 1841). Composition by N.V. Drukkerij G. J. Thieme, Nijmegen, The Netherlands Printed in the United States of America To the Memory of EDMUND B. WILSON wba fb : ' i a { ; ; a ae ee pe ddrd re ery Nii Wr otis —, Gent ¢ wiih ae ee a a a 2) ee = — Oy ay 44 ered ae ane fk | AX er va ye) eat) are nd ra tie ee ee . oy (Oey re je aRewrs 4 agtensy's faihi YU, an st Sos 4 ape PREFACE The sea urchin, Arbacia punctulata, which occurs along the Eastern coast of North America, has for many years provided material for experi- mental work on cells, done mostly at the Marine Biological Laboratory at Woods Hole, Mass. The animals are readily obtained and the eggs are produced in large quantities throughout the summer. This species is the American Arbacia and is not found in Europe, where another species, Arbacia lixula occurs. The latter species and many other genera of sea urchins have been used for experimental work at Naples, on | the Swedish coast, in the British Isles, France, Japan, and elsewhere. The eggs are fundamentally similar but differ in details. The Arbacia egg is an ideal cell. It is spherical, thus rendering changes in size easy to determine. It is fairly simple in comparison with most cells. It is quite hardy and can be subjected, without damage, to moderate changes in the sea water, produced by the addition of water, or salts, or anaesthetics, or other chemicals, and to changes in temperature, pressure, light, and other physical factors. Harmful ef- fects and recovery can be readily detected by fertilizing the egg and watching its development. The granules in the egg can be moved by centrifugal force, and the egg can be broken into halves and quarters containing different kinds of materials in definite amounts. The ex- perimental work on sea urchin eggs has included every line of approach, cytology, embryology, physiology, and biochemistry, and has been concerned with the solution of many fundamental problems. The earliest experiments on Arbacia punctulata eggs were those of Jacques Loeb at Woods Hole in 1892, who wrote a paper entitled “Investigations in Physiological Morphology. III. Experiments on Cleavage’’, published in the Journal of Morphology. It dealt with divi- sion of the nucleus without cleavage of the cell, caused by hypertonic sea water. The next paper was that of T.H. Morgan in 1893, published in the Anatomische Anzeiger, part of which was on the same subject, taking exception to some of Loeb’s results. Loeb and Morgan were succeeded by many well-known biologists. We have a fine heritage of experimental research on the Arbacia egg, and it is partly in an endeavor to gather together this work and make it more readily available to later investigators that this book has been written. The book is divided into four parts. The first part deals with sea urchins in general—their history, which begins before Aristotle and Vill PREFACE Pliny, their use as food, interesting facts concerning their mode of life and their habits, and other matters of general interest, including a classification of the Echinoidea mentioned in this monograph. The second part is devoted to the normal development of the Arbacta punctu- lata egg from fertilization through metamorphosis, with original photo- graphs of the different stages. The third part is concerned with centri- fuging, a subject which has particularly interested the author. The fourth part is a compilation of all other experimental work in which Arbacia eggs and sperm have been used since the pioneer work of Jacques Loeb in 1892 until 1954. This is arranged alphabetically according to subjects. The results of all investigators are given without any attempt to evaluate them. Together with the references to Arbacia there are given with each topic a few important references to other species. There is an extensive bibliography with complete titles. The illustrations are mostly original photographs by the author. Many subjects have been treated in the compilation, and many of my co-workers at Woods Hole and Princeton and others elsewhere have been consulted on subjects most familiar to them. I take this opportunity to thank most heartily all who have helped me by sup- plying information and by reading parts of the manuscript. Among these are: E. G. Ball, E. S. G. Barron, H. F. Blum, E. G. Butler, A. D. Chiquoine, the late H. L. Clark, G. H. A. Clowes, K. S. Cole, K. Dan, L. V. Heilbrunn, B. F. Howell, the late A. C. Johnson, L. H. Klein- holz, M. E. Krahl, G. G. Lower, the late B. Lucké, D. A. Marsland, D. Mazia, A. Monroy, A. R. Moore, I. Motomura, A. K. Parpart, J. S. Rankin Jr., S. Ranzi, J. Runnstrom, the late D’Arcy Thompson, A. Tyler, and C. A. Villee. My sister, Dr. Mary N. Browne, a classical scholar, has been most helpful with the section on history. I wish especially to thank my husband, E. Newton Harvey, who has been frequently consulted and has rendered invaluable assistance con- cerning topics of which he has expert knowledge. May 1955 ETHEL BROWNE HARVEY Princeton, N. J. CONTENTS \ 4 PART I. GENERAL SUBJECTS ES, Chapter 1. Etymology, nomenclature a. Echinus and sea urchin . b. Arbacia punctulata °. Chapter 2. Historical a. Greeks and Romans . b. Middle Ages : c. Rondelet to L. Agassiz d. Modern e. History of Embryology f. The Pluteus Chapter 3. Fossils . Chapter 4. Uses of sea urchins . As food. . As medicine . As food for other animals As cups, lamps, ink In art, on coins and as jewelry . As experimental material momo of Chapter 5. Description a. Size and shape b. Color c. Morphology Chapter 6. Natural history . Feeding habits . Growth and age . Regeneration . . Habitat, burrowing ee and collects . Phototaxis and light reaction Geotropism g. Locomotion an) ol oF eltome Chapter 7. Sex and ee a. Sex ratio b. Sex dimorphism (erie ae). c. Sex determination in Arbacia panera : d. Hermaphroditism . e. 1% g- h. CONTENTS Breeding season, shedding of eggs and sperm . Lunar periodicity (other species) Forced shedding . Removal of gonads Chapter 8. Species of Echinoidea near Woods Hole, and Arbacia lixula (pustulosa) Chapter g. Classification, including places of occurrence and size of eggs when available PART II. NORMAL DEVELOPMENT Chapter 10. Immature egg and maturation tae) ge fol, (ey cops te g. . Growth and differentiation of the oocyte . Structure of the oocyte . Reaction to sperm . Centrifuging and gravity. . Respiration : Permeability to water Polar body formation Chapter 11. Mature egg, unfertilized and fertilized om we Oy nO 1G Om ots . Quantity of eggs in an Arbacia . . Methods of estimating volume and aunty . Shape of eggs . . Size, weight and density abe, eggs. . Size of fertilized egg . Variations in size . . Aberrant sized eggs and eles ; . Effect of age, temperature, etc., on size aie eggs Structure of the egg . Membranes and layers . Diagrams of surface layers of Mebaeie and wibice eggs . . Granules Chapter 12. Sperm Wish) igar) (el for (oy tops tS) . Morphology . Swimming rate . Motility . Longevity . ; Injury: . Agglutination . . Chemotaxis CONTENTS h. Polyspermy i. Physiology . j. Seminal fluid . k. Techniques Chapter 13. Fertilization and Cleavage . Fertilization . Cleavage . Schedule of deeclepmens . Cleavage without membranes . Sperm entrance and cleavage planes The centriole . . Mid-bodies . Chromosomes . Visibility of the ere Fate: Isolation of mitotic apparatus . Elongation at cleavage Asynchrony in cleavage . . Micromeres . Absence of oxygen Refertilization ee. Sa p- Fertilization after parthenogenesis . O- Bis) fe re Fp OF. re ee Oo f Chapter 14. Blastula, gastrula and pluteus a. Blastula and gastrula. b. Pluteus, description ; c. Development when not fed . d. Food for pluteus . Chapter 15. Metamorphosis . a. Arbacia punctulata b. Other species PART Il]. CENTRIFUGED EGGS Chapter 16. Methods. a. Whole eggs b. Crushed eggs . Chapter 17. Stratification of the egg Leo , a. Unfertilized eggs . ; b. Vital staining of stratified egg c. Stained sections of stratified egg d. Stratification of other species Xl CONTENTS e. Stratification and polarity ; f. Redistribution of material and return to nsphere Chapter 18. Breaking of the egg: egg fractions and contents . Chapter 19. Factors affecting stratification and breaking. a. Centrifugal force . Si Whale b. Hypo- and hypertonic sea water c. Single salt solutions d. Temperature, pressure, ration e. Fertilization Chapter 20. Properties of egg fractions . Chapter 21. Development of centrifuged eggs and fractions . . Spherical centrifuged eggs . Elongate centrifuged eggs . White half-eggs . Red half-eggs, Perilized . avyalc . Red half-eggs, Unfertilized. Pariveae aed merogones Clear quarter . Sa ta: . Mitochondrial (granular) daarer . Yolk quarter Pigment quarter . Eggs fertilized, then centrifuged. Ee? TO fe@e] feel a (el te) tom [9 . Conclusions PART IV. COMPILATION OF EXPERIMENTAL WORK Ageing of eggs. Amoebocytes Amoeboid eggs Anaesthetics (narcotics) Calcium Carbohydrate ietabaligi Chromatophores Clear layer of centrifuged eggs Cleavage : ‘ Composition of egg Cortical layer . Cyanides Cytochrome and cytochrome audlace Cytolysis Density-Specific Se fe 128 . 128 3 129 Lor a ION ee et g2 7s 199 133 ~ 135 + 138 , 138 7138 - 139 . 140 + (540 2 142 . 148 . 143 e Sa) orga . 145 - 149 - 149 . 150 . 151 - 155 - 197 . 158 . 160 . wer « 166 HorG7 :. 169 Pog EE s 132 CONTENTS Echinochrome . : Electrical properties and feo Enzymes : Fertilization membeane Fertilizin and agglutinin . Heat production Hyaline layer . Hybrids Hydrogen ion concentration, ee Infrared light . Jelly layer . Leucocytes . Lipids . Lithium Magnesium Mitochondria . Nitrogen Nuclear division aout ear division Nucleoproteins Oil (see also lipids) Osmotic pressure . :. & Oxygen lack and low oxygen tension Parthenogenesis Perivisceral fluid . Perivitelline space. Permeability Phosphorus metabolism Plasma membrane Polyspermy Potassium . : Pressure, Gy drostatic : Proteins Radium Respiration : Rhythms of Pisce paige: Shell Agia ie Sodium. Tension at the Mek Twins, Triplets, Quadruplets Ultrasonic waves . se? Ultraviolet light xill . 173 Fabs ta Ls alg ions . 182 182 . 182 erase. 185 187 187 A nLao . 189 . 190 HOE 192 - 193 -, 193 - 194 - 195 « 196 - 197 . 198 « 20% . 202 ; 203 . 206 . 208 . 208 7/209 2720 e201 ae svete . 219 ~ 220 aoe ~2e2 igen of ee , 229 XIV CONTENTS Viscosity ‘ é Visible light and Bp ovodynemiol action . Vital dyes . Vitelline membrane X-rays . Yolk granules . Index Bibliography {approximately mean titles) 5 bea . 226 aor . 228 152290 231 » 235 . 289 . Breaking of unfertilized egg by centrifugal force . TEX® FIGURES . Sea urchin and hedgehog . Lantern of Aristotle . . Sea urchins in an emblem . . Rondelet’s figure of sea urchins . Sea otter eating sea urchins . Structure of Arbacia test. . Vertical section of Arbacia . . Young sea urchins in brood pouch of Goniocidaris canalt- culata . . Membranes and layers of unfertilized egg . Membranes and layers of fertilized egg . Arbacia spermatozoon y 24 PVE PLATES . Tests of Arbacia punctulata . Maturation of the egg; polar body formation . Normal development of the living egg from one cell to gastrula . Normal development, continued; gastrula to pluteus . Stained sections of developing eggs . Metamorphosis . Breaking of unfertilized egg by centrifugal force . Development of centrifuged eggs, elongate and spherical . Development of white half-egg . Development of red half-egg (fertilized) . Development of red half-egg (parthenogenetic) . Development of clear quarters and eggs centrifuged after fertilization . Stratification and size of half-eggs in relation to cen- trifugal force . Stratification and size of half-eggs in hypo- and hyper- tonic sea water . Stratification and breaking of eggs, centrifuged in single salt solutions Miscellaneous photographs PALES PLATES PLATE I. Test of Arbacia punctulata. Photograph 1. Aboral or dorsal view. 2. Oral or ventral view showing the teeth. 3. Side view. 4. Close-up of photograph 1 showing 4 anal and 5 genital plates, with genital pores; including the large madreporic plate. 5. Side view of the test without spines. 6. Aboral view with a light bulb inside the shell; this is ap- proximately natural size. Photographs 3 to 6 with the assistance of G. G. Lower. PLATE: it: Maturation of the egg; polar body formation. Living eggs except iW 18 which are stained sections. 1 to 6. Growth of the oocyte. 7, 8. Breaking of the germinal vesicle. 9, 10. First polar body. ‘11, 12. Second polar body; note flattening of the cell in 12. Photographs 18, 14, 15. Centrifuged oocytes. 16. Formation of blebs where spermatozoa hit the surface. 17. Stained section, showing a bleb and spermatozoa on the surface and inside the egg. 18. Section showing mitochondria (dark material) around the germinal vesicle. Magnified about 270 x except 17, 18, about 600 x. PLATE III. Normal development of living eggs, from one cell to the gastrula. Times after fertilization at 23° C are given under each photograph. Magnification about 270 x, the same throughout as nearly as possible. Photograph 1. Unfertilized egg. 2. Spermatozoa around the egg. 3. Fertilization cone above, center. 4. Fertilized egg. 5. Monaster. 6. “Streak” stage, narrow streak. 7. Broad streak fading out and enlarged nucleus. 8. Amphiaster. 9 to 11. First cleavage; note cells are separate in 10, close together in 11. Photograph 12. Four cells. 13. Eight cells. 14. Twelve cell stage, four colorless micromeres below. 15. Sixteen cells. 16. About thirty-two cells. 17 to 20. Blastulae. 21. Hatching from the fertilization membrane. 22. Late blastula. 23. Early gastrula. 24. Late gastrula, spicules forming. PLATE IV. Normal development, continued; gastrula to pluteus. Times after fertilization at 23° C are given under each photograph. Magnification (except 9) the same as Plate III, about 270 x, as nearly as possible the same throughout; Photograph 9 is approximately 180 x. Photograph 1. Late gastrula. 2. Same stage in dark field. 3. “Prism” stage. 4 to 6. Early pluteus. 7. Anal or ventral view of pluteus. 8. Oral or dorsal view of the same pluteus. 9. Side view of pluteus. PLATE V. Stained sections of developing eggs, fixed in Bouin, stained with Heidenhain’s haematoxylin. Photograph 1. Unfertilized egg. 2. Sperm and sperm aster. 3. Sperm nucleus fused with female nucleus. 4. “Streak” stage, with enlarged nucleus. 5. Prophase. 6. Metaphase. 7. Anaphase. 8. Telophase. 9, 10. Second cleavage. 11. Four cells. 12. Early blastula; note that the spindles have no asters. Magnified about 450 x. PLATE VI. Metamorphosis. All photographs (except 12) are to the same scale magnified about 24 x. Photograph 1 is of eggs at this magnification. 2. Three day plutei. 8. A new pair of arms with red tips forms after 1] days if the plutei are fed. 4. Another pair of arms, median, forms when about 3 weeks old. 5. Arms longer; four weeks old. 6. Arms begin to degenerate; four tubular processes, the auricles, are present; 5 weeks old; the body of the adult grows inside the pluteus. 7. Five tube feet are formed in a sort of pocket; 2 months old. 8 to 10. Fifteen primitive spines, three adjacent to each tube foot; 2% to 3 months. 11. More tube feet have formed; 3% months, the oldest raised in the laboratory; diameter 1 mm. including spines. 12. Smallest young Ar- bacia brought from the sea, about 6 mm. including spines. (Biol. Bull. 97:292, 1949, modified. ) PLATE VII. Breaking of the unfertilized egg by centrifugal force, 10,000 x g. Photograph 1. Stratified egg showing oil, clear layer, mitochondria, yolk and pigment. The nucleus is always in the clear layer under the oil cap. 2. White half, containing oil, clear layer, mitochondria, a little yolk and the nucleus. 3. White half, with further centrifuging. 4. Clear quarter, containing oil, clear layer and nucleus. 5. Mitochondrial or granular quarter, containing all the mitochondria and a little yolk. 6. Red half, containing yolk and pigment. 7. Red half with further centrifuging. 8. Yolk quarter, containing yolk. 9. Pigment quarter, containing a little yolk and all the pigment. 10. Eggs in three layers in sugar solution after centrifuging; white halves (top), unbroken whole eggs, and red halves (bottom). 11. Unfertilized eggs stratifying and breaking apart in the centrifuge microscope; photographed while rotating about 6,000 x g. 12. White halves, taken from the centrifuge tubes. 13. Red halves. Magnification of Photographs 1-9, 270 x. ( Photo- graphs 1-9 Biol. Bull. 79:167, 1940c. Photographs 10-13 Biol. Bull. 71:105, 1936, modified. ) PLATE VIII. Development of centrifuged eggs (at 23°). Photographs 1 to 8. Elongate eggs. 1. Unfertilized elongate egg stratified as in Plate VU; note cortical granules at periphery of the clear layer. 2. Usual two cell stage, 1% hours after fertilization at 23°. 3. Usual four cell stage, % hours. 4. Occasional four cell stage, 1% hours. 5. About 16 cells, 213 hours; note red cells are larger than the white ones. 6. About 32 cells, micromeres at left, 3 hours. 7. Slipper blastula, 5% hours. 8. Hatching from the fertilization membrane, 7 hours. Photographs 9 to 20. Spherical eggs. 9. Unfertilized egg; same layers as in Photo- graph 1. Photograph 10. Monaster. Note pigment being redistributed as the aster forms. 11. First cleavage, usually at right angles to the stratification. 12. First cleavage parallel with the stratification. 13. Four cell stage, 1% hours. 14. Eight cells, 2 hours. 15. Micromeres (without pigment) at the oil cap. 16. Micromeres (without pigment) at the centrifugal pole. 17. Blastula. 18. Late blastula, pigment still concentrated. 19, 20. Plutei with pigment still concentrated; in 19, near the mouth, in 20 in one arm, 24 hours. Magnification about 270 x. PLATE IX. Development of white half-egg. Times (at 23°) after fertilization given under each photograph. Photographs 1 to 4. Elongate egg. 5 to 21. Spherical egg. 1 and 5. Immediately after removal from the cen- trifuge. 6. Monaster (not well marked). 7. “Streak” stage, enlarged nucleus. 8. Breaking of nuclear membrane. 9. Amphiaster. 10. Just before cleavage. 11, 12. First cleavage in any plane. 13 to 17. Cleavages to form blastula; blurring in 17 caused by blastula swimming inside the fertilization membrane. 18. Hatching from the fertilization mem- brane. 19. Free-swimming blastula. 20. Gastrula. 21. Pluteus; note lattice-like skeleton in the arms. Magnification about 270 x. (Biol. Bull. 79:171 and 178, 1940c, modified. ) PLATE X. Development of red half-egg, fertilized; fertilized merogone. Times after fertilization at 23° are given under each photograph; 1 to 4, elongate eggs; 5 to 16, spherical eggs. Photographs | and 5. Unferti- lized eggs immediately after removal from the centrifuge. 2 to 4, Early cleavages. 6. Soon after fertilization, showing fertilization membrane and hyaline layer. 7. Male nucleus. 8. Monaster. 9. Amphiaster and cleavage furrow. 10 to 13. Cleavages. 14. Hatching from the fertiliza- tion membrane. 15. Free-swimming blastula. 16. Pluteus, well formed. 17 to 20. Less regular development with fertilization membranes rup- tured, so that cells are more scattered. 21 to 24. Division planes have failed to come in; nuclear division without cell division. 24. Egg pep- pered with small nuclei without cell division. Magnification about 270 x. (Biol. Bull. 79:177, 1940c. Photograph 16 has been replaced by a later photograph. ) PLATE XI. Development of red half-egg, parthenogenetic; parthenogenetic merogone. Times (at 23°) after activation are given under each photograph. Note the similarity of these photographs and those of the fertilized merogones on Plate X, facing this. Compare picture for picture. Photographs 1 to 4. Elongate eggs. 5 to 16. Spherical eggs. 1 and 5. Unactivated eggs immediately after removal from the centri- fuge. 2 to 4. Early cleavages. 6. Soon after activation, showing ferti- lization membrane and hyaline layer. 7. Clear sphere, simulating a nucleus. 8. Monaster. 9. Amphiaster and cleavage furrow. 10 to 13. Cleavages; note especially 13, quite regular many celled organism, similar to the same stage of a fertilized merogone on preceding plate. 14. Hatching from the fertilization membrane. 15. Blastula. 16. Non- cellular but healthy-looking parthenogenetic merogone of four weeks; there has been no differentiation. 17 to 20. Cleavages without fertili- zation membranes; cells are scattered. In Photograph 20, right, cell divisions have taken place in the light but not in the pigmented por- tion. 21 to 23. Multi-astral eggs with small spheres associated with the asters. 24. Many small spheres resembling nuclei. Magnification about 270 X (Biol. Bull. 79:179, 1940c. ) PLATE XII. Development (at 23°) of clear quarter-eggs and of eggs centrifuged after fertilization. Magnification about 270 x. Photographs 1 to 13. Development of clear quarter. Photograph 1. Fertilized clear quarter, showing fertilization mem- brane. 2. Monaster stage, 20 minutes after fertilization. 8. Two cells, two hours after fertilization. 4. Two cell stage of the clear quarter and about a 32-cell stage of the white half, two hours after fertilization. 5 and 6. Four cells, three hours after fertilization. 7. Eight cells, four hours after fertilization. 8 and 9. Sixteen cells, 5 hours after fertiliza- tion. 10. Clear quarter, about 32 cells, and white half photographed at the same time, further advanced, 5 hours after fertilization. 11. Early blastulae of clear quarter and white half from the same culture, about 6% hours after fertilization. 12. Free-swimming blastula of clear quar- ter, 20 hours after fertilization. 13. Pluteus of clear quarter, 10 days old. Photographs 14 to 17. Eggs centrifuged after fertilization. Photograph 14. Eggs centrifuged one minute after fertilization for six minutes at 10,000 x g; they break up into small pieces. 15. Egg cen- trifuged four minutes after fertilization for six minutes at 10,000 x g; it stratifies like the unfertilized eggs, but not nearly so well (Cf. with Plate VII, Photograph 1). 16. Egg broken apart by centrifuging for six minutes, thirty minutes after fertilization, the fertilization mem- brane having been removed previously; picture was taken 2% hours after fertilization. Note that the white half has cleaved, and red half has not. 17. Eggs form long streamers when centrifuged 6 to 20 min- utes after fertilization, the membranes having been removed just after fertilization. This photograph was taken while the eggs were rotating (about 6,000 x g). (Photographs 1-13 J. Exp. Zool. 102:269, 1946a. ) PLATE XIII. Stratification of unfertilized eggs and size of half-eggs with varying centrifugal forces. The first vertical column shows stratification. The second vertical column shows size of half-eggs. With low forces, the eggs are well stratified and the white half is much larger than the red half. With high forces, the eggs are not well stratified and the white half is small while the red half is very large. Photograph 5. Red half obtained with 10,000 x g and recentrifuged at 10,000 x g. Control to Photograph 12; note clear layer at top, due to further settling of gran- ules. Photograph 12. Red half obtained with 100,000 x g and recentri- fuged at 10,000 x g. The granules had not been segregated in the first centrifuging with very high forces, and when recentrifuged, they stratify as in the original egg. The mitochondrial layer has been stained with methyl green. (Biol. Bull. 80:357, 1941a.) PLATE XIV. Stratification of unfertilized eggs and size of half-eggs when centri- fuged in hypo- and hypertonic sea water (10,000 x g). Magnification about 270 x. In hypotonic sea water (Photographs 1 to 3 in 60% and 4 to 6 in 80%), the clear layer is large, with few granules; the heavy granules are well packed as compared with control eggs in sea water (Photographs 7 to 9). The white half-eggs (Photographs 2 and 5) are large, having the excess water. When centrifuged in hypertonic sea water (Photographs 10 to 12) the clear layer is small, the mitochondrial layer very thick and the heavy granules, yolk and pigment not well separated. The white half is small, containing less water as compared with the red half (Photograph 12). The red half remains about the same size in hypo- and hypertonic sea water, the change in water con- tent taking place in the white halves. The change in size of the nucleus is easily observed, since it lies in the clear layer in these centrifuged eggs. Compare Photographs 1, 2, with 10, 12. (Biol. Bull. 85:143, 1943. ) PLATE. XV. Stratification and breaking of unfertilized eggs when centrifuged in single salt solutions. The left column shows stratification of eggs cen- trifuged at 3,000 x g for two minutes in (1) NaCl or KCl; (3) sea water; (5) MgCl, or CaCl,. The right column shows amount of breaking apart at 10,000 x g for four minutes in (2) NaCl or KC]; (4) sea water; (6) MgCl, or CaCl,. The eggs stratify best in cal- cium, and least in potassium and sodium salts (Photographs 5 and 1). The order of decreasing stratification (increasing viscosity) is CaCl, > MgCl, > S. W. > NaCl > KCl. The ease with which they break into halves is in the reverse order, those in NaCl break most readily, those in CaCl, least readily (Photographs 2, 4, 6). The ease of breaking must be determined by the effect of the salts on the surface layers and not on the interior viscosity of the protoplasm. (Biol. Bull. 89:73, 1945.) PLATE XVI. Miscellaneous. Photographs 1, 2. Electrical method of determining sex, showing sperm and eggs shed from genital pores. 1. Male. 2. Female. 3. Egg showing jelly layer. 4. Jelly layer centrifuged off. 5. Amoebocytes, red and white. 6. Hyaline layer centrifuged off as a crescent near lower surface of egg. 7. Development without fertiliza- tion membrane. 8. Twins. 9. Cleavage in sea water lacking calcium, showing clear micromeres. 10. Bloated pluteus caused by not being fed. Photographs 11 to 13 of stained sections of developing eggs. 11. Mitotic figure of first cleavage of whole cell, metaphase. 14. Anaphase. 12. Mitotic figure of fertilized red half, metaphase. 15. Anaphase. 13. Mitotic figures of parthenogenetic merogone; note absence of spindle but well formed asters. In 11 and 14 there are two nuclei; in 12 and 15 one nucleus and in 13 no nucleus. (Photograph 6 Biol. Bull. 66:233, 1934 no. 2; Photograph 8 Biol. Bull. 78:205, 1940a no. 9; Photograph 10 Biol. Bull. 97:290, 1949 no. 14; Photographs 11-15 Biol. Bull. 79:185, 1940c nos. 136-140. ) Tests Plate |. rr? SS NEY RA 4 ty ga Kee vd SiGe Plate Il. Maturation of egg 13. 1% hrs. 14. 2 hrs, 10 min 15. 21% hrs 208 his Sere 39 15) hrs 24. JS ie Plate III. Normal development (living) 1. ee “. — : LS Tn acd : : ¥ foe oe * % Pe - 3th | ae oe She: ae ‘S | : 4 . 7 2 nse i232 ; # rie “oe ce -* fi a “s A 9 , . ‘ z 4 ig 4 ~- As 17 hrs 2 al/ahrs 3. 18 hrs a ae = P Aae ee: ae oo) yr yh 4 ae att 4 wile ot i a ae 4. 20 hrs ahr 9. 2 days 8. 2 days 7. 2 days Plate IV. Normal development (living), continued ca 10. 1% hrs 11. 1% hrs 12. 4 hrs Plate V. Normal development (sections) i) yi " ~ ae 10. 50 min 12. 56 min 7 > 14. 2 hrs 15. 22 hrs Pe 18. 9% hrs 19. 16 hrs 20. 1 day Plate 1X. White half, development 1. Unfertilized 2. 2 hrs 3. 3 hrs 4. 4 hrs 5. Unfertilized 6. 10 min 7. 20 min 8. 30 min a. fa 9. 1% hrs 10. 2 hrs 11. 3 hrs 13. 8 hrs. 14. 11 hrs 19. 4 hrs ‘ 21. 4 hrs 22. 6 hrs 234 7 hrs 24. 9 hrs Plate X. Red half, fertilized, development (Fertilized merogone) 1. Unfertilized 2. 3 hrs 3. 4 hrs 4. 18 hrs 4 7 9 eae . x x 4% ow ~—ailf », vn 7 << + he , e : 6. 8. 11% hrs 2 fa ‘ MOE me 9. 2% hrs 10. 3 hrs 11. 4% hrs 12. 5 hrs 13. 27 hrs ‘ >» 17. 4hrs 21. 3 hrs 22. 6 hrs 23. 10 hrs 24. 10 hrs Plate XI. Red half, parthenogenetic, development (Parthenogenetic merogone) ie | oie Plate XII. Clear quarter, and eggs centrifuged after fertilization 10,000 xg —p a” 80,000 x g 5 Amy dams 100,000 x g Plate XIII. Varying centrifugal force 125% Plate XIV. Centrifuged in hypo- and hypertonic sea water Va NaCl (KCI 5 Stratification MgCl, (CaCl,) Plate XV. Centrifuged in single salt solutions Breaking Plate XVI. Miscellaneous PART I GENERAL SUBJECTS CHP? TER’! Etymology, Nomenclature a. ECHINUS AND SEA URCHIN The sea urchin, Arbacia punctulata, belongs in the class of Echinoidea, this name coming from the Greek word éyivoc, meaning hedgehog. A sea urchin is a hedgehog of the sea, zovttog éyivoc, as distinguished from a land urchin or hedgehog of the land, yepoatog éyivoc, the word éytvos referring to the spiny nature of the animals. The name Echinus was used for the sea urchin by Aristotle and has been retained for the most common genus. The Engiish name “urchin” comes from the Middle English, urchon, which comes from the Old French, irechon or herichon (the modern French for hedgehog is hérisson), and this is derived from the Latin, ericius, meaning hedgehog. The scientific name for the hedgehog! which is common throughout Europe is Frinacius europaeus. Thus the words “urchin” and “‘echinoid”’ are derived, one from the Latin and the other from the Greek word for hedgehog. The Latin ericius is related to an older Greek word yp, also meaning hedgehog (cf. English “‘hirsute’’). The term urchin or street urchin for a small boy comes through the application of the term to a fairy or elf which often took the form of a hedgehog playing pranks on children; later it was applied to the small child himself. Urchin is used in the sense of elf or goblin by Shake- speare: “For this, be sure, tonight thou shalt have cramps, Side-stitches that shall pen thy breath up; urchins Shall, for that vast of night that they may work, All exercise on thee.” The Tempest I, ii, 325. (The notes say that urchins are hedgehogs or hobgoblins.) ’ “But they'll nor pinch, Fright me with urchin-shows, pitch me i’ the mire, ! The hedgehog is not found in America; the common Canadian and American porcupine (Erithizon dorsatum) is quite a different animal. 4 THE AMERICAN ARBACIA Nor lead me, like a firebrand, in the dark Out of my way, unless he bid ’em.”’ The Tempest II, ii, 4. ‘Like urchins, ouphes and fairies, green and white’’. The Merry Wives of Windsor IV, iv, 50. The country boys around Cambridge always call a hedgehog an urchin (D’Arcy Thompson, personal communication, February 1948). The French, oursin!, for sea urchin, has the same derivation (from the Latin ericius) as the English urchin, and is said to be a corruption of hérisson (hedgehog). It probably does not mean a little bear, as some- times stated. The Italian name for sea urchin, riccto di mare or riccio marino, is from the same Latin root ertcius, and means literally a hedge- hog of the sea. The Spanish word is similar, ertzo de mar. The German word for sea urchin, Seeigel, also means hedgehog (Jgel); the older German word was Meerigel. The modern Greek word for sea urchin is achinos, similar to the ancient Greek. In the earlier British literature, sea urchins were often referred to as sea hedgehogs, as for instance by Sir Thomas Browne (1658) in his Garden of Cyrus, where he philosophizes on the arrangement of the plates and organs in the sea hedgehog in series of five: ““By the same number doth nature divide the circle of the sea star, and in that order and number disposeth these elegant semicircles, or dental sockets and eggs in the sea hedgehog.” Sir John Hill (1752) also refers to the sea hedgehog. Sea urchins are still called sea hedgehogs in certain parts of England and Scotland. In A History of British Starfishes by the cele- brated British naturalist, Edward Forbes (1841, p. 141) there is a most interesting woodcut, showing two boys at the seashore with a sea urchin and a hedgehog, apparently greatly amused at their resem- blance (Fig. 1). Forbes calls them egg-urchins and sea-eggs, and they are still called sea-eggs in some regions of England and Scotland; in Jamaica, they are usually called sea-eggs. The term sea-egg probably does not refer to the eggs or to their use as food, but is rather based on the appearance and texture of the bleached, bare test as found on the beaches (H. L. Clark, 1933, p. 82). Sir Hans Sloane (1725) de- scribes the fossil shells in the chalk pits of Kent as becoming filled with a fine chalk and used as a medicine for digestive troubles, whence they are called chalk eggs. The fossil spines of a large species are known as Jewstones, or JFudeo de mer (see Oxford Dictionary). In an interesting 1 The French ours (bear) comes from the Latin ursus and this from a different root, &pxtoc. ETYMOLOGY, NOMENCLATURE 5 letter from Governor Winthrop of Connecticut in 1670 to the Royal Society of London concerning some strange animals found there, he refers to the sea urchin as an egg-fish or buttonfish. They are also known as sea chestnuts by the English because of their resemblance to the spiny burrs of the chestnut; they are also called sea thistles, needle shells and porcupine stones and, at Plymouth, whore’s eggs. The fishermen at Mousehole, in Cornwall, call them zarts, doubtless an old Fic. 1. Sea urchin and hedgehog, from a woodcut of Edward Forbes, A History of British Starfishes and other Animals of the Class Echinodermata (London, 1841). Cornish word (Trewavas, 1922). Sea urchins are called porcupines, chestnuts, burrs, spikes and whore’s eggs by the collectors and fisher- men.at Woods Hole, Mass. and at Beaufort, N. C. The French call them chdétaignes de mer (sea chestnuts) and the shells without spines oranges de mer and pommes de mer. The Germans, similarly call them See- Kastanien and See-Apfel. The Genoise call them zincin (Rondelet, 1554). In his Natural History of Chile, Molina (1787, p. 165) describes a black sea urchin, Echinus niger, later called Arbacia nigra or Tetrapygus niger (see Mortensen Monograph II, p. 582) which he says has black eggs and is called the devil’s hedgehog, and is never eaten. 6 THE AMERICAN ARBACIA b. ARBACIA PUNCTULATA The name of the genus, Arbacia, was given in 1835 by John Edward Gray, who removed it from the genus Echinus in which Linneus (1758) had included all the 17 species of sea urchins. The name, according to L. Agassiz in his Nomenclator Zoologicus (1842-1846) has no special derivation but is a “‘vox euphon’’. Bell (1889) and Mortensen (1935) call it a “nonsense”? name, having no significance. The name was probably derived from Arbaces (reigned 876-848 B.C.) who was, according to some historians (e.g., Ctesias'), the founder and first king of the Medean Empire which was started by the rebellion of the Medean general, Arbaces, against Sardanapalus, the last of the Assy- rian kings. The Medean dynasty lasted from 876 B.C. until its over- throw by Cyrus in 559 B.C. It seems likely that the name was sug- gested to Gray by the historical poem Sardanapalus by Lord Byron, in which Arbaces is a Medean satrap aspiring to the throne of Sardana- palus; this poem was published in 1821, a few years before Gray used the name. Another genus taken from Echinus at the same time by Gray was Salenia, possibly suggested by another character, Salemenes, in the same poem. An Egyptian character named Arbaces also occurs in Bulwer Lytton’s The Last Days of Pompeii, but this book was published Sept. 1834, after Agassiz (Feb. 1834) had used Gray’s name, so that this was probably not the source of the name Arbacia. Arbacia just missed being called Echinocidaris, a name given by Des Moulins independently and at almost the same time as Gray’s Arbacia. Gray’s paper On the genera distinguishable in Echinus was read April 28, 1835, and published July 17, 1835 in the Proceedings of the Coological Society of London (see Bell, 1889). The name had been adopted by L. Agassiz in his Prodrome which was read in February 1834 and published in July 1836; Agassiz had known of Gray’s nomenclature through correspondence with him in 1834 before its publication. Des Moulins, not aware of Gray’s work, published his Etudes sur les Echinides August 15, 1835, (dated July 1835), and gave the name Echinocidarts to what Gray called Arbacia. There seems no doubt that Gray’s Arbacia has precedence over Des Moulins’ Echinocidaris, and will not be changed as have so many other names of sea urchins. The altercation as to priority may be followed by reading L. Agassiz’s Monographies d’ Echino- dermes, 1838, p. 17, and Des Moulins’ Etudes, 1835-1837, p. 207, and the short note of Bell (1889). ' Herodotus (484-424 B.C.) does not mention Arbaces, and according to some modern historians Arbaces was purely legendary. ETYMOLOGY, NOMENCLATURE 7 The specific name punctulata was given by Lamarck in 1816, and retained by Gray (1835) when he named the genus Arbacia, removing it from Echinus. The word punctulata doubtless refers to the shagreen- like surface of the dried test, though this is characteristic also of other species (personal communication from H. L. Clark, 1947). CHAPTER 2 Historical a. GREEKS AND ROMANS Sea urchins were well known to the ancient Greeks and Romans, and have been frequently mentioned in their writings as a food, together with oysters, snails, and other sea food. Even before Aristotle, the Echint were well recognized as a food, e.g., by Epicharmus (born ca. 540 B.C.) in his comic poem The Marriage of Hebe, and Archippus in his comic play The Fishes, written ca. 415 B.C. Hippocrates (ca. 460- 377 B.C.) also mentions them in his De diaeta. (See D’ Arcy Thompson’s Greek Fishes, p. 72). But we owe to Aristotle (384-322 B.C.) the first very detailed de- scription of the sea urchin, some of which is quite correct. Aristotle writes in his Historia Animalium iv. 5 (translation by D’Arcy Thompson, 1910, p. 5302-5314): “The urchins are devoid of flesh, and this is a character peculiar to them... There are several species (yévn) of the urchin, and one of these is that which is made use of for food; this is the kind in which are found the so-called eggs, large and edible, in the larger and smaller specimens alike; for even when as yet very small they are provided with them. There are two other species, the spatan- gus, and the so-called bryssus; these animals are pelagic and scarce. Further, there are the echinometrae, or ‘mother urchins’, the largest in size of all the species. In addition to these there is another species, small in size, but furnished with large hard spines; it lives in the sea at a depth of several fathoms; and it is used by some people as a specific for cases of strangury. In the neighborhood of Torone! there are sea 1 Torone (now Toron) was a prominent ancient town near the tip of Sithonia (or Langos) , the middle of the three peninsulas projecting from Chalcidice, the southern part of Mace- donia, into the Aegean Sea, southeast of Salonika. Aristotle was born (384 B.C.) not far from here, at Stagira (or Stavros), also in Chalcidice, his father being physician to the king of Macedonia, the father of Philip. After studying with Plato in Athens (367-347), Aristotle returned to Macedonia to instruct the son of Philip, Alexander the Great, who was later of great assistance in providing him with money and collections of animals. Pliny says (vill. 17; Bostock and Riley, vol. 2, p. 265) that Alexander employed some thousands of men in every region of Asia and Greece to collect animals for Aristotle. HISTORICAL 9 urchins of a white color', shells, spines, eggs and all, and that are longer than the ordinary sea urchin... ‘All urchins are supplied with eggs, but in some of the species the eggs are exceedingly small and unfit for food, singularly enough, the urchin has what we may call its head and its mouth down below, and a place for the issue of the residuum up above. For the food on which the crea- ture lives lies down below; consequently the mouth has a position well adapted for getting at the food, and the excretion is above near to the back of the shell. The urchin also has five hollow teeth inside, and in the middle of these teeth a fleshy substance serving the office of a tongue. Next to this comes the oesophagus and then the stomach, divided into five parts and filled with excretion, all the five parts uniting at the anal vent, where the shell is perforated for an outlet. Underneath the stomach, in another membrane are the so-called eggs [ovaries] identical in number in all cases, and that number is always an odd number, to wit five... The urchin uses its spines as feet; for it rests its weight on these, and then moving shifts from place to place.” It is on account of this description that the dental apparatus has been known as “‘Aristotle’s lantern.”’ The passage however, is somewhat confused in the original Greek, some manuscripts reading +o odyx (the body), and others +6 ctow« (the mouth). In the former case, the whole shell is compared to a lantern. D’Arcy Thompson prefers the latter, and gives as a translation, together with the original Greek and a discussion, in his Greek Fishes (1947, p. 71): “The sea urchin’s mouth (or oral apparatus) is from beginning to end a continuous structure; but in surface view it is not continuous, but looks like a lantern with the horn-panes? left out all round.’ His drawing showing the com- parison of the sea urchin’s lantern of Aristotle, with an antique lantern is reproduced in Figure 2. Rondelet (1554), Gesner (1558), and Aldro- vandi (1606) speak of Aristotle’s comparison with a lantern, but do not call it ‘“‘Aristotle’s lantern.’ It seems to have been first called “‘Aristotle’s lantern,”’ laternam Aristotelis, by Klein in 1734 (p. 41-42) and Plate 31, Figs. a, b, c). Several standard works on zoology state that Aristotle’s lantern was so named by Pliny (e.g., Bronn’s Thier- Reich, Bd. II, Abt. 3, Buch IV, p. 1068), but I have found no reference to the lantern in Pliny. 1 There are several species of white sea urchins, one of which is Lytechinus variegatus, the former Toxopneustes variegatus which occurs at Beaufort, N. C. and was used by E. B. Wilson in his classical studies published in the Atlas of Fertilization. The same species in Bermuda is usually brown, occasionally white. In Bermuda, Tripneustes (Hipponoé) esculentus is known as the white urchin. 2 Horn was used in the place of glass. 10 THE AMERICAN ARBACIA B Fic. 2. (A.) The lantern of Aristotle. (B.) A Greek lantern, from D’Arcy Thomp- son, A Glossary of Greek Fishes (Oxford University Press, London, 1947). In his De Partibus Animalium (translation by Wm. Ogle, 1911, p. 680) Aristotle repeats much of this description of the sea urchin, noting especially its spherical shape and the radial symmetry of its organs and its edible eggs, and adds: “Though the ova are to be found in these animals even directly they are born, yet they acquire a greater size than usual at the time of the full moon; not as some think, because sea urchins eat more at that season, but because the nights are then warmer, owing to the moonlight. For these creatures are bloodless, and so are unable to stand cold and require warmth.”’ In his De Generatione Animalium (v. 3, translation by A. Platt, 1910, p- 783%) Aristotle’s speculations are interesting but completely un- scientific. The sea urchins “‘have large and hard spines because the sea in which they live is cold on account of its depth (for they are found in sixty fathoms and even more). The spines are large because the growth of the body is diverted to them, since having little heat in them, they do not concoct their nutriment and so have much residual matter and it is from this that spines, hairs and such things are formed ; they are hard and petrified through the congealing effect of the cold.” Pliny (23-79 A.D.) has repeated in his Natural History (ix. 51, trans- lation by Bostock and Riley, 1890-1900, vol. 2, p. 427) some of the description of Aristotle. Pliny says that the sea urchin has spines in- stead of feet, has a mouth in the middle of the body on the under side, and has five ovaries, and notes that the eggs are bitter. He also, like HISTORICAL er Aristotle, refers to the white urchins at Torone; then follows this curious passage which has been repeated by many of the older writers: “Tt is said that these creatures foreknow the approach of a storm at sea, and that they take up little stones with which they cover themselves, and so provide a sort of ballast..., for they are very unwilling by rolling along to wear away their prickles (Fig. 3). As soon as seafaring persons observe this, they at once moor their ship with several anchors.” This passage, no doubt, refers to those species like Lytechinus variegatus and Psammechinus miliaris which normally cover themselves with shells, algae, etc., probably for concealment or as protection against light (see under Phototaxis and Light Reaction, Chapter 6). Pliny also, like Aristotle, refers to the eggs occurring at full moon! (Pliny, ix. 74; Bostock and Riley, vol. 2, p. 465). According to Pliny (xxxii. various chapters; Bostock and Riley, vol. 6) sea urchins were used as an antidote for certain poisonous plants (dorycnium and carpathum) and as a remedy for many ills, ulcers, inflammatory tumors, scrofula, eye troubles, etc., and the spines for strangury (cf. Aristotle). In some cases the shells were ground up in wine or vinegar, sometimes burnt; in other cases the eggs were used. Kidney stones were treated ‘“‘by drinking sea urchins, pounded, spines and all, in wine; the due proportion being one semi-sextarius”’ (about a cup) “of wine for each urchin, and the treatment being continued till its good effects are visible. The flesh, too, of the sea urchin, taken as a food is very useful as a remedy for the same malady.” “They should be burnt with vipers’ skins and frogs, and the ashes sprinkled in the drink (vinegar); a great improvement of the eyesight being guaranteed as the sure result.’ For methods of cooking see Poissons et Animaux Aquatiques au temps du Pline by Cotte (1944, p. 237). It is of interest that several places in Greece derive their names from Echinus. Pliny mentions an island named Echinussa (now Kimolos), one of the Cyclades in the Aegean Sea, near Melos, where fossil specimens are still abundant on the coast (iv. 23; Bostock and Riley, vol. 1. p. 322, and footnote). He also mentions a group of islands called Echinades, also called Kurtzolari, in the Ionian Sea off Acarnania on the west coast of Greece, at the mouth of the Achelous River (iv. 19; Bostock and Riley, vol. 1, p. 310 and footnote). There was also a town in Acarnania named Echinos, probably now Ai Vasili (iv. 2; Bostock and Riley, vol. 1, p. 273 and footnote). There are many references to sea urchins by other Greek and Roman 1 The lunar periodicity has been especially studied by Fox (1924a) and is taken up in Chapter 7. 12 THE AMERICAN ARBACIA TUMIDIS NON MERGIMVR. VNDIS. Nase Lilla tole empaertacmns sie rental Nem \ A LA AF 3 y ~ ae 2555 awn BOs Difcemeoexemplo cafus pranoffe futuross Pravifaanteminns namgucpericlanocent, Fic. 3. Sea urchins covered with pebbles during a storm. From Joachim Camerarius, Symbolorum et Emblematum Cent. IV, Francofurti, 1654. Above the figure: ‘‘We are not submerged by the swollen waves’’. Below: “Learn by my example to become acquainted beforehand with the chances of the future; for dangers foreseen do less harm”’. HISTORICAL 13 writers, both before and after Pliny. These are usually concerned with their use as a food or medicine or in relation to phases of the moon (see Fox 1924a, b; Zirpolo 1929; D’Arcy Thompson 1947) or to the storm. Among these writers may be mentioned: Ennius (239-169 B.C.) who spoke in his Hedyphagetica (ed. Vahlen, p. 220) of the “‘dulces echini”’; Lucilius (ca. 148-103 B.C.) who said in his Satzrae “‘luna alit ostrea et implet echinos”; Horace (65-8 B.C.) who said in his Epodes (v. 28) “horret capillis ut marinus asperis echinus’’; Dioscorides (first century A.D., contemporary of Pliny), a botanist who said of Echinus marinus that “‘it is good for ye stomach, good for ye belly and ureticall; the raw shell of which, being roasted does well to be mixed amongst detergentia medicamenta made for ye psorae (itch). Being burnt it cleanseth foule ulcers and doth repress excrecencies of ye flesh.” Galen (130-200 A.D.) spoke of sea urchins both as food and medicine. Atha- naeus wrote (after 228 A.D.) in his Detpnosophistae or Banquet of the Learned (iii. g1) that echini are ‘‘tender, juicy, easily digested, and when eaten with honey, vinegar, parsley and mint they are whole- some, sweet and good-tasting. In some places they are rather bitter, and those in Sicily act as a laxative.” St. Augustine (354-431 A.D.) wrote in his De Civitate Dei (v. 6) “‘Et lunaribus incrementis atque decrementis augeri et minui quaedam genera rerum, sicut echinos et conchas.”’ b. MippLe AGEs Michael Glycas, in the twelfth century, in his annals of events from the beginning of the world speaks of the forecast of a storm by the sea urchins loading themselves with stones as described by Pliny. The poet Manuel Phile (ca. 1275-1340) also refers to this belief in one of his natural history poems, De Echino Aquatili, and Joachim Camerarius (1534-1598) speaks of it in his Symbolorum et Emblematum (Centuria quarta, p. 51) and shows a fine woodcut of a sea urchin with the pebbles on its back (Fig. 3). In his encyclopaedia, De Proprietatibus Rerum, Bartholomaeus (An- glicus) (ca. 1240) wrote concerning hedgehogs and sea urchins: ““The Urchin is a beast heled with pricks, hard and sharp, and his skin is closed about with pikes and pricks, and he closeth himself therewith. And he is a beast of purveyance”’ (i.e. the hedgehog). “And there is a manner kind of Urchins with a white shell and white pikes, and layeth many eggs... In Urchins is wit and knowing of coming of winds north or south... Also the Urchin breedeth five eggs better than other, and the eggs of some be much and great, and some be less; for some be 14 THE AMERICAN ARBACIA better to seething and to defying (i.e., digesting) than other... Also Urchins have a little body and many pikes” (this is, of course, the sea urchin). This is from the English translation of J. Trevisa (1398) from Seager’s Natural History in Shakespeare's Time (1896). Chaucer (1340?—1400) refers to sea urchins in his Boethius: “Sharpe fishes that highten echines”’ (Skeat ed., 1897, Bk. III, Meter VIII). c. RONDELET TO Louris AGASSIZ Rondelet (1507-1566) is probably the first writer after Pliny to give a description of the sea urchin all of which was not taken directly from Aristotle and Pliny. It is said that his figure (1554) of a sea urchin cut across ‘“‘so that it may be observed better” is the earliest figure of a dissected invertebrate (see Singer, 1931, p. 95) (Fig. 4). He also de- scribes the dental structure which, he says, was compared to a lantern by Aristotle; and he remarks that “‘there is nothing in the whole sea more elegant and pleasing to look at.” Belon (1553) added nothing to Aristotle’s account. Gesner (1558, 1563) and Aldrovandi (1606) have for the most part repeated Ronde- let’s description and reproduced his figure of a dissected sea urchin. Descriptions and classification of sea urchins made some progress through Rumphius (1705), Breynius (1732), and Klein (1734). The accepted binomial classification dates from Linneus (1758), though he grouped all the Echinoids under one genus, Echinus, in which he included 17 species; Klein had divided them into 24 genera and 60 species. Leske (1778) reintroduced Klein’s classification, using the binomial nomenclature. Lamarck in 1816 described the species Echinus punctulatus, which Gray in 1835 changed to a new genus Arbacia punci- ulata. Both Lamarck (1816) and Cuvier (1817) and many later writers included the Echinoderms among the Radiata together with Coelen- terates, Infusoria, etc. The term Echinodermata originated with Klein (1734) to include only the Echinoidea ; then the Asteroidea were added (by Bruguiéres 1789) and then the Holothuroidea and Crinoidea (by Lamarck 1816); see Cuvier (1834, p. 537). The Echinodermata were established as a primary division of the animal kingdom by Leuckart in 1848. But, though this classification was generally accepted, L. Agas- siz in his Essay on Classification (1857, p. 71) still maintained that “the undivided type of Radiata appears to me as one of the most natural branches of the animal kingdom, and I consider its subdivision into Coelenterata and Echinodermata as an exaggeration of the anatomical differences between them.” HISTORICAL [5 ———s SAAR, — WOO = —— tH) Fic. 4. Rondelet’s figure of a sea urchin, from De Piscibus Marinis, Lugduni, 1554. This is said to be the earliest figure of a dissected invertebrate. The history of the classification of the sea urchins, including Arbacra, and the chronology of the nomenclature (and a comprehensive biblio- graphy) are given in A. Agassiz’s classic monograph Revision of the Echini (1872-1874). A good historical review is given in Bronn’s Thter- Reich (1904, Bd. II, Abt. 3, B. IV, p. 1002, 1007, 1321-1338), and in Lambert and Thiéry (1909, p. 9-21); a short account is given in Lan- kester’s Treatise on Zoology (1900, Part 3, p. 282-285). Tiedemann (1816) added to our knowledge of the structure of the sea urchin. A very detailed description of the anatomy of Echinus lividus 16 THE AMERICAN ARBACIA has been given by Valentin (1841), in a monograph included in L. Agassiz’s Monographies d’ Echinodermes ; another of these monographs is that by L. Agassiz on Des Salénies (1838), in which he mentions the genus Arbacia as having been established by Gray. A most interesting book of this period is one referred to previously, by the celebrated marine naturalist, Edward Forbes, entitled A History of the British Star- fishes, and other Animals of the Class Echinodermata (1841), and dedicated to Louis Agassiz. It is a somewhat popular but scientific account of animals belonging to the five main classes of Echinoderms, including several different kinds of British sea urchins. He reckoned that in a moderate sized common British sea urchin, which he calls Echinus Sphera (E. esculentus), there are 1,860 suckers, and twice that number of pores (3,720) and about 600 plates “‘all dove-tailing together with the greatest nicety and regularity, bearing on thcir surfaces above 4,000 spines, each spine perfect in itself, and of a complicated structure, and having a free movement on its socket. Truly the skill of the Great Architect of Nature is not less displayed in the construction of a sea- urchin than in the building up of a world!” (p. 153). Especially inter- esting are the many woodcuts, some of anatomical features, some of scenes, and some imaginative; one of these has been reproduced in the present Monograph as Fig. 1. The great Swiss naturalist, Louis Agassiz (1807-1873) made im- portant contributions to our knowledge of the Echinoids, both living and fossil, his work being followed by that of his son Alexander Agassiz (1835-1910). The Jate R. T. Jackson (1861-1948) published in 1912 an important volume on the Phylogeny of the Echini and in 1927 Studies of Arbacia punctulata and Allies, a detailed study of the variation of the skeletal parts. d. MopERN The two recent authorities on living sea urchins are the late Danish investigator, Th. Mortensen (1868-1951) and the late American in- vestigator, Hubert Lyman Clark (1870-1947). The large Monograph of the Echinoidea by Mortensen (1928-1951), consisting of 16 quarto vo- lumes of text and plates and an index, was completed just before his death. It is a monumental piece of work devoted to the Echinoidea and a masterpiece of completeness and accuracy. It contains an excellent and very comprehensive account of Arbacia punctulata and its relatives (referred to here as M II: 529-580, Mortensen, vol. II, p. 529-580). These two eminent authorities are in general harmonious, but disagree on a few points of classification and relationships, e.g., Diadema and HISTORICAL i7 Centrechinus. Mortensen holds to the old name of Diadema, which Clark abandoned for the ‘‘proper’’ name of Centrechinus (Clark, 1925, p. 41; 1946, p. 278; Mortensen, 1940, M III, 1: 243).? e. HisrorY OF EMBRYOLOGY Fertilization (by artificial insemination) of the living sea urchin egg was first described by Derbés in 1847, in Echinus esculentus ; he also described and figured the cleavage and development to the well- developed pluteus of about two weeks; stages described as later than that are probably degenerative and not developmental forms. A little earlier in the same year, 1847, cleavage was described, probably for the first time, from observations made in 1845 on the same species, by von Baer of St. Petersburg; he described the development only to the free-swimming blastula just after hatching. Dufossé (1847), also before Derbés, described cleavage, hatching and early development of Echinus esculentus, but this he believed to be entirely radial, and his description is confused and difficult to follow without illustrations. Some years later, a detailed study was made of fertilization and early cleavage of the eggs of the starfish and the sea urchin, Toxopneustes lividus (Paracentrotus?) independently and almost simultaneously by O. Hertwig (1876) and Fol (1877, 1879), and of Toxopneustes variegatus by Selenka (1878). f THE PLoUrevs The name of pluteus for the larval form of some Echinoderms was originated by Johannes Miiller in 1846. He gave the name Pluteus paradoxus (1846a) to what he thought was a new animal which came from the North Sea near Helgoland. Later in the same year (1846b), he found this to be the larval form of an Ophiuran, later identified as Ophiura albida (Mortensen, 1921, p. 14). The Latin word pluteus he translated into German as Staffelei or Gestell, and the English have translated the German into easel. The word was chosen because of the resemblance of the larva, turned upside down (as Miiller always drew them) to an easel. ‘“‘Wir sehen ein Gestell vor uns aus 2 Seiten- leisten, die nach oben convergieren... nach unten divergieren und von 1 The long controversy between Mortensen and H. L. Clark (and others) concerning Diadema vs. Centrechinus was finally settled in November 1953 in favor of Diadema (Inter- national Commission on Zoological Nomenclature, Opinions and Declaration vol. 3, Opinion 206). The controversy began with R. T. Jackson in 1912. 18 THE AMERICAN ARBACIA denen jede noch ein FuBgestell von 2 Staben abgiebt... Da einmal alles einen Namen haben mul, so mag dieser Korper Pluteus heifen, was so viel als Staffelei oder Gestell bedeutet”’ (1846a, p. 108, 110). The Latin word, pluteus, however, is according to both Latin-German and Latin-English dictionaries usually applied to a military structure, shed or shelter (from pluvia, rain), or to a stand or bookcase. But, of course, the shape of a shelter might have suggested an easel to Miiller. The English word, easel, comes from the Latin asellus, or little ass, which carries things. Johannes Miller published many papers on the plutei of different species of Echinoderms and their metamorphosis between 1846 and 1855, including a series of nine in the Abhandlungen der Koniglichen Akademie der Wissenschaften zu Berlin ; these are classics and the illustra- tions are particularly fine. A very good list of studies on larval forms arranged by species, up to 1921 is given by Mortensen (1921, p. 12, and a list arranged phylogenetically by Fell, 1948). Among the most complete studies are those of Arbacia punctulata by Brooks (1882) and E. B. Harvey (1949); A. dixula and Paracentrotus lividus by von Ubisch (1913 a, b); Echinocyamus pusillus by Théel (1892); Echinus esculentus by MacBride (1903, 1914a. Text-book of Embryology vol. 1, p. 504, and by Shearer, De Morgan and Fuchs (1914); Enchinocardium cordatum, by MacBride (1914b); Heltoctdaris crassispina, Mespilia globulus, Strongy- locentrotus pulcherrimus and other Japanese forms by Onoda (1931, 1936). Especially notable are the Studies of the Development and Larval Forms of Echinoderms by Mortensen (19213; 1931, I and II; 1937, III; 1938, IV). This last series (I-IV) comprises studies made during expeditions to Kei Islands (Amboina), Java-S. Africa (Onrust, Mauritius) and Egypt (Ghardaqa). See p. 117. The pluteus of Arbacia is somewhat different from that of other sea urchins. The development of the three or four day pluteus of the Euro- pean species, A. lixula was described by Busch in 1849, and later stages by Johannes Miiller in 1854 under the name of Echinocidaris aequituber- culatus ; he figures the six-armed stage, which has been copied in several textbooks, and the later stages and metamorphosis. A more complete study of A. lixula has been made by von Ubisch (1913a, b, c, 1927, 1932). The late pluteus of A. stellata (incisa) from the Gulf of Panama, which is similar to that of A. punctulata and A. lixula, has been described and figured by Mortensen (1921 p. 29, Plate VII, Fig. 3). The development of Arbacia punctulata, the pluteus and metamor- phosis, is quite similar to that of A. lixula (see Chapter 15 and Plate VI of this Monograph). The pluteus was first raised by A. Agassiz at his HISTORICAL 19 laboratory at Newport, R. I. Several stages were described and figured by him in his Revision of 1872-1874, the six-armed stage (p. 729, Fig. 66), and the very young metamorphosed form three weeks older, with primitive spines and tube feet (p. 734, 735, Figs. 68, 69, and Plate V, Fig. 9). A more complete account has been given by Fewkes (1881), working in Agassiz’s laboratory, and a more detailed description of the late stages by Agassiz himself (1904, p. 53, and Plates 53, 54). Another group of investigators studied the development and metamorphosis of Arbacia punctulata at Beaufort, N. C. W. K. Brooks has published these observations together with excellent drawings in his Handbook of Invertebrate Zoology (1882), a laboratory manual written for his students at Beaufort. He has included observations and drawings of his students, Garman and Colton, published in 1883. A. Agassiz has selected the best figures of A. punctulata (Figs. 38-45) for Plate IX of his monograph of 1883 a from both groups of investigators, and figures of A. lixula (Figs. 20-25; 28-37) from Busch and J. Miiller. CHAP E Rs2 Fossils The oldest fossil sea urchin is probably Bothriocidaris'! from the Ordo- vician of Russia (Esthonia). This fossil was accepted as the ancestor of the Echinoidea until Mortensen (1928, 1930), the eminent Danish authority, considered it a cystoid and not an echinoid. Mortensen’s views were opposed by equally good authorities, Hawkins (1929, 1931), Jackson (1929), and H. L. Clark (1932, 1946), but Mortensen in his comprehensive monograph of 1928-1951 (1935, M II, p. 15) still holds to his own view. However, Jackson (1912, p. 208) has said: ““The most primitive type of Echini, I believe emphatically, is Bothriocidaris.”’ Though rare in the Ordovician, and few in the Silurian and Devo- nian, fossil sea urchins are abundant in all periods after the Devonian, and have been excellent material for the palaeontologist. There are about 6,000 fossil species (Grassé, 1948, p. 162; Clark, 1946, p. 277). Fossils of sea urchins were scattered over northern Europe during the glacial period, and these have played a part in folklore. They were regarded as thunder stones, fallen from heaven with the thunder, and thus supposed to protect against thunder. They were also used in prehistoric times as amulets (Mortensen, 19274, p. 9). Fossil spines of some large sea urchins are known as Jewstones or Lapides Fudaici. These were especially well known in Syria and Pale- stine, where they were used in medicine as a diuretic (Sir John Hill, 1751; Oxford Dictionary). They have been found also, together with the tests filled with chalk in the Chalk Pits of Kent. These “‘chalk-eggs”’ were used to cure digestive troubles and were therefore saved by the workmen (Sir Hans Sloane, 1725). Fossil sea urchins are used by oil companies to determine the age of the beds at certain depths. This is especially true of the Cretaceous of Texas. The family of Arbacitdae is known from the Tertiary to Recent (Jackson, 1912, p. 209) and there are many fossil genera in the family (Mortensen, 1935, M II, p. 548). Fossil tests and spines of Arbacia 1 The statement by Jackson (1912, p. 244) that Aldrovandi figured Bothriocidaris in 1606 is apparently a mistake (Mortensen, 1913a, 1928; Jackson, 1929). FOSSILS 21 punctulata (called Anapesus carolinus or Echinus punctulatus) and also of Lytechinus variegatus have been found well preserved in the post-Pliocene beds of South Carolina (Holmes, 1860). They were taken from excava- tions made for tidal drains in the upper part of the city of Charleston. A little earlier figure (without description) similar to the one of Holmes under the name “‘Echino-cidaris, species doubtful,”’ from the Pliocene of South Carolina is given by Tuomey and Holmes (1857). Mortensen (1935, M II, p. 566 and 1943, M III 2, p. 446) considers this fossil as possibly an ancestor of the present Arbacia punctulata, and the Ana- pesus carolinus of Holmes as identical with the present form. (See also A. Agassiz, 1883b, p. 85). Lytechinus fossils have been found in Bermuda when dredging during the construction of the air base at Castle Harbor in 1941-1943 (H. B. Moore and D. M. Moore, 1946). CHAPTER 4 Uses of Sea Urchins a. As Foop Sea urchins have been used by man as food since ancient times, especially around the Mediterranean and in tropical countries. Only cer- tain species are edible and only the gonads; both sexes are usually used, but in some places only one sex, usually the female. In general they are eaten raw, often with a little lemon, but in some places they are cooked. The ancient Greeks and Romans used sea urchins (echini) as food together with oysters and clams; they are mentioned by Epicharmus (b. ca. 540 B.C.), Archippus (ca. 415 B.C.), Aristotle (384-322 B.C.), Ennius (239-169 B.C.), Horace (68-8 B.C.), Pliny 23-79 A.D.), Athe- naeus (ca. 228 A.D.) and many others. Sea urchin shells have been found in the kitchens of Pompeii (Kellar, 1913). Sea urchins usually preceded the main course of a dinner, and were often highly seasoned “with honey, vinegar, parsley and mint” (Athenaeus, Deipnosophistae iil. g1). Several authors have written of the famous supper of Len- tulus when he was made priest of Mars, at which echini were the first dish (see Pennant, 1777, p. 68). They were also cooked, and several recipes have been handed down by the gourmet of ancient times, Apicius (80 B.C.-40 A.D.). Among them is the following: ‘Put the urchins singly in boiling water, cook. To the meat thus cooked, add a sauce made of bay leaves, pepper, honey broth, a little oil, bind with eggs, sprinkle with pepper and serve.” In Italy, sea urchins are known as ricci di mare, and together with oysters and other sea food as frutta di mare. In Naples they are sold in the markets and by the fishermen along the water front. Paracentrotus lividus is the form usually eaten; Sphaerechinus granularis is also eaten but mostly by the fishermen; it is difficult to get because it lives in deep water and occurs mostly only around Gajola. Psammechinus micro- tuberculatus is too small and Arbacia lixula is not eaten. The native fishermen have the curious idea that Arbacia are the males and not edible, and Paracentrotus are the females and edible, so that one asks for USES OF SEA URCHINS 23 females in the market. If the animals are in water too deep to be taken by hand, the fishermen dive and grasp the urchin with a “canna’’, a reed pole with a split end. Paracentrotus is eaten in other places in Italy and also in Sicily. In modern Greece, sea urchins are eaten raw or cooked with rice. They are also eaten in Portugal and in South Ame- rica, Loxechinus albus in southern Chili (Bernasconi, 1947). In France, Paracentrotus lividus is commonly used as food along the Mediterranean, and this species but not the large Echinus esculentus is eaten on the north coast (Roscoff) and is also sent to Paris, where one obtains them at oyster bars. Over a million sea urchins a year are brought into the fish markets of Marseilles, and sold (before 1904) for 20,000 francs (Bronn’s Thier-Reich, p. 1307). One also dips bread into them“ a la maniére des oeufs—a la mouillette”’ (Dujardin et Hupé, 1862, p. 458); this is called une oursinade. When cooked they are said to taste like crayfish (écrevisse). The early French writers, Belon (1553) and Rondelet (1554) called the edible sea urchins doulcins or doussins and the non-edible ones rascasses (a very bony fish, Scorpaena, used chiefly in making bouillabaisse). In England to-day sea urchins are not commonly eaten. Pennant in 1777, p. 68, said they “‘are eaten by the poor in many parts of England and by the better sort abroad.” According to several present-day British biologists, they are not eaten at the marine stations. They are not usually eaten by Americans. On the Pacific Coast, at Pacific Grove, both Strong ylocentrotus franciscanus and S. purpuratus are eaten, mostly by the Italian and other immigrants. However, some Americans have found ‘“‘the gonads of S. franciscanus, eaten a l’Italienne (raw) with French bread, very good—extremely rich, and possibly more subtle than caviar. If it were not for the fact that the race is already being depleted by the appreciative Italians, urchins could be highly recommended as a table delicacy” (Ricketts and Calvin, 1948, p. 58). Some years ago, the Chinese at Pacific Grove sent quantities of sea urchins to China to be used as food (Kellogg, 1899). S$. francts- canus is eaten by the Indians and Greeks in British Columbia. S. dréba- chiensis is eaten by the inhabitants of the Aleutian Islands and Alaska. Even in New York City, one finds sea urchins for sale in the markets and on the streets of Greenwich Village, where they are bought and eaten mainly by Italians; they cost about five cents apiece. They are also served in a few restaurants in New York. These are S. drébachiensts, and come from the northern waters around Rhode Island, Boston, and Maine. Arbacia punctulata and A. lixula are not usually eaten. In many tropical countries, sea urchin gonads are commonly eaten, 24 THE AMERICAN ARBACIA both raw and cooked. In the West Indies, the natives take the gonads of several individuals of the large Tripneustes and bake them in the half shell of one (H. L. Clark, 1933, p. 82). When fresh and properly cooked they are said to be as good as any fish roe. In the Barbadoes, it has been necessary to pass laws regulating the gathering and sale of Tripneustes, for the persistent demand threatened their extinction. The eggs of this form, called chardron blanc, are made into an omelet in Martinique (Cotte, 1944, p. 238). They are eaten by the Portuguese in Bermuda. The Malays near Singapore use Diadema saxatile (Centrechinus setosus) as food (Bedford, 1900). In Japan at least six species are edible, in the north (Asamushi), south (Kyushu), and at Misaki, and some have commercial value. They are expensive, costing about fifty cents apiece (Motomura, personal communication). The gonads are eaten raw, sometimes with a little lemon, and also cooked. They are boiled and eaten with a little lemon, or used as a soup. They are often placed on a shell and heated in a fire, and this dish is said to be very good (Motomura). A customary way of preparing the gonads is to mix them with about three times as much salt and make a paste. This is then stored in very attractive special jars and used, about a half teaspoonful at a time, as a relish. It is said to taste like caviar (Inoué, personal communication). b. As MEDICINE As a medicine Pliny recommended sea urchins, both raw and cooked for many ills, ulcers, tumors, kidney troubles, etc. (see under Historical). Galen, Athenaeus, and other writers also speak of their value as a medicine. In more modern times, it is said that in certain parts of the Midi of France, one drinks the perivisceral fluid to help digestion; the dose is half a glass a day (Mourson and Schlagdenhauffen, 1882). The use of ‘‘chalk eggs’ and Jewstones has been mentioned under Fossils. In Dalmatia also the shells are used as an astringent (Faber, 1883). For their use as food and medicine, see D’Arcy Thompson’s Greek Fishes and Tortonesi (1939). c. As Foop FoR OTHER ANIMALS Besides serving as food for man, sea urchins are eaten by many fish (Bronn’s Thier-Reich, p. 1302), and in Cape Cod waters Arbacia and Strongylocentrotus are devoured by the cod and haddock (A. Agassiz, 1872-1874, p. 707). Arbacia are consumed by starfish, spider crabs USES OF SEA URCHINS 25 and also by other Arbacia, even while still living. In northern regions, Strongylocentrotus driébachiensis is eaten by the arctic fox, sharks, sea otters, and sea birds (Mortensen, 1943, M III 3, p. 209). A picture of sea otters eating sea urchins while swimming is reproduced as Figure 5. Fic. 5. Sea otter eating sea urchins, after Barabash-Nikiforov, The Sea-Oiter, in Russian, 1933. From Mertens 1935. d. Usrt or SHELLS As Cups, LAmps, INK Shells of sea urchins were used in ancient times by apothecaries to hold drugs, as mentioned by Hippocrates, Lucilius, and others. In modern times shells of the large species are used as lamps with an electric bulb inside. In Italy, Sphaerechinus is so used and in Sweden and England, Echinus esculentus. In Monaco, the shells of Paracentrotus lividus are beautifully prepared at the Musée Océanographique (for sale) and are most striking with a small bulb inside making all the little holes for the tube feet light up in rows, as shown in Plate I, Photograph 6. In Japan the shells are filled with a fish oil and provided with wicks (Robins, 1939, p. 126). In some places in England (Robin Hood’s Bay), Echinus esculentus shells are used as flower pots, especially for cactus. In Maine, sand dollar shells (Echinarachnius parma) are ground up and used as an indelible ink (Verrill and Smith, 1874, p. 69). e. In ART, ON COINS AND AS JEWELRY Figures of sea urchins are to be found in ancient mosaics and on vases (Keller, 1913). A reproduction of a mosaic in the baths of Medeina showing various sea animals including (probably) a sea urchin, is to 26 THE AMERICAN ARBACIA be found in Dahremberg, Saglio, and Pottier (1904, t. III, part 2, p. 2116). Imprints of sea urchins were stamped on ancient coins, e.g., from Segesta and Teos (see Plate VIII, Figs. 42, 43 of Imhoof-Blumer und Keller, 1889). In architecture, a part of the capital of a Corinthian or other column is known as Echinus (see Oxford Dictionary). Necklaces of sea urchin shells were apparently used in olden times, as pictured by Klein (1734). Use of fossil sea urchins by oil companies and as amulets has been described under Fossils. f. Usk As EXPERIMENTAL MATERIAL Perhaps the most important use of sea urchins is as experimental material for biological work. The extensive use of the eggs and sperm is made evident by the large compendium which follows as Part IV of this Monograph; this deals mostly with only one species, Arbacia punct- ulata, and with investigations carried out almost exclusively at Woods Hole. Experiments on other species done in Naples, Sweden, Denmark, England and other places are equally if not more extensive. CHAPTER 5 Description a. SIZE AND SHAPE. PLATE I The shells (without spines) of Arbacia punctulata, collected at random at Woods Hole during many summers measure 0.6—5.6 cm. in dia- meter and 0.3-2.7 cm. in height. They are roughly twice as broad as high. The spines measure 0.35~-2.5 cm. in length. The overall dia- meter (including spines) is 1.3-10 cm. The average animal has a dia- meter of about 4 cm. without spines and about 8 cm. with spines, and a height of about 2 cm. The spines vary in length and in shape in different regions of the body. The spines on the oral surface are flat- tened and short, those on the aboral surface are long and pointed. Jackson (1927) measured 14,100 specimens of Arbacia at Woods Hole during the summers of 1913, 1914, and 1915 and his figures are quite similar to mine; his smallest one measured 0.5 cm. in diameter (with- out spines), and the largest ones 5.3 cm. The most numerous were 3-4 cm. in diameter. There is considerable variation in the shape of the shell, some shells being more flattened than others, and some quite conical. D’Arcy Thompson (1942, p. 944) in his Growth and Form has an interesting discussion on the shape of sea urchin shells, suggesting that they are similar to drops of liquid and flatten by their own weight, the small ones remaining spherical. The conical shells, he thinks, may be due to accumulation of lighter substances, such as oil in the eggs, in the upper part of the shell. Lowndes (1944b) has pointed out some of the fallacies in these arguments; he found that the shells will withstand heavy weights (4 kilos) placed on them, without injury, and calls attention to the fact that there is very little oil in the eggs. Measure- ments have been made of the diameter and height of a group of 26 dried shells of Arbacia of varying sizes and it was found that the ratio diameter/height in the largest shells (4.2 to 4.7 cm. diameter) was 1.96/1; in the medium sized shells (2.0 to 2.7 cm.) it was 1.956/1; in the smallest shells (1.53 to 1.95 cm.) it was 2.01/1. There is no flattening with age. Even in a newly metamorphosed animal, the body is not spherical, but somewhat flattened. 28 THE AMERICAN ARBACIA The Arbacia punctulata collected by me in 1942 at Beaufort, N. C. are considerably larger than those at Woods Hole, averaging 5 cm. in diameter (without spines) and 3 cm. in height; their spines are more slender and longer, measuring up to 3.5 cm. The eggs of the Beaufort form are also larger, 80 u in diameter as against 74 u for the the Woods Hole form. Jackson (1927) thinks that “the Florida material differs enough from the Woods Hole type so that it might well be considered a distinct local form” (p. 449). b. CoLor The color of Arbacia punctulata is quite variable, from reddish gray to reddish to purplish to brownish to almost black. Young individuals are inclined to be lighter. There is no change in color when kept in the light or dark for short periods, as shown by Parker (1931) for a period of 10 hours. I have confirmed this several times, and Kleinholz (un- pub.) has also found this. But they are affected by light if kept for longer periods. A group of six animals of medium coloration which I kept in the dark room at Woods Hole (in 1948) were decidedly lighter than a similar group kept in bright light in a laboratory room after a period of two months; there was a slight difference even after a week. I have carried out similar long-time experiments for several summers. Dark animals kept in the dark become darker (black) ; dark animals kept in the light become lighter (brownish). Light animals kept in the dark become greyish; light animals kept in the light be- come reddish or purplish. The results are complicated by the fact that there are at least two different kinds of pigment in the test and spines, a melanoid and an echinochrome, and that the test and spines are different in color. For pigments in A. lixula and P. lividus see Glaser and Lederer (1939). Another experiment was carried out (summer of 1954), using the same animals for light and dark exposure. A group of six Arbacia of different sizes were kept in an aquarium in a very bright room with sunlight (in the mornings) for a month, and kodachrome pictures were then taken. This same group was then kept in a dark room under exactly similar conditions (not fed), and completely shielded from any light, for a month. Kodachrome pictures were taken on the same film with exactly the same conditions of lighting, exposure, etc. When exposed to the light the animals were decidedly more red, and there was also more black pigment. Ultraviolet light causes the animals to become reddish (E. B. H. DESCRIPTION 29 unpub.). H. L. Clark (1933, p. 80) found a difference in color in northern and southern forms. ‘“‘While northern specimens have the test commonly a deep brown, with a reddish cast, and the spines lighter and often a dull brownish red, in the south there are two ex- tremes; on the one hand, the test is a light wood-brown, with spines very light (a dingy cream color) at the base, becoming dull reddish purple at the tip, and on the other, test and spines are deep reddish purple or almost black in certain individuals. Both these extremes are observed in specimens from Florida. Specimens from Cuba, Yu- catan, and Tobago, are very dark and have slender, relatively long, spines.” The color change in A. /ixula was studied many years ago in Naples by von Uexkiill (1896), who found that they became black in the light and brown in the dark. I confirmed this in Naples in 1934 (unpub.) finding that light specimens of A. lixula became darker in the light and dark specimens became lighter in the dark over a period of a month; in some individuals which were isolated, the change was noticeable within a day. Kleinholz (1938) found that from a group of 12 dark specimens of A. lixula at Naples the ones kept in the light for 6 hours remained dark, while those kept in the dark became brownish. Some dark individuals turned brownish in the dark in go minutes, and when brought into the sunlight became dark again in an hour. The two species of Arbacia react to light by change in color in much the same way, but A. /ixula reacts more rapidly. In nature A. lixula is much darker than A. punctulata. Centrostephanus longispinus (at Naples) reacts in much the same way, a dark animal turning gray in the dark within two hours (von Uexkiill, 1896; Kleinholz, 1938). Von Uexkiill found that the change in body color was due to changes in the chromatophores of the body skin which were expanded in the light and concentrated to small points in the dark. Kleinholz found that the tube feet isolated from an illuminated animal were brownish red while those from an animal kept in the dark were pinkish white, owing to the dispersed or contracted phase of the chromatophores. A still more rapid change in color occurs in Diadema (Centrechinus) antillarum in Jamaica, especially in the young animals; they become lighter, almost white in the dark (Millot, 1950). The change is due to changes in the large skin chromatophores, expanded in the light and contracted in the dark. Even a spot of light will cause the change. No albino specimens of Arbacia have been reported, but Prince (1913) has described an albino of Strongylocentrotus drobachiensis from 30 THE AMERICAN ARBACIA St. Andrews in New Brunswick, Canada; and H. L. Clark (1933, p. 79) states that albino or partially albino individuals of Centrechinus antil- larum are occasionally seen in the West Indies. Cuénot (1912) found entirely white specimens of Psammechinus miliaris at Arcachon “‘com- parable to albinos,”’ but he also found transitions to the usual color. Deep sea forms are often, according to Koehler (1927, p. 9) extremely brilliant. c. MorpHo.wocy. PLATE I At the aboral end of A. punctulata, surrounding the anus, there are (usually) four anal and five genital plates including the large madre- poric plate (Plate I and Figs. 6, 7). There may be only two or as many as fourteen anal plates; there are often three or five (Jackson, 1927, p- 464). Each of the five genital plates has typically one genital pore through which the eggs and sperm exude; there are frequently two genital pores in one plate, sometimes more, and in one case seven (Jackson, 1927, p. 458). Between and below the genital plates are five small ocular plates. There are considerable variations in the number and arrangement of plates (Osborn, 1898, 1901; Jackson, 1927). At the oral end project the five teeth of Aristotle’s lantern. The Jantern is composed of 40 calcareous pieces controlled by 60 muscles (Jackson, 1927, p. 483; Reid 1950). The test is made up of small pentagonal plates arranged in double rows, five narrow (ambulacral) and five broad (interambulacral) rows. These plates are studded with tubercles which bear the spines and pedicellariae. The pedicellariae! have been described by Agassiz and Clark (1908, p. 71). Tube feet project through a double row of pores arranged in ten lines, one on each side of the narrow (ambula- cral) row of plates. As growth occurs both by the increase in number of plates and by increase in size of each plate, the total number of plates depends on the size of the animal. It has been estimated that a large Arbacia (4.5 cm. diameter) has about 1,000 plates and a small Arbacia (2.5 cm. diameter) has about 700 plates. These figures are based on measurements made by the students in the invertebrate course at the Marine Biological Laboratory at Woods Hole in 1951, 1 The pedicellariae of Toxopneustes pileolus are poisonous (Fujiwara, 1935, Annot. Zool. Jap. 15 : 62-69; Mortensen, Monograph III 2, p. 469). The long pointed spines of the Diademas are poisonous (Mortensen, Monograph III 1 p. 249). See C. J. Fish and M. C. Cobb (Thayer), 1954, Noxious marine animals of the central and western Pacific Ocean. Research Report 36 of U. S. Fish and Wildlife Service. DESCRIPTION 31 under the direction of Dr. Ralph I. Smith of the Univ. of California. The plates of 72 animals were counted by members of the class, and there seems no doubt that the number of plates varies with the size of the animal. Jackson (1927, p. 471) states that the increase in size in Arbacia is attained more by the increase in size of individual plates than by increase in number; an animal 4.7 cm. in diameter had 16 plates in an interambulacrum, and one 1.4 cm. had to plates. A care- ful study of three Chinese (and Japanese) species, Temnopleurus toreu- maticus, T. hardwickit and Strong ylocentrotus pulcherrimus, by Hsai (1948) collected from Kiao-chow Bay, Tsingtao, showed that in young animals the number of plates is a function of the diameter of the test, but that as the animals approach maximal size, they stop forming new plates and growth takes place only by the enlargement of the plates. Many years ago, the celebrated naturalist Forbes (1841) estimated that in Echinus esculentus, a much larger form than Arbacia, there are in an animal of moderate size 600 plates, 3,720 pores, 1,860 suckers and 4,000 spines. There is a simple coiled alimentary canal, a water vascular system and a nervous system, but the main body of the animal (in season) is filled with the five ovaries or testes (Fig. 7). The ovaries are red, or brownish late in the season, and the testes are brownish white, the oozing sperm being white. The histological structure of the gonads and gonad walls of A. punctulata is described with drawings and photo- graphs by L. Palmer (1937) and L. Palmer Wilson (1940). A photo- graph of the ovary is given by Liebman (1950). In other species, the genital glands have been described by R. Koehler (1883) for A. lixula and many other species; by Hamman (1887) for many species; by Tennent, Gardiner, and Smith (1931) and Miller and Smith (1931) for Echinometra lucunter; by Lindahl (1932b) for Paracentrotus lividus ; by Aiyar (1938) for the Indian species, Salmacis bicolor ; and by Tennent and Ito (1941) for the Japanese species, Mespilia globulus. An excellent account of the structure of A. punctulata is given by Reid in Brown’s Selected Invertebrate Types (1950), written for the In- vertebrate Zoology course at Woods Hole (Figs. 6, 7). Other accounts of A. punctulata are those of: A. Agassiz (1872-1874) Revision of the Echini (pp. 263-266); H. L. Clark (1902) Echinoderms of the Woods Hole Region, p. 563; Coe (1912) Echinoderms of Connecticut, pp. 85-108; Jack- son (1912) Phylogeny of the Echini, (1927) Studies of Arbacia punctulata and Allies (external structure) ; Petrunkewitch (1916) Morphology of Inverte- brate Types, pp. 191-201. W. K. Brooks (1882) in his Handbook of Inver- tebrate Zoology describes and gives good drawings of the external and THE AMERICAN ARBACIA 32 ‘roystiqnd ay) pur “af ‘umoag “y ‘WP Jo Asoqanoo Aq psonpoiday ‘(OG61 ‘yI0X MAN ‘suog pur AattM uyoft) af ‘umoug ‘VW ‘a Aq porpa ‘sag aqDAQaysanUuy paj22Jas UL POY "WM Worg ‘oANjON.YS paftejap Moys 0} “paaouraa Ap}.red sourds ym ‘DIDINJIUNg DIIDGLY JO 489} IY) JO sooVJINS [VIOG" PUe [RIO ‘9 ‘OLT jOoy eqn} [ero avrtey[aotpad AOVANNS TVYORV HOVANNS TVYO oyed zed [eroeinqurezayur [ezoe—nqures9yur wosA SES oye[d [eueins e1od yey1ued 9joejue4 [etoe[nquie snue As, - By at vee Nt e yD 18 [eru0yst10d euBvIqUIAUL [erUIO\sIIEd 799 Oqny TeroeNqure 3 DESCRIPTION : ‘raystqnd oy) pue uf ‘umoig “y “4 Jo Asajinoo Aq paonpoiday *(oS61 ‘yIoX MaN ‘suog pue Aa[tAA uYyof) “af ‘UMoIg “Wy Aq partpa ‘sadly aypagapsoqur pajsapas’ UL Ploy “We°M Woy ‘suess0 yeusajUL MOYs 0} ‘DjDJNJIUNd DIIDG4P JO UOTNDIS [VOTNIIA ONeUIUILISeIG *L “Oy yynour yeueo [eIper ‘ p09 a@AIeU [eIper euvIquioU [eTUI0}sLIed U19}JUR] §,9]}0}SLIY Q[DISAA UBI[Og eueo Sut Aye} uegdi0 [exe Na! 2 peuos Youulo}s [euvo oLioderpeul 9UuT}so}UL e1od [eyTU98 snseydose ayt1oderpeur 34 THE AMERICAN ARBACIA internal anatomy and embryology of A. punctulata, having used this book as a laboratory guide for his students at Beaufort, before the Marine Biological Laboratory at Woods Hole was started. An ex- haustive monograph on sea urchins including the Arbaciidae has just been completed by the authority on the subject, Mortensen (1928— 1951); A. punctulata is in vol. II, pp. 573-575, 1935. For a good general description of the morphology of a sea urchin (Echinus esculentus) reference may be had to MacBride’s account in the Cambridge Natural History, vol. 1 (1906), or to Lankester’s Treatise on Xoology, part 3 (1900), or to the more recent volume of Grassé, Traité de Zoologie, t. 11 (1948). A more complete comparative account with experimental data and comprehensive bibliography is given in Bronn’s Thier-Reich Bd. II, Abt. 3, Buch IV (1904). The excellent volume on Echinodermata in the series of Invertebrates by Libbie H. Hyman (1955) was published after this book was sent to press. CHAPT E.R6 Natural History a. FEEDING HABITS Arbacia punctulata eats almost anything in an aquarium, and probably does so in its native habitat. They have been observed eating Fucus, Laminaria, Ulva, polyps of coral, sponges, mussels, sand dollars, and other Arbacia, both the soft parts and the shell. Parker (1932) observed and photographed two Arbacia eating a Fundulus which “may be caught and eaten by the sea urchins. The capture usually takes place at night, and the prey is almost always a partly spent fish. I doubt if a fully vigorous Fundulus is ever taken by a sea urchin. I have never witnessed the first steps in the capture”’ (Parker, 1932 p. 95; see also Gudger, 1933). Arbacia certainly cannot capture live fish, but eat them when moribund or dead (E. B. H. observations for many years). For feeding habits of A. punctulata and other sea urchins see van der Heyde (1922). Arbacia live well in aquaria supplied with Fucus and Laminaria and shells peppered with holes containing the sulphur boring-sponge, Cliona celata, and of course with running sea water; some animals and plants decay and pollute the water. Although they will eat almost anything that is at hand, they can get along without food, except for the small organisms brought in with the sea water, for several months. Two sets of six Arbacia were kept in aquaria (capacity about four gallons), one set in the dark and one set in the light, without being fed, for two months. All individuals of both sets were perfectly healthy after that period. The food for the developing pluteus is diatoms, especially Nitzschia closterium which can be cultured with Miquel’s solution (E. B. Harvey, 1949) or more simply by adding 0.002% Na,HPO,12H,O + 0.01%, KNO, to sea water (see Part II). When the shell is developing, the calcareous material can be supplied by the calcareous protozoon Trichosphaerium and later by the red alga Corallina, according to Shearer, de Morgan, and Fuchs (1914). 36 THE AMERICAN ARBACIA b. GROWTH AND AGE Very little is known of the growth and age of sea urchins, especially of A. punctulata. From fertilization through metamorphosis of A. punct- ulata takes (under laboratory conditions and about 23° C.) three or four months, and the animal is then about 1 mm. in diameter (Plate VI, Photo 11). The smallest sea urchin brought in from the outside by the collectors July 22, 1952, was 6 mm. in diameter with spines, 3.6 mm. without spines, and was 1.6 mm. high. (Plate VI, Photo 12). Smaller specimens, just metamorphosed, have not been found, though a careful search has been made on the stones and shells brought up from the beds and in the plankton above the beds. The newly metamorphosed young of most species do not seem to be common, but Mortensen (19274, p. 333) says the larvae and newly metamorphosed young of Echinocardium cordatum are sometimes found in enormous numbers in some places. Arbacia mature when very young; several about 1 cm. in diameter (without spines; 1.7 cm. with spines) had ripe functional eggs or sperm, and the eggs when fertilized developed into fine plutei. One that was 0.7 cm. (without spines) had ripe functional sperm, and one that was only 0.6 cm. had well developed ovaries containing immature eggs of all sizes, many of maximum size, and a few mature eggs, which, however, did not fertilize. Several small urchins, 2 cm. in diameter (without spines) brought in Aug. 1, 1951, contained all mature and no immature eggs. This early maturity has been observed in other forms. Psammechinus miliaris has been found with ripe eggs and sperm when 1.2 and 1.3 cm. and even when less than 1 cm., when they are less than a year old; and Echinus esculentus and E. acutus “‘when very small” (Shearer, de Morgan, and Fuchs, 1914, p. 270; Orton, 1920, p. 352 footnote). Sphaerechinus granularis is sexually mature when 0.8 cm. in diameter (Mortensen, 1927 a, p. 311). It is of interest that Aristotle noticed this in his sea urchins, for in the Historia Animalium he says “the eggs (ovaries) are found in the larger and smaller specimens alike; for even when as yet very small they are provided with them” (see under Historical). A. punctulata of all sizes have been brought in by the collectors, ranging from 3.6 mm. to 56 mm. in diameter (without spines), but until recently the small ones were found very rarely. Nothing is known as to the age of the large ones formerly brought in and used for experi- mental work. It seems probable that these came from old beds in which the young ones could not establish themselves, or perhaps were carried by currents, as plutei, elsewhere. The small Arbacia probably NATURAL HISTORY 37 come from newer beds recently discovered. A similar situation was found by H. B. Moore (1934) for Echinus esculentus near Port Erin. One bed, at Breakwater, had no small ones, none less than 7 cm. while two other beds, at Brest and Chickens had mostly small ones, under 4 cm. The best figures for the growth of sea urchins over a period of time are those of Elmhirst (1922) for the large urchin Echinus esculentus and of Bull (1939) for the smaller Psammechinus miliaris, given in the fol- lowing table: Diameter of test (without spines) in cms. Echinus esculentus. (Elmhirst, 1922 at Millport, Scotland; Mortensen, 1927 a, p. 299) Just 6mos tyr 2yrs 3 yrs 4 yrs 5 yrs 6 yrs 7-8 yrs (probably) metamorphosed 2 4 4-7 7-9 Q-II — — 15-16 mature Psammechinus miliaris. At Cullercoats, Northumberland (Bull, 1939) 0.1 0.33 2.0 2.62 2.92 3.03 3.7 3.87 mature Figures for the size of Psammechinus miliaris in different months and years are given by Lindahl and Runnstrém (1929). The Indian form Salmacis bicolor at Madras was found to measure 4-5 mm. at 3 months, 13 mm. at 6 months, and 16 mm. at 12 months (Aiyar, 1935). There are no figures published for Arbacia punctulata owing to the difficulty in keeping the animals over winter. The growth during the three month season (June to September) at Woods Hole was found to be only about 1 mm. even when well fed with Fucus and Cliona (E. B. H.) A group of ten, of approximately the same size, had an average diameter (test) of 22.4 mm. on June 23, 1951 and 23.5 mm. on Sept. 24, an increase of 1.1 mm. in three months.! These were kept at Woods Hole over the winter, but owing to lack of running sea water for several days only one survived. It measured 25 mm. on May 29, 1952, an increase of 1.5 mm. during the eight winter months. Judging from meager data, a medium sized Arbacia test grows about 3 mm. a year, and continuously through the year. A very young Arbacia, the smallest one yet collected, was 3.6 mm. (test) on July 23, 1952 and 5-2 mm. on Sept. 28, agrowthof 1.6mm. intwo months. The percentage increase in size is much greater in the small one, as one would expect. According to H. B. Moore (1934, 1935a, 1937), the growing period of Echinus esculentus is limited to the spring months, when spawning also takes place. The duration of life is 4-8 years. 1 Milligan (1916, Zoologist 20 : 399) found that Echinus (Psammechinus) miliaris (27 mm. diameter without spines) increased in the laboratory about 1 mm. in 5 months. 38 THE AMERICAN ARBACIA The age of an individual can be told in some cases by the rings of growth on each genital plate, these being marked by an annual deposit of pigment (Deutler, 1926; H. B. Moore, 1935a, 1937; Yonge, 1949, p. 166). Increase in size of the test is brought about both by accretion to the old plates and addition of new ones. c. REGENERATION Though regeneration is not so marked in sea urchins as in starfish, they can regenerate spines, tube feet, pedicellariae and spheridia, and even large fractures of the shell are healed (Mortensen, 1927a, p. 262; Grassé, 1948, p. 149). Chadwick (1929) reports a case of Echinus escu- lentus in the aquarium at Port Erin, which had lost all its spines, fallen to the bottom and seemed dead, but then regenerated its spines. I have observed many Arbacia with apparently regenerated spines, and have observed the process in a few cases; they regenerate fairly rapidly. A medium-sized animal measured 5.2 cm. with spines, the test without spines 2.8 cm.; the spines removed measured 1.2 cm. Three rows of spines were removed on June 18, 1952. They regenerated at the rate of a little over 1 mm. a week; ina month they measured 0.5 cm. and in two months (Aug. 17) they were almost completely regenerated. H. W. Jackson (1939), in a similar experiment with A. punctulata obtained similar results; the spines (10-12 mm.) regenerated 5 to 6 mm. in a month. For regeneration of spines in Strong ylocentrotus, and references in the literature, see E. F. Swan, 1952. The regenerative power of the pluteus of Arvbacia is great; an arm is regenerated in a few days (E. B. Harvey, 1949). d. Hasirat, BuRROwING Forms AND COLLECTING 1. — Rock-Boring Forms Arbacia punctulata, like most sea urchins, which are all sea forms, live in aggregations which are often widely separated. They live on rocky or shelly bottoms, or adhere to rocks, but do not make excavations in rocks as do several other species, especially those exposed to rough seas. One of the rock-borers is Echinometra lucunter in Bermuda, where it 1s known as the “rock” or “reef” urchin (H. L. Clark, 1899; Verrill, 1907; E. B. Harvey, 1947). Paracentrotus lividus is found in holes in the rocks in exposed places as observed and pictured long ago by Rondelet (1554). It is found especially on the west coast of France (Cailliaud, NATURAL HISTORY 39 1856; Fischer, 1864) and of Ireland as described and photographed by Southern (1915) in the rock pools of Clare Island (see also Forbes, 1841, p. 169 for its occurrence in Ireland). P. lividus is not found in holes around Naples nor is any other species, probably because there is little tide there. Psammechinus miliaris has been reported as rock- boring on the French coast (Cailliaud, 1856, and other older investi- gators), but Runnstrém says (personal communication) that it is not rock-boring on the Swedish coast. The California form, Strong ylocen- trotus purpuratus is rock-boring (A. Agassiz, 1872-1874, p. 706; Ricketts and Calvin, 1948, p. 129, and Plate X XVII). It has recently been reported that this form bores into steel piles and has caused damage to the piles driven by oil companies for their oil-well piers near Santa Barbara (Irwin, 1953). Fewkes (1890a, b) has reported that S. dréba- chiensis makes excavations in the rocks at Grand Manan in Canada, as well as two species, Eucidaris thouarsu and Echinometra vanbrunti, at Guaymas Harbor in Mexico. According to John (1889), Arbacia lixula in the Azores makes holes in the lava rocks. It is generally agreed that the boring is done with their spines and teeth, aided by waves and currents. As the animals grow, they often become imprisoned in their holes. For reviews of rock-burrowing Echinoids, see John (1889) and Otter (1932); they also discuss the methods of boring. Echinocardium cordatum is an interesting sand-burrowing form (Grassé, 1948, p. 198; Yonge, 1949, p. 238). 2. — Distribution Arbacia punctulata occurs along the North American east coast from the southern coast of Cape Cod to Florida; Woods Hole is probably its northern limit. It also occurs on the north coast of Cuba, in Yucatan, Curagao, Trinidad, and Tobago, but not in the Lesser Antilles, Jamaica, Puerto Rico, or Bermuda. (H. L. Clark, 1902, 1923, 1933; Mortensen, 1935, M II, p. 575). The species is supposed to have arisen together with the closely related A. spatuligera on the west coast of tropical America and to have reached its present home on the east coast while there was still open water between the Caribbean Sea and the Pacific Ocean; the original species then diverged into the present two species, A. punctulata and A. spatuligera. The origin of the Mediterranean form, A. lixula, cannot be traced. Around Woods Hole, many years ago (1874-1919) Arbacia were taken in many places in Buzzard’s Bay and Vineyard Sound, near Quisset, North Falmouth, North of Nashawena, Kettle Cove, off West Chop (Martha’s Vineyard), Sankaty Head (Nantucket), and around 40 THE AMERICAN ARBACIA the Fish Commission wharf (Verrill and Smith, 1874; Clark, 1902; Sumner, Osburn, and Cole, 1911; Allee, 1919, 19232, b, c). In more recent years, they have been obtained off Naushon Island, Hadley Harbor, Lackey’s Bay, Tarpaulin Cove, and off Nobska. They disappeared from Nobska about 1937, and later from other beds, until in 1948-1950 they were obtained from only two beds, Lackey’s Bay and Tarpaulin Cove, and here too they were becoming scarce. The scarcity of the urchins became so serious that a survey of the whole Woods Hole area was made in the summer of 1950 in an effort to discover new beds, under the direction of J. S. Rankin Jr., the Summer Naturalist at the M. B. L. For a period of six weeks, only one Arbacia was found, at Cotuit. Then in September, 1950, a good bed was found on Martha’s Vineyard between Menemsha Bight and Gay Head. Un- like the other beds, this bed had small as well as large urchins, and has subsequently become a source of supply, though they are difficult to collect and many are too small for experimental work. In 1951, while exploring further afield, many beds were found on the south-east coast of Fisher’s Island, off the south coast of Connec- ticut. These urchins were of all sizes, young ones as well as large ones. Unfortunately the distance from Woods Hole (62 miles) necessitates a two-day trip, often hampered by bad weather, but it is another source of supply of Arbacia. Also, in 1954, Arbacia were found near the “old Nobska’”’ bed, having returned there after an absence of 17 years. Some of the old beds which have been unproductive for some years have now been stocked with urchins, young and old from the newer beds, in the hope that they may again furnish many urchins in the future. 3. — Quantities Used Arbacia punctulata are found in shallow water and at depths down to 700 ft. They are obtained by dredging in water 20-90 ft. deep, usually with a tangle. The associations in which they are found around Woods Hole have been studied by Allee (1923 ,, b). In an ordinary summer, ten or twenty years ago, 20,000—25,000 animals were used at the Marine Biological Laboratory. The greatest number used in one summer (1933) was approximately 70,000. The table below shows the decrease in numbers used, in the last twenty years. In 1947, 45,000 were delivered to 18 investigators. In 1948, 15,000 were delivered to 20 investigators; 50,000 were ordered. In 1949, only 4,000 were delivered and in 1950 only 3,000. The following table is taken mostly from the report for 1950 of J. S. Rankin, Jr. NATURAL HISTORY 4I YEAR NUMBER ORDERED NUMBER COLLECTED 1933 and 1934 — 70,000 1939 21,000 29,000 1940 36,000 31,000 1941 31,000 28,000 1942-1946 (war years) 15,000 per year 13,000 per year 1947 54,000 45,000 (600 delivered daily to 18 investigators) 1948 50,000 15,000 (250 daily to 20 in- vestigators) 1949 70,000 4,000 (45 daily) 1950 is 3,000 (3-7 daily) In 1950, orders had to be kept over until they could be filled, owing to scarcity of animals. In 1951, 7,000 were brought in, from Menemsha Bight; 3,000 were returned to the sea as too small for laboratory use. In 1952, 11,000 were brought in from Fisher’s Island on 7 trips. The supply in 1953, 1954 and 1955 was adequate. The value of the Arbacia supplied may be judged by the fact that they sell for $13 a hundred at the Biological Supply House. As an interesting comparison, about 63,700 lbs. of lobster were brought in by local fishermen from the Woods Hole area in 1949; this is approxi- mately 56,000 lobsters. In 1950 there were 44,500 lbs. or 39,000 lob- sters. One lobster averages 1 1/8 lbs. (Data kindly supplied by Homer P. Smith, business manager of the M.B.L.). A lobster sells for $ .80 at Woods Hole, or $ 80 a hundred. The cause of the decreasing quantities of Arbacia around Woods Hole is not known. Depth bombing during the war years has been suggested, and also the increase in numbers of starfish, their especial enemy. The scarcity may be due to climatic conditions, such as hard winters and the two recent hurricanes (1938, 1944). But it seems probable that it has been caused by over-collecting; most animals which are system- atically taken for food or wearing apparel, e.g., lobsters, birds, seals, are protected by law in some way to prevent their extinction. Arbacia are not protected. Also the quantities of Arbacia needed for the newer chemical investigations of eggs and sperm are enormous in comparison with the few needed in the past, for morphological or even for physio- logical work. Arbacia have been recorded as scarce several times previously. A survey was made by the U. S. Bureau of Fisheries in 1874 (Verrill and Smith), and again in 1go2 (H. L. Clark) of the region around Woods 42 THE AMERICAN ARBACIA Hole and localities noted in which Arbacia and other Echinoderms occurred. It was found in a later report (Sumner, Osburn, and Cole, 1911) that Arbacia which had been abundant in the summer of 1903 were scarce after the severe winter of 1903-1904, in the summers of 1904 and 1905, and were not abundant for several years; in the sum- mers of 1908 and 1909 large quantities were again obtained in Vine- yard Sound. The following table is taken from the more complete table of Sumner, Osburn and Cole (1g11, p. 114). The numbers in the first column are station designations (l.c., p. 201). 1903 1904 7522 Nobska Light Many None 7523 5 33 Several I spine Wed, ss » Very abundant None F5ION 55 »» » West Chop Abundant None 7532 West Chop, Tarpaulin Cove Many Few spines 7549 Tarpaulin Cove, Nobska Many Few fragments & spines 7563 Gay Head, West Chop Many Fragments & spines In 1905, throughout Vineyard Sound as a whole, living Arbacia were taken only five times during the summer and never more than two at one time. Strong ylocentrotus drébachiensis and Echinarachnius parma were not similarly affected at this time (l.c., p. 115). In the summer of 1911, there was again a dearth of Arbacia (Morse, 1912). They were particularly abundant in the summer of 1917, being taken at Kettle Cove, Quisset, North Falmouth, and off Nobska (Allee, 1919, 1923c). But in 1918, following the severe winter of 1917— 1918, only a few were taken. They had recovered by 1920 (Allee, 1923a, c). The scarcity in 1949, however, followed a very mild winter, but the previous winter had been severe. The earliest indication of any decrease in the numbers of Arbacia was made by Loeb as far back as 1900 when he says ‘““The sea urchins have practically died out in the immediate neighborhood of the Woods Hole laboratory, and we have to send out the steam launch to collect them. For this reason even at the height of the spawning season there is little danger of the sea water containing spermatozoa in such quanti- ties as to interfere with experiments with unfertilized eggs (Loeb 1900a, P- 450).” A similar disappearance of the British sea urchin, Echinus esculentus, was noted in the deep water of Plymouth Sound (120 ft.) in 1899 after a south-west gale (MacBride, 1906, p. 504). Psammechinus miliaris dis- NATURAL HISTORY 43 appeared from Whitstable after an exceptionally cold winter of 1928- 1929; it had been abundant there (Yonge, 1949, p. 278). Temporary migrations have been reported for Echinus esculentus at Millport (Elmhirst, 1922; Stott, 1931). An inshore migration during the spawning season has been noted and a seaward movement caused by frosts and heavy weather. It has been suggested that migrations are due to food conditions (Gemmill, 1900). See also Deutler (1926, p.193). Nothing is known about any migrations of Arbacia. There seems no doubt that the abundance of sea urchins, the locali- ties in which they are found and the general condition of the animals vary from year to year; this applies especially to Arbacia punctulata. e. PHOTOTAXIS AND LIGHT REACTION Arbacia punctulata move away from the light and go to a shaded or darker region, by the combined action of spines and tube feet; certain individuals, however, go to the light (S. J. Holmes, 1912). Removal of the so-called eyes on the ocular plates does not change the reaction. Strong light on the tube feet causes them to be withdrawn. Many other experiments are described by Holmes. The very young animals are often found in empty bivalve shells, probably to get away from the light. The smallest one yet brought in by the collectors at Woods Hole (diameter 6 mm. with spines) was found in an empty Venus shell. When taken out it was observed always to move to a shaded area, often under the empty shell, sometimes at the rate of 1 cm. in 5 minutes (E. B. H.). Other species, Arbacia lixula, Sphaerechinus granularis, Centrostephanus longispinus, and Paracentrotus lividus, have also been found to move away from the light and seek the dark (von Uexkiill, 1896; Mangold, 1909); and also Psammechinus miliaris (Bolin, 1926; Lindahl and Runnstrom, 1929). However Romanes (1885) says that ‘“‘the Echint manifest a strong disposition to crawl toward and remain in the light” (p. 319). Reactions to changes in light intensity have been studied in A. /ixula, Centrostephanus longispinus and Lytechinus variegatus, especially with regard to the spines and pedicellariae, by von Uexkiill (1896, 1899, 1900, 1909), Mangold (1909), and Cowles (1911), and more recently Diadema anitil- larum has been studied by Millot (1950, 1954, Phil. Trans. Roy. Soc. London B 238: 187-220). The habit of “‘decorating”’ or ‘‘masking”’ themselves with bits of shell and seaweed, characteristic of certain littoral species of sea urchin, has been attributed by some observers to protection against light (H. L. 44 THE AMERICAN ARBACIA Clark, 1921; Lindahl and Runnstrém, 1929, p. 407; Mortensen, 1943, MeFi 3'p:*134) ‘A number of specimens (Psammechinus miliaris) were held some time in a basin with running water, alternately kept in light or in total dark- ness. When in the light, the specimens covered themselves with pieces of algae, etc., when in the darkness they dropped these covering pieces”” (Mortensen, 1943, M III, 3 p. 135). Several explanations for the habit other than light have been offered: concealment against enemies, camouflage, protection against drying in low water (Orton, 1929). Boone (1928) thought it must be to prevent detection by ene- mies or potential victims rather than protection against light, for he found in the case of Lytechinus that specimens kept in the laboratory at Miami, Fla. in relatively dark aquaria “for weeks at a time camou- flaged themselves quite as thoroughly and industriously as their rela- tives on the open reefs”’ (p. 21). This has been criticised by Mortensen (1943, M III 2, p. 443). The best known species having the habit of decorating themselves are Lytechinus variegatus (H. L. Clark, 1921; Boone, 1928; Mortensen, 1943 M III 2, p. 442); Psammechinus miliaris (Lindahl and Runnstrom, 1929; Orton, 1929; Mortensen, 1943 M III 3, p. 134); Psammechinus microtuberculatus (Noll, 1881); Paracentrotus lividus (Mortensen, 1927, p. 308; 1943, M III 3, p. 164); Sphaerechinus granularis (Toxopneustes brevi- spinosus) (Dohrn, 1875; Mortensen, 1943 M III 2, p. 522); Tripneustes esculentus (H. L. Clark, 1921), and the Japanese species Strongylocen- trotus pulcherrimus (Dan, personal communication). | The ancients had a curious explanation for this habit, given by Pliny and referred to by many of the older writers, even as late as Camerarius in 1654 in his book on symbols which has an interesting picture of an urchin covered with stones (Fig. 3). They thought that at the approach of a storm the sea urchins cover themselves with small stones to provide a sort of ballast to prevent them rolling around and wearing away their spines. When the urchins were covered with stones it was an indication to sailors that a storm was approaching and they should anchor their ships (see under Historical). f. GEOTROPISM No data have been found for geotropism in Arbacia, punctulata or lixula, but Diadema and Lytechinus are negatively geotropic, climbing up- wards in an aquarium, irrespective of dark and light and also of access to oxygen (Parker, 1922, 1936). Other urchins found to be negatively NATURAL HISTORY 45 geotropic are: Psammechinus microtuberculatus (Baglioni, 1905) and Psammechinus miliaris (Bolin, 1926; Lindahl and Runnstrém, 1929). The blastulae and gastrulae of Arbacia punctulata swim at the surface of the water; the plutei are scattered (Lyon, 1906b, 1907). The sur- face swimming is not due to light for it occurs in total darkness, nor is it due to oxygen supply for it takes place when the tube containing them is inverted. This is a true negative geotropism since the larvae are heavier than sea water; their specific gravity is about 1.06 (Lyon, 1906b, 1907). The blastulae from centrifuged eggs also come to the top, though the heavy pigment makes them heavier on one side (Lyon). g. LOCOMOTION Sea urchins move in any direction by means of their spines and tube feet (Holmes, 1912). According to A. Agassiz (1872-1874, p. 264) “The mode of moving of Arbacia is quite different from that of our common Strongylocentrotus ; instead of dragging itself along by means of the suckers of the actinal surface, it makes free use of its spines, and by a sort of tilting motion advances quite rapidly. The spathiform shape of the spines around the actinosome in species of this genus is undoubted- ly due to the wear and tear produced by this means of locomotion.”’ Specimens of Arbacia punctulata have been observed to move over the glass of an aquarium at the rate of 35-40 mm. per minute after being disturbed; they average 22.2 mm. per minute when undisturbed (H. W. Jackson, 1939). I observed a very small Arvbacia, 6 mm. in dia- meter (with spines) move at the rate of 3 mm. per minute. Echinus sphaera (esculentus) and lividus (probably Paracentrotus lividus) moved along a horizontal surface 6 in. per minute, and up on a vertical, surface I in. in 4 minutes (Romanes and Ewart, 1881; Romanes, 1885). Gemmill (1912) found that Echinus esculentus and miliaris usually moved, out of water, I in. in 5 minutes, the fastest being 3-4 in. in 5 minutes. Lytechinus variegatus ascended a glass plate with an average speed of 1.8 mm. per minute, and a maximum speed of 12 mm.; on a vertical surface it progressed by means of its tube feet exclusively. On a hori- zontal surface of sand it progressed by means of its spines exclusively at the rate of 82 mm. per minute, average, with a maximum of 137 mm. (Parker, 1936). The sand dollar, Echinarachnius parma, averaged 13.7 mm. per minute, with a maximum of 18 mm., when progressing in the sand (Parker, 1927). As early as 1712, the locomotion of sea urchins aroused the attention 46 THE AMERICAN ARBACIA of Réaumur, who noticed that they used their spines as feet and mar- velled at the number of muscles it must take to move 2,100 spines. Arbacia punctulata can “right”? themselves, i.e., turn over to the ven- tral side when placed on the dorsal side, easily and quickly, both large and small ones. In a flat-bottom glass dish, they raise themselves to their side sometimes within two minutes and then fall to the ventral side quickly by gravity (E. B. H.). Von Uexkiill (1909, p. 106) says A. lixula cannot turn over on a flat surface, only on a slanting surface, but A. punctulata seems to have no difficulty, if in water. It cannot turn over when out of water. The righting movements for Echinus esculentus and P. lividus have been described in detail with illustrations by Romanes and Ewart (1881) and Romanes (1885), and for Arbacta lixula by von Uexkill (1909, p. 106). Parker (1927) and Parker and Van Alstyne (1932) have described locomotion and righting movements in Echinarachnuus. Though locomotion is usually accomplished by the tube feet and spines, it can also be effected by the lantern as described by Romanes and Ewart (1881) and by Gemmill (1912) in Echinus. The lantern may be used both for progression and rotation when the animal is out of water. GiEGA Pel EeRoay Sex and Breeding a. SEX RATIO To investigators working on Arbacia eggs, it always seems as though there were many more males than females. An accurate count of the animals shows that this is not so, but they are approximately equal in number. Shapiro (1935a) found that throughout one season the ratio was I male to 1.03 females (2,358 animals). Ikeda (1931) found the ratio in Temnopleurus toreumaticus was 1 female to 1.018 males (2,093 animals). b. SEX DIMORPHISM (OTHER SPECIES) No external morphological difference between the sexes has been detected in Arbacia punctulata, and this is true of most sea urchins. There are, however, a few species in which the males and females differ or have been reported to differ in size or shape of the test, in the genital pores or genital papillae, and in the color of the tube feet. In Arbacia lixula, Cerami (1924, as quoted by La Cascia, 1930) thought that the male has a taller and rounder shell and a larger peristome and lantern, but La Cascia (1930), on measuring a greater number of specimens found no sex difference. There is some disagreement about Paracentrotus lividus. O. Schmidt (1878 in Brehm’s Thierleben Abt. 4, Bd. 2, Wirbellose Thiere) says that his boatman, in Lesina, could tell the males and females apart from the boat; the males were smaller, darker, and more spherical, the females flatter and more reddish violet. He says that his boatman was never deceived, but he had difficulty himself in distinguishing them. Camerano (1890) found no difference in color in the two sexes, but agreed with Schmidt that the males were smaller and the females flatter. However, La Cascia (1930), on making a more thorough study, concluded that one could not distinguish the males and females by shape or size. In the Japanese form, Temnopleurus toreumaticus, Ikeda (1931) found from a careful biometric study that the males are slightly 48 THE AMERICAN ARBACIA taller than the females; but this has been denied by Mortensen (1943, M. III, 2 p. 79 footnote). In Cidaris membranipora (nutrix), according to Studer (1880b) the shell of the female is flatter, while in Hemiaster (Abatus) cavernosus, the shell of the male is flatter. In Gonvocidaris canaliculata there is no differ- ence in external form, but there is a difference in the genital plates. The brood-pouches of these and several other forms also indicate the sex (Studer, 1880a) (Fig. 8). In some forms, genital papillae are sometimes found at the opening of the genital ducts (see Bronn’s Thier-Reich, 1904, Bd. II, Abt. 3, Buch IV, p. 1135), and are sometimes different in the two sexes. In Echinocardium mediterraneum the genital papilla of the female is thicker and shorter than that of the male (Hamann, 1887 and Plate XVIII, no. 7). In Echinocyamus pusillus it is much longer in the male, while in Psammechinus miliaris it is present in the male, often with red pigment, and is lacking in the female (Marx, 1929; see Grassé, vol. 11, p. 160). In this species, the difference between the males and females is suf- ficient for the sexes to be separated into different aquaria, as is done in Runnstrém’s laboratory in Kristineberg, though sometimes there is a mistake and the female tank becomes clouded with sperm (personal communication). M. M. Swann recently (1954) has found a slight difference in the sexes in Echinus esculentus, Paracentrotus lividus, Psammechinus microtuber- culatus, Ps. miliaris, and Sphaerechinus granularis. ““In the male the five genital pores are borne on short papillae... their edges are glistening white... In the female the genital apertures are not borne on papillae but are more or less sunk below the level of the surroundings... ‘They are usually somewhat smaller than in the male and often oval in shape. The glistening white edges characteristic of the male papillae are never visible... The descriptions given do not apply to Arbacia lixula. Here the genital apertures are sunk in circular pits, and papillae may be present or absent in either sex.”’ In Lytechinus anamesus and L. pictus, the gonopores of the female are larger than those of the male (Tyler, 1944 and verified by C. B. Metz in 1953). A rather striking sex dimorphism has been found by Motomura (1941 a) in the Japanese species, Strongylocentrotus pulcherrimus ; the tube feet on the oral side are yellow in the female and white in the male. SEX AND BREEDING 49 c. SEX DETERMINATION IN ARBACIA PUNCTULATA Though there is no morphological difference in the sexes in Arbacia there are several ways of distinguishing males and females. One method is by forced extrusion of gametes from a gonopore (E. B. Harvey, 1940d). With a Luer syringe and very fine (no. 27) hypodermic needle, insert a drop of sea water saturated with KCl, into one genital pore. The operation is most easily done with an electric light shining from above on the aboral surface of the animal so as to show up the genital pores, and under a binocular microscope. A fine glass pipette, of slightly smaller bore than the genital pore can be used but is liable to break. A few eggs or a little sperm will almost immediately begin to ooze out from this pore alone, and the sex can be distinguished by the color (red 2, white 3). The shedding is stopped at once by placing the animal in a jar of still, not running, sea water; leave for a few hours before returning to running sea water. The readiness with which the Arbacia may be thus “‘sexed’’ depends somewhat on the maturity of the animal and the time of year. In a second method a small amount of material is drawn out with a Luer syringe (and no. 27 needle), or fine glass pipette, applied at the opening of one of the genital ducts; it is examined in a drop of sea water under the microscope. As there is a sharp bend in the gonoduct just beneath the surface (see Fig. 7 from Brown’s Invertebrate Types), care must be taken not to insert the needle into the coelomic cavity instead of the gonoduct. Also the red color may be due not to eggs but to red amoebocytes present in both sexes. The electrical method of determining sex (E. B. Harvey, 1952, 1953, 1954) is by far the best for distinguishing male and female in Arbacia and other sea ur- chins. An alternating current of 10 volts is passed through the animal, which will at once shed its sperm or eggs. Ordinary 60-cycle alternating current can be used, with the 110-voltage, reduced to 10 volts by a transformer. Lead electrodes have been found best, as they are non-toxic and are easily made from lead tubing or from heavy lead wire. The electrodes are placed at any two points on the shell of the animal which lies, aboral side up, covered with sea water. Almost immediately after the current is passed, the eggs or sperm will exude from each of the five gonopores, the sperm in a thin white thread, and the eggs in a thicker red (in Arbacia) thread, later tending to clump (Plate XVI, Photographs 1, 2). When the current is stopped, the shedding ceases; but it begins again when the current is allowed to pass. In this way, the sex of the animal can be quickly determined, and a few eggs or a little sperm obtained without harming the animal; and the same animal can be used repeatedly. The eggs, if removed at once, fertilize perfectly and develop normally. The method is of great value in places where sea urchins have become scarce. The rapid response of the sea urchin is due to the presence in the walls of the ovary and testis of a layer of smooth muscle cells which are stimulated by the electric current, causing the walls to contract and force out the eggs or sperm. A much more elaborate electrical method was described several years ago by Iwata (1950) for Mytilus and for a Japanese species of sea ur- chin, Helvocidaris crassispina. The simple method described above has also been used successfully on the sand dollar and on several species of annelids. Sea urchins containing only immature eggs do not respond. In some species a higher voltage may be required. 50 THE AMERICAN ARBACIA d. HERMAPHRODITISM Hermaphroditism is quite rare in Arbacia, as also in other sea urchins, but it does occur. Among the many thousands of Arbacia opened in the course of twenty-five summers at Woods Hole, I have found only two cases of well marked hermaphroditism. One was fully described by me in 1939a. This animal appeared to have four red ovaries and one whitish testis with oozing sperm. On careful examination it was found that each of these gonads had a very slight amount of tissue of the oppo- site sex. The eggs from this animal were readily self-fertilized and developed normally into normal plutei. One summer (July 10, 1946, unpub.) an animal was opened which was to all appearances a female, but some of the eggs became fertilized without applying sperm, and no male had been opened, so that contamination was unlikely. On a care- ful examination, it was found that there were small bits of testis tissue in two of the ovaries, and sperm from this tissue, after becoming motile in sea water, would fertilize the eggs. It seems likely from this experien- ce, that most cases of supposed contamination resulting in accidental (and provoking) fertilization of eggs, such as many competent investi- gators have occasionally observed, are due not to carelessness in tech- nique but to a very slight hermaphroditism of the animal, the testis tissue being too small to be detected. Three other cases of hermaphroditism in Arbacia punctulata have been reported, two by Heilbrunn (1929). In one of these, there were four ovaries and one ovotestis, and in the other, two ovaries, two testes, and one ovotestis. Both of these gave rise to normal larvae when self- fertilized. Shapiro (1935a) described an animal with four testes and one ovary, but the development of the eggs when self-fertilized was abnormal and only a few developed to gastrulae. K. C. Fisher found a case of hermaphroditism (July 10, 1942 unpub.) among the Arbacia used in his physiology class at Woods Hole; this animal had three ovaries with some testis tissue and two testes. These eggs when self- fertilized developed normally into plutei. In 1954, Mrs. Cornman observed an Arbacia spawning spontaneously when the collector brought in the animals. Eggs came from four gonopores, sperm from one. The spawning could be controlled at will with the electric current applied to the shell (see under c. Sex Determination). References to hermaphroditism in other species of sea urchins and above data for Arbacia punctulata are given below. SEX AND BREEDING Arbacia punctulata Heilbrunn (1929); 49, ! 33 Bo 2 Sapl 3. Shapiro (1935a); 19,46. E. B. Harvey (19394); 5 3. (1946 unpub.) ; 3 9, 2 3 K. C. Fisher (1942, unpub.); 2 3, 3 3. M. E. Cornman (1954, unpub.) ; 4 9, 1 ¢. Arbacia lixula (pustulosa) J. Gray (1921); questionable. Reverberi (1940); 492,153 32,26: (1947))s Sissel. t 3: ID AS Pit Oo eG. 3c Neefs (1953); d-: we , tO, Dendraster excentricus Needham and A. R. Moore (1929); 23°, 23 4. Echinocardium cordatum Giard (1900) ; protandrous. H. B. Moore (1935b); sex not stated. Echinus esculentus H. B. Moore (1932); 4.9, 1 dg. Paracentrotus lividus Fuchs (19144); 3 9, 2 3. Herlant (1918); 4g, 1 3. & J. Gray (1921); same as Fuchs (19144). Drzewina and Bohn (1924); 49. 1d. Neefs (1937); d. Psammechinus microtuberculatus Herbst (1925 unpub. See Tab. Biol. VI, p. 501, 507); 3 2, 2 ¢. Reverberi (1947); 1 3, 4 3. Sphaerechinus granularis Viguier (1900) ; sex not stated. Neefs (1952); 3. Strong ylocentrotus drébachiensis Gadd (1907); 42,14. HI 52 THE AMERICAN ARBACIA e. BREEDING SEASON; SHEDDING OF EGGs AND SPERM With few exceptions, the unfertilized eggs and the sperm of sea urchins are shed in the sea water; the eggs are there fertilized and develop into free-swimming larvae or plutei which pass through a complicated meta- morphosis before acquiring the adult form. This is true of Arbacia _ punctulata. However, a few viviparous species of Echinoidea have been described, mostly from the southern hemisphere. The eggs which arc usually very large, 1-2 mm. (Hesse u. Doflein, 1914, II p. 619; Tab. Biol. VI, p. 503), develop in a sort of brood pouch formed by the long spines, either dorsally or ventrally. The larval stage is usually imper- fectly developed or omitted entirely and the animals come out as young adults. Among these may be mentioned: Anochanus sinensis from the China Sea, as described by Grube (1868); Hemiaster (Abatus) cor- datus, collected on the Transit of Venus expedition (1874-1875) by Dr. Jerome H. Kidder (1875-1876), near the Kerguelen Islands and described by A. Agassiz (1876) ; the same species and Cidaris nutrix and Goniocidaris canaliculata, collected on the Challenger expedition (1873- 1876) near the Kerguelen Islands and the Falkland Islands and de- scribed by Wyville Thomson (1877, 1878). One of his interesting woodcuts is reproduced as Fig. 8 in this monograph; several of them are reproduced in MacBride’s text-book (1906, p. 535, 555, 603). An account of these and other Echinoidea collected on the Challenger Expe- dition may be found in A. Agassiz’s report (1881). Hypstechinus corona- tus and Gontocidaris umbraculum and other species are described by Mortensen (1927a, p. 293; 1927b; 1931, p. 7). The breeding season for Arbacja punctulata at Woods Hole is from the middle of June till the middle of August, though the season varies from year to year according to temperature. During May and early June, the gonads are small and it is often difficult to tell whether they are ovaries or testes. The males ripen earlier and remain in good con- dition later than the females. The eggs mature in the ovary, so that when they are shed or procured from the ovary during the season most of them have already given off (and lost) their polar bodies, and the eggs are ready for fertilization and immediate development. For about a week before the middle of June, the eggs are ripening and all sizes of eggs in the germinal vesicle stage and all stages of polar body for- mation are then found. Some immature eggs are often found among the ripe eggs later on until towards the end of the season. There are usually many immature eggs late in the season in animals from the new beds at Menemsha Bight and Fisher’s Island. SEX AND BREEDING 53 Fic. 8. Young sea urchins in brood pouch of the Falkland Islands sea urchin, Goniocidaris canaliculata. After Sir C. Wyville Thomson in The Voyage of the “Challenger” The Atlantic. Vol. II, p. 224, 1877. Arbacia is different from the starfish, Astertas_ forbest, which sheds its eggs in the germinal vesicle stage, and maturation of the eggs takes place after they are laid. These (starfish) eggs should be fertilized during or just after the first maturation division, one to one and a half hours after removal from the animal. When Arbacia are brought in from the sea after the middle of August, many of them have already shed. By September first, most of the ani- mals have shed, although one finds a few with eggs and many with sperm. If, however, the animals are brought into the laboratory carlier in the season before they have shed, and kept in aquaria (capacity 16 gallons or more) with running sea water, they retain their eggs and sperm and remain in good condition through October (E. B. Harvey, 1939b). The animals require no special food, but apparently they eat 54 THE AMERICAN ARBACIA each other. It is quite essential that they are not overcrowded (150- 200 in an average sized aquarium, 16 gallons), that there is a steady flow of salt water, and that the water is kept unpolluted. Any unfavor- able condition will cause the animals to shed, and when one sheds the others are likely to shed also. Two lots of six animals each were kept, one set in the light and one set in the dark from July 1 to Sept. 16, 1948, and all were in perfect condition at the end of that time. The eggs from animals kept for some time in the aquaria are slightly different late in the season, taking longer to cleave, irrespective of tem- perature, and they are more viscous, taking longer to stratify and break apart with centrifugal force; also some of the red pigment granules remain in the white half. The rate of oxygen consumption is decreased (Shapiro, 1935c). The amount of carbohydrate and of phosphorus is greater and of nitrogen is less in the stored eggs (Hutchens, Keltch, Krahl, and Clowes, 1942; Crane, 1947). It seems probable that there are other differences of a physiological or chemical nature, but for many purposes the eggs are quite suitable to use as they develop nor- mally and give normal plutei. A difference between summer and winter eggs has been observed for Paracentrotus lividus at Naples (Horstadius, 1925). Some eggs obtained from A. punctulata which had been kept at Woods Hole until the middle of January appeared normal except that they were pale. These, when fertilized, developed to blastulae (A. E. Navez, personal communication, 1936). Good sperm has been obtained from animals kept over winter until March; the females from this lot had full ovaries, but only degenerate and abnormal eggs which had apparently not been shed the previous season (E. B. Harvey, 1942). It would seem that in general at Woods Hole under natural con- ditions, A. punctulata shed their eggs, when fully ripe, the latter part of the summer, then do not have ripe eggs again until the following summer. It is quite possible, however, under laboratory conditions for a ripe animal to shed some of its eggs at one time and more later on. The breeding season of A. punctulata at Beaufort, N. C., is con- siderably in advance of that at Woods Hole. They are fully ripe and in fine condition during May (E. B. H.). Some animals have ripe eggs and sperm in January (C. B. Metz, personal communication, Jan. 1951). In Florida, at Alligator Harbor, they are ripe from September until June and in fine condition in February, March, and April (C. B. Metz, personal communication, Jan. 1955). The closely related species, A. lixula, which is common at Naples, 1s ripe all the year according to Lo Bianco (1909), but the eggs are bad SEX AND BREEDING 55 at certain periods, especially in September (R. D. Allen, personal communication, Jan. 1955). It has been stated by Orton (1920) that in parts of the sea where conditions such as temperature do not change much, marine animals will breed continuously. This would apply to the tropics. He bases his conclusion on the statement of Semper (1881, p. 136) in Animal Life that in the Philippines he “could not detect a single species of which he could not at all seasons find fully grown specimens, young ones and freshly deposited eggs.’ Mortensen (1921, p- 245; 1938, p. 12), however, has found this not to be true of several species in the tropics which had no ripe eggs when he examined them, e.g., Diadema antillarum and Echinometra vanbrunti. He thinks it likely that some species in the tropics have more than one breeding season. O. Koehler (1916, p. 258) made a special study of Paracentrotus lividus at Naples, examining a group of 20 animals every five days for two months (November, February), and found no regular variation in size or content of the gonads. He had an ingenious method of cutting a window in the test to obtain a piece of gonad, then sealing it over with wax, using the same gonad for successive observations (p. 127). He found that during the summer in aquaria at Naples it took one and a half to two months to form ripe genital products (p. 257). Fox (19244), however, using Koehler’s window technique, found that at Roscoff during the summer (water temperature 17-19 °C.), Paracentrotus lividus which just after spawning contained no ripe eggs, gave ripe eggs in abundance after nine days. In the one form, Dzadema at Suez, that has been shown to have a lunar periodicity (see below), it must take about four weeks (temperature of sea water 26-29 °C.) to form new genital products (Fox 1924b). Monroy (personal communication) finds that at Naples the sea urchins usually shed after a storm or the scirocco, and it takes a week before one can obtain new eggs. Jacques Loeb (1915a) observed that at Pacific Grove, California, Strong ylocen- trotus purpuratus in a certain region (temperature 12-15 °C.) shed their eggs and sperm one day in March, and immature eggs began to appear during the next week and ripe eggs in about ten days. Similarly Ten- nent (1g10) observed that Lytechinus variegatus at Beaufort, N. C. again had ripe eggs a week after he had found them empty. Tyler (1949) obtains new batches of eggs from Lytechinus and Strong ylocentrotus of the west coast at two week intervals after forced shedding, during the breeding seasons. Whether the eggs obtained two weeks after forced shedding in Arbacia at Woods Hole are from a regenerated ovary as Tyler thinks, or are the eggs left from an incomplete previous shedding, it is difficult to say, though this could be determined by Koehler’s 56 THE AMERICAN ARBACIA window technique (1915, p. 127). I have found no evidence of imma- ture or maturing eggs, indicative of a regenerating ovary, either when examined fresh or in sectioned material a week or ten days after forced shedding from animals fully ripe; the ovaries are full of ripe mature eggs which were obviously not shed when the others were. f. LUNAR PERIODICITY (OTHER SPECIES) The idea that sea urchins and other sea animals are full of eggs at the period of full moon was quite generally held by the ancients. Both Aristotle and Pliny expressed this belief, and other classical writers, e.g., Lucilius and St. Augustine (see under Historical). The belief prevailed through the middle ages and is still held by the fishermen around the Mediterranean. No periodicity has been verified by in- vestigators at the Naples Station. However, one of the earliest workers on fertilization of sea urchin eggs, Fol (1879, p. 86), states that in Toxopneustes lividus (Paracentrotus lividus) and Sphaerechinus brevispinosus (S. granularis) at Messina, the sexual products are liberated each month at full moon, and the animals are then empty for a few days; there are more of the sexual products in spring and summer than in fall and winter; he says that the fishermen are aware of these facts, since sea urchins are used for the table. There is some evidence that Lytechinus ( Toxopneustes) variegatus at Beaufort is ripe at full moon and empty just after (Tennent, 1g10a, p. 658 footnote) but the evidence of lunar periodicity is not conclusive (Tennent, Gardiner, and Smith, 1931, p. 7). It is said that in Greece the gonads of sea urchins used for food appear on the market only during certain phases of the moon and at other times the animals are spent (Just, 1919, II, p. 17 footnote, quoting Tennent). A special study of lunar periodicity was made by Fox (19244, b) who found that there is a lunar periodicity in the Red Sea form Cen- trechinus (Diadema) setosus at Suez. At full moon during the breeding season, an animal spawns, becomes again full of ripe eggs or sperm at the next full moon. But at nearby Alexandria, there was no perio- dicity in the Mediterranean form Paracentrotus lividus and none in this species at Naples, Marseilles, or Roscoff. Fox is of the opinion that the belief in periodicity spread in ancient times from Suez, where it does occur, to Greece and the Mediterranean countries where it does not occur, and the belief persists to the present day (see also Zirpolo, 1929). Mortensen (1937, III) could not confirm Fox’s observations on Diadema setosum, finding that ripe specimens could be obtained irre- SEX AND BREEDING 57 spective of the full moon near Suez. In a later paper, however (1938, IV), he agrees with Fox with regard to Diadema, but for other echino- derms studied “‘there is not much support for a lunar periodicity,” and he considers lunar periodicity in the Echinoderms near Suez a rare exception (p. 12). ._ There is no evidence of periodicity in Arbacia punctulata, but the lunar periodicity of the worm Nereis limbata at Woods Hole is well established (Lillie and Just, 1913), also of the “‘fireworm”’ in Bermuda, of the pa- lolo worm and of many other animals. A good evaluation of lunar periodicity in sea urchins and other animals is given by Korringa, 1947, Ecological Monographs, 17 : 349-381. g. FORCED SHEDDING Arbacia punctulata may be induced to shed its eggs and sperm by cutting the animal or injuring it in other ways. Miss Palmer (1935, 1937) dis- covered that spawning may be induced by injecting tissue extract, KCl, or CaCl, into the perivisceral cavity through a slit in the test. I found that one has merely to inject with a pipette some KCl or NaCl solutions into the mouth, through the teeth to induce shedding (E. B. Harvey, 1939c). Tyler (1949a) advocates an injection of 0.5 cc. of 0.5 M KCl which is practically isosmotic, into the body cavity of an average sized Arbacia. The animal, after injection, is held over a dish of sea water into which the eggs or sperm are shed. Although eggs may be collected in this way and the animal remain uninjured, it has been my experience that sometimes the eggs are not fertilizable, even after repeated washings with sea water (E. B. Harvey, 1939b). This has also been the experience of many other investigators, especially during the summer of 1950 when the scarcity of Arbacia necessitated conservation of the animals. It would seem that there is some substance on the out- side of the shell, a dermal secretion perhaps, which is toxic to the eggs, rather than the perivisceral fluid which has been often considered toxic (see under Perivisceral Fluid, Part 1V, especially reference to Pe- quegnat, 1948). If one has a female half covered with sea water into which the eggs are being shed, one finds that the shed eggs will not form a fertilization membrane or be fertilized on addition of sperm, whereas the eggs from inside the animal give 100% fertilization. The same observations had been made previously by Ohshima (1921). Professor Runnstrém, during the summer of 1950 at Woods Hole, found that the toxic effect could be overcome by the addition of sodium periodate (5 x 10-5 M) to the sea water (personal communication). 58 THE AMERICAN ARBACIA There is of course a great advantage in procuring large quantities of eggs by the KCl method, without destroying the animals. Forced shedding by the electrical method has already been described in Section c of this chapter. h. REMOVAL OF GONADS The old method of obtaining eggs and sperm, practiced for many years, was to remove the ovaries and testes intact from the animal. If animals are not scarce, or if for some reason shedding with KCI or electricity is contra-indicated, the following procedure is advocated (E. B. Harvey, 1939b). Wash the animal with running cold fresh water for a moment or two to kill any sperm possibly adhering to the shell. Take the animal, oral (teeth) side up, and cut around the shell with scissors, at about its widest circumference; then remove the upper (oral) part of the shell. The five gonads are now in view in the lower part, the ovaries red, the testes white. To prepare the eggs for experimental work, run a pair of curved forceps gently under an ovary, to snip the duct, and then remove it intact to a finger bowl about a quarter full of fresh sea water. Remove the other four ovaries in the same way. Con- trary to the prevalent opinion, the fluid in the body cavity (in a small amount), is not toxic to the eggs. Let the bowl stand for five or ten minutes so that the ripe eggs may flow out of the ovaries. Then put a piece of cheese cloth whose holes are five to ten times the diameter of the egg (i.e. about 0.5 mm.) and which has been wet with sea water, over another finger bowl and pour the egg suspension through. The débris and pieces of tissue will be be held back and you will have eggs free and clean in the dish, ready for use. To keep the eggs for several hours, there should not be too many eggs in the dish, just enough to form a thin layer on the bottom. The bowls of eggs are best kept on the floor of the cement aquarium tables where cool water will flow around them, and they should be kept covered to prevent evaporation. The eggs treated in this way are suitable for use throughout the day though they change slightly on standing. Individual batches of eggs vary greatly in shape, percentage of fertilization, reaction to centrifugal force, etc. Any batch which does not give 98 % fertilization membranes or which shows abnormalities in cleavage should be discarded. The testes should be removed with curved forceps in the same way as the ovaries, but put into a small salt-cellar or a very small Stender dish (3 cm. diameter) without water. This dish should be covered and kept cool. The sperm are inactive when kept concentrated, but become active in sea water, soon wearing themselves out. If the dish of sperm is placed immediately in the refrigerator and kept at about &° C, the sperm will keep perfectly for 4-5 days. After dilution, the sperm are in optimum condition for only an hour or so. After opening a male, care should be taken to prevent contamination of females opened subsequently. Scissors and forceps should be put into a bowl of tap water and one’s hands should be thoroughly washed, immediately after opening an animal. COHVLAUP YE Eek ss Species of Echinoidea near Woods Hole, Mass, and Arbacia lixula (pustulosa) There are three species of Echinoidea which occur in the vicinity of Woods Hole. Arbacia punctulata is, or has been, the most common species and the one most used for experimental work as the eggs are ripe most all summer. The “green” urchin, Strongylocentrotus drobachiensis, has been taken in deep water in Vineyard Sound, off Gay Head, Nantucket, and many places off Cape Cod, Barnstable, Chatham, Provincetown, and off George’s Bank 75 miles east of Cape Cod. It is a northern species, being circumpolar in the Arctic, and occurs in the north on both the Atlantic and Pacific coasts of North America, on the east coast as far south as New Jersey. It breeds, however, in the early spring, and is spent and of no use for experimental work at Woods Hole during the summer. It occurs in great abundance at the Mt. Desert Laboratory at Salisbury Cove, Maine, and is ripe there also in the spring, March and April. It is full of (mostly) unripe eggs in January and February, full of ripe eggs in March and April, and is spent by May 15, having no ripe eggs in the summer and fall, though some males have ripe sperm. The animals were shipped to me to Princeton for several years (1936-1942) in the spring, for work on hybridization (E. B. Harvey, 1942). The eggs were fertilized and developed normally to plutei, provided they were kept in the cold, at about 10° C. The egg of Strongylocentrotus drobachiensis is 160 yw. in diameter, is un- pigmented and has a jelly coat about 96 pv thick. After fertilization, there is a large perivitelline space of about 30 uw; the hyaline layer is well formed after an hour (at 10° C.) when it is about 3 p thick. First cleavage takes place in 2-3 hours at 10° C. With centrifugal force, the unfertilized mature egg stratifies with oil at the centripetal pole, then yolk granules, clear layer, and mitochondria at the centrifugal pole; the nucleus (14 uw in diameter) lies under the oil cap. There is also, sometimes, a clear layer under the oil instead of, or in addition to, the clear layer beneath the yolk. The egg breaks witha centrifugal force of 12,000 X g for twelve minutes, just above the clear layer under the 60 THE AMERICAN ARBACIA yolk, into a large nucleate half of 152 w diameter and a very small lower half of 80 yp diameter. When fertilized, the lower non-nucleate half containing a little yolk, clear layer, and mitochondria, develops much better than the upper half containing oil and yolk and the nucleus. Further information concerning the distribution, morphology, and larval development may be found in Mortensen’s Monograph, 1943, vol. IIT, Part. 3; p..198. The sand dollar, Echinarachnius parma, is abundant at Woods Hole and breeds during the summer, so that it is excellent material for ex- perimental work at the Marine Biological Laboratory. The eggs are best early in the season, June and July. The egg is large, 145 p in dia- meter, and is unpigmented. It is surrounded by a jelly coat 95 yu thick or less, in which are imbedded large red pigment granules 6-8 yw in diameter. After fertilization there is a large perivitelline space 30 u across; the hyaline layer is so thin as to be unmeasureable at any time. Development is a little slower than in Arbacia; first cleavage occurs at 23° C. in 1} hours (Arbacia 50 min.), and they swim in 16 hours (Arbacia 8 hours). Echinarachnius parma from Maine is a larger animal, averaging 8 cm. in diameter while the one at Woods Hole averages 5 cm., but the egg measurements are the same. With centrifugal force, the mature unfertilized egg stratifies with yolk granules above, then the clear layer, then granules consisting of two kinds, the upper, finer granules being mitochondria, staining with methyl green and Janus green, and the bottom layer of coarser granules and staining with neutral red and methylene blue. Usually there is no oil cap, but sometimes there are a few oil drops at the centripetal pole; the nucleus lies as usual at the centripetal pole. There is sometimes a clear layer here as well as below, as in photograph 14 of Costello (1939). With sufficient force, 10,000 X g for twelve minutes, the egg breaks across the clear layer into a large upper half (126 » diameter) and a smaller lower half (100 » diameter). Both parts develop after fertilization. See Costello, Hoe ees Two other species of Echinoidea were dredged by fishermen in August 1954, about 600 ft. down, go miles south east of No Man’s Land. One was a large dull grey urchin with heavy spines, tentatively identified as Cidaris abyssicola A. Agassiz. The other was a rather beautiful smaller urchin, reddish with long spines, tentatively identified as Coelopleurus jloridanus A. Agassiz. These were quite new to the Woods Hole region. Both are described by Mortensen, the first in his Monograph I : 301, and the second in II : 612. There is another species of Arbacia, A. lixula (pustulosa), accessible SPECIES OF ECHINOIDEA AND ARBACIA LIXULA 61 to investigators who work at the Stazione Zoologica in Naples and some other marine stations in Europe. The eggs of the two species are similar, but that of A. lixula is a little larger (79 up diameter) than that of A. punctulata (74 w diameter), is more heavily pigmented, has a smaller perivitelline space (1-2), and a thicker hyaline layer (3 » on fertilization) E. B. Harvey, 1933a, 1934, 1938a. It stratifies similarly with centrifugal force and is broken into two halves with about the same force (10,000 x g for three minutes), but with this force into al- most equal halves, the upper half containing oil, clear layer, mitochon- dria, a little yolk, and the nucleus, and the lower half containing yolk and pigment (E. B. Harvey, 1933a, 1938a). See also Mortensen’s Monograph II : 566. There are four other species of Arbacia besides A. punctulata and A. lixula. These are: A. stellata, or incisa, A. spatuligera, A. crassispina, and A. dufresni. These are listed in the Classification, with localities where they occur and references to Mortensen’s Monograph. CHAPTER 9 Classification There are about 6,700 species of Echinoidea (Grassé, 1948, p. 162) of which about 600 species are living (H. L. Clark, 1946, p, 277), the rest fossil. In the family Arbacidae, there are 8 recent and 14 fossil genera (H. L. Clark, 1946, p. 306; Mortensen, 1935, M II, p. 547). In the genus Arbacia there are 6 species, all living. All Echinoderms live in salt water. The following classification of the Echinoidea is based on H. L. Clark’s Catalogue of the Recent Sea Urchins (1925), together with some later papers and personal communications with Prof. Clark in 1936 and 1947. He differs in certain cases from Th. Mortensen, whose classification is given in the Handbook of the Echinoderms of the British Isles (1927), and in his Monograph (1928-1951). For simplification, only species are here listed which occur in regions most visited by investigators from Woods Hole, or which are referred to in this Monograph. All species of the genus Arbacia are listed. Much of the data has been taken from Mortensen’s comprehensive Monograph of 1928-1951, referred to in the text as M followed by the appropriate volume and page; these references are given after each species, and the reader may here obtain practically all the available information, and references to all the literature relative to that parti- cular species. Many of the references on egg size are from sources not published but obtained through correspondence or from my own notes. When several authorities:agree on the size of egg, only one authority is given. CLASS ECHINOIDEA (OUTLINE) Order I Cidaroida Other genera (6) not described Family 1 Cidaridae Suborder C Camarodonta Order II Centrechinoida (Diadematoida !) Family 1 Temnopleuridae Suborder A Aulodonta Family 2 Echinidae Family 1 Centrechinidae (Diadematidae) Family 3 Strongylocentrotidae Suborder B Stirodonta Family 4 Echinometridae Family 1 Arbaciidae Order III Exocycloida Genus a Arbacia Suborder A Clypeastrina Genus b Tetrapygus Family 1 Clypeastridae 1 Diadematoida, Diadema, etc. should be used instead of Centrechinoida, etc. See foot- note in chapter 2d, p. 17. CLASSIFICATION 63 Family 2 Laganidae Suborder C Cassidulina Family 3 Fibulariidae Family 1 Neolampadidae Family 4 Scutellidae Suborder D Spatangina Suborder B Echinoneina Family 1 Hemiasteridae Family 1 Echinoneidae Family 2 Spatangidae CLAss ECHINOIDEA Order I Cidaroida Family 1 Cidaridae Cidaris cidaris (Linné) (Dorocidaris papillata). See MI : 300. Iceland, Norway, Southeast England, Ireland, Spain, Mediterranean, Naples, Malta, Azores, Madeira, Canaries, Cape Verde. Egg 160-180 u (Prouho, 1887). M I : 289. Stylocidaris affinis (Philippi) (Dorocidaris papillata). Confused, especially at Naples, with Cidaris cidaris. See Mortensen, M I, p. 293, 341. Florida, Ber- muda, West Indies, Gulf of Mexico, Mediterranean, Naples, Malta, Nice, Madeira, Canaries, Cape Verde. M I : 336. Eucidaris (Cidaris) tribuloides (Lamarck). ‘‘Slate pencil urchin.”’ South Caro- lina to Brazil, Bermuda, Bahamas, West Indies, Jamaica, Mexico, Azores, Africa, Cape Verde. Egg Bermuda 70 » (E.B.H., 1947, unpub.) ; Jamaica 70 wu (Tennent, 1922). M I : 400. Ctenocidaris (Cidaris, Eurocidaris, Stereocidaris) nutrix (Wyv. Thomson). Ker- guelen Island. Egg 2 mm. (Hesse and Doflein, 1910-1914, Bd. I : 583; Pt: G19..00 bs 126, Goniocidaris canaliculata A. Agassiz (Aporocidaris antarctica Mortensen). Falkland and Kerguelen Islands. M I : 116. Goniocidaris umbraculum Hutton. New Zealand. MI : 164. Order II Centrechinoida (Diadematoida) see footnote on preceding page. Sukorder A Aulodonta Family 1 Centrechinidae (Diadematidae, old name, still used by Mortensen) Centrechinus (Diadema) antillarum (Philippi). Florida, Bermuda, West Indies, Jamaica to Surinam, Mexico, Azores, Madeira, Canaries to Cape Verde. Very long spines, can be 40 cm. Egg Bermuda 71 uv (E.B.H., 1947 unpub.) ; Tobago 70 u (Mortensen, 1921). M III, 1 : 269. Centrechinus (Diadema) setosus (Jackson). Suez, Red Sea, African coast to Durban, Madagascar, Australia, Java, Amboina, South Seas, Japan, Misaki. Egg about 100 u, Amboina (Mortensen, 1931), 91.6 u Misaki (Dan, 1952 unpub.). M III, 1 : 256. Centrostephanus longispinus (einliopet N Sevaanaes: Naples, Nice, Mo- rocco to Cape Verde, Azores, Canaries. M III, 1 : 300. Centrostephanus coronatus (Verrill). Southern California, Corona del Mar, Santa Catalina Island, Gulf of California. M ITI, 1 : 314. Suborder B Stirodonta Family 1 Arbaciidae M II : 529 Genus a Arbacia Arbacia punctulata (Lamarck). East coast, Cape Cod to Florida, Woods Hole, Beaufort, Tortugas, Yucatan, Cuba, Curacao, Tobago, Trinidad; not Bermuda, Jamaica, Puerto Rico or Lesser Antilles. Egg Woods Hole 74 u (E.B.H. 1936); Beaufort 80 u (E.B.H., 1942 unpub.). M II : 573. Arbacia lixula (Linné) (Echinocidaris des Moulins, A. pustulosa (Leske, A. aequituberculata (Blainville), A. africana (Troschel). Mediterranean, Naples, Monaco, Nice, Marseilles, Spain, Atlantic Coast of North Africa, Guinea, Gold Coast, Azores, Madeira, Canaries, Brazil. Egg 79 vu (E.B.H., 1933a; 1938a). M II : 566. 64 THE AMERICAN ARBACIA Arbacia incisa or stellata (Blainville; ? Gmelin); incisa preferred by Clark, stellata by Mortensen; see Clark, 1948, p. 245. Lower California to Peru, Gulf of California, Mexico, Gulf of Panama. MII :575. Arbacia spatuligera (Valenciennes). West Coast of South America, Peru. M II : 577 Arbacia crassispina Mortensen. Only from Tristan da Cunha, Nightingale Island. M II : 580. Arbacia dufresnii (Blainville) (A. alternans (Troschel)). South Coast of South America, Chile, Strait of Magellan. M II : 579. Genus b Tetrapygus Tetrapygus (Arbacia, Echinocidaris) niger (Molina). South America, Peru, Chile, M II : 582. There are 6 other living genera and 14 fossil genera listed by Mortensen, 1935, M II : 547. See also Clark, 1946, p. 306. The living genera are: Arbaciella, Coelopleurus, and the rare genera, Pygmaeocidaris, Dialitho- cidaris, Habrocidaris, and Podocidaris. Suborder C Camarodonta Family 1 Temnopleuridae Temnopleurus toreumaticus (Leske). Indo-West Pacific, Japan to African East Coast, Amakusa, Misaki, Korea, China, Philippines, Singapore, East Indies, Australia, Ceylon, India, Iran. Egg 80 p, preserved (Onoda, 1936). M III, 2 : 76. Temnopleurus hardwickii (Gray). Japan, Hakodate, Asamushi, Misaki, Korea, China. Egg 115 wu. (Motomura, 1950 unpub.). M III, 2 : 84. Mespilia globulus (Linné). Japan, Seto, Misaki, Korea, Phillipines, Malay region, Samoa. Egg 80 wu (Onoda, 1936); 110.8 uw (Dan, 1952 unpub.). MPL Ei 2929177: Salmacis virgulata L. Agassiz. Java Sea, Singapore; Indian Ocean to Ceylon, Torres Str., East Australia. May be confused with Temnopleurus alexandri. NWETET, fo ad. Salmacis bicolor L. Agassiz. Indian Ocean, Java Sea. Egg 100 wu (Atyar, 1935): M III, 2: 112. Hypsiechinus coronatus Mortensen. Brood protecting. Southwest of Iceland, Denmark Strait. M III, 2 : 296. Family 2 Echinidae (Mortensen recognizes a family Toxopneustidae which seems superflous to Clark.) Echinus esculentus Linné (E. sphaera). England, Plymouth, Ireland, Scot- land, Denmark, Sweden, Norway, Iceland, France, Roscoff, to Portugal, southern limit. One of largest urchins may measure 20 cm. Egg 180 u Plymouth (Shearer, de Morgan, and Fuchs, 1914, p. 267); 145-9 “ Sweden (Borei, 1935 unpub.). M III, 3 : 25. Echinus melo Lamarck. Mediterranean, Naples, Portugal, West Africa to Cape Verde, Canaries, Azores, West Ireland. M III, 3 : 53. Echinus acutus Lamarck (E. flemingii). England, Plymouth, Ireland, Den- mark, Norway, Iceland, Mediterranean, Naples, Nice. Egg 130-140 u (Shearer, de Morgan, and Fuchs, 1914; p. 267). M III, 3 : 41. Psammechinus (Echinus, Parechinus) microtuberculatus (Blainville). West- ern Mediterranean, Naples, Adriatic, Trieste. Egg 102 » Naples (E.B.H., 1933a; 1938a). M III, 3 : 139. Psammechinus (Echinus, Parechinus) miliaris (P. L. S. Miiller; Gmelin). Two distinct types, S- and Z- (Lindahl and Runnstrém, 1929). Iceland, Norway, Sweden, Denmark, Scotland, Ireland, England, Plymouth, N. W. coast of Africa to Cape Verde Isl.; not Mediterranean. Egg: S-type 114.8 yp, Z-type 98.3 » (Lindahl and Runnstrém, 1929). Eggs variable in size (Borei, 1948). M III, 3 : 127. CLASSIFICATION 65 Lytechinus (Toxopneustes) variegatus (Lamarck). Typical form is West Indian (H: L. C.). Jamaica, Puerto Rico. M III, 2 : 437. Subspecies atlanticus (A. Agassiz). Bermuda. Usually brown, some are white. Egg 103 pv (E. B. H., 1947 unpub.). M III, 2 : 444. Subspecies carolinus (A. Agassiz). Beaufort, Tortugas. Usually white. Egg 112 u, Beaufort (E. B. H., 1942 unpub.). roo-120 pw, Tortugas (Tennent et al., 1929). M III, 2 : 444. Clark says Toxopneustes is a good but very different genus in the Pacific. Lytechinus anamesus H. L. Clark. Southern California, Corona del Mar., Lower California, Guadeloupe Island. Egg 111 w (Tyler, 1949 unpub.). M III, 2 : 452. Lytechinus pictus (Verrill). Southern California, Corona del Mar, Lower California, Gulf of California. Egg 111 uw (Tyler, 1949 unpub.). M III, 2: 450. Tripneustes (Hipponoé) esculentus (Leske), (T. ventricosus (Lamarck). See M III 2, p. 488, 497. Called ‘‘sea eggs” in West Indies. Large form, test may be 15 cm. Bermuda, Bahamas, Florida, Tortugas, West Indies, Ja- maica, Trinidad, to Brazil. W. coast of Africa. Egg 84 u (E. B. H., 1933 a). M III, 2 : 490. Tripneustes (Hipponoé) gratilla (Linné). Indo-Pacific, South Seas, Australia, Hawaii, Japan, Seto. Egg go uw (Onoda, 1936). M III, 2 : 500. Toxopneustes pileolus (Lamarck). Indo-West Pacific, Madagascar, Fiji, Japan, Misaki, Seto. Egg 80 u preserved (Onoda, 1936). M III, 2 : 472. Family 3 Strongylocentrotidae Paracentrotus (Strongylocentrotus) lividus (Lamarck). Mortensen places this in the Echinidae. Entire Mediterranean, Nice, Naples, Alexandria, Cana- ries, Madeira, Azores, African coast to Rio de Oro, Roscoff, Ireland and Scotland. Egg at Roscoff 100 u, at Naples go uw (Runnstrém, 1933); at Roscoff go uw (Lindahl and Lundin, 1948). (See also E. B. H., 1933; 1938a). MII, 3 : 157. Strongylocentrotus drébachiensis (O. F. Miiller). Named for Drébak, a sea- side town in Norway, near Oslo. Known as the “‘green urchin.’’ Circum- polar in the Arctic. Atlantic coast from Arctic to New Jersey, abundant in Maine, occurs at Woods Hole. North Pacific coast, Alaska, Aleutian Is- lands, Puget Sound, N. E. Russia, Norway, Sweden, Denmark, Scotland. Egg Maine 160 u (E. B. H., 1942). Sweden 136 uw (Vasseur, 1949). M III, Bg) 100. Strongylocentrotus franciscanus (A. Agassiz). Pacific Coast to Alaska, Puget Sound, Pacific Grove, Corona del Mar, Northern Japan, Hakodate. Egg 120 p (E. B. H., 1942). M III, 3 : 242. Strongylocentrotus purpuratus (Stimpson). Pacific Coast to Alaska, Corona del Mar, Pacific Grove. Egg 80 u (E. B. H., 1942). M III, 3 : 236. Strongylocentrotus (Allocentrotus) fragilis Jackson. Pacific Coast from Lower California to Vancouver, Corona del Mar. M III, 3 : 255. Strongylocentrotus (Hemicentrotus) pulcherrimus (A. Agassiz). Japan, Hako- date, Asamushi, Misaki, Seto, North coast of China. Egg 96.55 pu (J. C. Dan, 1952 unpub.). M III, 3 : 248. Strongylocentrotus nudus (A. Agassiz). Northern Japan, Asamushi, Vladi- vostok. M III, 3 : 232. Strongylocentrotus intermedius (A. Agassiz). Northern Japan, Hokaido, Asa- mushi, Onagawa, Vladivostok. M III, 3 : 225. Pseudocentrotus depressus (A. Agassiz). Placed by Mortensen in family Toxopneustidae. Southern Japan, Misaki. Egg 105.9 » (Endo, 1952 un- pub.). M III, 2 : 541. Heliocidaris (Anthocidaris) crassispina (A. Agassiz). Placed by Mortensen 66 THE AMERICAN ARBACIA in family Echinometridae. Southern Japan, Misaki. Egg 100 u (Moto- mura, 1950 unpub.); 95.5 uw (Dan, 1952 unpub.). M III, 3 : 328. Sphaerechinus granularis (Lamarck). Mortensen places this with Trip- neustes and Lytechinus in family Toxopneustidae. Large form, may be 16 cm. Mediterranean, Naples, Nice, Channel Isls. Guernsey to Cape Verde, Spain, Portugal, Azores, Madeira, Canaries. Egg 98 uw (E. B. H., 1933a; 1938a). M III, 2 : 515. Family 4 Echinometridae, usually elongate, elliptical Echinometra lucunter (Linné) (E. subangularis (Leske)). Known as ‘‘rock’’ or “reef”? urchin. Bermuda, West Indies, Jamaica, Florida to Brazil, Mexico, West Africa, Dakar to Angola. Egg 80 up, 95 uw Tortugas (Leitch, 1934a;1936); 85 uw and 97 uw Bermuda (E. B. H., 1947 unpub.); 120 u Tobago (Mortensen, 1921, p. 71). M III, 3 : 357. Echinometra viridis A. Agassiz. Southern Florida, Tortugas, Curacao, Vene- zuela, Greater Antilles, St. Thomas. M III, 3 : 368. Echinometra mathaei (Blainville). Clark says this is the most abundant sea urchin in the world. Indo-West Pacific, Suez to Hawaii, Egypt, E. African coast to Durban, Madagascar, Ceylon, Torres Strait, Murray Isl., Australia, Samoa, Japan, Seto, etc. M III, 3 : 381. Echinometra vanbrunti A. Agassiz. Rock-borer. West coast of North Ame- rica from central California to Peru, Mazatlan, Mexico; Gulf of California Gulf of Panama. M III, 3 : 373. Order III Exocycloida Suborder A Clypeastrina. Usually flattened. Family 1 Clypeastridae Clypeaster rosaceus (Linné). N. Carolina, Florida to Brazil, Tortugas, Baha- mas, West Indies, Puerto Rico, Jamaica, Barbadoes, Curagao. Egg 200 u (in Bouin) (Gardiner, 1927). M IV, 2 : 40. Clypeaster subdepressus (Gray). Florida, Tortugas, West Indies, Jamaica, Brazil. M IV, 2: 112. Clypeaster japonicus Déderlein. Southern Japan, Misaki. Egg 125 » (Moto- mura, 1950 unpub.); 115.3 u (Dan, 1952 unpub.). M IV, 2 : gg. Family 2 Laganidae Peronella (Laganum) lesueuri (A. Agassiz) or (Valenciennes). Confusion of species and several varieties. Australia, Torres Strait, Japan, Misaki, China. Egg 300-400 uw (Mortensen, 1921, p. 111; Tennent, 1924); 276 (Dan, 1952 unpub.). M IV, 2 : 263. Family 3 Fibulariidae Echinocyamus minutus (Pallas) (E. pusillus (O. F. Miller) is Mortensen’s preference). Iceland, Norway, Sweden, Scotland, England, Plymouth, Ireland, Mediterranean, Naples, Marseilles, North Africa, Azores. Egg 88 uw (Théel, 1892). M IV, 2: 178. Family 4 Scutellidae “‘Sand dollars”’ Echinarachnius parma (Lamarck). East Coast Labrador to Maryland, Maine, Woods Hole, West Coast Alaska to Puget Sound, Aleutian Islands, Japan. Egg 145 vu (E. B. H., 1942). M IV, 2 : 367). Dendraster (Echinarachnius) excentricus (Eschscholtz). Pacific Coast, Alaska to Lower California, Nanaimo. Egg Pacific Grove 120 up (Snyder, 1925; Chase, 1935); 115 u (Needham and Needham, 1930); Corona del Mar 114. u (Tyler, 1937). M IV, 2 : 382. Mellita quinquiesperforata (Leske) (M. pentapora (Gmelin), M. testudinata Klein). 5 holes in test. Known as the ‘“‘keyhole urchin.”” Vineyard Sound to Brazil, Florida, Bermuda, Jamaica, Puerto Rico, Mexico. Egg Ber- muda? 110u (Crozier 1918); Beaufort 150 u (E. B. H., 1942 unpub.). MeIV 25: 422% ‘ CLASSIFICATION 67 Mellita (Leodia) sexiesperforata (Leske) (M. hexapora (Gmelin), M. sex- foris (Lamarck)). 6 holes in test. “‘Keyhole urchin.”’ South Carolina to Uraguay, Bermuda, West Indies, Jamaica. Egg 260 uy (Crozier, 1918). Mr PV,.2) 2 429; Encope michelini L. Agassiz. Gulf of Mexico, Florida to Yukatan. M IV, 2: 441. Encope emarginata (Leske). East Coast, Florida to Argentina, West Indies, Trinidad. M IV, 2 : 438. Astriclypeus manni Verrill. Known in Japan as the “‘perforated pancake ur- chin.” Southern Japan, Misaki. Egg 182.1 uw (Dan, 1952 unpub.). M IV, 2: 416. Suborder B Echinoneina Family 1 Echinoneidae Echinoneus cyclostomus Leske (E. semilunaris (Gmelin)). Cosmopolitan in tropics, Tortugas, Bermuda, West Indies, Jamaica, Tobago, Indo-Pacific, Madagascar, Torres Strait, Australia, Hawaii. M IV, 1 : 75. Suborder C Cassidulina Family 1 Neolampadidae Anochanus sinensis Grube. Brood protecting. China Sea. M IV, 1 : 344. Suborder D Spatangina “‘Heart urchins” Family 1 Hemiasteridae Moira atropos (Lamarck). North Carolina, Beaufort, South Carolina, Flori- da, Texas, West Indies, Jamaica, Puerto Rico. M V, 2 : 329. Hemiaster (Abatus) cordatus Verrill, or cavernosus. Brood protecting. Kerguelen Island. Egg 1 mm. (Hesse and Doflein, 1910, Bd. 1, p. 583; 1914, Bd. II, p. G19). M V, 2 : 257. Family 2 Spatangidae Brissopsis lyrifera (Forbes). Norway, Sweden, Scotland, Ireland, England, Mediterranean, Naples, South Africa, Cape of Good Hope. M V, 2 : 380. Brissus brissus (Leske) (B. unicolor Klein). Tortugas, Bermuda, West Indies, Jamaica, Mediterranean, Naples, Cape Verde, Madeira, Azores. M V, 2 : 509. Meoma ventricosa (Lamarck). Southern Florida, Bahamas, West Indies, Jamaica. M V, 2 : 529. Spatangus purpureus O. F. Miiller. Norway, Sweden, Scotland, Ireland, England, Plymouth, Mediterranean, Naples, Malta, Algiers, Azores, M V;.2:: ro. Spatangus raschi Lovén. Norway, Ireland. M V, 2 : 14. Lovenia cordiformis A. Agassiz. Southern California, Corona del Mar, Gulf of California, Mexico, Panama, Galapagos Islands. M V, 2 : 104. Echinocardium (Amphidetus) cordatum (Pennant). Burrows. Norway, Sweden, England, Plymouth, Ireland, Mediterranean, Naples, Nice, Japan, Asamushi, Australia. Egg at Kristineberg, Sweden is pear shape, 125 u. (Gustafson, 1945; Runnstrém, 1948). “‘Ellipsoidal’’ at Millport, Scot- land (MacBride, 1914b); at Asamushi 110 up (Motomura, 1950 unpub.). M V, 2: 152. Echinocardium mediterraneum (Forbes). Mediterranean, Naples, Spain, Portugal. Egg 40 p (Hamman, 1887, p. 140). M V, 2: 162. Echinocardium pennatifidum Norman. Norway, Bergen, Faroe Islands to Channel Islands. M V, 2 : 163. u La (Need on im a - mun is ree c — ; | * PART II NORMAL DEVELOPMENT A Ss a, . < THIMAAL Sd, LAO. oe 7 a7 — oo a ‘ = = a’ : : c fi oO iy ; f hs nies Woe Ata: ¢ a Hpk om 1a i we - ly are i: 2 OW, “aay in i won! _ aie + oe ‘an Dahan is j Gil ALP ALE Re 310 The immature egg and its maturation a. GROWTH AND DIFFERENTIATION OF THE OocyTE. PLATE II The Arbacia egg matures in the ovary, so that when the eggs are shed they have lost their polar bodies and are fully ripe and ready for fertil- ization. The various stages of growth of the young oocyte and the polar body formation are best studied at Woods Hole in early or middle June. One can obtain immature eggs among the mature ones later in the summer, but they are usually not abundant. The very young oocytes, of about 14 y in diameter, have a very large germinal vesicle with a vesicular nucleolus, very little cytoplasm, sometimes granular, sometimes quite clear, and no pigment..(Plate IT, Photograph 1). From the data given in Table 1, it will be seen that at this stage the nuclear material (germinal vesicle) occupies about two thirds of the whole cell and that there is more than twice as much nuclear material as cytoplasmic. As the immature egg increases in size, the germinal vesicle also increases, but not so much as the cyto- plasm. The largest immature egg is the same size as the mature egg, but the mature nucleus is very much smaller than the germinal vesicle of the immature egg, about 1/40th the volume. The young oocytes are still without pigment till they reach a dia- meter of about 33 » (Photograph 3). They then become slightly pig- mented, the pigment granules, gradually increasing in quantity with growth. The oil, recognizable as an oil cap after centrifuging, appears about the same time as the pigment. The mitochondria appear around the germinal vesicle at about the same time, as can be determined by staining with methyl green. A few scattered mitochondrial granules are sometimes present in smaller eggs, of about 23 yv in diameter. Jelly is formed when the young oocyte is about 60 » in diameter (Photo- graph 6). The data are given in Table 1. No study has been made of the oogenesis and spermatogensis of Arbacia, but Tennent and Ito (1941) have published a very complete account of the oogenesis of the Japanese form, Mespilia globulus, which is probably similar, 72 THE AMERICAN ARBACIA TABLE 1 SIZE OF OOCYTE, GERMINAL VESICLE AND NUCLEOLUS DURING GROWTH CYTOPLASM STAGE OOCYTE calvieay fie NUCLEOLUS iva pares VESICLE COLUMN 4 ; FROM COLUMN 2) I 2 3 4 5 6 7 D(u) V (v8) Diu) V(u) Diy) V (ui) V (u?) Very young 14.3 1,531 12.8 1,098 4.8 57-9 433 Pigment present 33 18,820 22 5575 9.6 463.2 13,245 Jelly present 60 13,100 30 14,140 10 523.6 98,960 Largest 74 212.200 38.4 29,650 411.2 735.6 182,550 SIZE OF MATURE EGG AND NUCLEUS MATURE EGG NUCLEUS CYTOPLASM D 74 V 212,200 Di 115) V 1796.3 V 211,404 b. STRUCTURE OF THE OocyTE. PLATE II ‘The immature egg when fully developed consists of cytoplasm and germinal vesicle, usually excentric. The cytoplasm consists, like that of the mature egg, of a matrix or ground substance in which are scattered spherical oil droplets, yolk granules, spherical red pigment granules or vacuoles, mitochondria, and microsomes. All the granules are scat- tered through the cytoplasm except the mitochondria which form a thick layer around the outside wall of the germinal vesicle; this can easily be seen in eggs stained with methyl green. The fully formed germinal vesicle is about 38 uw in diameter, and contains a vesicular nucleolus, usually excentric, about 11 » in diameter, often with one or many small bodies or nucleolini within it (see also R. D. Allen, 1951b). When fixed in Bouin’s solution and stained with Heidenhain’s haemotoxylin, the nucleolus is quite black, and there is a black staining network through the germinal vesicle (E.B. Harvey and Lavin, 1944) ; (Photographs 17 and 18). Excellent photographs of sections of the immature Lytechinus (Toxopneustes) egg thus prepared are to be found in Wilson’s (1895) Atlas of Fertilization, Plate I. The appearance of the immature egg with ultraviolet light is similar to that in the stained preparations (E. B. Harvey and Lavin, 1944). The nucleolus and chromatin network are absorbing and appear black in photographs THE IMMATURE EGG AND ITS MATURATION 73 showing the presence of nucleic acid compounds. Casperson and Schultz (1940) think these are of the ribose type (in Psammechinus mili- aris). The appearance of the immature Arbacia egg with other stains is described and illustrated by E. B. Wilson (1926) ; Benda-osmic, Benda- Kull, Champy-Kull, Champy-osmic, thionin, toluidin blue have been used. In some of these figures, bodies which appear to be nucleolini inside the nucleolus show up clearly but are not mentioned in the text. Some cytochemical techniques have been described by Krugelis (1947b): Gomori, fast green, toluidin blue, and Feulgen. With certain intravitam stains, the immature egg stains more inten- sely than the fertilized egg, and this more intensely than the unfer- tilized (Lyon and Shackell, 1g10b; E. N. Harvey, tgtoc). The immature egg is surrounded, like the mature egg, by a layer of jelly. In the older oocytes it is the same in amount as in the mature egg and may be as much as 2 pu thick. It is thinner in younger oocytes; in an oocyte whose diameter was 60 yu, the jelly was only 3.2 uw. The jelly coat is discussed under the mature egg and further data may be found in Part IV, Jelly Layer. c. REACTION TO SPERM One of the most characteristic features of the immature egg is its reac- tion to sperm. The formation of papillae or blebs (Photograph 16) wherever sperm hit the surface is a well-known phenomenon in many echinoderms including Asterias and holothurians (Hobson, 1927). These papillae have many different forms; they may be blunt or sharply pointed and are sometimes branching; they are always hyaline, without granules. They have been carefully studied by Seifriz (1926) in the sand dollar. The sperm enter the immature Arbacia egg as (Photo- graph 17) they do other sea urchin eggs, e.g., Lytechinus as described by Wilson (1895) in his Aélas. But no fertilization membrane is raised. The papillae are considered similar to fertilization cones which form on mature eggs after fertilization. They are formed within two minutes after adding sperm, and they last for about 20 minutes and are gra- dually resorbed. They are formed on eggs with germinal vesicle intact, after the germinal vesicle has broken, during polar body formation and even an hour after the second polar body has been given off. They do not form in Ca-free sea water though the sperm are motile; this has been found also for Anthocidaris crassispina by Sugiyama (1938g) and for Psammechinus miliaris by Rothschild and Swann (1949). It is of interest that fertilization of mature eggs will not take place in Ca- 74 THE AMERICAN ARBACIA free sea water, though the sperm are motile (Loeb, 1915a; E. B. H., unpub.; see also Monroy, 1949 for Ps. microtuberculatus). The papillae do not form on immature eggs which have been in hypertonic sea water for 20 minutes and then returned to sea water, a method of producing parthenogenesis of mature eggs (E. B. Harvey, 1938a for Ps. microtuberculatus). Sugiyama states that the papillae do not form if the sperm have been treated with ultraviolet light, but this is not true for Arbacia (E. B. H., unpub.). d. CENTRIFUGING AND GRAVITY On centrifuging, about 10,000 x g for 3 minutes, the mature unfer- tilized egg is well stratified. The immature egg is only partially strati- fied, with a small oil cap at the centripetal pole and pigment at the centrifugal pole and above this the yolk but sometimes not well separ- ated from the pigment (Photograph 13). The clear layer is not well defined, and there is no mitochondrial layer. After centrifuging the mitochondria remain as a thick layer around the wall of the germinal vesicle as shown in Photograph 18 (not centrifuged). The egg does not elongate nearly as much as the mature egg, and does not break apart with the forces which are sufficient to break the mature egg into halves. The immature egg is thus shown to be much more viscuous than the mature egg. This has been noted by previous observers (Heilbrunn, 1928, p. 278; 1952, p. 85; Goldforb, 1935b). The germinal vesicle, in centrifuged eggs, lies at the centripetal pole under the oil, and the nucleolus is heavy and always lies at the bottom of the germinal vesicle (Photograph 13). Gray (1927b) has made some interesting studies on the effect of gravity on the nucleolus of the Echinus esculentus egg. He found that the nucleolus descends by gravity at the rate of 0.4 p per second (1.5 mm. per hour). The nucleolus in the eggs of some species can be driven by strong centrifugal force, with an air turbine, right through the germinal vesicle wall. Such is the case with Strong ylocentrotus drobachiensis (E. B. H., unpub.). e. RESPIRATION The rate of respiration is less in immature than in mature eggs ac- cording to Boell, Chambers, Glancy, and Stern (1940). Borei (1948) found it slightly higher in Psammechinus miliaris, and Lindahl and Holter (1941) in Paracentrotus lividus. THE IMMATURE EGG AND ITS MATURATION 75 f. PERMEABILITY TO WATER According to Churney (1941b, 1942), the permeability to water of the immature egg is the same as the mature egg; the germinal vesicle acts as a perfect osmometer, and the nucleolus swells and shrinks reversibly. g. PoLAR Bopy Formation. PLATE II After the immature egg has reached its full size, it throws off two polar bodies. The first step in this process is the approach of the germinal vesicle to the cell wall (Photograph 7). Then after a period of about 1} hours, during which the nucleolus has become smaller and dis- appeared, the wall of the germinal vesicle breaks down; the red pig- ment granules become more numerous and very bright in color in this region, just inside the cell wall near the breaking germinal vesicle (Photographs 7 and 8). The mitochondrial granules do not take part, but are heaped up on the opposite side of the breaking germinal vesicle, as can be seen in eggs stained with methyl green. A clear area is seen where the germinal vesicle broke down, and two or two and a half hours (23° C.) after the breakdown, the first polar body is given off. One to one and a half hours later the second polar body is given off. Then about two hours later, the mature nucleus is formed. During the formation of the polar bodies the egg becomes somewhat flattened in the axis of the polar bodies (Photograph 12). This lasts for about 7 minutes; then the egg becomes spherical again. By watching individual eggs fertilized at intervals (23° C.) after the second polar body has been given off, the following data have been obtained: 3 tor hr. No fertilization membrane, delayed and irregular cleavage, polyspermy, 3 cells at once. 1} to 13 hrs. Fertilization membrane, polyspermy, irregular cleay- age, normal swimming blastulae. 2 to 23 hrs. Cleavage delayed and slightly irregular, polyspermy, 4 cells at once. 3 to5 hrs. Slight delay in first cleavage; cleavages regular, mi- cromeres normal, normal plutei. There is apparently a sort of cytoplasmic maturation following nuclear maturation such as has been described for Paracentrotus lividus by Paspaleff (1927) and for Psammechinus miliaris by Runnstrom and Monné (1945) and Runnstrém (1948b). The eggs do not develop 76 THE AMERICAN ARBACIA perfectly until several hours after the second polar body has been given off. If Arbacia eggs are centrifuged at the time of polar body formation, it has been found that the polar bodies may come off in any relation to the stratification, in the yolk or pigment zone or even at the oil cap (E. B. H.) (Photographs 14 and 15). It seems that the original polarity of the egg must determine their position and not the polarity imposed by centrifugal force expressed by the stratification. Usually the polar bodies of Arbacia have been thrown off and dis- carded by the time the eggs are laid or taken from the ovary, but in some batches they are retained in the majority of the eggs. In such a batch, Hoadley (1934) found that they may lie in any position with regard to the (mature) nucleus, and I have confirmed this observation in other batches. The polar bodies in Paracentrotus lividus are given off in the micropyle (Boveri, 1901), and also in Lytechinus (Tennent, Taylor, and Whitaker, 1929); this is true also of Arbacia. The first cleavage plane passes through the region of the polar bodies. The micromeres are formed at the vegetal pole, opposite the polar bodies. Previous observers also found that the micromeres formed “‘approxi- mately opposite to the micropyle” in Arbacia (Morgan and Spooner, 1909, p. 116; Spooner, 1911; Ho6rstadius, 19374). OTHER SPECIEs (ADDITIONAL) AND GENERAL REFERENCES A. Brachet, 1922. Paracentrotus lividus, fertilization of immature eggs. J. Brachet, 1933. Paracentrotus lividus, Feulgen stain. Bryce, 1902. Polar bodies, sections. Derbés, 1847. ‘“Oursin comestible’’; earliest figure of immature egg. Harris, 1939. Echinus esculin'us, viscosity and polarity; fall of nucleolus. Lindahl and Holter, 1941. Paracentrotus lividus, respiration. Lyon and Shackell, 1910b. Toxopneustes variegatus, permeability. Runnstr6m, 1928c. Paracentrotus lividus, etc., papillae, surface. Selenka, 1878. Toxopneustes variegatus, early description. Skowron and Skowron, 1926. Sphaerechinus granularis, permeability. Tennent, Gardiner, and Smith, 1931. Echinometra lucunter, stains for micro-chemistry. von Ubisch, 1950. Paracentrotus lividus, general. Wilson, 1895. Atlas of Fertilization. Wilson, 1899. Protoplasmic structure. Wilson, 1925. The Cell, General. Wilson and Mathews, 1895. Toxopneustes variegatus, polarity. CRAP IE Rit The mature egg, unfertilized and fertilized a. QUANTITY OF EGGs IN AN ARBACIA The number of eggs obtained from one Arbacia varies considerably with the season. Even at the height of the season, some females have many more eggs than others. Usually, the eggs obtained from one female in the usual way, by allowing them to ooze out of the excised ovaries and then filtering through cheesecloth, will well cover the bottom of a finger bowl in a thin layer; from exceptionally good females, they cover the bottoms of two finger bowls. When allowed to settle for several hours by gravity, the volume of the eggs from an exceptionally good female amounts to 5 cc. With light centrifuging, this amounts to 3 cc., but many are now without jelly. The 5 cc. of settled eggs with jelly contain approximately 4,000,000 eggs since each egg has a volume of 1,260,300 uv? (diameter, egg 74 » + jelly 60 u, without interspaces be- cause the jelly of adjacent eggs is contiguous). The volume of the settled eggs is about equal to the volume of eggs left in the ovaries. One Arbacia, then, contains about 8,000,000 eggs. The same figure has been arrived at for the number of eggs shed either by KCl or by the electrical method. MacBride (1906, p. 529) states that a well grown Echinus esculentus contains 20,000,000 eggs; this compares fairly well with the figures for Arbacia since Echinus esculentus is a very much larger animal. QUANTITY OF EGGS IN I CC. SETTLED BY GRAVITY (E. B. HARVEY) Eggs with jelly 800,000 (vol. per egg 1,260,000 u3; there are no interspaces, eggs are contiguous). Eggs without jelly 3,500,000 (vol. per egg 212,200 uw; allowing 26% for interspaces). Eggs without jelly 4,700,000 (no allowance for interspaces). Other figures have been given by Krahl (1950, p. 177). Eggs per 10 c.mm. 46,500 Eggs per wet gram 4,300,000 Eggs per mg. dry weight 17,600 78 THE AMERICAN ARBACIA b. METHODs FOR EsTIMATING VOLUME AND QUANTITY Volume. 1. Direct measurement. Measure the diameters of many eggs of a sample, convert to volume}, and take average. 2. Diffraction method. Described by Lucké, Larrabee and Hartline, (1935). Used by Lucké e¢ a/. in subsequent papers; Korr (1937); Ballentine (1940b); et al. Number of eggs in a suspension. The main error in determining numbers of eggs is caused by the jelly coat which varies in different batches of eggs, and may be removed from some or all eggs by agitation, centrifuging, etc. The presence of the jelly coat makes a difference of about 60 yw in the diameter of each egg, and it has a volume of about 1,050,000 y'. The following methods have been reviewed and appraised by Shapiro (1935c). 1. Haemocytometer. Used by Tang and Gerard (1932), Gerard and Ruben- stein (1934); e¢ al. 2. Centrifuge, using haematocrit tubes. Used by Whitaker (1933a IV; 1935); Clowes et al. (1936, etc.); Mazia (1937); criticized by Gerard and Rubenstein (1934). 3. Dilution method of Parpart, described by Shapiro (1935c). This seems to be the best method. Immerse quickly a capillary of 1 mm. bore into a uniform sus- pension of experimental eggs, filling to a length of 8-10 cm. Lay the capillary on its side under a binocular microscope, and count the number of eggs. Do this three times. If the suspension is too thick, dilute to a suitable amount and allow for the dilution factor in calculation of number of eggs in original suspension. Used by Korr (1937); Ballentine (1940b) e¢ al. Number of cleaving eggs. Piace the eggs at the desired stage in a weak formol solution, 0.04 to 0.5%, and count at leisure for percentage (Morgan, 1895b and many others). c. SHAPE OF EGGs Arbacia eggs are usually spherical when shed or removed from the ovary. Sometimes they are aspherical due to crowding in the ovary, especially late in the season; they become spherical on standing or on fertilization (see also Goldforb, 1935a). They become amoeboid with ethyl urethane, urea, etc. See Amoeboid Eggs, Part. IV. They do not flatten by gravity (McCutcheon, Lucké, and Hartline, 1931; Cole, 1932; E. N. Harvey, 1933; E. B. Harvey, 1934). It was claimed by Chambers (1921 a) that Arbacia eggs do flatten by gravity, and by Vlés (1926) that Paracentrotus lividus eggs flatten. Rothschild and Barnes (1953) state that P. lividus eggs do not flatten by gravity. The egg of Echinocardium cordatum (Kristineberg, Sweden) is peculiar in being pear-shaped, but becomes spherical on fertilization (Gustaf- son, 1945; Runnstrém, 1948). MacBride (1914b) describes it as “‘ellip- soidal’’ at Millport, Scotland. The eggs of the sea urchins at Naples, Sphaerechinus granularis, Psammechinus microtuberculatus, and Paracentrotus lividus are much more aspherical than those of Arbacia punctulata at Woods Hole (E. B. Harvey, 19334). 1 A good conversion table from diameters to volumes of spheres is given in the Chemical Engineers’ Handbook, 3rd edition, 1950, p. 34, 35; 2nd edition, 1941, p. 90, 91. J. H. Perry, editor. THE MATURE EGG, UNFERTILIZED AND FERTILIZED 79 d. SizE, WEIGHT AND DENsITY OF EGG SIZE DIAMETER SURFACE AREA VOLUME Egg without jelly 74 w 17,200 wu? 212,200 3 Egg with jelly 134 uw (74 + 60) 56,430 wu? 1,260,000 3 Fertilized egg including fertili- zation membrane, without jelly 84 (74+ 10) 22,170 2? 310,300 w Nucleus 11.5 2 415.6 pw? 796 u3 WEIGHT I cc eggs, dry wt. 265 mg. (Ballentine, 19404) 10° eggs, dry wt. 58.0 mg. 10 c. mm. eggs, dry wt. 2.63 mg. (Krahl, 1950) IO c. mm. eggs, wet wt. 10.9 mg. Dry weight is approximately 24 % of wet weight (Krahl, 1950). DENSITY 1.081 to 1.087; same to 16 cell stage; blastula lighter than 16 cell stage, but heavier than sea water; plutei 1.055 to 1.066 (Lyon, 1907). 1.0485 to 1.0656, unfertilized (Heilbrunn, 1926a, 1928, p. 71). 1.090, unfertilized, with jelly (E. N. Harvey, 1931, 19324). 1.084, unfertilized, without jelly. e. SIZE OF FERTILIZED EGG It is generally accepted that the fertilized egg (without fertilization membrane) is the same size as the unfertilized (Whitaker, 1933b), though it is larger by the addition of the hyaline layer, which is extra- neous to the egg proper (E. B. Harvey, 1933 a for A. lixula). But Glaser (1913; 1914a, b, c; 1924) found the egg smaller by 2.5 u immediately after fertilization, then larger, and also R. S. Lillie (1916a) smaller by 1.0 u. Chambers (1921 a) found it slightly larger, and Shapiro (1948b) found an increase of 2.7°% in volume. There is no appreciable increase in diameter until the gut is complete in the gastrula.stage, about 17 hours after fertilization (E. B. Harvey, 1949). f. VARIATION IN SIZE Average size of unfertilized egg as recorded by some investigators is shown in the following tabulation. The figures in brackets are computed from the figure given by the author. 80 THE AMERICAN ARBACIA DIAMETER, VOLUME, v3 AUTHOR 74.1 (213,000) Glaser, 1914a 74.6 (217,400) Glaser, 1924 75.0 (220,900) Heilbrunn, 1915a 74.1 213,000 R. S. Lillie, 1916a 74.1 213,000 McCutcheon, Lucké, and Hartline, 1931 (72.4) 198,900 McCutcheon, Lucké, and Hartline, 1931 (75-4) 224,500 McCutcheon, Lucké, and Hartline, 1931 73 203,700 E. B. Harvey, 1932 74 212,200 E. B. Harvey, 1936, 1941a, 1946 (72.9) 202,640 Goldforb, 1935a Variation in size by one investigator is shown by: Goldforb’s (1935a) figures for 1,000 eggs from 25 females: 68.3 u to 77.4 w diameter. Shapiro’s (1935c) figures for eggs measured through summer of 1934: 64 » to 81 uw diameter. Variation in size has also been studied by Glaser (1914a, 1924) and by R. S. Lillie (1916a). Variation is great for different females but is quite small for the eggs of one female, as noted by many observers and studied especially by Goldforb (19354). g. ABERRANT SIZED Eccs AND NUCLEI Aberrant sized eggs and nuclei. Occur in some batches of normal eggs; are uniform in size with no gradations in any one batch (E. B. Harvey). 1. Giant egg with giant nucleus. Egg D. g1 p, Nucleus 14 p, (1939); in 1% of eggs; normal development. Egg D.'96 u, Nucleus 14.4 y; in a few eggs (1950). 2. Normal egg with giant nucleus, Egg D. 72 », Nucleus 17 w; in 1% of eggs; normal development (1940). Egg D. 71 », Nucleus 29 py; in .01% of eggs; abnormal development (1939). 3. Small egg with giant nucleus. Egg D. 57.6 u, Nucleus 16.5 yu; normal cleavage, blastulae, no plutei (1940). 4. Small egg with normal nucleus. Egg D. 50u, Nucleus 11.5 p (1940). 5. Normal egg with 2 nuclei, same volume together as a normal nucleus (1940). 6. Immature egg with two germinal vesicles (1943). THE MATURE EGG, UNFERTILIZED AND FERTILIZED 81 h. ErFEct oF AGE, TEMPERATURE, ETC. ON SIZE OF EGGs Effect of: Age. No effect for 4 hours (Glaser, 1914a). Increase in size for first 23-50 hrs., measurable by 3rd hr., then decrease (Goldforb, 1918a, b; 19354). Temperature. No effect 5.4—29.3 °C. (Lucké and McCutcheon, 1926a; 1935): pH. No effect 4.0-9.8° (Lucké and McCutcheon, 1926a, b). Amino acids. No swelling (Lucké and McCutcheon, 1926a). Ether. No effect unless injured (Lucké and McCutcheon, 1926a). Urethanes. No effect (Lucké, 1931). Oxygen lack. Become slightly smaller (Hunter, 1936). i. STRUCTURE OF THE EGG Like the immature egg, the mature egg contains oil drops, yolk gra- nules, mitochondria, red pigment granules, and microsomes, all scat- tered in a matrix or ground substance, and a small nucleus. The alveo- lar structure of the egg protoplasm has been described by E. B. Wilson (1899, 1926). Data on the various granules will be found in Part IV, under the appropriate heading. Photographs of the living unfertilized egg and of a stained section are shown on Plate III, Photograph 1, and Plate V, Photograph 1. Nucleus. — The nucleus of the mature egg is excentric, has a diameter of approximately 11.5 pu, is lighter than the cytoplasm, 1.e., goes to the centripetal pole when the egg is centrifuged. Not much structure is seen in the living nucleus, and it does not take any vital dyes (E.B.H., 1941 c). A network of chromatin material is seen in the nucleus of eggs sectioned and stained with Heidenhain’s haematoxylin (see Plate V). In photographs taken with ultraviolet light, the network is absorbing and appears black, showing the presence of nucleic acid compounds, generally considered to be of the desoxyribose type (E. B. Harvey and Lavin, 1944). The nucleus is permeable to water and swells and shrinks in hypo- and hypertonic sea water (E. B. Harvey, 1943) (Table 11; Plate XIV). In 100% sea water the volume is 796 uw® (diameter 11.5 uw); in 60% sea water the volume is 2,145 w? (diameter 16.0 w); in 125% sea water the volume is 382 u3 (diameter 9.6 y.). They recover perfectly and return to normal size when replaced in sea water. The swelling and shrinking of the germinal vesicle of the immature egg has been studied by Churney (1941 b, 1942). 82 THE AMERICAN ARBACIA Jelly. - The mature egg, like the immature, is surrounded by a jelly coat which may be as thick as 32 yu. It has the same refractive index as sea water and cannot be seen with a light microscope, dark field, or phase contrast microscope. It is readily demonstrated with a slight tinge of Janus green or toluidin blue in the sea water. The old method of Boveri (1901) may also be used. He rubbed a stick of India or Chi- nese ink in a drop of sea water to make a thick solution and added the eggs. The jelly is perfectly clear and the India ink particles remain in the surrounding sea water. Also when many sperm are added to eggs in a dish of sea water, many of them are caught in the jelly and form a halo around the eggs. The even spacing of eggs as they are viewed with the microscope is caused by the invisible jelly. If the eggs are contiguous, the jelly is absent and the eggs are in poor condition. The jelly coat may be made to disappear under many experimental con- ditions, e.g., acid, ultraviolet, x-rays, etc. (see under Jelly Layer in Part TV.) Micropyle. — A funnel shaped micropyle can sometimes be demon- strated in the jelly of both mature and immature eggs by the use of India or Chinese ink or the ink from living squid. I have found the squid ink the most satisfactory, and it is best to use very fresh eggs, not washed. The squid ink makes the jelly swell, often to 60 p, twice its normal thickness, almost equalling the diameter of the egg. (See under Felly Layer, Part IV). Originally observed by Boveri (1901) in Paracen- trotus lividus, the micropyle has been shown in colored drawings in Arbacia eggs, both normal and centrifuged, by Morgan and Spooner (1909) ; they state that it is difficult to detect after the early cleavages. It is much better seen in Psammechinus microtuberculatus and Paracentrotus lividus than in Arbacia, according to Plough (1929). Boveri (19014, b) and others found that the micropyle marks the place where the polar bodies were given off, the animal pole, and thought it marked the point of attachment of the egg to the ovarian wall. But Jenkinson (1911), Lindahl (1932b), and Hérstadius (1939), for Paracentrotus, and Tennent and Ito (1941) for Mesipilia have found that the polar bodies come off from the free end, and the egg is attached to the ovarian wall at the opposite or vegetal pole. The micropyle has been used as a landmark for the localization of materials in the Arbacia and other eggs in polarity studies. Morgan (1909) found that both in the normal and centrifuged egg of Arbacia the micromeres lie approximately opposite the micropyle. (See also Morgan and Spooner, 1911). Harnley, (1926); Tennent, Taylor, and Whitaker (1929), and Plough (1927, 1929) have also used the micro- THE MATURE EGG, UNFERTILIZED AND FERTILIZED 83 pyle in their studies on the localization of materials in the Arbacia egg. Though the micropyle functions as the place of entry of the sperm in some forms, this is not true for sea urchins, for the sperm can enter at any point. This can readily be determined in centrifuged, stratified eggs. j- MEMBRANES AND LAYERS. FIGURES 9 AND 10 1. Unfertilized Egg Egg diameter, without membranes, 74 vu. Jelly layer. On outside of egg. 28 to 32 u thick (E. B. H.). Plate XVI, Photograph 3. Vitelline membrane. Outside of plasma membrane; difficult to distin- guish from plasma membrane. Lifts off on fertilization to form fertili- zation membrane. Not measurable with light microscope; 25 mu with electron microscope (E. B. Harvey and Anderson, 1943). Plasma membrane. Lies over cortical layer. 10 mu (Danielli, 1942, p. 72). Located inside cortical layer according to Parpart and Laris (1954). Cortical layer. Location of cortical granules which disappear on fertili- zation. Thickness of layer in A. punctulata is 0.8 uw; 1 to 2 wv in other eggs (Monroy and Oddo, 1946; Runnstrém, 1949b; Mitchison, 1952). 2. Fertilized Egg Jelly layer. As in unfertilized egg. Fertilization membrane. 25 my. when first formed from vitelline mem- brane; becomes thicker and tougher (E. B. Harvey and Anderson, 1943). Less than .03 w with electron microscope (Hillier, Lansing, and Rosenthal, 1952). Perwitelline space. Between fertilization membrane and hyaline layer 3°to'5 w (E.-B.-H.). Hyaline layer (or ectoplasmic layer). An investing layer which binds the blastomeres together, 2 to 3 yu thick when fully formed, 15 to 20 minutes after fertilization (E. B. H.). Plasma membrane. As in unfertilized egg. Cortical layer. As in unfertilized egg but no cortical granules. Most of pigment granules located here after fertilization (McClendon, 1tg0gb et al.). Data on the various layers and membranes will be found under the appropriate heading in Part IV. 84 THE AMERICAN ARBACIA Jelly Vitelline membrane Cortical layer Cortical granules z Ose Oe SORES CietaneeS ? S088 granules Fic. 9. Diagram of membranes and layers of unfertilized egg of Arbacia punctulata. E. B. Harvey and K. Dan. The plasma membrane is not shown; it lies just outside the cortical layer, and inside the vitelline membrane; or, according to Parpart and Laris (1954), inside the cortical layer. Jelly Fertilization membrane space Hyaline layer Cortical layer Endoplasm iopesaese cs ° o oe oe@ Oe S96 29 02090, 79 020 sono, 280% 598 weahce oO? 2°9°0 te) °@°oO- 8 LF, 2°00 209% 0D: 3 BosSosensee cee, DaUR6 eo SIeg Os eP ese Oe TO cep Pigment e ° 070 Ok (@} 9 2 Oe”: a ePeO. Bs Botta 3°55 ON OE granules b 200% 29, 630 Fic. 10. Diagram of membranes and layers of fertilized egg of Arbacia punctulata. E. B. Harvey and K. Dan. Perivitelline THE MATURE EGG, UNFERTILIZED AND FERTILIZED 85 k. DIAGRAMS OF SURFACE LAYERS OF ARBACIA AND OTHER EGGs Chambers, 1938b. Am. Nat. 72 : 146. Fertilized sea urchin egg. Dan and Dan, 1940. Biol. Bull. 78 : 488. Fertilized Strong ylocentrotus pul- cherrimus egg. Danielli, 1942. Bourne’s Cytology p. 69. Fertilized Arbacia egg. Kopac, 1940a. Cold Spring Harbor Symp. 8 : 168. Unfertilized and fer- tilized Arbacia egg. Moser, 1939a. 7. Exp. ool. 80 : 432. Unfertilized Arbacia egg. Motomura, 1941b. Sci. Rep. Tohoku Imp. Univ. 16 : 355. Unfertilized eggs of S. pulcherrimus. Ohman, 1945. Archiv f. Zool. 36A, no. 7 : 39. Unfertilized sea urchin egg. Runnstrém, 1949 a. Adv. Enzymol. 9 : 245; 1949b. Pub. Staz. Zool. Nap. 21 : 13. Unfertilized sea urchin egg. Runnstr6m and Monné, 1945a. Ark f. ool. 36A, no. 18 : 2. Sea urchin oocyte. ]. GRANULES Oil globules. 0.6 to 1.0 uw (spherical) (E. B. H.). Mitochondria. 0.6 to 1.0 » (spherical) (E. B. H.). Yolk granules. 0.7 to 1.1 uw (irregular, polyhedral) (E. B. H.). Pigment vacuoles. All sizes up to 1.7 w (spherical). Unevenly spaced all through unfertilized egg; most go to periphery on fertilization (McClendon, 1909b; E. B. Wilson, 1926, Fig. 2; Cannan, 1927; Bere. Hepeial): Cortical granules. 0.8 » (spherical). (Moser, 1939a; E. B. H., 1946a; et al. At periphery in unfertilized eggs; disappear on fertilization. Microsomes. 0.4 » or less (E. B. H.) Very small granules not visible in normal living egg, but seen in the centrifuged egg throughout the clear layer when stained with Heidenhain’s haematoxylin. (Lyon, 1907; E. B. Harvey, 1g40c; E. B. Wilson’s 1925 The Cell p. 32). (Golgi bodies, described by E. B. Wilson (1926), but by no one else; Just (1927) thinks they are oil drops). Percentage of formed bodies based on measurements in centrifuged eggs (E. N. Harvey, 19324): Nucleus 0.4% Oil g obules no%, Mitochondria 4.8% Yolk 27.2% 86 THE AMERICAN ARBACIA Pigment vacuoles 5.5% Fluid GTI, Data on the various granules will be found in Part IV. GENERAL REFERENCES Wilson, 1925. The Cell; older literature. Runnstr6ém, 1952. Modern Trends in Physiology and Biochemistry ; recent literature on cytoplasm. Mazia, 1952. Modern Trends in Physiology and Biochemistry; recent literature on the nucleus. CO ACP IER. 12 Sperm a. MORPHOLOGY The spermatozoon of Arbacia punctulata consists of a pointed conical head, a middle piece the shape of a flattened cylinder and a long thin tail (Fig. 11). Measurements of the living sperm are (approximately) : HEAD MIDDLE PIECE TAIL Height or length 3.25 0.75 u 45.0 LZ Diameter (maximum) 2.0 pL 2.0! 0.2L Volume (calculated) 3:4 ps 2.4 ps 1.4 3 Total volume 7.2 “3 (See E. B. Harvey and Anderson, 1943). Since the volume of the egg is 212,000 », the sperm is only about 1/30,000th the volume of the egg. Using another egg and other data, Needham (1931, Chem. Emb. p. 1251) arrived at the same figure. The spermatozoon of Echinus esculentus, roughly the same shape, has been studied by Rothschild. He has calculated from photomicro- graphs that the volume of the whole sperm is about 18 w3, of the head and middle piece about 10 uw? and of the middle piece not more than 5 v.°; he found difficulties in measuring the radius of the sperm tail, but says it lies between 0.3 and 0.1 yw (Rothschild, 1950b, d; 19514; 1952). According to Pictet (1891) the diameter of the tail of all sea urchins is about 0.2 yw. It has been found, in general, that the species of sea urchin which have large eggs also have large sperm (E. B. Har- vey, 1942). The egg of Echinus esculentus has a diameter of 180 pu. and that of Arbacia punctulata 74 +, so that one would expect the spermatozoon of Echinus to be larger. The conical head of the spermatozoon of Arbacia punctulata appears to be a mass of chromatin, staining black with Heidenhain’s haem- atoxylin, without any special structure. The tip, the acrosome or per- feratorium, however, stains with methyl green and Janus green, mito- chondrial stains, and with methylene blue. Popa (1927, p. 251) says 88 THE AMERICAN ARBACIA Fic. 11. Drawing of Arbacia punctulata spermatozoon. “there is an exceedingly minute opening in the point of the head’, through which “one gets the impression that the spermatozoon elimi- nates very small amounts of an extremely sticky substance’’. The head of the sperm contains the haploid group of chromosomes which have a volume, if they are the sole components of the sperm head, of about 3.4%. A diploid group of 38 chromosomes from a first cleavage cell in a smear preparation stained with aceto-carmine has a volume of about 10.2 2° (average diameter of a spherical chromosome is about 0.8 pz), a little more than double that of the sperm head. The expected double relation is fairly close when one considers the uncertainty as to how much of the material is pure chromatin both in the diploid plate of chromosomes and in the haploid sperm head. The amount of DNA in the Arbacia sperm is 0.9 X 10~* micrograms, calculated as 1.3 to 1.4 u3 according to Mazia (personal communication, 1955), or about one third of the volume (or weight) of the head. Marshak and Marshak (1953) give 7.9 X 10-7? micrograms in one sperm. The middle piece of the spermatozoon of Arbacia punctulata contains two large spheres, probably centrosomes, especially well seen with the phase microscope. These bodies in the middle piece stain purple with methyl green and blue with Janus green, indicating the presence of SPERM 89 mitochondrial material. They also stain with methylene blue, not a mitochondrial stain. Rothschild (1952) thinks that the middle piece probably contains enzyme complexes, such as the cytochrome system and that this is suggested by the presence in it of mitochondria-like structures. The middle piece of Arbacia can be isolated by a special technique involving the removal of heads and tails (Di Stefano and Mazia, 1952); these authors think that the middle pieces are the center of ribonucleic acid activity. A preliminary paper on the isolated middle pieces has been published recently by Neff (1953). Popa (1927) also isolated certain components of the sperm by destroying others. The tail of the Arbacia spermatozoon consists of about ten separate fibrils of uniform thickness, each fibril having a diameter of about 50 mu (0.05 w), as shown in electron microscope pictures (E. B. Harvey and Anderson, 1943). Electron microscope pictures of sperm of other animals show that the fibrillar structure is characteristic of sperm tails, e.g., the squid (Schmitt, Hall, and Jakus, 1943) and the bull (Baylor, Nalbandov, and Clark, 1943). Many years ago, separate fibrils were observed (and figured) in the tails of many different kinds of sperm, of birds, insects, fish, amphibia, and reptiles, by Ballowitz (1888, 1890) in teased material, with an ordinary light microscope. In the chaffinch, he found 7-11 fibrils, somewhat as are found in Arbacza. The structure and reaction to stains of the sperm of Arbacia punctulata have been especially studied by Popa (1927). He was particularly interested in the distribution of fats, which he found present in the head, middle piece, and tail. The peculiar forms of the spermatozoa which he observed with different stains can be duplicated by treatment of living sperm with distilled water, drying, etc. (E. B. H.). In distilled water, the heads swell to double their volume (E. B. Harvey and An- derson, 1943). Figures of the spermatozoon of Arbacia lixula given by Pictet (1891) and by Retzius (1910) show it to be similar to that of A. punctulata. The acrosome or perferatorium and the two spherical bodies in the middle piece are figured by Retzius on his Plate XV, no. 2. The measurements of A. lixula sperm given by Field (1895) are similar to those of A. punctulata. Recent photographs of sperm of other species are those of Vasseur (1947) of Echinocardium cordatum; of Tyler (1949c) of Lytechinus pictus taken with the electron microscope; of Rothschild (1951 a) of Echinus esculentus ; of J. C. Dan (1952) of two Japanese species (also Dan, 1954, Biol. Bull. 107 : 335-349 and Afzelius, 1954, Zeit. f. Zellforsch. 42 : 134-148). go THE AMERICAN ARBACIA b. SwIMMING RATE The swimming rate of the Arbacia punctulata spermatozoon is, according to Grave and Downing (1928) and Grave (1934), about 1 mm. per minute at 22.5°C., or 20 times its length (50) per minute, and slightly faster at a higher temperature. Gemmill (1900) gives a little higher rate for Echinus esculentus. For Psammechinus miliaris, with a slightly different technique, Rothschild (1951a) estimates 200 » per second (translatory speed), or about 12 mm. per minute at 18° C. The human sperm swims, in the uterus and Fallopian tube (a distance of about 190 mm.), at the average rate of about 2.7 mm. per minute (R. L. Brown, 1944) or about 45 times its own length (60 y) in one minute. A good swimmer swims a mile in 20 minutes, so that a six-foot man swims 44 times his own length in one minute. A human sperm and a man thus swim at about the same rate, length for length (E. B. Harvey, 1946b). For a further comparison, a gastrula of Arbacia lixula, whose dia- meter is approximately 0.2 mm., swims in a tube of its own diameter according to Needham (1931, Vol. 2, p. 1247) at the rate of 1.17 meters per hour, or 20 mm. per minute, 100 times its diameter. The Arbacia lixula gastrula swims about 20 times as fast as the Arbacia punctulata sperm, or 5 times as fast, length for diameter. A-greater rate would be expected on account of the numerous organs of propulsion, the cilia, though it is not as streamlined. Paramoecium caudatum swims about 3 mm. per second or 15 times its length (ca. 200 ) according to Lud- wig (1928); this makes the rate about 180 mm. per minute or goo times its length. Paramoecium therefore swims nine times as fast as the gastrula of A. lixula. This greater rate might be expected since Para- moecium caudatum is streamlined. Other rates have been given for Para- moecium by other investigators. Tabulae Biologicae (IV p. 480, Metzner, 1927) give 1.3 mm. per second for P. caudatum, length 216 uw. Chase and Glaser (1930) give 833 u per second, species not mentioned but proba- bly P. caudatum. Wichterman (personal communication 1953) has found that for a given species, the rate varies with the phase in its growth and life cycle. c. MorTILitTy The effect of dilution in activating the sperm and the loss of fertilizing power with time have been studied in Arbacia punctulata by F. R. Lillie (1915b), though they were observed earlier in Echinus esculentus by SPERM gi Gemmill (1900). Cohn (1918) found that increased alkalinity in- creased the activity with a corresponding decrease in length of life; and that increased CO, decreased activity and increased length of life. In concentrated sperm, more CO, is produced which inactivates the sperm and they live longer. Increasing the temperature increases the activity as one would expect. KCN inactivates them and prolongs their life (Cohn, 1918). The motility of sperm is increased by egg water or egg extracts (F. R. Lillie, 1913b, 1915 a, 1919, p. 111, etc.; Woodward, 1918; Cohn, 1918; Sampson, 1922; et al). According to Hartmann et al. (1939), it is the echinochrome in the egg water of A. /ixula that causes the activation. Tyler (1939), however, found this not to be true for Strongylocentrotus purpuratus, and Cornman (1941) for A. punctulata. Other sperm-stimulating substances have been found by Clowes and Bachman (1921), including propyl, allyl, and cinnamyl alcohol, propylene, and a “‘volatile substance derived from marine eggs.”’ The sperm of Arbacia are immobile when in the testes, and become motile only when diluted. The sperm are immotile in absence of oxygen and therefore cannot fertilize the eggs (E. B. Harvey, 1930); they are also immotile in KCN (Cohn, 1918); Loeb (1915a) had found this for Strongylocentrotus purpuratus. Motility is also suppressed by halo- and nitrophenols (Clowes, 1951) and by dilute HgCl, (F. R. Lillie, 1921b), and by the SH reagent PCMB (p-chloro-mercuri-benzoate) (Runnstr6m, 1952). Sperm are more resistant to anaesthetics than are the eggs, but they can be anaesthetized. In 0.2 M ethyl urethane they lose their fertilizing power in } hour and their motility in 2 hours (E. B. H.). Chloretone is efficacious in quieting them. In Ca-free sea water, the sperm are motile and surround the eggs but will not fertilize them (E. B. H.; et al.); this was observed many years ago by Loeb (1915 a). d. LONGEVITY Although there are many references in the literature to increasing the life span of sperm, it would seem that the length of life depends on their activity, and this is dependent on many factors as described above, and especially on temperature. The less active they are, the longer they live. However, Budington (1935) says that acetylsalicylic acid (aspirin) causes sperm to retain their fertilizing power longer. Tyler (1950, 1955) found that various amino acids and peptides, ver- Q2 THE AMERICAN ARBACIA sene, and other metal-chelating agents extend the functional life span very greatly. For Lytechinus pictus and S. purpuratus see Tyler and Atkinson (1950). e. INJURY Arbacia sperm are injured by hypo- and hypertonic sea water; alcohol 1-12% added to sea water; sea water slightly acidified with HCl; ageing for 6-24 hours (Dungay, 1913). The injured sperm caused irregular cleavage, abnormal blastulae, gastrulae and plutei, the results being non-specific. The sperm are also harmed by CuCl, and HgCl, (ER Lilhey 1621 'b): f. AGGLUTINATION A very noticeable phenomenon in motile Arbacia sperm is agglutina- tion, the collection of numbers of sperm into clumps. This was ob- served by Buller (1g00b) in A. lixula sperm, and has been studied in- tensively in A. punctulata by F. R. Lillie (1912, 1913, b, 1914, 19154, 191Q, p. 112, etc.); and by Glaser (1914b); Cohn (1918); Woodward (1918); Sampson (1922); Popa (1927); et al. The substance in sus- pensions of eggs causing this reaction has been called by F. R. Lillie (1913 a) fertilizin and earlier was called iso-agglutinin (F.R. Lillie, 1912). Sperm extracts agglutinate the eggs and inactivate fertilizin (Frank, 1939). The agglutination is usually between the heads of the sperma- tozoa (F. R. Lillie, 1919, p. 119; Sampson, 1922; Tyler, 1948). Sec Part IV, Fertilizin and Agglutinin for recent reviews, especially Tyler (1948, 1949C¢). : g. CHEMOTAXIS The classical studies of Pfeffer (1884) showed without much question that the sperm (antherozoids) of ferns were attracted to the archego- nium (and egg), and that this was due to the malic acid and its salts occurring there, a definite chemotaxis. But whether the sperm of animals are attracted to the eggs by chemotaxis has never been proven. Buller (1900a, b, 1902), working in Pfeffer’s laboratory, confirmed the chemotaxis in the case of ferns, but when working on sea urchins, including Arbacia lixula, at Naples, could find no evidence of chemotaxis, and concluded that in these forms the eggs and sperm meet by chance. Other investigators (e.g. von Dungern, 1902) confirmed Buller’s re- SPERM 93 sults. However, F. R. Lillie (1913b, 1919 p. 102) stated that in Arbacia punctulata, the sperm are attracted to CO, and to egg water which contains “‘fertilizin’. But Loeb (1914c) thought that Lillie’s explan- ation of his experiments was incorrect, and that they did not neces- sarily show chemotaxis. Later, Loeb took a decided stand against chemotaxis of eggs and sperm and expressed his views in two of his books, The Organism as a Whole (1916, p. 92) and Forced Movements, Tropisms and Animal Conduct (1918, p. 139). The subject has been taken up again recently by Rothschild (1951a, b, 1952), who has come to the conclusion that “‘in the animal kingdom spermatozoa probably meet or collide with eggs by chance” (1952, p. 1), and that “‘chemo- taxis of spermatozoa toward eggs has never been observed with cer- tainty” (1951b, p. 40). For the older work see Morgan’s Experimental Embryology, Chapter II, 1927. h. PoLysPERMY See Part IV, Polyspermy. i. PHysIOLOGY For references to the physiology of the sperm of Arbacia punctulata, such as respiration, enzymes, etc., see under the appropriate topic in Part IV which treats of both eggs and sperm. j. SEMINAL FLUID The seminal fluid of Arbacia punctulata has been especially studied by Hayashi (1945, 1946) with the following results. Sperm are as motile in seminal fluid as in sea water; they retain their fertilizing power longer. The pH of the seminal fluid is 7.6—7.9; its osmotic pressure is 10% lower than sea water; it contains less than 10 yg reducing sugar in 5 Cc.; it contains 2.5 mg. protein per cc.; it does not act as a nutrient for the sperm. It does not contain anti-fertilizin, but increases agglu- tination. It delays the fall in respiration following the increase after dilution. The seminal fluid or “seminal plasma”’ of Echinus esculentus has been studied by Rothschild (1948b) with special regard to O, tension; he also noted the large amount of potassium in the seminal fluid. k. TECHNIQUES Method of keeping sperm Remove the testes from a freshly opened animal as intact as possible, and place them, undiluted, in a small covered stender dish (about 3 cm. diameter). Place 94 THE AMERICAN ARBACIA immediately in the refrigerator at about 8 °C. They keep perfectly for several (4-5) days and give nearly 100 % fertilizations. To Kill Sperm Formalin, 0.1 to 1 % (Tyler). Distilled water kills Arbacia sperm almost immedia- tely (E. B. H.). Keep at 40 °C. for 20 minutes (E. B. H.). Rothschild and Swann (1951) use for Ps. miliaris, hypotonic sea water, 45 % sea water with distilled water, to kill spermatozoa in presence of eggs and prevent fertilization without harmful effect on the eggs. To Prepare Sperm Suspensions for Fertilization One drop (0.1 cc.) of concentrated (‘‘dry’”’) sperm to 100 cc. sea water, then one drop of this to 10 cc. sea water which contains two drops of concentrated eggs; this is the amount for a Syracuse watch glass (E. B. H.). Just (1939a) recommends one drop of ‘“‘dry’”’ sperm to 10 cc. sea water, then two drops of this to inseminate eggs in 250 cc. sea water. The dilute suspensions of sperm last only a short time after preparation, owing to their activity, so that fresh dilutions must be made frequently. A simple and convenient method of fertilizing a small dish of eggs is to take a small amount of sperm on a toothpick and agitate the eggs with it. With experience, this becomes fairly accurate (E. B. H.). Determination of Number of Sperm in a Suspension Barron (personal communication) obtains a constant suspension by measuring the turbidity with a Coleman Junior spectrophotometer, and checks this against the dry weight. Mazia (personal communication) counts the number of sperm in a dilute suspension, fixed in 0.1 % formalin, with a haemocytometer. There are 5 cc. of concentrated sperm in one Arbacia, obtained by allowing the sperm from the five ruptured testes to settle. Tyler found 2 X 10!°spermatozoa in 1 cc, by haemocytometer measurements (per- sonal communication, July 1954). This is the same number as he had obtained from S. purpuratus and Lytechinus (Tyler and Rothschild, 1951; Tyler, 1953). The complete number of sperm in one Arbacia then, 1s 1011, approximately a million million; the number of eggs in one Ar- bacia is about 8,000,000. One sperm has been calculated to be about 1/30,000 the volume of the egg. The various methods of counting spermatozoa have been discussed and evaluated by Rothschild (1950a), who advocates a photoelectric absorptionometer. OTHER SPECIES (ADDITIONAL) AND GENERAL REFERENCES Bernstein and Mazia, 1953a. S. purpuratus, DNA. Fuchs, 1914c. Paracentrotus lividus, A. lixula, etc., fertilizing power increased by egg secretions. Gray, 1931. Experimental Cytology p. 408-421. References to older literature. Morgan, 1927. Experimental Embryology p. 15-93. Chemotaxis, activity etc. Rothschild, 1951a. Review of recent literature, with good bibliography, with titles. Runnstrém, 1949a.Many references to sperm in comprehensive review The Mechanism of Fertilization. Southwick, 1939. Echinometra subangularis, activity of sperm. Tyler, 1948. Review. CHAPTER 13 Fertilization and Cleavage a. FERTILIZATION. PLATEs III AND V The normal development of the Arbacia punctulata egg, from fertilization through the cleavages to the early pluteus of four days, is shown in a series of photographs of the living eggs and of stained sections (Plates III, IV, and V). This was done many years ago for Lytechinus ( Toxop- neustes) variegatus in the classic “‘Atlas of Fertilization” by E. B. Wilson (1895), and the two species are quite similar. For P. lividus see Boveri (1895) and Horstadius (1935). When a spermatozoon touches the surface of a mature Arbacia egg, after penetrating the jelly coat, it is engulfed, and a fertilization cone or entrance cone is formed within 20 seconds. The fertilization mem- brane rises over this and quickly spreads around the egg; this takes only about five seconds. The fertilization membrane lifts off, leaving a perivitelline space, at first very narrow, but gradually becoming wider so as to be 3-5 uw when fully formed. At first it is usually not equidistant from the surface of the egg, but later becomes so. The whole process from the time that the sperm touches the egg until the fertili- zation membrane is fully raised takes about two minutes at 23 °C. The fertilization membrane is now generally believed to form from the vitelline membrane with the addition of the cortical granule material. See Part IV under Fertilization Membrane, Vitelline Membrane, Cortical Layer. The fertilization cone which is raised at the point of entry of the sperm is at first conical or flattened, but later becomes flame-like (Plate III, Photograph 3). It may disappear quickly but often lasts 4 to 5 minutes, especially in eggs early and late in the season, and in eggs kept cold. In sectioned and stained material, Mathews (Wilson and Mathews, 1895) has described the rotation of the sperm head soon after its en- trance into the egg. It rotates through an angle of 180° so that the base containing the sperm aster becomes directed inwards, toward the egg nucleus. (See Wilson’s The Cell, 3 ed., p. 397). 96 THE AMERICAN ARBACIA The sperm aster becomes quite large, easily visible in the living egg, indicating the position of the male pronucleus as it approaches the female pronucleus. The small sperm nucleus flattens over and unites with the large egg nucleus and the astral rays spread through the egg; this is the monaster stage. The rays disappear and the centrosome (probably) divides, forming a curved disk over the nucleus; this is the “streak”’ stage (Plate III, Photograph 6; Plate V, Photograph 4). The cell wall at this stage is not smooth but somewhat crenate. The hyaline layer which starts to form soon after fertilization has now become 2 to 3 p thick. The nucleus enlarges from 11.5 u to 16 wu in diameter, nearly three times in volume; it becomes elliptical, and then the nuclear membrane breaks (Plate III, Photograph 7). The chromatin material, meanwhile, forms into chromosomes, and a spindle with an aster at each pole is formed on which the chromosomes lie, at first irregularly scattered (prophase), then lined up at the equator (metaphase) (Plate III, Photo- graph 8; Plate V, Photograph 6). Then the chromosomes divide, half of each chromosome going to each pole (anaphase). After reaching the pole, each chromosome becomes vesicular and the vesicles fuse (telophase). The centrosphere is largest and the astral rays most extended during the late anaphase and telophase (in Arbacia). b. CLEAVAGE. PuLaTtes III AnD V Now the egg elongates and the cleavage furrow comes in, usually asymmetrically, on one side first, the side nearest the excentrically placed spindle. There is a heaping up of the hyaline layer in the furrow and a corresponding thinning at the poles. The red pigment granules also tend to accumulate in the furrow, as noted by many observers, e.g., McClendon, 1g10b. See under Chromatophores, Part IV. The two cells are at first well separated but later become closely pressed together owing probably to the formation of the next mitotic figure (Plate III, Photographs 10, 11). The first cleavage plane passes through the polar axis, i.e., in the region where the polar bodies formed, the animal pole. The second cleavage plane is also meri- dional and at right angles to the first. The third cleavage plane is equatorial, and we have eight equal blastomeres. The fourth cleavage plane is differential, cutting off four small unpigmented cells, the mi- cromeres, at the vegetal pole (Plate III, Photographs 14, 15). More cleavages follow, and a blastula of many cells is formed. It will be noticed in the photographs that the spindles in the later cleavages have no asters, a characteristic of plant cells (Plate V, Photograph 12). FERTILIZATION AND CLEAVAGE Q7 c. SCHEDULE OF DEVELOPMENT A time table of cleavage in a normal batch of Arbacia eggs at 23 °C, is given in Table 2, and photographs on Plates III, [V and V. Other schedules of cleavage times in Arbacia punctulata have been given by Fry and Parkes (1934) ; Fry (1936); Hoadley and Brill (1937), and Blum and Price (1950b). The rates of first cleavage at different temperatures and the Q,, for cleavage as determined by Loeb and Wasteneys (1911a) and Loeb and Chamberlain (1915) are given in Table 3, together with a few figures by Fry (1936). The rate of the first three cleavages at different temperatures as obtained by Hoadley and Brill (1937) is given in Table 4. For a comparison of Arbacia punctulata with some other species, a few references for cleavage rates in other species are given in Table 5. TABLE 2 SCHEDULE OF DEVELOPMENT (E. B. HARVEY) Time Table of Cleavage and Development of Arbacia punctulata Eggs at 23 °C. Times after fertilization for 50 % of the eggs to reach the stage designated. Completionvof fertilization membrane: 62 3) a «= & 3 9 & 2 min. Sperm aster .. oN GE cates JE a si ame Se 8 min. Union of pronuclei Gngnetins eo) ol vag.) SO c= FE, Sa 10 min. Completion of hyaline layer (begins 2 min.) . . . . . . . . 20 min. Streak stage .. tes. 3 ORR OS Wa BD Bs ae sear Nuclear membrane tosis oe, Sart pas SP ee A os Coane eee 35 min. LOMAS ALS bata Sey Bee te) ues, ys buss os | Gl RAN ei ee Ce eee 35 min. Ara ASO Oe Oe Sey Me tenis, ok eet SL ae Sa) ne aa 40 min. PAUARASeEmA Gh 2 aiclane «Yoneda ates!) A SMS eae ee 42 min. PCIGMUASC re Ml bs hc ol ad! ith pba, “ah er ke Ler eee 45 min. FSEMECICAMARC uO -COU 2 eeda Vell. cos" Sb o> rey Foy ee SeR ae 50 min.. Qna.cleavage;. 4-cell. 2 | (... .f26soum-alter rst .. |. hl 78 min. Growcleavage, G-cell-... 1 .. 25 min. after’end . 4. .- . -... Soguasea 4th cleavage; (12-cell) micromeres; 27 min. after 3rd . . . . . 130 min. no-cell Lhe) eh") sotminsafter'erd”)) leat ia eR aga ean. Bthecleavage-a(20-cell) i... 22 min. after 4th.) nes ee (28-cell) joss poe yp qegO Mingaften4th ~, 2 :) 24), oageebRem: g2-cell ay ers or 92min. after.4th. ~- «+ paps eet “AGF Piatcmtasiplastuiae. ca. T.0G0%CElIS: 5. fc soy. US bas, we) ag Santucci ers ee Pe eR ee nse Seclorauebeminiswwe an, Way aise! ace cae: tee hh pe toe ky ees luted asec’. Pe ee ae me 1 day Plutei, maximum oe special ieee ee te a ee a) There is a slight variation in cleavage times in different batches of eggs and considerable variation at different times of the year, irrespective of temperature. The above table is for a standard batch in mid-season at 23 °C. It is based on both living eggs and stained sections. There is a greater variation in the later stages of development, e.g., time of hatching. 98 THE AMERICAN ARBACIA TABLE 3 TIME IN MINUTES FROM FERTILIZATION TO FIRST CLEAVAGE LOEB AND LOEB AND TEMPERATURE WASTENEYS (1911 a) CHAMBERLAIN FRY (1936) LOEB (19134) (1915) 7.0" 498.0 —- — 8.0° 410.0 411.0 — g.0° 308.0 297.5 —_ 10.0° 217.0 208.5 a WI. — 175.0 — 12.0° 147.0 148.0 — 13.0° a 129.0 es 14.0° — 116.0 as 15.0° 100.0 100.0 113.0 16.0° 85.5 — a= 17.5, 70.5 = = 18.0° 68.0 68.0 a 19.0° a 65.0 —_ 20.0° 56.0 56.0 67.0 21.0° — 53-3 — 22.0° 47.0 46.0 _ 23.0° = 45-5 = 24.0° — 42.0 — 25.0° 40.0 39.5 42.0 26.0° 33.5 — —- 27.5° 34.0 aa Te 30.0° 33.0 — — 31.0° 37.0 a — 32.0° no cl. — — TABLE 4 TIME IN MINUTES FROM FERTILIZATION TO FIRST, SECOND AND THIRD CLEAVAGE (FROM HOADLEY AND BRILL, 1937) TEMPERATURE IST CLEAVAGE 2ND. CLEAVAGE 3RD. CLEAVAGE g.6° 295 486 528? 12.0° 180 288 399 15.2° 110.5 174.5 239 Toate 78.5 126 172 21.3° 57-5 94 130 24.1° 46.5 72.5 99 26.9° 41.3 65.5 g2 30.0° 40 64 89 FERTILIZATION AND CLEAVAGE TABLE 5 99 TIME OF FIRST CLEAVAGE OF SOME SPECIES, IN MINUTES AFTER FERTILIZATION SPECIES LOCATION TEMP. TIME REFERENCE Arbacia punctulata Woods Hole 23° 50 Many Arbacia lixula Naples 16° 110 E. B. H., 1933a Naples 18° 99 Callan, 1949 Dendraster excentricus Pacif. Grove 20° 55 Moore, 1933 Pasadena 22° 47 Tyler, 1936a Echinometra lucunter Bermuda 25° go E. B. H., 1947 (unp.) Lytechinus variegatus Beaufort 22° 55 E. B. H., 1942 (unp.) Bermuda 24° 55 E. B. H., 1947 (unp.) Tortugas 28° 40 Tennent, 1g11a Paracentrotus lividus Roscoff 18° 71 Ephrussi, 1933 Naples 16° go E. B. H., 1933a Naples 18° 76 Callan, 1949 Ps. microtuberculatus Naples 16° 70 E. B. H., 1933a Naples 18° 61 Callan, 1949 Psammechinus miliaris Millport 17 67 Gray, 1927a Sweden 18° 56 Borei, 1948 Sphaerechinus granularis | Naples 16° 105 E. B. H., 1933a Naples 18° 100 Callan, 1949 S. franciscanus Pacif. Grove 20° 95 Moore, 1933 Pacif. Grove 19° 80 E. B. H., 1941 (unp.) S. purpuratus Pasadena 20° 77 Tyler, 1936a Pacif. Grove 19° 70 E. B. H., 1941 (unp.) Tripneustes esculentus Bermuda 22° go E. B. H., 1932 (unp.) Tortugas 26° 75 Tennent, 1gl1a It will be seen that the eggs of some species cleave more rapidly than those of Arbacia punctulata, some less rapidly. There seems to be no relation between the size of the egg and the rate of cleavage. One might think that the cleavage rate of a small egg would be faster than that of a large egg. But the small egg of Tripneustes esculentus, 84 u dia- meter, cleaves more slowly (go min. at 22 °C.) than the large egg of Dendraster excentricus, 114 ~ diameter which cleaves in 47 min. at 22 °C. It may be noted that large eggs may come from small species, (e.g., 100 THE AMERICAN ARBACIA Psammechinus microtuberculatus, and small eggs from large species, e.g., Tripneustes esculentus. Arbacia has a fairly small egg (for sea urchins) which cleaves at an intermediate rate. For sizes of eggs, see Classifica- tion, Part I, Chapter 9. Fox (1938) has found that the cleavage rate of some species (Para- centrotus lividus) is different in different localities. H6rstadius (1925) found that in the same locality, Naples, the cleavage rate of this species differs in different seasons of the year; winter eggs and summer eggs have a different rate when kept at the same temperature. This is true also, to a limited extent, of Arbacia punctulata ; the eggs obtained late in the season (September) are slower to cleave, irrespective of temper- ature, than those obtained at mid-season (July, August). d. CLEAVAGE Wi1THOUT MEMBRANES If the fertilization membranes are removed soon after fertilization, by shaking, the eggs develop quite normally, though somewhat spread out (Plate XVI, Photograph 7). They are held together by the hyaline layer. They cleave at about the same rate but become free-swimming earlier, since the fertilization membrane does not have to be dissolved by the “hatching enzyme’’. Normal plutei are formed. If the fertilization membranes are removed and the eggs placed in sea water without calcium, the hyaline layer does not form and the cells are no longer held together. The isolated cells cleave several times and among them, micromeres may be distinguished by their size (Plate XVI, Photograph g). Soon the isolated masses of cells go to pieces. e. SPERM ENTRANCE AND CLEAVAGE PLANES A sperm may enter at any point on the surface of the egg, as noted by many observers. This can be best demonstrated in centrifuged Arbacia eggs, where it may be observed to enter in any zone of the stratified egg, pigment, yolk, clear layer, or even the oil cap. The tail of the sperm has not been observed to enter the Arbacia egg, but is left outside, though the portion adjacent to the head is often seen within the fertilization membrane, in the perivitelline space. This portion as well as the distal end soon disappears as though dissolved. The tail is left outside in the Lytechinus ( Toxopneustes) egg (Wilson’s Atlas 1895, p. 14), and probably in other sea urchins, as stated by Wilson, more positively in the earlier editions of The Cell (Compare the first edition 1896, p. 136, 149 and second edition 1911, p. 188, 200 FERTILIZATION AND CLEAVAGE IOI with the third edition 1925, p. 395). Recently J. C. Dan (1950) has stated that in six species of Japanese sea urchins and starfish, the tail enters the egg, though none of her figures show it actually inside the egg, only in the perivitelline space, as frequently seen in Arbacia. The question does not seem to be of much importance since the tail has completed its function and is probably of no further use. The first cleavage plane has been observed to cut through without any relation to the entrance point of the sperm, not necessarily through it. This has been found to be the case also in Paracentrotus lividus by Horstadius (1928, 1939) who used the Vogt (1925) vital staining technique to locate the sperm entry. However, Wilson (Wilson and Mathews, 1895, p. 324) says that in Toxopneustes (Lytechinus) “the plane of first cleavage is in the great majority of cases at least approxi- mately through the entrance point of the sperm.” See also Wilson’s Cell, 1925, p. 1104. Though one sperm. usually enters the egg, the question has been raised whether fertilization is possible with only one sperm. Kite (1912) injected with a micropipette several sperm into the jelly of an Arbacia egg and obtained fertilization from a single sperm. Glaser (1915), how- ever, held that more than one sperm is required to bring about changes in the membrane necessary for one sperm to enter the egg. Tyler (1949c) likewise considers more than one sperm necessary. f. THE CENTRIOLE There is no definite single granule or centriole observable in the center of the aster in Arbacia punctulata, either in the sperm aster or the cleavage aster, but a group of small granules. This was stated many years ago by Mathews (Wilson and Mathews, 1895), and was believed to be true also for artificial astrospheres (cytasters) by Morgan (1899). See the discussion by Morgan (1899) and Wilson (1g01a). A very skep- tical view concerning the centriole in Echinoderm eggs, especially in Echinarachnius parma, has been held by Fry (1929 and many other papers). See also Wilson’s Cell, 1925, p. 677. g. Mip-Bopigs Mid-bodies (Zwischenk6rper), thickenings of the disappearing spindle fibers at the equator, are characteristic of plant cells and are very pro- minent in many animal cells, e.g., the spermatogonial cells of the Orthopteran, Rhomalium (see Fig. 60, p. 138 in Wilson’s Cell 1925). These mid-bodies are not conspicuous in Arbacia, though Fry (1937) 102 THE AMERICAN ARBACIA describes them as occurring in this form. They are mentioned as occurring in Lytechinus by Wilson in his Atlas (1895, p. 27 and Fig. XVII), but do not appear conspicuous or typical. h. CHROMOSOMES The chromosomes of Arbacia punctulata are small and crowded and difficult to count. The diploid number, in cleavage cells, is probably 38. This number, 38, is also given, with drawings, by Matsui (1924); and Morgan (1927, p. 627 footnote) says that 36-38 is recorded by E. B. Wilson and students. E. B. Harvey (1940c) gives 32~38 as the diploid number, and half that number for the parthenogenetic egg. Tennent (1912b, p. 397) says “‘about 40”’ (this article was incorrectly attributed to Jordan, 1912, in my tabulation, 1920, p. 12). In Arbacia lixula, the Naples species, Baltzer (1910) gives 40 as the number of chromosomes in cleavage cells. In Lytechinus ( Toxopneustes) variegatus which closely resembles Arbacia in cytological details, the number is 36 or 38 (E. B. Wilson, 1895, Aélas, p. 23; 1g01a, b; Ten- nent, 1912b). The chromosome numbers of the other Echinoidea are given in the tabulation of E. B. Harvey, 1920, p. 12-14. This list (including a mistake) has been copied in Tabulae Biologicae, vol. IV, 108 (1927) by Breslau-Harnish, and in vol. 18, p. 34 (1939) by McClung. Makino, 1951, has added four more references to this list, including Arbacia punctulata (38 chromosomes) and Echinarachnius parma (52 chro- mosomes) by Matsui, 1924; Clypeaster rosaceus (44 chromosomes) by Gardiner, 1927; and Strongylocentrotus intermedius (50 chromosomes) by Niiyama and Makino, 1947. To make the list complete there should be added Cidaris tribuloides with 37, 38 diploid, 18, 19 haploid, 19 parthenogenetic eggs (Tennent, 1922); and Mespilia globulus with 38 diploid, 19 haploid (Tennent and Ito, 1941). It will be seen that most sea urchins have 36-38 chromosomes, diploid, including Arbacia punctulata. There are apparently no sex chromosomes in Arbacia ; at least none have been reported. But there are sex chromosomes in Lytechinus varie- gatus and Tripneustes esculentus (Tennent, 1911 b, 19124, b, c, 1922); in Paracentrotus lividus and Psammechinus microtuberculatus (Baltzer, 1913); and in Cidaris tribuloides (Tennent, 1922). The digametic sex is the male - and not the female as was once thought. (Baltzer, 1909, 1910, 1913). The chromosomes of Arbacia are small and of different sizes and shapes; some are spherical and some rod-like (Tennent, 1912b; Matsui, 1924; E. B. Harvey, 1940c, Photograph 130 on Plate VIII). From FERTILIZATION AND CLEAVAGE 103 measurements of metaphase plates of first cleavage stained with aceto- carmine, a medium-sized, spherical chromosome has a diameter of ca. 0.8 u, giving a volume of ca. 0.268 »3. All the 38 chromosomes would have a volume of ca. 10.2 1°, or 1/20,000 the volume of the egg. The sperm head, containing the haploid number of chromosomes, has a volume of ca. 3.4 3, as calculated from the data (length 3.25 p, thickness 2 uw) of E. B. H. (see under Sperm, Chapter 12a). The volume of desoxyribose nucleic acid in the Arbacia sperm head is 1.3 to 1.4 3 calculated by Mazia (personal communication 1955). For Drosophila, Sturtevant (personal communication 1950) calculated that the diploid chromosomes have a volume of ca. 1.0 1°, and the sperm head (hap- loid) about one quarter of this. Other figures for Drosophila from which similar volumes can be calculated have been given by Muller (1929) and Gowen and Gay (1933). 1. VISIBILITY OF THE CLEAVAGE FIGURE In the living egg of Arbacia, asters are plainly visible, but spindles and chromosomes cannot be observed with the usual light microscope even with the best apochromatic objectives and compensating oculars. When no granules are present, as in the clear quarters of the centrifuged egg, the asters cannot be seen. In photographs taken with ultraviolet light, the chromatin material and chromosomes of the living egg appear as they do in fixed and stained sections (Harvey and Lavin, 1944). With infrared light, photographs of the living egg show the configuration of the mitotic figure in a striking manner as a brilliant white area against a dark granular cytoplasmic background (Harvey and Lavin, 1951b). The spindle of Arbacia has not been studied with a polarizing microscope, but the mitotic figures of other sea urchin eggs show a beautiful birefringence (Swann, 1951a,b; Innoué and Dan, 1951; see the fine photographs in these papers and Hughes’, 1952, book, Plate XIII). The structure of the mitotic figure is best studied in sectioned material. The best fixative is probably Bouin, although the egg shrinks from 74 to 50 yu; the most satisfactory stain is Heidenhain’s haema- toxylin. Very good total mounts can be made with aceto-earmine and acetic orcein. A photograph of a very thin section of a spindle taken with the electron microscope, has been made by Geren and McCulloch (1951). Fading out of cleavage figure or furrow (reversible) is caused by Colchicine, Nebel 1937; Nebel and Ruttle, 1938; Beams and Evans, 1940. 104 THE AMERICAN ARBACIA Cold, Heilbrunn, 1g20b. Ether, Heilbrunn, 1920b. See E. B. Wilson, 1g01b for Lytechinus. Hydrostatic pressure, Marsland, 1938, 1950, 1951 for A. punctulata ; 1939 for A. lixula. Micromanipulation, Chambers, 1919, 1938c, 1951. Oxygen-lack, Mathews, 1907; E. B. Harvey, 1927 for other species; 1930 for A. punctulata. Podophyllin, etc., Cornman and Cornman, 1951. Quinine, Mathews, 1907. Urethane, Painter, 1918; E. B. H., unpub. j. IsoLATION oF Mitotic APPARATUS An important method has been recently devised by Mazia for isolating the mitotic apparatus from the rest of cell and has been applied to Arbacia punctulata (Mazia and Dan, 1952; Dan, Ito, and Mazia, 1952). This technique, the details of which are given in their papers, allows a much better understanding of the structure, function, and chemistry of different parts of the mitotic figure. k. ELONGATION AT CLEAVAGE Elongation of the Arbacia egg at the time of cleavage is easily observed in the living egg, as seen in the photographs. Studies have been made on the elongation especially by Churney (1936, 1940). He found that with the fertilization membrane present, the egg elongates from 74 to 82.5 u, an elongation of 11.5% (at 22.2-26.0 °C.) ; without the fertili- zation membrane it elongates from 74 to 103.2 », an elongation of 39.4%; see also A. Scott (1946). For the effect of mechanical pressure, see Chambers (1946, 1951). A mathematical treatment of the elon- gation of the Arbacia egg has been made by Buchsbaum and William- son (1943). It will be observed from the photographs (Plate III) that the elongation takes place at telophase (see also Just, 1928b). A special study of elongation at cleavage in Echinus esculentus has been made by Gray (1931, p. 194). ]. ASYNCHRONY IN CLEAVAGE It will be noticed from the schedule of development (Table 2) and from the photographs that there is an asynchrony in cleavage begin- ning after the 8-cell stage, when one quartet (vegetal) divides horizon- FERTILIZATION AND CLEAVAGE 105 tally into 4 large cells, macromeres, and 4 small cells, micromeres, some 5 minutes before the other quartet (animal) divides meridionally into 8 equal cells, mesomeres (Plate III, Photographs 14, 15). There is thus a definite 12-cell stage preceding the 16-cell stage by about 5 minutes at 23 °C. The interval is somewhat variable in different bat- ches of eggs and very rarely (in one batch only out of many hundreds) does not occur at all, the 16-cell stage following directly on the 8-cell. In Lytechinus ( Toxopneustes) variegatus also this fourth cleavage is asyn- chronous according to Tennent (1g11a) and Tennent, Taylor, and Whitaker (1929), so that there is a definite 12-cell stage. In the next cleavage also, the three types of cells, mesomeres, macro- meres and micromeres divide asynchronously, so that there is a 20- and a 28-cell stage preceding the 32-cell stage by 10 and 2 minutes respec- tively (23 °C.). The 4 macromeres divide first, then the 8 mesomeres and then the 4 micromeres. It is difficult to follow the division of the different types of cells further, into the 64-cell stage, but sections of later cleavages definitely show that at any particular time the cells are in different stages of mitosis; that asynchrony continues in the later cleavages. There is, however, at least at first, a greater interval between the division of certain sets of cells than between others. There seems to be a major rhythm with considerable spread. Recently an abstract on this subject, also for Arbacia, has been pub- lished by Scott and Fox (1952) Their results are similar to mine, and they have carried the asynchrony through the sixth and seventh cleavages. It is of interest that many years ago Tennent (1911a) published a detailed table of the stages and times in the development of the Lytechinus egg, similar to the one for Arbacia presented here. He has given the times for asynchronous divisions up to 124 cells. The earlier asyn- chronous cleavages correspond exactly with those of Arbacia. The asynchrony in cleavage becomes important in studies of rhythms of oxygen consumption in relation to mitoses, such as those of Zeuthen (1951, etc.). It may be that in some species the rhythms are more marked than in Arbacia and Lytechinus, and Zeuthen (1951, p. 52) found that in Psammechinus microtuberculatus ‘‘the micromeres have multiplied slower than the rest of the cells.” , m. MIcROMERES The micromeres of Arbacia are colorless in contrast to the rest of the 106 THE AMERICAN ARBACIA cells which are pigmented. Apparently the pigment recedes from the lower parts of the lower four cells (vegetal) at the eight-cell stage (Morgan, 1893). There has never been a very good explanation for this, nor has any experimental work been done on it. McClendon (1910b, p. 243) thinks that “the pigment entirely disappears from the micromere pole, indicating spreading movements due to the surface tension being less here than in the region of the future cleavage furrow. Similar movements of granules have been observed in the cutting off of polar bodies in various eggs, and it may be concluded that for the separation of a very small cell from a large mass of protoplasm a very great difference in surface tension between the pole of the small cell and the cleavage furrow is required.” In some cases, it was found by Morgan (1893) that the retreat of pigment to form the micromeres takes place long before the actual cutting off of the cells—in the four or even two-cell stages. An early appearance of micromeres, in the 8-cell stage, was found by Painter (1915) in eggs treated with phenyl urethane. In eggs treated with mustard gas, the micromeres are formed early and one (colorless) micromere is often present in the two-cell stage (E. B. H., 1943 unpub.). According to most investigators, the micromeres of Arbacia, as in other sea urchins, come off at the vegetal pole, nearly opposite the funnel in the jelly which marks the position of the polar bodies (Morgan and Spooner, 1909; Spooner, 5911 ; Hérstadius, 1937a). The micromere- forming material was located by Harnley (1926) in the unfertilized Arbacia egg, between the nucleus and the center of the egg. But Ten- nent, Taylor, and Whitaker (1929) could not confirm this, and held that there was no localization of micromere-forming material in the unfertilized eggs, and that in the fertilized egg the micromeres formed at the cut surface of any fragment. Ho6rstadius (1937a) could not confirm this, nor could he confirm Harnley’s work. He believes that in Arbacia the micromere-forming material is in the vegetal half of a cut egg, and that the animal half never forms micromeres, the condi- tion he and others have found for other sea urchin eggs. But he admits that ‘“‘micromere formation is very sensitive to mechanical injury” and “In Arbacia fragments the micromere-formation seems to be inhibited very often’? (Hérstadius, 1937a, p. 304). Plough (1927) had, some years before, maintained that there was a localization of skeleton- forming material even before first cleavage in Arbacia eggs as well as in those of Echinarachnius, Echinus, and Paracentrotus (1927, 1929), just as maintained by Horstadius (1937a). A study of micromeres in egg fragments has been made by Tennent, Taylor, and Whitaker (1929). FERTILIZATION AND CLEAVAGE 107 In centrifuged whole eggs, after fertilization, the micromeres may come off at any place, even at the oil cap (E. B. H.). Their position is probably determined by the original polarity of the egg. They may be pigmented but are usually not. See Plate VIII, Photographs 5, 6, 15, 16. There may be not four micromeres, but three, two, one or none, and yet normal development may follow. This has been found by Tennent, Taylor, and Whitaker (1929) and also by Horstadius (19374). Horstadius also calls attention to the fact that not all small cells are micromeres, but that small cells may be formed as the result of entirely different factors from those leading to micromere formation. These facts must be born in mind in any investigations involving micromeres. n. ABSENCE OF OxYGEN Whether an egg can be fertilized in absence of oxygen has always been an intriguing question. Unfortunately for its answer, oxygen is neces- sary for motility of the sperm, so that in its absence they cannot swim to the eggs and fertilize them. If there is the slightest trace of oxygen so that a few sperm are very slightly motile, a fertilization membrane is thrown off, but no further development takes place (E. B. Harvey, 1930). That it is the lack of motility of the sperm and not the absence of oxygen is shown by an experiment of Kitching and Moser (1940). Arbacia eggs were kept in absence of oxygen beside a drop of a parthe- nogenetic agent, and then the two drops were mixed in absence of oxygen. A fertilization membrane was formed. o. REFERTILIZATION It has been the experience of many investigators that an egg, once fertilized, cannot be fertilized again, even if the fertilization membrane has been removed (Loeb, 1916, p. 85; F. R. Lillie, rg19, p. 161; e¢ al.). Until recently an only exception for sea urchins is the report of Bury (1913) that if the eggs of Strongylocentrotus lividus and Echinus micro- tuberculatus, already fertilized and with fertilization membranes, are kept in the cold, 0° C., they can be refertilized. Recently, Sugiyama (1947, preliminary; 1951) has found that in Strongylocentrotus pulcher- rimus and other Japanese species, after the fertilization membranes of fertilized eggs have been mechanically removed, and the eggs are washed in Ca-Mg-free sea water, they could be refertilized. The sperm penetrated the eggs and took part in the formation of the mitotic figures producing irregular cleavages characteristic of polyspermy ; the 108 THE AMERICAN ARBACIA fertilization membranes were not replaced. Refertilization could take place even without removing the original fertilization membrane if treated soon enough; and also in the 2-cell stage. Pp: FERTILIZATION AFTER PARTHENOGENESIS Loeb thought this possible if the fertilization membrane was removed from the parthenogenetic egg (Strong ylocentrotus purpuratus) ; he thought even blastomeres could be fertilized (Loeb, 19134, p. 234, 237; 1914b; 1915a, b). A reversal of parthenogenetic development and subsequent fertilization could take place also in Arbacia eggs (Loeb, 1913¢; Wasteneys, 1916). But others found that a subsequent fertilization was not possible unless parthenogenetic treatment was incomplete (GAR Moore,*r916, 1917; F. R: Lillie; 1910; ps 167; 1921 as Just, 19224; Lillie and Just, 1924, p. 502). However, more recently Ishida and Nakano (1947, 1950) have found that if the eggs of S. pulcherrimus were treated with a parthenogenetic agent (butyric acid) and the fert- ilization membranes removed mechanically, and were then placed in a Ca-Mg-free medium, they could be fertilized. Sperm entered and cleavage took place similar to that characteristic of polyspermy. GHAP TE R14 Blastula, Gastrula and Pluteus a. BLASTULA AND GASTRULA. PLATEs III AND IV By counting the number of cells at the periphery of an optical section of a living blastula, it has been calculated that there are approx- imately 1,000 cells in an Arbacia blastula just before hatching; this would represent approximately 2'° or ten cleavages. MacBride (1914) estimated 1,000 for Psammechinus microtuberculatus and Morgan (1895c) 500-525 for Sphaerechinus granularis, about nine divisions. Soon each cell acquires a cilium and rotates inside the fertilization membrane. About 8 hours after fertilization at 23° C. (74 to 93 hours in different batches) it breaks through the fertilization membrane and becomes free-swimming. This is done by means of a “hatching enzyme”’ which dissolves the membrane. Such an enzyme was found many years ago in fish eggs, in Lepidosiren by Kerr in 1900, and was especially studied in the Ascidians by Berrill (1929). In sea urchins its occurrence was reported by Ishida (1936) in Strongylocentrotus pulcherrimus, and later by Kopac (1941) in Arbacia punctulata. The Japanese species was studied again more recently by Sugawara (19434). The blastocoel of the Arbacia egg is usually small on hatching, in contrast to many other sea urchins, e.g., Strong ylocentrotus drébachiensis, Lytechinus variegatus. In Arbacia the blastocoel gradually becomes larger leaving only a thin layer of ciliated cells at the periphery of the blastula. The cilia are longer at the apical pole as early as hatching, forming the apical tuft. After the blastula has become free-swimming, it remains spherical for about seven hours, swimming very actively. Then it begins to invaginate (about 15 hours after fertilization) at the pole where the micromeres came off, the original vegetal pole, and opposite the pole where the polar bodies were given off, the animal pole (Morgan and Spooner, 1909; Horstadius, 1937a). This relationship was beautifully shown for the Paracentrotus lividus egg in the classic studies of Boveri (1go1, etc.) and is the same for Arbacia. The invagination continues until it approaches the anterior end I1O THE AMERICAN ARBACIA where the mouth is formed, making the gut complete (about 17 hours after fertilization). The original in-pocketing remains as the anus. Meanwhile (about 16 hours) the skeleton has appeared as a pair of triradiate spicules, one on each side of the (incomplete) gut. During this period there has been no appreciable increase in the size of the organism over that of the egg (without the fertilization membrane), and one would not expect an increase before the alimentary canal is complete and it can take in food from the outside. Then growth occurs and further differentiation. The axis of the larva changes, giving the ““‘prism’’ stage, and one of the prongs of the triradiate spicules elongates (dark field photograph, Plate IV, Photograph 2). With further elongation, these become rods on each side of the gut, and the early pluteus is formed, at first without arms (Photograph 4, about 20 hours after fertilization). At about this time, the large red pigment spots begin to appear. The arms then grow out, increasing in length with time. The pluteus is quite well formed a day after fertilization, and increases in size during the next two or three days (Plate IV, Photo- graphs 5-9). b. THE PLutTeus, DEscripTION. PLATE IV The pluteus which we are accustomed to see in our cultures three or four days after fertilization is roughly triangular in shape, swimming with its arms forward, and its pointed end behind, by means of cilia which cover the surface of the body. There are two pairs of arms, a shorter pair on the dorsal side near the mouth, the oral or dorsal arms; and a much longer pair on the ventral side, the anal or ventral arms which may measure 400 yu from base to tip (Plate IV, Photographs 7-9). The skeleton consists or rods running into the arms, thinner ones into the oral arms, and thicker ones into the anal arms; these long rods meet at the base in a heavy spiny mass. There is also a transverse connecting rod. The rods in the long anal arms of Arbacia are not solid, but fenestrate or ladder-like. Many other sea urchins have this same type of skeleton in their long arms, e.g., Tripneustes, Sphaerechinus, and Echinarachnius (sand dollar). In other sea urchins, the skeleton of the anal arm is a solid rod, e.g., Lytechinus, Psammechinus, Paracentrotus, Strong ylocentrotus. These two types of arm skeleton have been of great value in hybridizing experiments, in determining maternal and paternal inheritance. In Arbacia, another pair of arms which come in later, the postero-dorsal arms, are also fenestrate. According to Fell (1948), the fenestrate rods represent a primary structure since they are found in the larvae of the more primitive forms. BLASTULA, GASTRULA AND PLUTEUS III It was found many years ago by Pouchet and Chabry (1889) that calcium is necessary for the formation of the pluteus skeleton. Develop- ment would not take place in sea water without any calcium, since the cells break apart (Herbst, 1900). They found that if 1/1oth the normal amount of Ca in sea water was replaced by Na, no skeleton was formed. Loeb (1g00a) found that Mg and COQ, ions are also necessary for a normal skeleton. He could obtain plutei of Arbacia with normal skeleton in a solution of: 95 cc. 5/8 n NaCl + 1 cc. 10/8 n MgCl, + 1 cc. 5/8n KCl + 2cc. 10/8 n CaCl, + 1 cc. 1/8n Na,CO,. Herbst, in a series of papers (1892-1904) studied the relation of the composition of the sea water to the development of the pluteus. He showed that SO, is necessary for the development of the skeleton (1904). See J. D. Robertson’s review (1941). The development of the triradiate spicules in Echinus esculentus has been studied by Woodland (1906, 1907). The spicule arises in an early mesenchyme cell as a granule which becomes three-cornered. The digestive tract of the pluteus is J-shape consisting of a mouth on the dorsal side between the two shorter arms, an oesophagus, stomach, and intestine and ending in the anus between the two long arms on the ventral side (Plate IV, Photographs 7, 8). There are large red pigment spots scattered irregularly over the body and often more abundantly along the arms, especially at their tips. They are sometimes irregular in shape, sometimes spherical (E. B. H.). Each spot consists of 20 to 30 individual granules about 2 p in diameter. The pigment spot itself is variable in size, an average spherical one measuring about 7 uw in diameter. The red pigment is echinochrome, the same that is found in the chromatophores of the egg, having the same composition and absorption spectrum (Ball and Cooper, 1949). The granules swell in distilled water, like the chroma- tophores of the egg, having similar osmotic properties (E. B. H.). The permeability value has not been studied, and it would be interesting to compare this with the permeability value of the chromatophores as given by D. L. Harris (1943). The gradual decrease in number of the chromatophores and appearance of the pigment spots in the very early pluteus stage following the prism stage might also prove to be an inter- esting study. Photographs of a well developed pluteus are shown on Plate IV, Photographs 7-9. These are anal (ventral) and oral (dorsal) views of the same animal, and a side view. When photographed with ultraviolet light (2537 A), certain regions appear much darker than others (E. B. Harvey and Lavin, 19514). ii THE AMERICAN ARBACIA The most absorbing regions are the digestive tract and the two trans- verse ciliated bands, the oral band around the mouth and the postoral (or ventral) band between the two long(anal) arms above the anal opening. These regions are the most active physiologically, being con- cerned with procuring and digestion of food, and there may be some correlation between physiological activity and ultraviolet absorption. In fixed preparations stained with haematoxylin, these same regions, the alimentary canal and the two transverse ciliated bands, are deeply stained. When photographed by infrared light (8,o00—-10,000 A), the struc- tures of the pluteus appear as they do with visible light except that the red pigment spots are not distinguishable (E. B. Harvey and Lavin, 1951 a). An interesting though somewhat involved mathematical or geo- metrical explanation of the form of a pluteus larva may be found in D’Arcy Thompson’s Growth and Form (1948, p. 625). The normal pluteus is quite uniform in shape, both in different batches and in individuals of the same batch. There do, however, occur in some cultures, and usually in the entire culture, plutei of a different shape. Sometimes the anal arms are widely divergent, and sometimes they are close together. The cause for these abnormalities has not been determined. Such abnormalities can, however, be produced by experi- mental conditions, such as KCN, acids, alkalis, salts, alcohols, etc. These have been studied by Child (1916b, 1941, p. 197-211) with relation to axial gradients, and also by Medes (1917). Abnormal plutei have also been produced by acetylsalicylic acid (aspirin), probably an acid effect (Budington, 1935); dinitrophenol, iodoacetic acid, pyo- cyanine, methylene blue (Waterman, 1938); malonic acid (Rulon, 1948). Tennent (1910a) has made a statistical study of variations in Lytechinus plutei. Most substances have a harmful effect on the plutei, but M. M. Brooks (1943) reports that methylene blue increased the length of the anal arms; those treated with methylene blue averaged 420 » while the controls averaged 280 u. An interesting effect of KCN on the plutei has been reported by Lyon (1902). Ciliary motion is stopped, and when the larvae are re- turned to sea water, it starts up again, but the cells, either singly or in small masses, break loose and swim for a moment; then the pluteus disintegrates. The same result follows anaerobiosis caused by prolonged exposure to hydrogen gas. BLASTULA, GASTRULA AND PLUTEUS 113 c. DEVELOPMENT OF PLUTEUS WHEN Nor FEp At Woods Hole, the pluteus reaches its maximum size in three to four days, and will not continue to grow unless it is specially fed. At its maximum (without feeding), the long anal arms measure about 400 py; the longest ones in my cultures were 442 » from base to tip. Cultures of these plutei may be kept in the laboratory, if the sea water is changed every day or so, for three or four weeks. The plutei gradually get smal- ler by resorption of the arms, and the body takes on a bloated appear- ance (Plate XVI, Photograph 10). There is apparently sufficient food material in the sea water for them for three of four days after hatching, and there is considerable growth, but after this they degenerate unless supplied with additional food. It seems to make no difference in their growth if supplied with extra food before the fourth day. d. Foop For PLUTEUS The best food for sea urchin larvae has been found to be the diatom Nitzschia closterum, but they will grow on other diatoms, e.g., Lich- mophora (E. B. H.). The Nitzschias themselves must be raised in pure culture and require a special diet, Miquel’s solution’. The method has been worked out by Allen and Nelson (1g10) in Plymouth, England, and has been used by many investigators at the Plymouth laboratory. Shearer, de Morgan, and Fuchs (1914) have in this way succeeded not only in raising the normal plutei of several species of sea urchin to maturity, but have also raised some hybrid plutei to maturity. Fuchs (1914b) has even obtained the next or F, generation of these hybrids. 1 Miquel’s solution as modified by Allen and Nelson (1910), consists of: Solution A KNO, 20.2 gm. Distilled water 100 cc. Solution B Na,HPO,'12 H,O 4 gm. CaCl,-6 H,O 4 gm. FeCl, (melted) 21CG: HCI (concentrated) DECC. Distilled water 80 cc. To each liter of sea water add 2 cc. Solution A andi cc. Solution B, and sterilize by heating to 70° C. When cool, decant off the clear liquid from the precipitate, which will have formed when Solution B is added to the sea water. Ketchum and Redfield (1938) have used a slight modification. A very simple medium for growing Nitzschia has been used by John Ryther at the Woods Hole Oceanographic Institute (personal communication, Sept. 1954): Add to sea water at 20° C. or below Na,HPO,:12 H,O .002% KNO, 01% II4 THE AMERICAN ARBACIA Unfortunately the late larval characters of Echinus esculentus x E. acu- tus, from which the F, generation was obtained, are alike in the two species, so that no information as to the inheritance could be obtained; none of the F, hybrids between E. esculentus or E. acutus x Ps. miliaris which would have given the information, reached maturity.. Miss Gordon from MacBride’s laboratory raised some Arbacia plutei at Woods Hole in 1926, using this method, but she was particularly interested in the later development of the test, and gives no account of the changes in the pluteus in her publication (1929). There are several varieties of Nitzschia closterium. There is a very small form, Nitzschia closterium minutissima, the one used in the Plymouth laboratory and now cultured at the Oceanographic Institution at Woods Hole.! This is about 24 u long. A larger form from the New Jersey coast has been cultured at Rutgers University; this measures about 100 u. A still larger form grows in the Eel Pond at Woods Hole; this measures about 200 pu. In the small variety, there are three types of cells, the normal spindle-shaped cell, a triradiate cell and an oval cell (D. P. Wilson, 1946). The relation of these three types to each other and their division has been studied by Wilson. The method of feeding Echinoderm larvae with diatoms originated with Caswell Grave (1902a, b) at the Beaufort, N. C. laboratory, where he was rearing the sand dollar Mellita testudinata. The diatoms are swept into the mouth and oesophagus of the Arbacia pluteus by means of very active cilia. The plutei thrive equally well on the small and the large varieties of Nitzschia closterium (E. B. H.). The very young sea urchins just after metamorphosis thrive better on the calcareous protozoon Trichospherium, which furnishes the cai- careous matter for the shell; a little later they flourish on the red alga Corallina, according to Shearer, de Morgan, and Fuchs (1914, p. 276), for Echinus. These have not been tried with Arbacia punctulata. In some places the plutei do not require extra food for growth and development, but apparently obtain sufficient food from the sea water. This is the case with Arbacia punctulata at Beaufort, N.C. Brooks (1882), and two of his students, Garman and Colton (1883) apparently raised the plutei through metamorphosis without extra food. The sea water there is rich in diatoms. 1 In a recent paper of N. I. Hendey (1954) entitled Note on the Plymouth “‘Nitzschia’’ Culture in J. Marine Biol. Assoc’n, U. K. 33 : 335-339, the identification of the original culture of Nitzschia of Allen and Nelson (1910) has been questioned, and identified by him as Phaeo- dactylum tricornatum Bohlin, not a diatom but might be related to the Chrysophyceae. GEV ASP TE Rods Metamorphosis a. ARBACIA PUNCTULATA. PLATE VI After the 3 or 4 day old pluteus, raised in the laboratory, is fed Nitz- schias, it increases in size, and the anal arms grow to about 600 p (from 400 2) when a week old. Then little knobs appear toward the base on the pluteus, which, by the eleventh day have grown out into a new pair of arms extending backward, the postero-lateral (Mortensen) or ventral-lateral (Brooks) (Plate VI, Photograph 3). These arms have very red tips owing to accumulation of red pigment bodies, and they have much longer and stronger cilia than the other arms. These arms grow longer and another pair of knobs appears between the original anal arms and the new red-tipped arms (2 to 3 weeks); these become the postero-dorsal (Mortensen) or dorsal-lateral (Brooks) arms (Photo- graph 4). These arms have fenestrate rods like the anal arms, unlike the red-tipped arms which have solid rods. All the arms grow much longer, and the animal is easily visible to the naked eye, looking like a small spider (Photograph 5). The animal tumbles about on the tips of its arms and also swims by means of its cilia. The arms are variable in length individually and relatively to each other. They are very fragile and are easily broken off when the animal bumps into something or when transferred to another dish. They have great regenerative capacity, the arms growing out again when broken off. One pluteus from which I had cut off the red-tipped arm about half way down, had completely regenerated it together with the red pigment in five days, so that it looked exactly like its mate. When three or four weeks old, two pairs of tubular processes appear, two dorsal, two ventral, the auricular lobes (Photograph 6). Tho more pairs of arms arise in the head end, the antero-lateral and the antero-dorsal, so that there are now six pairs of arms, three long pairs and three shorter pairs. The body of the adult Arbacia is now seen as a yellowish green mass in the pluteus, the dark area in Photograph 6 and thereafter. There are areas of dark red pigment on the surface of the body. The young adult is formed in the body of the pluteus and grows at the expense of the pluteus. 116 THE AMERICAN ARBACIA When about two months old, five tube feet appear at one side of the body in a sort of pocket, but soon extend out radially; they have suckers at their extremities and are very active, expanding and con- tracting (Photograph 7). Within the next two weeks, 15 petal-like structures appear, three adjacent to each tube foot; these are the primi- tive spines (Photograph 8). The pluteus had already reached its max- imal development, and the arms their maximal length, about 1.6 mm.; the whole animal including arms was over 3 mm., maximal diameter. A good diagram of this stage is given by Gordon (1929, p. 29). The length of the anal arm at different ages is given in Table 6. A mathe- matical treatment of the data has been given by Glaser (1950), showing that they fit closely with a modified version of Huxley’s allo- metric equation. TABLE 6 APPROXIMATE LENGTH OF LONG (ANAL) ARM FROM BASE TO TIP (IN /L) ANAL ARM FED NOT FED 1 day 180 180 2 days 300 300 3 days 380 380 4 days 410 410 5 days 450 : 330 6 days 480 250 1 week 600 200 11 days 700 180 2 weeks 750 150 3 weeks 800 I month 1000 134 months 1300 2 months 1400 24 months 1600 The head of the pluteus has remained for some time. The arms now or sometimes before this begin to degenerate, the flesh peels off leaving the bare skeleton (Photograph 6), and they are gradually lost. The metamorphosed animal now consists of the greenish spherical body with 5 tube feet and 15 primitive spines (Photograph 9, 10). The tube feet soon increase in number, the newer ones being more slender (Photograph 11). This is the latest stage obtained in the laboratory; the animal measured about 1 mm. including spines, and was about 34 months old. The times given for different stages are only approxi- mate as they vary greatly in different lots. A very young adult Arbacia was found in July 1952 in a clam shell METAMORPHOSIS I17 brought in from the sea. This measured 6 mm. including spines. The spines are more numerous and more slender than in the metamor- phosed animal, but the structure of these spines and the primitive spines is the same. They are transparent, with veins running through them, something like an insect’s wing. The stages between the animal metamorphosed in the laboratory and the youngest animal found out- side are much desired. Plutei have not been found in the tow above the beds. The account given above has been revised from an earlier account (E. B. Harvey, 1949), which did not include the later stages with well formed primitive spines. Earlier studies on the Arbacia metamorphosis, as well as that of other forms have been treated in Part I (Chapter 2, Section f) under Historical. The best early study of Arbacia punctulata is that of Brooks (1882) and his students, Garman and Colton (1883), published by Brooks in his Handbook of Invertebrate Zoology (1883), in which excellent drawings of the different stages: are given. The works of Agassiz (1872-1874, 1883, 1904) and of Miss Gordon (1929) should also be consulted. b. OTHER SPECIES Many studies on the metamorphosis of other species of sea urchins have been made. Among the most complete studies are those on Echinus esculentus by MacBride (1903, 1914a Text-book of Embryology vol. 1, p- 504) and by Shearer, De Morgan, and Fuchs (1914); the develop- ment of Psammechinus miliaris and Ps. microtuberculatus is similar (Mac- Bride, 1914a); Arbacia lixula and Paracentrotus lividus by von Ubisch (1913 a, b, c, 1927, 1932, 1950); see also Busch (1849); and Miller (1854) ; Echinocyamus pusillus by Théel (1892) ; Echinocardium cordatum by MacBride (1914b); Salmacts bicolor by Aiyar (1935); Mespilia globulus, Strong ylocentrotus pulcherrimus, and other Japanese forms by Onoda (1931, 1936). A very good list of studies on larval forms is given by Mortensen (1921, p. 12), and a more recent list arranged phylogeneti- cally by Fell (1948). Studies made during expeditions to Kei Islands (Amboina), Java, S. Africa (Onrust, Mauritius) and Egypt were published by Mortensen (1931, I and II; 1937, III; 1938 IV); and there are many references in his Monograph (1928-1951). See also this Monograph under Historical, Part I, Chapter 2, Section f. (p. 18). A very good general treatise on larval development may be found in Grassé’s Traité de Zoologie, t. 11, p. 307-312, 1948. ig ih ent esi PARA, Itt CENTRIFUGED EGGS CHAP TE Role Methods a. WHOLE EGGs When Arbacia punctulata eggs are centrifuged in sea water, they are thrown to the bottom of the tube and crushed, because they are heavier than the sea water. They must be centrifuged in a medium of the same density as themselves, in which they will be suspended. The solution must also be of the same osmotic pressure as the eggs, so that they will not swell or shrink. Since individual eggs vary in density, it is best to centrifuge the eggs in a medium of graded density, made by partially mixing sea water and 0.85 M cane sugar. The sugar solution! is osmotic with but of slightly greater density than the eggs. Two parts of the cane sugar solution are placed in the bottom of a small slender centrifuge tube and one part of sea water containing the eggs on top. By a slight rotation of the tube the solution can be partly mixed and a density gradient established, so that when centrifuged, the eggs come to lie in a region of their own density. As they break apart during centrifuging, the half-eggs move to a new region equal to their density. They are consequently separated into layers, the heavy (red) halves at the bottom and the light (white) halves at the top (the unbroken eggs are in the middle), as shown in Plate VII, Photograph 10. With a fine pipette the eggs of each layer can be removed without conta- mination with the eggs of other layers, and large numbers can be collected for experimental work. Asmall electric centrifuge has been used with two (or four) glass tubes 6.5 cm. long and 0.4 cm. inside diameter, narrow in order to prevent mixing by convection currents. The centrifugal force, F, in terms of force of gravity (g), is given by the equation F = 0.04 x radius (in cm.) x (r.p.s.)!. A centrifugal force of 10,000 x g for four minutes 1 The 0.85 M sugar (molecular weight 342.24) solution is prepared by adding 29 gm. sugar (commercial samples are as good as ‘‘chemically pure’’) to sufficient tap water to make 100 cc. Tap water is used rather than distilled water as the slight alkalinity prevents stickiness of the eggs. I have found it best to weigh out several lots of 5.8 gm. sugar and to add tap water to make 20 cc. when needed; the sugar solution will keep for several days in the refrigerator (8 °C.), but becomes acid on standing at room temperature. 122 THE AMERICAN ARBACIA is sufficient to break the eggs into halves, and a much smaller force, 3,000 X g for two minutes will stratify them (Plate VII; also Fig. 12). In working with other eggs at other places, corrections must be made for differences in density of the sea water and the eggs. The sugar-sea water medium described, in which the eggs are centri- fuged, is quite harmless. Eggs may be kept in the solution for five hours, and, when returned to sea water, cleave and develop as well as the con- trols, and at the same rate. The eggs, however, cannot be fertilized in the sugar solution, though the sperm are active and surround the eggs as normally. This may be due to lack of sufficient calcium; it has been found that eggs cannot be fertilized in Ca-free sea water, though the sperm are active. In his pioneer experiments, Lyon (1907) .used gum arabic for sus- pending the eggs. He used a centrifugal force of 6,400 x g for one or two minutes, the eggs remaining spherical, though he apparently had a few eggs break into halves which he said could not be fertilized. b. CrRusHED Eccs. HOMOGENATES When Arbacia eggs are crushed in a mortar after they have been frozen, and then centrifuged, McClendon (1909a) found that the material separated into two layers, a fluid centripetal layer and a jelly-like centrifugal layer. The chemical composition of the layers is given in Table 7. References to more recent methods of obtaining egg homo- genates by freezing and thawing are given by Runnstrém (19354). Kopac (1943) has recommended the following treatment: . Remove jelly layer by washing in two or more changes of 0.52 M NaCl. . Transfer to 1.0 M solution of urea. . Within 1 or 2 minutes wash eggs free of urea with 0.53 M KCI. . Transfer eggs to a measured volume of citrated KCI solution containing 9 volumes 0.53 M KCl and 1 volume of 0.35 M Na-citrate. 5. Immediately disintegrate eggs by flushing in the above solution through a fine bore pipette. : ‘The resulting suspension which includes all granules and nongranular residue of the cells is then centrifuged gently to remove unbroken eggs and foreign particulate debris. The supernatent is again centrifuged, at high speed, to separate the granules and other formed elements. The sediment now contains pigment vacuoles, yolk granules, and some mitochondria. The oil globules collect at the meniscus of the centrifuge tube. In the absence of Ca ions, the granules are stable and may be pre- served intact for considerable periods. The more or less nongranular supernatent fluid may be separated from the sedimented granules by pipette transfer. This con- tains most of the residue of the cytoplasmic matrix.” PON 4 METHODS 123 TABLE 7 COMPOSITION OF LAYERS IN CRUSHED ARBACIA EGGS IN PERCENT OF WHOLE EGG (McClendon 1909 a) Centripetal Centrifugal Layer ane Gare Whole egg Water 28.6 53-3 81.9 Solids 3.9 14.2 18.1 Ether ext. 0.308 1.946 2.254 Pins ther: ext. 0.00154 0.06760 0.06914 Alcohol ext. 1.6 3.48 5.08 P in alcohol ext. 0.0434 0.0814 0.1248 Water ext. 0.78 1.42 2.20 P in water ext. 0.130 0.1822 0.3122 Residue of water ext. 1.309 7.29 8.599 P in residue 0.0392 0.1167 0.1559 N in residue 0.1625 0.775 0.9375 Ash in residue 0.0162 0.0264 0.0426 Total P in layers 0.21414 0.4478 0.66194 Many other methods of preparing a homogenate or a suspension of the contents of Arbacia eggs have been used. The important consider- ation in obtaining a particulate system is to break up the eggs in a medium devoid of calcium,. since the yolk and pigment granules disintegrate in its presence. The eggs may be ruptured by forcing through a hypodermic needle from a syringe (Keltch, Strittmatter, Walters, and Clowes, 1950), by grinding with sand in a mortar (Krahl, Keltch, Neubeck, and Clowes, 1941), or by use of a Waring blender. Subsequent centrifuging of the “‘brei’’ results in separation of the various constituents, which can then be studied separately. Monroy recommends for A. lixula, for jelly-free eggs: LiCl 1 M + NaHCO, 0.04 M + Na,CO, 0.01 M + Versene 0.01 M, in distilled water. Use a Potter homogenizer (Personal communication July 1955). ‘SUISNJIIIUID IasUOT YM saynuess oy Jo Surysed ssyinNyz 0 anp st afod jejedinuss ay) ie 4 ‘ON Ur vate Ieapd oY, -< off noge payruseyy ‘apeos 01 apqrssod se Ajayeindo¥ se apeul ‘sydersoioyd pure sayoioys epronyt pisWe) WOT a1 SSUIMLIP dT, “SxVatq W YOTYM our suaqenb pue saayey oy? pur (3 X OOO‘OT 3% soynuru § noqe) 99103 [eSnyrUa. Aq payne.ns ‘Dipjujound pivgip jo 88a pazi[Hsayun sy fT “St “OT pabrjiajnaras pabnjijnar bby 304m pabnjisynaras }1®H ® {eH ayrym THE AMERICAN ARBACIA 124 VIOVaEay fe AP oe Bie 17 Stratification of the egg granules a. UNFERTILIZED Ecos. PLATE VII The stratification of the well-centrifuged egg of Arbacia, beginning at the light (centripetal) pole, is (1) oil, (2) clear layer without visible granules (see Part IV, Clear Layer), (3) a thin finely granular layer of mitochondria, (4) a large yellowish heavy layer of larger yolk granules, (5) a red pigment layer at or near the (centrifugal) pole. The nucleus is always at the centripetal pole just under the oil cap. (E. B. Harvey, 1932, 1936, etc.) (Plate VII, Photograph 1 and Figure 12). In the older work on Arbacia eggs, only four layers were described, the mitochondrial layer being omitted, owing probably to insufficient force (Lyon, 1906a, 1907; Morgan, 1909). In still earlier work on Arbacia lixula, Sanzo (1904) obtained only two zones, a protoplasmic and a granular zone, and he figures an oil cap; this led him to believe that he had changed a homolecithal into a telolecithal egg. b. VITAL STAINING OF STRATIFIED EGG The different layers of the centrifuged egg stain differently with different vital dyes (E. B. Harvey, 1941c). A table is given (Table 8) of a number of vital dyes and their effect on the different layers. Many other stains were tried, especially acid dyes, without effect. Some dyes, e.g., rose bengal, stained only after the egg was dead. The nucleus does not stain, vitally, in any dye; and the cytoplasm only in basic dyes as noted long ago by Mathews (1907). Quinine, cinchonine, and cinchonadine stain the pigment almost black like methylene green, with no effect on development (E. B. H., unpub.). c. STAINED SECTIONS OF STRATIFIED EGGs Eggs were fixed in Bouin’s fluid, sectioned and stained with iron hematoxylin; some were counterstained with eosin and orange G (E. B. Harvey, 1940c). The clear protoplasmic layer which is optically 126 THE AMERICAN ARBACIA empty in the living egg, stains blue and consists of very small granules, the microsomes; this was described in the early paper of Lyon (1907). The yolk stains orange with a rose tinge and the pigment orange. The mitochondria can sometimes be distinguished as a darker bluish band between the protoplasm and the yolk. The oil cap does not show; it is probably dissolved in the fixing fluid. The two half-eggs show the same stratification except that one usually sees a blue cap of proto- plasm on the red half-egg, especially when well centrifuged so as to TABLE 8 VITAL DYES ON CENTRIFUGED UNFERTILIZED EGGS OF ARBACIA PUNCTULATA (From E. B. Harvey, 1941c, with a few additions) Dye Jelly Oil Clear Layer Mitochondria Yolk Pigment Remarks Bismarck brown oO oO Yellow (upper Yellow Yellow Brown Slightly part more soluble in intense) sea water Brilliant cresyl fo) fo) fo) fo) Blue Blue Very blue innocuous Chrysoidin fo) (0) Light yellow Light Yellow Reddish (upper part yellow brown more intense) Crystal violet oO oO fe) Purple o fo) Dahlia oO fo) fo) Purple oO oO Gentian violet oO fe) o Purple o oO Janus dark blue B Purple fe) to) fo) o fo) Janus green or Purple fo} (o) Blue Oo ° Rather toxic Janus green B Methyl green oO oO ° Purple Oo oO Methyl violet o o Upper part Purple Purple Purple violet (later) (later) Methylene blue (o) fo) fo) fo} Blue Blue Very innocuous Methylene green fe) fo) o fo) fo) Almost black Neutral red oO oO Pinkish yellow _ Pinkish Brick red Blood red, Slightly (lower part yellow almost soluble in more intense) black sea water Nile blue oO oO Light blue Light blue Blue Bluish Slightly sulphate (upper part brown to soluble in more intense) blue black sea water Rhodamine ° o Pink (upper Pink Pink Deep red Very part more innocuous intense) Safranin O Yellow ° fe) Pink (after fe) Blood red = Not soluble (few cases) 1-2 hours) in sea water Thionin Pinkish oO o Lavender fo) o Not soluble (few cases) (few cases) in sea water Toluidin blue Pinkish fo) Pinkish Lavender Lavender Purple to More intense lavender lavender blue black _ if stained alter cen trif. STRATIFICATION OF THE EGG GRANULES e257, allow further packing of the granules at the centrifugal pole. In the living egg this is seen as a clear layer (Plate XIII, Photograph 5; also Fig. 12, No. 7). d. STRATIFICATION OF OTHER SPECIES Diameters of the eggs in micra are given. The layers in square brackets may or may not be present. Data from E. B. Harvey (19334, 1938a, 1947, and unpublished) unless otherwise noted. Arbacia lixula (79). Stratifies like A. punctulata (E. B. H.; Callan, 1949, with diagram). Arbacia punctulata (74). Oil, clear, mitochondria, yolk, pigment. Echinarachnius parma (145). Oil, absent in some, [clear], yolk, clear, mitochondria, neutral red-staining granules (or methylene blue). Echinocardium cordatum (125). Yolk, clear, mitochondria; apparently no oil (Monné, 1944b). ’ Echinometra lucunter (85, 97). Oil, clear, mitochondria, yolk. Lytechinus ( Toxopneustes) variegatus (103, 112). Oil, (little), yolk, clear, mitochondria, [clear]. Mellita quinquiesperforata (150). Oil, clear, mitochondria, yolk. Paracentrotus lividus (go). Oil, [clear], yolk, clear, mitochondria, [clear] (E. B. H.; Callan, 1949, with diagram). Psammechinus microtuberculatus (102). Oil, [clear], yolk, clear, mitochon- dria (E. B. H.; Callan 1949, with diagram; Lindahl, 1932c). Psammechinus miliaris (98, 115). Oil, yolk, clear, mitochondria (Holter and Linderstrém-Lang, 1940; Runnstrém and Kriszat, 1950a, with figures). Sphaerechinus granularis (98). Oil, clear, mitochondria, yolk, [clear]. (E. B. H.; Callan, 1949, with diagram). Strong ylocentrotus drébachiensis (160). Oil, [clear], yolk, clear, mitochon- dria. Strong ylocentrotus franciscanus (120). Oil, [clear], yolk, clear, mitochon- dria, [clear]. Strong ylocentrotus purpuratus (80). Oil, [clear], yolk, mitochondria, clear. Tripneustes (Hipponoé) esculentus (84). Oil, [clear], yolk, [clear], mito- chondria, clear. In all the forms listed above, the nucleus is at the centripetal pole under the oil cap, except that Monné (1944b) shows it near the centri- fugal pole in Echinocardium cordatum. In Temnopleurus sp? Motomura (1935) states that it is at the centrifugal end. 128 THE AMERICAN ARBACIA e. STRATIFICATION AND POLARITY The Arbacia eggs fall at random in the centrifuge tubes. There is no orientation as they fall. If there is any polarity in the unfertilized egg, the stratification by centrifugal force bears no relation to it. This was stated by Morgan and Lyon (1907) and proven by Morgan (1909) by comparing the position of the micropyle, which indicates the original axis of the egg, with the stratification; there was no relation. The position of the polar bodies, if given off during centrifugation bears no relation to the stratification but they may be formed in any region, in the pigment zone or yolk or even at the oil cap (Plate II, Photographs 14, 15). f. REDISTRIBUTION OF STRATIFIED MATERIAL AND RETURN TO SPHERES If the eggs, after centrifuging, are returned to sea water, the stratifica- tion gradually disappears, and the granules are again scattered through- . out the egg. The first layer to be lost is the mitochondrial layer; this becomes indistinct almost immediately and cannot be distinguished after 10-15 minutes. The oil cap remains longest, often for 24 hours. If the eggs have become dumbbells when the centrifugal force is removed they gradually return to the original spherical shape. Shapiro (1941) has studied the kinetics of the return in sea water, and in sea water without calcium. In sea water they round up, at first rapidly (several minutes), later more slowly. In sea water without calcium they elongate more and contract more quickly. Normal eggs, uncentrifuged, when deformed by squeezing through a capillary tube (diameter 50 u; egg 74 u) round up too quickly to be accurately timed by eye. Moving pictures indicate that the return takes about 0.1 second (E. N. Harvey and Shapiro, 1941). If fertilized, the elongate eggs do not round up, but remain elongate during cleavages and as blastulae (Plate VIII, Photographs 1-8). CoH AGRE ESR 118 Breaking of the Egg Ege Fractions and Contents When centrifuged with low forces, 3000 x g for two minutes, the eggs remain spherical but become stratified. When centrifuged at 10,000 x g for four minutes, most batches of eggs stratify, become dumbbell- shaped and then break across the yolk into two slightly unequal spheres or “‘half-eggs” (Plate VII and Fig. 12). In the centrifuge tubes (Photo- graph 10) there are three distinct layers, the white half-eggs toward the top (centripetal end) of the tube, a pinkish layer of elongate un- broken eggs a little below, and a layer of red half-eggs at or near the bottom (centrifugal end) of the tube. In some cases there are five layers with this force due to the breaking of the red half-eggs into quarters. The layers now are: white half-eggs at the centripetal pole, unbroken whole eggs, yolk quarters, unbroken red halves, and pig- ment quarters at the centrifugal pole (E. B. Harvey, 1936, photo 6). These quarters can also be obtained by recentrifuging the red halves. The white half-eggs may also be broken into clear and mitochondrial quarters by pipetting them off and recentrifuging them in a slightly less dense sugar solution for about 45 minutes. The nucleus is always in the white half-egg and in the clear quarter-egg. It is lighter than the granules and always lies just under the oil cap at the centripetal pole. The white halves contain oil cap, nucleus, clear layer, mitochondria, and a little yolk; the slightly smaller red half contains most of the yolk and all the pigment granules; the clear quarter contains oil cap, nucleus, and clear layer; the mitochondrial quarter contains all the mitochondria and some yolk; the yolk quarter contains only yolk and the pigment quarter, all the pigment granules, and a little yolk. The sizes of the fractions obtained with a force of 10,000 x g are given in Table 9; Photographs 1-9, Plate VII, and a chart drawn from the photographs in Figure 12 (E. B. Harvey, 1932, 1936, 1940C¢, 1951). Although the size of the fractions obtained by centrifuging at 10,000 times g for 4 minutes is usually as given, there do occur batches of 130 THE AMERICAN ARBACIA TABLE 9 SIZE OF THE ARBACIA EGG AND ITS FRACTIONS IN MICRA WITH 10,000 X G E. B. Harvey, 1936 Approximate Parts Diameter Volume proportion of whole egg us per cent Whole egg (nucleate) 74 212,200 White half (nucleate) 62 124,800 58 Clear quarter (nucleate) 56 91,950 43 Mitochondrial quarter 40 33,510 15 Red half 56 91,950 43 Yolk quarter 52 73,620 35 Pigment quarter 32 17,160 8 Nucleus 11.5 796 0.4 apparently normal eggs which break differently. Sometimes the white half is much larger than normal and the red half correspondingly very small; rarely the white half is smaller than normal and the red half correspondingly large. Usually the entire batch breaks in the same way, but occasionally the eggs in one batch will break in the three different ways, without intermediates (E. B. Harvey, 1936, photos 7-9). © APT ER a9 Factors affecting stratification and breaking a. CENTRIFUGAL Force. PLATE XIII With low forces, it takes longer to break the eggs apart, and the eggs have become well stratified before breaking. The white half is much larger than the red half. With high forces obtained by the air turbine, the egg breaks quickly before it has become well stratified. The white half is much smaller than the red half. The red half can be restratified like the whole egg if recentrifuged (Plate XIII, Photograph 12). With intermediate forces, the time for breaking and the stratification is intermediate, and the white half and red half are more nearly equal in size. These results are given in Table ro and in Photographs on Plate XIII (see E. B. Harvey, 19414). b. Hypo- AND Hypertonic SEA WATER. PLATE XIV When eggs are kept and centrifuged in hypotonic sea water (60%, 80%), it takes longer to break them apart. The granules are well packed so that few remain in the white half, which is much larger than the red half. It is also much larger than the control (100% sea water), while the red half remains about the same size. When eggs are kept and centrifuged in hypertonic sea water (125%), the clear area is small, the mitochondrial layer very thick, being spread over a smaller area and in many cases well marked. The pigment is not well separated from the yolk, there being no clear line of demar- cation. It is inaccurate to speak of “‘well-stratified” eggs, since they may be well stratified with regard to one layer (mitochondria) and poorly stratified with regard to others (yolk and pigment). The white halves are much smaller than the controls, the red halves about the same size. The eggs break more readily in hypertonic than in normal sea water. These results are shown in Table 11 and on Plate XIV (see E. B. Harvey, 1943). 132 THE AMERICAN ARBACIA TABLE 10 EFFECT OF VARYING THE CENTRIFUGAL FORCE ON TIME TO BREAK AND SIZE OF HALVES E. B. Harvey, 1941a Whole egg Diameter () Volume (3) Ratio 74 212,000 (approx.) Minutes Force ‘ to break (x g) ba ¥ W E Wek 20 4,000 70 4! 180,000 36,000 Ret 4 10,000 62 56 125,000 92,000 423 I 60,000 59 59 107,000 107,000 ergs 80,000 56 62 92,000 125,000 Bt 4 100,000 4! 70 36,000 180,000 | a4 TABLE 11 SIZE OF HALVES WHEN CENTRIFUGED IN HYPO- AND HYPERTONIC SEA WATER E. B. Harvey, 1943 Whole egg White half Red half Nucleus Per Sea water Diam. Vol. Diam. Vol. Diam. Vol. Diam. Vol. cent u us u. ys u ue lu uw? broken Eggs in hypo- and hypertonic sea water, then centrifuged 10,000 X g for 4 minutes 60 % 82.4 (292,900) 70.4 (182,700) 58.0 (102,200) 16.0 (2,145) 10% 80% 74.9 (220,000) 62.1 (125,400) 56.3 (93,400) 12.8 (1,098) 20% 100 % 72.0 (195,400) 59.0 (107,500) 56.0 (91,950) 11-5 (796) 70% 125 % 66.6 (154,700) 51-7 (72,360) 53.8 (81,540) 9.6 (382) 98% Recovery in 100 per cent sea water 60 %-100 % 72.0 (195,400) 59.2 (108,600) 55.7 (90,480) 11.2 (736) 80 %-100 % 72.0 (195,400) 59.2 (108,600) 54.7 (86,170) 11.5 (796) 100 % 72.0 (195,400) 59.0 (107,500) 56.0 (91,950) 11.5 (796) 125 %-100 % 72.0 (195,400) 56.3 (93,940) 57.6 (100,060) 11.2 (736) The return of whole eggs to normal size is approximately perfect, as found also by McCutcheon, Lucké, and Hartline, 1931, p. 402. c. SINGLE SALT SOLuTIONS. PLATE XV Eggs were kept for 40 minutes in solutions of different salts isosmotic with the sea water, 0.52 M NaCl, 0.53 M KCl, 0.34 M CaCl,, 0.37 FACTORS AFFECTING STRATIFICATION AND BREAKING 133 M M¢gCl,, and centrifuged in fresh similar solutions with a measured amount of isosmotic sugar solution at the bottom of each tube to keep the eggs suspended (E. B. Harvey, 1945). It was found that they stratify with decreasing readiness (indicating increasing viscosity) in the following order: CaCl, > MgCl, > Sea water > NaCl > KCI. They break into “‘halves’” with decreasing readiness in the reverse order, those in CaCl, which stratify best break least readily. In the bivalent salts they stratify better and break less readily than in sea water, and in the monovalent salts they stratify less and break more readily than in sea water. The ease of breaking must be determined by an effect of the salts on the surface layers rather than by their effect on the interior viscosity. Heilbrunn (1923, 1928) gives a similar series for viscosity, except that Na and K are reversed. No change in size of halves was noted in the different salt solutions, but the percentage of eggs which broke into two when centrifuged at 10,000 x g for 4 minutes was as follows: KCl, 99%; NaCl, 90%; sea water, 50%; MgCl,, 20%; CaCl,, none. The rate of stratification was the reverse of the above, i.e., the eggs in CaCl, were best stratified (E. B. Harvey, 1945 and Plate XV). d. TEMPERATURE, PRESSURE, RADIATION According to Costello (1934, 1938) the amount of stratification de- creases with decrease in temperature, the eggs break less readily, and the red halves are smaller. I have obtained somewhat different results, due probably to difference in procedure. There seems no doubt, how- ever, that eggs kept for three or more hours at 8° C. stratify better and a larger percentage break into two than among those kept at 23°, when centrifuged at the same time. Moreover, eggs are usually con- siderably stratified by gravity on standing overnight at 8° C., but do not stratify at room temperature. Under increased hydrostatic pressure unfertilized eggs stratify more rapidly (Brown, 1934) and break into halves more easily (E. B. Harvey and Brown, unpub.). After treatment with x-rays or ultraviolet light the eggs break more readily (E. B. Harvey, 1940, 1950, unpub.). According to Cheney (1949a) they stratify less easily and break less readily in 0.1% caffeine than in sea water. e. FERTILIZATION When the eggs are fertilized first and then centrifuged, they stratify 134 THE AMERICAN ARBACIA like the unfertilized ones, but in not so well-defined layers (Plate XII, Photograph 15; cf. with Plate VII, Photograph 1). As is well known, they become more viscous on fertilization (Heilbrunn, 1915, 1920a, 1928, p. 264; E. B. Harvey, 1932, 1933b; Goldforb, 1935b). Except for a short period immediately after fertilization, they break apart into white and red halves like the unfertilized eggs; these are usually of the same relative size as the unfertilized halves, the white half a little larger than the red, except if centrifuged just before cleavage when the white half is quite small. If centrifuged one or two minutes after fertilization, the eggs break into very small pieces (E. B. Harvey, 1933b, 1940b) (Plate XII, Photograph 14). The fertilization membrane must be removed by vigorous shaking just after it is formed, in order to allow the eggs to elongate and break. If centrifuged during the monaster stage, 6-20 minutes after fertiliza- tion, the eggs form long streamers which retract into spheres on re- moval from the centrifuge. This is particularly noticeable with low forces and readily observed with the centrifuge microscope (E. B. Harvey, 1933b ) (Plate XII, Photograph 17). If centrifuged slowly as the fertilization membrane is forming, this may be greatly stretched to a length of 128 uw from a diameter of 80 uw, and the egg may break into two parts inside the stretched membrane. If the force is removed, the two parts may coalesce or may remain and develop separately; in the latter case, the female nucleus may be in the white half and the male nucleus in the red half; the red sphere with the male nucleus usually develops better than the white sphere with the female nucleus. When eggs are centrifuged after fertilization, the hyaline layer is thrown off as a ring or crescent into the perivitelline space. This is much more striking in eggs with a large perivitelline space, e.g., Psammechinus microtuberculatus, and is observed better in Arbacia punctul- ata if the fertilization membrane is broken (E. B. Harvey, 1934) (Plate XVI, Photograph 6). The hyaline layer is later replaced, and if the fertilization membrane remains intact, the egg develops normally. CHAP LER’ 20 Properties of egg fractions Egg fractions have been used to analyze the effect of ultraviolet light and x-rays, etc. on the egg cell (see Part IV); quite a number of studies have been made of the chemical and physical properties of the half-eggs in order to evaluate the part which the various granular layers may play in the life of the cell. Some of the results are given in condensed form below. Consistency. - When manipulated with a needle the red halves are found to be sticky and glutinous and can be pulled out in strands, while the white halves explode if punctured (E. B. Harvey, 1932; see Chambers, 1938a). The fertilization membrane which forms on the white half is much thinner than that on the red half. Density. — White half, 1.076; red half > 1.1 when obtained with 10,000 x g. Whole egg without jelly 1.084. (E. N. Harvey, 1932). Osmotic Behavior. — Closely parallels the whole egg. The value for osmotically inert material b is twice as great in the red half as in the white half, but computation of b magnifies experimental error and the difference in white and red halves may not be as great (Lucké, 1932). Actual values for the volume change in hypo- and hypertonic sea water are given in Table 12. Water enters white halves several times more rapidly after fertilization than before (Shapiro, 19394). Electrical Properties. — The electrical capacity of the white half and red half is the same as that of the whole egg, about 1 microfarad per cm?, On fertilization, the capacity of whole eggs and white halves in- creases 3—4 times (Cole and Curtis, 1938; Cole, 1941). The details are given in Part IV. The internal resistance of whole eggs and white halves, fertilized and unfertilized, is about the same, 180 ohms per cm’, but the internal resistance of red halves (unfertilized) is 3 to 6 times that of the whole egg (Cole and Curtis, 1938; Cole, 1941). The details are given in Part IV. The surface charge (zeta potential) of whole eggs is —30.8 + 0.54 millivolts; white halves, —20.9 + 0.69 millivolts; red halves, —27.6 + 0.35 millivolts (Dan, 1936, IV). 136 THE AMERICAN ARBACIA TABLE 12 OSMOTIC BEHAVIOR OF FRACTIONS E. B. Harvey, 1943 Whole Egg White Half Red Half Sea Wat : F ; oars mini u vol. u3 diam. pu vol. u3 diam. u vol. u3 Eggs centrifuged in sea water, then placed in hypo-hypertonic sea water 190 %- 60% 82.4 (292,900) 67.4 (160,300) 63.4 (133,400) 100 %- 80% 74.9 (220,000) 61.8 (123,600) 57.2 (97,990) 100 7, —_72.0 (195,400) 59.0 (107,500) 56.0 (91,950) 100 %-125% 66.6 (154,700) 54-4 (84,300) 51.2 (70,300) Lucké’s (1932) mean values for above 100 %- 60% (84.6) 317,380 (69.5) 175,560 (63.9) 136,700 100 %- 70% (80.6) 274,020 (66.3) 152,320 (61.7) 123,060 100% (72.2) 197,440 (59.2) 108,600 (55-7) 90,680 Only the volumes are given by Lucké; the diameters are calculated from the volumes. Lucké found the swelling of whole eggs and fractions completely reversible. Respiration. — ‘The white half consumes oxygen at about the same rate (0.135 cm3/hour/cm?® eggs) as the whole egg, while the red half has about twice that rate. On fertilization the rate for whole eggs and white halves increases about 2.5 times, while the red halves increase 1.2 times (Shapiro, 19356, 1939b). See Part IV for details. Addition of para- phenylene diamine to white and red halves causes a greater oxygen uptake by the white halves (Boell, Chambers, Glancy, and Stern, 1940). KCN does not affect oxygen uptake of white halves but decreases oxygen consumption of red halves and of both white and red halves after fertilization (Shapiro, 1940). Enzymes. — Peptidase is found mostly in the white half localized in the matrix; ratio of peptidase activity of white: red halves = 3.4 (Holter, 1936, 1949). Indophenol (cytochrome) oxidase is more abundant in the red half; ratio of activity white: red halves = 0.5 (Navez and E. B. Harvey, 1935). See Hutchens, Kopac, and Krahl (1942) for association with matrix in disintegrated eggs (see Part IV). Dehydrogenases (measured by reduction of ferricyanide) are in greater concentration” in the red half. Activity ratio of white to red halves 45/55. (Ballentine, 1940c). PROPERTIES OF EGG FRACTIONS 137 Desoxyribonuclease (DNase) activity of white halves is greater than red but not per unit volume. (Mazia, Blumenthal, and Benson, 1948; Mazia, 19494, b). Phosphatase activity (acid or alkaline) is not restricted to white halves (Mazia, Blumenthal, and Benson, 1948). For distribution of peptidase and catalase in fractions of other eggs (Paracentrotus lividus, Psammechinus miliaris, Echinarachnius, Dendraster) see Holter and Linderstrom-Lang (1940) and Holter (1949). Lipids. — 75% of free fats and sterols, 56°% of bound lipids, and 61.6% of total lipids are present in the red halves (Hunter and Parpart, 1946). CVHDA PVT E Roi Development of centrifuged eggs and fractions a. SPHERICAL CENTRIFUGED Ecos. PLATE VIII If the eggs of Arbacia are centrifuged with a low force (e.g., 3,000 times g for two minutes), they stratify but do not elongate or break apart; they remain spherical. When these spherical eggs are fertilized, the cleavages are quite normal (E. B. Harvey, 1932, 1940c, 1951). The first cleavage usually (but not always) comes through the axis estab- lished by centrifuging, at right angles to the stratification; the second cleavage is at right angles to the first and parallel to the stratification, and the third at right angles to the first two and to the stratification. The micromeres may come off anywhere without regard to the strati- fication, but approximately opposite the micropyle, which marks the original axis of the egg (Plate VIII, Photographs 15, 16). Plutei developed from these eggs, normal in every respect except that the pigment was localized somewhere, in any part of the larva. These facts were established by the early work of Lyon (1g06a, 1907) and Morgan and Lyon (1907). Further studies with regard to polarity and develop- ment were made by Morgan (1908, 1909), Morgan and Spooner (1909), and Spooner (ig11), and I can confirm them. A general sum- mary of cleavage in these eggs and in egg fractions will be found in the paper of E. B. Harvey (1951). A resumé and able discussion of polarity and centrifuged eggs is given in Morgan’s Experimental Embry- olozy, 1927, p. 496-500. b. ELONGATE CENTRIFUGED Ecos. PLATE VIII As mentioned, the elongate eggs when unfertilized resume their spher- ical shape within a few hours. If, however, they are fertilized immedi- ately in sea water they retain their elongate or dumbbell shape (E. B. Harvey, 1932, 1940c, 1951). The fertilization membrane follows the contour of the egg, but there is usually left a slight bulge of the mem- brane at the centripetal pole, owing to a slight shrinking of the egg immediately after the fertilization membrane has formed. There must be a gelation or setting of the protoplasm following fertilization, for DEVELOPMENT OF CENTRIFUGED EGGS AND FRACTIONS 139 if the membranes are removed by drawing up the eggs in a fine capil- lary, the eggs retain their aspherical shape (and develop). Cytological details can be observed in the living elongate egg: the sperm aster, union of the pronuclei, enlargement and breaking of the nucleus, cleavage spindle, can all be observed, except that astral radiations cannot be seen unless there are granules. The spindle forms in the long axis of the egg where there is room for it. The first cleavage plane almost always comes across the short axis of the egg, parallel with the stratification but not at the narrowest part, dividing the egg into a smaller clear cell and a larger granular cell. This is in contrast to the first cleavage of the spherical centrifuged egg described, where the first cleavage plane is usually perpendicular to the stratification. The shape of the egg determines the position of the first cleavage plane. The second cleavage plane usually comes in perpendicular to the first in the clear cell, and either perpendicular or parallel with the first in the granular cell. The second and following cleavages are asyn- chronous, the smaller clearer cells dividing more rapidly than the larger pigmented cells. A slipper-shaped blastula is formed with un- equal-sized cells. The pigmented area remains distinct through the cleavages and in the early pluteus as in the spherical egg. . Micromeres may be formed in any region of the elongate egg, even at the oil cap (E. B. H.) (Plate VIII, Photographs 5, 6). They probably form in relation to the original axis of the egg. They may be pigmented but usually are not. c. Wuire Hatr-Eccs. PLATE IX The white half-egg which has been allowed to stand in sea water after centrifuging, and become spherical, develops after fertilization, exactly like the whole uncentrifuged egg cleaving at the same rate, sometimes a little faster (E. B. Harvey, 1932, 1940¢, 1951). The nuclear phenomena accompanying fertilization can be observed in the living egg with great clearness, and they parallel those described by Wilson (1895, The Atlas of Fertilization) in the unpigmented egg of Lytechinus ( Toxo- pneustes) variegatus. In the clear, nongranular portion of the egg, how- ever, no astral rays can be seen. The first cleavage plane usually comes in through the oil cap, but may be parallel with the stratification, or at an angle. Following cleavages come in quite normally, but micro- meres have not been observed. Blastulae are often formed, normal except for size and color, and these may develop into plutei with well developed arms and normal skeleton but more slowly. In many of the cultures, however, the white halves develop abnormally, and especially 140 THE AMERICAN ARBACIA characteristic are the permanent blastulae with imperfect skeleton, the ‘“‘Dauerblastulae’’. There seems to be no constant percentage of normal development in any one batch of eggs, but certain whole batches develop better than others. Pigment granules characteristic of normal plutei appear in the white plutei after a few days. White half-eggs which are fertilized while still elongate after centri- fuging develop like the elongate whole egg. The first cleavage plane goes across the short axis and divides the egg unequally. By subsequent cleavages, a white slipper-shaped blastula is formed. White half-eggs will also develop parthenogenetically if treated with concentrated sea water, and develop into white plutei. d. Rep Hatr-Ecocs, FERTILIZED MEROGONES. PLATE X The red half-egg has no nucleus at the time of fertilization, but after fertilization the sperm nucleus with its accompanying aster may often be seen 15-20 minutes after the fertilization membrane has been given off. The fertilization membrane and the hyaline layer are much thicker than in the white half-eggs. The sperm nucleus is at first very small, but enlarges and is quite noticeable about 30 minutes after fertilization. The radiations disappear, the nucleus enlarges and breaks down about an hour after fertilization. A dumbbell-shaped nucleus or a very small amphiaster can sometimes be seen in the living egg, and then two nuclei (about 80 minutes after fertilization). In stained preparations one sees a very narrow spindle in metaphase and anaphase with chro- mosomes, and coarse astral fibers (Plate XVI, Photographs 12, 15). A two-celled stage may occur, especially in eggs fertilized while elongate, but often cell boundaries fail to come in. The first cleavage plane in the elongate egg comes across the short axis of the egg. Sometimes fairly regular cleavages result in a blastula of many cells with a small blastocoel, but often the blastula is multinucleate without cell bound- aries. Hatching has been observed 11 hours after fertilization, and they become active swimmers. Several perfectly normal plutei with lattice-like skeletons have been raised; they were very heavily pig- mented, and of course much smaller than those from whole eggs (see E. B. Harvey, 1932, 1940Cc, 1951). (Plate X, Photograph 16). e. Rep Ha.tr-Eccs, UNFERTILIZED. PARTHENOGENETIC MEROGONES. PLATE XI The red half-eggs, though having no nucleus, can be activated by a parthenogenetic agent, hypertonic sea water or distilled water. They DEVELOPMENT OF CENTRIFUGED EGGS AND FRACTIONS I4I develop quite like the fertilized red half. They develop best if activated just after centrifuging while still elongate, but spherical ones can also be activated. The fertilization membrane and hyaline layer are thick, as in the fertilized red halves. A clear sphere resembling a nucleus is often present 20 minutes after activation; it is difficult to say whether there is a membrane around it, but there seems to bea phase bound- ary. The monaster which is often very conspicuous, arises near the ‘““pseudonucleus.” The monaster stage is followed by an amphiaster, and a cleavage plane may come in between the two asters in typical fashion. It seems curious that there should be a monaster stage pre- ceeding the amphiaster when there is no nucleus. The first cleavage plane may divide the egg equally, in any relation to the stratification though more often parallel with it. In elongate eggs the first cleavage plane comes across the short axis as in other types of elongate eggs and half-eggs. Successive cleavages may be fairly regular and multicellular organisms ofsome 500 cells have been obtained. Often cell walls fail to come in, and in later stages the egg is peppered with asters and small spheres which resemble nuclei. Development is very slow and they hatch only after 24 hours, whereas the whole egg hat- ches in 8 to g hours; the fertilized red half hatches in 11 hours. No further development has been obtained; there is apparently no growth or differentiation without nuclear material, though these organisms have lived for a month; unfertilized eggs live at room temperature only a day or two. Many substances have been added to the sea water in an effort to get further development of the parthenogenetic merogones without success: killed Arbacia sperm, living frog nuclei, nuclei acid, phage, auxin, adenine, guanine, uracil. In stained sections there are of course no chromosomes, and there are no spindles, but there are large asters arranged in pairs with very coarse rays (Plate XVI, Photograph 13). The Feulgen reaction, specific for chromatin, was negative for the parthenogenetic merogones; there was no red staining material, whereas the fertilized merogones prepared in the same way at the same time showed it very clearly. A comparison of the blastula of a fertilized merogone and a parthenogenetic mero- gone in aceto-carmine preparations shows very clearly the presence of chromosomes in the former, and their absence in the latter (see E. B. Harvey, 1936, 1938a, 1940b, c, 1951, Figs. 72, 73). Compare photographs on Plate XI with corresponding ones on Plate X to see how similar is the development of parthenogenetic mero- gones and fertilized merogones up to a certain stage. 142 THE AMERICAN ARBACIA f. CLEAR QUARTER. PLATE XII The clear quarter can be obtained by centrifuging the white half-egg for 20-30 minutes at 10,000 Xx g; to break the whole egg into halves takes only about 4 minutes. It is best to remove the white halves from the first centrifuging and recentrifuge in a fresh graded sugar-sea water mixture of the same density as the halves. The clear quarters are easy to distinguish from the white halves in the one and two-cell stages, but later it is difficult to distinguish them; they should therefore be segre- gated from the white halves. The clear quarter has an oil cap but no granules; it contains the bulk of the ground substance or matrix (about two-thirds). The total liquid in the egg has been computed as 61.1% of the total volume (E. N. Harvey, 1932a). The clear quarter is about as near pure protoplasm, without granular inclusions, as it is possible to obtain in the living condition. Although the clear quarter-egg is, in the living condition, free of the usual granules, mitochondria, yolk, and pigment, one can observe a trace of granules appearing as a fine line across the egg, somewhat below the oil cap, if examined with high magnification (700 x) just after centrifuging. This line separates the clear area into two distinct portions. Sometimes small fibers are observ- able among the granules (E. B. Harvey, 1946a). These two portions of the clear layer stain with different intensity with many vital dyes (E. B. Harvey, 1941c). They have been studied by McCulloch (19524), and the fibers studied with the electron microscope. In order to get any development, it is necessary to allow the clear quarters to stand for an hour or two before they are fertilized. Other- wise the fertilization membrane is not formed or is so thin that it rup- tures. The stages following fertilization are exactly like those of a normal egg except that the fertilization membrane is thin, the perivitel- line space small, and the astral fibers are not visible because of lack of granules. The whole process is slower. A normal cleavage may take place in 2 hours instead of 50 minutes, which is the time for the whole egg and white half. The cleavage plane may be in any relation to the oil cap, so that one of the blastomeres contains all, none, or any portion of the oil. The second cleavage, resulting in four equal cells, usually takes place in about 3 hours instead of 14 hours as in the nor- mal egg. Successive cleavages follow, all delayed, till a normal blastula is formed. The blastula hatches from the fertilization membrane and becomes free-swimming in about 20 hours or longer (instead of 8 hours). These blastulae are much less granular than those f.om white halves. Some of these clear quarter blastulae have formed normal gastrulae DEVELOPMENT OF CENTRIFUGED EGGS AND FRACTIONS 143 and have developed into small normal plutei with short arms after about three days, with normal Arbacia skeleton with a spiny base and lattice-like arm skeleton but with short arms (Plate XII, Photograph 13). After about four days, pigment spots appear. These must be a new formation in both the clear quarter and white-half plutei, since the egg brought in no pigment. Many of the clear quarters develop abnormally as loose clusters of cells caused by the thin hyaline layer, and the nuclei often become very large, three times the normal dia- meter. Many of the plutei are abnormal with abnormal skeleton. There are no mitochondrial granules in the clear quarter-egg, and none are brought in by the sperm in any appreciable amount, as shown by staining with methyl green. The blastulae also have none, but the plutei have them, localized especially in the cells around the gut, as they are in plutei from whole eggs and white halves. These mitochon- dria must be a new formation, just as are the pigment granules which appear in the pluteus from the white half-egg. The slow development of the clear quarter, though having two nuclei, argues against Whitaker’s (1929) idea that the ratio of the amount of nuclear and cytoplasmic material is the determining factor in cleavage rate. When treated with a parthenogenetic agent, hypertonic sea water, a fertilization membrane may be formed, and early nuclear changes may occur, but no pluteus is formed as in the case of the white half. g. MirocHONDRIAL (GRANULAR) QUARTER The mitochondrial or granular quarter (lower part of the white half) contains all the mitochondria and some yolk and no nucleus. It may cleave after fertilization quite regularly, and typical 2, 4, 16, etc. cell stages have been obtained, but usually cleavage planes fail to come in, so that multi-astral and multinucleate cells are common. The devel- opment is a little slower than in normal eggs. Swimming blastulae have been formed in g hours, but no skeleton and no plutei. h. YOLK QUARTER The yolk quarter (upper part of the red half) contains only yolk granules and no nucleus. It develops when fertilized, like the mitochon- drial quarter with sometimes regular cleavages and sometimes it is multinucleate, lacking cleavage planes. It develops further, however, forming plutei with irregular skeletons and pigment, but no perfect 144 THE AMERICAN ARBACIA plutei have been obtained. There is more of the ground substance in these quarters than in the mitochondrial quarters, as may be ascer- tained by the clear layer often seen at the centripetal pole when the red half is recentrifuged (Plate XIII, Photograph 5). This quarter is also considerably larger than the mitochondrial quarter. i. PIGMENT QUARTER The pigment quarter, containing all the pigment and a little yolk, is very much smaller than any of the other fractions, only one twelfth the volume of the whole egg. It develops after fertilization like the other granular quarters, usually with multinucleate cells, though some regular 2-, 4-, 8-, 16 cell stages have been found. The development is very much delayed. Some swimmers were found after a day, but there was no further development. It is of interest that Morgan (1893) found that fragments of the Arbacia egg 1/70 the volume of the egg would cleave. These fragments were obtained by violent shaking of the whole eggs. According to Tennent, Taylor, and Whitaker (1929) for Lytechinus variegatus, fragments 1/50 the volume of the whole egg could not be fertilized, fragments 1/35 the volume could be fertilized but segmented irregularly, 1/24 gave regular cleavage, 1/17 formed blastulae with mesenchyme, 1/10 formed gastrulae and fragments about 1/4 the volume of the whole egg formed plutei. j. Eccs FERTILIZED, THEN CENTRIFUGED When the whole egg is fertilized and then centrifuged (but not elongat- ed) at any stage after fertilization, it may develop quite normally, and give rise to plutei normal in every respect except for pigmentation. The first cleavage plane usually passes through the oil cap, perpendicul- ar to the stratification but may be parallel with it (E. B. Harvey, 1934). If centrifuged after the spindle has formed, this is thrown to the centripetal pole, and the cleavage plane comes in through the oil. By observing with the centrifuge microscope, one can see the cleavage plane coming in during rotation at 6,000 x g (E. B. Harvey, 1933b). Spooner (1909) obtained the same percent of development, no matter at what stage after fertilization the eggs were centrifuged. When the fertilized eggs are broken apart after fertilization, at any stage, the white half, containing the combined ¢ and @ nuclei, may cleave and develop quite normally through the blastula stage. A few acquire a skeleton and later, pigment, and may become very abnormal DEVELOPMENT OF CENTRIFUGED EGGS AND FRACTIONS 145 plutei, but no normal plutei have been obtained (Plate XII, Photo- graph 16). They usually remain ““Dauerblastulae.’’ Often the first two white blastomeres develop independently forming twins; this is doubt- less due to the lack of the hyaline layer which is thrown off by centri- fugal force as a ring or crescent. This can be readily seen in the perivi- telline space if the fertilization membrane is ruptured (Plate XVI, Photograph 6). The red half does not develop. This is in marked con- trast to the red half obtained from unfertilized eggs which may cleave quite normally after parthenogenetic treatment, though having no nucleus or nuclear material. The red half from the fertilized egg has nevertheless been in contact with nuclear material. Parthenogenetic agents do not have any effect on this red half. It is onlyif the fertilized egg is broken apart before the g and 2 nuclei have united, that the red half may divide, owing to the presence of the j nucleus (E. B. Harvey, 1933b, 1940b). k. CONCLUSIONS An artificial distribution of granules by centrifugal force does not radi- cally change the course of development after the egg is fertilized. No special granules are necessary for cleavage and development (oil, yolk, pigment, and mitochondria), since any fraction may develop without one or more of these, and the clear quarter, which lacks all of them (except oil), may develop into a normal pluteus. As for the nuclei, it is apparent that the male nucleus is not necessary for cleavage and development, since parthenogenetic development takes place in many forms, and, in Arbacia results in a perfect pluteus in both the whole eggs and the white halves. The female nucleus is not necessary, since merogonic development of fertilized non-nucleate fragments takes place in many forms and may result in a perfect pluteus in the red halves of Arbacia. It has been shown that cleavage and early development may take place, in the parthenogenetic merogones, without any nucleus at all. The essential material for cleavage and early development, therefore, must be the matrix or clear material which is present in all the frac- tions. In the living state, this is optically empty, except for the thin line of granules segregated out under high centrifugal forces. When fixed and stained with haematoxylin, the matrix appears filled with very small granules, microsomes (Lyon, 1907, E. B. Harvey, 1940¢, Figure 124). It contains nucleoproteins, as shown by ultraviolet of a wave length of 2537 A (E. B. Harvey and Lavin, 1944). It contains the 146 THE AMERICAN ARBACIA greater part of the enzyme peptidase, as determined by Holter (1936), and probably other enzymes. It can be separated by centrifugal force into a lighter portion and a heavier portion, which react differently to various vital dyes (E. B. Harvey, 1941 c). The ordinary granules must have some importance and may be used in early development, but they are not essential and can be replaced. At least one nucleus seems to be necessary for differentiation and complete development, but the early stages of development involving cell multiplication can take place without any nucleus. PART TV COMPILATION OF EXPERIMENTAL WORK ARRANGED ALPHABETICALLY* * Statements always refer to Arbacia punctulata unless otherwise noted. Preliminary papers are not listed when followed by a complete paper. COMPILATION OF EXPERIMENTAL WORK AGEING OF EGGS Size.—Increase in volume with age (Goldforb, 1918a, b, 1935a; Smith and Clowes, 1924b). Decrease when still older (Goldforb, 1935 a). Shape.—More globular with age (Goldforb, 1935a). Jelly Layer —Disappears (Goldforb, 1918a, b, and many others). Vitelline Membrane.—Stretches more and bursts more rapidly in hypotonic sea water, with age (Goldforb and Schechter, 1932, Goldforb, 1937). Fertilization Membrane.—Retarded or lacking and closer to the egg, none after 36-52 hours at 20-22° (E. N. Harvey, 1g10b, 1914; Goldforb, 1918a, b; Tyler, Ricci, and Horowitz, 1938). Longevity.—Of unfertilized egg is indicated by ability to form fertilization mem- brane, i.e. 36 to 52 hours after shedding (See above). Longevity can be increased by (1) sterile conditions (Gorham and Tower, 1902; Tyler et al., 1938, to 10 days) ; (2) KCN (Loeb and Lewis, 1902, to 7 days; Loeb, 1911, 1913 a, p. 26); (3) 2% alcohol (Tyler et al., 1938, to double the time) ; (4) chloral hydrate (Loeb, 1913 a, p. 91); (5) dinitro-o-cresol (Clowes and Krahl, 1936a); (6) acid sea water of pH 5.8 to 6.0 (Smith and Clowes, 1924b) ; (7) low calcium content of sea water (Schechter, 1937) ; (8) anaerobiosis (Loeb and Lewis, 1902; Loeb, 1911, 1913 a, p. 26, slight increase). See Cyanides. Polyspermy.—Increases with age (Goldforb, 1918b; Smith and Clowes, 1924b). Oil Coalescence.—Increases (Chambers and Kopac, 1937,, Kopac and Chambers, 1937): Cytolysis—Increases with age (Goldforb, 1918a, b; Page, 19294). Agglutination—And fusion of eggs (Goldforb, 1918a, b, 1929b, c). Separation of Blastomeres.—(Goldforb, 1918b). Cleavage.—Slower, more irregular and fewer eggs cleave (Goldforb, 1918a, b; Smith and Clowes, 1924b). Respiration Increase (Wasteneys, 1916; Gerard and Rubenstein, 1934; Tyler, Ricci, and Horowitz, 1936, due to bacteria). Permeability!.—Increase (Goldforb and Schechter, 1932; Goldforb, 1935c). Viscosity.—Increase for 35 hours, then decrease (Goldforb, 1935b). Breaking with Centrifugal Force.—Less readily (Shapiro, 1935b; E. B. H. unpub.). Ageing of Eggs in Animal.—Due to lateness of season or to keeping animals in aquaria one to three months. Cleavage takes longer, irrespective of temperature; eggs are more viscous, take longer to stratify; break less readily with centrifugal force; some red granules remain in white half after centrifuging (E. B. Harvey, 1939b). Oxygen consumption is reduced (Shapiro, 1935c). Amount of carbohydrate and phosphorus is greater, and of nitrogen is less (Hutchens, Keltch, Krahl, and Clowes, 1942; Crane, 1947). Eggs lose pigment with time and become pale (E. B. H.). AGGLUTININ see Fertilizin AMOEBOCYTES (ELAEOCYTES) (See Chromatophores, Echinochrome, Perivisceral Fluid) Definition —Amoebocytes are small amoeboid cells in the perivisceral fluid, white and red, the red ones being filled with chromatophores containing echinochrome (Plate XVI, Photograph 5). 1 Permeability means permeability to water throughout this Compilation unless other- wise indicated. 150 ALPHABETICAL COMPILATION Historical—Observed by Valentin in 1841 (published in one of the Monographies d’Echinodermes of L. Agassiz) in Echinus lividus (Paracentrotus lividus) ; by Williams (1852) in E. spaera (E. esculentus) ; by Geddes (1880) and Gamgee (1880) in Arbacia lixula; by MacMunn (1883, 1885) in P. lividus; by Cuénot (1891a, b) in many species; et al. In Arbacia punctulata, McClendon (1912a) and McClendon and Mit- chell (1912) et al., refer to them as “‘elaeocytes.’’ This term really means oil or fat cells, and was originally applied to yellowish lymphocytes, not amoeboid, containing fat, in the coelomic fluid of certain oligochaetes (Rosa, 1896). Occurrence.—In Perivisceral Fluid, q.v., (Mathews, 1900; McClendon, 1g1ob, 1912a, McClendon and Mitchell, 1912; Kindred, 1921, 1926; H. V. Wilson, 1924; Donnellon, 1938; et al.). Throughout body, especially around testes and ovaries (E. B. H. unpub.). Color.—Red and white amoebocytes occur in about equal numbers. The red ones owe their color to Echinochrome, present in Chromatophores, q.v. Amount of Echinochrome.—In amoebocytes: 3.78 gm. per 100 cc. of packed volume of body cells; the eggs contain on the average 0.58 gm. of pigment per 100 cc. of eggs packed by centrifuging (Ball and Cooper, 1949). Pigment Released.—By water, tissue extracts, salts especially potassium, hypertonic solutions, fat solvents, mechanical and electrical stimulation, ultraviolet light, cold, heat; but it is not released if the body fluid is oxalated or citrated, under most of these conditions (Donnellon, 1938). Release by electric current was earlier observed by McClendon (1910b). Colorless Amoebocvtes.—Of body fluid swell and dissolve if NaCl, KCl or MgCl, are added to the sea water (Mathews, 1900). Shape.—Cylindrical or spherical. Decidedly amoeboid. Become spherical on addi- tion of water and with ultraviolet light and x-rays (E. B. H. unpub.), and with hydrostatic pressure (Marsland, 1938). Size.—Cylinders average 30 mw long by 7.3 uw diameter; volume 1256 y?. Spheres average 13.3 « diameter; volume 1232 yw? (E. B. H. unpub.). Stain.—Colorless amoebocytes are basophilic, red ones acidophilic (Kindred, 1926). Function.—Not certain. Red ones may be respiratory (Geddes, 1880; Gamgee, 1880; MacMunn, 1885; in other species). See under Echinochrome. Cause clotting of perivisceral fluid (Heilbrunn, 1928, p. 228; Donnellon, 1938). Fertilization.—Inhibited by amoebocytes (Pequegnat, 1948), probably due to echinochrome (Couillard, 1952). Other Species (additional) Davidson, 1953. Echinarachnius parma. Kindred, 1924. Strongylocentrotus drébachiensis, S. franciscanus, Echinarachnius excentricus. AMOEBOID EGGS Amoeboid Eggs.—These are caused by Standing overnight (E. B. H. unpub.). Urea (R. S. Lillie, 1903; Moser, 1940; Kitching and Moser, 1940; A. R. Moore, 1929, in S. purpuratus). Sucrose (Kitching and Moser, 1940). Ethyl] urethane, 0.4 M for 1 hr. (E. B. H. per E. N. Harvey, 1933). KCl (Churney, 1940). MgCl, (Loeb, 1g00a; Churney, 1940). Electric current, at anode (McClendon, 1910b). Photodynamic action, light + rose bengal or eosin (Alsup, 1941). OF EXPERIMENTAL WORK I5I Trypsin, in Dendraster excentricus (A. R. Moore, 1951 a). Hypertonic sea water, in Ps. microtuberculatus (E. B. Harvey, 1938a). Clear Quarter-Eggs.—Fertilized, become amoeboid if not cleaved (E. B. Harvey, 1946). Clear quarter-eggs and white half-eggs, made parthenogenetic with ultra- violet light, become amoeboid (E. B. Harvey and Hollaender, 1938). Arrest of Amoeboid Movement.—By high hydrostatic pressure on urea-treated eggs. By lack of oxygen (Kitching and Moser, 1940). Other Species (additional) Dendraster excentricus with trypsin (A. R. Moore, 1951 a). Paracentrotus lividus and Ps. microtuberculatus, upper half (with nucleus) of centrifuged egg (E. B. Harvey, 1934, p- 239)- Sphaerechinus granularis, whole centrifuged egg and lower (non-nucleate) half of centrifuged egg (E. B. Harvey, 1938a, p. 184). ANAEROBIOSIS See Oxygen-Lack ANAESTHETICS (NARCOTICS) Effect on Size of Egg.—Volume increase in 3 % ether (Heilbrunn, 1928, p. 203); in chloroform (Heilbrunn, 19154). Effect on Shape of Egg.—Eggs become amoeboid if kept in 0.2 M ethy] urethane for one hour (E. B. H. per E. N. Harvey, 1933). Elongate, centrifuged eggs also become amoeboid. These can be fertilized, the fertilization membrane following the irregular contour; they cleave in the irregular shape on return to sea water. If not fertilized the amoeboid eggs become spherical on return to sea water (E. B. H., 1931 unpub.). Fertilization —Eggs can be fertilized in alcohols, but do not cleave; cannot be fertilized in ethyl acetate though sperm surround the eggs (Blumenthal, 1928). Can be fertilized in 0.15 M ethyl urethane, sometimes forming a fertilization membrane (E. B. H.). Surface Precipitation ReactionMay be prevented by anaesthetics (Heilbrunn, 1934). Cleavage.—Effective concentrations of many anaesthetics for reversible arrest of cleavage are given in Table 13, from R. S. Lillie (1914b). Effective concentrations of some urethanes for reversible arrest of cleavage are given in Table 14 (E. B. H. per E. N. Harvey, 19324). Mitotic Figure.—Anaesthetics (many) may prevent formation of spindle and cause rays to disappear (Heilbrunn, 1920a, b). Urethanes cause astral eays to fade out and cleavage planes to disappear (Painter, 1918; E. B. H., 1930 unpub.). When placed in 0.2 M ethyl urethane at metaphase, cleavage planes fade out and when returned to sea water are replaced irregularly, but normal blastulae may be formed (E. B. H.). These phenomena are similar to those described and figured by me (1927, 1930) for eggs kept without oxygen. A similar disappearance of cleavage planes also is caused by high hydrostatic pressures (Marsland, 1938, 1939, 1950, 1951) and in Ps. microtuberculatus by mechanical pressure (Boveri, 1897). Disappear- ance of astral rays by various agents was first found by Q. and R. Hertwig in 1887 in Paracentrotus lividus, and very carefully studied by E. B. Wilson (1901b) in ether- ized eggs of Toxopneustes (Lytechinus) variegatus; he also studied obliteration of cleavage furrows. Monasters formed with chloral hydrate were described in P. lividus by O. and R. Hertwig (1887); in Lytechinus in etherised eggs (E. B. Wilson, 1g01b); in P. lividus and A. punctulata with phenyl urethane (Painter, 1915, 1916, 1918). 152 ALPHABETICAL COMPILATION TABLE 13 EFFECTIVE CONCENTRATIONS OF ANAESTHETICS TO ARREST CLEAVAGE OF ARBACIA PUNCTULATA EGGS From R. S. Lillie 1914b, 7. Biol. Chem. 17 : 139 Ethyl alcohol ca. 5 v. % (0.87 M) n-Propyl alcohol ca.2v. % (0.27 M) Iso-propyl alcohol C42 3°V. 7, (0.4) Mi) n-Butyl alcohol ca. 0.8 v. % (0.086 M) i-Amyl alcohol ca. 0.4 v. % (0.037 M) Capryl alcohol ca. 0.015 v. % (0.001 M) Methyl urethane 2-2.5 % (0.27—-0.33 M) Ethyl urethane 1.5-1.75 % (0.15-0.19 M) Phenyl urethane 0.08-0.1 % (0.005—0.006 M) Ethyl ether 0.5-0.6 v. % (0.05-0.06 M) Chloroform ca. 0.06 % (1/1. sat.) (0.005 M) Chloral hydrate 0.1I-0.12 % (0.006—0.007 M) Chloretone 0.2-0.25 % (0.008-0.01 M) Nitromethane ca. 2. v. % (0.42 M): unfavorable Acetonitrile ca. 2. v. % (0.5 M): unfavorable Ethyl] nitrate ca. 0.25 v. % (0.025 M): unfavorable Paraldehyde 2-3 v. % (0.15-0.2 M): unfavorable Chloralose ineffective in sat. sol. (ca. 0.6 %) Acetanilide ineffective in sat. sol. (ca. 0.5 %) Phenyl urea ineffective in sat. sol. (ca. 0.5 %) TABLE 14 CRITICAL ANAESTHETIC CONCENTRATIONS OF URETHANES FOR REVERSIBLY SUPPRESSING CLEAVAGE IN ARBACIA PUNCTULATA EGGS From E. B. Harvey per E. N. Harvey, 1932a, Biol. Bull. 52 : 151 Ethyl] urethane 0.15-0.2 M N-propyl urethane 0.07 M Isopropyl urethanes o.1 M N-butyl carbamate 0.025 M Isoamyl carbamate 0.01 M Phenyl] urethane 0.00125—0.0025 M Spiral asters formed in Paracentrotus lividus with phenyl urethane (Painter, 1916) ; with KCN (Runnstrém, 1930, p. 155). Spiral asters formed with high centrifugal force in Psammechinus microtuberculatus (E. B. Harvey, 1935a). See classical study of Mark (1881) on spiral asters normally occuring in Limax campestris. Rhythms.—In mitotic cycle, of sensitivity to anaesthetics. With ether (Spaulding, 1904); higher alcohols (Baldwin, 1920); many anaesthetics (Heilbrunn, 19204). Gastrulation.—Effect of alcohols (Waterman, 1936). Ciliary Activity—Of blastulae and plutei stopped by chloretone (chlorobutanol) ; use a small amount of saturated solution in sea water added to swimming larvae (E. B. H.). By magnesium sulphate (E. B. H.) ; widely used to quiet marine organisms (Mayor, 1909; R. S. Lillie, 1916c; Heilbrunn, 1920b, 1934, 1952, p. 528); mag- nesium anaesthesia antagonised by calcium (Heilbrunn, 1952, p. 589). OF EXPERIMENTAL WORK 153 Recovery from Anaesthetics.—Unfertilized eggs may be kept two hours in optimum concentration of any urethane listed in Table 14 and recover in sea water immediately ; fertilized eggs undergo slight development while in urethane and time of first cleavage on recovery is independent of duration of exposure, 40 minutes to two hours in ethyl urethane (E. B. H., 1930 unpub.). Similar results for alcohols (Blumen- thal, 1928). Differential susceptibility and recovery (with ethyl alcohol) with relation to axial gradients (Child, 1g16a, b). Acceleration of cleavage on return of anaesthetised eggs to sea water (Blumenthal, 1928). Prolongation of Life—Of unfertilized egg. By chloral hydrate (Loeb, 1913, p. 91). With 2% ethyl alcohol, life is doubled, and this is not due to bactericidal action (Tyler, Ricci, and Horowitz, 1938). See also under Cyanides. Salts and Anaesthetics—Antagonism (R. S. Lillie, 1912, 1914a, 1917). Effect of Temperature—On carbamate narcosis (Cornman, 1950b). Respiration and Cleavage.—Anaesthetics prevent cleavage with little effect on re- spiration. See Respiration A III 1. First shown by Warburg (1910) for Paracen- trotus lividus with phenyl urethane; 0.0005 M phenyl urethane blocks cleavage and reduces oxygen consumption to 80 % of normal. More fully studied in S. purpuratus with ethyl urethane and other anaesthetics by Loeb and Wasteneys (1913b). In Arbacia, 0.001 M phenyl urethane blocks cleavage and reduces oxygen consumption to 70 % of control (Clowes and Krahl, 1940, Krahl and Clowes, 1940). 0.1 M ethyl urethane stops cleavage and reduces oxygen consumption to 75 % (Fisher and Henry, 1944, Fisher, Henry, and Low, 1944). 0.0045 M chloral hydrate blocks cleavage, leaving 45% oxygen consumption, (and has practically no effect on unfertilized eggs) (Fisher and Henry, 1944). See Table VIII of Krahl (1950). For barbiturates and local anaesthetics (benzoates, etc.) on cleavage and oxygen consumption see Clowes and Krahl (1940); Krahl and Clowes (1940); Clowes Keltch and Krahl (1940) ; Krahl, Keltch and Clowes (19404) ; see Tables VI and VII of Krahl (1950). For urethane and methylene blue on respiration (Barron, 1929). Permeability—Decreased by anaesthetics (R. S. Lillie, 1912, 1914a, b, 1916c, 1918b, etc.; Lucké and McCutcheon, 1932, but see 1926 a and Lucké, 1931). Increase due to fertilization is prevented by anaesthetics (R. S. Lillie, 1918a, b; Lucké and McCutcheon, 1932. For Table of concentrations effective in preventing increase of permeability, which are similar to those arresting cleavage see R. S. Lillie (1918b, p. 426). According to Heilbrunn (1925c, 1943, pp- 144, 531), permeability is not decreased by ether. According to McClendon (1909b) permeability is increased by all agents causing parthenogenesis, including ether. Viscosity.—Decreased if anaesthetic is dilute, increased if concentrated (Heilbrunn, 19204, b, 1925c, 1927, 1928, p. 205; by centrifuge tests). See Table 15 from Heil- brunn (1928) giving concentrations and lethal doses of many anaesthetics, with regard to viscosity. Decrease in 2.5 % ether according to Heilbrunn (1920a), increase according to Chambers (1924, p. 250); but Heilbrunn (1925c¢, p. 474 and 1928, p. 207) thinks Chambers’ results were due to heating by microscope lamp. Decrease in 0.2 M ethyl urethane by centrifuge tests (E. B. H., 1930 unpub.). Chloretone increases viscosity (Heilbrunn, 1920a, b). Parthenogenesis.—Caused by chloroform, ether, alcohol (Mathews, 1900; McClen- don, 1909b, 1910b; Heilbrunn, 1928, p. 261). By acetone, chloretone, urethane, chloral hydrate, methyl acetate, ethyl acetate, ethyl butyrate, methyl salicylate (Heilbrunn, 1913; Just, 1929a for acetone). Parthenogenesis by hypertonic sea water prevented by anaesthetics (R. S. Lillie, 1914a, b, 1917; Heilbrunn, 19204). Reversal of parthenogenesis with chloral hydrate (or NaCN); (Loeb, 1913, 1914b, 1915b); this is denied by F. R. Lillie and Just (1924, p. 505). See Cyanides. Sperm Respiration.—Ethyl urethane, 0.1 M increases respiration by 23 %; 0.01 M 154 ALPHABETICAL COMPILATION TABLE 15 CONCENTRATIONS OF ANAESTHETICS FOR FERTILIZED ARBACIA PUNCTULATA EGGS VISCOSITY From Heilbrunn, 1928, Colloid Chemistry of Protoplasm, p. 206, amended from Heilbrunn, 1920b, p. 311 Concentration of Concentration of solution found to Length of solution found to Length of Reagent decrease viscosity exposure cause coagulation exposure Vol; Y in minutes Vol. % in minutes Anaesthetic conc. Lethal conc. Ether 2.5 9 3.5 404 Chloroform 0.13 63 1 (emulsion) 84 Chloral hydrate 0.08 13 I 35 Chloral hydrate 0.25 28 == = Nitromethane 2 244 3 16 Paraldehyde 4 7, 12} 8 63 Acetone 5 29 10 9 Ethyl nitrate 0.3 64 = aaa Ethyl nitrate 0.5 29 Ethyl acetate 3 414 5 194 Ethyl acetate 4 64 — = Ethyl butyrate 0.25 334 0.5 5 Ethyl butyrate 0.33 18 = — Acetonitrile 4 63 5 13} Propyl alcohol (n) I 10 — — Propyl alcohol (n) 1.33 25 — = Amy] alcohol 0.66 284 I 40 Phenyl] urethane 4/5 sat. 6 saturated 15 (= <0.5 %) Ethyl urethane 1.5 8 3 294 Ethyl urethane 2.5 18 — =— has no effect. See Respiration B I 8. Phenyl urethane saturated solution inhibits respiration 60%, one fourth saturated inhibits 12%, one tenth saturated has no effect (see Respiration B II 6) (Barron, Nelson, and Ardao, 1948). Sperm are more resistant to anaesthetics than eggs, but they can be anaesthetised (Blumenthal, 1928). Sperm lose fertilizing power after one half hour in 0.2 M ethyl urethane; lose motility after two hours (E. B. H., 1930 unpub.). Other Species (additional) and General References Cornman, 1950a. Tripneustes esculentus, Lytechinus variegatus with carbamates. Cornman, Skipper, and Mitchell, Jr., 1951. Tripneustes esculentus, urethane. Fisher, 1942. General. Fuhner, 1904. Paracentrotus lividus and Psammechinus miliaris with alcohols etc. R. S. Lillie, 1916c. Theory of Anaesthesia. Monné, 1947. Ps. miliaris, etc.; anaesthetics on structure of protoplasm. Runnstrém, 1928b, 1930. P. lividus, A. lixula with ethyl and phenyl urethanes. OF EXPERIMENTAL WORK 155 ARTIFICIAL PARTHENOGENESIS See Parthenogenesis CALCIUM Amount in egg.—1.g0 mg Ca per 108 eggs (10° eggs = 0.124 gm. dry weight) or 0.047 millimols (Page, 1927b). Total Ca is about 0.3 mg. per cc. eggs; same in unfertilized and fertilized eggs. Ex- changeable Ca per cell is 19.2 x 10~* mg. The concentration of free Ca in the unfer- tilized egg is of the order of 0.0005 M and increases by 0.001 M on fertilization; bound Ca decreases on fertilization by about 15 %. Ca release on fertilization and cytolysis (Mazia, 1933, 1937, 1940; Heilbrunn, Mazia, and Steinbach, 1934). Sea water.—At Woods Hole contains 0.428 gm. Ca per liter at 20° C. (Page, 1927C, 1928). Perivisceral Fluid—Contains 0.395 mg./cc. of Ca as against 0.41 mg./cc. in sea water (Schechter, 1937, from analysis by Mazia). CaCl,.—Isotonic with sea water at Woods Hole is 0.30 M (M. B. L. Chemical room) usually given as 0.34 M. Radioactive Ca (Ca*®).—Accumulated only by eggs with jelly coats and for only six hours after fertilization (Rudenberg, 1953). Surface Precipitation Reaction—With breakdown of pigment granules; calcium necessary (Heilbrunn, 1928, Chapt. 13; 1930, 1943, p. 86; 1952, p. 102; Costello, 1932; et al.). Effect of anaesthetics (Mg and ether) on surface precipitation reaction (Heilbrunn, 1934). For other references to calcium and breakdown of pigment granules see Gross (1951); and of other granules (yolk) see Costello (1932) and (cortical) see Moser (19394, b). Vitelline membrane.—Made brittle by Ca (Heilbrunn, 1928, p. 149; Chambers, 1944, 1949, 1950; see Kopac, 19404). Surface potential of egg.—Effect of Ca (Dan, 1936). Hyaline layer—tLack of calcium prevents formation of, or causes dissolution of, hyaline layer of fertilized egg so that blastomeres fall apart. First described by Herbst in 1900 in Echinus microtuberculatus and subsequently by many others. Effect of lack of Ca on hyaline layer in Arbacia punctulata and other species described by E. B. Harvey (1933b, 1934). See Hyaline Layer. (Plate XVI, Photograph 9). Jelly.—Dissolves in Ca-free sea water (E. B. H.). Ca Necessary for Fertilization.—No fertilization membrane is formed if fertilized in Ca-free sea water though sperm are motile (Loeb, 1915a; Glaser, 1915; E. B. H. unpub.; also Monroy, 1949 for Psammechinus microtuberculatus). But see Shapiro (1941). Cleavage.—Addition of calcium (3 times amount in artificial sea water) delays cleavage, lack of calcium accelerates cleavage (Shapiro, 1941). Delay and arrest with calcium (Schechter, 1937). Effect on furrowing (Scott and Pollen, 1951). Effect on Cytolysis —CaCl, prevents cytolysis (R. S. Lillie, 1g11a, b, 1912; Page, 1924; Schechter, 1936). When used for a long time, CaCl, causes cytolysis (Heil- brunn, 1928, p. 147). Antagonism.—Ca counteracts NaCl and other Na and K salts (Loeb, 1g00a; Mathews, 1905; R. S. Lillie, 1911 a, b, 1912, 1914a; McCutcheon and Lucké, 1928; Heilbrunn, 1943, p. 463). Ca antagonises Mg (Heilbrunn, 1934). Respiration.—For other species (Hultin and Vasseur, see list below). Permeability to water.—Decreased by isotonic CaCl, (R. S. Lillie, 1910; McCut- cheon and Lucké, 1928; Lucké and McCutcheon, 1929, 1932) ; no effect on fertilized eggs (R. S. Lillie, 1918b). Permeability to ethylene glycol not significantly changed by Ca (Stewart and Jacobs, 1936). Viscosity.—Decreased by isotonic CaCl, (Heilbrunn, 1923, 1928, p. 146, 232; 159 ALPHABETICAL COMPILATION 1943, p. 81; 1952, p. 96; E. B. Harvey, 1945). The cations arranged according to their effect in decreasing viscosity (better stratification) are Ca > Mg > sea water > Na > K (E. B. Harvey, 1945, p. 74) Ca > Mg > sea water > K > Na > NH, (Heilbrunn, 1928, p. 147). Added Ca decreases viscosity (Wilbur and Recknagel, 1943). Breaking of eggs.—Isotonic CaCl, causes eggs to break with centrifugal force less readily than in sea water; the better stratified, the less easily they break. The cations arranged for ease in breaking are in the reverse order to that given above for dc- creasing viscosity (E. B. Harvey, 1945). Calcium-Free Sea Water.—As compared with eggs in sea water, no difference in rate of stratification, indicating change in viscosity, could be detected by observation with a double head centrifuge microscope (E. B. Harvey 1933b) (Table 16). Wilbur and Recknagel (1943) found increased viscosity when calcium was removed from the sea water by potassium citrate. According to Kriszat and Runnstrém (1951) viscosity is increased in calcium-free sea water in Ps. miliaris. Unfertilized Arbacia eggs elongated more and broke more readily in calcium-free sea water, and fertilized eggs broke more readily than in sea water (E. B. Harvey, 1933b, 1945). Shapiro (1941) found that unfertilized eggs elongated more and rounded up more rapidly in calcium-free sea water, whereas they elongated less and rounded up more slowly in increased calcium (3 times amount in artifical sea water). Parthenogenesis.—Caused by adding CaCl, to the sea water (Loeb, 1900a, 19134, Pp. 59, etc.) ; by 0.3 M CaCl, (Hollingsworth, 1941). But R. S. Lillie (1910, 1g1 1a, b, 1914a) found that isotonic CaCl, did not activate, and prevented activation by NaCl. Ca is necessary for activation by ultraviolet light (Heilbrunn and Young, 1930; Heilbrunn and Mazia, 1936) and other parthenogenetic agents (Moser, 1939b). For the necessity of calcium for parthenogenesis in general see Tyler (1941 a, p- 322) and Heilbrunn (1943, p. 661). Longevity of Egg.—Increased by reduction of Ca (Schechter, 1937). TABLE 16 CALCIUM-FREE AND ARTIFICIAL SEA WATER M. B. L. at Woods Hole, Mass. as prepared in Chemical Room, for salinity 31 (grams per liter) NaCl KCl CaCl,.2H,O MgCl,.6H,O MgSO,.7H,O NaHCO- grams per liter 24.72 0.67 1.36 4.66 6.29 0.18 ml. of 1M solution* 423.0 9.00 9.27 22.94 25-50 2.15 pH of sea water at Woods Hole 8.14 (Ball and Stock 1937). A is 1.805. (Herbst (1900) at Naples, for salinity 38 (grams per liter) NaCl KCl CaCl, MgSO, MgHPO, 3%+0.07% 0.08% 0.13% 0.66 % in excess, to replace for CaCl, alkalinity Calcium can be removed from sea water with potassium oxalate (or citrate), about 1.5 gm. per liter of sea water. For other formulae for calcium-free sea water, see McClung’s Microscopical Technique, 3rd ed., P- 559, 1950; Horstadius, 1935 and Tyler, 1953. * Total mixture to be diluted to one liter. OF EXPERIMENTAL WORK 157 Agglutination.—Of sperm caused by calcium (Loeb, 1915a). See also Vasseur (1949 a) for S. drébachiensis. Formation of Skeleton—Calcium necessary for pluteus (probably P. lividus); if 1/10th of Ca in sea water is precipitated by sodium oxalate a recognizable alteration in development occurs (Pouchet and Chabry, 1889 a, b). Other Species (additional) and General References Bialaszewicz, 1927, 1929. A. lixula, Paracentrotus lividus ; electrolytes. Costello, 1932. Echinarachnius parma, surface precipitation reaction. Fukuda, 1934. Pseudocentrotus depressus, membrane and s.p.r. Heilbrunn, 1952. General Physiology, p. 530. Herbst, 1904. Salts necessary for development. Hultin, 1949b. Psammechinus miliaris, respiration of egg homogenates. Hultin, 1950a. Paracentrotus lividus, same. Hultin, 1950b. Paracentrotus lividus, viscosity of egg homogenates. Lepeschkin, 1941. A. lixula etc. s.p.r. etc. Monroy-Oddo, 1946. A. lixula, change of Ca content after fertilization. Orstrom and Orstrom, 1942. P. lividus, Ca content of eggs. Robertson and Webb, 1939; Robertson, 1941. Amount in sea water Ae body fluids. Rothschild and Barnes, 1953. P. lividus, amount in egg; table of salts and species. Rulon, 1941a. Dendraster excentricus, development in Ca-free sea water. Runnstr6ém, 1925. P. lividus, Ps. micr., Ps. miliaris, calctum-lack. Runnstr6ém and Kriszat, 1950a. Ps. miliaris, centrifuging. Sugiyama, 1938d. Pseudocentrotus depressus, S. pulcherrimus, fertilization membrane. Tyler, 1941a, p. 322. Parthenogenesis in general. Vasseur, 1949b. S. drébachiensis, E. esculentus, Ps. miliaris; oxygen uptake of sperm. CARBOHYDRATE METABOLISM Total Carbohydrate—Determined as glucose. Amount. About 50 mg. glucose per gm. egg protein (Perlzweig and Barron, 1928). About 110 mg. glucose per gm. egg pro- tein or 7 % of dry weight of egg, 65 % being protein; mostly in egg, little in jelly, (Hutchens, Keltch, Krahl, and Clowes, 1942). Glycogen.—Identified in eggs by Blanchard in 1927 as noted by Perlzweig and Barron (1928), published by Blanchard in 1935. Amount: 40-57 mg. glycogen per gm. egg protein in unfertilized eggs or 46 % of total carbohydrate (Hutchens e¢ al. 1942). Free Reducing Sugar.—Absent (Perlzweig and Barron, 1928; Hutchens, et al., 1942). Pyruvate-—Amount. 70 ug. pyruvate per gm. dry weight of eggs (Goldinger and Barron, 1946). Eggs utilize 64 wg. pyruvic acid per gm. dry weight per hour, unfer- tilized; 7 times as much (445 ug.), fertilized (Goldinger and Barron, 1946). 160 wg pyruvate per gm. dry weight per hour, unfertilized; 310 yg. fertilized, in cytolysates (Krahl, Jandorf, and Clowes, 1942). Lactic Acid —Amount. 3.14 mg. lactic acid per gm. protein in unfertilized eggs, increase to 5.68 mg. with KCN; slight increase 1-2 hours after fertilization, from 2.71 to 3.23 mg. (Perlzweig and Barron, 1928). Lactic acid negligible during first 24 hours of development (Hutchens, et al., 1942). Phosphorylation —Of carbohydrate intermediates (Keltch, e¢ al., 1950, 1951; Stritt- matter, et al., 1950; Clowes, et al., 1950, 1951a, b; Clowes, 1951): Todoacetate.—Affects carbohydrate metabolism (Runnstrém, 1935Cc). Other Species (additional) and General References Barron, 1952b. General. Cleland and Rothschild; 1952a, b. Echinus esculentus, glycolysis. 158 ALPHABETICAL COMPILATION Ephrussi, 1933. Paracentrotus lividus, amount of carbohydrate. Immers, 1952. Echinus esculentus, Echinocardium, etc., sugars. Krahl, 1950. Review. Lindberg and Ernster, 1948. Strongylocentrotus drébachiensis, phosphorylation. Monné and Slautterback, 1950. Staining of P. lividus. Orstrém and Lindberg, 1940. P. lividus, glycogen, etc., before and after fertilization. Rothschild and Mann, 1950. Echinus esculentus, sperm. Runnstr6m, 1949b. General. Spikes, 1949. S. purpuratus, Lytechinus pictus, sperm. Stott, 1931. Echinus esculentus. Zielinski, 1939. P. lividus. CHROMATOPHORES (PIGMENT GRANULES) See also Echinochrome Chromatophores.—Spherical vacuoles containing echinochrome (Chambers, 1935a, 1938a; D. L. Harris, 1943; et al.). See E. B. Wilson (1899, p. 7 and p. 26, Plate I, Fig. 2; 1926, p. 113), who described them as alveoli and showed them as hollow spheres. Occurrence.—In eggs, unfertilized and fertilized, plutei and in red Amoebocytes, q.v. Location.—In unfertilized eggs, they are distributed throughout the cytoplasm (Lyon, 1907; E. N. Harvey, 1909, 1910b; McClendon, 1gtob, 1912a; R. S. Lillie, 1911b; et al.). But they are not evenly distributed (Heilbrunn, 1926a; E. B. H. unpub.). On fertilization or parthenogenesis, they migrate to periphery of egg (McClendon, 1909b, 1910b, 1912a; E. N. Harvey, 1909, 1910b; Heilbrunn, 1928, p. 264; K. Dan, 19514; et al.). See E. B. Wilson (1926, Fig. 2a, b). In fertilized eggs, they are located especially at periphery and in cleavage furrow (Loeb, 1895, p. 274, 1905, p. 401; G. F. Andrews, 1897b, p. 85; Mathews, 1906b; McClendon, 1910b, 1912a; Cannan, 1927; e¢ al.). Return to uniform distribution after each cleavage (K. Dan, 19514, b). After 8-cell stage, they move away from the micromere pole, and the micromeres are therefore colorless (Morgan, 1893; Lyon, 1907; E. N. Harvey, 1909; McClendon, 1g10b; e¢ al.). Location in monaster eggs (Painter, 1918). In plutei, 20 to 30 chromatophores, each about 2 yw in diameter, are grouped together to form pigment spots, distributed irregularly over the body. These may be spherical or irregular in shape, and vary in size; the average spherical ones are about 7 w in diameter. They swell and break in distilled water (E. B. H. unpub.). Size.—Chromatophores are all sizes up to 1.7 w (E. B. H. unpub.). See also Harris (1943) who says 1 to 2 mw. Weight.—Heaviest bodies in the egg; go to centrifugal pole on centrifuging (Lyon, 1906a, 1907; McClendon, 1g0ga; E. B. Harvey, 1932, 1936, etc.; et al.). Specific Gravity—Approximately 1.1035; of whole egg is approximately 1.0485 (Heilbrunn, 1926a). Amount in Egg.—5.5 % (E. N. Harvey, 1932a); 10 % (Costello, 1939). Origin.—Pigment begins to form in immature egg when about 33 in diameter; younger oocytes are colorless (E. B. H. unpub.). Stain.—With vital dyes. Methylene blue, neutral red (McClendon, 1g09b, 19124; E. N. Harvey, 1g10c; Lucké, 1925; E. B. Harvey, 1941¢; et al.). Bismark brown, brilliant cresyl blue, chrysoidin, methyl violet, Nile blue, rhodamine, saffranin o, toluidin blue (E. B. Harvey, 1941c). They stain black with methylene green and also with quinine, cinchonine and cinchonidine, with no effect on development (E. B. H. unpub.). E. B. Wilson (1926) states that they stain intensely with Janus OF EXPERIMENTAL WORK 159 Green B, but I have not succeeded in staining them with any of many samples of Janus green and Janus green B. Table 8. With histological stains. Not stained with most protoplasmic stains (Lyon 1907; E. B. Wilson, 1926) ; not stained with iron haematoxylin, but are stained with orange G (E. B. Harvey, 1940c) ; stained with Delafield’s haematoxylin (McClendon, 1g09b, 1912a). Appearance in electron microscope preparations (McCulloch, 1952a). Ultraviolet Light—Somewhat absorbing (E. B. Harvey and Lavin, 1944). Release of Pigment from Chromatophores in Unfertilized Eggs.—On standing (F. R. Lillie, 1912; Glaser, 1914b, c, 1921a, 1924; Heilbrunn, 1928, p. 246; et al.). But Shapiro (1946) thinks not. Rupture or puncture of vitelline membrane (E. N. Harvey, 1910b; Heilbrunn, 1928, p. 70; Chambers, 1938a; Churney, 19414; D. L. Harris, 1943; et al.). Puncture of chromatophore membrane (Chambers, 1935a, 1938 a; D. L. Harris, 1943). Electrical, mechanical, or chemical stimulation (McClen- don, 1g10a, b, 1912a; Heilbrunn, 1928, p. 244; Chambers, 1938a). Heat (E. N. Harvey, 1910b; Chambers, 1938a). Hypotonic sea water or distilled water (E. N. Harvey, 1910b; and many others). Process described by D. L. Harris (1943). Hypertonic sea water (E. N. Harvey, 1910b; Glaser, 1914b). Parthenogenetic agents (McClendon, 1gogb; R. S. Lillie, 1910, 1911a, b; E. N. Harvey, 1910b). Calcium, and to a much less extent magne- sium and strontium (Heilbrunn, 1926a, 1928, p. 223, 1930, 1934, etc. in surface precipitation reaction; D. L. Harris, 1943; Gross, 1951, 1953). Acids (E. N. Harvey, 1910b; Barth, 1929). lodosobenzoic acid (Monroy and Runnstrém, 1950, 1952). Alkalis (D. L. Harris, 1943). Soaps (Heilbrunn, 1928, p. 242; Page, Shonle, and Clowes, 1933; Gross, 1951). Fat solvents (Heilbrunn, 1828, p. 242). Urea, acetamide, sucrose, ethylene glycol (D. L. Harris, 1943); but see Churney (1938) for urea. Anaerobiosis (Shapiro, 1946). Pigment Not Released in absence of calcium; chromatophores remain intact (Heilbrunn, 1926a, 1928, p. 224, etc.); D. L. Harris (1943) uses 0.35 M sodium citrate to keep chromatophores intact. In homogenates, chromatophores remain intact in absence of calcium (Gross, 1951). Release of Pigment on Fertilization or Parthenogenesis.—(McClendon, 1909b, 1910b, 1912a; McClendon and Mitchell, 1912; E. N. Harvey, 1909, 1910b,c; R. S. Lillie, 1910, 1911a, b, 1914b; Lyon and Shackell, 1910b; Glaser, 1914b, c, 1923, 1924; Just, 1922, 1; Shapiro, 1946; et al). Release of Pigment from Chromatophores in Fertilized Eggs.—On standing. More released from fertilized than from unfertilized eggs (Loeb, 1910; Lyon and Shackell, 1910b; Glaser, 1914b, c, 1923; Shapiro, 1946; et al.). Pressure (Churney 1941a). Mer- curic chloride, with clumping of pigment (Hoadley, 1930). Potassium chloride, oxalate and citrate, ammonium chloride, urea; but no effect on unfertilized eggs (Churney, 1938; Churney and Moser, 1940). During cleavage (R. S. Lillie, 1914b; see Churney, 1940). Effect on Fertilization Membrane.—Coagulating (Monroy and Runnstrém, 1950, 1952; Runnstrém, 1950-1951, p. 143; 1952b, p. 67). Osmotic Properties —Permeability constant: 0.38 cubic micra of water enter the vacuole per square micron of surface area per minute per atmosphere difference in osmotic pressure; higher than for the cell (D. L. Harris, 1943). Pigment vacuoles therefore do not constitute part of osmotic dead space postulated by Lucké, Hartline, and McCutcheon (1931). Coalescence of Chromatophores—With chloroform (E. N. Harvey, 1g10c). In pre- sence of calcium, magnesium or strontium ions (D. L. Harris, 1943). Movement.—Of individual chromatophores in unfertilized eggs. Observed by McClendon (1910b). With T. V. microscopy; translatory motion of 5 « per second at 24° C. (Parpart, 1953). Under hydrostatic pressure (D. E. S. Brown, 1934; Mars- land, 1950). Through cleavage (Dan, 19514, b). 169 ALPHABETICAL COMPILATION No Fading with Anaerobiosis (t.e. not reduced). —(Korr, 1939, p. 83 and Ball in same paper, p. 92). See under Echinochrome. Electric Charge.—Possibility of (E. N. Harvey 1909, 1910b; McClendon, 1g1ob, 1912a, Heilbrunn, 1926b). Contain Copper.—(Glaser, 1923). Other Species Fischel, 1g06b. A. lixula. E. B. Harvey, 1933a, 1938a. A. lixula. CLEAR LAYER OF CENTRIFUGED EGGS Position in Arbacia.—Layer in centrifuged eggs between oil (centripetal) and granules: mitochondria, yolk and pigment (centrifugal) ; contains nucleus (early investigators Lyon, 1906a, 1907; Morgan, 1909; McClendon, 190ga e¢ al.; later investigators E. N. Harvey, 19324; E. B. Harvey, 1932, 1936, 1940C¢, etc.; et al.). Structure.—Optically empty in living egg except after prolonged centrifuging. A narrow band of granules then appears across the clear layer of white halves and clear quarters, forming two zones (E. B. Harvey, 1946a; McCulloch, 1952). Birefrin- gent fibrils in lower part of clear zone (McCulloch, 1952a). Electron microscope preparations (McCulloch, 1952a; Lansing, Hillier, and Rosenthal, 1952). Some pigment granules remain in clear layer of eggs late in the season (E. B. H.). Amount.—61.1 % (E. N. Harvey, 19324); 45 % (Costello, 1939); these figures in- clude fluid between the packed granules; the difference between the two figures is probably due to the amount of centrifuging. About 2/3 of total fluid is in clear layer (E. B. Harvey, 1946a). Specific Gravity.—About 1.0358; of whole egg is about 1.0485 (Heilbrunn, 1926a). Viscosity.—Two centipoises, determined by centrifuge tests and return of granules (Heilbrunn, 1952, p. 81; see also Heilbrunn, 1926a, b, 1927, 1928, p. 67). Stain.—With vital dyes. Faint stain with Bismark brown, chrysoidin, methyl violet, neutral red, Nile blue, rhodamine, toluidin blue (E. B. Harvey, 1941 c). Two portions stain differentially (E. B. Harvey, 1946a). Table 8. Fixed material.—Layer is filled with very small granules which stain blue with iron haematoxylin (Lyon, 1907; Morgan and Lyon, 1907; E. B. Harvey, 1940c; E. B. Harvey and Lavin, 1944; et al.); these are probably the “‘microsomes”’ of Wilson (1925, p. 32), they measure about 0.2 mw in diameter (E. B. H.). Clear layer stains with protoplasmic stains, alum cochineal (Lyon,1907). Stains differentially (two kinds of material) with haematoxylin-orange-G-eosin (E. B. Harvey, 1946a, Figs. 32, 33). Ultraviolet Light—Absorbed by clear layer in formalin-fixed sections (E. B. Harvey and Lavin, 1944). Localization of Peptidase—In clear layer (Holter, 1936). Other Species In some other species, e.g., Sphaerechinus granularis, the clear layer is at the centripetal pole as it is in Arbacia punctulata and A. lixula, being lighter than the granules. In other species, e.g., Psammechinus microtuberculatus, it is below the yolk but above the mitochondria. In still other species, e.g., Tripneustes esculentus, it is beneath the yolk and mitochondria, being the heaviest material in the egg. In some species, e.g., Paracentrotus lividus, the clear material may be in more than one zone (E. B. Harvey, 1933a, 19382); Other references for clear layer: Harvey, E. B. per E. N. Harvey, 1939. Echinarachnius parma, Strongylocentrotus drobachiensis, etc. Harvey, E. B., 1947. Lytechinus variegatus and other Bermuda forms. OF EXPERIMENTAL WORK 161 Lindahl, 1932¢.Psammechinus microtuberculatus, fine colored picture, p. 330; probably Ps. miliaris is the same. Linderstrém-Lang, 1938-1939. Ps. miliaris, diagram. Monné, 1944b. Echinocardium caudatum, Ps. miliaris. See under Centrifuged Eggs, Part III, Chapter 17d, p. 127. CLEAVAGE, ACCELERATION Many of the substances listed under Cleavage are treated more fully. under Respiration Acetyl Choline.—Acceleration in low concentrations, inhibition in high (Balzer and Villee, 1951; Villee and Villee, 1952. Alcohols.—Slight acceleration (Blumenthal, 1928); in low concentrations (Water- man, 1936). " Alkali.—Slightly alkaline sea water accelerates cleavage 14 or 2 cc. of 1/10-normal NaOH to 100 cc. sea water (Loeb, 1898, but see 1913a, p. 35). Also see Glaser (1914b) and Medes (1917). Acceleration at pH 8.2 to 9.2, maximum at 8.8. Delay at pH 9.4 (Smith and Clowes, 1924c). For Echinus esculentus see B. Moore, Roaf and Whitley (1905). Calcium-free Sea Water.—Accelerates cleavage; excess Ca delays cleavage (Shapiro, 1941; Scott and Pollen, 1951). Carcinogenic Hydrocarbons.—(Choleic acid compounds). Acceleration of cleavage (Keltch, Krahl, and Clowes, 1937). Retarded and atypical cleavage (Lucké, Parpart, and Ricca, 1941). Copper Chloride.—Acceleration in very dilute CuCl,, 10-13 M (Finkel, Allee, and Garner, 1942; Allee, Finkel, et al., 1942). See under Delay. Crowding.—Early cleavages accelerated if eggs moderately dense, delayed if very dense (Allee and Evans, 19374, b, c). For S. drébachiensis see Frank and Kurepina (1930); for P. lividus see Maxia (1933); for Anthocidaris crassispina, Pseudocentrotus depressus, S. pulcherrimus see Sugiyama (1938e). Cyanide, Potassium.—Accelerates cleavage in very weak concentrations, 10-* M; arrests cleavage in stronger concentrations, 10-° M to 10-4 M (Lyon, 1902). See under Delay and Cyanides. Cystin.—Acceleration of cleavage and development (Mathews, 1909); not con- firmed by King (1912). Egg Extracts —‘‘Homotypic extracts.’’ Some accelerate cleavage, some do not (Allee, Finkel, et al., 1942). Acceleration if fats removed (Peebles, 1929). Heat.—Cleavage accelerated as temperature is raised up to about 31° C. (Loeb and Wasteneys, 1911a; Loeb, 1913a, p. 32; Loeb and Chamberlain, 1915; Hoadley and Brill, 1937). See Tables 3 and 4. Highest temperature permitting cleavage is about 28 °C.; killed at 32° C. (E. B. H.). For Ps. microtuberculatus and Sphaerechinus granularis see Peter (1905). For P. lividus see Ephrussi (1933); for S. purpuratus, Lytechinus anamesus and Dendraster excentricus see Tyler (1936a, b). See under Delay. Hydrogen Ion Concentration —See below under pH. Hypotonic Sea Water.—g4-98 %. Accelerates first cleavage 5% of normal time (Cornman, 1943). Mechanical Shocks and Vibrations—Accelerate cleavage (Meltzer, 1903; Mathews and Whitcher, 1903). Whitney (1906) thinks this is due to rise in temperature. Shaking accelerated cleavage in A. lixula also (Lyon 1903). Methylene Blue.—Cleavage accelerated with 5 x 10-° %, retarded with 5 x 10°*% (M. M. Brooks, 1933, 1943). Shapiro (1948a) finds delay; also Clowes and Krahl (1936a). See Krahl (1950, Tables VIII and IX). See under Delay. Oxygen Lack, Partial—May accelerate cleavage (Loeb, 1895a, 1905 translation). 162 ALPHABETICAL COMPILATION “IT will not commit myself definitely to the statement that in case of a partial lack of oxygen a transitory acceleration of cleavage occurs’’ (Loeb, 1905, p. 403). pH.—Acceleration at pH 8.2 to 9.2, maximum at 8.8. Delay above pH 9.4 and below 6.0. (Smith and Clowes, 1924C¢, d). Pilocarpine.—In low concentrations accelerates development, in high concentra- tions delays (Mathews, 1901a; Sollman, 1904a). See Balzer and Villee (1951) and Viilee and Villee (1952). For Dendraster excentricus see Rulon (1941b). Radium.—Alpha rays accelerate cleavage, beta rays delay (Packard, 1915, 1916). CLEAVAGE, DELAY OR ARREST Acetyl choline.—Delay in high concentrations, acceleration in low concentrations (Balzer and Villee, 1951; Villee and Villee, 1952). Acid.—Delay (Loeb, 1898, 19134, p. 35; Glaser, 1914b; Medes, 1917). Retarded below pH 6.0 (Smith and Clowes, 1924c). For Echinus esculentus see B. Moore, Roaf, and Whitley (1905). Ageing. —(Goldforb, 1918a, b; Smith and Clowes, 1924b). No delay if kept, for at least six hours (Fry, 1936). Alanin.—(King, 1912). Alcohols.—Delay except in very weak concentrations (Blumenthal, 1928; Water- man, 1936. See below under Anaesthetics. Alkali.—Acceleration in weak concentrations (Loeb, 1898, 19134, p. 35; Glaser, 1914b; Medes, 1917). Delay above pH g.4 (Smith and Clowes, 1924c). For Echinus esculentus see B. Moore, Roaf, and Whitley (1905). Ammonium chloride.—(Kopac, 1948b). Anaerobiosis.—See below under Oxygen-Lack. Anaesthetics—For anaesthetic and lethal doses of common anaesthetics see Table 13 (from R. S. Lillie, 1914b), Table 15 (from Heilbrunn, 1928, p. 206) ; for urethanes see Table 14 (E. B. Harvey per E. N. Harvey, 1932a). See below Barbiturates, Ben- zoates, Dithiocarbamates, Urethanes. See also Anaesthetics (Separate topic). Antibiotics.—Penicillin, aspergillus, etc. (Cornman, 1949); actinomycin, aureo- thrycin, etc. (Miura, 1953). Ascorbic Acid.—Or vitamin C (Shapiro, 1948c). Asparagine.—Development slow, cleavage normal (King, 1912). Aspartic acid.—King, 1912). Aspergillus Filtrates—Antibiotic (Cornman, 1949). Atropin.—(Mathews, 1901a; Sollman, 1904a; Villee and Villee, 1952). Azide.—Complete and reversible inhibition of cleavage, and respiration inhibited by 50% at 5 xX 10-3 M (Krahl, 1950, p. 195); also Krahl, Keltch Neubeck, and Clowes (1941); Fisher, Henry, and Low (1944). For Dendraster excentricus see Rulon (1950a). Barbiturates.—Or barbituric acid derivatives (Clowes and Krahl, 1940; Clowes, Keltch, and Krahl, 1940; Krahl, 1950, p. 200 and his Table VI). Benzoates.—And other local anaesthetics (Clowes and Krahl, 1940; Krahl, Keltch, and Clowes, 1940a; Krahl, 1950, p. 200 and his Table VII). Bile Salts.—(Genther and Schmidt, 1931). Brilliant Cresyl Blue.—(Shapiro, 1948a). Caffeine.—(Cheney, 1945, 1948). Calcium.—Added to sea water delays cleavage, calcium-free sea water accelerates (Shapiro, 1941). Carbamates.—See Urethanes. Carbon Dioxide.—Clowes and Smith, 1923; Smith and Clowes, 1924a; Haywood, 1927; Haywood and Root, 1930, 1932; Allee and Evans, 1937b; Allee, Finkel, e¢ al., 1942). OF EXPERIMENTAL WORK 163 Carbon Monoxide.—M. M. Brooks, 1933; Clowes and Krahl, 1940; Krahl and Clowes, 1940; Krahl, p. 194, 1950). For P. lividus see Runnstrém (1930); for Den- draster excentricus see Pease (1942C). Carcinogenic Hydrocarbons.—Choleic acid compounds. Delayed cleavage (Lucké, Parpart, and Ricca, 1941). Accelerated cleavage (Keltch, Krahl, and Clowes, 1937). Chloral Hydrate.—Fisher and Henry, 1944). See under Anaesthetics above and Tables 13 and 15. Chloretone.—Cleavage checked by 0.08 % (Heilbrunn, 19204, p. 233). Clavacin.—Antibiotic (Cornman, 1949). Colchicine.—About 10-4 M. Mitosis stopped in late prophase or metaphase (Nebel, 1937; Nebel and Ruttle, 1938; Beams and Evans, 1940; Wilbur, 1940; Cornman and Cornman, 1951). For Ps. microtuberculatus see Zeuthen (1951, p. 59). For a historical review, see Eigsti, Dustin and Gay-Winn (1949). Cold.—Progressive delay as temperature is lowered from about 31° C. (Loeb and Wasteneys, 1911a; Loeb, 1913a, p. 32; Loeb and Chamberlain 1915; Hoadley and Brill, 1937; Villee and Villee, 1952; Tables 3, 4, and 19). For Ps. microtuberculatus, Sphaerechinus granularis see Peter (1905); for P. lividus see Ephrussi (1933) ; for S. pur- puratus, Lytechinus anamesus and Dendraster excentricus see Tyler (1936a, b). Copper Chloride.—1 /62,500 CuCl, in sea water prevents cleavage; 1/500,000 pre- vents fertilization (F. R. Lillie, 1921b). See also Hoadley (1923); Glaser (1923); Waterman (1937). Runnstrém (1939) removes poisonous effect of copper by shaking the eggs. Acceleration in very dilute solutions, 1o-!* M (Finkel, Allee, and Garner, 1942; Allee, Finkel, et al., 1942). Corticotropin.—ACTH. (Menkin, 1952, 1953, Proc. Exp. Biol. Med. 82 : 189-194). Cortisone.—And desoxycorticosterone (Cornman, 1950c; Menkin, 1952). Cresols.—See Phenols. o-Cresol Indophenol.—(Clowes and Krahl, 1936a; Krahl, 1950, Table VIII). Crowding.—Cleavage delayed if eggs are very dense; accelerated if moderately dense (Allee and Evans, 19374). Cyanides.—Cleavage arrested by about 10-5 M to 10-4M (Krahl, 1950, p. 192 and his Table V). Data of different observers are given under Cyanides. The re- quired concentration depends on the pH (Krahl). Acceleration of cleavage in very weak concentrations, about 10-§ M (Lyon, 1902). Reversal of cleavage inhibition by adenosine triphosphate (ATP). (Barnett, 1953). Desoxycorticosterone.—(Cornman, 1950c; Menkin, 1952). Deuterium oxide.—See Heavy Water. Dimethyl-p-phenylene diamine—(Clowes and Krahl, 1936a; Krahl, 1950, Table VIII). For P. lividus see Runnstrém (1930, 1932). Dinitrophenols.—And dinitrocresols. See Phenols. Dinoflagellate Contaminated Sea Water.—See ‘‘Red tide.” Dithtocarbamates.—(Krahl, 1950, Table VIII). Echinochrome.—(Woodward, 1918). But Allee, Finkel, et al. (1942) find no effect. Egg or Embryo Extracts or ‘‘Water”’.—(Glaser, 1913, 1914b; F. R. Lillie, 1921b; Springer, 1922; Peebles, 1929; Allee and Evans, 1937b, c; R. D. Allen, 1951). Some extracts accelerate (Allee, Finkel, et al., 1942). Eserine.—Inhibition of cleavage (Villee and Villee, 1952). Stated to accellerate in low concentrations (Balzer and Villee, 1951). Gliotoxin.—Antibiotic (Cornman, 1949). Glutamic Acid.—(King, 1912). Glycerine.—(R. S. Lillie, 1903). Glycocol.—Cleavage normal, later development slow (King, 1912). Halophenols and Halocresols.—See Phenols. Heat.—Highest temperature permitting any cleavage lies between 30.4° C. and 32.7° C.; optimum 24° C. or 25° C. (Hoadley and Brill, 1937). See Acceleration. 164 ALPHABETICAL COMPILATION Heavy Water.—Deuterium oxide. Delay (Lucké and E. N. Harvey, 1935). Heparin.—And heparin-like substances (Gagnon, 1950; Heilbrunn and Kelly, 1950; C. V. Harding, 1951). Hydrogen Ion Concentration.—See acid, alkali, pH. Hypertonic Sea Water.—Loeb, 1892, 1904; Norman, 1896; Mathews, 1905; Medes, 1917; R. S. Lillie, 1918b; Heilbrunn, 1920a; Churney, 1940). Hypotonic Sea Water.—(Medes, 1917; Heilbrunn, 1920a, b; Just, 1929b; Churney, 1940). Retardation in 84 % sea water or less, acceleration if slightly hypotonic, 94 to 98 % (Cornman, 1943). Iodoacetate.—lodoacetic acid. Runnstrém (1935c) used 0.03 M iodoacetate for re- tarding development. See also Waterman (1938, 1941); Needham and Needham (1940); Clowes and Krahl (1940); Krahl and Clowes (1940); Krahl (1950, p. 199). For P. lividus, Ps. microtuberculatus, Ps. miliaris see Tchakotine (1938); for Dendraster excentricus see Pease (1941). Janus Green B.—(R. D. Allen, 1950). Leucin.—(Mathews, 1909). Delay and pecularities, of later development. (King, 1912). Leucotaxine.—(Menkin, 1940). Local Anaesthetics—See above under Benzoates. Malonic Acid.—Delay (Rulon, 1948). Krahl and Clowes (1940) found no effect with concentrations used. Malonate inhibition reversed by adenosine triphosphate, ATP (Barnett, 1953). For Dendraster excentricus see Pease (1941); Rulon (1951). Malononitrile.—(Villee, Lowens, et al., 1949). Mercuric Chloride.—1 /625,000 HgCl, in sea water prevents cleavage, 1/125,000 prevents fertilization (F. R. Lillie, 1921b; also Hoadley, 1923, 1930; Waterman, 1937; Barron and Seki, 1952). Cf. Copper chloride above. For Paracentrotus lividus see Rapkine (1931). Metal Salts (Heavy ).—For copper and mercury, see above. For effect of other heavy metal salts on cleavage and development, see Hoadley (1923); Waterman (1937). Metal Salts (Alkali and Alkaline Earth) —Toxic effects, Li > Na > Mg or Ca > K > Rb > Cs (Page, 1929b). See under Calcium, Lithium, Magnesium, Potassium, Sodium. Methylene Blue.—Cleavage delayed in 5 x 1074%, accelerated in 5 xX 107-5 % (M. M. Brooks, 1933, 1943). Delay (Clowes and Krahl, 1936a; Krahl, 1950, Tables VIII and IX; Shapiro, 1948a). Waterman (1938, 1941) finds delay except possibly in very weak concentrations. For effect on development of Dendraster excentricus and S. purpuratus, see Child (1950b). See under Acceleration. Morphine.—No effect with 2 x 10-2 M (Krahl, 1950, p. 206). Napthoquinones.—(Anfinsen, 1947). See Krahl (1950, p. 199). Necrosin.—(Menkin and Pirovane, 1949). Neutral Red.—(Clowes and Krahl, 1936a; Krahl, 1950, Table VIII). Nitrogen Mustards.—Delay at prophase (E. B. Harvey and Cannan, 1943, unpub. except as a confidential report of a war project, see Gilman, and Phillips 1946; E. B. Harvey, 1946a). Delay (Barron, Seegmiller, Mendes, and Narahara, 1948; Hut- chens and Podolsky, 1948; Villee, Lowens, et al., 1949; Cornman, 1950d; Krahl, 1950, p. 205). For A. lixula and Sphaerechinus granularis, see Tau and de Nicola (1949). Nitrophenols and Nitrocresols.—See Phenols. Nitrous Oxide.—Delay under pressure of 2.3 atmospheres. No delay with nitrogen or helium even under pressure of 61 atmospheres (Haywood, 1953). Osmotic Pressure.—Pressure less than 13.2 atmospheres or greater than 28.7 pre- vents cleavage (Churney, 1940). Oxygen-lack or Low Oxygen Tension.—See topic Oxygen-Lack and Low Oxygen Ten- sion. Cleavage retarded at 11 mm. Hg; arrested below 4 mm. Hg. (Amberson, 1928). Slight lack of oxygen may possibly accelerate cleavage (Loeb, 1895, 1905 OF EXPERIMENTAL WORK 165 translation, p. 403, see above under Acceleration. Inhibition reversed by adenosine triphosphate, ATP (Barnett, 1953). Parthenogenetic Treatment.—Delay; first cleavage (at 23° C.) occurs 14 to 5 hours after activaticn; fertilized eggs cleave 50 minutes after fertilization (E. B. Harvey, 1936, 1951; E. B. Harvey and Hollaender, 1938). Delay also noted by Loeb (1g00a, 1904; Moser (1940) and others. For general information about Arbacia and other species see Loeb’s Artificial Parthenogenesis (1913 a). Penicillin and Penichromin.—(Henry and Henry, 1945; Cornman, 1949). Slight in- hibition of cleavage with very weak concentrations of noncrystalline samples of penicillin, but virtually no effect with highly purified samples (Clowes and Keltch, 1946 unpub., per Krahl, 1950, p. 206). Perivisceral Fluid—(F. R. Lillie, 1913a, 1914, 1919, p. 173; A. E. Woodward, 1918; Just, 1922a, III; Goldforb, 1935b). pH.—Delay of cleavage below pH 6.0 and above g.4; acceleration pH 8.2 to 9.2 (Clowes and Smith, 1923; Smith and Clowes, 1924b, c,d). See under Acid and Alkali above and under Alkali in Cleavage: Acceleration. Phenols, Cresols and Related Substances——(Clowes and co-workers, 1934-1951, especially Clowes and Krahl, 1936a; Krahl and Clowes, 1936a, 1938; Clowes 1951). See the comprehensive review of Krahl (1950, p. 196 and his Tables III, IV). Also Waterman (1938, 1941); Villee, Lowens et al. (1949); A. Scott (1950). Incom- plete reversal of inhibition of dinitrophenol by ATP (Barnett, 1953). For Dendraster excentricus see Pease (1941). Phosphorus, P??.—(Green and Roth, 1950). Pilocarpine—In high concentrations, delay; in low concentrations accelerate cleavage (Mathews, 1901 a; Sollman, 1904a; Balzer and Villee, 1951, but see Villee and Villee, 1952). For Dendraster excentricus see Rulon (1941b); Pease (1942b). Podophyllin, Podophyllotoxin, Quercitin and Derivatives—(Cornman, 1947a; Cornman and Cornman, 1951). Polysaccharides.—Heparin, etc. (C. V. Harding, 1951). Pressure, Hydrostatic—(Marsland, 1938, 1950, 1951; Kitching and Moser, 1940; E. B. H., 1933 unpub.). For Arbacia lixula see Marsland (1939). “Purple X.”’—(Glaser, 1914c; A. E. Woodward, 1915, 1918). Pyocyanine.—(Runnstrém, 1935a; Clowes and Krahl, 1936a; Krahl, 1950, Table VIII; Waterman, 1938, 1941). For Dendraster excentricus see Pease (1942b); Moore, Bliss, and Anderson (1945) also for S. purpuratus. Quinine.—Very poisonous; one part of quinine sulphate in 17,000 parts sea water stops cleavage (Mathews, 1907). Poisonous action noted by O. and R. Hertwig in 1887. Radium.—Beta rays delay, alpha rays accelerate cleavage (Packard, 1915, 1916). “Red Tide.”—Dinoflagellate-contaminated sea water. (Cornman, 1947b). Rhodamine B and Light.—(L. B. Clark, 1940). Rotenone.—And related compounds (Cornman and Rogers, 1951; Rogers and Cornman, 1951). Season.—Cleavage delay late in the season (Medes, 1917; Woodward, 1918; Fry, 1936; E. B. Harvey, 1939b). Sperm Extracts —(Frank, 1939). Sugar.—(R. S. Lillie, 1903; Loeb, 1913a, p. 130 for Strong ylocentrotus purpuratus with good discussion of sugar effects; R. S. Lillie and Cattell, 1923). No fertilization in isotonic sugar solution, but cleavage when returned to sea water even after 5 hours in sugar (E. B. Harvey, 1932). Sulfide, Sodium.—(Krahl, Keltch, Neubeck, and Clowes, 1941; Krahl, 1950, P- 195). Sulphanilamide.—And related compounds (Fisher, Henry, and Low, 1944; Krahl, 1950, Table VIII). 166 ALPHABETICAL COMPILATION Sulphur, S**.—(Green and Roth, 1953, Biol. Bull. 105 : 364) Temperature—See under Cold, delay; and Heat, acceleration. Theobromine and Theophylline (Cheney, 1949b). Thiurea.—(Bevelander, 1946). For Dendraster excentricus see Rulon (1950b). Toluyene Blue.—(Shapiro, 1948a). Tyrosin.—(Mathews, 1909; King, 1912). Slight acceleration in some cases with weak solutions (Mathews, 1909). Ultraviolet Light.—See Ultraviolet Light. Cleavage and development delayed. Uranyl Nitrate-—(Villee, Lowens, et al., 1949). Urea.—(R. S. Lillie, 1903). Urethanes.—Carbamates. Anaesthetic doses given in Table 14. See under Anaes- thetics. See Krahl, 1950, p. 199, and his Table VIII. Usnic Acid.—Antibiotic (Marshak and Harting, 1948; Marshak, 1949a; Marshak and Fager, 1950). : Vitamin C.—Ascorbic acid (Shapiro, 1948c). Washing Eggs Repeatedly —Delays cleavage (F. R. Lillie, 1914; Woodward, 1918; et al.). X-Rays.—Cleavage and development delayed. See X-Rays. COELOMIC FLUID See Perivisceral Fluid COMPOSITION OF EGGS For general analyses of centrifuged layers of crushed eggs, see Table 7 (McClendon, 190Qa). Solids.— 26.5 % (Ballentine, 1940a) ; 23.9 % (Hutchens, Keltch, et al., 1942). Ash.—8.5—10 % dry weight eggs (Page, 1927b); 7-8 % (Blanchard in E. N. Har- vey, 19324). Electrolytes —In millimoles per kilogram water in eggs, calculated by Rothschild and Barnes (1953): Na 321, K 354, Ca 269, Mg 1044, Cl'30, sulphate 0.0301 (Page, 1927b); K 96, Ca 38, Mg 17 (Blanchard, quoted by E. N. Harvey, 1932a). For Arbacia lixula; Na 280, K 162, Ca 16, Mg 41, Cl 384 (Bialaszewicz, 1929); Ca 17, Mg 21 (Monroy-Oddo, 1946). For other species, especially Paracentrotus lividus, sec Rothschild and Barnes (1953). Sea Water Electrolytes—Millimoles per kilogram) for 19 per thousand salinity are: Na 475, K 10, Ca (+Sr) 10, Mg 54 Cl 554, sulphate 29 (Rothschild and Barnes, 1953). Iron.—o.030 mg. per 10® eggs; 0.0005 millimoles (Page, 1927b). Copper.—17 wg. percc. of unripe ovarian eggs; 175 ug. per cc. of unfertilized and 21 wg. per cc. of fertilized eggs (Glaser, 1923). Total Phosphorus.—In millimoles per kilogram water in eggs is 181 (Page, 1927b). For Arbacia lixula 132 (Bialaszewicz, 1929). For other species see Rothschild and Barnes (1953). See also Phosphorus Metabolism for phosphorus fractions of egg. Total Nitrogen.—o.107 mg. per mg. dry weight (Ballentine, 1940a); 0.10 mg. Ng per mg. dry weight (Hutchens, Keltch, et al., 1942). See Nitrogen. Total Protein —65 % dry weight (Hutchens, Keltch, e¢ al., 1942). See Protein and Nucleoprotein. Total Fat.—See Oil and Lipids. Carbohydrate.—50 mg. (Perlzweig and Barron, 1928); 110 mg. acid hydrolyzable carbohydrate determined as glucose (7 % of dry egg weight) per gm. egg protein, of which 46 % is glycogen (Hutchens, Keltch, et al., 1942). Practically none from jelly. Glycogen content, 50-80 mg. per gm. egg protein (Blanchard, 1935). See Carbohydrate Metabolism. OF EXPERIMENTAL WORK 167 Reducing Sugar.—Absent (Perlzweig and Barron, 1928; Hutchens, Keltch, et al., 1942). Cholesterol.—Present (Mathews, 1913). Lactic Acid.— 3.14 mg. per gm. egg protein. 19 % increase in 4-8 cell fertilized eggs (Perlzweig and Barron, 1928), not confirmed by Hutchens, Keltch, et al. (1942). Echinochrome.—See Echinochrome. Enzymes.—See Enzymes and Cytochrome. Other Species and General References Bialaszewicz, 1927, 1929. A. lixula and Paracentrotus lividus, electrolyte content. Ephrussi and Rapkine, 1928, and Ephrussi, 1933. Paracentrotus lividus, protein, fat, carbo- hydrate, ash. Harvey, -E. N., 1932a. Constants, general. Krahl, 1950. General. Leitch, 1934b, 1936. S. franciscanus, S. purpuratus, nitrogen, lipoid, ash. Lindberg, 1943. Echinocardium cordatum, phosphate fractions. Malm and Wachtmeister, 1950. Psammechinus miliaris, S. drébachtensis, potassium content. Mitchinson and Swann, 1953. Ps. miliaris, 25.5% solids. Needham, 1931 and 1942. General. Orstrém and Lindberg, 1940. Paracentrotus lividus, carbohydrate content and metabolism. Pantin, 1931. Body fluids, composition. Rothschild and Barnes, 1953. Electrolyte content of many species. Stott, 1931. Echinus esculentus, carbohydrates. Tennent, Gardiner, and Smith, 1931. Echinomeira lucunter, lipids and glycogen. Wetzel, 1907. Paracentrotus lividus, per cent of solid, fat, Nj, P, ash. Zielinsky, 1939. Paracentrotus lividus, phosphate fractions and carbohydrate metabolism. CORTICAL LAYER Position.—A layer just beneath the plasma membrane and above the mass of cyto- plasm (Figs 9, 10). According to Parpart and Laris (1954) it is outside the plasma membrane. Properties.—A relatively rigid gel, comparatively clear; may be liquified reversibly (Danielli, 1942, p. 86). Its thickness is 0.8 u. in A. punctulata; 1 u to 2 u in some forms (Runnstr6ém and Monné, 1945a; Mitchison, 1952). Cortex of centrifuged unfertil- ized egg is bright in dark field (Moser, 1939a) and is birefringent (McCulloch, 1952a). Cortical layer contains calcium proteinate (Heilbrunn, 1952, p. 465, 538). Bound calcium in cortex of fertilized eggs can be released by potassium (Churney and Moser, 1940). Liquefaction By Micromanipulation (Chambers, 1935b, 1938b). Hydrostatic pressure (Brown, 1934; Marland, 1938, 1950, 1951). Decreased temperature (Mars- land, 1950). Stiffens.—With calcium (Heilbrunn, 1952, p. 97). Cortical Granules.—In cortical layer, they disappear 20 seconds after fertilization or parthenogenesis and help form the fertilization membrane. The granules are approxi- mately 0.8 u in diameter and are not displaced by high centrifugal forces, 6,000 x g (Moser, 1939a, b, 1940); 10,000 x g (E. B. Harvey, 1946a). The cortical reac- tion takes about 10 seconds at 25.7° C. (Moser, 1939a). These granules were first observed and described in Arbacia by E. N. Harvey (1911, p. 523) as minute granules unmoved by the centrifuge, which disappear on fertilization and whose substance helps to form the fertilization membrane. These granules were observed and figured in the unfertilized egg of Echinus esculentus, and their absence in the fertilized egg was notcd by Gray (1924, p. 169; by an error they are called ‘“‘micromeres” instead 168 ALPHABETICAL COMPILATION of ‘“‘microsomes’’ in the legend. They were described in stained sections of the Lytechinus (formerly Toxopneustes) egg by Hendee (1931), a student of Tennent’s, and are beautifully shown in the color of the stain used. Their disappearance upon ferti- lization is shown in her pictures of unfertilized and fertilized eggs. Apparently these same granules were described as “‘Janus green”’ granules in S. pulcherrimus by Moto- mura (1936). More recently and apparently without knowledge of previous literature, the cor- tical granules have been re-discovered and carefully studied in Arbacia by Moser (1939a, b, 1940), especially with regard to fertilization and parthenogenesis and to the relation of the granules to the fertilization membrane. Similar studies have been carried on by the Swedish school (See Runnstr6ém, Monné, and Wicklund, 1946; Runnstrém, 1948c; etc.). It seems now to be generally agreed that the fertilization membrane is formed by a combination of the cortical granule material with the vitelline membrane. Before maturation, the cortical granules are located inside the egg, not at the periphery (McCulloch, 1952b). This has been found to be the case also in Psamme- chinus miliaris and Brissopsis lyrifera (Runnstr6m and Monné, 1945a; Monné and Harde, 1951), and in the Japanese species Strong ylocentrotus pulcherrimus (Motomura, 1936, 1941b). For the structure of the cortical granules in Arbacia punctulata as shown by the elec- tron microscope see McCulloch (19524, b); Lansing, Hillier, and Rosenthal (1952). The cortical granules of Arbacia punctulata stain with Janus green (Motomura, per- sonal communication 1954). Pigment Granules—(Chromatophores) which have migrated to the periphery on fertilization are located in the cortex (Brown, 1934 and see under Chromatophores, location). Cytolysis.—Of cortical layer believed by Loeb to cause activation and membrane formation (Loeb, 1913a, Chap. 17, e€tc.). Movement.—Of cortical layer in fertilized eggs (Dan, 19514, b.). Effect of Methylated Xanthine.—On cortical granules (Cheney, 1951). Ribonucleic Acid.—Present in cortex (Lansing and Rosenthal, 1949). Other Species and General References Endo, 1952. Japanese sea urchins, cortical granules. Just, 1939b. Biology of the Cell Surface. Mitchison and Swann, 1952. Psammechinus miliaris, Paracentrotus lividus, birefringence and light scattering. Monné, 1948. General. Other references to Monné and co-workers given here. Monroy, 1947. Ps. microtuberculatus, birefringence. Other references to Monroy and co- workers given here. Monroy and Montalenti, 1947. Ps. miliaris, birefringence. Moore, A. R., 1949a, b, 1951 b. Strongylocentrotus purpuratus, pro-membranes in cortex; and general. Rothschild and Swann, 1949. Ps. miliaris, cortical layer on fertilization. Runnstrém, 1948a, c. Ps. miliaris, membranes and cortical granules. Runnstrém, 1949a, b; 1952a. General. Other references to Runnstrém and co-workers given here. Runnstrom, Monné, and Broman, 1944. Ps. miliaris etc. birefringence. Runnstrém, Monné, and Wicklund, 1946. Ps. miliaris, Echinocardium cordatum, S. drébachiensis, membranes and cortical granules. OF EXPERIMENTAL WORK 169 CYANIDES Fertilization Eggs can be fertilized while in KCN (Blumenthal, 1927, 1928; Just, 1928a; A. Scott, 1950). Effect on fertilization membrane (Just, 1928a). Development.—Of fertilized eggs continues slightly in KCN (Heilbrunn, 1920a; Blumenthal, 1928, 1930; Just, 1928a). Cleavage.—a. Arrest. Effective concentration to arrest cleavage in Arbacia punctulata according to different observers: M/10,000 KCN (Lyon, 1902). Few drops of 1/10 % NaCN to 50 cc sea water (Loeb and Wasteneys, 1g11a, Loeb, 19134, p. 26. M/8,000 KCN (R. S. Lillie, 1914b). 0.000,625 % KCN (Heilbrunn, 19204). N/1,000 KCN, least concentration used (Blumenthal, 1928). 5 x 10-5 M KCN (M. M. Brooks, 1933). 1.6 x 10-4 M KCN (Clowes and Krahl, 1940). 1.5 M HCN arrested cleavage and reduced respiration to 35 % of control. See Krahl (1950, p. 192 and his Table V). Effect of Cyanides together with other substances affecting respiration: Methyl- ene blue and KCN (Barron, 1929; Runnstrém, 1935a; M. M. Brooks, 1943). Toluidin blue and KCN (Barron and Hamburger, 1932). Pyocyanine and HCN or KCN Barron and Hamburger, 1932; (Runnstrém, 1935a; Korr, 1937, with tem- perature effects). Cytochrome oxidase and NaCN or KCN (Krahl, Keltch, Neu- beck, and Clowes, 1941; Ball, 1942). Phenols and KCN (Clowes and Krahl, 1934, b, 1935, 1936b; Krahl and Cieccs 1940; Clowes, 1951). Respiration of unfertilized eggs. Not much affected by cyanide (Runnstrém, 1935a; Korr, 1937; Ball, 1942; M. M. Brooks, 1943, 1946b). Reduced to 50% of control for unfertilized eggs with 1.3 x 10-? M KCN, 25 % of conirol for fertilized eggs (Clowes and Krahl, 1934b). Residual oxygen consumption reduced to 43 % of control for unfertilized eggs with 10-* M HCN, 18 % of control for fertilized eggs (Robbie, 1946b). For pyocyanine and cyanide on unfertilized eggs, see Runnstrém, 1935a); Korr (1937). Respiration and Cleavage of half-eggs. Unfertilized white halves little affected; in fertilized white halves, O, uptake decreased and cleavage inhibited; in unfertilized and fertilized red halves, O, consumption decreased (Shapiro, 1940). Permeability Increase (McClendon, 1909b). KCN decreases, HCN increases permeability (Blumenthal, 1927, 1928). M/800 to M/1000 has no effect on increased permeability following fertilization, though cleavage is inhibited; high concentra- tions, M/200, prevent the increased pcr neability following fertilization (R. S. Lillie, 1918a, b; Lucké and McCutcheon, 1932). Vasey —Increase (Heilbrunn, 1920<, b). Parthenogenesis.—Caused by KCN (McClendon, 1909b, ‘oo M. M. Brooks, 1946a, fertilization membrane, no cleavage). KCN aids parthenogenesis started with isotonic salt solutions (R. S. Lillie, 1911 b). KCN does not prevent. partheno- genesis caused by salts (R. S. Lillie, 1914a; Heilbrunn, 1915a). NaCN does not prevent parthenogenesis caused by ultraviolet light (Loeb, 19144). Reversal of Parthenogenesis.—After parthenogenetic treatment, NaCN causes eggs to return to resting stage according to Loeb (1913 4, p. 234, 1913c, 1914b, 1915a, b 1916, p. 190, etc.) with decrease in oxidations (Wasteneys, 1916). These eggs can be fertilized, with increase of oxidations. These results ‘have been questioned by F. R. Lillie and Just (1924, p. 505) and others. See Parthenogenesis, and p. 108. Effect on Sperm.—KCN inactivates and prolongs life of sperm (Cohn, 1918). 1 X 10-4 M HCN inhibits completely sperm respiration (Barron, Nelson, and Ardao 1948). See Respiration B II 4. Other Species Drzewina and Bohn, 1912. Paracentrotus lividus, sperm. Lindahl, 1941. Paracentrotus lividus. Loeb, 1907. Strongylocentrotus purpuratus. Loeb and Wasteneys, 1913b. S. purpuratus. Orstrém, 1932, b. P. lividus. Pease, 1941. Dendraster excentricus. Robbie, 1948, 1949. Echinarachnius parma, Tripneustes esculentus. Runnstrém, 1928b, 1930. Arbacia lixula, P. lividus. OF EXPERIMENTAL WORK I71 CYTOCHROME AND CYTOCHROME OXIDASE Cytochromes a, b, c—Occur in sperm, not detectable in eggs, of Arbacia (Ball and Meyerhoff, 1940; Ball, 1942; Clowes and Krahl, 1940; Krahl, Keltch, Neubeck, and Clowes, 1941). Cytochromes a and b, found spectroscopically in unfertilized eggs of Ps. miliaris (Rothschild, 1949). No cytochrome c yet detected (Borei, 1951, in Echinus esculentus egg). Cytochrome Oxidase.—(Indophenol oxidase, probably ‘‘Atmungsferment”’ of War- burg). Occurs in sperm and eggs (Ball and Meyerhoff, 1940, eggs doubtful;, Ball, 1942; Krahl, Keltch, Neubeck, and Clowes, 1941). In unfertilized and fertilized eggs in about same amount (Krahl et al., 1941). Indophenol oxidase activity as determined by Nadi reaction (Navez and E. B. Harvey, 1935): Whole eggs unfertilized _1.0 Whole eggs fertilized 1.4 White halves unfertilized 2.4 Whole eggs stretched by Red halves unfertilized 4.8 centrifugal force 3.2 Part of oxidase activity may be due to oil (Navez, 1938). In disintegrated eggs, more cytochrome oxidase associated with matrix than with mitochondria, yolk or pigment granules (Hutchens, Kopac, and Krahl, 1942). For temperature effects see Korr (1937). Inhibited by cyanide, carbon monoxide in the dark (reversal with light), sodium azide, sodium sulfide, 0.6 M NaCl (Krahl, Keltch, Neubeck, and Clowes, 1941; Ball, 1942). Other Species (additional) and General References Brachet, 1950. Chemical Embryology (scattered references. Deutsch and Gustafson, 1952. Ps. miliaris, in development. Krahl, 1950. Review, p. 182. Rothschild, 1948a. Echinus esculentus, sperm. Rothschild, 1949. Ps. miliaris, light and carbon monoxide on eggs. Rothschild, 1951 b. General. Symposium on Respiratory Enzymes, 1942, Univ. Wisconsin. CYTOLYSIS Term first used for marine eggs (Strongylocentrotus purpuratus) by Loeb (1904), ac- cording to Heilbrunn (1928, p. 239). Caused by—1. Ageing of eggs. (Goldforb, 1918a, b; Goldforb and Schechter, 1932; Schechter, 1936; Page, 19294). . Calcium chloride (isotonic) for a long time. (Heilbrunn, 1928, p. 147). . Carcinogenic substances, dibenzanthrene. (Lucké, Parpart, and Ricca, 1941). . Electric current. (Lillie and Cattell, 1925; Heilbrunn, 1928, p. 244). . Fat solvents. Soaps, saponin, chloroform, alcohol, bile salts etc. (E. N. Harvey, 1910c; Page and Clowes, 1922; Page, 1929a; Page, Shonle, and Clowes, 1933; Heilbrunn, 1928, p. 242; et al.). 6. Heat. (Heilbrunn, 1928, p. 249; Dan, 1936). With S. purpuratus eggs (von Knaffl- Lenz, 1908; A. R. Moore, 1910, 1917). 7. Hypertonic sea water. (Loeb, 1913 a, p. 89 for S. purpuratus). 8. Hypotonic sea water, distilled or tap water. 25 cc. sea water + 75 cc. distilled water for 2 to 3 minutes (Glaser, 1913); 40 volumes sea water to 60 volumes tap water for $ hour (R. S. Lillie, 1916b); distilled water 3-} minutes (Schech- ter, 1936) ; also Page and Clowes (1922); Heilbrunn (1928, p. 242); Just(1928a) ; et al. On fertilized eggs (R. S. Lillie, 1916b; Page, 19294). g. Shaking. (Tang and Gerard, 1932). 10. Sodium chloride. (E. N. Harvey, 1910c; Loeb and Wasteneys, 1910; Loeb, 1913, p. 180; Heilbrunn, 1928, p. 251; Mazia, 1933; Dan, 1936; et al.). op OF N 72 ALPHABETICAL COMPILATION 11. Ultrasonic waves. (E. N. Harvey, E. B. Harvey, and Loomis, 1928; E. N. Harvey, 1930; E. N. Harvey and Loomis, 1931). 12. Ultraviolet light. (Lillie and Baskervill, 1922; Hinrichs, 1927; Heilbrunn and Young, 1930; E. B. Harvey and Hollaender, 1938). 13. Visible light and rhodamine B. (L. B. Clark, 1940). Prevention By CaCl, and MgCl, (R. S. Lillie, 1911 a, 1912; Page, 1924). De- creased by pretreatment with excess Ca and increased with diminished Ca (Schech- ter, 1936). Decreased by KCN, chloral hydrate, chloretone, and other anaesthetics (R. S. Lillie, 1912; Loeb, 1913, p. 91 for S. purpuratus). Increase of Free Ca.—In egg on cytolysis (Heilbrunn, Mazia, and Steinbach, 1934). Acid Formation.—(Runnstrém, 1935qd). : pH.—Of cytolysed eggs (acid of injury), 5.3 + 0.2; normal eggs, 6.8 + 0.2 (Pandit and Chambers, 1932). Methylene blue, etc.—Reduced by cytolysed eggs more rapidly (Ballentine, 1938). Respiration Increased (Loeb, 1913a, p. 14; Tang, 1931a; Whitaker, 1933a, p. 487, footnote; Tyler, Ricci and Horowitz, 1938, but they think it may be due to bacteria present). See Heilbrunn (1915b). Permeability —Increased (E. N. Harvey, 1910c; A. R. Moore, 1917 for S. purpu- ratus). Viscosity.—Increased (Heilbrunn, 1928, p. 245). But said to be decreased in S. purpuratus (von Knaffl-Lenz, 1908; Loeb, 19134, p. 188; A. R. Moore, 1917). Membrane Formation.—Caused by all cytolytic agents (Loeb, 1913a, p. 8, etc.). Parthenogenetic agents cause incipient cytolysis (Heilbrunn, 1943, p. 661). Fertilized Eggs—As compared with unfertilized eggs. Sometimes cytolyse more readily, sometimes less readily according to the agent, time after fertilization (R. S. Lillie, 1916b; Page, 1929a) and the species (compare last references with Loeb, 1913a, p. 92 and Page and Clowes, 1922). Dark (or Black) and Pale (or White) Cytolysis.—(Loeb, 1913 a, p. 89-91 and Chapt. 17 for S. purpuratus ; Goldforb, 1918b). Dark cytolysis in Arbacia with certain soaps (Page, Shonle, and Clowes, 1933); pale cytolysis (Heilbrunn, 1928, p. 232, 239). White and black cytolysis in Echinus esculentus (Rothschild, 1938). Nature of Cytolysis—(Loeb, 1913a, Chapt. 17; A. R. Moore, 1917; Heilbrunn, 1928, p. 238-254 and 1943, p. 661). Autolysis.—(Lyon and Shackell, 19104). Other Species There are many other references to cytolysis in Arbacia and other species scattered through the literature. Mention is here made only of Hobson (1932) for Psammechinus miliaris and Lord Rothschild (1938) concerning the changes in the egg surface in Echinus esculentus during cytolysis. DENSITY — SPECIFIC GRAVITY Unfertilized Egg.—Density 1.081-1.087 (Lyon, 1907). 1.0485 to 1.0656 (Heilbrunn, 1926a, 1928, p. 71). With jelly 1.090 (E. N. Harvey, 1931c, 1932a). Without jelly 1.084 (E. N. Harvey, as above). Density of unfertilized egg is approximately same as 3 parts isosmotic (0.85 M) cane sugar solution plus one part sea water (E. N. Harvey, 1931C). Granules.—In unfertilized egg 1.14 (Heilbrunn, 1926a, 1928, p. 72). Clear Protoplasm.—(Matrix) of unfertilized egg 1.04 (Heilbrunn, as above). White Half-Egg.—Separated by centrifugal force 1.076 (E. N. Harvey, 1931¢, 19324). Red Half-Egg.—Separated by centrifugal force > 1.100 (E. N. Harvey, as above). OF EXPERIMENTAL WORK 173 Nuclear Fluid—Density about 1.04 (Heilbrunn, 1928, p. 85). Nucleus less dense than cytoplasm, goes to centripetal pole on centrifuging. Nucleolus.—Density about 1.14 (Heilbrunn, 1928, p. 85). Fertilized Egg.—2-cell and 16-cell. Same density as unfertilized egg (Lyon, 1907). Blastula.—Density about 1.060; heavier than sea water, lighter than egg (Lyon, 1906b, 1907). Pluteus.—Density between 1.055 and 1.066 (Lyon, 1907). Sea Water. Density in different localities Woods Hole .024 at 19.5° C. (Lyon, 1907) .02426 at 21.5° C. (Garrey, 1915) .0238 at 20° C. (E. N. Harvey, 19314) Tortugas .0246 (McClendon, 1g10b) .0244-1.0253 (Leitch, 1934a) .0241 at 12.9° C. (Bolin, from records, 1952) .0266 at 11° C, (Lowndes, 19442) .0278 at 16° C. (E. B. Harvey, 19334) Pacific Grove Plymouth, Eng. Naples, Italy ee en | DENSITY OF EGGS OF OTHER SPECIES Arbacia lixula Unfertilized egg with jelly = lor (E. B. Harvey, 1933a) without jelly 1.096 Echinometra lucunter Unfertilized egg 1.04 (Leitch, 19344) Echinus esculentus Similar to Ps. miliaris (Lowndes, 1944) Paracentrotus lividus Unfertilized egg with jelly 1.083 (E. B. Harvey, 1933a) without jelly 1.079 Psammechinus microtuberculatus With jelly 1.074 (E. B. Harvey, 19334) without jelly 1.072 Psammechinus miliaris Unfertilized egg 1.0725 (Lowndes, 19444) Fertilized, 1 hr. 1.0736 Blastula, 14 hr. 1.0758 Blastula, 20 hr. 1.0793 Gastrula, late, 44 hr. 1.0839 Echinopluteus, early, 68hr. 1.09626 Sphaerechinus granularis Unfertilized egg with jelly 1.083 (E. B. Harvey, 1933a) without jelly 1.081 Str. pulcherrimus Without fert. mem. 1.0772 (Hiramoto, 1954) With fert. mem. 1.056 DYES See Vital Dyes ECHINOCHROME See Amoebocytes, Chromatophores Name.—Given by MacMunn (1883, 1885, 1889) for the coloring matter in peri- visceral fluid, ovaries and shell of Echinus esculentus (?), E. sphaera, Strong ylocentrotus (Paracentrotus) lividus and Amphidotus (Echinocardium) cordatus. Called ‘‘arbacin”’ by Vlés and Vellinger (1928) in Arbacia aequituberculata (A. lixula). Occurrence.—In test, eggs, and red amoebocytes of Arbacia punctulata (McClendon, 1912a; Cannan, 1927; et al.). In test is probably present as Ca salt or adsorbed on CaCoO,; not readily extracted by organic solvents in which echinochrome is soluble. Can be obtained in solution by digesting shells in acid (Ball, personal communica- 174 ALPHABETICAL COMPILATION tion, Aug. 1954). In amoebocytes, eggs and plutei, is in chromatophores, though R. S. Lillie (1911b) thought some is diffused through the cell. Occurs only in Echinoidea among the Echinoderms (Fox and Scheer, 1941). Amount.—In eggs, 0.58 gm. pigment per 100 cc. eggs packed by centrifuging. In amoebocytes, 3.78 gm. per 100 cc. of packed body cells; same from ¢ and Q. In test 0.19 grams echinochrome per 100 gm. Average 2 contains 38 gm. echinochrome, 3 half this (Ball and Cooper, 1949). Chemical Structure.—C,.H,,O, (Ball, 1936, Ball and Cooper, 1949). Same for A. lixula (Lederer and Glaser, 1938; Hartman, Schartau, Kuhn, and Wallenfels, 1939). It is a polyhydroxynaphthoquinone, which in the eggs is bound to proteins (Kuhn and Wallenfels 1939; Wallenfels and Gauhe, 1943 in A. lixula). According to some investigators echinochrome contains copper (Glaser, 1923) ; fats (McClendon, 1g90ga, 19124); fatty acids (Navez, 1939). Soluble—In acetone, absolute alcohol, ether (McClendon, 1912a); in chloro- form, ether, benzene; insoluble in petroleum ether (Ball, 1934, 1936). Same for A, lixula (Lederer and Glaser, 1938); same for S. purpuratus (Tyler, 1939). Adsorbed.—On charcoal (Hinrichs, 1927); on norite (Navez and DuBois, 1940). Extraction and Crystallization Of echinochrome in A. punctulata (McClendon, 1912a; Cannan, 1927; Ball, 1934, 1936; Ball and Cooper, 1949). Forms reddish or orange needle-like crystals (McClendon, 1912a; Ball, 1934). Same for A. lixula (Lederer and Glaser, 1938; Glaser and Lederer, 1939; Kuhn and Wallenfels, 1939) ; and for S. purpuratus (Tyler, 1939). Color Change.—Red or orange in acid, violet or green in alkali (McClendon, 1910a, b). Red to yellow; pK,’ value at 26 °C. calculated to be 6.38; unstable in alkaline ranges from pH 6.8 (Ball, 1936; Ball and Cooper, 1949). In sodium citrate at pH 7.4 turns dirty brown then clear green (D. L. Harris, 1943). Faded by ultra- violet (Hinrichs, 1927; E. B. H., 1950 unpub.). Change of color used as a natural indicator of pH in A. lixula (Vlés and Vellinger, 1938). Absorption Spectra.—Studied by MacMunn (1883, 1885) in other species. In A. punctulata by McClendon (19124); Ball (1936); Ball and Cooper (1949). Absorp- tion spectra same for eggs, amoebocytes, and tests (McClendon, 19124); also for spines and plutei (Ball and Cooper, 1949). Red acid form has peaks at 255, 335; and 475 my; yellow form at 4 275, 400 and 475 mu (Ball, 1936; Ball and Cooper, 1949). For absorption spectra of A. lixula see Runnstrém (1928b); Lederer and Glaser (1938); Kuhn and Wallenfels (1939). Oxidation-Reduction.—Echinochrome does not form a dissociable compound with oxygen, but can be reduced (with sodium hydrosulphite) and re-oxidized. Ey = +0.1995 volts; and E,’ at pH 7 and 30 °C. = —o.221 volts (Cannan, 1927). For A. lixula see Lederer and Glaser (1938). In eggs, it is not reduced by anaerobiosis (McClendon, 1910b, 1912a; Cannan, 1927; Korr, 1939, p. 83; and Ball, same paper, p. 92). With regard tv a species having very little pigment, Cannan (1927, p. 187) says “‘it would appear that E. esculentus holds its pigment in the partially reduced state, since the perivisceral fluid is almost colorless but rapidly turns red when removed from the animal. In Arbacia, the echinochrome is in the oxidized state and I know of no observation of the spontaneous decolorization of the cells in vivo.” Function.—Not known. Was thought to be an oxygen carrier in other species by Geddes (1880) ; MacMunn (1885); Griffiths (1892a) ; but this was denied by Cuénot (1891 a). Has been questioned for A. punctulata by McClendon (1912a); Cannan (1927). Stated to increase respiration, 16 times, in Paracentrotus lividus and Sphaerechinus granularis (Friedheim, 1932), but this was denied by Tyler (1939) for S. purpuratus. Stated to be a sperm activating agent in A. lixula (Hartman, Schartau, Kuhn, and Wallenfels, 1939), but this was denied by Tyler (1939) for Strong ylocentrotus purpuratus OF EXPERIMENTAL WORK 175 and by Cornman (1941) for A. punctulata. See Hartmann, Schartau, and Wallenfels (1940). Fertilization.—Inhibited by echinochrome from red amoebocytes; has no effect on cleavage if eggs exposed after fertilization; binds calcium; causes agglutination of sperm (Couillard, 1952). Allee et al. (1942) also found no effect on cleavage. For release.—Of echinochrome from chromatophores, see under Chromatophores. Other Species (additional) and General References Baldwin, 1952, p. 184 General. Fox, 1953. Biochromes. Fox and Scheer, 1941. Pacific coast forms. Goodwin, Lederer, and Musago, 1951. Spinochromes, nomenclature. Goodwin and Srisukh, 1950. E. esculentus, P. lividus, 5 pigments. Goodwin and Srisukh, 1951. Echinocardium cordatum, echinochrome almost absent. Krahl, 1950. Review. Lederer, 1940. Review. ECTOPLASMIC LAYER See Hyaline Layer ELECTRICAL PROPERTIES AND EFFECTS I. Electrical properties of eggs. A. Membrane capacity and internal resistance (From Cole’s 1941 Table in Tabulae Biologicae, vol. 19, Part II, p. 25). Membrane Capacity Internal Resistance uf/cm? ohm /cm? Suspensions Unfertilized ca. 1.0 go (Cole, 1928) Fertilized ca. 1.0 go Unfertilized 0.73 186 (Cole and Cole, 1936; Fertilized 3.1 186 Cole, 1937, 1938) Unfertilized 0.86 147 (Cole and Spencer, 1938) Fertilized a 180 Single eggs Unfertilized Tat 180 (Cole and Curtis, 1938) Fertilized 2.8 210 White halves Unfertilized 0.63 125 (Cole and Curtis, 1938) Fertilized 2.25 125 Red halves Unfertilized 0.62-2.3 360-775 (Cole and Curtis, 1938) B. Surface charge; Zeta potential (Dan, 1933; see also 1931). Unfertilized eggs with jelly —34.1 + 0.47 millivolts Unfertilized eggs without jelly —30.3 + 0.47 millivolts Fertilized eggs without jelly —28.7 + 0.42 millivolts Unfertilized eggs without jelly, in dead sperm suspension —26.7 + 0.56 millivolts Cleaving eggs without jelly —27.2 millivolts 176 ALPHABETICAL COMPILATION Centrifuged eggs and fractions without jelly (Dan, 1936, IV) Whole eggs —30.8 + 0.54 millivolts Light halves —20.9 +- 0.69 millivolts Heavy halves —27.6 + 0.35 millivolts Effect of dilution and medium on Zeta potential (Dan, 1936, III). C. Miscellaneous. We rie Membrane resistance, unfertilized and fertilized eggs (calculated) > 25 ohm/cm? (Cole and Curtis, 1938); “‘1,000 a reasonable value” (Cole, 1940); > 100 ohm/cm? (Cole 1941). Electrical properties of cell membrane (McClendon, 1g1ob; R. S. Lillie, 1911 b, 1916b). Granules have +charge, surface layer — charge (Heilbrunn, 1923, 1926b, 1928, p. 183). Electrical changes on stimulation and cleavage (R. S. Lillie, 1903, 1909, 1916b). Increase (about one fourth) of electric conductivity of eggs on fertilization or parthenogenesis (McClendon, 1910a, c, 1912b). Electrical properties of sperm. Negatively charged. Cataphoretic velocity 1.75 uw/sec. per volt/cm. Surface p.d. 22.0 millivolts (Mudd, Mudd, and Keltch, 1929). Electrical effects on A. Unfertilized eggs. Move to anode (Moser 1939b). See above under B. Surface charge (Dan). Disintegrate, at anode first; unfertilized before fertilized (McClendon, 1g10a, b, c; Moser, 1939b). Parthenogenesis caused by induction shocks (McClendon, 1909b, 1910b). Parthenogenesis and development not caused by direct current (R. S. Lillie and Cattell, 1925). Cortical response and membrane elevation caused by direct current, at side toward anode first (Moser, 1937, 1939b). Permeability increased (McClendon, 1g10a, b, c, 1914b). Viscosity; transitory decrease, then increase (Angerer, 1939). B. Fertilized eggs. No relation between electrical conductivity of medium and cleavage until con- centration reduced to 20 %, then cleavage slower (R. S. Lillie and Cattell, 1923). Eccentricity of egg in fertilization membrane, nearer the membrane at anode (McClendon, 1g10b, 1914a; Dan, 1933). C. Nucleus. Goes to anode (McClendon, 1g10b). D. Chromatophores. Lose pigment (McClendon, 1910a, b.) Other Species Cole, 1935. Tripneustes esculentus. Cole, 1941. Tabulation in Tabulae Biologicae. Dan, 1934, Echinarachnius, surface charge. Biol. Bull. 66 : 247-256. Gray, 1916. Arbacia lixula, Psammechinus miliaris etc. lida, 1943a, b, c. Pseudocentrotus depressus, Strongylocentrotus pulcherrimus, capacitance. McClendon, 1910b. Lytechinus variegatus, Tripneustes esculentus. Rothschild, 1938. Echinus esculentus, biophysics of cell surface. Rothschild and Swann, 1949. Psammechinus miliaris, action potential. Vlés, 1931. Paracentrotus lividus, lysis. OF EXPERIMENTAL WORK 177 ENZYMES The classification is based on Sumner and Somer’s book on Enzymes (1953) 1. Proteolytic Proteolytic enzyme in granules of unfertilized egg (A. A. Woodward, Jr., 1949). Peptidase in matrix not in granules (Holter, 1936, 1949). Trypsin and chymotrypsin. In Psammechinus miliaris and Echinocardium cordatum, dissolve the vitelline membrane of the unfertilized egg, so that when fertilized, no fertilization membrane is formed, but the egg develops (Monné and Broman, 1944; Runnstrém, 1948a). Trypsin was used for A. punctulata by Runnstrém in 1950 and by Dan and Mazia in 1951 as the best method of obtaining fertilized eggs without fertilization membranes. Dan used 4 mg. crystalline trypsin in 100 cc. sea water for 10 minutes. Effect on membrane was first observed by Kunitz in 1932 (See A. R. Moore, 1949a, footnote p. 243; 1949b, footnote p. 207). Papain dissolves hyaline layer of developing eggs; no effect of ficin or trypsin (Northrop, 1947; E. B. H.). See under Hyaline Layer. ‘“‘Hatching enzyme’’, a protease (Sugawara, 19434) dissolves fertilization mem- brane of normal blastulae, in S. pulcherrimus (Ishida, 1936; Sugawara, 19434, b). In A. punctulata (Kopac, 1941). For earlier work on other forms, especially Ascidians, see Berrill (1929). Autolysis. See Lyon and Shackell (19104). In sperm and egg extracts. Negative results (Gies, 1901). See Loeb (1901, 19134, chapter 19; older references given here). Also see Rothschild, 1952. 2. Nucleases Polynucleotidase; localized in nucleus of egg, also in sperm; present in white half-egg, not in red half (Mazia, 1941). Withdrawn by Mazia in 1950. Ribonuclease, RNase. Drop in activity after fertilization which is maintained 20 hours (Bernstein, 1949; Krahl, 1950). Desoxyribonuclease, DNase. In eggs, half-eggs and sperm; not restricted to nuc- leus but distributed in cytoplasm in unfertilized egg; about equal in the two half-eggs; no change in development to pluteus, but sedimentable fraction increases; activity in egg is 10 times that of mammalian tissue. Activity in sperm is 10 times that of egg per unit volume, 10~‘ times per cell (Mazia and Neff, 1947; Mazia, Blumenthal, and Benson, 1948; Mazia, 19494, b.). Inhibition of DNase by usnic acid (Marshak, 1949a; Marshak and Fager, 1950). 3. Esterases Cholinesterase, in Paracentrotus lividus (Augustinsson and Gustafson, 1949). Lipase, lipolysin (A. E. Woodward, 1918, 1921; Glaser, 1921b, 1922a, 1923). Just (1929a, 1930a) thinks this does not exist. Runnstrém (1949a) could not find it in A. lixula. Phosphatase. In unfertilized, fertilized and developing eggs (Mazia, 1941; Mazia, Blumenthal, and Benson, 1948; Krugelis, 1947b. In oocytes (Krugelis, 19474, b). Not localized in nucleus as indicated by half-eggs (Mazia, 1941; Mazia, Blumenthal, and Benson 1948). Adenosintriphosphatase, ATPase. In S. purpuratus (Conners and Scheer, 1947); in P. lividus (Runnstrém, 19494, b). 4. Carbohydrases Hyaluronidase, in A. lixula sperm (Monroy and Ruffo, 1947). Hyaluronidase? in A. punctulata eggs (Chambers, 1949). These have been questioned by Krauss (1950). See also A. Monroy, L. Tosi, G. Giardina and R. Maggio, 1954, Biol. Bull, 106 : 169-177; and Rothschild, le eS ; 1a (“4,1 BRAR - A Tae AY bey > ’ T7ae,% J 2 178 ALPHABETICAL COMPILATION 5. Oxidizing enzymes and coenzymes Cytochrome oxidase. See Cytochrome and Cytochrome Oxidase. Catalase. Greater amount following fertilization; more in sperm than in eggs, observation of A. P. Mathews (Lyon, 1909). Not more after fertilization, but respiration 4—6 times greater (Amberg and Winternitz, 1911). Present in matrix not in granules in Ps. miliaris (Holter, 1949). Present in sperm (Evans, 1947; Barron, Gasvoda and Flood, 1949). Dehydrogenases (Korr, 1937; Ballentine, 1940b, in half-eggs, 1940c). Succinic dehydrogenase present in sperm not in eggs (Ball and Meyerhoff, 1940). Does not appear in eggs even after 24 hours (Goldinger and Barron, 1946). Flavin-adenine-dinucleotide, FAD, coenzyme (Krahl, Keltch and Clowes, 1940b; Krahl, 1950, p. 183). Diphosphopyridine nucleotide, DPN, coenzyme (Jahndorf and Krahl, 1942; Krahl, 1950, p. 183). Diphosphothiamine (cocarboxylase), coenzyme (Krahl, Jahndorf, and Clowes, 1942; Goldinger and Barron, 1946; Krahl, 1950, p. 184). Enzymes for oxidative phosphorylation (Clowes, 1951; Clowes, Keltch, Stritt- matter, and Walters, 1950; Clowes, Keltch, and Walters, 1951a, b; Keltch, Smythe, and Clowes 1951; Keltch, Strittmatter, Walters, and Clowes; 1950, Strittmatter, Keltch, Walters, and Clowes, 1950); Krahl’s Review, 1950, pp. 184, 198). 6. Transferases Hexokinase, in homogenates of eggs and embryos (Krahl, Keltch, Walters, and Clowes, 1953). Other Species (additional) and General References Barron, 1952a. General. Bohus-Jensen, 1950. P. lividus, Sphaerechinus granularis, ficin, trypsin. Bohus-Jensen, 1953. Lytechinus vartegatus, Mellita sexiesperforata, Echinometra lucunter, trypsin on cross fertilization. Cleland and Rothschild, 19524, b. E. esculentus, glycolytic. Deutsch and Gustafson, 1952. Ps. miliaris, decrease of catalase during development. Doyle, 1938. Ps. miliaris, peptidase and catalase. Gustafson and Hasselberg, 1950, 1951. Ps. miliaris, P. lividus etc., enzymes on developing eggs. Holter, 1949. General. Holter and Lindahl, 1941. P. lividus, peptidase. Holter and Linderstrém-Lang, 1940. General. Krahl, 1950. Review. Linderstroém-Lang, 1939. General. Lundblad, 1950. A. lixula, P. lividus, proteolytic enzyme. Mazia, 1952. General on nucleus. A. R. Moore, 1951 a. Dendraster excentricus, trypsin. Rothschild, 1950b, c. Echinus esculentus, catalase in sperm and eggs. Rothschild, 1951 a, 1952. Review of sperm. Runnstrém, 1949a, b, c; 1950-1951. General. FERTILIZATION MEMBRANE Definition —The fertilization membrane is the membrane normally formed after fertilization or activation by a parthenogenetic agent. Its precursor is the vitelline membrane. Historical.—The fertilization membrane was first described by Derbés in 1847 in Echinus esculentus, and later by Fol (1877) in Asterias glacialis. Origin.—Many of the older investigators believed it arose from the preexisting OF EXPERIMENTAL WORK 179 vitelline membrane (R. S. Lillie, 1911 a, 1916b; Kite, 1912; Heilbrunn, 1913, 1915a, 1928, p. 259; Glaser, 1913, 1915, 1924; F. R. Lillie, 1914; Chambers, 19214, 1924; et al.). But some thought it arose de novo, by secretion of a membrane substance (E. N. Harvey, 1909, 1910b, 1914) or by precipitation of oppositely charged col- loids (McClendon, 1909b, 1911, 1914a; see Garrey, 1919). More recently it has been definitely shown that the fertilization membrane comes from the vitelline membrane, since it does not form when the vitelline membrane has been removed by KCl, urea, or trypsin. (See below under “‘Removable’’) ; development takes place without a fertilization membrane. (See under Vitelline Membrane). It has also been shown that the cortical granules which disappear on fertilization help in the formation of the fertilization membrane (Moser, 1939a; etc.). See under Cortical Layer. It is now generally accepted that the fertilization membrane is the pre-existing vitelline membrane whose properties have changed, together with cortical granule material, in Arbacia and in other species (See Runn- strom, 1952a, Chapt. VII ‘The Origin of the Fertilization Membrane’’). Formation.—The fertilization membrane starts to form at the sperm entry in about 20 seconds after it touches the surface (E. B. H.; see also Just, 1928a, 1939b, p. 105; Moser, 1939a; et al.). See Part II, Fertilization. This was first observed in Asterias glacialis by Fol (1877). It is fully elevated about two minutes after fertilization at 23 °C. (E. B. H.). According to Heilbrunn (1913, 1915a, 1924a), membrane elevation is due to a lowering of the surface tension, since all parthenogenetic agents do lower the sur- face tension. According to Loeb (1913a, p. 212; 1912a, p. 136, 150), it is due to swelling of a colloid and liquefaction of the surface. According to E. N. Harvey (1g910b) and R. S. Lillie (19114) it results from increase in permeability. Jelly is not necessary for its formation (E. N. Harvey, 1914; F. R. Lillie, 1914; F. R. Lillie and Just, 1924, footnote p. 453; et al.). Oxygen necessary for formation of fertilization membrane in fertilized eggs, because it is necessary for motility of sperm (E. B. Harvey, 1930; Barron, 1932). Not necessary for membrane formation in partheno- genetic eggs (Loeb, 1913a, p. 215; Kitching and Moser, 1940). Structure—No regular structure or pattern is shown by the electron microscope (E. B. Harvey and Anderson, 1943). This is also true of Ps. miliaris, according to Mitchison (1953). Hillier, Lansing, and Rosenthal (1952) say that the membrane, in Arbacia, is composed of a single layer of loosely packed particles. Thickness—Though readily visible, its thickness is not measureable with a light microscope. Measured with an electron microscope, it is 250 A when first elevated and dried (E. B. Harvey and Anderson, 1943). According to Hillier, Lansing, and Rosenthal (1952), it is less than 300 A thick. A recent measurement with the elec- tron microscope, of the fertilization membrane of a different species, Ps. miliaris, gives its thickness as 100 A (Mitchison, 1953). The dry thickness as measured with an interference microscope is given as about 160 A (Mitchison and Swann, 1953). The early membrane is easily ruptured (R. S. Lillie, 1916b; E. B. Harvey, 1933b, et al.). It becomes thicker and tougher after about five minutes (Heilbrunn, 1915a; Chambers, 1921a; E. B. Harvey, 1933b; E. B. Harvey and Anderson, 1943; et al.). In Ps. miliaris its thickness is given as about 1u (Runnstrém, Monné, and Wicklund, 1946). Specific Gravity.—Lighter than the eggs. If placed in distilled water immediately after they are formed, the membranes can be freed of the egg material and form a layer above the eggs (E. B. Harvey and Anderson, 1943). Whitaker (1933a) found that sometimes they were thrown off in the centrifuge and then formed a layer above the eggs. Elasticity—Can stretch with centrifugal force from 82 yu diameter (normal) to 140 u when first formed; they resist stretching after five minutes (E. B. Harvey, 1933b; E. B. Harvey and Anderson, 1943). Expansibility (Chambers, 1942). 180 ALPHABETICAL COMPILATION Chemical Properties—A “‘haptogen’’ membrane consisting mainly of protein (R. S. Lillie, 1909). Probably an ‘‘albuminoid’’; insoluble in concentrated H,SO,, HCl, KOH, NaOH, etc. (E. N. Harvey, 1g10b). Not a lipoid because not scluble in ben- zol, ether, alcohol, saponin, etc. (Loeb, 1913a, p. 214). A protein gel with little or no lipid (Heilbrunn, 1915a). Insoluble in KCl, urea or trypsin after hardening, soluble while elevating (Kopac, 1940a, 1941a; Chambers, 1942, 1944; A. R. Moore, 1949a, p. 243 footnote, re Kunitz, 1932 unpub.). For Psammechinus miliaris see Monroy and Runnstrém (1948). Contains ribonucleic acid (Lansing and Rosen- thal, 1949). Oil Coalescence.—Prevented by fertilization membrane (Kopac, 1940a, 19414; Chambers, 1944). Permeability.—Freely permeable to salts of sea water, relatively impermeable to sugar and proteins (E. N. Harvey, 1g10b). Freely permeable to salts, impermeable to colloids like egg albumen and difficultly permeable to sugar (R. S. Lillie, rg11a). Permeable to water, salts and sugar, impermeable to colloids (Loeb 19134, p. 208; 1916, p. 108). Permeable to electrolytes when fully formed (Heilbrunn 1915 a). Distance from Egg Surface.—Normally 3 to 5 » (E. B. Harvey per E. N. Harvey, 1932a; E. B. Harvey and Anderson, 1943; et al.). May be 6.5 u (E. B. H.). It may be closely adherent under various conditions so as to be difficult to detect (by cold, 32 °C., E. N. Harvey, 1910b; Just, 19284; et al.), or it may be widely separated (by urea, Moser, 1940). Collapses in 1 or 2 % egg albumen (Loeb, 1913 a, p. 208; Heil- brunn, 1915a, 1924a; R. S. Lillie, 1918b); in 2% Witte’s peptone (Garrey, 1919) ; with blood albumen (Chambers, 1942). . Longevity —Membranes obtained from hatching blastulae dissolve almost at once (due to hatching enzyme present). Membranes obtained in distilled water may re- main intact for 12 hours (E. B. Harvey and Anderson, 1943). Function.—Not to prevent other sperm from entering as was originally suggested by Fol (1877), and maintained by many others (e.g., Kite, 1912). It is not necessary for development (McClendon, 1912b; Glaser, 1913; E. N. Harvey, 1914; Chambers, 1930; Loeb, 1915c, though earlier, 1913a, p. 233, be thought it was necessary). It is probably protective. See Plate XVI, Photograph 7. A Second Fertilization Membrane.— Many investigators have found that a fertilized egg cannot be fertilized again, even if the fertilization membrane has been removed (Loeb, 1916, p. 85; F. R. Lillie, 1919, p. 25, 161). Loeb (1913a, p. 234) however, thought that fertilization could be superimposed on artificial parthenogenesis, and a second membrane would form after the one due to parthenogenesis had been shaken off (Loeb, 1913a, p. 234, 1914b, 1915a, b). This was shown not to be the case, but to be due to insufficient treatment with the parthenogenetic agent, by C. R. Moore (1916, 1917); F. R. Lillie (1914, 1919, p. 167, 1921a); Just (19224): F. R. Lillie and Just (1924, p. 502). But more recently, Sugiyama (1947, 1951) in the case of fertilized eggs (Strong ylo- centrotus pulcherrimus), and Ishida and Nakano (1947, 1950) in the case of partheno- genetic eggs, state that refertilization can take place if the eggs are washed in Ca-Mg-free sea water after (usually) shaking off the first membrane. See Part II, Fertilization, Chapter 13, sections o and p. (p. 107, 108). Removable by:—(1) Shaking (McClendon, 1g10b; E. N. Harvey, 1911; F. R. Lillie, 1914; Plough, 1927; Kopac, 19404; et al.). Shake the eggs immediately after the fertilization membranes have formed, in a test tube about one quarter full of eggs and sea water, violently, with thumb over open end, for about 30 seconds (E. B. H.). (2) Straining through bolting cloth (Just, 1939a, b, p. 199 footnote). (3) Sucking through a fine pipette (Plough, 1927; E. B. Harvey, 1932). (4) Micro- manipulation (Chambers, 1942). (5) Distilled water, one minute after formation of membranes; empty membranes are recoverable (E. B. Harvey and Anderson, 1943). (6) Sea water from around hatching blastulae which contains “hatching OF EXPERIMENTAL WORK 181 enzyme”’ (Kopac, 1941 a); see Part II; Chapter 14a, BLASTULA. (7) Isosmotic KCl while membrane is elevating, 1.5 minutes after insemination (Kopac, 1940a; Chambers, 1942, 1944). (8) 1.0 M urea while membrane is elevating (Chambers, 1940, 1942; Kopac, 1940a, 1943; Moser, 1940; A. R. Moore, 1930a for Strong ylo- centrotus purpuratus). (9) 4 mg. crystalline trypsin in 100 cc. sea water for 10 minutes, or 0.1 % noncrystalline trypsin of Merck, as membrane is elevating (Dan, 1951 unpub.; see Runnstrém and co-workers for other species, especially Runnstrém, 1948a). See under Vitelline Membrane. A method for removing fertilization membranes from large quantities of eggs by bolting silk has been described by Lindahl and Lundin (1948) for Paracentrotus lividus. Effect of Ageing or Repeated washings—Membrane retarded or prevented (E. N. Harvey, 1914, after 52 hours; Goldforb, 1918a, b, after 42 hours; F. R. Lillie, 1914, after 11 to 33 washings). Effect of Calcitum-free Sea Water—As medium. No membrane is formed and there is no cleavage though sperm are active (Loeb, 1915a; E. B. H. unpub.). Also for Ps. microtuberculatus (Monroy, 1949). Calcium.—In medium not necessary for hardening of membrane (Chambers, 1942). Effect of Heparin—No membrane formed (C. V. Harding, 1951). Effect of Iodosobenzoic Acid.—And cytoplasmic fraction. Membrane thicker and change of birefringence; hatching inhibited, membranes do not dissolve (Monroy and Runnstrém, 1952; Runnstrém and Kriszat, 1952a,c). Effect of other SH- reagents (Runnstrém and Kriszat, 19524). Effect of Electric Current—On fertilized egg. Fertilization membrane nearer the egg at anode (McClendon, 1g1ob, 1914a; Dan, 1933). Effect of Ultraviolet ‘‘Blitz’’.—On unfertilized egg. Fertilization membrane forms on one side only (E. N. Harvey, 1942). See Spikes (1944) for Lytechinus pictus. In centrifuged eggs and fractions—Membrane thinner at centripetal pole of whole egg and white halves; very thin on clear quarters and breaks at oil cap if fertilized immediately after centrifuging. Thicker and closely investing on red half, mitochon- drial quarter and pigment quarter (E. B. Harvey, 1933b, 1940c, 1946). Other Species and General References Carter, 1924. Sphaerechinus granularis. Chase, 1935. Strongylocentrotus furpuratus, Dendraster excentricus. Endo, 1952. Japanese species. Gray, 1922. Ps. miliaris. Hobson, 19324. Ps. miliaris. Hyman, 1923. S. purpuratus, S. franciscanus. Just, 1919. Echinarachnius parma. Just, 1939b. The Biology of the Cell Surface. General. F. R. Lillie, 1919. Problems of Fertilization. General. F. R. Lillie and Just, 1924. Fertilization in Cowdry’s General Cytology. Loeb, 1912a. The Mechanistic Conception of Life. Loeb, 1913 a. Artificial Parthenogenesis and Fertilization. Loeb, 1916. The Organism as a Whole. A. R. Moore, 1930a, b; 1949a, b; 1951a. S. purpuratus, Dendraster excentricus. Monroy, 1949. Psammechinus microtuberculatus. Other references may be found in review by Runnstr6m 1949a. Motomura, 1934a, 1941b, 1950c. S. pulcherrimus, etc. Runnstrém, 1949a, b, c; 1952a, b. General. Other references to Runnstrém, Monroy, and co-workers may be found especially in 1949a. 182 ALPHABETICAL COMPILATION FERTILIZIN AND AGGLUTININ This subject with its vast literature will not be covered in this Monograph. Excellent reviews have recently been published to which the reader is referred. Bielig, H. J. and F. Medem, 1949. Experientia 5 : 11-30. Rothschild, Lord, 1951a. Biol. Rev. 26 : 1-27. Runnstrom, J. 1949a. Adv. in Enzymol. 9 : 241-327. Tyler, A. 1948. Physiol. Rev. 28 : 180-219; 1949¢. Am. Nat. 83 : 195-219. Reviews of the earlier work which the reader may consult are: Just, E. E. 1930. Protoplasma 10 : 300-342. Lillie, F. R. 1919. Problems of Fertilization. Lillie, F. R. and E. E. Just, 1924. Fertilization, in Cowdry’s General Cytology, section VIII. HEAT PRODUCTION Eggs.—Unfertilized, 0.08 calories per hour per million eggs; fertilized, (2-8 cell), 0.52 calories. At instant of fertilization, rate of heat production is 10-12 times that of unfertilized eggs, then decreases for 20 minutes, then constant until first cleavage, then drops and remains constant to 8-cell stage (Rogers and Cole, 1925). Sperm.—‘‘The heat production of Arbacia sperm is similar to that of an exothermic chemical reaction of the first order’’ (Rogers and Cole, 1925, p. 352). Other Species Meyerhoff, 1911. Paracentrotus lividus, eggs and sperm. Shapiro, 1948d. Compilation in Tabulae Biologicae. Shearer, 1922b. Psammechinus miliaris, eggs. Trurnit, 1939. Ps. miliaris, change during cleavage. HYALINE LAYER Hyaloplasmic Layer, Ectoplasmic Layer Definition —Hyaline layer is an investing layer of the egg formed after fertilization or parthenogenetic treatment; binds blastomeres together. Historical—Though previously observed by O. Hertwig (1876), Fol (1877, 1879) and Selenka (1878) ; it was first described by Hammar in 1896 as an “‘ectoplasma- tische Schicht”’ in Echinus miliaris ; ‘‘a clear, colorless homogeneous layer.”” This was confirmed by E. A. Andrews in 1897. It was described for Arbacia by G. F. Andrews earlier in the same year. Its importance as a ‘‘Verbindungsmembran’’ was shown by Herbst in 1900 in Echinus microtuberculatus. Thickness.—Very thin, less than 0.5 u when first formed, becomes gradually thicker until after 20 minutes it is two to three u thick (E. B. Harvey, 1934). Its thickness varies in different species. It is thicker in Arbacia lixula than in A. punctulata, and is here measureable immediately after fertilization; it can be shown by measuring that it is added to the surface (E. B. Harvey, 1933a, 1934). It is very thin in the starfish egg, there being no appreciable hyaline layer (Chambers 1921 a, 1930, 1940, etc.). It is intermediate in strength between A. punctulata and Asterias in the sand dollar egg (Echinarachnius parma) (Chambers, 1940). It is very thin in Echinarachnius parma (E. B. H. unpub.) and in Dendraster excentricus (Moore, 1928a). It is thin on the centripetal pole of the centrifuged egg, on the white halves and clear quarters; thick over the centrifugal pole and in the red halves, yolk and pig- ment quarters (E. B. Harvey, 1932, 1940c, 1946a). It is thickened and wrinkled OF EXPERIMENTAL WORK 183 in the cleavage furrow (McClendon, 1g10b; Painter, 1918; Just, 1928b; E. B. Harvey, 1934; Chambers, 1938c). It is increased in thickness by hypertonic sea water (E. B. Harvey, 1940a, Photograph 1, p. 205; Gray, 1924, 1931, p. 198, in E. esculentus). Structure.—Gelatinous film (Loeb, 1913a, p. 19); tough, sticky, fibrous, elastic; can be torn with micro-needles (Chambers, 1921a, 1930, 1940). Is probably a calcium proteinate (A. R. Moore, 1928a, 1949b in S. purpuratus); see Chapter 6 of Gray’s Experimental Cytology (1931). Stains—With isamine blue, toluidin blue (Kite, 1912). It does not stain with methylene blue, brilliant cresyl blue or neutral red (E. B. Harvey, 1934). Function.—Binds blastomeres together (Herbst, 1900 in Ps. microtuberculatus). For Arbacia punctulata see McClendon, ig10b; Chambers, 1921a, 1930, 1938c, 1940; E. B. Harvey, 1946a; et al. Important in cell division (Just, 1928a; Gray, 1924, 1931, p. 196 in E. esculentus). See Plate XVI, Photograph g. Ca-free sea water.—Dissolves. See Calcium for preparation. For Arbacia see (E. B. Harvey, 1934; Chambers, 1940; et al.). Isosmotic KCl.—Dispersed by (Chambers, 1940, 1944; Kopac, 1940a, 1941a). By lithium (Chambers, 1940). NaCl + KCl.—o.52 M in proportions 19 : 1 at pH 7; is non-toxic (Chambers, 1938c, 1940; Kopac, 19404). Papain, Trypsin and Ficin.—Dissolved by enzyme papain, not by trypsin or ficin (Northrop, 1947; E. B. H.). Bohus-Jensen (1950) also found it not dissolved by trypsin or ficin (in Ps. miliaris, etc.). There is disagreement about trypsin; Runn- str6m, Monné, and Broman (1944) found that trypsin did dissolve the hyaline layer in Ps. miliaris; and A. R. Moore (1951 a) in Dendraster excentricus, but not (1949 a, b) in S. purpuratus. There may be differences in the trypsin used or in the reaction of different species, or differences due to the time when the trypsin is used. Aceto-carmine.—Dispersed by aceto-carmine, forming spheres, which unite and form striations in perivitelline space (E. B. Harvey, 1950, unpub.). Urea.—Prevents formation (A. R. Moore, 1930a, 19494, in S. purpuratus). Twins.—When dissolved, in 2-cell stage twins are formed (E. B. Harvey, 19404; 1935b in Ps. microtuberculatus ; Loeb, 1909b in S. purpuratus). Plate XVI, Photo. 8. Hatching Enzyme.—Not dissolved by (Kopac, 1941 a). X-ray Effect—(Kopac, 1941b). Replaced.—After removal (E. B. Harvey, 1934, 1935b; Kopac, 1940a; Gray, 1931, p. 212, in E. esculentus). Centrifugal force.—Hyaline layer is thrown off as a crescent in Arbacia, as a ring in Ps. microtuberculatus, lying in the perivitelline space at the centrifugal pole. (Plate XVI, Photograph 6). This can be dissolved in calcium-free sea water, and re-precipi- tated in sea water (E. B. Harvey, 1934). Coalescence.—With oil drops, none (Kopac, 1940a, 1941a; Chambers, 1944). Permeability.—Freely permeable to electrolytes (Chambers, 1940; Gray, 1931, p- 198 in Echinus esculentus). i Other Species (additional) and General References Dan and Ono, 1952. Mespilia globulus. Dan, Yanagita, and Sugiyama, 1937. Mespilia globulus. Goldschmidt and Popoff, 1908. P. lividus, Ps. microtuberculatus. Gray, 1924. Echinus esculentus. Gray, 1931. Experimental Cytology, general; chapt. 6 and 9g. Just, 1939b. The Biology of the Cell Surface. General. Moore, A. R., 1949a, b; 1951b. Review. Morgan, 1927. Experimental Embryology, p. 138. General. 184 ALPHABETICAL COMPILATION HYBRIDS Hybrids of Arbacia Punctulata.— Arbacia functulata $ x Asterias forbesti 9. Few gastrulae, not maternal (Morgan, 1893). Mathews, 1901c questions this hybridization. Tyler and: Metz (1954) found that Arbacia eggs would not cross fertilize with Asterias sperm with or without trypsin treatment. Arbacia punctulata 2 x Echinarachnius parma 3. Plutei, maternal (E. B. Harvey, 1942). Difficult cross (Just, 1919, Matsui, 1924). Arbacia punctulata $ x Echinarachnius parma 2. Blastulae, maternal (E. B. Harvey, 1942). Plutei, not described (Just, 1919). Plutei, maternal; cytology (Matsui, 1924). Arbacia punctulata 2 x Lytechinus ( Toxopneustes) variegatus 3. Few plutei, inter- mediate; cytology (Tennent, 1912b, c). Abnormal plutei (E. B. Harvey, 1942 unpub.). Arbacia punctulata $ x Lytechinus (Toxopneustes) variegatus 9. Few plutei, inter- mediate; cytology (Tennent, 1912b, c). Arbacia punctulata? x Mellita pentapora 3. Plutei, more maternal (Tennent, 1g10b). Arbacia punctulata 2 < Moira atropos 3. Plutei, more maternal; cytology (Tennent, 1908, 1910b). : Arbacia punctulata § < Psam- mechinus miliaris. Harvey, E. B., 1933a. Naples species. Harvey, E. B., 1942. Echinarachnius parma x S. drobachiensis, maternal; S. franciscanus < S. purpuratus, maternal, but see A. R. Moore, 1943. Harvey, E. B., 1947. Lytechinus variegatus < Tripneustes esculentus, TELS Herbst, oe Horstadius, 1936c. Psammechinus microtuberculatus picasa Ting” Hultin, 1948a, b. Naples species. Matsui, 1924. List of hybrids. Moore, A. R., 1949c. Portugaliae Acta Biologica, Ser. A. Morgan, 1927. Experimental Embryology. General. Nimann, 1933. Naples species. Runnstrém, 1949a, 1952a. General. Shearer, De Morgan, and Fuchs, 1914. Psammechinus miliaris, Echinus esculentus and E. acutus. Tabulae Biologicae, 1930, VI : 514. Grimpe. Tennent, rg1ob. List of hybrids. Tennent, 1912a. Lytechinus variegatus < Tripneustes esculentus. Tennent, 1922. Cidaris tribuloides. Tennent, 1923. Japanese species. von Ubisch, 1937. Echinocyamus pusillus < Ps. miliaris. von Ubisch, 1939. Echinocardium cordatum X Ps. miliaris. Wilson, 1925. The Cell. HYDROGEN ION CONCENTRATION, PH Sea Watzr at Woods Hole.—pH usually taken as approximately 8.0 (Heilbrunn, 1943, p- 473). Other values are 8.1 to 8.3 (Redfield, 1948, etc.). See Ball and Stock (1937). pH in relation to CO, content (Henderson and Cohn, 1916; McClendon, 1916). 186 ALPHABETICAL COMPILATION Cytoplasm of Arbacia punctulata.—By injection of indicators pH of unfertilized and fertilized egg 6.8 + 0.2 (Pandit and Chambers, 1932). Wiercinski (1944) gives: pro- toplasm pH 6.2 + 0.2, hyaline protoplasm (white half-eggs) 5.8 to 6.8, granular material 5.4, nucleus above 7.0. Change of Intracellular pH.—In salts of weak acids and weak bases (Jacobs, 1940). See Smith and Clowes, 1924; Haywood and Root, 1930, 1932 for GO,. For Echina- rachnius parma eggs with CO, and NHs, see Chambers, 1928. Other eggs.—Paracentrotus lividus and Echinocardium cordatum, pH of interior, unfer- tilized and fertilized eggs to 16 cell stage is 6.6 (Needham and Needham, 1926a). But according to Vlés (1924), Vlés and Vellinger (1928) et al., the pH of the eggs of Paracentrotus lividus and Arbacia aequituberculata (lixula) is about 5.5. Rapkine and Wurmser (1926) found the pH of cytoplasm and nucleus of P. lividus oocytes the same, around 7.0. For a discussion of the controversy see Reiss’ Monograph (1926) and Needham’s Chemical Embryology, Vol. II, p. 839-855 (1931). Blastocoel—pH of blastocoel of A. punctulata, in blastula, gastrula and pluteus is same as sea water (Chambers and Pollack, 1927). But according to Rapkine and Prenant (1925) the pH of blastocoel of P. lividus and Echinocardium cordatum is 7.0 to 7.3 in blastula and pluteus; 8.5 in early gastrula when spicules form. See Needham (1931, p. 846-849). Cytolysed eggs.—pH in A. functulata is 5.3 + 0.2 (Pandit and Chambers, 1932). Effect of pH of Surrounding Medium.—On hyaloplasm of egg, none unless injured (Wiercinski, 1944). On fertilization, block to fertilization in medium of pH 6.8 and below (Clowes and Smith, 1923, Smith and Clowes, 1924d). On cleavage and development, normal cleavage rate (if fertilized in sea water) at pH 8.2 to 5.8. Op- timum for fertilization, cleavage and viability pH 6.0 (Clowes and Smith, 1923, Smith and Clowes, 1924b, c). Acceleration of cleavage rate or development if medium is slightly alkaline (Loeb, 1898, but see 1913 a, p. 35; also Glaser, 1914b; pH 8.2 to 9.2 (Smith and Clowes, 1924c). Retardation of cleavage below pH 6.0 and above 9.4 (Smith and Clowes, 1924c, d). Effect of pH on pluteus (Child, 1916b, Medes, 1917). On polyspermy, maximum near pH 7.2, none at 7.4 to 9.8 (Clowes and Smith, 1923, Smith and Clowes, 1924d). On respiration, diminished by low pH (Root, 1920; Anfinson, 1947). Increased by alkalinity (Wasteneys, 1916). On permeability, no swelling till injury at pH 4.0; none at 9.8 (Lucké and McCutcheon, 1926b). See Permeability. On viscosity (Barth, 1929; Howard, 1931). On parthenogenesis, slight if pH more than 9.0 (Smith and Clowes, 1924b). On cytolysis, greatest in medium of pH 9.3 (Smith and Clowes, 1924b). On sperm, effects on (Cohn, 1918). E ffects of Substituted Phenols.—In relation to pH (Clowes and Krahl, 1936a; Krahl and Clowes, 1938; Hutchens, Krahl, and Clowes, 1939). Effects of Barbituric Acid Derivatives—In relation to pH (Clowes, Keltch, and Krahl, 1940; Krahl, 1950). Effect of Local Anaesthetic Bases.—In relation to pH (Krahl, Keltch, and Clowes, se Other Species (additional) and General References Ashbel, 1931. A. lixula and P. lividus. Gray, 1931. Experimental Cytology, p. 85-87, general. Heilbrunn, 1943. An Outline of General Physiology, p. 47-55; 473-476; enzymes, p. 199. Hirabayashi, 1937. Toxopneustes pileolus, blastocoel. Lison, 1935. General; 1941, Tabulation. Moore, B., Roaf and Whitley, 1905. Echinus esculentus, rate of cleavage increases with slight alkalinity. Needham, 1931. Chemical Embryology, vol. II, general. Needham and Needham, 1926b. Review. Reiss, 1926. Review Monograph. OF EXPERIMENTAL WORK 187 INFRARED LIGHT No experimental data for Arbacia punctulata, but in Strong ylocentrotus franciscanus and purpuratus, fertilizability decreased with wave lengths 0.8 to 1.2 uw (Nelson and S. C. Brooks, 1933). Photographs of eggs, half-eggs, and nuclei of A. punctulata with infrared light (E. B. Harvey and Lavin, 19514, b). Reference Giese, 1947. General on radiations. INTERFACIAL TENSION Oil-Protoplasm; Oil-Water. See under Oil (Coalescence) INTRAVITAM DYES See Vital Dyes JELLY LAYER Definition. A gelatinous layer on the outside of the egg, often called the zona pellucida or chorion (Plate XVI, Photograph 3 and Figs 9, 10). Observed in Echinus esculentus by Derbés in 1847, and called by him /a couche mucilagineuse. Funnel-shaped micropyle present in jelly. Visibility.—Not visible in sea water because of its refractive index, but its presence is indicated by the spacing between individual eggs. If eggs are contiguous, there is no jelly. Jelly and micropyle can be demonstrated by India or Chinese ink (old method of Boveri, 1901), or squid ink. The jelly is readily shown by a slight tinge to the sea water of Janus green or toluidin blue (cause shrinkage after some minutes) ; also by the halo of sperm caught in the jelly after a heavy insemination. It has been described as radially striated as it comes from the ovary, then becoming invisible (McClendon 19144); as a fibrillar network (Chambers, 1933). Thickn2ss.—Is 28 to 32 wp; the average volume ca. 1,050,000 u3. It swells to 60 u with squid ink (E. B. H.). The jelly of Ps. miliaris has been found to swell to double its width with rabbit serum and certain amino acids (Runnstrém, Monné, and Wicklund, 1946), and also with versene (Borei and Bjorklund, 1953) ; this also caused swelling of the Lytechinus egg jelly (Tyler, 1953). For the chemistry of squid ink, see Ball and Ramsdell (1940). ee Specific Gravity—Lighter than the egg, goes to centripefal pole on centrifuging (Shapiro, 1935c; E. B. H. unpub.) (Plate XVI, Photograph 4). Presence.—In oocytes as well as mature eggs (F. R. Lillie, 1914, 1919, p. 119). Not present in oocytes under 60 » diameter and may remain till hatching (E. B. H.). Not necessary for fertilization membrane (F. R. Lillie, 1914, 1915a; E. N. Harvey, 1914, et al.). Some early observers thought it necessary (McClendon, 1911, 19144). See F. R. Lillie and Just, 1924, p. 453, footnote. Electric Charge.—Negative, is acidic (McClendon, 1911, 1912b). For more recent work see Runnstrém, Tiselius, and Vasseur (1942). Permeability changes.—No effect (R. S. Lillie, 1917). Loss of Felly—On standing in sea water (many observers). Loss of jelly with age (R. S. Lillie, 1917; Goldforb, 1918a, b; et al.). Not Replaced.—When removed (R. S. Lillie, 1921; et al.). Contains Agglutinins.—(Fertilizin), but not in immature eggs (F. R. Lillie, 1914, 1915a, 1919, p. 117, etc.; Glaser, 1914b; Loeb, 1915a; Frank, 1939; e¢ al.). For 188 ALPHABETICAL COMPILATION presence of fertilizin in jelly of immature eggs of other species see Vasseur (1952, p. 26). Tyler (1941b) considers fertilizin identical with jelly in other species (S. pur- puratus) ; also Runnstr6m (19524, p. 45). Fertilization.—Aided by jelly (F. R. Lillie, 1914; Tyler, 1941b for S. purpuratus). Takes up Chlorine.—From sea water (Glaser, 1922b); takes up copper (Glaser, 1923). Takes up calcium (radioactive) (Rudenberg, 1952). CaCl,.—Makes it sticky (Page, 1929b). Is Precipitated.—By cytoplasmic fraction from frozen eggs (antifertilizin) (Monroy and Runnstrém, 1952), previously shown for S. purpuratus by Tyler (19404). Chemistry of Jelly—Jelly is a hyaline proteid dissolving in sea water; mucin (McClendon, 1909a, 1912b). For other species, Vasseur (1948a) gives about 20 % protein and 80% polysaccharide esterified with sulphuric acid; the composition varies in different species. Tyler (1949c) gives for the composition of fertilizin (jelly) of S. purpuratus approximately equivalent amounts of amino acids (> 20%), reducing sugar (> 25 %) and sulphate (23 %). Coalescence with Oil Drops.—Inhibited (Kopac, 19404). Stains.—With Janus green, Janus dark blue B, saffranin O, thionin, toluidin blue (E. B. Harvey, 1941c). According to McClendon (19144) it stains with methylene blue and neutral red. Stains with acridine orange, but this is very toxic. Table 8. Can Be Removed.—By washing and agitation (McClendon, 1914a; F. R. Lillie, 1914; E. N. Harvey, 1914; R. S. Lillie, 1917; e¢ al.). Straining through bolting silk (Just, 1928 a, 1939a; Shapiro, 1935c; et al.). Fine pipette. Acid; 1.4 cc. N/1o HCl + 50 cc. sea water (F.R. Lillie, 1915 a; et al.). I use one drop N/10 HCl, from a medium pipette, to 50 cc. sea water, then wash well (E. B. Harvey, 1941c). Alkali (McClen- don, 1909b; Barth, 1929). Calcium-free sea water (E. B. H.). NaCl, isosmotic (0.54 M) (R. S. Lillie, 1921; Kopac, 1940a). KCl, isosmotic (0.53 M) (E. B. H. unpub.; Page, 1929b). NaCl + CaCl, 17:1 (R. S. Lillie, 1921). NH,Cl (Kopac 1948 a). It is also removed by trypsin, chymotrypsin, and papain, in other species (Tyler, 1940b in S. purpuratus; Runnstrém, Tiselius, and Vasseur, 1942, and Min- ganti, 1953, in Ps. miliaris). Centrifuging (McClendon, 1914a; Shapiro, 1935c¢; E. B. Harvey, 1941); this is variable and not reliable. X-rays (E. B. Harvey, 1941 c; Evans, Beams, and Smith, 1941). Ultraviolet light (E. B. H., 1950 unpub.) ; in Para- centrotus lividus (Tchakotine, 1921 a). For centrifuging see Plate XVI, Photograph 4. Other Species (additional) and General References Hobson, 1927. FE. esculentus, Ps. miliaris. Monné, 1944a. Ps. miliaris, birefringence. Motomura, 1950b. S. pulcherrimus. Runnstrém, 19494. Review. Runnstrém, 1952a. Review in Symp. Soc. Exp. Biol. VI. Tyler, 1948. Review. Vasseur (et al.). Many papers summarized in independent publication printed by Kihl- stroéms Tryckeri, 1952; most of these are listed by Runnstrom 1952a. LEUCOCYTES See Amoebocytes, Perivisceral Fluid Occurrence.—In perivisceral fluid, together with amoebocytes (Geddes, 1880; Cuénot, 1891 a in other species; Kindred, 1921, 1926; H. V. Wilson, 1924; Donnellon, 1938 in A. punctulata). Two kinds distinguished by Liebman (1950), phagocytes and tre- phocytes. OF EXPERIMENTAL WORK 189 Shape.—Irregular with long filamentous processes or pseudopods (Kindred, 1921, 1926; H. V. Wilson, 1924; Liebman, 1950). Phagocytosis and Clotting —(Kindred, 1921, 1926; H. V. Wilson, 1924; Donnellon, 1938; Liebman, 1950). Trephocyte material taken up by oocytes (Liebman, 1950). Other Species Kindred, 1924. S. drébachiensis. LIPIDS See also Oil In eggs.—Cholesterol present in Arbacia egg, not in Astertas (Mathews, 1913; Page, 1923, 19274). Cephalin, more; lecithin, less in Arbacia than in Asterias (Page, 1927a). No change in lecithin (?) content between 2-cell stage and blastula (Shackell, 1911); this was criticised by Robertson and Wasteneys (1913) who found decrease in lecithin in Strong ylocentrotus purpuratus. Amount. A. In alcohol-ether extract, 8.3 gm. of oil from 183 million eggs; 1.539 gm. of acetone-insoluble material with high percentage of cephalin. Iodine number of oil 146-148, saponification value approximately 606 (Page, 19274). B. Total fat (all material soluble in petroleum ether) in a million unfertilized eggs is 5.65 mg; sterol 0.43 mg. or 7.5 % of total fat; phospholipid 2.17 mg. or 38 % of total fat. Total fat decreases up to time of hatching (8 hrs.), increases for 10 hrs., then decreases; sterol unchanged and phospholipid uncertain. Loss of total fat in 43 hrs. is 3.54 mg. per million eggs (Hayes, 1938). C. Total lipid (“‘lyophylled” eggs), 5.4% of whole egg, 26.9 % of solids in egg; 77 % of total lipid is probably bound to protein. No change in total or bound lipid in 5 hrs. development (Parpart, 1941). Crude oil obtained with fat solvents is reactive with Nadi reagent. I, index 180- 190, saponification index around 200. Sterols and phospholipids present, also fatty acids and glycerids (Navez, 1938, 1939; Navez and DuBois, 1940). In Centrifuged Eggs.—(Ether extract). Amount. Whole eggs 2.254 % total fat; cen- tripetal layer of centrifuged crushed eggs 0.3 %; centrifugal layer 1.946 % (McClen- don, 190ga). In Half-Eggs.—(Obtained by centrifuging). White half-eggs: free fats and sterols (ether extract) 2.2 mg. per million halves; bound lipids (alcohol-ether extract) g.6 mg. per million halves. Red half-eggs: free fats and sterols 6.6 mg. per million halves; bound lipids 12.2 mg. per million halves. 75 % of free fats and sterols, 56 % of bound lipids are in red half, or 61.6 % of total lipids (Hunter and Parpart, 1946). In Nucleoli.—Lipids and phospholipids not present in nucleoli of Arbacia, as deter- mined by staining (Gates, 1941). In Sperm.—Lipoids and lipoproteins present in acrosome, middle piece and tail, on the surface (Popa, 1927). Lipids in alcohol-ether extracts do not contain agglu- tinating substance; this is in protein residue (Frank, 1939). Effect on The Eggs:—Of fatty acids. Cause parthenogenesis, especially butyric (Loeb, 1913a, p. 71, etc., e¢ al., see under Parthenogenesis. Decrease viscosity (Howard, 1931). Of soaps. Different effects by different soaps on cytolysis, pigment discharge, stratification and breaking with centrifugal force and on response to microdissection (Page, Shonle, and Clowes, 1923). Other Species (additional) and General References Cleland and Rothschild, 1952a. Echinus esculentus, eggs, analysis. Ephrussi, 1933. Paracentrotus lividus, analysis. 1g0 ALPHABETICAL COMPILATION Leitch, 1934b. Strong ylocentrotus purpuratus, S. franciscanus, analysis. Needham, 1931. Chemical Embryology. General, vol. I : 310, 347; vol. II : 1244. Ohman, 1945. Ps. miliaris, Echinocardium cordatum, lipid layer, and analysis. Rothschild and Cleland, 1952. Echinus esculentus, sperm, analysis. Tennent, Gardiner, and Smith, 1931. Echinometra lucunter, microchemistry, and analysis. Wetzel, 1907. Paracentrotus lividus, analysis. LITHIUM Occurrence.—Lithium is present in tissues of Paracentrotus lividus and Echinus esculentus (Fox and Ramage, 1931). Li enters eggs and embryos of Sphaerechinus granularis (Ranzi and Falkenheim, 1937). LiCl Isotonic—With sea water at Woods Hole is approximately 0.56 M (Page, 1929). 0.60 M (M. B. L. Chemical Room). Effect of Li on Eggs.—General effect is exogastrulation and increase of entoderm (vegetalization) as first shown by Herbst (1892) for Echinus (Psammechinus) micro- tuberculatus, Strong ylocentrotus (Paracentrotus) lividus and Sphaerechinus granularis. Exo- gastrulation in Arbacia punctulata obtained with N/80-N/1oo LiCl (MacArthur, 1924); with 20 parts 0.54 M LiCl and 8o parts sea water for 4 hours (Costello, 1948). Toxicity.—Eggs will develop in 5 cc. of 2.6% LiCl + 100 cc. sea water for 20 hours; more toxic for Echinarachnius parma ; effect can be counteracted by potassium or pyocyanine (Runnstrém, 1935b). But Moore, Bliss and Anderson (1945) found this not true for S. purpuratus and Dendraster excentricus, that pyocyanine effect is addi- tive to Li effect. Toxic effect of Li is mitigated by K, Rb and Cs (Loeb, 1920). Hyaline Layer.—Is softened and dispersed (Runnstrém, 1935b; Chambers, 1940). Pigment Granules.—LiCl prevents breakdown of pigment granules by Ca (surface precipitation reaction) in order of NH, > Na > K > Li (Heilbrunn, 1928, p. 230). Respiration.—No references found for Arbacia. Li reduces respiration in Paracen- trotus lividus (Lindahl, 1936; Lindahl and Ohman, 1938); in S. purpuratus and Den- draster excentricus (Moore, Bliss, and Anderson, 1945). Permeability.—Increase (Lucké and McCutcheon, 1929). Parthenogenesis.—Slight stimulation with LiCl (Loeb, 1g00a; Hollingsworth. 1941). Replacement.—Lithium can be replaced by many substances in causing exogastru- lation, e.g., NaCl, CuCl,, NiCl,, methylene blue and other dyes and substances. For specific references see Child’s Patterns and Problems of Development (1941, Pp. 222 footnote) ; and Child (1948). Other Species (additional) and General References Child, 1936a, 1940, 1941. S. franciscanus, S. purpuratus, Dendraster excentricus ; axial gradient. Herbst, 1892, 1893, 1896. Ps. microtuberculatus, P. lividus, Sphaerechinus granularis. Horstadius, 1936a, b. P. lividus. Lindahl, 1942. Review. MacArthur, 1924. S. franciscanus, Echinarachnius parma. Moore, A. R., 1930c; Moore, Bliss, and Anderson, 1945. S. purpuratus, Dendraster excentricus ; lithium and pyocyanine on development and respiration. Needham, 1942. Biochemistry and Morphogenesis, p. 485; general. Rulon, 1946. Dendraster excentricus ; lithium and sodium thyocyanate. Runnstrém, 1928a. P. lividus. von Ubisch, 1929. Echinocyamus pusillus. Waterman, 1932. P. lividus. OF EXPERIMENTAL WORK IgI MAGNESIUM Amount in Egg.—4.48 mg. magnesium per 10° eggs (10° eggs = 0.124 gm. dry weight), or 0.182 millimoles (Page, 1927b). Amount in Sea Water—At Woods Hole 1.3004 gm. per liter at 20° C. (Page, 1927C, 1928). MgCl, Isotonic.—With sea water at Woods Hole is 0.30 M (M. B. L. Chemical Room). Also given as 0.37 M. MgSO, isotonic with sea water at Woods Hole may be 0.81 M (M. B. L. Chemical Room). Also given as 0.52 M. Surface Precipitation Reaction —Mg acts like Ca but is less potent (Heilbrunn, 1930, 1934, 1943, P- 470, 538). Cytolysis.—Prevented by MgCl, (R. S. Lillie, 1911a, b; Page, 1924). Mg Counteracts NaCl.—And other Na and K salts (Loeb, 1900a; Mathews, 1905; R. S. Lillie, t911a, b, 1914a; McCutcheon and Lucké, 1928). Mg is antagonized by Ca (Heilbrunn, 1934; Hollingsworth, 1941). Colorless Amoebocytes of Body Fluid.—Dissolved by MgCl, (Mathews, 1900). Amoeboid Eggs.—Caused by MgCl, (Loeb, 1900a; Churney, 1940). Effect of Mg on Fertilized Eggs.—Nuclear division without cell division (Loeb, 1895b, 1900a; Norman, 1896). Astropheres present also (Morgan, 1899). Mg as an Anaesthetic.—MgSO, widely used to quiet marine organisms (Mayor, 1909; R. S. Lillie, 1910, 1916c; Heilbrunn, 1934, 1943, p. 460; 1952, p. 528). First used by Tullberg in 1892 (see last reference of Heilbrunn). Mg anaesthesia counter- acted by Ca (Heilbrunn, 1943, p. 531). Magnesium sulfate can be used on blastulae and plutei of Arbacia. ‘‘Methocoel’’, methyl cellulose, also quiets cilia (Marsland, 1943) ; also Chloretone. See McClung’s Microscopical Technique, 1950, p. 436. Respiration —No references found. Permeability.—Decrease (R. S. Lillie, 1910; McCutcheon and Lucké, 1928; Heil- brunn, 1943, p- 531). Viscosity.—(Better stratification with decreased viscosity). MgCl, decreases visco- sity (Heilbrunn, 1923, 1928, p. 147, 1943, p. 81; E. B. Harvey, 1945). Isosmotic solutions arranged in order of their effect in decreasing viscosity (better stratification): CaCl, > MgCl, > sea water > NaCl > KCI (E. B. Harvey, 1945). Heilbrunn 1928, p. 147) gives them in the same order except that K and Na are reversed. Breaking with Centrifugal Force.—Break less readily in MgCl, than in sea water. Isosmotic solutions arranged in order of their effectiveness in causing breaking into halves, proceed in the reverse order from that given above for decrease in viscosity ; the greater the viscosity (less stratification), the more readily they break (E. B. Harvey, 1945). Parthenogenesis.—MgCl, added to sea water, used first by Morgan (June, 1899, 1900a, b) for parthenogenesis, then by Loeb (October, 1899, 1g900a, b); plutei first obtained by Loeb (1899; see Loeb 19134, p. 53, 57, etc.). R. S. Lillie (1g10, 1g11a, 1914a) found isotonic MgCl, did not activate. Hollingsworth (1941) obtained slight parthenogenesis with isotonic MgCl,, but inhibition of its activation by CaCl). Other Species (additional) and General References Bialazewicz, 1927, 1929. A. pustulosa (lixula) and Paracentrotus lividus, electrolytes. Heilbrunn, 1952, p. 528. General Physiology, general. Herbst, 1904. Salts necessary for development. Loeb, 1913 a. Artificial Parthenogenesis, especially for S. purpuratus and S. franciscanus. Robertson and Webb, 1939. Amount of Mg in sea water and body fluids. Rothschild and Barnes, 1953. P. lividus, amount in egg; table of salts and species. Wilson, 1901 a. Toxopneustes (Lytechinus) variegatus, cytology. 192 ALPHABETICAL COMPILATION METABOLISM See Respiration MITOCHONDRIA Size.-—Smallest granules that can be moved by centrifugal force, 0.6 to 1.0 u (E. B. H. per E. N. Harvey, 1932a; E. B. Harvey, 1936). With electron microscope 0.3 to 0.5 up, probably (McCulloch, 19524). Density.—Lighter than yolk and pigment granules; form a narrow band above the yolk in centrifuged eggs (first described by E. N. Harvey, 19322 as the “‘fifth layer’; E. B. Harvey, 1932, 1936, etc.; e¢ al.). It is the last layer to form with centrifuging, and the first layer to redistribute, disappearing five to ten minutes after centrifuging (BE. B. H:)- Amount in Egg.—4.8 % (E. N. Harvey, 1932a); 5 % (Costello, 1939). Origin.—Normally mitochondria appear in the immature egg when it is about 33 w in diameter, at the same time as the pigment and oil; a few scattered granules sometimes appear when a little smaller, 23 u. In the immature egg of all sizes, the mitochondrial granules are concentrated around the germinal vesicle, and are not displaced by strong centrifugal force (10,000 x g) (E. B. H.). They appear de novo in the clear quarter egg in the pluteus. This quarter contains no mitochondria after being separated off from the unfertilized egg by centrifugal force, and there are none throughout cleavage nor in the blastulae, but they do occur in the pluteus. This is true also of the pigment granules in the clear quarter. There are no pigment granules until the pluteus is about 4 days old. (E. B. Harvey, 1946a). Stain.—Best vital stain is methyl green, stains violet. Janus green stains them green but is rather toxic (E. B. Harvey, 1932, 1941c). Other vital stains are: Bismark brown, chrysoidin, gentian violet, methyl violet, neutral red, Nile blue, rhodamine, saffranin O (later), thionin (few cases), toluidin blue (E. B. Harvey, 1941c). Also crystal violet, dahlia B (E. B. H. unpub.). Fixed material. Mitochondria stain red with Benda-Kuhl (E. B. Wilson, 1926). Slightly stained with iron haematoxylin (E. B. Harvey, 1940c). Table 8. Other Species (additional) and General References In some other species, e.g., Sphaerechinus granularis, the mitochondria are the lightest granules as in Arbacia punctulata and A. lixula (E. B. Harvey, 1933 a, 1938 a). In some species they are the heaviest material in the egg, e.g., Psammechinus microtuberculatus (E. B. Harvey, 1933a, 1938a, and see colored picture of Lindahl, 1932, p. 330) and Ps. miliaris (Linder- strém-Lang, 1938-1939). In still other species, they are the heaviest granules, but lighter than the clear material, e.g., Tripneustes esculentus (E. B. Harvey, 19334, 1947)- Gustafson, 1952. General. Gustafson and Lenicque, 1952. Ps. miliaris, distribution of mitochondria at different stages of development. Harvey, E. B., 1933a, 1938a. Naples species. Harvey, E. B., 1947. Bermuda species. Runnstrém, 1952b. General, in Barron’s Modern Trends. NARCOTICS See Anaesthetics OF EXPERIMENTAL WORK 193 NITROGEN Nitrogen by weight, times 6.25 is usually assumed to give protein present. Amount.—Of nitrogen in eggs (Ballentine, 19404): 0.107 mg. nitrogen per mg. dry weight 265 mg. dry weight per cm.? cells 58.0 mg. dry weight per 108 cells 26.8 mg. nitrogen per cm.° cells 5.86 mg. nitrogen per 10° cells. Similar figures have been given by Hutchens, Keltch, Krahl and Clowes (1942) ; they report also 10 % of nitrogen is in the jelly. Increase.—In soluble nitrogen on autolysis of unfertilized and fertilized eggs, in acid solution; increase in soluble nitrogen on autolysis of sperm in neutral or alkaline media; about one sixth of soluble nitrogen from autolysed and control eggs is of protein origin (Lyon and Shackell, 1gtoa). Other Species Ephrussi, 1933. Paracentrotus lividus, 10.7% nitrogen dry weight. Gustafson and Hjelte, 1950. P. lividus, amino acid—N. Hultin, 1950c. P. lividus, uptake of *N-labeled ammonia. Wetzel, 1907. P. lividus, 1.6% nitrogen wet weight, 7.2% dry weight. NUCLEAR DIVISION WITHOUT CELL DIVISION Historical._—First obtained by O. and R. Hertwig in 1887 in Paracentrotus lividus with nicotine, chloral hydrate, etc. In Arbacia punstulata, with salts on fertilized eggs, with NaCl by Loeb (1892). Questioned by Morgan (1893, 1896). Loeb’s results were confirmed by Norman (1896) with MgCl,, and restated by Loeb (1895b, 1g00a). Morgan later (1899) agreed that nuclear division could take place without cell division, but was irregular and accompanied by artificial astropheres. Urea.—(R. S. Lillie, 1903). Cold.—O*° to 2 °C., then room temperature (Lyon, 1904b). Acid.—And alkali (Smith and Clowes, 1924Cc). Carbamates.—(Cornman, 1950b). Nitrous Oxide.—(Haywood, 1953). Normally in many fertilized red half-eggs, granular, yolk and pigment quarter- eggs obtained by centrifugal force (E. B. Harvey, 1932, 1940¢, 1951, and this Mono- graph). For obliteration of cleavage plane, leaving nuclear without cell division see Anaesthetics, Hydrostatic Pressure, Oxygen-Lack. Other Species (additional) and General References Boveri, 1897. Echinus (Psammechinus) microtuberculatus, pressure and cold. Driesch, 1892. Same species, pressure and heat. Godlewski, 1908. Same species, CO,. Korschelt and Heider, 1902, p. 215. General. Moore, Roaf, and Whitley, 1905. Echinus esculentus, alkali. Polowzow, 1924. P. lividus, alcohol. Sugawara, 19434, b. S. pulcherrimus, “hatching enzyme’’, trypsin, pepsin, papayotin, CeCl,, ageing. Sugiyama, 1938a, b, c. S. pulcherrimus, Pseudocentrotus depressus, Mespilia globulus etc., egg albumen, heparin, hirudin; oxygen consumption. E. B. Wilson, 1901 a. Toxopneustes (Lytechinus) variegatus, parthenogenetic eggs with MgCl,. E. B. Wilson, 1go01b. Fertilized eggs with shaking and ether. E. B. Wilson, 1925. The Cell, p. 174. Analysis, separability of factors. Ziegler, 1894. Echinus (Ps.) microtuberculatus, pressure. 194 ALPHABETICAL COMPILATION NUCLEOPROTEINS See also Proteins and Phosphorus Metabolism Historical—M athews (1897) discovered a substance in Arbacia lixula sperm, not a protamine, but similar to a histone, in combination with nucleic acid, which he called ‘‘arbacin’’. Mathews (1915, p. 174) extracted a very small amount of nucleic acid (?) from unfertilized Arbacia punctulata eggs. Amount in Eggs and Embryos.—Blanchard (1935) obtained 12.75 gm. of crude nucleic acid from 4,820 gm. of unfertilized eggs; 1.08 gr. of desoxyribose nucleic acid (DNA) and about same amount of ribose nucleic acid, its pentose derivative (RNA). For more recent figures on amounts of DNA and RNA in eggs and embryos see Table 17 under Phosphorus Metabolism. Amount of RNA phosphorus in unfertilized egg is 20 x 10-* micrograms; DNA phosphorus 0.7 to 1 x 10 micrograms. DNA/RNA = 0.05 in unfertilized egg, 0.17 in 8-hour embryo (blastula), 0.46 in 24-hour embryo (pluteus). RNA in un- fertilized egg does not change in early development, but DNA increases during cleavage until pluteus stage; hence DNA of fertilized egg probably does not come from RNA of unfertilized egg as earlier postulated by Brachet in other species (Schmidt, Hecht, and Thannhauser, 1948; see also Villee, Lowens, Gordon, Leonard and Rich, 1949; Villee, Villee and LaPlace, 1953). DNA of blastula is 10 x RNA (Abrams, 1951; see Schmidt et al., 1948). Marshak and Marshak (1953) give amounts in an unfertilized egg as RNA 2.4 x 107° micrograms, DNA 8.1 x 1078 micrograms, and state that there is no detectible Feulgen-positive material in the nucleus of the mature ovum. Location in Egg.—In other species (Paracentrotus lividus, Psammechinus miliaris, etc.) it has been shown that DNA is located in the nucleus, but there is also some RNA; RNA is mostly in cytoplasm near nuclear membrane and in germinal vesicle of immature egg (work of Brachet and Casperson). See Runnstrém in Modern Trends (1952, p. 65); Brachet’s Chemical Embryology (1950, p. 212); Brachet (1947, 1952); Casperson and Schultz (1940). In A. punctulata.—Location of RNA-proteins in eggs (Tsuboi, 1953). Location of nucleic acid compounds in immature, mature and centrifuged eggs and half-eggs as photographed by ultraviolet light (E. B. Harvey and Lavin, 1944, 1951 a). Feulgen reaction negative in parthenogenetic merogones, positive in fertilized merogones (E. B. Harvey, 1940c, p. 186). RNA present in cell cortex, vitelline and fertilization membranes (Lansing and Rosenthal, 1949, 1952). Feulgen Reaction.—For determination of DNA. See Gray’s Experimental Cytology (1931, p. 84); Brachet (1933, 1937, 1950); Pasteels and Lison (1950, p. 448); McClung’s Microscopical Technique (1950, p. 135); Gomori (1952). For recent apprai- sals of the Feulgen reaction see Brachet (1952, p. 176); Lesler (1953); Lumb (1950). Amount and Location in Sperm.—Nucleic acid 29.66 % (in A. lixula, Mathews, 1897). RNA 1 % of amount in egg; DNA 3 % of amount in egg (Schmidt, et al., 1948). Sperm contributes 1/3oth as much DNA to fertilized egg as does the egg (Mazia, 1949b). DNA, about 0.9 x 10~§ micrograms in one Arbacia sperm (Mazia, personal communication, July 1951). Marshak and Marshak (1953) give 7.9 < 1077 micro- grams DNA in one sperm. RNA activity localized in middle piece of sperm (Di Stefano and Mazia, 1952). Two DNA fractions in sperm (Barton, 1951, 1952). Analysts and Synthesis.—Methods of extraction and analysis (Schmidt and Thann- hauser, 1945; Schmidt, et al., 1948; Villee, et al., 1949; Mazia, 1949b; Abrams, 1951, with isotope tracers; Marshak and Vogel, 1951). Analysis of phosphorus fractions (Crane, 1947; Schmidt, et al., 1948; Villee, et al., 1949, 1950, see Table 17 under Phosphorus Metabolism). Purines, adenine and guanine extracted from unfertilized eggs (Blanchard, 1935). Purines and pyrimidines, adenine, cytosine, thymine ex- OF EXPERIMENTAL WORK 195 tracted from sperm nucleic acid (Daly, Allfrey and Mirsky, 1950). From sperm and eggs, but no uracil in sperm, no thymine in eggs (Marshak and Vogel, 1951). Synthesis of nucleic acid purines, adenine and guanine (Abrams, 1951). See Marshak and Marshak, 1953. Effect of Various Drugs.—On nucleic acid metabolism (Villee, et al., 1949; Villee and Villee, 1952). Inhibition of DNA and DNase by usnic acid (Marshak and Fager, 1950). Other Species (additional) and General References Bernstein and Mazia, 19534, b. S. purpuratus. Brachet, 1947, 1952. General. Brachet, 1950. Chemical Embryology, Chapt. 6, general. Callan, 1949. Naples species, RNA in eggs. Casperson and Schultz, 1940. Psammechinus miliaris, ultraviolet. Elson and Chargaff, 1952a, b. Paracentrotus lividus, DNA in eggs and sperm; PNA in embryos. Hultin, 1949a. A. lixula, Ps. miliaris etc., agglutination by nucleoproteins. Krahl, 1950. Review, p. 187. Lison and Pasteels, 1951. P. lividus, amount DNA at different stages. Loeb, 1907. Nuclein synthesis, general. Masing, 1910. A. lixula, no nuclein synthesis in development. Mirsky and Ris, 1951. Echinometra and general. Needham and Needham, 1930. Dendraster excentricus, nuclein synthesis. Rothschild, 1951a. Purines and pyrimidines in sperm of Echinus esculentus. Runnstr6m, 1952. General, in Barron’s Modern Trends. Symposium on nucleic acids, 1947. Cold Spring Harbor Symposia, vol. 12. Symposium on nucleic acid, 1947. Symposia of Society for Experimental Biology, no. 1. Symposium (Discussion) on nucleic acids, 1951. J. Cell. and Comp. Physiol. 38, supplement no. I. Vendreley, C. and R., 1949. A. lixula, P. lividus. OIL See also Lipids Size.—Of oil globules in egg. Diameter 0.6—1.0 u (E. B. H. per E. N. Harvey, 19324; E. B. Harvey, 1936). Smaller than 1 » (Chambers, 1938 a); e¢ al. Density.—Lightest material in egg; goes to centripetal pole in centrifuged eggs, forming an oil cap (Lyon, 1906a, 1907); McClendon, 190ga; E. B. Harvey, 1932, 1936, etc.; et al.). Amount.—Of oil in oil cap. 1 % of egg (E. N. Harvey, 19324); 2 % (Costello, 1939). In Lytechinus variegatus there is usually no oil cap but sometimes a few oil drops at centripetal pole; good oil cap in dilute (80 %) sea water (E. B. H.). Decrease.—In number of oil drops (in sections) from unfertilized egg to 4-cell stage, slight increase in blastula (Pelluet, 1938). Stain.—Oil cap does not stain with any vital dyes tried (E. B. Harvey, 1941c). In fixed preparations; oil cap does not blacken much with osmic acid and does not stain (Lyon, 1907). Black with osmic acid, subsequently bleaches with turpentine; black with Benda-Kuhl (E. B. Wilson, 1926) ; cf. Pelluet (1938). Oil cap does not show in preparations fixed in Bouin but does after formalin and Flemming (E. B. Harvey, 1940c; E. B. Harvey and Lavin, 1944). Ultraviolet Light.—Oil cap is slightly absorbing (E. B. Harvey and Lavin, 1944). Effect of Ammonium Salts—‘‘Lipophanerosis” (see Needham, 1942, p. 206), in- crease in size of oil cap, probably due to release of bound oil (Heilbrunn, 1936; Wiercinski, 1944; cf. Kopac (1948 a) ; see also Navez 1938; Navez and Du Bois, 1940; 196 ALPHABETIC AL COMPILATION Parpart (1941) found no decrease in bound lipid). Effect of CaCl,.—On oil cap, agglutination; of NaCl and KCl, dispersion (Cham- bers, 1938 a). Coalescence.—Of oil globules of centrifuged egg into a single mass by heat or formalin then compression (Chambers, 19334). Coalesceace of egg with oil drops on the surface and interfacial tension of oil-protoplasm and oil-water (Chambers, 1935b, 1936, 1937a, 1938b; Chambers and Kopac, 1937a; Kopac, 1938, 1939a, b; 1940a, b, 1943, 1944, 1948a, 1950; Kopac and Chambers, 1937, 1938). Other Species Chambers and Kopac, 1937c, Lytechinus variegatus, Echinometra lucunter, coalescence of oii drops. Tennent, Gardiner, and Smith, 1931. Echinometra lucunter, staining of oil. OSMOTIC PRESSURE Freezing Point Depression. A, of a gram molecular solution of a nonelectrolyte = 1.86°C. and corresponds to an osmotic pressure of 22.4 atmospheres at 0 °C. Since osmotic pressure varies directly with the freezing point depression, 22.4/1.86 (or 12) x A gives the osmotic pressure. Osmotic pressure of sea water at Woods Hole is 21.7 atmospheres at o °C. (Chemical room, M. B. L.). According to Garrey (1915) it 1s 21.9 atmospheres. Osmotic pressure of eggs is same as sea water. Freezing point depression, A, of sea water in different localities in —°C.: Beaufort, N. C. —2.04 (Garrey, 1915) Naples, Italy — 2.29 (Botazzi, 1897) —2.2-2.3 (E. B. Harvey, 19334) — 2.2-2.43 (D’Amora, 1937) New York (old aquarium) —1.85 (W. H. Cole, 1940, per H. W. Smith) Pacific Grove, Cal. —1.905 (Garrey, 1905, 1915) Salisbury Cove, Me. —1.759 (W. H. Cole, 1940) Tortugas, Florida —2.03 (McClendon, 1g10b) Woods Hole, Mass. —1.805 (Garrey, 1905; Chemical room, M. B. L.) Osmotic pressure calculated for a molecular solution does not always equal osmotic pressure as ascertained by measuring volume changes of the eggs, e.g., sugar, CaCl, (Loeb, 1913a, p. 130; Heilbrunn, 1952, p. 127). Salt Solutions —Isomotic with the sea water and eggs at Woods Hole (salinity 31, A 1.805 °C.). Solutions used in Chemical room of M. B. L.: (Kindness of G. M. Cavanaugh). NaCl 0.52 M LiCl 0.60 M KCl 0.53 M CsCl 0.53 M CaCl, 0.30 M RbCl 0.58 M MgCl, 0.30 M NaBr 0.53 M MgSO, 0.81 M? Na,HPO, 0.40 M NH,Cl 0.53 M (Heilbrunn) Na,SO, 0.53 M Cane Sugar Solution.—Isosmotic with the eggs at Woods Hole, as ascertained by swelling and shrinking: 0.85 M (E. B. H.). Loeb (1913a, p. 130) gives 0.75 M and Garrey (1915) gives 0.73 M. For centrifuging use # isosmotic sugar solution to $ eggs in sea water. See Part III, Centrifuging. Cane sugar solution isosmotic with eggs in sea water at Naples is 1.0 molar (E. B. Harvey, 19334). Dextrose Solution —Isosmotic with Woods Hole sea water is 0.95 molal (Lucké, 1931). Cleavage.—Is arrested if osmotic pressure is less than 13.2 atmospheres or more than 28.7 (Churney, 1940). OF EXPERIMENTAL WORK 197 OXYGEN CONSUMPTION See Respiration OXYGEN-LACK (ANAEROBIOSIS) AND LOW OXYGEN TENSION Size of Egg.—Slightly smaller in absence of oxygen (Hunter, 1936), but not statistic- ally significant (Keckwick and E. N. Harvey, 1934). Life-Span.—Of unfertilized egg increased, slightly, much less than with KCN (Loeb and Lewis, 1902; Loeb, 1911, 1913a, p. 26). Unfertilized Egg —Remains normal for 8 hours: in absence of oxygen, with slight delay in formation of fertilization membrane and cleavage when fertilized (in air) after 3 or more hours without oxygen (E. B. Harvey, 1930); normal for 5 hours (Barron, 1932). Fertilization Membrane.—No fertilization membrane formed if fertilized in absence of oxygen, but sperm are not motile (E. B. Harvey, 1930; see Barron, 1932). Fertil- ization membrane is formed on parthenogenetic eggs in absence of oxygen (Kitching and Moser, 1940). Cleavage Arrested.—Reversibly (Loeb, 1895, translated 1905; 1913a, p. 25; Lyon, 1902, susceptible period 10-12 minutes after fertilization; E. B. Harvey, 1927, for P. lividus and Ps. microtuberculatus, susceptible period just before cleavage; E. B. Harvey, 1930, for A. punctulata; Amberson, 1928; Tang and Gerard, 1932; Runn- str6m, 1935a, even with pyocyanine; Clowes and Krahl, 1940). Low Oxygen Tension.—On respiration and cleavage. Oxygen consumption practic- ally constant between 228 and 20 mm. Hg, reduced below 20 mm. Hg. Cleavage not retarded until below 11 mm. Hg, and arrested below 4 mm. Hg (Amberson, 1928). Similar results by Tang and Gerard (1932) ; Kitching and Moser 1940; Clowes and Krahl (1940). Reversed by ATP (Barnett, 1953). On respiration of unfertilized eggs. Oxygen consumption begins to fall at 20 mm. Hg (Tang 19314). Asters.—Not formed, or disappear in absence of oxygen; reversible (Mathews, 1907; E. B. Harvey, 1921, 1930). Cleavage Planes.—Obliterated by oxygen-lack, but on admitting air, irregular cleavage planes come in and go (Loeb, 1905, p. 401; E. B. Harvey, 1927, 1930) and result in normal blastulae and plutei. Similar results with urethane and ether (see Anaesthetics), and high hydrostatic Pressures, q.v.). Embryos.—Disintegrate in lack of oxygen (Lyon, 1902). X-Rayed Eggs.—Have less cleavage delay in absence of oxygen (Anderson, 1939) Toxicity.—Of salts, etc. reduced (Loeb, 1910). Amoeboid Motion Produced by Urea.—Is arrested; also arrested by high hydrostatic pressures (Kitching and Moser, 1940). Echinochrome.—Is released from unfertilized eggs in O, lack (Shapiro, 1946). Echinochrome not changed to the reduced form by O, lack. See under Echino- chrome. Permeability—To water and ethylene glycol not affected by oxygen-lack (Hunter and E. N. Harvey, 1936; Hunter, 1936, correcting Keckwick and E. N. Harvey, 1934, Hunter and E. N. Harvey, 1935). Parthenogenesis—Caused by oxygen-lack (Mathews, 1900; McClendon, 1909b, 1910b). Sperm.—Immobilized by absence of oxygen, reversible till after 3 to 4 hours expo- sure. Fertilizing power lost and no recovery after 4 hours (E. B. Harvey, 1930; see also Barron, 1932)..Motility and fertilizing capacity maintained by glycine and other amino acids (Tyler and Lord Rothschild, 1951; Tyler, 1953). 198 ALPHABETICAL COMPILATION Other Species and Reviews Loeb, 1913a, and many other references. Strong ylocentrotus purpuratus. Tang, 1933, 1941. Reviews. PARTHENOGENESIS (ARTIFICIAL PARTHENOGENESIS) Historical—A. Natural parthenogenesis of Echinoderms (to gastrula stage) was first descriked by Greeff in 1876 in Asteracanthium rubens (Asterias glacialis) ; though doubted by some, this was generally conceded correct (O. Hertwig, 1890, p. 304). It was described in several species of sea urchins, including Arbacia lixula by Viguier (1900a, b), working in Algiers. He claimed that the eggs even developed to plutei in normal sea water. This was questioned by Loeb (1901) and others. Lyon (1903) found that A. lixula had a natural tendency to parthenogenesis, but only after 20 to 24 hours in sea water. Loeb (19004, p. 469) states that A. punctulata eggs reach a 2- or 4-cell stage after standing about 24 hours in sea water. Mathews (1907, p. 107) says that Arbacia has a typical non-parthenogenetic ovum, and Asterias is almost parthenogenetic. B. Artificial parthenogenesis in sea urchins was first studied by O. and R. Hertwig in 1887, who found that eggs of P. lividus formed a membrane when shaken with chloroform. R. Hertwig (1895, 1896) obtained 2-cell stages with strychnine. In Arbacia punctulata, cleavages to about 64 cells, were obtained first by Morgan who read a paper before the Morphological Society Dec. 26, 1897, and published his results in Feb. 1898 in a preliminary paper, and in June 1899 in a complete paper; he used NaCl, KCl or MgCl, in sea water. Loeb’s first paper was published in Oct. 1899; he was the first to obtain parthenogenetic plutei (Arbacia punctulata), using MgCl, in sea water. He extended the work to other species, especially the Californian Strong ylocentrotus purpuratus, and devised many other methods, seeking a physico- chemical explanation of development. He published about 75 papers on this sub- ject, and his book on Artificial Parthenogenesis (1909, translated in 1913) is a classic. Experimental.—A. Best method for Arbacia punctulata. This is hypertonic sea water for about 20 minutes. The sea water is made hypertonic either by boiling to half its volume, or by adding 30 gm. NaCl per liter. Different batches respond differently, as also do the different egg fractions. A slight variation in time of exposure may give better results in some batches (E. B. Harvey, 1936, 1940c). Some of the methods listed give only fertilization membranes, others give a few cleavages and others give normal plutei. B. Methods which have proved effective for Arbacia punctulata a. Physical. 1. Mechanical agitation (McClendon, 1909b, 1g10b). Effective for Asterias Lut not for Arbacia (Mathews, 1901b, 1907). 2. Puncture (Moser, 1939b; Kitching and Moser, 1940). 3. Electricity. Induction shocks (McClendon, 1g909b, t1g10b). Direct current (Moser, 1939b). No effect of electric current (R. S. Lillie and Cattell, 1925). => 4 Heat, around 32 °C. (Mathews, 1900; McClendon, 1909b, 1910b; Loeb, 1913 a, p. 185; Heilbrunn, 1925a, 1928, p. 262). —> 5. Cold, o&=10 °C. (Morgan, 1g00b; Greeley, 1902; McClendon, 1gogb, 1910b; E. B. Harvey, 1936, 24 hrs. at 8°). 6. Photodynamic action (visible light) (R. S. Lillie and Hinrichs, 1923; Hinrichs, 1926a; + rose bengal or eosin, Alsup, 1941). 7. Ultraviolet light (Loeb, 1914a; R.S. Lillie and Baskervill, 1922; Heilbrunn and Young, 1930; E. B. Harvey and Hollaender, 1937, 1938; Nebel, E. B. OF EXPERIMENTAL WORK 199 Harvey, and Hollaender, 1937; Hollaender, 1938; Moser, 1939b; E. N. Har- vey, 1942). 8. X-rays (E. B. Harvey unpub.; negative, Richards, 1915). b. Chemical. = 1. Oxygen lack or diminished oxygen (Mathews, 1900; McClendon, 1909b, 1910b). . Hypertonic sea water, by evaporation (S. J. Hunter, 1901, 1903; Greely, 1903; McClendon, 1g09b, 1910b; Glaser, 1913; Just, 1928c; E. B. Harvey, 1936, 1940C). 3. Hypotonic sea water or distilled water (McClendon, 1909b, 1910b; “Glaser, 1913; Just, 1928a, 1939a, p. 45, distilled water 15 seconds; 1939b, 4. Salts. NaCl (Morgan, 1898, 1899, 1900a, b; Loeb, 1g00b, 1901, 1913a, p. 60, etc.; Greeley, 1903; McClendon, 1909b, 1910b; Heilbrunn, 1915a, 1928, p. 261; C. R. Moore, 1917; Just, 1922a I, 19394, b, p. 224; E. B. Harvey, 1936, 1940c; et al.). NaCl, NaBr, NaNO;, NaI, NaCNS (R. S. Lillie; 1910, 1911b). Na,SO, (Just, 1929a). KCl (Morgan, 1899, 1900b; Loeb, 1900a, b, 1901, 1913a, p. 60, etc.; Just, 1922a I, 19394, b, p. 224; E. B. Harvey, 1936). KI, KCNS (R. S. Lillie, 1g10, 1911b). CaCl, (Loeb, 1g00a, 19194, p. 59, etc.; Hollingsworth, 1941). or “MgCl, (Morgan, 1899, 1900a, b; Loeb, 1899, 1900a, b, 1901, 19134, p. 57, etc.; Greeley, 1903; Hollingsworth, 1941; e¢ al.). BaCl, (Just, 1929a; Hollingsworth, 1941). SrCl, (Hollingsworth, 1941; see Heilbrunn, 1915a). HgCl, (F. R. Lillie, 1921b; Hoadley, 1923, 1930; Heilbrunn, 1925d). 5. Acids. HCl, HNO,, feeble; H,SO,, negative (Loeb, 1g00a, 1901, 19134, p. 57, 138). CO, (McClendon, 1909b, 1910b; McClendon and Mitchel, 1912; Jacobs, 1922). Acetic acid (McClendon, 1g09b, 1910b; D. Harding, 1951). Lactic, phosphoric, butyric acids and injury substances (D. Harding, 1951). ‘> Butyric acid, alone. Fertilization membrane only (Loeb, 1913a, p. 71, 19154; F. R. Lillie, 1914; Heilbrunn, 1915a; C. R. Moore, 1916; Just, 1939b, p. 222). 2-cell (A. R. Moore, 1915). Et al. = Butyric acid + hypertonic sea water, Loeb’s double method (Loeb, 1913a, p- 71, 1916, p. 99: 50 cc. sea water + 2 cc. N/10 butyric acid for 2-4 min.; sea water 10-15 min.; 50 cc. sea water + 8 cc. 24 m NaCl for 174-224 min.; sea water; at 23 °C.) Heilbrunn (1915a, p. 170) advises only $ min. in 50 cc. sea water + 2.8 cc. N/10 butyric, then hypertonic sea water. This method has been used by Just (1939b, p. 222) and many others, but hypertonic sea water alone is much simpler and gives good results. Many other fatty acids have been used by Loeb (1913 a, pp. 67, 134, 185) for Strong ylocentrotus purpuratus, and would probably work for Arbacia. 6. Alkalis and amines. NaOH, KOH, NH,OH (Loeb, 1900a, 1913a, p. 57; McClendon, 1909b, 1910b); membranes form in solutions more alkaline than pH g.o (Smith and Clowes, 1924.b). NH,OH (weak base) better than NaOH, KOH or tetraethyl- ammonium hydroxide (strong bases) (Loeb, 1912b, 1913a, p. 147). Amines (butylamine, benzylamine, protamine (Loeb, 19134, p. 149). 7. Fat solvents, esters and narcotics. Toluol (Heilbrunn, 1915a, 1928, p. 261; Heilbrunn and Young, 1930; 200 ALPHABETICAL COMPILATION Moser, 1939b). Chloroform (Mathews, 1900; Heilbrunn, 1915a, 1928, p. 261). Ether (Mathews, 1900; McClendon, 1g09b, 1910b). Alcohol (Mathews, 1900). Benzol, toluol, amylene, chloroform, aldehyde, salicylaldehyde, ether, alcohol, propyl alcohol, given by Loeb, 1913a, pp. 181-183, probably refer to Strong ylo- centrotus purpuratus, but some may have been tried also on Arbacia. Acetone (Heilbrunn, 1913; Just, 19294). Chloretone, urethane, chloral hydrate, esters: —methyl acetate, ethyl acetate, ethyl butyrate, methyl salicylate (Heilbrunn, 1913). 8. Detergents. Bile salts and soap (Loeb, 1913a, p. 177, probably S. purpuratus). ““Dreft’’ (E. B. Harvey unpub.). g. Glucosides Saponin (Heilbrunn, 1915a, 1928, p. 261; Moser, 1939b; Kitching and Moser, 1940). Saponin, solanin, digitalin (Loeb, 1913a, p. 174, probably refers to Strong ylo- centrotus purpuratus). 10. Alkaloids. Strychnine (Morgan, 1g00a, b; Mathews, 1900). Quinine, pilocarpine (Mathews, 1900). 11. Nonelectrolytes. Ngucrose (Loeb, 1g00b, 1913a, p. 60; E. B. Harvey, 1936; Moser, 1940). C. D. Urea (Loeb, 1g00b, 19134, p. 60; Moser, 1940); Kitching and Moser, 1940). Thiurea, glycerine (Moser, 1940). Acetamide (Heilbrunn, 1913). 12. Proteins, enzymes, organ extracts. Egg albumen (Heilbrunn, 1915a, 1921, 1928, p. 261). Blood serum (Loeb, 1912b, 1913a, p. 194 footnote; see Heilbrunn, 19154). Thrombin from ox blood (Just, 1929 a). Ovarian extract (Glaser, 1913, 1914C). Sperm extract (Sampson, 1926, but see Loeb, 1901, 1913a, p. 201; Gies, 1901; Frank, 1939). Extract of injured tissues (D. Harding, 1951). Lipolysin (A. E. Woodward, 1918). Hirudin (Just, 19294). Papain? (Loeb, 1901). 13. Miscellaneous. KCN (McClendon, 1909b, 1910b; see Heilbrunn, 1915a; M. M. Brooks, 1946a, b, Lyon, 1903 for P. lividus. Picric acid (trinitrophenol) (Heilbrunn, 1913). Chlorine (Heilbrunn, 1925d). Iodine (Woodward and Hague, 1917). Tannine and ammonia (R. S. Lillie, 1910; McClendon, 1g1o0b). ““Hexaresorcinol”’ (E. B. Harvey unpub.). Vitamin K (2-methyl-1,4-naphthoquinon) (Halaban, 1949). Fertilization after parthenogenesis. See Part II, Chapter 13, paragraph f, p. 108. Development of parthenogenetic eggs. Some batches develop much better than others. Development same as in fertilized eggs, but slower (many observers) ; first cleavage 14 to 5 hrs. for parthenogenetic, 50 min. for fertilized (E. B. Harvey, 1936, and unpub., E. B. Harvey and Hollaender, 1938). Parthenogenetic plutei of A. punctulata have been obtained in early stages but not carried through metamorphosis, though this could undoubtedly be done. Shearer and Lloyd (1913) raised 15 parthe- nogenetic Echinus esculentus through metamorphosis; this took 8 weeks, whereas OF EXPERIMENTAL WORK 201 normal eggs take 5-6 weeks; they died soon after. Delage (1909) raised two partheno- genetic Paracentrotus (Strong ylocentrotus) lividus through metamorphosis (60 days) to the young adult of 18 months; they then measured 3.1 and 3.6 cm. overall, the tests 1.5 and 2.2 cm. One was a ¢ with ripe sperm, the other immature, but probably 3. The ¢ is digametic in sea urchins (Tennent, 1g11b, 1912a, b, c in Toxopneustes and Hipponoé, and Baltzer, 1913, in P. lividus and Ps. microtuberculatus). E. Physiology of parthenogenetic eggs. Same as fertilized eggs for increase of oxi- dation rate (McClendon and Mitchell, 1912; Keltch 4nd Clowes, 1947; Warburg, 1910 for S. lividus); for increase of viscosity (Heilbrunn, 1915a, 1928, p. 261), for increase of permeability (R. S. Lillie, 1916a) etc. F. Cytology of parthenogenetic eggs. Different from fertilized eggs on account of absence of ¢ nucleus (Morgan, 1899, 1900b; E. B. Harvey and Hollaender, 1937, 1938; Nebel, E. B. Harvey, and Hollaender, 1937). See careful study of Toxopneustes (E. B, Wilson, 1901 a); also Hindle (1910 for S. purpuratus). Half number of somatic chromosomes (16-18) in early cleavage (E. B. Harvey, 1940c); 18 in Toxopneustes (E. B. Wilson, 1901a) and in S. purpuratus (Hindle, 1910). G. Parthenogenetic merogones. Development (parthenogenetic) of non-nucleate half and quarter eggs, broken by centrifugal force; also of parthenogenetic nucleate fractions (E. B. Harvey, 1936, 1940b, c, 1946a, 1951; E. B. Harvey and Hollaender, 1937, 1938). See Part III, Chapt. 21 e and Plate XI. Other Species and General Rerefences Bronn’s Thier-Reich, 1904, pp. 1213-1223. Review and methods. Dalcq, 1928. Les Bases Physiologiques de la Fécondation et de la Parthénogénése. Delage, 1901. 1908. Paracentrotus lividus ; methods and cytology. E. B. Harvey, 1938a. Naples species, parthenogenetic merogones. E. N. Harvey, 1910a, b. Methods; also Lytechinus variegatus. Heilbrunn, 1913, p. 349. Methods; 1915a. General. F. R. Lillie, 1919. Problems of Fertilization. R. S. Lillie, 1941. Review. Loeb, 1913a. Arlificial Parthenogenesis and Fertilization. Loeb, 1916. The Organism as a Whole, pp. 95-127. Lyon, 1903. Naples forms, including A. lixula. McClendon, 1g910b, p. 245. Methods. Morgan, 1927. Experimental Embryology, pp. 537-593- Tabulae Biologicae, 1927, Bd. IV, p. 216. Tyler, 1941a. Review. Vandel, 1931. La Parthénogénése. E. B. Wilson, 1925. The Cell, pp. 467-487. PERIVISCERAL FLUID (COELOMIC FLUID, BLOOD) See Amoebocytes, Leucocytes Historical.—Studied in other species by Quatrefages (1850); Williams (1852) who called it “‘cyclaqueous fluid’’; Geddes (1880); Gamgee (1880); MacMunn (1883, 1885); Cuénot (1891a, b). Referred to in Arbacia punctulata by Mathews (1900) ; McClendon (1g10b), etc. Contains.—Amoebocytes, Leucocytes (McClendon, 1912a; McClendon and Mit- chell, 1912; Kindred, 1921, 1926; Donnellon, 1938; et al.). Also other kinds of cells (H. V. Wilson, 1924). Inorganic Composition —Identical with sea water in Echinus esculentus (J. D. Robert- son, 1939; he thinks the small differences found by Bethe and Berger, 1931, due to inaccurate methods). Identical with sea water in Strong ylocentrotus drébachiensis and 202 ALPHABETICAL COMPILATION Echinarachnius parma (W. H. Cole, 1940). For calcium in A. punctulata Schechter (1937) gives, from analysis by Mazia, 0.395 mg./cc. of calcium in coelomic fluid against 0.41 mg./cc. in sea water. Chemistry —For A. punctulata, see Van der Hyde (1922). For Paracentrotus lividus see Mourson and Schlagdenhauffen (1882). Toxicity to Eggs.—Perivisceral fluid harmful to eggs (Fol, 1879, p. 86). Plasma (filtered) inhibits fertilization (F. R. Lillie, 1914, 1919, p. 173; Lillie and Just, 1924; Just, 1922a, II1; A. E. Woodward, 1918). But others found it is not the serum in the perivisceral fluid but material from the pigmented amoebocytes (echino- chrome?) that prevents fertilization (Pequegnat, 1948; Couillard, 1952). Still others found that the toxic substance is not the perivisceral fluid but a dermal secretion or material from the outside of the shell (Ohshima, 1921; E. B. Harvey, 1939b). The toxic substance was found to have no effect on cleavage if eggs were exposed after fertilization (Ohshima, 1921; Pequegnat, 1948; Couillard, 1952). Runnstrém (1950- 1951) says toxic effect can be removed by sodium periodate. Toxicity to Sperm.—Serum not harmful (F. R. Lillie, 1912, 1919, p. 174; Just, 19224, III; Lillie and Just, 1924, p. 495; Ohshima, 1921). Contains Agglutinin.—(F. R. Lillie, 1912, 1914; Couillard, 1952). Increases Viscosity of Eggs.—(Goldforb, 1935b). Clotting —Caused by cells or cell extracts, not plasma (Heilbrunn, 1928, p. 228; Donnellon, 1938). Caused by leucocytes (Kindred, 1921; H. V. Wilson, 1934). For effect of various substances on clot formation (Donnellon, 1938). Other Species (additional) and General References Barnes and Rothschild, 1950. Echinus esculentus, copper content. Bialaszewicz, K., 1933. P. lividus, Sphaerechinus granularis, mineral content. Bogucki, 1930. Paracentrotus lividus, re harmful action. Davidson, 1952. Echinarachnius parma, clotting. Ephrussi, 1925. P. lividus, fertilization membrane. Grassé, 1948. Traité de Zoologie, general. Kindred, 1924. S. drébachiensis, S. franciscanus, Echinarachnius excentricus ; cellular elements. Pantin, 1931. General. Robertson and Webb, 1939. Estimation of inorganic content. Tyler, 1946. S. franciscanus, S. purpuratus, Lytechinus pictus, Dendraster excentricus; heteroaglu- tinins. Webb, 1937. E. esculentus, P. lividus; inorganic content. ‘ PERIVITELLINE SPACE AND CONTENTS Definition.—The perivitelline space is the space between the egg surface or vitelline membrane and the fertilization membrane. Width.—3 to 5 u (E. B. Harvey per E. N. Harvey, 19324). May be 6.5 u (E. B. H.). Decreased under various conditions so as to be practically obliterated (E. N. Harvey, 1910b; Just, 1928a; et al.). With 1 to 2 % egg albumin (Loeb, 19134, p. 208; Heil- brunn, 1915a; R. S. Lillie, 1918b). With 2% Witte’s peptone (Garrey, 1919). Increased, 2 or 3 times, with urea treatment (Moser, 1940). Differs in width in different species (E. B. Harvey, 19334, 1934; et al.). Contents.—Chiefly sea water (E. N. Harvey, 1910b). Sea water and a colloid (Loeb, 1913a, p. 207; 1916, p. 108). A liquid probably containing colloids (R. S. Lillie, 1g11a; Garrey, 1919; Glaser, 1924: et al.). A jelly-like liquid, as concluded by Fol in 1879 (E. B. Wilson, 1925, p. 413). A gel containing colloids; appears striated when under tension or when extended by electric current (McClendon, 1910b, 19144). Striated with aceto-carmine; striations are formed by coalescence of spheres from hyaline layer (E. B. Harvey unpub.). OF EXPERIMENTAL WORK 203 Liquid removed with pipette from perivitelline space by Chambers causing col- lapse of membrane (Garrey, 1919). Fine suspension of carbon in sea water injected in perivitelline space (Chambers, 1942). Electrical Properties—Bears positive charge (McClendon, 1g1ob, 1912b, 1914a). Other Species (additional) Gray, 1927b. E. esculentus, Psammechinus miliaris. Hiramoto, 1954. Hemicentrotus (Strongylocentrotus) pulcherrimus. Hobson, 1927. E. esculentus, Ps. miliaris. Mitchison and Swann, 1953. Ps. miliaris, colloid. PERMEABILITY A. Unfertilized Egg.— 1. Penetration of water; osmotic properties. Change in size in hypo- and hyper- tonic sea water (Sollman, 1904b; R. S. Lillie, 1910-1918; Loeb, 1913a, p. 61; Lucké, et al. including McCutcheon, Hartline, Larrabee, Ricca, Parpart, 1926- 1951; Northrop, 1927; Jacobs, 1933a, b, c; Stewart and Jacobs, 1936; E. B. Harvey, 1943. See Table 12; Shapiro, 1948b, et al. Reviews by Lucké and McCutcheon, 1932; Lucké, 1940; Wilbrandt, 1941 in Tabulae Biologicae, Vol. 19 (Pt. 2), pp. 371- 389 for tables. See Plate XIV. Equilibrium size given by (V, — b)P, = (V.,— b)P.,; whee V, = volume in sea water, P, = osmotic pressure sea water, V,, = volume in concentrated or diluted sea water, P,, — osmotic pressure in concentrated or diluted sea water, b = osmotically inactive material (Lucké and McCutcheon, 1932; Lucké, 1940). Complete recovery on return to sea water. Temperature has no effect on equilibrium (Lucké, 1935). Osmotically inactive material b, 6-20 %, average 12 % (McCutcheon, Lucké, and Hartline, 1931; Lucké, Larrabee, and Hartline, 1935). Increase on fertilization (Shapiro, 1948b). Rate of water penetration. Permeability to water is defined as: oy = kA(P — P,,), where dV/dt = rate of change in volume, A = surface area, P = osmotic pressure at time t, P,, = osmotic pressure within egg or of solution with which cell is in equilibrium, and k the permeability constant. For integrated equations and discus- sion of derivation see Lucké and McCutcheon, 1932, Lucké, 1940. Endosmosis and exosmosis. For water entering eggs at 20 °C.,k = 0.087 u3 per u? surface per atmosphere difference of pressure per minute; for water leaving eggs, k = 0.141 p3. At 15 °C., k = 0.05 — 0.06 for endosmosis and 0.07—0.08 for exos- mosis. Values are independent of osmotic pressure but depend on kind and propor- tions of salt in medium, injury, narcotics, temperature, etc. (Lucké, Hartline, and McCutcheon, 1931). At 22 °C. by diffraction method of study, k = 0.106 for endos- mosis and 0.127 for exosmosis (Lucké, Larrabee, and Hartline, 1935); also Stewart and Jacobs, 1936. 2. Penetration of heavy water same as water (Lucké and E. N. Harvey, 1935). 3. Penetration of non-electrolytes. Permeability to a solute (S) may be defined as $a (co—§) where dS/dt = rate of change of amount of solute; A = surface area; Cs = external concentration; V = volume of the egg., k = number of moles that will penetrate 204 ALPHABETICAL COMPILATION 1 uw? of surface in 1 minute with a concentration difference between exterior and interior of 1 mole per liter (Jacobs and Stewart, 1932). For additional methods of study see Jacobs, 1933a, b, c. Values of k: ethylene glycol, 3.6 x 10-15; acetamid, 5.8 x 10-); propionamid, 14.2 x 107; butyramid, 36.6 x 10-5; glycerol, 05 x 10-15 (Stewart and Jacobs, 1932a). For large effect of temperature, see Stewart and Jacobs, 1932b. At 21.5-23 °C. k for diethylene glycol, 2.6 x 10715; ethylene glycol 4AexX 107; propylene glycol 7.7 x 1o- (Stewart and Jacobs, 1936). Ethylene glycol k at 24° by diffraction method, 4.0 x 10~- (Lucké, Larrabee, and Hartline, 1935); See also Stewart, 1931a and Lucké, Hartline, and Ricca, 1939. No effect of lack of oxygen on penetration of ethylene glycol (Hunter, 1936). 4. Penetration of ammonium salts. Salts of strong acids do not penetrate while salts of weak acids penetrate due to entrance of undissociated acid and ammonia (Stewart, 1931 b, Jacobs and Stewart, 1936). 5. Penetration of fatty acids and their salts. Deduced from effect on viscosity (Howard, 1931). See also Hydrogen Ion. 6. Penetration of various agents active in suppressing cleavage and other egg activities, see Krahl review, 1950, pp. 189-192. 7. Penetration of ions. Potassium (Shapiro and Davson, 1941). Radioactive phos- phate (Na,HPO,); P®? absorption is connected with cell activity, 40 times greater in fertilized egg (Abelson, 1947, 1948). 8. Penetration of dyes. See Vital Dyes. g. Factors affecting permeability. Age. Increase (Goldforb, 1935c). Anaesthetics and narcotics. For general statements see R. S. Lillie, 1912, 1gI4a, b, 1916c, 1917, 1918a, b, and Heilbrunn, 1925c. Urethanes, decrease in isotonic ea cose, no change in sea water (Lucké and McCutcheon, 1926a, 1932, Lucké, 1931). See Anaesthetics. Caffeine. No effect (Cheney, 1948). Carcinogens. Choleic acids of 10-methyl benzanthrene, 20-methylcholanthrene, 1, 2, 5, 6 dibenzanthrene do not affect k for water although they retard or prevent cleavage (Lucké, Parpart, and Ricca, 1941). Cyanide. McClendon, 1g09b; R. S. Lillie, 1918a, b; HCN increases, KCN decreases (Blumenthal, 1927, 1928). Electric current. 60 cycle A. C. has no effect in sea water but causes a slight de- crease in isotonic glucose containing small amounts of NaCl, KCl and CaCl, (Fowler, 1934). Electrolytes. For general statement see Mathews, 1905; R. S. Lillie, 1910, 1gt1a, b, 1912, 1914a; Lillie and Baskervill, 1921, 1922; McClendon, 1910a; Lucké and McCutcheon, 1926a, b, 1929, 1932. Absence of ions (glucose solution) increases k for water from 0.05 to 0.1 at 12 °C, and 0.0001 M CaCl, or MgCl, added to glucose solution maintains k same as in sea water (McCutcheon and Lucké, 1928). Cations decrease permeability to water, the effectiveness increasing with the valence of the cation. In 0.38 M dextrose solution containing 0.005 M K, citrate (in which solution cells have high water permeability), the following concentrations of cobaltamine chlorides were required to reduce permeability to the value ob- tained in sea water: —o0.00005 M of the 6 valent salt, more than twice as much of the 4 valent salt, more than eight times as much of the 3 valent, and 64 times as much of the 2 valent salt, while this amount of the 1 valent salt was incompletely effective. Temperature 12° + 0.5 °C. (Lucké and McCutcheon, 1929). Anions increase permeability to water, the effectiveness increasing rapidly with the valence of the anions. In 0.38 M dextrose solution containing 0.0005 M CaCl,, 0.001 M of potassium ferrocyanide was required to definitely increase permeability, twice as much ferricyanide, four times as much potassium sulphate, and eight times OF EXPERIMENTAL WORK 205 as much chloride. Temperature 12° + 0.5 °C. (Lucké and McCutcheon, 1929). H-ion concentration. No effect on penetration of water (Lucké and McCutcheon, 1926b). See also Hydrogen Ion. For the effect of pH on penetration of many active compounds which are salts of weak acids or bases, see Smith and Clowes, 1924, and Haywood and Root, 1930, 1932, for bicarbonates; see Krahl and Clowes, 1938 and Hutchens, Krahl, and Clo- wes, 1939, for substituted phenols; see Krahl, 1940, Clowes, Keltch, and Krahl, 1940 for barbiturates and see Krahl, Keltch, and Clowes, 1940a for local anaes- thetics. Injury. Increase (Lucké and McCutcheon, 1926a, b, 1930, 1932; decrease, Gold- forb, 1935C). Jelly coat. No effect (R. S. Lillie, 1917). Leucotaxine. Increases k for water from 0.12 to 0.19 (Menkin, 1940). Non-electrolytes. Increase k for water from 0.05 for sea water to 0.097 for glucose, 0.103 for saccharose, and 0.142 for glycocoll (McCutcheon and Lucké, 1928). See also McClendon, 1g10a. Organic extracts. Arbacia egg extracts increase (Glaser, 1914c); manmalian testis and spleen increase (Favilli, 1932). Oxygen lack. Slight increase in k to water, no effect on osmotic equilibrium (Keckwick and E. N. Harvey, 1934); no effect on k for water or ethylene glycol but slight decrease in volume of egg (Hunter and E. N. Harvey, 1936; Hunter, 1936). Sea water concentration. No effect on k for water, as was once supposed (McCut- cheon and Lucké, 1926; Lucké and McCutcheon, 1927, 1932; Lucké, Hartline, and McCutcheon, 1931; Lucké, Larrabee, and Hartline, 1935. Temperature. Higher temperatures greatly increase k to water. Q 19, 2 togandu values, 13000-17000 (Lucké and McCutcheon, 1926a, 1932; McCutcheon and Lucké, 1926, 1927, 1932; Lucké, Hartline, and McCutcheon, 1931). No effect on equilibrium (Lucké, 1935). k for ethylene glycol, propionamid and butyramid also greatly increased by rise of temperature (Stewart and Jacobs, 1932b). Ultraviolet light. Increase (Heilbrunn and Mazia, 1936, p. 650). No effect with eggs of Strong ylocentrotus purpuratus (Reed, 1948). See Heilbrunn, 1952, p. 164 and Part IV. X-rays. No effect (Lucké, Ricca, and Parpart, 1951). B. Egg Fractions —White and red halves. See Chapter 20. (Lucké, 1932; Shapiro, 1939a; E. B. Harvey, 1943, and Table 12 and Plate XIV). C. Fertilized Eggs and Parthenogenetic Eggs.—Increase in permeability as com- pared with unfertilized eggs (McClendon, 1g0gb, 1g10a, b; E. N. Harvey, 1909, 1g10c; R.S. Lillie, 1910, 1911 a, b; Lyon and Shackell, 1910b; Glaser, 1913; Loeb, 1913a, p. 92, 1916, p. 119; Heilbrunn, 1915). k for penetration of water increases about 4 times (R. S. Lillie, 1g16a, 1918a; McCutcheon and Lucké, 1932; Lucké and McCutcheon, 1932. k for penetration of ethylene glycol increases three times after fertilization and somewhat less after distilled water activation (Stewart and Jacobs, 1932a); k for diethylene glycol and propylene glycol doubled in fertilized eggs (Stewart and Jacobs, 1936). ~ Change in osmotically inert fraction on activation from 7.3 to 27.4% (Shapiro, 1948b). For permeability rhythms see R. S. Lillie, rg10, 1g11a, b, 1914.b, 1916b, 1917. D. Nucleus of Unfertilized Egg —See Table 11 and Plate XIV for changes in size in hypo- and hypertonic sea water (E. B. Harvey, 1943). E. Cytoplasmic Granules—Yolk granule behavoir complicated. Pigment granules act like leaky osmometers with k for water eee somewhat higher than for cell (Harris, 1943). For release of pigment on zation, see Chromatophores. The large pigment spots of plutei swell from g (E.'B. .H.). 206 ALPHABETICAL COMPILATION F. Immature Egg.—More permeable than mature (Lyon and Shackell, 1g10b). G. Germinal Vesicle—Approximates a perfect osmometer in behavoir (Churney, 1942). H. Sperm.—The volume doubles in distilled water before bursting (E. B. Harvey and Anderson, 1943). See Shapiro, 1948e. Proc. Soc. Exp. Biol. Med. 67 : 180-182. I. Amoebocytes.—Become spherical in distilled water before bursting (E. B. H.). See Amoebocytes. Other Species (additional) and General References Brooks and Brooks, 1941. The Permeability of Living Cells. General. Brooks and Chambers, 1948. S. franciscanus, S. purpuratus, P 3? uptake. Chambers and White, 1949. S. purpuratus, P?® uptake. Chambers, Whiteley, Chambers, and Brooks, 1948. Lytechinus pictus, P?* uptake. Davson and Danielli, 1952. The Permeability of Natural Membranes. General. Dorfman, 1932, 1933. Strong ylocentrotus drébachiensis, rnythmic changes in osmotic properties. Fukuda, 1935. Anthocidcris crassistina and Pseudocentrotus depressus, water penetration and ionic composition of medium. Herlandt, 1914, 1918b, 1920. Paracentrotus lividus, Sphaerechinus granularis, rhythmic changes in permeability. Hobson, 1932a. Psammechinus miliaris, permeability to water after fertilization. Jacobs, 1924. Cowdry’s General Cytology. General; 1952a in Modern Trends. General. Krahl, 1950. General. Leitch, 1931, 1934a, b, 1936. S. franciscanus, S. purpuratus, Echinometra lucunter, Dendraster ex- centricus, permeability to water and non-solvent volume. Lindberg, 1949. Psammechinus miliaris, P?? uptake. Lucké and McCutcheon, 1932. General. Lyon and Shackell, rg10b. Lytechinus, dyes. Skowron and Skowron, 1926. Sphaerechinus granularis, immature egg. Thoérnblom, 1932. Paracentrotus lividus, change in permeability on fertilization. Whitaker, 1936. S. franciscanus, water and dyes after fertilization. Wilbrandt, 1941. General in Tabulae Biologicae 19, pt. 2. PHOSPHORUS METABOLISM See also Nucleoproteins Amount.—Of phosphorus in unfertilized Arbacia egg.s 0.9064 mg. total phosphate _per 108 eggs; 0.0291 millimoles; 10° eggs = 0.124 gm. dry weight (Page, 1927b). In centrifuged eggs (McClendon, 1909a). Table 17. 130 mg. phosphorus per gram nitrogen or 0.31 mg. per cent of wet weight (Crane, 1947). 96 * 10~® micrograms phosphorus per egg (Schmidt, Hecht, and Thannhauser, 1948). Of phosphorus in fertilized Arbacia eggs. Same in 2-4 cell and blastula (Shackell, 1911). This was criticised by Robertson and Wasteneys (1913) for S, purpuratus, and answered by Masing (1914). Uptake of P*? by fertilized egg is 40 times that by the unfertilized; 1.0 x 10-* mg. per million eggs one hour after fertilization. At 10 °C. 1/7th amount phosphorus taken up as at 23° (Abelson, 1947, 1948). Uptake of P*? increases rapidly on fertilization and rate of uptake is not affected by x-rays (Evans, 1950). Analysis—Of phosphorus fractions. Table 7 in centrifuged eggs (McClendon, 1902a) and Table 17 (Villee et al., 1949). Amount of Phosphorus in Sperm.— 2.86 % (in A. lixula, Mathews, 1897). Phospholipids.— (Lecithin) in unfertilized eggs. 2.17 mg. per 10% eggs or 38 % of total fat which is 5.65 mg. per 108 eggs (Hayes, 1938) ; he calculates 3.84 mg. phos- pholipid per 10* eggs from McClendon’s (190ga) data and 8.4 mg. from Page’s OF EXPERIMENTAL WORK 207 (1927a, b) data. For later references, see Table 17 of Analyses of phosphorus frac- tions. ATP and ADP.—In eggs (Abelson, 1948; see Whiteley, 1949). ATP restores division rate reduced to 50 % by lowered oxygen tension (Barnett, 1953). Radioactive Phosphate P??.—Used for metabolism studies by Brooks (1940, 1943; Abelson (1947, 1948); Villee, et al. (1949, 1950, 1952, 1953)- Effect of P3?.—On division rate; delay (Green and Roth, 1950). — Phosphorus Metabolism.—Inhibited by (1) usnic acid (Marshak and Harting, 1948) ; (2) dinitrophenol, malononitrile, uranyl nitrate, nitrogen mustard, cold; high con- centrations of pilocarpine, atropine, eserine, acetylcholine, with an acceleration of phosphorus metabolism using low concentrations (Villee, et al., 1949, 1952). See also Abelson (1947). Phosphorylation.—(Clowes, 1951; Clowes, Keltch, Strittmatter, and Walters, 1950; Clowes, Keltch, and Walters, 1951a, b; Keltch, Smythe, and Clowes, 1951; Keltch, Strittmatter, Walters, and Clowes, 1950; Strittmatter, Keltch, Walters, and Clowes, 1950; Krahl’s Review, 1950, pp. 184, 198). TABLE 17 ANALYSES OF THE PHOSPHORUS FRACTIONS OF ARBACIA EGGS In milligrams P per gram wet weight of egg or embryo (from Villee, Lowens, Gordon, Leonard, and Rich, 1949, p. 98 Acid sol. Phospho- Acid ine DNA RNA Phospho- Investigator Stage P lipid P_ sol. P Pr P;, .protein: P Crane (1947) | Unfertilized _—_1.26 1.26 0.84 0.034 0.63 0.155 eggs Schmidt, Hecht Unfertilized — 1.52 1.06 0.039 0.924 0.108 and Thann- eggs hauser (1948) Villee et al. 3 hr. 1.42 — 0.86 0.05 0.74 0.06 (1949) embryos See also Abelson (1947, 1948). The above table is given in a slightly modified form by Krahl (1950, p. 188). Another table including Arbacia and other sea urchins is given by Whiteley (1949). Other Species (additional) and General References Brooks and Chambers, 1948. S. purpuratus, S. franciscanus. Chambers and Mende, 1953. S. drébachiensis. Krahl, 1950. Review, p. 187. Lindberg, 1949, 1950. ATP in Ps. miliaris and P. lividus. Masing, 1910, 1914. A. lixula. Needham and Needham, 1930. Dendraster excentricus. Rothschild and Barnes, 1953. Phosphorus fractions of Paracentrotus lividus. Rothschild and Mann, 1950. ATP in sperm of Echinus esculentus. Runnstrém, 1952b. General. Wetzel, 1907. P. lividus. Whiteley, 1949. Lytechinus pictus and General. Zielinski, 1939. P. lividus. 208 ALPHABETICAL COMPILATION PHOTODYNAMIC ACTION See Visible Light PIGMENT GRANULES See Chromatophores, Echinochrome PLASMA MEMBRANE Definition—Plasma membrane is the membrane at the surface of the protoplasm,* over the cortical layer, supposed to be responsible for the permeability of the cell. The older investigators made no distinction between plasma membrane and vitelline membrane, calling the two together the “‘pellicle.’’ It is difficult to distinguish the two optically in an unfertilized Arbacia egg. It has been called the ‘‘luminous’’ layer by Runnstr6m and Monné (1945a) in Psammechinus miliaris, since it is luminous in dark field. See Figs 9 and 10. Thickness—Less than 10 my. (Danielli, 1942, 1951b, p. 154). The dried plasma membrane of the red blood cell is 50--S0 A (Parpart and Ballentine, 1952; Hillier and Hoffman, 1953). Structure.—A liquid film (Chambers, 1935 b, 1938b, 1944, 1949). Fatty (Chambers, 1935b). Lipoid with adsorbed protein (E. N. Harvey and Danielli, 1938; Danielli 1951b, p. 151; Davson and Danielli, 1952, p. 57). Oil Coalescence.—Coalescence of egg with oil drops on the surface (Chambers, 1935b, 1944; Chambers and Kopac, 1937a; Kopac and Chambers, 1937; Kopac, 1940a, 1943; etc.). Other Prcperties.—Very delicate, easily ruptured, becomes more fluid on churning with needle and with calcium; cannot be removed; is necessary for life of cell; can repair itself (Chambers, 1938b, 1940, 1944, 1949, 1950; Kopac, 19404). Other References (General) Just, 1939b. The Biology of the Cell Surface. Runnstrém, 19494. POLYSPERMY Definition.—Polyspermy is the fertilization of an egg by two or more sperm, the egg dividing into three or more cells at first cleavage. Historical—Observed by Fol (1877) and studied by O. and R. Hertwig (1887) in Paracentrotus lividus. A good account of the early work of the Hertwigs, Boveri (1902, 1907), etc. is given in Morgan’s (1927) Experimental Embryology, Chapt. VII. It was concluded that “in the sea urchin the division of the protoplasm is strictly regular, but the chromosomal distribution is disturbed”’ (p. 87) and “‘swimming blastulae develop from these eggs, but only very rarely a normal embryo”’ (p. 86). See also Wilson’s (1925). The Cell, p. 416 and 917. Occurs.—In Arbacia in unripe eggs fertilized soon after the extrusion of the second polar body (E. B. H.). See Part II, Chap. 10, sect. g. Caused.—In Arbacia by Stale eggs, i.e., eggs left standing in sea water (Hoadley 1923; J. M. Clark, 1936, after 16 hours). Shaking (Morgan, 1893, 1895b). Cold * Recent investigations of A. K. Parpart and P. C. Laris with a television microscope, indicate that the plasma membrane of the unfertilized Arbacia egg lies inside the cortical, layer which is between the vitelline membrane and the plasma membrane (Biol. Bull. 107 : 301, 1954). OF EXPERIMENTAL WORK 209 5 °C. (Just, 1939b, p. 202). Excess sperm (F. R. Lillie, 1919, p. 260; J. M. Clark, 1936). But Just (1928a) thinks not. At pH 7.4—-6.8, none at 7.4-9.8 (Clowes and Smith, 1923, Smith and Clowes, 1924d; J. M. Clark, 1936); A. Scott, 1946). Organic acids; aspartic (King, 1912); butyric (Just. 1939b, p. 202). SH-reagent, p-chloromercuri- benzoate (Runnstrém and Kriszat, 1952a). Substances used by Hertwigs (1887) : chloral hydrate 0.2 %; cocaine hydrochloride 0.025 %; nicotine 1 drop to 200 cc.; morphine sulphate 0.6%; strychnine sulphate 0.1 %; quinine sulphate 0.05 %. (J. M. Clark, 1936). Salts of Na, K, Ca, Mg (J. M. Clark, 1936). Fat solvents, alcohol, ether, chloroform, ethyl urethane (J. M. Clark, 1936). Cleavage of polyspermic eggs (Scott, 1946). Other Species (additional) Baltzer, 1908. Paracentrotus lividus, Psammechinus microtuberculatus. Brachet, 1922. P. lividus, during, maturation. Bury, 1913. P. lividus, Ps. microtuberculatus, cold. Ishida and Nakano, 1950. Strong ylocentrotus pulcherrimus, fertilization after parthenogenesis. Rothschild, i953a. P. lividus, nicotine; 1954, Quart. Rev. Biol. 29: 332-342. Rothschild and Swann, 1950, 1951. Ps. miliaris, nicotine. Runnstr6ém and Monné, 1945a. Ps. miliaris, Brissopsis lyrifera, during maturation. Sugiyama, 1947, 1951. S. pulcherrimus etc., after refertilization. Wilson and Mathews, 1895. Lytechinus variegatus. POTASSIUM Amount in Egg.—2.445 mg. potassium per 108 eggs (108 eggs = 0.124 gm. dry weight) or 0.063 millimoles (Page, 1927b). Amount in Sea Water.—At Woods Hole. 0.412 gm. per liter at 20 °C. (Page, 1927Cc). K : Na in Eggs.—As 1.90 : 1; in sea water K : Na as 0.0213 : 1 (Blanchard per Howard, 1931). Twenty times as much K in eggs as in sea water; about same amount in unfertilized and fertilized eggs (Shapiro and Davson, 1941). KCl Isotonic.—With sea water at Woods Hole is 0.53 M (M, B. L. Chemical Room). Loss of K.—By eggs in sea water and accumulation of K in sea water with excess K. (Shapiro and Davson, 1941). Uptake.—And loss of K4? by unfertilized and fertilized eggs (E. L. Chambers, White, Jeung, and S. C. Brooks, 1948; E. L. Chambers, 1949; Chambers and Chambers, 1949); also for S. purpuratus. Replacement.—Of K ion by rubidium and cesium, but not by thorium and uranium (R. F. Loeb, 1920). Chemical Character.—And physiological action of potassium ion (J. Loeb, 1920). Toxicity.—KCl less than NaCl (Loeb, 1g00a; R. S. Lillie, 1910, 1g11a, b, 1912; Page, 1924, 1929b; Chambers and Chambers, 1938; et al.). For toxicity on Arbacia eggs, Page (1929b) arranges cations thus: Li > Na > Mg or Ca > K > Rb > Gs, used as chlorides. Toxic effect can be counteracted by CaCl, and MgCl, and certain anaesthetics (R. S. Lillie, 1911 b, 1912). Isotonic K Cl has little effect on unfertilized eggs, and only in susceptible periods on fertilized eggs; also for other K salts and for S. purpuratus (Chambers and Chambers, 1938, 1949). Pigment Granules.—In unfertilized egg are not affected by isotonic KCl; in fer- tilized egg they break down after monaster stage (Churney and Moser, 1940). Isotonic KCl prevents action of Ca on pigment granules which causes them to break down in the surface precipitation reaction (Heilbrunn, 1928, p. 230). Hyaline Layer.—Absent in isotonic KCl (Chambers and Chambers, 1949). Sperm.—Immobilised in KC] at pH 6.0 (Chambers and Chambers, 1949). 210 ALPHABETICAL COMPILATION Clotting of Body Fluid and Liberation of Pigment.—Hastened in the order of SO, < Cl < NO, < SCN (Donnellon, 1938). Colorless Amoebocytes of Body Fluid.—Dissolved by KCl. (Mathews, 1900). Amoeboid Eggs.—Caused by KCl (Churney, 1940). Respiration—No references found. Permeability.—K causes increase of permeability in the order of KC] < KBr < KNO,; < KCNS < KI (R. S. Lillie, 1910, 1911a, b). Increase of permeability with increase in valence of anions (McCutcheon and Lucké, 1928; Lucké and McCutcheon, 1929). Increased permeability can be counteracted by CaCl,, MgCl, and certain anaesthetics (R. S. Lillie, 1911 b, 1912; McCutcheon and Lucké, 1928, by CaCl, and MgCl,.) Viscosity.—KC] increases viscosity in the order: KCl > NaCl > sea water > MgCl, > CaCl, (E. B. Harvey, 1945). Heilbrunn gives the same order except that KCl and NaCl are reversed (Heilbrunn, 1923, 1928, p. 146, 1943, p. 81). Breaking with Centrifugal Force.—Break more readily than in sea water in the order of increasing viscosity as above (reverse order of stratification), that is, the most viscous (least stratified) break most readily (E. B. Harvey, 1945). Parthenogenesis.—Caused by KCl and other K salts. KCl added to sea water was first used by Morgan (18gg) as a parthenogenetic agent and subsequently by many others. K salts listed above (under permeability) by R. S. Lillie, cause partheno- genesis, effective in the order given, but are more effective if followed by hypertonic sea water (R. S. Lillie, 1910, 19114, b.). Other Species (additional) and General References Bialaszewicz, 1927, 1929. A. lixula, P. lividus; electrolytes in eggs. Heilbrunn, 1952. General Physiology, p. 523. Herbst, 1904. Salts necessary for development. Loeb, 1913a. Artificial Parthenogenesis. S. purpuratus, S. franciscanus, especially. Malm and Wachtmeister, 1950. Ps. miliaris, S. drébachiensis ; amount K in unfertilized and fertilized eggs. Oddo and Esposito, 1951. A. lixula, P. lividus ; changes in K content after fertilization. Robertson, 1939; Robertson and Webb, 1939; Webb, 1939. Inorganic composition of sea water and body fluids. Rothschild, 1948b. E. esculentus ; K in seminal, perivisceral fluids and sea water. Rothschild and Barnes, 1953. P. lividus; inorganic constituents of eggs; table of salts and species. PRESSURE (HYDROSTATIC, INTERNAL AND MECHANICAL) A. Hydrostatic (External) Pressure. Unfertilized eggs.—Decrease in viscosity as shown by stratification of centrifuged eggs, 408 atmospheres (about 6,000 lbs/in?) with force of 7,200 x g (Brown, 1934). Eggs break into halves more readily, with centrifugal force (E. B. H., 1933 unpub.). No ill effects if compressed to 680 atmospheres (10,000 lbs/in?) for several minutes (Kitching and Moser, 1940). Eggs which had been made amoeboid (by urea) stop movement at 340 atmospheres; also stop in O, lack (Kitching and Moser, 1940). Fertilized eggs.—Decrease in viscosity especially of the cortical zone as shown by displacement of peripheral pigment granules by centrifuging at 408 atmospheres with force of 7,200 X g (Brown, 1934). Pressure arrests and obliterates cleavage furrow reversibly, at 450 atmospheres (Marsland, 1938, 1942, 1950, 1951). Effect similar to Oxygen-Lack, q.v., and urethanes and ether (see Anaesthetics). Causes unequal first cleavage of centrifuged spherical eggs along the stratification (E. B. H., 1933 unpub.); without pressure first cleavage of spherical eggs is equal OF EXPERIMENTAL WORK PIU and usually perpendicular to stratification. Delays cleavage (E. B. H., 1933 unpub.; Marsland, 1938; Kitching and Moser, 1940). Temperature and pressure.—Lowered temperature acts like increased pressure (Mars- land, 1950); see Brown (1934). Adenosine triphosphate—Counteracts inhibiting effects of pressure (Marsland, Landau, and Zimmerman, 1953). Insemination.—Prevented at 6,000 Ibs/in?, but sperm are active (Marsland, 1948). Amoebocytes.—Become spherical at 400 atmospheres (Marsland, 1938). Moderately High Pressures.—(61 atmospheres) of nitrogen and helium have no effect on cleavage; nitrous oxide delays cleavage at 2.3 atmospheres (Haywood, 1953). B. Internal Pressure. Internal Pressure.—Of unfertilized egg is 40 dynes per cm.” (Cole, 1932). Internal pressure of unfertilized egg necessary to rupture the membrane, is of order of 1/100 atmosphere. Effect of hypotonic sea water, salts, pH, anaesthetics, ultraviolet light, trypsin on resistance to internal pressure (Rieser, 1950). Internal pressure.—Of fertilized egg, calculated from Chambers’ experiment of rupturing one of two blastomeres, 62 dynes per cm.” (Sichel and Burton, 1936; see Chambers, 1938c). , C. Mechanical Pressure. Method.—Cover slip or compressorium. Position of Cleavage Planes and Micromeres.—Changed, cells forming flat plates (Morgan, 1893). Obliteration of Cleavage Plane.—In Psammechinus microiuberculatus (?) (Boveri, 1897). Other Species (additional) and General References Driesch, 1892. Echinus (Psammechinus) microtuberculatus, mechanical pressure. Lepeschkin, 1941 a. A. lixula etc., mechanical pressure. Marsland, 1939, 1951. A. lixula, hydrostatic pressure. Marsland, 1942. General. Marsland and Landau, 1950. Echinarachnius parma, hydrostatic pressure. Morgan, 1927, p. 468. Experimental Embryology. Mechanical pressure, general. Pease, 1942a. Strong ylocentrotus purpuratus, hydrostatic pressure. : Ziegler, 1894. Echinus (Psammechinus) microtuberculatus, mechanical pressure. PROTEINS See aiso Nucleoproteins Protein is assumed to be 6.25 times the nitrogen present, by weight. Amount in Egg.—About 65 % of egg dry weight is protein (Hutchens, Keltch, Krahl, and Clowes, 1942). Change on Fertilization.—12 °% of total protein becomes insoluble 3 to 10 minutes after fertilization (Mirsky, 1936; known as ‘“‘Mirsky protein’’). See Monroy and Oddo (1951) for A. punctulata and A. lixula. Amount in Seminal fluid——About 2.5 mg. per cc. undiluted seminal fluid (Hayashi, 1945, 1946). On Sperm Surface.—(Hayashi, 1945, 1946; Popa, 1927, lipoprotein). Basic Proteins.—(Protamines and histones) extracted from sperm, cause agglutin- ation (Metz, 1949); see Frank (1939). Other Species and General References Conners and Scheer, 1947. S. purpuratus, analysis of protein. Ephrussi, 1933. P. lividus, protein is 66.88% of dry weight. Hultin, 1949a. A. lixula, P. lividus, Ps. miliaris, Echinocardium cordatum, basic proteins. 212 ALPHABETICAL COMPILATION Kavanau, 1953. S. purpuratus, amino acids in development. Leitch 1934b. S. franciscanus, S. purpuratus, amount of protein. Lindvall and Carsj6, 1951. E. esculentus, protein fractions. Monroy, 1950. P. lividus, protein fractions by electrophoresis. Runnstrém, 1949a. General. Tyler, 1948. Agglutinins. RADIUM Effect on Cleavage.—Beta rays retard, gamma rays accelerate cleavage; most suscept- ible in metaphase (Packard, 1915, 1916). Rays cause cytolysis.—(Packard, 1916). Other Species Bohn, 1903. Strong ylocentrotus lividus. Hertwig, G., 1912. Parechinus miliaris. Miwa, Yamashita, and Mori, 1939, 1940, 1941. Pseudocentrotus depressus and Strong ylocentrotus pulcherrimus. Reiss, 1925. Probably Paracentrotus lividus. RESPIRATION, OXYGEN CONSUMPTION, METABOLISM Index A. Eggs I. Increase of O, consumption (Table 18). II. Increase of O, consumption with reversible blocking or delay of cell division. III. Practically no effect on O, consumption with blocking (sometimes revers- ible) of cell division. IV. Decrease or inhibition of O, consumption and of cell division. V. Half-eggs (Table 18). VI. Homogenates (cell-free system). VII. Carbon dioxide production. VIII. Respiratory quotient, RQ . IX. Temperature coefficient, Q ,,, Table 19. B. Sperm I. Increase of O, consumption. II. Decrease or inhibition of O, consumption. III. Carbon dioxide production. IV. Respiratory quotient. Location in Text. A. Eggs Anaesthetics, A III 1; see also topic Anaesthetics. Azide, A III 6. Barbiturates A III 1 (Barbituric acid). Benzoic acid A III 1 (Local anaesthetic bases). Caffeine A IV 8. Carbamates (Urethane). Carbon dioxide A IV 2. Carbon monoxide A IV 3. Centrifuged eggs A I 7 (Stretched). Chloral hydrate A III 1. Cleavage A I 3. OF EXPERIMENTAL WORK 213 Cresol-indophenol A I 8. (O-cresol-indophenol). Cyanides A IV 4; see topic Cyanides. Cytolysis A I 6. Development, during A I 3. Dimethyl paraphenylenediamine A II 2. Dyes A I 8. (Redox indicators). Fertilization, after, A I 1. Halophenols A II 3. Hexose phosphate A I to. Hydrogen peroxide A II 4, IV 9. Hydroxyl ions A I g (OH). Immature eggs A I 2. Indophenol A I 8 (O-cresol-indophenol). Iodoacetate A IV 5 (Iodoacetic acid). Local anaesthetic bases A III 1 (Benzoic acid). Low oxygen tension A IV 1 (See also topic Oxygen-Lack). Malononitrile A IV 7. Maturation of egg, after, A I 2. Mercuric chloride A IV g, II 4. Methylene blue A I 8. Naphthoquinones A I 12, IV 6. Narcotics A III 1; see also topic Anaesthetics. Neutral red A I 8. Nitrogen mustards A III 2. Nitrophenols A II 3. OH-ions A I g (Hydroxyl ions). Oxygen-lack; see topic Oxygen-Lack. Oxygen tension, low A IV 1. Paraphenylenediamine A II 2. (Dimethylparaphenylenediamine). Parthenogenesis, after, A I 1. (Sodium chloride); A I 11. Penicillin A III 3. Phenols A II 3 (Halophenols, nitrophenols). Phosphate A I to. Pyocyanine A II 1. Redox indicators A I 8. Shaking A I 5. Sodium chloride A I 11, A I 1 (Parthenogenesis). Standing A I 4. Stretched eggs A I 7 (Centrifuged). Sulfide, sodium A III 7. Sulphanilimide A III 5. Toluidin blue A I 8. Unfertilized eggs A I 1. Urethanes A III 1 (Anaesthetics). Usnic acid A IIT 4. X-rays A II 4, IV 9g. Location in Text. B. Sperm Cyanides B II 4. Dilution B I 1. Egg water B II 1. Hydrogen peroxide B I 5, II 2. Iodoacetate B I 3. Malonate B I 3. 2A) ALPHABETICAL COMPILATION Nitrogen mustards B I 7. Seminal fluid B I 2. Sulfhydryl groups B I 4, II 7. Uranyl nitrate B II 5. Urethane B I 8, II 6. Usnic acid B I 6, II 3. X-rays B I 5, II 2. TABLE 18 OXYGEN CONSUMPTION (CM?/HOUR/CM® EGGS) From Ballentine 1940b Species Unfert; Wert) 1 /Unts) emp: Reference Arbacia'punctulata 0.158 0.790 5.0 25° Tang, 1931 0.550 25° Tang and Gerard, 1932 0.040 0.200 5.0 21° Whitaker, 1933a, b 0.038 0.148 3.9 24° Rubenstein and Gerard, 1934 0.023 0.101 4.4 21° Rubenstein and Gerard, 1934 0.149 0.385 2.6 26° Shapiro, 1935b 0.060 0.320 5-3 21° Korr, 1937 0.090 0.450 5.0 25° Korr, 1937 0.100 0.450 4.5 25° Ballentine, 1940b Arbacia lixula 0.028 0.168 6.0 20° Warburg, 1908 Psammechinus miliaris 0.021 0.79 3.8 15° Shearer, 1922b Paracentrotus lividus 0.036 0.216 6.0 23° Warburg, 1915 0.216 21° Runnstrém, 1930 Asterias 0.080 0.080 1.0 23° Tang, 1931b Arbacia punctulata, Shapiro, 1935b, 1939b half-eggs Whole egg 0.149 0.385 2.6 26° Light half 0.123 0.349 2.37 Heavy half 0.281 0.266 1.2 To these references should be added: Shapiro (1935c, tor eggs kept four weeks in the laboratory; Clowes and Krahl (1936a), Keltch and Clowes (1947) who give for both fertilized and parthenogenetic eggs of A. punctulata 2.7 times the unfertilized; Robbie (1946b) who gives F/Unf. = 4.5 at 23°; Shearer (1922a) for Echinus micro- tuberculatus. A list similar to Ballentine’s is given by Krahl (1950, Table I). For Psammechinus miliaris, immature, unfertilized and fertilized see Borei (1948). RESPIRATION A, Eggs I. Increase of O, Consumption (Table 18) 1. After Fertilization and Parthenogenesis.—First described by Warburg (1908) for Arbacia lixula as 6 to 7 times more for the fertilized than for the unfertilized egg. For A. punctulata, 3 to 4 times (Loeb, 1910; Loeb and Wasteneys, 1911a; Loeb, 1913a, p. 27; etc.). For the parthenogenetic egg of Strong ylocentrotus lividus as about OF EXPERIMENTAL WORK 215 the same as for the fertilized egg (Warburg, 1910); of A. punctulata as double (McClendon and Mitchell, 1912). Later, more accurate data were tabulated by Ballentine (1940b), Table 18. He has converted some of the original data, given in millions of eggs into cm of eggs for uniformity in the equation: QO, = cm® O, con- sumed per hour per cm’ cells. 2. After Maturation—Mature egg consumes more oxygen than immature (Boell, et al., 1940). But Lindahl and Holter (1941) found that the oocytes of P. lividus con- sumed more oxygen than the unfertilized or fertilized eggs; Borei (1948) found that the oocytes of Ps. miliaris consumed more than the unfertilized eggs. 3. During Development.—Increase hourly to fifth hour after fertilization (Loeb, 1913a, p. 29). Increase 1.6 to 4.5 hours (Chesley, 1934). Gradual increase to 95 minutes (Whitaker, 19334). Increase to 22nd hour (Tyler, Ricci, and Horowitz, 1938). Gradual increase till hatching, then more marked increase (Hutchens, Keltch, et al., 1942; Krahl, 1950). Tang (1931) found no change through third cleavage, but he (1948) found mitotic rhythms. Rhythms have also been found in Ps. miliaris, etc. by Zeuthen (1947, 1949, 1950, 1951). For increased respiration in later stages see M. M. Brooks (1943). 4. On Standing.—(Wasteneys, 1916; Gerard and Rubinstein, 1934, p. 376 foot- note; et al.). Tyler, Ricci and Horowitz (1938) find increase is due to bacteria; see also Gorham and Tower (1902). 5. On Shaking.—(Whitaker, 1933a, p. 488 footnote; Velick, 1941, also for cen- trifuged eggs). 6. On Cytolysis.—(Loeb, 1913 a, p. 14; Tang, 1931; Whitaker, 19334, p. 487 foot- note; Tyler, Ricci, and Horowitz, 1938; et al.). Decrease found by Heilbrunn (1915b). See Rubenstein and Gerard (1934) and Ballentine (1940c). 7. In Stretched Eggs.—By centrifuging, 60 to 100 % greater oxygen consumption (Velick, 1941). Cytochrome oxidase activity 3.2 times greater (Navez and E. B. Harvey, 1935, see under Cytochrome Oxidase). 8. By Redox Indicators.—Methylene blue or toluidin blue (Barron, 1929; Barron and Hamburger, 1932; Chesley, 1934; Runnstrém, 1935a; Clowes and Krahl, 1936a; Ballentine, 1938, 1940b). Increased oxidation with block or delay of cell division (Clowes and Krahl, 1936a, Shapiro, 1948a). But M. M. Brooks (1943) found increased oxidation with acceleration of development and larger plutei. Neutral red (Clowes and Krahl, 1936a). O-cresol-indophenol (Clowes and Krahl, 1936a). See Krahl, (1950, Table VIII). 9. By OH Ions.—(Loeb and Wasteneys, 1911b, 1915; Wasteneys, 1916, see Loeb, 1913a, p. 37; McClendon and Mitchell, 1912). 10. By Hexose Phosphate——(Runnstr6m, 19354). 11. By Sodium Chloride.—And other parthenogenetic agents (Mitchell and McClen- don, 1911; McClendon and Mitchell, 1912; Keltch and Clowes, 1947). 12. By Naphthoquinones.—In low concentrations; decrease of respiration in higher concentrations (Anfinsen, 1947). II. Increase of O, Consumption with Reversible Blocking or Delay of Cell Divi- sion by 1. Pyocyar 2e.—Increased O, consumption, cell division reversibly blocked in high concentraticas; iron-containing enzyme not involved (Barron and Hamburger, 1932; Runnstrém, 1935a; Clowes and Krahl, 1936a; Korr, 1937). Also in thawed frozen eggs (Runnstrém, 19354). 2. Dimethylparaphenylenediamine.—Cell division reversibly blocked in high concen- trations (Runnstrém, 1935a; Clowes and Krahl, 1936a). Effect on O,uptake of half-eggs (Boell, Chambers, Glancy, and Stern, 1940). 3. Nitro- and Halophenols—And related compounds; cell division reversibly blocked at optimum respiratory concentration (Clowes and Krahl, 1934a, b, 1935, 216 ALPHABETICAL COMPILATION 1936a, b, c; Krahl and Clowes, 1935, 1936a, b, 1938, 1940; Hutchens, Krahl, and Clowes, 1939; Krahl, Keltch, and Clowes, 1937; Clowes, 1951). On parthenogenetic eggs (Keltch and Clowes, 1947; Keltch, Walters, and Clowes, 1947). On cell-free system, see under VI. See Krahl (1950, p. 196 and his Table IV.) 4. Mercuric Chloride, Hydrogen Peroxide and X-rays.—In small doses increase respir- ation with delay of cell division; large doses decrease respiration with delay of cell division (Barron, Flood, and Gasvoda, 1949; Barron and Seki, 1952; Barron, Seki, and Johnson, 1952). 5. Methylene blue—See above A I 8. III. Practically No Effect on O, Consumption, or Slight Decrease, with Blocking (Sometimes Reversible) of Cell Division by 1. Anaesthetics.—(Narcotics). See topic Anaesthetics. 2. Nitrogen Mustards.—(Barron, Seegmiller, Mendes, and Narahara, 1948; Hut- chens and Podolsky, 1948). 3. Penicillin —(Henry and Henry, 1945). See Krahl (1950, p. 206). 4. Usnic Acid.—Antibiotic (Marshak and Harting, 1948). 5. Sulphanilamide.—(0.04, M) blocks cleavage and reduces oxygen consumption to 55 %. No effect on unfertilized eggs (Fisher, Henry, and Low, 1944). (See Krahl, 1950, Table VIII). 6. Sodium Azide.—(5 < 10~? M) cell division blocked while respiration is inhibited 50 % (Krahl, Keltch, Neubeck, and Clowes, 1941; Fisher, Henry, and Low, 1944). 7. Sodium Sulfide—(2 x 10-4 M) cell division blocked while respiration is in- hibited 50 % (Krahl, Keltch, Neubeck, and Clowes, 1941). IV. Decrease or Inhibition of O, Consumption and of Cell Division 1. Low oxygen Tension—O, consumption practically constant from 228 to 20 mm. Hg., then decreases; same for cell division; no cleavage below 4 mm. Hg. (Amberson, 1928). See also Tang (1931, 1933, 1941; Gerard (1931); Tang and Gerard (1932) ; Clowes and Krahl (1940); Krahl (1950). For unfertilized eggs, see Tang (19314). 2. Carbon Dioxide.—(Root, 1930; Haywood and Root, 1930, 1932). 3. Carbon Monoxide.—Action reversed by light (Clowes and Krahl, 1940; Krahl, 1950). On cytochrome oxidase (Krahl, Keltch, Neubeck, and Clowes, 1941). See M. M. Brooks (1943). 4. Cyanides.—See under topic Cyanides. 5. Iodoacetate.—(Iodoacetic acid). (Runnstrém, 1935c; Clowes and Krahl, 1940; Krahl and Clowes, 1940). 6. Naphthoquinones.—Inhibit respiration and cleavage in high concentrations, stimulate respiration in low concentrations (Anfinsen, 1947). 7. Malononitrile—Depresses respiration, inhibits cleavage (Villee, et al., 1949). 8. Caffeine.—(Cheney, 1945, 1946). g. Mercuric Chloride, Hydrogen Peroxide and X-rays.—In large doses decrease respi- ration with delay of cell division; in small doses increase respiration with delay of cell division (Barron, Flood, and Gasvoda, 1949; Barron and Seki, 1952; Barron, Seki, and Johnson, 1952). For x-rays see also Chesley (1934) and Evans (1950). V. Oxygen Consumption of Half-Eggs Unfertilized white half, same as whole egg; unfertilized red half 88 % in excess of whole egg; fertilized white half 2.7 fold increase at 25.9 °C, like whole egg; fertilized red half same as unfertilized (Shapiro, 1935b, 1939b). See under A I, 1 Table 18. (Also Navez and E. B. Harvey, 1935; Ballentine, 1940c). VI. Oxygen Consumption in Cell-Free System In homogenates or in thawed frozen eggs (Runnstr 6m, 19354, also with pyocyanine or methylene blue). Oxidase activity but no respiration (Boell, Chambers, Glancy, and Stern, 1940). Cytochrome oxidase activity and content of fractions (Krahl, OF EXPERIMENTAL WORK 217 Keltch, Neubeck, and Clowes, 1941; Hutchens, Kopac, and Krahl, 1942). Oxygen consumption of cell-free system of unfertilized eggs about three times that of same weight of intact unfertilized eggs and about same as that of equal weight of fertilized eggs (Keltch, Strittmatter, Walters, and Clowes, 1950). Slightly different figures given by Crane and Keltch (1949). Stimulation by dinitrocresol and phosphate; also methods (Crane and Keltch, 1949). Oxidative phosphorylation (Keltch, et al., 1950; Clowes, Keltch, et al., 1950; Strittmatter, et al., 1950; Clowes, et al., 1951 a, b; Keltch, et al., 1951). VII. Carbon Dioxide Production Nearly equivalent increase on increase of O, consumption (Clowes and Krahl, 1936a). See Ballentine (1940b, c). Carbon dioxide production occurs in rhythms in segmenting eggs, greatest at time of active division (Lyon, 19044, b). VIII. Respiratory Quotient RQ, of fertilized eggs 0.78 (Amberson, 1928); 0.71 (Root, 1930). Of eggs one to 25 hours after fertilization 0.86 (Hutchens, Keltch, Krahl, and Clowes, 1942; Krahl, 1950). IX. Temperature Coefficient, Q ,, (Table 19) Fertilized eggs, about 2 (3°-27 °C.) (Loeb and Wasteneys, 1911a; Loeb, 1913 a, Pp. 33)- Unfertilized eggs, 4.1; fertilized eggs 1.8; cytolysed eggs 1.9 (13°—30 °C.). Increase of O, consumption on fertilization depends on temperature, 8 times as great at 11 °C., and twice as great at 29.9 °C.; there should be no increase at 32 °C. (Ruben- stein and Gerard, 1934). TABLE 19 TEMPERATURE COEFFICIENTS, Q,9 FOR VELOCITY OF OXIDATIONS AND OF FIRST CLEAVAGE OF ARBACIA PUNCTULATA Loeb and Wasteneys, 1911a; Loeb, 1913a, p. 33; Loeb and Chamberlain, 1915 Interval of Qio for Q 9 for cleavage temperature respiration of Q 40 for (Loeb and fertilized egg cleavage Chamberlain 1915) 3-137 2.18 = ites 5-15, 2.16 nae =e a 2.0 7.3 = 8-18° — 6.0 6.0 9-19" = >4.0 4.5 10—20° 27, 3-9 3.7 II—21° = ian 3.3 12—22° a Sao Bes 13-23" 2.45 = 2.8 —

NaCl > sea water > MgCl, > CaCl, (E. B. Harvey, 1945). Heilbrunn gives the same order except that KCl and NaCl are reversed (Heilbrunn, 1923, 1928, p. 146, 1943, p. 81). Breaking with Centrifugal Force.—Break more readily than in sea water in the order of increasing viscosity (reverse order of stratification), that is, the most viscous (least stratified) break most readily (E. B. Harvey, 1945). Parthenogenetic Agent.—NaCl added to sea water; first used by Morgan (1898, 1899, 1g00a, b, etc.); subsequently by Loeb, R. S. Lillie, and many others. NaOH has also been used (Loeb, 19134, p. 148). The best parthenogenetic agent for Arbacia punctulata is: 30 gm. NaCl to one liter of sea water for 20 minutes, then sea water. Or increase the salt content of sea water by boiling to half its volume, leave eggs in this solution for 20 minutes, then sea water (E. B. Harvey, 1936). Results vary with different batches of eggs, and one can vary the solution and time slightly. Other Species and General References Bialaszewicz, 1927, 1929. A. lixula, P. lividus; electrolytes in eggs. Harvey, E. B., 1938a. Parthenogenetic agent in Naples sea urchins. Heilbrunn, 1952. General Physiology, p. 519. Herbst, 1904. Salts necessary for development. Loeb, 1913a. Artificial Parthenogenesis. S. purpuratus, S. franciscanus, especially. Malm and Wachtmeister, 1950. Ps. miliaris, S. drébachiensis, amount Na in unfertilized and fertilized eggs. Morgan, 1896. Sphaerechinus granularis, astrospheres. Robertson, 1939; Robertson and Webb, 1939; Webb, 1939. Inorganic composition of sea water and body fluids. Rothschild and Barnes, 1953. P. lividus, inorganic constituents of egg; table of salts and species. SPECIFIC GRAVITY See Density SURFACE FORCES See Tension at the Surface SURFACE TENSION See Tension at the Surface TENSION AT THE SURFACE Surface Tension, Surface Forces Unfertilized Egg.—o.2 dyne/cm., by centrifuge method (E. N. Harvey, 1931, 19324, b, 1937, 1938). 0.08 dyne/cm., by compression method; internal pressure 40 dynes/ cm.” (Cole; 1932). Agreement with Harvey and Cole, by stretching (Norris, 1939). Fertilized Egg.—(Without fertilization membrane). Same as unfertilized till just before cleavage, then increase. 0.03-0.05 dyne/cm. (for eggs late in season) (Cole and Michaelis, 1932). Cleaving Egg.—o.og dyne/cm.; 62 dynes/cm.? excess internal pressure when one of two blastomeres is punctured (Sichel and Burton, 1936). Fertilization Membrane.—Formation due to lowered surface tension (Heilbrunn, 1913, 1915 a, 1924a, 1925d). Cytolysis.—Due to lowering of surface tension of plasma membrane (Heilbrunn, 1915 a). OF EXPERIMENTAL WORK 223 Cell Division Explained by change in surface tension (R. S. Lillie, 1903, 1909; McClendon, 1910b; see Wilson’s The Cell, 1925, p. 158, 192—197 for general refer- ences and discussion; also Gray, 1931, p. 214). For interfacial tension, oil-protoplasm, oil-water, see under Oil, references to Chambers and Kopac. Other Species and Reviews Danielli, 1942, Bourne’s Cytology and Cell Physiology. Review. E. N. Harvey, 1954. Protoplasmatologia. Review. E. N. Harvey and Danielli, 1938. Review. Vlés, 1926. Paracentrotus lividus. TWINS, TRIPLETS, QUADRUPLETS Twins, triplets, quadruplets may be produced through destruction of hyaline layer which binds blastomeres together by: Shaking.—In Echinus (Driesch, 1891, and later). Hypotonic Sea Water.—(Loeb, 1905), p. 303, translation of 1894 paper). See Just, 19394, Pp. 13. Hypertonic Sea Water.—Just after cleavage is best method. (E. B. Harvey, 19404). (Plate XVI, Photograph 8). MgCl,.—Added to sea water (Loeb 1900a). Lack of K.—Na or Ca in sea water, in S. purpuratus, (Loeb, 1909b, 1912, p. 204). Ethyl Urethane.—(0.2 M) 3 to 60 min. then sea water and fertilize; fertilization membrane pinches the egg into two parts (E. B. H. unpub.). Centrifuging.—In 2-cell stage, in Ps. microtuberculatus (E. B. Harvey, 1935b); in Arbacia (1940a). Hyaline layer is removed by centrifugal force. Each twin has two micromeres (E. B. H. unpub.). Natural twins formed in Prionocidaris baculosa by separation of first two blastomeres which develop separately into half sized larvae. (Mortensen, 1938, p. 14). Twins, triplets and quadruplets from a single egg may all develop into perfect dwarf plutei (E. B. Harvey, 1940a). Plate XVI, Photograph 8. ULTRASONIC WAVES High frequency sound waves on Arbacia punctulata eggs (E. N. Harvey, E. B. Harvey, and A. L. Loomis, 1928; E. N. Harvey, 1930). High speed photographs show eggs can be cytolysed in less than 1/1200 second (E. N. Harvey and Loomis, 1931). Other Forms Schmidt, F. O., 1929, California starfish. ULTRAVIOLET LIGHT Cleavage and Development (Eggs or Sperm Radiated).—Delayed cleavage and ab- normal development (Child, 1924, p. 109 footnote; Hinrichs, 1926b, c, 1927; Nebel, E. B. Harvey, and Hollaender, 1937; E. N. Harvey, 1942; Giese, 1946). Delayed cleavage and enhanced recovery with visible light (Blum, ef al., 1949a, b, 1950a, c, d, 1951; Marshak, 1949b, c). Of centrifuged eggs. Delay greater if irradiated through oil cap, less if through pigment (C. V. Harding and Thomas, 1950). Of half-eggs. Delay in cleavage of white halves; and of red (non-nucleate) halves ex- cept when exposed before fertilization, to wave lengths of 2700-3130 A (Blum, et al., 1949a, 1950C, d, 1951), Photorecovery of white halves (Blum, et al., 1949a, 1950c, d, 1951; Marshak, 1949b, c). 224 ALPHABETICAL COMPILATION Parthenogenetic Agent.—(Loeb, 1914a; R. S. Lillie and Baskervill, 1922; Heilbrunn and Young, 1930; E. B Harvey and Hollaender, 1937, 1938; Moser, 1939b). For cytology, multipolar and anastral mitoses see Nebel, E. B. Harvey, and Hollaender (1937); E. B. Harvey and Hollaender (1938). For method, Hollaender (1938). Fertilization membrane on one side (Moser, 1939b; E. N. Harvey, 1942); also Spikes (1944) for Lytechinus pictus and Reed (1943, 1948) for S. purpuratus. Both white and red (non-nucleate ) half-eggs are activated with wave lengths of 2260-2480 A, and the red halves with 2650-3300 A also (E. B. Harvey and Hollaender, 1937, 1938). Cytolytic Agent.—R. S. Lillie and Baskervill, 1922; Heilbrunn and Young, 1930). Permeability—Uncertain (See Heilbrunn’s 1952 General Physiology, p. 164). In- crease in P. lividus (Tchakhotine, 1921b) and in Lytechinus pictus (Spikes, 1944), but Reed (1948) found no change in permeability in S. purpuratus. Viscosity.—Decrease for 15 minutes then increase (Heilbrunn and Young, 1930). Breaking with Centrifugal Force.—Break more readily after radiation (E. B. H., 1950 unpub.). Loss of Fertilizin—And agglutinating power (Hinrichs, 1926c, 1927). Echinochrome.—Fades (Hinrichs, 1927). Pigment granules in egg become clumped (E. B. H., 1950 unpub.). Loss of Felly.—(E. B. H., 1950 unpub.). Sperm.—Reduced motility and fertilizing power and agglutination (Hinrichs, 1926b, c, 1927). Sperm more sensitive than eggs (Giese, 1946). Sperm treated with ultraviolet before fertilization delay cleavage; no photorecovery if outside the egg (Marshak, 1949b, c; Blum, et al., 1950c, 1951). Amoebocytes—They round up and red ones become pale (E. B. H., 1950, unpub.). Chromatin.—As photographed by ultraviolet light (E. B. Harvey and Lavin, 1944). Eggs.—Half-eggs and plutei as photographed by ultraviolet light (E. B. Harvey and Lavin, 19514). Ultraviolet and Heat.—(Hinrichs, 1927; Hutchings, 1948). Other Species (additional) and General References Casperson and Schultz, 1940, Psammechinus miliaris, absorption spectra. Chase, 1938. Dendraster excentricus. Giese, 1939. Strong ylocentrotus purpuratus, sperm. Giese, 1945, 1946, 1947, 1949, 1950. General and reviews; see especially for references to Giese on Pacific Coast forms. Giese and Wells, 1952. S. purpuratus. Heilbrunn and Mazia, 1936. Review in Duggar’s Biological Effects of Radiation, vol. I, p. 625-676. Hollaender, 1954. Radiation Biology. Review. Tchakhotine, 1921b. Paracentrotus lividus, puncture method. Tchakhotine, 1937. P. lividus, parthenogenesis, change in permeability. Vlés and Gex, 1928, 1934. P. lividus. Wells and Giese, 1950. S. purpuratus, photoreactivity. — VISCOSITY Water.—At 20 °C. = 1 centipoise. Clear Protoplasm.—Of Arbacia egg = 3 centipoises; determined by centrifuging granules and by Brownian movement of granules in centrifuged egg. (Heilbrunn, 1926a, b, 1927, 1928, p. 67, 1943, p. 69). Entire Protoplasm.—Equals 6—7 centipoises (Heilbrunn, 7bzd.). Nuclear Fluid.—About 10 centipoises (Heilbrunn, 1943, p. 73). OF EXPERIMENTAL WORK 225 x Immature Egg.—Is more viscous than mature. (Heilbrunn, 1921, 1928, p. 278, 1943, p. 70; Goldforb, 1935b; E. B. H.). Maturation.—During, decrease in viscosity (Goldforb, 1935b; E. B. H.). Fertilized Egg —More viscous than unfertilized. (Heilbrunn, 1915, 1g20a, 1928, p. 264; E. B. Harvey 1932, 1933b). Prophase.—After breaking of nuclear membrane, increase (E. B. H.). Parthenogenetic Egg.—More viscous than unfertilized. (Heilbrunn, 1915, 1928, p. 261). Stages in Mitosis—Changes in viscosity (Heilbrunn, 1920a, 1921, 1927, 1928, p. 265, 1943, p. 653; Chambers, 1919, 1924; Fry and Parks, 1934; Fry, 1936; Page, 19294). Red Halves —More viscous than white halves (E. B. Harvey, 1932; Chambers, 1938 a). Decrease in Viscosity by Ageing. Decrease after 35 hours (Goldforb, 1935b). Alkalies (Barth, 1929). Anaesthetics. Ether (Heilbrunn, 1920a and b, 1925c, 1927, 1928, p. 205, 288; see Chambers, below under Increase). Ether and other anaesthetics (Heilbrunn, 1920a and b, 1928, p. 206). See Table 15. Carbon dioxide (Howard, 1931, 1932; Jacobs, 1922, after short exposure). Colchicine (Beams and Evans, 1940, prevents gelation and aster disappears after fertilization; Wilbur, 1940). Electric current; direct and alternating; transitory decrease, then increase (Angerer, 1939). Fatty acids and salts of fatty acids (Howard, 1931). Heparin and heparin-like substances (Heilbrunn and Kelly, 1950). Hypotonic sei water (Heilbrunn, 1920a and b, 1928, p. 211; Chambers, 1924; E. B. Harvey, 1943). Mechanical agitation (Chambers, 1924). Nitrogen mustards (E. B. H. and Cannan, unpub.). : Pressure, hydrostatic; on unfertilized eggs and on cortex of fertilized eggs (Brown, 1934; Marsland, 1938, 1939, 1942, 1950, 1951.) Salts. CaCl, (Heilbrunn, 1923, 1927, 1928, p. 146, 1943, p. 81; E. B. Harvey, 1945; see Chambers, 1949). MgCl, (Jbid). Aluminum chloride (Heilbrunn, 1925b, 1928, p. 151). Copper chloride; latent period 21 min, then decrease, then increase (Angerer, 1937). (See also Heilbrunn, 1928, p. 143). Temperature. Heat, decreasing viscosity with increasing temperature —1.8 °C. to +28 °C. (Costello, 1934). Cold, about —3 °C. (Heilbrunn, 1920a and b, 1928, p. 106). Eggs stratify by gravity if kept overnight at 8° but not at 21.5°. They break more readily on centrifuging at low temperatures (E. B. H.); Costello (1938) found the reverse. Ultraviolet rays, decrease at first, then increase. (Heilbrunn and Young, 1930). Increase in Viscosity by Acids (Barth, 1929; list of acids and effective pH on p. 510). Ageing. Viscosity increases for first 35 hrs., then decreases (Goldforb, 1935b). Anaesthetics. Chloretone (Heilbrunn, 1920a, b). Ether (Chambers, 1924; see Heilbrunn and above under Decrease). Body fluid (Goldforb, 1935b). Carbon dioxide (Jacobs, 1922 after long exposure). Cytolysis (Heilbrunn, 1928, p. 245). Electric current; direct and alternating; transitory decrease, then increase (Ange- rer, 1939). Formalin (Heilbrunn, 1927). Hypertonic sea water (Heilbrunn, 1915, 1920a, 1943, p. 81; Chambers, 1924; * E. B. Harvey, 1943). 226 ALPHABETICAL COMPILATION Injury substances (D. Harding, 1951). Light with dyes, eosin and rose bengal (Alsup, 1941). Potassium cyanide (Heilbrunn, 19204, b). Salts. NaCl (Heilbrunn, 1923, 1927, 1928, p. 146, 1943, p. 81; E. B. Harvey 1945; see Chambers, 1949). KCl (Jbid.). Potassium citrate (Wilbur and Recknagel, 1943). NH,Cl (Heilbrunn, 1923, 1928, p. 146). CuCl,; increase after 20 min. (Heilbrunn, 1928, p. 143; see also Angerer, 1937): CuSO, (Heilbrunn, 1927, 1928, p. 143). HgCl, (Ibid. ). Saponin (Heilbrunn, 1915). Temperature. Cold, o °C. to 5 °C. (Payne, 1928, 1930). Cold, increasing viscosity with increasing cold 28 °C. to —1.8 °C. (Costello, 1934). Cold, extreme, below —3 °C. (Heilbrunn, 1920b). Heat, 31.5 °C. to 32.9 °C. (Heilbrunn, 1925a, 1928, p. 116). Ultraviolet rays, decrease of interior at first, then increase. (Heilbrunn and Young 1930). X-rays, increased viscosity following fertilization is prolonged; no effect on unfer- tilized eggs (W. L. Wilson, 1950). No Effect on Viscosity of External pH (Heilbrunn, 1923, pH 7-9; Goldforb, 1935b). Shearing (centrifugal) forces (Heilbrunn, 1928, p. 47, 1943, p. 70; Howard, 1932). X-rays on unfertilized eggs (W. L. Wilson, 1950). Other Species and General References J. E. Harris, 1939. Echinus esculentus, nucleus. Heilbrunn, 1928, 1943, 1952. General. Hyman, 1923. Strong ylocentrotus franciscanus and S. purpuratus. Marsland and Landau, 1950. Echinarachnius parma. Mitchell, 1941. General. Runnstrém, 1928c, d. Echinocardium, Paracentrotus, Psammechinus microtuberculatus, Ps. miliaris, Arbacia lixula. See his literature list for other references. Runnstr6m and Kriszat, 1950a. Ps. miliaris. Seifriz, 1920, 1924. Tripneustes, Echinarachnius ; 1929. General. VISIBLE LIGHT AND PHOTODYNAMIC ACTION Visible Light.—On cleavage, little effect (Giese, 1947; Marshak, 1949c). On parthe- nogenesis. Induces membrane formation (R. S. Lillie and Hinrichs, 1923; Hinrichs, 1926a). Photosensitization.—Visible light and dyes. On cleavage and development (eggs or sperm treated), abnormal development or delayed cleavage with Benzoflavin (Hinrichs, 1926a, b). Eosin (Hinrichs, 1923; 1926a, b.; Child, 1924, p. 109 footnote; Pereira 1925). Methylene blue (Hinrichs, 1926a, b). Neutral red (Hinrichs, 1926a, b). Rhodamine B (L. B. Clark, 1940). Riboflavin, on sperm (Marshak, 1949c). Rose bengal (Alsup, 1941). Causes parthenogenesis with Eosin (R. S. Lillie and Hinrichs, 1923; Alsup, 1941) and Rose bengal (Alsup, 1941). Causes cytolysis with rhodamine (L. B. Clark, 1940), Photoreactivity.—Enhanced recovery with visible light after ultraviolet light (Blum et al., 1949a, b, 1950c, d, 1951; Marshak, 1949b, c). OF EXPERIMENTAL WORK 227 Reversal.—Inhibition by carbon monoxide of cytochrome oxidase is reversed by light (Krahl, Keltch, Neubeck, and Clowes, 1941). Viscosity.—Increase with eosin or rose bengal (Alsup, 1941). Other Species Bohn and Drzewina, 1923. Strong ylocentrotus lividus, neutral red and light. A. R. Moore, 1928b. S. purpuratus, eosin and light. Rethschild, 1949. Psammechinus miliaris, light and CO, Tennent, 1942. Lytechinus variegatus, many dyes and light. Also, many short articles in this literature list. Wells and Giese, 1950. S. purpuratus, on sperm. VITAL DYES On Stratified, Centrifuged Eggs.—See Part III on Centrifuging, Chapter 17 and Table 8. (Also E. B. Harvey, 1941Cc). On granules of egg.—See under Chromatophores, Clear Layer, Mitochondria, Yolk Granules. (This section). Immature eggs.—( Toxopneustes) stain with certain intravitam dyes more than fer- tilized and fertilized more than unfertilized (Lyon and Shackell, tg10b; E. N. Har- vey, I9I10C). Increased Oxygen Consumption—With methylene blue, toluylene blue and the effect of KCN and urethanes (Barron 1929, Barron, and Hamburger, 1932). Methylene blue, neutral red, o-cresol-indophenol, dimethyl paraphenylenediamine, with block of cell division (Clowes and Krahl, 1936a: Krahl, 1950, Tables VIII, IX). Methyl- ene blue, effect on cleavage and development (Waterman, 1938, 1941). Methylene blue with acceleration of development; effect of KCN and CO (M. M. Brooks, 1943). Methylene blue, toluylene blue, brilliant cresyl blue with delay of first cleavage (Shapiro, 1948a). Methylene blue; x-rays have no effect (Chesley, 1934). With pyocyanine not affected by KCN (Barron and Hamburger, 1932). More in- crease in respiration in unfertilized than fertilized eggs; more with pyocyanine than with methylene blue; effect of HCN and of lithium with pyocyanine (Runnstrém, 1935a, b). Increase of respiration with block to cell division (Clowes and Krahl, 1936a; Krahl, 1950, his Table VIII). Effect of KCN and temperature (Korr, 1937). Effect on cleavage and development (Waterman, 1938, 1941). Janus Green B.—Delays or inhibits cleavage and development. (R. D. Allen, 1950). Photosensitizing Action—Neutral red and methylene blue (Hinrichs, 1926a, b); rhodamine B (L. B. Clark, 1940). See extensive study on Lytechinus variegatus by Tennent (1942; and many short papers). Exogastrulae.—Produced by two indigo sulphonate dyes, not by methylene blue (M. M. Brooks, 1951). Vital Staining.—Of parts of eggs or embryos to trace development (Vogt method) ; not used for Arbacia but for other sea urchin eggs by von Ubisch (1925), Lindahl (1932c) and Hérstadius (1935), et al.; see article by Hérstadius (1950) in McClung’s Microscopical Technique, 3rd ed., p. 561-563. Injection. Of vital dyes for pH determination, see under Hydrogen Ion Concen- tration. General References Standard books on cytological technique: , Conn’s Biological Stains, 1946, 5th ed. / & Lee’s Vade Mecum, 1950, 11th ed. ; McClung’s Microscopical Technique, 1950, 3rd ed / 228 ALPHABETICAL COMPILATION Technical books on dyes: Rowe, 1924, Editor of Color Index. Schultz, 1928-1934, Farbstofftabellen. Vital staining, general: von Mollendorff, 1920, 1928. Other Species Bank, 1933. Arbacia lixula; nucleus. Becker, 1936. Review. ; Child, 1936b. S. purpuratus, S. franciscanus, Dendraster excentricus; dyes and axial gradients. Gellhorn, 1931. S. purpuratus ; dyes and permeability. Gersch and Ries, 1937. A. lixula, Sphaerechinus granularis, Paracentrotus lividus, Psammechinus miliaris ; determination. Lepeschkin, 19415. P. lividus; neutral red. Monné, 1945. Ps. miliaris, Echinocardium cordatum etc.; eggs centrifuged and stained. Also other papers. Moore, Bliss, and Anderson, 1945. S. purpuratus, Dendraster excentricus ; pyocyanine. Orstrém, 1932a. P. lividus ; dimethylparaphenylenediamine. Ranzi and Falkenheim, 1937. Sph. granularis ; determination. Good literature list with titles. Runnstr6m, 1930, 1932. P. lividus; methylene blue and dimethylparaphenylenediamine; 1935b. Echinarachnius parma; pyocyanine. Runnstrém and Thérnblom, 1938. P. lividus; pyocyanine. VITELLINE MEMBRANE Definition.—It is the membrane on the exterior of the egg proper, outside the plasma membrane. The older investigators made no distinction between plasma membrane and vitelline membrane referring to them together as a ‘“‘pellicle’’. It is difficult to distinguish the two optically in an unfertilized Arbacia egg. The vitelline membrane is now generally believed to elevate on fertilization or parthenogenesis, and become, somewhat modified, the fertilization membrane (Chambers, i942, 1944; Kopac, 1940a). See under Fertilization Membrane and Fig, 9 and ro. Thickness.—Readily visible but not measurable with a light microscope. As deter- mined by the electron microscope, it is (dried) about 250 A thick just after being elevated as the fertilization membrane (E. B. Harvey and Anderson, 1943). This is considerably thicker than the dried membrane of the red blood cell which according to the latest data is 50-60 A (Parpart and Ballentine, 1952; Hillier and Hoffman, 1953). However, Mitchison (1953) has recently given a lower figure for the (ferti- lization) membrane of Psammechinus miliaris, 100 A, with electron microscope. Sec Fertilization Membrane. Structure.—According to Heilbrunn (1915a, 1926b, it is a protein gel with little or no lipid, slightly rigid. According to Kopac (19404), it is soft, plastic and gela- tinous, a delicate film-like membrane. In electron microscope photographs, no struc- ture is evident (E. B. Harvey and Anderson, 1943). Centrifuging.—Causes it to flow toward centrifugal pole, becoming thinner at cen- tripetal pole (E. B. Harvey, 1932). Microdissection—Can be torn or pulled out with a needle, or removed (Kite, 1912; Chambers, 1921a, 1930, 1942, 1949; Kopac, 19404). Re-Formed.—When slightly broken (Heilbrunn, 1915a; Chambers, 1917. 19214). Elasticity—(Heilbrunn, 1928, p. 99, 250; 1943, p. 70, 92; Cole, 1932; E. N. Harvey, 1936, 1937; Chambers, 1942). On the unfertilized egg it stretches from the diameter of a sphere, 74 up, to the length of a spheroid, 140 py, on centrifuging (E. B. Harvey and Anderson, 1943). About 25 % increase in surface on centrifuging (E. N. Harvey, 1931c). Can be stretched between two needles (Norris, 1939). OF EXPERIMENTAL WORK 229 Tension at Surface.—o.2 dyne/cm. (E. N. Harvey, 1931 c, 1937; Norris, 1939), when stretched; 0.08 dyne/cm. (Cole, 1932); 0.09 dyne/cm. (Sichel and Burton, 1936). Tension of unfertilized and fertilized eggs, without fertilization membranes, (until just before cleavage) is the same (Cole and Michaelis (1932). See Tension at the Surface, and E. N. Harvey (1954). Electrical Properties Of two membranes together (McClendon, ig1ob; R. S. Lillie, 1911 b, 1916b; Heilbrunn, 1923, 1926b, 1928, p. 183). Membrane resistance is > 100 ohms/cm? (Cole, 1941; see also 1940 and Cole and Curtis, 1938). Capacity about one microfarad/cm? (Cole, 1941). Zeta potential without jelly, about 30.3 millivolts (Dan, 1933). See Electrical Properties. Oil Coalescence.—(Kopac and Chambers, 1938; Kopac, 1940a; Chambers, 1944). Removal.—By (1) micromanipulation (Kite, 1912; Chambers, 1921 a, 1930, 1938b, 1942; Chambers and Kopac, 1937a; Kopac and Chambers, 1938). (2) Washing in isosmotic NaCl or KCl when membrane is being lifted off to form the fertilization membrane (transitional membrane), 1.5 minutes after insemination (Chambers, 1942, 1944; Kopac, 1940a). (3) Urea (1 M) (Chambers, 1940, 1942; Kopac, 1940a, 1943; Moser, 1940). Pioneer work on development without fertilization mem- brane by action of urea on sirface of egg (Strong ylocentrotus purpuratus) was done by A. R. Moore (1929, 1930a). He used a molar solution of urea at pH 7 for two minutes, then fertilized the eggs in sea water. Moser (1940) and others (Kopac, 1940a) have used a similar technique for Arbacia. (4) Trypsin, used for A. punctulata by Runnstrém in 1950 and by Dan and Mazia in 1951. Dan used (1951 unpub.) 0.1 % non-crystalline trypsin of Merck in sea water for a few minutes or 4 mg. crystalline trypsin in 100 cc. sea water for 10 minutes; this dissolves the vitelline membrane, and is probably the best way of obtaining fertilized eggs without fertil- ization membranes. Trypsin had been used previously for Psammechinus miliaris and Echinocardium cordatum (Runnstrém, Monné, and Broman, 1944). In 1932 (unpub.) Kunitz had found that trypsin had an effect on the precursor of the fertilization membrane in Arbacia, preventing its appearance (See A. R. Moore, 1949 a, footnote, P- 243; 1949b, footnote, p. 207). Best medium for keeping denuded eggs is mixture of NaCl and KCl in proportion of 19 to 1 in concentrations isotonic with sea water and pH 7.0 (Chambers, 1940, 1944). Effect of Calcium.—Brittleness increased (Heilbrunn, 1928, p. 149; Chambers, 1944, 1949, 1950; see Kopac, 19404). X-Rays.—(Kopac, 1940c, 1941b). Anaesthetics.—(R. S. Lillie, 1914b). Ageing.—(Goldforb, 1937). Centrifugal Force on Breaking.—Break more readily when vitelline membrane is removed (Solano and Mazia, 1953). Copper.—Present and absorbed (Glaser, 1923). Ribonucleic Acid.—Present (Lansing and Rosenthal, 1949). Strength.—Vitelline membrane of different species differs in strength; that of Arbacia is weaker than that of Echinarachnius and Asterias (Kopac, 19404). Other Species (additional) and General References Carter, 1924. Sphaerechinus granularis. Chase, 1935. Strong ylocentrotus purpuratus, Dendraster excentricus. Hobson, 1932b. Psammechinus miliaris. Hultin, 1948a, b. Ps. miliaris, Sphaerechinus granularis, Paracentrotus lividus, A. lixula; trypsin and urea, cross fertilization. Hyman, 1923. S. franciscanus, S. purpuratus, and review of earlier work. Just, 1939b. The Biology of the Cell Surface, general. Moore, A. R., 1930b. Dendraster. 230 ALPHABETICAL COMPILATION Moore, A. R., and M. M. Moore, 1931. Paracentrotus lividus. Motomura 1941b. S. pulcherrimus. Runnstrém, 1948a. Ps. miliaris, Echinocardium cordatum, trypsin. Runnstrém, 1949a, 1952a. General. Runnstrém and Monné, 1945a. Ps. miliaris, Echinocardium cordatum ; trypsin. Runnstr6ém, Monné, and Broman, 1944. Ibid. Runnstr6m, Monné, and Wicklund, 1946. Jbid. X-RAYS Cleavage and Development (Eggs or Sperm Radiated).—Delayed cleavage and devel- opment (Mavor and De Forrest, 1924; Henshaw, et al., Francis, C. T. Henshaw, Cohen, 1932-1941; Chesley, 1934; Heilbrunn and Young, 1935; Evans and Beams, 1939; Little and Evans, 1940; Evans, 1940, 1942, 1947, 1950; Rugh, 1949; Lucké, Ricca, and Parpart , 1951; Barron and Seki, 1952). Cleavage delay occurs mostly in prophase (Henshaw, 1938c, 1940 II; Henshaw and Cohen, 1940, see Henshaw, 1940 IV). Multipolar cleavage (Henshaw, 1938b, 1940 VI, 1941). Enlarged nuclei with nucleoli in prophase with delay in cleavage (E. B. Harvey, 1946a). Effect on gastrulation, form exogastrulae (Waterman, 1934). Recovery (Henshaw, 1932, 1938b, etc.; White, 1938; Evans, 1950). Recovery not affected by visible light, thus differing from ultraviolet (Blum, et a]., 1949a, 1950Cc, 1951). Delay greater with addition of ovarian tissue (Heilbrunn and Young, 1935); with lowered temperature (Henshaw, 1940 V). Delay inhibited by potassium citrate (Wilbur and Recknagel, 1943) ; delay lessened by packing of eggs (Cohen, 1940) ; by oxygen-lack (Anderson, 1939). Delay in cleavage of white halves but not of (non- nucleate) red halves if radiated before fertilization (Henshaw, 1938a; Blum, et al. 1950C, 1951). Enlarged nuclei with nucleoli in early prophase of white halves (E. B. Harvey, 1946a). Parthenogenetic Agent.—In whole eggs, white halves and (non-nucleate) red halves (E. B. Harvey, 1940 unpub., but see reference in Giese, 1947, p. 269, line 33 of first column). Cause Cytolysis.—(Lucké, Ricca, and Parpart, 1951). Permeability.—No effect (Richards, 1915; Lucké, Ricca and Parpart, 1951). Respiration. Of eggs. No effect (Chesley, 1934). Slight effect (Evans, 1940). Of sperm. Inhibition (Barron, Gasvoda, and Flood, 1949; Barron, Flood, and Gasvoda, 1949). Of eggs and sperm, inhibition in large doses, increase in small (Barron and Seki, 1952). Viscosity—No effect on unfertilized eggs (Wilbur and Recknagel, 1943; W. L. Wilson, 1950). Period of increased viscosity after fertilization continues longer (W. L. Wilson, 1950). Breaking with Centrifugal Force.—Break more readily after radiation (E. B. H., 1940 unpub.). Loss of Fertilizin—And agglutinating power (Richards and Woodward; 1915; Evans, Beams, and Smith, 1941; Metz, 1942). Loss of Jelly—(Evans and Beams, 1939; Evans, 1940b; M. E. Smith and Evans, 1940; Evans, Beams, and Smith, 1941; E. B. H., 1940 unpub.). Sperm.—Loss of motility and fertilizing power (Evans and Beams, 1939; Evans, 1942, 1947); effect lessened by proteins, egg albumen, etc., in the sea water (Evans and Slaughter, 1941; Evans, Slaughter, Little, and Failla, 1941). No recovery (Henshaw, 1936, 1938b). Sperm is more sensitive than the egg to x-rays (Mavor and De Forrest, 1924; Henshaw, 1936). Vitelline Membrane.—(Kopac, 1940c, 1941b). Amoebocytes.—Become spherical (E. B. H., 1940 unpub.). OF EXPERIMENTAL WORK 231 Other Species and Reviews Giese, 1947. Review. Hollaender, 1954. Radiation Biology. Review. Langendorff, 1931. Psammechinus miliaris. Lee, 1947. Review. Miwa, Yamashita, and Mori, 1939, 1940, 1941. Pseudocent:otus depressus, Strong ylocentrotus pulcherrimus. Reiss, 1925. Probably Paracentrotus lividus. YOLK GRANULES Size.—0.7 to 1.1 u diameter (E. B. H. per E. N. Harvey, 19324, E. B. Harvey, 1936) ; 0.3 u (Heilbrunn, 1926a); 1.0 to 1.5 with electron microscope (McCulloch, 19524). Shape.—Irregular or polyhedral (E. B. H. per E. N. Harvey, 19324, E. B. Harvey, 1936). Elliptical (McCulloch, 19524). Density.—Lighter than chromatophores; form a layer above pigment layer and below mitochondrial layer in centrifuged eggs (E. N. Harvey, 1932a; E. B. Harvey, 1932, 1936, etc.). Approximately 1.1035; the whole egg is approximately 1.0485 (Heilbrunn, 1926a). Amount in Egg.— 27.2% (E. N. Harvey, 1932a). 38 % (Costello, 1939). Stain.—With vital stains: Bismark brown, brilliant cresyl blue (faint), chrysoidin, methyl violet (later), methylene blue\ (faint), neutral red, Nile blue, rhodamine, toluidin blue (E. B. Harvey, 1941c). Fixed material. Yolk granules darken with osmic acid (Lyon, 1907; E. B. Wilson, 1926); not stained with most protoplasmic stains (Lyon, 1907; E. B. Wilson, 1926); pale blue with Benda-Kuhl (E. B. Wilson, 1926) ; stain with orange G but not iron haematoxylin (E. B. Harvey, 1940c). Table 8. Break Down.—In hypotonic sea water (D. L. Harris, 1943). On rupture of vitelline membrane, and with cytolytic agents, e.g. saponin; no break down in aksence of calcium (Costello, 1932). BIBLTOGRAREY BIBLIOGRAPHY The following list includes all the important papers and books written on Arbacia punctulata and also many on other sea urchins during the last century, from about 1850 until 1954. Every paper and book listed has been looked over by the author except that of W. Busch (1849) which was inaccessible BIBLIOGRAPHY ABELSON, P. H. 1947. Permeability of eggs of Arbacia punctulata to radioactive phos- phorus. Biol. Bull. 93 : 203 (Abstract). . 1948. Studies of the chemical form of P%* after entry into the Arbacia egg. Biol. Bull. 95 : 262 (Abstract). ABRAMS, R. 1951. Synthesis of nucleic acid purines in the sea urchin embryo. Exp. Cell Res. 2 : 235-242. Agassiz, A. 1867 (1864). On the embryology of the Echinoderms. Mem. Amer. Acad. Arts and Sci. 9 : 1-30. . 1869. On the young stages of Echini. Bull. Mus. Comp. Zool. Harvard 1 : 279- 296. . 1872-74. Revision of the Echini. Mem. Mus. Comp. Zool. Harvard 3, no. 7 : 11-762. . 1876. On viviparous Echini from the Kerguelen Islands. Proc. Amer. Acad. Arts and Sci. 11 : 231-236. . 1881. Report on the Echinoidea, dredged by H. M. S. Challenger 1873- 1876. Report on the scientific results of H. M. S. Challenger, Zoology III, Part IX : 1-321. . 1883. a. Selections from embryolog- ical monographs. If. Echinodermata. Mem. Mus. Comp. Zool. Harvard 9, no. 2 : I-45. . 1883. b. Reports on the results of dredging by the U. S. Coast Survey Steamer Blake. XXIV Part I. Report on the Echini. Mem. Mus. Comp. Zool. Har- vard 10, no. I : I-94. . 1904. The Panamic deep sea Echini. Mem. Mus. Comp. Zool. Harvard 31 : 53-55 and Plates 53, 54. and Crark, H. L. 1908. Hawaiian and other Pacific Echini. Mem. Mus. Comp. Kool. Harvard 34 : 43-133. Acassiz, L. 1836. Prodrome d’une mono- graphie des radiares. Mém. Soc. Sci. Nat. Neuchatel 1 : 168-199. . 1838. Monographies d’Echinodermes. Des Salénies. Neuchatel. . 1842-1846. Nomenclator Zoologicus. Soloduri; Jent et Gassman.. . 1857. Essay on classification. Contri- butions to the Natural History of the U.S.A. Vol. 1. Boston. Little, Brown & Co. Aryar, R. G. 1935. Early development and metamorphosis of the tropical Echinoid Salmacis bicolor Agassiz. Proc. Indian Acad. Sci. Sect. B, 1 : 714-728. . 1938. Salmacis. Indian Zoological Mem., ed. by K. N. Bahl, VII. Lucknow Pub. House. ALDROVANDI, U. 1606. De Reliquis Animalibus. Lib. III. De testaceis, sive conchyliis. Cap. XL : 400-416. De Echinis. Bononiae. 235 ALLEE, W. C. 1919. Note on animal distri- bution following a hard winter. Biol. Bull. 36 : 96-104. . 1923. a. Studies in marine ecology. I. The distribution of common littoral in- vertebrates of the Woods Hole region. Biol. Bull. 44 : 167-191. . 1923. b. Studies in marine ecology. III. Some physical factors related to the distribution of littoral invertebrates. Biol. Bull. 44 : 205-253. . 1923. c. Studies in marine ecology: IV. The effect of temperature in limiting the geographical range of invertebrates of the Woods Hole littoral. Ecology 4 : 341- 354- and Evans, G. 1937. a. Some effects of numbers present on the rate of cleavage and early development in Arbacia. Biol. Bull. 72 : 217-232. and Evans, G. 1937. b. Further studies on the effect of numbers on the rate of cleavage in eggs of Arbacia. 7. Cell. and Comp. Physiol. 10 : 15-28. and Evans, G. 1937. c. Certain ef- fects of numbers present on the early de- velopment of the purple sea urchin, Ar- bacia punctulata: a study in experimental ecology. Ecology 18 : 337-345. , Finket, A. J., GARNER, H. R., Evans, G. and Merwin, R. M. 1942. Some effects of homotypic extracts on the rate of cleavage of Arbacia eggs. Biol. Bull. 83 : 245-259. ALLEN, E. J. and NEtson, E. W. 1910. On the artificial culture of marine plankton organisms. Q. 7. M.S.55 : 361-431. Also J. Marine Biol. Assoc. 8 : 421-474 (1910). ALLEN, R. D. 1950. The effect of Janus green B on cleavage and protoplasmic viscosity. Biol. Bull. 99 : 353 (Abstract). . 1951. a. Antimitotic substances sec- creted from eggs. Biol. Bull. 101 : 214 (Abstract). . 1951. b. The role of the nucleolus in spindle formation. Biol. Bull. 101 : 214 (Abstract). Ausup, F. W. 1941. Photodynamic studies on Arbacia eggs. Biol. Bull. 81 : 297 (Ab- stract). AMBERG, S. and WinTeErRnITz, M. C. IgIt. The catalase of sea urchin eggs before and after fertilization with especial reference to the relation of catalase to oxidation in general. 7. Biol. Chem. 10 : 295-302. AmBerson, W. R. 1928. The influence of oxygen tension upon the respiration of unicellular organisms. Biol. Bull. 55 : 79- gl. ANDERSON, R. S. 1939. The X-ray effect on the cleavage time of Arbacia eggs in the 236 absence of oxygen. Biol. Bull. 77 : 325 (Abstract). AnpreEws, E. A. 1897. Hammar’s Ecto- plasmic layer. Amer. Natur. 31 : 1027- 1032. AnpDREws, G. F. 1897. a. Some spinning activities of protoplasm in starfish and sea- urchin eggs. 7. Morph. 12 : 367-389. . 1897. b. The living substance as such: and as organism. 7. Morph. 12, suppl. to Part 2 : 1-176. ANFINSEN, C. B. 1947. The inhibitory action of naphthoquinones on respiratory pro- cess: the inhibition of cleavage and respi- ration in the eggs of Arbacia punctulata. J. Cell. and Comp. Physiol. 29 : 323-332. ANGERER, C. A. 1937. The effect of salts of heavy metals on protoplasm. I. The ac- tion of cupric chloride on the viscosity of sea urchin eggs. 7. Cell. and Comp. Physiol. 10 : 183-197. . 1939. The effect of electric current on the relative viscosity of sea urchin egg protoplasm. Biol. Bull. 77 : 399-406. Apicius, M. G(astus). 80 B.C.-A.D. 40? De re coqguinaria. Trans. as Cookery and Dining in Imperial Rome by J. D. Vehling. 1936. W. M. Hill Pub., Chicago. ARCHIPPUS, written ca. 415 B.C. The Fishes. ARISTOTLE. 384-322 B.C. Historia Anima- lium. The works of Aristotle trans. into English under the editorship of J. A. Smith and W. D. Ross, Vol. IV. Trans. by D’Arcy Thompson. 1910. Oxford, Clarendon Press. . De Partibus Animalium. The works of Aristotle trans. into English under the editorship of J. A. Smith and W. D. Ross, Vol. V. Trans. by Wm. Ogle, 1911. Ox- ford, Clarendon Press. . De Generatione Animalium. The works of Aristotle trans. into English under the editorship of Jj. A. Smith and W. D. Ross, Vol. V. Trans. by A. Platt. 1910. Oxford, Clarendon Press. ASHBEL, R. 1930. Sul quoziente respiratorio della uova fecondate e non fecondate dei ricci di mare (Arbacia pustulosa Gray). Boll. Soc. Biol. Sperim. 5 (2) : 72-74. . 1931. L’azione della concentrazione degli idrogenioni sulla respirazione della uova fecondate e non fecondate dei ricci di mare. Pub. Staz. Zool. Napoli 11 : 194—- 203. ATHENAEvs. After A.D. 228. Deipnosophistae, or Banquet of the Learned. Trans. by C. D. Yonge. London. H. G. Bohn. 1854. 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Fisica, Math. e Sct. Nat., Napc- fertilized eggs of the sea urchin (Paracen- li Ser. 2, vol. 3 : 393-402. SUBIECE TN DEX SUBJECT INDEX The Compilation of Experimental Work in Part IV contains prac- tically everything known about the subjects and is arranged alpha- betically, with subheads in logical sequence, in index form. The main topics are given with page numbers in the Index but the subheads (italicized in text) are indexed only when not included in a main topic. Subheads under “Cleavage” and “Respiration” are arranged alphabetically in the text and “Respiration” has a special index to various factors influencing metabolism (p. 212). Many substances not listed in the Index may be found under these main topics, and also under ‘“‘Parthenogenesis.” References to “Other Species” occur at the end of each topic. These species are not indexed individually. Abatus, see Hemiaster Acetamide, on chromatophores, 159 Acetyl choline, cleavage, 161; phosphorus metabolism, 207 Achinos, 4 Acids, on chromatophores, 159; on cleav- age, 162; on nuclear without cell divi- sion, 193; as parthenogenetic agents, 199; OM Viscosity, 225 Adenosine triphosphate, ATP, overcomes inhibition of cleavage by KCN, 163; phenols, 165; by oxygen-lack, 197, 207; by pressure, 211; malonate, 164 Age and growth, 36-38 Ageing of eggs, see Compilation, 149; on size, 81 Agglutination, 92, 149, 182 Agglutinin, see Fertilizin; on sperm, 92 Albinos, 29-30 Alimentary canal, 31; Fig. 7 Alkali, cleavage, 161, 162; nuclear with- out cell division, 193; parthenogenetic agents, 199; Viscosity, 225 Ambulacral plates, 30, 31; Plate I and Fig. 6 Ammonium salts, chromatophores, 159; cleavage, 162; oil, 195; permeability, 204; rhythms of susceptibility, 219 Amoebocytes, see Compilation, 149-150; Plate XVI, 5 Amoeboid eggs, see Compilation, 150-151 Amulets, 20 Anaerobiosis, see Oxygen-lack Anaesthetics, see Compilation, Tables 13-15 Anal plates, 30; Plate I and Fig. 6 Anapesus carolinus, 21 Anaphase, 96; Plate XVI, 13, 14, 15 151-154; Anastral cleavage, 96; Plate V, 12 Anions, permeability, 204 Anochanus sinensis, brood pouch, 52; clas- sification, 67 Anthocidaris crassispina, papillae in im- mature eggs, 73 Antibiotics, penicillin, penichromin, glio- toxin, aspergillus, actinomycin, aureo- thrycin, clavacin, usnic acid, on cleavage, 162, 163, 165 Anus, Figs. 6, 7; gastrula, 110 Arbaces, 6 Arbacia incisa, see A. Stellata Arbacia lixula (pustulosa), metamorphosis, 18, 19, 117; food, 22; pigment, 28; color change, 29; gonads, 31; rock-borer, 39; phototaxis, 43; righting movements, 46; sex dimorphism, 47; hermaphrodite, 51; ripe, 54; egg, 61, 63; centrifuging, 61, 127; Classification, 63; sperm, 89; swim- ming rate of gastrula, go; echinochrome on sperm, 91; chemotaxis of sperm, 92; cleavage, 99; chromosomes, 102; pres- sure, 104; “Mirsky protein,” 211; many references in Compilation, mostly under Other Species after each topic Arbacia niger, see Tetrapygus niger Arbacia punctulata, origin of genus and species, 6, 7; classification, 63; Plate I; other references are too numerous to be indexed under this heading Arbacia spatuligera, origin, 39; classifica- tion, 64 Arbacia stellata (incisa), pluteus, 18; clas- sification, 64 Arbaciidae, fossils, 20; classification, 62, 63 290 Aristotle’s lantern, 9, 14, 30; Figs. 2, 7; in locomotion, 46 Artificial parthenogenesis, see Partheno- genesis Aster, 95, 96; spiral, 152; not formed in O,-lack, 197 Asterias, maturation of egg, 53; blebs on immature egg, 73 Asteroidea, 14 Asynchrony in cleavage, 104-105 Atropine, cleavage, 162; phosphorus metab- olism, 207 Azide, sodium, on cleavage, 162; on cyto- chrome oxidase, 171 Barbiturates, see Anaesthetics, 162, 186 Bermuda, fossils, 21; food, 24 Bile salts, on cleavage, 162; cytolysis, 171; parthenogenesis, 200 Blastocoel, 109; pH, 186 Blastomeres separated with age, 149 Blastula, 109; Plates III, V Blebs (papillae) on immature egg with sperm, 73; Plate II, 16, 17 Blood, see Perivisceral fluid Body fluid, see Perivisceral fluid Bothriocidaris, 20 Breaking of egg with centrifugal force, see Egg fractions Breeding season, at Woods Hole, 52-56; at Beaufort, N.C., 54 Brood pouches, 52; Fig. 8 Burrowing sea urchins, 38, 39 Buttonfish, 5 153, Caffeine, on stratification and breaking, 133; permeability, 204; on respiration and cleavage, 216 Calcium, see Compilation, shell, 221 Calcium-free sea water; see Compilation under Calcium, 156; Table 16; no papil- lae on oocyte, 73; fertilization prevented, g1, 181; on sperm, g1; on hyaline layer, 155, 183; on viscosity, 156; on cleavage, 161 Capacity, electrical, 175 Carbohydrate and carbohydrate metab- olism, see Compilation, 157-158; see also sugar; in late eggs, 149; in eggs, 166 Carbon dioxide, attracts sperm, 93; cleav- age, 162; respiration, 216, 217; rhythms, 217, 219; sperm, 218; viscosity, 225 Carbon monoxide, cleavage, 163; on cyto- chrome oxidase, 171, 216; respiration, 216 Carcinogens (dibenzanthrene), cleavage, 163; cytolysis, 171; permeability, 204 155-1573 in SUBJECT INDEX Cations, permeability, 204; toxicity; 209; see also individual salts Centrechinus, and Diadema, 17 footnote; albino, 30; classification, 63; see Diadema Centrifuged eggs, 121-146; Fig. 12; Plate VII; stain, 125, 126, Table 8; other eggs, 127; development, 138-139, Plate VIII; centrifuged after fertilization, 133-134, 144; Plate XII, 14-17 Centrifuged egg fractions, development, 138-145; conclusions, 145-146 Centrifuging, methods, 121-122 Centriole, 101 Centrostephanus longispinus, color change, 29; phototaxis, 43; classification, 63 Cesium, and potassium, 209 Chalk eggs, 4, 20 Challenger expedition, 52 Chemotaxis of sperm, 92 Chlorine, taken up by jelly, 188; parthe- nogenesis, 200 Chromatophores, see Compilation, 158- 160; see also Echinochrome (pigment granules); in skin, 29; in plutei, 111, 158 Chromosomes, sperm, 88, 103; egg, 96; cleavage, 102; number and size, 102; sex chromosomes, 88, 102; parthenogenetic egg, 102; in other Echinoidea, 102 Cidaris, sex dimorphism, 48; brood pouch, 52; Cidaris abyssicola, 60; classification, 63; egg size, 63; chromosomes, 102; see Compilation for Other Species Cilia quieted, anaesthetics, 152; KCN, 169; MgSO,, 191; methocel, 191 Cinchonidine, stain, 125, 158 Cinchonine, stain, 125, 158 Citrate (sodium), for chromatophores, 159 Classification, history of, 14, 15; of Echi- noidea, 62-67 Clavacin (antibiotic), cleavage, 163 Clear layer, see Compilation, 160; imma- ture egg, 74; mature egg, 125; stain, 126; Table 8; in white half, 126, 129; in red half, 126; Fig. 12; Plate VII; other eggs, 127 Clear quarter-egg, 129, 130, 142-143; Fig. 12; Plates VII, XII; Table 9 Cleavage, acceleration, see Compilation, 161-162; cleavage, delay or arrest, see Compilation, 162-166; history, 17; de- scription, 96-100; time schedule, 97; Tables 2-5; Plates IJI-V; without mem- branes, 100; Plate XVI, 7, 9; in centri- fuged eggs, 138-139; Plate VIII; with oxygen-lack, 161, 164, 197; with pres- sure, 165, 210-211; see Respiration Cleavage furrow, 96; pressure, 155, 158, 210 Cleavage plane and sperm entry, 100 SUBJECT INDEX Cliona celata, 35 Clypeaster rosaceus, classification, 66; egg size, 66; chromosomes, 102 Coelenterata, 14 Coelomic fluid, see Perivisceral fluid Coelopleurus floridanus, 60; classification, 64; see Mortensen II, 612 Co-enzymes, 178 Coins, 26 Colchicine, cleavage figure fading out, 103; rhythms, 163, 219; viscosity, 225 Color and color change of A. punctulata, 28, 29; of A. lixula, Centrostephanus and Diadema, 28, 29; ultraviolet, 28 Composition of eggs, see Compilation, 166- 167 Cooking sea urchins, 11, 13, 22, 23, 24 Copper, in chromatophores, 160; in eggs, 166; copper chloride, cleavage, 161, 163; exogastrulation, 190; viscosity, 225 Cortical granules, 85; Figs. 9, 10; 167-168 Cortical layer, see Compilation, 167-168; 83; Figs. 9, 10 Corticotropin, ACTH, cleavage, 163 Cortisone, cleavage, 163 Cretaceous, 20 Crinoidea, 14 Crushed eggs, see Homogenates; Table 7 Cyanides, see Compilation, 169-170; cleav- age, 161, 163 Cystoid, 20 Cytochrome and cytochrome oxidase, see Compilation, 171; in sperm, 89 Cytolysis, see Compilation, 171-172 “Decorate” with pebbles, etc., 43, 44; see Storm forecast Dendraster excentricus, hermaphroditism, 51; Classification, 66; egg size, 66; cleav- age, gg; lithium, 190; see Compilation, Other Species Density (specific gravity), see Compilation, 172-173; 79 Derbés, 17 Description of sea urchin, historical, 8-16; modern, 27-34; Figs. 6, 7; Plate I Desoxycorticosterone, cleavage, 163 Development, 95-117; Plates III, IV, V, VI; Tables 2-5 Devonian, 20 Diadema (Centrechinus), name, 16, 17; see footnote p. 17; color change, 29; albino, 30; reaction to light, 43; lunar periodicity, 55, 56; classification, 63; size of egg, 63 Dibenzanthrene, see Carcinogens 2g1 Dimethylparaphenylenediamine, cleavage, 163; respiration, 215, Distribution, 39, 40 Drosophila, chromosomes, 103 Dyes, see Vital dyes Easel, 17-18 Eating, see Feeding habits Echinarachnius parma, use of shell, 25; locomotion, 45; righting movement, 46; description, 60; hyaline layer, 60; cen- trifuging, 60, 127; size of egg, 60, 66; classification, 66; papillae in oocyte, 73; centriole, 101; chromosomes, 102; many references in Compilation, mostly under Other Species after each topic Echinidae, classification, 62, 64 Echinocardium (Amphidetus) cordatum, metamorphosis, 18, 36, 117; sand bur- rowing, 39; hermaphrodite, 51; classifica- tion, 67; size and shape of egg, 67, 78; sperm, 89; many references in Compila- tion, mostly under Other Species after each topic Echinocardium mediterraneum, sex di- morphism, 48; classification, 67; egg size, 67 Echinochrome, see Compilation, 173-175; see also Chromatophores, Pigment granules; motility of sperm, g1; in pluteus, 111; in late eggs, 149 Echinocidaris, 6, 18, 21, 63 Echinocyamus pusillus, metamorphosis, 18, 117; sex dimorphism, 48; classifica- tion, 66; size of egg, 66; see Compilation, Other Species Echinodermata, échinodermes, 14, 16; ori- gin of term, 14 Echinoidea, name, 3; classification, 14-16, 62-67; fossil, 20; Challenger expedition, viviparous, 52; Woods Hole, 59-60; num- ber of species, 62; chromosome numbers, 102 Echinometra, old term, 8; gonads, 31; “reef urchin,” rock-boring, 38, 39; clas- sification, 66; egg size, 66; cleavage, 99; see Compilation, Other Species Echinus, name, 3, 6; historical, 9-16; geo- graphical names, 11; architecture, 26 Echinus acutus, mature, 36; classification, 64; egg size, 64; hybrids in nature, 184 Echinus esculentus, number of plates, pores, etc., 16, 31; embryology, 17; use of shell, 25; mature, 36; growth, 37; duration of life, 37; disappearance, 42; migrations, 43; locomotion, 45, 46; right- ing reaction, 46; sex dimorphism, 48; hermaphrodite, 51; classification, 64; size 292 of egg, 64; sperm, 87, 89, go; seminal fluid, 93; spicules, 111; metamorphosis, 117; lithium, 190; many references in Compilation, mostly under Other Spe- cies after each topic Echinus niger, see Tetrapygus niger Ectoplasmic layer, see Hyaline layer Egg albumin, on perivitelline space, 202 Egg extracts, cleavage, 161, 163; enzymes, 177; parthenogenesis, 200; permeability, 205 Egg-fish, egg-urchin, 4, 5 Egg fractions, 121, 129-130; Table 9; Fig. 12, Plate VII; size and centrifugal force, 131; Table 10; Plate XIII; size when centrifuged in hypo-hypertonic sea water, 131; Table 11; Plate XIV; size when centrifuged in single salts, 132-133; Plate XV; effect of temperature, pressure, radiation, 133; properties, 135-137; Os- motic behavior, 136, Table 12; develop- ment, 139-144; size necessary for devel- opment, 144; enzymes, 177; lipids, 189 Egg, structure, immature, 72; mature, 81 Elaeocytes, see Amoebocytes, 149, 150 Electrical method of sex determination, 49 Electrical properties and effects, see Com- pilation, 175-176 Electrolytes, in eggs, 166; in sea water, 166; permeability, 204 Embryology, history, 17; development, 95- 11 nae cone, see Fertilization cone Enzymes, see Compilation, 177-178 Ericius, 3, 4 Erinacius europaeus, 3 Erizo de mar, 4 Eserine, cleavage, 163; phosphorus metab- olism, 207 Esterases, 177, 178 Ether, on cleavage figure, 104; see Anaes- thetics in Compilation Ethylene glycol, on chromatophores, 159 Etymology, 3-7 Eucidaris, rock-boring, 39; ‘‘slate pencil” urchin, 63; classification, 63; egg size, 63 Feeding habits, 35; of plutei, 113 Fertilization, history, 17; description, 95- g6; Plates III and V; oxygen lack, 107; after fertilization and parthenogenesis, 107-108, 180; inhibited by red amoe- bocytes, 150, 175; pressure, 211 Fertilization cone (entrance cone), 95; Plate III, 3 Fertilization membrane, see Compilation, 178-181; 83, Fig. 10 Fertilizin, see Compilation, 182; 92 SUBJECT INDEX Feulgen reaction, 141, 194 Fireworm, lunar periodicity, 57 Fisher’s Island sea urchins, 40, 52 Flattening of egg, 78 Flavin enzymes, 178 Folklore, 20 Food for man, in Greece and Rome, 8, 13, 22; Pompeii, 22; Italy, 22; France, 23; England, 23; N. America and S. America, 23; West Indies, 24; Bermuda, 24; Japan, 24; food for animals, 24, 25; Fig. 5 Forced shedding, 57-58 Formalin, on viscosity, 225 Fossils, 4, 11, 20-21; see Jewstones, chalk eggs, folklore Fractions, see Egg fractions Fragment (size), capable of development, 144 Freezing point depression, 196 Frutta di mare, 22 Fucus, 35 Fundulus, 35 Gastrula, gastrulation, 109-110; Plate III, 23, 24; alcohols, 152 Genital plates, 30; Plate I and Fig. 6 Genital pores, 30; Plate I and Fig. 6; sex dimorphism, 48 Geographical names derived from Echinus, in Greece, 11 Geotropism, 44; blastulae and gastrulae, 45 Germinal vesicle, 71, 72; Table 1; Plate II; centrifuged, 74; permeability, 81 Gestell, 17, 18 Glycerine, cleavage, 163; parthenogenesis, 200 Glycocol, cleavage, 163 Goblin, 3 Golgi bodies, 85 Gonads, 31; Fig. 7; gonad wall, 31; re- moval, 58 Goniocidaris, sex dimorphism, 48; brood pouch, 52, 53; Fig. 8; classification, 63 Gonopores, see Genital pores Granules in egg, 85; Figs. 9, 10; in centrifuged egg, 125, Fig. 12; Plate VII; stained, 125, 126, Table 8; see under oil, mitochondria, yolk, pigment Greek, Greeks, 3, 8-13; food, 13, 22 Growth and age, 36-38 Habitat, 38 Hatching, 97, 109 Hatching, enzyme, 109, 177 Heat production, see Compilation, 182 Heavy water, cleavage, 164; permeability, 203 SUBJECT INDEX Hedgehog, 3, 4, 5, 13; Fig. 1 Heliocidaris crassispina, development, 18; sex determination, 49; classification, 65- 66; egg size, 66 Helium, pressure on cleavage, 211 Hemiaster (Abatus), sex dimorphism, 48; brood pouch, 52; classification, 67; egg size, 67 Heparin, cleavage, 164, 165; fertilization membrane, 181; viscosity, 225 Heérisson, 3, 4 Hermaphrodites, 50-51; list, 51 Hipponoé, see Tripneustes Historical, 8-19; embryology, 17 Holothuroidea, 14 Homogenates, methods of preparation, 122-124; centrifuged, composition of layers, 122; Table 7; chromatophores, 159; cytochrome oxidase, 171 Hyaline layer, see Compilation, 182-183; 83; Fig. 10; 96, 97; centrifuged off, 145; Plate XVI, 6; in Ca-free sea water, 155, 183; Plate XVI, 9; with KCl, 209 Hyaloplasmic layer, see Hyaline layer Hybrids, see Compilation, 184-185; arm skeleton, 110 Hydrocarbons, carcinogenic, cleavage, 161 Hydrogen ion concentration, see Compila- tion, 185-186 Hydrostatic pressure, see Pressure, hydro- static Hypo-hypertonic sea water, germinal vesi- cle, 75, 81; nucleus, 81; centrifuging, 131, Table 11, Plate XIV; amoebocytes, 150; amoeboid eggs, 151; chromato- phores, 159, twins, 223 Hypsiechinus coronatus, brood protecting, 52; Classification, 64 Immature eggs, electrical method, 49; oc- currence, 52; see oocyte Impedance, electrical, 175, 176 Infrared light, see Compilation, 187 Injury substances, parthenogenesis, 200; permeability, 205; viscosity, 226 Ink, made from sand dollar shells, 25 Interambulacral plates, see Ambulacral plates Interfacial tension, see Oil coalescence and tension at the surface Intravitam dyes, see Vital dyes Iodoacetate, Iodoacetic acid, on plutei, 112; carbohydrates, 157; cleavage, 164; respiration, 216 Iodosobenzoic acid, chromatophores, 159; fertilization membrane, 181 Iron in eggs, 166 =93 Jelly layer, see Compilation, 187-188; in immature egg, 72; in mature egg, 82, 83; Plate XVI, 3; Figs. 9, 10; destroyed, 82; stain, 82; Table 8 Jewelry, 26 Jewstones, Judeo de mer, lapides judaict, 4, 20, 24 Koehler’s window technique, 55 Laminaria, 35 Lamps, 25 Lantern, see Aristotle’s lantern Late eggs, 54 Latin, 3 Layers (surface) of egg, 83; Figs. 9, 10; list of diagrams, 85; in centrifuge tubes, 129, Plate VII, 10; centrifuged eggs, see stratification Lentulus, supper of, 22 Leucocytes, see Compilation, 188-189 Lichmophora, 113 Life, duration in Echinus esculentus, 37 Lipids, see Compilation, 189; see also Oil Lithium, see Compilation, 190 Lobsters, 41 Lipophanerosis, 195 Locomotion, 45, 46; Réaumur, 46; by lan- tern, 46 Longevity of egg, see Compilation under Ageing, 149; see also chloral hydrate, 153; Ca, 156; KCN, 169; oxygen-lack, 197; Of sperm, amino acids, etc., 91-92; KCN, 170; of fertilization membrane, 180 Low oxygen tension, see Oxygen-lack Lunar periodicity, 10, 11, 13, 56-57 Lytechinus anamesus, sex dimorphism, 48; classification, 65; egg size, 65 Lytechinus pictus, sex dimorphism, 48; classification, 65; egg size, 65; sperm, 89 Lytechinus (Toxopneustes) variegatus, white, 9; decorates itself, 11, 44; fossil, 21; reaction to light, 43; locomotion, 45; renewed ovaries, 55; Classification, 2 sub- species, 65; egg size, 65; oocyte, 72, 73; number of sperm, 94; development, 95; first cleavage, 99, 101; sperm tail, 100; mid-bodies, 102; chromosomes, 102; blas- tocoel, 109; jelly, 188; lithium, 190; many references in Compilation, mostly under Other Species after each topic Madreporic plate, 30, Plate I and Fig. 6 Magnesium, see Compilation, 191 Malonic acid, cleavage, 164 Malononitrile, cleavage, 164; phosphorus metabolism, 207 294 Maturation, 71-76; Plate II; see oocyte and polar bodies; cytoplasmic maturation, 75 Maturity and size, 36 Mechanical shocks, or stimulation, chro- matophores, 159; cleavage, 161; cytolysis, 171; parthenogenesis, 198; susceptibility, 220; viscosity, 225 Medicine, 11, 13, 20, 24 Membranes and layers of egg, 83; Figs. 9, 10; list of diagrams, 85 Mercuric chloride, chromatophores, 159; cleavage, 164 Merogones, see Hybrids, 184; homospermic, 184, Plate X, XVI, 12, 15; heterospermic, 184; parthenogenetic, 140-141, Plates XI and XVI, 13 Mespilia globulus, development, 18, 117; gonads, 31; classification, 64; size of egg, 64; oogenesis, 71; chromosomes, 102; see Compilation, Other Species Metabolism, see Respiration Metal salts, cleavage, 164 Metamorphosis, 18, 19, 115-117; Plate VI; Other Species, 18, 117 Metaphase, 96; Plate XVI, 11, 12, 13 Methylated xanthine, on cortical layer, 168 Micromanipulation, fading of cleavage figure, 104; cortical layer liquefied, 167; hyaline layer, 183 Micromeres, 76, 105-107, 138, 139, 158; Plate VIII, 5, 6, 15, 16; pressure, 211 Micropyle, 76, 82-83, 138 Microsomes, 85, 160 Mid-bodies, 101; in Rhomalium, 101 Migration of sea urchins, 43 Miquel’s solution, 35, 113 Mitochondria, see Compilation, 192; im- mature egg, 71, 74; mature egg, size and amount, 85; mature egg centrifuged, 125; stain, 126, Table 8; in sperm, 89 Mitochondrial (granular) quarter-egg, 129; Table g; Fig. 12, Plate VII; develop- ment, 143 Mitotic cycle, see Rhythms of susceptibility Mitotic figure, 96, 97; of whole and half eggs, Plate XVI, 11-15; visibility, 103; structure, 103; fading out, 103-104; iso- lation, 104; anaesthetics, 151; hydro- static and mechanical pressure, 151; oxygen-lack, 151 Monaster, 96, Plate III, 5, Plate V, 3; with anaesthetics, 151; chromatophores, 158 Morphine, cleavage, 164 Morphology, 14, 16, 30-34; literature, 31, 34; Figs. 4, 6, 7, Plate I Mosaics, 25 SUBJECT INDEX Multi-astral and multi-nucleate eggs, 140, Plate X; 141, Plate XI; 143, 144 Mustard gas, see Nitrogen mustards Names for sea urchins, in English, Latin, Greek, Italian, French, German, Spanish, 377 Naphthoquinones, cleavage, 164; respira- tion, 215, 216 Narcotics, see Anaesthetics Necrosin, cleavage, 164 Nereis, periodicity, 57 Nickel chloride, exogastrulation, 190 Nitrogen, see Compilation, 193; in late eggs, 149; in eggs, 166; pressure, 211 Nitrogen mustards, micromeres, 106; cleav- age, 164; phosphorus metabolism, 207; respiration of eggs, 216; of sperm, 218; rhythms, 220; viscosity, 225 Nitrophenols and nitrocresols, see Phenols Nitrous oxide, cleavage, 164; nuclear divi- sion without cell division, 193; pressure, 211 Nitzschia closterium, 35, 113 Nomenclature, 3-7 Nuclear division without cell division, see Compilation, 103 Nucleases, 177 Nucleic acid, see Nucleoproteins Nucleolini, 72, 73 Nucleolus in immature egg, 72, 73; ultra- violet, 72; RNA, 73; gravity and cen- trifugal force, 74; density, 173; lipids, 189 Nucleoproteins, see Compilation, 194-195; see also Proteins and Phosphorus metab- olism; RNA in nucleolus, 73; DNA in nucleus, 81; DNA and RNA in sperm, 88, 89, 103; RNA in cortex of egg, 168 Nucleus, size, 79, 81; aberrant size, 80; structure, 81; dyes and stains, 81; ultra- violet, 81; size in hypo- and hypertonic sea water, 81, Table 11; Plate XIV; en- largement and breaking of membrane, 96; Plates III 7, V 4, 5; density, 173; viscosity, 224 Ocular plates, 30; Fig. 6 Oil, see Compilation, 195-196; see also Lipids; in immature egg, 71, 74; in ma- ture egg, size and amount, 85; in cen- trifuged egg, 125; Fig. 12; Plate VII; stain, 126, Table 8 Oil coalescence, 149, 196 Oil companies, 20, 39 Oil-protoplasm, see Oil coalescence Oil-water, see Oil coalescence Oocyte, growth and differentiation, 71; SUBJECT INDEX Table 1 and Plate II; structure, 72; stain and ultraviolet, 72; jelly, 73; reaction to sperm, 73; centrifuging and gravity, 74, 76; respiration, 74; permeability to water, 75; polar body formation, 75, 76, Plate II Oogenesis, Mespilia, 71 Ophiura albida, 17 Oranges de mer, pommes de mer, 5 Ordovician, 20 Osmotic behavior, Tables 11, 12; Plate XIV; see Compilation under Permea- bility Osmotic pressure, see Compilation, 196 Oursin, 4 Ovary, see Gonad Oxidizing enzymes, 178 Oxygen consumption, see Respiration Oxygen-lack, see Compilation, 197-198; sperm, 91; cleavage figure, 104; fertiliza- tion, 107; chromatophores, 160 Papillae on immature egg, see Blebs; geni- tal, 48; sex dimorphism, 48 Paracentrotus (Strongylocentrotus) lividus, development, 18, 95, 117; as food, 22, 23; use of shell, 25; pigment, 28; gonads, 31; burrowing, 38; phototaxis, 43; dec- orates itself, 44; locomotion, 45; righting movements, 46; sex dimorphism, 47, 48; hermaphrodite, 51; summer and winter eggs, 54; ripe eggs, 55; lunar periodicity, 56; classification, 65; egg size, 65; respira- tion of oocyte, 74; cytoplasmic matura- tion, 75; polar bodies, 76; first cleavage, 99; cleavage rate, 100; chromosomes, 102; refertilization, 107; many references in Compilation, mostly under Other Species, after each topic Paramoecium, swimming rate, go Paraphenylinediamine, see Dimethylpara- phenylinediamine Parthenogenesis, see Compilation, 198-201; fertilization after, 108, 170; cleavage delay, 165 Parthenogenetic merogones, i40-141; Plates XI, XVI, 13; 201 Pebbles on shell, see Storm forecast, and Decorate Pedicg}lariae, 30; Fig. 6 Penicillin, cleavage, 162, 165 Periodate (sodium), 202 Perivisceral fluid, see Compilation, 201-202 Perivitelline space, see Compilation, 202- 203; 83, 95 Permeability, see Compilation, 203-206; see also Osmotic behavior PH, see Hydrogen ion concentration =00 Phenols, on sperm, g1; plutei, 112; cleav- age, 165; phenols and pH, 186; on phos- phorus metabolism, 207; 215, 220 Phospholipids, 206, 207 Phosphorus metabolism, see Compilation, 206-207; see also Nucleoproteins; Table 17; in late eggs, 149; in eggs, 166; P® cleavage, 165 Phosphorylation, see Carbohydrate metab- olism, 157; enzymes, 178; phosphorus metabolism, 207, 217 Photodynamic action, see Visible light Phototaxis, 43 Pigment granules, see Chromatophores, Echinochrome, test and spines, 28; im- mature egg, 71, 74; mature egg, 85, 86; cleavage, 96; mature centrifuged egg, 125; Fig. 12, Plate VII; stain, 126, Table 8; in pluteus, 110, 111; effect of potas- sium and calcium, 209 Pigment quarter-egg, 129, Table 9, 144, Fig. 12, Plate VII Pigment spots, in pluteus, 110, 111 Pilocarpine, cleavage, 162, 165; phorus metabolism, 207 Plasma membrane, see Compilation, 208; 83, Figs. 9, 10 Pliocene, post-Pliocene, 21 Pluteus, origin of term, 17, 18; description, 110-112; Plate IV; skeleton, 110-111; pig- ment spots, 111; ultraviolet photographs, 111; infrared, 112; abnormal, 112; meth- ylene blue, 112; KCN, 112; develop- ment, not fed, 113, Plate XVI 10; when fed, 116; length of arm in growth, 116, Table 6 Pluteus paradoxus, 17 Plymouth, 113 Podophyllin, podophyllotoxin, quercitin, fading of cleavage figure, 104; cleavage, 165 Polar bodies, formation, 75-76; Plate II; centrifuging, 76; lost or retained, 52, 76; stratification, 128 Polarity of egg, 76, 128, 138, 139 Polyspermy, see Compilation, 208-209; in old eggs, 149; in immature egg, 75 Porcupine, porcupine stones, 3, 5 Potassium, see Compilation, 209-210 Pressure, hydrostatic, internal, mechani- cal, see Compilation, 210-211; on cleay- age figure, 104, 151 Prism larva, 110; Plate IV, 3 Prophase, 96, Plate V, 5 Proteins, see Compilation, 211-212; see also Nucleoproteins, 194-195 Psammechinus microtuberculatus, food, 22; covers with pebbles, 44; geotropism, phos- 296 45; sex dimorphism, 48; hermaphrodite, 51; Classification, 64; size of egg, 64; first cleavage, 99; chromosomes, 102; refertilization, 107; development, 117; pressure, 211; many references in Com- pilation, mostly under Other Species Psammechinus miliaris, cover with peb- bles, 11, 44; albino, 30; mature, 36; growth and size, 37; rock boring, 39; disappearance, 42; phototaxis, 43; geo- tropism, 45; sex dimorphism, 48; clas- sification, 64; two types, S and Z, 64; size of egg, 64; RNA in immature egg, 73; papillae, 73; respiration of imma- ture eggs, 74; cytoplasmic maturation, 75; swimming rate of sperm, go; first cleavage, 99; development, 117; many references in Compilation, mostly under Other Species after each topic Pseudocentrotus depressus, see Compila- tion, Other Species Pseudonucleus, 141 Pyocyanine, see Vital dyes; plutei, 112; cleavage, 165, 197; respiration, 170, 215; lithium, 190 Quadruplets, see Compilation, 223 Quantities of Arbacia used, 40, 41 Quantity of eggs, 77-78, 94; of sperm, 94 Quercitin, cleavage, 165 Quinine, cleavage, 104, 165; stains chroma- tophores, 125, 158; parthenogenesis, 200; polyspermy, 209 Radiata, 14 Radium, see Compilation, 212; cleavage, 162, 165 Red _ half-egg, 129, 130, 140-141; Fig. 12; Plates VII, X, XI; Table 9; properties, 135-137; see Parthenogenetic merogone Reef urchin, 38, see Echinometra lucunter Regeneration, spines, etc., 38; pluteus, 115 Removal of gonads, 58 Resistance, electrical, 175, 176 Respiration, oxygen consumption, metab- olism, see Compilation, 212-219 Rhomalium, mid-bodies, 101 Rhythms of susceptibility during mitotic cycle, see Compilation, 219-220 Riccio di mare, riccio marino, 4, 22 Righting reaction, 46 Rings of growth, 38 Ripe eggs, Arbacia, 52; Paracentrotus lividus, 55; Lytechinus, 55; S. purpura- tus, 55 Rock-borers, 38-39 Roentgen rays, see X-rays Roman, Romans, 8-13; food, 22 SUBJECT INDEX Rondelet, historical, 14; Fig. 4; food, 23 Rotation of sperm head, 95 Rotenone, cleavage, 165 Rubidium, and potassium, 209 Salmacis bicolor, gonads, 31; classification, 64; egg size, 64; metamorphosis, 117 Salts, see Na, K etc.; parthenogenetic agents, 199; viscosity, 226 Sand-burrower, Echinocardium cordatum, Sardanapalus, 6 Scarcity of Arbacia, 40-42; of other sea urchins, 42, 43 Sea chestnuts, see Kastanien, chdtaignes de mer, 5 Sea eggs, sea thistles, 4, 5 Season, cleavage, 165 Seeigel, 4, see Apfel, 5 Seminal fluid, 93 Sex determination, morphological, 47; electrical method, 49 Sex dimorphism, 47-48 Sex ratio, 47 Shape of Arbacia, 27 Shape of eggs, 78; Echinocardium, 178; flattening, 78; amoeboid, 78 Shedding of eggs and sperm, 52-58; forced shedding, 57-58 Shell, see Compilation, 221; see also test; uses, 25-26; skeleton of pluteus, 110, 157; chemical composition, 221 Silurian, 20 Size of Arbacia, 27, 28; increase, 31; when mature, 36 Size of eggs, given in classification, 63-67; size of unfertilized egg, 78, 79; varia- tion in size, 79, 80; aberrant size, 80; effect of age, temperature, etc., 81; fer- tilized egg, 79; size and cleavage rate, 99; in hypo-hypertonic sea water, 132; Table 11, Plate XIV Skeleton, Ca, 157; arm skeleton in hybrids, 110 Sodium, see Compilation, 221-222 Sodium periodate, 57 Specific gravity, see Density Sperm, 87-94; morphology, 87-89; Fig. 11; stains, 88; swimming rate, go; motility, g0, 91; echinochrome, 91; oxygen-lack on motility, 91; Ca-free sea water on motility and fertilization, 91; anaes- thetics, 91; longevity, 91; injury, 92; ag- glutination, 92; chemotaxis, 92; poly- spermy, 93; see Compilation; techniques, 93-94; number of sperm, 94; entrance and cleavage plane, 100; number for fertilization, 101; KCl on, 209 SUBJECT INDEX Sperm aster, 96 Sperm extracts, agglutination, 92; cleav- age, 165; enzymes, 177; parthenogenesis, 200 Sperm head, size and stain, 87; chromo- somes, DNA, 88, 103; rotation, 95 Sperm middle piece, 87, 88; stain, 88; isolation, 89 Sperm tail, 87, 89 Sphaerechinus granularis, as food, 22; ma- ture, 36; phototaxis, 43; decorates itself, 44; sex dimorphism, 48; hermaphrodites, 51; lunar periodicity, 56; classification, 66; egg size, 66; first cleavage, 99; many references in Compilation, mostly under Other Species after each topic Spines, use, 9, 46; number, 16; fossil, 20; size and shape, 27, 28, 32; pigment, 28; tubercles, 30 Staffelei, 17, 18 Stored eggs (in aquarium), 54 Storm forecast by pebbles on sea urchin, 11, 12, 13; Fig. § Stratification, in immature egg, 74, 76; Plate II, 13-15; and polarity, 76, 128, 138; mature egg, 125, 126; Fig. 12; Plate VII; staining, 125-126; Table 8; other eggs, 127; with different forces, 131; Plate XIII; in hypo-hypertonic sea water, 131; Plate XIV; in different salts, 132; Plate XV; effect of temperature, pressure, radiation, 133 Streak stage, 96, Plate III, 6, 7; Plate V, 4 Strongylocentrotus drébachiensis, as food, 23, 25; albinos, 29; rock boring, 39; hermaphrodite, 51; general, 59; egg, 59, 65; centrifuged egg, 59, 60, 127; clas- sification, 65; blastocoel, 109; many ref- erences in Compilation, mostly under Other Species after each topic Strongylocentrotus franciscanus, as food, 23; classification and egg size, 65; first cleavage, 99; see Compilation, Other Species Strongylocentrotus intermedius, classifica- tion, 65; chromosomes, 102 Strongylocentrotus (Hemicentrotus) pul- cherrimus, development, 18, 117; plates and size, 31; decorates itself, 44; sex dimorphism, 48; classification and egg size, 65; refertilization, 107; fertilization after parthenogenesis, 108; see Part IV, Other Species Strongylocentrotus purpuratus, as food, 23; rock-boring, steel-boring, 39; classifica- tion and egg size, 65; forced shedding, 55; echinochrome on sperm, 91; KCN 297 on sperm, 91; number of sperm, 94; first cleavage, 99; lecithin, 189; lithium, 190; many references in Compilation, mostly under Other Species after each topic Strontium, on chromatophores, 159 Structure of unfertilized egg, 81; Plate III, » and Plate V; 1 Sugar (sucrose), see Carbohydrate; amount for centrifuging, 121, 196; amoeboid eggs, 150; chromatophores, 159; cleavage, 165; parthenogenesis, 200 Sulphanilamide, cleavage, 165 Sulphide, sodium, cleavage, 165; on cyto- chrome oxidase, 171 Sulphydryl group (PCMB, 91), motility, 91; polyspermy, 209 Sulphur, S®*, cleavage, 166 Supersonic sound waves, see Ultrasonic waves Superstition, see Storm forecast Surface forces, see Tension at the surface Surface layers of egg, see Layers Surface precipitation reaction, anaesthet- ics, 151; calcium, 155; chromatophores, 159; Mg, 191; KCl, 209 Surface tension, see Tension at the surface Susceptibility, see Rhythms of Swimming rate, sperm of Arbacia and other species, human sperm, Paramoe- cium, gastrula, man, go sperm Teeth, 9, 30, 32, 33 Telophase, 96 Temnopleurus plates and size, 31; sex ratio, 47; sex dimorphism, 47; classifica- tion, 64; egg size, 64 Temperature, cleavage figure, 104; stratifi- cation and breaking, 133; amoebocytes, 150; carbamate narcosis, 153; chroma- tophores, 159; cleavage, 161, 163; cya- nides, 169; cytolysis, 171; nuclear and cell division, 193; parthenogenesis, 198; phosphorus metabolism, 207; pressure, 211; respiration and cleavage, 217; rhythms, 219; viscosity, 225; Tables 3, 4, 19 Tension at the surface, Surface forces, Surface tension, see Compilation, 222- 223 Tertiary, 20 Test, size and shape, 27-28, 36; Plate I; Figs. 6, 7; increase in size, 30-31, 37-38; pigment, 28 Testis, see Gonad Tetrapygus niger (Echinus niger, Arbacia niger, 5; Classification, 64 Theobromine and Theophylline, cleavage, 166 298 Thiurea, cleavage, 166; parthenogenesis, 200 Thorium, and potassium, 209 Thunder stones, 20 Time schedule of development, Tables 2, 3, 4, Plates III-V Torone, 8, 11 Toxopneustes variegatus, see Lytechinus variegatus Transferases, 178 Triplets, see Compilation, 223 Tripneustes (Hipponoé) esculentus, white urchin, 9; as food, chardron blanc, “sea eggs,” 4, 24; decorates itself, 44; classifica- tion, 65; egg size, 65; first cleavage, 99; chromosomes, 102; many references in Compilation, mostly under Other Species after each topic Tube feet, 30, 31; Fig. 6; sex dimorphism, 48 Tubercles, 30 Twins, triplets, quadruplets, see Compila- tion, 223 97-98, Ultrasonic waves, see Compilation, 223 Ultraviolet light, see Compilation, 223- 224; on animal, 28; on egg, sperm and development, 223-224; viscosity, 225 Uranium, and potassium, 209 Uranyl nitrate, cleavage, 166; phosphorus metabolism, 207 Urchin, term, 3, 4 Urea, amoeboid eggs, 150, 151, 197; Cleav- age, 152, 166; chromatophores, 159; pre- vents fertilization membrane, 179, 181; prevents hyaline layer, 183; partheno- genesis, 200 Urethane, on sperm, 91; on cleavage SUBJECT INDEX figure, 104; see Anaesthetics in Com- pilation Uses of sea urchins, 22-26 Usnic acid (antibiotic), cleavage, 166; DNA, 195; on phosphorus metabolism, 207 Variation of skeletal parts, 16 Viscosity, see Compilation, 224-226 Visible light and Photodynamic action, see Compilation, 226-227 Vital dyes, see Compilation, 227-228; on stratified eggs, 125-126; Table 8 Vitamin C (ascorbic acid), cleavage, 166 Vitelline membrane, see Compilation, 228- 230 Washing eggs (repeatedly), effect on cleav- age, 166; on fertilization membrane, 181 Weight of egg, 79 White half-egg, 129, 130, 139-140; Fig. 12; Plates VII, IX; Table 9; properties, 135- 137 White sea urchins, 9, 11, 13 Whore’s eggs, 5 X-rays, see Compilation, 230-231 Yolk granules, see Compilation, 231; im- mature egg, 74; mature egg, 85; cen- trifuged egg, 125; stain, 126; Fig. 12; Plate VII; Table 8 Yolk quarter-egg, 129, 130, 143; Table 9; Fig. 12; Plate vil Zarts, 5 Zeta potential, 175, 176 Zincin, 5 crits a & . aap 7 a Pi : b> vans wat - - : ° ra Ta eceh i ‘4 1; a. : a oe Te ae % » ¥ r i . : . ~ ’ ~— y 1] — vi a ; a , i = ress tik abs a cibater athe ote bok) Bete te ee