Handle with EXTREME CARE This volume is damaged or brittle and CANNOT be repaired! * photocopy only if necessary * return to staff * do not put in bookdrop ._ Gerstein Science Information Centre j Digitized by the Internet Archive in 2009 with funding from University of Toronto htto://www.archive.org/details/journalofexperim12broo Y\ THE JOURNAL OF EXPERIMENTAL ZOOLOGY EDITED BY WiuiaM E. Caste FRANK R. LILuiE Harvard University University of Chicago Epwin G. ConxKLIN Jacques Logs Princeton University Rockefeller Institute CuHar_es B. DAVENPORT Tuomas H. Morcan Carnegie Institution Columbia University Horacr JAYNE GrorGe H. Parker The Wistar Institute Harvard University HersBenrt S. JENNINGS Epmunp B. Witson, Johns Hopkins University Columbia University and Ross G. HARRISON, Yale University Managing Editor = VOLUME 12. sh ie 1912 cat THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. ee i old Oh, > Wk wy he Cage Fy, ¥ y j 7 \ oN : \ v “| ti r CONTENTS 1912 No. 1. JANUARY 5 Epwin G. Conxuin. Cell size and nuclear size. Thirty-seven figures...... 1 Raymonp Peart aNp Maynie R. Curtis. Studies on the physiology of reproduction in the domestic fowl. V. Data regarding the physiology PmUHeROVIOICtee HOUT HEUTOS > <\-- sae eee 543 rae AUTHOR AND SUBJECT INDEX DAPTATION of fish (Fundulus) to higher temperatures. On the 543 Alcohol, nicotine, tobacco smoke and caffeine on white mice. Comparative studies of the effects of I. Effects on reproduction and growth. 133 Amphibians. Studies on sex-determination tn. V. The effects of changing the water content of the egg at or before the time of fertilization on the sex-ratio of Bufo lentiginosus. 319 Arbacla-Toxopneustes crosses. Studies in cytology. I. A further study of the chromosomes of T. variegatus. II. The behavior of the chromo- somes in 391 ANCROFT, Franx W. Heredity of pigmenta- tion in Fundulus hybrids. 153 Bancrort, FI. W., Lops, Jacques anv. Can the spermatozoon develop outside the egg? 381 AFFEINE on white mice. Comparative stud- jes on the effects of alcohol, nicotine, tobacco smoke and I. Effects on reproduction and growth. 133 Carbon dioxide on various protozoa. Studies on the physiological characters of species. I. The effects of 519 Cell size and nuclear size. 1 Characters of species. Studied on the physiologi- eal. I. The effects of carbon dioxide on various protozoa. 519 Chemical properties of hay infusions with special reference to the titratable acidity and Its rela- tion to the protozoan sequence. 265 Chromosomes of Toxopneustes variegatus. II. The behavior of the chromosomes in Arbacia-Toxo- meustes crosses. Studies in cytology. I. A urther study of the 391 Conkuin, Epwin G. Cell sizeand nuclearsize. 1 CoprLanp, Manton. The olfactory reactions of the puffer or swellfish, Speroides maculatus (Block and Schneider). 363 Curtis, Maynie R., Peart, RayMonp and Stud- jes on the physiology of reproduction in the domestic fowl. V. Data regarding the phys- jology of the oviduct. 99 Cytology. Studies in I. A further study of the chromosomes of Toxopneustes variegatus. The behavior of the chromosomes in Arbacia- Toxopneustes crosses. 391 )eces: Size inheritance in 369 ERTILIZATION in Hyatina senta. Rein- vigoration produced by cross 337 Fertilization in Nereis. Studies of III. The mor- phology of the normal fertilization of Nerels. V. The fertilization power of portions of the spermatozoon. 413 Fixe, Morris S. Chemical properties of hay In- fusions with special reference to the titratable acidity and its relation to the protozoan se- quence. 265 Fowl. Studies on the physiology of reproduction in the domestic WV. Data regarding the physi- ology of the oviduct. 99 FYowls. An experiment dealing with sex-linkage in 599 Vv Fundulus hybrids. Heredity of pigmentation in 153 Fundulus to higher temperatures. On the adap- tation of fish 543 ROWTH. Comparative studies on the effects of aleohol, nicotine, tobacco smoke and caf- feine on white mice. I. Effects on reproduc- tion and 133 EREDITY of pigmentation in Fundulus hybrids. 153 Hybrids. Heredity of pigmentation in Fundu- lus 153 Hydatina senta. Reinvigoration produced by cross fertilization in 337 Hydatina senta. Studies in the life cycle of. III. Internal factors influencing the proportion of male-producers. 283 NFUSIONS. Observations on the origin and sequence of the portozoan fauna of hay 205 Infusions with special reference to the titratable acidity and its relation to the protozoa sequence. Chemical properties of hay 265 Inheritance in ducks. Size 369 Internal factors influencing the proportion of male- producers. Studies in the life cycle of Hydatina senta. 283 ACOBS, Merxet Henry. Studies on the physiological characters of species. I. The effects of carbon dioxide on various proto- zoa. 519 ING, Heten Dean. Studies on sex-deter™ mination in Amphibians. V. The effects of changing the water content of the egg at or before the time of fertilization on the sex- ratio of Bufo lengitinosus. 319 IFE cycle of Hydatina senta. Studies in the. TIT. Internal factors influencing the propor- tion of male-producers. 283 Litire, FRANK R. Studies of fertilization in Nereis. III. The morphology of the normal fertiliza- tion of Nereis. IV. The fertilization power of portions of the spermatozoon. 413 Loren, Jacques, and Bancrort, F. W. Can the spermatozoon develop outside the egg? 381 Lorn, Jacques, and WasTENEYs, HarpotpH. On the adaptation of fish (Fundulus) to higher temperatures. 543 Longevity in Saturniid moths; an experimental study. 179 N ALE-PRODUCERS. Studies in the life cycle ih of Hydatina senta. III. Internal factors influencing the proportion of 283 Male-producing eggs of Phylloxerans. The elimi- nation of the sex-chromosomes from the 479 Mice. Comparative studies on the effects of alcohol, nicotine, tobacco smoke and caffeine on white. I. Effects on reproduction and growth. 133 Moraan, THomas H. The elimination of the sex- chromosomes from the male-producing eggs of Phylloxerans. ~ 479 Moths; an experimental study. Longevity in Satur- niid 179 vil AUTHOR AND SUBJECT INDEX EREIS. Studies of fertilization in. III. The morphology of the normal fertilization of Nereis. IV. The fertilization power of por- tions of the spermatozoon. 413 Nice, L. B. Comparative studies on the effects of alcohol, nicotine, tobacco smoke and caffeine on white mice. I. Effects on reproduction and growth. 133 Nicotine, tobacco smoke and caffeine on white mice. Comparative studies on the effects of alcohol, I. Effects on reproduction and growth. 133 Nuclear size. Cell size and 1 Oe. reactions of the puffer or swell- fish, Speroides maculatus (Block and Schnej- der). The 363 Oviduct. Studies on the physiology of reproduction in the domestic fowl. V. Data regarding the physiology of the 99 EARL, Raymonp, and Curtis, Maynie R. Studies on the physiology of reproduction in the domestic fowl. V. Data regarding the physiology of the oviduct. 99 Paruips, JoHN C. Size inheritance in ducks. 369 Phylloxerans. The elimination of the sex-chromo- somes from the male-producing eggs of 479 Pigmentation in Fundulus hybrids. ° Protozoa. Studies on the physiological characters of species. I. The effects of carbon dioxide on various 519 Protozoan fauna of hay infusions. Observations on the origin and sequence of the 205 sequence. Chemical properties of hay in- fusions with special reference to the titratable acidity and its relation to the 265 R2& NewLui£, Por Rav, and. Longevity in Saturniid moths; an experimental study. 179 Reproduction and growth. Comparative studies on the effects of alchohol, nicotine, tobacco smoke and caffeine on white mice. I. Effects on 133 Reproduction in the domestic fowl. Studies on the physiology of V. Data regarding the phy- siology of the oviduct. 99 Serene moths; an experimental study. Longevity in 179 Sex-chrosomomes from the male-producing eggs of of Phylloxerans. The elimination of the 479 Heredity 153 Sex-determination in Amphibians. Studieson. V. The effects of changing the water content of the egg at or before the time of fertilization on the sex-ratio of Bufo lengitinosus. 319 Sex-linkage in fowls. An experiment dealing with 599 Sex-ratio of Bufo lentiginosus. Studies on_sex- determination in Amphibians. Y. The effects of changing the water content of the egg, at or before the time of fertilization on the 319 Suuut, A. FRANKLIN. Studies in the life cycle of Hydatina senta. III. Internal factors influenc- ing the proportion of male-producers. 283 Species. Studies on he physiological characters of. I. The effects of carbon dioxide on various protozoa. 519 Spermatozoon develop outside the egg? Can the 381 ——Studies of fertilization in Nereis. III. The morphology of the normal fertilization of Nereis. IV. The fertilization power of por- tions of the 413 Spheroides maculatus (Block and Schneider). The olfactory reactions of the puffer or swellfish. 363 Sturtevant, A. H. An experiment dealing with sex-linkage in fowls. 599 Swellfish, Speroides maculatus (Block and Schnet- aan): The olfactory reactions of _ the pul f reste eeaee On the adaptation of fish (Fundulus) to higher 543 Tennant, Davin H. Studies in cytology. I. A further study of the chromosomes of Toxo- pneustes variegatus. II. The behavior of the chromosomes in Arbacia-Toxopneustes cr es. 3) Tobacco smoke and caffeine on white mice. Com- parative studies on the effects of aleohol, nico- tine, I. Effects on reproduction and growth.133 Toxopneustes variegatus. II. The behavior of the chromosomes in Arbacia-Toxopneustes crosses. Studies in cytology. I. A further study of the chromosomes of 391 ASTENEYS, Harpotpx, Loes, Jacques, and On the adaptation of fish Cone to higher temperatures. 543 Water content of the egg at or before the time of fertilization on the sex-ratio of Bufo lengiti- nosus. Studies on sex-determination in Am- phibians. V. Theeffectsofchangingthe 319 WuitNEY, Davin Day. Reinvigoration produced by cross fertilization in Hydatina senta. 337 Wooprurr, LorRanpE Loss. Observations on the origin and sequence of the protozoan fauna of hay infusions. 205 CELL SIZE AND NUCLEAR SIZE EDWIN G. CONKLIN From the Department of Biology, Princeton University THIRTY-SEVEN FIGURES CONTENTS PART I. CELL SIZE AND NUCLEAR SIZE IN NORMAL DEVELOPMENT............ 4 I. Unequal cell division.......... 2 ah a a ae eS re 4 1. The maturation divisions. . SPER een cer ese Ac te dc 4 ; OO EC. oe 5 ea es ee 6 j 5h Significance of the yolk lobe. . SS ren ee Ee ea ee ae 9 \ II. Cell size and nuclear size in eggs and blastomeres................ . 212 1. Cell size and nuclear size in the cleavage of Crepidula plana.... 14 2. Cell size and nuclear size in the cleavage of Fulgar carica....... 23 lL. Cell size and nuclear size in adult tissue cells...................... 25 Wen he inciting causes of cell division. ..........6..60cesee esse eee eeee 29 VY. Growth of protoplasm during cleavage...................... a8,r GA VI. Rate of nuclear growth during cleavage. panty e sk: hap ena 36 1. Nuclear growth during the cleavage of the egg of Crepidula.... 38 2. Nuclear growth during the cleavage of the egg of Fulgar....... 40 3. Nuclear growth during the cleavage of other animals. ..... 42 4. Growth of different nuclear constituents............... Sine a. Nuclearsap........... Be ht A ORE SC ate 44 Joy, LOT eae LS TE ee ce 46 ¢-;Ghromatinis..<...... -< ee PN ee eee, soe eee aie Breed oi) d. Chromosomes.......... BN NAS OAS ee ee 48 e. Plasmasomes............ Rieke Bev eae ey ae nee 51 Pe GeantrosOmeR And spheres... .... 25. cecccsec se tee esters 53 5. Conclusions as to nuclear growth stoma cleavage..........-- 54 6. Comparison of growth of chromatin with increase of chemical substances and processes during cleavage............-.----- 56 VII. Senescence, rejuvenescence, and the ratio of nucleus to plasma..... 57 PART II. EXPERIMENTAL STUDY OF CELL SIZE AND NUCLEAR SIZE IN THE EGGS OF CREPIDULA PLANA...... : SR Pe ee ere ere Ose 63 I. Nuclear size and chromosome nates ee eR RR ite Mic 63 II. Nuclear size and cell size in centrifuged eggs of Crepidula.......... 64 1 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 JANUARY, 1912 bo EDWIN G. CONKLIN III. General results of these experiments .................2..2+-e+020e8 75 i” Nuclearsizeanicentrihuredierocmassspne cee meee eee - =a. 75 2. The sizes of spindles, centrosomes, spheres and asters........ 77 3. The rhythm of division in centrifuged eggs Secon Sete 4. Growth of cytoplasm at the expense of yolk................... 77 5. Unequal and differential cell divisions ayer aes » ers 6. Regulation in the cleavage process Ste ay Soe ad 81 General summary and index................. PRG oc Boe CORSE ob tee RS 83 Literature cited Bo ee Lhe Si eau afin 00s Cyne MOET AT RE ha ae 88 In the development of all organisms considerable differences of size appear, sooner or later, among constituent cells; sometimes the blastomeres of the cleaving egg differ in size, in other cases these differences appear only later during the blastula, gastrula, or larval stages. Endoderm cells are usually larger than those of the ectoderm, ciliated cells are generally larger than non-ciliated ones, muscle and nerve cells are usually larger than epithelial or mesenchyme cells. These differences in the size of cells may be due to unequal cell division, to unequal rate of division, or to unequal growth of cells after division, and in some eases all of these factors may be represented in the same egg or embryo. It is frequently assumed that unequal cell divisions are caused by the accumulation of metabolic substances, such as yolk at one side of a cell, and the crowding of the protoplasm and nucleus to the opposite side. Such unequal divisions are frequently found in yolk-laden eggs, and may be artificially produced at will by centrifuging the yolk to one pole or the other of a dividing cell. But in many cases this is not the cause of unequal cell division; the yolk may be uniformly distributed with regard to the poles of the spindle and yet the cleavage, may be unequal, or unequal division may take place in purely protoplasmic cells, in which the eccentric position of the spindle is not due to pressure. Innumerable cases of this sort have been found both in normal and in experimentally altered conditions. Often such unequal divisions are associated with visible histological differences in the resulting cells. The study of cell-lineage has shown that in some cases a particular cell is distinguished from the time of its formation by its size, proto- plasmic structure, rate of division, prospective significance and CELL SIZE AND NUCLEAR SIZE 3 potency. In such cases differences in the sizes of cell are associ- ated with some of the earliest differentiations of the developing egg. But differences in the size of cells may be due, not to unequal cell division, but to unequal rates of division, or to unequal growth of cells subsequent to division. In some instances cells divide rarely and consequently become large, while adjoining cells divide frequently and therefore remain small. The fact that cells are not always of the same size at the time of division is one of capital importance for it shows conclusively that the factors which bring about cell division may be separated from those which cause growth. In connection with the size of cells as a whole may be considered the sizes of many of their constituent parts, such as nuclei, chromosomes, plasmasomes, centrosomes, ete. The size relations which exist between these parts of the cell and the plasma should throw light upon the interrelation between these cell constituents in other respects than size. The quantitative relations of differ- ent cell constituents at various phases of activity should be of significance in the study of many fundamental problems of growth, differentiation and cellular physiology. Within the past few years several contributions on this sub- ject have appeared, principally from Boveri and R. Hertwig, and their students. In so far as these works have dealt with the development of the egg they have been based on a study of eggs of ‘indeterminate cleavage’ in which it is not possible to trace individual blastomeres throughout the cleavage period; the re- sults have therefore been mass results, based on averages of cells of a given stage. In the study of vital phenomena it is frequently important to deal with individual rather than with average results; in the following pages I have attempted to apply the method of the quantitative study of cells and of cell constit- uents to individual blastomeres at various stages of the cleavage. 4 EDWIN G. CONKLIN PART I CELL SIZE AND NUCLEAR SIZE IN NORMAL DEVELOPMENT I. Unequal cell divisions 1. The maturation divisions. The most unequal of all cell divisions are those which give rise to the polar bodies. The actual diameter of the first polar body and of the egg, and the relative volumes of the two, are here given for a number of dif- ferent animals. These measurements were made on eggs which had been fixed, stained, and mounted in balsam. TABLE 1 Sizes of polar bodies and eggs SPECIES DIAMETER FIRST DIAMETER RELATIVE VOLUMES POLAR BODY OF EGG Me a Cumingia tellinoides. ... : 6 45 1 : 421.8 Amphioxus lanceolatus....... 6 108 1 : 5832.0 Cynthia partita....... 9 105 1 : 1560.8 Cerpidula plana... . 12 136 1 : 1442.8 Crepidula fornicata. ... 12 182 1 : 3443.0 Crepidula convexa.... : 15 280 1 : 6434.5 Crepidula adunea..... : 15 410 1 : 20123.6 Fulgur ecarica...... 3 15 1600 i eine) (0) In many other cases, such as the eggs of selachians, amphib- ians and birds, the disproportion between the polar body and the egg is much greater than in the cases here measured. The sig- nificant thing here is not merely the degree of inequality, but also the relative uniformity in size of the polar bodies as compared with the egg. Although the eggs of different animals vary enor- mously in size, the polar bodies vary relatively little, and it is safe to conclude, both from observation and experiment, that the polar bodies are in general the smallest cells which can be formed from egg cells by the process of normal cell division. In spite of this very great inequality of the daughter cells, the mitotic figure in the first maturation division of Crepidula and of many other animals is the largest in the whole life eyele. When CELL SIZE AND NUCLEAR SIZE oO first formed this spindle lies near the middle of the egg, and if the division wall were to form while the spindle lies in this position a polar body would be formed whose diameter would be to that of the egg as 1:1, 1 : 2, or at the least 1:3. Later the spindle moves toward the periphery until one pole comes into contact with the cell membrane. The membrane then protrudes over this pole and into this protrusion the end of the spindle moves; at the same time the spindle itself constantly grows shorter, until finally the spindle is but little more than double the diameter of the polar body, and in the separation of the polar body the division wall passes through the equator of the spindle. In Crepidula plana the first ‘maturation spindle shortens to about half its original length; during the metaphase its maximum length is about 42u, at the time when the polar body is being separated it is only 24u long. By means of centrifugal force it is possible to prevent the spindle from moving from its first position and also from short- ening, and under these circumstances giant polar bodies are formed, sometimes quite as large as the remainder of the egg. In all such cases the division wall passes through the equator of the spindle. Evidently the factors which bring about this most unequal of all cell divisions are (1) the eccentricity and (2) the shortening of the maturation spindle. The second polar body is but slightly smaller than the first, nevertheless the spindle is much smaller, its maximum length in Crepidula plana being 184; correspondingly it shortens much less in the anaphase than the first polar spindle, being almost as long when the division wall begins to form as in the metaphase. Though the second polar spindle may appear at some distance from the point at which the first polar body was formed, and although its axis may lie at right angles to that of the first polar spindle, it invariably rotates into the axis of the latter and the whole spindle moves toward the surface until its outer pole comes to lie immediately under the first polar body, and here the second polar body is pushed out. In this case the principal factor which causes the inequality of division is the eccentricity of the spindle. If the spindle is prevented by pressure or cen- 6 EDWIN G. CONKLIN trifugal force from taking this eccentric position the resulting cell division may be nearly equal, or a giant second polar body may be formed (fig. 11). 2. Cleavage. The first cleavage of Crepidula and of Fulgur is approximately equal. The pronuclei lie near the animal pole of the egg, the egg nucleus lying somewhat nearer the polar bodies than the sperm nucleus. The first cleavage spindle is oriented so as to lie at right angles to the egg axis, but it is im- possible in these eggs to determine whether the spindle lies in a particular cross axis or not. However in typical cases the spindle invariably lies at right angles to the chief axis with its equator in that axis, and the protoplasm and yolk are divided by the first cleavage plane with strict equality. The same is true of the second cleavage which in all these regards resembles the first. It has been generally assumed that equal cleavages, alternately at right angles, are due to simple mechanical conditions, such as the greatest diameter of the protoplasmic mass, and that they require no further explanation. As a matter of fact equal cleay- ages, and successively alternating ones, cannot be explained in so simple a manner. The fact that the first cleavage spindle invariably stands at right angles to the chief axis of the egg and with its equator in that axis shows that there is here some orienting power of the highest significance. It is well known that there is considerable variation in the path which the sper- matozoon takes through the egg, and in its manner of meeting with the egg nucleus; there is also much variation in the actual positions of the cleavage centrosomes and in the initial position of the first cleavage spindle, without any corresponding variation in the final position of the spindle or of the cleavage plane. As a result of the study of large numbers of eggs of many different animals, under both normal and experimental conditions, its seems to me necessary to conclude that the same factor which brings about an unequal division of an egg such as that of Unio, operates to cause the equal division of an egg like that of Crepid- ula; ‘this factor is to be found in the polarity and symmetry of the egg itself. In Unio, where the first cleavage is very unequal, CELL SIZE AND NUCLEAR SIZE 7 Lillie (01) has shown that the spindle oscillates in the cell before coming to rest in its eccentric position; and in many cleavages in Crepidula the spindle may at first lie out of its normal position and may move later into its proper place; and this applies not only to the eccentricity but also to the axial position of the spin- dle. Something outside of the spindle itself determines the posi- tion which it shall take in the cell, and this is as true of equal and alternating cleavages as of unequal and non-alternating ones. TABLE 2 Sizes of macromeres and micromeres in Crepidula and Fulgur SPECIES MACROMERES DIAMETER MICROMERES DIAMETER Parte Ke M 1A-1D 81 la-ld 30 ca.19.9 31 2A-2D | 80 2a-2d 36 ca. 10.6 :1 3A-3D | 76 3a-3d 33 Core de I | Gaplang....... ANDY 66 4d 38 ca. 4.9:1 4A-4C 60 4a4te 42 Ca. 2.0) suk 5A, 5B 75 5a, 5b 60 ea: 1:9)271 5C, 5D 68 5e, 5d 68 ca. aL 1A-1D 195 la-ld 69 | ca. 22.0 :1 C. convexa.... 2A-2D 195 2a-2d 50 | ca. 59.3 :1 3A-3D 195 3a-3d 50 Ca.09e ot | 1A-1D 800 la-ld 0) ca. 1000 : 1 | 2A-2D S00 2a-2d 80 ca. 1000 : 1 Fulgurearica.. {) 3A-3D 800 3a-3d 80 ca. 1000 : 1 4D 780 4d 130 ca. 216 :1 ca ord 4A4C 740 4a-4te 370 The third, fourth, and fifth cleavages of the macromeres of Crepidula and of other gasteropods are successively alternating in direction, and are notably unequal in the size of the daughter cells; while the sixth and all subsequent divisions of the macro- meres are more nearly equal than the preceding ones. The diameters of the cells formed by these cleavages and their approxi- mate ratios, in Crepidula and Fulgur, are shown in Table 2. In the structure of the macromeres there is no visible organi- zation which would explain why the first two cleavages of the egg are equal, the three following ones very unequal and subse- 8 EDWIN G. CONKLIN quent cleavages more nearly equal again, and yet it is certain that some such organization must be present. It is generally believed that the inequality of macromeres and micromeres is due to the quantity of yolk contained in the former and where the quantity of yolk is extremely great, as in Fulgur, this is undoubtedly one of the causes of the great difference in the sizes of the macromeres and micromeres; but that it is not the only cause of the inequality is shown by experiments in which by centrifuging eggs at the first or second cleavage two of the macromeres come to contain no yolk, while the other two contain all of the yolk; in the macro- meres which are purely protoplasmic and contain no yolk the subsequent cell divisions are still unequal, protoplasmic micro- meres of the usual size being separated from the protoplasmic macromeres, (see p. 81). The study of normal as well as of artificially altered cleavage points unmistakably to the conclu- sion that the position and axis of each spindle is fixed by the structure of the cell protoplasm, and since the position and axis of the spindles change regularly in successive divisions this protoplasmic structure must change regularly in successive cell generations. Boveri (’05) says that the position of the spindle is not due to a permanent cell structure, but that the constitution of the egg undergoes progressive alterations, which then react on the division centers. Among the micromeres certain cell divisions are quite unequal, and here there can be no question that this inequality of division is In no way associated with the presence of yolk, since the micro- meres are purely protoplasmic. In Crepidula the first and second subdivisions of the first quartet cells (figs. 3, 8). which give rise respectively to the ‘turret’ cells and the ‘apical rosette’ cells, are very unequal; as is also the division of the second quartet cells which give rise to the ‘tip’ cells of the arms of the ectodermal cross.. The diameters of the two daughter cells in each of these divisions, and the approximate ratio of one to the other, are as follows in Crepidula plana: la-ld,? 25y,la2-Id2, I13p..... Saasioss See Baas > eee Ratio2 : lat-—1d?-1, 30u, Iat?-1d?-* 18u...... ina a Ge Sone eee Ratio5 :3 2al-12d!"1, 20u, 2al-2-2Qd?-*, 15y .. . (aot eee Ratio 2:1 = CELL SIZE AND NUCLEAR SIZE 9 In the case of normal eggs it cannot be demonstrated that the inequality may not be due to mutual pressure among the cells, but in certain experiments which will be described in another paper, this factor may be entirely eliminated, isolated blastomeres showing the same inequalities of division as do those in the cell complex. In all of these cases of definite types of cleavage, the position of the spindle, and consequently the direction of division and the relative size of the daughter cells, is determined by some structural peculiarity of the protoplasm and not by the presence of metabolic substances within the cell or by pressurefrom without. 8. Significance of the yolk lobe. Under normal conditions the line of intersection of the first and second cleavage planes marks the chief axis of the egg, its two ends being the animal and vegetal poles. In eggs in which the cleavages are unequal, the chief axis, thus defined, runs from the animal pole, which is marked by the position of the polar bodies, to a point more or less removed from the diametrically opposite pole. Is this chief axis predetermined in the egg or is it established by the positions of the first and second cleavage planes? Observ- ation alone affords no positive answer to this question, but the fact that the spindle takes a definite and characteristic position in the egg indicates that something outside the spindle determines its position, and points to the conclusion that the chief axis is already present in the egg, as a structural differentiation before cleavage begins. This conclusion is well supported by experi- ment, as will be shown later. In this connection the significance of the so-called ‘yolk lobe’ is interesting. As is well known this lobe is found in many eggs, especially in those in which the first and second cleavages are unequal. It is present however in minute form in such eggs as those of Crepidula and Fulgur in which the first two cleavages are approximately equal, but in cases in which these cleavages are unequal it is much larger, and in general the size of the yolk lobe is proportional to the inequality of division. In all cases so far as I am aware the yolk lobe lies diametrically opposite the animal pole, and if detached from the egg at the time when it is fully formed, the egg divides into equal blastomeres, as Wilson 10 EDWIN G. CONKLIN (04) found in Dentalium; if it remains attached it fuses, at the close of the cleavage, with one of the cells, which then becomes larger than the other one. In this case the cleavage spindle is not eccentric and the furrow cuts down through the center of the egg until it reaches the yolk lobe when it turns to one side of the lobe leaving it attached to one of the cells. In this way a cleavage which began as an equal one becomes unequal. Where the spindle is eccentric from the start and the furrow does not pass through the center of the egg the yolk lobe is not prominent. In this way inequality of division may arise through the eccen- tricity of the spindle, or through the formation of a yolk lobe which remains connected with one of the two daughter cells, which would otherwise be equal. One cannot study the eggs of different animals without being much impressed with the fact that the distribution of yolk to the four macromeres is highly characteristic of different species and orders. Thus among prosobranchs the yolk is distributed either equally to all the macromeres, as in Crepidula, Fulgur, Trochus. ete., or if one of the macromeres is larger than the other three it is the left posterior macromere, D, as in Nassa, Urosalpinx, Tritia, ete. Among opisthobranchs, if the macromeres are unequal in size it is one or both of the anterior ones, A or B, which is the larger. Among pulmonates, so far as I recall, the macromeres are always equal in size. The fact that there are these characteristic differences in the sizes of the macromeres of different orders indicates that they have some characteristic cause; and the fact that in nearly re- lated species the macromeres may be equal or unequal indicates that in this case the cause is not a very general one. If one considers that the first and second cleavages normally pass through the egg axis, and that their position is determined by this structural feature, the unequal distribution of yolk to the four macromeres may be due to the localization of the yolk in different parts of the ovarian egg,—on the posterior side of the chief axis in prosobranchs, on the anterior side in opisthobranchs; while a larger or smaller yolk lobe would determine the degree of inequality of the macromeres in the different species. CELL SIZE AND NUCLEAR SIZE 11 Apart from the relation of the yolk lobe to unequal cleavage Wilson (’04) has shown that it bears some relation to the forma- tion of the pretrochal region in the larva of Dentalium; when the lobe was removed the pretrochal organs failed to develop. What the morphogenetic factors are, which are located in the yolk lobe, is not known, but the significance of the lobe can scarcely be for the formation of the pretrochal region, since in animals with no lobe or with a very minute one these regions form quite as well as in those with a large lobe. These explanations refer to the “prospective significance” of the yolk lobe, and I_ know of no certain evidence as to the cause of its formation. The fact that such a lobe is present in almost all gasteropod eggs, differing only in size in different species, and that it is present in the eggs of annelids and a large number of other animals, indicates that it has some cause of general occur- rence. In 1897 I suggested that the yolk lobe marks the point of attachment of the ovarian egg to the follicular wall. At this point there is left a little mass of protoplasm on the surface of the egg, and here there is a weak spot in the protoplasmic pellicle which surrounds the egg. If the egg is put under pressure the yolk may be caused to flow out at this point, and in the increased tension which accompanies mitosis a yolk lobe is often pushed out at this spot. On the whole then, it seems probable that the yolk lobe rep- resents a temporary extrusion of egg substance during mitotic pressure at the former point of attachment to the ovarian wall, and that as a result of the presence of a large lobe of this kind, the first and second cleavages may be rendered unequal though the intersection of the furrows may lie in the egg axis and in the polar diameter of the egg. In this connection one recalls the ‘ Dotterball’ and the ‘Granula- ball’ observed by Hogue (710) and Boveri (710) in centrifuged eggs of Ascaris. Boveri comes to the conclusion that these are formed because they lie outside the influence of the asters or spheres: ‘‘Man kénnte vielleicht sagen:—der von einen Sphiire eingenommene Plasmabezirk sucht sich von allem was ausserhalb dieses Wirkungskreises liegt, abzuschniiren,” (p. 123). He sup- 2 EDWIN G. CONKLIN poses that when the spindle, lying at right angles to the egg axis, is pushed far toward the animal or vegetal pole, a ‘ball’ is formed at the opposite pole. Whether this ‘ball’ is homologous with the yolk lobe I shall discuss in another paper in which the arti- ficial production of such ‘balls’ will be considered, but I wish to point out here that although the first and second cleavage spindles in the large eggs of Crepidula and Fulgur le near the animal pole, and far from the vegetal, the yolk lobe in these forms is very small, whereas in the minute eggs of the oyster and the clam, where the spindles are much nearer the vegetal pole, the yolk lobe is relatively very large. If, as I believe, this lobe is the result of an unsymmetrical distribution of yolk and egg substance with reference to the egg axis, or in the case of Ascaris with reference . to the normal division plane, the great size of the lobe in some cases and its minute size in others, in which the area lying outside the ‘‘ Wirkungskreise” of the spheres is much greater than in the former, would find a ready explanation. IT. Cell size and nuclear size in eggs and blastomeres Strasburger (93) was the first to show by detailed measure- ments that a fairly definite ratio exists between the nuclear size and the cell size in the embryonic cells of any given species of plant. He gives tables of measurements of the sizes of nuclei and cells in some forty different species, the nuclei ranging in diameter from 16y to 3y, and the cells from 24 to 5u. . In gen- eral he found that the ratio of nuclear diameter to cell diameter is approximately as 2 to 3; and the ratio which exists in any case, is held to be due in general to the ‘working sphere of the nucleus,’ i.e., to the extent to which the metabolic interchange between nucleus and cytoplasm can reach. Gerassimoff (’01, ’02) found, in the cell division of Spirogyra, that when both daughter nuclei were caused to remain in one of the daughter cells, that cell grew to a larger size than normal, and he therefore concluded that the nuclear size determines the cell size. CELL SIZE AND NUCLEAR SIZE 13 Boveri (’02, ’05) found that the size of the nuclei in sea urchin larvae is dependent upon the number of chromosomes which enter into the nuclei; in parthenogenetic or hemikaryotic eggs the nu- clei are smaller than in fertilized (amphikaryotic) ones, and they are smaller in the latter than in diplokaryotic eggs in which the number of chromosomes is greater than normal. Furthermore he found that nuclei with a small number of chromosomes are not only smaller than those containing a larger number but that the cells in which they lie are also smaller, owing to the occurrence of a larger number of cell divisions in cells with small nuclei than in cells with large ones. Boveri’s work was based primarily on his studies of echinoderm ‘development and some of his conclusions are not applicable, without modification, to the eggs and larvae of other forms, especially forms in which there are great inequalities of cleavage and in which various cells of the larva differ markedly from one another in size. Thus his generalization, sometimes mentioned as ‘Boveri’s Law,’ viz., ‘‘Die Grésse der Larvenzellen ist eine Funktion der in ihnen enthaltenen Chromatinmenge, und zwar ist das Zellvolumen der Chromosomenzahl direkt proportional,”’ could not apply, without modification, to eggs or larvae in which various cells differ greatly in size without any corresponding difference in the number of chromosomes. Unequal cell divisions are frequently found in the development of mollusks, annelids and ascidians, where purely mechanical causes, such as mutual pressure between cells or the pressure of yolk within cells are not involved; in such eases the sizes of the nuclei invariably become proportional to that of the plasma, though the number of chro- mosomes remains the same in every nucleus. Similarly, many cells at first equal in size become unequal through dissimilar growth, and their nuclei then become unequal also. Finally, in each of the animal groups named, cells at first equal in size may become unequal through dissimilar rates of division. In all such cases the number of chromosomes appears to be, and pre- sumably is, the same in every nucleus of a given egg or embryo. Evidently in cases of normal development the number of chromo- somes does not determine the varying sizes of cells and nuclei. 14 EDWIN G. CONKLIN R. Hertwig (’03, ’08) as a result of his earlier work (’89) upon protozoa, has laid especial emphasis upon the fundamental sig- nificance of the ratio of nuclear size to cell size. He says (’03, p. 56): Wir haben im vorhergehenden sehr komplizirte Wechselwirkung zwischen Kern und Protoplasma kennen gelernt. Verkleinerung der Kernmasse fiihrt zu Verkleinerung der Zellgrésse (Boveri), Vergréss- erung der Kernmasse zu einer Vergrésserung der Zelle (Gerassimoff, Boveri). Andererseits kann aber auch Schwund des Plasmas zu einer Reduktion des Kernmaterials Veranlassung werden. Diese Verhilt- nisse kann man nur erkliren, wenn man die oben vertretene Annahme macht, das jeder Zelle normalerweise eine bestimmte Korrelation von Plasma- und Kernmasse zukommt, welche wir kurz die “ Kernplas- marelation” nennen wollen. More recently Hertwig and his students have made many notable contributions to these ‘“‘new problems of the cell theory,” as Hertwig (’08) calls them. It has long been known that large cells have large nuclei, small cells small nuclei: Das Neue, welches in der Lehre von der Kernplasma-Relation gegeben ist, ist der Gedanke, dass das Massenverhiltnis von Kern zu Proto- plasma, der Quotient k/p, d.h. Masse der Kernsubstanz dividiert durch Masse des Protoplasma, ein gesetzmiissig regulierter Factor ist, dessen Grosse fiir alle von Kerne beeinflusten Lebensvorgiinge der Zelle, fiir Assimilation und organisierende Tatigkeit, fiir Wachstum und Teil- ung, von fundamentaler Bedeutung ist. Hertwig calls attention to the fact that the Kernplasma-Rela- tion differs in different phases of cell life, and he chooses for meas- urement that phase when the cell has come out of division and begins to nourish itself and to grow. This condition is known as the Kernplasma-Norm, and departures from it constitute what he calls Kernplasma-Spannung. This work of Hertwig and his school will be discussed more fully after the presentation of my observations. 1. Cell size and nuclear size in the cleavage of Crepidula plana. In my work on Karyokinesis and Cytokinesis in Crepidula (’02) I showed that the sizes of nuclei, spheres and asters, centrosomes, chromosomes, and plasmasomes are correlated with the quantity of cytoplasm in the cell, and the following pages constitute an CELL SIZE AND NUCLEAR SIZE 15 elaboration and further extension of that work. The egg of Crepidula plana is a particularly favorable object for the study of such a subject. The eggs may be stained and mounted entire in such manner that all of these cell constituents show with great distinctness, and the advantage of seeing whole eggs and nuclei in making such measurements is sufficiently obvious. A further advantage of the study of whole eggs is found in the fact that the exact stage in the cell cycle is more easily determined in whole eggs than in sections. My work has shown that it is most important in comparing the sizes of cell constituents to compare precisely corresponding stages, and accordingly I have chosen for measurement stages of the maximum and minimum sizes of the nuclei. The growth of the nucleus is more rapid in the last stage of the resting period preceding mitosis (‘Kernteil- ungswachstum’ of Hertwig) than at any other time in the cell cycle, and in order to find the maximum nuclear size it is neces- sary to measure the nuclei just before the nuclear membrane dis- appears. Such stages are easily selected by looking for eggs in which part of the nuclei of a certain generation of cells are divid- ing while others have not yet begun to divide, as in figs. 1 and 2. At this stage there is great uniformity in the dimensions of the nuclei of particular blastomeres, and as the nuclei at this stage are regular spheres, it is easy to calculate their volumes. The cell dimensions are more difficult to determine than are those of the nucleus. In cells which contain yolk and in cells of irregular shape it is not possible to determine the volume of the plasma with accuracy. After the first cleavage the plasma and yolk are sufficiently well separated so that the dimensions of the cytoplasm can be fairly well observed; before the first cleavage the plasma is so mixed with the yolk that this can not be done and I have here had recourse to the method of centrifuging the yolk out of the egg, leaving only the nucleus and plasma which can then be easily measured. Wherever it could be done, I have chosen cells for measurement which were as nearly spherical as possible, but where the dimensions in different axes differed con- siderably I have determined the mean diameter, which is the one recorded. 16 EDWIN G. CONKLIN It is well known that during mitosis the general surface tension of a cell increases, and the cell tends to become spherical in shape. In measuring the maximum cell size, I have usually taken the stage immediately after the nuclear membrane disappears, and when the cell approaches a spherical shape. Similarly the mini- mum cell size has been determined by measuring the daughter cells during the telophase when they are approximately spherical. I have confidence in the substantial accuracy of my measurements of these maximum and minimum sizes of the purely protoplasmic micromeres. The volume of plasma in the yolk-containing macromeres is merely an approximation. All measurements were made with Zeiss 1/1 micrometer eye- piece and 3 mm. homogeneous immersion objective. In all cases enough cells and nuclei were measured to give a fair average, though there is relatively little variation in the sizes of particular cells and cell constituents at corresponding stages in the cell cycle. All the eggs studied were fixed, stained and mounted in the same manner, so that alterations due to shrinkage should be approximately the same in all. It is evident from table 3 that while large cells have larger nuclei than small cells, the relation of nuclear volume to cell volume is not constant. In different blastomeres of the same egg the Kernplasma-Relation, measuring nuclei and cells at their maximum size, varies from 1 :14.5 to 1:0.37; even in purely protoplasmic cells it varies from 1 : 14.5 to 1:8.7. In cells con- taining yolk the ratio of nuclear volume to cell volume (includ- ing the yolk) varies from 1 :89.5 to 1 :34.8. In the different blastomeres of this egg there is no constant nuclear-plasmic ratio, or Kernplasma-Norm. However in different eggs corresponding blastomeres have the same Kernplasma-Relation, when measured at corresponding stages. The volumes of the protoplasm and of the nucleus show little variation in any given blastomere and the Kernplasma-Relation of each of the blastomeres named in table 3 is practically the same in all eggs. Since many of these blasto- meres are peculiar in odplasmic constitution and prospective significance it is not improbable that the peculiarities in their CELL SIZE AND NUCLEAR SIZE 17 TABLE 3 Maximum nuclear size and cell size in the blastomeres of Crepidula plana; (measured just before nuclear membrane dissolves) DIAME oR VOLUME OF | BEeemM) jesse | vouwies ao tal ae | Tae Pome a BLASTOMSBES | (O25 ENE I INCLUDING | NUCLEUS NUCLEUS (VOLUME OF RELATION NUCLEUS | NUCLEUS | | | , be cubic bh “ | 7 cubic cubic i Before maturation. —_150 1,755,000 | ca.64* | 42 32,409 97,1381 | 1:3 Before first cleay- | 930+ oe: SRC RCE ORNS 142 | 1,488,910 ca. 65° = |*o'24=34.5 21,375 121,430 1:5.6 AB, CD, before sec- | ond cleavage. re} 106 619,329 ca. 51* | 24 7,238 61,741 | 1:8.5 A, B, C, D, before | third cleavage... 82 286,712 ca. 44° 22 | 5,775 38,570 1:6.6 1A-1D, before | fourth cleavage. . . 81 276,350 | ca. 40 21 | 4,849 28,431 1:58 Ia-ld, before divi- BIO asics ass 30 4 1,437 12,603 | 1:8.7 2A-2D, before fifth cleavage.......... 80 266,240 | ca. 36 18 3,055 21,196 Sy 2a-2d, before divi- MlOQueeape sas. <<. 36 15 | 1,767 22,484 1:12.7 lal--Idt-, before di- WABI OM Seaeeietata)a cats « 30 12 | 905 13,135 1:14.5 1a?-—-1d2-, before divi- SOT mene a's(2'x 15 7 180 1,587 1:88 3D, before sixth cleavage.......... 76 228,288 ca. 30 16 2,145 11,895 1:5.5 3A-3C, before sixth cleavage.......... 76 228,288 | ca. 22 16 2,145 3,430 | 1:1.6 3a-3d, before divi- IDI aeeie cd ocsie ve 33 l4 1,437 19,250 1:13.3 2at-—2dl., before divi- POM etme rie iad ale! (0's 30 l4 1,437 12,603 1:8.7 2a2.—-2d?., before divi- CG) Sane ecncee ne | 30 14 1,437 12,603 1:8.7 4d, before seventh’ cleavage.......... 38 28,533 ca. 22 il 697 4,878 i by 4A-4D, before sev- | enth cleavage... 60 112,320 | ca. 20 18 3,055 1,134 | 1:20.37 da-4e, before sey- | enth cleavage..... 42 32,409 ca. 14 12 | 905 532 | 1: 0.58 * After yolk has been centrifuged out of egg. In normal eggs yolk and proto- plasm are not well segregated at this stage. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 18 EDWIN G. CONKLIN Kernplasma-Relation may be the result of differentiations ale present in the blastomeres. In table 3 only maximum nuclear and cell dimensions are given for the different blastomeres. Results would undoubtedly differ ereatly if the minimum nuclear and cell dimensions were taken instead of the maximum. Accordingly in table 4 the minimum nuclear and cell dimensions fort the various blastomeres of Cre- pidula plana are given, together with the Kernplasma-Relation of each. It is well known to cytologists that in cells undergoing regular division the minimum size of the nucleus is reached in the late anaphase, when the individual chromosomes have contracted to their smallest size and when they are most closely crowded together. A little earlier than this stage the chromatic plate is wider and the spaces between individual chromosomes greater; a little later the chromosomes begin to absorb achromatin and to swell up to form the chromosomal vesicles. At this stage of greatest nuclear contraction the chromosomal plate has approxi- mately the form of a disk or short cylinder, and although the polar ends of the chromosomes are closer together than the equatorial ends, the disk being like a truncated cone, rather than a cylinder, we shall not greatly err if we treat this chromosomal disk as a short cylinder, rather than as a truncated cone. In table 4, in the column giving the dimensions of the chromosomal disk the first number is the diameter of the disk, the second its thickness. Popoff (’08) has found in Frontonia that immediately after cell-division there is a diminution of the nucleus, which is then followed by a slow growth (‘Funktionelles Wachstum’ of Hert- wig), and this by a much more rapid growth of the nucleus pre- ceding division (‘Teilungswachstum’ of Hertwig). Both the functional growth and the divisional growth occur in the cleavage of Crepidula, but there is no diminution of the nucleus following division as in Frontonia. On the other hand, the minimum nu- clear size is reached in the anaphase just before division of the cell body, as has been explained. In the early telophase the chromosomal plate is drawn close to, and moulded over, the centrosome, and consequently the shape CELL SIZE AND NUCLEAR SIZE 19 of the chromosomal plate, its degree of curvature and width, is dependent in part upon the size of the centrosome. I found in 1893 that in unequal cell division the centrosomes and asters become unequal before the cell division is finished, though in the earlier stages of mitosis the centrosomes and asters at the two poles of the spindle are equal in size; only after the cell division is finished do the daughter nuclei become unequal. The present work has confirmed these earlier conclusions and has shown in addition that the shape of the chromatic plate at the ends of the spindle is influenced by the size of the centrosomes, and hence by the equality or inequality of the division. If the centrosome is large the chromosomes form a slightly arched plate on its surface; if it is small the plate is highly arched. In the former case the plate remains relatively wide and the daughter nuclei when they are formed are disk-shaped; in the latter the plate and the daughter nuclei become more nearly spherical. Therefore, in comparing the sizes of chromatic plates it is necessary to meas- ure them before this difference in shape appears, i.e., in the late anaphase. But even when all these precautions are taken the probable error in measuring objects of such small dimensions is considerable, but at least these measurements give the relative order of magnitude of the chromosomal disks in the different blastomeres. The minimum cell dimensions occur in the early telophase, when the daughter cells first separate; at this stage the cells are nearly spherical in form and it is not difficult to calculate their volumes with substantial accuracy. While the minimum cell size does not occur at precisely the stage when the nuclei are smallest, it occurs so soon thereafter that it can make but little difference in the determination of the Kernplasma-Relation. In short the Kernplasma-Relation, when plasma and nuclei are measured at their minimum sizes, varies in different blasto- meres from 1:29 to 1: 285.6. Except in the division of certain cells in the fourth and fifth cleavages (2A—2D and 2a—2d, 2a'-2d' and 2a*-2d?) there is no appearance of a constant ratio between nucleus and plasma in these different blastomeres. In general the dimensions of the nuclear plates decrease with every cleav- 20 EDWIN G. CONKLIN TABLE 4 Minimum nuclear size and cell size in the blastomeres of Crepidula plana. (Nu- clear plate measured in the late anaphase; cell diameter in early telophase) 2 Ee 4 g 5 « & moO on Zz im? D; STAGE BLASTOMERES c oon 2 fe 5 = pealcheraa| oGe : nae : g gaze pie ge oar Bs a 235 a bs B<,, Zo 2 2m zz an aR ee a a a > > Ct Me “ “ cubic cubic fh 2 cells AB, CD..| 105 ca. 46, 9x3 190.5 30,504 1 : 160. 4 cells A,B,C,D,..| 78 ca. 40| 8x3 150.6 30,695 1 : 203.8 Aerie 1A-1D... 75 ca. 36] 6x3 $4.6 24,166 1 : 285.6 : \la-Id .. PH, 27 6x3 84.6 10,235 1 3120 12 cells 2A-2D....| 72 ca. 30, 6x3 84.6 13,955 1 65 2a-2d.... 30 30 | 6x3 84.6 13,955 6s 16 cells 2 lal-ld! ..| 30 30 5563} 58.8 13,955 1 223% ~ \1a2-1d? 15 15 ‘a15.60) 58.8 1,708 1:29 90 cells 3A-3D... 72 Can ib 4x3 By st) 1,729 1:46 < NSRESil oe cll) BE 25 | 4x38 37.5 8,087.5 | 1: 215.6 24 cells 2a1-2d1...) 24 24 4x3 37.5 7.200 1 +195 25 cells {4D ae 60 5x3 58.8 (Ad errr ec cok 30 Byes} || 58.8 2a7-2d2...| 24 24 | 4x3 | 31.50 7,200 13195 29 cells { lat-1d!1 15 15) 4x3 3f.0 1,729.5 | 1 : 46 lal2Idl-2 | 24 24) 4x3 | By nta 7,200 1:195 39 cells JAA ae: | 4x3 37.5 ~ |4a-4e..... | 4x3 a0-0) age, while all the cells of the same generation have nuclear plates of about the same size, though their protoplasmic volumes vary widely. There is a great difference between the maximum and mini- mum volumes of the same nucleus, ranging from 1:27 to 1:38, while there is a relatively slight difference between the maximum and minimum sizes of the plasma and accordingly the Kern- plasma-Relation of any blastomeres varies continuously from the stage of minimum nuclear volume to that of maximum nuclear volume. Hertwig (08) has chosen as the stage showing the Kernplasma-Norm, ‘‘das Verhalten der jugendlichen Zelle, welche eben aus der Teilung hervorgegangen ist und nun anfingt sich CELL SIZE AND NUCLEAR SIZE 21 von neuem zu ernihren, um abermals heranzuwachsen und sich zu teilen.”’ I have tried to make measurements at the stage described by Hertwig, but find that in these segmenting eggs it is too ill defined to be safely used. Between successive divisions the nuclei are growing continuously and rapidly and there is no clearly marked pause in the nuclear growth; accordingly slight differences in the stages chosen for measurement show relatively large differences in the sizes of the nuclei. In order to take a stage intermediate between the two ex- tremes of nuclear size, and in which the nuclei may be regarded as having reached a normal functional condition, not related primarily to the preceding or succeeding division, I have chosen that stage when the nuclei first become regularly spherical in shape. For a considerable part of the resting period the nuclei are elongated at right angles to the previous spindle axis, and in the plane of the chromatic plate of the previous anaphase; during the latter part of the resting period they grow very rapidly in preparation for the succeeding division (‘ Kernteilungswachs- tum’ of Hertwig). The stage when the nuclei first become spher- ical lies somewhere between these two phases and may therefore be considered to represent the mean nuclear size. However this stage is not so precisely defined as are the stages of maximum and minimum nuclear size, and therefore the nuclear dimensions are likely*to be more variable. It is obviously more difficult to determine the volume of the cleavage cells during the resting period, when they are pressed into irregular and polygonal forms, than during mitosis when they approach a spherical shape. However the approximate accuracy of the cell dimensions recorded in table 5 may be judged by com- paring them with the maximum and minimum cell dimensions, given in tables 3 and 4. The Kernplasma-Relatior. varies about as much for mean dimensions of the nucleus and cell as for maximum ones, though not as much as for minimum dimensions. In yolk-containing cells it varies from 1:1.1 to 1: 27.5 and in purely protoplasmic cells from 1:7 to 1:35.7. 22 EDWIN G. CONKLIN TABLE 5 Mean nuclear size and cell size in the blastomeres of Crepidula plana. (Measured when nuclei first become spherical after division) Te 5 a cg ce ne & 58 me | a z STAGE BLASTOMERES “ Ae ° | fe 2 m as % | ° u a B28 || g 2 20 BER : : Be a a a > 4 Mw BL Mu cubic cubic ps f 918 Before cleavage.... 136 ca. 60} 4 12 3,960 109,137 1 32765 2 cells AB,CD...|105 | ca, 44| 18 3,055 41,240 | 1:13.5 4 cells A,B,C,D.| 78 | ca. 40] 16 2,145 31,185 | 1:14.5 Pee UDean 1) | cies 2s 15 | 1,767 22,484 | 1:12:7 ee ere Nie deeaee aSO 30 he 905 13,185 | 1:14.5 2 cells (2A 2D:-+-| 72 | ca. 36) 15 1,767 29.484 | soa 5 oe On oder tea 36 12 905 23,346 | 1:25.6 , _filatd'...| 30 30 | 9 382 13,658 | 1:35.7 16 cells ee ee = Ss rs la2-1d?...) 15 15 6 115 1,654 1:14.6 20 cells (24 3D----| 72 | ca. 18 14 1,437 1,618 | 1:11 pare tears Wei necyshe gs! G8) 30 12 905 13,135 | 1:14.5 : SERGE Al) Di) 27 12 905 9,330 | 1:10.3 DAT cellar aes a a x : 2a-2d?...| 27 27 12 905 9)330) | LT =aOkS 25 cells | fe De casrterons 60 Oo sae pet ae 34 9 382 DOL ealieh eee te 15 6 113 1,654 |1:14.6 aes att diez |e 24 12 905 6,333 | 1:7 NSC) 2 15 1,767 aie J SECs 9 382 Incidentally the interesting fact appears that nuclei in large cells do not become spherical until they have reached a larger size than their sister nuclei in small cells; for example in the micro- meres Ja—1d and 2a—2d the nuclei become spherical when they are about 12 in diameter, in the macromeres 1A—1/.D and 2A-2D their sister nuclei are about 15y in diameter before they become spherical. Since the same amount of chromatin goes into each of the sister nuclei, the difference in the size of the nuclei when they first become spherical must be found in some other factor. It seems probable that it is due to the shape of the chromosomal disk, which remains flattened, or slightly arched, in large cells CELL SIZE AND NUCLEAR SIZE 23 and is highly arched in small ones, owing to the greater or smaller size of the centrosomes, as explained on p. 19. The flattened chromosomal plate gives rise to a disk-shaped nucleus, which only later becomes spherical, whereas the highly arched plate sooner gives rise to a spherical nucleus. 2. Cell-size and nuclear size in the cleavage of Fulgur carica. The eggs of Fulgur carica are the largest gasteropod eggs of which I know, while the eggs of Crepidula plana are among the smallest. It will be instructive therefore to compare the Kernplasma- Relation in these two cases. Table 6 gives the mean nuclear size and cell size of the blastomeres of Fulgur. The eggs measured were fixed, stained and mounted entire, as in the case of Crepi- dula plana. Owing to the great size of these eggs it was necessary TABLE 6 Mean nuclear size and cell size in the blastomeres of Fulgur carica be mF fie 2 : aa) eb Sg 35 Be | Ge | ge BS 2B ze a=) eS 2 a6 | 25 | 3% 3% 3 ue Me BM cubic cubic Macromeres AB, CD, before second cleavage. . 1200 200 40 33,280 4,126,720 | 1 : 124 Beeb, LD; peters fined cleavage. . ; 800 160 32 17,040 2,112,880 | 1 : 124 1A-1D, Barore Reunth clear Nail, 35 acne ee eee 800 | 160 | 32 17,040 | 2,112,880 | 1 :124 2A-2D, before fifth cleav- Woh 4 Sea Aiea 800 160 3o2 17,040 2,112,880 | 1 :124 3D, before sixth cleavage.) 800 160 32 17,040 2,112,880 | 1 : 124 3A-3C, before sixth cleav- age.. 800 160 48 57,508 2,062,412 | 1:35.8 4\-4D, “before” seventh cleavage................| 768 | 160 96 460,063 1,669,857 | 1 :3.6 Micromeres la-ld. Seco | 80 16 2,145 264,095 | 1 : 127.7 HE css wont nase ras ae | 80 | 16 | 2,145 264,095 | 1 : 127.7 31310) ae Po Soen | 8O 16 2,145 264,095 | 1 : 127.7 Unni ho Ry eae ee Rees Ss 8O 16 2,145 264,095 | 1 : 127.7 1: 124 RNC p yes a) 2.0.0 spel ma S ; 40 8 266 33,014 24 EDWIN G. CONKLIN to make the measurements under a relatively low power, the 8 mm. apochromat objective and the 1/1 micrometer eyepiece of Zeiss. Owing to the relatively low magnification the probable error is greater than in the measurements of the eggs of C. plana. With the exception of the cells $A—3C and 4A—-4D the Kern- plasma-Relation is in this case practically constant, varying only from 1: 124 to 1:127. In view of the fact that I can find no constant Kernplasma-Relation in the blastomeres of Crepidula this result in the case of Fulgur is unexpected. I am sure that my measurements in the case of Fulgur are not so accurate as in Crepidula, the number of eggs measured being relatively small and the magnification used low, so that each interval of the scale stood for 8%, The uniformity in the measurements of the differ- ent blastomeres and nuclei of Fulgur may be due in part to this fact; on the other hand this would only account for the lack of minor variations and would not explain the general uniformity. There is no doubt that the micromeres of Fulgur are more uni- form in size, than those of Crepidula; also the whole cleavage process is very much slower, and (with the exception of the macro- meres 3A-3C and 4A—4D) the divisions are more nearly synchro- nous in the different cells than in Crepidula. It seems probable that these two facts are connected with the more uniform Kern- plasma-Relation of the different blastomeres of Fulgur. This conclusion is rendered still more probable by a consider- ation of the two generations of cells in which there is a wide de- parture from the usual Kernplasma-Relation, viz. 3A—3C and 4A-4D. In these cases, as in the same cells in Crepidula the resting stage is particularly long, lasting in the case of 44-4D until all the organs of the embryo are outlined and more than one thousand cells are present; consequently the nuclei grow to an enormous size so that the Kernplasma-Relation falls in one case to 1 : 35.8 and in the other to 1 : 3.6. In the corresponding cells in Crepidula the ratio is 1: 1.6 and 1 : 0.37; the volume of the nucleus in the last named case being about three times that of the plasma. The Kernplasma-Relation of the cells 4a-4e is 1 : 0.58 in Crepidula, the nuclei being about twice as voluminous as the plasma; in the corresponding cells of Fulgur this ratio cannot be CELL SIZE AND NUCLEAR SIZE 25 readily determined since the nuclei undergo several divisions, though the cell body does not divide. From these measurements it may be concluded that when cell division takes place at regular intervals the Kernplasma-Relation is fairly constant; when it takes place at irregular intervals this ratio is variable. The longer the resting period the larger the nucleus becomes, and in extremely long resting periods the greater part of the plasma may be taken up into the nucleus. These observations are in full agreement with experiments on the eggs of Crepidula which will be described later. They are not antagonistic to Boveri’s conclusions as to the correlation be- tween chromosome number and nuclear size; on the other hand my own experiments show that the size of the nucleus is depend- ent, in part, upon the number of chromosomes which enter into its formation. But in normal cells all of which contain the same number of chromosomes differences in nuclear size must be due to some other factor. The results of my measurements do not indicate that the Kern- plasma-Relation of Hertwig is either a constant or self regulating ratio in the blastomeres of these eggs; on the other hand it appears to be a result rather than a cause of the rate of cell division, and consequently it is a variable rather than a constant factor. Furthermore the size of the nucleus, in these eggs, is dependent upon at least three factors: (1) The initial quantity of chromatin (number of chromosomes) which enter into the formation of the nucleus (Boveri). (2) The volume of the protoplasm in which the nucleus lies. (3) The length of the resting period. III. Cell size and nuclear size in adult tissue cells It is generally believed that embryonic cells differ greatly from adult tissue cells in their ‘““Kernplasma-Relation.’’ In a series of thoughtful and suggestive works Minot (’90, 795, ’08) has main- tained that differentiation, senescence and finally death are the accompaniments, if not the results, of an increase of protoplasm as compared with nucleus. It is well known that embryonic cells of plants are more purely protoplasmic than adult cells, 26 EDWIN G. CONKLIN which are frequently filled with vacuoles and sap so that the size of the cell gives no true idea of the volume of the cytoplasm. Among animals adult tissue cells often become filled with the products of differentiation or metabolism, such as fibers, granules, secretions, oil, ete., which greatly increase the cell dimensions. It is evidently a difficult if not impossible task to determine the quantity of real protoplasm in such cells and thus to discover the true “‘Kernplasma-Relation.’”’ However in certain less highly differentiated cells, especially in epithelial and glandular tissue, the true Kernplasma-Relation may be established with a fair degree of accuracy. Unquestionably the physiological state of a cell has much to do with its nuclear-plasmic ratio. Hodge (’92) found the nuclei of nerve cells shrunken after extreme stimulation, and it has been long known that the same is true of gland cells. In Crepidula the liver cells, when active, are filled with secretion and are among the largest in the body, but when the secretion has been discharged and they have returned to an inactive condition, the cell body is much smaller and the nucleus larger. I have measured the cells and nuclei of a number of tissues of Crepidula plana, derived from the three germ layers, and the results are given in table 7. Since these cells vary in shape to a great extent, and in order to facilitate comparison of cell diam- eter and nuclear diameter, cells were chosen for measurement which were as nearly as possible spherical or cubical in shape. In all elongated cells the long axis and one cross axis were measured and it was assumed that the other cross axis was of the same di- mensions as the one observed. It is evident that in these tissue cells of Crepidula plana there is no marked increase of protoplasm over nucleus as compared with the blastomeres of the same species; throughout the cleavage, with the exception of the cells 3A-3D and 4A—4D, the average Kernplasma-Relation for nuclei and cells of mean size is about 1 : 15, for nuclei and cells of maximum size about 1 : 6; the aver- age ratio in adult tissue cells, which are not filled with metabolic products, is about 1 :10.5. In the case of the ganglion cells the nuclei are relatively and absolutely larger than in the other tissues, CELL SIZE AND NUCLEAR SIZE 27 TABLE 7 Cell size and nuclear size in tissue cells of sexually mature individuals of Crepidula plana DIMENSIONS OF DIAMETER VOLUME OF ee EE DURA DEC ELS CELL OF NUCLEUS NUCLEUS ae Begeae | Kh “ cubic cubic Intestinal epithelium....) 11 x 11 x 12 6 113 1,339 |1:11.8 Gastric epithelium..... ..| 10x10 x 36 8 68 3.3392) |e =oi4 Liver duct epithelium.... | 10x10 x18 6 113 1,628 | 1:14.4 Liver cells (filled with secretion products)..... 15x 15 x 45 6* 113 10,012 1 : 88.6 Liver cells (without secre- tion products)....... 14 x 14 x 30 9 382 5,498 |1:14.4 Kidney cells Redutainiic secretion products).... 15 x15x 15 6 113 3,262 | 1:28.8 Ectodermal epithelium (near anus).. ; fa bw 4 33 342 12083 Gill chamber peihelani. 6x 6x12 4 33 405 Saye Gill filament epithelium (le Mh 3s Kt) 4 33 408 | 1:12.3 Epithelium from foot 6x 6x15 5 65.4 474.6) 1:7.1 Ganglion cell (large)... 17 Spee 12 905 Seed. OU Grd Ganglion cell (large)....... 10 x 10 x 20 9 382 1.618) le 2 Oéeytes I (before yolk formation)......... 123 7 180 836 | 1:4.6 Oécytes I (before volk for MAGTLON)kc)ese a's « 114 7 180 791 1:3.4 Oécytes I (before Role fon MeBtION)......:-. 10 6 113 407 1:3.6 Oécytes I (before y a for PEINOID) Som cle cea aac os 8 5 65.4 203 oS i | Oécytes I (before yolk for- EEVENETOIN) Rites ciaee chic 5 5 64 4 33 111 1 33.3 “Nucleus shrunken and very irregular in sh ape. the Kernplasma-Relation being about 1:5; however in this case the nerve fiber is not added to the cell body and this would doubtless greatly increase the volume of the plasma. Muscle cells in Crepidula are long, slender and crooked and I have found it impracticable to estimate their volumes with any degree of accuracy. Doubtless the plasma, including the contractile sub- stance, is here relatively much more abundant than in embryonic or epithelial cells. In the epithelial and gland cells of adult 28 EDWIN G. CONKLIN Crepidula the embryonic ratio of nucleus to plasma is main- tained with little change. In all the o6cytes up to the time that yolk formation begins the nuclei are relatively large, the ratio of nucleus to plasma being about 1 : 3.6, and in the younger and smaller odcytes the nuclei are relatively larger than in the older and larger ones. Eycleshymer (’04) found that the volume of the plasma in the striated muscle cells of Necturus increased about ten times as much as the nuclear volume, during development from the 8 mm. embryo to the adult condition. There is, therefore, in these later stages a notable shifting of the Kernplasma-Relation in favor of the plasma. It is probable however that the contractile substance which makes up the larger part of the muscle cell, does not con- tribute to the growth of the nucleus as does the protoplasm of embryonic cells—that so far as the growth of the nucleus is con- cerned it acts as does yolk, oil, membranes, fibers and other products of metabolism and differentiation. If only the sarco- plasm of the muscle cell and not its contractile substance is able to contribute to the growth of the nucleus, the small volume of the nuclei as compared with the entire cell would find a ready explan- ation. There can be no doubt that the plasma is the chief seat of differentiation, as Minot has emphasized, and that highly differentiated cells, such as muscle, nerve, and some kinds of connective tissue, have a larger amount of plasma and its products, relative to the nucleus, than have embryonic cells. In the case of fiber cells, fat cells, and probably muscle cells, the cell body becomes filled with the products of differentiation and metabo- lism, which like the yolk in egg cells, or the secretion products in liver cells cannot enter the nucleus and consequently do not influ- ence its size. In such tissue cells the cell body is relatively much greater as compared with the nucleus, than in purely protoplas- mic cells, but I have been unable to find any evidence that the ratio of protoplasm (using this term in its usual sense) to the nucleus is greater in tissue cells of Crepidula than in the blas- tomeres. CELL SIZE AND NUCLEAR SIZE 29 IV. The inciting causes of cell division The relative sizes of cells and of nuclei are dependent, in part, upon the rate of cell division. Cells which divide infrequently are larger, other things being equal, than those which divide often. The turret cells (1a*-/d?) of Crepidula are the smallest cells in the entire embryo at the time of their formation (figs. 3, 4); however they divide but twice during the whole of the cleavage period, and consequently they grow to be very large; whereas each of the apical cells from which they were derived, gives rise during the cleavage period to twelve cells the combined volume of which is not much greater than that of one full-grown turret cell. Evidently the factors which bring on or delay cell-division have much to do, indirectly, with the sizes of cells and nuclei. Strasburger (’93) supposed that cell division occurred when the ratio of the cell body to the nucleus increased beyond a certain point, which might be regarded as marking the limit of the ‘work- ing sphere of the nucleus;’ with the division of the cell the normal ratio was once more restored. Boveri (’04) sought to find the inciting cause of cell division in the chromosomes. He believed that the chromosomes divide when they have reached a size double that which they had at the close of the preceding division. At the same time he showed that the rhythm of the division of the centrosomes may be inde- pendent of that of the chromosomes and that division of the cell depends upon the centrosomal rhythm rather than upon the chro- mosomal rhythm. That there is a rhythm of division for chromosomes and centro- somes seems to be well established by Boveri’s work, but this rhythm in the case of the chromosomes is not determined by the time when they have grown to double their size at the close of the preceding division. Marcus (’06) and Erdmann (08) have shown that the chromosome size throughout the cleavage of Strongy- locentrotus is a constantly decreasing one. Baltzer (’08) admits that the chromosomes do not double in size at each cycle of divi- sion; he does not find any great diminution in chromosome size up to the 16-cell stage, though the chromosomes in the blastula 30 EDWIN G. CONKLIN stage are undoubtedly smaller than those of early cleavage stages. In Crepidula the chromosomal plate decreases in size in successive cleavages, though by no means uniformly; but at no time during the cleavage period do the chromosomes grow to their original size at the beginning of the cleavage. Boveri’s view, therefore, finds no support in the cell-divisions of the cleavage period. R. Hertwig (’03, ’08) finds the inciting cause of division in a ‘Kernplasma-Spannung,’ due to the unequal growth of nucleus and plasma: Die Kernplasma-Relation muss eine Verschiebung erfahren zuungun- sten des Kernes, es muss sich eine Kernplasma-Spannung entwickeln, welche allmihlich zunimmt, bis schliesschlich ein Grad erreicht wird, den ich friiher Kernplasma-Spannung in engeren Sinne genannt habe. In dieser Spannung erblicke ich die Ursache der Teilung. Ich nehme an, dass, wenn ein Héhepunkt der Kernplasma-Spannung erreicht wird, der Kern die Fahigkeit gewinnt, auf Kosten des Protoplasma zu wachsen, und das die hierbei sich vollziehenden Stoffumlagerungen zur Teilung der Zelle fiihren. Zum funktionellen Wachstum gesellt sich das Teilungs- wachstum des Kernes, um die Kernplasma-Norm wiederherzustellen.” (p. 20) Relative Zunahme der Kernsubstanz, gleichgiiltig, ob dieselbe durch Vergrésserung des Kerns bei gleichbleibender Protoplasmamenge oder Verringerung des Protoplasma bei gleichbleibender Kerngrésse her- beigefiihrt wird, miisste eine Verlangsamung der Teilung und im ersten Fall eine Steigerung der Teilgrésse zur Folge haben; umgekehrt miisste relative Abnahme der Kernmasse den Eintritt der Kernteilung be- schleunigen, die Teilgrésse herabsetzen (p. 23). Hertwig holds that his own work on Infusoria, and that of Gerassimoff on Spirogyra, show that an increase of nuclear mass leads to a slowing of divisions and an increase of the division size of the cell; and that the process of the segmentation of the animal egg shows that a great reduction of nuclear mass leads to a high degree of divisional activity. He says that many external and internal conditions influence the Kernplasma-Relation and he expresses the hope that his theory may not be cast aside because here and there a fact may be found which cannot be brought under it, without further consideration. As we have seen the Kernplasma-Relation varies widely in certain blastomeres of Crepidula and Fulgur. In these cases wide departures from the Kernplasma-Norm have not brought CELL SIZE AND NUCLEAR SIZE 31 on cell division, and if Kernplasma-Spannung is a cause of cell- division it must be a minor factor in this case. It seems to me probable from my observations and experiments on segmenting eggs, that the Kernplasma-Relation in these blastomeres is a re- sult rather than a cause of the rhythm of cell division, and that the factors which bring on cell division are to be found in some intrinsic condition in the nucleus or centrosome, rather than in the maintenance of a constant ratio of nuclear volume to cell volume. Support is lent to this view by the phenomena of odgenesis, for we have in the germinal vesicle the largest nucleus in the entire life cycle, following upon the longest resting period, while the second maturation division follows immediately upon the first, usually before a resting nucleus is formed. The long delay in the appearance of the first maturation division, as well as the short period intervening between the first and second maturation divisions, must both be attributed, as it seems to me, to intrinsic conditions in the cell, other than ‘Kernplasma-Spannung.’ In the cleavage of the egg the rate of division seems to depend, in part, on the quantity of protoplasm present. As long as a con- siderable quantity of plasma is present in the blastomeres the rate of division is rhythmical, but when the macromeres have given off almost all the plasma inthe formation of the three quartets of ecto- meres, a long resting period follows. The first of these macro- meres to divide, giving rise to the fourth quartet, is the one with the largest amount of plasma, viz., 3D, while the cells 3A—3C normally divide much later. However if, by centrifuging at the right stage, 3C is caused to contain more plasma than usual it may divide at the same time as 3D, as shown in fig. 37. The cells 4A-4D, in which the resting period is particularly long, contain very little plasma, and this appears to be absorbed by the nucleus almost as fast as it is formed. The micromere /d is slightly smaller than its fellows, /a-/c, and it divides later than the latter. The ‘turret’ cells, /a?-1d?, are the smallest cells in the egg, when they are formed, and they have the longest resting period. In spite of this evidence that the quantity of protoplasm has to do with the rate of division, there is other conflicting evidence which is hard to harmonise with it; thus, these same ‘turret’ cells, 32 EDWIN G. CONKLIN which are at first so small and have so long a resting period, be- come much larger than adjoining cells before they divide. R. Lillie (10) maintains that ‘‘the primary change in the initiation of cell division and development is an increase in the permeability of the plasma membrane.”’ It is well known that the general surface tension of the cell increases during mitosis, and I have found that the tension of the cell membrane is locally reduced at the two poles of the cell before and during division (see Conklin, 02, p. 94; also this paper, p. 82). It is quite possible that. this polar reduction in surface tension before mitosis begins may have something to do with initiating division. On the whole it seems probable that the time of cell division is dependent upon the coincidence of several more or less independ- ent factors. Boveri has shown that the division phases in nu- cleus and centrosome may be more or less independent of each other, though complete cell division depends upon the coincidence of the two. To these factors may, perhaps, be added the quantity of protoplasm, and thus indirectly the ‘Kernplasma-Relation’ and perhaps also increased permeability of the cell membrane, and a local reduction of surface tension at the poles of the cell. Gurwitsch (08) maintains that the blastomeres are ready for division at all times, and that only “Kernplasma-Koinzidenz’ or ‘Zustands-Koinzidenz,’ is necessary to start division. He sug- gests that a coincidence of polarity of nucleus and plasma may be necessary, and he concludes from the apparently accidental occurrence of divisions in different parts of an egg or embryo, that several independently variable factors may be concerned, the coincidence of which is necessary to bring on cell division. The latter part of this conclusion seems to me to be justified by the facts which I have presented. V. Growth of protoplasm during cleavage It is well known that the egg as a whole does not increase in vol- ume until after the cleavage period. Indeed Godlewski (’08) finds that there is in Echinus and in Strongylocentrotus, no change in the quantity of plasma at the 64-cell stage, as compared with the 2 CELL SIZE AND NUCLEAR SIZE 33 unsegmented egg; however, in the blastula there is an actual loss, the total volume of plasma being about one-third less than in the unsegmented egg; during this period the nuclear material has increased in volume at the expense of the plasma. Whether the plasma actually increases during cleavage at the expense of the yolk has not been determined, so far as I am aware, in any case. By means of the centrifuge it is possible to throw the yolk out of the egg before cleavage and during the early cleavage stages, leaving the plasma which can then be readily measured. In later cleavage stages I have not been able to throw the yolk out of the small blastomeres by means of the centrifuge; but on the other hand the protoplasm and yolk are normally segregated in these stages so that it is possible to determine the approximate dimensions of both without having recourse to the centrifuge. The following table gives the total maximum volumes of all the nuclei, protoplasm and yolk in the eggs of Crepidula plana at various cleavage stages. Following Popoff (’08), I have deter- mined the coefficients of growth of the nucleus and of the pro- toplasm for each stage; these coefficients are obtained by dividing the volume of a later stage by that of an earlier one, and they represent the growth in ‘times,’ or multiples of the initial quantity. In the first half of each column of coefficients the earlier stage is the one before maturation, while in the second half of each column it is the one before the first cleavage. The coefficient of growth of TABLE 8 Total maximum volumes of nuclei, protoplasm and yolk in the eggs and cleavage stages of Crepidula plana | | COEFFICI- | COHFFICIENTS| TOTAL Ife TOTAL | YOLUME OF STags NUCLEI Santana Ne zy eg os meee Sees aes ieee | is GROWTH GROWTH RELATION cubic uh | cubic mM | cubic u cubic Before ma- turation. . 32,409 | 97,131 1,625,460 1,755,000 1.0 1.0 1:3 Before first | cleavage. 21,375 121,480 | 1,346,105 | 1,488,910 0.65 1.0 1.25 | 1.0 15.6 2 cells...... 14,476 123,482 1,100,700 | 1,238,658 0.45 | 0.67 1.27 1.02 1:8.5 4cells...... 23,100 154,280 969,468 | 1,146,848 0.71 | 1.08 1.58 | 1.27 1:6.6 mmells:.. .... 25,144 164,136 972,320 | 1,161,600 0.77 | 1.17 1.68 1.35 1:6.5 16 cells..... 23,628 233,608 | 980,156 | 1,237,392 0.72) 1.10) 2.45 1.92 1:9.8 24 cells... .. 30,164 258,897 } 890,727 1,179,788 0.92 | 1.41 2.66 2.13 1:8.6 (231,000) | (1,151,891) (2.35) \(1.90)! (1: 7.7) THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. | 34 EDWIN G. CONKLIN any stage, less the coefficient of the initial stage, viz. unity, gives the percentage of growth of that stage, as compared with the ini- tial stage. Since the ectoderm at the 24-cell stage is a plate of purely protoplasmic cells, nearly square, about 80 on each side and 36u thick its volume is about 230,400 cubic y; subtracting the volumes of the nuclei of the plate, 21,584 cubic yp, leaves 208,816 cubie p as the volume of the cytoplasm! of the ectodermal plate. Adding to this the volume of the protoplasm in the macromeres 3A—3D, viz. 22,185 cubic yu, we have as the total volume of the proto- plasm at the 24-cell stage 231,000 cubic ». This figure is 27,897 cubic uv less than the volume of protoplasm at the 24-cell stage given in the table, which was calculated from the dimensions of each individual cell, rather than from those of the entire ecto- dermal plate. It is highly probable that the lower figure is nearer correct than the higher one, since minor errors in the measure- ments of individual cells are greatly magnified in determining the total volumes of these cells. The same remark applies to the total volume of protoplasm in the 16-cell stage, which is probably actually less than the volume given in the table; and if the total volume of the protoplasm is less than the amount given in the table the total volume of the yolk in these stages is of course increased correspondingly. But assuming that the smaller number (in brackets) represents the actual volume of the protoplasm in the 24-cell stage of Crepi- dula plana we must admit that there has been a great growth in the plasma at the expense of the yolk during the cleavage. The coefficient of protoplasmic growth (i.e., the volume of protoplasm of any stage divided by the volume of protoplasm of the stage just before maturation) is given in the next to the last column of the table; and a glance at this shows that the protoplasm at the 24-cell stage is at least 24 times as voluminous as in the maturation stage, while the yolk is correspondingly less voluminous. The volume of the entire egg, also, is considerably less in the 24-cell stage than at the beginning of development. Indeed there has been a gradual decrease in the volume of the entire egg during 'The words ‘cytoplasm’ and ‘protoplasm’ are used synonomously throughout this paper. CELL SIZE AND NUCLEAR SIZE 35 the early cleavages. These results show a general agreement with those of Godlewski. The growth of plasma at the expense of yolk during the matur- ation and the cleavage period, was shown to occur in my studies of the effects of centrifugal force on the eggs of Lymnaea and Physa (Conklin, 710). In the living eggs of these animals the substances may be stratified by centrifugal force into a gray (light) zone, a clear (middle) zone and a yellow (heavy) zone; the gray and clear zones constitute what I have here regarded as proto- plasm, while the yellow zone is in large part composed of yolk. “Before the first maturation the yellow substance composes at least one-half of the entire egg; just before the first cleavage it composes only about one-eighth of the egg. The clear and gray substances, which together constitute about one-half of the egg in the earlier period, form seven-eighths of the egg in the later period,” (p. 436). In the normal eggs of Lymnaea and Physa, which have not been centrifuged, the clear and yellow substances are easily recog- nizable, and the stages in the transformation of the latter into the former have been studied in the paper mentioned, from which the following summary is quoted: In the course of development, from the maturation of the egg to the gastrulation, the relative quantities of clear (plasma) and yellow sub- stance (yolk) are reversed. At the beginning the clear substance is small in quantity, and is chiefly visible in the germinal vesicle (though experiments show that some of it is distributed through the yellow sub- stance) and at this stage the entire cell body is yellow in color. With the establishment of the germinal layers the yellow substance is limited to the few cells constituting the endoderm and mesoderm, while all the rest of the embryo, by far the larger part, is composed of clear substance. This change in the relative quantities of these two substances is due in part to their separation and segregation during the course of develop- ment, but in much greater part to the transformation of yellow sub- stance (yolk) into the clear (plasma). It is a phenomenon of general occurrence among many animals that the clear protoplasm of the egg is very small in quantity before the dissolution of the germinal vesicle and that it gradually increases in quantity after that stage. This is doubtless due in large part to the dissolving of yolk and its conversion into clear protoplasm, and it is a significant fact that this process takes place most rapidly after the breaking down of the wall of the germinal vesicle and the escape of a large part of the nuclear contents into the cell body (p. 423). 36 EDWIN G. CONKLIN There are no eggs wholly without yolk and probably in all of them plasma is formed at the expense of yolk during the cleavage period. This probability is of great significance, for all studies which have had to do with the relative quantities of protoplasmic and nuclear materials during these early stages of development have dealt only with the entire cell contents without attempting to determine what part of this is plasma. In many cases, the great disproportion between cell volume and nuclear volume at the beginning of developemnt is due to the fact that a large part of the cell volume is made up of yolk; if the volume of the plasma only is compared with that of the nucleus it is found that the relative quantity of plasma is actually less at the beginning of development, than in the later cleavages, with the single excep- tion of those blastomeres which have unusually long resting periods. In Crepidula there is no excess of plasma over nuclear material in the early stages, in comparison with the later ones, as Minot and others have assumed, and the process of cleavage is not in this case a method of restoring the Kernplasma-Norm, or of rejuvenating senile cells, by an enormous increase of nuclear material as compared with the plasma. As a matter of fact the plasma increases almost as rapidly as the nuclear material during the cleavage of this egg, and even adult tissue cells have a Kernplasma-Relation but little different from that of the blastomeres, (see p. 25). VI. Rate of nuclear growth during cleavage It is well known that during cleavage there is usually no in- crease in the volume of the egg, but it is generally held that the increase in the nuclear substance is very great. In his book on “‘Age, Growth and Death” Minot (’08) says: ‘‘The nuclei multiply (in cleavage); they multiply at the expense of the pro- toplasm. ‘They take food from the material which is stored up in the ovum, nourish themselves by it, grow and multiply until they become the dominant part in the structure’ (p. 166). He suggests that this nuclear increase during cleavage is a process of rejuvenation, though he admits that the relative increase of nu- CELL SIZE AND NUCLEAR SIZE . 37 clear material as compared with protoplasmic may be prolonged beyond the period of segmentation (p. 167). But although he emphasizes the growth of the nuclear material as a whole during the cleavage, he specifically recognizes the fact that there is a rapid reduction in the size of individual nuclei in the early stages (pp. 174, 179). Hertwig (’03) also has emphasized this great growth of the nuclear material during the early stages of develop- ment. He says (p. 116): There is an enormous disproportion of nucleus and protoplasm at the beginning of cleavage, and this disproportion is gradually equalized by the transformation of cell substance into nuclear substance. The man- ner of this may be imagined by supposing that resting protoplasm con- tains chromatin and achromatic material and that at every cell division it is analysed into these constituents serving for the growth of the nucleus. Loeb (09) also has called attention to the doubling of nuclei at each division, with the consequent increase of nuclear material in a geometric ratio, and the resemblance which this bears to autokatalytic reactions. The great increase in the nuclear substances during cleavage has been commented upon by many writers, and the references cited have been chosen rather because of the theories which have been based upon this phenomenon than because they represent an unusual opinion as to the phenomenon itself. At the time when the following computations of the rate of nuclear growth during cleavage were made, I was unaware that anyone had made computations of a similar sort. Since my material afforded an unusually good opportunity for making such computations, I carefully measured the diameters of the germinal vesicle, of the ege and sperm nuclei, of all the nuclei up to the 24-cell stage, of those of the 42-cell stage, and of the 70-cell stage,—every nucleus being measured at its maximum size, so far as possible,—with the results given in the following tables. These results have been as surprising to me as they are likely to be to any of my readers. After this work was completed I became acquainted with the _work of Godlewski (08) and Frl. Erdmann (’08) on the sizes of nuclei and of individual chromosomes of the blastomeres of 38 EDWIN G. CONKLIN Echinus and Strongylocentrotus. Godlewsky found that from the 1-cell to the 64-cell stage the nuclear substance grows nearly in geometric ratio; from the 64-cell stage to the blastula, with about 1256 cells, there is little increase in the nuclear substance, but since he supposes that the number and size of the chromo- somes in the later stages remain the same as in the earlier ones, the nuclei must become richer in chromatin in the later stages. He finds that the volume of the plasma in the blastula stage is about one-third less than in the unsegmented egg and he con- siders that a large part of this lost plasma has been converted into chromatin. Erdmann (’08) has made a careful computation of the volume of the resting nuclei and of individual chromosomes in the early cleavage stages, and in the blastula and gastrula of Strongylocentrotus. She finds that the chromosomes of the pluteus period have only about one-fortieth the volume of those of the first spindle, but though the individual chromosomes grow smaller continually, the total nuclear volume increases at the expense of the plasma up to the late blastula stage. 1. Nuclear growth during the cleavage of the egg of Crepidula. The maximum, minimum and mean volumes of the nuclei at different stages of the cleavage of Crepidula plana are given in tables 3 to 5 and the coefficients of growth of all the nuclei are given in table 8. It remains only to summarize the facts there presented and to give the nuclear volumes and the rate of growth in certain later stages of the cleavage. This has been done in table 9, where the maximum, miminum and mean nuclear vol- umes of every nucleus from the 2-cell to the 32-cell stage is given, together with the coefficient of growth for each stage. Since this table starts with the 2-cell stage the coefficients of growth are different from those given in table 8, where subsequent stages are compared with the germinal vesicle or with the germ nuclei. For the purpose of determining the usual rate of growth for each cycle of cell division during the cleavage it is desirable to start with the 2-cell stage. The germinal vesicle is an extraordinarily large nucleus, and since two nuclei are present in the egg before the first cleavage the nuclear condition at this stage is unusual; on this account the rate of nuclear growth during cleavage is CELL SIZE AND NUCLEAR SIZE 39 TABLE 9 Rate of nuclear growth during the cleavage of Crepidula plana == MAXIMUM (COEFFICIENT MEAN COEFFICIENT) MINIMUM (COEFFICIENT STAGE BLASTOMERES NUCLEAR OF NUCLEAR OF | NUCLEAR OF VOLUMES | GROWTH | VOLUMES | GROWTH | VOLUMES | GROWTH | | cubic cubic cubic bw SROGUA PAIS, CD iiss ccvenencae 14,476 1.0 6,110 1.0 381 1.0 cells A) B,C, D........... 23,100 1.6 8,580 1.4 602.4 1.58 8 cells DAUD) bere cc orcistac oiniac'e 19,396 7,068 338.4 NA ec detsjarsio sivas pi0:a.< 5,748 3 620 338.4 25,144 1.73 10.688 1.74 676.8 1.77 BOD Wiese sisies cav's's 12,220 7,068 338.4 7,068 3,620 338.4 po eicalls 3,620 1,528 235.2 720 452 ‘ 235.2 23,628 1.63 12,668 | 2.07 1147.2 3.01 8,580 5,748 150.0 5,748 3,620 150.0 24 cells 5,748 3,620 150.0 5,748 3,620 150.0 3,620 1,528 235.2 720 452 235.2 30,164 2.08 18,588 3.04 1070.4 2.80 4A-4D 12,220* 7,068 171.3 A ein races sieieie'n cae 697 382 58.8 MBACP ataercle 'claic aes» 2,715 1,146 | 112.5 BOO bedieoe Sane eee 5,748 3,620 150.0 32 cells ; 2a!-2d!.............. 5,748 3,620 | 150.0 5,748 3,620 | | 150.0 3,620 452 150.0 lat.2-1dl.2......, re 2,095 3,620 150.0 Jat1d?i..isci0% Go 720 452 235.2 39,311 2.71 23,980 3.92 | 1327.8 3.48 Total growth in thirty divi- PNOUE sh rata'ainlek pialalsluteterersie ere ats 24,835 2.715 17,870 3.92 946.8 3.48 Average growth for each diyi- WW Wanita canannposa acer Sinod | MELAS 1.05(=5%)| 595.6 1.09(=9%) 31.5 1.08(=8%) * This volume is reached only at a much later stage, shortly before the closure of the blastopore (fig. 6). During this same period from the 2-cell to the 32-cell stage the coefficient of growth of maximum nuclear surfaces is 4.28, or an average increase of about 11 per cent for each division. 40 EDWIN G. CONKLIN best determined by comparing subsequent stages with the 2-cell stage. Furthermore the nuclear volume in the 2-cell stage is less than at any other stage, and it consequently forms a good starting point for the study of nuclear growth. Finally, the volume of all the nuclei in the 70-cell stage, without attempting to determine the maximum volume of each nucleus, is shown in table 10. At the 70-cell stage the ectomeres are already closing over the yolk on the oral hemisphere, and it may be assumed that the cleavage will show no new tendencies as to the growth of nuclear substance until the embryo as a whole begins to grow. Whether nuclei are measured at either their maximum size, their minimum size or at a size intermediate between these two extremes, the rate of growth during cleavage is found to fall far short of a doubling or increase of 100 per cent at each division. The average nuclear growth during early cleavage is not more than 5 to 9 per cent for each division, and in the later cleavage it falls as low as 1 per cent for each division. A growth of nuclear substance at this rate scarcely deserves to be designated as ‘phe- nomenal’ or ‘colossal.’ On the other hand, the protoplasm which is generally supposed to remain fixed in quantity during cleavage, increases at a more rapid rate than the nuclei, from the 1-cell to the 24-cell stages, as shown in table 8. In view of the facts here presented, even though it be for only a single species, the generally accepted conclusion as to the great increase of nuclear substance during cleavage, as contrasted with the lack of growth of the protoplasm, evidently needs revision, as do also the theories which have been founded upon this supposed fact. 2. Nuclear growth during the cleavage of the egg of Fulgur. While my results are based largely upon the study of Crepidula plana they are not limited entirely to this species. The fol- lowing measurements of the nuclei of Fulgur carica are prob- ably not very accurate since they had to be made under a rela- tively low power objective (8 mm. apochromat) and since the material at my command did not permit the study of a large num- ber of eggs, and the selection of nuclei at maximum size. Never- ——o CELL SIZE AND NUCLEAR SIZE ‘ 41 TABLE i0 Actual nuclear diameters and volumes in the 70-cell stage of Crepidula plana | COEFFICIENT OF GROWTH | NUCLEAR TOTAL NUCLEAR JEL 2 DIAMETER VOLUME Ni aaleas Wicker, volume surfaces rm Be PHC CMISUHPC REE eee rcs fos canes beaacs| A 14,476 1 1 (225290 oe See ae 16 8,579 (leh eo are | 10 1,571 11 Entomeres El, E2., aes 7 359 Git CE ok 5 See Been 6 226 UNI SY EE aes ee 0) 1,047 eer enomeres ae 7 Sane eee 9 763 First quartet 4 Apicals, lat-“-Id'). 00, 10 2,094 3 Basals, lat-?--1¢!-?1,... 2... 9 1,145 1 Basal, 1G LC, 2 cons ees cae } > a2 905 3 Middles, la!:?-2-1e!-22,. 02... | 12 2,714 Aeiimrete; Vaz ld*, oo... ee ww el 7 718 Second quartet a 3 Tip cells, 2al-2et!.. 2... | 6 339 © |1 Tip cell, Bite IO onal A RA | 10 524 § (4 Girdle cells, 2a!-?-2d'-?1........| 9 1,527 2 4 Girdle cells, 2a!-?->2d!-?-2,.......! 10 2,094 f) |4 Girdle cells, 2a2-1--2d?-1-1.... 2... a 1,527 19 | 4 Girdle cells, 2a?1-2-2d?1-2,.......| 9 1,527 4 Girdle cells, 2a?-2-2d?"?,..........| 5 262 Third quartet 4 Girdle cells, 3a!--3d!"!........... 10 2,094 4 Girdle cells, 8a!-2-3d!? .......... 9 1,527 4 Girdle cells, 3a?-8d?............ 6 452 | 4 Girdle cells, 3a?-*-3d2"?........... 6 452 70 cells. Total nuclear volume........ 32,446 2.24 5.30 The total volume of these 70 nuclei is almost exactly the same as the volume of the germinal vesicle, about 50 per cent more than the volume of the germ nu- clei, and 35 per cent more than the mean nuclear volume of the 32-cell stage, with which mean volume, rather than with the maximum, this actual volume of the nuclei of the 70-cell stage should be compared. In the 38 nuclear divisions lead- ing from the 32-cell stage to the 70-cell stage the nuclear material has increased at an average rate of less than 1 per cent for each division. 42 EDWIN G. CONKLIN TABLE 11 Diameters and volumes of the nuclei, 2-cell to 16-cell stages of Fulgur carica STAGE BLASTOMERES TNT CERTE Eman NMS ccleree eee tercsceteteetre eee E ML | Baan i SicellseA BC Die eenee eeeenee 40 67,020 | 1.0 AcellshACiBe CAD ee ene 32 | 68,628 1.02 | | Seige CASED Aen ee 32 68,628 la-Id 16 8,576 | 77,204 1.15 2A-2D... < aE 32 | 68,628 f2'celisiy 2a-2d) ne ee eee 16 8,576 laid: cee ee 16 | 8,576 | 85,780 1.28 | | 3423) pe ee 32 | 68,628 T& cali’) 98-04 o> een eee eee 16 | 8,576 Sa-0d: «ee ee 16 | 8,576 (etd. eee 16 | 8,576 | 94,356 1.40 theless they indicate the general rate of nuclear growth in this prosobranch, In fourteen nuclear divisions there has been an increase in neuclear substance of 40 per cent, or an average increase for each division of 2.8 per cent. The rate of nuclear growth is practically the same in the other species of Crepidula as in C. plana; and in all prosobranchs the nuclear material increases but slightly during the cleavage period. 3. Nuclear growth during the cleavage of other animals. From a casual examination of the segmenting eggs of nematodes, echin- oderms, amphioxus and ascidians, as well as from a study of the figures of various authors, it is evident that the nuclear growth in these forms is greater during the early cleavages than in the gastropods. In all of these forms the germinal vesicle is relatively much larger and the egg and sperm nuclei much smaller than in the gastropods, while the decrease in nuclear size in the early CELL SIZE AND NUCLEAR SIZE 43 cleavages is not so marked as in the gastropods, though of neces- sity the nuclei must grow smaller in all animals as cleavage progresses. In the ascidian, Styela (Cynthia) partita, the maximum nuclear diameters and volumes in the different cell generations are shown in table 12: TABLE 12 Maximum nuclear diameters and volumes in Styela (Cynthia) partita 1 T | | COEFFICIENTS OF GROWTH AVERAGE DIAMETER TOTAL VOLUME - ——— —————— EO} NUCLEUS OF NUCLEI — Nuclear volume pune “ cubic Before first ma- furation.......| 54 82,448 1.0 Before first cleavage....... 912+ 712 1,809 0.02 | 1.0 } aTNES a Aces l6u 4,289 0.05 |) 2.37 1.0 1.0 chiGIEURE @ paeeee 14 5,748 0.06 3.17 1.34 PAGEL R ee sere idly oc iW 3 9,203 0.11 | 5.08 2.14 TGicelleva. ss... Ml 11,1738 0.13 6.17 2.60 BEICOUIB es cscs + 10 16,755 0.20 | 9.26 3.90 64cells.......... 8 17,152 0.20 | 9.48 4.00 | WARIGEIIS: cine)eea0\<- 6.5 18,406 0.22 10.17 4.29 POOCEUS. 0.200. 5.25 19,395 0.23 10.72 4.52 | 1G) The nuclei of different blastomeres of the same generation vary considerably in size, and I have not attempted to measure each individually, as in the case of Crepidula, nevertheless the measurements given represent approximately the average nuclear diameters for each generation of blastomeres. When the cells become very numerous a very slight error in the measurement makes a big difference in the results, and the total nuclear volume in the later stages may not be very accurate. Nevertheless the table does give a true idea of the order of magnitude of the nuclei in the different generations. In comparing this table with those for Crepidula it will be seen at once that the germinal vesicle is relatively larger, the germ nuclei smaller and the growth of the nuclear material in the early stages greater in Styela than in Crepidula. The volume of the egg and sperm nuclei represents a loss of 98 per cent as com- 44 EDWIN G. CONKLIN pared with that of the germinal vesicle; and even in the 256-cell stage the volume of all the nuclei is 77 per cent less than that of the germinal vesicle. Comparing the nuclear volumes of subse- quent stages with that of the germ nuclei, we find that up to the 32-cell stage there is an increase of 826 per cent, or an average for the first 31 nuclear divisions of 26 per cent for each division; from the 32-cell stage to the 256-cell stage there is an increase of 146 per cent, or an average increase of 0.6 per cent for each divi- sion. Since the germinal vesicle is unusually large and the germ nuclei unusually small, a better idea of the rate of nuclear growth in the egg will be obtained by comparing the nuclear volumes of later stages with that of the two cell stage, as was done in the case of Crepidula. Such a comparison is given in the last column of Coefficients in table 13. From this it appears that the nuclear growth from the 2-cell stage to the 32-cell stage is 290 per cent or an average increase for each of 30 divisions of 9.6 per cent; from the 32-cell stage to the 256-cell stage the nuclear volume in- creases 62 per cent, or an average increase for 224 divisions of 0.27 per cent for each division. In the cleavage of the eggs of amphioxus and of echinoderms the rate of nuclear growth is essentially similar to that of the ascidians. Here also the germinal vesicle is very large and the total volume of the nuclei at the close of cleavage is much less than the volume of the germinal vesicle, though decidedly greater than the volume of the germ nuclei at the beginning of cleavage. In all of these cases the nuclei in the early cleavages contain little chromatin and much achromatin; while they are more densely chromatic in the later stages, showing that the chromatin has increased in quantity relatively more than the achromatin. This is probably due to the fact that the chromosomes take up less cytoplasmic substance in the smaller cells than in the larger ones, the amount of achromatin in the nucleus depending in aE part upeE the quantity of cytoplasm in the cell. . Growth of different nuclear constituents. a. Nuclear sap. ii of the substances within a nucleus do not increase at the same rate. The most abundant constituent of a fully formed nucleus is nuclear sap, and this is scarcely present at all in the earliest stages of the nuclear cycle. During each resting period the nu- CELL SIZE AND NUCLEAR SIZE 45 clear sap increases in amount from zero until it forms the principal bulk of the nucleus, and when mitosis comes on it passes into the cell body, and as a constituent of the nucleus sinks again to zero. The substance which forms the nuclear sap is absorbed by the nucleus from the cell body throughout the whole of the resting period, only to be thrown out into the cell body again at the end of that period. Consequently the nuclear sap is no more a nuclear constituent than a protoplasmic one, belonging to both nucleus and protoplasm. Studies on the growth of nuclear material should therefore be confined to the growth of the chromatin, but the difficulty of measuring the amount of chromatin at different stages will be appreciated without further comment. Also the fact that so large a part of the nuclear material belongs also to the protoplasni should be taken into account in experiments dealing with the isolation of nuclei from protoplasm; evidently the only satisfactory way in which such isolation can be accom- plished is by isolating chromosomes, rather than resting nuclei. There is good reason for believing that the nuclear sap contrib- utes to the nourishment and growth of the chromatin and linin, and that it in turn receives substances from these, so that the materials which pass into the cell body when the nuclear mem- brane dissolves, are not wholly the same as those which were taken up by the nucleus from the cell body. I have elsewhere (02) called attention to the fact that the escaping nuclear sap stains more deeply than the cell protoplasm and may therefore be called ‘chromatic sap.’ As to the mechanism of this intake of protoplasmic substance into the nucleus there is every visible evidence that it is of the nature of osmosis. The nucleus becomes spherical in shape un- less subjected to outside pressure, or to the action of substances which cause plasmolysis. The nuclear membrane remains entire and distinct until the last phase of nuclear growth, immediately preceding mitosis, when the nucleus swells very rapidly and the nuclear membrane becomes thin and then disappears. The measurements given in the preceding section show that the total quantity of the more fluid part of the nucleus, the nuclear 2Watase (1893) says,—-‘“The structure known as the nucleus contains a great deal of cytoplasmic substance.” 46 EDWIN G. CONKLIN sap, does not increase in quantity during the cleavage of the egg; we have seen that the total volume of all the nuclei of Crepidula at the 70-cell stage is about equal to that of the germinal vesicle, while in Styela the volume of all the nuclei at the 256-cell stage is 77 per cent less than the volume of the germinal vesicle. The conclusion is justified, therefore, that the more fluid constituent of the nucleus decreases greatly in volume during the early cleavage stages, and that the nuclei therefore become denser during this period. b. Linin. Just as the nuclear sap is proportional in volume to the volume of the nucleus as a whole, so also it is evident that the linin is more abundant in large nuclei than in small ones. Evi- dently it is not possible to determine the volume of linin in a resting nucleus, but since the spindle fibers are composed largely of linin it is possible by measuring the size of spindles to determine, at least in a general way, the relative quantities of linin in different nuclei. In the following table the length of the spindle from TABLE 13 Length of spindle in the maturation and cleavage of Crepidula plana _ DIAMETER OF PRECEDING STAGE LENGTH OF SPINDLE NUCLEUS I Me irstimaturationie +.) eee 42 42 Second maturation............ 18 = Hirsticleavageseoes eee eee 30 34.5 Second cleavage, AB, CD...... 30 24 Third cleavage, A, B,C, D.... 27 22 Fourth cleavage, 1A-1D.... 25 21 Fourth cleavage la-ld......... 21 12 Fifth cleavage 2A-2D......... 24 18 21 15 Fifth cleavage 2a-2d.......... centrosome to centrosome is given for successive cleavages of C. plana, the measurements being made in each case in the stage of the metaphase. The diameter of the nucleus is also given for comparison with the spindle length (table 13). In general the diameter of the spindle at its equator is, in the prophase and metaphase, about the same as the diameter of the nucleus from which it came. Spindles in the protoplasmic ecto- meres are relatively larger than the size of the nucleus would lead CELL SIZE AND NUCLEAR SIZE 47 one to expect and this probably is due to the fact, which I (’05) have established in the ascidians, that the polar partsof the spindle are not derived from the nucleus but from the protoplasm. With this proviso, it is true that, within the same species, large nuclei give rise to larger spindles than do small ones and this may be held to indicate that the linin is more abundant in the former than in the latter. The fact that the spindle fibers of ascidians are composed of equatorial and polar parts, the former derived from the nucleus and the latter from the protoplasm, and the fact that these two portions of the spindle, and also the polar fibers, are fundamentally alike, indicates that the linin, like the nuclear sap, is a constit- uent which belongs both to the nucleus and to the protoplasm. ce. Chromatin. The amount of chromatin undoubtedly in- creases during the cleavage; the resting nuclei in the later stages being more densely chromatic than those of the earlier stages. In each cell the chromatin is smallest in quantity when the daugh- ter chromosomes are first separated, and it grows in quantity during the resting period. Not all of the chromatin of the resting stage goes into the formation of the chromosomes of the next mitosis, but some of it in the form of granules (oxychromatin) or chromatic sap escapes into the cell body on the dissolution of the nuclear membrane. The larger the nucleus is and the longer the resting period through which it has come, the greater the quantity of chromatin which thus escapes at mitosis. Gardiner (98) estimated that the amount of chromatin which thus escaped into the cell body at the first maturation division of Polychaerus was five hundred times as great as that which went to form chromosomes, and conditions are similar in Styela, Crepidula, and many other forms. Consequently the volume of the chromo- somes in successive stages cannot be used as a measure of the growth of the chromatin. Nevertheless the growth of the chro- mosomal mass, as well as the growth of the entire nuclear volume, will give some idea as to the growth of the chromatin during cleav- age. Table 9, giving as it does the volumes of the nuclei and chromosomal plates at various stages, furnishes data upon which an opinion as to the growth of the chromatin of the resting stages 48 EDWIN G. CONKLIN may be based. From the 2-cell to the 32-cell stages the growth in volume of the resting nuclei lies between 171 per cent for maximum nuclear size, and 292 per cent for mean nuclear size while the growth of the chromosomal plates is 248 per cent. It seems very probable therefore that the growth of the chromatin during these stages lies somewhere between 171 per cent and 292 per cent, or an average increase for each of the 30 divisions rep- resented of from 5.7 per cent to 9.7 per cent. In all cases the growth of the chromatin falls far short of 100 per cent, or a doub- ling, in each division cycle. In Strongylocentrotus, Erdmann (08) finds that the ratio of chromatin to plasma is seven times greater in the pluteus than at the beginning of development, and she points out that this means that plasma contributes to the growth of the chromatin. While the chromatin as such is peculiar to the nucleus, there can be no doubt that large quantities of chromatin escape into the protoplasm. Such chromatin usually loses its distinctive staining reaction and presumably suffers chemical change. On the other hand we know that chromatin grows at the expense of substances received from the protoplasm. The work of Masing (10) on the nucleinie acid content of the egg indicates that this important constituent of chromatin is about as abundant in early stages as in later ones; he supposes that it exists in the pro- toplasm. d. Chromosomes. What is true of the quantitative relations of the chromatin as a whole is true also of the individual chromo- somes; those formed from large nuclei are larger than those from small ones; the chromosomes do not double in volume in each successive cleavage, but they become individually smaller as cleavage progresses. These facts are not difficult to demonstrate, but they are difficult to express in any numerical proportion, owing to the irregular shape and small size of the chromosomes, which make it very difficult to determine their volume. In Crepidula the chromosomes are very small and numerous, the full number being probably 60, and they are usually crowded together so that it is difficult to photograph them, or even to draw their outlines accurately, and since they are so small it is CELL SIZE AND NUCLEAR SIZE 49 not practicable to measure them directly with the 1/1 micrometer eyepiece. Nevertheless by selecting sections in which only a part of the chromosomes are shown I have been able to sketch the outlines of many of them with what I believe to be substan- tial accuracy. For the purpose of comparing the sizes of chromo- somes from different cleavages I have chosen two generations of blastomeres in which the difference in the size of the nuclei is at a maximum, the nucleus in one cell being about twice the di- ameter of that in the other; these blastomeres are the macromeres AB and CD, and the micromeres 1/a—/d (figs. 7 and 8). In the former the diameter of the nucleus just before division is about 24u, in the latter about 14u. When the nuclei of the cells in question had begun to divide and the mitotic figures were in the equatorial plate stage, the chromosomes from a number of these spindles were drawn as accurately as possible with a camera lucida. In order to be certain that the stage of division was the same in each case only longitudinal sections through the spindle were chosen; and in order to avoid as far as possible individual differences in the sizes of chromosomes, only the largest and most isolated chromosomes were drawn. Fig. 9 shows chromosomes from four different spindles of the second cleavage; fig 10 shows chromosomes from the first division of the first quartet cells (1a-1d), also from four different spindles. In all cases the chro- mosomes are magnified 2000 diameters. It is plain from these figures that the chromosomes from the larger nuclei are larger than those from the smaller ones, though the difference in the diameters and volumes of the chromosomes are not as great as the difference in the volumes of the nuclei from which they came. The average volume of the chromosomes from the large nuclei is about 5.2 cubic » and of those from the small nuclei about 2.6 cubic x. While the volumes of the nuclei as a whole are to each other about as 5 : 1, the volumes of their individual chromosomes are to each other as 2:1. In the case of nuclei which differ but slightly in volume it is not possible to be certain that the chromosomes differ in size, but in all cases in which the differences in the size of nuclei is considerable it can THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 50 EDWIN G. CONKLIN be seen that the larger nuclei give rise to larger chromosomes than do the smaller ones. Since the probable error is much greater in the measurement of individual chromosomes than of whole chromosomal plates, I have not attempted to measure individual chromosomes in each stage of the cleavage; on the other hand the dimensions of the chromosomal plates are given in table 4 for each cell up to the 32-cell stage. These measurements show that from the 2-cell to the 32-cell stage the chromosomal mass increases in volume 248 per cent or an average of 8 per cent for each of 30 divisions. The chromosomal plates, and consequently the individual chro- mosomes, grow smaller as cleavage advances, but in the same gen- eration of cells small nuclei have smaller chromosomes than large ones. In short, the size of the chromosome is dependent upon the size of the nucleus from which it comes, rather than upon the cell generation to which it belongs. In the main these observations are in harmony with those of Erdmann, and Baltzer, to which reference has already been made. In Crepidula, as in the echinids studied by the authors named, the individual chromosomes grow smaller as the cleavage advances, but this is causally related to the decrease in the size of the nuclei and of the cells, and where, in later cleavage stages, the nuclei and cells remain large, there the chromosomes also are larger than in smaller sister cells. Just as the size of the nucleus is con- nected with the volume of the cytoplasm in which it lies, so the size of the chromosomes is connected with the volume of the nucleus from which they come. Montgomery (’10) has found that the sperm cells of Euschistus are of two sizes and he concludes (p. 127), that “‘it is probable that the large sperm possess no more chromatin than the small, though the heads in the former are much larger. The dimegaly expresses itself accordingly in differences of amount of karyolymph and of the substance (linin) that composes the mantle fibers, but much more markedly in the amount of cytoplasm.” He finds also that the mitochondria (idiozome) increase directly with the amount of cytoplasm. According to my observations chromo- somes from large nuclei are larger than those from small ones of CELL SIZE AND NUCLEAR SIZE 51 the same generation, though naturally it is more difficult to detect size differences in objects as small as chromosomes than in entire nuclei. Where the differences in nuclear volumes are great one can always detect corresponding differences in chromosome vol- umes. The chromosomes of the spermatid are usually smaller than those of the o6tid, but when the chromosomes of the first cleavage spindle appear, those from the sperm nucleus are usually as large as those from the egg. The reason for this is to be found in the fact that both grow, after fertilization, in the same medium, the egg plasma, and for approximately the same length of time. e. Plasmasomes. The conclusion that large nuclei have large chromosomes, and vice versa, also applies to the sizes of nucleoli (plasmasomes) ; they are larger in large nuclei than in small ones. However in this case another factor is involved for the size of nucleoli is not only dependent upon the size of the nucleus, but also upon the length of the resting period; indeed the latter seems to be the more important factor of the two. The largest of all nucleoli is the one found in the germinal vesicle, at the close of the longest resting period in the entire life cycle. In these gasteropod eggs the next largest nucleoli are found in the cells 44—4D and 4a—4e (fig. 6) in which the resting stage is particularly long. The nuclei of the cells 44—4D are of the same size as those of 2A—2D viz. 184 in diameter, but the nucleoli of the former have about three times the diameter of those of the latter. In earlier stages of cleavage where the blastomeres are dividing rapidly it is difficult to compare the sizes of nucleoli, not only because their number varies considerably, but also because each plasmasome is usually surrounded by a layer of chromatin gran- ules which renders exact measurements difficult. The number of plasmasomes appears to depend to a large extent upon the degree of fusion of an originally large number of separate plasmasomes. When chromosomes are isolated so that each gives rise to a dis- tinct vesicle, each may contain a minute plasmasome, and there may be as many of these as there are chromosomal vesicles. In Crepidula the number is always greatest during the earlier stages of the resting period; during the later stages they appear to fuse by EDWIN G. CONKLIN together becoming fewer and larger as the individual chromosomal vesicles fuse. For a considerable period two nucleoli are com- monly found in each nucleus, one in each gonomere, or nuclear half. However, when the resting stage is long, these two fuse into a single large plasmasome. While the nucleus continues to grow in size up to the time of the dissolution of the nuclear membrane, the plasmasome usually disappears before the formation of the spireme. In comparing the relative sizes of nucleoli it is important to compare corre- sponding stages; accordingly in my measurements they were measured when they had reached approximately their maximum size, and before the nucleus had reached its maximum. Nucleoli differ more or less in size even in different cells of the same genera- tion, owing perhaps to the more or less complete fusion of the many original nucleoli; it is significant in this connection that after a long resting period they are much more uniform in size and constant in number than when the resting period is short. The following table gives the diameters of nuclei and nucleoli (plasmasomes) in various blastomeres of Crepidula: TABLE 14 Mazimum nucleolar size and nuclear size in the blastomeres of Crepidula plana DIAMETER VOLUME NUMBER DIAMETER VOLUME NUCLEAR- STAGE BLASTOMERES OF OF OF OF OF NUCLEOLAR NUCLEUS NUCLEUS NUCLEOLI NUCLEOLI NUCLEOLI RATIO “ cubic i cubic lcell,beforematuration 42 32,409 1 12 905.0} 35:1 2 cells, AB, €D..... 20 4,189 2 3 28.0 | 149 :1 A:cells ANB Cu peer 15 1,767 2 21, 13 TT \\220 a 8 cells f1A-1D. eee) © 719) 3,591 2 gy 28.0 | 128 :1 i Wales oe 5 52 13 1,150 2 2,13 7.0} 264551 .. Uf DAE ID Perse 14 1,437 | 2 2 8.3 | 180 31 12cells 4 “ ie F (2a-2drer see 15 1,767 2 2 8.3 | 220 21 . (et Sldieenee 12 905 2 Bh 133)) 2OORea 1G celle eee 7 130 2 1 1.0 180:1 r (3A-3D. 15 1,767 1 73 221.0 Sol 2 cele aeode 15 1,767 | 2 3 28.0 63:1 25 cells Adie ee 9 382 1 3 14:0))) 2731 32 cells, 4a-4e.......... 13 905 1 6 113.0 Seal Ca. 100 cells, 4A-4D.... 15 1,767 1 9 382.0 | 4.6 :1 Fulgur carica: Ca. 1000 cells, 4A-4D 96 462,192 1 27 10306.0 44:1 CELL SIZE AND NUCLEAR SIZE 53 In eggs in which the nuclear division has been greatly delayed, if not entirely stopped, by the use of hypertonic salt solutions the nucleoli become much larger than in normal eggs. Thus in the eggs of Crepidula plana treated with 4 per cent NaCl solution for two hours, and then put into normal sea water for six hours, the sizes of nuclei and nucleoli are as follows: TABLE 15 Nucleolar size and nuclear size in eggs of Crepidula plana in hypertonic sea water DIAMETER VOLUME NUMBER DIAMETER VOLUME NUCLEAR- STAGE BLASTOMERES OF OF OF oF OF NUCLEOLAK NUCLEUS NUCLEUS NUCLEOLI NUCLEOLI NUCLEOLI RATIO “ cubic js “ cubic ph . 1cell, pronuclei...... {|e 24 eee - “2 aS oa id 21 4,849 1 10 524 9:1 Dicdllg A BsCD..ss0.0.. 24 7,238 1 9 382 19:1 4cells, A, B,C,D...... 15 1,767 1 9 BRP ae Gia BcelswtA-lD.s.......| 18 3,055 1 Oye eesse S31 The great size of the single nucleolus in each of these nuclei is probably due to the fact that division has been delayed and the resting period prolonged. f. Centrosomes and spheres. Finally we may consider in this connection the sizes of centrosomes, and spheres though they are not parts of the nucleus. In general in Crepidula, large cells contain large centrosomes and spheres, while small cells contain small ones. The maximum diameters of centrosomes in the cleavage of C. plana, vary from 2u to 7u, the measurements being made during the telophase of division. The maximum diameters of the sharply defined spheres, during the resting stages, vary from 5u to 12u; and in all cases, so far as I have observed, the largest centrosomes and spheres occur in the cells which have the largest amount of protoplasm, while the smallest occur in the cells with the least amount of protoplasm. The centrosomes and spheres are the cell constituents which first become unequal in an unequal cell division. As soon as the spindle becomes eccentric, the centrosome and sphere which lies farthest from the center of the cell becomes smaller than the one 54 EDWIN G. CONKLIN at the opposite pole. Only after the division wall forms do the daughter nuclei become unequal. 5. Conclusions as to nuclear growth during cleavage. The rate and amount of nuclear growth during cleavage is much less than is generally believed. Whether the nuclear volume is taken when the nuclei are at their maximum, mean, or minimum size, the nuclear growth is far from 100 per cent, or a doubling, in each division. In Crepidula the nuclear growth is not more than 5 per cent to 9 per cent for each division from the 2-cell to the 32-cell stage, and less than 1 per cent for each division after the 32-cell stage. At the 2-cell stage the nuclear volume is least and up to the 32-cell stage the chromatin increases at an average rate of about 8 per cent for each division. The stage when the volume of protoplasm is least, after the egg has reached its full size, is just before the first maturation division; between the first maturation and the 24- cell stage the protoplasm increases at an average rate of nearly 6 per cent for each division. At the end of cleavage the ratio of nuclear material to protoplasmic differs but little from the ratio at the beginning. In Fulgur the nuclear growth from the 2-cell stage to the 16-cell stage averages only 2.8 per cent for each divi- sion, and the general Kernplasma-Relation remains unchanged. In Styela the nuclear growth from the 2-cell to the 32-cell stage averages 9.6 per cent for each division; from the 32-cell stage to the 256-cell stage it averages only 0.27 per cent for each division. Such a rate of growth is not significant and indicates that the meaning of cleavage is to be found in something other than the increase of nuclear material as compared with the plasma. In general the growth of each of the different nuclear constit- uents parallels the growth of the nuclear material as a whole, though this is not true of the nuclear sap, which belongs to both cytoplasm and nucleus. During cleavage the fluid content of the egg as a whole decreases, the o6plasm becoming more consistent in later stages than in earlier ones. The total fluid content of the nuclei in the early cleavage stages is much less than that of the germinal vesicle; even in the later cleavages the nuclear sap is not so abundant, in some animals, as in the germinal vesicle. In Crepidula the volume of all the nuclei at the 70-cell stage is CELL SIZE AND NUCLEAR SIZE 55 only equal to that of the germinal vesicle, though the volume of the chromosomal plates has increased 250 per cent; in Styela the volume of all the nuclei of the 256-cell stage is 77 per cent less than that of the germinal vesicle, though the total chromoso- mal volume has increased many fold during this period. Linin is a nuclear constituent which is found also in the proto- plasm, and during cleavage it grows in quantity at about the same rate as the nuclear and protoplasmic materials as a whole. The polar parts of the spindle and the astral rays arise in the pro- toplasm outside the nucleus, while the equatorial portion of the spindle comes from the nucleus, as is shown with great clearness in the cleavage mitoses of ascidians. Correspondingly the size of the spindle is a resultant of the volume of the nucleus and of the protoplasm. Chromatin is more distinctively a nuclear substance than the nuclear sap or linin, though it undoubtedly grows at the expense of substance received from the protoplasm and in turn contributes material to the protoplasm. From the 2-cell to the 32-cell stage in Crepidula the growth, of the chromatin amounts to between 6 per cent and 10 per cent for each division, and as the fluid con- tents of the nuclei do not increase during cleavage the nuclei become more chromatic in later stages than in earlier ones. Chromosomal material, as represented in the condensed chro- mosomal plates of the anaphase, increases in volume 248 per cent from the 2-cell to the 32-cell stages of Crepidula, or an average growth of about 8 per cent for each division. Individual chromo- somes grow smaller as cleavage advances, but this is due to the smaller size of the nuclei from which they come rather than to the cell generation to which they belong; nuclei of the same genera- tion which differ greatly in size produce chromosomes which differ in size, the larger nucleus producing larger chromosomes than the smaller one. In the blastomeres of Crepidula the size and number of nucleoli (plasmasomes) are influenced by the size of the nucleus and the length of the resting period. In most of the nuclei there are two nucleoli, but when the resting period is long, these fuse into a single one. In experiments, anything which prolongs the resting 56 EDWIN G. CONKLIN period leads to an increase in the size of the nucleoli. During the normal cleavage of Crepidula the ratio of the nuclear volume to the nucleolar volume varies from 220 :1 to 4.6 :1. Centrosomes and spheres are proportional in size to the volume of the protoplasm in which they lie; they are always larger in large cells than in small ones and hence they grow progressively smaller as cleavage advances. In general the volume of each of the nuclear constituents named is influenced by the volume of protoplasm of the cell, and by the length of the resting period. The protoplasm contributes sub- stances to the growth of each of these constituents, and the more abundant it is the larger they grow, provided the period of growth is the same in all cases. Where the growth peroid (interkinesis) is very long the nuclei becomes unusually large and may ulti- mately absorb the greater part of the protoplasm. 6. Comparison of growth of chromatin with increase of chemical substances and processes during cleavage. Loeb in several import- ant papers has shown that the nucleus is the oxidizing center of the cell, and that the chromatin is chiefly concerned in bringing about oxidations. Warburg (’08) found the oxidative power of the egg to increase at a relatively slow rate during cleavage. More recently, in view of the oft-repeated assertion that the chro- matin doubles at each division, Loeb (’09) concluded that the supposed growth of chromatin in geometric ratio indicates that nuclear synthesis is of the nature of an autokatalytic reaction. Masing (10) has shown that in the eggs of Arbacea pustulosa the nucleinie acid in the fertilized but unsegmented egg is as great as in the ‘morula’ with 500 to 1000 cells. He concludes that, ‘“‘the colossal increase of nuclear mass in the cleavage leads to no perceptible increase of nucleinic acid in the germ. A corollary of this must be that the total quantity of nucleinic acid necessary to build up the nuclear apparatus of the germ must be preformed in the protoplasm” (quoted from Godlewski, ’11). Shackell (11) has reached a similar conclusion with regard to the nuclein content of the egg and blastula of Arbacea punctulata. The results of my observations as to the rate of the growth of chromatin is especially significant when compared with the work CELL SIZE AND NUCLEAR SIZE 57 of Warburg. I find that the chromosomal mass grows at the rate of 8 per cent for each division up to the 32-cell stage. It is difficult to connect this rate of growth of the chromosomes with the lack of growth in the nucleinic acid content as shown by Mas- ing, or with the lack of growth of the nuclein content as shown by Shackell, and it seems necessary to assume as both of these in- vestigators have done, that these substances are already pre- formed in the protoplasm. If this be true, I venture the sug- gestion that the large amount of chromatin (oxychromatin) which escapes into the cell body when the germinal vesicle dis- solves may constitute the nuclein and nucleinie acid which is distributed through the cell body. VII. Senescence, rejuvenescence, and the ratio of nucleus to plasma. It is well known that Minot (’90, ’95, ’08) maintains that the cause of senescence is the increase of plasma and its products at a rate greater than that of the nucleus. According to his view the egg at the beginning of development is in a senile condition, “in which there is an excessive amount of protoplasm in propor- tion to the nucleus, and in order to get anything which is young a process of rejuvenation is necessary . . . . During the segmentation of the ovum the condition of things has been re- versed so far as the proportions of nucleus and protoplasm are concerned. We have nucleus produced, so to speak, to excess. The nuclear substance is increased during the first phase of de- velopment. Hence our conclusion:—Rejuvenation is accom- plished chiefly by the segmentation of the ovum.”’ He sums up his views on this subject in his four laws of age (’08, p. 250), the first two of which are: 1. ‘‘Rejuvenation depends on the in- crease of the nuclei. 2. Senescence depends on the increase of the protoplasm, and on the differentiation of the cells.” Richard Hertwig’s views (’89, 03, ’08) are apparently diamet- rically opposed to those of Minot, though I do not find them so definitely expressed. He finds that senescence, or rather ‘de- pression’ and ‘physiological degeneration,’ are accompanied by an enormous growth of the nucleus. As a result of his work on 58 EDWIN G. CONKLIN Actinosphaerium and Infusoria, which had been overfed for a long time, he found that there was an enormous growth of the nucleus followed by physiological degeneration. The animals which saved themselves from this condition did it by the reduction of their nuclei, either by eliminating nuclear substance directly, or by the loss of the greater part of the nuclear material during conjugation, after which normal nuclear conditions were restored. He regards the immature egg cell, with its great nucleus, as in a condition of depression similar to that found in the protozoa named. By the processes of maturation and fertilization this nuclear material is greatly reduced: ‘‘Beim Beginn der Fur- chung und auch spiter ein enormes Missverhiltniss von Kern und Protoplasma vorhanden ist, und dieses Missverhiltniss allmihlich eine Ausgleich erfihrt, indem Zellsubstanz in Kern- substanz umgewandelt wird,” (03, p. 116). Apparently then, in Hertwig’s view, senescence or depression, is accompanied by too great an amount of nuclear material, which is then reduced, by maturation in the case of the egg cell, to such an extent that this enormous disproportion of nucleus to protoplasm appears; later, by means of the process of cleavage, during which the nuclear material grows at the expense of the protoplasm, the normal relations of nucleus to protoplasm are restored. Popoff (’08) accepts Hertwig’s view in all essential respects. He adds the interesting suggestion that in their period of depres- sion preceding maturation the sex cells are so weakened that they are unable to assimilate nutriment, and they consequently store up food as yolk. The formation of yolk, glycogen and fat are, according to this author, not indications of increased activity of cells, but of incapacity to carry the organic synthesis to its end, viz., the formation of plasma. While Minot’s hypothesis differs fundamentally from Hert- wig’s as to the cause of senescence, the former holding that it depends upon the increase of protoplasm over nucleus, the latter that it is accompanied by an increase of nucleus over protoplasm, both agree that in the segmentation of the egg there is an enor- mous growth of the nuclear material as compared with the pro- toplasm. CELL SIZE AND NUCLEAR SIZE 59 Neither Minot nor Hertwig took account of the fact that a large part of the nuclear contents belongs to both nucleus and proto- plasm. The ‘Kernplasma-Relation’ depends very largely upon the quantity of protoplasmic material temporarily in the nucleus; in the 4-cell stage of Crepidula the ratio of nuclear volume to protoplasmic volume is 1 : 6.6 when the nuclei are measured at their maximum size, but 1 : 203.8 when they are measured at their minimum size. Neither of the authors named, in describ- ing the enormous growth of the nuclear material during cleavage, took account of the growth of the protoplasm during cleavage at the expense of the yolk. My observations on Crepidula have yielded the following re- sults, which bear upon the hypothesis under discussion: (1) While the germinal vesicle is absolutely the largest nucleus in the early stages of development, it is not so large with reference to the protoplasm, and hence according to Hertwig, not in so deep a depression, as the nuclei of certain blastomeres, which ex hypo- these should be undergoing restoration to normal conditions. (2) The growth of nuclear material during cleavage is not nearly so great as has been assumed, averaging not more than 10 per cent for each division up to the 32-cell stage, and not more than 1 per cent for each division after that stage. (38) The growth of proto- plasm at the expense of yolk during maturation and early cleay- age is considerable, averaging about 6 per cent for each division up to the 24-cell stage. (4) The ‘Kernplasma Relation,’ while constant for specific blastomeres, is by no means uniform for all the blastomeres of a given stage, but may vary from 1 : 1 to 1 : 14 in different blastomeres of the same generation. (5) The ‘Kern- plasma-Relation’ in adult epithelial cells of all three germ layers is about the same as in the majority of the blastomeres. (6) The absolute size of the nucleus depends upon the quantity of proto- plasm in the cell and the length of the resting period (interkinesis). (7) The greater part of the nuclear volume consists of material which belongs to the protoplasm as much as to the nucleus; during the resting period this is taken in osmotically through the nuclear membrane, and is given out again at mitosis by the dis- solution of that membrane. (8) The immature egg cell, which 60 EDWIN G. CONKLIN according to Popoff is so weakened that it is unable to assimilate nutriment, and consequently can only store up food instead of making protoplasm, does as a matter of facet form protoplasm throughout the whole of the growth period. So far as they go, therefore, these results do not support the view that senescence is due to either an increase or to a decrease of nuclear volume as compared with that of the protoplasm. But I think that this conflict between my results and those of Minot and Hertwig is, after all, confined to details, and that in the fundamental conception of the causes of senescence and rejuven- escence they may be brought into harmony. With the general thesis that senescence is associated with the accumulation in the cell of the products of metabolism and differentiation, and that rejuvenation consists in a return to a condition in which these products are largely eliminated, as Minot and Hertwig have urged, I am in hearty agreement; their assumption that changes in the nucleus-plasma ratio are the causes of these phenomena seems to me to be merely an error of detail. In a very suggestive paper, Child (’11) has recently maintained that senescence and rejuvenescence are caused by a decrease or an increase in the fundamental metabolic reactions. Anything which decreases the rate of metabolism, such as ‘‘decrease in permeability, increase in density, accumulation of relatively inactive substances, etc.,’’ will lead to senescence. ‘‘Rejuven- escence consists physiologically in an increase in the rate of metabo- lism and is brought about in nature by the removal in one way or another of the structural obstacles to metabolism” (p. 609). This hypothesis finds much support in the phenomena con- nected with the early development of the egg. It is well known that construct ve metabolism takes place only in the presence of nuclear material, and it has long been known that the nuclei of various kinds of gland cells give off substances which play an important part in the metabolism of the cell. Loeb (799) has shown that the nucleus is the oxidative center of the cell; Mathews identifies oxidase with chromatin; R. Lillie (’02) finds that oxidation takes place most rapidly in the immediate vicinity of the nucleus. If the rate of metabolism is associated with sen- CELL SIZE AND NUCLEAR SIZE 61 escence or rejuvenescence, as Child maintains, anything which facilitates the nterchange between nucleus and protoplasm should lead to rejuvenescence, anything which decreases it should lead to senescence. During cleavage the increase in nuclear surfaces is much greater than the increase in nuclear volumes. While the increase in max- ‘mum nuclear volumes up to the 32-cell stage of Crepidula is about 5 per cent for each division, the growth in the maximum nuclear surfaces during this period is about 11 per cent for each division. From the 2-cell to the 70-cell stage the nuclear volume increases only 2.24 times, while the nuclear surfaces increase 5.30 times. In Styela the nuclear volume increases from the 2- cell stage to the 256-cell stage only 4.52 times, the nuclear sur- faces increase 13.75 times. Unquestionably this greater growth of nuclear surfaces as compared with nuclear volumes, facilitates the interchange between nucleus and protoplasm. There is also a considerable increase of cell membranes during cleavage, but most of this increase is confined to surfaces of contact between cells, and free surfaces show but little growth. My observations teach that there is little, if any, interchange of materials through partition walls separating cells. ; Another and much more efficient means of facilitating the inter- change between nucleus and protoplasm is found in the mitotic division of the nucleus. During the cycle from one division to the next the nucleus absorbs materials from the cell body, only to throw back into the cell body these and other materials when the nuclear membrane dissolves in mitosis. The chromatin is thus brought into the most intimate relations with the proto- plasm. There is thus a sort of ‘‘diastole and systole of the nu- cleus’? (Conklin, ’02), by which the interchange between nucleus and protoplasm is greatly hastened. Indeed in the paper just referred to I suggested that this function of mitosis may be quite as important as the division and separation of the chromosomes, which is usually supposed to be the one function of mitosis. The hypothesis that the more rapid interchange between nu- cleus and protoplasm is associated with increased metabolism is supported by some very significant physiological work on the 62 EDWIN G. CONKLIN maturation, fertilization and cleavage of the egg. Loeb first showed that the immature egg, with germinal vesicle intact, is metabolically inactive; it absorbs but little oxygen and gives off little carbon dioxide. On the other hand when the membrane of the germinal vesicle dissolves, metabolic activity increases, and unless the egg is started in the process of development, by fertilization or other means, it soon dies. Lyon (’04) found that during the cleavage of the sea urchin egg the evolution of carbon dioxide is more rapid during the periods of division than during those of rest. Warburg (08) found that the fertilized sea-urchin egg uses six to seven times as much oxygen as the unfertilized egg. It is well known that the condensed chromatin of the chro- mosomes is brought into intimate relation with the protoplasm during mitosis, and of course the same is true of the condensed chromatin of the sperm head following fertilization. We may conclude, I think, that mitosis increases metabolism by facili- tating the interchange between nucleus and protoplasm, and particularly by setting free chromatin in the protoplasm, either by the dissolution of the nuclear membrane, or by the introduction of the sperm head in fetilization. Rapid and intimate interchange between the chromatin and the protoplasm is the condition of rapid metabolism, and ex hypothese of rejuvenescence; slow interchange is the condition of slow metabolism, and of senescence. Such a view has many points in common with the hypotheses of Minot and Hertwig, while it avoids many of the serious difficulties which those hypoth- eses encounter. It is thus evident that one may hold, with Minot and Hertwig, that the germ cells before maturation are senescent, and that maturation, fertilization and cleavage rep- resent a rejuvenescence, without necessarily connecting these processes with the nucleus-plasma ratio. 3R. Lillie (1910) holds that this is due to increased permeability of the plasma membrane during division. CELL- SIZE AND NUCLEAR SIZE 63 PART II EXPERIMENTAL STUDY OF CELL SIZE AND NUCLEAR SIZE IN THE EGGS OF CREPIDULA PLANA I. Nuclear size and chromosome number In Crepidula the relation of nuclear size to chromosome number is the same as in the Echinid larvae studied by Boveri (’05). By the use of various hypertonic salt solutions abnormal mitoses may be produced in Crepidula eggs; one of the most common of these abnormalities consists in the scattering of the chromo- somes, so that they do not fuse together to form two daughter nuclei, one in each cell, but many small nuclei. Indeed there may be almost as many small nuclei as there are chromosomes, every isolated chromosome being capable of producing a s° all nuclear vesicle. In all such cases the nuclear vesicles formed from a small number of chromosomes always remain smaller than those formed from a larger number. (In any given species the size of the nucleus is proportional to the number of chromosomes which go into its formation, providing the other factors which control nuclear size, viz., quantity of cytoplasm and length of resting period, are the same. On the other hand the size of the cell body is not dependent upon the size of the nucleus inthe early cleavages of Crepidula, as Gerassimoff (’02) found to be the case in Spirogyra and as Boveri determined in the case of Echinid larvae, but the reverse is true. In the eggs of Crepidula which have been treated with salt solutions the cell body frequently does not divide at all and many nuclei may be left in a single cell; where the cell itself divides there is a tendency for the blastomeres to divide in normal fashion, giving rise to macromeres or micromeres as in the normal egg, even though polyasters and abnormal mitoses are present. Con- sequently these eggs afford no evidence that the size of the nucleus has an influence on the size of the cell body. 64 EDWIN G. CONKLIN IT. Nuclear size and cell size in centrifuged eggs of Crepidula While the size relations of cells and of their various constituents may be readily observed in normal eggs, it is especially in eggs which have been centrifuged at various stages of development that the factors which determine these various size relations can be most satisfactorily studied. The various constituents of a cell may be moved by centrifugal force to one pole or another, according to their specific weights, and the axis of centrifuging. In this way the yolk, the cytoplasm, the nuclei and the centro- somes, may be caused to take very abnormal positions in the cell. Even the mitotic figure may be moved out of its ordinary position in the earliest stages of its formation, but after it has reached the metaphase it can be moved only with great difficulty; from this stage on it is anchored, probably to the cell membrane by the astral radiations, while the other constituents of the cell are free to move under the influence of centrifugal pressure. In this way it happens that the cytoplasm may be centrifuged away from the spindle and the latter left in a dense mass of yolk; or the normal relations of cytoplasm and yolk to the poles of the spindle may be completely changed; or the normal size relations of the daughter cells may be quite reversed. As illustrating these changed rela- tions, due to centrifuging, a few eggs are shown in figs. 11-37, selected from a great number which are similar to these. These eggs were centrifuged on a centrifugal machine run by water pressure, at the rate of 2000 revolutions per minute; the radius of rotation was 6 em., consequently the centrifugal pres- sure was nearly 270 times that of gravity. Eggs were centrifuged at this rate for varying lengths of time, after which they were removed from the machine and either fixed at once, or left for a longer or shorter time in sea water before fixation. All eggs were fixed in Kleinenberg picro-sulphuric mixture, were preserved in 70 per cent alcohol only long enough to wash out the fixing fluid, and were then stained in my modification of Delafield’s haematoxy- lin and mounted entire in balsam, in the manner described in previous papers (Conklin, ’02 et seq.) CELL SIZE AND NUCLEAR SIZE 65 In fig. 11 an egg is shown which was centrifuged for ten minutes after the formation of the first polar body and before the formation of the second, the axis of centrifuging being such that the lighter protoplasm was thrown to the vegetative pole and the heavier yolk to the animal pole, thus reversing the normal positions of these substances. After centrifuging, the egg was left in sea water for three hours before being fixed. The first polar body, which has partially divided, lies at the animal pole; the second matura- tion spindle has been greatly elongated and its axis has been turned somewhat, its lower pole having been moved to the right in the figure. The egg has begun to constrict opposite the equa- tor of the spindle, thus leading to the formation of a giant sec- ond polar body. The nucleus of this second polar body consists only of a compact mass of chromosomes surrounded by yolk; the sphere connecting these chromosomes with the egg membrane is much elongated. The egg nucleus and sphere at the lower pole of the spindle are in contact with the field of cytoplasm and are much larger than those at the upper pole. The sperm nucleus and sphere, lying in the cytoplasmic field, are much the largest in the egg. In normal condition these relations are reversed, the sperm nucleus lying in the yolk, while the egg nucleus is in the cytoplasmic field; and in such cases the egg nucleus and sphere are larger than those of the sperm; however as the sperm nucleus approaches the egg nucleus and thus moves up into the cytoplasm it continually grows larger until, at the time the two meet, the sperm nucleus is almost as large as the egg nucleus. {The fact that the normal size relations of these two nuclei may be reversed by reversing the positions of the cytoplasm and yolk, furnishes con- clusive evidence of the fact that the relative sizes of the egg and sperm nuclei and asters are dependent upon the quantity of eyto- plasm in which they lie. Furthermore fig. 11 shows that the spindle itself is a structure composed of fibers more firm than the surrounding substance, and is not merely an arrangement of the granules, which happen to be present in a field of force, into lines, like iron filings in a mag- netic field. The spindle remains fixed in position when all sur- rounding substances change position, and the spindle fibers, THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 1 66 ‘ EDWIN G. CONKLIN though much elongated preserve their usual appearance. In this regard my work confirms the conclusions of Morgan (’10) as to the nature of the spindle in Cerebratulus, and is at variance with the work of Lillie (09) on Chaetopterus. In fig. 12 an egg is shown which was centrifuged for fifteen minutes during the first cleavage and was then left for three hours in sea water. The axis of centrifuging is indicated here, as elsewhere, by the lighter vacuolated substance at one pole and the heavier yolk at the opposite pole; this axis is also marked by an arrow, the head of the arrow marking the distal pole during centrifuging, the tail of the arrow the central pole. In figs. 12 to 15 the first cleavage plane does not pass through the animal pole, which is marked by the polar bodies, but is displaced to one side, and the cleavage is not meridional, as in normal eggs; furthermore the cleavage is not equal, quantitatively and qualita- tively, as in normal eggs, but is markedly unequal, most of the cytoplasm having gone into the smaller one of the two daughter cells, while the larger one contains little cytoplasm and much yolk. This is evidently due to the fact that the greater mass of yolk in the larger cell has displaced the cleavage plane to one side of its normal position. Corresponding to this difference in the quantity of cytoplasm in the first two blastomeres of these eggs, there is a decided dif- ference in the size of the nuclei and spheres, the latter always being proportional in size to the quantity of cytoplasm in which they lie. The smaller cells with the larger quantity of cytoplasm thus have larger nuclei and spheres than the larger cells, which have a smaller quantity of cytoplasm. The eggs represented in figs. 13, 14, and 15 were centrifuged for five hours during the first cleavage and were then fixed at once. It is evident that division took place while the eggs were on the centrifugal machine and that the daughter nuclei have grown to the size shown while the eggs were still being centrifuged. Other eggs centrifuged for the same length of time were allowed to develop further after being removed from the centrifuge, and they show that in most cases the eggs were still alive after cen- trifuging and not seriously injured. Fig. 13 shows a very note- CELL SIZE AND NUCLEAR SIZE 67 worthy fact to which attention will be devoted in a future paper, viz., that the cell axis, which is marked by the line passing through the nucleus and sphere (and centrosome), remains unchanged after centrifuging. In the stage shown in fig. 13, the spheres lie between the nuclei and the polar bodies in normal eggs, and al- though the positions of cytoplasm and yolk, and of the first cleav- age plane have been changed in this egg, this cell polarity remains unchanged. Fig. 16 represents an egg which was centrifuged thirty minutes and then left in sea water for twenty hours. Neither this egg nor any others of this lot developed far after being centrifuged; it is possible that the eggs were injured in some way so that noneof them _ developed, or it is barely possible that the record of the experiment is wrong. In all the eggs of this lot the appearance is that of eggs which had been under normal conditions for about three or four hours after being removed from the centrifuge. This egg was evidently centrifuged during the first cleavage, which was very unequal, practically all of the cytoplasm having gone into the smaller of the two daughter cells. The nucleus and sphere in this smaller cell are enormous, whereas in the larger yolk cell they are extremely small, indeed no larger than in the anaphase stage of division. The chromosomes form a compact mass which stains deeply and contains no achromatic material. The sphere is small also but the fact that it holds its normal position with respect to the nucleus shows not only that the pol- arity of the cell remains unchanged, but also that the material of the sphere is different from the ordinary cytoplasm. In many cases similar to fig. 16 cytoplasm slowly forms around the chromo- somes in the yolk cell and ultimately such a cell may develop in a normal manner. There is no evidence that cytoplasm ever passes through the cell membrane from one cell to another, and there is positive evidence that this does not occur. The formation of cytoplasm around a mass of chromosomes in a yolk field is therefore an occurrence of more than ordinary importance. The question has been asked frequently whether the nucleus alone ean form cytoplasm or the eytoplasm alone a nucleus. It is known that the latter never happens; a mass of cytoplasm without 68 EDWIN G. CONKLIN a nucleus may live for some time and show certain vital functions, but it is unable to grow or to regenerate lost parts. It is much more difficult to test the former question, for it is usually impos- sible to separate the nucleus completely from the cytoplasm and yet leave it in a medium in which growth would be possible. Verworn (’91) succeeded in shelling the nucleus out of Thalas- sicolla, but found that the isolated nucleus was unable to grow a new cell body; but apart from the objection that the resting nucleus contains a large amount of cytoplasmic substance, this experiment is not conclusive for it is possible that the failure to grow a cell body was due to the lack of a proper nutrient medium in which the nucleus could operate. The present experiment is free from most of these objections, though it must be confessed that one objection still remains, viz., it is not possible to be certain that every trace of cytoplasm has been removed from the yolk cell. Nevertheless the amount of cytoplasm left in the cell is very small and is quite indistinguish- able, the only visible constituents of the cell being chromosomes, sphere and yolk. In the growth of cytoplasm in such a cell there first appears a very thin layer of cytoplasm around the chromo- somes, then the yolk in the immediate periphery of this begins to dissolve and the cytoplasm increases in amount. Coincidently the chromosomes swell up, absorbing achromatic material from the cytoplasm, and in later stages the growth of both cytoplasm and nucleus goes forward at an increasing rate. The formation of cytoplasm takes place only in the presence of chromatin and in its immediate vicinity; on the other hand the chromosomes grow only when surrounded by cytoplasm. ‘This indicates that some influence, probably of a chemical nature, goes out from the chro- mosomes and leads to the solution of yolk and the formation of cytoplasm. Whether this influence from the chromosomes may act directly upon the yolk, or only indirectly through the medium of a minimal quantity of cytoplasm, is not certain, but it seems probable that the latter is the case. After cytoplasm has been formed around the chromosomes, but not before, the chromosomes themselves begin to swell up, absorbing achromatic material from the cytoplasm, and the chromatin grows in quantity. Cyto- CELL SIZE AND NUCLEAR SIZE 69 plasm is essential to the growth of the nucleus and of the chroma- tin; on the other hand chromatin is essential to the growth of cytoplasm, or to the conversion of yolk or food substances into cytoplasm. The life of the cell consists in an interchange of materials between the nucleus and the cytoplasm; the one cannot grow in the absence of the other. This conclusion agrees with the generalization of Godlewski (10): ““Zuerst das Bildingsmate- rial geliefert und von den betreffenden Regeneratskomponenten zum Protoplasm assimiliert wird, dass dagegen in der zweiten Regenerationsphase dieses Protoplasma sich wenigstens teilweise zur Kernsubstanz transformiert”’ (p. 88). The question has been much discussed as to whether the nuclei, and more particularly the chromosomes of the germ cells, are the sole ‘bearers of heredity,’ as Weismann, and many others, have maintained. We have experimental evidence that the cytoplasm cannot form chromatin in the absence of preéxistent chromatin. On the other hand there is no certain evidence that the chromatin can form cytoplasm in the absence of preéxisting cytoplasm. The experiment described above is not entirely conclusive, for while chromosomes in a yolk field form cytoplasm, it is probable that a minimal amount of cytoplasm is left in the yolk field, and it may be said that this merely grows by assimila- tion of yolk. On the other hand my experiments show that where we have equal division of the chromosomes and unequal division of the protoplasm we may have regulation and normal development; whereas this never follows abnormal distribution of the chromosomes; in other words protoplasmic abnormalities are capable of regulation when the nucleus is normal, but the re- verse is not the case. The nucleus is the regulating center of the cell, and it is probably also the assimilating center. And since both of these functions are involved in inheritance, to this extent at least the nucleus may be said to be the inheritance center. Fig. 17 represents an egg which was centrifuged for four hours during the first cleavage and was then placed under normal con- ditions for six hours before being killed. The polar body marks the original animal pole and in the centrifuging most of the yolk was thrown to this pole, most of the cytoplasm to the opposite 70 EDWIN G. CONKLIN pole. The first cleavage plane is nearly equatorial in position, and one of the cells contains most of the cytoplasm. The spindles for the second cleavage have formed and the spindle in the cell containing the larger amount of cytoplasm is distinctly larger than the one in the other cell; each is proportional in size to the resting nucleus from which it came and to the volume of cytoplasm in the cell. The fact that the polarity of the cells has not been changed by the abnormal position of the first cleavage plane is indicated by the fact that the spindles are parallel to each other, but not to the plane of cleavage, as in normal eggs. In short there is evidence that the spindles here attempt to take up the positions which they would have occupied in a normal egg, with meridional cleavage. Fig. 18 represents an egg from a lot which was centrifuged fifteen minutes in gum arabic, as recommended by Lyon (’04), and which was fixed three hours after removal from the centrifuge. Fig. 19 shows an egg which was centrifuged thirty minutes, and was fixed six hours later. In both cases the centrifuging took place during the first cleavage, as is shown by the unequal distribution of cytoplasm and yolk on both sides of the first cleavage plane. In the second cleavage, which evidently occurred after the eggs were removed from the centrifuge, the cytoplasm was distributed equally to the daughter cells. In fig. 18 the second cleavage took place a little earlier in the cell rich in cytoplasm (AB) than in the other (CD), but the smaller size of the nuclei in the latter is probably due in part to the fact that these cells are poor in cytoplasm. In fig. 19 the inequality in the distribution of cyto- plasm at the first cleavage is much greater than in fig. 18; never- theless the second cleavage occurred in the cell poor in cytoplasm (CD) at nearly the same time as in the other cell (AB). Although the nuclei in the cells C and D are much smaller than those in A and B, their structure shows that they are in nearly the same stage of the cell cycle. Their smaller size is due to the smaller quantity of cytoplasm in which they lie. Figs. 17-19 indicate that the absolute size of the nucleus has little to do with the time of its division; small nuclei in yolk-rich cells divide almost as rapidly as large nuclei in cells rich in cytoplasm. CELL SIZE AND NUCLEAR SIZE 71 Figs. 20 to 28 show eggs which were centrifuged during the second cleavage. The first and second cleavages may always be distinguished by the fact that the polar furrow bends to the right in the first cleavage and to the left in the second (Conklin 97). In fig. 20 the distribution of cytoplasm and yolk to the daughter cells was equal in the first cleavage but unequal in the second, and the daughter nuclei are proportional in size to the volume of the cytoplasm in which they lie. In fig. 21, which represents an egg which was centrifuged for 30 minutes and fixed at once, the second cleavage is very unequal, two of the macromeres (B and C’) being small protoplasmic cells, which resemble micromeres in appearance, but which behave like macromeres as the study of later stages (figs. 24 to 28) shows. Fig. 22 represents an egg which was centrifuged for thirty minutes during the second cleavage and then kept under normal conditions for twenty-one hours before being fixed. The second cleavage was suppressed although the nucleus divided in the upper cell, AB, but not completely in the lower one, CD. These nuclei have given rise to spindles for the third cleavage, there being two independent spindles in the cell AB, and two spindles which are fused at one pole in the cell CD, thus forming a triaster. The degree of abnormality in this case is indicated by the fact that the development has been halted at this stage, although a normal egg would have reached the 20-cell stage at least, in the time which elapsed after centrifuging. With the exception of fig. 26, all of the figs. from 23 to 28 were drawn from the same lot of eggs which were centrifuged for thirty minutes in the 2-cell stage, and then kept for six hours under normal conditions before being fixed. In all of these eggs the second cleavage was made very unequal by the centrifuging. Two of the macromeres are not only much smaller than the other two, but are composed entirely of cytoplasm, whereas the two larger macromeres contain all of the yolk. Nevertheless the behavior of these two small, protoplasmic macromeres is almost identically like that of the large, yolk-rich macromeres; the micro- meres are given off from both the protoplasmic and the yolk laden macromeres at practically the same time and in the same 72 EDWIN G. CONKLIN direction; the micromeres formed from these abnormal macro- meres are the same as in normal eggs in which all the macromeres are of the same size and contain the same quantity of cytoplasm and yolk. In short there is here a form of regulation which leads to the formation of normal micromeres from abnormal macro- meres, and the exact manner in which this cellular regulation takes place is of fundamental importance, and will be discussed later. Fig. 23 represents an egg similar in many respects to fig. 21, but of a later stage. The smaller protoplasmic macromeres preserve their original polarity as is shown by the fact that the spheres lie between the nuclei and the polar bodies. On the other hand each of the large macromeres contains a tetraster; the spin- dles are those of the third cleavage. Fig. 24 represents an egg of the same type as the preceding, after the third cleavage; each macromere has given rise to a micromere which is normal in form, position, constitution and size, although the macromeres are very abnormal in these regards, two of them containing all of the yolk and very little protoplasm, and the other two being small and purely protoplasmic. Indeed the macromeres 7A and 1D nearly exhausted all the cytoplasm which they contained in order to form cytoplasmic micromeres of normal size; on the other hand, the size of the micromeres 1b and /c is not influenced by the fact that the macromeres from which they come are small and are purely protoplasmic. Fig. 25 is a drawing of an egg in a slightly older stage than fig. 24; the large macromeres, 1B and 1C, are giving off the second set of micromeres, 2b and 2c, while two of the first set of micro- meres, /b and /c, are just beginning to divide. The four small cells which lie to the left of the polar bodies are the macromeres 1A and 1D and the micromeres 1a and /d; these cells are purely protoplasmic and are very small, all four of them being no larger than one of the micromeres, 1b or Jc, in the other quadrants. Nevertheless these minute ‘macromeres’ have each given rise by an equal cleavage, to a micromere as large as itself. Although these micromeres are much smaller than those in the other quad- rants, they are the largest that could be formed from the macro- CELL SIZE AND NUCLEAR SIZE 73 meres in question without making the macromeres smaller than the micromeres, thus reversing the usual inequality of this divi- sion; in short the division of these cells represents the nearest possible approach to normal conditions. Figs. 27 and 28 show eggs of the same type as the preceding, but at a stage after the formation of the second set of micro- meres (2a—2d) and during the division of the first set (/a—/d). Here also these micromeres are normal in size, although the size relations, and the cytoplasmic or yolk content of the macromeres from which they came, are very abnormal. In the cleavages which follow after the centrifuging, complete regulation has occurred, so far as this is possible. It is not possible for regulation to take place by the redistribution of cytoplasm and yolk by passage through a cell membrane. Fig. 26 represents an egg which was centrifuged for thirty minutes during the second cleavage, and then fixed twelve hours later. At the time of centrifuging the nuclear division in the second cleavage was complete, but the division of the cell body was suppressed. Consequently each of the blastomeres, AB and CD, contained two nuclei, which by subsequent division in the manner indicated in fig. 22 have given rise to two sets of micromeres, la-Id, and 2a-2d. Both sets of micromeres have divided, as indicated by the connecting bonds, thus forming a somewhat ab- normal cap of sixteen micromeres. The nuclei of the macromeres are indicated by the reference lines from the letters 2A—2D. Other cases similar to this one will be shown and described in another paper, but this one egg shows that it is possible for both the nuclei of a binucleate cell to divide at the same time and to give rise to separate cells, each with a single nucleus, and that such cells may approximate in form and position normal blastomeres. Fig. 29 represents an egg which was centrifuged for four hours at the close of the second cleavage, and fixed at once after centri- fuging. The yolk has been forced out into lobes, which are still connected with the protoplasmic portions of the cells except in the case of one cell, where the lobe has been completely separated. It is a significant fact that the point at which the lobe forms, and consequently the point where the cell membrane is weakest lies 74 EDWIN G. CONKLIN at the outer pole of the axis which passes through the centrosome and nucleus and these axes mark the position of the spindles of the third cleavage. Here, as in every other instance, the smallest nucleus is found in the cell which has the smallest amount of cytoplasm. Fig. 30 is a drawing of an egg which was centrifuged ten min- utes in gum arabic, during the first cleavage, and fixed four hours later during the third cleavage. Macromeres A and B are richer in cytoplasm and poorer in yolk than C and D, and correspondingly the spindles and asters are larger in the former than in the latter. Figs. 31 and 32 represent eggs which were centrifuged four hours during the first cleavage, and were fixed six hours later. In both eggs the macromeres A and B are richer in cytoplasm and poorer in yolk than C and D. In fig. 31 the cells A and B con- tained more cytoplasm and divided earlier than C and D; at least one-half of the cytoplasm in the latter cells has gone into the formation of the micromeres, which are still, however, smaller than normal. The first cleavage in this egg did not pass through the animal pole, marked by the polar bodies, but was displaced to one side, and the spiral form of the cleavage is not clearly preserved in the cells C and D. While the regulation in the size of these micromeres is not complete, the tendency to approach the normal condition is evident. Fig. 32 is similar to fig. 31, though the macromeres C and D of this egg contained a larger amount of cytoplasm than in fig. 31, and the regulation in the size of the micromeres is complete. Fig. 33 shows an egg, from the same slide as fig. 30, which was centrifuged ten minutes in gum arabic and fixed four hours later. The macromeres /A and /B contain more cytoplasm and are dividing earlier than 1C and 1D, but the micromeres from the former are no larger than those from the latter. Fig. 34 represents an egg which was centrifuged for two and one-half hours during the first cleavage, and was fixed twenty-one hours later. The macromeres 2A and 2B contain much cyto- plasm, while 2C and 2D contain little and yet the micromeres formed from the latter are almost as large as those from the former. CELL SIZE AND NUCLEAR SIZE 75 Figs. 35, 36, 37 represent eggs, from the same experiment, which were centrifuged thirty minutes during the first cleavage, and were fixed twelve hours later. The size of the micromeres of the first, second or third sets is but little influenced by the quan- tity of cytoplasm in the macromeres; the size regulation of the micromeres is here practically complete. In fig. 37 the cell 4c forms at the same time as 4d, though in normal eggs it does not form until much later; the precocious formation of this cell is probably due to the fact that the amount of cytoplasm in macro- mere C was larger than normal. III. General results of these experiments The results of these experiments, which have been described in the order of development from the earlier to the later stages without reference to a logical presentation of general questions, may now be classified and compared with the observations on cell size and nuclear size given in Part I of this paper. In gen- eral these experiments support in every detail the conclusions based upon the study of normal eggs and blastomeres. 1. Nuclear size in centrifuged eggs. In centrifuged eggs, as in normal ones, the size of the nucleus is always dependent upon the quantity of cytoplasm surrounding the nucleus and upon the length of the resting period. Nuclei which are normally large may be caused to remain small, and nuclei which are normally small may be rendered large by merely changing the positions of the yolk and cytoplasm in the cell. In normal eggs of Crepidula the egg nucleus lies in a protoplas- mic field near the animal pole of the egg, while the sperm nucleus enters the egg near the vegetal pole 4nd moves up toward the animal pole through a field of yolk. As long as the sperm nucleus is in this yolk it remains very small, and only when it emerges into the protoplasmic field near the egg nucleus does it begin to grow rapidly. The egg nucleus on the other hand, grows rapidly and becomes much larger than the sperm nucleus. If now an egg is centrifuged during the formation of the second polar body so as to throw the yolk to the animal pole and the cytoplasm to 76 EDWIN G. CONKLIN the vegetal pole, the normal size relations of the germ nuclei is reversed, the sperm nucleus becoming larger than the egg nucleus as shown in fig. 11. Godlewski (’08) holds that the size of the sperm nucleus depends upon the time which elapses before its union with the egg nucleus; it also depends, as I have shown, upon the quantity of cytoplasm in which it les. We conclude therefore, that in all animals the relative sizes of egg and sperm nuclei are dependent upon the amount of cytoplasm in which they lie, and upon the length of the growth period (interkinesis). In this connection it may be worth while to remark that one reason why the rhythm of cleavage, in Boveri’s, Driesch’s, and Godlewski’s experiments, follows the maternal rather than the paternal type may be found in the fact that the rate of growth of the nucleus is dependent upon the quantity and quality of the protoplasm of the egg. In the cleavage of the egg the size of the nucleus is dependent upon the quantity of protoplasm in which it lies, as shown by figs. 12 to 20. In eggs subjected to strong centrifugal force the egg contents separate into three zones, a yellow zone of yolk at the distal (heavy) pole, a gray zone of oily and watery substance at the central (light) pole, and a clear zone of protoplasm between these two. It is the latter substance which contributes to the growth of the nucleus, as is shown by such cases as fig. 16 in which the gray substance was centrifuged out of the egg and practically all of the yolk thrown into one of the blastomeres, and most of the clear protoplasm into the other; the nucleus in the blastomere which contains yolk but little or no protoplasm has scarcely grown at all, the one in the cell containing the clear protoplasm, but without the gray substance, has grown enormously. Sim- ilar, though less striking, differences in the sizes of nuclei, depend- ing upon the quantity of clear protoplasm in the cell, are found in all the eggs figured. In centrifuged eggs the nucleus always occupies the middle zone, and as I have just shown it grows at the expense of substance received directly from this zone. The fact that the specific gravity of the nucleus and of this middle zone are the same, is probably due to the fact that so much of the absorbed nuclear material is from this zone. CELL SIZE AND NUCLEAR SIZE WG 2. The sizes of spindles, centrosomes, spheres and asters. The study of centrifuged eggs shows, as was observed in the ease of normal eggs, that the sizes of spindles, centrosomes, spheres and asters are dependent upon the quantity of cytoplasm in which they lie. The size of the spindle is also related to the size of the nucleus, as I have already shown, but as this, in turn, is dependent upon the quantity of cytoplasm of the middle zone, it follows that the size of the spindle as well as that of the centrosome and sphere is related to the quantity of cytoplasm in which they lie. Fig. 17 shows spindles in sister cells which are quite different in size owing to the different amounts of cytoplasm in these two cells; while figs. 11, 12, and 16 show centrosomes and sphere which vary in size depending upon the quantity of cytoplasm surround- ing them. In this connection attention should be called to the fact that the spindles from the stage of the metaphase to the end of mitosis are anchored in the cell, and can be moved only with much difficulty. The spindle fibers are tougher and more con- sistent than the surrounding plasm, and they are not a mere arrangement of granules in the lines of force as Lille (’09) has maintained for Chaetopterus. 8. The rhythm of division in centrifuged eggs. The rhythm of division is not dependent solely upon nuclear size, nor cell size, nor the ratio of one to the other (Kernplasma-Relation), though it may be influenced by the absolute amount of cytoplasm present in the cell. Cleavage cells which contain a large amount of cyto- plasm, and which therefore have large nuclei, usually divide a little earlier than cells poor in cytoplasm, and with small nuclei, though this is not always the case, as is shown by fig. 17, in which the large and the small nuclei divide at the same time. Nuclei which differ greatly in size may still be in the same stage of the nuclear cycle, as shown in fig. 19, and may divide at the same time. On the other hand, figs. 25, 31, 34 and 37 show cases in which nuclei of the same generation divide earlier in cells rich in cytoplasm than in cells which are poor in this substance. 4. Growth of cytoplasm at the expense of yolk. Centrifuged eggs afford an excellent opportunity of studying the way in which 78 EDWIN G. CONKLIN cytoplasm grows at the expense of yolk. In cases in which the centrifuging occurred after the spindle was anchored in the cell, but before the division wall had formed, the cytoplasm may be thrown almost entirely to one pole of the spindle and the yolk to the other; accordingly when division occurs one of the daughter cells will contain almost all the cytoplasm, the other all the yolk, while both cells will receive the same number and mass of chro- mosomes, fig. 16. The chromosomes which are left in the yolk field remain small and compact since there is no cell substance which they can absorb. After some time the yolk in the vicinity of the chromosomes may begin to disappear and cytoplasm to appear in its place. It can scarcely be doubted that some sub- stance, probably an enzyme, is given off by the chromosomes and dissolves the yolk, and that this dissolved yolk is then converted into cytoplasm through the influence of the chromosomes. Once a small field of cytoplasm is formed around the chromosomes, they begin to abSorb it and to become vesicular. The process of form- ing cytoplasm may then go forward rapidly and in the end the yolk cell may give rise to protoplasmic micromeres in a normal manner (fig. 31). It is probable that a small amount of cyto- plasm, which cannot be displaced by centrifuging, is left in the yolk cell, and it is possible that the formation of new cytoplasm would not take place in the absence of this small remnant, but it can be proved conclusively that this formation of cytoplasm takes place only in the vicinity of the chromosomes, and that in the absence of this chromatic material it never occurs at all. Under these circumstances the conclusion seems justified that the chro- matin has the power of forming cytoplasm when placed in a suit- able nutrient medium, such as yolk, and that the cytoplasm in turn contributes to the growth of the nucleus and of the chromatin. 5. Unequal and differential cell divisions. By centrifuging, the size and constitution of the blastomeres may be changed; divi- sions which are normally equal may be made unequal, and vice versa; cells which are normally protoplasmic may be filled with yolk and vice versa. In this way both the cell size and the cell content may be controlled experimentally. CELL SIZE AND NUCLEAR SIZE 79 Acknowledgedly the position of the spindle conditions the plane of the cleavage, the division wall passing through the equator of the spindle. When by any means the spindle is displaced from its normal position the division plane is displaced. In this way giant polar bodies may be formed, as shown in fig. 11, or macro- meres may be formed which are small and free from yolk, as shown in figs. 16, 21-28, e al. Are the inequalities and differentiations of normal cleavage due to similar causes, viz., external or internal pressure? Clearly external pressure cannot be involved in the unequal division of free cells, such as the maturation divisions of the egg; and the fact that isolated blastomeres of the 4-cell stage divide in the normal manner into small protoplasmic micromeres and large yolk-rich macromeres, shows that these unequal divisions during the cleavage period cannot be explained as the result of reciprocal pressure among cells. On the other hand, the forma- tion of micromeres of normal size and constitution from purely protoplasmic macromeres, as shown in figs. 24, 27, 28, 33, 36, et al., indicates that this inequality of division cannot be due to the crowding of the spindle to one side of the cell by internal pressure, such as might come from the presence of a mass of yolk—because in the eases cited, little or no yolk is present in the macromeres. If internal pressure is involved in the unequal division of these protoplasmic cells it must be pressure of a very different sort from that involved in the presence of a mass of metabolic products at one side of the cell. While the spindle may be pressed out of position by external or internal pressure this will not serve to explain the eccentric position of the spindle in such cases as I have described. A satisfactory explanation of unequal and differential cell divi- sion must also be able to be applied to equal and non-differential cleavage, for the causes of the latter are not simple mechanical conditions, such as pressure. In the case of cleavages which are normally equal, if the spindle and yolk are moved to eccen- tric positions in the cell, they come back, if possible, to their normal positions when the pressure is removed; indeed they some- times seem to come back against considerable pressure, as when 80 EDWIN G. CONKLIN a spindle moves out of a protoplasmic field into the yolk in order to reach its normal position in the cell. When eggs like the one shown in fig. 11 are removed from the centrifuge, the egg and sperm nuclei, together with the cytoplasm surrounding them move up through the yolk until they ultimately le in their normal position on the animal side of the egg, beneath the polar bodies. How- ever far the germ nuclei or the first cleavage spindle may be re- moved from the chief axis of the egg, they invariably come back to their normal positions, with the equator of the spindle in the egg axis, and the long axis of the spindle at right angles to the egg axis, unless the spindle is held so long in its abnormal position that it is caught in that position by the divisional processes. The same is true also of the nuclei and spindles of the 2-cell stage; when moved out of the median plane of the cell they come back to that median plane, unless the cells are injured or the spindles are held in their abnormal position until the metaphase or a little later. Evidently the cause of equal cell division, such as the first and second cleavages of Crepidula, is not so simple as those have assumed who have attributed it to pressure, the line of least resistance, or the long axis of the protoplasmic mass. Not only the eccentricity or lack of eccentricity, but also the axis of the spindle is of great importance in determining the char- acter of the cleavage. While the former is associated with the equality or inequality of division, the latter conditions its differ- ential or non-differential character. The polar differentiation of the egg is the first visible morphogenetic differentiation, and it is not without significance that in the first and second cleavages of the egg the spindles are at right angles to the egg axis, while in the third, fourth and fifth cleavages they are more nearly par- allel with that axis. I have hitherto spoken of the position of the spindle as if it were the one cause of equal or unequal, differential or non-differ- ential cleavage; but for many reasons it is evident that the posi- tion of the spindle is itself the result of the structure or organiza- tion of the protoplasm, and that in this organization polarity and symmetry play an important part. Many years ago (’93) I showed that even before the spindle is formed, the shape of the CELL SIZE AND NUCLEAR SIZE 81 cell may indicate the position and direction of the coming cleav- age, and I maintained then and in subsequent papers (’97, 99, 02) that the position of the spindle and the size, position, and histological character of the daughter cells is the result of the structure of the protoplasm, and particularly of the polarity and symmetry of the cell. These conclusions have been confirmed by muchexperimental work on cell division, which I have completed but have not yet published. The position of the spindle and the plane of cleavage may be greatly changed, but the polarity and organization of the protoplasm remain unchanged, as I shall show in a future paper. Indeed it is very difficult to alter the polarity of any cell as Lillie (706, 09) has shown, and one reason for this is to be found in the fact, as I have discovered in Crepidula, that the cell axis, i.e., the axis connecting nucleus and centrosome, can rarely be changed by artificial means. 6. Regulation in the cleavage process. Evidently connected with this persistent organization of the cell is the power of regu- lation which is shown in the cleavage of the egg as well as in the regeneration of adult parts. Whenever the size or constitution of blastomeres of Crepidula have been changed, or when cleavages have been suppressed, subsequent cleavages come back to the normal form so far as this is possible. The original disturbance can be righted only very gradually if at all, since neither yolk, cytoplasm nor nuclei can pass through cell membranes, and the only redistribution of substances possible is by means of new cell divisions. But in Crepidula the divisions following upon such a disturbance of the usual cleavage process are almost if not en- tirely normal. This is very evident in the divisions following upon disturbances of the first two cleavages. All of the yolk may be centrifuged into two of the macromeres and practically all of the cytoplasm into the other two, as in figs. 16, 19, 21, 23, et. al; two of the ‘macromeres’ may be very small and two very large, as in figs. 16, 21, 23 to 28; or one of these first two cleavages may be suppressed, as in figs. 22 and 26; but if such abnormal eggs are allowed to develop under normal conditions, the micromeres are formed in normal manner, as is shown in figs. 24 to 28 and 32 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 1 82 EDWIN G. CONKLIN to 37. Whatever the content of the different macromeres may be, whether purely protoplasmic or entirely yolk the micromeres are always protoplasmic, even though division must be delayed until the cytoplasm which goes into the micromeres can be formed from yolk (figs. 24, 31, 35); whatever the size of the macromeres, the micromeres formed from them are approximately normal in size, even though yolk-rich cells must give up most of their cyto- plasm (figs. 24, 31, 35), or protoplasmic micromeres must divide equally (figs. 25, 28), in order to give rise to micromeres of the usual size. Such regulations of cleavage are probably caused, in the case of Crepidula, by the persistent polarity of each cell, which in turn leads to the localization of the spindle in a definite axis, with its pole at a definite distance from the surface of the cell. In what manner the polarity of the cell may cause the localiza- tion of the spindle is clearly shown in the cleavage of Crepidula. In former publications (’99, ’02) I have called attention to the fact that definite movements of cell substance take place in divid- ing cells, and that these movements serve to orient the spindles; these movements are always related to the polarity of the cell and to that of the entire egg. Furthermore, I have elsewhere (02) called attention to the fact that the cell membrane is weakest opposite the poles of the spindle. I was formerly of the opinion that this was due to some influence of the spindle on the cell membrane, but a further study shows that these weak places in the cell membrane are present before the spindle forms and can not therefore be caused by the spindle. In the egg shown in fig. 29 the places of reduced tension on the cell membrane are indicated by the lobes of yolk attached to the cells, and a line drawn through the centrosome, nucleus and lobe indicates the precise position which the spindle will take at the next cleavage. The axes of the third cleavage spindles are here marked out long before the spin- dles are formed; the weak spot in the cell membrane is not caused by the position of the spindle, but the latter is the result of the former. Experiments on eggs in the 2-cell, 4-cell and 8-cell stages of cleavage show that the positions of the points where the mem- brane is weakest, change in each cell generation and that they CELL SIZE AND NUCLEAR SIZE 83 always mark out the position of the spindle. These lobes are formed only when the egg is subjected to pressure and then only at those points on the cell surface which mark the position which will be taken by the poles of the spindle. Since the spindle axes change in successive cleavages it follows that this point of reduced tension also changes in successive cell generations. I conclude therefore that the position of the spindle, and all the morphogenetic results which follow from this, is dependent upon the polarity of the cell; which polarity manifests itself not only in the localization of cytoplasmic substances, but also, and more fundamentally, in definite movements of the odplasm and in reduced tension of the cell membrane at the poles of the cell. GENERAL SUMMARY AND INDEX Part I. Observations 1. The equality or inequality of cell division in normal cleavage is due to internal causes, rather than to the presence of metabolic substances, such as yolk, within the cell or to pressure from with- out. These internal causes are to be found in the polarity of the cell, in movements of the cytoplasm, and in the structure of the cellmembrane. Since the position and axes of the spindles change regularly in successive divisions this protoplasmic organization must also change regularly (pp. 6-9). 2. The yolk-lobe is a temporary extrusion of yolk or odplasm during mitotic pressure, at the former point of attachment to the ovarian wall and a little to one side of the vegetative pole. If this lobe is large, the resulting cleavage is unequal, although the furrow cuts through the chief axis and the center of the egg. The degree of inequality of the first and second cleavages is measured by the size of the yolk-lobe. The yolk-lobe is the result of an unsymmetrical distribution of yolk or egg substance with refer- ence to the egg axis (pp. 9-11). 3. In Crepidula plana the Kernplasma-Relation varies greatly in different blastomeres and at different stages, depending chiefly upon the length of the resting period (interkinesis). In cases 84 EDWIN G. CONKLIN where nuclei and cells are measured at their maximum size it varies from 14.5 to 0.37; at mean size from 35.7 to 1.1; at minimum nuclear and cell size it varies from 285 to 29. In protoplasmic blastomeres, which contain no yolk, the Kernplasma-Relation varies from 14.5 to 8.7, when the nuclei are at their maximum size; and from 35.7 to 7, when the nuclei are at mean size. In Fulgur, at mean size, it varies from 127.7 to 3.6 (pp. 16-24). 4. In different eggs, corresponding blastomeres have approxi- mately the same Kernplasma-Relation; but in different blasto- meres of the same egg or of different eggs the Kernplasma-Relation is neither a constant nor a self regulating ratio. It appears to be a result rather than a cause of the rate of cell division, and con- sequently a variable rather than a constant factor (pp. 24-25). 5. In the tissue cells of adult Crepidulas there is no marked increase of cytoplasm over nucleus, as compared with the blasto- meres. The Kernplasma-Relation of various adult epithelial cells, not filled with metabolic products, varies from 28 to 7; in oécytes and ganglion cells it varies from 6 to 3 (pp. 25-28). 6. The size of the nucleus is dependent upon at least three factors: (a) The initial quantity of chromatin (Boveri); (b) The volume of the cytoplasm; (c) The length of the resting period (p. 25). 7. The inciting cause of cell division in Crepidula is not found solely in the limitations of the working sphere of the nucleus (Strasburger), nor in the doubling of the volume of the chromo- somes (Boveri), nor in a Kernplasma-Spannung (Hertwig), but rather in the coincidence of centrosomal, chromosomal and cyto- plasmic rhythms, which are probably connected with the rate and nature of metabolism in the cell (pp. 29-82). 8. During the cleavage of the egg of Crepidula plana the volume of the cytoplasm more than doubles between the 1-cell and the 24-cell stage the average growth for each division being about 6 per cent; the yolk decreases in volume by nearly one-half and the entire egg is smaller at the 24-cell stage than at the 1-cell stage. This can only mean that the yolk contributes to the growth of cytoplasm during the cleavage period (pp. 32-36). CELL SIZE AND NUCLEAR SIZE 85 9. The average nuclear growth during cleavage is not more than 5 per cent to 9 per cent for each division up to the 32-cell stage and it may fall as low as 0.3 per cent to 1 per cent for each division after that stage; and in every case it falls far short of a doubling, or increase of 100 per cent, for each division (pp. 36— 44, 54, 55). 10. Both nuclear sap and linin belong to the cytoplasm as well as to the nucleus. The chromatin is the most distinctive nuclear substance. All of these constituents are more abundant in large cells than in small ones. The mitotic spindle is of both nuclear and cytoplasmic origin and its size depends upon the volume of both nucleus and cytoplasm (pp. 44-47, 55). 11. The average growth in volume of chromatin from the 2- cell to the 32-cell stage is about 8 per cent for each division period, being about the same as the growth of the nucleus as a whole (pp. 47-48, 55). 12. The chromosomes become individually smaller as cleavage progresses, and in general small nuclei give rise to smaller chromo- somes than do large nuclei (pp. 48-51, 55). 13. The size of the nucleoli (plasmasomes) depends upon the size of the nucleus and the length of the resting period; the larger the nucleus and the longer the resting period, the larger the plas- masomes become (pp. 51-53, 55-56). 14. Centrosomes and spheres of large cells are larger than those of smaller ones (pp. 53, 56). 15. The rate of growth of chromatin during the early cleavages of Crepidula (8 per cent for each division) harmonizing with the slight rate of increase of the oxidative power of the egg as de- termined by Warburg (p. 56). 16. My observations do not support the view that senescence is due to a decrease (Minot), or an increase (Hertwig) of nuclear, as compared with protoplasmic material; nor that rejuvenescence is accomplished during cleavage by the great increase of nuclear material relative to the protoplasm. On the other hand senes- cence seems to be associated with a decrease, rejuvenescence with an increase of metabolism (Child). Anything which decreases the interchange between nucleus and cytoplasm, such as products 86 EDWIN G. CONKLIN of differentiation and metabolism within the cell, or a dense nu- clear membrane, decreases metabolism and leads to senescence; anything which facilitates this interchange increases metabolism and leads to rejuvenescence. It is suggestive that in early devel- opment increased oxidation is associated with fertilization and mitosis (Loeb, Lyon, Warburg) (pp. 57-62). Part II. Experiments 17. By centrifugal force the substance of the eggs and blasto- meres of Crepidula may be stratified into a zone of heavy yolk at one pole, a zone of lighter oil and water at the other pole, and a zone of clear cytoplasm between these two; and since these eggs orient but slightly if at all while being centrifuged, the axis of centrifuging and of stratification may form any angle with the egg axis. In the early development of Crepidula the volume of yolk is much greater than the volume of cytoplasm and conse- quently the latter may be displaced to any side of the center of the egg or blastomere (p. 64). 18. On the other hand the mitotic figure, after the prophase, can be moved only with great difficulty, and owing to this fact the substances of a cell can be distributed in very atypical man- ner with respect to the poles of the spindle and the resulting daugh- ter cells. In this way all the yolk present in a dividing cell may be thrown into one of the daughter cells, and almost all of the cytoplasm into the other (p. 64). 19. These experiments show that the spindle is a specific structure and not merely a dynamic expression of lines of force. It remains in position and functions normally when the substance in which it usually lies is completely replaced by other substance. The spindle fibers are denser than the general cytoplasm and may be stretched, shortened or bent by pressure (p. 65). 20. If centrifuging occurs during the second maturation divi- sion, when the poles of the egg are clearly marked, the yolk may be driven to the animal pole and the cytoplasm to the vegetal pole, the spindle may be much elongated and a giant polar body may be formed,(fig.11). In such cases the sperm nucleus, which CELL SIZE AND NUCLEAR SIZE 87 enters the egg near the vegetal pole, lies in a cytoplasmic field, the egg nucleus in a yolk field, and the former grows more rapidly than the latter, thus reversing the usual size relations of the germ nuclei. The relative size of the germ nuclei is dependent upon the volume of the cytoplasm in which they lie as well as upon the length of time that the sperm nucleus has been in the egg (pp. 67, 75). 21. If centrifuging occurs during the cleavage almost all the yolk present may go into one daughter cell, almost all the cyto- plasm into the other (figs. 16, 19). Under these circumstances the subsequent growth of the daughter nuclei is proportional to the volume of the cytoplasm of the middle zone in which they lie. Neither the yolk nor the substances of the lighter zone con- tribute directly to the growth of the nucleus (pp. 75-76). 22. The size of spindle, centrosome, and sphere in any cell is not definitely fixed, but may be modified by altering the quantity of cytoplasm; the larger the quantity of cytoplasm in a cell, the larger are all the structures named (p. 77). 23. The rhythm of division may be modified, but only to a slight extent, by altering the quantity of cytoplasm in a cell. In general, cells rich in cytoplasm divide a little earlier than those poor in this substance; but though the quantity of cytoplasm in a cell and the size of its nucleus may be greatly changed by cen- trifuging, the rhythm of cleavage is but slightly changed (p. 77). 24. When the daughter chromosomes at one pole of a spindle are left in a cell composed almost entirely of yolk, they do not form a vesicular nucleus until yolk has been dissolved and a certain amount of cytoplasm has been formed around the chromo- somes. It is evident that something, perhaps an enzyme, is given off from the chromosomes or chromatin, which leads to the trans- formation of yolk into cytoplasm; this cytoplasm is in turn taken up by the chromosomes and ultimately contributes to the growth of the chromatin, (pp. 77-78). 25. The typical size, position and constitution of blastomeres, and consequently the type of cleavage, do not depend upon exter- nal or internal pressure, but upon a definite polarity, symmetry and movement of the cell contents, and upon reduced surface 88 EDWIN G. CONKLIN tension at the poles of the cell. Therefore, the causes of equal or unequal, differential or non-differential divisions are intrinsic rather than extrinsic (pp. 78-81). 26. Whenever the size, constitution or number of blastomeres is changed from the typical condition, subsequent cleavages come back to the normal form so far as this is possible. This regula- tion in cleavage is connected with a persistent polarity of the cell, which is not changed by centrifuging, and which manifests it- self in a definite cell axis passing through nucleus and centrosome, in typical movements and localizations of cell contents, and in reduced tension of cell membrane at the poles of the cell (pp. 81-83). LITERATURE CITED Battzer, F. 1908 Die Chromosomen yon Strongylocentrotus lividus und Echi- nus microtuberculatus. Arch. f. Zellforschung, Bd. 2. Boveri, Tu. 1902 Ueber mehrpolige Mitosen als Mittel zur Analyse des Zell- kerns. Verh. d. Phys. med. Gess. Wirzburg., Bd. 35. 1904 Ergebnisse iiber die Konstitution der chromatischen Substanz des Zellkerns. Jena. 1905 Zellenstudien V. Ueber die Abhangigkeit der Kerngrésse und Zellenzahl der Ausgangszellen. Jena. 1910 Ueber die Teilung centrifugierter Eier von Ascaris megalocephala. Arch. f. Entw. Mech., Bd. 30. Conxkuin, E. G. 1893 The fertilization of the ovum. Woods Hole Lectures. Ginn and Co., Boston. 1897 The embryology of Crepidula. Jour. Morph., vol. 13. 1899 Protoplasmic movement as a factor in differentiation. Woods Hole Lectures. 1902 Karyokinesis and cytokinesis in the maturation, fertilization and cleavage of Crepidula. Jour. Acad. Nat. Sciences of Philadelphia, vol. 12. 1905 The organization and cell-lineage of the ascidian egg. Idem, vol. 13. 1910 The effects of centrifugal force on the organization and develop- ment of the eggs of fresh water pulmonates. Jour. Exp. Zool., vol. 9. * Cuitp, C. M. 1911 A study of senescence and rejuvenescence based on experi- ments with Planaria dorotocephala. Arch. f. Entw. Mech., Bd. 31. ErpMANN, Rh. 1908 Experimentelle Untersuchung der Massenverhiltnisse von Plasma, Kernund Chromosomen in dem sich entwickelnden Seeige- lei. Arch. f. Zellforschung, Bd. 2. CELL SIZE AND NUCLEAR SIZE 89 *. EycitesHyMer, A. 1904 The cytoplasmic and nuclear changes in the striated muscle cell of Necturus. Am. Jour. Anat., vol. 3. GarpineER, E.G. 1898 The growth of the ovum, etc., in Polychaerus. Jour. Morph., vol. 15. GerassimorF, J. 1900 Ueber die Lage und die Function des Zellkerns. Bull. Soc. imp. Natur. Moscou, 1899. 1901 Ueber den Einfluss des Kerns auf das Wachstums der Zelle. Idem. 1902 Die Abhingigkeit der Grosse der Zelle von Menge ihrer Kern- masse. Zeitsch. f. allgem. Physiol., I. GoptewskI, H. 1908 Plasma und Kernsubstanz in der normalen und der durch aussere Faktoren veriinderten Entwicklung der Echiniden. Arch. f. Entw. Mech., Bd. 26. 1910 Plasma und’ Kernsubstanz bei der Regeneration der Amphibien. Arch. f. Entw. Mech., Bd. 30. Gurwitscu, A. 1908 Ueber Primissen und anstossgebende Faktoren der Fur- chung und Zellvermehrung. Arch. f. Zellforschung, Bd. 2. Hertwic, R. 1889 Ueber die Kernkonjugation der Infusorien. Abh. Bayer. Akad. Wiss., I] KI., Bd. 17. 1903 Ueber Korrelation von Zell- und Kerngrésse und ihre Bedeutung fiir die geschlechtliche Differenzierung und die Teilung der Zelle. Biol. Centralb., Bd. 22. 1908 Ueber neue Probleme der Zellenlehre. Arch. f. Zellforschung, Ba: Hoper, C. F. 1892 A microscopical study of the changes due to functional activity of nerve cells. Jour. Morph., vol. 7. Hogur, Mary J. 1910 Ueber die Wirkung der Centrifugalkraft auf die Eier von Ascaris megalocephala. Arch. f. Entw. Mech. Bd. 29. Liniiz, F. R. 1901 The organization of the egg of Unio, based on a study of its maturation, fertilization and cleavage. Jour. Morph., vol. 17. +1902 Differentiation without cleavage in the egg of the annelid Chae- topterus. Arch. f. Entw.-Mech., Bd. 14. 1906 Observations and experiments concerning the elementary phe- nomena of embryonic development in Chaetopterus. Jour. Exp. Zool., vol. 3. 1909a Polarity and bilaterality of the annelid egg. Experiments with centrifugal force. Biol. Bull., vol. 16. 1909b Karyokinetic figures of centrifuged eggs. An experimental test of the center of force hypothesis. Biol. Bull., vol. 17. Lituir, R.S. 1902 On the oxidative properties of the cell nucleus. Amer. Jour. Physiol., vol. 7. 1909 The general biological significance of changes in the permeability of the surface layer or plasma membrane of living cells. Biol. Bul., vol. 17. 1910 Physiology of cell division, II. Amer. Jour. Physiol., vol. 26. 90 EDWIN G. CONKLIN Lorg, J. 1899 Warum ist die Regeneration kernléser Protoplasmastiicke un- moglich oder erschwert? Arch. f. Entw. Mech., Bd. 8. 1909 Die chemische Entwicklungserregung des tierischen fies. Ber- lin, 1909. 1910 Ueber den autokatalytischen Charakter der Kernsynthese bei der Entwickelung. Biolog. Centralb., Bd. 30. Lyon, E. P. 1904 Rhythms of susceptibility and of carbon dioxide production in cleavage. Am. Jour. Physiol., vol. 11. 1907 Results of centrifugalizing eggs. Arch. f. Entw. Mech., Bd. 28. Masina, HE. 1910 Ueber das Verhalten Neucleinsiure bei der Furchung Seeig- leies. Hoppe-Seylers Zeitschrift. Bd., 67. Review by Godlewski, Arch. f. Entw. Mech., Bd. 31. Minor, GC. 8. 1890 On certain phenomena of growing old. Proc. Amer. Ass’n Adv. Sci., vol. 29. 1895 Ueber die Vererbung und die Verjungung. Biol. Central., Bd. 15. 1908 Age, growth and death. Putnams, New York. Montcomery, T. H. 1910 On the dimegalous sperm and chromosomal variation of Huschistus, with reference to chromosomal continuity. Arch. f. Zellforschung, Bd. 5. Moraan, T. H. 1910 Experiments bearing on the nature of the karyokinetie figure. Proc. Soc. Exp. Biol. and Medicine, vol. 7. Perer, K. 1906 Der Grad der Beschleunigung tierischen Entwicklung durch erhdhte Temperatur. Arch. f. Entw. Mech., Bd. 20. Pororr, M. 1908 Experimentelle cytologische Studien. Arch. f. Zellforschung, Bd. 1. SHackett, L. F. 1911 Phosphorus metabolism during early cleavage of the echinoderm egg. Science, vol. 34, no. 878. SrTrRAsBuRGER, E. 1893 Ueber die Wirkungssphire der Kerne und die Zell- grosse. Histolog. Beitrige, Bd. 5. Verworn, M. 1891 Die physiologische Bedeutung des Zellkernes. Arch. f. d. ges. Physiol., Bd. 51. WarTasez, 8. 1893 On the nature of cull organization. Woods Hole Lectures. Witson, E. B. 1904 Experimental studies on germinal localization. Jour. Exp. Zool., vol. 1. DESCRIPTION OF FIGURES All figures (with the exception of figs. 9 and 10) represent entire eggs of Crepid- ula plana, fixed, stained, and mounted on slides. They were drawn with the aid of a camera lucida under Zeiss Apochromat 3 mm., Ocular 4, and represent a mag- nification of 333 diameters. In the centrifuged eggs, the axis of centrifuging is, in many cases, indicated by an arrow, the head of the arrow marking the distal (heavy) pole and the tail of the arrow the central (light) pole. In figs. 12 to 19, and 29 to 37 the first cleavage is in the long axis of the page, the second cleavage (figs. 18 and 19) is at right angles to this. In figs. 21 to 28 the first cleavage runs across the page, the second, lengthwise of it. CELL SIZE AND NUCLEAR SIZE 91 Figs. 1-6 Successive stages in the development of the egg of C. plana, showing the maximum sizes of the nuclei of the macromeres. Fig. 1, 4-cell, just before third cleavage; fig. 2, 8-cell, just before fourth cleavage; fig. 3, 16-cell, just before fifth cleavage; fig. 4, 24-cell, just before sixth cleavage in macromere 3D; fig. 5. 42-cell, just before sixth cleavage in macromeres 3A-3C; fig. 6, Gastrula, just before seventh cleavage of the macromeres. 92 EDWIN G. CONKLIN ny é . ( SSA gt) al : > cf : VIN ECan i 2 aT » = (% re o Ne 9 LO Fig. 7 2-cell stage of C. plana. The nuclei just before the second cleavage are 24u in diameter. Fig. 8 12-cell stage of C. plana. The nuclei in the first quartet of micromeres, la-ld, three of which are dividing, are 14u in diameter at their maximum size. Fig.9 Chromosomes from four different spindles of the second cleavage, all in the metaphase and all magnified 2000 diameters. Fig. 10 Chromosomes from four different spindles of the cells la-1d, all in the metaphase and all magnified 2000 diameters. Fig. 11 Egg centrifuged ten minutes after formation of first polar body and during formation of second; fixed three hours after centrifuging. Telophase of second maturation division; indication of formation of enormous second polar body. The size of nuclei is dependent upon quantity of cytoplasm in which they lie. Fig. 12. Centrifuged fifteen minutes in gum arabic, fixed three hours later. Evidently centrifuged during first cleavage; almost all of the cytoplasm is in the smaller cell. The size of the nuclei is proportional to the quantity of cytoplasm. CELL SIZE AND NUCLEAR SIZE 93 Fig. 13 Centrifuged five hours (2000 revolutions per minute) during the first cleavage; fixed at once; structure similar to preceding. Fig. 14 From the same experiment as the preceding. Size of nuclei is propor- tional to the quantity of clear (granular) cytoplasm; yolk and oily or watery con- stituents of the cytoplasm do not influence nuclear size. Fig. 15 From the same experiment as the preceding, and showing similar results. Fig. 16 Centrifuged thirty minutes; fixed twenty hours after centrifuging. Egg has not developed. Enormous difference in the size of sister nuclei. 94 EDWIN G. CONKLIN Fig. 17 Centrifuged four hours (2000 revolutions per minute); fixed six hours after. Evidently centrifuged during first cleavage. The cleavage plane does not pass through the polar axis. The spindles are proportional to the size of nuclei from which they were formed, and to the volume of cytoplasm in which they lie. They are not parallel to the plane of the first cleavage, which is here out of its normal position. Fig. 18 Centrifuged fifteen minutes in gum arabic; fixed three hours after. Evidently centrifuged during first cleavage. The second cleavage appeared earlier in the more protoplasmic cells (A and B), than in the others. Fig. 19 Centrifuged thirty minutes; fixed six hours later. Evidently centri- fuged during the first cleavage. The size of the nuclei is plainly dependent upon the volume of the cytoplasm in which they le. Tig. 20 Centrifuged four hours, (2000 revolutions per minute); fixed at once. Evidently centrifuged during the second cleavage; the daughter nuclei are pro- portional in size to the volume of cytoplasm in which they lie. CELL SIZE AND NUCLEAR SIZE 95 Fig. 21 Centrifuged thirty minutes; fixed at once. Centrifuged during the second cleavage, which was thus made very unequal, two of the macromeres (A and D) containing all the yolk and the other two (B and C) being small and purely protoplasmic. Fig. 22 Centrifuged thirty minutes; fixed twenty-one hours later. The second cleavage was suppressed. Two spindles for the third cleavage are present in each cell, but the cell body shows no signs of division. Fig. 23 Centrifuged thirty minutes, during the second cleavage; fixed six hours later; two of the macromeres are small and protoplasmic; tetrasters are present in the other two. Fig. 24 Same slide as preceding. All of the macromeres have given rise to normal micromeres of similar size, although two of the macromeres are small and purely protoplasmic while the other two are large and contain much yolk and little cytoplasm. 96 EDWIN G. CONKLIN Fig. 25 Same slide as preceding. The minute protoplasmic ‘macromeres’ (A and D) have divided equally into the macromeres 1A and 1D and the micro- meres Ja and id. The other macromeres (B and C) have given rise to micromeres somewhat larger than usual. Fig. 26 Centrifuged for thirty minutes during the second cleavage; fixed twelve hours later; the nuclear divisions of the second cleavage were completed, but the cell divisions were suppressed. Each of these two binucleate macromeres has given rise to two first, and two second quartet cells, just as if four macromeres were present, and each of these micromeres has subdivided in approximately nor- mal manner and is uni-nuclear. Fig. 27 Centrifuged for thirty minutes in 2-cell stage; fixed six hours later. Micromeres formed from protoplasmic macromeres are of the same size as those formed from large yolk macromeres. Fig. 28 Same as preceding. The regulation in the formation of micromeres is complete. Fig. 29 Centrifuged four hours (2000 revolutions per minute); fixed at once. The yolk was thrown out into lobes, one of which has been detached; the smaller nuclei are in the smaller cells. Fig. 30 Centrifuged ten minutes in gum arabic during first cleavage; fixed four hours later. Asters and spindles are proportional to the volume of the cytoplasm. Fig. 31 Centrifuged four hours during the first cleavage; fixed six hours later. Most of the cytoplasm is in the smaller macromeres and these have divided earlier than the larger ones. At least one-half of the cytoplasm in the larger macromeres goes into the micromeres. The first cleavage is not strictly meridional and the spiral form of division is lost. (For explanation of figs. 32 and 33, see p. 98). THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 97 98 EDWIN G. CONKLIN Fig. 32 From the same slide as the preceding. Though the macromeres differ in size and protoplasmic content, the micromeres are all of the same size. Fig. 33 Centrifuged ten minutes in gum arabic; fixed four hours later. The protoplasmic macromeres are dividing earlier than the others. The size of the micromeres does not depend upon the quantity of cytoplasm in the macromeres from which they came. Fig. 34 Centrifuged two and one-half hours; fixed twenty-one hours later. The size of the micromeres is almost irrespective of the size of the macromeres; also it is nearly independent of the amount of cytoplasm in the macromeres. Fig. 35 Centrifuged thirty minutes during the first cleavage; fixed twelve hours later. The micromeres from the protoplasmic macromeres are but little larger than those from the yolk cells. Fig. 36 From the same slide as the preceding, showing essentially the same conditions. Fig. 37 From the same slide as the preceding. The cell 4c forms at the same time as 4d, though in normal eggs it is formed much later. STUDIES ON THE PHYSIOLOGY OF REPRODUCTION IN THE DOMESTIC FOWL V. DATA REGARDING THE PHYSIOLOGY OF THE OVIDUCT! RAYMOND PEARL anp MAYNIE R. CURTIS FOUR FIGURES INTRODUCTION The oviduct of a laying hen is divided into five main parts, readily distinguishable by gross observation. Beginning at the cranial end of the organ these parts, in order, are: (a) the infundibulum, or funnel, (b) the albumen secreting portion, (c) the isthmus, (d) the uterus or ‘shell gland’ and (e) the vagina. Each of these parts is generally supposed (teste the existing liter- ature) to play a particular and exclusive réle in the formation of the protective and nutritive envelopes which surround the yolk in the complete egg as laid. Thus the funnel grasps the yolk at the time of ovulation; the glands of the albumen region secrete the different sorts of albumen (thick and thin) found in the egg; the shell membranes are secreted in the isthmus; and finally the glands of the uterine wall secrete the calcareous shell. This is in brief the classical picture of the physiology of the oviduct. The gross anatomical appearance and relation of the several parts of the oviduct of the fowl are shown in fig. 1. ‘Papers from the Biological Laboratory of the Maine Experiment Station, No. 33. The previous papers in this series of ‘Studies on the Physiclogy of Reproduction in the Domestie Fowl” are: 1. Regulation in themorphogenetic activity of the oviduct. Jour. Exp. Zodl., vol. 6, pp. 839-359, 1909. ir. Data on the inheritance of fecundity obtained from the records of egg pro- duction of the daughters of ‘200-egg’ hens. Maine Agricultural Experiment Station, Annual Report for 1909, pp. 49-S4. ur. A ease of incomplete hermaphroditism. Biological Bulletin, vol. 17, pp. 271-286, 1909. tv. Data on certain factors influencing the fertility and hatching of eggs. Maine Agricultural Hxperiment Station, Annual Report for 1909, pp. 105-164. 99 100 RAYMOND PEARL AND MAYNIE R. CURTIS Fig. 1 Photograph of a hen’s oviduct which has been removed, slit longitudi nally throughout its length and opened out flat in order to show the gross anatomy. In order to get the whole duct on the photographic plate it was necessary to tran- sect it at about the middle. A, the infundibulum. Note muscle fibers in wall and absence of any extensive gland development. £, albumen secreting portion; note heavy glandular development. The albumen portion ends and the isthmus begins at z. The line of demarcation is very distinct in the freshly prepared ovi- duct. C, the isthmus. D, the uterus or shell gland. £, the vagina; about one- third natural size. PHYSIOLOGY OF THE OVIDUCT 101 For some years past experiments and observations have been systematically carried on in this laboratory with the object of acquiring: a more extended and precise knowledge of the physi- ology of the hen’s oviduct than is to be gained from the literature. It is the purpose of this paper to present a certain part of the results obtained bearing upon the physiology of two of the lower (caudal) morphological divisions of the duct, namely, the isthmus and the uterus. Our results indicate that these portions of the oviduct perform certain functions which have not hitherto been observed or described. So far as we are able to learn from the existing literature the opinion has been held by all who have worked upon the subject that the particular functional activity of each portion of the oviduct (as above described) is limited to that portion. Thus it is commonly held that when an egg in its passage down the oviduct leaves the albumen portion it has all the albumen it will ever have; when it leaves the isthmus it has all its shell mem- branes; and when it leaves the uterus all its shell. On this pre- vailing view there are in the albumen portion only albumen secreting glands; in the isthmus only membrane secreting glands; and in the uterus only shell secreting glands. We were first led to doubt the entire adequacy of this assumption by the observa- tion, frequently made in connection with routine autopsy work, that eggs in the isthmus with completely formed shell membranes, and eggs in the uterus bearing in addition to the complete shell membranes a partially formed shell, weighed considerably less than the normal average for laid Barred Plymouth Rock eggs. This observation led to an inquiry as to whether (a) this apparent lower weight of presumably completed, but not laid eggs, as compared with those which had been laid, was a real phenomenon of general occurrence, and (b) if so, to what it was due. Does the egg increase in weight after the formation of shell membranes and shell merely by the absorption of water, or by the actual addition of new albumen? These are the problems with which the present paper has to do. We may now turn to the consideration of the observational and experimental data. 102 RAYMOND PEARL AND MAYNIE R. CURTIS THE DISTRIBUTION OF THE DIFFERENT KINDS OF ALBUMEN IN THE EGG In the normal egg of the hen there are certainly three and possibly four different albumen layers which can easily be dis- tinguished on the basis of physical consistency. These are: (A) the chalaziferous layer. This is a thin layer of very dense albuminous material which lies immediately outside the true yolk membrane. It is continuous at the poles of the yolk with the chalazae, and is undoubtedly found in connection with those structures. It is so thin a layer that it might well be, and often has been, taken for the yolk membrane. (B) The inner layer of fluid (thin) albumen. This layer is only a few millimeters in thickness and there is some doubt as to its existence as a sep- arate, distinct layer. (C) The dense albumen. This is the layer which makes up the bulk of the ‘white’ of the egg. It is com- posed of a mass of dense, closely interlaced albumen fibres, with some thin fluid albumen between the meshes of the fibrous net- work. The dense albumen as a whole will not flow readily, but holds itself together in a flattened mass if poured out upon a plate. (D) The outer layer of fluid albumen. This is the prin- ciple layer of thin albumen, which makes up the fluid part of the ‘white’ observed when an egg is broken. Three of these layers, A, C, and D are readily demonstrable and there can be no question whatever as to their existence. Regarding the existence of B as a separate and distinct layer there is more doubt. Gadow? definitely asserts the existence of such a layer in the following words: ‘‘Dicht auf der Dotterhaut befindet sich eine diinne Lage des fliissigen Eiweisses.”” It is possible that what has been taken by previous observers to form this layer B is only a little thin albumen squeezed out of the meshes of the dense layer (C) when the egg is broken. Let us now consider the distribution of the different sorts of albumen in eggs at different stages in their passage down the oviduct. The following extracts from autopsy protocols are to the point here. ° Gadow, H., Vogel (Anatomischer Theil); in Bronn’s Klassen und Ordnungen des Thier-Reichs. Leipzig, 1891, p. 869. PHYSIOLOGY OF THE OVIDUCT 103 Autopsy No. 370. Hen No. 952. March 19, 1910 Egg found in the albumen portion of the oviduct 11 cm. in front of the cranial end of the isthmus. This egg consisted of a yolk surrounded by thick albumen (layer C) but with no trace whatever of the albumen layer D. Not yet having entered the isthmus the egg lacked a shell membrane. Autopsy No. 332. Hen No. 420. December 22, 1909 This case was similar to that just cited. Here an egg was in the albumen portion of the duct with its caudal end 6 em. in front of the cranial end of the isthmus. The egg consisted of yolk surrounded by dense albumen (layer C). There was no trace of the thin albumen (layer D) to be observed. The egg had no shell membrane. Autopsy No. 366. Hen No. 276. March 18, 1910 Egg in albumen portion of oviduct with its caudal end 4 cm- in front of the cranial end of the isthmus. This egg had no shell membrane. The yolk was surrounded by thick albumen (layer C). The egg bore no trace of the thin albumen (layer D), even though it was only this short distance (4 em.) from the point where the ‘albumen secreting’ portion of the duct was finally to be left. Autopsy No. 301. Hen No. E39. July 14, 1909 When this bird was killed an egg was found at the lower end of the albumen portion of the oviduct just about to enter the isthmus. Not yet having entered the isthmus the egg had no shell membrane upon it. It consisted merely of a yolk surrounded by albumen. The outermost layer of this albumen was dense and corresponded to layer C described above. There was no trace of thin albumen (layer D) on this egg although it was just on the point of leaving the so-called albumen region of the oviduct. 104. RAYMOND PEARL AND MAYNIE R. CURTIS Autopsy No. 369. Hen No. 1154. March 19, 1910 Egg in the lower end of the albumen portion of the oviduct just at the point of entering the isthmus. This egg had no mem- brane. The yolk was surrounded by albumen layers A, B, and C. No trace of the outer thin albumen (layer D) was to be found. All these cases agree in showing that the egg does not receive the outer layer of thin fluid albumen (layer D) during its sojourn in the so-called albwmen secreting portion of the oviduct. While but five specific autopsy records are cited here it is only fair to say that this result is confirmed by all our experience with eggs in the albumen secreting portion of the oviduct. This experience covers many more than the five cases given here. These cases are chosen as particularly significant, however, because in them we have definite quantitative records of the exact location of the egg in the albumen portion. The successive autopsy records show that beginning with an egg 11 cm. away in front of the isthmus and going downwards in the duct until the actual boundary of the isthmus is reached, there is no qualitative change in the albu- men secretion. Whatever albumen is added to the egg imme- diately prior to the formation of the shell membrane, is of the dense fibrous variety (layer C), so far as direct observation indi- cates. The fact that there is no thin albumen on the egg when it enters the isthmus is shown in fig. 2. In this figure A shows an egg of hen No. 8018 which was removed from the upper part of the isthmus. The thin membrane which had been formed was removed; the egg placed in a Petri dish and photographed. For comparison a normal laid egg from hen 8018 was broken into a Petri dish in the same way. Its photograph is shown in fig. 2, B. It is at once apparent that there is a great difference between the two eggs in respect to amount and consistency of albumen. In the egg which had just left the albumen portion of the oviduct (ege A) the albumen is of firm consistency and retains its shape, forming a compact mass about the yolk. In the laid egg (egg B) the albumen is much thinner, and does not hold its shape, but flows out over the bottom of the dish. PHYSIOLOGY OF THE OVIDUCT 105 8018, with p bird, B isthmus of the oviduct of hen No. For further explanation see text. aid egg of the same al size. an egg taken from the Showing A 106 RAYMOND PEARL AND MAYNIE R. CURTIS Crucial evidence is here afforded by those cases occasionally to be observed, where the egg is Just entering the isthmus, and has one end in the albumen portion of the duct and the other end inthe isthmus. It was first pointed out by Coste? that the forma- tion of the shell membrane at the upper end of the isthmus is a discrete process. That is, as the end of the egg advances from the albumen portion into the isthmus, membrane is deposited upon it. The membrane is complete over the whole egg only after the egg has entirely passed into the isthmus. This account of membrane deposition we have confirmed by direct observation in this laboratory. Now in cases where one-half of the egg lies within the isthmus and bears a membrane while the other half is in the albumen portion and has no membrane it can plainly be seen that the shell membrane is deposited directly on the outer surface of the thick albumen (layer C) and that no trace of the thin albumen (layer D) is present at the time the membrane is formed. It might be contended that the thin albumen which is to form layer D is really present at the time the membrane is deposited, but that instead of forming a separate outer layer it is held by adhesion or otherwise within the meshes of the fibrous network of the dense albumen of layer C. On this view it might be sup- posed that this more fluid albumen passes out of the network to form a definite and separate layer at some time after the mem- brane is laid down. This contention, however, cannot be correct, because, as will be demonstrated in the next section of the paper, the egg does not have its full complement of albumen by weight at the time when the shell membrane is formed. The fluid albumen of layer D should weigh just as much, whether in the interstices of a fibrous meshwork, or forming a separate layer. Yet the facts show that after a thin albumen (layer D) has been visibly formed the egg contains by weight about 50 per cent more albumen than it did before this layer was visibly formed. 8 Coste, M., Histoire du développement des corps organisés, tome 1, p. 295, 1847. PHYSIOLOGY OF THE OVIDUCT 107 THE PROPORTIONATE WEIGHT OF YOLK AND ALBUMEN IN EGGS IN DIFFERENT STAGES OF FORMATION Having learned by direct observation, as set forth in the preceding section that the egg as it enters the isthmus does not visibly bear the outer layer of thin albumen, the next step in the analysis is to determine whether the amount of albumen (by weight) in the egg definitely increases during its sojourn in the isthmus and uterus, and if so to what extent. In order to do this it is necessary to take eggs at successive intervals after they have entered the isthmus, separate and weigh yolk and albumen each by itself, and then compare the weights so obtained with the weights of yolk and albumen in normal, completely formed and laid eggs produced by the same individual birds. Experiments of this kind we have carried out with the results described in this section of the paper. It should be said that the technique followed in the separating and weighing of the eggs to furnish these data is that described by one of the authors in another place.‘ Table 1 gives data regarding the weight of yolk and albumen in eggs which have completed their passage through the albumen secreting portion of the oviduct, and have advanced varying distances into the isthmus and shell gland. The data here given are extracted from the more detailed table exhibited in the Appen- dix of this paper. The plan of table 1 is to compare the weights of the parts of a series of eggs taken from different levels of the oviduct with the weights of the same parts in normal laid eggs of the same birds. Owing to the considerable individual variability in the weights of eggs it is only by such comparisons as this that reliable results may be reached. To determine the means for the normal laid egg varying numbers of eggs were used in different cases. In one instance (item 3) only one laid egg was available for compari- son. In all other cases the mean of two or more complete nor- mal, laid eggs are used. It will be noted that in somecases ‘Curtis, M. R., Annual Report Maine Agricultural Experiment Station, 1911, pp. 93-112. 108 RAYMOND PEARL AND MAYNIE R. CURTIS (items 3, 4, 6, 9, and 15) the weights are given only to one decimal place (tenths of a gram). These were eggs studied in the early stage of the investigation, and mostly are cases in which the data were taken in connection with other studies in progress in the laboratory, for which finer weighing was not essential. These cases are to be regarded as giving a much rougher sort of data than the others tabled, where the weighings are accurate to hundredths of a gram. In no instance, however, does one of these ‘rough’ eases stand alone. That is, there are one or more eges for which finer weighing are tabled from each of the levels of the oviduct wherefrom a roughly weighed egg was taken. These ‘rough’ cases then serve merely to confirm evidence ob- tained from more precise weighing. All differences in the table are given the + sign when the oviduct egg or its part is greater than the laid egg or its part. The differences are taken — when oviduct egg or its part is smaller. The last column of the table gives the percentage which the weight of albumen in the oviduct egg at the specified level is of the mean total weight of albumen in the normal laid egg of the same bird. This last column then shows directly what proportion of the total albumen which the egg is to have has been laid down at each specified level of the oviduct. From table 1 the following points are to be noted: 1. When the egg leaves the albumen portion of the oviduct it weighs roughly only about half as much as it does when it is laid. Nearly all of this difference is in the albumen. Thus these weighings fully confirm the conclusion reached from direct exam- ination of the eggs, as described in the preceding section. The evidence thus far presented shows that the egg gets all of its thin albumen (layer D), which constitutes nearly 60 per cent by weight of the total albumen, only after it has left the supposedly only albumen secreting portion of the oviduct, and has acquired a shell membrane, and the shell is in process of formation. The fact that the egg increases considerably in size after it enters the isthmus is obvious from simple visual comparison of egg from this region of the oviduct with normal laid eggs of the same bird even though no weights whatever are taken. PHYSIOLOGY OF THE OVIDUCT 109 ‘ TABLE 1 Data showing the increase in absolute and relative weight of albumen after the egg has passed through the so-called albumen secreting part of the oviduct a | z S ag = ZZ a 5<2 B aS Bisa ae LOCATION OF EGG IN OVI{DUCT ES pes , eriind 33 | 2S A hes he mE * Bb = offs on oR x | °a a me mS a) ae aN 3 Bisa Bue a< Ss ae & z z 2 = 2 | grams grams | grams grams 1. At caudal end of albu- men portion. No mem- brane (Hen8009)...... 27.20 0 15.38 11.82 76.9 | 34.2 Mean of the four previ- ously laid eggs of same INES TURP aytet oiFs sous he ys ag 57.57 6.23 16.7! 34.55 205.8 WMifference!. . <2 22) sn)- —30.37 -—6.23 —1.41 —22.73 | —128.9 2. At caudal end of albu- men portion. No mem- brane (Hen 8005)........ 29.53 0 15.87 13.66 86.1} 43.6 Mean of the nine pre- viously laid eggs of same IGM, oo o68d6 eoenapaepaee |e ett | SB Iak) 15.67 31.34) 200.0 | _ Difference RCo Ss Cob ren —23.28 —5.79 +0.20 —17.68 | —113.9 | 3. Just entering isthmus, — : little cap of membrane on caudal tip. (Hen OB on mre ravers teid syatarare: Aaah s 29.5 0 16.0 13.5 84.4) 43.5 Mean of two previously laid eggs of samehen... 56.25 9.0 | 16.25 31.0 190.8 | Difference........... eg | eter O00 ee 0-201) —17ep0 ||) —10b |e 4, Entering isthmus. Cov- ered with membrane ex- cept for a little of crani- al tip. (Hen 266F).... 31.0 14.5 16.5 113.8 53.2 Normal egg laid by hen RETO GEN 2 SBopmaOosenene 51.0 6.0 14.0 31.0 221.4 TOURELEN CON. 6s ola bess —20.0 0.0) jo 4-5) 1076 ‘ 5. Entering isthmus. Covered with membrane exceptfor alittle of crani- al tip. (Hen 8027)......| 32.44 0.24 15.69 16.51 105.2} 51.5 Mean of the four pre- viously laid eggs of same GIANT 2S. k acces en 54.69 5.76 16.87 32.07 190.2 = = —— Difference..............| —22.25| —5.52 —1.18 | —15.56 —85.0 110 RAYMOND PEARL AND MAYNIE R. CURTIS TABLE 1—Continued = = eee = : - & nD 2 Fs ee rs 2 623 5 8% Ar ad LOCATION OF EGG IN OVIDUCT = nits an as 215 ZZ 3 Baz eae Ba Ms 8B < 228 Ae 2a % AG! 8 am ae - S|) ae grams grams grams grams 6. In upper part of isth- : mus. Membrane com- plete, but thin. (Hen DOA) 'S Acpsie ie siete ee olet he 32.0 16.5 15.5 93.9 | 48.1 Mean of two previously, | laideggsofsamehen....| 60.5 8.75 | 19.5° 32:25) 165.4 Differencenss eee e —28.5 | =) || N50) | 7. In upper part of isth- mus. Membrane thin. | (Elen 8008) ee tepec ae eleezonoo 0.28 12.57 15.48 123.2} 49.5 Mean of the five previously, laideggsofsamehen....) 50.20 5.54 13.39 31.27 DBRT) _ Difference....... eee —21.87 | —5.26| —0.82 | —15.79 | —110.3 8. Three cm. below begin- ning of isthmus. Mem- | brane thin. (Hen 1367). 32.08 | 0.39 16.12 15.57 96.6 56.9 Mean of nine previously _ laid eggs of same hen.... 48.45 5.40 15.70 27.34 | 174.1 Difference te eee SiGe | 3.00 ey 2a aa | 9. Two em. above caudal end of isthmus. (Hen : 4G) ccs Seebia.c2 = eerie: | 31.0 2.0 16.0 13.0 81.3] 50.5 Mean of two previously laideggsofsamehen.... 47.5 7.0 | 14.75 25.75 174.6 _ Differences... ee: | —16.5 —5.0 +1.25 | —12.75| —93.3 | 10. In lower part of isth- mus. (Hen 8010)....... 30.00 0.53 14.04 15.43 109.9} 51.3 Mean of eleven previously | laideggsofsamehen.... 51.54 5.77 | 15.72 30.06 | 191.2 | Difference. Bape ore —21.54 5.24 1.68 14.63 | —81.3 11. In lower part of isth- mus. (Hen8018)........ 37.27 0.58 16.37 20.32} 124.1 | 56.4 Mean of four previously laideggsofsamehen..... 60.10 | 6.63 17.42 36.05 206.9 —1.05 | -15.73 | —82.8 | Difference:.. 722.52 ceeer |=222°83 —6.05 5 This is too high a value, probably arising from errors in separation of yolk and white. These particular data were taken before the refinement of method used in later studies had been worked out. PHYSIOLOGY OF THE OVIDUCT 111 TABLE 1—Continued g af | | LOCATION OF BGG IN OVIDUCT e Pare} Hy az 315 za eae cea g 5 a8 xh aus 5 ana am ax S aS a E z = S a grams grams grams grams 12. In uterus, but no shell found. Egg surrounded - by fluid in uterus. (Hen BOSS) Beret i tett ase 3505 os, + 43.93 0.76 17.30 25.87 149.5 | 71.1 Mean of four previously laideggsofsamehen....| 60.92 6.76) 17.77 | 36.39 204.8 __ ADT ONC Cscpccnbancease =16.99 | —6.00| —0.47 | —10.52| —55.3 13. In uterus, but no vis- ible shell formed. (Hen 208 Ue gba ADE eaeor naa 45.15 0.96 17.25 26.94 156.2 75.5 Mean of the six previously | laideggsofsamehen....} 59.22 6.22} 17.34 35.66 205.7 Difference: 0. cc. vue oe —14.07 —5.26 0.09 8.72 49.5 14. In uterus but no vis- ible shell formed. Some fluid in uterus. (Hen USO) Metach trate sianse-ne ope ers 46.67 0.88} 19.13 26.66 139.4 | 80.7 Mean of two previously laideggsofsamehen....| 58.99 6.91 19 04 33.04 173.5 Wifferences:.). .-c6< .0 =~ —12.32 -—6.03| +0.09| -—6.38 | —34.1 15. In uterus, small amount of shell formed. (TenSLOGT) rer epie tert 45.00 4.50 15.50 25.00 161.3 87.0 Mean of two previously laideggsofsamehen....) 52.00 7.50 15.75 28.75 182.5 ID TiC Ra eee —7.00| —3.00}| —0.25| -—3.75 | —21.2 16. In uterus, some shell formed. (Hen 8021)....| 43.66 1.35 15.18 Bias 178.7 | 95.1 Mean of three previously | laid normal eggs of same |nYoi hs Stee aoe oN gese See 49 26 5.18 15.56 28 .52 183.3 Difference..........-... —5.60| —3.83| —0.38, -1.39| —4.6 112 RAYMOND PEARL AND MAYNIE R. CURTIS Thus in fig. 3 are shown (1) a membane covered egg taken from the isthmus of the oviduct of hen No. 8018 shortly before it would have entered the uterus, and (2) two normal laid eggs of the same bird. The larger size of the latter is obvious. The isthmus egg is the one at the extreme left in the picture. 2. It is apparent from examination of the differences in the columns giving albumen weights and albumen-yolk ratios that in general the farther down the oviduct the egg proceeds the more albumen it gets. Very nearly one-half the total weight of albu- men of the completed egg is added in the uterus, an organ hither- to supposed to be entirely devoted to shell formation. Clearly very much more albumen is added to the egg in the uterus than in the isthmus. This, of course, does not necessarily mean any more rapid rate of secretion in the uterus, because of the time element involved. The egg stays much longer in the uterus than in the isthmus. 3. This brings us to a consideration of the question of the rate of secretion of albumen in different positions of the oviduct. We have attempted to approach this problem by the graphical method. The results obtained are not to be regarded as highly accurate in respect to minute details. It is an exceedingly diffi- cult matter to get very precise data in the individual instances regarding time relations in the physiology of the oviduct. We must therefore depend upon average results. The attempt has been made in fig. 4 to show graphically the net average results from the data collected in this laboratory regarding the time taken in the passage of the egg through the several portions of the oviduct and the rate of secretion of albumen in the same portions. As a measure of the albumen is taken the percentage of the total albumen of the laid egg which has been acquired at each specified level of the duct. The time is plotted as abscissa, and the per- centage of albumen as ordinate. It is not possible to recount here in detail all the evidence on which the points in this diagram are based. It would involve the presentation of considerable material which has no direct bearing on the subject of the present paper. We shall, therefore, be obliged to state only briefly, and in some degree categorically, PHYSIOLOGY OF THE OVIDUCT 113 the ind two normal laid eggs of left isthmus (hen SO18) at the from the LZ covered ¢ natural a membrane Showing yird for comparison Pig 114 RAYMOND PEARL AND MAYNIE R. CURTIS a =) TONES OF TOTAL ALBWITEN =) N 8 J HOURS O / 2 é ALBUMEN PORTION Us UTERUS Fig.4 Diagram showing what percentage of the total amount of albumen present in the normal laid egg of the domestic fowl is present at successive levels in the oviduct. The smooth curve is the parabola for which the equation is given in the text. the manner in which the plotted points were determined. In the first place it has been found in our work here that when a hen is laying regularly one egg per day ovulation occurs at approximately the same time as laying. That is the odcyte which will be laid as a completed egg tomorrow enters the infundibulum at the time when today’s passes through the vagina and is laid. This then is taken as a fundamental datum in the calculation of the rate of passage of the egg down the oviduct. The mean of all available observations made in this laboratory gives 3.2 hours as the time required for the passage of the egg through the ‘albumen portion’ of the oviduct. This includes the total time from the entrance of the egg into the infundibulum to its entrance into the isthmus. This agrees very well with the statements of earlier workers® who generally give the time spent in the albumen portion of the duct as ‘about’ three hours. 6 Cf. Lillie, F. R. The Development of the Chick. New York, 1908, pp. 23-25. PHYSIOLOGY OF THE OVIDUCT 115 In regard to the time taken by the egg in passing through the isthmus our observations are far from agreeing with the state- ments on this point in the literature. Taking the mean of all available data 0.6 of an hour is found to cover the time during which the egg is in the isthmus. All our observations agree well amongst themselves, and we are convinced that this figure is substantially correct for the breed of fowls here used (Barred Plymouth Rocks). This is a much shorter time than earlier workers have estimated. Thus Gadow’ says: ‘‘Im Isthmus soll das Ei ungefaéhr 3 Stunden lang verweilen.”’ Lillie’ gives the same estimate on the authority of Kélliker. Patterson? who has published most recently on this matter, while reducing some- what the time for passage through the isthmus, still gives a value considerably higher than that found in the work of this laboratory. His statement is as follows (loc. cit., p. 105): ‘‘The writer finds that in a hen kept under normal conditions, the egg traverses the entire length of the oviduct in about twenty-two hours. The time occupied in the different portions of the oviduct is as follows: Glandular portion, three hours; isthmus, two to three hours; uterus and laying sixteen to seventeen hours.” With all parts of this statement except that relating to the isthmus our results are in entire agreement. At an early stage of the studies in this laboratory on the physiology of the oviduct we were of the same opinion as Patterson as to the time taken in passing through the isthmus. More extended observations, covering a fairly wide range of conditions has convinced us that, as already stated, the egg normally takes less than one hour in passing through the isthmus. It is, of course, possible that there are breed differences in respect to the time the egg stays in the isthmus, and that Barred Plymouth Rocks are strikingly exceptional in this regard but this hardly seems probable. It is more likely that the estimate of earlier workers has been somewhat too large. 7 Gadow, H., loc. cit., p. 872. § Loc. cit. ® Patterson, J. T., Journal of Morphology, vol. 21, pp. 101-134, 1910. 116 RAYMOND PEARL AND MAYNIE R. CURTIS As stated in the preceding paragraphs the points on the time or abscissal axis represent the mean or average results of fairly extensive experimental data, some of which are not included in the present paper. Now we may consider the determination of the points plotted as ordinates. At the outstart it should be said that no observations have yet been made on the rate of secretion of albumen at different levels of the ‘albumen portion’ itself. Therefore from the zero point at ‘ovulation’ when the naked yolk enters the infundibulum to the beginning of the isthmus we have connected the points with a dotted line to indicate that in this region no direct observations are available. The first plotted point (40.4) at the beginning (cranial end) of the isthmus is the mean albumen percentage of three eggs which were taken from this point in the duct.. The next point plotted is the mean albu- men percentage (50.6) of four isthmus eggs which were taken from the upper part of the isthmus. The next point (53.8) is the mean albumen percentage of four isthmus eggs taken from the lower part of the isthmus. The last six points are based on the obsery- vations of single eggs which have been in the uterus the indicated length of time. The smooth curve which graduates these observations is the parabola y = 17.5915z — 0.81712? — 0.4164, in which y denotes percentage of albumen and z time in hours during which the egg has been in the oviduct. The origin of x is taken atO (ovulation). The parabola was fitted to the observa- tions by the method of least squares. The diagram shows clearly that there is scarcely any diminu- tion in the rate of secretion of albumen until nearly the total amount has been acquired by the egg. There is not the slightest evidence of any break in the rate of secretion of albumen after the egg leaves the so-called ‘albumen portion’ of the duct. From the time the yolk enters the upper end of the ‘albumen portion’ there is a gradual diminution of the rate of secretion of albumen, giving rise to the parabolic curve. But plainly there is no sudden change. The egg gets more than half of its total albumen after PHYSIOLOGY OF THE OVIDUCT ilil7¢ it leaves the ‘albumen portion’ of the duct and it takes this at nearly the same rate as it did the earlier part. It is of interest to note the similarity of this curve showing the rate of increase of albumen in the formation of the individual egg to the curve previously published by one of the writers!® for the increase in weight of egg (which is quite closely corre- lated with amount of albumen) with increasing amount of yolk, as measured by number of yolks. 4. It will be noted that the differences in the column headed ‘weight of yolk’ are in the majority of cases negative. In other words, in these instances the yolk of the laid egg is heavier than the yolk of the oviduct egg. Now, of course, yolk as such is not added during the passage of the egg down the oviduct. This being so one would expect that in the long run the yolk of any given laid egg would be as often in defect as in excess of the weight of the yolk of any given oviduct egg. There is some indication that this is not strictly true, but that the ‘laid’ yolk tends to be heavier. Such a phenomenon would be in accord with the fact definitely demonstrated by Greenlee," in his study of cold stor- age eggs, that in the normal, unboiled egg there is a continuous transfer of water from albumen to yolk by osmosis. Certain of Miss Curtis’” earlier results had suggested that this was possibly the case. THE ABSOLUTE AND RELATIVE AMOUNT OF NITROGEN IN THE ALBUMEN OF EGGS IN DIFFERENT STAGES OF FORMATION While the two lines of evidence presented in the preceding sec- tions of the paper amply demonstrate that the thin albumen is added to the egg after it leaves the albumen portion of the duct, it seemed advisable, because of the novelty of the results to col- lect still further evidence of another kind. This evidence, which will be set forth in the present section of the paper, has to do with 10 Pearl, R., Zoologischer Anzeiger, Bd. 35, pp. 417-423, 1910. 1 Greenlee, A. D., U.S. Department Agriculture Bureau of Chemistry, Circu- lar 83, pp. 1-7, 1911. 12 Curtis, M. R., loc. cit. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 118 RAYMOND PEARL AND MAYNIE R. CURTIS the nitrogen content of the albumen in eggs taken from different levels of the oviduct. Not only do these chemical data confirm the results obtained from the other lines of evidence, thus demon- strating still more thoroughly and conclusively the main point under discussion, but they also throw light on certain matters which could not be elucidated by other than chemical methods of attacking the problem. The analytical work on which the data of this section are based was performed in the Department of Chemistry of the Maine Experiment Station, by Mr. H. H. Hanson, Associate Chemist. It is a pleasure to express our obligation to him for coming to the ald of the present investigation in this way. It should be stated that the nitrogen determinations were made by the modified Kjeldahl method, as used by the Association of Official Agricultural Chemists. The chemical data are exhibited in condensed form in table 2. The complete figures for the same eggs are given in table A of the Appendix. It is evident that the data set forth in table 2 confirm the re- sults previously obtained. Thus, to consider first moisture con- tent of the albumen, it is seen that the albumen of the normal laid egg contains between 87 and 88 per cent of moisture (mean 87.40). This value agrees very well with those obtained by Willard and Shaw" and Pennington.’ But when the egg enters the uterus its albumen has a water content of only about 80 per cent. Or, the albumen of the egg at this level of the oviduct has by actual weight some 15 grams less water than the laid egg. The longer the egg stays in the uterus the ‘thinner’ the albumen becomes (ef. hen 387 in the above table), i.e., the higher its water content. This, of course, is the same thing which has been shown above, namely, that most of the thin albumen is added in this region. The percentage content of the albumen in nitrogen brings out again the same point. From a nitrogen content of about 4 per 4 Willard, J. T. and Shaw, R. H., Kansas Agricultural Experiment Station, Bulletin 159, pp. 143-177, 1909. 1s Pennington, M. E., Journal of Biological Chemistry, vol. 7, pp. 109-132, 1910. PHYSIOLOGY OF THE OVIDUCT 119 cent at the upper end of the isthmus, the relative amount of this element in the albumen diminishes steadily till the egg is laid. The point of greatest interest and importance in connection with these chemical data, hinges upon the absolute amount of nitrogen in the albumen. Since it is solely the thin albumen layer which is added after the egg leaves the albumen portion of the oviduct the possibility is at once suggested that what happens in the lower portions of the duct is not a true secretion of another albumen layer but merely a taking up of water from the blood by osmosis, and a dilution or partial solution of the dense albumen already present. Such a view assumes in other words that all that is added to the albumen after the egg enters the isthmus is water. Clearly the only way to test finally the validity of this idea is to carry out such chemical determinations as are tabled above. The last column of table 2 shows the available evidence, which appears reasonably clear in its significance, though because of the minute absolute amount of nitrogen in the white of an egg the case is not a simple one. What the figures from the analyses of thirteen oviduct eggs show is that with four exceptions, the oviduct egg has absolutely less nitrogen in its albumen than the normal laid egg of the same hen. This, of course, is what would be expected if there is an actual secretion of albumen by the glands of the oviduct, and this secretion is added to the egg. It means that these oviduct eggs have been removed before they received their full amount of albumen. If it were the case, on the contrary, that only water was added to the egg after it left the albumen portion of the duct, it would be expected that the amount of nitrogen would be the same in an oviduct egg from the isthmus or uterus as in the normal laidegg. The chemical data clearly indicate that there is a definite addition of albumen to the egg in the isthmus and shell gland, and that the thin albumen layer does not represent solely a dilution of the dense layer. The four cases in which the analyses furnish an exception to this rule are undoubtedly to be explained as the result of fluctuation in the absolute size of the egg. It will be noted that each of these four eggs have been in the uterus some time and would therefore 120 RAYMOND PEARL AND MAYNIE R. CURTIS TABLE 2 Data showing the relative and absolute amounts of nitrogen in the albumen of eggs taken from different levels of the oviduct, and in normal laid eggs | | & z & SH | Sey 1 shy | Ske | seq LOCATION OF EGG IN OVIDUCT 5 a a ae a rs as A oom & me = mou ga 2eis 238 mae ges feiaes pay 2 Ear g a Ez At caudal end of albumen por- | tion. No membrane. (Hen S005) Sat Somer nen wanna eae 13.66 4.13 0.5635 Mean of normal laid eggs of Bame hey Pap eies at Aare eles 31.34 1.98 0.6188 Wifference <4 fies. enone —17.68 |. +2.15 | —0.0558 Entering isthmus........ oh cace Covered with membrane except) for a little of cranial tip. (Hen BOLT) i carat Ne. tt seu seer 16.51 | 3.78 0.6233 Mean of normal laid eggs of same hens eecree ie oce Go eee 32.07 2.00 0.6381 Ditferenceee.-- see eee eae '—15.56 +1.78 | —0.0148 Entering isthmus. Covered with membrane except for a little! } of cranial tip. (Hen168)...... 21.4015) 79.45 17.0036 3.04 0.6515 Normal laid egg of same hen. .... 38.4895| 87.44 33.6536 1.78 0.6860 Difference:...:. ... apemoobuacoote —17.0880) —7.99 '-16.6500 +1.26 0.0345 In upper part of isthmus. Mem- brane thin. (Hen8008)........ | 15.48 3.47 0.5372 Mean of normal laid eggs of same| ene ese aes ase asesewnaeses |_ 31.27 2.06 0.6407 Difference...........-........|—15.79 +1.41 | —0.1035 In lower part of isthmus. (Hen) SOLO) Etc 2c. see eee 15.43 3.30 0.5092 Mean of normal laid eggs of same I Moceerep eae nee cocsocooEccr | 30.06 | | | 1.90 0.5894 Difference. <2. sasaceeee eee —14.63 +1.40 | —0.0802 In lower part of isthmus. (Hen) 8018) i550 Sod doc ee Dae eel 20.32 | | 3.34 0.6756 Mean of normal laid eggs of same 11\-) eee One oor ss cS n> uo 36.05 | wale 1.89 0.6960 Differencé:./1... eee ee 15.73 | | +1.45 | —0.0204 13 In calculating the absolute amount of nitrogen in the normal laid eggs the mean albumen weight for the whole number of such eggs available for each hen has been used. PHYSIOLOGY OF THE OVIDUCT TABLE 2—Continued 124 z LOCATION OF EGG IN OVIDUCT E 5 5 é 5 & z 3 a= RES REE e i) | x In uterus, but no visible shell fonmeds) (Henly) "2.22... 18.841 | 79.58 | 14.9943) 3.03 0.5711 Normal laid egg ofsamehen.....)_ 34.419 | 87.46 | 30.1016 1.79 0.6154 MD TMETEO CO eels ccc cysiv fas sees .-|—15.578 —7.88 |—15.1073) +1.24 —0_0443 In uterus, but no shell formed. Egg surrounded by fluid in uterus. (Hen 8038).. .| 25.87 | 2.78 0.7179 Mean of normal laid Cans ofe same) TERE aaa tibet: Saver crneearciaierets |) seOLoo 1.78 0.6492 Difference... .. deseguuare +++ + -|=10.52 _| +1.00 | +0.0687 In uterus, but no visible shell formed. (Hen8030).......... 26.94 eRe) 0.6727 Mean of normal laid eggs of same, hen.. = .| 35.66 | 1.79 0.6485 ae +0.71 | +0.0242 In uterus, but no visible shell formed. Some fluid in uterus. (Hen 8033). . : .| 26.66 2.30 0.6118 Mean of saga lad eggs fai same NG: wane Oe RA Set Re eee 33.04 1.68 0.5534 DTeEnences - fecckone scene 2 —6.38 +0.62 | +0.0584 In uterus, small amount of shell.) (Hen 200). . : Foo eles yAl 82.89 17.7154 2.51 0.5371 Normal laid egg Bok same hen. Gels es 27.608 86.80 23.9627; 1.88 0.5197 Dis ere | —6.237 | —3.91 | —6.2473| +0.68 | +-0.0174 In uterus. Small amount of shell. (Hen387)..............| 29.9665) 86.62 25.9559) 1.92 0.5741 Normal laid egg of same hen... . | 34.9305 87.89 | 30.7005 L 0.5997 IDES OCC eee | —4.9640) —1.27 | —4.7446) +0.20 | —0.0256 In uterus. Some shell formed. (Hen 8021)... a 27.138 2.07 0.5616 Mean of senate! [aad eggs eine: same Renee ene ee lnoge5e | 1.96 0.5653 IDETEN CE. ako ies wool: —1.39 +0.11 | —0.0037 122 RAYMOND PEARL AND MAYNIE R. CURTIS have received nearly their full amount of albumen. It can readily be seen that if the oviduct egg happens to be an excep- tionally large one, relative to the other eggs of the same bird, it may have a slightly greater absolute amount of nitrogen though not yet laid, than another relatively or absolutely smaller laid ege taken for acontrol. It seems quite clear, in the light of data collected in this laboratory on the fluctuation in size and proportions of the parts of eggs'® that this is the correct explanation of these apparent exceptions in the chemical analyses. It will be noted that in these four cases it is only the absolute amounts of nitrogen (and not the percentages) which furnish exceptions to the rule. Supplementary evidence There is available evidence of still other sorts to indicate that there is a real addition of albumen to the egg after it leaves the so-called ‘albumen’ portion of the oviduct. In the first place a histological study of the oviduct which has been made in this laboratory by Dr. Frank M. Surface!” shows that histologically the same kind of glands which are found in the so-called albumen portion of the duct, are also found in the isthmus and uterus. The differences between the glands of the different regions are quantitative not qualitative. In removing eggs from the uterus it is frequently found that the egg is surrounded by a thin fluid which has evidently been secreted and is in process of being taken into the egg by osmosis. A case of this sort is described in the following autopsy record. Autopsy No. 523. Hen 8038. Killed March 28, 1911 for data This hen laid at 9 a.m. and was killed at 4.15 p.m., or 73 hours after laying. There was an egg in the uterus. The uterus was much larger than the egg. When a cut was made in this organ a small amount of clear fluid flowed out. The cut was clamped off 16 Cf. No. 1 of these ‘Studies,’ loc. cit. supra. 17 Reported at the Ithaca meeting of the American Society of Zoologists, Eastern Branch, December, 1910, but not yet published. PHYSIOLOGY OF THE OVIDUCT 125 and about 2 cc. of the fluid drained into a bottle. This fluid was analysed, the following being the chemist’s report: Amount taken: 1.9860 grams being all of sample. Nitrogen found: 0.22 per cent. From this record it is clear that the fluid taken up by the egg in the uterus, is far from being water. It carries more than a fifth of 1 per cent of nitrogen. In other words it is a dilute albu- men. That the egg does not take water from the blood by osmosis, and in this way dilute the dense albumen to form the thin is further evidenced by the fact, shownby Atkins'* that in the domes- tic fowl the osmotic pressure of the blood is very considerably higher (nearly two atmospheres) than the osmotic pressure of the egg. In any osmotic exchange under these conditions water would tend to pass from the egg to the blood and not in the other direction. SUMMARY OF RESULTS Putting all the evidence together, the following account of the processes by which the hen’s egg acquires its protective and nutritive coverings summarizes the results of the present study. Certain of these results are novel and others confirm the experi- ence of earlier workers. 1. After entering the infundibulum the yolk remains in the so-called albumen portion of the oviduct about three hours and _ in this time acquires only about 40 to 50 per cent by weight of its total albumen and not all of it as has hitherto been supposed. 2. During its sojourn in the albumen portion of the duct the eges acquires its chalazae and chalaziferous layer, the dense albumen layer, and (if such a layer exists as a distinct entity, about which there is some doubt) the inner fluid layer of albumen. 3. Upon entering the isthmus, in passing through which portion of the duct something under an hour’s time is occupied instead of three hours as has been previously maintained, the egg receives its shell membranes by a process of discrete deposition. 18 Atkins, W. R. G., Scientific Proceedings of the Royal Dublin Society, vol. 12 (N. 5.) pp. 123-130, 1909. Cf. also Biochemical Journal, vol. 4, pp. 480-484, 1909. 124 RAYMOND PEARL AND MAYNIE R. CURTIS 4. At the same time, and during the sojourn of the egg in the uterus, it receives its outer layer of fluid or thin albumen which is by weight 50 to 60 per cent of the total albumen. 5. This thin albumen is taken in by osmosis through the shell membranes already formed. When it enters the egg in this way it is much more fluid than the thin albumen of the laid egg. The fluid albumen added in this way dissolves some of, the denser albumen already present, and so brings about the dilution of the latter in some degree. At the same time, by this process of dif- fusion, the fluid layer is rendered more dense, coming finally to the consistency of the thin layer of the laid egg. The thin albumen layer, however, does not owe its existence in any sense to this dilution factor, but to a definite secretion of a thin albu- men by the glands of the isthmus and uterus. 6. The addition of albumen to the egg is completed only after it has been in the uterus from five to seven hours. 7. Before the acquisition of albumen by the egg is completed a fairly considerable amount of shell substance has been deposited on the shell membranes. 8. For the completion of the shell and the laying of the egg from twelve to sixteen, or exceptionally even more, hours are required. or PHYSIOLOGY OF THE OVIDUCT 12 APPENDIX The following table gives in extenso the original data on which this paper is based. It should be said that the weights are in grams. “ So rns CURTIS A A aN Sos Sst meee ee RAYMOND PEARL AND MAYNIE R. 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EFFECTS ON REPRODUCTION AND GROWTH L. B. NICE From the Biological Laboratory of Clark University ONE FIGURE CONTENTS PMT OULU LON Meanie nar etatereerck aah ieee sl ake esis Re tad ae Sey eae Mei uea Oo: JNICOLICI| Sa Sa BRIE PS GCE eee ORIN Ce ave - PEE ee .. 183 Nicotine and tobacco smoke................... 3 .. 135 NO HET OIG acer racine ret A caiaaisie A> oi eer .. 135 Methods. . : 7 135 Effects on the werent and health ar the adult MIG: +). 02 = .. 138 Effects on the fecundity of the adults and viability of the 3 young. . 139 Growth of the young subjected to the same conditions as their parents... 139 Growth of the young not subjected to the drugs....... 145 Comparison of the growth of mice given drugs with Poe: not given drugs.. 146 SIDA guaae dis SUIS Soren Ee epee Caen oe ee : Peres Biol leyasyel anita tile pap aROnne REneOse ‘ as 149 INTRODUCTION Alcohol The effects of aleohol on animal offspring has been shown in several investigations to be injurious. Laitinen (31) found that alcoholized rabbits and guinea pigs had more stillborn young than the controls; and that the growth of the living young was retarded. Of 23 pups from a pair of alcoholic dogs in Hodge’s (28) experiments, 8 were deformed, 9 were born dead, and only 4 were viable. From the control pair 4 were deformed, none were born dead and 41 were viable. Forty-three per cent of the eggs THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 1 133 134 L. B. NICE of Ceni’s (10) aleoholized fowls developed normally under ordi- nary conditions in comparison to 77% per cent of the controls. A fluctuating temperature at the beginning of incubation pre- vented all alcoholic eggs from developing perfectly while 27 per cent of the control eggs were normal. Todde (55) aleoholized 15 roosters from two to four months and concluded that alcohol causes a torpidity of the testicles, acting probably through its affinity for the central nervous system. Ber- tholet (5) found in 37 out of 39 cases of aleoholism in men more or less of an atrophy of the testicles. Ceni’s (10) three alecoholized hens laid less than half the average number of eggs, but since fowls are abnormally developed in this respect we might antici- pate that anything which injured their general health would disturb the laying function without drawing the conclusion that in other animals alcohol would promote sterility. Laitinen (31 and 33) found that aleoholized rabbits and guinea pigs had more young than the controls. The investigations of the Eugenics Laboratory (4, 18 and 48) and of Laitinen (33) demonstrate that more children are born to alcoholic than to sober parents. As to the effect of alcohol on human offspring, Demme (16) compared 10 non-alcoholic with 10 alcoholic families and found that 82 per cent of the children of the former and only 17.5 per centof the latter were normal. Sollier (53) states that it is demon- strated that progeny conceived during drunkenness is doomed to idiocy. Morel (41) gives the history of an alcoholic family through four generations to final extinction, concluding that alcoholic intoxication produces degeneracy, depravity and idiocy. On the other hand Pearson, Elderton and Barrington (48, 18 and 4) believe that their statistical data prove that extreme alco- holism is a result, not a cause of degeneracy and that the abnor- mal pedigrees are due to defective stock and not to alcohol. In an investigation of about four thousand school children of alco- holic and sober parents they could not find that parental alcohol- ism had any unfavorable effect on the offspring. —e EFFECTS OF DRUGS ON WHITE MICE 135 Nicotine and tobacco smoke Tobacco infusion injected into young rabbits by Richon and Perrin (46) had a decided stunting effect; however, if the injec- tions were stopped, the animals gained rapidly and soon equalled the controls. Rabbits subjected to the fumes of tobacco by Zebrowski (60) for six to eight hours daily for several months suffered from loss of appetite and great emaciation, losing from 20 to 47 per cent of their weight. Nicotine injected into eggs by Féré (19) seemed to be a poison to some and a stimulant to others. Fleig (21) subjected guinea pigs to heavy inhalations of tobacco smoke, to injections of extract of smoke or injections of nicotine, and obtained abortions, still-births, under-sized, weak and stunted young. Unfortunately he fails to give definite details and condi- tions of his experiments. Thirteen cases of sexual impotence caused by the abuse of tobacco are reported by Cannata (9); abstinence from smok- ing restored the men to a normal condition. Hitchcock (52) at Amherst College and Seaver (52) at Yale found by measuring students that the smokers developed less in height and lung capac- ity during their college course than the nonsmokers. Meylan (39), however, at Columbia could not find that smoking had any decided effect on growth. Caffeine No experiments could be found on the effects of caffeine on growth and reproduction. Rivers and Weber (47) show that caffeine increases capacity for muscular work. The investiga- tions of Crimer (12), Pincussohn (44), Sasaki (50), Schulz- enstein (51) and Voit (57) indicate that coffee and tea retard digestion. METHODS A comparative study was undertaken to test the effects of alcohol, nicotine and caffeine on the offspring of animals, when fed in small enough amounts so as not to injure their health. White mice were chosen for the experiments since they breed rapidly, so that the results could be based on large numbers, and a 136 LG: BS NICE second generation could be quickly obtained to compare with the first. The experiments were started with 30 female and 15 male white mice about four months old. They were bought froma dealer and guaranteed not to be brothers and sisters. Four hun- dred and forty-one young were born of the first generation in seven months. Twenty females and 12 males of this number were bred at the age of two and a half months, care being taken to prevent inbreeding. These had 230 young in four months. Five lines were carried; one was given alcohol, another nicotine, a third was subjected to the fumes of tobacco smoke, a fourth received caffeine and the fifth line was carried for controls. Two females and one male were kept in a cage; each female was moved to a separate cage before her young were born and remained there as long as they were suckling, which lasted from twenty- five to thirty days. The cages were of wire mesh 6 inches wide, 5 inches deep and 12 inches long. The regular diet of all the mice was buckwheat, and crackers soaked in milk; every few days they were given carrots, grass, meat, ete. The alcohol line The first generation in this line consisted of 6 females and 3 males, and in the second generation, the offspring of the first, of 5 females and 3 males. Two cubie centimeters per mouse of 35 per cent alcohol were added daily to the crackers and milk of these mice. Instead of water they drank 35 per cent alcohol which was placed in bottles containing siphons; so the animals drank directly from the bottles, and evaporation was prevented. The mice were first given 10 per cent alcohol which was gradually increased to 45 per cent. Thirty-five per cent was found to be a safe medium since stronger percentages sometimes intoxicated the mice. All the mice in this line except 16 received alcohol, the young beginning to take it at the age of three weeks. Sixteen young were given no alcohol themselves although their mothers received it for sixteen days after the birth of the young. EFFECTS OF DRUGS ON WHITE MICE 137 The nicotine line The first generation in this line consisted of 6 females and 3 males, and the second generation, the offspring of the first, of 3 females and 2 males. This line had 2 ce. per mouse of 1:1000 nicotine solution added to their crackers and milk daily, and the same solution was sub- stituted for drinking water in bottles with siphons. Different strengths of nicotine had been tested on white mice from 1:2000 to 1:500 solution. The last was fatal but on 1:1000 they remained in good health, although this solution was found to kill grey rats in nine days. All the mice in this line except 6 received nicotine, the young beginning to take it at the age of three weeks. Six young were given no nicotine themselves although their mothers received it for sixteen days after the birth of the young. The line subjected to the fumes of tobacco The first generation in this line consisted of 6 females and 3 males, and the second generation, the offspring of the first, of 8 females and 4 males. These mice were subjected to tobacco smoke for about five minutes at a time and then aired for five or ten minutes; this was repeated for two hours each day. The smoke chamber was a bell jar made air tight by being placed on glass and moistened around the bottom. About 4 grams of Connecticut leaf tobacco were burned each day in a clay pipe inserted in a rubber cork in the top of the bell jar. The smoke was drawn through the jar by an air pump, the tube for this purpose being in the rubber cork. All the mice of this line except 9 were subjected to the fumes of tobacco, the young being put in the smoke chamber from the time they were a few days old. Nine young were not subjected to the fumes of tocacco, although their mothers were each day while they were suckling. 138 L. B. NICE The caffeine line This line consisted of 6 female and 3 male mice. No second generation was obtained for breeding since many of the young died and others were eaten by their parents. Each mouse had 2 ce. of 1:100 caffeine citrate solution added to his crackers and milk daily, and drank this solution instead of water. The caffeine citrate was first given in a 1:500 solution which was gradually increased to a 1:50 solution. Since they did not seem to be thriving on this strength it was decreased to 1:100, on which they remained in good health. All the mice in this line except 8 received caffeine, the young beginning to take it at the age of three weeks. Eight young were given no caffeine themselves, although their mothers received it for sixteen days after the birth of the young. The control line The first generation of control mice consisted of 6 females and 3 males. Since these mice had few young and many were eaten there were none of the second generation ready for breeding three months after the experiments were started. Therefore 6 young females and 3 males were obtained from the same source as the first lot of mice so as to serve as controls for comparison with the second generations of the other lines. EFFECTS ON THE WEIGHT AND HEALTH OF THE ADULT MICE The females were weighed each time after they had given birth to a litter, and the males from time to time. During the course of the experiments all the mice gained in weight; in the first genera- tion, carried seven months, the alcohol mice gained 6 grams each on an average, and the others gained 2 grams; in the second genera- tion carried four months, the aleohol mice gained 2 grams and the others 1 gram. This would indicate that the mice remained healthy and that alcohol has a fattening effect. EFFECTS OF DRUGS ON WHITE MICE 139 EFFECTS ON FECUNDITY OF THE ADULTS AND VIABILITY OF THE YOUNG _A record was kept of the litters of each female. Many of the young in all the lines were eaten. In the tables 1 to 13 only those young that died from lack of vitality are recorded. TABLE 1 Record of the young of each female Control line. First generation | 5 oa -OUN: FEMALE NUMBER OF NUMBER OF | NUMBER OF NUMBER OF YOUNG MONTHS OBSERVED LITTERS YOUNG BORN | THAT DIED IN 3. OE RRS ERD 7 3 18 0 13. (ron eRe 7 2 12 0 (C.. e 7 2 16 0 1D): 0 ee ee iii 2 v/ 0 1D | 3 1 7 0 IN. BROGAN ciiic. <4: 7 10 60 0 * Female E died at the end of three months. No pathological condition could be found. t Female F was killed by accident at the beginning of the experiments. TABLE 2 Alcohol line. First generation : ae =a = FEMALE | NUMBER OF NUMBER OF NUMBER OF NUMBER OF YOUNG i" MONTHS OBSERVED LITTERS YOUNG BORN THAT DIED PAWN E eer ciok %,: 7 2 17 1 1. 7 2 1 (GE 437 See | a 4 2 1 1D) 4%, ORR 7 4 ) 2 Dini ee aBee tf 2 4 i055 Ase eRe Motali, ac... 5] vi 14 $1 9 1 | 3 3% wks. * Female F died three and a half weeks after the experiments were started. No pathological conditions could be found. 140 te) BS oNIGE TABLE 3 Nicotine line. First generation 7 NUMBER OF NUMBER OF NUMBER OF NUMBER OF YOUNG SE MONTHS OBSERVED LITTERS YOUNG BORN THAT DIED dN Boece toes RE 7 3 13 1 Be ae ler eres u 2 7 0 Cathe 7 1 4 4 De ayer 7 | 2 13 2 eae Ae ee 7 5 36 | 2 oy 7 | 4 31 9 Motalics -.<.16 | 17 104 18 TABLE 4 Line subjected to tobacco fumes. First generation FEMALE MONTHS OBSERVED) winsks «|| gouNagomn ||) atcn ayia Ae See 7 4 23 | 9 BAS ore. 7 4 | 30 5 (Geowese. 7 3 22 6 1D Bear ace cS Eeaa 7 4 21 | 7 Be. 4 3t 15 | 14 1D Patent: ratte d ine —— s = zs Totally as-eare: 7 18 lil | 41 1 4 * E was killed by accident at the end of three months. + F died at the beginning of the experiments. ‘ { One of these litters was an abortion. TABLE5 * Caffeine line. First generation NUMBER OF NUMBER OF NUMBER OF _ | NUMBER OF YOUNG seers MONTHS OBSERVED | LITTERS YOUNG BORN THAT DIED AN. ase ace nee 7 3 15 0 Bina ki aca erneeae 7 3 16 8 Care eee rns 7 2 11 5 Dis tae 7 3 27 2 Bh osc Braetnseee Uf 2 7 4 1a 7 3 9 3 Total 6 7 16 85 22 EFFECTS OF DRUGS ON WHITE MICE TABLE 6 Control line. Second set ree eee | Pesee on | meee oe | roumme or somme Jari SBS Onn 4 1 i 0 LE eke Soe tCek 4 2 12 0 (Cocco am emeeee 4 2 4) 0 1D)... cou hops Oeeee 4 2 9 0 Dit 6 peace eee 4 1 2 0 4 1 4 0 TABLE 7 Alcohol line. Second generation 5 ans co} é a Py aie ce Ba © °o | | ae 0 i) an 8 ene ae a Tea ee “UMTERS | YouN@poRN | THaT DIED Ce a ee ee : 4 1 5 0 + 2 13 1 4 2 16 2 4 2 13 | 3 2 1 9 1 _| 4 8 56 7 2 TABLE 8 Nicotine line. Second generation ] aaa MONTHS oBarRvED| Lirrens | YouNasoRN | Har DIED. Dn ee 4 2 il 5 BRM atch Se cece 3 4 2 13 0 (Os. Aone eee 4 1 6 0 Total.. 4 4 5 30 | on 142 L. B. NICE TABLE 9 Line subjected to tobacco fumes. Second generation FEMALE NUMBER OF NUMBER OF NUMBER OF | NUMBER OF YOUNG MONTHS OBSERVED LITTERS YOUNG BORN THAT DIED AM saattentts aoa coe 4 3 21 3 Bit og snc aee 4 3 15 1 Cer ee eee nae 4 2 11 2 ID Ras ceo oer 4 4 22 9 er Be Eee 4 2 12 3 eee 4 1 8 1 RO hee Aes e 3 1 6 1 15 ey ere ee 3 1 6 6 Total =6 4 17 101 26 2 3 TABLE 10 Record of the young of the first generation Summary of tables 1, 2, 3, 4 and & NUMBER OF < NUMBER OF uve | *uupenor | “Goan” | “gaaen or | xonmes or | souna mat Control......... ‘ 3 f ; 10 60 0 AlGoholicasnccs 5 7 14 81 9 Nicotine......... 6 7 17 104 18 Smoked......... f4 7 18 1 41 \1 af @affeineyae cess 6 7 16 | 85 22 TABLE 11 Record of the young of the second generation Summary of tables 6, 7, 8 and 9 NUMBER OF es NUMBER OF sxe | -Nuamenor | “Sowans” | “Tunenor | wuwmen oy | Zouwa uaz Controle 6 = 9 43 0 Alicoholyereeiee f A 5 ; 56 7 Nicotine........: 3 4 5 30 | 5 Smoked. pees ec [6 4\ 17 101 | 26 \2 3 f © EFFECTS OF DRUGS ON WHITE MICE 143 TABLE 12 Average of one female of the first generation for seven months S eae Se i | AVERAGE NUMBER OF AVERAGE NUMBER OF PER CENT OF YOUNG LINE | LITTERS YOUNG THAT DIED @onttroliewecscneady Ar « 2.2 13.3 0 JAK Yo) a0) Be Gea aOR Ey seem 2.8 16.1 leit NMG OMAS. oat upbasboceao 2.8 17.3 Vike BOAOKC Mes frenetic = clare 4.0 24.6 37.0 @ameme... cc. ca ee: 2.7 14.1 25.3 TABLE 13 Average of one female of the second generation for four months LINE AVERAGE NUMBER OF AVERAGE NUMBER OF PER CENT OF YOUNG 9 LITTERS YOUNG THAT DIED (Gia) hihg0) ease O eee Eee 1 (foal 0 PAN CON Olin jaiiieyoelsys a's 1.8 12.4 12.5 INGO UIC ccttecrers sci sicias 1.66 10.0 16.6 ISIMOKEE cou ctseislyiewiee 2.26 13.4 26.0 In both generations the mice subjected to tobacco fumes had more young than any of the other lines, whereas the controls had the fewest young. Tobacco fumes had a marked effect on the viability of the young, since 37 per cent of the first generation died from lack of vitality and 26 per cent of the second. One abortion occurred in this line. Caffeine was also injurious to the young, 25 per cent dying. Nicotine and alcohol had a less notice- able influence. None of the control young died in either gener- ation. GROWTH OF THE YOUNG SUBJECTED TO THE SAME CONDITIONS AS THEIR PARENTS The young were either weighed singly or in litters at birth and every week for eight weeks. Since it was not always possible to weigh them before they suckled, the variations in the birth weights have little significance. The records were not carried beyond eight weeks for the young females often become pregnant at that time. 144 L. B. NICE TABLE 14 Weekly growth of the young of the first generation subjected lo the same conditions as | their parents & re | )ae & a Gla @ Hm | & ae eo = | 3 o | Ha IA Uwe) mie Dn Hg Da iS lee Pavey SS elicisy) BR ee Nie me Ba |S |B | a" |agleslaelae| Bf | Be | ae | BF Beata eee se as FRIFFIP alr) FE Fe | Fz Lars) LINE aZe | ash lL el ae, |aalaciabiap| af ay a 8 aa OA4/ 044 EG) oc SzZIOslonlao| oF ok oF of 4&8) 228 |82) = hi Ey | mn! | n n & eae le je a eae) & & & = Belo |e |Eulm@l go | p4 | Ge | oe | Be Sele |e, (See Se) WSS a SE | ei Aa a lp a ag/aR| ae | as ag ag ae Fa, |FelCales |ERIEF) Fa eo EE aes Ey LINE ase |e Al Bl ae, |eaiao] af | ab aa By ae Oak ORB o| Ckm |SZIOE) Ge | Go og on oa ) 7) SS \\ on 0 3 3 Fig. 1 Pure F. heteroclitus, four days old, showing first chromatophores. X 25. Fig. 2. Pure F. heteroclitus, three and one half days old, circulation started not more than a few minutes before drawing began. X 25. Fig. 3 Brain of same embryo drawn in fig. 2 but three and one half hours later. X 25. 172 FRANK W. BANCROFT Fig. 4 Pure F. heteroclitus, five days old, showing the brain chromatophores and half of the yolk chromatophore ring about the embryo. H=heart. 25. Fig.5 Pure F. heteroclitus, six days old, showing the first crop of brain chro- matophores well expanded. Red brain chromatophores not drawn. 25. Fig. 6 Same embryo drawn in fig. 5, but nineteen hours later showing the first appearance of the second crop of brain chromatophores. The four cells of the first crop still recognizable. > 25. Fig. 7 Same embryo drawn in fig. 6, but forty-eight hours later. Shows the fusion of the head chromatophores. HEREDITY OF PIGMENTATION 173 Fig. 8 Pure I’. heteroclitus, three and one-half days old. Heart beating but no circulation. Shows the yolk chromatophore ring about the edge of the hollow vesicle under the embryo. X 20. Vig. 9 F. majalis egg hybrid, seven days old. Shows both crops of head chro- matophores. X 25. Fig. 10 Pure F. majalis, seven days old. Belongs to same series as embryo of fig 9. Shows absence of the first crop of head chromatophores; also characteris- tic shape of black yolk chromatophores. 25. Fig. 11 F. majalis egg hybrid, six days old. Shows shape and arrangement of yolk chromatophores. 19. Fig. 12. Pure F. majalis, six days old. Shows the absence of yolk chromato- phores on the part of the yolk away from the embryo. Many chromatophores near the embryo were not drawn as they were much obscured by the yolk globules. 19. Fig. 13 Pure F. heteroclitus, four days old. There were no blood vessels near the cells figured. Shows characteristic early chromatophore group, before they have migrated onto the blood vessels. Embryo same stage as fig. 2. %& 114. Fig. 14 F. heteroclitus egg hybrid, four days old; has good circulation. Em- bryo same stage as fig. 2. Shows characteristic group of yolk chromatophores on the side of the yolk sac, opposite to the embryo, where the blood vessels have just become established. > 114. 174 HEREDITY OF PIGMENTATION Wi) 18 19 Vig. 15 Pure F. heteroclitus, twelve days old. Fully developed red chroma- tophore near the eye of embryo. Dotted lines indicate blood vessels. > 75. Fig. 16 I. heteroclitus egg hybrid, twelve days old. Fully developed red yolk- chromatophore in center of clear space in black chromatophore reticulum. X 75. Fig. 17 Pure F. majalis, thirteen days old. Characteristic red yolk chroma- tophore on blood vessel. X 75. Fig. 18 Pure F. heteroclitus, six days old. Group of chromatophores begin- ning to form chromatophore reticulum on blood vessels near left eye. % 114. Fig. 19 IF. heteroclitus egg hybrid, six days old. Same series and same age as embryo of fig. 18. Group of chromatophores on blood vessel near left eye. > 114. 176 FRANK W. BANCROFT 24 23 22 Fig. 20 Pure F. heteroclitus, seven days old. Same embryo as in fig. 18. Chromatophores on blood vessel near left eye. To show increase in size and com- plexity during last twenty-four hours. X 114. Fig. 21. F. heteroclitus egg hybrid. Same embryo as in fig. 19. Chromato- phores on blood vessel near left eye. Note increase in size of red chromatophores and the beginning of the small processes of black chromatophores. > 114. Fig. 22. Pure F. heteroclitus, eleven days old. Typical fully developed red chromatophore among the black chromatophore reticulum. 114. Fig. 23 F. heteroclitus egg hybrid, eleven days old. Of same age and series as embryo in fig. 22, with which it is to be compared. Note double layer of chro- matophores and fine black reticular processes. > 114. Fig. 24 Pure F. heteroclitus. Most of the eggs of this lot had hatched, but in this embryo the circulation had stopped though the heart was still beating, and the blood vessels distinct and full of blood. The yolk chromatophores have begun to lose their arrangement on the blood vessels and have developed much longer branches than any normally seen in this species. 114. HEREDITY OF PIGMENTATION 177 27 Fig. 25 Pure F. majalis, thirty-five days old, just hatched. Shows the line of chromatophores along the dorsal surface of the nerve cord, N...N, which were expanded; and the contracted chromatophores of the lateral line, L...L. A...A= Aorta, V...V=Ventral vein. Neither the chromatophores on these last two ves- sels nor on the dorsal surface of the fish have been drawn in. X 25. Fig. 26 IF. heteroclitus egg hybrid, sixteen days old, just hatched. S...S line of chromatophores under dorsal skin, partly contracted by the nareotization. N...N chromatophores on nerve core, partly contracted; L...Z lateral line. All the red chromatophores contracted, black chromatophores partly contracted at left where they formed a complete line when the drawing was begun. In the cen- ter of the lateral line the chromatophores are expanded; and on the right some of them are completely contracted on account of another dose of narcotic. A...A =Aorta V...V=Ventral vein. Chrematophores on these last two vessels drawn in from another uncontracted fish. X 25 Fig. 27 Pure F. heteroclitus, just hatched. Shows red opaque chromatophores of the lateral line. X 127. 178 FRANK W. BANCROFT 30 Fig. 28 F. majalis egg hybrid, four days old. Shows position of the heart which has just begun to beat, and the shallow vesicle, V. underneath the embryo. Fig. 29 Pure F. majalis, four days old. Same series and age as embryo in fig. 28. To show the greater depth of the vesicle, V. under the embryo and the normal position of head and heart. Heart has just begun to contract. Fig. 30 F. majalis egg hybrid, twenty-four days old. Dissected out of the egg shell at a time when a few fish of the same lot had hatched and when many had died. Shows vesicle V. under the embryo and the greatly stretched heart within it. LONGEVITY IN SATURNIID MOTHS: AN EXPERIMENTAL STUDY PHIL RAU anp NELLIE RAU FIVE CHARTS INTRODUCTION The observations and experiments herein recorded upon the longevity of some of the Saturniid moths were undertaken in order to discover the value of some of the theories that have been advanced to account for the duration of life. Much of the theorizing is based upon the insufficient and inaccurate knowl- edge of the ages attained by different organisms and the rela- tion of such length of life to their reproductive function, as well as to their environmental conditions. The attempt to place our knowledge of the duration of life upon a scientific basis demands’ the gathering of many data on many species, and on the mated and unmated individuals of both sexes. With these needs in view we found some members of the family Saturniidae in sufficient numbers to suit our purpose. A more important reason for this choice was the fact that they have aborted mouth-parts, and the adult insects take no nourishment. This would eliminate the probabilities of curtailment of life due to insufficient or improper food. The cocoons were gathered and carefully strung to trees where they could be subjected to the natural changes of the weather conditions during the winter. Just previous to the emerging time they were taken into a shed, the temperature of which varied but little from that of the outside. The imagines were kept under ordinary dome-shaped, wire dish-covers, which varied from 22 to 32 inches in circumference. 179 180 PHIL RAU AND NELLIE RAU This work falls into the following divisions: The duration of life in: 1. Samia cecropia. 1910—178 insects from St. Louis cocoons. 2. Samia cecropia. 1911—112 insects from St. Louis cocoons. Cocoons placed in incubator. 3. Samia cecropia. 1911—42 insects from St. Louis cocoons. Imagines placed in ice-box. 4. Samia cecropia. 1911—283 insects from St. Louis cocoons. 5. Samia cecropia. 1911—133 insects from Long Island cocoons. 6. Callosamia promethea. 1911—170 insects from Creve Coeur Lake, Missouri, cocoons. 7. Tropaea luna. 1911—60 insects from St. Louis and Pike County, Missouri, cocoons. 8. Telea polyphemus. 1911—19 insects from St. Louis and Pike County cocoons. REVIEW OF THE THEORIES Before taking up the details of the work, it would be well to rehearse here briefly the various theories which have been advanced to account for the duration of life. First among these is the theory of Weismann, that the dura- tion of life of an organism be ges is really dependent upon adaptation to external condi- tions, that its length, whether longer or shorter, is governed by the needs of the species, and that it is determined by precisely the same mechan- ical process of regulation as that by which the structure and functions of an organism are adapted to its environment.! I consider that death is not a primary necessity, but that it has been secondarily acquired as an adaptation. I believe that life is endowed with a fixed duration, not because it is contrary to its nature to be unlimited, but because the unlimited existence of individuals would be a luxury without any corresponding advantage. The above-mentioned hypothesis upon the origin and necessity of death leads me to believe that the organism did not finally cease to renew the worn-out cell material because the nature of the cells did not permit them to multiply 1 Essays upon Heredity, 2 ed., vol. 1, p. 9, 1891. LONGEVITY IN SATURNIID MOTHS 181 indefinitely, but because the power of multiplying indefinitely was lost when it ceased to be of use.” In answering the question (continues Weismann, loc. cit., p. 20), as to the means by which the lengthening or shortening of life is brought about, our first appeal must be to the process of natural selection. Duration of life, like every other characteristic of an organism, is sub- ject to individual fluctuations. . . . As soon as the long-lived individuals in a species obtain some advantage i in the struggle for exist- ence, they will probably become dominant and those with the short- est lives will be exterminated. Lankester,* according to Romanes, has pointed out ‘‘a highly remarkable correlation between potential longevity in the indi- vidual and frequency of parturition.” ‘This correlation he attributes to generative expenditure acting directly to the cur- tailment of life.”’ Romanes,* who like Weismann sees in the duration of life an adaptation, does not agree with Lankester that it is the genera- tive expenditure ‘‘that causes the curtailment of life, but that it is the curtailment of life by Natural Selection, which causes the high generative expenditure within the lessened period.” In opposition to the utilitarian theory of Weismann is that of Gotte,® that oe all animals are mortal, and reproduction is in itself the cause of death. Reproduction in Protozoa is preceded by encystation. In this condition the organism passes into a non-living condition, from which it revives with renewed youth and renewed life; a similar condi- tion occurs in the egg of Metazoa, during a certain period in which it forms an unorganized, non-living body, composed of organic substances. Eimer’s® own opinion is that in the Metazoa as well as in the “Protozoa the germ cells are immortal; only the soma dies. “The latter is not really an end in itself, but rather its principal fure- tion is to ensure the maintenance of orzaniec life, by favoring reproduc- tion, by sheltering the germ-cells till their maturity, and in order to deposit them repeatedly; further, by the dispersal of the same in space, 2'Tioe: clt-, p: 20: %’ Comparative Longevity, 1870, quoted by Romanes, Monist, vol. 5, p. 163, 1895. 4 Monist, vol. 5, p. 163, 1895. 5 Quoted by Eimer, Organic Evolution, p. 67, 1890. 5 Loc. cit., p. 68-69. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 2 182 PHIL RAU AND NELLIE RAU by incubation in the widest sense, and so on. Further it has the fune- tion of strengthening the power of endurance of the species by she inher- itance of acquired characters.’ “Reproduction is unending growth. Not reproduction is the essential cause of death. . . . . the soma is not really an end in itself, but rather its principal function is to ensure the maintanence of organic life by tavoring reproduction. Minot? seems to conclude that ‘‘the duration of life depends. upon the rate of cytomorphosis. If that cytomorphosis is rapid the fatal condition is reached soon, if it is slow the fatal condition is postponed.”’ Flourens® thinks that the length of life of an animal is equival- ent to five times its period of growth, while Nageli® thinks that “natural death does not exist in nature, for trees more than a thousand years old perish not by natural death, that is to say natural decay of their vitality, but by some catastrophe.” Metchnikoff® says: ‘‘It is impossible to regard natural death, if indeed it exist, as the product of natural selection for the bene- fit of the species. In the press of the world natural death could hardly come into operation because maladies or the voracity of animals so frequently cause natural death.” While Morgan" says that “in some cases the length of life and the coming to maturity of the germ-cell may be, in some way, physiologically connected seems not improbable, but that this relation has been regulated by the competition of species with each other can scarcely be seriously maintained,” he will ‘not pretend to say whether the mutation theory can or cannot be made to appear to give the semblance of an explanation of the length of life in each species.” Other theories of less importance are reviewed by Metchnikoff in “‘The Prolongation of Life’ and “The Nature of Man.” 7 The Problem of Age, Growth and Death, p. 228, 1908. § Quoted by Metchnikoff, Prolongation of Life, p. 40, 1908. ® Quoted by Metchnikoff, The Nature of Man, p. 265. 19 Loc. cit., p. 267. 11 Evolution and Adaptation, p. 371, 1908. LONGEVITY IN SATURNIID MOTHS 183 OBSERVATIONS In 1909 notes were made upon the duration of life in the Cecro- pia moth.“ The observations were made upon only a small number however, so it was decided to carry the work on during 1910, on a much larger scale. New factors of much interest were discovered entering into the work of that year, so it was deemed best to continue an experimental investigation through a third year in order to get adequate and conclusive data on some of the phenomena appearing. These three consecutive years of observations on the Cecropia were made upon material gathered from the same locality, River des Peres, St. Louis. The parallel work was carried on upon the Cecropia moths from Long Island, New York, in order to ascer- tain whether there were any unusual phenomena in the St. Louis material due to purely local conditions. TABLE 1 Mean duration of life a 2 a a *o 2 o a & a a 2 3 LOT hoses a = a ois z E & = a & a & 3a 5a % o Bo) ip orate ts Seat: a E | af = 3 : e < 4% < Zz Sa | Ss eS isha sd |Po7s"| ote | = a nu E | [] 26 | a ‘. = = a ee —_ [ee 916 —— 2S = = P [ince 1% = = —"\ —s . Se = mn 50% | ei =IA a m = = 5 - April 4 52 49 April 29 76 70 May 27 71 65 5 59 48 30 71 52 29 78 65 6 69 53 May 1 63 60 30 71 67 if 75 72 2 69 65 | 31 73 67 8 80 71 3 75 69 June 1 76 70 9 76 62 4 76 67 3 77 66 10 62 52 7 85 | 67 4 75 70 11 56 50 8 74 64 5 70 67 12 | 59 | 50 OM |) 72 |) 61 6 68 | 66 13 60 54. 10 64 60 3} 75 7 14 66 58 ll 66 64 10 74 65 15 78 62 12 73 60 ll 79 75 16 72 56 iG} 74 63 12 74 68 17 65 55 14 75 66 | 13 77 70 18 75 59 || 15 71 70 14 75 70 19 83 75 16 79 69 15 78 | 74 20 82 61 17 72 65 16 79 70 21 65 57 18 66 60 17 80 70 22 67 61 20 68 52 18 74 70 23 78 65 21 67 58 19 73 66 24 63 53 | 22 61 57 20 72 68 25 78 ol | 23 69 59 23 85 72 26 60 58 24 79 71 25 | 92 83 27 73 54 26 78 64 26 89 81 220 LORANDE LOSS WOODRUFF dition of, for example, the A / cultures at the end of the first fifteen days, with the A J/I cultures at the end of the same length of time without taking the temperature into account. However, it is fair to compare the relative time of appearance of the various organisms in A J and the relative time of appearance of the var- ious organisms in A ///; but even here an error is undoubtedly present, though, it is believed, it is not sufficiently marked to appreciably influence the general results though minor variations which occurred in particular infusions may well be due to it. This error arises from the fact that the different species of organ- isms in the infusions undoubtedly have their own optimum tem- perature for development and consequently it may be supposed that a particular form, which has a comparatively high optimum temperature, may reach its maximum later than another with a slightly lower optimum temperature, in the cultures existing during the early part of April when the general average tem- perature was lower, while it may attain its maximum earlier than the latter in the cultures which reached a corresponding stage of their development when the temperature was generally higher. As already stated, the first intention was to follow the entire fauna and flora which developed in the infusions, but this involved more labor than could be performed accurately by one observer. Consequently although a record was kept of all the animals and plants which actually were observed, these data will not be pre- sented because I am not satisfied that they are sufficiently accur- ate or comprehensive. One who has not attempted to follow in detail a series of cultures, started in the manner described, has not, I think, an adequate realization of the wealth of forms which will develop. Some of the forms appear and disappear with such marvellous rapidity that if they are not immediately identified, in many eases it is impossible to do it later. Therefore, I repeat that the description which follows simply affords the data col- lected in regard to certain well-known genera and groups of Pro- tozoa, which appeared in sufficient numbers, in a large majority of the infusions, to render their study of value in attempting to reach some general conclusions as to their sequence in such in- fusions under the conditions of the experiment. It is believed that the concentration of attention on these few forms is prefer- PROTOZOAN FAUNA OF HAY INFUSIONS 221 able to a wider consideration of many transient species which appear apparently at random, for, if it is possible to reach any conclusions of value from the study of these few dominant forms, it may open the way for an explanation of the seemingly fortuitous distribution of the remaining species. A tabulation of the fauna of the infusions showed that the first analysis of the results should consider the following groups and genera of Protozoa: Monads, Colpoda, Oxytricha and various closely related hypotrichous forms, Paramaecium, Vorticella, and Amoéba, because all these organisms were present in practically every infusion. The term ‘monads’ is used in a broad sense to include several different genera and a multitude of species of small flagellate Protozoa usually classified under the generic names Oikomonas, Monas, Bodo, ete. Colpoda cucullus is the most common member of the genus Colpoda which has appeared in the infusions. Occasionally the form of the organism has not agreed exactly with the specific description usually given, and it may well be that some of these organisms properly rank as other species of the genus, but as this could be determined only by following out the life history of the animals, it was necessary to assign the forms merely to the genus. In a number of cases species of Colpidium was found intermixed with the Colpoda. Colpoda and Colpidium are apparently adapted to practically identical conditions of the infusions and consequently it matters little which form is chosen for study. Since Colpoda has usually appeared in greater abun- dance than Colpidium, it has been selected, as the representative of this type of ciliate, for detailed study in this work. Among the hypotrichous ciliates which appeared, Oxytricha was prob- ably the most common, but closely associated with this genus was Stylonychia, Urostyla, Gastrostyla, ete., and therefore the various species of these genera were considered as a unit and are designated in this work as ‘Hypotrichida.’ Also several members of the Vorticellidae appeared, nearly all of the genus Vorticella. The term ‘Vorticella’ accordingly is used to include all true mem- bers of this genus regardless of species. The same is true of the term ‘Amoeba’ as here employed, this name being used to include such forms as Amoeba guttula, radiosa, etc., as well as typical Amoeba proteus. ‘Paramaecium’ is applied to two species, De, LORANDE LOSS WOODRUFF aurelia and caudatum, indiscriminately. It is apparent, then, that no attempt has been made to identify the various species, as this would necessitate a large amount of labor entirely incom- mensurate with the value of the information gained for the prob- lem in hand. All of the forms included together are adapted to the same general environment (as the results which follow show), and therefore it is logical to consider them together as a unit with- out regard to the taxonomic variations of the individual moieties of which it is composed. B. GENERAL OBSERVATIONS ON THE COURSE OF DEVELOPMENT OF HAY INFUSIONS In infusions (A) made with boiled hay, which is allowed to remain in the jar, most of the hay sinks quickly to the bottom and remains there. In the cultures (C) made with unboiled hay most of the material floats near the surface for four or five days and then begins to sink gradually to the bottom. It is usually all at the bottom within two weeks. When the hay is allowed to remain in the infusion (A, C) this slowly disintegrates and is reduced to a more or less amorphous mass by the end of the sec- ond month. The rapidity of these changes, however, varies con- siderably with the temperature to which the cultures are subjected. When hay and water are combined the liquid rapidly becomes straw colored, and within the first few days bubbles of gas appear entangled amongst the hay at the bottom, and these rise by degrees to the surface. At comparatively high initial tempera- tures the gas will frequently disturb the hay and sometimes raise it to the surface. Peters’ observations show that this gas is chiefly CO:. By the third or fourth day the color of the culture liquid appears darker and this becomes increasingly pronounced until finally the liquid is of a dark brownish color. One familiar with infusions can, of course, readily tell the approximate age of a culture by its color. Fine’s studies on these infusions show that the light and yellowish shades of color are due to relatively high acidity; the darker and brownish shades to relatively low acidity. 10 Fine. Loc. cit. PROTOZOAN FAUNA OF HAY INFUSIONS 22a When the infusions are first made up, the liquid, though col- ored, is transparent, but within forty-eight hours it becomes markedly turbid due to the development of countless bacteria. The bacteria at this time are equally distributed throughout the medium but on the third day a ‘zoogloea’ begins to be established and gradually increases in amount until it finally falls to the bot- tom and another is formed. In some cases, however, the ‘zoo- gloea,’ after reaching its maximum thickness, at approximately the end of thirty days, gradually thins out and practically dis- appears in situ. These variations in the transformation of the ‘zoogloea’ introduce a complicating factor in the study of the protozoan life of infusions, because in the cases in which it falls to the bottom, it changes the center of population of certain types quite suddenly, and thus causes a redistribution of some forms. Thebacteria, then, at first are equally distributed through- out the fluid, then the largest number is at the bottom and top, while in the center of the volume of liquid there are comparatively few. The hay and smaller amount of oxygen at the bottom, and the more abundant supply of oxygen at the top, offer attractions for different forms with the result that apparently approximately the same number are to be found in each region. After the ‘zoogloea’ has fallen or disappeared the center of bacterial life is again at the bottom amongst the remnants of the disintegrating hay. As soon as the bacteria have become numerous, and their action on the hay has put a certain amount of it in a form avail- able for animal life, then occurs the great growth of Protozoa, comprising saprophytic, herbivorous, carnivorous and omnivorous forms, and this phase of the life of the infusions we shall consider in detail. After the period of greatest protozoan fauna has passed, roti- fers become numerous, and as the diatoms, desmids, and filamen- tous eyanophyceae and chlorophyceae flourish, under proper conditions of illumination, several species of Anguillula, copepods, ete., are more or less abundant. This condition of the fauna and flora merges imperceptibly into what may be called a condition of nearly stable equilibrium, in which green plants and animals, under 224 LORANDE LOSS WOODRUFF optimum conditions of light and temperature, are so adjusted that for a considerable period a practically self-supporting and self-suf- ficient microcosm exists—but with the balance of nature estab- lished neither the Protozoa nor bacteria can ever again attain their maximum abundance. C. THE A, B AND C GROUPS OF INFUSIONS All three types of infusions (A, B,C) which were made up gave the same general cycle of events, but the A and C series were slightly slower in development (as one would expect from the presence of the hay) than the B series. The cycle of the C series was essentially the same as that of the A series except that it pro- geressed somewhat more slowly until the hay became thoroughly soaked. A practical disadvantage of the C’ series is presented by the fact that the unboiled hay, containing considerable air, has a tendency to float and so changes somewhat the distribution of the organisms until it sinks to the bottom at about the end of two weeks. This nuisance may be avoided by weighting the hay with glass. So far as length of cycle is concerned, however, both the A and the C series offered equal advantages for study, but the cycle of the B series (without hay) being considerably shorter, . the sequence of the different types of organisms was more rapid, the number of organisms present was much smaller, and stable equilibrium of the infusions was attained sooner (cf. figs. 5, 6, 7). However, since the richness of the animal life was seriously de- creased, this series did not prove to be the best for study, and accordingly such a method of making up cultures is not recom- mended for investigations of this character. Nevertheless, the results derived from all three types of cultures will be given here. The data from each of the twenty-six cultures have been re- corded (as already described), then these data from each culture of each set of experiments of the A, B, and C series, started at the same time, have been averaged together. Therefore, in discuss- ing these data, I shall refer (unless it is specifically noted to the contrary) to the average number of organisms, time of appearance, etc., in infusions comprising each group as follows: PROTOZOAN FAUNA OF HAY INFUSIONS 225 A-1, A-2, A-3, A-4, A-5, A-6, averaged and designated A J A-21, A-22 averaged and designated A II A-31, A-32 averaged and designated A I// A-41, A-42 averaged and designated A IV. B-1, B-2" averaged and designated BI B-21, B-22 averaged and designated BIT ° B-31, B-32 averaged and designated B III B-41, B-42 averaged and designated BIV C-1, C-2, C-3 averaged and designated C J C-31 designated C TIT C-41, C-42 averaged and designated C JV This method of treating the data was decided upon because it gives, it is believed, the fairest picture of the protozoan sequence in the infusions. As a matter of fact the individual infusions of the respective groups presented comparatively unimportant variations—except in certain cases which are mentioned. Three of the six infusions composing group A J were discontinued at the end of the first month because the variations between the indi- vidual infusions was not sufficient to warrant the study of so many. For a record of the surface sequence of a single infusion, reference should be made to C /// (fig. 10). For the data of a single form (Paramaecium) at the bottom of two infusions com- prising a single group, see fig. 12. D. _ TIME OF APPEARANCE, MAXIMUM NUMBER AND DISAPPEARANCE OF REPRESENTATIVE PROTOZOAN FORMS AT THE SURFACE OF THE INFUSIONS 1. Monad A I group. Monads were the first animals to appear in con- siderable numbers and their maximum was attained on the 7th day when there were about 5200 per ee. Their decline was equally rapid and by the 20th day of the life of the infusions none were observed in the samples studied. A II group. These forms were the first to appear, reach their maximum of 2000 per ee. on the 4th day, and miminum on the Sth day. 226 LORANDE LOSS WOODRUFF A III group. Monads were practically absent from the two cultures of this group, and this is the only instance in which they did not appear in numbers sufficient to be considered. There were, perhaps, 100 per cc. at several counts. This dearth is accounted for, I think, by an exceptionally heavy growth of Col- poda, which occurred in this group very early (cf. table 2 and fig: 3). A IV group. The monads appeared on the 2nd day, attained their maximum of 1000 per ec. on the 4th day and reached their minimum on the 6th day. BI group. ‘These forms appeared on the 2nd day, attained a maximum of 4200 per cc. on the 8th day, declined to 500 per ce. on the following day and then gradually became less and less until by the 23rd day their number was negligible. On the 36th day, however, they reappeared, attained the number of about 2000 per ec. on the 40th day, and reached extinction on the 60th day. BIT group. On the 4th day there were 1200 monads per cc., and on the 8th day they had entirely disappeared. BIII group. In this group the monads attained a maximum of 5000 per ec. by the 9th day, and by the 12th day there were none remaining. BIV group. A maximum of 1400 per ce. was reached on the 3rd day of the life of the cultures, and then a rapid decline resulted in extinction by the end of the first week. C I group. In these three cultures the average maximum number of monads, nearly 8000 per cc., occurred on the 15th day, and was followed by an abrupt decline ending with their disap- pearance on the 20th day. The maximum of Colpoda occurred on the same day as that of the Monads. C III group. In this group, represented by a single infusion (C-31), the monads attained a maximum of over 8000 per cc. on the Sth day, declined rapidly to 2500 per ec. on the 13th day, and reached a minimum of practically zero on the 24th day. C IV group. Here the monads rose to the number of 5000 per ec. on the 17th day, declined to about 2500 per cc. on the 21st day, then rose to their maximum of 7600 on the 27th day, and by the 32nd day had reached a minimum. SIONS PROTOZOAN FAUNA OF HAY INFU (‘¢zq ‘d “jo spoqjour jo sjrejop 10g) *“— - - — = Bqoaoury | *— — = B]ja04104 {——— = UMN D9vUIBIG ‘—-— = BpryouyodAy !-—-— = wpodjop!:---= pwuoyy ‘suorsnjur oy4 Jo aouaystxo ayy Jo SAvp Jo s9quinu oy} poyjoyd st wsstosqu oy} UG “aovJANS oY} 48 *00 tod smSTURd.IO Jo JaquInu oY} OJWoOIpU soyRuIpIgQ “dnoas 7] yp [BLT 02 0) 000¢ OO00E o0zs 228 LORANDE LOSS WOODRUFF From the study of the monads in all the cultures it is clear that in every instance they were the first type of protozoon to appear and the first to reach a maximum. This is undoubtedly to be explained by the fact that these forms, combining holozoic and saprozoic methods of nutrition, are able to feed on the bacteria which are developing so rapidly at this period, and also to absorb various substances entering into solution from the hay. The monads under consideration are also the first forms to decline and practically disappear, and this is probably due, in part, to the rapid decrease in numbers of the bacteria brought about by the monads themselves and by the rising generations of Colpoda. 2. Colpoda A I group. Colpoda was the second protozoon to appear in considerable numbers and its maximum was attained on the 14th day when there were about 2500 per cc. Its decline was equally rapid and by the 25th day very few active individuals were seen. Beginning at about the 30th day, however, more were observed and on the 37th day there were about 600 per ce. This second rise in numbers was followed by a more gradual decline which ended in the extinction of this form by the 66th day of the life of the infusions. A ITI group. This form was the second to appear and very slowly attained its maximum of 1000 per ee., which took place on the 27th day, then it fell in number to about 200 per ce., rose again to about 500 per ec. on the 44th day, and then became ex- tinct on the 49th day. A IIT group. Colpoda was the second protozoon to appear in considerable numbers in these infusions, the eyele of the monads being apparently aborted. Colpoda arose abruptly to the great number of 15,000 per ec. on the 10th day, fell to about 11,000 per ec. on the following day, and by the 15th day very few active forms were observed. However, almost immediately it had an- other period of reproductive activity which brought up the num- ber to about 4000 per ce. on the 29th day. After this second high point it decreased in number, but persisted until the 63rd day PROTOZOAN FAUNA OF HAY INFUSIONS 229 of the infusion’s life. The growth of Colpoda in this group of infusions is remarkable for its abundance and persistence, for during the greater part of the life of the infusion, Colpoda was the form which dominated. A IV group. Colpoda was the second form to attain its max- imum, which occurred on the 13th day with 2500 per ec. present. This number persisted to the 17th day, and then a very quick decline ended in the extinction of the form four days later. BTIgroup. Colpoda was the third to attain its maximum, being preceded by the monads and. the hypotrichida. Its maximum occurred on the 14th day and this was followed by a slow decline resulting in the disappearance of Colpoda on the 30th day. BIT group. Colpoda attained its maximum abundance on the 6th day, then rapidly proceeded to its extinctionon the 15th day. The notably small development of Colpoda in this group of in- fusions is paralleled by that of all the other organisms in B /T. B III group. In this group of infusions Colpoda rose rapidly to a maximum of 8000 per ce. on the 18th day, and then fell even more rapidly to extinction on the 29th day. _ In this series of infusions Colpoda was again the dominant form, greatly outnum- bering the hypotrichida and paramaecia whose small maxima occurred before its own. BIV group. The appearance of Colpoda occurred relatively late, none being observed until the 6th day, and its maximum growth occurred on the 12th day, and its extinction on the 16th day. In this series it was the fourth form in point of time to reach its greatest abundance. CI group. In these three cultures the average maximum num- ber of Colpoda, 4500 per ec., occurred on the 15th day, after a rapid rise from the 7th day. Then there was an equally sudden decline to about 40 per ec. by the 22nd day, and this number gradually decreased until it became negligible at the 46th day. C III group. Again in this culture the growth of Colpoda over-shadowed that of all the other forms. Appearing on the 4th day it gradually increased until a maximum of about 15000 per cc. was attained on the 33rd day. This was sustained for four days and then a remarkable decrease brought it down to about THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 2 WOODRUFF LORANDE LOSS 230 BSSTOSq® oy} UO Bpodjog {: +--+ = peuoyw ‘“— ++ — = sqoouy {+ — — = vyjoomio, { — - — = vprtyongodsé q \ [9h A Pryor H ‘SUOISNJUT OY} JO doUdySIxX9 oY} Jo SAup Jo acaquinu oY} poqyord st ‘9dBJANS OY} 7B “od Iod sustuvs10 JO Loquinu oy} oyworpursoyvuIpIQ, “dnows yy py Z'3y PROTOZOAN FAUNA OF HAY INFUSIONS 231 600 per cc. on the 48rd day, and 60 per ce. on the 49th day. Approximately this number persisted until the end of the ob- servations on the 76th day. CIV group. Colpoda developed in greater abundance in this group than in any other, attaining, comparatively gradually, a maximum of 25,000 per ce. on the 32nd day, falling to 15,000 on the 37th day, and to 100 per ec. by the 44th day, and practically reaching extinction by the 56th day. An analysis of the above data in regard to Colpoda shows that this form is adjusted to those surface conditions of infusions which exist when the monads have about run their course. 3. Hypotrichida AI group. Representatives of the hypotrichida were the third to appear in considerable number and their maximum of about 2400 per cc. was attained on the 51st day after a long gradual rise. Their decrease in number was somewhat more abrupt, resulting in their extinction by the 85th day of the life of the infusions. A ITI growp. These forms were the second to appear and the third to reach a maximum growth. This was attained on the 39th day after a long gradual increase. A rapid decline reduced them to about 40 per cc. on the 49th day, and they persisted in this number to the 74th day. There was a slight increase in number on the 75th day, at which time the observations were dis- continued. A III growp. A few hypotrichida appeared on the 6th day, gradually increased until there were about 400 per ec. on the 15th and 16th days, then declined to the 24th day. Their maximum of 700 per cc. was reached on the 29th day, after which they de- clined until, on the 43rd, they became extinct. A IV group. The maximum consisting of 1600 per ce. fol- lowed those of the monads and Colpoda and occurred on the 26th day. (0) 0) co Pe = = ° fo) 3 aa oS Se tie N — 2 o 2 x NE sal Sp 3 ae ort Zz +Z = . wo N N = p N Serene | Gey mice avOl os OF” O€ ” Oo do jo abeyus.198q Ajiwe 4 UI S|ENPIAIPU] JO ‘ON 290 A. FRANKLIN SHULL > 3 = oO Ww 59 oO =) zB 2c, uM iS 8 283 wo ilintoes 1 to 15 21 to 25 31 to 35 41 to 46 Number of Generation Fig. 6 be compared with fig. 2, which shows the proportion of male- producers for the same lines. Fig. 6 represents line 5 of table 1, and is to be compared with fig. 3. Both curves show a decrease in size of family, which is especially marked in fig. 6. It does not follow, however, that there is any relation between the two phe- nomena, namely, the decrease in the size of family and the decrease in the proportion of male-producers with long-continued parthenogenesis. To‘discover whether the great fluctuations in the proportion of male-producers has any relation to size of family, several lines that showed the greatest fluctuations have been examined in detail. While in some cases the families, or groups of families, showing a great increase in the proportion of male-producers over the preceding generations also showed a great increase in the size of the family, this was not true in a number of other cases. One need not conclude from this that there is no relation between size of family (vigor of line) and proportion of male-producers. There are many accidents which might happen to a female whose family includes many male-producers. I have known a number of females to die as a result of accidentally (?) starting to devour a large Paramecium or a fiber in the water. The small families produced by such females may have included mostly male- producers, so that a study of individual families can hardly be LIFE CYCLE OF HYDATINA SENTA 291 expected to show a correspondence between ‘vigor’ and the pro- portion of male-producers. Effect of inbreeding on the proportion of male-producers In the second of these studies (Shull, 711 a) I deseribed experi- ments in which two distinct lines of rotifers, yielding different proportions of male-producers, were crossed, the zygotes giving rise to lines in some crosses yielding more male-producers than either parent line, in other crosses a proportion of male-producers intermediate between those of the parent lines. In order to explain these phenomena, an attempt was made to inbreed the same lines that were used in the earlier crossing experiments. Females were paired with males of the same line, and a large number of fertilized eggs was secured. These eggs, however, did not hatch before it was necessary to discontinue the experi- ments. Ihave now, however, in other lines, succeeded in obtain- ing viable offspring from females paired with their own nephews or cousins, and give the results in the following experiments. Experiment 1. Inbreeding. Some winter eggs were collected in the spring and kept in an ice-chest or in cold running water until September. The eggs were then brought to room tempera- ture and began to hatch in five days. From one of the females thus obtained was reared the line of rotifers used in this and the following inbreeding experiments. Between October 7 and October 20, many females of the parthe- nogenetic line just mentioned were paired with males of the same line. Of 1099 eggs obtained, one hatched October 22, and from her another parthenogenetic line was bred. The number of male- and female-producers in this inbred line is compared, in table 2, with the corresponding data from that part of the original line which was reared at the same time. It appears from the table that the inbred line yielded 16.7 per cent of male-producers, the original line only 10.6 per cent. In such an experiment, however, the original line is necessarily further removed from the fertilized egg than is the inbred line. We have learned above that there may be a progressive decrease 292 A. FRANKLIN SHULL TABLE 2 Showing the number of male- and female-producers in two lines of Hydatina senta, one line being the result of inbreeding in the other line, that is, being derived from the offspring of a male and female both from the other line. Male-producers are designated 32, female-producers 9 9. ORIGINAL LINE INBRED LINE | DEG atae Number of 7) Number of 2 9 Date of first’ | Number of o?) Number of 2 9 young | young | October October 24 1 8 24 0 28 26 | 4 30 25 10 36 28 0 28 27 2 21 29 0 48 29 | 2 17 31 5 40 30 2 43 November — November | | 2 | 0 19 1 | Sie 44 4 1 15 3 | 7 38 6 u 33 5 | 2 45 8 8 38 6 2 44 10 9 36 8 0 26 12 0 22 10 | 14 36 14 1 38 12 13 9 16 | 2 46 14 6 37 18 5 27 16 3 30 20 | 8 18 18 10 9 22 2 31 20 0 23 25 6 39 ae, 1 11 26 2 28 24 4 10 28 5 | 23 26 1 ai 30 4 4 28 } 9 25 December 30 0 41 2 0 27 December 4 3 40 2 Phe 33 7 3 23 4 1 43 9 rat 4 6 | 20 20 12 1 22 9 | ul 10 13 0 5 17 2 0 29* 11 2 13 15 0 15* 13 0 17 15 3 14* = = — ~ if Motalenaer 88 736 147 732 Per cent of opel Badaose 10.6 16.7 * Remainder of family not recorded. LIFE CYCLE OF HYDATINA SENTA 293 in the proportion of male-producers, such that a comparison of two lines may show fewer male-producers in the older line, although the two lines would have been equal had each been taken at the same number of generations after the fertilized egg. An application of this discovery is found in the present experiment. The original line in this experiment is line 8 of table 1. It will be seen in that table that the early generations of the line had a larger number of male-producers than the subsequent genera- tions. The original line passed through ten generations before the inbred line started. If these ten generations, comprising 147 male-producers and 315 female-producers, be added to the 27 generations given in table 2, then the total for the original line shows 18.2 per cent of male-producers, or a higher percentage than that of the inbred line. We may not be justified in including the first ten generations, as I have just done, but in view of the decrease in the proportion of male-producers in the later genera- tions, it seems to me unsafe to ignore the early generations. Incidentally it may be mentioned that the average size of family in the entire original line, exclusive of the last two families which were not fully recorded, was 34.5 as compared with 30.7 in the inbred line. Experiment 2. Twice inbreeding. Females of the inbred line of the preceding experiment were paired with males of the same line. Of 144 eggs obtained, one hatched November 26, and became the parent of the line in table 3 designated ‘twice inbred.’ This line is compared with those parts of its parent (also inbred) line and the original (‘grandparental’) line that were reared at the same time. Here the difference between the two inbred lines is not great, while the percentage of male-producers in the original line is con- siderably less. Since all three of the lines are at different ‘ages,’ that is, a different number of parthenogenetic generations has been passed through in each line since the fertilized egg from which the line was derived, it is of interest to note the proportion of male-producers in the whole lines, and not merely those parts that were bred simultaneously. In the original line, including 28 generations reared previously to the beginning of this experi- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 2 294 A. FRANKLIN SHULL TABLE 3 Showing number of male- and female-producers in three lines of Hydatina senta, the line in the second column being inbred from that in the first column, and the line in the third column from that in the second (see text) ORIGINAL LINE } INBRED LINE | TWICE INBRED LINE | Or Or Or Or | | oO Oo HOt 1 sos ‘o o | lato o Date of first | 2 | © Date of first s © | Date of first | . % young amen Eas young ral a || young | ae) 3 | 5 g ee || | 8 E Zz Z| Zz Zz | ie Z ss = ae ay oa he ie a hs — vo 12, n 4 PLATE 6 EXPLANATION OF FIGURES 18to25 Show the effects of removal of the jelly by centrifuging on the spermato- zoon attached to the egg. It willbe noticed that the cone in this and the following plates, though just as well defined as the preceding, stains differently. The behavior of the cone is the same, however. 18 Entire spermatozoén present, drawn out to band. History: centrifuged 7200 revolution in thirty-five seconds, fifty minutes after insemination; preserved immediately. 19 The middle-piece and part of the base of the spermatozoén have been removed by the jelly. The protoplasm surrounding the cone has been raised in a protuberance, which happens not infrequently (cf. fig. 22). History same as fig. 18. 20to23 These figures show removal of increasingly large portions of the sperm head by the jelly. Each drawing from a single section of aseparateegg. History same as fig. 18. 24to25 To show effects of removal of jelly by centrifuging after penetration has begun. History same as fig. 18. 466 FERTILIZATION FRANK IN K. NEREIS LILLIE ATI THE PLATE 7 EXPLANATION OF FIGURES 26 Removal of external part of the spermatozoon by centrifugingin an advanced stage of penetration (cf. fig. 7). History: centrifuged 7200 revolutions in forty seconds, fifty minutes after insemination. Preserved fifteen minutes later. 27to31 Toshow early penetration of injured or partialspermatozoa. History: centrifuged 7200 revolutions in forty seconds, fifty minutes after insemination. Preserved fifteen minutes later. 27 Part of the spermatozoon has entered. The remainder is shown external to themembrane. The internal part is definitely dividedintwo. Rotationis begin- ning. 28 The part of the spermatozoén external to the membrane is nearly separated from the internal part, which is itself definitely divided in two. All parts a little swollen as shown by the tone of the stain. The rotation of the cone is beginning. 29 The internal part is apparently breaking off from the much larger external part of the spermatozoén. Cone in process of rotation. 30 A case in which only a small part of the spermatozoén has entered; the rest of the spermatozoon is lost. It represents alater stage of an injury similar to that shown in figs. 20 or 21. ‘ 31 A case similar to fig. 30. 32 A somewhat later state of rotation of the cone than shown in preceding fig- ures. History the same. 33aand33b Twosuccessive sections of the same egg; the parts of the spermato- zoon shown in the two sections are entirely separate. The proximal larger part (33 a) is proceeding with its rotation and development, leaving the base of the sperm head and the middle piece behind. History same as figs. 27-31. 34 Penetration stage of a sperm remnant preserved fifteen minutes after centri- fuging. 40 Two partial sperm nuclei with asters associated with a single cone. Prob- ably a later stage of a condition like that shown in figs. 27 or 28. 468 FERTILIZATION IN NEREIS FRANK RK, LILLIE PLATE 7 26 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No, 4. Kenji Toda, Del. 469 PLATE 8 EXPLANATION OF FIGURES 35to 87 To show origin of asters in connection with partial sperm nuclei. In each case a remnant of the spermatozo6én on the surface guarantees the partial nature of these sperm nuclei; note the variation in size. History: Centrifuged 7200 revolutions in forty seconds, fifty minutes after insemination; preserved fif- teen minutes later. Fig. 36 is a combination of three sections. 470 sz FERTILIZATION IN NEREIS FRANK R. LILLIE PILATE 8 Kenji Toda, Del vou. 1%, No. 4. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, PLATE 9 EXPLANATION OF FIGURES 38 and 39 Two unusually small sperm nuclei with their asters, separating from the cones. From two eggs;compare the size of the entire sperm nucleus at this stage (fig. 11). History same as figs. 35 to 37. 41 Two sperm nuclei of unequal size from the same egg. Compare size of cen- trosomes and asters. History: Centrifuged 7200 revolutions in thirty-six seconds, forty-four minutes after insemination; preserved ninety-two minutes after insemi- nation. Reconstruction of three sections. 472 NEREIS LILLIE IN RK, RTILIZATION FE FRANK K \ OF EXPERIMENTAL. ZOOLOGY JOURNAL THE PLATE 10 EXPLANATION OF FIGURES 42 Five successive sections showing the entire germ nuclei of one egg. The male nucleus has five nucleoli, the female has thirteen. The line of apposition of the germ nuclei is seen in the third section. History: Centrifuged 7200 revolu- tions in thirty-six seconds, forty-four minutes after insemination. Preserved sixty-four minutes later. 474 FERTILIZATION IN NEREIS FKANK K. LILLIE PLATE 10 THE JOUKNAL OF EXPERIMENTAL. ZOOLOGY, vot. 12, No. 4 Kenji Toda, Del PLATE 11 EXPLANATION OF FIGURES 43 Three successive sections showing the entire egg nucleus of an egg from which the spermatozoon was entirely removed by centrifuging. Both polar bodies formed. Fourteen chromosomes indicated. History: Centrifuged 7200 revolu- tions in thirty-seven seconds, forty-two minutes after insemination. Preserved sixty-five minutes later. 44 Three successive sections showing the entire egg nucleus of an egg from which the spermatozoén was entirely removed by centrifuging. The first polar body was not formed in this case, and a monaster arises around the egg-chromo- some group. History same as fig. 43. 476 FERTILIZATION IN NEREIS FRANK K. LILLIE 43a 43h 43¢ THE JOURNAL OF EXPERIMENTAI ZOOLOGY, PILATE 11 dda 44h, t4¢ “ te THE ELIMINATION OF THE SEX CHROMOSOMES FROM THE MALE-PRODUCING EGGS OF PHYLLOXERANS T. H. MORGAN From the Zoélogical Laboratory, Columbia University TWENTY-NINE FIGURES My studies of the life cycle of the phylloxerans of the hickories have shown first ('08) why the fertilized eggs produce only females,! and second (’09) that the production of the males is caused by the elimination of a chromosome from the male-pro- ducing egg.2 One essential point in the life eycle still remained unexplained; namely, the cause of the production of small male- and large female-producing eggs. The differentiation of these two kinds of eggs precedes, in the life cycle, the formation of the true males and sexual females. Jt may appear therefore that the question of the sex determination antedates those changes that lead to the elimination of a chromosome from the male-produc- ing egg, and, uf so, the real question of sex determination might seem to lie deeper than the manewvres of the sex chromosomes. Until this point is cleared up the value of the chromosome hypothesis in sex determination may seem to hang in the balance. IT am now able to bring forward certain evidence which I believe throws light on this important topic and I am prepared to offer an hypothesis based on the new evidence, which, if true, substan- tiates the view that one of the essential changes in the formation of the large and the small eggs is connected with changes in the sex chromosomes. 1 Proc. Soc. Exp. Biology and Medicine, vol. 5, 1908, and Science, vol. 29, 1909. ? Proc. Soc. Exp. Biology and Medicine, vol. 7, 1910. 479 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 4 480 T. H. MORGAN The main points that were described in my previous papers may be summarized as follows: 1. Two classes of sperm are produced in the male differing in the presence and absence of a pair of chromosomes. One class of sperm degenerates. It corresponds to the male-producing class of otherinsects. The other class produces functional spermatoza which entering the egg give rise to females only. These sperm correspond to the female-producing class of other insects. 2. The male-producing egg contains one less chromosome after the extrusion of its single polar body than it contained before this event. In a preliminary note (710) I have stated how this elimination takes place and in the present paper I bring forward the evidence on which this statement was based. 3. The difference in size between the male-producing and the female-producing egg, before the former has extruded its polar body, proves that the predetermination of the males ante- dates the extrusion of the chromosome in the polar body of the (smaller) male-producing egg. 4. More male-producing individuals are the descendants of each stem-mother than female-producing individuals. The stem- mother must give rise to two kinds of eggs, i. e., they must be different either before or after the polar body is extruded. The factor that differentiates these two kinds of eggs, was not dis- covered. It is this point that the evidence now brought forward may I hope help to elucidate. THE DIFFERENCES IN THE CHROMOSOME GROUPS IN THE POLAR SPINDLES OF THE STEM-MOTHER’S EGG AND OF THE MALE AND FEMALE-PRODUCING EGGS In my former paper (’09) I have figured ten equatorial plates of polar spindles of the eggs produced by the stem-mother. In all of these the sex chromosomes are of nearly the same size. In two other plates one chromosome is much smaller than the others, which is probably due to this chromosome having been cut by the knife. Failure to find the missing piece in the next section would not be significant, since it might be very difficult to find such a piece in the egg filled with yolk granules of about the same ELIMINATION OF SEX CHROMOSOMES 481 size as the chromosomes. Side views of the polar spindle, of which four are given, each with three chromosomes, show these to be of equal size. One of two cases of an egg nucleus (just prior to the formation of the spindle) also shows six equal chro- mosomes; the other case shows four equal chromosomes and one of double size that no doubt-represents two chromosomes overlapping or else stuck together. When these chromosome plates are compared with those of the female-producing egg shown in fig. LX, page 255 of my paper (’09), the size relations seem to be about the same. Only four plates of these eggs were found. I suspect that one of the equatorial plates that is assigned to a male-producing egg, namely, fig. LX; K, really belongs to a female-producing egg. Occasionally it is difficult, owing to the obliquenessof the sections, to make sure that a particular egg is a large or a small one. If this egg is excluded, or referred to the female-producing series, there remain fourteen equatorial plates of male-producing eggs. In all of them one chromosome is noticeably smaller than the rest. I can now add four more chromosome groups of male-pro- ducing eggs to this list, figs. 1, 2,3,4. Three of these are equa- torial plates or just prior to that phase, and the fourth shows the chromosomes in the nucleus just prior to the spindle stage. They all contain five larger chromosomes, and one much smaller than the other five. It is true that there is some variation in the relative size of the chromosomes in all of these figures, which makes it difficult to express exactly the relative sizes of the different chromosomes, and therefore I am aware of the danger of attempting to distin- guish between the plates of the male- and female-producing eggs; yet the presence of the very small chromosome is so distinctive of the smaller eggs that I believe no error is committed in attribut- ing to this difference at least a real significance. If the two kinds of chromosomal groups just specified are significant one should expect to find a similar difference in the somatic cells of the individuals that give rise to these eggs, for since each of these individuals produces only one kind of egg (all the eggs found in one individual are male-producing or female- 482 T. H. MORGAN producing) this difference should be apparent on inspection of the somatic cells of these individuals. In my paper I have, in fact, given nine plates taken from young stages of the devel- opment of the embryo. Some of these figures, notably fig. VLII C, E, I, show six nearly equal chromosomes, while five of them, notably fig. VIII A, B, D, F, G show five larger and one smaller chromosomes. When these drawings were made the importance Eee Tart ee SS i, 3 of the size relations was not appreciated, and the number of cases is too small to be of great value, but it is significant, I think, that the two kinds of chromosome groups required by the hypoth- esis are actually represented in these figures. It appears, then, probable that after the extrusion of the polar body of the egg of the stem-mother a change has taken place in those individuals that become the male-egg-producers. One chromosome has become smaller. ELIMINATION OF SEX CHROMOSOMES 483 THE DIVISION OF THE POLAR SPINDLE IN THE MALE- AND IN THE FEMALE-PRODUCING EGGS A brief abstract of the results given in this section was pub- lished in the Proceedings of the Society of Experimental Biology and’ Medicine? for May, 1910. In order to study the division of the polar spindle a large amount of new material was collected in the summer of 1909 which was cut and studied during the following winter. It has been most laborious to find eggs in which the polar spindle was in the process of division, and I wish to express my obligations to my assistant, Miss E. M. Wal- lace, who has found most of the new cases here figured. In my former paper (’09) I described the anaphase of two eggs that seemed to be female eggs (see below), but none of the male eggs; and it is the latter that would be expected to give the critical evidence. This evidence was briefly stated in my prelim- inarynotein 1910. I shall nowgive drawings of several anaphases of male eggs that show beyond doubt that a lagging chromosome is present; that it passes to the outer pole, and forms a separate vesicle in the polar body. The first case is shown in fig. 5 representing an anaphase of the polar spindle. Five chromosomes lie at the outer pole and five at the inner pole. In the middle of the spindle lies a double chromosome. It is relatively large and its two halves appear somewhat unequal. For reasons that appear later I shall speak of this as a single chromosome that has already divided into halves. The second ease is shown in fig. 6. Here also five chromosomes, somewhat elongated, lie at the outer pole and five at the inner pole of the spindle. ‘In the middle of the spindle there is a double chromosome, its halves equal as far as can be determined. The third case is shown in fig. 7. It represents a later stage; the polar body being in process of constricting from the egg. The group of chromosomes at the outer pole is now in process of division. Five chromosomes can be recognized, two dividing, and three having completed their division. At the inner pole ‘Proc. Soc. Exp. Biology and Medicine, vol. 7, 1910. 484 T. H. MORGAN ELIMINATION OF SEX CHROMOSOMES 485 the nucleus has begun to form. It contains five distinct chro- mosomes. Midway between the poles is the lagging chromosome completely divided into equal or nearly equal parts. A few traces of the spindle fibers are discernible. This is the clearest case that I have found and shows very distinctly the conditions at this stage. The next case, fig. 8, is not so instructive, since the chromo- somes in the inner nucleus have in one place seemingly stuck together so that only four bodies are seen. The lagging chro- mosome could not be found but the five outer chromosomes are distinct. Fig. 9 shows the polar body nearly constricted off. The five inner chromosomes are clearly seen. The lagging chromosomes were not found and may have been fused with the lump of chro- matin‘in the polar body that represents the massed chromosomes. ‘ In fig. 10 (and in figs. 9, 11, 12 and 13) the egg nucleus is represented nearer the surface than in the actual section. The nucleus of the polar body contains a fused mass of chromatin. What appears to be the lagging chromosome lies on its outer wall, and is partially constricted into halves. The inner nucleus shows five equal or nearly equal chromosomes. As this is the only case observed where the lagging chromosome lies on the outer wall of the nucleus of the polar body, and as it is difficult to see how the chromosome could have reached this position; and moreover since the double body is smaller than the lagging chro- mosome in the other cases; it may be that this deeply staining body is not the lagging chromosome at all but a pair of displaced yolk granules. This interpretation is supported by the next case. In this instance fig. 11 the inner nucleus is well formed and its chromosomes diffused or at least not stained. In the polar body there is a nucleus in which four chromosomes can be made out with the double lagging chromosome lying on the inner side of the nucleus. 10 gray @ 8 red 9 III. Brown Leghorn @ X Red F, 2 ——> 3 redo’ 3 red 9 IV. Gray F; @ (from II) X Brown Leghorn 2 = Saas 5 duckwing # 5 redo 7 gray @ 1 silver gray 9 1 brown duckwing 9 2 red 9 2 brown 2? EXPLANATION OF RESULTS As I pointed out in my preliminary note, there is here at least one sex-linked factor, which I then called G, causing the two types of F; females. The F, generation agrees with this, mating III giving no grays, and mating IV both gray and non-gray males and females. But there are obviously other factors con- tributing to the F. result, which is decidedly complex, as has so often been found to be the case in experiments with fowls. I do not, therefore, feel justified in giving more than a tentative explanation of the results, since the numbers are small, and only a few of the many crosses which would be required to test any explanation have been made. The following will, however, cover the results obtained, and is the simplest scheme that I have been able to work out. Let us assume that the Columbian Wyandotte carries an inhibitor, 7, for red in all parts of the body, with the exceptions noted below. This is the G of my earlier paper. The Wyan- dotte also carries another sex-linked inhibitor, V, which prevents the production of red in the neck (and saddle of the male), not affecting the other parts. This is probably the factor described by Davenport (’11) as found in Dark Brahmas. Birds not earry- 504 A. H. STURTEVANT ing these two factors have these regions of a red or reddish color, so that the Brown Leghorn, and probably also the Columbian Wyandotte, must carry a factor for red, R. The factors J and N do not completely inhibit R, since most of my J-bearing birds show traces of brown here and there, and all the white necked males are very ‘brassy’ (yellowish). Both these characters some- times appear in pure Columbian Wyandottes. Apparently there is also a Leghorn pattern factor, L,? causing black breast and black on the wing coverts in the male, and black stippling and salmon breast in the female, the latter effect appearing even in the presence of J. The factor L is hypostatic to another pat- tern factor, P, which is carried by the Columbian Wyandotte, and which inhibits all the colors just mentioned, as caused by L, leaving the color of the part dependent upon F and its inhib- itors. But one dose does not completely inhibit the stippling of the female. An alternative view, equally as satisfactory, I think, is that there is no inhibitor P, but that the Wyandotte has no L, and that the absence of this factor is dominant to its presence, heterozygous females being distinguishable by the stippling. On the first view L is probably present in all my birds. The con- stitution of the various types would then be as follows: Columbian Wyandotte INRPL Brown Leghorn inRpL Gray INRPL or InRPL Red inRPL Silver gray INRpL, or nRpL Brown duckwing iNRpL One other combination is possible—iNRPL. This should give a bird with Columbian pattern, white or straw neck, and red body. It is possible that such would have appeared had more birds been raised, but I know of no variety having any similar color combination, and have never observed it in a cross-bred 2This is probably one of the components of the J (Jungle pattern factor) of Davenport (09). SEX-LINKAGE IN FOWLS 505 fowl. This seems to me to be one of the most difficult points in connection with the hypothesis here given to explain my results. It may be that there is some interaction between N and P, such that when both are present N cannot produce its effect. Then iNRPL would give red. This could be tested by raising large enough numbers from mating IV to find out the real F; proportions, or by testing a number of reds with Brown Leghorns and seeing if any of them gave brown duckwings in F; or F,.. My principal evidence indicating that N is sex-linked was the fact that the females from mating II had red necks. But since they also had P, if the above hypothesis is correct, the only good reason for making N sex-linked is that it is probably identical with the factor described by Davenport (’11) as being sex-linked. If it is not so linked, then some of the reds from mating III should also carry it, and that mating should, eventually, produce some brown duckwings.® Since I have no evidence that R or L is missing in any of my birds I shall simplify the following formulae by omitting them. In these formulae MM represents a male, Mm a female. I. Columbian Wyandotte « INPM INPM Brown Leghorn @ inpM inpm INPM inpM — gray? INPM inpm. — gray 2 II. Brown Leghorn @ inpM inpM Columbian Wyandotte 2 INPM inPm inpM INPM —gray & inpM inPm —red 92 III. Brown Leghorn inpM inpM Red ? (gametes) inPM inpMinPm inpm inpM inPM —red@& inpM inpM —brown & (not seen) inpM inPm —red 9? inpM inpm —brown @ (not seen) ’The real solution of this difficulty may be that J and N are coupled. 506 A. H. STURTEVANT IV. Gray o& (gametes) INPM INpM InPM InpMiNPMiNpM inPM inpM Brown Leghorn 2 inpM inpm INPM inpM —gray¢# INpM inpM —duckwing 7 InPM inpM —gray¢ InpM inpM ~—duckwing © (silver gray) iNPM inpM —red & (white-necked?) iNpM inpM —duckwing 7 inPM inpM —red@ inpM inpM —brown ¢& (not seen) INPM inpm —~gray 2 INpM inpm —silver gray @ InPM inpm —~gray 2 InpM inpm —silver gray 2 iNPM inpm —red 92 (white-necked?) iNpM inpm —brown duckwing ? inPM inpm —red 9 inpM inpm —brown @ OTHER EXPERIMENTS DEALING WITH THE SAME COLORS Bateson (’02, ’09) and Punnett (’05) have given some facts regarding the duckwing color. When the Brown Leghorn was crossed with the White Dorking or White Leghorn, they obtained in F, some silver gray females. Two of these mated to a pure Silver Gray Dorking male gave only silver grays, and of these, four females to a male gave only silver grays. From these facts Bateson (’09) infers that the replaced red and yellow of the Brown Leghorn probably depends upon a separate factor, which his white breeds lacked. It seems to me more probable that this factor, which I have called R, was present in all three breeds, and that the two white breeds carried also the factor I. Any F, female showing the silver gray color would then be as pure for J as a pure Dorking, the factor being sex-linked, which explains why they had no trouble in getting a dominant F, to breed true. Mr. T. Reid Parrish, a Columbian Wyandotte breeder, has published in advertising circulars and in poultry journals (e.g., Parrish, ’11) detailed accounts of how he originated a strain of Columbian Wyandottes (probably not the one used in my ex- periments). According to this account he used Light Brahma SEX-LINKAGE IN FOWLS 507 females with a White Wyandotte male. The Light Brahma has exactly the color of the Columbian Wyandotte. The breed seems to have been brought from the Orient in something like its present form, so that its history as to the origin of its color must probably remain a matter of conjecture. The White Wyan- dotte was derived directly from the Silver Laced Wyandotte, and is still, or was comparatively recently, a not uncommon sport from that variety (see McGrew, ’01, and poultry literature generally). This would seem to indicate that it is a recessive white, probably due to the dropping out of a color producer. Mr. Parrish’s statements support this view, as he says he obtained, in the F, generation from his cross, silver laced, barred, and Columbian birds—apparently no whites. These F; silvers he says were not typical, some of them having nearly white breasts, ‘‘yet showing a trace of lacing throughout the plumage.’’ This sounds as though they were much like the birds I obtained in my cross between silvers and Columbians (Sturtevant, ’11). From the result of that cross it would appear that silver is incompletely hypostatie to Columbian. Mr. Parrish says of his F, barred birds mentioned above that they ‘‘showed stronger Brahma markings than the silvers, but there was unmistakable barring throughout the plumage, being especially noticeable in tail and wing, some specimens showing barring in every section.’’ This is a most interesting statement, in view of the work of Spillman (09), Goodale (’09, ’10), Pearl and Surface (’10), and Daven- port (’06, 09) on barring. In this connection it is worth noting that occasionally a few barred feathers occur in pure Columbian Wyandottes, especially in the tail coverts of young males, and that one of my F, males (a duckwing) in the experiment described above has some barring in his hackle. Mr. Parrish states that he mated his F, Columbians with White Wyandottes, reciprocally. From Columbian female he obtained the same three classes as in F,, but we are not told whether or not whites appeared. The mating with Columbian male gave whites and Columbians, but he doesn’t say what else, if anything. 508 A. H. STURTEVANT The only conclusions which I feel safe in drawing from these data are that the White Wyandotte is a recessive white, lacking a color producer, and that it carries a silver laced determiner. Bateson (’02) gives confirmatory evidence for the first of these conclusions. SEX FORMULAE It will be noted that I have used above the MM, Mm scheme for sex formulae in preference to the more usual Ff, ff formula (see Morgan, 711). I have made the change because the formula used here gives a mechanism which allows both complete sex- linkage, and also incomplete association with the sex-determiner. I shall now present the evidence which has led to this view. It seems to me that the evidence now before us warrants the conception of the chromosomes as the carriers of Mendelian factors or genes, as a working hypothesis. This conception is especially helpful in considerations of sex-linkage and the other forms of gametic coupling or associative inheritance. The recent hypothesis put forward by Morgan (’11 a, ’11 b, ’11 ¢) to explain these phenomena seems to me to overcome the old difficulties encountered by the chromosome hypothesis of Mendelian inher- itance. I shall make this conception the basis of my argument in favor of the MM, Mm scheme. Sex-linked inheritance of the type concerned here has now been known for some time, and has been recognized in Lepi- doptera and in birds, as follows: Abraxas (Doncaster and Raynor, 06; Doneaster, ’08), canaries (Durham and Marryatt, ’08), fowls (Bateson, ’09; Spillman, ’09; Goodale, ’09, 710; Pearl and Sur- face, 10; Bateson and Punnett, ’11; Davenport, ’11; Sturtevant, "11), and ducks (Goodale, 711). Since my argument for the MM, Mm sex formula depends largely upon certain cases which I believe to represent partial sex-linkage, it will perhaps be well to present in some detail the evidence for the existence of this phenomenon. In this category I have included three cases of the Abraxas type (one in the fowl, one in the canary, and one in Aglia tau), and one, which I shall describe later, in the Drosophila type of sex-linkage. SEX-LINKAGE IN FOWLS 509 Bateson and Punnett (11) describe certain exceptions occur- ring in their sex-linkage experiment with fowls, which they sug- gest may be due to a failure of the usual association between the sex-linked facotr and the sex-determiner, i.e., to ‘crossing over’ in the female. This is what I mean by partial sex-linkage. The sex-linked factor in canaries transforms pink eyes to black, and may then be represented by the symbol B. The following crosses have been reported by Durham and Marryatt (’08): Black 7 BM BM Pink ? bM bm BMbM —black @ 19 BM bm —black @ 7 F, black @ BMbM Black 9 BM bm BM BM—pblack @& \ o BMbM —black oJ ~ BMbm —black 9 18 bM bm —pink @ 13 Fi black @ BMbM Pink ? bM bm BM bM —black & 24 bM bM —pink ¢& 21 BMbm —black bM bm —pink @ 19 Pink bM bM Fi black 92 BM bm bM BM—black @ 4 bM bm —pink @ 6 It will be seen that all the above crosses give the typical Abraxas results if B and M be assumed to be completely cou- pled,* but I have purposely omitted one cross: 4 Only in the second and fourth matings is there any opportunity for crossing over, and in those two a total of only seventeen birds that would be affected by such crossing over. 510 A. H. STURTEVANT Pink @ bM bM Black @ BM bm BMbM —black @ bM bm —pink 9 black 9 It is the last class of four black females which is of interest in this connection, and is inexplicable on the current scheme. I see only two explanations of this class—either the sire was, through some mistake, really black-eyed, which I mention only because it presents itself as a possible way out of the difficulty; or else, as I think probable, we have here anexample of partial sex-linkage, B ordinarily being coupled with M., If this coupling be incom- plete, then black females are to be expected from the last mating, as the following analysis will show: Gametes of pink 7 bM bM Gametes of black 2 BM bm (Bm bM) BM bM —black 7 bm bM —pink @ (bM bM —pink & ) (Bm bM —black ? ) This hypothesis could be easily tested. If it is correct, then the cross just discussed should, if large enough numbers be reared, produce as many pink males as black females. Furthermore, if these black females be bred to pink males, there should arise a race which would be dimorphic—black-eyed females and pink- eyed males—except for the occasional ‘crossing’ back of B, which would now be coupled with m, and m occurs only in the female. Such a relation, if it be shown to exist, would be highly interesting in its bearing upon the problem of secondary sexual characters. The third case which I have interpreted as partial sex-linkage is that of the moth Aglia tau reported by Standfuss (’96), and discussed by Castle (03). The variety lugens of this species is dominant to the type. Its gene may be designated L. My interpretation of this case is that L and M are associated in such degree that ‘crossing over’ occurs in about one-third, instead of the usual one-half of the cases. The analysis of the matings SEX-LINKAGE IN FOWLS uel! then is as follows. It is obvious that all the lugens moths used were heterozygous. Lug.o LM IM TauQ IM Im LM IM — lug. ¢& 31 IM IM — tau o 14 LM Im — lug. 9? 13 IM Im — tau 9? 28 The results of this one cross are not in accord with my hypoth- esis, since all four classes should be equal, but I think the num- bers are rather too small to be very significant. The reciprocal cross gives: . Tau co IM IM Gametes, lug. 9 2 LM2Im 11M 1Lm 21M LM —lug. o 26 11M IM —tau o@ 13 21M Im —tau 9 25 11M Lm —lug. @ 11 This case comes out as I expect, but since the numbers are no larger and no more disproportionate than those in the first cross, I must rest my case on the third: Lug. @ LM IM Gametes, lug. 9 2 LM1IM2 lm Lm 2LMLM } 2 LM IM r>—5 lugo 129 11M LM } 11M IM —ltauc 16 1 LM Lm 11M Lm }—4lug. 9 9% 2ILMIm J 21M Im —2tau 9 36 The relative size of the classes is perhaps as near the expected proportion as could be looked for, and becomes still nearer expec- tation if the coupling strength be increased slightly.°. So much, then, for the experimental evidence bearing upon the case. 5Standfuss (710) has published more data on this cross, but unfortunately has not reported the sex ratios obtained. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 4 512 A. H. STURTEVANT The cytological evidence relating to birds and Lepidoptera is not very helpful. Guyer’s (09, ’09 a) reports on guinea-fowls and chickens are directly opposed to the experimental evidence, in that they make the male heterozygous for sex. Since a re-ex- amination of the fowl case by other cytologists has so far failed to convince them that Guyer’s view is correct, I think we may for the present disregard this evidence, at least in so far as it concerns the fowl. Several observers (see Stevens, ’06; Dederer, 07; Cook, 710; Doneaster, ’11) have studied the spermatogene- sis of Lepidoptera, and in some cases have seen what they sus- pected to be an equal pair of idiochromosomes. I do not know of any further cytological evidence in these two classes of animals. F i M M 100 OU UE 20 oe 2 Cf Q Ch 2 3 4 The cytological evidence indicates that, in the Lepidoptera at least, the male has two equal idiochromosomes. Judging by the experimental evidence, this must also be the case in birds, and the female must, of course, have at least one similar chro- mosome (see figs. 2 to 4). These three are the carriers of the genes for sex-linked factors. The doubtful point is the mate of this chro- mosome, present in the female-producing egg. If the sex formula be Ff, ff, as we have been supposing, then this chromosome would be, visibly or imperceptibly, larger than the ‘male’ (f- bearing) chromosome, since it would have one factor, 7, not present in that chromosome (fig. 2). In this case it would seem that complete sex-linkage, such as that found in Abraxas and in barred fowls, would occur not at all, or at least only rarely, since every part of the f-chromosome would have a homologous part in the F-chromosome, and crossing over would thus be pos- sible. It might be that the process of reduction is such that no crossing over is possible in oogenesis, but if the cases of par- SEX-LINKAGE IN FOWLS oltes tial sex-linkage be admitted, such crossing over must be possible.® If, on the other hand, we use the MM, Mm scheme we meet with no such difficulties. In this case the heterochromosome contained in the female-producing egg is smaller than its mate in the male-producing egg, since it lacks the factor M (fig. 3),’ and the sex-formula is MM, Mm, two F’s being always present in both sexes, and contained, presumably, in some other pair of chromosomes. This would allow complete sex-linkage, for genes in that part of the M-chromosome having no homologue in the m-chromosome, and incomplete sex-linkage, for genes in the part having such a homologue. One might, of course evade this conclusion by assuming the condition represented in fig. 4. In this case the chromosomes would not conjugate evenly, and the part marked ZL could carry such factors as are completely sex-linked. Of the converse case, where the male is heterozygous for sex there are not so many examples known. In Drosophila Mor- gan (’10, ’11, ’11 a, ete.) has reported numerous sex-linked factors. Miss Stevens (’08) has found here exactly the cytological con- ditions demanded by the experimental evidence. In man color- blindness follows this same scheme of inheritance, as do appar- ently several diseases (Bateson, ’09; Morgan, 11). Here, too, Guyer (’10) has reported cytological evidence that it is the male which is heterozygous. Finally we have the case of partial sex- linkage referred to above. Miss Stevens (’11) has reported hetero- chromosomes in the male guinea-pig, and as that animal has been experimentally bred quite extensively I was led to look for sex-linkage in it. Perhaps the dwarf form studied by Miss Sollas (09) is such a case. This is a recessive form, and has not been reared to maturity, or had not been in 1909, so that the case is not thoroughly worked out, but it seems to be most easily explainable as a case of partial sex-linkage. If we rep- 6It should be noted that, if my view is correct, th sex chromosomes under dis- cussion are homologues, not of the X and Y of Diptera, Hemiptera, ete., but of the M-bearing chromosomes in those groups. 7 On this scheme two F’s are to be assumed to be always present in both sexes, probably situated in another pair of chromosomes. 514 A. H. STURTEVANT resent the factor for normal size, carried by the heterochromo- somes, by \, then in ordinary guinea-pigs this is present in both gametes of each sex. But in a certain strain used by Miss Sollas, from which all her dwarfs descended, it had dropped out of the odd chromosome. ‘This strain would go on breeding true, then, for some time, according to this scheme: NF NF normal ? NF nf normal @ NF NF normal @? NF nf normal @ But now suppose crossing over sometimes occurred, and we should have: NF NF normal ? NF nf (nF Nf) normal &@ (gametes) NF NF normal ? NF nf normal 7 (NF nF normal ?) (NF Nf normal ) We should still have no dwarfs, but if the heterozygous female were mated to a male of her own race we should get dwarfs, thus: NF nF normal 2? NF nf normal 7 NF NF normal 2 NF nF normal NF nf normal ¢@ nF nf dwarf of This explains why Miss Sollas should obtain a great majority of male dwarfs, and how, as seems to have occurred, the pecu- liarity may descend through the female. But she does get some dwarf females from normal parents. These are to be expected, since they would appear in the last mating above if crossing over again occurred in the male, thus: SEX-LINKAGE IN FOWLS 515 NF nF normal @? NF nf (nF Nf) normal &@ (gametes) NF NF normal 92 NF nF normal 2 NF nf normal @ nF nf dwarf ¢ (NF nF normal 9) (nF nF dwarf 92) (NF Nf normal @) (nF Nf normal ~) The actual numbers for families containing dwarfs is as follows: Normal 9 normal @ dwarf ? dwari @ 25 49 5 20 This gives a preponderance of normal males as against dwarf males, but it should be remembered that for every dwarf female due to crossing over there is a normal male produced, and sec- ondly, the mutant is obviously not a very viable one, so that a shortage is perhaps not surprising, especially in such compar- atively small numbers. The fact that there are nearly as many dwarf males as normal females is obviously due to the cireum- stance that the males of both kinds taken together are more than twice as numerous as the females. In plants Correns (’07) and Shull (10, *11) have shown that in certain dioecious species of Bryonia and Lychnis it is the male which is heterozygous. In the absence of cytological evidence or sex-linkage phenomena a chromosome interpretation of these eases would perhaps be out of place. SUMMARY There is a sex-linked factor carried by the Columbian Wyan- dotte—an inhibitor for red in the plumage. This breed proba- bly also carries another sex-linked factor, an inhibitor for red in the neck. It apparently carries a pattern factor inhibiting the breast color, and, in the female, the stippled back of the Brown Leghorn. The silver gray color is probably epistatic to the Jungle fowl or brown color. 516 A. H. STURTEVANT The white Wyandotte is a silver laced breed with a color pro- ducer dropped out. An attempt is made to explain three sets of phenomena, in fowls, in canaries, and in Aglia tau respectively, as cases of partial sex- linkage. Using this explanation, it is argued that the sex for- mula for birds and Lepidoptera is probably; 7, MM, FF; ¢, Mm, FF. The case of the dwarf guinea-pig is explained as per- haps representing partial sex-linkage in a form where the male is heterozygous for sex. February, 1912 LITERATURE CITED Bateson, W. 1902 Rep. Evol. Comm. vol. 1. 1909 Mendel’s principles of heredity. Cambridge. Bateson, W. AND PuNnNETT, R. C. 1905 Rep. Evol. Comm., vol. 2. 1911 Jour. Genet., vol. 1, p. 185. CastLe, W. E. 1903 The heredity of sex. Bull. Mus. Comp. Zool., Harvard, vol. 40, no. 4. Cook, M. H. 1910 Spermatogenesis in Lepidoptera. Proc. Acad. Nat. Sci., Philadelphia, April, p. 294. Correns, C. 1907 Die Bestimmung und Vererbung des Geschlechtes, nach neuen Versuchen mit héheren Pflanzen. Berlin. Davenport, C. B. 1906 Inheritance in poultry. Carnegie Inst. Washington Publ. 52. 1909 Inheritance of characteristics in domestic fowl. Carnegie Inst. Washington Publ. 121. 1911 Another case of sex-limited heredity in poultry. Proce. Soc. Exp. Biol. Med., vol. 9, p. 19. Deperer, P. H. 1907 Spermatogenesis in Philosamia cynthia. Biol. Bull. vol. 13, p. 94. Doncaster, L. 1908 Rep. Evol. Comm., 4 1911 Some stages in the spermatogenesis of Abraxas grossulariata and its variety lacticolor. Jour. Genet., vol. 1, p. 179. Doncaster, L. anp Raynor, G. H. 1906 Breeding experiments with Lepid- optera. Proc. Zool. Soc., London, vol. 1, p. 125. SEX-LINKAGE IN FOWLS by bs Duruam, F. M., anp Marryarr, D. C. E. 1908 Notes on the inheritance of sex in canaries. Rep. Evol. Comm., vol. 4, p. 47. Goopats, H. D. 1909 Sex and its relation to the barring factor in poultry. Science. n. s., vol. 29, p. 1004. 1910 Breeding experiments with poultry. Proc. Soc. Exp. Biol. Med., vol. 7, p. 178. 1911 Studies on hybrid ducks. Jour. Exp. Zool., vol. 10, p. 241. Guyrr, M. F. 1909 The spermatogenesis of the domestic guinea (Numida meleagris Dom.). Anat. Anz., Bd. 34, p. 502. 1909 a The spermatogenesis of the domestic chicken (Gallus gallus Dom.). Anat. Anz., Bd. 34, p. 573. 1910 Accessory chromosome in man. Biol. Bull., vol. 19, p. 219. McGrew, T. F. 1901 The Wyandotte. U.S. Dept. Agr., An. Ind. Bur. Bull., : no. 31. Morean, T. H. 1910 Sex-limited inheritance in Drosophila. Science, n. s. vol. 32, p. 120. 1911 The application of the conception of pure lines to sex-limited inheritance. Am. Nat., vol. 45, p. 65. 191la An attempt to analyze the constitution of the chromosomes on the basis of sex-limited inheritance in Drosophila. Jour. Exp. Zool., vol. 11, p. 365. 1911b Random segregation versus coupling in Mendelian inherit- ance. Science, n.s., vol. 34, p. 384. 1911¢ Chromosomes and associative inheritance. Ibid, vol. 34, p. 636. Parrisu, T. R. 1911 Catalogue, Parrish strain Columbian Wyandottes. Nash- ville. Peart, R., aND SurracE, F. M. 1910 Further data regarding the sex-limited inheritance of the barred color pattern in poultry. Science, n. s., vol. 32, p. 870. Suu, G. H. 1910 Inheritance of sex in Lychnis. Bot. Gaz. 49, p. 110. 1911 Reversible sex-mutants in Lychnis dioica. Bot. Gaz., vol. 52, p. 329. Soutas, I. B. J. 1909 Report Evol. Comm., vol. 5, p. 51. Spituman, W.J. 1909 Barringin Barred Plymouth Rocks. Poultry, vol. 5, p.7. Sranpruss, M. 1896 Handbuch der paliarktischen Grosschmetterlinge. Jena. 1910 Chaerocampa (Pergesa) elpenor L. ab. daubii Neip. und einige Mitteilungen iiber Wesen und Bedeutung der Mutationen illustriert an Aglia tau L. Iris, Bd. 24, p. 155. (See also a review, by H. Fed- erley; Arch. Rass. u. Gesellsch. Biol., Bd. 7, p. 755.) R 518 A. H. STURTEVANT Stevens, N.M. 1906 Studies in spermatogenesis. IT. Carnegie Inst. Washing- ton Publ., 36, p. 33. 1908 A study of the germ-cells of certain Diptera. Jour. Exp. Zool., vol. 5, p. 359. 1911 Heterochromosomes in the guinea-pig. Biol. Bull., vol. 21, p. 155. Sturtevant, A. H. 1911 Another sex-limited character in fowls. Science. Mees vole SB 1s shi STUDIES ON THE PHYSIOLOGICAL CHAR ACTERS OF SPECIES I. THE EFFECTS OF CARBON DIOXIDE ON VARIOUS PROTOZOA MERKEL HENRY JACOBS From the Zoological Laboratory, University of Pennsylvania CONTENTS I. Introduction. . : Rene Aa Mew Ee ete Dnt ae IT. Material and methods: Be caret oct III. Observations and experiments......... A. Ciliates. . P Aare HG Parameciinne: auch ye cghaek 2. Paramecium aurelia..... 3. Paramecium bursaria 4. Colpidium colpoda.... 5. Coleps hirtus.......... 6. Blepharisma lateritia.. 7. Euplotes patella.... 8. Vorticella nebulifera....... B. Flagellates. . : 4 ite Peceneas a tric ewharin.: 2. Euglena viridis (?).. 3. Chilomonas paramecium..... 4. Entosiphon sulcatum.... IV. Discussion of results........ V. Summary....... Sdiciace CARE ORR tr, ISIDWORT ADI. cpicckeoce en erates. + os I. INTRODUCTION Considering its importance in connection with many aspects of modern biological research, the question of the physiological characters of species, as opposed to their morphological ones, This con- dition has doubtless resulted partly from the fact that physio- logical characters, on account of their less definite and tangible has received a surprisingly small amount of attention. 519 520 MERKEL HENRY JACOBS nature, are more difficult to deal with than morphological ones, and partly from the fact that single physiological characters at least, are notoriously unreliable guides in the questions of classifi- cation and phylogeny that up until the present day have occupied so large a share of the attention of working biologists. The latter objection, however, no longer holds today, at least to the same extent that it formerly did. Modern zoology is not so much interested in finding out what are the probable rela- tionships of a given animal as in learning what it is, and especially what it does. This is the physiological point of view, whichis uppermost in the minds of most biologists today. No data which throw light on what goes on in the living organism are any longer considered unimportant; indeed, they are coming to be recognized as a vital necessity. If our knowledge of comparative physiology . were as complete as our knowledge of comparative morphology, for example, there is not a single one of the more modern develop- ments of biological science that would not have its possibilities enormously extended. It is therefore a matter of increasingly great importance to accumulate accurate data on the physio- logical characters of organisms, to determine which ones are fundamental, and which accidental, which are constant in a given species, or larger group, and which vary in different individuals of the same species, or perhaps in the same individual at different times; in short to obtain as full and comprehensive a knowledge as possible of the physiological characters of organisms. Perhaps the day may come when it will be possible to define any species in physiological and chemical terms in the same way in which it is now defined in morphological ones, and when no description of an organism will be considered complete which does not include its chief physiological peculiarities along with its structural one. The biologists of that day will be able successfully to attack prob- lems that for the present must remain untouched on account of lack of the proper kind of knowledge. It is needless to state that many observations of the sort sug- gested have already been made. Not to mention the more or less scattered ones made on many widely separated groups of organisms, we already have a considerable knowledge of the EFFECTS OF CARBON DIOXIDE 521 physiological characters of many of the bacteria, a group in which, for obvious reasons, our physiological knowledge has far outstripped our morphological knowledge. Botanists have also accumulated an enormous fund of knowledge relating to the com- parative physiology of the green plants, while in such special fields as the study of the blood sera of the higher vertebrates, to give but one example, encouraging progress has been made. Nevertheless, it is apparent that very little has been done in the way of systematic studies along the lines suggested, with the ulti- mate object of making the physiological characters of each organ- ism as well known as its morphological ones. Such an under- taking is not the work of one man or of one generation. Many years must elapse before our knowledge will be anything but exceedingly fragmentary and scattered. The following paper is therefore a very modest contribution to so large a subject. It deals merely with the effects of a single common and important substance, carbon dioxide, on a number selected protozoan forms, with especial reference to their movements and general vitality. It will be followed at intervals by other papers on the effects of various other substances, so far as possible on the same forms. It is not claimed that the results are, or will be, complete or exhaus- tive; still it is hoped that they may not be without interest and a certain amount of value. II. MATERIAL AND METHODS The forms studied were various of the most common ciliate Infusoria and flagellates, i.e., Paramecium caudatum, P. aurelia, P. bursaria, Colpidium colpoda, Coleps hirtus, Blepharisma lateritia, Euplotes patella, Vorticella nebulifera, Peranema tri- chophorum, Euglena viridis (?), Chilomonas paramecium, and Entosiphon suleatum. In the case of all the forms mentioned except the last one, observations were made on individuals from several different cultures of different origin, the intention being to obtain, so far as possible, data which would apply to the species as a whole and not simply to a particular race. Of course it will be necessary to extend the observations still further before draw- 522 MERKEL HENRY JACOBS ing absolutely final conclusions; it is thought, however, that fur- ther work will not materially alter the results arrived at in this paper. The general method employed in studying the effects of carbon dioxide on the forms in question was to subject them, in a drop of culture fluid, to a continuous stream of this gas in an Engelmann gas chamber. The drop of liquid containing them was placed on a slide or cover glass and the latter inverted in the usual way over the opening of the gas chamber, the joints being made air- tight with vaseline. The observations were made entirely with the compound microscope, chiefly with a Leitz 3-objective, although in doubtful cases the 7-objective was also employed. The points especially noted were the time required to stop normal locomotion, the time required completely to stop the beat of the cilia, flagella, ete., and the longest possible exposure after which recovery is possible when normal conditions are restored. In addition, incidental observations were made on the general behay- ior of the organisms and the visible structural changes produced in the cell by carbon dioxide. The gas used in the experiments was generated in the apparatus designed by McCoy, from marble and C. P. hydrochloric acid diluted in the proportion of one part of acid to four of water. Before coming in contact with the animals it was passed through two wash bottles filled with a solution of sodium carbonate to remove any traces of hydrochloric acid that might be present and also to ensure thorough saturation with water vapor. That no appreciable amount of hydrochloric acid was left in the gas was shown by conducting it into a silver nitrate solution, which in the course of two hours showed no traces of a precipitate or even of a cloudiness. The gas after being thus purified waS conducted successively through four Engelmann chambers, each placed on the stage of a microscope, and connected by rubber tubing in such a way that the same gas passed through all of them. This arrange- ment was found very useful, not only in making comparisons between different species under as nearly identical conditions as possible, but also in facilitating a larger number of independent observations on individuals belonging to the same species. EFFECTS OF CARBON DIOXIDE HOS Preliminary experiments showed the necessity of observing a number of precautions. The first of these is that the rate of evolution of the carbon dioxide gas shall be approximately the same in different experiments which it is desired to compare, since it was found that, other things being equal, the slower the stream of carbon dioxide passing over the drop the longer the animals survive. This is probably due to the fact that in a rapid stream the air is removed from the gas chamber and the drop more quickly, and the animals have less time to adjust themselves to the new conditions than in the case of a slow stream. By using all four of the gas chambers in one experiment it was found easy to compare a considerable number of forms with this factor constant. Frequently, indeed, a number of forms were present in the same drop and thus subjected to exactly the same condi- tions. In order to be able to compare experiments made on different days the attempt was made always to have the gas evolved at the rate of approximately 100 ce. per minute. This it was found possible to do within the necessary degree of accu- racy by proper regulation at the beginning of the experiment of the apparatus, which is automatic when once started. A second and most important point to be considered is the temperature, which has a marked effect on the time in which death occurs. A preliminary experiment on the three species of Para- mecium showed that at 22°C. death occurs in roughly half the time in which it does at 12°C. In order that this factor might be made constant, all of the experiments here recorded were made at, or very near, the first mentioned temperature, which is slightly above ordinary room temperature. A third point that cannot be neglected is the size of the drop containing the animals. Preliminary experiments showed that this has an appreciable effect on the results obtained, especially when the drop is very small. In one such experiment in a very small drop the average time of death of a certain ‘pure’ race of Paramecium aurelia was seventeen minutes while the average for the same race in a rather large drop was thirty minutes. In two medium sized drops in the same experiment the times were twenty-eight and twenty-nine minutes respectively. It will be 524 MERKEL HENRY JACOBS seen therefore that while there is not very much difference between the medium sized drops and the large one, the small one shows a decided difference. This is probably due to the greater sudden- ness with which the animals were subjected to the carbon dioxide in the latter case rather than to any difference in its concentra- tion ultimately in the drops, since these probably all became prac- tically fully saturated long before the end of the experiment. In order to guard against this source of error the drops were all made as nearly the same size as possible, the standard being a drop about 8 mm. in diameter with a moderate curvature. Results obtained in this way were constantly compared with those obtained when a number of the forms in question were present in the same drop. The final precaution has already been mentioned, namely, not to base far-reaching conclusions on results obtained from a single culture. In the case of some of the forms studied more than a dozen cultures were employed; in all of them except one form, obtained very late, the number of cultures was at least three. Ill. OBSERVATIONS AND EXPERIMENTS A. Ciliates 1. Paramecium caudatum. The effects of carbon dioxide on this form are briefly as follows. Immediately after the current of gas has been turned on, the animals exhibit a general restlessness and begin to seek the center of the drop, or rather, to avoid its edges, doubtless because the concentration of the carbon dioxide is greatest there. Inside a minute, as a rule, in a drop of the size used in these experiments, they have collected in its central and thickest part. Here they swim about actively but in very short paths, since the ‘avoiding reaction’ occurs whenever their move- ments have a tendency to carry them into the thinner and con- sequently more saturated part of the drop. Soon, however, generally within two or three minutes, they cease to be able to discriminate between the concentrations in different parts of the drop and spread out again until they are uniformly distributed. Sometimes toward the end of the experiment, for unknown reasons, they collect about its edge. Previous workers have noticed the EFFECTS OF CARBON DIOXIDE 525 behavior just described. Loeb and Hardesty (95) mention that Paramecium aurelia remains in the center of the drop fifteen min- utes. In all probability they were dealing with a larger drop _than the one used in these experiments, where in no case did either P. aurelia or P. caudatum require more than a few minutes to become adjusted to the new condition. After this primary response, the animals swim about in a more or less normal manner but more and more slowly, until they finally come to rest in a time that may vary from twenty or twenty-five minutes to several hours. Even after locomotion has ceased the cilia continue to beat for some time. As death approaches they beat slowly and irregularly and frequently show visible signs of injury. Often a group of them may keep on beating after the others have come to rest. There seems to be nothing constant about the part of the body where movements persist longest. After the cilia have completely stopped it seems to be impossible to start them again even by prolonged exposure to the air. The animals are to all intents and purposes dead. The same thing is true of the other ciliates studied, with the exception of Vorticella, whose membranelles are ofteti stopped before the animal is seri- ously injured and consequently can be started again. In the majority of ciliates, however, the vibratile structures are among the most resistant parts of the cell and when they have finally succumbed the life of the rest of the cell is practically extinct. In the meantime, certain other changes have been occurring. Among the mos: striking of these is the change in shape of the body, which becomes shorter and thicker. Some of the increase in the thickness is doubtless due to the shortening, but this does not account for all of it, and it is probable that an actual increase in volume occurs by the absorption of water. This phenomenon is more strikingly shown in some of the other forms studied than in P. caudatum. About the time that the swelling becomes notice- able, the nuclei begin to be very clearly visible, standing out sharply from the rest of the protoplasm by their greater opaque- ness. This is perhaps due to the acid nature of the medium sur- rounding the cell since other acids produce the same phenomenon. ' A furthereffect of the carbon dioxide often is apparent in the burst- 526 MERKEL HENRY JACOBS ing of the pellicle and the flowing out of droplets of clear proto- plasm. This may occur either before or after the cilia have stopped beating, but is not so constant in P. caudatum as in some of the other forms studied. There is reason to believe that this result is at least partly due to actual injury of the pellicle and not merely to an increase of internal pressure, since the bursting some- times occurs when the cell has not markedly swollen and also never appears until late, while the cell may reach almost its maxi- mum volume quite early. Furthermore, after one droplet has formed, thus supposedly relieving the internal pressure, others may form in quick succession at other parts of the cell boundary. This effect is not a specific one of carbon dioxide since Budgett (98) noted the same phenomenon in Paramecium and other pro- tozoa when merely deprived of oxygen in a stream of hydrogen, and high temperatures also cause the same result. The time that elapses from the beginning of the experiment until the death of the animal varies considerably with cireum- stances. The lowest average obtained in a single experiment was about twenty minutes, the highest over three-and-a-half hours, more cultures, however, approaching the latter value than the former. In an effort to determine to what extent P. caudatum can be said to have a specific resistance especial attention was paid to the question of the amount of variation shown by differ- ent individuals, races and cultures. A large mass of data was accumulated which will be made the basis of another paper on a somewhat different subject. It may not be out of place, however, to say here that while there is some evidence that different races may have different powers of resistance, these differences are insignificant compared with the enormous changes in resistance that a single race may undergo under appropriate changes in the culture medium. It is possible artificially to change the resistance very greatly, and such changes also occur naturally during the ageing of the culture, an old culturein general having a high resist- ance, and animals kept in the laboratory for a time: being more resistant than ‘wild’ ones. Great as these variations are, however, they have their limits and in the dozens of cultures and thousands of individuals studied none were found which had as low a resist- EFFECTS OF CARBON DIOXIDE 527 ance as the average Coleps hirtus for instance, or as high a one as the average Colpidium colpoda. Furthermore in a given culture, if P. caudatum has a higher resistance than usual, the other forms present also will, and their relative resistance remains practically constant. It is possible, therefore, to attribute to P caudatum as well as to the other forms a specific resistance, remembering only that its absolute value is somewhat subject to variation under different conditions and that in comparing differ- ent forms it is well to have them either from the same culture or at least to make a considerable number of independent observa- tions on different cultures. 2. Paramecium aurelia. A comparison of this form with the preceding one will illustrate the statement just made. P. aurelia is in general considerably less resistant than P. caudatum. When the two forms are present in the same culture the former is always killed sooner than the latter, though rarely, when different cul- tures are studied, some strains of P. aurelia are encountered which show a higher resistance than some of the most susceptible strains of P. caudatum. In general, however, P. aurelia is killed in less than a half hour while P. caudatum nearly always survives several times as long. The average time of death in the two extreme experiments on P. aurelia was a little over ten minutes on the one hand and over two hours on the other, in most of the experiments, however, lying, as already stated, below thirty minutes. Loeb and Hardesty state that P. aurelia is killed by carbon dioxide in two-and-a-half to three-and-a-half hours. Perhaps their culture was an abnormally resistant one, or possibly the application of the gas was slower than in these experiments, or the temperature lower In view of the fact, however, that the figures given by them are quite typical of the rather more common P. caudatum at ordinary room temperature, and also that the distinction be- tween caudatum and aurelia formerly was not very sharply drawn, it is possible that they were dealing with the former rather than the latter species. The difference in size between the two forms is probably not the reason for their different powers or resistance, since in the same species no constant relation could be found between the time of death and the size of the animal Further- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 4 528 MERKEL HENRY JACOBS more, the larger P. busaria is less resistant than P. aurelia and the somewhat smaller Colpidium colpoda far more resistant. The cause of the difference is evidently of a more deep seated nature. In the general effects produced on it by carbon dioxide, P. aurelia closely resembles the preceding species. It shows the same negative response at first, which disappears in about the same time. The body swells in about the same way. The nuclei often become very distinct, and this fact frequently renders an accurate identification possible without staining. The interval between the time when locomotion ceases and the cilia stop beat- ing is both relatively and absolutely shorter than in P. caudatum There is also a much greater tendency for the pellicle to rupture, this occurring in some cultures in almost every individual. Per- haps this apparently greater delicacy of the body wall may be correlated with the lower powers of resistance of this form. 3. Paramecium bursaria. In a number of the cultures used in these experiments this species was found associated with the two preceding ones and therefore a favorable opportunity was presented to compare it with them. Such a comparison shows that it is the least resistant of the three. The average time of death was generally ten to twenty minutes, though in a number of cultures it was less than five and only rarely ran as high as thirty. The most resistant individual found lived over an hour but this was a most exceptional case. (It was in this culture that P. aurelia also showed its highest resistance—over two hours.) When the three forms in question are present in the same culture in every case observed the relative resistance was: bursaria, aurelia, caudatum, and might perhaps be represented numerically very roughly as 1: 2:4. The general effects of the carbon dioxide on this form are on the whole s milar to those already described in the case of the other two species. The pellicle apparently is very delicate and nearly always ruptures while the cilia are more markedly affected than those of the other species; as their move- ments cease they become matted together and very quickly be- come partly disintegrated, being represented only by an indis- tinet zone about the animal. It is rather interesting that this green form is less resistant than the colorless ones. Doubtless EFFECTS OF CARBON DIOXIDE 529 on account of the presence of chlorophyll in its body it is accus- tomed to rather a low concentration of carbon dioxide, since this substance is constantly being removed by it from the surrounding medium. Experiments to determine whether it was more, or less, resistant in bright light than in the dark have as yet not given very positive results, chiefly on account of the difficulty of controlling the temperature factor. 4. Colpidium colpoda. This in every case proved to be the most resistant species studied, living long after the other forms in the same drop had succumbed. The general effects on it of the carbon dioxide are as follows. When the stream of gas is turned on a strong negative response is shown and the animals collect in the center of the drop in the same manner as the forms already mentioned; soon, however, they become uniformly distributed and thereafter behave normally almost to the time of death which hardly ever occurs in less than six hours, and may take place much later. In anumber of experiments the carbon dioxide was allowed to flow for six or seven hours and was then shut off without how- ever admitting the air, and the animals were found to be in a normal condition the next day. In one such experiment they remained alive for a week, but this experiment was somewhat vitiated by the fact that a small quantity of chlorophyll derived from disintegrated Euglena cells was present in the drop, and during the hours of daylight could have furnished a certain amount of oxygen, though only a small portion of the carbon dioxide in the chamber could have been gotten rid of in thisway. However, even if these last results be discarded the fact remains that Col- pidium is exceedingly resistant to carbon dioxide and may remain alive in a drop saturated with it for many hours. Prowazek (’03) found in an experiment of a different sort that Colpidium survives a simple lack of oxygen far better than Paramecium caudatum. Carbon dioxide is not without its effects on Colpidium, how- ever. Locomotion, while not so markedly affected as in most of the other forms, nevertheless becomes less active than normally. Before death occurs the animals become quiescent, often at the, edge of the drop, and the cilia beat more and more slowly until’ they stop. One point of interest is that the animals, while they 530 MERKEL HENRY JACOBS move about in a fairly normal manner appear to take no food, and the food vacuoles present at the beginning of the experiment gradually are lost until the bodies of the organisms become re- markably transparent. For along time there is no swelling of the body, but rather a narrowing, doubtless due to the loss of the food vacuoles; towards the last there may be a certain amount of swelling but the pellicle very rarely ruptures as it does in various species of Paramecium. 5. Coleps hirtus. This species forms a marked contrast with the preceding, being the least resistant of all the ciliates studied. Even the most resistant individuals hardly approach the least resistant ones of the other forms. The effect of the carbon diox- ide is seen almost instantly. The animals show no decided nega- tive reaction, apparently being overcome too quickly for such to occur. The normal, rather active movements of locomotion cease within a few seconds and thereafter only irregular ‘vibrating’ movements occur up to the time of death, of which the average is well under five minutes. Even the most resistant individuals do not live for ten minutes. The visible effects of the carbon dioxide are greater on this form than any of the others studied. The barrel-shaped body swells until it is broadly elliptical or even circular in outline, the increase in volume being very decided. At the same time the plates of the armature become indistinct and disappear, giving the appearance at least of actually being dissolved away. In consequence the body becomes very trans- parent and the protoplasm may be seen to undergo coagulation phenomena. The cilia sometimes beat after the armature has partly or entirely disappeared but usually their movements cease early. The cell in most cases bursts, generally at one of the ends; sometimes this occurs in two or three minutes, before much swell- ing has taken place. 6. Blepharisma lateritia. This on the whole is a very resistant form, being second only to Colpidium in this respect. Itshows, however, rather more individual variation in the same culture than most of the other forms studied, isolated individuals some- ‘times succumbing quite early. Strangely enough, in spite of its general resistance its movements are very quickly affected. At EFFECTS OF CARBON DIOXIDE 531 first it exhibits a slight negative response which, however does not last very long and is not so decided as in the case of many of the other forms. Very soon its movements, never very active, become markedly slowed, and for long periods there is either no locomotion at all or this is very slow. The membranelles and cilia, however, keep on beating up to the time of death, which in the majority of cases occurs in from three to six hours. These two structures in Blepharisma seem to have about the same resistance. Two phenomena which are particularly characteristic of this species are the marked change in form which occurs and the tend- ency towards the formation of large vacuoles in the protoplasm. The body, originally lanceolate, within an hour or two often becomes very broadly ovate or sometimes almost circular in out- line. Corresponding to the general variability of the species in other respects, there is a great individual variation in this regard also. ‘Towards the end, considerable distortion of the body occurs but bursting is rare. 7. Euplotes patella. This form, on account of its peculiar cuirass-like modification of the pellicle might be expected to show a high resistance, but such is not the case. Its resistance in general is somewhat below that of Paramecium aurelia. The effect of the carbon dioxide on it becomes apparent very early. It is not markedly stimulated, though it does show a negative response at first. The normal movements of locomotion dis- appear as a rule after five to ten minutes, although the membra- nelles sometimes keep on moving an hour longer. However, in most cases the average time of death is considerably less than this—generally under half-an-hour. The cell very early becomes much distorted and the pellicle of the ventral surface ruptures, allowing the escape of drops of clear protoplasm, while the macro- nucleus at the same time becomes very distinct and granular. Even after these changes have occurred, however, the membra- nelles continue their beat for a considerable time; the cirri are almost as resistant, though their movements are very irregular and uncoordinated towards the last. The average time required for all movements to cease varied in different experiments from about twenty-five minutes to a little over an hour. Rossbach 532 MERKEL HENRY JACOBS (72) claims that not only Euplotes but also Stylonychia and Chil- odon and higher animals as well were completely killed by carbon dioxide in three minutes. Such low figures give grounds for the suspicion that his gas was not pure or that some other disturbing factor was present. 8. Vorticella nebulifera. This in many respects is the most interesting form studied. It presents a case where both vibratile structures (membranelles) and contractile ones (myonemes of the ‘bell’ and contractile filament of the stalk) are highly devel- oped, and it is a point of considerable interest to compare the effect of carbon dioxide on these two classes of structures in the same cell. Its effects are as follows. Almost instantly when the gas is turned on the animal is strongly stimulated and makes perhaps half-a-dozen violent contractions of the stalk, which then slowly relaxes, and thereafter so long as the gas is allowed to flow neither contracts spontaneously nor can be made to do so by mechanical stimuli. The time required to produce this paralysis of the stalk varies from thirty to sixty seconds. The myonemes of the ‘bell’ are similarly affected and the animal remains fully expanded throughout the experiment. If the gas is not allowed to act too long, full and speedy recovery may occur on removal to the air. For instance, in one experiment after a five minutes’ exposure to carbon dioxide and a subsequent five min- utes’ exposure to the air the animals were all normal in every respect. If the exposure to the gas is longer continued, however, permanent injury to the stalk results. After fifteen minutes the animals show a strong tendency to drop off of their stalks and the latter can be seen to be altered in appearance, the contractile filament becoming broken up into irregular refractive fragments and droplets. If the detached animals recover, they regenerate a new stalk. The effect on the membranelles is almost the reverse of that on the contractile structures. At first they may show a temporary cessation of movement but usually inside a few moments they begin beating again and may continue to do so forthree-quarters of an hour or more. Sometimes they stop temporarily and then start again even in the carbon dioxide while, removed to the air, EFFECTS OF CARBON DIOXIDE 533 they show considerable powers of recovery after a lengthy period of rest. In this respect they differ from the membranelles of Euplotes and Blepharisma which in these experiments never could be made to resume their beat after having having completely stopped. Doubtless this difference is due to the fact that in Vor- ticella they are accustomed to stopping every time the disc is retracted, while in the other two forms they normally remain in continuous motion and are stopped by nothing short of actual injury to the cell. The powers of recovery of Vorticella after all movements have ceased are quite considerable. In one experiment after three- quarters of an hour practically all of the individuals had become quiet, and many had been in this condition for half-an-hour or more. Five hours after exposure to the air in a moist chamber about half of them had recovered and many had begun to regen- erate the missing stalk, which was already one-tenth to one-half the length of the body. By the next day these stalks were one- half to two times the body length. In this experiment about 50 per cent of the individuals never recovered and this is typical of a number of experiments that were tried. It may be said, there- fore, that certain of the movements of Vorticella are almost in- stantly affected and others only after a much longer time. Even after all visible movements have ceased the powers of recovery of the animals are considerable. In a few instances individuals were observed which, before the beginning of the experiment, had formed the circlet of cilia used in the free-swimming existence. Such cilia were about as resist- ant as the membranelles, though they were not observed to beat again after having stopped. In no ease did individuals which broke from their stalks during the course of the experiment on account of the effect of the carbon dioxide, form such cilia, but their locomotion was entirely by means of their membranelles. The Vorticella cell apparently shows no tendency to burst and form droplets of protoplasm, but a considerable change in form may occur. In an atmosphere of carbon dioxide V. nebulifera inside a few minutes tends to assume the more rounded form characteristic of V. campanula. In the individuals which are 534 MERKEL HENRY JACOBS killed the protoplasm becomes brownish and opaque, doubtless due to coagulation phenomena. B. Flagellates 1. Peranema trichophorum. This form shows conditions which suggest those already noted in Vorticella. As is well known, Peranema has a single very prominent flagellum, which in loco- motion is directed straight forward. Ordinarily it is quite rigid except the tip, whose movements cause a slow forward progres- “sion of the animal. During progression, and also when otherwise at rest, the body shows very decided changes in form, these ‘meta- bolic’ movements, or contortions, being one of the most striking characteristics of the species. When exposed to carbon dioxide Peranema responds perhaps as quickly as any of the forms studied. Its forward progression ceases almost instantly and after a few preliminary, and rather vigorous contortions, the body suddenly becomes paralyzed in whatever state of contraction it may hap- pen to be in at the time. Only rarely does the animal have time to contract to a spherical form before being overtaken with this paralysis; consequently the typical appearance of the body is an irregular mass which does not change its form so long as the stream of carbon dioxide is allowed to flow. The flagellum responds differently. Contact with the carbon dioxide causes it to beat with a swinging motion in which the proxi- mal as well as the distal portion is concerned. Sometimes these movements are rather vigorous, but they never give rise to loco- motion. They may continue with more or less uniformity for a half or even three-quarters of an hour; at the end of that time the flagellum gradually comes to rest. All visible movements have now ceased, but the animals are not necessarily dead. Experi- ments were tried to determine what powers of recovery Peranema possesses after becoming perfectly motionless. It was found that when the drop containing them is removed from the gas chamber and placed in contact with the air, a considerable percentage of the individuals may recover after an exposure of an hour and a quarter to carbon dioxide, even though all movements have for a EFFECTS OF CARBON DIOXIDE 535 long time ceased. In the case of individuals exposed for two hours, however, no recoveries occurred although the drop was kept under observation for twenty-four hours. The time required for recovery to occur depends on the length of time the gas has acted. After an exposure of five minutes, metabolic movements of the body begin in less than a half minute after contact with the air, and the animals may be entirely normal in ten minutes. After a longer exposure the time required is much greater. In .one experiment after an exposure of thirty-four minutes, but few of the animals were in a state of normal activity after an hour-and- a-half in the air, though at the end of four hours most of them showed no signs of injury and were normal in every respect. Peranema therefore represents a form in which the effects of carbon dioxide on locomotion and the contractile movements of the body are almost instantaneous, but which is killed only after a prolonged exposure. The point of greatest interest is that while certain movements of the body are brought to a standstill in a few seconds the flagellum may continue to beat for half-an- hourormore. Weare therefore dealing with structures concerned in producing movements in the same cell which show a consider- able physiological difference. 2. Euglena viridis(?). This form is in some respects more resistant and in others less resistant than Peranema. The time required for locomotion to cease is longer and the powers of _ recovery after an extended exposure greater, but the flagellum is much more sensitive and the length of time required to bring to an end all visible movements is considerably less. Like Pera- nema, Euglena shows no decided negative reaction to the gas as do many of the forms studied, though locomotion may persist for a few moments. Often the first effect of the gas is to cause a short temporary cessation of all movements, which quickly reap- pear. Soon, however, movements of progression cease andthe organisms show signs of life only by vibrating movements which are due to the abnormal beat of the flagellum. These gradually cease and the animals sink to the bottom of the drop motionless and perfectly extended. The time required to produce cessation of all movement varies from two or three to ten minutes. The 536 MERKEL HENRY JACOBS particular Euglena studied, which was close to, but probably not identical with, E. viridis, was not one which very actively changes its form, and consequently was not a very favorable one in which to observe the effect of carbon dioxide on the contractile move- ments of the body. It may be said, however, that while ‘eugle- noid’ movements were observed in many individuals before the beginning of the experiment and also after recovery, they never occurred during its progress, consequently the conditions here probably are the same, even if less striking, than those found in Peranema. After the organisms have settled to the bottom of the drop and become motionless the only change that can be observed is a gradual slow swelling of the body. At the same time there is a slight shortening which, however, is not sufficient to account for the greater thickness of the organisms as careful measurements show. This swelling continues until the shape of the body has changed from cylindrical to broadly elliptical in outline and the chlorophyll bodies appear forced apart from each other. In extreme cases the cell may appear to be at the point of rupture, though this rarely occurs, the pellicle being very tough and elastic. Although all movements cease in Euglena in ten minutes or less, it requires a much longer time to kill the organisms. Even after an exposure of three hours about a third of the individuals eventually recovered, though the time required was considerable. The recovery of Euglena is far slower than that of Peranema. After an exposure of seven minutes no recoveries could be notiecd a half hour after removal to the air, although they began to occur soon after that, and in an hour and a quarter practically all the individuals were normal. After an exposure of two or three hours, the time required for recovery is three or four hours or more. 3. Chilomonas paramecium. This form shows great individual and also cultural variation. While in a few cases the animals become motionless in fifteen or twenty minutes, the average time required generally is three-quarters of an hour or more. Many resistant individuals retain their movements for several hours. In general, therefore, this may be said to be a form with a high resistance. Unlike the two previous flagellates, Chilomonas EFFECTS OF CARBON DIOXIDE 537 shows most decided reactions to the presence of carbon dioxide. The first effect is often to cause a temporary cessation of motion which lasts however only for a few seconds, after which the ani- mals are remarkably active. They show a striking tendency to seek the center of the drop at first, later becoming uniformly dis- tributed again. Their motions at first are normal but gradually the animals come to rest and give evidences of life only by a slow rotation or by quick darting movements which they occasionally make. In practically every case the cell becomes circular in out- line and if the experiment be long continued may actually burst. Chilomonas like the preceding form also has considerable powers of recovery after all motion has ceased. In one experiment even after an exposure to carbon dioxide of two-and-a-half hours about 75 per cent of the individuals eventually recovered after exposure to the air. In other cases, however, even after a shorter exposure the mortality is greater. 4. Entosiphon sulcatum. This form unfortunately was studied in only one experiment in which, however, a considerable number of individuals was present. Judging from these rather incom- plete data, it is by far the most resistant of the flagellates exam- ined. After an exposure of five hours it not only was alive,but the movements were not very markedly affected. Both flagella continued to beat, though in rather a stiff and jerky fashion, and slow forward progression continued. How long it would have survived cannot be said, but probably the time would have been considerably above that mentioned, since when the experiment had to be ended none of the animals had as yet been killed. IV. DISCUSSION OF RESULTS From the results given it is apparent that all of the forms studied are injured and eventually killed by pure carbon dioxide, but that the resistance of the different forms is very different. Colpidium colpoda can withstand without injury an exposure of many hours, while Coleps hirtus is killed in three or four minutes. Sometimes the time of cessation of visible movements and the point at which the cell is so severely injured that recovery cannot occur, may coincide, as in most of the ciliates (in Euplotes patella 538 MERKEL HENRY JACOBS irreparable injury probably occurs before the membranelles cease beating) while in other cases, the animal is capable of full recovery long after all movements have ceased, e.g., flagellates and Vorti- cella. Some animals which are otherwise fairly resistant to car- bon dioxide, as shown by their powers of recovery after 4 pro- tracted exposure to it, or by the long continuation of visible movements, show its effects very quickly by their inability to carry on normal locomotion in its presence. Peranems is the most striking example of this condition, Euglena and Euplotes also being relatively quickly affected. In their primary response the different forms also show distinct differences. The three species of Paramecium studied as well as Colpidium, and Chilo- monas show a decided negative reaction and an effort to escape from it. This reaction is less marked in Blepharisma and Eu- plotes, while in the other forms it is practically lacking, probably because normal movements are so quickly interfered with. (En- tosiphon was not studied in this connection.) It will be seen, therefore, that the different forms studied show certain charac- teristic differences in reactions and general resistance to carbon dioxide. It has already been pointed out that there is a certain amount of individual and cultural variation in the same species, which makes it impossible to put in exact quantitative form the time in which death oceurs, etc. Nevertheless the relative resistance of each form as compared with other forms from the same culture is fairly constant and furthermore it is at least possible to say that certain forms always have a high, and others always a low resist- ance. While some forms may ‘overlap,’ others, as for example Colpidium and Coleps, never do. ; The observations here recorded are not the first that have been made on the differences in resistance to carbon dioxide shown by different organisms. Frinkel (’88) studied the effect of this substance on various bacteria with the result that some were found to thrive almost as well as in air, others had their development checked but were not killed, while others were quickly destroyed. Lopriore (’95) also, in his careful experiments on the effects of carbon dioxide on the spores and mycelia of fungi and the pollen EFFECTS OF CARBON DIOXIDE 539 tubes of angiosperms, found decided specific differences. Many other scattered observations exist, which however, it is difficult to compare on account of the different methods employed in obtaining them. It is interesting to consider the results here obtained in con- nection with those of Jennings and Moore (’01) on the chemotactic effect of carbon dioxide on various protozoa. Of the four forms mentioned by them as being attracted by this substance, three (i.e., Paramecium caudatum, Colpidium colpoda, and Chilomonas paramecium) have been studied in these experiments and all show marked powers of resistance, as well as a strong negative response when the concentration is suddenly made high in the edges of the drop. Of the forms found by them to be indifferent, unfortu- nately only two genera (Euglena and Euplotes) were available, but these both showed a relatively low resistance as compared with related forms, at least so far as the continuance of locomotion is concerned. It would be interesting to study the other members of their list in this connection. Doubtless many other facts of behavior could be brought into line with such physiological pecu- liarities as the one under consideration. One of the most interesting results that appears from these experiments is the striking difference that seems to exist between the contractile elements of the cell—the myonemes—on the one hand, and the vibratile ones—cilia, membranelles, and flagella on the other. The former are very quickly thrown out of func- tion while the latter continue their normal movements for a long time. The best illustration of this point is Vorticella, in which the contractile filament of the stalk, and the myonemes, after a primary stimulation, are inside a minute or less completely para- lyzed, while the membranelles perhaps after stopping for a short time continue to beat for three-quarters of an hour. Such results are in agreement with those obtained by other workers. Nere- sheimer (’03), for example, found that the myonemes and mem- branelles of Stentor are differently affected by substances like morphin, which paralyze the former and do not affect the latter. Lillie (12) has observed that the cilia of Arenicola larvae continue their acvitity for hours in isotonic sugar or magnesium chloride 540 MERKEL HENRY JACOBS solutions, or in solutions of chloroform or ether, which prevent completely all muscular movements. Recently Mayer (711) has found that the effects of many ions on ciliary and muscular move- ments are exactly opposite those that depress the one stimulating the other. He found that the ciliary movements of trochophore larvae at first cease in water charged with carbon dioxide but later start again. It is well known that carbon dioxide at first stimulates and later depresses muscular movements in the higher vertebrates (Lee, ’07). In Vorticella in the same cell this antag- onistic action appears very clearly. In other forms it is harder to demonstrate a primary depression of ciliary activity, possibly because of the response of the organism as a whole in an adaptive way. In Chilomonas, however, the primary depression of the movements of the flagella nearly always occurs. V. SUMMARY 1. Each of the twelve forms studied reacts to carbon dioxide in a characteristic way and has a characteristic resistance, which is highest in Colpidium colpoda, which remains alive many hours, and lowest in Coleps hirtus, which is killed in a few minutes. A certain amount of individual and cultural variation may occur which prevents the expression of the resistance of the species in absolute terms. Compared with other forms under the same conditions, however, the relative resistance is fairly constant. 2. Some forms are killed outright very quickly (Coleps hirtus and Paramecium bursaria). In others all movements are stopped in a few minutes but death occurs relatively late, the powers of recovery being high (Euglena). In still others, locomotion ceases very promptly but movements of the cilia, flagella, etc., may per- sist for a long time (Peranema trichophorum, Euplotes patella, ete.). In theremainder, more or less normallocomotion continues for a considerable time (most of the ciliates, Chilomonas and Entosiphon). The result in all cases, however, if the experiment be long enough continued, is cessation of movements and death. 3. In the same cell the contractile elements are usually quickly paralyzed (Vorticella and Peranema) while the vibratile structures (cilia, membranelles, flagella) are much more resistant. In some EFFECTS OF CARBON DIOXIDE 541 cases (Vorticella) the action of carbon dioxide on these two classes of structures is exactly opposite, the contractile elements being first stimulated and then paralyzed and the vibratile ones often temporarily stopped and then started again. 4. Ordinary cilia and their modifications, membranelles and cirri when present in the same cell show approximately the same resistance. Flagella show great variation, that of Euglena being paralyzed in a few minutes and those of Chilomonas and Ento- siphon remaining active for several hours. 5. In ciliates in general, with the exception of the specialized Vorticella, recovery after complete cessation of movement is impossible; in the flagellates, movement cease long before the cell is permanently injured. 6. The general effects of carbon dioxide on the cell are to cause (a) cessation of movement, (b) absorption of water and consequent swelling, (c) injury to the cell wall, (d) death and coagulation of the protoplasm. BIBLIOGRAPHY Bupcertr, 8. P. 1898 On the similarity of structural changes produced by lack of oxygen and certain poisons. Am. Jour. Physiol., vol. 1, pp.210-214. FRANKEL, C. 1888 Einwirkung der COs auf die Lebenstitigkeit der Mikro- organismen, Zeitschr. f. Hygiene, vol. 5, pp. 332-362. Jennines H. 8S. and Moors, E. M. 1901 Studies on reactions to stimuli in unicellular organisms. VIII. On the reactions of Infusoria to carbonic and other acids with especial reference to the causes of the gatherings 929 spontaneously formed. Am. Jour. Physiol., vol. 6, pp. 2383-250. Lez, F. S. 1907 The action of normal fatigue substances on muscle. Am Journ. Physiol., vol. 20, pp. 170-179. Litiiz, R. 8. 1912 Antagonism between salts and anaesthetics. Amer. Jour. Physiol, vol. 29, pp. 372-397. Logs, J. and Harpesty, I. 1895 Ueber die Localization der Athmung in der Zelle. Pfliig. Arch., vol. 61, pp. 583-594. Lopriore, G. 1895 Ueber die Einwirkung der Kohlensiiure auf das Protoplasma der lebenden Pflanzenzelle Jahrb. wiss. Bot., vol. 28, pp. 531-626. Mayer, A. G. 1911 The converse relation between ciliary and neromuscular movements. Papers from the Tortugas Laboratory of the Carnegie Institution of Washington, vol. 3. 542 MERKEL HENRY on” ie pee - Neresnemer, E. 1903 Ueber die Hohe histo hi logischer Differe Ale heterotrichen Ciliaten. Arch. f. Protistenkunde vol. Prowazex, S. von 1903 Studien zur Biologie der Zelle. Zeitse Phys., Bd. 2. : gt ; Rosspacu, M. J. 1872 Die rhythmischen Bewegungserscheinunge _ fachsten Organismen und ihr Verhalten gegen physikalische A und Arzneimittel. Verh. d. phys. med. Gesell. zu Wiirzburg vol. 11, pp. 179-242. : . 2 é 1 ) \ ON THE ADAPTATION OF FISH (FUNDULUS) TO HIGHER TEMPERATURES JACQUES LOEB ann HARDOLPH WASTENEYS From the Rockefeller Institute, New York I. INTRODUCTION It is a well known fact that organisms can stand a higher tem- perature if the latter is raised gradually than if it is raised sud- denly. This phenomenon is referred to in biology as a case of adaptation. Dallinger states that he succeeded in adapting cer- tain protozoa to a temperature of 70° by gradually raising their temperature during several years. Schottelius had found that colonies of Micrococcus prodigi- osus when transferred from a temperature of 22° to that of 38° no longer formed pigment and trimethylamin. When transferred back to the temperature of 18° to 22° the formation of pigment and of trimethylamin was resumed. After the cocci had been cultivated for ten or fifteen generations at 38° they failed to form pigment even when transferred back to 22°. These experiments became the starting point for similar experi- ments by Dieudonné. He used Bacillus fluorescens putidus which forms a fluorescein pigment and trimethylamin. The optimal temperature for this bacillus is 22°. At 35° it grew but did not form pigment or trimethylamin. At 37°.5 growth ceased. Re- transferred to 22° pigment and trimethylamin were again formed. Dieudonné! exposed a culture of this bacillus to 35°. After twenty-four hours a second culture was taken from this and also kept at 35°, and this process was repeated each day. The fif- teenth generation thus cultivated at 35° began to form some pig- ment and from the eighteenth generation on, at 35° the formation ! Dieudonné, Arb. aus dem kaiserl. Gesundheitsamte, vol. 9, p. 492, 1894. 543 THE JOURNAL OF EXPERIMENTAL ZOGLOGY, VOL. 12, No. 4 544 JACQUES LOEB AND HARDOLPH WASTENEYS of pigment and trimethylamin had become as good as that in the cultures kept at 22°. The organisms had, therefore, become ‘adapted’ to a temperature of 35° which at first was unfavorable. Dieudonné did not succeed in causing pigment formation in this bacillus at a higher temperature than 35° although the bacil- lus finally grew at a temperature of 41°.5. He obtained similar results in experiments on other pigment-forming bacteria. Daven- port and Castle? made experiments with tadpoles of frogs. One lot of eggs and tadpoles was kept at about 15°, a second lot for twenty-eight days at 25°C. While the tadpoles raised and kept at 15° went into heat rigor at 40°.3C., those kept for twenty-eight days at 25° were not affected by this temperature but went into heat rigor at 43°.5. Their resistance to high temperature had therefore risen 3°.2. When the latter tadpoles were put back for seventeen days to a temperature of 15° they had lost their resist- ance to high temperature to some extent but not completely, since they went into heat rigor at 41°.6. The authors suggest that this adaptation to a higher temperature is due to a loss of water on the part of the protoplasm. They assume that the rise in temperature causes a comparative acceleration in the excretion of water on the part of the tadpoles. The hypothesis of these authors is based upon the fact, that spores of bacteria are more resistant to high temperatures than the bacteria. While this is a fact, nothing in the experiments of Davenport and Castle proves that the amount of water in the tadpoles is diminished by the rise in temperature. The idea of Davenport and Castle was put to a direct test by Kryz.? He kept frogs, toads and salamanders at room temperature, and at temperatures as high as 40°C., for a number of days or weeks and tested the coagulation temperature of the muscle plasma for both ranges of temperatures. He found that the coagulation temperature was identical for the animals kept at low and those kept at high temperatures. The following observations by Kammerer‘ indicate also an after- effect of the raising of temperature. Lacerta muralis from the 2 Davenport and Castle, Arch. f. Entwickelungsmechanik, vol. 2, p. 227, 1896. 3 Kryz, Arch. f. Entwickelungsmechanik, vol. 23, p. 560, 1907. 4 Kammerer, Arch. f. Entwickelungsmechanik, vol. 30, p. 379, 1910. ADAPTATION OF FISH TO TEMPERATURE 545 cooler climate of Nieder Oesterreich darkens already at a tempera- ture of 25° and becomes perfectly black at a slightly higher tem- perature. Lacerta muralis from the warmer climate of Italy remains perfectly normal at 25° and darkens only at 37°. Kam- merer points out that the higher temperature at which the latter lizards had lived prevented the effects which the same rise in tem- perature produced in lizards that had previously lived at a lower temperature. Il. THE INFLUENCE OF THE CONCENTRATION OF THE SOLUTION UPON THE RESISTANCE OF FUN- DULUS TO HIGH TEMPERATURE We used as material for our experiment half-grown Fundulus which were caught in Long Island Sound in January and kept in a room in the laboratory, the temperature of which varied a little around 10°C. Two fish were put into each of the following solu- tions: H.O, m/128, m/64, m/32, m/16, m/8, m/4, NaCl + KCl+ CaCl, in the usual proportion. It was found that the higher the concentration of the solution—up to a certain limit—the longer the fish could survive in a given high temperature. The method consisted in putting a number of battery jars with 500 ce. of the solution into a large water bath the temperature of which was constantly watched and kept constant. When all the solu- tions had reached the desired temperature the fish were introduced. In a number of cases the experiment was continued for several days in a bacteriological thermostat. Table 1 gives the duration of life for these fish in each of the solutions for various tempera- TABLE 1 DURATION OF LIFE OF FUNDULUS IN a TEMPERA- 5 Se } Rot TURE | | H:0 m/128 | m/64 m/32 m/16 m/8 m/4 im | CaCl | : | ea 1 25° 4hrs. indef. indef. -indef. indef. indef. indef. indef. 27° | lhr. | 2hrs.| 2hrs.)indef. indef. indef. indef. | indef. BOP i cae Sie 1/50! 62’ 90’ indef. indef. indef. 31° | 43? | 25’ Vit 50’ = indef. | indef. | indef. 33° | 2’ 6’ 9’ 7 9’ 80’ 546 JACQUES LOEB AND HARDOLPH WASTENEYS tures. The term ‘indefinite’ means that the fish were alive and apparently normal at the end of the experiment. These experiments were repeated with the same material with approximately identical results; 33° was as a rule the upper limit ' the fish could resist during the month of January. Occasionally a fish would survive a sudden exposure to a temperature of 33° in a m/4 solution of NaCl + KCl + CaCl, but such fish were no longer normal and would swim on their side. It was next ascertained in which concentration the fish could resist the highest temperature. It was found that this concentra- tion was m/4. In m/2 and 3 m they were not able to resist as high a temperature as in m/4 or 2m solution of NaCl + KCI + CaCl.. When sea-water was substituted for Ringer’s solution (NaCl + KCl + CaCl.) the results were the same as represented in table 1. TABLE 2 DURATION OF LIFE OF FUNDULUS IN | TEMPERA- : the DEX- TURE TROSE H:.0 m/32 m/16 m/8 m/4 im m/2 gm 29 40 40 30 40 100 40 7 40 31° 16’ | 20’ 166 || 20% iy ||) BAP BR 4 ie! The fact that the temperature which the fish could resist was the higher, the higher the concentration of the solution, suggested the possibility that the loss of water on the part of the fish in- creased their resistance. This explanation, however, does not agree with the observation made by Sumner and corroborated by us that Fundulus undergoes practically no change in weight when put into distilled water or when put back into sea-water. The idea that loss of water made Fundulus more resistant to heat could be tested by the substitution of sugar solution for Ringer’s solution or sea-water. It was found that dextrose solutions were not able to protect the fish, in fact such solutions were little if any better than H.O (table 2). The duration of life of the fish in the dextrose solutions is about identical with that in distilled water. This excludes the sugges- tion that osmotic phenomena determine the influence of the con- s "a ADAPTATION OF FISH TO TEMPERATURE 547 centration found in the experiments with Ringer solution or with sea-water; especially if we consider the fact that Loeb found that Fundulus ean live for over a month in an m/8 solution of dextrose at ordinary room temperature (if the solution is renewed every day/- The difference between the results expressed in tables 1 and 2 suggested that the protective action of the Ringer solution was of the nature of a specific salt action. We, therefore, tested the idea whether or not other salt solutions, e.g., NaCl or CaCl, could afford the same protection. It was found that this was true to a slight degree in the case of NaCl but not in the case of CaCls. Table 3 may serve as an example. TABLE 3 DURATION OF LIFE OF FUNDULUS IN TEMPERA- _ > —— SS | z LURE NaCl H.0 m/64 m/32 m/16 m/8 m/4 im m/2 | — es | — — 29° By) Boy! 40/ 50° indef. 120’ 120’ 30° | H20 | m/64 m/32 m/16 m/8 m/4 im } CaCh 29° | 20/ “109 60' 90' 60/ 30’ 10’ In an m/8 NaCI solution some but not all of the fish could live indefinitely at 29°; in an m/8 or m/4 solution of sea-water or Ringer solution they could all live indefinitely at 29°. We must therefore conclude that the protection which sea-water or a Ringer solution gives Fundulus against a high temperature is due to a specific effect of the combination of the three salts NaCl, KCl and CaCl in the right proportion. The idea presented itself that this protective action of the salts was the expression of an antagonism between the salts and a substance produced at a great velocity at a higher temperature, e.g., an acid. Experiments, to be discussed a little further on, on the immunization of fish against a high temperature eliminate this possibility. Experiments were tried on tadpoles and on a species of fresh water fish to ascertain whether these animals could resist high 548 JACQUES LOEB AND HARDOLPH WASTENWYS temperatures better in an m/8 Ringer solution than in tap water or weaker Ringer solutions. No positive results were obtained. Ill. THE ADAPTATION OF FUNDULUS TO HIGH TEMPERATURES Fish from the cold room (10° to 14°C.) were kept for lengths of time varying from one hour to several hours at 27°C., in an m/4 Ringer solution, and then put into H.O, m/64, m/32, m/16, and m/8 Ringer solution at 31°. It was found that the longer they stayed at a temperature of 27° the more resistant they became to . the temperature of 31°, so that finally they survived at that tem- perature even in distilled water. One sees that fish that had been kept in m/4 Ringer solution for seventy-two hours lived indefinitely in 31° even in distilled TABLE 4 PREVIOUSLY EXPOSED TO DURATION OF LIFE OF FUNDULUS AT 31° IN 27° In m/4 RINGER . SEE ae =a, SS Ip a BENGE BOO EO NEO x H:0 m/64 m/32 m/16 m/8 CO 0 hour 13! 40/ 43' 120’ or indef. indef. 1 hour 30! 95’ 150’ 102 indef. | 4 hours 62’ indef. indef. indef. indef. 23 hours 180’ indef. indef. indef. indef. 72 hours | indef. indef. indef. indef. indef. | water. Itshould be stated that each experiment was accompanied by a control experiment with animals that had not been immun- ized and in all cases the fish die in less than an hour in 31° in solu- tions below m/8. Fish kept in the cold room (10° to 14°C.) and put from there directly into a solution of 35° neafly all died in a few minutes even in the optimal solution of m/4 sea-water or Ringer. Experi- ments were undertaken to ascertain the minimum time the fish had to be kept at 27° in m/4 sea-water in order to be rendered immune to a sudden transfer into m/4 Ringer solution at 35°. It should be stated, however, that the fish that had been kept at 27° for sixteen and twenty-one hours did not all survive in 35° ADAPTATION OF FISH TO TEMPERATURE 549 TABLE 5 PREVIOUSLY IN THERMOSTAT AT 27° DURATION OF LIFE OF FUNDULUS AT 35° 0 hour BY 1 hour of 3 hours 3” 6 hours oe 8 hours 6’ 16 hours indefinitely 21 hours indefinitely 44 hours indefinitely while all the fish that had been kept at 27° for forty-four hours survived. ; This experiment was repeated with a second set of fish with the results shown in table 6. = TABLE 6 PREVIOUSLY IN THERMOSTAT AT 27° FOR DURATION OF LIFE OF FUNDULUS AT 35° m/4 RINGER 1 hour rid ss 3 hours 2 6 hours | . 8 hours partly ndefinite 16 hours 21 hour | . | partly indefinite, but better than pre 24 hours vious group 32 hours 40 hours | 48 hours } in efinite 65 hours 72 hours It seems that if Fundulus are kept more than twenty-four hours in a temperature of 27° they can with impunity be put into an m/4 Ringer solution of 35°. If fish are kept from six to sixteen hours at 27° and then suddenly transferred to 35° (in m/4 Ringer solution) some of them die and some survive; and the tendency to survive increases the longer the fish are previously kept at 27°. ee 550 JACQUES LOEB AND HARDOLPH WASTENEYS A series of experiments was carried out in which fish were kept ina thermostat at 30°C. for various lengths of time to test whether this accelerated their adaptation to a temperature of 35°. This was true only to a slight extent. In all these experiments the fish were suddenly transferred to an m/4 Ringer solution at 35°. We were curious to know if these animals could also survive if suddenly transferred to a temperature of 35° in distilled water. This is indeed the case as table 7 shows. Fundulus can become adapted to a temperature of 35° in dis- tilled water if they are kept for two days or longer at a tempera- ture of 27°. It seemed to make no difference whether the fish had been kept at 27° in m/4 sea-water or in distilled water. TABLE 7 > E L PREVIOUSLY EXPOSED TO 27° FOR DURATION OF LIFE OF FUNDULUS ROE aa PUT INTO DISTILLED WATER OF 0 days (control) 4’ 2 days indefinite 3 days indefinite Finally experiments were made to see to how high a tempera- ture these fish could be adapted in a week. By keeping the fish at a temperature of 27° over night and raising them during the day to a gradually higher temperature we found that they could be kept at the end of the week at a temperature of 40°C., for two hours without apparent injury. At a temperature of 41° they soon suffered in their power to maintain their equilibrium. They were immune to a temperature of 40° not only in an m/4 Ringer solution, but also in an m/64 solution. The lot which was in dis- tilled water died early during the experiment through an accident. It is probable that Fundulus once adapted to a certain tempera- ture can stand this temperature in any concentration of a Ringer solution below m/4. ADAPTATION OF FISH TO TEMPERATURE 551 IV. THE SUMMATION OF THE IMMUNIZING EFFECTS OF SHORT PERIODS OF EXPOSURE TO HIGH TEMPERATURE In the immunization experiments described thus far the fish had been exposed continuously for a rather long period of time to a temperature of 27°. We wanted to know if it was possible to immunize them for a higher temperature by exposing them only a short period of time each day and keeping them in the interval at a temperature of from 10° to 14°C. This would mean that the immunizing effect produced in the animal during a short exposure to a high temperature would be preserved at least twenty- four hours until the next exposure to a high temperature took TABLE 8 DATE DURATION AND TEMPERATURE OF EXPOSURE March 7 1 hour from 17° to 31° 2 hours at 31° 8 1 hour from 17° to 33° 2 hours at 33° 9 1 hour from 18° to 35° 2 hours at 35° 11 3 hours from 11° to 37° 12 2 hours from 17° to 37° 13 2 hours from 16° to 37° 14 2 hours from 17° to 37° 15 2 hours from 17° to 37° 2 hours at 37° 16 2 hours from 17° to 37° 2 hours at 37° 18 2 hours from 17° to 38° 14 hours at 38° 19 2 hours from 18° to 38° 2 hours at 38° 20 2 hours from 19° to 39° 2 hours at 39° place; and would be added to the immunizing effect of the next exposure to a high temperature. Table 8 gives the periods of exposure. Most of the fish died on the third day when the temperature was raised only to 35°. For this reason we did not dare to expose the fish for more than a few minutes to a temperature of 37° on the 11th, 12th, 13th and 14th of March. The fish were ex- posed to a higher temperature for not more than four hours on one day. We have seen that an exposure of four hours in itself does not suffice to create immunity to a temperature of 35° or above. Hence the fact that these fish were finally able to resist a 552 JACQUES LOEB AND HARDOLPH WASTENEYS temperature of 39° indicates a cumulative effect of the different exposures to a higher temperature. In other words, each heating increased the immunity and this gain was not lost during one or two days. V. THEIMMUNITY TO A HIGH TEMPERATURE IF'‘ONCE ACQUIRED IS KEPT FOR MORE THAN FOUR WEEKS In order to prove this, fish were put for various lengths of time into a thermostat at 27°, tested in regard to their immunity against high temperature and then put back into the cold room and tested again. A few examples will illustrate this. Five fish were immun- ized to a temperature of 39° by exposing them daily for a number of hours to an increasing temperature, until they could live in a temperature of 39°C. (in m/4 Ringer solution). The process of acclimatization extended over a period of twelve days (see pre- vious experiment). After this they were kept for eight days con- stantly at a temperature of from 10° to 14°C. On the eleventh day they were put suddenly into a temperature of 31° and the tempera- ture of the water in which they were, was brought, inside of two hours, to a temperature of 39°, and then keptatthisheight. A con- trol experiment was carried on simultaneously with fish taken from the same cold room, which had not been acclimated. The solu- tions used were m/4 Ringer. The control fish that had not been acclimated to high temperature were dead in one and a half hour when the temperature had reached 36°. The acclimated fish kept alive for over an hour at 39° when the experiment was discontinued. In eight days, therefore, the immunity of the fish to high tem- perature had not diminished. Four lots of fish had been immunized to a temperature of 35° by keeping themtwenty-four ,thirty-two, forty and seventy-two hours respectively at a temperature of 27°. After this the fish were put into the cold room and kept there at a temperature rang- ing from 10° to 15°C., for twenty-eight days. They were then put into an m/4 Ringer solution at a temperature of 85°. Simul- taneously six fish of the same lot, which had not been immunized but kept in the cold room permanently, were put into the same temperature and the same solution. Four of the latter fish died ADAPTATION OF FISH TO TEMPERATURE 553 within two minutes, the rest were dead forty minutes later. The fish ‘that had been immunized before were, with the exception of two individuals, all alive and normal after five hours. Yet, the fact that two of the fish suecumbed may be an indication that their resistance to 35° was less than immediately after immunization. It is quite possible, however, that these two fish which had been kept in small dishes for such a long time had suffered through this captivity. This idea is supported by the fact that in a third experiment fish had kept their immunity to high temperature for thirty-three days after immunization against 35°. The immunization con- sisted in exposing the fish for three and six days respectively to 27°. After that they were kept in the cold room for thirty-three days. When after that time subjected to a temperature of 35° they remained perfectly normal for five and a half hours, when the experiment was discontinued. We made a large number of experiments in which the duration of the immunity against high temperatures was tested sooner after the process of immunization than in the above mentioned experi- ments. In all these experiments it was found that the fish did not lose their immunity against temperatures of 39° and 35° respectively if they were put into the cold room for a period of thirty-three days or less after immunization. VI. EXPERIMENTS WITH FISH KEPT AT A CONSTANT TEMPERATURE OF 0°.4 C. The fish which we used for this experiment were caught in January and kept since that time in a cold room in which the temperature varied between 10° and 14°C. Our experiments showed, that these fish died in a rather short time when suddenly put into a diluted Ringer solution or diluted sea-water of 31°, provided that the concentration of the solution was below m/8. In an m/8 or m/4 Ringer solution or sea-water they were able to resist the temperature of 31° without any previous immunization. We put a large number of these fish in an ice chest in which the temperature remained constantly at 0.°4, and investigated at various intervals whether the resistance of these animals to a 554 JACQUES LOEB AND HARDOLPH WASTENEYS temperature of 31° differed from that of the fish kept at from 10° to 14° (cold room). The fish put in the ice chest had previously been at a temperature of 10° for several weeks. If we consider the behavior of the fish in an m/8 solution at 31° we notice a steady diminution of resistance among those kept at 0°.4; while among those kept at 10° to 14° the resistance increased somewhat. Thelatter resultisnotaccidental. Wemustremember that the fish were taken in January when the temperature of the water was not far from 0°C. The long exposure to a temperature of from 10° to 14° had therefore a slight immunizing effect. Our next task was to ascertain whether fish immunized to resist a sudden transfer to a temperature of 35° kept this immunity if put on ice just as well as if kept at a temperature of 10° for the same period. Our experiments thus far cover only an exposure of fourteen days on ice (T. = 0°.4). During this time the immu- nity was not diminished, as the following example will show. Fish were immunized to a sudden transfer to 35°C. by keeping them for two days at 27°. They were then put into a thermostat with a constant temperature of 0°.4 for fourteen days and put directly TABLE 9 DURATION OF LIFE AT 31° IN “ ore) we 2 Pre RINGER KEPT aT 0.4° C. t SOvURION, H:0 m/32 m/16 m/8 | = Sy ——— 7 days 26’ 104’ indef. 19 days 13’ 42’ ily some indef. 33 days 24’ 30’ 69’ 80’ 41 days 22 34’ 22' 8’ | ! < +— “ = | DURATION OF LIFE AT 31° IN | = = | “RINGER . 0° (or —— — —— —- KEPT AT 1 SOLUTION H:0 m/32 m/16 m/8 * = ———— E = Over 7 days | 20’ 135’ indef. Over 19 days | 13’ 26’ Dan indef Over 33 days | 2! 86 some indef. indef. Over 41 days 41’ some indef. some indef. indef. ADAPTATION OF FISH TO TEMPERATURE 595 into a m/4 Ringer solution of a temperature of 35° (A). Simul- taneously fish which had been immunized for 35° by keeping them three days at 27° and which had then been kept at between 10° and 14° for nineteen days were also put into a m/4 Ringer solution of 35° (B). In addition two controls were made: Fish kept on ice at 0°.4 for twenty days but not previously immunized (C), and fish not previously immunized kept for several weeks at a temperature of from 10° to 14° (D) were also suddenly transferred to a temperature of 35°. Table 10 gives the result. TABLE 10 DURATION OF LIFE OF FISH AT 35°C. IN m/4 RINGER SOLUTION B. Immunized but C. Not immunized . A. Immunized but kept k ° . D. Not immunized aoe ept at 10° for nine- kept on tice for a - 3 on ice for fourteen days teandays twenty days kept in cold room Alive after3 hours One alive after 3 Die in 2’ Die in 2’ hours The experiment was repeated with the same result, only those in lot A and B remained all alive. It is therefore obvious that the resistance acquired for a higher temperature (35°) is not lost or diminished if the fish are kept for two weeks on ice. THEORETICAL The phenomenon of adaptation considered in this paper is the fact that fish can resist a high temperature better if the latter is raised gradually than when it is raised suddenly. Physics offers us an analogy to this phenomenon in the experience that glass vessels which burst easily when their temperature is raised sud- denly, remain intact when the temperature is raised gradually. This phenomenon finds its explanation in the fact that glass is a poor conductor of heat and that when the temperature is raised suddenly, e.g., inside a glass cylinder, the inner layer of the cylinder expands while the outer layer, on account of the slowness of the conduction of heat, does not expand equally and the cylinder bursts. 556 JACQUES LOEB AND HARDOLPH WASTENEYS The following idea for the explanation of the mechanism of adaptation suggests itself. The rise in temperature brings about certain changes especially in the surface of the cells or the body of the animal, whereby the latter loses its protective impermea- bility. If the rise in temperature occurs gradually the blood (and especially the salts of the blood or of the surrounding solu- tion or of both) has time to repair the damage. If the rise, how- ever, occurs suddenly then the damage done cannot be repaired quickly enough by the blood, or the salts of the surrounding solu- tion, to prevent the death of the cell or the animal. The peculiar influence of the concentration and nature of the surrounding solu- tion described in this paper would harmonize with this suggestion. A second possible suggestion is that under the influence of the higher temperature a substance is formed in the animal which protects it against the effects of high temperature. The formation of this substance is also a function of time and for this reason an animal can keep alive if the temperature is increased gradually but cannot keep alive if it is increased rapidly. Both suggestions would explain the fact that if an animal is once immunized against a high temperature it will keep this im- munization, for some time at least, even if kept at a low tempera- ture or onice. Further experiments with which we are occupied may decide between these and other possible suggestions. SUMMARY 1. Experiments were made with Fundulus which were caught in the winter and kept at a low temperature (from 10° to 14°C.), to find out the maximum temperature into which they could, with impunity, be transferred suddenly. It was found that the maxi- mum temperature varied with the concentration of the sea-water or a Ringer solution; being about 25°C. for a concentration of m/128 or m/64; 27°C. for a concentration of m/32; 31°C. for a concentration of m/8, and almost 33°C. for a concentration of m/4. The latter concentration was the optimum, the resistance to high temperature decreasing again with a further rise in con- centration. ; ADAPTATION OF FISH TO TEMPERATURE 557 2. It was found that dextrose solutions were not able to afford any protection against the effects of a sudden rise in temperature. From these and similar experiments with CaCl, solutions it fol- lows that the protective action of sea-water or a Ringer solution against high temperature is not an osmotic but a specific effect of the salts of the sea-water. 3. It was ascertained how long it takes to immunize the fish against the harmful effects of a sudden transfer to a temperature of 35°C. It was found that by keeping the fish for thirty hours or more at a temperature of 27° they were immunized against a tem- perature of 35°. Often a noticeable immunizing effect was pro- duced already by an exposure of sixteen hours or even a little less to a temperature of 27°. Fish kept for two days at 27° were able to survive if suddenly transferred to distilled water of 35°C. 4. The immunity against a temperature of 35° acquired by keep- ing the fish for two days at 27° is not lost or weakened if the fish are afterwards kept as long as thirty-three days at a temperature of from 10° to 14°. Our experiments have not been extended beyond this period of time. 5. The immunity against a temperature of 35°C. is also main- tained if the fish are kept after the two days’ exposure to 27° for two weeks at a temperature of 0°.4 C. 6. Fish immunized against a temperature of 39° and then kept at a temperature of from 10° to 14° for eleven days did not lose their immunity. 7. A longer exposure of fish to a temperature of 0°.4 may finally lower their resistance to high temperature. 8. In order to immunize fish to a temperature of 39° it is not necessary to expose them continuously to a higher temperature. An intermittent exposure to a higher temperature during a number of hours each day will bring about the same effect. 9. Various suggestions for a possible theory of these phenomena are made. 7 — ‘ s 7 - oe 7 : ? ® as : ' ® 2 * x. : ® fe BINDING <=_T. 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