Spek rae et & or3* “$76 z ‘4 ies Lots oe ore seit mx x see AAPL : x we henl { © ry 4 x “ Oye c " ee oe: eo é seit elite Na rad vr 7 reetatae ys eresseetcs At Ly 5eaos! a o Cre ve Sani = ete s a os es reek ad ath SE tee oe eee es Sih et ee ee on oe 5 959 1058 ee BoE Te Test ses we tf Sa Tad oe Se os ee ceri tte salt repos : ; ae r A 7 "yy v ea ay ee ion ALS ke * ct Ay th ane rh ~. retin Hee a ery ~ e523 he. & EOL eas tote Mf he Tatoo! © ee, re Stites ores tet aes of ct *< eh 5G aes at . es eri ee te €) - Set ies esepdetaelesear stare, btyee" >&* je certs i se teacacs < is setity 485 7; pesihrbek orate 3. agepti : a5 apie lek z 7 Pao Ses es <3i¢ 4°, ae vt ). wae ee 12 | 136 1 : 1442.8 Crepidulavfornicatase....{¢2 eee 12 | 182 1 : 3443.0 Crepidulavconwexas can... ae 15 280 1 : 6434.8 Crepidulayaduncay. s.r! el eee 15 | 410 1 : 20123 .6 IDiUIIA DOORN AMOR, oi Ho mchaomed ehh ate alan cs oe. | 15 | 1600 iL SPA eis 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 cycle. When ~ CELL SIZE AND NUCLEAR SIZE 2) first formed this spindle hes 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 18u; 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 les 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 cleav- 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 ff 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 le 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 Raetiea Me | Me (| 1A4-1D 81 la-Id 30 Cantor cet 2A-2D 80 2a-2d 36 ca. 10.6: 1 3A-38D 76 | 3a-3d 33 Canh2 mle @Aplanaey seer 4D 66 4d | 38 Geis 25.0) gal 4A-4C 60 | 4a-4e 42 GH. Bae oil 5A, 5B 75 metay: Fir) 0) 60 Gis 1h.@) gu 5C, 5D 68 5e, 5d 68 ca. il gi ( 1A-1D 195 la-ld 69 ¢a, 22.0) 31 (Caconvexaaaee 2A-2D 195 2a-2d 50 CH G8)3) oil 3A-38D 195 3a-3d 50 Car OO ons L (|) 1A-1D 800 la-ld 80 | ea. 1000 : 1 2A-2D 800 2a-2d 80 ea. 1000 : 1 Fulgur carica. . 3A-3D 800 3a-3d 80 can OOO Rt 4D 780 4d 130 Cas eZGucel | 4A4C | 740 4a—te 370 Can sonee! 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 ege 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: PSC Roe Oya iicylck lei eomcodom abacus ay pom nda acacH a: iat lO) cell Lal Git 3 0) dated 1Sp Ney ee eee Ratio5 :3 PER ey, me (vec Warne Ronen ons a gsnananaedncndeaee 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. 3. 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. etc., 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 Bb, 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 til 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 (10) 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 k6énnte vielleicht sagen:—der von einen Sphire eingenommene Plasmabezirk sucht sich von allem was ausserhalb dieses Wirkungskreises liegt, abzuschniiren,” (p. 123). He sup- 12 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. II. 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 16 to 3y, and the cells from 24y 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,’ 1e., 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, pe Sb)e 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 Lebensvorginge 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 tS 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 ege 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 odplasmie constitution and prospective significance it is not improbable that the peculiarities in their CELL SIZE AND TABLE 3 NUCLEAR SIZE Le Maximum nuclear size and cell size in the blastomeres of Crepidula plana; (measured just before nuclear membrane dissolves) STAGE BLASTOMERES | DIAMETER OF CELL Before maturation. Before first cleav- APOE Gee eh Oe fae | AB, CD, before sec- ond cleavage. ...| A, B, C, D, before third cleavage. ..| 1A-1D, before fourth cleavage... la-ld, before divi- 2A-2D, before fifth cleavage.......... 2a-2d, before divi- lal--ld!-, before di- WISIOAL soadegecasee | 3D, before sixth cleayage--..... 3A-3C, before sixth cleavage.......... 3a-3d, before divi- 4d, before seventh cleavage.......... | 4A-4D, before sev-, enth cleavage..... 4a-4c, before sev- enth cleavage...... 106 80 76 76 38 60 42 VOLUME OF CELL cubic 1,755,000 1,488,910 619,329 286,712 276,350 266,240 228,288 228,288 28,533 112,320 32,409 a DIAMETER | | VOLUME OF f Or | DIAMETER VOLUME (PROTOPLASM,) KERN- PROTOPLASM OF OF | LESS PLASMA- INCLUDING NUCLEUS NUCLEUS | VOLUME OF | RELATION NUCLEUS | NUCLEUS be | be cubic uw | cubic pu ca. 64* 42 32,409 Q7lstew | W3 | 930+- ca. 65* |*o124=34.5| 21,375 121,430 | 1:5.6 ca. 51* 24 7,238 61,741 |1:8.5 i] ca. 44* | 22 5,775 | 38,570 1:6.6 | | | | ca. 40 DA 4,849 28,481 | 1:58 30 14 1043 Zen e603 ee ele S27 ca. 36 | 18 3,055" | 920,196) 4/127 | | 36 | 15 LTT Wh OesSe we Nie e1D) 7 | | Sema 12 905 13,135 | 1:14.5 15 | 7 180 PSST lASes cao il) Gs 2,145 11,895 |1:5.5 ca. 22 16 2,145 3,430 |1:1.6 33 14 1,437 19,250 | 1:13.3 30 14 1,437 12,608 |1:8.7 30 14 1,437 12,603 |1:8.7 ca. 22 ll 697 ARQT Sienna a7 ca. 20 18 3,055 1,134 | 1:0.37 ca. 14 12 905 532 | 1:0.58 * After yolk has been centrifuged out of egg. plasm are not well segregated at this stage. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 In normal eggs yolk and proto- 18 EDWIN G. CONKLIN Kernplasma-Relation may be the result of differentiations already present in the blastomeres. In table 3 only maximum nuclear and cell dimensions are given for the different blastomeres. Results would undoubtedly differ greatly if the minimum nuclear and cell dimensions were taken instead of the maximum. Accordingly in table 4 the minimum nuclear and cell dimensions for 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 detérmination 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 (2A4—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) ee et Chk eae eae | 8. | Bec zeae ee | 83 : hes a 8 |< 5 | ol 2 a Op Z Am B D STAGE BLASTOMERES | © Cie oe fe BAR 5 | 5 Boe 58 ae | Bae < Stipes 28 B ates eevee weet i eS Ss tae nea aa | as ae a | a | Aa > | > td | ob be | Me cubic mh | cubic pL | 2) cells’ AB, CD..|M105) |) ican 4Gn Oech eLoOR 30,504 | 1 : 160. 4 cells A,B,C,D,..| ‘78 | ca. 40| 8x3 | 150.6 30,695 | 1: 203.8 Seibel) oe | ca. 36) 6x3 84.6 | 24,166 | 1 : 285.6 la-ld ...| 27 | 27 | 6x3 84.6 1023510 |aeat20 1 delle eee e|? 72 | can 80) Gees ita 13,955 | 1:165 \a=2dee ee s0ra 30| 6x3 | 8416 J) SB@5R 9) demas fe teene le ae 20 30| 5x3 58.8 0 '|) 13,9055) Sees \da%Id? .., 15 | 15 | 5x3 5Sas = yell Om Os 1, $29 Ree sD sy 2, | eee 15| 4x38 37. by) Wyle 720) a alee SES wae | al 25| 4x3 37.5 8,087.5 .| 1 : 215.6 24 cells 2al-2d!...| 24 DAN Ax3 A 3T 8 7200)" 7 195 oS cella to GON | 5x3 588) | Alby thasy wo | 30 | 5x3 58.8 | DO NN 24| 4x3 37.5 7,200 | 1:195 29 cells ¢lat!-Id'! | 15 15 | 4x3 37.5) y= e72Ueon Welk 46 eee | 24 24| 4x3 Bab h ua Nt), oT 200 Mailer RTOS ANEAC, Si, Are Vl B75) anal | ce pees ee | | 4x3 37.5 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 anfangt 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-Relation 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) 1 iS n 2} fa as! ke A Am 8 4 STAGE BLASTOMERES © OF ° i me A ees es as re ° ofA & S Bsa A a asp | 3 Bo. Bas : B Bae 2% a | SE 4 5 BRS ae A a a > > 4 Me be a, cubic pL | cubic | f 218 | ‘ Before cleavage... .| 136 ca. 60 \ o12 3,960 109,137 ee at O ovellilsy INES (CID). «|| OS ca. 44 18 | 3,055 41,240 Inelaee 13.5 4. ‘cells Aj B,C, Dal. 78 ca. 40 16 | 2a SE 135. uly. Peale PASM. 4). ao ca. 36 15 1,767 4 22,484 | 1:12.7 > itastde..2-) 30 30 | 12 905 » 13,885, >| alae oleate 2A-2D....) 72 ca. 36 Sy | AGC | 22, A84A- NGL Fae one NDe- Oden ) 905 6,333 iL 37 4A-4C.... il) MevAsyy 32 , ace ae sy | 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 1 A—1D 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 s§ | 68 | 8 . ce p m ee |e B og Of0n8 26 e ag | ae ep he CLOVES IT Arey ga] ge | $8 Eon eee ee hose <0 dp | <2 Aa BaP A i pe a A | A 5 | = g | - | = be iy Nf cubic | cubic [ Macromeres ; AB, CD, before | | | | aecond cleavage. . .| 1200 | 200 | 40 33,280 | 4,126,720 | 1 :124 iN, 1335 (G3. JD), erate ‘third | | cleavage. . : 800 | 160 32 | 17,040 | 2,112,880 | 1 : 124 1A-1D berore fourth cleave | | | | EYES eater ee coicen Catto tad, orcas | 800) 160 | 32 17,040) | 21125880) | ll 2 124 2A-2D, before fifth cleav- | | age.. 800 | 160 | 32 17,040 | 2,112,880 | 1 : 124 3D, heron ae elena: 800 | 160 | 32 17,040 | 2,112,880 | 1 : 124 3A-8C, before sixth cleav- | | | age.. Breese Jett SOO) | 160 AS) | 5G,008) |) 2062412) le Bors Atay, before” geieuiln | Cleavavewncy tee 2) 768, 160 96 | 460,063 | 1,669,857 [ies 3.6 Micromeres | | | | | | lacidl, Sk Oe ae 80 |, ae. 2,145 | 264,095 | 1 : 127.7 Fa ee | 80 | 16 2,145 264,095 | 1 : 127.7 SASSY, oc pit Ret aes ee a | 80 16 2,145 264,095 | 1 : 127.7 RES Go te A eee SOs Gas 2,145 264,095 | Ped | 1 : 124 Wea Ue ite epic & os & alls 2G A0e a 8h 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 3A—-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 8u. 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 Ba 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. (8) The length of the resting period. ITI. 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, ’95, ’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, etc., 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 axisand 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 TABLE 7 Cell size and nuclear size in tissue cells of sexually mature individuals of Crepidula plana | VOLUME OF DIMENSIONS OF | DIAMETER lsontzaaesl ama naes |) eS= nafs shana) (pnts CELL OF NUCLEUS) NUCLEUS posemnics See be Me cubic cubic jh Intestinal epithelium..... 11 x 11 x 12 6 Mise Petes.) lest s8 Gastric epithelium....... | 10 x 10 x 36 8 68 | 3,382 | 1: 12.4 Liver duct epithelium.... | 10x10x 18 6 ait etG28. 01) 1 ni4e4 Liver cells (filled with, | | | | secretion products)..... ING) 5315) se etay | 6* 113 TOON eee S86 Liver cells (without secre- | | | | LOMO CICS) see eee 14x 14 x 30 9 | 382 5,498 }1:14.4 Kidney cells (containing | secretion products)....) 15 x 15x 15 6 113 Saya WL Sesyrss Eetodermal epithelium | | | | | (Meat ANUS). eel ca ts. I 5x 5x15 4 33 342 |: 10:3 Gill chamber epitheium..| 6x 6x 12 4 33 405 eal ee Gill filament epithelium... 7x 7x 9 | + SOM ee LOS nal lana 223 Epithelium from foot....| 6x 6x15 5 65.4 474.6/1:7.1 Ganglion cell (large)...... Ig se it 22233 12 905 5,724 LG He Ganglion cell (large)...... 10 x 10 x 20 | 9 382 | 1,618 eal Oécytes I (before yolk | HOMINENENOI\)). scnococeadeor 123 | a 180 836 | 1:4.6 Oécytes I (before volk for- MATION) 4 y eee tee 113 eer ol To TQM lid ike Odécytes I (before yolk for- | LINCOM) peste saben eee 10 6 113 407 Le S.@ Odcytes I (before yolk for- Mla aIOM|) eg poate cere ee 8 | 5 65.4 2OSy Tilecrord Odécytes I (before yolk for- | | TEWMIOM))s sco ceccemece son 63 4 | 33 111 12383 *Nucleus shrunken and very irregular in shape. 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 odcytes 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?-1d?) 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. e 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 allmahlich 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, gleichgtiltig, ob dieselbe durch Vergrosserung 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 $1 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 in the 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?-/d?, 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, 3} 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 CELL SIZE AND NUCLEAR SIZE ao 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 | OTA COEFFICI- | COBFFICIENTS | TOTAL STAGE VOLUME OF} VOLUME OF |VOLUME OF VOLUME OF ENTS OF OF KERN- | NUCLEI PROTOPLASM YOLK NUCLEAR | PROTOPLASMC | PLASMA- | EGG GROWTH GROWTH RELATION | =I n | cubic w cubic cubic 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 | 121,430 1,346,105 | 1,488,910 |0.65/1.0 | 1.25 | 1.0 | 1:5.6 2 cells...... | 14,476 123,482 1,100,700 | 1,238,658 | 0.45|0.67| 1.27 | 1.02| 1:8.5 4 cells...... 23,100 154,280 969,468 | 1,146,848 | 0.71| 1.08] 1.58 | 1.27) 1:6.6 S cells... ...2. | 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 | 1,237,302 | 0.72) 1.10] 2.45 | 1.92] 1:9.8 24 cells.....| 30,164 258,897 390,727 | 1,179,788 | 0.92] 1.41] 2.66 | 2.13) 1:8.6 | ___ (231,000) (1,151,891) | | | (2.35) 11.90)! (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 36h thick its volume is about 230,400 cubic u; subtracting the volumes of the nuclei of the plate, 21,584 cubic p, leaves 208,816 cubic pz 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 », we have as the total volume of the proto- plasm at the 24-cell stage 231,000 cubic ». This figure is 27,897 cubic yu 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 3y5) 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 eray 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 on 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 egg 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 TABLE 9 NUCLEAR SIZE 39 Rate of nuclear growth during the cleavage of Crepidula plana MAXIMUM |COEFFICIENT) MEAN (|COEFFICIENT| MINIMUM |COEFFICIENT STAGE BLASTOMERES | NUCLEAR or NUCLEAR OF NUCLEAR Or VOLUMES | GROWTH | VOLUMES | GROWTH | VOLUMES | GROWTH cubic cubic wu cubic Z)cellanwAB CD sscdnecseeiye 14,476 1.0 6,110 1.0 381 1.0 Sicelisy Ay By CD asoocr tees 23,100 1.6 8,580 1.4 602.4 1.58 cate 10.513 DSaaeanioecnoeees 19,396 7,068 338.4 Va—Udlet/ischesaeics adapecee 5,748 3 620 338.4 25,144 1.738 10.688 1.74 676.8 WaT DAH ID Seve eyaclene 12,220 7,068 338.4 16 Galle DADs Mee eons 7,068 3,620 338.4 Senate Tee nen eee 3,620 1,528 235.2 1 Vo LD re ee 720 452 235.2 23,628 1.63 12,668 2.07 1147.2 3.01 GASB ID) re ectemeielsosss 8,580 5,748 150.0 DAO casa et tess eats 5,748 3,620 150.0 24 cell Pal diss she aenataeree 5,748 3,620 150.0 GH De oa lec 5,748 3,620 150.0 NEVES Kc BRS eet ae 3,620 1,528 235.2 Laz ld2a sss. sere 720 452 235.2 30,164 2.08 18,588 3.04 1070.4 2.80 UNE DY. aa ons soo 12,220* 7,068 171.3 0 ee a Ee 697 382 58.8 AAC OGER wept th aati 2,715 1,146 112.5 Sa Sd aera 5,748 3,620 150.0 32 cells 4 2al—2d!. 1... eee ee 5,748 3,620 150.0 Data De ia cinco 5,748 3,620 150.0 IW Ne Le aaadoe 3,620 452 150.0 Pat 2 Vdh.2 eis actcis 3 2,095 3,620 150.0 IGE 0 Cae anne 720 452 235.2 39,311 2.71 23,980 3.92 1327.8 3.48 Total growth in thirty divi- SIONS Sas star atoyeycyar oie. ejace ies ie eiatels 24,835 LS 17,870 3.92 946.8 3.48 Average growth for each divi- ; SUM Ear Teyersiss ciate sersiatelatererecs 827.8 1.05(=5%)| 595.6 1.09(=9%) Bile) 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- CELL SIZE AND NUCLEAR SIZE 41 TABLE i0 Actual nuclear diameters and volumes in the 70-cell stage of Crepidula plana NUCLEAR |TOTAL NUCLE comment ia ORE een cea pak te | VOCUME | Nuclear Nuclear | | volume | surfaces | a Ml | DICE list ae Gremmr te teehee na ratte e sie lbeen 4: 14,476 | 1 1 (AAR re ea eho 16 8,579 BAC eee eh og 10 1,571 11 Entomeres E!, E2 ; 7 359 RetRezamees Ales oh se ur ae od 2260 4 Mesomeres fae os CO te x | eee | | First quartet | | 4 Apicals; lat oldu, say oe ls. HOM») 2525004 3 Basals, lal-?-1-1e2-21, aa allay, | 1 Basal, Hae tao, & a a 905 | | 3 Middles, la!:2:2-1el-2-2,.......... 12 | 2,714 | APAvurnetslia2—ld2 05 sae ee ee 7 718 | Second quartet | | -- Selupicellsh Zatti 2el La ane 6 | 339 Or aempcelll: PL NE ass ce MR i 10 524 4 4 Girdle cells, 2a!-2-1-2d!:2-1,.. 11... 9 1527 S |4 Girdle cells, 2a!-2-2-2d!-2-2,,......| 10 2,094 f] |4 Girdle cells, 2a2-1--2d2-1-1,.00 0... 9 1,527 as) 4;Girdlecells\2a2 = 2 2d7-125. 9 1,527 | 4 Girdle'cells; 2a?:2-2d?*2,.......... 5 | 262 Third quartet | | 4 Girdle cells, $a!--3dt-).. 20... 10 | 2,094 4 Girdle cells, 3a!:2-3d!2 .......... 9 1,527 4. Girdle'cells; 3a?-—302-)5.. 1.22... 6 | 452 (4 Girdle cells, 3a2-?-3d2-2........... 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 88 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 nuclet, 2-cell to 16-cell stages of Fulgur carica | | DIAMETER OF TOTAL VOLUME OF COEFFICIENT OF SNE TERA NSO OBE TEOES) NUCLEUS NUCLEI NUCLEAR GROWTH i ath a : M . cubic PACES AMS | CAD id westiererie scheiele eye 40 67,020 | 1.0 Aceliis AWB Gu): scans 32 68,628 | 1.02 TAS TID Ar te aan deat 32 68,628 8 cells { 3 ot \issid.\’.) eee 16 8,576 77,204 1.1155 DIASO DE: ete eee nee By 68,628 12 cells’ a2 ye eee 16 8,576 ar 6 IN re de oe a 16 8,576 85,780 1.28 | SASS Die eee ee: | 382 68,628 Sa S diecaes ee ee | 16 8,576 | 16 ll | ) | aia pies te oa aes | 16 8,576 Lis has] io ka Sapeh ene re Bh | 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. 83. Nuclear growth during the cleavage of other anvmals. 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 COEFFICIENTS OF GROWTH Seen le ata | occas mn | Nuclear volume Dee be cubic Mb | Before first ma- turation.......| 54 824437 1) Poc0 Before first | cleavage....... | 912+ 12 S09") 4\)) 0202 1.0 Deelisnsee a 16u [ie EDISTO CONEY IN SDE 1.0 1.0 Acellsn eee aan 14 (eee 74S:. | SO06s ame seley 1.34 Sicelles eee cae: 13 fem O208 6° *| + OUTenn5 "Os 214 Aocellase eae 11 TS! Oe Galan i260 2 cellser Sue oe, ha vata) | 16,755 | 0.20 | 9.26 3.90 GAicellsaen ee eee 8 In E52 | OL20Pa ona 4.00 ISicellsyyseees 6.5 18406. li .OL22eRtOntz 4.29 256 cellsian see. 5.25 19,395 | Ob23 5 101,72 4.52 | 13.75 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 large part upon the quantity of cytoplasm in the cell. 4. Growth of different nuclear constituents. a. Nuclear sap. All 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.2. 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 protoplasm 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 | | | . nos | | M Me Pirstimaturation..t6¢.. 0.0054 42 42 Second maturation............ 18 == Binstyclempare wes saree ace aa 30 34.5 Second cleavage, AB, CD......| 30 24 Third cleavage, A, B,C, D.... 27 22 Fourth cleavage, LA-1D....... 25 21 Fourth cleavage la-Id.........| 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. e. 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 nucleinic 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 ja-—/d (figs. 7 and 8). In the former the diameter of the nucleus just before division is about 24u, in the latter about 144. 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 w and of those from the small nuclei about 2.6 cubic yu. 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 o6dtid, 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 4A—4D and 4a—4¢ (fig. 6) in which the resting stage is particularly long. The nuclei of the cells 4A—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 52 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 Maximum nucleolar size and nuclear size in the blastomeres of Gini plana | DIAMETER | VOLUME NUMBER | DIAMETER VOLUME | NUCLEAR- STAGE BLASTOMERES Or | OF | OF OF | OF NUCLEOLAR | NUCLEUS NUCLEUS | NUCLEOLI | NUCLEOLI | NUCLEOLI RATIO ML cubic | : a cubic pL lcell,beforematuration 42 | 32,409 | 1 12 905.0 35:1 ZcellayA By CD rants 20 4,189 2 3 28.0 | 149: 1 A cells) Ass © 2D a5 ae. 15 1,767 | 2 25, 14 Meee 220) So Scalia PAID: al “19 3,091 | 2 3 | 28.0 | 128-3 1 Haldar | lS. 1,150 | 2 Dales 7.0 | 164 :1 2A-2D...5.:.) 14 1,437 2 2 S75) 180521 12cells 2 } | Oy Ge sate bh \15 tener. 2 2 8.3 | 220:1 Geechee Taide... 12 905 | 2 332 18.3 | 50 al! Lat-td? ees. 7 180 2 1 1.0 | 180 :1 3A-3D.. ge 1,767 1 74 221'0)| S31 20cells 2 2 | 2 Car Nsesodam 15 1,767 | 2 3 28.0 63:1 25 cells ric ea Pee | 9 382 | 1 3 14:0 | 2/7 32 cells, 4a-4chs,-co5- 50 13 905 1 6 113.0 Sica 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 ie 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 | NUCLEOLAR NUCLEUS | NUCLEUS | NUCLEOLI | NUCLEOLI | NUCLEOLI RATIO a cubic | MU cubic 1 cell, pronuclei...... [\9 24 7,238 | : 12 905 8:1 Vo 21 4,849 1 10 524 9:1 DicellseAiBe OD) wascen 24 7,238 1 9 382 OB! dicells, Al BY C,D...... 15 1,767 1 9 382 | 4.6:1 8 cells, 1A-1D.......... 18 | 3,055 1 9 382 Sune 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 7yu, the measurements being made during the telophase of division. The maximum diameters of the sharply defined spheres, during the resting stages, vary from 5yu 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 eytoplasm and nucleus. During cleavage the fluid content of the egg as a whole decreases, the odplasm 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 lhe; 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 o: 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 nucleinic 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 yf 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 nucleinic acid which is distributed through the cell body. VII. Senescence, reyuvenescence, 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 Missverhdéltniss von Kern und Protoplasma vorhanden ist, und dieses Missverhaltniss allmiihlich eine Ausgleich erfaihrt, 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. (3) The growth of proto- plasm at the expense of yolk during maturation and early cleav- 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 fact 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 (99) 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 ege 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. 3. Lillie (1910) holds that this is due to increased permeability of the plasma membrane during division. CELL SIZE AND NUCLEAR SIZE 63 PARA £1 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 HEchinid 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 wn 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 cm., 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 cyto- 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 eges 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. 18, 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 can form cytoplasm or the cytoplasm 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 1ts 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 reeommended 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 a 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 Jc 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, 7B 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 Ja and Jd; these cells are purely protoplasmic and are very small, all four of them being no larger . than one of the micromeres, /b 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 Oe 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 (la—1d). 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-1d, 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 24—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 /C 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 wo 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 and 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 lies. 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 Tea. 2. The sizes of spindles, centrosomes, spheres and asters. The study of centrifuged eggs shows, as was observed in the case 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 Lillie (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, ef 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 cases 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 SO 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 lie 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 cleay- 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, 709) has shown, and one reason for this is to be found in the fact, as I have discovered in Crepidula, that the cell axis, 1.e., the axis connecting nucleus and centrosome, can rarely be changed by artificial means. 6G. 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 odcytes 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; (ce) The length of the resting period (Dp. 2 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. 29-32). 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. Ove, 0): 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 Bautzer, F. 1908 Die Chromosomen von 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 tiber 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. Conkuin, E. G. 1893 The fertilization of the ovum. Woods Hole Lectures. 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Mech., Bd. 31. Minot, C. S. 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. Monteomery, T. H. 1910 On the dimegalous sperm and chromosomal variation of Euschistus, with reference to chromosomal continuity. Arch. f. Zellforschung, Bd. 5. Morean, T. H. 1910 Experiments bearing on the nature of the karyokinetic figure. Proc. Soc. Exp. Biol. and Medicine, vol. 7. Peter, K. 1906 Der Grad der Beschleunigung tierischen Entwicklung durch erhéhte Temperatur. Arch. f. Entw. Mech., Bd. 20. Pororr, M. .1908 Experimentelle cytologische Studien. Arch. f. Zellforschung, 1BYele ale SHACKELL, L. F. 1911 Phosphorus metabolism during early cleavage of the echinoderm egg. Science, vol. 34, no. 878. STRASBURGER, E. 1893 Ueber die Wirkungssphire der Kerne und die Zell- grésse. Histolog. Beitrige, Bd. 5. VerRworn, M. 1891 Die physiologische Bedeutung desZellkernes. Arch. f. d. ges. Physiol., Bd. 51. WatTase, 8. 1893 On the nature of cull organization. Woods Hole Lectures. Wiutson, 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 8 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 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-1d, 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. 18 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 lie. Fig. 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 Jd. 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 Domestic Fowl’’ are: 1. Regulation in the morphogenetic activity of the oviduct. Jour. Exp. Zodl., vol. 6, pp. 389-359, 1909. 11. 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. 49-84. ur. A case of incomplete hermaphroditism. Biological Bulletin, vol. 17, pp. 271-286, 1909. Iv. Data on certain factors influencing the fertility and hatching of eggs. Maine Agricultural Experiment 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. 8, 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 orshell gland. E, 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 les 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. 3870. Hen No. 952. March 19, 1910 Egg found in the albumen portion of the oviduct 11 em. 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 cm. 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 albumen 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 ege 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 (egg 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. 105 PHYSIOLOGY OF THE OVIDUCT Jepun poyde 7x0} 90S UOTJVUB[dxe IOyJIN}] IOg ‘aZIS [BANZvU {SUOT}IPUOD [BOTJUApT Is0joyd ‘pig owes ayy JO 850 pIv] [BUlIOU B& g pUe ‘paAoUIad OURIGUIOUT [JAYS S4T YM ‘gTO8 “ON Us JO JONpIAO oY JO snuUIyysT oY} WOT] UDyey B30 UL P Burmoyg 7% ‘BLT q Vv 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 in the 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’ cases 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 ege 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 TABLE 1 109 Data showing the increase in absolute and relative weight of albumen after the egg 5 Nn | | lees ze | |B a 5<< 5 6 B | i ud LOCATION OF EGG IN OVIDUCT | i= aia Hy ae lI 3 x b= ae BE a 38 Ka Ba B g BS a gy aq BI S| Ks BI = z E - =} ry 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 GIs rs Sete nets cbse oa ea 20h 57.57 6.23 16.79 34.55 205.8 | Difference. ...)5.6..2.% a —30.37 | —6.23 | —1.41 | —22.73 | —128.9 : 2. At caudal end of albu- | men portion. No mem- | | | brane (Hen 8005)........ ee a2eu53 | 0 | 15.87 13.66 86.1] 43.6 Mean of the nine pre- | | viously laid eggs of same | Wena eas cohen SOD SLE 5.79 | 15.67 | 31.34 200.0 Diticrewce shes | —23.28 | —5.79) +0.20 | —17.68 | —113.9 3. Just entering isthmus, | | little cap of membrane | on caudal tip. (Hen | OS eres coat iee cr neice. | 29.5 | 0 VIGOR imager 84.4 | 438.5 Mean of two previously | | | laid eggs of same hen... P25 | 9.0 | 16.25 31.0 190.8 i Difference.......... Pet 20) |) 9-00) 0,2an eon | Oo 4. Entering isthmus. Cov- | | | ered with membrane ex- | | cept for a little of crani- | al tip. 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EFFECTS ON REPRODUCTION AND GROWTH L. B. NICE From the Biological Laboratory of Clark University ONE FIGURE CONTENTS MET OAUIC ETO Mets Se oe ele feaeicee zeta ORE Sees So? hs ates ER hr 133 ENC aK) Leet ere eee ees At nicl tse Oat er a SNe ENS ore mie lat aha aes 133 INiGotimevandstobaccormokeseereeeriate] a: ee ene ee eee eee 135 ( OR TIET VE) pee ies: OP a AROS Lt A eee IRC SAS So: id.c Coho nee 135 IVE EO Cas ay see eee Re aaa a 6) 3 snd A eR Ate Ee Effects on the weight and health of the adult mice......................... 138 Effects on the fecundity of the adults and viability of the young........... 139 Growth of the young subjected to the same conditions as their parents..... 159 Growth of the young not subjected to the drugs...:..:i......--.-.s.-7---.: 145 Comparison of the growth of mice given drugs with those not given drugs.. 146 SSS UTR RPV aca hs Sd hal oP SRN Ss set’ sain Lae ee ee. Eas 147 Billoo ora isyfarey ae on iy capo se tera oat: fc Re A re Rae ca ates 149 INTRODUCTION Alcohol The effects of alcohol on animal offspring has been shown in several investigations to be injurious. Laitinen (31) found that aleoholized 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 alecoholic- 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 i. BS oNICE of Ceni’s (10) alcoholized 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) alcoholized 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 alcoholized 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 EKugenics Laboratory (4, 18 and 43) and of Laitinen (33) demonstrate that more children are born to alcoholic than to sober parents. As to the effect of aleohol 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 aleoholic intoxication produces degeneracy, depravity and idiocy. On the other hand Pearson, Elderton and Barrington (43, 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. 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 Cramer (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 L. B. 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 cubic 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 ee. 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. Hight 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 FEMALE | NUMBER OF | NUMBER OF | NUMBER OF NUMBER OF YOUNG | MONTHS OBSERVED LITTERS | YOUNG BORN | THAT DIED oes i ; | \e ee ee ee 7 3 | 18 | 0 Soo oe eee ii 2 | 12 | 0 CP esc o i 2 | 16 | 0 OY 3s Beas eee ae 7 2 | A | 0 TO, 8 ee a 3 1 | 7 | 0 Bee | Total 4 | 7 10 | 60 0 i} * Female E died at the end of three months. No pathological condition Conia be found. t Female F was killed by accident at the beginning of the experiments. TABLE 2 Alcohol line. First generation | NUMBER OF NUMBER OF NUMBER OF NUMBER OF YOUNG gH uEe | MONTHS OBSERVED LITTERS | YOUNG BORN THAT DIED | f= kt ee et aaa . Neat ise) 2 7 2 17 1 Sere Neer ‘4 2 | 12 1 CSS eh) | 7 4 | 23 1 1D eee i 4 | 15 2 I AR 3 A. | "6 2 | 14 4 F*, Motale sas. 5) i 14 | 81 | 9 1 34 wks. | | * Female F died three and a half weeks after the experiments were started. No pathological conditions could be found. 140 Nicotine line. LL. Be NICE TABLE 3 First generation FEMALE NUMBER OF NUMBER OF NUMBER OF Ma Oe DEINE MONTHS OBSERVED | LITTERS YOUNG BORN THAT DIED NE erie erie Ai oi | 3 13 1 1B aA cid eae eee eeee ii 2 7 0 Oye a tee a 7 il 4 4 1D cee ea eee 7 2 13 | 2 1 ee hoe Reet 7 | 5 36 | 2 1D is | 4 31 9 dotaleeeer 6 a | 17 104 18 TABLE 4 Line subjected to tobacco fumes. First generation NUMBER OF NUMBER OF NUMBER OF | NUMBER OF YOUNG BME MONTHS OBSERVED LITTERS YOUNG BORN THAT DIED JRC Oe SAS ae af 4 23 | 9 Behe ete PKs, if + 30 | 5 Os eae eee 7 3 22 6 iD ea geod wc eee a 4 21 7 IDSs 9h ee eee 4 Sut 15 14 yr esac See Total JA 7 18 Titit 41 1 4 * EK was killed by accident at the end of three months. + F died at the beginning of the experiments. t One of these litters was an abortion. Caffeine line. TABLE 5 First generation NUMBER OF NUMBER OF NUMBER OF NUMBER OF YOUNG EPEC MONTHS OBSERVED | LITTERS YOUNG BORN THAT DIED TAN Ay aly aa a 3 15 0 Bit ieee oe Uf 3 16 8 a oes Eee 7 2 IL 5 1] Bee. 8,53 ae een 0 3 Df 2 o Rae eles eS i | 2 7 4 ss. 7 | 3 9 3 Total 6 7 16 2? EFFECTS Control line. TABLE 6 OF DRUGS ON WHITE MICE Second set 141 FEMALE NUMBER OF NUMBER OF NUMBER OF NUMBER OF YOUNG MONTHS OBSERVED LITTERS YOUNG BORN THAT DIED 6 ink eee 4 1 ii 0 LR St ee ee 4 2 12 0 era pee sone 4 2 9 0 1D Red ae arene 4 2 9 0 Be geal ona Aee ae 4 1 2 0 4 1 4 0 Totaly 225-0 4 9 43 0 TABLE 7 Alcohol line. Second generation FEMALE NUMBER OF NUMBER OF NUMBER OF NUMBER OF YOUNG | MONTHS OBSERVED | LITTERS YOUNG BORN THAT DIED Pe WS Ce ct 4 1 5 0 Be Re 4 2 13 il hoe Oe ae 4 2 16 2 ig tags geieaonete 4 2 13 3 3 Sol Rane ere koe 2 1 9 1 Motalenci.cc 4 4 8 56 a 1 2 TABLE 8 Nicotine line. Second generation FEMALE NUMBER OF NUMBER OF NUMBER OF NUMBER OF YOUNG MONTHS OBSERVED LITTERS YOUNG BORN THAT DIED Sis 6 BELG eee 4 2 11 5 aoe Cee eee 4 2 13 0 BP aren Gre i. 4 1 6 0 Motalessea. - 4 4 5 30 5 142 L. B. NICE TABLE 9 Line subjected to tobacco fumes. Second generation FEMALE MONTHS OBSHEVED| ELETHRE!) GoUNGsodN) | “mzan Dimp DNS Sey ye a on 4 3 21 3 Ie aes: ore: 4 3 15 1 (CE Fh San eee 4 2 6 2 1) er ee aaa ramen 4 4 22 9 12): ayaa aby ase 4 2 12 3 eee 4 1 8 1 GRE OS emo 3 1 6 1 15 Meenas 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 we | EMBERoY | “Slowons” | “uumup or | auMpne ox | rouwe-naat Controlen 65. {4 7\ 10 60 0 ; \1 3 f Alcohnolerriassee. 5 7 14 81 9 INGCoOtIMe Ne 6 u 17 104 18 Sralecd eee J4 cn 18 11 41 \1 a Caffeine......... 6 a 16 85 22 TABLE 11 Record of the young of the second generation Summary of tables 6,7, 8 and 9 NUMBER OF NUMBER OF LINE “yematey, | (MONTHS | “Uiveens | young pon | YOUNG THAT Controlasneeaose 6 4 9 43 0 INCOME 54 soca fa 4 | 8 56 Ne ip a2 Nicotimes. .:---4: 3 4 5 30 Smoked../...... [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 LINE | AVERAGE NUMBER OF | AVERAGE NUMBER OF PER CENT OF YOUNG LITTERS YOUNG | THAT DIED Controle santas eon cc ee 2.2 13.3 0 AIG OH ON Meas tie teh 2.8 16.1 Til al INTC OAM. .cce re reas ater 2.8 17.3 17.3 SLINOLAEC | Soop coro ami cool 4.0 24.6 37.0 Guiheimen. eo seso ins cee: | Psat 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 LITTERS YOUNG THAT DIED Controle 4.5. -ose oes 5) tal 0 INGO Voll, Season wae shee dol 1.8 12.4 12.5 INT COLIN ere 1.66 10.0 16.6 Smokedee ee. Aa 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 is) Bs aOR TABLE 14 Weekly growth of the young of the first generation subjected to the same conditions as their parents | | | a ae |e le le Blea En ces tere | aM by H lo | Be IH Velie ele S| oe mn i i 2 2 2 See IAs Sie el) Se mie se Se AA eI bp | aA Be eS ee aSs BD aaa ae ae Fool Fan leo) Eo |FE/FFIFRIE.| & Bay | (es) | ere LINE ase lage | Flag. |aajaclaziap| af a a8 a & aa az /e2) goa S| on ml > 4 > 5 ow o Bo) OR |S BISBIGRiGo| oF oh OB og ae caso0e can aaah Wess || AAW IO] Tbs PAE ta yy tS COO) | TO) 4] TB Odes 15), (0) Smoked...............| 20.0 | 20) 261.2 (2.44.5) 5.3 ee WO) |) TPA TE, e385 } | | One hundred and thirty-three young mice of the first genera- tion and 63 of the second were subjected to the same conditions as their parents. Although there was much variation in the same line or even in the same litter, on the whole the five lines in both generations grew at about the same rate. The alcohol young excelled all the others. There seems to be no constant correlation between the weight of the parent and growth of the young. For instance the alcohol adults are large in the first generation and the controls are small, EFFECTS OF DRUGS ON WHITE MICE 145 while the reverse is true of the second generation; yet the alcohol young are larger in both generations. The caffeine adults are large but their young are the smallest of all. GROWTH OF THE YOUNG NOT SUBJECTED TO THE DRUGS The following Table (Table 16) shows the growth of the off- spring of drugged parents, not themselves subjected to the drugs: TABLE 16 Growth of the young not subjected to the drugs | LINE AT THREE WEEKS AT FOUR WEEKS AT FIVE WEEKS AT SEVEN WEEKS AT EIGHT WEEKS | AT BIRTH PER MOUSE AT SIX WEEKS WEIGHED AT ONE WEEK NUMBER YOUNG AVERAGE WEIGHT AVERAGE WEIGHT AVERAGE WEIGHT AT TWO WEEKS A WEIGHT | AVERAGE WEIGHT AVERAGE WEIGHT AVERAGE WEIGHT AVERAGE WEIGHT AVERAGE WEIGHT grams | grams | grams | grams | grams | grams | grams grams | grams Conitnol=see44- 33 | 1.35 | 2.85 | 4.0 6.2) | 7205) MOO Zar | 1428" 1628 Mleoholen ks a. 1G) ed) lise4 abe.) 73), Meas IA eon Gal Leonel O52 Nicotine .=e-..-| 6 1.5 | 2139 4.7 | 6.0 | 8.5 ) 1000314 16-0), 18:0 Smoked (..2/2.: OP ete 2280" 4K 257) 5.1) | Mel Onan al see 14 S54 @aiteine:.-..-. Se tG, ja2e2y 40) 5.4) e8z0R) Be Aza() | | WAOKO aero: | 5 * The weights of the controls are based on both sets of controls in tables 14 and 15. From this it is seen that the young of the alcohol line de- cidedly excelled all the others in growth. COMPARISON OF THE GROWTH OF MICE GIVEN DRUGS WITH THOSE NOT GIVEN DRUGS The young of nicotine parents that were not given nicotine themselves excelled somewhat in weight those young that were given nicotine. Tobacco fumes had no appreciable effect, the smoked and non-smoked mice growing at about the same rate. The young of caffeine parents that were not given caffeine them- 146 L. B. NICE TABLE 17 Comparison of the growth of mice given drugs with those not given drugs ; 2 : aid m Dm mM ) Q m 5 a aa a & Bp ae ae a8 aE ae mit lsseeaa es | ee | 2 Fe | Fe | Fe MICE ng |8@a| as ae ag ap a @ a af | af Bo | Onn | O4 oF oe oko) og ok OB oe Bea eee cee | a) ee ee ae |de2| ge | gs | ge | ae | ae | Be | ae | ag Mwah ee ieee Lo | eau | grams | grams | grams | grams | grams | grams | grams | grams | grams NO OMETOLA Cie veh: 33) iso) 2285 400 6:2 | 7207 AOLOn 1207 | 14.8)| 1628 Without | alcoholt.....| 16 sierra 9) UlRes4 (3 14 ae 36.| 176") tOr2 With alcohol* .| 39 | 1.32 |3.3 | 4.97 | 6.5] 9.6 | 12.1 | 14.0 | 16.2 | 17.5 Without mcotine;.. 6-6 om aon ivacer, 6.0] 8.5 | 10.9 | 13.4 | 16.0 | 18.0 With nicotine*} 42 | 1.33 /3.0 | 4.3 5.5 | 8.0] 10.5 | 12.6 | 14.4 | 15.1 Nonsmoked{..| 9 | 1.1 | 2.89| 4.25] 5.1 | 7.1 | 10.5 | 13.2 | 14.8 | 15.4 Smoked™ 23... Ot, 22a eee 5.7 | 8.0 | 10.5 | 13.0 | 14.4 | 15.3 Without | caffeine} ..... So Nel Ge Mesa ae@ 5-4) 8.0 | 10.97) 13570) 215.247 20 With caffeine..| 27 | 1.3 | 3.0 | 4.2 5:3 | Cad | LORI LON ea 339 * These weights are the averages of the two generations from tables 14 and 15. + These weights are from table 16. The parents were subjected to the drugs. selves grew faster than those given caffeine. Although the young of the aleohol mice when given alcohol themselves excelled all the other mice in growth, other young of these same mice when not given alcohol grew even faster. The control mice grew faster than the caffeine mice, were excelled by the alcohol mice but grew at about the same rate as the nicotine and smoked mice. I wish to thank Dr. C. F. Hodge for criticism, Dr. Louis N. Wilson, librarian of Clark University, for securing the literature, and my wife, Margaret Morse Nice for aid in keeping the records and preparing the manuscript. ‘SUMMARY 1. The control mice had the fewest young of any of the lines; the fecundity of the alcohol, nicotine and caffeine mice was some- what greater, while the mice subjected to tobacco fumes in both generations had almost twice as many young as the controls. EFFECTS OF DRUGS ON WHITE MICE 147 2. The mice subjected to tobacco fumes had the largest pro- portion of young that died from lack of vitality, 37 per cent dying in the first generation, and 26 per cent in the second. Caffeine also had an injurious effect, on the viability of the young, 25 per- cent dying. Nicotine and alcohol had a less noticeable influence, 17.3 per cent and 11.1 per cent respectively died in the first gener- ation, and 16.6 per cent and 12.5 per cent in the second. None of the control young died in either generation. 3. Out of 707 young born none were deformed, none were born dead, and only one abortion occurred. This took place in the smoke chamber. 4. When adult white‘mice were subjected to alcohol, nicotine, tobacco smoke and caffeine the growth of their offspring was not affected unfavorably. 5. When both adults and young were subjected to the drugs, caffeine had a slightly retarding influence on growth nicotine and tobacco smoke had no appreciable effect while the alcohol mice grew faster than the controls. 6. In all the experiments the young of the alcohol mice sur- passed all the others in weight. They grew most rapidly when they themselves received no alcohol. @ Fig. 1. Curve showing the growth of the mice. The abscissas represent the age of the mice in weeks, the ordinates their weight in grams. Control line 33 mice —-— — — Alcohol line 16 mice not given alcohol themselves —-——— Alcohol line 39 mice given alcohol Bh SOB — Nicotine line 6 mice not given nicotine themselves ei ne Nicotine line 42 mice given nicotine —— —— ~— — Smoked line 9 mice not subjected to tobacco fumes themselves — — — Smoked line 57 mice subjected to tobacco fumes —_.—— Caffeine line 8 mice not given caffeine themselves — —- — Caffeine line 27 mice given caffeine 148 bo 10 ial 13 14 15 16 EFFECTS OF DRUGS ON WHITE MICE 149 BIBLIOGRAPHY Appot, A. C. 1896 The influence of acute alcoholism on the normal vital resistance of rabbits. Jour. of Ex. Med., New York, vol. 1, pp. 447- 481. Apter, I. 1902 Preliminary note on some effects of tobacco on the tissue of rabbits. J. Med. Research, vol. 3, no. 2. ApuER, I. and Hensget,O. 1906 Intravenous injections of nicotine and their effects upon the aorta of rabbits. J. Med. Research, vol. 10. 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Vort, Kart Untersuchungen iiber den Einfluss des Kochsalzes, des Kaffees und der Muskelbewegungen auf den Stoffwechsel. Miinchen Cotta, 253 pp. Wuitney, D.D. 1911 The poisonous effects of alcoholic beverages not pro- portional to their alcoholic contents. Science, N. S8., vol. 33, no. 850, April 14, pp. 587-590. ZEBROWSKI, E. V. 1907 Zur Frage iiber die Wirkung des Tabak-rauches auf die Blutgefiisse bei Tieren. Centralblatt fiir allg. Path. u. path. Anatomie, Jena., vol. 18, no. 9. 1908 Zur Frage vom Einfluss des Tabakrauches auf Tiere. Cen- tralblatt fiir allg. Path. u. path. Anat., Bd. 19, no. 15. HEREDITY OF PIGMENTATION IN FUNDULUS HYBRIDS FRANK W. BANCROFT From the Department of Experimental Biology, Rockefeller Institute, New York THIRTY FIGURES Ever since the rediscovery of Mendel’s law, students of heredity who have investigated characters in the adult have habitually sought to determine whether or not these characters were inherited according to the Mendelian formula. Those, however, who have investigated the inheritance of larval and embryonal characters have usually not considered the facts from the Mendelian point of view but have sought to determine whether the inheritance was maternal or paternal. While it is possible that this point of view may be the most fruitful one from which to regard hybrids of distantly related species; still the work of Loeb, King, and Moore,! showed that for the larvae of closely related sea-urchins, at any rate, the Mendelian point of view could be adopted with profit. They found that in the larvae obtained by crossing Stronglyo- centrotus purpuratus and S. franciscanus certain characters of each species were dominant over the allelomorphic character in the other species, and the result was the same no matter whether the character in question was maternal or paternal. On account of the wealth of teleost material in Woods Hole and its favorable character for such investigations it was suggested by Dr. Loeb, whose constant helpfulness during the course of the work I wish to acknowledge, that I take up the study of inheritance in Fundu- lus from the Mendelian and the physiological points of view. Previous work on Mendelian inheritance of this form is limited to the paper by Newman? who came to the conclusion that 1 Arch. f. Entwick-mech. 1910, Bd. 29, p. 354. 2 Jour. Exp. Zool., vol. 5, p. 503. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 2 FEBRUARY, 1912 153 154 FRANK W. BANCROFT although some characters, such as the size and shape of the chro- matophores, and the color pattern on head and body, show Mendelian dominance, that ‘‘nearly all of the characters observed may be classed as examples of blended inheritance of one sort or 3 another. . METHODS Reciprocal crosses between Fundulus heteroclitus and Fundulus majalis were made in the usual way; both eggs and sperm from a number of individuals of each species being mixed for each exper- iment. The developing embryos were kept in Syracuse watch- glasses and fingerbowls, loosely covered with glass. The water was frequently changed and the eggs were carefully separated from the masses to insure a maximum supply of oxygen. After hatching the young fish were usually kept isolated in fingerbowls. They were fed with what they could pick off of foul eelgrass, and small pelagic organisms obtained by towing. Later fish flesh, flies, and liver were used for food. Some of the little fish were put out in the eel-pond in cloth cages of various kinds, but though they grew much faster than any that were kept in the laboratory so many of the cages got broken that only one of these fish was kept until the middle of September when they were transferred from Woods Hole to New York. INHERITANCE OF COLOR CHARACTERS Three kinds of chromatophores were observed, all three of them occurring in all the embryos of both pure species, and of both kinds of hybrids. They are: 1. Black opaque chromatophores. These are the first to appear, and apparently persist throughout the life of the fish. 2. Red opaque chromatophores, usually of a brick red or deep yellow color, showing white or creamy with a closed diaphragm. They sometimes take on a white or creamy color but can usually be easily distinguished from other tissues by their conspicuous white color when the light is turned off. They are nearly as 3 Jour. Exp. Zool., vol. 5, p. 355. ’ HEREDITY OF PIGMENTATION 155 large as the black chromatophores, appear in early embryonal life at about the same time as the black or but shortly after them, and disappear completely within a few days after hatching. 3. Small lemon yellow, or greenish yellow chromatophores. The pigment in these is transparent and it is only by carefully observing them in the most favorable locations such as the fins that the branched processes and the chromatophore-like shape of the cell can be made out. They first appear a few days before hatching and persist as long as the fish have been observed. As all three kinds of chromatophores were found in all the embryos, and as no small differences in the color of the chroma- tophores that seemed to be significant were seen, the color differ- ences observed were due to the variation of the chromatophores in (1) number, (2) size and shape, (8) location or arrangement, (4) rate of appearance and development. Table 1 gives a summary of the main features of the pigmenta- tion in the four forms studied. An inspection of the table will show that in the first four char- acters (viz.: the red and black yolk chromatophores, head chro- matophores, and red chromatophores of the lateral line) all of which are not concerned with the rate of development there is a well marked Mendelian dominance, while in the last two charac- ters (viz.: the arrangement and time of appearance of the yolk chromatophores) both of which are concerned with the rate of development the dominance is not so evident. The characters will be considered seriatim. 1. Red yolk chromatophores The number of the red yolk chromatophores of the pure F. heteroclitus and both kinds of hybrids is about the same. No attempt was made to count them accurately but in all these three forms the red yolk chromatophores are very conspicuous features. In F. majalis, on the other hand, these chromatophores are so few that in one of the series they could not be found at all. In other series the first red yolk chromatophores appeared near the embryo from the fifth to the ninth day and increased slightly in BANCROFT FRANK W. 156 *‘SUIIOJ I9YJO OY} jo Aue ul uvy} J0}R] Iwodde soroydoyeumorys YLOA 4SAy ‘oArquie oy} 03 oy1s0ddo e1oydsturey oy} ul01y yuosqe Ajoirjyue oie pue ‘odaquie oy} Jo sapis oy} ye avodde qsig sosoydoyvumoryo ylO XR ‘suryoyey uodn soroydoyeuoryos §=yorytq 09 I0 (0G YNOGR YAM our] [eR.107e'T ‘soroydoyeur -O1YD Pot JNOYITM UT] [k.10}R'T ‘quoesqe so1oyd -OVBUIOIYD poy Jo dow 4saty “sngIpoo19joy “iT aind ul uvyy soyouvag 1asu0y puv o10UL YIM puB ‘1o]]/euUIS seroydozeumoryo yjOoA yoRlq "‘SUILOJ aI} I9qj0 UL UBY} poyoursq sso] PUB Jo][BUIS OS]TR JaMey Yonur soroydoyeumoiryos =YylOA pay! SITVf{VW ‘af GYOd “ULLOJ SUTMOT[OJ UI UBY} JoI[Ive ynq ‘SnNoTA -oid ut uvYyy 103%] «vodde soroydoyeumorys YylLOA ysaty ‘oAIquia oy} 09 a}1soddo OBS YIOA ayy Jo Javed 4eyy ur Jamo} A[poeploep 10 yuesqe qsiy 4B seroydoyvuo1yo YOR ‘saroyd -OJVUIOIYD Yovlq Moj ATOATY -vIvdu0d YAIM oul] [V10qyeT ‘snjlpoo1ojey “yy oand ur sv OWLS OY} Joe oury [e10,e'T ‘yuoseid seroyd -oyeuo1yo pvey Jo doa 4saty ‘s0084S ATIBO UT SN4I[DOIOJZ0Y ‘q oind jo esoyy 0} «e]TUITS soroydoyeuoryo = YyyoA youlg "SngIpoo1ejzoy “q oind ul uevy} poyouraq Sse] puBv ‘1aT[[BVUS 9794T] B ynq “JuBpuNqe puUv oF1e] © SOLIIDOUGTLAH “a & 6 SITIVIVN ‘a ‘SULIOJ JUONDesqns UI UBYY JeljIva ynq snota -oid ul ueyy Joye] avodde so1oydoyeurorygos Y [OA 4Satyy "SSBlO SNOTAeId UT SB poynqiaystp ATUeAV ynoqe 4siy ye soroydoyevuroryo YO X "SNgIpoo0.107,0Y “q oand ut ueyy Ssurgoyey uodn saroydoyeuoryo youyq O10 MOF BV YYIM OUT] [B10 BT “19UOOS Op] 04 uIseq soroydoyeumoryo pot ay} yNq ssByo snorAeid ut sev oUIBS olf} JNoqe ouly, [v10} eT *yuasoid soroyd -oyvUIOLYyO pBoy jo dodo ysaty *sassao -o1d oynutur Auvur YyIM pue IoT[VUIS 9]}IT] B 9Nq sseRyo snotaoid Jo asoy} 0} «e]TUUIS soroydoyeuory9s9 =yoA youyg “ULO} SNOTAIIA UL UBvY} JoT[eUIS ynq odevys ULES JO “VUBPUNG’ puv 95.10] sotoydoyeumorys YTOA poy dIwd#AH DOA SOLIIDOURLAH “A © SITVLVW ‘a X 4 SOLITOOURLAH “a “SULIOJ JOYJO oY} Jo Auv UL UBvYy} Jor[ee reodde soroydo,BuIoryo YJOA 4ST T “yaed 1ay}0 Aue uO 8B OAIGUIA BY} 04 eytsoddo ows yjoA 944 jo soBjims oy} uo AuBUI se AyIvou 10 o3Inb ae e104} Ivedde say soroyd -oPBULOIYD Y[OA oy} Woy, “SUT _-yoyey uodn sortoydozeur -O1YO YORTG MOF ynq 10 euouU YIM OUT] [B10}RrTT ‘DUTYOYBY VLOJoq pue 48 so1oydozvur -O1Y9 pod JO aul] SnonutTy -u00 B YIM OUT] [B10] eT ‘yueseid so1oydoyeur -o1yo pvey jo dowd 4sany “SUL -jueM ATTensn sessodord ‘advys [vuos4 [od [v1oues B Jo pue os51B] saroyd -oywuIOIyD Y[OA youylg “quepunqgse pue ds1vT so1oydoyeumoryo ylOA poy lo SOLIIOOURLETH “od AUAd sprigh UD SUuLOf aNd Uaanjag sadualaff{ip 40709 jod190UIL UaqGny 13q ‘ LIU he Tl HTaViL HEREDITY OF PIGMENTATION 157 number though stillremaining in the vicinity of the embryo. After a few days their number again decreased markedly, apparently by incorporation within the embryo, so that they could only be found in a small proportion of the eggs, and in these not more than two or three cells to each yolk sac. There is no doubt then, that, so far as the number of these cells is concerned, there is a well marked Mendelian dominance, but as it is almost impossible to count them accurately I cannot be sure that in the hybrids the number of red yolk chromatophores is not slightly less than in pure F. heteroclitus. An examination of the red yolk chromatophores in the four forms for size showed that the chromatophores of F. majalis were usually much smaller than those of any of the other forms; but that these cells in the F. majalis egg hybrid were slightly smaller and in the F. heteroclitus egg hybrid considerably smaller than in the pure F. heteroclitus (compare figs. 13 and 14; 15, 16, and 17; 18 and 19; 20 and 21; 22 and 23). As in most cases where the hybrids showed a condition intermediate between those of the two pure species it was the F. heteroclitus egg hybrid which was most like the pure F. heteroclitus; this case in which the F. majalis egg hybrids were most like the pure F. heteroclitus deserves special emphasis. In shape the red yolk chromatophores of both hybrids approxi- mated closely to the dominant condition of the pure F. hetero- clitus. When they first appeared, these chromatophores in all four forms had about the shape of the red yolk chromatophore in fig. 13. In the pure F. majalis the red yolk chromatophores even at the period of their maximum complexity (fig. 17) did not progress far beyond this early shape; while the chromatophores of the three other became much larger and more complex. The F’. heteroclitus egg hybrids usually had red yolk chromatophores of exactly the same shape as the pure F. heteroclitus (compare figs. 18 and 19, 20 and 21, 22 and 23). The F. majalis egg hy- brids also in many cases had cells of exactly the same shape and almost the same size, as those of the pure F. heteroclitus. In some cases, however, the processes of these cells in the hybrids were fewer and the central body less extensive (fig. 9). 158 FRANK W. BANCROFT 2. Black yolk chromatophores For the first few days of their existence the black yolk chroma- tophores of both hybrids were identical with those of the pure F. heteroclitus in shape and the differences in size could appar- ently be entirely accounted for by differences in the ages of the cells. Compare figs. 18 and 14, 18 and 19 for the resemblance between the pure F’. heteroclitus and the F. heterolcitus egg hybrid and figs. 8 and 11 for the resemblance between the pure F’. heter- oclitus and the F. majalis egg hybrid. In the pure F. majalis these cells were on the whole smaller and with more and longer processes than in the other three forms (see figs. 10, 12). This difference in F. majalis was most pronounced in those cells which were nearest to the embryo, as in fig. 10; the cells that were far- ther from the embryo (fig. 12) differed less in shape and size from those of F. heteroclitus. We have seen that for the early stages there was complete dom- inance of the F. heteroclitus condition, as has been stated by Newman.‘ With further development, however, characteristic differences appeared so that each form could be distinguished by the appearance of the black yolk chromatophores alone. In the pure F. heteroclitus these chromatophores soon crept onto the blood vessels, forming a coarse reticulum in which the clear spaces between the chromataphores were in general equal to or wider than the space occupied by the chromatophores themselves. There were very few branches traversing these clear spaces. In the F. heteroclitus egg hybrid the chromatophores did not hug the vessels so closely so that the width of the colored meshes : was usually about twice the thickness of the clear spaces between them. These clear spaces were also usually thickly traversed by a feltwork of fine processes (fig. 23). The color of this reticulum was also paler than in the pure F. heteroclitus, apparently because the chromatophores were spread over a greater area. In the F. majalis egg hybrid there is, on account of the greater size of the egg, agreater space to be covered by thechromato- phores. Together with this factor there was a much less pro- 4 Jour. Exp. Zool., vol. 5, p. 550. HEREDITY OF PIGMENTATION 159 nounced tropism of the chromatophores for the blood vessels, so that hereno characteristic chromatophore reticulum was formed. In addition there was much variation in the shape of the chroma- tophores during these later stages; some of them still retained their previous characteristic polygonal shape, while others devel- oped long processes like the red yolk chromatophores or the black yolk chromatophores in the pure F. mayjalis. In pure F. majalis the black yolk chromatophores were charac- terized by their long and numerous processes also during these later stages. In considering these later deviations from the complete domi- nance of the early stages it might be thought that we had here two intermediate conditions manifesting themselves in the hybrids due to a delayed influence of some ‘branching factor’ derived from F. majalis. For in both cases the deviations are in the direction of the more profuse branching characteristic of F. majalis. I think, however, that in this case the deviations from complete dom- inance are due mainly to a diminution of the tropism of the chro- matophores for the blood vessels discovered by Loeb.> This reduced chemotropism is probably due to a change in the contents of the blood vessels. resulting from the diminished yolk assimi- lation in the hybrids which will be discussed later. However, no matter what its cause, there can be no doubt that in both the hybrids the yolk chromatophores are less closely approximated to the blood vessels than in either of the pure forms. Now when in 5 Jour. Morph. 1898, viii, 161; Arch. f. d. ges. Physiol. liv, 525. Loeb at first thought that this tropism was chemotropism due principally to oxygen. Later he was inclined to consider that it might be stereotropism. Among the embryos in this series it was noticed in a number of cases that after the circulation had be- come well established and the chromatophore network was well formed that the blood accumulated in some one region and stopped circulating through the ves- sels, although the heart still continued beating normally for several days. When this stoppage of the circulation took place in a comparatively early stage it was seen on several occasions that the chromatophores left the vessels and became again uniformly distributed over the surface of the yolk. As the vessels were still present it must be concluded that chemotropism and not stereotropism is responsible for the creeping of the chromatophores on to the blood vessels. When the circulation stopped in later embryonal life the chromatophores usually con- tinued to remain on the blood vessels even several days after the blood had ceased to circulate. 160 FRANK W. BANCROFT pure F. heteroclitus this chemotorpism of the chromatophores for the blood vessels is destroyed by the stoppage of the circulation the black chromatophores of this form also develop more branches than usual and may even in extreme cases (such as fig. 24) simu- late closely the chromatophores of the pure F. majalis. This instance shows very clearly the importance of an analysis of the mechanism of heredity insisted upon by Loeb in 1898* and more recently by Newman. 3. Head chromatophores One of the factors which influenced Newman in arriving at the conclusion that in the hybrids between majalis and hetero- clitus, blended inheritance predominates is the manner of the appearance of the head pigmentation. Newman found, and I have confirmed his results, that the head pigment appears first on the pure F’. heteroclitus; next on the F. heteroclitus egg hybrid; next on the F’. majalis egg hybrid; and last on the pure F. majalis. Now, while this is true, still a closer analysis of this process has furnished what is probably the clearest case of Mendelian domi- nance encountered in this whole study. In the pure F. heteroclitus, and in both hybrids the black head chromatophores appeared in two crops separated by an interval of about two days. In the pure F. majalis, on the other hand, this first crop was wanting and the second crop appeared at about the same time that it did in the pure F. heteroclitus. The first head chromatophores appeared on the first or second day after the first appearance of the yolk chromatophores. They were first seen on the sides of the brain, having probably migrated in from the yolk, and wandered onto the dorsum of hind and mid- brain, only in rare cases reaching the fore-brain. Various stages in the development of these cells are shown in figs. 1 to 6. As soon as they reached the dorsum of the brain they began to expand and finally became very conspicuous objects (fig. 5). Figures 2 and 3 which show these chromatophores before and after an interval of three and a half hours give some idea of the rapidity with which this migration and expansion takes place. 6 Marine Biol. Lab. Lectures for 1897 and 1898, pp. 227-229. HEREDITY OF PIGMENTATION 161 In both hybrids these head chromatophores appear in essen- tially the same way that they do in the pure F. heteroclitus; there are, however, minor differences. In the hybrids they appear a little later, and they remain for a day or more on the sides of the brain without migrating onto its dorsum instead of only for several hours as in the case of the pure F. heteroclitus. Even after the chromatophores of the hybrids have reached the dorsum of the brain they do not look like any stage in the development of the same cells in the pure F. heteroclitus, for they are smaller and there are more of them. The comparative numbers of these cells for ten fish each of the pure F. heteroclitus and the F. heter- oclitus egg hybrid are given in table 2. The differences while not great are, I think, significant. In the F. majalis egg hybrid the size and numbers of these cells are essentially similar. TABLE 2 ° NUMBERS OF HEAD CHROMATOPHORES AVERAGE Pigeelm heserocktus fon mid-brain..| 2 | 0 | 0 | 1 | 5) 1) 2 | 0 | 0 | AN ssl , \on hind-brain. ein eae: | 4/413 } 2/0) 5 3.4 | | | | | | ¥. heteroclitus egg {on mid-brain..|6|6|5/1)3|0/6|5 [10 | 4 4.6 hybrid......... (on hind-brain.| 5 | 4 | 2/3 | 4.7 | 4 | 6|3/4 4.2 In the pure F. majalis, on the other hand, there was no sign of this first crop of head chromatophores except in one case, out of several hundred embryos examined; and in this case the first crop was represented only by a single cell. With this single exception the first chromatophores to appear on the head of F. mayalis were similar in all respects to the second crop of the other forms. The second crop of head chromatophores in the pure F. heter- oclitus and the two hybrids always appeared at a distinct interval after the first crop. This interval varied from one to four days. In the hybrids this crop usually appeared one or more days later than in the pure F. heteroclitus; but in the pure F. majalis it appeared at the same time as in the other pure form. In all four forms the method of development of this crop was the same, and was different from that of the first crop. As far as could be seen 162 FRANK W. BANCROFT these cells developed in situ; appearing first as faint, grey, thin and well branched cells scattered all over the dorsum of the brain. Soon after their first appearance their color became much deeper and they began to expand rapidly, so that after several days it was no longer possible to distinguish them from the chromato- phores of the first crop. Fig. 6 shows the appearance of this second crop of head chromatophores in the same embryo which is represented nineteen hours earlier in fig. 5. A little later stage after the chromatophores of the second crop have become so large that they cannot always be certainly distinguished from those of the first crop is shown for the F. majalis egg hybrid of series 7 in fig. 9. To be compared with this last figure is fig. 10 taken from an embryo of the pure F. majalis also of series 7 and having the same age as the embryo figured in fig. 9. The entire absence of anything at all corresponding to the first crop of chromatophores is very evident. It is very clear then that the F. heteroclitus character ‘presence of the first crop of head chromatophores’ dominates over the F. majalis character ‘absence of this crop.’ Furthermore, since the second crop of chromatophores appears in F. majalis synchronous- ly with its appearance in F. heteroclitus, the delayed appearance of both crops in the hybrids cannot be considered a condition intermediate between that of the two pure forms. 4. Red chromatophores of the lateral line In the pure F. heteroclitus and the F. heteroclitus egg hybrids at the time of hatching and the F. majalis egg hybrids (which usually do not hatch) at about the same time the lateral line is mapped out by a series of about twenty conspicuous red chroma- tophores the characteristics of which are shown in figure 27. In all of these three forms these cells were very similar in appear- ance, and there can be no doubt that they are essentially the same kind of cells as the red yolk chromatophores. In the pure I. majalis these red chromatophores were not present on the lateral line at hatching time. There was, however, aseries of very HEREDITY OF PIGMENTATION 163 indistinct pale cells showing white with a closed diaphragm which fora time I took to represent the redchromatophores. Later, how- ever, it was found that ‘these cells were probably the processes of the black chromatophores from which the pigment had with- drawn itself. For, all over fishes that were slightly older similar cells were found, with beautiful branched processes showing white by reflected light, and at the center of almost every one a small mass of contracted black pigment. We have then at the time of hatching what appears to be a clear case of the domi- nance of the F. heteroclitus character ‘presence of red chromato- phores’ over the F. majalis character ‘absence of red chromato- phores.’ Immediately after hatching, however, this state of affairs began to change, for the red chromatophores began to fade, and when the fish were fed well had entirely disappeared in three or four days. When the fish were starved these chromatophores were visible in some cases for several days longer. These pig- ment cells did not contract or die but usually remained well branched and expanded as long as they were visible. The pig- ment, however, faded until it could only be seen in a few of the cell processes, usually lasting longest at the tips of these processes. Then it became practically invisible by transmitted, though still visible with reflected light; and finally could not be made out at all. The possibility that in order to be visible this pigment needs something that it had been obtaining from the yolk but which is absent in the ordinary food naturally suggested itself, and per- haps receives some support from the fact that the pigment faded sooner when the fish was fed. But on the other hand, this rapid fading might equally well have been due to a general accelera- tion of development due to the feeding. An attempt was made to test the matter by feeding yolk but the close of the breeding season prevented conclusive results. 164 FRANK W. BANCROFT 5. Black chromatophores of the lateral line The pure F. majalis upon hatching and shortly before hatch- ing had a series of from 40 to 60 black chromatophores along the lateral line (fig. 25). There were usually two chromatophores to the segment. One of these was near the surface of the fish and expanded in a plane parallel to the surface. The other was situ- ated farther from the surface upon the septum in the frontal plane separating the dorsal from the ventral musculature. This second chromatophore expanded in the plane of this septum. The pure F. heteroclitus at hatching time and before, usually had no black chromatophores at all along the lateral line. Per- haps ten per cent had one or two black chromatophores along the lateral line, and a much smaller percentage had more. But none were seen which had more than ten or twelve black chromato- phores in this place. Upon the first day after hatching, however, 80 per cent or 90 per cent of the fish were found to have black . chromatophores varying in number from 1 to 26 and averaging about 8. During the next few days this increase continued until all the fish had from 20 to 30 black chromatophores along the lateral line. The F. heteroclitus egg hybrids were on the whole similar to the pure F. heteroclitus, but exhibit a slightly intermediate con- dition as the black chromatophores begin to appear on the lateral line a little earlier than in the pure F. heteroclitus. Thus at hatching time the hybrids usually had enough chromatophores so that above the anus, when expanded, they made a continuous line of black, with scattered black cells posterior to this region. Fig. 26 represents part of a fish of this kind, in which, however, the chloretone given to quiet the animal has caused the chroma- tophores to contract slightly and thus break up the continuous black line which was originally present. In the same series a comparison of the lateral lines a few days after shows that at this time also the hybrids maintained their lead in the develop- ment of the black chromatophores along the lateral line. At that time ten of the hybrids averaged 29.2 black chromatophores to a HEREDITY OF PIGMENTATION 165 lateral line with the extremes at 21 and 34; while ten of the pure form averaged 18.6 with extremes at 3 and 29. In this series the F. heteroclitus egg hybrids hatched at the same time as the pure F. heteroclitus so that these comparisons were made at the same age as well as the same stage and the difference in favor of the hybrids cannot be due to their greater age. In the F. majalis egg hybrids the black chromatophores on the lateral line behaved quite similarly to those of the F. heterocli- tus egg hybrid. But as these hybrids usually never do hatch a precise determination of the hatching time could not be made. We have then so far as this character is concerned a well marked difference between the two species at the time of hatching and an incomplete dominance of the F. heteroclitus condition in the hybrids; but since the stages before and after hatching have not been sufficiently studied it cannot be told whether both species go through exactly the same series of changes, merely differing in their rate, or to what extent the hybrids are intermediate between the two pure forms. 6. Distribution of yolk chromatophores At the time of their first appearance both kinds of yolk chroma- tophores in the pure F. heteroclitus and the F. heteroclitus egg hybrids were found to be distributed over the whole surface of the yolk, and the region opposite to the embryo had quite as many or nearly as many chromatophores as any other region. In the pure F. majalis, on the other hand, almost the whole of the yolk hemisphere opposite to the embryo was free from chro- matophores; and it took a number of days before the migration of pigment cells into this region became noticeable. The F. majalis egg hybrids presented an intermediate condition, for most of them had a small chromatophore free area in the region opposite to the embryo, and the others had fewer chromatophores than usual in this region. Figs. 11 and 12 show something of the differences between these last twoforms. Later on the migration of chromatophores filled these empty areas with pigment cells and obliterated the difference between the various forms. 166 FRANK W. BANCROFT 7. Rate of development of yolk chromatophores In the pure F. heteroclitus the black yolk chromatophores first appeared when the heart was beginning to beat and before a cir- culation had been established; also before the fore-brain had acquired any lumen. The embryo had about twelve somites. The red chromatophores could usually not be seen until the next day. In the F. heteroclitus egg hybrids the black yolk chromato- phores also appeared at the time when the heart was first beating and before the circulation had started. In this form, however, the heart-beat and circulation started a little later than in the pure F. heteroclitus. At this time the fore-brain of the embyro usually had something of a lumen (condition was intermediate between figs. 1 and 2). Accordingly in this form the first appear- ance of the yolk chromatophores was later in time and also at a later stage in the development of the embryo. In the F. majalis egg hybrids the yolk chromatophores did not appear until after the circulation was established, and until the embryo had a large lumen in the fore-brain like fig. 4. In the pure F. majalis the yolk chromatophores appeared twelve to twenty-four hours later than in the F. majalis egg hybrids, at a time when the embryo was in a stage about half way between those represented in figs. 4 and 5. Thus it is seen that both with respect to the time, and with respect to the development of the embryo, the hybrids had rates of development intermediate between those of their parent forms, and there was no indication of Mendelian dominance. The dis- covery, however, of factors which necessitated a Mendelian inter- pretation of the development of the head pigmentation, which, at first sight appeared exactly similar to this case of yolk pigmen- tation, makes one suspect that more study may result also in a Mendelian interpretation of the rate of development of the yolk chromatophores. Although in this case the intermediate position of the hybrids and the lack of dominance is most evident, the same phenomenon is seen to a less degree in the development of the black chroma- HEREDITY OF PIGMENTATION Loa tophores of the lateral line, and the arrangement of the black yolk chromatophores, where incomplete dominance is associated with differences in rate of development. Newman’s results also point in the same direction, though he does not mention this con- trast between the absence of dominance in characters connected with the rate of development and the presence of dominance in other characters. Thus most of the characters which Newman investigated were concerned with the rate of development and in most of them he found ‘blended heredity.’ Thus we see that in general characters connected with the rate of development show blended heredity, and it may be that such characters are so intimately connected with extra nuclear substances such as the yolk that complete dominance is not obtainable. LATER DEVELOPMENT This later development concerns only the two parent species and the: F. heteroclitus egg hybrid for the F. majalis egg hybrid was never found to live longer than two days after hatching. Newman found that none of this form hatched; but in these exper- iments, usually a few fish hatched in each series, perhaps a dozen in all. In all of these the hatching seemed premature, the yolk sac had not been absorbed, and the fish died a little later. In the other forms the chromatophores began to contract shortly after hatching, probably because they then came under the influ- ence of the nervous system. As the amount of contraction varied with the environment, and with the condition of the fish, close comparison of color patterns, and of size and shape of the chroma- tophores was no longer possible. The motions of the fish, for it was usually not safe to risk narcotization, also made exact comparisions difficult, I think, however, that it may be safely said that the only changes that have taken place since hatching are all in the direction of making the three forms more like each other, until at the present time, three months after hatching I can find no characters which will distinguish any one form from the other two. They all have developed much more pigment of the black and greenish yellow kinds, especially in the dorsal region; 168 FRANK W. BANCROFT and they all have developed from three to six or seven trans- verse black bands due both to an increased number of chromato- phores and an increased expansion of the chromatophores in the region of the bands. Between the bands there are at present usually not more than one black chromatophore to the scale, while in the more pronounced bands there may be as many as four or five chromatophores to the scale. The present appearance of the fish is much like that of the females of F. heteroclitus shortly after the breeding season, when indistinct transverse dark bands may be seen. CHARACTERS OTHER THAN THOSE OF PIGMENTATION As regards the rate of the development of the embryo my obser- vations confirm those of Newman on most points. The devel- opment of the F. heteroclitus egg hybrid was slower than that of - its maternal parent; and the development of the F. majalis egg hybrid, during the early stages was faster than that of the pure F. majalis. After hatching the F. heteroclitus egg hybrid seemed more vigorous and grew faster under like conditions than either of the pure forms. The failure of the F. majalis egg hybrids to develop well during the later stages seemed to depend primarily upon the poor diges- tion of the yolk in this form. The first considerable difference that could be seen between the pure F. majalis and the F. majalis egg hybrid was that in the hybrid the yolk was not digested away from under the embryo as rapidly as in the pure form. A result of this appeared to be that, when the heart was forming, the vesicle ’ underneath the embryo was very shallow and the normal anterior curvature of the head did not take place (compare figs. 28 and 29). These two factors seemed to be responsible for the fact that when the heart first began to beat in the hybrid it was closely pressed to the ventral side of the embryo (fig. 28) and did not extend across the vesicle making an angle of nearly 90° with the embryo as in the pure form (fig. 29). Consequently from the very first the heart in the hybrid was much stretched and on the next day when the circulation had become established the heart HEREDITY OF PIGMENTATION 169 in the hybrid was much longer, narrower, and less efficient than in the pure form. In the hybrid the heart never did get over this initial handicap but was stretched farther as development proceeded until finally shortly before the embryos died it had assumed the appearance shown in fig. 30, and at each beat was propelling only a very small amount of blood through the vessels. It seems then that a slight retardation in the digestion of the yolk led to such an increase in the distance between the points of attachment of the heart to yolk sac and embryo, that the heart could not grow fast enough to catch up, but remained permanently disabled. SUMMARY 1. While it had been generally assumed that in hybrid embryos the inheritance was either maternal or paternal, Loeb, King and Moore have ealled attention to the fact that for the hybrid sea- urchin embryo we find dominance of individual characters as in the adult. For Fundulus hybrids Newman found a dominance of a few individual characters, but usually found the characters inter- mediate between those of the two parent species. In this study, which is concerned mainly with the pigment characters of Fun- dulus heteroclitus, F. majalis, and their hybrids, dominance of individual characters has been found in most cases, as in the fol- lowing characters: a. The character—presence of many large red yolk chromato- phores (F. heteroclitus condition) is dominant over the charac- ter—presence of few small red yolk chromatophores (F. majalis condition). _ b. The size and shape of the black yolk chromatophores of F. heteroclitus is dominant over the size and shape of these same cells characteristic of F. majalis. c. The presence of a first crop of head chromatophores appear- ing before the majority of the head chromatophores (I. hetero- clitus condition) is dominant over the absence of this crop of head chromatophores (F. majalis condition). d. The presence of red chromatophores along the lateral line at hatching time, or shortly before it (F. heteroclitus condition) THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 2 170 FRANK W. BANCROFT is dominant over the absence of red chromatophores at the same time (F. majalis condition). 2. In all of the above characters which concern mainly the presence or absence and not the time relations of the pigment characters the dominance is very evident, though often to a certain extent incomplete. 3. In the following characters, however, which are mainly concerned with the time relations the dominance is much less complete, or wanting altogether. a. At hatching time F. majalis has a row of 50 or 60 black chromatophores along the lateral line, while in F. heteroclitus there are usually no chromatophores on the lateral line until hatching time when they begin to appear and gradually increase in number. The hybrids are intermediate, having at hatching time about 15 or 20 black chromatophores on the lateral line, and developing additional cells more rapidly than in the pure F. heteroclitus. b. When the yolk chromatophores in F. heteroclitus first appear they are evenly distributed over the whole yolk sac; while in F. majalis they are absent from the yolk hemisphere farthest from the embryo. The F. heteroclitus egg hybrid is like its maternal species, while the F. majalis egg hybrid is inter- mediate having, on the side of the yolk sac opposite to the embryo, a small area in which the chromatophores are either absent or fewer than elsewhere. . ec. A more perfect case of blended inheritance exists, as New- man has already shown, for the time of appearance of the yolk chromatophores. In F. heteroclitus these cells appear much earlier (both with respect to time, and with respect to the stage of development of the embryo) than in F. majalis. In the hybrids the time of appearance of these cells is intermediate, but each hybrid resembles its maternal more than its paternal parent. 4. It appears then that the presence of certain pigment char- acters dominates over their absence or lesser development; while for the time relations of these pigment characters blended hered- ity holds. This difference may be fundamental or due to an incomplete analysis of the time relations. HEREDITY OF PIGMENTATION 171 5. A similar ease of blended inheritance, described by Newman for the time of first appearance of head pigmentation, was found to be actually a case of the combination of two crops of head chro- matophores. The second crop appears in both species and hy- brids The first crop is present in F. heteroclitus and both hy- brids and hence its presence is a dominant in the Mendelian sense. 6. Immediately after hatching the characters which have served to distinguish the four forms begin to disappear so that after a few months both pure species and the hybrids look practically alike. . All camera drawings from living fish. The opaque red chromatophores are figured in red. Dotted lines indicate blood vessels. H=heart. Fig. 1 Pure F. heteroclitus, four days old, showing first chromatophores. S< 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. * 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 vas Fig. 8 Pure F. 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 25. Fig.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. X 25. Fig. 11 F. majals 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. X19. 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, oppcsite to the embryo, where the blood vessels have just become established. 114. 174 SI On HEREDITY OF PIGMENTATION ] 18 19 Fig. 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 F. 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. > 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 F. 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. 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. I14. 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. X 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 Mee ee eee 7 F ee, 26 fa Re AS 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 lateralline, 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. 25, Fig. 26 F. heteroclitus egg hybrid, sixteen days old, just hatched. S...S line of chromatophores under dorsal skin, partly contracted by the narcotization. N...N chromatophores on nerve core, partly contracted; L...L lateral line. All tite red chromatophores contracted, bisick Ghiomenoonores asta: 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. Chrcematophores on these last two vessels drawn in from another uncontracted fish. 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 sueh 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. 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 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 5 4 Gaedeane 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 furc- tion is to ensure the maintenance of organic 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, 2FOCarclite ps 20s 3 Comparative Longevity, 1870, quoted by Romanes, Monist, vol. 5, p. 168, 1895. 4 Monist, vol. 5, p. 163, 1895. ® 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 func- tion of strengthening the power of endurance of the species by ‘he 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, physiologieally 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. 1OTOC, Cite, Da Love 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 | | | | | 2 ne) 8 3 D D a) & A 5 5 i} | 3 Ce S ae 28 an a BUA LOT a 40 zo cS ano) a < a < a ee se) S ne 5 a a = < < g 2 S| ge | 36 3 3 za = p = p < < 4 4 2 | | S. cecropia. 1910. | | | St. Louis (early). .| 17.40 17.67) 14.83 16.80) 15.77) 17.29) 17.59) 15.79} 16.65 S. cecropia. 1910. | | | St. Louis (late)... .| 10.62) 10.52) 9.46 10.50. 9.90 10.51 10.56 9.76} 10.14 S. cecropia. 1911. — | | | | Sty lowiss.)...9.- 7. 52\) 7.738) 6:96) -7.90| 7-20) 787-70) 7, 71) 771 S. cecropia. 1911. | | | | ING wa Orkerr sete 7.90| 8.54) 6.62] 8.25) 7.25 8.42) 8.37) 7.65) 8.06 S. cecropia. 1911. | | Ineubatoneces. 2-4) 8.16) Sad) (7.03 8.80 7.40 8.48 SA BW S00 S. cecropia. 1911. | | | ees Boxe scenes. 4 | | 17.58} 19.39] 18.60 C. promethea. 1911.) 3.74) 4.21| 5.18} 6.54) 4.51} 4.91) 4.13) 6.21] 4.82 T. luna. 1911........| 4.60) 6.07] 6.60] 5.80] 5.60, 5.96 5.86, 5.96/ 5.90 T. polyphemus. 1911. | | | 5.33] 9.29] 6.79 Table 1 brings together the means of all the lots of material, and will be referred to frequently for comparison. 2 Trans. Acad. Sci. St. Louis, vol. 19, pp. 21-48, 1910. 184 PHIL RAU AND NELLIE RAU The St. Louis Cecropias will be discussed in detail, and the other groups will be taken up later only for comparative evidence. The mean duration of life of all the St. Louis Cecropias (under normal conditions only) for the three years was 10.61, 13.73 and 7.71 days. We are at once struck with the great variation, for in so brief a life a day is as a decade in the life of man. If now we can detect the reasons for these variations from year to year in the life of the population, it may lead us toward the discovery of the factors controlling the duration of life of the species. In 1910 notes were based upon 178 insects froni the 205 which emerged, (101 males and 104 females). Hardly was the work begun when a marked difference in the date of emergence was observed. The insects of that year began to emerge on April 13, a month earlier than in the year before or after. Table 2 shows at a glance the marked correlation between the date of emergence and the duration of life of the animals; those which emerged early lived distinctly longer lives than those which appeared late in the season. The duration of life of the entire population varies from 5 to 25 days. The table clearly shows how all of the early emerging insects segregate to the long-lived lot, while those emerging late in the season fall under short lives. In fact, May 14 seems to be a distinct dividing line between the early emerging or long-lived groups, and the late or short-lived group. The late lot, taken separately, has practically the same dates of emergence and duration of life as the 1909 population. It certainly seems remarkable that the population should split up in this fashion; the problem is most perplexing. The ques- tions at once arise in our minds: Is long or short life hereditary? Is it regulated by climatic conditions? Are these results due to local conditions, and would the same be seen in material from other localities and in other species of the same family? Has each organism an ‘“‘allotment”’ of a certain number of days, i.e., from the time of the fertilization of the egg to the death of the adult, and is a longer or shorter period in one of the early stages correlated with a shorter or longer life in the imago? A cause for this early emergence is very uncertain to deter- mine without tracing the duration of the different stages of the whole life eyele of each insect. But the fact that this abnormally LONGEVITY IN SATURNIID MOTHS TABLE 2 185 DURATION OF LIFE IN DAYS EMERGENCE DATES OF 5.5 6.5 7 7.5 1 _ a3 ap) he! fo! pel he} fel fe} pe} fel fel fe! he! ho 00 ]00/o2 | 3}S]S fy lea lea /e0 |00 ft Ht fd 0 cS | Joo lS lalaslolo Pee Feel Reh Real hea AM tal Ih HU RN a hE He UE US HOS 1 1 1 | 1 | 1 1 2) 1 Y1 2) 1 1 1 1) 2) 1 1 1 1 PA) PA) al 1 yah 1) 1 2 2 4) 1) ah) i 2 1 1 1 LA 1) 1 TH 2 3 1 1) 1 1} 1) 2) 2) 1 1 2 2 1 1} 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 2 1 1 1 1 1 1 2 1 1 1 1) 1 lj 2 2 1 1 1 1 1 alate atiat 1 2 1 yj 1 1) I 2 1 A) i 1 1) 3 1 1 1 1 aT 1 Soc oceaoee ooo ose soso 9} 8 6) 8) 5} 7| 1)13) 8) 9) 6) 6 3) 4] 5) 4) 5) 6} 7 4) 4) 5] 6) 4) 3 | 21 21.5 22 22.5 | 23 | 23.5 | 24.5 [25 RRR co | le i or orN ~eChs) fo olan) me bob Re 2/178 186 PHIL RAU AND NELLIE RAU early emergence followed the exceptionally warm month of March at once leads one to suspect that the high temperature at that stage of their pupal development may have accelerated growth. The mean temperature for March, 1910, was 57.5° F., as contrasted with 44° and 47° for the other two years, andthe maximum reached was 87°.18 It was noticed that during some of the cold days of April and May the animals showed signs of extreme sluggishness, and while Cecropias in confinement seem to be inactive only during the day, those which were on hand when the cold snap came were extremely sluggish during both day and night. It was thought that the cold had a direct effect upon the duration of life, for when the animals were inactive, little or no reserve nutriment was consumed and this saving of vital energy, which is never replenished, may have prolonged their lives. A correlation clearly exists between temperature and longevity. We find that almost all of the long- lived insects (table 2) emerged between April 13 and May 10. These died at intervals between April 27 and May 29. The aver- age of the daily mean temperatures for this period of 47 days was 57.5° F. The short-lived ones emerged between May 11 and June 17, and died between May 21 and June 28; the average of the daily mean temperature for this period of 49 days was 68.1° F. Therefore the average of the mean temperature was lower by 11° during the time when the long-lived insects existed. Now when we tabulate these two groups separately (tables 3 and 4), we find the mean duration of life of the early lot to be 16.65 days, while that of the late ones which ran intowarm weather, is only 10.14 days. 13 To quote from the Monthly Meteorological Summary of the United States Weather Bureau: ‘‘The weather for March was very unusual. The mean tem- perature was 57.5° which is 3.2° higher than for any previous March and the tem- perature was continuously above the normal except on the 9th, 10th, 14th and 15th. The maximum temperature for the month was 87° and this has been exceeded but once in March in the history of the station. Freezing temperatures occurred on three days only. . . . . The number of clear days, 22, is the highest ever observed in March, and the number of cloudy days, 2, the lowest. The sunshine was 79 per cent of the possible amount and was greatest for March since the records began. All temperatures given in this paper are quoted from these reports. LONGEVITY IN SATURNIID MOTHS 187 The desire to test further the influence of temperature upon length of life led us to (1) the Incubator Experiments, and (2) the Ice-Box Experiments. Early emerging, long-lived Cecropias, 1910 TABLE 3 DAYs | ey CLASS | fo] ho 19! fe! he| 19 | 19| fol fe} pe} fe! fo] fo] ho {2 MEAN S| 0 ea eS Sa at A iu Vol Mated @s............ 1 Hs lal 2 | a {al 2tal | | | fal {ala} {asl 17.40 Unmated o’s......... 1 21 | a 4 | | 3 3) 1) 1 4) 3) | 2 a 3 2) 2 a af | a) | 1182) 17.67 Meiied @iete-ceseecceeee| f(t a21 ol] d) Bes inate} ay 2h | Al si a a | |26| 14.83 Unmated 9’s......... 1 1} | 1 | 1) 2) 1) 2) 1 2) 3) 2 a2)" la} 2) 1) | 1/25) 16.80 All mated insects ......| 1) 1} 1) 1) 3 1 1) 1 3} 3} | 3} 2} 1) 2 | 3} 1) 4} 2) 3) 1 | 1) 1) 41) 15.77 All unmated insects... 1) | 1/ 1) | 3) 1 1) 3 2 2) 1) 3 3 3 4) 3) 3) | 3) 3 3 3 2) 1) 1) 2 2 2157] 17.29 INE US ee } a) | a} | a} 3} a | aja] | 2a al aaa 14/3 3 2 2) 1 2) | 2/4) 147 17.59 NOS Ee ae oe 1) 1} 4] 2} 2 1) 1] 2 5} 4] 2) 2) 2) a} 4! 3} 3} a) 3 1] 3} a) a) | | | 2 a) | 1151] 15.79 Whole population .... 2| 1223 4 2) 2) 6) 5) 2) 4) 5) 4 5 4 6) 4) 4) 5) 6) 4) 3) 2) 1) 2) 2) 3) 1 2/98) 16.65 TABLE 4 Late emerging, short-lived Cecropias, 1910 DAYS | GUNES ho} |rof aco 19| fo) fro) fro] fro] fre] fro) fo] fo) fro a [PES eee carRercrecesseeessseseses| 9 es el ee RR | | | | Menace ree eh te | fa} fal | [aaa 4 [3 1| 18 | 10.62 Wmmieated GUS. sstewcses ss ear ne 2 koi eLe 12 RS eaiesiieg| He e200 TORS 2 Mattes Ocaae ai egies: 1) | 3i a} a} | taal at a ala) | of atal fal | 1} | 30| 9.46 Wramated® Q’s\ ss tee. UES oe] | teat lee 1 1) | 12) 10.50 All mated insects............... | 2} | 3) 2) 2) 1) 2) 4 5 5] 3 3} 1] | 6 2) 4) | 1 1| 1) 48) 9.90 All unmated insects............ | | 2) | 5) 5 1) | 3) | 3) 1) 3) 43 1 1) | 32} 10.51 J CR ee ee | 1 1) 1) 1) 2) 3) 3 2) 4 1) 3) | 7] 2 6 | 1) 38 | 10.56 IIT Coli ie ae ee ee nee | 3) | 3) 1) 3) | 5] 6 3) 3) 2) 2) 1) 1) 2 4} a) | a) 2) | 42] 9.76 Whole population...............| 2) 3 2 4| 1) 7] 9) 6 5) 6| 3) 4) 1) 9 67 1) 1 2} 1) 80} 10.14 INCUBATOR EXPERIMENTS, CECROPIA 1911 After the work of 1910 had given a clue that the climatic con- ditions may have been a factor in regulating the duration of life, it was thought that if by some method the cocoons could be con- trolled so that the imagines could be gotten in January or Febru- ary and subjected to the climatic conditions of that season, some interesting results would be obtained. Some 300 cocoons were placed in an incubator on September 20, the temperature of which was regulated to about 70° F. 188 PHIL RAU AND NELLIE RAU (about the mean temperature for the period in 1910 when the moths emerged). Up to the 10th of January, nothing had emerged. At this time a living-room was obtained with a fairly even temperature. Up to the middle of February no results were obtained. It then became necessary to remove the entire lot to the basement, the temperature of which, while not recorded, was moderately uniform and distinctly higher than that out-of- doors. They were sprinkled with water at intervals of a few days. These details would not be worth recording but for the fact that it was expected that under these conditions the insects would probably emerge somewhat earlier than the normal time. On the contrary however, the 162 insects which emerged (72 males, 90 females), left the cocoon between June 5 and July 9, the latest period yet met with in Cecropia work. This lot was gathered early and placed in the incubator very soon after pupation, while probably some of the cocoons still contained larvae. It seems that the constant even temperature conditions at this stage made the animals lethargic and indiffer- ent to the normal development, when only a year before an unu- sually warm March had probably caused many to emerge sooner. The warm March of 1910 caused an earlier emergence, but warmth was furnished to the insects at a period of their develop- ment when they were susceptible to its accelerating influences, but when it was given them at an early stage of their pupal devel- opment, counter results were obtained. Can it be that they spun their cocoons in preparation for the cold of winter, but just about the time or even before they had left the larval stage their summer (i.e., high temperature) was resumed so they lacked the stimulus (cold) to start them promptly in their pupal development? If we had reason to believe that the first animals to pupate are the last to emerge, this would easily explain in part the lateness of these in emerging, for they were all gathered very early in the pupating season, while many caterpillars were yet to be seen. We cannot believe, however, that this can be a full explanation for the phenomenon, for the lateness of these toc far exceeds that of any others observed from the same region, either in cap- tivity or free. LONGEVITY IN SATURNIID MOTHS 189 Another interesting feature was the degree of pigmentation displayed by these insects. While no measurement was made of the color or its distribution, the general darkness of this lot was clearly evident. Out of the 162 insects which emerged from the incubator lot, records were kept on the duration of life of 112. The mean length of life for this lot compares well (table 1) with the results from the New York and St. Louis 1911 material. The following table gives the details of the duration of life of this lot. TABLE 5 Incubator Cecropias DAYS CLASS it aes l (ldenat a oe ; | T. |MEan £9) 05 WIG NE 68) 9 | 20H) Lie) 12 esa as le | | } Metiedeis Se... hid caay st eee et |! Bh oc RG 4 20 | 8.10 Whamiated UB: ca2// 3 bnes yose cowie eden ee) ie alee Uy ines es Wee oe 39 | 8.31 Werpeclk Ons ee een fee nee ee ecg (Pty; Sul Os| ye Our 2418 Fe ihe eB ees fn ated yQ.'S- waste onc bones vie a [Norte asa a5 2} 1] 2) 4 | | 20 | 8.80 Allimated imsewpsi....2.<- 628. -5--%-| 1 5/1318) ACTEM RT at | | | 53 | 7.40 All unmated insects.................. | 4| 5] 8/44) is] 6) #] 4) a) | | 59 | 8.48 NU a eee | 5 T| 8/12/14) 4] 7) 2| | | | 59 | 8.24 INI 69, cereal ear ee be eae re ected bata t/a [as 5/13] 2] 1] 2] PT SES ania Misuleipopulationy: co... --.4e.chn- 4) 2) 9) U8 20 17 [27 Co) ae a | 112 | 8.00 | | | | ICE-BOX MATERIAL, CECROPIA 1911 Finding it impossible to get the incubator material to emerge during the winter, and wishing to ascertain definitely whether the duration of life is influenced by low temperature, a number of cages containing two insects each were placed in an ordinary household ice-box, the temperature of which varied from 9° to 12° C., and on a few occasions 15°. Forty-two insects were used in this experiment, and the duration of life varied from 6 to 32 days. Had the temperature been uniform in all parts of the ice-box, the 18 insects which lived 13 days or less would probably have lived longer. As it was, most of those which were kept on the top shelf nearest the ice compartment lived the longest. Corre- lation table 7 shows that their contemporaries lived the normal number of days. Lack of facilities and material made it impossible to make more extended observations. This number, however, gives suf- 190 PHIL RAU AND NELLIE RAU ficient evidence that long life in this case is a matter dependent upon climatic conditions. TABLE 6 Ice-box Cecropias | DAYS CLASS Th, Aa) celle jaan irae al RTT ATV eV | fe) | MIEAGN | 6 7] 8 9 10 11 121314 15 16 17 18 1920 21 222324 25/26 27 28 29 303132 ee eS LA eg I et ae PS PP fA de | || Res | | ‘| | EAE eric 3. gure earee ANN GM RR Ph | 1} 2} 1) 1) 1] | 1/2 1) |) 1 4) ae anes Bemaleqeecs sth bss. || a3) | |") fa a ls F At dle 18 19.39 SAN ee Me ae dae | 1 | 32, 338 | [2 2 EEEEEE 4) 1) 1} 42 | 18.60 | | | | | | This ice-box material compares well in the duration of life with the early lot of 1910, but the fact must not be forgotten that in spite of being kept in the ice-box at a temperature of 9 to 11° C., the insects were not so sluggish as they were during some of the much colder days of 1910. Could the refrigerator have been properly regulated, no doubt a greater period of life could have been attained. That the animals were far from inactive was evident from the worn condition of the wings. Copulation and oviposition also occurred while under these conditions. The activities of these may not have been normal; still the profound sluggishness which was observed during the cold spells of the year before did not occur. ST. LOUIS MATERIAL, CECROPIA 1911 The object of this work was to see if the population would split up into long- and short-lived groups as it did in 1910. This lot comprised 339 insects, 171 males and 168 females. Notes on the duration of life were made on 283 of this number. They emerged between May 8 and June 14. The 1911 population was tabulated in a correlation table (7) similar to the one for 1910. These emerged at the same time of year as did the late group of 1910, and the duration of life was practically the same. A chance break in the continuity of emer- gence (fig. 2) is probably due to the drop in temperature during those few days. This is also true for the break of only one day, June 7. LONGEVITY IN SATURNIID MOTHS 191 TABLE 7 ay E § is DAYS ag == — <8 | | | | As| 3/ 4) 5] 6| 7| 8 9 10 | 11 | 12 | 13 | 14 5-8 | 1 ha 9 ity | 2 10 itl! a) Bi } | 8 11 | 1 | AA aul}. iat | 11 12 | 1 1 | 2 13 CU OR ate eh aed) al ie al 14 14 Hat Re Sah Sani 1 12 15 2 oF ath al 6 16 cAlie | 4 17 2 ait 5 18 1 | 1 19 1 1 | | 2 20 | 21 | 22 23 eae 24 3 | 2] 2) 9 9 25 1 | | Hp! 26 1 peal 1 | es 27 tI) Blt at | 8 B3 | at a Th Te) Yeh) aw | 13 29 mil a4]. 8 ta 6 30 | Qi ol) A She eh Bal a | 18 31 Sh ieeaa les Sale SulinG | 25 6-1 Ti) a) GI B® | 12 2 Rl it) Ql ws ly 15 oiitesa 6H] MSile 2 ey |) L2Nne2 | 33 aid Billeesi|y eal 2) || 1 | 25 5 2 | 2/2) 2 | 8 6 Ml) Bl atl) & | 2 | 12 q | Io a Se) Wil Uy) a) al, sl 1 | 9 Ole 3 1 1 | 1 7 10 1 | 2 3 11 3 10 nets | stelle 7 12 | 13 14 2 2 aS SN SE 0 # e 6| 6| 32) 29] 56] 60 41 34/10) 4] 4] 1] 283 In comparing the means for the different classes of this lot (table 1) with that of the late emerging group 1910, we find the duration of life shorter in all cases in 1911. Since this period was warmer in 1911 than in 1910, this only adds one more bit of evidence to our temperature hypothesis. A comparison of table 8 with table 1 will show how in every case a variation in the length of life accords with a simultaneous variation in tem- perature. 192 PHIL RAU AND NELLIE RAU TABLE 8 Mean temperatures 1969 1910 1911 IMamehe ot aes ae 44 58 AT PACT Tell Denar eeey ee acai Sear 54 56 54 INTE G ches Se a ei CO, 64 61 71 75 | 72 79 TINT Psy eee ae te te Thus the warm March 1910 brought forth the insects at an abnormally early date; a warm May and June 1911 was asso- ciated with shorter lives of the animals, while for the same period in 1910, a slightly longer duration of life was associated with the lower temperature. It will be seen that none of the animals emerged at an abnor- mally early period and that none lived an unusually long number of days. The table below gives further details on this lot. TABLE 9 St. Louis Cecropias, 1911 DAYS CLASS Sd = me , a, aris Sa a6 | 7 | 8 | 9) | 100) 11) 22) 13} 14 WW icnrol etic Gnaspacecene ae PaeAcao Mock 1 4 | 3 An) Govan! | 21 eae Winmateditoustconss cece a eeere see) One eal kOe Lon ic One 24al neo om eo mere i) 121 } 7273 Misaite di QUsis oe corde sfatstatcisie alert stanpeeaters Cale elt LO es tee: 1 | 28 | 6.96 Unmated Qs Foes Seo De aU TSE aL) ey 3 a) we) DN A) SSO 1) e600 Alllomatediimsects: 2 ey) ase ce ei -ecul V1) Sy 10!) SOs} 5; | 49 | 7.20 Alllinimmatedhinsectsas see eee see sae 5 | 6 | 21 | 24] 46 | 52] 32] 29] 10|| 4) 4] 1 234 | 7.81 UN bkopht Shas c a cia etna tite eee Ont tn, = 4| 4/ 12) 16) 26) 28 | 26 | 19 | Dales | 142 | 7.70 AED is rice aes rapere leceenteecs oe lol ete: = eee 2} 22) } 20))| 13/30), 32)) 15) 155), Sy) 2 | AS 141 real 6 | 41| 34| 10] 4| i Wiholeipopulationce..-casee eno oeeeea nO wo to i) © | on for) far) =} rc — ww NEW YORK MATERIAL, CECROPIA 1911 To ascertain just how foreign material would compare with St. Louis material, both in time of emergence and duration of life, 200 Cecropia cocoons were procured™ from Queens County, Long Island, N. Y. These arrived during the latter part of March, and between May 15 and June 3, 139 imagines emerged, 79 males and 60 females. Notes on the duration of life were made 1 Prom the American Entomological Company, New York. LONGEVITY IN SATURNIID MOTHS 193 on 133 of these. The mean duration of life of these compares well (table 1) with the data for the St. Louis material for the same year. ! TABLE 10 New York Cecropias DAYS | CLASS — | T. |MEAN 3 | 4 5) 6/7) 8] 9 10 | 11 | 12| 13 | 14 | Matedeirctsts ty. (eae e mame aa relate eval feral eae | 4 20 | 7.90 Ummnatediot eine ee ce ere are-| Da eats Salil oa) 6) || 02) | 4 ase al 56 | 8.54 MamedbOuses Start coMseac gh eee 1| Dural <2) 8 iaeth | ea 21 | 6.62 Wrmeitods Oana stae/ inno he koh afel Bora Sl |) We leet [te 36s ie8e25 Aulimvatedbinsects:eae. ase sees eee: | a) Bhai ksh LET yell) i) 2 } al | AL 725 All unmated insects...........-...... PHY 2) BS) Pei exob |) ka penile fealisss| iy ti) 928) \ (8342 AMIBCUBS, Potente Pesan ke ade WN: Di 25) a 11510) TM) IPTG Ih by pat eon eZ Ba eS AOU S Seincrasins site aie syey2 aiden icte cn > 2 #1) 15.) 1G V8) 16 aaa et YY) GP GE Wiroletpopulation: ascent osm fy 4 err eeenie es |leOnt Loui wedaul) div) | DALE 210 pea les 1} 133 | 8.06 CALLOSAMIA PROMETHEA 1911 To compare the longevity of the Cecropia moth with that of others of the same family, some 300 cocoons of Callosamia pro- methea were obtained from Créve Coeur Lake region, St. Louis, early in the spring. These brought forth 183 imagines, 116 males and 67 females. .Notes on the duration of life could be made on 170 of this number. It will be noticed (table 1) that the mean duration of life varies greatly in the sexes, and that they do not attain the age reached by the Cecropias. TABLE 11 Prometheas DAYS | | CLASS : SEE ee RTE | NTA Be | 53> Macalsalag em seleg) adon | aa | TERA ey ence eee ee a oe NP OU G) | Greate | 1} 19 37 ORTON GLH ceo es 6 20/28/2041 9 3 90 | 4.21 Marted Ol see ne ae ees eo ee te Lae Gl i Se) By, GL) 22 | 5.18 Wramated soars cere Nasr, Fete 8 OMIM eal) OUEST Gh la 39 | 6.54 Allgmatedsinsects: fens ce. eee tac. cess 2) 15 | Sal Gi] Shi) ZEN ae) al ip) A eae oil Alitummatedunsectse- eee arrest 8 | 22] 30| 26] 17) 14) 6] 6 129 | 4.91 LU cits plead, ke a 8 | 29/34] 25| 9] 3 | 1| 109 | 4.13 PAIN bS} Acide ie Philosophical transactions, Royal Society, London, vol. 23, 284, 1703, p. 1366. This communication is accompanied by the first published figure of Paramaecium. From the description in the text, however, it is evident that the author at times confused Paramaecium and certain hypotrichous forms. These same figures are reproduced by Baker, in his treatises on the microscope. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 2 ‘ 214 LORANDE LOSS WOODRUFF Depuis l’instant de sa préparation, une infusion change incessamment, et plus ou moins vite, suivant la température; elle montre seulement d’abord le Bacterium termo, puis quelqu’autre Bacterium et le Vibrion linéole, puis des Monades, des Amibes et quelques autres Vibrions ou Spirillum; un peu plus tard, les Enchelys et les Trichodes commencent as’y montrer avec des Kolpodes qui, grossissant rapidement, se montrent conformes au type nommé Kolpoda cucullus; enfin, viennent les Trach- elius, les Loxodes, les Coccudina ou Ploesconia, les Paramécies, les Kérones, les Glaucomes et les Vorticelles, soit tous ensemble, soit séparément; mais toujours 4 peu prés des mémes animalcules, de ceux que Joblot nommait d’une maniére trés-significative les Cornemuses, les petites Huitres, les Chaussons, que Gleichen appelait les gros et petits Ovales, les Pendeloques et les animalcules pantoufles. Le nombre en est assez restreint, et c’est & peine si les quinze genres que nous venons de citer fournissent en tout quarante ou cinquante espéces. Si les infu- sions sont conservées pendant longtemps, elles changent tout 4 faitde nature; pourvu que le fiquide soit en quantité suffisante, la substance mise 4 infuser devient un sol sur lequel peuvent se développer des végéta- tions, ainsi que sur la paroi du vasc; si la lumiére est assez intense, on observe méme des végétations vertes; alors, avec d’autres Infusoires on peut rencontrer dans les liquides des Systolides st des Diatomées.® It is obvious, however, from the preliminary experiments out- lined in this paper in regard to the origin of protozoan fauna of hay infusions, that the Protozoa which appear, when laboratory water is added to ordinary hay, are insufficient in variety to ren- der their study profitable from the standpoint of the sequence of forms, because, to determine a sequence of any general interest, it is necessary that a large number of species be present initially so that the dominating forms may be selected for particular study. It would clearly be easier to work out the sequence of forms en- eysted on the hay, but by doing this a sequence would be obtained which would represent merely that of a special group of forms and this would obviously vary more or less with each lot of hay. Again, since paramaecia cannot be secured from dried grass, this form would not appear in the series. It was necessary then to employ other means of making up and seeding the infusions, so that there would be no doubt but that all the more common protozoan forms were present at the begin- ning. It was also necessary to start as many infusions as could be carefully studied simultaneously, in order to have the record 6 Histoire naturelle des Zoophytes. Infusoires. Paris, 1841, pp. 173-174. PROTOZOAN FAUNA OF HAY INFUSIONS 25 sufficiently comprehensive to rule out as far as possible individual variations and give final results of some general applicability; for, as Dujardin quaintly expressed his own experience with infusions :7 Rien de plus simple que de préparer des infusions et d’y voir se pro- duire les Infusoires; mais rien de plus difficile que d’obtenir des résultats semblables de deux infusions préparées en apparence dans les mémes conditions: c’est qu’en effet les circonstances ne peuvent jamais étre exactement semblables. En supposant que la dose des ingrédients et la qualité de ces ingrédients soient les mémes, la température, l’état hygrométrique et l’état électrique, ainsi que l’éclairage, et l’agitation ou le renouvellement de lair, n’auront pas pu étre les mémes ou varier de la méme maniére dans les deux cas. Or, toutes les causes exercent sur le développement des Infusoires une influence qui, pour n’étre pas scien- tifiquement déterminée, n’en est pas moins bien réelle et souvent bien considérable. A. EXPERIMENTS Twenty-six infusions were made up with nearly pure timothy hay and laboratory tap water. In every case 20 grams of hay and 5 liters of water were put into a glass battery jar with a capacity of about 53 liters. Each was loosely covered with a plate of glass to prevent undue evaporation and the entrance of dust. The jars were situated in a small room with windows on three sides so that all the infusions received practically the same illumina- tion. The temperature was recorded with a maximum and mini- mum thermometer. With this as the general plan, three methods of procedure were followed, giving three types of infusions desig- nated respectively, A, B and C. A Infusions. In this series the hay was boiled for five minutes in approximately 250 cc. of water and then sufficient tap water was added to make 5 liters. This infusion was then ‘seeded’ with 5 ce. of material from laboratory infusions and aquaria rich in animal and plant life. The ‘seed’ used in this series and in the following B series was thoroughly mixed in a flask before being added, so that each was seeded as nearly the same as possible. B Infusions. These were made up exactly the same as the A series, except that the hay was removed from the infusion by “Loc. cit., pp. 170-171. 2G LORANDE LOSS WOODRUFF straining it through cheese cloth. This eliminated all but an insignificant number of the smallest fragments. C Infusions. To make up this set, 20 grams of hay was put into five liters of tap water. It was neither boiled nor strained. A few drops of ‘seed’ was added, thus insuring the presence of all the chief forms seeded into the A and B infusions. The twenty-six infusions were made up at intervals and were designated as follows: April 1st: A-1, A-2, A-3, A-4, A-5, A-6, B-1, B-2, C-1, C-2, and C-3. April 13th: A-21, A-22, B-21, and B-22. April 24th: A-31, A-32, B-31, B-32, and C-31. May ist: A-41, A-42, B-41, B-42, C-41, and C-42. Hach of the infusions existing during April was studied daily from its inception to May Ist. After this date the observations were made for a while at forty-eight hour intervals, and then at somewhat longer intervals depending on the rapidity of change in the respective cultures. The last regular count was made on June 26th, 1909, but since that time up to the present (Oct., 1911) the infusions have been kept under general observation. The methods of study consisted of an examination of samples of the liquid taken from the top, middle and bottom of the jars, and the enumeration of the different Protozoa, Rotifera, Algae, etc., which were present. The liquid was removed from the jar for study with a 5 ce. pipet. The ‘surface’ medium studied was taken from three points in the jar just under the surface film; one at the side nearest to the chief source of light, another at the side farthest from the chief source of light, and the third directly at the centre of the surface of the infusion. The ‘middle’ medium was taken from this portion of the infusion by inserting the point of the pipet quickly to the region, while the other end of the pipet was closed with the finger. The ‘bottom’ medium was taken in a similar manner. In ‘middle’ and ‘bottom’ counts care was exercised to move the tip of the pipet through the re- spective regions in order to get a representative sample. Only one pipetful was taken in each of these counts because of the possi- bility that a few organisms might get into the pipet when it was passing through the upper portion of the fluid on its downward PROTOZOAN FAUNA OF HAY INFUSIONS 217 course, and such error as existed from this would only be augmen- ted by passing the pipet more than once through this region. Various methods were tried to avoid this error entirely. For example, when the study of a sample suggested that possibly some of the organisms observed might have entered from the surface fluid, another sample was taken with a pipet in the tip of which a cork was inserted. When the pipet in this condition had reached the point from which the sample was desired, a wire was inserted through the pipet and the cork pushed out. The pipet, of course, immediately filled with water up to the level of the surrounding infusion and the cork itself rose to the surface. In the great majority of cases it was found that samples taken by this latter method simply corroborated those taken by the more expeditious means, and consequently it is believed that the data secured with the method generally used in the work possesses an error which is negligible. ; After a sample of the infusion had been removed it was imme- diately put into a watch glass and stirred, and then 1 ec. was taken with a pipet and put into a Sedgwick-Rafter counting cell. As is well known, this consists of a glass slide upon which is ce- mented a metal rectangle. The dimensions of the space enclosed by the rectangle is 50 x 20 mm., and, as the metal is 1 mm. thick, when the rectangle supports a large cover glass it forms a cell which has a capacity of exactly 1 cc. The sample to be examined, then, was spread out on the slide to a depth of 1 mm., and pre- sented to view a total of 1000 cubic mm. The contents of this cell was then at once examined under a microscope which was provided with an ocular micrometer so ruled that, with lenses and tube length properly adjusted, a square of the micrometer just covered 1 sq. mm. of the field, and by focussing through the depth of the liquid enclosed by the square, a volume of the sample equal to 1 cu. mm. was under observation. By counting the organisms which were included, during a unit of time, in the 1 cu. mm. under observation, and multiplying this by 1000, the number of organ- isms in the cell could be ascertained. Usually ten such counts, * For a detailed description of the apparatus, cf. Whipple: The microscopy of drinking water, 2d ed. 1910. 218 LORANDE LOSS WOODRUFF each of about one minute duration, were made for each sample and their average taken. This was the general method of observa- tion employed, but in samples in which only a few comparatively large forms were present the number of each species was counted directly under a dissecting lens. Again, in cases in which myriads of the tiniest active monads were present it was impossible to count them satisfactorily and accordingly it was necessary to esti- mate the number present on the basis of the experience gained by the use of the exact counting system. In addition to the ob- servations made with the compound microscope, in nearly every case the sample was also examined with a lens magnifying about ten diameters, in order that a comprehensive view of the slide could be secured which would serve to indicate the general dis- tribution of the organisms on the slide, and act as a check on the more exact observations. Accordingly, while the enumeration of the organisms varied as exigencies demanded, all the counts were made by one person and consequently the personal equation of the observer, which must influence to some extent the data collected from such a series of observations, remained the same. It is believed that the data secured are sufficiently comprehensive to give accurately the relative number and to show approximately the actual number of the various organisms present. It is obvious, of course, that the method employed does not give data which show the presence in the infusions of one or a dozen organisms. Therefore the terms employed, ‘time of appearance’ and ‘time of disappearance,’ indicate simply the presence or absence of a sufficient number of animals to be detected by the method. More than this, I believe, could not be secured without the expenditure of more labor than one individual could devote to it daily for a period of three months. Obviously the rate of development of an infusion will depend upon the temperature to which it is subjected, and, within limits, the higher the temperature the more rapidly the sequence of forms will proceed.’ The ideal way, therefore, to conduct such a series of experiments as these under consideration would be to ° Wocdruff and Baitsell: The temperature coefficient of the rate of reproduc- tion of Paramaecium aurelia, Am. Jour. Physiol., vol. 29, no. 2, 1911. PROTOZOAN FAUNA OF HAY INFUSIONS 219 maintain a constant temperature throughout the work. This was impracticable when the observations were made and consider- able fluctuations in temperature occurred. However, all the in- fusions of the same set were subjected to the same temperature and consequently the relative time of appearance of the different forms in these is directly comparable. As the work progressed, from April to June, the average temperature of the room increased (cf. table 1), and consequently the infusions made later than April Ist, were subjected from the start to higher temperatures than the former. Thus it is impossible to compare accurately the con- TABLE I | TEMPERATURE | TEMPERATURE | | TEMPERATURE (F.) | (F.) | el (F.) DATE, 1909 eS z DATE, 1909 S | z DATE, 1909 | a z Si 5 3 5 = 5 April 4 52 49 April 29 76 70 May 27 |) “71 65 See 9506 eS 30 71 52 | 29 78 | 65 Gay, GO ea May 1 63 60 30 vill 67 iN 72 2 69 | 65 31 73-1 Gn Sa IO) seer al 3 75 | 69 June 1 iG" 70 9 76 62 4 76 67 Br (i778 ll 66 10 | 62 | 52 7 85 | 67 4 75 | 70 Tan | bGee 50 8 74 | 64 || 5 70" |e 67 12) 59) |) 50 9 AP Bil 6 68 66 13° | 60" | 154 10 64 | 60 8 75 | 70 14 | 66 | 58 11 66 | 64 10 74 | -65 Sal 78) ML 162 12 73 | 60 11 79 1S 1Gog2- alk 256 13 74) Gs 12 74 | 68 17 |) 65%) | "55 14 75 | 66 13 Tv | 70 Sie et Toe 15 71) Ome 14 75 | 70 1OMt CS2R 75 16 79 | 69 15 Hom eevee oh 82 Gl 17 725 |-AGs 16 79 | 70 OT al G5 457 18 66 | 60 17 80% | "70 DOANE GT. N61 20 68° | 620i 18 74 | 70 O30 78.265 21 Giaulas os 19 73 | 66 PA | 63..| 53 22 61 57 20 72 | 68 Deh TS. | 51 23 69 | 59 23 S50 ie 72 DGeRIR NGOS ||! 15S 24 TOR wa 25 92° 1-483 D780 ll <54 26: i 78iyh 64en 26) 895) 8S 28 70 59 | | 220 LORANDE LOSS WOODRUFF dition of, for example, the A J cultures at the end of the first fifteen days, with the A JI/J 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 cases 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 papal | 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 Amoeba, 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, 222 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. Loe. cit. PROTOZOAN FAUNA OF HAY INFUSIONS 223 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 cyanophyceae and chlorophyceae flourish, under proper conditions of illumination, several species of Anguillula, copepods, etc., 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 4A, 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 shghtly 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- gressed 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 eycle 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 (ef. 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, ete., in infusions comprising each group as follows: LS) Or PROTOZOAN FAUNA OF HAY INFUSIONS Di 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 JI A-31, A-32 averaged and designated A I/T A-41, A-42 averaged and designated A JV B-1, B-2 averaged and designated B J B-21, B-22 averaged and designated BIT B-31, B-32 averaged and designated B JIT B-41, B-42 averaged and designated BIV C-1, C-2, C-3 averaged and designated C J C-31 designated OP JUUE C-41, C-42 averaged and designated C IV 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 JI/ (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 cc. Their decline was equally rapid and by the 20th day of the life of the infusions none were observed in the samples studied. A ITI group. These forms were the first to appear, reach their maximum of 2000 per cc. on the 4th day, and miminum on the Sth day. 226 LORANDE LOSS WOODRUFF A ITI 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 ce. 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 ce. on the 8th day, declined to 500 per ec. 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 cc. on the 40th day, and reached extinction on the 60th day. B II group. On the 4th day there were 1200 monads per ce., and on the 8th day they had entirely disappeared. BIITI group. In this group the monads attained a maximum of 5000 per ee. by the 9th day, and by the 12thday there were none remaining. BIV group. A maximum of 1400 per ee. 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 ce., 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 8th day, declined rapidly to 2500 per ee. on the 13th day, and reached a minimum of practically zero on the 24th day. CIV 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. FAUNA OF HAY INFUSIONS PROTOZOAN uINO9BVUIBIBg ‘— -— ayy peqyoyd st B ssTosqe (‘egg -d ‘Jo spoyjou jo srejop 1oq) *“— - - — = Bqoomy ! *— — = e]]e0I}404 -——— = epryowjoddéy {—-—-— = vpodjog!:----= peuoyy ‘“suorsnjut oy} Jo 90U04sTx9 oy} Jo SAvp Jo coquinu ay} UQ ‘sdBjAMS oY} 7B “09 Jed suISTUBSIO Jo JoquINU dy} 9}BOTpUI SeywUIPIQ “dnois 7 yp T ‘Sy OL Og OS O 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 ec. 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 cc. 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 IT group. This form was the second to appear and very slowly attained its maximum of 1000 per ce., which took place on the 27th day, then it fell in number to about 200 per cc., rose again to about 500 per cc. on the 44th day, and then became ex- tinct on the 49th day. A III group. Colpoda was the second protozoon to appear in considerable numbers in these infusions, the cycle of the monads being apparently aborted. Colpoda arose abruptly to the great number of 15,000 per ce. on the 10th day, fell to about 11,000 per ee. 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 cc. 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 ce. present. This number persisted to the 17th day, and then a very quick decline ended in the extinction of the form four days later. BIgroup. 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. BII group. Colpoda attained its maximum abundance on the 6th day, then rapidly proceeded to its extinetionon 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. BIII group. In this group of infusions Colpoda rose rapidly to a maximum of 8000 per cc. 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 cc. 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 LORANDE LOSS WOODRUFF 230 f-— — = epodjog ‘:-:- = peuojy = eqooumy { - — — = BTfeomio, $— - — = BpryormodAy ‘SUOISNJUT OY} JO ddU4STX9 oy} Jo SABp Jo Aoquinu oy} poqjo[d sT BSSTOSGBsy}ZUQ ‘soRJANs ayy YB ‘90 Jod SUISTULSIO JO JOqUINU dy} 9} BOIPUT SoPVUIpIC) ‘dnois JJ V 2 sly OOOL 0002 OOO€ PROTOZOAN FAUNA OF HAY INFUSIONS Zou 600 per cc. on the 43rd day, and 60 per cc. 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 ec. on the 32nd day, falling to 15,000 on the 37th day, and to 100 per ce. 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 Al 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 along 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 II group. 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 group. Monad.........|10 10 10 versus Colpodare. 4-4: 0 | O IL (5378) Colpodares- 8 | 6 8 versus | | Hypotrichida.. 3 (A IJ, BIT, BIV) | 4(B), BUI,BIV,CUE)| 2 (A IH, B II) Hypotrichida. v7 | 7 4 versus | Paramaecium. .| 1 (A JV) | O 0 Paramaecium. .| 8 | 7 2 versus | Vorticella...... all (GxAV9) | 2 CARTIER CoTaVa) 0 Paramaecium. . 7 let 0 versus | | Amoeba... | 1e(CLV) le (CeLVe) 6 Viorticellas.... | 7 | 4 (A IT, Al TEE CARE (BT) CIV) versus | Amoeba....... \ouCAGh a Bag CDV). a From the above tables the most frequent sequence for the entire series of infusions is found to be as follows: APPEARANCE MAXIMUM DISAPPEARANCE (1) Monad (1) Monad (1) Monad (2) Colpoda (2) Colpoda (2) Colpoda (3) Hypotrichida (3) Hypotrichida (3) Hypotrichida (4) Paramaecium (4) Paramaecium (4) Amoeba (5) Vorticella (5) Amoeba!” (5) Paramaecium (6) Amoeba (6) Vorticella (6) Vorticella 11 Cases in which both of the organisms being compared attained the condition on the same day are not included. The fact that, both the organisms frequently survived the period of observation (except in Series B) is responsible for the rela- tively few cases included in the third column. 2 The figures for Amoeba versus Vorticella are so nearly the same that the variation is well within the error of the experiments. 244 LORANDE LOSS WOODRUFF A similar analysis of the data of the A, B, and C series of infu- sions separately shows the following sequence: A Series APPEARANCE MAXIMUM (1) Monad (1) Monad (2) Colpoda (2) Colpoda (8) Hypotrichida (8) Hypotrichida (4) Paramaecium (4) Paramaecium (5) Vorticella Vorticella (6) Amoeba i‘ (5) cine B Series APPEARANCE MAXIMUM (1) Monad (1) Monad Colpoda Colpoda 2) ee ee (2) awe a. (3) Paramaecium (3) Paramaecium (4) Vorticella (4) Amoeba (5) Amoeba (5) Vorticella C Series APPEARANCE MAXIMUM (1) Monad (1) Monad (2) Colpoda (2) Colpoda (8) Hypotrichida (8) Hypotrichida (4) Paramaecium (4) Paramaecium (5) Vorticella (5) Vorticella (6) Amoeba (6) Amoeba V. PROTOZOAN FAUNA AT THE MIDDLE OF THE INFUSIONS It is evident, from the observations on these infusions, that the protozoan fauna of the middle of the infusions is meager, com- pared with that of the top and bottom. Practically all the organ- isms which have been observed at either the top or bottom have been found in the middle counts; but either in such small numbers, or so irregularly, as to make a detailed tabulation of the records of little value. Therefore they are not presented here. Bio- logically, the middle of the infusion clearly offers a less favorable environment than either the top or the bottom, and is therefore tenanted chiefly by a free-swimming population brought there by an overcrowding at the top or bottom, and by forms emigrat- ing from the top to the bottom as the cycle proceeds. Naturally 245 PROTOZOAN FAUNA OF HAY INFUSIONS ‘++ — = Bqoouly {+ — — = B][A0I}I0A f — = unnosmeisg {— -— = epiysjodAq { - —-- = epodjop (...+ = peuoy ‘SUOISNJUT OY} Jo voMI4STxO oY} Jo SABp Jo ToquInU sy} pe}jo]d ST BssTOsqV oY} UQ ‘dOBJANS oy} 4B SUISTUBSIO Jo IequINU oY} JUdSeIded soyeuIpIo oy, “dnows PJ] q 1 ‘SW OL 09 O€ | 0 OOO! 0002 OOOE 0008 000g THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 2 246 LORANDE LOSS WOODRUFF those protozoa, like Paramaecium, which are strong swimmers are most frequently found in this region. VI. PROTOZOAN FAUNA AT THE BOTTOM OF THE INFUSIONS On account of the marked difference in the bottom fauna of the A, B, and C infusions, it is more convenient to consider each of these types of infusions separately. 1. A Infusions Monad. The types of monads recorded in the surface fauna were observed in inappreciable numbers at the bottom, so that it is evident that when these forms disappear from the surface their cycle is over. Certain other species of monads appeared irreg- ularly in comparatively small numbers at the bottom, but it is unnecessary to recount them here. Colpoda. In groups I and II Colpoda did not appear at all at the bottom. In group III comparatively few Colpoda (approx- imately one-twenty-fifth as many as at the top) appeared just during the top maximum. Group IV showed a maximum of 500 per ee., which coincided with the top maximum of 2500 per ce. Hypotrichida. These forms occurred in negligible number in groups II, II] and IV. In group I there was a small maximum of 60 per ce. on the 38th day. Paramaecium. Practically no paramaecia appeared at the bottom in any of the A cultures except A2, where, toward the end of the observations, one count of 300 per cc. was taken. Vorticella. Vorticella were not observed in group I until near the end (76th day) when a maximum of 40 per cc. was attained. Group II, however, showed the largest number for the A series, with a maximum of 400 per cc. on the 72nd day, 1.e., near the end of the observations. In groups III and IV Vorticella was not observed until nearly the end of the study when maxima of about 40 per ce. were reached. Amoeba. In all the groups of infusions, amoebae were in greater abundance at the bottom than at the top. A maximum of 3000 per ce. occurred from the 55th to the 60th day in A J; a maximum of 6000 per cc. on the 76th day in A JJ; a maximum of 2000 per PROTOZOAN FAUNA OF HAY INFUSIONS 247 ec. from the 36th to the 50th day in A JIT; and a maximum of 6500 per cc. on the 23rd day in A IV. 2. B Infusions Monad. In groups BI and B JI monads were practically absent. B III had none at the bottom during their presence at the top, but later a few were observed at the bottom from the 35th to the 55th day. In B IV monads appeared in numbers be- Fig.8 BIV group. The ordinates represent the number of organisms at the surface. On the abscissa is plotted the number of days of the existence of the in- fusions. Monad = ----; Colpoda = —--—- ; Hypotrichida = —-—; Vor- ticella = — — - ; Amoeba = —.-- —. tween 500 and 100 per ce. from the 20th to the 35th day. Their appearance was coincident with the descent of paramaecia. It will be recalled that few monads were observed at the surface of the infusions of this group. Colpoda. Considerable diversity existed between the Colpoda in the B groups. In BI and B III they attained a temporary maximum of approximately 15000 per cc., just after their dis- appearance from the top (ef. fig. 13). In B IJ and B IV prac- tically none were seen at the bottom. 248 LORANDE LOSS WOODRUFF Hypotrichida. ‘These forms appeared in negligible numbers at the bottom in groups II and IIT, while in group I the largest bottom count was taken, i.e., 110 per ce. on the 33rd day (cf. fig. 14). Group IV also showed a relatively large bottom count as compared with the top count, having a maximum of 80 per ce. on the 24th day. Paramaecium. B I showed a larger number of Paramaecia at the bottom than at the top. Conjugating specimens were seen at the bottom only (ef. figs 5 and 12). B II showed practically no paramaecia at the bottom, but this is explained by the fact that Didinium early exterminated them in this group. A heavy growth appeared in B JI/ after conjugation was prevalent at the top (ef. fig. 13). B IV had very few paramaecia at the bottom until the 34th day and then there appeared about 500 per ce. All disappeared by the 45th day. Their appearance at the bot- tom was coincident with a decline at the top which was brought about by Didinium. Vorticella. In group I this form attained a maximum of 175 per ce. on the 38th day, while in group II they reached a maximum of 240 on the 20th day. Vorticella appeared in group III in small numbers at both top and bottom, the bottom maximum being 60 per ce. on the 31st day. In group IV, however, the larg- est bottom count was recorded, i.e., 600 per ec. on the 28th day (clase. 115). Amoeba. There was a great difference in the amoeba fauna of B1 and B2 of group I, so that it is better to present these separ- ately. Bi had a maximum of 10000 per ce. on the 58rd day, while B2 contained practically no amoebae at any time. The B II group showed a large growth which attained a maximum of 2500 per ce. on the 50th day and terminated on the 58th day. There was a relatively small maximum of 250 per cc. on the 37th day in BIIJ and the amoebae had disappeared by the 45th day. A maximum of 2000 per ec. was in existence in B IV from the 35th to the 45th day, when the last regular observation was made. This culture, however, supplied countless Amoeba proteus for class use for two years thereafter. 249 PROTOZOAN FAUNA OF HAY INFUSIONS ‘— ++ — = Bqooury: - — — = B][90T}104 ‘———._ = ummtoovweieg :—:+— = epryoujodxAy { --- = epodjog {--- - = peuopy “SUOISNJUL OY} JO 9DU94STX9 OY} JO SABp Jo JaquInu oy} po}jo[d st vsstosqe oy} UD “a0BsAMS 9Y} YB “0d 10d suISTUvS.IO Jo IequINU oY} YUeSeddor so}BUIpIO oY, ‘dnoas ED Bal O€ O OOOL 0002 250 LORANDE LOSS WOODRUFF 3. C Infusions Monad. Monads appeared only in inappreciable numbers in all the groups of infusions. In group IV, however, a number of monad forms, other than those included in the top counts, ap- peared in considerable numbers for a time. Colpoda. Practically no Colpoda were recorded for groups I and III. Group IV showed a brief maximum of 2500 per ce. which coincided with that of the top. Hypotrichida. The hypotrichous fauna was practically zero. Paramaecium. Paramaecia were not observed, except in group I where 100 per cc. were recorded for five days after a rapid decline at the top. Vorticella. Practically no Vorticella appeared in the bottom counts. Amoeba. In group I a few amoebae appeared on the 45th day and reached a maximum of 250 per ce. within the next five days, and then disappeared with equal rapidity. A heavy growth of 10000 tiny amoebae was attained in group III by the twenty- fifth day, and all were practically gone within ten days. In group IV a maximum of 10000 tiny amoeba was recorded on the 20th day and from this time the number gradually decreased until the 47th day when very few were observed. This decline was followed by a rapid rise to about 2000 per cc. on the 56th day when the last count was taken. V1l. DISCUSSION AND CONCLUSIONS FROM THE OBSERVATIONS ON THE SEQUENCE OF THE SURFACE, MIDDLE AND BOTTOM FAUNA 1. Surface fauna. These extended observations on the protozoa of typical labor- atory infusions, made up by several different methods, clearly indicate a definite succession of certain representative forms at the surface of the water.¥ 13 T amindebted to Mr. T. 8. Painter, one of my students, who made for me a careful study of a number of similar infusions in the Yale Laboratory and also at his home in Salem, Virginia. His observations show an essentially comparahle PROTOZOAN FAUNA OF HAY INFUSIONS 251 The close agreement both of the sequence of appearance and of maximum numbers in all three series (A, B, C) is striking (cf. p. 244) and indicates that the sequence is not merely the result of factors incidental to the methods employed. The data in regard to the time of disappearance is relatively meagre for the A and C series because many of the typical forms studied survived the period of the last observation. Consequently the sequence of time of disappearance is based chiefly on data from the B series, which, on account of the removal of the hay, passed through its cycle much more rapidly. It is remarkably suggestive that the sequence (derived from the entire series of infusions) of all the forms at the time of appearance and at the time of maximum numbers and at the time of disap- pearance is zdentical, with the exception of Amoeba. The data indicate that the Amoeba cycle in the infusions is comparatively short since the position of Amoeba in the series advances progres- sively forward: it being last at the time of appearance, next to last (before Vorticella) at the time of maximum and third from last (before Paramaecium and Vorticella) at the time of disappear- ance. However, as has been already pointed out, the data is not sufficient to positively establish the relative position of Amoeba and Vorticella at the period of maximum numbers. A study of the curves plotted from the surface counts of single infusions or groups of infusions reveals the fact that when once a great development is attained by a particular form, this maxi- mum is seldom approached again. There are, however, some striking exceptions to this as, for example, Colpoda in group A III (cf. fig. 3) and the Hypotrichida in group C IV (ef. fig. 11). The curves further show that the major rise and fall in numbers are usually of about equal rapidity, though the final complete disappearance of an organism from the infusion may be long deferred. Careful searching in many of the A and B infusions sequence of forms with the one here described. Among the monads, however, he found a large development of Chilomonas, while this form was. relatively scarce in my infusions. Also, his Amoeba fauna was partially replaced by a consider- able growth of Arcella. This latter result is interesting since it shows that some- what closely related rhizopods fill substantially the same place in the economy of the infusions. LORANDE LOSS WOODRUFF 252 "—-++— = eqoouly {.— — = eya0r10A {——— = umrovureieg ‘—-— = epryojodséy ‘ - -- = epodjop {...+ = peuoy, “WOISNJUTOY} Jo soUa}sSTx9 oY} Jo SABp Jo JequInu oy} pezzoTd SI Bsstosqe oy} UO “Q0BjANS 9Y} 9B °90 tod SuIsTUBSIO Jo IaquINuU oy} YUdSeIdad SoyvuUTpIO oY, “dnows 777 DO OL “BI OOo! 000¢ OO00€ PROTOZOAN FAUNA OF HAY INFUSIONS PDS) 7600 25000 1000 Fig. 11 CIV group. The ordinates represent the number of organisms per ec. at the surface. On the abscissa is plotted the number of days of the existence of the infusions. Monad = ee =--- peorichids =—-—} Paramaecium = ae icelle: = = Amoeba — i 254 LORANDE LOSS WOODRUFF after a lapse of nearly three years showed a few survivors of nearly all the chief forms, mostly at the bottom among the algae and débris. 2. Middle fauna It is impossible to determine any definite sequence of forms for the middle of the infusions—this region being, as already pointed out, amore or less neutral territory which is encroached upon from time to time by organisms from the top and bottom as conditions in these regions vary. 3. Bottom fauna The bottom fauna also has not exhibited a definite succession similar to that of the top. A study of the data already presented shows that the protozoan forms under consideration, with the exception of many amoebae, are essentially surface dwellers and seldom resort to the bottom except during or after a period of great development at the top. However, there is no invariable correla- tion between a fall in numbers at the top and a rise in numbers of the same species at the bottom, and it seems clear that, in the majority of cases, when a species declines in one region, most of the animals encyst or die. The latter is certainly true for Para- maecium because many hundreds of passive and dying individuals, affording a feast for Coleps, may sometimes be seen among the débris at the bottom. Again, myriads of cysts of hypotrichous forms are frequently found at the bottom as the surface decline proceeds. Amoebae, among the protozoa under consideration, appear to give some evidence of migrating from the surface to the bottom which is their chief abode. The data on amoebae give the impression that some forms first appear in the infusions as amoebo-flagellates which gradually increase in size and before long are unable to assume the flagellated phase. The pesudo- podia of these are first of the guttula type but become more and more long and slender until many typical radiosa forms are present, and these in turn give place to typical large A. proteus. Only in certain infusions has it been possible to trace such a series, but PROTOZOAN FAUNA OF HAY INFUSIONS 209 in these it has been quite striking, and in one of the later infusions I was able to predict correctly that declining amoebo-flagellates would be replaced by typical amoebae. Such a cycle, of course, would not be remarkable in view of the results of some investi- gations on amoebae.“ Although the data from these infusions by no means prove that the forms represented in this cycle are stages in the life history of a single species, nevertheless I lean toward the view that such will prove to be the case (ef. p. 211). Taken as a whole, the study of the bottom fauna has proved to be less interesting than was anticipated, as I had expected to find Fig. 12 Comparison of the Paramaecia fauna at the bottom of infusion Bi ( ) and infusion B2 (- - -). x = point at which an epidemic of conjugation occurred in B1. a much closer correlation between declines at the top and rises at the bottom, and vice versa. Apparently the bottom forms are largely independent of those at the surface, and the protozoan types under consideration, with the exception of the amoebae, are represented at the bottom by considerable numbers of active indi- viduals chiefly when some sudden change, such as the falling of the zoogloea, brings them down, or by stragglers which manage to exist by avoiding the competition at the top. It is nearly always possible, by careful searching, to find at the bottom a few strug- gling individuals which have survived from an earlier prosperous surface population. 14 For example, cf. Metcalf: Studies upon Amoeba. Jour. Exp. Zool., vol. 9, 1910. bo Or o> LORANDE LOSS WOODRUFF 4. Factors determining the sequence The problem becomes enormously complex when an attempt is made to decide upon the chief determining factors of the observed sequence of organisms at the surface of the infusions, and is en- tirely beyond our power of analysis from the data extant. There- fore, I believe, it is preferable at this time not to enter into an extended discussion of this question. I shall, however, briefly mention some points which seem to indicate suggestive lines for future study. There is experimental evidences that, broadly speaking, the potential of division decreases from monads to paramaecia; that is, for example, paramaecia, under optimum conditions, divide less frequently than the majority of the hypotrichida, and simi- larly, the latter divide less rapidly than Colpoda. In regard to Vorticella and Amoeba, however, sufficient data are not at hand to make a definite statement. With this in mind a series of experiments were made on the time of appearance of maximum numbers of Monads, Colpoda, Hypo- trichida and Paramaecium in separate flasks of infusion which were seeded with a single individual of one species. The multi- plication of the respective forms in the various flask cultures was observed, and the results showed remarkable agreement with the sequence of maximum numbers as determined for these same forms in the regular infusions. Consequently it appears that the number of specimens of any particular organism initially intro- duced into the large infusions, or the time of emergence of en- cysted forms has not had an important influence on the sequence of maximum numbers in these infusions as determined for the complete series. It may well, however, account for at least some of the individual variations in the sequence of appearance in numbers sufficient to be included in the samples studied, and of maximum numbers, which are apparent in particular groups of in- fusions. Again the interaction of the different forms would appear, at first glance, not to be a crucial factor in the sequence of maximum numbers since, in the experiments cited, the ‘se- ‘quence’ was duplicated, when only one species of organisms was PROTOZOAN FAUNA OF HAY INFUSIONS 257 in each flask of infusion. This conclusion nevertheless, does not necessarily follow from the data, because all of the forms under consideration can flourish on a bacterial diet, which, of course, was supplied in each case. The interaction of the various forms clearly plays a part in the duration of the maximum andthe rapidity of the decline. Experiments by the slide method of cul- ture, which I have employed in my pedigree culture work, show that in culture medium which is the same from day to day prac- tically the same ‘sequence’ of maximum numbers occurs and in this case it is apparent that chemical changes in the environment are not responsible for the results. Further, it is possible to carry all the forms under consideration for at least one hundred genera- tions by this slide method, and this is sufficiently long to show that enough organisms can be produced in a medium which is chemically constant to supply, many times over, the number of organisms recorded at the maxima.n the regular infusions. Con- sequently I think that these observations indicate that the rela- tive potential of division of the four forms under discussion is adequate, under certain conditions at least, to establish the ob- served sequence of maximum numbers, and clearly suggest that it may be an important factor in large infusions. The data from these infusions lead me to believe that the strictly biological factors are of greatest importance, and that it is neces- sary to look to somewhat subtle chemical changes in the medium for the important chemical factors in the environment. Fine’s studies! on these infusions are in accord with this view and indi- cate that such general chemical changes-in the environment as, for example, titratable acidity are not determining factors, at least for these particular species. My work on the excretion prod- ucts of Paramaecium shows," however, that such substances have an inhibiting influence on the reproduction of this form, and it is quite probable that these products affect the sequence, maximum numbers, and decline of the various species. In fact Shelford, in hisstudies on the ecological succession of fish in ponds, believes that 1siCf. Hine: loess cits 16 The effect of excretion products of Paramaecium on its rate of reproduction. Jour. Exp. Zool., vol. 10, no. 4, 1911. LORANDE LOSS WOODRUFF 258 ‘QOVJANS OY} YB patind900 uoTyVSnfuod Jo dTULEp -Ido uv yorym ye yuiod = x °*--- + - = WO}}0q oY} 4B pues ——— = 90¥]jINS 904} 3B WNTIDevUI -BIBqd :— + — = W0}}0q 94} ¥B puw - — — = voBJINS 9Y} 4B Bpodjog ‘dnoiws TT _g El “BUY OOO€ 0001 0008 PROTOZOAN FAUNA OF HAY INFUSIONS 259 his data show that the succession of those forms is not deter- mined by the kind of available food but to an unused increment of ‘decomposition and excretory materials which, in the last analy- sis, affects breeding.” 5. Decline in numbers Closely involved with the problem of the sequence of appear- ance and maximum of the various forms is that of their more or less rapid decline in numbers. Here again the accumulated data do little more than establish the fact. The decline of the monads is quite clearly due, in part at least, to the evident variation in the amount of food in solution and to the rising hosts of Colpoda. The decline of Colpoda may be similarly ascribed to the domi- nance of the hypotrichida. Most of the hypotrichous forms were literally filled with ingested Colpoda which formed their staple diet. The relations of Paramaecium and Vorticella to their pre- decessors, successors and to each other is not so apparent, but their abundance may well be influenced by a succession of the bacterial flora, for example, which unfortunately could not be followed in these studies, as well as to the host of other protozoan species. The competition between the various forms is so keen and the cycle is so rapid that even daily observations are, at times, insufficient to reveal the kaleidoscopic changes. Now and then, however, some prominent case of competition, such as that be- tween Paramaecium and Didinium, is forced upon the attention and the reason for the extinction of one form is clear. Didinium, in fact, so quickly exterminated the paramaecia in groups A [[ and B IV that it was necessary to omit the records of paramaecia in the table of sequence of these infusions (cf. table 2). In B I also the paramaecia cycle was considerably aborted by Didinium. Among other instances of a similar nature, the destruction of hosts of Colpoda by the suctorian Podophrya may be mentioned. In other words, one who closely follows a series of infusions dayby day cannot but be impressed with the intense struggle for food and the eternal warfare in this microcosm, and become con- 17 Biological Bulletin, vol. 22, no. 1, 1911. 260 LORANDE LOSS WOODRUFF vineed, though he cannot prove, that in the final anaylsis the paramount factor is food, though many other factors, such as excretion products, etc., may play a not unimportant part. Bio- metrical study of variation in certain Protozoa shows that the average size of the population is smaller after their period of ereatest abundance in an infusion and that ‘‘there can be little doubt that one of the chief factors which induce saprophytes like Chilomonas to disappear from a culture is that the medium no longer furnishes proper food (either in amount or kind, or both).’’#8 Fig. 14 BT group. Hypotrichous fauna at the bottom. VIIl. CONJUGATION Comparatively few epidemics of conjugation were observed in this entire study, and these were chiefly among paramaecia, so that the data in this connection are quite meager. It therefore has not been possible to make any definite correlations between the presence of the phenomenon and the fate of the conjugating forms in the infusions. However, a study of the records in regard to Paramaecium seems to show that conjugation usually occurs when a comparatively large number of individuals are present and that immediately following an epidemic there is a temporary decline in the number of specimens observed. After this decline there may or may not be a large increase in the number of animals. It seems clear that in many cases conjugation is coincident with sudden changes in the environment. In fact the phenomenon may occur in certain cases almost solely among individuals which have been carried to the bottom with falling zoogloea. But that this does not of necessity bring about conjugation is shown by in- fusions Bi and B2. Conjugating paramaecia were not seen at 18 Pearl: Variation in Chilomonas under favorable and unfavorable conditions, Biometrika, vol. 5, 1906-1907. PROTOZOAN FAUNA OF HAY INFUSIONS 261 the surface of either of these infusions, and at the bottom it was only observed in BI. At the point marked x (fig. 12) fully 95 per cent of the animals were conjugating. Nevertheless the par- amaecia fauna at the bottom ran practically the same course in each infusion, in fact it survived somewhat longer in the infusion in which conjugation was not observed. This culture also illus- trates a case in which a temporary decline in numbers occurred immediately after an epidemic of conjugation (ef. Mmgst2)p Fig. 15 BIV group. Vorticella fauna at the surface (----- ) and bot- tom ( Ne In group B III, in which there was an exceptionally large bottom fauna, conjugation was observed among the paramaecia at the surface and bottom simultaneously, but was somewhat more prevalent at the top. Fig. 13 shows that the epidemic occurred at the period of the surface maximum and that the ensuing decline at the top was coincident with a remarkably large increase in the bottom growth. Apparently many species of infusoria do not resort to conju- gation, to sustain rapid. cell division when the environment is slowly changing and the data give no reason for believing that THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 2 262 LORANDE LOSS WOODRUFF conjugation effects ‘rejuvenation.’ In many cases encystment occurs and the organisms remain at the bottom when conditions become somewhat unfavorable; but undoubtedly the majority die after their period of maximum abundance. My experience with these cultures leaves me with the impression that conjuga- tion will be found to be a means resorted to by many species to survive acute changes in the environment, which, for example, preclude encystment. It is suggestive in this connection that in forms like the hypotrichida, which, as is well known, have a de- cided tendency to encyst, and the cysts of which were observed in great abundance at the bottom of these infusions after the active forms passed their maximum, not a single syzygy was observed; while in Paramaecium, in which the power of encystment has never been established, conjugation is recorded comparatively fre- quently. However, it is also clear from this work that a condi- tion which will induce conjugation in one race of Paramaecium will not always induce it in another, as epidemics have occurred between small races, while among giant races intermingled with them syzygies were not seen. This, of course, is in accord with Jennings’ studies on Paramaecium.!® The problem of the con- ditions inducing conjugation, and also of the effect of conjugation has recently become so complex from our increasing knowledge of the life history of various paramaecium genotypes, that the observations here recorded are interesting chiefly as throwing a side light on certain factors of the phenomenon as they appear in large cultures. IX. SUMMARY The following points may be emphasized: 1. Ordinary hay added to tap water will not produce an infusion which is productive of a sufficient number of representative proto- zoan forms to make it profitable for the study of protozoan se- quence. 2. Air, water, and hay are all sources from which the protozoa of infusions are derived, and increase in importance in the order 19 What conditions induce conjugation in Paramaecium? Jour. Exp. Zeol., vol. 9, no. 2, 1910. PROTOZOAN FAUNA OF HAY INFUSIONS 263 given. Of these three, however, air is practically a negligible factor in seeding infusions. 3. In hay infusions, seeded with representative forms of the chief groups of Protozoa, there is a definite sequence of appear- ance of the dominant types at the surface of the infusion, i.e., Monad, Colpoda, Hypotrichida, Paramaecium, Vorticella and Amoeba. 4. The sequence of maximum numbers and of disappearance is identical with that of appearance, except that apparently the position of Amoeba advances successively from the last (sixth) place to the fifth place and then to the fourth place. 5. A definite sequence of forms is not apparent at the middle or bottom of the infusions. 6. The middle of the infusions is tenanted chiefly by a free- swimming population brought there by an overcrowding at the top or bottom. 7. All of the protozoan forms considered (except Amoeba) are chiefly surface dwellers and it is evident that when they pass their greatest development at the surface this maximum is seldom approached again, and their cycle is practically over. | 8. The major rise and fall in numbers are usually about equally rapid, though the final disappearance of an organism may be long deferred. 9. The appearance of any of the protozoan forms under con- sideration (excepting Amoeba) in appreciable numbers at- the bottom is most often coincident with or immediately subsequent to its surface maximum, and portends its more or less rapid elimi- nation as an important factor in the life of the infusion. 10. Numerous abnormal individuals and cysts are frequently to be found at the bottom in great abundance immediately after the surface maximum. 11. There is some evidence that amoebae migrate from the sur- face to the bottom which is their chief abode. 12. The observations give the impression that some amoebae appear as amoebo-flagellates which gradually increase in size and finally assume the form of typical A. proteus. 264 LORANDE LOSS WOODRUFF 13. There is some evidence that the relative potential of divi- sion of the various forms may have an appreciable influence on the sequence of the maxima. 14. Emphasis is pit upon the strictly biological interrelations (e.g., those involving food and specific excretion products) of the various forms as the most important determining factors in the observed sequence. 15. The observations suggest that conjugation will be found to be a means resorted to by many species to survive acute changes in the environment, which, for example, preclude encystment. CHEMICAL PROPERTIES OF HAY INFUSIONS WITH SPECIAL REFERENCE TO THE TITRATABLE ACIDITY AND ITS RELATION TO THE PROTO- ZOAN SEQUENCE MORRIS S. FINE From the Sheffield Biological Laboratory, Yale University FIVE FIGURES In the preceding paper! are presented the results of a study of the succession of the protozoan fauna of a series of hay infusions. The present paper gives the results of some chemical investiga- tions on the infusions employed by Professor Woodruff with a view to correlating, if possible, certain chemical conditions with the protozoan sequence there shown to occur. Although one cannot hope to obtain a complete analysis of the chemical factors involved, there are a limited number of deter- minations which can be made with some degree of ease and accu- racy, and which may quite reasonably be expected to throw light upon this problem. In the present work a preliminary survey was made to determine those estimations which would be likely to yield satisfactory results, with the view of instituting an inten- sive study of such factors. The determinations? thus first made were: (1) Phenolphthalein acidity, (2) Methyl-orange alkalinity, (3) Oxygen consumed, (4) Chlorides, and (5) Solids (total, organic and inorganic). The ‘phenolphthalein acidity’ was obtained by titrating 5 ce. of infusion with 0.01 N NaOH, using phenolphthalein as indicator. 17, L. Woodruff: Jour. Exp. Zool., vol. 12, No. 2. The present work was under- taken at the suggestion of Professor Woodruff, to whom I am indebted for suggestions and criticism. 2 Cf. A. W. Peters: Amer. Journ. Physiol., vol. 17, p. 454, 1907. 265 266 MORRIS S. FINE For the ‘methyl-orange alkalinity’ 5 ee. of infusion were titrated with 0.01 N HCl with methyl-orange as the indicator. As suggested by Peters,? the samples were titrated under xylol, thus retarding the loss of volatile matter during the process. In this manner was obtained the titratable acidity which, however, as Peters‘ points out, is probably not a correct expression of the con- in centration of H ions with which the organisms are in contact. For the third and fourth determinations recourse was had to methods employed in sanitary water analysis. To determine the oxygen consumed, 5 ce. of infusion, filtered clear, and diluted to 200 ec. with distilled water, were treated with 10 cc. of 50 per cent H.SO, and 0.01 N KMn0O,, the excess of the permanganate being titrated back with 0.01 N oxalic acid. For the chloride determination, 5 cc. infusion, filtered clear, were titrated with 0.01 N AgNOs, using a solution of K, CrO, as an indicator. Total solids were obtained by evaporating 25 cc. of the filtered infusion to dryness. The residue was ignited, thus furnishing the data for the inorganic solids. The difference between these two last values is the solid organic matter. The three latter determinations are recorded in table 1. Infu- sion 2 suggests a general increase in oxidized material. The data, however, are scant and lack uniformity. The figures for the chlorides show a rise to a maximum with a subsequent fall. What significance, if any, may be placed upon these data it is impossible Oreee natlge indicate, as is to be expected, organic matter a general trend toward mineralized material. Of all the prelim- inary data, those for ‘phenolphthalein acidity’ appeared to be most characteristic and constant. Moreover this determination lent itself very readily to serial estimation. It was therefore planned to study this factor in considerable detail for a large number of infusions prepared in various ways. tosay. The ratios soc: cit., p. 463% 4 Loe. cit., p. 464. CHEMICAL PROPERTIES OF HAY INFUSIONS TABLE 1 PRELIMINARY INFUSION 1* PRELIMINARY INFUSION 2* aa eee ; i : 2 a SOLIDS meer a SOLIDS Q Zap = =) | Zp =” — =| a ee | a- | Be | Sl Fos Grams per 100 cc. Bie || 9 aS Grams per 100cc. | 2|& mw. | KO infusion Sie ish i) da infusion | aye SPP? iz PAS aL Les AES! Z 5/5 BSa /ES2 | gle |[ES2 |8Se Ae jogs jogs 4\z |o48 |e | 2/2 gE |82e laze | SP lee [ale sisae len8 2 aa Pel Alga.digana) = Sh ae | ° lgensigses) Eh Reyes ae Blackulscaa § S e | 2& |Soaascaal & 6 By ass <|O ID = RS fe) a lO iS | & fe o) Pe | | | | ae a iz | se 1 | 8 0.034 0.010 0.024 0.42 8 0.068) 0.014) 0.054 0.26 a 14 264 0.044) 0.016 0.028, 0.57 14 422 0.078) 0.024| 0.054 0.44 8 | 12 236 | 0.044) 0.020) 0.024 0.83 16 | 356 | 0.071) 0.024 0.047, 0.51 UN a) 194 | 0.048 0.021 0.027 0.78 16 324 0.076) 0.026 0.050 0.52 15, 14 192 | 0.051) 0.023) 0.028) 0.82 16 290 | 0.078) 0.028) 0.050) 0.56 Le 12, 188 | 0.058) 0.026) 0.032, 0.81 | 16 254 | 0.081) 0.033) 0.048) 0.70 22)| 12 214 | 0.058) 0.024 0.034 0.71 16 | 214 | 0.078) 0.028 0.050, 0.56 25 | 12 | 194 | 0.052) 0.024 0.028 0.86 | 14 216 0.075) 0.030 0.045 0.67 29 14 | 260 0.068 0.032 0.036 0.90 14 174 | 0.084) 0.036) 0.048 0.75 32 | 14 | 210) 0.062) 0.024 0.038 0.63 | 14 226 | 0.073) 0.024 0.049} 0.49 36 | 12 206 | 0.068 0.025 0.043) 0.60 12 | 230 | 0.084; 0.024 Ode 0.40 39 10 | 204 | 0.069) 0.031) 0.088 0.82 8 | 224 | 0.083} 0.030 0.053 0.57 43 6 212 | 0.081) 0.030) 0.051 0.60, 10 206 | 0.082) 0.032 0.050, 0.64 * These infusions were prepared in the same manner as the typical members of series A (see below). PREPARATION OF INFUSIONS® Series A: Typical members. Infusions A-1, A-2, A-3, A-4, A-5, A-6, A-21, A-22, A-31, A-32, A-41, A-42, A-51° were made essentially like those prepared by Peters. hay in about 250 ee. of water were boiled five minutes. In each case 20 grams Timothy Both infusion and solid hay were then transferred to a battery jar con- taining some unboiled water and the volume made up to 5 These infusions were than equally seeded with samples taken from several cultures of varying ages, in order to insure the liters. 5 For further details see Woodruff, loc cit. 6 The protozoan count was omitted in A-51. 268 MORRIS Ss. FINE presence, initially, of as large a number of representative proto- zoan forms as possible. Atypical member. Infusion D-1 was prepared exactly as the above with the exception that the water to which the boiled hay and infusion were transferred had been heated to about 90° C. and no protozoa were introduced. By this means was obtained a protozo6n-free culture fluid.’ Series B: Typical members. Infusions B-1, B-2, B-21, B-22, B-31, B-32, B-41, and B-428 were prepared by boiling 20 grams Timothy hay for five minutes, just as in series A, and then strain- ing into unboiled water so that a final volume of 5 liters was obtained. These were seeded exactly as in series A. Atypical members. Infusion E-1 was prepared just like the typical members of this series except that the water into which the boiled infusion was strained had been raised to a temperature of 90° C. We have thus a protozoén-free infusion ‘of series B, just as D-1 is a protozoén-free infusion of series A. Infusions BB-1, BB-2, BB-3, BB-4, BB-5, and BB-6 were prepared exactly like the typical members of this series but in addition were treated in various ways. BB-1 and BB-2 were left unchanged, serving as controls. To BB-3 and BB-4 were added 5 and 20 grams of dextrose respectively. BB-5 was kept practically neutral to phenolphthalein by adding, when necessary, the calculated amount of NaOH. This necessitated stirring at each addition of alkali, and hence, as a check, BB-6 was stirred at the same time. Series C: Typical members. Infusions C-1, C-2, C-3, C-3]1, C-41, C-42, and M-1 each consisted of 20 grams unboiled hay with 5 liters unboiled water. To this was added a small amount of seed. With certain exceptions, mentioned elsewhere, the hay was kept continuously at the bottom of the infusion. Atypical member. Infusion 8-1 was prepared by heating 20 grams of dry hay in an autoclave and adding 5 liters of water which had been warmed to 90° C. S-1 is therefore a protozoén- free infusion of series C. 7 No attempt was made subsequently to keep the infusion free from bacteria. 8 Infusions B-41 and B-42 were subjected to a chemical examination so infre- quently that the results are omitted from table 3. CHEMICAL PROPERTIES OF HAY INFUSIONS 269 Bacterial infusions. Eight infusions were prepared as follows: In each of eight cotton plugged flasks, 2.8 grams of hay and 700 cc. of water were placed and the mixtures sterilized in an autoclave. They were allowed to cool and were then treated in various ways: two were kept sterile; four were inoculated with a pure culture of B. coli; and two were inoculated with a pure culture of B. subtilis. On the foregoing cultures records were obtained for the ‘phenol- phthalein acidity’ and ‘methyl-orange alkalinity.’ In the ideal experiment, the temperature should have been maintained con- stant throughout the period during which the infusions were under observation; or, at least, all infusions should have been subjected to the same changes in temperature. Neither of these conditions could be conveniently brought about.? RESULTS The data secured in this study may be most readily presented by tables and curves. In tables 2, 3, 4, and 5 are recorded the results of the ‘phenolphthalein acidity’ and ‘methyl-orange alka- linity’ determinations, expressed in cubic centimeters of 0.01 N NaOH or 0.01 N HCl per 100 ce. of infusion. Peters has made the observation that the acidity becomes greater as the depth of the infusion increases and in order to give this quantitative expres- sion, titrations were made on samples taken from the bottom of the infusion at frequent intervals during its history. These results are given in italics immediately above the figures for the top, and are expressed as so many cubic centimeters of 0.01 N NaOH or 0.01 N HCl per 100 ce. of infusion greater (or less) than the titrations for samples taken from the top. The principal points of interest, brought out in these tables, are illustrated in figs. 1,2,3,and4. In calculating average curves only the ‘typical members’ of the three series were included. As a matter of fact, as far as the actual titrations are concerned, some of the ‘atypi- cal members’ might have been included, e.g., D-1, E-1 and 8-1 9 For a discussion of the influence of temperature on these infusions, cf. Wood- ruff, loc. cit., p. 218 and also p. 274 of the present paper. 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Pt | | | 8I | IZ | ¥% SI | ieee et] r+ | | | | oF | | ae) Re 6+ 7 ¥ ouiel| vq} | | | OF [rr | et | ar | | 6 | 02 | 2 | 8% | | | | | ker | | OF] | Oa) OF FT Seta) ats e 9 ¢ 6 [2t\et| |ST ST PLE \otST | 91 ET et I |x| ot | et | os | 2 |e |9% | 82 | oe | ve | 08 | 2 | | o* | 0=| OF O= OF OF| OF] OF| BI+| est | | | ) € 9¢ ¢| |s| | |or QT| gT/6r[at}oz| €z /€2/@t| 6r bz GT St | Or | ST | Bt | Bt | OT Zt | zt | ST | st or 8 ¢ lai (4 ze | #2 | 9% | 02 | SI | e+ s+ st+| stort) e+ te+| Ol € ¢ cI 9% a | 2 6 | OT Lt | 91 | I+ e+} ss] s9+ 96+) 86+ Asi 98T+ 8 9 g | joc} | | 02 92 61 61 gt | ST Ae Pe PO IE | NY I+) | joe] | lrtist| lect) lost| = rert\er+ ast eoi+ ost list ioett eri est+ 7 | t | | |e 91 STST \LT0Z | 92 22 ST SE Sh Vere er Ch cn seeks WE eh OR cer | | | | | OF) OF o=| 8—| sc+| t+] o9+| a9+) 9+|99+ | 9+) s+) 99+ G02 661 GOLIST FLT E9T ZB 180269 1S FF IF OF GE SE CE TEOE6zRs LZ) 92 ahs eG \cs TZ 0 GI ST LT OT) St | FL T | GT | IT | OF \e@ We Wee ON eh Wz 272 Sava (uoIsnyut *99 QOT 10d FOVN N 10'0 Jo saoqourtyuse o1qny)) Q sarwagy ‘fqypron uraypyzydjouay P ATaVL CHEMICAL PROPERTIES OF HAY INFUSIONS Die TABLE 5 Methyl Orange Alkalinity (Cubic centimeters of 0.01 N HCl per 100 cc. infusion) DAYS | | (ee | Fan mieea es (ROW te|n2 | 3/4/5/6|7/8/|9 [10 | 11) 12] 13) 14] 15 16 17 18| 19 20} 21| 22} 23| 24| 25| 26) 27| 28 / +-4|+4|0 +0 +0\—2| 0-4/0 | | A-1....;8|9/6/4]6/8)| 8 10) 10) 12) 10) | | | | laeeila'e | +i +e+020+2-+9+0-9 40-2 ect eet ae) | A-2 | 7} 10) 6) 4) 8) 8 | 10) 12) 12) 14 14) 12) 14) 14) 14 16 | 20 | : | i} —2| | | RECA 83) Sic8 Wealea| | | | | 16 | le | Paci A-5....| 8 | 10) 6 | | We" | | | | +0) —9 | | | | | | A-6....| 4 | Kea |pelate ey | | | +0 —2\+0 +0 +2 +0 —2/+0 lies | | | B-....|7/8|4|6|6|8|6| 10 19 12/12/12 8/19 [12 | | | 12 ete +0) | | Wana B-2....| 7 | 8 |\4 | | | | Te | | +8 +2)+2/+2)+2-42\—2+4 | | pes} | beth | C-1....) § | 10] 6 | 6 | 6 | 10] 10) 12| 12) 8 | 12] 12) 8 14 | | 16 | | 22 | He | | | +4 +2) | | Hes Oi C2k 16118 | 10 10 | 10 Keil | | | +9)+0—-2+0 | Wwe lah} | C-3....| | 10] 6 10 10 10 10 | la at | | | | | do not differ materially from the ‘typical members’ of their respective groups. From the average of series C in addition to the ‘atypical member’ S-1, certain ‘typical members’ C-1, C-3 and M-1 were also omitted—C-1 because on the fifth day it was slightly stirred; C-8 because during the early part of its history the hay was at the top; and M-1 because it was stirred at certain intervals for a definite purpose, as explained in another place. In boiled infusions (series A and B) we can corroborate Peters’!° results in regard to the ‘phenolphthalein acidity,’ i.e., a rapid rise in which a maximum is reached in from two to six days, followed by a more gradual decline; the lowest point being reached fifteen to twenty days earlier in series B than in series A (fig. 1). The curve for series C is somewhat different; a maximum being attained much more gradually. Further reference will be made to this below. 10 Peters: Amer. Journ. Physiol., vol 18, p. 330, 1907. 274. MORRIS S. FINE Fig. 1 ‘Phenolphthalein acidity’ for infusions of series A, B, and C; samples taken from top of media. Ordinates represent number of cubic centimeters of 0.01 N NaOH per 100 ce. of infusion. Upper curves: series A, average acidity = ; Maximum acidity = .---.----- ; minimum acidity = - ---- ‘ Middle curve: series B, average acidity. Lower curve: series C, average acidity. Curves for maximum and minimum acidity of series B and C are not given as the variations are not important. It would seem that for the ‘phenolphthalein acidity’ at least, the inconstant temperature was not an important consideration. Infusions of the entire series A (infusions started at different times and therefore experiencing different temperature changes) show no greater individual variations in acidity than does the group A-l . . . . A-6 (infusions started on the same day and hence subjected to the same changes in temperature). This is illustrated in table 2. For this reason, when calculating average curves, I have not considered it necessary to exclude infusions merely because they were subjected to different temperature variations. As to the factors underlying the production of acid, I agree with the view of Peters!® viz., that bacterial fermentation mainly is CHEMICAL PROPERTIES OF HAY INFUSIONS 275 responsible. That the protozoa play a relatively small part in the acid production is shown in infusions D-1, E-1, and S-1, in which throughout their history practically no protozoa were present, and yet their acidity curves do not differ materially from the others of their respective series (tables 2, 3, 4). In order further to demonstrate that the bacteria are almost entirely responsible for the acid production, hay infusions were prepared and inoculated with pure cultures of bacteria as described on page 269. The results for ‘phenolphthalein acidity’ are recorded in table 6 (also fig. 5). It is apparent (especially for the B. coli infusions) that the acidity curves do not show any striking variations from those obtained from cultures promiscu- ously seeded with various forms of protozoa and bacteria. TABLE 6 Phenolphthalein acidity. Bacterial infusions (Cubie centimeters 0.01 N NaOH per 100 ce. infusion) DAYS o| 1| 2 3) 4| 5| 6) 7] 8 9/10/11 12 19 2848 Sreriletles ten tome Ie? lial alee tye fetal nee 7 6 6 7 Peres Died ? € Oo re =) os oO =} 3 2 Ww 72} (9-2) £ is Ze wo Saiktor 11 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 fibér 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 described 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. I have 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 7 9@, female-producers 9 @. | ORIGINAL LINE 1 INBRED LINE DISS | Number of 9| Number of 2 Pa EENGO! Eos | Number of o&' 9} Number of 9 2 young | | young October October | 24 1 8 24 | 0 28 26 4 30 25 10 36 28 0 28 | Pil 2 Zit 29 0 48 | 29 2 17 * Sil 5 40 | 30 2 43 November | | | November 2 | 0 193) 1 3 44 4 1 15 3 a 38 6 | 7 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 3, val 20 0 23 25 6 39 | 22 1 11 26 2 28 24 4 10 28 iR 2B 26 1 7 30 4 | 4 28 9 25 December | 30 0 41 By 0 27 _ December 4 3 40 2 2 33 7 3 23 | 4 | 1 43 9 | 11 4 \ 6 20 20 12 1 | 22 9 | it 10 13 0 5 i 2, 0 29* 11 2 13 1{:55 0 15e 13 0 17 15 3 14* Rotalersm: 88 736 le 7 732 Per cent of CoG Bootes 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 | o OF OF ea Oo OF % em il D oa || % Or Date of first = = || Date of first 6 ig | Date of first | © Ss young 5 & young 38 1 young B & 3 g 5 g g Z Z | Z Z Zi | Z November | November | November | | 28 5 23 || 28 9 25 28 i; @ 15 30 4 4 | 30 0; 41] 30 ee) 20 December | December | December 2 | 0 27 || 2 2 ile 33a 2 7 39 4 | a || 2 a) 4 1 43 || 4 2 18 1. | 3 23 || 6 20 20 || 6 2 31 9 11 4 | 9 11 10 8 26 12 1 22 | if | 2 || 9 1 23 13 0 | 5 11 2 13 || 11 | 5 8 0 29*| 13 0 17 | 13 vara 16 115) | 0 15%) 15 By alae] 15 0 3 | | 9 3% Motal.>......| 27 | 199% 65 | 218 | 50 | 202 i“ | Per cent of & Q| 12.3 22.9 * Remainder of family not recorded. ment, there were 18.2 per cent of male-producers; and in the inbred line, including 19 earlier generations, 16.7 per cent. In the ‘twice inbred’ line, which is given in full in table 3, there were 19.8 per cent of male-producers. It may be pointed out incidentally that in the original line as a whole, not including the last two generations which were incom- pletely recorded, the average size of family was 34.5, in the inbred line as a whole 30.7, and in the ‘twice inbred’ line 24.0. This seems to indicate a loss of vigor, perhaps due to inbreeding. Experiment 3. Inbreeding in another line. In this experiment the comparative ages of the inbred line and the line from which it was derived are not known. The line designated A in table LIFE CYCLE OF HYDATINA SENTA 295 TABLE 4 Showing number of male- and female-producers in two lines of Hydatina senta, one line being derived from the other by inbreeding, as in table 2 | LINE A | INBRED LINE mate of rst Number of o) Number of 2 9| Date of first Number of o'9| Number of ? 2 young | | young | June | October | 17 7 14 12 35 16 19 11 | 17 | 14 | 23 25 21 20 22 | LG 0 21 22 25 | 11 i 18 33 16 23 25 | 24 } 20 | 1 11 25 1900" 9 | | 30 22 26 21 | 14 | 22 | 43 8 27 29 19 | 27 | 0 16 | | 29 23 14 eee. = | } | — ALO Gall terate'a's 157 130 | | 188 149 : az =m |p I Per cent of | Sot ee 54.7 | B57 4 was derived from a female taken from a culture in the labora- tory about June 1. This culture originated from a collection of rotifers taken at Grantwood, New Jersey, in the latter part of March, 1911, and transplanted to manure cultures in Ann Arbor shortly afterwards. These manure cultures were maintained without observation until June, when the female that became the parent of line A was isolated. Females of line A were paired with males of the same line about June 25 to June 27, and fertilized eggs obtained. These eggs were allowed to dry in the dishes and to remain dry until the last of August, when they were again covered with water. Females began to hatch from them in a week, and from one of them the ‘inbred line’ of table 4 was started. Completed records of this line were not kept until October 12, after which date eight com- plete generations were recorded. The two lines in table 4 may not be of the same age; either one may be the older. Furthermore, since they were not bred simul- taneously, the conditions may not have been the same. How- 296 A. FRANKLIN SHULL ever, as the food cultures were frequently changed, each of these recorded lines must have been fed from three or more cultures. The averages of these three or more cultures should, I believe, be nearly equal. The evidence, though not complete, is presented for what it is worth. Line A included 54.7 per cent of male- producers, the inbred line 55.7 per cent. Incidentally it may be pointed out that the average size of family in line A is 35.8, in the inbred line 37.4. On the whole it seems that inbreeding does not markedly alter the proportion of male-producers, though perhaps had the inbred lines been bred as long as were the original lines, the decrease in the proportion of male-producers which sometimes accompanies long-continued parthenogenesis might have shown aslight decrease in that proportion as a result of inbreeding. Effect of long duration of the fertilized egg stage on the proportion of male-producers 7 DURATION OF THE FERTILIZED Eaa Stace. Whereas parthe- nogenetic eggs hatch pretty uniformly in twelve to fourteen hours after laying, great variability has been found in the length of time which fertilized eggs from the same source, or from different sources, spend in-the egg stage. Thus, in the cross between New York females and Baltimore males described in an earlier paper (Shull, ’11 a), 408 eggs were obtained from 38 matings made from May 14 to May 17, inclusive. On May 24, three of these eggs hatched; and each day thereafter, with four exceptions, up to June 10, one or more eggs hatched. In the seventeen days from May 24 to June 10, 53 eggs, laid by 19 out of the 38 females, hatched. The remaining 355 eggs were kept two months longer, to August 10, but no more of them hatched, and the lot was then discarded. In no case did all the eggs laid by one female hatch, the highest record being six out of seven; while 19 of the females laid eggs none of which hatched within twelve weeks. It is prob- able that many of these would never have hatched. About the same time, May 12 to May 15, 25 females of the Bal- timore line were inbred, that is, paired with males of the same line. LIFE CYCLE OF HYDATINA SENTA 297 They laid 298 eggs, none of which hatched before August 10, at which time they were discarded. ©) (OE 10} ©) (CP (GN LON) OTS FON MONON 1) XO} 10} (0) 19," 19) (0) 8) ©) ODIO) GE ‘oA OM oPaci ie) HoMiey (cater roo} CMC! o/s ep to} Ho TRO Mron fesse oii) HO) 0) <2) 5 2 OQ ©) © 2) ©) Erie O GS) © WN Moo) fe} () (0) te} (©) ©) © (9) © © CS) C) ©) BVO) @ CONCH CN) CC) 1) ©) © @ 0 © 1) OO) © © 6 POV GH Oop!) WS) 19) N19) (2) Oh VC 7 PQPPPAHAAHAAPASPPSPAAPAA SPSS LPSRLPASPRAVPSRLP LALLY Lo} 10} 19} (0) (9) 19) OG} 19) @) @ ) OC) O} (9) TABLE 9 Showing the number, and order of production, of male- and female-producers of Hydat- ina senta, in seven families, all of which were reared to maturity in spring water. The parents are sisters of the parents in table 8. The vertical line divides each family at the point where, in the corresponding family in table 8, the female was trans- ferred to manure solution. Male-producers are designated by &, female-pro- ducers by @. NUMBER OF FAMILY Je SISAL OS OSS SOAS SOS OO ome odo Pict 22 PVA? Zn POMP PPISLLQIPAAIP AA III SHI SISA AGS SLAG LE LY ; | PPPQBQMPVEESEEQSLE 3 POPP LPPPLPLPAHL LMG AMIS LR PPL IAPAHAHAHAA EAP PLY | 22 Eee 4 OPP PPPASPASPAPAHAP PS VPPMPIAIPSPPSPPLPP PP LPLAGAAGA | PI AAARS CSS S ISM Mo sR Mo ero Motto op lowretictiotiot ot opomoslomosrop Ca PF ORO 2 Oia" OO PO icion Lichen eS OS SIN EN Giro CARON ON CMON Sul fo i} (0519) (0) Terie) (oh fousey se) j © “I o> Cr | 2999 999999999999 306 A. FRANKLIN SHULL line at the point where, in the corresponding family in table 8, the female was transferred to manure solution. The two tables (8 and 9) together show in an unmistakable manner that male-producers have been quickly excluded from the latter part of the families in table 8 by transferring the parents to manure solution. Only one of the young produced in the man- ure solution was a male-producer, and that one hatched from the very first egg laid after the transfer. This one case is important as indicating that the nature of a female is determined prior to the laying of the egg from which she hatches; or if that determina- tion is a gradual process, it has proceeded so far prior to the lay- ing of the egg that manure solution is unable to reverse it. Experiment 7. In this experiment a line was bred in manure solution. From each generation two sisters were reserved for breeding. One of these females was kept throughout life in manure solution, and all her offspring were reared to maturity in the same solution. The line thus reared consisted of 121 individuals, all female-producers, showing that the manure solu- tion was strong enough to exclude male-producers from the fami- lies then being reared. The other female, of the two mederved for breeding, was kept in manure solution until she had laid from 1 to 16 eggs, and was then transferred to spring water, where she produced the rest of her family. The eggs laid in manure solution were hatched, and the young reared to maturity, in m&nure solution. The eggs laid in spring water were hatched, and the young reared to matur- ity, in spring water. The details of this experiment are given in table 10. The vertical line divides each family at the point where the parent was transferred to spring water. Many of the females hatched from eggs laid in spring water were male-producers, notwithstanding that their parents had previously been in manure solution strong enough to exclude male- producers. This indicates that the nature of a female is not determined in the very early (odgonial) stages of the egg from which she hatches. Of particular interest in this connection is the second family of table 10, from which it appears that the very first egg laid after the mother was transferred to spring water LIFE CYCLE OF HYDATINA SENTA 307 yielded a male-producer. It is quite possible that an error was made in determining the relative ages of these first individuals, for, as stated above, the relative ages of the young rotifers iso- lated at one time was determined from their relative sizes. When the oldest rotifers were much alike it was sometimes difficult to determine relative ages. But in any case, I do not think it is possible that I have misplaced this individual by more than one step in order of age. That is, the first male-producer in the sec- ond family of table 10 can hardly have been later than the second young produced after the mother was transferred to spring water. TABLE 10 Showing number, and order of production, of male- and female-producers in seven families of Hydatina senta, in which the early eggs were laid and hatched, and the young reared to maturity, in manure solution, the later eggs laid and hatched, and the young reared to maturity, in spring water & oe LAID, HATCHED AND REARED IN MANURE LAID, HATCHED, AND REARED IN SPRING ga SOLUTION WATER a 1 Coll Ko mo Momo) cro propte) (ol Key tool cued oploNTors | fe) omen Oooo 2at\\ PPO QVPPQPASPPVIMSF SLAP APH AAA PLL PLL LAE | 229229999 3 Lo) May toyster" {oo} ey (0) Con Koy fo Lolaony Konmon to (fe) (0)"(9) (2) 4 POP PPP ViPVPVPVPVVPVSVPeVeePeee 5 POPOV OVOP SPQVVPMIPRAP PLASM LAH APPL AAGA EP 6 PO QPVPWPPMPAPAVPOPVPVPAMHP MSG ASPLRASPASP SPS APAS LLP LP ASL LAA OO OVORO OOF OTO O IOE OF ONS uf POO QPPWPHPRPAVPWMHSRASPVPMIVPSP ASS SRLPASPSLPSP HS LPL LAA | FIPFLAPLPAHPQLPAGIAS LAA The average number of eggs laid by these females on the days when they were transferred to spring water was 14.4 per day. That is, an interval of 1.66 hours elapsed between the laying of two successive eggs. Since the first male-producer in the second family in table 10 was not later than the second one produced after its mother was transferred to spring water, the nature of this female was not determined until within 2 x 1.66 hours, or 3.32 hours, before laying. 308 A. FRANKLIN SHULL Experiments 6 and 7 together indicate that the nature of a female (with respect to the kind of offspring she will produce) is determined before the egg from which she hatches is laid, but not until within several hours of the time when the egg is laid. Or, if this determination is a gradual process, it has proceeded so far before the egg is laid that manure solution can not reverse it, but has not proceeded so far until within several hours of lay- ing, but that manure solution can reverse it. Microscopic exami- nation of the living animals, which are so transparent that the eggs and odgonia may readily be seen, shows that the last several hours of the egg stage, within the parent’s body, includes the entire growth period. DISCUSSION The decrease in the proportion of male-producers with long- continued parthenogenesis, which was shown to occur in some parthenogenetic lines of Hydatina, is of interest from several - points of view. First, may not this decrease account for part of the differences observed between parthenogenetic lines in cases where the ages of the lines are not known? If one line, started immediately from a fertilized egg, be compared with another removed by a hundred generations from the fertilized egg, the latter line might be expected to show fewer male-producers, even if in their early generations both lines -had been equal. If differences between parthenogenetic lines may thus be sec- ondarily produced, how does this phenomenon affect the results of crossing reported in my earlier paper (Shull, ’11 a)? That depends on the relative ages of the lines. The Baltimore line was started from a female collected in March. The winters are sufficiently rigorous in Baltimore, I think, to prevent continued reproduction during that season. A female collected in spring, therefore, must descend from a fertilized egg that hatched prob- ably not earlier than February of the same year. The Balti- more line can hardly have been more than a month or two old when I obtained it. Regarding the age of the New York line there is less certainty. The parent of this line was found in Janu- ary in a culture in the laboratory, which had been stocked with LIFE CYCLE OF HYDATINA SENTA 309 rotifers more than two years before, and to which none had been added since. This culture had been examined many times for rotifers, but none were seen until the single specimen which pro- duced the line recorded as the New York line was found. I am inclined to think, therefore, that this female had recently hatched from a fertilized egg, and that the New York line was accordingly about a month older than the Baltimore line. Whether this difference inage may account forthe difference in the proportion of male-producers between the two lines is uncertain. That differences in the proportion of male-producers not depend- ent on differences in age may exist between two lines is shown, however, by another experiment, in which the F, line was crossed back to the New York line, and in several other cases not recorded in that paper. In the cases to which I refer, the older line pro- duced more male-producers than the younger line. Uncertainty as to the age of the original lines, therefore, can not invalidate the conclusion that differences dependent on an internal agent do exist between parthenogenetic lines; it merely modifies our conception of the nature of those differences, a sub- ject that is discussed elsewhere. Some of the long parthenogenetic lines recorded in table 1, it is to be noted, do not show an evident decrease in the proportion of male-producers; nor do they show an increase. Each of these lines began with a low percentage of male-producers, and could not have decreased much. These lines showed considerable fluctuations in the proportion of male-producers, periods of few male-producers being followed by periods of many. If such a fluctuating line began with a long period of few male-producers, a decrease in the proportion of male-producers.could only be discovered by breeding it through a large number of generations, including several waves of male-producers, and finding that suc- cessive waves were less marked. Forty-six generations are hardly enough for this. The progressive decrease in the proportion of male-producers is so marked in some cases, and its absence in some lines so easily explained, that I am inclined to regard it as a general phenomenon. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 2 310 A. FRANKLIN SHULL Just such a progressive change occurs in daphnians (Wol- tereck, ’11), but here the number of sexual individuals increases with the age of the line, instead of decreasing as in Hydatina. That the change should be in the opposite direction in the roti- fers and daphnians need hardly surprise one, since other phenom- ena are reversed in the two groups. For example, late females in a family of daphnians produce more sexual daughters than do their older sisters, while late females in a family of Hydatina produce fewer male-producers (sexual females) than do the early females. The progressive decrease in the proportion of male-producers may also have a bearing on pure line* work in general. So far as we know there is no method by which parthenogenesis may change the genotypic constitution of a line, yet parthenogenetic lines of Hydatina do change. May not pure lines suffer progres- sive change, notwithstanding they are composed of homozygous individuals? If so, there can be differences between two pure lines having the same genotypic constitution, even when both are reared under the same external conditions. If this were found to be true, it would not invalidate the conclusion that pure line differences exist, but would modify our explanation of them and their apparent behavior in inheritance. How this progressive decrease in the proportion of male-pro- ducers is brought about is not known. At first, it seemed that the proportion of male-producers might be determined by the vigor of the parthenogenetic line. The view that long-continued reproduction, whether bi-sexual, parthenogenetic, or vegetative, without the introduction of new ‘blood’ in crosses, is detrimental to vigor, is often expressed, even if not always correct. Such a loss of vigor seems to occur in Hydatina, as evidenced by the decrease in the size of family in successive parthenogenetic gen- 3 In an earlier paper I have spoken of a series of parthenogenetic generations in Hydatina senta as a pure line. While parthenogenetic species do not meet the requirement of Professor Johannsen’s definition of a pure line, there seems to be no abuse of the fundamental conception of pure lines in applying the term to par- thenogenetic species. The term klon, orclone, used by plant geneticists to denote vegetatively produced varieties, can hardly be used for Hydatina, since there is a wide step between parthenogenesis and vegetative reproduction. Under these circumstances I have preferred to use the term parthenogenetic line in the present paper. LIFE CYCLE OF HYDATINA SENTA St erations (figs. 5 and 6). The simultaneous decrease of vigor and of the proportion of male-producers could be readily ‘explained’ in part by assuming that the two phenomena are correlated. Opposed to this view is the fact that inbreeding in Hydatina seems on the whole, to result in a diminution of vigor, whereas the proportion of male-producers appears on the average to be unaltered by inbreeding. Such could not be the case if any true correlation between the two phenomena existed. A further objec- tion to the view that vigor and the proportion of male-producers are correlated is found in the results of experiments (3 and 4) in which lines derived rom females that remained long in the fer- tilized egg are compared with lines derived from females that hatched quickly from the egg. If the duration of the egg stage is inversely proportional to vigor, as one might expect, a correla- tion between vigor and the proportion of male-producers would result in a lower percentage of male-producers in parthenogenetic lines derived from late hatching females. This appears not to be the case. The decrease in vigor due to inbreeding may be ac- counted for if we assume that vigor is due to the degree of hetero- zygosis of the individuals, as has been found to be the case with corn (G. H. Shull, 708). But this assumption will not explain the decrease of vigor with long-continued parthenogenesis (figs. 5 and 6). We have seen that the internal nature of a parthenogenetic line of Hydatina is subject to some degree of change dependent on the age of the line, but that initial differences also exist among differ- ent lines. We may thus conceive the internal nature, as far as it concerns the life cycle, to be composed of at least two parts: First, the genotypic constitution, determined at the moment of fertilization, and, barring irregularities partaking of the nature of mutations, remaining constant through many generations; and second, a changeable element which is probably to be included in Woltereck’s reaction-norm. We may either add to these a third element which causes the great fluctuations (‘waves’) in the pro- portion of male-producers, or assume that the reaction-norm is itself very variable. Si A. FRANKLIN SHULL Of the second factor, little can be said except in a descriptive way. In Hydatina it progressively changes so that the propor- tion of male-producers decreases with the age of the partheno- genetic line: Whether this change is due to continued breeding under uniform conditions, or to some other cause, is not known. Little more can be said of the variable element, whether separate from or only a featureof the progressive one. Fluctuations in the ‘sexuality’ of daphnians occur, such’ that periods of few sexual forms may alternate with periods in which sexual individuals are numerous. Woltereck (11) attributes the form of the cycle to antagonistic substances, now the one, now the other gaining the ascendancy in a rhythmical manner. I have found in Hydatina just such fluctuations, which I have not been able to trace to any external agent. Nevertheless, it appears that the extent of the fluctuations is not independent of external conditions. Thus, in my earlier starvation experiments (Shull, ’10, fig. 1), both the starved and the well-fed lines show simultaneous fluctuations in the same direction, but in every case the wave is more marked in the well-fed line than in the starved. Even if the external con- ditions (chemical substances in the water, for example) are not the cause of this fluctuation, they do modify its amplitude. Regarding the first element of the internal nature of Hydatina, the genotypic constitution (zygotic constitution of Punnett, ’06), we fortunately have more evidence. The crossing experiments described in my former paper (Shull, ’11 a), together with the results of inbreeding described in this article, enable us at least to eliminate certain possible views regarding the internal cause of the form of the life cycle. The proportion of male-producers can not be dependent on the simple quantity of some substance present. For it is difficult to see why, in some crosses, the F, line should be intermediate in the proportion of male-producers between its parent lines, while in other crosses the proportion in F,; should exceed not only that of either parent line alone, but of both parent lines combined (op. cit., Experiments 36 and 35). Among Mendelian explanations, it can not be assumed that the life cycle in a given line is dependent on a single gene or a pair of LIFE CYCLE OF HYDATINA SENTA ils genes, representing a certain proportion of male-producers. For this explanation could not account for intermediate F, in some eases, and an F, higher than both parent lines in other cases. If we assume that many genes participate in the production of the cycle, many of the results so far obtained are easily explained. If we think of these genes for male-producers as being all alike, equipotent, and additive in their effects, so that six genes pro- duce twice as many male-producers as three genes; we should then have to assume, in order to explain the crosses described in my former paper, that the percentage of male-producers is pro- portional to the number of genes for which the line is heterozygous. This explanation seemed plausible when it was thought that vigor and the life cycle were correlated; for Mm corn it seems probable that vigor is dependent on the degree of heterozygosis. The crux of this explanation is found in the results of inbreeding. If the per- centage of male-producers is proportional to the number of genes for which the line in question is heterozygous, inbreeding, by reducing the number of genes for which the line is heterozygous, should rapidly reduce the proportion of male-producers. This it does not do. Inbreeding results in a line that includes practi- cally the same proportion of male-producers as the line from which it was derived. Even twice inbreeding, or inbreeding a line itself the result of inbreeding, does not certainly show a reduction in the percentage of male-producers. It can not be assumed, therefore, that the genes for the propor- tion of male-producers are all alike and effective in proportion to their numbers. Instead, we may assume that the life cycle is dependent on a number of genes not all alike, some being more effective than others, and some combinations producing more male-producers than other combinations, even when these combi- nations involve the same number of genes. That something akin to segregation of these representatives of the cycle occurs, is made probable by the fact that crosses between the same par- thenogenetic lines are not equal with respect to the proportion of male-producers. That the genes are not alike nor additive in their effects is shown by the fact that a cross may result in a higher proportion of male-producers than in both parent lines combined. 314 A. FRANKLIN SHULL The effect of crossing on the cycle can not be predicted, therefore, from the form of the cycle in the lines to be crossed, but only after tests are made by experiment. Whatever be the nature of the genotypic constitution, the form of the cycle in a parthenogenetic line having a given constitution is dependent in part upon the environment. It was earlier shown that certain chemical substances were capable of reducing the proportion of male-producers. From evidence presented in this paper, we may now conclude that the effect of these substances is felt only during the growth period of the egg. Once the egg has reached its full growth, or at least after it has been laid, chemical substances which, when applied throughout life, exclude male-producers are powerléss to change the nature of the female hatching from the egg. In like manner, these substances are powerless to affect the nature of a female before the egg from which she hatches begins its growth. So far as these chemical substances are concerned, the fate of an egg is irrevocably determined in its growth period. Since the maturation spindle is formed in these eggs before they are laid (Whitney, ’09), it is not impossible — that the influence of external agents is limited to the matura- tion period. This localization of ‘sex-determination’ in the growth period is of interest in several connections. First, it shows why the starvation experiments of Punnett (’06) and Whitney (07) did not result in an increased proportion of male-producers, as did the experiments of Nussbaum (’97) and myself (Shull, 710). Even if starvation, as carried out by the former two investigators, so altered the chemical composition of the water that a change in the life cycle might have been expected, nevertheless it was not applied at a timewhen it might havebeen effective. Inthe experi- ments of Punnett and Whitney, the females were starved only during the first few hours after hatching, not when their eggs were in their growth period. The localization of the period susceptible to external agents also goes to disprove my former explanation of the observed fact that late daughters of a family yielded fewer male-producers than did their sisters of the early part of the family. I assumed that LIFE CYCLE OF HYDATINA SENTA 315 the accumulation of certain chemical substances in the cultures as these became old might cause the offspring of the late females to be more largely female-producers than the offspring of early females. In the light of the discovery that the period suscepti- ble at least to certain chemical substances is limited to the growth period, my former explanation regarding the later females of a family would account for a preponderance of female-producers in the last part of the family, but not for a preponderance of females _ that produce female-producers. A preponderance of female- producers in the last part of the family, as compared with the early part, does not occur, as was shown by compiling data from 349 families (Shull, 710). A comparison with the Cladocera with respect to the suscepti- ble period will be of interest. The Cladocera do not lend them- selves to an inquiry of this kind as readily as do the rotifers, for the offspring of a daphnian are not all of one sex. However, according to Woltereck (11), Daphnia has two ‘labile’ periods, one just before the eggs enter the brood chamber, the other very much earlier, in the odgonial stages. It seems not improbable that the labile period immediately prior to the entrance of the eggs into the brood chamber falls within the growth period, as in Hydatina. And finally, not the least valuable result of the discovery that manure solution is effective only in the growth period of the egg, is that a way now seems open to discover the manner in which chemical substances affect the life cycle. The question whether these substances alter the events in a given cell, or whether they merely decide which of two already differentiated classes of cells shall develop, bids fair to be answered. If there are two classes of cells already differentiated, and manure solution prevents one of them from developing; and if eggs may come to the growth period before being affected by manure solution; then females from a line producing many male-producers, if placed in manure solution, should frequently show traces of degenerating eggs, or of eggs that do not develop and must be pushed aside to make room for cells of the other class. Observations on this point are now in progress. S16 A. FRANKLIN SHULL SUMMARY A progressive decrease in the proportion of male-producers with long-continued parthenogenesis occurs in some lines of Hydatina, perhaps in all. It is not improbable that differences between parthenogenetic lines may thus secondarily arise, which are independent of both genotypic constitution and the immediate external environment. A progressive decrease in the size of family with long-continued parthenogenesis occurs in some lines. There is apparently no correlation between decrease in size of family (decrease of vigor) and decrease in proportion of male-producers. The time required by fertilized eggs to hatch varies from a few days to many weeks. The length of time required for a fertilized egg to hatch is prob- ably not correlated with the proportion of male-producers in the parthenogenetic line derived from the egg. Parthenogenetic lines derived from fertilized eggs that require a long time to hatch may be less vigorous (as measured by size of family) than those from early hatching eggs. Individuals hatching from fertilized eggs are not only all females, as previously known, but are all female-producers. Whether a female is to be a male-producer or a female-pro- ducer is irrevocably decided (so far as manure solution is con- cerned) in the growth period of the parthenogenetic egg from which the female hatches. Sex is determined a generation in advance. BIBLIOGRAPHY Maupas, E. 1891 Sur la déterminisme de la sexualité chez l’Hydatina senta. Comp. Rend. Acad. Sci., Paris, T. 113, pp. 388-390. Nusssaum, M. 1897 Die Entstehung des Geschlechtes bei Hydatina senta. ° Arch. f Mikr. Anat. u. Entw., Bd. 49, pp. 227-308. Punnett, R. C. 1906 Sex-determination in Hydatina with some remarks on parthenogenesis. Proc. Roy. Soc., B, vol. 78, pp. 223-231. LIFE CYCLE OF HYDATINA SENTA 317 Suuut, A. F. 1910 Studies in the life cycle of Hydatina senta. 1. Artificial control of the transition from the parthenogenetic to the sexual method of reproduction. Jour. Exp. Zool., vol. 8, no. 3, June, pp. 311-354. 1911 a wu. The rdle of temperature, of the chemical composition of the medium, and of internal factors upon the ratio of parthenogenetic to sexual forms. Ibid., vol. 10, no. 2, February, pp. 117-166. 1911 b The effect of the chemical composition of the medium on the life cycle of Hydatina senta. Biochem. Bull., vol. 1, no. 2, December, pp. 111-136. Suut, G. H. 1908 The composition of a field of maize. Amer. Breed. Assn., vol. 4. Wuitney, D. D. 1907 Determination of sex in Hydatina senta. Jour. Exp. Zool., vol. 5, no. 1, November, pp. 1-26. 1909 Observations on the maturation stages of the parthenogenetic and sexual eggs of Hydatina senta. Ilbid., vol. 6, no. 1, pp. 137-146. Wotrtereck, R. 1909 Weitere experimentelle Untersuchungen iiber Artver- anderung, speziell ttber das Wesen quantitativer Artunterschiede bei Daphniden. Verh. d. deutsch. zool. Gesell., pp. 110-172. 1911 Uber Veridnderung der Sexualitit bei Daphniden. Experimen- telle Untersuchungen tiber die Ursachen der Geschlechtsbestimmung, Internat. Rev. d. ges. Hydrobiol. u. Hydrogr., Bd. 4, Heft 1 and 2. April and June, pp. 91-128. ? i i ay tee a ro . « ‘ "i Aaa hy P wor j ‘ f re rhe ' e574 ae pt ’ ‘ Pale ’ 7 « * S ae ¥ a A r ior ls ha” Pah / 4 é ; x 7 7 e , ; i : ¢ f , mi i rT Aes he ee ae wae By , a ae v8 Ps s.. * ~ 3 - “ALF 4 wb 4 5 STUDIES ON SEX-DETERMINATION 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 LENTIGINOSUS HELEN DEAN KING From The Wistar Institute of Anatomy and Biology The investigations recorded in the present paper are a contin- uation of those that have been carried on for several years. past in an attempt to ascertain whether external factors can influence the determination of sex in the toad, Bufo lentiginosus. All of the experiments were made with the eggs from two females, a and b; the eggs from each female being fertilized with sperm from the same male. The individuals derived from the eggs of female a are considered to belong to the ‘series A’ group of experiments; while ‘series B’ refers collectively to the indi- viduals that developed from the eggs of female 6. It was not possible to note the exact number of eggs used in ‘any experi- ment, but an attempt was made to use approximately the same number of eggs in each case, and to estimate, as accurately as possible, the number of eggs that failed to develop. The apparatus that was used in rearing the tadpoles was de- scribed in detail in a previous paper (King, 711). As this appara- tus has its limits of capacity, it was not possible to use all of the embryos that developed from each lot of eggs. Definite numbers of individuals, forming in every case except the acid experiments at least 75 per cent of the total number of eggs that had been experimented upon, were taken at random as they emerged from their jelly like membrane three days after the experiments were begun. In the various tables in this paper the figures given in the column headed ‘total number of individuals’ refer, there- 319 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 3 APRIL, 1912 320 HELEN DEAN KING fore, to the number of tadpoles taken for rearing, and not to the number of eggs that had been used in making the experiment. The tadpoles lived chiefly on spirogyra, nitella, and various other water plants taken from ponds in which toads normally breed each year. Occasionally they were fed on finely ground fish or frogs’ muscle; but food of this kind that was not eaten within two or three hours was removed, as it fouled the water very quickly and so increased the mortality. Experience has shown that water plants, with their accompanying hordes of micro-organisms, form a food supply for toad tadpoles much superior to that used in any former experiments. (King, ’07 b, 09 SAO): In Bufo the sexes cannot be distinguished until the tadpoles are approaching metamorphosis, and even when young toads live until their tails have been absorbed it is necessary to section the gonads in a considerable number of cases in order to ascertain the sex. Several attempts were made in past.years to feed young toads so that they might live until their gonads were well differ- entiated; but such attempts were failures, owing to the difficulty of obtaining a sufficient supply of small insects to feed a large number of individuals. Last spring it was found that young toads would eat various species of aphids and grow rapidly on a diet composed chiefly of these insects. As this food could be obtained in considerable abundance, nearly all of the individuals used in the various series of experiments were kept alive for about three weeks after they had completed their metamorphosis. By this time the sex glands were so well differentiated that the sex could readily be ascertained by examining the gonads in toto under a dissecting lens. The period of metamorphosis seems to be a critical one in the life history of toads reared under artificial conditions, as there is always an increased mortality at this time. All of the individu- als that died at this stage of development were preserved in Tellyesnicky’s fluid, which has been found far superior to corro- sive-acetic as a fixative for the gonads of Bufo, and their sex ascertained by means of sections. SEX-DETERMINATION IN AMPHIBIANS a yal Altogether 4224 tadpoles were used in the various experiments, and of this number 3784 individuals were carried through to metamorphosis and their sex ascertained. The mortality in the entire series was, therefore, only 10.41 per cent, which is much less than the mortality that must occur in any lot of eggs laid under natural conditions. Whatever explanation may be offered for the unusual sex ratios obtained in some of these experiments it is evident that they cannot be ascribed to selective mortality. According to Davenport (’97), water forms from 60 per cent to 90 per cent of the whole mass of protoplasm in nearly all kinds ‘of cells and is of the utmost importance for the various chemical processes taking place in the living organism. It is conceivable, therefore, that placing eggs under conditions that would alter their water content just before or during the time of fertilization might favor the development of one sex or the other, if it be that the sex of an individual depends upon some definite metabolic process occurring during the fertilization period. Some investi- gations made along this line last year gave such suggestive results that this past spring I confined my experiments with the eggs of Bufo to an attempt to ascertain whether the normal proportion of the sexes would be altered if the water content of the eggs was changed at or before fertilization. For convenience in description these experiments are divided into two classes: (1) those in which the water content of the unfertilized eggs was affected; (2) those in which the eggs were subjected to conditions that altered their water content during the fertilization period. 1. EXPERIMENTS ON THE UNFERTILIZED EGG Former experiments in which unfertilized eggs of Bufo were sub- jected to the action of hypertonic solutions of salt and of sugar gave results which strongly suggested that the normal sex ratio had been altered by the treatment which the eggs had received previous to their fertilization (King, 711). Unfortunately the mortality at the time the eggs were fertilized, and also among the tadpoles during the early stages of their development, was ore HELEN DEAN KING very great. One could not exclude the possibility, therefore, that selective mortality was responsible for the results, even if there is no evidence that mortality is ever selective in amphibian tad- poles reared under artificial conditions. In making the experiments mentioned above it was found that solutions of salt and of sugar as strong as 24 per cent could not be used on the unfertilized eggs for more than five minutes without rendering the great majority of them incapable of being fertilized. In continuing these experiments very weak solutions were em- ployed so that the mortality at the time of fertilization might be decreased. A batch of about 400 eggs, taken from female a, was placed in a 2 per cent solution of cane sugar; another batch of approxi- mately the same number of eggs was put in a 2 per cent solution of NaCl. Each lot of eggs remained in the solution for ten min- utes, and was then quickly washed off in running water and fer- tilized in tap water. At least 95 per cent of the eggs that had been subjected to the action of the salt solution segmented nor- mally. Comparatively few of the tadpoles died during the early stages of development, and the entire number of individuals in which sex was not ascertained was only 12 per cent. The results obtained with the eggs that had been placed in the sugar solution were even more satisfactory. Not more than 2 per cent of the eggs failed to develop, and only 7 per cent of the 300 individuals that were taken for rearing died before it was possible to ascer- tain their sex. In each of these lots, as shown in table 1, a per- centage of females was obtained that was considerably higher than that found among the toads that served as control for the exper- iments in this series. The latter individuals were developed from eggs of female a which had been fertilized in tap water with sperm from the same male that was used in the fertilization of all of the other eggs taken from this female. Eggs, taken from female 6, were subjected to the action of a 2 per cent solution of sugar for twenty minutes and were then fertilized in tap water; only about 5 per cent of these eggs failed to segment. In 53, or 22.40 per cent, of the 250 tadpoles that were taken for development, the sex was not ascertained. This SEX-DETERMINATION IN: AMPHIBIANS 323 loss was due, in great part, to an accidental contamination of the water in two of the dishes containing the tadpoles and not to the treatment that the eggs received at the time that they were ready for fertilization. The lot of toads carried through metamorphosis gave a percentage of females nearly 10 points above that in the control for the series, and slightly greater than that found among the individuals belonging to the corresponding experiment in series A (table 1). Owing to the fact that solutions of NaCl are much more injurious to the eggs of Bufo than are sugar solutions, the ex- periment in which eggs from female b were immersed for twenty TABLE 1 Eggs treated with hypertonic solutions before fertilization | iB om ‘. & fe 4 SERIES SOLUTION USED < A a oe Z is A iy | § aS ERS a of as z a a a a x ua Bs a o* ee < B a Be >] a a a | ps as an min. | A 2 percent sugar | 10 300 279 | 131 | 148 03.04 | 88.51 A 2 per cent salt 10 200 176 70 =| 106 60.34 66.03 B 2percentsugar | 20 250 =| 194 86 | 108 55.66 | 79.62 A | Control 350 | 322 | 169 | 153 | 47.50 | 110.45 B | Control 350 334 | 178 | 156 46.40 114.10 minutes in a 2 per cent solution of NaCl before fertilization was a failure. Only a few of the eggs segmented, and as all but twelve of the embryos died during gastrulation the experiment had to be abandoned. In each of the experiments outlined above there was found among the individuals carried through to metamorphosis a per- centage of females considerably above that in the control lot. These results accord well with those obtained in former experi- ments with hypertonic solutions (King, 711), although the per- centages of females are somewhat lower, owing possibly to the fact that the eggs were treated with weaker solutions. 324 HELEN DEAN KING Taking the sex ratio for any lot of individuals as the number of males to each 100 females, it is found that the toads derived from eggs that were subjected to the action of the salt solution before fertilization give a much lower sex ratio than that occurring among the individuals developed from eggs which had been treated with sugar solution (table 1). If this difference can be attributed to the fact that the osmotic action of salt is several times greater than that of sugar, it follows that the more water that is extracted from the egg just before fertilization the greater becomes its tendency to produce a female rather thanamale. On this assump- tion it is the egg, and not the sperm, that contains the sex-deter- mining mechanism. In Bufo, therefore, as in the sea-urchins according to the recent investigations of Baltzer (’09), the female is heterozygous as regards sex and the male is homozygous. The above interpretation of these results is not the only one that can be given, although it seems to me to be the most plausible. Selective mortality cannot be held responsible for the sex ratios obtained, since in none of the experiments was the mortality sufficiently great, either at the time that the eggs were fertilized or during the development of the tadpoles, to have appreciably affected the results. There are two possible explanations for these results that do not involve the admission that external fac- tors can influence sex. It is conceivable that subjecting eggs to the action of hypertonic solution just before their fertilization may have rendered them more easily penetrated by spermatozoa that were female-producing than by those that were male-pro- ducing, assuming that the spermatozoan determines sex as the current chromosome sex theory demands. This means, however, that fertilization must here be considered as selective, though Wilson (’10) has recently shown that selective fertilization is most improbable in any form. A study of the spermatogenesis of Bufo (King, ’07) has not shown any dimorphism of the spermato- zoa that might be associated with sex-determination; neither has such a dimorphism been found in the spermatozoa of any am- phibian so far investigated. It seems somewhat absurd, there- fore, to assume the existence of dimorphic spermatozoa in Bufo SEX-DETERMINATION IN AMPHIBIANS ozo in order that the result of these experiments may be ascribed to selective fertilization. There remains the possibility that the sex ratios in these lots of individuals were chance variations in the normal sex ratio, and that the treatment to which the eggs were subjected, previous to their fertilization, had nothing whatever to do with the sex of the future embryos. In table 2 is given a summary of the pro- portion of the sexes and of the sex ratios in various lots of indi- viduals that have served as controls for different series of experi- ments made during the past six years. The 500 young toads examined in 1904 were obtained from the banks of the Susque- hanna River at Owego, New York, shortly after they had com- pleted their metamorphosis under natural conditions. All of the other individuals used in computing the table were developed from the eggs of females obtained in the vicinity of Philadel- phia, Pa. In every case the eggs were normally or artificially fertilized in laboratory tap water, and the tadpoles reared under very uniform external conditions. No lots of individuals have been included which developed from eggs that were subjected to any abnormal treatment, at or before the time of fertilization, TABLE 2 Sex ratios in various control lots of individuals yeag | OF | acemenacran | (Mates | tuMtared | VU ruene Sup cine 1904 500) 241 | 259) ates 7) 93.05 00% I | °259 | 341 ie os6 33 75.92 | | 651 292 359 554) 1) | 81.38 1908 140 | 64 76 54.28 | 84.21 1909 S230) ci) 157) | seiGomaeemolesS 94.56 210° 134 65. | 69 | 51.41 | 94:20 cate 1259 aia | - 372 || “408 52.00 | 92.25 201 TAO 8 565 75 53.57 86.66 200i) 2794 , eats 53700, |; 188.67 er S50 men mene S22 169 153 47.50 | 110.45 350 334 178 | 156 | 46.40 | 4114.10 Total 4119 | 1956 21638 Ml 52-51, — |), 90-42 326 HELEN DEAN KING or in which the tadpoles were exposed to unusual conditions of temperature or of nutrition during the course of their develop- ment, although in many such eases the sex ratios obtained were very similar to those of control lots. In the various lots of individuals whose sex data are included in table 2 the number of males to each 100 females varies from 75.92 to 114.10; when the percentages of females are compared there is found to be a difference of 10.43 points between the extremes of the series. These figures indicate that normally there is but little variation in the proportion of the sexes in different lots of toads. Table 2 shows also that there is no marked seasonal variation in the sex ratio of Bufo, such as Pfliiger (’82) and von Griesheim (’81) claim is the case with frogs. The latter investi- gators based their conclusions on the sex ratios in adult frogs col- lected from different localities in different years. My investi- gations have been confined entirely to the sex ratios in young toads that have recently completed their metamorphosis. Judging from the proportion of the sexes in several hundred adult toads that I have collected at various times during the past ten years, the sex ratio in adult individuals is very different from that in the young, since among adults there appears to be a considerable excess of males which is particularly noticeable during the breed- Ing season. The sex ratios in the two lots of individuals derived from eggs that were subjected to the action of sugar solution before fertil- ization fall within the limits of normal variation in the sex ratio (table 2). There is, therefore, some ground for an assumption that these cases afford no evidence that the normal proportion of the sexes was altered by the treatment which the unfertilized eggs received. The sex ratio found in the individuals that developed from eggs that were treated with salt solution is considerably lower than that in any control lot so far examined; but this may, perhaps, be considered as an exceptional variation. If these sex ratios are mere chance deviations from the normal, it certainly seems very remarkable that all three of them should show such a high percentage of females. “I SEX-DETERMINATION IN AMPHIBIANS 32 2. EXPERIMENTS ON THE FERTILIZED EGG If the sex of an embryo is not definitely fixed by the character of the spermatozoan that fertilizes the egg, it is possible that the zygote is a sex-hybrid and that external conditions, acting during the early stages of development, may turn the balance in favor of one sex or the other. Several different experiments were made this year to see whe- ther changing the water content of the zygote would have any effect on the sex ratio. ‘These experiments may be divided into two groups: (A) those in which an attempt was made to cause the eggs to absorb an increased amount of water during the fer- tilization period; (B) those in which eggs were made to lose water during this time. With increased absorption of water According to Loeb (’06), eggs can be made to take up water by placing them in weak solutions of acid or of alkali, the quantity of water absorbed depending on the strength of the solution used. Former experiments have shown that the eggs of Bufo are very sensitive to the action of acid and of alkaline solutions, and that it is not possible to subject them to the action of a solution stronger than 0.01 per cent without rendering the great majority incapable of development. Last year seven lots of eggs, from four different females, were fertilized in weak solutions of acetic acid (0.0025 per cent to 0.01 per cent), and in every instance the percentage of females obtained was from 10 per cent to 20 per cent lower than that in the control lot. Unfortunately no definite conclusions could be drawn from these experiments, since in every case the mortality was very great both at the time that the eggs were fer- tilized and during the growth of the tadpoles. I planned to repeat these experiments on a large scale this past spring in the hope that definite conclusions would be possible from the results obtained. To my great surprise, however, I found that it was not possible to obtain any considerable number of eggs that would develop normally after being fertilized in solu- tions of acetic acid. Altogether twenty batches of eggs, from 328 HELEN DEAN KING five different females, were experimented upon, and in no case did more than one-tenth of the eggs segment even when the solu- tion used had a strength of only 0.0025 per cent. The failure of these experiments cannot be due to the chance selection of a particularly bad lot of eggs and sperm, since eggs from two of the five females were used for all of the other experiments that were made, and the very great majority of them developed normally although they were fertilized under very unusual conditions. The only explanation that I can offer for this very unexpected result is that, when fertilization was attempted, the eggs happened to be in a physiological condition that rendered them particularly sensitive to the action of acid solutions. This past spring no toads were obtained until the seventh of April, and each of the five females used for these experiments had already laid a portion of her eggs before she was brought into the laboratory; the eggs were, therefore, very ripe. In 1910, females were obtained the latter part of March, and as none of them had laid any of their eggs when captured, the eggs were presumably in an early stage of ripening when they were experimented upon. According to Hertwig (06), the physiological condition of amphibian eggs varies considerably at different phases of their ripening, and it may be, therefore, that very ripe eggs are more easily injured by acid solutions than are eggs that are in an earlier stage of development. The individuals belonging to only one of the acid series were saved. In each experiment in the series about 400 eggs, taken from female b, were fertilized in solutions of acetic acid and re- moved to fresh water at the end of one-half hour. The strengths of solutions used, which were the same as those employed last year, are shown in table 3. Many eggs that segmented in a more TABLE 3 Eggs fertilized in solutions of acetic acid | | | NUMBER SEX | | PER CENT NUMBER MALES STRENGTHS TOTAL NUMBER D8 sae ty ee | asceRTaInED | MALES | BeeaNeaS FEMALES to 100 FEMALES — | ae | ~ — —— per cent | 0.01 46 42 22 20 ATE G2 an el OROO 0.0050 51 42 | 26 16 38.19 | 162.50 0.0025 7 | 19 [ea sie Be |e Sggrotal T8000 SEX-DETERMINATION IN AMPHIBIANS 399 * or less normal manner died during the gastrulation period, so the number of tadpoles that could be taken for rearing was very small. The sex data obtained in this series are shown in table 3. No conclusions can be drawn from these results since there were so few individuals in the various lots. The experiments have been recorded simply because the sex ratios found agree with those obtained in similar experiments made last year. Altogether ten different experiments have been made in which various lots of eggs have been fertilized in acid solutions, and in each case a very low percentage of females has been obtained. Such a consistent series of results, in so many different cases, strongly suggests that the acid solutions have increased the tendency of the eggs to produce males rather than females, presumably by causing them to absorb an increased amount of water during the fertilization period. In all of these experiments, however, the mortality was very great, so it is possible that selective mortality was respon- sible for the results; though why acid solutions should invariably be more injurious to young females than to young males is not at all clear. It will be necessary to repeat these experiments on eges that are in a physiological condition to withstand the injuri- ous action of acid solutions before any definite conclusions are possible regarding the effects of such solutions on the sex ratio of Bufo. In another experiment eggs were fertilized in water that had been distilled in glass, in the hope that the zygote would absorb an increased amount of water and thus tend to produce a male rather than a female. The eggs, which were taken from female a, remained in the distilled water for thirty hours, the water being changed three times during this period. Practically all of the eggs experimented upon segmented in a normal manner and con- tinued their development. Not many of the 400 tadpoles taken for rearing died during their early development, and the entire loss was only 18.75 per cent. The 345 individuals that were carried through to metamorphosis were found to consist of 189 males and 156, or 45.21 per cent of females. In this instance the sex ratio of 121.15 males to 100 females differs so little from that in the control for the series (table 1) that evidently the nor- mal proportion of the sexes was not appreciably altered by the 330 HELEN DEAN KING conditions to which the eggs were subjected during the early stages of their development. The results obtained in this experiment might seem to indicate that increasing the amount of water in the egg at the time of fertilization has no influence whatever on the process of sex- determination; but there is another possible interpretation of them which seems worth considering. The ripe eggs of the toad are surrounded by two membranes and embedded in a thick, jelly like substance. It is therefore possible that when eggs are fertilized in distilled water the osmotic pressure on them is, for some little time, practically the same as that to which eggs are subjected when they are fertilized under natural conditions. If this be so, the results of this experiment give no evidence what- ever regarding the effects on the sex ratio of increasing the water content of the eggs during the period of fertilization. Although the sex-determining mechanism was not affected by the distilled water, some change was produced in the eggs which had a decided influence on their later development. The tad- poles belonging to this lot were very small, and their development, although apparently normal, was so retarded that they were the last of all of the individuals in the various series of experiments to undergo metamorphosis. None of the experiments in which an attempt was made to in- crease the water content of the zygote have given results that could be considered as conclusive. It is suggestive, perhaps, that in every instance a relatively low percentage of females has been obtained; but other methods of experimentation will have to be employed before it will be possible to determine whether increas- ing the amount of water in the eggs at the time of fertilization really leads to an alteration of the sex ratio. With loss of water It would seem to be an easy matter to reduce the water content of the zygote by fertilizing the eggs in a hypertonic solution and allowing them to remain in the solution for a considerable length of time. Unfortunately the spermatozoa of Bufo are very easily injured, and even 1 per cent solutions of salt or of sugar render the great majority of them incapable of fertilizing the eggs. In SEX-DETERMINATION IN AMPHIBIANS Boll continuing experiments of this kind it was considered necessary, therefore, to use very weak solutions in order that the mortality among the spermatozoa might be greatly reduced. One batch of about 400 eggs, taken from female a, was placed with spermatic fluid in a 4 per cent solution of cane sugar; an- other batch of eggs from the same female was fertilized in a 4 per cent solution of NaCl. Each lot of eggs remained in the solu- tion for one-half hour and was then transferred into fresh water. The mortality at the time of fertilization was slightly greater where salt solution was used, but in this ease it was not more than 10 per cent. Since a former study of the fertilization of the egg of Bufo (King, ’01) has shown that the egg is normally penetrated by the spermatozoan within three or four minutes after it has been deposited, it is evident that in these experiments the solutions acted chiefly on the zygote and not on the unfertilized egg. In each case 300 embryos were taken for rearing, and the greater number of these, as shown in table 4, were carried through to metamorphosis and their sex ascertained. Each lot gave a percentage of females higher than that in the control for the series, but well within the limits of normal variation in the per- centages of females as shown in table 2. It is doubtful, there- fore, if in either case the normal proportion of the sexes was altered by the treatment which the eggs received at the time that they were fertilized. One lot of eggs, taken from female 6, was fertilized in a + per cent solution of salt, and another lot was fertilized in a } per cent TABLE 4 Eggs fertilized in hypertonic solutions 1 1 n nm eas .\)2 fa | 38 | a8 | i) BH wa | | a S < 5 2) Qa< is) a | i Sy | 5 25 | ae | | ee ial Ses ZEO SERIES | SOLUTION USED <4 Bay) | i een) Bs = fe ZS < | ra] a | zS ate Es ° en | e 5 | n e) So 3 AS a) a a ea! sa | & S| sees 30 S06 a 5 =} | 50 a a Qs 5a bH~ a a | & fh a Cy Z a i — | | | min. | | | | | | | | | | PD x3 | A 2 per cent sugar 30 | 300 | 246 | 112 | 184 | 54.47) 83.58 110.45 A | dper cent salt 30 300 | 272) 130| 142 | 52.20) 91.54! B | dpercent sugar 60 400 | 367 | 185) 182 | Hien 114.10 B 7 per cent salt 60 450 | 391) 215 | 176 | 45.26)121.59| | | | | | | Bo HELEN DEAN KING solution of sugar; in each case the eggs remained in the solution for one hour before being transferred into tap water. These eggs reacted very differently from those that were fertilized in acid solutions (table 3), although they were taken from the same female and fertilized with sperm from the same male. In neither lot was the mortality at the time of fertilization greater than 1 per cent, and only a small number of tadpoles died in the early stages of their development. As shown in table 4, the sex ratio in the individuals that were carried through to metamorphosis was in each instance practically the same as that in the control for the series. These results indicate unmistakably that the solutions in which the eggs were fertilized had no effect on the sex of the tadpoles, although they continued to act on the zygote for nearly an hour. As indicated in table 4, the results obtained in these experiments offer no evidence that the sex ratio in Bufo can be altered by fertilizing the eggs in hypertonic solutions. This negative result may, possibly, be due to the fact that it is not possible to fertilize the eggs in hypertonic solutions that are strong enough to pro- duce any appreciable change in the osmotic pressure. Keeping eggs out of water for some time after their fertilization was another means employed to cause the zygote to lose water, or at least to prevent its absorption of water, during the early stages of development. This method of experimentation has the very great merit that the eggs are not subjected to the action of any chemical substance that might possibly produce changes in them that would lead to abnormal development and to the early death of the embryos. The technique employed in the two experiments that were made this year was as follows: On their removal from the uterus of the female the eggs were placed on filter paper and a few drops of water containing spermatozoa were distributed over them with a pipette in as uniform a manner as possible. The excess of water was then quickly drained off, and the eggs were transferred into a moist chamber where they remained for a number of hours be- fore they were allowed to continue their development in water. With very few exceptions all of the eggs from female a that were experimented upon were fertilized, and they began segmenting SEX-DETERMINATION IN AMPHIBIANS 333 fully ten minutes before there was any indication of a division in the eggs of the control lot for the series. When the embryos were removed from the moist chamber and placed in water, seventy- seven hours after the experiment was begun, all of the jelly that had surrounded the eggs had disappeared and the embryos were lying on the nearly dry filter paper from which it took some time to float them off. Out of about 450 embryos that were taken from the moist chamber, 400 were selected for rearing. The tadpoles in this lot were noticeably larger than were any other tadpoles in the series, and they began metamorphosing less than five weeks after the experiment was started. The mortality during the development of the tadpoles was remarkably low, only 4.75 per cent, so that selective mortality could have had very little influence on the proportion of the sexes in the lot of individuals carried through to metamorphosis. In the 381 individuals in which sex was ascertained 275, or 72.33 per cent, were females. This percentage of females is nearly 25 points higher than that in the control for the series (table 1), and much too high to be considered as a chance variation in the normal proportion of the sexes. In this, as in the other series of investigations made last spring, the experiment was repeated with the eggs from a different female in order to avoid the possibility of drawing conclusions from an unusual sex ratio that might be merely a chance variation. As a check for the experiment made with the eggs from female a, a lot of about 400 eggs, taken from female }, was fertilized out of water and kept in a moist chamber for fifty hours. In this in- stance, also, practically all of the eggs were fertilized, and the development of the tadpoles was similar in every respect to that of the tadpoles belonging to the corresponding experiment in series A. The mortality among these tadpoles also was very slight (6.50 per cent), and 374 individuals were carried through to meta- morphosis and their sex ascertained. This lot of individuals, as shown in table 5, contained 77.27 per cent of females, which is 30.87 points above that in the control for the series. The sex ratio in this instance, 29.41 males to 100 females, falls far below that in any lot of toads so far examined. 334 HELEN DEAN KING TABLE 5 Eggs fertilized out of water | TOTAL | NUMBER | | PER CENT “hase NUMBER MALES SERIES ENE WANS SHS eer MALES | FEMALES | PPMALES | 100 TO 100 FEMALES | DIVIDUALS | TAINED | | | FEMALES (CONTROL LOT) { =e | = Sea ijn A 400@ | 381 \) 106 —|) 275 | 72033 93aI54. || 110245 B | 400; |). 374 ~)| 05/80; 2890 Gea | | 29-41) 114 10 In none of the experiments that have been made with the eggs of Bufo in order to study the problem of sex-determination have the sex ratios obtained been any where near as low as those indi- cated in table 5. These results cannot be ascribed to an error in distinguishing the sexes, since the gonads in all of the individuals that were killed three weeks after they had completed their metamorphosis were well differentiated and the sex of the few individuals that died during metamorphosis was shown unmis- takably by sections. It is evident that whatever part selective mortality may have had in producing the unusual sex ratios obtained in various former experiments, it cannot be held responsible for these last results. Had all of the individuals in which sex was not ascertained been males, which of course is very improbable, the resultant sex ratios would still be very much lower than any of those indicated in table 2. The individuals in series A would contain 45.45 males to 100 females; while among the individuals belonging to series B there would be 38.40 males to 100 females: no control lot of indi- viduals so far examined has given a sex ratio of less than 81 males to 100 females. In these experiments the eggs were not subjected to the action of any chemical substance, but were merely kept out of water for some hours after their fertilization. It is evident, therefore, that the only change that could have been produced in the eggs was a diminution in their water content during the.early stages of their development. The eggs probably lost but little water from evap- oration during the fertilization period, as they were kept in a moist atmosphere in a closed vessel; but normally, as shown by the investigations of Bialaszewiez (’08), amphibian eggs absorb a considerable amount of water before the appearance of the first SEX-DETERMINATION IN AMPHIBIANS ooo cleavage plane, and such an absorption of water was not possible under the conditions to which these eggs were subjected. Unless by chance, therefore, in picking out the individuals to be reared, I selected in each case tadpoles that would give a great majority of females when developed, I can see no alternative but to as- sume that sex in Bufo can be altered by changing the water con- tent of the eggs at the time of fertilization. The weight of recent experimental and cytological evidence is, however, decidedly against the view that external factors can have any influence what- ever in determining sex. In making these experiments the spermatic fluid was distributed over the eggs within two or three minutes after they had been taken from the female. It is probable, therefore, that each egg was fertilized by the first spermatozoan that reached it, since in such a short space of time the well protected eggs could not lose sufficient water from evaporation to make selective fertilization possible, unless it be that fertilization is normally selective when the eggs of Bufo are fertilized. If the male is responsible for sex, each batch of eggs might have been expected to give a nearly equal proportion of the sexes, regardless of the external conditions to which they were subjected at the time of their fertilization; for former experiments have shown that, if the spermatozoa of Bufo are dimorphic, both kinds of spermatozoa must be produced in approximately equal numbers in each testicle of every normal male (King, 711). In each case, however, as indicated in table 5, the individuals carried through to metamorphosis contained a proportion of females greatly in excess of that in any control lot as yet examined and much beyond the limits of probable normal variation. The chromosome theory of sex-determination does not, therefore, offer a satisfactory explanation of these results, unless one arbitrarily assumes that the lot of spermatozoa used in fertilizing each lot of eggs happened to contain a much greater number of female-producing spermatozoa than of those that were male-producing. The results of the experiments in which eggs were fertilized out of water, taken in connection with those obtained when eggs were subjected to the action of hypertonic solutions before fertilization, THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 3 336 HELEN DEAN KING strongly suggest that in Bufo sex does not depend exclusively on the spermatozoan, but that it is determined by the egg alone, or by both egg and sperm. It would appear, also that sex can be influenced by decreasing the water content of the egg at or before the time of fertilization. Hertwig’s (’06) theory that sex is determined by the mass rela- tion between the chromatin and the cytoplasm seems to offer a tentative explanation of these results, if it is applied to the con- ditions existing in the zygote and not to those in the ripe, unfer- tilized egg. Until these experiments have been repeated and extended, however, it will be useless to attempt the formulation of a theory of sex-determination that will explain these results and bring them in harmony with those that have been obtained by other investigators in this field. LITERATURE CITED BautzEr, F. 1909 Die Chromosomen von Strongylocentrotus lividus und Hchi- nus microtuberculatus. Arch. Zellforsch., Bd. 2. Brauaszewicz, K. 1908 Beitrige zur Kenntniss der Wachstumvorgiinge bei Amphibienembryonen. Bull. Inter. de l’Akad. Sci. de Cracovie. Math.-Natur. Classe. Davenport, C. B. 1897 Experimental morphology. The Macmillan Company. vVoN GriesHEIM, A. 1881 Ueber die Zahlenverhiltnisse der Geschlechter bei Rana fusca. Arch. gesammte Physiol., Bd. 26. Hertwia, R. 1906 Weitere Untersuchungen ueber das Sexualitaitsproblem. Verhandl. deutsch. zo6l. Gesellsch. Kine, H. D. 1901 The maturation and fertilization of the eggs of Bufo lentigi- nosus. Jour. Morph., vol. 17. 1907 a The spermatogenesis of Bufo lentiginosus. Amer. Jour. Anat., vol. 7. © 1907 Food as a factor in the determination of sex in amphibians. Biol. Bull., vol. 18. 1909 Studies on sex-determination in amphibians. II. Ibid., vol. 16. 1910 Temperature as a factor in the determination of sexin amphibians. Ibid., vol. 18. 1911 Studies on sex-determination in amphibians. IV. The effects of external factors, acting before or during the time of fertilization, on the sex ratio of Bufo lentiginosus. Ibid., vol. 20. Logs, J. 1906 The dynamics of living matter. The Macmillan Company. Pritcer, E. 1882 Ueber die das Geschlecht bestimmenden Ursachen und die Geschlechtsverhiltnisse der Frosche. Arch. gesammte Physiol., Bd. 29. Witson, E. B. 1910 Selective fertilization and the relation of the chromosomes to sex-determination. Science, vol. 32. REINVIGORATION PRODUCED BY CROSS FERTIL- IZATION IN HYDATINA SENTA! DAVID DAY WHITNEY From the Biological Laboratory, Wesleyan University The full significance of fertilization is far from being clear not- withstanding a vast amount of speculation and observation upon both plants and animals. Darwin observed self-fertilized and cross fertilized plants for several generations and determined that cross fertilization is generally beneficial and self fertilization is injurious. ‘‘This is shown by difference in height, weight, con- stitutional vigor, and fertility of offspring from crosses and self- fertilized flowers, and in the number of seeds produced by the parent plants.” He also collected considerable data from breed- ers showing that the majority of them were of the opinion that cross breeding between individuals of the same race which lived in separated localities, caused an increase of constitutional vigor in the resulting race. Later Biitschl regarded conjugation in the Protozoa as a process involving rejuvenation and considered fertilization in the Metazoa in the same light. He was followed by Maupas and finally by Calkins who has found that the conjugation of two in- dividuals in a weak race of Paramoecia caused a reinvigoration of the race to such an extent that it was able to pass through another cycle of at least 376 generations before it became as weak as the original race from which the two conjugating indi- viduals were taken. 1T am greatly indebted to the Directors of the Biological Laboratory of the Brooklyn Institute of Arts and Science, Cold Spring Harbor, N. Y., and of the Marine Biological Laboratory, Woods Hole, Mass., for their courtesies and for placing at my disposal private rooms and laboratory facilities during the summers of 1909 and 1911 respectively. 337 338 DAVID DAY WHITNEY Although considerable work on the problem of rejuvenescence by fertilization has been done on plants, nevertheless experiments and observations on the multicellular animals in connection with the reinvigoration of the race by fertilization are as yet very few and inconclusive. The purpose of this present paper is to demon- strate that a great amount of rejuvenescence occurs when two weak races are cross bred and that only a small amount of re- juvenescence takes place when each weak race is inbred with itself. On October 6, 1908, a fertilized egg from a wild culture of the rotifer, Hydatina senta, was put into some fresh culture water and on October 12, 1908, a young female hatched from the egg. - A pedigreed parthenogenetic culture or race was started from this female and was called race A. In the 59th generation of this race A, on February 24, 1909, two parthenogenetic sisters were iso- lated. One became the mother of what has been called the 60th . generation of race A and the other became the mother of what has been called the 60th generation of race B. In other words at the 59th generation the race was split into two sister races. One was still called race A and the other was called race B. These two sister parthenogenetic races A and B were kept in syracuse watch glasses. Usually once in forty-eight hours ten daughter-females of each race were isolated, each daughter-female being placed in a separate watch glass. They produced the young females of the succeeding generation. Both races were always fed from the same food culture jars made from a culture of horse manure and water inoculated with bacteria and protozoa. During the first fifteen months, until January, 1910, these food cultures contained a miscellaneous assortment of protozoa but in January, 1910, pure food cultures of the flagellate, Polytoma, in horse manure solu- tions were started and proved so successful that they have been continued to the present time. The special method of making these cultures has been described in a former paper. The two pedigreed sister parthenogenetic races were continued up to March 3, 1911, at which time race B apparently from exhaus- tion died out in the 384th parthenogenetic generation. How- ever, some fertilized eggs of this race were saved which had been REINVIGORATION PRODUCED BY CROSS FERTILIZATION 339 produced in some minor experiments which had been performed at about this time in February. In this way the race was pre- served and used in later experiments in connection with the problem of in- and cross-breeding. ‘The parthenogenetic race A is alive at the present time in the 503rd generation, but is in a very exhausted condition. During the whole period in which the two races were con- ducted in parallel generations the external factors or environment were as identical as possible. The individuals of each generation were isolated at the same time, put into the same kind of dishes, with approximately the same amount of tap water to which was added the same kind and amount of food from the same food culture jars. They were always kept side by side in the stacked watch glasses, at the same room temperature, and in the same illumination. Some of the time they were kept in a dark room and some of the time in a well lighted room. The greater part of the time they werein Middletown, Connecticut, but during a few weeks of the summer of 1909 they were at Cold Spring Harbor, New York, and also in Vermont. The summer of 1910 they were only out of Middletown two or three weeks when in Vermont. In the summer of 1911 race A was in Woods Hole, Massachusetts, and in Vermont for a few weeks. The criterion selected for deciding whether the races were strong or weak was the rate of parthenogenetic reproduction. This was selected because of simplicity of observation together with its fun- damental importance in connection with growth and metabolism. In order to determine the comparative vigor of the two races A and B their rates of parthenogenetic reproduction were obtained and compared with the'rates of reproduction of two other parthe- nogenetic races Cand D. Race C was started about nine months later than races A and B, from a parthenogenetic egg of a wild individual which was isolated from a general wild culture of roti- fers supposed to have started a few months previously from a fertilized egg. Race D was started at the time of the experiments from a fertilized egg of another wild unpedigreed general culture of rotifers which was begun in October of 1908. 340 DAVID DAY WHITNEY In some of the experiments the eggs of the females from ‘the different races were counted at frequent intervals in order to determine whether all the females of the various races produced the same number of eggs in the same period of time. This was not found to be the case for the females of some of the races pro- duced eggs faster than the females of other races but as the eggs of all females of all races hatched in about the same length of time after they were laid, the rates of reproduction were deter- mined by counting the young females in a dish with their mother after a definite period of time had elapsed since the mother was first isolated. The first series of observations were made during the period in which race B was becoming extinct. Many young partheno- genetic females of approximately the same size were isolated from each of the four races at the same time, placed under identical external conditions and their rates of reproduction recorded. Table 1 shows the general results. Race B was unmistakably the weakest in that only one female out of sixty isolated was able to live and reproduce, while twenty others lived the normal length of time for individuals of the race, but never laid any eggs. These twenty females developed and produced many eggs in their ovaries but never laid them. The eggs remained inside the body of the female and ultimately seemed to fill the entire animal, crowding and concealing all the internal organs from view. After a time some of these eggs were observed to start development into embryos; but soon the embryos died and many of the egg mem- branes ruptured and the body of the female became filled with a mass of egg materials from the broken and decomposing eggs. These females finally became larger than normal females; due to this accumulation of unlaid eggs which crowding out the wall of the animal caused the large size. Such females are designated as sterile females. Thirty-nine of the remaining females did not live to maturity probably because of their weak condition. In race A forty-six of the young females matured and produced daughter-females at a higher rate than the one female of the Brace. In race C fifty-three of the young females matured and had a higher reproduction rate than either of the races A REINVIGORATION PRODUCED BY CROSS FERTILIZATION 34] “6061 ‘FZ Areniqay ‘edv1 F ay} Jo uOT}BIOUT Y36¢ 94} WOIy UDHBYT, GE ET | 982 6¢ 0 I 6¢ 09 | IEG "‘SHM ¢ 07 Z 9-€ 03 91-z IT.-€1-z 330 *[97aq | a 099 | Tg€ €¢ 0 L €¢ 09 | 16-692 ‘SOUlg ‘IAT 9-€ 091-2 | 60,-91-9 880 ‘uoyqaeg |-°--* 7 re) i I 02 6e IZ 09 8e-08e | ‘sourg‘siXz | g-gorgT-z | g0,-9-0T 83 “THseq | +d 1 F O61 OF 0 as OF 09 P8E-6LE "SOUL G ‘SIA Z | 9-§ 0} 91[-Z | 80,-9-01 B30 ‘[Iq1eq | Vv NViS Pod ees oe $6 ee pete ee Preis (TI61) | Ta woud ae ones | -Oud ONION | oars Tea GOV" TO ADV INAWIHaaXa OILANAD aqadoigaga | govu | hsp EOL Soy 84 DILANETDONGHLYVd -ONTHLNVd | aL “outed cE | younca 910994 S900 ay) au fo yjbua) quarayfns v 10f panurjuoo fi PUD 1ap]O saworaq 99D4 ay} SD sasDa.Iap woujonpoidas d4auUabouayjlvd fo ays ay) JOY) Snoys OS]D 7] “Sa9D4 9Yjauabhouayj.od paaibrpad ayy fo anof fo sobva aaryoivdwuos ay} ‘sajp4 aayonpoisdas ay) fiq pun sabo ay) Burnoyy T ATaVL 342 DAVID DAY WHITNEY and B. In race D fifty-nine of the young females matured and the reproduction rate was twice that of race C and three times that of race A. When the two races A and B were in the 60th to 80th genera- tions their rates of reproduction were probably very much like that of race D although no exact data were taken in this period. However, from general observations made at this time it was clearly seen by the observer that each young female in both of these races under ordinary conditions matured and produced ten or more young daughter-females in forty-eight hours. It was customary in these early generations to take ten young daughter- females from a single mother with which to form the succeeding generation. Later as the generations increased this became im- possible and for the isolation of ten young daughter-females at the end of forty-eight hours two mothers were required and later still three mothers were required. At the same time it was noticeable that the females of race B in the same length of time were producing fewer daughter-females than the femalesof race A. During the summer of 1910 this was so apparent that difficulty was experienced in being able to isolate ten young daughter- females from both races which were of the same age and size at the end of forty-eight hours. In order to continue these two _ races in a parallel series of generations by isolations of young females of the same size from both races daughter-females of race A were isolated which were produced later in a family, from the tenth to the thirtieth, and the daughter-females from several mothers of race B were isolated which were the earliest ones pro- duced in each family. Thus by isolating the daughter-females . from near the middle of a family from race A and the ‘first born’ daughter-females from race B it was possible to keep the genera- tions of both races parallel. From table 1 and from these general observations it is readily seen that as the parthenogenetic race became older the rate of reproduction decreased very decidedly and also that the chances for each young female to grow to maturity were lessened. This decrease in the rate of reproduction may not necessarily be due to long continued parthenogenetic reproduction, but rather to REINVIGORATION PRODUCED BY CROSS FERTILIZATION 343 the constant environment of the horse manure food cultures. The influence of the environment upon the race will be considered in a subsequent paper when certain experiments which are in progress now shall have been completed. At present it is useless to discuss this point because of the lack of sufficient data. From the evidence it is also concluded that race B has’ become the weakest or the most exhausted in its general vigor, while race D is the strongest and most vigorous. After the general vigor or vitality of the parthenogenetic races A and B had been ascertained it was decided to determine whether fertilization within each race would increase its general vitality. Several females from each race were placed in separate new cultures made in small battery jars and allowed to live in them two to three weeks. During this time males appeared and fertilized eggs were produced. After a short time these fertilized eggs from both races A and B were hatched and a series of paral- lel observations were made upon the rates of reproduction of the races, from the fertilized eggs, from the original parthenogenetic race, and from the new race D. ‘Tables 2 and 3 show the negative influence of inbreeding once in race A. Table 4 shows the same result in race B. After these results were obtained it was thought best to ascer- tain whether or not a second inbreeding of the races which had _already been inbred once would reinvigorate them, perhaps by an accumulation of stimuli of some sort which were too weak in the first fertilization to give apparent results. Table 5 gives the data and results of the second successive inbreeding of race 4. The new race D was used as the control or normal race as has been done in the former experiments. ‘Table 6 shows the results ob- tained from fifty-one females each of which developed from a different fertilized egg of race B which had already been inbred once. In neither table is there found any very marked increase of the rate of reproduction. These two races, both of which re- sulted from a second successive inbreeding of the original races, were continued. Later in the year race A produced fertilized eggs which resulted from the third successive inbreeding of the race, and race B even was allowed to produce fertilized eggs which 344 DAVID DAY WHITNEY resulted from the fourth successive inbreeding of the race. Pre- vious to this time race D had been destroyed and consequently a new race, H, was started from a fertilized egg from the same wild general culture of rotifers from which race D had been started. This new race was used as the control. Certain obvious parts of tables 9, 10,11 and 12 give the data and results of these observations. In these tables it is noticeable that the rates of reproduction of races A and B have not risen to any marked extent although a slight increase in the rates is appar- ent. The conclusion may be safely drawn that successive in- breeding of such weak races does not increase their general con- stitutional vigor to any considerable degree, even though this successive inbreeding is allowed to occur four times, as in race B. As the two sister parthenogenetic races have been demon- strated to be in a weakened state and this weakness has been shown to continue in each race after several successive cross fertilizations have taken place it now remains to show what results are obtained when these two weakened races are allowed to cross breed and reciprocal cross fertilization of the eggs occurs. The first series of observations on the crossing of the two races A and Bis recorded in table 8. A few females from each of these weakened races were put together into one battery jar which contained a new food culture. Many males soon appeared and after several days eighteen fertilized eggs were taken out and. hatched after resting a few days. The rates of reproduction of seventeen of these females were determined and compared with the reproduction rates of ten different females of the new race D. The average reproduction rate of these seventeen females was much higher than either of the average reproduction rates of the two parent races A and B which have been compared with the reproduction rate of race D in tables 1 to 4. In fact 1t even ap- proached closely to the reproduction rate of race D. If the records of three of these seventeen females which were probably not the result of a cross fertilization are eliminated the two average repro- duction rates are much closer. This great increase in the repro- duction rate and its close approximation to that of the control was assumed to be due to a reinvigoration caused by cross fertili- zation. REINVIGORATION PRODUCED BY CROSS FERTILIZATION 345 ‘(Sh 'p) SopBures 10yYSnep oonpoid yey} sayeuraqg—s 4 4 ) 66'S 6II 02 Mg 8% ord 19 Lb “ AreurUINg A 9 Fe F 0 0 ¢ aT em gee ah ics eee | =C “oA AI gg ied ¥ 0 0 g eT gI | Or ie ae ra 9 og ¢ z'0 I pe ALT 9 6 | Be ie i | ¢ ie ee 8S a eee. IVT Or 6 | a F I | 6 8 z ¥% at ¢ Hey 91 6 | oe one | = oa: ——— suor} | 86 ‘Pp poonpoid | pezB]OSt 86 "Pp peonpoid peye[ost sé ‘p peonpoid PeyB[OSt | ~B19Udr) ‘ou ‘AY 86 ‘Pp 8466 ‘ou ‘AW 86 ‘p S66 ‘Ou ‘AV 86 ‘"p S66 | TT6T OWL Vf DOA GAZITILYAA woud da ‘NOILVUANAD HLCF-HLOF LAOdV NI GOVY AIIM OILANADONAHLUVd MOAN V “JL AYD.LOBLAULAL JOU SA0p aDU0 W aan Burpaauqur DY} SajD4 wor1jonpoudas aarjouvdwod ay} fig Buamoys 2190, AOVU VW 40 OLE NOILVUANAD OILANADONAHLUVd ONIGAAUANI WOUT DOA AaAZITILUAA ist V GOVU V JO 0] f-9OP SNOILVUANAYD OILANADONTHLUVA Vv ¢ ATaAVL L DAVID DAY WHITNEY 346 68 igat ST GLE 0€ 8 6L°0 } = 46 Fe | | | L 86 6F | i} g°0 & 61-9 “OAH 9 OF-9) oA 9-9) eA L 86 6F $ 99 °0 j GI-9 “OAH g OT-9 ‘eA 9-9 “PAM 9 0 0 6-9 PA ] 8-9 “9AM 9-9 “OA tf) 0 0 | @}-9 eAu G 8-9 8AM | 9-9 SA i | 0 0 TI-9 “Au g L-9 AGH 7-9 SA € | 0 0 TI-9 “840 ¢ } ONG a) SENG G GL | 9€ | $ GL é 08 8 VP | G6: 6-9 “9AM ¢ | 1-9 OA P90) 840 I —= | — -— } a —- > ----- | a 84'p peonpoid | poyeost 84'p poonpoid | payzepost eG i : | ; | ‘ou ‘AW 86 "Pp | 866 ‘ou ‘AW sé ‘p | sé} OLEAN ON IT6] SUL oN | TI6T OUT, TT6] ony | ON 3 : So] BUIOy Geis o2 pPeyB[Ost S852 Z B) ac) SHA ae cae aes a ce a Josysnep jo sudsyO saPBULo} Joy SNC, eo ae s 3 iy iene NOILVUANAD HL0G GHL LAOAV NI aovu SCAT OUNCE ae NOM i G 1g NOILVHANAD OILANADONAHLYVd DNIGAAUANI WOUd SPOT GAZI a1IM OILANADONGHIUVd MAN “VEENaS oe oe MELE Is, V da “VL ayn.Lobrvauras JOU sa0p 90U0 YW aans Burpaasqur jDY) sajna UorYonposdas aarnupdwmos ay) fig Buinoys a1qQv,[, € WITAVL REINVIGORATION PRODUCED BY CROSS FERTILIZATION 347 NOIL -VUANGD | | yp | OBI 4 6el | GF +010 z 61 RD ei en male eget: Areurung 89 73 Gee | Coa Scream umes 0 0 ¢ eae Brae ll Te | 9I-FI-F FA Comal =e) lh 6 0) 6. el 10} 0 Oe eles 0 Oe 0) eI-TI-F Pe lil SAE ¢ I Or Or Os 0) 1 84 ade =| or O-8-F | Pee es ge | 9% Or Oe) z ee £8 Ole | 8-9-F Race. | evi ¢ 2 02 Or 0 Poel eee ee eeu | 9-F-F Be ae appa eee eee cag eeloe! |p SOR) = enrages Gee Ele aia Pay ‘OU CA : . q 2 Z | “AW ~ ' tS) V | jo-on V | sé -p Vos. -p 9 V_ |286.p aaTaeanat ze: a al A eee ae =| : agovu g ao | Spans linea ae peace AOVU Cy AO [62-18% SNOIL 082 SNOILVUANGD OIL IOF-L6¢ SNOIL | pee aes MON -VUANAD JDILANADONAHLUVd -ANADVONAHLYVd DNIGAAUA | -VUANAD DILANGTDONAHLYUVd a 18) -NI WOUdT DOH AAZITILYAA V Ist qf ‘Jl aDLOBLAUIAL JOU $a * ATAVL op a0u0 g a0n4 Burpaaqui yoy) sans worjonposdas aarjoivdwuod ay? fig Buinoyy 348 DAVID DAY WHITNEY TABLE 5 Showing by the comparative reproduction rates that inbreeding race A two successive times does not reinvigorate it | D ioe NEW PARTHENOGE- FERTILIZED EGGS FROM INBREEDING A 1st be aa -- GENERATIONS g ‘ | ge i F SP ae douse Penaies Offspring of daughter | Parthenogenetic ized eggs | isolated females age MN 5, ae No. Time 1911 | Time 1911 | No. | Time 1911 | No. | 2%" |, 4.434! pro Pine: | duced | 1 | A.M. 6-20 | Eve. 6-24 eee 9 dead. | No young. | Sterile 2 | A.M. 6-22 | A.M. 6-24] 4 | A.M. 6-26 | 28 |7 10 aeTONe\ eee 3 | A.M. 6-22| A.M. 6-24) 2 | A.M. 6-26) 14 |7 | 10 70 7 4 | A.M. 6-22 | A.M. 6-27 | Large! 9 dead. | No young. Sterile 5 | A.M. 6-23 | Eve. 6-28 | Large @ dead. | No young. | Sterile 6 | A.M. 6-23 | Eve. 6-28 | Large 9 dead. | No young. | Sterile 7 | A.M. 6-23} Eve. 6-28 | Large) 9 dead. | No yjoung. Sterile 8 | A.M. 6-23 | Eve. 6-28 | Large 9 dead. | No yjoung.| Sterile 9 | A.M. 6-23 | A.M. 6-25| 4 |A.M.6-27| 18 [4.5 | 5 | 55 | 11 10 | A.M. 6-23 | A.M. 6-26| 5 | A.M.6-28| 28 [5.6 | 5 68 | 13.6 11 A.M. 6-24 | A.M. 6-26 4 | A.M. 6-28 | 29 {7.25 5 68 13.6 12 A.M. 6-24 | Eve. 6-27| 3 Eve. 6-29 All |dead. | 13 | A.M. 6-25 | Eve. 6-27| 5 | Eve. 6-29 | 30 16 | 4 | 52 14 14 | A.M. 6-26 | Eve. 6-27| 4 | Eve. 6-29 20 |5 se GO) |) 1 15 | A.M. 6-26 | A.M. 6-28 | 5 | A.M.6-30 | 37 |7.4 65 13 16 | A.M. 6-26 | Eve. 6-29 | Large 9 dead. | No young. Sterile 17 | Eve. 6-26 | A.M. 6-28) 4 | A.M. 6-30| 34 [8.5 5. |) eb uleas 18 Eve. 6-26 | A.M. 6-29 4 | Eve. 6-30 | 10 |2.5 | 6 39 6.5 19 | A.M. 6-27) A.M. 6-29} 3 | Eve. 6-30] 5 |1.66+ 6 39 6.5 20 | Eve. 6-27 | Eve. 6-30| 2 |Eve.7-2 | 4 2 | 2 Ppp | alt 21 Eve. 6-27 | Eve. 6-29 4 | Eve. 6-30 10 |2.5 6 39 6.5 22 Eve. 6-27 | Eve. 6-29| 3 Eve. 6-380 | 6 2 6 39 6.5 23 | Eve. 6-28 | A.M. 7-2 | 4 | A.M. 7-4 | 22 |5.5 5 63 12.6 24 | Eve. 6-28 | A.M. 7-2 | Large Q dead. | No young Sterile 25 | Eve. 6-28 | A.M. 7-2 4 | A.M. 7-4 1 0.25 | 5 63 12.6 26 | Eve. 6-28 | A.M. 7-2 | Large 9 dead. | No yjoung.) Sterile 27 | Eve. 6-28 | A.M. 7-2 | Large 9 dead. | Noyloung.| Sterile 28 | Eve. 6-28 | A.M. 7-2 | 5 | A.M. 7-4 | 30 |6 | 5 63 12.6 29 | Eve. 6-30 | A.M. 7-2 5. | A.M. 7-4 30 |6 oO] 63 12.6 30 | Eve. 6-30 | A.M. 7-2 | Aa PAVIV Ieee _ 35 8.75 5 63 12.6 31 | Eve. 6-30 | A.M. 7-2 | 3 | A.M. 7=4 | J5 |5 5 | 63 12.6 32 | Eve. 6-30 | AM. 7-2 | = | #9 | 33 | Eve. 6-30 | A.M. 7-2 | 3 | A.M. 7-4 | 5 |1.66+} 5 63 12.6 34 | A.M. 7-2 | Eve. 7-3 3 | Eve. 7-5 | 13 433+) 4 44 11 85 | A.M. 7-2 | Eve. 7-3 3 | Eve. 7-5 26 |8.66++| 4 44 11 36 | A.M. 7-2 | Eve. 7-3 5 | Eve. 7-5 42 |8.4 | 4 44 | il 37 | A.M. 7-2 |AM.7-4 | 5 | A.M. 7-6 | 44 (8.8 4 50 | 12.5 | 38 | AM.7-2 |A.M.7-4 | 4 | AM. 7-6 | 27 [6.75 4 50 | 12.5 39 A.M. 7-3 | P.M. 7-5 3 A.M. 7-7 0 |0 5 61 12.2 40 | A.M. 7-3 | P.M. 7-5 2) || ASME 7=7 ieee | 5 61 1232; 41 A.M. 7-3 P.M. 7-5 | 3 A.M. 7-7 | 5 |1.66+] 5 61 12.2 42) A Mog3 SPM 76 |) 2) | ACM ova Onl) 45 61) |) 1922 | | | REINVIGORATION PRODUCED BY CROSS FERTILIZATION 349 TABLE 5—Continued NOx , NEW ke maunoce: FERTILIZED EGGS FROM INBREEDING A IST eS ee amare + GENERATIONS i s | 3 or i - . Bee nce Aor ae ted OFSprine Gl dauebter Parthenogenetic ized eggs isolated HENS | d. Qs | No. | Time 1911 | Time 1911 | No. | Time 1911} No. | AY- | 88 Al po ae | | ‘ a 43 | A.M. 7-3 | P.M. 7-5 ANNAN, YEE || TBE 5 Gil |) 1) 44 | A.M. 7-3 | P.M. 7-5 2 | A.M. 7-7 0 0 5 61 | 12.2 45 | A.M. 7-3 | P.M. 7-5 Qe PAUN7=7 pie 1525 4 47 | 11.75 46 | Eve. 7-3 | A.M. 7-5 5 | A.M. 7-7 | 43 18.6 4 47 | 11.75 47 | Eve. 7-3 | A.M. 7-5 5 | A.M. 7-7 | 31 |6.2 4 47 | 11.75 48 | Eve. 7-3 | A.M. 7-5 5 | A.M. 7-7 | 30 |6 4 47 | 11.75 49 Eve. 7-3 | A.M. 7-5 5 | A.M. 7-7 | 24 |4.8 4 47 | 11.75 50 | Eve. 7-3 | A.M.7-5 | 5 |A.M.7-7-| 19 8.8 | 4 AT ties 51 | A.M. 7-4 | Eve. 7-5 APM = Teast Wia75) | 4 43 | 10.75 52 | A.M. 7-4 | Eve. 7-5 | 4 | P.M. 7-7 | 17 (4.25 4 43 | 10.75 53 | A.M. 7-4 | Eve.7-5 | 4 | P.M. 7-7 | 20 [5 4 43 | 10.75 54 | A.M. 7-4 | Eve. 7-5 2. | P.M. 7-7 9 4.5 4 43 | 10.75 55 | A.M. 7-4 | Eve. 7-5 4 | P.M. 7-7 Sele 4 43 | 10.75 | 56 | A.M. 7-4 | Hve. 7-5 | 3 | P.M. 7-7 | 16 |5.38+/ 4 | 43 | 10.75 (| 57 | A.M. 7-4 | Eve. 7-5 4 | P.M. 7-7 | 20 |5 4 | S4artor75 | | | 57 | 172 864 5.02 217 | 2373 | 10.92} Summary 45 | 172 864 5.02 68 | 739 | 10.86; Summary of | | | the exact no. | of fertile in- dividuals } used. | TABLE 6 Showing by the comparative reproduction rates that inbreeding race B two successive times does not reinvigorate it | Cc A B 2np D PARTHENOGE- | FERTILIZED EGG | Sm eae NEW PARTHENO- NETIC GENERA- | FROM INBREED- 114... 303-306 GENETIC WILD TIONS 413-416 | ING B Ist | onmanl Oi aer RACE GENS = 5 = || ; ATION | | | | | | | eee ERE Sa RO pgkegbad ds es lated duced d.?s lated duced} d.?s lated duced d.?s/ lated duced d.2s_ — = — | | | - —___—_—} __— —— — BeaGe ee 10a ete tte) 2) |) ty lo 10 | 35 13.5 Sie e125 ea) WT 5/16-18 =| 8 | 25 |3.12} 3 | 1 |0.33+) 9 | 58 (6.44 BM Ly sey xe) PR 5/ 18-20 7 oN The |) eT he 9 | 87 |9.66+/ 5 53 10.6 II 5/ 10-22 | 8 28 | 3.5 4 | 13 |3.33+) 9 72 8 5 64 | 12.8 | II\/ x i = es = |e Summary.... 33 | M1 | 2.15) 13 | 23 [1.76 | 37 | 252 6.81 20 | 176 | 8.8 350 DAVID DAY WHITNEY TABLE 7 Showing by the comparative reproduction rates that inbreeding race B two successive times does not reinvigorate it D | NEW PARTHENOGE- l B 2NnpD | NETIC WILD RACE FERTILIZED EGGS FROM INBREEDING B Isr Young 99s __ Parthenogenetic Offspring of daughter , 7 | . 7 each tae tories frarnalss | Parthenogenetic | =. le | 99s d. 9s Av.no | No. Time 1911 | Time 1911 | No. | Time 1911 | No. ‘Ay. N0-|i) 5 5 80 | 16 26 | A.M. 6-14 Eve. 6-17 | 9 A.M. 6-20 1 | 0.5 5 80 16 27 A.M. 6-14 | A.M. 6-20 | Large} 9 dead. | No young. | Sterile 28 A.M. 6-14 A.M. 6-20 Large 9 dead. | No young. | | Sterile 29 | A.M. 6-14 | A.M. 6-20 | Large| 9 dead. | No yjoung. | | | Sterile 30 | A.M. 6-14 | A.M. 6-20 | Largel @ dead. | No young. | Sterile 31 | A.M. 6-16 | A.M. 6-18 3 | AM. (6=20))) 25) 8. 33-1. | 5 67 5.4 32 | A.M. 6-16 | A.M. 6-18| 4 | A.M. 6-20 | 11 | 2.754 | i 67 13.4 33 A.M. 6-16 | A.M. 6-18 4 | A.M. 6-20 | DOM OAZD 5 (He) asia! 34 A.M. 6-16 | A.M. 6-19 5 P.M. 6-21 0 0 5 83 16.6 35 A.M. 6-16 | A.M. 6-19 4) >) PM6—215 020 5 83 16.6 36 A.M. 6-16 | Eve. 6-21 Large) 9 dead. No young. Sterile 37 A.M. 6-16 Eve. 6-21 Large 9 dead. No young. | Sterile 38 A.M. 6-16 | Eve. 6-21 | Large, 9 dead. No young. Sterile 39 A.M. 6-16 | Eve. 6-21 | Large 9 dead. | No young. | Sterile 40 | A.M. 6-16 | Eve. 6-21 | Large) 9 dead. {No young. | Sterile 41 Eve. 6-17 | A.M. 6-19 3 P.M. 6-21 | 23 7.66+- 5 83 16.6 42 | Eve. 6-17| A.M. 6-19| 3 | P.M. 6-21| 32 |10.66+ 5 83 | 16.6 | 43 Eve. 6-17 | A.M. 6-20 5 P.M. 6-22) 31 6.2 4 62 15.4 | 44 Eve. 6-17 | A.M. 6-20 4 P.M. 6-22 7 ETA) 4 62 | 15.4 45 Eve. 6-17 | A.M. 6-22 Large 9 dead. No young. | Sterile REINVIGORATION PRODUCED BY CROSS FERTILIZATION 351 TABLE 7—Continued B 2npD | FERTILIZED EGGS FROM INBREEDING B Isr | D NEW PARTHENOGE- | NETIC WILD RACE Young 29s Parthenogenetic | Offspring of daughter from different daughter females | Parthenogenetic fertilized eggs | isolated females | No.| Time 1911 | Time 1911 | No. | Time 1911 | No. | Av.no.|, 2 28 ea une. Copan’ ; | |AV-DO-lisolated| d. 9s duced | | P| 46 | Eve. 6-17 | A.M. 6-22 | Large) 9 dead. | No young. Sterile 47 | A.M. 6-19 | Eve. 6-21 3 | Eve. 6-23 OFF 10 5 66 13.2 48 | A.M. 6-19 | Eve. 6-21 5 | Eve. 6-23 39 7.8 5 66 13,2 49 | A.M. 6-19 | Eve. 6-21 3 | Eve. 6-23 0 0 5 66 13.2 50 | A.M. 6-19 | Eve. 6-21 4 | Eve. 6-23 26 6.5 5 66 13.2 51 | A.M° 6-19 | Eve. 6-21 4 | Eve. 6-23 33 8.25 5 66 13.2 51 | | 127 345 | 2.71+ | 159 1622 | 10.2 | Summary 32 127 (23 ||P) se |) oa | Sina (oe | | the exact | | number of | fertile indi- | viduals used. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 3 aon DAVID DAY WHITNEY TABLE 8 Showing by the comparative reproduction rates that crossing the two races A and B causes a reinvigoration of the ensuing hybrid race ° A 408 X B 2NnD FERTILIZED EGGS FROM THE PROBABLE CROSSING OF RACE A, | D PARTHENOGENETIC GENERATION 408 + AND RACE B, NEW PARTHENOGE- AFTER INBREEDING TWICE, IN A MIXED CULTURE NETIC WILD RACE OF THE TWO RACES IN A BATTERY JAR : | ae ee Bee Peis Offspring of daughter | Parthenogenetic fertilized eggs | isolated eee No. | Time 1911 | Time 1911 | No. | Time 1911 No.| AY: |, 228 ae an | | no. Oo sigs || ce Oe 1 |M. 5-22 | Eve. 5-24| 5 |AM.5-27| 53/106 | 5 62 | 12.4 2|M. 5-22|Eve. 5-24) 5 |AM.5-27| 62 /12.4 | 5 62 | 12.4 3 | Eve. 5-22 | Eve. 5-24 5 | AM. 5-27 | 54 /10.8 | 5 62 12.4 4 | Rive. 5-22 | Eve. 5-24 | 3 |AM 507/422 114 | 5 62 12.4 5 | Eve. 5-22 | Eve. 5-24 | 5 | A.M. 5-27| 46 | 9.2 | 5 62 12.4 6 | Eve. 5-23 | Eve. 5-27 | 5 | Eve. 5-29 | 15 3 5 40 8 a al Eve. 5-23 | Eve. 5-27 | 5 | Eve. 5-29 31 6.2 5 | 40 8 8 | Eve. 5-23 | Eve. 5-27 | 5 | Eve. 5-29 | 48 | 9.6 5 | 40 8 9 | Eve. 5-24 | Eve. 5-27 5 | Eve. 5-29 31 6.2 5 40 8 10 | Eve. 5-24 | Eve. 5-27 | 5 | Eve. 5-29 | 40 | 8 ee 8 11 | Eve. 5-24 | Eve. 5-27 | 3 | Eve. 5-29 @ i 2 Sian) 8 12 | Eve. 5-24 | Eve. 5-27 | 3 | Eve. 5-29 21 Uf 5 40 8 13. | Eve. 5-24 | Eve. 5-27 4 | Eve. 5-29 35 8.75 5 40 8 } 14 | Eve. 5-24 | Eve. 7-28 | Large 9 alive. | No young. | | Sterile 15 | Eve. 5-24 | Eve. 5-27 2 | Eve. 5-29 15 7.5 5 | 40 8 16 | Eve. 5-24 | Eve. 5-27 4 | Eve. 5-29 24 6 5 40 8 17 | Eve. 5-24 | Eve. 5-27 | 3 | Eve. 5-29 17 5.66+- 5 40 8 18 | Eve. 5-24 | Eve. 5-27 5 | Eve. 5-297) 11 30) 5 40 | 8 | s | feos’ 18 72 551 7.6+ | 85 790 9.2+ | Summary | | | | | 17 he) 551 7.6+ 10 102) 11052 Summary | of exact | number of individuals | | used 14 59 519 8.79 10 102 10.2 Summary after elimi- | nation of | nos: Gl and 18. 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