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THE JOURNAL 


OF 


EXPERIMENTAL ZOOLOGY 


EDITED BY 


WiuiaM E. Caste FRANK R. LILuiE 

Harvard University University of Chicago 
Epwin G. ConxKLIN Jacques Logs 

Princeton University Rockefeller Institute 
CuHar_es B. DAVENPORT Tuomas H. Morcan 

Carnegie Institution Columbia University 
Horacr JAYNE GrorGe H. Parker 

The Wistar Institute Harvard University 
HersBenrt S. JENNINGS Epmunp B. Witson, 

Johns Hopkins University Columbia University 

and 


Ross G. HARRISON, Yale University 
Managing Editor 


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VOLUME 12. sh ie 
1912 cat 


THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY 
PHILADELPHIA, PA. 


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CONTENTS 
1912 
No. 1. JANUARY 5 
Epwin G. Conxuin. Cell size and nuclear size. Thirty-seven figures...... 1 


Raymonp Peart aNp Maynie R. Curtis. Studies on the physiology of 
reproduction in the domestic fowl. V. Data regarding the physiology 
PmUHeROVIOICtee HOUT HEUTOS > <n. s0.00s.a0e sae veneaacls so be Pace argo eden 99 


L. B. Nicz. Comparative studies on the effects of alcohol, nicotine, tobacco 
smoke and caffeine on white mice. I. Effectson reproduction and growth. 
(CBGRGRARC «Gade ini: eae Ree en aie ee ees cece ren ene merc or 133 


No. 2. FEBRUARY 10 


Frank W. Bancrorr. Heredity of pigmentation in Fundulus hybrids. 
SRRTTTRUAT NCEE BS 2 a gas gw wise a ayes cinieinieys » Coe, ce Sees oer ee else 153 


Put Rav anp Nettie Rav. Longevity in Saturniid moths; an experimental 
Ree MUR VORID TINO SEs (52 ck, -19' ates ais «wie oe Sel stisiee tls Salsa ie cess ee 179 


LoranvE Loss Wooprurr. Observations on the origin and sequence of the 
protozoan fauna of hay infusions. Fifteen figures..............--. .. 205 


Morais S. Fine. Chemical properties of hay infusions with special reference 
to the titratable acidity and its relation to the protozoan sequence. Five 


A. FRANKLIN SHULL. ‘Studies in the life cycle of Hydatina senta. III. 


Internal factors influencing the proportion of male-producers. Six 
EROS. oe EM = «5. 5 wail cin cen Slee soe 5 ne ye oe Cyelsmsiericl sans 283 


\ 


iV CONTENTS 


No. 3. APRIL 5 


Hevten Dean Kina. 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...................... 319 


Davin Day Wuitney. Reinvigoration produced by cross fertilization in 
Joh iZek:ntitsid:(-) 0k: pene CC eee eee aS occ con hoduaas soabee 337 


Manton Copretanp. The olfactory reactions of the puffer or swellfish, 
Spheroides maculatus (Bloch and Schneider)........................04. 363 


Joun C. Puituies. Size inheritance in ducks................ achoooddooede 369 


Jacques Logs anp F. W. Bancrorr. Can the spermatozo6n develop outside 
theiegg? Mleventfigures: «2 <.<..2: ooo. ++ + «seen eer Eee .- 381 


Davin H. TENNENT. Studies in cytology. I. A further study of the chro- 


mosomes of Toxopneustes variegatus. II. The behavior of the chromo- 
somes in Arbacia-Toxopneustes crosses. Twenty-one figures.......... 391 


No. 4. MAY 20 


Frank R. Livite. Studies of fertilization in Nereis. III. The morphology 
of the normal fertilization of Nereis. IV. The fertilization power of por- 
tions of the spermatozoén. Forty-eight figures........................ 413 


T. H. Morean. The elimination of the sex-chromosomes from the male- 
producing eggs of Phylloxerans. Twenty-nine figures.................. 479 


A. H. Sturtevant. An experiment dealing with sex-linkage in fowls. Four 
Merket Henry Jacogps. Studies on the physiological characters of species. 
I. The effects of carbon dioxide on various protozoa.................... 519 


Jacques LorB AND HArRDOLPH WASTENEYS. On the adaptation of fish (Fun- 
dulus)) to bigher temperatures......--.2- seeps. - > <\-- sae eee 543 


rae 


AUTHOR AND SUBJECT INDEX 


DAPTATION of fish (Fundulus) to higher 
temperatures. On the 543 
Alcohol, nicotine, tobacco smoke and caffeine on 
white mice. Comparative studies of the effects 

of I. Effects on reproduction and growth. 133 
Amphibians. Studies on sex-determination tn. V. 
The effects of changing the water content of the 

egg at or before the time of fertilization on the 
sex-ratio of Bufo lentiginosus. 319 
Arbacla-Toxopneustes crosses. Studies in cytology. 
I. A further study of the chromosomes of T. 
variegatus. II. The behavior of the chromo- 
somes in 391 


ANCROFT, Franx W. Heredity of pigmenta- 
tion in Fundulus hybrids. 153 


Bancrort, FI. W., Lops, Jacques anv. Can the 
spermatozoon develop outside the egg? 381 


AFFEINE on white mice. Comparative stud- 
jes on the effects of alcohol, nicotine, tobacco 
smoke and I. Effects on reproduction and 
growth. 133 
Carbon dioxide on various protozoa. Studies on 
the physiological characters of species. I. The 
effects of 519 
Cell size and nuclear size. 1 
Characters of species. Studied on the physiologi- 
eal. I. The effects of carbon dioxide on various 
protozoa. 519 
Chemical properties of hay infusions with special 
reference to the titratable acidity and Its rela- 
tion to the protozoan sequence. 265 
Chromosomes of Toxopneustes variegatus. II. The 
behavior of the chromosomes in Arbacia-Toxo- 
meustes crosses. Studies in cytology. I. A 
urther study of the 391 
Conkuin, Epwin G. Cell sizeand nuclearsize. 1 
CoprLanp, Manton. The olfactory reactions of 
the puffer or swellfish, Speroides maculatus 
(Block and Schneider). 363 
Curtis, Maynie R., Peart, RayMonp and Stud- 
jes on the physiology of reproduction in the 
domestic fowl. V. Data regarding the phys- 
jology of the oviduct. 99 
Cytology. Studies in I. A further study of the 
chromosomes of Toxopneustes variegatus. 
The behavior of the chromosomes in Arbacia- 
Toxopneustes crosses. 391 


)eces: Size inheritance in 369 


ERTILIZATION in Hyatina senta. Rein- 
vigoration produced by cross 337 
Fertilization in Nereis. Studies of III. The mor- 
phology of the normal fertilization of Nerels. 

V. The fertilization power of portions of the 
spermatozoon. 413 
Fixe, Morris S. Chemical properties of hay In- 
fusions with special reference to the titratable 
acidity and its relation to the protozoan se- 
quence. 265 
Fowl. Studies on the physiology of reproduction 
in the domestic WV. Data regarding the physi- 


ology of the oviduct. 99 
FYowls. An experiment dealing with sex-linkage 
in 599 


Vv 


Fundulus hybrids. Heredity of pigmentation in 


153 
Fundulus to higher temperatures. On the adap- 
tation of fish 543 


ROWTH. Comparative studies on the effects 

of aleohol, nicotine, tobacco smoke and caf- 
feine on white mice. I. Effects on reproduc- 
tion and 133 


EREDITY of pigmentation in Fundulus 
hybrids. 153 


Hybrids. Heredity of pigmentation in Fundu- 
lus 153 
Hydatina senta. Reinvigoration produced by cross 
fertilization in 337 
Hydatina senta. Studies in the life cycle of. III. 
Internal factors influencing the proportion of 
male-producers. 283 


NFUSIONS. Observations on the origin and 
sequence of the portozoan fauna of hay 205 


Infusions with special reference to the titratable 
acidity and its relation to the protozoa sequence. 
Chemical properties of hay 265 

Inheritance in ducks. Size 369 

Internal factors influencing the proportion of male- 
producers. Studies in the life cycle of Hydatina 
senta. 283 


ACOBS, Merxet Henry. Studies on the 
physiological characters of species. I. The 
effects of carbon dioxide on various proto- 
zoa. 519 


ING, Heten Dean. Studies on sex-deter™ 


mination in Amphibians. V. The effects 
of changing the water content of the egg at 
or before the time of fertilization on the sex- 
ratio of Bufo lengitinosus. 319 


IFE cycle of Hydatina senta. Studies in the. 
TIT. Internal factors influencing the propor- 

tion of male-producers. 283 
Litire, FRANK R. Studies of fertilization in Nereis. 
III. The morphology of the normal fertiliza- 
tion of Nereis. IV. The fertilization power of 
portions of the spermatozoon. 413 
Loren, Jacques, and Bancrort, F. W. Can the 
spermatozoon develop outside the egg? 381 
Lorn, Jacques, and WasTENEYs, HarpotpH. On 
the adaptation of fish (Fundulus) to higher 


temperatures. 543 
Longevity in Saturniid moths; an experimental 
study. 179 


N ALE-PRODUCERS. Studies in the life cycle 
ih of Hydatina senta. III. Internal factors 


influencing the proportion of 283 
Male-producing eggs of Phylloxerans. The elimi- 
nation of the sex-chromosomes from the 479 


Mice. Comparative studies on the effects of alcohol, 
nicotine, tobacco smoke and caffeine on white. 
I. Effects on reproduction and growth. 133 
Moraan, THomas H. The elimination of the sex- 
chromosomes from the male-producing eggs 
of Phylloxerans. ~ 479 
Moths; an experimental study. Longevity in Satur- 
niid 179 


vil AUTHOR AND SUBJECT INDEX 


EREIS. Studies of fertilization in. III. The 
morphology of the normal fertilization of 
Nereis. IV. The fertilization power of por- 
tions of the spermatozoon. 413 

Nice, L. B. Comparative studies on the effects 
of alcohol, nicotine, tobacco smoke and caffeine 
on white mice. I. Effects on reproduction and 

growth. 133 

Nicotine, tobacco smoke and caffeine on white mice. 

Comparative studies on the effects of alcohol, 

I. Effects on reproduction and growth. 133 

Nuclear size. Cell size and 1 


Oe. reactions of the puffer or swell- 
fish, Speroides maculatus (Block and Schnej- 
der). The 363 
Oviduct. Studies on the physiology of reproduction 
in the domestic fowl. V. Data regarding the 
physiology of the 99 


EARL, Raymonp, and Curtis, Maynie R. 
Studies on the physiology of reproduction 

in the domestic fowl. V. Data regarding the 
physiology of the oviduct. 99 
Paruips, JoHN C. Size inheritance in ducks. 369 
Phylloxerans. The elimination of the sex-chromo- 
somes from the male-producing eggs of 479 
Pigmentation in Fundulus hybrids. 


° 
Protozoa. Studies on the physiological characters 
of species. I. The effects of carbon dioxide 
on various 519 
Protozoan fauna of hay infusions. Observations 
on the origin and sequence of the 205 
sequence. Chemical properties of hay in- 
fusions with special reference to the titratable 
acidity and its relation to the 265 


R2& NewLui£, Por Rav, and. Longevity in 
Saturniid moths; an experimental study. 179 


Reproduction and growth. Comparative studies 
on the effects of alchohol, nicotine, tobacco 
smoke and caffeine on white mice. I. Effects 
on 133 

Reproduction in the domestic fowl. Studies on 
the physiology of V. Data regarding the phy- 
siology of the oviduct. 99 


Serene moths; an experimental study. 
Longevity in 179 


Sex-chrosomomes from the male-producing eggs of 
of Phylloxerans. The elimination of the 479 


Heredity 
153 


Sex-determination in Amphibians. Studieson. V. 
The effects of changing the water content of 
the egg at or before the time of fertilization on 
the sex-ratio of Bufo lengitinosus. 319 

Sex-linkage in fowls. An experiment dealing with 599 

Sex-ratio of Bufo lentiginosus. Studies on_sex- 
determination in Amphibians. Y. The effects 
of changing the water content of the egg, at or 
before the time of fertilization on the 319 

Suuut, A. FRANKLIN. Studies in the life cycle of 
Hydatina senta. III. Internal factors influenc- 
ing the proportion of male-producers. 283 

Species. Studies on he physiological characters of. 
I. The effects of carbon dioxide on various 
protozoa. 519 

Spermatozoon develop outside the egg? Can the 381 

——Studies of fertilization in Nereis. III. 
The morphology of the normal fertilization 
of Nereis. IV. The fertilization power of por- 
tions of the 413 

Spheroides maculatus (Block and Schneider). The 
olfactory reactions of the puffer or swellfish. 363 

Sturtevant, A. H. An experiment dealing with 
sex-linkage in fowls. 599 

Swellfish, Speroides maculatus (Block and Schnet- 

aan): The olfactory reactions of _ the pul 


f reste eeaee On the adaptation of fish 
(Fundulus) to higher 543 


Tennant, Davin H. Studies in cytology. I. 
A further study of the chromosomes of Toxo- 
pneustes variegatus. II. The behavior of the 
chromosomes in Arbacia-Toxopneustes cr 
es. 3) 

Tobacco smoke and caffeine on white mice. Com- 
parative studies on the effects of aleohol, nico- 
tine, I. Effects on reproduction and growth.133 

Toxopneustes variegatus. II. The behavior of the 
chromosomes in Arbacia-Toxopneustes crosses. 
Studies in cytology. I. A further study of 
the chromosomes of 391 


ASTENEYS, Harpotpx, Loes, Jacques, 

and On the adaptation of fish Cone 

to higher temperatures. 543 

Water content of the egg at or before the time of 
fertilization on the sex-ratio of Bufo lengiti- 
nosus. Studies on sex-determination in Am- 
phibians. V. Theeffectsofchangingthe 319 
WuitNEY, Davin Day. Reinvigoration produced 
by cross fertilization in Hydatina senta. 337 
Wooprurr, LorRanpE Loss. Observations on the 
origin and sequence of the protozoan fauna of 
hay infusions. 205 


CELL SIZE AND NUCLEAR SIZE 


EDWIN G. CONKLIN 


From the Department of Biology, Princeton University 


THIRTY-SEVEN FIGURES 


CONTENTS 
PART I. CELL SIZE AND NUCLEAR SIZE IN NORMAL DEVELOPMENT............ 4 
I. Unequal cell division.......... 2 ah a a ae eS re 4 
1. The maturation divisions. . SPER een cer ese Ac te dc 4 ; 
OO EC. oe 5 ea es ee 6 j 
5h Significance of the yolk lobe. . SS ren ee Ee ea ee ae 9 \ 
II. Cell size and nuclear size in eggs and blastomeres................ . 212 
1. Cell size and nuclear size in the cleavage of Crepidula plana.... 14 
2. Cell size and nuclear size in the cleavage of Fulgar carica....... 23 
lL. Cell size and nuclear size in adult tissue cells...................... 25 
Wen he inciting causes of cell division. ..........6..60cesee esse eee eeee 29 
VY. Growth of protoplasm during cleavage...................... a8,r GA 
VI. Rate of nuclear growth during cleavage. panty e sk: hap ena 36 
1. Nuclear growth during the cleavage of the egg of Crepidula.... 38 
2. Nuclear growth during the cleavage of the egg of Fulgar....... 40 
3. Nuclear growth during the cleavage of other animals. ..... 42 
4. Growth of different nuclear constituents............... Sine 
a. Nuclearsap........... Be ht A ORE SC ate 44 
Joy, LOT eae LS TE ee ce 46 
¢-;Ghromatinis..<...... -< ee PN ee eee, soe eee aie Breed oi) 
d. Chromosomes.......... BN NAS OAS ee ee 48 
e. Plasmasomes............ Rieke Bev eae ey ae nee 51 
Pe GeantrosOmeR And spheres... .... 25. cecccsec se tee esters 53 
5. Conclusions as to nuclear growth stoma cleavage..........-- 54 
6. Comparison of growth of chromatin with increase of chemical 
substances and processes during cleavage............-.----- 56 
VII. Senescence, rejuvenescence, and the ratio of nucleus to plasma..... 57 
PART II. EXPERIMENTAL STUDY OF CELL SIZE AND NUCLEAR SIZE IN THE EGGS 
OF CREPIDULA PLANA...... : SR Pe ee ere ere Ose 63 
I. Nuclear size and chromosome nates ee eR RR ite Mic 63 
II. Nuclear size and cell size in centrifuged eggs of Crepidula.......... 64 
1 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 
JANUARY, 1912 


bo 


EDWIN G. CONKLIN 


III. General results of these experiments .................2..2+-e+020e8 75 

i” Nuclearsizeanicentrihuredierocmassspne cee meee eee - =a. 75 

2. The sizes of spindles, centrosomes, spheres and asters........ 77 

3. The rhythm of division in centrifuged eggs Secon Sete 

4. Growth of cytoplasm at the expense of yolk................... 77 

5. Unequal and differential cell divisions ayer aes » ers 

6. Regulation in the cleavage process Ste ay Soe ad 81 

General summary and index................. PRG oc Boe CORSE ob tee RS 83 
Literature cited Bo ee Lhe Si eau afin 00s Cyne MOET AT RE ha ae 88 


In the development of all organisms considerable differences 
of size appear, sooner or later, among constituent cells; sometimes 
the blastomeres of the cleaving egg differ in size, in other cases 
these differences appear only later during the blastula, gastrula, 
or larval stages. Endoderm cells are usually larger than those 
of the ectoderm, ciliated cells are generally larger than non-ciliated 
ones, muscle and nerve cells are usually larger than epithelial 
or mesenchyme cells. 

These differences in the size of cells may be due to unequal cell 
division, to unequal rate of division, or to unequal growth of 
cells after division, and in some eases all of these factors may be 
represented in the same egg or embryo. It is frequently assumed 
that unequal cell divisions are caused by the accumulation of 
metabolic substances, such as yolk at one side of a cell, and the 
crowding of the protoplasm and nucleus to the opposite side. 
Such unequal divisions are frequently found in yolk-laden eggs, 
and may be artificially produced at will by centrifuging the yolk 
to one pole or the other of a dividing cell. But in many cases this 
is not the cause of unequal cell division; the yolk may be uniformly 
distributed with regard to the poles of the spindle and yet the 
cleavage, may be unequal, or unequal division may take place in 
purely protoplasmic cells, in which the eccentric position of the 
spindle is not due to pressure. Innumerable cases of this sort 
have been found both in normal and in experimentally altered 
conditions. Often such unequal divisions are associated with 
visible histological differences in the resulting cells. The study 
of cell-lineage has shown that in some cases a particular cell is 
distinguished from the time of its formation by its size, proto- 
plasmic structure, rate of division, prospective significance and 


CELL SIZE AND NUCLEAR SIZE 3 


potency. In such cases differences in the sizes of cell are associ- 
ated with some of the earliest differentiations of the developing 
egg. 

But differences in the size of cells may be due, not to unequal 
cell division, but to unequal rates of division, or to unequal growth 
of cells subsequent to division. In some instances cells divide 
rarely and consequently become large, while adjoining cells divide 
frequently and therefore remain small. The fact that cells are 
not always of the same size at the time of division is one of capital 
importance for it shows conclusively that the factors which bring 
about cell division may be separated from those which cause 
growth. 

In connection with the size of cells as a whole may be considered 
the sizes of many of their constituent parts, such as nuclei, 
chromosomes, plasmasomes, centrosomes, ete. The size relations 
which exist between these parts of the cell and the plasma should 
throw light upon the interrelation between these cell constituents 
in other respects than size. The quantitative relations of differ- 
ent cell constituents at various phases of activity should be of 
significance in the study of many fundamental problems of growth, 
differentiation and cellular physiology. 

Within the past few years several contributions on this sub- 
ject have appeared, principally from Boveri and R. Hertwig, 
and their students. In so far as these works have dealt with the 
development of the egg they have been based on a study of eggs 
of ‘indeterminate cleavage’ in which it is not possible to trace 
individual blastomeres throughout the cleavage period; the re- 
sults have therefore been mass results, based on averages of 
cells of a given stage. In the study of vital phenomena it is 
frequently important to deal with individual rather than with 
average results; in the following pages I have attempted to apply 
the method of the quantitative study of cells and of cell constit- 
uents to individual blastomeres at various stages of the cleavage. 


4 EDWIN G. CONKLIN 


PART I 
CELL SIZE AND NUCLEAR SIZE IN NORMAL DEVELOPMENT 
I. Unequal cell divisions 


1. The maturation divisions. The most unequal of all cell 
divisions are those which give rise to the polar bodies. The 
actual diameter of the first polar body and of the egg, and the 
relative volumes of the two, are here given for a number of dif- 
ferent animals. These measurements were made on eggs which 
had been fixed, stained, and mounted in balsam. 


TABLE 1 


Sizes of polar bodies and eggs 


SPECIES DIAMETER FIRST DIAMETER RELATIVE VOLUMES 


POLAR BODY OF EGG 
Me a 

Cumingia tellinoides. ... : 6 45 1 : 421.8 
Amphioxus lanceolatus....... 6 108 1 : 5832.0 
Cynthia partita....... 9 105 1 : 1560.8 
Cerpidula plana... . 12 136 1 : 1442.8 
Crepidula fornicata. ... 12 182 1 : 3443.0 
Crepidula convexa.... : 15 280 1 : 6434.5 
Crepidula adunea..... : 15 410 1 : 20123.6 
Fulgur ecarica...... 3 15 1600 i eine) (0) 


In many other cases, such as the eggs of selachians, amphib- 
ians and birds, the disproportion between the polar body and the 
egg is much greater than in the cases here measured. The sig- 
nificant thing here is not merely the degree of inequality, but also 
the relative uniformity in size of the polar bodies as compared 
with the egg. Although the eggs of different animals vary enor- 
mously in size, the polar bodies vary relatively little, and it is 
safe to conclude, both from observation and experiment, that the 
polar bodies are in general the smallest cells which can be formed 
from egg cells by the process of normal cell division. 

In spite of this very great inequality of the daughter cells, the 
mitotic figure in the first maturation division of Crepidula and 
of many other animals is the largest in the whole life eyele. When 


CELL SIZE AND NUCLEAR SIZE oO 


first formed this spindle lies near the middle of the egg, and if the 
division wall were to form while the spindle lies in this position 
a polar body would be formed whose diameter would be to that 
of the egg as 1:1, 1 : 2, or at the least 1:3. Later the spindle 
moves toward the periphery until one pole comes into contact 
with the cell membrane. The membrane then protrudes over 
this pole and into this protrusion the end of the spindle moves; 
at the same time the spindle itself constantly grows shorter, 
until finally the spindle is but little more than double the diameter 
of the polar body, and in the separation of the polar body the 
division wall passes through the equator of the spindle. In 
Crepidula plana the first ‘maturation spindle shortens to about 
half its original length; during the metaphase its maximum length 
is about 42u, at the time when the polar body is being separated 
it is only 24u long. 

By means of centrifugal force it is possible to prevent the 
spindle from moving from its first position and also from short- 
ening, and under these circumstances giant polar bodies are 
formed, sometimes quite as large as the remainder of the egg. 
In all such cases the division wall passes through the equator 
of the spindle. Evidently the factors which bring about this 
most unequal of all cell divisions are (1) the eccentricity and (2) 
the shortening of the maturation spindle. 

The second polar body is but slightly smaller than the first, 
nevertheless the spindle is much smaller, its maximum length in 
Crepidula plana being 184; correspondingly it shortens much less 
in the anaphase than the first polar spindle, being almost as long 
when the division wall begins to form as in the metaphase. 
Though the second polar spindle may appear at some distance 
from the point at which the first polar body was formed, and 
although its axis may lie at right angles to that of the first polar 
spindle, it invariably rotates into the axis of the latter and the 
whole spindle moves toward the surface until its outer pole 
comes to lie immediately under the first polar body, and here 
the second polar body is pushed out. In this case the principal 
factor which causes the inequality of division is the eccentricity 
of the spindle. If the spindle is prevented by pressure or cen- 


6 EDWIN G. CONKLIN 


trifugal force from taking this eccentric position the resulting 
cell division may be nearly equal, or a giant second polar body 
may be formed (fig. 11). 

2. Cleavage. The first cleavage of Crepidula and of Fulgur 
is approximately equal. The pronuclei lie near the animal pole 
of the egg, the egg nucleus lying somewhat nearer the polar 
bodies than the sperm nucleus. The first cleavage spindle is 
oriented so as to lie at right angles to the egg axis, but it is im- 
possible in these eggs to determine whether the spindle lies in 
a particular cross axis or not. However in typical cases the 
spindle invariably lies at right angles to the chief axis with its 
equator in that axis, and the protoplasm and yolk are divided 
by the first cleavage plane with strict equality. The same is 
true of the second cleavage which in all these regards resembles 
the first. 

It has been generally assumed that equal cleavages, alternately 
at right angles, are due to simple mechanical conditions, such as 
the greatest diameter of the protoplasmic mass, and that they 
require no further explanation. As a matter of fact equal cleay- 
ages, and successively alternating ones, cannot be explained in 
so simple a manner. The fact that the first cleavage spindle 
invariably stands at right angles to the chief axis of the egg 
and with its equator in that axis shows that there is here some 
orienting power of the highest significance. It is well known 
that there is considerable variation in the path which the sper- 
matozoon takes through the egg, and in its manner of meeting 
with the egg nucleus; there is also much variation in the actual 
positions of the cleavage centrosomes and in the initial position 
of the first cleavage spindle, without any corresponding variation 
in the final position of the spindle or of the cleavage plane. As 
a result of the study of large numbers of eggs of many different 
animals, under both normal and experimental conditions, its 
seems to me necessary to conclude that the same factor which 
brings about an unequal division of an egg such as that of Unio, 
operates to cause the equal division of an egg like that of Crepid- 
ula; ‘this factor is to be found in the polarity and symmetry of 
the egg itself. In Unio, where the first cleavage is very unequal, 


CELL SIZE AND NUCLEAR SIZE 7 
Lillie (01) has shown that the spindle oscillates in the cell before 
coming to rest in its eccentric position; and in many cleavages 
in Crepidula the spindle may at first lie out of its normal position 
and may move later into its proper place; and this applies not 
only to the eccentricity but also to the axial position of the spin- 
dle. Something outside of the spindle itself determines the posi- 
tion which it shall take in the cell, and this is as true of equal and 
alternating cleavages as of unequal and non-alternating ones. 


TABLE 2 


Sizes of macromeres and micromeres in Crepidula and Fulgur 


SPECIES MACROMERES DIAMETER MICROMERES DIAMETER Parte 
Ke M 

1A-1D 81 la-ld 30 ca.19.9 31 
2A-2D | 80 2a-2d 36 ca. 10.6 :1 
3A-3D | 76 3a-3d 33 Core de I | 
Gaplang....... ANDY 66 4d 38 ca. 4.9:1 
4A-4C 60 4a4te 42 Ca. 2.0) suk 
5A, 5B 75 5a, 5b 60 ea: 1:9)271 
5C, 5D 68 5e, 5d 68 ca. aL 
1A-1D 195 la-ld 69 | ca. 22.0 :1 
C. convexa.... 2A-2D 195 2a-2d 50 | ca. 59.3 :1 
3A-3D 195 3a-3d 50 Ca.09e ot 
| 1A-1D 800 la-ld 0) ca. 1000 : 1 
| 2A-2D S00 2a-2d 80 ca. 1000 : 1 
Fulgurearica.. {) 3A-3D 800 3a-3d 80 ca. 1000 : 1 
4D 780 4d 130 ca. 216 :1 
ca ord 


4A4C 740 4a-4te 370 


The third, fourth, and fifth cleavages of the macromeres of 
Crepidula and of other gasteropods are successively alternating 
in direction, and are notably unequal in the size of the daughter 
cells; while the sixth and all subsequent divisions of the macro- 
meres are more nearly equal than the preceding ones. The 
diameters of the cells formed by these cleavages and their approxi- 
mate ratios, in Crepidula and Fulgur, are shown in Table 2. 

In the structure of the macromeres there is no visible organi- 
zation which would explain why the first two cleavages of the 
egg are equal, the three following ones very unequal and subse- 


8 EDWIN G. CONKLIN 


quent cleavages more nearly equal again, and yet it is certain that 
some such organization must be present. It is generally believed 
that the inequality of macromeres and micromeres is due to the 
quantity of yolk contained in the former and where the quantity 
of yolk is extremely great, as in Fulgur, this is undoubtedly one 
of the causes of the great difference in the sizes of the macromeres 
and micromeres; but that it is not the only cause of the inequality 
is shown by experiments in which by centrifuging eggs at the 
first or second cleavage two of the macromeres come to contain 
no yolk, while the other two contain all of the yolk; in the macro- 
meres which are purely protoplasmic and contain no yolk the 
subsequent cell divisions are still unequal, protoplasmic micro- 
meres of the usual size being separated from the protoplasmic 
macromeres, (see p. 81). The study of normal as well as of 
artificially altered cleavage points unmistakably to the conclu- 
sion that the position and axis of each spindle is fixed by the 
structure of the cell protoplasm, and since the position and axis 
of the spindles change regularly in successive divisions this 
protoplasmic structure must change regularly in successive cell 
generations. Boveri (’05) says that the position of the spindle 
is not due to a permanent cell structure, but that the constitution 
of the egg undergoes progressive alterations, which then react 
on the division centers. 

Among the micromeres certain cell divisions are quite unequal, 
and here there can be no question that this inequality of division 
is In no way associated with the presence of yolk, since the micro- 
meres are purely protoplasmic. In Crepidula the first and second 
subdivisions of the first quartet cells (figs. 3, 8). which give rise 
respectively to the ‘turret’ cells and the ‘apical rosette’ cells, 
are very unequal; as is also the division of the second quartet 
cells which give rise to the ‘tip’ cells of the arms of the ectodermal 
cross.. The diameters of the two daughter cells in each of these 
divisions, and the approximate ratio of one to the other, are as 
follows in Crepidula plana: 


la-ld,? 25y,la2-Id2, I13p..... Saasioss See Baas > eee Ratio2 : 
lat-—1d?-1, 30u, Iat?-1d?-* 18u...... ina a Ge Sone eee Ratio5 :3 
2al-12d!"1, 20u, 2al-2-2Qd?-*, 15y .. . (aot eee Ratio 2:1 


= 


CELL SIZE AND NUCLEAR SIZE 9 


In the case of normal eggs it cannot be demonstrated that the 
inequality may not be due to mutual pressure among the cells, 
but in certain experiments which will be described in another 
paper, this factor may be entirely eliminated, isolated blastomeres 
showing the same inequalities of division as do those in the cell 
complex. In all of these cases of definite types of cleavage, the 
position of the spindle, and consequently the direction of division 
and the relative size of the daughter cells, is determined by some 
structural peculiarity of the protoplasm and not by the presence 
of metabolic substances within the cell or by pressurefrom without. 

8. Significance of the yolk lobe. Under normal conditions 
the line of intersection of the first and second cleavage planes 
marks the chief axis of the egg, its two ends being the animal and 
vegetal poles. In eggs in which the cleavages are unequal, the 
chief axis, thus defined, runs from the animal pole, which is marked 
by the position of the polar bodies, to a point more or less removed 
from the diametrically opposite pole. 

Is this chief axis predetermined in the egg or is it established 
by the positions of the first and second cleavage planes? Observ- 
ation alone affords no positive answer to this question, but the 
fact that the spindle takes a definite and characteristic position 
in the egg indicates that something outside the spindle determines 
its position, and points to the conclusion that the chief axis is 
already present in the egg, as a structural differentiation before 
cleavage begins. This conclusion is well supported by experi- 
ment, as will be shown later. 

In this connection the significance of the so-called ‘yolk lobe’ 
is interesting. As is well known this lobe is found in many eggs, 
especially in those in which the first and second cleavages are 
unequal. It is present however in minute form in such eggs as 
those of Crepidula and Fulgur in which the first two cleavages 
are approximately equal, but in cases in which these cleavages 
are unequal it is much larger, and in general the size of the yolk 
lobe is proportional to the inequality of division. In all cases 
so far as I am aware the yolk lobe lies diametrically opposite the 
animal pole, and if detached from the egg at the time when it is 
fully formed, the egg divides into equal blastomeres, as Wilson 


10 EDWIN G. CONKLIN 


(04) found in Dentalium; if it remains attached it fuses, at the 
close of the cleavage, with one of the cells, which then becomes 
larger than the other one. In this case the cleavage spindle 
is not eccentric and the furrow cuts down through the center of 
the egg until it reaches the yolk lobe when it turns to one side of 
the lobe leaving it attached to one of the cells. In this way a 
cleavage which began as an equal one becomes unequal. Where 
the spindle is eccentric from the start and the furrow does not 
pass through the center of the egg the yolk lobe is not prominent. 
In this way inequality of division may arise through the eccen- 
tricity of the spindle, or through the formation of a yolk lobe which 
remains connected with one of the two daughter cells, which 
would otherwise be equal. 

One cannot study the eggs of different animals without being 
much impressed with the fact that the distribution of yolk to the 
four macromeres is highly characteristic of different species and 
orders. Thus among prosobranchs the yolk is distributed either 
equally to all the macromeres, as in Crepidula, Fulgur, Trochus. 
ete., or if one of the macromeres is larger than the other three it 
is the left posterior macromere, D, as in Nassa, Urosalpinx, 
Tritia, ete. Among opisthobranchs, if the macromeres are 
unequal in size it is one or both of the anterior ones, A or B, 
which is the larger. Among pulmonates, so far as I recall, the 
macromeres are always equal in size. 

The fact that there are these characteristic differences in the 
sizes of the macromeres of different orders indicates that they 
have some characteristic cause; and the fact that in nearly re- 
lated species the macromeres may be equal or unequal indicates 
that in this case the cause is not a very general one. If one 
considers that the first and second cleavages normally pass 
through the egg axis, and that their position is determined by 
this structural feature, the unequal distribution of yolk to the 
four macromeres may be due to the localization of the yolk in 
different parts of the ovarian egg,—on the posterior side of the 
chief axis in prosobranchs, on the anterior side in opisthobranchs; 
while a larger or smaller yolk lobe would determine the degree 
of inequality of the macromeres in the different species. 


CELL SIZE AND NUCLEAR SIZE 11 


Apart from the relation of the yolk lobe to unequal cleavage 
Wilson (’04) has shown that it bears some relation to the forma- 
tion of the pretrochal region in the larva of Dentalium; when the 
lobe was removed the pretrochal organs failed to develop. What 
the morphogenetic factors are, which are located in the yolk lobe, 
is not known, but the significance of the lobe can scarcely be for 
the formation of the pretrochal region, since in animals with no 
lobe or with a very minute one these regions form quite as well 
as in those with a large lobe. 

These explanations refer to the “prospective significance” of 
the yolk lobe, and I_ know of no certain evidence as to the cause 
of its formation. The fact that such a lobe is present in almost 
all gasteropod eggs, differing only in size in different species, and 
that it is present in the eggs of annelids and a large number of 
other animals, indicates that it has some cause of general occur- 
rence. In 1897 I suggested that the yolk lobe marks the point 
of attachment of the ovarian egg to the follicular wall. At this 
point there is left a little mass of protoplasm on the surface of 
the egg, and here there is a weak spot in the protoplasmic pellicle 
which surrounds the egg. If the egg is put under pressure the 
yolk may be caused to flow out at this point, and in the increased 
tension which accompanies mitosis a yolk lobe is often pushed 
out at this spot. 

On the whole then, it seems probable that the yolk lobe rep- 
resents a temporary extrusion of egg substance during mitotic 
pressure at the former point of attachment to the ovarian wall, 
and that as a result of the presence of a large lobe of this kind, 
the first and second cleavages may be rendered unequal though 
the intersection of the furrows may lie in the egg axis and in the 
polar diameter of the egg. 

In this connection one recalls the ‘ Dotterball’ and the ‘Granula- 
ball’ observed by Hogue (710) and Boveri (710) in centrifuged 
eggs of Ascaris. Boveri comes to the conclusion that these are 
formed because they lie outside the influence of the asters or 
spheres: ‘‘Man kénnte vielleicht sagen:—der von einen Sphiire 
eingenommene Plasmabezirk sucht sich von allem was ausserhalb 
dieses Wirkungskreises liegt, abzuschniiren,” (p. 123). He sup- 


2 EDWIN G. CONKLIN 


poses that when the spindle, lying at right angles to the egg axis, 
is pushed far toward the animal or vegetal pole, a ‘ball’ is formed 
at the opposite pole. Whether this ‘ball’ is homologous with 
the yolk lobe I shall discuss in another paper in which the arti- 
ficial production of such ‘balls’ will be considered, but I wish to 
point out here that although the first and second cleavage spindles 
in the large eggs of Crepidula and Fulgur le near the animal 
pole, and far from the vegetal, the yolk lobe in these forms is 
very small, whereas in the minute eggs of the oyster and the clam, 
where the spindles are much nearer the vegetal pole, the yolk lobe 
is relatively very large. If, as I believe, this lobe is the result of 
an unsymmetrical distribution of yolk and egg substance with 
reference to the egg axis, or in the case of Ascaris with reference . 
to the normal division plane, the great size of the lobe in some 
cases and its minute size in others, in which the area lying outside 
the ‘‘ Wirkungskreise” of the spheres is much greater than in the 
former, would find a ready explanation. 


IT. Cell size and nuclear size in eggs and blastomeres 


Strasburger (93) was the first to show by detailed measure- 
ments that a fairly definite ratio exists between the nuclear size 
and the cell size in the embryonic cells of any given species of 
plant. He gives tables of measurements of the sizes of nuclei 
and cells in some forty different species, the nuclei ranging in 
diameter from 16y to 3y, and the cells from 24 to 5u. . In gen- 
eral he found that the ratio of nuclear diameter to cell diameter 
is approximately as 2 to 3; and the ratio which exists in any case, 
is held to be due in general to the ‘working sphere of the nucleus,’ 
i.e., to the extent to which the metabolic interchange between 
nucleus and cytoplasm can reach. 

Gerassimoff (’01, ’02) found, in the cell division of Spirogyra, 
that when both daughter nuclei were caused to remain in one of 
the daughter cells, that cell grew to a larger size than normal, and 
he therefore concluded that the nuclear size determines the cell 
size. 


CELL SIZE AND NUCLEAR SIZE 13 


Boveri (’02, ’05) found that the size of the nuclei in sea urchin 
larvae is dependent upon the number of chromosomes which enter 
into the nuclei; in parthenogenetic or hemikaryotic eggs the nu- 
clei are smaller than in fertilized (amphikaryotic) ones, and they 
are smaller in the latter than in diplokaryotic eggs in which the 
number of chromosomes is greater than normal. Furthermore 
he found that nuclei with a small number of chromosomes are 
not only smaller than those containing a larger number but that 
the cells in which they lie are also smaller, owing to the occurrence 
of a larger number of cell divisions in cells with small nuclei than 
in cells with large ones. 

Boveri’s work was based primarily on his studies of echinoderm 
‘development and some of his conclusions are not applicable, 
without modification, to the eggs and larvae of other forms, 
especially forms in which there are great inequalities of cleavage 
and in which various cells of the larva differ markedly from one 
another in size. Thus his generalization, sometimes mentioned 
as ‘Boveri’s Law,’ viz., ‘‘Die Grésse der Larvenzellen ist eine 
Funktion der in ihnen enthaltenen Chromatinmenge, und zwar 
ist das Zellvolumen der Chromosomenzahl direkt proportional,”’ 
could not apply, without modification, to eggs or larvae in which 
various cells differ greatly in size without any corresponding 
difference in the number of chromosomes. Unequal cell divisions 
are frequently found in the development of mollusks, annelids 
and ascidians, where purely mechanical causes, such as mutual 
pressure between cells or the pressure of yolk within cells are not 
involved; in such eases the sizes of the nuclei invariably become 
proportional to that of the plasma, though the number of chro- 
mosomes remains the same in every nucleus. Similarly, many 
cells at first equal in size become unequal through dissimilar 
growth, and their nuclei then become unequal also. Finally, 
in each of the animal groups named, cells at first equal in size may 
become unequal through dissimilar rates of division. In all 
such cases the number of chromosomes appears to be, and pre- 
sumably is, the same in every nucleus of a given egg or embryo. 
Evidently in cases of normal development the number of chromo- 
somes does not determine the varying sizes of cells and nuclei. 


14 EDWIN G. CONKLIN 


R. Hertwig (’03, ’08) as a result of his earlier work (’89) upon 
protozoa, has laid especial emphasis upon the fundamental sig- 
nificance of the ratio of nuclear size to cell size. He says (’03, 
p. 56): 


Wir haben im vorhergehenden sehr komplizirte Wechselwirkung 
zwischen Kern und Protoplasma kennen gelernt. Verkleinerung der 
Kernmasse fiihrt zu Verkleinerung der Zellgrésse (Boveri), Vergréss- 
erung der Kernmasse zu einer Vergrésserung der Zelle (Gerassimoff, 
Boveri). Andererseits kann aber auch Schwund des Plasmas zu einer 
Reduktion des Kernmaterials Veranlassung werden. Diese Verhilt- 
nisse kann man nur erkliren, wenn man die oben vertretene Annahme 
macht, das jeder Zelle normalerweise eine bestimmte Korrelation von 
Plasma- und Kernmasse zukommt, welche wir kurz die “ Kernplas- 
marelation” nennen wollen. 


More recently Hertwig and his students have made many 
notable contributions to these ‘“‘new problems of the cell theory,” 
as Hertwig (’08) calls them. It has long been known that large 
cells have large nuclei, small cells small nuclei: 


Das Neue, welches in der Lehre von der Kernplasma-Relation gegeben 
ist, ist der Gedanke, dass das Massenverhiltnis von Kern zu Proto- 
plasma, der Quotient k/p, d.h. Masse der Kernsubstanz dividiert durch 
Masse des Protoplasma, ein gesetzmiissig regulierter Factor ist, dessen 
Grosse fiir alle von Kerne beeinflusten Lebensvorgiinge der Zelle, fiir 
Assimilation und organisierende Tatigkeit, fiir Wachstum und Teil- 
ung, von fundamentaler Bedeutung ist. 


Hertwig calls attention to the fact that the Kernplasma-Rela- 
tion differs in different phases of cell life, and he chooses for meas- 
urement that phase when the cell has come out of division and 
begins to nourish itself and to grow. This condition is known as 
the Kernplasma-Norm, and departures from it constitute what he 
calls Kernplasma-Spannung. This work of Hertwig and his 
school will be discussed more fully after the presentation of my 
observations. 

1. Cell size and nuclear size in the cleavage of Crepidula plana. 
In my work on Karyokinesis and Cytokinesis in Crepidula (’02) 
I showed that the sizes of nuclei, spheres and asters, centrosomes, 
chromosomes, and plasmasomes are correlated with the quantity 
of cytoplasm in the cell, and the following pages constitute an 


CELL SIZE AND NUCLEAR SIZE 15 


elaboration and further extension of that work. The egg of 
Crepidula plana is a particularly favorable object for the study 
of such a subject. The eggs may be stained and mounted entire 
in such manner that all of these cell constituents show with great 
distinctness, and the advantage of seeing whole eggs and nuclei 
in making such measurements is sufficiently obvious. 

A further advantage of the study of whole eggs is found in the 
fact that the exact stage in the cell cycle is more easily determined 
in whole eggs than in sections. My work has shown that it is 
most important in comparing the sizes of cell constituents to 
compare precisely corresponding stages, and accordingly I have 
chosen for measurement stages of the maximum and minimum 
sizes of the nuclei. The growth of the nucleus is more rapid in 
the last stage of the resting period preceding mitosis (‘Kernteil- 
ungswachstum’ of Hertwig) than at any other time in the cell 
cycle, and in order to find the maximum nuclear size it is neces- 
sary to measure the nuclei just before the nuclear membrane dis- 
appears. Such stages are easily selected by looking for eggs in 
which part of the nuclei of a certain generation of cells are divid- 
ing while others have not yet begun to divide, as in figs. 1 and 2. 
At this stage there is great uniformity in the dimensions of the 
nuclei of particular blastomeres, and as the nuclei at this stage 
are regular spheres, it is easy to calculate their volumes. 

The cell dimensions are more difficult to determine than are 
those of the nucleus. In cells which contain yolk and in cells of 
irregular shape it is not possible to determine the volume of the 
plasma with accuracy. After the first cleavage the plasma and 
yolk are sufficiently well separated so that the dimensions of the 
cytoplasm can be fairly well observed; before the first cleavage the 
plasma is so mixed with the yolk that this can not be done and I 
have here had recourse to the method of centrifuging the yolk 
out of the egg, leaving only the nucleus and plasma which can 
then be easily measured. Wherever it could be done, I have 
chosen cells for measurement which were as nearly spherical as 
possible, but where the dimensions in different axes differed con- 
siderably I have determined the mean diameter, which is the one 
recorded. 


16 EDWIN G. CONKLIN 


It is well known that during mitosis the general surface tension 
of a cell increases, and the cell tends to become spherical in shape. 
In measuring the maximum cell size, I have usually taken the 
stage immediately after the nuclear membrane disappears, and 
when the cell approaches a spherical shape. Similarly the mini- 
mum cell size has been determined by measuring the daughter 
cells during the telophase when they are approximately spherical. 
I have confidence in the substantial accuracy of my measurements 
of these maximum and minimum sizes of the purely protoplasmic 
micromeres. The volume of plasma in the yolk-containing 
macromeres is merely an approximation. 

All measurements were made with Zeiss 1/1 micrometer eye- 
piece and 3 mm. homogeneous immersion objective. In all cases 
enough cells and nuclei were measured to give a fair average, 
though there is relatively little variation in the sizes of particular 
cells and cell constituents at corresponding stages in the cell 
cycle. All the eggs studied were fixed, stained and mounted in 
the same manner, so that alterations due to shrinkage should be 
approximately the same in all. 

It is evident from table 3 that while large cells have larger 
nuclei than small cells, the relation of nuclear volume to cell 
volume is not constant. In different blastomeres of the same 
egg the Kernplasma-Relation, measuring nuclei and cells at their 
maximum size, varies from 1 :14.5 to 1:0.37; even in purely 
protoplasmic cells it varies from 1 : 14.5 to 1:8.7. In cells con- 
taining yolk the ratio of nuclear volume to cell volume (includ- 
ing the yolk) varies from 1 :89.5 to 1 :34.8. In the different 
blastomeres of this egg there is no constant nuclear-plasmic ratio, 
or Kernplasma-Norm. However in different eggs corresponding 
blastomeres have the same Kernplasma-Relation, when measured 
at corresponding stages. The volumes of the protoplasm and of 
the nucleus show little variation in any given blastomere and the 
Kernplasma-Relation of each of the blastomeres named in table 
3 is practically the same in all eggs. Since many of these blasto- 
meres are peculiar in odplasmic constitution and prospective 
significance it is not improbable that the peculiarities in their 


CELL SIZE AND NUCLEAR SIZE 17 


TABLE 3 


Maximum nuclear size and cell size in the blastomeres of Crepidula plana; (measured 
just before nuclear membrane dissolves) 


DIAME oR VOLUME OF | 
BEeemM) jesse | vouwies ao tal ae | Tae Pome a 
BLASTOMSBES | (O25 ENE I INCLUDING | NUCLEUS NUCLEUS (VOLUME OF RELATION 
NUCLEUS | NUCLEUS | 
| | 
, be cubic bh “ | 7 cubic cubic 
i 
Before maturation. —_150 1,755,000 | ca.64* | 42 32,409 97,1381 | 1:3 
Before first cleay- | 930+ 
oe: SRC RCE ORNS 142 | 1,488,910 ca. 65° = |*o'24=34.5 21,375 121,430 1:5.6 
AB, CD, before sec- | 
ond cleavage. re} 106 619,329 ca. 51* | 24 7,238 61,741 | 1:8.5 
A, B, C, D, before | 
third cleavage... 82 286,712 ca. 44° 22 | 5,775 38,570 1:6.6 
1A-1D, before | 
fourth cleavage. . . 81 276,350 | ca. 40 21 | 4,849 28,431 1:58 
Ia-ld, before divi- 
BIO asics ass 30 4 1,437 12,603 | 1:8.7 
2A-2D, before fifth 
cleavage.......... 80 266,240 | ca. 36 18 3,055 21,196 Sy 
2a-2d, before divi- 
MlOQueeape sas. <<. 36 15 | 1,767 22,484 1:12.7 
lal--Idt-, before di- 
WABI OM Seaeeietata)a cats « 30 12 | 905 13,135 1:14.5 
1a?-—-1d2-, before divi- 
SOT mene a's(2'x 15 7 180 1,587 1:88 
3D, before sixth 
cleavage.......... 76 228,288 ca. 30 16 2,145 11,895 1:5.5 
3A-3C, before sixth 
cleavage.......... 76 228,288 | ca. 22 16 2,145 3,430 | 1:1.6 
3a-3d, before divi- 
IDI aeeie cd ocsie ve 33 l4 1,437 19,250 1:13.3 
2at-—2dl., before divi- 
POM etme rie iad ale! (0's 30 l4 1,437 12,603 1:8.7 
2a2.—-2d?., before divi- 
CG) Sane ecncee ne | 30 14 1,437 12,603 1:8.7 
4d, before seventh’ 
cleavage.......... 38 28,533 ca. 22 il 697 4,878 i by 
4A-4D, before sev- | 
enth cleavage... 60 112,320 | ca. 20 18 3,055 1,134 | 1:20.37 
da-4e, before sey- | 
enth cleavage..... 42 32,409 ca. 14 12 | 905 532 | 1: 0.58 


* After yolk has been centrifuged out of egg. In normal eggs yolk and proto- 
plasm are not well segregated at this stage. 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 


18 EDWIN G. CONKLIN 


Kernplasma-Relation may be the result of differentiations ale 
present in the blastomeres. 

In table 3 only maximum nuclear and cell dimensions are given 
for the different blastomeres. Results would undoubtedly differ 
ereatly if the minimum nuclear and cell dimensions were taken 
instead of the maximum. Accordingly in table 4 the minimum 
nuclear and cell dimensions fort the various blastomeres of Cre- 
pidula plana are given, together with the Kernplasma-Relation 
of each. 

It is well known to cytologists that in cells undergoing regular 
division the minimum size of the nucleus is reached in the late 
anaphase, when the individual chromosomes have contracted to 
their smallest size and when they are most closely crowded 
together. A little earlier than this stage the chromatic plate is 
wider and the spaces between individual chromosomes greater; 
a little later the chromosomes begin to absorb achromatin and to 
swell up to form the chromosomal vesicles. At this stage of 
greatest nuclear contraction the chromosomal plate has approxi- 
mately the form of a disk or short cylinder, and although the polar 
ends of the chromosomes are closer together than the equatorial 
ends, the disk being like a truncated cone, rather than a cylinder, 
we shall not greatly err if we treat this chromosomal disk as a 
short cylinder, rather than as a truncated cone. In table 4, 
in the column giving the dimensions of the chromosomal disk the 
first number is the diameter of the disk, the second its thickness. 

Popoff (’08) has found in Frontonia that immediately after 
cell-division there is a diminution of the nucleus, which is then 
followed by a slow growth (‘Funktionelles Wachstum’ of Hert- 
wig), and this by a much more rapid growth of the nucleus pre- 
ceding division (‘Teilungswachstum’ of Hertwig). Both the 
functional growth and the divisional growth occur in the cleavage 
of Crepidula, but there is no diminution of the nucleus following 
division as in Frontonia. On the other hand, the minimum nu- 
clear size is reached in the anaphase just before division of the 
cell body, as has been explained. 

In the early telophase the chromosomal plate is drawn close to, 
and moulded over, the centrosome, and consequently the shape 


CELL SIZE AND NUCLEAR SIZE 19 


of the chromosomal plate, its degree of curvature and width, is 
dependent in part upon the size of the centrosome. I found in 
1893 that in unequal cell division the centrosomes and asters 
become unequal before the cell division is finished, though in the 
earlier stages of mitosis the centrosomes and asters at the two 
poles of the spindle are equal in size; only after the cell division 
is finished do the daughter nuclei become unequal. The present 
work has confirmed these earlier conclusions and has shown in 
addition that the shape of the chromatic plate at the ends of the 
spindle is influenced by the size of the centrosomes, and hence 
by the equality or inequality of the division. If the centrosome 
is large the chromosomes form a slightly arched plate on its 
surface; if it is small the plate is highly arched. In the former 
case the plate remains relatively wide and the daughter nuclei 
when they are formed are disk-shaped; in the latter the plate and 
the daughter nuclei become more nearly spherical. Therefore, 
in comparing the sizes of chromatic plates it is necessary to meas- 
ure them before this difference in shape appears, i.e., in the late 
anaphase. But even when all these precautions are taken the 
probable error in measuring objects of such small dimensions is 
considerable, but at least these measurements give the relative 
order of magnitude of the chromosomal disks in the different 
blastomeres. 

The minimum cell dimensions occur in the early telophase, when 
the daughter cells first separate; at this stage the cells are nearly 
spherical in form and it is not difficult to calculate their volumes 
with substantial accuracy. While the minimum cell size does 
not occur at precisely the stage when the nuclei are smallest, 
it occurs so soon thereafter that it can make but little difference 
in the determination of the Kernplasma-Relation. 

In short the Kernplasma-Relation, when plasma and nuclei 
are measured at their minimum sizes, varies in different blasto- 
meres from 1:29 to 1: 285.6. Except in the division of certain 
cells in the fourth and fifth cleavages (2A—2D and 2a—2d, 2a'-2d' 
and 2a*-2d?) there is no appearance of a constant ratio between 
nucleus and plasma in these different blastomeres. In general 
the dimensions of the nuclear plates decrease with every cleav- 


20 EDWIN G. CONKLIN 


TABLE 4 


Minimum nuclear size and cell size in the blastomeres of Crepidula plana. (Nu- 
clear plate measured in the late anaphase; cell diameter in early telophase) 


2 Ee 4 g 5 « 
& moO on Zz im? D; 
STAGE BLASTOMERES c oon 2 fe 5 = 
pealcheraa| oGe : nae : 
g gaze pie ge oar Bs 
a 235 a bs B<,, Zo 
2 2m zz an aR ee 
a a a > > Ct 
Me “ “ cubic cubic fh 
2 cells AB, CD..| 105 ca. 46, 9x3 190.5 30,504 1 : 160. 
4 cells A,B,C,D,..| 78 ca. 40| 8x3 150.6 30,695 1 : 203.8 
Aerie 1A-1D... 75 ca. 36] 6x3 $4.6 24,166 1 : 285.6 
: \la-Id .. PH, 27 6x3 84.6 10,235 1 3120 
12 cells 2A-2D....| 72 ca. 30, 6x3 84.6 13,955 1 65 
2a-2d.... 30 30 | 6x3 84.6 13,955 6s 
16 cells 2 lal-ld! ..| 30 30 5563} 58.8 13,955 1 223% 
~ \1a2-1d? 15 15 ‘a15.60) 58.8 1,708 1:29 
90 cells 3A-3D... 72 Can ib 4x3 By st) 1,729 1:46 
< NSRESil oe cll) BE 25 | 4x38 37.5 8,087.5 | 1: 215.6 
24 cells 2a1-2d1...) 24 24 4x3 37.5 7.200 1 +195 
25 cells {4D ae 60 5x3 58.8 
(Ad errr ec cok 30 Byes} || 58.8 
2a7-2d2...| 24 24 | 4x3 | 31.50 7,200 13195 
29 cells { lat-1d!1 15 15) 4x3 3f.0 1,729.5 | 1 : 46 
lal2Idl-2 | 24 24) 4x3 | By nta 7,200 1:195 
39 cells JAA ae: | 4x3 37.5 
~ |4a-4e..... | 4x3 a0-0) 


age, while all the cells of the same generation have nuclear plates 
of about the same size, though their protoplasmic volumes vary 
widely. 

There is a great difference between the maximum and mini- 
mum volumes of the same nucleus, ranging from 1:27 to 1:38, 
while there is a relatively slight difference between the maximum 
and minimum sizes of the plasma and accordingly the Kern- 
plasma-Relation of any blastomeres varies continuously from the 
stage of minimum nuclear volume to that of maximum nuclear 
volume. Hertwig (08) has chosen as the stage showing the 
Kernplasma-Norm, ‘‘das Verhalten der jugendlichen Zelle, welche 
eben aus der Teilung hervorgegangen ist und nun anfingt sich 


CELL SIZE AND NUCLEAR SIZE 21 


von neuem zu ernihren, um abermals heranzuwachsen und sich 
zu teilen.”’ I have tried to make measurements at the stage 
described by Hertwig, but find that in these segmenting eggs it 
is too ill defined to be safely used. Between successive divisions 
the nuclei are growing continuously and rapidly and there is no 
clearly marked pause in the nuclear growth; accordingly slight 
differences in the stages chosen for measurement show relatively 
large differences in the sizes of the nuclei. 

In order to take a stage intermediate between the two ex- 
tremes of nuclear size, and in which the nuclei may be regarded 
as having reached a normal functional condition, not related 
primarily to the preceding or succeeding division, I have chosen 
that stage when the nuclei first become regularly spherical in 
shape. For a considerable part of the resting period the nuclei 
are elongated at right angles to the previous spindle axis, and 
in the plane of the chromatic plate of the previous anaphase; 
during the latter part of the resting period they grow very rapidly 
in preparation for the succeeding division (‘ Kernteilungswachs- 
tum’ of Hertwig). The stage when the nuclei first become spher- 
ical lies somewhere between these two phases and may therefore 
be considered to represent the mean nuclear size. However this 
stage is not so precisely defined as are the stages of maximum 
and minimum nuclear size, and therefore the nuclear dimensions 
are likely*to be more variable. 

It is obviously more difficult to determine the volume of the 
cleavage cells during the resting period, when they are pressed 
into irregular and polygonal forms, than during mitosis when they 
approach a spherical shape. However the approximate accuracy 
of the cell dimensions recorded in table 5 may be judged by com- 
paring them with the maximum and minimum cell dimensions, 
given in tables 3 and 4. 

The Kernplasma-Relatior. varies about as much for mean 
dimensions of the nucleus and cell as for maximum ones, though 
not as much as for minimum dimensions. In yolk-containing 
cells it varies from 1:1.1 to 1: 27.5 and in purely protoplasmic 
cells from 1:7 to 1:35.7. 


22 EDWIN G. CONKLIN 


TABLE 5 


Mean nuclear size and cell size in the blastomeres of Crepidula plana. (Measured 
when nuclei first become spherical after division) 


Te 5 a 
cg ce ne 
& 58 me | a z 
STAGE BLASTOMERES “ Ae ° | fe 2 
m as % | ° u 
a B28 || g 2 
20 BER : : Be 
a a a > 4 
Mw BL Mu cubic cubic ps 
f 918 
Before cleavage.... 136 ca. 60} 4 12 3,960 109,137 1 32765 
2 cells AB,CD...|105 | ca, 44| 18 3,055 41,240 | 1:13.5 
4 cells A,B,C,D.| 78 | ca. 40] 16 2,145 31,185 | 1:14.5 
Pee UDean 1) | cies 2s 15 | 1,767 22,484 | 1:12:7 
ee ere Nie deeaee aSO 30 he 905 13,185 | 1:14.5 
2 cells (2A 2D:-+-| 72 | ca. 36) 15 1,767 29.484 | soa 
5 oe On oder tea 36 12 905 23,346 | 1:25.6 
, _filatd'...| 30 30 | 9 382 13,658 | 1:35.7 
16 cells ee ee = Ss rs 
la2-1d?...) 15 15 6 115 1,654 1:14.6 
20 cells (24 3D----| 72 | ca. 18 14 1,437 1,618 | 1:11 
pare tears Wei necyshe gs! G8) 30 12 905 13,135 | 1:14.5 
: SERGE Al) Di) 27 12 905 9,330 | 1:10.3 
DAT cellar aes a a x : 
2a-2d?...| 27 27 12 905 9)330) | LT =aOkS 
25 cells | fe De casrterons 60 
Oo sae pet ae 34 9 382 
DOL ealieh eee te 15 6 113 1,654 |1:14.6 
aes att diez |e 24 12 905 6,333 | 1:7 
NSC) 2 15 1,767 
aie J 
SECs 9 382 


Incidentally the interesting fact appears that nuclei in large 
cells do not become spherical until they have reached a larger 
size than their sister nuclei in small cells; for example in the micro- 
meres Ja—1d and 2a—2d the nuclei become spherical when they 
are about 12 in diameter, in the macromeres 1A—1/.D and 2A-2D 
their sister nuclei are about 15y in diameter before they become 
spherical. Since the same amount of chromatin goes into each 
of the sister nuclei, the difference in the size of the nuclei when they 
first become spherical must be found in some other factor. It 
seems probable that it is due to the shape of the chromosomal 
disk, which remains flattened, or slightly arched, in large cells 


CELL SIZE AND NUCLEAR SIZE 23 


and is highly arched in small ones, owing to the greater or smaller 
size of the centrosomes, as explained on p. 19. The flattened 
chromosomal plate gives rise to a disk-shaped nucleus, which 
only later becomes spherical, whereas the highly arched plate 
sooner gives rise to a spherical nucleus. 

2. Cell-size and nuclear size in the cleavage of Fulgur carica. 
The eggs of Fulgur carica are the largest gasteropod eggs of which 
I know, while the eggs of Crepidula plana are among the smallest. 
It will be instructive therefore to compare the Kernplasma- 
Relation in these two cases. Table 6 gives the mean nuclear 
size and cell size of the blastomeres of Fulgur. The eggs measured 
were fixed, stained and mounted entire, as in the case of Crepi- 
dula plana. Owing to the great size of these eggs it was necessary 


TABLE 6 


Mean nuclear size and cell size in the blastomeres of Fulgur carica 


be mF fie 2 
: aa) eb Sg 35 
Be | Ge | ge BS 
2B ze a=) eS 2 
a6 | 25 | 3% 3% 3 
ue Me BM cubic cubic 
Macromeres 
AB, CD, before 

second cleavage. . 1200 200 40 33,280 4,126,720 | 1 : 124 
Beeb, LD; peters fined 

cleavage. . ; 800 160 32 17,040 2,112,880 | 1 : 124 
1A-1D, Barore Reunth clear 

Nail, 35 acne ee eee 800 | 160 | 32 17,040 | 2,112,880 | 1 :124 
2A-2D, before fifth cleav- 

Woh 4 Sea Aiea 800 160 3o2 17,040 2,112,880 | 1 :124 
3D, before sixth cleavage.) 800 160 32 17,040 2,112,880 | 1 : 124 
3A-3C, before sixth cleav- 

age.. 800 160 48 57,508 2,062,412 | 1:35.8 

4\-4D, “before” seventh 

cleavage................| 768 | 160 96 460,063 1,669,857 | 1 :3.6 

Micromeres 
la-ld. Seco | 80 16 2,145 264,095 | 1 : 127.7 
HE css wont nase ras ae | 80 | 16 | 2,145 264,095 | 1 : 127.7 
31310) ae Po Soen | 8O 16 2,145 264,095 | 1 : 127.7 
Unni ho Ry eae ee Rees Ss 8O 16 2,145 264,095 | 1 : 127.7 
1: 124 


RNC p yes a) 2.0.0 spel ma S ; 40 8 266 33,014 


24 EDWIN G. CONKLIN 

to make the measurements under a relatively low power, the 
8 mm. apochromat objective and the 1/1 micrometer eyepiece 
of Zeiss. Owing to the relatively low magnification the probable 
error is greater than in the measurements of the eggs of C. plana. 

With the exception of the cells $A—3C and 4A—-4D the Kern- 
plasma-Relation is in this case practically constant, varying only 
from 1: 124 to 1:127. In view of the fact that I can find no 
constant Kernplasma-Relation in the blastomeres of Crepidula 
this result in the case of Fulgur is unexpected. I am sure that 
my measurements in the case of Fulgur are not so accurate as in 
Crepidula, the number of eggs measured being relatively small 
and the magnification used low, so that each interval of the scale 
stood for 8%, The uniformity in the measurements of the differ- 
ent blastomeres and nuclei of Fulgur may be due in part to this 
fact; on the other hand this would only account for the lack of 
minor variations and would not explain the general uniformity. 
There is no doubt that the micromeres of Fulgur are more uni- 
form in size, than those of Crepidula; also the whole cleavage 
process is very much slower, and (with the exception of the macro- 
meres 3A-3C and 4A—4D) the divisions are more nearly synchro- 
nous in the different cells than in Crepidula. It seems probable 
that these two facts are connected with the more uniform Kern- 
plasma-Relation of the different blastomeres of Fulgur. 

This conclusion is rendered still more probable by a consider- 
ation of the two generations of cells in which there is a wide de- 
parture from the usual Kernplasma-Relation, viz. 3A—3C and 
4A-4D. In these cases, as in the same cells in Crepidula the 
resting stage is particularly long, lasting in the case of 44-4D 
until all the organs of the embryo are outlined and more than one 
thousand cells are present; consequently the nuclei grow to an 
enormous size so that the Kernplasma-Relation falls in one case 
to 1 : 35.8 and in the other to 1 : 3.6. In the corresponding cells 
in Crepidula the ratio is 1: 1.6 and 1 : 0.37; the volume of the 
nucleus in the last named case being about three times that of the 
plasma. The Kernplasma-Relation of the cells 4a-4e is 1 : 0.58 
in Crepidula, the nuclei being about twice as voluminous as the 
plasma; in the corresponding cells of Fulgur this ratio cannot be 


CELL SIZE AND NUCLEAR SIZE 25 


readily determined since the nuclei undergo several divisions, 
though the cell body does not divide. 

From these measurements it may be concluded that when cell 
division takes place at regular intervals the Kernplasma-Relation 
is fairly constant; when it takes place at irregular intervals this 
ratio is variable. The longer the resting period the larger the 
nucleus becomes, and in extremely long resting periods the greater 
part of the plasma may be taken up into the nucleus. 

These observations are in full agreement with experiments on 
the eggs of Crepidula which will be described later. They are 
not antagonistic to Boveri’s conclusions as to the correlation be- 
tween chromosome number and nuclear size; on the other hand 
my own experiments show that the size of the nucleus is depend- 
ent, in part, upon the number of chromosomes which enter into 
its formation. But in normal cells all of which contain the same 
number of chromosomes differences in nuclear size must be due 
to some other factor. 

The results of my measurements do not indicate that the Kern- 
plasma-Relation of Hertwig is either a constant or self regulating 
ratio in the blastomeres of these eggs; on the other hand it appears 
to be a result rather than a cause of the rate of cell division, and 
consequently it is a variable rather than a constant factor. 
Furthermore the size of the nucleus, in these eggs, is dependent 
upon at least three factors: (1) The initial quantity of chromatin 
(number of chromosomes) which enter into the formation of the 
nucleus (Boveri). (2) The volume of the protoplasm in which 
the nucleus lies. (3) The length of the resting period. 


III. Cell size and nuclear size in adult tissue cells 


It is generally believed that embryonic cells differ greatly from 
adult tissue cells in their ‘““Kernplasma-Relation.’’ In a series of 
thoughtful and suggestive works Minot (’90, 795, ’08) has main- 
tained that differentiation, senescence and finally death are the 
accompaniments, if not the results, of an increase of protoplasm 
as compared with nucleus. It is well known that embryonic 
cells of plants are more purely protoplasmic than adult cells, 


26 EDWIN G. CONKLIN 


which are frequently filled with vacuoles and sap so that the size 
of the cell gives no true idea of the volume of the cytoplasm. 
Among animals adult tissue cells often become filled with the 
products of differentiation or metabolism, such as fibers, granules, 
secretions, oil, ete., which greatly increase the cell dimensions. 
It is evidently a difficult if not impossible task to determine the 
quantity of real protoplasm in such cells and thus to discover the 
true “‘Kernplasma-Relation.’”’ However in certain less highly 
differentiated cells, especially in epithelial and glandular tissue, 
the true Kernplasma-Relation may be established with a fair 
degree of accuracy. 

Unquestionably the physiological state of a cell has much to 
do with its nuclear-plasmic ratio. Hodge (’92) found the nuclei 
of nerve cells shrunken after extreme stimulation, and it has been 
long known that the same is true of gland cells. In Crepidula 
the liver cells, when active, are filled with secretion and are among 
the largest in the body, but when the secretion has been discharged 
and they have returned to an inactive condition, the cell body is 
much smaller and the nucleus larger. 

I have measured the cells and nuclei of a number of tissues of 
Crepidula plana, derived from the three germ layers, and the 
results are given in table 7. Since these cells vary in shape to a 
great extent, and in order to facilitate comparison of cell diam- 
eter and nuclear diameter, cells were chosen for measurement 
which were as nearly as possible spherical or cubical in shape. 
In all elongated cells the long axis and one cross axis were measured 
and it was assumed that the other cross axis was of the same di- 
mensions as the one observed. 

It is evident that in these tissue cells of Crepidula plana there 
is no marked increase of protoplasm over nucleus as compared 
with the blastomeres of the same species; throughout the cleavage, 
with the exception of the cells 3A-3D and 4A—4D, the average 
Kernplasma-Relation for nuclei and cells of mean size is about 
1 : 15, for nuclei and cells of maximum size about 1 : 6; the aver- 
age ratio in adult tissue cells, which are not filled with metabolic 
products, is about 1 :10.5. In the case of the ganglion cells the 
nuclei are relatively and absolutely larger than in the other tissues, 


CELL SIZE AND NUCLEAR SIZE 27 


TABLE 7 


Cell size and nuclear size in tissue cells of sexually mature individuals of Crepidula 


plana 
DIMENSIONS OF DIAMETER VOLUME OF ee EE 
DURA DEC ELS CELL OF NUCLEUS NUCLEUS ae Begeae 
| Kh “ cubic cubic 
Intestinal epithelium....) 11 x 11 x 12 6 113 1,339 |1:11.8 
Gastric epithelium..... ..| 10x10 x 36 8 68 3.3392) |e =oi4 
Liver duct epithelium.... | 10x10 x18 6 113 1,628 | 1:14.4 
Liver cells (filled with 
secretion products)..... 15x 15 x 45 6* 113 10,012 1 : 88.6 
Liver cells (without secre- 
tion products)....... 14 x 14 x 30 9 382 5,498 |1:14.4 
Kidney cells Redutainiic 
secretion products).... 15 x15x 15 6 113 3,262 | 1:28.8 
Ectodermal epithelium 
(near anus).. ; fa bw 4 33 342 12083 
Gill chamber peihelani. 6x 6x12 4 33 405 Saye 
Gill filament epithelium (le Mh 3s Kt) 4 33 408 | 1:12.3 
Epithelium from foot 6x 6x15 5 65.4 474.6) 1:7.1 
Ganglion cell (large)... 17 Spee 12 905 Seed. OU Grd 
Ganglion cell (large)....... 10 x 10 x 20 9 382 1.618) le 2 
Oéeytes I (before yolk 
formation)......... 123 7 180 836 | 1:4.6 
Oécytes I (before volk for 
MAGTLON)kc)ese a's « 114 7 180 791 1:3.4 
Oécytes I (before Role fon 
MeBtION)......:-. 10 6 113 407 1:3.6 
Oécytes I (before y a for 
PEINOID) Som cle cea aac os 8 5 65.4 203 oS i | 
Oécytes I (before yolk for- 
EEVENETOIN) Rites ciaee chic 5 5 64 4 33 111 1 33.3 


“Nucleus shrunken and very irregular in sh ape. 


the Kernplasma-Relation being about 1:5; however in this 
case the nerve fiber is not added to the cell body and this would 
doubtless greatly increase the volume of the plasma. Muscle 
cells in Crepidula are long, slender and crooked and I have found 
it impracticable to estimate their volumes with any degree of 
accuracy. Doubtless the plasma, including the contractile sub- 
stance, is here relatively much more abundant than in embryonic 
or epithelial cells. In the epithelial and gland cells of adult 


28 EDWIN G. CONKLIN 


Crepidula the embryonic ratio of nucleus to plasma is main- 
tained with little change. In all the o6cytes up to the time that 
yolk formation begins the nuclei are relatively large, the ratio 
of nucleus to plasma being about 1 : 3.6, and in the younger and 
smaller odcytes the nuclei are relatively larger than in the older 
and larger ones. 

Eycleshymer (’04) found that the volume of the plasma in the 
striated muscle cells of Necturus increased about ten times as 
much as the nuclear volume, during development from the 8 mm. 
embryo to the adult condition. There is, therefore, in these later 
stages a notable shifting of the Kernplasma-Relation in favor of 
the plasma. It is probable however that the contractile substance 
which makes up the larger part of the muscle cell, does not con- 
tribute to the growth of the nucleus as does the protoplasm of 
embryonic cells—that so far as the growth of the nucleus is con- 
cerned it acts as does yolk, oil, membranes, fibers and other 
products of metabolism and differentiation. If only the sarco- 
plasm of the muscle cell and not its contractile substance is able 
to contribute to the growth of the nucleus, the small volume of the 
nuclei as compared with the entire cell would find a ready explan- 
ation. There can be no doubt that the plasma is the chief seat 
of differentiation, as Minot has emphasized, and that highly 
differentiated cells, such as muscle, nerve, and some kinds of 
connective tissue, have a larger amount of plasma and its products, 
relative to the nucleus, than have embryonic cells. In the case 
of fiber cells, fat cells, and probably muscle cells, the cell body 
becomes filled with the products of differentiation and metabo- 
lism, which like the yolk in egg cells, or the secretion products in 
liver cells cannot enter the nucleus and consequently do not influ- 
ence its size. In such tissue cells the cell body is relatively much 
greater as compared with the nucleus, than in purely protoplas- 
mic cells, but I have been unable to find any evidence that the 
ratio of protoplasm (using this term in its usual sense) to the 
nucleus is greater in tissue cells of Crepidula than in the blas- 
tomeres. 


CELL SIZE AND NUCLEAR SIZE 29 


IV. The inciting causes of cell division 


The relative sizes of cells and of nuclei are dependent, in part, 
upon the rate of cell division. Cells which divide infrequently 
are larger, other things being equal, than those which divide often. 
The turret cells (1a*-/d?) of Crepidula are the smallest cells in 
the entire embryo at the time of their formation (figs. 3, 4); 
however they divide but twice during the whole of the cleavage 
period, and consequently they grow to be very large; whereas each 
of the apical cells from which they were derived, gives rise during 
the cleavage period to twelve cells the combined volume of which 
is not much greater than that of one full-grown turret cell. 
Evidently the factors which bring on or delay cell-division have 
much to do, indirectly, with the sizes of cells and nuclei. 

Strasburger (’93) supposed that cell division occurred when the 
ratio of the cell body to the nucleus increased beyond a certain 
point, which might be regarded as marking the limit of the ‘work- 
ing sphere of the nucleus;’ with the division of the cell the normal 
ratio was once more restored. 

Boveri (’04) sought to find the inciting cause of cell division in 
the chromosomes. He believed that the chromosomes divide 
when they have reached a size double that which they had at the 
close of the preceding division. At the same time he showed 
that the rhythm of the division of the centrosomes may be inde- 
pendent of that of the chromosomes and that division of the cell 
depends upon the centrosomal rhythm rather than upon the chro- 
mosomal rhythm. 

That there is a rhythm of division for chromosomes and centro- 
somes seems to be well established by Boveri’s work, but this 
rhythm in the case of the chromosomes is not determined by the 
time when they have grown to double their size at the close of the 
preceding division. Marcus (’06) and Erdmann (08) have shown 
that the chromosome size throughout the cleavage of Strongy- 
locentrotus is a constantly decreasing one. Baltzer (’08) admits 
that the chromosomes do not double in size at each cycle of divi- 
sion; he does not find any great diminution in chromosome size 
up to the 16-cell stage, though the chromosomes in the blastula 


30 EDWIN G. CONKLIN 


stage are undoubtedly smaller than those of early cleavage stages. 
In Crepidula the chromosomal plate decreases in size in successive 
cleavages, though by no means uniformly; but at no time during 
the cleavage period do the chromosomes grow to their original 
size at the beginning of the cleavage. Boveri’s view, therefore, 
finds no support in the cell-divisions of the cleavage period. 

R. Hertwig (’03, ’08) finds the inciting cause of division in a 
‘Kernplasma-Spannung,’ due to the unequal growth of nucleus 
and plasma: 

Die Kernplasma-Relation muss eine Verschiebung erfahren zuungun- 
sten des Kernes, es muss sich eine Kernplasma-Spannung entwickeln, 
welche allmihlich zunimmt, bis schliesschlich ein Grad erreicht wird, 
den ich friiher Kernplasma-Spannung in engeren Sinne genannt habe. 
In dieser Spannung erblicke ich die Ursache der Teilung. Ich nehme 
an, dass, wenn ein Héhepunkt der Kernplasma-Spannung erreicht wird, 
der Kern die Fahigkeit gewinnt, auf Kosten des Protoplasma zu wachsen, 
und das die hierbei sich vollziehenden Stoffumlagerungen zur Teilung 
der Zelle fiihren. Zum funktionellen Wachstum gesellt sich das Teilungs- 
wachstum des Kernes, um die Kernplasma-Norm wiederherzustellen.” 
(p. 20) 

Relative Zunahme der Kernsubstanz, gleichgiiltig, ob dieselbe durch 
Vergrésserung des Kerns bei gleichbleibender Protoplasmamenge oder 
Verringerung des Protoplasma bei gleichbleibender Kerngrésse her- 
beigefiihrt wird, miisste eine Verlangsamung der Teilung und im ersten 
Fall eine Steigerung der Teilgrésse zur Folge haben; umgekehrt miisste 
relative Abnahme der Kernmasse den Eintritt der Kernteilung be- 
schleunigen, die Teilgrésse herabsetzen (p. 23). 


Hertwig holds that his own work on Infusoria, and that of 
Gerassimoff on Spirogyra, show that an increase of nuclear mass 
leads to a slowing of divisions and an increase of the division size 
of the cell; and that the process of the segmentation of the animal 
egg shows that a great reduction of nuclear mass leads to a high 
degree of divisional activity. He says that many external and 
internal conditions influence the Kernplasma-Relation and he 
expresses the hope that his theory may not be cast aside because 
here and there a fact may be found which cannot be brought under 
it, without further consideration. 

As we have seen the Kernplasma-Relation varies widely in 
certain blastomeres of Crepidula and Fulgur. In these cases 
wide departures from the Kernplasma-Norm have not brought 


CELL SIZE AND NUCLEAR SIZE 31 


on cell division, and if Kernplasma-Spannung is a cause of cell- 
division it must be a minor factor in this case. It seems to me 
probable from my observations and experiments on segmenting 
eggs, that the Kernplasma-Relation in these blastomeres is a re- 
sult rather than a cause of the rhythm of cell division, and that 
the factors which bring on cell division are to be found in some 
intrinsic condition in the nucleus or centrosome, rather than in the 
maintenance of a constant ratio of nuclear volume to cell volume. 
Support is lent to this view by the phenomena of odgenesis, for 
we have in the germinal vesicle the largest nucleus in the entire 
life cycle, following upon the longest resting period, while the 
second maturation division follows immediately upon the first, 
usually before a resting nucleus is formed. The long delay in 
the appearance of the first maturation division, as well as the 
short period intervening between the first and second maturation 
divisions, must both be attributed, as it seems to me, to intrinsic 
conditions in the cell, other than ‘Kernplasma-Spannung.’ 

In the cleavage of the egg the rate of division seems to depend, 
in part, on the quantity of protoplasm present. As long as a con- 
siderable quantity of plasma is present in the blastomeres the rate 
of division is rhythmical, but when the macromeres have given off 
almost all the plasma inthe formation of the three quartets of ecto- 
meres, a long resting period follows. The first of these macro- 
meres to divide, giving rise to the fourth quartet, is the one with 
the largest amount of plasma, viz., 3D, while the cells 3A—3C 
normally divide much later. However if, by centrifuging at the 
right stage, 3C is caused to contain more plasma than usual it 
may divide at the same time as 3D, as shown in fig. 37. The cells 
4A-4D, in which the resting period is particularly long, contain 
very little plasma, and this appears to be absorbed by the nucleus 
almost as fast as it is formed. The micromere /d is slightly 
smaller than its fellows, /a-/c, and it divides later than the latter. 
The ‘turret’ cells, /a?-1d?, are the smallest cells in the egg, when 
they are formed, and they have the longest resting period. 

In spite of this evidence that the quantity of protoplasm has 
to do with the rate of division, there is other conflicting evidence 
which is hard to harmonise with it; thus, these same ‘turret’ cells, 


32 EDWIN G. CONKLIN 


which are at first so small and have so long a resting period, be- 
come much larger than adjoining cells before they divide. R. 
Lillie (10) maintains that ‘‘the primary change in the initiation 
of cell division and development is an increase in the permeability 
of the plasma membrane.”’ It is well known that the general 
surface tension of the cell increases during mitosis, and I have 
found that the tension of the cell membrane is locally reduced at 
the two poles of the cell before and during division (see Conklin, 
02, p. 94; also this paper, p. 82). It is quite possible that. this 
polar reduction in surface tension before mitosis begins may have 
something to do with initiating division. 

On the whole it seems probable that the time of cell division is 
dependent upon the coincidence of several more or less independ- 
ent factors. Boveri has shown that the division phases in nu- 
cleus and centrosome may be more or less independent of each 
other, though complete cell division depends upon the coincidence 
of the two. To these factors may, perhaps, be added the quantity 
of protoplasm, and thus indirectly the ‘Kernplasma-Relation’ 
and perhaps also increased permeability of the cell membrane, 
and a local reduction of surface tension at the poles of the cell. 
Gurwitsch (08) maintains that the blastomeres are ready for 
division at all times, and that only “Kernplasma-Koinzidenz’ 
or ‘Zustands-Koinzidenz,’ is necessary to start division. He sug- 
gests that a coincidence of polarity of nucleus and plasma may be 
necessary, and he concludes from the apparently accidental 
occurrence of divisions in different parts of an egg or embryo, that 
several independently variable factors may be concerned, the 
coincidence of which is necessary to bring on cell division. The 
latter part of this conclusion seems to me to be justified by the 
facts which I have presented. 


V. Growth of protoplasm during cleavage 


It is well known that the egg as a whole does not increase in vol- 
ume until after the cleavage period. Indeed Godlewski (’08) finds 
that there is in Echinus and in Strongylocentrotus, no change in 
the quantity of plasma at the 64-cell stage, as compared with the 


2 
CELL SIZE AND NUCLEAR SIZE 33 


unsegmented egg; however, in the blastula there is an actual loss, 
the total volume of plasma being about one-third less than in the 
unsegmented egg; during this period the nuclear material has 
increased in volume at the expense of the plasma. Whether the 
plasma actually increases during cleavage at the expense of the 
yolk has not been determined, so far as I am aware, in any case. 
By means of the centrifuge it is possible to throw the yolk out 
of the egg before cleavage and during the early cleavage stages, 
leaving the plasma which can then be readily measured. In 
later cleavage stages I have not been able to throw the yolk out 
of the small blastomeres by means of the centrifuge; but on the 
other hand the protoplasm and yolk are normally segregated in 
these stages so that it is possible to determine the approximate 
dimensions of both without having recourse to the centrifuge. 
The following table gives the total maximum volumes of all the 
nuclei, protoplasm and yolk in the eggs of Crepidula plana at 
various cleavage stages. Following Popoff (’08), I have deter- 
mined the coefficients of growth of the nucleus and of the pro- 
toplasm for each stage; these coefficients are obtained by dividing 
the volume of a later stage by that of an earlier one, and they 
represent the growth in ‘times,’ or multiples of the initial quantity. 
In the first half of each column of coefficients the earlier stage is 
the one before maturation, while in the second half of each column 
it is the one before the first cleavage. The coefficient of growth of 
TABLE 8 


Total maximum volumes of nuclei, protoplasm and yolk in the eggs and cleavage 
stages of Crepidula plana 


| | COEFFICI- | COHFFICIENTS| TOTAL 
Ife TOTAL 
| YOLUME OF 


STags NUCLEI Santana Ne zy eg os meee Sees aes ieee 
| is GROWTH GROWTH RELATION 
cubic uh | cubic mM | cubic u cubic 
Before ma- 
turation. . 32,409 | 97,131 1,625,460 1,755,000 1.0 1.0 1:3 
Before first | 
cleavage. 21,375 121,480 | 1,346,105 | 1,488,910 0.65 1.0 1.25 | 1.0 15.6 
2 cells...... 14,476 123,482 1,100,700 | 1,238,658 0.45 | 0.67 1.27 1.02 1:8.5 
4cells...... 23,100 154,280 969,468 | 1,146,848 0.71 | 1.08 1.58 | 1.27 1:6.6 
mmells:.. .... 25,144 164,136 972,320 | 1,161,600 0.77 | 1.17 1.68 1.35 1:6.5 
16 cells..... 23,628 233,608 | 980,156 | 1,237,392 0.72) 1.10) 2.45 1.92 1:9.8 
24 cells... .. 30,164 258,897 } 890,727 1,179,788 0.92 | 1.41 2.66 2.13 1:8.6 
(231,000) | (1,151,891) (2.35) \(1.90)! (1: 7.7) 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. | 


34 EDWIN G. CONKLIN 


any stage, less the coefficient of the initial stage, viz. unity, gives 
the percentage of growth of that stage, as compared with the ini- 
tial stage. 

Since the ectoderm at the 24-cell stage is a plate of purely 
protoplasmic cells, nearly square, about 80 on each side and 36u 
thick its volume is about 230,400 cubic y; subtracting the volumes 
of the nuclei of the plate, 21,584 cubic yp, leaves 208,816 cubie p 
as the volume of the cytoplasm! of the ectodermal plate. Adding 
to this the volume of the protoplasm in the macromeres 3A—3D, 
viz. 22,185 cubic yu, we have as the total volume of the proto- 
plasm at the 24-cell stage 231,000 cubic ». This figure is 27,897 
cubic uv less than the volume of protoplasm at the 24-cell stage 
given in the table, which was calculated from the dimensions of 
each individual cell, rather than from those of the entire ecto- 
dermal plate. It is highly probable that the lower figure is nearer 
correct than the higher one, since minor errors in the measure- 
ments of individual cells are greatly magnified in determining 
the total volumes of these cells. The same remark applies to the 
total volume of protoplasm in the 16-cell stage, which is probably 
actually less than the volume given in the table; and if the total 
volume of the protoplasm is less than the amount given in the 
table the total volume of the yolk in these stages is of course 
increased correspondingly. 

But assuming that the smaller number (in brackets) represents 
the actual volume of the protoplasm in the 24-cell stage of Crepi- 
dula plana we must admit that there has been a great growth in 
the plasma at the expense of the yolk during the cleavage. The 
coefficient of protoplasmic growth (i.e., the volume of protoplasm 
of any stage divided by the volume of protoplasm of the stage 
just before maturation) is given in the next to the last column of 
the table; and a glance at this shows that the protoplasm at the 
24-cell stage is at least 24 times as voluminous as in the maturation 
stage, while the yolk is correspondingly less voluminous. The 
volume of the entire egg, also, is considerably less in the 24-cell 
stage than at the beginning of development. Indeed there has 
been a gradual decrease in the volume of the entire egg during 


'The words ‘cytoplasm’ and ‘protoplasm’ are used synonomously throughout 
this paper. 


CELL SIZE AND NUCLEAR SIZE 35 


the early cleavages. These results show a general agreement 
with those of Godlewski. 

The growth of plasma at the expense of yolk during the matur- 
ation and the cleavage period, was shown to occur in my studies 
of the effects of centrifugal force on the eggs of Lymnaea and 
Physa (Conklin, 710). In the living eggs of these animals the 
substances may be stratified by centrifugal force into a gray 
(light) zone, a clear (middle) zone and a yellow (heavy) zone; the 
gray and clear zones constitute what I have here regarded as proto- 
plasm, while the yellow zone is in large part composed of yolk. 
“Before the first maturation the yellow substance composes at 
least one-half of the entire egg; just before the first cleavage it 
composes only about one-eighth of the egg. The clear and gray 
substances, which together constitute about one-half of the egg 
in the earlier period, form seven-eighths of the egg in the later 
period,” (p. 436). 

In the normal eggs of Lymnaea and Physa, which have not been 
centrifuged, the clear and yellow substances are easily recog- 
nizable, and the stages in the transformation of the latter into 
the former have been studied in the paper mentioned, from which 
the following summary is quoted: 


In the course of development, from the maturation of the egg to the 
gastrulation, the relative quantities of clear (plasma) and yellow sub- 
stance (yolk) are reversed. At the beginning the clear substance is 
small in quantity, and is chiefly visible in the germinal vesicle (though 
experiments show that some of it is distributed through the yellow sub- 
stance) and at this stage the entire cell body is yellow in color. With 
the establishment of the germinal layers the yellow substance is limited 
to the few cells constituting the endoderm and mesoderm, while all the 
rest of the embryo, by far the larger part, is composed of clear substance. 
This change in the relative quantities of these two substances is due in 
part to their separation and segregation during the course of develop- 
ment, but in much greater part to the transformation of yellow sub- 
stance (yolk) into the clear (plasma). It is a phenomenon of general 
occurrence among many animals that the clear protoplasm of the egg 
is very small in quantity before the dissolution of the germinal vesicle 
and that it gradually increases in quantity after that stage. This is 
doubtless due in large part to the dissolving of yolk and its conversion 
into clear protoplasm, and it is a significant fact that this process takes 
place most rapidly after the breaking down of the wall of the germinal 
vesicle and the escape of a large part of the nuclear contents into the 
cell body (p. 423). 


36 EDWIN G. CONKLIN 


There are no eggs wholly without yolk and probably in all of 
them plasma is formed at the expense of yolk during the cleavage 
period. This probability is of great significance, for all studies 
which have had to do with the relative quantities of protoplasmic 
and nuclear materials during these early stages of development 
have dealt only with the entire cell contents without attempting 
to determine what part of this is plasma. In many cases, the 
great disproportion between cell volume and nuclear volume at 
the beginning of developemnt is due to the fact that a large part 
of the cell volume is made up of yolk; if the volume of the plasma 
only is compared with that of the nucleus it is found that the 
relative quantity of plasma is actually less at the beginning of 
development, than in the later cleavages, with the single excep- 
tion of those blastomeres which have unusually long resting 
periods. In Crepidula there is no excess of plasma over nuclear 
material in the early stages, in comparison with the later ones, 
as Minot and others have assumed, and the process of cleavage 
is not in this case a method of restoring the Kernplasma-Norm, 
or of rejuvenating senile cells, by an enormous increase of nuclear 
material as compared with the plasma. As a matter of fact 
the plasma increases almost as rapidly as the nuclear material 
during the cleavage of this egg, and even adult tissue cells have 
a Kernplasma-Relation but little different from that of the 
blastomeres, (see p. 25). 


VI. Rate of nuclear growth during cleavage 


It is well known that during cleavage there is usually no in- 
crease in the volume of the egg, but it is generally held that the 
increase in the nuclear substance is very great. In his book 
on “‘Age, Growth and Death” Minot (’08) says: ‘‘The nuclei 
multiply (in cleavage); they multiply at the expense of the pro- 
toplasm. ‘They take food from the material which is stored up 
in the ovum, nourish themselves by it, grow and multiply until 
they become the dominant part in the structure’ (p. 166). He 
suggests that this nuclear increase during cleavage is a process of 
rejuvenation, though he admits that the relative increase of nu- 


CELL SIZE AND NUCLEAR SIZE . 37 


clear material as compared with protoplasmic may be prolonged 
beyond the period of segmentation (p. 167). But although he 
emphasizes the growth of the nuclear material as a whole during 
the cleavage, he specifically recognizes the fact that there is a 
rapid reduction in the size of individual nuclei in the early stages 
(pp. 174, 179). Hertwig (’03) also has emphasized this great 
growth of the nuclear material during the early stages of develop- 
ment. He says (p. 116): 


There is an enormous disproportion of nucleus and protoplasm at the 
beginning of cleavage, and this disproportion is gradually equalized by 
the transformation of cell substance into nuclear substance. The man- 
ner of this may be imagined by supposing that resting protoplasm con- 
tains chromatin and achromatic material and that at every cell division 
it is analysed into these constituents serving for the growth of the 
nucleus. 


Loeb (09) also has called attention to the doubling of nuclei 
at each division, with the consequent increase of nuclear material 
in a geometric ratio, and the resemblance which this bears to 
autokatalytic reactions. 

The great increase in the nuclear substances during cleavage 
has been commented upon by many writers, and the references 
cited have been chosen rather because of the theories which have 
been based upon this phenomenon than because they represent 
an unusual opinion as to the phenomenon itself. At the time 
when the following computations of the rate of nuclear growth 
during cleavage were made, I was unaware that anyone had made 
computations of a similar sort. Since my material afforded an 
unusually good opportunity for making such computations, I 
carefully measured the diameters of the germinal vesicle, of the 
ege and sperm nuclei, of all the nuclei up to the 24-cell stage, of 
those of the 42-cell stage, and of the 70-cell stage,—every nucleus 
being measured at its maximum size, so far as possible,—with the 
results given in the following tables. These results have been 
as surprising to me as they are likely to be to any of my readers. 

After this work was completed I became acquainted with the 
_work of Godlewski (08) and Frl. Erdmann (’08) on the sizes of 
nuclei and of individual chromosomes of the blastomeres of 


38 EDWIN G. CONKLIN 


Echinus and Strongylocentrotus. Godlewsky found that from 
the 1-cell to the 64-cell stage the nuclear substance grows nearly 
in geometric ratio; from the 64-cell stage to the blastula, with 
about 1256 cells, there is little increase in the nuclear substance, 
but since he supposes that the number and size of the chromo- 
somes in the later stages remain the same as in the earlier ones, 
the nuclei must become richer in chromatin in the later stages. 
He finds that the volume of the plasma in the blastula stage is 
about one-third less than in the unsegmented egg and he con- 
siders that a large part of this lost plasma has been converted into 
chromatin. Erdmann (’08) has made a careful computation of 
the volume of the resting nuclei and of individual chromosomes 
in the early cleavage stages, and in the blastula and gastrula of 
Strongylocentrotus. She finds that the chromosomes of the 
pluteus period have only about one-fortieth the volume of those 
of the first spindle, but though the individual chromosomes grow 
smaller continually, the total nuclear volume increases at the 
expense of the plasma up to the late blastula stage. 

1. Nuclear growth during the cleavage of the egg of Crepidula. 
The maximum, minimum and mean volumes of the nuclei at 
different stages of the cleavage of Crepidula plana are given in 
tables 3 to 5 and the coefficients of growth of all the nuclei are 
given in table 8. It remains only to summarize the facts there 
presented and to give the nuclear volumes and the rate of growth 
in certain later stages of the cleavage. This has been done in 
table 9, where the maximum, miminum and mean nuclear vol- 
umes of every nucleus from the 2-cell to the 32-cell stage is given, 
together with the coefficient of growth for each stage. Since this 
table starts with the 2-cell stage the coefficients of growth are 
different from those given in table 8, where subsequent stages are 
compared with the germinal vesicle or with the germ nuclei. 
For the purpose of determining the usual rate of growth for each 
cycle of cell division during the cleavage it is desirable to start 
with the 2-cell stage. The germinal vesicle is an extraordinarily 
large nucleus, and since two nuclei are present in the egg before 
the first cleavage the nuclear condition at this stage is unusual; 
on this account the rate of nuclear growth during cleavage is 


CELL SIZE AND NUCLEAR SIZE 39 
TABLE 9 
Rate of nuclear growth during the cleavage of Crepidula plana 
== 
MAXIMUM (COEFFICIENT MEAN COEFFICIENT) MINIMUM (COEFFICIENT 
STAGE BLASTOMERES NUCLEAR OF NUCLEAR OF | NUCLEAR OF 
VOLUMES | GROWTH | VOLUMES | GROWTH | VOLUMES | GROWTH 
| | 
cubic cubic cubic bw 
SROGUA PAIS, CD iiss ccvenencae 14,476 1.0 6,110 1.0 381 1.0 
cells A) B,C, D........... 23,100 1.6 8,580 1.4 602.4 1.58 
8 cells DAUD) bere cc orcistac oiniac'e 19,396 7,068 338.4 
NA ec detsjarsio sivas pi0:a.< 5,748 3 620 338.4 
25,144 1.73 10.688 1.74 676.8 1.77 
BOD Wiese sisies cav's's 12,220 7,068 338.4 
7,068 3,620 338.4 
po eicalls 3,620 1,528 235.2 
720 452 ‘ 235.2 
23,628 1.63 12,668 | 2.07 1147.2 3.01 
8,580 5,748 150.0 
5,748 3,620 150.0 
24 cells 5,748 3,620 150.0 
5,748 3,620 150.0 
3,620 1,528 235.2 
720 452 235.2 
30,164 2.08 18,588 3.04 1070.4 2.80 
4A-4D 12,220* 7,068 171.3 
A ein races sieieie'n cae 697 382 58.8 
MBACP ataercle  'claic aes» 2,715 1,146 | 112.5 
BOO bedieoe Sane eee 5,748 3,620 150.0 
32 cells ; 2a!-2d!.............. 5,748 3,620 | 150.0 
5,748 3,620 | | 150.0 
3,620 452 150.0 
lat.2-1dl.2......, re 2,095 3,620 150.0 
Jat1d?i..isci0% Go 720 452 235.2 
39,311 2.71 23,980 3.92 | 1327.8 3.48 
Total growth in thirty divi- 
PNOUE sh rata'ainlek pialalsluteterersie ere ats 24,835 2.715 17,870 3.92 946.8 3.48 
Average growth for each diyi- 
WW Wanita canannposa acer Sinod | MELAS 1.05(=5%)| 595.6 1.09(=9%) 31.5 1.08(=8%) 


* This volume is reached only at a much later stage, shortly before the closure 
of the blastopore (fig. 6). 
During this same period from the 2-cell to the 32-cell stage the coefficient of 
growth of maximum nuclear surfaces is 4.28, or an average increase of about 11 
per cent for each division. 


40 EDWIN G. CONKLIN 


best determined by comparing subsequent stages with the 2-cell 
stage. Furthermore the nuclear volume in the 2-cell stage is 
less than at any other stage, and it consequently forms a good 
starting point for the study of nuclear growth. 

Finally, the volume of all the nuclei in the 70-cell stage, without 
attempting to determine the maximum volume of each nucleus, 
is shown in table 10. 

At the 70-cell stage the ectomeres are already closing over the 
yolk on the oral hemisphere, and it may be assumed that the 
cleavage will show no new tendencies as to the growth of nuclear 
substance until the embryo as a whole begins to grow. 

Whether nuclei are measured at either their maximum size, 
their minimum size or at a size intermediate between these two 
extremes, the rate of growth during cleavage is found to fall far 
short of a doubling or increase of 100 per cent at each division. 
The average nuclear growth during early cleavage is not more 
than 5 to 9 per cent for each division, and in the later cleavage it 
falls as low as 1 per cent for each division. A growth of nuclear 
substance at this rate scarcely deserves to be designated as ‘phe- 
nomenal’ or ‘colossal.’ On the other hand, the protoplasm which 
is generally supposed to remain fixed in quantity during cleavage, 
increases at a more rapid rate than the nuclei, from the 1-cell 
to the 24-cell stages, as shown in table 8. In view of the facts 
here presented, even though it be for only a single species, the 
generally accepted conclusion as to the great increase of nuclear 
substance during cleavage, as contrasted with the lack of growth 
of the protoplasm, evidently needs revision, as do also the theories 
which have been founded upon this supposed fact. 

2. Nuclear growth during the cleavage of the egg of Fulgur. 
While my results are based largely upon the study of Crepidula 
plana they are not limited entirely to this species. The fol- 
lowing measurements of the nuclei of Fulgur carica are prob- 
ably not very accurate since they had to be made under a rela- 
tively low power objective (8 mm. apochromat) and since the 
material at my command did not permit the study of a large num- 
ber of eggs, and the selection of nuclei at maximum size. Never- 


——o 


CELL SIZE AND NUCLEAR SIZE ‘ 41 


TABLE i0 


Actual nuclear diameters and volumes in the 70-cell stage of Crepidula plana 


| COEFFICIENT OF GROWTH 
| NUCLEAR TOTAL NUCLEAR 


JEL 2 DIAMETER VOLUME Ni aaleas Wicker, 
volume surfaces 
rm Be 
PHC CMISUHPC REE eee rcs fos canes beaacs| A 14,476 1 1 
(225290 oe See ae 16 8,579 
(leh eo are | 10 1,571 
11 Entomeres El, E2., aes 7 359 
Git CE ok 5 See Been 6 226 
UNI SY EE aes ee 0) 1,047 
eer enomeres ae 7 Sane eee 9 763 
First quartet 
4 Apicals, lat-“-Id'). 00, 10 2,094 
3 Basals, lat-?--1¢!-?1,... 2... 9 1,145 
1 Basal, 1G LC, 2 cons ees cae } > a2 905 
3 Middles, la!:?-2-1e!-22,. 02... | 12 2,714 
Aeiimrete; Vaz ld*, oo... ee ww el 7 718 
Second quartet 
a 3 Tip cells, 2al-2et!.. 2... | 6 339 
© |1 Tip cell, Bite IO onal A RA | 10 524 
§ (4 Girdle cells, 2a!-?-2d'-?1........| 9 1,527 
2 4 Girdle cells, 2a!-?->2d!-?-2,.......! 10 2,094 
f) |4 Girdle cells, 2a2-1--2d?-1-1.... 2... a 1,527 
19 | 4 Girdle cells, 2a?1-2-2d?1-2,.......| 9 1,527 
4 Girdle cells, 2a?-2-2d?"?,..........| 5 262 
Third quartet 
4 Girdle cells, 3a!--3d!"!........... 10 2,094 
4 Girdle cells, 8a!-2-3d!? .......... 9 1,527 
4 Girdle cells, 3a?-8d?............ 6 452 | 
4 Girdle cells, 3a?-*-3d2"?........... 6 452 
70 cells. Total nuclear volume........ 32,446 2.24 5.30 


The total volume of these 70 nuclei is almost exactly the same as the volume 
of the germinal vesicle, about 50 per cent more than the volume of the germ nu- 
clei, and 35 per cent more than the mean nuclear volume of the 32-cell stage, with 
which mean volume, rather than with the maximum, this actual volume of the 
nuclei of the 70-cell stage should be compared. In the 38 nuclear divisions lead- 
ing from the 32-cell stage to the 70-cell stage the nuclear material has increased 
at an average rate of less than 1 per cent for each division. 


42 EDWIN G. CONKLIN 


TABLE 11 


Diameters and volumes of the nuclei, 2-cell to 16-cell stages of Fulgur carica 


STAGE BLASTOMERES TNT CERTE Eman NMS ccleree eee tercsceteteetre eee 
E ML | Baan i 
SicellseA BC Die eenee eeeenee 40 67,020 | 1.0 
AcellshACiBe CAD ee ene 32 | 68,628 1.02 
| 
| 
Seige CASED Aen ee 32 68,628 
la-Id 16 8,576 
| 77,204 1.15 
2A-2D... < aE 32 | 68,628 
f2'celisiy 2a-2d) ne ee eee 16 8,576 
laid: cee ee 16 | 8,576 
| 85,780 1.28 
| 
| 3423) pe ee 32 | 68,628 
T& cali’) 98-04 o> een eee eee 16 | 8,576 
Sa-0d: «ee ee 16 | 8,576 
(etd. eee 16 | 8,576 
| 94,356 1.40 


theless they indicate the general rate of nuclear growth in this 
prosobranch, 

In fourteen nuclear divisions there has been an increase in 
neuclear substance of 40 per cent, or an average increase for each 
division of 2.8 per cent. The rate of nuclear growth is practically 
the same in the other species of Crepidula as in C. plana; and 
in all prosobranchs the nuclear material increases but slightly 
during the cleavage period. 

3. Nuclear growth during the cleavage of other animals. From 
a casual examination of the segmenting eggs of nematodes, echin- 
oderms, amphioxus and ascidians, as well as from a study of the 
figures of various authors, it is evident that the nuclear growth 
in these forms is greater during the early cleavages than in the 
gastropods. In all of these forms the germinal vesicle is relatively 
much larger and the egg and sperm nuclei much smaller than in 
the gastropods, while the decrease in nuclear size in the early 


CELL SIZE AND NUCLEAR SIZE 43 


cleavages is not so marked as in the gastropods, though of neces- 
sity the nuclei must grow smaller in all animals as cleavage 
progresses. 

In the ascidian, Styela (Cynthia) partita, the maximum 
nuclear diameters and volumes in the different cell generations 


are shown in table 12: 
TABLE 12 


Maximum nuclear diameters and volumes in Styela (Cynthia) partita 


1 T 
| | 

COEFFICIENTS OF GROWTH 
AVERAGE DIAMETER TOTAL VOLUME - ——— —————— 


EO} NUCLEUS OF NUCLEI — 
Nuclear volume pune 
“ cubic 

Before first ma- 

furation.......| 54 82,448 1.0 
Before first 

cleavage....... 912+ 712 1,809 0.02 | 1.0 } 
aTNES a Aces l6u 4,289 0.05 |) 2.37 1.0 1.0 
chiGIEURE @ paeeee 14 5,748 0.06 3.17 1.34 
PAGEL R ee sere idly oc iW 3 9,203 0.11 | 5.08 2.14 
TGicelleva. ss... Ml 11,1738 0.13 6.17 2.60 
BEICOUIB es cscs + 10 16,755 0.20 | 9.26 3.90 
64cells.......... 8 17,152 0.20 | 9.48 4.00 | 
WARIGEIIS: cine)eea0\<- 6.5 18,406 0.22 10.17 4.29 
POOCEUS. 0.200. 5.25 19,395 0.23 10.72 4.52 | 1G) 


The nuclei of different blastomeres of the same generation 
vary considerably in size, and I have not attempted to measure 
each individually, as in the case of Crepidula, nevertheless the 
measurements given represent approximately the average nuclear 
diameters for each generation of blastomeres. When the cells 
become very numerous a very slight error in the measurement 
makes a big difference in the results, and the total nuclear volume 
in the later stages may not be very accurate. Nevertheless the 
table does give a true idea of the order of magnitude of the nuclei 
in the different generations. 

In comparing this table with those for Crepidula it will be seen 
at once that the germinal vesicle is relatively larger, the germ 
nuclei smaller and the growth of the nuclear material in the 
early stages greater in Styela than in Crepidula. The volume of 
the egg and sperm nuclei represents a loss of 98 per cent as com- 


44 EDWIN G. CONKLIN 


pared with that of the germinal vesicle; and even in the 256-cell 
stage the volume of all the nuclei is 77 per cent less than that of 
the germinal vesicle. Comparing the nuclear volumes of subse- 
quent stages with that of the germ nuclei, we find that up to the 
32-cell stage there is an increase of 826 per cent, or an average 
for the first 31 nuclear divisions of 26 per cent for each division; 
from the 32-cell stage to the 256-cell stage there is an increase of 
146 per cent, or an average increase of 0.6 per cent for each divi- 
sion. Since the germinal vesicle is unusually large and the germ 
nuclei unusually small, a better idea of the rate of nuclear growth 
in the egg will be obtained by comparing the nuclear volumes of 
later stages with that of the two cell stage, as was done in the case 
of Crepidula. Such a comparison is given in the last column of 
Coefficients in table 13. From this it appears that the nuclear 
growth from the 2-cell stage to the 32-cell stage is 290 per cent 
or an average increase for each of 30 divisions of 9.6 per cent; 
from the 32-cell stage to the 256-cell stage the nuclear volume in- 
creases 62 per cent, or an average increase for 224 divisions of 
0.27 per cent for each division. 

In the cleavage of the eggs of amphioxus and of echinoderms 
the rate of nuclear growth is essentially similar to that of the 
ascidians. Here also the germinal vesicle is very large and the 
total volume of the nuclei at the close of cleavage is much less 
than the volume of the germinal vesicle, though decidedly greater 
than the volume of the germ nuclei at the beginning of cleavage. 
In all of these cases the nuclei in the early cleavages contain little 
chromatin and much achromatin; while they are more densely 
chromatic in the later stages, showing that the chromatin has 
increased in quantity relatively more than the achromatin. This 
is probably due to the fact that the chromosomes take up less 
cytoplasmic substance in the smaller cells than in the larger ones, 
the amount of achromatin in the nucleus depending in aE part 
upeE the quantity of cytoplasm in the cell. 

. Growth of different nuclear constituents. a. Nuclear sap. 
ii of the substances within a nucleus do not increase at the same 
rate. The most abundant constituent of a fully formed nucleus 
is nuclear sap, and this is scarcely present at all in the earliest 
stages of the nuclear cycle. During each resting period the nu- 


CELL SIZE AND NUCLEAR SIZE 45 


clear sap increases in amount from zero until it forms the principal 
bulk of the nucleus, and when mitosis comes on it passes into the 
cell body, and as a constituent of the nucleus sinks again to zero. 
The substance which forms the nuclear sap is absorbed by the 
nucleus from the cell body throughout the whole of the resting 
period, only to be thrown out into the cell body again at the end 
of that period. Consequently the nuclear sap is no more a nuclear 
constituent than a protoplasmic one, belonging to both nucleus 
and protoplasm. Studies on the growth of nuclear material 
should therefore be confined to the growth of the chromatin, but 
the difficulty of measuring the amount of chromatin at different 
stages will be appreciated without further comment. Also the 
fact that so large a part of the nuclear material belongs also to 
the protoplasni should be taken into account in experiments 
dealing with the isolation of nuclei from protoplasm; evidently 
the only satisfactory way in which such isolation can be accom- 
plished is by isolating chromosomes, rather than resting nuclei. 

There is good reason for believing that the nuclear sap contrib- 
utes to the nourishment and growth of the chromatin and linin, 
and that it in turn receives substances from these, so that the 
materials which pass into the cell body when the nuclear mem- 
brane dissolves, are not wholly the same as those which were 
taken up by the nucleus from the cell body. I have elsewhere 
(02) called attention to the fact that the escaping nuclear sap 
stains more deeply than the cell protoplasm and may therefore 
be called ‘chromatic sap.’ 

As to the mechanism of this intake of protoplasmic substance 
into the nucleus there is every visible evidence that it is of the 
nature of osmosis. The nucleus becomes spherical in shape un- 
less subjected to outside pressure, or to the action of substances 
which cause plasmolysis. The nuclear membrane remains entire 
and distinct until the last phase of nuclear growth, immediately 
preceding mitosis, when the nucleus swells very rapidly and the 
nuclear membrane becomes thin and then disappears. 

The measurements given in the preceding section show that 
the total quantity of the more fluid part of the nucleus, the nuclear 


2Watase (1893) says,—-‘“The structure known as the nucleus contains a great 
deal of cytoplasmic substance.” 


46 EDWIN G. CONKLIN 


sap, does not increase in quantity during the cleavage of the egg; 
we have seen that the total volume of all the nuclei of Crepidula 
at the 70-cell stage is about equal to that of the germinal vesicle, 
while in Styela the volume of all the nuclei at the 256-cell stage 
is 77 per cent less than the volume of the germinal vesicle. The 
conclusion is justified, therefore, that the more fluid constituent 
of the nucleus decreases greatly in volume during the early 
cleavage stages, and that the nuclei therefore become denser 
during this period. 

b. Linin. Just as the nuclear sap is proportional in volume to 
the volume of the nucleus as a whole, so also it is evident that the 
linin is more abundant in large nuclei than in small ones. Evi- 
dently it is not possible to determine the volume of linin in a 
resting nucleus, but since the spindle fibers are composed largely 
of linin it is possible by measuring the size of spindles to determine, 
at least in a general way, the relative quantities of linin in different 
nuclei. In the following table the length of the spindle from 

TABLE 13 
Length of spindle in the maturation and cleavage of Crepidula plana _ 


DIAMETER OF PRECEDING 


STAGE LENGTH OF SPINDLE NUCLEUS 
I Me 
irstimaturationie +.) eee 42 42 
Second maturation............ 18 = 
Hirsticleavageseoes eee eee 30 34.5 
Second cleavage, AB, CD...... 30 24 
Third cleavage, A, B,C, D.... 27 22 
Fourth cleavage, 1A-1D.... 25 21 
Fourth cleavage la-ld......... 21 12 
Fifth cleavage 2A-2D......... 24 18 
21 15 


Fifth cleavage 2a-2d.......... 


centrosome to centrosome is given for successive cleavages of 
C. plana, the measurements being made in each case in the stage 
of the metaphase. The diameter of the nucleus is also given for 
comparison with the spindle length (table 13). 

In general the diameter of the spindle at its equator is, in the 
prophase and metaphase, about the same as the diameter of the 
nucleus from which it came. Spindles in the protoplasmic ecto- 
meres are relatively larger than the size of the nucleus would lead 


CELL SIZE AND NUCLEAR SIZE 47 


one to expect and this probably is due to the fact, which I (’05) 
have established in the ascidians, that the polar partsof the spindle 
are not derived from the nucleus but from the protoplasm. With 
this proviso, it is true that, within the same species, large nuclei 
give rise to larger spindles than do small ones and this may be 
held to indicate that the linin is more abundant in the former than 
in the latter. 

The fact that the spindle fibers of ascidians are composed of 
equatorial and polar parts, the former derived from the nucleus 
and the latter from the protoplasm, and the fact that these two 
portions of the spindle, and also the polar fibers, are fundamentally 
alike, indicates that the linin, like the nuclear sap, is a constit- 
uent which belongs both to the nucleus and to the protoplasm. 

ce. Chromatin. The amount of chromatin undoubtedly in- 
creases during the cleavage; the resting nuclei in the later stages 
being more densely chromatic than those of the earlier stages. 
In each cell the chromatin is smallest in quantity when the daugh- 
ter chromosomes are first separated, and it grows in quantity 
during the resting period. Not all of the chromatin of the resting 
stage goes into the formation of the chromosomes of the next 
mitosis, but some of it in the form of granules (oxychromatin) 
or chromatic sap escapes into the cell body on the dissolution of 
the nuclear membrane. The larger the nucleus is and the longer 
the resting period through which it has come, the greater the 
quantity of chromatin which thus escapes at mitosis. Gardiner 
(98) estimated that the amount of chromatin which thus escaped 
into the cell body at the first maturation division of Polychaerus 
was five hundred times as great as that which went to form 
chromosomes, and conditions are similar in Styela, Crepidula, 
and many other forms. Consequently the volume of the chromo- 
somes in successive stages cannot be used as a measure of the 
growth of the chromatin. Nevertheless the growth of the chro- 
mosomal mass, as well as the growth of the entire nuclear volume, 
will give some idea as to the growth of the chromatin during cleav- 
age. Table 9, giving as it does the volumes of the nuclei and 
chromosomal plates at various stages, furnishes data upon which 
an opinion as to the growth of the chromatin of the resting stages 


48 EDWIN G. CONKLIN 


may be based. From the 2-cell to the 32-cell stages the growth 
in volume of the resting nuclei lies between 171 per cent for 
maximum nuclear size, and 292 per cent for mean nuclear size 
while the growth of the chromosomal plates is 248 per cent. It 
seems very probable therefore that the growth of the chromatin 
during these stages lies somewhere between 171 per cent and 292 
per cent, or an average increase for each of the 30 divisions rep- 
resented of from 5.7 per cent to 9.7 per cent. In all cases the 
growth of the chromatin falls far short of 100 per cent, or a doub- 
ling, in each division cycle. In Strongylocentrotus, Erdmann 
(08) finds that the ratio of chromatin to plasma is seven times 
greater in the pluteus than at the beginning of development, and 
she points out that this means that plasma contributes to the 
growth of the chromatin. 

While the chromatin as such is peculiar to the nucleus, there 
can be no doubt that large quantities of chromatin escape into 
the protoplasm. Such chromatin usually loses its distinctive 
staining reaction and presumably suffers chemical change. On 
the other hand we know that chromatin grows at the expense of 
substances received from the protoplasm. The work of Masing 
(10) on the nucleinie acid content of the egg indicates that this 
important constituent of chromatin is about as abundant in 
early stages as in later ones; he supposes that it exists in the pro- 
toplasm. 

d. Chromosomes. What is true of the quantitative relations 
of the chromatin as a whole is true also of the individual chromo- 
somes; those formed from large nuclei are larger than those from 
small ones; the chromosomes do not double in volume in each 
successive cleavage, but they become individually smaller as 
cleavage progresses. These facts are not difficult to demonstrate, 
but they are difficult to express in any numerical proportion, owing 
to the irregular shape and small size of the chromosomes, which 
make it very difficult to determine their volume. 

In Crepidula the chromosomes are very small and numerous, 
the full number being probably 60, and they are usually crowded 
together so that it is difficult to photograph them, or even to 
draw their outlines accurately, and since they are so small it is 


CELL SIZE AND NUCLEAR SIZE 49 


not practicable to measure them directly with the 1/1 micrometer 
eyepiece. Nevertheless by selecting sections in which only a 
part of the chromosomes are shown I have been able to sketch 
the outlines of many of them with what I believe to be substan- 
tial accuracy. For the purpose of comparing the sizes of chromo- 
somes from different cleavages I have chosen two generations of 
blastomeres in which the difference in the size of the nuclei is at 
a maximum, the nucleus in one cell being about twice the di- 
ameter of that in the other; these blastomeres are the macromeres 
AB and CD, and the micromeres 1/a—/d (figs. 7 and 8). In the 
former the diameter of the nucleus just before division is about 
24u, in the latter about 14u. When the nuclei of the cells in 
question had begun to divide and the mitotic figures were in the 
equatorial plate stage, the chromosomes from a number of these 
spindles were drawn as accurately as possible with a camera 
lucida. In order to be certain that the stage of division was the 
same in each case only longitudinal sections through the spindle 
were chosen; and in order to avoid as far as possible individual 
differences in the sizes of chromosomes, only the largest and most 
isolated chromosomes were drawn. Fig. 9 shows chromosomes 
from four different spindles of the second cleavage; fig 10 shows 
chromosomes from the first division of the first quartet cells 
(1a-1d), also from four different spindles. In all cases the chro- 
mosomes are magnified 2000 diameters. 

It is plain from these figures that the chromosomes from the 
larger nuclei are larger than those from the smaller ones, though 
the difference in the diameters and volumes of the chromosomes 
are not as great as the difference in the volumes of the nuclei 
from which they came. The average volume of the chromosomes 
from the large nuclei is about 5.2 cubic » and of those from the 
small nuclei about 2.6 cubic x. While the volumes of the nuclei 
as a whole are to each other about as 5 : 1, the volumes of their 
individual chromosomes are to each other as 2:1. In the case of 
nuclei which differ but slightly in volume it is not possible to be 
certain that the chromosomes differ in size, but in all cases in 
which the differences in the size of nuclei is considerable it can 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 


50 EDWIN G. CONKLIN 


be seen that the larger nuclei give rise to larger chromosomes than 
do the smaller ones. 

Since the probable error is much greater in the measurement of 
individual chromosomes than of whole chromosomal plates, I 
have not attempted to measure individual chromosomes in each 
stage of the cleavage; on the other hand the dimensions of the 
chromosomal plates are given in table 4 for each cell up to the 
32-cell stage. These measurements show that from the 2-cell 
to the 32-cell stage the chromosomal mass increases in volume 
248 per cent or an average of 8 per cent for each of 30 divisions. 
The chromosomal plates, and consequently the individual chro- 
mosomes, grow smaller as cleavage advances, but in the same gen- 
eration of cells small nuclei have smaller chromosomes than large 
ones. In short, the size of the chromosome is dependent upon 
the size of the nucleus from which it comes, rather than upon the 
cell generation to which it belongs. 

In the main these observations are in harmony with those of 
Erdmann, and Baltzer, to which reference has already been made. 
In Crepidula, as in the echinids studied by the authors named, the 
individual chromosomes grow smaller as the cleavage advances, 
but this is causally related to the decrease in the size of the nuclei 
and of the cells, and where, in later cleavage stages, the nuclei 
and cells remain large, there the chromosomes also are larger than 
in smaller sister cells. Just as the size of the nucleus is con- 
nected with the volume of the cytoplasm in which it lies, so the 
size of the chromosomes is connected with the volume of the 
nucleus from which they come. 

Montgomery (’10) has found that the sperm cells of Euschistus 
are of two sizes and he concludes (p. 127), that “‘it is probable that 
the large sperm possess no more chromatin than the small, 
though the heads in the former are much larger. The dimegaly 
expresses itself accordingly in differences of amount of karyolymph 
and of the substance (linin) that composes the mantle fibers, but 
much more markedly in the amount of cytoplasm.” He finds 
also that the mitochondria (idiozome) increase directly with the 
amount of cytoplasm. According to my observations chromo- 
somes from large nuclei are larger than those from small ones of 


CELL SIZE AND NUCLEAR SIZE 51 


the same generation, though naturally it is more difficult to detect 
size differences in objects as small as chromosomes than in entire 
nuclei. Where the differences in nuclear volumes are great one 
can always detect corresponding differences in chromosome vol- 
umes. 

The chromosomes of the spermatid are usually smaller than 
those of the o6tid, but when the chromosomes of the first cleavage 
spindle appear, those from the sperm nucleus are usually as large 
as those from the egg. The reason for this is to be found in the 
fact that both grow, after fertilization, in the same medium, the 
egg plasma, and for approximately the same length of time. 

e. Plasmasomes. The conclusion that large nuclei have large 
chromosomes, and vice versa, also applies to the sizes of nucleoli 
(plasmasomes) ; they are larger in large nuclei than in small ones. 
However in this case another factor is involved for the size of 
nucleoli is not only dependent upon the size of the nucleus, but 
also upon the length of the resting period; indeed the latter seems 
to be the more important factor of the two. The largest of all 
nucleoli is the one found in the germinal vesicle, at the close of the 
longest resting period in the entire life cycle. In these gasteropod 
eggs the next largest nucleoli are found in the cells 44—4D and 
4a—4e (fig. 6) in which the resting stage is particularly long. The 
nuclei of the cells 44—4D are of the same size as those of 2A—2D 
viz. 184 in diameter, but the nucleoli of the former have about 
three times the diameter of those of the latter. 

In earlier stages of cleavage where the blastomeres are dividing 
rapidly it is difficult to compare the sizes of nucleoli, not only 
because their number varies considerably, but also because each 
plasmasome is usually surrounded by a layer of chromatin gran- 
ules which renders exact measurements difficult. The number of 
plasmasomes appears to depend to a large extent upon the degree 
of fusion of an originally large number of separate plasmasomes. 
When chromosomes are isolated so that each gives rise to a dis- 
tinct vesicle, each may contain a minute plasmasome, and there 
may be as many of these as there are chromosomal vesicles. In 
Crepidula the number is always greatest during the earlier stages 
of the resting period; during the later stages they appear to fuse 


by EDWIN G. CONKLIN 


together becoming fewer and larger as the individual chromosomal 
vesicles fuse. For a considerable period two nucleoli are com- 
monly found in each nucleus, one in each gonomere, or nuclear 
half. However, when the resting stage is long, these two fuse 
into a single large plasmasome. 

While the nucleus continues to grow in size up to the time of 
the dissolution of the nuclear membrane, the plasmasome usually 
disappears before the formation of the spireme. In comparing 
the relative sizes of nucleoli it is important to compare corre- 
sponding stages; accordingly in my measurements they were 
measured when they had reached approximately their maximum 
size, and before the nucleus had reached its maximum. Nucleoli 
differ more or less in size even in different cells of the same genera- 
tion, owing perhaps to the more or less complete fusion of the 
many original nucleoli; it is significant in this connection that 
after a long resting period they are much more uniform in size 
and constant in number than when the resting period is short. 
The following table gives the diameters of nuclei and nucleoli 
(plasmasomes) in various blastomeres of Crepidula: 


TABLE 14 
Mazimum nucleolar size and nuclear size in the blastomeres of Crepidula plana 


DIAMETER VOLUME NUMBER DIAMETER VOLUME NUCLEAR- 


STAGE BLASTOMERES OF OF OF OF OF NUCLEOLAR 
NUCLEUS NUCLEUS NUCLEOLI NUCLEOLI NUCLEOLI RATIO 
“ cubic i cubic 
lcell,beforematuration 42 32,409 1 12 905.0} 35:1 
2 cells, AB, €D..... 20 4,189 2 3 28.0 | 149 :1 
A:cells ANB Cu peer 15 1,767 2 21, 13 TT \\220 a 
8 cells f1A-1D. eee) © 719) 3,591 2 gy 28.0 | 128 :1 
i Wales oe 5 52 13 1,150 2 2,13 7.0} 264551 
.. Uf DAE ID Perse 14 1,437 | 2 2 8.3 | 180 31 
12cells 4 “ ie F 
(2a-2drer see 15 1,767 2 2 8.3 | 220 21 
. (et Sldieenee 12 905 2 Bh 133)) 2OORea 
1G celle eee 7 130 2 1 1.0 180:1 
r (3A-3D. 15 1,767 1 73 221.0 Sol 
2 cele aeode 15 1,767 | 2 3 28.0 63:1 
25 cells Adie ee 9 382 1 3 14:0))) 2731 
32 cells, 4a-4e.......... 13 905 1 6 113.0 Seal 
Ca. 100 cells, 4A-4D.... 15 1,767 1 9 382.0 | 4.6 :1 


Fulgur carica: 
Ca. 1000 cells, 4A-4D 96 462,192 1 27 10306.0 44:1 


CELL SIZE AND NUCLEAR SIZE 53 


In eggs in which the nuclear division has been greatly delayed, 
if not entirely stopped, by the use of hypertonic salt solutions the 
nucleoli become much larger than in normal eggs. Thus in the 
eggs of Crepidula plana treated with 4 per cent NaCl solution 
for two hours, and then put into normal sea water for six hours, 
the sizes of nuclei and nucleoli are as follows: 


TABLE 15 


Nucleolar size and nuclear size in eggs of Crepidula plana in hypertonic sea water 


DIAMETER VOLUME NUMBER DIAMETER VOLUME NUCLEAR- 

STAGE BLASTOMERES OF OF OF oF OF NUCLEOLAK 
NUCLEUS NUCLEUS NUCLEOLI NUCLEOLI NUCLEOLI RATIO 

“ cubic js “ cubic ph . 

1cell, pronuclei...... {|e 24 eee - “2 aS oa 
id 21 4,849 1 10 524 9:1 
Dicdllg A BsCD..ss0.0.. 24 7,238 1 9 382 19:1 
4cells, A, B,C,D...... 15 1,767 1 9 BRP ae Gia 
BcelswtA-lD.s.......| 18 3,055 1 Oye eesse S31 


The great size of the single nucleolus in each of these nuclei 
is probably due to the fact that division has been delayed and the 
resting period prolonged. 

f. Centrosomes and spheres. Finally we may consider in this 
connection the sizes of centrosomes, and spheres though they are 
not parts of the nucleus. In general in Crepidula, large cells 
contain large centrosomes and spheres, while small cells contain 
small ones. The maximum diameters of centrosomes in the 
cleavage of C. plana, vary from 2u to 7u, the measurements being 
made during the telophase of division. The maximum diameters 
of the sharply defined spheres, during the resting stages, vary 
from 5u to 12u; and in all cases, so far as I have observed, the 
largest centrosomes and spheres occur in the cells which have 
the largest amount of protoplasm, while the smallest occur in 
the cells with the least amount of protoplasm. 

The centrosomes and spheres are the cell constituents which 
first become unequal in an unequal cell division. As soon as the 
spindle becomes eccentric, the centrosome and sphere which lies 
farthest from the center of the cell becomes smaller than the one 


54 EDWIN G. CONKLIN 


at the opposite pole. Only after the division wall forms do the 
daughter nuclei become unequal. 

5. Conclusions as to nuclear growth during cleavage. The rate 
and amount of nuclear growth during cleavage is much less than 
is generally believed. Whether the nuclear volume is taken when 
the nuclei are at their maximum, mean, or minimum size, the nuclear 
growth is far from 100 per cent, or a doubling, in each division. In 
Crepidula the nuclear growth is not more than 5 per cent to 9 per 
cent for each division from the 2-cell to the 32-cell stage, and less 
than 1 per cent for each division after the 32-cell stage. At the 
2-cell stage the nuclear volume is least and up to the 32-cell stage 
the chromatin increases at an average rate of about 8 per cent for 
each division. The stage when the volume of protoplasm is 
least, after the egg has reached its full size, is just before the first 
maturation division; between the first maturation and the 24- 
cell stage the protoplasm increases at an average rate of nearly 
6 per cent for each division. At the end of cleavage the ratio of 
nuclear material to protoplasmic differs but little from the ratio 
at the beginning. In Fulgur the nuclear growth from the 2-cell 
stage to the 16-cell stage averages only 2.8 per cent for each divi- 
sion, and the general Kernplasma-Relation remains unchanged. 
In Styela the nuclear growth from the 2-cell to the 32-cell stage 
averages 9.6 per cent for each division; from the 32-cell stage to 
the 256-cell stage it averages only 0.27 per cent for each division. 
Such a rate of growth is not significant and indicates that the 
meaning of cleavage is to be found in something other than the 
increase of nuclear material as compared with the plasma. 

In general the growth of each of the different nuclear constit- 
uents parallels the growth of the nuclear material as a whole, 
though this is not true of the nuclear sap, which belongs to both 
cytoplasm and nucleus. During cleavage the fluid content of the 
egg as a whole decreases, the o6plasm becoming more consistent 
in later stages than in earlier ones. The total fluid content of 
the nuclei in the early cleavage stages is much less than that of 
the germinal vesicle; even in the later cleavages the nuclear sap 
is not so abundant, in some animals, as in the germinal vesicle. 
In Crepidula the volume of all the nuclei at the 70-cell stage is 


CELL SIZE AND NUCLEAR SIZE 55 


only equal to that of the germinal vesicle, though the volume of 
the chromosomal plates has increased 250 per cent; in Styela 
the volume of all the nuclei of the 256-cell stage is 77 per cent 
less than that of the germinal vesicle, though the total chromoso- 
mal volume has increased many fold during this period. 

Linin is a nuclear constituent which is found also in the proto- 
plasm, and during cleavage it grows in quantity at about the 
same rate as the nuclear and protoplasmic materials as a whole. 
The polar parts of the spindle and the astral rays arise in the pro- 
toplasm outside the nucleus, while the equatorial portion of the 
spindle comes from the nucleus, as is shown with great clearness 
in the cleavage mitoses of ascidians. Correspondingly the size 
of the spindle is a resultant of the volume of the nucleus and of 
the protoplasm. 

Chromatin is more distinctively a nuclear substance than the 
nuclear sap or linin, though it undoubtedly grows at the expense 
of substance received from the protoplasm and in turn contributes 
material to the protoplasm. From the 2-cell to the 32-cell stage 
in Crepidula the growth, of the chromatin amounts to between 
6 per cent and 10 per cent for each division, and as the fluid con- 
tents of the nuclei do not increase during cleavage the nuclei 
become more chromatic in later stages than in earlier ones. 

Chromosomal material, as represented in the condensed chro- 
mosomal plates of the anaphase, increases in volume 248 per cent 
from the 2-cell to the 32-cell stages of Crepidula, or an average 
growth of about 8 per cent for each division. Individual chromo- 
somes grow smaller as cleavage advances, but this is due to the 
smaller size of the nuclei from which they come rather than to the 
cell generation to which they belong; nuclei of the same genera- 
tion which differ greatly in size produce chromosomes which differ 
in size, the larger nucleus producing larger chromosomes than the 
smaller one. 

In the blastomeres of Crepidula the size and number of nucleoli 
(plasmasomes) are influenced by the size of the nucleus and the 
length of the resting period. In most of the nuclei there are two 
nucleoli, but when the resting period is long, these fuse into a 
single one. In experiments, anything which prolongs the resting 


56 EDWIN G. CONKLIN 


period leads to an increase in the size of the nucleoli. During 
the normal cleavage of Crepidula the ratio of the nuclear volume 
to the nucleolar volume varies from 220 :1 to 4.6 :1. 

Centrosomes and spheres are proportional in size to the volume 
of the protoplasm in which they lie; they are always larger in 
large cells than in small ones and hence they grow progressively 
smaller as cleavage advances. 

In general the volume of each of the nuclear constituents named 
is influenced by the volume of protoplasm of the cell, and by the 
length of the resting period. The protoplasm contributes sub- 
stances to the growth of each of these constituents, and the more 
abundant it is the larger they grow, provided the period of growth 
is the same in all cases. Where the growth peroid (interkinesis) 
is very long the nuclei becomes unusually large and may ulti- 
mately absorb the greater part of the protoplasm. 

6. Comparison of growth of chromatin with increase of chemical 
substances and processes during cleavage. Loeb in several import- 
ant papers has shown that the nucleus is the oxidizing center of 
the cell, and that the chromatin is chiefly concerned in bringing 
about oxidations. Warburg (’08) found the oxidative power of 
the egg to increase at a relatively slow rate during cleavage. 
More recently, in view of the oft-repeated assertion that the chro- 
matin doubles at each division, Loeb (’09) concluded that the 
supposed growth of chromatin in geometric ratio indicates that 
nuclear synthesis is of the nature of an autokatalytic reaction. 
Masing (10) has shown that in the eggs of Arbacea pustulosa the 
nucleinie acid in the fertilized but unsegmented egg is as great 
as in the ‘morula’ with 500 to 1000 cells. He concludes that, 
‘“‘the colossal increase of nuclear mass in the cleavage leads to no 
perceptible increase of nucleinic acid in the germ. A corollary 
of this must be that the total quantity of nucleinic acid necessary 
to build up the nuclear apparatus of the germ must be preformed 
in the protoplasm” (quoted from Godlewski, ’11). Shackell 
(11) has reached a similar conclusion with regard to the nuclein 
content of the egg and blastula of Arbacea punctulata. 

The results of my observations as to the rate of the growth of 
chromatin is especially significant when compared with the work 


CELL SIZE AND NUCLEAR SIZE 57 


of Warburg. I find that the chromosomal mass grows at the 
rate of 8 per cent for each division up to the 32-cell stage. It is 
difficult to connect this rate of growth of the chromosomes with 
the lack of growth in the nucleinic acid content as shown by Mas- 
ing, or with the lack of growth of the nuclein content as shown by 
Shackell, and it seems necessary to assume as both of these in- 
vestigators have done, that these substances are already pre- 
formed in the protoplasm. If this be true, I venture the sug- 
gestion that the large amount of chromatin (oxychromatin) 
which escapes into the cell body when the germinal vesicle dis- 
solves may constitute the nuclein and nucleinie acid which is 
distributed through the cell body. 


VII. Senescence, rejuvenescence, and the ratio of nucleus to plasma. 


It is well known that Minot (’90, ’95, ’08) maintains that the 
cause of senescence is the increase of plasma and its products at 
a rate greater than that of the nucleus. According to his view 
the egg at the beginning of development is in a senile condition, 
“in which there is an excessive amount of protoplasm in propor- 
tion to the nucleus, and in order to get anything which is young 
a process of rejuvenation is necessary . . . . During the 
segmentation of the ovum the condition of things has been re- 
versed so far as the proportions of nucleus and protoplasm are 
concerned. We have nucleus produced, so to speak, to excess. 
The nuclear substance is increased during the first phase of de- 
velopment. Hence our conclusion:—Rejuvenation is accom- 
plished chiefly by the segmentation of the ovum.”’ He sums up 
his views on this subject in his four laws of age (’08, p. 250), the 
first two of which are: 1. ‘‘Rejuvenation depends on the in- 
crease of the nuclei. 2. Senescence depends on the increase of 
the protoplasm, and on the differentiation of the cells.” 

Richard Hertwig’s views (’89, 03, ’08) are apparently diamet- 
rically opposed to those of Minot, though I do not find them so 
definitely expressed. He finds that senescence, or rather ‘de- 
pression’ and ‘physiological degeneration,’ are accompanied by 
an enormous growth of the nucleus. As a result of his work on 


58 EDWIN G. CONKLIN 


Actinosphaerium and Infusoria, which had been overfed for a 
long time, he found that there was an enormous growth of the 
nucleus followed by physiological degeneration. The animals 
which saved themselves from this condition did it by the reduction 
of their nuclei, either by eliminating nuclear substance directly, 
or by the loss of the greater part of the nuclear material during 
conjugation, after which normal nuclear conditions were restored. 
He regards the immature egg cell, with its great nucleus, as in a 
condition of depression similar to that found in the protozoa 
named. By the processes of maturation and fertilization this 
nuclear material is greatly reduced: ‘‘Beim Beginn der Fur- 
chung und auch spiter ein enormes Missverhiltniss von Kern 
und Protoplasma vorhanden ist, und dieses Missverhiltniss 
allmihlich eine Ausgleich erfihrt, indem Zellsubstanz in Kern- 
substanz umgewandelt wird,” (03, p. 116). Apparently then, 
in Hertwig’s view, senescence or depression, is accompanied by 
too great an amount of nuclear material, which is then reduced, 
by maturation in the case of the egg cell, to such an extent that 
this enormous disproportion of nucleus to protoplasm appears; 
later, by means of the process of cleavage, during which the 
nuclear material grows at the expense of the protoplasm, the 
normal relations of nucleus to protoplasm are restored. 

Popoff (’08) accepts Hertwig’s view in all essential respects. 
He adds the interesting suggestion that in their period of depres- 
sion preceding maturation the sex cells are so weakened that they 
are unable to assimilate nutriment, and they consequently store 
up food as yolk. The formation of yolk, glycogen and fat are, 
according to this author, not indications of increased activity 
of cells, but of incapacity to carry the organic synthesis to its 
end, viz., the formation of plasma. 

While Minot’s hypothesis differs fundamentally from Hert- 
wig’s as to the cause of senescence, the former holding that it 
depends upon the increase of protoplasm over nucleus, the latter 
that it is accompanied by an increase of nucleus over protoplasm, 
both agree that in the segmentation of the egg there is an enor- 
mous growth of the nuclear material as compared with the pro- 
toplasm. 


CELL SIZE AND NUCLEAR SIZE 59 


Neither Minot nor Hertwig took account of the fact that a large 
part of the nuclear contents belongs to both nucleus and proto- 
plasm. The ‘Kernplasma-Relation’ depends very largely upon 
the quantity of protoplasmic material temporarily in the nucleus; 
in the 4-cell stage of Crepidula the ratio of nuclear volume to 
protoplasmic volume is 1 : 6.6 when the nuclei are measured at 
their maximum size, but 1 : 203.8 when they are measured at 
their minimum size. Neither of the authors named, in describ- 
ing the enormous growth of the nuclear material during cleavage, 
took account of the growth of the protoplasm during cleavage at 
the expense of the yolk. 

My observations on Crepidula have yielded the following re- 
sults, which bear upon the hypothesis under discussion: (1) 
While the germinal vesicle is absolutely the largest nucleus in the 
early stages of development, it is not so large with reference to the 
protoplasm, and hence according to Hertwig, not in so deep a 
depression, as the nuclei of certain blastomeres, which ex hypo- 
these should be undergoing restoration to normal conditions. 
(2) The growth of nuclear material during cleavage is not nearly 
so great as has been assumed, averaging not more than 10 per cent 
for each division up to the 32-cell stage, and not more than 1 per 
cent for each division after that stage. (38) The growth of proto- 
plasm at the expense of yolk during maturation and early cleay- 
age is considerable, averaging about 6 per cent for each division 
up to the 24-cell stage. (4) The ‘Kernplasma Relation,’ while 
constant for specific blastomeres, is by no means uniform for all 
the blastomeres of a given stage, but may vary from 1 : 1 to 1 : 14 
in different blastomeres of the same generation. (5) The ‘Kern- 
plasma-Relation’ in adult epithelial cells of all three germ layers 
is about the same as in the majority of the blastomeres. (6) The 
absolute size of the nucleus depends upon the quantity of proto- 
plasm in the cell and the length of the resting period (interkinesis). 
(7) The greater part of the nuclear volume consists of material 
which belongs to the protoplasm as much as to the nucleus; 
during the resting period this is taken in osmotically through the 
nuclear membrane, and is given out again at mitosis by the dis- 
solution of that membrane. (8) The immature egg cell, which 


60 EDWIN G. CONKLIN 


according to Popoff is so weakened that it is unable to assimilate 
nutriment, and consequently can only store up food instead of 
making protoplasm, does as a matter of facet form protoplasm 
throughout the whole of the growth period. 

So far as they go, therefore, these results do not support the 
view that senescence is due to either an increase or to a decrease 
of nuclear volume as compared with that of the protoplasm. 
But I think that this conflict between my results and those of 
Minot and Hertwig is, after all, confined to details, and that in the 
fundamental conception of the causes of senescence and rejuven- 
escence they may be brought into harmony. With the general 
thesis that senescence is associated with the accumulation in the 
cell of the products of metabolism and differentiation, and that 
rejuvenation consists in a return to a condition in which these 
products are largely eliminated, as Minot and Hertwig have 
urged, I am in hearty agreement; their assumption that changes 
in the nucleus-plasma ratio are the causes of these phenomena 
seems to me to be merely an error of detail. 

In a very suggestive paper, Child (’11) has recently maintained 
that senescence and rejuvenescence are caused by a decrease or 
an increase in the fundamental metabolic reactions. Anything 
which decreases the rate of metabolism, such as ‘‘decrease in 
permeability, increase in density, accumulation of relatively 
inactive substances, etc.,’’ will lead to senescence. ‘‘Rejuven- 
escence consists physiologically in an increase in the rate of metabo- 
lism and is brought about in nature by the removal in one way 
or another of the structural obstacles to metabolism” (p. 609). 

This hypothesis finds much support in the phenomena con- 
nected with the early development of the egg. It is well known 
that construct ve metabolism takes place only in the presence 
of nuclear material, and it has long been known that the nuclei 
of various kinds of gland cells give off substances which play 
an important part in the metabolism of the cell. Loeb (799) 
has shown that the nucleus is the oxidative center of the cell; 
Mathews identifies oxidase with chromatin; R. Lillie (’02) finds 
that oxidation takes place most rapidly in the immediate vicinity 
of the nucleus. If the rate of metabolism is associated with sen- 


CELL SIZE AND NUCLEAR SIZE 61 


escence or rejuvenescence, as Child maintains, anything which 
facilitates the nterchange between nucleus and protoplasm should 
lead to rejuvenescence, anything which decreases it should lead 
to senescence. 

During cleavage the increase in nuclear surfaces is much greater 
than the increase in nuclear volumes. While the increase in max- 
‘mum nuclear volumes up to the 32-cell stage of Crepidula is 
about 5 per cent for each division, the growth in the maximum 
nuclear surfaces during this period is about 11 per cent for each 
division. From the 2-cell to the 70-cell stage the nuclear volume 
increases only 2.24 times, while the nuclear surfaces increase 
5.30 times. In Styela the nuclear volume increases from the 2- 
cell stage to the 256-cell stage only 4.52 times, the nuclear sur- 
faces increase 13.75 times. Unquestionably this greater growth 
of nuclear surfaces as compared with nuclear volumes, facilitates 
the interchange between nucleus and protoplasm. There is also 
a considerable increase of cell membranes during cleavage, but 
most of this increase is confined to surfaces of contact between 
cells, and free surfaces show but little growth. My observations 
teach that there is little, if any, interchange of materials through 
partition walls separating cells. ; 

Another and much more efficient means of facilitating the inter- 
change between nucleus and protoplasm is found in the mitotic 
division of the nucleus. During the cycle from one division to 
the next the nucleus absorbs materials from the cell body, only 
to throw back into the cell body these and other materials when 
the nuclear membrane dissolves in mitosis. The chromatin 
is thus brought into the most intimate relations with the proto- 
plasm. There is thus a sort of ‘‘diastole and systole of the nu- 
cleus’? (Conklin, ’02), by which the interchange between nucleus 
and protoplasm is greatly hastened. Indeed in the paper just 
referred to I suggested that this function of mitosis may be quite 
as important as the division and separation of the chromosomes, 
which is usually supposed to be the one function of mitosis. 

The hypothesis that the more rapid interchange between nu- 
cleus and protoplasm is associated with increased metabolism 
is supported by some very significant physiological work on the 


62 EDWIN G. CONKLIN 


maturation, fertilization and cleavage of the egg. Loeb first 
showed that the immature egg, with germinal vesicle intact, is 
metabolically inactive; it absorbs but little oxygen and gives off 
little carbon dioxide. On the other hand when the membrane 
of the germinal vesicle dissolves, metabolic activity increases, 
and unless the egg is started in the process of development, by 
fertilization or other means, it soon dies. Lyon (’04) found that 
during the cleavage of the sea urchin egg the evolution of carbon 
dioxide is more rapid during the periods of division than during 
those of rest. Warburg (08) found that the fertilized sea-urchin 
egg uses six to seven times as much oxygen as the unfertilized 
egg. It is well known that the condensed chromatin of the chro- 
mosomes is brought into intimate relation with the protoplasm 
during mitosis, and of course the same is true of the condensed 
chromatin of the sperm head following fertilization. We may 
conclude, I think, that mitosis increases metabolism by facili- 
tating the interchange between nucleus and protoplasm, and 
particularly by setting free chromatin in the protoplasm, either by 
the dissolution of the nuclear membrane, or by the introduction 
of the sperm head in fetilization. 

Rapid and intimate interchange between the chromatin and 
the protoplasm is the condition of rapid metabolism, and ex 
hypothese of rejuvenescence; slow interchange is the condition of 
slow metabolism, and of senescence. Such a view has many 
points in common with the hypotheses of Minot and Hertwig, 
while it avoids many of the serious difficulties which those hypoth- 
eses encounter. It is thus evident that one may hold, with 
Minot and Hertwig, that the germ cells before maturation are 
senescent, and that maturation, fertilization and cleavage rep- 
resent a rejuvenescence, without necessarily connecting these 
processes with the nucleus-plasma ratio. 


3R. Lillie (1910) holds that this is due to increased permeability of the plasma 
membrane during division. 


CELL- SIZE AND NUCLEAR SIZE 63 


PART II 


EXPERIMENTAL STUDY OF CELL SIZE AND NUCLEAR SIZE IN 
THE EGGS OF CREPIDULA PLANA 


I. Nuclear size and chromosome number 


In Crepidula the relation of nuclear size to chromosome number 
is the same as in the Echinid larvae studied by Boveri (’05). 
By the use of various hypertonic salt solutions abnormal mitoses 
may be produced in Crepidula eggs; one of the most common 
of these abnormalities consists in the scattering of the chromo- 
somes, so that they do not fuse together to form two daughter 
nuclei, one in each cell, but many small nuclei. Indeed there 
may be almost as many small nuclei as there are chromosomes, 
every isolated chromosome being capable of producing a s° all 
nuclear vesicle. In all such cases the nuclear vesicles formed from 
a small number of chromosomes always remain smaller than 
those formed from a larger number. (In any given species the 
size of the nucleus is proportional to the number of chromosomes 
which go into its formation, providing the other factors which 
control nuclear size, viz., quantity of cytoplasm and length of 
resting period, are the same. On the other hand the size of the 
cell body is not dependent upon the size of the nucleus inthe 
early cleavages of Crepidula, as Gerassimoff (’02) found to be the 
case in Spirogyra and as Boveri determined in the case of Echinid 
larvae, but the reverse is true. 

In the eggs of Crepidula which have been treated with salt 
solutions the cell body frequently does not divide at all and many 
nuclei may be left in a single cell; where the cell itself divides 
there is a tendency for the blastomeres to divide in normal fashion, 
giving rise to macromeres or micromeres as in the normal egg, 
even though polyasters and abnormal mitoses are present. Con- 
sequently these eggs afford no evidence that the size of the nucleus 
has an influence on the size of the cell body. 


64 EDWIN G. CONKLIN 


IT. Nuclear size and cell size in centrifuged eggs of Crepidula 


While the size relations of cells and of their various constituents 
may be readily observed in normal eggs, it is especially in eggs 
which have been centrifuged at various stages of development 
that the factors which determine these various size relations can 
be most satisfactorily studied. The various constituents of a 
cell may be moved by centrifugal force to one pole or another, 
according to their specific weights, and the axis of centrifuging. 
In this way the yolk, the cytoplasm, the nuclei and the centro- 
somes, may be caused to take very abnormal positions in the cell. 
Even the mitotic figure may be moved out of its ordinary position 
in the earliest stages of its formation, but after it has reached the 
metaphase it can be moved only with great difficulty; from this 
stage on it is anchored, probably to the cell membrane by the 
astral radiations, while the other constituents of the cell are free 
to move under the influence of centrifugal pressure. In this way 
it happens that the cytoplasm may be centrifuged away from the 
spindle and the latter left in a dense mass of yolk; or the normal 
relations of cytoplasm and yolk to the poles of the spindle may be 
completely changed; or the normal size relations of the daughter 
cells may be quite reversed. As illustrating these changed rela- 
tions, due to centrifuging, a few eggs are shown in figs. 11-37, 
selected from a great number which are similar to these. 

These eggs were centrifuged on a centrifugal machine run by 
water pressure, at the rate of 2000 revolutions per minute; the 
radius of rotation was 6 em., consequently the centrifugal pres- 
sure was nearly 270 times that of gravity. Eggs were centrifuged 
at this rate for varying lengths of time, after which they were 
removed from the machine and either fixed at once, or left for a 
longer or shorter time in sea water before fixation. All eggs were 
fixed in Kleinenberg picro-sulphuric mixture, were preserved in 
70 per cent alcohol only long enough to wash out the fixing fluid, 
and were then stained in my modification of Delafield’s haematoxy- 
lin and mounted entire in balsam, in the manner described in 
previous papers (Conklin, ’02 et seq.) 


CELL SIZE AND NUCLEAR SIZE 65 


In fig. 11 an egg is shown which was centrifuged for ten minutes 
after the formation of the first polar body and before the formation 
of the second, the axis of centrifuging being such that the lighter 
protoplasm was thrown to the vegetative pole and the heavier 
yolk to the animal pole, thus reversing the normal positions of 
these substances. After centrifuging, the egg was left in sea water 
for three hours before being fixed. The first polar body, which 
has partially divided, lies at the animal pole; the second matura- 
tion spindle has been greatly elongated and its axis has been 
turned somewhat, its lower pole having been moved to the right 
in the figure. The egg has begun to constrict opposite the equa- 
tor of the spindle, thus leading to the formation of a giant sec- 
ond polar body. The nucleus of this second polar body consists 
only of a compact mass of chromosomes surrounded by yolk; 
the sphere connecting these chromosomes with the egg membrane 
is much elongated. The egg nucleus and sphere at the lower pole 
of the spindle are in contact with the field of cytoplasm and are 
much larger than those at the upper pole. The sperm nucleus 
and sphere, lying in the cytoplasmic field, are much the largest 
in the egg. In normal condition these relations are reversed, 
the sperm nucleus lying in the yolk, while the egg nucleus is in 
the cytoplasmic field; and in such cases the egg nucleus and sphere 
are larger than those of the sperm; however as the sperm nucleus 
approaches the egg nucleus and thus moves up into the cytoplasm 
it continually grows larger until, at the time the two meet, the 
sperm nucleus is almost as large as the egg nucleus. {The fact that 
the normal size relations of these two nuclei may be reversed by 
reversing the positions of the cytoplasm and yolk, furnishes con- 
clusive evidence of the fact that the relative sizes of the egg and 
sperm nuclei and asters are dependent upon the quantity of eyto- 
plasm in which they lie. 

Furthermore fig. 11 shows that the spindle itself is a structure 
composed of fibers more firm than the surrounding substance, 
and is not merely an arrangement of the granules, which happen 
to be present in a field of force, into lines, like iron filings in a mag- 
netic field. The spindle remains fixed in position when all sur- 
rounding substances change position, and the spindle fibers, 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 1 


66 ‘ EDWIN G. CONKLIN 


though much elongated preserve their usual appearance. In this 
regard my work confirms the conclusions of Morgan (’10) as to 
the nature of the spindle in Cerebratulus, and is at variance with 
the work of Lillie (09) on Chaetopterus. 

In fig. 12 an egg is shown which was centrifuged for fifteen 
minutes during the first cleavage and was then left for three 
hours in sea water. The axis of centrifuging is indicated here, 
as elsewhere, by the lighter vacuolated substance at one pole 
and the heavier yolk at the opposite pole; this axis is also marked 
by an arrow, the head of the arrow marking the distal pole during 
centrifuging, the tail of the arrow the central pole. In figs. 12 
to 15 the first cleavage plane does not pass through the animal 
pole, which is marked by the polar bodies, but is displaced to one 
side, and the cleavage is not meridional, as in normal eggs; 
furthermore the cleavage is not equal, quantitatively and qualita- 
tively, as in normal eggs, but is markedly unequal, most of the 
cytoplasm having gone into the smaller one of the two daughter 
cells, while the larger one contains little cytoplasm and much yolk. 
This is evidently due to the fact that the greater mass of yolk 
in the larger cell has displaced the cleavage plane to one side of 
its normal position. 

Corresponding to this difference in the quantity of cytoplasm 
in the first two blastomeres of these eggs, there is a decided dif- 
ference in the size of the nuclei and spheres, the latter always 
being proportional in size to the quantity of cytoplasm in which 
they lie. The smaller cells with the larger quantity of cytoplasm 
thus have larger nuclei and spheres than the larger cells, which 
have a smaller quantity of cytoplasm. 

The eggs represented in figs. 13, 14, and 15 were centrifuged for 
five hours during the first cleavage and were then fixed at once. 
It is evident that division took place while the eggs were on the 
centrifugal machine and that the daughter nuclei have grown to 
the size shown while the eggs were still being centrifuged. Other 
eggs centrifuged for the same length of time were allowed to 
develop further after being removed from the centrifuge, and 
they show that in most cases the eggs were still alive after cen- 
trifuging and not seriously injured. Fig. 13 shows a very note- 


CELL SIZE AND NUCLEAR SIZE 67 


worthy fact to which attention will be devoted in a future paper, 
viz., that the cell axis, which is marked by the line passing through 
the nucleus and sphere (and centrosome), remains unchanged 
after centrifuging. In the stage shown in fig. 13, the spheres lie 
between the nuclei and the polar bodies in normal eggs, and al- 
though the positions of cytoplasm and yolk, and of the first cleav- 
age plane have been changed in this egg, this cell polarity remains 
unchanged. 

Fig. 16 represents an egg which was centrifuged thirty minutes 
and then left in sea water for twenty hours. Neither this egg nor 
any others of this lot developed far after being centrifuged; it is 
possible that the eggs were injured in some way so that noneof them 
_ developed, or it is barely possible that the record of the experiment 
is wrong. In all the eggs of this lot the appearance is that of eggs 
which had been under normal conditions for about three or four 
hours after being removed from the centrifuge. 

This egg was evidently centrifuged during the first cleavage, 
which was very unequal, practically all of the cytoplasm having 
gone into the smaller of the two daughter cells. The nucleus and 
sphere in this smaller cell are enormous, whereas in the larger 
yolk cell they are extremely small, indeed no larger than in the 
anaphase stage of division. The chromosomes form a compact 
mass which stains deeply and contains no achromatic material. 
The sphere is small also but the fact that it holds its normal 
position with respect to the nucleus shows not only that the pol- 
arity of the cell remains unchanged, but also that the material 
of the sphere is different from the ordinary cytoplasm. In many 
cases similar to fig. 16 cytoplasm slowly forms around the chromo- 
somes in the yolk cell and ultimately such a cell may develop in 
a normal manner. There is no evidence that cytoplasm ever 
passes through the cell membrane from one cell to another, and 
there is positive evidence that this does not occur. The formation 
of cytoplasm around a mass of chromosomes in a yolk field is 
therefore an occurrence of more than ordinary importance. The 
question has been asked frequently whether the nucleus alone 
ean form cytoplasm or the eytoplasm alone a nucleus. It is 
known that the latter never happens; a mass of cytoplasm without 


68 EDWIN G. CONKLIN 


a nucleus may live for some time and show certain vital functions, 
but it is unable to grow or to regenerate lost parts. It is much 
more difficult to test the former question, for it is usually impos- 
sible to separate the nucleus completely from the cytoplasm and 
yet leave it in a medium in which growth would be possible. 
Verworn (’91) succeeded in shelling the nucleus out of Thalas- 
sicolla, but found that the isolated nucleus was unable to grow 
a new cell body; but apart from the objection that the resting 
nucleus contains a large amount of cytoplasmic substance, this 
experiment is not conclusive for it is possible that the failure to 
grow a cell body was due to the lack of a proper nutrient medium 
in which the nucleus could operate. 

The present experiment is free from most of these objections, 
though it must be confessed that one objection still remains, 
viz., it is not possible to be certain that every trace of cytoplasm 
has been removed from the yolk cell. Nevertheless the amount 
of cytoplasm left in the cell is very small and is quite indistinguish- 
able, the only visible constituents of the cell being chromosomes, 
sphere and yolk. In the growth of cytoplasm in such a cell there 
first appears a very thin layer of cytoplasm around the chromo- 
somes, then the yolk in the immediate periphery of this begins to 
dissolve and the cytoplasm increases in amount. Coincidently 
the chromosomes swell up, absorbing achromatic material from 
the cytoplasm, and in later stages the growth of both cytoplasm 
and nucleus goes forward at an increasing rate. The formation 
of cytoplasm takes place only in the presence of chromatin and 
in its immediate vicinity; on the other hand the chromosomes grow 
only when surrounded by cytoplasm. ‘This indicates that some 
influence, probably of a chemical nature, goes out from the chro- 
mosomes and leads to the solution of yolk and the formation of 
cytoplasm. Whether this influence from the chromosomes may 
act directly upon the yolk, or only indirectly through the medium 
of a minimal quantity of cytoplasm, is not certain, but it seems 
probable that the latter is the case. After cytoplasm has been 
formed around the chromosomes, but not before, the chromosomes 
themselves begin to swell up, absorbing achromatic material 
from the cytoplasm, and the chromatin grows in quantity. Cyto- 


CELL SIZE AND NUCLEAR SIZE 69 


plasm is essential to the growth of the nucleus and of the chroma- 
tin; on the other hand chromatin is essential to the growth of 
cytoplasm, or to the conversion of yolk or food substances into 
cytoplasm. The life of the cell consists in an interchange of 
materials between the nucleus and the cytoplasm; the one cannot 
grow in the absence of the other. This conclusion agrees with 
the generalization of Godlewski (10): ““Zuerst das Bildingsmate- 
rial geliefert und von den betreffenden Regeneratskomponenten 
zum Protoplasm assimiliert wird, dass dagegen in der zweiten 
Regenerationsphase dieses Protoplasma sich wenigstens teilweise 
zur Kernsubstanz transformiert”’ (p. 88). 

The question has been much discussed as to whether the nuclei, 
and more particularly the chromosomes of the germ cells, are 
the sole ‘bearers of heredity,’ as Weismann, and many others, 
have maintained. We have experimental evidence that the 
cytoplasm cannot form chromatin in the absence of preéxistent 
chromatin. On the other hand there is no certain evidence that 
the chromatin can form cytoplasm in the absence of preéxisting 
cytoplasm. The experiment described above is not entirely 
conclusive, for while chromosomes in a yolk field form cytoplasm, 
it is probable that a minimal amount of cytoplasm is left in the 
yolk field, and it may be said that this merely grows by assimila- 
tion of yolk. On the other hand my experiments show that 
where we have equal division of the chromosomes and unequal 
division of the protoplasm we may have regulation and normal 
development; whereas this never follows abnormal distribution 
of the chromosomes; in other words protoplasmic abnormalities 
are capable of regulation when the nucleus is normal, but the re- 
verse is not the case. The nucleus is the regulating center of the 
cell, and it is probably also the assimilating center. And since 
both of these functions are involved in inheritance, to this extent 
at least the nucleus may be said to be the inheritance center. 

Fig. 17 represents an egg which was centrifuged for four hours 
during the first cleavage and was then placed under normal con- 
ditions for six hours before being killed. The polar body marks 
the original animal pole and in the centrifuging most of the yolk 
was thrown to this pole, most of the cytoplasm to the opposite 


70 EDWIN G. CONKLIN 


pole. The first cleavage plane is nearly equatorial in position, 
and one of the cells contains most of the cytoplasm. The 
spindles for the second cleavage have formed and the spindle 
in the cell containing the larger amount of cytoplasm is distinctly 
larger than the one in the other cell; each is proportional in size 
to the resting nucleus from which it came and to the volume of 
cytoplasm in the cell. The fact that the polarity of the cells has 
not been changed by the abnormal position of the first cleavage 
plane is indicated by the fact that the spindles are parallel to 
each other, but not to the plane of cleavage, as in normal eggs. 
In short there is evidence that the spindles here attempt to take 
up the positions which they would have occupied in a normal 
egg, with meridional cleavage. 

Fig. 18 represents an egg from a lot which was centrifuged fifteen 
minutes in gum arabic, as recommended by Lyon (’04), and which 
was fixed three hours after removal from the centrifuge. Fig. 
19 shows an egg which was centrifuged thirty minutes, and was 
fixed six hours later. In both cases the centrifuging took place 
during the first cleavage, as is shown by the unequal distribution 
of cytoplasm and yolk on both sides of the first cleavage plane. 
In the second cleavage, which evidently occurred after the eggs 
were removed from the centrifuge, the cytoplasm was distributed 
equally to the daughter cells. In fig. 18 the second cleavage took 
place a little earlier in the cell rich in cytoplasm (AB) than 
in the other (CD), but the smaller size of the nuclei in the latter 
is probably due in part to the fact that these cells are poor in 
cytoplasm. In fig. 19 the inequality in the distribution of cyto- 
plasm at the first cleavage is much greater than in fig. 18; never- 
theless the second cleavage occurred in the cell poor in cytoplasm 
(CD) at nearly the same time as in the other cell (AB). Although 
the nuclei in the cells C and D are much smaller than those in A 
and B, their structure shows that they are in nearly the same 
stage of the cell cycle. Their smaller size is due to the smaller 
quantity of cytoplasm in which they lie. Figs. 17-19 indicate 
that the absolute size of the nucleus has little to do with the time 
of its division; small nuclei in yolk-rich cells divide almost as 
rapidly as large nuclei in cells rich in cytoplasm. 


CELL SIZE AND NUCLEAR SIZE 71 


Figs. 20 to 28 show eggs which were centrifuged during the 
second cleavage. The first and second cleavages may always 
be distinguished by the fact that the polar furrow bends to the 
right in the first cleavage and to the left in the second (Conklin 
97). In fig. 20 the distribution of cytoplasm and yolk to the 
daughter cells was equal in the first cleavage but unequal in the 
second, and the daughter nuclei are proportional in size to the 
volume of the cytoplasm in which they lie. 

In fig. 21, which represents an egg which was centrifuged for 
30 minutes and fixed at once, the second cleavage is very unequal, 
two of the macromeres (B and C’) being small protoplasmic cells, 
which resemble micromeres in appearance, but which behave like 
macromeres as the study of later stages (figs. 24 to 28) shows. 

Fig. 22 represents an egg which was centrifuged for thirty 
minutes during the second cleavage and then kept under normal 
conditions for twenty-one hours before being fixed. The second 
cleavage was suppressed although the nucleus divided in the upper 
cell, AB, but not completely in the lower one, CD. These nuclei 
have given rise to spindles for the third cleavage, there being two 
independent spindles in the cell AB, and two spindles which are 
fused at one pole in the cell CD, thus forming a triaster. The 
degree of abnormality in this case is indicated by the fact that 
the development has been halted at this stage, although a normal 
egg would have reached the 20-cell stage at least, in the time which 
elapsed after centrifuging. 

With the exception of fig. 26, all of the figs. from 23 to 28 were 
drawn from the same lot of eggs which were centrifuged for thirty 
minutes in the 2-cell stage, and then kept for six hours under 
normal conditions before being fixed. In all of these eggs the 
second cleavage was made very unequal by the centrifuging. 
Two of the macromeres are not only much smaller than the other 
two, but are composed entirely of cytoplasm, whereas the two 
larger macromeres contain all of the yolk. Nevertheless the 
behavior of these two small, protoplasmic macromeres is almost 
identically like that of the large, yolk-rich macromeres; the micro- 
meres are given off from both the protoplasmic and the yolk 
laden macromeres at practically the same time and in the same 


72 EDWIN G. CONKLIN 


direction; the micromeres formed from these abnormal macro- 
meres are the same as in normal eggs in which all the macromeres 
are of the same size and contain the same quantity of cytoplasm 
and yolk. In short there is here a form of regulation which leads 
to the formation of normal micromeres from abnormal macro- 
meres, and the exact manner in which this cellular regulation 
takes place is of fundamental importance, and will be discussed 
later. 

Fig. 23 represents an egg similar in many respects to fig. 21, 
but of a later stage. The smaller protoplasmic macromeres 
preserve their original polarity as is shown by the fact that the 
spheres lie between the nuclei and the polar bodies. On the other 
hand each of the large macromeres contains a tetraster; the spin- 
dles are those of the third cleavage. 

Fig. 24 represents an egg of the same type as the preceding, 
after the third cleavage; each macromere has given rise to a 
micromere which is normal in form, position, constitution and 
size, although the macromeres are very abnormal in these regards, 
two of them containing all of the yolk and very little protoplasm, 
and the other two being small and purely protoplasmic. Indeed 
the macromeres 7A and 1D nearly exhausted all the cytoplasm 
which they contained in order to form cytoplasmic micromeres 
of normal size; on the other hand, the size of the micromeres 
1b and /c is not influenced by the fact that the macromeres from 
which they come are small and are purely protoplasmic. 

Fig. 25 is a drawing of an egg in a slightly older stage than fig. 
24; the large macromeres, 1B and 1C, are giving off the second 
set of micromeres, 2b and 2c, while two of the first set of micro- 
meres, /b and /c, are just beginning to divide. The four small 
cells which lie to the left of the polar bodies are the macromeres 
1A and 1D and the micromeres 1a and /d; these cells are purely 
protoplasmic and are very small, all four of them being no larger 
than one of the micromeres, 1b or Jc, in the other quadrants. 
Nevertheless these minute ‘macromeres’ have each given rise by 
an equal cleavage, to a micromere as large as itself. Although 
these micromeres are much smaller than those in the other quad- 
rants, they are the largest that could be formed from the macro- 


CELL SIZE AND NUCLEAR SIZE 73 


meres in question without making the macromeres smaller than 
the micromeres, thus reversing the usual inequality of this divi- 
sion; in short the division of these cells represents the nearest 
possible approach to normal conditions. 

Figs. 27 and 28 show eggs of the same type as the preceding, 
but at a stage after the formation of the second set of micro- 
meres (2a—2d) and during the division of the first set (/a—/d). 
Here also these micromeres are normal in size, although the size 
relations, and the cytoplasmic or yolk content of the macromeres 
from which they came, are very abnormal. In the cleavages which 
follow after the centrifuging, complete regulation has occurred, 
so far as this is possible. It is not possible for regulation to take 
place by the redistribution of cytoplasm and yolk by passage 
through a cell membrane. 

Fig. 26 represents an egg which was centrifuged for thirty 
minutes during the second cleavage, and then fixed twelve hours 
later. At the time of centrifuging the nuclear division in the 
second cleavage was complete, but the division of the cell body was 
suppressed. Consequently each of the blastomeres, AB and CD, 
contained two nuclei, which by subsequent division in the manner 
indicated in fig. 22 have given rise to two sets of micromeres, 
la-Id, and 2a-2d. Both sets of micromeres have divided, as 
indicated by the connecting bonds, thus forming a somewhat ab- 
normal cap of sixteen micromeres. The nuclei of the macromeres 
are indicated by the reference lines from the letters 2A—2D. 
Other cases similar to this one will be shown and described in 
another paper, but this one egg shows that it is possible for both 
the nuclei of a binucleate cell to divide at the same time and to 
give rise to separate cells, each with a single nucleus, and that such 
cells may approximate in form and position normal blastomeres. 

Fig. 29 represents an egg which was centrifuged for four hours 
at the close of the second cleavage, and fixed at once after centri- 
fuging. The yolk has been forced out into lobes, which are still 
connected with the protoplasmic portions of the cells except in 
the case of one cell, where the lobe has been completely separated. 
It is a significant fact that the point at which the lobe forms, and 
consequently the point where the cell membrane is weakest lies 


74 EDWIN G. CONKLIN 


at the outer pole of the axis which passes through the centrosome 
and nucleus and these axes mark the position of the spindles 
of the third cleavage. Here, as in every other instance, the 
smallest nucleus is found in the cell which has the smallest amount 
of cytoplasm. 

Fig. 30 is a drawing of an egg which was centrifuged ten min- 
utes in gum arabic, during the first cleavage, and fixed four 
hours later during the third cleavage. Macromeres A and B 
are richer in cytoplasm and poorer in yolk than C and D, and 
correspondingly the spindles and asters are larger in the former 
than in the latter. 

Figs. 31 and 32 represent eggs which were centrifuged four 
hours during the first cleavage, and were fixed six hours later. 
In both eggs the macromeres A and B are richer in cytoplasm and 
poorer in yolk than C and D. In fig. 31 the cells A and B con- 
tained more cytoplasm and divided earlier than C and D; at least 
one-half of the cytoplasm in the latter cells has gone into the 
formation of the micromeres, which are still, however, smaller 
than normal. The first cleavage in this egg did not pass through 
the animal pole, marked by the polar bodies, but was displaced 
to one side, and the spiral form of the cleavage is not clearly 
preserved in the cells C and D. While the regulation in the size 
of these micromeres is not complete, the tendency to approach 
the normal condition is evident. Fig. 32 is similar to fig. 31, 
though the macromeres C and D of this egg contained a larger 
amount of cytoplasm than in fig. 31, and the regulation in the 
size of the micromeres is complete. 

Fig. 33 shows an egg, from the same slide as fig. 30, which was 
centrifuged ten minutes in gum arabic and fixed four hours later. 
The macromeres /A and /B contain more cytoplasm and are 
dividing earlier than 1C and 1D, but the micromeres from the 
former are no larger than those from the latter. 

Fig. 34 represents an egg which was centrifuged for two and 
one-half hours during the first cleavage, and was fixed twenty-one 
hours later. The macromeres 2A and 2B contain much cyto- 
plasm, while 2C and 2D contain little and yet the micromeres 
formed from the latter are almost as large as those from the former. 


CELL SIZE AND NUCLEAR SIZE 75 


Figs. 35, 36, 37 represent eggs, from the same experiment, 
which were centrifuged thirty minutes during the first cleavage, 
and were fixed twelve hours later. The size of the micromeres of 
the first, second or third sets is but little influenced by the quan- 
tity of cytoplasm in the macromeres; the size regulation of the 
micromeres is here practically complete. In fig. 37 the cell 4c 
forms at the same time as 4d, though in normal eggs it does not 
form until much later; the precocious formation of this cell is 
probably due to the fact that the amount of cytoplasm in macro- 
mere C was larger than normal. 


III. General results of these experiments 


The results of these experiments, which have been described 
in the order of development from the earlier to the later stages 
without reference to a logical presentation of general questions, 
may now be classified and compared with the observations on 
cell size and nuclear size given in Part I of this paper. In gen- 
eral these experiments support in every detail the conclusions based 
upon the study of normal eggs and blastomeres. 

1. Nuclear size in centrifuged eggs. In centrifuged eggs, as in 
normal ones, the size of the nucleus is always dependent upon 
the quantity of cytoplasm surrounding the nucleus and upon the 
length of the resting period. Nuclei which are normally large 
may be caused to remain small, and nuclei which are normally 
small may be rendered large by merely changing the positions of 
the yolk and cytoplasm in the cell. 

In normal eggs of Crepidula the egg nucleus lies in a protoplas- 
mic field near the animal pole of the egg, while the sperm nucleus 
enters the egg near the vegetal pole 4nd moves up toward the 
animal pole through a field of yolk. As long as the sperm nucleus 
is in this yolk it remains very small, and only when it emerges 
into the protoplasmic field near the egg nucleus does it begin to 
grow rapidly. The egg nucleus on the other hand, grows rapidly 
and becomes much larger than the sperm nucleus. If now an 
egg is centrifuged during the formation of the second polar body 
so as to throw the yolk to the animal pole and the cytoplasm to 


76 EDWIN G. CONKLIN 


the vegetal pole, the normal size relations of the germ nuclei is 
reversed, the sperm nucleus becoming larger than the egg nucleus 
as shown in fig. 11. Godlewski (’08) holds that the size of the 
sperm nucleus depends upon the time which elapses before its 
union with the egg nucleus; it also depends, as I have shown, 
upon the quantity of cytoplasm in which it les. We conclude 
therefore, that in all animals the relative sizes of egg and sperm 
nuclei are dependent upon the amount of cytoplasm in which 
they lie, and upon the length of the growth period (interkinesis). 
In this connection it may be worth while to remark that one 
reason why the rhythm of cleavage, in Boveri’s, Driesch’s, and 
Godlewski’s experiments, follows the maternal rather than the 
paternal type may be found in the fact that the rate of growth 
of the nucleus is dependent upon the quantity and quality of the 
protoplasm of the egg. 

In the cleavage of the egg the size of the nucleus is dependent 
upon the quantity of protoplasm in which it lies, as shown by figs. 
12 to 20. In eggs subjected to strong centrifugal force the egg 
contents separate into three zones, a yellow zone of yolk at the 
distal (heavy) pole, a gray zone of oily and watery substance at 
the central (light) pole, and a clear zone of protoplasm between 
these two. It is the latter substance which contributes to the 
growth of the nucleus, as is shown by such cases as fig. 16 in which 
the gray substance was centrifuged out of the egg and practically 
all of the yolk thrown into one of the blastomeres, and most of 
the clear protoplasm into the other; the nucleus in the blastomere 
which contains yolk but little or no protoplasm has scarcely 
grown at all, the one in the cell containing the clear protoplasm, 
but without the gray substance, has grown enormously. Sim- 
ilar, though less striking, differences in the sizes of nuclei, depend- 
ing upon the quantity of clear protoplasm in the cell, are found 
in all the eggs figured. In centrifuged eggs the nucleus always 
occupies the middle zone, and as I have just shown it grows at the 
expense of substance received directly from this zone. The fact 
that the specific gravity of the nucleus and of this middle zone are 
the same, is probably due to the fact that so much of the absorbed 
nuclear material is from this zone. 


CELL SIZE AND NUCLEAR SIZE WG 


2. The sizes of spindles, centrosomes, spheres and asters. The 
study of centrifuged eggs shows, as was observed in the ease of 
normal eggs, that the sizes of spindles, centrosomes, spheres 
and asters are dependent upon the quantity of cytoplasm in which 
they lie. The size of the spindle is also related to the size of the 
nucleus, as I have already shown, but as this, in turn, is dependent 
upon the quantity of cytoplasm of the middle zone, it follows that 
the size of the spindle as well as that of the centrosome and sphere 
is related to the quantity of cytoplasm in which they lie. Fig. 
17 shows spindles in sister cells which are quite different in size 
owing to the different amounts of cytoplasm in these two cells; 
while figs. 11, 12, and 16 show centrosomes and sphere which 
vary in size depending upon the quantity of cytoplasm surround- 
ing them. 

In this connection attention should be called to the fact that 
the spindles from the stage of the metaphase to the end of 
mitosis are anchored in the cell, and can be moved only with 
much difficulty. The spindle fibers are tougher and more con- 
sistent than the surrounding plasm, and they are not a mere 
arrangement of granules in the lines of force as Lille (’09) has 
maintained for Chaetopterus. 

8. The rhythm of division in centrifuged eggs. The rhythm of 
division is not dependent solely upon nuclear size, nor cell size, 
nor the ratio of one to the other (Kernplasma-Relation), though 
it may be influenced by the absolute amount of cytoplasm present 
in the cell. Cleavage cells which contain a large amount of cyto- 
plasm, and which therefore have large nuclei, usually divide a 
little earlier than cells poor in cytoplasm, and with small nuclei, 
though this is not always the case, as is shown by fig. 17, in which 
the large and the small nuclei divide at the same time. Nuclei 
which differ greatly in size may still be in the same stage of the 
nuclear cycle, as shown in fig. 19, and may divide at the same 
time. On the other hand, figs. 25, 31, 34 and 37 show cases in 
which nuclei of the same generation divide earlier in cells rich 
in cytoplasm than in cells which are poor in this substance. 

4. Growth of cytoplasm at the expense of yolk. Centrifuged 
eggs afford an excellent opportunity of studying the way in which 


78 EDWIN G. CONKLIN 


cytoplasm grows at the expense of yolk. In cases in which the 
centrifuging occurred after the spindle was anchored in the cell, 
but before the division wall had formed, the cytoplasm may be 
thrown almost entirely to one pole of the spindle and the yolk 
to the other; accordingly when division occurs one of the daughter 
cells will contain almost all the cytoplasm, the other all the yolk, 
while both cells will receive the same number and mass of chro- 
mosomes, fig. 16. The chromosomes which are left in the yolk 
field remain small and compact since there is no cell substance 
which they can absorb. After some time the yolk in the vicinity 
of the chromosomes may begin to disappear and cytoplasm to 
appear in its place. It can scarcely be doubted that some sub- 
stance, probably an enzyme, is given off by the chromosomes and 
dissolves the yolk, and that this dissolved yolk is then converted 
into cytoplasm through the influence of the chromosomes. Once 
a small field of cytoplasm is formed around the chromosomes, they 
begin to abSorb it and to become vesicular. The process of form- 
ing cytoplasm may then go forward rapidly and in the end the 
yolk cell may give rise to protoplasmic micromeres in a normal 
manner (fig. 31). It is probable that a small amount of cyto- 
plasm, which cannot be displaced by centrifuging, is left in the 
yolk cell, and it is possible that the formation of new cytoplasm 
would not take place in the absence of this small remnant, but 
it can be proved conclusively that this formation of cytoplasm 
takes place only in the vicinity of the chromosomes, and that in the 
absence of this chromatic material it never occurs at all. Under 
these circumstances the conclusion seems justified that the chro- 
matin has the power of forming cytoplasm when placed in a suit- 
able nutrient medium, such as yolk, and that the cytoplasm in 
turn contributes to the growth of the nucleus and of the chromatin. 

5. Unequal and differential cell divisions. By centrifuging, the 
size and constitution of the blastomeres may be changed; divi- 
sions which are normally equal may be made unequal, and vice 
versa; cells which are normally protoplasmic may be filled with 
yolk and vice versa. In this way both the cell size and the cell 
content may be controlled experimentally. 


CELL SIZE AND NUCLEAR SIZE 79 


Acknowledgedly the position of the spindle conditions the plane 
of the cleavage, the division wall passing through the equator of 
the spindle. When by any means the spindle is displaced from 
its normal position the division plane is displaced. In this way 
giant polar bodies may be formed, as shown in fig. 11, or macro- 
meres may be formed which are small and free from yolk, as 
shown in figs. 16, 21-28, e al. 

Are the inequalities and differentiations of normal cleavage 
due to similar causes, viz., external or internal pressure? Clearly 
external pressure cannot be involved in the unequal division 
of free cells, such as the maturation divisions of the egg; and 
the fact that isolated blastomeres of the 4-cell stage divide 
in the normal manner into small protoplasmic micromeres and 
large yolk-rich macromeres, shows that these unequal divisions 
during the cleavage period cannot be explained as the result of 
reciprocal pressure among cells. On the other hand, the forma- 
tion of micromeres of normal size and constitution from purely 
protoplasmic macromeres, as shown in figs. 24, 27, 28, 33, 36, 
et al., indicates that this inequality of division cannot be due to 
the crowding of the spindle to one side of the cell by internal 
pressure, such as might come from the presence of a mass of 
yolk—because in the eases cited, little or no yolk is present in 
the macromeres. If internal pressure is involved in the unequal 
division of these protoplasmic cells it must be pressure of a very 
different sort from that involved in the presence of a mass of 
metabolic products at one side of the cell. While the spindle 
may be pressed out of position by external or internal pressure 
this will not serve to explain the eccentric position of the spindle 
in such cases as I have described. 

A satisfactory explanation of unequal and differential cell divi- 
sion must also be able to be applied to equal and non-differential 
cleavage, for the causes of the latter are not simple mechanical 
conditions, such as pressure. In the case of cleavages which 
are normally equal, if the spindle and yolk are moved to eccen- 
tric positions in the cell, they come back, if possible, to their 
normal positions when the pressure is removed; indeed they some- 
times seem to come back against considerable pressure, as when 


80 EDWIN G. CONKLIN 


a spindle moves out of a protoplasmic field into the yolk in order 
to reach its normal position in the cell. When eggs like the one 
shown in fig. 11 are removed from the centrifuge, the egg and sperm 
nuclei, together with the cytoplasm surrounding them move up 
through the yolk until they ultimately le in their normal position 
on the animal side of the egg, beneath the polar bodies. How- 
ever far the germ nuclei or the first cleavage spindle may be re- 
moved from the chief axis of the egg, they invariably come back 
to their normal positions, with the equator of the spindle in the 
egg axis, and the long axis of the spindle at right angles to the egg 
axis, unless the spindle is held so long in its abnormal position 
that it is caught in that position by the divisional processes. The 
same is true also of the nuclei and spindles of the 2-cell stage; 
when moved out of the median plane of the cell they come back 
to that median plane, unless the cells are injured or the spindles 
are held in their abnormal position until the metaphase or a little 
later. Evidently the cause of equal cell division, such as the first 
and second cleavages of Crepidula, is not so simple as those have 
assumed who have attributed it to pressure, the line of least 
resistance, or the long axis of the protoplasmic mass. 

Not only the eccentricity or lack of eccentricity, but also the 
axis of the spindle is of great importance in determining the char- 
acter of the cleavage. While the former is associated with the 
equality or inequality of division, the latter conditions its differ- 
ential or non-differential character. The polar differentiation 
of the egg is the first visible morphogenetic differentiation, and it 
is not without significance that in the first and second cleavages 
of the egg the spindles are at right angles to the egg axis, while 
in the third, fourth and fifth cleavages they are more nearly par- 
allel with that axis. 

I have hitherto spoken of the position of the spindle as if it 
were the one cause of equal or unequal, differential or non-differ- 
ential cleavage; but for many reasons it is evident that the posi- 
tion of the spindle is itself the result of the structure or organiza- 
tion of the protoplasm, and that in this organization polarity and 
symmetry play an important part. Many years ago (’93) I 
showed that even before the spindle is formed, the shape of the 


CELL SIZE AND NUCLEAR SIZE 81 


cell may indicate the position and direction of the coming cleav- 
age, and I maintained then and in subsequent papers (’97, 99, 
02) that the position of the spindle and the size, position, and 
histological character of the daughter cells is the result of the 
structure of the protoplasm, and particularly of the polarity and 
symmetry of the cell. 

These conclusions have been confirmed by muchexperimental 
work on cell division, which I have completed but have not yet 
published. The position of the spindle and the plane of cleavage 
may be greatly changed, but the polarity and organization of the 
protoplasm remain unchanged, as I shall show in a future paper. 
Indeed it is very difficult to alter the polarity of any cell as Lillie 
(706, 09) has shown, and one reason for this is to be found in the 
fact, as I have discovered in Crepidula, that the cell axis, i.e., 
the axis connecting nucleus and centrosome, can rarely be changed 
by artificial means. 

6. Regulation in the cleavage process. Evidently connected 
with this persistent organization of the cell is the power of regu- 
lation which is shown in the cleavage of the egg as well as in the 
regeneration of adult parts. Whenever the size or constitution 
of blastomeres of Crepidula have been changed, or when cleavages 
have been suppressed, subsequent cleavages come back to the 
normal form so far as this is possible. The original disturbance 
can be righted only very gradually if at all, since neither yolk, 
cytoplasm nor nuclei can pass through cell membranes, and the 
only redistribution of substances possible is by means of new cell 
divisions. But in Crepidula the divisions following upon such 
a disturbance of the usual cleavage process are almost if not en- 
tirely normal. This is very evident in the divisions following upon 
disturbances of the first two cleavages. All of the yolk may be 
centrifuged into two of the macromeres and practically all of 
the cytoplasm into the other two, as in figs. 16, 19, 21, 23, et. al; 
two of the ‘macromeres’ may be very small and two very large, 
as in figs. 16, 21, 23 to 28; or one of these first two cleavages may 
be suppressed, as in figs. 22 and 26; but if such abnormal eggs 
are allowed to develop under normal conditions, the micromeres 
are formed in normal manner, as is shown in figs. 24 to 28 and 32 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 1 


82 EDWIN G. CONKLIN 


to 37. Whatever the content of the different macromeres may 
be, whether purely protoplasmic or entirely yolk the micromeres 
are always protoplasmic, even though division must be delayed 
until the cytoplasm which goes into the micromeres can be formed 
from yolk (figs. 24, 31, 35); whatever the size of the macromeres, 
the micromeres formed from them are approximately normal in 
size, even though yolk-rich cells must give up most of their cyto- 
plasm (figs. 24, 31, 35), or protoplasmic micromeres must divide 
equally (figs. 25, 28), in order to give rise to micromeres of the 
usual size. 

Such regulations of cleavage are probably caused, in the case 
of Crepidula, by the persistent polarity of each cell, which in 
turn leads to the localization of the spindle in a definite axis, 
with its pole at a definite distance from the surface of the cell. 
In what manner the polarity of the cell may cause the localiza- 
tion of the spindle is clearly shown in the cleavage of Crepidula. 
In former publications (’99, ’02) I have called attention to the 
fact that definite movements of cell substance take place in divid- 
ing cells, and that these movements serve to orient the spindles; 
these movements are always related to the polarity of the cell 
and to that of the entire egg. Furthermore, I have elsewhere 
(02) called attention to the fact that the cell membrane is weakest 
opposite the poles of the spindle. I was formerly of the opinion 
that this was due to some influence of the spindle on the cell 
membrane, but a further study shows that these weak places in 
the cell membrane are present before the spindle forms and can 
not therefore be caused by the spindle. In the egg shown in fig. 
29 the places of reduced tension on the cell membrane are indicated 
by the lobes of yolk attached to the cells, and a line drawn through 
the centrosome, nucleus and lobe indicates the precise position 
which the spindle will take at the next cleavage. The axes of the 
third cleavage spindles are here marked out long before the spin- 
dles are formed; the weak spot in the cell membrane is not caused 
by the position of the spindle, but the latter is the result of the 
former. Experiments on eggs in the 2-cell, 4-cell and 8-cell stages 
of cleavage show that the positions of the points where the mem- 
brane is weakest, change in each cell generation and that they 


CELL SIZE AND NUCLEAR SIZE 83 


always mark out the position of the spindle. These lobes are 
formed only when the egg is subjected to pressure and then only at 
those points on the cell surface which mark the position which 
will be taken by the poles of the spindle. Since the spindle axes 
change in successive cleavages it follows that this point of reduced 
tension also changes in successive cell generations. 

I conclude therefore that the position of the spindle, and all 
the morphogenetic results which follow from this, is dependent 
upon the polarity of the cell; which polarity manifests itself 
not only in the localization of cytoplasmic substances, but also, 
and more fundamentally, in definite movements of the odplasm 
and in reduced tension of the cell membrane at the poles of the 
cell. 


GENERAL SUMMARY AND INDEX 
Part I. Observations 


1. The equality or inequality of cell division in normal cleavage 
is due to internal causes, rather than to the presence of metabolic 
substances, such as yolk, within the cell or to pressure from with- 
out. These internal causes are to be found in the polarity of the 
cell, in movements of the cytoplasm, and in the structure of the 
cellmembrane. Since the position and axes of the spindles change 
regularly in successive divisions this protoplasmic organization 
must also change regularly (pp. 6-9). 

2. The yolk-lobe is a temporary extrusion of yolk or odplasm 
during mitotic pressure, at the former point of attachment to the 
ovarian wall and a little to one side of the vegetative pole. If 
this lobe is large, the resulting cleavage is unequal, although the 
furrow cuts through the chief axis and the center of the egg. The 
degree of inequality of the first and second cleavages is measured 
by the size of the yolk-lobe. The yolk-lobe is the result of an 
unsymmetrical distribution of yolk or egg substance with refer- 
ence to the egg axis (pp. 9-11). 

3. In Crepidula plana the Kernplasma-Relation varies greatly 
in different blastomeres and at different stages, depending chiefly 
upon the length of the resting period (interkinesis). In cases 


84 EDWIN G. CONKLIN 


where nuclei and cells are measured at their maximum size it 
varies from 14.5 to 0.37; at mean size from 35.7 to 1.1; at minimum 
nuclear and cell size it varies from 285 to 29. In protoplasmic 
blastomeres, which contain no yolk, the Kernplasma-Relation 
varies from 14.5 to 8.7, when the nuclei are at their maximum size; 
and from 35.7 to 7, when the nuclei are at mean size. In Fulgur, 
at mean size, it varies from 127.7 to 3.6 (pp. 16-24). 

4. In different eggs, corresponding blastomeres have approxi- 
mately the same Kernplasma-Relation; but in different blasto- 
meres of the same egg or of different eggs the Kernplasma-Relation 
is neither a constant nor a self regulating ratio. It appears to 
be a result rather than a cause of the rate of cell division, and con- 
sequently a variable rather than a constant factor (pp. 24-25). 

5. In the tissue cells of adult Crepidulas there is no marked 
increase of cytoplasm over nucleus, as compared with the blasto- 
meres. The Kernplasma-Relation of various adult epithelial 
cells, not filled with metabolic products, varies from 28 to 7; in 
oécytes and ganglion cells it varies from 6 to 3 (pp. 25-28). 

6. The size of the nucleus is dependent upon at least three 
factors: (a) The initial quantity of chromatin (Boveri); (b) 
The volume of the cytoplasm; (c) The length of the resting period 
(p. 25). 

7. The inciting cause of cell division in Crepidula is not found 
solely in the limitations of the working sphere of the nucleus 
(Strasburger), nor in the doubling of the volume of the chromo- 
somes (Boveri), nor in a Kernplasma-Spannung (Hertwig), but 
rather in the coincidence of centrosomal, chromosomal and cyto- 
plasmic rhythms, which are probably connected with the rate and 
nature of metabolism in the cell (pp. 29-82). 

8. During the cleavage of the egg of Crepidula plana the volume 
of the cytoplasm more than doubles between the 1-cell and the 
24-cell stage the average growth for each division being about 
6 per cent; the yolk decreases in volume by nearly one-half and 
the entire egg is smaller at the 24-cell stage than at the 1-cell 
stage. This can only mean that the yolk contributes to the 
growth of cytoplasm during the cleavage period (pp. 32-36). 


CELL SIZE AND NUCLEAR SIZE 85 


9. The average nuclear growth during cleavage is not more 
than 5 per cent to 9 per cent for each division up to the 32-cell 
stage and it may fall as low as 0.3 per cent to 1 per cent for each 
division after that stage; and in every case it falls far short of a 
doubling, or increase of 100 per cent, for each division (pp. 36— 
44, 54, 55). 

10. Both nuclear sap and linin belong to the cytoplasm as well 
as to the nucleus. The chromatin is the most distinctive nuclear 
substance. All of these constituents are more abundant in large 
cells than in small ones. The mitotic spindle is of both nuclear 
and cytoplasmic origin and its size depends upon the volume of 
both nucleus and cytoplasm (pp. 44-47, 55). 

11. The average growth in volume of chromatin from the 2- 
cell to the 32-cell stage is about 8 per cent for each division period, 
being about the same as the growth of the nucleus as a whole 
(pp. 47-48, 55). 

12. The chromosomes become individually smaller as cleavage 
progresses, and in general small nuclei give rise to smaller chromo- 
somes than do large nuclei (pp. 48-51, 55). 

13. The size of the nucleoli (plasmasomes) depends upon the 
size of the nucleus and the length of the resting period; the larger 
the nucleus and the longer the resting period, the larger the plas- 
masomes become (pp. 51-53, 55-56). 

14. Centrosomes and spheres of large cells are larger than those 
of smaller ones (pp. 53, 56). 

15. The rate of growth of chromatin during the early cleavages 
of Crepidula (8 per cent for each division) harmonizing with the 
slight rate of increase of the oxidative power of the egg as de- 
termined by Warburg (p. 56). 

16. My observations do not support the view that senescence 
is due to a decrease (Minot), or an increase (Hertwig) of nuclear, 
as compared with protoplasmic material; nor that rejuvenescence 
is accomplished during cleavage by the great increase of nuclear 
material relative to the protoplasm. On the other hand senes- 
cence seems to be associated with a decrease, rejuvenescence with 
an increase of metabolism (Child). Anything which decreases 
the interchange between nucleus and cytoplasm, such as products 


86 EDWIN G. CONKLIN 


of differentiation and metabolism within the cell, or a dense nu- 
clear membrane, decreases metabolism and leads to senescence; 
anything which facilitates this interchange increases metabolism 
and leads to rejuvenescence. It is suggestive that in early devel- 
opment increased oxidation is associated with fertilization and 
mitosis (Loeb, Lyon, Warburg) (pp. 57-62). 


Part II. Experiments 


17. By centrifugal force the substance of the eggs and blasto- 
meres of Crepidula may be stratified into a zone of heavy yolk 
at one pole, a zone of lighter oil and water at the other pole, and 
a zone of clear cytoplasm between these two; and since these 
eggs orient but slightly if at all while being centrifuged, the axis 
of centrifuging and of stratification may form any angle with the 
egg axis. In the early development of Crepidula the volume of 
yolk is much greater than the volume of cytoplasm and conse- 
quently the latter may be displaced to any side of the center of 
the egg or blastomere (p. 64). 

18. On the other hand the mitotic figure, after the prophase, 
can be moved only with great difficulty, and owing to this fact 
the substances of a cell can be distributed in very atypical man- 
ner with respect to the poles of the spindle and the resulting daugh- 
ter cells. In this way all the yolk present in a dividing cell may 
be thrown into one of the daughter cells, and almost all of the 
cytoplasm into the other (p. 64). 

19. These experiments show that the spindle is a specific 
structure and not merely a dynamic expression of lines of force. 
It remains in position and functions normally when the substance 
in which it usually lies is completely replaced by other substance. 
The spindle fibers are denser than the general cytoplasm and 
may be stretched, shortened or bent by pressure (p. 65). 

20. If centrifuging occurs during the second maturation divi- 
sion, when the poles of the egg are clearly marked, the yolk may 
be driven to the animal pole and the cytoplasm to the vegetal 
pole, the spindle may be much elongated and a giant polar body 
may be formed,(fig.11). In such cases the sperm nucleus, which 


CELL SIZE AND NUCLEAR SIZE 87 


enters the egg near the vegetal pole, lies in a cytoplasmic field, 
the egg nucleus in a yolk field, and the former grows more rapidly 
than the latter, thus reversing the usual size relations of the germ 
nuclei. The relative size of the germ nuclei is dependent upon 
the volume of the cytoplasm in which they lie as well as upon the 
length of time that the sperm nucleus has been in the egg (pp. 
67, 75). 

21. If centrifuging occurs during the cleavage almost all the 
yolk present may go into one daughter cell, almost all the cyto- 
plasm into the other (figs. 16, 19). Under these circumstances 
the subsequent growth of the daughter nuclei is proportional to 
the volume of the cytoplasm of the middle zone in which they 
lie. Neither the yolk nor the substances of the lighter zone con- 
tribute directly to the growth of the nucleus (pp. 75-76). 

22. The size of spindle, centrosome, and sphere in any cell is 
not definitely fixed, but may be modified by altering the quantity 
of cytoplasm; the larger the quantity of cytoplasm in a cell, the 
larger are all the structures named (p. 77). 

23. The rhythm of division may be modified, but only to a 
slight extent, by altering the quantity of cytoplasm in a cell. 
In general, cells rich in cytoplasm divide a little earlier than those 
poor in this substance; but though the quantity of cytoplasm in 
a cell and the size of its nucleus may be greatly changed by cen- 
trifuging, the rhythm of cleavage is but slightly changed (p. 77). 

24. When the daughter chromosomes at one pole of a spindle 
are left in a cell composed almost entirely of yolk, they do not 
form a vesicular nucleus until yolk has been dissolved and a 
certain amount of cytoplasm has been formed around the chromo- 
somes. It is evident that something, perhaps an enzyme, is given 
off from the chromosomes or chromatin, which leads to the trans- 
formation of yolk into cytoplasm; this cytoplasm is in turn taken 
up by the chromosomes and ultimately contributes to the growth 
of the chromatin, (pp. 77-78). 

25. The typical size, position and constitution of blastomeres, 
and consequently the type of cleavage, do not depend upon exter- 
nal or internal pressure, but upon a definite polarity, symmetry 
and movement of the cell contents, and upon reduced surface 


88 EDWIN G. CONKLIN 


tension at the poles of the cell. Therefore, the causes of equal 
or unequal, differential or non-differential divisions are intrinsic 
rather than extrinsic (pp. 78-81). 

26. Whenever the size, constitution or number of blastomeres 
is changed from the typical condition, subsequent cleavages come 
back to the normal form so far as this is possible. This regula- 
tion in cleavage is connected with a persistent polarity of the cell, 
which is not changed by centrifuging, and which manifests it- 
self in a definite cell axis passing through nucleus and centrosome, 
in typical movements and localizations of cell contents, and in 
reduced tension of cell membrane at the poles of the cell (pp. 
81-83). 

LITERATURE CITED 


Battzer, F. 1908 Die Chromosomen yon Strongylocentrotus lividus und Echi- 
nus microtuberculatus. Arch. f. Zellforschung, Bd. 2. 

Boveri, Tu. 1902 Ueber mehrpolige Mitosen als Mittel zur Analyse des Zell- 
kerns. Verh. d. Phys. med. Gess. Wirzburg., Bd. 35. 
1904 Ergebnisse iiber die Konstitution der chromatischen Substanz 
des Zellkerns. Jena. 
1905 Zellenstudien V. Ueber die Abhangigkeit der Kerngrésse und 
Zellenzahl der Ausgangszellen. Jena. 
1910 Ueber die Teilung centrifugierter Eier von Ascaris megalocephala. 
Arch. f. Entw. Mech., Bd. 30. 

Conxkuin, E. G. 1893 The fertilization of the ovum. Woods Hole Lectures. 
Ginn and Co., Boston. 
1897 The embryology of Crepidula. Jour. Morph., vol. 13. 
1899 Protoplasmic movement as a factor in differentiation. Woods 
Hole Lectures. 
1902 Karyokinesis and cytokinesis in the maturation, fertilization 
and cleavage of Crepidula. Jour. Acad. Nat. Sciences of Philadelphia, 
vol. 12. 
1905 The organization and cell-lineage of the ascidian egg. Idem, 
vol. 13. 
1910 The effects of centrifugal force on the organization and develop- 
ment of the eggs of fresh water pulmonates. Jour. Exp. Zool., vol. 9. 

* Cuitp, C. M. 1911 A study of senescence and rejuvenescence based on experi- 

ments with Planaria dorotocephala. Arch. f. Entw. Mech., Bd. 31. 

ErpMANN, Rh. 1908 Experimentelle Untersuchung der Massenverhiltnisse 
von Plasma, Kernund Chromosomen in dem sich entwickelnden Seeige- 
lei. Arch. f. Zellforschung, Bd. 2. 


CELL SIZE AND NUCLEAR SIZE 89 


*. 
EycitesHyMer, A. 1904 The cytoplasmic and nuclear changes in the striated 
muscle cell of Necturus. Am. Jour. Anat., vol. 3. 
GarpineER, E.G. 1898 The growth of the ovum, etc., in Polychaerus. Jour. 
Morph., vol. 15. 
GerassimorF, J. 1900 Ueber die Lage und die Function des Zellkerns. Bull. 
Soc. imp. Natur. Moscou, 1899. 
1901 Ueber den Einfluss des Kerns auf das Wachstums der Zelle. Idem. 
1902 Die Abhingigkeit der Grosse der Zelle von Menge ihrer Kern- 
masse. Zeitsch. f. allgem. Physiol., I. 
GoptewskI, H. 1908 Plasma und Kernsubstanz in der normalen und der durch 
aussere Faktoren veriinderten Entwicklung der Echiniden. Arch. f. 
Entw. Mech., Bd. 26. 
1910 Plasma und’ Kernsubstanz bei der Regeneration der Amphibien. 
Arch. f. Entw. Mech., Bd. 30. 
Gurwitscu, A. 1908 Ueber Primissen und anstossgebende Faktoren der Fur- 
chung und Zellvermehrung. Arch. f. Zellforschung, Bd. 2. 
Hertwic, R. 1889 Ueber die Kernkonjugation der Infusorien. Abh. Bayer. 
Akad. Wiss., I] KI., Bd. 17. 
1903 Ueber Korrelation von Zell- und Kerngrésse und ihre Bedeutung 
fiir die geschlechtliche Differenzierung und die Teilung der Zelle. 
Biol. Centralb., Bd. 22. 
1908 Ueber neue Probleme der Zellenlehre. Arch. f. Zellforschung, 
Ba: 
Hoper, C. F. 1892 A microscopical study of the changes due to functional 
activity of nerve cells. Jour. Morph., vol. 7. 
Hogur, Mary J. 1910 Ueber die Wirkung der Centrifugalkraft auf die Eier 
von Ascaris megalocephala. Arch. f. Entw. Mech. Bd. 29. 
Liniiz, F. R. 1901 The organization of the egg of Unio, based on a study of its 
maturation, fertilization and cleavage. Jour. Morph., vol. 17. 
+1902 Differentiation without cleavage in the egg of the annelid Chae- 
topterus. Arch. f. Entw.-Mech., Bd. 14. 
1906 Observations and experiments concerning the elementary phe- 
nomena of embryonic development in Chaetopterus. Jour. Exp. Zool., 
vol. 3. 
1909a Polarity and bilaterality of the annelid egg. Experiments with 
centrifugal force. Biol. Bull., vol. 16. 
1909b Karyokinetic figures of centrifuged eggs. An experimental 
test of the center of force hypothesis. Biol. Bull., vol. 17. 
Lituir, R.S. 1902 On the oxidative properties of the cell nucleus. Amer. Jour. 
Physiol., vol. 7. 
1909 The general biological significance of changes in the permeability 
of the surface layer or plasma membrane of living cells. Biol. Bul., 
vol. 17. 
1910 Physiology of cell division, II. Amer. Jour. Physiol., vol. 26. 


90 EDWIN G. CONKLIN 


Lorg, J. 1899 Warum ist die Regeneration kernléser Protoplasmastiicke un- 
moglich oder erschwert? Arch. f. Entw. Mech., Bd. 8. 

1909 Die chemische Entwicklungserregung des tierischen fies. Ber- 
lin, 1909. 

1910 Ueber den autokatalytischen Charakter der Kernsynthese bei 
der Entwickelung. Biolog. Centralb., Bd. 30. 

Lyon, E. P. 1904 Rhythms of susceptibility and of carbon dioxide production 
in cleavage. Am. Jour. Physiol., vol. 11. 

1907 Results of centrifugalizing eggs. Arch. f. Entw. Mech., Bd. 28. 

Masina, HE. 1910 Ueber das Verhalten Neucleinsiure bei der Furchung Seeig- 
leies. Hoppe-Seylers Zeitschrift. Bd., 67. Review by Godlewski, Arch. 
f. Entw. Mech., Bd. 31. 

Minor, GC. 8. 1890 On certain phenomena of growing old. Proc. Amer. Ass’n 
Adv. Sci., vol. 29. 

1895 Ueber die Vererbung und die Verjungung. Biol. Central., Bd. 15. 
1908 Age, growth and death. Putnams, New York. 

Montcomery, T. H. 1910 On the dimegalous sperm and chromosomal variation 
of Huschistus, with reference to chromosomal continuity. Arch. f. 
Zellforschung, Bd. 5. 

Moraan, T. H. 1910 Experiments bearing on the nature of the karyokinetie 
figure. Proc. Soc. Exp. Biol. and Medicine, vol. 7. 

Perer, K. 1906 Der Grad der Beschleunigung tierischen Entwicklung durch 
erhdhte Temperatur. Arch. f. Entw. Mech., Bd. 20. 

Pororr, M. 1908 Experimentelle cytologische Studien. Arch. f. Zellforschung, 
Bd. 1. 

SHackett, L. F. 1911 Phosphorus metabolism during early cleavage of the 
echinoderm egg. Science, vol. 34, no. 878. 

SrTrRAsBuRGER, E. 1893 Ueber die Wirkungssphire der Kerne und die Zell- 
grosse. Histolog. Beitrige, Bd. 5. 

Verworn, M. 1891 Die physiologische Bedeutung des Zellkernes. Arch. f. 
d. ges. Physiol., Bd. 51. 

WarTasez, 8. 1893 On the nature of cull organization. Woods Hole Lectures. 

Witson, E. B. 1904 Experimental studies on germinal localization. Jour. 
Exp. Zool., vol. 1. 


DESCRIPTION OF FIGURES 


All figures (with the exception of figs. 9 and 10) represent entire eggs of Crepid- 
ula plana, fixed, stained, and mounted on slides. They were drawn with the aid 
of a camera lucida under Zeiss Apochromat 3 mm., Ocular 4, and represent a mag- 
nification of 333 diameters. In the centrifuged eggs, the axis of centrifuging is, 
in many cases, indicated by an arrow, the head of the arrow marking the distal 
(heavy) pole and the tail of the arrow the central (light) pole. In figs. 12 to 19, 
and 29 to 37 the first cleavage is in the long axis of the page, the second cleavage 
(figs. 18 and 19) is at right angles to this. In figs. 21 to 28 the first cleavage runs 
across the page, the second, lengthwise of it. 


CELL SIZE AND NUCLEAR SIZE 91 


Figs. 1-6 Successive stages in the development of the egg of C. plana, showing 
the maximum sizes of the nuclei of the macromeres. Fig. 1, 4-cell, just before 
third cleavage; fig. 2, 8-cell, just before fourth cleavage; fig. 3, 16-cell, just before 
fifth cleavage; fig. 4, 24-cell, just before sixth cleavage in macromere 3D; fig. 5. 
42-cell, just before sixth cleavage in macromeres 3A-3C; fig. 6, Gastrula, just 
before seventh cleavage of the macromeres. 


92 EDWIN G. CONKLIN 


ny 
é 


. ( SSA gt) al 
: > cf : VIN ECan 
i 2 
aT » = 
(% re o Ne 


9 LO 


Fig. 7 2-cell stage of C. plana. The nuclei just before the second cleavage are 
24u in diameter. 

Fig. 8 12-cell stage of C. plana. The nuclei in the first quartet of micromeres, 
la-ld, three of which are dividing, are 14u in diameter at their maximum size. 

Fig.9 Chromosomes from four different spindles of the second cleavage, all 
in the metaphase and all magnified 2000 diameters. 

Fig. 10 Chromosomes from four different spindles of the cells la-1d, all in the 
metaphase and all magnified 2000 diameters. 

Fig. 11 Egg centrifuged ten minutes after formation of first polar body and 
during formation of second; fixed three hours after centrifuging. Telophase of 
second maturation division; indication of formation of enormous second polar 
body. The size of nuclei is dependent upon quantity of cytoplasm in which they 
lie. 

Fig. 12. Centrifuged fifteen minutes in gum arabic, fixed three hours later. 
Evidently centrifuged during first cleavage; almost all of the cytoplasm is in the 
smaller cell. The size of the nuclei is proportional to the quantity of cytoplasm. 


CELL SIZE AND NUCLEAR SIZE 93 


Fig. 13 Centrifuged five hours (2000 revolutions per minute) during the first 
cleavage; fixed at once; structure similar to preceding. 

Fig. 14 From the same experiment as the preceding. Size of nuclei is propor- 
tional to the quantity of clear (granular) cytoplasm; yolk and oily or watery con- 
stituents of the cytoplasm do not influence nuclear size. 

Fig. 15 From the same experiment as the preceding, and showing similar 
results. 

Fig. 16 Centrifuged thirty minutes; fixed twenty hours after centrifuging. 
Egg has not developed. Enormous difference in the size of sister nuclei. 


94 EDWIN G. CONKLIN 


Fig. 17 Centrifuged four hours (2000 revolutions per minute); fixed six hours 
after. Evidently centrifuged during first cleavage. The cleavage plane does not 
pass through the polar axis. The spindles are proportional to the size of nuclei 
from which they were formed, and to the volume of cytoplasm in which they lie. 
They are not parallel to the plane of the first cleavage, which is here out of its 
normal position. 

Fig. 18 Centrifuged fifteen minutes in gum arabic; fixed three hours after. 
Evidently centrifuged during first cleavage. The second cleavage appeared 
earlier in the more protoplasmic cells (A and B), than in the others. 

Fig. 19 Centrifuged thirty minutes; fixed six hours later. Evidently centri- 
fuged during the first cleavage. The size of the nuclei is plainly dependent upon 
the volume of the cytoplasm in which they le. 

Tig. 20 Centrifuged four hours, (2000 revolutions per minute); fixed at once. 
Evidently centrifuged during the second cleavage; the daughter nuclei are pro- 
portional in size to the volume of cytoplasm in which they lie. 


CELL SIZE AND NUCLEAR SIZE 95 


Fig. 21 Centrifuged thirty minutes; fixed at once. Centrifuged during the 
second cleavage, which was thus made very unequal, two of the macromeres 
(A and D) containing all the yolk and the other two (B and C) being small and 
purely protoplasmic. 

Fig. 22 Centrifuged thirty minutes; fixed twenty-one hours later. The second 
cleavage was suppressed. Two spindles for the third cleavage are present in each 
cell, but the cell body shows no signs of division. 

Fig. 23 Centrifuged thirty minutes, during the second cleavage; fixed six hours 
later; two of the macromeres are small and protoplasmic; tetrasters are present in 
the other two. 

Fig. 24 Same slide as preceding. All of the macromeres have given rise to 
normal micromeres of similar size, although two of the macromeres are small and 
purely protoplasmic while the other two are large and contain much yolk and little 
cytoplasm. 


96 EDWIN G. CONKLIN 


Fig. 25 Same slide as preceding. The minute protoplasmic ‘macromeres’ 
(A and D) have divided equally into the macromeres 1A and 1D and the micro- 
meres Ja and id. The other macromeres (B and C) have given rise to micromeres 
somewhat larger than usual. 

Fig. 26 Centrifuged for thirty minutes during the second cleavage; fixed twelve 
hours later; the nuclear divisions of the second cleavage were completed, but the 
cell divisions were suppressed. Each of these two binucleate macromeres has 
given rise to two first, and two second quartet cells, just as if four macromeres 
were present, and each of these micromeres has subdivided in approximately nor- 
mal manner and is uni-nuclear. 

Fig. 27 Centrifuged for thirty minutes in 2-cell stage; fixed six hours later. 
Micromeres formed from protoplasmic macromeres are of the same size as those 
formed from large yolk macromeres. 

Fig. 28 Same as preceding. The regulation in the formation of micromeres 
is complete. 


Fig. 29 Centrifuged four hours (2000 revolutions per minute); fixed at once. 
The yolk was thrown out into lobes, one of which has been detached; the smaller 
nuclei are in the smaller cells. 

Fig. 30 Centrifuged ten minutes in gum arabic during first cleavage; fixed four 
hours later. Asters and spindles are proportional to the volume of the cytoplasm. 

Fig. 31 Centrifuged four hours during the first cleavage; fixed six hours later. 
Most of the cytoplasm is in the smaller macromeres and these have divided earlier 
than the larger ones. At least one-half of the cytoplasm in the larger macromeres 
goes into the micromeres. The first cleavage is not strictly meridional and the 
spiral form of division is lost. (For explanation of figs. 32 and 33, see p. 98). 

THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 


97 


98 EDWIN G. CONKLIN 


Fig. 32 From the same slide as the preceding. Though the macromeres differ 
in size and protoplasmic content, the micromeres are all of the same size. 

Fig. 33 Centrifuged ten minutes in gum arabic; fixed four hours later. The 
protoplasmic macromeres are dividing earlier than the others. The size of the 
micromeres does not depend upon the quantity of cytoplasm in the macromeres 
from which they came. 

Fig. 34 Centrifuged two and one-half hours; fixed twenty-one hours later. The 
size of the micromeres is almost irrespective of the size of the macromeres; also 
it is nearly independent of the amount of cytoplasm in the macromeres. 

Fig. 35 Centrifuged thirty minutes during the first cleavage; fixed twelve hours 
later. The micromeres from the protoplasmic macromeres are but little larger 
than those from the yolk cells. 

Fig. 36 From the same slide as the preceding, showing essentially the same 
conditions. 

Fig. 37 From the same slide as the preceding. The cell 4c forms at the same 
time as 4d, though in normal eggs it is formed much later. 


STUDIES ON THE PHYSIOLOGY OF REPRODUCTION 
IN THE DOMESTIC FOWL 


V. DATA REGARDING THE PHYSIOLOGY OF THE OVIDUCT! 
RAYMOND PEARL anp MAYNIE R. CURTIS 
FOUR FIGURES 
INTRODUCTION 


The oviduct of a laying hen is divided into five main parts, 
readily distinguishable by gross observation. Beginning at the 
cranial end of the organ these parts, in order, are: (a) the 
infundibulum, or funnel, (b) the albumen secreting portion, (c) 
the isthmus, (d) the uterus or ‘shell gland’ and (e) the vagina. 
Each of these parts is generally supposed (teste the existing liter- 
ature) to play a particular and exclusive réle in the formation 
of the protective and nutritive envelopes which surround the 
yolk in the complete egg as laid. Thus the funnel grasps the 
yolk at the time of ovulation; the glands of the albumen region 
secrete the different sorts of albumen (thick and thin) found in 
the egg; the shell membranes are secreted in the isthmus; and 
finally the glands of the uterine wall secrete the calcareous shell. 
This is in brief the classical picture of the physiology of the oviduct. 

The gross anatomical appearance and relation of the several 
parts of the oviduct of the fowl are shown in fig. 1. 


‘Papers from the Biological Laboratory of the Maine Experiment Station, 
No. 33. The previous papers in this series of ‘Studies on the Physiclogy of 
Reproduction in the Domestie Fowl” are: 

1. Regulation in themorphogenetic activity of the oviduct. Jour. Exp. Zodl., 
vol. 6, pp. 839-359, 1909. 

ir. Data on the inheritance of fecundity obtained from the records of egg pro- 
duction of the daughters of ‘200-egg’ hens. Maine Agricultural Experiment 
Station, Annual Report for 1909, pp. 49-S4. 

ur. A ease of incomplete hermaphroditism. Biological Bulletin, vol. 17, pp. 
271-286, 1909. 

tv. Data on certain factors influencing the fertility and hatching of eggs. 
Maine Agricultural Hxperiment Station, Annual Report for 1909, pp. 105-164. 

99 


100 RAYMOND PEARL AND MAYNIE R. CURTIS 


Fig. 1 Photograph of a hen’s oviduct which has been removed, slit longitudi 
nally throughout its length and opened out flat in order to show the gross anatomy. 
In order to get the whole duct on the photographic plate it was necessary to tran- 
sect it at about the middle. A, the infundibulum. Note muscle fibers in wall 
and absence of any extensive gland development. £, albumen secreting portion; 
note heavy glandular development. The albumen portion ends and the isthmus 
begins at z. The line of demarcation is very distinct in the freshly prepared ovi- 
duct. C, the isthmus. D, the uterus or shell gland. £, the vagina; about one- 
third natural size. 


PHYSIOLOGY OF THE OVIDUCT 101 


For some years past experiments and observations have been 
systematically carried on in this laboratory with the object of 
acquiring: a more extended and precise knowledge of the physi- 
ology of the hen’s oviduct than is to be gained from the literature. 
It is the purpose of this paper to present a certain part of the 
results obtained bearing upon the physiology of two of the lower 
(caudal) morphological divisions of the duct, namely, the isthmus 
and the uterus. Our results indicate that these portions of the 
oviduct perform certain functions which have not hitherto been 
observed or described. 

So far as we are able to learn from the existing literature the 
opinion has been held by all who have worked upon the subject 
that the particular functional activity of each portion of the 
oviduct (as above described) is limited to that portion. Thus 
it is commonly held that when an egg in its passage down the 
oviduct leaves the albumen portion it has all the albumen it will 
ever have; when it leaves the isthmus it has all its shell mem- 
branes; and when it leaves the uterus all its shell. On this pre- 
vailing view there are in the albumen portion only albumen 
secreting glands; in the isthmus only membrane secreting glands; 
and in the uterus only shell secreting glands. We were first led 
to doubt the entire adequacy of this assumption by the observa- 
tion, frequently made in connection with routine autopsy work, 
that eggs in the isthmus with completely formed shell membranes, 
and eggs in the uterus bearing in addition to the complete shell 
membranes a partially formed shell, weighed considerably less 
than the normal average for laid Barred Plymouth Rock eggs. 
This observation led to an inquiry as to whether (a) this apparent 
lower weight of presumably completed, but not laid eggs, as 
compared with those which had been laid, was a real phenomenon 
of general occurrence, and (b) if so, to what it was due. Does 
the egg increase in weight after the formation of shell membranes 
and shell merely by the absorption of water, or by the actual 
addition of new albumen? These are the problems with which 
the present paper has to do. 

We may now turn to the consideration of the observational 
and experimental data. 


102 RAYMOND PEARL AND MAYNIE R. CURTIS 


THE DISTRIBUTION OF THE DIFFERENT KINDS OF ALBUMEN IN 
THE EGG 

In the normal egg of the hen there are certainly three and 
possibly four different albumen layers which can easily be dis- 
tinguished on the basis of physical consistency. These are: 
(A) the chalaziferous layer. This is a thin layer of very dense 
albuminous material which lies immediately outside the true 
yolk membrane. It is continuous at the poles of the yolk with 
the chalazae, and is undoubtedly found in connection with those 
structures. It is so thin a layer that it might well be, and often 
has been, taken for the yolk membrane. (B) The inner layer 
of fluid (thin) albumen. This layer is only a few millimeters in 
thickness and there is some doubt as to its existence as a sep- 
arate, distinct layer. (C) The dense albumen. This is the layer 
which makes up the bulk of the ‘white’ of the egg. It is com- 
posed of a mass of dense, closely interlaced albumen fibres, with 
some thin fluid albumen between the meshes of the fibrous net- 
work. The dense albumen as a whole will not flow readily, but 
holds itself together in a flattened mass if poured out upon a 
plate. (D) The outer layer of fluid albumen. This is the prin- 
ciple layer of thin albumen, which makes up the fluid part of 
the ‘white’ observed when an egg is broken. 

Three of these layers, A, C, and D are readily demonstrable 
and there can be no question whatever as to their existence. 
Regarding the existence of B as a separate and distinct layer 
there is more doubt. Gadow? definitely asserts the existence of 
such a layer in the following words: ‘‘Dicht auf der Dotterhaut 
befindet sich eine diinne Lage des fliissigen Eiweisses.”” It is 
possible that what has been taken by previous observers to form 
this layer B is only a little thin albumen squeezed out of the 
meshes of the dense layer (C) when the egg is broken. 

Let us now consider the distribution of the different sorts of 
albumen in eggs at different stages in their passage down the 
oviduct. The following extracts from autopsy protocols are to 
the point here. 


° Gadow, H., Vogel (Anatomischer Theil); in Bronn’s Klassen und Ordnungen 
des Thier-Reichs. Leipzig, 1891, p. 869. 


PHYSIOLOGY OF THE OVIDUCT 103 


Autopsy No. 370. Hen No. 952. March 19, 1910 


Egg found in the albumen portion of the oviduct 11 cm. in 
front of the cranial end of the isthmus. This egg consisted of 
a yolk surrounded by thick albumen (layer C) but with no trace 
whatever of the albumen layer D. Not yet having entered the 
isthmus the egg lacked a shell membrane. 


Autopsy No. 332. Hen No. 420. December 22, 1909 


This case was similar to that just cited. Here an egg was in 
the albumen portion of the duct with its caudal end 6 em. in 
front of the cranial end of the isthmus. The egg consisted of 
yolk surrounded by dense albumen (layer C). There was no 
trace of the thin albumen (layer D) to be observed. The egg had 
no shell membrane. 


Autopsy No. 366. Hen No. 276. March 18, 1910 


Egg in albumen portion of oviduct with its caudal end 4 cm- 
in front of the cranial end of the isthmus. This egg had no shell 
membrane. The yolk was surrounded by thick albumen (layer 
C). The egg bore no trace of the thin albumen (layer D), even 
though it was only this short distance (4 em.) from the point 
where the ‘albumen secreting’ portion of the duct was finally 
to be left. 


Autopsy No. 301. Hen No. E39. July 14, 1909 


When this bird was killed an egg was found at the lower end of 
the albumen portion of the oviduct just about to enter the isthmus. 
Not yet having entered the isthmus the egg had no shell membrane 
upon it. It consisted merely of a yolk surrounded by albumen. 
The outermost layer of this albumen was dense and corresponded 
to layer C described above. There was no trace of thin albumen 
(layer D) on this egg although it was just on the point of leaving 
the so-called albumen region of the oviduct. 


104. RAYMOND PEARL AND MAYNIE R. CURTIS 
Autopsy No. 369. Hen No. 1154. March 19, 1910 


Egg in the lower end of the albumen portion of the oviduct 
just at the point of entering the isthmus. This egg had no mem- 
brane. The yolk was surrounded by albumen layers A, B, and 
C. No trace of the outer thin albumen (layer D) was to be found. 

All these cases agree in showing that the egg does not receive the 
outer layer of thin fluid albumen (layer D) during its sojourn in 
the so-called albwmen secreting portion of the oviduct. While but 
five specific autopsy records are cited here it is only fair to say 
that this result is confirmed by all our experience with eggs in 
the albumen secreting portion of the oviduct. This experience 
covers many more than the five cases given here. These cases are 
chosen as particularly significant, however, because in them we 
have definite quantitative records of the exact location of the egg 
in the albumen portion. The successive autopsy records show 
that beginning with an egg 11 cm. away in front of the isthmus 
and going downwards in the duct until the actual boundary of 
the isthmus is reached, there is no qualitative change in the albu- 
men secretion. Whatever albumen is added to the egg imme- 
diately prior to the formation of the shell membrane, is of the 
dense fibrous variety (layer C), so far as direct observation indi- 
cates. The fact that there is no thin albumen on the egg when 
it enters the isthmus is shown in fig. 2. In this figure A shows an 
egg of hen No. 8018 which was removed from the upper part of 
the isthmus. The thin membrane which had been formed was 
removed; the egg placed in a Petri dish and photographed. For 
comparison a normal laid egg from hen 8018 was broken into a 
Petri dish in the same way. Its photograph is shown in fig. 2, B. 
It is at once apparent that there is a great difference between the 
two eggs in respect to amount and consistency of albumen. In 
the egg which had just left the albumen portion of the oviduct 
(ege A) the albumen is of firm consistency and retains its shape, 
forming a compact mass about the yolk. In the laid egg (egg B) 
the albumen is much thinner, and does not hold its shape, but 
flows out over the bottom of the dish. 


PHYSIOLOGY OF THE OVIDUCT 105 


8018, with 


p 


bird, 


B 
isthmus of the oviduct of hen No. 
For further explanation see text. 


aid egg of the same 


al size. 


an egg taken from the 


Showing A 


106 RAYMOND PEARL AND MAYNIE R. CURTIS 


Crucial evidence is here afforded by those cases occasionally 
to be observed, where the egg is Just entering the isthmus, and 
has one end in the albumen portion of the duct and the other end 
inthe isthmus. It was first pointed out by Coste? that the forma- 
tion of the shell membrane at the upper end of the isthmus is a 
discrete process. That is, as the end of the egg advances from 
the albumen portion into the isthmus, membrane is deposited 
upon it. The membrane is complete over the whole egg only 
after the egg has entirely passed into the isthmus. This account 
of membrane deposition we have confirmed by direct observation 
in this laboratory. Now in cases where one-half of the egg lies 
within the isthmus and bears a membrane while the other half 
is in the albumen portion and has no membrane it can plainly 
be seen that the shell membrane is deposited directly on the outer 
surface of the thick albumen (layer C) and that no trace of the thin 
albumen (layer D) is present at the time the membrane is formed. 

It might be contended that the thin albumen which is to form 
layer D is really present at the time the membrane is deposited, 
but that instead of forming a separate outer layer it is held by 
adhesion or otherwise within the meshes of the fibrous network 
of the dense albumen of layer C. On this view it might be sup- 
posed that this more fluid albumen passes out of the network to 
form a definite and separate layer at some time after the mem- 
brane is laid down. This contention, however, cannot be correct, 
because, as will be demonstrated in the next section of the paper, 
the egg does not have its full complement of albumen by weight 
at the time when the shell membrane is formed. The fluid 
albumen of layer D should weigh just as much, whether in the 
interstices of a fibrous meshwork, or forming a separate layer. 
Yet the facts show that after a thin albumen (layer D) has been 
visibly formed the egg contains by weight about 50 per cent more 
albumen than it did before this layer was visibly formed. 


8 Coste, M., Histoire du développement des corps organisés, tome 1, p. 295, 1847. 


PHYSIOLOGY OF THE OVIDUCT 107 


THE PROPORTIONATE WEIGHT OF YOLK AND ALBUMEN IN EGGS 
IN DIFFERENT STAGES OF FORMATION 


Having learned by direct observation, as set forth in the 
preceding section that the egg as it enters the isthmus does not 
visibly bear the outer layer of thin albumen, the next step in 
the analysis is to determine whether the amount of albumen (by 
weight) in the egg definitely increases during its sojourn in the 
isthmus and uterus, and if so to what extent. In order to do this 
it is necessary to take eggs at successive intervals after they have 
entered the isthmus, separate and weigh yolk and albumen each 
by itself, and then compare the weights so obtained with the 
weights of yolk and albumen in normal, completely formed and 
laid eggs produced by the same individual birds. Experiments 
of this kind we have carried out with the results described in 
this section of the paper. 

It should be said that the technique followed in the separating 
and weighing of the eggs to furnish these data is that described 
by one of the authors in another place.‘ 

Table 1 gives data regarding the weight of yolk and albumen 
in eggs which have completed their passage through the albumen 
secreting portion of the oviduct, and have advanced varying 
distances into the isthmus and shell gland. The data here given 
are extracted from the more detailed table exhibited in the Appen- 
dix of this paper. 

The plan of table 1 is to compare the weights of the parts of 
a series of eggs taken from different levels of the oviduct with the 
weights of the same parts in normal laid eggs of the same birds. 
Owing to the considerable individual variability in the weights 
of eggs it is only by such comparisons as this that reliable results 
may be reached. To determine the means for the normal laid 
egg varying numbers of eggs were used in different cases. In 
one instance (item 3) only one laid egg was available for compari- 
son. In all other cases the mean of two or more complete nor- 
mal, laid eggs are used. It will be noted that in somecases 


‘Curtis, M. R., Annual Report Maine Agricultural Experiment Station, 1911, 
pp. 93-112. 


108 RAYMOND PEARL AND MAYNIE R. CURTIS 


(items 3, 4, 6, 9, and 15) the weights are given only to one decimal 
place (tenths of a gram). These were eggs studied in the early 
stage of the investigation, and mostly are cases in which the data 
were taken in connection with other studies in progress in the 
laboratory, for which finer weighing was not essential. These 
cases are to be regarded as giving a much rougher sort of data 
than the others tabled, where the weighings are accurate to 
hundredths of a gram. In no instance, however, does one of 
these ‘rough’ eases stand alone. That is, there are one or more 
eges for which finer weighing are tabled from each of the levels 
of the oviduct wherefrom a roughly weighed egg was taken. 
These ‘rough’ cases then serve merely to confirm evidence ob- 
tained from more precise weighing. All differences in the table 
are given the + sign when the oviduct egg or its part is greater 
than the laid egg or its part. The differences are taken — when 
oviduct egg or its part is smaller. The last column of the table 
gives the percentage which the weight of albumen in the oviduct 
egg at the specified level is of the mean total weight of albumen 
in the normal laid egg of the same bird. This last column then 
shows directly what proportion of the total albumen which the 
egg is to have has been laid down at each specified level of the 
oviduct. 

From table 1 the following points are to be noted: 

1. When the egg leaves the albumen portion of the oviduct it 
weighs roughly only about half as much as it does when it is 
laid. Nearly all of this difference is in the albumen. Thus these 
weighings fully confirm the conclusion reached from direct exam- 
ination of the eggs, as described in the preceding section. The 
evidence thus far presented shows that the egg gets all of its thin 
albumen (layer D), which constitutes nearly 60 per cent by weight 
of the total albumen, only after it has left the supposedly only 
albumen secreting portion of the oviduct, and has acquired a 
shell membrane, and the shell is in process of formation. The 
fact that the egg increases considerably in size after it enters the 
isthmus is obvious from simple visual comparison of egg from this 
region of the oviduct with normal laid eggs of the same bird even 
though no weights whatever are taken. 


PHYSIOLOGY OF THE OVIDUCT 109 
‘ 
TABLE 1 
Data showing the increase in absolute and relative weight of albumen after the egg 
has passed through the so-called albumen secreting part of the oviduct 


a | z 
S ag = 
ZZ 

a 5<2 B aS Bisa ae 
LOCATION OF EGG IN OVI{DUCT ES pes , eriind 33 | 2S 

A hes he mE * Bb 

= offs on oR x | °a 

a me mS a) ae aN 

3 Bisa Bue a< Ss ae 

& z z 2 = 2 

| grams grams | grams grams 


1. At caudal end of albu- 
men portion. No mem- 
brane (Hen8009)...... 27.20 0 15.38 11.82 76.9 | 34.2 
Mean of the four previ- 
ously laid eggs of same 
INES TURP aytet oiFs sous he ys ag 57.57 6.23 16.7! 34.55 205.8 


WMifference!. . <2 22) sn)- —30.37  -—6.23  —1.41 —22.73 | —128.9 
2. At caudal end of albu- 

men portion. No mem- 

brane (Hen 8005)........ 29.53 0 15.87 13.66 86.1} 43.6 
Mean of the nine pre- 

viously laid eggs of same 


IGM, oo o68d6 eoenapaepaee |e ett | SB Iak) 15.67 31.34) 200.0 | 
_ Difference RCo Ss Cob ren —23.28 —5.79 +0.20 —17.68 | —113.9 | 


3. Just entering isthmus, — : 
little cap of membrane 
on caudal tip. (Hen 


OB on mre ravers teid syatarare: Aaah s 29.5 0 16.0 13.5 84.4) 43.5 
Mean of two previously 

laid eggs of samehen... 56.25 9.0 | 16.25 31.0 190.8 | 

Difference........... eg | eter O00 ee 0-201) —17ep0 ||) —10b |e 
4, Entering isthmus. Cov- 

ered with membrane ex- 

cept for a little of crani- 

al tip. (Hen 266F).... 31.0 14.5 16.5 113.8 53.2 
Normal egg laid by hen 

RETO GEN 2 SBopmaOosenene 51.0 6.0 14.0 31.0 221.4 

TOURELEN CON. 6s ola bess —20.0 0.0) jo 4-5) 1076 ‘ 
5. Entering isthmus. 

Covered with membrane 


exceptfor alittle of crani- 

al tip. (Hen 8027)......| 32.44 0.24 15.69 16.51 105.2} 51.5 
Mean of the four pre- 

viously laid eggs of same 

GIANT 2S. k acces en 54.69 5.76 16.87 32.07 190.2 


= = —— 


Difference..............| —22.25| —5.52 —1.18 | —15.56 —85.0 


110 RAYMOND PEARL AND MAYNIE R. CURTIS 
TABLE 1—Continued 
= = eee = : - 
& nD 2 
Fs ee rs 
2 623 5 8% Ar ad 
LOCATION OF EGG IN OVIDUCT = nits an as 215 ZZ 
3 Baz eae Ba Ms 8B 
< 228 Ae 2a % AG! 
8 am ae - S|) ae 
grams grams grams grams 
6. In upper part of isth- : 
mus. Membrane com- 
plete, but thin. (Hen 
DOA) 'S Acpsie ie siete ee olet he 32.0 16.5 15.5 93.9 | 48.1 
Mean of two previously, | 
laideggsofsamehen....| 60.5 8.75 | 19.5° 32:25) 165.4 
Differencenss eee e —28.5 | =) || N50) | 
7. In upper part of isth- 
mus. Membrane thin. | 
(Elen 8008) ee tepec ae eleezonoo 0.28 12.57 15.48 123.2} 49.5 
Mean of the five previously, 
laideggsofsamehen....) 50.20 5.54 13.39 31.27 DBRT) 
_ Difference....... eee —21.87 | —5.26| —0.82 | —15.79 | —110.3 
8. Three cm. below begin- 
ning of isthmus. Mem- | 
brane thin. (Hen 1367). 32.08 | 0.39 16.12 15.57 96.6 56.9 
Mean of nine previously 
_ laid eggs of same hen.... 48.45 5.40 15.70 27.34 | 174.1 
Difference te eee SiGe | 3.00 ey 2a aa | 
9. Two em. above caudal 
end of isthmus. (Hen : 
4G) ccs Seebia.c2 = eerie: | 31.0 2.0 16.0 13.0 81.3] 50.5 
Mean of two previously 
laideggsofsamehen.... 47.5 7.0 | 14.75 25.75 174.6 
_ Differences... ee: | —16.5 —5.0 +1.25 | —12.75| —93.3 | 
10. In lower part of isth- 
mus. (Hen 8010)....... 30.00 0.53 14.04 15.43 109.9} 51.3 
Mean of eleven previously | 
laideggsofsamehen.... 51.54 5.77 | 15.72 30.06 | 191.2 | 
Difference. Bape ore —21.54 5.24 1.68 14.63 | —81.3 
11. In lower part of isth- 
mus. (Hen8018)........ 37.27 0.58 16.37 20.32} 124.1 | 56.4 
Mean of four previously 
laideggsofsamehen..... 60.10 | 6.63 17.42 36.05 206.9 
—1.05 | -15.73 | —82.8 | 


Difference:.. 722.52 ceeer |=222°83 


—6.05 


5 This is too high a value, probably arising from errors in separation of yolk 
and white. These particular data were taken before the refinement of method 
used in later studies had been worked out. 


PHYSIOLOGY OF THE OVIDUCT 111 
TABLE 1—Continued 
g af | | 
LOCATION OF BGG IN OVIDUCT e Pare} Hy az 315 za 
eae cea g 5 a8 xh aus 
5 ana am ax S aS 
a E z = S a 
grams grams grams grams 
12. In uterus, but no shell 
found. Egg surrounded - 
by fluid in uterus. (Hen 
BOSS) Beret i tett ase 3505 os, + 43.93 0.76 17.30 25.87 149.5 | 71.1 
Mean of four previously 
laideggsofsamehen....| 60.92 6.76) 17.77 | 36.39 204.8 
__ ADT ONC Cscpccnbancease =16.99 | —6.00| —0.47 | —10.52| —55.3 
13. In uterus, but no vis- 
ible shell formed. (Hen 
208 Ue gba ADE eaeor naa 45.15 0.96 17.25 26.94 156.2 75.5 
Mean of the six previously | 
laideggsofsamehen....} 59.22 6.22} 17.34 35.66 205.7 
Difference: 0. cc. vue oe —14.07 —5.26 0.09 8.72 49.5 
14. In uterus but no vis- 
ible shell formed. Some 
fluid in uterus. (Hen 
USO) Metach trate sianse-ne ope ers 46.67 0.88} 19.13 26.66 139.4 | 80.7 
Mean of two previously 
laideggsofsamehen....| 58.99 6.91 19 04 33.04 173.5 
Wifferences:.). .-c6< .0 =~ —12.32 -—6.03| +0.09| -—6.38 | —34.1 
15. In uterus, small 
amount of shell formed. 
(TenSLOGT) rer epie tert 45.00 4.50 15.50 25.00 161.3 87.0 
Mean of two previously 
laideggsofsamehen....) 52.00 7.50 15.75 28.75 182.5 
ID TiC Ra eee —7.00| —3.00}| —0.25| -—3.75 | —21.2 
16. In uterus, some shell 
formed. (Hen 8021)....| 43.66 1.35 15.18 Bias 178.7 | 95.1 
Mean of three previously | 
laid normal eggs of same 
|nYoi hs Stee aoe oN gese See 49 26 5.18 15.56 28 .52 183.3 
Difference..........-... —5.60| —3.83| —0.38, -1.39| —4.6 


112 RAYMOND PEARL AND MAYNIE R. CURTIS 


Thus in fig. 3 are shown (1) a membane covered egg taken from 
the isthmus of the oviduct of hen No. 8018 shortly before it 
would have entered the uterus, and (2) two normal laid eggs of 
the same bird. The larger size of the latter is obvious. The 
isthmus egg is the one at the extreme left in the picture. 

2. It is apparent from examination of the differences in the 
columns giving albumen weights and albumen-yolk ratios that 
in general the farther down the oviduct the egg proceeds the more 
albumen it gets. Very nearly one-half the total weight of albu- 
men of the completed egg is added in the uterus, an organ hither- 
to supposed to be entirely devoted to shell formation. Clearly 
very much more albumen is added to the egg in the uterus than 
in the isthmus. This, of course, does not necessarily mean any 
more rapid rate of secretion in the uterus, because of the time 
element involved. The egg stays much longer in the uterus than 
in the isthmus. 

3. This brings us to a consideration of the question of the rate 
of secretion of albumen in different positions of the oviduct. We 
have attempted to approach this problem by the graphical 
method. The results obtained are not to be regarded as highly 
accurate in respect to minute details. It is an exceedingly diffi- 
cult matter to get very precise data in the individual instances 
regarding time relations in the physiology of the oviduct. We 
must therefore depend upon average results. The attempt has 
been made in fig. 4 to show graphically the net average results 
from the data collected in this laboratory regarding the time taken 
in the passage of the egg through the several portions of the 
oviduct and the rate of secretion of albumen in the same portions. 
As a measure of the albumen is taken the percentage of the total 
albumen of the laid egg which has been acquired at each specified 
level of the duct. The time is plotted as abscissa, and the per- 
centage of albumen as ordinate. 

It is not possible to recount here in detail all the evidence on 
which the points in this diagram are based. It would involve 
the presentation of considerable material which has no direct 
bearing on the subject of the present paper. We shall, therefore, 
be obliged to state only briefly, and in some degree categorically, 


PHYSIOLOGY OF THE OVIDUCT 113 


the 


ind two normal laid eggs of 


left 


isthmus (hen SO18) at the 


from the 


LZ 


covered ¢ 


natural 


a membrane 


Showing 


yird for comparison 


Pig 


114 RAYMOND PEARL AND MAYNIE R. CURTIS 


a 
=) 


TONES OF TOTAL ALBWITEN 
=) 


N 
8 


J 


HOURS O / 2 é 
ALBUMEN PORTION Us UTERUS 


Fig.4 Diagram showing what percentage of the total amount of albumen 
present in the normal laid egg of the domestic fowl is present at successive levels 
in the oviduct. The smooth curve is the parabola for which the equation is given 
in the text. 


the manner in which the plotted points were determined. In the 
first place it has been found in our work here that when a hen is 
laying regularly one egg per day ovulation occurs at approximately 
the same time as laying. That is the odcyte which will be laid 
as a completed egg tomorrow enters the infundibulum at the 
time when today’s passes through the vagina and is laid. This 
then is taken as a fundamental datum in the calculation of the 
rate of passage of the egg down the oviduct. 

The mean of all available observations made in this laboratory 
gives 3.2 hours as the time required for the passage of the egg 
through the ‘albumen portion’ of the oviduct. This includes the 
total time from the entrance of the egg into the infundibulum to 
its entrance into the isthmus. This agrees very well with the 
statements of earlier workers® who generally give the time spent 
in the albumen portion of the duct as ‘about’ three hours. 


6 Cf. Lillie, F. R. The Development of the Chick. New York, 1908, pp. 23-25. 


PHYSIOLOGY OF THE OVIDUCT 115 


In regard to the time taken by the egg in passing through the 
isthmus our observations are far from agreeing with the state- 
ments on this point in the literature. Taking the mean of all 
available data 0.6 of an hour is found to cover the time during 
which the egg is in the isthmus. All our observations agree well 
amongst themselves, and we are convinced that this figure is 
substantially correct for the breed of fowls here used (Barred 
Plymouth Rocks). This is a much shorter time than earlier 
workers have estimated. Thus Gadow’ says: ‘‘Im Isthmus 
soll das Ei ungefaéhr 3 Stunden lang verweilen.”’ Lillie’ gives 
the same estimate on the authority of Kélliker. Patterson? who 
has published most recently on this matter, while reducing some- 
what the time for passage through the isthmus, still gives a value 
considerably higher than that found in the work of this laboratory. 
His statement is as follows (loc. cit., p. 105): ‘‘The writer finds 
that in a hen kept under normal conditions, the egg traverses 
the entire length of the oviduct in about twenty-two hours. 
The time occupied in the different portions of the oviduct is as 
follows: Glandular portion, three hours; isthmus, two to three 
hours; uterus and laying sixteen to seventeen hours.” With all 
parts of this statement except that relating to the isthmus our 
results are in entire agreement. At an early stage of the studies 
in this laboratory on the physiology of the oviduct we were of 
the same opinion as Patterson as to the time taken in passing 
through the isthmus. More extended observations, covering 
a fairly wide range of conditions has convinced us that, as 
already stated, the egg normally takes less than one hour in 
passing through the isthmus. It is, of course, possible that 
there are breed differences in respect to the time the egg stays in 
the isthmus, and that Barred Plymouth Rocks are strikingly 
exceptional in this regard but this hardly seems probable. It is 
more likely that the estimate of earlier workers has been somewhat 
too large. 


7 Gadow, H., loc. cit., p. 872. 
§ Loc. cit. 
® Patterson, J. T., Journal of Morphology, vol. 21, pp. 101-134, 1910. 


116 RAYMOND PEARL AND MAYNIE R. CURTIS 


As stated in the preceding paragraphs the points on the time 
or abscissal axis represent the mean or average results of fairly 
extensive experimental data, some of which are not included in 
the present paper. Now we may consider the determination of 
the points plotted as ordinates. At the outstart it should be said 
that no observations have yet been made on the rate of secretion 
of albumen at different levels of the ‘albumen portion’ itself. 
Therefore from the zero point at ‘ovulation’ when the naked yolk 
enters the infundibulum to the beginning of the isthmus we have 
connected the points with a dotted line to indicate that in this 
region no direct observations are available. The first plotted 
point (40.4) at the beginning (cranial end) of the isthmus is the 
mean albumen percentage of three eggs which were taken from 
this point in the duct.. The next point plotted is the mean albu- 
men percentage (50.6) of four isthmus eggs which were taken from 
the upper part of the isthmus. The next point (53.8) is the mean 
albumen percentage of four isthmus eggs taken from the lower 
part of the isthmus. The last six points are based on the obsery- 
vations of single eggs which have been in the uterus the indicated 
length of time. 

The smooth curve which graduates these observations is the 
parabola 


y = 17.5915z — 0.81712? — 0.4164, 


in which y denotes percentage of albumen and z time in hours 
during which the egg has been in the oviduct. The origin of x 
is taken atO (ovulation). The parabola was fitted to the observa- 
tions by the method of least squares. 

The diagram shows clearly that there is scarcely any diminu- 
tion in the rate of secretion of albumen until nearly the total 
amount has been acquired by the egg. There is not the slightest 
evidence of any break in the rate of secretion of albumen after the 
egg leaves the so-called ‘albumen portion’ of the duct. From the 
time the yolk enters the upper end of the ‘albumen portion’ there 
is a gradual diminution of the rate of secretion of albumen, giving 
rise to the parabolic curve. But plainly there is no sudden 
change. The egg gets more than half of its total albumen after 


PHYSIOLOGY OF THE OVIDUCT ilil7¢ 


it leaves the ‘albumen portion’ of the duct and it takes this at 
nearly the same rate as it did the earlier part. 

It is of interest to note the similarity of this curve showing the 
rate of increase of albumen in the formation of the individual 
egg to the curve previously published by one of the writers!® 
for the increase in weight of egg (which is quite closely corre- 
lated with amount of albumen) with increasing amount of yolk, as 
measured by number of yolks. 

4. It will be noted that the differences in the column headed 
‘weight of yolk’ are in the majority of cases negative. In other 
words, in these instances the yolk of the laid egg is heavier than 
the yolk of the oviduct egg. Now, of course, yolk as such is not 
added during the passage of the egg down the oviduct. This 
being so one would expect that in the long run the yolk of any 
given laid egg would be as often in defect as in excess of the weight 
of the yolk of any given oviduct egg. There is some indication 
that this is not strictly true, but that the ‘laid’ yolk tends to be 
heavier. Such a phenomenon would be in accord with the fact 
definitely demonstrated by Greenlee," in his study of cold stor- 
age eggs, that in the normal, unboiled egg there is a continuous 
transfer of water from albumen to yolk by osmosis. Certain of 
Miss Curtis’” earlier results had suggested that this was possibly 
the case. 


THE ABSOLUTE AND RELATIVE AMOUNT OF NITROGEN IN THE 
ALBUMEN OF EGGS IN DIFFERENT STAGES OF FORMATION 


While the two lines of evidence presented in the preceding sec- 
tions of the paper amply demonstrate that the thin albumen 
is added to the egg after it leaves the albumen portion of the duct, 
it seemed advisable, because of the novelty of the results to col- 
lect still further evidence of another kind. This evidence, which 
will be set forth in the present section of the paper, has to do with 


10 Pearl, R., Zoologischer Anzeiger, Bd. 35, pp. 417-423, 1910. 

1 Greenlee, A. D., U.S. Department Agriculture Bureau of Chemistry, Circu- 
lar 83, pp. 1-7, 1911. 

12 Curtis, M. R., loc. cit. 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 1 


118 RAYMOND PEARL AND MAYNIE R. CURTIS 


the nitrogen content of the albumen in eggs taken from different 
levels of the oviduct. Not only do these chemical data confirm 
the results obtained from the other lines of evidence, thus demon- 
strating still more thoroughly and conclusively the main point 
under discussion, but they also throw light on certain matters 
which could not be elucidated by other than chemical methods of 
attacking the problem. 

The analytical work on which the data of this section are based 
was performed in the Department of Chemistry of the Maine 
Experiment Station, by Mr. H. H. Hanson, Associate Chemist. 
It is a pleasure to express our obligation to him for coming to the 
ald of the present investigation in this way. 

It should be stated that the nitrogen determinations were made 
by the modified Kjeldahl method, as used by the Association of 
Official Agricultural Chemists. 

The chemical data are exhibited in condensed form in table 2. 
The complete figures for the same eggs are given in table A of the 
Appendix. 

It is evident that the data set forth in table 2 confirm the re- 
sults previously obtained. Thus, to consider first moisture con- 
tent of the albumen, it is seen that the albumen of the normal laid 
egg contains between 87 and 88 per cent of moisture (mean 87.40). 
This value agrees very well with those obtained by Willard and 
Shaw" and Pennington.’ But when the egg enters the uterus 
its albumen has a water content of only about 80 per cent. Or, 
the albumen of the egg at this level of the oviduct has by actual 
weight some 15 grams less water than the laid egg. The longer 
the egg stays in the uterus the ‘thinner’ the albumen becomes 
(ef. hen 387 in the above table), i.e., the higher its water content. 
This, of course, is the same thing which has been shown above, 
namely, that most of the thin albumen is added in this region. 

The percentage content of the albumen in nitrogen brings out 
again the same point. From a nitrogen content of about 4 per 


4 Willard, J. T. and Shaw, R. H., Kansas Agricultural Experiment Station, 
Bulletin 159, pp. 143-177, 1909. 
1s Pennington, M. E., Journal of Biological Chemistry, vol. 7, pp. 109-132, 1910. 


PHYSIOLOGY OF THE OVIDUCT 119 


cent at the upper end of the isthmus, the relative amount of this 
element in the albumen diminishes steadily till the egg is laid. 

The point of greatest interest and importance in connection 
with these chemical data, hinges upon the absolute amount of 
nitrogen in the albumen. Since it is solely the thin albumen 
layer which is added after the egg leaves the albumen portion of 
the oviduct the possibility is at once suggested that what happens 
in the lower portions of the duct is not a true secretion of another 
albumen layer but merely a taking up of water from the blood 
by osmosis, and a dilution or partial solution of the dense albumen 
already present. Such a view assumes in other words that all 
that is added to the albumen after the egg enters the isthmus is 
water. 

Clearly the only way to test finally the validity of this idea is to 
carry out such chemical determinations as are tabled above. 
The last column of table 2 shows the available evidence, which 
appears reasonably clear in its significance, though because of 
the minute absolute amount of nitrogen in the white of an egg 
the case is not a simple one. What the figures from the analyses 
of thirteen oviduct eggs show is that with four exceptions, the 
oviduct egg has absolutely less nitrogen in its albumen than the 
normal laid egg of the same hen. This, of course, is what would 
be expected if there is an actual secretion of albumen by the glands 
of the oviduct, and this secretion is added to the egg. It means 
that these oviduct eggs have been removed before they received 
their full amount of albumen. If it were the case, on the contrary, 
that only water was added to the egg after it left the albumen 
portion of the duct, it would be expected that the amount of 
nitrogen would be the same in an oviduct egg from the isthmus or 
uterus as in the normal laidegg. The chemical data clearly indicate 
that there is a definite addition of albumen to the egg in the isthmus 
and shell gland, and that the thin albumen layer does not represent 
solely a dilution of the dense layer. 

The four cases in which the analyses furnish an exception to this 
rule are undoubtedly to be explained as the result of fluctuation 
in the absolute size of the egg. It will be noted that each of these 
four eggs have been in the uterus some time and would therefore 


120 RAYMOND PEARL AND MAYNIE R. CURTIS 


TABLE 2 


Data showing the relative and absolute amounts of nitrogen in the albumen of eggs 
taken from different levels of the oviduct, and in normal laid eggs 


| | & z 
& 
SH | Sey 1 shy | Ske | seq 
LOCATION OF EGG IN OVIDUCT 5 a a ae a rs as A oom 
& me = mou 
ga 2eis 238 mae ges 
feiaes pay 2 Ear g a Ez 
At caudal end of albumen por- | 
tion. No membrane. (Hen 
S005) Sat Somer nen wanna eae 13.66 4.13 0.5635 
Mean of normal laid eggs of 
Bame hey Pap eies at Aare eles 31.34 1.98 0.6188 
Wifference <4 fies. enone —17.68 |. +2.15 | —0.0558 
Entering isthmus........ oh cace 
Covered with membrane except) 
for a little of cranial tip. (Hen 
BOLT) i carat Ne. tt seu seer 16.51 | 3.78 0.6233 
Mean of normal laid eggs of same 
hens eecree ie oce Go eee 32.07 2.00 0.6381 
Ditferenceee.-- see eee eae '—15.56 +1.78 | —0.0148 
Entering isthmus. Covered with 
membrane except for a little! } 
of cranial tip. (Hen168)...... 21.4015) 79.45 17.0036 3.04 0.6515 
Normal laid egg of same hen. .... 38.4895| 87.44 33.6536 1.78 0.6860 
Difference:...:. ... apemoobuacoote —17.0880) —7.99 '-16.6500 +1.26 0.0345 
In upper part of isthmus. Mem- 
brane thin. (Hen8008)........ | 15.48 3.47 0.5372 
Mean of normal laid eggs of same| 
ene ese aes ase asesewnaeses |_ 31.27 2.06 0.6407 
Difference...........-........|—15.79 +1.41 | —0.1035 
In lower part of isthmus. (Hen) 
SOLO) Etc 2c. see eee 15.43 3.30 0.5092 
Mean of normal laid eggs of same 
I Moceerep eae nee cocsocooEccr | 30.06 | | | 1.90 0.5894 
Difference. <2. sasaceeee eee —14.63 +1.40 | —0.0802 
In lower part of isthmus. (Hen) 
8018) i550 Sod doc ee Dae eel 20.32 | | 3.34 0.6756 
Mean of normal laid eggs of same 
11\-) eee One oor ss cS n> uo 36.05 | wale 1.89 0.6960 
Differencé:./1... eee ee 15.73 | | +1.45 | —0.0204 


13 In calculating the absolute amount of nitrogen in the normal laid eggs the 
mean albumen weight for the whole number of such eggs available for each hen 


has been used. 


PHYSIOLOGY OF THE OVIDUCT 


TABLE 2—Continued 


124 


z 
LOCATION OF EGG IN OVIDUCT E 5 5 é 5 & z 3 
a= RES REE 
e i) | x 
In uterus, but no visible shell 
fonmeds) (Henly) "2.22... 18.841 | 79.58 | 14.9943) 3.03 0.5711 
Normal laid egg ofsamehen.....)_ 34.419 | 87.46 | 30.1016 1.79 0.6154 
MD TMETEO CO eels ccc cysiv fas sees .-|—15.578 —7.88 |—15.1073) +1.24 —0_0443 
In uterus, but no shell formed. 
Egg surrounded by fluid in 
uterus. (Hen 8038).. .| 25.87 | 2.78 0.7179 
Mean of normal laid Cans ofe same) 
TERE aaa tibet: Saver crneearciaierets |) seOLoo 1.78 0.6492 
Difference... .. deseguuare +++ + -|=10.52 _| +1.00 | +0.0687 
In uterus, but no visible shell 
formed. (Hen8030).......... 26.94 eRe) 0.6727 
Mean of normal laid eggs of same, 
hen.. = .| 35.66 | 1.79 0.6485 
ae +0.71 | +0.0242 
In uterus, but no visible shell 
formed. Some fluid in uterus. 
(Hen 8033). . : .| 26.66 2.30 0.6118 
Mean of saga lad eggs fai same 
NG: wane Oe RA Set Re eee 33.04 1.68 0.5534 
DTeEnences - fecckone scene 2 —6.38 +0.62 | +0.0584 
In uterus, small amount of shell.) 
(Hen 200). . : Foo eles yAl 82.89 17.7154 2.51 0.5371 
Normal laid egg Bok same hen. Gels es 27.608 86.80 23.9627; 1.88 0.5197 
Dis ere | —6.237 | —3.91 | —6.2473| +0.68 | +-0.0174 
In uterus. Small amount of 
shell. (Hen387)..............| 29.9665) 86.62 25.9559) 1.92 0.5741 
Normal laid egg of same hen... . | 34.9305 87.89 | 30.7005 L 0.5997 
IDES OCC eee | —4.9640) —1.27 | —4.7446) +0.20 | —0.0256 
In uterus. Some shell formed. 
(Hen 8021)... a 27.138 2.07 0.5616 
Mean of senate! [aad eggs eine: same 
Renee ene ee lnoge5e | 1.96 0.5653 
IDETEN CE. ako ies wool: —1.39 +0.11 | —0.0037 


122 RAYMOND PEARL AND MAYNIE R. CURTIS 


have received nearly their full amount of albumen. It can 
readily be seen that if the oviduct egg happens to be an excep- 
tionally large one, relative to the other eggs of the same bird, it 
may have a slightly greater absolute amount of nitrogen though 
not yet laid, than another relatively or absolutely smaller laid 
ege taken for acontrol. It seems quite clear, in the light of data 
collected in this laboratory on the fluctuation in size and proportions 
of the parts of eggs'® that this is the correct explanation of these 
apparent exceptions in the chemical analyses. It will be noted 
that in these four cases it is only the absolute amounts of nitrogen 
(and not the percentages) which furnish exceptions to the rule. 


Supplementary evidence 


There is available evidence of still other sorts to indicate that 
there is a real addition of albumen to the egg after it leaves the 
so-called ‘albumen’ portion of the oviduct. In the first place a 
histological study of the oviduct which has been made in this 
laboratory by Dr. Frank M. Surface!” shows that histologically 
the same kind of glands which are found in the so-called albumen 
portion of the duct, are also found in the isthmus and uterus. 
The differences between the glands of the different regions are 
quantitative not qualitative. 

In removing eggs from the uterus it is frequently found that the 
egg is surrounded by a thin fluid which has evidently been secreted 
and is in process of being taken into the egg by osmosis. A case 
of this sort is described in the following autopsy record. 


Autopsy No. 523. Hen 8038. Killed March 28, 1911 for data 


This hen laid at 9 a.m. and was killed at 4.15 p.m., or 73 hours 
after laying. There was an egg in the uterus. The uterus was 
much larger than the egg. When a cut was made in this organ a 
small amount of clear fluid flowed out. The cut was clamped off 


16 Cf. No. 1 of these ‘Studies,’ loc. cit. supra. 
17 Reported at the Ithaca meeting of the American Society of Zoologists, Eastern 
Branch, December, 1910, but not yet published. 


PHYSIOLOGY OF THE OVIDUCT 125 


and about 2 cc. of the fluid drained into a bottle. This fluid 
was analysed, the following being the chemist’s report: 


Amount taken: 1.9860 grams being all of sample. 
Nitrogen found: 0.22 per cent. 


From this record it is clear that the fluid taken up by the egg 
in the uterus, is far from being water. It carries more than a 
fifth of 1 per cent of nitrogen. In other words it is a dilute albu- 
men. 

That the egg does not take water from the blood by osmosis, 
and in this way dilute the dense albumen to form the thin is 
further evidenced by the fact, shownby Atkins'* that in the domes- 
tic fowl the osmotic pressure of the blood is very considerably 
higher (nearly two atmospheres) than the osmotic pressure of the 
egg. In any osmotic exchange under these conditions water 
would tend to pass from the egg to the blood and not in the other 
direction. 

SUMMARY OF RESULTS 


Putting all the evidence together, the following account of the 
processes by which the hen’s egg acquires its protective and 
nutritive coverings summarizes the results of the present study. 
Certain of these results are novel and others confirm the experi- 
ence of earlier workers. 

1. After entering the infundibulum the yolk remains in the 
so-called albumen portion of the oviduct about three hours and 
_ in this time acquires only about 40 to 50 per cent by weight of its 
total albumen and not all of it as has hitherto been supposed. 

2. During its sojourn in the albumen portion of the duct the 
eges acquires its chalazae and chalaziferous layer, the dense 
albumen layer, and (if such a layer exists as a distinct entity, 
about which there is some doubt) the inner fluid layer of albumen. 

3. Upon entering the isthmus, in passing through which portion 
of the duct something under an hour’s time is occupied instead 
of three hours as has been previously maintained, the egg receives 
its shell membranes by a process of discrete deposition. 


18 Atkins, W. R. G., Scientific Proceedings of the Royal Dublin Society, vol. 12 
(N. 5.) pp. 123-130, 1909. Cf. also Biochemical Journal, vol. 4, pp. 480-484, 1909. 


124 RAYMOND PEARL AND MAYNIE R. CURTIS 


4. At the same time, and during the sojourn of the egg in the 
uterus, it receives its outer layer of fluid or thin albumen which 
is by weight 50 to 60 per cent of the total albumen. 

5. This thin albumen is taken in by osmosis through the shell 
membranes already formed. When it enters the egg in this 
way it is much more fluid than the thin albumen of the laid egg. 
The fluid albumen added in this way dissolves some of, the denser 
albumen already present, and so brings about the dilution of the 
latter in some degree. At the same time, by this process of dif- 
fusion, the fluid layer is rendered more dense, coming finally 
to the consistency of the thin layer of the laid egg. The thin 
albumen layer, however, does not owe its existence in any sense 
to this dilution factor, but to a definite secretion of a thin albu- 
men by the glands of the isthmus and uterus. 

6. The addition of albumen to the egg is completed only after 
it has been in the uterus from five to seven hours. 

7. Before the acquisition of albumen by the egg is completed 
a fairly considerable amount of shell substance has been deposited 
on the shell membranes. 

8. For the completion of the shell and the laying of the egg from 
twelve to sixteen, or exceptionally even more, hours are required. 


or 


PHYSIOLOGY OF THE OVIDUCT 12 


APPENDIX 


The following table gives in extenso the original data on which 
this paper is based. It should be said that the weights are in 
grams. 


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COMPARATIVE STUDIES ON THE EFFECTS OF 
ALCOHOL, NICOTINE, TOBACCO SMOKE 
AND CAFFEINE ON WHITE MICE 


I. EFFECTS ON REPRODUCTION AND GROWTH 


L. B. NICE 


From the Biological Laboratory of Clark University 


ONE FIGURE 


CONTENTS 
PMT OULU LON Meanie nar etatereerck aah ieee sl ake esis Re tad ae Sey eae Mei uea Oo: 
JNICOLICI| Sa Sa BRIE PS GCE eee ORIN Ce ave - PEE ee .. 183 
Nicotine and tobacco smoke................... 3 .. 135 
NO HET OIG acer racine ret A caiaaisie A> oi eer .. 135 
Methods. . : 7 135 
Effects on the werent and health ar the adult MIG: +). 02 = .. 138 
Effects on the fecundity of the adults and viability of the 3 young. . 139 
Growth of the young subjected to the same conditions as their parents... 139 
Growth of the young not subjected to the drugs....... 145 
Comparison of the growth of mice given drugs with Poe: not given drugs.. 146 
SIDA guaae dis SUIS Soren Ee epee Caen oe ee : Peres 
Biol leyasyel anita tile pap aROnne REneOse ‘ as 149 


INTRODUCTION 
Alcohol 


The effects of aleohol on animal offspring has been shown in 
several investigations to be injurious. Laitinen (31) found that 
alcoholized rabbits and guinea pigs had more stillborn young 
than the controls; and that the growth of the living young was 
retarded. Of 23 pups from a pair of alcoholic dogs in Hodge’s 
(28) experiments, 8 were deformed, 9 were born dead, and only 4 
were viable. From the control pair 4 were deformed, none were 
born dead and 41 were viable. Forty-three per cent of the eggs 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 1 


133 


134 L. B. NICE 


of Ceni’s (10) aleoholized fowls developed normally under ordi- 
nary conditions in comparison to 77% per cent of the controls. 
A fluctuating temperature at the beginning of incubation pre- 
vented all alcoholic eggs from developing perfectly while 27 per 
cent of the control eggs were normal. 

Todde (55) aleoholized 15 roosters from two to four months and 
concluded that alcohol causes a torpidity of the testicles, acting 
probably through its affinity for the central nervous system. Ber- 
tholet (5) found in 37 out of 39 cases of aleoholism in men more or 
less of an atrophy of the testicles. Ceni’s (10) three alecoholized 
hens laid less than half the average number of eggs, but since 
fowls are abnormally developed in this respect we might antici- 
pate that anything which injured their general health would 
disturb the laying function without drawing the conclusion that 
in other animals alcohol would promote sterility. Laitinen (31 
and 33) found that aleoholized rabbits and guinea pigs had more 
young than the controls. The investigations of the Eugenics 
Laboratory (4, 18 and 48) and of Laitinen (33) demonstrate that 
more children are born to alcoholic than to sober parents. 

As to the effect of alcohol on human offspring, Demme (16) 
compared 10 non-alcoholic with 10 alcoholic families and found 
that 82 per cent of the children of the former and only 17.5 per 
centof the latter were normal. Sollier (53) states that it is demon- 
strated that progeny conceived during drunkenness is doomed to 
idiocy. Morel (41) gives the history of an alcoholic family 
through four generations to final extinction, concluding that 
alcoholic intoxication produces degeneracy, depravity and idiocy. 
On the other hand Pearson, Elderton and Barrington (48, 18 
and 4) believe that their statistical data prove that extreme alco- 
holism is a result, not a cause of degeneracy and that the abnor- 
mal pedigrees are due to defective stock and not to alcohol. In 
an investigation of about four thousand school children of alco- 
holic and sober parents they could not find that parental alcohol- 
ism had any unfavorable effect on the offspring. 


—e 


EFFECTS OF DRUGS ON WHITE MICE 135 


Nicotine and tobacco smoke 


Tobacco infusion injected into young rabbits by Richon and 
Perrin (46) had a decided stunting effect; however, if the injec- 
tions were stopped, the animals gained rapidly and soon equalled 
the controls. Rabbits subjected to the fumes of tobacco by 
Zebrowski (60) for six to eight hours daily for several months 
suffered from loss of appetite and great emaciation, losing from 20 
to 47 per cent of their weight. Nicotine injected into eggs by 
Féré (19) seemed to be a poison to some and a stimulant to others. 
Fleig (21) subjected guinea pigs to heavy inhalations of tobacco 
smoke, to injections of extract of smoke or injections of nicotine, 
and obtained abortions, still-births, under-sized, weak and stunted 
young. Unfortunately he fails to give definite details and condi- 
tions of his experiments. 

Thirteen cases of sexual impotence caused by the abuse of 
tobacco are reported by Cannata (9); abstinence from smok- 
ing restored the men to a normal condition. Hitchcock (52) at 
Amherst College and Seaver (52) at Yale found by measuring 
students that the smokers developed less in height and lung capac- 
ity during their college course than the nonsmokers. Meylan 
(39), however, at Columbia could not find that smoking had any 
decided effect on growth. 


Caffeine 


No experiments could be found on the effects of caffeine on 
growth and reproduction. Rivers and Weber (47) show that 
caffeine increases capacity for muscular work. The investiga- 
tions of Crimer (12), Pincussohn (44), Sasaki (50), Schulz- 
enstein (51) and Voit (57) indicate that coffee and tea retard 
digestion. 

METHODS 


A comparative study was undertaken to test the effects of 
alcohol, nicotine and caffeine on the offspring of animals, when 
fed in small enough amounts so as not to injure their health. 
White mice were chosen for the experiments since they breed 
rapidly, so that the results could be based on large numbers, and a 


136 LG: BS NICE 


second generation could be quickly obtained to compare with the 
first. 

The experiments were started with 30 female and 15 male 
white mice about four months old. They were bought froma 
dealer and guaranteed not to be brothers and sisters. Four hun- 
dred and forty-one young were born of the first generation in 
seven months. Twenty females and 12 males of this number 
were bred at the age of two and a half months, care being taken to 
prevent inbreeding. These had 230 young in four months. 
Five lines were carried; one was given alcohol, another nicotine, 
a third was subjected to the fumes of tobacco smoke, a fourth 
received caffeine and the fifth line was carried for controls. 

Two females and one male were kept in a cage; each female was 
moved to a separate cage before her young were born and remained 
there as long as they were suckling, which lasted from twenty- 
five to thirty days. The cages were of wire mesh 6 inches wide, 
5 inches deep and 12 inches long. 

The regular diet of all the mice was buckwheat, and crackers 
soaked in milk; every few days they were given carrots, grass, 
meat, ete. 


The alcohol line 


The first generation in this line consisted of 6 females and 3 
males, and in the second generation, the offspring of the first, 
of 5 females and 3 males. 

Two cubie centimeters per mouse of 35 per cent alcohol were 
added daily to the crackers and milk of these mice. Instead of 
water they drank 35 per cent alcohol which was placed in bottles 
containing siphons; so the animals drank directly from the bottles, 
and evaporation was prevented. The mice were first given 10 
per cent alcohol which was gradually increased to 45 per cent. 
Thirty-five per cent was found to be a safe medium since stronger 
percentages sometimes intoxicated the mice. 

All the mice in this line except 16 received alcohol, the young 
beginning to take it at the age of three weeks. Sixteen young 
were given no alcohol themselves although their mothers received 
it for sixteen days after the birth of the young. 


EFFECTS OF DRUGS ON WHITE MICE 137 


The nicotine line 


The first generation in this line consisted of 6 females and 3 
males, and the second generation, the offspring of the first, of 
3 females and 2 males. 

This line had 2 ce. per mouse of 1:1000 nicotine solution added 
to their crackers and milk daily, and the same solution was sub- 
stituted for drinking water in bottles with siphons. Different 
strengths of nicotine had been tested on white mice from 1:2000 
to 1:500 solution. The last was fatal but on 1:1000 they remained 
in good health, although this solution was found to kill grey rats 
in nine days. 

All the mice in this line except 6 received nicotine, the young 
beginning to take it at the age of three weeks. Six young were 
given no nicotine themselves although their mothers received it 
for sixteen days after the birth of the young. 


The line subjected to the fumes of tobacco 


The first generation in this line consisted of 6 females and 3 
males, and the second generation, the offspring of the first, of 
8 females and 4 males. 

These mice were subjected to tobacco smoke for about five 
minutes at a time and then aired for five or ten minutes; this 
was repeated for two hours each day. The smoke chamber was 
a bell jar made air tight by being placed on glass and moistened 
around the bottom. About 4 grams of Connecticut leaf tobacco 
were burned each day in a clay pipe inserted in a rubber cork in 
the top of the bell jar. The smoke was drawn through the jar 
by an air pump, the tube for this purpose being in the rubber cork. 

All the mice of this line except 9 were subjected to the fumes of 
tobacco, the young being put in the smoke chamber from the time 
they were a few days old. Nine young were not subjected to the 
fumes of tocacco, although their mothers were each day while 
they were suckling. 


138 L. B. NICE 


The caffeine line 


This line consisted of 6 female and 3 male mice. No second 
generation was obtained for breeding since many of the young 
died and others were eaten by their parents. 

Each mouse had 2 ce. of 1:100 caffeine citrate solution added 
to his crackers and milk daily, and drank this solution instead 
of water. The caffeine citrate was first given in a 1:500 solution 
which was gradually increased to a 1:50 solution. Since they did 
not seem to be thriving on this strength it was decreased to 1:100, 
on which they remained in good health. 

All the mice in this line except 8 received caffeine, the young 
beginning to take it at the age of three weeks. Eight young were 
given no caffeine themselves, although their mothers received it 
for sixteen days after the birth of the young. 


The control line 


The first generation of control mice consisted of 6 females and 
3 males. Since these mice had few young and many were eaten 
there were none of the second generation ready for breeding three 
months after the experiments were started. Therefore 6 young 
females and 3 males were obtained from the same source as the 
first lot of mice so as to serve as controls for comparison with the 
second generations of the other lines. 


EFFECTS ON THE WEIGHT AND HEALTH OF THE ADULT MICE 


The females were weighed each time after they had given birth 
to a litter, and the males from time to time. During the course 
of the experiments all the mice gained in weight; in the first genera- 
tion, carried seven months, the alcohol mice gained 6 grams each on 
an average, and the others gained 2 grams; in the second genera- 
tion carried four months, the aleohol mice gained 2 grams and the 
others 1 gram. This would indicate that the mice remained 
healthy and that alcohol has a fattening effect. 


EFFECTS OF DRUGS ON WHITE MICE 139 


EFFECTS ON FECUNDITY OF THE ADULTS AND VIABILITY OF THE 
YOUNG 


_A record was kept of the litters of each female. Many of the 
young in all the lines were eaten. In the tables 1 to 13 only 
those young that died from lack of vitality are recorded. 

TABLE 1 
Record of the young of each female 


Control line. First generation 


| 5 oa -OUN: 
FEMALE NUMBER OF NUMBER OF | NUMBER OF NUMBER OF YOUNG 


MONTHS OBSERVED LITTERS YOUNG BORN | THAT DIED 

IN 3. OE RRS ERD 7 3 18 0 
13. (ron eRe 7 2 12 0 
(C..  e 7 2 16 0 
1D): 0 ee ee iii 2 v/ 0 
1D | 3 1 7 0 
IN. 

BROGAN ciiic. <4: 7 10 60 0 


* Female E died at the end of three months. No pathological condition could be 
found. 


t Female F was killed by accident at the beginning of the experiments. 


TABLE 2 


Alcohol line. First generation 


: ae =a = 
FEMALE | NUMBER OF NUMBER OF NUMBER OF NUMBER OF YOUNG 
i" MONTHS OBSERVED LITTERS YOUNG BORN THAT DIED 
PAWN E eer ciok %,: 7 2 17 1 
1. 7 2 1 
(GE 437 See | a 4 2 1 
1D) 4%, ORR 7 4 ) 2 
Dini ee aBee tf 2 4 
i055 Ase eRe 
Motali, ac... 5] vi 14 $1 9 
1 | 3 


3% wks. 


* Female F died three and a half weeks after the experiments were started. No 
pathological conditions could be found. 


140 te) BS oNIGE 


TABLE 3 


Nicotine line. First generation 


7 
NUMBER OF NUMBER OF NUMBER OF NUMBER OF YOUNG 


SE MONTHS OBSERVED LITTERS YOUNG BORN THAT DIED 
dN Boece toes RE 7 3 13 1 
Be ae ler eres u 2 7 0 
Cathe 7 1 4 4 
De ayer 7 | 2 13 2 
eae Ae ee 7 5 36 | 2 
oy 7 | 4 31 9 
Motalics -.<.16 | 17 104 18 
TABLE 4 
Line subjected to tobacco fumes. First generation 
FEMALE MONTHS OBSERVED)  winsks «|| gouNagomn ||) atcn ayia 

Ae See 7 4 23 | 9 
BAS ore. 7 4 | 30 5 
(Geowese. 7 3 22 6 
1D Bear ace cS Eeaa 7 4 21 | 7 
Be. 4 3t 15 | 14 

1D Patent: ratte d 

ine —— s = zs 
Totally as-eare: 7 18 lil | 41 

1 4 


* E was killed by accident at the end of three months. 
+ F died at the beginning of the experiments. ‘ 
{ One of these litters was an abortion. 


TABLE5 * 


Caffeine line. First generation 


NUMBER OF NUMBER OF NUMBER OF _ | NUMBER OF YOUNG 
seers MONTHS OBSERVED | LITTERS YOUNG BORN THAT DIED 
AN. ase ace nee 7 3 15 0 
Bina ki aca erneeae 7 3 16 8 
Care eee rns 7 2 11 5 
Dis tae 7 3 27 2 
Bh osc Braetnseee Uf 2 7 4 
1a 7 3 9 3 
Total 6 7 16 85 22 


EFFECTS OF DRUGS ON WHITE MICE 


TABLE 6 


Control line. Second set 


ree eee | Pesee on | meee oe | roumme or somme 
Jari SBS Onn 4 1 i 0 
LE eke Soe tCek 4 2 12 0 
(Cocco am emeeee 4 2 4) 0 
1D)... cou hops Oeeee 4 2 9 0 
Dit 6 peace eee 4 1 2 0 

4 1 4 0 


TABLE 7 


Alcohol line. Second generation 


5 
ans 
co} é 
a Py 
aie 
ce Ba 
© 
°o | 
| 
ae 0 i) an 8 ene ae a 


Tea ee “UMTERS | YouN@poRN | THaT DIED 
Ce a ee ee 
: 4 1 5 0 
+ 2 13 1 
4 2 16 2 
4 2 13 | 3 
2 1 9 1 
_| 
4 8 56 7 
2 
TABLE 8 
Nicotine line. Second generation 
] 
aaa MONTHS oBarRvED| Lirrens | YouNasoRN | Har DIED. 
Dn ee 4 2 il 5 
BRM atch Se cece 3 4 2 13 0 
(Os. Aone eee 4 1 6 0 
Total.. 4 4 5 


30 


| on 


142 L. B. NICE 


TABLE 9 


Line subjected to tobacco fumes. Second generation 


FEMALE NUMBER OF NUMBER OF NUMBER OF | NUMBER OF YOUNG 
MONTHS OBSERVED LITTERS YOUNG BORN THAT DIED 
AM saattentts aoa coe 4 3 21 3 
Bit og snc aee 4 3 15 1 
Cer ee eee nae 4 2 11 2 
ID Ras ceo oer 4 4 22 9 
er Be Eee 4 2 12 3 
eee 4 1 8 1 
RO hee Aes e 3 1 6 1 
15 ey ere ee 3 1 6 6 
Total =6 4 17 101 26 
2 3 
TABLE 10 
Record of the young of the first generation 
Summary of tables 1, 2, 3, 4 and & 
NUMBER OF < NUMBER OF 
uve | *uupenor | “Goan” | “gaaen or | xonmes or | souna mat 
Control......... ‘ 3 f ; 10 60 0 
AlGoholicasnccs 5 7 14 81 9 
Nicotine......... 6 7 17 104 18 
Smoked......... f4 7 18 1 41 
\1 af 
@affeineyae cess 6 7 16 | 85 22 
TABLE 11 
Record of the young of the second generation 
Summary of tables 6, 7, 8 and 9 
NUMBER OF es NUMBER OF 
sxe | -Nuamenor | “Sowans” | “Tunenor | wuwmen oy | Zouwa uaz 
Controle 6 = 9 43 0 
Alicoholyereeiee f A 5 ; 56 7 
Nicotine........: 3 4 5 30 | 5 
Smoked. pees ec [6 4\ 17 101 | 26 
\2 3 f 


© 


EFFECTS OF DRUGS ON WHITE MICE 143 


TABLE 12 


Average of one female of the first generation for seven months 
S eae Se i 
| AVERAGE NUMBER OF AVERAGE NUMBER OF PER CENT OF YOUNG 


LINE 


| LITTERS YOUNG THAT DIED 
@onttroliewecscneady Ar « 2.2 13.3 0 
JAK Yo) a0) Be Gea aOR Ey seem 2.8 16.1 leit 
NMG OMAS. oat upbasboceao 2.8 17.3 Vike 
BOAOKC Mes frenetic = clare 4.0 24.6 37.0 
@ameme... cc. ca ee: 2.7 14.1 25.3 
TABLE 13 
Average of one female of the second generation for four months 
LINE AVERAGE NUMBER OF AVERAGE NUMBER OF PER CENT OF YOUNG 
9 LITTERS YOUNG THAT DIED 
(Gia) hihg0) ease O eee Eee 1 (foal 0 
PAN CON Olin jaiiieyoelsys a's 1.8 12.4 12.5 
INGO UIC ccttecrers sci sicias 1.66 10.0 16.6 
ISIMOKEE cou ctseislyiewiee 2.26 13.4 26.0 


In both generations the mice subjected to tobacco fumes had 
more young than any of the other lines, whereas the controls had 
the fewest young. Tobacco fumes had a marked effect on the 
viability of the young, since 37 per cent of the first generation 
died from lack of vitality and 26 per cent of the second. One 
abortion occurred in this line. Caffeine was also injurious to the 
young, 25 per cent dying. Nicotine and alcohol had a less notice- 
able influence. None of the control young died in either gener- 
ation. 


GROWTH OF THE YOUNG SUBJECTED TO THE SAME CONDITIONS 
AS THEIR PARENTS 


The young were either weighed singly or in litters at birth and 
every week for eight weeks. Since it was not always possible to 
weigh them before they suckled, the variations in the birth weights 
have little significance. The records were not carried beyond 
eight weeks for the young females often become pregnant at that 
time. 


144 


L. B. NICE 


TABLE 14 


Weekly growth of the young of the first generation subjected lo the same conditions as 


| 


their parents 


& re | )ae & a Gla @ Hm | & ae eo 

= | 3 o | Ha IA Uwe) mie Dn Hg Da 

iS lee Pavey SS elicisy) BR ee Nie me 

Ba |S |B | a" |agleslaelae| Bf | Be | ae | BF 

Beata eee se as FRIFFIP alr) FE Fe | Fz Lars) 

LINE aZe | ash lL el ae, |aalaciabiap| af ay a 8 aa 

OA4/ 044 EG) oc SzZIOslonlao| oF ok oF of 

4&8) 228 |82) <olee\<Biee| 2e linha =p <A 

See| Sse (28) Ges |SeGclaclae| Se | Se | Be | Be 

Bon | Fon pF ood Sees aS a SS aa SS] Sey 

< | <4 | [RE digi 7] }) < < 
| | | | | 
= = =| +— 4 . = 
| grams | grams grams gm.gm. gm. \gm.| grams | grams | grams | grams 
Control... .:.......|/ 24-8 20.7 || 17) 124 2.74.05.6/6.9 10.1 | 14.6 | 15.7 | 16.8 
Aleohol..... 30.6 | 24.5 | 2G 1.30 3.15.06.49.7| 12.0 | 14.0 | 16.4 | 17.5 
7 ; 7419956 ‘ oF le ai Pa ao = E 
Nicotine?.ce..s..- 21.4 | 22.2 oF 1.35 3-14 2'5.5)8..0) 10.5 | 13.0 | 12.5 | 15.1 
‘ € | 92 |c le ne « 

Smoked....... | 23.9 | 23.6 | 31) 1.23 2.94 .016.08.2) 10.5 | 13.3 | 14.9 | 15.3 
; a € ¢ | al | | 
Caffeine. ......... 27.9 | 22.2 | 27) 1.30 3.04.2)5.3/7.7| 10.1 | 10.7 | 12.1 | 18.9 
| | | | | 
} i } i] } if 

TABLE 15 


Weekly growth of the yo 


ung of the second generation 


as their parents 


subjected to the same conditions 


> = hi Ey | 

mn! | n n 

& eae le je a eae) & & & 

= Belo |e |Eulm@l go | p4 | Ge | oe | Be 

Sele |e, (See Se) WSS a SE | ei 

Aa a lp a ag/aR| ae | as ag ag ae 

Fa, |FelCales |ERIEF) Fa eo EE aes Ey 

LINE ase |e Al Bl ae, |eaiao] af | ab aa By ae 

Oak ORB o| Ckm |SZIOE) Ge | Go og on oa 

<me8 |<S/85) 96 |solee) <8 | <& <5 <a <a 

ee RR Be BLO Rela. Be | we Pe me ae 

Bon |PaiSF| Pa |B </R<) Bx ale ie Bs Bs 

= j<°lzZ | < < |< a = % a = 

| | = 
| | 

grams gm. grams gm.gm.| grams | grams | grams | grams | grams 
Controleves. rece eee 29.5 | 25) 14) 1.47 3.04.0} 6.9 | 7.1 | 9.9 | 10.9 | 14.0 
Alconolvesn.eteaee 23.3 | 22) 13] 1.35 |2.84.9| 6.75 | 9.6 | 12.2) 14.0 | 16.0 
Nicotinesa.rs..ee: 20.5 | 23) 10} 1.3 |2.94.5) 5.5 | 8.00 | 10.5 | 13.0 | 15.0 
Smokedee cent 20.0 | 20) 26) 1.2 |2.44.5)5.3 | 7.5 | 10.5 | 12:7 | 13:8 


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, 


——— oie” as 


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 4 young nol subjected to the drugs 


n a = to] 
a | eo | oe | eo | es I Ex 
: | S4 | 2 | 2 | 22 | 2 | gs ea 
2 ae ae ag | ae ae aE 
Sa EE | EE = eee || ee 5 
LINE + gn 2° 25 af 20 a= 
ty Oz oe 20 oF on om 
z= so | 36 s= | 3% | 22 28 
Ze } 82 | Bs aS | #& | 3& BE 
Zz | < < < < < < 
grams | grams grams grams | grams | grams | grams | grams | grams 
Gontrolay..... ; 33 | 1.35 | 2.85 | 4.0 6.2'| 7.0} 1070'| 12.7 | 14.8 | 16.8 
Aleohol........ 16 |0.5 |3.4 |5.2 | 7.3] 11.4 | 14.7] 15.6 | 17.6 | 19.2 
Nicotine.......) -6 | 1.5 | 2.38 14.7 | 6.0] 8.5} 10.9 | 13.4 | 16.0 | 18.0 
Smoked........ 9 |} 1.1 | 2.89 | Aa Sek) | | LOLD | 1802") 14289] 15.4 
Caffeine....... 8 126) [222 4-0 5.41 8.0] 10.9 | 13.0} 15.2 | 17.0 


* The Weinhtaok the controls are based on both sets of controls in tables 14 and 15. 


From this it is seen that the young of the a line de- 
eidedly 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 
: See. a eine. breebeles wie 


& & & iI Hm & ef | oe 
Sa BE | BE) Bed be | GP) be) Sea 
MICE ms 28 ao aa ae ai ea al | ad 
ae 25] $e se0\ ge | se | So | cele 
25 ef | e8|¢e| es | es | as | 38 | ag 
Z 4 20 loess % Z x | 2 
| grams | grams grams | grams | grams | grams | grams | grams grams 
Control*....... 33 | 1.35|2.85| 4.0 | 6.2] 7.0] 10.0) 12.7 | 14.8 | 16.8 
Without 
aleoholt.....| 16 |1.5' | 3.4 |5.2 | 7.3] 11.4 | 14.7] 15.6 | 17.6 | 19.2 
With alcohol*.| 39 | 1.32|3.3 |4.97| 6.5| 9.6| 12.1 | 14.0| 16.2] 17.5 
Without | | | 
nicotine}... 6 |1.5 |2.3 |4.7 | 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) 18.2 | 14.8 | 15.4 
Smoked*....... 57 |1.22)2.7 |}4.2 | 5.7] 8.0] 10.5 | 13.0| 14.4] 15.3 
Without | 
caffeine} ..... | 8 |1.6 |2.2 |4.0 | 5.4] 8.01 10.9 | 13.0 | 15.2) 17.0 
With caffeine..| 27 11.3 {3.0 |4.2 | 5.3|-7.7| 10.1 | 10.7 | 12.1 | 13.9 


* These weights are the averages of the two generations from tables 14 and 15. 
7 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 alcohol 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 aleohol 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. 


20 


19 


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 
Bs Pe ee Nicotine line 6 mice not given nicotine themselves 
SS ene 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 


13 


14 


15 


16 


EFFECTS OF DRUGS ON WHITE MICE 149 


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_ 19 


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31 


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ks 


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EFFECTS OF DRUGS ON WHITE MICE 151 


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60 


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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 §. 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 
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, (3) 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. 


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HEREDITY OF PIGMENTATION 157 


number though still remaining 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 isnot 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. 13 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 here no 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. majalis. 

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. 1893, 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 
im the development of these cells are shown in figs. 1 to 6. As 
soa) 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 thus migration and expansion takes place. 


6 Marine Biol. Lag. 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 


Pure F. heteroclitus { 


on mid-brain.. 2.0 0 1 
(on hind-brain. 5 


5 2 4 : 
413/)4/4)/4/3/2/0/5 3.4 


F. heteroclitus egg | on mid-brain.. 6 6 
hybrid.... . (on hind-brain. 5 4 


bo or 
ca | 
~ 
es 
~ 
~ 
bo 


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. majalis 
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 
F. 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 hed 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 | 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 
latera! 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 ef 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 two forms. 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 167 


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 rhay 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 seale, 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 (¢ompare 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 (F. 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. 

ce. 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 case 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. 


1 
2 
WR 
a | >) 
7) SS 
\\ on 
0 3 


3 


Fig. 1 Pure F. heteroclitus, four days old, showing first chromatophores. 
X 25. 

Fig. 2. Pure F. heteroclitus, three and one half days old, circulation started 
not more than a few minutes before drawing began. X 25. 

Fig. 3 Brain of same embryo drawn in fig. 2 but three and one half hours later. 
X 25. 


172 FRANK W. BANCROFT 


Fig. 4 Pure F. heteroclitus, five days old, showing the brain chromatophores 
and half of the yolk chromatophore ring about the embryo. H=heart. 25. 

Fig.5 Pure F. heteroclitus, six days old, showing the first crop of brain chro- 
matophores well expanded. Red brain chromatophores not drawn. 25. 

Fig. 6 Same embryo drawn in fig. 5, but nineteen hours later showing the first 
appearance of the second crop of brain chromatophores. The four cells of the 
first crop still recognizable. > 25. 

Fig. 7 Same embryo drawn in fig. 6, but forty-eight hours later. Shows the 
fusion of the head chromatophores. 


HEREDITY OF PIGMENTATION 173 


Fig. 8 Pure I’. heteroclitus, three and one-half days old. Heart beating but 
no circulation. Shows the yolk chromatophore ring about the edge of the hollow 
vesicle under the embryo. X 20. 

Vig. 9 F. majalis egg hybrid, seven days old. Shows both crops of head chro- 
matophores. X 25. 


Fig. 10 Pure F. majalis, seven days old. Belongs to same series as embryo of 
fig 9. Shows absence of the first crop of head chromatophores; also characteris- 
tic shape of black yolk chromatophores. 25. 

Fig. 11 F. majalis egg hybrid, six days old. Shows shape and arrangement 
of yolk chromatophores. 19. 

Fig. 12. Pure F. majalis, six days old. Shows the absence of yolk chromato- 
phores on the part of the yolk away from the embryo. Many chromatophores near 
the embryo were not drawn as they were much obscured by the yolk globules. 19. 

Fig. 13 Pure F. heteroclitus, four days old. There were no blood vessels 
near the cells figured. Shows characteristic early chromatophore group, before 
they have migrated onto the blood vessels. Embryo same stage as fig. 2. %& 114. 

Fig. 14 F. heteroclitus egg hybrid, four days old; has good circulation. Em- 
bryo same stage as fig. 2. Shows characteristic group of yolk chromatophores on 
the side of the yolk sac, opposite to the embryo, where the blood vessels have just 
become established. > 114. 

174 


HEREDITY OF PIGMENTATION Wi) 


18 19 


Vig. 15 Pure F. heteroclitus, twelve days old. Fully developed red chroma- 
tophore near the eye of embryo. Dotted lines indicate blood vessels. > 75. 

Fig. 16 I. heteroclitus egg hybrid, twelve days old. Fully developed red yolk- 
chromatophore in center of clear space in black chromatophore reticulum. X 75. 

Fig. 17 Pure F. majalis, thirteen days old. Characteristic red yolk chroma- 
tophore on blood vessel. X 75. 

Fig. 18 Pure F. heteroclitus, six days old. Group of chromatophores begin- 
ning to form chromatophore reticulum on blood vessels near left eye. % 114. 

Fig. 19 IF. heteroclitus egg hybrid, six days old. Same series and same age as 
embryo of fig. 18. Group of chromatophores on blood vessel near left eye. > 114. 


176 FRANK W. BANCROFT 


24 23 22 


Fig. 20 Pure F. heteroclitus, seven days old. Same embryo as in fig. 18. 
Chromatophores on blood vessel near left eye. To show increase in size and com- 
plexity during last twenty-four hours. X 114. 

Fig. 21. F. heteroclitus egg hybrid. Same embryo as in fig. 19. Chromato- 
phores on blood vessel near left eye. Note increase in size of red chromatophores 
and the beginning of the small processes of black chromatophores. > 114. 

Fig. 22. Pure F. heteroclitus, eleven days old. Typical fully developed red 
chromatophore among the black chromatophore reticulum. 114. 

Fig. 23 F. heteroclitus egg hybrid, eleven days old. Of same age and series 
as embryo in fig. 22, with which it is to be compared. Note double layer of chro- 
matophores and fine black reticular processes. > 114. 

Fig. 24 Pure F. heteroclitus. Most of the eggs of this lot had hatched, but 
in this embryo the circulation had stopped though the heart was still beating, and 
the blood vessels distinct and full of blood. The yolk chromatophores have begun 
to lose their arrangement on the blood vessels and have developed much longer 
branches than any normally seen in this species. 114. 


HEREDITY OF PIGMENTATION 177 


27 


Fig. 25 Pure F. majalis, thirty-five days old, just hatched. Shows the line 
of chromatophores along the dorsal surface of the nerve cord, N...N, which were 


expanded; and the contracted chromatophores of the lateral line, L...L. A...A= 
Aorta, V...V=Ventral vein. Neither the chromatophores on these last two ves- 
sels nor on the dorsal surface of the fish have been drawn in. X 25. 


Fig. 26 IF. heteroclitus egg hybrid, sixteen days old, just hatched. S...S line 
of chromatophores under dorsal skin, partly contracted by the nareotization. 
N...N chromatophores on nerve core, partly contracted; L...Z lateral line. All 
the red chromatophores contracted, black chromatophores partly contracted at 
left where they formed a complete line when the drawing was begun. In the cen- 
ter of the lateral line the chromatophores are expanded; and on the right some of 


them are completely contracted on account of another dose of narcotic. A...A 
=Aorta V...V=Ventral vein. Chrematophores on these last two vessels 


drawn in from another uncontracted fish. X 25 
Fig. 27 Pure F. heteroclitus, just hatched. Shows red opaque chromatophores 
of the lateral line. X 127. 


178 FRANK W. BANCROFT 


30 


Fig. 28 F. majalis egg hybrid, four days old. Shows position of the heart 
which has just begun to beat, and the shallow vesicle, V. underneath the embryo. 

Fig. 29 Pure F. majalis, four days old. Same series and age as embryo in 
fig. 28. To show the greater depth of the vesicle, V. under the embryo and the 
normal position of head and heart. Heart has just begun to contract. 

Fig. 30 F. majalis egg hybrid, twenty-four days old. Dissected out of the 
egg shell at a time when a few fish of the same lot had hatched and when many 
had died. Shows vesicle V. under the embryo and the greatly stretched heart 
within it. 


LONGEVITY IN SATURNIID MOTHS: AN 
EXPERIMENTAL STUDY 


PHIL RAU anp NELLIE RAU 
FIVE CHARTS 
INTRODUCTION 


The observations and experiments herein recorded upon the 
longevity of some of the Saturniid moths were undertaken in 
order to discover the value of some of the theories that have 
been advanced to account for the duration of life. Much of 
the theorizing is based upon the insufficient and inaccurate knowl- 
edge of the ages attained by different organisms and the rela- 
tion of such length of life to their reproductive function, as well 
as to their environmental conditions. The attempt to place our 
knowledge of the duration of life upon a scientific basis demands’ 
the gathering of many data on many species, and on the mated and 
unmated individuals of both sexes. 

With these needs in view we found some members of the family 
Saturniidae in sufficient numbers to suit our purpose. A more 
important reason for this choice was the fact that they have 
aborted mouth-parts, and the adult insects take no nourishment. 
This would eliminate the probabilities of curtailment of life due 
to insufficient or improper food. 

The cocoons were gathered and carefully strung to trees where 
they could be subjected to the natural changes of the weather 
conditions during the winter. Just previous to the emerging 
time they were taken into a shed, the temperature of which varied 
but little from that of the outside. The imagines were kept under 
ordinary dome-shaped, wire dish-covers, which varied from 22 
to 32 inches in circumference. 


179 


180 PHIL RAU AND NELLIE RAU 


This work falls into the following divisions: The duration of 
life in: 

1. Samia cecropia. 1910—178 insects from St. Louis cocoons. 

2. Samia cecropia. 1911—112 insects from St. Louis cocoons. 
Cocoons placed in incubator. 

3. Samia cecropia. 1911—42 insects from St. Louis cocoons. 
Imagines placed in ice-box. 

4. Samia cecropia. 1911—283 insects from St. Louis cocoons. 

5. Samia cecropia. 1911—133 insects from Long Island 
cocoons. 

6. Callosamia promethea. 1911—170 insects from Creve 
Coeur Lake, Missouri, cocoons. 

7. Tropaea luna. 1911—60 insects from St. Louis and Pike 
County, Missouri, cocoons. 

8. Telea polyphemus. 1911—19 insects from St. Louis and 
Pike County cocoons. 


REVIEW OF THE THEORIES 


Before taking up the details of the work, it would be well to 
rehearse here briefly the various theories which have been 
advanced to account for the duration of life. 

First among these is the theory of Weismann, that the dura- 
tion of life of an organism 


be ges is really dependent upon adaptation to external condi- 
tions, that its length, whether longer or shorter, is governed by the needs 
of the species, and that it is determined by precisely the same mechan- 
ical process of regulation as that by which the structure and functions 
of an organism are adapted to its environment.! 

I consider that death is not a primary necessity, but that it has been 
secondarily acquired as an adaptation. I believe that life is endowed 
with a fixed duration, not because it is contrary to its nature to be 
unlimited, but because the unlimited existence of individuals would be a 
luxury without any corresponding advantage. The above-mentioned 
hypothesis upon the origin and necessity of death leads me to believe 
that the organism did not finally cease to renew the worn-out cell 
material because the nature of the cells did not permit them to multiply 


1 Essays upon Heredity, 2 ed., vol. 1, p. 9, 1891. 


LONGEVITY IN SATURNIID MOTHS 181 


indefinitely, but because the power of multiplying indefinitely was 
lost when it ceased to be of use.” 

In answering the question (continues Weismann, loc. cit., p. 20), as 
to the means by which the lengthening or shortening of life is brought 
about, our first appeal must be to the process of natural selection. 
Duration of life, like every other characteristic of an organism, is sub- 
ject to individual fluctuations. . . . As soon as the long-lived 
individuals in a species obtain some advantage i in the struggle for exist- 
ence, they will probably become dominant and those with the short- 
est lives will be exterminated. 


Lankester,* according to Romanes, has pointed out ‘‘a highly 
remarkable correlation between potential longevity in the indi- 
vidual and frequency of parturition.” ‘This correlation he 
attributes to generative expenditure acting directly to the cur- 
tailment of life.”’ 

Romanes,* who like Weismann sees in the duration of life an 
adaptation, does not agree with Lankester that it is the genera- 
tive expenditure ‘‘that causes the curtailment of life, but that 
it is the curtailment of life by Natural Selection, which causes 
the high generative expenditure within the lessened period.” 

In opposition to the utilitarian theory of Weismann is that of 
Gotte,® that 


oe all animals are mortal, and reproduction is in itself the 
cause of death. Reproduction in Protozoa is preceded by encystation. 
In this condition the organism passes into a non-living condition, from 
which it revives with renewed youth and renewed life; a similar condi- 
tion occurs in the egg of Metazoa, during a certain period in which it 
forms an unorganized, non-living body, composed of organic substances. 


Eimer’s® own opinion is that in the Metazoa as well as in the 
“Protozoa the germ cells are immortal; only the soma dies. 


“The latter is not really an end in itself, but rather its principal fure- 
tion is to ensure the maintenance of orzaniec life, by favoring reproduc- 
tion, by sheltering the germ-cells till their maturity, and in order to 
deposit them repeatedly; further, by the dispersal of the same in space, 


2'Tioe: clt-, p: 20: 

%’ Comparative Longevity, 1870, quoted by Romanes, Monist, vol. 5, p. 163, 
1895. 

4 Monist, vol. 5, p. 163, 1895. 

5 Quoted by Eimer, Organic Evolution, p. 67, 1890. 

5 Loc. cit., p. 68-69. 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 2 


182 PHIL RAU AND NELLIE RAU 


by incubation in the widest sense, and so on. Further it has the fune- 
tion of strengthening the power of endurance of the species by she inher- 
itance of acquired characters.’ “Reproduction is unending growth. 
Not reproduction is the essential cause of death. . . . . the 
soma is not really an end in itself, but rather its principal function is 
to ensure the maintanence of organic life by tavoring reproduction. 


Minot? seems to conclude that ‘‘the duration of life depends. 
upon the rate of cytomorphosis. If that cytomorphosis is rapid 
the fatal condition is reached soon, if it is slow the fatal condition 
is postponed.”’ 

Flourens® thinks that the length of life of an animal is equival- 
ent to five times its period of growth, while Nageli® thinks that 
“natural death does not exist in nature, for trees more than a 
thousand years old perish not by natural death, that is to say 
natural decay of their vitality, but by some catastrophe.” 

Metchnikoff® says: ‘‘It is impossible to regard natural death, 
if indeed it exist, as the product of natural selection for the bene- 
fit of the species. In the press of the world natural death could 
hardly come into operation because maladies or the voracity of 
animals so frequently cause natural death.” 

While Morgan" says that “in some cases the length of life 
and the coming to maturity of the germ-cell may be, in some way, 
physiologically connected seems not improbable, but that this 
relation has been regulated by the competition of species with 
each other can scarcely be seriously maintained,” he will ‘not 
pretend to say whether the mutation theory can or cannot be 
made to appear to give the semblance of an explanation of the 
length of life in each species.” 

Other theories of less importance are reviewed by Metchnikoff 
in “‘The Prolongation of Life’ and “The Nature of Man.” 


7 The Problem of Age, Growth and Death, p. 228, 1908. 

§ Quoted by Metchnikoff, Prolongation of Life, p. 40, 1908. 
® Quoted by Metchnikoff, The Nature of Man, p. 265. 

19 Loc. cit., p. 267. 

11 Evolution and Adaptation, p. 371, 1908. 


LONGEVITY IN SATURNIID MOTHS 183 


OBSERVATIONS 


In 1909 notes were made upon the duration of life in the Cecro- 
pia moth.“ The observations were made upon only a small 
number however, so it was decided to carry the work on during 
1910, on a much larger scale. New factors of much interest were 
discovered entering into the work of that year, so it was deemed 
best to continue an experimental investigation through a third 
year in order to get adequate and conclusive data on some of 
the phenomena appearing. 

These three consecutive years of observations on the Cecropia 
were made upon material gathered from the same locality, River 
des Peres, St. Louis. The parallel work was carried on upon the 
Cecropia moths from Long Island, New York, in order to ascer- 
tain whether there were any unusual phenomena in the St. Louis 
material due to purely local conditions. 


TABLE 1 
Mean duration of life 


a 2 a 
a *o 2 o a & 
a a 2 3 
LOT hoses a = a ois z E & = 
a & a & 3a 5a % o 
Bo) ip orate ts Seat: a E | af 
= 3 : e 
< 4% < Zz Sa <a 2 < 
a b = b < < < < 


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. 

DiMLOUIRE. caeeineeOel eto Otol weO0r “720 7.81) (7.70) 7.71) 7271 
S. cecropia. 1911. 

New) Works. os: «. 7.90| 8.54) 6.62} 8.25) 7.25 8.42 8.37 7.65 8.06 
S. cecropia. 1911. 

Incubator..........| 8.10) 8.31] 7.03} 8.80) 7.40 8.48 8.24 7.73) 8.00 
8. cecropia. 1911. 

WeeeBoxseen.sn oa 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 


33) 9.29) 6.79 


or 


T. polyphemus. 1911. | 


Table 1 brings together the means of all the lots of material, and will be referred to frequently 
for comparison. 


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 from 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 
loeal 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 cycle of each insect. But the fact that this abnormally 


oy et Locd BU Ua val vet Cia -_ OC Oe OOD te 1D OD OO HH OLD 00 red 
Te 2 ae oS IPE cs eae BS Ee 
ER TENS : z aE c | 
TM = : Br a : = SS Sarg | | 
Gaat S =—s3 es ial SSS a Pe ae ae ae a 
Be | 2 >| Ss eS isha sd |Po7s"| 
ote | = a nu E | [] 
26 | a ‘. = = a ee —_ [ee 
916 —— 2S = = P [ince 
1% = = —"\ —s . Se = 
mn 50% | ei =IA a m = = <a SS 
H oe | ee ie 7 pa | 2° 
. em ~” = Cc 
(©) ger | = aoe e =n a aa ies +| 
3 6r ae. = = z se ge ak 
e j2e can ae 
B ee | 
a [ 
Z, z If: 
eli | 
P at | 
2 a| 
_ a z I 
A E 
<4 gre 
a 9928) 
a =o 
Lee Es 
S z 
Sa! 
a i 
4 0 
lo} 0 
4 ih 
6 
"8 
8 
i) 
L 
‘9 
9 
| "¢ 
|e ae 
| DONGONANa 


|"foemuva, | [*2SSS2RRARARRHRRS TAPP Sula AS ShSARAARARRARRSS [OPT en mograaeeSs 


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, and the maximum reached 
was 87°. 

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. 

TABLE 3 
Early emerging, long-lived Cecropias, 1910 


DAYs 
a . 
CLASS | fio{ leo jo fie!) fol fie! fis} feo! fea} feo} fio} fro! feo] fro) JT MEAN 
lalaiccoeninm awa inino on rina cao Oon te NN Mm 
| eistotshstetel ie SK eRe Re eRe eS Oe NNANAANANI AC 
Siclarvenele ta na Atlas 
| |. | 11) | | 2 aE 1| 2) 2 1) | 1) 1) [15) 17.40 
a | 21) | 44 3) 3} 1) 1) 11.3) | 2} 1) 3 2 2) 3) a) | a) | 1132) 17.67 
voes| |] a) 2) | 1) 3,3) a) faa a 26, 14.83 
Unmated 9’s.........../ 1) | | 1) |! 2} 1}2a] | [aaa | jaja ja 2} 1\ | 1125) 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) 1) /41| 15.77 
All unmated insects ...,| 1| | 1| 1) | 3] 1) 1| 3| 2) 2| 1) 3 3) 31 4| 3/3 | 3133, 3) 2 1) 1) 2) 2) ‘| 2157 17.29 
SMIRK sc ceicisisin'sicis siesta | Yy |131 11 2} 3 3) 1) 1) 3} 3} 1) 4) 3) 3 2) 2 1) 2 2) 1) 1/47, 17.59 
PATIN QUE os ic afc se ciate +} YY) Y) 2) 2) 1) 1) 2) 8) 4) 2 22 1) 4 39) 3 1 311 2) 1) | 1/51] 15.79 
Whole population ...... 2) 1) 2] 2) 3 4) 2 26 5) 2 45) 4 5 4) 6 4) 4) 5 6 4 3) 2 12 2 3 1) 298 16.65 
TABLE 4 
Late emerging, short-lived Cecropias, 1910 
DAYS 
ea 19} ‘era | lies] Folehalika Shall balatciabe Lo) ys T. |MEAN 
esesrheeccssanassss=sesscs 
Pa eal leh lect sala mee iW ety 
IEREACUCUA Wc wctiicisecis te sis esiste| A |} 1 Yo} }2422 1 4 | § 118 10.62 
Unmated o"s............. }y ; 33H [2 13 13) 2 3 } 20 | 10.52 
Mated 9’s........ } } 3) 2 | 2433121 391 1 1 | 30) 9.46 
(Uiimated Q'eiesccs<<a cass esse] || } 1) | 3 2 1 | 1) | 2 1 1) | 12) 10.50 
All mated insects............-..| 2 | 3) 2} 2) 1) 2) 4) 5,5, 33 1) | 6 2/4) | 1 1 1/48) 9.90 
All unmated Insects... . | 7/351 13 | 318 4 3 1 1) } 32} 10.51 
JAN eel Beas on On GaARGA 1 } 11233 2413 7 2/6 1 38 10.56 
JN QS Vaos diye Aan SS eEDS 1) | 3) 1) 3) | 86 3/3, 2) 2) 1) 1) 2) 4) a) | 11 |2] | 42) 9.76 
Whole population.......... | 2} | 3) 2) 4) 1) 7/9 6 5 6 3) 4/1/96 7 | 11 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 faetor 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 i Tr ] T. |MEAN 
4 5 6 7 8 9 | 10} 11 | 12} 13 | 14} 15 

Mated o’s....... Seo & kl G 4 20 «8.10 
Unmated os..... 8B} 4] 4) 11] 8) 4 2 39 | 8.31 
Mated 9’s..... I 3/10) 9| 2) 8 33 | 7.03 
Unmated 9's........ WY a3 GR StS Ry PH | 8 | 20 8.80 
All mated inseets. .... Pell bits: des) Si!) 14 4 53 | 7.40 
All unmated insects. . 4/ 5| 8 | 14/13] 6) 4] 4] 1 59 | 8.48 
JAIN CSU RES RALOEpac 5] 7] 8112) 14) 4) 7} 2 59 | 8.24 
All s..Ber........ Wes) TL 13) 6113) 2) 1) 2] 1 53 | 7.73 
Whole population J] 9) 18) 21) 17) 27) 6) 8) 4) 1 112 | 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 = — 


| ~ = ay MEAN 
6 7| 8 91011 1213/14 15 1617 18 19 20 21 22 23 24125 26 27 28 29 3031 32 
| fe] he 
Males... Fes! la} | | 22423 1) 2) 4) 1 1] | 1) 2 1] | 1) 1] 1) -24 | 17.58 
Teenie | | a) | aaa y.} 4) 13} | aaa 3 18 | 19.39 
3] 2) 3] 3) 6 2 1) 33) 3,1 4) 1 41/1) 42 | 18.60 


All 30 : 1 


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 


DAYS 


DATE OF 
EMBERGENCE 


13° 14 


(iis 
~ 
on 
3 
x 
oe 
=) 
= 
—) 
= 
_ 
) 


nt 
fo) 


CS) 
_ 


nee 
ee 
a Oe a ee 


_ 
a 
rm 


Ke rmnmeb 
_ 


to to 
o = 
_ 
oo 
i) 
bo 
is) 


wrwr 
mawrnre 


Cn a 
to 
r<) 
w 
eo 


ee a ad 
AWnanranwnwore 


_ 
no 
to 


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 


1909 1910 1911 
March. F P 44 58 47 
April... 38 : 54 56 54 
May.. 64 61 71 
June...... 75 72 79 


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 = ea = = — T MEAN 
3) 4 | 5/6’) 7 | 8 | 9) | 10] 11 | 12) 13 | 14 

Mated o's... ee 1 4| 3 4) 5| 4 2 eiap2 
Unmated 2's _ 3| 4| 8113) 26) 24] 21/15) 5| 2 121 | 7.73 
Mated 9's. e200) |) 1401) Al a 28 | 6.96 
Unmated 9s 2| 2) 13/11) 20)-28| 11) 14) 5) 2\) 4] 1] 113 | 7.90 
All mated insects 1 11] 5/10} 8| 9) 5 49 | 7.20 
All unmated insects. . 5| 6)| 21/ 24| 46) 52| 32) 29) 10| 4] 4 1| 234 | 7.81 
All @'s.:..... 4| 4) 12] 16) 26| 28] 26] 19) 5) 2 wo 177 
All 9's 2'| 21 20 | 13 || 30 |'32) 15) 15) 5] Bi) 4) 1) ra Nivea 
Wholelpooulation 6 6 32 29, 56. 60 34-4 4| 4| 1) 283 | 7.71 


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 


“ From 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 <= == Tr. MEAN 


INA est paar atiber ioc Dan omuceates Dt) 7) oi eee 1 20 | 7.90 
PTI ATCA VAL awe asec puiea daa pee 2 1 3/12] 6] 12)14) 5] 1 56 | 8.54 
APOE SUS roe fiero re, diz cts co 1 | 2 7 7) 2 1 1 21 6.62 
Unmated 2's........ 1 BNA 38 A) aes 4 1| 36 | 8.25 
All mated insects.................- 1 3) 8) 14! 7) SY] 2 1 41 | 7.25 
All unmated insects. . 1) 2| 3} 7| 20) 10) 19) 19) 8) 2 1| 92 | 8.42 
I UECHES pes etatsteteteeleles 2} 2) 4119) 11) 16) 15] 5) 2 76 «8.37 
PUPS otsice) ei Mats forefereisie eitielciei fale sivahr ss 2 4/11/15] 6 8 6 3 1 1 37 7.65 
Whole population... 2) 2) 6] 15) 34] 17) 24| 21) 8} 3 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 I1 


Prometheas 
DAYS 
CLASS Tv. MEAN 
Aine pA eo} 6) 7 8 9/10) 11 

MALOU ECM Bec tacenias ec: man eease sec | ey ONG aC | 1) 19 | 3.74 
Unmated o's. 6| 20] 28) 24) 9| 3 90 | 4.21 
Mated 9's.... Ges oe sie a ot 1 22 | 5.18 
Unmated 9’s.......... ; ioe See Shae 6:1 6 39 | 6.54 
All mated insects....... 2/15) 8) 6] 3) 4) 1 1 1} 41 | 4.51 
All unmated insects... 8/22] 30/ 26/17/14] 6) 6 129 | 4.91 
AUS URS Sper eee § | 29) 34] 25; 9) 3 1 109 | 4.13 
Ait te Tae ct 2| 8 t 7} 11) 15 7 7 61 6.21 
Whole populatio .--| 10! 37 | 38 | 32| 20/ 18] 7 ) 2 


1 
1 

i 

| 9 


194 PHIL RAU AND NELLIE RAU 


Mayer found that most of the C. promethea lived about 3 
days. Table 11 shows that the life of these insects varied from 
2 to 11 days, with the mean falling at 4.8 days. These were kept 
in confinement; Mayer does not state the source of his data. 

The accompanying table correlating the length of life with the 
time of emergence shows no relation between these two factors. 


TABLE 12 

me 
a DAYS 
Et i—— — — = 
ag 2| 3) 4) 5| 6] 7} 8| 9/10/11 
5-8 eG 1 
9 | | | 
10 ail 1 | 3 
rr | ae 
12 1 1 
13 | | | 
14) 0) 2) | Le} 6 
15 ese a 1] 5 
16 1] 2/5 2) 3] | 1 14 
17 1) 22: |(et| | 6 
18 2| 4 5| 1| bole 
19 sO) 53] Py) 65/1 2 
20 | 1) 4) 7) 2) 1 15 
21] 1] | 2] 5] 2] | | 14 13 
22 Sil eet edn 1} 7 
23) 4| 5) 2 1| 12 
24 Sid } 2 | 15 
25| 2| 3| 5| 2} 2 ie 8 
26 | 5 jai 1| 1 Allg 
27 Bi Si |i rill oles 
28 1 | 3| 3) 1 8 
29° «1 | 1| | 2 
30 | 2] 2 
31 ‘ip | at 1| 1 | 4 
6-1 ee | ; 1 ; 2 
hpi) a) | 1 
3 ener 
4 ih | | 
5 | | 
6) | | 
7 | | | | | 
8. | 1} | leet 
9 
10 | 
u| | | | 4 | ks 

10 | 37 | 38| 32] 20] 18| 7 7| | 1170 


18 Mayer, A. G., Psyche, vol. 9, p. 16, 1900. 


LONGEVITY IN SATURNIID MOTHS 195 


TROPAEA LUNA 1911 


To gather more data on other species of this family, cocoons of 
Tropaea luna were obtained. Sixty-two imagines (36 males and 
26 females) emerged between May 10 and June 4, with one excep- 
tion on April 23. Records were kept on 60 of these. 

It will be seen that the length of life of males and females was 
almost equal and that the mean for the entire population was 
greater than that for C. promethea. Table 13 gives the details 
of this work and is self-explanatory. 


TABLE 13 
Luna 
DAYS 
CLASS —_—_—— T. MEAN 
3| 4!) 5 7} 8| 9 
BUM CNCIE Ratpicle as/oi-1aje!s cielalolniv's cis sle\sicwvic a-claaipiaie wie Sa ciseisis erainbia [mu 1 2 1 5 4.60 
Unmated o’s........ at stasiorsistat Drie! w/aparciaiatara' sre aieiale ss : 3} 3] 3] 8] 8] 3) 2] 30 | 6.07 
Mated 9's.. Scone Eveitetathaiawieletersters pisler tie rats oy Lt 5 6.60 
Unmated 9's.... Witdannngoccont 1}; 5} 4} 3] 2] 4] 1] 20 | 5.80 
All mated insects LY} Dae 1 10 5.60 
All unmated Insects 4} 8) 7/11)10] 7| 3) 50 | 5.96 
TN oO ae en 4 4 5 9 8 3 2 35 5.86 
All 9's 1 5 4 6 3 5 1 25 5.96 
Whole population... 5/°9/] 9/15) 11}] 8! 3] 60 | 5.90 


TELEA POLYPHEMUS 1911 


Twenty-one cocoons of T. polyphemus gave 13 males and 8 
females. Notes on longevity were made on 19 of these. None 
of this species mated in confinement. Table 14 shows a remark- 
able difference in the life of the sexes, the male varying from 3 
to 7 days, and the female from 7 to 12 days. The duration of 
life of the entire population was 6.79 days. 


TABLE l4 
Polyphemus 
DAYS 
CLASS ] T MEAN 
3| 4) 5] 6) 7| 8] 9| 10) 12] 12 
TEL) oo uaSS ae aR Re AB =e es Lees 12 | 5.33 
Females....... oPPe: : SN GES 1 4) 1 1 7 | 9.29 
Whole population DU) rj io Gl ae Ge 4) 1 1. 19 | 6.79 


1°6 PHIL RAU AND NELLIE RAU 


PROPORTION OF SEXES 


The accompanying table shows the proportion of sexes of the 
different lots. The grand total shows the males to be in excess, 
and especially so in the Prometheas. Here the proportion cor- 
responds well to the 111 males and 65 females noted by Mayer 
in 1899.16 

TABLE 15 


Proportion of sexes 


YEAR SPECIES MALE FEMALE TOTAL 
1909 SS. (CECKODIA.- eee ee 43 25 68 
1909 S. ceeropia...2.....- Sere Si: 22 13 35 
1910 SAI CECLOD1 An eee eee 101 104 205 
1911 S: CECrOpla: 2 eens eee 171 168 339 
1911 SI CECTOPIE IN Gwe eee 79 60 139 
1911 S. cecropia, incubator......... 72 90 162 
1911 C. promethea...... pitas. eet 116 67 183 
1911 Tuna. eee 36 oe 36 26 62 
1911 t..polyphemus!=..-. = 42a: 13 8 21 


Potal..d.ctc..215-Pe oer Ree : 653 561 1214 


PRIORITY OF MALE EMERGENCE 


The evidence gleaned from this material substantiates Dar- 
win’s conclusion that the males are the first to emerge. In every 
lot excepting the Lunas this was apparent, and in nearly every 
case where a break in the continuity of emergence occurred, the 
first subsequently to emerge were also males. 

In order to get some tangible proof of this priority, the mean 
date of emergence was gotten for the males and females of the 
different species. The difference between these dates would ob- 
viously be the mean priority of all of one sex over the other. The 
differences given in table 16 are in favor of the males in all cases 
excepting the Lunas. 

The emergence of the insects on each day is shown in the accom- 
panying curves. Graphs were made for only those species of 
which we had sufficient numbers to make the curves reliably 


16 Psyche, vol. 9, p. 15, 1900. 


9b 


} 


g. 3 The emergence of 79 males and 60 females of Samia cecropia; New 


York, 1911 material 


i 


F 


Fig. 1 The emergence of 101 males and 104 females of Samia cecropia; St. Louis, 1910 material. 


ial. 


Louis, 1911 materia! 


Fig. 2. The emergence of 171 males and 168 females of Samia cecropia; St. 


aoreeneranneseaoeroarovnenne 


SSSSISRRRRRK 


Tiny 


DUNES genet 4 | vi 
Vga elininamyensiess orange Yeates te cate geal 


| i } Ww \ 
svat ob iain mene 
a Tl ‘ 
i . Pall i i ey i 
in 1 || he , 1 


i 
Ty alae Gah eh 


Pay aie he? yin 
! phantom at ay la er Drennan ages he» oo a AA +e 
| t ht we oe es 4 
Henke A aE MER Ty 8 
e Bee covet, away 
ae ee Le Rrt ae! 
} ’ 
hans 2! 
| r 
| : 
rr i 
| \ t iF 
i yh) 
i ay a 
; i ‘i co t 
t = tH as, 
. 
eT I fi pe 
i wate 
me SMe) i 
; Hy) tis 
ee ka Me 3 ae 
i of? Siaeeviere wave. tO. Bel Aatiek 
f + mie iy mew eh SM athe dee a " 
y d a \ 
" ive oe 
t y 
be 
: ‘ ae ex 
ae f a a 
| i ie Al i 
‘i Pe at Fal a 
yee’ iy : i 
hl aK jee ; 
ft ‘ 
j 
; i ‘ yt re 
4 ‘ay nee r 4 Ma ky oe shine f wg ae ai a . 
| ae hae ca 4 ¥ ‘ bi Ft Mi ‘ hitee 
(fe i ny redpaticy Ait Nees daha Mah Mace ta 
' au au i Pa ybe ty : * ay : 
HAND : 
4 


LONGEVITY IN SATURNIID MOTHS 197 


TABLE 16 


Days 
CGacropia,y1 910 kes wearer te ae eerie ays cops orempiehe 2 Sree iclels a <n ...1.02 
Weeropias 19 lilo eee see ae cing dete «oc o/ds,siealeleiy siz ois vole «is eee 23580 
CeCropia wINCMDA Olesen ete ea eae cerns Fetes sees 4.51 
@ecropias NGweVOEKM@ane. fac swae ince hee etie Gaerne < beeen aes 2.20 
[Prometheameece cere case ok oR od Mee ane ticle tc bares Fayrcs She aw 2.68 
Teo halide seo 5 oe ORen Benepe PO CeRIOS Ce See See aCe eens 2.60 
Tibi sOBie acisk iouat dade Sakae 6 eee aOehee aaeR neo Octe HIM eee aes 1.33 


significant. Solid lines are for the males; dotted lines are for the 
females, and the dash and dot lines represent temperature. 
The figures on the base line are the dates when emergence 
occurred, the small numerals on the left are for the number of 
individuals, and the large figures are degrees of temperature in 
Fahrenheit. One can see at a glance how the males throughout 
keep slightly in advance of the females. 

An attempt was made to find whether any correlation exists 
between temperature and rate of emergence. No absolute state- 
ment will be ventured upon this point—the data are submitted 
for the readers’ own judgment—but to us it seems highly improb- 
able that such a close agreement as appears in a large part of the 
data should be no more than coincidence. <A careful inspection 
will reveal a closer agreement than might at first be thought, for 
of course it must not be expected that at the extremes of the sea- 
son, when very few individuals emerge, there will be a marked 
appearance of such a correlation. 

In justice to the evidence, however, attention should be called 
to the fact in the case of those which began to emerge about May 
8, 1911, that although the temperature was then only 71°, yet 
that was a decided leap above the temperature of the preceding’ 
week, which was below 55, or about the same as April. The 
temperature for the balance of the month of May continued abnor- 
mally high. 

Darwin mentions!’ that this male priority is true of frogs, 
toads and the majority of salmon, ‘“‘and throughout the class 
of insects the males are almost always the first to emerge from 
the pupal stage.’’ Whether or not this holds throughout the 


17 Descent of Man, p. 240. A. L. Burt’s reprint from 2 ed. n. d. 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 2 


198 PHIL RAU AND NELLIE RAU 


class of insects does not seem to be fully known, but in this mate- 
rial it is clearly evident. 

An explanation of this early male emergence was attempted by 
Darwin. ‘The cause of the difference between the males and 
females in the period of arrival at maturity is sufficiently obvious. 
Those males which annually first migrated into any country, or 
which in the spring were first ready to breed, or the most eager, 
would leave the largest number of offspring and these would tend 
to inherit similar instincts and constitutions.” 

This explanation is far from clear when we try to apply it to 
Saturnid material. A more probable explanation would be that 
the males emerge first merely because they do not require-such 
a long period of development to mature their gonads. The 
female has a large number of ova to nourish, probably requires 
a greater amount of food in the larval stage and a greater period 
of pupation, and late emergence follows. 

But the item of interest is to find whether early emergence of 
the males is an adaptation, as Darwin seemed to think, for the 
benefit of the species. From the data of 1910 it seems that 
‘adaptation’ gives us males at one time, and when these are almost 
dead or too old to mate then a succession of females, many of 
which die without leaving issue owing to the lack of mates. To 
illustrate this let us say that with 101 good males and 104 females 
in 1910, 52 males (more than one half) could not be mated, and 
even though we called to aid some of the 69 stray males which were 
attracted to the laboratory, still it was possible to supply only 
65 of the females with mates. It would seem that for the benefit 

* of the race both sexes should emerge at about the same time (since 
both are mature for mating at about the same age) in order to 
eliminate any expenditure of energy in finding each other, and 
to eliminate any possibility of a great proportion of the males 
and females dying without having mated, in spite of the fact that 
the sexes may be equal.!s 

It was suggested that the early maturity of the male may be 
due to a shortened period of development. This is deserving of 


18 Tt may be that the males of one area migrate to another territory to accom- 
plish mating, but we have no proof to that effect. 


LONGEVITY IN SATURNIID MOTHS 199 


careful investigation on the length of pupal, larval and egg stages 
of both sexes, and might lead to a simple and proper explanation 
of this phenomenon. 


COMPARISON OF THE LENGTH OF LIFE OF THE SEXES 


In 1909 it was found that the males of the Cecropia were longer 
lived than the females. In comparing the duration of life of the 
sexes in table 1 we find the males to have lived longer in the 1910 
Ceeropias, the New York, and the Incubator Cecropias. In the 
Lunas and the 1911 St. Louis Cecropias there was not a signifi- 
eant difference. In the Polyphemus and Prometheas, the oppo- 
site is true; we find a great difference in favor of the females 


COMPARISON OF THE LENGTH OF LIFE OF MATED AND UNMATED 
INSECTS 

In the entire Cecropia material and also the Prometheas, we 
find no appreciable difference in the length of life of the mated and 
unmated males In the females of the six lots, however, we see 
a significant difference apparently resulting from this condition, 
the unmated females being the longer lived. It is very interesting 
that mating may be such a tax upon the females (it cannot be the 
Ovipositing, for the unmated females also experience that) as to 
effect a curtailment of life. No such curtailment due to mating is 
detected in the males. In the Lunas, we find a very slight differ- 
ence in favor of the mated females. This result, however, is derived 
from a small number. Inspection of the two columns of table 1, 
all mated and all unmated insects, shows that the balance swings 
in every case toward longer lives in the unmated insects. 


LAPSE OF TIME BETWEEN LAST EGG LAYING AND DEATH 


According to Weismann’s theory one would expect that in a 
monogamous species the males would die soon after mating, 
while the females would live long enough to completely oviposit. 

In the five lots of Cecropias we find the mean duration of life 
to be even greater in the mated males than in the mated females. 
Surely the continuation of such a long, useless life in the male 
cannot be an adaptation for the good of the species. In both 


200 PHIL RAU AND NELLIE RAU 


the 52 fertilized and the 28 unfertilized females of 1910 we found'® 
that life was cut short while most of the insects retained many 
eggs. Now if the duration of life be an adaptation for the good 
of the species, why were not such lives permitted to continue? 
In many cases also a lapse of time, sufficient for completing oyvi- 
position, intervened between the last egg-laying and death. 
Tables 17 and 18 shows the number of eggs each female retained 
and the number of hours it lived after ceasing to oviposit.?° 


TABLE 17 


Fertilized females 


EGGS HOURS EGGS HOURS EGGS HOURS EGGS HOURS 
240 0 41 16 12 ah, 14 3 0 
165 8 41 ih 10 | O 3 30 
151 0 34 0 9 40 2 0 
119 15 31 14 9 ? 1 7 
105 i 29 14 | 8 0 1 15 
101 16 o7 | 6 || Sime = 238 1 14 
95 38 22 | Zo MAS a | ? 0 96 
94 6 20) | |yena0 | 7 Peon tal nl axial 
93 60 20 0 -| 6ic pW 07 4 4 
SON al, ly 18 15 M5 Zit NOs |) 9 38 
72 | 0 15 FI | 5 | 0 | 0 8 
56: «|| je al2 14 38 5 12 0 38 
aN aa 13 15 3 4 0 2 
TABLE 18 


Unfertilized females 


EGGS HOURS i EGGS HOURS EGGS HOURS 
270 2 103 14 40 2. 
27 | = A 102 2 37 0 
247 ? 100 ? 32 24 
189 | 12 85 6 31 30 
1799 Ml 0 80 0 23 ? 
144 7 | 68 0 21 0 
137 | 6a” ih Pace 2 17 ? 
135 Zoe yall ok 653 0 2 ? 
153° S| aelOe es 48 36 2 ? 
110 ? 


19 Trans. Acad. Sci., St. Louis, vol. 20, p. 314-315, 1911. 
20 Less than six hours is designated by 0. 


LONGEVITY IN SATURNIID MOTHS 201 


Here we may see many insects, both mated and unmated, 
dying in the very midst of egg-laying. Then again we see others 
which perfectly or almost perfectly oviposited, continuing a useless 
life in some cases up to 38 or even to 96 hours. 

The long life of some females after completely ovipositing, and 
the4duration of life of others to [insufficient Jcomplete egg-laying, 
and the long useless life of the male all lead ‘to the belief that the 
duration of life is not, as Weismann says, an adaptation for the 
good of the species which came about through Natural Selection. 

It seems more natural to assume that the duration of life de- 
_ pends upon the amount of reserve nutriment which the insects 
_ acquire at an earlier stage, the activity of the insects, and the 

climatic conditions. The climatic conditions seem to influence 

_ the insects’ activity; the activity effects the expenditure of reserve 
nutriment, and this expenditure controls the length of life, for 
the nutrition of the imago depends wholly upon this reserve. 
In short, it seems that the length of life depends to a degree upon 
physiological processes. 

In 1911 data were gathered on the completeness of oviposition 
and the lapse of time between last egg-laying and death in C. 
promethea,?! and the same facts were found to hold for this spe- 
cies as for 8. cecropia. In Promethea it was not definitely ascer- 
tained whether or not they are monogamous. 


THE RELATION OF LONGEVITY TO THE REPRODUCTIVE FUNCTION 


Weismann says: “‘No better arrangement for the maintenance 
of the species . . . . can be imagined than that supplied 
by diminishing the duration of life and simultaneously increasing 
the rapidity of reproduction.” If this were true of the Cecropia 
moth we should find this monogamous species living only long 
enough to carry on the function of reproduction, i.e., males dying 
soon after mating and the females living long enough completely 
to oviposit. This we by no means find. Table 1 shows that the 
males live about as long as the females, even though they are of 
no further use to the species. We have shown that among the 


21 Details are in course of preparation for publication. 


202 PHIL RAU AND NELLIE RAU 


females some lived a considerable time after all eggs were depos- 
ited, others died retaining many eggs although they had ample 
time to completely oviposit, and still others died in the midst of 
ovipositing. In 1910 it was found that there was absolutely no 
relation between completeness of oviposition and (1) long life, 
(2) the longer or shorter time spent in copulo, or (3) the age of 
the insects at mating. The mated females, however, oviposited 
more completely than the unmated ones, although they had less 
time to devote to it owing to the long time spent in mating. 

In considering whether or not the length of life is an adapta- 
tion for the good of the species, the completeness of oviposition 
seems to be a factor worthy of attention. The discoveries in the 
relation of long life to completeness of oviposition, as well as the 
long, useless life of the males, do not seem to substantiate this 
theory. 


CONCLUSION 


When the facts gleaned from these observations are compared 
with the various theories which have been advanced to account 
for the duration of life, one becomes conscious of the fact that 
much direct work must be done before any broad generalizations 
can be applied. Very little is accurately known of the normal 
ages of the different members of the animal and vegetable king- 
doms, and still less on the relation of longevity to reproduction— 
why it is that one organism lives for a certain period while another, 
which may mature in the same length of time, or may attain the 
same size, or live in the same environment, attains an entirely 
different age. 

Weismann, as we have already stated, thinks that the dura- 
tion of life is regulated solely by the needs of the species, and that 
this came about through natural selection. Eimer hints about 
the inheritance of acquired characters. Morgan seems to think 
that the problem of the duration of life is one for physiological 
investigation, but goes on cautiously to mention the mutation 
theory. Thus it continues, each man finding in the duration of 
life a conformity to his own already formulated theories. Whether 
it is a subject to be considered in any theory of evolution 


LONGEVITY IN SATURNIID MOTHS 203 


will be known only after much work has been done upon the 
relation of longevity to the function of reproduction in a vast 
variety of species. 

Our work leads us to believe that there are still factors unknown 
which influence the duration of life. One of these, the influence 
of climatie conditions upon the length of life in the Cecropia 
moth, we have been fortunate to discover. If it were only adverse 
conditions curtailing life, no great significance could be attached 
to it, but when favorable, though abnormal. conditions were dis- 
covered and supplied, life and activity continued far beyond what 
was thought to be possible. This has hitherto not been considered 
in any of the theories, and the experimental work here recorded 
shows this factor to be of importance. There are no doubt many 
other influences awaiting discovery, which will have to be con- 
sidered before we dare philosophize upon the subject. : 

It would be well to suggest some of the phenomena yet to be 
investigated upon a wide variety of material before a theory can 
be firmly established to account for the duration of life. 

1. The difference in the duration of life of the sexes. 

2. The duration of life of mated and unmated individuals of 
both sexes. 

3. The proportionate numbers of the sexes. 

4. The maturing of one sex before the other. 

5. Monogamy or polygamy. 

6. The relation of longevity of the female to the welfare of 
the young. In some forms where the parents do not care for 
the young, is the duration of life sufficient for bringing forth all 
the young (e.g., complete oviposition or seed-bearing), and in 
those which care for the offspring, is the length of life adequate 
for the necessary care of the young (e.g., food and flying in birds)? 

7. Number of potential ova that the organism may possess 
when overtaken by death. 

8. Does old age cause the inability of the individual to bring 
forth the young, although sexually capable? (e.g., lapse of time 
after incomplete OViposition in the Cecropia). 

9. What proportion of the eggs deposited by a mated female 
are fertile? 


204 PHIL RAU AND NELLIE RAU 


10. In animals (similar to those considered in this paper), 
in which the female is fertilized once for all, is there any relation 
between time spent in copulo and the fertility of the eggs; i.e., 
does a long period of copulation insure the fertility of all the eggs? 

11. Is copulation itself correlated with longer or shorter life? 

12. The effects of climate. 

13. Food conditions of the adult; its effect upon the present 
and the subsequent generations. 

14. Nutrition and environment in developmental stages. 

These ideas, which make no claim of completeness, were gleaned 
principally from work with these insects, hence many are inade- 
quate or cannot apply to other forms. It is true that many organ- 
isms will not permit of such investigation, and that confinement 
in some cases will cause marked changes. It may be years before 
sufficient and conclusive data can be had for the solution of the 
problem, but the vital importance of the phenomena of longevity 
in relation to the interests of the human race, as well as biologically, 
should at once arouse investigators to the accumulation of such 
data, even though it be only as by-products to their chosen line of 
work. 


St. Louis, Mo., October 30, 1911. 


OBSERVATIONS ON THE ORIGIN AND SEQUENCE 
OF THE PROTOZOAN FAUNA OF HAY INFUSIONS 


LORANDE LOSS WOODRUFF 
From the Sheffield Biological Laboratory, Yale University 


FIFTEEN FIGURES 


CONTENTS 

me General introductions tietmat cases fects ces ses -2 as os sacs Sree 206 
u. Origin of the protozoan fauna of hay infusions. .... “ae 5 Picts eee 
BSS HXPOrIMe Nts raecsyeir He vise - SO ee ne ae eC 208 
Bes OSUltgesirassrrs yade cte cies ai a aS AeSe Pig 210 
GC OnclUsIOUN ee ceemeet: cet etree ee flees ure se aacce ced 213 

mr. Relative number and sequence of repr Eentotive acleroni FESS in hay 

NOAUBLONG': sfeters cic ardsren caste use a Tas EER icant 5 2 

A. Experiments. . See Mires aero 215 

B. General Sbservations on the course of dev elopment of hay infus- 
ions. AF ie Perro STONERS taki ee en erie : 222 
C. The a: B end Cp groups ae safiatons ts. . 224 

D. Time of appearance, maximum number and disappearance of rep- 
resentative protozoan forms at the surface of the infusions. .. 225 
Me VIO ACNE eo nets Stn tee ae » oS oe I erry ee a 
2 CGO POd atu iyahs ak: take sn rs oe Ae en |. aici Sia RO 
Sa LLY PO LLC DG Avascniey. sue cles ees ots ae Dee Can fire 

4 (PATAMACCWUMG «< . .sieie5 so Noble: ite 220 Cog eg ee ea a ee ee 

RISA VOLUICOL ERS hrs shee. ny. Kian cicteetn arierce soe eee Me tose oO 
Gap AM ORDA tens ct eis acess csleen wie ET Nia chia a abe 3 TS 2o5 
iv. Summary of surface observations....................--- 8 est aa EAL 
v. Protozoan fauna at the middle of the infusions............ ee i .. 244 
vi. Protozoan fauna at the bottom of the infusions.............. Re eels 
LAE OSIMEUSL ODN eee ete ee Ao eMe es crisie eh Ceili a as ne woawgscle Je tee AG 
2 wv BP NbUsIONRe ecco cee eens as Sf he, ARO SEEE 247 
eon Conlin UISTOUS uiitety Wat cate totes ic Ge echis ec. es chu s 250 

vu. Discussion and conclusions from the observations on the sequence of the 
surface, middle and bottom fauna............ ... 250 
USER OR AOR ort op Ben CRORE ee ae eee z chnaeeed Mees 250 
ey. Middleviaun arcs ces dc ainee caw ce 2 eee Poke deer Oe 
ee OULOMMIDONIN emInoar eee rei lee's, tic care ae - DAL a ee 
4, Factors determining the sequence........................ ots «ofe2OG 
DeMDECLIME MINIM DELS Safes. cidaicnc c cwac sats toMelelee « slenwe ws ose 0209 
SV irem CLOTH TU EL OLIN chiara TICs chia MaIa en cle eRRiSS foc vis crarafardlanicrw s/eiabate 260 
Tek, SWNT ac oo Qagete ant 6 COROT OnO OS Ge eT OGT Te Cee Ean See ateCtonrr: 262 


206 LORANDE LOSS WOODRUFF 


I. GENERAL INTRODUCTION 


Although hay infusions have been one of the chief means of 
providing organisms for microscopists from the early days of 
Leeuwenhoek, there are comparatively few published data which 
have been secured through a careful study of the origin, relative 
number, and sequence of the various organisms which abound in 
them. It is a well known fact that a hay infusion presents a 
kaleidoscopic series of phenomena from its inception until it 
finally reaches a stage of sterility, or, in the presence of sunlight, 
of practically stable equilibriumin which animalsand green plants 
become so adjusted that a veritable microcosm exists; and it is 
also generally accepted, largely on the basis of casual observation 
of infusions made up for one purpose or another, that the organ- 
isms appear and disappear in quite a regular sequence. 

It seemed desirable, accordingly, to attempt to study the fauna 
and flora of representative infusions by some comparatively exact 
methods. The first intention was to make a comprehensive tabu- 
lation of the entire animal and plant life of the infusions studied, 
including bacterial counts, as well as to follow the chemical and 
physical changes in the medium. This proved to be impossible 
without the aid of more assistance than was available.t Conse- 
quently the study was chiefly confined to a careful observation of 
the Protozoa which appeared, and especially to certain charac- 
teristic forms which were present in large numbers in practically 
all the infusions studied. 

This general problem has been considered by Peters,? but more 
data were needed for points of attack on the biological effects of 
one type of organism on another, and this survey of the protozoan 


1 Certain chemical analyses, chiefly in regard to the acidity of the infusions, were 
made by Dr. M.S. Fine, and his results are published independently in the follow- 
ing paper, in this journal, entitled: Chemical Properties of Hay Infusions with 
Special Reference to the Titratable Acidity and its Relation to the Protozoan 
Sequence. 

2 Metabolism and division in Protozoa. Proc. Amer. Acad. Arts. and Sci., vol. 
39, no. 20, 1904. Chemical studies on the cell and its medium. 1. Methods for 
the study of liquid culture media. Amer. Journ. Physiol., vol. 17, no. 5, 1907; 
Il. Some chemico-biological relations in liquid culture media. Amer. Jour. 
Physiol., vol. 18, no. 3, 1907. 


PROTOZOAN FAUNA OF HAY INFUSIONS 207 


fauna of hay infusions is preliminary to further studies on the 
interactions of particular species on each other. 


ll. ORIGIN OF THE PROTOZOAN FAUNA OF HAY INFUSIONS 


The point first considered was the source or sources from which 
the protozoa which appear in infusions of hay are derived. This 
general problem has, of course, been treated at length in the long 
series of experiments on spontaneous generation which occupied 
the attention of biologists for several centuries. The present 
experiments were planned to determine the best method of making 
up infusions for the purpose of the study of their biological cycle, 
and incidentally to show the relative importance of air, water and 
hay, and whether some forms appear in infusions chiefly through 
one of these channels and others through another. 

Hay is generally considered the chief source of the protozoan 
life of infusions. Kent? in 1879 studied the question 


from whence (are) derived all these myriad organisms frequently pro- 
duced in such abundance as to literally jostle each other for room in 
every drop of water extracted forexamination? . . . . hayfrom 
different localities was placed in maceration and examined continuously 
from its first contact with the fluid medium, from periods varying in 
duration from a few days only to several weeks. The water added to 
the hay was of the purest possible description, and was frequently boiled 
for some time to prevent the introduction of extraneous germs. In all 
instances, the results obtained were broadly and fundamentally the same, 
and differed only with respect to the specific types found living together 
in the separate infusions. Even here, however, the general dominance 
of two or more special forms was notably apparent. 


Kent was satisfied, then, that the organisms were derived from 
the hay, and microscopical examination of the mode of distribu- 
tion of the cysts upon the lowermost blades, colored brown or 
yellow from incipient decay, led him to conclude that ‘‘all the 
essential conditions of their life cycle had been passed in close 
connection with it.” He put this conclusion to a practical test 
by gathering grass saturated with dew during a heavy fog and 
studying it without the addition of any water. 


+A manual of the infusoria. London, 1880, pp. 135-141. 


208 LORANDE LOSS WOODRUFF 


In every drop of water examined, squeezed from the grass or obtained 
by its simple application to the glass slide, animalcules in their most 
active condition were found to be literallyswarming . . . Their 
purpose in life, as in the case of the animalcules “inhabiting artificial 
infusions, is to break down and convert into new protoplasmic matter 


this otherwise waste product . . . . Tomaintainthe balance here, 
however, and to check the too rapid increase of the various herbivorous 
monads, we find other types . . . . developed side by side with 


and feeding in turn upon the plant-eating species. 


Following Kent’s method, I have examined grass from the cam- 
pus wet with dew and light rain, and have obtained substantially 
the same results. Active forms of various flagellates, chiefly 
monads, and ciliates such as Colpoda, Chilodon, ete., were ob- 
served swimming in the moisture on the blades of grass. I have 
not found them in such great abundance as described by Kent, 
but still in sufficient numbers to make an interesting demonstra- 
tion. Goodey, also, in his recent study of the Protozoa of the 
soil found a number of active forms among the surface vegeta- 
tion.4 


A. EXPERIMENTS 


Twenty-four hay infusions were made up, and kept in a well 
lighted room in the laboratory at room temperature. Twelve 
contained hay cut near the laboratory, and twelve practically 
pure timothy hay from a farm near New Haven. These two sorts 
of hay were designated, for convenience, Y and T respectively. 
Each infusion consisted of about 5 grams of hay in 1 liter of tap 
water, and was contained in a flask with a capacity of 1500 ce. 
These infusions were divided into four groups of from four to eight 
infusions, and in each of these four groups, half of the flasks con- 
tained Y,and half Thay. The four groups of infusions were desig- 
nated by the letters, A, W, H and WH respectively. 

A Infusions. In this group of eight infusions the hay was put 
into the various flasks and then subjected to seven pounds pres- 
sure of steam for one hour in an autoclave. The water was like- 
wise subjected to the same conditions and when it had again 


+A contribution to our knowledge of the Protozoa of the soil. Proc. Royal 
Society, Series B, vol. 84, 1911. 


PROTOZOAN FAUNA OF HAY INFUSIONS 209 


reached the room temperature it was added to the sterile hay 
in the flasks. Four of these infusions were left exposed to the 
air, and four were plugged with cotton, sterilized dry at a temper- 
ature of 180° C. for one hour. 

W Infusions. The hay in these six infusions was sterilized — 
exactly as in the case of the A series, but the water was not ster- 
ilized. All the flasks were plugged with sterile cotton. 

H Infusions. The water used in this series of six infusions was 
sterilized as in the case of the A series, and to this water was 
added fresh hay. Sterile cotton plugs were inserted in each 
flask. 

WH Infusions. These infusions, four in number, were made by 
simply adding fresh hay to ordinary tap water. The tops of the 
flasks were covered with inverted beakers. 

The twenty-three infusions may be tabulated as follows: 


A Series—to determine the organisms derived from the atmosphere. 
Atl, At2, Ay1, Ay? = sterilized hay and water. Exposed to the air. 
Al3 = control; sterilized hay and water. Not exposed to the air, but plugged 
with cotton. 
At4, Ay4 = control; sterilized hay and water. Inoculated with pure cultures 
of Paramaecium aurelia and caudatum, and also with Oikomonas. Plugged. 
W Series—to determine the organisms derived from tap water. 
Wt1, Wt2, Wt3, Wy1, Wy2, Wy3 = sterilized hay and fresh water. All the 
flasks plugged with sterile cotton. 
H Series—to determine the organisms derived from the hay. 
Ht1, Ht2, Ht8, Hy1l, Hy2, Hy3 = sterilized water and fresh hay. Flasks 
plugged. 
WH Series—control. 
WHt1, WHt2, WHy1, WHy2 = fresh water and fresh hay. Mouth of flask 
covered with inverted beaker. 


The experiments were started on July 29th. The infusions 
were examined at intervals of approximately one week during 
the following six weeks, and at irregular intervals thereafter until 
November 11th, when the remaining infusions were destroyed. 
It was planned to carry the observations for six weeks, but at the 
end of this time it seemed advisable to continue certain ones 
longer. 


210 LORANDE LOSS WOODRUFF 


B. RESULTS 


A Series. The A series gives evidence as to the general influ- 
ence of the atmosphere as a source of protozoan life in labor- 
atory infusions. The mouth of the containing flasks measured. 
13 inches in diameter and thus afforded ample exposure to the 
air without rendering rapid evaporation troublesome. The 
flasks stood during most of the time in a room in which hay was 
being used for various purposes, and consequently there was 
ample opportunity for the air to be contaminated with cysts, ete. 
Also, during the day time the windows at either end of the room 
allowed a considerable current of air to pass over the flasks. Again, 
certain flasks were placed on a shelf outside of the window where 
they were exposed to the air of the campus. 

The results derived from this series are as follows: <Ay1 
remained free from protozoa from the start to October 31st, at 
which time it was seeded with Paramaecium and Oikomonas. 
Two days later it showed a good growth of each of these forms, 
thus proving that it was a favorable fluid for protozoa. Ay2 was 
sterile in regard to protozoa until September 24th when a very 
few tiny amoebae appeared, and remained until October 31st, 
when the culture was destroyed. Ati contained on August 26th 
a few small hyaline bodies which seemed to be cysts. A week 
later there appeared a few tiny amoebae, and on September 
24th a heavy growth of monads was observed which persisted to 
the discontinuance of the infusion on October 31st. At2 remained 
sterile until September 24th when tiny amoebae appeared and 
continued to be present to the end. On October 31st the infusion 

yas seeded with paramaecia and these had greatly increased in 
number by November 2nd when the infusion was destroyed. 
Al3, which was kept plugged as a control, was first examined on 
September 2nd and was sterile. It was discontinued at this time. 
The At4 and Ay4 cultures were seeded at the beginning with 
Paramaecium and Oikomonas and showed heavy growths from 
the start—thus proving that the media, from the inception of the 
experiments, offered favorable conditions for protozoan life. 
These two infusions were discontinued on November 2nd. 


PROTOZOAN FAUNA OF HAY INFUSIONS 211 


It is believed that in this series of experiments exceptional 
opportunities were offered for infection of the culture medium by 
air borne cysts, etc., to occur, and the resulting protozoan fauna 
shows that the atmosphere is a negligible factor in the seeding of 
hay infusions used for laboratory study. 

W series. The data from the W group of infusions show the 
protozoan life which was introduced with the laboratory tap 
water. Wy showed from the start heavy growths of Chilo- 
monas, Oikomonas, and Chilodon, and these persisted in varying 
numbers until November 9th. At this time the culture was 
seeded with paramaecia and two days later there was a con- 
siderable increase in their number. The culture was discontinued 
at this time. In the Wy2 infusion there appeared several species 
of monads, including Oikomonas and Bodo. A rotifer (Rotifer 
vulgaris) was observed on August 12th and increased in numbers 
until there were about 2000 per ce. at the top of the infusion, when 
the culture was discontinued on November 11th. The culture was 
seeded toward the end with paramaecia which multiplied rapidly. 
Wt1 and Wt2 developed numerous species of monads and also 
considerable growths of a tiny amoeba. Wi/ had as many as 
5000 per ec. when it was lost by an accident on September 2nd. 
Wt2 on the same day had 20,000 amoebae per cc., and on Septem- 
ber 24th these were succeeded by myriads of Amoeba radiosa. 
The culture was seeded with paramaecia on November 9th, and 
was destroyed on November 11th when it contained a good cul- 
ture of this animal. Wy3 and W3 remained plugged, as a con- 
trol, until November 9th and when examined on this day they con- 
tained practically the same fauna as the other cultures of the W 
series as described above. A point worthy of special note, how- 
ever, is that Wt3 showed, in addition to many tiny amoebae, 
about twenty-five Amoeba proteus per cc. of the fluid at the top 
of the culture. It is interesting that in certain cultures heavy 
growths of tiny amoebae appeared; that in one culture these gave 
place to radiosa forms; and in a third, Amoeba proteus appeared. 
This suggests the possibility that Amoeba proteus was introduced 
in the form of extremely minute spores which became apparent as 
tiny amoebae, later became amoebae of the radiosa type, and in 


212 LORANDE LOSS WOODRUFF 


one culture, developed as far as the typical proteus form (ef. 
De2o9) 

Obviously tap water will vary from time to time throughout 
the year, and no emphasis is placed on the completeness of the 
experiment in respect to the species which can be introduced 
through this channel. However, the work is extensive enough 
to clearly show that an insufficient number of species of Protozoa 
is introduced with ordinary tap water to make this a practical 
method for seeding infusions for study. 

H Series. The organisms which appeared in these cultures 
must have been encysted on the dry hay with which the infusions 
were made, and therefore they represent at least some of the 
forms which one may secure in the laboratory through this source. 
Hy1, Hy2, Ht1, and Ht2 showed a closely similar series of forms, 
including all those which have been noted in the previously des- 
cribed cultures except Chilomonas and typical Amoeba proteus, 
and in addition several species of Colpidium, Colpoda, Oxytricha, 
and other hypotrichous forms, Glaucoma, Holophrya, Spathidium, 
Bursaria, etc. All of these infusions were seeded with Paramae- 
cium on November 9th, and when discontinued there was a heavy 
growth of this organism in each, thus proving that a favorable 
medium was present for Paramaecium. Hy3 and Ht3, served as 
a control, and were not examined until the end of the experiments 
when they contained essentially the same forms as the other 
members of the H series. 

WH Series. This group of infusions, consisting of fresh hay 
and water partially exposed to the atmosphere, was carried as a 
control for the above experiments. The protozoan fauna which 
developed was somewhat more meager than that developed by the 
H series. The explanation of this fact is not at once apparent 
since the hay and the water employed came from the same source 
as that used in making the other infusions. It was evident that 
the cycle of the infusions of this series developed more rapidly 
than those of the other series, and a possible explanation is that 
the bacteria introduced with the water so.augmented the initial 
processes of decay with their attendant phenomena that a medium 
less favorable for large growths of various protozoan forms was 


PROTOZOAN FAUNA OF HAY INFUSIONS 213 


produerd. These data, though too meager to be conclusive, 
suggest that sterile water added to fresh hay may prove to be a 
better medium for the development of the protozoa encysted on 
the hay. 

C. CONCLUSIONS 


Viewed in their entirety, these twenty-three infusions indicate 
that: (1) Ordinary hay added to tap water usually will not 
produce an infusion which is productive of a sufficient number of 
representative forms to make it profitable for a study of proto- 
zoan sequence. (2) Air, water, and hay are all sources from 
which the Protozoa are derived, and increase in importance in 
the order given. Of these three, however, air is practically a 
negligible factor in seeding infusions. 


III. RELATIVE NUMBER AND SEQUENCE OF REPRESENTATIVE PRO- 
TOZOAN FORMS IN HAY INFUSIONS 


A somewhat regular sequence of organisms in infusions of one 
kind or another attracted the attention of the early devotees of 
the simple microscope, as is shown, for example, by the following 
paragraph from a letter written in September, 1702, by an anony- 
mous person who was led by the writings of Leeuwenhoek to 
make such studies: 


In my observations of the Animalcula in Waters I have seen many of 
the same species in the several infusions, and even in Waters that have 
been exposed (especially at this time of the year) any time without any 
particular mixture, such as you find in the hollow of a Cabbage-leaf, or 
on the Dipsacus, ete., and I am confident that many of these are the 
same Creatures under different dresses. For I have noted such a regular 
process in them, and such a constant order of their appearance, that I 
am of opinion most of them are the product of the Spawn of some invis- 
ible Volatile Parents® 


Nearly a century and a half later Dujardin, from his experience 
with infusions, wrote: 


5 Philosophical transactions, Royal Society, London, vol. 23, 284, 1708, 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 4 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 a faitde 
nature; pourvu que le liquide 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 vase; 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- 
cysted 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 


® Histoire naturelle des Zoophytes. Infusoires. Paris, 1841, pp. 173-174. 


PROTOZOAN FAUNA OF HAY INFUSIONS PAS) 


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, |’état 
hygrométrique et l’état électrique, ainsi que l’éclairage, et l’agitation ou 
le renouvellement de l’air, 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 54 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 ce. 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 


7 Loe. cit., pp. 170-171. 


216 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 Ist: 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, C41, and C-42. 

Each 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, 
ete., which were present. The liquid was removed from the 
jar for study with a 5 ec. 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 ce. 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 ee. 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, 

8 Por 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 


° Woodruff 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 
(ef. 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- 


ad 


TABLE I 
TEMPERATURE | TEMPERATURE TEMPERATURE 
(F.) | (e.) (F.) 
DATE, 1909 = ] DATE, 1909 ait 3 DATE, 1909 = 3 
3 5 5 > 5 - 
April 4 52 49 April 29 76 70 May 27 71 65 
5 59 48 30 71 52 29 78 65 
6 69 53 May 1 63 60 30 71 67 
if 75 72 2 69 65 | 31 73 67 
8 80 71 3 75 69 June 1 76 70 
9 76 62 4 76 67 3 77 66 
10 62 52 7 85 | 67 4 75 70 
11 56 50 8 74 64 5 70 67 
12 | 59 | 50 OM |) 72 |) 61 6 68 | 66 
13 60 54. 10 64 60 3} 75 7 
14 66 58 ll 66 64 10 74 65 
15 78 62 12 73 60 ll 79 75 
16 72 56 iG} 74 63 12 74 68 
17 65 55 14 75 66 | 13 77 70 
18 75 59 || 15 71 70 14 75 70 
19 83 75 16 79 69 15 78 | 74 
20 82 61 17 72 65 16 79 70 
21 65 57 18 66 60 17 80 70 
22 67 61 20 68 52 18 74 70 
23 78 65 21 67 58 19 73 66 
24 63 53 | 22 61 57 20 72 68 
25 78 ol | 23 69 59 23 85 72 
26 60 58 24 79 71 25 | 92 83 
27 73 54 26 78 64 26 89 81 


220 LORANDE LOSS WOODRUFF 


dition of, for example, the A / cultures at the end of the first fifteen 
days, with the A J/I cultures at the end of the same length of 
time without taking the temperature into account. However, 
it is fair to compare the relative time of appearance of the various 
organisms in A J and the relative time of appearance of the var- 
ious organisms in A ///; but even here an error is undoubtedly 
present, though, it is believed, it is not sufficiently marked to 
appreciably influence the general results though minor variations 
which occurred in particular infusions may well be due to it. 
This error arises from the fact that the different species of organ- 
isms in the infusions undoubtedly have their own optimum tem- 
perature for development and consequently it may be supposed 
that a particular form, which has a comparatively high optimum 
temperature, may reach its maximum later than another with 
a slightly lower optimum temperature, in the cultures existing 
during the early part of April when the general average tem- 
perature was lower, while it may attain its maximum earlier than 
the latter in the cultures which reached a corresponding stage of 
their development when the temperature was generally higher. 
As already stated, the first intention was to follow the entire 
fauna and flora which developed in the infusions, but this involved 
more labor than could be performed accurately by one observer. 
Consequently although a record was kept of all the animals and 
plants which actually were observed, these data will not be pre- 
sented because I am not satisfied that they are sufficiently accur- 
ate or comprehensive. One who has not attempted to follow in 
detail a series of cultures, started in the manner described, has 
not, I think, an adequate realization of the wealth of forms which 
will develop. Some of the forms appear and disappear with such 
marvellous rapidity that if they are not immediately identified, 
in many eases it is impossible to do it later. Therefore, I repeat 
that the description which follows simply affords the data col- 
lected in regard to certain well-known genera and groups of Pro- 
tozoa, which appeared in sufficient numbers, in a large majority 
of the infusions, to render their study of value in attempting to 
reach some general conclusions as to their sequence in such in- 
fusions under the conditions of the experiment. It is believed 
that the concentration of attention on these few forms is prefer- 


PROTOZOAN FAUNA OF HAY INFUSIONS 221 


able to a wider consideration of many transient species which 
appear apparently at random, for, if it is possible to reach any 
conclusions of value from the study of these few dominant forms, 
it may open the way for an explanation of the seemingly fortuitous 
distribution of the remaining species. 

A tabulation of the fauna of the infusions showed that the first 
analysis of the results should consider the following groups and 
genera of Protozoa: Monads, Colpoda, Oxytricha and various 
closely related hypotrichous forms, Paramaecium, Vorticella, and 
Amoéba, because all these organisms were present in practically 
every infusion. The term ‘monads’ is used in a broad sense to 
include several different genera and a multitude of species of 
small flagellate Protozoa usually classified under the generic names 
Oikomonas, Monas, Bodo, ete. Colpoda cucullus is the most 
common member of the genus Colpoda which has appeared in the 
infusions. Occasionally the form of the organism has not agreed 
exactly with the specific description usually given, and it may well 
be that some of these organisms properly rank as other species of 
the genus, but as this could be determined only by following out 
the life history of the animals, it was necessary to assign the forms 
merely to the genus. In a number of cases species of Colpidium 
was found intermixed with the Colpoda. Colpoda and Colpidium 
are apparently adapted to practically identical conditions of the 
infusions and consequently it matters little which form is chosen 
for study. Since Colpoda has usually appeared in greater abun- 
dance than Colpidium, it has been selected, as the representative 
of this type of ciliate, for detailed study in this work. Among 
the hypotrichous ciliates which appeared, Oxytricha was prob- 
ably the most common, but closely associated with this genus 
was Stylonychia, Urostyla, Gastrostyla, ete., and therefore the 
various species of these genera were considered as a unit and are 
designated in this work as ‘Hypotrichida.’ Also several members 
of the Vorticellidae appeared, nearly all of the genus Vorticella. 
The term ‘Vorticella’ accordingly is used to include all true mem- 
bers of this genus regardless of species. The same is true of the 
term ‘Amoeba’ as here employed, this name being used to include 
such forms as Amoeba guttula, radiosa, etc., as well as typical 
Amoeba proteus. ‘Paramaecium’ is applied to two species, 


De, LORANDE LOSS WOODRUFF 


aurelia and caudatum, indiscriminately. It is apparent, then, 
that no attempt has been made to identify the various species, 
as this would necessitate a large amount of labor entirely incom- 
mensurate with the value of the information gained for the prob- 
lem in hand. All of the forms included together are adapted to 
the same general environment (as the results which follow show), 
and therefore it is logical to consider them together as a unit with- 
out regard to the taxonomic variations of the individual moieties 
of which it is composed. 


B. GENERAL OBSERVATIONS ON THE COURSE OF DEVELOPMENT 
OF HAY INFUSIONS 


In infusions (A) made with boiled hay, which is allowed to 
remain in the jar, most of the hay sinks quickly to the bottom 
and remains there. In the cultures (C) made with unboiled hay 
most of the material floats near the surface for four or five days 
and then begins to sink gradually to the bottom. It is usually 
all at the bottom within two weeks. When the hay is allowed to 
remain in the infusion (A, C) this slowly disintegrates and is 
reduced to a more or less amorphous mass by the end of the sec- 
ond month. The rapidity of these changes, however, varies con- 
siderably with the temperature to which the cultures are subjected. 

When hay and water are combined the liquid rapidly becomes 
straw colored, and within the first few days bubbles of gas appear 
entangled amongst the hay at the bottom, and these rise by 
degrees to the surface. At comparatively high initial tempera- 
tures the gas will frequently disturb the hay and sometimes raise 
it to the surface. Peters’ observations show that this gas is 
chiefly CO:. By the third or fourth day the color of the culture 
liquid appears darker and this becomes increasingly pronounced 
until finally the liquid is of a dark brownish color. One familiar 
with infusions can, of course, readily tell the approximate age of 
a culture by its color. Fine’s studies on these infusions show that 
the light and yellowish shades of color are due to relatively high 
acidity; the darker and brownish shades to relatively low acidity. 


10 Fine. Loc. cit. 


PROTOZOAN FAUNA OF HAY INFUSIONS 22a 


When the infusions are first made up, the liquid, though col- 
ored, is transparent, but within forty-eight hours it becomes 
markedly turbid due to the development of countless bacteria. 
The bacteria at this time are equally distributed throughout the 
medium but on the third day a ‘zoogloea’ begins to be established 
and gradually increases in amount until it finally falls to the bot- 
tom and another is formed. In some cases, however, the ‘zoo- 
gloea,’ after reaching its maximum thickness, at approximately 
the end of thirty days, gradually thins out and practically dis- 
appears in situ. These variations in the transformation of the 
‘zoogloea’ introduce a complicating factor in the study of the 
protozoan life of infusions, because in the cases in which it falls 
to the bottom, it changes the center of population of certain 
types quite suddenly, and thus causes a redistribution of some 
forms. Thebacteria, then, at first are equally distributed through- 
out the fluid, then the largest number is at the bottom and top, 
while in the center of the volume of liquid there are comparatively 
few. The hay and smaller amount of oxygen at the bottom, and 
the more abundant supply of oxygen at the top, offer attractions 
for different forms with the result that apparently approximately 
the same number are to be found in each region. After the 
‘zoogloea’ has fallen or disappeared the center of bacterial life 
is again at the bottom amongst the remnants of the disintegrating 
hay. 

As soon as the bacteria have become numerous, and their 
action on the hay has put a certain amount of it in a form avail- 
able for animal life, then occurs the great growth of Protozoa, 
comprising saprophytic, herbivorous, carnivorous and omnivorous 
forms, and this phase of the life of the infusions we shall consider 
in detail. 

After the period of greatest protozoan fauna has passed, roti- 
fers become numerous, and as the diatoms, desmids, and filamen- 
tous eyanophyceae and chlorophyceae flourish, under proper 
conditions of illumination, several species of Anguillula, copepods, 
ete., are more or less abundant. This condition of the fauna and 
flora merges imperceptibly into what may be called a condition of 
nearly stable equilibrium, in which green plants and animals, under 


224 LORANDE LOSS WOODRUFF 


optimum conditions of light and temperature, are so adjusted that 
for a considerable period a practically self-supporting and self-suf- 
ficient microcosm exists—but with the balance of nature estab- 
lished neither the Protozoa nor bacteria can ever again attain their 
maximum abundance. 


C. THE A, B AND C GROUPS OF INFUSIONS 


All three types of infusions (A, B,C) which were made up gave 
the same general cycle of events, but the A and C series were 
slightly slower in development (as one would expect from the 
presence of the hay) than the B series. The cycle of the C series 
was essentially the same as that of the A series except that it pro- 
geressed somewhat more slowly until the hay became thoroughly 
soaked. A practical disadvantage of the C’ series is presented by 
the fact that the unboiled hay, containing considerable air, has 
a tendency to float and so changes somewhat the distribution of 
the organisms until it sinks to the bottom at about the end of two 
weeks. This nuisance may be avoided by weighting the hay with 
glass. So far as length of cycle is concerned, however, both the 
A and the C series offered equal advantages for study, but the 
cycle of the B series (without hay) being considerably shorter, . 
the sequence of the different types of organisms was more rapid, 
the number of organisms present was much smaller, and stable 
equilibrium of the infusions was attained sooner (cf. figs. 5, 6, 7). 
However, since the richness of the animal life was seriously de- 
creased, this series did not prove to be the best for study, and 
accordingly such a method of making up cultures is not recom- 
mended for investigations of this character. Nevertheless, the 
results derived from all three types of cultures will be given here. 

The data from each of the twenty-six cultures have been re- 
corded (as already described), then these data from each culture 
of each set of experiments of the A, B, and C series, started at the 
same time, have been averaged together. Therefore, in discuss- 
ing these data, I shall refer (unless it is specifically noted to the 
contrary) to the average number of organisms, time of appearance, 
etc., in infusions comprising each group as follows: 


PROTOZOAN FAUNA OF HAY INFUSIONS 225 


A-1, A-2, A-3, A-4, A-5, A-6, averaged and designated A J 
A-21, A-22 averaged and designated A II 

A-31, A-32 averaged and designated A I// 

A-41, A-42 averaged and designated A IV. 

B-1, B-2" averaged and designated BI 

B-21, B-22 averaged and designated BIT ° 
B-31, B-32 averaged and designated B III 

B-41, B-42 averaged and designated BIV 

C-1, C-2, C-3 averaged and designated C J 

C-31 designated C TIT 

C-41, C-42 averaged and designated C JV 


This method of treating the data was decided upon because it 
gives, it is believed, the fairest picture of the protozoan sequence 
in the infusions. As a matter of fact the individual infusions of 
the respective groups presented comparatively unimportant 
variations—except in certain cases which are mentioned. Three 
of the six infusions composing group A J were discontinued at the 
end of the first month because the variations between the indi- 
vidual infusions was not sufficient to warrant the study of so 
many. For a record of the surface sequence of a single infusion, 
reference should be made to C /// (fig. 10). For the data of a 
single form (Paramaecium) at the bottom of two infusions com- 
prising a single group, see fig. 12. 


D. _ TIME OF APPEARANCE, MAXIMUM NUMBER AND DISAPPEARANCE 
OF REPRESENTATIVE PROTOZOAN FORMS AT THE SURFACE OF 
THE INFUSIONS 


1. Monad 


A I group. Monads were the first animals to appear in con- 
siderable numbers and their maximum was attained on the 7th 
day when there were about 5200 per ee. Their decline was equally 
rapid and by the 20th day of the life of the infusions none were 
observed in the samples studied. 

A II group. These forms were the first to appear, reach their 
maximum of 2000 per ee. on the 4th day, and miminum on the 
Sth day. 


226 LORANDE LOSS WOODRUFF 


A III group. Monads were practically absent from the two 
cultures of this group, and this is the only instance in which they 
did not appear in numbers sufficient to be considered. There 
were, perhaps, 100 per cc. at several counts. This dearth is 
accounted for, I think, by an exceptionally heavy growth of Col- 
poda, which occurred in this group very early (cf. table 2 and fig: 3). 

A IV group. The monads appeared on the 2nd day, attained 
their maximum of 1000 per ec. on the 4th day and reached their 
minimum on the 6th day. 

BI group. ‘These forms appeared on the 2nd day, attained a 
maximum of 4200 per cc. on the 8th day, declined to 500 per ce. on 
the following day and then gradually became less and less until 
by the 23rd day their number was negligible. On the 36th day, 
however, they reappeared, attained the number of about 2000 
per ec. on the 40th day, and reached extinction on the 60th day. 

BIT group. On the 4th day there were 1200 monads per cc., 
and on the 8th day they had entirely disappeared. 

BIII group. In this group the monads attained a maximum 
of 5000 per ec. by the 9th day, and by the 12th day there were none 
remaining. 

BIV group. A maximum of 1400 per ce. was reached on the 
3rd day of the life of the cultures, and then a rapid decline resulted 
in extinction by the end of the first week. 

C I group. In these three cultures the average maximum 
number of monads, nearly 8000 per cc., occurred on the 15th day, 
and was followed by an abrupt decline ending with their disap- 
pearance on the 20th day. The maximum of Colpoda occurred on 
the same day as that of the Monads. 

C III group. In this group, represented by a single infusion 
(C-31), the monads attained a maximum of over 8000 per cc. on 
the Sth day, declined rapidly to 2500 per ec. on the 13th day, and 
reached a minimum of practically zero on the 24th day. 

C IV group. Here the monads rose to the number of 5000 per 
ec. on the 17th day, declined to about 2500 per cc. on the 21st 
day, then rose to their maximum of 7600 on the 27th day, and by 
the 32nd day had reached a minimum. 


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PROTOZOAN FAUNA OF HAY INFU 


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228 LORANDE LOSS WOODRUFF 


From the study of the monads in all the cultures it is clear that 
in every instance they were the first type of protozoon to appear 
and the first to reach a maximum. This is undoubtedly to be 
explained by the fact that these forms, combining holozoic and 
saprozoic methods of nutrition, are able to feed on the bacteria 
which are developing so rapidly at this period, and also to absorb 
various substances entering into solution from the hay. The 
monads under consideration are also the first forms to decline 
and practically disappear, and this is probably due, in part, to 
the rapid decrease in numbers of the bacteria brought about by the 
monads themselves and by the rising generations of Colpoda. 


2. Colpoda 


A I group. Colpoda was the second protozoon to appear in 
considerable numbers and its maximum was attained on the 14th 
day when there were about 2500 per cc. Its decline was equally 
rapid and by the 25th day very few active individuals were seen. 
Beginning at about the 30th day, however, more were observed 
and on the 37th day there were about 600 per ce. This second 
rise in numbers was followed by a more gradual decline which 
ended in the extinction of this form by the 66th day of the life 
of the infusions. 

A ITI group. This form was the second to appear and very 
slowly attained its maximum of 1000 per ee., which took place on 
the 27th day, then it fell in number to about 200 per ce., rose 
again to about 500 per ec. on the 44th day, and then became ex- 
tinct on the 49th day. 

A IIT group. Colpoda was the second protozoon to appear in 
considerable numbers in these infusions, the eyele of the monads 
being apparently aborted. Colpoda arose abruptly to the great 
number of 15,000 per ec. on the 10th day, fell to about 11,000 per 
ec. on the following day, and by the 15th day very few active 
forms were observed. However, almost immediately it had an- 
other period of reproductive activity which brought up the num- 
ber to about 4000 per ce. on the 29th day. After this second high 
point it decreased in number, but persisted until the 63rd day 


PROTOZOAN FAUNA OF HAY INFUSIONS 229 


of the infusion’s life. The growth of Colpoda in this group of 
infusions is remarkable for its abundance and persistence, for 
during the greater part of the life of the infusion, Colpoda was 
the form which dominated. 

A IV group. Colpoda was the second form to attain its max- 
imum, which occurred on the 13th day with 2500 per ec. present. 
This number persisted to the 17th day, and then a very quick 
decline ended in the extinction of the form four days later. 

BTIgroup. Colpoda was the third to attain its maximum, being 
preceded by the monads and. the hypotrichida. Its maximum 
occurred on the 14th day and this was followed by a slow decline 
resulting in the disappearance of Colpoda on the 30th day. 

BIT group. Colpoda attained its maximum abundance on the 
6th day, then rapidly proceeded to its extinctionon the 15th day. 
The notably small development of Colpoda in this group of in- 
fusions is paralleled by that of all the other organisms in B /T. 

B III group. In this group of infusions Colpoda rose rapidly 
to a maximum of 8000 per ce. on the 18th day, and then fell even 
more rapidly to extinction on the 29th day. _ In this series of 
infusions Colpoda was again the dominant form, greatly outnum- 
bering the hypotrichida and paramaecia whose small maxima 
occurred before its own. 

BIV group. The appearance of Colpoda occurred relatively 
late, none being observed until the 6th day, and its maximum 
growth occurred on the 12th day, and its extinction on the 16th 
day. In this series it was the fourth form in point of time to 
reach its greatest abundance. 

CI group. In these three cultures the average maximum num- 
ber of Colpoda, 4500 per ec., occurred on the 15th day, after a 
rapid rise from the 7th day. Then there was an equally sudden 
decline to about 40 per ec. by the 22nd day, and this number 
gradually decreased until it became negligible at the 46th day. 

C III group. Again in this culture the growth of Colpoda 
over-shadowed that of all the other forms. Appearing on the 4th 
day it gradually increased until a maximum of about 15000 per 
cc. was attained on the 33rd day. This was sustained for four 
days and then a remarkable decrease brought it down to about 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 2 


WOODRUFF 


LORANDE LOSS 


230 


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q \ [9h A Pryor H 


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‘9dBJANS OY} 7B “od Iod sustuvs10 JO Loquinu oy} oyworpursoyvuIpIQ, “dnows yy py Z'3y 


PROTOZOAN FAUNA OF HAY INFUSIONS 231 


600 per cc. on the 48rd day, and 60 per ce. on the 49th day. 
Approximately this number persisted until the end of the ob- 
servations on the 76th day. 

CIV group. Colpoda developed in greater abundance in this 
group than in any other, attaining, comparatively gradually, a 
maximum of 25,000 per ce. on the 32nd day, falling to 15,000 on 
the 37th day, and to 100 per ec. by the 44th day, and practically 
reaching extinction by the 56th day. 


An analysis of the above data in regard to Colpoda shows that 
this form is adjusted to those surface conditions of infusions which 
exist when the monads have about run their course. 


3. Hypotrichida 


AI group. Representatives of the hypotrichida were the third 
to appear in considerable number and their maximum of about 
2400 per cc. was attained on the 51st day after a long gradual rise. 
Their decrease in number was somewhat more abrupt, resulting 
in their extinction by the 85th day of the life of the infusions. 

A ITI growp. These forms were the second to appear and the 
third to reach a maximum growth. This was attained on the 
39th day after a long gradual increase. A rapid decline reduced 
them to about 40 per cc. on the 49th day, and they persisted 
in this number to the 74th day. There was a slight increase in 
number on the 75th day, at which time the observations were dis- 
continued. 

A III growp. A few hypotrichida appeared on the 6th day, 
gradually increased until there were about 400 per ec. on the 15th 
and 16th days, then declined to the 24th day. Their maximum 
of 700 per cc. was reached on the 29th day, after which they de- 
clined until, on the 43rd, they became extinct. 

A IV group. The maximum consisting of 1600 per ce. fol- 
lowed those of the monads and Colpoda and occurred on the 26th 
day. <A continuous decline brought the number down to 100 per 
cc. by the 48rd day; and this number persisted to the 50th day, 


232 LORANDE LOSS WOODRUFF 


after which there was a slight increase up to the end of the obser- 
vations on the 57th day. 

BI group. These forms attained a maximum of 560 per ce. on 
the 11th day, and then very gradually declined until they became 
practically extinct on the 55th day. 

BIT group. In this group the maximum of the hypotrichida 
was reached on the 11th day, and extinction on the 29th day. 

B III group. The hypotrichida were practically negligible 
as the maximum number which occurred on the 10th day was less 
than 40 per cc., and the animals disappeared completely by the 
15th day. 

BIV group. Here these forms reached their greatest abun- 
dance, 250 per ce., on the 7th day, declined to about 20 per ce. by 
the 11th day, gradually rose to 150 per ce. on the 26th day, and 
disappeared by the 35th day. 

C I group. In these three cultures the average maximum of 
about 360 per ee. occurred on the 19th day, and was followed by 
a continuous decline until the 49th day, when about 40 per ce. 
were seen. From this time on slight fluctuations in numbers oc- 
curred, and at the last observation on the 86th day, about 40 
per cc. were still to be seen. 

C ITI group. In this group, represented by a single infusion, 
these forms first appeared on the 20th day, reached their greatest 
abundance, 300 per cc., on the 24th day, then declined to about 40 
per ce. on the 28th day, and persisted in approximately this 
number to the end of the observations. 

CIV group. Here the hypotrichous forms reached a maximum 
of 500 per ec. on the 32nd day, declined to about 40 per cc. on 
the 37th day, rose to 400 per cc. on the 44th day, declined again 
to about 20 per ce. on the 51st day, and remained at about this 
number to the last observation on the 56th day. 


From the study of the hypotrichous ciliates which appeared 
in all the cultures, it is clear that these forms are adjusted to 
conditions at the surface of the infusions which are present, in 
most cases, after the monads have nearly disappeared and Colpoda 
has passed its period of greatest abundance. 


233 


PROTOZOAN FAUNA OF HAY INFUSIONS 


‘— ++ — = nqoouly 
', — — = eyoomao, {——— = wintoovureing !—-— = vpryaujoddAyy {--- = wpodjop !:-- - 
= pRuoyy ‘suoIsnjut oy} Jo doUAaXSTXO 949 JO SAupP Jo JoquMU oY} pozjoO[d st Bsstosqu oy} UG 
‘advJANS OY} YB ‘09 tod suIsTUBZIO Jo JequNuU 94} O}BOIpUT SoywuIpso oy, “dnoaws P77 Wg “AMT 


0002 


000” 


bo 
JU) 
= 


LORANDE LOSS WOODRUFF 


4. Paramaecium 


A I growp. Paramaecium appeared in considerable numbers 
about the 14th day and after various fluctuations in numbers 
reached a maximum of 1100 per ce. on the 51st day. This was 
succeeded by a general decline in numbers to the 73rd day when 
only about 40 per ec. were present; but after this their number 
increased to about 700 per cc. on the 79th day and continued so to 
the end of the observations on the 86th day. 

A IT group. The data for the paramaecia of these two cul- 
tures will be considered later, because the presence of Didinium 
so altered the paramaecium cycle that it cannot be fairly com- 
pared with that in the other cultures. 

A III group. Paramaecium made its appearance here on the 
7th day and gradually increased to its greatest abundance on the 
48th to 50th days, at which time about 3700 individuals per ce. 
were present. After this the number rapidly fell to 300 per ce. 
on the 64th day when the last count was made. 

ATV group. This organism appeared on the 4th day, attained 
the number of 2500 per ce. on the 31st day, and this maximum con- 
tinued for fourteen days after which there was an abrupt decline 
to about 50 per ce. on the 57th day, when observations were sus- 
pended. 

BI group. Paramaecium appeared on the 10th day, attained 
a low maximum of less than 100 per ec. on the 20th day and then 
very gradually reached extinction by the 65th day. 

B II group. This form was present in small numbers prac- 
tically from the start (8rd day) and reached a small maximum of 
about 160 per ce. on the 7th day, from which time they gradually 
decreased until they became extinct on the 24th day. This cul- 
ture also was influenced by the activities of Didinium, and this 
will be discussed later. 

BITII group. Paramaecium appeared on the 11th day, reached 
its maximum on the 17th day when 160 per ec. were counted. 
From this time to the 65th day, when the last count was taken, 
it continued to persist in varying numbers under 100 per cc. 


PROTOZOAN FAUNA OF HAY INFUSIONS 235 


BIV group. The cycle of Paramaecium will not be considered 
at this time as it was so altered by Didinium that it is not at all 
comparable with that of the other cultures. 

CI group. Paramaecium appeared on the 21st day, gradually 
multiplied until 600 per ce. were counted on the 40th day, then 
increased rapidly to 2500 per ec. on the 45th day, fell to 1200 per 
ec. on the 48th day, and to 200 per cc. on the 61st day. From 
this time there was a gradual decline to the last count on the 86th 
day, when about 80 per ec. were present. 

C III group. In this group, comprising but a single infusion, 
paramaecia appeared on the 23rd day, increased to about 440 
per cc. on the following day and fell in number until only about 10 
per ee. were observed on the 34th day. From this point they 


\; 


30 “40 50 60 


4 
0 


Fig.4 ATJV group. The ordinates indicate the number of organisms per cc. 
at the surface. On the abscissa is plotted the number of days of the existence of 
the infusions. Monad =--- - ; Colpoda = —---; Hypotrichida = — - —; 
Paramaecium = ; Vorticella = — — - ; Amoeba = —.- - —. 


236 LORANDE LOSS WOODRUFF 


rose to a maximum of about 500 per cc. on the 37th to the 42nd day 
after which there was a general decline to 200 per cc. at the last 
observation on the 64th day. 

C IV group. This organism appeared on the 3lst day, at- 
tained its greatest abundance on the 44th day, with about 600 
per ec. present, and then declined to 200 per ce. on the 65th day, 
when the final examination was made. 


The results given above, when compared with those of the hypo- 
trichida, show that Paramaecium usually attains its maximum 
numbers at the surface when the hypotrichous ciliates have passed 
their period of greatest abundance. 


5. Vorticella 


ATgroup. Vorticella made its appearance on the 30th day and 
increased gradually in numbers until the 65th day when about 
100 per ce. were seen. Then it rose rapidly to a maximum of 
700 per cc. on the 73rd day, declined equally rapidly to 200 per 
ec. by the 79th day, and then rose again to nearly 700 per ce. on 
the 86th day when the last observation was made. 

A ITI group. This form appeared on the 5th day and persisted 
in small numbers to the 27th day. From this point it rose to 3000 
per ce. on the 44th to the 49th day, and then quite rapidly de- 
clined to about 80 per ce. on the 67th day. Another rise to about 
500 per ce. occurred on the 75th day when the culture was dis- 
continued. 

A TIII group. Vorticella appeared on the 8th day and continued 
in almost negligible numbers until the 25th day when it began to 
multiply rapidly until the maximum of 700 per cc. occurred on the 
29th day. It then decreased to less than 20 per ec. by the 33rd 
day and remained in about this number to the 50th day, after 
which it again increased to 600 per cc. on the 57th day and declined 
to about 120 per ce. on the 64th day, when the last count was 
taken. 

A IV group. This form made its appearance on the 7th day 
and persisted in comparatively small numbers until the 26th day, 


PROTOZOAN FAUNA OF HAY INFUSIONS 237 


when it began a period of great abundance, reaching a maximum 
of 3500 per cc. from the 43rd to the 50th day. It then declined 
to about 1600 per ee. on the 57th day when the last count was 
taken. Vorticella attained a higher maximum than any other 
protozoon in this group of infusions. 

BI group. Vorticella appeared on the 18th day, rose to about 
200 per ee. on the 21st day and fell to 20 per cc. on the 26th day. 
It continued in about this abundance up to the 34th day when it 
multiplied rapidly and produced a maximum of about 1000 
per cc. by the 37th day. This was followed by a rapid decline for 
a few days and then a slow decline to the 67th day when the form 
became extinct. 

BII group. Vorticella appeared on the 5th day and fluctuated 
in numbers under 200 per ce. until the 45th day when the maxi- 
mum of 300 per ce. occurred. This was followed by a rapid de- 
cline resulting in extinction by the 50th day. 

B III group. This genus appeared on the 16th day and _ per- 
sisted in very small numbers, reaching its maximum of 120 per 
ec. on the 59th day. It had decreased somewhat by the 64th day 
when the last observation was made. 

BIV group. In this group Vorticella appeared on the 2nd day 
and persisted in numbers less than 200 per ce. until the 11th day, 
then arose abruptly to 3500 per ce. on the 16th day, and fell 
almost equally abruptly to about 20 per ce. by the 26th day. It 
persisted in approximately this number to the last count on the 
36th day. 

CI group. In this group of three infusions, the curve for Vor- 
ticella shows a peculiar series of fluctuations. The form appeared 
on the 44th day, rose to 500 per ce. by the 57th day, fell to 20 
per ee. by the 67th day, rose again to practically 500 per ec. by the 
74th day, fell again to about 20 per ce. by the Slst day and then 
had still another rise which brought the organism to its maximum 
on the 86th day, when the last observation was taken. 

CITI group. Vorticella appeared and attained its maximum of 
100 per ee. on the 38th day in this infusion. From this time it 
very gradually decreased in numbers until the 65th day when the 
final count was made which showed about 50 per cc. 


238 LORANDE LOSS WOODRUFF 


C IV group. Again in this series Vorticella passed through a 
series of fluctuations, beginning on the 19th day, reaching a 
maximum on the 32nd day of approximately 500 per cc., falling 
to nearly zero on the 37th day, and thereafter increasing in num- 
ber until there were about 240 per ce. present at the final count on 
the 56th day. 


A study of the data presented above shows that Vorticella 
usually attains its greatest abundance later than Paramaecium. 


6. Amoeba 


A I group. Amoeba was first seen on the 47th day, rose to a 
maximum of about 1000 per cc. by the 56th day and decreased 
until none were present on the 67th day. 

A II group. This form appeared on the 21st day and fluctuated 
in numbers until the 50th day when it completely disappeared. 
It reappeared again on the 66th day and reached a maximum of 
about 1000 per ec. on the 75th day when the last count was made. 

A IIT group. Amoebae appeared on the 17th day and after 
various fluctuations attained a maximum of 2700 per ce. on 
the 57th day. After this they declined rapidly and had disap- 
peared by the 64th day when the last count was taken. 

A IV group. These forms were first seen on the 5th day and 
continued to be present, though in practically negligible numbers, 
until the 21st day. Then they began to multiply rapidly and 
attained a maximum of 2500 per cc. on the 32nd day. A sudden 
decline brought the number down to about 40 per ec. on the 36th 
day and zero was reached by the 50th day. 

BI group. Amoebae did not appear at all in one of the cultures 
of this group, and in the other a total of 22 amoebae were observed 
at different times from the 40th to the 87th day. 

BIT group. Starting on the 23rd day, this form reached a per- 
iod of greatest abundance on the 45th day when about 100 per 
cc. were counted. Extinction occurred by the 55th day. 

BIII group. This form appeared on the 14th day and reached 
a maximum of 1000 per ee. on the 25th day. They completely 
disappeared by the 37th day. 


239 


: 


PROTOZOAN FAUNA OF HAY INFUSION 


‘ 


“OOBJANS 


= *B]ooyI0A f— = wnbermeivg ! /— = epiyouyodaéyy {- - —- = vpodjop 


+ = pBRuojy “suorsnjur ay} jo AIUIISTXY OY} jo sAup jo Joqumu a peyold st BSSIOSqe ayy uc 


ay} ye ‘oo aod suIstuRsI10 Jo JoquNU oY} a}RoIpUr soywuIpio oT, “dnoww yy gq ¢ “AT 


e) 


240 LORANDE LOSS WOODRUFF 
BIV group. About 80 amoebae per cc. were observed on the 


7th day, and from this they gradually decreased until the 18th 
day when the last individuals were seen. 


1000 


Fig.6 8B II group. The ordinates indicate the number of organisms per ec. 
at the surface. On the abscissa is plotted the number of days of the existence of 
theinfusions. Monad =- - - -;Colpoda = ~ — —; Hypotrichida = — - —; Para- 
maecium = ; Vorticella = — — -; Amoeba = —- - —. 


C I group. Amoeba was first seen on the 46th day, reached its 
highest point one day later and maintained this maximum of 
nearly 300 per cc. for five days, after which a slow but steady 
decline brought it to extinction by the 86th day. 

C III group. This form appeared on the 42nd day, reached its 
greatest abundance, 120 per ec., on the following day, and dis- 
appeared by the 53rd day. 

CIV. group. In this culture amoebae reached a greater devel- 
opment than in any of the others. They were first seen on the 
6th day and reached 5000 per ce. from the 18th through the 22nd 
day. Then they declined quickly to about 500 per ce. on the 27th 
day, only to rise again almost as fast to their maximum of 5700 
per ce. on the 38th day. From this point they fell to 200 per ce. - 
by the 51st day, and at the last observation, on the 56th day, 
about 1000 per cc. were again present. 


A careful analysis of the above data shows that amoebae 
attained their greatest development slightly later than Paramae- 
cium and earlier than Vorticella at the surface of the infusions. 


PROTOZOAN FAUNA OF HAY INFUSIONS 


IV. SUMMARY OF SURFACE COUNTS 


fionadsieeerme me ae ah 
Colpod any presenters oe 
Eiypotrichtdaemmies.' es). 
IPSramMeaAecniMa nee ws) es 
Worticelliany-eesc<cce,« Stier. 
AIMOCDSE te eaiteae aces ace ess 


Colpodai teciom ytntwe seme 
Hypotrichida.c 2.22... 
IPATAMIACCIUM Ns gage ste 
Wionticelllaty sete creer e seeeels 
JAIN OCD Se tease soak tae oo Sysie me 


IMamAA Roraee. fh eche veers ate 
Golpod aan. nce sm -tameserecre 
1alyigeo elite Clap We S66 eosin 
Paramaécium.<..........- 
WOLtIGEl Aas -caivacietae eter 
PATRORD Sine tarsscteh: see ate 


Monsdiwa cmc se eeee ee 
@olpadaee = fy-taccstan tn 
yp otrchidaianest cnr: 
Paramaecium............. 
WOLTICOLA teres acne) mcrefenle 
PAINOGD BT tice scche paises ¥ 507 


A III 


A IV 


PMonadsie. iscsi coee8 <1 


Paramaeciumy a... .---.- 
WoOnbicell as jensca6 os civics 
AM OSD AS etree. vets as os te 


TABLE 2 


DAY OF 
APPEARANCE 


—_ b 
Non ots or 


cost eS Or bo 


DAY OF MAXIMUM 


DAY OF 
DISAPPEARANCE 


20 
66 
85 


* A dashin this column indicates that the organism was still present when the 
. 


last observation was made. 


7 Omitted because Didinium affected the sequence. 


{Disturbed by Didinium. 


242 LORANDE LOSS WOODRUFF 


TABLE 2—Continued 


DAWOE DAY OF MaXIMUM | PAY OF 
APPEARANCE | DISAPPEARANCE 

Monads 2 4 8 

@ol pod ate sn erent renee 5 6 15 

B II iy popmichidan ss...) 3 11 29 
Paramaecium........ 13 7 24 

| Vorticella.......... 5 45 50 
[PAmoeb adie: aun: ol osc. 23 45 55 
MonadSe emer seiccr tls 5 9 12 
@olpodavepere tener 5 18 29 

BIII Eiypotmichida. 4.622. vase | 8 10 15 
Rananiae crim: vent eee 11 17 = 
Worticella ns. s-= miter 16 59 = 
(PArmoebare scence = se 14 25 37 
((Monads:t sacs -5 Pere 2 3 7 
Golpodates- eer name 6 12 16 

B IV Ey potnichidaeeeee sen eee 3 7 35 
Paramaecium.............. 70 0 0 
Worticellatic.n tes «crite: 2 16 36 
(PAmoebare cee sti. ene a. fi 7 18 
NVIOTIAGSY e236 epssperi e eiecetns 6 | 15 20 
Colpodanmen er na. seer rr | 8 15 | 46 

C y | Hypotrichida AM Ee 16 19 | — 
eRe am ae ciinmeretees tere: 21 45 == 
Worticellaren see eee. si ae 44 86 = 
pAmne babes near pee serie ara 46 47 86 
Mona sss cee aes 3 8 24 
(Colpodaeere ast ss- ne 4 33 —_ 

CI: Ey potrichid a) Here r= a. 20 24 | = 
|(Paramaecium: (7. -- 2 o---- 23 37 = 
laWiorticellasess= sn oom 38 38 = 
tAmoebareesrc. ese =n 42 43 | 53 
Monadseny tree =| 2 | 27 | 32 
Colpod areca: ce. rireee 5 32 | 56 
CIV? Eiypobrichid deer 2 effete 18 32 | = 
Paramaecium. ............ 31, i == 
Vionticellanees see. ase 19 32 = 
IAmioe ba cette rrs eye aceneel- 6 38. = 


PROTOZOAN FAUNA OF HAY INFUSIONS 


243 


TABLE 34 
TIMES ATTAINED MAXIMUM TIMES DISAPPEARED 
TIMES APPEARED FIRST 
FIRST FIRST 

Monad.........|10 10 10 

versus 
Colpoda....... 0 0 1 (BI) 
Colpoda.-...:. 8 6 8 

versus - 


Hypotrichida. .| 3 (A IJ, BIT, BIV) | 4(B), BUI,BIV,CI1)| 2 (A II, B IIT) 


Hypotrichida. Aig 7 4 
versus : 
Paramaecium. .| 1 (A/V) 0 0 
Paramaecium.. 8 vi 2 
versus 
Vorticella......] 1(C ZV) 2) (A TEE EG DYV) 0 
Paramaecium. . 7 7 0 
versus 
Amoeba....... | 1 (CIV) 1 (CIV) 6 
Vorticella..... ei A (ADE, ARE Commie WCB TD) 
CIV) 
versus 
Amoeba....... | 8(AIV, BIT, CIV) 5 7 


From the above tables the most frequent sequence for 
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 


the 


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. 


12 The figures for Amoeba versus Vorticella are so nearly the same that the 


yariation 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 
(83) Hypotrichida (3) Hypotrichida 
(4) Paramaecium (4) Paramaecium 
(5) Vorticella _. { Vorticella 
(6) Amoeba (5) aan 
B Series 
APPEARANCE MAXIMUM 
(1) Monad (1) Monad 
a Colpoda 3 Colpoda 
(2) Hypotrichida @) Hypotrichida 
(3) Paramaecium (3) Paramaecium 
(4) Vorticella (4) Amoeba 
(5) Amoeba (5) Vorticella 
C Series 
APPEARANCE MAXIMUM 
(1) Monad (1) Monad 
(2) Colpoda (2) Colpoda 
(3) Hypotrichida (3) 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 


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'+— — = 8/9010, ‘———. = umtoovurvavg {— -— = epryomjodéq { - -- = epodjoy 
fs +++ = pBUO]Y ‘“SUOISNJUT oY} Jo adue}SIxa oY} Jo SAup Jo 1oquInu oYy pozyo]d st BSSTOSGR oY} 
UO “SORJINS OY} 9B SUISTUBSIO Jo IoquINU 94} yuoseidor soyeUIpsO oY, “dnows Pg LB 


4 


OL 09 OS Ov O& 02 Ol 


0002 


o000€ 


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 ce., which coincided with the top maximum of 2500 per ce. 

Hypotrichida. These forms occurred in negligible number in 
groups II, III] 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 ec. was taken. 

Vorticella. Vorticella were not observed in group I until near 
the end (76th day) when a maximum of 40 per ec. was attained. 
Group II, however, showed the largest number for the A series, 
with a maximum of 400 per ec. on the 72nd day, i.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 cc. 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 cc. occurred from the 55th to the 60th day in A J; a maximum 
of 6000 per ec. 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 I/T; and a maximum of 6500 
per cc. on the 23rd day in A JV. 


2. B Infusions 


Monad. In groups BI and B II 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 JV monads appeared in numbers be- 


3500 


3000 


1000 


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 BJ and B I/I they attained a temporary 
maximum of approximately 15000 per cc., just after their dis- 
appearance from the top (ef. fig. 13). In B JI 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 TI and IIT, while in group I the largest 
bottom count was taken, i.e., 110 per ec. 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 JI 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 attaimed a maximum of 175 
per ee. on the 88th day, while in group IT 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 cc. on the 28th day 
(cf. fig. 15). 

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 53rd 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 ec. on the 37th 
day in B III and the amoebae had disappeared by the 45th day. 
A maximum of 2000 per ec. was in existence in B JV 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. 


iz _—o—_—= eqooury ! _— — = BI[PTJAIOA ‘—_—— = TINO 9B ULB.LG 


{—-+— = epryomjodéy { --- = vpodjop !- +++ = peuojy ‘suorsnyur oy} Jo o0ua4sIxo ayy Jo sep Jo oquinu ayy 
= po}jold st vsstosqe oy} UG *eoBsANS oY} 4B ‘D0 19d suIsTUBTIO Jo JoquInU oY} Yueserdos soyvuIpsO oY, “dnows 7 QD 6 ‘IT 


06 Ol O 


Ooo! 


0002 


0008 


PROTOZOAN FAUNA OF HAY INFUSIONS 


O00v 


000¢ 


——- 


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 ec. 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 ce. 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 ee. 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.¥ 


18 T am indebted 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 comparable 


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 eyele 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 identical, 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 I/I 
(ef. fig. 3) and the Hypotrichida in group C JV (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 


“_.+— = Bqoouly 

.— — = Byeon10q {——— = wmpovurieg {‘—-— = epryomyodaAyy ‘ --- = epodjop 
/ = pBUoy “UOTSNJUTOY} Jo ooUa}sIxe ayy Jo SABp Jo Laquinu 94} payyoTd SI Bsstosqe oy} UO 

“aoBJINS OY} YB "99 Tod SuIsTUvBIO Jo LaquINU oYy JUEsetdad soywuIpAO oy, “noid 77 DO OT SM 


OL eX) OG eh O€ 0¢ Ol 


PROTOZOAN FAUNA OF HAY INFUSIONS 253 


7600 25000 


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 = wocacurd =---;Hy pesaciie =—-—} 
Paramaecium = - Worticalla, =—— Pee reba =—--— 


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 255 


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 ease (cf. 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 


eck 
as 


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. 


400 


200 


2 i Oieeaggna70 


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 Wor example, cf. Metcalf: Studies upon Amoeba. Jour. Exp. Zool., vol. 9, 
1910. 


256 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. 2 

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 PAB 


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 in 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 


18 Cf. Fine: loc. cit. 
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 


“QOBJANS OY} 9B poatind00 uoTesntuoo JO olulop 


-1do ue yor ye gurod =*x *- + + » + = w0}}0q oY} yw pues ——— = 9dov]ns oy} YB LAINTDOVUT 
-eivg {— - — = wojj0q oy} ye puw - — — = aovjJans oy} ye vpodjog ‘dno’ 777g gt 3 
OL 09 ele} Ov 


oe 02 Ol 0 


ae 


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." 


§. 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 IJ 
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 II 
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 


vinced, 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 
greatest 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). 


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. 

Tt 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 B1. At the pomt 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 (cf. fig. 12). 


Fig. 15 BIV group. Vorticella fauna at the surface (- - - -- ) and bot- 
tom ( i 


In group B JII, 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. 


1X. 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. Zool., 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, Le., 
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 put 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, 
(8) 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. 


1L. 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 cc. 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- 
+ 


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 cc. of infusion, filtered clear, and diluted 
to 200 cc. with distilled water, were treated with 10 ec. of 50 per 
cent H.SO, and 0.01 N KMn0O,, the excess of the permanganate 
being titrated back with 0.01 N oxalie acid. For the chloride 
determination, 5 ce. 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 
tosay. The ratios ~OTRANS? meates -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. 


3 Loc. cit., p. 463. 4 Loc. cit., p. 464. 


CHEMICAL PROPERTIES OF HAY INFUSIONS 267 


TABLE 1 
PRELIMINARY INFUSION 1* | PRELIMINARY INFUSION 2* 
8 a | SOLIDS | | Hy a SOLIDS 
a ze | a 2 ——— = = 
2Z An | fe! | 2 an ce! 
S 22% | Grams per 100 ce. Bi 3 Sis Grams per 100 ce. ae 
re HO | infusion e/e = Ho infusion 6/e 
Bild lepine SIE Base ey ee | 
as |e | ol lade & | | 
so8 [see | Ble |aee |see | z\2 
E25 \EGe | £3 i245 BGs | gs 
a isi an eS 2 Sis nies = = 2 S16 
%|8@8 |328 | a | 2 | 8 8 328 2 | ¢ |#® 
Alexi geang| = & E 9 |2an 885-8) = & = < 
B\Ssaulscanl € 6 Sy E-|SSsaasoaa 3 5 & I 
<0 jo BA | 4 S) a |O S a 5 3 c 
1 | 8 0. 034 0.010 0.024 0.42 8 0.068 0.014) 0.054 0.26 
4| 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 
11} 16 194 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 
18| 12 188 0.058 0.026 0.082 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 
20 | 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 0.060 0.40 
39} 10 204 | 0.069 0.031 0.038 0.82. 8 224 0.083 0.030 0.053 0.57 
6 0.030 0.051 0.60 | 10 206 0.082 0.032 0.050 0.64 


43 | 212 | 0.081 


* These infusions were prepared in the same manner as the typical members of serles A (see below). 


PREPARATION OF 


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. In each case 20 grams Timothy 
hay in about 250 ce. of water were boiled five minutes. Both 
infusion and solid hay were then transferred to a battery jar con- 
taining some unboiled water and the volume made up to 5 
liters. These infusions were than equally seeded with samples 
taken from several cultures of varying ages, in order to insure the 


INFUSIONS® 


5 For further details see Woodruff, loc cit. 
6 The protozoan count was omitted in A-951. 


268 MORRIS S. 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-42% 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 protozo6n-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-31, 
C-41, C-42, and M-1 each consisted of 20 grains 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 S-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. §-1 is therefore a protozo6n- 
free infusion of series C. 

7 No attempt was made subsequently to keep the infusion free from bacteria. 


® /nfusions B-41 and B-42 were subjected to a chemical examination so infre- 
quently that the results are omitted from tabie 3. 


CHEMICAL PROPERTIES OF HAY INFUSIONS 269 


Bacterial infusions. Eight infusions were prepared as follows: 
In each of eight cotton plugged flasks, 2.8 gramsof hay and 700 ce. 
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 S-1 


® For a discussion of the influence of temperature on these infusions, ef. Wood- 
ruff, loc. cit., p. 218 and also p. 274 of the present paper. 


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271 


CHEMICAL PROPERTIES OF HAY INFUSIONS 


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CHEMICAL PROPERTIES OF HAY INFUSIONS 273 


TABLE 5 


Methyl Orange Alkalinity 
(Cubic centimeters of 0.01 N HCl per 100 ce. infusion) 


DAYS 
| | | | | it " 
}0|1 2) 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 
SE a a a i i a a bd bs 
| +4)=4)£0 =0)=0—2/+0 —4/=0 
A-l....)8|9| 6/4] 6|8 | 8| 10 10 12 10 6 
+4\+2]+0+0\+9\+2+0\—2) |+0—2 
A-2...., 7/106) 4|8/8 10 12\ 12) 14) | 14 121 14\ 14) 14 16 20 
+2|+0\+0/+0-+4 =#=0|+0 
A383 |8|9]8 6} 8| 10 6 14 10 10 
8 | | 
A-4....|8|8| 8 | 16 
A-5.... 8 | 10) 6 | | 
+0) —2 an . 
A-6....| 4 | | | 44 
| £0) —2)£0/+0-+2/+0—2)+0 | = hal 
B-1....| 7] 8} 4|6|6|8| 6| 10 10 12) 12) 128) 10 | 12 12 12 
| ml In} | 
B-2. =f 718/4| | | | | | 
+8\+28)+2/+2\+2/+2\—2-+4 hat 
C-1....| 8] 10 6 | 6 | 6 | 10 10 12) 12 8 | 12 12 8 4 16 22 
| |e | | | M4! |+2 
C2...)6)8) | el | 10) 10 | 10 
| |42\-0\—2+0| | 
, CB...) | 10 6 19 19 10) 10) | | 


do not differ materially from the ‘typical members’ of their 
respective groups. From the average of series C in addition to 
the ‘atypical member’ 8-1, 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-3 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 cwrves: series A, average acidity = 

; maximum acidity = -:-------- ; minimum acidity = —---——--— 5 
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 | 28) 48 
Mmhenilendetcns bocce een |e 4 | 4 {iat RAN: Baty / 
VEU DM yng sre crams vice 
GME eee cals eis cc ce care eal |) LO] L816} 13 11 10 8} Sr 8} 15 
GoliH2 se... F ie 21) 16) 14) 13 10 9 1°40 G& 5 
(COTES Eager Eon 19] 23} 24) 22 22 20 20 19 17 10 
(Cah ae | | 20] 21120) | 20| | 19 17, 16 14 7 
Subtilis-l....... 4/12) 10) 9 7 6 5 6 6 6 6 


SUDDUIS=2.5, 2 -1.- 8 » seers | 8} 10) 8| 7 6 5 6 6 7 6 


From figs. 2 and 3, it is evident that the maximum difference in 
top and bottom acidity for series A is considerably less than the 
minimum difference for series C. Fig. 4 illustrates the maximum 
difference in series C. From table 3 it is apparent that in series 
B there is no essential difference between top and bottom acidity. 

The difference between acidity at the top and bottom of the 
infusion is probably to be referred to the unequal bacterial food 
supply in these two regions. In cultures of series B the food 
supply is uniform throughout the media, and hence acidity dif- 
ferences are not apparent. Where, as in infusions of series A, 


276 MORRIS S. FINE 


Fig. 2 Infusion A-2. illustrating maximum difference in cultures of series A 
between ‘phenolphthalein acidity’ of top and bottom of infusion. Ordinates 
represent number of cubic centimeters of 0.01 N NaOH per 100 ce. of infusion. 
Top = ———; bottom = ----- 4 


days 10 20 30 40 205 


Fig. 3 Infusion C-2, illustrating minimum difference in cultures of series C 
between ‘phenolphthalein acidity’ of top and bottom of infusion. Ordinates 
represent number of cubic centimeters of 0.01 N NaOH per 100 cc. of infusion. 
Top = ————-; bottom = —----— . 


at the bottom is hay, whose constituents are dissolving con- 
tinuously, differences of this nature are manifest. However, in 
the course of preparation, a considerable portion of material has 
already entered solution, equilibrium is thus quickly reached and 
acidity differences—never very great—soon disappear. The infu- 


CHEMICAL PROPERTIES OF HAY INFUSIONS 277 


120 


100 


80 


ie} 
days 10 20 30 40 205 


Fig. 4 Infusion C-31, illustrating maximum difference in cultures of series C 
between ‘phenolphthalein acidity’ of top and bottom of infusion. Ordinates 
represent number of cubic centimeters of 0.01 N NaOH per 100 cc. of infusion. 
Top = —————; bottom = ----- 4 


sions of the C series present quite a different condition. Here 
untreated hay is covered with clear water. There is the oppor- 
tunity for the formation of a concentrated solution of hay con- 
stituents at the bottom, which serve for the development of a 
high degree of acidity. During the first few days there is a rela- 
tively concentrated solution at the bottom with a corresponding 
high acidity, while at the top of the infusion both these conditions 
arereversed. As diffusion proceeds, the concentration approaches 
uniformity and hence acidity differences tend to disappear. Uni- 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 2 


278 MORRIS S. FINE 


Oye 10 20 30 40 50 


Fig. 5 Bacterial infusions, average ‘phenolphthalein acidity.’ Ordinates 
represent number of cubic centimeters of 0.01 N NaOH per 100 ce. of infusion. 
Upper curve, coli. Lower curve, subtilis. 


form acidity is of course attained when the rate of diffusion of hay 
constituents balances that of their solution. The present study 
contains many data illustrative of the above explanation. 

Peters is of the opinion that the deeper layers of the infusion 
owe their higher acidity to greater concentrations of CO... ‘‘Ow- 
ing to the slowness of diffusion, each upper layer of the liquid 
protects the adjacent lower layer from rapid interchange with the 
air and enables the deeper layer to maintain a higher acidity.”! 
This factor is, I believe, probably only of secondary importance. 
The greater acidity in the lower regions of the hay infusion is 
to be attributed primarily to their greater concentration of acid- 
yielding hay constituents, as explained above. Peters’ explana- 
tion would not entirely account for the fact that infusions of the 
B series show uniform acidity throughout the cultures, while the 
acidity differences in an infusion of series C' is very great. 

In the early history, at least of the typical members of series 
C, the increase in acidity is not uniform with increase in depth. 
Until the immediate region of the hay is reached, the acidity may 
be practically uniform. To illustrate this: on the fifth day a 
sample taken from just above the hay in infusion C-2” titrated 
exactly the same as a sample from the top, while the difference 
between the acidity at the top and extreme bottom was 58. On 


11 Loc. cit., p. 332: 12 See table 4. 


CHEMICAL PROPERTIES OF HAY INFUSIONS 279 


the sixth day a sample taken from just above the hay in infusion 
C-31" titrated 20, against 136 an inch below (in the midst of 
the hay), and 10 at the top of the infusion. It is thus clear why 
the tops of typical infusions of series C are so tardy in reaching 
their maximum acidity (fig. 1). 

Infusion M-51" on the second day of its history was showing 
an acidity difference of 41 between the top and bottom. It was 
then thoroughly stirred after which samples from both top and 
bottom titrated 20. By the next day an acidity difference had 
again developed to the extent of 20. It was again thoroughly 
mixed (both top and bottom then titrating 29), and two days later, 
an acidity difference (to the extent of 9) was once more apparent. 
From here on, however, equilibrium was soon reached. This 
further illustrates the point made that acidity does not increase 
uniformly with the depth. On the second day of this infusion 
the top and bottom titrated 15 and 56 respectively. When thor- 
oughly mixed the infusion gave a uniform titration of 20 instead 
of the average 35. 

In the early history of the typical infusion of series C, the liquid 
in the immediate region of the hay is highly colored, while the 
liquid above is almost colorless. The approach to equality in 
acidity can be followed roughly by the gradual diffusion of this 
colored material. 

The observations on acidity differences may be summarized 
as follows: 

1. The greatest acidity is found near the source of supply of 
soluble material upon which the bacteria can act. 

2. The difference between top and bottom acidity depends 
upon the relative concentrations of acid-yielding hay constitu- 
ents in these regions. 

3. When there is uniform concentration, or when diffusion keeps 
pace with solution, there is no essential difference between top 
and bottom acidity. 

From table 5 we see that the curve for ‘methyl-orange alkalin- 
ity’ follows an irregular course with a general upward tendency, 
as also noted by Peters. The latter’s figures for these determina- 
tions are from three to ten times as great as here recorded. This 


280 MORRIS S. FINE 


may possibly be accounted for by the difference in the quality 
of the tap waters used in the two investigations. For example, 
the tap water employed in our experiments titrated 6 ec. 0.01 
N HCl per 100 ce. of sample, while water from a neighboring 
well titrated 22 cc. per 100 ec. of sample. 


RELATION OF TITRATABLE ACIDITY TO PROTOZOAN SEQUENCE® 


The organisms make their appearance at about the same time 
in the three types of infusions. The protozoa of the B series run 
their course more rapidly than those of the A and C series, the 
two latter not varying markedly in this respect. These time 
relations are probably in great part dependent upon the food sup- 
ply. The latter is the determining factor for acidity. However, 
the degree of titratable acidity existing in an ordinary hay infu- 
sion has no important bearing on the duration and character of 
the cycle taking place, for in the three types of infusions, the 
sequence of the dominant protozoan forms studied was the same, 
although these culture fluids show characteristic differences in 
the degree of acidity. Furthermore, the cycle normally occurring 
in the B series was not influenced when the acidity was purposely 
increased to two or three times that ordinarily obtained.!¢ When, 
however, the acidity was raised to seven or eight times that of the 
typical B infusion,“ the development of the protozoan fauna 
was greatly retarded. Decreased acidity, likewise, has no appre- 
ciable influence upon the protozoan cycle: BB-5 had an almost 
negligible acidity” throughout its history, yet the sequence of 
organisms did not differ in any important respect from that 
found in the control infusion, BB-6, or other typical infusions of 
this series. 

While certain species of protozoa are particularly abundant 
during the period of high acidity and others during low acidity, 


18 Cf. Woodruff, loc. cit. 

4 This was accomplished by adding dextrose to the infusion. Cf. p. 268 and 
table 3. 

16 Brought about by adding alkali when necessary. Cf. p. 268 and table 3. 

16 The biological examination of infusions BB-l . . . . BB-6 was made 
by Dr. Woodruff but not reported in his paper. 


CHEMICAL PROPERTIES OF HAY INFUSIONS 281 


detailed correlations are not possible. It is my opinion from this 
study that even did we know exactly the course of the curves 
illustrating mineralization, oxygen consumed, acidity, alkalinity 
and other information of a general character—with such data 
alone nothing but the most superficial correlations could be 
expected. It is not improbable that a partial explanation of the 
sequence of protozoa occurring in hay infusions may be found in 
the influence of certain excretory products of an organism upon 
others of its own or other species. That this is a consideration of 
some importance is brought out in a recent paper by Woodruff,?’ 
in which it is shown that the excretory products of paramaecia 
inhibit the reproduction of these organisms. 


SUMMARY 


The acidity of hay infusions is essentially due to bacteria, 
their efficiency in producing acid being governed by the concen- 
tration of the infusion in acid-yielding materials. The protozoa 
play a relatively small part in the production of acid. The 
sequence of protozoa and the course of the titratable acidity possess 
no intimately mutual relation. Either may vary within wide 
limits without appreciably influencing the course of the other. 


17 Woodruff: Jour. Exp. Zool., vol. 10, p. 557, 1911. 


STUDIES IN THE LIFE CYCLE OF HYDATINA SENTA 


Ill. INTERNAL FACTORS INFLUENCING THE PROPORTION OF 
MALE-PRODUCERS! 


A. FRANKLIN SHULL 


SIX FIGURES 


NTPROGU GIONS. see cach ase iieeioai ae 6+ ba elelo(d a smie\n a sds aera aged en OPO LT OES 283 
Observations and experiments. . « StReeRyeae ones 284 
Decrease in the proportion vol. male- produce ers with loner soutinued ¥ par- 

thenogenesis EE Renan Martie ors de nia was sled ahs | eet «Ot 
Decrease in size of family with long-continued part ihenbeta nesis. .. . 288 
Effect of inbreeding on the proportion of male-producers.... 291 
Effect of long duration of the fertilized egg stage on the proportion of 
male-producers....... : 296 
Period at which the nature of a ‘female i is determined 301 
MO IRGUSSION Score ois lpelersic erases ale ite tare apetelSotcl- xia ; Bi tessa 308 
Stink Gagede bemoans eiocc Oc aparece nie Miso Bike, e/g 315 
iss Toi heCneyo) WB aacaseancnead 2 de nedc oda aern ar ieealeapaare t aah 316 
INTRODUCTION 


The existence of internal* factors affecting the life cycle of Hy- 
datina senta was demonstrated in my earlier experiments (Shull, 
11 a) in which two or more parthenogenetic lines of rotifers were 
bred under the same external conditions, and yielded different 
proportions of male-producers. Two such lines were crossed, 
and several new lines thus produced. One of these new lines was 
crossed back to one of the parent lines, and another new line 


1 Contributions from the Zoological Laboratory of the University of Michigan, 
No. 138. 

2 In a recent suggestive paper, Woltereck (’11) has included me among those 
who hold that the life cycle of Hydatina is determined by external agents, not- 
withstanding that a large section of the paper which Woltereck cites (Shull, ’11 a) 
was devoted exclusively to internal factors. Inthe hope of correcting this impres- 
sion of Woltereck’s which may be shared by others, the present article will be made 
to include only evidence regarding internal agents, leaving to a future paper the 
further work that has been done or is in progress on external agents. 


283 


284 A. FRANKLIN SHULL 


derived from their union. These various lines reacted to the 
same environment by yielding different proportions of male- 
producers. Such differences could only be explained by assum- 
ing that each line possessed an internal nature different from 
that of the others. 

The nature of the internal agent or agents was not discussed in 
connection with the earlier experiments, since what I hoped 
might prove a crucial test of their nature, namely, the results of 
inbreeding, was not available because the fertilized eggs obtained 
by inbreeding did not hatch. I have now obtained offspring by 
inbreeding, and the evidence from this source, together with that 
from the former crosses, serves at least to eliminate some possible 
conceptions of the internal agents. The inbreeding experiments 
are described and their bearing discussed in the following pages. 
The question whether the internal factor, whatever its nature, 
is constant, or whether it may undergo progressive change, is also 
taken up. And finally, experiments which seem to determine 
the time at which external agents may affect the cycle are 
described. 


OBSERVATIONS AND EXPERIMENTS 


Decrease in the proportion of male-producers with long-continued 
parthenogenesis 


From observations incidentally made on several lines of roti- 
fers bred through a considerable number of generations, I gained 
the impression that there was a progressive decrease in the pro- 
portion of male-producers with long-continued parthenogenesis. 
Such a condition, if proven to exist, besides being of considerable 
theoretical interest, would be of importance in judging the results 
of breeding experiments in which two distinct or unrelated lines 
are compared with one another. Inasmuch as a number of my 
experiments involved such a comparison, I have compiled data 
from a number of lines which were reared through a number of 
generations large enough to give results of value. There are 
eight of these lines, each including 46 or more generations, which 
are given in table 1. 


LIFE CYCLE OF HYDATINA SENTA 285 


TABLE 1 


Showing proportion of male-producers in eight parthenogenetic lines of Hydatina 
senta in successive generations. The generations are taken two by two, and the 
proportions are given in per cents. 


GENERATION 


1-2| 3-4) 5-6) 7-8 | 9-10 | 11-12; 13-14, 15-16) 17-18) 19-20 21-22 23-24 


42.2 49.1 | 66.2 | 75.9 | 64.0 | 31.8) 5.1) 47.4 | 77.3) 64.2 | 36.4 | 18.9 
19.1} 12.5 | 28.1) 0.0) 2634 | 62.5 | 25.4 | 79.2 | 20.4) 4.4] 0.0} 32.2 
69.5 | 50.0 | 48.7| 6.8 | 42.1| 12.2) 30.3 | 33.8} 0.0/ 42.3 | 36.3 
27.4 1.7) 1.4) 27.8) 9.57 7.1 7.0} 5.0; 8.7 | 45.5] 10.3 
29.8 | 47.0 | 25.4 | 54.2 | 53.2 | 49.4 | 33.3 | 37.2 | 34.8] 9.0] 48.7 
4.0) 0.0) 1.1) 10.2) 35.4} 0.0) 0.0] 0.0) 17.6) 4.9] 2.9 
| 18.9) 0.0} 2.2) 1.2] 2.0} 45.8) 2.6) 27.1 | 1.3] 9.6| 1.9 
56.1 | 33.6| 15.8| 9.2] 11.6| 0.0] 7.8| 14.2] 18.6| 1.6 8.7 
| 
33.4 | 28.3 | 21.3 | 25.0 | 31.0 | 18.1 | 26.0 | 26.9} 18.7 18.7 | 20.0 
—o GENERATION 
25-26 | 27-28 29-30) 31-32, 33-34) 35-36 37-38 39-40 41-42 43-44 45-46 
-0.0;} 2.5} 8.4] 6.8; 3.4) 0.0] 9.5 | 48.5) 26.3 66.2 
52.3 | 17.5 | 14.9} 25.3} 12.0) 4.5/15.3) 5.5} 0.0! 4.6 
42.8 | 24.4) 23.8 | 45.5 | 40.6 | 32.3 | 38.9 34.4) 50.0, 3.4 
53.9 43.0 62.6 | 13.0 16.3 | 17.6) 15.1 3.3/17.0 2.6 
| 19.3] 0.0) 13.3 | 40.8 | 18.4 | 22.9 | 43.1) 16.2} 10.9 25.0 
| 22.4] 13.6| 5.4] 14.2/ 17.8) 0.0) 5.0) 23.4) 8.0 3.4 
10.2) 0.0} 9.6| 6.6) 31.5) 0.0) 0.0) 1.5} 0.0) 0.0 
10.6 | 25.0| 4.2 | 34.1 1.7; 2.8) 7.8) 12.1] 0.0; 0.0 
PASVETOGO Sb aicje'e tise sais naive’ | 24.2 | 15.8 | 17.8 | 23.3 | 17.7 | 10.0 | 16.8 | 18.1 | 14.0 | 13.2 


to 
= 
| ie 


In compiling the data, I have combined the generations two 
by two, partly to save labor in computing percentages, partly 
to smooth out the great fluctuations that often occur from one 
generation to the next. Two methods of handling the data would 
have been practicable. All the male-producers and the female- 
producers, respectively, of corresponding generations in all eight 
lines could be added together, and the percentage of male-pro- 
ducers computed for the whole group. Or the percentages could 
be determined for each line separately, and then an average of 
the eight percentages in corresponding generations computed. 
The former method would give to a line producing large families 
much greater weight in determining the end result than to a line 
that produced small families; the latter method makes all the 
lines of equal importance, regardless of the number of individuals. 


286 A. FRANKLIN SHULL 


Since my incidental observations had been made on lines produc- 
ing large families, it seemed more conservative to adopt the latter 
method, of computing the percentages in each line separately, 
and taking the mean of the eight percentages. This method has 
the further advantage, as we shall see later, of enabling us to 
analyze the result. 

Most of the lines in table 1 included more than 46 generations. 
In such eases, the first 46 generations are given. In two cases 
the lines included 92 or more generations; these were divided into 
two parts, the first to the 46th generation constituting one line 
as given in table 1, the 47th to the 92d another line, while the 
remainder were omitted. Thus, lines 1 and 2 are parts of the 
same line, as are also lines 6 and 7. Generations not fully re- 
corded have been omitted, except where they were necessary (as 
they were in one case) to complete the 46 generations. The one 
case of incomplete records can hardly invalidate the results. 

These lines have been, for the most part, recorded in detail in 
my former papers (Shull, 710, 711 a), or are given in the following 
pages. Lines | and 2 are found almost complete in table 1 of those 
papers, line 3 in table 3 (right column), line 4 in table 34 (right 
column), line 5 in part in table 37 (middle column), lines 6 and 
7 almost complete in tables 20 and 34 (left column in both), 
and line 8 in part in tables 2 and 6 of this paper. As the details 
of the families may be had in the places cited, they are not 
repeated here. 

The mean percentages of male-producers in the various pairs of 
generations, given at the bottom of table 1, show great fluctua- 
tions; but the earlier generations have plainly more male-produc- 
ers than the later generations. When they are represented by a 
curve, as in fig. 1, it is obvious at a glance, notwithstanding the 
fluctuations, that there is a progressive decrease in the proportion 
of male-producers from the first generation to the last. 

If the eight lines be examined separately, it is seen that most 
of this decrease in the proportion of male-producers is due to 
three lines (1, 2, and 5), and perhaps a fourth (line 8). Lines 1 
and 2, which are two parts of the same line, are plotted in fig. 2. 
Because we have here only a single line, the fluctuations are so 


287 


CYCLE OF HYDATINA SENTA 


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1 to 5 11 to 15 21 to 25 31 to 35 41 to 46 
Number of Generation 
Fig. 3 


great that I have taken the generations in groups of five. Despite 
the enormous differences among the groups of five generations, 
there is seen to be a general diminution in the proportion of male- 
producers through the series. In like manner, line 5 (fig. 3) 
shows such a decrease in a most unmistakable manner. 

The remaining five lines, including line 8 which would show a 
considerable decrease in the early generations, are combined and 
plotted in fig. 4. Here it would perhaps not be justifiable to 
assume a decrease in the proportion of male-producers in the 
lines as a whole. A possible explanation of these differences 
between different lines, and the importance of the phenomenon 
of decrease in the proportion of male-producers where it occurs, 
are discussed elsewhere. 


Decrease in size of family with long-continued parthenogenesis 


It became apparent in several lines of rotifers that the families 
became gradually smaller, and that it was increasingly difficult 
to maintain the animals in a healthy and vigorous condition. 
To determine whether there were any possible connection between 
the diminution in the size of family (reduction of vigor) and the 
decrease in the number of male-producers, I have plotted the size 
ot family in those lines that showed the decrease in the propor- 
tion of male-producers. 

For the sake of comparison, the generations are again taken in 
groups of five. Fig. 5 represents lines 1 and 2 of table 1, and may 


LIFE CYCLE OF HYDATINA SENTA 289 


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290 A. FRANKLIN SHULL 


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Number of Generation 
Fig. 6 


be compared with fig. 2, which shows the proportion of male- 
producers for the same lines. Fig. 6 represents line 5 of table 1, 
and is to be compared with fig. 3. Both curves show a decrease 
in size of family, which is especially marked in fig. 6. It does not 
follow, however, that there is any relation between the two phe- 
nomena, namely, the decrease in the size of family and the 
decrease in the proportion of male-producers with long-continued 
parthenogenesis. 

To‘discover whether the great fluctuations in the proportion of 
male-producers has any relation to size of family, several lines 
that showed the greatest fluctuations have been examined in 
detail. While in some cases the families, or groups of families, 
showing a great increase in the proportion of male-producers 
over the preceding generations also showed a great increase in 
the size of the family, this was not true in a number of other cases. 
One need not conclude from this that there is no relation between 
size of family (vigor of line) and proportion of male-producers. 
There are many accidents which might happen to a female whose 
family includes many male-producers. I have known a number 
of females to die as a result of accidentally (?) starting to devour 
a large Paramecium or a fiber in the water. The small families 
produced by such females may have included mostly male- 
producers, so that a study of individual families can hardly be 


LIFE CYCLE OF HYDATINA SENTA 291 


expected to show a correspondence between ‘vigor’ and the pro- 
portion of male-producers. 


Effect of inbreeding on the proportion of male-producers 


In the second of these studies (Shull, 711 a) I deseribed experi- 
ments in which two distinct lines of rotifers, yielding different 
proportions of male-producers, were crossed, the zygotes giving 
rise to lines in some crosses yielding more male-producers than 
either parent line, in other crosses a proportion of male-producers 
intermediate between those of the parent lines. In order to 
explain these phenomena, an attempt was made to inbreed the 
same lines that were used in the earlier crossing experiments. 
Females were paired with males of the same line, and a large 
number of fertilized eggs was secured. These eggs, however, 
did not hatch before it was necessary to discontinue the experi- 
ments. Ihave now, however, in other lines, succeeded in obtain- 
ing viable offspring from females paired with their own nephews 
or cousins, and give the results in the following experiments. 

Experiment 1. Inbreeding. Some winter eggs were collected 
in the spring and kept in an ice-chest or in cold running water 
until September. The eggs were then brought to room tempera- 
ture and began to hatch in five days. From one of the females 
thus obtained was reared the line of rotifers used in this and the 
following inbreeding experiments. 

Between October 7 and October 20, many females of the parthe- 
nogenetic line just mentioned were paired with males of the same 
line. Of 1099 eggs obtained, one hatched October 22, and from 
her another parthenogenetic line was bred. The number of male- 
and female-producers in this inbred line is compared, in table 2, 
with the corresponding data from that part of the original line 
which was reared at the same time. 

It appears from the table that the inbred line yielded 16.7 per 
cent of male-producers, the original line only 10.6 per cent. In 
such an experiment, however, the original line is necessarily 
further removed from the fertilized egg than is the inbred line. 
We have learned above that there may be a progressive decrease 


292 A. FRANKLIN SHULL 


TABLE 2 
Showing the number of male- and female-producers in two lines of Hydatina senta, 
one line being the result of inbreeding in the other line, that is, being derived from 
the offspring of a male and female both from the other line. Male-producers are 
designated 32, female-producers 9 9. 


ORIGINAL LINE INBRED LINE 
| 
DEG atae Number of 7) Number of 2 9 Date of first’ | Number of o?) Number of 2 9 
young | young | 
October October 
24 1 8 24 0 28 
26 | 4 30 25 10 36 
28 0 28 27 2 21 
29 0 48 29 | 2 17 
31 5 40 30 2 43 
November — November | | 
2 | 0 19 1 | Sie 44 
4 1 15 3 | 7 38 
6 u 33 5 | 2 45 
8 8 38 6 2 44 
10 9 36 8 0 26 
12 0 22 10 | 14 36 
14 1 38 12 13 9 
16 | 2 46 14 6 37 
18 5 27 16 3 30 
20 | 8 18 18 10 9 
22 2 31 20 0 23 
25 6 39 ae, 1 11 
26 2 28 24 4 10 
28 5 | 23 26 1 ai 
30 4 4 28 } 9 25 
December 30 0 41 
2 0 27 December 
4 3 40 2 Phe 33 
7 3 23 4 1 43 
9 rat 4 6 | 20 20 
12 1 22 9 | ul 10 
13 0 5 17 2 
0 29* 11 2 13 
15 0 15* 13 0 17 
15 3 14* 
= = — ~ if 
Motalenaer 88 736 147 732 
Per cent of 


opel Badaose 10.6 16.7 


* Remainder of family not recorded. 


LIFE CYCLE OF HYDATINA SENTA 293 


in the proportion of male-producers, such that a comparison of 
two lines may show fewer male-producers in the older line, 
although the two lines would have been equal had each been taken 
at the same number of generations after the fertilized egg. An 
application of this discovery is found in the present experiment. 
The original line in this experiment is line 8 of table 1. It will 
be seen in that table that the early generations of the line had a 
larger number of male-producers than the subsequent genera- 
tions. The original line passed through ten generations before 
the inbred line started. If these ten generations, comprising 
147 male-producers and 315 female-producers, be added to the 
27 generations given in table 2, then the total for the original line 
shows 18.2 per cent of male-producers, or a higher percentage 
than that of the inbred line. We may not be justified in including 
the first ten generations, as I have just done, but in view of the 
decrease in the proportion of male-producers in the later genera- 
tions, it seems to me unsafe to ignore the early generations. 

Incidentally it may be mentioned that the average size of family 
in the entire original line, exclusive of the last two families which 
were not fully recorded, was 34.5 as compared with 30.7 in the 
inbred line. 

Experiment 2. Twice inbreeding. Females of the inbred line 
of the preceding experiment were paired with males of the same 
line. Of 144 eggs obtained, one hatched November 26, and 
became the parent of the line in table 3 designated ‘twice inbred.’ 
This line is compared with those parts of its parent (also inbred) 
line and the original (‘grandparental’) line that were reared at the 
same time. 

Here the difference between the two inbred lines is not great, 
while the percentage of male-producers in the original line is con- 
siderably less. Since all three of the lines are at different ‘ages,’ 
that is, a different number of parthenogenetic generations has 
been passed through in each line since the fertilized egg from which 
the line was derived, it is of interest to note the proportion of 
male-producers in the whole lines, and not merely those parts 
that were bred simultaneously. In the original line, including 
28 generations reared previously to the beginning of this experi- 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 2 


294 A. FRANKLIN SHULL 


TABLE 3 


Showing number of male- and female-producers in three lines of Hydatina senta, the 
line in the second column being inbred from that in the first column, and the line in 
the third column from that in the second (see text) 


ORIGINAL LINE } INBRED LINE | TWICE INBRED LINE 
| Or Or Or Or | | oO Oo 
HOt 1 sos ‘o o | lato o 
Date of first | 2 | © Date of first s © | Date of first | . % 
young amen Eas young ral a || young | ae) 3 
| 5 g ee || | 8 E 
Zz Z| Zz Zz | ie Z 
ss = ae ay oa he ie a hs — <r. Ge aod = | 
November | | November | | November | 
28 5 |\" S23 ens 9 | 25:1 ©9928 1) Gneaaentes 
30 4 4480 +] @ |, 41s 880, = | onan 
December | December | | December | 
2 LO eee) 2 | 2) 33 2 lina 39 
4 3 40 4 (ect 43 || 4 | 2 18 
7 Ba) 223i 6 | 20 20 | 6 2 31 
9 11 4] 9 11 10 | 38 26 
12 1 22 || eli 2 | 9 eet 23 
13 0 5 | 11 lee 13 | 11 ooo 8 
0 29*! 13 eo 17 | 13 1 16 
15 0 15* 15 83 14*| 15 0 3 
| | | 9 Bie 
Totalescoe: 27 | 192 | 65 | 218 50 | 202 
| 
Per cent of 7 9 12.3 22.9 | 19.8 


* 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 29 


or 


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 
Beciot rat | nberotas|Numbeot oe) Due Came | ot90| Number of 99 
young | young 
June | October 
17 7 14 | 12 35 16 
19 11 17 | 14 23 25 
21 20 ie | 22 17 0 21 
22 25 11 18 33 16 
23 2 | 24 20 1 11 
25 19 | 9 30 22 
26 25 4 a 43 8 
27 29 19 27 0 16 
29 23 14 
Motals ns. 157 130 188 149 
Per cent of 
Oko) See 54.7 55.7 


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 aécompanies 
long-continued parthenogenesis might haveshownaslight decrease 
in that proportion as a result of inbreeding. 


Effect of long duration of the fertilized egg stage on the proportion of 
male-producers 


DURATION OF THE FERTILIZED Eco 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, 711 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. A portion of this lot of eggs was 
kept cool (11° C.) for over seven weeks and then brought to room 
temperature; and another portion was frozen for thirty-three 
hours and then gradually thawed out. But none of these eggs 
hatched within twelve weeks, after which time observations 
ceased, 

The F; females mentioned in the same paper (op. cit.) were 
inbred, but of 146 eggs laid, none hatched in seven weeks. Like- 
wise, in the cross between F; and the Baltimore parent line, 
which were related lines, of 179 eggs none hatched in seven weeks. 
In the cross between F, and the New York parent line (also re- 
lated lines), of 814 eggs, two hatched in about a week, while 
no more hatched in the next seven weeks. 

In the above cases there is great variability in the duration of 
the egg stage in eggs coming from the same source; and there is 
considerable difference between eggs coming from one source 
and those from another source. Thus, crosses between unrelated 
lines yielded the greatest percentage of viable eggs; crosses 
between related lines yielded few or no viable eggs, while inbreed- 
ing failed to produce eggs that would hatch at all. 

Since that time I have obtained numerous viable ‘inbred’ 
eggs, and crosses that produced a much higher percentage of 
viable eggs; but in none of these cases were complete records 
kept, hence comparative figures are not available. 

It has already been demonstrated (Shull, ’11 a) that fertilized 
eggs from different sources may yield parthenogenetic lines 
including different proportions of male-producers. The great 
variability in the duration of the egg stage in eggs from the same 
source, described above, suggested that this variability might 
be related to the proportion of male-producers in the partheno- 
genetic lines derived from those eggs; that is, that parthenogenetic 
lines derived from eggs that hatched quickly might include more 
or fewer male-producers than lines derived from late-hatching 
eggs. The following experiments were performed to test this possi- 
bility. The eggs used are the ‘inbred’ eggs obtained by pairing 
males and females of the same line (the ‘original line’ in Experi- 
ment 1). 


298 A. FRANKLIN SHULL 


Experiment 38. A fertilized egg which hatched in not less than 
7 nor more than 14 days became the parent of the line in the first 
column of table 5, while the second column represents the line 
obtained from an egg that hatched in about four weeks. 

The individual from the early-hatched egg yielded a line with 
22.3 per cent of male-producers, the late-hatched only 15.4 per 
cent, notwithstanding that the ‘early-hatched’ line is the older 
line by two or three weeks and might therefore be expected to 
have decreased somewhat in the proportion of male-producers. 


TABLE 5 


Showing number of male- and female-producers in two lines of Hydatina senta, one 
derived from a fertilized egg that hatched in two weeks or less after laying, the other 
from an egg that hatched in four weeks 


EARLY HATCH LATE HATCH 
Date of first Number of 72 Number of 2 2 RL Number of o' 9: Number of 2 9 
young young 
November November 
22 1 11 22 2 11 
24 4 10 24 4 17 
26 1 7 26 4 28 
28 9 25 28 6 37 
30 0 4] 30 1 , 24 
December | December | | 
2 2 33 2 1 | 18 
4 1 43 4 ie | 22 
6 20 20 5 uf 27 
9 | BI 10 8 1 40 
17 2 10 11 0 
11 | 2 13 8 25 
13 0 if 15 1 8* 
15 3 14* 
ROGAI See 71 246 7 257 
Per cent of 


Gp OM ec ete 22.3 15.4 


* Remainder of family not recorded. 


LIFE CYCLE OF HYDATINA SENTA 299 


This possible decrease can hardly be urged in this case, however, 
as a reason for assuming that the effect of late hatching is even 
greater than table 5 shows it to be. For the ‘early-hatched’ 
line is here taken at a period of many male-producers. Prior 
to the opening of this experiment, the early hatched line included 
16 generations. If these 16 generations be included with the 
12 generations in table 5, the total for the early hatched line is 
147 male-producers and 732 female-producers, or 16.7 per cent of 
male-producers. This is not greatly in excess of the 15.4 per cent 
in the late hatched line. 

It may be pointed out incidentally that the average size of 
family in the late hatched line exclusive of the last family which 
was not fully recorded, is 26.8. In the early hatched line, if we 
count only the generations given in table 5, the average size of 
family is 25; but if the 16 earlier generations be included, the 
average size of family is 30.7. There is perhaps here a decrease 
of vigor associated with duration of the egg stage. 

Experiment 4. Of the same lot of fertilized eggs as that used 
in the preceding experiment, one hatched after not less than 75 
days nor more than 91 days in the egg stage, which is a much 
longer time than most of the other eggs of the same lot required. 
A line of 12 generations was bred from this individual, and is 
given in the right column of table 6. No line derived from an 
early hatching egg of this same lot was in existence at that time 
to compare with the late hatching line. The ‘original line’ (of 
.Experiment 1) from which the lot of inbred eggs was obtained 
was still being reared, and that part of it which occurred simul- 
taneously with the late hatched line in table 6 is givenfor com- 
parison; but the original line isso much older than the late hatched 
line that we should expect it to have fewer male-producers even 
if it were at first equal to the late hatching line in the proportion 
of male-producers. This expectation is justified by the percent- 
ages in the table, where the original line gives only 9.4 per cent 
of male-producers, whereas the entire original line, including 39 
generations previous to the seven here given, gave 17.3 per cent 
of male-producers. This is only a little less than the percentage 
obtained from the late hatching egg. It should also be recalled 


300 A. FRANKLIN SHULL 


TABLE 6 


Showing the number of male- and female-producers in two lines of Hydatina senta, 
the one being reared from a resting egg that remained unhatched seventy-five to 
ninety-one days, the resting egg having been obtained from inbred female of the other 
line in this table. 


ORIGINAL LINE | LATE HATCHING INBRED 
Date of first Number of o&'9| Number of ? 2 atone | Number of o& 9 Number of 2 2 
young young 
January January 
9 1 7 9 2 14 
2 16 11 6 21 
12 1 11 13 3 10 
1 6 0 8 
14 2 2 15 0 21 
0 7 17 3 8 
0 2 1 10 
1 8 19 7 24 
18 0 | 3 21 1 35 
Te ey: 23 2 24 
21 0 2, 25 0 21 
30 0 | 5 26 12 22 
0 } 2 28 15 4 
February | | O) 25 
1 0 | 2 February | 
Liedelll : 1 Nee ae 14 
Totaleteee oe 8 | (ea 58 261 
Per cent of 


of Bas Oe 9.4 18.1 


that the three other inbred lines recorded in the preceding experi- * 
ments yielded 19.8 per cent, 16.7 per cent, and 15.4 per cent of 
male-producers respectively. 

These percentages are so nearly equal that, on the whole, it 
seems pretty certain that there isno connection between thelength 
of time spent in the resting egg, and the proportion of male-pro- 
ducers. 

It may be pointed out incidentally that in the entire original 
line, including 38 generations previous to the seven recorded in 
table 6, but excluding those not recorded in full, the average size 
of family was 26.3, while the average size of family in the late 


LIFE CYCLE OF HYDATINA SENTA 301 


hatched line here shown is only 21.2. How much of this decreased 
vigor may be correlated with long duration of the resting egg 
stage, and how much with inbreeding, can not be ascertained 
from the experiment. , 


Period at which the nature of a female is determined 


At what time in the life of an individual is it determined 
whether she will bea male- ora female-producer? Is it determined 
at a definite period, or is it a gradual process covering a long 
interval? These questions were answered variously by the early 
students of Hydatina. Nussbaum (97) believéd that a young 
female if treated properly could be made to produce either males 
or females, at the will of the experimenter. A young female 
that was starved, became according to his view, a male-producer. 
Maupas (791), on the other hand, concluded that the nature of 
the female was determined in the egg from which she hatched, 
a conclusion which he expressed by saying that the sex of the off- 
spring is determined in the body of the grandmother. Though 
Woltereck (711) apparently accepts Maupas’s view, partly because 
a female produces only males or only females, partly perhaps 
because of some provisional statements of my own (Shull, ‘10), 
statements which were confirmed in a paper subsequent to Wol- 
tereck’s (Shull, 711 b), hitherto there has been no satisfactory 
proof of either proposition. 

It has now been found possible to determine the time, or at 
least one time, at which the character of a female is decided, in a 
manner that seems to me conclusive. Both fertilized eggs and 
parthenogenetic eggs afford evidence on this point. 

EviIpENCE FROM FERTILIZED Eaes. It has long been known 
that fertilized eggs of Hydatina produce only females. I find 
no statement, however, as to whether these females are always 
female-producers, or whether they may be of both kinds. They 
could not be all male-producers, or the species would have per- 
ished long ago. 

In my breeding experiments I have reared to maturity 469 
females from resting eggs, and every one has been a female-pro- 


302 A. FRANKLIN SHULL 


ducer. They were reared under the same conditions as other par- 
thenogenetically produced females, many of which were male- 
producers. It is safe to conclude, therefore, that at the moment 
of fertilization it is determined not only that the immediate 
offspring shall be female, but that the individuals of the next 
generation shall be females. Whether this may be spoken of as 
sex determination a whole generation in advance, or not, is not 
clear; for no matter whether a male egg develops parthenogeneti- 
cally and produces a male, or is fertilized and yields a female, the 
next generation in the direct line, if there be any, is necessarily 
always female. Aside, however, from the use of the word ‘sex- 
determination’ there can be no doubt that in this case the nature 
of the females of the first generation is determined in the fertilized 
eggs from which they hatch, and before those eggs are laid. There 
is no a priori reason, therefore, for supposing that the nature of 
other females may not also be determined in the parthenogenetic 
eggs from which they hatch, and before those eggs are laid. 

EvIDENCE FROM PARTHENOGENETIC Eaeas. The discovery 
that rotifers bred in a fairly strong solution of horse manure may 
be made to yield only female-producers, as pointed out in my 
earlier article (Shull, 710), was employed in the following experi- 
ments. 

Experiment 5. A line of rotifers was reared in spring water. 
When the first members of a new generation were isolated, two 
were reserved for further breeding. One of these, together with 
all her offspring, was kept in spring water to continue the line. 
The other was reared to maturity in spring water, being examined 
every twelve hours, at 9 a.m. and 9 p.m., daily. As soon as she 
was observed to have laid eggs, she was transferred to a new dish 
of spring water, in which she remained during the next twelve 
hours; while the water on the eggs already laid was removed, and 
replaced with filtered, undiluted manure solution. Every twelve 
hours thereafter the female was transferred to fresh spring water, 
while the eggs laid in the preceding twelve-hour period were placed 
in manure solution. The eggs were allowed to hatch in manure 
solution and the young were reared to maturity in the same solu- 
tion. Accordingly, every egg was laid in spring water, and (with 


LIFE CYCLE OF HYDATINA SENTA 303 


the exception of three eggs that hatched in less than twelve hours) 
was hatched, and the young reared to maturity in manure solu- 
tion. That the manure solution used was strong enough to ex- 
clude male-producers was shown by rearing five successive gener- 
ations in it. These five generations comprised 185 individuals, 
all female-producers, while the sister line in spring water, as shown 
in table 7, included many male-producers. 


TABLE 7 


Showing number of male- and female-producers in two series of generations bred 
from sister individuals of Hydatina senta, in one of which the eggs were laid and the 
young reared to maturity in spring water, in the other the eggs were laid in spring 
water, but hatched and the young reared to maturity in manure solution. 


HATCHED AND REARED IN SPRING WATER HATCHED AND REARED IN MANURE SOLUTION 
SEE TE 
f | Yate of firs 
DEOG EE | Number of o'9 Number of ? 2 ies Gace Number of o'? Number of ? 
young young 
February | February 
§ 25 15 Ss 9 35 
9 | 19 30 9 38 9 
11 | 8 31 13 26 
12 | 18 31 Z 7 33 
MROt All he <<a: 70 107 67 106 


Per cent of 
oe ike 39.5 38.7 

If the nature of a female is not determined before the twelfth 
hour of the egg stage, the treatment just described should exclude 
male-producers from the one line. If the nature of a female is 
determined at some time between the first and twelfth hours of 
the egg stage, the treatment described should cause a reduction 
in the proportion of male-producers in the one line; and the later 
the determination occurs, the greater should be that reduction. 

From table 7 it appears that the young rotifers reared, from the 
egg stage on, in manure solution, comprise approximately as many 
male-producers as those reared in spring water. The fact that 
there is little or no reduction in the proportion of male-producers 
in manure solution seems to indicate that the nature of the female 


304 A. FRANKLIN SHULL 


is determined at, or prior to, an early egg stage; or if this determi- 
nation is a gradual process, it has proceeded so far before the early 
egg stages that manure solution is unable to reverse it. 

Experiment 6. In this experiment, a line was reared in spring 
water, and as in the preceding experiment two sister females were 
reserved from each generation for the purpose of breeding. One 
female was kept in spring water all her life, and all her offspring 
were reared to maturity in spring water. The other female was 
kept in spring water until she had laid 13 to 18 eggs, and was 
then transferred to filtered, undiluted manure solution. She was 
transferred to fresh manure solution every twelve hours thereafter, 
to prevent accumulation of bacteria, so that all of her eggs after 
the thirteenth to eighteenth were laid in manure solution. All 
the eggs laid in spring water were hatched, and the young reared 
to maturity, in spring water. All the eggs laid in manure solu- 
tion were hatched, and the young reared to maturity, in manure 
solution. 

That the manure solution used in this experiment was strong 
enough to exclude male-producers was shown by rearing in it 
five successive generations of all female-producers, as described 
in the preceding experiment. Further proof of its effectiveness 
is found in the additional control presently to be described. 

The details of this part of the experiment are given in table 8, 
where the rotifers of the early part of the family, that were 
reared in spring water, are given to the left of the vertical line, 
those reared in manure solution to the right of that line. The 
offspring are recorded in the order in which they hatched, this 
order being determined fairly accurately, I believe, by the rela- 
tive sizes of the young rotifers when they were isolated. Only 
one male-producer hatched from an egg laid in manure solution, 
and that came from the very first egg laid after the transfer of the 
female to the manure solution. 

The control of this experiment, that is, those families bred 
entirely in spring water, is given in table 9. Each family was 
reared from a sister of the parent of the corresponding family 
in table 8. These control families are divided by the vertical 


LIFE CYCLE OF HYDATINA SENTA 305 


TABLE 8 


Showing the number, and order of production, of male- and female-producers of 
Hydatina senta, in seven families, the early eggs of which were laid and hatched, 
and the young reared to maturity, in spring water, the later eggs laid and hatched, 
and the young reared in manure solution. Male-producers are designated by &, 
female-producers by. 


& 

iC) 

aig LAID, HATCHED, AND REARED IN SPRING LAID, HATCHED, AND REARED IN MANURE 

gS: WATER SOLUTION 

zZ 

1 PPVPAPPASAPIPASHPLASLH MPL PLE 

2 NOTE ORORO cl LONG, OO Orch OO) O12 O08) O19 O19 O98 2999 

co) fos ietgel eile) £6) selene se peter. 

3 DIST OTE OU OTOROSO OF OS1S" OF ONO'S OVO! Or Or O19 9 2'9'9 

4 IFOVPLPAPAPPPPAFPMLPSLSLPSIPLL LHL IL 

5 PRE One QS 2 SF PSD V8.2 89 8 
| 29929999999 999929929 

6 DISH Hae oe Dia Oia i9 

7 PPQPPIOPPHVSAPSASATPAIS LSS SPSHSSS SHS LISS HSS PPY 
Be Cee eae 2 Ee Ye 


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 9. 


NUMBER OF 
FAMILY 


1 POVHIAPAPL VALI PAAA AAP IIA PIAA IP LAPP IAPS OS 
Mase ooo o 
| PPLLPLPLLPLPAPAAAIPAIAIPAAAGATITFIAF LLG 
| | PASMPLPYSPPLPIPPILAH PP 
3 POSIPPPAMALSSIGISLLLGPAATAAT PTL LGS 
222999 
4 |PPAPPPASPPAPASHPHFAMPVASSLVPPASL IP IPPLGLLGIAIAS 
IIAP AAPIAPIAI SPSS ASE 
POPQAPQLILIPPPAA PI TIAPIPSI PII IS 
SFIPPPPAIVPPISPPMAHFIAPPAL 
PPOLPPLPFAVLSLIPPAAPYPSSP PSAP LAPP LLLP 
PPOPSPSLYVPAVPIPLILI IY 


“10 cr 


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 reserved 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 manure 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 (o6gonial) 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 


4 
° 
ra LAID, HATCHED AND REARED IN MANURE LAID, HATCHED, AND REARED IN SPRING 
a4 SOLUTION WATER 
5 & | 
z 
—=—— — —| 
1 VPPPQOPAAPA SLES LAS LSPS 
| Lo Ls he re gle Me Hk. 
2 | Syne) CVT ete) Looe} geiwrence\ ete MeTe MMe dope Mo MI Moa 
co ike oes cube de o| 
3 PP AG OTIS FOTO GLO RO O14 919 .O)919 29 
4 OLIGO O19) 9) 9 1019) 9. 019)918.0),.0' 99:9) 995919 
5 POO POPP ASSVS VPS AMSASSLIEDAM ASHES PPS EAA P 
6 POOP OPPS APPS S SSP AMS SSS SSS SSL HS LSPL LIISA 
CPOPSOPPVPQIEAHMO LEP 
7 POOP PAPO SPASMS MPMSHS ASP ASPAPASPSS ASS SLMS SSL IAAF 
1 MAP LPALS ESP AAA ISASP 


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 for the 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 sue- 
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- 
thenogenetie 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 311 


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 ege 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, ’08). 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. 


312 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 (711) 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, 710, 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 313 


genes, representing a certain proportion of male-producers. For 
this explanation could not account for intermediate F, in some 
cases, 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 in 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. hat 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 powerless 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, 10). 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. In theexperi- 
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. 


316 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. 


Nusspaum, M. 1897 Die Entstehung des Geschlechtes bei Hydatina senta. 
Arch. f Mikr. Anat. u. Entw., Bd. 49, pp. 227-308. 


Punnetr, 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 uw. The rédle 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. 


Suutz, G. H. 1908 The composition of a field of maize. Amer. Breed. Assn., 
vol. 4. 


Watney, 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. lbid., vol. 6, no. 1, pp. 137-146. 


Wo.utereck, R. 1909 Weitere experimentelle Untersuchungen iiber Artver- 
ainderung, speziell tiber das Wesen quantitativer Artunterschiede bei 
Daphniden. Verh. d. deutsch. zool. Gesell., pp. 110-172. 


1911 Uber Veriinderung der Sexualitiit bei Daphniden. Experimen- 
telle Untersuchungen iiber die Ursachen der Geschlechtsbestimmung, 
Internat. Rev. d. ges. Hydrobiol. u. Hydrogr., Bd. 4, Heft 1 and 2. 
April and June, pp. 91-128. 


La 
» 
7 
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= 


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 6; 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 b. 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, 11). 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, 
ANS), “Al, LD) 

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 thistime. 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 321 


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, ’11). Unfortunately the 
mortality at the time the eggs were fertilized, and also among 
the tadpoles during the early stages of their development, was 


322 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 shewn 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 b, 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 


: e . ae 

g | aa | ts =3 

§ as a a A 2a 

SHRIES SOLUTION USED i | 5a = o a a 

fecatese |) as a | oa re 

a | z 8 = a = 2 gy3 

2 a 5° P| a 2a Se 

& & Zz 3 ta fe % 
min. 

A | 2percentsugar| 10 300 279 | 1381 | 148 53.04 88.51 
A 2percentsalt | 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, ’11), 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 (710) 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 dimorphie spermatozoa in Bufo 


SEX-DETERMINATION IN AMPHIBIANS 325 


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 


TOTAL NUMBER 


eb omer) ars mane | ee 
1904 500 241 259 51.80 93.05 
1907 600 259 341 56 83 75.92 
(651 292 359 55.14 81.33 
1908 140 64 76 54.28 84.21 
1909 323 157 166 51.39 94.56 
210 (134 65 69 51.41 94.20 
1910 1259 ) 775 372 403 52.00 92.25 
: 201 ) 140 65 75 53.57 86.66 
(200 94 106 53.00 88.67 
i911 4 350 (322 169 153 47.50 110.45 
350 \334 178 156 46.40 114.10 


Total... . 4119 1956 2163 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 cases 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 10.48 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.¢ertainly 
seems very remarkable that all three of them should show such 
a high percentage of females. 


SEX-DETERMINATION IN AMPHIBIANS 327 


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, apd 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 6, 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 


STRENGTHS TOTAL NUMBER 2 ten S : : 

BENGTRED NUMBER SEX PER CENT NUMBER MALES 

Ores OUUIIONE) Cs ASCERTAINED Ae DSU NEES) FEMALES TO 100 FEMALES 
USED INDIVIDUALS 

~ _| = ee 

per cent 
0.01 46 42 22 20 47.62 110.00 
0.0050 51 42 26 16 38.19 | 162.50 


0.0025 27 19 14 5 26.31 280.00 


SEX-DETERMINATION IN AMPHIBIANS 329 


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 
eggs 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 13.75 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 331 


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 } per cent solution of cane sugar; an- 
other batch of eggs from the same female was fertilized in a 3 
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 case 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 b, 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 


me ’ ' on 2 a 
| ° 3 ] ae a 
| S me R Pa we <) 3 
) = nt Ka rH <5 <A 
| §& =D 38 = 32 afc 
SERIES SOLUTION USED } < 58 Bi = a fal ial 
lage |) Sie Pama # | o8 | &S |] ase 
° 25 ne g 4 = am amo 
B | sa |a8| 3 a | na | ge] pee 
5 & Z 2 ie Sy z % 
min. 
A |4percentsugar | 30 | 300) 246 112 134 54.47) $3.58\ 119 45 
A } per cent salt 30 300 | 272) 180! 142 | 52.20) 91.54 
B }percentsugar | 60 400 | 367 | 185 182  49.59101.64\ 114 19 
B } per cent salt 60 450 391 235 176 | 45.26)121.59)) 


Bor 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 pots 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 


FUMBER 


TOTAL NUMBER - 5 NUMBER MALES 
SERIES |NUMBERIN-SEX ASCER-- MALES | FEMALES ee emi 00) TO ‘to 100 FEMALES 
DIVIDUALS TAINED | FEMALES (CONTROL LOT) 
| — | = --- — = 
A | 400 281 106 275 W233 ||) 8804 | Wil0v45 
127, 29.41 114.10 


Bi) fy 400) pi) 374 85 289), 4) iar! 

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 Bialaszewicz (’08), amphibian eggs absorb a 
considerable amount of water before the appearance of the first 


SEX-DETERMINATION IN AMPHIBIANS 335 


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, ’11). In each ease, 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 Echi- 
nus microtuberculatus. Arch.» Zellforsch., Bd. 2. 

Biauaszewicz, K. 1908 Beitrige zur Kenntniss der Wachstumvorginge bei 

- Amphibienembryonen. Bull. Inter. de Akad. Sci. de Cracovie. 

Math.-Natur. Classe. 

Davenport, C. B. 1897 Experimental morphology. The Macmillan Company. 

VON GrRigsHEIM, A. 1881 Ueber die Zahlenverhiltnisse der Geschlechter bei 
Rana fusca. Arch. gesammte Physiol., Bd. 26. 

Hertwic, R. 1906 Weitere Untersuchungen ueber das Sexualititsproblem. 
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. 13. 
1909 Studies on sex-determination in amphibians. JI. 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. 

Prutcer, 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 Biitschli 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 #f 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 ino@ulated 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 avery 
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 were in 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 341 


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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 froma 
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, #, 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 it 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 


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348 DAVID DAY WHITNEY 


TABLE 5 


Showing by the comparative reproduction rates that inbreeding race A two successive 
times does not reinvigorate it 


D 
one NEW PARTHENOGE- 
FERTILIZED EGGS FROM INBREEDING A Ist RE ae eae 
| + GENERATIONS 
erp | aparthene aired Offsp bing pf daughter Parthenogenetic 
ized eggs isolated a | 
= 7 | | Ay. 5 | d- 28 -no. 
No. | Time 1911 | Time 1911 | No. | Time 1911 | No. | = Pa aly pre, sane 
1 | A.M. 6-20 | Eve. 6-24 | Large 9 dead. | No young. | Sterile 
2 | A.M. 6-22 | A.M. 6-24 ae] A.M. 6-26 28 \7 10 | 7 7 
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 9 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 2 dead. No young. | Sterile 
8 A.M. 6-23 | Eve. 6-28 Large 9 dead. No young. | Sterile 
9 | A.M. 6-23 | A.M. 6-25 4 | A.M. 6-27 18 4.5 be 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 |6 | 4 52 14 
14 | A.M. 6-26 | Eve. 6-27 4 | Eve. 6-29 | 20 |5 5 60 12 
15 | A.M. 6-26) A.M. 6-28 5 | A.M.6-30 | 37 |7.4 5 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 i) 65 13 
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 22 il 
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. 6-30 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 9 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 @ dead. | No yjoung.| } Sterile 
27 Eve. 6-28 | A.M. 7-2 | Large 9 dead. No young. 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 5 63 12.6 
30 6-30 A.M. 7-2 4 A.M. 7-4 35 8.75 5 63 12.6 
31 6-30 | A.M. 7-2 3 | A.M. 7-4 15 5 | 5 63 12.6 
32 6-30 | A.M. 7-2 = | a? 
33 6-30 | A.M. 7-2 3 | A.M. 7-4 5 |1.66+ 5 63 12.6 
34 7-2 |Bve. 7-3 | 3 |Eve. 7-5 | 18 /4.33+| 4 44 | 11 
35 7-2 | Eve. 7-3 3 | Eve. 7-5 26 (8.66+ 4 44 ll 
36 7-2 | Eve. 7-3 5 | Eve. 7-5 42 |8.4 4 44 11 
37 | 7-2 | A.M. 7-4 5 | A.M. 7-6 448.8 4 50 12.5 
38 | 7-2 A.M. 7-4 | 4 | A.M. 7-6 27 (16.75 | 4 50 12.5 
39 | 7-3 | P.M. 7-5 3 | A.M. 7-7 0 0 5 61 12.2 
40 | 7-3 | P.M. 7-5 | 2 | A.M. 7-7 2} 5 61 12.2 
41 7-3 | P.M. 7-5 3 | A.M. 7-7 5 1.66+) 5 61 12.2 
42 7-3 | P.M. 7-5 2 | A.M. 7-7 0 \0 5 61 2.2 


REINVIGORATION PRODUCED BY CROSS FERTILIZATION 349 


TABLE 5—Continued 


D 
NEW PARTHENOGE- 


| 
= 


Summary 


A 2np 
FERTILIZED EGGS FROM INBREEDING A 1sT seth hag ag 
+ GENERATIONS 
Young ? 9s from Parthenogenetic | rid - 
different fertil- | daughter females | Oe pring oF asian | Parthenogenetic 
ized egge isolated | eee a 
No. Time 1911 | Time 1911 | No. | Time 1911 | No PAY: | 298 | 4,8? | Av.no. 
oe made e Z © “| no. isolated dasa d. 9s 
43 | A.M. 7-3 | P.M. 7-5 | 4 | A.M. 7-7 15 3.75 5 61 12.2 
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 2 A.M. 7-7 ll 5.5 4 47 11.75 
46 | Eve. 7-3 | A.M. 7-5 5 | A.M. 7-7 43 8.6 | 4 47 11.75 
47 | Eve. 7-3 | A.M. 7-5 5 | A.M. 7-7 si 16.2 | 4 47 11.75 
48 | Eve. 7-3 | A.M. 7-5 5 | A.M. 7-7 30 66 | 4 47 11.75 
49 | Eve. 7-3 | A.M. 7-5 | 5 | A.M. 7-7 24 64.8 04 | 47 11.75 
50 | Eve. 7-3 | A.M. 7-5 | 5 | A.M. 7-7 19 3.8 | 4 47 11.75 
51 | A.M. 7-4 | Eve. 7-5 4 | P.M. 7-7 317.75 | 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 8 2 4 43 10.75 
56 | A.M. 7-4 | Eve. 7-5 3 | P.M. 7-7 16 5.33+ 4 43 10.75 
57 A.M. 7-4 | Eve. 7-5 4 | P.M. 7-7 20 5 1 43 10.75 
—| I casl Ls 
57 172 864 5.02 217 2373 10.92 
45 172 864 5.02 68 739 10.86 
| 
= 
TABLE 6 


Summary of 
the exact no. 
of fertile in- 
dividuals 
used. 


Showing by the comparative reproduction rates that inbreeding race B two successive 


Cc 


} 


_times does nol reinvigorate it 


A B 2np - D 
PARTHENOGE- | FERTILIZED EGG | \)ANTHENOGE’ | NEW PARTHENO- 
NETIC GENERA- | FROM INBREED- “71... 393-306 GENETIC WILD 
TIONS 413-416 inG B Isr eo Soe RACE GENERA- 
—— | ————__—- = ATION 
Time 1911 99s/d.9s| Av. | 99s/d.9s| Av. | 9 9sid.9s| Av. | 99s/d.?@s Av. 
iso- pro- no. iso- | pro-| no. iso- | pro- | no. | iso-  pro- no. 
lated duced d.9s lated duced) d.?s lated Nie d.Qs lated duced d.?s 
5/ 14-16 10 ll ital 2 1 (0.5 10 35 3.5 | 5 12 2.4 vt 
5/16-18 8 25 | 3.12 3 1 /0.33+) 9 58 6.44 5 47 9.4 II 
5/18-20 7 te |i 4 8 /2 9 87 (9.664) 5 5310.6 Ill 
5/10-22 8 28 | 3.5 4 13 |3.33+) 9 72 8 5 64 | 12.8 IV 
Summary.... 33 | 71 =| 2.15] 13 | 23 {1.76 37-2526. 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- | 
NETIC WILD RACE 


B 2np | 
FERTILIZED EGGS FROM INBREEDING B Isr 


Young 2? 9s Parthenogenetic 
from different | daughter females | 
fertilized eggs isolated 


Offspring of daughter 


Females Parthenogenetic 


== | 
] d. 9s | | 


No | | =) = 29s | Av.no. 
No.) Time 1911 Time 1911. No. | Time 1911 No. | Av. no. lsolated Pa akon 


| 


fo | FF, 
| 


1 | Eve. 6-12 | A.M. 6-15 5 | A.M. 6-17} 0 |0 5 25 5 

2 | Eve. 6-12 | A.M. 6-15 5 | A.M. 6-17 2 | 0.4 5 25 5 

3 | Eve. 6-12 | A.M. 6-15 4 | A.M. 6-17} 0 |0 5 25 ) 5 

4 Eve. 6-12) A.M. 6-15 5 | A.M. 6-17| 12 | 2.4 Tie |) 25 | 36 

5 | Eve. 6-12 Eve. 6-17 Large) 2 dead. No y oung. | Sterile 
6 Eve. 6-12 Eve. 6-17. Large 9 dead. No young. Sterile 
7 | A.M. 6-13 | Eve. 6-15 4 | Eve. 6-17 9 2.25 5 20 | 4 

8 A.M. 6-13 Eve. 6-15 5 | Eve. 6-17 | 11 | 2.2 5 20 | 4 

9 | A.M. 6-13 | Eve. 6-15 4 Eve. 6-17 8 | 2 5 20 | 4 

10 A.M. 6-13 | A.M. 6-17 3 | A.M. 6-19, 0 0O 5 52 10.4 

11 A.M. 6-13 | A.M. 6-18 | Large| 9 dead. No young. | | Sterile 
12 A.M, 6-13 | A.M. 6-19 Large 9 dead. | No young. Sterile 
13 Eve. 6-13 | A.M. 6-16 2 } A.M. 6-18} 5 | 2.5 5 o2, | 6.4 | 

14 Eve. 6-13 | A.M. 6-16 5 | A.M. 6-18} 15 | 3 5 32 6.4 

15 Eve. 6-13 A.M. 6-16 4 |A.M.6-18| 4 1 5 32 6.4 

16 Eve. 6-13 A.M. 6-20 Large @ dead. | No young. Sterile 
17 Eve. 6-13 A.M. 6-20 Large 9 dead. No young. Sterile 
18 Eve. 6-13 | A.M. 6-20 Large 9 dead. | No young. | Sterile 
19 Eve. 6-13 A.M. 6-20 | Large 9 dead. No young. | Sterile 
20 A.M. 6-14 Eve. 6-16 5 | Eve. 6-18 | 6 1.2 5 21 4.2 | 

21 A.M. 6-14 Eve. 6-16 3 Eve. 6-18 | 0 0 5 21 4.2 

22. A.M. 6-14 Eve. 6-16 4 | Eve. 6-18 1 | 0.25 5 21 4.2 

23 A.M. 6-14 | A.M. 6-17 5 A.M. 6-19 2 | 0.4 5 52 10.4 | 

24 A.M. 6-14 A.M. 6-17 5 | A.M. 6-19 2 | 0.4 5 52 | 10.4 

25 A.M. 6-14 Eve. 6-17 3 | A.M. 6-20} 15 | 5 5 80 16 

26 A.M. 6-14 Eve. 6-17 2 | 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. 1 Sterile 
29 A.M. 6-14 A.M. 6-20 | Large 9 dead. No young. | | Sterile 


30 A.M. 6-14 A.M. 6-20 Large 9 dead. No young. | ; Sterile 
67 13. 


31 A.M. 6-16 | A.M. 6-18 3 | A.M. 6-20) 25 | 8.33+ 5 4 

32 A.M. 6-16 | A.M. 6-18 4 A.M. 6-20) 11 | 2.754 5 | 67 13.4 

33. A.M. 6-16 | A.M. 6-18 4 | A.M. 6-20} 25 | 6.25 5 67 13.4 

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 | P.M. 6-21 0 0 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 2 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=- bya || 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 1.75 4 62 15.4 

45 Eve. 6-17 A.M. 6-22 Large @ dead. No young. Sterile 


—< 


REINVIGORATION PRODUCED BY CROSS FERTILIZATION 351 


TABLE 7—Continued 


D 
NEW PARTHENOGE- 
NETIC WILD RACE 


B2np 
FERTILIZED EGGS FROM INBREEDING B Ist 


Young ° 9s Parthenogenetic 
from different daughter females: 


Offspring of daughter 


feavinlin Parthenogenetic 


fertilized eggs isolated 
| | 99s | a 88 |av.no,| 
No. Time 1911 | Time 1911 | No. Time 1911 No. Ay. NO. | olated aaa ld. 98 
—— = — — 4 — - _ | — = 
| | | 
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 | 0 0 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 
eS ee ee eee = ———— 
51 | 127 | 345 | 2.71+ | 159 1622 10.2 Summary 
' | i | 
— . = i | | . : 
32 | 127 345 | 2.71+) 54 508 9.4 | Summary of 
| the exact 
| | number of 
fertile indi- 
| viduals used. 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 3 


302 


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 


AFTER INBREEDING TWICE, IN A MIXED CULTURE 


A 408 X B 2np 
FERTILIZED EGGS FROM THE PROBABLE CROSSING OF RACE A, 
PARTHENOGENETIC GENERATION 408 + AND RACE B, 


OF THE TWO RACES IN A BATTERY JAR 


D 


NEW PARTHENOGE- 
NETIC WILD RACE 


| | 
ae a a aa Cesare of ee Parthenogenetic 
fertilized eggs _ isolated | emanes 
| | | | S Av. 
No. Time 1911 | Time 1911 No. | Time 1911 | No. AY 9.98, oe | no. | 
| | | | duced d. 9s 
1 |M. 5-22 | Eve. 5-24 5 | A.M. 5-27} 53 | 10.6 5 62 12.4 
2 |M. 5-22 | Eve. 5-24 5 | A.M. 5-27} 62 | 12.4 5 62 12.4 
3 | Eve. 5-22 | Eve. 5-24 | 5 | A.M. 5-27) 54 | 10.8 5 62 12.4 
4 | Eve. 5-22 | Eve. 5-24} 3 | A.M. 5-27/| 42 | 14 5 62 12.4 
5 | Eve. 5-22 | Eve. 5-24 | 5 | A.M. 5-27| 46 | 9.2 eh GPs 12.4 
6 | Eve. 5-23 | Eve. 5-27) 5 | Eve. 5-29 15 3 | 5 | 40 8 
7 | 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 oH et) 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 | 5 40 8 
11 | Eve. 5-24 | Eve. 5-27 | 8 | Eve. 5-29}; 6 | 2 | 5 | 40 8 
12 | Eve. 5-24 | Eve. 5-27 3 | Eve. 5-29 PAU lye | 5 | 640 8 | 
13 | Eve. 5-24 | Eve. 5-27 4 | Eve. 5-29) 35 | 8.75 | 5 | 640 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 Toi} 6 | 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. 5-29 ‘| 11 22) | 5 40 8 
i] 
| = | | 
18 | 72 551 | 7.6+| 85 | 790 | 9.24+) Summary 
17 | 72 551 | 7.6+| 10 | 102 | 10.2 | Summary 
of exact 
| number of 
| individuals 
| used 
= =| 
| 
14 | 59 519 8.79 10 102 10.2 Summary 
| after elimt- 
| nation of 
nos. 6, 11, 
| and 18. 


REINVIGORATION PRODUCED BY CROSS FERTILIZATION 353 


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358 DAVID DAY WHITNEY 


Later this assumption was doubted and the possibility was 
recognized that this reinvigoration might be due to external con- 
ditions. Consequently further experiments entailing great care 
were carried out in which every fertilized egg obtained was known 
for a certainty to have been fertilized by the sperm of the other 
race. Separate new cultures of the two races were made in 
several battery jars at the same time. After a few days many 
males appeared in both races. Then several thousand females 
of each race were isolated in separate watch glasses and soon 
produced both female and male parthenogenetic eggs. These 
eggs were transferred to other watch glasses containing food 
culture water and allowed to hatch. At this period of hatch- 
ing the eggs of both races were continuously watched and the 
young females and the young males were isolated as soon as 
they left the egg membrane thus preventing any fertilization by 
males of the same race. Scores of young females and dozens 
of young males were isolated from each race and then the young 
males of one race were placed in the dish containing the young 
females of the other race. Copulation soon took place and later 
some of the females at maturity produced the thick-shelled con- 
spicuous fertilized eggs. Such females were readily distinguish- 
able, by the general appearance of the enclosed eggs, from the 
parthenogenetic females, and were isolated in separate dishes 
containing food and allowed to produce as many fertilized eggs 
as possible. By this method several dozens of cross fertilized 
eggs were obtained. Eggs of race A were fertilized by the sperm 
of race B and eggs of race B were fertilized by the sperm of race A. 

Twenty-four of these cross fertilized eggs were hatched thus 
starting twenty-four separate races. The immediate offspring 
of each of these races consisting of parthenogenetic females were 
cared for and the reproduction rates of eighty-four parthenoge- 
netic daughter-females from these were determined. At the same 
time the rates of reproduction of the A and B race after the third 
and fourth successive inbreeding respectively were determined 
and also that of the control race, #. All fertilized eggs in these 
reciprocal crossbreeding and the third and fourth successive 
inbreeding experiments of races A and B were produced in the 


REINVIGORATION PRODUCED BY CROSS FERTILIZATION 359 


same week, rested from two to three weeks, and then were hatched 
in the same period of time, two to five days. Tables 9, 10 and 
11 give the details of the experiments and table 13 shows the 
general results in a summary of these three tables together with 
table 8. From these tables it is seen that the reproduction rate 
of the two crossed races irrespective of the manner of the cross 
approaches very closely to the reproduction rate of race £; 
while the reproduction rates of each of the two races which had 
been inbred were very much lower. This seems to be positive 
proof that in crossing two weak races the resulting race which 
develops from the cross-fertilized egg has its general vigor or vital- 
ity greatly increased. 

The high reproduction rates of the two wild parthenogenetic 
races D and # and of the parthenogenetic races A, B and C at 
their beginning, is probably due to the cross-breeding of different 
racesin the same jar. Races A and B were sister races developing 
from the same parthenogenetic mother which originally devel- 
oped from a fertilized egg. If after an interval of about three 
hundred generations each of these two sister races becomes a 
distinct race, as is shown by their different rates of reproduction 
and also by the effects of in- and cross-breeding, it is reasonable 
to suppose that in a general culture jar standing for two to three 
years many different races are constantly appearing and inbreed- 
ing. Consequently all fertilized eggs taken from such a jar will 
develop into races each having a high rate of reproduction. It 
has been previously stated that races A and B originally devel- 
oped from one fertilized egg taken from a wild culture jar. Race 
C was taken from a wild culture jar and races D and F developed 
from fertilized eggs which were taken from the same general wild 
rotifer culture jar that was made in October of 1908. All of these 
races used came from a general culture of rotifers which was orig- 
inally collected in a certain ditch in Grantwood, New Jersey, in 
October of 1906. 

Shull in his experiments in crossing the New York race with 
the Baltimore race of Hydatina senta caught sight of this same 
fact that cross breeding two races increases the rate of reproduc- 
tion but he was not certain of its validity. His table 37 prob- 


360 DAVID DAY WHITNEY 


ably proves it; but as only the descendants of one crossfertilized 
egg were studied and since there was a lack of all time data and 
a failure to record the number of parthenogenetic mothers used 
in each generation, this table can not be taken as very conclusive 
evidence. This tentative conclusion of Shull’s was not known 
until after nearly all the above results of this paper were ascer- 
tained. 

Castle and his collaborators in breeding experiments with Dro- 
sophila state that: 


A cross between two races, one inbred for thirty or more generations 
and of low produetiveness, the other inbred for less than ten generations 
and of high productiveness, produced offspring like the latter in produc- 
tiveness but not superior to it. The same two races crossed after an 
additional year of inbreeding (about twenty generations) produced 
offspring superior to either pure race in productiveness. 


This seems to be in the final results a case parallel to that of 
Hydantina senta. When two races have been closely inbred, 
like brother and sister for many generations or even when two 
races have been bred parthenogenetically, which is the extreme 
of inbreeding, for many generations they show in cross-breeding 
a great increase in productiveness of offspring which is superior 
to that of either of the parent races. 

The effect of inbreeding among animals has been of consider- 
able interest and is of great practical importance; but even at the 
present time there is much diversity of opinion in regard to the 
matter. The relation of inbreeding to sterility has been observed 
in experiments upon mammals by Crampe, Bos, and Guaita; 
upon birds by Fabre-Domengue. They all found the relation to 
be a causal one, continuous inbreeding, as of brothers and sisters, 
resulting in a decrease of fertility, accompanied more or less by 
lack of vigor, diminution in size, partial or complete sterility, and 
pathological malformations. 

In inbreeding experiments upon the pomace-fly, Drosophila, 
Castle and his collaborators state that ‘“‘inbreeding probably re- 
duces very slightly the productiveness of Drosophila.” 

Moenkhaus has recently completed inbreeding experiments 
upon Drosophila in which he has inbred brothers and sisters for 


REINVIGORATION PRODUCED BY CROSS FERTILIZATION 361 


seventy-five generations and found no increase in sterility or a 
decrease in vigor. Perhaps if these inbreeding experiments can 
be carried on for two to three hundreds of generations there may 
appear an increase in sterility and general debility. In Hydatina 
senta at the 75th parthenogenetic generation there was no notice- 
able decrease of vigor; but much later it gradually appeared as the 
generations increased and the race became older. 


SUMMARY 


1. Two distinct sister parthenogenetic races of Hydatina senta 
characterized by the difference in their rates of reproduction and 
general vigor were developed from one original parthenogenetic 
race under identical external conditions. 

2. Races of Hydatina senta allowed to reproduce parthenoge- 
netically for 384 generations, extending through a period of 
twenty-nine months under identical environments, showed a 
gradual decrease in their rates of reproduction. This was as- 
sumed to signify a decrease in the general constitutional vigor or 
vitality of the race. 

3. Successive inbreedings of the weakened parthenogenetic 
sister races, one to four times, caused a slight increase in their 
reproduction powers. 

4. Reciprocal cross fertilization or cross-breeding of such weak- 
ened parthenogenetic sister races of Hydatina senta caused a 
sudden and very pronounced increase in the reproduction rate 
of the ensuing race. This shows that cross fertilization of the 
two weakened races greatly reinvigorated both races and prob- 
ably restored them to their normal vigor which they possessed 
when they started from the original fertilized egg. 

5. The high reproduction rates of new races of Hydatina senta, 
developed from fertilized eggs which were taken from the same 
general wild culture jar that had been standing at least for 
twenty-nine months is due probably to cross-breeding of different 
races. These different races may have been introduced into the 
culture when it was started or they may have developed since 
from the original race. 


January 2, 1912. 


362 DAVID DAY WHITNEY 


BIBLIOGRAPHY 


Bos, J.R. 1894 Untersuchungen iiber Folgen der Zucht in engster Blutver- 
wandtschaft. Biol. Centralbl., Bd. 14, pp. 75-80. 


Birscuur, O. 1876 Studien iiber d. erst. Entwicklungsvorg. d. Eizelle d. 
Zelltheilung u. die Conjugation der Infusorien. Abhand.d. Senken- 
berg. naturfor. Gesell. Fr. a. M., Bd. 10, pp. 213-452. 


Caukins, G. N. 1904 Studies on the life history of Protozoa. tv. Jour. Exp. 
Zool., vol. 1, pp. 423-461. 

CastTLe, W. E., CARPENTER, F. W., Cuark, A. H., Mast, S. O., and Barrows, W.M. 
1896 The effects of inbreeding, cross-breeding and selection upon the 


fertility and variability of Drosophila. Proc. Amer. Acad. Arts and 
Sciences, vol. 41, no. 33, pp. 731-786. 


Cramer, H. 1883 Landwirtsch. Jahrbiicher, Bd. 12, p. 421. 

Cutt, 8. A. 1907 Rejuvenescence as the result of conjugation. Jour. Exp. 
Zool., vol. 4, pp. 85-89. ; 

Darwin, C. The effects of cross and self fertilization in the vegetable kingdom. 
The variation of animals and plants under domestication. 


FaBrRE-DOMENGUE, P. 1898 Unions consanguines chez les Co'ombins. L’In- 
termédiaire des Biologistes, tom. 1, p. 203. 


Guaita, G. von. 1898 Versuche mit Kreuzungen von verschiedenen Rassen 
der Hausmaus. Ber. naturf. Gesellsch. zu Freiburg, Bd. 10, pp. 317- 
332. 


Mauvpas, E. 1889 Le rajeunissement karyogamique chez les ciliés. Archives de 
zoologie experimentale et génerale, Tome 7, pp. 149-517. 


Moenxkuaus, W.J. 1911 The effects of inbreeding and selection on the fertility, 
vigor and sex-ratio of Drosophila ampelophila. Jour. Morph., vol. 22, 
pp. 123-154. 


Suu, A.F. 1911 Studies in the life cycle of Hydatinasenta. Jour. Exp. Zodl., 
vol. 10, pp. 117-166. 


Suuty, G.H. 1908 The composition of afield of maize. Report Amer. Breeders’ 
Association, vol. 4, pp. 296-301. 


1909 A pure-line method in corn breeding. Report Amer. Breeders’ 
Association, vol. 5, pp. 51-59. 


1910 Hybridization methods in corn breeding. Am. Breeders’ Maga- 
zine, vol. 1, no. 2. 


1911 Genotypes of maize. Amer. Nat., vol. 45, pp. 234-252. 
1911 Experiments with maize. Bot. Gaz., vol. 52, no. 6, pp. 480-485. 


Wuirney, D. D. 1910 The influence of external conditions upon the life cycle 
of Hydatina senta. Science, N. S., vol. 32, no. 819, pp. 345-349. Sep- 
tember. 


THE OLFACTORY REACTIONS OF THE PUFFER OR 
SWELLFISH, SPHEROIDES MACULATUS (BLOCH 
AND SCHNEIDER) 


MANTON COPELAND 


Bowdoin College, Brunswick, Maine 


The opinion has long been held that the olfactory organs of 
fishes are concerned with a sense of smell, and that many species 
make considerable use of this sense in locating food. Only 
recently, however, has this view been substantiated by physio- 
logical evidence. Parker (’10) was the first to describe reactions 
of fishes which were unquestionably dependent on the stimulation 
of the olfactory apparatus by odorous substances. The species 
studied was the fresh water catfish, Amiurus nebulosus. The 
following year Parker (’11) tested the common killifish, Fundulus 
heteroclitus, and Sheldon (’11) the smooth dogfish, Mustelus 
canis, for the sense of smell, and obtained marked response to 
olfactory stimuli from each. A few weeks spent at the Biological 
Laboratory of the United States Bureau of Fisheries at Woods 
Hole, Massachusetts, afforded me opportunity to study the sense 
of smell in the puffer, Spheroides maculatus, with the following 
results. I wish to express my thanks to Dr. F. B. Sumner, Direc- 
tor of the laboratory, for many kindnesses received during my 
stay. 

The olfactory apparatus of the puffer is not of the type most 
commonly seen in the higher fishes. Each nasal chamber occu- 
pies the interior of a papilla which rises about 4 mm. above the 
upper surface of the snout, antero-mediad to the eye, and is pro- 
vided with two small circular apertures, one anterior in position, 
and the other situated at the end of a rather poorly marked cylin- 
drical extension directed laterad. Both apertures seem to be con- 
stantly open. 

363 


364 MANTON COPELAND 


By testing a resting fish with carmine suspended in water, I 
was unable to discover any evidence of a current passing through 
the olfactory chambers, neither of the intermittent type dependent 
on respiratory movements common in many fishes, nor of the 
continuous kind produced by cilia, as described by Parker (710) 
in the catfish. If; however, a colored solution is gently forced into 
one of the olfactory apertures by means of a pipette, it readily 
passes through the chamber and out the other aperture. I am 
led to conclude, therefore, that the forward locomotion of the 
puffer forces water through the anterior openings of the nasal 
chambers and out the lateral ones, and at that time conditions are 
most favorable to the stimulation of the olfactory cells by odorous 
substances. That, in truth, the puffer is seldom at rest, when in 
captivity at least, is readily apparent after a few hours observa- 
tion of its habits in a large aquarium. The elevated position of 
the nasal chambers is well adapted to the formation of water 
currents through them by forward locomotion. When a fish is 
swimming rapidly back and forth in an aquarium the olfactory 
organs become directed backward slightly, as it progresses through 
the water, but become erect as it turns in its course. At first, I 
believed that this inclination of the nasal organs was indicative 
of the force of impact of the water against them. I subsequently 
discovered, however, that this is not necessarily so, as the same 
result could be obtained by making a threatening gesture in front 
of the fish. 

Preliminary tests to determine whether the puffer would react 
to concealed food were begun upon eight to twelve fishes, which 
occupied one of the large observation aquaria of the Station. The 
method of experimentation was essentially like that of Parker 
(710, 711), and Sheldon (’11). Two cheese cloth packets of similar 
appearance, one containing meat of the smooth dogfish, Mustelus 
canis, and the other filled with cheese cloth, were suspended some 
distance apart in the aquarium. The presence of food in one of 
the packets could be detected by the fish only through the stimu- 
lation of its chemical sense organs by material emanating from 
the meat. In several tests the packet containing meat was 
quickly seized and bitten open, whereas the other, although some- 


OLFACTORY REACTIONS OF SPHEROIDES 365 


times bitten, received less attention and never was opened. When 
packets made of cotton cloth were substituted for those of cheese 
cloth, similar results were obtained; i.e., the one with meat was 
bitten open, the powerful jaws of Spheroides cutting through the 
cloth as if it had been tissue paper. From these tests it became 
evident that, in order to obtain any extended series of reactions 
to the packets, they must be constructed to withstand a severe 
biting as well as permit the escape of odorous material. This 
end was accomplished by covering with cheese cloth a pair of 
tea strainers made of tin and fine wire netting, one of which was 
filled with dogfish meat, and the other with cheese cloth. The 
fish were not adverse to biting such an object, which was at the 
same time flexible and impossible to tear apart, and, accordingly, 
they were used throughout the experiments to be described. 

With two packets reconstructed in this manner I again pro- 
ceeded to test the fish, about a dozen in number. For fifteen 
minutes after the packets were suspended in the aquarium, the 
puffers bit actively at both, but decidedly more at the one con- 
taining meat. At the end of that time, they were observed for 
one hour, and a record was kept of the number of times each 
packet was bitten, the relative positions of the two being changed 
every fifteen minutes. The packet containing meat received 
42 bites, the one with cheese cloth 4. These tests show con- 
clusively that the puffer is able to discover concealed food. 

That sight plays an important part in the search for edible 
substances is made clear from the fact that the packet of cheese 
cloth is occasionally seized by the hungry fish. Moreover, if a 
wad of filter paper attached to the end of a wire is drawn through 
the water, it is pursued and taken into the mouth as eagerly as 
if it were meat. But, whereas the meat is always swallowed, the 
filter paper, although often drawn into the mouth several times, 
is ultimately discarded. Similar reactions were observed by 
Parker (711) in the killifish. That sight will not explain their 
final discrimination between edible and inedible material is quite 
evident. 

I next planned a series of experiments to ascertain the part 
played by the olfactory apparatus in the reactions of the fish to 


366 MANTON COPELAND 


hidden food. Four puffers were isolated in an aquarium, and 
their normal reactions to the two packets were first recorded. As 
in previous experiments, fresh dogfish meat was always used for 
food, and the positions of the baited and unbaited packets were 
exchanged every fifteen minutes. During the first test hour, 
when the fish were very hungry, the packet with meat was bitten 
119 times, and the cheese cloth packet 18 times. At the end of 
the experiment the fish were fed, and three days later they were 
again tested for an hour: 67 bites at the baited packet, and 8 at 
the other one resulted. It now became necessary to eliminate the 
olfactory organs, to repeat the tests with the packets, and com- 
pare the results with those set forth above. To render function- 
less the olfactory apparatus of Spheroides was a comparatively 
easy task, involving no cutting of nerves or stitching together 
of nares. A silk thread, tied by a single knot around each organ, 
contracted the olfactory chambers so as to prevent effectually any 
flow of water through them. 

About two hours after the close of the test last described, the 
nasal organs of the four puffers were tied in this manner. An 
hour later they were snapping small pieces of dogfish meat from 
the end of a wire in perfectly normal fashion, and soon afterward 
I tested them for an hour with the two packets. At no time did 
they pay any attention to either, although they eagerly seized 
small pieces of meat dropped into the aquarium, or offered to them 
on the end of a wire. Eighteen hours later the test was repeated 
with similar results. As the packets were being suspended in 
the aquarium, the one containing cheese cloth was bitten twice by 
one of the fish, an evident visual reaction, but at no other time 
during the hour was either touched. The fish swam about in a 
characteristic manner, and, on being tested with meat fragments, 
showed they were hungry. There was nothing in their behavior 
to indicate that the contracted state of their olfactory organs was 
in itself at all disturbing. At the conclusion of the test the threads 
were removed, and, as might be expected, the nasal organs 
appeared considerably distorted. On the following day the fish 
failed to react to the packets. Two days later, however, after 
sufficient time had elapsed for the recovery of the injured parts, 


- 


OLFACTORY REACTIONS OF SPHEROIDES 367 


reactions to the packets again resulted, characterized by normal 
discrimination between the two. In a fifteen minute test the 
baited packet was bitten 28 times, and the other one twice. 

These experiments show that reactions to concealed food cease 
when the olfactory apparatus is rendered inoperative, and are 
resumed only when the organs again become functional. 

A week later the foregoing experiments were repeated, and an 
attempt made to hasten the recovery of the olfactory organs by 
shortening the time during which they were to be tied. Two of 
the four puffers previously tested were isolated in an aquarium 
and allowed to become hungry. They then reacted in an essen- 
tially normal way to the two packets, biting the one with meat 34 
times, and the other 10 times in one hour. For eleven minutes 
after the packets had been suspended in the aquarium, no appar- 
ent attention was given them, although both fish were passing 
and repassing them constantly. Suddenly one of the puffers ap- 
proached the packet containing meat and bit it. It was a reaction 
which any unprejudiced observer would have called olfactory. 
After this test, the nasal organs were tied, and about two hours 
later the fish were again tested for an hour. The thread which 
surrounded one of the organs dropped off shortly before the test 
was made. Neither of the packets was touched, although before 
and after the test both fish ate pieces of meat from the end of a 
wire in their habitual way. The threads were immediately re- 
moved, and, on the following day, they were given two one-hour 
tests with the packets. No reactions to either resulted. Forty 
hours after untying the threads they were again tested. One of 
the fish bit 8 times at the packet with meat and 5 times at the 
one made of cheese cloth, whereas the other fish ignored both. 
Each ate actively from the end of a wire at the conclusion of the 
test. 

Subsequent examination of the condition of the nasal organs 
of these two puffers showed that such behavior might have been 
expected, if concealed food is scented by means of these organs. 
Both organs of the fish which failed to react were so badly crushed 
by the second tying that they could not have been functional after- 
ward. The second puffer, however, showed one organ substan- 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 3 


368 MANTON COPELAND 


tially normal, the other somewhat distorted, a condition of the 
olfactory apparatus which might well allow stimulation of suffi- 
cient strength to call forth a response as described above; one 
weak in character, in which discrimination between the two pack- 
ets was less marked than normally. 

The tests here recorded show that the puffer approaches and 
bites a packet containing concealed food many more times than 
it does one filled with cheese cloth; secondly, that these reac- 
tions cease when the olfactory apparatus is rendered inoperative 
(although pieces of meat are eagerly taken when seen) and, thirdly, 
that the ability to discriminate between the two packets is re- 
gained when the olfactory apparatus again becomes functional. 

I, therefore, conclude that Spheroides maculatus responds to a 
stimulation of its olfactory apparatus by substances in dilute 
solution emanating from concealed dogfish meat, and, that it 
discovers hidden food by the sense of smell. 


BIBLIOGRAPHY 
Parker, G. H. 1910 Olfactory reactions in fishes. Jour. Exp. Zoél., vol. 8, 
pp. 535-542. 


1911 The olfactory reactions of the common killifish, Fundulus hetero- * 
clitus (Linn.). Jour. Exp. Zodél., vol. 10, pp. 1-5. 


Sueupon, R. E. 1911 The sense of smell in selachians. Jour. Exp. Zodl., vol. 
10, pp. 51-62. 


SIZE INHERITANCE IN DUCKS 


JOHN C. PHILLIPS 


From the Laboratory of Genetics of the Bussey Institution.’ 


The inheritance of size in animals is a question of great theo- 
retical interest, but difficult to analyze. 

Lock (’06) in the case of maize showed that height of the plant 
is not inherited as a simple Mendelian character. Castle (’09) 
showed the same to be true of the weight and of various skeletal 
dimensions of rabbits, and characterized such inheritance as 
blending. 

Ghigi (09) referring to a cross which he made between Paduan 
fowls and bantams stated that in size of body and of eggs produced 
the (F) eross-bred individuals were intermediate between the 
parent races, and that later generations showed no tendency to 
return to the conditions found in the parent races. The number 
of animals studied by Ghigi was small and no great stress was 
laid upon the point of non-segregation. 

Emerson (10), however, after a more detailed and exact study 
of the inheritance of height in maize, and of several other size and 
shape characters in gourds, found that while F2 was strictly inter- 
mediate between the parents and no more variable, F. showed a 
greatly increased variability, which he interpreted as ‘‘ merely 
a mathematical way of expressing the fact that the F, individuals 
exhibit marked segregation of size and shape characters.”” Such 
an interpretation was made possible by the discovery by Nillson- 
Ehle (09) and by East (10) that a Mendelizing character may 
have multiple germinal representation, in which case, though 
physiologically a single unit-character, it may produce dihybrid, 
trihybrid, or even more complex Mendelian inheritance ratios 


1 Contribution No. 12. 


369 


370 JOHN ©. PHILLIPS 


in F,. If in such cases dominance is lacking, an apparently blend- 
ing inheritance results, attended in F; by no increase of varia- 
bility, but in F, by greatly increased variability. 

Now this, as Emerson has shown, is exactly what happens in 
size inheritance in plants. Such cases are therefore open to in- 
terpretation as Mendelian inheritance without dominance, in 
which more than a single unit character is involved. The theo- 
retical aspects of the matter have been fully discussed by Lang 
(10) and Castle (711). 

The following preliminary results (summarized in table 1) from 
a cross between two different size races of ducks show indications 
of segregation in body weight. As it will be some time before 
further data can be added, it is thought worth while to record the 
experiment as far as it has progressed. 

An experiment of this sort, based, as it must be, on the perfect 
health of the animals, is necessarily subject to a serious source of 
error. Larger numbers than have yet been obtained are there- 
fore desirable, and it is hoped that these may be had in future 
seasons. The season of 1911 was very dry and hot and may have 
had some effect on the variability of the F, generation. It was 
certainly not favorable toward fertility in duck eggs. The F, 
generation is much too small and an attempt will be made to 
obtain larger numbers, using as mothers random females from the 
small race. Tarsus and bill measurements will also be taken to 
supplement the weights of the animals. 


MATERIAL 


Ducks were chosen for this purpose because it is possible among 
them to obtain two races greatly different in size, and yet produc- 
ing fertile hybrids. Important also is the fact that the young can 
be raised in large numbers during a single definite growth season. 
Maturity is reached quickly, and entire lots can be killed and ex- 
amined at the same age. It is possible also to raise different 
groups or generations during the same season, thus pr Gyan B) 
check upon environmental effects. 

The large French Rouen duck was chosen for the large parent, 
and the common domestic mallard for the small parent. 


371 


SIZE INHERITANCE IN DUCKS 


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The original Rouen stock consisted of two males and four fe- 
males. They were obtained from a dealer in April, 1910, but were 
not weighed until the following fall. During the summer of 
1910 two males and five females were raised from the original 
stock, while in 1911 six males and nine females were brought to 
maturity from the same source. These twenty-two birds (sec- 
ond and third generation, Rouen stock, table 1) were killed and 
weighed at the same time as the F’’s and F,’s, so that a definite 
control might be obtained. The mean weight of the Rouen 
race was calculated from these twenty-two individuals. For 
the males, it was found to be 2321 grams; for the females, 2237 
grams. The original Rouens were not included in this calculation, 
because when weighed for the first time, at one and one-half 
years of age, they were very fat and not directly comparable with 
their offspring. Their weight was 16 per cent greater than the 


TABLE 2 


Weights of pure large race, Rowen 


MALES FEMALES 

Age Grams Grams 

( 2600 2220 

Original stock........... of 4 15 years 280 ae 
{ 2870 

f 2470 2220 

| | 2600 2300 

Second generation (1910)........ eps. | o> months 2370 
2420 

2670 

[ 1980 1885 

| 2162 1990. 

| 2252 2095 

2260 2105 

Third generation (Gl) eeeeeeeee cscs , 5 months 2335 2175 
2512 2200 

2202 

2230 


2460 


SIZE INHERITANCE IN DUCKS 373 


mean of all their offspring. It seems probable that the mean 
weight of the Rouen stock as here used is rather too small. Ma- 
turity is probably not reached quite as soon by this large breed 
as by the smaller breeds and the very vigorous F, cross-bred birds. 

The mallard stock was obtained from one hundred and twenty- 
five eggs received from a game preserve in New Jersey in the spring 
of 1909. From these eggs sixty-five birds were raised to maturity. 
In 1910 part of this original stock produced one hundred and sixty 
mallards which were raised to maturity. Most of these were 
killed off and weighed in three lots, October 13, October 22 and 
November 10 (table 3).: A comparison of the weights showed that 


TABLE 3 


Weight of pure small race, mallard 


MALES FEMALES 
AGE “: 
Grams Grams 
( 845 819 
854 864 
892 S74 
896 855 
910 895 
916 896 
934 970 
944 994 
973 1005 
1024 1039 
1064 1048 
}months..... Sid AR Aa ee aE 1074 
1094 
1096 
1100 
1112 
1115 
1140 
1174 
1180 
1222 
1313 


INCOM Ps cena trig sil osteicidie Sei kers ta aed sie ahs 1039 935 


374 JOHN C. PHILLIPS 


TABLE 3—Continued 


MALES 
AGE 
. r Grams 


f 870 


AS NMONUDS eee rece ker Pes etd ts 1110 


Se, MODES se ee ae eet 4 


Meats2c:-c8 coche peces te EEE Meare 1089 


FEMALES 


Grams 


770 
855 
855 
840 
860 
875 
890 
942 
945 
950 
952 
960 
963 
970 
70 
985 
993 
1002 
1020 
1040 
1045 
1090 
1090 


Meéaniallages:) o:¢ :.:.cnccs eee eee 1068 


SIZE INHERITANCE IN DUCKS 375 


the birds had practically gained their adult size by October 22. 
The mean weights of the three lots is as follows. 


SEX OCTOBER 13 OCTOBER 22 NOVEMBER 10 
IMiALGS Et men. < acn2EEe : 1039 1076 1089 
RO MNALESE: « «os seaaeee 935 937 967 


This stock of mallards is the pure semi-domestic Anas boschas 
used in the English game preserves. They are perhaps slightly 
larger than pure wild mallards, which average about 1000 grams 
for the males, depending somewhat on food supply; (no large 
series of the weights of wild mallards is at hand). 


MATINGS 


In the spring of 1910 three female mallards taken at random 
from the original stock were mated with one of the Rouen stock 
males. At the same time two female Rouens were mated with 
a male mallard, but the two settings of eggs which were obtained 
from this last mating proved tobe entirely sterile, due undoubtedly 
to the small size of the male bird. While on the subject of this 
attempted reciprocal cross, it might be well to state that there is 
probably little or no error introduced into the experiment on ac- 
count of a difference in size of the eggs in the two races. The 
lengths of seven Rouen and seventeen mallard eggs were measured 
in 1909. There is a good deal of variation in the eggs of both 
strains and an overlapping of the two variation curves. The 
Rouen eggs are roughly 2.40 inches in length and the mallard eggs 
2.25 inches in length. 

The F, eggs from the mating male Rouen xX female mallard 
were set in two lots and hatched on the same date, May 29. They 
were reared under hens in exactly the same place and under the 
same conditions as the two parent classes. Both this group and 
the F, birds were banded. These F,; birds, ten males and three 
females, were weighed October 29, at the age of five months. 
The class is obviously too small, but as far as it goes it shows a 
remarkable uniformity. Between the largest and smallest males 


376 JOHN C. PHILLIPS 


there is a difference of only 200 grams. The coefficient of varia- 
bility (c. v) is 5.82 for the males and 3.43 for the females. 

In 1911 an attempt was made to raise another lot of F,’s but 
only two fertile eggs were obtained and these were discarded. 

The F, generation (table 4) was obtained by mating the three 
F, females, Nos. 83, 101 and 106, to male No. 105. This male 
is slightly above the mean size of the F; males, which fact perhaps 
accounts, in part at least, for the larger size of the F, mean, as 
compared with that of the F;. The eggs from this mating were 
set in three lots, May 29, June 8 and June 15. The entire class 
was killed and weighed on October 29. The ages therefore varied 
from one hundred and thirty-four to one hundred and fifty-one 
days. The mean weight of the seven youngest males, hatched 
June 15, is 1738 grams, as against 1863 grams for nine males 
hatched May 29, a difference of about 7 per cent. This shows 
that adult weight is very nearly reached at four and one-half 
months of age. 

A glance at table 4 shows the very remarkable variation which 
these F,’s exhibit. There is an actual range of variation in weight 
of 887 grams among the males and of 650 grams among the fe- 
males. The smallest male, No. 158, is of substantially the same 
weight as the largest pure mallard. The largest F, male, No. 175, 
‘is only slightly smaller than the mean of the pure Rouen males for 
1911, the difference being only 2 per cent. 

The F, coefficient of variation is 12.07 for the males as against 
5.82 for the F, males, while the F, females have a coefficient of 
variation of 11.07 as against 3.43 for the F, females. 

It is interesting to note that the coefficient of variation is larger 
in the males than in the females, in all groups except the pure 
large race, where the reverse is the case. 


GENERAL CONSIDERATIONS 


A fuller statistical analysis of the foregoing data may help 
to make their significance plain. For this purpose the observed 
weights may be classified in a variation table. Since males are 
on the average heavier than females, it is evident that separate 
tables must be made for males and females. Let us take the mean 


SIZE INHERITANCE IN DUCKS 377 


TABLE 4 
Weights of F, Animals when five months old, 1910 


MALES FEMALES 
No. Grams No. Grams 
84 1470 83 1570 
85 1660 101 1530 
86 1600 106 1660 
87 1670 
100 | 1740 
102 1740 
103 1650 
104 | 1770 
105 1720 
107 1630 
Migan!.2 <2 Soa 1665 1587 
TABLE 5 


Weights of F, Animals, 1911. 


MALES | FEMALES 

No. Age, days Grams | No. Age, days Grams 
158 151 1320 | 161 151 1325 
164 151 1967 165 151 1527 
166 151 2060 167 151 1875 
170 151 1885 | 168 [wast 1697 
171 151 2085 | 169 151 1975 
175 151 2207 172 151 1295 
176 151 1815 173 151 1603 
177 151 1sa7 WV) 1180 151 1895 
ee "|| “150, ° || desae | 481 151 1650 
189 lol | 1725 182 151 1675 
200 141 1482 183 151 1645 
214 141 2007 186 151 1717 

15 141 1583 187 151 1672 
217 | BE | 1760 199 141 1410 
223 134 1667 292 134 1572 
224 134 1660 229 134 1615 


303 134 1720 


pe sraysvatel uate stitstarere <1 1781 lime 1634 


378 JOHN C. PHILLIPS 


weights of each pure race as class means in a variation table, which 
shall contain a convenient number of intermediate classes, say 
seven. Tables may thus be constructed separately for each sex 
(tables 6 and 7). 

In both tables the variation of the pure races is seen to be about 
classes 2 and 10 as their respective modes. Indeed the tables 
were constructed with that end in view, the observed means of 
the weights being made the means for classes 2 and 10. Since 
further the number of intervening classes is the same in both 
tables, these tables may legitimately be combined, class by class, 
to obtain a table of weight distribution for both sexes, which 
however will be free from any serious error? due to the fact that 
the two sexes differ in size. Such a combination table is table 8. 

From an examination of these tables it is clearly seen that (1) 
the F, animals vary closely about the middle class, 6, exactly 
intermediate between the parent races. The extent of variation 
of the F; animals is small. (2) the F, animals vary about the 
same intermediate class, 6. The amplitude of variation of the F, 
animals is greater than that of the F; animals, but does not ex- 
tend beyond the nearer limit of the respective grand-parental 
races. In the 33 F, animals studied no variate occurs which is as 
small as the mean of the small race or as large as the mean of the 
large race. No case of complete segregation occurs. (3) The in- 
creased variability of F, as compared with F; may be regarded as 
due to partial segregation of genes having multiple representation 
in the gamete, or as due to modification of gametes in other ways 
as a result of their association in F, zygotes. The evidence at 
present available is insufficient to decide between these contrasted 
views. 

In conclusion, I wish to express special thanks to Professor 
Castle for valuable help in correlating the results of this exper- 
iment. 

2 This difficulty was met by Galton (’89, Natural inheritance) in a different 
way, which however would for our present purpose, it is thought, be less accurate 
as well as more laborious. Finding that the average male measurement was 
greater than the corresponding average female measurement he ascertained the 


* ratio of the two to each other and then converted all female measurements into 
equivalent male measurements by multiplying by this ratio. 


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TABLE 8 


Weight distribution of male and female ducks combined 


Class numibertvqccnme er eet = barrie C8] 2 lesen | Sal Belmvanes | 9 | 10 | 11 12 | 13 

- = = = — | | | — - 

FROUCT ee eee eee NEMS Feet eee Alas By) wet al 

Mallardy seep epics flake ck + sc 23 54 23) 2 | | | 

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BIBLIOGRAPHY 


CastLe, W. E. 1909 Studies of inheritance in rabbits. Publication no. 114, 
Carnegie Institution of Washington. 


1911 Heredity. D. Appleton and Co., New York. 


East, E. M. 1910 A Mendelian interpretation of variation that is apparently 
continuous. American Naturalist, vol. 44, pp. 65-82. 


Emerson, R. A. 1910 The inheritance of sizes and shapes in plants. American 
Naturalist, vol. 44, pp. 739-746. 


GuiGci, ALESSANDRO 1909 Ricerche di sistematica sperimentali sul genere 
Gennaeus wagler. Bologna. p. 38. 


Lane, A. 1910 Die Erblichkeitsverhiltnisse der Ohrenlinge der Kaninchen 
nach Castle und das Problem der intermediaren Vererbung und Bildung 
konstanter Bastardrassen. Zeit. f.ind. Abstammungs- und Vererbungs- 
lehre, Bd. 4, pp. 1-23. 


Lock, R. H. 1906 Studies in plant breeding in the tropics 11. Experiments 
with maize. Annals Roy. Bot. Gardens, Perademja, vol. 3, part 2, 
pp. 95-184. 


Nitson-Euie, H. 1909 Kreuzungsuntersuchungen an Hafer und Weizen. Lund 
Universitets Arsskrift, N. F., Afd. 2. Bd. 5. - 


CAN THE SPERMATOZOON DEVELOP OUTSIDE 
THE EGG? 


JACQUES LOEB anv F. W. BANCROFT 


From the Rockefeller Institute, New York 
ELEVEN FIGURES 
I. INTRODUCTION 


The experiments on artificial parthenogenesis have shown that 
an egg which naturally cannot develop without sperm can be 
caused artificially to develop without being fertilized by sperm. 
It is natural to raise the question whether a spermatozo6n can 
be caused to develop into a larva without an egg. The experi- 
ments on merogony prove that a fragment of the egg deprived of 
a nucleus can develop into a larva if a spermatozo6n enters the 
egg; this shows that the egg nucleus is not necessary for the devel- 
opment. The phenomena of merogony do not yet prove that 
the spermatozo6n alone can give rise to an embryo. There are 
several reasons for doubting this possibility. In the first place 
we have reason to assume that the protoplasm of the egg is the 
embryo itself. If this be correct we can understand that the 
spermatozo6n might be able to transmit a number of characters 
to the offspring, without possessing the possibility of becoming 
an embryo or creating one outside of an egg. 

In the second place it is possible that only the protoplasm con- 
tains the apparatus necessary for nuclear and cellular division and 
that a spermatozo6n is not able to create this apparatus. In the 
third place it is possible that the egg protoplasm of each species 
contains nutritive material and enzymes of so specific a character 
that it is not likely that we are able to imitate it in the near future. 
The attempt at causing the spermatozoén to develop without an 


381 


382 JACQUES LOEB AND F. W. BANCROFT 


egg is at the same time an attempt to test the validity of the 
reasons which seem to speak against this possibility. 

There is only one paper dealing with this problem, by J. de 
Meyer.! He raised the question whether it is necessary that the 
spermatozo6n should come in contact with the cytoplasm of 
the egg in order to undergo the first phases of its normal evolu- 
tion. He used the sperm of Echinus microtuberculatus, which he 
placed in sea-water containing an extract of the eggs of the same 
species and found that under these conditions the spermatozoa 
swelled so as to lose completely their normal appearance. The 
tail remained unchanged, but the cytoplasmic covering of the 
head, the middle piece, and the chromatic portion of the head all 
seemed to swell; and in some eases an indistinct vesicular strue- 
ture was seen which stained a little stronger than its surroundings, 
and seemed to be a nucleus. He concludes that incomplete as 
his results may be, they give a right to conclude, ‘‘that the male 
just as the female cell is capable of evolution under the influence 
of external agencies” (p. 94). 


Il. MATERIAL AND METHODS 


Our own experiments were carried on on the sperm of the fowl. 
The sperm was removed aseptically. Only the sperm contained 
in the lower portion of the vas deferens was used. It was kept 
in a sterilized moist chamber at about 39° C., but was always 
used soon after its removal from the animal, not later than 
three hours after it was taken out. The media used for the 
culture of the spermatozo6n were: egg yolk, egg albumen, chicken 
blood serum and § and zy Ringer solutions. Slides, cover 
glasses and instruments were sterilized in a flame and small 
hanging drops of the various media were inoculated with the 
spermatozoa. The cover glasses were inverted over hollow slides 
and sealed with a vaseline and paraffine mixture. In a few cases 
the eggs were broken into glass vessels and small quantities of 
sperm injected into the yolk with a capillary pipette. After 
stated intervals yolk and sperm were taken out for examination 
with a capillary pipette. 


1 J. de Meyer, Arch. de Biologie, vol. 26, p. 65, 1911. 


CULTURES OF SPERMATOZOA 383 


III. OBSERVATIONS ON LIVING MATERIAL 


When the spermatozoa of the fowl are observed in a hang- 
ing drop of white of egg, kept at about 40° C., the first change is 
seen after fifty or sixty minutes. It consists in the collection of 
a small amount of some substance having a low refractive index 
about the middle pieces of some of the spermatozoa. In favor- 
able cases as many as 60 per cent of the spermatozoa may undergo 
this change. At this time many of these spermatozoa are still 
swimming. During the course of the next few hours these lowly 
refractive areas increase in size until they are about half as long 
as the sperm head and acquire a fairly distinct ellipsoidal outline. 
Then in many cases the sperm head can be seen to be bent in a 
horse shoe or spiral shape, and to be included in the wall of the 
vesicle, which has now become spherical, while the tail of the sper- 
matozo6n still remains unchanged or has disappeared without 
taking any part in the transformation. The next change is an 
increasing indistinctness in the sperm head, and an increasing 
refractive power of the whole vesicle so that it can hardly be dis- 
criminated at all in the albumen. It is not possible to follow the 
process farther in unstained material. 

In some ‘cases these vesicles instead of being spherical stretch 
out along the whole side of the sperm head, or may become en- 
tirely disconnected from the spermatozo6n. 

If yolk is used as a culture medium for the sperm essentially 
the same phenomena occur; and in the various Ringer solutions 
vesicles containing the sperm heads are also formed, but in the 
Ringer solution, as a rule, the steps in the formation of these ves- 
icles could not be seen without staining. ; 


IV. OBSERVATIONS ON PRESERVED MATERIAL 


When the hanging drops are fixed in Flemming’s fluid and 
stained and examined in Herla’s vesuvin and malachite-green 
mixture, it can be seen that in its early stages the vesicle has dis- 
tinct walls and a homogeneous unstained fluid of a low refractive 
index in its interior. This fluid is possibly water and this would 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 3 


384 JACQUES LOEB AND F. W. BANCROFT 


account for the fact that the vesicle is conspicuous in albumen 
and yolk, but invisible in Ringer solution. 

The vesicle seems to be formed by the imbibition of water by 
the very thin protoplasmic envelope of the sperm head and middle 
piece. For after the formation of the vesicle, head, tail and 
middle piece are, so far as can be seen, unchanged (fig. 1). In 
many cases the vesicle is seen at the front end of the spermatozo6n. 
Such cases result from the bending of the spermatozo6n in the 
middle piece region, as the series illustrated in figs. 1, 2 and 3 
show. The maximum development of the unchanged vesicle is 
shown in fig. 4. 

When the vesicle has reached its full size the material of which 
its surface is composed seems to wet the sperm head very easily. 
For in the next stage the sperm head is in contact with the wall 
of the vesicle along its whole length, and the vesicle has usually 
assumed a more or less spherical shape (figs. 5 to 8). 

Up to this point the transformations were found to take place 
in the same way in all the media employed, but in the various 
Ringer solutions the transformation went no farther than this 
even when the spermatozoa were left in the solutions for forty- 
eight hours and longer. 

In the yolk and albumen, however, the development toward the 
formation of a nucleus went a little farther. In these media solu- 
tion of the sperm head took place, which seemed to begin as soon 
as the head was drawn into the vesicle. For in these media stages 
like figs. 7, a, c, d, e, were quite difficult to find, while in the Ringer 
solutions such stages, and those in which the sperm head within 
the vesicle was entirely unchanged were the most frequent trans- 
formations observed (figs. 5 and 6). 

In preparations that were fixed after the spermatozoa had been 
in contact with the yolk or albumen only two or three hours the 
most frequent transformation of the vesicles observed is one in 
which the head has entirely disappeared while the whole vesicle 
takes a rather dilute nuclear stain. In a few cases nuclei of this 
type still show remnants of the sperm head as in fig. 7, 6 and ec. 
These appearances would seem to indicate a solution of the chro- 
matin of the sperm head in the contents of the vesicle. 


CULTURES OF SPERMATOZOA 385 


After the spermatozoa have been left in contact with the cul- 
ture medium for about eighteen hours no more, or but very few, 
of these uniformly stained vesicles are to be found. But there 
are many fairly normal looking nuclei in which the chromatin is 
all present in the shape of discrete particles resting on the nuclear 
wall, and in which no linin, or but very small amounts of it, can 
be seen (figs. 8 to 11). The prineipal reason for believing that 
a certain amount of linin is present, even if it is obscured by the 
chromatin, is that some vesicles are seen, which seem to have 
broken away from the spermatozoa before they came in contact 
with the sperm head, and which do not contain any chromatin 
at all. In these a few strands of linin-like substance may usually 
be seen traversing the interior of the vesicle; and it is likely that 
these strands are also present in the other vesicles but cannot be 
made out on account of the chromatin. 

It would seem probable that the chromatin in these nuclei is 
derived by a condensation of the uniformly distributed chromatin 
of the previous stage, though it is possible that in a certain num- 
ber of cases the sperm head breaks up into chromatin particles 
without a previous complete solution. 

Ordinarily no signs of either protoplasm or sperm tails are to be 
seen in connection with these nuclei but occasionally both may 
be observed, as in figs. 8b and 10a; and in several cases it was seen 
that the middle piece had not been incorporated within the nucleus 
or vesicle but could be distinctly made out in the tail attached to 
the vesicle. 

When the preparations are fixed in Flemming’s fluid, stained 
with Czaplewsky’s carbolic gentian violet, dehydrated and 
mounted in balsam, clearer pictures of the completed nuclei were 
obtained (figs. 9 to 11) but the series of intermediate stages in 
the formation of these nuclei seemed to be entirely different. It 
was possible to make out a connected series of transformations 
of the sperm head into nuclei, but since this series contained none 
of the vesicles so characteristic for the living and glycerine mate- 
rial it must be concluded that this series is composed mainly of 
artefacts resulting from the shrinkage of the vesicles. 


386 JACQUES LOEB AND F. W. BANCROFT 


In these balsam preparations the first change which seemed to 
take place was a broadening of the sperm head with a decided 
increase in staining power. Then the sperm head gradually 
seemed to shorten and assume various irregular and sometimes 
angular shapes, still retaining its high staining power. Finally 
these small deeply staining heads seemed to become larger and 
vesicular, and typical nuclei were formed. Accordingly it would 
seem that when first formed these vesicles are so delicate that 
they cannot stand this technique without shrinking all out of 
shape. 

From these experiments we must conclude that in yolk and 
white of egg the spermatozo6n undergoes the transformation into 
a nucleus. We have not noticed any mitosis or asterformation 
and we are, therefore, not yet in a position to state that the 
spermatozo6n can undergo mitosis outside the egg. 


PLATE 1 
EXPLANATION OF FIGURES 


All the figures where drawn with a camera from preparations of chicken sperma- 
tozoa and nuclei into which the spermatozoa were transformed. 

The vesicles in figs. 1 to 6 all stain more lightly than the substance surrounding 
them, as is represented in fig. 6. To represent the preparations faithfully figs. 1-5 
should also have a background similar to that in fig, 6. All except fig. 4 are from 
preparations fixed in Flemming’s fluid. 

1 Chicken spermatozoa showing an early stage in the vesicle formation, 
and the beginning of the bending of the spermatozo6én in the middle-piece region. 
Sperm kept in white of egg two hours, forty-five minutes. Stained and examined 
in Herla’s malachite green and vesuvin mixture. X 1620. 

2 Spermatozoa with vesicles as in fig. 1 but more eompletely bent on them- 
selves in the middle-piece region. All of the loops contain vesicles which are seen 
exactly from the side in a and c and slightly from the edgeinb. From same prep- 
aration as fig. 1. X 1620. 

3 Spermatozoa similar to those in fig. 2 but seen edge on. From same prep- 
aration as figs. land2. > 1620. ¥ 

4 Spermatozoén showing the maximum development of the vesicle before 
the bending of the sperm head begins. Sperm kept in yolk for about four hours. 
Killed, stained and examined in Herla’s. X 640. 


CULTURES OF SPERMATOZOA PLATE 1 
JACQUES LOEB AND F. W. BANCROFT 


o- CZ 


PLATE 2 
EXPLANATION OF FIGURES 


5 Sperm heads which have been bent so that they are either within or in con- 
tact with the vesicle throughout their entire length. In 6 the flagellum and 
middle piece of the tail are still visible, but in a and ¢ these can no longer be 
made out. Sperm in™ Ringer solution + 0.35 per cent “ NaHCO; for five hours. 
X 1750. 

6 Sperm heads contained in vesicles; a still showing remains of tail; b 
shows maximum development that the vesicles attain in Ringer solution. Sperm 
in *. Ringer solution four hours. Dark background due to coagulated egg albu- 
men which was mixed with the Ringer solution immediately before fixing to pre- 
vent the vesicles being washed off the cover glass Herla’s. 1620. 

7 Early stages in the solution and transformation of the sperm heads 
within the vesicle. From same preparation as figs. 1, 2 and 3. 

8 Vesicles in which part of the chromatin is present as discrete granules, 
and part ean still be recognized as the portion of the sperm head which adjoins the 
tail. In 6 two masses of cytoplasm can be seen anteriorly. Herla’s. 1620. 

9 Sperm heads transformed into nuclei. Chromatin as far as could be 
determined all next tothe membrane. Sperm in egg albumen twenty-three hours. 
Gentian violet and balsam. 5000. 

10 Spermatozoa transformed into nuclei. a@ also appears to have some 
vacuolated protoplasm Sperm in egg albumen twenty-six hours. Herla’s. 
« 3100. 

11 Spermatozoa transformed into nuclei. Chromatin all on nuclear mem- 
brane. Gentian violet and balsam. 3100. 


388 


CULTURES OF SPERMATOZOA 
JACQUES LOEB AND F. W. BANCROFT 


5 


a O08 oa (Ss 


b c 


PLATE 2 


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8 
# #h rs 2 : 
} ¥ - @ } < 6 
A 
/ 
(e 


1a 


it . Le 
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389 


STUDIES IN CYTOLOGY 


I. A FURTHER STUDY OF THE CHROMOSOMES OF TOXOPNEUSTES 
VARIEGATUS 


Il. THE BEHAVIOR OF THE CHROMOSOMES IN ARBACIA-TOXO- 
PNEUSTES CROSSES 


DAVID H. TENNENT 
From the Zoélogical Laboratory, The Johns Hopkins University 
TWENTY-ONE FIGURES 
I 


In 1910 Miss Heffner made a study of straight fertilized Toxo- 
pneustes eggs andreached the conclusion that there were two classes 
of zygotes, one with two and one with three V-shaped chromo- 
somes in each anaphase plate. This condition showed a simi- 
larity to the one described by Baltzer (’09) for Echinus and was 
regarded as an indication that its significance was the same, 
namely that in Toxopneustes there were two kinds of unfertilized 
eggs. 

My observations on Hipponoé crosses ('11—12) taken in con- 
nection with those of Miss Pinney (11) on straight fertilized 
Hipponoé eggs led me to believe that possibly there might be 
another interpretation for Toxopneustes and it occurred to me 
that the facts might be determined most readily by studies of the 
chromosomes of the egg in chemically fertilized eggs and of the 
chromosomes of the spermatozoan in fertilized enucleated egg 
fragments. The question to be decided was whether the un- 
paired V in zygotes containing three V-shaped chromosomes, was 
to be associated with the egg or with the spermatozoan. 

The work upon which this paper is based was begun in the Laboratory of the 
Bureau of Fisheries at Beaufort, N. C. I am indebted to the Hon. George M. 
Bowers, Commissioner of Fisheries, for the privilege of working in this labora- 
tory and to Mr. Henry D. Aller, Director of the laboratory, for many courtesies. 

391 


a 


392 DAVID H. TENNENT 


A. Chemically fertilized eggs 


The method of chemical fertilization that I used was that of 
Loeb (09), with a very slight modification. The eggs were 
placed in a butyric acid mixture (6 ce. + butyric acid + 94 ee. 
sea water), for from one and one-half to two and one-half minutes, 
transferred from this to sea water to which an amount of + NaOH 
solution calculated to be sufficient to neutralize the amount of 
butyric acid which had been carried over in the pipette, had been 
added; allowed to remain in this for twenty minutes; transferred 
to hypertonic sea water, (13 ec. 24 m. NaCl + 87 cc. sea water), 
for fifteen to twenty-five minutes; and finally transferred to sea 
water. 

A larger percentage of eggs formed membranes when placed 
in sea water plus NaOH, upon removal from the butyric acid 
mixture, than when placed in straight sea water. 

With this sea urchin, Toxopneustes, it is possible to determine 
the proper length of time that the eggs should remain in the hyper- 
tonic sea water by observing nuclear changes in the egg. Hindle, 
(10), in describing the parthenogenetic development of Stron- 
gylocentrotus purpuratus says, in one place, that no apparent 
changes ‘“‘beyond a slight reduction of the clear zone of hyalo- 
plasm surrounding the nucleus’? may be observed while the eggs 
are immersed in the hypertonic solution, and in another place, 
‘during the treatment with hypertonic salt solution there is a 
slight increase in the size of the nucleus and the clear zone almost 
disappears.” 

In Toxopneustes the nucleus and the cytoplasm immediately 
surrounding it may be seen to be in a state of activity. From 
the surface of the nucleus, processes seem to push out and then 
retract, the nucleus meantime enlarging in volume, while in the 
cytoplasm, currents showing a movement of substance toward the 
nucleus may be seen. 

If the eggs be transferred from the hypertonic sea water at the 
time when, in the greatest number, the nucleus has reached its 
maximum size, just prior to the bursting of the membrane, it 
will be found that a larger percentage of regularly segmenting 


STUDIES IN CYTOLOGY 393 


eggs may be obtained than if the transfer be made at any other 
time. 

It should be remembered that Hindle and I have worked with 
different eggs and that the processes are not necessarily exactly 
the same in both. I am inclined to believe, however, that the 
greater opacity of the Strongylocentrotus egg has prevented an 
observation similar to the one deseribed here. 

In practice I found it convenient to divide the eggs into two 
portions as they were removed from the butyric acid mixture, 
placing those removed during the interval one and one-half to 
two minutes in one dish and those removed during the interval 
two to two and one-half minutes in another. Sometimes one 
lot, sometimes the other was better. In the work I used finger- 
bowls of about 400 ee. capacity. Probably the greatest advantage 
accruing from the use of the NaOH was that of being able to 
use smaller amounts of sea water and thus concentrating the mass 
of eggs. 

A considerable number of experiments for the determination 
of the most favorable hypertonic sea water were made. Theo- 
retically it would seem as though a hypertonic solution containing 
all of the salts of sea water might be more favorable than sea 
water increased in one salt only. However, neither the hyper- 
tonic solutions made by the evaporation of sea water, nor the 
use of hyper molecular Van’t Hoff solutions offered any advan- 
tages over the solution mentioned above. 

It seems scarcely necessary to note that the experiments were 
carried out with extreme care. Sterilized dishes and pipettes, and 
sea water heated to 70° C. cooled and filtered, were used through- 
out the work. Controls to indicate whether chance fertilization 
had taken place were kept throughout. 

My observations on the external and internal changes in the 
egg agree, except as above noted, with the descriptions of Loeb, 
Hindle and Wilson, and therefore need no discussion here. 

At first thought it might seem that after the exceedingly careful 
work that has been done on the cytology of artificially partheno- 
genetic Toxopneustes eggs nothing would be gained by a further 
study of such eggs. It must be remembered however, that the 


394 DAVID H. TENNENT 


work which has been done up to this time has gone no further, 
with respect to the chromosomes, than the determination of the 
presence of the haploid number and that in general the chromo- 
somes were rod-like in form. 

Within the past five years, partly from the knowledge that in 
Echinoderms chromosomes of different form are associated with 
different species and partly from the knowledge of chromosomes 
showing peculiarity of form in other phyla, the necessity of a fur- 
ther insight into the conditions existing in chemically fertilized 
eggs has arisen. 

This new study of the chromosomes in artificially partheno- 
genetic eggs has brought out the fact that all of the eggs are alike 
in that in each anaphase plate there are two V-shaped chromo- 
somes. 

It must be mentioned again that in many instances it is ex- 
tremely difficult to determine the V-shaped elements in the spin- 
dle. The arms of the V are frequently in contact with each other, 
giving the chromosome the appearance of a very thick rod. In 
some cases it is possible to ascertain that the rod is actually double; 
in others it becomes a matter of deduction. The conclusions 
reached are based on the study of a large number of spindles and 
where it has been necessary to make an interpretation of the 
nature described, it has been made in accordance with the evi- 
dence given by the clearer spindles and not with a preconceived 
idea of what the conditions should be. 

It is noteworthy that the best chances for an exact determina- 
tion of this form of the chromosomes is afforded by eggs in which 
the number of asters is above the normal. This is due to the 
fact that in such eggs the chromosomes are widely scattered. 

In fig. 1 A and B a multipolar spindle with apparently six asters 
is shown. There are eight V’s (four pairs) shown in this egg, 
three passing to pole A, three to pole B, one to pole # and one to 
pole F. The most probable explanation of the condition shown 
here lies in the assumption of the doubling in number of the chro- 
mosomes by a monaster division, followed by the multipolar 
divisions shown in the illustration. Such a method of behavior 
may be seen in many of the living eggs during the early develop- 


STUDIES IN CYTOLOGY 395 


ment. In addition this conclusion is practically the only one 
which can be reached in view of the now well established fact of 
the doubling of the number of chromosomes during monaster 
formation. 

Fig. 2 would be difficult to interpret, were it considered on its 
own merits alone. The long rod in each anaphase plate is evident 
The V’s appear here simply as thickened rods; 19 chromosomes 
are shown in each plate. Fig. 3 shows a similar state of affairs. 
Fig. 4 is similar with the exception that the V’s in 4 A are evident 
as such. 

The description for these figures may be taken as typical of the 
large number studied in which the chromosomes could be made out 
clearly. I must also state that in my preparations, representing 
sixteen very successful chemical fertilizations, the number of 
abmodal chromosomes is very high. There is nothing like the 
almost uniformly even and regular series of division figures that 
may be seen in sections of normally fertilized eggs, nor do the 
figures approach in regularity those which I obtained from the 
artificially parthenogenetic starfish egg in 1906. The evi- 
dence here seems to be a confirmation of the statement made by 
Nemec (711) that the form of the chromosomes may be changed 
by reagents although the processes of mitosis may be unmodified. 


B. Fertilized enucleated egg fragments 


‘ 

The eggs were broken into fragments by being shaken in a test 
tube with broken bits of cover glass. For one series this mass was 
turned out in a flat dish and enucleated fragments picked out under 
the microscope with a pipette. The fragments were then fer- 
tilized with Toxopneustes sperm. Fertilization membranes were 
formed. The tempo of cleavage was the same as in eggs witha 
nucleus. Other series were prepared by shaking the eggs into 
fragments as above noted and fertilizing all of the fragments. In 
these the distinction between fragments with the double number 
of chromosomes and those with the half number is easily seen. 
The study of fresh material stained with Schneider’s aceto-carmine 
did not give especially useful results. 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 3 


396 DAVID H. TENNENT 


The study of the sections of the preserved material was more 
easily made than was the study of the artificially parthenogenetic 
material. The evidence from this material seems to show con- 
clusively that there are two classes of spermatozoa with respect 
to the V-shaped chromosomes, one class with one Vv, the other 
with two. 

In fig. 5 one V and the long rod are shown. The number of 
chromosomes in each plate is 18. In fig. 7 two V’s and the long 
rod are evident, 19 chromosomes are present. In fig. 8, one V 
with the arms separated, one V with the arms in contact and the 
long rod. Probably 19 chromosomes are present. 


Il 


While working with the phase of the question just described 
it occurred to me that possibly conclusive evidence might be given 
by segmenting eggs from crosses between Arbacia punctulata, in 
which as I showed in 1907, the chromosomes are all small, and 
Toxopneustes, in which the chromosomes are larger. 

I succeeded in making this cross for the first time during the 
past summer (’11). Up to that time I had not been able to secure 
a usable percentage of fertilizations by either the Hertwig method 
or the Loeb method, but by combining the two methods I 
obtained fertilization in a fair number. 

The Arbacia eggs were allowed to stand in sea water for four 
hours, then transferred to 400 cc. sea water + 8 cc. % NaOH for 
five minutes, and then Toxopneustes sperm added.  Fertiliza- 
tion membranes were formed on from 50 per cent to 90 per cent 
of the eggs, in different lots. 

Toxopneustes eggs were allowed to stand in sea water for two 
hours, then placed in 400 ec. sea water + 6 ec. 4 NaOH for five 
minutes and Arbacia sperm then added. Fertilization membranes 
were formed on about 10 per cent of the eggs. 

Numerous experiments showed the durations given to be the 
best. From these fertilizations only a few embryos developed to 
the pluteus stage, and all of these showed their hybrid origin. 
The skeleton was of an intermediate type. Most of the embryos 


STUDIES IN CYTOLOGY 397 


develop very irregularly, pronounced abnormalities appearing 
during the blastula stage. : 

The correlation between development and the behavior of the 
chromosomes is very striking. In a few instances the mitosis of 
the first segmentation is regular, with very little elimination of 
chromosomes. In the large majority of instances the behavior 
of the chromosomes is irregular from the beginning, many of the 
chromosomes being massed together and eliminated from parti- 
cipation in further mitosis. 

It has been suggested that the reason that the chromosomes of 
one species or the other lag during division in some crosses lies 
in the differences in normal division rate. It is interesting, in 
this connection that in the Arbacia ¢ x Toxopneustes = cross the 
Toxopneustes chromosomes lag although the division rate in this 
species is more rapid than in Arbacia. It is clear that the cause 
for lagging must be some other than that of difference in division 
rate. -I have previously pointed out that in any Hipponoé-Tox- 
opneustes cross cleavage is hastened by the Toxopneustes sperm, 
i.e., that of the quicker species. 


ARBACIA @ X TOXOPNEUSTES oc CROSS 


The chromosomes in the zygotes of “Arbacia punctulata are 
in general short and rod-like in form. Owing to their extremely 
small size I have made no prolonged effort to make out individu- 
ality of form. Fig. 6 represents one of three sections passing 
througha mitotic figure in anaphase. Thetotalnumber of chromo- 
somes is about forty. There is some variation in length, the 
longest chromosomes in Arbacia exceeding in length the shortest 
in Toxopneustes. 

Figs. 10-17 represent anaphases of the first division of the 
crosses. These represent the most nearly normal figures that 
were found. 

Fig. 10 A, B and C represent the most normal division figure 
found. Of the Toxopneustes chromosomes the long rods and the 
V’s may be recognized. Counting all of the chromosomes present 
the formula would be 33; those lagging at the center probably 
would have failed to be included in the daughter nuclei. Most of 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 3 


398 DAVID H. TENNENT 


these lagging chromosomes may be seen by their size and form 
to be Toxopneustes chromosomes. 

Fig. 11 A, B and C, chromosome formula #4. Long rods and 
V’s in anaphase plates; some Toxopneustes chromosomes lagging 
between the plates. 

Fig. 12 A, B and C, chromosome formula #?. Some of the 
chromosomes in 12 Care probably fragments of those in lower 12 B 
Toxopneustes V’s not present. Apparently there has been an 
elimination of Arbacia chromosomes. 

Fig. 13 A and B, chromosome formula #3. One Toxopneustes 
V and the long rod present. Lagging chromosomes probably of 
Toxopneustes. There seems to have been an elimination of Arba- 
cia chromosomes. 

Fig. 14 A and B, chromosome formula 33. Long Toxopnuestes 
rod absent. Some of both Arbacia and Toxopneustes chromo- 
somes have been eliminated. 

Fig. 15 A and B, chromosome formula 37. Toxopneustes V’s 
not present. Full number of Arbacia chromosomes does not seem 
seem to be present. 

Fig. 16 A, B and C, chromosome formula #}. Toxopneustes 
V’s not present. There has been an elimination of both Toxo- 
pnuestes and Arbacia chromosomes. 

Fig. 17 A, B and C, chromosome formula #2. Pronounced lag- 
ging of Toxopneustes chromosomes between daughter plates. 


THE TOXOPNEUSTES ? X ARBACIA oc CROSS 


Passing now to the reciprocal cross, figs. 18-21, we notice at 
once a difference in the appearance of the figures, the number of 
chromosomes present in each plate being less than in the plates 
of the reciprocal cross. 

‘Fig. 18 A and B, chromosome formula 33. Both of the Toxo- 
pneustes V’s and the long rod are present. Most of the Arbacia 
chromosomes have failed to enter the division figure. 

Fig. 19 A and B, chromosome formula 73. Both of the Toxo- 
pneustes V’s and the long rod are present. Nearly all of the 
Arbacia chromosomes have been rejected. 


STUDIES IN CYTOLOGY 399 


Fig. 20 A and B, chromosome formula 77. Almost a typical 
Toxopneustes thelykaryotic figure. Possibly one or two Arbacia 
chromosomes present. 

Fig. 21 A and B, tripolar figure. Total number of chromo- 
somes present about 85. Four Toxopneustes V’s (two pairs) 
present. If this egg was fertilized by two Arbacia spermatozoa 
about half of the Arbacia chromosomes have been eliminated. 

I have already pointed out the fact that few of the Arbacia- 
Toxopneustes hybrids pass through the gastrula stage. From 
my experiments I have obtained very few plutei indeed, and 
these few plutei showed evidence of their hybrid origin. If we 
examine some of the more irregular division figures, not only of 
the first cleavage but of the later cleavages, we shall find many 
very pronounced abnormalities; we shall find that not only may 
the chromosomes of the sperm be involved but that those of the 
egg as well fail to take their places in the spindle, the result 
being that the full haploid number of neither parent is present. 
This fact may well be intimately associated with the abnormal 
development during the blastula stage. 

The only cytological investigation having immediate connec- 
tion with these crosses is that of Baltzer ('10), on reciprocal 
Arbacia pustulosa crosses. Baltzer has shown that the form of 
the chromosomes in this species is like those of Arbacia punctu- 
lata which I described in 1907. Baltzer made six Arbacia crosses. 

Strongylocentrotus ¢ < Arbacia = 
Sphaerechinus ¢ X Arbacia 2 
Echinus ¢ X Arbacia ¢ 

Arbacia ¢ X Strongylocentrotus ¢ 
Arbacia @ X Sphaerechinus 2 
Arbacia ¢ X Echinus 2 

TIn.all of these crosses the results are in general like those which 
I have just described for Arbacia punctulata. In all of the 
crosses, in most individuals, there is an ‘Erkrankung’ before or 
during the blastula stage, this indisposition being so marked as to 
resemble a sudden poisoning of the embryos. 

In Baltzer’s Arbacia ¢ by Strongylocentrotus = or Echinus ¢ 
crosses from 8 to 10 chromosomes were eliminated. In the Arba- 


400 DAVID H. TENNENT 


cia ° by Sphaerechinus ~ cross about 18 chromosomes were 
eliminated (Baltzer *10, text figs. 18 and 19). In Baltzer’s 
Echinus ¢, Strongylocentrotus ¢ and Sphaerechinus ¢ x Arba- 
cia « crosses two types of behavior were apparent. In the first 
two cases elimination of chromosomes occurred during the blastula 
stage; in the last case the chromosomes were retained. In all of 
these cases the skeleton of the plutei was nearer the maternal 
type than the paternal, but the hybrid nature was evident. 

My Arbacia @ & Toxopneustes ~ cross is almost identical in 
its behavior to Baltzer’s Arbacia ¢ * Echinus ¢ or Strongylo- 
centrotus ¢ cross. My Toxopneustes ¢ % Arbacia ¢ cross is 
unlike any of Baltzer’s Arbacia ~ crosses, in that elimination of 
chromosomes took place during the cleavage. I shall return to 
these points later. 


SUMMARY 


The results of this investigation may be summarized as follows: 

1. The study of artificially parthenogenetic eggs has shown 
that the eggs of Toxopneustes are all alike in that each contains 
two V-shaped chromosomes. 

2. The study of fertilized enucleated fragments of Toxopneustes 
eggs has shown that Toxopneustes spermatozoa are of two classes, 
one class containing one V-shaped chromosome, the other contain- 
ing two-Vshaped chromosomes. 

3. The study of the Arbacia ¢ x Toxopneustes < cross has 
shown that elimination of both Arbacia and Toxopneustes chro- 
mosomes may take place during cleavage. 

4. The study of the Toxopneustes ¢ x Arbacia @ cross has 
shown that nearly all of the Arbacia chromosomes may be elim- 
inated in the early cleavage. 


DISCUSSION 


The first point requiring consideration jis in connection with the 
results of the study of parthenogenetic eggs and of fertilized enu- 
cleated egg fragments of Toxopneustes. I have shown here that 
Toxopneustes is in agreement with Hipponoé (11, ’12), in that the 


STUDIES IN CYTOLOGY 401 


female is homogametic and the male digametic rather than with 
Strongylocentrotus (Baltzer, ’09),in which the female is digametic. 

I still feel that we are not yet able to apply one of the now 
accepted sex formulae to either Hipponoé or Toxopneustes. It 
is futile, at this time, to attempt to make a definite count of the 
chromosomes in these eggs. I feel reasonably sure that the num- 
ber in the zygotes is 37 and 38; but granting this to be true, no 
reliable conclusion can be drawn until we know the facts concern- 
ing synapsis in the formation of the germ cells. It is possible that 
the question can be settled only by the study of the odgenesis and 
spermatogenesis in these forms, but so much has been determined 
by the experimental method that one may hope that some Echino- 
derm may be found which will afford material favorable enough 
for deciding even this point. 

It is interesting in this connection to note that since the females 
of Hipponoé and Toxopneustes are homogametic we should 
obtain uniformly individuals of the same sex from chemically 
fertilized eggs. The conditions in Hipponoé indicate that these 
individuals would be female. 

It must also be noted that my results are not quite in accord 
with those of Miss Heffner. My observations would give us two 
classes of Toxopneustes zygotes, one with three and one with four 
V-shaped chromosomes rather than one with two and one with 
three. 

My investigation has shed no further light on the nature of 
these idiochromosomes; we do not know whether they are com- 
pound, whether they are single and have this individual form, or 
whether the form is due simply to the place of attachment of the 
spindle fiber. The last idea seems untenable in the light of their 
definite numerical occurrence. 

Turning from these purely cytological considerations to some 
of the facts concerning heredity in Echinoderms we must first 
notice that if one wishes to speak with accuracy it is now impos- 
sible to make the broad statement that all of the individuals of 
a given cross are maternal or paternal, or even intermediate, in 
a strict sense, in character. 


402 DAVID H. TENNENT 


We are at a point now where we may, gather together the many 
different accounts that have been given and restore order in a 
field which has been in a state of disorder. We have conclusive 
evidence that under certain conditions, most of the embryos 
of a given cross will have a skeleton of a definite type and we also 
know that a smaller number may depart radically from this type. 
Thus we may obtain from eggs of one female fertilized by sperm 
from one male a complete series of skeletons ranging from the 
purely maternal to the purely paternal form. 

When we compare these series numerically with a similar series 
of zygotes from some species, in the cleavage and slightly later 
stages we find the same sort of variation in the number of retained 
maternal and paternal chromosomes. In other words, there is 
as great a variation, of its kind, in the kind of chromosomes in 
cross fertilized eggs as there is in the kind of skeleton in hybrid 
plutei. This is not true for all species. In some hybrids all of 
the chromosomes are retained and a dominance of one kind of 
skeleton over another is exhibited. 

The facts regarding the retention or elimination of chromosomes 
and the character of the ensuing pluteus are of interest. Pre- 
sented briefly they are: 

1. Elimination of no chromosomes and dominance of one species 
over the other with respect to the character of the skeleton. 


Examples: Toxopneustes ¢ x Hipponoé ¢ 
Echinus ¢ x Antedon ¢ (Baltzer) 
Strongylocentrotus ¢ x Antedon ~ (Baltzer) 
2. Elimination of part of the chromosomes and dominance of 
one species over the other with respect to the character of the 
skeleton. 


Examples: Hipponoé ¢ x Toxopneustes 7 
Echinus 2° X Sphaerechinus < (Baltzer) 
Strongylocentrotus ¢ X Sphaerechinus ~ (Baltzer) 
3. Elimination of no chromosomes and skeleton of intermediate 
character. 


Examples: Sphaerechinus ¢ X Strongylocentrotus ¢ (Baltzer) 
Sphaerechinus ¢ xX Arbacia ~ (Baltzer) 


STUDIES IN CYTOLOGY 403 


4. Elimination of part of the chromosomes and skeleton of inter- 
mediate character. 


Examples: Toxopneustes ° & Hipponoé 4 
Arbacia @ X Echinus ¢ (Baltzer) 
Arbacia ¢  Toxopneustes # 
Toxopneustes 9 & Arbacia ¢ 
5. Elimination of part of both maternal and paternal chromo- 
somes and inhibition of development. 


Examples: Arbacia ¢  Toxopneustes 2 
Toxopneustes ¢ x Arbacia ¢ 

This list of examples is not meant to be exhaustive but is given 
simply as a means of illustration. 

It is evident that in a single series there is a correlation between 
the types of larvae exhibited and the behavior of the chromosomes. 
Tt is further evident, that taking echinoid hybrids as a whole, there is 
a series of well defined grades, from retention of all chromosomes and 
preponderance of one type over another, through retention of all chro- 
mosomes and a blending of type, to a rejection of more than the half 
number and failure of development. 

It must be pointed out that such examples as Echinus ¢ xX 
Antedon ~ are in a way misleading to one who does not remember 
that the crinoid larva of early age has no skeleton. We should not 
expect a spermatozoan having no determinant for a skeleton to 
produce much effect in an egg having such a determinant or 
determinants. 

It should further be pointed out that such cases as those coming 
under paragraph 2, e.g., Hipponoé ¢ x Toxopneustes < as well 
as the fertilizations of the echinoid egg by the sperm of annelids 
and molluses (Godlewski, ’11, Kupelwieser, ’09), and the fertiliza- 
tion of eggs which have been given a certain impulse to partheno- 
genetic development by means of chemicals, give as these authors 
have pointed out, thelykaryotic larvae. Practically larvae de- 
rived from such crosses inherit from the egg parent alone just as 
strictly as if the eggs had been caused to develop from the first 
by artificial chemical fertilization. 


404 DAVID H. TENNENT 


These facts represent a definite advance in our ideas concerning 
the relation of chromosomes to somatic characters. They do not 
however aid us in deciding the question as to whether the nucleus 
is the sole bearer of the determinants of one character or the other. 
Godlewski (711), in his expression that neither the nucleus alone, 
nor the protoplasm alone, but both parts of the cell body are 
concerned, in the determination of hereditary characters, and that 
for the development of the paternal characters an interaction be- 
tween paternal nucleus and protoplasm is indispensable, raises 
an old objection in a new form and one which it is exceedingly diffi- 
cult to deny. As our knowledge of the interaction of nucleus and 
cytoplasm increases, particularly along the lines of nuclear syn- 
thesis, we have convincing evidence that the chromatin requires 
a very definite environment in order that it may increase. Conk- 
lin’s (12) observations on the fate of chromosomes centrifuged 
into the yolk in Crepidula is one case in point. It is evident that 
Godlewski’s position is correct in so far as concerns the persistence 
and growth of the paternal nuclear material, and this material 
must persist and increase if it is to influence development. How 
much farther than that we may go in insisting on the necessity of 
all parts of the paternal germ cell body is somewhat doubtful. 


February 14, 1912. 


STUDIES IN CYTOLOGY 405 


LITERATURE CITED 


BaurzeErR, F. 1909 Die Chromosomen von Strongylocentrotus lividus und Echi- 
nus microtuberculatus. Arch. f. Zellforsch., Bd. 2. 


1910 Uber die Beziehung zwischen dem Chromatin und der Entwick- 
lung und Vererbungsrichtung bei Echinodermenbastarden. Arch. f. 
Zellforsch., Bd. 5. 


Conxuin, E.G. 1912 Cell size and nuclear size. Jour. Exp. Zool., vol. 12. 


Gop.tewskl, E. 1911 Studien iiber die Entwicklungserregung. Arch. Ent. 
Mech. der Organ. 


Herrner, B. 1910 A study of chromosomes of Toxopneustes variegatus which 
show individual peculiarities of form. Biol. Bull., vol. 19. 


Hrinpuez, E. 1910 A eytological study of artificial parthenogenesis in Strongylo- 
centrotus purpuratus. Arch. Ent. Mech. der Organ., Bd. 31. 


Kuretwieser H. 1909 Entwicklungserregung bei Seeigeleiern durch Mollusk- 
ensperma. Arch. Ent. Mech. der Organ., Bd. 27. 


Logs, J. 1909 Die Chemische Entwicklungserregung des tierischen Eies. Ber- 
lin. 


Nemec, B. 1911 Das Problem der Befruchtungsvorginge. Berlin. 


Pinney, E 1911 A study of the chromosomes of Hipponoé esculenta and Moira 
atropos. Biol. Bull., vol. 21. 


TENNENT, D. 191] A heterochromosome of male origin in Echinoids. Biol 
Bull., vol. 21. 


1912 The behavior of the chromosomes in cross fertilized Echinoid 
eggs. Jour. Morph., vol. 23. 


PLATE 1 


EXPLANATION OF FIGURES 


All of the figures were drawn from sections with the aid of a camera lucida at 
a magnification of 1500 diameters. They were then doubled in size by means of a 
drawing camera and compared with the original preparations. The figures have 
subsequently been reduced one half in publication. 

14 From chemically fertilized eggs 

1A and B Six poled division figure. 

2 Lateral view anaphase plate of first cleavage. 

3 Lateral view anaphase plate of first cleavage. 

4A and B Lateral view anaphase plate of first division. 

Arbacia 

6 Lateral view of one section of an anaphase plate of a straight fertilized 

Arbacia egg. 
From fertilized enucleated egg fragments 

5A, Band C Three sections of same mitotic figure in anaphase. One V and 
one long rod. 

7A, Band C Lateral view of anaphase plates as in fig. 5. Two Vs and one 
long rod. 


PLATE 1 


STUDIES IN CYTOLOGY 
DAVID H. TENNENT 


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THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No.3 
APRIL, 1912 
407 


PLATE 2 


EXPLANATION OF FIGURES 


8A, Band C Lateral view as above. Two Vs and one long rod. 
9A and B Lateral view of anaphase plate. Tripolar figure. 


Arbacia 2 X Toxopneustes o cross 


10-17 Lateral views of anaphases of the first division. 


10A, Band C The Vs and long rod characteristic of Toxopneustes may be 
seen. 


11A, B and C Long rod and Vs. Some of Toxopneustes chromosomes lag- 
ging. 

12A, Band C Toxopneustes Vs not present. Probably some of the Arbacia 
chromosomes have been eliminated. 


13A and B- One Toxopneustes V and long rod. Lagging and elimination as 
in fig. 12. 


408 


STUDIES IN CYTOLOGY 
DAVID H. TENNENT 


PLATE 2 
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409 


PLATE 3 
EXPLANATION OF FIGURES 


144A and B No long Toxopneustes rod. Some of both Toxopneustes and 
Arbacia chromosomes have been eliminated. 

15A and B- No Toxopneustes Vs. Some of the Arbacia chromosomes seem 
to be absent. 

16A, Band © NoToxopneustes Vs. Some of both Toxopneustes and Arbacia 
chromosomes have been eliminated. 

17A, Band C Pronounced lagging of Toxopneustes chromosomes. 


Toxopneustes 9 & Arbacia @ cross 


1SA, B and C Lateral view of anaphase plates. Two Toxopneustes Vs and 
the long rod. Few Arbacia chromosomes. 

19A and B Lateral view of anaphase plates. Two Toxopneustes Vs and the 
long rod. Few Arbacia chromosomes. 

20A and B. Nearly all of the Arbacia chromosomes are absent. 

21A and B Tripolar figure. Two pairs of Toxopneustes Vs are present. 
Probably about half of the Arbacia chromosomes have been rejected. 


410 


STUDIES IN CYTOLOGY PLATE 3 
DAVID H. TENNENT 


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STUDIES OF FERTILIZATION IN NEREIS 


Ill. THE MORPHOLOGY OF THE NORMAL FERTILIZATION OF NEREIS 
IV. THE FERTILIZING POWER OF PORTIONS OF THE SPERMATOZOON 


FRANK R. LILLIE 


From the Marine Biological Laboratory, Wood’s Hole, Mass., and the Hull 
Zoological Laboratory, University of Chicago 


FORTY-EIGHT FIGURES 


ELEVEN PLATES 


In the preceding numbers of these studies dealing with the 
cortical changes of the egg, and with partial fertilization, refer- 
ence has been made to certain phases of the normal fertilization. 
_A detailed study was found to be necessary to clear the way for 
further considerations. The results of this detailed study are 
presented in the first part of the present paper. The second part 
deals with the fertilizing power of portions of the spermatozo6n 
and with the theory of fertilization. 


Ill. THE MORPHOLOGY OF THE NORMAL FERTILIZATION OF NEREIS 
1. The spermatozoén: (Fig. 1, a, b, c, d) 


The spermatozoa are unusually large. The head terminates 
in a very delicate perforatorium which possesses a slight distal 
enlargement.! Between the base of the perforatorium and the 
chromatin of the head is some differentiated material forming a 
head-cap (h. c.). This is very distinctly differentiated in position 
and staining reaction from the chromatin of the head. The chro- 
matin of the head is not so condensed as in most spermatozoa; 


‘In the living spermatoén the perforatorium is shorter and shaped more like 
the spike of a helmet. 


413 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 4 
may 1912 


414 FRANK R. LILLIE 


it is often arranged in two masses as shown in fig. 1 with some 
karyolymph; sometimes the arrangement is more reticular, but 
the karyolymph is always in evidence in good preparations. The 
middle-piece is very distinctly set off from the head; it is ring- 
shaped, very broadly attached to the base of the head. The 
attachment of the tail is to the margin of the ring, and is there- 
fore excentric to the axis of the head. There is a decided asym- 
metry, particularly illustrated in b, c, d, of fig. 1. 


2. The ovum 


The ovum has been described in the preceding parts (Lillie, 
11) of this study. A brief description will therefore serve here. 
It is about 100u in greatest diameter, somewhat flattened in a 
polar direction, and girdled by a double zone of large oil drops 
embedded in the yolk-bearing protoplasm (text fig. 1). It is 
bounded by a vitelline membrane and possesses a coarsely alveolar 
cortical layer about 7 in thickness external to the yolk-bearing 
layer. 


3. Observations on fertilization in the living egg 


If the eggs remain unfertilized in the sea-water, maturation 
does not take place, and the egg remains unchanged with intact 
germinal vesicle. 

a. The cortical changes. When insemination takes place a 
large number of spermatozoa become attached to the ovum if 
the sperm is present in excess. In about two to three minutes 
all spermatozoa, with the exception of one, which is alone con- 
cerned in the subsequent fertilization, begin to be carried away 
from the surface of the egg by an outflow of jelly from the ovum. 
This is more particularly described in the first of these studies 
(F. R. Lillie, ’11). The jelly is formed from the alveolar contents 
of the cortical layer, which gradually disappears, until in the 
course of about fifteen minutes the original cortical layer is repre- 
sented only by the perivitelline space and the delicate walls of 
the original alveoli crossing this space to the plasma membrane 
(see study 1). 


STUDIES OF FERTILIZATION 415 


Text fig. 1 Three photographs of eggs of Nereis in ink before ins il 
b, three minutes after insemination; c, twelve minutesafter i 
b were taken with direct sunlight and short exposure (one second with diffuse 
light and longer exposure (ten seconds See text for descriptior 


The formation of the jelly may be most readily observed if the 
eggs are inseminated in sea-water, into which India-ink has been 
ground so as to make a black suspension. I here reproduce three 
photographs showing the formation of the jelly under such cir- 
cumstances (text fig. 1, a, b, c); ais an egg taken before insemina- 
tion; the germinal vesicle and the oil globules stand out very 
distinctly; the cortical layer may be seen on the left side of the 
figure, but it does not come out very distinctly because it is seen 
through a layer of ink between the cover slip, which touches the 


416 FRANK R. LILLIE 


top of the egg, and the equator of the egg. Fig. 1 b shows the 
amount of jelly formed about three minutes after insemination; 
the cortical layer is already much reduced. Fig. 1 ¢ was taken 
twelve minutes after insemination; it shows a great increase in 
amount of jelly due not only to continued secretion from the egg, 
but also, presumably, to swelling of the jelly already formed. The 
walls of the alveoli which contained the jelly-forming spherules 
may be seen on the right side of this photograph crossing the peri- 
vitelline space to the plasma membrane. On the upper side may 
be seen the fertilization cone and external to it outside the vitel- 
line membrane the spermatozoon. <A conical pointer of ink is 
directed to the spermatozo6n. This forms regularly in eggs in- 
seminated in ink and is due to the ink particles pressing in along 
the tail of the spermatozo6n embedded in the jelly, owing to their 
Brownian movements. This ink cone remains even after the 
spermatozo6on has penetrated and thus serves as indicator of the 
point of entrance of the spermatozo6n up to the time of cleavage. 
A study of the relation of the point of entrance to the first cleavage 
plane by one of my students is now under way (see Just, ’12). 

Text fig. 2 is a drawing from the living egg based on such a pho- 
tograph as 1c. The details are all clearly shown here and are 
described in the legend of the figure. 

b. The fertilization cone. The spermatozoon, that remains 
after the first jelly-formation, is attached by its perforatorium to 
the vitelline membrane. The protoplasm of the ovum immedi- 
ately beneath now forms a conical elevation (fertilization cone) 
which crosses the perivitelline space and becomes attached to the 
vitelline membrane beneath the spermatozo6n (text figs. 1 ¢ 
and 2). The fertilization cone then gradually retracts and dis- 
appears, drawing the vitelline membrane with it so as to form a 
depression in which the spermatozo6n is included. At this stage 
one may easily imagine that the spermatozo6n has been taken into 
the egg, as it is apt to be concealed in the depression of the mem- 
brane; but this is not the case. The stage of greatest develop- 
ment of the depression, corresponding to the complete retraction 
of the fertilization cone, is about twenty-two to twenty-five min- 
utes after insemination (see figs., Lillie °11, p. 369). 


STUDIES OF FERTILIZATION AIT 


The fertilization cone is no longer seen in the living egg. But 
the sections show that its substance has become so modified as to 
form a definite cell-organ, which can be followed for some time 


after penetration of the spermatozoon. 


Ege of Nereis fifteen minutes after insemination i he 


Text fig 
outer circle represents the ink, the clear area between this and the egg is the jelly 
nes of the pte 


c.l., cortical layer showing the radiating | 


zation cone; g.v., germinal vesicle, drawn perhaps a little too strong t begins 
to break down about this time; 7. ¢., ink cont oil drops | membrane 
$p., Spermatozoon; v.m., vitelline membrane yolk sp lies 

c. Penetration of the spermatozoén. Following the development 


of the depression in the vitelline membrane consequent upon 
the retraction of the fertilization cone, the perivitelline space 


418 FRANK R. LILLIE 


narrows around the entire egg, the depression in the vitelline mem- 
brane therefore disappears and the spermatozo6n again becomes 
prominent externally. It remains external until about forty to 
fifty minutes from the time of insemination and then disappears 
rather abruptly within the egg. Its penetration coincides with 
the late anaphase or telophase of the first maturation division, or 
with the extrusion of the first polar body. 

A definite granule to which the tail is attached always remains 
on the membrane at the point of penetration. The cytological 
study shows this to be the middle piece, which does not enter the 
egg. Not only have I repeated this observation frequently in 
two successive seasons, but it has been demonstrated to classes of 
students at Woods Hole and to some of the investigators there. 
The middle pieceand tail of the spermatozoén do notenter in the ferti- 
lization of the egg of Nereis. In view of the emphasis which has 
recently been put on cytoplasmic contributions by the spermato- 
zoon in fertilization, I paid particular attention to this point in 
the summer of 1911, and was able to determine not only that it 
fails to enter at the same time as the sperm-head, but that it can 
be demonstrated external to the membrane up to the time of the 
first cleavage at least (cf. also Just, ’12). 

I have seen the penetration of the spermatozo6n take place in 
all positions from the animal to the vegetative pole, and I cannot 
say that there is any preferred point of entrance with reference to 
the poles. The existence of polyspermy, which is not infrequent, 
proves that there is no preferred meridian for penetration. So it 
would appear that any point on the surface of the ovum may be 
used for penetration. 


4. The phenomena of fertilization as seen in sections 


a Before penetration. Technique: The eggs are fixed for an 
hour in Meves’ modification of Flemming’s fluid made as follows: 
Chromic acid 0.5 per cent, 15 cc.; osmic acid, 2 per cent, 3.5 
cc.; glacial acetic acid, three drops. Staining in iron haematoxy- 
lin alone. 


STUDIES OF FERTILIZATION 419 


a. The spermatozoon is readily found in sections prior to pene- 
tration, and all parts can frequently be recognized, though the 
tail is usually invisible owing to extraction of the stain. The 
middle piece is always distinctly differentiated, but it is rather 
variable in appearance. Its ring-form is usually distinguishable 
in sections and in some cases one or more minute granules may be 
distinguished in it. The head stains solid black in the iron haema- 
toxylin. Fifteen minutes after insemination (fig. 2, a, b, c) the 
perforatorium has penetrated the vitelline membrane and is in 
contact with the fertilization cone. 

The fertilization cone itself presents a rather remarkable appear- 
ance, and it deserves very careful attention, owing to the rdéle 
that it plays in the penetration of the spermatozodn. Fifteen 
minutes after insemination it projects somewhat above the con- 
tour of the egg, and its substance, which is practically homogene- 
ous, stains much more deeply in the iron haematoxylin than the 
neighboring cytoplasm. This difference in staining reaction so 
suddenly acquired indicates a modification of its substance which 
might be conceived as due to slight coagulation by a fluid intro- 
duced by the spermatozo6n. In any event the change in stain 
is due to its relation to the spermatozo6n, and it is the first indi- 
cation of its conversion into a specialized cell-organ. 

A fertilization cone has been described in various eggs (echinids, 
asteroidea, etc.), but in all cases previously described its signifi- 
‘ance has been, apparently, merely temporary and local, a reac- 
tion of the ovum to the spermatozo6n with no definable function 
of its own. The case of Nereis, however, is very different, as will 
be seen from the following description. 

At twenty-seven minutes after insemination the fertilization 
cone no longer forms any projection (fig. 3); it is usually, on the 
contrary, somewhat depressed in the center. The perforatorium 
stains somewhat more strongly than before. 

At thirty-four minutes after insemination the perforatorium has 
entered the surface of the fertilization cone to a slight extent 
(fig. 4), and a small granule appears at its tip, surrounded by a 
slight clear area. <A little later (fig. 5, a, b, c, d, thirty-seven 
minutes after insemination) there is a group of three or four black 


420, FRANK R. LILLIE 


granules, which we shall name atlachment granules, at the tip of 
the perforatorium embedded near the surface of the fertilization 
cone. In one ease (fig. 5 a) the perforatorium is seen to be double, 
a condition that I have also observed once in a preparation of 
spermatozoa alone. The perforatorium now stains very strongly. 

The spermatozo6n is now, therefore, actually anchored in the 
substance of the fertilization cone by the perforatorium and 
attachment granules noted above. I interpret the increased 
strength of stain in the perforatorium as due to the flow of sub- 
stance through it from the spermatozoén to form the granules 
found at its tip in the fertilization cone. 

One can give only a more or less probable interpretation of 
these phenomena. The extreme delicacy of the perforatorium 
and the presence of a distal enlargement on it make it improbable 
that it bores through the membrane in a merely mechanical way; 
and the improbability is strengthened by the fact that the sper- 
matozo6n is absolutely immobile after attachment to the membrane. 
It seems probable, therefore, that the vitelline membrane is weak- 
ened at the point of application of the perforatorium, presumably 
by a fluid flowing through the perforatorium, though on account 
of its extreme delicacy it is impossible to be certain that the per- 
foratorium is tubular. The staining reaction and differentiation 
of the fertilization cone could then be explained by the assump- 
tion that it is affected by a fluid furnished by the spermatozo6n, 
and the appearance of the granules within the fertilization cone 
as due to inflow from the spermatozoén through the perfora- 
torium. The mechanism of the spermatozo6n certainly appears 
more complex than we have hitherto suspected. I would assume, 
therefore, that the material of the head cap, forming a reservoir 
of material at the base of the perforatorium, functions in relation 
to penetration. 

b. Penetration. The actual penetration of the head of the 
spermatozo6n is shown in figs. 6, 7 and 8, all taken from material 
preserved forty-eight and one-half minutes after insemination. 
The complex made up of the fertilization cone and the head of 
the spermatozo6n acts asa unit. The cone retreats into the inte- 
rior of the protoplasm and the head of the spermatozo6n becomes 


STUDIES OF FERTILIZATION 421 


a narrow chromatin band as it enters through the minute aper- 
ture in the vitelline membrane. Figs. 9 a and 9 6 (fifty-four 
minutes after insemination) show penetration completed and it 
will be noted that the middle piece remains external. 

Examining now the details of this process, every stage of which 
is found in the preparations, we may note: (1) The sperm nuc’eus 
enters through an aperture in the membrane much smaller than 
itself, and is hence drawn out into a strand. (2) The inner end 
of the entering sperm nucleus begins to swell, by absorption 
of fluid soon after its entrance has begun (figs. 6, 7, 8). (3) 
Striae appear in the protoplasm between the fertilization cone 
and the surface of the egg. (4) When penetration has actually 
begun (fig. 6) the distance, corresponding to the length of the 
perforatorium, which originally separated the chromatin of the 
spermatozo6n from the attachment granules in the fertilization 
cone has become very much reduced. The sperm nucleus is now 
almost or actually in contact with the fertilization cone (figs. 6, 
a Se 

If we inquire into the mechanism of this process, it is quite 
obvious that the initiative, so to speak, is on the side of the ovum. 
It is inconceivable that the spermatozo6n should be the primum 
movens and push the fertilization cone before it into the proto- 
plasm without being coiled up or bent within the protoplasm. 
But, in the many instances observed, it is invariably as straight 
as shown in the figures. The fertilization cone indeed appears 
to actively penetrate, or to be engulfed by the egg protoplasm, 
and to draw the sperm nucleus after it through the narrow aper- 
ture in the vitelline membrane. 

The final stage of penetration is shown in figs. 9a and96. The 
entire sperm nucleus is now within the egg cytoplasm, some dis- 
tance from the periphery, and it will be seen that the middle 
piece has remained without on the egg membrane. This is per- 
fectly characteristic, if not invariable, and the same observa- 
tion was made repeatedly on the living egg as already stated. 
The picture is perfectly clear as shown under a very high magni- 
fication in fig. 9b, and not only is the middle piece plainly external, 
but there is no sign whatever of any sperm-component at the base 


422 FRANK R. LILLIE 


of the sperm head. An assumption that the spermatozo6n intro- 
duces any differentiated structure at the base of the head could, 
in this case, be due only to a preconception in its favor. 

c. Revolution of the sperm-nucleus and origin of the sperm-aster. 
The original orientation of the sperm nucleus within the egg is 
perfectly apparent, owing to the association of its apex with the 
fertilization cone from the very beginning of penetration. The 
whole complex of fertilization cone and sperm nucleus now rotates 
180 degrees, so that the fertilization cone, which was placed cen- 
trally to the sperm nucleus, comes to lie peripheral to it. In fig. 
10 a (fifty-four minutes after insemination) the revolution is shown 
nearly completed. Fig. 10 6 shows the cone-nucleus-aster com- 
plex of the same egg more highly magnified; and figs. 10 ¢ and 
10 d show the entrance point of the spermatozo6n involved, with 
tail (10 c) and middle piece (10 d) external to the vitelline mem- 
brane. The sperm asteris beginning toform in this stage opposite 
to the fertilization cone, thus in the position of the original middle 
piece, which, as we have seen, was left on the egg membrane. A 
minute granule, actually in the substance of the nucleus (fig. 10 6) 
is the point of focus of the rays. In fig. 11 (sixty-four minutes 
after insemination) this granule, now more clearly recognizable, 
is still in contact with the sperm nucleus. The rotation of the 
sperm nucleus is invariable, and no exception has been seen in 
the numerous cases observed; the point of origin of the sperm 
aster is Just as invariable. 

The fact that the sperm aster arises from the base of the sperm 
nucleus as it is oriented in the spermatozo6n, not only in Nereis 
but in all other forms accurately determined, is unquestionably 
of fundamental significance. I may, however, be permitted to 
point out that the usual interpretation, which connects the sperm 
aster with the centrosome of the spermatid (occupying this posi- 
tion in the spermatozo6n) is not the only possible interpretation. 
It is important to emphasize this point because such an interpre- 
tation ascribes the fertilizing power of the spermatozo6n in the 
last analysis, not to the spermatozo6n as a whole, but to a minute 
part of it, the centrosome; and it involves a whole theory not only 
of fertilization, but of cell division in general, and much of cellular 


STUDIES OF FERTILIZATION 423 


physiology, by ascribing certain very general functions and powers 
to this minute cell element. 

The alternative interpretation, which I would present, is that 
the appearance of the sperm.aster in a position definitely oriented 
with reference to the sperm nucleus may be a nucleo-plasmic 
reaction localized by polarity of the nucleus. 

In any case, if the centrosome theory is to be retained for Nereis, 
it becomes necessary to assume that, in this form, the centrosome 
is contained, not in the middle piece, but in the nucleus of the 
spermatozoon. In the experimental study, which forms the 
second part of the present paper and study number 4 of the series, 
I shall present crucial evidence against this assumption; discus- 
sion of the matter may therefore be postponed. 

After the appearance of the sperm aster, the nucleus penetrates 
yet more deeply within the egg and leaves the fertilization cone 
behind near the inner margin of the yolk (fig. 11), where it soon 
disappears. So far as I know, it is a perfectly unique cell organ, 
except in its earliest stages, and it is certainly surpyising to find 
it in so highly differentiated a condition, and with apparently so 
active a function in the penetration of the spermatozo6n, as it is 
in Nereis. 

d. Division of the sperm aster. The sperm nucleus moves 
towards the center of the egg, and the aster separates from it a 
little and divides, forming an amphiaster. A secondary spindle 
then arises between one or both of the sperm-centrosomes and the 
inner centrosome of the second maturation spindle (figs. 12 and 
13). This has been very fully described by Bonnevie (’10). Shortly 
after the sperm amphiaster is formed, it is noticeable that one of 
the asters and centrosomes is decidedly larger than the other 
(figs. 13 and 14a). Bonnevie has also called attention to, this 
fact, but has not, as I believe, sufficiently emphasized the fact 
that it is a secondary condition. I must dissent a little from her 
view that the sperm aster does not always divide prior to the com- 
pletion of the second maturation division; in my material, at least, 
the amphiaster is invariably formed prior to this time. 

e. The germ nuclei and origin of the cleavage centers. After the 
second polar body is formed, the egg aster gradually dwindles and 


424 FRANK R. LILLIE 


disappears (figs. 14a, 146 and 15). There can be not the least 
doubt of this fact in my material, though Bonnevie, working on 
the same form, but with different cytological methods, remains 
doubtful on this point. I shall not attempt an elaborate cytologi- 
cal analysis of the matter here (though the evidence from this 
side is, I believe, conclusive), because in eggs from which the 
sperm nucleus has been removed experimentally the gradual 
disappearance of the egg aster can be traced with complete cer- 
tainty, owing to the absence of any possible confusion with the 
sperm aster (see second part of this paper, p. 440). The fully 
formed egg nucleus in such eggs never has a trace of the aster 
(see studies 2 and 4). The chromosomes of the egg swell and 
form vesicles (figs. 14 a, 14 6, 15), which gradually fuse together 
and establish the egg nucleus. At the same time the sperm 
nucleus begins to enlarge (fig. 14 b) and the sperm amphiaster 
becomes less distinct than before and its radiations less extensive 
(figs. 18, 14 and 15). The sperm amphiaster continues to wane 
and when the two germ nuclei have come together, the smaller 
aster has become indistinguishable (fig. 15). The larger aster 
can, however, always be distinguished through the stages of the 
germ nuclei, and can be seen to become the larger aster of the 
first cleavage spindle after the partition wall between the germ 
nuclei has disappeared (figs. 16 a, 16 b, 17). At this time a 
much smaller aster arises opposite the larger (fig. 17), like it in 
the plane of apposition of the germ nuclei. The first cleavage 
spindle is thus heterodynamie from its inception, and the first 
cleavage of the egg is strikingly unequal, as is well known from 
Wilson’s study (Wilson, 794). 

In the preceding paragraph I have summarized a very comple 
cated period of the fertilization process to which I have given 
much study, and which exhibits many interesting cytological 
details, as for instance, the accumulation of granules in the neigh- 
borhood of the germ nuclei (figs. 15 and 16 a) which almost 
certainly eseape from the latter (fig. 16 a). As regards the 
main point, the origin of the cleavage centers, there can be no 
doubt that the larger one is derived from the larger aster of the 
sperm amphiaster, for it ean be followed continuously and is 


STUDIES OF FERTILIZATION 425 


never absent. The simplest interpretation of the smaller cleavage 
center is that it represents the smaller sperm aster, although 
there is a brief period when it is not demonstrable, owing (possibly) 
to defect in the cytological technique. It is my opinion, then, 
that the sperm amphiaster becomes the cleavage amphiaster in 
Nereis, as in so many other animals. 


5. Discussion 


If any apology is needed for presenting so strictly a morphologi- 
eal study of such an apparently threadbare subject as the fertili- 
zation of the ovum, I might say that the impulse to make it came 
from an experimental study, and that it'is necessary to the experi- 
mental results which follow. It has, moreover, yielded some 
details of observation which deserve to go on record on their 
own account. 

The conclusion that the cleavage centers arise from the sperm 
centers is in agreement with many other studies. But I am 
unable to accept the usual conclusion that the sperm centers 
arise around a centrosome introduced by the spermatozo6én into 
the egg, and that, therefore, the sperm centrosome is the fertiliz- 
ing agent of the spermatozo6n, and the sperm nucleus concerned 
exclusively with amphimixis. The crux of the problem is pre- 
cisely here on the question of the origin of the sperm centers. 
The fact that the middle piece of the spermatozo6n which usually 
includes the spermatid centrosome does not penetrate the egg in 
Nereis is evidence of a certain amount of value only. Defects in 
cytological technique may always be invoked to explain failure 
to observe the introduction of a centrosome by the spermatozoon. 
Little as I may be inclined to admit this, it is necessary to grant 
some force to this objection where such delicate cytological 
details are involved. It would, however, I believe, be recognized 
as crucial evidence that the sperm centrosome is not necessary to 
fertilization, if a distal fraction of the sperm head alone were 
proved to form a sperm aster, a certain portion of the base of the 
sperm head as well as the middle piece being prevented from 
entering. Such results are described in the second part of this 
paper. 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 4 


426 FRANK R. LILLIE 


There has been much discussion during the past year concern- 
ing a cytoplasmic basis of certain aspects of inheritance, and a 
consequent re-investigation of the penetration of the spermato- 
zoon. Following Meves’ study (711) of the réle of plastochondria 
in the fertilization of Ascaris, in which he concluded that ‘‘the 
plastosomes represent the hereditary substance of the cytoplasm 
as the chromatin does that of the nucleus,’’ a number of authors 
investigated the penetration of the spermatozo6n in echinids. 
Dantan (711) asserts that in Paracentrotus lividus and Psam- 
mechinus miliaris, the entire spermatozo6n enters the egg, and he 
concludes that fertilization should be defined as the union of two 
complete gametes which fuse nucleus to nucleus and cytoplasm 
to cytoplasm. Witschi(’11) describes a case in Strongylocentrotus 
in which the tail of the spermatozo6n entered in fertilization, but 
he thinks it probable that in this form the tail is oftener left on 
the membrane. Ries (’11) describes a curious shedding of an 
involucre in the penetration of the spermatozo6n in Strongylo- 
centrotus, but he believes that the axial structures of head, middle 
piece and tail enter. His account must, however, be accepted 
with considerable reserve until confirmed. Finally, Meves (’11a) 
has studied the relation of the middle piece in the fertiliza- 
tion of Parechinus miliaris, and believes as the result of his obser- 
vations that it probably furnishes plastochondria. He says noth- 
ing about penetration of the tail, so it is fair to assume that it 
does not occur in Parechinus. 

The classical accounts of the penetration of the spermatozoon 
in sea-urchins, according to which the tail is left on the membrane 
and only head and middle piece enter seem to be on the whole 
confirmed so far as the main principle (i.e., the non-essential 
character of the tail in fertilization) is concerned, since both 
entrance and non-entrance have been observed. The tail cannot 
therefore be regarded as supplying a cytoplasmic basis for inherit- 
ance in sea-urchins. 

I have shown that in Nereis the middle piece of the spermato- 
zoon is likewise left on the membrane, so we cannot look to it as a 
cytoplasmic basis for inheritance in this form as Meves does in the 
sea-urchin. On the other hand it is possible that the fixation 


STUDIES OF FERTILIZATION 427 


granules introduced by the spermatozo6n represent a cytoplasmic 
element (whether concerned in inheritance or not), but of this we 
cannot be certain until their derivation is better known. 

The only characteristic thing about the cytoplasmic elements 
introduced by the spermatozo6n is their great variability as to 
quantity and character in different animals. In Ascaris a very 
large quantity of cytoplasm containing characteristic plastosomes 
is introduced, as Meves has shown. In many, probably most, 
forms with flagellated spermatozoa, the entire spermatozo6n 
enters; in some echinids the tail is left without, and in Nereis 
both tail and middle piece fail to enter; and turning to plants, 
in phanerogams apparently nothing but the nucleus is eventually 
eoncerned. There is nothing on the cytoplasmic side to corre- 
spond with the regularity of the nuclear phenomena in both ani- 
mals and plants. In such precise phenomena as those of inherit- 
ance a mechanism of equal precision is to be expected, and it 
must be admitted that on the cytoplasmic side no such mechan- 
ism has been discovered. Moreover, as the laws of inheritance 
are the same for animals and plants, a similar mechanism must 
exist for both, and such has been discovered only in the nuclei of 
the gametes. There is bad logic in the assumption that what- 
ever parts of the spermatozoén enter the egg are necessarily 
concerned in the mechanism of transmission in inheritance, and 
the view that the cytoplasmic elements of the male gamete are 
concerned primarily in accessory functions of fertilization, such 
as locomotion and penetration, is still logically well founded. 


IV. THE FERTILIZING POWER OF PORTIONS OF THE SPERMATOZOON 
1. Introduction and methods 


In the second of these studies (F. R. Lillie, ’11) it was shown 
that the stimulus of the spermatozo6n in fertilization involves 
two phases: (1) an external phase, prior to entrance of the sper- 
matozoon but after its attachment to the egg, in which certain 
cortical changes are induced, jelly is secreted by the egg and the 
mechanism of maturation of the ovum is released; and (2) an 
internal phase beginning after the entrance of the spermatozoon, 


428 FRANK R. LILLIE 


which is necessary if cleavage of the ovum is to take place. The 
present study is a contribution to the analysis of the latter phase 
based on observations concerning the fertilizing power of por- 
tions of the spermatozoon. 

It is questionable whether any direct and universal method for 
such an experiment could be devised, for one would have to over- 
come the difficulties of isolating a spermatozo6n, of operating on 
it, and of ensuring the entrance of a part into the ovum, under 
precautions that would preclude the possibility of fertilization by 
an intact spermatozoén. These difficulties might be overcome 
by an instrument sufficiently delicate to enable one to amputate 
parts of the attached spermatozo6n before its entrance into the 
egg. The same result has been obtained in Nereis by a method 
that enables one to operate in bulk, to remove fractions of the 
attached sperm head of varying size, to observe the entrance of 
the part remaining attached to the egg, and to study its fertiliz- 
ing effect, at least to a certain extent. 

In brief, the method consists in centrifuging the inseminated 
eggs of Nereis at five minute intervals before penetration and pre- 
serving the centrifuged eggs at appropriate times. The effective- 
ness of the method depends on conditions already described (see 
part 1 of the present paper, study 3 of the series) which may be 
summarized briefly as follows: The spermatozo6n remains exter- 
nal to the vitelline membrane with its perforatorium embedded 
in the entrance cone for about fifty minutes, more or less, depend- 
ing on the temperature, after insemination. It is embedded inthe 
thick viscous jelly secreted by the egg (text figs. 1 and 2). If, 
now, a quantity of eggs be centrifuged with sufficient force, they 
first accumulate at the distal ends of the tubes in a mass which 
becomes closely packed together. The jelly, which is of less 
specific gravity than the eggs, then separates from the latter and 
forms a layer above the eggs. In squeezing through the narrow 
interstices between the closely packed eggs the jelly rubs over 
the surface of each egg and in many cases carries the attached 
spermatozo6n away with it, leaving, however, the perforatorium 
and attachment granules in the cone as evidence of its former 
presence. In other cases, especially if the eggs be centrifuged 
shortly before the time of penetration of the spermatozoon, it 


STUDIES OF FERTILIZATION 429 


draws out the substance of the head of the spermatozoén, which 
is very ductile at this time, into a strand, and in numerous cases 
it carries away the tail and middle piece or variable portions of the 
head in addition. Partial sperm heads of all sizes are therefore 
left attached to the egg by the perforatorium. Such partial 
sperm heads then penetrate, if the eggs be left to develop in sea- 
water, and their behavior may be studied. 

In each experiment 8 or 9 stages in the process of fertilization 
were centrifuged at five minute intervals, in order to be sure that 
the entire period of penetration of the spermatozo6n was covered, 
because particularly striking results were to be expected from 
removal of the external part of the spermatozo6n after a certain 
amount had entered (figs. 6 to 8). Several hundred eggs were 
centrifuged each time. One has to be sure that neither too little 
or too much centrifuging is done, and it was only as a result of 
considerable experience extending over three years that sixty 
revolutions of the handle of the centrifuge in about forty seconds, 
giving 7200 revolutions of the tubes at a radius of 6 em., was 
selected as most favorable. 

The following protocol of an experiment will show exactly how 
the experiments are carried out and the material secured for 
examination. 

EXPERIMENT 16 


September 21, 1911 


To preserve a series immediately after centrifuging and thirty minutes later 
Eggs fertilized at 8:28 a.m. Temperature of air, 18°C.; of water, 18°C. 


OBSERVATIONS ON PER CENT 
DESIGNATION CENTRIFUGING PRESERVATION OF SEGMENTED EGGS AMONG 
THOSE REMAINING 


a.m. a.m. a.m. a.m. 


(35-5) eee 8:58 16.1.1, 8:59 16.1.2} 9:29 35 per cent 10.47 
UGP2 eae. | X 60) 9:03 16.2.1) 9:04 16.2.2) 9:34 15 per cent 10.48 
NG recs, oe X 60} 9:08 16.3.1; 9:09 16.3.2 | 9:39 10 per cent 10.52 
LG Maser, <5 | X 60} 9:18 |16.4.1) 9:14 16.4.2 | 9:42 5 per cent 10.54 
1ersee |X 60| 9:18 16.5.1 9:19 16.5.2) 9:49 (25-30 percent! 10.55 
GAGi sho. Vase 60) 9:23 |16.6.1) 9:24 16.6.2 9:53 65 per cent 10.56 
I SEY eee X 60} 9:28 |16.7.1) 9:29 16.7.2 | 9:58 75 per cent 10.57 
MGS ieretacerar= X 60} 9:33 116.8.1 9:34.5 16.8.2 | 11:03 85 per cent 11.02 
MGSO eso. X 60} 9:38 16.9.1) 9:39.5 16.9.2 | 11:09.5 | 90+ percent | 11.03 


Controls—preserved at 8:57, 9:12.5, 9:35 a.m. .. 90+ per cent 11.05 


430 FRANK R. LILLIE 


The eggs preserved immediately after centrifuging enable one 
to study the immediate effects of the separation of the jelly on the 
spermatozo6n, and those preserved later show what portions of 
spermatozoa remaining attached after centrifuging have entered 
the egg and what their fertilizing power has been up to the time of 
preservation. Some of the eggs were kept living for estimation 
of the per cent of eggs that undergo segmentation (last column). 
In other experiments the eggs were preserved at different periods 
following centrifuging, because no single experiment gives a suffi- 
cient quantity of material for preserving a complete series of 
stages. In this way from a considerable number of experiments 
a very complete set of stages was secured. 

Each lot of eggs preserved included several hundred which were 
embedded together in paraffine and cut in serial sections which 
would usually cover four or five slides under a 50 by 25 mm. 
cover, if all the material were cut. On one such slide I estimated 
by counting that there were 374 eggs present; and the 1911 mate- 
rial alone made 376 slides. Only about a fourth of the slides 
contain the desired stages, and the figures are given only to show 
that a large quantity of material has actually been under review to 
give the results. However, I may say that figs. 18 to 23 are all 
from a single slide, and other interesting stages occurred on the 
same slide; it is possible in fact to demonstrate the whole set of 
phenomena from a few slides of the large number prepared. 

The clew to the whole set of phenomena was given by the dis- 
covery of very minute sperm nuclei in the material used for the 
second of these studies (F. R. Lillie, ’11). In the effort to dis- 
cover the origin of these minute, and presumably partial, sperm 
nuclei, the whole history was gradually worked out as given here. 

The centrifugal force employed causes a very complete segre- 
gation of the oil and granules of the egg into four zones which are 
illustrated in text fig. 3, taken from a section fixed in Meves’ 
modification of Flemming’s fluid and stained in iron haematoxylin. 
These zones are, beginning with the constituents of least specific 
gravity; (1) the zone of the oil drops; (2) the hyaline zone in which 
the smaller basophile granules gather for the most part; (3) the 
zone of the yolk-spheres; (4) a zone consisting of small hyaline 


STUDIES OF FERTILIZATION 431 


spheres not stained by the osmic acid or haematoxylin, inter- 
spersed with basophile granules larger than those of zone 2. The 
penetration and rotation phenomena appear in the hyaline zone 
even more clearly than in the normal egg, hence most of the figures 
illustrating these phenomena are taken from the hyaline zone. 


2. The effects on the spermatozoa 


a. Before the beginning of penetration. ‘The effects of the re- 
moval of the jelly on the spermatozo6n have been studied especi- 
ally on stages shortly before penetration begins, both because these 
are the stages most affected as shown by the effects on cleavage 


Text fig. 3 The zones of the centrifuged egg of Nereis 


(stages 16.3 and 16.4, in the preceding table) and also because 
spermatozoa injured at this time would presumably have a better 
chance to enter, being so near to the actual time of penetration. 

Figs. 18 to 23 show successive degrees of injury to spermatozoa 
from a single experiment (6.5 of 1911). The eggs were preserved 
immediately after centrifuging 7200 revolutions at a radius of 6 
em. in thirty-five seconds, fifty minutes after insemination, on 
June 19, 1911, when the temperature of the water was still quite 
low and the processes correspondingly slow. In fig. 18 the entire 


432 FRANK R. LILLIE 


head of the spermatozo6n is present as shown by the middle 
piece, but it has been drawn out to a band and shows its granu- 
lar structure. Fig. 19 shows a case in which the middle piece 
and a small part of the base of the sperm head has been entirely 
removed. Figs. 20, 21, 22 and 23 show the removal of increas- 
ingly large portions of the sperm head. Cases could be illus- 
trated beyond either end of this series in which on the one hand 
the spermatozo6n is entirely uninjured, and on the other hand 
even the perforatorium is pulled out of the entrance cone. The 
cases illustrated are merely selections from a very much larger 
set of observations. 

It is shown, therefore, that practically any degree of injury to 
the spermatozo6n may be produced prior to its entrance. Cases 
in which the delicate perforatorium is broken next the head leay- 
ing only the cone and attachment granules are the most common 
as is to be expected; but the other classes of injury bear witness to 
the tenacity of the hold of the spermatozo6n on the egg. 

b. Injuries to the spermatozodn after penetration has begun. 
This class of injuries is relatively rare because the actual process 
of penetration requires only about two minutes, and the chances 
of involving it are therefore correspondingly few. However, I 
have found a considerable number of such cases in the prepara- 
tions. At first I looked to such injuries as the only source of the 
partial sperm nuclei already observed in the egg, but as I made the 
observations described in the preceding section, I realized that 
they were not the only or indeed the chief source of such partial 
sperm nuclei. Figs 24 and 25 illustrate two cases from the same 
experiment (6.5.1, 1911) from which figs. 18 to 23 are taken. In 
fig. 24 it will be seen that penetration has already begun (cf. fig. 6), 
and the external part of the spermatozo6n is ravelled out and the 
middle piece, at least, lost. Fig. 25 is a little more complicated; 
in this case penetration had probably reached a condition inter- 
mediate between figs.6and7. The centrifugal force has doubled 
up the part of the sperm in the egg, and drawn out the external 
part removing the middle piece. Fig. 26, finally, is a clear cut 
case, the external part alone being removed entirely, and the 
remainder showing a clear penetration picture. This is the prob- 


STUDIES OF FERTILIZATION 433 


able interpretation of this picture, although it was taken from a 
preparation killed fifteen minutes after centrifuging; further pene- 
tration following centrifuging was probably prevented in this case 
by the large oil drops which were driven around the enteringsperm. 


8. Penetration of injured spermatozoa 


Injured or partial spermatozoa may enter the egg, demon- 
strating that penetration of the spermatozo6n, after attachment 
is once secured, is an active function of the egg and not at all of 
movements: of the sperm itself, if any further evidence is needed 
on this point. But if the entire spermatozo6n be removed, the 
cone remains superficial and does not penetrate; at least I have 
repeatedly found it in a superficial position fifteen to thirty or 
more minutes after the normal time of penetration, and I have 
never found it actually penetrated unless accompanied by all 
or a part of the sperm nucleus. 

The evidence for the entrance of injured or partial sperm heads 
is furnished by cytological study of eggs fixed at definite periods 
after centrifuging. Particularly clear evidence is furnished by one 
series of nine stages fixed fifteen minutes after centrifuging. In 
the fifth stage of this series centrifuged fifty minutes after insemi- 
nation and fixed fifteen minutes later, early stages of penetration 
are very abundant, and among them are some that show unequi- 
vocal evidence of being partial sperm nuclei, for there are numer- 
ous cases in which an injured part of the sperm head is left out- 
side on the membrane and thus guarantees the partial nature of 
the sperm nucleus within. Some such cases are illustrated in 
figs. 27 to 32. 

Fig. 27 shows a case killed fifteen minutes after centrifuging 
in which the connection between the internal and external parts 
of the spermatozo6n is entirely broken. The partial sperm nu- 
cleus within the egg and the cone have already begun to rotate. 
In fig. 28 we have a similar case, except that there still remains 
a delicate connection between internal and external parts of the 
spermatozo6n, and this condition would lead, by rupture of this 
connection, to the condition shown in fig. 27. It would seem, 


434 FRANK R. LILLIE 


then, that the spermatozoén may be so injured in the process of 
centrifuging that it ruptures in the process of penetration, and we 
thus learn that partial sperm nuclei may come from spermatozoa 
merely injured and not actually broken in the process of centri- 
fuging. It should also be noted that in each of these cases the 
internal part of the spermatozoon is divided in two parts; presum- 
ably after penetration, by constriction around an injury. Fig. 
29 shows a condition similar in many respects to figs. 27 and 28, 
in that the entire spermatozo6n is present, and a large external 
part is separating from a smaller internal part. Figs. 30 and 31 
show the early penetration of parts of spermatozoa, recognizable 
as such merely by their small size in the absence of the external 
part. The cases illustrated in figs. 30 and 31 are such as would 
be derived from injuries similar to those shown in figs. 20 and 21. 

Rotation of the sperm cone complex has already begun in figs. 
27 to 31, while the cone is much nearer the surface of the egg than 
in normal fertilization. Rotation begins immediately after pene- 
tration is completed, and hence takes place nearer to the surface 
when portions only of the sperm are concerned than when the 
whole sperm is concerned. In this connection the readershould 
compare figs. 9 and 10. 


4. Stages of rotation of the partial sperm nuclei and origin of the 
4 g y I L g ) 
sperm aster 


Convincing evidence of the partial nature of sperm nuclei in 
later stages of rotation and origin of the aster is difficult to secure, 
in spite of the fact that the control consisting of part of the same 
lot of eggs preserved immediately after centrifuging may show 
numerous instances of partial sperm heads like those illustrated 
in figs. 18 to 26. The difficulty of securing unequivocal evidence 
arises from the fact that mere size, except in extreme cases, is no 
longer a safe guide; in the first place the diameters of the nuclei 
vary only as the cube root of their volumes, hence considerable 
differences in volume may be represented by undetectable differ- 
ences of diameter. In the second place the sperm nuclei are not 
round and all are not in favorable positions for comparable meas- 


STUDIES OF FERTILIZATION 435 


urements. In the third place the volume of the sperm nuc eus 
normally increases considerably for some time after entrance, and 
the difficulty of deciding whether comparable stages are involved 
is sometimes great. 

In spite of these difficulties, however, I have found sperm 
nuclei which must be interpreted as partial on the basis of their 
size alone, especially in later stages. 

Fortunately however, it is not necessary to rely on size differ- 
ences alone for in some of these stages, as in the case of the pene- 
tration stages just considered, a remnant of the sperm head may 
be found adhering to the membrane at the point of entrance, 
guaranteeing the partial nature of the sperm nucleus within. A 
few of these stages may be considered first: 

Fig. 32 shows a case immediately continuing those deseribed in 
Section 3. Here the rotation had already begun, as evidenced 
by the position of the cone, a very delicate connection still remain- 
ing with the external part of the sperm head impedes the rotation 
of the sperm nucleus within. Fig. 33 a and b shows a more ad- 
vanced case; in fig. 33 a the cone and sperm nucleus are shown 
almost half rotated. One would not be able to decide from 
the size that it was a partial sperm nucleus, but the next section, 
shown in 33 b contains a considerable portion of the sperm head 
still connected with the middle piece which has remained without 
on the egg membrane. This is a very critical case, demonstrating 
that a partial sperm nucleus will rotate like a complete one. The 
portion in 33 b is entirely disconnected from the nucleus shown in 
33 a; they are in the very act of separation. In fig. 35 we have a 
very fortunate section in which a completely rotated fertilization 
complex: cone, sperm nucleus and aster, is present, and a con- 
siderable portion of the same sperm head is found on the mem- 
brane outside the egg; the external portion is entirely comparable 
to the condition shown in figs. 19, 24 and 25 and there can be no 
question about its interpretation. Undoubtedly, the condition 
came from one essentially similar to that shown in fig. 25; it is,in 
fact, exactly what one would predict a later stage of thecondi- 
tion of fig. 25 to be, assuming a break to occur between the parts 
within and without the membrane. 


436 FRANK R. LILLIE 


Fig. 36 shows a case in which part of a sperm head has been left 
behind in the peripheral protoplasm at the point of entrance, and 
we have the completely rotated complex of cone, sperm nucleus 
and aster within. Another similar case is illustrated in fig. 37, 
and still other illustrations could be given. 

It will be noted that there are very considerable size differences 
in the sperm nuclei of figs. 35 to 37, and all of these are distinctly 
smaller than the normal comparable stage shown in fig. 11. But 
in these cases it is not necessary to rely on the distinctly smaller 
size as evidence of their partial nature, for we have the evidence 
of sperm remnants left at the point of penetration. In figs. 38 
and especially 39, however, we have cases in which the undeniably 
minute size of the sperm nuclei alone is sufficient evidence, taken 
in connection with the records of injury to the external sperm 
head by removal of the jelly illustrated in figs. 18 to 26, to prove 
that they have been derived from a mere fragment of an entire 
sperm head probably (in the case of fig. 39) not exceeding one- 
sixth to one-eighth of the bulk of the entire head, although the 
external part was entirely lost in this case. 

It is, perhaps, not necessary, but it may be well to emphasize 
the fact that the cases selected for illustration are all from com- 
plete series of sections, and that the neighboring sections were 
always consulted for possible parts of the sperm nucleus. The 
small size is not due to division by the microtome knife. The cases 
described are selections from a much greater number of records. 

It will be noted that if small fragments of a sperm head can 
produce aster formation in the egg, the possibility of polyspern y 
with parts of a single spermatozo6n is given. This condition, 
which I anticipated on theoretical grounds, was finally found 
and is illustrated in fig.40. There are two sperm nuclei, each with 
an aster, associated with a single cone. The small size of these 
nuclei marks them as partial, and their connection with a single 
cone as parts of a single spermatozoon. The only alternative 
explanation of this figure would be that two spermatozoa had 
become implanted so close together as to produce a single cone, 
and that they had received comparable injuries in the process of 
centrifuging. Against this explanation are the results of many 


STUDIES OF FERTILIZATION 437 


observations of polyspermy in none of which was implantation 
nearly close enough together to produce a single cone; in one series 
that I possess many eggs have thirty or more spermatozoa im- 
planted, but the points of insertion are always separate. More- 
over, in such a case one would expect the cone to be larger than 
usual, but in this case it is a little below the average size; one 
would also expect to find two equal groups of implantation 
granules with separate attachments of the nuclei, but only one 
group and one attachment is found here, viz., in the nucleus to the 
left; the granule near the right nucleus is too small to represent a 
separate implantation group, and moreover, it has no connection 
with the neighboring nucleus. Finally, it will be seen that con- 
ditions such as those illustrated in figs. 27 and 28 must inevitably 
lead to the condition of fig. 40 if the fragments of the sperm head 
do not reunite. 

There is thus every reason for interpreting these two nuclei 
as parts of a single one. This rare find smply emphasizes the 
conclusions already reached concerning the fertilizing power of 
portions of the sperm head. 

Without attempting at this place to discuss the results fully, I 
would, nevertheless, emphasize the two facts of greatest impor- 
tance already brought out. In the first place it is shown that an 
apical fragment of the sperm head is able to produce an accompany- 
ing aster in the egg cytoplasm; the sperm aster has therefore no 
necessary relation to the middle piece of the spermatozoén, or to the 
centrosome of the spermatid. In other words, using the formation 
of a sperm aster as criterion, the fertilizing power of the spermato- 
zoon is not localized in the middle piece, as supposed by Boveri 
and others, but is a function of even small fragments of the sperm 
nuc’eus alone. In the second place the great beauty of this 
material is that the orientation of the sperm nucleus, whether 
entire or partial, is preserved until after the origin of the sperm 
aster, and this enables one to determine that the sperm aster 
always arises in relation to the most basal point of the sperm 
nucleus. Altogether, I have observed well over one hundred 
cases of entire and partial sperm nuclei in these stages, and have 
never found any exception to the rule that the sperm aster arises 


438 FRANK R. LILLIE 


at the point of the sperm nucleus farthest from the cone. In 
other words, the position of origin of the sperm aster is a function of 
polarity of the sperm nucleus, and it is this which explains its invari- 
able origin, so far as has been recorded in the literature, in the 
position of the middle piece of the spermatozo6n; and the theory 
that a centrosome introduced by the spermatozo6n is necessary 
for such formation is therefore shown to be incorrect. 

The results so far show that sperm fragments, even of very 
minute size, may enter the egg in conjunction with the cone, rotate 
in the normal manner and produce an aster in the egg-cytoplasm. 
The question now arises, what is the ultimate fate of such frag- 
ments? Is their fertilizing power adequate to produce segmen- 
tation of the egg? 


5. The later history of the partial sperm nuclei 


Partial sperm nuclei separate from the cone and penetrate 
towards the center of the egg like normal ones (figs. 38 and 39). 
The sperm aster divides and forms an amphiaster in the stage of 
the anaphase of the second maturation division characterized by 
inequality of the two poles as in the normal. But apparently the 
size of the centrosomes and the extent of the astral radiations 
are directly proportional to the mass of the sperm nucleus con- 
cerned. This is brought out very well in fig. 41, which is a recon- 
struction from three successive sections showing two sperm nuclei 
of unequal size in the same egg. The egg in question had been 
centrifuged forty-four minutes after insemination and was pre- 
served forty-seven minutes later in the stage of the telophase of 
the second maturation division. It will be observed that the 
larger nucleus (left) is accompanied by a larger centrosome and 
aster than the smaller one (right), and it should be stated that the 
aster in each case is the larger one of an amphiaster. Here, where 
direct comparison between nuclei of unequal size and their accom- 
panying asters within the same egg is possible, the proportional 
size of asters to nuclei is obvious. I do not mean of course to 
assert that the proportions are mathematically accurate for this 
would be impossible to determine. 


STUDIES OF FERTILIZATION 439 


In general, larger sperm nuclei are accompanied by larger 
asters and smaller nuclei by smaller, roughly proportional, asters, 
after they are fully formed. Exceptions to this rule are certainly 
rare. I have, however, found a very few cases, two or three in 
all, in which a very small sperm nucleus is accompanied by < 
disproportionately large aster. The explanation of such cases is 
uncertain, but I am inclined to attribute it to a secondary reduc- 
tion of the sperm nucleus after penetration and aster formation, 
such as might conceivably result from some form of injury received 
in centrifuging. 

The significance of this proportional relation is at once apparent; 
if the aster is a product of a nucleo-cytoplasmic reaction of some 
kind, as we have already seen reason to believe, there must be a 
quantitative relation between the product (aster) on the one hand, 
and the reacting elements (nucleus and cytoplasm) on the other, 
and this is what we find. 

After maturation is completed and the germ nuclei are formed, 
we have to find a new criterion for partial sperm nuclei. Com- 
parison of size of the egg and the sperm nucleus alone is not very 
satisfactory, because both nuclei are swelling very rapidly at this 
time and they may meet and begin to fuse before their enlarge- 
ment is complete, so that complete identity in size of egg and 
sperm nucleus prior to fusion is not invariable in the normal fer- 
tilization. But a valid criterion may be found in the following 
phenomena: the germ nuclei are formed by chromosomal vesicles, 
one for each chromosome, and in each vesicle a sharply marked 
chromatic nucleolus arises before the separate vesicles fuse. Fu- 
sion begins very early and growth of the nucleoli accompanies 
it; however, an elimination or dissolution of the nucleoli begins 
before fusion is complete, so that their number is rapidly reduced 
again, and they entirely disappear before the actual prophases 
of the first cleavage spindle. Fusion of the two germ nuclei 
with one another may also begin before the fusion of the chromo- 
somal vesicles in each is complete. Under normal conditions the 
number of the chromatic nucleoli is probably the same in each 
germ nucleus in the early stages. I therefore looked for cases of 
striking disparity in number between the chromatic nucleoli of 


440 FRANK R. LILLIE 


the egg and sperm nuclei. Some such cases were found, but they 
were much fewer in number than expected on the basis of the 
number of partial sperm nuclei found in earlier stages. I was 
therefore led to suspect that the smallest sperm nuclei might dis- 
integrate prior to this time. However, I have failed to find 
direct evidence for this. It may be, therefore, that the failure 
to find the expected number of partial sperm nuclei in the stage in 
question is due to the fact that the critical period for such deter- 
mination is of very brief duration. 

Fig. 42 illustrates a case of disparity between the two germ 
nuclei. Five sections are involved, and the male and female 
nuclei are indicated by the appropriate signs. The male com- 
ponent, distinguished by its accompanying aster, exhibits five 
nucleoli and the female thirteen. The volume of the female 
component is also much greater than that of the male compo- 
nent. I believe, therefore, that we have here an undeniable case 
of fusion of a partial sperm nucleus with an entire egg nucleus. 

In the stage of the first cleavage spindle of eggs centrifuged 
just before penetration of the spermatozo6n we have three classes 
of eggs, aside from a very few polyspermic eggs: (1) Some with a 
more or less normal cleavage spindle; (2) some with a monaster 
centered in a group of chromosomes; (3) some without any trace 
of astral radiations although chromosomes are formed. In a 
particular lot of eggs (7.4 of 1911) of which 10 per cent segmented, 
at the stage of the first cleavage the first class is rare, the second 
class is quite common, and the third is the most frequent condi- 
tion. The first class evidently corresponds to the 10 per cent of 
eggs that segmented. 

The two latter classes are illustrated in figs. 43 and 44. Fig. 
43 is a camera drawing of the three sections of the nucleus of a 
single egg belonging to the third class. The eggs of the first class 
of the same lot were in various stages of the anaphase and telo- 
phase of the first cleavage. There appear to be fourteen chro- 
mosomes, the number usually found in the maturation spindles; 
the position near the animal pole proves it to be the egg nucleus. 
The absence of the sperm nucleus is readily demonstrated. There 
is an entire absence of all radiations in the cytoplasm. Both 


STUDIES OF FERTILIZATION 44] 


polar bodies are present. This condition, as I have already said, 
is the commonest condition in such a lot of eggs, and both polar 
bodies are invariably present. 

Attention may be directed to the fact that each chromosome 
is embedded in a homogeneous ground substance of about the 
same tint, in the iron haematoxylin stain employed, as the cyto- 
plasm. Evidently, each chromosome with its surrounding ma- 
trix corresponds to a single chromosomal vesicle of the early egg 
nucleus; the numbers are the same. The chromosome of the 
succeeding cell generation arises within the substance of the chro- 
mosome of the preceding generation in this case. 

The second class of cases, monasters, is illustrated in fig. 44. 
I was at first inclined to think that these might be due to fertili- 
zation with partial sperm nuclei, especially as the degree of devel- 
opment of the monaster shows a wide range of variation. But 
careful study of the eggs concerned showed that the first polar 
body had invariably failed to form, and the second was always 
present alone. About twenty-five cases of this kind have been 
examined without a single exception occurring. 

In the second study of this series (Lillie, 11) I have described 
the cause of failure of the first polar body. This condition 
occurs in eggs centrifuged just before the formation of the 
first polar body. The first maturation division may then take 
place within the egg forming two nuclei, and the second matura- 
tion spindle which involves both nuclei is a tetraster (Lillie, ’11, 
fig. 6). The second polar body is formed from one pole of the 
tetraster and three nuclei are left in the egg, which soon unite. 
Under these circumstances, if the sperm nucleus be absent, a 
more or less feeble monaster may develop at the time of the 
first cleavage; though in a few cases where only the second polar 
body was formed, no signs of radiations were found. 

There is thus a more or less striking difference in the behavior 
of the egg nucleus in those cases where both polar bodies are 
formed and those in which the first polar body is suppressed. It 
is a very interesting problem whether the formation of the mon- 
aster in the latter case is a purely quantitative relation, due to 
the larger number of chromosomes present in such cases? There 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 4 


442 FRANK R. LILLIE 


is considerable variation in such cases in the size of the second 
polar body and the quantity of the chromatin which it contains, 
and corresponding differences in the number of chromosomes 
left in the egg. But the degree of development of the monaster is 
not a function of the number of chromosomes in the egg; and there 
are cases in which no aster formation is associated with a larger 
number of chromosomes in such eggs, and a well developed mon- 
aster with a smaller number. It is possible that there may be a 
qualitative relation depending on what chromosomes are extruded 
in the second polar body; but in view of the complicating consider- 
ations resulting from possible injurious effects of centrifuging 
itself, no definite conclusion on this point seems possible. 

It is, in any event, certain that the sperm nucleus is absent in 
both the second and third classes of eggs. 

As regards the effect of the partial sperm nuclei on the cleavage 
process we are therefore reduced to the class of cases in which a 
cleavage spindle is actually formed. In observing the living eggs 
I was struck with the fact that the cleavage of many centrifuged 
eggs tends to be irregular or partial, especially of those centri- 
fuged at the time when injuries to the spermatozo6n were to be 
expected. And in the sections I find many cases of partial cleay- 
age. The cleavage of many also stops in the two-cell stage. It 
is natural to suppose that such partial cleavages are the result 
of fertilization with partial sperm nuclei, seeing that we know 
from the data recorded above that there is not even the least 
indication of cytoplasmic cleavage in the entire absence of the 
sperm nucleus. A rigorous demonstration of such a conclusion 
would, however, require a cytological analysis in which the num- 
ber of chromosomes in the different cleavage spindles of normal 
and partially segmenting eggs should be compared; the relative 
sizes of the karyokinetic figure, and possibly other data, should 
also be taken into account. Unfortunately, the material pre- 
served for this study is in too advanced a stage to make an exhaus- 
tive study of these relations, and this part of the investigation 
must therefore be postponed. 

It must be admitted that other causes than fertilization with 
partial spermatozoa might be responsible for the partial cleavage, 


STUDIES OF FERTILIZATION 443 


e. g., injury of certain kinds caused by the centrifuging, possibly 
abnormal maturation, or a general systemic disturbance of the 
cytoplasm. But this does not seem very probable, since eggs 
centrifuged at times when injury to the spermatozo6n is not to 
be anticipated do not exhibit the partial cleavage, at least to the 
same extent. I would therefore regard it.as probable, though 
not proved, that partial sperm nuclei tend to produce more or 
less defective cleavage. 


GENERAL DISCUSSION 
1. The centrosome theory of fertilization 


The centrosome theory of fertilization is still accepted by most 
morphologists in spite of the doubts that have been thrown on 
the theory of the permanence and genetic continuity of the cen- 
trosome by the production of asters in unfertilized eggs (Morgan, 
Wilson, ete.), and by the studies in artificial parthenogenesis. 
The theory that the spermatozoén introduces an extra-nuclear 
centrosome destined to become the organ of cell division of the 
odsperm is confronted for the first time, in the results of the fore- 
going study, with a crucial experimental test. And the result 
that even small parts of the sperm head produce a typical centro- 
some and aster in the egg cytoplasm conclusively demonstrates 
the inadequacy of this conception of fertilization. Solongassimilar 
experiments on other forms are lacking, there is no reason to 
believe that the production of the sperm centrosome depends 
upon any different principle in Nereis than in other forms. 

Meves (11a) demonstrates that the sperm aster does not arise 
in connection with the so-called middle piece of the spermato- 
zo6n in the ease of the sea-urchin, but at the base of the sperm 
nucleus itself, the middle piece being already separated from the 
nucleus and lying to one side at the time that the sperm as- 
ter arises. These results, while inconclusive in themse ves, are 
nevertheless distinctly unfavorable to the existence of an extra- 
nuclear centrosome as the cause of formation of a sperm aster in 
this classical case. Further, one can say without fear of success- 
ful contradiction that in no animal has it been shown that the 


444 FRANK R. LILLIE 


sperm aster arises around an extranuclear centrosome of spermatic 
origin. There is, therefore, no reason for assuming that the 
experiments on Nereis have revealed an exceptional condition. 
Either, then, the centrosome theory of fertilization must be 
rejected in toto, or the sperm nucleus must be regarded as a cen- 
tronucleus in Boveri’s sense (Boveri ’00), 1. e., as including the 
centrosome. If we examine the latter point of view we see that 
it would be necessary to assume that centrosomes exist in the 
sperm nucleus at every level, for the sperm aster can form at any 
level as demonstrated by the experiments. The diagram (text fig. 


Text fig. 4 


4) will make this clear. It represents the sperm nucleus in the 
form that it possesses as penetration is nearly completed 
(ef. fig. 8); normally the sperm aster arises at point a, but if ab be 
removed then at point 6, if ac be removed at c, if ad be removed 
at d,ete. But the assumption that centrosomes exist through- 
out the nucleus and condition this phenomena would seem to be 
merely an exaggerated morphological point of view with refer- 
ence to a problem that is, after all, fundamentally physiological, 
viz.: by virtue of what properties does the sperm nucleus exercise 
this effect? If the great mass of experimental cytological data 
favored the view of the permanence of the centrosome, the con- 


STUDIES OF FERTILIZATION 445 


ception of the centronucleus might legitimately be extended to 
cover the present case, but this cannot be said to be true; and in 
the present state of our knowledge such an explanation would 
appear forced and merely formal. 

The formation of the sperm aster takes place on the boundary 
between nucleus and cytoplasm, and as we have seen (fig. 41), 
there are very definite quantitative relations between the bulk 
of the partial sperm nuclei and the degree of development of the 
aster and the size of the centrosome. ‘This relation leads to the 
conclusion that if intranuclear centrosomes are the causes of the 
formation of the sperm aster, not only must they exist at every 
level, but also that they must decrease in size from the base to 
the apex of the sperm nucleus! The formation of the sperm aster 
on the boundary between nucleus and cytoplasm and the quanti- 
tative relations existing between size of the nucleus and of the 
aster demonstrate, it seems to me, that the centrosome and aster 
owe their existence to an interaction between nucleus and cyto- 
plasm, and not to any third element. All the observed relations 
in the case’of Nereis harmonize with this point of view. 

The production of astrospheres remote from the nucleus in the 
experiments of Morgan (’96 and ’99) and others show, it is true, 
that the nucleus is not necessary for the production of such phe- 
nomena. These asters are apparently very temporary formations, 
and evidence that their central bodies may divide like the centro- 
some of the sperm aster, or other centrosomes associated with 
nuclei, is lacking. Nevertheless, it seems probable that funda- 
mentally similar physiological causes are at the foundation of 
both sets of phenomena, and we can only assert our profound 
ignorance of what these causes really are. 


2. The polarity of the sperm-nucleus 


We may use the term polarity to describe the fact that the 
sperm aster arises invariably at the most basal point of the sperm 
nucleus, whether it be entire or partial. This phenomenon cor- 
responds accurately to the general features of polarity of ova or 
lower organisms, as, for instance, the formation of oral organs 
at the oral end of a cut piece, etc. 


446 FRANK R. LILLIE 


It might, perhaps, seem possible at first thought that the aster 
appears in this position with reference to the partial sperm nuclei 
because we have here a broken surface; this is at least a condition 
in which this particular point of the surface of the partial nuclei 
differs from the remainder of its surface. But in the entire sperm 
nucleus, where there is no broken surface, the position is always 
the same. Moreover, some ten or fifteen minutes elapses after 
entrance of the sperm head before the aster becomes visible, and 
in this time the nucleus has changed form so as to produce a 
pointed extremity (figs. 33 a, 36, 37, 40, etc.) in the position 
where the aster is to appear whether the nucleus be entire or 
partial. This is perhaps sufficient evidence of repair of the 
wounded surface. 

We have to seek some more profound cause for this localization, 
and I believe that it must be regarded as a special case of organic 
polarity to be explained like other cases on the basis either of 
gradation or orientation of materials. From this point of view 
the nucleus would possess an immanent structure determining 
the location of aster formation and therefore the plane of division 
of the nucleus. 


3. Theory of fertilization 


I have pointed out repeatedly in these studies that fertiliza- 
tion involves two phases, viz.: an external phase prior to entrance 
of the spermatozo6n, in which certain cortical changes are pro- 
duced in the egg, and an internal phase, following penetration, 
involving a complex series of phenomena. As I pointed out in 
the introduction, this paper is a contribution to the analysis of 
the second phase. On the basis of experiments on artificial par- 
thenogenesis and hybrid fertilization, Loeb has made a similar 
distinction of two phases, and so far the results of what we may 
call the biological and the physico-chemical analyses of fertili- 
zation are in accord. 

If we reject the centrosome theory of fertilization, as I believe 
we are compelled to do, what point’ of view from the side of the 
biological analysis shall we put in its place? The theory of the 


STUDIES OF FERTILIZATION 447 


internal phase of fertilization must proceed from the funda- 
mental fact of the difference in behavior of the sperm nucleus 
and the egg nucleus in the cytoplasm of the egg. The former 
induces aster formation and karyokinesis; the latter does not. 
Let us recall the facts in the case of Nereis briefly again: (1) 
Even minute fragments of the sperm nucleus cause the formation 
of an aster withacentrosome capable of division. (2) If the sperm 
nucleus be prevented from entering, the egg nucleus may indeed 
form chromosomes but no aster arises, provided that both polar 
bodies are formed.2 (3) We may add from experiments on other 
forms that in the absence of the egg nucleus the sperm nucleus 
behaves the same as in its presence. 

Clearly, then, there is some difference, associated with their 
sex-origin, between these two nuclei; and the most direct form 
of interpretation of this difference is that which identifies it with 
the fundamental sex characters which inhere in every cell. In 
other words, the sperm nucleus has the character maleness, what- 
ever that may be, and neither the egg nucleus nor cytoplasm pos- 
sesses this character. It makes no difference that half the sper- 
matozoa may carry the factor for femaleness and half for maleness. 
The distinction between character and factor is clear. It may be, 
on the other hand, that ova and spermatozoa acquire in the course 
of gametogenesis special differentiating properties that are the 
cause of the fertilizing power of the spermatozo6n. 

However we may conceive the demonstrated difference between 
the sperm nucleus and the egg nucleus, it is obvious that there is a 
lack of interchange between the egg nucleus and the egg cytoplasm 
that conditions the inhibition of the unfertilized egg. In some 
way, then, the maturation divisions of the egg must have removed 
certain reacting constituents of the germinal vesicle, or have 
brought about certain cytoplasmic changes in the egg, because 
we have perfect karyokinetic phenomena in the maturation divi- 
sions and a sudden cessation thereafter. 


* The monaster that arises after suppression of the first polar body with preven- 
tion of entrance of the sperm nucleus forms a special problem which we need not 
consider here. 


448 FRANK R. LILLIE 


But it is clear at least that' the maturation of the egg does not 
differ from the maturation of the spermatozoon in this respect. 
In both cases capacity for further cell-division is lost after the 
second maturation division, and it is quite natural, certainly, to 
postulate similar causes for this phenomenon in both sexes. If, 
as the results of the present study indicate, karyokinesis is the 
result of a certain qualitative nucleo-plasmic relation (to be dis- 
tinguished from R. Hertwig’s quantitative nucleo-plasmic rela- 
tion) we have to postulate in both cases a disturbance of this 
relation. And as this relation must be conceived: as a chemical 
interaction of some kind, precipitated possibly by rhythmical 
changes of permeability of the nuclear membrane, the alteration 
in question must involve either the establishment of an imperme- 
able condition of the nuclear membrane or a chemical change in 
nucleus or cytoplasm. But we have seen in Nereis, that, even 
when the membrane of the nucleus of the mature egg breaks down, 
no karyokinetic phenomena follow, unless the egg is fertilized. 
So the phenomenon of cessation of division can hardly be con- 
ceived as conditioned by the membrane alone. 

The egg-cell and the spermatid are not the only cells that lose 
the capacity for division in the course of development. In the 
course of senescence all cells lose this capacity, and studies in 
cell-lineage have shown that certain cells entirely lose the capacity 
for division in very early stages. I need cite only the case of the 
so-called turret cells in Crepidula (Conklin ’97), which are formed 
in the sixteen cell stage, and which divide only twice during the 
cleavage period. Mead has called attention to similar cases in 
his studies of cell-lineage in Annelids (Mead ’98). Cessation of 
division cannot be a problem of centrosome or no centrosome in 
such cases; nor yet in the case of the spermatid. A much more 
profound physiological cause must be involved. 

Constructive metabolism has come essentially to a standstill 
in the mature gametes; the rate of metabolism in the mature un- 
fertilized egg as tested by oxygen consumption is many times less 
than that of the fertilized egg (Warburg, ’05). Child (11) cites, 
as conditions that lower the rate of metabolism, decrease in per- 
meability, increase in density, accumulation of relatively inactive 


STUDIES OF FERTILIZATION 449 


substances, etc., but we know that constructive metabolism is 
also impossible in the absence of a nucleus, and we may conclude 
from many facts, as Conklin (712) expresses it, that ‘‘rapid and 
intimate interchange between the chromatin and the protoplasm 
is the condition of rapid metabolism and ex hypothese of rejuven- 
escence; slow interchange is the condition of slow metabolism, and 
of senescence.” It is on account of the slowness of such inter- 
change between nucleus and cytoplasm, as I believe, that the 
unfertilized egg is inhibited from development. The internal 
function of the spermatozo6n in development is to restore the 
condition of active and intimate interchange between nucleus 
and cytoplasm. Aster formation and karyokinesis are evidences 
of such restoration. The sperm nucleus and egg cytoplasm are 
immediately capable on union of such interchange, and as the 
fertilization process proceeds the egg nucleus is drawn in. 

We are led, then, to the following point of view with reference 
to the internal phenomena of fertilization, viz.: in both the sperm 
and the egg cell as the result of maturation the capacity for the 
nucleo-plasmic interaction necessary for construction metabolism 
has been lost. But such interaction takes place between the 
sperm nucleus and egg cytoplasm, and this initiates the internal 
phenomena of fertilization. ‘The egg nucleus also is drawn into 
the karyokinetic phenomena in one of two ways, either that the 
sperm nucleus has so altered the egg-cytoplasm that karyokinetic 
reaction between the egg-nucleus and its own cytoplasm can now 
follow, or that copulation of the germ nuclei results in a change in 
the egg nucleus that restores its capacity for the necessary nucleo- 
plasmic reaction. 

In his experiments on constricting fertilized eggs of the sea- 
urchin between the germ nuclei, so that the copulation of the 
latter was prevented, Ziegler (’98) has shown that the egg nucléus 
becomes surrounded by cytoplasmic..radiations which rhyth- 
mically appear and disappear synchronously with disappearance 
and reappearance of the nuclear membrane. These observations 
indicate a change produced by the sperm nucleus throughout the 
egg cytoplasm, inducing partially but not completely the rhyth- 
mical series of successive karyokinetic divisions. Other obser- 


450 FRANK R. LILLIE 


vations too numerous to mention demonstrate a very profound 
effect of the sperm nucleus on the egg cytoplasm, perhaps none 
more strikingly than Herlant’s (’11) recent observations on the 
control of definite cytoplasmic areas (spermatic energids) by sper- 
matozoa in di- and tri-spermic eggs of the frog. 

Ziegler’s observations then indicate that the egg nucleus reacts 
to the egg cytoplasm when altered by the spermatozo6n, but 
incompletely. It seems probable, therefore, that copulation of 
the germ nuclei also involves an interaction between them that 
completes the fertilization phenomena. It is interesting to note 
that, though the chromosomes form in Nereis from the egg nucleus 
after the spermatozo6n has been removed, they are not set free 
in the cytoplasm as they are after copulation with the sperm 
nucleus, but each is embedded in a matrix, and thus presents quite 
a different appearance from the normal. We may perhaps find in 
this fact, indicating lack of reaction between the chromatin and 
the cytoplasm, some evidence that the completion of fertiliza- 
tion involves interaction between the germ nuclei also. 

It remains to inquire briefly how this analysis compares with 
the analysis of fertilization given by experiments in artificial par- 
thenogenesis? In the first place we may note again that there is 
perfect agreement in the general fundamental distinction of two 
phases in the fertilization process as made first by Loeb, viz.: 
the cortical change which may be induced before penetration, 
and the internal changes, which follow penetration. As regards 
the cortical change, the view of Loeb (09) that it is essentially 
a cytolytic change appears to me less fundamental than the view 
of R.S. Lillie (11), that it is essentially an increase of permeabil- 
ity. One can readily understand that cytolysis should follow 
very rapidly on an increase of permeability induced by chemical 
means, which may be much greater than that normally induced 
by the spermatozo6n, if such increase be not secondarily regulated. 
And in any event, if interchange between the egg and its medium 
be set up by increase of permeability, in a condition of inactivity 
of the nucleus, such as exists in the unfertilized egg, the resulting 
metabolism must be of a destructive character and so lead to a 
relatively rapid death of the egg as compared with eggs in which 


STUDIES OF FERTILIZATION 451 


the cortical changes have not been induced. The conception of 
Bataillon (’10), moreover, that the egg excretes certain inhibiting 
substances contained in its cortex, as a result of the cortical 
change, is quite readily included in this point of view; indeed, 
the jelly excreted by the egg of Nereis as a result of the external 
stimulus of the spermatozo6n would obviously hinder free inter- 
change between the egg and the medium so long as it exists within 
the egg as a thick cortical layer. 

The second phase in fertilization has been treated by Loeb 
and R. 8. Lillie. Loeb’s interpretation is that the second agent 
in artificial parthenogenesis serves to check the tendency to cytoly- 
sis set up by the first agent; and he extends this point of view to 
the two phases of normal fertilization. In this opinion he is 
followed by Godlewski (711), who has shown that the cytolysis, 
which follows on fertilization of sea-urchin eggs with sperm of 
Chaetopterus, can be checked, and parthenogenetic development 
induced, by a brief treatment of such cross fertilized eggs with 
hypertonic sea-water. R. S. Lille (’11) regards ‘‘the critical 
change in the egg, to which membrane formation and the initia- 
tion of cleavage are due, as a well marked and rapid increasein 
the permeability of the plasma-membrane.’’ This tends ‘‘to 
destroy the normal osmotic equilibrium and allow abnormal dif- 
fusion of substances into and out of cells’”’ leading to derangement 
and eventual destruction of the chemical organization of the 
latter. And he regards it as an unavoidable conclusion that one 
essential effect of the after treatment with hypertonic sea-water 
is to restore the normal permeability. 

There is considerable similarity in these points of view; both 
regard the second agent jn parthenogenesis essentially as a regula- 
tory factor. Godlewski’s very striking results on the combination 
of cross fertilization and artificial parthenogenesis lead to the 
same kind of conclusion; to quote this author (711): 

Wir haben gesehen, dass weder die Kreuzbefruchtung allein, noch die 
so kurz dauernde Exposition in hypertonischer Lésung allein ausreicht, 
um die Entwicklung auszulésen. Erst durch die Kombination der 
beiden Faktoren ist der ausreichende Anstoss zur Entwicklung gegeben. 


Ich sehe in diesen Tatsachen die Bestitigung der von J. Loeb aufgestell- 
ten Hypothese, dass der Process der Entwicklungserregung sich in zwei 


452 FRANK R. LILLIE 


Momente zerlegen lisst—im ersten findet die Cytolyse, im zweiten die 
Regulierung der im ersten Akt bereits eingetretenen inneren Reaktionen 
im Elorganismus statt. 


There is nothing in the results of these authors inconsistent 
with the idea that the regulating effect of the second agent in 
artificial parthenogenesis may be attained through the re-estab- 
lishment of normal interchange between the nucleus and the cyto- 
plasm, which is: the view to which I have been led by the results 
described in this series of papers. And this suggestion contains 
the possibility of a complete accord between the results of the 
analysis of fertilization by methods of artificial parthenogenesis, 
and the more direct method of analysis contained in my papers. 

If the egg be put in a healthy metabolic state by the resump- 
tion of normal nucleo-plasmic relations following the penetration 
of the spermatozoon, it can no doubt regulate its own external 
affairs. And so it seems to me that the regulation of cortical 
permeability and cytolysis is probably a secondary effect of the 
re-establishment of normal nucleo-plasmic interchange. Even 
in artificial parthenogenesis, where the sperm nucleus is lacking, 
the action of the second agent may proceed in the same way, 
by causing the re-establishment of normal interchange between 
the egg nucleus and cytoplasm, restoring thus healthy conditions, 
which then regulate the cortical changes. But whether the tend- 
ency to cytolysis be checked thus secondarily, or by some direct 
effect of the second agent on the cytoplasm, development certainly 
cannot proceed without the establishment of normal metabolic 
interchange between the nucleus and the cytoplasm, and this must 
certainly be regarded as a fundamental function of the second 
agent in artificial parthenogenesis. 

To sum up the conclusions in a sentence we may say: the action 
of the spermatozo6n in fertilization involves two distinct phases, 
the first of which may be effected before penetration and brings 
about a sudden and marked increase in permeability of the egg- 
membrane; the second, which follows after penetration, consists 
essentially in the establishment of normal interchange between 
nucleus and cytoplasm, and consequent normal regulation of all 

* the activities of the cell. 


STUDIES OF FERTILIZATION 453 


LITERATURE CITED 


Bataituon, E. 1910 Le probléme de la fécondation circonscrit par l’imprégna- 
tion sans amphimixie et la parthénogenése traumatique. Arch. de 
Zool. Exp. et Gén., 5 Sér., Tom. 6. 


BonneEvIE, Kristine 1910 Ueber die Rolle der. Centralspindel wihrend der 
indirekten Zelltheilung. Arch. fiir. Zellforschung. Bd. 5. 


Boveri, Th. 1900 Zellen-Studien, Heft 4. Ueber die Natur der Centrosomen. 
Jena, Gustav Fischer. 


1907 Zellen-Studien. Heft 6. Die Entwicklung dispermer Seeigeeleier. 
Ein Beitrag zur Befruchtungslehre und zur Theorie des Kerns. Jena, 
Gustav Fischer. 


Cuitp, C. M. 1911 A study of senescence and rejuvenescence based on experi- 
ments with Planaria dorotocephala. Arch. Entw.-Mech., Bd. 31. 


Conkuin, E.G. 1897 The embryology of Crepidula, a contribution to the cell- 
. lineage and early development of some marine gastropods. Jour. 
Morph., vol. 13. 


1912 Cell size and nuclear size. Jour. Exp. Zodl., vol. 22. 


Danan, J. L. 1911 La fécondation chez le Paracentrotus lividus (Lam.) et le 
Psammechinus miliaris (Mill.). Comptes rend. hébd. des Sci. de 
Vacad. des Sci., Tom. 152 Paris. 


Gop.ewsk!, Emin Jun. 1911 Studien iiber die Entwicklungserregung. 1. Kom- 
bination der heterogenen Befruchtung mit der kiinstlichen Partheno- 
genese. wu. Antagonismus der Hinwirkung des Spermas von verschie- 
denen Tierklassen. Arch. Entw-Mech., Bd. 33. 


Hervant, Maurice 1911 Recherches sur les oeufs di-et-trispermiques de Gre- 
nouille. Arch. de Biol., Tom. 26. 


Hertwia, R. 1908 Ueber neue Probleme der Zellenlehre. Arch. fiir Zellfor- 
schung, Bd. 1. 


Just, Ernest E. 1912 The relation of the first cleavage plane to the entrance 
point of the sperm. Biol. Bull., vol. 22. 


Liu, F. R. 1911 Studies of fertilization in Nereis. 1. The cortical changes 
in theegg. 1. Partial fertilization. Jour. Morph., vol. 22. 


Liturz, R. S. 1911 The physiology of cell-division. 1v. The action of salt 
solutions followed by hypertonic sea water on unfertilized sea-urchin 
eggs and the réle of membranes in mitosis. Jour. Morph., vol. 22. 


Logs, J. 1909 Die chemische Entwicklungserregung des tierischen Eies. Ber- 
lin, Julius Springer. 
Meap, A.D. 1898 The rate of cell division and the function of the centrosome. 


Biol. Lectures delivered at the Mariné Biological Laboratory of Woods 
Hole, 1896-1897. Boston, Ginn and Company. 


454 ' FRANK R. LILLIE 


Meves, Frreprich 1911 Ueber die Beteiligung der Plastochondrien an der 
Befruchtung des Eies von Ascaris megalocephala. Arch. mikr. Anat., 
Bd. 76. 


191la Zum Verhalten des sogenannten Mittelstiickes des Echiniden- 
spermiums bei der Befruchtung. Anat. Anz., Bd. 40. ; 


Minor, C.S. 1908 Age, growth and death. Putnams, New York. 


Moraan, T. H. 1896 The production of artificial astrospheres. Arch. Entw.- 
Mech., Bd. 3. 


1899 The action of salt solutions on the unfertilized and fertilized eggs 
of Arbacia and of other animals. Arch. Entw.-Mech., Bd. 8. 


Ries, Jutrus 1910 Kinematographie der Befruchtung und Zelltheilung. Arch. 
mikr. Anat., Bd. 74. 


Scutcxine, A. 1903 Zur Physiologie der Befruchtung, Parthenogenese und 
Entwicklung. Arch. ges. Physiol., Bd. 97. 


WarsureG, O. 1908 Beobachtungen tiber die Oxydationen in Seeigelei.. Zeit- 
schr. fiir physiol. Chemie., Bd. 57. 


Witson, E. B. 1892 The cell-lineage of Nereis. Jour. Morph., vol. 6. 


Witscui, Emin. 1911 Ueber das Eindringen des Schwanzfadens bei der Be- 
fruchtung von Seeigeleiern. Biol. Centralb!., Bd. 31. 


ZieGuLER, H. E. 1898 Experimentelle Studien iiber die Zelltheilung. 1. Arch. 
Entw.-Mech., Bd. 16. 


DESCRIPTION OF PLATES 


All figures were drawn with the camera at stage level with Zeiss Apochromat 
1.5 mm. oil immersion objective, and No. 12 compensating ocular, except where 
otherwise stated. All figures, except 1, from sections of inseminated eggs of 
Nereis limbata. All sections from eggs killed in Meves’ fluid and stained in 
iron haematoxylin. Plates 1 to 5 illustrate the third study; 6 to 11 the fourth 
study. 


PLATE 1 


EXPLANATION OF FIGURES 


1 Spermatozoa of Nereis from preparations fixed in Gilson’s fluid and stained 
in safranin and Siure-violet. 1a, the entire spermatozoén; 1b and 1c show the 
excentric attachment of the tail to the middle piece; 1 d, basal view of the sperm- 
head showing ring-shaped middle piece and attachment of tail. h.c., head-cap; 
m.p., middle-piece; p, perforatorium. It may be noted here that the form of the 
perforatorium in the living spermatozoon is more like the spike of a helmet. 

2a,2b,2c From three eggs fifteen minutes afterinsemination. The entrance 
cone is well developed and stains dark, homogeneous. The perforatorium has 
pierced the membrane, at least in 2 a and 2c, but is not embedded in thecone. Note 
variations in appearance of the middle-piece. Tail not seen. 

3a,3b From two eggs twenty-seven minutes afterinsemination. The coneis 
retracted, but stains as before. The perforatorium stains more strongly than in 
fig. 2, but it has not yet entered the substance of the cone. The tails were very 
distinctly seen in these preparations. 

4 Thirty-four minutes afterinsemination. The perforatorium has now entered 
the substance of the cone, and granules are beginning to appear in a smaller 
lighter area of the cone surrounding its tip. 

5 a,5 6 From two eggs thirty-seven minutes after insemination. The clear 
space in the cone is now larger and the granules at the tip of the perforatorium 
more numerous; 5 a, from an almost tangential section, So that the vitelline mem- 
brane was obscured. Note thedouble perforatorium; 5 b, middle piece shows no 
granules; cf. 2 b. 


456 


FERTILIZATION IN NEREIS 
FRANK RK. LILLIE 


THE JOURNAL OF EXPERIMENTAI 


PLATE 2 


EXPLANATION OF FIGURES 


5candid From two eggs thirty-seven minutes after insemination; ef. 5 a and 
5b. 

6 Forty-eight and one-half minutes after insemination. The entrance cone 
has begun to sink into the egg, drawing the head of the spermatozoon after it. 

7 Forty-eight and one-half minutes after insemination. Later stage of pene- 
tration of the spermatozo6n. 

8 Forty-eight and one-half minutes after insemination. Still later stage of 
penetration. 

9aand9b. Two drawings of the same section; 9 a drawn with Zeiss 2 mm. 
apochromatic objective and no. 4 compensating ocular; 9b, the part of 9 a contain- 
ing the spermatozoon, drawn with Zeiss 1.5 mm. apochromatic objective and no. 
12 compensating ocular. Fifty-four minutes afterinsemination. The sperm head 
has completed its penetration, and its base is some distance from the periphery; 
but the middle-piece remains outside. Anaphase of the first maturation division. 


FERTILIZATION IN NEREIS 
FRANK R, LILLIE PLATE 2 


THE JOURNAL OF EXVERIMENTAI OOLOGY, VOL. 1 N 4 


PLATE 3 


EXPLANATION OF FIGURES 


10a, 106,10c,10d Four drawings from the same section; 10a drawn with Zeiss 
2 mm. apochromatic objective and No.6 compensating ocular; 106, 10 c, 10d 
drawn with Zeiss 2mm. apochromat and no. 12 compensating ocular; 10 6 is the 
sperm mucleus of the same egg; 10 ¢ and 10 d are from the following sections to 
show the entrance point with tail and middle piece on the membrane. Fifty-four 
minutes after insemination. Rotation of the sperm head and cone, and origin of 
the sperm aster from the pole of the nucleus opposite to the cone. Prophase of 
the second maturation division. 

11 Sixty-four minutes after insemination. The sperm head has penetrated 
within the layer of yolk, and has separated from the cone, which does not appear 
after this stage. In the next section another sperm centrosome and aster appear; 
the sperm centrosome has divided. 

12 Sixty-seven minutes after insemination. Sperm amphiaster with equal 
poles; spindle formation beginning between egg and sperm centrosomes. Ana- 
phase of second maturation spindle. Drawn at stage level with Zeiss 2 mm. 
apochromatic objective and compensating ocular no. 6. 


460 


THE 


FERTILIZATION IN NEREIS 
FRANK kK. LILLIE 


JOURNAL OF EXVERIMENTAL. Zoi 


£6 1 


PLATE 4 


EXPLANATION OF FIGURES 


13 Sixty-seven minutes after insemination. Later stage of sperm amphiaster 
with unequal centrosomes and asters. Double spindle formation between egg 
center and sperm centers. Drawn at stage level with Zeiss 2mm. apochromat 
objective and compensating ocular no. 6. 

14aand14b Seventy-seven minutes after insemination. Two successive sec- 
tions of the same egg. The second polar body is just formed. The egg aster is 
beginning to degenerate. The sperm asters are also less developed than pre- 
viously, especially in the case of the smaller one. Sperm amphiaster in 14 a; 
sperm nucleus in 146. Drawn at stage level with Zeiss 2mm. apochromat objec- 
tive and compensating ocular no. 6. 


462 


FERTILIZATION IN NEREIS 
FRANK R. LILLIE 


PLATE 4 


THE JOURNAL OF EXPERIMENTAL. ZOOLOGY. VOL. 12 No, 4 


PLATE 5 


EXPLANATION OF FIGURES 


15 Seventy-seven minutes after insemination. The two nuclei to the right 
above are parts of the egg nucleus not yet fused together. The sperm nucleus to 
the left below. The aster is the larger sperm aster; the smaller one could not be 
found. Drawn at stage level with Zeiss 2mm. apochromat objective and compen- 
sating ocular no. 6. 

16aand16b Twosuccessive sections of the germ nuclei, seventy-seven minutes 
after insemination. There is but a single aster derived from the larger sperm 
aster. Note the black granules in the neighborhood of the germ nuclei and aster. 
Drawn at stage level with Zeiss 2mm. apochromat objective and compensating 
ocular no. 6. 

17 Origin of the cleavage centers. The partition between the germ nuclei 
has disappeared. The larger aster is derived from the larger sperm aster. Drawn 
at stage level with Zeiss 2mm. apochromat objective and compensating oculer 
no. 6. 


464 


FERTILIZATION IN NEREIS 
FRANK R. LILLIE 


PLATE 5 


THE JOURNAL OF EX!VERIMENTAL. 20% > vo 12, n 4 


PLATE 6 


EXPLANATION OF FIGURES 


18to25 Show the effects of removal of the jelly by centrifuging on the spermato- 
zoon attached to the egg. It willbe noticed that the cone in this and the following 
plates, though just as well defined as the preceding, stains differently. The 
behavior of the cone is the same, however. 

18 Entire spermatozoén present, drawn out to band. History: centrifuged 
7200 revolution in thirty-five seconds, fifty minutes after insemination; preserved 
immediately. 

19 The middle-piece and part of the base of the spermatozoén have been 
removed by the jelly. The protoplasm surrounding the cone has been raised in a 
protuberance, which happens not infrequently (cf. fig. 22). History same as fig. 
18. 

20to23 These figures show removal of increasingly large portions of the sperm 
head by the jelly. Each drawing from a single section of aseparateegg. History 
same as fig. 18. 

24to25 To show effects of removal of jelly by centrifuging after penetration has 
begun. History same as fig. 18. 


466 


FERTILIZATION 


FRANK 


IN 


K. 


NEREIS 
LILLIE 


ATI 


THE 


PLATE 7 


EXPLANATION OF FIGURES 


26 Removal of external part of the spermatozoon by centrifugingin an advanced 
stage of penetration (cf. fig. 7). History: centrifuged 7200 revolutions in forty 
seconds, fifty minutes after insemination. Preserved fifteen minutes later. 

27to31 Toshow early penetration of injured or partialspermatozoa. History: 
centrifuged 7200 revolutions in forty seconds, fifty minutes after insemination. 
Preserved fifteen minutes later. 

27 Part of the spermatozoon has entered. The remainder is shown external to 
themembrane. The internal part is definitely dividedintwo. Rotationis begin- 
ning. 

28 The part of the spermatozoén external to the membrane is nearly separated 
from the internal part, which is itself definitely divided in two. All parts a little 
swollen as shown by the tone of the stain. The rotation of the cone is beginning. 

29 The internal part is apparently breaking off from the much larger external 
part of the spermatozoén. Cone in process of rotation. 

30 A case in which only a small part of the spermatozoén has entered; the rest 
of the spermatozoon is lost. It represents alater stage of an injury similar to that 
shown in figs. 20 or 21. ‘ 

31 A case similar to fig. 30. 

32 A somewhat later state of rotation of the cone than shown in preceding fig- 
ures. History the same. 

33aand33b Twosuccessive sections of the same egg; the parts of the spermato- 
zoon shown in the two sections are entirely separate. The proximal larger part 
(33 a) is proceeding with its rotation and development, leaving the base of the 
sperm head and the middle piece behind. History same as figs. 27-31. 

34 Penetration stage of a sperm remnant preserved fifteen minutes after centri- 
fuging. 

40 Two partial sperm nuclei with asters associated with a single cone. Prob- 
ably a later stage of a condition like that shown in figs. 27 or 28. 


468 


FERTILIZATION IN NEREIS 
FRANK RK, LILLIE PLATE 7 


26 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No, 4. Kenji Toda, Del. 


469 


PLATE 8 


EXPLANATION OF FIGURES 


35to 87 To show origin of asters in connection with partial sperm nuclei. In 
each case a remnant of the spermatozo6én on the surface guarantees the partial 
nature of these sperm nuclei; note the variation in size. History: Centrifuged 
7200 revolutions in forty seconds, fifty minutes after insemination; preserved fif- 
teen minutes later. Fig. 36 is a combination of three sections. 


470 


sz 


FERTILIZATION IN NEREIS 


FRANK R. LILLIE PILATE 8 


Kenji Toda, Del 


vou. 1%, No. 4. 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, 


PLATE 9 


EXPLANATION OF FIGURES 


38 and 39 Two unusually small sperm nuclei with their asters, separating from 
the cones. From two eggs;compare the size of the entire sperm nucleus at this 
stage (fig. 11). History same as figs. 35 to 37. 

41 Two sperm nuclei of unequal size from the same egg. Compare size of cen- 
trosomes and asters. History: Centrifuged 7200 revolutions in thirty-six seconds, 
forty-four minutes after insemination; preserved ninety-two minutes after insemi- 
nation. Reconstruction of three sections. 


472 


NEREIS 


LILLIE 


IN 


RK, 


RTILIZATION 


FE 


FRANK 


K 


\ 


OF EXPERIMENTAL. ZOOLOGY 


JOURNAL 


THE 


PLATE 10 


EXPLANATION OF FIGURES 


42 Five successive sections showing the entire germ nuclei of one egg. The 
male nucleus has five nucleoli, the female has thirteen. The line of apposition of 
the germ nuclei is seen in the third section. History: Centrifuged 7200 revolu- 
tions in thirty-six seconds, forty-four minutes after insemination. Preserved 
sixty-four minutes later. 


474 


FERTILIZATION IN NEREIS 
FKANK K. LILLIE PLATE 10 


THE JOUKNAL OF EXPERIMENTAL. ZOOLOGY, vot. 12, No. 4 Kenji Toda, Del 


PLATE 11 


EXPLANATION OF FIGURES 


43 Three successive sections showing the entire egg nucleus of an egg from 
which the spermatozoon was entirely removed by centrifuging. Both polar bodies 
formed. Fourteen chromosomes indicated. History: Centrifuged 7200 revolu- 
tions in thirty-seven seconds, forty-two minutes after insemination. Preserved 
sixty-five minutes later. 

44 Three successive sections showing the entire egg nucleus of an egg from 
which the spermatozoén was entirely removed by centrifuging. The first polar 
body was not formed in this case, and a monaster arises around the egg-chromo- 
some group. History same as fig. 43. 


476 


FERTILIZATION IN NEREIS 
FRANK K. LILLIE 
43a 
43h 
43¢ 


THE JOURNAL OF EXPERIMENTAI 


ZOOLOGY, 


PILATE 11 


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44h, 


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THE ELIMINATION OF THE SEX CHROMOSOMES 
FROM THE MALE-PRODUCING EGGS 
OF PHYLLOXERANS 


T. H. MORGAN 


From the Zoélogical Laboratory, Columbia University 
TWENTY-NINE FIGURES 


My studies of the life cycle of the phylloxerans of the hickories 
have shown first ('08) why the fertilized eggs produce only 
females,! and second (’09) that the production of the males is 
caused by the elimination of a chromosome from the male-pro- 
ducing egg.2 One essential point in the life eycle still remained 
unexplained; namely, the cause of the production of small male- 
and large female-producing eggs. The differentiation of these 
two kinds of eggs precedes, in the life cycle, the formation of 
the true males and sexual females. Jt may appear therefore 
that the question of the sex determination antedates those changes 
that lead to the elimination of a chromosome from the male-produc- 
ing egg, and, uf so, the real question of sex determination might 
seem to lie deeper than the manewvres of the sex chromosomes. 
Until this point is cleared up the value of the chromosome 
hypothesis in sex determination may seem to hang in the balance. 

IT am now able to bring forward certain evidence which I believe 
throws light on this important topic and I am prepared to offer 
an hypothesis based on the new evidence, which, if true, substan- 
tiates the view that one of the essential changes in the formation 
of the large and the small eggs is connected with changes in the 
sex chromosomes. 


1 Proc. Soc. Exp. Biology and Medicine, vol. 5, 1908, and Science, vol. 29, 1909. 
? Proc. Soc. Exp. Biology and Medicine, vol. 7, 1910. 
479 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 4 


480 T. H. MORGAN 


The main points that were described in my previous papers 
may be summarized as follows: 

1. Two classes of sperm are produced in the male differing in 
the presence and absence of a pair of chromosomes. One class of 
sperm degenerates. It corresponds to the male-producing class 
of otherinsects. The other class produces functional spermatoza 
which entering the egg give rise to females only. These sperm 
correspond to the female-producing class of other insects. 

2. The male-producing egg contains one less chromosome after 
the extrusion of its single polar body than it contained before 
this event. In a preliminary note (710) I have stated how this 
elimination takes place and in the present paper I bring forward 
the evidence on which this statement was based. 

3. The difference in size between the male-producing and 
the female-producing egg, before the former has extruded its 
polar body, proves that the predetermination of the males ante- 
dates the extrusion of the chromosome in the polar body of the 
(smaller) male-producing egg. 

4. More male-producing individuals are the descendants of 
each stem-mother than female-producing individuals. The stem- 
mother must give rise to two kinds of eggs, i. e., they must be 
different either before or after the polar body is extruded. The 
factor that differentiates these two kinds of eggs, was not dis- 
covered. It is this point that the evidence now brought forward 
may I hope help to elucidate. 


THE DIFFERENCES IN THE CHROMOSOME GROUPS IN THE POLAR 
SPINDLES OF THE STEM-MOTHER’S EGG AND OF THE 
MALE AND FEMALE-PRODUCING EGGS 


In my former paper (’09) I have figured ten equatorial plates 
of polar spindles of the eggs produced by the stem-mother. In 
all of these the sex chromosomes are of nearly the same size. In 
two other plates one chromosome is much smaller than the others, 
which is probably due to this chromosome having been cut by 
the knife. Failure to find the missing piece in the next section 
would not be significant, since it might be very difficult to find 
such a piece in the egg filled with yolk granules of about the same 


ELIMINATION OF SEX CHROMOSOMES 481 


size as the chromosomes. Side views of the polar spindle, of 
which four are given, each with three chromosomes, show these 
to be of equal size. One of two cases of an egg nucleus (just 
prior to the formation of the spindle) also shows six equal chro- 
mosomes; the other case shows four equal chromosomes and 
one of double size that no doubt-represents two chromosomes 
overlapping or else stuck together. 

When these chromosome plates are compared with those 
of the female-producing egg shown in fig. LX, page 255 of my 
paper (’09), the size relations seem to be about the same. Only 
four plates of these eggs were found. I suspect that one of the 
equatorial plates that is assigned to a male-producing egg, 
namely, fig. LX; K, really belongs to a female-producing egg. 
Occasionally it is difficult, owing to the obliquenessof the sections, 
to make sure that a particular egg is a large or a small one. 
If this egg is excluded, or referred to the female-producing series, 
there remain fourteen equatorial plates of male-producing eggs. 
In all of them one chromosome is noticeably smaller than the 
rest. I can now add four more chromosome groups of male-pro- 
ducing eggs to this list, figs. 1, 2,3,4. Three of these are equa- 
torial plates or just prior to that phase, and the fourth shows 
the chromosomes in the nucleus just prior to the spindle stage. 
They all contain five larger chromosomes, and one much smaller 
than the other five. 

It is true that there is some variation in the relative size of 
the chromosomes in all of these figures, which makes it difficult 
to express exactly the relative sizes of the different chromosomes, 
and therefore I am aware of the danger of attempting to distin- 
guish between the plates of the male- and female-producing eggs; 
yet the presence of the very small chromosome is so distinctive 
of the smaller eggs that I believe no error is committed in attribut- 
ing to this difference at least a real significance. 

If the two kinds of chromosomal groups just specified are 
significant one should expect to find a similar difference in the 
somatic cells of the individuals that give rise to these eggs, for 
since each of these individuals produces only one kind of egg 
(all the eggs found in one individual are male-producing or female- 


482 T. H. MORGAN 


producing) this difference should be apparent on inspection 
of the somatic cells of these individuals. In my paper I have, 
in fact, given nine plates taken from young stages of the devel- 
opment of the embryo. Some of these figures, notably fig. VLII 
C, E, I, show six nearly equal chromosomes, while five of them, 
notably fig. VIII A, B, D, F, G show five larger and one smaller 
chromosomes. When these drawings were made the importance 


Eee Tart ee SS 


i, 3 


of the size relations was not appreciated, and the number of 
cases is too small to be of great value, but it is significant, I think, 
that the two kinds of chromosome groups required by the hypoth- 
esis are actually represented in these figures. 

It appears, then, probable that after the extrusion of the polar 
body of the egg of the stem-mother a change has taken place in 
those individuals that become the male-egg-producers. One 
chromosome has become smaller. 


ELIMINATION OF SEX CHROMOSOMES 483 


THE DIVISION OF THE POLAR SPINDLE IN THE MALE- AND 
IN THE FEMALE-PRODUCING EGGS 


A brief abstract of the results given in this section was pub- 
lished in the Proceedings of the Society of Experimental Biology 
and’ Medicine? for May, 1910. In order to study the division 
of the polar spindle a large amount of new material was collected 
in the summer of 1909 which was cut and studied during the 
following winter. It has been most laborious to find eggs in 
which the polar spindle was in the process of division, and I 
wish to express my obligations to my assistant, Miss E. M. Wal- 
lace, who has found most of the new cases here figured. 

In my former paper (’09) I described the anaphase of two 
eggs that seemed to be female eggs (see below), but none of the 
male eggs; and it is the latter that would be expected to give the 
critical evidence. This evidence was briefly stated in my prelim- 
inarynotein 1910. I shall nowgive drawings of several anaphases 
of male eggs that show beyond doubt that a lagging chromosome 
is present; that it passes to the outer pole, and forms a separate 
vesicle in the polar body. 

The first case is shown in fig. 5 representing an anaphase of 
the polar spindle. Five chromosomes lie at the outer pole and 
five at the inner pole. In the middle of the spindle lies a double 
chromosome. It is relatively large and its two halves appear 
somewhat unequal. For reasons that appear later I shall speak 
of this as a single chromosome that has already divided into 
halves. 

The second ease is shown in fig. 6. Here also five chromosomes, 
somewhat elongated, lie at the outer pole and five at the inner 
pole of the spindle. ‘In the middle of the spindle there is a 
double chromosome, its halves equal as far as can be determined. 

The third case is shown in fig. 7. It represents a later stage; 
the polar body being in process of constricting from the egg. 
The group of chromosomes at the outer pole is now in process 
of division. Five chromosomes can be recognized, two dividing, 
and three having completed their division. At the inner pole 


‘Proc. Soc. Exp. Biology and Medicine, vol. 7, 1910. 


484 T. H. MORGAN 


ELIMINATION OF SEX CHROMOSOMES 485 


the nucleus has begun to form. It contains five distinct chro- 
mosomes. Midway between the poles is the lagging chromosome 
completely divided into equal or nearly equal parts. A few 
traces of the spindle fibers are discernible. This is the clearest 
case that I have found and shows very distinctly the conditions 
at this stage. 

The next case, fig. 8, is not so instructive, since the chromo- 
somes in the inner nucleus have in one place seemingly stuck 
together so that only four bodies are seen. The lagging chro- 
mosome could not be found but the five outer chromosomes are 
distinct. 

Fig. 9 shows the polar body nearly constricted off. The five 
inner chromosomes are clearly seen. The lagging chromosomes 
were not found and may have been fused with the lump of chro- 
matin‘in the polar body that represents the massed chromosomes. 
‘ In fig. 10 (and in figs. 9, 11, 12 and 13) the egg nucleus is 
represented nearer the surface than in the actual section. The 
nucleus of the polar body contains a fused mass of chromatin. 
What appears to be the lagging chromosome lies on its outer 
wall, and is partially constricted into halves. The inner nucleus 
shows five equal or nearly equal chromosomes. As this is the 
only case observed where the lagging chromosome lies on the 
outer wall of the nucleus of the polar body, and as it is difficult 
to see how the chromosome could have reached this position; and 
moreover since the double body is smaller than the lagging chro- 
mosome in the other cases; it may be that this deeply staining 
body is not the lagging chromosome at all but a pair of displaced 
yolk granules. This interpretation is supported by the next case. 

In this instance fig. 11 the inner nucleus is well formed and 
its chromosomes diffused or at least not stained. In the polar 
body there is a nucleus in which four chromosomes can be made 
out with the double lagging chromosome lying on the inner side 
of the nucleus. <A yolk granule lies on the outer wall of the 
nucleus. 

A similar stage is shown in the next figure, fig. 12. The inner, 
or egg nucleus shows its contained chromosomes in process of 


486 T. H. MORGAN 


becoming diffuse. The polar body is cut off from the egg; its 
nucleus contains five chromosomes, and lying near the nucleus 
is the large lagging chromosome divided into two parts. 

In the next figure, fig. 13, the chromosomes, both in the polar 
body and in the egg nucleus, have fused. The preservation may 
have been poor. The lagging chromosome lies in a vesicle of 
its own to one side of the polar body nucleus. 

The polar body of another egg, fig. 14, shows five chromo- 
somes in its nucleus, three of which at least are elongated as 
though dividing. The lagging chromosome lies outside. Its two 
halves are separated and each half is slightly dumb-bell shaped. 

Three other polar bodies are shown in figs. 15, 16, and 17. 
Each shows the lagging chromosome outside of the nucleus; and 
in two cases surrounded by a partial vacuole of its own. 

In addition to these new cases I have studied and redrawn 
some of the figures given in myformer paper, restaining when neces- 
sary to better bring out the chromosomes. One of these, fig. 18, 
shows the equatorial plate of a male egg with one large chro- 
mosome (partly constricted), four intermediate, and one small 
chromosome. Two eases, figs. 19, 20, show polar bodies and 
their contained nucleus and the lagging chromosome outside. 
The third figure, fig. 21, shows the anaphase of an egg that I 
now interpret asa male egg. The interpretation of this anaphase 
figure is difficult because of the presence of a stained body near 
the center of the spindle. After staining and restaining several 
times it seems to me probable that this body is in reality a chro- 
mosome and not a yolk sphere as I formerly thought probable. 
Another sphere lies beyond the outer chromosome group and 
near it another body (in outline in the figure). Both of these 
seem to be in yolk spheres. At the inner pole five distinet chro- 
mosomes are present. If the stained body in the center of the 
spindle be interpreted as a chromosome the spindle bears a close 
resemblance to the spindle of the male-producing egg, but the 
egg is large, and mainly for this reason I was formerly inclined 
to think it a female egg. The case is doubtful and can not be 
interpreted with certainty. 


ELIMINATION OF SEX CHROMOSOMES 487 


I have tried to find again all the polar body stages figured in 
my former paper in order to reéxamine them, but it has not been 
possible to discover several of them owing to the fading of the 
stain. I wished especially to reéxamine figs. XY; N, P, Q, which 
show the lagging chromosomes passing into the polar bodies, 
but have not succeeded in finding them again. All three rep- 
resent the same spindle under different conditions. While these 
figures were drawn with as much care as possible I now realize 
that some of them might receive a clearer interpretation in the 
light of the information that I have gained from a new study 
of the polar spindle. One point is especially clear that in most 
cases the circle enclosing the chromosomes of the polar bodies 
of the male-producing egg was drawn so as to include the lagging 
chromosome, while in reality the ordinary chromosomes lie in 
one vesicle and the accessory in another clear area at the side 
Gtieune latters eehus im fies, X- C0, G Lf, £, M, RR, S, 7, U, the 
sixth chromosome generally shown as bifid represents the lagging 
chromosome. 

In the new preparations I have found only one anaphase of 
a female-producing egg, fig. 22. Fortunately this is a very clear 
case..- Six chromosomes lie at each pole, and there is no lagging 
chromosome present. Three of the outer chromosomes are 
dumb-bell shaped. The only other cases of this kind of egg are 
the doubtful case, here refigured, fig.21, and the outer pole shown 
in my former paper in fig. XY; K. 


Summary 


The evidence shows that four of the five chromosomes of 
the male-producing egg divide equally when the polar body is 
formed. One chromosome lies in the middle of the spindle and 
becomes divided into equal parts. It finally passes out into 
the polar body, just before the latter cuts off, and fails in con- 
sequence to become incorporated in the nucleus of the polar body. 
In all these respects its behavior is closely similar to that of the 
lagging chromosome described by McClung for this chromosome 
in the spermatocytes of orthoptera. 


488 T. H. MORGAN 


The six chromosomes of the female-producing egg appear to 
divide equally, so that the outer and the inner pole of the spin- 
dle get six chromosomes each. 

One point of especial importance I have not been able to settle 
satisfactorily, namely, the fate of the smallest chromosome. The- 
oretically I should expect it to be the lagging chromosome. But 
the figures show the lagging as large as the rest. On the other 
hand it is equally clear that none of the others can be identified 
as the smallest—they appear to be of equal size. The evidence 
is therefore inconclusive either way. If the smallest is the lagging 
it must increase in size before it divides so that its size relations 
are changed. 


THE POLAR SPINDLES IN THE SEXUAL EGG 


Two questions of theoretical interest are involved in the antic- 
ipated reduction in the number of chromosomes in the sexual 
egg of the phylloxerans, namely, the number of the reduced 
chromosomes and their size relations. Whether the reduced 
number would be four or three could not be prophesied from 
the behavior of the chromosomes in the parthenogenetic series, 
for if, as I think, there ate two small 2-chromosomes attached 
to the two large X’s, the former might separate at the ‘reduction 
period’ to form a smaller pair, giving four chromosomes or else 
remaining attached to‘the larger X there would appear only 
three chromosomes. Six cases have been found showing clearly 
that the number of chromosomes in the sexual egg is three. I 
have found two eggs that show equatorial plates, figs. 23, 26; 
two eggs that show the chromosomes in the nucleus just before 
its resolution, figs. 27, 28; and two eggs that show side views 
figs. 24,25. There can be no question that the reduced number 
is three. The size relations are more difficult to determine. 
In general it may be said that they are all of nearly the same 
size, although one of them generally appears larger than the other 
two. There is no such disparity in size between the largest 
and the smallest as that observed in the male-producing egg, 
which is a strong argument against my earlier suggestion that 
the smallest chromosome in the equatorial plate of the male- 


ELIMINATION OF SEX CHROMOSOMES 489 


producing egg is formed by the union of the two small z’s that 
leave their larger partners at this time and fuse in order that 
‘reduction’ may occur. For, should this happen in the male- 
producing egg in order to insure the separation of the two small 


a ———— 
+. = i a aa 


ge fe iw 


23 
24 25 


eo —— 
@ 


8 as 


26 


28 


27 


x’s one might anticipate a similar change in the sexual egg. We 
must conclude, therefore, that one of the pairs in the sexual 
egg represents the fusion of the two large X’s and their attached 
small 2’s. One large X and one small x would therefore be left 


490 T. H. MORGAN 


in the egg. The female-producing sperm brings in during ferti- 
lization a large X and a small x which brings the number back 
to the six chromosomes (in reality eight since two are double, 
X and x) of the stem-mother’s soma. 


THEORETICAL INTERPRETATION OF THE RESULTS 


From the results given in preceding sections I draw the follow- 
ing conclusions. The cells of the stem-mother (that comes from 
the fertilized egg) contain six equal or nearly equal chromosomes. 
Two of these, that I call X, have attached to them two smaller 
chromosomes that I call small «. The stem-mother’s cells have 
therefore in reality eight chromosomes. The eggs produced 
by the stem-mother also contain these six (or eight) chromosomes 
that appear in the equatorial plate of the polar body. One polar 
body is extruded. The division of the chromosomes has not 
been observed. I assume that at this time all of the chromosomes 
divide equally, except in the case of those eggs that will become 
male-ege producers. In these eggs one of the small 2’s passes 
undivided into the polar body. Presumably it passes out at- 
tached to the outgoing half of the larger X, with which it has been 
fused. Unless it separated from the large X it might not 
appear as a lagging chromosome at the time; if it became detached 
it might appear as a lagging chromosome; or both the large out- 
going half of the large X with its attached small 2 might lag behind 
the rest. Further work will be necessary to settle this point. 
This kind of egg, after the polar body is extruded, will contain 
six chromosomes, one having been reduced in size by the loss of 
the small z. This group appears in the equatorial plate of the 
polar spindle of the small ‘male’ egg. The difference in the size 
relations of the chromosomes observed in the polar spindle of 
this egg, as compared with the size relations of the chromosomes 
in the stem-mother’s egg is accounted for by the loss of one chro- 
mosome—the small x. If eight chromosomes are present in the 
stem-mother’s cells and eggs there are only seven in the body 
cells and in the eggs of the male-producers. When the polar 
body of the male-producing egg is formed all the chromosomes 
divide except one, which, lagging on the spindle, finally passes 


ELIMINATION OF SEX CHROMOSOMES 49] 


Cluonvosomes of Stem 


mothers frclar shurtdle 


Two Types (A.B) of divis- 
ton of above To produce the 


female line Aand the male linen 


Polar spindle of femate 
preducing egg, Ay and male 


Preducing egg. B 


Diyiston of last 


Somatic Calls of female 


A and male B 


Reduced numler of 
Chiemeserus an Sexual GOA; 


and anv Sfevmatocyte B 


Duister of Pelaw spindle 
ww Sexual tqq A Furst 


duisen ef Spermalecyte B 


« 
a 
2° @ 


0-000 ~~ " ©0002 000 
00006 ~~ c02000 


seo .° 
dl did 0.@ 


DIAGRAM 1 


492 T. H. MORGAN 


out bodily into the polar body dividing at this time as do 
also the members of the outer group of chromosomes. The 
result is that five visible chromosomes remain in the male egg, 
in reality six chromosomes, since the large X and the small « 
are attached to each other. In the body cells of the male these 
two X’s often remain united, but sometimes partially separate. 
When reduction takes place three visible chromosomes are pres- 
ent in the spermatocytes (one of these three is the fused pair). 
Two of these divide equally at the first division, and one (the 
fused pair) lags behind and finally passes to the female-producing 
sperm. ‘Toward the end of this division the large X and the 
small x, not infrequently partially or even completely separate. 
In the second spermatocyte division all the chromosomes of 
the female-producing cell divide equally, giving rise to two func- 
tional sperm containing three visible chromosomes, or four in 
reality since one is double. The class of cells without the X’s 
degenerate. ‘ 
Returning again to the stem-mother in order to trace the history 
of the female line I assume that when the polar spindle divides 
all of the eight chromosomes divide, leaving eight in the egg (six 
visible) of which there are two pairs, each containing a large X 
and its attached small z. The eggs develop into the female 
producers, whose polar spindles contain six equal chromosomes 
like those of the polar spindle of the stem-mother. The larger 
number of chromosomes in the female-producing egg accounts 
for the larger size of the egg, as compared with the male-pro- 
ducing egg, which, as shown above, contains one less chromosome 
(the small 2). When the polar body is set free the chromosomes 
divide equally, six (in reality eight) passing out, and six (or eight) 
remaining in the egg. The body cells of the sexual female con- 
tain therefore six (eight) chromosomes. A reduction division 
occurs in the sexual eggs, so that three (in reality four) chromo- 
somes appear. When the polar bodies are formed—there are 
two of them to judge by analogy with the aphids—three whole 
chromosomes (in reality four) are given off at one of the divisions 
so that three (in reality four) remain in the egg. The female- 
producing spermatozoon introduces into this egg during fertili- 


ELIMINATION OF SEX CHROMOSOMES 493 


zation the same chromosome group, which brings back the number 
of chromosomes to that characteristic of the stem-mother, and 
starts the same cycle again in the next year. 

The attachment of the two small x’s to the two large X’s, that 
is assumed to occur throughout this series, except when one of 
the small «’s is supposedly lost in the male-producing line of the 
stem-mother’s egg, and the loss of one large X when the male egg 
extrudes its polar body, may seem to be the most doubtful points 
in the preceding account. That a small x is actually present is 
shown clearly in the spermatogenesis, and in some of the somatic 
cells of the male. It is, therefore, highly probable that the other 
large X, found in the stem-mother and female line, has also a small 
xattached. Otherwise asymmetrical distribution of the chromo- 
somes can not take place. But my assumption that one small 
x is eliminated from those eggs of the stem-mother that give 
rise to the male line may appear more problematical. I readily 
grant that this is hypothetical. There are two facts, however, 
that give the hypothesis some probability. First, by means 
of this hypothesis the change in observed size-relations that takes 
place in the chromosome group of the male-producing egg can 
be accounted for. Second, the apparent absence of the small 
chromosome, in the lagging chromosome of the polar spindle of 
the male-producing egg, supports this view. On the basis of 
these two observations I have ventured to offer the above hypoth- 
esis, especially as it seems to give a consistent view of the changes 
that take place at the most critical stage in the life cycle when 
two lines are produced. In my former paper I have pointed 
out that there is no external condition that appears adequate to 
account for this dichotomy, and, if this is correct, we are warranted 
in looking for an internal factor that produces the result. The 
assumption moreover is in accord with the view, now well estab- 
lished, that the production of males is associated with the absence 
of certain chromatin in the egg. From this point of view the 
male-egg-producer—the winged migrant—is half a step towards 
the production of a male; the final step is taken when the other 
X is eliminated, which demonstrably occurs at the next stage 
when the polar body of the male egg is eliminated. 


494 T. H. MORGAN 


That an XY chromosome may be present attached to another 
chromosome has been shown in recent years by Boveri, Boring 
and Gulick in several species of Ascaris. The same looseness 
of attachment that I have observed has also been found in the 
eggs of Ascaris where in certain individuals and at certain times 
a separation has been recorded, while in other individuals the X 
chromosome remains completely united with its larger compan- 
ion. In my own ease the attachment is between two X chromo- 
somes while in the other cases the attachment is between an XY 
and what is apparently an ordinary chromosome. 


OTHER POSSIBLE INTERPRETATIONS CONSIDERED 


In my former paper when dealing with the differences in the 
chromosomal groups in the equatorial plates of the polar spindles 
of the male- and female-producing eggs I have suggested that 
the change could be accounted for if in the male egg the two small- 
est chromosomes (the two small x’s) each left its larger partner 
and fused together while the two larger also fused. This would 
give the same actual number as before, but a relative difference 
in size would result. The logical conclusion from the assumption 
would be that when the polar bodies are given off the large X’s 
separate (reduction division for the pair) and the small 2’s 
also separate, the other chromosomes dividing equationally. This 
assumption was necessary for the large X and the small x pair 
because in the spermatogenesis a large Y and a small x are still 
present. 

If this view were correct the lagging chromosomes in the ana- 
phase of the male egg should consist of a large and a small chro- 
mosome. The facts show that two chromosomes do actually 
lag but unfortunately for the assumption they are equal and rel- 
atively large. The doubleness of this lagging body can be better 
explained by a precocious division into equal parts, since the 
other chromosomes that pass out into the polar body also show 
signs of division at this time. It is clear that my former inter- 
pretation must be abandoned. 

There is another interpretation that might be considered, namely, 
that the lagging body is really a large Y and small x closely united 


ELIMINATION OF SEX CHROMOSOMES 495 


and the division is equal in both as in the other chromosomes 
that pass out. This would leave a similar pair (large X and 
small x) in the male-producing egg which becomes the lagging 
pair in the spermatogenesis. This hypothesis works out con- 
sistently but it leaves unexplained the observed size differences 
that appear in the chromosome groups of the male egg; it also 
leaves unaccounted for the production of the smaller male egg 
and it ‘explains away’ the observed size relations in the lagging 
chromosomes of the male egg: Hence I think this view must 
also be put aside. 

Again we might assume that the large X is the sex chromosome 
and the small chromosome attached to it (its synaptic mate) 
is in reality not an X at all, but a Y chromosome. Were this 
the case the Y should pass into the male-producing sperm since 
this is the characteristic behavior of Y in other insects. As it 
does not do so there is no basis of fact to support such an inter- 
pretation. 

Lastly, one may ask whether the two large X’s in the stem- 
mother’s egg are of the same size and also whether the two small 
x’s present are of the same size. Assume for instance that the 
two smaller x’s are unequal in size. If the larger of them should 
pass out into the polar body of the stem-mother’s egg the egg might 
become a male-egg-producer, if the smaller passes out the egg 
might become a female-egg-producer. In this way the two 
lines become differentiated. But this would leave in the female 
line two X’s and one small a (the larger one). We should have 
to assume then that the sexual egg eliminates one large X and 
retains the other large X and the other x (its companion). In 
other words a second differential division must be assumed. In 
the absence of evidence we are scarcely justified in making two 
such assumptions. Moreover, if this view were correct we should 
expect to find a chromosome group in the polar spindle of the 
female producing egg like that in the male-producing egg, but this 
is what we do not find. Of course if the larger of the small 2's 
that is assumed to be left in the female-egg-producer were much 
larger than that left in the male-egg-producer the size differences 
might not be so marked, but until this ean be established we are 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, No. 4 


496 T, H. MORGAN 


scarcely justified inmaking thisassumption even although by doing 
so we may seem to give an attractive explanation of the splitting 
into the male and female lines. 

Other combinations will suggest themselves but offer no advan- 
tages I believe over the one that I have suggested. 


COMPARISON BETWEEN PHYLLOXERA CARYAECAULIS AND 
PHYLLOXERA FALLAX 


It may be worth while to compare briefly the conclusions 
reached for P. caryaecaulis with the results described in my former 
paper for another species, P. fallax. The latter has twelve chro- 
mosomes in the equatorial plate of the egg laid by the stem- 
mother. After the extrusion of the polar bodies I have described 
twelve chromosomes as the number for the cells of the embryo. 
If as in P. caryaecaulis there are two pairs of X’s present that 
are united this number would still be found, but if this view were 
correct for the male line we should expect to find six in the male 
spermatocytes of which one is double. Five should divide equally 
and the double one should Jag with or without showing its double- 
ness at this time. In reality only four divide equally and two 
whole chromosomes that were separate in the equatorial plate 
of the spermatocyte come near together and become the lagging 
chromosomes of this division. There can be no question of their 
relations in the spermatocytes since the chromosomes are per- 
feetly distinct and hundreds of such stages have been studied. 
It seems necessary therefore to recast this first view and to reéx- 
amine the facts. In fig. Il; A-F of my paper six chromosome 
groups of polar spindles of male- and female- producing eggs are 
drawn. Of these groups three show twelve chromosomes, two 
show eleven, and one is doubtful. In fig. Il a; C-T there are nine 
groups showing twelve chromosomes, one is doubtfully twelve 
or eleven, one shows eleven, and one shows ten. These are all 
from winged individuals. These retain their eggs for some days 
and several ripe eggs are found in the body of each individual, 
while the wingless individuals, which have replaced largely the 
winged in this species, bring to maturity only one egg at a time 
which is laid as soon as it is ripe. It is therefore more difficult 


fod 


ELIMINATION OF SEX CHROMOSOMES 497 


to get polar body spindles in this type. The winged individuals 
produce only male eggs, so far as I have found. Therefore the 
fifteen equatorial plates of the winged migrants supposedly belong 
to male eggs. Now it is well recognized that the large number 
of chromosomes is more likely to represent the typical number, 
for, when one or two are lacking they may be in other sections, 
or be cut, or obscured by the other chromosomes. It appears 
then that twelve chromosomes are present in the male eggs. 

I found only two eases in wingless individuals in which the 
number of chromosomes could be clearly counted, fig. IIa U and 
W. In both ten chromosomes are present, but two are of double 
size and probably represent the four X’s fused together in two 
pairs. There is no visible evidence therefore of the loss of one 
chromosome in the eggs that give rise to the male-producers as 
I have postulated for P. caryaecaulis. 

After the polar bodies have been given off from the large and 
the small eggs—unfortunately I have never found a spindle in 
process of division—two kinds of embryos are found, namely, those 
containing twelve and those containing ten chromosomes. It 
appears therefore that two chromosomes are lost from the smaller 
egg. Morever only ten chromosomes appear in the sperma- 
togonial cells. The evidence therefore may seem to point to the 
conclusion that in this species there is usually no loss of chro- 
mosomes in any of the eggs (at least none that can be pointed 
out) of the stem-mother at the time when the polar bodies are 
formed. Hence the separation into the male-produceys and the 
female-producers at this time can not be shown to be due to the 
loss of one of the twelve visible chromosomes. 

It may appear therefore that the evidence contradicts the 
hypothesis offered to account for the change in the other species, 
but it will be noticed that the comparison rests in the assumption 
that the two equal X chromosomes in the spermatocytes of P. 
fallax correspond to the large X and the small xof P. caryaecaulis. 
But it may equally well be true that there are two large X’s 
with two small z’s attached (that do not become visible) in P. 
fallax. In other words not only are there twice as many ordi- 
nary chromosomes but twice as manysex chromosomes also. The 


498 T. H. MORGAN 


total number of one species is double that of the other. If this 
is the case the loss of the two smaller z’s might take place in 
those eggs of the stem-mother that give rise to the male pro- 
ducers and its loss might not be apparent unless its mate were 
sufficiently reduced in size to make its loss visible. It has not 
been possible to make out the size relations of the chromosomes 
in. P. fallax with sufficient clearness for the evidence to be of any 
value one way or the other. The question for this species must 
be left unanswered, but it should at least be noticed that the two 
large lagging chromosomes of P. fallax behave like the single 
lagging chromosomes of P. caryaecaulis, and not like the large 
X and the small one of the latter form. 


AN EXPERIMENT DEALING WITH SEX-LINKAGE 
IN FOWLS 


A. H. STURTEVANT 


From the Zoological Laboratory, Columbia University 
FOUR FIGURES 


In 1911 I published a preliminary report (Sturtevant, ’11) of 
an experiment showing sex-linked inheritance in fowls. The 
more significant data for the second generation can now be given. 


DESCRIPTION OF PARENT BREEDS 


The two breeds used in this experiment were the Columbian 
Wyandotte and the Brown Leghorn. The Columbian Wyan- 
dotite (fig. 1) is chiefly white, but with the tail, primaries, upper 
web of secondaries, median stripe to neck feathers, and some 
tail coverts black, in both sexes. 

The Brown Leghorn is nearly the color of the wild Gallus 
bankiva.! The sexes are strongly dimorphic. The males have 
reddish yellow edging to the neck and back feathers, some red 
on the shoulders and wing coverts, and a rather yellowish brown 
lower web to the secondaries (wing-bay of the fanciers), the 
rest of the plumage being typically black. The female has the 
tail, primaries, upper web to secondaries, and stripe in neck 
feathers black. Her breast is salmon yellow, and the neck feath- 
ers are edged with yellow. The rest of the plumage is finely 
stippled or mossed with yellowish brown and black. 


1A very good illustration showing male and female of this color is given by Bate- 
son (’09, pl. 4, figs. 3-4, opp. p. 103). 
499 


500 A. H. STURTEVANT 


DESCRIPTION OF CROSS-BRED BIRDS 


In F, the males are all alike, whichever way the cross is made. 
They are of fairly typical Columbian color, but perhaps rather 
darker than the parent stock, showing black stripes in some 
back feathers and black ticking (small specks) elsewhere. These 
characters, however, may occasionally appear in a pure Colum- 
bian male. There are two types of F; females, depending on 
the direction of the cross. When a Columbian male is used 
they approach the Columbian color, but differ in having coarse 
irregular stippling of the Leghorn type, in the same places as 
in the Leghorn female. These and the cross-bred males of the 
type described above I shall, for convenience, call grays. The 
F, red females obtained fromthe Brown Leghorn male and Colum- 
bian Wyandotte female (called browns in my earlier paper) 
are simply grays with the white replaced by a uniform red, about 
the shade of the breed known as Rhode Island Red. The F, 
results have shown that they are not to be considered as browns, 
genotypically like the Brown Leghorns, as I had supposed they 
were. 

In F; occurred several new types, which I shall describe before 
proceeding to the analysis. Red males, like red females, are 
simply grays with red substituted for white. 


SEX-LINKAGE IN FOWLS 501 


Another quite new type of males occurred—the duckwing. 
This type has the Leghorn or Jungle fowl pattern, with black- 
striped straw-colored neck and saddle, white wing-bay, red 
shoulders and back, black primaries, tail, and wing coverts, 
and mainly black breast, though a little white and some brown 
shows here. Now pure Brown Leghorn males occasionally show 
some brown on the breast, and some of the white on the only 
specimen of the above class examined in adult plumage is proba- 
bly due to the fact that he was sick when young, and did not 
grow well (but see letter from Mr. Westfall quoted below). 
Therefore I believe this male is a brown with white or straw- 
white substitut d for red and yellow in neck, saddle, and win - 
bay. 

There are two types of duckwing females. One, the silver 
gray, is the color of the Brown Leghorn female, with all the 
brown or yellow except that of the breast, replaced by white 
or light gray. This replacement is, however, incomplete on the 
back and wings. This is the color of the Silver Duckwing Game or 
Leghorn, and of the Silver Gray Dorking. I have seen brown in 
the above mentioned regions on exhibition specimens of two of 
these breeds. What I have called the brown duckwing female 
has the yellow replaced by white only in the neck. I know of 
no breed where this condition appears (but see statement in 
letter from Mr. Westfall, quoted below.) 

The presence of these two types of females suggests that there 
should be two types of duckwing males. Perhaps one type 
should be like the Silver Gray Dorking or the Dark Brahma 
males, which have the Brown Leghorn color with all brown and 
red replaced by white. Or perhaps there should have been 
golden duckwings, that is some with straw-colored wing-bay, 
as well as neck, back, saddle, and wing-bow. I had five duck- 
wing males, but raised only one to maturity. The last time I 
examined the others they were not quite three months old. At 
that time all five looked about alike in color, and my notes regard- 
ing them are as follows: necks black and white; backs red, 
slightly stippled with black, straw-colored saddle feathers begin- 


502 A. H. STURTEVANT 


ning to appear; wing-bows red and black; coverts and wing-bays 
brown stippled; breasts and tails black. In this connection 
it may be of interest to note what Mr. Henry Hales, breeder 
of Silver Gray Dorkings, says (in a letter dated December 28, 
1911) in regard to the juvenile plumage of males in that breed: 


The males have not the stippling like the females at any age: before 
adult feathering the cockerels have the coloring mixed up with white, 
black, and brown. One would hardly believe they would have the 
decided colors of the full plumage of the cocks. One cannot judge what 
the young ones are going to be until they are fully feathered. 


Tn view of this statement,it may be that my duckwing male was 
not far enough advanced when killed to show quite the adult 
plumage. Mr. Watson Westfall, another breeder of Silver Gray 
Dorkings, wrote to me, under date of December 29, 1911, giving 
a somewhat different statement, as follows: 


The Silver Gray female was once quite a brown colored hen and the 
males did not have white saddles and hackles but were straw color with 
some red across the shoulders. At this time they were known as Gray 
Dorkings, but by continual selection the English bred this brown out 
of them, and when the saddle, back, and hackle of the male was quite 
free of red and straw color and the female more gray the word Silver 
was added, making the name Silver Gray as we have it now. As the 
chicks hatch now with the very pure white top male as sire all the cock- 
erels show themselves plainly at once by being whiter on the head than 
the pullets, but as soon as they begin to feather they soon become very 
closely alike and remain so until the adult feathers appear, when the 
males will show black on breast while the females will begin to show 
red. Butlike all such varied colored fowls the female gets her real color 
long before the male. At two months old and up until the time the 
adult feathers begin to show on the cockerels they have a lot of stippled 
feathers on wings and back, but not many are as evenly stippled as 
the pullets, and where they are so much stippled and there is no brown at 
all to show on the wings, such specimens are very apt to be splashed with 
white on the breast. The red doesn’t all want to be lost in the chick 
feathers or else there will be failure in breast coloring. 


Specimens representing the grays (one 7 and one ¢), reds 
(one ~), duckwings (one ¢ and one @¢ ), and extracted browns 
(one ¢) are deposited with the Zoélogical Laboratory of Colum- 
bia University. 


SEX-LINKAGE IN FOWLS 503 


The matings made and the offspring produced were as follows: 


I. Columbian Wyandotte @ < Brown Leghorn 9—— 9 gray 7 
3 gray 9 


‘IL. Brown Leghorn & Columbian Wyandotte 2 ——> 10 gray @ 
8 red 9 


III. Brown Leghorn @ X Red F, 2 ——> 3 redo’ 
3 red 9 


IV. Gray F; @ (from II) X Brown Leghorn 2 = Saas 
5 duckwing # 
5 redo 
7 gray @ 
1 silver gray 9 
1 brown duckwing 9 
2 red 9 
2 brown 2? 


EXPLANATION OF RESULTS 


As I pointed out in my preliminary note, there is here at least 
one sex-linked factor, which I then called G, causing the two 
types of F; females. The F, generation agrees with this, mating 
III giving no grays, and mating IV both gray and non-gray 
males and females. But there are obviously other factors con- 
tributing to the F. result, which is decidedly complex, as has 
so often been found to be the case in experiments with fowls. 
I do not, therefore, feel justified in giving more than a tentative 
explanation of the results, since the numbers are small, and only 
a few of the many crosses which would be required to test any 
explanation have been made. The following will, however, 
cover the results obtained, and is the simplest scheme that I 
have been able to work out. 

Let us assume that the Columbian Wyandotte carries an 
inhibitor, 7, for red in all parts of the body, with the exceptions 
noted below. This is the G of my earlier paper. The Wyan- 
dotte also carries another sex-linked inhibitor, V, which prevents 
the production of red in the neck (and saddle of the male), not 
affecting the other parts. This is probably the factor described 
by Davenport (’11) as found in Dark Brahmas. Birds not earry- 


504 A. H. STURTEVANT 


ing these two factors have these regions of a red or reddish color, 
so that the Brown Leghorn, and probably also the Columbian 
Wyandotte, must carry a factor for red, R. The factors J and 
N do not completely inhibit R, since most of my J-bearing birds 
show traces of brown here and there, and all the white necked 
males are very ‘brassy’ (yellowish). Both these characters some- 
times appear in pure Columbian Wyandottes. Apparently there 
is also a Leghorn pattern factor, L,? causing black breast and 
black on the wing coverts in the male, and black stippling and 
salmon breast in the female, the latter effect appearing even 
in the presence of J. The factor L is hypostatic to another pat- 
tern factor, P, which is carried by the Columbian Wyandotte, 
and which inhibits all the colors just mentioned, as caused by 
L, leaving the color of the part dependent upon F and its inhib- 
itors. But one dose does not completely inhibit the stippling of 
the female. 

An alternative view, equally as satisfactory, I think, is that 
there is no inhibitor P, but that the Wyandotte has no L, and 
that the absence of this factor is dominant to its presence, 
heterozygous females being distinguishable by the stippling. On 
the first view L is probably present in all my birds. The con- 
stitution of the various types would then be as follows: 


Columbian Wyandotte INRPL 


Brown Leghorn inRpL 
Gray INRPL or InRPL 
Red inRPL 
Silver gray INRpL, or nRpL 
Brown duckwing iNRpL 


One other combination is possible—iNRPL. This should 
give a bird with Columbian pattern, white or straw neck, and red 
body. It is possible that such would have appeared had more 
birds been raised, but I know of no variety having any similar 
color combination, and have never observed it in a cross-bred 


2This is probably one of the components of the J (Jungle pattern factor) of 
Davenport (09). 


SEX-LINKAGE IN FOWLS 505 


fowl. This seems to me to be one of the most difficult points 
in connection with the hypothesis here given to explain my results. 
It may be that there is some interaction between N and P, such 
that when both are present N cannot produce its effect. Then 
iNRPL would give red. This could be tested by raising large 
enough numbers from mating IV to find out the real F; proportions, 
or by testing a number of reds with Brown Leghorns and seeing 
if any of them gave brown duckwings in F; or F,.. My principal 
evidence indicating that N is sex-linked was the fact that the 
females from mating II had red necks. But since they also had 
P, if the above hypothesis is correct, the only good reason for 
making N sex-linked is that it is probably identical with the 
factor described by Davenport (’11) as being sex-linked. If 
it is not so linked, then some of the reds from mating III should 
also carry it, and that mating should, eventually, produce some 
brown duckwings.® 

Since I have no evidence that R or L is missing in any of my 
birds I shall simplify the following formulae by omitting them. 
In these formulae MM represents a male, Mm a female. 


I. Columbian Wyandotte « INPM INPM 
Brown Leghorn @ inpM inpm 


INPM inpM — gray? 
INPM inpm. — gray 2 


II. Brown Leghorn @ inpM inpM 
Columbian Wyandotte 2 INPM inPm 


inpM INPM —gray & 


inpM inPm —red 92 
III. Brown Leghorn inpM inpM 
Red ? (gametes) inPM inpMinPm inpm 
inpM inPM —red@& 
inpM inpM —brown & (not seen) 
inpM inPm —red 9? 
inpM inpm —brown @ (not seen) 


’The real solution of this difficulty may be that J and N are coupled. 


506 A. H. STURTEVANT 


IV. Gray o& (gametes) INPM INpM InPM InpMiNPMiNpM inPM inpM 
Brown Leghorn 2 inpM inpm 


INPM inpM —gray¢# 
INpM inpM —duckwing 7 
InPM inpM —gray¢ 
InpM inpM ~—duckwing © (silver gray) 
iNPM inpM —red & (white-necked?) 
iNpM inpM —duckwing 7 

inPM inpM —red@ 

inpM inpM —brown ¢& (not seen) 
INPM inpm —~gray 2 

INpM inpm —silver gray @ 

InPM inpm —~gray 2 

InpM inpm —silver gray 2 

iNPM inpm —red 92 (white-necked?) 
iNpM inpm —brown duckwing ? 

inPM inpm —red 9 

inpM inpm —brown @ 


OTHER EXPERIMENTS DEALING WITH THE SAME COLORS 


Bateson (’02, ’09) and Punnett (’05) have given some facts 
regarding the duckwing color. When the Brown Leghorn was 
crossed with the White Dorking or White Leghorn, they obtained 
in F, some silver gray females. Two of these mated to a pure 
Silver Gray Dorking male gave only silver grays, and of these, 
four females to a male gave only silver grays. From these facts 
Bateson (’09) infers that the replaced red and yellow of the 
Brown Leghorn probably depends upon a separate factor, which 
his white breeds lacked. It seems to me more probable that 
this factor, which I have called R, was present in all three 
breeds, and that the two white breeds carried also the factor 
I. Any F, female showing the silver gray color would then be 
as pure for J as a pure Dorking, the factor being sex-linked, 
which explains why they had no trouble in getting a dominant 
F, to breed true. 

Mr. T. Reid Parrish, a Columbian Wyandotte breeder, has 
published in advertising circulars and in poultry journals (e.g., 
Parrish, ’11) detailed accounts of how he originated a strain of 
Columbian Wyandottes (probably not the one used in my ex- 
periments). According to this account he used Light Brahma 


SEX-LINKAGE IN FOWLS 507 


females with a White Wyandotte male. The Light Brahma has 
exactly the color of the Columbian Wyandotte. The breed 
seems to have been brought from the Orient in something like 
its present form, so that its history as to the origin of its color 
must probably remain a matter of conjecture. The White Wyan- 
dotte was derived directly from the Silver Laced Wyandotte, 
and is still, or was comparatively recently, a not uncommon 
sport from that variety (see McGrew, ’01, and poultry literature 
generally). This would seem to indicate that it is a recessive 
white, probably due to the dropping out of a color producer. 
Mr. Parrish’s statements support this view, as he says he obtained, 
in the F, generation from his cross, silver laced, barred, and 
Columbian birds—apparently no whites. These F; silvers he says 
were not typical, some of them having nearly white breasts, ‘‘yet 
showing a trace of lacing throughout the plumage.’’ This sounds 
as though they were much like the birds I obtained in my cross 
between silvers and Columbians (Sturtevant, ’11). From the 
result of that cross it would appear that silver is incompletely 
hypostatie to Columbian. Mr. Parrish says of his F, barred 
birds mentioned above that they ‘‘showed stronger Brahma 
markings than the silvers, but there was unmistakable barring 
throughout the plumage, being especially noticeable in tail and 
wing, some specimens showing barring in every section.’’ This 
is a most interesting statement, in view of the work of Spillman 
(09), Goodale (’09, ’10), Pearl and Surface (’10), and Daven- 
port (’06, 09) on barring. In this connection it is worth noting 
that occasionally a few barred feathers occur in pure Columbian 
Wyandottes, especially in the tail coverts of young males, and that 
one of my F, males (a duckwing) in the experiment described 
above has some barring in his hackle. 

Mr. Parrish states that he mated his F, Columbians with 
White Wyandottes, reciprocally. From Columbian female he 
obtained the same three classes as in F,, but we are not told 
whether or not whites appeared. The mating with Columbian 
male gave whites and Columbians, but he doesn’t say what else, 
if anything. 


508 A. H. STURTEVANT 


The only conclusions which I feel safe in drawing from these 
data are that the White Wyandotte is a recessive white, lacking 
a color producer, and that it carries a silver laced determiner. 
Bateson (’02) gives confirmatory evidence for the first of these 
conclusions. 


SEX FORMULAE 


It will be noted that I have used above the MM, Mm scheme 
for sex formulae in preference to the more usual Ff, ff formula 
(see Morgan, 711). I have made the change because the formula 
used here gives a mechanism which allows both complete sex- 
linkage, and also incomplete association with the sex-determiner. 
I shall now present the evidence which has led to this view. 

It seems to me that the evidence now before us warrants the 
conception of the chromosomes as the carriers of Mendelian 
factors or genes, as a working hypothesis. This conception is 
especially helpful in considerations of sex-linkage and the other 
forms of gametic coupling or associative inheritance. The recent 
hypothesis put forward by Morgan (’11 a, ’11 b, ’11 ¢) to explain 
these phenomena seems to me to overcome the old difficulties 
encountered by the chromosome hypothesis of Mendelian inher- 
itance. I shall make this conception the basis of my argument 
in favor of the MM, Mm scheme. 

Sex-linked inheritance of the type concerned here has now 
been known for some time, and has been recognized in Lepi- 
doptera and in birds, as follows: Abraxas (Doncaster and Raynor, 
06; Doneaster, ’08), canaries (Durham and Marryatt, ’08), fowls 
(Bateson, ’09; Spillman, ’09; Goodale, ’09, 710; Pearl and Sur- 
face, 10; Bateson and Punnett, ’11; Davenport, ’11; Sturtevant, 
"11), and ducks (Goodale, 711). 

Since my argument for the MM, Mm sex formula depends 
largely upon certain cases which I believe to represent partial 
sex-linkage, it will perhaps be well to present in some detail the 
evidence for the existence of this phenomenon. In this category 
I have included three cases of the Abraxas type (one in the fowl, 
one in the canary, and one in Aglia tau), and one, which I shall 
describe later, in the Drosophila type of sex-linkage. 


SEX-LINKAGE IN FOWLS 509 


Bateson and Punnett (11) describe certain exceptions occur- 
ring in their sex-linkage experiment with fowls, which they sug- 
gest may be due to a failure of the usual association between 
the sex-linked facotr and the sex-determiner, i.e., to ‘crossing 
over’ in the female. This is what I mean by partial sex-linkage. 

The sex-linked factor in canaries transforms pink eyes to black, 
and may then be represented by the symbol B. The following 
crosses have been reported by Durham and Marryatt (’08): 

Black 7 BM BM 

Pink ? bM bm 
BMbM —black @ 19 
BM bm —black @ 7 


F, black @ BMbM 
Black 9 BM bm 


BM BM—pblack @& \ o 
BMbM —black oJ ~ 
BMbm —black 9 18 
bM bm —pink @ 13 
Fi black @ BMbM 
Pink ? bM bm 
BM bM —black & 24 
bM bM —pink ¢& 21 


BMbm —black 
bM bm —pink @ 19 


Pink bM bM 
Fi black 92 BM bm 
bM BM—black @ 4 


bM bm —pink @ 6 


It will be seen that all the above crosses give the typical 
Abraxas results if B and M be assumed to be completely cou- 
pled,* but I have purposely omitted one cross: 


4 Only in the second and fourth matings is there any opportunity for crossing 
over, and in those two a total of only seventeen birds that would be affected by 
such crossing over. 


510 A. H. STURTEVANT 


Pink @ bM bM 
Black @ BM bm 
BMbM —black @ 
bM bm —pink 9 
black 9 


It is the last class of four black females which is of interest in 
this connection, and is inexplicable on the current scheme. I see 
only two explanations of this class—either the sire was, through 
some mistake, really black-eyed, which I mention only because 
it presents itself as a possible way out of the difficulty; or else, 
as I think probable, we have here anexample of partial sex-linkage, 
B ordinarily being coupled with M., If this coupling be incom- 
plete, then black females are to be expected from the last mating, 
as the following analysis will show: 


Gametes of pink 7 bM bM 
Gametes of black 2 BM bm (Bm bM) 


BM bM —black 7 
bm bM —pink @ 

(bM bM —pink & ) 
(Bm bM —black ? ) 


This hypothesis could be easily tested. If it is correct, then the 
cross just discussed should, if large enough numbers be reared, 
produce as many pink males as black females. Furthermore, 
if these black females be bred to pink males, there should arise 
a race which would be dimorphic—black-eyed females and pink- 
eyed males—except for the occasional ‘crossing’ back of B, which 
would now be coupled with m, and m occurs only in the female. 
Such a relation, if it be shown to exist, would be highly interesting 
in its bearing upon the problem of secondary sexual characters. 

The third case which I have interpreted as partial sex-linkage 
is that of the moth Aglia tau reported by Standfuss (’96), and 
discussed by Castle (03). The variety lugens of this species 
is dominant to the type. Its gene may be designated L. My 
interpretation of this case is that L and M are associated in such 
degree that ‘crossing over’ occurs in about one-third, instead 
of the usual one-half of the cases. The analysis of the matings 


SEX-LINKAGE IN FOWLS uel! 


then is as follows. It is obvious that all the lugens moths used 
were heterozygous. 


Lug.o LM IM 
TauQ IM Im 


LM IM — lug. ¢& 31 
IM IM — tau o 14 
LM Im — lug. 9? 13 
IM Im — tau 9? 28 


The results of this one cross are not in accord with my hypoth- 
esis, since all four classes should be equal, but I think the num- 
bers are rather too small to be very significant. The reciprocal 
cross gives: . 


Tau co IM IM 
Gametes, lug. 9 2 LM2Im 11M 1Lm 


21M LM —lug. o 26 
11M IM —tau o@ 13 
21M Im —tau 9 25 
11M Lm —lug. @ 11 


This case comes out as I expect, but since the numbers are no 
larger and no more disproportionate than those in the first cross, 
I must rest my case on the third: 


Lug. @ LM IM 

Gametes, lug. 9 2 LM1IM2 lm Lm 
2LMLM } 
2 LM IM r>—5 lugo 129 
11M LM } 
11M IM —ltauc 16 
1 LM Lm 
11M Lm }—4lug. 9 9% 
2ILMIm J 
21M Im —2tau 9 36 


The relative size of the classes is perhaps as near the expected 
proportion as could be looked for, and becomes still nearer expec- 
tation if the coupling strength be increased slightly.°. So much, 
then, for the experimental evidence bearing upon the case. 


5Standfuss (710) has published more data on this cross, but unfortunately has 
not reported the sex ratios obtained. 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 4 


512 A. H. STURTEVANT 


The cytological evidence relating to birds and Lepidoptera 
is not very helpful. Guyer’s (09, ’09 a) reports on guinea-fowls 
and chickens are directly opposed to the experimental evidence, 
in that they make the male heterozygous for sex. Since a re-ex- 
amination of the fowl case by other cytologists has so far failed 
to convince them that Guyer’s view is correct, I think we may 
for the present disregard this evidence, at least in so far as it 
concerns the fowl. Several observers (see Stevens, ’06; Dederer, 
07; Cook, 710; Doneaster, ’11) have studied the spermatogene- 
sis of Lepidoptera, and in some cases have seen what they sus- 
pected to be an equal pair of idiochromosomes. I do not know 
of any further cytological evidence in these two classes of animals. 


F i M M 


100 OU UE 20 oe 


2 Cf Q Ch 


2 3 4 


The cytological evidence indicates that, in the Lepidoptera 
at least, the male has two equal idiochromosomes. Judging 
by the experimental evidence, this must also be the case in birds, 
and the female must, of course, have at least one similar chro- 
mosome (see figs. 2 to 4). These three are the carriers of the genes 
for sex-linked factors. The doubtful point is the mate of this chro- 
mosome, present in the female-producing egg. If the sex formula 
be Ff, ff, as we have been supposing, then this chromosome 
would be, visibly or imperceptibly, larger than the ‘male’ (f- 
bearing) chromosome, since it would have one factor, 7, not 
present in that chromosome (fig. 2). In this case it would seem 
that complete sex-linkage, such as that found in Abraxas and 
in barred fowls, would occur not at all, or at least only rarely, 
since every part of the f-chromosome would have a homologous 
part in the F-chromosome, and crossing over would thus be pos- 
sible. It might be that the process of reduction is such that 
no crossing over is possible in oogenesis, but if the cases of par- 


SEX-LINKAGE IN FOWLS oltes 


tial sex-linkage be admitted, such crossing over must be possible.® 
If, on the other hand, we use the MM, Mm scheme we meet 
with no such difficulties. In this case the heterochromosome 
contained in the female-producing egg is smaller than its mate in 
the male-producing egg, since it lacks the factor M (fig. 3),’ 
and the sex-formula is MM, Mm, two F’s being always present 
in both sexes, and contained, presumably, in some other pair 
of chromosomes. This would allow complete sex-linkage, for 
genes in that part of the M-chromosome having no homologue 
in the m-chromosome, and incomplete sex-linkage, for genes in 
the part having such a homologue. One might, of course evade 
this conclusion by assuming the condition represented in fig. 4. 
In this case the chromosomes would not conjugate evenly, and 
the part marked ZL could carry such factors as are completely 
sex-linked. 

Of the converse case, where the male is heterozygous for sex 
there are not so many examples known. In Drosophila Mor- 
gan (’10, ’11, ’11 a, ete.) has reported numerous sex-linked factors. 
Miss Stevens (’08) has found here exactly the cytological con- 
ditions demanded by the experimental evidence. In man color- 
blindness follows this same scheme of inheritance, as do appar- 
ently several diseases (Bateson, ’09; Morgan, 11). Here, too, 
Guyer (’10) has reported cytological evidence that it is the male 
which is heterozygous. Finally we have the case of partial sex- 
linkage referred to above. Miss Stevens (’11) has reported hetero- 
chromosomes in the male guinea-pig, and as that animal has 
been experimentally bred quite extensively I was led to look 
for sex-linkage in it. Perhaps the dwarf form studied by Miss 
Sollas (09) is such a case. This is a recessive form, and has 
not been reared to maturity, or had not been in 1909, so that 
the case is not thoroughly worked out, but it seems to be most 
easily explainable as a case of partial sex-linkage. If we rep- 


6It should be noted that, if my view is correct, th sex chromosomes under dis- 
cussion are homologues, not of the X and Y of Diptera, Hemiptera, ete., but of 
the M-bearing chromosomes in those groups. 

7 On this scheme two F’s are to be assumed to be always present in both sexes, 
probably situated in another pair of chromosomes. 


514 A. H. STURTEVANT 


resent the factor for normal size, carried by the heterochromo- 
somes, by \, then in ordinary guinea-pigs this is present in both 
gametes of each sex. But in a certain strain used by Miss Sollas, 
from which all her dwarfs descended, it had dropped out of the 
odd chromosome. ‘This strain would go on breeding true, then, 
for some time, according to this scheme: 


NF NF normal ? 
NF nf normal @ 
NF NF normal @? 
NF nf normal @ 


But now suppose crossing over sometimes occurred, and we 
should have: 


NF NF normal ? 
NF nf (nF Nf) normal &@ (gametes) 


NF NF normal ? 
NF nf normal 7 
(NF nF normal ?) 
(NF Nf normal ) 


We should still have no dwarfs, but if the heterozygous female 
were mated to a male of her own race we should get dwarfs, thus: 


NF nF normal 2? 
NF nf normal 7 


NF NF normal 2 
NF nF normal 
NF nf normal ¢@ 
nF nf dwarf of 


This explains why Miss Sollas should obtain a great majority 
of male dwarfs, and how, as seems to have occurred, the pecu- 
liarity may descend through the female. But she does get some 
dwarf females from normal parents. These are to be expected, 
since they would appear in the last mating above if crossing 
over again occurred in the male, thus: 


SEX-LINKAGE IN FOWLS 515 


NF nF normal @? 
NF nf (nF Nf) normal &@ (gametes) 


NF NF normal 92 
NF nF normal 2 
NF nf normal @ 
nF nf dwarf ¢ 
(NF nF normal 9) 
(nF nF dwarf 92) 
(NF Nf normal @) 
(nF Nf normal ~) 


The actual numbers for families containing dwarfs is as follows: 


Normal 9 normal @ dwarf ? dwari @ 
25 49 5 20 


This gives a preponderance of normal males as against dwarf 
males, but it should be remembered that for every dwarf female 
due to crossing over there is a normal male produced, and sec- 
ondly, the mutant is obviously not a very viable one, so that a 
shortage is perhaps not surprising, especially in such compar- 
atively small numbers. The fact that there are nearly as many 
dwarf males as normal females is obviously due to the cireum- 
stance that the males of both kinds taken together are more than 
twice as numerous as the females. 

In plants Correns (’07) and Shull (10, *11) have shown that 
in certain dioecious species of Bryonia and Lychnis it is the male 
which is heterozygous. In the absence of cytological evidence 
or sex-linkage phenomena a chromosome interpretation of these 
eases would perhaps be out of place. 


SUMMARY 


There is a sex-linked factor carried by the Columbian Wyan- 
dotte—an inhibitor for red in the plumage. This breed proba- 
bly also carries another sex-linked factor, an inhibitor for red 
in the neck. It apparently carries a pattern factor inhibiting 
the breast color, and, in the female, the stippled back of the 
Brown Leghorn. 

The silver gray color is probably epistatic to the Jungle fowl 
or brown color. 


516 A. H. STURTEVANT 


The white Wyandotte is a silver laced breed with a color pro- 
ducer dropped out. 

An attempt is made to explain three sets of phenomena, in fowls, 
in canaries, and in Aglia tau respectively, as cases of partial sex- 
linkage. Using this explanation, it is argued that the sex for- 
mula for birds and Lepidoptera is probably; 7, MM, FF; ¢, 
Mm, FF. The case of the dwarf guinea-pig is explained as per- 
haps representing partial sex-linkage in a form where the male 
is heterozygous for sex. 


February, 1912 


LITERATURE CITED 


Bateson, W. 1902 Rep. Evol. Comm. vol. 1. 
1909 Mendel’s principles of heredity. Cambridge. 
Bateson, W. AND PuNnNETT, R. C. 1905 Rep. Evol. Comm., vol. 2. 
1911 Jour. Genet., vol. 1, p. 185. 


CastLe, W. E. 1903 The heredity of sex. Bull. Mus. Comp. Zool., Harvard, 
vol. 40, no. 4. 


Cook, M. H. 1910 Spermatogenesis in Lepidoptera. Proc. Acad. Nat. Sci., 
Philadelphia, April, p. 294. 


Correns, C. 1907 Die Bestimmung und Vererbung des Geschlechtes, nach 
neuen Versuchen mit héheren Pflanzen. Berlin. 


Davenport, C. B. 1906 Inheritance in poultry. Carnegie Inst. Washington 
Publ. 52. 


1909 Inheritance of characteristics in domestic fowl. Carnegie Inst. 
Washington Publ. 121. 


1911 Another case of sex-limited heredity in poultry. Proce. Soc. 
Exp. Biol. Med., vol. 9, p. 19. 


Deperer, P. H. 1907 Spermatogenesis in Philosamia cynthia. Biol. Bull. 
vol. 13, p. 94. 


Doncaster, L. 1908 Rep. Evol. Comm., 4 


1911 Some stages in the spermatogenesis of Abraxas grossulariata 
and its variety lacticolor. Jour. Genet., vol. 1, p. 179. 


Doncaster, L. anp Raynor, G. H. 1906 Breeding experiments with Lepid- 
optera. Proc. Zool. Soc., London, vol. 1, p. 125. 


SEX-LINKAGE IN FOWLS by bs 


Duruam, F. M., anp Marryarr, D. C. E. 1908 Notes on the inheritance of 
sex in canaries. Rep. Evol. Comm., vol. 4, p. 47. 


Goopats, H. D. 1909 Sex and its relation to the barring factor in poultry. 
Science. n. s., vol. 29, p. 1004. 
1910 Breeding experiments with poultry. Proc. Soc. Exp. Biol. 
Med., vol. 7, p. 178. 
1911 Studies on hybrid ducks. Jour. Exp. Zool., vol. 10, p. 241. 
Guyrr, M. F. 1909 The spermatogenesis of the domestic guinea (Numida 
meleagris Dom.). Anat. Anz., Bd. 34, p. 502. 
1909 a The spermatogenesis of the domestic chicken (Gallus gallus 
Dom.). Anat. Anz., Bd. 34, p. 573. 
1910 Accessory chromosome in man. Biol. Bull., vol. 19, p. 219. 
McGrew, T. F. 1901 The Wyandotte. U.S. Dept. Agr., An. Ind. Bur. Bull., 
: no. 31. 
Morean, T. H. 1910 Sex-limited inheritance in Drosophila. Science, n. s. 
vol. 32, p. 120. 
1911 The application of the conception of pure lines to sex-limited 
inheritance. Am. Nat., vol. 45, p. 65. 
191la An attempt to analyze the constitution of the chromosomes 
on the basis of sex-limited inheritance in Drosophila. Jour. Exp. 
Zool., vol. 11, p. 365. 
1911b Random segregation versus coupling in Mendelian inherit- 
ance. Science, n.s., vol. 34, p. 384. 
1911¢ Chromosomes and associative inheritance. Ibid, vol. 34, 
p. 636. 
Parrisu, T. R. 1911 Catalogue, Parrish strain Columbian Wyandottes. Nash- 
ville. 


Peart, R., aND SurracE, F. M. 1910 Further data regarding the sex-limited 
inheritance of the barred color pattern in poultry. Science, n. s., 
vol. 32, p. 870. 


Suu, G. H. 1910 Inheritance of sex in Lychnis. Bot. Gaz. 49, p. 110. 


1911 Reversible sex-mutants in Lychnis dioica. Bot. Gaz., vol. 52, 
p. 329. 


Soutas, I. B. J. 1909 Report Evol. Comm., vol. 5, p. 51. 
Spituman, W.J. 1909 Barringin Barred Plymouth Rocks. Poultry, vol. 5, p.7. 
Sranpruss, M. 1896 Handbuch der paliarktischen Grosschmetterlinge. Jena. 


1910 Chaerocampa (Pergesa) elpenor L. ab. daubii Neip. und einige 
Mitteilungen iiber Wesen und Bedeutung der Mutationen illustriert 
an Aglia tau L. Iris, Bd. 24, p. 155. (See also a review, by H. Fed- 
erley; Arch. Rass. u. Gesellsch. Biol., Bd. 7, p. 755.) R 


518 A. H. STURTEVANT 


Stevens, N.M. 1906 Studies in spermatogenesis. IT. 


Carnegie Inst. Washing- 
ton Publ., 36, p. 33. 
1908 A study of the germ-cells of certain Diptera. Jour. Exp. Zool., 
vol. 5, p. 359. 
1911 


Heterochromosomes in the guinea-pig. Biol. Bull., vol. 21, 
p. 155. 


Sturtevant, A. H. 1911 Another sex-limited character in fowls. Science. 
Mees vole SB 1s shi 


STUDIES ON THE PHYSIOLOGICAL CHAR ACTERS 


OF SPECIES 


I. THE EFFECTS OF CARBON DIOXIDE ON VARIOUS PROTOZOA 


MERKEL HENRY JACOBS 


From the Zoological Laboratory, University of Pennsylvania 


CONTENTS 


I. Introduction. . : Rene Aa Mew Ee ete Dnt ae 


IT. Material and methods: Be caret oct 
III. Observations and experiments......... 
A. Ciliates. . P Aare 
HG Parameciinne: auch ye cghaek 
2. Paramecium aurelia..... 
3. Paramecium bursaria 
4. Colpidium colpoda.... 
5. Coleps hirtus.......... 
6. Blepharisma lateritia.. 
7. Euplotes patella.... 
8. Vorticella nebulifera....... 
B. Flagellates. . : 4 
ite Peceneas a tric ewharin.: 
2. Euglena viridis (?).. 
3. Chilomonas paramecium..... 
4. Entosiphon sulcatum.... 
IV. Discussion of results........ 
V. Summary....... Sdiciace CARE ORR tr, 
ISIDWORT ADI. cpicckeoce en erates. + os 


I. INTRODUCTION 


Considering its importance in connection with many aspects 
of modern biological research, the question of the physiological 
characters of species, as opposed to their morphological ones, 
This con- 
dition has doubtless resulted partly from the fact that physio- 
logical characters, on account of their less definite and tangible 


has received a surprisingly small amount of attention. 


519 


520 MERKEL HENRY JACOBS 


nature, are more difficult to deal with than morphological ones, 
and partly from the fact that single physiological characters at 
least, are notoriously unreliable guides in the questions of classifi- 
cation and phylogeny that up until the present day have occupied 
so large a share of the attention of working biologists. 

The latter objection, however, no longer holds today, at least 
to the same extent that it formerly did. Modern zoology is 
not so much interested in finding out what are the probable rela- 
tionships of a given animal as in learning what it is, and especially 
what it does. This is the physiological point of view, whichis 
uppermost in the minds of most biologists today. No data which 
throw light on what goes on in the living organism are any longer 
considered unimportant; indeed, they are coming to be recognized 
as a vital necessity. If our knowledge of comparative physiology . 
were as complete as our knowledge of comparative morphology, 
for example, there is not a single one of the more modern develop- 
ments of biological science that would not have its possibilities 
enormously extended. It is therefore a matter of increasingly 
great importance to accumulate accurate data on the physio- 
logical characters of organisms, to determine which ones are 
fundamental, and which accidental, which are constant in a given 
species, or larger group, and which vary in different individuals 
of the same species, or perhaps in the same individual at different 
times; in short to obtain as full and comprehensive a knowledge 
as possible of the physiological characters of organisms. Perhaps 
the day may come when it will be possible to define any species 
in physiological and chemical terms in the same way in which it is 
now defined in morphological ones, and when no description of 
an organism will be considered complete which does not include 
its chief physiological peculiarities along with its structural one. 
The biologists of that day will be able successfully to attack prob- 
lems that for the present must remain untouched on account of 
lack of the proper kind of knowledge. 

It is needless to state that many observations of the sort sug- 
gested have already been made. Not to mention the more or 
less scattered ones made on many widely separated groups of 
organisms, we already have a considerable knowledge of the 


EFFECTS OF CARBON DIOXIDE 521 


physiological characters of many of the bacteria, a group in 
which, for obvious reasons, our physiological knowledge has far 
outstripped our morphological knowledge. Botanists have also 
accumulated an enormous fund of knowledge relating to the com- 
parative physiology of the green plants, while in such special 
fields as the study of the blood sera of the higher vertebrates, to 
give but one example, encouraging progress has been made. 
Nevertheless, it is apparent that very little has been done in the 
way of systematic studies along the lines suggested, with the ulti- 
mate object of making the physiological characters of each organ- 
ism as well known as its morphological ones. Such an under- 
taking is not the work of one man or of one generation. Many 
years must elapse before our knowledge will be anything but 
exceedingly fragmentary and scattered. The following paper 
is therefore a very modest contribution to so large a subject. It 
deals merely with the effects of a single common and important 
substance, carbon dioxide, on a number selected protozoan forms, 
with especial reference to their movements and general vitality. 
It will be followed at intervals by other papers on the effects of 
various other substances, so far as possible on the same forms. It 
is not claimed that the results are, or will be, complete or exhaus- 
tive; still it is hoped that they may not be without interest and a 
certain amount of value. 


II. MATERIAL AND METHODS 


The forms studied were various of the most common ciliate 
Infusoria and flagellates, i.e., Paramecium caudatum, P. aurelia, 
P. bursaria, Colpidium colpoda, Coleps hirtus, Blepharisma 
lateritia, Euplotes patella, Vorticella nebulifera, Peranema tri- 
chophorum, Euglena viridis (?), Chilomonas paramecium, and 
Entosiphon suleatum. In the case of all the forms mentioned 
except the last one, observations were made on individuals from 
several different cultures of different origin, the intention being 
to obtain, so far as possible, data which would apply to the species 
as a whole and not simply to a particular race. Of course it will 
be necessary to extend the observations still further before draw- 


522 MERKEL HENRY JACOBS 


ing absolutely final conclusions; it is thought, however, that fur- 
ther work will not materially alter the results arrived at in this 
paper. 

The general method employed in studying the effects of carbon 
dioxide on the forms in question was to subject them, in a drop of 
culture fluid, to a continuous stream of this gas in an Engelmann 
gas chamber. The drop of liquid containing them was placed 
on a slide or cover glass and the latter inverted in the usual way 
over the opening of the gas chamber, the joints being made air- 
tight with vaseline. The observations were made entirely with 
the compound microscope, chiefly with a Leitz 3-objective, 
although in doubtful cases the 7-objective was also employed. 
The points especially noted were the time required to stop normal 
locomotion, the time required completely to stop the beat of the 
cilia, flagella, ete., and the longest possible exposure after which 
recovery is possible when normal conditions are restored. In 
addition, incidental observations were made on the general behay- 
ior of the organisms and the visible structural changes produced 
in the cell by carbon dioxide. 

The gas used in the experiments was generated in the apparatus 
designed by McCoy, from marble and C. P. hydrochloric acid 
diluted in the proportion of one part of acid to four of water. 
Before coming in contact with the animals it was passed through 
two wash bottles filled with a solution of sodium carbonate to 
remove any traces of hydrochloric acid that might be present and 
also to ensure thorough saturation with water vapor. That no 
appreciable amount of hydrochloric acid was left in the gas was 
shown by conducting it into a silver nitrate solution, which in the 
course of two hours showed no traces of a precipitate or even of a 
cloudiness. The gas after being thus purified waS conducted 
successively through four Engelmann chambers, each placed on 
the stage of a microscope, and connected by rubber tubing in such 
a way that the same gas passed through all of them. This arrange- 
ment was found very useful, not only in making comparisons 
between different species under as nearly identical conditions as 
possible, but also in facilitating a larger number of independent 
observations on individuals belonging to the same species. 


EFFECTS OF CARBON DIOXIDE HOS 


Preliminary experiments showed the necessity of observing a 
number of precautions. The first of these is that the rate of 
evolution of the carbon dioxide gas shall be approximately the 
same in different experiments which it is desired to compare, 
since it was found that, other things being equal, the slower the 
stream of carbon dioxide passing over the drop the longer the 
animals survive. This is probably due to the fact that in a rapid 
stream the air is removed from the gas chamber and the drop 
more quickly, and the animals have less time to adjust themselves 
to the new conditions than in the case of a slow stream. By 
using all four of the gas chambers in one experiment it was found 
easy to compare a considerable number of forms with this factor 
constant. Frequently, indeed, a number of forms were present 
in the same drop and thus subjected to exactly the same condi- 
tions. In order to be able to compare experiments made on 
different days the attempt was made always to have the gas 
evolved at the rate of approximately 100 ce. per minute. This 
it was found possible to do within the necessary degree of accu- 
racy by proper regulation at the beginning of the experiment of 
the apparatus, which is automatic when once started. 

A second and most important point to be considered is the 
temperature, which has a marked effect on the time in which death 
occurs. A preliminary experiment on the three species of Para- 
mecium showed that at 22°C. death occurs in roughly half the 
time in which it does at 12°C. In order that this factor might be 
made constant, all of the experiments here recorded were made 
at, or very near, the first mentioned temperature, which is slightly 
above ordinary room temperature. 

A third point that cannot be neglected is the size of the drop 
containing the animals. Preliminary experiments showed that 
this has an appreciable effect on the results obtained, especially 
when the drop is very small. In one such experiment in a very 
small drop the average time of death of a certain ‘pure’ race of 
Paramecium aurelia was seventeen minutes while the average 
for the same race in a rather large drop was thirty minutes. In 
two medium sized drops in the same experiment the times were 
twenty-eight and twenty-nine minutes respectively. It will be 


524 MERKEL HENRY JACOBS 


seen therefore that while there is not very much difference between 
the medium sized drops and the large one, the small one shows 
a decided difference. This is probably due to the greater sudden- 
ness with which the animals were subjected to the carbon dioxide 
in the latter case rather than to any difference in its concentra- 
tion ultimately in the drops, since these probably all became prac- 
tically fully saturated long before the end of the experiment. In 
order to guard against this source of error the drops were all made 
as nearly the same size as possible, the standard being a drop about 
8 mm. in diameter with a moderate curvature. Results obtained 
in this way were constantly compared with those obtained when 
a number of the forms in question were present in the same drop. 
The final precaution has already been mentioned, namely, not 
to base far-reaching conclusions on results obtained from a single 
culture. In the case of some of the forms studied more than a 
dozen cultures were employed; in all of them except one form, 
obtained very late, the number of cultures was at least three. 


Ill. OBSERVATIONS AND EXPERIMENTS 


A. Ciliates 


1. Paramecium caudatum. The effects of carbon dioxide on 
this form are briefly as follows. Immediately after the current of 
gas has been turned on, the animals exhibit a general restlessness 
and begin to seek the center of the drop, or rather, to avoid its 
edges, doubtless because the concentration of the carbon dioxide 
is greatest there. Inside a minute, as a rule, in a drop of the size 
used in these experiments, they have collected in its central and 
thickest part. Here they swim about actively but in very short 
paths, since the ‘avoiding reaction’ occurs whenever their move- 
ments have a tendency to carry them into the thinner and con- 
sequently more saturated part of the drop. Soon, however, 
generally within two or three minutes, they cease to be able to 
discriminate between the concentrations in different parts of the 
drop and spread out again until they are uniformly distributed. 
Sometimes toward the end of the experiment, for unknown reasons, 
they collect about its edge. Previous workers have noticed the 


EFFECTS OF CARBON DIOXIDE 525 


behavior just described. Loeb and Hardesty (95) mention that 
Paramecium aurelia remains in the center of the drop fifteen min- 
utes. In all probability they were dealing with a larger drop 
_than the one used in these experiments, where in no case did either 
P. aurelia or P. caudatum require more than a few minutes to 
become adjusted to the new condition. 

After this primary response, the animals swim about in a more 
or less normal manner but more and more slowly, until they finally 
come to rest in a time that may vary from twenty or twenty-five 
minutes to several hours. Even after locomotion has ceased the 
cilia continue to beat for some time. As death approaches they 
beat slowly and irregularly and frequently show visible signs of 
injury. Often a group of them may keep on beating after the 
others have come to rest. There seems to be nothing constant 
about the part of the body where movements persist longest. 
After the cilia have completely stopped it seems to be impossible 
to start them again even by prolonged exposure to the air. The 
animals are to all intents and purposes dead. The same thing is 
true of the other ciliates studied, with the exception of Vorticella, 
whose membranelles are ofteti stopped before the animal is seri- 
ously injured and consequently can be started again. In the 
majority of ciliates, however, the vibratile structures are among 
the most resistant parts of the cell and when they have finally 
succumbed the life of the rest of the cell is practically extinct. 

In the meantime, certain other changes have been occurring. 
Among the mos: striking of these is the change in shape of the 
body, which becomes shorter and thicker. Some of the increase 
in the thickness is doubtless due to the shortening, but this does 
not account for all of it, and it is probable that an actual increase 
in volume occurs by the absorption of water. This phenomenon 
is more strikingly shown in some of the other forms studied than 
in P. caudatum. About the time that the swelling becomes notice- 
able, the nuclei begin to be very clearly visible, standing out 
sharply from the rest of the protoplasm by their greater opaque- 
ness. This is perhaps due to the acid nature of the medium sur- 
rounding the cell since other acids produce the same phenomenon. 

' A furthereffect of the carbon dioxide often is apparent in the burst- 


526 MERKEL HENRY JACOBS 


ing of the pellicle and the flowing out of droplets of clear proto- 
plasm. This may occur either before or after the cilia have 
stopped beating, but is not so constant in P. caudatum as in some 
of the other forms studied. There is reason to believe that this 
result is at least partly due to actual injury of the pellicle and not 
merely to an increase of internal pressure, since the bursting some- 
times occurs when the cell has not markedly swollen and also 
never appears until late, while the cell may reach almost its maxi- 
mum volume quite early. Furthermore, after one droplet has 
formed, thus supposedly relieving the internal pressure, others 
may form in quick succession at other parts of the cell boundary. 
This effect is not a specific one of carbon dioxide since Budgett 
(98) noted the same phenomenon in Paramecium and other pro- 
tozoa when merely deprived of oxygen in a stream of hydrogen, 
and high temperatures also cause the same result. 

The time that elapses from the beginning of the experiment 
until the death of the animal varies considerably with cireum- 
stances. The lowest average obtained in a single experiment 
was about twenty minutes, the highest over three-and-a-half 
hours, more cultures, however, approaching the latter value than 
the former. In an effort to determine to what extent P. caudatum 
can be said to have a specific resistance especial attention was 
paid to the question of the amount of variation shown by differ- 
ent individuals, races and cultures. A large mass of data was 
accumulated which will be made the basis of another paper on a 
somewhat different subject. It may not be out of place, however, 
to say here that while there is some evidence that different races 
may have different powers of resistance, these differences are 
insignificant compared with the enormous changes in resistance 
that a single race may undergo under appropriate changes in the 
culture medium. It is possible artificially to change the resistance 
very greatly, and such changes also occur naturally during the 
ageing of the culture, an old culturein general having a high resist- 
ance, and animals kept in the laboratory for a time: being more 
resistant than ‘wild’ ones. Great as these variations are, however, 
they have their limits and in the dozens of cultures and thousands 
of individuals studied none were found which had as low a resist- 


EFFECTS OF CARBON DIOXIDE 527 


ance as the average Coleps hirtus for instance, or as high a one 
as the average Colpidium colpoda. Furthermore in a given 
culture, if P. caudatum has a higher resistance than usual, the 
other forms present also will, and their relative resistance remains 
practically constant. It is possible, therefore, to attribute to 
P caudatum as well as to the other forms a specific resistance, 
remembering only that its absolute value is somewhat subject to 
variation under different conditions and that in comparing differ- 
ent forms it is well to have them either from the same culture or 
at least to make a considerable number of independent observa- 
tions on different cultures. 

2. Paramecium aurelia. A comparison of this form with the 
preceding one will illustrate the statement just made. P. aurelia 
is in general considerably less resistant than P. caudatum. When 
the two forms are present in the same culture the former is always 
killed sooner than the latter, though rarely, when different cul- 
tures are studied, some strains of P. aurelia are encountered which 
show a higher resistance than some of the most susceptible strains 
of P. caudatum. In general, however, P. aurelia is killed in less 
than a half hour while P. caudatum nearly always survives several 
times as long. The average time of death in the two extreme 
experiments on P. aurelia was a little over ten minutes on the one 
hand and over two hours on the other, in most of the experiments, 
however, lying, as already stated, below thirty minutes. Loeb 
and Hardesty state that P. aurelia is killed by carbon dioxide in 
two-and-a-half to three-and-a-half hours. Perhaps their culture 
was an abnormally resistant one, or possibly the application of 
the gas was slower than in these experiments, or the temperature 
lower In view of the fact, however, that the figures given by 
them are quite typical of the rather more common P. caudatum 
at ordinary room temperature, and also that the distinction be- 
tween caudatum and aurelia formerly was not very sharply drawn, 
it is possible that they were dealing with the former rather than 
the latter species. The difference in size between the two forms 
is probably not the reason for their different powers or resistance, 
since in the same species no constant relation could be found 
between the time of death and the size of the animal Further- 


THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 4 


528 MERKEL HENRY JACOBS 


more, the larger P. busaria is less resistant than P. aurelia and the 
somewhat smaller Colpidium colpoda far more resistant. The 
cause of the difference is evidently of a more deep seated nature. 

In the general effects produced on it by carbon dioxide, P. 
aurelia closely resembles the preceding species. It shows the same 
negative response at first, which disappears in about the same 
time. The body swells in about the same way. The nuclei 
often become very distinct, and this fact frequently renders an 
accurate identification possible without staining. The interval 
between the time when locomotion ceases and the cilia stop beat- 
ing is both relatively and absolutely shorter than in P. caudatum 
There is also a much greater tendency for the pellicle to rupture, 
this occurring in some cultures in almost every individual. Per- 
haps this apparently greater delicacy of the body wall may be 
correlated with the lower powers of resistance of this form. 

3. Paramecium bursaria. In a number of the cultures used 
in these experiments this species was found associated with the 
two preceding ones and therefore a favorable opportunity was 
presented to compare it with them. Such a comparison shows 
that it is the least resistant of the three. The average time of 
death was generally ten to twenty minutes, though in a number of 
cultures it was less than five and only rarely ran as high as thirty. 
The most resistant individual found lived over an hour but this 
was a most exceptional case. (It was in this culture that P. 
aurelia also showed its highest resistance—over two hours.) 
When the three forms in question are present in the same culture 
in every case observed the relative resistance was: bursaria, 
aurelia, caudatum, and might perhaps be represented numerically 
very roughly as 1: 2:4. The general effects of the carbon dioxide 
on this form are on the whole s milar to those already described 
in the case of the other two species. The pellicle apparently is 
very delicate and nearly always ruptures while the cilia are more 
markedly affected than those of the other species; as their move- 
ments cease they become matted together and very quickly be- 
come partly disintegrated, being represented only by an indis- 
tinet zone about the animal. It is rather interesting that this 
green form is less resistant than the colorless ones. Doubtless 


EFFECTS OF CARBON DIOXIDE 529 


on account of the presence of chlorophyll in its body it is accus- 
tomed to rather a low concentration of carbon dioxide, since this 
substance is constantly being removed by it from the surrounding 
medium. Experiments to determine whether it was more, or 
less, resistant in bright light than in the dark have as yet not 
given very positive results, chiefly on account of the difficulty of 
controlling the temperature factor. 

4. Colpidium colpoda. This in every case proved to be the 
most resistant species studied, living long after the other forms in 
the same drop had succumbed. The general effects on it of the 
carbon dioxide are as follows. When the stream of gas is turned 
on a strong negative response is shown and the animals collect in 
the center of the drop in the same manner as the forms already 
mentioned; soon, however, they become uniformly distributed 
and thereafter behave normally almost to the time of death which 
hardly ever occurs in less than six hours, and may take place much 
later. In anumber of experiments the carbon dioxide was allowed 
to flow for six or seven hours and was then shut off without how- 
ever admitting the air, and the animals were found to be in a 
normal condition the next day. In one such experiment they 
remained alive for a week, but this experiment was somewhat 
vitiated by the fact that a small quantity of chlorophyll derived 
from disintegrated Euglena cells was present in the drop, and 
during the hours of daylight could have furnished a certain amount 
of oxygen, though only a small portion of the carbon dioxide in 
the chamber could have been gotten rid of in thisway. However, 
even if these last results be discarded the fact remains that Col- 
pidium is exceedingly resistant to carbon dioxide and may remain 
alive in a drop saturated with it for many hours. Prowazek (’03) 
found in an experiment of a different sort that Colpidium survives 
a simple lack of oxygen far better than Paramecium caudatum. 

Carbon dioxide is not without its effects on Colpidium, how- 
ever. Locomotion, while not so markedly affected as in most of 
the other forms, nevertheless becomes less active than normally. 
Before death occurs the animals become quiescent, often at the, 
edge of the drop, and the cilia beat more and more slowly until’ 
they stop. One point of interest is that the animals, while they 


530 MERKEL HENRY JACOBS 


move about in a fairly normal manner appear to take no food, 
and the food vacuoles present at the beginning of the experiment 
gradually are lost until the bodies of the organisms become re- 
markably transparent. For along time there is no swelling of the 
body, but rather a narrowing, doubtless due to the loss of the 
food vacuoles; towards the last there may be a certain amount of 
swelling but the pellicle very rarely ruptures as it does in various 
species of Paramecium. 

5. Coleps hirtus. This species forms a marked contrast with 
the preceding, being the least resistant of all the ciliates studied. 
Even the most resistant individuals hardly approach the least 
resistant ones of the other forms. The effect of the carbon diox- 
ide is seen almost instantly. The animals show no decided nega- 
tive reaction, apparently being overcome too quickly for such to 
occur. The normal, rather active movements of locomotion cease 
within a few seconds and thereafter only irregular ‘vibrating’ 
movements occur up to the time of death, of which the average is 
well under five minutes. Even the most resistant individuals do 
not live for ten minutes. The visible effects of the carbon dioxide 
are greater on this form than any of the others studied. The 
barrel-shaped body swells until it is broadly elliptical or even 
circular in outline, the increase in volume being very decided. 
At the same time the plates of the armature become indistinct 
and disappear, giving the appearance at least of actually being 
dissolved away. In consequence the body becomes very trans- 
parent and the protoplasm may be seen to undergo coagulation 
phenomena. The cilia sometimes beat after the armature has 
partly or entirely disappeared but usually their movements cease 
early. The cell in most cases bursts, generally at one of the ends; 
sometimes this occurs in two or three minutes, before much swell- 
ing has taken place. 

6. Blepharisma lateritia. This on the whole is a very resistant 
form, being second only to Colpidium in this respect. Itshows, 
however, rather more individual variation in the same culture 
than most of the other forms studied, isolated individuals some- 

‘times succumbing quite early. Strangely enough, in spite of its 
general resistance its movements are very quickly affected. At 


EFFECTS OF CARBON DIOXIDE 531 


first it exhibits a slight negative response which, however does 
not last very long and is not so decided as in the case of many of 
the other forms. Very soon its movements, never very active, 
become markedly slowed, and for long periods there is either no 
locomotion at all or this is very slow. The membranelles and cilia, 
however, keep on beating up to the time of death, which in the 
majority of cases occurs in from three to six hours. These two 
structures in Blepharisma seem to have about the same resistance. 
Two phenomena which are particularly characteristic of this 
species are the marked change in form which occurs and the tend- 
ency towards the formation of large vacuoles in the protoplasm. 
The body, originally lanceolate, within an hour or two often 
becomes very broadly ovate or sometimes almost circular in out- 
line. Corresponding to the general variability of the species in 
other respects, there is a great individual variation in this regard 
also. ‘Towards the end, considerable distortion of the body occurs 
but bursting is rare. 

7. Euplotes patella. This form, on account of its peculiar 
cuirass-like modification of the pellicle might be expected to show 
a high resistance, but such is not the case. Its resistance in 
general is somewhat below that of Paramecium aurelia. The 
effect of the carbon dioxide on it becomes apparent very early. 
It is not markedly stimulated, though it does show a negative 
response at first. The normal movements of locomotion dis- 
appear as a rule after five to ten minutes, although the membra- 
nelles sometimes keep on moving an hour longer. However, in 
most cases the average time of death is considerably less than 
this—generally under half-an-hour. The cell very early becomes 
much distorted and the pellicle of the ventral surface ruptures, 
allowing the escape of drops of clear protoplasm, while the macro- 
nucleus at the same time becomes very distinct and granular. 
Even after these changes have occurred, however, the membra- 
nelles continue their beat for a considerable time; the cirri are 
almost as resistant, though their movements are very irregular 
and uncoordinated towards the last. The average time required 
for all movements to cease varied in different experiments from 
about twenty-five minutes to a little over an hour. Rossbach 


532 MERKEL HENRY JACOBS 


(72) claims that not only Euplotes but also Stylonychia and Chil- 
odon and higher animals as well were completely killed by carbon 
dioxide in three minutes. Such low figures give grounds for the 
suspicion that his gas was not pure or that some other disturbing 
factor was present. 

8. Vorticella nebulifera. This in many respects is the most 
interesting form studied. It presents a case where both vibratile 
structures (membranelles) and contractile ones (myonemes of 
the ‘bell’ and contractile filament of the stalk) are highly devel- 
oped, and it is a point of considerable interest to compare the 
effect of carbon dioxide on these two classes of structures in the 
same cell. Its effects are as follows. Almost instantly when the 
gas is turned on the animal is strongly stimulated and makes 
perhaps half-a-dozen violent contractions of the stalk, which 
then slowly relaxes, and thereafter so long as the gas is allowed to 
flow neither contracts spontaneously nor can be made to do so by 
mechanical stimuli. The time required to produce this paralysis 
of the stalk varies from thirty to sixty seconds. The myonemes 
of the ‘bell’ are similarly affected and the animal remains 
fully expanded throughout the experiment. If the gas is not 
allowed to act too long, full and speedy recovery may occur on 
removal to the air. For instance, in one experiment after a five 
minutes’ exposure to carbon dioxide and a subsequent five min- 
utes’ exposure to the air the animals were all normal in every 
respect. If the exposure to the gas is longer continued, however, 
permanent injury to the stalk results. After fifteen minutes the 
animals show a strong tendency to drop off of their stalks and the 
latter can be seen to be altered in appearance, the contractile 
filament becoming broken up into irregular refractive fragments 
and droplets. If the detached animals recover, they regenerate 
a new stalk. 

The effect on the membranelles is almost the reverse of that on 
the contractile structures. At first they may show a temporary 
cessation of movement but usually inside a few moments they 
begin beating again and may continue to do so forthree-quarters 
of an hour or more. Sometimes they stop temporarily and then 
start again even in the carbon dioxide while, removed to the air, 


EFFECTS OF CARBON DIOXIDE 533 


they show considerable powers of recovery after a lengthy period 
of rest. In this respect they differ from the membranelles of 
Euplotes and Blepharisma which in these experiments never 
could be made to resume their beat after having having completely 
stopped. Doubtless this difference is due to the fact that in Vor- 
ticella they are accustomed to stopping every time the disc is 
retracted, while in the other two forms they normally remain in 
continuous motion and are stopped by nothing short of actual 
injury to the cell. 

The powers of recovery of Vorticella after all movements have 
ceased are quite considerable. In one experiment after three- 
quarters of an hour practically all of the individuals had become 
quiet, and many had been in this condition for half-an-hour or 
more. Five hours after exposure to the air in a moist chamber 
about half of them had recovered and many had begun to regen- 
erate the missing stalk, which was already one-tenth to one-half 
the length of the body. By the next day these stalks were one- 
half to two times the body length. In this experiment about 50 
per cent of the individuals never recovered and this is typical of a 
number of experiments that were tried. It may be said, there- 
fore, that certain of the movements of Vorticella are almost in- 
stantly affected and others only after a much longer time. Even 
after all visible movements have ceased the powers of recovery 
of the animals are considerable. 

In a few instances individuals were observed which, before the 
beginning of the experiment, had formed the circlet of cilia used 
in the free-swimming existence. Such cilia were about as resist- 
ant as the membranelles, though they were not observed to beat 
again after having stopped. In no ease did individuals which 
broke from their stalks during the course of the experiment on 
account of the effect of the carbon dioxide, form such cilia, but 
their locomotion was entirely by means of their membranelles. 

The Vorticella cell apparently shows no tendency to burst and 
form droplets of protoplasm, but a considerable change in form 
may occur. In an atmosphere of carbon dioxide V. nebulifera 
inside a few minutes tends to assume the more rounded form 
characteristic of V. campanula. In the individuals which are 


534 MERKEL HENRY JACOBS 


killed the protoplasm becomes brownish and opaque, doubtless 
due to coagulation phenomena. 


B. Flagellates 


1. Peranema trichophorum. This form shows conditions which 
suggest those already noted in Vorticella. As is well known, 
Peranema has a single very prominent flagellum, which in loco- 
motion is directed straight forward. Ordinarily it is quite rigid 
except the tip, whose movements cause a slow forward progres- 
“sion of the animal. During progression, and also when otherwise 
at rest, the body shows very decided changes in form, these ‘meta- 
bolic’ movements, or contortions, being one of the most striking 
characteristics of the species. When exposed to carbon dioxide 
Peranema responds perhaps as quickly as any of the forms studied. 
Its forward progression ceases almost instantly and after a few 
preliminary, and rather vigorous contortions, the body suddenly 
becomes paralyzed in whatever state of contraction it may hap- 
pen to be in at the time. Only rarely does the animal have time 
to contract to a spherical form before being overtaken with this 
paralysis; consequently the typical appearance of the body is an 
irregular mass which does not change its form so long as the stream 
of carbon dioxide is allowed to flow. 

The flagellum responds differently. Contact with the carbon 
dioxide causes it to beat with a swinging motion in which the proxi- 
mal as well as the distal portion is concerned. Sometimes these 
movements are rather vigorous, but they never give rise to loco- 
motion. They may continue with more or less uniformity for a 
half or even three-quarters of an hour; at the end of that time the 
flagellum gradually comes to rest. All visible movements have 
now ceased, but the animals are not necessarily dead. Experi- 
ments were tried to determine what powers of recovery Peranema 
possesses after becoming perfectly motionless. It was found that 
when the drop containing them is removed from the gas chamber 
and placed in contact with the air, a considerable percentage 
of the individuals may recover after an exposure of an hour and 
a quarter to carbon dioxide, even though all movements have for a 


EFFECTS OF CARBON DIOXIDE 535 


long time ceased. In the case of individuals exposed for two 
hours, however, no recoveries occurred although the drop was kept 
under observation for twenty-four hours. The time required 
for recovery to occur depends on the length of time the gas has 
acted. After an exposure of five minutes, metabolic movements 
of the body begin in less than a half minute after contact with the 
air, and the animals may be entirely normal in ten minutes. After 
a longer exposure the time required is much greater. In .one 
experiment after an exposure of thirty-four minutes, but few of 
the animals were in a state of normal activity after an hour-and- 
a-half in the air, though at the end of four hours most of them 
showed no signs of injury and were normal in every respect. 

Peranema therefore represents a form in which the effects of 
carbon dioxide on locomotion and the contractile movements of 
the body are almost instantaneous, but which is killed only after 
a prolonged exposure. The point of greatest interest is that 
while certain movements of the body are brought to a standstill 
in a few seconds the flagellum may continue to beat for half-an- 
hourormore. Weare therefore dealing with structures concerned 
in producing movements in the same cell which show a consider- 
able physiological difference. 

2. Euglena viridis(?). This form is in some respects more 
resistant and in others less resistant than Peranema. The time 
required for locomotion to cease is longer and the powers of _ 
recovery after an extended exposure greater, but the flagellum 
is much more sensitive and the length of time required to bring 
to an end all visible movements is considerably less. Like Pera- 
nema, Euglena shows no decided negative reaction to the gas as 
do many of the forms studied, though locomotion may persist for 
a few moments. Often the first effect of the gas is to cause a 
short temporary cessation of all movements, which quickly reap- 
pear. Soon, however, movements of progression cease andthe 
organisms show signs of life only by vibrating movements which 
are due to the abnormal beat of the flagellum. These gradually 
cease and the animals sink to the bottom of the drop motionless 
and perfectly extended. The time required to produce cessation 
of all movement varies from two or three to ten minutes. The 


536 MERKEL HENRY JACOBS 


particular Euglena studied, which was close to, but probably not 
identical with, E. viridis, was not one which very actively changes 
its form, and consequently was not a very favorable one in which 
to observe the effect of carbon dioxide on the contractile move- 
ments of the body. It may be said, however, that while ‘eugle- 
noid’ movements were observed in many individuals before the 
beginning of the experiment and also after recovery, they never 
occurred during its progress, consequently the conditions here 
probably are the same, even if less striking, than those found in 
Peranema. 

After the organisms have settled to the bottom of the drop and 
become motionless the only change that can be observed is a 
gradual slow swelling of the body. At the same time there is a 
slight shortening which, however, is not sufficient to account for 
the greater thickness of the organisms as careful measurements 
show. This swelling continues until the shape of the body has 
changed from cylindrical to broadly elliptical in outline and the 
chlorophyll bodies appear forced apart from each other. In 
extreme cases the cell may appear to be at the point of rupture, 
though this rarely occurs, the pellicle being very tough and elastic. 

Although all movements cease in Euglena in ten minutes or 
less, it requires a much longer time to kill the organisms. Even 
after an exposure of three hours about a third of the individuals 
eventually recovered, though the time required was considerable. 
The recovery of Euglena is far slower than that of Peranema. 
After an exposure of seven minutes no recoveries could be notiecd 
a half hour after removal to the air, although they began to occur 
soon after that, and in an hour and a quarter practically all the 
individuals were normal. After an exposure of two or three hours, 
the time required for recovery is three or four hours or more. 

3. Chilomonas paramecium. This form shows great individual 
and also cultural variation. While in a few cases the animals 
become motionless in fifteen or twenty minutes, the average time 
required generally is three-quarters of an hour or more. Many 
resistant individuals retain their movements for several hours. 
In general, therefore, this may be said to be a form with a high 
resistance. Unlike the two previous flagellates, Chilomonas 


EFFECTS OF CARBON DIOXIDE 537 


shows most decided reactions to the presence of carbon dioxide. 
The first effect is often to cause a temporary cessation of motion 
which lasts however only for a few seconds, after which the ani- 
mals are remarkably active. They show a striking tendency to 
seek the center of the drop at first, later becoming uniformly dis- 
tributed again. Their motions at first are normal but gradually 
the animals come to rest and give evidences of life only by a slow 
rotation or by quick darting movements which they occasionally 
make. In practically every case the cell becomes circular in out- 
line and if the experiment be long continued may actually burst. 
Chilomonas like the preceding form also has considerable powers 
of recovery after all motion has ceased. In one experiment even 
after an exposure to carbon dioxide of two-and-a-half hours about 
75 per cent of the individuals eventually recovered after exposure 
to the air. In other cases, however, even after a shorter exposure 
the mortality is greater. 

4. Entosiphon sulcatum. This form unfortunately was studied 
in only one experiment in which, however, a considerable number 
of individuals was present. Judging from these rather incom- 
plete data, it is by far the most resistant of the flagellates exam- 
ined. After an exposure of five hours it not only was alive,but 
the movements were not very markedly affected. Both flagella 
continued to beat, though in rather a stiff and jerky fashion, and 
slow forward progression continued. How long it would have 
survived cannot be said, but probably the time would have been 
considerably above that mentioned, since when the experiment 
had to be ended none of the animals had as yet been killed. 


IV. DISCUSSION OF RESULTS 


From the results given it is apparent that all of the forms 
studied are injured and eventually killed by pure carbon dioxide, 
but that the resistance of the different forms is very different. 
Colpidium colpoda can withstand without injury an exposure 
of many hours, while Coleps hirtus is killed in three or four minutes. 
Sometimes the time of cessation of visible movements and the 
point at which the cell is so severely injured that recovery cannot 
occur, may coincide, as in most of the ciliates (in Euplotes patella 


538 MERKEL HENRY JACOBS 


irreparable injury probably occurs before the membranelles cease 
beating) while in other cases, the animal is capable of full recovery 
long after all movements have ceased, e.g., flagellates and Vorti- 
cella. Some animals which are otherwise fairly resistant to car- 
bon dioxide, as shown by their powers of recovery after 4 pro- 
tracted exposure to it, or by the long continuation of visible 
movements, show its effects very quickly by their inability to 
carry on normal locomotion in its presence. Peranems is the 
most striking example of this condition, Euglena and Euplotes 
also being relatively quickly affected. In their primary response 
the different forms also show distinct differences. The three 
species of Paramecium studied as well as Colpidium, and Chilo- 
monas show a decided negative reaction and an effort to escape 
from it. This reaction is less marked in Blepharisma and Eu- 
plotes, while in the other forms it is practically lacking, probably 
because normal movements are so quickly interfered with. (En- 
tosiphon was not studied in this connection.) It will be seen, 
therefore, that the different forms studied show certain charac- 
teristic differences in reactions and general resistance to carbon 
dioxide. 

It has already been pointed out that there is a certain amount 
of individual and cultural variation in the same species, which 
makes it impossible to put in exact quantitative form the time 
in which death oceurs, etc. Nevertheless the relative resistance 
of each form as compared with other forms from the same culture 
is fairly constant and furthermore it is at least possible to say that 
certain forms always have a high, and others always a low resist- 
ance. While some forms may ‘overlap,’ others, as for example 
Colpidium and Coleps, never do. ; 

The observations here recorded are not the first that have been 
made on the differences in resistance to carbon dioxide shown 
by different organisms. Frinkel (’88) studied the effect of this 
substance on various bacteria with the result that some were found 
to thrive almost as well as in air, others had their development 
checked but were not killed, while others were quickly destroyed. 
Lopriore (’95) also, in his careful experiments on the effects of 
carbon dioxide on the spores and mycelia of fungi and the pollen 


EFFECTS OF CARBON DIOXIDE 539 


tubes of angiosperms, found decided specific differences. Many 
other scattered observations exist, which however, it is difficult 
to compare on account of the different methods employed in 
obtaining them. 

It is interesting to consider the results here obtained in con- 
nection with those of Jennings and Moore (’01) on the chemotactic 
effect of carbon dioxide on various protozoa. Of the four forms 
mentioned by them as being attracted by this substance, three 
(i.e., Paramecium caudatum, Colpidium colpoda, and Chilomonas 
paramecium) have been studied in these experiments and all show 
marked powers of resistance, as well as a strong negative response 
when the concentration is suddenly made high in the edges of the 
drop. Of the forms found by them to be indifferent, unfortu- 
nately only two genera (Euglena and Euplotes) were available, but 
these both showed a relatively low resistance as compared with 
related forms, at least so far as the continuance of locomotion is 
concerned. It would be interesting to study the other members 
of their list in this connection. Doubtless many other facts of 
behavior could be brought into line with such physiological pecu- 
liarities as the one under consideration. 

One of the most interesting results that appears from these 
experiments is the striking difference that seems to exist between 
the contractile elements of the cell—the myonemes—on the one 
hand, and the vibratile ones—cilia, membranelles, and flagella 
on the other. The former are very quickly thrown out of func- 
tion while the latter continue their normal movements for a long 
time. The best illustration of this point is Vorticella, in which 
the contractile filament of the stalk, and the myonemes, after a 
primary stimulation, are inside a minute or less completely para- 
lyzed, while the membranelles perhaps after stopping for a short 
time continue to beat for three-quarters of an hour. Such results 
are in agreement with those obtained by other workers. Nere- 
sheimer (’03), for example, found that the myonemes and mem- 
branelles of Stentor are differently affected by substances like 
morphin, which paralyze the former and do not affect the latter. 
Lillie (12) has observed that the cilia of Arenicola larvae continue 
their acvitity for hours in isotonic sugar or magnesium chloride 


540 MERKEL HENRY JACOBS 


solutions, or in solutions of chloroform or ether, which prevent 
completely all muscular movements. Recently Mayer (711) has 
found that the effects of many ions on ciliary and muscular move- 
ments are exactly opposite those that depress the one stimulating 
the other. He found that the ciliary movements of trochophore 
larvae at first cease in water charged with carbon dioxide but 
later start again. It is well known that carbon dioxide at first 
stimulates and later depresses muscular movements in the higher 
vertebrates (Lee, ’07). In Vorticella in the same cell this antag- 
onistic action appears very clearly. In other forms it is harder 
to demonstrate a primary depression of ciliary activity, possibly 
because of the response of the organism as a whole in an adaptive 
way. In Chilomonas, however, the primary depression of the 
movements of the flagella nearly always occurs. 


V. SUMMARY 


1. Each of the twelve forms studied reacts to carbon dioxide 
in a characteristic way and has a characteristic resistance, which 
is highest in Colpidium colpoda, which remains alive many 
hours, and lowest in Coleps hirtus, which is killed in a few minutes. 
A certain amount of individual and cultural variation may occur 
which prevents the expression of the resistance of the species in 
absolute terms. Compared with other forms under the same 
conditions, however, the relative resistance is fairly constant. 

2. Some forms are killed outright very quickly (Coleps hirtus 
and Paramecium bursaria). In others all movements are stopped 
in a few minutes but death occurs relatively late, the powers of 
recovery being high (Euglena). In still others, locomotion ceases 
very promptly but movements of the cilia, flagella, etc., may per- 
sist for a long time (Peranema trichophorum, Euplotes patella, 
ete.). In theremainder, more or less normallocomotion continues 
for a considerable time (most of the ciliates, Chilomonas and 
Entosiphon). The result in all cases, however, if the experiment 
be long enough continued, is cessation of movements and death. 

3. In the same cell the contractile elements are usually quickly 
paralyzed (Vorticella and Peranema) while the vibratile structures 
(cilia, membranelles, flagella) are much more resistant. In some 


EFFECTS OF CARBON DIOXIDE 541 


cases (Vorticella) the action of carbon dioxide on these two classes 
of structures is exactly opposite, the contractile elements being 
first stimulated and then paralyzed and the vibratile ones often 
temporarily stopped and then started again. 

4. Ordinary cilia and their modifications, membranelles and 
cirri when present in the same cell show approximately the same 
resistance. Flagella show great variation, that of Euglena being 
paralyzed in a few minutes and those of Chilomonas and Ento- 
siphon remaining active for several hours. 

5. In ciliates in general, with the exception of the specialized 
Vorticella, recovery after complete cessation of movement is 
impossible; in the flagellates, movement cease long before the cell 
is permanently injured. 

6. The general effects of carbon dioxide on the cell are to cause 
(a) cessation of movement, (b) absorption of water and consequent 
swelling, (c) injury to the cell wall, (d) death and coagulation of 
the protoplasm. 


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of oxygen and certain poisons. Am. Jour. Physiol., vol. 1, pp.210-214. 


FRANKEL, C. 1888 Einwirkung der COs auf die Lebenstitigkeit der Mikro- 
organismen, Zeitschr. f. Hygiene, vol. 5, pp. 332-362. 


Jennines H. 8S. and Moors, E. M. 1901 Studies on reactions to stimuli in 
unicellular organisms. VIII. On the reactions of Infusoria to carbonic 
and other acids with especial reference to the causes of the gatherings 


929 


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Lez, F. S. 1907 The action of normal fatigue substances on muscle. Am 
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Litiiz, R. 8. 1912 Antagonism between salts and anaesthetics. Amer. Jour. 
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Logs, J. and Harpesty, I. 1895 Ueber die Localization der Athmung in der 
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Mayer, A. G. 1911 The converse relation between ciliary and neromuscular 
movements. Papers from the Tortugas Laboratory of the Carnegie 
Institution of Washington, vol. 3. 


542 MERKEL HENRY 


on” ie 
pee - 
Neresnemer, E. 1903 Ueber die Hohe histo 


hi logischer Differe Ale 
heterotrichen Ciliaten. Arch. f. Protistenkunde vol. 


Prowazex, S. von 1903 Studien zur Biologie der Zelle. Zeitse 
Phys., Bd. 2. : gt ; 


Rosspacu, M. J. 1872 Die rhythmischen Bewegungserscheinunge 
_ fachsten Organismen und ihr Verhalten gegen physikalische A 
und Arzneimittel. Verh. d. phys. med. Gesell. zu Wiirzburg 

vol. 11, pp. 179-242. : . 2 


é 


1 


) 


\ 


ON THE ADAPTATION OF FISH (FUNDULUS) TO 
HIGHER TEMPERATURES 


JACQUES LOEB ann HARDOLPH WASTENEYS 


From the Rockefeller Institute, New York 
I. INTRODUCTION 


It is a well known fact that organisms can stand a higher tem- 
perature if the latter is raised gradually than if it is raised sud- 
denly. This phenomenon is referred to in biology as a case of 
adaptation. Dallinger states that he succeeded in adapting cer- 
tain protozoa to a temperature of 70° by gradually raising their 
temperature during several years. 

Schottelius had found that colonies of Micrococcus prodigi- 
osus when transferred from a temperature of 22° to that of 38° 
no longer formed pigment and trimethylamin. When transferred 
back to the temperature of 18° to 22° the formation of pigment 
and of trimethylamin was resumed. After the cocci had been 
cultivated for ten or fifteen generations at 38° they failed to form 
pigment even when transferred back to 22°. 

These experiments became the starting point for similar experi- 
ments by Dieudonné. He used Bacillus fluorescens putidus which 
forms a fluorescein pigment and trimethylamin. The optimal 
temperature for this bacillus is 22°. At 35° it grew but did not 
form pigment or trimethylamin. At 37°.5 growth ceased. Re- 
transferred to 22° pigment and trimethylamin were again formed. 

Dieudonné! exposed a culture of this bacillus to 35°. After 
twenty-four hours a second culture was taken from this and also 
kept at 35°, and this process was repeated each day. The fif- 
teenth generation thus cultivated at 35° began to form some pig- 
ment and from the eighteenth generation on, at 35° the formation 


! Dieudonné, Arb. aus dem kaiserl. Gesundheitsamte, vol. 9, p. 492, 1894. 


543 


THE JOURNAL OF EXPERIMENTAL ZOGLOGY, VOL. 12, No. 4 


544 JACQUES LOEB AND HARDOLPH WASTENEYS 


of pigment and trimethylamin had become as good as that in 
the cultures kept at 22°. The organisms had, therefore, become 
‘adapted’ to a temperature of 35° which at first was unfavorable. 

Dieudonné did not succeed in causing pigment formation in 
this bacillus at a higher temperature than 35° although the bacil- 
lus finally grew at a temperature of 41°.5. He obtained similar 
results in experiments on other pigment-forming bacteria. Daven- 
port and Castle? made experiments with tadpoles of frogs. One 
lot of eggs and tadpoles was kept at about 15°, a second lot for 
twenty-eight days at 25°C. While the tadpoles raised and kept 
at 15° went into heat rigor at 40°.3C., those kept for twenty-eight 
days at 25° were not affected by this temperature but went into 
heat rigor at 43°.5. Their resistance to high temperature had 
therefore risen 3°.2. When the latter tadpoles were put back for 
seventeen days to a temperature of 15° they had lost their resist- 
ance to high temperature to some extent but not completely, 
since they went into heat rigor at 41°.6. The authors suggest 
that this adaptation to a higher temperature is due to a loss of 
water on the part of the protoplasm. They assume that the rise 
in temperature causes a comparative acceleration in the excretion 
of water on the part of the tadpoles. The hypothesis of these 
authors is based upon the fact, that spores of bacteria are more 
resistant to high temperatures than the bacteria. While this is 
a fact, nothing in the experiments of Davenport and Castle proves 
that the amount of water in the tadpoles is diminished by the rise 
in temperature. The idea of Davenport and Castle was put to 
a direct test by Kryz.? He kept frogs, toads and salamanders 
at room temperature, and at temperatures as high as 40°C., for 
a number of days or weeks and tested the coagulation temperature 
of the muscle plasma for both ranges of temperatures. He found 
that the coagulation temperature was identical for the animals 
kept at low and those kept at high temperatures. 

The following observations by Kammerer‘ indicate also an after- 
effect of the raising of temperature. Lacerta muralis from the 


2 Davenport and Castle, Arch. f. Entwickelungsmechanik, vol. 2, p. 227, 1896. 
3 Kryz, Arch. f. Entwickelungsmechanik, vol. 23, p. 560, 1907. 
4 Kammerer, Arch. f. Entwickelungsmechanik, vol. 30, p. 379, 1910. 


ADAPTATION OF FISH TO TEMPERATURE 545 


cooler climate of Nieder Oesterreich darkens already at a tempera- 
ture of 25° and becomes perfectly black at a slightly higher tem- 
perature. Lacerta muralis from the warmer climate of Italy 
remains perfectly normal at 25° and darkens only at 37°. Kam- 
merer points out that the higher temperature at which the latter 
lizards had lived prevented the effects which the same rise in tem- 
perature produced in lizards that had previously lived at a lower 
temperature. 


Il. THE INFLUENCE OF THE CONCENTRATION OF THE 
SOLUTION UPON THE RESISTANCE OF FUN- 
DULUS TO HIGH TEMPERATURE 


We used as material for our experiment half-grown Fundulus 
which were caught in Long Island Sound in January and kept in a 
room in the laboratory, the temperature of which varied a little 
around 10°C. Two fish were put into each of the following solu- 
tions: H.O, m/128, m/64, m/32, m/16, m/8, m/4, NaCl + KCl+ 
CaCl, in the usual proportion. It was found that the higher 
the concentration of the solution—up to a certain limit—the 
longer the fish could survive in a given high temperature. The 
method consisted in putting a number of battery jars with 500 ce. 
of the solution into a large water bath the temperature of which 
was constantly watched and kept constant. When all the solu- 
tions had reached the desired temperature the fish were introduced. 
In a number of cases the experiment was continued for several 
days in a bacteriological thermostat. Table 1 gives the duration 
of life for these fish in each of the solutions for various tempera- 


TABLE 1 
DURATION OF LIFE OF FUNDULUS IN a 
TEMPERA- 5 Se } Rot 
TURE | 
| H:0 m/128 | m/64 m/32 m/16 m/8 m/4 im | CaCl 
| 

: | ea 1 
25° 4hrs. indef. indef. -indef. indef. indef. indef.  indef. 
27° | lhr. | 2hrs.| 2hrs.)indef. indef. indef. indef. | indef. 
BOP i cae Sie 1/50! 62’ 90’ indef. indef.  indef. 
31° | 43? | 25’ Vit 50’ = indef. | indef. | indef. 


33° | 2’ 6’ 9’ 7 9’ 80’ 


546 JACQUES LOEB AND HARDOLPH WASTENEYS 


tures. The term ‘indefinite’ means that the fish were alive and 
apparently normal at the end of the experiment. 

These experiments were repeated with the same material with 
approximately identical results; 33° was as a rule the upper limit 
' the fish could resist during the month of January. Occasionally 
a fish would survive a sudden exposure to a temperature of 33° in 
a m/4 solution of NaCl + KCl + CaCl, but such fish were no 
longer normal and would swim on their side. 

It was next ascertained in which concentration the fish could 
resist the highest temperature. It was found that this concentra- 
tion was m/4. In m/2 and 3 m they were not able to resist as 
high a temperature as in m/4 or 2m solution of NaCl + KCI + 
CaCl.. When sea-water was substituted for Ringer’s solution 
(NaCl + KCl + CaCl.) the results were the same as represented 
in table 1. 


TABLE 2 
DURATION OF LIFE OF FUNDULUS IN | 
TEMPERA- : the DEX- 
TURE TROSE 
H:.0 m/32 m/16 m/8 m/4 im m/2 gm 
29 40 40 30 40 100 40 7 40 


31° 16’ | 20’ 166 || 20% iy ||) BAP BR 4 ie! 

The fact that the temperature which the fish could resist was 
the higher, the higher the concentration of the solution, suggested 
the possibility that the loss of water on the part of the fish in- 
creased their resistance. This explanation, however, does not 
agree with the observation made by Sumner and corroborated by 
us that Fundulus undergoes practically no change in weight when 
put into distilled water or when put back into sea-water. The 
idea that loss of water made Fundulus more resistant to heat 
could be tested by the substitution of sugar solution for Ringer’s 
solution or sea-water. It was found that dextrose solutions were 
not able to protect the fish, in fact such solutions were little if 
any better than H.O (table 2). 

The duration of life of the fish in the dextrose solutions is about 
identical with that in distilled water. This excludes the sugges- 
tion that osmotic phenomena determine the influence of the con- 


s "a 


ADAPTATION OF FISH TO TEMPERATURE 547 


centration found in the experiments with Ringer solution or with 
sea-water; especially if we consider the fact that Loeb found that 
Fundulus ean live for over a month in an m/8 solution of dextrose 
at ordinary room temperature (if the solution is renewed every 
day/- 

The difference between the results expressed in tables 1 and 2 
suggested that the protective action of the Ringer solution was 
of the nature of a specific salt action. We, therefore, tested the 
idea whether or not other salt solutions, e.g., NaCl or CaCl, 


could afford the same protection. It was found that this was true 


to a slight degree in the case of NaCl but not in the case of CaCls. 
Table 3 may serve as an example. 


TABLE 3 


DURATION OF LIFE OF FUNDULUS IN 


TEMPERA- _ > —— SS | z 
LURE NaCl 
H.0 m/64 m/32 m/16 m/8 m/4 im m/2 | 
— es | — — 
29° By) Boy! 40/ 50° indef. 120’ 120’ 30° 
| H20 | m/64 m/32 m/16 m/8 m/4 im } CaCh 


29° | 20/ “109 60' 90' 60/ 30’ 10’ 


In an m/8 NaCI solution some but not all of the fish could live 
indefinitely at 29°; in an m/8 or m/4 solution of sea-water or Ringer 
solution they could all live indefinitely at 29°. We must therefore 
conclude that the protection which sea-water or a Ringer solution 
gives Fundulus against a high temperature is due to a specific effect 
of the combination of the three salts NaCl, KCl and CaCl in the 
right proportion. The idea presented itself that this protective 
action of the salts was the expression of an antagonism between 
the salts and a substance produced at a great velocity at a higher 
temperature, e.g., an acid. Experiments, to be discussed a little 
further on, on the immunization of fish against a high temperature 
eliminate this possibility. 

Experiments were tried on tadpoles and on a species of fresh 
water fish to ascertain whether these animals could resist high 


548 JACQUES LOEB AND HARDOLPH WASTENWYS 


temperatures better in an m/8 Ringer solution than in tap water 
or weaker Ringer solutions. No positive results were obtained. 


Ill. THE ADAPTATION OF FUNDULUS TO HIGH TEMPERATURES 


Fish from the cold room (10° to 14°C.) were kept for lengths 
of time varying from one hour to several hours at 27°C., in an m/4 
Ringer solution, and then put into H.O, m/64, m/32, m/16, and 
m/8 Ringer solution at 31°. It was found that the longer they 


stayed at a temperature of 27° the more resistant they became to . 


the temperature of 31°, so that finally they survived at that tem- 
perature even in distilled water. 

One sees that fish that had been kept in m/4 Ringer solution 
for seventy-two hours lived indefinitely in 31° even in distilled 


TABLE 4 


PREVIOUSLY EXPOSED TO DURATION OF LIFE OF FUNDULUS AT 31° IN 


27° In m/4 RINGER . SEE ae =a, SS Ip a BENGE 


BOO EO NEO x H:0 m/64 m/32 m/16 m/8 CO 
0 hour 13! 40/ 43' 120’ or indef. 
indef. 
1 hour 30! 95’ 150’ 102 indef. | 
4 hours 62’ indef. indef. indef. indef. 
23 hours 180’ indef. indef. indef. indef. 


72 hours | indef. indef. indef. indef. indef. | 


water. Itshould be stated that each experiment was accompanied 
by a control experiment with animals that had not been immun- 
ized and in all cases the fish die in less than an hour in 31° in solu- 
tions below m/8. 

Fish kept in the cold room (10° to 14°C.) and put from there 
directly into a solution of 35° neafly all died in a few minutes 
even in the optimal solution of m/4 sea-water or Ringer. Experi- 
ments were undertaken to ascertain the minimum time the fish 
had to be kept at 27° in m/4 sea-water in order to be rendered 
immune to a sudden transfer into m/4 Ringer solution at 35°. 

It should be stated, however, that the fish that had been kept 
at 27° for sixteen and twenty-one hours did not all survive in 35° 


ADAPTATION OF FISH TO TEMPERATURE 549 


TABLE 5 
PREVIOUSLY IN THERMOSTAT AT 27° DURATION OF LIFE OF FUNDULUS AT 35° 

0 hour BY 
1 hour of 
3 hours 3” 
6 hours oe 
8 hours 6’ 

16 hours indefinitely 

21 hours indefinitely 

44 hours indefinitely 


while all the fish that had been kept at 27° for forty-four hours 
survived. ; 
This experiment was repeated with a second set of fish with the 


results shown in table 6. = 
TABLE 6 
PREVIOUSLY IN THERMOSTAT AT 27° FOR DURATION OF LIFE OF FUNDULUS AT 35° m/4 RINGER 
1 hour rid 
ss 3 hours 2 
6 hours | . 
8 hours partly ndefinite 
16 hours 
21 hour | . | partly indefinite, but better than pre 


24 hours vious group 


32 hours 
40 hours | 
48 hours } in efinite 
65 hours 
72 hours 


It seems that if Fundulus are kept more than twenty-four hours 
in a temperature of 27° they can with impunity be put into an 
m/4 Ringer solution of 35°. If fish are kept from six to sixteen 
hours at 27° and then suddenly transferred to 35° (in m/4 Ringer 
solution) some of them die and some survive; and the tendency to 
survive increases the longer the fish are previously kept at 27°. 


ee 


550 JACQUES LOEB AND HARDOLPH WASTENEYS 


A series of experiments was carried out in which fish were kept 
ina thermostat at 30°C. for various lengths of time to test whether 
this accelerated their adaptation to a temperature of 35°. This 
was true only to a slight extent. In all these experiments the fish 
were suddenly transferred to an m/4 Ringer solution at 35°. 

We were curious to know if these animals could also survive if 
suddenly transferred to a temperature of 35° in distilled water. 
This is indeed the case as table 7 shows. 

Fundulus can become adapted to a temperature of 35° in dis- 
tilled water if they are kept for two days or longer at a tempera- 
ture of 27°. It seemed to make no difference whether the fish 
had been kept at 27° in m/4 sea-water or in distilled water. 


TABLE 7 


> E L 
PREVIOUSLY EXPOSED TO 27° FOR DURATION OF LIFE OF FUNDULUS ROE aa 


PUT INTO DISTILLED WATER OF 


0 days (control) 4’ 
2 days indefinite 
3 days indefinite 


Finally experiments were made to see to how high a tempera- 
ture these fish could be adapted in a week. By keeping the fish 
at a temperature of 27° over night and raising them during the 
day to a gradually higher temperature we found that they could 
be kept at the end of the week at a temperature of 40°C., for two 
hours without apparent injury. At a temperature of 41° they 
soon suffered in their power to maintain their equilibrium. They 
were immune to a temperature of 40° not only in an m/4 Ringer 
solution, but also in an m/64 solution. The lot which was in dis- 
tilled water died early during the experiment through an accident. 
It is probable that Fundulus once adapted to a certain tempera- 
ture can stand this temperature in any concentration of a Ringer 
solution below m/4. 


ADAPTATION OF FISH TO TEMPERATURE 551 


IV. THE SUMMATION OF THE IMMUNIZING EFFECTS OF SHORT 
PERIODS OF EXPOSURE TO HIGH TEMPERATURE 


In the immunization experiments described thus far the fish 
had been exposed continuously for a rather long period of time to a 
temperature of 27°. We wanted to know if it was possible to 
immunize them for a higher temperature by exposing them only 
a short period of time each day and keeping them in the interval 
at a temperature of from 10° to 14°C. This would mean that 
the immunizing effect produced in the animal during a short 
exposure to a high temperature would be preserved at least twenty- 
four hours until the next exposure to a high temperature took 


TABLE 8 
DATE DURATION AND TEMPERATURE OF EXPOSURE 
March 
7 1 hour from 17° to 31° 2 hours at 31° 
8 1 hour from 17° to 33° 2 hours at 33° 
9 1 hour from 18° to 35° 2 hours at 35° 
11 3 hours from 11° to 37° 
12 2 hours from 17° to 37° 
13 2 hours from 16° to 37° 
14 2 hours from 17° to 37° 
15 2 hours from 17° to 37° 2 hours at 37° 
16 2 hours from 17° to 37° 2 hours at 37° 
18 2 hours from 17° to 38° 14 hours at 38° 
19 2 hours from 18° to 38° 2 hours at 38° 
20 2 hours from 19° to 39° 2 hours at 39° 


place; and would be added to the immunizing effect of the next 
exposure to a high temperature. Table 8 gives the periods of 
exposure. 

Most of the fish died on the third day when the temperature 
was raised only to 35°. For this reason we did not dare to 
expose the fish for more than a few minutes to a temperature of 
37° on the 11th, 12th, 13th and 14th of March. The fish were ex- 
posed to a higher temperature for not more than four hours on 
one day. We have seen that an exposure of four hours in itself 
does not suffice to create immunity to a temperature of 35° or 
above. Hence the fact that these fish were finally able to resist a 


552 JACQUES LOEB AND HARDOLPH WASTENEYS 


temperature of 39° indicates a cumulative effect of the different 
exposures to a higher temperature. In other words, each heating 
increased the immunity and this gain was not lost during one or 
two days. 


V. THEIMMUNITY TO A HIGH TEMPERATURE IF'‘ONCE ACQUIRED 
IS KEPT FOR MORE THAN FOUR WEEKS 


In order to prove this, fish were put for various lengths of time 
into a thermostat at 27°, tested in regard to their immunity against 
high temperature and then put back into the cold room and tested 
again. A few examples will illustrate this. Five fish were immun- 
ized to a temperature of 39° by exposing them daily for a number 
of hours to an increasing temperature, until they could live in a 
temperature of 39°C. (in m/4 Ringer solution). The process of 
acclimatization extended over a period of twelve days (see pre- 
vious experiment). After this they were kept for eight days con- 
stantly at a temperature of from 10° to 14°C. On the eleventh day 
they were put suddenly into a temperature of 31° and the tempera- 
ture of the water in which they were, was brought, inside of two 
hours, to a temperature of 39°, and then keptatthisheight. A con- 
trol experiment was carried on simultaneously with fish taken from 
the same cold room, which had not been acclimated. The solu- 
tions used were m/4 Ringer. The control fish that had not been 
acclimated to high temperature were dead in one and a half hour 
when the temperature had reached 36°. The acclimated fish kept 
alive for over an hour at 39° when the experiment was discontinued. 
In eight days, therefore, the immunity of the fish to high tem- 
perature had not diminished. 

Four lots of fish had been immunized to a temperature of 35° 
by keeping themtwenty-four ,thirty-two, forty and seventy-two 
hours respectively at a temperature of 27°. After this the fish 
were put into the cold room and kept there at a temperature rang- 
ing from 10° to 15°C., for twenty-eight days. They were then 
put into an m/4 Ringer solution at a temperature of 85°. Simul- 
taneously six fish of the same lot, which had not been immunized 
but kept in the cold room permanently, were put into the same 
temperature and the same solution. Four of the latter fish died 


ADAPTATION OF FISH TO TEMPERATURE 553 


within two minutes, the rest were dead forty minutes later. The 
fish ‘that had been immunized before were, with the exception of 
two individuals, all alive and normal after five hours. Yet, the 
fact that two of the fish suecumbed may be an indication that their 
resistance to 35° was less than immediately after immunization. 
It is quite possible, however, that these two fish which had been 
kept in small dishes for such a long time had suffered through 
this captivity. 

This idea is supported by the fact that in a third experiment 
fish had kept their immunity to high temperature for thirty-three 
days after immunization against 35°. The immunization con- 
sisted in exposing the fish for three and six days respectively to 
27°. After that they were kept in the cold room for thirty-three 
days. When after that time subjected to a temperature of 35° 
they remained perfectly normal for five and a half hours, when the 
experiment was discontinued. 

We made a large number of experiments in which the duration 
of the immunity against high temperatures was tested sooner after 
the process of immunization than in the above mentioned experi- 
ments. In all these experiments it was found that the fish did 
not lose their immunity against temperatures of 39° and 35° 
respectively if they were put into the cold room for a period of 
thirty-three days or less after immunization. 


VI. EXPERIMENTS WITH FISH KEPT AT A CONSTANT 
TEMPERATURE OF 0°.4 C. 


The fish which we used for this experiment were caught in 
January and kept since that time in a cold room in which the 
temperature varied between 10° and 14°C. Our experiments 
showed, that these fish died in a rather short time when suddenly 
put into a diluted Ringer solution or diluted sea-water of 31°, 
provided that the concentration of the solution was below m/8. 
In an m/8 or m/4 Ringer solution or sea-water they were able to 
resist the temperature of 31° without any previous immunization. 
We put a large number of these fish in an ice chest in which the 
temperature remained constantly at 0.°4, and investigated at 
various intervals whether the resistance of these animals to a 


554 JACQUES LOEB AND HARDOLPH WASTENEYS 


temperature of 31° differed from that of the fish kept at from 10° 
to 14° (cold room). The fish put in the ice chest had previously 
been at a temperature of 10° for several weeks. 

If we consider the behavior of the fish in an m/8 solution at 31° 
we notice a steady diminution of resistance among those kept at 
0°.4; while among those kept at 10° to 14° the resistance increased 
somewhat. Thelatter resultisnotaccidental. Wemustremember 
that the fish were taken in January when the temperature of the 
water was not far from 0°C. The long exposure to a temperature 
of from 10° to 14° had therefore a slight immunizing effect. 

Our next task was to ascertain whether fish immunized to resist 
a sudden transfer to a temperature of 35° kept this immunity if 
put on ice just as well as if kept at a temperature of 10° for the 
same period. Our experiments thus far cover only an exposure 
of fourteen days on ice (T. = 0°.4). During this time the immu- 
nity was not diminished, as the following example will show. Fish 
were immunized to a sudden transfer to 35°C. by keeping them 
for two days at 27°. They were then put into a thermostat with 
a constant temperature of 0°.4 for fourteen days and put directly 


TABLE 9 


DURATION OF LIFE AT 31° IN 


“ ore) we 2 Pre RINGER 
KEPT aT 0.4° C. t SOvURION, 
H:0 m/32 m/16 m/8 | 
= Sy ——— 
7 days 26’ 104’ indef. 
19 days 13’ 42’ ily some indef. 
33 days 24’ 30’ 69’ 80’ 
41 days 22 34’ 22' 8’ | 
! 
< +— “ = 
| DURATION OF LIFE AT 31° IN | 
= = | “RINGER 


. 0° (or —— — —— —- 
KEPT AT 1 SOLUTION 


H:0 m/32 m/16 m/8 * 

= ———— E = 

Over 7 days | 20’ 135’ indef. 
Over 19 days | 13’ 26’ Dan indef 
Over 33 days | 2! 86 some indef. indef. 


Over 41 days 41’ some indef. some indef. indef. 


ADAPTATION OF FISH TO TEMPERATURE 595 


into a m/4 Ringer solution of a temperature of 35° (A). Simul- 
taneously fish which had been immunized for 35° by keeping them 
three days at 27° and which had then been kept at between 10° 
and 14° for nineteen days were also put into a m/4 Ringer solution 
of 35° (B). In addition two controls were made: Fish kept on 
ice at 0°.4 for twenty days but not previously immunized (C), 
and fish not previously immunized kept for several weeks at a 
temperature of from 10° to 14° (D) were also suddenly transferred 
to a temperature of 35°. Table 10 gives the result. 


TABLE 10 
DURATION OF LIFE OF FISH AT 35°C. IN m/4 RINGER SOLUTION 
B. Immunized but C. Not immunized . 
A. Immunized but kept k ° . D. Not immunized 
aoe ept at 10° for nine- kept on tice for a - 3 
on ice for fourteen days teandays twenty days kept in cold room 
Alive after3 hours One alive after 3 Die in 2’ Die in 2’ 


hours 


The experiment was repeated with the same result, only those 
in lot A and B remained all alive. It is therefore obvious that 
the resistance acquired for a higher temperature (35°) is not lost 
or diminished if the fish are kept for two weeks on ice. 


THEORETICAL 


The phenomenon of adaptation considered in this paper is the 
fact that fish can resist a high temperature better if the latter 
is raised gradually than when it is raised suddenly. Physics offers 
us an analogy to this phenomenon in the experience that glass 
vessels which burst easily when their temperature is raised sud- 
denly, remain intact when the temperature is raised gradually. 
This phenomenon finds its explanation in the fact that glass is 
a poor conductor of heat and that when the temperature is raised 
suddenly, e.g., inside a glass cylinder, the inner layer of the cylinder 
expands while the outer layer, on account of the slowness of the 
conduction of heat, does not expand equally and the cylinder 
bursts. 


556 JACQUES LOEB AND HARDOLPH WASTENEYS 


The following idea for the explanation of the mechanism of 
adaptation suggests itself. The rise in temperature brings about 
certain changes especially in the surface of the cells or the body 
of the animal, whereby the latter loses its protective impermea- 
bility. If the rise in temperature occurs gradually the blood 
(and especially the salts of the blood or of the surrounding solu- 
tion or of both) has time to repair the damage. If the rise, how- 
ever, occurs suddenly then the damage done cannot be repaired 
quickly enough by the blood, or the salts of the surrounding solu- 
tion, to prevent the death of the cell or the animal. The peculiar 
influence of the concentration and nature of the surrounding solu- 
tion described in this paper would harmonize with this suggestion. 

A second possible suggestion is that under the influence of the 
higher temperature a substance is formed in the animal which 
protects it against the effects of high temperature. The formation 
of this substance is also a function of time and for this reason an 
animal can keep alive if the temperature is increased gradually 
but cannot keep alive if it is increased rapidly. 

Both suggestions would explain the fact that if an animal is 
once immunized against a high temperature it will keep this im- 
munization, for some time at least, even if kept at a low tempera- 
ture or onice. Further experiments with which we are occupied 
may decide between these and other possible suggestions. 


SUMMARY 


1. Experiments were made with Fundulus which were caught 
in the winter and kept at a low temperature (from 10° to 14°C.), 
to find out the maximum temperature into which they could, with 
impunity, be transferred suddenly. It was found that the maxi- 
mum temperature varied with the concentration of the sea-water 
or a Ringer solution; being about 25°C. for a concentration of 
m/128 or m/64; 27°C. for a concentration of m/32; 31°C. for a 
concentration of m/8, and almost 33°C. for a concentration of 
m/4. The latter concentration was the optimum, the resistance 
to high temperature decreasing again with a further rise in con- 
centration. ; 


ADAPTATION OF FISH TO TEMPERATURE 557 


2. It was found that dextrose solutions were not able to afford 
any protection against the effects of a sudden rise in temperature. 
From these and similar experiments with CaCl, solutions it fol- 
lows that the protective action of sea-water or a Ringer solution 
against high temperature is not an osmotic but a specific effect 
of the salts of the sea-water. 

3. It was ascertained how long it takes to immunize the fish 
against the harmful effects of a sudden transfer to a temperature 
of 35°C. It was found that by keeping the fish for thirty hours or 
more at a temperature of 27° they were immunized against a tem- 
perature of 35°. Often a noticeable immunizing effect was pro- 
duced already by an exposure of sixteen hours or even a little less 
to a temperature of 27°. Fish kept for two days at 27° were able 
to survive if suddenly transferred to distilled water of 35°C. 

4. The immunity against a temperature of 35° acquired by keep- 
ing the fish for two days at 27° is not lost or weakened if the fish 
are afterwards kept as long as thirty-three days at a temperature 
of from 10° to 14°. Our experiments have not been extended 
beyond this period of time. 

5. The immunity against a temperature of 35°C. is also main- 
tained if the fish are kept after the two days’ exposure to 27° for 
two weeks at a temperature of 0°.4 C. 

6. Fish immunized against a temperature of 39° and then kept 
at a temperature of from 10° to 14° for eleven days did not lose 
their immunity. 

7. A longer exposure of fish to a temperature of 0°.4 may finally 
lower their resistance to high temperature. 

8. In order to immunize fish to a temperature of 39° it is not 
necessary to expose them continuously to a higher temperature. 
An intermittent exposure to a higher temperature during a number 
of hours each day will bring about the same effect. 

9. Various suggestions for a possible theory of these phenomena 
are made. 


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