THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
p
GARY N. CALKINS, Columbia University FRANK R. LlLLEE, University of Chicago
E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago
E. N. HARVEY, Princeton University GEORGE T. MOORE, Missouri Botanical Garden
SELIG HECHT, Columbia University T. H. MORGAN, California Institute of Technology
M. H. JACOBS, University of Pennsylvania G. H. PARKER, Harvard University
H. S. JENNINGS, Johns Hopkins University W. M. WHEELER, Harvard University
E. E. JUST, Howard University EDMUND B. WILSON, Columbia University
ALFRED C. REDFffiLD, Harvard University
Managing Editor
VOLUME LVIII
FEBRUARY TO JUNE, 1930
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Entered October 10, 1902, at Lancaster, Pa., as second-class matter under
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CONTENTS
No. 1. FEBRUARY.
HOBER, RUDOLPH
The First Reynold A. Spaeth Memorial Lecture. The Pres-
ent Conception of the Structure of the Plasma Memhrane ..... 1
MONTGOMERY, HUGH
The Copper Content and the .Minimal Molecular Weight of
the Hemocyanins of Busycon canalicnlatum and of Loli-o
pealei ................................................. IS
FAURE-FREMIET, K.
(irowth and Differentiation of the Colonies of Xoothamninm
alternans (Clap, and Lachm. ) ............................ JS
I IAI.L, F. G., and ROOT. R. \\".
The Influence of Humidity on the Bodv Temperature of Cer-
tain Poikilotherms ...................................... 5_'
MORGAN, T. H., and TVI.KR, AI.KKRT
The Point of Entrance of the Spermato/oon in Relation to tin-
Orientation of the Emhryo in E.i^s with Spiral Cleavage ..... 31'
Lrrz. BKENTOX R.
The Effect of Low < >xy^cn Tension on the Pulsations of the
Isolated Holothurian Cloaca .............................. 74
Ar.pATOv, \Y. \\'.
Phenotypical Variation in Body and Cell Si/.e of I )ro>ophila
melanogaster ........................................... S3
JACOBS, M. H.
Osmotic Properties of the Erythrocytc. I. Introduction. A
Simple Method for Studying the Rate of I leniolysis ......... In4
No. 2. APRIL,
I IOADLKV. LKK.H
Some Effects of H^CL on Fertili/.ed and I'nfi-rtili/.ed I;.^.L:- of
. \rhacia punctulata ...................................... 1 -'.^
\\'nriAKER. Dorci.A.s. and MORCAN. T. II.
The Cleavage o! Polar and . \ntipolar MaKe^ nf the 1-".^^ of
( 'haeti i] >t<.-ru> ........................................... 145
iii
iv CONTENTS
PAGE
REDFIELD, ALFRED C.
The Absorption Spectra of Some Bloods and Solutions Con-
taining Hemocyanin 150
CONKLIN, CECILE
Anoplophrya marylandensis n. sp., a Ciliate from the Intes-
tine of Earthworms of the Family Lmnbricidae 176
DEMPSTER, W. T.
The Growth of Larvae of Ambystoma maculatum under Nat-
ural Conditions 182
SMITH, DIETRICH C.
The Effects of Temperature Changes upon the Chromato-
phores of Crustaceans 193
No. 3. JUNE, 1930
COE, WESLEY R.
Unusual Types of Nephridia in Nemerteans 203
GRAY, I. E., and HALL, F. G.
Blood Sugar and Activity in Fishes with Notes on the Action
of Insulin 217
BLUM, HAROLD F.
Studies of Photodynamic Action. I. Hemolysis by Previously
Irradiated Fluorescein Dyes 224
REDFIELD, ALFRED C.
The Equilibrium of Oxygen with the Hemocyanin of Limulus
polyphemus determined by a Spectrophotometric Alethod 238
HOADLEY, LEIGH
Polocyte Formation and the Cleavage of the Polar Body in
Loligo and Chaetopterus 256
PICKFORD, GRACE EVELYN
The Distribution of Pigment and other Morphological Con-
comitants of the Metabolic Gradient in Oligochaets 265
SIVICKIS, P. B.
Distribution of Setae in the Earthworm, Pheretima bengueten-
sis Beddard 274
JAHN, THEODORE L.
Studies on the Physiology of the Euglenoid Flagellates. II.
The Autocatalytic Equation and the Question of an Autocatalyst
in Growth of Euglena 281
CONTENTS v
PAGE
HARVEY, ETHEL BROWNE
The Effect of Lack of Oxygen on the Sperm and Unfertilized
Eggs of Arbacia punctulata, and on Fertilization 288
RAFFEL, DANIEL
The Effect of Conjugation within a Clone of Paramecium
anrelia 293
SMITH, GEORGE MILTON
A Mechanism of Intake and Expulsion of Colored Fluids by
the Lateral Line Canals as Seen Experimentally in the Goldfish
(Carassius auratus) 313
VATNA, SUP.
Rat Vas Deferens Cytology as a Testis Hormone Indicator and
the Prevention of Castration Changes by Testis Extract Injec-
tions 322
STUNKARD, H. W., and NIGRELLI, R. F.
On Distomum vibex Linton, with Special Reference to its
Systematic Position 336
CHAMBERS, ROBERT .
The Manner of Sperm Entry in the Starfish Egg 344
INDEX 370
Vol. LVIII, No. 1 FEBRUARY, 1930
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
Woods Hole, Massachusetts
THE FIRST REYNOLD A. SPAETH MEMORIAL LECTURE1
THE PRESENT CONCEPTION OF THE STRUCTURE OF
THE PLASMA MEMBRANE
RUDOLPH HOBER
PHYSIOLOGISCHES INSTITUT, KIEL
Ladies and gentlemen: I feel in this moment that more than ever
since the beginning of my scientific life, I have sympathies with this
country, where the modern view of general physiology, to which I
myself have devoted my life's work, has been developed with perhaps
greater success than anywhere else. More than ever here in Woods
Hole I feel the genius of Jacques Loeb, who, as no one else since the
days of Claude Bernard, taught us so impressively that the most
important task of physiology lies in recognizing the general properties
of living matter, and who spent here in this place the happiest days of
his life doing research work. And sadly here too I remember at this
hour my friend, Reynold Spaeth, in whose memory I have the honour
to give you this lecture today. Sixteen years ago he came to Kiel
with his young wife, an enthusiastic young scientist, eagerly longing
to take up physical chemistry as his weapon with which to advance
into the undiscovered land of science. And then after his return from
Germany, like his great idol Jacques Loeb and many of Loeb's students,
he learned to love above all the scientific atmosphere of Woods Hole,
—he who was destined to leave us so early, disregarding in his intense
eagerness for research the dangers of the tropics.
The genius loci of Woods Hole, who apparently holds his protecting
hand over general physiology with particular kindness, also moves
me to take as the theme of this lecture the present conception of the
structure of the plasma membrane. I am sure that with this theme
1 Delivered at the Marine Biological Laboratory, Woods Hole, on September 9,
1929. The announcement of the foundation of the Spaeth Memorial Lecture will
be found in the Report of the Director of the Laboratory for 1928 (Biol. Bull., 1929,
57: 22).
1
1
RUDOLPH HOBER
I shall enter the sphere of interest of many people who have performed
and are still performing physiological studies at Woods Hole. At
once, with this theme I recall to mind the investigations which Reynold
Spaeth put forward with so much skill, perseverance and enthusiasm
during his short residence in my laboratory.
Ladies and gentlemen, many of you will agree with me that the
problem of cell permeability belongs among the most urgent questions
of general physiology, I daresay perhaps of special physiology too.
For the working out of a theory of permeability is intimately joined
with the understanding of many fundamental phenomena of life, such
as nutrition, secretion, absorption, excretion, growth and irritability.
Hence let us begin to follow a little the development of the doctrines
of the permeability of cells.
In 1855 Naegeli described the phenomenon of plasmolysis of the
plant cell, consisting in a persisting retraction of the protoplast from
the cell wall, if the cell is bathed in what we call today a hypertonic
solution. Pfeffer in 1877 gave an explanation of the permanent
plasmolysis, comparing the plant cell with the "Traubesche Zelle"
made up by a precipitation membrane, for instance, by a copper
ferrocyanide membrane. He suggested that the protoplast is sur-
rounded by some limiting layer on the outer surface, the plasma
membrane having, like the inorganic precipitation membrane, the
property of semipermeability, that is, permeability to water but im-
permeability to such dissolved substances as produce the plasmolysis.
Some time later, Overton gave an unquestionable proof that Pfeffer's
assumption was correct; making use of a series of organic compounds,
he showed that, in full harmony with the theory of solutions based by
van't Hoff upon the experiments of Pfeffer, all solutions which produce
the beginning of plasmolysis have the same molecular concentration.
Thus at first one was compelled to assume that the interior of the
living cell was shut off from dissolved substances and that only water
was able to enter.
The next important step in recognizing the nature of the limiting
membrane of the cell was the discovery of Klebs and de Vries that
besides the dissolved substances which cause permanent plasmolysis,
there exist some others, — for instance glycerol and urea, which given
in hypertonic solution, instead of bringing about the permanent
plasmolysis, only produce the initiation of shrinking, which is followed
sooner or later by deplasmolysis. Furthermore, Overton and others
have discovered a great many substances that do not plasmolyse at all.
It was only a logical outcome of the theory of the plasma membrane to
explain all this by the assumption that for such substances the mem-
STRUCTURE OF THE PLASMA MEMBRANE
brane is not impermeable, but allows them to pass faster or slower.
These conclusions could be often established beyond doubt by chemical,
optical and other forms of analysis of the contents of the cells.
But the question now arises as to whether the inorganic precipi-
tation membranes behave in perhaps the same manner, that is, whether
or not they are permeable to the same substances to which the plant
cells, as indicated by the plasmolysis experiments, are permeable.
Curiously enough, this question, which is derived so easily from the
experiments, has been answered only recently by the systematic experi-
ments of Collander.2 This author showed that the copper ferrocyanide
membrane behaves also in a very different manner in relation to a
great number of organic non-electrolytes, allowing some to pass not
at all, others to pass slowly, and still others quickly. But the laws
governing the speed of permeation through the precipitation membrane
differ widely from those which hold good in the case of the plasma
membrane, as is illustrated by Table I.
TABLE I
Substance
Relative per-
meability of
Rhcco discolor
Permeability
of Copper
Ferrocyanide
Molecular
Volume
Relative
Solubility
in Ether
Methyl alcohol .
125
+ + + + +
8 2
0 273
Ethyl alcohol . .
71
+ + + +
12 8
1 86
Valeramide
69
+ +
28 7
0 170
Ethyl urethane. .
59
0 637
Ethylene glycol
4.4
+ + + +
144
0 0068
Diethylurea. . . .
2.0
0 0185
Glycerol .
1.3
+ + +
206
00012
Methylurea
1.2
00012
Urea . ....
1.1
+ + + + +
13 7
00005
Glucose . .
1.02
+
37 5
<0 0001
Glycocoll .
1.0
+ + + +
17.1
< 00001
Saccharose. ...
1.0
+
704
<0 0001
The table contains data with respect to the behavior of twelve
organic non-electrolytes. The first series of numbers shows the various
speeds of permeation as related to the epidermis cells of Rhvo discolor,
the second the speeds of permeation in relation to the copper ferro-
cyanide membrane. It can be easily seen that between both there
exists no parallelism at all. The third series indicates the molecular
volumes calculated by Collander from the values of molecular re-
fraction; the fourth series gives their relative solubilities in ether.
Now comparing the second and third series, we recognize clearly that
the velocity of permeation of the precipitation membrane is a function
- Collander, Kolloidchem. Beihefte, 19, 72, 1924 and 20, 273, 1925.
4 RUDOLPH HOBER
of molecular volume. This governing rule being established, the
character of the membrane is immediately revealed. It behaves as a
sieve for molecules so that the size of its pores determines whether
or not the dissolved substance can permeate. Such a membrane
is semipermeable as soon as the diameter of the molecules of the
solution surpasses a certain size. The passage is then allowed only
to water, for its molecules are characterized by an especially small
volume. And since it is highly probable that the pores of the mem-
brane are not all of the same size, the molecules with a diameter
below the limiting value have a greater chance to slip through, as
they are smaller. Furthermore, the fourth series of numbers shows
that the permeability of the plasma membrane might depend upon
quite another principle, that is, the principle of solubility in the sub-
stance of which the membrane is composed or, briefly, the principle
of selective solubility. Thus we come to speak of the first compre-
hensive theory of cell permeation, the lipoid theory of Overton.
It is a well-known fact that the lipoid theory has been supported
by a large amount of powerful arguments, but it is also well-known
that one has struggled sharply against it, very often, I believe, with
insufficient arguments. But even today the theory cannot con-
clusively be judged for the simple reason that the physico-chemical
foundation is partly too narrow and partly too uncertain. Professor
Jacobs was indeed completely right when he wrote a short time ago:
"It may be emphasized that what is most needed in the field of cell
permeability at the present day is facts." As everybody knows,
Overton based his theory in the first place on the comparison between
the speed of penetration of substances and their relative solubility
in oil. Collander, recently reviewing most carefully the experiments
of Overton on plant cells, advocated especially the correspondence
between permeability and solubility in ether.3 Both these authors
are quite clear about the limited value of their comparison, and the
table also shows that the parallelism is fairly incomplete. Therefore
the lipoid theory is still nowadays a petitio principii. However, the
thesis that the permeability to the organic non-electrolytes is to be
compared to the solubility in organic solvents agrees so often with
the experimental data, that I myself have practically no doubt that
it is only necessary to discover such solvents as might be still better
suited to comparison with the material of the plasma membrane than
oil or ether. It is really astonishing that since the lipoid theory was
set up more than thirty years ago, so little systematic research work
3 Collander and Barlund, Soc. scient. fenn., 2, 9, 1926; Barlund, Ada botan.fenn.,
5, 1929.
STRUCTURE OF THE PLASMA MEMBRANE
has been done on the relative solubility of organic compounds in
different organic solvents comparable to the lipoids of Overton, in order
to get a firm basis for the theory. It is well known that an interesting
attempt to find a better model was made by Nirenstein some years
ago.4 He showed that several exceptions to the rule previously given
by Paul Ehrlich, that the vital colors which enter easily into the
living cell dissolve in oil, could be removed by trying to imitate the
plasma membrane with a mixture of an oil with a fatty acid and an
organic fat-soluble base. Table II shows experiments by which I
was able to compare the relative solubility of acid dyes in the above-
mentioned oil mixture with the relative absorption of colors by red
blood corpuscles.5
TABLE II
Sulfonic Acid Dyes
•
Relative
Solubility
in Oil
Mixture
Relative
Absorption
by Blood
Corpuscles
Sulfonic Acid Dyes
Relative
Solubility
in Oil
Mixture
Relative
Absorption
by Blood
Corpuscles
Wollgrun, Licht-
Tropaeolin 1
30
1-3
grun
0
0
Tropseolin 2
30
4-8
Cyanol, EriocyaninJ
Orange R . ...
85
7-16
Azofuchsin I .
< 1
0.3-1
Brilliant orange R
71
7-16
Azofuchsin G
< 1
<1
Metanil yellow
94
10-16
Bromophenol blue . .
27
1-3
It can be seen that there exists a parallelism between solubility and
absorption, and it is especially noteworthy that this similarity means
not only intensity of staining, but means permeability; for it follows
from the table that dyes which are not dissolved in the oil mixture
do not enter the blood corpuscles at all. However, such experiments
with dyestuffs do not come out quite satisfactorily, as I can easily
show. Therefore it is necessary to collect further experimental data
to get a clear understanding, inasmuch as the cell permeability is a
solution permeability.
But there can be no doubt that the cell permeability is not only
a solution permeability with regard to an oil-like solvent within the
plasma membrane. In the first place in this connection it is a very
striking fact that water enters the cell usually with remarkable
speed, because it is impossible to reconcile this entrance with the
supposition that the membrane consists entirely of an oily phase.
Secondly there exists an apparent permeability of certain kinds of
cells to inorganic anions, though the inorganic salts are generally not
4 Nirenstein, Pfliiger's Arch., 179, 233, 1920.
5 Unpublished experiments.
6 RUDOLPH HOBER
in the least soluble in organic solvents. Thirdly, there are important
nutritive materials which cannot get into the cell in any way, but
belong also to those substances that are nearly or entirely insoluble
in the organic solvents.
Now we are able to interpret the first and second points by re-
turning to the already-mentioned sieve theory of permeability of the
precipitation membranes, and we will see that on that account the
comprehension of the structure of the plasma membrane receives a very
important supplement. More than thirty years ago Koeppe, Giirber
and Hamburger made the discovery, — which has often been verified
since, — that the red blood corpuscles have a selective permeability
for anions. It is well known that this property has the greatest
importance for the buffer capacity of the blood; but it seemed for a
long time to be a strange unicum, for which there existed hardly any
physical parallel. Otherwise it might have been possible to construct
a model to imitate the peculiarities of the membrane of the blood
corpuscles. Today the matter is practically clear; the well-known
experiments of Michaelis and Collander with artificial membranes,
especially with dried collodion membranes, enabled us to understand
the singular phenomenon. Michaelis proved that these membranes
are, under certain circumstances, the seat of a great potential difference,
whose direction and amount is an obvious sign that the membranes
are exclusively cation-permeable.6 Therefore there is an analogy
between the cation-permeable collodion membrane and the anion-
permeable blood corpuscle membrane. At the collodion membrane the
anion plays no role, whereas a cation gives rise to an electromotive
force which increases as its migration velocity increases or as its
diameter decreases. On account of these facts Michaelis has proposed
the following hypothesis: the membrane allows only the cation to pass
through it as through a sieve; the ions with the smallest diameter
pass with the greatest speed, and the entrance of ions into the pores
of the membrane is prevented if their diameter exceeds a certain value.
That is apparently the reason why, for instance, the earth-alkali ions
are unable to pass some collodion membranes characterized by rather
narrow pores. In this way we may understand that a quantity of an
ion sufficient to'be detected by chemical methods can penetrate only
if there is present another cation on the other side of the membrane,
so that an exchange can take place. That is exactly the same as
with the red blood corpuscles, where from the beginning the demon-
stration of the selective anion-permeability depended upon the fact
that as long as there exist differences of concentration in the proper
6 Michaelis, Naturwissenschaften 1926, 14: 33.
STRUCTURE OF THE PLASMA MEMBRANE
direction, the anions of the surrounding solution can be exchanged
against the anions of the interior of the cell.
And now the question arises, how it is to be understood, that
in the case of the collodion membrane the pore permeability is limited
to the cations and, in the case of the red blood corpuscles, to the
anions. Michaelis had already turned his mind to the fact that the
substance of the cation-permeable membrane itself is negatively
charged, and he connected this idea with the well-known membrane
studies of Bethe and Toropoff 7 and the experiments on the reversal
of membrane potentials in gelatine discs, which have been established
by Matsuo in my own laboratory.8 As a matter of fact it can be
proved that this idea is right. There exists a relation between the
electric charge of the membrane material and the faculty of the ions
with opposite electric charge to pass. In my laboratory Mond
succeeded in demonstrating that if the negative charge of the collodion
is changed to a positive charge by addition of a basic dye, for instance
by rhodamin, the membrane thus formed, instead of being exclusively
cation-permeable is changed into a membrane of selective anion-
permeability.9 Table III illustrates the resulting conditions.
TABLE III
Membrane Potentials in Rhodamin-collodion Membranes
— Cl
0.1M NaCl ^~
0.1M NaCl
0.1M NaCl
0.1M NaCl
0.1M NaCl
0.1M NaCl
SCN > I > Br > Cl > SO4
Cations without effect
The dotted arrows show the direction of the movement of the
chlorine ions; their length is a measure of the potential dependent on
the velocity of the ions. The arrows drawn refer in a corresponding
manner to the anions of the opposite side of the membrane. The
electromotive forces decrease from -f- 60 millivolts to - - 3.8 millivolts
along the series of anions: thiocyanate, iodine, bromine, chlorine,
sulfate. The cations are without any effect. The membrane potential
is therefore approximately zero if there is sodium chloride and
7 Bethe and Toropoff, Zeitschr. f. physik. Chemie, 88, 686, 1914 and 89, 597,
0.1M NaSCX
+ 60 millivolts
0.1M Nal
+ 33
0.1M NaBr
+ 20
0.1M NaCl
0
0.1M Na2SO4
- 3.8
0.1M KC1
+ 2
1915.
8 Matsuo, Pfliiger's Arch., 200, 232, 1923.
9 Mond and Hoffmann, Pfliiger's Arch., 220, 194, 1928.
RUDOLPH HOBER
potassium chloride in the same concentration on each side of the
membrane.
There can be no doubt that these experiments demonstrate on
the one hand in a very conclusive manner the existence of anion-
permeability on a membrane originally cation-permeable, but they
reveal on the other hand some difficulties in our understanding of
these and, as we shall see, of other alterations of the ion-permeable
membranes. The membrane potential of the rhodamin collodion
membrane does not increase with increasing migration velocity of
the effective ion, as has been found by Michaelis with the cation-
permeable collodion membrane, but the potential changes according
to the lyotropic series. This seems to point to some kind of relation
of ion-permeability to the colloidal state of the membrane, which is
known to depend in an especially characteristic manner on the lyotropic
properties of the ions.
However, before discussing this question more amply, we will
look at a remarkable consequence of the membrane experiments just
described. Mond, supposing that the membrane material of the red
blood corpuscles is electropositive, suggested that their natural anion-
permeability might be turned into cation-permeability, if one succeeds
in giving the membrane substance a negative charge.10 This actually
happens by the addition of a suitable amount of hydroxyl ions. As
soon as the reaction in the surrounding medium of the blood corpuscles
is made more alkaline than pH 8, the usual selective anion-permeability
is displaced by selective cation-permeability, so that now an exchange
between the potassium ions of the interior with the sodium ions of the
environmental solution begins, while the chlorine and bicarbonate ions
present in both serum and corpuscles, which were up to this point
able to pass through, are now fixed. Mond has advocated the view
that the decisive constituent of the plasma membrane, to which the
opposite charge is to be attributed, has ampholyte character and might
be globine, that is, a protein body, because the reaction by which
this reversal of anion-permeability into cation-permeability takes
place conforms with the isoelectric point of the globine, which is
pH 8.1.
In this way we come to a conception, similar to the well-known
hypothesis of Nathansohn, that the cell surface is comparable to a
mosaic of both lipoids and proteins. Apparently the plasma mem-
brane of the red blood corpuscles consists of at least two constituents,
a lipoid phase, whose existence enables the lipoid-soluble substances
to enter, and a protein phase, which is pore-permeable, so that water
10 Mond, Pflilger's Arch., 217, 618, 1927.
STRUCTURE OF THE PLASMA MEMBRANE
as well as dissolved substances, whose molecular size is small enough,
can pass through. As to the character of the structure of the cation-
permeable membranes, which we will now discuss, the opinion is not
yet substantiated enough.
TABLE IV
Resting Potentials of the Sciatic Nerve
Time
Potential
Solution
Time
Potential
Solution
millivolts
millii'ctis
3:21
20.6
Ringer
3:20
27 .4
Ringer
3:53
20.5
Ringer
3:52
27.5
Ringer
3:55
Ringer with 0.08 % KC1
3:54
Ringer with 0.08% KC1
+ 0.1%CaCl2
4:03
19.0
4:02
27.7
( i
4:28
17.2
4:27
28.5
i (
5:18
16.5
5:17
28.6
1 1
It seems that the cation-permeable membranes exist more fre-
quently than anion-permeable membranes. As Bernstein and I have
pointed out twenty-five years ago, the hypothesis of selective cation-
permeability gives a good explanation of the electro-negativity that
results from injury and of negativity resulting from activity. But
if we produce a difference of potential in uninjured tissues such as
muscles, liver and apple by joining their surfaces at two different
points with two different salt solutions, then we see that, although
the cations are better enabled to produce an electric current (ap-
parently in connection with the negative character of the membrane
colloids), the anions have an action too, the strength of which is
correlated with their position in the lyotropic series: sulfate, chlorine,
bromine, nitrate, iodine, thiocyanate. But the cations also do not
act exactly according to the series of the size of the ions: csesium,
rubidium, potassium, sodium, lithium, but succeed each other as
potassium, rubidium, csesium, sodium, lithium, — a series met with
rather often, as I have found in relation to changing the hydrophilic
colloidal state, and characterized by the peculiar dislocation of caesium.
And finally, regarding the membrane potentials of muscle and nerve,
we encounter a cation antagonism, for example that between potassium
and calcium, which might be explained by assuming an influence on
the state of membrane colloids, whereas it is difficult to explain it by
supposing the existence of a sieve-like membrane, the size of whose
pores remains unchangeable. Table IV gives as an example the
behavior of the sciatic nerve of a frog.11
11 Hober and Strobe, P finger's Arch., 222, 71, 1929.
10 RUDOLPH HOBER
As the experiment on the left side demonstrates, the resting
potential falls if the uninjured surface of the nerve is brought into
contact with a Ringer's solution in which the percentage of potassium
chloride is raised to more than 0.08. If we increase not only the con-
centration of potassium chloride but also of calcium chloride to 0.01,
the alteration of the initial potential, as is to be seen in the experiment
on the right side of the figure, does not take place. So we notice
again in regard to the membrane potentials the well-known antagonism
between potassium and calcium, and since there is hardly any doubt
that the permeability of the plasma membrane due to its porous
structure plays a significant role, we concluded that this permeability
is, according to the nature of the composing material, much more
variable than the pore-permeability of the artificial ion-permeable
membranes, especially of the collodion membranes.
Before leaving the interesting question of ion-permeability, I wish
to direct your attention to a membrane with very curious qualities.
Last year I set up and examined a membrane which was a patch-
work of cation-permeable pieces of collodion and anion-permeable
pieces of rhodamin collodion.12 Figure 1 gives the scheme. This
membrane must have the following qualities, and in fact it does
have them. If we place a salt solution on one side of it, for instance,
a solution of potassium chloride, and on the other side water, the salt
cannot diffuse into the water, although the membrane is as permeable
for the potassium ions as for the chlorine ions, because a passage in
chemically detectable quantity would be possible only if it could
happen at just the same place in equivalent amounts of cation and
anion, or in other words, because one ion can move only at an
infinitesimal distance from the opposite. However, the passage of
the potassium chloride is rendered possible as soon as a salt, whose
ions can interchange through the membrane with the potassium and
the chlorine ions, is placed on the other side of the membrane. It
seems to me that membranes of this kind, which, in spite of their
permeability for anion and cation, are able to entirely prevent the
escape of salts, have been realized by nature and play an important
role.
Now the question arises as to whether, in addition to the water,
only inorganic ions take the way through the pores of the plasma mem-
brane. Logically the answer is no. For if there are molecules whose
volume is of the same order as that of the permeating ions, then they
naturally must take the way through the pores, regardless of the
possibility of their passing equally well through the membrane by
12 Hober and Hoffmann, Pfliiger's Arch., 220, 558, 1928.
STRUCTURE OF THE PLASMA MEMBRANE
11
selective solution. Of course it has been pointed out by'Michaelis that
the collodion membrane, if it is dried enough to establish selective ion-
permeability and therefore to give the maximum electromotive effect,
allows those molecules to pass whose diameter is about the same as that
KCI
14-1-M-l
water
NaBr
FIG. 1.
of glucose.13 A similar behavior is met with in the plasma membranes.
It will be noted that among the organic non-electrolytes entering into
the cell, there are some which permeate more quickly than might be
expected in relation to their relative lipoid-solubility, or, more cor-
rectly, in respect to their relative solubility in ether, supposing that
the relative solubility in ether is to be acknowledged as a likely measure
of the physiological phenomenon. Some of these substances are
characterized by a relatively small molecular volume, for instance,
ethylene glycol and glycerol. Therefore Collander may be quite
right in considering their comparatively rapid permeation into plant
cells as due to the porosity of the plasma membrane.14 In other
cases where a disagreement occurs between velocity of penetration
and solubility in ether, for example with urea and its derivatives,
even the view of a sieve-like property fails to overcome the difficulties.
But here we can see, as I have found with Watzadse, that the difficulties
will be removed if, instead of the solubility in ether, the solubility in
the previously mentioned oil mixture of Nirenstein will be correlated
with the physiological phenomenon.15
13 Loc. cit.
14 Loc. cit.
15 Watzadse, Pfliiger's Arch., 222, 640, 1929.
12
RUDOLPH HOBER
The assumption of the porosity of the plasma membrane in this
manner being justified in several ways, it will be necessary to study
as intimately as possible the properties of the artificial porous mem-
branes and especially, because of their great stability, those of dried
collodion membranes. Therefore perhaps it is not too audacious to
consider the possibility, in relation to physiological conditions, that
certain molecules with a diameter not too great and not too small might
be stopped in the pores and obstruct them in the same manner that
ultramicroscopic particles are not only kept back by an ultrafilter, but
finally also obstruct its pores.
FIG. 2. Diffusion of thiocyanate in 15', retarded by urethanes.
From this point of view Anselmino has made experiments in my
laboratory. He favored the obstruction of the pores by using
narcotics, because they can be adsorbed by the collodion.16 The
result was that the collodion membrane was obstructed to such a
degree that the osmotic movement of water as well as the diffusion of
molecules of small size was strongly retarded. Figure 2 reproduces a
striking experiment. You see that the diffusion of thiocyanate is
reversibly slowed by several urethanes, and that the homologous
urethanes exert their influence characteristically so that the longer
their carbon chains are, the smaller their limiting concentration will be,
in the same way that we usually observe in narcosis.
16 Anselmino, Pfliiger's Arch., 220, 524, 1928.
STRUCTURE OF THE PLASMA MEMBRANE 13
It will be necessary to find out still more exactly which substances
are suitable for the obstruction of the pores and which are not.
Michaelis has recently found that the speed of diffusion of glucose
through a collodion membrane of suitable pore size decreases with time
more and more, and he regards this too as effectuated by an obstruction
of the pores.17 But in this case we do not have to deal with an adsorb-
able substance. It still remains an open question for the future, how
far the decrease of cell-permeability during narcosis, so often already
observed, is to be attributed to the porosity of the plasma mem-
brane. If this really happens, our ideas as to the nature of narcosis
would be greatly supplemented.
Now we will consider an especially difficult matter. As we have
seen, the entrance of numerous organic non-electrolytes into the
living cell may be considered as a matter of lipoid-solubility; the
entrance of other organic non-electrolytes, of some ions and of water
may be considered as a matter of diffusion through the pores of the
plasma membrane. But there is a group of substances of a very
remarkable physiological significance, which can neither directly enter
by dissolving in the oily phase nor by migrating through the porous
phase, but which, nevertheless, do obviously enter. To this group
belong substances which constitute a considerable proportion of the
nutritive material, such as many sugars and amino-acids. There can
be no doubt that this passage is not merely a simple form of permeation,
in the sense that it depends on a certain permanent and invariable
physicochemical behavior of a membrane. Either the plasma mem-
brane must change under definite conditions in such a way that a
temporary removal of the barrier to diffusion is brought about, or, — as
has often been supposed, — reversible chemical reactions of the food-
stuffs occur even in the surface of the cell, so that either the products of
reaction are enabled to pass through or a more or less complicated
series of single reactions is terminated by the appearance of the food-
stuffs inside the cell wall.
Adhering to the physicochemical character of this lecture, we will
discuss, basing our remarks on experimental data, only one of these
forms of the ingestion of the nutritive substances, namely the alteration
of the plasma membrane in such a wray that for a short time it becomes
permeable to substances for which it is otherwise not permeable.
As a matter of fact, there is a well-known form of intake of nutritive
material which can be considered as an opening of the plasma mem-
brane, that is, phagocytosis. For as the protoplasm is flowing around
the particles of food in order to incorporate them, the superficial layer
17 Michaelis and Weech, Jour, of Gen. Physiol, 12, 55, 1928.
14 RUDOLPH HOBER
necessarily must be partly destroyed. On the other side there exist
further conditions for naturally opening the plasma membrane in a
reversible manner, particularly as a so-called functional increase of
permeability, that is, as an increase of permeability accomplished by
function, or better, by excitation as preparation for function. I am
not able to give an extended review of our knowledge of functional
increase of permeability within the limits of this lecture, but I shall
relate one striking demonstration of the bringing about of a reversible
increase of permeability. If one brings Spirogyra cells into a solution
of cyanol, a well-diffusing blue sulfonic acid dye, the protoplasts will
remain unstained for several weeks. Some years ago in my laboratory
Banus observed that while sending an alternating current of appropri-
ate strength through the threads of algae, the blue dye would pass out of
the solution into the interior of the cell, namely into the sap of the
vacuoles.18 After this, the current being stopped, the algae were left
for some time in the blue solution; then they were taken out and
washed with pure water. It resulted that the vacuole retained the
blue dye in spite of its diffusibility, the dye which entered being
imprisoned as long as the cell was alive. Apparently the electric
current had opened the plasma membrane, a substance to which the
interior of the cell is closed under natural conditions had penetrated,
and behind it the plasma membrane had shut up. In this way an
event was produced, owing to experimental conditions, that is never
realized in nature; but a natural phenomenon, the reversible increase
of permeability, had been reproduced, possibly in a somewhat crude
manner. Perhaps there occurred only a regeneration after an injury
generated by the current. But, examining the conditions more
closely, we may recognize that nature may sometimes duplicate them.
For, in regard to the well-known studies of Bethe and ToropofT on
gelatine diaphragms, it is highly probable that the flow of an electric
current is accompanied by changes of hydrogen and hydrox}^ ion
concentration on the cell boundary so that these active ions, either by
hydration and liquefaction or by aggregation of the surface colloids,
can amplify or narrow the paths to be taken by diffusing substances
and can in this way produce reversible changes in permeability.
Thus we learn more and more to regard the plasma membrane as a
formation with varying properties so that its permeability exhibits
different degrees succeeding one another in time. But the plasma
membrane does not only vary in one and the same object temporarily,
but, — and this shall be the last point to be discussed in this lecture, -
it varies also in one and the same kind of cell from species to species.
18 Banus, Pfliiger's Arch., 202, 184, 1924.
STRUCTURE OF THE PLASMA MEMBRANE 15
I shall only demonstrate this with one especially simple object, namely,
the red blood corpuscles again, and with this object I wish to demon-
strate further in what direction the research into the nature of cell
permeability is to be extended. Finally I return in this way once
more to the phenomena of porous permeability and of solution per-
meability of the cells.
As we have seen before, the limiting membrane of the blood
corpuscles, according to the electropositive charge in the wall of its
pores, allows only the anions to exchange by diffusion from one side of
the membrane to the other. Further, it has been pointed out by
different authors that each anion passes through the corpuscle mem-
brane with a specific velocity. Now Mond in my laboratory has
raised the question of the existence of differences in the relative
velocities from species to species as evolving from the different sizes
of the holes in the sieve-like membrane, and in order to decide this
question, he examined the exchange of chlorine ions against sulfate
ions, which are known to wander especially slowly.19 Mond actually
found considerable differences in the different animals. The inter-
change is quickest in the blood corpuscles of man, then there follow
pig, horse, cattle. The conclusion that we have to come to in the
experiment just described with differences in pore size has been
supported by Mond by comparing the sulfate ion with the tetrahydric
alcohol erythritol as a non-electrolyte which is insoluble in lipoids
and which is known to penetrate into the blood corpuscles and other
cells as slowly as the sulfate ion. The same result occurred, namely,
the speed of permeation was greatest with the corpuscles of man and
the least with those of cattle.
But there exist not only differences from animal to animal in the
porous permeability of the cells; the same state of affairs holds for
the solution permeability. It is well-known that almost every basic
dye enters the living cell, but there are rather few acid dye-stuffs that
are suitable to it. As to the sulfonic acid dyes, evidently only those
enter which dissolve in the oil mixture worked out by Nirenstein and,
as has been illustrated by a table in the beginning of my lecture, the
dyes enter the cells the more as their relative solubility in the oil
mixture is greater. Now I have discovered that the partition
coefficient of blood corpuscles to surrounding solution differs under
the same conditions from one species to the other; for example, the
coefficient is greater with the blood corpuscles of the pig than with
those of cattle and sheep, and with these greater than with those of
the horse.20 This is demonstrated for two dyes in Table Y.
19 Mond and Gertz, Pfl tiger's Arch., 221, 623, 1929.
20 Unpublished experiments.
16
RUDOLPH HOBER
It appears at once that two explanations may be attempted: either
we have to assume that the blood corpuscles of all four animals contain
lipoids of the same quality, on which the dyes are distributed, but the
quantity is greatest in the corpuscles of the pig and is smallest in
the corpuscles of the horse; or we have to do with nearly the same
quantity of the lipoids in every kind of corpuscle, but the lipoids
differ qualitatively as to their power to dissolve dyes, the power
being greatest with the pig and smallest with the horse. It is my
opinion that we must prefer the second explanation; for whenever
the passage of the dyes is dependent here upon the lipoid solubility,—
and unquestionably this is the case, — then we must expect that a dye-
stuff penetrating into the blood corpuscles of the pig will also get
through the corpuscles of the horse, even if their lipoid phase is
very small; but I have found that, on the contrary, the corpuscles
of the horse are nearly impermeable to several of the staining sub-
stances examined. Thus we conclude that not only the properties
of the porous phase of the cell boundary, but also its dissolving
properties, vary from animal to animal.
TABLE V
Partition of Dyestuffs to Blood Corpuscles
Kind of
Corpuscle
Dye
Initial Con-
centration
Final Con-
centration
Partition
Coefficient
Horse
Tropsolin 1
0.0025
0.0017
1.9
Cattle
Tropsolin 1
0.0025
0.0015
2.7
Pie
Tropseolin 1
0.0025
0.0013
3.7
Horse
Bromophenol blue
0.0025
0.0022
0.55
Cattle
Bromophenol blue
0.0025
0.0020
1.0
Pig
Bromophenol blue
0.0025
0.0015
2.7
Ladies and gentlemen, I have come to the end and I shall repeat
now the previously quoted words of Professor Jacobs: "It may be
emphasized that what is most needed in the field of cell permeability at
the present day is facts." And in relation to that I wish to add to what
I have already said to you a quotation from Professor Ralph Lillie's
lecture concerning the scientific view of life. He said: 'What is
required is the imagination or construction of some model that will
reproduce in intelligible form the essential features of the phenomenon
under consideration. Intelligibility is the essential criterion of the
scientific view; it aims at making phenomena intellectually compre-
hensible." We perceive better than anywhere else the striking ad-
vantage of using models in the development of our knowledge of cell
permeability, beginning with Traube and Pfeffer and passing from
STRUCTURE OF THE PLASMA MEMBRANE 17
Overton to Collander and Michaelis. It is very peculiar that in this
direction the physical chemists have realized almost nothing from
these weary, but very fascinating and instructive studies of what is
required as a model of the cell membrane. This is to their own dis-
advantage, I believe, because they here overlooked fundamental
problems worthy of pursuit by the methods of exact science which
are applicable to the membranes, the qualities of which have been
discussed in this lecture. Under these circumstances the physiologist
is constrained, and will be constrained still more in the future, to leave
his proper work, as he must for a shorter or even for a longer time put
away physiology, and become pure physicist or pure physical chemist
in order to answer preliminary questions of great importance to
physiology. Otherwise he will be open to the great danger of fabri-
cating hypotheses. But whoever among the physiologists resolves to
leave his physiological studies, he may encourage himself by re-
membering that it was Jacques Loeb who, feeling obliged to do so in
regard to his science and to himself, created in the last years of his
life a monumental work on the physical chemistry of the protein bodies.
In this way he manifested anew his perseverance and his enthusiasm,
both properties which distinguished also Reynold Spaeth, whose
memory is with us today.
THE COPPER CONTENT AND THE MINIMAL MOLECULAR
WEIGHT OF THE HEMOCYANINS OF BUSYCON
CANALICULATUM AND OF LOLIGO PEALEI
HUGH MONTGOMERY
(From the Department of Physiology, Harvard Medical School, Boston, and the
Marine Biological Laboratory, Woods Hole)
The view, originally put forward by Fredericq (1878), that copper
is a normal constituent of hemocyanin and that it has a significance in
the respiratory function of this protein similar to that of iron in
hemoglobin has been substantiated by later investigations, particularly
those of Begemann (1924) and Redfield, Coolidge and Montgomery
(1928), which show that the combining ratio of copper to oxygen is
the same in the blood of a large number of invertebrates. A knowledge
of the quantity of copper in hemocyanin consequently provides
significant information with regard to its respiratory function. Inas-
much as the amount of copper in the various hemocyanins does not
appear to be the same, such data gives unequivocal evidence of the
specific character of the respiratory pigments in the different groups
of invertebrates. Furthermore, because of the very small number
of copper atoms in the hemocyanin molecule, the copper content is a
most valuable basis from which to estimate the minimal molecular
weights of these proteins.
In this paper an investigation of the hemocyanin of the whelk,
Busy con canaliculatum, and of the squid, Loligo pealei, is described.
Mendel and Bradley (1906) studied the respiratory protein of the
blood of the whelk, which they called hemosycotypin, — a name derived
from the then current generic name of this form, Sycotypus. They
report that it contained zinc as well as copper.1 They concluded that
copper composed only 0.043 per cent of the weight of the molecule, a
value very much smaller than that obtained in the case of other
hemocyanins and one which leads to very high estimates of the protein
content of the blood when the oxygen capacities demonstrated by
1 It seems preferable to include "hemosycotypin" among the hemocyanins
because it has been demonstrated that the combining ratios of copper and oxygen
are the same in this case as in that of other hemocyanins and because recent obser-
vations in this laboratory appear to make it doubtful whether the zinc is a true
constituent of the protein molecule. Inasmuch as specific differences appear to
exist between the hemocyanins of different groups of animals, confusion will be apt
to result if each hemocyanin is given a different specific name.
18
COPPER CONTENT OF HEMOCYANIN 19
Redfield, Coolidge and Montgomery (1928) are taken into account.
The copper content of the hemocyanin of the squid does not appear to
have been previously examined.
The copper content of these hemocyanins has been determined on
material purified according to several standard procedures applicable
to protein substances. Analyses for copper were made by the method
described by Redfield, Coolidge and Shotts (1928). Between 10 and
20 c.c. of the hemocyanin solutions were used in each sample. The
samples were dried in an oven at 100-110° C. for 48 hours, cooled in a
dessicator and weighed. This procedure was repeated daily until
successive weights did not vary more than i mgm. The samples of
dried hemocyanin weighed between 100 and 300 mgm. Digestion,
the electrolytic separation of copper, and its estimation were carried
out exactly as described, except that in the titration 15 drops of
potassium iodide were used instead of 10, as this modification was
found to sharpen the end point.
We have not succeeded in producing definitely crystalline prepara-
tions of the hemocyanin of Busycon canaliculatum by methods which
have been found applicable in other cases. Dhere, Baumeler and
Schneider (1929) have also been unsuccessful in crystallizing this
hemocyanin. However, on prolonged dialysis against distilled water
a precipitate is formed which appears to be composed of short rods
and which gives a silky sheen on shaking similar to that characteristic
of crystalline protein preparations.2 Busycon hemocyanin appears
to be a globulin, as it is insoluble in the region of its isoelectric point in
salt solutions of sufficient dilution. This property has been used in
purifying our material as well as the usual procedure of salting out with
ammonium sulphate, employed by Redfield, Coolidge and Shotts
(1928) in the preparation of Limulus hemocyanin.
2 In an attempt to produce crystals, a number of preparations of hemocyanin,
all of which showed a silky sheen on shaking, have been made by different methods
from several species. The precipitated particles were too small, however, to be
recognized under the microscope as definite crystals, though a very fine rod shape
was observed in many cases. By the addition of 2 drops of serum to 1-2.5 c.c. of
0.05M acetate buffer solution of pH 4 to pH 5, the hemocyanins of Busycon canalicu-
latum and of Busycon carica were precipitated and showed a sheen on shaking.
In the case of the bloods of the eight different species; Limulus polyphemus (horse-
shoe crab), Busycon canaliculatum, Busycon carica, Libinia emarginata (spider crab),
Loligo pealei, Homarus americanus (lobster), Callinectes sapidus (blue crab), and
Ovalipes ocellatus (lady crab), the hemocyanin was precipitated by diluting the
serum 20 to 200 times and adding a few drops of 0.006 per cent acetic acid to 5 c.c.
of the diluted serum. The acid must be added slowly or a precipitate will be formed
which will show no sheen. Too much acid redissolves the precipitate.
In several cases these hemocyanin precipitates were concentrated by centrifuging
and redissolved, whereupon the solutions appeared distinctly blue. This color dis-
appeared when the solution was reduced with sodium hydrosulfite so that evidently
the hemocyanin was not denatured by the process.
20
HUGH MONTGOMERY
TABLE I
Copper Content of Hemocyanin of Busycon canaliculatum
Specimen No.
Method of Preparation
Dry Weight
Copper
Copper
grams
me,m.
per cent
IVa
Three washings at isoelectric point
0.2071
0.496
0.240
0.2053
0.496
0.242
0.2067
0.492
0.238
0.2070
0.482
0.234
IVb
Four additional washings at isoelectric
0.1541
0.366
0.237
point
0.1538
0.378
0.245
0.1544
0.371
0.240
0.1554
0.378
0.243
VI
Three washings at isoelectric point
0.0914
0.225
0.246
0.0919
0.204
(0.227)
0.0916
0.217
0.238
0.0917
0.230
0.238
0.2694
0.642
0.238
0.2699
0.634
0.235
0.2698
0.633
0.235
0.1816
0.437
0.241
0.1821
0.433
0.239
VII
Salting out and dialysis
0.1694
0.440
0.260
0.1680
0.436
0.260
0.1700
0.441
0.260
0.1699
0.443
0.260
0.1693
0.436
0.258
0.1693
0.438
0.258
0.1693
0.434
0.256
0.1693
0.440
0.260
0.1693
0.441
0.260
0.1693
0.434
0.256
VIII
Salting out and dialysis under con-
0.1075
0.263
0.242
ditions leading to precipitation
0.2130
0.517
0.242
0.2120
0.530
0.250
X
Salting out and dialysis
0.3967
0.948
0.239
0.3948
0.944
0.238
0.3965
0.950
0.238
0.3978
0.948
0.237
0.6492
1.535
0.236
0.6485
1.554
0.240
0.6487
1.534
0.236
XI
Salting out and dialysis
0.5494
1.318
0.240
0.5481
1.308
0.238
0.5483
1.309
0.239
0.5478
1.311
0.239
0.5472
1.315
0.240
COPPER CONTENT OF HEMOCYANIN 21
Specimen IVa was made from blood which had been preserved
with toluene in the cold room for two weeks. It was diluted with
ten times its volume with distilled water and brought into the region
of its isoelectric point by the careful addition of 0.01N HC1. The
precipitate resulting was separated by centrifuging and put into
solution in the original volume of water by the addition of an amount
of sodium hydroxide equivalent to the hydrochloric acid previously
added. This process was twice repeated. The precipitate finally
obtained was washed with distilled water. The final product con-
tained only a trace of chloride. Whenever acid or alkali was added,
it was run in through a glass tube which had been drawn to a fine
point while the hemocyanin was being vigorously stirred. In order to
determine whether further purification of this product could be
obtained, the entire process of purification was repeated four more
times on a portion of Specimen IVa, the resulting preparation being
designated Specimen IVb. Specimen VI was made in a manner
similar to Specimen IVa. Specimen VII was made from blood which
had been preserved half-saturated with ammonium sulphate for a
month. The precipitated hemocyanin was separated by centrifuging
and dissolved in a large volume of 5 per cent saturated solution of
ammonium sulphate. The solution was centrifuged in order that a
small amount of insoluble material might be discarded, and the
solution was reprecipitated by the addition of saturated ammonium
sulphate. This process was repeated twice. The solution was then
dialyzed against 0.001N sodium hydroxide under 20 cm. Hg reduced
pressure for two weeks, at the end of which time it was free of sulphate.
The preparation of Specimen VIII included the same steps as Specimen
VII, except that it was dialyzed against 0.001N sodium hydroxide for
five weeks at atmospheric pressure. At the end of the fifth week a
precipitate appeared in the solution which gave on shaking a silky
sheen similar in appearance to that produced by protein crystals.
The precipitate consisted of rod-shaped particles about 2 IJL in length.
The solution still contained traces of sulphate and was consequently
centrifuged and the precipitate washed three times with a large
volume of distilled water. The sulphate test was then negative.
Specimens X and XI were prepared from material which had been
kept over two years precipitated in half saturated ammonium sulphate.
They were purified by reprecipitation with ammonium sulphate
(pH 8.0), repeated three times, followed by dialysis against 0.0001
sodium hydroxide for 18 days. The preparation and analysis of
Specimens X and XI were made by Miss Elizabeth Ingalls.
The results of the analyses of these preparations are given in
HUGH MONTGOMERY
Table I. The copper content obtained in the case of preparations
made in the various ways is very nearly the same. This fact may be
taken as evidence that fairly pure preparations of the protein have
been obtained. The fact that the copper content of Specimen IVb
was not materially increased over that in Specimen IVa by additional
washing is further evidence for the adequacy of the method of purifi-
cation employed.
The best representative value of the copper content of Busycon
canaliculatum hemocyanin appears to be 0.24 per cent. Specimen
VII yields consistent values 0.02 per cent higher than this. Inasmuch
as Specimens VIII, X and XI, prepared by the same general method,
agree with the general series, it is probable that the high value obtained
in the case of Specimen VII should be attributed to some systematic
analytical error rather than to superiority in the method of prepa-
ration.
Two specimens, which were obtained by the dialysis of fresh blood
without other attempt at purification, yielded a product which con-
tained about 0.22 per cent copper. This material was free of chloride
and had the same nitrogen content per unit weight as the others.
The result would appear to indicate that another protein may be
present in the blood, but that if so, it exists only in small amounts.
In the case of Limnlus, the hemocyanin appears to account for about
95 per cent of the protein of the serum. In order to investigate this
possibility further an attempt has been made to determine how far
the nitrogen content of the blood of Busycon canaliculatum may be
accounted for by the hemocyanin contained in it as estimated from
the quantity of copper present. The nitrogen content of Specimen X
was determined by the Kjeldahl method. Successive analyses yielded
15.6; 15.5; 15.7; 15.5; 15.4; 15.7; mean 15.5 grams nitrogen per 100
grams dry weight. The copper content of Specimen X was 0.238
grams per 100 grams dry weight. One part of copper consequently
corresponds to 65.2 parts of nitrogen. Two specimens of blood were
analyzed for copper and nitrogen. The first contained 0.074 mgm.
copper per c.c. and 4.92 mgm. nitrogen per c.c. From the copper
content it may be estimated that it contained 4.84 mgm. nitrogen as
hemocyanin. The second specimen of blood contained 0.066 mgm.
copper per c.c. and 4.14 mgm. nitrogen per c.c. The hemocyanin
concentration as estimated from the copper content would account
for 4.3 mgm. nitrogen. It is evident from these measurements that
hemocyanin will account approximately for all of the protein nitrogen
in Busycon blood.
One preparation of the hemocyanin of the allied species, Busycon
COPPER CONTENT OF HEMOCYANIN
carica, was made. The blood had been preserved in a precipitated
condition in half-saturated ammonium sulphate for one year in the
cold room. The hemocyanin was separated, purified by the procedure
employed in the case of Bnsycon canaliculatum Specimen X. Analysis
of the copper content of the purified material yielded the following
values: 0.217, 0.235, 0.238 per cent. The copper content of the
hemocyanin of this species appears to be approximately the same as
that of Busy con canaliculatum.
The hemocyanin of the squid, Loligo pealei, may be readily
crystallized by methods similar to those first employed by Henze
(1901) in preparing crystalline Octopus hemocyanin, and consequently
lends itself well to purification. Squid hemocyanin is insoluble in
solutions containing high concentrations of ammonium sulphate. It
was found that if enough saturated ammonium sulphate solution is
added to the blood to form a very slight cloud of precipitated
hemocyanin, a fuller precipitation in the form of crystals can then be
produced by several procedures designed to decrease the solubility of
the hemocyanin in the solution. These were: (1) the careful addition
of increasing quantities of ammonium sulphate, (2) increasing the
hydrogen ion concentration as in the Hopkins-Pinkus (1898) method
of crystallizing albumen, or (3) raising the temperature. These
methods can be used with success in combination. Crystallization by
raising the temperature, which is presumably due to increasing the
"salting out" effect of the ammonium sulphate at the higher tempera-
ture is particularly efficacious and has the advantage that it involves
the addition of no reagents and may consequently be accomplished
slowly so as to favor the formation of crystals. It was found that by
raising the temperature from 0° C. to 30° C., a heavier crystalline
precipitate is produced than by raising it to room temperature only.
A temperature change within a range which will not denature the
protein did not crystallize all the hemocyanin that was in the solution.
Consequently, the yield may be increased by combining the tempera-
ture method with the addition of ammonium sulphate or of acid.
When crystallization is produced in this manner, there is formed first
a fine precipitate, visible under the microscope but apparently
amorphous. This changes in a few minutes to fine rods and then to
bundles of needles and finally to large needles. The process is much
like that described in the case of Eledone moschata hemocyanin by
Robert (1903). The appearance of the crystalline rods is similar to
that figured by Dhere (1919, figure 4), in the case of the oxyhemocyanin
of Helix pomatia formed in the presence of sodium sulphate. If large
excess of reagents are added suddenly, the precipitate produced is
24
HUGH MONTGOMERY
amorphous. Crystallization of squid hemocyanin was obtained more
readily from fresh blood than from preparations which had been
preserved in a precipitated condition in concentrated ammonium
sulphate or from previously crystallized hemocyanin. Crystals which
had been kept for a year in the cold room in their mother liquor (half
saturated ammonium sulphate), were found to have become insoluble
in distilled water. This phenomenon was observed by Craifaleanu
(1919) i.n the case of crystals of the hemocyanin of Octopus vulgaris.
Craifaleanu called this form "para-hemocyanin."
TABLE II
Copper Content of Hemocyanin of Loligo pealei
Specimen No.
Method of Preparation
Dry Weight
Copper
Copper
grams
mgm.
per cent
I
Salting out and dialysis
0.1485
0.384
0.258
0.1486
0.371
0.250
0.1502
0.388
0.257
0.1490
0.376
0.252
II
Crystallization and dialysis
0.0785
0.194
0.244
0.1620
0.386
0.238
0.1624
0.390
0.242
V
Salting out and dialysis
0.4579
1.155
0.252
0.4594
1.161
0.254
0.4593
1.178
0.256
0.4601
1.159
0.252
0.4592
1.154
0.252
Analyses of the copper content of the hemocyanin of Loligo pealei
have been made upon three preparations. Specimens I and V were
prepared from blood which had been precipitated by the addition of
ammonium sulphate to half saturation and kept in the cold room at
about 5° C. for two years. The material had a fishy odor, which dis-
appeared when it was shaken with air and from which the final prepa-
rations were entirely free. The precipitate was separated from the
supernatant fluid with the centrifuge and was dissolved with a small
volume of 5 per cent ammonium sulphate. The solution was again
centrifuged to throw down any insoluble material, and the fluid was
drawn off and reprecipitated by the addition of saturated ammonium
sulphate. This process was repeated twice. The solution was finally
dialyzed until it was found to be free of sulphate. Specimen II was
prepared by crystallization from fresh blood. The blood was chilled
to 0°, and then sufficient saturated ammonium sulphate was added to
COPPER CONTENT OF HEMOCYANIN
25
produce a very slight precipitation of hemocyanin. The temperature
was then raised from 0° to 20°, when full precipitation was obtained.
The precipitate was in the form of needle-shaped crystals about ten ju
in length. The crystals were separated from the mother liquor by
centrifuging and dissolved with 5 per cent saturated ammonium
sulphate. Insoluble material was removed by centrifuging, and the
hemocyanin was then reprecipitated as before. This second pre-
cipitate was not crystalline, however. The preparation was then
dialyzed against water until free of ammonium sulphate. All threp
preparations had a clear blue-green color and became colorless in the
characteristic way upon reduction with sodium hydro-sulphite.
Table II contains the data obtained from analyses of these prepa-
rations of squid hemocyanin, which all yield values for the quantity
of copper in the molecule close to 0.25 per cent.
It is interesting to compare the values obtained for the copper
content of the hemocyanin of Busycon and Loligo with those previously
reported for other species, particularly with regard to their systematic
relationships. In Table III are collected the various determinations
TABLE III
Copper
Author
Cancer
per cent
0.32
Griffiths (1892).
Homarus. . .
0.34
1 t
Sepia
0.34
U
Octopus vulgaris
0.38
Henze (1901).
Loligo pealei
0.25
Helix pomatia
0.25
Burdel (1922).
11 U
0.29
Begemann (1924).
Busycon canaliculatum . . .
Limulus polyphemus
0.24
0.173
Redfield, Coolidge and Shotts (1928).
of the copper content of hemocyanin which occur in the literature.
It is noteworthy that the value obtained in the case of Busycon
canaliculatum and Busycon carica does not differ greatly from those
attributed to the other gastropod, Helix pomatia. The value obtained
for Helix pomatia by Begemann, whose method of copper analysis we
have employed, exceeds the value obtained with Busycon by an amount
well in excess of the apparent experimental errors. These hemocyanins
appear also to differ in certain other respects. Busycon hemocyanin
cannot be crystallized by methods which succeed in the case of Helix
(Dhere, Baumeler and Schneider, 1929). Busycon hemocyanin is
insoluble in the region of its isoelectric point in the presence of quite
^V08 "^
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(uui LIBRARY
1C — ;
26 HUGH MONTGOMERY
appreciable amounts of salt. Helix hemocyanin, on the other hand,
appears to be readily dissolved by very small concentrations of salt
under these circumstances (Svedberg and Heyroth, 1929).
It is surprising that such a great difference exists between the
copper content of the hemocyanin of the squid and that of the octopus.
Inasmuch as the properties of the respiratory pigments in these two
cephalopods appear to be very similar, we believe it to be desirable to
redetermine these values by methods of preparation and analysis
which are strictly comparable.
The weight of hemocyanin containing one atom of copper is given
by dividing the atomic weight of copper, 63.57, by the fraction of
the weight of hemocyanin due to this element. In the case of Busycon
canaliculatum this fraction is 0.25 X 10~2. The minimal molecular
weight of Busycon hemocyanin thus appears to be approximately
26,500, when estimated upon the basis of its copper content. It has
been shown, however, by Redfield, Coolidge and Montgomery (1928),
that when hemocyanin becomes associated with oxygen to form
oxy hemocyanin, one molecule of oxygen is combined with a quantity
of hemocyanin containing two atoms of copper. Inasmuch as it
appears highly unlikely that the oxygen molecule is dissociated into
its constituent atoms in its reaction with the respiratory protein, it
seems safe to assume that each molecule of oxyhemocyanin is com-
bined with not less than one molecule of oxygen. The hemocyanin
molecule must consequently contain at least two atoms of copper.
Estimated on this basis, the minimal molecular weight of Busycon
hemocyanin is approximately 53,000. In a similar way it may be
calculated that the minimal molecular weight of the hemocyanin of
Loligo pealei, estimated on the basis of its copper content, is 25,400,
and when the oxygen-combining relations are taken into account,
the combining weight appears to be approximately 51,000.
SUMMARY
The hemocyanin of Busycon canaliculatum contains 0.24 per cent
of copper and 15.8 per cent of nitrogen. Its minimal molecular weight
is approximately 53,000.
The copper content of the hemocyanin of Busycon carica appears
to be the same.
The hemocyanin of Loligo pealei contains 0.25 per cent of copper
and has a minimal molecular weight of approximately 51,000.
COPPER CONTENT OF HEMOCYANIN
REFERENCES
BEGEMANN, H., 1924. Over de ademhalingsfunctie van haemocyanine, thesis,
Utrecht; for abstract see Jordan, H., 1925. Zeitschr.f. vergl. Physiol., 2: 381.
BURDEL, A., 1922. Contribution a 1'etude des hemocyanines, thesis, Fribourg.
CRAIFALEANU, A., 1919. Boll. Soc. Natur. Napoli, Anno 32: 88.
DHERE, C., 1919. Jour, physiol. et path, gen., 18: 503.
DHERE, C., BAUMELER, C., AND SCHNEIDER, A., 1929. Compt. rend. Soc. de biol.,
101: 759.
FREDERICQ, L., 1878. Arch, de Zool. esp. et gen., 7: 535.
GRIFFITHS, A. B., 1892. Compt. rend. Acad., 114: 496.
HENZE, M., 1901. Zeitschr. physiol. Chem., 33: 370.
HOPKINS, F. G., AND PINKUS, S. N., 1898. Jour. Physiol., 23: 130.
ROBERT, R., 1903. Arch. f. ges. Physiol., 98: 411.
MENDEL, L. B., AND BRADLEY, H. C., 1906. Am. Jour. Physiol., 17: 167.
REDFIELD, A. C., COOLIDGE, T., AND MONTGOMERY, H., 1928. Jour. Biol. Chem.,
76: 197.
REDFIELD, A. C., COOLIDGE, T., AND SHOTTS, M., 1928. Jour. Biol. Chem., 76:
185.
SVEDBERG, T., AND HEYROTH, F. F., 1929. Jour. Am. Chem. Soc., 51: 539.
GROWTH AND DIFFERENTIATION OF THE COLONIES OF
ZOOTHAMNIUM ALTERNANS (CLAP. AND LACHM.)
E. FAURE-FREMIET
COLLEGE DE FRANCE, PARIS
INTRODUCTION
In a preceding publication (1922) I have insisted on the fact that
colonial Vorticellidse constitute an intermediary step between a popula-
tion of like cells (cultures of free Infusoria) and a multicellular organ-
ism; unlike free cells with unlimited power of division, whose population
growth theoretically follows a geometrical progression. The col-
onies of Epistylis, of Carches'mm, or of Zoothamnmm generally have
a limited growth, following a special cycle, independent of a possible
sexual cycle. In these colonies the lineage of each cell is perfectly de-
nned by dichotomous ramifications of a common peduncle, and it is pos-
sible to show in a large number of cases the existence of somewhat dif-
ferential divisions giving two sister cells whose power of multiplication
is different. In certain species (Eplstylis arenicolce, Epistylis Perrieri}
the first divisions can be dichotomous and equal, so that the mass growth
of a number of individuals follows a geometrical progression ; but soon
the sister cells resulting from each division multiply unequally, and the
growth approaches more or less an arithmetical progression.
On the other hand, the study of the growth of the common peduncle,
which is considered as a product of the protoplasmic activity, shows
that the latter may decrease in course of time. But the Vorticellida*
colonies form, from time to time, migrating individuals which may be of
the same size as the other individuals (Carchesium, Epistylis} or more
voluminous (some Epistylis, some Zoothamniuni, called heteromorphic).
In these individuals, and in these only, appear secretory granules already
observed by Engelmann and more recently (1926) by Wesenberg-Lund,
which seem to be connected with the formation of the peduncle, and one
can consider the hypothesis of an active substance, or of a transformable
substance, produced in a definite quantity and periodically, by certain
individuals, which is divided among the descendants of the latter and at
the same time is diminished little by little.
It appears then that the growth of a group of cells may be limited by
28
ZOOTHAMNIUM ALTERNANS 29
factors somewhat internal but altogether independent of the hypothetical
notion of a " factor of senescence " 1
The Zoothamnium called heteromorphic, about which I have given
some detail in my paper of 1922, seems to give the most typical ex-
amples as to the role of these internal, or properly cellular factors, in
the general form of growth of a colony and its limitation.
Claparede and Lachmann described in 1858 a marine species,
Zoothamnium altcrnans (described later by Mobius under the name of
Z. Cienkowskii) ; the aspect of the colonies, they say, is that of " un
arbre a branches courtes et tres regulierement alternantes. La forme
de ces families a sa cause dans un arret de division spontanee qui frappe
en general 1'un des deux individus issus de chaque division. Lorsqu'un
individu A se divise en deux individus B et B1 ', 1'un des deux, B par
exemple, ne se forme qu'un pedoncule fort court et son developpement
reste stationnaire a partir de ce moment, tandis que 1'autre, B', secrete
un pedoncule plus long, puis se divise en deux nouveaux individus, C et
C', dont le premier, qui est tou jours du cote de la branche opposee a
celui ou se trouvait 1'individu B, ne forme qu'un pedoncule tres court
et ne se divise pas davantage tandis que C' forme un pedoncule plus long
et se divise en deux individus D et D' et ainsi de suite."
That is not all ; in Z. altcrnans and in Z. arbuscula Ehrb. or Z.
genlculatwn Ayrton (see Wesenberg-Lund, 1925, and Furssenko, 1925)
the migrating individuals which' will be the origin of new colonies and
will thus begin a new cycle, are distinguished not only by a few mor-
phological characters, but also by their voluminous size and the well-
determined place where they originate in the colony, generally at the
junction of the main branches. These large migrating individuals are
the " ciliospores " of Wesenberg-Lund or " macrozoides " of Furssenko,
much larger than the " trophozoides " or " microzoides " which consti-
tute the most numerous individuals of the colony.
Ehrenberg had observed these individuals in Z. arbuscula, and had
noticed that they result from the growth of an individual not unlike the
others, but always situated at the junction of a branch. This author
admits that one of the two individuals issued from a bipartition on the
branch while the other grows without dividing, thus being, he says " the
aunt " of the individuals of the branch. Claparede and Lachmann find
this same condition in Z. altcrnans, but sometimes this growing indi-
1 In other publications (1925-26) I tried to show that in several very different
cases the idea of a factor of senescence could be replaced either by the hypothesis
of differing speeds in a group of transformations necessary to cellular activity,
or by the assumption of a "probability" of transformation which would be too
long to develop here. (See Faure-Fremiet and Laura Kaufman, 1928, and Faure-
Fremiet and H. Garrault, 1928.)
30 E. FAURfi-FREMIET
vidual may undergo a division. Zootliainnimn alternans (Claparede
and Lachmann) is found frequently on the coasts of Brittany ; I have
found it in abundance in Woods Hole and was able to follow the dif-
ferent stages of the colony cycle and of the formation of the " cilio-
spores." I observed a few phenomena of conjugation, quite sporadic,
but I have not observed a sexual cycle analogous to the one discovered
by Wesenberg-Lund in Z. gcniculatum or described by Furssenko in
Z. arbuscula.
TECHNIC
In order to follow the complete evolution in a large number of
colonies, I have used numbered slides, ruled in squares with a diamond
point. These slides were first placed in a crystallization dish containing
numerous colonies of Z. altcrnans. After several hours, they were re-
moved and placed in a Petri dish containing sea water and examined
under a binocular microscope. All individuals recently attached were
carefully located and designated in numeral order ; those whose peduncle
had already developed or had already given the first division were
removed with a needle.
After this operation, the slides were placed vertically on frames
floating in an aquarium through which ran a strong current of sea
water ; this was done to avoid the deposit of particles and of microorgan-
isms. The slides were then examined periodically and the different
stages of the development of each colony were carefully recorded in
function of time.
When the cytological examination of a colony is necessary, it is
always easy to detach this colony with a fine pipette, in order to study it
under the high power, in vivo, or after fixation.
The best technic for the study of the nuclear apparatus is the
fixation by OsO4 for a short time followed by boracic carmine stain.
The presence (generally in the Vorticellidae) of a cuticle and the con-
tractability of a peduncle constitute two technical difficulties which are
not easy to overcome ; it may be necessary to cut the colony with a fine
scalpel in order to isolate certain individuals which it is necessary to fix
and stain.
STRUCTURE OF THE COLONIES
The appearance of colonies of Z. altcrnans is very nearly that of a
palm (Fig. 1) ; they have a main trunk and oblique branches placed
alternately in the same plane, on right and left of the axis; the main
trunk always bears at the top a terminal individual of rather large size ;
the lateral oblique branches bear a variable number of small individuals ;
ZOOTHAMNIUM ALTERNANS
31
finally along the trunk, at the juncture of the lateral branches, are
found the voluminous migrating- individuals either macrozooids or
macrospores.
Trt
FIG. 1. A young colony of Zootliaiiniinin altcrnans (Clap, and Lachm.),
showing the main trunk and the alternate lateral branches. TM, terminal macro-
zooid ; Ci, ciliospores at different stages of growth, located on the anterior side
of the colony at the first division of each branch D, E, G, H. The branch F , in
this case, bears, at the same place, only two microzooids apparently identical with
the others.
The lateral branches of the colony observed in extension are almost
always slightly curved in, and most of the individuals borne by these
branches are inclined toward the outside of the curvature. The two
sides of the palm are thus different, and one can define at the same time
a base and a summit, an anterior and a posterior side.
The elements of symmetry of such a colony are a main axis repre-
sented by the trunk, and a median plane, antero-posterior, separating the
two halve^ right and left.
As for all the other species of the genus Zoothainnhtm, the colonial
peduncle bears an elastic tube whose role is passive, and a continuous
" cordon central," dichotomically ramified, which represents the pro-
32
E. FAURfi-FREMIET
longation of the lower extremity of each individual ; this central cordon
has itself a protoplasmic tube (/) limited by a fine film and surrounding
a muscular fiber which terminates at the basal part of each individual
by a conical group of myonemes.2 The migrating individuals, or
" ciliospores," when liberated swim rapidly with their posterior ciliary
crown. They are large individuals, flattened in the antero-posterior
direction, and look like a top. They attach themselves by means of the
scopula (/) and begin to secrete the peduncle. At the same time they
lose their posterior ciliary crown and progressively take on again the
ordinary subconical form.
. 10 [»
100 p
Time :
15 m.
ZH.
10 H.
FIG. 2. Fixation of the ciliospore and construction of the peduncle. At first
the top-like ciliospore turns quickly on the slide, then the building of the peduncle
begins ; the same individual is shown fifteen minutes after fixation. The ciliary
crown slows down and disappears while the peduncle grows (Epistylis stage) dur-
ing a short time (two hours) ; finally, one can see the differentiation of the " cor-
don central" and the muscular fiber (ten hours).
The peduncle is at first a solid cylindrical body of a fibrillar structure
which grows rapidly ("Epistylis stage") ; after two hours it reaches
2 For the structure of the Vorticellidse in general, and of the peduncle in par-
ticular, see Faure-Fremiet (1906).
ZOOTHAMNIUM ALTERNANS
a length of about 250 //.. The secretion then begins to slow down and a
section of the peduncle is ring-like; there is a central canal, at the bot-
tom of which remains attached a part of the body of the infusorian,
which from now on will lengthen itself along with the tube of the
peduncle and become differentiated in a central cordon with the muscular
fiber or " spasmoneme " (Fig. 2).
Six or seven hours (at the temperature of 21° C.) after the start of
the secretion of the peduncle, the original individual undergoes a first
unequal division which gives a macrozooid and a microzooid ; the plane
passing through these two zooids and the common peduncle is the median
plane of symmetry of the future colony. The large cell remains clearly
axial after this first division and continues to form actively the prin-
cipal peduncle of the colony. After four to seven hours it undergoes a
second unequal division; the interval between the following divisions is
longer, from ten to sixteen hours; but always during the growth of the
colony the terminal individual is a macrozooid. each division of which
separates a microzooid in the median plane of the colony. The succes-
sive series of terminal microzooids constitutes a main strain perfectly
schematized by the axial trunk of the colony.
\\c. shall designate each cell of this series by a Roman numeral
representing the division which started it ; we shall have then the origi-
nal individual, or ciliospore, then the series of macrozooids, I, II, III,
... X, etc.
^Ye shall designate with capital letters the corresponding series of
median microzooids detached from the main strain (microzooids of first
order), A, B, C, . . . J, etc. Each branch of the colony is started by
the division, alternately at the right and at the left of the median plane
of each microzooid of the first order. But, in accordance with the dia-
gram of Claparede and Lachmann, only one of the two cells resulting
from such a division is the origin of a lateral limb ; we shall designate it
by a small letter preceded by the coefficient 1 ; the other cell remains
median and will be designated by its capital letter preceded by the same
coefficient 1.
At the beginning of the formation of the fifth branch, for example,
we shall have first the division of the terminal macrozooid IV, which
will give a new terminal macrozooid V and a median microzooid E.
The latter will divide in a perpendicular plane to that of the division of
IV, and will give two individuals, one of which, IE, remains in the
median plane while the other, \c, situated for example at the right of
this plane, will be the origin of the branch (Fig. 3).
Each branch has also a main axis and lateral branches but does not
have a well-defined median plane nor median individuals. The division
3
34
E. FAURfi-FREMIET
of \c, for instance, gives rise to two cells apparently similar, 2e* and
2c'-. The individual 2el remains in the axis and gives at the new divi-
sion 3cl (axial) and 3r2 (lateral) ; 3cl will give 4cl (axial) and 4c2
(lateral), etc.
IV
FIG. 3. Scheme of the branch E and the basis of the branch F, showing the
lineage of the median microzooids IE and \F and the different microzooids.
Likewise the individuals 2c2, 3c2, and 4c2 will give successively two
or three generations, the elements of which we shall designate by the sym-
bols 2c21, 2e22, 3c21, 3e22, etc; according to the rule of Claparede and
Lachmann 3c22 does not divide, but 3r21 gives 3c211 and 3e212 ; the num-
ber of generations formed by the lateral branches seems to be always
rather limited.
The median individuals of the second generation: \A, IB, 1C . . .
IE, etc., can divide once and give IA1 and 1A2 for example. But while
\A, IB, 1C, and their two immediate descendants remain microzooids
identical to these designated by the small letters, ID, IE and the follow-
ing ones, or the two cells of the second generation, ID1, ID2; IE1, IE2..
etc., undergo a considerable growth and are transformed into ciliospores,
or migrating macrozooids, which soon detach themselves from the com-
mon trunk to swim freely and to attach themselves later on.
It appears clearly then that during the growth of a colony of
Zoothanmiuni altcrnans the two cells resulting from the division of one
initial cell are never equivalent as to their " potentialities." But in con-
firming the observations of Ehrenberg and of Claparede and Lachmann,
we may now make them more precise by showing that the progressive
segregation of the power of multiplication and of the power of growth
is very rigorously tied up with the respective position of the individual
separated by the successive divisions. It seems then that a certain
ZOOTHAMNIUM ALTERNANS
number of divisions at least must be considered as differential divisions.
The cytological examination confirms this interpretation.
FIRST DIVISION OF THE INITIAL MACRGZOOID
The first division is characterized, in a rigorously constant manner,
by the unequal division of the macronucleus and of the protoplasm of
the initial individual of the colony (Fig. 4) between the first two cells,
the macrozooid / and the microzooid A (Fig. 5). A short time before
this division, the macronucleus, which takes the shape of a long twisted
rod, enlarges at one of its extremities in a compact mass. The other
extremity is thin and often flattens slightly, and becomes elongated in
the median plane of the individual. The two edges of this flat portion
are often slightly thickened, so that a side view gives the impression of a
structure in a horseshoe shape. The micronucleus remains near the
thick extremity and soon lengthens into a spindle. Meanwhile the
peristome and the scopula divide as well as the central cordon of the
peduncle and soon an upper and a lower furrow, growing in depth
toward each other, begin to separate two cells of very unequal size.
The micronucleus completes its own division, then the macronucleus is
divided unequally at the time when the two furrows join; the macro-
zooid (which remains the terminal individual on the axis of the colony)
retains the thickened part of the macronucleus and a micronucleus ; the
microzooid (which becomes the first median individual A} retains the
thin part of the macronucleus and a micronucleus (Fig. 6).
Considering the irregular shapes of the body and of the macro-
nucleus in Z. alternans, it is impossible to calculate the corresponding
volume and to establish the values of the nucleoplasmic relation. Never-
theless, it is clearly evident that the ratio N/P is greater in the micro-
zooids than in the macrozooids, i.e., the macronucleus is divided into
two daughter cells even more unequally than the cytoplasm.
It is difficult to establish whether there exists a difference in com-
position between the two unequal extremities of the macronucleus
divided between / and A. The " nuclear reaction '' of Feulgen does not
show any difference between these two parts, and their structure differs
very little. Most frequently one can observe a linear orientation, in a
continuous and parallel line of the chromatin granules (microsomes)
in the thin part of the macronucleus which will be distributed by the
division. On the other hand, the voluminous mass which remains in
the macrozooid / shows an irregular distribution of its microsomes.
This mass behaves as a chromatin reserve which would not be affected
at all by the phenomena of division.
Supposing that the terminal condensation of the macronucleus repre-
36
E. FAURE-FREMIET
seats a kind of segregation of the chromatin material, we shall describe
this first unequal division as a differential quantitative and qualitative
division.
FIG. 4. Ciliospores at the beginning of the peduncle's formation, showing the
appearance of the macronucleus before the first division.
FIG. 5. First cleavage of the ciliospore, giving the terminal macrozooid / and
the median microzooid A. The figure shows the differential division of the macro-
nucleus (figured by dotting) and the apparently equal division of the micronucleus
(black — spindle stage).
FIG. 6. Later stage of the first cleavage, showing the terminal macrozooid /
and the median microzooid A ; macronucleus figured by dotting ; resting micro-
nucleus black.
FIG. 7. Fourth cleavage on the main strain giving the terminal macrozooid
V and the median microzooid E. The qualitative equal division of the macro-
nucleus (figured by dotting) is shown.
LATER DIVISIONS OF THE INDIVIDUALS OF THE MAIN STRAIN
The division of the individuals / and // presents exactly the same
differential character as that of the initial individual. It is different at
the time of division of the individual ///. In the latter, the macro -
nucleus shows at the outset of the repartition a symmetrical thickening
at each of its granular extremities which appear entirely homologous.
ZOOTHAMNIUM ALTERNANS 37
The median part, finely striated, is divided, however, into two unequal
parts by the division of the protoplasmic body, which isolates here again
an axial and terminal macrozooid, V. and a median microzooid E (Fi<y
7).
All the later divisions of the individuals from the main strain, i.e.,
IV, V , VI . . . X etc., are of the same type, and we shall consider these
divisions as quantitatively differential only.
DIVISION OF THE MEDIAN MICROZOOIDS
The median microzooids, A, B, and C, which have received only the
thin extremity of the initial macronucleus, undergo an almost equal
division which gives for example \A^ (median) and la2 (lateral) of the
same dimension and of the same structure, both having a thin and
twisted macronucleus, as well as the descendants of Irt2, 1£>2, and If2
(Fig. 8).
On the other hand, the median microzooids, D , E, F, and the follow-
ing undergo an unequal division, quantitatively and qualitatively dif-
ferential, like that of the first three individuals : the ciliospores / and
//. A short time before the division, when the median individual begins
to lengthen in the transverse plane, its macronucleus takes the shape of
an elliptic blade, presenting in a marginal point a large subspherical
thickening. This thick part of the macronucleus, on the other hand,
lengthens at the time of division and is divided between the two in-
dividuals \D, Id, IE and \c, etc. (Fig. 9).
These facts indicate that the differential division takes place at
two different times from the fourth generation of the axial cells. For
instance, when the division of /// divides into IV and D, the microzooid
D has a little less than a half macronucleus ; but this half macronucleus
is qualitatively similar to that of the macrozooid IV, having a granular
terminal thickening. However, the microzooid D shows a nucleoplasmic
relation, a ratio N/P superior to that of macrozooid IV, for the proto-
plasm has divided much more unequally than the macronucleus. It is
a small individual with a large macronucleus.
When the microzooid D divides, the cytoplasmic division is almost
equal, but the division of the macronucleus is qualitatively differential,
because the thickened and granular part does not divide but goes whole
to the median individual ID. The outcome is that the ratio N/P is
still increased in this individual.
The axial microzooids ID, IE, etc., can undergo a division and give
for instance ID1 and ID2; but these two individuals, which remain
median, soon begin to enlarge without dividing any further.
38
E. FAURfi-FREMIET
The microzooids Id, Ic, etc., as said above, go through a series of
divisions which always give individuals with long and slender macro-
nuclei.
m
8
FIG. 8. Cleavage of the median microzooid B, giving, with equal division of
the macronucleus, the microzooids 1&1 and lb-. Comparison between the terminal
macrozooid /// and the median microzooid C (resting stage).
FIG. 9. Cleavage of the median microzooid D, giving the future median
macrozooid ID (ciliospore) and the microzooid Id, with a differential division of
the macronucleus.
FIG. 10. One median macrozooid (\G for example) at the beginning of its
growth, and one microzooid of the corresponding branch. The large difference
in size of the macronucleus is to be noted.
FIG. 11. Two median macrozooids during the time of growth. In the macro-
nucleus, numerous large nucleoli are to be seen (figured as vesicles on the
drawing).
GROWTH OF THE MEDIAN MICROZOOIDS AND FORMATION OF THE
« CILIOSPORES
The median microzooids of the fourth generation (D or ID1 and
ID2} and of the following generations (E, F, G, etc.) increase rapidly
until they reach a length of about 55 p to 70 /A, in one day, two days, or
two and a half days.
ZOOTHAMNIUM ALTERNANS
39
The macronucleus, already voluminous, begins to grow and forms
a very large horseshoe-shaped body. The micronucleus situated at
the lower part in a slight depression lengthens into a spindle as in
preparation for the division. While the macronucleus increases, rather
refringent nucleoli appear in the midst of the chromatic granulations,
not giving the reaction of Feulgen (Fig. 10).
Soon, while the protoplasmic growth goes on, it seems that the
nuclear growth stops. The very numerous nucleoli alone still increase
in volume (Fig. 11). Then the outline of the macronucleus disappears,
the nucleoli project on the surface of the chromatic mass, and one can
observe very numerous stages of disintegration and of degeneration of
the macronucleus and of its fragments (Fig. 12).
12
14
15
FIG. 12. Later stage of the median macrozooid's growth. Disintegration and
disappearance of the macronucleus.
FIG. 13. One median macrozooid almost ready to leave the colony : p, posterior
ciliary crown; Ma and Mi, macronucleus and micronucleus of the new nuclear
apparatus ; r, residual mass of chromatin.
FIG. 14. Top view of a median macrozooid (same stage as that shown in
Fig. 12).
FIG. IS. Terminal macrozooid making the posterior ciliary crown and soon
ready to leave the colony.
40 E. FAUR&-FREMIET
Finally, one sees in the center of the cytoplasmic mass containing a
rather larger number of residual masses, a short macronucleus, arched,
staining very intensely, containing only very small nucleoli, and accom-
panied by a resting spherical micronucleus (Figs. 13 and 14). This
aspect, frequently observed, is that of a nuclear apparatus of new for-
mation, and it is probable that the changes just described represent a
phenomenon of endomixis. I was, however, unable to follow in the
individuals stained in toto the fate of the spindle-shaped micronucleus
observed in the preceding stages. It probably divides and makes up the
new nuclear apparatus ; but this stage was not observed in my set of
preparations. At the end of the protoplasmic growth and when the
nuclear changes are completed, a furrow appears around the median
individual, at about the posterior third. It is the future ciliary crown,
whose vibratile elements appear soon afterward. At the same time
the organism flattens in the antero-posterior direction, and takes the
shape of a top. The cytoplasm is filled with diverse inclusions, a great
number of which are probably nuclear residue. In the posterior region,
above the " scopula," appear very numerous inclusions which are not
very refringent. Neutral red in I'tvo colors them a brownish red.
These inclusions correspond to the secretion granules whose existence
I have already mentioned in the migrating individuals of different
Vorticellidse.
There are still a few lipoid granules, and, toward the middle of the
body, numerous small inclusions fixing neutral red in an intense red
color. Iodine fixation gives a mahogany color, but the latter is not any
stronger than for the microzooids.
The " ciliospore " which has thus been formed becomes almost lens-
shaped. The peristome remains closed and the posterior ciliary fringe
is animated with active movements which soon determine the liberation
of the migrating individual (Fig. 15).
GROWTH OF THE COLONIES OF ZOOTHAMNIUM ALTERNANS
At a temperature of 21° C., in an aquarium with running water, the
growth of the colonies of Z. alter nans goes on very regularly for a
period of eight to ten days. Hence it is easy, by periodic examinations
of a specific colony, to follow the increase in number of the individuals
as a function of time. We have then a measure of the colony's growth.
This measure is not very exact, because certain individuals grow without
dividing and their mass is clearly larger than that of the others.
However, the group of large cells given by the terminal macrozooid and
the ciliospores is always rather restricted, and one can admit that the
ZOOTHAMNIUM ALTERNANS
41
appearance of the development is rather well represented by the varia-
tion in number of the individuals. A more important error may arise
from the fact that some parasitic Infusoria (Acineta) very often get
into the microzooids (especially the microzooid of the first branch) and
multiply in this individual, which does not divide and soon falls off.
Because of this, it is necessary at every investigation to trace a total
scheme of the colony studied, indicating the place of each individual,
which with some practice, may be quickly made by examining the colo-
nies in extension in a thin water layer with a low power objective. By
this means it is possible to keep an account of the accidental influences ;
but when the number of individuals increases too much, beyond the
eighth day, for example, this method of pointing becomes very difficult
and soon impossible to use with precision.
TABLE I
Numbers of Colonies Examined
Dafp
T '
1
2
3
4
5
6
7
8
9
10
11
12
July 14
11A.M.
0
0
0
0
0
0
0
0
0
0
0
0
15
11A.M.
III
III
III
III
II
III
III
III
II
III
III
16
12 M.
V
V
V
IV
IV
III
IV
IV
III
IV
IV
17
4:30 P.M.
VII
VIII
VII
V
VI
V
VII
VI
V
VI
18
9A.M.
VIII
IX
IX
VII
VII
VII
IX
VIII
VI
19
10A.M.
IX
XII
VIII
VIII
VIII
XI
X
20
11:30 A.M.
XI
XIV
XII
X
IX
XIII
21
9 P.M.
XII
XVI
XIV
XV
22
9 P.M.
XVII
XII
Numbers of Colonies Examined
T '
13
14
15
16
17
18
19
20
21
22
23
11-
July 14
11 A.M.
0
0
0
0
0
0
0
0
0
0
0
15
11 A.M.
11
III
III
II
IV
I
III
II
III
16
12 M.
IV
V
V
III
V
V
IV
IV
17
4:30 P.M.
VI
VII
V
V
VII
VI
VI
18
9A.M.
VIII
VIII
VII
IX
VII
19
10A.M.
20
11:30 A.M.
21
9 P.M.
22
9 P.M.
The simultaneous study of the growth of the various colonies placed
in apparently identical conditions, on the same slide or on adjacent slides,
shows at first that the speed of growth is not the same for all the
colonies. We have already seen that the interval between two divisions
varies in rather large proportion, in the same stage, in two different
colonies (i.e. four hours to seven hours between the division of / and
that of //; ten hours to sixteen hours between the division of // and that
of///).
42
E. FAURfi-FREMIET
Table I shows the records of twenty-three colonies (experiment
commenced on the 14th of July) ; the figure 0 indicates the initial
macrozooid at the beginning of the peduncle formation, and the Roman
numbers indicate the number of the terminal individual on the main
strain ; we see, thus, that on the fourth day, there may be a difference
of two generations between different colonies and that on the eighth day
the difference may be four generations. The whole number of indi-
viduals borne by each colony differs, of course, proportionally.
N
130
no
no
wo
90
80
70
60
SO
40
30
20
10
ll'f
96
168
193. H.
FIG.
and 7) ;
16. Curves of growth from four colonies of Z. altcnians (Nos. 1, 2, 4,
number of the individuals in ordinates ; time (in hours) in abscissae.
The data relative to colonies Nos. 1, 2, 4, and 7 are plotted in the
curve of Fig. 16. These are only gross numbers, there being no cor-
rection for some microzooids parasitised or dropped out. Besides, these
various curves show that for each colony the rate of growth varies itself
in the course of the growth; but it is difficult to determine the part of
the accidental factors already mentioned and capable of introducing
some disturbance.
Fig. 17 represents in function of time the genealogical and complete
view of a colony having given sixteen generations on the main strain.
ZOOTHAMNIUM ALTERNANS
43
The essential data are given by the successive records of colony No. 2,
completed, as regards the incomplete branches sprung from A, B, and
C, by the data furnished by other colonies studied in the same experi-
ment (3, 5, 20, etc.). Furthermore, the periods of some divisions have
been settled according to the survey of the successive and periodical ex-
aminations of colony No. 2 with interpolations ; I have kept account, in
this case, of the interval settled with more precision than in other ex-
periments in which either the first stages of the colony or the growth of
a branch were connected at intervals of time most closely approached
from hour to hour.
The curve represented in Fig. 18 is drawn according to this scheme.
The daily increase of the number of individuals shows the following
numbers :
Time
(in hours)
Number of
individuals
Increase of the unity
of mass in 12 hours
Number of zooids
made in 24 hours
0
1
1
12
2
2
24
4
2
3
36
9
2.25
48
15
1.66'
11
60
23
1.58
72
31
1.34
16
84
41
1.32
96
55
1.34
24
108
66
1.20
120
84
1.27
29
132
104
1.23
144
122
1.17
38
156
137
1.12
168
147
1.06
25
The first part of this tabulation shows a rather regular increase and
such that the number of the individuals, i.e., approximately the whole
protoplasmic mass, doubles at regular intervals, from twelve hours to
twelve hours.
Of course, we find again here, at first the geometrical progression of
the ratio 2 which characterized the multiplication by bipartition of a
mass of cells which keep always the same speed of growth. If we
choose for unity of time this period of twelve hours, we see, however,
that after the second day the rate of growth of the unity of mass, which
averaged about 2, slows down progressively from 1.66 to 1.58 and 1.34,
then persists for some time at a median and constant level: 1.32, 1.34,
1.20, 1.27.
Then, in a last period, this rate of growth again slows down with the
* Y
44
E. FAURfi-FREMIET
values 1.23, 1.17, 1.12, 1.06; but the difficulty in obtaining an exact
enumeration does not permit a determination of its values when the
colony approaches its greatest size.
Then it appears that the growth progressively slackens in the whole
of the colony ; the time necessary to double the protoplasmic mass grows
as the protoplasmic mass increases; it is a limiting factor of the growth.
But it is evident that this factor (or limiting factor), in the case of
Z. alternans, is not a factor of senescence which affects equally all the
individuals, and involves a sort of progressive segregation, whose nuclear
phenomena give a parallel objective picture.
JULY
1
>
I
01
*•— M
VI
JJ|
XvJ
XIV 1
JN • |
xu J V
T
-168
-Iff
-14-*,
\ \ 1 .131
XII 1
-ItO
L
v .
K
XI
1 ' '
-/at
i
X J
•* >
i
IX
-9s
'-
L
vm H — ' -J
. gt
vn
?!
- fz
vi F — 1
- to
'
W D
1 _ 3t>
c
m
n
-24
B
A
i
_ It
Ci
. 0
15
H,
FIG. 17. Genealogical view of a colony at the sixteenth generation; time
in abscissae; lineage of each individual in ordinates.
This leads us to examine the case of the main strains. After the
second division, which takes place rapidly, four to seven hours after the
first one and at a temperature of 21° C., the rhythm of the bipartitions
of the axial macrozooid slows down, (sixteen to seventeen hours
between second and third divisions), then remains sensibly constant.
During the entire growth of the colonies, more than twenty bipartitions
of the axial macrozooid succeeded each other at intervals of ten to six-
teen hours. The growth of the axial peduncle was fairly constant.
It seems then that during eight to ten days at least, the functional
activity and the power of growth of the axial macrozooid remain con-
stant, and, in the colonies already developed, one can observe the for-
ZOOTHAMNIUM ALTERNANS 45
mation of a posterior ciliary crown around the terminal individual.
Thus the axial macrozooid can become a migrating individual equivalent
to a ciliospore, but one never observes in this case the endomictic trans-
formation of the nuclear apparatus. We have seen how the nuclear
segregation which is established during the differential divisions seems
to determine the characteristic features of the median individuals and
of the microzooids. However, we must admit that the later divisions of
the microzooids are still different, although they are not accompanied
by a visible nuclear segregation.
According to the rule of Claparede and Lachmann, we can still
distinguish in one branch one main strain and lateral strains.
The fourth branch, for instance, after the differential divisions
which separate ID and Id may be represented as follows: Id gives 2d-
and 2dl. Let us give the exponent 1 to the main strain of this branch;
2d'2 gives 2rf21 and 2d" which do not divide any further ; 2rf1 on the
contrary gives 3d- and 3d1. The smaller branch issued from 3d2 has r.
principal axis, but the number of generations is reduced. The first
division separates 3d", which does not divide any further, and 3d"\
which gives 3d'21- and 3d211 without descendants. The individual 3d--
gives 4dl and 4r/2 ; 4d~ gives 4d'22 without descendants and 4d21, which
still gives 4d'21- and 4(/211 without descendants. The individual 4o!1
tf.
ISO
no
ISO
/to
I/O
100
go
so
1°
60
SO
1,0
30
SO
w
FIG. 18. Curve of growth of Z. altcrnans colony drawn from Fig. 17.
46 E. FAURfi-FREMIET
finally gives 5d'2 without descendants, and Srf1 which divides into 6dl
and 6d- without descendants.
The interval which separates the microzooid divisions is at first of
the same order (or even more rapid) than the interval which separates
the divisions of the axial individual ; but it increases progressively and
in such a colony, for example, the individuals of the sixth branch will
represent six successive generations from the cell F, while its sister cell
VI will have given during the same length of time ten successive genera-
tions.
We can see from Fig. 17, for instance, that the microzooids 2d~- and
2d21 live more than three days and a half without bipartition ; such a
fact is more typical with some microzooids of the earlier branches, A and
B, which maintain themselves for more than five days without division.
But after this time (corresponding to ten generations on the main
strain of the branch) these individuals do not appear larger than the
others ; yet they feed and their protoplasm contains many digestive
vacuoles. The decrease of the power of growth which characterizes
these individuals is not dependent upon their age — and for this reason
we cannot admit the notion of the factor of senescence — but of their
position in the colony, as if the differential divisions assured the pro-
gressive segregation of a factor of growth. But we can still notice that
this segregation, as it may be seen by the form of the growth of the
branch D, for instance, is yet continued during the divisions of the
microzooids which show no longer a differential appearance.
In short, if we bear in mind the main axis of the colony, its branches
and its boughs, we see that the power of growth, and of multiplication,
decreases according to a kind of gradient, in proportion with its removal
from the main strain.
The differential character of the cellular divisions seems to be the
essential condition which slows down and restrains the growth of the
colonies of Z. altcmans. But, theoretically at least, this restricted
growth should go on indefinitely. It is not the case here. Secondary
factors play here an important role; the development of different para-
sites (Protozoa, Protophytes) make it impossible to obtain a normal
growth of the colonies beyond ten days, under ordinary laboratory con-
ditions or in a natural marine environment ; soon the last surviving in-
dividuals leave the common peduncle. The microzooids often form in
this case a posterior ciliary wreath ; their fate has not been determined.3
3 A few cases of conjugation have been observed between a terminal macro-
zooid and a migrating microzooid. These cases were rare ; the later phenomena
were not followed.
s
ZOOTHAMNIUM ALTERNANS 47
CONCLUSIONS
The sexual cycle described by Furssenko and by Wesenberg-Lund
in the voluminous species of Zoothanin'mm (Z. arbuscula Ehrb., Z.
gcniculatuui Ayrton) is rather special and with Z. alternant (Clap, and
Lach.) I have never observed anything- similar, either on the Britanny
coast or in my cultures at Woods Hole; I will not, then, attempt to
compare the evolutionary cycle of these different species. The objective
that has led me into the minute study of these colonies of Vorticellidae is
the cyclical evolution — generally considered — of an initial cell's lineage,
which is here the foundation macrozooid or the " ciliospore."
The growth of colonies of Z. alternans is limited, in a great meas-
ure, by external agents such as parasitic infections, or the growth of
animal and vegetable microorganisms which change the surrounding
conditions of a specific colony.
In the cultures watched as described above, these various circum-
stances, somewhat accidental, are much reduced ; yet the growth of each
colony appears to be limited in itself ; I have taken the common in-
dividual— the microzooid — as unity of mass, and I have observed that
the rate of growth decreases in function of time for the whole of each
colony studied ; at the same time, some particular migrating individuals
are formed and become the source of new colonies ; it is precisely this
" cyclical " appearance of growth in the colonies of Vorticellidre that 1
have described in an earlier paper ( Faure-Fremiet, 1922) ; I have con-
sidered two different hypotheses : ( 1 ) the formation during the evolu-
tion of the migrating individuals of a limited stock of an hypothetical
" active substance " which divides and becomes increasingly smaller
with each generation of daughter-cells, or (2) a progressive modification
of the intimate composition of the cells, variations which would lie
" corrected " only during the evolution of their own migrating cells.'
In any case, this cyclical and limited evolution gives to the colonies
of Vorticellidce (Epistylis, Carchcsiuni, ZootJiaiiniiiun) somewhat of an
individualized character. In this regard, the case of Z. alternans is
very striking. At first, the successive divisions of the cells derived
from the first individual and the regularity with which they follow one
another in exactly determinate planes which fix the general features of
the colony, closely recall the process of a strictly predetermined cleavage,
but one which would be complicated with a continuous growth.
Secondly, the existence in these colonies of a main strain and of
secondary strains characterized by different nuclear qualities and dif-
ferent evolutionary properties recall in a certain measure the separation
4 These suppositions have been examined and criticized in a very interesting
work of G. Teissier (1928).
48 E. FAUR£-FREMIET
of the germinative and somatic strains during the cleavage of an Ascaris
egg-
Thirdly and finally, we can characterize the individuality of the
colony by the repartition of the power of growth and the power of
multiplication of its cells according to a certain gradient.
In connection with another species of ZootJictiniiiiini Wesenberg-
Lund also considers the notion of the individuality of the colony, for
the various individuals are tied by the continuous protoplasmic, thread
of the ramified peduncle and this brings about in their mass rather a
physiological unity. But the above-indicated characteristics are again
met, more or less accentuated, in other colonial Vorticellidse in the
species Epistylis and Carchcsium, for example, which do not show any
protoplasmic connection between the zooids.
The case of these colonies is then nearer that of a " population " of
cells, and their cyclical evolution appears very similar to populations of
free Infusoria, studied by so many authors.
The case of Z. alternans is still, from this point of view, particularly
interesting. In these species, the Claparede and Lachmann rule shows
that two daughter-cells have not necessarily the same power of growth
and of proliferation. I found the same rule (1922) in some species of
the genera Carchesium and Epistylis, and more especially with Epistylis
arcnicola (n. sp).
Here there seemed to exist in the course of the successive biparti-
tions a kind of progressive segregation of the power of growth, but
we find in Z. alternans, as an objective support of this hypothesis, the
differential divisions, which are produced at the origin of each lateral
branch and which indicate a kind of nuclear segregation.
In this species the main strain's cells which keep a constant nuclear
appearance, keep also a constant rate of growth and, apparently, an
indefinite multiplicative power. We witness, then, a cytological
mechanism, probably independent of the external factors which rule
the functional differentiation of the cells belonging to the same family,
in a process of growth.
This cytological factor, or those which are superimposed upon it,
rules at the sa.me time the family's general mode of growth ; it intervenes
as a limiting factor, independent of the colony's age, and quite distinct,
by this fact, from a factor of senescence in the true meaning of this
word. However, the colony's initial individuals, the " ciliospores," ap-
pear to be characterized by a kind of " physiological potential " greater
than that of the main strain's common individuals.
As in all the colonial Vorticellidas that I have previously studied,
they are characterized by large size and by the presence of definite
ZOOTHAMNIUM ALTERNANS 49
granulations connected with the secretion of the basic peduncle's inert
substance.
During their particular growth, accompanied by a complete changing
of the nuclear apparatus, the cells acquire these properties and we can
thus show that near the end of the colony's cycle of growth an endomic-
tic cycle exists, closely comparable to that observed in a population of
free Infusoria.
But we must remark that, here again, the particular evolution of
these " ciliospores " and the endomictic phenomena of which they are
the seat, are determined, not by their age, but by their place in the
colony's plan, just as if this evolution were still connected with the same
mechanism of differential division and of nuclear segregation.5
I am very glad to be able here to express my thanks to the Inter-
national Education Board, to my American colleagues who made my
residence at Woods Hole so profitable for me, and, very particularly, to
Dr. Calkins and Mrs. Harnley, who have helped me in translating this
paper.
SUMMARY
1. The first division of the initial macrozooid (or ciliospore) deter-
mines the median antero-posterior plane of the colony; the subsequent
cleavages of the daughter individuals are brought about according to
equally determined schemes, which give the main strain (or axial trunk)
and the lateral branches, alternately at right and at left.
2. The individuals constituting the main strain are of a rather
large size (axial macrozooids) ; their cleavage is always accompanied by
a differential division giving rise to a new axial macrozooid and a median
microzooid.
3. The differential divisions are characterized by an unequal division
of the protoplasmic mass, accompanied either by a sensibly equal di-
vision of the macronucleus (division supposed to be quantitatively
differential), or by the unequal division of the macronucleus in which
the larger mass (delicately granular) remains in the larger individual,
while the thinner part (often of fibrillar structure) goes to the micro-
zooid (division supposed to be qualitatively differential).
4. The cleavages of the ciliospores and those of the axial macro-
zooids, I, II, and III are always differential as regards the protoplasm
and the nucleus. The cleavages of the macrozooids IV and after give a
cytoplasmic differential division and an equal nuclear division; the dif-
5 Long ago I mentioned an apparently differential division in Lagcnophrys, in
which one of the individuals remained sedentary, while the other migrated and
secreted a new shell (1904).
4
50 E. FAURfi-FREMIET
ferential division of the macronucleus is carried back to the cleavage of
the corresponding- median microzooids.
5. The common microzooids have a limited power of growth and of
multiplication.
6. The median individuals having a large macronucleus after the dif-
ferential division of the median microzooids D and progeny begin an
active period of growth accompanied or unaccompanied by only one
ulterior division : these forms constitute the median macrozooids or
" ciliospores."
7. The growth of the ciliospores is accompanied by an important
hypertrophy of the macronucleus followed at first by a disintegration,
then by a reconstitution through an endomictic process.
8. During the growth of the median macrozooids, some grains of se-
cretion accumulate at the individual's posterior end, then the ciliary
crown grows, the ciliospore breaks away, swims freely, then settles down
on a substratum and becomes the source of a new colony.
9. The character of the differential divisions on the main strain
seems to determine the individual's differentiation of the colony; this
differentiation depends not only on the individual's size, but also on
its physiological potencies.
10. Independently of the obviously differential divisions, it is shown
that the power of growth is divided among the microzooids according to
a gradient, so to speak.
11. The unequal power of growth of the various individuals of a
colony gives to its whole growth a behavior which approaches the be-
havior of an organism. This unequal share constitutes for the growth
of the whole a limiting factor very unlike a factor of senescence.
LITERATURE CITED
CLAPAREDE AND LACHMANN, 1858-1861. Etudes sur les Infusoires et les Rhizo-
podes. Memoires dc I'lnstitnt Gcnez'ois, 5, 6, 7 and 8.
FAURE-FREMIET, E., 1904. Epuration et rajeunissement chez les Vorticellidse.
Compt. rend. Soc. Biol, 57: 428.
FAURE-FREMIET, E., 1905. Structure de 1'appareil fixateur chez les Vorticellides.
Arch. f. Protistcn, 6: 207.
FAURE-FREMIET, E., 1910. La fixation chez les Infusoires cilies. Bull. Sclent.
France et Bclglque, 44: 27.
FAURE-FREMIET, E., 1922. Le cycle de croissance des colonies de Vorticellides.
Bull. Sclent. France et Belgiquc, 56: 427.
FAURE-FREMIET, E., AND GARRAULT, H., 1928. La courbe de decroissance de
ponte chez " Margaropus Australis." Ann. Ph\siol. ct Ph\slcochlmle
Biol., 6.
FAURE-FREMIET, E., AND GARRAULT, H., 1925. La Cinetique de Developpement.
Coll. Les Problemes Biologiques. Press. Univ. France.
FURSSENKO, A., 1924. Zur Konjugation von Zoothamnium arbuscnla Ehrbg.
Trav. Soc. Naturalistes de Leningrad, 54: fasc. I.
ZOOTHAMNIUM ALTERNANS 51
FURSSENKO, A., 1924. Zur Biologic von Zoothamnium arbuscula Ehrenbcrg.
Arch. Russes de Protistol., 3: 75.
FURSSENKO, A., 1929. Lebenscyclus und Morphologic von Zoothamnium arbus-
cula Ehrb. Arch. f. Protistenk., 67: 376.
TEISSIER, G., 1928. Croissance de population et croissance des organismes.
Examen historique et critique de quelques theories. Ann. Physiol. et
Physicocliimie Biol., 4: 343.
WESENBERG-LUND, C., 1925. Contribution to the biology of Zoothamnium geni-
culatum Aryton. D. Kgl. Danske Vidensk, Selsk. Skriftcr, naturvidensk.
og mathcm. Afd., 8 Raekke X, 1.
THE INFLUENCE OF HUMIDITY ON THE BODY TEM-
PERATURE OF CERTAIN POIKILOTHERMS
F. G. HALL AND R. W. ROOT
(From the Zoological Laboratory, Duke University)
Poikilothermic animals are commonly accredited with possession
of a body temperature closely approximating that of their environment.
In general this appears to be true. However, there are some cases
where the body temperature of certain " cold blooded " animals may be
very unlike that of their surroundings. Such examples are given by
Rogers and Lewis (1916) in a table which they have compiled from the
investigations of numerous workers. It shows that not all investigators
agree even as to the temperature of the same species. It is probable
that much of the discrepancy is due to different types of method. On
the other hand, a more careful examination of the conflicting results of
various authors as to the correspondence between body and environ-
mental temperature shows that the greatest variations occur when ani-
mals are subjected to atmospheric conditions.
The factors which influence the temperature of animals may 'be
classified as follows : intrinsic — those that lie within the organism and
act to produce a temperature different from that of the environment ;
extrinsic — those imposed on the animal from without. The extrinsic
factors are (1) conduction and convection, (2) radiation, (3) evapora-
tion of water. A discussion of the role played by each factor is given
by Pearse and Hall ( 1928) . It is the purpose of this paper to study the
influence of the third factor, namely, the evaporation of water, on the
body temperature of various poikilo therms.
EXPERIMENTAL METHODS
Apparatus. — The apparatus employed consisted of an air pump,
several gas washing bottles — some containing concentrated sulfuric acid,
others water — a chamber in which animals under experimentation were
placed, temperature-measuring instruments, which included a potenti-
ometer, a high sensitivity suspension galvanometer, and a copper-con-
stantin thermocouple.
The air pump was adjusted to supply air at a constant rate of 22.6
liters per minute through two possible air leads. One lead was through
52
INFLUENCE OF HUMIDITY ON BODY TEMPERATURE
53
four wash bottles containing concentrated sulfuric acid and the other
through four similar wash bottles containing pure water. The amount
of air passing proportionally through each lead was controlled by screw
pinch cocks. Thus air of any desired humidity from 7 per cent to 100
per cent could be obtained. The relative humidity of the air was
measured by a calibrated hair hygrometer suspended in an enclosed jar
through which all the air passed before entering the experimental animal
chamber.
B
30 T
T
FIG. 1. Apparatus used to determine the influence of relative humidity on
the body temperature of animals.
The experimental chamber in which animals were placed is shown
in Fig. 1. A cylindrical percolator (A} was immersed in a constant
temperature bath (B). Animals were tied to a sliding rack (R) which
was so arranged that only a small portion of the animals' bodies was
in contact with it, thus allowing a maximum surface to be exposed to
the moving air. The end of this rack closed the mouth of the percola-
tor. Two precision thermometers (T) were inserted through the rack,
one in the upper, the other in the lower portion of the percolator. The
thermocouple lead wires (C~) also passed through the end of the rack.
The mouth of the percolator was packed with cotton to lower the rate
of conduction. The direction of air flow is shown by arrows. The
temperature in all experiments was maintained at 20° C.
Experimental Animals. — The species chosen for this investigation
were: Amphibians — the frog, Rana pipicns Schreber; the salamander,
PletJicdon glntinosiis Green ; the toad, Bufo fowleri Carman. Reptiles—
the lizard, Sccloporus nndulatns Latreille ; the " horned toad," Pliry-
uosoina corniiium Harlan; the turtles, Terrapene Carolina Carolina
Linn., Cistudo major Agassiz, Chrysemys marginata Agassiz ; the alii-
54
F. G. HALL AND R. W. ROOT
gator, Alligator mississippiensis Daudin. All animals were kept under
good laboratory conditions, and were alive and active at the end of each
experiment. Individuals were weighed at the beginning and end of
each experiment. From four to ten individuals of each species were
used and several determinations were made on each individual.
Temperature Records. — Environmental temperatures were recorded
by use of precision thermometers placed in the experimental chamber.
The body temperature was determined with a thermocouple inserted
through the anus well up into the animal's body. Each thermocouple
used was calibrated against a precision thermometer (previously cali-
brated by the U. S. Bureau of Standards). The temperature readings
are believed to be accurate to ± 0.01° C. Records of the temperature
of each animal and its environment were made at the following relative
humidity points: 7 per cent, 25 per cent, 50 per cent, 75 per cent and
95-100 per cent.
TABLE I
Showing Variations in Body Temperature of Several Species of
Poikilothcrms from Environmental Temperatures in At-
mospheres of Different Relative Humidities
Species
Relative Humidity
7
25
50
75
95-100
Salamander
-9.21
-8.60
-7.33
-0.70
-0.37
-0.72
-0.34
-0.39
-6.34
-6.75
-5.31
-0.70
+0.02
-0.57
-0.23
-0.26
-4.62
-4.68
-3.98
-0.15
+0.11
-0.52
-0.11
-0.15
-2.54
-3.01
-2.48
+0.30
+0.19
-0.41
-0.03
-0.08
-0.29
-0.13
-0.74
+0.64
+0.38
-0.12
+0.15
+0.18
Frog .
Toad
Lizard
Horned "Toad"
Turtle water
Turtle, land .
Alligator
Plus signs signify a higher body temperature than that of the environment;
minus signs indicate a depression in body temperature below that of the environment.
RESULTS
Amphibians. — The body temperature of the salamander, frog, and
toad very closely approximated that of their environment when the
surrounding atmosphere was saturated, or nearly so, with water vapor.
In atmospheres of low humidity, however, a considerable depression in
the body temperature below that of the environment was obtained.
Salamanders showed the most marked depression, toads the least
marked. The average results obtained are shown in Table I. Con-
INFLUENCE OF HUMIDITY ON BODY TEMPERATURE
55
siderable weight loss was suffered by these animals. At low humidities
their skins appeared dry and their bodies emaciated.
Reptiles. — The response of reptiles to atmospheres of varying hu-
midity was quite unlike that of amphibians. Whereas amphibians
showed great depression in body temperature when exposed to a dry
environment, reptiles showed only slight depression. In fact, if the
relative humidity be maintained between 90 and 100 per cent, many rep-
tiles will show a body temperature slightly higher than that of their sur-
°c
10
8
'-£ 6
o>
«j
o>
o>
Ol
o:
-2
\
Amphib i a
f
\
Reptiha
0
20
40
60
80 100%
Relative Humidity
FIG. 2. Graph showing relation of body temperature to environmental tem-
perature of amphibians and reptiles when subjected to different relative humidities.
roundings. Lizards and water turtles (Chryscinys marginata) were
influenced the most by low humidity. Apparently the water turtle is
slightly more susceptible to the influence of humidity than the land form.
Weight loss in the reptiles was practically nil. Subjection to low hu-
56 F. G. HALL AND R. W. ROOT
midity for long periods of time showed no apparent injurious effect.
Table I contains the average results obtained on all forms summarized
to show the difference in response to surroundings of varying relative
humidity. Fig. 2 shows the comparison of the response of amphibians
as a group with that of reptiles, and shows the variation in the change of
body temperature from that of the environment at similar relative
humidities.
DISCUSSION
It is apparent from the results obtained that in atmospheres of low
relative humidity, amphibians will have a much lower body temperature
than that of their environment. Such a condition results from the
evaporation of water from the surface of the body. The body tem-
perature of reptiles is but slightly changed by similar conditions. Thus
it is clearly indicated that the difference in response of these two classes
lies in the type of integument. The amphibians with moist skin will
readily lose water by evaporation. They have little means of retaining
water as has been shown by Gray (1928). The moisture of their
integument is in dynamic equilibrium with the water content of their
environment. The inner tissues supply water when that at the surface
has been evaporated (Hall, 1922). Thus, for example, a salamander
behaves physically very much like a wet bulb thermometer. The de-
pression in temperature is not as great, probably because water is not
transported to the surface as rapidly as in the wick of a wet bulb ther-
mometer.
Amphibians are limited in their habitat to moist places. They
possess a "reaction pattern" (Pearse, 1922), which permits them to
live only under damp logs and stones or in marshes or other watery
places. Thus they become more conspicuous on rainy days when the
atmosphere offers a more favorable and less restricted environment for
their activities. It is perhaps interesting to speculate that a frog may
have a lower body temperature on a dry, sunny day than on a somewhat
colder, rainy day.
The possession of a scaled integument, characteristic of the reptiles,
greatly increases the power of water retention. Reptiles give up water
very slowly and will resist desiccation for long periods of time (Hall,
1922). Not only by possession of an integument, but by certain in-
ternal physiological processes, such as the elimination of nitrogenous
wastes as uric acid instead of urea, they conserve water. In conse-
quence many reptiles live in very dry surroundings.
Perhaps the principal explanation of the discrepancies in reports
by many investigators of the correspondence between body and en-
INFLUENCE OF HUMIDITY ON BODY TEMPERATURE
vironmental temperatures is that they are due to a lack of control or
record of humidity. In the light of these experiments any results ob-
tained without knowledge of the relative humidity of the surroundings
in which an animal's temperature is taken would seem meaningless.
A further observation seems to indicate that the influence of changes
o
in humidity on the body temperature of these animals decreases as
animals higher in the phylogenetic series are used. It appears that
t; 10-
o>
xxo
CD
8-
6- ^
4-
o>
o>
0>
0
«0
00
Spec ies
FIG. 3. Showing a comparison of the change in temperature of the body of
each species studied when the humidity was lowered from 100 per cent to 7 per
cent saturation.
amphibians as they progress in evolution show a decrease in their sus-
ceptibility to humidity variations. The same fact apparently holds for
the reptiles. Fig. 3 represents the results arranged to show the maxi-
mum change in body temperature relative to environmental temperature
in each of the species used, the salamander showing the greatest change,
the alligator the least. The reptiles seem to have a more stable body
temperature than amphibians because they are less influenced by en-
vironmental factors. Possibly the increased ability of water retention
evolved in the reptiles is a " milestone " on the road to homoiothermism.
58 F. G. HALL AND R. W. ROOT
SUMMARY
1. Amphibians show marked response in body temperature to en-
vironmental variations in relative humidity. When subjected to an
atmosphere of 7 per cent relative humidity at 20° C., a depression of
several degrees centigrade may occur in their body temperature.
2. Reptiles show very little response to variations in relative hu-
midity. The integument apparently prevents the evaporation of mois-
ture from the surface of the body.
3. It is suggested that the evolution of the scaly integument of
reptiles from the slimy and moist skin of amphibians, with the con-
comitant power of water retention, is perhaps an important step in the
evolution of homoiothermism.
BIBLIOGRAPHY
GRAY, J., 1928. The Role of Water in the Evolution of Terrestrial Vertebrates.
Brit. Jour. E.rpcr. Biol, 2: 26.
HALL, F. G., 1922. The Vital Limit of Exsiccation of Certain Animals Biol.
Bull., 61 : 31.
PEARSE, A. S., 1922. The Effects of Environments on Animals. Am. Nat.
56: 144.
PEARSE, A. S., AND HALL, F. G., 1928. Homoiothermism. New York.
ROGERS, C. G., AND LEWIS, E. M., 1916. The Relation of the Body Temperature
of Certain Cold Blooded Animals to that of their Environment. Biol.
Bull, 21: 1.
THE POINT OF ENTRANCE OF THE SPERMATOZOON IN
RELATION TO THE ORIENTATION OF THE EM-
BRYO IN EGGS WITH SPIRAL CLEAVAGE
T. H. MORGAN AND ALBERT TYLER
(From the Marine Biological Laboratory, Woods Hole, and the William G.
Kcrckhoff Laboratories of the Biological Sciences,
California, Institute of Technology)
If the entrance of the spermatozoon into the egg is instrumental
in determining the planes of cleavage, and the cleavage planes bear a
definite relation to the embryonic axes, it would still remain important
to find out whether the side of the egg on which the sperm enters is
a factor in locating the dorsal (or ventral) side of the embryo. In
some eggs having an equal first cleavage, such as the frog, the ascidian
and the sea-urchin, observations of this kind have been reported, and
a distinct relation has been found between the side of the egg on
which the sperm enters and the future dorso-ventral axis of the em-
bryo. Curiously enough, despite the large number of careful observa-
tions on the cell-lineage of eggs with a spiral type of cleavage, there
is only one set of observations on the relation of the entering point
to the first cleavage plane, and even here we do not know whether
the side on which the sperm enters becomes the dorsal or the ventral
side.
In the course of our work another relation was found that is both
novel and has a bearing on the interpretation of the so-called law of
alternate right- and left-cleavage in spiral types. In Cumingia it was
discovered that two types of second cleavage occur in equal numbers,
one of which in ordinary parlance would be called a right-handed, the
other a left-handed spiral, yet in both cases the third cleavage was
found to be always dexiotropic. As a consequence of this relation it
follows that in one case the first plane of cleavage corresponds to the
median plane of the embryo, and in the other case the second plane of
cleavage corresponds to the median plane, provided the later sequence
of events is the same for both types.
A third relation has not, so far as we know, been carefully studied,
namely, whether in eggs with an unequal first cleavage, the plane of
cleavage passes through the pole or consistently to the side. Without
exception our observations show that the plane passes to the side on
59
60 T. H. MORGAN AND ALBERT TYLER
which the smaller cell comes to lie, but the relations here are not the
same in the three types examined, nor are the succeeding events always
the same. However, these relations will be shown to have a significant
bearing on the location of the median plane of the body.
The Cleavage of Cumingia
The early cleavage of the egg of the bivalve mollusk Cumingia
tell'moides has been described by Morgan (1910) and Browne (1910).
The following observations were made in the summer of 1929 at
Woods Hole, Mass. The eggs and sperm were obtained by the usual
method of isolating individuals in small dishes of sea water. The eggs
were washed and samples removed for fertilization at once or soon after
deposition. A square of vaseline was laid down on a slide and two
fragments of No. 2 cover slips placed on the vaseline for additional
support. A drop of eggs was placed in the square and a small drop
of very dilute sperm-suspension was added. A cover slip was placed
on the preparation and the slide was examined at once under the micro-
scope. The eggs were brought under observation in less than thirty
seconds after insemination. To some of the eggs one or more spermat-
ozoa were already attached ; to others they soon became attached. Only
those cases in which one or a few spermatozoa were attached were fol-
lowed— if the insemination had been too heavy the slide was rejected.
The egg of Cumingia is about 66 micra in diameter, and with the jelly
about 107 micra. The glass supports were about 140 micra in thick-
ness, which with the further help of the vaseline sufficed to prevent
compression of the eggs.
The pole of the egg of Cumingia can readily be identified by a clear
area free from pigment. The outer pole of the first maturation spindle
lies in the center of this area. The identification of the pole is later
checked by the point of extrusion of the polar bodies. The sperm enters
at any point of the periphery of the egg. On attaching itself to the
egg the spermatozoon becomes immotile, its tail extending radially
from the surface. About 30 seconds after attachment the egg rather
suddenly becomes distinctly ovoid in shape, with the more pointed end
at the point of attachment. This change in shape lasts 30 seconds or
less. As the egg rounds out again the sperm enters. This phenom-
enon enables one to identify the particular sperm that will enter, even
before the sperm-head has penetrated. Other sperms in the jelly, ap-
parently even touching the surface of the egg, do not call forth this
striking reaction. The change in shape is something more than the
formation of a fertilization cone, since it involves a change in form
of the whole egg. Unless the entering sperm is exactly on the horizon,
ENTRANCE POINT OF SPERM AND CLEAVAGE
61
the change in form of the egg may not he observed; also there seems
to be some difference in different sets of eggs as to its appearance.
The first polar body appears five or six minutes after fertilization.
In making observations, all the sperms at or near the periphery of
the egg were located on a drawing, and their relative position in three
dimensions noted. Those that did not enter served as markers.
Spermatozoa that are too far above or below the optical section of the
egg cannot always be seen. When the polar body appears, the pole can
be more accurately located in relation to the position of the entrance
point. As a rule only one egg in each preparation was followed. The
observations were made under magnifications of 284 and 440 diameters.
The first cleavage appeared about 50 minutes after fertilization.
The location of the plane was noted in the drawing with respect to
the point of entrance. This was checked as far as possible by the posi-
tion of the markers, since, if any shifting of the egg occurred, their
positions would change. The first division; Fig. 1, a, b, is unequal.
a' b
FIG. 1. The first and second cleavages of Cumingia showing the two pos-
sible types of 4-cell stage. In a the C-cell comes off " counterclockwise " ; in b'
" clockwise."
The smaller blastomere, following the convention for this type of egg,
will be called AB, and the larger blastomere CD. The second cleavage,
Fig. 1, a', b', divides AB equally (A and B), and CD into unequal
parts (C and D) ; the C-blastomere being smaller and approximately
62 T. H. MORGAN AND ALBERT TYLER
the size of A or B. Theoretically the C-cell might form from either
side of CD (Fig. 1, a', b'). It is obvious, then, that there would be
two possible configurations or arrangements of the blastomeres after
the division that are mirror figures of each other (Fig. 1, a', and Fig.
I, b'). As will be shown, it is important at this stage not to identify
these two types as dextral or sinistral cleavages, although this would
be the usual interpretation.
The clockwise sequence ABCD may seem to imply that the second
cleavage has been leiotropic and the third will be dexiotropic, or con-
versely for the counter-clockwise sequence DCS A ; but by utilizing
the usual lettering we do not wish here to commit ourselves to such
an implication. The reasons for this will appear later.
Entrance Point of Spermatozoon in Relation to the First Cleavage
in Cumin gia
Ninety-eight cases were recorded in which the relation of the en-
trance point to the first cleavage plane was definitely ascertained. In
77 cases there was strict coincidence between the plane of the first di-
vision and the entrance point. In 13 the entrance point was less than
45° from the cleavage plane. In 8 cases the divergence was greater
than 45° and less than 90°. Whether the expectation of close coin-
cidence should be 100 per cent and the departures be considered as
due to abnormalities, or as due to errors of observation may be briefly
considered.
Polyspermy might introduce a complication, but it can be detected
either by the presence of extra pronuclei, or by irregularities in the
cleavage. Compression of the egg might be one of the factors de-
termining the position of the cleavage plane. To avoid this, the sup-
ports were made so thick that the space between the slide and the cover
slip was greater than the diameter of the egg plus the jelly. If the
sea water evaporates, the retreating edge of water may cause the egg
to move, and the hypertonicity might cause irregularities in cleavage.
This was avoided to a large extent by the wall of vaseline ; also eggs
were selected that lay in the centre of the drop. Any movement of
the eggs can be detected by their position with respect to neighboring
eggs. The change in shape that the egg undergoes before cleavage is
not a serious source of error, especially if checked by the presence of
" markers " on the egg, but during division the change in shape of the
egg may cause slight changes in position. Therefore, whenever pos-
sible, the egg was constantly watched throughout this period. In some
cases when the cleavage is horizontal the egg may roll over. This is
prevented to some extent by avoiding jarring of the table etc. When
ENTRANCE POINT OF SPERM AND CLEAVAGE
63
one or more of these factors was observed to come into play, the egg
under observation was rejected.
The first cleavage plane does not pass through the pole (as deter-
mined by the position of the attached polar bodies), but slightly to
one side. When considered from the entrance point of the sperm, the
pole of the egg being up, this plane may be said to pass to the right
or to the left of the pole. Whenever the first plane passes to the
right of the pole, the AB-ce\\ comes to lie to the right of the entrance
point (Fig. 2, a) ; whenever it passes to the left of the pole, the AB
FIG. 2. The cleavage planes of Cumingia with respect to the entrance point
of the spermatozoon, a, 2-cell stage with AB to right of sperm-entrance point ;
b and c, the two possible types of second cleavage.
comes to lie to the left (Fig. 3, a). This simple relation, which is
constant in all the eggs examined, has apparently been overlooked by
earlier observers in eggs of this type. The polar bodies adhere to the
surface of the CD blastomere, and are carried into the furrow during
the first division. Of the 77 cases of coincidence between the entrance
point and the first plane, the AB was to the right in 40 cases and to
the left in 37 cases. It appears that the chances are equal that the
smaller cell lies to the right or to the left of the entrance point. The
bearing of these two possibilities on the location of the plane of bilateral
symmetry will be considered presently.
It is obvious that when the small cell (AB) lies to the right of the
entrance point there are two possible types of second cleavage (Fig.
T. H. MORGAN AND ALBERT TYLER
64
2, b, and Fig. 2, c) ; similarly when the small cell (^/£) lies to the
left (Figs. 3, b, and 3, c). As a matter of fact it was found in these
77 cases of coincidence that when the AB was to the right, only one
of the two theoretical types appeared, namely, that shown in Fig. 2, b.
When the AB was to the left, again only one of the two theoretical
types appeared, namely, that shown in Fig. 3, b. Ordinarily the cleav-
FIG. 3. The same as Fig. 2, except that the AB-ce\l lies to the left of the
sperm-entrance point.
age giving the first type (Fig. 2, b) would be called a leiotropic second
cleavage, implying that the third would be dexiotropic. The second
type, Fig. 3, b, would be called a dexiotropic second cleavage, implying
a leiotropic third. However, a study of the third cleavage of Cumingia
has shown that the division is always dexiotropic. This information
was obtained from eggs preserved at the time of the oncoming third
cleavage. The orientation of the spindles with respect to the poles
was determined in 84 eggs, and in every case they showed the cleavage
to be dexiotropic (Fig. 5). The observation shows in the first place
that it would have been erroneous to conclude that because the third
cleavage is dexiotropic, the second must have been leiotropic. It
would have been equally erroneous to have concluded from the two
types of four-cell stages that the direction of the spiral would be dif-
ferent in the two types. By parity of reasoning it would seem unjusti-
fiable to infer that because a given egg shows a leiotropic second cleav-
age, the first cleavage must have been dexiotropic, and thus to designate
the egg as a dexiotropic egg. .
ENTRANCE POINT OF SPERM AND CLEAVAGE
65
Such reasoning might have led one to infer that a dexiotropic third
cleavage in Cumingia means that the second cleavage must have been
leiotropic. A study of preserved eggs in the anaphase of the second
division gave no indication of a spiral arrangement of the spindle.
The spindles in the CD- and AB -cells appear to lie in the same hor-
izontal plane (Fig. 4, a, b, c, d~), instead of being tilted in opposite
directions, as has been described for other eggs at this division (Mead,
Conklin). Of course it is possible that the tilting of the spindles in
the Cuniingia egg is too slight to be visible, but nevertheless it is in-
FIG. 4. Two-cell stages of Cumingia showing the positions of the spindle
for the second cleavage, a and b, polar views ; in a the C-cell will come off
clockwise, in b the C-cell will come off counterclockwise, c and d, antipolar views ;
in c the C-cell will come off counterclockwise, in d, clockwise. The two poles
of the spindles appear to lie at the same level in all cases.
teresting to note that in this egg in which two different types of four-
cell stages occur the spindles do not show a visible tilting. The spindles,
in the AB- and CD-cells, are horizontal as shown in the figures (Fig.
4, a, b, c, d). However, they are not parallel, but, especially in the
CD-cell, the spindle makes an angle with the plane of division.
In order to answer the question, if it should arise, as to whether
both types of cleavage in Cumingia produce normal embryos, a few
eggs of each type were isolated. Normal embryos developed from each.
The normal trochophore swims in a dexiotropic spiral. This also
occurred in the embryos from these two types. Moreover, all the em-
5
66
T. H. MORGAN AND ALBERT TYLER
bryos from a culture swim in the same kind of spiral. In adult Cumin-
gia the two valves of the shell are different in the articulation joint on
the median dorsal side. All shells examined were alike, i.e., not right
or left, but all the same.
Location of the D-Ccll in Relation to the Entrance Point
It has been found that when the first plane passes to the right of
the pole (Fig. 2, a) the next division is always of such a sort that the
D-cell is later away from the point of entrance of the sperm (Fig. 2. M.
Similarly when the first plane passes to the left of the pole (Fig. 3. «)
the next division is always of such a sort that the future Z)-cell is again
away from the point of entrance (Fig. 3, b). The records from living
eggs show that in 32 cases in which the cleavage plane passed to the
right of the pole, the L>-cell lay on the side opposite the entrance point,
giving the arrangement of the blastomeres shown in Fig. 2, b. In 30
FIG. 5. Four-cell stages of Cuniiiigia showing the position of the spindles
for the next division. In all cases the spindles show that the next division will
be dexiotropic.
cases in which the plane passed to the left, the .D-cell also lay on the
side opposite the entrance point, as in Fig. 3, b. No exceptions to
this rule are found.
So far the description has been restricted to those cases where the
first cleavage plane coincided very nearly with the entrance point. In
addition there were a few other cases, as reported above, where the
coincidence was not so close and where there were no reasons to sup-
ENTRANCE POINT OF SPERM AND CLEAVAGE
67
pose that errors of observation were made. There were 13 such cases
recorded in which the cleavage plane was less than 45° from the en-
trance point. If the entrance point is arbitrarily brought to the nearest
point of the actual cleavage plane, then there are 3 cases in which AB
is to the right of the entrance point, and 10 cases in which AB is to the
left. The same relations of Z)-cell to entrance point obtain for both
of these sets of cases as for those in which there was strict coincidence.
There were also 8 cases in which the first cleavage plane was more
than 45° from the entrance point. This divergence is too great to
make a comparison profitable.
Relation of the Entrance Point of the Sperm to the Plane of Bilateral
Symmetry
The evidence reported above has an important bearing on the re-
lation of the point of entrance of the sperm to the plane of bilateral
symmetry of the body. It has been shown that in 78 per cent of
the cases close coincidence was observed between entrance point and
first cleavage plane. In about half of these the first cleavage passed
to the right of the pole (Fig. 2 a), giving the type of 4-cell stage shown
in Fig. 2, b. At the next cleavage, the third, the 1-d micromere forms
dexiotropically (Fig. 5). If from this point onwards the cleavages
FIG. 6. Diagrams indicating the location of the 4-d cells in the two types of
cleavage shown in Fig. 2, b, and in Fig. 3, b.
alternate, left and right, the 4-d cell will come off leiotropically and will
lie next to the second plane of cleavage as shown in Fig. 6, a. It has
been shown (Lillie, 1895) for at least one pelecypod (Unio) that the
4-d blastomere gives rise to the larval mesoblast. and establishes the
plane of bilateral symmetry. This means that the second plane of
cleavage coincides approximately with the median plane of the body.
In the other half of the recorded cases the first cleavage passed to
the left of the pole (Fig. 3. a) giving the type of 4-cell stage sho\v;
68 T. H. MORGAN AND ALBERT TYLER
in Fig. 3, b. The l-d again forms dexiotropically, Fig. 5. It follows
from the same reasoning that the 4-d micromere comes off leiotropically,
and will here lie next to the first plane of cleavage as shown in Fig. 6, b,
and this plane of cleavage will now approximate the median plane of
the body.
It may seem, then, that either the first or the second plane of cleav-
age may become the median plane of the body. This follows only
on the assumption made above, which, although known to be true for
other eggs, has not been entirely shown in this case. It is possible,
for example, that the second somatoblast which determines the median
plane may be formed at different divisions in the two cases. If, for
example, in the type shown in Figs. 3, a, and 3, b, the second soma-
toblast appeared one division earlier or one division later, the median
plane would be the same as in the other case (Fig. 2, a, and 2, b). As
shown by the evidence, when the first cleavage plane passes to the
right of the pole, the plane of bilateral symmetry coincides with the
second cleavage plane, and when it passes to the left, with the first cleav-
age plane. What determines the passage of the first cleavage plane
to the right of the pole in some cases and to the left in others is un-
known. The fact that about 50 per cent of each type occurs suggests
that it is merely a matter of chance. If we assume that the unfertilized
egg has its materials radially arranged around the polar axis, and that
the entering sperm determines through movements of the contents of
the egg (or otherwise) that materials correlated with the determination
of the D-cell come to lie opposite the entrance point of the sperm ; and
furthermore, that the cleavage plane does not pass through this ma-
terial, then a possible interpretation suggests itself. It is obviously
not necessary to make this assumption in quite the same crude form
as suggested above in order to express these relations, for, at the time
of the first division, all of the egg appears to be involved in the process.
The risk of making such a generalization will be apparent when another
egg, Chatoptcrus, is examined.
The Cleavage of C licet opterus
The eggs were washed in sea water, and allowed to stand about
20 to 30 minutes during which time the first polar spindle forms. A
drop of eggs was put onto a slide prepared in the same way as for
Cumingia. The egg measures 106 micra in diameter, without the jelly,
and 111 micra with the jelly. The same thickness of cover slip sup-
port etc. was used as for Ciuningia. A very small drop of very dilute
sperm-suspension was added to the eggs which were examined immedi-
ately. In most cases the spermatozoa were already attached as though
ENTRANCE POINT OF SPERM AND CLEAVAGE 69
the combination had been made almost instantaneously. The sper-
matozoon enters 15 to 30 seconds after insemination and may be missed
unless the preparation is examined very quickly. The pole of the egg
can be identified by the clear area in which the spindle for the first
maturation division lies. The sperm enters at any point, and a slight
fertilization cone appears at the point of entrance. The extra sperm
which do not enter remain attached, and serve as markers. The exact
position of the pole is given by the location of the polar bodies.
The cleavage of the egg of Chatopterus has been described by Mead,
Wilson, and Lillie, and the relation of the median plane of the body
to the first cleavage plane determined, but so far no one has examined
the relation of the entrance point of the sperm to the first cleavage.
The third cleavage of the egg is dexiotropic, and the fourth leiotropic,
so that 2-d (the first somatoblast) comes off near the second cleavage
plane, and 4-d (the second somatoblast) is similarly placed. This de-
termines that the median plane of the body lies near the second cleavage
plane.
The Relation of the Entrance Point of the Sperm to the First Cleavage
Plane
As in Cniningia the location of the sperm that had entered was
recorded on the drawing, and the individual eggs watched until the
cleavage furrow appeared. In 48 eggs there was a fairly strict co-
incidence; in 35 eggs the entrance point was less than 45° from the
plane of the first division, and both to the right and left of the plane.
In 33 eggs it was more than 45° and less than 90° to the right and
left. Thus in only 41 per cent of the cases was there a close agree-
ment between entrance point and cleavage plane ; but if the entrance
point is not in some way correlated with the direction of the first
cleavage plane, even this percentage of coincidence would not be ex-
pected. Taking first the cases where coincidence occurs, it was found
that in 23 cases the first plane passed to the right of the pole, which
means that the AB-cel\ lay to the right of the entrance point as in
Fig. 7, a. In 25 cases it passed to the left of the pole, thus placing
the AB-cell to the left of the entrance point as in Fig. 7, b. In
both cases, however, the second cleavage gave the same arrangement
of cells, namely, that shown in Figs. 7, a', b'. According to the usual
convention these four-cell stages would be obtained from leiotropic
second cleavages (which is actually true for the Ch&topterus egg), but
in one type, Fig. 7, a', the Z7-cell would lie away from the entrance
point of the sperm, and in the other type near the entrance point (Fig.
7,6')-
70
T. H. MORGAN AND ALBERT TYLER
The third cleavage in all cases observed, both in the living and
in the preserved eggs, was dexiotropic. If the subsequent cleavages
alternate left and right, the 4-d cell in both types will come to lie near
to the second plane of cleavage (Fig. 7, a', b'). This means that the
second plane coincides with the median plane of the body, although
in one type the entrance point of the sperm would be to the right of
the median plane, and in the other it would be to the left.
4d
FIG. 7. Diagrams indicating the position of the first cleavage with respect
to the polar body, and the entrance point of the spermatozoon ; also the location
of the 4-d cell. In a' the position of the 4-rf resulting from the type of first
division in a is shown, in b' that in b.
The Cleavage of Nereis
The egg of Nereis is particularly well suited for a study of relation
of entrance point to cleavage, not only because the slow entrance of
the sperm makes for accuracy of observation, but also because after
the sperm-head has entered, a portion is left sticking to the fertilization
membrane, and, if exactly on the horizon, may be still seen at the time
when the cleavage begins. The technique was the same as for the
Cumingia eggs, but since the egg is larger, thicker supports made from
ENTRANCE POINT OF SPERM AND CLEAVAGE 71
glass tubing were used. Owing to the great thickness of the jelly a
relatively large space between the cover and slide is essential. The
location of the first cleavage with respect to the entrance point of the
sperm has been studied by Just. The observations reported here were
made to determine not only the constancy of the relation, but also to
determine whether the AB-ce\\ always forms to one side of the en-
trance point — a relation not previously reported. It was found that
whereas the AB lay to the right in a very large number of cases, there
were a few cases where it lay to the left. Nevertheless, at the four-
cell stage only one arrangement of blastomeres was found (even in
those with AB to the left), namely, that shown in Figs. 7, a', or 7, b'.
The first plane of cleavage coincided with the entrance point in 33
cases. In 17 cases it was less than 45°. In 14 cases it was more than
45° and less than 90°. It is apparent from these observations that the
agreement (51 per cent) is far from perfect.
Of the 33 cases of close coincidence, the first plane passed to the
right of the pole in 28 cases, and in five cases to the left. Of the 17
cases less than 45° away, it passed to the right in 11 cases, and to
the left in 6 cases. This conclusion was reached by arbitrarily shifting
the entrance point to the nearest surface point in the cleavage plane.
Here again there were more cases where AB lay to the right than to
the left.
The configuration of the cells after the second cleavage is always
of the same type (Fig. 7, a', or /, //), whether the first cleavage passes
to the left or to the right of the pole. In the 28 cases in which the
first plane passed to the right, the Z)-cell formed away from the en-
trance point and in the five cases in which it passed to the left the
.D-cell formed near the entrance point.
The third cleavage of Nereis, as is well known, is always dexiotropic.
The succeeding divisions of the egg alternate left and right. Hence,
in both sets of cases the 4-d cell conies to lie near the second cleavage
plane, which Wilson has shown to be near the median plane of the body.
In 1912 Just reported results of experiments on Nereis eggs, in
which the entrance point was marked by the path of India ink in the
ielly. He found coincidence varying from 50 per cent in one set to
60, to 80, to 95 per cent in other sets. He placed emphasis on those
sets in which the greatest amount of agreement occurred. The excep-
tions he supposed were due to errors of technique, since by a change
in technique he found in one set of 60 eggs, 100 per cent coincidence.
Our own results gave only 51 per cent exact coincidence. That the
vaseline we used was not injurious was shown by removing the eggs
from the slide after the 4-cell stage and finding that they produced nor-
72 T. H. MORGAN AND ALBERT TYLER
mal trochophores. We tried the India ink method in the hope of ob-
taining a large number of observations from a single preparation, but
abandoned it because of the uncertainty in many cases of following
the marker exactly to the surface of the membrane, and unless this can
be done with absolute certainty there remains too great a chance of
making a wrong inference, especially when the coincidence is not quite
exact. In our opinion continuous observations on single eggs, while
much more tedious, are safer.
DISCUSSION
The main interest in these observations concerns the two types of
the four-cell stages in Cumingia. As pointed out, one type arises in
eggs in which the ^5-cell forms to the right of the entrance point,
and the other where it forms to the left. Since these two types give
rise to two different planes of bilateral symmetry, on the assumption
made, the problem of the determination of these planes seems to resolve
itself into the problem of what determines that the cleavage plane lies
to one or to the other side of the pole. Since these two types appear
with equal frequency in Cumingia, it may seem that it is only a matter
of " chance " to which side of the pole it passes. In Nereis there is
only one type of four-cell stage and the AB-cc\] in the majority of
cases (85 per cent) forms to the right of the entrance point. To this
extent it conforms to the rule found for Cumingia. Since the AB-ce.ll
of Nereis lies to the right of the entrance point in 85 per cent of cases,
its location does not here seem to be a matter of chance. In Chatopterus
there is again only one type of four-cell stage, but here the AB-ce\\
lies equally often to the right or to the left of the entrance point. Since
there are here three different types of behavior leading to the forma-
tion of normal embryos, it may be inadvisable at present to try to re-
duce them all to one mechanism. The spiral type of cleavage common
to all these eggs might incline one to attempt to find an explanation
of the fact that the first cleavage plane passes to the right (with respect
to entrance point) or to the left consistently in the different types. In
Cumingia the egg regulates according to whether the AB-cd\ lies to
the right or to the left of the entrance point. In Chatopterus, although
the ^!5-cell again may lie either to the right or left of the pole there
is no regulation, because the second cleavage plane coincides with the
median plane. In Nereis no regulation is necessary, in this sense, in
the majority of cases because these all conform to the same rule, but
in the few exceptional cases the result is the same as in Chcctopterus.
As already stated, an examination of the second cleavage spindle
of Cumingia has not shown a spiral arrangement of the spindles. It
ENTRANCE POINT OF SPERM AND CLEAVAGE 73
is equally obvious, however, that, just prior to the division, the spindle
in the CD-cell lies well to one side, indicating the future position of the
C-cell. After the division, the A and C blastomeres approach each
other, more nearly in the polar than in the antipolar hemisphere in both
types, while the B and D cells meet in a straight line at or near the
antipole. If this be taken as evidence for a spiral second cleavage, then
there are both leiotropic and dexiotropic second cleavages in Cnuiingia.
Since the third cleavage is always dexiotropic this would contradict
the " law " of alternating spiral cleavages.
It has been pointed out in the text that the two types of cleavage
of Ciiniingia give rise to two different planes of bilateral symmetry.
In one type the median plane coincides with this first cleavage plane,
and in the other type with the second. This conclusion, however, is
based on the assumptions that the law of alternating cleavage holds
from the third cleavage on, and that the 4-d blastomere gives rise to
the germ bands.
BIBLIOGRAPHY
BROWNE, E. N., 1910. Effects of Pressure on Cumingia Eggs. Arch. f. Ent-
wickelungsincclianick d. Organ., 29.
CONKLIN, E. G., 1902. Karyokinesis and Cytokinesis in the Maturation, Fertiliza-
tion and Cleavage of Crepidula and other Gasteropoda. Jour. Acad.
Nat. Sci. of Phila., 12.
JUST, E. E., 1912. The Relation of the First Cleavage Plane to the Entrance
Point of the Sperm. Biol. Bull, 22.
LILLIE, F. R., 1895. The Embryology of the Unionidae. A Study in Cell Lineage.
Jour. Morph., 10.
LILLIE, F. R., 1906. Observations and Experiments concerning the Elementary
Phenomena of Embryonic Development in Chastopterus. Jour. Ex per.
ZooL, 3. ,
MEAD, A. D., 1897. The Early Development of Marine Annelids. Jo-ur. Morph.,
13.
MORGAN, T. H., 1910. Cytological Studies of Centrifuged Eggs. Jour E.vper.
ZooL, 9.
WILSON, E. B., 1883. Observations on the Early Developmental Stages of Some
Polychaetous Annelids. Stud. Biol. Lab., Johns Hopkins Unir., 2.
WILSON, E. B., 1892. The Cell Lineage of Nereis. Jour. Morph., 6.
THE EFFECT OF LOW OXYGEN TENSION ON THE PULSA-
TIONS OF THE ISOLATED HOLOTHURIAN CLOACA
BRENTON R. LUTZ
(From the Bermuda Biological Station for Research,'1 the Mount Desert Island
Biological Laboratory, and the Physiological Laborator\
of Boston University School of Medicine)
The sequence of events in the respiration of Sticliopns niocbii
Semper has been adequately set forth by Crozier (1916). In laboratory
aquaria the rhythmic activity of the cloaca is distinctly periodic. A
series of several pulsations is followed by a pause during which water
is expelled from the respiratory tree. Then another series of inspira-
tions begins. The number of inspirations in a series was found by
Crozier (1916) to range from five to eleven, the greatest number being
found in the largest animal. Pearse (1908) pointed out that, if the
respiratory pulsations of Thyonc briareus are prevented for some time
by repeated mechanical stimulations, the contractions which ensue when
stimulation ceases are greatly augmented in amplitude.
Oxygen deficiency has often been associated with periodicity and
augmentation of response in various tissues. Douglas and Haldane
(1909) have described periodic breathing in man under low oxygen
tensions, and Douglas (1910) found the same type of breathing at
high altitudes. Magnus (1904) and Frey (1923) reported that a stop-
page of the oxygen supply to beating smooth muscle results immediately
in an increase in amplitude. The present paper deals with the phe-
nomena which have been observed on decreasing the oxygen available
to a rhythmically beating isolated strip of circular muscle from the cloaca
of Stichopus niocbii Semper. This holothurian is found in great num-
bers in the shore waters at the Bermuda Biological Station. During
the summer of 1927 the author repeated some of the experiments on
a ring preparation from the cloaca of Cucumaria frondosa, very abun-
dant at the Mount Desert Island Biological Laboratory, Maine.
METHOD
Crozier (1916) has shown that the cloaca in situ in the isolated
posterior end of Stichopus will maintain its pulsations for many hours.
No reference to the use of an isolated strip of this organ could be
1 Contribution number 158.
74
HOLOTHURIAN MUSCLE AND OXYGEN LACK 75
found in the literature. The present work was carried out with an
opened ring of the circular muscle of the cloaca. A cloacal-end prep-
aration was first made similar to that described by Crozier (1916). The
cloaca was then excised by cutting the radial muscles with a scalpel
and freeing the organ from the anal rim by a transverse cut. From
the muscular tube thus obtained a strip was made, one to two centi-
meters broad, and from four to six centimeters long. This strip was
suspended vertically in a vessel of sea water by means of an L-shaped
glass rod and a counterbalanced aluminum lever. A 250 cc. graduated
cylinder cut off to hold about 125 cc. was found convenient as a vessel
to hold measured amounts of sea water, or through which sea water
could be made to flow continuously. The temperature of the water was
recorded and found to vary little during an experiment, or from day
to day. Therefore no special precautions for maintaining constant
temperature were necessary.
RESULTS
Records were taken from strips of Siiclwpits cloaca beating under
the following conditions: (1) in a continuous flow of sea water, (2)
in a limited amount of sea water, (3) in boiled sea water with added
carbon dioxide, (4) in boiled sea water of various degrees of aeration,
and (5) in normal sea water with potassium cyanide added.
Continuous Floiv of Sea }\\itcr. — When sea water was made to flow
continuously through the vessel at the rate of about 100 cc. a minute,
the strip beating therein gave a tracing which was exceedingly uniform
over a period of several hours, as may be seen in Fig. 1. Both am-
plitude and tone increased during the first hour. This condition was
maintained for an hour or more. Then the tone began to fall very
gradually while the amplitude remained about the same. After five
to seven hours from the beginning, the amplitude began to decrease
slightly. The rhythm was exceedingly regular and no indications of
periodicity appeared. The rate of beat decreased slowly from the start,
in one case almost 50 per cent after seven hours and forty-one minutes ;
but the preparation was still vigorous and regular.
Limited Amount of Sea ITater. — When a strip was allowed to beat
in a limited amount of sea water, that is in 100 cc. without change, the
amplitude began to increase in about three hours and distinct periodicity
developed as seen in Fig. 2. The increase in amplitude continued for
an hour or more, becoming 230 per cent in one case. The tone was
maintained until the increase in amplitude occurred, when it gradually
fell ; but the increase in amplitude was not entirely due to a decrease
in tone since the contractions of the strip raised the lever a greater
76
BRENTON R. LUTZ
N 3
HOLOTHURIAN MUSCLE AND OXYGEN LACK
77
distance above the base line than in the beginning. Finally both am-
plitude and tone fell markedly. The rate of beat decreased constantly
from five or six at the beginning to two or three per minute during
the periods of beating. The length of the periods of inhibition of beat
gradually increased to three or four minutes.
Boiled Sea Water. — Sea water which had been boiled in a narrow-
necked flask and cooled to laboratory sea water temperature (28° C.
or 29° C. ) was used. When the muscle strip was immersed in 100 cc.
of this water, the first two or three beats usually increased in amplitude,
but both tone and amplitude almost immediately fell and the strip ceased
beating in from three to five minutes as shown in Fig. 3, A. If the
FIG. 3. A. Cloaca! strip in 100 cc. of boiled sea water. pH 8.4. B. Cloacal
strip in 100 cc. of boiled sea water treated with c.arbon dioxide, pH 5.8. Aeration
at X. C. Cloacal strip in 100 cc. of boiled sea water, pH 8.3. Aeration at A'.
pH 8.2 immediately after aeration. pH 8.2 after 102 minutes.
water was aerated within three minutes by sucking it into a hypodermic
syringe and squirting it back forcibly, a partial recovery occurred, which
showed periodicity at first but later an uninterrupted rhythm (see Fig.
3, C). Several attempts to bring about recovery after waiting a longer
period failed. The pH of the boiled sea water (indicator method)
was sometimes as high as 8.8 as compared with 8.1 to 8.3, the pH
for unboiled sea water in this region.
78 BRENTON R. LUTZ
Boiled Sea Water with Added Carbon Dioxide. — Inasmuch as boil-
ing removed the carbon dioxide as well as the oxygen, the former was
replaced by means of a carbon dioxide generator. This resulted in
boiled sea water ranging from pH 5.8 to 7.7. At the latter value the
strip ceased to beat in three and one-half minutes and at the former
value cessation occurred in three minutes. Aeration of the water after
a three minute period of cessation failed to induce recovery (see Fig.
3, B). It seems therefore that neither the lack of carbon dioxide
in the boiled sea water nor the increased alkalinity was the cause of the
cessation of the pulsations.
A moderate excess of carbon dioxide was produced by treating
125 cc. of unboiled sea water with carbon dioxide until the pH was 7.0.
This procedure was brief and probably did not remove much oxygen.
In experiment 75 (Fig. 4) the amplitude began to decrease slowly after
an hour, the rate decreasing gradually from the beginning. Neither
augmentation of amplitude nor periodicity had appeared when the ex-
periment was stopped after two hours and fifty-six minutes. When,
however, an excess of carbon dioxide was produced by adding a few
drops of N/10 HC1 to a preparation beating in 100 cc. of unboiled
sea water, there was an immediate rise in tone and increase in am-
plitude which soon gave way to a fall of tone and amplitude and finally
to cessation of beat. It is therefore not probable that an accumulation
of carbon dioxide in the immersion fluid as a result of tissue activity
in a limited volume of water is the cause of the appearance of perio-
dicity although it might be called upon to account for the increase in,
amplitude.
Boiled Sea Water of Various Degrees of Aeration. — When a de-
creased oxygen content of the sea water was produced, either by mix-
ing boiled sea water with unboiled sea water or by partial aeration
of boiled sea water, the augmentation and periodicity appeared much
sooner than when a limited volume of unaltered sea water was used,
the onset varying from a few minutes to two hours, according to the
degree of oxygen lack. In one case the boiled sea water had been
stored for several hours in a narrow-necked flask with only a few
square centimeters of water surface exposed to the air. A strip beating
in 100 cc. of this water became periodic at once and each successive
period showed an increase in amplitude which finally amounted to
about 200 per cent. The tone and the rate of beat, however, fell rapidly.
In another experiment in which 100 cc. of boiled sea water had
been partially aerated, wave-like variations in amplitude appeared 13
minutes after immersion, and gradually developed into periodicity 53
minutes after the start. The amplitude increased from 10 mm. to 24
HOLOTHURIAN MUSCLE AND OXYGEN LACK
79
80 BRENTON R. LUTZ
mm. and was still high when the experiment was stopped at the end
of 93 minutes. The tone fell rapidly during the first five minutes and
then more slowly during the next ten minutes after which it was un-
changed. The rate decreased about fifty per cent during the first half
hour and then remained constant.
In experiment 62 (Fig. 5) the boiled sea water (100 cc.) was par-
tially aerated. Before boiling the pH was 8.3, but after boiling and
partial aeration it was 8.4. The amplitude of a strip beating in this
water decreased at first with a tendency to form waves. Then for a
period of 80 minutes the amplitude remained constant, but at the end
of this period the amplitude began to increase, becoming 65 per cent
greater than that during the previous period of uniform amplitude.
Periodicity appeared in about two hours from the beginning of the
experiment, the number of beats in each period ranging from ten to
sixteen, while the period of interruption varied from one minute and
a quarter to three minutes. The pH was still 8.4 about forty-five
minutes after periodicity and augmentation were well developed. Ap-
parently these phenomena were not due to increased acidity of the sur-
rounding medium, nor was the cessation of beat in the cases of extreme
oxygen lack due to an increase in the concentration of the salts resulting
from boiling.
When 75 cc. of boiled sea water were mixed with 25 cc. of unboiled
water, the pH of the mixture was 8.8. The first few contractions
increased in amplitude about 20 per cent, lasting for about three minutes.
Then a fall occurred, and the amplitude remained uniform in height
until waves in amplitude appeared in one hour indicating the onset of
periodicity, which became well marked about half an hour later. At
this time the pH was still 8.8.
Effect of Potassium Cyanide. — When ten drops (about 0.5 cc.) of
.M/10 potassium cyanide were added to 100 cc. of sea water in which
a strip had been beating for a few minutes, the results were similar
to those obtained with partial aeration. An increase in amplitude oc-
curred within two minutes which varied in different cases from 18 to
400 per cent. The tone increased at about the same time. Periodicity
occurred within fifteen minutes. In one case it began in three minutes,
and the rate of beat was increased about one beat per minute for a
brief period after the addition of potassium cyanide. Finally the
tone and amplitude fell and the strip ceased to beat (see Fig. 6).
An examination of the results presented above suggests that lack
of oxygen is responsible for the appearance of the two chief phenomena
noted. Since augmentation and periodicity did not occur with a con-
tinuous flow of water but did occur in three hours when the amount
HOLOTHURIAN MUSCLE AND OXYGEN LACK
81
of water was limited to 100 cc., one might expect that one or more
of several factors were responsible, such as, an increase in carbon di-
oxide, an increase in unoxidized acids, a depletion of essential ions,
or a depletion of oxygen. However, when the carbon dioxide content
of sea water was increased at the beginning, the phenomena did not
FIG. 6. Experiment 82. Cloacal strip in 100 cc. of sea water. Time of im-
mersion 4:28. At A", 4:35, 20 drops of A//10 potassium cyanide added.
appear, although a temporary increase in amplitude and tone could
be produced upon addition of hydrochloric acid. Moreover, in the
experiments in which the phenomena did appear, the pH of the sea
water was either unchanged or decreased very slightly. Since both
augmentation and periodicity were made to appear much sooner when
the water was partly depleted of oxygen at the beginning, or when
potassium cyanide was added, the inference is that oxygen lack was
either directly or indirectly responsible.
When a ring preparation made from the cloaca of Cucninarid
frondosa was allowed to beat in a limited volume of sea water, namely,
25 or 30 cc., periodicity appeared in 50 minutes on the average in eight
out of ten preparations. Two showed no periods. Augmentation
of amplitude occurred in five 'cases. When boiled sea water was used
the periodicity appeared in 25 minutes on the average in 14 out of 15
preparations. One showed no periods. Augmentation of amplitude
occurred in 13 preparations. These results, especially when considered
in the light of the results on Stichopus, indicate that lack of oxygen
is a factor tending toward an early development of periodicity and
augmentation.
DISCUSSION
Periodicity is a part of the normal respiratory sequence of a holo-
thurian, the rhythmical contractions of the cloaca being inhibited while
the body muscles squeeze out the sea water from the respiratory tree
6
BRENTON R. LUTZ
through the relaxed anal valve. Crozier (1916), however, found no
evidence of periodicity in the cloacal-end preparation of Sticlwpus and
came to the conclusion that the stimulus for spouting has its origin
outside the cloaca. It should be noted that he used larger volumes
of water than were used in the work reported in this paper. Appar-
ently no oxygen deficiency existed in his preparations, in which the
cloacal pumping probably produced a sufficient movement of water to
keep it aerated beyond the needs of the preparation. Since the isolated
cloacal strip will exhibit regular periods of inhibition, the inference is
that a part of the normal mechanism for spouting lies within the cloacal
muscle. Since periodicity is lacking with sufficient aeration and appears
quickly under conditions of oxygen deficiency, one is inclined to be-
lieve that low oxygen tension is a factor in determining the normal
respiratory sequence in the holothurian.
Periodicity is commonly observed in the respiratory activity of
vertebrates, as for example in the breathing of hibernating animals,
in Cheynes- Stokes respiration, and in respiration at high altitudes.
The causes of this phenomenon are usually associated with the chemical
conditions in the respiratory center. Most authors have offered ex-
planations which concern directly or indirectly the hydrogen ion con-
centration of the blood or fluid surrounding the cells. Gesell (1925),
however, has called attention to the hydrogen ion concentration within
the cells of the former, pointing out that when oxygen is present carbon
dioxide is formed, but if oxygen is lacking lactic acid results. In
either case the activity of the center increases as the acidity rises. As-
suming a critical level, one needs further to call upon a mechanism
for altering either the level or the acidity to account for periodic in-
hibition.
The augmentation of amplitude observed with a decrease in the
available oxygen is in accord with the work of Magnus (1904) and
of Frey (1923), who worked on vertebrate smooth muscle. Gross
and Clark (1923), in an investigation on the influence of the oxygen
supply on the response of the isolated intestine to drugs, stated that
cutting off the oxygen resulted in a decrease in amplitude and tone.
They did not comment on the immediate brief increase in amplitude
and tone shown in their published graphs. The literature offers many
additional observations which indicate that a certain degree of oxygen
lack results in increased activity of tissue. Kaya and Starling (1,909)
found that lowering the oxygen tension resulted in excitation in the
whole nervous system. Sherrington (1910) found that a certain degree
of asphyxia favored the elicitation of the scratch reflex, and sug-
gested that the hyperexcitability of the reflex was due to oxygen
HOLOTHURIAN MUSCLE AND OXYGEN LACK
lack. Mathison (1911) showed by the use of hydrogen, nitrogen,
and carbon monoxide that the initial effect of oxygen lack on the medul-
lary centers is clearly stimulating. Gasser and Lovenhart (1914) found
by the use of carbon monoxide and sodium cyanide that decreased oxi-
dation stimulated the medullary centers at first but later depressed them.
Kellaway (1919) demonstrated that lack of oxygen may lead to stim-
ulation of the adrenal glands, and Lutz and Schneider (1919) have
observed a dilatation of the pupil in men during a period of breathing
nitrogen. They also presented evidence to show that the cardiac and
the respiratory medullary centers in man respond very quickly to
changes in the partial pressure of oxygen. A decrease in oxygen ten-
sion increased the activity of these centers, while an increase in oxygen
tension decreased their activity. Glazer (1929) found that intravenous
injection of sodium cyanide in a dog increases the reflex response of
the anterior tibial muscle, and Winkler (1929) obtained a similar effect
with low alveolar oxygen tension.
In the muscle-and-nerve-net preparation reported in the present
paper, it appears that the carbon dioxide content and the acidity of
the surrounding fluid are not primary factors in controlling its activity.
This conclusion is supported by the work of Hogben (1925) who found
that, on adding acid to the perfused heart of Mala and of Homants,
the pH could be lowered from 7.0 to 5.6 without producing a change
in the mechanical phenomena. Reduction beyond this point produced
an immediate effect on the character of the rhythm. Nor could any
alteration be noticed in the beating of the smooth muscle of Helix and
of Aplysla on changing the pH from 7.0 to 6.0. In fact it is possible
that the pH outside of the cell may vary markedly without greatly al-
tering that inside of the cell. The oxygen tension appears to have some
influence on tissue acidity. Frey (1923) presented evidence which
shows that without oxygen the tissue rather than the surrounding fluid
first changes its hydrogen ion concentration, and if this approaches
the optimal value, an increased ability to respond ensues. The anaerobic
production of acid in cellular activity and the role of oxygen in the re-
covery process suggest that oxygen lack is acting indirectly when cellular
activity is first increased and is subsequently depressed.
SUMMARY
1. An isolated muscle strip from the cloaca of Stlchopns mocbll
Semper and a ring of muscle from the cloaca of Cncuinariu- frondosa
were used in sea water as rhythmically beating preparations.
2. In a continuous flow of sea water the contractions (Stichopus)
were nearly uniform in rate and amplitude over a period of several
hours, but a gradual decrease in both finally occurred.
84 BRENTON R. LUTZ
3. In a limited volume of sea water (100 cc.) the amplitude (Stich-
opus} began to increase after three hours and a distinct periodicity of
the regular rhythm developed. In the case of the cloacal ring of
Cucumaria beating in 25 or 30 cc. of sea water, periodicity appeared
in 50 minutes on the average.
4. In boiled sea water the strip (Stichopus) ceased beating in from
three to five minutes, but partial recovery took place if the water was
aerated within three minutes. If the carbon dioxide was replaced in
the boiled sea water, cessation of beat occurred as before. In 25 or
30 cc. of boiled sea water the ring of cloacal muscle from Cncumaria
developed periodicity in 25 minutes on the average.
5. A moderate excess of carbon dioxide in sea water (pH 7.0) did
not bring on augmentation nor produce periodicity.
6. In boiled sea water of various degrees of aeration the augmenta-
tion of amplitude and the periodicity appeared sooner than in unboiled
sea water. When little oxygen was present both phenomena appeared
almost immediately, while the pH of the surrounding fluid was un-
changed.
7. When potassium cyanide was added to the sea water an increase
in amplitude and tone occurred and periodicity appeared.
8. Evidence from the literature is cited supporting the view that
decreased oxygen tension results at first in increased activity of mus-
cular and nervous tissues. This view is further supported by the evi-
dence presented in this paper.2
BIBLIOGRAPHY
CROZIER, W. J., 1916. Jour. Ex per. Zool, 20: 297.
DOUGLAS, C. G., 1910. Jour. Physiol, 40: 454.
DOUGLAS, C. G., AND HALDANE, J. S., 1909. Jour. Physiol., 38: 401.
FREY, W., 1923. Zeitschr. f. gcs. E.vpcr. Med., 31: 64.
GASSER, H. S., AND LOEVENHART, A. S., 1914. Jour. Pharm. E.rpcr. Thcrap.,
5: 239.
GESELL, R., 1925. Physiol. Rev., 5: 551.
GLAZER, W., 1929. Am. Jour. Physiol., 88: 562.
GROSS, L., AND CLARK, A. J., 1923. Jour. Physiol., 57: 457.
HOGBEN, L. T., 1925. Quart. Jour. E.rper. Physiol., 15: 263.
KAYA, R., AND STARLING, E. H., 1909. Jour. Physiol., 39: 346.
KELLAWAY, C. H., 1919. Proc. Physiol. Soc., Jow. Physiol, 52, Ixiii.
LUTZ, B. R., AND SCHNEIDER, E. C., 1919. Am. Jour. Physiol., 50: 327.
MAGNUS, R., 1904. Pfliiger's Arch., 102: 123.
MATHISON, G. C, 1911. Jour. Physiol, 42: 283.
PEARSE, A. S., 1908. Biol. Bull, ~15: 259.
SHERRINGTON, C. S., 1910. Quart. Jour. E.rper. Physiol, 3: 213.
WINKLER, A. W., 1929. Am. Jour. Physiol. 89: 243.
2 The writer wishes to express his thanks to Dr. Edward L. Mark, who
generously accorded the privileges of the Bermuda Biological Station, and to
Dr. H. V. Neal for the many courtesies extended at the Mount Desert Biological
Laboratory.
PHENOTYPICAL VARIATION IN BODY AND CELL SIZE
OF DROSOPHILA MELANOGASTER
W. W. ALPATOV
(From the Institute for Biological Research, Johns Hopkins University)
I.
The purpose of this paper is to contribute to the solution of the
question of the relationship of the cell size and body size, using well-
known and standard material. The literature devoted to this question
is very extensive, but most of the work done cannot be considered to
fulfil the requirements of exact experimental investigation, in regard
either to the control of conditions, or the homogeneity of the material,
or the precision and accuracy of the treatment. Comparatively modern
compilations of the data available have been made by Levi (1906) and
Martini (1924).
Concerning the more limited problem of the correlation of body size
and cell size in Diptera there have been two recently published papers.
Loewenthal (1923) attacks a problem which corresponds to one part
of the present investigation, namely the influence of underfeeding on
the body and cell size of the blow-fly. The first criticism which may
be made of Loewenthal's work is that he does not give any indication
of the ages of the normal and underfed maggots. It therefore is not
clear whether the observed smaller size of the hypodermis cells is due
to differences in the age of larvae or in the feeding. At the same time
Loewenthal does not find any difference in the cell size of the gonad
rudiments, in spite of their difference in size. The following conclu-
sion is reached (p. 91) :" Danach ist die Korpergrosse der ausgebilcleten
Imagostadiums unabhangig von der Zellgrosse und allein bedingt von
der mehr oder minder grossen Zellanzahl." Further a totally incorrect
statement is made concerning the absence of cell divisions during the
larval life (p. 92) : " Mit Abschluss der Embryonalentwicklung stellen
die larvalen Zellen ihre Vermehrungstatigkeit ein, das ganze Wachstum
der Larve von wenigen mm Lange nach dem Schliipfen aus dem Ei
bis zur Lange von 2 cm einer verpuppungsreifen Ruhelarve beruht allein
— wenn man von den wahrend der Larvalperiode fiir^lie Gesamtgrosse
nicht ins Gewicht fallenden Imaginalanlagen*absieht — auf dem Gros-
senwachstum der Zellen." Przibram's and Megusar's (1912) investi-
gations showed that this is not the case in the postembryonal develop-
85
86 W. W. ALPATOV
ment of Sphodromantis (Orthoptera, Mantidae) and I (1929) have
shown also that the metamorphosis of Drosophila is connected with six
simultaneous divisions of the cells of the whole body.
The same subject of the relationship of the size of an organ and
the size of the cell has been touched upon by Bridges (1921, 1925).
In both of his papers differences in the cell structure, namely, nuclear
structure, are shown to be connected with the size of the whole body
and its organs. It was discovered that these intersex-producing females
(triploid) could be identified by their somatic characters, namely, large
coarse bristles and large roughish eyes (1921, p. 253). In the second
paper it is the size of the ommatidia which is shown to be different in
flies having different chromosomal complexes. ' The cells of triploid
individuals are readily seen to be larger than the cells of diploids, and
correspondingly their facets are larger" (Bridges, 1925, p. 709).
I became interested in the problem of body size and cell size years
ago while working on the oceanographic expedition of the Floating
.Marine Scientific Institute to the Russian arctic seas. The first ex-
pedition in 1921 gave ver)^ impressive material on the geographical
variation in the dimensions of the body of different marine animals.
It could be particularly easily shown on such a group of animals as
Isopoda, which have a postembryonal development ending with a definite
imaginal stage analogous to that of insects. Extensive biometrical data
on variation of Isopoda, taken from localities with different tempera-
tures, showed perfectly that the colder regions (for instance, the Kara
Sea) are populated by races which have a larger body size than regions
with warmer water temperature (Barents Sea). On the second expe-
dition I strove to collect some material on the histology of local races
of some of the species of Isopoda. But the severe conditions of naviga-
tion during this and following summers did not allow the accomplish-
ment of this intention. During the winter of 1927-28, working at
this Institute, I succeeded in working out a more or less accurate method
of producing Drosophila imagoes of different sizes, using two factors,
temperature and underfeeding. The method of counting the number
of hairs on the wings of Drosophila as a method of estimating the
number of cells on a certain surface of the wing was discovered by a
friend, Dr. Th. Dobzhansky (1929), who was kind enough to explain
it to me. I have the pleasure to express also my deepest gratitude to
Dr. Raymond Pearl for criticism and valuable suggestions.
II.
Two factors have been used in producing flies of an abnormal size.
It was shown in an earlier paper that the first of them was the low tern-
DROSOPHILA MELANOGASTER 87
perature, which decreases the rate of development and produces flies of
a larger size (see Alpatov and Pearl, 1929). The method of collecting
new-born larvae has already been described (Alpatov, 1929) . Flies
belonging to Wild Line 107 have been taken for parents of our ex-
perimental animals, the collected larvae being 0-2 hours old at the
moment of putting them on food. The bottles had been planted with
yeast 2 hours before the putting on of larvae, and watered with a few
drops of distilled water. Electric and low temperature Hearson incu-
bators were used for keeping the bottles with flies. Five bottles with
50 larvae each were kept at 18° C, five others at 28° C. The develop-
ment from the moment of the populating of the bottle till the moment
of the pupation was more than twice as long in the cold series as in
the warm. It is unnecessary to discuss here at length the question of
temperature and development rate, this having been done in another
paper (Alpatov and Pearl, 1929). The technique of breeding in the
experiment with underfeeding was the same except for the fact that
the yeast was put in the bottles with synthetic medium at the moment
of populating the bottles with larvae.
A method of getting undernourished larvae by taking larvae from
the food before the normal end of feeding has been used by various
workers, for instance, Ezhikov (1917, 1922), Smirnov (1926, 1927),
Cousin (1926), Herms (1928) and others. Most of these authors did
not attempt to determine with sufficient accuracy the moment of taking
the larvae from the food, Herms being in that respect an exception. In
the present investigation, larvae were taken from the food exactly 48
hours after the moment of populating the bottles with 0-2 hour-old
larvae. Larvae which reached the desired age were taken from bottles
and placed in half-pint bottles containing plain agar. The mouths of
the bottles were covered with 40 mm. watch glasses and sealed with
plastaline used in modelling. This was done in order to prevent the
larvae, which become very active, from crawling out. The day after
the larvae had turned into pupae the watch glasses were replaced by the
usual cotton stoppers.
Table I shows that the larvae with a subnormal period of feeding
pupate earlier than normally fed ones. This can be compared with
Kopec's (1924) statement that ". . . if we begin to apply starvation to
older specimens during developmental stages . . . the transformation
of these animals is accelerated." A little longer prepupal development
of the normally fed larvae, those which served as controls to the underfed
being compared with the 28° flies of the early October experiment,
cannot be very easily interpreted. It might exist in a difference in
conditions — perhaps a difference in yeast growth which lengthened the
duration of development of larvae in the second set of experiments.
88
W. W. ALPATOV
TABLE I
Data on the Conditions of the Development of Flies Reared for the Study of
the Problem of Cell Size
Temperature
limits
of variation
Average
Time of the
beginning of the ex-
periment
Time from egg
until pupation,
in hours
Time
of
feeding
Underfed flies
Kept at 28°
—
October 24, 1928
80.39±.50
48 hours
Normally fed
flies
Kept at 28°
.
October 24, 1928
93.16±.74
Until normal
leaving of
the food
28° flies
27.1-28.9°
28.2°
October 8, 1928
87.40±.36
Until normal
leaving of
the food
18° flies
17.0-20.0°
18.2°
October 8, 1928
200.86±.89
Until normal
leaving of
the food
The flies have been collected in 70 per cent alcohol and measured
in glycerine under a cover glass. The following characters on the wings
of collected flies have been studied : the length and width of the wing,
and the number of hairs on a surface equal to 0.1 square mm. on the
lower surface of the wing. Fig. 1 represents the points of measure-
ment and the place where the hairs have been counted.
Region of
6r/stte counting
FIG. 1. Measurements of the wing. AB, length of the wing, CD, width of
the wing. The square shows the area of the bristle countings.
For the measurements the following optical systems were chosen :
Spencer 25.4 mm. objective and a micrometer ocular in a No. 2 ocular.
The countings of the hairs of the lower surface of the wing were done
in a way approved by Th. Dobzhansky. Pieces of paper with squares
DROSOPHILA MELANOGASTER
89
representing 0.1 square mm. at a given magnification have been pre-
pared by projecting through an Abbe camera lucida 0.1 mm. from
an object micrometer placed on the microscope stage. A Spencer
microscope was used with objective 4 mm. and ocular [ 10. The hairs
have been projected by means of the camera lucida and drawn with a
sharp pencil. Only hairs whose bases happened to fall inside the square
have been counted (Fig. 2).
A/or/na/Sy fed a £o/d fe/nperafare \
OOO_ T /<OO_
FIG. 2. This figure represents the bristles on the surface of 0.1 mm.2 in the
lower surface of the wings of underfed, normally fed, and cold temperature fe-
males. The bristles which have a line across their middle have been counted,
those without lines had their basis outside the limits of the 0.1 mm.2 and have
not been counted.
We did not consider it wise to count the hairs exactly at a certain
point (in so many parts of a millimeter from a certain vein) as has
been done by Dobzhansky. There are two reasons for not doing so.
First of all the distribution of hair on that part of the wing is more
or less uniform. On the other hand, the wings of underfed and normal
are so different in size that a distance expressed in absolute measurement
would show morphologically quite different regions. Fifty specimens
of each set of underfed, normal fed and 18° flies were studied in regard
to the density of the hairs. Dr. Th. Dobzhansky succeeded in finding
that on the wings each hair corresponds to a separate cell. This can
be seen on specimens of flies just emerged from the pupae. The wings
look opaque and the cells can be distinctly seen. It is very likely that
the tiny hair covering the thorax of Drosophila corresponds also to
hypodennis cells, and their density may also be used as a method of
studying the size of the hypodermal cells.
III.
It is desirable at this stage to digress briefly to consider a matter
which arose as an extension of the original problem. It is the question
90
W. W. ALPATOV
of functional relation between the time of larval feeding and the final
size of the flies. First of all I reinvestigated the data published by
Herms (1928) and found that when plotted on a diagram they reveal
a very interesting picture.
I
t
Fema/es y =3.387 + 0.976* -O./22xz +0.0047**
0-.
Mates y=3.773 + 0.67<3x-O.044j(2+O.OOyx*
«8
5V 60 66 72 78 8V 3O
/yours
FIG. 3. The relation between the wing length and the length of the feeding
period in Lucilia scricata. Data from Herms (1928).
Fig. 3 represents Herms' data and two cubic parabolas which I fitted
to the observed points. Up to the 78 hour point the trend of the curves
represents the upper part of a typical growth curve. There cannot
be any doubt that this trend corresponds exactly to the upper branch
of the logistic curve which can be fitted to the growth of Drosophila
larvse of the third instar (see Alpatov, 1929). But the decline after
78 hours is quite remarkable. Going back to my paper on larval growtn
in Drosophila I was able to find in Fig. 13 particularly a slight indication
as to an analogous decline of the size of the larvse killed at the latter
end of the life of the culture. It was therefore decided to clear up
this question on specially collected material. This was done in April
1929. Forty bottles containing 0.500 grams of Magic yeast with 25
drops distilled water were populated by 80 larvse each. Five drops of
water were added every day during the larval growth. The experiment
was run at a temperature of 25° C.
Table II contains data on the sex relations in the material studied.
Let us first compare the percentage of males emerged from larvse taken
from the food at 48-80 hours, which is equal to 102.6, with that of
DROSOPHILA MELANOGASTER
91
males emerged from larvae taken from food at the age of 84-96 hours,
in which case the percentage is 89.9. This difference finds its explana-
tion in the fact that male larvse in our case started pupation earlier than
females, which is shown by the very high percentage of males among
TABLE II
Absolute and Relative Numbers of Larva, Pitpcc and Adult Flics in the
Experiment on Underfeeding of Larva
Hours from the
beginning of
feeding
Number
of larvae
taken
from the
food
Number
of the
pupae ob-
served
Number
of pupae
unable to
produce
flies
Number of flies emerged
Total
In per
cent of
the larvae
Male
Female
Male in
per cent
of female
48 ...
151
170
152
158
162
170
151
135
150
i .
17
7
11
12
2
7
12
3
1
30
80
87
123
154
153
117
127
142
19
47.1
57.2
77.8
95.1
90.0
77.6
94.1
94.7
18
44
37
62
72
85
69
56
70
12
36
50
61
82
68
48
71
72
150.0
122.2
74.0
101.6
87.8
125.0
143.8
78.9
97.2
52
56
60
64
68
72
76
80
Total 48-80
— •
—
— •
—
—
513
500
102.6
84
86
137
111
110
95
85
36
12
30
46
61
73
2
4
11
3
10
6
78
115
100
100
79
75
90.7
83.9
90.1
90.1
83.2
88.2
39
62
56
45
30
27
39
53
44
55
49
48
100.0
117.0
127.3
81.8
61.2
56.3
88
90
92
94
96
Total 84-96
—
—
—
—
— •
259
288
89.9
Flies emerged from pupae at 84-96 hours
84
25
11
227.3
88 ....
.
.
.
,
6
5
120.0
90 ....
.
=
,
23
8
287.5
92
.
,
30
12
250.0
94
,
^
32
23
139.1
96
,
45
27
166.1
Total 84-96
_
161
86
187.2
larvae pupated naturally at the age of 84-96 hours — 187.2 per cent.
On the whole the group of bottles which was taken to get larvse fed
84-96 hours shows a percentage of males equal to 109.1. Comparing
it with the sex proportion in normal undisturbed bottles where we had
92
W. W. ALPATOV
356 males and 415 females, we find that the normal percentage of males
is 85.8. We can therefore draw the conclusion that there is a definite
preponderance of males among flies emerged from the underfed larvae.
In other words it seems that a selective process makes the male more
resistant to underfeeding.
TABLE III
Wing Length, Width and Relative Width of the Flies Emerged from Larva
taken from the Food at Different Hours
Hours
Males
Females
Length
Width
Index
Number
Length
Width
Index
Number
48
1.107
.6490
58.6
17
1.207
.6972
57.8
12
52
1.164
.6847
58.8
25
1.239
.7154
57.7
25
56
1.331
.7847
59.0
25
1.413
.8034
56.9
25
60
1.321
.7697
58.3
25
1.493
.8459
56.7
25
64
1.394
.8145
58.4
25
1.572
.8898
56.6
25
68
1.409
.8289
58.8
25
1.588
.9102
57.3
25
72
1.406
.8428
59.9
25
1.561
.9083
58.2
25
76
1.371
.7983
58.2
25
1.511
.8493
56.2
25
80
1.412
.8261
58.5
25
1.586
.8938
56.4
25
84
1.476
.8833
59.8
25
1.673
.9349
55.9
25
88
1.440
.8516
59.1
25
1.641
.9321
56.8
25
90
1.472
.8777
59.6
25
1.646
.9255
56.2
25
92
1.423
.8468
59.5
25
1.614
.9032
56.0
25
94
1.426
.8457
59.3
25
1.613
.9077
56.3
25
96
1.444
.8686
60.2
25
1.608
.9083
56.4
25
TABLE IV
Wing Length, Width and Relative Width of flic Flics Emerged from
Pupated at a Given Hour, and of Those Emerged from Pupa
Pupated during the Whole Pupation Period
Pupa:
Hour
Males
Females
Length
Width
Index
Number
Length
Width
Index
Number
84
1.484
1.490
1.493
1.456
1.455
1.463
1.475
.8805
.8887
.8876
.8516
.8499
.8544
.8745
59.3
59.6
59.5
58.5
58.4
58.4
59.3
24
6
15
25
25
25
40
1.709
1.728
1.715
1.649
1.644
1.672
1.673
.9901
.9944
1.007
.9312
.9389
.9536
.9668
57.9
57.5
56.7
56.5
57.1
57.0
57.8
11
4
8
12
23
25
40
88
90
92
94
96
Normal pupation. .
Tables III and IV give the average length and width of wings of
our material. The wing length is graphically represented in Fig. 4.
DROSOPHILA MELANOGASTER
93
With the exception of some cases (72, 76 and 80 hours) the ma-
terial confirms what could he seen on curves based on Herms' data.
The most interesting thing is the declining slope of the curves toward
the end. It is not only with underfed flies that this decline is noticeable,
V8
S6 60
6V 68 72 76 8O 8V 88 SO 92 9V 96
Mours of /arra/ feeding
FIG. 4. This figure represents the relation of the length of the wing and
the length of the larval feeding in Drosophila melanogaster. The triangles in-
dicate the length of wings of flies pupated at certain hours.
but the flies normally pupated in the beginning of the pupation period
had longer wings {i.e., larger bodies) than flies in which pupation has
been delayed.
Table V gives the statistical proof of this conclusion. It can be
definitely seen that in males and females without regard to whether
the pupation is going naturally or the flies emerge from larvae taken
from the food, those which pupate first are larger than those which
pupate later. We may express the observed phenomenon in a little
different form. There is a negative correlation between the duration
of larval life and the size reached during growth. The faster the larva
grows the sooner it reaches the pupal stage. We take the liberty of
comparing our case with the experiments on Ciicumis mclo described
by Pearl in his book, The Rate of Living, (1928). The larvae which
reach a larger size in a short time have naturally a higher rate of growth
than larvae which remain small for a longer time. Therefore the state-
ment brought forward by Pearl (p. 139) that ''between growth rate
and duration of life to the beginning of death the correlation is negative
and significant in degree " can be perfectly well applied to our case.
We do not know whether these differences arise really as a result
of inherent vitality or are the result of differences of treatment of
larvae during the population of the bottle. Further experiments have
to solve this question. Our results are very close to Kopec's discovery
(1924) of the negative correlation between the duration of larval period
94
W. W. ALPATOV
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W. W. ALPATOV
and the weight of the chrysalids in Lynmntria disbar (L.). This nega-
tive correlation found in twelve experimental groups out of sixteen is
particularly well expressed in males.
s/./
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Length of Me using /n /n/cro/neter diws/ons
FIG. 5. Correlation between the length of the wings and the number of
bristles per 0.1 mm.2 on the lower surface of the wings of the male.
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length of the w/ng in m/crome/er af/^/j/o/?s
FIG. 6. Correlation between the length of the wings and the number of the
bristles per 0.1 mm.2 on the lower surface of the wings of the females.
DROSOPHILA MELANOGASTER
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IV.
Correlation tables shown in Figs. 5 and 6 contain the basic data
on the number of hairs on 0.1 mm.2 and length of the wings. The
horizontal axis gives the wing length in divisions of the ocular microm-
eter, each division being equal to 28.333 microns. Table VI represents
constants derived from Figs. 5 and 6 with the addition of wing length
of 28° flies. The wing length is expressed in millimeters.
TABLE VII
Average Width of the Wings and Width Index, i.e., Width Expressed in Per
Cent of the Length
Underfed
flies
Normally fed
flies
28° flies
18° flies
Width
of the
Index
Width
of the
Index
Width
of the
Index
Width
of the
Index
wing
wing
wing
wing
Males . . .
.7151
58.23
.8970
59.33
.8871 ±.0021
59.62±.ll
1.024
58.85
Females .
.7253
57.29
1.015
58.30
1.004 ±.002
58.51±.12
1.102
57.31
Let us discuss the influence of the factor under consideration on
the wing as a whole. Table VII gives us the constants for the width
in millimeters as well as the width in percentage of the length. There
is a pronounced sex difference in the size of the wing, the females in
all groups being larger than the males. The relative width of the wing
is larger in the males, as can be seen by comparing males and females
in all groups, and particularly those of the 28° group. The difference
is 6.9 times larger than its probable error. (The indices in this case
have been calculated by the use of Pearson's formula.) Another point
of interest concerning the relative width of the wing is that in the fe-
males as well as in the males the underfed and 18° flies seem to have
narrower wings than the " normal " 28° flies. The sex difference is
also influenced by abnormal conditions. Table VIII shows that in
" normal " 28° conditions the sex difference is the greatest, while un-
derfeeding and low temperature reduce the difference. The lower line
in Table VIII contains recalculated data from the experiment described
in a former paper (Alpatov and Pearl, 1929). The effect of low tem-
perature and consequently of the slow development can be seen in this
case also. It is difficult to find an adequate explanation of this phe-
nomenon, which very likely is connected with certain differences in male
and female postembryonal development, that is, with different time of
the manifestations of different characters during the larval or pupal life.
DROSOPHILA MELANOGASTER
99
Turning our attention to the main problem of our investigation,
one glance at the correlation tables shows that the larger the size of
the wing of the corresponding group of flies, the smaller the number
of cells on the area of 0.1 mm.2 In other words, the larger flies, con-
sidering inter-group variation, have also larger cells. The coefficients
of correlation for each of the six groups of flies have been calculated
separately. They are given in Table VI. Only in the case of underfed
males and females is the correlation significant and negative. The con-
clusion is that in underfed flies the size of the body is negatively cor-
related with the number of cells on a definite surface of the wing. A
possible but very dubious explanation of the absence of such correlation
in the case of normally fed and cold temperature flies might be that the
variation in the wing length of Drosophila developed from normally
fed larvae is so small that the correlation could not manifest itself.
TABLE VIII
Sex-Index of the ll'imj Lcntjth, i.e., Male Wing Length Expressed in Per Cent
of the Female
When
studied
Underfed flies
Normally fed
flies (28°)
28° flies
18° flies
1928
95.33±.54
86.76±.15
86.71±.15
90.61 ±.26
1927
—
—
88.18±.16
91.93±.18
So far as the variation of the flies belonging to different groups is
concerned, it can be seen that the coefficient of variation of the number
of cells does not show any definite difference in different groups. At
the same time the variation of underfed flies in the length of the wing
is much greater than that of the flies which had a normal feeding, no
matter at what temperature. Previous investigators who have worked
on variation of flies under conditions of under-feeding have also de-
scribed the increasing variation of experimental animals (see Smirnov
and Zhelochovtsev, 1926).
We have now to approach the problem of the actual surface-size of
the cells and its relationship to the size of the whole organ. Table IX
represents all the calculations relating to this question. The surface of
a cell in square microns was determined by dividing 10,000 microns
(0.1 mm.2) by the number of hairs on that surface. It can easily he
seen that the larger the flies the greater the surface of the cell. An-
other point of interest is the pronounced sex difference in the size of
the cells, the females having much larger cells than the males. This
has been pointed out by Dobzhansky (1929). The next step was to
100
W. W. ALPATOV
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DROSOPHILA MELANOGASTER
101
come from the surface values to linear values which has been done by
calculating the length of the cell, which was obtained by taking a square
root of the surface of the cell. The data on wing length gave the
possibility to calculate the percental decrease in the wing length taking
the wing length of 18° flies as a basis. Multiplying by this percental
decrease in wing length the number giving the " length " of cell in cold
temperature (18°) flies, we obtained the figures represented in our
table under the heading " Calculated length of cells." Comparing them
with the dimensions obtained by taking the square root, we can easily
see that the assumption that the wing length varies proportionally to
the length of its constituents does not hold true. The three columns
on the right of Table VII represent the changes in wing size and cell
size expressed in per cent of 18° (cold) flies. The same relationship
between these two characteristics is shown in a percental scale on Fig.
7, the diagonal line represents the relationship in case of a proportional
change in wing length and cell length ; the dotted line shows the actual
percental decrease in cell size in different groups of our flies.
SO 6O 7O SO SO /OO /O 2O ~30
/O 20 JO
SO 6O 7O 8O SO SOO
Length of Me
Length of the
FIG. 7. The dotted lines represent the relationship between the percentage
of decrease of the wing length and the percental decrease in the corresponding
percental length of the cells calculated by taking the square root of the surface
of the cells.
The general conclusion of all these calculations is that the reduced
size of cells alone cannot explain the reduction of the organ. The only
possible way to explain it is the assumption that the decrease in the
organ size — in our case in wing size — is not the result of a decreased
size of its cells alone, but also of a reduced number of cells. This last
conclusion has a certain bearing upon the problem of the cell constancy
in the organism.
102 W. W. ALPATOV
If our discussion is correct, the organism can evidently react to
the factor decreasing in size not only by decreasing the size of the cells
but also the number of cell divisions. The present limited material
does not warrant further discussion, but it may be hoped that other
investigations in the field of cell-biometry may create a similar basis
for understanding the variation of the whole organism as Die Zellulars
Pathologic of Virchow did for the interpretation of the pathology of
the whole organism.
SUMMARY
1. Dobzhansky's method to determine the number of cells under
the surface of the wing membrane of Drosopliila melauogastcr by count-
ing the number of hairs has been used in the present investigation of
the relationship of the organ size to the size of its cells.
2. Underfeeding and development at low temperature have been the
factors to produce flies under and above the normal size.
3. The functional relation between the time of feeding of larvae
and the size of the wings of larvae being the expression of the upper
part of the logistic larval growth of the third larval instar can be
expressed by a cubic parabola.
4. There is a definite tendency for large larvae (i.e., fast-growing
ones) to pupate earlier, which finds a certain analogy with Pearl's cor-
relation that " between growth rate and duration of life (in this case,
duration of larval life) to the beginning of death the correlation is
negative and significant in degree."
5. As far as all three groups of flies (underfed, normal and cold
flies) are concerned the size of the wings is negatively correlated with
the number of hairs on a definite surface of the wing when the groups
are considered as ^vholcs (inter-group correlation). The existence of
such a negative correlation could be shown also within the group of
underfed females and males, but not within the other groups.
6. Expressing in per cent the increase in size of the whole organ
and the increase of the linear dimensions of the cells there is a dis-
crepancy in the rate of changes. This leads to the conclusion that the
changes in size of the wing cannot be accounted solely by the changes
in the size of the cells. The number of cells must play also a certain
iole in this process.
LITERATURE
ALPATOV, W. W., 1929. Growth and Variation of the Larvae of Drosopliila
wclanogaster. Jour. E.rp. ZooL, 52: 407.
ALPATOV, W. W., AND PEARL, RAYMOND, 1929. On the Influence of Temperature
during the Larval Period and Adult Life on the Duration of the Life
of the Imago of Drosopliila melanogaster. Am. Nat., 63: 37.
DROSOPHILA MELANOGASTER 103
BRIDGES, CALVIN B., 1921. Triploid Intersexes in Drosophila mclatwgaslcr.
Science, 54: 252.
BRIDGES, CALVIN B., 1925. Haploidy in Drosophila mclanogastcr. Proc. Nat.
A cad. Science, 11: 706.
COUSIN, G., 1926. Influence du temps reserve a la nutrition sur les phases du
cycle evolutif et les metamorphoses de Calliphora erythrocephala. Com[>t.
rend. Soc. Biol, 95: 565.
DOBZHAXSKY, TH., 1929. The Influence of the Quantity and Quality of the
Chromosomal Material on the Size of the Cell in Drosophila melanog aster.
Arch. f. Entit'icklngsnicch. d. Ore;., 115: 363.
EZHIKOV, J., 1917. Influence de 1'inanition sur la metamorphose des mouches
a ver. Rev. Zool. Rnssc, 3.
EZHIKOV, J., 1922. Uber anatomische Variabilitiit iiber dirckt Wirkung ausserer
Einfliisse. Rev. Zool. Russc, 3.
HERMS, W. B., 1928. The Effect of Different Quantities of Food during Larval
Period on the Sex Ratio and Size of Luc ilia scricata Meigen and Thco-
baldia mad ens (Thorn). Jour. Econ. Entom., 21: 720.
KOPEC, S., 1924. Experiments on the Influence of the Thyroid Gland on Meta-
morphosis and Weight of Insects. Mcmoircs de I'lnstltut national poJonais
d'economie rnrale a Pullaivy, 5: 356.
LEVI, G., 1906. Studi sulla grandezza della cellule. Arch. Ital. </i Anal, c di
EmbrioL, 5.
LOEWENTHAL, H., 192'3. Cytologische Untersuchungen an nonnalen und exper-
imentell beeinflusstcn Dipteren (Calliplwra erythrocephela) . Arch /.
Zcllforsclning, 17: 86.
MARTINI. E., 1924. Die Zellkonstanz und ihre Beziehungen zu anderen zoologischen
Vorwiirfen. Ztschr. /. Anat. mid Entuncklnr/sgcs., 70: 179.
PEARL, RAYMOND, 1928. The Rate of Living. New York.
PRZIBRAM, H., AND MEGUSAR, F., 1912. Wachstumsmessungcn an Sphodromantis
bloculata Burm. Arch. f. Entwick., 34: 680.
SMIRNOV, E., AND ZHELOCHOVTSEV. A., 1926. Veranderung der Merkmale bei
Calliphora erythrocephala Mg. unter dem Einfluss verkiirzter Ernahrungs-
periode der Larve. Arch. f. Entu'icklngstncch.. 108: 579.
SMIRNOV, E., AND ZHELOCHOVTSEV, A. N.. 1927. Einwirkung der Nahrungsmenge
auf die Merkmale von Drosophila funcbris Fbr. Zool. Anz., 70: rS.
OSMOTIC PROPERTIES OF THE ERYTHROCYTE
I. INTRODUCTION. A SIMPLE METHOD FOR STUDYING THE RATE OF
HEMOLYSIS
M. H. JACOBS
(From the Department of Physloloyy, University of Pennsylvania, and the Marine
Biological Laboratory, Woods Hole, Massachusetts)
I.
There is almost no other single type of animal cell which has been
so extensively employed in experimental work in the fields of osmotic
phenomena and of cell permeability as the mammalian erythrocyte.
From the time of the early studies of Hamburger (1886) down to the
present day it has been recognized as possessing a number of peculiar
advantages as experimental material. Thus, it can be obtained at all
times and places in what for practical purposes are unlimited quantities;
indeed, the investigator himself carries about with him wherever he
goes a never-failing supply of absolutely fresh and normal erythrocytes,
ready for use at a moment's notice. Because of the remarkable con-
stancy of its natural environment — the mammalian body — the erythro-
cyte, unlike certain other cells frequently used for similar studies, may
be expected to show only relatively slight variations in its physiological
properties from day to day and from season to season. Furthermore,
its simple structure and low rate of metabolism prevent complications
which are frequently troublesome with other types of material. Re-
moved from the body it can be kept, if not in an unaltered, at least in a
usable condition for a longer time than almost any other kind of animal
cell. Finally, there are available for its study methods of great sim-
plicity which are not only quantitative but which are also statistical to
an extent perhaps nowhere else realized with physiological material.
Because of these striking, and to a considerable extent unique, ad-
vantages the erythrocyte would appear to be an almost ideal type of
material for studies in which a high degree of quantitative accuracy is
desired. A survey of the literature, however, reveals all too frequently
a disappointing failure on the part of investigators to obtain results
of this character. Not only is there a very common lack of agreement
between the conclusions reached by different workers, but even the same
investigator is not infrequently forced to acknowledge an inability on
repeating his experiments to obtain consistent and reproducible data.
104
OSMOTIC PROPERTIES OF ERYTHROCYTE 105
The erythrocyte, in spite of its apparent simplicity, behaves, in fact,
as if it were either naturally a highly variable and capricious type of
material, or — what is more likely — as if it were peculiarly sensitive to
certain environmental factors which with other types of cells are much
less troublesome.
In the course of work which has occupied the author for several
years and which will be reported in detail in the series of papers of
which the present one is the first, the general conclusion has been reached
that the erythrocyte is indeed a highly suitable form of material for
many types of experimental work and that accurately reproducible re-
sults may be obtained with it, but that such results are possible only
with a more careful attention to details than is needed with most other
forms of physiological material. As a matter of fact, the very sim-
plicity of the mammalian erythrocyte, which in its mature condition is
perhaps only questionably to be called a living cell at all, prevents the
maintenance by it in a changing environment of the relative internal
constancy which is so characteristic of more complicated cells and of
entire organisms. The simplicity of the erythocyte is, therefore, rather
paradoxically, actually a source of complexity for the experimenter.
Furthermore, there are certain special reasons, closely connected with
the functions which the erythrocyte has to perform, why its osmotic
properties, in particular, are of necessity far more profoundly affected
by slight environmental changes than are those of perhaps any other
known type of cell. These reasons will be discussed in the second paper
of this series.
In general, the relation which the erythrocyte, considered as ex-
perimental material, appears to bear to other types of cells is much
the same as that which a canoe bears to boats of more stable design.
Both the erythrocyte and the canoe when properly handled have very
definite and characteristic advantages, but both have the tendency to
penalize any carelessness in their management in a prompt and unmis-
takable manner. Perhaps at some future clay this peculiarity of the
erythrocyte may be considered rather as an advantage than a disad-
vantage.
II.
Before considering certain of the peculiarities of the erythrocyte
itself it seems advisable to deal with some of the methods which have
been employed in the past in studying the osmotic properties of this
type of cell, and, in particular, with the one which has been gradually
developed by the author and has been used in the experimental work
upon which all of the papers of the present series are based. By giving
106 M. H. JACOBS
a single description of the method at this point, unnecessary repetitions
may later be avoided.
Osmotic changes in the erythrocyte are, in general, always associated
with volume changes. This is true whether the changes are of the
simple sort produced by the passage of water alone between the cell
and its surroundings or of the more interesting and complicated type,
so useful in studies of cell permeability, where the movement of water
depends upon osmotic inequalities set up by the passage of dissolved
substances across the cell boundary. Any quantitative study of osmotic
phenomena will therefore involve the measurement of the amount of
volume change which occurs in a given experiment, or the rate of this
change, or both.
In the case of the erythrocyte there are available two remarkably
simple methods for studying volume changes. The first is the hemato-
krit method introduced by Hedin (1891). By means of it the total
volume of all of the cells in a sample of a given suspension is meas-
ured, the cells being tightly packed together in a fine graduated tube
by centrifugal force. The advantages of this method are, first, its
simplicity and, second, its statistical nature, by which the variability
of the millions of individual cells is averaged out. Its greatest disad-
vantage— and this, unfortunately, is a fatal one in many cases — is that
the time required to pack the cells into a mass free from intercellular
fluid is so great, even with the most powerful centrifugalization avail-
able, that the method can be used only to obtain final end points or,
at most, to follow volume changes of extreme slowness. For this rea-
son, in the present series of studies, it has been possible to use it only
rarely.
A second method, of even greater simplicity, is that of hemolysis.
This method, first systematically employed by Hamburger (1886), de-
pends on the fact that when an erythrocyte in swelling reaches a certain
volume, which varies not only with the species of animal but also prob-
ably with the individual erythrocyte, it loses a sufficient part of its hemo-
globin to become invisible, or almost so, both as viewed singly under
the microscope or in the aggregate in a suspension in a test tube. In
some cases it is possible by appropriate treatment to restore the invisible
corpuscles to visibility ; in other cases it is not.
The term hemolysis is sometimes applied to the mere disappearance
of erythrocytes ; at other times it is used to describe their more complete
destruction. This double use of the term, while unfortunate, is perhaps
unavoidable at present and every author should therefore designate the
sense in which he employs it. It will here be used, for convenience, to
apply to what for practical purposes is the easier and more certain
OSMOTIC PROPERTIES OF ERYTHROCYTE 107
end point to observe, namely, the disappearance of the erythrocyte from
visibility rather than its more or less complete destruction, concerning
which there is usually much greater uncertainty. This usage is further
justified by the fact that in "osmotic hemolysis " complete destruction
is apparently very difficult to obtain. Thus, Adair, Barcroft, and Bock
(1921) were unable with water alone to separate the hemoglobin from
the cells containing it sufficiently to obliterate certain effects believed
to be due to the cells themselves, though this could be done after the
addition of ether, which presumably completed the destruction of the
cells.
The hemolysis method for studying the swelling of erythrocytes
and, indirectly, therefore, the penetration of dissolved substances, pos-
sesses the advantage of extreme simplicity. With no apparatus other
than a test tube, very fair ideas as to many problems of cell-permeability
may be obtained. The apparatus here to be described refines the method
to an extent which permits the experimenter to secure results of a really
high degree of accuracy. An even greater advantage of the hemolysis
method, however, is that it is available for the study of rates of swelling,
even in experiments of very short duration. In the present series of
papers no experiments of a total duration of less than one second will
be reported, but the author has pointed out elsewhere (1927) that a
principle used with conspicuous success for another purpose by Hart-
ridge and Roughton (1923) can be adapted to the study of hemolytic
processes whose duration is only a fraction of a second as is the case,
for example, with the hemolysis of the erythrocytes of the sheep in
distilled water. In its adaptability to problems involving rapid rates of
swelling, and consequently some of the most interesting problems of
cell physiology, the hemolysis method is, in fact, of unique importance.
On the other hand, the method possesses at least two disadvantages
which must be frankly admitted and then dealt with as adequately as
circumstances permit. The first is that hemolysis may be caused or
influenced by various factors other than osmotic ones. The disappear-
ance of an erythrocyte does not necessarily indicate that it has by swell-
ing reached some definite hemolytic volume, V,,, though this is fre-
quently the case. It is important, therefore, that certain control experi-
ments shall always be performed before inferences concerning the rate
of swelling are drawn from observations on the rate of hemolysis.
These control experiments may take various forms. Thus, in cases
where osmotic factors alone are involved, it should be possible to show :
(1) that the substance or substances present in the solution in which
hemolysis occurs have no observable hemolytic effect when added in
varying amounts, up to and preferably exceeding those employed in the
108 M. H. JACOBS
experiments, to an isotonic solution of NaCl or some similar non-
penetrating substance; (2) that the process of hemolysis by a pure
solution of the substance in question may be stopped at will at any de-
sired point by the addition in osmotically suitable amounts of NaCl, sac-
charose, etc.; or (3) that if a solution of NaCl be chosen which is suf-
ficiently hypotonic to cause the hemolysis of some but not all of the
erythrocytes in a given sample of blood, the addition of the substance
to the partially hemolyzed suspension causes no increase in the degree
of hemolysis. The last mentioned test is a very delicate one, though
it is somewhat difficult to employ for reasons to be discussed in the
following paper of this series.
A second disadvantage of the method is that even in cases where it
is reasonably certain that the occurrence of hemolysis is due to the at-
tainment of a definite volume, Vh, this volume represents merely one
point on the swelling curve. As compared with the egg of Arbacia
(Lillie, R. S., 1916), (McCutcheon, M., and Lucke, B., 1926) whose
volume changes can be measured continuously, the erythrocyte appears
capable at best of supplying to the investigator only very meagre in-
formation about the course of the swelling process.
This disadvantage, however, is not so serious as it might at first
sight appear to be. There is reason to believe that the course of the
swelling of the erythrocyte can be represented by a fairly simple equa-
tion (Jacobs, M. H., 1928) which permits the entire curve to be calcu-
lated approximately when one point on it is known. This question will
be dealt with more fully in a later paper. Furthermore, in perhaps
most experiments, what is desired is not so much the entire curve of
•swelling as some general measure of the velocity of the swelling process
under various experimental conditions, and this may frequently be ob-
tained by a comparison of the times required under the conditions in
question to reach the same state of swelling in each case. For work
of this type the critical hemolytic volume, Vh, when such a volume
exists, is a very satisfactory and convenient criterion for comparison.
One important additional point connected with the use of the hemo-
lysis method remains to be mentioned. Both this and the hematokrit
methods are statistical in the sense that* millions of cells are employed
with each. But whereas the latter measures the total volume of all of
Ihe cells together without separating them into groups, the former is
complicated by the fact that different individual cells hemolyze with
different degrees of readiness, and in determining the time of hemolysis,
the cells must, in effect, be divided into groups for separate time-meas-
urements. The size of these groups will depend upon the delicacy of
the method employed, \Yhen a distinction can be made between, for
OSMOTIC PROPERTIES OF ERYTHROCYTE 109
example, 75 per cent and 76 per cent apparent hemolysis, as is the case
with the method about to be described, then the time of hemolysis for
the group of cells lying between these limits and consisting of one per
cent of the total number may be taken as approximately the arithmetical
mean of the times at which the above-mentioned degrees of hemolysis
are attained. With a cruder method, or in the region of five or ten
per cent hemolysis, where measurements are much more difficult to
make, the groups dealt with are of necessity larger and a mere averaging
of two times gives correspondingly less accurate results.
Because of the heterogeneous nature of any collection of erythro-
cytes, it is impossible to speak simply of the " time of hemolysis " for
a given sample of blood. Different times must be measured for different
groups of cells, or, if desired, a single group may be arbitrarily selected
for a given experiment by determining in advance for what particular
degree of hemolysis the time shall be measured. In any case, the prob-
lem is a much more complicated one than if the blood contained only
erythrocytes of uniform physiological properties.
On the other hand, a certain degree of heterogeneity may in some
respects be an advantage. Assuming that the different degrees of os-
motic resistance of the various cells are dependent chiefly on different
individual values of the critical hemolytic volume, Vh, which is a plaus-
ible, though as yet an entirely unproved assumption, a possible means
is suggested for obtaining more information about the course of the
entire swelling curve than could be furnished by a perfectly homo-
geneous group of cells. The details of such a method still remain to
be worked out.
A much more definite advantage of the heterogeneity of a given
population of erythrocytes is the following. It is frequently necessary
to find a solution of " critical concentration " for a group of cells, i.e.,
which is just at the point of being able to hemolyze these cells without
actually doing so. Cells in such a solution are extremely sensitive test
objects for studying the effects of such factors as pH, temperature, etc.,
as will be pointed out in greater detail in a later paper. If the cells in
such a group possessed identical properties, it would require many trials
to find the appropriate concentration to the desired degree of accuracy
(i.e., to less than 0.001M). With as heterogeneous a group, however,
as the erythrocytes in ordinary blood, any concentration within fairly
wide limits may be selected' with the certainty that there will be present
in the blood a group of cells which will exactly " fit " the concentration
so chosen. In later papers frequent applications of this principle will
be mentioned.
110 M. H. JACOBS
TIL
A method suitable for the study by the hemolysis method of the
osmotic properties of the erythrocyte should possess the following char-
acteristics. It should allow the degree of hemolysis to be estimated
more accurately and more rapidly than the usual laborious and not very
exact methods of making cell counts or of making hemoglobin de-
terminations after a preliminary centrifugalization. It should permit
the time required for the attainment of a given percentage of hemolysis
to be measured accurately, even when the total duration of the experi-
ment is only a few seconds. The usual methods are entirely useless
in such cases, and this is perhaps the reason why little work has as yet
been done on the rates of any except very slow types of hemolysis.
The method should, in the third place, provide not merely for the
measurement of the time required to reach some single percentage of
hemolysis but for that required for the attainment of many different
percentages ; otherwise, the heterogeneous nature of a population of
erythrocytes may give rise to a type of difficulty that will be discussed
in a later paper. Finally, though less essential than the characteristics
already mentioned, simplicity of the apparatus itself and convenience
in its use would be highly desirable features.
The method here described possesses all of these characteristics.
It permits successive determinations of the relative concentrations of
cells in different suspensions, as well as of apparent percentages of
hemolysis, to be made in a few seconds each, which under favorable
conditions are reproducible to one or two per cent. It may be used
for the study of all rates of hemolysis where the time measured is more
than one second. Furthermore, it permits the measurement not merely
of the time required to reach some arbitrarily selected degree of hemo-
lysis but also of the times corresponding to all percentages from zero to
upwards of 90 per cent. These measurements, which are extremely
easy to make, take the form of permanent .kymograph tracings where
mistakes in instrumental readings or in the recording of them by the
observer are impossible, and where all of the details of the experiment
are presented in a way that facilitates ready interpretation. Finally,
the apparatus is very simple and inexpensive. A crude but satisfactory
form of it can be constructed in an hour out of materials available in
any laboratory, and its operation can be mastered in a few minutes.
The variety of uses to which it can be put and the degree 'of accuracy
which can be secured with it will be made more evident in the later
papers of this series.
In principle, the method is not new. It involves merely the meas-
urement of the turbidity of a suspension of erythrocytes by determining
OSMOTIC PROPERTIES OF ERYTHROCYTE
111
the maximum depth of the suspension through which the image of the
glowing filament of a carbon lamp is visible. It is to be noted that
what is observed is a distinct image rather than the total amount of
transmitted light, as is the case, for example, with the methods of Ponder
( 1923, 1927) or with the nephelometer. Methods similar to the present
one for the study of suspensions have been used or suggested by Vies
(1921), Holker (1921) and others, but they lack certain of its most
useful features.
The source of the image is the filament of an old-fashioned carbon
lamp. The brightness of the filament is kept constant by the use of
a milliammeter to measure and a sliding rheostat to regulate the current
flowing through it. For the particular lamp employed, a current oi
200 milliamperes has proved to be a suitable one and has been every-
where used except where otherwise specified. If desired, the depth
of the suspension may be kept constant and the current measured which
under the given conditions makes the filament visible. This method,
however, is inferior to the one adopted in being less sensitive and in
involving more difficult calibrations.
10
FIG. 1. A simple form of the apparatus, described in detail in the text.
A form of the apparatus somewhat simpler than the one actually
employed, but which shows more clearly in a photograph its most es-
sential parts is illustrated in Fig. 1. The image of the filament, 1, is
reflected upward by the mirror. 2, through the vessel, 3. in which the
112 M. H. JACOBS
suspension to be examined is placed. This vessel is in the form of
a tube 2.5 cm. in diameter with a funnel-like expansion above and closed
below by a glass plate cemented to the tube with deKhotinsky cement.
In cases where it is not necessary to keep the whole apparatus in a water
bath for temperature control a separate glass funnel may be substituted.
Into the vessel, 3, plunges a tube, 4, coated internally with a dead-
black varnish and closed at its lower end by a small coverglass cemented
to it with deKhotinsky cement. It is best always to cover with paraffin
any such cement which can come in contact with the solutions used in
the experiments. The position of the plunger, 4, is adjusted by means
of the rack and pinion of an ordinary microscope, 5, to the tube of which
it is attached by the arm, 6. Attached to the microscope are also the
pointer, 7, which gives readings on a millimeter scale and the writing
point, 8, which touches the smoked paper of a kymograph (not shown).
Any movement of 4 is therefore recorded by the kymograph, while at
the same time its exact setting can be read from the scale. At the
beginning of an experiment the apparatus is adjusted so that a scale
reading of zero corresponds to close contact between the bottom of
3 and that of 4. The necessary adjustments of 4 are facilitated by the
screws, 9 and 10. Where greater simplicity is desired, a satisfactory
substitute for the arm, 6, can be improvised from several ordinary metal
clamps.
Since in osmotic experiments on the erythrocyte (as will be pointed
out elsewhere) accurate temperature control is essential, the vessel, 3,
is usually immersed almost to the top of the funnel in a covered water-
bath (not shown in Fig. 1) with blackened interior to cut off all light
except that passing through a glass window in its bottom. For the
design of this water-bath and for several other features of the apparatus
the author is indebted to his assistants, Mr. Arthur K. Parpart and
Mr. Wilbur A. Smith.
When the apparatus has been set for a series of experiments it
is desirable not to disturb it in changing solutions. This is easily
avoided by emptying the vessel, 3, through a removable glass tube (not
shown) attached to a filter pump. Another fine-pointed glass tube,
also not shown, is usually allowed to dip into the solution in 3. This
tube is connected with the compressed air supply and provides in short
experiments for rapid and uniform mixing of the blood and the solu-
tions introduced into 3, while in longer ones the current of air may
be used as desired to prevent any settling of erythrocytes on the bottom
of the vessel. Other tubes connected with the compressed air supply
and also not shown provide for the stirring of the water in the water-
bath and the prevention of condensation of moisture on the window
in its bottom when it is employed at low temperatures.
OSMOTIC PROPERTIES OF ERYTHROCYTE 113
In using the instrument in an ordinary hemolysis experiment, the
procedure is as follows. The tube, 4, is elevated until it is considerably
above the position of the expected initial reading. The desired quantity
of blood (usually one carefully formed drop from a special pipette)
is placed on a small removable paraffin-coated shelf, 11, which is sus-
pended from the side of the funnel. The kymograph is started and
the compressed air turned on. Then, as the solution is suddenly poured
upon the blood with one hand, the tube is lowered by the other until
the image of the filament just appears. The beginning of the experi-
ment is therefore shown by a sudden drop in the line made by the
writing point. When the image is seen to increase slightly in brightness,
the tube is quickly raised a few millimeters, causing it to disappear.
When it again appears the tube is again raised, and this process is
repeated until the tube emerges from the liquid. With the apparatus
employed by the author and with 25 c.c. of liquid, which is a convenient
quantity, this occurs at a scale reading of approximately 60 mm. rep-
resenting, for samples of blood, in the proportions used, between 80
and 90 per cent apparent hemolysis.
FIG. 2. Typical record of the course of hemolysis of ox blood in 2M ethylene
glycol. The time intervals are 5 seconds with every twelfth signal omitted.
The type of record obtained in an experiment of this sort is il-
lustrated in Fig. 2. This particular record gives 11 points on a curve
representing hemolysis of ox blood in 2M ethylene glycol. The time
intervals marked on the record are of five seconds each with every
twelfth one omitted. The slow fall of the curve prior to the sudden
rise which indicates hemolysis is due to the gradual recovery by the
erythrocytes, with the penetration of the solute, of their initial volumes,
and their subsequent further swelling, after a pronounced shrinking
114 M. H. JACOBS
has been produced by the concentrated solution employed. Even with
solutions of penetrating substances isosmotic with blood, the swelling
that precedes hemolysis is usually indicated by a slight fall in the curve.
By using greater dilutions of blood so that the readings appear higher
on the scale these effects can be considerably magnified and used to
FIG. 3. Typical record of the partial hemolysis of ox blood in 0.082M NaCl.
The marks on the curve indicate 30 second intervals.
good advantage in studying volume changes rather than hemolysis.
As would be expected, swollen corpuscles produce lower and shrunken
ones higher readings than normal ones, a fact already noted by Holker
(1921).
In experiments of longer duration, where the method described is
wasteful of kymograph paper and fatiguing to the eye of the observer,
it is preferable to make readings at regular intervals marked by the
writing point itself, allowing the drum to move only enough each time
to record the level of the reading. A record of this sort covering 18.5
minutes with readings every 30 seconds is reproduced in Fig. ' 3. It
represents the partial hemolysis of ox corpuscles in O.OS2M NaCl
slightly buffered for pH 7.4 with phosphates.
When the duration of the experiments is very short, i.e., less than
perhaps 10 seconds, kymograph records become difficult to make.
Fairly complete and accurate hemolysis curves may be obtained, how-
ever, in such cases by setting the instrument in advance at any selected
point and determining with a stop-watch the time required to reach
this point. The vessel is then emptied and the experiment repeated
with a different setting of the instrument, and so on, as many times
as desired. The complete curve may then be plotted from the separate
points obtained.
With experiments of such extremely short duration (i.e. less than
OSMOTIC PROPERTIES OF ERYTHROCYTE 115
perhaps 1.5 seconds) that the time required for the uniform mixing
of the blood and the solution becomes significant, it is scarcely profitable
to attempt to obtain times corresponding to the lower scale readings.
Fair accuracy, however, may be secured with sufficiently high settings
so that most of the suspension is under the bottom of the inner tube,
in which case imperfect mixing is much less serious than otherwise.
It is for this reason, as well as because of the fact that the accuracy
of the instrument is greater for the higher scale readings, that the author
has chosen 75 per cent apparent hemolysis of an approximately 1 : 500
suspension as a very convenient criterion for comparison when for
any reason it is necessary to select some single degree of hemolysis
for this purpose. With the apparatus used and with most samples of
blood this point usually corresponds to a scale reading in the vicinity
of 40 mm.
In the use of the instrument several precautions may be mentioned.
The only subjective feature of the method is the decision by the ob-
server as to when the filament may be said to be visible. This decision
is made with different degrees of readiness and constancy by different
persons. The author finds it most convenient so to place the lamp and
the mirror that what is seen in the tube is a single small loop of the
filament. A reading is taken when the exact form of the entire loop
is visible. To secure the greatest sensitiveness of the eye, readings
should always be approached from the side of the' invisibility rather
than from that of the visibility of the filament. In any case, it is im-
portant to work fairly rapidly. The image should be approached with-
out hesitation and the reading made without an attempt by moving
the tube up and down unnecessarily to secure exactly the right degree
of distinctness. What might otherwise be gained in this way is more
than lost by the changes that are caused in the sensitiveness of the
eye of the observer.
In general, the experience of 'each individual will soon teach him
under what conditions he can secure the most reproducible results.
Fortunately, the method permits no possible bias to enter into the meas-
urements, since the observer is unable while making a reading to see
the record on the drum, which reproduces with strict fidelity the results
of his judgment. It is therefore a very simple matter for anyone using
the method to obtain in this way, on a drum moved for the purpose
by hand, a series of readings at different levels, which, when subse-
quently measured, will give exact information as to the reliability of
his readings. The readings of the author, in a test of this sort, rarelv
show a variation of more than 0.3 mm. for a scale reading of 10 mm.
or of more than 1.5 mm. for a scale reading of 50 mm. For a sus-
116
M. H. JACOBS
pension whose initial reading is 10 mm. these variations correspond
to differences in the estimated percentages of hemolysis of approx-
imately three and less than one per cent, respectively. By averaging
a number of readings for a single point, such errors can be still further
reduced. The method is therefore seen to be capable of yielding results
of a high degree of accuracy.
IV.
The question of the relation between the observed scale readings
and the corresponding degrees of hemolysis may now be discussed.
Changes in the opacity of the suspension are due primarily to changes
in the number of cells which it contains and secondarily to changes in
R
60
50
30
20
10
3
6
8
'/c
FIG. 4. Effect of dilution of blood on scale reading. Curve A represents
dilution with 0.9 per cent NaCl of a suspension of ox erythrocytes. Curve B
represents dilution of a similar but originally less concentrated suspension (ap-
proximately 1 :500) with a solution containing hemoglobin in the proper amount
to give standards representing different degrees of apparent hemolysis ; 7? — scale
reading in millimeters and \/C = reciprocal of concentration in arbitrary units.
the properties of the individual cells and of the surrounding medium.
Since the first mentioned factor is by far the most important, it may
be considered first by itself as uncomplicated by, for example, the state
OSMOTIC PROPERTIES OF ERYTHROCYTE H7
of swelling of the cells or the presence of hemoglobin in the surrounding
solution.
The relation between the concentration of cells in a given suspension
and the scale reading of the instrument may readily be obtained by a
simple calibration experiment in which a geometrical series of dilutions
of an original suspension is used for purposes of standardization. For
example, beginning with 125 c.c. of a fairly concentrated suspension
of cells in 0.9 per cent NaCl, 25 c.c. are removed for the first measure-
ment and are replaced by 25 c.c. of the salt solution. After thorough
mixing this process is then repeated for any desired number of times,
a series of suspensions each four-fifths as concentrated as the one
preceding it being obtained. Frequently, a factor of dilution of three-
fourths or even one-half will give results which are entirely satisfactory
with correspondingly less labor.
If now the scale readings so obtained are plotted against the recip-
rocals of the concentrations, as has been done in the graph labelled
A in Fig. 4, it will be seen that the points lie almost on a straight line,
indicating that the relation between the scale reading R, and the con-
centration C may be represented approximately by the rectangular
hyperbola,
CR =- a constant.
Actual calculations show that the errors introduced by estimating the
relative number of cells in a given suspension, as compared with a
standard, by means of this simple relation are usually insignificant.
Thus, Table I, from which the data used in constructing graph A of
Fig. 4 were obtained, shows in columns 1 and 4, respectively, the relative
concentrations of cells as determined by actual dilution and as calculated
from the relation,
CR = R0,
R0 being the scale reading for the original suspension whose concen-
tration is taken as unity.
It will be observed that the differences amount in no case to more
than one per cent, though in other similar experiments differences of
two per cent, or rarely more, have been obtained. In general, the dif-
ferences are greater for low scale readings where the errors of observa-
tion are relatively large. Because of the important effect of slight er-
rors in determining the initial scale reading, R0, an average value for
the constant in the equation may, if desired, be obtained from all of
the CR products. For comparison with the figures already mentioned
there are given in column 5 concentration values calculated in this way.
On the whole, they are seen to agree very closely with the values in
columns 1 and 4.
118
M. H. JACOBS
As to the simple mathematical relationship found to exist between
the number of cells and the observed scale-reading, it may be stated
that much the same relation has been reported by Vies (1921) and
Holker (1921) who used methods somewhat similar in principle to the
present one, though differing from it in a number of respects, for meas-
uring the opacity of various cell suspensions.
TABLE I
Relation between Scale Readings and Concentration of Erytlirocytes in Suspensions
Concentration
in arbitrary
units = C
Scale readings in
millimeters = R
(each figure is the
average of 10
readings)
Product
CR*
Concentration
calculated from
initial reading
Ro = 5.0
Concentration
calculated from
average of CR
products = 4.85
1.00
5.0
5.0
—
—
.80
6.3
5.0
.79
.77
.64
7.7
4.9
.65
.63
.51
9.6
4.9
.52
.51
.41
12.0
4.9
.42
.40
.33
15.0
5.0
.33
.32
.26
18.3
4.8
.27
.27
.21
22.6
4.7
.22
.21
.17
28.6
4.8
.17
.17
.13
35.3
4.7
.14
.14
.11
43.6
4.7
.11
.11
* This product was calculated from more accurate values of C than those in
column 1, which are rounded off to two places of decimals only.
The exactness with which relative numbers of cells can be estimated
from scale readings, either by calculation or by the use of appropriate
standards, particularly for readings above 20 mm., suggests the possi-
bility of using the apparatus, though it was designed primarily for
studies of hemolysis, for making the ordinary red-cell counts so fre-
quently needed in physiological and in medical work and for which the
laborious and not very accurate hemocytometer method is commonly
employed. Preliminary experiments in this direction have shown that
by first diluting the blood so that the resulting suspension gives a reading
on the more sensitive part of the scale, successive independent deter-
minations differing from one another by no more than one or two
per cent may be obtained at will. The time required for each deter-
mination, exclusive of that required for cleaning and drying the blood
pipette is approximately 15 seconds. With the enormously more la-
borious hemocytometer method, successive counts, as is well known,
usually vary by at least five per cent. Of course, the method gives
only relative and not absolute numbers of cells (though it can be made
OSMOTIC PROPERTIES OF ERYTHROCYTE 119
absolute within the limits of the hemocytometer method itself by
means of one preliminary cell count) and the readings obtained with
it are affected by any variation in the size and shape of the erythrocytes
in different samples of blood, as well as by their numbers. The errors
to be expected from these sources, however, under the usual physio-
logical conditions are not likely to be as great as those constantly and
unavoidably associated with the far more difficult method now almost
universally employed.
In using the instrument to estimate percentage of hemolysis, several
factors in addition to the concentration of cells must be considered.
In the first place, during hemolysis not only do the cells decrease in
number, but the hemoglobin liberated from them and contained in the
surrounding solution absorbs light and therefore tends to produce lower
scale readings than correspond to the mere number of cells. This com-
plication may be dealt with readily, however, by making the dilutions
in the calibration series with a solution containing the concentration
of hemoglobin that would result from complete hemolysis of the cells.
Such a solution is readily prepared by adding to distilled water twice
the quantity of blood contained in the same volume of the standard
suspension and then, after complete hemolysis has occurred and the
solution is entirely transparent, mixing with it an equal volume of
sodium chloride solution of twice the concentration of that desired.
As a matter of fact, it turns out that with the dilution of blood that
is otherwise most convenient to work with (approximately one part of
blood to five hundred of solution) the effect of the hemoglobin on the
reading of the instrument, while detectable, is, practically, almost neg-
ligible. Under these circumstances the product :
(100 — per cent hemolysis) X scale reading
proves to be almost constant, as is indicated in graph B of Fig. 4 where
the scale readings plotted against the reciprocals of the percentage of
unhemolyzed cells lie almost on a straight line.
The theoretical apparent percentages of hemolysis represented in
the prepared standards in this particular experiment and the corre-
sponding figures as calculated by the equation
per cent hemolysis = - 100 ( 1 - • -77° 1
are given in Table II in columns 1, 3 and 4, respectively, and are seen
to be in better agreement than might, from the nature of the case,
reasonably have been expected.
For many purposes, therefore, with a very fair degree of accuracy,
120
M. H. JACOBS
apparent percentages of hemolysis may simply be calculated from initial
scale readings as if the presence of hemoglobin in the external solution
could be disregarded. For such calculations, a graphic method, which
perhaps requires no explanation here, has been found to save much
time. In cases where higher concentrations of erythrocytes are em-
ployed or where special accuracy is required, however, appropriate
standards for calibration should be prepared.
TABLE II
Relation between Scale Readings and Apparent Percentages of Hemolysis
Apparent percentage
of hemolysis repre-
sented by standard
Scale reading (each
figure is the average
of 5 readings)
Percentage hemolysis
calculated from initial
scale reading
Percentage hemolysis
calculated from average
of CR products
0
8.6
— .
—
20
10.4
17
20
36
13.0
34
36
49
16.6
48
50
59
20.6
58
60
67
25.7
67
68
74
32.2
73
74
79
39.2
78
79
83
48.5
82
83
87
59.6
86
86
The assumption has so far tacitly been made that a given per cent
of hemolysis may be represented by a mixture of unaltered cells and
of completely hemolyzed cells. This is, unfortunately, not strictly true.
In the first place, any solution which is sufficiently dilute to cause os-
motic hemolysis of any of the cells must of necessity cause swelling
of all of the unhemolyzed cells. In the second place, the possibility
must be considered that cells which have not as yet undergone hemolysis
may have, nevertheless, given up some of their hemoglobin to the sur-
rounding solution. Both of these factors might be expected to have
optical effects which would considerably complicate the situation as
so far outlined.
With regard to the first factor, an approximate allowance may read-
ily be made for it by taking as the initial reading for purposes of
calculation, not that for a given suspension in 0.9 per cent NaQ, but
that for a similar suspension in a solution which is decidedly hypotonic
though not quite sufficiently so to cause any hemolysis. This concen-
tration may readily be determined by experiment ; for ox blood it is
usually in the vicinity of M/& NaCl. Figures obtained in this way
by calculation or by calibration with standards made up as before but
with the use of hypotonic instead of isotonic solutions, are undoubtedly
OSMOTIC PROPERTIES OF ERYTHROCYTE 121
more accurate than those secured with the neglect of this precaution.
It is impossible, however, because of the heterogeneous nature of the
material dealt with, to prepare by a simple method of mixtures standards
which reproduce with complete fidelity the conditions in a partly hemo-
lyzed sample of the blood.
Even more troublesome is the second difficulty mentioned above.
If osmotic hemolysis is, as is maintained by Saslow (1929) an "all or
none " phenomenon, then the preparation of standards representing
fairly well a given degree of hemolysis is perhaps possible. If, on the
other hand, as is believed by Baron (1928), this is not the case, but
in a given mixture of hemolyzing cells some have undergone complete
hemolysis (in the sense of becoming completely invisible), while others
have lost lesser amounts of hemoglobin which can be expected to vary
greatly with the conditions of the experiment, then not only is it im-
possible to prepare standards representing accurately different per-
centages of hemolysis, but the term percentage of hemolysis itself ceases
to have any very exact meaning.
Under these circumstances, and until there is more general agree-
ment than there is at present as to whether osmotic hemolysis is or
is not an " all or none " phenomenon, it is perhaps unprofitable to try
to introduce into our methods refinements which may have little real
significance. It seems preferable merely to speak, as has already been
done, of an apparent or an approximate percentage of hemolysis, using
for our estimations some convenient though arbitrary type of standard.
Figures of this sort will have a considerable value, if used with a recog-
nition of their limitations. In any case, regardless of the type of
standard employed, such figures will usually involve an uncertainty
of only a few per cent in the assumed degree of hemolysis.
In the absence of any general agreement at present as to a precise
definition of percentage of hemolysis, the especial value of a method
such as the one here described becomes apparent. The kymograph
tracings obtained with it are exact and unequivocal. There may be
doubt as to whether a certain point on the record indicates 75 per cent
or 78 per cent hemolysis, but the point itself is not in doubt. In most
experiments what is desired is not so much to know how long it re-
quires to reach, for example, exactly 75 per cent hemolysis, assuming
that this expression has any precise meaning, but rather how long it
requires under the chosen conditions to reach a point on the hemolysis
curve which can be represented by some reproducible standard. This
is possible with the present method with a high degree of accuracy.
122 M. H. JACOBS
SUMMARY
1. A simple method is described by which it is possible to measure
with a very satisfactory degree of accuracy the rate of hemolysis where
the time involved exceeds approximately one second. If the duration
of the experiment is ten seconds or more, a complete graphic record
of the entire process up to an apparent degree of hemolysis of between
80 and 90 per cent may be obtained.
2. The method may also be used for the accurate determination of
the relative numbers of erythrocytes in different suspensions and, as-
suming a satisfactory definition for the expression ;' percentage of
hemolysis," for the rapid estimation of the latter, within the range most
useful for experimental purposes, with an error of no more than one
or two per cent.
BIBLIOGRAPHY
ADAIR, G. S., BARCROFT, J., AND BOCK, A. V., 1921. Jour. PhvsioL, 55: 332.
BARON, J., 1928. Pfliigcr's Arch., 220: 242'.
HAMBURGER, H. J., 1886. Archir. f. Anat. u. PhysioL, 476.
HARTRIDGE, H., AND ROUGHTON, F J. W., 1923. Proc. Roy. Soc. (B) 94: 336.
HEDIN, S. G., 1891. Skand. Archil', f. PhysioL, 2: 134.
HOLKER, J., 1921. B'wchcm. Jour., 15: 226.
JACOBS, M. H., 1927. Am. Jour. PhysioL, 81: 488.
JACOBS, M. H., 1928. The Han'cy Lectures, 22: 146.
LILLIE, R. S., 1916. Am. Jour. PhysioL, 40: 249.
McCuTCHEON, M.,AND LUCRE, B., 1926. Jour. Gen. PhvsioL, 9: 697.
PONDER, E., 1923. Proc. Nov. Soc. (B),95: 382.
PONDER, E., 1927. Proc. Roy. Soc. (B), 101: 193.
SASLOW, G., 1929. Quart. Jour. Ex per. PhysioL, 19: 329.
VLES, F., 1921. Arch, dc ph\swl. bioL, 1: 189.
Vol. LVIII, No. 2 APRIL, 1930
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
SOME EFFECTS OF HgCl, ON FERTILIZED AND UNFER-
TILIZED EGGS OF ARBACIA PUNCTULATA
LEIGH HOADLEYi
( From the Zoological Laboratory, Harvard University and the Marine Biological
Laboratory, Woods Hole.)
In connection with a study of the fertilization reaction in Arbacia
punctulata, the writer reported, in a previous number of this journal
(Hoadley, 1923), some observations on the relationship between the
concentration of salts of the heavy metals and frequency of cleavage
in the egg of that form. In the course of these experiments it was
found that mercury had certain effects upon cleavage which appeared
to be indicative, though sufficient information was not available at that
time to warrant any discussion of the phenomena involved. During
the past summer, a study of these effects has been made. The results
of this investigation will be presented below.
In a series of the heavy metals, HgCL stands apart from the other
chlorides tested in respect to its action on cleavage and membrane
elevation. Thus it is found that there is no concentration of the mer-
curic salt which inhibits membrane elevation while permitting cell
division. Quite on the contrary, the presence of the mercuric ion ap-
pears at certain concentrations, to favor membrane elevation, resulting
in much wider membranes than is the case in sea-water alone. Cleav-
age, however, is completely inhibited in solutions of relatively low salt
content. When a series of concentrations is tested, it is found that
the inhibition of cleavage is associated with certain changes in the
egg which are peculiar to mercuric chloride, not being identified with
the action of any of the other salts studied. The definite nature of
these changes is most evident in solutions of HgCU between M 30,000
and M/50,000. These concentrations are from three to six times as
great as those required to suppress cleavage completely in eggs exposed
1 This investigation was aided by grant from the Milton Fund.
123
9
124 LEIGH HOADLEY
to the solution from the time of complete membrane elevation on. I
would like at this point to describe very briefly the general picture
presented by eggs under such conditions.
Eggs of Arbacia, transferred to a solution of HgCL in sea-water
(M/40,000) five minutes after insemination in sea- water, show well-
elevated membranes. The nuclear region is just becoming visible in
the living egg in the form of a bright spot. The pigment is composed
of very small granules which are evenly distributed throughout the
cortical portion. After approximately thirty minutes the entire picture
is changed, being very different from that found in the control eggs.
The pigment is clumped at one side of the egg, where it is bulging
outward as though the egg were cleaving into two parts, a larger one
containing little or no pigment and a smaller containing all of the
pigment which appeared in the cortical portion at the time of transfer.
After 10 minutes in this condition, the pigment again becomes distrib-
uted throughout the entire cytoplasmic portion of the egg which pro-
ceeds to cytolyze. The somewhat clumped pigment loses its red color
and fades materially early in the process. The pseudo-cleavage and
the behavior of the pigment prior to and after the pseudo-cleavage and
the accompanying cytoplasmic phenomena involved are of especial
interest.
EXPERIMENTS
In the present study, as in that cited above, the egg of Arbacia was
used. Solutions of HgCU were made in sea-water from an M/10 stock
solution in distilled water. The sea-water used in the dilutions had
been standing in the room for a considerable period of time to bring
it to the temperature of the air. Because of previous experience, at-
tention was focused on the behavior of fertilized eggs in HgCL, con-
centrations between M/30,000 and M/50,000.
The description of the experimental results will be made according
to increase in the concentration of mercuric salt in the solution em-
ployed. It should be borne in mind that the weakest, M/50,000. is a
concentration which is three times as great as that necessary to com-
pletely suppress cleavage in fertilized eggs placed in it directly after
complete membrane elevation. The experiments were performed in
the following way.
Sperm and eggs were obtained from fresh Arbacia after washing
the individuals and all instruments in running fresh water to kill ad-
hering gametes, and then in sea-water to guard against anv effects of
hypotonicity. The eggs were obtained by straining broken ovaries
through cheese-cloth. They were subsequently washed in three changes
of sea-water to carry off body fluid and small pieces of the gonads.
EFFECT OF HgCls ON EGGS OF ARBACIA
125
The eggs were then examined to be sure none had membranes elevated
and to ascertain the condition of the gametes. No batches of eggs
were used which showed one per cent in germinal vesicle stages. With
due precaution no eggs will be found with membranes elevated as a
result of sperm infection or hypotonicity. The eggs were inseminated
20 minutes (ca.) after the removal of the gametes from the ovary.
A sperm suspension, made by adding one drop of dry sperm to 25 cc.
of sea-water having been prepared, 7 drops of this were added to
50 cc. of sea-water into which the eggs from a single female had
been placed. One control of uninseminated eggs in sea- water, and
one in the HgCl2 solution were reserved for subsequent examination.
Observations were then made to determine the fertility of the eggs.
No batch with less than 98 per cent of the membranes elevated was
employed. The eggs were then allowed to stand in sea-water until 5
minutes after insemination, when the transfer to the HgCl2 was made.
When insemination is carried out as stated above it will be described
in the following way: 7(1 : 25) : 50.
The transfer of eggs to the HgCU solutions and the retransfer to
sea-water were accomplished as follows. Five minutes after insemina-
tion, 5 drops of the inseminated eggs were transferred to 10 cc. of
HgClo in sea-water and the eggs evenly arranged in the dish. This
last precaution is very necessary in order to assure comparable results,
for crowding affects not only the respiratory activities of the eggs, but
also the concentration of the Hg++ ion in the immediate neighborhood,
the initial amount being very small and the eggs containing substances
which, as we shall see, are mercury-avid. The retransfers to sea-water
were made at 3-minute intervals. One drop of eggs from the mercury
solution was placed in 7.5 cc. of sea-water. All dishes were kept
covered to prevent evaporation, and were also kept out of direct sun-
light to prevent much change in temperature. The results obtained
at any one concentration are so nearly the same that but one series
of experiments will be described under each.
The information obtained in the present experiments may be pre-
sented in two ways, either of which would yield comparable data. We
may consider each of the molecular concentrations of the salts, varying
the period of exposure, or we may compare similar periods of ex-
posure, varying the concentration of the solutions employed. Both
methods of procedure have been employed. In general, however, the
first method leads to greater simplicity, and will therefore be used in
the description. A word should be said about the differences in re-
sponse in batches of eggs from different females. This is, of course,
to be expected, and might lie satisfactorily explained could the relative
126 LEIGH HOADLEY
amount of pigment be computed as well as allowance made for differ-
ences in the states of the gametes when used.
M/50,0000 HgCL. — This concentration was made up in sea-water
from a M/10 HgCL solution in distilled water. The temperature of
the solution was 23° C. Eggs and sperm were prepared as described
above and insemination made 7(1: 25): 50. Uninseminated controls
in sea- water showed zero per cent membranes after 15 minutes. In-
seminated controls showed 100 per cent membrane elevation 5 minutes
after insemination. Uninseminated eggs in the HgCl2 showed 0.5 per
cent membrane elevation after 20 minutes' exposure. Five minutes
after the sperm were added to the eggs, 5 drops of eggs were put into
the M/50,000 HgCL solution. This constituted the stock from which
transfers were made to sea-water at 3-minute intervals up to 35 min-
utes. We shall now consider the effect of the solution on the eggs
remaining in it, the changes on the return of the eggs to sea-water,
the rate of cleavage in the eggs after each exposure, and the extent
of their development.
Mercuric chloride of this strength has a very definite effect on the
rate with which nuclear changes occur in the inseminated eggs. It
FIG. 1. Egg in the germinal vesicle stage showing the long processes ex-
pelled when egg is placed in the HgCL solution.2
is not as marked as in more concentrated solutions, but, over an ex-
tended period, e.g., 25 to 30 minutes, it is evident that the whole suc-
cession of nuclear phases is retarded. In this connection it should be
mentioned that the effects are immediately made manifest in eggs in
germinal vesicle stages (see Fig. 1 and note) and in eggs approaching
the period of division. Save for the slight retardation, there is no
- It is true that when Arbacia eggs are placed in sea-water a certain number
of the eggs in the germinal vesicle stage will throw out processes similar in
every way to those pictured here, but it is not true of all of the eggs in this
condition. In the mercuric chloride solution it is the case with all of them.
Whether this is a direct action of the solution on the more superficial regions of
the egg or whether this is due to the indirect action of the salt on the membrane
and subsequently the balance between the physical states of the cytoplasm and
the medium is not clear.
EFFECT OF HgCl* ON EGGS OF ARBACIA
127
evidence of any alteration of the eggs in the toxic solution until from
27 to 29 minutes, when 10 per cent of the eggs showed localization
of pigment (cf. Fig. 2). With the increase in exposure time, the
number of eggs showing the direct effect of the solution mounts thus.
32 minutes — 30 per cent, and 35 minutes — 40 per cent. With the
localization of the pigment, cytolysis may be seen to be taking place
around the clumped granules. The rest of the cortical portion of the
egg remains intact. After long exposure, the intact eggs swell greatly,
the contents being visibly coagulated and quite free of pigment. Frag-
mentation may be observed in some of the eggs in which pigment
FIG. 2. Series showing the progressive effect that a series of exposure times
has upon the localization of the pigment and the accompanying local cytolysis.
In h may be seen the general picture presented by an egg undergoing " pseudo-
cleavage." For explanation see text,
localization takes place. This is much more common in some of the
experiments to be described below. No true cleavage ever is seen in
eggs which remain in the HgCU solution.
The effect of solutions of mercuric salt is not immediately apparent
on the eggs. This is particularly noticeable in cases where the eggs
are returned to sea-water after short exposures. It may be seen in
the behavior of the pigment and subsequently in the rate and percentage
of cleavage, as well as in the viability of the cleaving stages. It will
128 LEIGH HOADLEY
be recalled that in the HgCl2 solution, noticeable clumping of pigment
appears at from 28 to 29 minutes. In the transferred eggs, however,
some 3 per cent show a great deal of pigment localization with pseudo-
cleavage at an age of 45 minutes, even when the transfer to sea-water
has taken place after 18 minutes' exposure. Thirteen minutes later,
these same eggs showed 55 per cent localized pigment. In eggs which
were transferred after 15 minutes' exposure, while no pseudo-cleavage
was found, 48 per cent showed localized pigment with cytolysis after
57 minutes. In eggs which have been exposed to the HgCL solution
for longer periods of time, the percentage showing pseudo-cleavage and
localized pigment increases directly with the length of exposure. This
last involves not only the extent of the action in individual eggs, but
also the number of eggs affected. Generally speaking however, the
extent of the phenomenon in individual eggs is directly comparable
after similar exposure times. This varies slightly with different batches
of eggs employed.
The extent to which pigment clumping takes place indicates a
mechanism responding in a purely quantitative fashion. In Fig. 2
may be seen a series of effects produced in eggs on return to sea-water
after successively longer periods of exposure to M/50,000 HgCl... The
clumping itself is but the initial step in a series of changes which will
be described here. Before the actual accumulation of the pigment
takes place, there is little evidence of migration within the cytoplasm
of the egg. In other words, the pigment which takes part in this
action does not form larger masses which move through the cortex,
but the massing occurs only at the point of final grouping. The result
is that there is no apparent local depletion of pigment, but rather, a
general depletion which takes place evenly over the entire cortex.
After a short exposure this may be similar to that shown in Fig. 2, a,
while after longer exposure it is more extensive, as shown in subse-
quent figures. As might be expected, with the increase in the amount
of the pigment activated, the area of the cortical region involved is
increased. The clumped pigment, however, is evidently destined to ex-
pulsion by the cytoplasm. As it reaches the surface in the particular
area shown, it breaks through the membrane as globules. These im-
mediately swell and undergo changes which are customarily described
in eggs as cytolytic. The area of " cytolysis " is directly referable to
that of pigment localization, the remainder of the cortex remaining
intact. In extreme cases where exposure is long (e.g. 30 minutes),
all of the visible pigment may be removed from the cytoplasm, and
instead of a " local cytolysis " of the area involved, this area may be
budded off as seen in Fig. 2, h. If this budding is extensive it may
EFFECT OF HgCl3 ON EGGS OF ARBACIA 129
result in what I have called a pseudo-cleavage; if it is not as extensive,
the budding may not be complete, the pigment may again enter the
pigment-free portion, and cytolysis follow around each of the pig-
ment masses. The most interesting observations associated with this
action are, that pigment is affected evenly throughout the cortex ; that
it is eventually localized at one region of the cortex ; and that it is
eliminated at this point, the elimination being accompanied by cytolytic
changes. It would be of great interest to determine at just what point
in the cortex the accumulation of the pigment takes place. Is it in
any constant relation to the polar axis of the egg? This is of even
greater importance after a consideration of certain facts concerning the
subsequent cleavage. There is, however, no definite evidence available
on this point. An hypothetical determination might very easily lead
to great error. This will be mentioned again in connection with
cleavage.
Not all of the eggs which show the effects of mercury action fail
to cleave. It has been mentioned above that no cleavage takes place
in HgCL solutions employed, so that we may confine our consideration
to those eggs which have been returned to sea-water. Two sorts of
data, both dependent on cell division, may be obtained from the exper-
iment. The solution reduces the total number of eggs cleaving, and,
in addition, it increases the length of the period between fertilization
and cleavage. In the series already considered above, the percentage
TABLE I
Effect of Ml 50,000 HgCl* on the time of the appearance of the first cleavage in
eggs exposed to the solution for various periods of time. Time in minutes after insemi-
nation.
,, First egg observed in
ExP°sure first cleavage
minutes
0 42 minutes
3 48.5
6 50.
9 53.
12 55.
15 68.
18 71.
21 74. (attempted)
of cleavage drops constantly from 100 per cent after 9 minutes' ex-
posure, to zero per cent after 24 minutes' exposure. There were only
a few eggs which showed any cleavage after 21 minutes' exposure, and
in those cases the retardation was very great, the time not being re-
corded because of the fact that all eggs were thought to be dead. The
length of period required for the cleavage shows much variation, but
the figures to be seen in Table I, for example, represent the time of
130
LEIGH HOADLEY
appearance of the first cleaving egg seen in each group of one batch.
The retardation is marked. The interpretation of conditions within
any one dish of eggs is further complicated by the fact that not all
of the eggs which show the localized cytolysis around the pigment,
cytolyze completely. In many cases in which cytolysis does not become
more extensive than is shown in Fig. 2, a, b, c, and even d, the elimina-
tion of pigment is not fatal to the egg, which subsequently divides.
This recovery, if it may be called such, appears to be more complete,
and sooner complete, the less pigment is involved in the cytolysis.
Thus, in one case, after an 84 per cent localized pigment cytolysis of
the type shown in Fig. 2 b, 94 per cent of the eggs cleaved. Such
behavior suggests the question as to possible relation between point
of cytolysis and plane of cleavage, a point which I shall discuss here
for all of the material considered.
There is a definite relation between the cleavage planes and the
point of pigment accumulation and cytolysis. Whether this indicates
an orientation of this point to polar orientation of the egg, or an effect
of the localization of the pigment clumping on the orientation of the
cleavage spindle, is not indicated in any of the experiments. In either
case, the subsequent cleavages occur as shown in Fig. 3, a, b, and c.
FIG. 3. Sketches to show the relationship between the point of pigment ex-
trusion and the first, second and third cleavage planes. For explanation see text.
The relation of the extruded pigment is plainly visible because it remains
attached to the blastomeres for a considerable length of time. All
evidence obtained seems to point to the probability that this pigment
is of relatively little significance as far as the early development of the
germ is concerned. Cleavages follow as in typical development and
motile larvae are formed. A more complete discussion of this point
will be found below.
The number of motile forms produced after exposure of eggs to
EFFECT OF HgCU ON EGGS OF ARBACIA 131
the mercuric chloride also expresses the toxicity and inhibition already
observed above. Table II is a record of conditions in the cultures 18
hours after insemination. Any exposure up to 9 minutes may be
seen, on reference to the table, to have relatively little effect on future
development. With the advance to 12 minutes' exposure, however,
the number of viable individuals has dropped to 60 per cent, and these
show marked retardation. After 15 minutes' exposure the retardation
TABLE II
Effect of M 150,000 HgCl? on the rate of development of larvae after exposure of the
eggs for various times as indicated. The examination of the culture was made eighteen
hours after insemination of the eggs.
Exposure Motile Extent ,£ntdevelop-
minutes per cent
0 100 Young larvae
3 100
6 100
9 90
12 60 Very late gastruke
15 40 Early gastrulae
18 30 Blastuke
21 1 Early blastulae
24 0
is even greater, as is also the case after 18 minutes. Viability also
decreases. The possible relationship between the retardation and the
action of the pigment will be considered below.
M/45,000 HgCL2. — As in the experiment just described, this con-
centration of HgCL was made up in sea-water from an M/10 stock so-
lution in distilled water. The temperature of the sea-water used in
the dilution was 22° C. Eggs and sperm were prepared with the cus-
tomary precautions. After controls of the uninseminated eggs had been
set aside, the balance of the gametes were placed in 50 cc. of sea-water
to which sperm suspension was added according to the formula
7(1: 25): 50. The eggs were all mature and showed 100 per cent
membrane elevation 5 minutes after the addition of the sperm. The
uninseminated eggs in sea-water showed zero per cent membrane 20
minutes later. No membranes were observed on uninseminated eggs
in HgCL 20 minutes after transfer. Five minutes after insemination,
5 drops of the eggs were added to 10 cc. of the A I 45,000 HgCL solu-
tion and these were evenly distributed on the bottom of the container.
From this lot, samples were returned to sea-water at 3-minute intervals
until 10 lots were available for study. The results of the observations
follow.
The mercuric chloride in this concentration has a far greater in-
132 LEIGH HOADLEY
hibiting effect than in that dealt with in the previous section. This
is noted first on observation of the nuclear changes in the egg. After
about six minutes in the solution, the eggs show the monasters charac-
teristic of the earliest phases and with this the progression of stages
stops. If the eggs are observed at intervals one finds that gradually
changes occur which concern the distribution of the pigment in the
cortical cytoplasm, but these do not involve further changes in the
nucleus. Those eggs in which the pigment does not clump gradually
swell and eventually show a coagulated cytoplasm within a much bloated
membrane. In by far the greater number of the eggs the swelling is
preceded by a pigment clumping which is much more extensive in this
case than in that previously described. After 24 minutes in the solu-
tion, about eight per cent of the eggs show a localization of the pigment
which is not always confined to one spot as was the case in the weaker
solution, but may be located at two or three regions, immediately below
FIG. 4. Sketches to show more extensive action of the solution on the eggs.
In a and c may be seen various degrees of multiple pigment foci. In b is an
egg with purely cytoplasmic cleavage, a process resembling fragmentation. In
d is a "polar " view of an egg to show the general picture presented by the
clumped pigment. For explanation see text.
the egg membrane (Fig. 4, a). This action is associated in a very small
number of the eggs with a pseudo-cleavage of the type described above,
or with a constriction of the pigment- free portion of the egg such as
may be seen in Fig. 4. b. The extent of the occurrence of such forms
increases with the length of the exposure as does also the frequency
of the eggs with multiple pigment centers, so that after some 40 min-
utes, when all of the eggs are swelling and none of them appear at
all normal, some eggs may be found with as many as six of these
EFFECT OF HgCls ON EGGS OF ARBACIA 133
centers arranged as in Fig. 4, c. These forms are always much rarer
than those of the pseudo-cleavage type.
It is not as easy to follow the course of the pigment clumping in
eggs in this concentration as it was in the previous one. This is true
for two reasons. A difference of 3 minutes in the exposure time in
the higher concentration is equal in its effects to that of a 12-minute
interval in the lower concentration. Moreover, the pigment changes
occur very rapidly after the eggs are returned to the sea-water, and
the duration of the intermediate stages is shortened. Hence their de-
termination is rendered more difficult. Associated with this more rapid
reaction within the egg after its return to sea-water, we find an increase
in the number of eggs in which the clumping of the pigment is com-
plete. Thus there is a rise to 20 per cent of pseudo-cleavage in eggs
that have been exposed to the mercuric solution for 24 minutes. This
complete isolation of the pigment appears for the first time in eggs
which have had a 15-minute exposure to the solution. Some effect
on the pigment is found even in the 6-minute eggs. It is not extensive,
resembling in most of its details the picture presented by the eggs in
Fig. 2, a. Of these eggs, 40 per cent were so affected while 98 per cent
of them cleaved. In the 9-minute eggs the condition is much more
advanced. Pigment clumping is accompanied by local cytolysis as in
the previous cases. The situation is very much more general here,
being found in 96 per cent of the eggs. There is a 100 per cent local
cytolysis in the 12-minute eggs which is far more extensive in its
nature than anything described in the discussion of the first series.
A rather typical egg ma}' be seen in Fig. 4, d. Further exposure tends
to produce even more extensive clumping of the pigment, with pseudo-
cleavage, and with the multiple pigment centers. These last are found
in eggs which appear for the most part to have a coagulated cytoplasm.
It looks very much as though pigment localization having been initiated,
coagulation had set in so that migration was stopped, leading to pig-
ment clumping around a number of centers. Local cytolysis occurs
around each of the pigment masses and swelling follows. None of
these eggs ever show any further developmental changes.
The effect of the HgCl2 on cleavage is much more extensive in
these concentrations than in the weaker ones. Immediately following
the retransfer of the eggs to sea-water, there is a rapid change in the
nuclear components, so that bar and streak stages are formed. This
takes place only in those eggs which have been in the solution for 15
minutes or less, though an occasional egg is found which will proceed
after 18 minutes in the toxic solution. There is a progressive retarda-
tion as the length of exposure increases. Thus, the 3-minute group
134 LEIGH HOADLEY
cleaved 9 minutes after the controls, while the 15-minute group was
retarded 75 minutes. In the IS-minute group, only an occasional egg
cleaved, and then 135 minutes after division in the controls. The effect
may most readily be seen in the percentage of cleaving eggs. This
drops from 98 per cent after 3 and 6 minutes' exposure, to 90 per
cent after 9 minutes', 72 per cent after 12 minutes', 18 per cent after
15 minutes', and less than one per cent after 18 minutes'. The per-
centage of cleavage is, therefore, inversely proportional to the length
of the exposure to the mercuric solution. Inasmuch as there is a 40
per cent local cytolysis in the 6-minute eggs, 96 per cent in the 9-minute
eggs, and 100 per cent in the 12-minute eggs, it is evident that many
of those which have shown local cytolysis must cleave. This has been
observed here as in the cases described above. The attached extruded
cytolysing pigment can be traced through at least the 16 and 32-cell
stages, as was the case above (cf. Fig. 3). Additional information on
viability is available for these eggs, however, for observations were
made periodically up to the time of pluteus formation.
Motile larvae were produced by cultures of eggs removed from the
HgCU solution after 3, 6, 9, and 12 minutes' exposure. Of these, only
the first three continued in their development to the formation of
plutei. Forty-eight hours after insemination in the first two cultures,
i.e., in those of 3 and 6 minutes' exposure to the solution, the plutei
produced appeared typical in every way. In the 9-minute culture, on
the other hand, the few plutei formed were much retarded. The arms
were short and the body heavy. A relatively small number of the
larvae continued their development to this point. It will be recalled in
this connection that before cleavage, 96 per cent of these eggs showed
localized pigment with an associated cytolysis which was quite extensive.
In the 6-minute group, the localized cytolysis and pigment clumping had
affected 40 per cent of the eggs. While a definite statement cannot
be made about the 9-minute culture, it was perfectly evident that over
60 per cent of the 6-minute eggs had formed plutei. The experiment
shows that typical plutei can be formed from the eggs which have
undergone the pigment localization and localized cytolysis produced by
short exposure to M/45,000 HgCl2 solutions.
M/40,000 HgCl.2. — The temperature of this concentration, which
was made up in sea-water from the M/10 stock solution of the salt
was 21.3° C. Eggs and sperm were prepared as before and insemina-
tion carried out according to the same formula, 7(1: 25): 50. The
usual controls of uninseminated eggs were set aside. That in sea-water
showed no membranes elevated after a period of 30 minutes. In HgCU
solution, examination of the material showed 8 eggs with wide mem-
EFFECT OF HgCL ON EGGS OF ARBACIA 135
branes (less than one per cent) after 15 minutes' exposure. Insem-
inated controls showed 100 per cent membrane elevation after 5 min-
utes and 100 per cent cleavage at 45 minutes. Five minutes after in-
semination, 10 drops of eggs were added to 10 cc. of the HgCl., solu-
tion. These were returned to sea-water at 3-minute intervals until
10 transfers had been made. The results of the experiment are similar
in many ways to those already described above, save that the modifica-
tions in the eggs appear earlier. After longer exposures, there are
some differences which will be discussed below.
The transfer of the eggs to the HgCL solution is immediately fol-
lowed by an increase in the width of the space formed by the elevation
of the membrane. Results of a similar nature were obtained in previous
experiments already referred to in the introduction. The increase is
much greater than any noted at weaker concentrations. In the mean-
time, the nuclear elements appear as monasters. This is as far as the
nuclear changes go in the solution, no bar or streak being formed.
The eggs remain in this condition until the total exposure amounts
to approximately 24 minutes, when the pigment begins to show the
clumped effect typical of longer exposures in less concentrated solu-
tions. Subsequently the eggs appear to be damaged rapidly, swelling
and coagulation being evident in a large percentage. The most marked
effects appear in the eggs which have been transferred to sea- water
after exposure to the HgCL.
There is little evidence of any modification in the development of
eggs exposed to the solution for three minutes save in a slight increase
in the length of time elapsing between insemination and the first cleav-
age. This does not markedly reduce the viability of the individuals
over a long period of time, however, for most of them continue in
their development and form subsequent stages at approximately the
same rate as the controls. After 6 minutes' exposure, the results are
essentially the same save that but 90 per cent of the eggs form blastulse,
a reduction of 6 per cent of the total number cleaving. The viability
is good. Nine minute eggs show a marked effect which is evident
first as the localized cytolysis described above, and later in the reduc-
tion of the percentage of cleaving eggs. Subsequently, further evidence
of the solution's action is seen in the retardation of development and
in the lowered viability of eggs in later cleavage stages. In many, de-
velopment stops after the first, second, or third cleavage. The per-
centage of dead individuals in the cultures is continually increasing.
As a result, relatively few reach even the earl}- gastrula stage. These
few show a marked retardation when compared with the control embryos
or with those of cultures of 3 and 6 minutes' exposure.
136
LEIGH HOADLEY
The condition just described is much further developed in the 12-
minute eggs. At least 10 per cent show a local cytolysis which involves
a greater portion of the cortical region than is concerned in the 9-minute
eggs. Cleavage is retarded far more and, when some of the eggs finally
do cleave, the total number is but 20 per cent, whereas it was 96 per
cent after 9 minutes' exposure. The lower percentage of the cleaving
eggs is apparently related to an increase in the amount of the pigment
clumping, which here shows the form pictured in Fig. 4, a, c, and d.
Several eggs had cleaved in the cytoplasmic portion (cf. Fig. 4. b), a
phenomenon much more frequently encountered in cultures of eggs
exposed for longer intervals. It will be described in more detail below.
In addition, some of the eggs show the isolation of the pigment as in
Fig. 2. Examination of the culture after 24 hours showed but an
occasional embryo still living, and in those development had been
very atypical.
The analysis of material removed from the toxic solution after
longer exposures is greatly complicated by the appearance of more of
the type of egg shown in Fig. 4, b than were found in the 12-minute
FIG. 5. Drawings to show the relation between the completely isolated pig-
ment and the cytoplasmic fragmentation or cleavages which are found after ex-
posures to more concentrated solutions of the salt. The cleavage in such cases
does not involve the nucleus. For explanation see text.
group. In addition, eggs are frequent in which the pigment-free
cytoplasmic portion is divided into even more units. Some of these
are shown in Fig. 5, a, b. c, and d. It is always accompanied by the
EFFECT OF HgCL ON EGGS OF ARBACIA
137
accumulation of all of the pigment in one region of the egg. In the
initial stages it looks much like a typical cleavage but, inasmuch as it
is followed by coagulation and swelling and nuclear changes are not
visible, it is evidently not a true cleavage but rather a purely cyto-
plasmic phenomenon. This conclusion is confirmed by the appearance
of the eggs after fixation. The number of the eggs undergoing typical
division falls abruptly. This may be seen in Table III. Many readings
TABLE III
Effect of M/-fO,000 HgClz on rate and percentage of cleavage of eggs after various
periods of exposure. Readings taken 227 minutes after insemination. Where the ex-
posure is more than six minutes the viability of the eggs is poor.
Exposure
minutes
0. . .
3. . .
6. . .
9.. .
12. . .
15. . .
18.. .
21.
Cleaved
per cent
. 100
. 98
. 96
. 94
. 20
4 (ca.)
0
0
Extent of develop-
ment
64-celI
64-cell
90% 64-cell
32-cell (many dead )
8-cell (cyt.)
Complicated by cleavages
as in
Fig. 4, b.
of the eggs have to be made to separate those with the cytoplasmic
division from those showing true cleavage following rather extensive
pigment localization. Approximately 50 per cent of the eggs show the
cytoplasmic division. The majority of the remainder show the type of
pigment localization pictured in Fig. 4, c, though there may be many
more of the isolated pigment masses than are shown there. Tn the 10
per cent (ca.) of the eggs which eventually undergo a true cleavage
the viability is very poor. As a result, less than one per cent go as
far as the 32-cell stage and these die shortly afterward. Death is ac-
companied by swelling and evident coagulation of the cytoplasm.
M/35,000 HgCL. — Experiments with this concentration of the
HgCL, when compared with those of less salt content, serve for the
most part to demonstrate the increased toxicity of the solution on
eggs after shorter periods of exposure. The solution was prepared
in the same way as those above. Controls were set aside for future
examination. Eggs were obtained from one female and sperm from
one male. The insemination was made according to the formula.
5 ( 1 : 25 ) : 50. The uninseminated control in sea-water showed no
membrane elevation after 20 minutes while the uninseminated control
in the HgCL solution showed a little less than one per cent after but
7 minutes' exposure, a percentage which increased with the length of
138 LEIGH HOADLEY
exposure. The inseminated eggs showed 100 per cent membrane ele-
vation at the end of 5 minutes, when transfer to the toxic solution
took place. In the mercuric solution there was a noticeable widening
of the cortical space between the fertilization membrane and the egg.
The nucleus forms the monaster, but no further nuclear changes take
place. As in previous cases, transfer to sea- water followed at 3-minute
intervals. For the most part the results obtained in the experiment
are directly comparable to those already described above, save that the
action of the salt is evident after shorter exposure times. The greatest
difference is seen in the occurrence of eggs with cytoplasmic division
after as short an exposure as 18 minutes, and the relatively large num-
ber of eggs with multiple pigment loci. In this concentration, the effect
is quite evidently one of cytoplasmic coagulation. The description of
the first four groups of eggs, i.e., those exposed for 3, 6, 9, and 12
minutes respectively deserves especial mention.
There is no localization of pigment in any of the eggs which have
been in the HgCL solution for but 3 minutes. One hundred per cent
of the eggs cleave regularly, though in this experiment the cleavage
takes place after a period of 53 minutes rather than 42 minutes after
insemination, as in the controls. No other effects of the exposure ap-
pear up to the time of larva formation. The cleaving eggs go through
the early developmental stages but slightly retarded, and form motile
larvse similar to the controls. This is not true of the eggs which have
been exposed for 6 minutes. This group shows a marked localization
of the pigment followed by extensive local cytolysis, (94 per cent of
the eggs at 60 minutes). Quite a large amount of the cortical region
of the egg is involved. The condition resembles that shown in Fig.
2. In spite of this 98 per cent of the eggs cleaved, though the cleavage
did not start in any of the eggs for 63 minutes, 10 minutes later than
division in the 3-minute culture, and 21 minutes later than the controls.
The deleterious effects of the exposure are evident in each of the fol-
lowing exposures. Three and one-fourth hours after insemination,
when the eggs of the control and the 3-minute group are in 16 and 32-cell
stages, this culture shows not only 16-cell stages but also 2, 4, and 8-cell
stages. Fifty per cent of the eggs are alive, while the rest have cleaved
once, twice, or three times, where the development stopped. None
of the eggs develop motile larvse. It is quite evident, therefore, that
the viability of the eggs has been markedly decreased by this short
exposure to the action of the mercuric salt.
Quite in contrast to the rest of the experiments described, some
few of the eggs which were in the 9-minute group show a pseudo-
cleavage of the type pictured in Fig. 2. In no other case is this evident
EFFECT OF HgCla ON EGGS OF ARBACIA 139
after such short exposure. Localization of pigment takes place in
all of the eggs after 60 minutes and in the majority is to be found in
numerous foci (vid. supr. : multiple localization). The few eggs not
showing either of these phenomena retained, theoretically at least, the
possibility of dividing. No cleavage was observed in these 3 hours
after insemination. Fifteen minutes later approximately 2 per cent
of the eggs had divided. The viability of these few was so diminished,
however, that none of them went beyond the 4- cell stage. The poison-
ing is complete in the 12-minute eggs, where no division takes place.
This is probably intimately associated with the increase in the per-
centage of pseudo-cleavages and the marked extensive local cytolysis
which takes place immediately upon the return of the eggs to the sea-
water. All of the eggs eventually swell and cytolyze, though there is
a marked differential effect in different eggs.
M/ 30, 000 HgCl.,. — This experiment was set up in the same way
as those described above. Uninseminated controls showed no mem-
branes after 20 minutes when in sea-water, and over one per cent
wide membranes when in HgCl2 solution. One hundred per cent mem-
branes were elevated on insemination and this was followed in sea-water
by cleavage at 46 minutes. The temperature of the solutions at the
initiation of the experiment was 22° C. There are a number of ways
in which the eggs here differ from those described in former experi-
ments, some of which involve merely an intensification of a previously
noted action of the salt, while others introduce new phenomena. The
first indication of the effect of the solution appears as a clumping of
the eggs. This has not been noted in any of the previous experiments.
There is evidently a modification of the quality of the surface membrane
of such a nature that the eggs adhere to each other. Some modification
or solution of the jelly layer investing the egg must be involved, for
control eggs showed the jelly layer. This is followed by a pseudo-
cleavage in the mercuric solution after 12 minutes' exposure. Such
eggs cytolyze at the point of pigment accumulation after 3 to 6 minutes.
After 30 minutes 80 per cent of the eggs show a cytolysis of the cortical
region associated with an accumulation of pigment. The eggs trans-
fered to sea-water show much the same sort of behavior. Even in eggs
which are transferred after 3 minutes' exposure there is 100 per cent
local cytolysis 63 minutes after insemination. Much of the cytolysis
is very extensive. The pigment accumulation in the region of the
cytolysis is a little greater than that described above for 6-minute eggs.
Eggs which have been exposed to the mercuric solution for longer
periods of time show further effects of the action of the salt. In many,
coagulation appears very early, so that further cytoplasmic changes and
10
140 LEIGH HOADLEY
pigment migration stop, the eggs merely swelling and cytolyzing. Some
of the most interesting modifications take place in the nninseminated
eggs which were placed in the HgCU solution. The pigment is clumped,
but in a way visibly different from that in inseminated eggs. In ad-
dition, there is a cytoplasmic cleavage or fragmentation which is partial,
involving one side of the egg only. This appears in but a very small
percentage of the eggs. It may be associated with those in which
membrane elevation has been produced by the solution, though no direct
evidence on this point is available. No nuclear changes are involved.
In previous experiments nuclear changes proceed after inseminated
eggs which have been in HgCl2 solutions are returned to sea-water.
It will 1)e recalled that in the more concentrated solutions, the nucleus
remains in the monaster stage during the time that the egg is in the
toxic solution, further progress appearing only after retransfer. The
nuclear changes in the solution of M/30,000 concentration proceed very
slowly to the monaster stage, even this change occurring only in a small
percentage of the eggs which are not greatly effected, after 18 minutes
in the solution. When these are returned to sea- water, the changes
proceed very slowly. Only those eggs which have been exposed for
but 3 minutes show any advance over the monaster. These eggs are
greatly retarded, showing only an early bar 27 minutes after insemina-
tion. The evidence of extensive injury is marked, therefore, not only
in the behavior of the cytoplasm and its inclusions, but also in the
nucleus. The depression of the viability of the eggs is so great in this
experiment that none of the eggs divided, even when the exposure is
as short as 3 minutes. This may be associated with the relation be-
tween nuclear and cytoplasmic phenomena.
DISCUSSION
From the results obtained and presented above, we find that the
mercuric chloride solution produces several specific changes in the egg,
all of which vary directly with the concentration of the solution and
the length of the exposure. A number of these phenomena deserve
special consideration in the discussion. The " activating " influence of
the HgCl, at certain concentrations will concern us first.
It has previously been reported (Lillie, 1921; Hoadley, 1923) that
in relatively high concentrations of mercuric chloride, not only is the
elevation of membranes wider on inseminated eggs than on inseminated
sea-water controls, but uninseminated controls show a certain per-
centage of eggs with membranes. The percentage of uninseminated
eggs elevating membranes on exposure to the solution varies directly
with the concentration of the salt. The production of the membranes
EFFECT OF HgCl, ON EGGS OF ARBACIA 141
is not immediate. They appear on certain eggs after as short an ex-
posure as five minutes, in others the membranes do not elevate for a
much greater period of time. There is no possibility of either hvpotonic
or hypertonic activation in any of the experiments cited. Although the
subsequent development of eggs showing membranes elevated after ex-
posure to mercuric chloride solutions has not been investigated, this
must, I think, be interpreted as an initiation of development in the
gametes. The evidence for this statement is derived entirely from
close observation of the pigment behavior and the subsequent cytolysis
of such eggs as compared first with that of membraneless eggs, and
second with that of eggs with fertilization membranes elevated before
exposure, both lots being left in the mercuric chloride solution during
the observations. The eggs with membranes elevated after exposure
to the HgCL resemble the inseminated eggs in every way. There must
be some alteration of the cortical membrane of the egg induced by the
solution which is similar to that produced at fertilization, and which
results in the elevation of a membrane and, subsequently, the like pen-
etration of the mercuric ion. As a result, the reaction to the HgCL
is that of an inseminated rather than of an uninseminated egg.
As would be expected in the case of an extremely toxic solution,
the effect is next evident in the cortical cytoplasm of the egg. The
pigment granules are visibly affected first and most extensively. This in
turn appears to be due to a specific mercury-avid property of the
pigment itself. The degree to which the total amount of pigment in
the egg is involved in this action is apparently of a quantitative nature
as is evidenced by the experiments. Similar effects are produced by
short exposure to concentrated solutions and longer exposures to less
concentrated solutions. An indication of the extent of the action may
be obtained by examining the behavior of the pigment subsequently
accumulating at the point of the egg at which extrusion and local
cytolysis occur. The very fact that after the extrusion of the pigment
which is localized, as aforesaid, cleavage may take place and larvae
may be formed indicates that the cytoplasm as a whole is not greatly
affected by short exposure to the mercury. Longer exposure has a
more extensive effect, so that coagulation appears and the eggs are
much damaged, further development not taking place. If, as would
seem legitimate, one may regard the pigment as a mercury-avid sub-
stance, a conceivable mechanism is available by which the mercuric ion
which has entered the egg may be bound and removed. Hence the egg
is enabled to continue its development. The clumping of the pigment
is very evidently not primarily a local response to the presence of the
mercuric salt. Rather, as is evident in many of the experiments, the
142 LEIGH HOADLEY
pigment is equally affected throughout the entire cortical region of the
cytoplasm. At a later time, the pigment becomes clumped or localized
at some single point near the surface of the egg. Where the pigment
clumps in a single mass, no coagulation of the egg cytoplasm appears,
as is the case on exposure for longer periods of time or to more con-
centrated solutions. Where coagulation takes place before the migra-
tion of the pigment is complete, there are a number of small foci, iso-
lated one from the other. Apparently the migration of the pigment has
been arrested by the coagulation of the surrounding cytoplasm. It is
also interesting to note that in the majority of cases where coagulation
does not occur, cleavage may follow the expulsion of the single pigment
focus. There is no evidence of any correlation between the site of
pigment accumulation and the .original polarity of the egg in Arbacia.
It is possible that an examination of the behavior of other forms may
yield valuable information concerning this point.
The elimination of the pigment in the eggs and their continued
development is of interest from yet another standpoint. Pigment in
eggs has been regarded as of developmental significance by many
workers. By some it has been thought to be of importance because of
its association with oxidative processes within the germ. Warburg
(1914), in experiments with Strongylocentrotus liridus, found that
oxidations in the egg were associated with the granular portion. We
might expect that if oxidative processes are intimately associated with
the granular portion of the egg, and hence, in part at least, with the
pigment granules, loss of the pigment granules would have a definite
effect on the future development of the egg. It has already been
shown by a number of workers that oxidative processes and develop-
mental rate over short periods of time are discrete in their action.
This is emphasized by Whitaker (1929) in a report of observations
on the relative rate of development in pigmented and unpigmented frag-
ments of Arbacia eggs. In the experiments reported above there is
an evident retardation in the developmental rate and also a marked re-
duction of the viability of the gametes. In view of Whitaker's results,
however, we must look in another direction for the interpretation of the
facts.
The retardation in developmental rate is evident in all cases in
which eggs are exposed to mercuric chloride solutions. The extent
of both the slowing down of the cleavage rate and the loss of viability
is directly dependent on the strength of the solution and the length of
the exposure. Apparently it is more extensive after pigment has been
extruded than when it has not, but this is always attendant on a longer
exposure period or a greater concentration of the solution. There is a
EFFECT OF HgCl2 ON EGGS OF ARBACIA 143
perceptible lag of the effect on the viability over the effect on the rate
of development. They show a correlation in that they both increase
with an increase in the effective period of the exposure. Eventually
the interpretation of the two is confused by the fact that only one or
two cleavages take place, the individuals then dying. This is especially
marked after even short exposures to high concentrations of the salt.
The amount of the pigment extruded in such cases is not very much
greater than that at lesser concentrations, and besides, we are dealing
with the early stages in development when oxidation rate and develop-
mental rate are quite independent. For that reason it appears that
the mercuric ion, both in retarding development and in lowering the
viability, acts directly on the cytoplasm of the egg rather than indirectly,
through the medium of the pigment granules.
Two types of cytolysis are associated with the action of the mercury
on the egg. In all cases where the eggs are allowed to remain in the
solution, they eventually coagulate and swell, no cleavage taking place.
The picture in the eggs which are retransf erred to sea- water is quite
different. There, the pigment accumulates and the cytolysis which fol-
lows involves only the region of pigment clumping and extrusion. The
rest of the cytoplasmic portion of the egg is little affected, as has been
mentioned above, so that cleavage and larva- formation follow. The
phenomena involved in the local cytolysis associated with the pigment
elimination lead to the conclusion that the product of the mercury bound
pigment is responsible for this action. The mechanism involved is
not clear.
The results of the investigation may be briefly summarized as
follows :
1. Mercury has an effect upon the egg of the sea-urchin Arbacia
which is unlike that found in the case of any of the other metallic
chlorides investigated.
2. Acting first on the cortical region it activates membrane elevation.
3. After longer exposures it has a direct effect upon the pigment
which has mercury-avid properties. The pigment reacts to the mercuric
solution by accumulation and subsequent extrusion at a localized point
(or points) on the surface of the egg.
4. The extrusion of the pigment is accompanied by a cytolysis of
the pigment granules and the associated cytoplasm.
5. The development of the zygote is retarded and its viability is
lowered bv the action of the mercuric solution.
144 LEIGH HOADLEY
PAPERS CITED
HOADLEY, LEIGH, 1923. Certain Effects of the Salts of the Heavy Metals on
the Fertilization Reaction of Arbacia punctulata. Biol. Bull., 44: 255.
LILLIE, F. R., 1921. Studies of Fertilization, X. The Effects of Copper Salts
on the Fertilization Reaction in Arbacia and a Comparison of Mercury
Effects. Biol. Bull., 41: 125.
WARBURG, OTTO, 1914. Zellstructur und Oxydationsgeschwindigkeit nach Ver-
suchen am Seeigelei. P finger's Arch., 158: 189.
WHITAKER, D. M., 1929. Cleavage rates in Fragments of Centrifuged Arbacia
Eggs. Biol. Bull., 57: 159.
THE CLEAVAGE OF POLAR AND ANTIPOLAR HALVES OF
THE EGG OF CH/ETOPTERUS
DOUGLAS WHITAKER AND T. H. MORGAN
(From the Marine Biological Laboratory, Woods Hole and the William G. Kcrck-
hoff Laboratories of the Biological Sciences, California
Institute of Technology.}
The following experiment was carried out as part of a program
to study the phenomenon of yolk-lobe formation that occurs in the eggs
of certain annelids and molluscs. This peculiar phenomenon simulates
cell-division to a striking degree, yet at the final moment, when the
yolk-lobe is attached only by its stalk to one of the, cells, a reversed
reaction takes place and the material of the lobe is absorbed by the
egg, or by the blastomere to which it is attached.
There were two relations that we wished to examine by cutting the
egg in two : First, to find out whether the polar lobe develops on both
fragments or only on one of them; second, whether the unequal first
cleavage is dependent on or the result of the presence of an antipolar
yolk field, or independent of its presence. In addition there were one
or two other questions that we hoped to clear up : e.g., whether there
is any relation between the appearance of the pear-shaped form that
the undivided egg assumes just prior to the appearance of the yolk-
lobe and the mitotic phenomenon ; whether the surface of the egg in
the region of the constriction that produces the yolk-lobe changes before
or during the formation of the lobe; and whether the condition of the
mitotic figure at the moment of the lobe's formation bears any causal
relation to the formation of the lobe. The experiments were made in
the summer of 1928 at Woods Hole.
Eggs taken from parapodia were put into sea water. The germinal
vesicle breaks down in a few minutes, and a spindle, pointing to the
pole, is formed. The polar region can be made out owing to the clear
region around the spindle. The egg remains in this condition until
fertilized. Just before cleavage the egg becomes pear-shaped with the
apex at the pole. A little later the egg begins to bulge at the antipole
to form the yolk-lobe that does not constrict off from this egg as
markedly as it does in some other eggs, such as Ilyaiiassa and Deiitaliiun.
Sections of the preserved egg during these periods show that the pear-
shaped stage first appears when the two pronuclei have come together
and the walls are disappearing. The two asters of the future division-
US
146 DOUGLAS WHITAKER AND T. H. MORGAN
figure are present and well developed. Just what internal condition
leads to the change in shape is not shown from these relations. One
might surmise, however, that the collapse of the very large pronuclei
with the resulting distribution of their fluid contents, or possibly the
nearer approach of the two astral fields resulting from the collapse of
the nuclei, may be connected with the change in shape of the whole egg.
Almost immediately the egg becomes rounded again and begins
to elongate in a direction at right angles to the plane of the oncoming
first division. At this time a bulging around the antipole indicates
the development there of the antipolar lobe. As the cleavage proceeds
the lobe becomes more conspicuous, and later becomes constricted at
the point of contact with the egg. The constriction becomes deeper,
giving the lobe an oblong or even rounded appearance, but in Chcetop-
terus the constriction is never carried as far as in the eggs of some
molluscs. At this time the cleavage furrow is progressing, but, from
the first, does not give so much the appearance of cutting through the
lobe as in Dentalium. As the furrow deepens it passes to one side of
the lobe. The point of attachment of the lobe remains on the larger
or CD-blastomere.
Sections of eggs that have been preserved in picro-formalin, or
Flemming's osmic acetic, stained in iron hsematoxylin and counter-
stained in erythrocene, do not reveal any unusual changes in the anti-
polar region during this period. Some of the yolk (and its surrounding
protoplasm) simply protrudes into the lobe. This yolk is a part of
the cup-shaped mass that lies over the lower hemisphere with the edges
of the cup extending toward the polar field. The yolk that goes into
the lobe is not discontinuous with the rest of the material. One gets
the impression that it is squeezed into the protrusion or bulges into it
as the lobe develops. As the base gradually constricts there is, in
sections, visible in the superficial layers nothing that is peculiar or
different from the rest of the neighboring surface. The 'impression
that one gets from sections is that the rounding up of the materials
that become the two blastomeres does not include the antipolar field,
and that the lobe is a by-product, so to speak, of these changes, and
not in itself actively engaged in the process of its formation.
This interpretation may appear at first sight to be in contradiction
to the observation that Wilson has made of the behavior of the yolk-
lobe of Dentalium when severed from the CD-blastomere. He found
that the isolated lobe showed alternate periods of rest and activity that
were synchronous with those of the next two divisions of the egg
when the yolk-lobe reappeared. In some cases he observed indications
that the yolk-lobe itself formed a lobe. These observations may not
CLEAVAGE OF EGG OF CH/ETOPTERUS 147
appear to harmonize with the supposition offered above, that the lobe,
as such, is a passive factor in the result, and its development the re-
sultant of the mitotic constrictions about the division centres ; but if, as
is not impossible, a rhythmical impetus, or something of the sort, is
initiated in the cytoplasm, it might conceivably be supposed to affect
even the isolated lobe, or even involve the formation of a cytoplasmic
aster in the lobe. Until observations are made on the interior of the
isolated lobe at the time of its activities we can only speculate as to
the causes of this remarkable phenomenon.
The operations on the egg were made before fertilization. The
outer membrane is very tough, even before fertilization, so that it is
difficult to sever it completely without destroying the egg within. This
difficulty is increased by the tendency of the Chcctopterus egg to burst,
and care is necessary in cutting in order to avoid great injury. It can
be done, however, with a 'quartz needle and the micro-dissection in-
strument. The egg is least injured if slowly pinched apart, and if the
membrane is gently twisted by rolling the needle back and forth. Dam-
age to the fragments is further reduced if the outer membrane is not
entirely severed after the egg is well cut apart. The results obtained
from a few eggs with membranes entirely severed were the same as
those obtained from the eggs whose fragments remained in the same
membrane. It does not seem likely that the membrane connection be-
tween the fragments has any effect. Independent fertilization of both
fragments takes place. All of the operations here described were made
either in the equatorial plane or parallel to it.
The polar fragment becomes pear-shaped at about the time after
fertilization when the egg passes through this stage. It then elongates
for the first cleavage, but a yolk-lobe does not appear at the antipolar
surface of the fragment. It is true that sometimes a bulging, or other
irregularity, appears in the region where the cut was made, especially
in the fragments most damaged in cutting, but it does not have the
distinctive shape of the normal lobe. It seems rather to be due to some
weakness at the cut surface, and as a result, perhaps, of changes of
tension within the egg or at the surface.
The first cleavage of the polar fragment is into unequal parts, which,
in general, have the same relative size as have the first two cells of the
normal egg. Since the yolk region has been removed, and still the
unequal cleavage appears in the fragment, the inequality cannot be
explained in the normal egg as due to the presence of yolk or yolk-lobe
material in the antipolar hemisphere.
The antipolar half of the egg, which contains only a sperm-nucleus,
does not assume a pear-shape prior to the first cleavage, but at the
148 DOUGLAS WHITAKER AND T. H. MORGAN
time when the cleavage is about to begin a typical antipolar lobe ap-
pears. The size of the lobe is approximately proportionate to the size
of the fragment. If at the time of the operation all of the material
that normally goes into the antipolar lobe is already present in the
antipolar hemisphere, it would appear that the formation of the antipolar
lobe is not simply the extrusion of a given amount of inert material
around the antipole, but is correlated with the size of the dividing ma-
terials and possibly with the size of the astral spheres.
The first cleavage is into unequal cells. The antipolar lobe comes
to lie in the larger cell into which it is later absorbed. Whether this
difference in size of the first two cells is in the same ratio as in the
polar fragment cannot be stated positively, but there is certainly no
striking difference in the two cases. Here, again, it may be pointed
out that the unequal cleavage is not dependent on the amount of yolk
in the dividing cell. Were it so, the smaller cell might be expected to
be disproportionately smaller in the basal fragment.
These general statements may be supplemented by the following
numerical data. In 19 cases both halves developed and conformed to
the above description. In 3 cases the polar half conformed; the anti-
polar was somewhat abnormal. In 3 cases both approximately con-
formed but were somewhat abnormal. In 5 cases the polar conformed,
the anti-polar did not develop ; in one case neither developed. In ad-
dition there were two cases in which the antipolar lobe was not seen
on the basal half, and in one case the antipolar half formed a lobe but
did not divide; the polar half failed to develop.
Mead ('98) described the early cleavage stage of the egg of Chcetop-
terus with numerous drawings of the mitotic figure during cleavage. At
the time when the yolk-lobe appears, the " rays " from the astrosphere
of the CD-cell are represented as extending into the polar lobe. The
antipolar cleavage plane is represented as lying at the crossing-point
of the rays of the two blastomeres to one side of the lobe. From the
figures, which appear partly schematized, it is not certain whether these
rays are at their outer ends anything more than lines resulting from the
arrangement of the peripheral yolk granules.
Lillie ('06) has also described the early cleavage stages of the
Ch&topterus egg. He found that after centrifuging the yolk is driven
away from the antipolar field in some of the eggs that have fallen in
the centrifuge with the polar hemisphere turned outward. When these
eggs cleave the yolk-lobe may contain the oil field, or the clear middle
zone, proving that the yolk as such is not essential to the formation
of the polar lobe. The more superficial layers of the egg are little,
if at all, distributed by the centrifuging, and what is here more to
CLEAVAGE OF EGG OF CH^TOPTERUS
the point, the mitotic figure occupies the normal position in such eggs
with respect to the pole and to the antipolar field.
Wilson ('29) has recently described the cleavage and development
of egg-fragments of the Chcctoptcrus obtained by centrifuging. The
eggs, for the most part, seem to fall at random in the centrifuge tube,
and are stratified without regard to the polar axis — at least, all possible
relations may be found. Strong centrifuging causes the eggs to elongate
and often the clear (centripetal) end constricts off from the yolk-bearing
end. The nucleated fragment, that lies nearer to the pole, can be
identified because such a fragment gives off the polar bodies. Unless
it could be shown that the spindle is also displaced at times by the
amount of centrifuging here used, this result makes the identification
of the polar fragment certain, regardless of whether it contains the
yolk or the oil cap. After fertilization both fragments may cleave.
Wilson finds that the unequal first and second cleavages are char-
acteristic of both fragments. In general, only those fragments that
do not give off polar bodies develop a yolk-lobe. Our own results
confirm entirely these conclusions. Their only merit is that they give,
perhaps, more accurate information regarding the regional origin of
the fragments, and make possible comparisons between fragments of
equal sizes of the same egg whose interior has not been disturbed by
centrifuging.
If, then, as these experiments appear to indicate, the antipolar lobe-
formation is not an essential part of the cleavage pattern but a by-
product of that pattern, its absence from the polar fragment and its
presence in the antipolar fragment remains to be explained. It is rea-
sonably certain from Lillie's and Wilson's centrifuging experiments
that this lobe is not directly caused by the presence of a particular kind
of yolk material at the antipole, but the occurrence of this material
might, by influencing the location of the mitotic figure, determine the
extra-territorial region that becomes the lobe. If so, the relatively
greater development of the mitotic figure in the polar fragment might
in itself account for the absence in it of a lobe at the two-cell stage.
Conversely for the antipolar fragment. But there is an alternative
possibility, namely, the relative location (and size) of the first spindles
in the two cases. If, for instance, it could be shown that in the polar
fragment the spindle is relatively nearer the centre and in the antipolar
fragment relatively nearer the polar side of the fragment, the two re-
sults would be in accord with the hypothesis suggested above.
THE ABSORPTION SPECTRA OF SOME BLOODS AND
SOLUTIONS CONTAINING HEMOCYANIN
ALFRED C. REDFIELD
(From the Department of Physiology, Harvard Medical School, Boston and
the Marine Biological Laboratory, Woods Hole)
The present study of the absorption of light by hemocyanin was
undertaken in the course of developing a spectrophotometric method
for the determination of the quantity of oxygen combined by the blood
of invertebrates which contain this pigment. The data obtained pro-
vide a precise description of the color characteristic of the body fluids
of the various animals examined. Attention has been directed not only
to the spectrum of oxygenated blood, which has already been examined
with precision in the case of a number of organisms by Dhere and his
collaborators (1919, 1920, 1929), Begemann (1924) and Quagliariello
(1922). but also to the apparent absorption of light by reduced blood.
The latter observations have led to the conclusion that a very consid-
erable fraction of the light passing through a hemocyanin solution may
be scattered by the hemocyanin molecules. The extent of this scattering-
determines in large part the color characteristic of the various bloods
when examined either by reflected or transmitted light. By taking ac-
count of the amount of light scattered by the reduced solution, it has
been found possible to determine the characteristic absorption spectrum
of the molecular complex responsible for the bluish color developed
when the hemocyanins combine with oxygen. In this way some in-
formation is obtained on the specificity of the oxygen-combining mech-
anism in the blood of different animals.
METHOD
Observations have been made upon the blood of the conch, Busycon
canaliciilatnm, the horse-shoe crab, Limulus polyphemus. the squid.
Loligo pealei, and the lobster, Homarus americanus. The bloods have
been drawn by methods previously described (Redfielcl, Coolidge and
Hurd, 1926), and preserved in the cold with toluene until prepared
for observation. Under these conditions they may be kept with little
change for many days. The bloods have been diluted to concentrations
appropriate for the methods involved with sea water, distilled water,
or various salt solutions, after which they have been allowed to stand
150
ABSORPTION SPECTRA OF HEMOCYANIN 151
overnight in the ice box in order to permit equilibrium with the modified
environment to be reached. The material has then been filtered and
placed in specially constructed tonometers in which it could be brought
into equilibrium with various mixtures of gases. Each tonometer con-
sisted of a cylindrical pyrex glass bottle of 200 cc. capacity, to the bottom
of which a T-tube was sealed. The ends of the T were ground parallel
to one another and were closed with optically flat glass plates sealed
in position with DeKhotinsky cement. A chamber was thus provided,
having an inside diameter of approximately one centimeter and a length
which was in most cases exactly 3.3 centimeters. Following equilibra-
tion with the gas mixture, the sample of solution could be run down
into the T-tube and the intensity of the light transmitted through it,
measured. The specimens were oxygenated by filling the tonometer
with oxygen or, in those cases where the character of the oxygen dis-
sociation curve permitted, with air. Solutions containing reduced
hemocyanin were prepared by evacuating the bottles after the intro-
duction of the solution and refilling them with hydrogen. The bottles
were then rotated for 15 minutes, -after which the bottles were re-
evacuated and again filled with hydrogen and equilibrated for an ad-
ditional period of 25 minutes. The precision of the measurements is
affected if the solutions are not perfectly clear. For this reason, the
greatest care is necessary in filtering the solutions and in being sure
that the dissolved materials are -in equilibrium with their environment
before filtration occurs, as otherwise small amounts of precipitated ma-
terial may appear in the solutions before the photometric measurements
are made. Reduced hemocyanin solutions are particularly troublesome
because small amounts of material become insoluble during the me-
chanical disturbances incidental to evacuation and equilibration of the
solutions. Under favorable circumstances, the insoluble particles pro-
duced in this way settle out if the specimens are allowed to stand for
an hour or more prior to making the measurements. Under other cir-
cumstances the solutions remain slightly cloudy and the precision of
the measurements is seriously interfered with. The second difficulty
is in obtaining complete reduction of the solutions. Reduction appears
to be satisfactorily attained by the method outlined above in the case
of the bloods. In solutions of purified hemocyanin, because of the
change in the shape and position of the oxygen dissociation curve, com-
plete reduction is much more difficult to obtain. Further repetition
of the processes of evacuation and equilibration with hydrogen would
undoubtedly achieve the desired effect, but unfortunately such repetition
increases the amount of insoluble material formed in such solutions
and thus defeats its purpose. The use of chemical reducing agents has
152 ALFRED C. REDFIELD
not been employed as those which have been tried have led to pro-
gressive changes in the color of the reduced material, which again de-
feats the objects of the experiments.
Measurements of the absorption of light by these solutions have
been made with a Konig-Martens spectrophotometer constructed by
Schmidt and Haensch. The light source of the instrument was il-
luminated by a Mazda projection bulb, the intensity of whose light
could be controlled by a rheostat. The width of the slits was kept at
0.2 millimeters except at wave lengths less than 480 m/t, when it was
increased to 0.4 or 0.6 millimeters as required in order to secure suf-
ficient illumination. The calibration of the wave length scale of the
instrument was checked from time to time and was found at all times
to be accurate within 1 m/x. The precision of the instrument was also
checked by the determination of the absorption of two colored glass
filters, which had been standardized by the U. S. Bureau of Standards.
The absorption of light is indicated by the following equation :
/o tan2 a0
7 ='
where /0 is the intensity of incident light, / the intensity of transmitted
light, a{ the angle of the analyzing prism at which the fields match when
a tube containing the solvent is placed in one of the beams of light ; a0
is this angle when the tube containing the solution is placed in this beam.
In all cases, ai was determined with the absorption vessel filled with dis-
tilled water. Test showed that the result was the same, within the
limits of observational error, in whichever beam the absorbing solutions
were placed. In order to obtain results which might be compared with
one another after the blood of different animals was examined, the
results have been expressed in terms of the extinction coefficient, E,
characteristic of each wave length as defined by
-£ = 10-™ (2)
•*0
where d is the length in centimeters of the column of fluid. It follows
that the extinction coefficient, E, is given by:
2 (log tan a0 — log tan aQ ,-,
~d~
In dealing with the absorption of light by hemocyanin, one is concerned
particularly with the absorption of light by the complex formed when
oxygen unites with hemocyanin. In this union it has been demonstrated
that one atom of oxygen is combined for each atom of copper contained
ABSORPTION SPECTRA OF HEMOCYANIN
153
in the hemocyanin. The union appears to depend upon some grouping
in the hemocyanin molecule, of which the copper forms an essential
part. For convenience we will refer to this arrangement as the
" chromatic group." For purposes of comparison it is interesting to
determine the absorption of light in relation to the number of chromatic
groups present. According to Beer's Law, the extinction coefficient of
a substance in solution is proportional to its concentration. We have
consequently expressed the absorption of light by the hemocyanin so-
lutions in terms of E/c, where c is the concentration of copper in the
solution expressed as milligram atoms per liter. An advantage of this
notation also lies in the fact that the concentration of copper in serum
may be readily obtained without the necessity of determining the number
of grams of hemocyanin which are present, an investigation which
cannot be made unless the hemocyanin of the species has been isolated
and properly studied.
THE APPLICATION OF BEER'S LAW TO HEMOCYANIN SOLUTIONS
The foregoing treatment assumes explicitly that in the absorption
of light by hemocyanin solutions Beer's Law is valid and that in con-
sequence E/c is a constant characteristic of the substance at each wave
/.o
OB
I S 3
CONCEN TffA T/ON
FIG. 1. Extinction coefficient of purified hemocyanin solutions of various
concentrations.
A. Busycon canaliculatum at 570 mju.
B. Limulus polyphemus at 580 m/u.
C. Limuhis polyphemus at 480 m/u.
length. Quagliariello (1922) and Svedberg and Heyroth (1929) both
present evidence that Beer's Law does not apply in the case of hemo-
cyanin solutions. We have consequently examined this question care-
154
ALFRED C. REDFIELD
fully and have found no indication that Beer's Law is not valid when
applied to such solutions and to such concentrations and at such wave
lengths as we have employed. In Fig. 1 is shown the relation between
the extinction coefficient of solutions of purified hemocyanin of two
species made at various concentrations. In the case of Busycon and
of Limulus the measurements were made at the wave length of max-
imal absorption and in the case of Limulus also at the wave length
at which the absorption is minimal. In all three cases the relation be-
tween extinction coefficient and concentration is linear within the ac-
curacy obtainable with photometric measurements on solutions of this
I.O
o.e
O.6
0.4
o.a
CONCENTRA T/ON
FIG. 2. Extinction coefficient of oxygenated and reduced solutions containing
hemocyanin at various concentrations.
A. Busycon canaliculatnm serum oxygenated. Dilution with 2.5 per cent
NaCl. W'ave length 570 mM.
B. The same, reduced.
C. Busycon canalicnlatnm hemocyanin in potassium phosphate buffer solution
oxygenated. Wave length 570 m/x. Dilution with phosphate buffer, 0.178 mo-
lecular phosphate; ionic strength 0.55; molecular fraction as K.jHPO4 0.90.
D. The same, reduced.
character. Quagliariello's measurements were made upon native blood
diluted with 2.5 per cent sodium chloride. It seemed possible that his
anomolous results were due to alterations in the environment of the
hemocyanin as the result of dilution, which might possibly affect the
ABSORPTION SPECTRA OF HEMOCYANIN
155
degree of scattering of light by the protein, to be subsequently discussed.
We have therefore made observations on the serum of Bitsycon canal-
iculatum similarly diluted with 2.5 per cent sodium chloride and have
measured the extinction coefficient not only of the oxygenated but of
O9
OB
O.7
C/c
O /
45O
5OC
55O GOO
WAVE LENGTH
65O
7OO
FIG. 3. Absorption spectra of blood of Busycon canaliculatum. Upper curve,
oxygenated blood ; lower curve, reduced blood ; intermediate curve, corrected
spectrum of chromatic group. Copper content of blood 0.066 mgrn. per cc. ;
dilution, 10 parts blood plus 18 parts H,O plus 2 parts 0.1N NaOH ; pH 9.0 ; ^
length of absorption vessel 3.3 cm.
the reduced solutions. The results are shown in Fig. 2, curves A and
B. Again it appears that the relation between extinction coefficient
and concentration is practically linear. As a further test we have made
observations upon a solution of purified Busycon canaliculatum hemo-
11
156
ALFRED C. REDFIELD
cyanin dissolved in potassium phosphate buffer and diluted carefully
with a similarly buffered solution so as to maintain constant ionic
strength. Measurements were made upon both the oxygenated and
reduced solutions which again conform closely to the requirements of
Beer's Law (Fig. 2, C and D). We consequently conclude that the
assumption of Beer's Law is valid in connection with the observations
discussed in this paper.
THE ABSORPTION SPECTRA OF NATIVE BLOOD
The typical spectra of the oxygenated and reduced bloods of
Limulus, Loligo, Busycon and Hoinarns are presented in Figs. 3, 4, 5
and 6. Detailed descriptions of the solutions will be found in the
45O
5OO
5 SO 5OO
WAVE LENGTH
650
7OO
FIG. 4. Absorption spectra of blood of Limulus polyphcmus. Upper curve,
oxygenated blood; lower curve, reduced blood; intermediate curve, corrected
spectrum of chromatic group. Copper content of blood 0.081 mgm. per cc. ;
dilution, 20 parts blood plus 35 parts sea water plus 5 parts 0.08N HC1 ; pH
6.05 ; length of absorption vessel 3.3 cm.
ABSORPTION SPECTRA OF HEMOCYANIN
157
legends of these figures. The upper curve in each case represents the
absorption of light by the oxygenated blood, the lower curve by the
reduced solution. A glance at the curves descriptive of the oxygenated
blood serves to show a very considerable difference in the shape of each
curve and in the general magnitude of the absorption. The curves
do not differ markedly from those described by Quagliariello and others
0.7
06
45O
5OO
55O BOO
WAVE LENGTH
650
TOO
FIG. 5. Absorption spectrum of blood of Loligo pealci. Upper curve, oxy-
genated blood ; lower curve, reduced blood ; intermediate curve, corrected spectrum
of chromatic group. Copper content of blood 0.249 mgm. per cc. ; dilution,
one part blood plus 6 parts sea water; pH 8.11 ; length of absorption vessel 3.3 cm.
in the case of European forms belonging to related groups. The curves
are alike in displaying a broad band of maximal absorption in the yellow
with more or less increased transmission in the region of blue-green.
It is in the relative values of the absorption in the blue-green and in
the yellow regions that the curves differ characteristically, the species
falling in the order Busycon, Linndus, Loligo, Hoinanis as the ab-
sorption in the blue-green region decreases. It is, of course, this differ-
ence which determines the observed colors of the different bloods.
158
ALFRED C. REDFIELD
SPECTRA OF REDUCED BLOODS
The spectra of the reduced bloods described by the lower curves
in Figs. 3, 4, 5 and 6 deserve particular attention. It may be noted in
each case, except that of the 'lobster, that these curves are similar in
sweeping with gradual ascent uninterrupted by any obvious absorption
bands as one passes from longer to shorter wave lengths. Comparing
these curves for the different species, it may be noted that the ab-
sorption of light by the reduced blood is greatest in those forms in
which the absorption by the oxygenated solution at the blue end of
the spectrum is relatively high, the order being again Busy con, Limulus,
Loligo. This fact may also be related to the observation of Redfield,
Coolidge and Hurd (1926) that the Tyndall effect of the bloods studied
06
O5
C/c
45O
5OO
55O BOO
LENGTH
65O
70O
FIG. 6. Absorption spectrum of blood of Homarus amcricanus containing
natural pigments. Upper curve, oxygenated blood ; lower curve, reduced blood ;
intermediate curve, corrected spectrum of chromatic group. Copper content of
blood 0.0505 mgm. per cc. ; dilution, 2 parts of blood plus one part of solution
containing 0.4 mols NaCI, 0.01 mols KG, 0.02 mols CaCl, per liter; pH 7.87;
length of absorption vessel 3.3 cm.
by them decreases in the order Busycon, Limulus, Loligo and suggests
that the absorption of light by reduced bloods may be due almost
entirely to the scattering of light by the solution. The absence of
ABSORPTION SPECTRA OF HEMOCYANIN
159
definite absorption bands in the reduced blood of these three species
supports this hypothesis.
According to Lord Rayleigh (Strutt. 1871), when a beam of light
passes through a medium containing particles small when compared
with the wave length, the light of various wave lengths is scattered
in proportion to the reciprocal of the fourth power of the wave length.
The light, which is scattered at an angle of 90° from the incident beam,
may be expected to be completely polarized provided the particles are
spherical. Observation of the Tyndall beam emitted by he.mocyanin
solutions shows indeed that the Tyndall light is polarized, and inasmuch
tf 7
06
•45O
O.S
01
FIG. 7. Extinction coefficients, E/c, of reduced blood plotted against the
reciprocal of the fourth power of the wave length, 1/X*. For data regarding
Busycon, Loligo and Homarus see legends to Figs. 3, 5 and 6. The data for
Limulus is presented under Fig. 11 at pH 8.77. Concentrations, c, are expressed
as milligram atoms of copper per liter.
as the beam disappears entirely when viewed through a properly oriented
Nicol prism, the polarization must be very nearly complete. Rayleigh
deduces that the attenuation undergone by the beam as the result of
160 ALFRED C. REDFIELD
scattering can be expressed by the equation
/ = I0e-™ «*, (4)
where x is the thickness of the scattering medium, A is the wave length,
and K is a constant characteristic of the solution in question. The
validity of this equation was demonstrated in the case of mastic solu-
lutions by Abney and Festing (1886). Mecklenburg (1915) has shown
that solutions of colloidal sulfur scatter light in proportion to the
reciprocal of the fourth power of the wave length when the diameter
of the particles falls between 5 and 93 m^. For larger particles the
relation no longer holds. The radius of the molecules of hemocyanin
of Helix and Limulus, according to Svedberg and Hey roth (1929),
are of the order of 10~c centimeters or 10 m/x, so that we may expect
the Rayleigh equation to apply in their case. From inspection of equa-
tions 2 and 4, it is obvious that for any given solution E or E/c should
be proportional to I/A4. We may consequently test the hypothesis
that the apparent absorption of light by bloods containing reduced
hemocyanin is due to the scattering of light by the hemocyanin molecules
by determining whether E/c at each wave length is proportional to the
reciprocal of the fourth power of the wave length. In Fig. 7 the values
of E/c for the various reduced bloods are plotted against I/A4. The
lines so formed in the case of Busycon, Limulus and Loligo are straight
lines which on exterpolation converge toward and meet at the origin,
indicating that the Rayleigh formula does in effect describe the phe-
nomena observed. It may be concluded consequently that the apparent
absorption of light by the reduced blood of Busycon, Limulus and Loligo
is to be attributed to the scattering of light by the dissolved hemocyanin.
THE CORRECTED SPECTRA OF THE CHROMATIC GROUPS
The absorption of light by oxygenated blood must now be attributed
to at least two components : the apparent absorption due to scattering
and the true absorption due to the chromatic group. If these are the
only factors involved, and if it be assumed that the scattering of light
by the hemocyanin molecule is unaltered by the process of oxygenation,
it is possible to correct the absorption spectra of the oxygenated bloods
for the apparent absorption due to scattering and obtain a corrected
spectrum of the chromatic group itself. If the attenuation undergone
by the beam of light as the result of scattering is given by
~1 = \C\-Erd,
f »
Jo
where 7X is the intensity of " unscattered " light which would emerge
ABSORPTION SPECTRA OF HEMOCYANIN 161
were no other factors involved, and Er is the extinction coefficient
characteristic of the reduced material ; and the further attenuation due
to absorption by the chromatic groups is indicated by
h = 10-**"
-/I
where /„ is the final intensity of the emerged beam and Ex is the ex-
tinction coefficient expressing the effect of the chromatic group, then
— = -\r\-(Ex+Er)d
T
Jo
The total absorption of light, however, is given by
h = io-*o*
Jo
where E0 is the extinction coefficient of the oxygenated solution. Con-
sequently,
EO = Ex + Er.
The extinction coefficient of the chromatic group at unit concentration
is consequently obtained by subtracting the value of E/c for the re-
duced solution from the value of E/c for the oxygenated solution at
each wave length. This has been done, and the results are indicated
by the intermediate curves in Figs. 3, 4, 5 and 6.
THE SPECTRA OF BLOOD CONTAINING OTHER PIGMENTS
The blood of the lobster requires special consideration because in
addition to hemocyanin, this blood, in common with that of other crusta-
ceans, contains the pigment tetronerythrin described by Halliburton
(1885). Consequently the reduced blood of this species usually has
a pinkish color and the bluish hue of the oxygenated blood has a more
neutral color than that of the other forms if the pigment is present
in sufficient amounts. As the result of the presence of this pigment,
the spectrum of reduced lobster blood does not conform to the Rayleigh
equation, as the lower curve in Fig. 7 shows. The tetronerythrin may
be extracted from the blood by shaking the blood with chloroform.
In Fig. 8 the absorption spectrum of the pigment extracted with chloro-
form is illustrated, the absorption of the dissolved pigment being com-
pared with the absorption when the vessel is filled with chloroform.
This substance possesses a maximal absorption at a wave length of
490 m/A and transmits nearly all of the incident light at wave lengths
greater than 600 m/^. The apparent absorption of light due to scattering
by the reduced blood of the lobster may consequently be arrived at
162
ALFRED C. REDFIELD
approximately. By considering the absorption spectrum of the reduced
blood at wave lengths greater than 600 m/x, it may be observed from
Fig. 7 that these points fall along a straight line drawn from the origin
of the diagram. Extending this line beyond 600 m/x indicates the de-
gree of apparent absorption due to scattering at these wave lengths.
The presence of tetronerythrin. or similar pigments, the color of
which is unaffected by the oxygenation of the blood, does not interfere
oso
O./6
0.03
0.04
45O 5OO 55O GOO
WAVE LENGTH
65O
TOO M/j.
FIG. 8. Absorption spectrum of solution of the pigment extracted from
lobster blood with chloroform. Concentration unknown ; length of absorption
vessel 3.3 cm.
with the determination of the corrected spectrum of the chromatic
group. This may be demonstrated by examining the spectrum of blood
from which the tetronerythrin has been extracted by chloroform. The
spectra of oxygenated and reduced lobster blood so treated are illus-
trated in Fig. 9. It may be observed that the spectrum of the reduced
solution no longer shows the irregularity due to the pigment. The
corrected spectrum of the chromatic group may be seen to be almost
identical with that obtained from the normal serum illustrated in Fig. 6.
A COMPARISON OF THE SPECTRA OF THE CHROMATIC GROUPS OF
DIFFERENT HEMOCYANINS
It is a question of considerable interest to what extent the various
respiratory proteins may be regarded as distinct " inventions of Na-
ture," especially in that it is desirable to know whether the possession
ABSORPTION SPECTRA OF HEMOCYANIN
163
of similar or identical respiratory pigments 'indicates a generic relation
between the groups of organisms possessing them. Recently much evi-
dence has accumulated establishing the fact that the various hemocyanins
are specifically different substances. This evidence consists in the dem-
onstration of distinctive differences in the physical and chemical prop-
erties of these proteins. On the other hand, the evidence regarding
the ratio between oxygen-combining power and copper content of the
hemocyanins indicates that these substances have certain points in com-
mon, at least with regard to the portion of the molecule concerned with
O.6
£/c
450
50O
55O BOO
LENGT/i
65O
7OO
FIG. 9. Absorption spectrum of blood of Homants americanus after extract-
ing the pigment with chloroform. Upper curve, oxygenated blood ; lower curve,
reduced blood ; intermediate curve, the spectrum of the chromatic group. Copper
content of blood 0.0522 mgm. per cc. ; dilution, 2 parts of blood plus one part
of solution containing 0.4 mols NaCl, 0.01 mols KC1, 0.02 mols CaCL per liter;
pH 8.05 ; length of absorption vessel 3.3 cm.
this function. To this complex when combined with oxygen we have
applied the designation " chromatic group." A comparison of the
spectra of the chromatic groups of different forms should consequently
give evidence regarding the similarity of the chromatic groups in the
164
ALFRED C. REDFIELD
hemocyanins of different classes of animals. In Fig. 10 the corrected
spectra of the chromatic groups of the four species which we have
studied are collected. It may be seen that on the whole the curves are
strikingly alike, not only with regard to their shape, but also in relation
to the actual quantity of light absorbed by equal numbers of chromatic
O.B
o.s
o./
45O
5OO
55O 6OO
LENGTH
S5O
7OO
FIG. 10. Absorption spectra of chromatic groups of blood of Busycon,
Limulus, Loligo and Homarus. For data see Figs. 3, 4, 5 and 6.
groups. One is forced to the conclusion that the complexes responsible
for these spectra are very much alike in each case. On the other hand,
there are unquestionable differences between the spectra in the different
cases.
FACTORS AFFECTING THE ABSORPTION OF LIGHT BY THE CHROMATIC
GROUP
A comparison of the chromatic groups of different species raises
the question as to whether the differences observed may be attributed
to differences in the chemical make-up of the body fluids in question.
ABSORPTION SPECTRA OF HEMOCYANIN
165
It is consequently desirable to examine the effect of the nature of the
solvent upon the absorption of light by hemocyanin solutions.
Hydrogen Ion Concentration. — The first point to be considered is
the influence of hydrogen ion concentration upon absorption and scat-
tering. When specimens of Limulus blood, to which various amounts
0.3
OB
O7
45O
5OO
55O GOO
HAVE LENGTH
650
7OO
FIG. 11. Absorption spectra of blood of Limulus polyphcmns at different
hydrogen ion concentrations. Upper curves, oxygenated blood at pH 8.77 (hollow
circles) and pH 9.42 (dots) ; lower curves, the same after reduction; intermediate
curve, the spectrum of chromatic group, which is identical in both cases. Copper
content 0.081 mgm. per cc. ; length of absorption vessel 3.3 cm. Dilution which
gave pH 8.77: 20 parts blood, 35 parts sea water. 5 parts 0.04N NaOH ; dilution
which gave pH 9.42: 20 parts blood, 35 parts sea water, 5 parts 0.1N NaOH.
of acid or alkali have been added, are examined, it is obvious to the
eye that the color of the solution more alkaline than about pH 9 is
different from the others. This difference is evident not onlv in the
166
ALFRED C. REDFIELD
oxygenated, but also in the reduced solutions, the oxygenated solution
being a purer blue beyond pH 9 and the reduced solution having a
fainter yellow color. In Fig. 11 are illustrated absorption spectra of
specimens of oxygenated and reduced Limulus blood which were diluted
with sea water, to which small quantities of sodium hydroxide had
been added so that the solutions were at pH 8.77 and 9.44 respectively.
With these curves the data presented in Fig. 4 should be compared,
as the latter was obtained from the same blood brought to pH 6.05
O.6
O.5
0.4
O3
o.e
OJ
\
45O 5OO 53O GOO
LENGTH
S50
TOO M/j
FIG. 12. Spectra of hemocyanin of Busycon canallculatum. Upper curve ,
oxygenated ; lower curve, reduced ; intermediate curve, spectrum of the chromatic
group. Hemocyanin purified by precipitating four times- with saturated ammonium
sulfate followed by dialysis. It contained 0.129 grams hemocyanin per cc. and
0.308 mgm. Cu per cc. Dilution, 2 parts hemocyanin solution plus 14 parts H2O
plus one part 0.1N NaOH ; pH 9.16; length of absorption vessel 3.3 cm.
by the addition of sea water containing small quantities of hydrochloric
acid. The spectra illustrated in Figs. 4 and 11 account for the observed
differences in color. The more alkaline specimen absorbs less light
than the others in both the oxygenated and the reduced conditions.
ABSORPTION SPECTRA OF HEMOCYAN1N
167
It is clear also that the more alkaline solution scatters less light than
do the others. Comparison of the corrected spectra of the chromatic
groups shows, on the other hand, that the true absorption of light
is not changed to a detectable degree by alterations in the hydrogen
ion concentration. The differences in the spectra of the oxygenated
bloods are sufficiently accounted for by the differences in scattering.
Salts. — A more profound alteration in the solvent may be obtained
by purifying the hemocyanin so that it may be dissolved in water
•450 50O
55O GOO
WAVE LENGTH
650
7OO
FIG. 13. Spectra of hemocyanin of Limulus Polyphemus. Upper curve, oxy-
genated ; lower curve, reduced ; intermediate curve, spectrum of the chromatic
group. Hemocyanin purified by precipitating four times with saturated ammonium
sulphate followed by dialysis. It contained 0.109 grams hemocyanin per cc. and
0.184 mgm. Cu per cc. Dilution, 5 parts hemocyanin solution plus 12.5 parts
H2O plus 2.5 parts 0.1N NaOH ; pH 9.10; length of absorption vessel 3.3 cm.
practically free of salts or other substances. By this means it is pos-
sible to compare the spectra of the chromatic groups of the different
hemocyanins in solutions which are more or less identical. When solu-
tions of pure hemocyanin are compared, it may be observed that the
Tyndall phenomenon has undergone great diminution. Dilute solutions
of reduced hemocyanin are practically colorless. The oxygenated so-
lutions are of a purer blue color than when these substances are dis-
solved in the blood. These characteristics are all accounted for by an
examination of the absorption spectra of the solutions, in which it may
be observed that the reduced solutions appear to absorb very little light
168
ALFRED C. REDFIELD
and to absorb only slightly more light at the violet end of the spectrum
than at the red end. Similarly the transmission of light in the blue-
green region of the spectrum of the purified oxygenated hemocyanin
is much greater than in the case of blood, and the absorption spectrum
•of the oxygenated solutions does not differ greatly from those of the
corrected spectra of the chromatic groups. Spectra of purified hemo-
cyanin solutions of Busycon, Limulus and Homarus are illustrated in
OS
0.3
O.I
45O
5OD
55O GOO
WAVE LENGTH
65O
7OO
FIG. 14. Spectra of hemocyanin of Homarus amcrlcamis. Upper curve,
oxygenated ; lower curve, reduced ; intermediate curve, spectrum of the chromatic
group. Hemocyanin, purified by dialysis. Solution contained 0.1185 grams dry
solids per cc. and 0.196 mgm. Cu per cc. Dilution : one part hemocyanin solution
plus 3 parts H2O ; pH 8.10; length of absorption vessel 3.3 cm.
Figs. 12, 13 and 14, together with the corrected spectra of the chromatic
groups.
Comparison may now be made between the spectra of the chromatic
groups of the purified hemocyanin and of the native blood. This is
done in the case of these three species in Tables I, II and III. For
accurate comparison the value of E/c for each wave length in the case
of the purified hemocyanin is compared with its value in the case of the
native blood. If the spectra of the chromatic groups are identical, this
ratio should be the same at all wave lengths and have the value 1.0.
ABSORPTION SPECTRA OF HEMOCYANIN
169
Examination of the tables showed that the ratio is not quite constant
in each case at different wave lengths. The divergences are not large,
but appear to be reproducible and indicate that the spectrum of the
chromatic groups undergoes certain small changes as the result of the
process of purification. The ratio also deviates from the value of 1.0
in each case. With Busy con and Homarus the purified material absorbs
only slightly less light at each wave length than does a like concentration
TABLE I
Comparison of absorption of light by chromatic groups of blood and purified hemo-
cyanin of Busy con canaliculatum.
Wave Length
Hemocyanin
Blood
Ratio
WM
460
E/c
0.216
E'c
0.238
0.908
480
0.230
0.260
0.885
500
0.296
0.333
0.890
520
0.406
0.436
0.932
540
0.497
0.530
0.938
560
0.546
0.580
0.942
580
0.546
0.575
0.950
600
0.506
0.537
0.942
620
0.442
0.473
0.935
640
0.380
0.403
0.942
660
0.325
0.348
0.935
680
0.280
0.304
0.923
700
0.242
0.268
0.904
of hemocyanin in native blood. In the case of Limulus the discrepancy
is much greater, amounting to about 30 per cent. These differences
might be due to an alteration in the absorption of light by each chromatic
group. On the other hand, they might be adequately accounted for
on the assumption that as the result of the process of purification a
certain quantity of the hemocyanin has lost the ability to combine with
oxygen, which is necessary in order that the chromatic group be formed.
The difference in the case of Limulus is sufficiently large to allow this
possibility to be tested by a determination of the oxygen-combining
power of the solution. The hemocyanin solution employed in this case
contained 1.93 milligram atoms of copper per liter and might be ex-
pected to have an oxygen capacity of 1.93 milligram atoms of oxygen
per liter. Actual analyses of the oxygen content of this solution when
equilibrated with air yielded the values, 1.94, 1.95, 1.90 (mean 1.93)
milligram atoms of oxygen per liter. Allowing 0.50 milligram atoms
of oxygen per liter dissolved in the solution, one obtains 1.43 milligram
170
ALFRED C. REDFIELD
atoms as the actual oxygen-combining capacity. This value is 74 per
cent of the theoretical, indicating that 26 per cent of the hemocyanin had
lost its ability to combine with oxygen. The absorption of light by
this solution is approximately 70 per cent of the absorption to be ex-
pected from the observations on hemocyanin as it occurs in native blood
as Table II shows. It seems clear that in the case of this specimen
at least, the discrepancy between the spectrum of blood and of the puri-
TABLE II
Comparison of absorption of light by chromatic groups of hemocyanin and blood of
Limulus polyphemus.
Wave Length
Hemocyanin
Blood
Ratio
WM
460
E/c
0 165
E/c
0 244
0 677
480
0 144
0 220
0 655
500
0 168
0 257
0 655
520. .
0 231
0 338
0 683
540 . .
0 298
0 438
0 680
560. .
0345
0 506
0 683
580
0365
0 528
0 692
600
0362
0 518
0 699
620
0344
0486
0 708
640
0 314
0 446
0 705
660. .
0 284
0 403
0 705
680. .
0 256
0 362
0 708
700
0 231
0324
0 714
fied hemocyanin solution is clue in large part to the modification of a
portion of the hemocyanin in the process of preparation or preservation.
The hemocyanin from which this specimen was prepared had been
preserved for many months precipitated in half -saturated ammonium
sulfate prior to preparation, and unfortunately we have not had an
opportunity of re-examining this question with freshly collected hemo-
cyanin.
These results lead to the conclusion that the observed differences
in the extinction coefficients of hemocyanin in blood and in purified
solutions may be accounted for largely by the denaturation of the
hemocyanin in the process of preparation. They do not demonstrate
that some difference in the absorption of light by the chromatic groups
does not occur. Unfortunately the precision of the available methods
for measuring oxygen capacity in these solutions is so low that changes
cannot be detected unless they are relatively large. It may be con-
cluded, however, that the spectra of the chromatic groups vary very
ABSORPTION SPECTRA OF HEMOCYANIN
171
little as the result of freeing the solutions from electrolytes and other
impurities.
A comparison of the absorption of light by the reduced solutions
of purified hemocyanin illustrated in Figs. 12, 13 and 14, with the
curves for the absorption of light by the reduced serum of the corre-
sponding species, shows that in the purified preparations, the scattering
of light is much less than in the native blood. In the case of the lob-
ster, the values of E/c characteristic of each wave length are, in the
TABLE III
Comparison of absorption of light by chromatic groups of hemocyanin and blood of
Homarus americamis.
Wave Length
Hemocyanin
Bloorl
Ratio
nifji
460
E/c
0.232
E/c
0.240
0.968
480
0.196
0.202
0.972
500
0.237
0.244
0.972
520
0.317
0.332
0.956
540.
0.393
0.410
0.959
560. ..
0.435
0.447
0.975
580. .. .
0.444
0.451
0.984
600. .
0.421
0.432
0.976
620. .
0.391
0.402
0.973
640. ..
0.358
0.366
0.979
660. .
0.327
0.333
0.984
680. .
0.295
0.300
0.984
700. .
0.270
0.268
1 .007
purified serum, about one-half those characteristic of the reduced blood.
In the blood of Busycon and Linuilits, the scattering of light is many
times greater than in the purified preparations.
The effect of purification upon the scattering of light may be
shown to be due primarily to the removal of electrolytes from the solvent
of the hemocyanin. By adding salt to purified hemocyanin solutions.
the scattering effect is greatly increased. At the same time, the spec-
trum of the oxygenated solution approaches more nearly that ot native
blood. The spectrum of the chromatic group, however, appears to re-
main unchanged. These facts are illustrated by the data in Table IV.
in which the values of E/c for oxygenated and reduced solutions oi
Busycon hemocyanin are compared when it is dissolved in water and
when it is dissolved in a solution of potassium phosphate of an ionic
strength approximately equal to that of native blood.
12
172
ALFRED C. REDFIELD
It may be concluded from the foregoing that the spectrum of the
chromatic group is a relatively constant characteristic of hemocyanin
solutions, influenced little if at all by the composition of the solvent
provided that this does not interfere with the oxygenation of the
material. On the other hand, the apparent absorption of light due to
scattering varies greatly with the nature of the solvent and particularly
with its salt content and hydrogen ion concentration. These facts are
essential to the use of photometric methods in examining these solutions.
They demonstrate that the measure of the absorption of light by the
TABLE IV
Absorption of light by hemocyanin of Busy con canaliculatum dissolved in potassium
phosphate buffer; phosphate concentration, 0.357 molar; pH, 7.7.
Wave
Length
Oxygenated
in
Phosphate
Reduced
in
Phosphate
Chromatic
Group in
Phosphate
Chromatic
Group —
Salt-Free
Ratio
»«M
460
E/c
0 559
E/c
0 345
Elc
0 ?14
Etc
0.216
1 009
480
0 514
0 288
0 2?6
0 ?30
1 017
500
0 544
0 237
0 307
0 296
0 964
520
0 625
0 208
0417
0 406
0 974
540
0 691
0 178
0 513
0 497
0 969
560
0 722
0 131
0 567
0 546
0963
580.
0 695
0 132
0 563
0 546
0 971
600 . .
0 632
0 118
0 514
0 506
0 985
620. .. .
0.560
0 105
0455
0 44?
0 972
640. ..
0.477
0092
0385
0 380
0 987
660
0.415
0.078
0337
0 325
0965
680
0.361
0.071
0 290
0 280
0 966
700
0.324
0.069
0 255
0 242
0 950
chromatic group may be a reliable index of the concentration of oxy-
hemocyanin. They also make it clear that in such measurements every
precaution must be taken to control and take account of the degree
of absorption due to the scattering of light.
In a preliminary report on the present investigation (Redfield, 1929)
it was suggested that the relative size of the particles of hemocyanin
could be deduced from the scattering of light with the aid of the Ray-
leigh theory. However, Raman (1927) has developed a theory of
scattering by colloidal solutions, in accordance with which it appears
possible to relate the observed optical phenomena to the osmotic pres-
sure of the solutions. The experiments of Loeb on gelatin indicate
that the variations in osmotic pressure of protein solutions induced
by altering the nature of the solvent, which he accounted for by the
ABSORPTION SPECTRA OF HEMOCYANIN
173
considerations involved in Donnan membrane equilibria, are in the
necessary direction and have sufficient magnitude to account for the
observed variations of scattering in terms of Raman's theory. Until
this possibility is examined critically, it is improper to draw inferences
/oo
BO
so
45O
5OO
55O GOO
WAVE LENGTH
B5O
1OO
FIG. 15. Absorption spectra of chromatic groups of purified hemocyanins of
Busy con, Limulus, and Homarus. The ordinate is an arbitrary scale such that
the value of E/c for each spectrum is- 100 at the wave length of maximal ab-
sorption. For data see Figs. 12, 13 and 14.
concerning the degree of aggregation of hemocyanin in blood from
the phenomenon of scattering.
COMPARISON OF THE CHROMATIC GROUPS OF PURIFIED HEMOCYANIN
IN AQUEOUS SOLUTIONS
In order to compare the spectra of the chromatic groups of the
different purified hemocyanins it is necessary to employ some method
which disregards the errors due to the denaturation of a certain portion
of the hemocyanin in the process of purification, as the foregoing
discussion indicates that data may not give us accurate information
with regard to the concentrations of oxygenated hemocyanin in the
various preparations. The spectra of the chromatic groups of the
different hemocyanins described by Figs. 12, 13 and 14 have conse-
quently been reduced to an arbitrary scale in which the maximal in-
174 ALFRED C REDFIELD
tensity of absorption in the yellow region has been taken as 100. The
data so obtained are plotted in Fig. 15. Comparing these curves, it is
evident that even in aqueous solutions the spectra of the chromatic
groups are markedly different. One may conclude consequently that
the characteristics of these spectra are not dependent upon the chemical
peculiarities of the body fluids of the different animals but on specific
differences in the chromatic groups themselves or on the influence of
the specific characteristics of the hemocyanin molecule as a whole upon
that portion which is concerned with the transport of oxygen.
SUMMARY
1. The absorption of light by the blood and by purified preparations
of the hemocyanin of the conch, Busycon canaliculatum, the horse-shoe
crab, Limulus polyphemus, the squid, Loligo pcalci, and the lobster,
Homarus ainericanus, has been studied. It is shown that the absorption
of light by solutions containing oxygenated hemocyanin may be re-
solved into two components: (a) that due to the true absorption by
the chromatic group formed by the union of oxygen with the portion
of the molecule containing copper and (b) that due to the scattering
of light by the dissolved protein.
2. In the analysis of the spectrum of the blood of the lobster, the
absorption of light by the pigment tetronerythrin has been taken into
account.
3. The spectrum of the chromatic group of a given species varies
very little, if at all, as the result of alterations in the hydrogen ion
concentration and salt content of the solution.
4. The spectra of the chromatic groups of the different species
display a considerable similarity, indicating a close chemical relation-
ship. There exist, however, definite differences in the spectra of each
species which persist after the process of purification and indicate defi-
nite specific differences in the various hemocyanins.
5. The scattering of light varies widely among the different species
and is responsible in large part for the difference in appearance of the
bloods, particularly when viewed by reflected light. The scattering is
modified greatly by changes in the composition of the solution, being
diminished in the more alkaline solutions and particularly in solutions
free from electrolytes.
REFERENCES
ABNEY AND FESTING, 1886. Proc. Roy. Soc., London. 40: 238.
BEGEMANN, H., 1924. Over de ademhalingsfunctie van haemocyanine. thesis,
Utrecht; for abstract see Jordan, H.. 1925. Zeitschr. f. vcrgl. Physiol,
2: 381.
ABSORPTION SPECTRA OF HEMOCYANIN 175
DHERE, C. AND BUKDEL, A., 1919. Jour, physiol. el. path, gen., 18: 685.
DHERE, C., 1920. Jour, physiol et path, gen., 19: 1081.
DHERE, C., BAUMELER, C, AND SCHNEIDER, A., 1929. Comfit, rend. Soc. de biol.,
101: 759.
HALLIBURTON, W. D., 1885. Jour. Physiol.. 6: 300.
MECKLENBURG, W., 1915. Zcitschr. f. Chemic und Industrie der Kollodt. 16: 97.
QUAGLIARIELLO, G., 1922. Pubblicaztoni della Stasione Zoologica di Napoli, 1: 57.
RAMAN, C. V., 1927. Indian Jour. Physics, 2: 1.
REDYIELD, A. C., 1929. Am. Jour. Physiol., 90: 489.
REDFIELD, A. C., COOLIDGE, T., AND HURD, A. L., 1926. Jour. Biol. Chem., 69: 475.
STRUTT, J. W., 1871. Phil. Mag.. 41: 447.
SVEDBERG, T., AND HEYROTH, F. F., 1929. Jour. Am. Chem. Soc., 51: 539.
ANOPLOPHRYA MARYLANDENSIS N.SP., A CILIATE
FROM THE INTESTINE OF EARTHWORMS
OF THE FAMILY LUMBRICID^
CECILE CONKLIN
(From the Department of Biology, Gouchcr College and the Department of Proto-
zoology, School of Hygiene and Public Health, Johns Hopkins University)
MATERIAL AND METHODS
Anoplophrya mar y land ensis, a new species of astomatous ciliate, was
discovered in the intestine of Lumbricus terrcstris (Linn.. 1758) and
Hclodrilus caUginosus (Savigny, 1826). Many of the infected hosts
were immature forms, making identification uncertain. Assistance in
identifying the earthworms given by Dr. Frank Smith, formerly of the
University of Illinois, is gratefully acknowledged.
The hosts infected with this form were obtained from a limited
area in the city of Baltimore. Hosts of the same species obtained from
three other localities within the city failed to show this form. Those
from two of the other regions showed no intestinal ciliates. In the
infected area the incidence of infection was 29.13 per cent.
The parasites were usually numerous in the infected worms. They
were found only in the anterior third of the intestine, and were most
numerous just in back of the gizzard. The organisms were obtained
after anesthetizing the host with chloretone. The body of the worm
was slit along the mid-dorsal line exposing the intestine into which
short incisions were made in different regions. The contents of the
intestine were removed from these regions with a tooth-pick and
smears were made in physiological salt solution. Smears were fixed
with Schaudinn's fixative and were stained with Heidenhain's iron
hsematoxylin. Parasites were studied in the living condition by placing
the contents of the intestine into a watch glass of physiological salt
solution.
DESCRIPTION
This new species was uniformly ciliated and flattened. The body
was extremely thin and did not appear to be more than one-fifteenth
its width in thickness. Stained specimens were not found in such a
position that thickness could be measured. The dorsal and ventral
surfaces of specimens just removed from the intestine appeared to be
perfectly flat. Some of them became slightly rounded after they had
176
ANOPLOPHRYA MARYLANDENSIS
177
been in the physiological salt solution for a few minutes. The change
in form was evidently due to a difference in osmotic pressure. The
body was rounded at the posterior end and slightly pointed at the
anterior end ; it was broader at the anterior end than at the posterior.
The greatest breadth was just anterior to the center of the body.
One hundred specimens were measured with an ocular micrometer
at a magnification of 1000. The following dimensions and biometrical
data were entered as being typical.
Breadth in
Length in microns
microns
16
36
40
44
48
52
56
60
64
68
72
1
1
2
20
24
28
32
36
40
1
5
3
9
1
2
5
5
9
1
2
1
26
1
7
2
1
2
1
3
2
19
1
1
2
7
8
5
3
27
1
4
5
3
13
1
1
2
4
3
9
14
12
18
15
13
11
t
3
2
^a — ; —
100
Length • ^Breadth
Range 36 to 72 M 16 to-42 P
Mean 52.7 ± 0.6/t 28.6 ± 0.4^
Standard Deviation 8.6 db 0.4 n 5.6 ± 0.3 n
Coefficient of Variation 16.4 ± 1.0% . 19.8 ± 1.0%
The body was covered with long cilia arranged in longitudinal rows
which were close together. The number of rows varied from 31 to
40 in five specimens. These longitudinal rows of cilia converged
slightly at each end of the parasite. The cilia of twelve specimens
averaged 7.3 /x in length. The average length of these twelve parasites
was 50.8 p. In general the length of the cilia varied directly with the
size of the parasite, the larger parasites having the longer cilia.
There was a thin pellicle which covered a transparent layer of
ectosarc. The ectosarc was confined to a thin layer except at the
anterior end where it made up the greater part of the pointed region.
The endosarc was very granular when stained with Heidenhain's iron
178 CECILE CONKLIN
haemotoxylin and it appeared to have many chromatin granules scat-
tered throughout.
There were two nuclei, a large ribbon-like macronucleus and a
small spherical micronucleus. The macronucleus extended through
the long axis of the body and was nearly as long as the body itself.
In 25 specimens the average length of the macronucleus was 43.1 //.
while the average length of these same parasites was 55.5 /A. The av-
erage width of the macronucleus was 5.9 /A while the average width of
the parasites was 32.7 p. The outline of the macronucleus was very
irregular and several showed fine projections extending into the endo-
sarc. (Fig. D.) Often a clear space appeared around the macro-
nucleus in stained specimens. This was probably due to shrinkage.
The micronucleus was very small and appeared to be spherical in
specimens not undergoing division. It was surrounded by a layer
of clear protoplasm, which may also have been due to shrinkage, for
the micronucleus could only be seen in stained specimens. The micro-
nucleus was found about mid-way between the anterior and posterior
ends of the body and was lateral to the macronucleus. It was always
found on the side of macronucleus opposite to that of the row of
contractile vacuoles.
If the parasites were numerous in the host, about every tenth one
was in the act of dividing. Division was transverse. Figs. B, C, D,
and E show different stages in transverse fission. The micronucleus
underwent a mitotic division with the formation of a spindle and
chromosomes. There were apparently only a few chromosomes but
their extremely small size made them very difficult to count. Fig. C
shows four chromosomes that have just divided and the two groups
are separating from each other. In the prophase of division the
chromatin granules appeared to be lined up in two strings with suc-
cessive enlargements which made them look like two strings of beads.
Tarnogradsky (1914) and Cepede (1909) have described posterior
budding in other species, but in the present study no specimens in
unequal division have been found nor did any have other individuals
attached to them.
No conjugating individuals were seen as has been described by
Collin (1909) in A. brasili Leger and Duboscq.
There was a single longitudinal row of contractile vacuoles along
one side of the macronucleus. The number varied from two to five.
The vacuoles in even a single specimen were of widely different sizes.
As many as five different specimens have been watched for one half
hour at a time and never have any of the vacuoles been seen to contract.
The specimens have been placed in a suspension of India ink but no
ANOPLOPHRYA MARYLANDENSIS 179
expulsion of fluid from the vesicles was seen. Lankester (1870) de-
scribed the contraction of vacuoles in an Opalina which has since been
classified as Anaplophrya naidos by Kent (1880) and more recently
as Biitschliella naidos by Mackinnon and Adam (1924). Lankester
said the contractile vacuoles in this form contracted very suddenly,
slowly reappearing in the same place. He found that the collapse oc-
curred a little less frequently than twice a minute. Tarnagradsky
(1914) found that the period of contraction of the contractile vacuoles
in A. inermis Stein was from 1.5 to 15 minutes. Cepede (1910) found
that the contractile vacuoles of A. alluri failed to contract if the animals
were removed from their normal habitat.
BEHAVIOR
Parasites could not be kept alive more than twenty- four hours out-
side the host. Physiological salt solution and various dilutions of it
with distilled water to 0.25, 0.5. 0.375, 0.625, and 0.75 of its normal
strength were used. The parasites lived longest in the normal solu-
tion. Those in the less concentrated solutions soon developed large
blisters on the body and died.
The parasite turned on its long axis as it swam, frequently making
complete turns but often making only a half turn from a horizontal
position to a horizontal position and back again. It was observed to
make this half turn even when the liquid in which it was confined
was sufficiently deep so that the animal was not cramped nor was it
prevented from making a complete turn. When it did make a com-
plete turn it was in a clockwise direction.
The parasite swam rapidly and underwent no deformation due to
the mechanical action of movement, though the body seemed flexible
enough to bend when it struck an obstruction.
DIFFERENTIAL DIAGNOSIS
As far as available literature goes to show, Atwplophrya inarvland-
ensis n.sp.,1 can with one exception be distinguished from all other
species in the genus which fall within its size range (36 to 72 //, in
length) by the fact that it has one row of contractile vacuoles instead
of two rows. The exception is A. parva Rossolimo, from an aquatic
oligochate and is separated from A. maryland ensis by the position of
the micronucleus. In A. maryland en sis, the micronucleus occurs upon
the side of the macronucleus opposite the contractile vacuoles; in A.
parva, both micronucleus and contractile vacuoles are on the same side
1 A type specimen of this species has been deposited in the National Museum
at Washington, D. C.
180
CECILE CONKLIN
- C.V.
B
C D
EXPLANATION OF FIGURES
All specimens stained with Heidenhain's iron haemotoxylin. All drawings
made with camera lucida at a magnification of X 750.
FIG. A. Anoplophrya marylandensis n.sp., normal resting individual. Mac.,
rnacronucleus ; mic., micronucleus ; c.v., contractile vacuole.
FIGS. B, C, D, E represent successive stages in division of A. marylandensis.
B, prophase, C, metaphase, D and E, telophase.
ANOPLOPHRYA MARYLANDENSIS 181
of the macronucleus. Furthermore, A. marylandensis is much broader
(averaging about 28 ^ in breadth) in comparison to A. parva (breadth,
17 At).
REFERENCES
BHATIA, B. L., AND GUTALI, A. N., 1927. On Some Parasitic Ciliates from
Indian Frogs, Toads, Earthworms and Cockroaches. Arch. f. Protistcnk.,
57:85.
CEPEDE, C, 1910. Recherches sur les Infusoires astomes. Theses published in
Arch. ZooL e.rper. ct gen., 5th Series, 3: 341.
COLLIN, B., 1909. La Conjugaison d'Anoplophrya branchiarum (Stein). Arch.
zool. cxper. et gen., 5th Series, 1 : 345.
DELPHY, JEAN, 1922. Infusoires parasites de Lombriciens limicoles. Bull. Mus.
d'Hlst. Nat., 7: 530.
GHOSH, E., 1918. Studies on Infusoria. Rec. hid. Mus., 15: 129.
LANKESTER, E. R., 1870. Remarks on Opalina and its Contractile Vesicles etc.
Quart. Jour. Mic. Sci., 10: 143.
MACKINNON, D. L., AND ADAM, D. I., 1924. Notes on Four Astomatous Ciliates
from Oligochaete Worms. Quart. Jour. Mic. Sci., 68: 211
ROSSOLIMO, L. L., 1926. Uber einige neue und wenig bekannte Infusoria-Astomata
aus den Anneliden des Russischen Nordens. ZooL Anzcig., 68: 52.
TARNOGRADSKY, M. D., 1914. Sur Anoplophrya inermis Stein, Infusoire parasite
de Helobdella stagnalis L. Ass. franc,, p. V nuance, d. sc., 43me Session:
546.
THE GROWTH OF LARV^ OF AMBYSTOMA MACULATUM
UNDER NATURAL CONDITIONS
W. T. DEMPSTER
(From the Zoological Laboratory of the University of Michigan)
INTRODUCTION
Attempts to describe the increment in length and weight during the
larval history of amphibians are confronted with either of two difficul-
ties. If the animals are raised in the laboratory at a constant temper-
ature, the normal or optimum food conditions cannot be duplicated
readily; if animals are collected periodically from their natural habitat,
the conditions of life there are so variable as to produce data difficult
to describe. Accordingly the literature is neither extensive nor con-
sistent.
Davenport (1897, 1899) presented a few data upon the weight
increment of larvae, of " the common frog " under laboratory conditions.
Three stages of growth are recognized; first a period of slow growth
accompanied by abundant cell division, then a period of rapid growth
due to imbibed water, and finally a period of equally rapid growth in
which the increment is due to increase both in organic substance and
water.
Schaper (1902) has provided data still more complete on the larval
growth of Rana esculenta under laboratory conditions. The daily
growth in weight and volume of these tadpoles is slight at first, gradu-
ally becoming greater and attaining a maximum value at about eighty
days. During the following week these values fall to about half as
the animal undergoes metamorphosis. During the first fourteen days
of development the organic matter and ash remain constant (13 mgm.
and 1 mgm. respectively). Weight increment during this period is due
solely to imbibition of water. The percentage of solid then increases
until maximum size is attained ; during metamorphosis this percentage
is further increased. Schaper's data on length increase cannot be easily
interpreted, probably because too few specimens were considered.
Robertson (1923) apparently unaware of Schaper's work has at-
tempted with indifferent success to convert Davenport's data into a
mathematical expression. He believed that the weight increment in
the frog tadpole could be expressed by a single symmetrical sigmoid
curve.
182
AMBYSTOMA MACULATUM 183
Studies on salamanders have shown variable rates of growth. When
the length-age data of Eycleshymer and Wilson (1910) on Necturus
under laboratory conditions and those of Bishop (1926) dealing with
the animal under natural conditions are plotted it may be seen that
during the first three (or four) months of development (except for
a period before the embryonic axis is straight) the length increases
at a constant rate.1 The nearly uniform yearly growth to the period
of sexual maturity, which Bishop records, suggests that aside from
thermal variations the rate of linear growth may be constant from
year to year.
Wilder (1924), on the other hand, has shown that under natural
conditions the rate of linear growth varies at different times during
the larval history in Spelerpes bislineata. Although she disregarded
the embryonic development, subsequent growth stages are recorded.
The post-embryonic stage, until the yolk is absorbed, is the period of
most rapid growth. The typical larval period during the fall and winter
of the first year is a time of slow growth. The latter part of this period
during the spring of the second year is characterized by rapid growth.
The premetamorphic stage during the fall and winter of the second
year involves a period of slow growth, then a period of fluctuating
growth. The metamorphic stage in the summer of the third year is
a period in which the catabolic changes are more pronounced than the
anabolic.
It must be noted that Spelerpes does not become terrestrial until
it has spent two years of its life in an aquatic habitat; Necturus is
permanently aquatic. It seems likely that a more typical method of
growth would be found in salamanders which have an aquatic stage
lasting for only a single season. The increase in length of Ambystoma
has been studied by two observers. Uhlenhuth (1919), who has stud-
ied A. opacnui under constant conditions, stated that the rate of growth
seems to be proportional to the velocity of metamorphosis (rate of
growth X age at metamorphosis — constant). He does not, however,
describe the growth rate of various periods of development.
Patch (1927) has described the length increase in three groups of
Ambystoma as consisting of two sigmoid curves, one embryonic and
the other larval. The point of junction of these two curves is 15.61
mm. in A. inaculatuin, 14.07 mm. in A. tigriiniiii, and 11.96 mm. in the
axylotl.
In order for curves of length increase to represent a fundamental
1 Actually, Bishop's data shows a slight variation from the constant growth
rate over a period of two weeks in July. The climatological records of the
U. S. Weather Bureau for the Saegertown, Pa. region give a rise in temperature
during this period which undoubtedly accounts for the fluctuation.
184 \V. T. DEMPSTER
phase of growth, the relation between weight and length for successive
stages must be constant, as Miss Patch has assumed. In view, how-
ever, of the marked changes in body form during the embryonic life
of Ambystoma it seems unlikely that the " indices of build " are uni-
form. It is necessary to have data on both length and weight, at least
to the point in development where the body form assumes nearly con-
stant larval proportions, in order to correctly appreciate the body in-
crement.
MATERIAL AND METHODS
During the spring of 1928, the author located a salamander pond
sufficiently well populated with spawning Ambystoma maculatum to
indicate that eggs and larval specimens could be obtained throughout
the season. The present study involving about 1700 specimens is the
outcome of two years of systematic collection from that habitat.
Delhi Pond is a shallow, sheltered, leafy-bottomed forest pond in
the environs of Ann Arbor. It has a maximum area of one-twelfth
acre and a maximum depth of about four feet ; it is ordinarily a per-
manent pond but became dry during the season of 1929. In addition
to A. maculatum, the usual invertebrate fauna, A. tigrinum and Ran a
cantabrigensis were present. -
From the time when eggs were first observed until the salamanders
metamorphosed, periodic random collections were made, the intervals
between successive collections being seldom more than a week. Thirty
to forty specimens formed a sample although occasional collections,
particularly during the early stages, amounted to more than a hundred
specimens. Specimens were collected by dredging the bottom of the
pond with a hand net formed of a yard of wire netting stretched be-
tween two poles. All parts of the pond were sampled so that the col-
lection is quite representative. The maximum-minimum temperatures
of the pond were recorded at the time of collection.
The specimens were brought to the laboratory alive, anesthetized
and measured, mutilated specimens having been discarded. During
the non-motile stages anesthesia was not required. The eggs and
embryos up to the period of hatching were placed for measurement
upon the stage of a binocular microscope fitted with a camera lucida.
2 Although B. G. Smith (1911) stated that Ambystoma tigrinum and A.
maculatum are not found in the same habitat in the Ann Arbor region, the author
found both salamanders in abundance in the present location. The two popula-
tions are more or less independent of one another and it is not evident that
the larger salamander through its predatory activities- seriously affects the A.
maculatum population. The eggs and larvae of the two species could not be
confused easily since the egg clutches, the time of hatching, the size and appear-
ance of embryos and larvae are distinctly different.
AMBYSTOMA MACULATUM 185
The image of the embryo could thus be superimposed on a properly
calibrated scale. The larger, later specimens were measured by means
of drafting calipers and millimeter rule. In the blastula and non-
motile stages the maximum diameter or length irrespective of the
curvature of the body axis in relation to the yolk mass was recorded.
The length taken in later stages when the axis was linear was the
maximum length.
The average weight at different developmental stages was also de-
termined : The anesthetized salamanders were placed in a tared crucible
and weighed after the excess water had been absorbed by pipette and
filter paper. They were then dehydrated for several days in a drying
oven at 95-97° C. and the dry weight determined. Following this the
sample was incinerated over a Meeker burner for two to ten hours
and the ash weighed.
RESULTS
Changes in Weight to the Period of Metamorphosis. — During the
year 1928, the first eggs were found on April 3. On August 18, many
specimens had begun to metamorphose at a weight of about 1200 mgm.
A week later there were relatively few specimens in the pond. During
the year 1929 the first eggs were collected on March 27, and on August
14, a number of specimens, at approximately 800 mgm. had metamor-
phosed. When the average weight of a sample of salamander eggs,
embryos or larvae is plotted against the age, as in Figs. 1 and 2, the
rate of growth may be expressed as a curve. The weight increment
was slow at first, gradually increasing to the middle of June when the
rate of increase became more and more rapid. By the middle of
July the rate of growth was at its maximum. In the first week of
August the growth rate was markedly reduced. Finally growth became
negligible and metamorphosis occurred. The weight increase may be
thus described as a single sigmoid curve. Under natural conditions
the first stages of growth were considerably prolonged, due to the low
water temperatures of spring. During the larval and premetamorphic
stages the temperatures are more nearly the same. The terminal period
of growth is very brief, so short in fact, that the curve of Ainbystoina
increment shows a marked variation from the curves of autocatalytic
growth in other animals. During both years the same general type
of sigmoid curve is demonstrated, although the actual weight values
are considerably different.
Linear Increase to the Time of Metamorphosis. — The curve formed
by plotting length against age shows a slight deviation during the first
four or five weeks of development. From this point on the curve is
sigmoid. Growth increment is gradually increased to a period within
186
W. T. DEMPSTER
three to four weeks of metamorphosis when the rate of increase is con-
siderably lowered, and finally becomes negligible. The deviation during
the early stages of development, which Miss Patch has interpreted
as a distinct period of growth, involving the typical sigmoid growth
rates, is due to the form changes of the embryo. The embryonic axis
from the neurula to the early limb bud stages is curved around the
iaoo
,' : \
it 'l
!\ **/ V
1 ' *-. '
/ '• / \ >
siETArraRpt-taaia
e« urn
eon
1928
FIG. 1. A curve showing the relation between average length and weight of
larval specimens of A. maculatum at various ages. Data of 1928.
yolk mass. Increase in length of this axis during the early growth
stages does not result in equivalent increase in the total length of the
embryo. It is not until the embryonic axis becomes straightened that
the total length shows marked increase.
AMBYSTOMA MACULATUM
187
That the departure from a single sigmoid curve during the early
development is not a distinct phase of growth may be demonstrated.
When an " index of build " (Length VWeight) is computed (Table I),
it is clearly indicated that length and weight are not directly associated
during the early stages. This index varies constantly to a period
shortly before hatching when the embryonic axis becomes linear. It
is fairly constant, however, for the free living larval stages.
A group of experiments carried under approximately constant tem-
perature conditions gives indication that the degree of curvature may
vary under environmental circumstances. Four groups of salamanders
FIG. 2. Curve of growth showing weight and linear increment. Data of
1929.
at the neurula stage were placed at approximately constant temperatures
of 4° C. ± 1, 13° C. db 1. 19° C. ± 1 and 27° C. ± 1. When the
four groups of length-age data acquired from these animals were
plotted in such a way as to rule out the change in growth rate due
to temperature, that is, when all the data, after allowance is made for
appropriate thermal coefficients of growth, are plotted as though the
animals were raised at 19° C., the shape of the curve is not the same
for each group. The linear increase in the 4° sample was practically
n
188
W. T. DEMPSTER
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AMBYSTOMA MACULATL'M 189
a straight line growth. Some deviation from this type of growth was
found in the 13° data, more in the 19° data and still more deviation in
the 27° data. It is very unlikely that the weight differs in these groups.
High and low temperatures apparently affect the efficiency of the cardio-
vascular mechanism so that atypical individuals are eventually produced.
Under high temperature conditions the embryo folds around the yolk,
develops rapidly and eventually straightens its axis ; under the low
temperature yolk is not readily utilized and the head and tail buds
from the time of their formation extend away from the yolk mass rather
than lie close to it.
In all the data provided by salamander collections under natural
conditions, it may be noted that the " probable error of length " has a
more or less constant ratio to the average length determinations of
the various stages. The population may thus be considered to be
fairly homogeneous concerning the individual growth rates.
Relation of Weight and Length. — Aside from the deviation between
length and weight in the early embryonic stage, due to embryonic fold-
ings, there are certain other fluctuations. Occasional samples from
two other ponds compared with 1928 curves indicated that for a certain
weight, there were considerable variations in length. In the period
before the animals began to feed there was little difference in these
values but later the differences were marked. When the 1929 curves
are superimposed on the 1928 curves this relation is brought out clearly.
The curves of linear growth and the weight curves practically coincide
to the point X of Fig. 1. From this point to the period of metamor-
phosis the variation is great. A higher average " index of build " is
found for the data of the first year as compared with that of 1929.
Time of Metamorphosis. — It seems quite probable that the actual
time of metamorphosis under natural conditions is associated with the
conditions of life in the pond. In August, 1929, the pond under con-
sideration became dry. The growth weight as evidenced by the curve
showed a marked slowing down toward the end of July, while in the
previous year, under more favorable conditions, this was not evident
until the first week in August. During this first year, in fact, there
were specimens in the pond for at least two weeks after most of the
salamanders had metamorphosed.3 This laggard group was formed of
3 R. G. Harrison (Correlation in the development and growth cf the eye,
etc. Arch, f. Entw.-Mech., Bd. 120, 1929) has figured three curves for the
post-embryonic linear growth of A. maculatum larvae under laboratory conditions.
These curves, which are sigmoid, indicate that the rate of growth is accelerated
with increased feeding and, in contrast to the present data, that the length at
metamorphosis is constant (47-50 mm.) under different feeding conditions. A
similar curve is given by L. S. Stone (Heteroplastic transplantation of eyes
between the larvae of two species of Amblystoma. Jour. £.r/>. Zool.. Vol. 55, 1930).
190
W. T. DEMPSTER
relatively large specimens (Fig. 1, a, a', b, b'}. It seems quite prob-
able that these specimens had not yet entered the third period of growth,
i.e. the terminal period of slow growth. During the second year, when
the pond became dry, two records, one before the pond became dry
and the other immediately afterward, are available on the length and
weight of recently metamorphosed specimens. In the first of these
both values are higher than in the second. The second group was un-
doubtedly " forced " by the drying of the pond to metamorphose before
reaching the stage at which the first group metamorphosed.
Alice (1911), who has studied the seasonal succession of pond
fauna, has indicated 'that there is a periodic change in numbers of
species and individuals found in forest ponds. There is an increase
in numbers of species which is slow during the spring months and rapid
in early summer, less marked in July and in late August the number
falls to the spring value. There seems to be a correlation between the
period of highest productivity of the pond as reported by Allee and
the period of rapid growth of the salamanders recorded here.
1929
FIG. 3. Graph showing the relative percentage of water and organic sub-
stance in larval salamanders of different ages. Data of 1929.
Relations of Water, Solids and Ash to Growth. — The eggs shortly
after they were laid had a weight of 7.32 mgm. consisting of 4.98 mgm.
of water and 2.34 mgm. of solid, of which .097 mgm. was ash. During
the embryonic period the dry weight was fairly constant. Actual in-
crease was associated with increase in inorganic matter and water
(Table I). The ash percentage, however, was practically constant
while the water increased in this period from 68 to 94 per cent. After
the animals began to eat, the dry weight increased considerably so
AMBYSTOMA MACULATUM
191
that the percentage of water decreased. To the period of metamor-
phosis there was a gradual increase of inorganic matter from 1 to 2
per cent. Water per cent decreased from 94 to 85 per cent and the
percentage dry weight increased from 6 per cent to 15 per cent. This
relationship is brought out in Fig. 3. Until the animals began to feed,
growth was purely a process of hydration ; afterwards it was due both
to imbibition of water and to increase in organic and inorganic ma-
terials. These findings are in accord with the work of Davenport and
Schaper on the Anura. Recently metamorphosed specimens showed
still further decrease in the percentage of water content. Data on
the percentage of water in older metamorphosed specimens (Table II)
show that this early decrease may be later compensated. The pro-
portion of dry weight, ash and water, however, seems to be variable
for the land forms. Two specimens from an indoor aquarium in
December showed a decrease in water content to 80 per cent body weight
and an increase in inorganic matter to 4 per cent.
TABLE II
Showing the relative content of water, solids and ash in terrestrial stages of A .
maculatum.
LENGTH
WET
WEIGHT
LENGTH3
WATER
DRY
WEIGHT
ASH
WATER
DRY
WEIGHT
ASH
WEIGHT
mm.
grams
grams
grams
grams
per cent
per cent
per cent
September 1929
50.5
.603
214
.491
.112
.011
81.51
18.49
1.77
71
1.659
218
1.412
.247
.031
85.12
14.88
1.86
76
2.504
177
2.206
.298
.032
88.10
11.90
1.27
82
4.616
120
4.063
.553
.084
88.02
11.98
1.82
88
4.709
146
4.012
.697
.101
85.10
14.80
2.14
93
5.602
144
4.955
.647
.102
88.45
11.55
1.82
104
5.875
193
4.939
.936
.124
84.07
15.93
2.11
December 1928
136
9.432
269
7.848
1.584
.427
83.21
16.79
4.53
138
8.792
301
7.044
1.748
.366
80.12
19.88
4.16
SUMMARY
1. Growth in weight of embryonic and larval Ambystoma maculatum
from the time that eggs are deposited to the period of metamorphosis
may be expressed as a single sigmoid curve.
2. The length curve, except for a short period before hatching when
the embryonic axis is curved, is likewise sigmoid.
3. The Ambystoma population of a pond is quite homogeneous,
the specimens metamorphosing at approximately the same time.
192 W. T. DEMPSTER
4. Under natural conditions the relation between weight and length
from year to year seems to be constant during the stages before feeding.
Later the relationships are variable because of feeding differences.
5. Growth to the time of food ingestion is associated with imbibition
of water. Later growth to the time of emergence of the salamanders
is correlated with a process in which the percentage of water content
decreases. During this period the inorganic constituents gradually
increase.
LITERATURE CITED
ALLEE, W. C., 1911. Seasonal Succession in Old Forest Ponds. Trans. III. Acad.
Sci., 4: 126.
BISHOP, S. C, 1926. Notes on the Habits and Development of the Mudpuppy,
Necturus maculosus (Rafinesque). Bull. N. Y. State Mus.. No. 268: 5.
DAVENPORT, C. B., 1897. The role of water in growth. Proc. Boston Soc. Nat.
Hist., 28: 73.
DAVENPORT, C. B., 1899. Experimental Morphology. Part 2. Effect of Chem-
ical and Physical Agents on Growth. Macmillan and Company.
EYCLESHYMER, A. C., AND WILSON, J. M., 1910. Normal Plates of the Develop-
ment of Necturus maculosus. Normcn. zur. Entivick. dcr Wir. Heraus.
v. F. Keibel. Gustav Fischer.
PATCH, E. M., 1927. Biometric Studies upon Development and Growth in Amby-
stoma punctatum and tigrinum. Proc. Soc. E.vp. Biol. and Mcd., 25: 218.
ROBERTSON, T. B., 1923. The Chemical Basis of Growth and Senescence. J. B.
Lippincott Co.
SCHAPER, A., 1902. Beitrage zur Analyse des thierischen Wachstums. Arch,
f. Entzv.-Mcch., 14: 307.
SMITH, B. G., 1911. Notes on the Natural History of Ambystoma jeffcrsonwnum,
A. punctatum and A. tigrinum. Bull. Wis. Nat. Hist. Soc., 9: 14.
UHLENHUTH, E., 1919. Relation between Thyroid Gland, Metamorphosis, and
Growth. Jour. Gen. Physiol.. 1: 473.
WILDER, I. W., 1924. The Relation of Growth to Metamorphosis in Eurycea
bislineata (Green). Jour. E.rp. Zoo/., 40: 1.
THE EFFECTS OF TEMPERATURE CHANGES UPON THE
CHROMATOPHORES OF CRUSTACEANS
DIETRICH C. SMITH i
(From the Harvard Biological Station, Soledad, Cienfucgos, Cuba and the
Zoological Laboratory, Harvard University.}
Temperature changes as they affected the chromatophores of crus-
taceans were not neglected in the researches of early investigators; those
of Jourclain ( 1878 ) being the first recorded in the literature to consider
the possible influence of this factor. At 5°-6° C., according to his
observations, the rapidity at which color changes occurred in Nica
cdnlis was appreciably reduced, ceasing entirely as the temperature ap-
proached nearer to zero. At this point the animals were almost trans-
parent, except for areas partly covered with matted white spots. Jour-
dain removed the eyes of those crustaceans and noted that the reddish
color assumed at room temperatures, under such circumstances, dis-
appeared entirely when the temperature of the water was lowered only
to reappear again on the restoration of the temperature to its former
level. Matzdorff (1883) observed no effect whatever of either high
or low temperatures upon the chromatophores of Idotea tricuspidata.
Somewhat later however, Gamble and Keeble (1900), after a few ob-
servations upon Hippolyte varians, reported observable color response
following exposure to heat and cold. Their specimens in common with
most other crustaceans possessed several differently colored pigments,
reds and yellows predominating, located with one exception in discrete
bodies or chromatophores. During the day the reds and yellows were
usually expanded, but at night these pigments were retracted into their
chromatophore centers and if it were not for a blue pigment, diffused
at this time throughout the tissues and free from any chromatophore.
the animals would be colorless. Gamble and Keeble selected three of
these transparent blue prawns, which they called " nocturnes," and
placed one in water at 15.5° C. (60° F.), one in water at 8° C. and
the last in water at 32° C. (93° F.). The first animal, in reality the
control as it was kept under normal temperature conditions, turned
greenish-brown as was to be expected. The second one at 8° C. main-
tained the nocturnal blue color, showing after thirty-five minutes some
traces of recovery, though one hour later this was still incomplete.
1 National Research Fellow in the Biological Sciences.
193
194 DIETRICH C SMITH
The prawn placed in 32° C. was almost immediately killed by the heat,
but remained nevertheless a brilliant nocturne for several hours, even
though during the first five minutes of this experiment the temperature
descended to 28° C. (83° F.).
Menke (1911) experimenting with Idotca, produced a contraction
of the chromatophores in about 15 minutes by raising the temperature
of the water from 11.5° C. to 20.5° C. This contraction was sustained
for about one hour when the pigment partially re-expanded. Five and
one-half hours later on, lowering the temperature to 12° C., the chro-
matophores again became completely expanded. But if at this time,
instead of lowering the temperature of the water, it was raised to 30°
C., the chromatophores also re-expanded completely. Complete ex-
pansion was also produced by lowering the temperature from 14° C.
to 4° C. Doflein (1910), working with Lcander xiphias placed several
specimens in complete darkness at 5°-8° C. for two to three days. At
the lapse of this time the chromatophores and the tissues of the animal
were completely impregnated with blue pigment, all other pigments
being completely retracted into their respective centers. But as Fuchs
(1914) points out, these results might follow either from continued
exposure to cold or to darkness. Megusar (1911) working with
Gelasimus, Potamobius, Palceinonetes, and Palcstnon, observed an ex-
pansion of the chromatophores on the sudden transfer of any of these
animals from water at 16°-18° C. to water at 25°-30° C. Similarly
a contraction of the chromatophores followed a sudden transfer from
water at 25°-30° C. to cooler water at 16°-18° C.
The results of these experiments are admittedly confusing, though
as Fuchs (1914) observed, no reasonable doubt can be entertained as
to the ability of temperature changes to produce an effect of some
sort upon the pigmentary responses of the crustaceans. Further in-
vestigation of the subject was thought desirable in the hope of ascer-
taining, if possible, just how important a factor the action of heat
and cold is in determining the distribution of the chromatophore pig-
ment of this group. For this purpose a fresh water shrimp, kindly
identified for me as Macrobrachiuni acanthurus Wiegmann by Dr. W.
L. Schmitt of the United States National Museum, was selected as the
subject of the experiments. These shrimps were obtainable in large
numbers from the Arimao river and its immediate tributaries in the
vicinity of the Harvard Biological Station, Cienfuegos. Cuba. I am
happy to acknowledge my thanks and appreciation to Dr. Thomas Bar-
bour for his assistance in putting the facilities of the Harvard Cuban
Station at my disposal.
When caught, the chromatophore pigments of these shrimps were
EFFECTS OF TEMPERATURE ON CHROMATOPHORES 195
more or less extended, giving the animal a reddish-brown color. This
color varied somewhat with the size of the animal, the smallest being
the lightest. As collected, the shrimps ranged from 2 cm. to 10 cm. in
length, measured from rostrum to telson. Males varying from 2 cm.
to 3 cm. in length were selected for the experiments. Females were
rejected, as at this time their abdomens were practically opaque owing
to the fact that they were carrying their eggs.
A word or two regarding the color changes of Macrobrachiwn will
be an aid to the understanding of what is to follow. Taken to the
laboratory and placed in white glazed porcelain bowls, the shrimps in
daylight soon became transparent and colorless ; microscopical examina-
tion of the abdomen and telson showing the chromatophores to be
completely contracted. If such animals were placed upon a black back-
ground, they assumed a dark reddish color with the chromatophores
well expanded. A somewhat superficial examination disclosed the
presence of two types of chromatophore pigments, both apparently
located in the same chromatophore, one being reddish-brown in color
and the other yellow. These facts were derived from a microscopical
examination of the living pigmentary units. Detailed histological study
of the chromatophores was not attempted.
Occasionally under somewhat varying conditions, animals were seen
with an unmistakable bluish color observed both in the light and in the
dark. The blue pigment producing this color when examined under
the microscope was clearly not confined to the chromatophores, but was
free in the tissues, though its concentration did appear greater about
the processes of the pigmentary centers. Gamble and Keeble (1900)
reported that a blue color was the regular accompaniment of the noc-
turnal phase of Hippolyte varians, a phase characterized by the retrac-
tion of all other pigments into their respective centers. According to
their statement, the blue pigment in Hippolyte responsible for the noc-
turnal coloration arises as a discharge product of the chromatophores,
leaving these organs on the contraction of the yellow and red pigments,
and apparently being derived from them. Left free in the tissues, the
blue pigment is permanently divorced from its point of origin and
persists in coloring the body of the prawns until it eventually disappears.
In these experiments upon Macrobrachium acanthurus determina-
tions were first made of the action of heat and cold upon the color
changes in normal shrimps. The method used was as follows : Two
or three animals were placed in white porcelain bowls and covered
with water at room temperatures. To this was added either warm or
cold water, as desired, until the particular temperature demanded by
the experiment was reached. Here it was either kept constant or
196 DIETRICH C SMITH
altered as necessary. The responses of the shrimps to temperature
changes when kept upon a black background were tested in the same
manner.
Numerous experiments with normal shrimps adapted to white back-
grounds demonstrated conclusively that such animals darkened when
exposed to temperatures either high enough or low enough to stimulate
the chromatophores. Surprising as it may seem, once the response
was complete, no criteria of any sort could be established separating
the darkening produced by heat from that produced by cold. The color
assumed in either circumstance was a deep red-brown, while micro-
scopical examination showed the pigments of the chromatophores to
be equally well extended at high and low temperatures. The protocols
of the two following experiments may be taken as typical of many
others :
2:13 — 28° C. Two colorless shrimps previously kept on a white background
for a day were placed in a white porcelain bowl and small pieces
of ice added to the water.
2 : 16—10° C. No change in color.
2:18 — 10° C. Shrimps appear slightly reddish.
2:22 — 15° C. Shrimps somewhat darker.
3:00 — 15° C. Shrimps a pronounced brown.
3:25—28° C. Shrimps still brown.
9 : 30—28° C. Shrimps colorless.
2 : 13 — 28° C. Two colorless shrimps previously kept on a white background
for a day were placed in a white porcelain bowl and warm water
gradually-added.
2 : 16—36° C. No change in color.
2:18—36° C. Shrimps faintly reddish.
2 : 21 — 36° C. Shrimps pronouncedly brown.
3:00—28° C. Shrimps colorless.
In all of the experiments the appearance of the red-brown color
was more rapid at high temperatures than at low. With heat only
10-15 minutes were necessary to make the animal completely dark,
while with cold 30-45 minutes were required. But regardless of
whether the shrimps were exposed to heat or to cold, once the point
of maximal darkening was reached, the intensity of the color was equal
in both cases.
When the animals were subjected to warmth the lowest temperature
capable of expanding the chromatophores was found to be 35° C., while
temperatures as high as 40° C. could be withstood without subsequent
death, though at this temperature and slightly below it, the shrimps re-
mained motionless, and with the exception of gill movements showed
no signs of life. Therefore, within the range of 35° C. to 40° C.
the color of the shrimp is determined by the temperature of its en-
EFFECTS OF TEMPERATURE ON CHROMATOPHORES 197
vironment rather than the type of background on which it happens to
be. Similarly shrimps placed in water colder than 6° C. died immedi-
ately, while any temperature above 15° C. and, of course, below 35°
C., failed to produce an expansion of the chromatophore pigment.
Therefore, between 6° C. and 15° C. the color of the shrimp is also
determined by temperature rather than background. It might be well
to mention here that the temperature of the water in which the shrimps
normally lived ranged from 25° C. to 30° C.
As a check upon these results and to determine whether there was
any possibility of temperature changes producing a contraction of the
chromatophore pigment, experiments similar to those just described
were performed upon dark shrimps while they were upon a black back-
ground. But such animals when exposed to various temperatures rang-
ing between 6° C. and 40° C. showed no alteration whatever in the
expanded condition of their chromatophores.
Recovery of normal color and activity was the rule when shrimps
subjected to effective temperatures were returned to water at about
28° C. But this recovery was more rapid in shrimps treated with
warmth than those treated with cold. The former required but 30 to
40 minutes, and the latter 6 to 7 hours before normal temperatures and
a white background again brought their chromatophore pigment to
complete contraction.
Tests were also made of the responses of blinded shrimps to tem-
perature changes, blinding being accomplished by cutting off the eyes
at the base of the eye stalk. Shortly after this operation the pigment
of the chromatophores began to expand and within an hour or so, re-
gardless of background, this expansion was complete and the animals
were red-brown in color. Shrimps in this condition placed in warm
and cold water and left so for an appreciable length of time — two to
three hours — showed no color change of any sort. Similarly shrimps
anaesthetized with 0.05 per cent chloretone. failed to show color re-
sponses to either heat or cold. Neither high nor low temperatures are
then capable of exerting any contracting effect upon the pigment of
the chromatophores, even when these organs are removed from the in-
fluence of any stimuli directly or indirectly produced by the retina.
As Perkins (1928) has shown in Pahcnwnetcs, the withdrawal
of pigment into the centers of the crustacean chromatophore is con-
trolled by a hormone elaborated in the eye stalks, a fact which was
later substantiated by Roller (1928) on Crangon and Lcandcr. Pos-
sibly then, as temperature changes acted to expand the chromatophore
pigment, there was an inhibition by heat or cold of the mechanism
controlling the production of this contracting secretion. Before such
198 DIETRICH C. SMITH
an hypothesis could be tested, it was necessary to ascertain definitely
whether or not such a secretion played a part in governing the chro-
matic responses of Macrobrachinin. Consequently Perkins' experi-
ments were repeated upon this animal. Five or six shrimps were
paled by placing them upon a white background for a day or more,
after which their eyes were removed and thoroughly macerated in 2
cc. of 0.7 per cent NaCl. One tenth cc. of the resulting solution was
then injected into the abdomens of several shrimps in the dark condition
and with well expanded chromatophores. In all cases the following
reactions were noted : Shortly after injection, 5-10 minutes, the shrimps
began to assume a bluish color which gradually increased in intensity
until within 30 minutes it had reached its maximum ; this was followed
by a gradual retraction of the pigment into the chromatophore centers,
a retraction which persisted until the shrimps had assumed a transparent
blue color. These results closely parallel the effects reported by Perkins
in Paltzmonetes, even to the formation of the blue color, and offer com-
plete substantiation of his findings. As control experiments 0.1 cc.
of the extract was injected into the abdomens of several shrimps in
the light condition with no observable effect. Similarly injection of
0.1 cc. of 0.7 per cent NaCl into blinded shrimps produced no pig-
mentary response.
The existence of a hormone produced by the action of light upon
the retina and released into the circulation to affect a contraction of the
chromatophore pigment is then demonstrated in the shrimps used in
these experiments. Is the formation of this hormone in any way in-
hibited by either high or low temperatures? Apparently not, as the
following experiments show. Two sets of extracts were prepared,
one from the eyes of shrimps darkened on a white background by warm
water (37° C.) and the other from the eyes of shrimps darkened on a
white background by cold water (15° C.), both groups being subjected
to their respective temperatures for the same length of time, namely
45 minutes. Two sets of blinded shrimps were then selected, one set
being injected with 0.1 cc. of one extract and the other set with the
same amount of the other extract. These animals were then replaced
in water at room temperature and the results noted. In both cases these
darkened shrimps paled within the specified length of time, but with
this difference,- — the blue color previously described appeared in only
one out of three of the shrimps injected with the extract prepared from
the eyes of animals kept at low temperatures, while it appeared in all
of the shrimps injected with the extract prepared from the eyes of
animals kept at high temperatures. Neglecting for the present the
significance of this variation, it is obvious that extreme temperatures
EFFECTS OF TEMPERATURE ON CHROMATOPHORES 199
in no way inhibit the manufacture or even the potency of the chro-
matophore-contracting hormone elaborated by the eye stalks.
This gives us a clue as to the manner in which heat and cold affect
the chromatophores of crustaceans. Unfortunately, these experiments
cannot give us a conclusive solution to this problem, though the data
at hand strongly indicate a direct effect. Positive information is not
to be derived from experiments on limbs or bits of integument isolated
from the bodies of these shrimps, as the chromatophores of such ex-
cised pieces expand at once. Consequently subjecting such preparations
to temperature variations accomplished no change in the distribution
of their expanded chromatophore pigment. But since experiments on
blinded and chloretonized shrimps give no evidence of any other type
of response to temperature changes than those seen in normal light
shrimps, and since neither heat nor cold affect the secretion of the
chromatophore-contracting substances elaborated in the eye stalks, it
seems reasonable to assume that the responses of the chromatophore
pigment of crustaceans to high and low temperatures are direct.
A word or two in regard to the blue color and its relation to tem-
perature changes. Keeble and Gamble (1903) state that the blue color
observed in nocturnal Hippolyte disappears completely at 60° C.,
while, as shown in an earlier paper (Gamble and Keeble, 1900), this
color is maintained at 8° C. under conditions that in other prawns kept
at a somewhat higher temperature (15° C.) produce its loss. This
latter observation is in accord with the experiments of Doflein (1910)
on the occurrence of a blue color in Lcandcr when the animals were
kept for an extended period in darkness and cold. But aside from
this, it is perhaps worthy of note that, as already mentioned, blinded
animals injected with the extracts prepared from the eyes of shrimps
subjected to cold showed only in one third of the cases a visible blue
color, whereas blinded shrimps injected with an extract from the eyes
of animals kept at high temperatures never failed to become pro-
nouncedly blue. Furthermore, throughout the course of these experi-
ments the blue color was repeatedly observed in connection with
shrimps subjected to high temperatures, while the records disclose only
one instance where it was seen in connection with shrimps exposed to
low temperatures ; a case where an animal kept at 6° C. for about 30
minutes turned blue when returned to 28° C. Perhaps this indicates a
relationship between changes in temperature and the appearance of the
blue color worthy of further investigation.
A survey of the work of previous investigators dealing with the
action of heat and cold upon the crustacean chromatophore reveals a
wide divergence of opinion. As we have already seen, Gamble and
200 DIETRICH C. SMITH
Keeble (1900) claimed that both high and low temperatures produce
or at least maintain a retraction of the pigment, a statement with which
Jourdain (1878) and Doflein (1910) are in agreement as far as low
temperatures are concerned. Menke (1911), on the other hand, reports
that in Idotca extreme high and low temperatures tend to produce an
expansion of the chromatophore pigment, though moderately high
temperatures (20°-25° C.) lead to a contraction. Megusar (1911),
however, observed an expansion of the chromatophore pigment with
heat and a contraction with cold, though this author apparently did not
subject his animal to temperatures lower than 15° C. In the most re-
cent communication Roller (1927) maintains that temperature changes
have no effect whatever upon the distribution of the chromatophore
pigment in Cranyon.
The results of the present writer's investigations are more in accord
with those of Menke than with those of other workers, since Menke
also observed an expansion of the pigment at both ends of the effective
temperature scale. Macrobrachiitui acanthtints is a semi-tropical form,
habituated to water normally remaining at 25°— 30° C. the year around.
Therefore, the response to temperature changes of such forms might
reasonably be expected to vary somewhat from those seen by Menke
in Idotca, a form adapted to life in cooler waters. Consequently we
need not be greatly concerned when Idotea responds to temperatures
of 20°-25° C. and Cuban shrimps do not. For the latter such tem-
peratures are obviously not warm. The important feature is that for
both types an expansion of the chromatophores is produced on exposure
to temperatures either extremely high or extremely low.
Among the lizards and amphibians high temperatures as a rule
produce a contraction of the chromatophores and low temperatures
an expansion. Among the vertebrates in general, variations from this
scheme are found in certain amphibians whose chromatophores are ap-
parently insensitive to heat and among the fishes, where innervated
melanophores react to warmth by expansion and to cold by contraction.
The denervated melanophores of fishes respond, however, to tempera-
ture changes as do the chromatophores of lizards and amphibians.
Since in these last two groups such reactions are presumably direct,
and since they are certainly direct in denervated fish melanophores, it
is permissible to say that among the vertebrates the independent re-
sponse of the chromatophore to heat is a contraction and to cold an
expansion. In the crustacean chromatophore where there is a high
probability that reactions to temperature variations are direct, though
this is admittedly not certain, an expansion of the chromatophore pig-
ment is produced both by heat and cold. On the basis of our present
EFFECTS OF TEMPERATURE ON CHROMATOPHORES 201
knowledge then there seems to be little resemblance between the pig-
mentary reactions to heat and cold in the vertebrates and the crustaceans.
Among the vertebrates, especially in the lacertilians, the ability of
the pigment cells to respond to temperature changes is sometimes given
a thermo-regulatory significance. But the crustacean chromatophorc
can certainly serve no such purpose, especially as the chromatic re-
sponses of this group are controlled by factors other than heat and
cold. It is inconceivable, for instance, that the form used in these
experiments would ever encounter in its usual environment temperatures
high enough or low enough to bring about changes in the distribution
of its chromatophore pigment other than the distribution determined
by background or light intensity.
SUMMARY
1. Expansion of the chromatophores of Macrobrachium acaiit hunts.
a Cuban shrimp, follows immersion of these animals in fresh water
at any temperature between 6° and 15° C. or between 35° and 40° C.
This reaction occurs regardless of the background upon which the
shrimp is placed. Between 15° C. and 35° C. the chromatophores of
this shrimp expand when the animal is placed upon a black background
and contract when the animal is placed upon a white background.
2. In blinded and chloretonized shrimps, the chromatophores are
expanded and this expansion is in no way altered by changes in back-
ground or temperature.
3. Neither high nor low temperatures have any effect upon the
potency or manufacture of the chromatophore-contracting substance
elaborated by the eye stalks.
BIBLIOGRAPHY
DOFLEIN. F., 1910. Lebensgewohnheiten und Anpassungen bei dekapoden Krebsen.
Festschrift. Hertwig, 3: 215.
FUCHS, R. F., 1914. Der Farbenwechsel und die chromatische Hautfunktion der
Tiere. Wintcrstein's Handbuch der vcryl. Physiol., Jena. 3: Halfte 1,
Teil 2, 1344.
GAMBLE, F. W., ANL KEEBLE, F. W., 1900. Hippolyte varians : a Study in Colour-
change. Quart. Jour. Micr. Sci.. 43: 589.
JOURDAIN, M. S., 1878. Sur le changements du couleur de Nika edulis. Cornet.
rend. Acad. Sci.. Paris, 87: 302.
KEEBLE, F. W., AND GAMBLE, F. W., 1903. The Color Physiology of Higher
Crustacea. Phil. Trans. Roy. Soc.. London, 196B: 295.
ROLLER, G., 1927. Uber Chromatophorensystem, Farhensinn und Farbwechsel bei
Crangon vulgaris. Ztschr. vcrgl. Physiol.. 5: 191.
ROLLER, G., 1928. Versuche uber die inkretorischen Vorgange beim Garneelenfarb-
wechsel. Ztschr. rergl. Physiol., 8: 601.
202 DIETRICH C. SMITH
MATZDORFF, C., 1883. Ueber die Farbung von Idotea tricuspidata Desm. Jena.
Ztsclu: f. Mcdizin u, Naturw., 16: 1.
MEGUSAR, F., 1911. Experimente iiber den Farbwechsel der Crustaceen. (I.
Gelasimus. II. Potamobius. III. Paljemonetes. IV. Palsemon.) Arch.
f. Entiv.-Mcch. d. Org., 33: 462.
MENKE, H., 1911. Periodische Bewegungen und ihr Zusammenhang mit Licht
und Stoffwechsel. Arch. gcs. Physio!., 140: 37.
PERKINS, E. B., 1928. Color changes in Crustaceans, especially in Palaemonetes.
Jour. Exp. Zool, 50: 71.
Vol. LVIII, No. 3
JUNE, 1930
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
UNUSUAL TYPES OF NEPHRIDIA IN NEMERTEANS
WESLEY R. COE
OSBORN ZOOLOGICAL LABORATORY, YALE UNIVERSITY
The earlier investigators on the morphology of the nemerteans
failed to find in any of the species of the family CephalotrichicUe the
pair of longitudinal nephridial ducts which are so conspicuous in most
nemerteans, and some of them came to the erroneous conclusion that
in this family the nephridia are absent. Wijnhoff (1910) corrected
this error, proving that a well-developed excretory system is actually-
present in the females of several species, but of a different nature
than had been found up to that time in any nemertean. Instead of
having all the terminal organs connected with a single longitudinal
canal of comparatively large size, each end bulb has its own efferent
duct leading to the exterior of the body.
Wijnhoff was unable to determine the exact configuration of the
organs or the details of their histological structure, although she de-
scribes and figures the terminal organs in their relation with the lateral
blood vessels and shows the groups of granular cells adjacent to the
end bulbs.
METANEPHRIDIA IN CEPHALOTHRIX MAJOR
On the coast of California occurs a species of Cephalothrix (C.
major Coe), in which the worms reach a size many times larger than
those of other known species of the genus and in which the histological
structure of the extremely complex excretory organs is clearly shown.
In this species, as in those studied by Wijnhoff, there is a series of
isolated nephridia in close contact with the lateral blood lacuna- on
each side of the body. Each nephridium consists of a multinucleate
terminal organ, or end bulb, with slender flagella on its free border,
and with a narrow canal leading to an enlarged glandular and con-
voluted tubule and thence by an efferent duct to a minute pore on the
dorsolateral aspect of the body (Figs. 1, 4, 5, 8, 9).
203
14
ebL
tern
VV f **/••, O t, '« *• ,. ri
FIGS. 1-4. Metanephridia of C. major. FIG. 1. Entire nephridium with
widely opened efferent duct. FIG. 2. Terminal organ (nephrostome) associated
with a large area of gelatinous parenchyma. FIG. 3. Terminal organ close be-
neath epithelial lining of blood lacuna. FIG. 4. Diagram of portion of body near
anterior end of intestinal region, showing position of nephridium (nc) and efferent
duct; bl, blood lacuna; bm, basement membrane of body wall; con, convoluted
tubule; ebl, epithelium of blood lacuna; ictn, hn, ocm, inner circular, longitudinal
and outer circular musculatures ; iep, intestinal epithelium ; in, integument ; In,
lateral nerve; ncd, efferent duct; nc[>, nephridiopore ; par, parenchyma; ps, probos-
cis sheath; re, rhynchocoel ; tc, terminal chamber; to, terminal organ.
TYPES OF NEPHRIDIA IN NEMERTEANS
205
The number of such independent nephridia is very large, more than
300 being found on each side of the body in an adult worm measuring
a meter or more in length. All of them are found in the anterior
half of the body. The most anterior ones border the blood lacunae
anterior to the mouth, the others being situated beside the lateral
lacuna? in the region of the foregut and extending posteriorly beyond
the anterior limits of the gonads. Although the nephridia are not
paired on the two sides of the body, there is more or less regularity
in their arrangement. Anteriorly they are more widely spaced than
somewhat farther back, and they are most closely placed and of max-
imum size in the region where the foregut opens into the intestine, that
is, in the region somewhat posterior to the most anterior gonads. More
posteriorly they are not only farther apart, but are appreciably smaller
and with fewer nuclei.
\ ebi
• . -.. 7| J
FIGS. 5-8. C. major. FIG. 5. Nephrostome (ne) imbedded in bulbous mass
of gelatinous parenchyma (par). FIG. 6. Nephrostome close beneath epithelium
of blood lacuna (cbl). FIG. 7. Transverse section of nephrostome, showing
outer circle of nuclei (n) belonging to the flagella-bearing cells and the inner
circle of smaller nuclei (n") lining the end canal. FIG. 8. Diagram of nephridium
in longitudinal section.
The actual distance between adjacent nephridia is commonly from
0.1 mm. to 0.2 mm. in the mounted sections, although some are sep-
arated by only 0.05 mm. or twice the diameter of the terminal bulb.
All are placed in a very similar situation with regard to the blood
lacunae and the nerve cords, always lying near the lumen of the blood
space in the angle adjacent and somewhat dorsal to the nerve cord
(Fig. 4). In many cases the terminal organ is situated on a low pa-
pilla, formed of the endothelium of the blood lacuna and its underlying
basement membrane. This papilla projects somewhat into the lumen
of the blood space, so that the greater part of the surface of the ne-
phridium comes in close proximity to the blood (Figs. 5, 6).
206
WESLEY R. COE
Each nephridium consists of three principal parts, (o) the terminal
bulb, (b*) the convoluted tubule, and (c) the efferent duct (Figs. 1,
8, 17).
-bl
tc-
bl
SLcon
con
-tc
,-nep
FIG. 9. Diagram of nephridium of C. major, showing slender flagella in
lumen of convoluted tubule (con) ; 9«, small portion of convoluted tubule with
flagella.
FIG. 10. Diagram of nephridium of C. spirali-s, showing both nephrostome
and convoluted tubule in bulbous projections on wall of blood lacuna (bl).
FIG. 11. C. spiralis. Section of nephrostome (tc) and loops of convoluted
tubule (con) in single bulbous projection of wall of blood lacuna (bl).
(a) The Terminal Bulb {Nephrostome). — This lies in all cases in
close proximity to one of the lateral blood lacunae, which are usually
much distended throughout the nephridial region (Fig. 4). Sometimes
the bulb occupies a small papilla projecting somewhat into the lumen
of the blood space and separated from the latter only by a thin covering
of parenchyma and the endothelial lining of the blood vessel (Fig. 3).
More often it lies deeper in the tissues and separated from the blood
vessel and from the surrounding tissues by the mass of gelatinous
parenchyma in which it is always imbedded (Fig. 2).
TYPES OF NEPHRIDIA IN NEMERTEANS 207
The terminal bulb is a mushroom-shaped structure with an ex-
tremely fine tubular stalk and with the free convex surface projecting
into a hemispherical chamber (Fig. 3). Occasionally it can be demon-
strated beyond question that a number of delicate flagella project freely
into the terminal chamber and that the lumen of the latter is continuous
with the slender tubule which pierces the stalk of the mushroom-shaped
end bulb (Figs. 5, 8). The flagella are obviously projections from
the free surface of the end bulb, and their vibration in life doubtless
serves to draw into the tubule the fluid which collects in the vacuole.
The wall of the chamber consists of a very delicate membrane with
one or two oval nuclei on its inner surface (Fig. 5). Two types of
cells are found in the terminal organ, (a) those which compose the
mushroom-shaped body and bear the flagella and (b) those belonging
to the tubule of the stalk. In neither part are there distinct cell
boundaries, but in the former the nuclei are much larger than in the
latter (Figs. 5, 6). The cytoplasm on the hemispherical free surface
of the terminal organ is dense and firm, forming a suitable support
for the flagella. In the deeper part of this cytoplasm upwards of 20
oval nuclei are imbedded, six to eight of these being seen in a single
longitudinal section (Figs. 5, 6, 8). In a cross section, however, the
entire number may be shown (Fig. 7).
The canal in the tubular stalk is very slender and only in exceptional
cases is the lumen demonstrable, due to the state of contraction at the
moment of preservation. The nuclei of this canal are often only half
the diameter of those of the terminal organ, or even less (Figs. 7, 8).
Their number seldom exceeds a dozen. The size of the terminal bulb
varies considerably as may be seen from figures 5-8, which are all
drawn to the same scale, the transverse diameter being usually from
0.024 to 0.027 mm., although the smallest are only 0.018 mm. across
and the largest as much as 0.03 mm.
(b) The Convoluted Tubule. — The slender canal in the stalk passes
through the parenchyma surrounding the terminal bulb and then en-
larges suddenly into a coiled tubule of much greater diameter and
often with a conspicuous lumen (Figs. 1, 3, 9). This part of the
nephridium is imbedded in a restricted mass of parenchyma more or
less continuous with that surrounding the terminal bulb and extending
into the inner portion of the longitudinal muscular layer. The con-
figuration of the convolution is quite variable, as a comparison of the
various figures will show. Sometimes there is but a single loop, but
usually the tubule twists spirally in an irregular manner, parts of it
appearing in four or more serial sections. The cytoplasm is coarsely
granular, with numerous inclusions, but the nuclei are not separated
208 WESLEY R. COE
by distinct cell boundaries (Fig. 9). Tbis part of tbe nephridium
closely resembles in structure the main longitudinal canal in those forms
having compound nephridia (protonephridia), and its coarsely granular
and vacuolated cytoplasm indicates that it has an important excretory
function.
Long slender cilia project from the inner walls of the convoluted
tubule ; giving the appearance of fine threads lying lengthwise in the
lumen and extending in the direction of the efferent duct (Figs. 9, 9a).
(c) The Efferent Duct. — The convoluted tubule leads directly into
an extremely slender efferent duct which passes radially, that is, dorsally
and laterally, in one of the connective tissue dissepiments separating
the bundles of longitudinal muscles. It then pierces the outer circular
musculature, the basement membrane and the integument, to open by a
minute pore on the dorsolateral surface of the body (Figs. 1, 4, 9).
The course of the duct may be so perfectly straight that nearly the
entire length may be contained in one or two of the serial sections, but
it is naturally seldom that the plane of the section coincides exactly
with that of the duct.
The wall of the duct is extremely thin, but the cytoplasm bears
numerous oval nuclei throughout its entire length. Even where the
duct pierces the integument it has its independent nucleated lining (Fig.
1), as Wijnhoff (1910) has already demonstrated for other species.
DISCUSSION
Excretory organs of this type have not been described for any
of the other groups of Plathelminthes. In some of the Annelids,
however, organs of somewhat similar structure are found, each with
a ciliated funnel (nephrostome) opening into the body cavity and with
a convoluted tubule, often of great complexity.
In the nephridium of Cephalothri.v the mushroom-shaped end bulb
is apparently homologous with the nephrostome of the annelid and may
be so designated. The terminal chambers in Ccplialothri.\- then repre-
sent minute ccelomic cavities, the fluid contents of which are in com-
munication with the outside world through the nephridia, exactly as
in annelids.
This type of excretory organ may be designated a metanephridium
in order to distinguish it from the more usual type, protonephridium,
found in nemerteans (Fig. 17, 5), where each of the numerous end
bulbs consists of a single flagellated cell imbedded in the body paren-
chyma and with its free border directed toward the efferent duct.
TYPES OF NEPHRIDIA IN NEMERTEANS 209
PHYSIOLOGY OF THE METANEPHRIDIUM
The process of excretion by this type of nephridium is presumably
accomplished by the withdrawal of waste-containing fluids from the
surrounding gelatinous parenchyma, and thus indirectly from the nearby
blood, by means of the ciliary action of the nephrostome. These fluids
then pass to the convoluted tubule, the cells of which are specialized
for the excretion of additional waste materials or for the absorption
of any contained nutrients, or both ; after which the remaining fluid
is forced through the efferent duct to be discharged through the ne-
phridiopore. The movement of the fluid in the convoluted tubule is
doubtless facilitated by the slender flagella with which it is provided.
The numerous granules and minute vacuoles in the cytoplasm of this
part of the nephridium are indicative of its excretory function, as
Strunk (1930) has recently demonstrated experimentally for Annelids.
EXCRETORY SYSTEMS IN CEPHALOTHRIX SPIRALIS
In another species of the genus, C. spiralls Coe (formerly considered
specifically identical with C. lincaris Oersted of Europe) of the New
England coast, the excretory system of the female is likewise of the
metanephridial type. In the two sexually mature males of this species
which were available for study, however, no metanephridia were found,
the excretory system consisting of a pair of clusters of protonephridia
situated on the median walls of the cephalic blood lacunae (Figs. 13, 15).
The meaning of this apparent sexual dimorphism is by no means clear
and will require further investigation on immature forms of both sexes.
It may be remembered in this connection, however, that a somewhat
similar condition prevails for the reproductive organs of some of the
bathypelagic nemerteans, the males of which have only a few pairs
of spermaries (and these are situated in the head), while the females
are provided with numerous ovaries on each side of the body in the
intestinal region (Coe, 1920). It will be recalled also that in the
Annelids and other groups of invertebrates the larval excretory system
is frequently of the protonephridial type, and is later replaced by the
metanephridia. It seems possible that the sexual dimorphism in Ccph-
alothrix may be similarly accounted for, assuming that the males have
retained the primitive protonephridia, and that these are replaced in
the females by the more complicated, and presumably more efficient
metanephridia. Studies on immature individuals of both sexes will
be made in the near future.
210
WESLEY R. COE
METANEPHRIDIUM
The metanephridium of the female C. spiralis is similar to that of
C. major, but is considerably larger in proportion to the size of the
body and is more intimately associated with the lumen of the blood
lacuna (Figs. 10, 11, 12, D, E, F). The average diameter of the
nephrostome in this smaller species is about 0.023 mm., with some as
small as 0.012 mm., as compared with 0.018 to 0.03 mm. in C. major.
bl
bl
'•;"••'••: tei
B
FIG. 12. Diagrams of the various types of nephridia found in nemerteans,
showing the relation of each to the blood lacuna; A, protonephridium, characteristic
of most nemerteans, imbedded in parenchyma close beneath blood lacuna ; B,
protonephridium of C. spiralis, male, hanging free in blood lacuna ; C, protone-
phridium of Geonemertes, imbedded in parenchyma; D, E, F, metanephridia of
C. spiral is, female, in successive stages of differentiation.
The shape as well as the position of the nephrostome varies consid-
erably in the same individual. Only occasionally is the organ circular
in surface view, with the opening of the end canal in the center. More
often the opening is considerably eccentric, showing more nuclei on
one side than on the other in vertical section (Figs. 10, 11, 12, F).
In some cases the organ is heart-shaped or distinctly bilobed, with the
opening in the indentation (Fig. 12, D, E).
In regard to their position relative to the blood lacunae, both the
nephrostome and the entire convoluted tubule may lie in the paren-
chyma beneath the epithelium and make no encroachment whatever on
the lumen of the blood space or both may form bulbous projections
into the lumen (Figs. 10, 11, 12, D, E, F). As a general rule, how-
ever, the terminal chamber projects freely into the blood space, while
considerable gelatinous parenchyma lies between the convoluted tubule
and the epithelial lining of the lacuna.
In the females a single pair of metanephridia is situated on the
TYPES OF NEPHRIDIA IN NEMERTEANS
211
dorsolateral borders of the cephalic lacunae not far anterior to the
mouth. The convoluted tubule of this nephriclium is greatly elongated
anteroposteriorly, with the slender efferent duct at its posterior end.
Anterior to the midgut the nephritlia are widely scattered, increasing
in abundance in the anterior portion of the gonad region and becoming
less numerous beyond the end of the proboscis sheath. At least a
hundred pairs are found in an individual of moderate size. In the
mounted sections the distance between adjacent nephriclia is usually
0.1 to 0.2 mm. in the anterior midgut region.
The nephrostome is frequently situated on a horizontal level with
the lateral nerve cord, with the convoluted tubule either anterior or
posterior and slightly dorsal thereto, but sometimes the nephrostome
is found much nearer the ventral side of the body. In the latter case
the efferent duct passes dorsally above the level of the nerve cord before
leading radially to the nephridiopore on the dorsolateral surface of
the body (Fig. 14).
13
FIG. 13. C. spiralis. Portion of transverse section through head of male,
showing terminal chambers (tc) of nephridium on median wall of blood lacuna
(/>/); con, convoluted tubule opening to surface through efferent duct (ncd) ;
hn, buccal nerve; cm and hn, circular and longitudinal musculatures; In, lateral
nerve cord ; p, proboscis.
FIG. 14. C. spiral is. An unusually large nephridium from the intestinal
region posterior to the end of the proboscis sheath, showing the terminal chamber
(tc) adjacent to the blood lacuna (cbl) and the voluminous convoluted tubule
(con) leading dorsally to join the slender efferent duct (ncd) ; in, integument;
lu. lateral nerve; hn, ocm, longitudinal and outer circular musculatures.
212 WESLEY R. COE
The nephrostome is evidently capable of considerable change of
shape by contraction and extension, for the mouth of the end canal
joining the terminal chamber may be widely opened (Figs. 10. 11, 14)
or it may be almost completely closed. The terminal chamber also
may be distended with fluid and thus widely separated from the ciliated
surface of the nephrostome (Fig. 10) or the fluid may be withdrawn,
allowing the thin wall of the chamber to lie close upon the nephrostome.
A few double nephridia were found, and Wijnhoff (1910) observed
the same condition in one of the species which she studied. The
twinning may involve only the terminal organ and its accompanying
end canal or it may include also the entire convoluted tubule. In the
latter case two complete nephridia join a single efferent duct.
PROTONEPHRIDIUM
Mention has been made of the fact that metanephridia have been
found thus far only in the females of the several species studied. Only
two sexually mature males of C. spiralis with suitable fixation have
been available for study and both of these were provided with ex-
cretory organs of the protonephridial type (Figs. 13, 15).
Each of the two individuals had a single pair of these organs situ-
ated on the median borders of the cephalic blood lacunse between the
brain and the mouth. Each nephridium consists of a cluster of fifty
or more end organs connected with a branched collecting tubule which
leads dorsally along the median face of the lacuna (Figs. 13, 15). On
the dorsomedian angle of the lacuna the collecting tubule opens into
the convoluted tubule, from which the efferent duct leads to the ne-
phridiopore on the dorsolateral border of the head (Fig. 13).
Each of the end organs consists of a single cylindrical or goblet-
shaped cell (flame cell) attached to the wall of the lacuna and more or
less completely surrounded by the blood. The cytoplasm of the cell
and the cell membrane are extended to form a central oval cavity in
which the slender flagella may swing freely (Figs 12, B, 15). The
proximal end of the flame cell is narrowed to a slender canal (end
canal) which joins with others to form the collecting tubule.
Such an intimate association of flame cells with the blood is known
for other species of nemerteans, but in no case is there any direct
communication between the blood and nephridial systems. In order
for fluid to pass from the blood to the excretory canal it must be
filtered through the osmotic membranes and cytoplasmic extensions of
the excretory cells.
TYPES OF NEPHRIDIA IN NEMERTEANS
213
Uebl
-Pc
cLj
Lee
-ned
FIG. 15. C. spiral is. Diagram of cephalic protonephridium of male, showing
the isolated flame cells (fc), with the terminal chambers (tc) leading to the
slender end canals (cc) and thence to the collecting tubule (ct), convoluted tubule
and efferent duct ; ebl, epithelial lining of cephalic blood lacuna.
FIG. 16. Gconcmcrtcs agricola. Diagram of single nephridium with a cluster
of slender terminal chambers (tc) and binucleate flame cells (fc) leading by
the narrow end canals (cc) to a thick-walled convoluted tubule (con) and thence
to the efferent duct (ncd) ; /, tuft of long cilia; ntc, nucleus of terminal chamber;
circular bars on wall of terminal chamber.
COMPARISON WITH OTHER FORMS
With the exception of the metanephridia of the females of species
belonging to the family Cephalotrichidae, the excretory systems of all
nemerteans in which such organs have been discovered are of the
protonephridial type. In the numerous species of hathypelagic nemer-
teans, as well as in the littoral Prosadcnoponis, no trace of an excretory
system has yet been found.
Characteristic of the vast majority of species is a system of simple
214
WESLEY R. COE
flame cells (Fig. 12, A; Fig. 17, B) imbedded in gelatinous parenchyma
in close proximity to a blood space. Slender end canals from the
flame cells lead to profusely branched collecting tubules and thence to
a single thick-walled longitudinal canal on each side of the body. One
or more slender efferent ducts lead from the longitudinal canal to the
exterior of the body (Fig. 17, B). Occasionally, also, some of the
efferent ducts open into the esophagus (Coe, 1906).
In such a system the ciliary action of the flame cells may withdraw
fluids from the surrounding parenchyma and thence from the contiguous
A
FIG. 17. Diagrams showing comparison between a simple metanephridium
(A) of Cephalothriv and the multiple protonephridium (B) more typical for the
nemerteans ; con, convoluted tubule; ct, collecting tubule; cbl, epithelial lining
of blood lacuna ; Ic, main longitudinal canal ; ned. efferent duct ; ncf>, nephridiopore,
to, terminal organ.
TYPES OF XEPHRIDIA IN NEMERTEAXS 215
blood space. After passing through the collecting tubules the fluid
enters the longitudinal canal with its thick walls of granular and vacuo-
lated cytoplasm, indicative of the secretory or excretory function of
this part of the system. After receiving the contributions from the
cells of the longitudinal canal, and possibly also returning to those cells
any nutrient materials that it may contain, the fluid is discharged
through the efferent ducts. The movement of fluids through the sys-
tem is facilitated by the delicate cilia with which the longitudinal canal
is provided (Fig. 17, B).
This system is commonly limited to the region of the body lying
between the mouth and the midgut, where the blood spaces are vol-
uminous and thin-walled, but in some cases it extends through other
regions of the body. In the fresh-water Prostoina, for example, ne-
phridia extend the entire length of the body, being separated into several
independent groups in the adult, but connected together in early life.
In the terrestrial nemerteans, Gconeniertcs, there are many isolated
groups of flame cells, each group with a convoluted tubule similar to
the longitudinal canal in nature, and with its own efferent duct (Fig.
16). The number of such isolated nephridia may be very great and
their extent may cover the greater part of the body. They are found
not only in the vicinity of the lateral blood vessels but also in the
parenchyma beneath the intestine and beside the proboscis sheath (Coe,
1929). As many as 35,000 are estimated to be present in one of the
terrestrial forms which has a body length of only 35 mm. (Schroder,
1918). The terminal chamber in these forms is relatively large and
its wall of much complexity (Figs. 12, C '; 16).
Although the terminal organ of Geoneuiertcs is composed of a
binucleate flame cell and a cylindrical collar cell, we know of no transi-
tion stage between this protonephridium and the multinucleate metane-
phridium of the female Ccphalothri.r. And although the convoluted
tubule of the latter is apparently homologous with the longitudinal
canal of the protonephridium, the terminal organs of the two types
seem to have originated independently, somewhat as have the larval
protonephridia and the adult metanephridia of the Annelids.
But the question as to whether the metanephridium of Cephalothrix
is preceded in the life history by an earlier excretory system of the
protonephridial type remains at present unanswered.
LITERATURE
COE, W. R., 1906. A Peculiar Type of Nephridia in Nemerteans. Biol. Bui!..
11: 47.
COE, W. R., 1920. Sexual Dimorphism in Nemerteans. Biol. Bull.. 39: 36.
COE, W. R., 1928. A New Type of Nephridia in Nemerteans. .-Inat. Rcc.. 41: 57.
216 WESLEY R. COE
COE, W. R., 1929. The Excretory Organs of Terrestrial Nemerteans. BioL
Bull., 56: 306.
SCHRODER, O., 1918. Beitrage zur Kenntniss von Geonemertes palaensis Semper.
Scnckcn. natur. Gcscllschaft., 35: 155.
STRUNK, CARMEN, 1930. Beitrage zur Excretions-Physiologic der Polychaten
Arenicola marina und Stylarioides plumosus. Zool. Jahrb., Abt. f. allg.
Zool. u. Physio!, d. Tiere., 47: 259.
WIJNHOFF, G., 1910. Die Gattung Cephalothrix und ihre Bedeutung fur die
Systematik der Nemertinen. Zool. Jahrb., Abt. f. Anat., 30: 427.
BLOOD SUGAR AND ACTIVITY IN FISHES
WITH NOTES ON THE ACTION OF INSULIN"
I. E. GRAY AXD F. G. HALL
(From the Zoological Laboratory, Duke University)
The blood sugar of fishes has been studied by numerous investigators
and great variations in amount have been reported for different species.
In most cases a given observer has worked on one or a very few species.
and correlations between the amount of sugar and the habits of the
fishes have not been attempted. Furthermore, it is difficult to compare
the results of different authors, since so many methods of determining
blood sugar haAre been employed. Macleod (1926), has suggested that
the more active fishes have higher blood sugar than do the more slug-
gish forms. One of us (Gray, 1929), has also pointed out a correlation
between activity and blood sugar, but detailed data were not given.
Among the mammals, Shirley (1928) hints at a tendency for low
blood sugar to accompany high activity. She, however, did not make
a comparative study, but limited her observations to a single species.
In a previous paper (Hall and Gray, 1929), a correlation was
pointed out between the habits of marine fishes and their hemoglobin
concentration. It was shown that among fifteen species of marine
teleosts, in general, the most active had the highest iron values, while
the blood of sluggish fishes had low iron content. The fishes with the
highest iron content were surface feeding forms with similar habits,
and fishermen consider them among the fastest swimmers. The highest
hemoglobin was noted among members of the families Scombridse and
Clupeidse, examples of which are, respectively, the mackerels and men-
haden. These fishes feed largely on plankton and small fishes, which
they can only obtain by constantly keeping in motion. At the other
extreme are the bottom feeders, such as the goosefish, toadfish, and
sand dab, which are very sluggish and have extremely low hemoglobin.
These forms remain quiescent on the bottom for long periods of time.
Between the two extremes are found the majority of fishes.
In the present paper it is shown that correlations similar to those
between hemoglobin and activity exist also between blood sugar and
activity.
This work was carried on at the United States Fisheries Station
at Woods Hole, Massachusetts.
217
218 I. E. GRAY AND F. G. HALL
MATERIALS AND METHODS ,
Blood sugar determinations were made on fifteen different SQ^cies
of teleosts, representing thirteen families, as shown in Table I.^Blie
fishes were obtained from commercial fish traps and were caremlly
placed in large floating " live cages," where they were kept free froi
asphyxial conditions for at least twenty- four hours before use. T1
importance of keeping the fishes free from asphyxial conditions cannot
be overemphasized. In a previous paper (Hall, Gray, and Lepkovsky,
1926), the changes that take place in the concentration of the blood con-
stituents of fishes under asphyxia were pointed out. Other workers
(McCormick and Macleod, 1925; Simpson, 1926; and Menten. 1927)
have noted that asphyxia tends to raise the blood sugar. The time re-
quired for fishes brought to the laboratory to recover from the partial
asphyxia to which they have been subjected incidental to capture and
transportation varies, of course, with the different species and with
the methods of handling, both before and after they are placed in the
" live cage." McCormick and MacLeod found that it required from
two to four days for asphyxial hyperglycemia of the sculpin to subside.
With our methods and facilities it was found that one full day was
generally enough time to allow for recovery from any asphyxia to which
the fishes might have been subjected. Menton (1927), concludes that
the variation in sugar content of a species is governed largely by the
amount of food ingested. In our experiments the food factor was
reduced to a minimum by not using the fishes for a day or more after
placing them in the " live cage."
The methods of procedure were similar to those employed in pre-
vious studies. The puffers, toadfish, and goosefish were bled from
the heart with a hypodermic needle. The other fishes were bled In-
severing the tail and collecting the blood from the caudal vessels in a
small Erlenmeyer flask. Lithium oxalate was used as an anti-coagulant.
The blood sugar was determined by Folin's modification of the Folin-\Yu
method (Folin, 1926; Folin and Svedberg, 1926). A large percentage
of the determinations was made on the same sample of blood used for
the iron determinations (Hall and Gray, 1929), to which reference has
previously been made. One fish was used for each determination.
During the study of the action of insulin the fishes were kept in
hatchery boxes, one fish to each box. Insulin from Eli Lilly and Com-
pany was used throughout. The insulin was administered by intra-
peritoneal injections, in doses of five to fifteen units, depending on
the size and species of fish. If the action of insulin in fishes is similar
to its action in mammals, overdoses were given in each case.
m
BLOOD SUGAR AND ACTIVITY IN FISHES
TABLE I
The Blood Sugar of Marine Fishes
219
No. of
Sugai
• per 100 c
c. of Blood
nations
Low
High
Average
Group I
Bull's eye mackerel
(Pneionatophorus colias)
Scombridae
10
Mg.
60 ?
Mg.
160.0
Mg.
90.7
Butterfish
(Poronottis triacanthus)
Menhaden
(Brevoortia tyrannies)
Stromateidae
Cltipeidae
8
30
57.5
s? q
113.6
151.5
79.4
75.2
Rudderfish
(Palinurichthys perciformis) . . .
Common mackerel
(Scomber scombrns)
Centrolophidae
Scombridae
7
9
54.9
48.5
83.3
76.6
67.7
63.5
Eel
(Anguilla, rostrata)
Anguillidae
4
40.6
67.6
59.0
Bonito
(Surda, sarda)
Scombridae
3
48 ,S
62.7
55.1
Scup
(Stenotomus chrvsops)
Sparidae
46
SS ^
81.4
52.6
Silver hake
(Merhiccius InMnearis)
Merlucciidae
9
?S 3
85.4
48.2
Group II
Sea robin
(Pnonotus carolinus)
Triglidae
9
?08,
60.9
37.4
Sand dab
(Lopliopsettu tnaculatd)
Pleu ronect idae
4
?4 6
42.5
31.0
Cunner
(Tauto^olabrus adspersus)
Puffer
(Spheroides maculatits)
Labridae
Tetraodontidae
4
15
13.4
45
35.1
41.3
25.2
23.1
Toadfish
(Opsanus tau)
Batrachoididae
6
102
22.3
15.4
Goosefish
(Lophius piscatorius)
Lophiidae
11
00
10.3
5.6
RESULTS AND DISCUSSION
The results of the blood sugar determinations of the fifteen species
of marine teleosts are given in Table I. The fishes were kept under
conditions approximating the normal as nearly as possible. The high
and the low blood sugar values are given together with the average to
show the individual variation within the same species. The high and
the low values may seem exceedingly far apart in a few cases, and
without explanation may be misleading. The great majority of blood
15
220 I. E. GRAY AND F. G. HALL
sugar determinations gave results near the average ; it was only occa-
sionally that a very high or exceedingly low value was obtained. There
appears to be, however, a relatively greater individual variation among
fishes of the same species kept under the same conditions, than among
mammals.
Blood sugar, like hemoglobin, appears to be correlated in a general
way with the habits and activity of the fishes. The bull's eye mackerel,
butterfish, menhaden, rudderfish, common mackerel, eel, bonito, scup,
and silver hake are not only more active fishes than the others given
in the table, but also have higher blood sugar. For convenience of
discussion the fishes are arbitrarily divided into groups I and II. There
is no sharp dividing line, however, between the two groups.
Group I consists, for the most part, of aggressive fishes that depend
on their own activities in obtaining food. They feed largely on plank-
ton, small fishes, or other small animals that require the expenditure
of considerable effort to obtain. Members of the Scombridie and
•
Clupeidas are especially noted for their great activity. Individuals of
these families are kept in captivity only with great difficulty even when
placed in large " live cages " where they have plenty of room for their
constant movements. It is doubtful if the bonito, mackerels, and men-
haden ever cease their movements.
Some fishes of group I, for example, the scup and hake, might well
be classed as intermediate in regard to their activity. They are not
always in motion, nor do they pursue their food with the aggressiveness
shown by the Scombridse and Clupeidse.
The fishes are arranged in the table, not in the order of their activity,
but according to their blood sugar content. The correlation between
activity and blood sugar is not absolute but occurs in most cases. If
arranged according to relative activity the bonito would be at or near
the top. The blood sugar of this species may not be strictly comparable
with that of the other fishes since it was impossible to keep the bonito
alive in captivity. Consequently the only data obtainable were deter-
minations made on three small specimens, bled as soon as brought from
the traps.
Some of group I are excellent migrating fishes that move rapidly
through the water in large schools. Mostly they are adapted for fast
movement by being " stream-lined " with body-form either fusiform or
laterally compressed.
In group II are the relatively inactive and sluggish fishes. In con-
trast to the majority of group I, these fishes are the less aggressive
bottom feeders that are adapted to life on the bottom by having the
body- form angular or depressed. The dinner, although having a body-
BLOOD SUGAR AND ACTIVITY IN FISHES
221
form resembling members of group I and being found in a variety of
habitats, seems to prefer the rocky bottom and does not roam over
wide areas in search of food.
It will be noted that the average blood sugar of the members of
this group is considerably lower than that of group I. The goosefish
and the toadfish are two of our most sluggish fishes and have very
low blood sugar. Many determinations of the goosefish blood showed
merely faint traces of sugar. The goosefish, although it feeds indis-
criminately on other fishes, does not as a rule pursue its food. It is
one of the anglers and attracts its prey by a lure on one of the dorsal
fin-rays. All of the fishes of this group are known to remain quiet
on the bottom for long periods of time, which habit is in sharp contrast
to the activities of the mackerels and menhaden.
TABLE II
The Effect of Insulin on the Blood Sugar of Fishes
No. of
Units of
Time for
ShnrL' tn
Sugar per IOC
) cc. of Blood
nations
Given
Appear
Normal
After Insulin
Group I
Menhaden
6
5
Hours
11- 3
Mgs.
75.2
A/gs.
8.6-20.2
Common mack-
erel
13
5
1- 4
63.5
9.4-31.2
Bull's eye mack-
erel
5
5
3- 6
90.7
9.4-11.1
Scup
20
5-10
10-23
52.6
0.0-15.3
Group II
Sea robin .
10
5-15
no shock
37.4
8.8-32.5
Puffer . . .
9
5-15
no shock
23.1
0.0-13.5
Toadfish
15
5-15
no shock
15.4
1.5-22.9
We may say, then, that there appears to be a general correlation
between the amount of sugar of the blood, the hemoglobin, the body-
form, the activitv, and the habits of marine fishes. Activitv is ex-
^ ' -•
pressed here qualitatively. There appears to be a dearth of quantitative
determinations of metabolic activity in fishes. The oxygen consumption
of the scup, puffer, and toadfish has been studied (Hall, 1929), and
the results bear out our estimate of the activity of these fishes. Under
the same conditions the oxygen consumption of the puffer was found
to be intermediate between the relatively high consumption of the scup
and the extremely low oxygen consumption of the toadfish. Because
of their great activity a comparable basal oxygen consumption of the
Scombridae and Clupeidse, fishes more active than the scup, could not
be determined.
I. E. GRAY AND F. G. HALL
A further interesting relation to activity was noted through a com-
parative study of the action of insulin on fishes. At a temperature
of 21° C. and under similar conditions, it was found that the very active
fishes, menhaden, common mackerel, and bull's eye mackerel, showed
insulin shock in a much shorter time than did the moderately active
scup. This was perhaps to be expected. Huxley and Fulton (1924),
and Olmsted (1924), have pointed out that the rate of action of insulin
is dependent upon the metabolic rate of the animal itself. The more
sluggish bottom feeders, sea robin, puffer, and toadfish. showed no
external evidences of the effects of insulin. As has been previously
noted, the normal blood sugar of these sluggish fishes is much lower
than that of the more active ones. In some cases, such as the toadfish.
the normal sugar concentration is not as high as the insulin-reduced
sugar concentration of the more active fishes. A condensed summary
of the action of insulin on fishes is given in Table II.
Insulin appears to reduce the blood sugar concentration of fishes
in much the same manner as in mammals, except that a longer time is
required for the action to take place. Although the number is limited,
at least some of each species whose blood was analyzed showed reduced
sugar concentration following insulin administration. The mackerels,
menhaden, and scup, if bled during convulsions, showed reduced sugar
content in each case. There is considerable individual variation in
the time required for the sugar content to be reduced ; and since the
sluggish fishes showed no convulsions, it was difficult to estimate the
length of time to allow for insulin action. Puffers, sea robins, and
toadfish, bled at various intervals between twenty and forty hours after
insulin injection, showed blood sugar values ranging from the normal
to mere traces. Since some of each of these species showed reduction
of sugar content, it is thought that in those cases wrhere, after insulin
administration, the blood sugar was within the normal range of varia-
tion, either enough time had not elapsed for the insulin to reduce the
sugar concentration, or else too much time elapsed and the fishes re-
gained the normal sugar content.
The time required for the blood sugar content to be reduced ap-
peared to be considerably greater in these sluggish forms than in the
more active fishes. It seems improbable that the failure to get insulin
shock could be due to insufficient insulin. Toadfish were given re-
peated injections of from five to fifteen units of insulin over a period
of several days with no visible signs of disturbed metabolism. With
the mackerels, menhaden, or scup a single five unit injection usually
resulted in death unless glucose was administered.
Insulin convulsions in fishes do not necessarily indicate that the
BLOOD SUGAR AND ACTIVITY IN FISHES
blood sugar concentration is reduced to its lowest level. The rate of
reduction of the blood sugar values following insulin injection has
been worked out for the scup and will be published later. Here it is
sufficient to say that the blood sugar content may be reduced in six to
eight hours in this fish. In a few cases mere traces of sugar remained
in the blood after eight hours and yet in no case were convulsions ap-
parent sooner than ten hours. In other words, a few scup had lower
blood sugar before reaching the convulsive stage than did other scup
in the midst of convulsions. Furthermore, the fact that sluggish fishes,
as the toadfish and puffer, have their blood sugar concentration reduced
without showing any shock at all, indicates that insulin shock in fishes
does not have as much significance as has been attributed to insulin
convulsions in mammals.
SUMMARY
1. Correlations between the blood sugar, hemoglobin, body- form,
activity and habits of fifteen species of marine teleosts are pointed out.
2. The fishes that show the greatest activity, those that feed at the
surface or are aggressively predaceous, have the highest blood sugar
concentration. The sluggish bottom feeders have low sugar content
in the blood.
3. Insulin shock may be easily produced in active species of fishes.
In sluggish forms no external evidence of the action of insulin could
be detected.
4. The blood sugar of fishes is reduced by the action of insulin.
Less time is required for reduction of sugar content to take place in
the active fishes than in the sluggish forms, due probably to differences
in the metabolic rate of the different species. In the sluggish forms
the sugar content may be reduced without convulsions or shock being
apparent.
5. The normal sugar of some of the sluggish fishes is often lower
than the insulin-reduced sugar of the more active fishes.
STUDIES OF PHOTODYNAMIC ACTION
I. HEMOLYSIS BY PREVIOUSLY IRRADIATED FLUORESCEIN DYES
HAROLD F. BLUM
DEPARTMENT OF PHYSIOLOGY, HARVARD MEDICAL SCHOOL x
The hemolysis of red blood cells by the combined action of light
and certain photoactive substances was first described by Sacharoff and
Sachs in 1905. Such hemolysis occurs in a very short time when red
blood cells are exposed to sunlight in dilute concentrations of the
photoactive substance. Sunlight alone does not produce hemolysis
provided the ultra-violet spectrum is screened out by exposing the
cells in glass, nor does the photoactive substance in equal concentration
in the dark. This is only one of a wide range of similar phenomena
brought about under similar conditions in other cells and tissues, which
are generally described collectively under the term photodynamic action
or photodynamic sensitisation. The photoactive substances which bring
these phenomena about include a large number of compounds, most of
which are fluorescent dyes. It is generally assumed that such effects
are not produced if the solution of the photodynamic substance is sep-
arately irradiated, the erythrocytes or other cells being added subse-
quently in the dark (see Clark 1922, p. 288). There are, however, a
few recorded experiments which indicate that this is possible.
Ledoux-Lebard (1902) found that eosine which had been previously
exposed to sunlight killed and cytolyzed paramecia; whereas non-
irradiated eosine of the same concentration did not. He suggested,
therefore, that photodynamic action is due to the formation of a toxic
eosine compound by the action of sunlight. Jodlbauer and Tappeiner
(1905) found that this did not occur if the eosine solution was neutral-
ized after irradiation and before the addition of the paramecia. They
claimed, therefore, that the Ledoux-Lebard effect was due to the forma-
tion of acid concomitant with the bleaching of the dye ; the acid being
the toxic agent. They did not consider this as a photodynamic effect.
Sacharoff and Sachs (1905) described hemolysis by previously irradiated
£-(o-nitrophenyl)— /Miydroxyethyl methyl ketone [" o-Nitrophenyl-
milchsaureketon "]. They were unable to produce hemolysis with pre-
viously irradiated eosine or erythrosine, however, and preferred to
1 Preliminary experiments for these studies were carried out in the Depart-
ment of Animal Biology, University of Oregon.
224
HEMOLYSIS BY PREVIOUSLY IRRADIATED DYES 225
consider their one positive result as not belonging to the typical photo-
dynamic phenomena. Fabre and Simonnet (1927) were able to pro-
duce hemolysis with lecithin which had been irradiated together with
hematoporphyrin by light from a mercury vapour arc. Moore (1928)
found that previously irradiated cosine killed the eggs of the sea urchin
Strongylocentrotus purpuratiis but did not cytolyze them; whereas when
the eggs were irradiated together with the dye, they were completely
cytolyzed. Moore hypothecates the formation of a toxic cosine com-
pound which produces cytolysis upon further irradiation after it has
entered the cell. On the other hand, Raab (1900) was unable to pro-
duce killing of paramecia by previously irradiated acridine solutions.
Hausmann was unable to produce killing of paramecia or hemolysis
with previously irradiated solutions of chlorophyll (1909) or hemato-
porphyrin (1910) ; although similar solutions produced these effects
when irradiated together with the cells. Hasselbach (1909) could not
produce hemolysis with previously irradiated solutions of several photo-
dynamically active substances including cosine and erythrosine. Pereira
(1925) found that Arbacia larvae were not killed by previously irra-
diated cosine in sea water.
The writer has found that it is possible, under carefully controlled
conditions, to bring about hemolysis with previously irradiated solutions
of the three fluorescein dyes which he has investigated, fluorescein, eosine
and erythrosine. This is of considerable interest because of its bearing
on certain theories of photodynamic action which will be discussed later
in this paper.
EXPERIMENTAL
Hemolysis by previously irradiated fluorescein, cosine, and erythro-
sine.— The writer's first attempts to produce hemolysis with previously
irradiated eosine solutions met with apparent success in only a few in-
stances. These were thought at first to be accidental, but with more
careful control of conditions it was found possible to obtain consistently
reproducible results. The successful technique required the selection
of proper hydrogen ion concentration and dye concentration.
The hydrogen ion concentration must be carefully buffered, since
unbuffered solutions tend to increase in acidity during irradiation.
This increase in acidity may inhibit the production of hemolysis by
bringing about fixation of the cells as will be pointed out in a later
paper. To insure the maximum obtainable buffering capacity, it was
found convenient to make up the dye in solutions of primary and sec-
ondary sodium phosphate mixtures. In order to insure a medium of
proper osmotic pressure for the blood cells, the phosphate mixtures were
226 HAROLD F. BLUM
calculated to have the same osmotic pressure as a 0.15 M sodium chloride
solution. This was done by assuming that the primary phosphate dis-
sociates into two ions, the secondary phosphate into three. The mol
fractions of the two salts required for a given hydrogen ion concentra-
tion were estimated by the use of Cohn's data for potassium phosphates
(see Clark, 1928, pp. 216-220). ~ The hydrogen ion concentrations
of the solutions were checked by means of the hydrogen electrode.
Such solutions proved rather unsatisfactory in the case of fluorescein,
and a solution containing 10 per cent of the phosphate mixture and
90 per cent 0.15 M sodium chloride, was used instead in most experi-
ments with this dye. The concentration of phosphate in this solution
is still many times that of fluorescein in most of the dye concentrations
which were used, and affords an adequate buffer.
The optimal concentration of the dye varies with a number of con-
ditions; some of which, as for example the intensity of irradiation, it
was impossible to control. It was found expedient, therefore, to use
a series of dilutions of the dyes ; usually consisting of ten dilutions
from 1 per cent to 0.002 per cent." These were exposed to the sunlight
for a given period of time. Blood cells were then added to the ir-
radiated solutions and also to a control consisting of a corresponding
series of non-irradiated dye solutions. Both series were then placed
in a dark room where the temperature was in the region of 20° C.
Observation of the tubes for hemolysis was made at intervals after the
addition of the cells. It was found that in most cases six hours sufficed
for the hemolysis to reach a maximum. Since the temperature during
irradiation could not be controlled, time was allowed, when necessary,
for the irradiated tubes to come to the same temperature as the controls
before adding the cells. The solutions were exposed in small test tubes
(10x75 mm.), each containing 2 cc. of the solutions. The blood cells
were added to each tube in the quantity of 0.02 cc. of a 50 per cent
suspension in 0.15 M sodium chloride, by means of a blood pipette.
This method avoids any appreciable dilution of the irradiated solution
upon the addition of the cells. This precaution has not been observed
by most of the investigators who have attempted to produce hemolysis
with previously irradiated substances. Human blood cells were used
2 Dr. G. Payling Wright and the writer have found that rabbit blood cells
suspended in such solutions show a variation in volume of approximately twelve
per cent over the range of hydrogen ion concentration between pH 7.7 and pH
6.0, and have approximately the same volume as cells in serum.
3 The dyes used were Fluorescein, sodium salt (Uranine), from the National
Aniline and Chemical Company, Erythrosine B (sodium salt of tetra-iodo-
fluorescein) also from the National Aniline and Chemical Company, and Eosine
Y (sodium salt of tetra-brom-fluorescein) from Coleman and Bell.
HEMOLYSIS BY PREVIOUSLY IRRADIATED DYES
227
in most of the experiments. They were washed by centrifuging three
times from suspension in 0.15 M sodium chloride to free them from
serum. It is advisable to have the cells as free from serum as possible,
since serum is effective in preventing photodynamic hemolysis (Busck,
1906). The intensity of the radiation could not be accurately estimated,
but it was found practicable to expose the solutions to bright midday
sunlight for one to two hours. Too long continued exposure causes
bleaching of the dye, resulting in a lowered concentration of the active
dye.
TABLE I
Hemolysis by Previously Irradiated Fluorescein
Solutions exposed to sunlight 90 minutes (2:00-3:30 P.M., September 29, 1929). All solutions
contain 10 per cent of sodium phosphate buffer, pH 6.4, isosmotic with 0.15 M NaCl, plus 90 per cent
of 0.15 M NaCl. Observations made after 16 hours in dark following addition of red blood cells.
H = complete hemolysis, (H) = partial hemolysis, and the dash is used when there is no detectable
hemolysis.
Concentration of
Irradiated solution. Red
fluorescein
blood cells added after
Non-irradiated solution
(control)
per cent
45 minutes
4 hours
1.0
—
—
0.5
—
—
. — -
0.25
—
—
. —
0.125
(H)
• —
—
0.062
(H)
(H)
— •
0.031
H
(H)
—
0.015
H
H
—
0.007
(H)
(H)
—
0.004
(H)
(H)
—
0.002
—
—
—
0.00
—
—
—
Tables I, II, and III show the results of typical experiments with
fluorescein, cosine and erythrosine respectively. In these tables, H
represents complete hemolysis (i.e. hemochromolysis and stromatolysis)
as well as can be judged by the naked eye, (//) represents partial
hemolysis, and the dash no detectable hemolysis. These classifications
are arbitrary, but since comparison can always be made with the control
tubes, there can be no doubt of the general validity of the observations.
An examination of Tables I, II, and III demonstrates quite clearly that
previously irradiated solutions of these dyes bring about hemolysis in
concentrations at which non-irradiated solutions do not. Some bleach-
ing of the dye takes place upon irradiation and this raises the question
whether the hemolysis may not be due to the products of this bleaching.
228
HAROLD F. BLUM
It has been found, however, that completely bleached solutions have
no hemolytic action.
Non-irradiated cosine and erythrosine produce hemolysis in suf-
ficiently high concentration, as is shown in Tables II and III. This
was described by Sacharoff and Sachs (1905) and studied by Tappeiner
(1908). It is apparently not due to irradiation during the preparation
of the solutions ; since in these experiments the results were the same
when the solutions were carefully prepared in the dark room under
red light, which is outside the absorption range of these dyes, as when
TABLE II
Hemolysis by Previously Irradiated Eosine
Solutions exposed to sunlight for 105 minutes (11:45A.M.-1:30 P.M., September 10, 1929). All
solutions contain sodium phosphate buffer, pH 7.0, isosmotic with 0.15 M NaCl. Observations made
5 hours after addition of red blood cells. The symbols are the same as those in Table I.
Concentration of
eosine
Irradiated solution. Red blood
cells added after
Non-irradiated solution
(control). Red blood
cells added after
per cent
45 minutes
2-'i4 hours
5 hours
45 minutes
5 hours
1.0
H
H
H
H
H
0.5
0.25
H
H
H
H
H
H
(H)
(H)
0.125
H
H
H
—
— .
0.062
H
H
H
—
—
0.031
0.015
0.007
H
(H)
(H)
(H)
H
(H)
—
—
0.004
. —
• —
—
—
—
0.002
—
—
—
—
—
0.0
—
—
—
—
—
prepared with ordinary precautions in the diffuse light of the laboratory.
The effect of short exposure to diffuse light is thus within the accuracy
of the observations described here. The absence of hemolysis in the
higher concentrations of the irradiated dye in Table I is probably due
to fixation of the cells. This phenomenon will be discussed in a later
paper. The marked effect of hydrogen ion concentration upon the
hemolytic activity of irradiated and non-irradiated dyes will also be
discussed in that paper ; the hydrogen ion concentrations for the ex-
periments here described have been chosen as those at which the differ-
ence in hemolytic activity between previously irradiated and non-irradi-
ated solutions could be most clearly demonstrated.
It will be noted in Tables I, II, and III that the results are changed
HEMOLYSIS BY PREVIOUSLY IRRADIATED DYES
229
very little when the exposed solutions are allowed to remain in the
dark for as much as four or five hours after irradiation, before the
addition of the cells. This shows very conclusively that the increased
hemolytic activity of the irradiated solutions cannot he due to their
having a greater temperature than the controls because of the absorption
of heat during the period of exposure, since ample time is allowed for
the two series of solutions to come to the same temperature. It also
demonstrates that whatever change occurs in the course of irradiation
is not rapidly reversible in the dark. Moore (1928) observed, similarly,
TABLE III
Hemolysis by Previously Irradiated Erythrosine
Solutions exposed to sunlight for one hour (11:00 A.M.-12:00 M., September 28, 19291. All
solutions contain sodium phosphate buffer, pH 6.5, isosmotic with 0.15 M NaCl. Observations made
6 hours after addition of red blood cells. The symbols are the same as those in Tables I and II.
Concentration of
Irradiated solution. Red blood
erythrosine
cells added after
Non-irradiated solution
(control)
per cent
45 minutes
1M hours
5 hours
1.0
H
H
H
H
0.5
H
H
H
H
0.25
H
H
H
H
0.125
H
H
H
H
0.062
H
H
H
(H)
0.031
H
H
H
(H)
0.015
(H)
(H)
(H)
—
0.007
• —
• —
—
—
0.004
—
. —
—
—
0.002
. —
—
—
—
0.00
—
—
—
—
that in the case of the killing of sea urchin's eggs by previously irradiated
cosine, the solution retained its toxic properties after six hours in the
dark.
DISCUSSION
Numerous hypotheses have been developed to explain the mechanism
of photodynamic action, most of which contain the assumption that the
photodynamic substance and substrate (e.g. cells) must be irradiated
together. This is true of the theory of Tappeiner (1909) which he
outlines as follows : The presence of the photodynamic substance merely
accelerates the action of visible light. The split products of this reac-
tion are removed through oxidation by molecular oxygen. Ordinarily
LIB
RARY!
/ •
230 HAROLD F. BLUM
these products accumulate and inhibit the reaction, but the combined
action of light and a photodynamic substance accelerates their removal
and consequently the total reaction. Another conception, based on the
fact that most of the photodynamic substances are fluorescent, is that
the photodynamic effects are due to the action of fluoresced radiation
upon the protoplasm. Since the fluoresced light is only a more or less
polarized radiation from a particular region of the visible spectrum
characteristic of the substance concerned (Pringshein 1928, p. 195), it
can hardly be expected to have such destructive effects. Moreover,
Raab (1900) showed that paramecia are not damaged when exposed
to the fluoresced radiation from a solution of fluorescent substance
with which they are not in contact ; and likewise, Sacharoff and Sachs
(1905) showed that red blood cells exposed under the same conditions
are not hemolyzed. Nevertheless, this concept remains current to a
certain extent. Schanz (1921) suggests from studies on the photo-
electric effect in albumin, and albumin plus fluorescein dyes, that the
changes brought about in the cell constituents are due to the absorption
of electrons emitted by the dye during irradiation. Clark (1922, pp.
302-303) suggests that the photodynamic substance shifts the photo-
electric threshold of the cell constituents from the ultra-violet into longer
wave lengths. Metzner (1924) claims that the photodynamic effects
are brought about by an action within the cell dependent upon the com-
bination (adsorption) of the dye with the protoplasm. Jodlbauer
( 1926) assumes that the dye must be adsorbed by the cell, and that only
those dyes are photodynamically active which retain their ability to
be activated by light while in combination with the cell substance. Such
theories demonstrate how firmly the idea is established that the photo-
dynamic substance and substrate must be irradiated together. Obvi-
ously all such explanations of photodynamic action must be discarded
or modified, in light of the fact that hemolysis may be brought about
by previously irradiated photodynamic substances.
EXPERIMENTAL
Evidence that Oxidation Is a Factor in Photodynamic Hemolysis. —
A theory of direct oxidation of cell constituents by the action of light
and the photodynamic substance was put forward by Straub (1904a).
His hypothesis was founded principally upon the analogy between the
photodynamic action of cosine upon cells and its ability to oxidize iodide
ion in the presence of light. He found (1904^) that, in proper concen-
tration, cosine may oxidize many times its equivalency of iodide when
the two substances are exposed to sunlight together in solution. He
conceived that the cosine is changed to an cosine peroxide by the action
HEMOLYSIS BY PREVIOUSLY IRRADIATED DYES 231
of light; and that this peroxide brings about the oxidation of an
equivalent amount of iodide ion, being returned in so doing to the
original cosine form. The cosine may then proceed to the oxidation
of another quantity of iodide, thus acting in a sense as a catalyst. He
could not, however, demonstrate the existence of an intermediate per-
oxide, being unable to obtain conclusive evidence of the oxidation of
iodide ion by the action of previously irradiated cosine (1904a).
The writer finds that previously irradiated cosine will oxidize iodide
ion, as shown by a positive starch reaction after adding potassium
iodide in the dark. The oxidation proceeds rather slowly immediately
after the addition of the potassium iodide, which may account for
Straub's failure to observe it in his experiments. Table IV" presents
some quantitative results obtained when (1) fluorescein dyes and po-
tassium iodide were irradiated in solution together, and (2) when the
iodide was added to the previously irradiated dyes. The determinations
were made by titration of the free iodine formed due to the oxidation
of iodide ion, with 0.001 N sodium thiosulfate against starch indicator.
When potassium iodide is added to the previously irradiated dye and
the mixture placed in the dark, the oxidation takes place quite slowly,
reaching a maximum after about three hours. The titrations were,
therefore, performed after the elapse of this time. The accuracy of
determination of iodine in such small concentrations is, of course, sub-
ject to some error. In order to determine the magnitude of this error,
solutions containing quantities of iodine of the same order as those
represented in Table IV were titrated. The solutions were of the same
volume, contained the same concentration of dye and of potassium
iodide, and were buffered at the same hydrogen ion concentration as
the experimental solutions. With concentrations of iodine correspond-
ing to the lowest values in Table IV, the determinations were con-
sistently 10 to 15 per cent lower than the theoretical. Writh quantities
of iodine corresponding to the highest values the error was not greater
than one per cent. The 0.001 N thiosulfate solution was always freshly
prepared by dilution from a 0.1 N stock solution.
The experiments described in Table IV represent conditions in the
region of the optimal for the reaction of the iodide with each clye.
The extent of these reactions seems to be greatly affected by the hy-
drogen ion concentration, and by other factors, which will not be dis-
cussed here. Controls containing the same concentration of potassium
iodide, but no dye, never showed more than a trace of free iodine when
exposed to sunlight simultaneously with the potassium iodide-dye mix-
tures. Likewise, solutions of the dye containing potassium iodide
showed no trace of free iodine after many hours in the dark.
232
HAROLD F. BLUM
TABLE IV
Oxidation of Potassium Iodide by Irradiated Fluorescein, Eosine, and Erythrosine
Fluorescein
KI
PH
Volume
of
Solution
Duration
of
Irradiation
KI
Added after
Irradiation
Volume
of
Na2S2O3
Mols of
Dye
Mols of
Iodide
Oxidized
0.00 IN
per
cent
Cc.
Hours
per cent
Cc.
0.0005 M
1.0
6.0
6.0
8
0.0
18.6
3 X 10-6
18.6 X 10~6
0.0005 M
1.0
6.0
6.0
8
0.0
17.1 *
3 X 10-6
17.1 X 10-"
0.0005M
1.0
6.0
6.0
0
0.0
0.0
3 X IQ-o
0.0
0.0005M
0.0
6.0
6.0
8
3.0
1.0 f
3 X 10-«
1.0 X 10-6
0.0005 M
0.0
6.0
6.0
8
3.0
0.5 |
3 X lO-6
0.5 X 10-«
0.0
1.0
6.0
6.0
8
0.0
0.4
0.0
0.4 X 10~6
Eosine
per
cent
Cc.
Hours
per cent
Cc.
0.001 M
3.0
6.0
6.0
6
0.0
14.5
6 X 10-6
14.5 X 10-6
0.00 1M
3.0
6.0
6.0
6
0.0
14.5 *
6 X 10-6
14.5 X lO-6
0.001 M
3.0
6.0
6.0
0
0.0
0.0
6 X 10-«
0.0
0.001 M
0.0
6.0
6.0
6
3.0
3.2 f
6 X lO-6
3.2 X 10~6
0.001 M
0.0
6.0
6.0
6
3.0
3.0 }
6 X 10-6
3.0 X 10-6
0.001 M
0.0
6.0
6.0
0
3.0
0.0
6 X 10-6
0.0
Erythrosine
per
cent
Cc.
Hours
per cent
Cc.
0.001M
3.0
6.0
6.0
6
0.0
19.1
6 X 10~6
19.1 X 10-fi
0.001 M
3.0
6.0
6.0
6
0.0
19.5 *
6 X 10-6
19.5 X 10-6
0.00 1M
3.0
6.0
6.0
0
0.0
0.0
6 X 10~6
0.0
0.001M
0.0
6.0
6.0
6
3.0
1.3 t
6 X 10-6
1.3 X 10-6
0.001 M
0.0
6.0
6.0
6
3.0
1.0}
6 X 10-6
1.0 X lO-6
0.001 M
0.0
6.0
6.0
0
3.0
0.0
6 X 10~6
0.0
* Titration after 3 hours in dark.
f KI added immediately after irradiation with titration after 3 hours in dark.
J KI added after 3 hours in dark following irradiation; titration 3 hours later.
Table IV shows that iodide ion equivalent to several times the quan-
tity of dye present may be oxidized when exposed together with the
dye (equivalency considered as one mol of iodide ion per mol of dye).
Straub (1904b) was able, in fact, to oxidize a quantity of iodide ion
sixty-five times as great as the quantity of dye present. On the other
hand, when the dye alone is irradiated and the potassium iodide added
subsequently in the dark, the quantity of iodide ion oxidized is always
less than that equivalent to the dye present. In the latter case it was
never found possible, in a considerable number of experiments under
varving conditions, to oxidize more iodide than a quantity equivalent
HEMOLYSIS BY PREVIOUSLY IRRADIATED DYES 233
to the quantity of dye present. When irradiated in the absence of
a readily oxidizable substance, such as iodide ion, a certain amount of
the dye is oxidized, as is indicated by bleaching. Thus the transforma-
tion of all the dye to the active form cannot be expected, and we should
expect that less iodide would be oxidized than a quantity equivalent to
the quantity of the dye originally present. This appears to be the case.
When the dye is exposed with a readily oxidizable substance, no bleach-
ing occurs, indicating that this substance is oxidized instead of the dye.
All these facts lend support to Straub's hypothesis. They demonstrate
at least that a substance is formed upon irradiation of the dye solution
which is capable of oxidizing substances which the non-irradiated dye
cannot, and indicate that this is an intermediate substance in the oxida-
tions brought about by the action of the dye and light.
The quantity of iodide ion oxidized is not greatly altered if the dye
is allowed to remain in the dark for several hours after irradiation
before potassium iodide is added. This shows that the change brought
about by irradiation is not rapidly reversible in the dark. This is ex-
actly parallel to the case of hemolysis where, as we have seen, hemolysis
is brought about by previously irradiated dye solutions which have re-
mained in the dark for several hours after irradiation before addition
of the cells. Substances produced in the bleaching of the dye are not
responsible for the oxidation of iodide ion, since completely bleached
solutions do not bring about this oxidation. This is again parallel
to the case of hemolysis, since as stated above, hemolysis is not produced
by completely bleached dyes. These latter facts suggest very definitely
that the substance in irradiated solutions of a fluorescein dye which
brings about hemolysis is the same as that which brings about the
oxidation of iodide ion ; and that, therefore, the former process is prob-
ably dependent upon an oxidation.
If it is true that the hemolysis of blood cells by irradiated dyes in-
volves the oxidation of cell constituents in a manner similar to the oxi-
dation of iodide ion, we should expect, parallel to the above observations,
more extensive oxidation and thus greater hemolysis when the dye is
irradiated together with the cells than when previously irradiated. In
the former case the dye may, presumably, act in a catalytic sense, thus
oxidizing several times its molecular equivalency of cell constituents ;
whereas in the latter case the amount of oxidation is limited by the
quantity of dye present. The data presented in Tables V, VI and VII,
appears to confirm this prediction ; the hemolytic action seems to be
quantitatively much greater when the dye and cells are irradiated to-
gether than when the dye is irradiated alone and the cells added later
in the dark. The statement that hemolvsis is more readily produced
234
HAROLD F. BLUM
TABLE V
Comparison of Hemolytic Activity of Fluorescein Irradiated With and Without Blood
Cells
Solutions exposed to sunlight for one hour and 30 minutes. All solutions contain sodium phos-
phate buffer, pH 6.5,isosmotic with 0.15 M NaCl. Observations made after 20 hours in dark following
addition of blood cells. Symbols as in preceding tables. P = precipitate.
Concentration
Fluorescein Solution
Fluorescein Irradiated
Fluorescein
of
Irradiated
Alone.
Not
Fluorescein
with Cells
Cells Added in Dark
Irradiated
per cent
1.0
P
(H)
—
0.5
P
(H)
—
0.25
P
(H)
—
0.125
H
(H)
—
0.062
H
(H)
—
0.031
H
(H)
—
0.015
H
(H)
—
0.007
H
—
— •
0.004
H
—
—
0.002
H
—
—
0.0
—
—
—
when the dye is irradiated together with the cells than when irradiated
separately is a generalization to which many exceptions occur, due chiefly
to the complicating factor of fixation which will be considered in a
later paper. That hemolysis may proceed farther in the former case
than in the latter, in conditions where fixation is not a complicating
TABLE VI
Comparison of Hemolytic Activity of Eosine Irradiated With and Without Blood Cells
Solutions exposed to sunligh tfor one hour and 30 minutes. All solutions contain sodium phos-
phate buffer, pH 6.9, isosmotic with 0.15 M NaCl. Observations made after 7 hours in dark following
irradiation. Symbols as in the preceding tables.
Concentration
of
Eosine
Eosine Solution
Irradiated
with Cells
Eosine Irradiated
Alone.
Cells Added in Dark
Eosine
Not
Irradiated
per cent
1.0
H
H
H
0.5
(H)
H
(H)
0.25
H
H
—
0.125
H
H
—
0.062
H
H
—
0.031
H
H
—
0.015
H
(H)
—
0.007
H
—
—
0.004
H
• —
—
0.002
H
—
—
0.0
• —
—
'
HEMOLYSIS BY PREVIOUSLY IRRADIATED DYES
235
factor, seems justified by all the writer's observations on red blood
cells under the two conditions. It would be, of course, absurd to at-
tempt an exact quantitative comparison between the results in Table
IV and those in Tables V, VI, and VII, since we do not know in the
case of the blood cells, what substances may be subject to oxidation,
or what their oxidation-reduction potentials may be.
The action of non-irradiated dyes, previously mentioned, is in all
probability not an oxidative process, since oxidation of iodide ion by
these dyes does not take place in the dark. Whatever the nature of
this process, however, when hemolysis occurs after irradiation in a
concentration of dye which does not produce hemolysis when not ir-
radiated, we are justified in the assumption that the changes bringing
about hemolysis may be oxidative, since we know that the oxidizing
power of the dye solution has been increased by irradiation.
TABLE VII
Comparison of Hemolytic Activity of Erythrosine Irradiated With and Without Blood
Cells
Solutions exposed to sunlight for one hour. All solutions contain sodium phosphate buffer. pH 7.0,
isosmotic with 0.15 M NaCl. Observations made after 6 hours and 20 minutes in dark following
irradiation. Symbols as in preceding tables.
Concentration
of
Erythrosine
Erythrosine Solution
Irradiated
with Cells
Erythrosine Irradiated
Alone.
Cells Added in Dark
Ervthrosine
" Not
Irradiated
per cent
1.0
H
H
H
0.5
H
H
H
0.25
H
H
H
0.125
H
H
H
0.062
H
H
H
0.031
H
H
—
0.015
0.007
H
H
(H)
—
0.004
H
—
—
0.002
H
—
—
0.0
—
—
• —
DISCUSSION
Further evidence that oxidation is an important factor in photo-
dynamic processes is not lacking. Oxygen is known to be necessary
for a number of photodynamic effects (Straub, 1904a; Jodlbauer and
Tappeiner, 1905), since they do not take place in its absence. Spe-
cifically as regards hemolysis, Hasselbach (1909) found that hemolysis
by light and certain photodynamic substances, including cosine and
16
236 HAROLD F. BLUM
erythrosine, did not take place in a vacuum, and Schmidt and Norman
(1922) found that hemolysis by cosine and sunlight did not occur in
hydrogen. Sacharoff and Sachs (1905) showed that the presence of
the reducing substance sodium sulfate may prevent hemolysis by ir-
radiated erythrosine. Noack (1920) showed that a number of in-
organic reducing agents may inhibit photodynamic effects, and Schmidt
and Xorman (1922) found that a number of readily oxidizable organic
and inorganic substances will prevent hemolysis by cosine and light.
Noack (1920) has also shown quite definitely that certain plant pig-
ments can be oxidized by various photodynamic substances and light,
and gives evidence that these phenomena involve the formation of in-
termediate peroxides.
CONCLUSIONS
The demonstration of the formation of an intermediate substance
in the process of photodynamic hemolysis by fluorescein dyes offers
quite conclusive evidence against the sensitization theory of Tappeiner
and other theories which assume that photodynamic substance and sub-
strate must be irradiated together. The demonstration that a definite
increase in the oxidizing power of solutions of these dyes is brought
about by irradiation, together with the accumulation of other evidence
pointing toward an oxidative process, makes it necessary to consider
the oxidation of cell constituents as a probable underlying factor in
photodynamic hemolysis. Likewise, such oxidations must be considered
as a possible factor in all photodynamic processes.
SUMMARY
1. Hemolysis may be produced by previously irradiated fluorescein,
cosine and erythrosine.
2. Similarly, previously irradiated fluorescein, cosine and erythrosine
oxidize iodide ion.
3. These findings render untenable the sensitization theory of Tap-
peiner and other theories which necessitate the simultaneous action of
light and the photodynamic substance, while supporting Straub's theory
of direct oxidation of cell constituents.
4. Oxidation must be considered as a probable underlying cause
in photodynamic hemolysis and all other photodynamic phenomena.
BIBLIOGRAPHY
BUSCK. G., 1906. Die Photobiologischen Sensibilisatoren und ihre Eiweissverbin-
dungen. Biochcm. Zcitschr., 1: 425.
CLARK, J. H., 1922. The Physiological Action of Light. PJiysioL Rer., 2: 277.
HEMOLYSIS BY PREVIOUSLY IRRADIATED DYES 237
CLARK, W. M., 1928. The Determination of Hydrogen Ions. Third Edition.
Baltimore.
FABRE, R., AND SIMONNET, H., 1927. Contribution a 1'etude de 1'hemolyse par
action photosensibilisatrice de l'hematoporphyrine. Compt. rend. Acad.
Sci, 184: 707.
HASSELBACH, K. A., 1909. Untersuchungen iiber die Wirkung des Lichtes auf
Blutfarbstoffe und rote Blutkorperchen wie auch iiber optische Sensi-
bilisation fur diese Lichtwirkungen. Biochem. Zcitschr., 19: 435.
HAUSMANN, W., 1909. Die photodynamische Wirkung des Chlorophylls und
ihre Beziehung zur photosynthetischen Assimilation der Pflanzen. Jahrb.
f. wiss. Botanik., 46: 599.'
HAUSMANN, W., 1910. Die sensibilisierende Wirkung des Hamatoporphyrins.
Biochem. Zcitschr., 30: 276.
JODLBAUER, A., 1926. Die physiologischen Wirkungen des Lichtes. Handbuch.
dcr norm. it. path. Physio!., 17: 305.
JODLBAUER, A., AND TAPPEINER, H., 1905. Die Beteiligung des Sauerstoffs bei
der Wirkung fluorescierender Stoffe. Dcntschcs Arch. f. klin. Med.,
82: 520.
LEDOUX-LEBARD, 1902. Action de la Lumiere sur la To.xicite de 1'Eosine. Ann.
lust. Pasteur., 16: 587.
METZNER, P., 1924. Zur Kenntnis der photodynamischen Erscheinung. III.
Biochem. Zcitschr., 148: 498.
MOORE, A. R., 1928. Photodynamic Effects of Eosine on the Eggs of the Sea
L'rchin, Strongylocentrotus purpuratus. Arch. di. Sci. BioL. 12: 231.
NOACK, K., 1920. Untersuchungen iiber lichtkatalytische Vorgange. Zcitschr.
f. Botanik, 12: 273.
PEREIRA, J. R., 1925. On the Combined Toxic Action of Light and Eosin. Jour.
E.vper. Zool, 42: 257.
PRINGSHEIN, P., 1928. Fluorescenz u. Phosporescenz. Berlin.
RAAB,' O., 1900. Ueber die Wirkung fluorescirender Stoffe auf Infusorien.
Zcitschr. f. BioL, 39: 524.
SACHAROFF, G., AND SACHS, H., 1905. Ueber die hamolytische Wirkung der
photodynamischen Stoffe. Miiiich. Med. Woch., 52: 297.
SCHANZ, F., 1921. Die physikalischen Vorgange bei der optischen Sensibilisation.
Pfliiger's Arch., 190: 311.
SCHMIDT, C. L. A., AND NORMAX, G. F., 1922. Further Studies on Eosin
Hemolysis. Jour. Gen. Physiol.. 4: 681.
STRAUB, W.. 1904<7. Ueber chemische Vorgange bei der Einwirkung voti Licht
auf fluoreszierende Substanzen. Miinch. Med. Woch.. 51: 1093.
STRAUB, W., 1904??. Uber den Chemismus der Wirkung belichteter Eosinlosung
auf oxydable Substanzen. Arch, e.vper. Path. u. PhannakoL, 51: 383.
TAPPEIXER, H., 1908. Untersuchungen u'ber den Angriffsort der fluorescierenden
Substanzen auf rote Blutkorperchen. Biochem. Zcitschr., 13: 1.
TAPPEINER, H., 1909. Die photodynamische Erscheinung. Ergebnisse der Physi-
ologic, 8: 698.
THE EQUILIBRIUM OF OXYGEN WITH THE HEMOCY-
ANIN OF LIMULUS POLYPHEMUS DETERMINED
BY A SPECTROPHOTOMETRIC METHOD
ALFRED C. REDFIELD
(From the Department of Physiology. Harvard Medical School, Boston, and
the Marine Biological Laboratory, Woods Hole)
The respiratory proteins, including hemoglobin, hemocyanin, chlo-
rocrurin and hemerythryn, are unique in combining with and dissociat-
ing from oxygen at pressures which fit them for the physiological trans-
portation of this gas. The factors which determine the condition of
equilibrium between oxygen and the pigment are of interest not only
because of the evident physiological relationship between the charac-
teristics of the oxygen dissociation curves of the blood of various or-
ganisms and the pressures of oxygen in the environment, but because
of the interesting physico-chemical problem which the phenomena pre-
sent. The hemocyanins appear to possess certain advantages for the
study of these problems. Not only do these proteins exist naturally in
solution in the blood so that the complications which arise from dealing
with corpuscles are avoided, but they are relatively stable compounds
which lend themselves without difficulty to purification and preserva-
tion. The hemocyanins of different species appear, in addition, to ex-
hibit very considerable differences in their physical and chemical prop-
erties; and consequently, one has the advantage in their study of being
able to resort to the comparative method in testing generalizations.
Finally, from the technical point of view, the hemocyanins which are
essentially colorless when reduced become strongly colored in the oxy-
genated state and consequently lend themselves to the employment of
colorimetric methods for the determination of the degree of oxygenation
of the solutions.
The present paper contains an account of a spectrophotometric
method for the determination of the degree of oxygenation of hemo-
cyanin solutions. The method is applied to an examination of the
equilibrium between oxygen and a purified salt-free preparation of the
hemocyanin of the horse-shoe crab, Linmlns [>oly[>licuu<s, at different
hydrogen ion concentrations.
THE SPECTROPHOTOMETRIC METHOD
The color of hemocyanin solutions has been taken as an indication
of the degree of oxygenation of the protein and used as the basis for
238
EQUILIBRIUM OF OXYGEN WITH HEMOCYANIN 239
constructing oxygen dissociation curves by Pantin and Hogben (1925)
and Redfield and Hurd (1925). Tbe method has an advantage over
the usual methods of gas analysis in that it requires no correction for
the oxygen dissolved in the solution — a correction which is relatively
large in comparison to the oxygen content in the case of hemocyanin
solutions and which is difficult to determine in a satisfactory manner.
When submitted to the proper controls, the method has the advantage
that it measures oxyhemocyanin directly by the employment of the
spectrophotometer to determine the absorption of monochromatic light
of suitable wave-length. Measurements can be made with an ease and
accuracy not obtained in the available methods of gas analysis when
applied to hemocyanin solutions.
The study of the absorption of light by hemocyanin solutions (Red-
field, 1930) affords the essential basis for the employment of the spec-
trophotometric method. It was shown that the absorption spectra may
be analyzed into two components. One is that due to the scattering
of light by the solution, the other is that attributable to the true absorp-
tion by the chromatic group formed when oxygen unites with the hemo-
cyanin molecule to form oxyhemocyanin. In addition, with the blood
of certain animals, the presence of other coloring matters must be taken
into account. The component due to the scattering of light is variable
depending upon the composition of the solution. It may, however, be
readily determined by the study of reduced solutions. The component
due to true absorption by the chromatic group was found to be a con-
stant, characteristic of the amount of oxyhemocyanin present, and to
vary little if at all with changes in the solution. The presence of other
pigments in the blood does not offer complications to the spectrophoto-
metric determination of the absorption of light by the chromatic group,
provided these pigments do not undergo change in color with oxygena-
tion ; and their influence upon the measurements may be largely avoided
by selecting for measurement wave-lengths which are little absorbed
by these pigments.
In order to confirm the assumption that the color of a hemocyanin
solution is an index of the quantity of oxygen combined with the protein
(a supposition heretofore entirely unsupported by exact experiment),
the degree of oxygenation of the serum of the horse-shoe crab, Limuhis
pol\plicinus, was determined simultaneously by the colorimetric method
and with the Van Slyke blood-gas analyzer, at a series of oxygen pres-
sures insufficient to produce complete saturation. To 90 cc. of fresh
serum. 10 cc. of 0.05 NaOH were added. The pH value of this solu-
tion was pH 8.3; the oxygen content was 1.4 volumes per cent when
equilibrated with air. Specimens of 10 cc. of serum were equilibrated
240
ALFRED C. REDFIELD
with air in tonometers evacuated to varying degrees, after the method
described by Pantin and Hogben (1925). The tonometers consisted
of 250 cc. cylindrical vessels provided with a small test tube sealed
on at one end. The other end was closed with a rubber stopper pro-
vided with a two-way glass stopcock. When equilibration was complete,
the hemocyanin was run down into the small test tube and its color
compared with a series of standards made up by diluting the original
serum as described by Pantin and Hogben. The tonometers were con-
nected with a reservoir of hydrogen and this gas was allowed to flow
in until the pressure was raised to that of the atmosphere. The sample
was now withdrawn from the tonometer into a pipette and transferred
to a Van Slyke blood-gas analyzer, with which its oxygen content was
measured. In estimating the oxygen dissolved in the samples, the solu-
bility coefficient was taken to be 0.0235 (Redfield, Coolidge and Mont-
gomery, 1928), a value closely checked by direct measurements on
this specimen of serum.
TABLE I
Comparison of Colorimetric and Gasometric Determination of Degree of Saturation of
Limulus Serum with Oxygen
Oz Pressure
Oi Content
O'i Dissolved
O2 Combined
Saturation
Color
mm. Hg
vol. per cent
vol. per cent
vol. per cent
per cent
per cent
3.1
0.082
0.010
0.072
7.8
10
4.5
0.152
0.014
0.138
15.0
20
5.3
0.235
0.016
0.219
23.8
25
10.8
0.399
0.034
0.365
39.7
40
13.8
0.484
0.043
0.441
48.0
50
15.9
0.548
0.049
0.499
54.4
55
21.7
0.653
0.067
0.586
63.8
65
34.4
0.953
0.106
0.847
92.0
90
155.0
1.400
0.480
0.920
100.0
100
The results of this experiment are recorded in Table I. It may be
seen that the degree of saturation of the solution as estimated from
its color agrees closely with that determined from the direct measure-
ment of the oxygen combined with the hemocyanin. The use of the
spectrophotometer would markedly improve the precision of the colori-
metric estimations in this experiment. The errors inherent in the gaso-
metric measurements are, however, so large that the significance of the
comparison would not be increased by the further refinement of this
part of the experiment.
While the foregoing affords practical demonstration of the utility
of colorimetric methods for determining the oxygenation of hemocyanin
solutions, confidence in the more precise measurements obtained with
the spectrophotometer must be based upon the theoretical adequacy of
the procedure.
EQUILIBRIUM OF OXYGEN WITH HEMOCYANIN 241
The analysis of the ahsorption of light by hemocyanin solutions
indicated that, for any wave-length of light
E0----Ex + Er, (1)
where E0 is the extinction coefficient of the oxygenated solution, Ex is
the extinction coefficient characterizing the absorption of light by the
chromatic groups in the oxygenated solution, and Er is the extinction
coefficient of the reduced solution. It was shown that Beer's law ap-
plies to hemocyanin solutions in both the oxygenated and reduced con-
dition. One may consequently write :
E0 = cK0,
ET = cKr,
Ex = cKx,
where c is the concentration of hemocyanin and K0, K,-, and K.e are the
extinction coefficients at unit concentration. It follows that
E0 = cKx + cKr. (2)
If E,, be the extinction coefficient characteristic of a mixture of oxygen-
ated and reduced hemocyanin in which the concentration of oxygenated
hemocyanin is yc and that of reduced hemocyanin is (1 - -y)c,
Ey = y(cKx + cKr) + (1 - y)cKrt (3)
Ey = ycKx + cKr. (4)
Substituting Er for cKr in equations (2) and (4), dividing and rear-
ranging,
F F
r
F - F
J—IQ J—Jr
This result is obtained without any explicit assumption regarding
the cause for the absorption of light measured by Er, and the equation
may consequently be applied in determining the degree of saturation of
solutions in which other pigments as well as scattering effects are re-
sponsible for the value of this term. It may also be derived by means
of a slightly different argument for cases such as that exhibited by
hemoglobin solutions, in which the prosthetic group absorbs considerable
but different quantities of light in the oxygenated and reduced condition.
It is assumed in the foregoing that an incompletely saturated solution
of hemocyanin is a mixture of completely reduced and completely oxy-
genated elements. No account is taken of the possibility that incom-
pletely oxygenated molecules may occur which possess absorption
spectra different from that of the completely oxygenated solution.
242
ALFRED C. REDFIELD
This possibility cannot be ignored in view of the success which theories
of intermediate degrees of oxygenation have met in explaining the char-
acteristics of the oxygen dissociation curves of hemoglobin (Adair,
1925; Ferry and Green, 1929) , even though the experiments of Conant
and McGrew (1930) failed to demonstrate the existence of such in-
termediate compounds. The high molecular weights reported for
hemocyanins (Svedberg and Chirnoaga, 1928; Svedberg and Heyroth,
1929) definitely indicate that many oxygen molecules may combine
with each hemocyanin molecule. If the chromatic group undergoes
intermediate degrees of oxygenation, this will affect the foregoing
deduction only in so far as the spectrum of the partially oxygenated
chromatic group differs from that of the completely oxygenated chro-
matic group. The spectrum of the chromatic group in a partially sat-
urated solution has consequently been determined and compared with
that of the completely oxygenated solution. The result is tabulated in
Table II. The ratio of the extinction coefficients of the chromatic group
of fully and partially oxygenated solutions is practically the same at all
wave lengths, which indicates that the partially saturated solution does
not contain intermediate compounds which differ in their spectral char-
acteristics from the fully oxygenated solution.
TABLE II
Comparison of Spectrum of Fully Oxygenated and Partially Oxygenated Hemocyanin
of Limuliis. Concentration. .0258 grams per cc. ; length of tube, 3.3 cm.; pH = 7.43;
"Salt Free."
Wave-
Length
Equilibrated
with
726 mm. O*
Equilibrated
with
2.6 mm. O2
Equilibrated
with
T T
Oxygenated
Chromatic
Group
Partially
Oxygenated
Chromatic
Group
y
mn
Eo
Ev
Er
E0 Er
Ey-Er
Ey-Er
E0-Er
460
0.157
0.100
0.034
0.123
0.066
.536
480
0.136
0.086
0.034
0.102
0.052
.510
500
0.151
0.094
0.030
0.121
0.064
.529
520
0.1Q9
0.120
0.028
0.171
0.092
.538
540
0.253
0.150
0.026
0.227
0.124
.546
560
0.288
0.170
0.026
0.262
0.144
.550
580
0.303
0.178
0.027
0.276
0.151
.547
600
0.302
0.172
0.025
0.277
0.147
.530
620
0.285
0.168
0.025
0.260
0.143
.550
640
0.265
0.155
0.024
0.241
0.131
.544
660
0.244
0.143
0.028
0.216
0.115
.532
680
0.216
0.127
0.028
0.188
0.099
.528
700
0.200
0.117
0.022
0.178
0.095
.530
EQUILIBRIUM OF OXYGEN WITH HEMOCYANIN 243
THE DETERMINATION OF THE OXYGEN DISSOCIATION CURVE
Measurement of Degree of Oxygcnation. — The extinction coefficients
of the solutions were measured with the aid of a Konig-Martens spec-
trophotometer. They are given by equations of the type
2 (log tan a0 - log tan «,)
E° = ~~
where a0 is the angle of the analyzing Nicol prism when oxyhemocyanin
is measured ; at is the angle when the absorption of light by the solvent
is determined ; and d is the length of the absorbing column. Similarly,
expressing the angular reading characteristic of an incompletely satur-
ated solution as a,, and that o'f the reduced solution as a,-, E,, and E,-
are obtained. Substituting in (5) the degree of saturation, v is given
by the expression
2, 2.
-ilog tan ciy — - log tan ar
a (t fff\
-V = 2~ ~T~ " (7)
- log tan a0 - - log tan ar
a (i
Since the corrections for the absorption of light by the solvent cancel
out, they need not be measured. In practice the greatest accuracy is
obtained at wave-lengths giving the greatest difference between the
values of E0 and E,. In the case of hemocyanin solutions, this occurs
in the yellow region of the spectrum. This fact is fortunate in that this
is the region in which readings can be made with the greatest accuracy.
It is also a practical advantage that the scattering of light is small at
these wave lengths and variations due to changes in the physical con-
ditions of the solution are consequently minimal. In preparing solu-
tions, the greatest precision is obtained by adjusting the concentration
and the length of the absorbing chamber so that the angle a0 is as large
as is compatible with precise measurement, that is, about 75°.
Equilibration with Oxygen. — In order to equilibrate the solutions
with oxygen of known pressure, tonometers such as those illustrated
in Fig. 1 were employed. These consist of cylindrical bottles of Pyrex
glass, having a capacity of 250 cc., to one end of which is sealed a
T-tube having an internal diameter of approximately one centimeter.
The ends of the T are ground parallel to one another and are closed with
flat glass disks sealed on with DeKhotinsky cement. The mouth of
the bottle is closed with a rubber stopper in which a two-way glass stop-
cock is inserted. About five cc. of the solution to be measured is placed
in the tonometer, which is then evacuated and refilled with a gas mixture
244
ALFRED C. REDFIELD
containing a convenient proportion of oxygen. In the experiments de-
scribed in this paper, nitrogen containing 2 to 5 per cent of oxygen
has been convenient ; for other solutions, air will serve or pure oxygen
FIG. 1. Arrangement for adjusting pressure of gas in tonometers. A,
tonometer in position for measuring pressure after equilibration. B, tonometer
in thermostat during settling of sediment. M, mercury manometer. R, reservoir
which serves to slow rate of evacuation of tonometers.
may be necessary. The tonometer is now evacuated to some definite
pressure and the solution equilibrated with the gas at this pressure, ro-
tating the tonometer in a horizontal position for fifteen minutes in a
water bath at constant temperature. The tonometer is next returned
to a vertical position and evacuated and filled again with the gas mixture
and pumped out to the same reduced pressure. The solution is further
equilibrated for twenty-five minutes and then, without removal from
the water bath, is turned into the vertical position and connected with
the manometer after setting the pressure in the system to that expected
to obtain in the tonometer. The passage connecting the tonometer and
manometer with the pump and reservoir is now closed. The stopcock
leading into the tonometer is opened, and the pressure obtaining in
the tonometer is carefully measured and recorded. The stopcock of
the tonometer is closed again and disconnected from the pump. The
tonometer is carefully dried and placed in an air-chamber inserted into
the water bath where it is kept for a period of approximately one hour
in order that the small particles of denatured protein which almost in-
variably form during the process of evacuation and equilibration may
EQUILIBRIUM OF OXYGEN WITH HEMOCYANIN 245
settle out. Following this, the absorption of light by the hemocyanin
solution is measured by placing the tonometer with the T-tube in the
path of one of the beams of the spectrophotometer. If the solutions
are not clear at the time when the measurements are made, the results
should be rejected.
The partial pressure of oxygen in the tonometer, />, is given by the
expression
p = (B - P - aq) /,
where B is the barometric pressure, P is the pressure in the tonometer
recorded at the end of equilibration, aq is the tension of aqueous vapor
at the temperature of the water bath, and / is the fraction of oxygen
in the gas mixture.
To obtain complete oxygenation, the tonometer is simply evacuated
and filled with pure oxygen gas prior to equilibration. To obtain com-
plete reduction is difficult under those circumstances in which the affinity
of hemocyanin for oxygen is great. We have not found the employ-
ment of chemical reducing agents satisfactory, as certain of these tend
to influence the color of the solution and others must be employed in
such concentrations that they may affect the scattering of light on which
the absorption by the reduced solution depends. The most satisfactory
procedure is to employ hydrogen to wash out the tonometer after the
oxygen is freed from the hemocyanin under low pressure. The solu-
tions are accordingly evacuated, equilibrated for twenty minutes, filled
with hydrogen, re-evacuated, again equilibrated, allowed to settle, and
then measured. Further repetition of the process does not lead to lower
readings, although it is doubtful whether, under certain circumstances,
this process removes the last traces of oxy hemocyanin. The reason for
this is that, in the process of evacuation and equilibration, small quan-
tities of denatured material are formed which fail to settle out com-
pletely when the solutions are allowed to stand. The formation of pre-
cipitates of this sort, which goes on more readily in the reduced solu-
tions, constitutes the principal limit to the precision of the method.
We have recently constructed tonometers which can be placed in the
cups of a large centrifuge and which make it possible to remove these
troublesome precipitates. Such tonometers have not been employed
in the experiments described in this paper.
The Preparation of the Hemocyanin Solutions. — The hemocyanin
employed in the present investigation was prepared from material ob-
tained during the summer of 1928. It was preserved in the precipitated
state by adding 350 grams of ammonium sulphate to each liter of serum.
The material was purified some months later by repeated salting out
246 ALFRED C. REDFIELD
followed by dialysis, against dilute sodium bydroxide, as described by
Redfield, Coolidge and Sbotts (1928). Tbree preparations were ob-
tained, having' the following characteristics : Specimen 18 A, dry weight
0.1031 gram per cc., combined base 19.4 X 10~5 mols per gram; Speci-
men 18 B, dry weight 0.1255 gram per cc., copper 0.0208 milligram
per cc. or 0.168 gram per 100 grams dry substance, combined base
19.1 ; ' 10 5 mols per gram; Specimen 18 C, dry weight 0.097 gram
per cc.. copper 0.16 milligram per cc. or 0.165 gram per 100 grams
dry weight, combined base 21.6 X 10^5 mols per gram. These solutions
were preserved with toluene at a low temperature. The day before
measurements were to be made, they were further diluted by the addition
of distilled water containing amounts of hydrochloric acid or sodium
hydroxide appropriate to secure the desired hydrogen ion activity and
to reduce the hemocyanin to a concentration favorable for the measure-
ments; that is, to about 2.5 per cent. After standing all night, the
solutions were filtered and then employed for the determination of the
oxygen dissociation curves. A portion of the solution was also reduced
by equilibration with hydrogen and used for the determination of the
hydrogen ion concentration by means of the hydrogen electrode.1
Measurements were made upon solutions at several hydrogen ion
activities between pH 7.4 and pH 10.4, and upon a solution at pH
4.5. At hydrogen ion activities intermediate between pH 4.5 and about
pH 6.8, Liundiis hemocyanin is insoluble in distilled water, and solu-
tions of sufficient clarity cannot be obtained. At reactions more acid
than pH 4.5, a colorless modification of L'unulus hemocyanin is formed
(Redfield and Mason, 1928). The characteristics of the oxygen dis-
sociation curve at these hydrogen ion activities will be dealt with in
a subsequent paper.2
DATA ON OXYGEN DISSOCIATION
The results of the series of measurements which have been made
upon purified solutions of Limulus hemocyanin are recorded in Table
III. The first column contains a description of the material employed
in each case ; the second column, the partial pressure of oxygen in the
tonometer at the completion of equilibration ; the third column, the value
of the extinction coefficient of the solution (2/d log tan a,/), as meas-
ured with the spectrophotometer, employing light of the wave-length
1 In the case of the two solutions prepared from Specimen 18 C, the pH value
was somewhat less than that to be expected from the amount of NaOH .added,
as judged from the titration curve published by Redfield, Humphreys and Ingalls
(1929). In the other solutions the agreement is good.
2 I am indebted to Miss Elizabeth Ingalls for technical assistance in con-
ducting the experiments and for preparing the hemocyanin solutions employed.
EQUILIBRIUM OF OXYGEN WITH HEMOCYANIN 247
590 m/j.. This measurement is not corrected for absorption by the
solvent. The fourth column records the extinction coefficient of the
oxygenated chromatic groups, 2/W(log tan a,, --log tan a-,-) ; the fifth
column, the value of v as defined in equation (7). The equilibration
was carried out in a water bath at 25° C. The length of the T-tube
of the tonometer in which the absorption of light was measured, d, was
usually 3.3 centimeters. In the case of a few measurements, tubes
were employed which differed slightly from this length (3.15 to 3.60
cm.).
THEORY OF OXYGEN EQUILIBRIUM
In oxyhemocyanin, one molecule of oxygen is bound by a quantity
of hemocyanin containing two atoms of copper. The reversible reac-
tion may consequently be indicated by the equation
where n represents the number of mols of oxygen bound by each mol
of hemocyanin. In treating the equilibrium according to the mass law,
as was done by Hiifner (1901) and later by Hill (1910), in the case
of hemoglobin, the result is
in which k is the equilibrium constant of the reaction. If v is the
fraction of hemocyanin in the oxygenated condition, 1 - -y is the reduced
fraction and, putting p, the partial pressure of oxygen in mm. of mer-
cury, in place of the oxygen concentration, equation (8) may be written
(9)
1-v
or,
log -— = log K + n log p. (10)
In this form the equation is convenient for graphical solution for n and K.
In Fig. 2 is reproduced the data recorded in Table I, arranged in
the form indicated by equation (10). The lines drawn through the
points in each case are straight lines indicating the linear relationship
demanded by the equation. The slope of the lines drawn through the
points, determining the value of n, is 1.0. The values of K correspond-
ing to the positions of the lines drawn in Fig. 2 are indicated in Table
III. Employing these values of K and taking ;/ as equal to 1.0 in each
case, the values of v may be calculated and are indicated in column 6 of
248
ALFRED C. REDFIELD
Table III for comparison with the observed values. It appears that
the theoretical treatment from which equation (10) is derived is ade-
quate to account for the shape of the oxygen dissociation curve at least
pn 743
pn
pn gsi
109 /i
FIG. 2. Logarithmic plot of data of oxygen dissociation curve of hemocyanin
of Ln;n</;M- polyplicwus at various pH values. Temperature, 25° C. ; y, fraction
of hemocyanin in oxygenated condition ; f>, oxygen pressure in mm. Hg.
as a first approximation, and to provide a single series of constants
to define the effect of hydrogen ion activity upon the equilibrium.3
Careful scrutiny of the data in Table III reveals a tendency for the
low values of v to be slightly greater than the calculated values and high
values to be slightly less than the theoretical. In order to make vivid
the adequacy of the theory for treating the entire set of observations,
in Fig. 3 the values of y obtained at each pH value are plotted against
Kf> in the usual form of the oxygen dissociation curve, and a line corre-
sponding to the theoretical treatment is drawn through the points, «
again being taken as 1.0.
3 It should be emphasized that the pH values are determined on reduced solu-
tions. No account has been taken of possible change in pH with oxygenation.
According to Redfield, Humphreys and Ingalls (1929), the effect may be expected
to be small.
EQUILIBRIUM OF OXYGEN WITH HEMOCYANIN
249
TABLE III
Data of Oxygen Dissociation Curves of Limn! us Hemocyanin. Temperature, 25° C.;
Wave-length, 590 HIM-
Description
P
2
-7 log tan ay
- loe tan a"
y
y
d '°g tan ar
mm. Hg
(observed)
(calculated)
Specimen 18 A
0
0.034
0
0
0
0.16
0.068
0.034
0.117
0.074
Concentration:
0.56
0.106
0.072
0.248
0.218
0.0258 grams per
0.80
0.133
0.099
0.342
0.286
cc.
1.24
0.149
0.115
0.397
0.382
Combined acid :
1.65
0.166
0.132
0.455
0.452
20 X 10~5 mols
2.34
0.194
0.160
0.552
0.540
per gram
2.74
0.204
0.170
0.586
0.578
pH 4.52
3.92
0.225
0.191
0.659
0.662
6.20
0.252
0.218
0.752
0.757
K = 0.500
7.28
0.259
0.225
0.776
0.784
8.65
0.269
0.235
0.810
0.812
11.9
0.281
0.247
0.852
0.856
14.8
0.283
0.249
0.859
0.881
17.6
0.291
0.257
0.886
0.898
20.6
0.298
0.264
0.910
0.913
24.5
0.299
0.265
0.914
0.924
27.4
0.299
0.265
0.914
0.933
39.0
0.305
0.271
0.935
0.952
744
0.324
0.290
1.00
1.00
Specimen 18 A
0
0.034
0
0
0
0.39
0.092
0.058
(0.265)
0.171
Concentration:
0.80
0.119
0.085
0.305
0.276
0.0258 grams per
1.49
0.158
0.124
0.441
0.415
cc.
2.61
0.188
0.154
0.548
0.554
Combined base:
3.74
0.207
0.173
0.616
0.640
19 X 10~5 mols
4.78
0.223
0.189
0.672
0.695
per gram
5.96
0.239
0.205
0.730
0.739
pH 7.43
6.83
0.253
0.219
0.780
0.766
8.38
0.258
0.224
0.797
0.800
£.' = 0.476
16.6
0.282
0.248
0.882
0.888
26.4
0.300
0.266
0.946
0.927
37.7
0.297
0.263
0.936
0.948
152
0.310
0.276
0.982
0.987
740
0.315
0.281
1.00
1.00
250
ALFRED C. REDFIELD
TABLE III (continued)
Data of Oxygen Dissociation Curves of Limulns Hemocyanin. Temperature, 25° C. ;
Wave-length, 590 m/i.
Description
P
-j log tan av
2 10B tan ^
y
y
d tan ar
mm. Hg
(observed)
(calculated)
Specimen 18 B
0
0.046
0
0
0
0.84
0.078
0.032
0.166
0.213
Concentration:
1.22
0.103
0.057
0.295
0.282
0.0208 grams per
1.98
0.123
0.077
0.399
0.390
cc.
3.15
0.150
0.104
0.539
0.504
Combined base:
4.40
0.158
0.112
0.580
0.587
39 X 10~5 mols
9.30
0.185
0.139
0.720
0.750
per gram
12.80
0.201
0.155
0.803
0.805
pH 8.51
17.7
0.204
0.158
0.818
0.852
28.1
0.217
0.171
0.886
0.900
K = 0.322
39.0
0.219
0.173
0.902
0.927
751
0.239
0.193
1.00
1.00
Specimen 18 C
0
0.047
0
0
0
0.63
0.082
0.035
0.155
0.177
Concentration:
0.97
0.093
0.046
0.204
0.217
0.0242 grams per
1.64
0.128
0.081
0.358
0.319
cc.
4.12
0.167
0.120
0.531
0.541
Combined base:
6.38
0.197
0.150
0.654
0.646
63 X lO"5 mols
8.94
0.209
0.162
0.717
0.719
per gram
11.5
0.216
0.169
0.748
0.767
pH 9.71
16.8
0.230
0.183
0.810
0.828
21.8
0.228
0.181
(0.801)
0.862
K = 0.286
26.7
0.250
0.203
0.898
0.885
724
0.273
0.226
1.00
1.00
Specimen 18 C
0
0.066
0
0
0
0.61
0.084
0.018
0.092
0.098
Concentration :
1.10
0.088
0.022
0.112
0.164
0.0242 grams per
1.79
0.124
0.058
0.296
0.242
cc.
4.15
0.155
0.089
0.454
0.426
Combined base:
6.53
0.177
0.111
0.566
0.538
77 X 10~5 mols
9.04
0.183
0.117
0.597
0.618
per gram
12.2
0.202
0.136
0.694
0.686
pH 10.42
17.1
0.242
0.176
(0.898)
0.754
22.6
0.225
0.159
0.812
0.802
K = 0.178
27.2
0.224
0.158
0.806
0.830
751
0.262
0.196
1.00
1.00
EQUILIBRIUM OF OXYGEN WITH HEMOCYANIN
251
DISCUSSION
In the forty years since Hiifner suggested the application of the
mass law to the equilibrium between oxygen and hemoglobin, numerous
investigations have indicated that equations similar to those employed
in the present treatment are more or less adequate to describe the data
in hemoglobin solutions free of electrolytes (Barcroft, 192cS). Un-
certainty has sometimes accompanied the results of such investigations
because of the instability of purified hemoglobin solutions (Ferry, 1924;
Hecht, Morgan and Forbes cited by Barcroft, 1928) . In the presence
of electrolytes and in blood, the dissociation curves of hemoglobin in-
variably have a sigmoid shape, requiring some additional assumptions
for their explanation.
/o
y
,y»< t
• -pM -ass K-OSOO
Q-ptl 743 K-O475
&-f>H B3/ K-G3SS
+ -pn 3 71 K-OSB6
y.-prt ID <se f<-o. 1 70
IB
FIG. 3. Data of oxygen dissociation curves of hemocyanin of Liuiiihis
polyphemus plotted to show the similarity of shape at various pH values, y is
fraction of hemocyanin present as oxyhemocyanin ; p is oxygen pressure in ram.
Hg ; temperature, 25° C. The curve corresponds to equation (9) when K = \,
»= 1.
In the case of hemocyanin solutions, Stedman and Stedman ( 1928)
found that the respiratory pigment of the snail, Hcli.r poinatla. com-
bines oxygen in accordance with the mass law, as expressed in equation
(8), n being taken to be 1.0. The hemocyanin of the Crustacea, Houi-
arus I'ulgaris and Cancer pac/unis, according to these investigators (1926
a, 1926 b) is characterized by oxygen dissociation curves of a more
complex nature when examined in dialyzed solution. The present in-
vestigation of Linntliis hemocyanin indicates that this substance, when
in " salt free " solutions, resembles the hemocyanin of Hcliv in its con-
formity to the mass law.
Stedman and Stedman, in discussing their observations on HeH.v
17
252 ALFRED C. REDFIELD
hemocyanin, conclude from the fact that the value of n is 1.0, that this
hemocyanin is dispersed in solution in such a way that each hemocyanin
molecule unites with but a single oxygen molecule. It is tempting to
draw the same conclusion with regard to Limulus hemocyanin, for the
investigations of Redfield, Coolidge and Shotts (1928) indicated that
the probable molecular weight of this protein is 73,400 and that each
molecule contains two atoms of copper. The measurements of Red-
field, Coolidge and Montgomery (1928) demonstrate further that such
a hemocyanin molecule would bind but a single oxygen molecule. The
value of ;; established in this investigation follows as a prediction from
these considerations. It must be recalled, however, that Svedberg and
Heyroth (1929) obtained much larger values for the molecular weight
of Linntlns hemocyanin by the employment of the ultra-centrifugal
method. In view of the uncertainty regarding the size of the hemo-
cyanin molecule, reserve is required in interpreting the data of the oxy-
gen equilibrium. If one goes back to the kinetic basis of the mass law
equation (8), it may be noted that the fundamental assumptions concern
the probability of the union of an oxygen molecule with the respiratory
protein and the probability of the dissociation of such a union. Where
expressions arise giving values of n greater than 1.0, or more complicated
equations, it is through the assumption that some relation exists be-
tween the combination of oxygen by contiguous groups ; either that
they unite with oxygen simultaneously as pairs or larger groups, or
that they combine in successive steps so that one cannot react until after
others have done so. All that can safely be concluded from a demon-
stration that hemocyanin unites with oxygen as though it were dis-
persed in molecules each combining with but a single oxygen molecule
is that it behaves as though this were the case. That is to say, the oxy-
gen dissociation curve is such as would be obtained if the various oxygen
binding groups reacted independently of one another so that the oxy-
genation of any one did not influence the probability of oxygenation or
reduction of any other. That this may be the case in a molecule con-
taining a number of oxygen-binding groups does not seem altogether
impossible when it is recalled that the molecular weight of such a mole-
cule would be 73,400 times the number of groups. It should be recalled
that in combining with acid, the molecule of Llimilus hemocyanin, which
binds at least 117 equivalents of acid, behaves as though each acid-
binding group reacted independently of every other (Redfield and
Mason, 1928). In this regard the behavior of this protein is not ex-'
ceptional.
The measurements recorded in Table III make it clear that as alkali
EQUILIBRIUM OF OXYGEN WITH HEMOCYANIX 253
is added to solutions of purified Linmlns hemocyanin the equilibrium
constant of oxygenation decreases progressively, indicating that greater
pressures of oxygen are required to produce any given degree of oxy-
genation. No suggestive relationship is apparent between the values
of K, and either the quantity of alkali added or the hydrogen ion activity
of the solution. It is noteworthy that the phenomena exhibited by these
purified solutions of hemocyanin differ markedly from those obtaining
in the native serum of Liinnliis. As pointed out briefly by Hogben
and Pinhey (1927) an extensive series of measurements on the oxygen
dissociation curves of Limulus serum (which we have not pub-
lished) demonstrate that at pH values up to about 8.3 the oxygen pres-
sure requisite to produce a given degree of oxygenation increases. At
higher pH values these pressures decrease again, much as is the case
with Helix aspersa blood (Hogben and Pinhey, 1926).
In their investigation of the dialyzed hemocyanin of Helix pomatia,
Stedman and Stedman (1928) report that no detectable change in the
curve with change in pH was observed. Experiments now in progress
with the purified hemocyanin of Busycon canaliculatum agree closely
with the findings in the case of Limulus, indicating a definite decrease
in the value of K with diminishing hydrogen ion activity. Inasmuch
as the results with Heli.v are otherwise very similar to those obtained
with Limulus and Busycon hemocyanin, we have reexamined the Sted-
mans' data and find evidence suggesting that, with this material, there
may be a small effect of hydrogen ion concentration upon the value of
K. In a set of curves defined by equation (9), differing only in the
value of K and where n equals 1.0, the greatest differences in y obtain
between degrees of saturation of 0.40 and 0.80. It is in this range that
differences in the curves mav be most readily detected. We have con-
sequently evaluated K on the basis of their data selected between these
degrees of saturation. The results are presented in Table IV. The
average value of K for the data selected is 0.250. Of the nine meas-
urements made on solutions more alkaline than pH 7, the mean value
is 0.217 and the highest value is 0.236. Of the ten measurements made
on solutions more acid than pH 7, the average value is 0.270, and only
two values are less than 0.236. This result indicates that a small but
definite change in the value of K may occur in the case of Helix hemo-
cyanin with change in hydrogen ion concentration, and that the phe-
nomena in this case may not differ qualitatively from that obtaining
with Limulus and Busvcon.
254
ALFRED C. REDFIELD
TABLE TV
•
The Equilibrium Constant of Oxygenation of HELIX POMATIA Hemocyanin at Various
pH Values Calculated from the Data of Stedman and Stedman (1928)
pH
P
Per cent
Saturation
(corrected)
Per cent
Unsaturated
K
mm. Hg
100 X y
100 X (1 - y)
4.04
2.89
47.9
52.1
0.318
8.38
70.3
29.7
0.282
11.61
72.4
27.6
0.226
4.79
2.74
40.4
59.6
0.247
6.73
67.9
32.1
0.314
6.25
2.85
46.7
53.3
0.307
6.45
64.5
35.5
0.282
11.13
70.8
19.2
0.331
6.35
3.79
47.9
52.1
0.243
11.30
62.6
37.4
0.148
7.81
2.83
39.8
60.2
0.234
7.00
57.1
42.9
0.190
7.96
62.8
37.2
0.212
8.74
2.90
40.6
59.4
0.236
6.15
59.1
40.9
0.235
10.04
65.8
34.2
0.191
9.02
2.50
37.0
63.0
0.235
6.00
55.6
44.4
0.209
7.51
62.1
37.9
0.218
SUMMARY
A spectrophotometric method for measuring the equilibrium of
hemoc.yanin and oxygen is described.
The oxygen dissociation curves of purified hemocyanin of Limulus
in the absence of salts and at various hydrogen ion activities are de-
termined.
It is shown that the equilibrium between oxygen and these hemo-
cyanin solutions is defined, as a first approximation, by the mass law
on the assumption that the various oxygen- combining groups react in-
dependently of one another in their combination with oxygen.
The value of the equilibrium constant of the oxygenation reaction
decreases as the pH value increases from 4.5 to 10.4.
BIBLIOGRAPHY
ADAIR, G. S., 1925. Jour. Bwl. Chew.. 63: 529.
ADAIR, G. S., 1925. Proc. Roy. Soc., London. Series A, 109: 292.
BARCROFT, J., 1928. The Respiratory Function of the Blood. Part II. Hemo-
globin. Cambridge.
CONANT, J. B., AND McGREW, R. V., 1930. Jour. Bwl Chan., 85: 421.
EQUILIBRIUM OF OXYGEN WITH HEMOCYANIN 255
FERRY, R. M., 1924. Jour. Biol. Client., 59: 295.
FERRY, R. M., AND GREEN, A. A.. 1929. Jour. Biol. Client., 81: 175.
HILL, A. V., 1910. Jour. Physio!., 40: iv.
HOGBEX, L. T., AXD PIN HEY, K. F., 1926. Brit. Jour. Ex per. Biol., 5: 55.
HUFNER, G., 1901. Arch. f. Anal. u. Physiol. Supp.-Band, 5: 187.
PAN-TIN, C. F. A., AND HOGBEN, L. T., 1925. Join: Marine Biol. Assn. United
Kingdom, 13: 970.
REDFIELD, A. C., 1930. Biol. Bull, 58: 150.
REDFIELD, A. C., COOLIDGE, T., AND MONTGOMERY, H., 1928. Jintr. Biol. Chem.,
76: 197.
REDFIELD, A. C., COOLIDGE, T., AND SHOTTS, M. A., 1928. Jour. Biol. Chem.,
76: 185.
REDFIELD, A. C, AND KURD, A. L., 1925. Proc. Nat. Acad. Sci., 11: 152.
REDFIELD, A. C, AND MASON, E. D., 1928. Jour. Biol. Chem., 77: 451.
STEDMAN, E., AND STEDMAN, E., 1926a. Biochem. Jour. 20: 938.
STEDMAN, E., AND STEDMAN, E., 1926/;. Biochem. Jour., 20: 949.
STEDMAX, E., AND STEDMAN, E., 1928. Biochem. Jour., 22: 889.
SVKDBERG, T., AND CniRNOAGA, E., 1928. Jour. Am. Chem. Soc., 50: 1399.
SVEDBERG, T., AND HEYROTH, F. F., 1929. Jour.Mtn. Chan. Soc., 51 : 550.
POLOCYTE FORMATION AND THE CLEAVAGE OF THE
POLAR BODY IN LOLIGO AND CHJETOPTERUS
LEIGH HOADLEYi
(From the Department of Zoology, Harvard University, and the Marine Biological
Laboratory, Woods Hole, Massachusetts)
The polar bodies which arise during the maturation of the eggs of
animals may be considered from two points of view. Most attention
has in the past been paid to the first, that of their meaning in the matura-
tion of the nucleus, which we will but mention here. These phenomena
are similar to those appearing in the maturation of the sperm cells and
hence, to generalize, the maturation of the egg cell may be said to
parallel that of the sperm as far as the reduction and equation divisions
are concerned. In addition there is the second point, that which con-
cerns the function and fate of the polar bodies (aborted ova, Mark
1881). This question has been shown by Conklin to be intimately as-
sociated with the history of the ovum and the relation of its maturation
phenomena to the penetration of the spermatozoon. In addition, it ap-
pears that one must consider the constitution of the polar body itself,
for in some cases the protoplasm extruded from the egg includes more
of the cytoplasmic regions (qualitatively) than in others. It is evident
that this qualitative difference in the division of the egg would be of
more significance than the mere quantitative division, though great
quantitative discrepancy in the equivalence of the daughter cells would
also be of great importance.
Observations have been made during the past summer which confirm
in the living egg the observations recorded by Lillie in experiments on
Choetopterus that the polar body of that form does not include all of
the constituent parts of the egg which continues to develop. These
results, when compared with some obtained in the study of the fate
of the polar bodies in Loligo, give a clew as to some of the factors
important to the non-developmental capacity of this structure. The
polar body of Loligo may divide several times as will be described below.
In Chcetopterus, the polar body does not divide and in this connection
it should be noted that in its formation, none of the cortical region
of the egg is involved so that the polar body represents only endoplasmic
cytoplasm and nucleus. This point would seem to be of great impor-
1 This investigation was aided by grant from the Milton Fund.
256
POLOCYTE FORMATION AND CLEAVAGE OF POLAR BODY 257
tance as far as the developmental capacity of the gamete is concerned.
We will consider the observations on these forms first and return to
the consideration of their significance and the relation between them
and other data already obtained below.
Loligo pealli
Eggs and sperm of the squid, Loligo pcalii, may be obtained in
large numbers from animals during the month of July at Woods Hole.
The mature eggs are found in the body cavity of the females and may
best be obtained in an uninseminated condition by opening the animal
on the posterior side. The eggs are clear and show a very distinct
micropyle as has been described by Watase (1891). Sperm may be
obtained from spermatophores produced in large numbers by the male.
Several of the spermatophores may be placed in a small amount of
sea-water and then broken or cut so that the sperm flow out into the
medium. This fluid may then be used to inseminate the eggs, which
should be placed in a small amount of sea-water. Owing to the large
size of both the eggs and the sperm and the clarity with which the well-
formed micropyle may be seen, the entire course of the sperm through
the micropyle and to the egg may be observed. As the sperm pene-
trates the egg, the polar cap may be seen to elevate, the cytoplasm
streaming to that portion of the egg directly under the inner opening
of the micropyle where it forms the blastodisc. In the squid, the pene-
tration of the spermatozoon normally takes place before the formation
of either of the polar bodies.
After the polar cap is well elevated, the first polar body appears.
This is relatively small though actually much larger than in Chatopterus,
for example. It appears first as a slight elevation on the surface of
the egg, which rapidly pinches it off. The polar body maintains its
approximate position on the surface of the egg, however, so that after
a short interval it serves as a locus in determining the site of the forma-
tion of the second polar body. This forms and the first polar body
divides. At that time the male pronucleus may be seen in the cytoplasm
of the egg as a bright spot near, but not in contact with, the female
pronucleus. The two gradually approach and are then lost to view
in the living egg. The egg then continues in its development, cleaving,
as has been described by Watase, to form a blastodisc covering first the
polar end of the egg and subsequently spreading around the yolk from
this point.
If. during the period immediately following the juxtaposition of the
two pronuclei, we observe the polar bodies, we find that these may
258 LEIGH HOADLEY
continue their development for a short time. Not only may the first
polar body divide, as is the case in many forms, but the second may
also divide or the first may show a division in one or both of its daughter-
cells. This is not always the case but is found in a large number of the
eggs of this form. In Fig. 1 may be seen a number of cases in which
there are four or five of the cells produced by the polar bodies attached
to the cortical portion of the egg and forming a nest of cells there.
When observed both in their division and afterwards, these are evi-
dently not the result of fragmentation of the cytoplasmic portion of
the polar body, but are real products of the division of these units.
Xo observations have been made in which more than six cells were
counted which resulted from the division of the polar bodies. Eggs
with five cells are not rare and those with four are common.
FIG. 1. Sketches of eggs of Lolic/o showing four and five polar bodies at-
tached to the surface.
The next series of observations on the extrusion of the polar bodies
in Loli(jo is difficult to make because of certain optical requirements.
The formation of the polar bodies was observed with the cardioid con-
denser. In the use of this, the object observed must be a specific dis-
tance from the condenser, which, owing to the great thickness of the
squid egg, requires the use of a cover glass as a slide and some manip-
ulation of the material. In the course of a large number of attempts,
however, certain of them were successful, so that the following de-
scription is based on a number of observations of the phenomenon.
As is the case with Cluetoptenis, which will be considered below, the
cytoplasmic portion of the egg of Loll go is covered with a thin cortical
zone. This zone is thinner than in Chcztopterus but similar in that
structurally it differs from the deeper layers. When the site of polar
body formation has been determined by the approach of the nucleus
and the elevation of the small mound from which it arises, it can be
seen that the cortical region is contained in the part elevated. In other
words, the portion of the cytoplasmic cap which goes into the polar
body is composed of a thin cortical region and deeper endoplasmic
POLOCYTK FORMATION AND CLEAVAGE OF POLAR BODY
portion. In addition, this receives, of course, a share of the maturation
nucleus. The second polar body is formed in essentially the same
manner. This fact is, I think, of great significance in the consideration
of the subsequent cleavage of the polocytes.
Clttrtoptcnts pcrgaincntoccns
The discussion and description of the formation of the polar bodies
of Ch(ctof>terns consists, insofar as the description of the living and
fixed egg is concerned, merely in a confirmation of the observations
of Lillie (1906). In addition, the egg has been studied by means of
the cardioid condenser, which enables the investigator to trace the cor-
tical, or as it is called in the above paper, the ectoplasmic portion of
the egg. and to distinguish it from the endoplasmic portion during the
maturation stages. The observations are entirely in accord with those
cited above, but will be described here in order that they may be re-
ferred to in the discussion.
FIG. 2. Photograph of a section of a Cluctoptcnts egg at the mesophase of
the first maturation division, showing the " ectoplasmic defect " and the arrange-
ment of adjacent parts.
FIG. 3. Sketch of an egg of Chcctoptcnis at the mesophase of the first matura-
tion division as it appears by dark field illumination with the cardioid condenser
to show the " ectoplasmic defect " as the polar elevation is forming.
As is well known, the egg of Ch(ctoptcnts is penetrated by tin
spermatozoon before the formation of the polocytes. Thus activated,
the cortical or ectoplasmic portion withdraws from the animal pole
of the egg, and the spindle of the first oocytic division approaches the
cell membrane remaining surrounded by the endoplasmic cytoplasm.
It becomes attached to the outer zone in the region of the ectoplasmic
260 LEIGH HOADLEY
defect and there forms the first polar body. This has been described
by Lillie (cf. Fig. 2) and may be seen in living eggs by means of the
cardioid condenser (Fig. 3). It is composed entirely of endoplasmic
substance without deutoplasm. The polar body must, therefore, repre-
sent ground substance plus some of the residual substance of the
germinal vesicle. In addition there is nuclear chromatin from the first
maturation division of the egg nucleus. The polocyte is entirely re-
leased from the egg, which immediately begins the second maturation
division, the ectoplasmic defect remaining till this is complete. During
this time the first polar body does not divide, as is the case in Loligo,
but remains inactive adjacent to the outer membrane covering the egg.
As in the case of the first polar body, the second contains none of the
cortical ectoplasmic material but only endoplasmic cytoplasm and nu-
cleus. The course as described here may be traced with ease by use
of the cardioid dark field apparatus, as the egg is relatively small and
there is an evident optical difference in the appearance of the cortical
ectoplasmic and the deeper endoplasmic layers. Associated with this
lack of the cortical layer is the fact that neither of the polocytes of
Chcctoptcrus divide as is the case of those produced by the egg of Loligo.
Discussion
As is well known, the relation between the formation of the polar
bodies and the penetration of the spermatozoon varies in different
groups of animals. In some forms the spermatozoon penetrates the
egg before either of the polar bodies is formed. In other cases the
first polar body is completed before penetration, but the second is de-
pendent upon the activation of the egg at fertilization. In still another
group the second polar body is also formed before penetration. It is
evident that conditions controlling the development of the polar body
must vary according to the conditions existing in different animals.
In the case of the first group, the polocyte is a part of the egg at the
time of the activation and therefore must initially be activated in the
same way. In the second group the second polar body must be activated
while the first is not. The first, in order to simulate the conditions
existing in the polar body of the first group, must be penetrated by a
sperm cell or be activated in some other fashion. In this group the
second polar body might conceivably develop save for the fact that
the cell organs introduced by the sperm are not present (see below).
In the third group of eggs, neither of the polar bodies is a part of the
egg at the time of sperm penetration and hence has fulfilled none of
the conditions attendant on its activation. In order to show any de-
POLOCYTE FORMATION AND CLEAVAGE OF POLAR BODY 261
velopment, therefore, it would have to be activated. In addition, as
has already been mentioned by Conklin (1915) both of the polar bodies
of the first group would be protected against further activation by
sperm because of cortical modifications attendant upon the previous
insemination of the egg. The same would be true of the second polar
body in the second group, In order to explain the lack of development
in these, it would be necessary to demonstrate either a consistent lack
of some significant part of the nuclear or cytoplasmic portion of the
entering spermatozoon or of the egg at the time of polocyte formation.
In the same way, development of the second polocyte of the second
group and both of the polocytes of the third group must be dependent
on subsequent activation. The last statement is dependent on another
condition which would seem in the light of the present observations
to be of primary importance. The polocyte must contain all of the
constituents of the egg essential both to its own activation, and to the
activation of the spermatozoon.
Unfortunately, there is little information as to the actual constitution
of the cytoplasmic portion of the polar body available in literature on
the subject. These structures have been considered as aborted ova by
Mark in his monograph on Liina.v coinpcstris (1881 ). In that form the
polar bodies do not develop. It would now be of great interest to know
the actual constitution of the polocyte.
In isolated cases the polar bodies have been observed to develop
to some extent at least. Lefevre (1907) cites the case of the polar
body of Thalasscina mclllta, which is formed in response to the acid
activation (artificial parthenogenesis) of the egg. Subsequent cleavage
of the extruded cell gives rise to a ' morula,' which later dies. In this
case also there is some confusion in that the actual composition of the
polocytes which develop is not known. In no case did they develop
beyond this early stage, however, though that may conceivably be due
to quantitative deficiencies which do not immediately concern us here.
The polar nuclei such as are formed in some insects and Crustacea
do not come under the realm of our discussion, but. inasmuch as they
sometimes cleave, 'we should mention them. The polar nuclei may fuse
and in some cases they divide to form what appear to be accessory em-
bryonic structures. This is the case in the hymenopter, Litomastix
(Silvestri, 1908).
The most extensive development of the polocyte is to be found
recorded in the paper of Francotte (1898) on the polyclads. in which
he records the formation of the gastrula by fertilized first polar bodies
of ProstheccrcEus vittatits. The argument is slightly different from
that in other cases, though it will concern us in a moment. We shall
262 LEIGH HOADLEY
therefore consider it here. The egg of this form produces a polar
body at the first maturation division, which may be as much as one-
fourth as large as the remaining portion of the egg. Subsequently
this may be fertilized, as is the egg itself, and then both of the units
give rise to one polar body and continue in their development. Fran-
cotte described the development of these forms only as far as the
gastrula. It may be assumed, however, that all of the conditions for
cell division and development of any other than a quantitative nature
are fulfilled. We shall return to a consideration of this case in con-
nection with the fate of the so-called giant polar bodies of other forms.
In connection with the impregnation of the polar bodies after their
extrusion, mention should be made of certain observations of Fol.
This investigator has reported sperm penetration of polocytes in echino-
derms. No mention is made of the reaction of the sperm or of the
egg, so that it is not known as to whether the conditions of activation,
as expressed morphologically, are complete or not. It is conceivable
that in this case the very small size of the polar body would preclude
its further development. It is also possible that the polar body of the
echinoderm egg does not include all of the parts of the egg requisite
to development.
It is in this last statement that we find a possible explanation of the
difference between the behavior of the polar bodies in Loligo and Chcc-
toptents. It can be demonstrated that in the formation of the polar
bodies in Chcctoptcrns, while the majority of the deutoplasm remains
within the egg and the polocyte consists mainly of hyaloplasm, only
the deeper endoplasmic portion of the cytoplasm is extruded, and the
polocyte does not contain all of the zones characteristic of the egg
which develops. It might be emphasized here that hyaloplasm and
ectoplasm are not identical. In complete accord with this lack of
ectoplasm, there is no second division of the primary polar body. In
Loligo, on the other hand, the extruded polar body contains not only
the endoplasmic region, but also some of the outer cortical part of the
cytoplasm. In this case the polocytes develop for a little while. A
striking; difference between the two types of polar body is to be found
in the presence or absence of the cortical ectoplasmic portion, which
may be assumed, therefore, to play an important role in future events.
Certain observations made by Conklin (1915, 1917) on the behavior
of artificial giant polocytes in Crepidula are very important in the con-
sideration of the phenomenon. In this paper, Conklin changes his
previous views on the subject and states that in such cases the lack
of the sperm aster may be the important factor in the non-development
of the large polar body. These were obtained by centrifuging the eggs
POLOCYTE FORMATION AND CLEAVAGE OF POLAR BODY 263
during the formation of the polar body. In those cases in which the
rotation of the egg was such that the spindle was in the line of the
gravitational pull and at the outer end, huge polar bodies were formed
which appeared to contain all of the egg substances and yet did not
develop. Following certain previous conceptions. Conklin differentiates
between those phenomena of fertilization leading to activation of the
cytoplasm and to the development of the egg. The polocyte. having
been activated with the rest of the egg by insemination, forms, but.
lacking the aster which he considers as the part essential to further de-
velopment, fails to divide. In view of the experiments of Lillie (1906)
on Chcetopterus eggs during maturation phases it would appear that
there may be a question as to the composition of the polar body. It"
one is to examine Fig. 24 (p. 185) in Lillie's report, one sees that
when the Chcetopterus egg is centrifuged, certain substances of the egg
are more or less free and that one of these is the endoplasmic material.
As a result, the endoplasmic material takes its position according to
the force of gravity. The ectoplasmic cortical material, on the other
hand, is fixed, not being displaced by the centrifugal force. If, then,
the force of gravity were applied in such a way that the endoplasmic
material were all pushed against the region of the ectoplasmic defect
or pole at which the spindle is attached, the result would be that it
would bulge outside of the ectoplasmic portion at this point and that,
when polar body formation was completed, a great quantity of this
material would be separated from the egg and follow the polar body.
This would not necessarily involve the inclusion of any of the ecto-
plasmic material in its formation. If this should prove to be the case,
the non-development of the polar body of Crcpidula, when present
as the giant polar body, might be due to the same factors which seem
at present to be responsible for the non-development of the pnlocyte
of Chcetopterus. In this connection it is of interest to note that the
first polocyte of Crcpidnla may divide once by mitosis and subsequently
several times by amitosis (Conklin, 1902, page 21, Fig. 41, etc.). This
does not occur in the second polocyte. There may be a discrepancy in
their constitution or in the quantitative relationships between the
amounts of substances of cytoplasmic nature present. In this way the
first polocyte may resemble that of Loligo, while the second may be like
that of Chcetopterus. In any event, the conditions present in Cluctop-
tcnis do not obtain for Loligo and the results differ accordingly. The
conception of the great importance of the cortical portion of the egg
to the phenomena of early development is not new as it has been stressed
in a number of places by several investigators, notably by Chambers
(1921) and Just (1923). These authors show by their experiments
264 LEIGH HOADLEY
that there is a real distinction morphologically and functionally between
cortex and endoplasm.
In conclusion I would like to suggest that in Loligo and Chcetopterus
and possibly in other forms, the development of the polar body is de-
pendent on the presence of cortical ectoplasmic material in that organ
or at least on the ratio between the amount of the ectoplasmic substance
and the remaining cytoplasmic and nuclear material.
LITERATURE CITED
CHAMBERS, ROBERT, 1921. Studies on the Organization of the Starfish Egg.
Jour. Gen. Physiol., 4: 41.
CONKLIX, E. G., 1902. Karyokinesis and Cytokinesis in the Maturation, Fertili-
zation, and Cleavage of Crepidula and other Gastropods. Jour. Phila.
Acad. Nat. Sci, 12: part 1.
CONKLIX, E. G., 1915. Why Polar Bodies do not Develop. Proc. Nat. Acad.
Scl., 1: 491.
CONKLIX, E. G., 1917. Effects of Centrifugal Force on the Structure and De-
velopment of the Eggs of Crepidula. Jour. Ex per. Zodl., 22: 311.
FRANCOTTE, P., 1898. Recherches sur la maturation, la fecondation et la seg-
mentation chez les Polyclades. Arch. d. Zodl., 6: 189.
JUST, E. E., 1923. The Fertilization-Reaction in Echinarachnius parma. VI.
The Necessity of the Egg Cortex for Fertilization. Biol. Bull., 44: 1.
LEFEVRE, G., 1907. Artificial Parthenogenesis in Thalassema Mellita. Jour. Ex-
per. Zodl., 4: 91.
LILLIE, F. R., 1906. Observations and Experiments Concerning the Elementary
Phenomena of Embryonic Development in Chaetopterus. Jour. Exper.
Zodl., 3: 153.
MARK, E. L., 1881. Maturation, Fecundation, and Segmentation of Lima.v cam-
pcstris Binney. Bull. Mus. Compar. Zodl., Cambridge, Mass., 6: 173.
WATASE, S., 1891. Studies on Cephalopods. I. Cleavage of the Ovum. Jour.
.Mor ph., 4: 247.
THE DISTRIBUTION OF PIGMENT AND OTHER MORPHO-
LOGICAL CONCOMITANTS OF THE METABOLIC
GRADIENT IN OLIGOCH^TS
GRACE EVELYN PICKFORD
OSBORN ZOOLOGICAL LABORATORY, YALE UNIVERSITY
INTRODUCTION
The form of the antero-posterior metabolic gradient of the Oligo-
cluets has now been well established by many workers and by almost
as many different methods. Hyman (1916) investigated the gradient
of susceptibility to KCN in many lower Oligochaets and distinguished
two types, a primary gradient found only in the primitive /Eolosomatidse
and young zooids of the Naididae in which the susceptibility decreased
progressively from head to tail, and a widely distributed secondary
type in which the susceptibility again rose at the posterior end. In
Lumbriculus rarians measurements of the oxygen intake of different
regions of the body by the Winkler technique (Hyman and Galigher,
1921) showed a secondary metabolic gradient; the accurate manometric
determinations of Shearer ( 1924) on the " earthworm " confirm the
primary but throw no light on the secondary gradient, since the experi-
ments were only made on head and tail portions. The early work of
Morgan and Dimon (1904) on the potential gradient showed that in
Lituibn'cns tcrrestris and Allolobophora foetida the head and tail were
electronegative to the middle region, while Moore and Kellogg (1914)
found that in an electric field Lumbricus oriented itself in the form of
a U with head and tail towards the cathode. Hyman and Bellamy
(1922) confirmed these results and correlated them with the metabolic
gradient. Hatai ( 1924) showed that in two Japanese species of Phcrc-
t'una (incorrectly named Perich&ta) the amount of heat required to
produce initial heat rigor in the muscles of the body wall was greatest
at the anterior and posterior ends and least in the middle region of the
body. He correlated these results with the percentage water content
of the body wall, which is inversely proportional to the temperature
required to produce initial heat rigor. Watanabe (1928) found that
in P. coiniininissiiiia the potential gradient is on the average of the
secondary type, although dorsally it is perhaps of the primary type.
Recently Perkins (1929) has published a short note in which he
265
266 GRACE EVELYN PICKFORD
claims that in earthworms the gradient of extractable reduced sulphydryl
reaches a maximum in the mid anterior region of the body. Perkins
summarizes his results as follows, " In earthworms I find that the
gradient of growth corresponds with the gradients of total iodine
equivalence, extractable sulphydryl, and total sulphur (gravimetric)
and not with the gradient of total metabolism observed by the oxygen
uptake ; the last, therefore, includes other oxidation systems which it
is legitimate to suppose result in katabolism rather than the anabolism
of growth. It is interesting to find that gradients in the earthworm
have a summit at about that point whence a divided worm grows for-
wards or backwards according to the aspect of the cut surface." x
As regards the dorso-ventral gradient very little work has been
done, although Hatai (1924) states that the temperature necessary to
produce initial heat rigor is greater for dorsal than for ventral and
intermediate for lateral portions of the body wall.
Little attention has been paid to the morphological concomitants
of the metabolic gradient. Hess (1924) showed that the sensitivity of
Linnbrictts terrcstris to light is greatest at the anterior end and least in
the mid region of the body and that except on the first five and last
two segments it is confined to the dorso-lateral regions ; he also noticed
that the distribution of pigment corresponds rather closely to the light
sensitivity. In a later paper Hess (1925) showed that the distribution
of the photo-receptor organs coincides with the distribution of the photo-
sensitive regions, thus putting the gradient of sensitivity to light on a
morphological basis. Nomura (1926) has extended the work of Hess,
showing that in the ventral nerve cord of Allolobophora foctlda Sav.
there is an axially graded distribution of photic response ; negative
orientation, which also characterizes the brain, increasing posteriorlv
and positive orientation anteriorly, while the supposed neurones causing
backward crawling are apparently restricted to the anterior end op-
posing the brain, which controls forward crawling.
DISTRIBUTION OF PIGMENT
Many species of earthworm are pallid and others may be colored
green, blackish, or yellow by as yet uninvestigated pigments, but by
far the most commonly occurring coloration is due to a reddish or
purplish-brown pigment which has been shown in some species (see
Kobayashi, 1928) to be a porphyrin allied to some derived from chloro-
phyl. This reddish pigment is characteristically distributed on the
dorsal side and is most intense at the anterior end. A typical case can
be found in the well-known species Luinbricus terrcstris Linn. Indi-
: References to text figure omitted.
METABOLIC GRADIENT IN OLIGOCH^TS 267
vicluals of this species will be found to vary somewhat in the intensity
and exact extent of pigmentation, but the following description taken
from a specimen recently caught near this laboratory will serve as an
example: "Intensely pigmented dorsally at the anterior end, the pig-
mentation extending laterally to about cd (the line of the lateral setse),
the first three segments also slightly pigmented ventrally; posterior to
the clitellum the lateral extent of the dorsal pigmentation becomes re-
duced until only a mid-dorsal line is left which persists throughout the
posterior half of the body; at the extreme posterior end there is again
an increase in intensity and extent of pigmentation (except on the
terminal segment which is small and pale) which extends laterally to
below the setal line cd on the seventh to the second last segments and
even faintly on the ventral side of the second, third, and fourth seg-
ments from the end." In this case the distribution of pigment follows
the secondary type of gradient, and it may be said in general that when-
ever a species of earthworm exhibits this red-brown pigmentation (pre-
sumed, but of course not proved in most genera to be due to a porphyrin
allied to that of Lnmbricus and Allolobophora (Eiscnia) foctida Sav..
it will be distributed according to the primary if not the secondary
type of gradient. Hatai (1924) noticed that in " Perichata " niegas-
colidioides Goto et Hatai the dorsal side was more pigmented than the
ventral, though curiously enough he did not correlate this with the
dorso-ventral gradient. My own investigations have so far been con-
fined to a systematic examination of South African species of the genera
Chilota and Acanthodrilus. In these genera every gradation from
total pallor to intense pigmentation can be found ; some of the most
interesting cases are those species, or varieties of otherwise pallid or
pigmented species, in which pigmentation is only found on the first
or last few segments. For example, the Cape Flats species Acantho-
drilns antiidinis Bedd. is pigmented dorsally on the first and last four
or five segments but more intensely on the latter, while in many unde-
scribed species of Cliilota only a few of the anterior segments are pig-
mented. When the dorsal pigmentation is intense and occurs along
the whole length of the body it is usual to find that the first five to ten
segments are deeply pigmented ventrally, while in many cases pigment
is deposited on the thickened septae and generally on the inner side
of the body wall at the anterior end. A more complete discussion will
be given in my forthcoming paper on the South African Acanthodrilinae.
The distribution of pigment in Oligochsets may be compared with
that described by Faris (1924) for Amblystoma embryos. In this case
the pigment is apparently a melanin and is deposited in regions of tissue
differentiation as opposed to regions of proliferation. If the intensity
18
268 GRACE EVELYN PICKFORD
of pigmentation in Oligochcets is really a function of the metabolic rate,
it seems possible that highly pigmented species would have a higher
oxygen intake than pallid ones. It is hoped to investigate this point
shortly on a large number of species. If this view is correct, and it
is supported by the fact that pallid species are more sluggish in their
movements and less irritable to handling than pigmented ones (com-
pare Allolobophora (Eisenia) rosea Sav. with species of Luinbricns),
it would seem unlikely that the porphyrin is merely derived from the
food of the worm, as has been suggested, and more probable that it is
a breakdown product of the worm's own haemoglobin.
MULTIPLICATION AND REDUCTION OF SET.E
As regards the more specifically morphological concomitants of the
axial gradient, certain stages in the reduction and multiplication of setal
numbers are significant. In the primitive lumbricine condition there
are two pairs of setae per segment except on the first, which never has
setae; a reduction in numbers sometimes takes place as in species of
the Microchcutiis bciihaini group where setse are absent on the first
six or seven segments of the adult (frequently only the lateral pair
are absent on segment 6). This trend to reduction finds an extreme
case in Tritoyciiia crassa Mchlsn., in which only the ventral setae of
the clitellar region persist.
In the Enchytrseidae parallel cases can be found; in the genus
Distichopus only ventral setae are present. In the genus Michaelsena
transitional species occur from M. inangcri Mchlsn., in which dorsal
and ventral setae are present throughout and M. principissce Mchlsn.,
in which the ventral setae commence on segment 3 and the dorsal on
segment 14, to M. iionnani Mchlsn., which has ventral setae from
segment 3 onwards but dorsal setae only on segments 4-6, and M.
subtilis Ude., in which dorsal setae are absent and ventral setae occur
only on segments 4-6. In the genus Achccia setae are totally absent.
These cases may be compared with the phenomenon of cephalization
in the Naididse (Stephenson, 1912 and 1923), in which certain anterior
segments are devoid of dorsal (i.e. lateral) setse. Hyman (1916)
found a very peculiar gradient in the Naid Chcetogastcr diaphamis,
in which the susceptibility was least at the head end. In this genus
dorsal setae are totally absent and ventral setae though present on seg-
ment 2 are absent on segments 3, 4, and 5.
The tendency to setal multiplication is a very widely distributed
phenomenon, and the perichaetine condition has apparently arisen in-
dependently many times in various families of the terrestrial or Neo-
Oligochaets (see Stephenson 1921, 1923 for a discussion of this and
METABOLIC GRADIENT IN OLIGOCH^ETS 269
other trends in the evolution of the Indian Oligochaeta). The multi-
plication of setae varies from a condition in which six or eight pairs
occur instead of four per segment to the purely perichaetine condition
in which each segment has a complete ring, but the most interesting
cases are those in which a transitional condition exists. In Mcgascolc.\~
zvilleyi Mchlsn. there are eight setae per segment at the anterior end and
twelve in the middle and posterior regions; in M. vilpattiensis Mchlsn.
there are eight setae in four pairs on segments 2 and 3, eight or nine
on segment 4, circa 11 on segment 13, circa 24 on segment 26, and circa
26 at the posterior end. In general in transitional species the smaller
number and 1 or more primitive paired condition persist at the an-
terior end. Sufficient data are unfortunately not available as to the
extreme posterior end, so that it is not possible to state whether the
smaller number also persists there in these intermediate forms. Hatai
(1924) has investigated the setal numbers in the purely perichaetine
species " Pe ricliccta " (Plicrctiina) inegascolidioidcs Goto et Hatai.
He finds that the number of segments is extremely constant and bears
no relation to the size of the worm and that the total number of setae
per worm does not vary very greatly. The number of setae per segment
increases from segment 2-25, remains about constant up to segment
100 and then decreases again, thus exhibiting a curve comparable with
the secondary type of oligochaet gradient. From a survey of the avail-
able data it would thus seem as if setal multiplication were correlated
with a lower and setal reduction with a higher metabolic rate. The
case of Acantlwbdclla pclcdina Grube, an aberrant parasitic form re-
garded until recently as a leech, must not be overlooked, although the
evidence (c.f. Clicctogastcr) cannot be interpreted until the form of
the metabolic gradient has been investigated. In this species setae are
present only ventrally on the first five segments.
MULTIPLICATION AND REDUCTION OF NEPHRTDIA
The trend to setal multiplication is paralleled and usually accom-
panied by the multiplication of the nephridia, primitively one pair per
segment. Unfortunately the whole subject of nephridial multiplication
stands in need of a thorough revision since the publication of Bahl's
admirable series of studies on Phcrctiuia (1919 and 1922), Lanipito
(1924) and Woodwardia (1926). The brief descriptions of system-
atists who classified their species as '' micronephridial," " megane-
phridial," and " mixed mega-and-micronephridial " are now shown
to be totally inadequate. Nevertheless, what little can be judged from
the existing knowledge yields points of considerable interest. In the
first place, loss or reduction of nephridia when it occurs seems to take
270 GRACE EVELYN PICKFORD
place at the anterior end, e.g. in Pontodrttus, Sparganophilus and
Diporochceta pcllitcida Bourne (re last species see Stephenson, 1925).
Bahl considers that the first step in nephridial multiplication was the
separation of the nephrostome, which then either disappeared or formed
with accompanying nephridial cells a separate septal meganephridium
opening into the gut, while the main mass of the nephridium hroke up
to form funnel-less integumentary nephridia. In Pheret'una the septal
nephridia have also undergone multiplication to the micronephridial
condition. If this view be provisionally accepted, the two trends, sep-
aration of the nephrostome and multiplication, may be considered in-
dependently. As regards the former, numerous cases can be found
in the literature in which " meganephridia " occur only in the middle
and posterior regions of the body. In "Lampito" (Mcgascolex}
trilobata Steph. and " L." niauritii Kinb., Bahl found that the septal
meganephridia commenced in segment 19, while in Woodwardia bahli
Steph. they commence at 24/25. Benham (1905) describes two species
of Spenceriella, — " Diporochata " gigantca and " D." shakes pearl, which
are " micronephric " but retain large paired nephrostomes in each seg-
ment. Unfortunately he does not say how far forward these occurred.
In Comarodrilus grai'd\i Steph. " micronephridia " occur in the anterior
part of the body as far back as segment 12; behind this "megane-
phridia " only. In the development of Octochcrtus multiponts, Beddard
found (1892) that the nephrostomes degenerate after their separation
from the nephridial mass, but that they may persist in the posterior
segments. These cases appear to be merely examples of a very gen-
eral phenomenon, viz., the tendency for the nephrostomes to disappear
anteriorly. An interesting case is that of Hou>ascolc.r corethntnis
Mchlsn., a species which is transitional both for perichaetine and micro,
nephridial conditions. The setae are lumbricine in the anterior and
middle regions and perichaetine posteriorly, while " meganephridia "
displace the " micronephridia " posteriorly.
The case of nephridial multiplication seusu strict o requires a sta-
tistical investigation, but observations such as those of Bahl on
" Lampito " and Pheret'una spp. and of Stephenson on Hoplochatella
kiuneari Steph. indicate that a great multiplication in numbers of micro-
nephridia in the clitellar region may be a general phenomenon.
While there is thus considerable evidence that nephridial and neph-
rostomal reduction follows the primary metabolic gradient, occurring
first at the anterior end, the case of nephridial multiplication is not
at all clear cut and the issue is frequently confused by the occurrence
of pharyngeal nephridia (tufts of funnel-less nephridia opening into
the pharynx) in the most anterior segments. The clitellar region, which
METABOLIC GRADIENT IN OLIGOCH^TS 271
is sometimes the region of greatest multiplication (ride supra}, is not
known to be the region of lowest metabolism, since the physiological
gradient has not been investigated for the species concerned, but evi-
dence from other species suggests that the clitellar region is too far
forward to coincide with the region of lowest metabolism. If Perkins'
(1929) speculations as to the anabolic gradient are well founded, it is
possible that certain morphological features such as nephriclial multi-
plication in the clitellar region might be interpreted more readily by a
correlation with this rather than with the total metabolic gradient. Ex-
amples have been cited above in which " micronephridia " are replaced
by or co-exist with " meganephridia " in the posterior part of the body.
Sometimes, c.</., in Notoscole.r pahiiensis Steph., these " meganephridia "
are definitely stated to be enlarged " micronephridia " without funnels
(Stephenson, 1924). Cases of nephridial multiplication without sep-
aration of the nephrostome are extremely rare. Bahl (1926) has de-
scribed the case of " Lainpito " diibins Steph., and apparently a similar
phenomenon occurs in the genus Tritogenia, which has two pairs of
nephridia per segment. In " Lauipito " dnbiits there are five pairs of
septal exonephridia per segment except anteriorly, where there may
be only three pairs. On the whole there is a suggestion that nephridial
multiplication is less pronounced in the regions of highest metabolism.
f
HOMCEOSIS
Finally, I should like to draw the attention of zoologists who have
not made a study of oligochaet systematics to the very general occurrence
of homreosis, not merely as occasional variations (Bateson, 1894) but
as normal subspecific, specific, generic and family characters, the seg-
mental shifting forwards and backwards of various organs, e.g. the
clitellum, genital openings, and accessory glands, gizzard, etc. being of
prime taxonomic importance. An excellent example may be taken from
the genus Acanthodriliis, which normally possesses paired male pores
on segment 18 and two pairs of prostatic pores on segments 17 and 19;
there may, however, be a backward shifting (Michaelsen, 1913) as in
Ac. coneensis Mchlsn. and Ac. uatalicius Mchlsn. with the male pores
on segment 19 and prostatic pores on 18 and 20 or Ac. roit.vi Mchlsn.
with the male pores on segment 20 and the prostatic pores on 19 and
21. A similar phenomenon occurs in undescribed South African species
of Chilota.
My best thanks are due to Dr. J. \Y. Buchanan of this laboratory
for his kindly advice and criticism.
272 GRACE EVELYN PICKFORD
SUMMARY
In Oligochffita the distribution of the photoreceptor organs and of
porphyrin pigmentation as well as the tendencies to reduction and
multiplication in numbers of setae and of nephridia per segment appear
as morphological concomitants of the metabolic gradient.
BIBLIOGRAPHY
BAHL, K. N., 1919. On a New Type of Nephridia Found in Indian Earthworms
of the Genus Pheretima. Quart. Jour. Mic. Sci., 64: 67.
BAHL, K. N., 1922. On the Development of the ' Enteronephric ' Type of
Nephridial System Found in Indian Earthworms of the Genus Pheretima.
Quart. Jour. Mic. Sci., 66: 49.
BAHL, K. N., 1924. On the Occurrence of the "Enteronephric" Type of
Nephridial System in Earthworms of the Genus Lampito. Quart. Jour.
Mic. Sci., 68: 67.
BAHL, K. N., 1926. The Enteronephric System in Woodwardia, with Remarks
on the Nephridia of Lampito dubius. Quart. Jour. Mic. Sci., 70: 113.
BATESON, W., 1894. Materials for the Study of Variation. MacMillan and
Company. London.
BEDDARD, F. E., 1892. Researches into the Embryology of the Oligochseta. I.
Quart. Jour. Mic. Sci., 33: 497.
BENHAM, W. B., 1905. An Account of Some Earthworms from Little Barrier
Island. Trans. N.Z. hist., 38: 248.
FARIS, H. S., 1924. A Study of Pigment in Embryos of Amblystoma. Anat.
Rec., 27: 63.
HATAI, S., 1924. Contributions to the Physiology of Earthworms. II. The
Effect of Temperature on the Shortening of the Body and the Content
of Water in the Body of Earthworms. Sci. Rep. Tohoku Imp. Univ.,
4th Series, 1: 3.
HATAI, S., 1924. Reply to the Remarks of Prof. Wilhelm Michaelsen Concerning
the Perichseta megascolidioides Goto and Hatai, and Further Observations
Made on this Species on the Relation of Body Length to the Number
of Segments and of Setae. Sci. Rep. Tohoku Imp. Univ., 4th Series,
1: 23.
HESS, W. N., 1924. Reactions to Light in the Earthworm, Lumbricus terrestris L.
Jour. Morph., 39: 515.
HESS, W. N., 1925. Photoreceptors of Lumbricus terrestris, with Special Refer-
ence to their Distribution, Structure and Function. Jour. Morph., 41: 63.
HYMAN, L. H., 1916. An Analysis of the Process of Regeneration in Certain
Microdrilous Oligochsetes. lour. Expcr. Zool., 20: 99.
HYMAN, L. H., AND BELLAMY, A. W., 1922. Studies on the Correlation between
Metabolic Gradients, Electrical Gradients, and Galvanotaxis. I. Biol.
Bull, 43: 313.
HYMAN, L. H., AND GALIGHER, A. E., 1921. Direct Demonstration of the Exist-
ence of a Metabolic Gradient in Annelids. Jour. E.rpcr. Zool.. 34: 1.
KOBAYASHI, S., 1928. The Spectroscopic Observations on Porphyrin Found in
the Integument of Earthworm, Allolobophora fcetida (Sav.). Sci. Rep.
Tohoku Imp. Unit'., 3, No. 3, Fasc. 2.
MICHAELSEN, W., 1913. Die Oligochaten von Neu-Caledonien und den benach-
barten Inselgruppen. F. Sarasin and J. Roux, Nova Caledonia, Zoologie,
Vol. 1, L. 3, No. 5.
MOORE, A. R., AND KELLOGG, F. M., 1916. Note on the Galvanotropic Response
of the Earthworm. Biol. Bull., 30: 131.
METABOLIC GRADIENT IN OLIGOCH/ETS 273
MORGAN, T. H., AND DIMON, A. C, 1904. An Examination of the Problems of
Physiological " Polarity " and Electrical Polarity in the Earthworm.
Jour. Expcr. Zobl., 1: 331.
NOM.URA, E., 1925. Effect of Light on the Movements of the Earthworm, Al-
lolobophora foctida (Sav.). Sci. Rep. Tohoku 7w/>. Univ., 1: 293.
PERKINS, M., 1929. Growth-gradients and the Axial Relations of the Animal
Body. Nature, 124: 299.
SHEARER, C, 1924. On the Oxygen Consumption Rate of Parts of the Chick
Embryo and Fragments of the Earthworm. Proc. Roy. Soc. London,
96: 146.
STEPHENSON, J., 1912. On a New Species of Branchiodrilus and Certain Other
Aquatic Oligochaeta, with Remarks on Cephalization in the Naididse.
Rcc. Ind. Mus., 7: 219.
STEPHENSON, J., 1921. Contributions to the Morphology, Classification, and
Zoogeography of Indian Oligochseta II. On polyphyly in the Oligochreta.
Proc. Zo'ol. Soc. London, Part 1, p. 103.
STEPHENSON, J., 1923. The Fauna of British India ; Oligoch.Tta. Taylor and
Francis, London.
STEFHENSON. J., 1924. On some Indian Oligochsta, with a Description of Two
New Genera of Ocnerodrilins. Rcc. Ind. Mus., 26: 317.
STEPHENSON, J., 1925. On some Oligochjeta Mainly from Assam, South India,
and the Andaman Islands. Rcc. Ind. Mus., 27: 43.
WATANABE, Y., 1926. On the Electrical Polarity in the Earthworm, Pcrichccta
communissima Goto et Hatai. Sci. Rep. Tohoku /;;;/>. Unir., 3. No. 2.
p. 139.
DISTRIBUTION OF SETsE IN THE EARTHWORM, PHERE-
TINIA BENGUETENSIS BEDDARD 1
P. B. SIVICKIS
(From the Zoological Laboratories, University of the Philippines, Manila and
Lietuvos Universitctas, Kaunas, Lithuania.)
The oligochaet genus, Phcretima, which occurs abundantly in the
Philippines and other oriental countries, is characterized by the pres-
ence of a large number of seta? on each segment except the most an-
terior. Taxonomists have regarded the distribution and number of
setae as specific characteristics, but apparently have observed that the
number varies on different segments, since they usually specify the
segment for which the number of setae is given (Michaelsen, 1900;
Stephenson, 1923). No data have been found, however, concerning
variation in number of setae on a particular segment. Counts of setae
made by the writer show a considerable range of variation, both in the
number of setae on corresponding segments of different individuals and
on different segments of the same individual. Moreover, the numbers
of setse on different segments of the same individual vary along the
axis in a way which suggests a relation to the longitudinal physiological
gradients. Data are given below concerning these variations.
MATERIAL AND METHODS
Pherftima benguetensis Beddard, the species on which the counts
were made, is common in the Philippines. During the greater part of
the rainy season the worms are found in large numbers near or on
the surface of the ground. By the end of the rainy reason they become
heavily parasitized by gregarines and later disappear almost completely,
but whether the disappearance is due to death or to movement away
from the surface of the ground is not known.
Counts of setae were made on one hundred animals. Fifty of these
were collected on the campus of the University of the Philippines and
fifty from the town of Pasig near Manila. The latter were somewhat
larger than the former, but their general specific characteristics indi-
cated that both lots belonged to the same species.
1 The data presented in this paper were obtained while the writer was a
member of the Department of Zoology of the University of the Philippines.
Acknowledgments are due to Miss Paz Lorenzo, Mr. D. Quajunco and Mr. G.
T. Lantin for assistance. My thanks are due to Prof. C. M. Child for critical
review of this paper.
274
SET.E IN EARTHWORM 275
The counts were made on animals preserved in formalin. For
counting they were opened along the mid-dorsal line, the internal organs
were removed, and the body wall was cut into pieces of a size con-
venient for microscopic examination between two slides. Counts of
such pieces were either made at once or the two slides with the piece
between them were tied together and placed in a hot one per cent solu-
tion of KOH for five hours or more, until they became transparent,
but were removed before maceration had proceeded so far that the
setae were freed from the tissue. A section along the dorsal mid-
line is more satisfactory for such preparations than a section elsewhere
because the dorsal wall is thicker than in other regions, and since the
KOH attacks the edges of the preparation first, the thicker dorsal wall
is not destroyed before the other parts have become sufficiently trans-
parent. After maceration the pieces were mounted in glycerol and all
the setae on the segments selected were counted under a low power
of the compound microscope with the aid of a mechanical stage. Par-
ticular care was taken to make certain that all setae on each segment
selected were included in the counts. In the region of the clitellum
counts are less readily made than elsewhere because the thickening of
the body wall in this region makes it difficult to see the setae.
Since there are no setae on the first segment, counts were begun
with the second, and further counts were made on the fifth, tenth, fif-
teenth, etc., that is, on every fifth segment up to the sixtieth. In order
to minimize possible errors which, however, proved to be less than was
feared, in counts on the fifteenth segment, a segment of the clitellum.
counts were made on the segment next anterior (13) and the segment
next posterior (17) to the clitellum. Counts from the posterior di-
rection began with the last posterior segment and were made on every
fifth segment until the sixty-fifth segment from the posterior end was
reached. This procedure leaves a short middle region uncounted in
some animals with a large number of segments, but since the mean
number of segments can readily be extrapolated in this region, the
results are not seriously affected.
The method of making counts in two directions from each end of
the body is regarded as preferable to that of making counts from an-
terior to posterior end, because by the latter method the most pos-
terior segment counted is rarely the last segment of the body and rep-
resents different levels in different cases.
DISTRIBUTION AND SIZE OF SET.£
Each segment except the most anterior possesses a large number
of setae more or less uniformlv distributed about the circumference,
276 P. B. SIVICKIS
but with occasional gaps and occasional duplications. The setae are
less than a millimeter in length, and taper slightly from the base to
a blunt tip.
1 io H « 5i Jo J! Jo v! ;o 55 to £5 to 5! 50 ~ ^o 35 ' jo 25 Jo is 10 5
FIG. 1. Graph from the data of Table I showing the variation in numbers
of setse along the main axis of the body in Phcretima bcnguctcnsis. Ordinates
represent the mean numbers of setse (M') on particular segments; abscissae repre-
sent segment numbers. The anterior end is at the left.
Setse from three regions of the body have been isolated by boiling
in KOH pieces of the body wall from the selected regions and have
been measured with an ocular micrometer. The data of such measure-
ments are as follows :
On segments 2-5, length 0.6 mm. ; diameter 0.08 mm.
36-40, 0.3 mm.; 0.05 mm.
Last ten segments, 0.5 mm. ; 0.03 mm.
These measurements indicate the variation in size of the setae. The
longest setae are found on the anterior and posterior segments, the
shortest in the middle regions. From the anterior end the seta- very
gradually decrease in size to the clitellum. For some distance posterior
to the clitellum the setae are only about half the length and little
more than half the diameter of those on the anterior segments. Pos-
terior to the middle of the body they begin to increase in size and for
the most posterior segments they are almost as long, though less in
diameter than those at the anterior end. In general the length of the
setae varies inverselv as their number.
IN EARTHWORM
277
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oo'"?
SO IO
10 O>
&*>.
sOio
^00
a^
"O i/l
1^3 O1
06^
O so
00 ^
0^.
SO 10
0°.
278 p. B. SIVICKIS
COUNTS OF SETVE
The numerical data for the first ten and the last ten animals of the
hundred counted are recorded in full in Table I as a sample indicating
how the counts run. Animals 1-10 of the table are from those col-
lected on the University campus, animals 91-100 from those collected
at Pasig. The first vertical column gives the number of the individual
in the series, the following column the number of setae counted on cor-
responding segments. The first vertical section of the table gives
counts from the anterior end to the sixtieth segment, the second sec-
tion, counts from the posterior end to the sixty-fifth segment from
that end. The last column gives the number of segments in each
animal. In the last two horizontal lines of the table are given the
mean values (-M) and the standard deviation (o-) as calculated by
the standard formulae for corresponding segments of all animals
counted, that is, each value of M and a given is the value for one
hundred corresponding segments. The variations of M at the differ-
ent body levels are plotted in the graph (Fig. 1).
Examination of the data recorded in the table shows that in spite
of a considerable variation in the number of setae per segment of any
individual, the general course of the variations in different regions is
well expressed by the means. The number of setae is relatively small
on the anterior segments, but increases rapidly to the twentieth seg-
ment, beyond this more slowly to the thirtieth segment, where the
maximum number of setae per segment is attained. Posterior to this
segment the number of setae decreases gradually to the posterior end
of the body.
DISCUSSION
The very definite course of variation in number of setae along the
body of Phcrctiina suggests that it must be correlated with regional
physiological differences of some sort, and since it is gradual and in
opposite directions in anterior and posterior regions, the possibility
that it may be in some way correlated with the longitudinal physiological
gradient in the body is also suggested. Nothing is known concerning the
gradients in Pherctima, but in most other oligochaets examined a double
gradient has been found. Hyman (1916) has found in most of the
microdrilous oligochaets a decrease in susceptibility from the anterior end
posteriorly to a certain level and an increase from this level to the pos-
terior end. Hyman and Galigher (1921) found a similar double gradi-
ent in oxygen consumption in Lumbnciihis and Nereis. Perkins ( 1929),
investigating oxygen consumption, total iodine equivalence, amount of
glutathione and total sulphur content in different regions of the body
IN EARTHWORM 279
of an earthworm (unnamed), also finds differentials which vary in two
directions. If such a double gradient exists in Phcrctinia, as is prob-
able, the smaller numbers of setae occur at the higher, and the larger
numbers at the lower levels of the respiratory gradient. We know
nothing at present concerning the nature of the relations between
gradients and setae, but it may be provisionally suggested that a develop-
ing seta sac in the regions of more intense metabolism inhibits the de-
velopment of other seta sacs over a greater distance than in regions
of lower metabolism, consequently at the higher gradient levels fewer
setae develop on the circumference of the segment than at lower levels.
Such an inhibiting action of a developing part or organ on other similar
organs within a certain distance from it is very generally recognized
by both botanists and zoologists, and in various cases the range of
this effect appears to be very definitely associated with the intensity
of metabolism in the part concerned. Whether this suggestion of a
possible relation between the numbers of setae on different regions of
the body is correct must remain for further investigation to determine.
In addition to the regional variations in numbers of setae, individual
variations in number on corresponding segments appear in the table.
The standard deviation a is lowest in the anterior region of the body.
This is particularly evident anterior to the tenth segment. The highest
value of a appears in the posterior region, particularly in the ten pos-
terior segments. Between these extremes a fluctuates between 4.62
and 6.00. The relatively low <r of the anterior region suggests physio-
logical stability in this region, and this is in accord with the fact that
it develops first and represents a dominant or relatively dominant
region. It is much less affected by parts posterior to it than they are
by it.
With respect to the practise of taxonomists of considering the num-
ber of setae on a particular segment as a specific character, it may be
noted that the data presented in the table show a very considerable
individual variation in these numbers and a high value of <r. Appar-
ently counts on many individuals would be necessary to make these
numbers reliable for species determination. Smaller numbers may,
however, be considered as possessing a certain diagnostic value when
considered together with other characters.
Some observations on Phcrctinia posthunia (P. incerta Beddard)
indicate that with certain limitations similar relations exist in that
species.
280 P. B. SIVICKIS
SUMMARY
The numbers of setae on particular segments of Pheretima bcnguc-
tcnsis vary in definite directions in different regions of the body. The
number is lowest on the most anterior segments, increases posteriorly
to a maximum at a level just posterior to the reproductive organs, and
then decreases gradually to the posterior end. A relation between this
course of variation and the physiological gradients is suggested. The
standard deviations for corresponding segments indicate that the num-
ber of setae on a particular segment should be used for determination
of species only in connection with other characters.
LITERATURE CITED
HYMAN, L. H., 1916. An Analysis of the Process of Regeneration in Certain
Microdrilous Oligochsetes. Jour. E.r/>. Zool.. 20: 99.
HYMAN, L. H., AND GALIGHER, A. E., 1921. Direct Demonstration of the Ex-
istence of a Metabolic Gradient in Annelids. Jour. Exp. Zool., 34: 1.
MICHAELSEN, W., 1900. OHgochaeta. Das Tierreich, No. 10.
PERKINS, M., 1929. Growth-Gradients and the Axial Relations of the Animal
Body. Nature. 124: 299.
STEPHENSON, J. F., 1923. Oligochaeta. Fauna of British India. London.
STUDIES ON THE PHYSIOLOGY OF THE EUGLENOID
FLAGELLATES
II. THE AUTOCATALYTIC EQUATION AND THE QUESTION OF AN
AUTOCATALYST IN GROWTH OF Eliglcna
THEODORE L. JAHN
BIOLOGICAL LABORATORY, UNIVERSITY COLLEGE, NEW YORK UNIVERSITY
The theory of a catalyst of growth, as proposed by Robertson, has
been the stimulus for a number of investigations to determine the pres-
ence or absence of an autocatalyst in protozoan cultures. The earlier
investigations have been reviewed previously (Jahn, 1929). and it
was shown experimentally at this time that the growth rate of Eitglcna
in mass cultures of high concentrations of organisms was not higher
than in cultures of relatively low concentrations, but that in most cases
the reverse was true.
It is the purpose of the present paper to reanalyze the experimental
data previously obtained (Jahn, 1929) from the point of view of rela-
tive rate of division at various times during the period of observation.
It will be shown, first, that the division rate, as calculated from the
autocatalytic formula used by Robertson in his work on ciliates, is a
progressively decreasing quantity, and hence that this autocatalytic
equation can not be interpreted to involve an autocatalyst which effects
an increase in division rate ; and second, that, on the basis of experi-
mental evidence, the autocatalytic equation may fit the growth curve
of Eliglcna cultures. On the basis of the experimental evidence, it is
believed that Robertson's theory of an autocatalyst of growth is un-
necessary to an interpretation of the experimental data obtained in the
case of Eliglcna.
The writer is deeply indebted to Professor R. P. Hall for sug-
gestions offered during the preparation of this paper.
THE DIVISION RATE AS DERIVED FROM THE AUTOCATALYTIC EQUATION
The equation for an autocatalytic chemical reaction has been ap-
plied by Robertson (1923) to the rate of growth of ciliates in isolation
cultures. The differential form of the equation is
doc
-j- = Kx(A • x), (Equation 1)
281
THEODORE L. JAHN
where .r is the number of organisms, A the maximum number attainable
in a given amount of medium in question, and t is time. When in-
tegrated this becomes
log -j-2— = AK(t - /,), (Equation 2)
A X
where t\ is the time when x = A/2.
The differential equation states that the rate of increase in number
of organisms at any time is proportional to the number present at that
time and to the difference between that number and the maximum
number attainable. Or one may let A equal the original (and also
total) food supply. If this is measured in units, such that one organism
utilizes on the average exactly one unit of food between divisions, then
at any time the amount of food consumed will be equal to the number
of divisions that have taken place. After the first few divisions the
number of divisions which have occurred is approximately equal to
the number of organisms present. Therefore A-x may be regarded as
the available food supply, and in this sense the equation means that
the rate of increase in number is proportional to the number present
and to the amount of free food material. In either case dx/dt is the
rate of increase of the total number of organisms as related to time.
This, however, is interpreted incorrectly by Robertson as the rate of
division of the organisms. The actual rate of division, that is, the
average frequency of division (or fission) per unit time, is not d.\'/dl
but — — , or the rate of increase of the total number of organisms
x
divided by the number (x) present at any given time (t). This we
may represent by D, and then we may restate the division rate as
D = dx/dt = KX(A - X] = _ x} (Equation 3)
x x
The division rate therefore varies directly as A-x. Since A is constant,
and .r is continually increasing, and A-x therefore decreasing, it can
readily be seen that the division rate as derived from the autocatalytic
equation is a decreasing linear function of x. If the division rate is
plotted against time, the result will be a sigmoid curve with a negative
slope, practically the same as the original integral curve except that the
abscissa will be shifted and the ordinate sign reversed (Fig. 1). Since
the division rate is continually decreasing, the intervals between divisions
will become progressively longer as the culture is continued.
More recently Robertson (1928) has proposed a new equation for
STUDIES ON EUGLENOID FLAGELLATES
283
the growth of Metazoa. The differential form of this equation is
dx _ kp , .
dt 1 +p
V)(A •-*),
(Equation 4)
where p is the constant proportion between nuclear and cytoplasmic
increment and b/p is the excess of nuclear material which is present
at the initiation of development, that is at the moment of fertilization,
Graph A
Graph C
GrapK B
D
FIG. 1. Type curves computed from the autocatalytic equation.
A. Differential curve showing dx/dt, the rate of increase in numbers, plotted
against time (t).
B. Integral curve showing x, the number of organisms, plotted against t; .v
approaches the value x = a as an asymptote.
C. Division rate (D) plotted against time. D approaches zero as an asymptote.
D. Division rate plotted against numbers of organisms (.r). D is a decreasing
linear function of x, becoming zero when x is equal to a. The scale of t is the
same in graphs A, B, and C.
b being the same quantity translated into its cytoplasmic equivalent
through multiplication by the proportionality factor p. Whatever
meaning p could assume in a protozoan culture is difficult to state, but
the division rate as calculated from this new equation is also a de-
creasing quantity as in the previous set of equations.
D =
dxfdt kpA b
kp
x
(1 + p)* 1 + p
(A • - b - - .T) . (Equation 5)
The division rate as calculated from the new equation is equal to the
sum of two quantities, one of which varies as the reciprocal of .v and
19
284
THEODORE L. JAHN
the other as a decreasing linear function of x. The sum of these two
quantities is, of course, a decreasing function. Therefore, the division
rate, whether calculated from the new or from the old equation, is a
decreasing function.
The above modification (equation 3) of the autocatalytic equation
has been expressed by Brody (1927), who states, "It signifies that
the relative rate of growth is directly proportional to the growth im-
pulse," (a-x}. Since Brody was considering the application of the
formula to Metazoa, he did not express the idea that the modification
might be used to represent division rate of cells, or that under such
conditions it would indicate a decrease in division rate.
Snell (1929) points out that since the volume of a growing organism
changes, equations derived from the law of mass action can not be ap-
plied to growth without considerable modification. The value of —
Jv
calculated from the modification he proposes is also a decreasing func-
tion as in the preceding equations.
A
SenesV
.6
.2
0
T
>eries
days
FIG. 2. Graphs showing the division rate (D) plotted against time for the
three cultures of Series V and a composite curve for the three cultures of Series
VI. Values of D were computed from the equation
dx/dt
The rate of increase of the total, dx/dt was determined by the graphical differ-
entiation of the experimental growth curves. These values were then divided by
the corresponding values of x to give the values of D for the times (0 under
consideration. It is to be noted that this is a descending sigmoid curve such
as is to be expected if the autocatalytic equation is applicable to the case. (See
also graph C, Fig. 1.)
STUDIES ON EUGLENOID FLAGELLATES
285
THE DIVISION RATE OF Euglena
The growth curves of Euglena from four series (I, III, V, and VI)
previously described by Jahn (1929) have been differentiated graph-
ically to give values of d.r/dt for various values of t. If the values
of dtf/dt are divided by corresponding values of x, one may arrive at
values of - -, or D, for the values of t considered. If D is plotted
X
against t, the result is a descending sigmoid curve. The division rate
curves for Series V and VI are shown in Fig. 2; the curves for the
other series are similar in form.
In Table I are shown the values of the division rate as computed
from analysable data.
TABLE I
. dx/dt
X
Culture number
Day
i
2
3
4
5
I, 1.
63
36
33
31
Ill, 1
80
59
44
V, 1.
1 00
80
50
40
V, 2
80
73
50
43
34
V, 3
.72
.69
55
40
37
VI, 1, 2, and 3 (averaged). .
.83
79
66
55
50
DISCUSSION
The results of the above analyses demonstrate a decreasing sigmoid
division-rate curve for cultures of Euglena. This indicates that the
growth rate of Euglena closely simulates the reaction rate expressed
by the autocatalytic curve, and that the periods between cell divisions
in a single line of cells become progressively longer as the culture is
continued.
The writer's observations on Euglena thus differ from those of
Robertson (summary, 1924), who maintains that his experiments also
show the growth of Infusoria (Enchdys) to be autocatalytic, since
in isolation cultures the division rate is low at first but becomes pro-
gressively higher with each successive division. The autocatalytic
formula, as stated, can be adopted only on the assumption that the
food supply is limited from the beginning and is therefore continuously
being decreased by the growth of the organisms. In Robertson's ex-
286 THEODORE L. JAHN
periments the available food supply was not decreasing during the period
of observation but was increasing due to bacterial growth, and Rob-
ertson's cultures also show an increase in division rate and not a de-
crease as required for the application of the autocatalytic equation.
Hence, it is obvious that the autocatalytic growth curve cannot be ap-
plied to such experiments with ciliates.
The experiments of the writer with Euglcna were conducted under
more readily controlled conditions than were previous experiments
with ciliates. Since bacteria are not a source of food for Euglcna,
it is safe to assume that the few bacteria present did not accelerate
appreciably the division rate of the organisms. Therefore, the food of
the flagellates was limited to the inorganic salts initially present in the
medium and the carbon dioxide dissolved in the water. Since the
primary physical factors (light and temperature) affecting growth were
constant, and the chemical substances (carbon dioxide and inorganic
salts) entering the reaction were continuously decreasing as the reaction
progressed, the experiment may be considered as more nearly re-
sembling a closed system — such as that to which the autocatalytic equa-
tion is applied in chemistry — in which the variables are food, flagellates,
and waste products of flagellates, the food being converted into more
flagellates and waste products. The available food material was con-
tinually decreasing as the organisms increased in number. Therefore,
the autocatalytic equation may be applied to the experiments of the
writer; whereas, it cannot, as previously explained, be applied to ex-
periments of other workers with ciliates.
Richards (1928) has shown that the division rate of yeast cells
in a limited volume of medium is a decreasing quantity; and further-
more, that when the medium was changed frequently, the division rate
remained practically constant. Hence, neither his results nor those of
the writer furnish a basis for the assumption of an autocatalyst capable
of accelerating division rate.
Robertson's concept of autocatalysis in Protozoa has, of course,
grown out of his numerous applications of the autocatalytic equation
to growth curves of plants, of man, and of other animals. As pointed
out above, the division rate of Protozoa in cultures, as calculated from
the autocatalytic equation, is an ever decreasing rather than a progres-
sively increasing quantity. In metazoan growth Robertson was not
measuring division rate of cells, but rather the increase in weight (or
increase in total number of cells') of a many-celled body. In Protozoa,
on the other hand, it was the rate of cell division as well as the rate
of increase of total number which he measured, and he assumed that
an increase in the latter necessarily involved an increase in the former.
STUDIES ON EUGLENOID FLAGELLATES
The rate of increase of the total number of cells in a metazoan or in
a protozoan culture is accelerated during the early phase or phases of
growth, but if the growth is autocatalytic, the rate of cell division is
continually decreasing. In either case, the rate of increase in total
number of cells (provided the increase follows the autocatalytic curve)
is accelerated because the number of growing units is increasing— not be-
cause of an acceleration of the growth rate of the individual units, but
in spite of a decrease in the growth rate of these units. The rate of
increase of the total number of cells and the division rate of the in-
dividual cells are two distinct conceptions which should not be confused.
BIBLIOGRAPHY
BRODY, SAMUEL, 1927. Growth and Development with Special Reference to Do-
mestic Animals. III. Growth rates, their evaluation and significance.
University of Missouri, Agr. E.r[>. Station. Bulletin 97..
CUTLER, D. W., AND CRUMP, L. M., 1924. The Rate of Reproduction in Artificial
Culture of Col Indium col fro da. Part III. Blochem. Jour., 18: 905.
JAHX, T. L., 1929. Studies on the Physiology of the Euglenoid Flagellates.
I. The relation of the density of population to the growth rate of
Euglena. Biol. Bull, 57: 81.
JAHX, T. L., 1929. The Autocatalytic Equation and the Question of an Auto-
catalyst in the Growth of Euglena. Anat. Rcc., 44: 224.
RICHARDS, OSCAR W., 1928. The Rate of Multiplication of Yeast at Different
Temperatures. Jour. Phys. Chcm., 32: 1865.
ROBERTSOX, T. B., 1923. The Chemical Basis of Growth and Senescence. Phila-
delphia and London.
ROBERTSOX, T. B., 1924. Principles of Biochemistry. Philadelphia and New York.
ROBERTSOX, T. B., 1928. The Dynamics of Growth and Differentiation. Arch.
Sci, Biol. (Napoli), 12: 235.
SNELL, GEORGE D., 1929. An Inherent Defect in the Theory that Growth Rate
is Controlled by an Autocatalytic Process. Proc. Nat. Acad. Sci., 15:
274.
THE EFFECT OF LACK OF OXYGEN ON THE SPERM AND
UNFERTILIZED EGGS OF ARBACIA PUNCTULATA,
AND ON FERTILIZATION
ETHEL BROWNE HARVEY
(From the Washington Square College, New York University and the Marine
Biological Laboratory, Woods Hole)
It has been shown in a former paper (Harvey, 1927) that when
fertilized eggs are deprived of oxygen, development is arrested, and
the eggs remain in whatever phase of division they were in when oxygen
was taken away ; they gradually resume development and pass through
subsequent phases of division when oxygen is readmitted. The experi-
ments were performed on two species of sea-urchin occurring at Naples,
Strongylocentrotiis (Paracentrotus) I'nidus and Echinus microtubcr-
culatus. Some of these experiments have been repeated on the Woods
Hole species, Arbacia punctulata, and have given the same results.
The present paper deals with the effect of lack of oxygen on the un-
fertilised eggs and the sperm of Arbacia punctulata, and on the fertiliza-
tion process in these eggs. The work was done during the summer of
1929 at the Marine Biological Laboratory of Woods Hole. I wish to
thank the Director for the facilities of the laboratory.
The experiments on unfertilized eggs and sperm were carried out
for the most part by bubbling hydrogen through a suspension of eggs
or sperm in sea-water in a closed glass vessel, from which they could
be drawn off at desired intervals for observation. The connection be-
tween the hydrogen tank and the glass vessel included a quartz tube
containing platinized asbestos which was kept heated to redness to
remove the last traces of oxygen ; from here to the glass vessel, the
connection was entirely of metal and glass, sealed with De Khotinsky
cement, to avoid the leakage of air which takes place through rubber
connections. The length of time for complete removal of air and re-
placement by hydrogen, of course, depends on size of vessel, amount
of sea-water, rate of bubbling, etc., but under the conditions of the
experiments it required approximately twenty minutes. That a state
of complete anaerobiosis obtained was shown by the fact that under
similar conditions the luminescence of luminous bacteria was stopped,
as ascertained by E. N. Harvey.
When unfertilized eggs are thus kept without oxygen, they are very
288
EFFECT OF OXYGEN LACK ON EGGS OF ARBAC1A 289
little affected. During a period of exposure of 8 hours, one can ob-
serve no difference in appearance between the eggs when drawn from
the hydrogen chamber and the control unfertilized eggs ; and the ex-
posed eggs can be fertilized and develop normally. For the first 3
hours, the eggs when withdrawn from the hydrogen chamber can be
fertilized with as much ease and as rapidly as eggs kept in air ; the
fertilization membrane comes off at the same time (1-2 minutes) and
the first cleavage plane comes in at exactly the same time (about 50
minutes) as in the control lots. When, however, eggs which have been
exposed over 3 hours to hydrogen are withdrawn and fertilized, there
is a slight lag (^r^Vz minutes) in the formation of the fertilization
membrane, a tendency of the membrane to adhere to the egg, a slight
crenulation of the egg surface, and a lag of from 2 to 5 minutes in
occurrence of the first cleavage. This was not due to the bubbling,
for when air in place of hydrogen was bubbled for the same length of
time through the same amount and concentration of eggs, these eggs
when fertilized showed no lag in the formation of the fertilization
membrane nor in time of cleavage over eggs kept at the same time
undisturbed in watch glasses and fertilized. When eggs, which have
been kept in hydrogen for three or more hours, are withdrawn and
left in air unfertilized for 45 minutes and are then fertilized, they
show no lag in membrane formation or in time of cleavage. The lag
evidently represents the recovery time from exposure to the oxygen -
free atmosphere. The unfertilized eggs have therefore a very short
recovery period after a prolonged exposure to hydrogen, and recover
instantly after a shorter exposure. They are thus in marked contrast
to fertilized eggs, which require a comparatively long period (!/o hour
to 1 hour) for recovery from exposure to hydrogen lief ore resuming
development. It may be that the longer recovery period of the fer-
tilized eggs from the effects of lack of oxygen is related to their greater
oxygen consumption as compared with that of the unfertilized eggs.
After exposure for 6 or 8 hours to either hydrogen or air (in the ap-
paratus used) some of the eggs become cytolyzed, owing probably to
the mechanical disturbance of the bubbling; the effect increases with
time until, after about ten hours, practically all the eggs are cytolyzed.
Whether, therefore, the life of the unfertilized egg is prolonged by
lack of oxygen could not be determined by these experiments. Loeb
and Lewis (1902) found that unfertilized eggs would live somewhat
longer in absence of oxygen (64 hours) than in air (48 hours), and
very much longer in a weak concentration (N/1000) of KCN (112
hours). This latter effect may, however, be due to destruction of
harmful bacteria by the KCN as pointed out by Gorham and Tower
(1902).
290 ETHEL BROWNE HARVEY
For experiments on sperm cells of Arbacia, a fairly concentrated
suspension was used, one drop of fresh undiluted sperm to 10 cc. of
sea-water {i.e., about .6 per cent), giving a decidedly milky appearance.
In such a concentration sperm live longer and retain their fertilizing
power for a longer time than in a more dilute suspension, probably
owing to COL> production as shown by Cohn (1918). When hydrogen
is bubbled through the sperm suspension for about two hours, the
sperm are motile immediately on withdrawal from the hydrogen cham-
ber, or at least as quickly as they can be observed under the micro-
scope. The lots of eggs into which they are immediately drawn form
fertilization membranes and cleave at the same time as the controls.
After an exposure of 2 to 3 hours, the sperm recover motility within
a few seconds and fertilize eggs with a very slight lag over the controls.
After an exposure of more than 3 hours, some of the sperm do not
recover motility and only a fraction of the eggs to which they are added
are fertilized. After 4 hours, the sperm are all inactive, do not fertilize
the eggs and never recover. A control experiment in which air in
place of hydrogen was bubbled through a similar amount and concen-
tration of sperm showed that the deleterious effect is due to lack of
oxygen and not to the mechanical agitation, since these sperm were
just as active and potent for fertilization even after 9 hours of bubbling
as are fresh sperm. It is interesting to note that the prevention of
oxidations by means of a hydrogen atmosphere gives a different result
from that obtained by the use of cyanides. Drzewina and Bohn ( 1912)
found that the sperm of Stronglyocentrotus would survive and remain
potent for 48 hours in KCN (1 : 1,000,000), and that when they were
subjected to KCN for long periods (1 to 10 hours) they caused a more
normal development of eggs than when subjected for a short period
(30 minutes to 1 hour). Cohn (1918) found that KCN rendered
Arbacia sperm inactive and prolonged their life, and in fact suggested
that " whatever decreases the activity increases the length of their life."
This is certainly not true for hydrogen. It may be, however, that
some other factor associated with the absence of oxygen, such as the
lack also of CO, is responsible for the death of the sperm in my ex-
periments.
A study was made of individual sperm cells in the absence of oxygen
by using a modified Engelmann chamber to which hydrogen was ad-
mitted and the sperm kept in a hanging drop (see Harvey, 1927). It
was found that in many cases enough oxygen leaked through the vaseline
seal with which the cover was mounted on the chamber to enable the
sperm to keep their motility for several hours. By entangling the sperm
in platinized asbestos threads, it was possible in some cases to keep
EFFECT OF OXYGEN LACK ON EGGS OF ARBACIA 291
them absolutely oxygen- free, and they became motionless within a half
hour. If air was then admitted, the sperm immediately became motile.
Even if the bubbling of hydrogen was stopped, within a very few min-
utes the sperm became active. It apparently requires a very minute
amount of oxygen for motility of the sperm. When sperm are kept
in an Engelmann chamber without oxygen for two hours, they clo not
recover motility on admission of air. They are killed by the absence
i if oxygen even more quickly than when the experiments are done in
bulk.
The most interesting question in connection with lack of oxygen
on eggs and sperm is whether fertilization can take place and the
fertilization membrane be thrown off during complete absence of oxy-
gen. An attempt to answer the question was made by keeping un-
fertilized eggs in one drop and sperm in another drop very close together
in an Engelmann chamber. Hydrogen was sent through for a half
hour, then the chamber was shaken so as to make the drops coalesce
and the sperm come in contact with the eggs, still keeping hydrogen
passing through the chamber and the seal intact. It was found that
when the sperm are completely immotile, they do not fertilize the eggs,
probably because they cannot get to the surface of the egg; they go
in currents around and past the eggs ; in no case is a fertilization mem-
brane thrown off. On admission of air the sperm become motile and
the membranes of the eggs are thrown off in 1 to 2 minutes as normally.
If there is the slightest trace of air leaking in the chamber, sufficient
for a few only of the sperm to be very slightly motile, some of the eggs
are fertilized on mixing the drops, and fertilization membranes are
thrown off, but no further development takes place until more air is
admitted. The question, therefore, whether oxygen is necessary for
membrane formation has not been answered. If there is absolutely
no oxygen, the sperm are absolutely immotile and cannot fertilize the
eggs, probably owing to mechanical difficulties, and no membranes are
given off. Loeb also found that if the sperm cells of Strongylocentrotus
were made immotile by NaCN, they were unable to fertilize the eggs
even when squirted on eggs with jelly removed. If in my experiments,
there is the slightest trace of oxygen, a few sperm remain motile and
fertilize eggs which throw off membranes. If membrane formation
does require oxygen, it is in an almost infinitesimal amount. It requires
more oxygen for the development of fertilized eggs than it does for
motility of sperm, fertilization of the egg and the formation of the
fertilization membrane.
ETHEL BROWNE HARVEY
SUMMARY
1. Unfertilized eggs of Arbacia are not visibly affected by complete
lack of oxygen for a period of 8 hours. After an exposure of 3 hours
they recover immediately on admission of air ; after a longer exposure,
when air is readmitted and the eggs are fertilized, there is a slight lag
in the formation of the fertilization membrane and in time of cleavage.
2. Sperm of Arbacia are rendered motionless by lack of oxygen, but
are otherwise unaffected for 2 hours. They recover immediately on
admission of air. After 3 hours some of the sperm are irreversibly
injured, and after 4 hours they are all killed.
3. When sperm are added to unfertilized eggs, both being in com-
plete absence of oxygen, fertilization does not take place, and the fertili-
zation membrane is not thrown off because the sperm are not motile, and
cannot get to the surface of the egg. The membrane is thrown oft"
immediately on admission of air. If there is the slightest trace of air,
which may leak through the vaseline seal to the chamber, sufficient for
only a few sperm to be very slightly motile, the eggs with which they
come in contact throw off fertilization membranes, but do not develop
further until more air is admitted. If oxygen is necessary for mem-
brane formation, it is in an almost infinitesimal amount.
LITERATURE
COHN, E. J., 1918. Studies in the Physiology of Spermatozoa. Biol. Bull, 34:
167.
DRZEWINA, A., AND BOHN, G., 1912. Effets de 1'inhibition des oxydations sur
les spermatozo'ides d'oursin et, par leur intermediare, sur le developpement.
Compt. rend. Acad. Sci., 154: 1639.
GORHAM, F. P., AND TOWER, R. W., 1902. Does Potassium Cyanide Prolong
the Life of the Unfertilized Egg of the Sea Urchin? Am. Jour. PhysioL,
8: 175.
HARVEY, E. B., 1927. The Effect of Lack of Oxygen on Sea Urchin Eggs.
Biol. Bull., 52: 147.
LOEB, J., 1915. On the Nature of the Conditions which Determine or Prevent
the Entrance of the Spermatozoon into the Egg. Am. Nat., 49: 257.
LOEB, J., AND LEWIS, W., 1902. On the Prolongation of the Life of the Un-
fertilized Eggs of Sea Urchins by Potassium Cyanide. Am. Jour.
Physio!., 6: 305.
THE EFFECT OF CONJUGATION WITHIN A CLONE OF
PARAMECIUM AURELIA
DANIEL RAFFEL
(From the Zoological Laboratory of the Johns Hopkins University)
INTRODUCTION
On the effects of conjugation in paramecium, particularly in re-
lation to the production of inherited variations, the results of investi-
gators are in conflict. Jennings (1913), working with both Para-
mecium aurelia and Paramecium caudatum, reported that conjugation
increased inherited variations : that it caused the production of diverse
biotypes. The members of a clone — a population derived by fission
from a single individual, whether an ex-conjugant or not — remained
nearly or quite uniform in their inherited characteristics so long as
conjugation did not occur among them. But after conjugation within
such a clone, the inherited characteristics of descendants of the different
ex-conjugants had become diverse. Thus by conjugation many dif-
ferent biotypes had been produced, the descendants of each ex-conjugant
constituting a single uniform biotype.
Calkins and Gregory (1913), on the other hand, reported that
there is in Paramecium caudatum as much variation among the de-
scendants of the four individuals produced by the first two fissions of a
single ex-conjugant as was found between the progeny of different ex-
conjugants. They conclude that, " The results of this study show that
physiological and morphological variations in the progeny of a single
ex-conjugant of Paramecium caudatum are fully as extensive as the
variation between the progenies from different ex-conjugants " (p.
523).
Jennings (1916, p. 528, and 1929, p. 188) has tried to show that
the results of Calkins and Gregory are invalidated by uncontrolled
sources of error. On the one hand, he holds that their method of
culture permitted continuing environmental differences between their
different populations, such as would give rise to differences that
would appear to be hereditary, although they were not. On the other
hand, he notes the occurrence of conjugation within some of their
cultures and the fact that this might readily have occurred undetected.
This would vitiate their conclusions.
293
294 DANIEL RAFFEL
Obviously, the situation calls for a new investigation of the matter,
in which such methods shall be employed as shall certainly exclude
the possibility that environmental differences affect the results, while
at the same time the occurrence of unobserved conjugation is ex-
cluded. It is such an investigation that is here presented. In order
to assure a uniform environment for all the lines of descent an elab-
orate technique was employed. This is described on later pages.
The method involved, first, the use of a synthetic culture medium
of known composition, with pure cultures of food organisms and
uniform glassware; second, continuation of the uniform conditions
by the cultivation of the paramecia under aseptic conditions; third,
frequent testing of the culture fluid in which the organisms have
lived in order to ascertain whether the uniformity of the environment
has been maintained. In addition, the organisms are cultured singly
and transferred daily to new drops of culture fluid, so that it is im-
possible for conjugation to occur. Continuing diversities between
lines cultivated simultaneously under such conditions can be inter-
preted only as caused by constitutional differences among the organ-
isms, not as due to diversities in food or cultural conditions, or other
extrinsic factors.
Taking these precautions, two comparisons are made. First, a
population descended from different ex-con jugants is compared with
a population derived by fission from non-con jugants of the parent
clone. Second, four lines descended from each ex-con jugant are
compared with one another, and the several such different clones are
similarly compared. In this way it is possible to determine whether
increased hereditary variation and differentiation into diverse bio-
types are produced by conjugation.
The investigation was suggested to me by Professor H. S. Jen-
nings, and my thanks are clue to him for assistance throughout the
work. I am also indebted to Rose Mahr Raffel, who assisted in
the carrying out of the experiment, and without whose aid cultures
of this magnitude, using the elaborate technique here employed, could
not have been carried through.
MATERIALS AND METHODS
In this investigation an elaborate technique was used in order to
subject all of the lines to identical environmental conditions. Great
care was taken to eliminate any possible sources of variation. To this
end the culture fluid, the food supply and the glassware used were
standardized to as great an extent as was possible. The work which
has been carried on for several years by Hartmann and his associates
EFFECT OF CONJUGATION OF PARAMECIUM 295
at the Kaiser- Wilhelm Institut fur Biologic has made possible the use
of synthetic culture fluids and pure cultures of food organisms for
the cultivation of protozoa. The use of pure cultures of unicellular
algae as food organisms appears first in the work of Luntz (1926) on
the rotifer Ptcrodina clliptica and more recently in the work of Adolph
(1929) with the ciliate Colpoda. The results of the work of Hartmann
and his associates are given in a recent paper of Belar (1928). The
following pages contain a detailed account of the methods used to obtain
uniformity in the environmental conditions throughout this experiment.
1. Culture Fluid
The culture medium used was a physiological salt solution of known
composition. After many attempts to find a solution in which the race
of Paramcciuui aurclia which was used would live, it was found that
if the solution described by Pringsheim (1928) for the cultivation of
algoe was altered so as to be neutral, it furnished an excellent medium
for this organism. This modification was obtained by replacing the
KH2PO4 used by Pringsheim by an equal molar concentration of
K2HPO4. The composition of the solution was KNO, 0.5 gram,
K2HPO4 0.06 gram, MgSO4 0.02 gram, FeCL, 0.001 gram, water 1000
grams. The water used in making this solution was redistilled from
a still made of Pyrex glass and had in all cases a conductivity less than
1.05 X 10 6 mho. This solution was made up in quantities of one
liter. It was then divided into portions of approximately 15 cc. in test
tubes. These test tubes were plugged with non-absorbent cotton and
the solution was sterilized in the autoclave for 15 minutes under 15
pounds of steam pressure. The solution was kept in this way for
periods varying from a few days to two months before it was used.
Tubes tested at intervals showed no bacteria and no measurable altera-
tions in composition.
2. Food Organism
The food organism used was a unicellular green alga, Stichococcus
bacillaris.1 This was cultivated on 0.05 per cent Benecke's agar com-
posed of water, 1000 grams; Agar-Agar, 15 grams; NH4NO3, 0.2
gram; CaCL, 0.1 gram; MgSO4.7H2O, 0.1 gram; and K,HPO4, 0.1
gram. The components of this agar were boiled together until the
agar-agar was all dissolved. Five cc. portions were poured into test-
tubes which were then sterilized in the autoclave under 15 pounds of
steam pressure for fifteen minutes. These tubes were then " slanted "
1 1 am indebted to Professor W. R. Taylor of the University of Pennsylvania
for the identification of this organism.
296 DANIEL RAFFEL
in order to obtain a large, easily accessible surface. Twenty of these
tubes were seeded from a pure culture of the alga on successive days.
After this the slants were used in the order in which they had been
seeded and as they were used they were replaced by new tubes seeded
from them. The tubes in which the alga was cultivated were kept
constantly before a north window in order to obtain sufficient light.
Each da}- the tube of Stichococctis to be used that day and a fresh
tube of the culture fluid were opened close to a flame into which their
open ends were immediately thrust. Then a small quantity, approx-
imately 5 cm., was scraped from the agar with a platinum loop which
had just been sterilized in the flame. This small quantity of the alga
was then quickly suspended in the solution and both tubes were im-
mediately restoppered. Then a new tube of agar was seeded from
the same tube and replaced in its proper place in the rack. Many tests
of the suspension were made from time to time and in no case was
any bacterial contamination found. An effort was made to have the
suspension of alga in the solution always of the same density. How-
ever, no method more accurate than a comparison of the appearance
of the tubes was found for determining the success of this effort. For
this reason, preliminary experiments were performed in order to de-
termine whether or not the quantity of algae used affected the rate of
reproduction of the paramecia. It was found that sufficient algae to
produce a slight greenish tinge in the suspension furnished enough
food for these organisms. Greater densities than this had no effect
on the rate of reproduction even when they were far in excess of any
used in the actual experimental work. At all times an excess of algae
was assured and the drops containing the paramecia always showed
a large number of the algae at the end of the period during which the
organisms remained in them.
It was found, however, that if the paramecia were kept in this so-
lution with this single food organism, they were unable to live and re-
produce. If a very slight trace of a Bacillus candicans was present, this
difficulty was eliminated.2 Attempts were made to cultivate the par-
amecia on this bacillus in the absence of the alga. All such attempts
failed, and when a mixture of the two food organisms was used, the
food vacuoles were dark green in color — indicating that the food supply
was composed mainly of the alga. After a slight trace of this bacterium
was once introduced into a culture of paramecium, it was perpetuated
in the transfers of the organisms. As far as it was possible to de-
2 I am indebted to Professor W. W. Ford, Professor of Bacteriology in
the School of Hygiene and Public Health of the Johns Hopkins University, for
the identification of this bacillus.
EFFECT OF CONJUGATION OF PARAMECIUM 297
termine by plating in the usual way, this bacterium was present in ap-
proximately the same quantity from day to day in all of the many cases
tested at random. It was thought advisable, however, to determine
whether or not differences in the quantity of this organism present af-
fected the rate of fission of the paramecia. There was no difference
in the effect produced by the presence of any quantity of the bacterium
less than that required to make the drops of culture fluid appear milky.
At no time during the course of this investigation was this condition
approached.
3. Glassware
The various lines of paramecium used in this investigation were
cultivated on slides with two concavities. It was found from prelim-
inary work that different slides affected the paramecia differently. On
some slides representatives of all the lines tested reproduced more
rapidly than did other representatives of the same lines on other slides.
The pH of drops of culture fluid which had remained on the different
slides was tested and was found to vary greatly. Drops of fluid which
had been identical when placed on the slides were found to vary by a
whole pH unit within twenty- four hours. This showed that the glass
of the various slides differs in solubility. New slides were then ob-
tained, all of the same kind of glass. These slides were of French
origin. After two days the organisms grown on these slides died out.
No amount of washing the slides with various kinds of solvents made
it possible to cultivate organisms on them. Investigation disclosed that
this French glass is made by a process involving the use of lead. It
appears that the presence of this element was responsible for the toxic
effects of these slides on the organisms. When this was discovered,
new slides were obtained which were of white glass and were all pro-
duced by the same manufacturer. These slides were the only ones
used in this investigation. Before they were used they were thoroughly
washed in running water. Then they were washed in ether and 95 per
cent alcohol in order to remove any organic matter with which they
might have been contaminated. They were again thoroughly washed
with running water, rinsed in several changes of tap water and finally
rinsed in hot distilled water. Each day the slides were thoroughly
washed in the following manner. First they were held, individually,
in running tap water and the depressions were rubbed well with the
thumb. They were then placed in a receptacle containing clean tap
water. In this receptacle they were rinsed three times. Then, after
the last tap water was thoroughly drained off, the slides were covered
with hot distilled water. Thev were then dried on racks.
298 DANIEL RAFFEL
In order to prevent contamination of the cultures by bacteria in
the air, Petri dishes 100 mm. in diameter and 15 mm. deep were used
as moist chambers. This made it possible to transfer the organisms
with a minimum of exposure to the air. The dishes contained water
at the bottom ; the two slides to each dish were supported above this on
strips of glass. After the Petri dishes, the slides, and the glass plates
were assembled, they were heated in the hot air sterilizer for one hour
at 150° C. In order to facilitate the handling of the numerous dishes
which were used, baskets were made from ^ inch wire netting which
held a dozen Petri dishes in four tiers of three dishes each.
The organisms were transferred by means of capillary pipettes.
Each of these contained a plug of cotton inserted into its wide end.
This is a precaution necessary to prevent contamination of the cultures
by microorganisms which would otherwise be introduced by the rubber
bulbs used on the ends of the pipettes. The glass part of the pipettes
with their cotton plugs were kept in large museum jars, in which they
were heated in the hot air sterilizer for one hour at 150° C. before
each time they were used.
4. Method of Transferring Organisms
Before the daily transfers were made, the Petri dishes were removed
from the hot air sterilizer. Then two drops of the culture suspension
were dropped into each concavity. Large pipettes which were drawn
out until the ends were 2 mm. in diameter were used for this purpose.
These pipettes, like the ones used for transferring the paramecia, were
protected by cotton plugs and were sterilized before each time that they
were used. The mouth of the test tube containing the suspension of
culture fluid was sterilized in the Bunsen flame each time that it was
opened. The tops of the successive Petri dishes were then raised on
one side, the pipette was introduced and two drops were allowed to
fall into each concavity. Four dozen dishes were prepared in this
manner at one time. From time to time bacteriological plates were
prepared from culture medium which was treated in the manner de-
scribed above, after it was left for twenty-four hours. In every case
the plates were negative, thus indicating that the technique was ab-
solutely dependable.
In transferring the animals a Petri dish containing the two slides
was placed on the stage of the binocular microscope. Another dish
containing new culture fluid was placed at the experimenter's right.
One organism was then taken from each concavity and transferred to
the corresponding concavity of the new dish. This was done very rap-
idly, using a clean pipette that had just been removed from the jar of
EFFECT OF CONJUGATION OF PARAMECIUM 299
sterile pipettes. A separate pipette was used for the organisms of each
dish. The new dishes were then removed to the constant temperature
chamber, in which they were left at a temperature of approximately
24° C. (There was in the history of the cultures variation in temper-
ature from 22.2 °-26.2° C.)
5. Isolation and Sterilization of the Clone
The various lines of Parainecinui aurelia used in this investigation
are the descendants of a single individual which was isolated from a
mass culture in the laboratory on July 29, 1929.
Parpart (1928) has shown that spores of bacteria may be, and often
are, carried within paramecium and that in washing these organisms,
precaution must be taken to eliminate these spores as well as the bacteria
external to the paramecium. For this reason, when the individual
which was used to start the clone for this investigation was washed, the
precautions suggested by Parpart were observed. The individual was
first washed successively in five concavities containing sterile culture
fluid. Then at intervals of one hour it was washed through five more
similar quantities of fresh culture fluid. It was then placed in a con-
cavity containing the regular culture suspension described above in
which there was a slight trace of the Bacillus caudicans. No bacteria
were added at any later time. From time to time throughout the course
of the experiment bacteriological plates were made from drops from
which the paramecia had been removed. Several dishes containing both
ex-con jugant and non-con jugant lines were taken at random for this
purpose. At no time did any plate made in this way indicate the pres-
ence of any bacteria except the bacterium which had been introduced
at the beginning.
THE EXPERIMENT
1. Plan
The plan of the experiment was as follows : A clone was obtained
by allowing a single individual of Paramecium aurelia to multiply. A
portion of the clone was induced to conjugate, while another portion was
kept without conjugation. The former, after the separation of the
pairs, yields lines of descent that constitute the ex-conjugant population,
the latter the non-conjugant population. These two populations are
later compared as to their mortality, fission rate, variation and the
inheritance of the variations.
For comparison with the results of Calkins and Gregory, a method
similar to theirs was employed for the grouping and subdivision of
20
300 DANIEL RAFFEL
the ex-conjugant lines. Each of the two members of a pair was al-
lowed after separation to divide twice, yielding four individuals of
common origin, the four quadrants. From each quadrant a line of
descent was obtained. Each set of four quadrants derived from a
single ex-conjugant is called, for convenience, a tetrad. The variation
within single tetrads is compared with the variation among lines belong-
ing to different tetrads (and so derived from different ex-con jugants).
This tests whether the diversity among the descendants of a single ex-
conjugant is as great as that between those of different ex-con jugants
(as is maintained by Calkins and Gregory).
2. Description
The experiment was begun with the isolation of a single organism
on July 29, 1929. The progeny of this individual were propagated
on slides by daily transfer until August 5, 1929. By this time there
were approximately 1500 individuals present. On the morning of
August 5, all of the individuals, except one, from each concavity, were
transferred to two small sterile culture dishes contained within Petri
dishes. No fresh culture fluid was added to those culture dishes and the
least possible quantity was carried over with the organisms. The other
organisms were transferred to clean slides in the usual manner. These
latter ones were the source from which the non-con jugant lines used
in this experiment were obtained. The process of transferring this
number of animals occupied several hours. Before all the organisms
had been transferred conjugation had begun in the two culture dishes.
One hundred and twenty pairs of con jugants were removed from the
culture dishes and numbered in order of their removal. The next morn-
ing the pairs had separated. The two members of each pair were trans-
ferred to the two concavities of a clean slide. The non-con jugants, one
from each dish which had been transferred to slides on August 5, were
transferred to clean slides until 112 non-con jugants had been trans-
ferred. The number of fissions was recorded in the case of the non-
conjugants. On August 7-8 the ex-conjugants completed their first
two divisions, giving rise to the four lines or quadrants from each of
the ex-conjugants which were to be propagated in this experiment.
On August 7th and 8th the non-con jugant lines and the ex-conjugant
lines were so distributed that no two non-con jugant lines or lines from
the same tetrad were cultivated in the same Petri dish. This was done
so that if any correlation was found between the quadrants of a tetrad
or between lines of the non-con jugant population, it could not be the
result of cultivation on the same slides or in the same dishes.
EFFECT OF CONJUGATION OF PARAMECIUM 301
From August 6th to September 10th inclusive, each line was trans-
ferred each day (except on August 8th and 10th as described below).
On August 8th and August 10th the amount of work was so great that
it was not possible to complete the transferring until after midnight.
The lines which were not transferred until after midnight on these days
were not transferred again for approximately 36 hours. On August
llth all of the lines which were incomplete because of losses were dis-
carded. When this was done, the number of lines retained was the
maximum number that two persons could transfer once daily, using this
involved technique. From August llth to the close of the experiment
on September 10th, all the lines surviving were transferred daily.
The actual numbers isolated at the beginning of the experiment were
for the non-con jugants 112; for the ex-conjugants 405 lines or " quad-
rants " derived from 105 different ex-conjugants, belonging to 58 dif-
ferent pairs. The numbers were reduced by accident or death of lines,
so that the actual numbers of lines available for comparison were, for
the first ten days of the experiment, 66 non-con jugants, 324 ex-con-
jugants ; for the first twenty days, 64 non-con jugants, 295 ex-conjugants ;
for the entire period of 36 days, 46 non-conjugants, 115 ex-conjugants.
During the first week following the beginning of the experiment
a rather large number of deaths occurred among the non-con jugant
lines. After this period there occurred a period of about three weeks
during which deaths among the non-con jugant lines were rare. Many
of the deaths which occurred during the early part of this period were
lines that had stopped dividing during the earlier period. On the
twenty-fifth day of the experiment (August 29th), the rate of mortality
among the ex-con jugant lines increased rapidly. This was followed
two days later by an increase in the rate of mortality among the non-
conjugants. This high rate of mortality continued for nearly ten days.
The occurrence of this high rate of mortality in the ex-conjugant lines
beginning twenty-five days after conjugation was accompanied by a
general depression in all the lines. This fact and the occurrence of
two such periods in the non-con jugant lines, separated by a period of
about twenty-five days, led to the suspicion that these were periods
of endomixis. On September 6th many of the excess animals from
the non-con iugant and ex-conjugant lines were stained and mounted
for stud}'. The individuals from the ex-conjugant lines showed in
many cases the conditions of late stages of endomixis. Numerous frag-
ments of macronuclei were present, and in one case the organism was
found to be at the climax of the endomictic process. The represen-
tatives of the non-conjugant lines showed on the whole the conditions
302 DANIEL RAFFEL
typical of earlier stages of endomixis. Large irregular macronuclei
were found, often accompanied by large fragments. It seems clear,
therefore, that the periods of high mortality were periods of endomixis :
a relation which other investigators have observed.
On September 10th, thirty-six days after conjugation had occurred,
the experiment was discontinued. At this time 46 lines of non-
conjugants and 115 lines of ex-conjugants were still in existence.
3. Results
The experiment was designed to supply data mainly upon the rate
of reproduction, its variability and the inheritance of the variations, in
the ex-conjugants and non-conjugants. It yields also certain data on
comparative mortality, which will be given first.
A. Mortality
A considerable number of the lines of both the non-conjugants and
ex-conjugants died out during the thirty-six days of culture. The
percentages surviving in each group at certain periods after the be-
ginning of the experiment, are the following:
After
20 days
25 days
35 days
Non-con jugant lines
73.0
68.6
51.7
Ex-conjugant lines
79.2
67.9
30.8
Thus on the whole the mortality is much greater among the lines
descended from the ex-conjugants. At the end 51.7 per cent of the
non-conjugant lines were alive as against 30.8 per cent of the ex-
conjugant lines.
B. Rate of Reproduction
The basic data as to comparative rate of reproduction in the non-
conjugants and ex-conjugants are given in Table I. The number of
fissions for both groups is reckoned from August 7th, on which day
all of the ex-conjugants divided once or twice. Thus the statistics are
not affected by any delay in fission due to the process of conjugation
itself.
Table I shows at A the number of fissions for the different lines
for the 20 days of culture beginning August 7th and ending August
26th; throughout this period there were 64 lines of non-conjugants
and 295 lines of ex-conjugants. the latter derived from 99 different ex-
conjugants and so forming 99 tetrads. At B are shown the distribution
EFFECT OF CONJUGATION OF PARAMECIUM
303
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\O CM O
<r> — i
•o
00
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o
00
IO
r^
f*> •*
O
i^
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ro •* r^
>-i CN
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-O
in o^ <^>
*-> CN —i
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OO -H VO
J^ »-H
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rvl O ON
IT> T-H
x
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•*
t-~ VO tN
<n cs
'to
to
E
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Tf
fO T*t -H
fN -H
_>,
'is
to
m
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rt
o
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c» -^
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S
"O
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»-i IO <*5
4—4
o
(N
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^f
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^— i
Non-conjugants
Ex-conjugants
Tetrads
304 DANIEL RAFFEL
of the numbers of fissions for the 46 surviving non-conjugant and 115
ex-con jugant lines that survived throughout the entire period of 36
days, August 6th to September 10th. At C in Table I are shown the
mean fission rates for the total period of survival, for all the lines that
lived more than 10 days.
Mean Rate. — As Table I shows, the mean rate of reproduction for
the non-conjugant and con jugant groups did not differ greatly, although
in every case the mean rate for the non-con jugants is higher by a small
but significant amount. The mean fission rate for all non-conjugant
lines is 1.58 ± 0.01 ; for all ex-conjugant lines, 1.48 =iz 0.01.
C. Variation in Fission Rate
But it is in the variation of the fission rate that the difference be-
tween the non-con jugants and ex-conjugants is striking. An inspection
of Table I shows at once that the variation in the ex-conjugant lines is
much greater than that in the non-conjugant lines. For the first twenty
days, the number of fissions in the non-conjugant lines varies from 28
to 37, a range of 10. For the ex-conjugant lines, in the same period
the range is from 16 to 37, a range of 22, more than double that for
the non-con jugants. For the entire 35 days, the non-conjugant lines
range from 47 to 61, the ex-conjugant lines from 38 to 61. The mean
daily fission rates (C, Table I) vary in the non-con jugants from 1.25
to 1.75; in the ex-conjugants from 0.85 to 1.85. The range for the
former is 0.55; for the latter 1.05, or nearly double that for the non-
conjugants. The fission rate for the lowest lines of ex-conjugants is
far below that for the lowest non-con jugants, and the highest ex-
conjugant line is above the highest non-conjugant. Conjugation within
the clone has caused a wide spreading out of the fission rates ; it has
produced stocks with lower, and with higher, rates than any found in
the clone before it has conjugated.
Computation of the standard deviations and coefficients of variation
shows the same great increase in variation as a consequence of conjuga-
tion. The means, standard deviations and coefficients of variation,
computed from the data shown in Table I, are given in Table II.
As Table II shows, the coefficient of variation of the ex-conjugant
lines is for the first 20 days 158 per cent of that of the non-con jugants ;
for the entire 35 days it is 187 per cent of that of the non-conjugants.
For the mean daily fission rates of the different lines, the coefficient of
variation for the ex-conjugants (10.14) is 139 per cent of that of
the non-conjugants (7.28).
EFFECT OF CONJUGATION OF PARAMECIUM
305
The comparative distribution of the fission rates of non-con jugants
and ex-con jugants, as shown in Table I, are worthy of notice. In the
first 20 days (A. Table I) 22 lines of ex-conjugants, or 7.4 per cent
of all, show fewer fissions than any of the non-con jugants. In the entire
35 days (B, Table I), the proportion is nearly the same: 7.8 per cent
of the ex-conjugant lines lie below all of the non-conjugant lines. At
the opposite extreme the two sets are alike ; the highest lines have the
same number of fissions in the two cases. In mean daily fission rates,
18 lines of ex-conjugants, or 5.6 per cent of all, lie below all of the
non-conjugant lines, while one ex-conjugant line lies above all the non-
conjugant lines.
TABLE II
Means, standard deviations and coefficients of variation of non-conjugant and
ex-conjugant lines, for the numbers of fissions during certain periods; and for the mean
daily fission rates of the different lines. Based on the data given in Table I.
A. Numbers of Fissions
First 20 Days
Total 35 Days
Mean
Stan. Dev.
Coef. Var.
Mean
Stan. Dev.
Coef. Var.
Non-conjug'ts
Ex-conjugants
32.9±0.2
31.3±0.1
1.88 ±0.11
2.83 ±0.08
5.70±0.34
9.02 ±0.25
56.1 ±0.3
53.3±0.3
3.19±0.22
4.49±0.20
5.68±0.40
8.42 ±0.38
B. Mean Daily Fission Rates of the Different Lines
Mean
Stan. Dev.
Coef. Var.
Non-conjugants. . .
Ex-conjugants. . . .
1.58 ±0.01
1.48 ± 0.01
0.12 ±0.01
0.15 ±0.00
7.28 ± 0.43
10.14 ±0.27
It is clear, therefore, that conjugation within the clone has much
increased the variability of the fission rate, and that one of the factors
in the increased variability is the production of a considerable number
of ex-conjugant lines that have a lower fission rate than any lines
among the non-conjugants.
D. Variation among Quadrants Derived from a Single Ex-conjugant,
Compared with Variation Between Lines Derived
from Different Ex-conjugants
Calkins and Gregory (1913) reached the conclusion that the varia-
tion between different quadrants (the four lines derived from a single
ex-conjugant) was as great as that between lines derived from diverse
ex-conjugants. Lines derived from a single ex-conjugant constitute,
306 DANIEL RAFFEL
of course, a clone within which conjugation has not occurred; so that
according to this result, there is no increase of variation in consequence
of conjugation within the clone. To test this particular matter, the
variation between the different quadrants of the same tetrads (each
tetrad derived from a single ex-conjugant) was compared with the
variation among progeny of the different ex-con jugants. For each
tetrad records of only two to four lines are available, so that the coef-
ficients of variation within the tetrad are not statistically adequate, but
the general result is of interest. For the number of fissions during
the first 20 days of the experiment, the mean coefficient of variation
for the lines constituting a single tetrad was 4.53 ; for the means of
the diverse tetrads (progeny of the diverse ex-con jugants), the coef-
ficient of variation was 8.32. For the average daily fission rate, the
mean coefficient of variation for the lines constituting a single tetrad was
5.22; for the diverse tetrads it was 8.57.
As will be seen by comparison with Table II, the mean variation
within tetrads (4.53) is of a similar order to the mean variation for
non-conjugant lines of a clone (5.70) (that is. to the variation within
a clone in which conjugation has not occurred). On the other hand,
the variation when the different tetrads are compared (8.32) is much
greater, and is similar to the variation (9.02) when all the lines derived
from ex-conjugants are compared. This indicates strongly that the
four quadrants produced by the first two divisions of an ex-conjugant
do not differ in any general way from any other products of fission
of a single individual. Further, the similarity between the coefficients
of variation for all ex-conjugant lines taken separately, and that for
the means of the diverse tetrads, indicates that the variation among
the ex-conjugant lines is due mainly to the inherent differences between
the ex-conjugants.
The higher variation among diverse tetrads, as compared with less
variation between the quadrants belonging to the same tetrads, may
be further shown by comparing the maximum differences found ( 1 )
between any two lines of the original non-conjugant population ; (2)
between quadrants belonging to a single tetrad; (3) between the means
of diverse tetrads ; and (4) between any two ex-conjugant lines. These
comparisons are given in Table III.
This table shows that the maximum difference within any of the 99
tetrads and the maximum difference between any two non-conjugant
lines of the original population are very nearly identical. On the
other hand, the maximum differences between any two ex-conjugant
lines are only slightly greater than the maximum differences between
EFFECT OF CONJUGATION OF PARAMECIUM 307
any two of the tetrads. (It is to he expected that the maximum dif-
ferences between two tetrads would he slightly smaller than that be-
tween the two ex-conjugant lines which differ most, since the fissions
for tetrads are usually the means of two to four lines.) Thus the
single tetrads do not significantly differ in their variability from the
general non-conjugant population, while the variation between the dif-
ferent tetrads is much greater than that within the tetrads.
TABLE III
Maximum Differences Bet-ween Lines Having Different Relations to Each Other uith
Respect to Conjugation
Total Fissions
First 20 Days Average Daily Fission Rate
Maximum difference between two non-con-
jugant lines of the original population ... 9 0.50
Maximum difference within any tetrad .... 10 0.58
Maximum difference between two means of
tetrads 17.75 0.84
Maximum difference between two ex-con-
jugant lines 21 1.00
The matter may be tested further by determining whether there is
correlation in fission rates between the members of the tetrads. If the
different quadrants within the tetrads differ as much as do the members
of different tetrads, there should be no correlation between the members
of the tetrads. If, on the other hand, the different quadrants of the
single tetrads show a significant correlation, this will demonstrate that
such quadrants are not so unlike as are different lines of the ex-con j ugar.t
population taken at random.
The data for this comparison are shown in Table IV, based on the
numbers of fissions during the first 20 days of the experiment. The
fissions for each quadrant of each tetrad are entered as X against the
fissions for each other quadrant of that same tetrad as Y. As some
of the tetrads had but two surviving lines, others three or four, the
total number of entries in the table is 332 pairs. Since the members
of the tetrads are like variates, the correlation must be computed as for
a symmetrical table in which each pair is entered twice, in reverse order
(see Jennings, 1911. for the method of computation).
The coefficient of correlation between the fission rates of the quad-
rants of the same tetrad, obtained from this table, is very high, amount-
ing to 0.854 ± 0.007. Beyond question, therefore, the quadrants de-
rived from a single ex-con jugant are much more alike in their fission
rates than quadrants derived from diverse ex-conjugants.
308 DANIEL RAFFEL
All the four lines of evidence thus agree in showing- clearly that a
population composed of different ex-conjugants of a clone has a higher
variation in fission rate than do the offspring of single ex-conjugants.
(1) The coefficient of variation is much higher for the ex-conjugant
population than for the non-conjugant. (2) The coefficients of varia-
tion for quadrants belonging to single tetrads is much less than the
coefficient of variation for the means of diverse tetrads. (3) The max-
imum differences between lines within tetrads are much less than the
maximum differences between means of different tetrads. (4) There
is a very high correlation (0.854) between the lines or quadrants de-
rived from the same ex-conjugant. These four lines of evidence es-
tablish firmly the fact that conjugation within a clone causes increase
of variation.
TABLE IV
Paramecium aurelia. Correlation between total number of fissions, August 7-26,
of each member of the tetrad with every other member.
18 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
16
1
1
22
1
1
2
23
1
1
24
1 1
1
3
25
2
2
26
1
1
2
27
1
3
2 1
7
28
2 2
1
1
3 2
1
1
13
29
1
1
2
3 6
7
3
2
3
28
30
1
2
6
6 8
6
3
1
1
1
35
31
1
4 2
5
7 14
5
5
7
3
4
57
32
1
1
4
5
6 7
7
10
5
2
1
49
33
1 1
6
8 10
11
10
10
1
58
34
1
3
1 9
3
16
6
3
1
43
35
3
7
3
4
1
1
19
36
1
1 1
1 1
4
1
10
37
1
1
2
11226
11
7 10
28
41 61
48
50
39
14
10 1
332
r = +0
854
± 0.007
E. Inheritance of the Diverse Fission Rates
In order to determine whether the diverse fission rates observed are
hereditary, the number of fissions which occurred in each line during
the first ten days after the ex-conjugants began to divide was cor-
related with the number of fissions during the following ten days.
These coefficients of correlation were obtained (a) for all of the non-
conjugant lines which lived from August 6th-26th, (b) for all of the
EFFECT OF CONJUGATION OF PARAMECIUM
309
ex-con jugant lines which lived through the same period, and (c) for
the means of all the tetrads of which one or more '' quadrants " sur-
vived until August 26th. The correlation tables from which these
coefficients of correlation were calculated are given in Tables V, VI and
VII. No correlation was found among the non-con jugant population
(Table V). The coefficient of correlation obtained was -f- 0.016 ±
0.084. Thus the differences in fission rate (which are not very great,
as Table V shows) are not inherited differences.
TABLE V
Paramecium aurelia. Non-conjugant lines. Correlation of number of fissions,
August 7-16 and August 17-26.
13
14
15
16
18
19
20
13
1
1
14
1
1
2
4
15
1
1
3
3
8
16
1
1
8
6
1
17
17
1
4
2
5
2
14
18
2
2
7
3
1
15
19
1
2
3
20
1
1
2
1 4
10
22
20
7
64
r =
+ 0
016 ± .084
TABLE VI
Paramecium aurelia. Ex-conjugant lines. Correlation of number of fissions,
August 7-16 and August 17-26.
6 9 10 11 12 13 14 15 16 17 18 19
10
1 1
1
3
11
1
1
1
3
12
1
1
2
2
6
13
2
1
3
3
4
1
14
14
1
2
3
9
7
6
28
15
2
1
11
9
16
11
2 1
53
16
1 1
1
3
8
24
20
19
6
83
17
1
4
8
10
17
18
2 1
61
18
1
1
1
6
10
10
3
32
19
1
1
1
1
5
1
10
20
1
1
2
2115
5
15
38
63
78
71
14 2
295
r = +
0.327 ±
0.035
310
DANIEL RAFFEL
For all the ex-conjugant lines, each taken separately (Table VI),
there is a well-defined positive correlation of -f- 0.327 ± 0.035. Thus
many of the diversities between these are inherited. When, however,
the means of the separate tetrads are taken, and the fissions for the
first ten days are tabulated against those of the second ten days (Table
VII), the correlation rises to +0.651 ±0.039. In such a table, the
process of averaging the different quadrants of the tetrad smoothes out
in large measure the accidental differences between the diverse lines,
leaving mainly the intrinsic differences, which are inherited ; hence the
high coefficient of correlation.
TABLE VII
Paramecium aurelia. Means of tetrads. Correlation of number of fissions, August
7-16 and August 17-26.
o o
o o
o *•>• if~-
10.50
1
11.00
1
12.00
1
13.50
1
14.00
1 1 1
1 2
14.25
1
14.33
1
14.50
1 1
14.67
1
1
14.75
1
15.00
1 121
2 1
15.25
1 1 1
15.33
1 1 1
15.50
1 1 1
1 1
15.67
1 2
1 1
15.75
1 1 1
1
16.00
1 112 1
5 1 2
16.25
1 11 1
1
16.33
1
1
16.50
2 2
1 1 1
16.67
1 1
1 1 1
16.75
1
1
17.00
1
2 2 1
17.25
1
2 1
17.33
1
1 1
17.50
1
1 1
17.67
1 1 1
112111171212 11 53636
164 103 2 5 1 1 2
r = +0.651 ±0.039
1
1
1
1
6
1
1
2
2
1
8
3
3
5
5
4
14
5
2
7
5
2
6
4
3
3
3
99
Thus in a non-con jugant population there is no indication of hered-
itary diversities between the different lines, while among the lines de-
EFFECT OF CONJUGATION OF PARAMECIUM 311
rived from different ex-conjugants, hereditary diversities are clearly
present. A population of ex-conjugant lines consists of diverse bio-
types produced by conjugation from a homogeneous clone.
F. Similarity Between the Lines Derived from the Two Members of
a Pair of Conjugants
Jennings (1913) and Jennings and Lashley (1913) found that the
lines derived from the two members of a pair of conjugants resembled
each other more than do the progeny of ex-conjugants derived from
different pairs. An attempt was made to determine whether this re-
lation holds for the population studied in this investigation. This was
done by correlating the mean number of fissions for twenty days, and
the mean average daily fission rate of all lines which lived for more
than ten days, of the two tetrads derived from each pair of conjugants.
The coefficients of correlation which were obtained were -4- 0.102 ±
0.073 and -- 0.188 ± 0.070. Because of the small number of pairs in-
volved and the large probable error obtained, these coefficients of cor-
relation are of uncertain significance. Another experiment using a
large number of pairs is planned, in order to investigate this matter
further.
SUMMARY AND DISCUSSION
This paper gives an account of an investigation designed to test
critically the question whether conjugation produces inherited variation
within a clone of Pamnieciuui aurclia. An elaborate technique was
devised and carried through, to prevent the occurrence of environmental
diversities among the lines of descent tested : a synthetic culture fluid
was employed ; pure cultures of food organisms used, and the glassware
standardized to the highest possible degree.
Cultivated in this way, ex-conjugant lines of descent were compared
with non-con jugant lines from the same parent clone, with respect to
the rates of fission. The results are :
(1) Conjugation greatly increased the variation in fission rate.
The population composed of lines descended from ex-conjugants
showed a much greater range of variation and a much greater coefficient
of variation than did the population derived from non-conjugants. The
variation was extended by conjugation mainly in the direction of lowered
fission rate. A considerable proportion of the ex-conjugant lines had
a lower daily fission rate than any of the non-conjugant lines. Others
had as high a fission rate as the non-conjugants (see Table I).
(2) The four quadrants derived from the first two divisions of
single ex-conjugants showed when compared only such variation as is
312 DANIEL RAFFEL
found in non-con jugants ; not at all such extreme variations as are
found between lines derived from diverse ex-conjugants. The four
quadrants from a single ex-con jugant are highly correlated in their
fission rates, showing a correlation coefficient of 0.854 dr 0.007. Such
quadrants derived from a single ex- con jugant are thus much more
alike in their fission rates than are lines derived from diverse ex-
conjugants. There is no indication that the first two fissions occurring
after conjugation have any effect in segregating diverse lines, or that
they differ in their effects from any other fissions.
(3) The diverse fission rates of lines or populations derived from
different ex-conjugants are in large measure inherited, while the differ-
ing rates of non-conjugant lines are not inherited.
The work therefore leads to the following conclusions: Conjugation
within a clone of Parameciuni aurelia produces diverse biotypes, having
different inherited fission rates. The fissions of a single ex-conjugant
do not give origin to diverse biotypes ; this is as true of the first two
fissions after conjugation as of later fissions.
LITERATURE CITED
ADOLF, E. F., 1929. The Regulation of Adult Body Size in the Protozoan Colpoda.
Jour. Expcr. Zool., 53: 269.
BELAR, K., 1928. Untersuchung der Protozoen. Methodik iviss. BloL, 1: 735.
CALKINS, G. N., AND GREGORY, L. H., 1913. Variations in the Progeny of a
Single Ex-conjugant of Paramecium caudatnm. Jour. Ex per. Zool., 15:
467.^
JENNINGS, H. S., 1911. Computing Correlation in Cases where Symmetrical
Tables are Commonly Used. Am. Nat., 45: 123.
JENNINGS, H. S., 1913. The Effect of Conjugation in Paramecium. Jour. Exper.
Zool., 14: 279.
JENNINGS, H. S., 1916. Heredity, Variation and the Results of Selection in the
Uniparental Reproduction of Difflugia corona. Genetics, 1: 407.
JENNINGS, H. S., 1929. Genetics of the Protozoa. Bibliographic a Gcnctica,
5: 105.
JENNINGS, H. S., AND LASHLEY, K. S., 1913. Biparental Inheritance and the
Question of Sexuality in Paramecium. Jour. Ex per. Zool., 14: 393.
LUNTZ, A., 1926. Untersuchungen iiber den Generationwechsel der Rotatorien.
Biol Zcntralb., 46: 233.
PARPART, A. K., 1928. The Bacteriological Sterilization of Paramecium. Biol.
Bull.. 55: 113.
PRINGSHEIM, E. G.. 1928. Algenreinkulturen. Eine Liste der Stamme, welche auf
Wunsch abgegeben wurden. Arch. f. Protisk., 63: 255.
A MECHANISM OF INTAKE AND EXPULSION OF COL-
ORED FLUIDS BY THE LATERAL LINE CANALS
AS SEEN EXPERIMENTALLY IN THE
GOLDFISH (CARASSIUS AURATUS)
GEORGE MILTON SMITH
ANATOMICAL LABORATORY, SCHOOL OF MEDICINE, YALE UNIVERSITY
In the course of studies of lateral line canals of the goldfish, it
seemed advisable to observe possible reactions of the canals of the
lateral line organs to absorption of coloring substances held in sus-
pension by water. To accomplish this purpose, goldfishes were im-
mersed in various weak solutions containing lampblack, India ink, ver-
milion and Berlin blue, and allowed to live over periods of time vary-
ing from a week to two months. From time to time the fishes were
examined and it was found that actually small amounts of these col-
oring substances had been taken up by the lateral line canals of the
head or trunk. Such small patches of absorbed pigment occasion-
ally found caught in the lumen of the lateral line canals gave, how-
ever, unsatisfactory evidence of any mechanism of absorption or ex-
pulsion of fluids into the system of canals. Finally, by using more
highly concentrated solutions of some of these same substances, in which
the fishes, temporarily, were allowed to swim, a very striking outline of
the lateral line canal system filled with coloring substance was obtained ;
and there was also offered an opportunity of directly observing the
intake and expulsion of these colored fluids through the pores dis-
tributed along the canal system.
An illustrative experiment is as follows : goldfish, length 5 cm. from
tip of snout to base of tail, whitish color. Solution : India ink 20 cc.,
water 200 cc., temperature 20° C. Preliminary examination of fish
showed normal-looking lateral line canals of head and of body. The
fish was placed in the India ink solution for thirty seconds, rinsed in
water, and changed to a shallow dish of water for examination under
the dissecting microscope. The canal system of each side of the head
was sharply outlined in black, the supra and infraorbital, the hyo-
mandibular and the supra-temporal canals were deeply injected, and
appeared as sharp black lines. The absorbed India ink extended to
about one-fourth of the adjacent region of the lateral line of the. trunk.
After the lapse of over one half minute, there was noted black coloring
313
314 GEORGE MILTON SMITH
matter, stringy as if mixed with mucus, first at one and then another
of the pores of either side of the head. The canals a few minutes
later, began to assume here and there a clearer, grayish appearance.
Bits of coloring matter were wiped away with cotton swabs from the
pores and were followed by fresh extrusion of delicate shreds of
darkly colored mucus. The fish was now allowed to swim in a large
jar of clear water at room temperature. At the end of 10 minutes the
hyomandibular, supra and infraorbital canals were clear of India ink.
At the end of 15 minutes the lateral canals of the trunk had nearly
cleared. At the completion of 30 minutes, only the supra-temporal
canal showed the remains of India ink in the form of a faint gray line.
The supra-temporal canal was cleared of the remaining India ink when
35 minutes had elapsed, so that all canals now contained a clear, limpid,
normal-looking mucus with no evidence of previous staining (Figs.
1-6).
The immediate penetration of colored fluids into the canal system
may be observed under a dissecting microscope by applying drops of
India ink, by means of a finely drawn pipette, over the pores of any
part of the canal system of the head or trunk. There follows a rapid
intermingling of India ink with the mucous contents of the canals
and a consequent spread of India ink along the canals in either direction
from the point of application of ink at the pores of the surface. If
the application of India ink is continued, adjacent communicating
branches of the canals soon become injected with the black coloring-
substance. When the application of India ink is discontinued, expulsion
of the India ink, mixed with mucus, begins and can be seen leaving
the canals at the. pores which furnish communicating passages between
the canals and surface. Elimination of India ink, mingled with mucus,
continues until the canals are entirely cleared and appear normal.
It is essential to employ healthy, active goldfishes for experiments
of this character. Dying fish take up coloring substances in an ir-
regular manner. It was found that in the dead goldfish a penetration
of coloring substances occurred to some extent. This seemed to be
less intense and more irregular and patchy than in the living fish and,
of course, there was not the immediate elimination of coloring sub-
stance by the flow of mucus from the canals. At times no penetration
of the coloring substances occurred in the case of the dead fish, possibly
on account of the lack of mucus in the canals.
Experiments such as these mentioned above were repeated many
times in different ways with evidence of intake and expulsion whenever
coloring substances were brought into contact with the lateral line
canals. This evidence occurred also in the experimentally-blinded fish
and in fishes with nares destroyed by cautery.
INTAKE AND EXPULSION OF COLORED FLUIDS
315
"
D A
5 / ^
4.
5.
6.
FIGS. 1, 2, 3, 4, 5 AND 6. Diagrammatic drawings of lateral line canals of
goldfish as seen from above, illustrating intake and expulsion of a solution of
India ink, 20 cc. ; water, 200 cc. Fig. 1, A. Lateral line canal of trunk; B. C. D,
and E, supra-temporal, hyomandibular, infraorbital and supraorbital canals, re-
spectively, previous to intake. Fig. 2. Filling of canals, after 30 seconds of im-
mersion in India ink solution indicated by black dots in canals. Fig. 3. Clearing of
supraorbital, infraorbital, and hyomandibular canals 10 minutes after fish was
placed in clear water. Figs. 4 and 5 show progress of clearing after 15 and
30 minutes respectively. Fig. 6 shows canal system entirely cleared after 35
minutes.
21
316 GEORGE MILTON SMITH
India ink and Berlin blue acted as coloring agents most favorable
for the experiments. Vermilion in suspension in water was useful for
the studies over longer periods of time when certain symmetrical dis-
tributions of absorbed coloring matter occurred. Lampblack was not
found satisfactory. It rarely gained entrance into the canals, possibly
because the conglomerate and adherent particles formed were too large
to permit of entrance into the pores of the canal system.
The complete elimination of absorbed coloring substances from
the lateral canals varied in different animals over a considerable range
of time. Such a difference in the elimination of India ink from the
lateral canals was noted in the following experiments, carried on simul-
taneously with two fishes of different size:
Two goldfishes, A and B, 4l/2 cm. and 7 cm. respectively in length ;
fluid for immersion : India ink, 100 cc. ; water, 500 cc. ; temperature,
20° C.
1 :27 P.M. Both fishes placed in India ink solution.
1 :30 P.M. Both removed and examined. In both, all branches of
lateral line system of head were colored black. The lateral lines
of the 'trunk were black in the proximal or cephalic third in
both fishes.
1 :31 P.M. Placed in tanks of fresh water. Both fishes, from now on,
examined under the binocular microscope every 10 minutes.
1 :41 P.M. In both fishes the lateral canals of the body were cleared
of black color, and in both the nasal parts of the supraorbital
canals and the submaxillary parts of the hyomandibular canals
were clear.
2:11 P.M. Clearing of canals had proceeded to a point where A
showed only a moderate amount of India ink in the supra-
temporal canal ; and B showed a very slight staining of the supra-
temporal, both infraorbitals and the posterior part of the hyo-
mandibular on the right and left sides.
2:31 P.M. Fish A showed only a slight amount of staining in the
occipital canal, while fish B had all canals perfectly cleared and
translucent.
2:51 P.M. Fish A had canals now entirely cleared of India ink, hav-
ing taken twenty minutes longer than fish B.
Apparently, with a change in environment, the lateral canals of the
goldfish were placed in operation as forms of testing apparatus. If the
fish was changed from one colored solution to another of different color,
directly, or with an opportunity of cleaning the canals in fresh water,
the lateral line canals took up the colored fluid of the new environment.
INTAKE AND EXPULSION OF COLORED FLUIDS 317
An experiment illustrating a change involving intake and expulsion
of three different colored solutions is the following-:
o
Goldfish, 514 cm. in length ; markings : whitish with slight black
pigment above eyes.
6:41 P.M. Placed in a dish containing vermilion, 20 grams; water,
500 cc.
6:54 P.M. Left supraorbital canal was brilliantly injected with ver-
milion and there was a small amount of vermilion in the right
hyomandibular canal near the angle of the jaw.
7 :00 P.M. Same distribution of vermilion as at previous reading.
Fish changed to clear water.
7:15 P.M. Left supraorbital canal clear of vermilion. Minute plug
of vermilion in right submaxillary region.
7:15 P.M. Fish placed in a dish containing Berlin blue. 5 grams;
water, 500 cc.
7:18 P.M. After 3 minutes taken out of Berlin blue solution. Both
infraorbital canal and submaxillary parts of hyomandibular canal
showed as bright blue.
7 :19 P.M. Placed in fresh water.
7 :29 P.M. Canals cleared of all traces of Berlin blue while in fresh
water for 10 minutes.
7 :30 P.M. Placed in a solution containing India ink, 100 cc. ; water,
500 cc. for one minute.
7:31 P.M. Removed from India ink solution (1 minute). All lateral
line canals of head and side were black.
7:31 P.M. Fish placed in clear water.
8:40 P.M. Canals of head and trunk now appeared completely cleared
of India ink, the lateral line canals having absorbed and ex-
pelled three different colored solutions in the space of two hours.
Similar results were noted in another fish (5 cm. in length; whitish
silvery color) which had been placed, six weeks previously, in a solu-
tion of Berlin blue, 5 grams ; water. 2500 cc. With all canals deeply
stained blue this fish was changed directly to a solution of India ink
20 cc. ; water, 200 cc. After one minute in India ink the nasal parts
of the supraorbital canals and the anterior regions of the hyomandibular
canals on both sides were black and readily distinguishable from the
adjacent blue. Returned to the same Berlin blue solution in which it
had been swimming for six weeks, the India ink in the above-mentioned
canals could no longer be recognized at the expiration of 20 minutes ;
all the canals of the head and trunk were again stained a bright blue.
When goldfishes were kept in an environment of colored fluid for
318 GEORGE MILTON SMITH
longer periods of time, such as one week to two months, the absorbed
coloring matter in the lateral canal system varied from a condition of
complete filling of the canals to one where only certain branches were
incompletely filled. Occasionally no coloring appeared in any of the
canals. In other words, the mucous secretion of the canals may clear
away previously absorbed coloring substance and keep the canals partly
or completely clear in spite of the fact that the fish is living in a
colored solution.
In the following experiment a goldfish was allowed to remain for
one month in a solution of Berlin blue. When placed in fresh water
at the expiration of that time, clearing of the canal system seemed
unusually long (3 hours and 20 minutes.) Tested immediately after-
wards for elimination of India ink, this substance was also slowly ex-
pelled (3 hours and 8 minutes). The time of intake did not seem to
be affected.
Experiment: 11/30/29. Goldfish, whitish silver in color; 4 cm. in
length was placed in a jar containing Berlin blue, 5 grams; water,
2500 cc. The lateral line canals of the head and body were stained
a vivid blue in 30 seconds. Examined from time to time during the
first three weeks, the fish showed variations in distribution of blue in
different branches. Examined daily for the last seven days of a thirty-
day period, all lateral line canals of head and trunk were intensely
stained with blue.
12/30/29. After a month immersed in Berlin blue solution, with
all the canals deeply stained blue, the fish was placed in clear water.
In 3 hours and 20 minutes, all the canals of the head and body were
clear of blue color. Changing the environment now to one of India
ink (20 cc. ; water, 200 cc., the canals became quickly and completely
stained black in 30 seconds. Returned to clear water, the canals were
freed of India ink in 3 hours and 8 minutes. Returned finally to the
original Berlin blue solution where the fish had lived previous to the
present experiment for a period of one month, the canals took up an
intense blue stain in 30 seconds.
In goldfishes kept in a solution containing vermilion, the intake
of red-pigmented particles was more leisurely performed, appearing
in small patches in the course of the first twenty- four hours. Two
fishes which were examined from day to day, during a period of two
months, showed various branches irregularly filled with vermilion
mixed with mucus contained in the canals. Not infrequently the ab-
sorbed vermilion was bilateral in distribution and symmetrically ar-
ranged in the different canals of the head and trunk. This symmetrical
INTAKE AND EXPULSION OF COLORED FLUIDS
319
distribution of vermilion in the lateral canal system of a fish kept in
a solution of vermilion, 10 grams ; water, 3000 cc. for two months
is indicated in the accompanying figures (7-11) based on daily ob-
servation for 5 days when a symmetrical pattern of intake happened
to be present.
DISCUSSION AND SUMMARY
It is not essential for the present purpose to state in detail the his-
torical data of the lateral line canals and organs. It may not be amiss,
I
8.
a
10.
FIGS. 7, 8, 9, 10 AND 11. Diagrammatic representation of canal system of
goldfish kept in a solution of vermilion, 10 grains ; water, 3000 cc., for a period
of two months. Absorbed pigment, although usually irregularly distributed in
lateral line canals, appeared symmetrically arranged in this instance during a
period of five consecutive days. The canals dotted in black contained absorbed
vermilion.
320 GEORGE MILTON SMITH
however, to recall that the presence of lateral canals in fishes, as cited
by Fuchs (1895) was known and described by at least three anatomists
of the seventeenth century, — Nicolas Stenonis (1664), Lorenzini
(1678), Rivinus (1687).
The lateral line canals were generally regarded as mucous canals
or Schleimkanale until the time of Leydig (1850-51), whose careful
histological studies of the contained end organs led him to the con-
clusion that the lateral organs were sensory organs. Since that time
a vast amount of data has accumulated as the result of the work of
many investigators, and reviews on the subject appear in connection
with the important works of Ayers (1892), Fuchs (1895), Allis (1904)
and Johnson (1917). From the functional standpoint, Lee (1898)
has stated that there has been no concensus of opinion as to the exact
function or mode of action of the lateral line sensory organs. His own
conclusions were that the lateral lines have a sensory function which
is closely connected with the motor organs and is analagous to the
function of the ear, and hence they may 'be regarded as organs of
equilibrium. Schulze (1870) had suggested earlier that this sense
perception was possibly an appreciation of mass movement of the
water or of movement of the body through the water; whereas Fuchs
(1895), from carefully conducted researches, was led to the conclusion
that the lateral line sensory organs gave sensory impressions of changes
in hydrostatic pressure. Hofer ( 1908) believes from his studies that
the lateral line organs are stimulated alone by weak currents of water.
Parker (1918), in the course of researches conducted on the auditory
apparatus, finds that the lateral lines respond to water vibrations which
are slower than those which affect the auditory mechanism.
Recent views of the lateral line sense organs place their function,
according to Herrick (1927) intermediate between tactile and auditory
organs. Their nerve supply, he states, is from the lateralis roots of the
seventh and tenth cranial nerves. He points out the intimate associa-
tion with the eighth nerve supplying the internal ear, and the termination
of these nerves in the acoustico-lateral area of the medulla. According
to Herrick (1927), the structure of the end organs of the lateral line
system and those of the human ear are of the same type.
From the experiments carried out on the goldfish cited in the present
communication, it would seem that there is in the lateral line canals
of the goldfish, demonstrable by the experimental use of colored fluid,
a mechanism of intake and expulsion of fluids. The intake is rapid
and seems to vary from a few seconds to a few minutes. The elim-
ination from the canals is slower and more deliberate, taking from fif-
teen minutes to one hour or more. Colored fluids in passing through
INTAKE AND EXPULSION OF COLORED FLUIDS 321
the pores of the lateral canals mix rapidly with mucus existing in the
canals, the mucus acting possibly as a diluent. The discharge of col-
oring substance from the canals is effected by an outward discharge
of mucus through the pores of the canals. The mixture of colored
material and mucus appears in the form of delicate colored shreds or
plugs as they are expelled. These colored mucous shreds quickly
wash away in surrounding water.
Therefore, experiments, such as these described, where lateral line
canals take up and expel different coloring substances in suspension
when the fish is changed to solutions of different color, suggest that
the lateral canals of the goldfish function, in part, at least, as sensory
testing mechanisms for chemical or physical changes in environment ;
and that the ready flow of mucus from the canals furnishes an efficient
means of eliminating fluids that have been tested by the end organs
of the canal system.
LITERATURE CITED
ALLIS, E. P., 1904. The Latero-Sensory Canals and Related Bones in Fishes.
Intcrnat. Monat. Anat. u. Phys., 21: 401.
AYERS, H., 1892. Vertebrate Cephalogenesis. II. A Contribution to the Morph-
ology of the Vertebrate Ear, with a Reconsideration of its Functions.
Jour. Morph., 6: 1.
FUCHS, S., 1895. Ueber die Function der unter der Haut liegenden Canalsysteme
bei den Selachiern. Plugcr's Arch., 59: 454.
HERRICK, C. J., 1927. An Introduction to Neurology. (See pages 124 and 233.)
HOFER, BRUNO, 1908. Studien iiber die Hautsinnesorgane der Fische. Berichte
aus der Kgl. Bayenschen Biologischen Versuchsstation in Miinchen,
Vol. 1, p. 115.
JOHNSON, S. E., 1917. Structure and Development of the Sense Organs of the
Lateral Canal System of Selachians (Mustelus canis and Squalus acan-
thias). Jour. Com par. New., 28: 1.
LEE, F. S., 1898. The Functions of the Ear and the Lateral Line in -Fishes.
Am. Jour. Physiol., 1: 128.
LEYDIG, F., 1850. Ueber die Schleimkanale der Knochenfische. Arch. f. Anat.
Physiol. u. Wis. Medicin.., p. 171.
LEYDIG, F., 1851. Ueber die Nervenknopfe in den Schleimkanalen von Lepi-
doleprus, Umbrina und Corvina. Arch. f. Anat. Physiol. u. Wis. Medicin.
Mcd., p. 235.
LORENZINI, S., 1678. Observazioni intorno alle Torpedini fatte da Stephano
Lorenzini Fiorentioni e dedicate al serenissimo Ferdinando III Principe
di Toscanio Firenze. Quoted by Fuchs, S.
PARKER, G. H., 1904. The Function of the Lateral Line Organs in Fishes.
Bull, of Bur. Fisheries, 24: 183.
RIVINUS, 1687. Observatio anatomic circa poros in piscium cute notandos. acta
erudit. Lipsiae. (Quoted by Fuchs, S.)
SCHULZE, F. E., 1870. Ueber die Sinnesorgane der Seitenlinie bei Fischen und
Amphibien. Arch. f. mikr. anat., 6: 62.
STENONIS, NICHOLAS, 1664. De musculis et glandulis observationem specimen
cum epistolis duabus anatomicis. Amstelodami. p. 54. (Quoted by Fuchs,
S.)
RAT VAS DEFERENS CYTOLOGY AS A TESTIS HORMONE
INDICATOR AND THE PREVENTION OF CASTRATION
CHANGES BY TESTIS EXTRACT INJECTIONS1
SUP VATNA
HULL ZOOLOGICAL LABORATORY, THE UNIVERSITY OF CHICAGO
I. INTRODUCTION
The cytological and histological changes in the prostate glands and
the seminal vesicles of the rat following castration have been worked
out by Moore, Price and Gallagher (1930) and Moore, Hughes and
Gallagher (1930) respectively, and it was found that there are some
dependable criteria, by which one can tell whether the sex hormone is
present or absent. It is desirable to know what other organs may be
affected and if the changes will be consistent enough to serve as a sex
hormone indicator. This paper will deal with the study of the vas
deferens of the white rat in its normal state and after different periods
of castration, and the effects of subcutaneous injections of extracts
from the testicle upon the castrate condition.
This study was suggested to me by Prof. Carl R. Moore as another
unit in the program of sex studies now being carried on in the De-
partments of Zoology and of Physiological Chemistry and Pharmacol-
ogy. I am grateful to him for advice and assistance given to me
throughout the course of the work. I will show in this paper that
the structure of the vas deferens is controlled by the internal secretion
of the testes and furthermore that this control can be maintained in
the castrated animals by means of subcutaneous injections of the ex-
tracts of bull testes. A preliminary account of the findings has already
appeared (Moore, Vatna and Gallagher, 1930). The numbered prep-
arations of bull testis extract were supplied in strengths unknown to
us until after assay. They were prepared by Mr. T. F. Gallagher under
the direction of Professor F. C. Koch in the Department of Physi-
ological Chemistry and Pharmacology, to both of whom is expressed
a debt of gratitude. The earlier papers from these laboratories (McGee ;
McGee, Juhn and Domm ; Moore and McGee ; Moore and Gallagher ;
Moore, Price, Hughes, Gallagher ; Gallagher and Koch ; Moore, Gal-
1 This investigation has been aided by a grant from the committee on research
in problems of sex of the National Research Council ; grant administered by
Prof. F. R. Lillie.
322
RAT VAS DEFERENS CYTOLOGY 323
laghcr and Koch) have presented the biological test methods previously
employed, and the methods of hormone extraction, and the reader is
referred to them for details.
Other laboratories have recently reported positive results from at-
tempted hormone extraction from the testis of various mammals and
the urine of men (Martins and Rocha e Silva. 1929; Loewe and Voss,
1929: Funk, Harrow and Lejwa, 1929, 1930).
II. MATERIAL AND METHOD
White rats were used in this experiment. The stud)' involves the
examination of the vas deferens from about thirty normal animals of
varying ages, thirty-five castrated, and fifty castrated injected animals.
Castration was performed through a mid- ventral abdominal incision.
In some cases the body of the epididymis was cut through, leaving the
tail of the epididymis attached to the vas deferens. With others the
entire epididymis was removed with the testis.
The proximal, or urethral end of the vas deferens presents a struc-
ture that shows more marked effects from castration than does the
distal, or epididymal end, hence the proximal two-thirds of this re-
productive tube has usually been the part that has received the greatest
attention.
The tissues were fixed for histological study in Benin's fluid and
Zenker formol mixture. Bouin's fluid was found to be the better of
the two, and therefore was used throughout the work. The sections
were cut at 4p. thickness and were stained in such mixtures as Delafield's
haematoxylin with eosin as a counter stain, or iron haematoxylin, or
Mallory's triple stain.
Mann's osmo-sublimate fixative was also used to demonstrate the
Golgi apparatus. The technique employed was that of Ludford's (1925,
1926) modification of the Mann-Kopsch method. Briefly, the vas was
cut into small pieces of about three mm. in length and fixed in a freshly
prepared mixture of an equal volume of one per cent osmic acid in
distilled water and a saturated solution of mercuric chloride in normal
salt solution, for about twenty hours. The tissue was then washed in
two changes of distilled water for about thirty minutes, and placed in
two per cent osmic acid solution in quantities sufficient to cover it, after
which it was placed in the dark at room temperature for about seven
days. At this time the osmic acid solution was discarded, the tissue
washed once in distilled water, and transferred in distilled water, to
an oven at about 35° C. for four days. The tissue was next washed
in running tap water over night, and then put through the ordinary his-
tological procedures, such as dehydration, clearing, imbedding, and sec-
324 SUP VATNA
tioning. The sections were bleached in a solution of hydrogen peroxide
in 95 per cent alcohol.
TIL THE STRUCTURE OF THE NORMAL VAS DEFERENS
The vas deferens of the rat is more or less spindle-shaped in ex-
ternal form. Between the urethral end and the middle of the vas, is
an elongated swollen region, from the distal end of which the tube
tapers toward the epididymis and from the proximal end toward the
urethra.
In the normal, the vas is always full of spermatozoa. This can be
detected with the naked eye because of the milky white streak which
is present in the middle throughout its length. The swollen region is
especially distended by spermatozoa.
The normal vas deferens has been studied both from animals sacri-
ficed for the purpose and from animals after unilateral castration of
varying periods. The latter type has been used in order to see whether
the spermatozoan content in any way modified the structure of the
epithelial lining. In the mammals there is no question now as to the
ability of one testis to keep up the normal state of the accessory repro-
ductive organs. The vas deferens from the latter group is preferred
for the sake of comparison, although there is no essential difference
between the normal histology of the vas from the two sources mentioned,
except when the spermatozoa have collected in an unusually large quan-
tity. Then the height of the epithelium may be slightly lowered due
to the distention of the lumen in general, but the arrangement of the
nuclei of the epithelium is not at all disturbed. The cilia may be some-
what distorted from normal shape. However, in all cases examined,
their appearance is decidedly not that of a castrate type.
The vas deferens of most mammals, as generally known, is not cil-
iated ; some species, however, are well furnished with cilia. The mouse
and the rat belong to the latter group. The word " cilia " in connection
with the vas deferens, Benoit ( 1926) thought should be " stereocils "
or " poils," on account of their non-vibratile nature. The short term
" cilia " will be used in this paper to mean " cilia-like " structures.
The histology of the vas deferens is a very simple one. The tube
consists of three easily distinguishable layers, the outside muscular layer,
the mucous, and the epithelial or inner layer. The outer coat covered
by peritoneum consists of longitudinal and circular muscle layers, and
makes up approximately four-fifths of the thickness of the walls of the
tube. Internal to the muscular layer is the so-called mucous layer com-
posed primarily of connective tissue-like cells and blood vessels. This
layer is sensitive to operative manipulation which is in no way related
RAT VAS DEFERENS CYTOLOGY 325
to hormone control. From an unoperated animal, it is narrow and
the cells are more or less tightly packed together, whereas the vas from
a unilaterally castrated animal has a much broader mucous layer and
the cells are rather scattered. The internal epithelial layer bordering
the small lumen is definitely separated from the mucous layer by a very
thin cord of about one or two cells in thickness. This cord of cells
forms the outline of the basal part of the epithelium, and will be re-
ferred to in this paper as the " basement-cell layer."
The epithelial layer is composed of tall columnar cells, resting upon
a distinct basement-cell layer, and the free end of the cell is covered
by a heavy mass of cilia-like structures projecting into the lumen. The
nuclei of the cells are generally oval in shape and variable in chromatin
constituents. They vary slightly in position in the vasa of different
animals, but in any one animal they occupy the same relative position
in all of the cells. Thus the nuclei are seen to form a definite layer
paralleling the basement-cell layer (see Figs. 1 and 5). At many places
in a section one observes a few nuclei that seem to be differentiating
from the basement cells, with others present above the nuclear layer
apparently migrating toward the lumen. In the lumen itself, one often
finds a group of epithelial cells in various stages of degeneration.
These findings suggest a series of changes in the normal vas deferens,
wherein cells are added to the epithelium from the basement-cell layer,
and at the same time others having functioned actively for a certain
time, are thrown off into the lumen, where degeneration occurs.
Between the nucleus and the ciliated border of each cell, the cyto-
plasm is of a condensed homogeneous granular character, whereas that
basal to the nucleus is much less dense and is fibrillar in character. The
difference between the distal and proximal ends of the epithelial cell is
very marked. The finely granulated material in the distal portion is
believed to be made up of secretory products (Myers-\Yard, 1897:
Benoit, 1920). Benoit (1926) by the use of a special technique found
certain definite lipoid bodies which he called " parasomes " in the epi-
thelial cells of the vas deferens of the mouse and rat. These " para-
somes " were believed to be the product of protoplasmic differentiation.
They first appear when the animals are about fifteen days old, and
in the adult they are found scattered throughout the cell. He suggests
that the " parasomes " normally undergo some sort of dissolution and
contribute to the formation of a liquid product of secretion. The in-
vestigation reported here has not involved a study of these " para-
somes."
There is always a small amount of secretion present in the lumen
of the vas in normal animals. This secretion forms a finely granular
homogeneous mass and stains with eosin.
326 SUP VATNA
The vas deferens prepared by the Mann-Kopsch technique reveals
definite, well-formed Golgi bodies in the epithelial cells. The Golgi
bodies are located approximately midway between the nucleus and the
lumen end of the cell and are of the reticular type. Their charac-
teristic shape is shown most clearly in slightly under-impregnated sec-
tions, in which case the threads making up the reticulum will be black-
ened only on the outside, thus giving a double-lined appearance. The
size of the Golgi bodies in the normal is about that of the nucleus,
though they may be somewhat larger in some cases.
IV. CHANGES IN THE VAS DEFERENS FOLLOWING CASTRATION
In order to determine whether the vas deferens was affected by
castration. I have studied preparations from animals in a closely graded
series from three days up to seven months after testis removal.
The tissue prepared from animals sacrificed at 3, 5, 6, 7, and 9
days after castration is essentially normal. The gross size, relative
thickness of the layers, the character of the epithelium and the condition
of the cilia do not differ markedly from the normals.
The Golgi bodies, however, begin to show some differences for
they become smaller in comparison to the size of the nuclei, and. more
striking, the reticulum breaks up to form a group of crooked rods or
coarse granules.
At 10 and 15 days after castration, the gross size as well as the
histological structure of the vas of some animals shows a decided
change, characteristic of a longer time castrate. The vas deferens be-
comes smaller and the epithelium may be typical of a 20-day castrate.
However, other animals castrated for this period may retain essentially
the normal condition in the vas.
The Golgi bodies after ten to fifteen days of castration have under-
gone a marked fragmentation. The portion of the cells where the Golgi
bodies are normally found will be seen to be full of scattered osmiophilic
granules. These granules may clump together, but the structure does
not suggest a normal Golgi apparatus.
Twenty clays after testis removal the vas deferens characteristically
shows the effects of castration. This period is of special importance
inasmuch as many of the effects of testis extract injection have been
studied for this period of time after operation.
The size of the vas is now noticeably smaller, due to the degenera-
tion of the muscular layer, which normally makes up almost the whole
thickness of the tube. The morphological structure of the mucous
layer has no constant bearing upon castration.
The most apparent changes occur in the epithelial layer. The ab-
RAT VAS DEFERKNS CYTOLOGY 327
solute height of the epithelium from the basement-cell layer to the
luminal border is slightly reduced. The cell walls are no longer clearly
visible, and the nuclei instead of forming a well-defined layer paralleling
the basement-cell layer are now more closely aggregated in an irregular
distribution giving the appearance of pseudostratification. The epi-
thelium now appears as a syncytium.
The nuclei show little, if any, reduction in size, but because of the
reduction in the amount of cytoplasm in the cells, they now lie close
to the basement-cell layer. The cytoplasm between the nucleus and
the lumen end of the cell is likewise greatly reduced.
The ciliary border of the epithelium also differs greatly from the
normal. The cilia are in most cases completely absent from the vas
deferens of 20-day castrate animals (see Fig. 6). In a few others
they may still be present but greatly reduced both in number and length
and present often an interwoven, irregularly twisted condition.
The secretion found in the lumen does not seem to be changed in
quality, but is much reduced in quantity following castration. However,
even after long-time castration, there is always a small amount of secre-
tion present. Benoit (1926) reports from his study on mice and rats
that the parasomes, the bodies responsible for the formation of secretory
products, disappear completely after thirty days of castration. From
our own study on the rat, we have been unable to confirm the statement
regarding the absolute cessation of secretion.
The Golgi bodies too are decidedly different from the normal at
this period of castration. Their gross size, relative to the si/e of the
nucleus, is very much reduced. The former reticular arrangement has
usually changed to a granular one, and these granules sometimes form
an irregular cap over the end of the nucleus.
The typical condition of the twenty-day castrate animal given above
holds for the majority of animals castrated for this period, but oc-
casionally slightly different conditions may be encountered. A few
apparently more resistant animals have suffered less from castration
than others and appear almost normal, except for a lower epithelium
and a slight crowding and displacement of the nuclei.
The typical degenerate condition of the vas deferens at twenty days
after testis removal represents, with some exceptions, essentially the
condition that is to be found in later castrates. The series which I
have studied includes animals castrated for periods of 21, 25. 30, 33,
40, 50, 60, 80, 110, 150, and 210 clays. As the age of castration in-
creases there is little, if any, increase in the amount of involution.
Fig. 7 shows the condition of the vas deferens in an animal castrated
for two hundred and ten days and in comparison with the normal (see
328 SUP VATNA
Fig. 5), clearly shows the absence of a ciliary border of the epithelium,
the lowered height of this layer, the apparent stratification of the
nuclei, the involuted mucous layer and the reduced muscular layer.
Fig. 2 in comparison with Fig. 1 demonstrates clearly the difference
between a five-month castrated vas and the normal.
It is apparent, therefore, that castration leads to a marked degenera-
tion of the vas deferens. Since this influence is to be attributed to
the endocrine influence of the testis rather than to the gametogenetic
influence, we have in this degeneration a means of testing the effective-
ness of preparations of testicular extracts. If testis removal is fol-
lowed by the injection of the testis extracts and the vas deferens re-
mains in a normal condition, it will be apparent that the extracts ex-
ercise an influence similar to that of the internal secretion of the testis.
My observations on this point are described in the following section.
V. THE EFFECTS OF TESTIS EXTRACT INJECTIONS
In the preceding section, definite changes have been described for
the various parts of the vas deferens. These are : Decrease in gross
size, involution of the muscular layer, slight lowering of the epithelium,
the syncytial character of the cells, pseudostratified appearance of
the nuclei, loss of the cilia, and reduction in size of the Golgi bodies,
with accompanying fragmentation.
Early work from these laboratories supplies proof that the active
principle of the internal secretion of the testes is contained in suitably
prepared lipoid extracts of the glands of the bull. In the course of
this study, many samples of the extracts have been used for injection
on over fifty castrated males. Some of these were less potent than
others, depending on the preparation methods and the dilution of the
samples. The results, therefore, are of a wide range. The typical
positive cases to be described were chosen from animals having re-
ceived appropriate strength of the hormone solutions.
Since twenty days was found to be the period at which the degen-
erative changes of the epithelium reach their height, it was selected as
minimal length of time for testing the hormone extracts. Animals
have been injected daily immediately after castration in order to see
whether the effects of testis removal could be indefinitely postponed.
In addition to this procedure, other animals have been castrated and
permitted to develop the castration condition with subsequent injection
to test the capability of the extracts to restore the degenerate to a normal
condition. This latter procedure has been followed in the case of
animals castrated as adults as well as those castrated before puberty.
RAT VAS DEFERENS CYTOLOGY
329
p
0
Cross-sections of rat vas deferens. Photomicrographs of Bouin-haematoxylin
preparations. About 50 X before reduction. (All photomicrographs were made
by Mr. Kenji Toda.)
1. From a normal animal.
2. From a five month castrate.
3. From a 110-day prepubertal castrate.
4. From a 110-day prepubertal castrate, given forty daily injections of bull
testis extract.
330 SUP VATNA
1. The Maintenance Experiment
In this series, the animals were given twenty daily injections, or
more in some cases, immediately after castration to maintain the normal
condition.
•
The histological study of such injected castrates shows a normal
structure of the vas. The epithelium is simple columnar and abundantly
supplied with cilia, and the nuclei have the simple regular arrangement,
typical of the normal. The Golgi bodies are approximately normal.
2. The Repair Experiment
a. Prepubertal castrates
Two series of prepubertally castrated animals have been utilised
for injection. The first group of four animals was castrated at four-
teen days after birth and the second group of five animals was cas-
trated at forty days of age. The second one is more instructive, hence
it will be described in detail as to the procedures. Five animals of
the same litter were castrated at forty days after birth, and at one
hundred days after castration, four animals were injected with the testis
extracts No. 8922, — one-half cc. being injected daily. When the in-
jections had been given for ten days, one of the four injected animals
was killed, and at the same time the uninjected control was also killed.
At twenty days after the injections, one of the three was killed. The
next one was killed after having received thirty daily injections, while
the last one was killed at forty days.
The results of the study of the experimental series are as follows :
The uninjected control showed every sign of a castrated condition
(see Fig. 3), with the typical loss in gross size, changes in nuclear ar-
rangement, lowering of the epithelium, etc.
The vas deferens of the 10-day injected animal resembles the castrate
type except that it shows an increase in the height of the epithelium
with a partial disappearance of the pseudostratified effect. The secre-
tion in the lumen and in the distal ends of the epithelial cells is greater
in amount.
In the 20-day injected animal the vas is nearer normal in that it
shows a strikingly high epithelium, with a fair amount of cilia.
The 30-day vas is indistinguishable from that of a normal, as far
as the structure of the epithelium is concerned. The size of the vas
as a whole is considerably larger than its castrate control but not as
large as the normal.
The vas deferens from the 40-day injected prepubertal castrate is
RAT VAS DEFERENS CYTOLOGY
331
normal both in structure and size. The diameter of the entire vas
is now double that of the control (Fig. 4).
This study shows that the prepubertal castrated vas deferens re-
sponds definitely to the introduced testis extract as do the adult castrates
and returns to the normal condition in forty days despite its undeveloped
state for a period of about one hundred and ten days.
Cross-sections of rat vas deferens. Photomicrographs of Bouin-haematoxylin
preparations. About 650 X before reduction.
5. Portion of Fig. 1. (Normal animal.)
6. From a 20-day castrate.
7. From animal No. 96 — tissue removed seven months after castration.
8. From same animal (No. 96), \\hich hadi received thirty daily injections
of bull testis extract after the removal of the tissue shown in Fig. 7.
b. Adult Castrates
A number of adult animals were castrated and allowed to remain
for various periods of time before injections were begun. The intro-
duction of testis extracts has always served to return the vas deferens
to the normal condition provided the concentration of the lipoid ex-
tract was sufficiently great.
22
332 SUP VATNA
The results of injecting the extract into long time castrates will he
illustrated by reference to one animal (No. 96). This animal was cas-
trated and seven months later was operated upon for removal of one
vas deferens to serve as the control, and its condition is shown in Fig.
7. The animal was then subjected to testis extract injection daily for
a period of thirty days; one-half cc. was injected subcutaneously each
day. It was killed and the opposite vas deferens removed to show the
effects of the injection. A cross section of the vas after injection is
shown in Fig. 8, and should be compared with its mate removed before
injections were begun (in Fig. 7). It can be seen clearly that whereas
the seven month castrated vas deferens is in a highly degenerate state,
its partner has been returned to the normal condition by means of the
injections. A second animal treated similarly, but injected for a period
of only twenty days, showed that the vas deferens had returned to an
almost normal condition within this period. When castration has been
of shorter duration, injections have been followed by similar return to
the normal condition.
VI. DISCUSSION
In this study we have demonstrated that the vas deferens is also
under the control of the sex hormone for its normal maintenance, as
was shown to be the case for the prostates and the seminal vesicles by
Moore, Price and Gallagher (1930) and Moore, Hughes and Gallagher
(1930) respectively. If the hormone-producing glands — the testes—
are removed, certain definite degenerative changes set in, and these
changes are maximal by about twenty days after testis removal.
The vas reacts more slowly to castration than do the seminal vesicles
and prostates of the rats and therefore has not provided as delicate a
method for hormone assay, nor one as easily read as the light area of
the prostates or the secretion granules of the seminal vesicles. Al-
though the changes following castration do not appear as rapidly in the
vas, they are as definite as those that appear in the other accessory re-
productive glands that have been studied. The vas responds positively
to potent injections of testis extract, therefore it provides a supple-
mentary test for the presence of the male hormone.
In other sections of this paper, data have been presented showing
that by injections (1) vasa of castrated animals have been maintained
at the normal level, (2) vasa that had been allowed to regress for seven
months after castration have been built up to normal, and (3) vasa of
prepubertally castrated animals have been allowed to regress for one
hundred and ten days and have been built up to a normal functioning-
state in forty days.
RAT VAS DEFERENS CYTOLOGY
One experiment was described in detail in which a rat was castrated
and after seven months one vas was removed and the other remained
to lie removed after thirty days of injections. The former was a typical
castrate, and the latter showed a condition normal in every respect.
From these data, there can be no doubt that the active principle of
the testis has been supplied by testis extract injections.
With varying potencies of hormone, the results of injections varied
from negative effects to complete replacements of the vas to the normal
state. The epithelium itself is more sensitive and responds more readily
to hormone injection than does the muscular layer and consequently
the vas may return to an approximately normal condition while the.
gross size is below that of the normal. This same condition obtained
in the prostate and the seminal vesicles.
Since, by testis extract injection, the vas can be maintained in a
normal state as is proved by histological and cytological study, it pro-
vides us with another male hormone indicator method to add to those
already developed — the spermatozoon motility test, the electric ejacula-
tion test, the seminal vesicle test, the prostate cytology test, and the
capon comb growth test.
VII. SUMMARY AND CONCLUSIONS
1. The vas deferens can be used as a male hormone indicator because
it is under the control of the internal secretion of the testis.
2. After castration, definite regressive changes take place within
twenty days in all animals.
3. These changes involve :
a. Reduction in gross size through regression of the muscular layer
of the vas.
b. Diminution of the amount of secretion in the lumen.
c. Reduction in epithelial height.
d. Loss of the cilia covering the epithelium.
c. Crowding together of the cells and obliteration of the cell walls.
/. Stratification of the nuclei.
g. Great reduction in the amount of cytoplasm in the cells.
//. Changes in the Golgi bodies involving loss in gross size and frag-
mentation of the Golgi material into rods or granules instead of the
typical reticulum of the normal.
4. All these changes can be prevented from developing in the cas-
trated animal by daily injections of suitably potent male hormone pre-
pared from the lipoid fraction of fresh bull testes and dissolved in
olive oil.
• "N. /
I
(u-i L I * • * i
/'
334 SUP VATNA
5. If the changes have been allowed to develop, the vas can be
built up to normal by daily injections of testis extracts.
6. In animals castrated before puberty and allowed to regress for
one hundred and ten days the vas can be built up to a normal func-
tioning state by injections ; a process which involves bringing the un-
differentiated duct to a normal adult state.
7. Injections of pure olive oil fail to prevent castration changes,
therefore the potent factor lies in the hormone itself.
LITERATURE CITED
BENOIT, J., 1920. Sur 1'existence de phenomenes secretaires dans le canal de-
ferent. Compt. rend. Soc. de BioL, 83: 1640.
BENOIT, J., 1926. Recherches anatomiques, cytologiques et histophysiologiques
sur les voies excretrices du testicule, chez les mammiferes. Arch, d'anat..
d'liist. ct d'cmbryol., 5: 176.
FUNK, C, AND HARROW, B., 1929. The Male Hormone. Proc. Soc. Expcr.
Biol. and Mcd., 26: 569.
FUNK, C. B., HARROW, B., AND LEJWA, A., 1929. The Male Hormone II. Proc.
Soc. Exper. Biol. and Mcd.. 26: 569.
FUNK, C, HARROW, B., AND LEJWA, A., 1930. The Male Hormone. Am. Jour.
Physio!., 92: 440.
GALLAGHER, T. F., AND KOCH, F. C., 1929. The Testicular Hormone. Jour.
Biol. Chem., 84: 495.
LOEWE, S., AND Voss, H. E., 1929. Gewinnung, Eigenschaften und Testierung eines
miinnlichen Sexualhormons. Sits. Akad. Wiss. U'icn. Math. Naturw.
KL. Oct. 24, 1929.
LUDFORD, R. J., 1925. Some Modifications of the Osmic Acid Methods in Cyto-
logical Technique. Jour. Roy. Mic. Soc., Part 1, p. 31.
LUDFORD, R. J., 1926. Further Modifications of the Osmic Acid Methods in
Cytological Technique. Jour. Roy. Mic. Soc., 46: 107.
McGEE, L. C., 1927. The Effect of the Injection of a Lipoid Fraction of Bull
Testicle in Capons. Proc. Inst. Med. Chicago, 6: 242.
McGEE, L. C., JUHN, MARY, AND DOMM, L. V., 1928. The Development of
Secondary Sex Characters in Capons by Injections of Extracts of Bull
Testes. Am. Jour. PhysioL, 87: 406.
MARTINS, T., AND ROCHA E SILVA, A., 1929. The Seminal Vesicles of the Cas-
trated Mouse, Test for the Testicular Hormones. Suf>pL d. Mem. Inst.
Oswaldo Cruz, 9: 196. Rio de Janeiro.
MOORE, C. R., AND McGEE, L. C, 1928. On the Effects of Injecting Lipoid Ex-
tracts of Bull Testes into Castrated Guinea Pigs. Am. Jour. PhysioL,
87: 436.
MOORE, C. R., AND GALLAGHER, T. F., 1929. On the Prevention of Castration
Effects in Mammals by Testis Extract Injections. Am. Jour. Physiol.,
89: 388.
MOORE, C. R., HUGHES, WINIFRED, AND GALLAGHER, T. F., 1930. Rat Seminal
Vesicle Cytology as a Testis Hormone Indicator and the Prevention of
Castration Effects by Testis Extract Injections. Am. Jour. Anat., 45:
109.
MOORE, C. R., AND GALLAGHER, T. F., 1930. Seminal- Vesicle and Prostate Func-
tion as a Testis-Hormone Indicator; the Electric Ejaculation Test. Am.
Jour. Anat., 45: 39. •
RAT VAS DEFERENS CYTOLOGY
MOORE, C. R., PRICE, DOROTHY, AND GALLAGHER, T. F., 1930. Rat-Prostate
Cytology as a Testis-Hormone Indicator and the Prevention of Castra-
tion Changes by Testis-Extract Injections. Am. Jour. Anal., 45: 71.
MOORE, C. R., GALLAGHER, T. F., AND KOCH, F. C., 1929. The Effects of Ex-
tracts of Testis in Correcting the Castrated Condition in the Fowl and
in the Mammal. Endocrinology, 13: 367.
MOORE, C. R., VATNA, S., AND GALLAGHER, T. F., 1930. Rat Vas Deferens
Cytology as a Testis Hormone Indicator and the Prevention of Castration
Changes by Testis Extract Injections. Anat. Rcc. (In press.)
MYERS-WARD, C. F., 1897. Preliminary Note on the Structure and Function of
the Epididymis and Vas Deferens in the Higher Mammalia. Jour. Anat.
London, 32: 135.
ON DISTOMUM VIBEX LINTON, WITH SPECIAL REFER-
ENCE TO ITS SYSTEMATIC POSITION
H. W. STUNKARD AND R. F. NIGRELLI
BIOLOGICAL LABORATORY, UNIVERSITY COLLEGE, NEW YORK UNIVERSITY
Distoinniii vibcx was described by Linton (1900, 1901, 1905), from
the pharynx and intestine of the smooth puffer, Spheroides maculatus.
For many years this species has been studied as the representative of
digenetic trematodes by the classes in Invertebrate Zoology at the Marine
Biological Laboratory of Woods Hole. Since the early and brief re-
ports of Linton, little or no research has been done on the parasite.
The purpose of this study is, therefore, to supplement the earlier de-
scriptions of its morphology and to allocate the species in the system
of classification of the digenetic trematodes.
LINTONIUM NEW GENUS
Distoinniii Diesing 1850 is the equivalent of Distoina Retzius 17SJ.
a name proposed as a substitute for Fasciola Linnaeus 1758 — and con-
sequently a synonym. Looss (1899) showed that Distomum is not
a generic but a group name, and with the subdivision and disappearance
of the previously accepted genus Distoinniii, the proper generic name
and systematic position of D. ribc.v has remained an open question.
Since Distomum is not a valid generic name, and since the species can
not be assigned to any existing genus, we propose the new genus
Lintonium to contain it.
The distribution of Lintonium i<ibcx, so far as has been determined,
appears to be limited to the species Spheroides maculatus, commonly
found off the coasts of New Jersey and New York and as far north as
Maine. Primarily, however, the members of the group of " swell-
fishes " are inhabitants of warmer waters, and the relatives of Lintonium
vibex are presumably to be found, if at all, in species of Spheroides
which inhabit warmer seas. According to Linton, the largest worms
are found in the pharynx, attached to the walls around the entrance
to the pouch. Young specimens, however, were encountered in the
intestine.
Except for certain details, which appear in the text, our observa-
tions agree with those of Linton. The parasites are so variable in
size and form that precise measurements are difficult to make. Fixed
336
DISTOMUM VIBEX LINTON 337
and stained sexually mature specimens vary from 2 to 7 mm. in length.
0.7 to 2 mm. in width, and 0.266 to 0.912 mm. in thickness. In living
worms, the region anterior to the acetahulum is very mobile and may
be elongated into a neck-like structure, one and one-half times the length
of the body posterior to the ventral sucker. In fixed specimens the
acetabulum is located at the posterior end of the anterior third of the
body. It is considerably larger than the oral sucker, oval to spherical
in shape, and measures from 0.4 to 1.3 mm. in diameter. The suckers
are powerful adhesive organs and the parasites are removed from their
attachments only with difficulty.
The body wall is strongly developed and the specimens are very
muscular. The cuticular covering measures from 0.021 to 0.032 mm.
in thickness and is much heavier on the dorsal than on the ventral
surface. When the worm is contracted the cuticula is thrown into
convolutions that give it a " ringed '" appearance, although it is not
provided with either scales or spines. The muscular layers of the body
wall consist of an external circular, an intermediate longitudinal, and
an internal oblique laver of fibers. In the anterior part of the bodv
especially, the parenchyma is traversed by well-developed fibers. These
are not arranged in definite layers and have branched or diffuse origins
and attachments. Immediately below the muscular wall there are many
glandular cells which probably secrete the cuticula. Inside the nuclear
zone, on the ventral side of the body, there is a well-developed series of
longitudinal muscles that extend from the body wall in the region behind
the genital pore to the region of the acetabulum, and others that extend
on the region of the ootype.
The mouth opening is subterminal and the oral sucker, spherical
to oval in shape, measures from 0.23 to 0.57 mm. in diameter. The
pharynx, situated immediately behind the oral sucker, measures from
0.10 to 0.19 mm. in diameter. Following the pharynx there is an ap-
parent esophagus of varying width and diameter. Histologically, how-
ever, this structure resembles the digestive ceca ; it is lined with epi-
thelium and should properly be regarded as a portion of the intestine.
Two simple intestinal crura pass posteriad in the dorsal and lateral
regions of the body, terminating blindly about the middle of the pos-
terior third of the worm.
The excretory pore is situated at the posterior tip of the body. It
opens from a small vesicle which is lined with cuticula. From the
vesicle two collecting tubes pass forward, dorsal and median to the
intestinal ceca to the level of the acetabulum where they cross to the
extracecal region and continue to the level of the pharynx. The col-
lecting vessels are variable in shape and size and the walls consist of
338
H. \V. STUNKARD AND R. F. NIGRELLI
— OS
- cs
C.c
ov--
Lintonium vibe.v, ventral view X 40. ac, acetabulum ; cs, cirrus sac ; gp. gen-
ital pore; in, intestine; mg, Mehlis' gland; os, oral sucker; ov, ovary; ph, pharynx;
ts, testis, lit, uterus; i<d, vitelline duct; vt, vitellaria.
DISTOMUM VIBEX LINTON
a basement membrane bearing a layer of flattened epithelial cells.
Further details of the system have not been worked out.
The testes are lateral, situated just behind the middle of the body.
Oval in shape, with their longest axis directed anteroposteriorly, they
measure from 0.15 by 0.22 mm. in small worms to 0.6 by 0.8 mm. in
the largest ones. From the anterior tip of each a vas deferens passes
forward on the dorsal side of the body and empties into the seminal
vesicle located in the caudal end of the cirrus sac. The cirrus sac is
situated on the dorsal side of the body in the region between the bifur-
cation of the alimentary tract and the anterior border of the acetabulum.
The sac has a well-developed fibromuscular wall, containing both circular
and longitudinal muscle fibers, and measures about 0.35 mm. in length
by 0.22 mm. in width. The seminal vesicle is somewhat coiled, and
in some whole mounts gives the appearance of being composed of two
parts : a small, oval, caudal portion and a much larger anterior portion.
From the vesicle a narrow duct, 0.06 to 0.07 mm. in diameter, leads
to the common genital pore. This duct is usually S-shaped and is
lined with columnar epithelium. Both vesicle and duct are surrounded
by prostate cells.
It is interesting to note that in one instance, a worm was found
with a single testis and vas deferens. Otherwise the specimen appeared
to be perfectly normal.
The ovary is trilobed ; it consists of one large dorsal and two smaller
ventral lobes. It is situated on the dorsal side of the body, at the right
of the median plane, in front of the testes, and behind the acetabulum.
It is slightly longer than broad, measuring from 0.15 to 0.54 mm. in
length and from 0.15 to 0.43 mm. in width. The oviduct arises at the
posterior tip of the dorsal lobe and just after entering the ootype, gives
off Laurer's canal. Laurer's canal passes forward in a winding course
and opens to the dorsal surface above the anterior margin of the ovary.
It traverses a distance of approximately 0.2 mm., measures about 0.015
mm. in diameter, and is lined with cuticula. After the origin of Laurer's
canal, the female duct passes posteriad and ventrad where it receives a
common vitelline duct and then turns dorsad and anteriad, to open into
the uterus. There is no seminal receptacle. The ootype is enclosed
in the cells of Mehlis' gland, which lies posterior and ventral to the
ovary. From the ootype the uterus extends laterally and forward.
This portion is filled with sperm and light-colored eggs with deeply
staining contents. The vitellaria consist of six lobes on each side of
the body. They lie in the extracecal area, from the level of the ovary
to the caudal ends of the intestinal ceca. Collecting ducts pass forward
along the medial face of the five caudal lobes and bend mediad in
front of the testes. The cephalic lobes have their own ducts, which
340 H. W. STUNKARD AND R. F. NIGRELLI
discharge into the main longitudinal ducts as they turn mecliad. These
ducts meet in the median line to form a common vitelline duct that
passes through Mehlis' gland to empty into the ootype. No vitelline
receptacle was observed. The uterus passes backward on the left side
of the body to the caudal end and then forward, and fills the intercecal
area behind the ovary with masses of complicated coils. In front of
the ovary the uterus continues in the dorsal portion of the body to
the genital pore, situated immediately behind the bifurcation of the
alimentary tract. The metraterm is short, and there is a small genital
sinus into which the male and female ducts open.
The uterus is filled with enormous numbers of eggs. They are
ovate in shape, with an operculum at the narrow end of the shell. They
measure from 0.045 to 0.054 mm. in length by 0.023 to 0.027 mm. in
width.
From the above description the genus Lintonium may be character-
ized as follows: small to medium sized distomes ; suckers powerful,
acetabulum larger than the oral sucker ; strongly muscular bodies, preace-
tabular region especially mobile ; esophagus short or absent, pseudo-
esophagus short, lined with digestive epithelium ; intestinal ceca extend
posterior to the testes ; excretory vesicle almost Y-shaped with short
stem, lateral crura extend to the region of the pharynx ; genital pore
ventral, immediately behind the bifurcation of the alimentary tract;
cirrus sac oval, preacetabular, enclosing seminal vesicle and cirrus ; testes
lateral, postovarian ; ovary postacetabular, lateral and pretesticular ;
uterine coils extend to posterior end of body, filling the intercecal area
behind the ootype ; eggs ovate, operculum at the smaller end ; vitellaria
lateral, postovarian.
In morphological features Lintonium agrees more closely with
Stcringotreina Odhner 1911 than with any other known genus.1 The
genus Steringotretna was proposed to contain a species described by
Nicoll (1909) as Steringophorus cluthcnsis, since the form could not
properly be retained in the genus Steringophorus because of differences,
especially in the form of the excretory vesicle. Lintonium differs from
Stcringotreina in several distinct morphological features. The acetab-
ulum, ovary, and testes are much farther forward, and there are differ-
ences in the form and location of the vitellaria.
Odhner (1911) proposed a new family, Steringophoridae, with two
subfamilies, Steringophorinae and Haplocladinas. In the former he in-
cluded Steringophorus Odhner 1905, Fellodistomum Stafford 1904, and
the two new genera, Rhodotrema and Steringotrema. It should be
noted, however, that Nicoll (1909) had erected the subfamily Fellodis-
1 According to Odhner, 1928 (Arkiv. f. Zoologi, Vol. 20). Stcringotrcma
puh'linun S. J. Johnston 1913 is identical with Gastris censors Liihe 1906.
DISTOMUM VIBEX LINTON 341
tominae to include Fellodistomum and Steringophorus. Consequently,
since the two groups are co-extensive, the proposal of the subfamily
Steringophorinae was a deliberate renaming of a previously validly
named subfamily. Odhner's reasons for changing the name are stated
as follows: " Wenn ich fur diese Unterfamilie den von Xicoll (1909,
S. 472) vorgeschlagenen Namen Fellodistominae verwenden wiircle,
miisste ich die ganze Familie Fellodistomidae nennen, was mir bei dem
Umstande, dass nur ein einziger Vertreter derselben mit cler Galle etwas
zu tun hat, allzu sinnlos erscheint. In Steringophorus erblicke ich
weiter diejenige Gattung, welche den Typus der ganzen Familie am
reinsten verkorpert ; wahrend die typische Art der Gattung Fcllodis-
touiuui, F. fcHis, entschieden als der am wenigsten typische Vertreter
der ganzen Unterfamilie bezeichnet werden darf. Aus diesen Griinden
erscheint es mir als richtig, den Namen Fellodistominae beiseite zu
schieben, und ich trage hierbei um so weniger Bedenken da sich dieser
Name als erst jiingst geschaffen noch nicht weiter eingebiirgert hat."
Commenting on Odhner's action, Woodcock (1912) stated that,
"... this change in name appears to contravene the usually accepted
rules," and referring to the family name this author observed that
". . . the name should be Fellodistomidae as the author (Odhner) him-
self recognizes." Nicoll (1913) further stated, " It is obvious that the
name Steringophorinae cannot stand but must give place to the earlier
Fellodistominae. The name of the family should consequently be
changed to Fellodistomidae." In a later paper, Nicoll (1915) used the
family name Fellodistomidae without comment.
F'oche (1925) attempted to justify Odhner's change of name but
his argument appears to be beside the point as will be shown later.
Fuhrmann (1928) adopted Odhner's classification and in the subfamily
Steringophorinae included Steringophorus Odhner, Fellodistomum Staf-
ford, Rhodotrcma Odhner, Steringotrema Odhner (syn. Pycnadena
Linton), Didyinorchis Linton, and Bacciffer Nicoll. It should be
pointed out that Didyinorchis Linton 1910 was preoccupied, and the
following year Linton (1911) proposed the name Pycnadena for it.
There appear to be too many differences between Steringotrema and
Pycnadena to regard them as identical, and Fuhrmann's statement of
synonymy is probably an error.
It will be noted that in Odhner's arrangement, Steringophorus is
named not only as type of the subfamily but of the family as well and
that Steringophorinae is designated as type subfamily. Poche based his
argument on the provision in the rules of nomenclature that the name
of a family or subfamily is to be changed when the name of the type
genus is changed. It is obvious, however, that the name of the type
342 H. W. STUNKARD AND R. F. NIGRELLI
genus of Nicoll's subfamily Fellodistominse was not changed in Odhner's
arrangement. Instead, another genus was selected as type. The
opinion of Professor Ch. W. Stiles was asked concerning the status
of Odhner's action and the validity of the subfamily name Sterin-
gophorinse. In a personal communication he makes the following state-
ment, ' Steringophorinae is a deliberate renaming of the subfamily
Fellodistominae.
" On page 98, Odhner gives a footnote in which he explains why
he renamed the subfamily. His explanation shows that he confused
two elements, namely, the genus which forms the nomenclatorial type
and the genus which he looked upon as the anatomical norm. This is
not an uncommon confusing which occurs in systematic zoology and
is due to the fact that the word " type " is used in so many different
senses. According to Odhner, Fellodistomum, the nomenclatorial type
of Fellodistominae, represents a peripheral genus from his point of view,
while Steringophorus represents the anatomical norm. This, of course,
is a point of view, but in the last analysis, is somewhat subjective and
may be changed by a division of the subfamily by some future author.
' The important point is that Fellodistomum is the nomenclatorial
type of the first available subfamily name.
' If Odhner's method of nomenclature were applied generally to
zoology, there would be numerous unnecessary changes in family and
subfamily names. On basis of Odhner's statements, Steringophorinae
is subjective synonym of Fellodistominae. It is subjective rather than
objective because it has a different type genus. I would not hesitate
an instant in this case, I would use Fellodistominae."
The analysis and decision of Professor Stiles is so incisive and
pertinent that its publication is a valuable contribution to zoological
literature. It outlines correct procedure and stands in contrast to the
confused and irrelevant argument of Poche. Since Fellodistominae is
accepted as the type subfamily of the family to which it belongs, the
family name must be Fellodistomidae. So far as has been determined,
the subfamily includes the following genera: Fellodlstounnn Stafford
1904, Stcrinyophorns Odhner 1905, Pycnadcna Linton 1911, Rhodotreina
Odhner 1911, Steringotrema Odhner 1911, Bacclgcr Nicoll 1914, and
Lintoniuni, gen. nov.
SUMMARY
Additions are made to the description of Distoinuin vibe.v Linton.
Since Distoiniun is not a valid generic name, and since the species cannot
be assigned to any known genus, the new genus Lintonium is erected
to contain it. The genus belongs to the subfamily Fellodistominse, Fam-
ily Fellodistomidae (Syn. Steringophoridae).
DISTOMUM VIBEX LINTON 343
BIBLIOGRAPHY
FUHRMANN, O., 1928. Trematoda. Kukenthal's Handbuch der Zoologie. Berlin
and Leipzig.
LINTON, E., 1900. Fish Parasites Collected at Woods Hole in 1898. Bull. U. S.
Fish Commission for 1899, 19: 267.
LINTOX, E., 1901. Parasites of Fishes of the Woods Hole Region. Bull. U. S.
Fish Commission for 1899, 19: 405.
LINTON, E., 1905. Parasites of Fishes of Beaufort, North Carolina. Bull. Bureau
of Fisheries, 24: 321.
LINTON, E., 1910. Helminth Fauna of the Dry Tortugas. II. Trematodes.
Pub. No. 133, Carnegie Institution of Washington, p. 11.
LINTON, E., 1911. Trematode.s of the Dry Tortugas. Science, 33: 303.
Looss, A., 1899. Weitere Beitrage zur Kenntniss der Trematoden-Fauna Aegyp-
tens, zugleich Versuch einer natiirlichen Gliederung des Genus Distomum
Retzius. Zoo/. Jahrb. abt. f. Svstcmatik, Geographic n. Biol. der Thicre,
12: 521.
NICOLL, W., 1909. Studies on the Structure and Classification of the Digenetic
Trematodes. Quart. Jour. Mic. Sci,, 53: 391.
NICOLL, W., 1913. Trematode Parasites from Food-fishes of the North Sea.
Parasit., 6: 188.
NICOLL, W., 1915. A List of the Trematode Parasites of British Marine Fishes.
Parasit., 7: 339.
ODHNER, T., 1911. I. Wissenchaftliche Mitteilungen. 1. Zum natiirlichen System
der digenen Trematoden. III. ZooV. Anzcig., 38: 97.
POCHE, F., 1925. Das System der Platodaria. Arch. f. Natttracs., Abt. B., 91: 1.
WOODCOCK, H. M., 1912. VI. Vermidea. Zoological Record, p. 32.
THE MANNER OF SPERM ENTRY IN THE STARFISH EGG
ROBERT CHAMBERS
(1-rotn the Ell Lilly Kesearcli Division, Woods Hole, and Washington Square
College, Nczv York Unirersity)
In an article published several years ago (Chambers, 1923) I de-
scribed some morphological aspects of the insemination of the starfish
egg. A peculiar feature in this process, the interpretation of which
has been adversely criticized (Lillie and Just, 1924; Just, 1929) is the
apparently passive and relatively slow travel of the blunt-headed sper-
matozoon through the jelly which surrounds the egg.
There is a striking contrast between the arrangement of the sper-
matozoa about freshly inseminated starfish (Asterias) and sea-urchin
(Arhacia) eggs. In Arbacia the pointed, narrow-headed sperm quickly
pass through the jelly surrounding the eggs and, within a few seconds
after insemination, are on the surface of the egg. In Asterias the blunt,
ovoid sperm penetrate very little into the jelly and collect on its outer
border far from the surface of the eggs. By careful observation, one
is able to detect a spermatozoon, advancing through the jelly by a pe-
culiar gliding movement to the egg. As described in my previous paper,
the moment when the spermatozoon starts to migrate through the jelly,
it is seen to be connected by a tenuous filament to a conical elevation
on the surface of the egg. The spermatozoon advances as the filament
progressively shortens until the head of the spermatozoon finally reaches
the cone into which it sinks. From there it travels into the main body
of the egg.
Fol, who was among the first to describe the penetration of a sper-
matozoon into an animal ovum (Fol, 1877) made an extensive study
of the process in Asterias and To.ropneitstes (Fol, 1879). In his studies
on the starfish he was struck by the peculiar directive movement of the
spermatozoon through the jelly to a conical elevation on the surface
of the. egg and considered the possibility that the progress was due to
the retraction of a filament, connecting the spermatozoon with the cone.
Fig. 1. He dismissed the idea that the filament is an outgrowth of the
spermatozoon, since he observed no diminution in volume of the head.
He also suggested that protoplasmic filaments may pre-exist extending
from the egg through the jelly and that a sperm, coming into contact with
one of these filaments, may be drawn in by a reaction on the part of
the egg. Not being able to observe such a filament except as a com-
paratively short extension of the cone, Fol concluded that the initial
344
•; ;
. .1*
. . fit
.. .&•
f^^^^^^T *%£f^\;$?$R^f
-*8j w
fy 2
*&%^W?$*W? ^>-Vr V tK^^I
^^W« " «WS*c5ftS»?
>s
tf-..
FIG. 1. Photographic reproduction of part of Phite III from Fol's paper
(1879) on Astcrias i/lucialis, the drawings of which were from the living egg.
In Fig. 1, a. b, and c are three successive phases of the same zoospcrm, zc. An
extension of the entrance cone is at Sa. The phase in which the zoosperm entered
is omitted here. In Fig. 2, a, />, c (d omitted) c, f, g, (It omitted) and i are
seven views of the same objects; in Fig. 2, b, c, a /oosperm, ,;c, is approach ing.
In c and / a second zoosperm, .;", is approaching. In <y and i are extensions,
Sc' and Sc", of the "cone d'exudation." Fig. 3, a, b, shows the approach of a
zoosperm to an exceptionally large cone. Fig. 4, a, b, c, slum's a zoosperm en-
tering near region of polar bodies, Cr.
346 ROBERT CHAMBERS
travel of the sperm is due to an attraction exerted by the cone from
a distance. A photographic reproduction of a part of Fol's illustrations
is shown in Fig. 1.
The results presented in this paper constitute a critical re-examina-
tion of the phenomenon and are based upon observations made at dif-
ferent periods every summer since the publication of my original article.
During the summer of 1929 at the Marine Biological Station, Ros-
coff, France, I was able to confirm the observations of Fol on the species
he used, Astcrias glacialis. Fol's article is remarkable for its wealth
of detailed description and should be referred to by any one interested
in the subject.
METHODS AND MATERIAL
Observations were made at Woods Hole on the ova of Aster las
rubcns, the common starfish, during all the summer months from June
to September. The ova were obtained both by allowing a ripe female
to shed the eggs naturally in sea-water and also by removing and cutting
up ripe ovaries in bowls of sea-water.
The insemination process was observed with a 3 mm. apochromatic
objective in both immature and mature ova at various times before,
during and after completed polar-body formation. The temperature
of the water in which the inseminations were made varied at different
times of the summer (from 15° C. to 20° C.). A preliminary insemina-
tion of a sample lot of the eggs was always made under the conditions
of the final experiments and only those kept for a study of the normal
process when normal fertilization membranes developed within a few
minutes on a minimum of 90 per cent of the eggs. For the crucial
experiments precautions were taken to make adequate dilutions of the
sperm-suspensions in order to procure maximum fertilization with a
minimum of sperm present. Heavily inseminated specimens were also
studied.
In all the cases in which the penetration of the spermatozoon was
observed, the manner of its entry proved to be essentially the same
irrespective of variations in temperature, age of eggs or amount of
sperm present.
Fol used the following excellent method for observing insemination.
He placed a drop of sperm-suspension on the slide of a compressorium
on the stage of the microscope and a hanging drop of sea-water contain-
ing the eggs on the coverslip of the cap of the compressorium, which
was inverted over the slide. After bringing the sperm-suspension into
the field of the microscope, he carefully lowered the cap of the com-
pressor until the two drops touched. The eggs, being heavier than the
water in which they were suspended, fell through the liquid, while the
SPERM ENTRY IN THE STARFISH EGG 347
sperm rose and encountered the eggs under conditions approaching
the normal.
The compressorium used by Fol may be dispensed with if a cover-
slip be mounted on feet of soft clay and the two drops brought together
by pressing down on the coverslip. Owing to the fact that the starfish
eggs react relatively slowly (15-45 seconds), the sperm can also be
mixed with the eggs in a dish. A drop of the mixture is then placed
on a slide and covered for observation. With a little practice one is
able to bring the eggs into view under an oil immersion objective within
5-10 seconds. Some of my studies were made with the use of the
micromanipulator, the sperm-suspension being microinjected into a
hanging drop containing the eggs already under view in the microscopic
field. With this method the entire sequence of events could be ob-
served from the moment that the sperm arrived in the vicinity of the
eggs.
Experiments were also made in which the microneedle was used to
operate on the surface of the egg and to seize entering spermatozoa.
For this purpose it was essential to have two observers using a demon-
stration ocular, one observer maintaining the spermatozoon in focus,
while the other observer operated the microneedles. I wish to take
this opportunity of expressing my appreciation to Dr. G. H. Faulkner
of the University of London, who was of the greatest assistance to me
in this way.
The time relations of the several steps in the penetration of the
spermatozoon vary within certain limits. Spermatozoa taken directly
from the testis are sluggish and frequently motionless, but become active
when diluted in sea-water. As long as they are actively motile, the
spermatozoa of different batches seem to be similar in their behavior
toward eggs of one lot. On the other hand, with eggs of different lots
and ages, considerable time-variations occur, although the consecutive
steps of the insemination process are the same. Immature eggs, as well
as eggs which have maturated and have stood for hours in sea-water
can be readily inseminated.
In immature eggs the penetration of a spermatozoon does not always
cause the vitelline membrane to rise so as to form the fertilization
membrane and, if plenty of sperm be present, the sperm will keep on
penetrating until the egg is fairly riddled with them. Polyspermy is
also the rule for mature eggs aged for three to five hours.
In freshly maturated eggs the peculiar reaction which prevents
polyspermy occurs within an average time of 45 seconds and the fer-
tilization membrane rises rapidly. In some batches of eggs the time
23
348 ROBERT CHAMBERS
limit of sperm-penetration may be only 75 seconds, although the usual
limit is two minutes.
EXPERIMENTAL
A. Observational Studies
1. The Jelly Around the Starfish Egg
The clear jelly which surrounds the egg swells in sea- water to form
a layer approximately % the diameter of the egg. The outer border
of this jelly can be shown by the well-known method of placing the
eggs in sea- water containing a suspension of India ink. In accordance
with Fol's findings, the jelly appears to be principally a matting of
delicate fibrillae arranged in radial lines. Its density is greatest close
to the egg and progressively loosens on approaching its external border.
Fol used an ingenious method to demonstrate the radial structure by
placing eggs in sea-water containing rod-shaped bacteria. The bacteria
implanted themselves in the jelly and always in lines perpendicular
to the egg's surface.
In the immature condition the jelly is bounded externally by a thin
cellular membrane which breaks up as the jelly swells in the water.
When this membrane is present the spermatozoa do not adhere to it.
As soon, however, as the membrane disrupts, the spermatozoa readily
accumulate in the peripheral meshes of the exposed jelly.
The density of the jelly is such that the starfish spermatozoa with
their blunt heads remain entrapped in its outermost zone while their tails
continually lash to and fro. On the other hand, the narrow-headed
sand-dollar and sea-urchin sperm can work their way quite through
the jelly of the starfish egg. Their progress is somewhat impeded
the farther they penetrate, but they arrive at the surface of the egg
within one or two minutes. This is in striking contrast to the few
seconds which it takes them to go through the looser jelly of both sand-
dollar and sea-urchin eggs.
The jelly of the starfish egg cannot be removed entirely by mechan-
ically shaking the eggs, although such a procedure is frequently success-
ful for sea-urchin and sand-dollar eggs.
2. Insemination of the Freshly Maturated Egg
In an inseminated preparation of eggs in sea- water a microscopic
examination will show the spermatozoa adhering to the outer borders
of the sticky egg- jelly. As long as the spermatozoa do not touch the
jelly they are as likely to swim away from the egg as towards it.
Fig. 2 (A—Q) represents seventeen successive steps in the passage
SPERM ENTRY IN THE STARFISH EGG
349
A1,-/ so" BA/ so" 0 no" Q
IS/ri
1 , 1 1 1
1
I V Z' J
K 9-9" ^
r\ 2 2 ^;,/1;-
^S£:g; . I
2'3" N '0 '2'5" p 5'3Q 6'
FIG. 2. Seventeen successive steps in the insemination of a starfish egg.
For description see text.
350 ROBERT CHAMBERS
of a spermatozoon through the egg-jelly and into the egg until the
diminutive sperm-aster becomes appreciable. The drawings were made
mostly from observations on one specimen obtained from freshly ma-
tured eggs shed naturally in a tank and inseminated with a minimum
dilution of spermatozoa to ensure proper insemination. The prepara-
tion was brought under observation (with a 3 mm. apochr. objective)
within 10 seconds after mixing the eggs with the spermatozoa.
In Fig. 2, A several spermatozoa are shown in the outer border
of the jelly. When first observed, the head of one of these was already
connected by means of a distinctly appreciable but tenuous filament ex-
tending through the jelly to a hyaline, conical papilla on the surface
of the egg.1
Twenty seconds later the spermatozoon had moved about halfway
in, Fig. 2, B. Its progress was steady and in a straight line, while the
tail stretched out motionless behind and only occasionally gave a
spasmodic twitch. The fertilization membrane was already to be seen
beginning to rise from the cone at the base of the filament. The suc-
cessive steps in the advance of the spermatozoon to the summit of the
cone (Fol's cone d'attraction) are shown in C to H. While this was
occurring, wave-like quivers (see D to G) passed over the cone and
the adjacent surface of the egg. When the spermatozoon reached the
summit of the cone, there was an appreciable pause of 30 seconds, after
which the sperm-head narrowed at its tip and lengthened out as it
slipped through the fertilization membrane to round out again after
it has passed into the underlying cone (/ to K). The changes in the
shape of the head of the spermatozoon suggest the existence of a pore
in the rising membrane through which the filament had previously ex-
tended and which is now the means of ingress for the spermatozoon.
When once the spermatozoon started to enter, it slipped through rapidly
and, within 2-3 seconds, passed definitely into the egg, where its progress
(TV— 0) could be followed along an ever-deepening, hyaline pathway
caused by a recession of the cytoplasmic granules. As the sperm-head
advanced in the egg it became increasingly difficult to see. Within 6
minutes after insemination, the diminutive sperm-aster (P and Q),
became evident at the bottom of the pathway. The path gradually
disappeared as granules moved back into it. Usually it is visible for
8 to 10 minutes after insemination.
During the progress of the spermatozoon through the jelly the
sperm-tail is relatively inactive. Frequently a spermatozoon moves all
the way to the insemination cone without a single twitch of its tail. A
1 Some of the best observations I have made of this phase were with dark-
ground illumination.
SPERM ENTRY IN THE STARFISH EGG
351
pronounced lashing of the tail occurs only during the pause after the
spermatozoon has reached the cone. Fig. 2, H, and while it is passing
through the fertilization membrane, Fig. 2, /-/•". As long as there is
a continuity between the tail and the advancing head within the egg,
the tail keeps on feebly lashing. When the connection with the sperm-
head is lost, the tail becomes motionless, but can be recognized for a
long time (ten to fifteen minutes), extending outward from the fer-
tilization membrane, Fig. 2, N-Q.
The fertilization membrane usually becomes evident in the region
v^iS^
r\ sK wg5js<
cavrspc.. :7>iy*, ,^:.\f<
FIG. 3. Progressive changes in the form of an exudation cone.
of the cone before the spermatozoon has migrated halfway through
the jelly, Fig. 2, B. Its complete elevation over the egg occurs within
5 to 20 seconds later.
The conversion of the entrance cone into the exudation cone (Fol's
cone d'exudation) takes place after the spermatozoon has passed into
the egg. Ever-changing, flame-like processes develop on the cone,
Fig. 2, M, N, which finally withdraw and the cone disappears, frequently
leaving behind minute globules, Fig. 2, O—Q, which become dispersed
in the space between the fertilization membrane and the egg. A varia-
tion of the exudation cone is shown in Fig. 3.
In over-inseminated eggs several spermatozoa may become attached,
Fig. 4, A, each to the tip of a filament extending from the egg. Al-
FIG. 4, A. Two spermatozoa migrating together into an over-inseminated egg.
B. One lost its attachment and was discarded, while the other successfully entered
the egg.
352
ROBERT CHAMBERS
though these spermatozoa begin to move through the jelly, there is a
tendency for only the most advanced one to reach and penetrate the
egg. The others, before reaching the egg, tend at one time or another
to lose connection with their filaments. Such released spermatozoa,
after a spasmodic twitch or two, remain permanently motionless. Fig.
3, B, in the jelly. The filaments which have lost their spermatozoa
are quickly withdrawn and, together with their cones, soon sink into
the egg.
'The filaments, extending from a cone to a spermatozoon, are usually
at right angles to the egg's surface. That this is not always the case
is shown in Fig. 5, where two convergent filaments are shown. This
argues against the pre-existence of definite radial canals in the egg-
jelly through which the spermatozoa might be supposed to move.
The shape of the head of the spermatozoon, as already commented
upon by Fol, occasionally changes considerably as the head moves
through the jelly. The change seems to be due mainly to a bulging of
the neck-piece on one or both sides of the head. Fig. 6, A, B (cf. Fig.
" 40" 60"
FIG. 5
FIG. 6
FIG. 5. Two spermatozoa attached to insemination filaments which are
convergent and not radial as usual.
FIG. 6. A. Sketches to show variations in shape of the heads of spermatozoa
migrating through the egg-jelly. B. Changes in shape of the head of one sper-
matozoon at intervals of 20, 40, and 60 seconds.
5). In Fig. 6, B are three sketches of a single spermatozoon, at in-
tervals of 20, 40 and 60 seconds after insemination. The impression
that the head of the spermatozoon is bent to one side may be due to the
distorted shape of the neck-piece. Occasionally, a spermatozoon ap-
pears to be carried through the jelly with the base of its tail at right
angles to the attachment of the insemination filament, while the rest
of the tail is curved so as to trail behind.
SPERM ENTRY IN THE STARFISH EGG
353
Figures 7—10 represent variations. Fig. 7 shows a sperm-head which
was unusual in performing active, wriggling movements for fully one
minute after having penetrated the egg while the tail hung motionless
outside. During these movements the sperm-head left the usual hy-
aline pathway and could be seen jostling and pushing aside the cyto-
plasmic granules encountered.
Fig. 8 shows a spermatozoon whose head, after passing through
^•% •>- IS I 1
^v;y^ ^x 2& ^ ^ SC to
FIG. 7. Four successive steps in the progress of an unusually active sperm-
head after it had penetrated an egg.
ri.-,
.+•*••-(., \-^.._ ••*•*> j:
- •' . i"- - . •* '4
-.<,':, ., - -V-i - T
-yU.% ; (J ' B -• -•'
':
O
B 1'4S" C
4' 15"
FIG. 8
FIG. 8. A spermatozoon which on entering an egg left its neck-piece outside
the fertilization membrane.
the fertilization membrane, broke away from its neck-piece which was
left outside with the tail.
Figs. 9 and 10 show the reactions of late arriving spermatozoa.
Fig. 9 shows a spermatozoon which succeeded in passing through an
already lifted fertilization membrane. During the process the cone
changed shape and flattened out, while the fertilization membrane be-
came appreciably indented. In Fig. 10 the spermatozoon reached the
cone, A-C, but failed to enter. The fertilization membrane wrinkled
and the cone formed accessory elevations, D—F, but, when the cone
finally withdrew from the membrane, the spermatozoon was left out-
354
ROBERT CHAMBERS
side. The head of the spermatozoon then sprang back for a short dis-
tance where it remained motionless and attached to the membrane by
a slender thread, G, nine minutes after insemination.
mmi ^spf; ',>^« *m^,
£$$&$$! Iplp jjlJ®3im "!:^-?'M>''; ^'•'?-:+' jj^'^iJI
B |'2S'
2'ifS" 0
E JIS"
3' 25"
FIG. 9. Delayed entry of a spermatozoon through a fertilization membrane
formed by the penetration into the egg of another spermatozoon not shown in
the figure.
3. The Origin of the Insemination Filament
The insemination filament is so fine that it is practically invisible
except when the cone at one end and the sperm-head at the other end
are brought simultaneously into focus. Considerable practice is re-
quired to detect the sperm at the moment when it is beginning to mi-
grate into the jelly. In the outer border of the jelly among several
spermatozoa whose heads are moving to and fro while their tails lash
about, one's attention becomes attracted to a sperm-head which has
ceased its side-to-side movements and, instead, is moving steadily and
in a straight line into the depths of the jelly. By looking along the
direction of its movement, a cone on the egg's surface becomes apparent
and, between the cone and the sperm, is to be seen the delicate, tenuous
insemination filament. In fresh maturing eggs I have never been able
to see the cone without also seeing the advancing sperm and the filament
connecting the two. The formation of the filament is apparently too
rapid. In immature eggs the cone is relatively much larger and as
already described (Chambers, 1923) I have several times observed a
tapering extension grow out from it until contact is made with a sperm,
whereupon the extending portion retracts and draws the sperm in with it.
In mature eggs which have been standing in sea-water for 2 to 4
hours there is frequently a greater response to multiple cone formation
than in fresh, maturing eggs and consequently the chances are better
to catch the initial stages. Eggs, 3 hours old, were placed in a shallow
hanging drop in a moist chamber and, after being brought under ob-
servation, a suspension of sperm was blown into one side of the
field by means of a micro-pipette. The spermatozoa quickly spread
in the interstices between the eggs and several became attached to the
SPERM ENTRY IN THE STARFISH EGG
355
outer border of the jelly of the egg in view. Within 10 seconds a
number of minute, conical, blister-like elevations developed on the
egg's surface opposite the sperm. A delicate membrane appeared as if
it were being lifted off the egg's surface by the rising cones. A few
of the hyaline cones increased in size and, during the several succeeding
seconds, there was no sign of any connection between them and the
sperm lying on the periphery of the jelly. One cone increased ap-
1 I
I I
C ' !' 1'20" b'1 3'' 40"
iiaaf!"-v~f"-v2ji.'5-.v5.'
FIG. 10. Attempted penetration of a delayed spermatozoon which was finally
discarded.
preciably in size and suddenly, within an instant, a distinct line could
be seen connecting its tip with the head of a spermatozoon. The other
spermatozoa remained on the surface of the jelly while the spermatozoon
in question began to migrate inward. While this was occurring, the
rounded surface of the cone tapered more and more and the ever-
shortening filament became appreciably thicker.
A curious phenomenon which may be of significance is the fact that,
in the majority of cases, the insemination filament always connects with
a spermatozoon. Because of this one is almost inclined to believe in
356 ROBERT CHAMBERS
a specific attraction such as Fol suggested. I may cite, for example,
a case in which about 30-50 spermatozoa were blown on the surface
of an egg. Most of the spermatozoa immediately became attached to
a restricted region on the outer border of the jelly. One, however,
wandered off a short distance and suddenly a cone appeared with a
_.V.7 ::-v...// ,- • , ,. , - , i y, • -
^•..\>'1' !"]"><•' •' '-
».-^^^]r^^Lj^.\-'.'^.
V/;-^
'&.**
V;v
FIG. 11 FIG. 12
FIG. 11. Polyspermy in an egg 5 hours old. The egg nucleus and two polar
bodies show prominently in the middle of the figure. Sperm at x, although more
advanced, entered later than sperm at y.
FIG. 12. Polyspermy in an immature egg.
tenuous filament extending to the spermatozoon diagonally through the
jelly. The filament then retracted with the spermatozoon on its tip and
insemination resulted.
4. Insemination of Immature and of Aged Eggs
Eggs aged by standing in sea- water lose their protective reaction
against polyspermy. Fig. 11 represents an egg which was inseminated
after it had been standing in sea-water for five hours, which is over
four hours longer than is usual for normal fertilization. Within one
minute numerous cones formed on the egg. The figure shows the egg
with six attached spermatozoa, all of which were taken in. Owing
to the rapidity of the procedure and the variations in the angles of
direction which the filaments take, it was impossible to ascertain whether
or not the cones in the figure which show no filaments did in reality
possess filaments with spermatozoa attached to them.
There is often a lack of uniformity in the sequence of the sperm
entries. In Fig. 1 1 the spermatozoon at x was in advance of its neighbor
at y. In spite of this, spermatozoon y entered before x.
One egg, two hours after maturation, formed two cones with in-
SPERM ENTRY IN THE STARFISH K<i<;
357
semination filaments at an interval of two minutes. Both successfully
drew in their spermatozoa. One minute later another cone and filament
developed. Its spermatozoon began to be drawn in, but the rising
fertilization membrane had appreciably formed and the spermatozoon
was discarded.
Another egg, 5 hours old, formed a large number of cones so close
together that, as they enlarged, they became more or less confluent and
spermatozoa kept migrating into them in large numbers, Fig. 13.
J
V
FIG. 13. Excessive over-insemination with formation of confluent cones.
The lack of a protective reaction against polyspermy in old, mature
eggs obtains also for immature eggs. This is shown in Fig. 12. The
entrance cones which form on the immature egg are distinctly larger
than those of the mature egg.
As the sperm passes into an immature egg no hyaline pathway is
formed such as occurs in the mature egg. The spermatozoon is quickly
lost to view among the cytoplasmic granules and no aster ever develops.
Also the exudation cone which forms at the site of the disappearing
entrance cone usually develops into a strikingly large prominence with
elongated flame-like processes. A membrane similar to the fertilization
membrane of mature eggs forms about an immature egg upon insemina-
tion. In fresh, immature eggs the membrane seldom rises. It simply
toughens as can be demonstrated by the microneedle. In old eggs,
which remain immature by maintaining an intact germinal vesicle, the
membrane frequently lifts off upon insemination.
24
ROBERT CHAMBERS
5. Time Relationships in the Insemination Process
The time relations of events in the insemination process are shown
in the accompanying tahle, in which records are given on observations
of a number of individual eggs.
After the eggs and sperm are mixed there is always an appreciable
time of 20 to 35 seconds before the first spermatozoon begins definitely
to migrate into the jelly. The average time to pass through the jelly
is 60 seconds. The spermatozoon remains on the surface of the en-
trance cone for about 25 seconds, after which it rapidly penetrates
the cone and passes into the interior of the egg. The diminutive sperm-
aster becomes appreciable within 5 to 6 minutes after insemination.
Within certain limits the sequence of events for fresh, maturing eggs
is fairly uniform. The greater variability in old eggs may be due to
the fact that aging eggs permit polyspermy and hence the data probably
include records on the penetration of late as well as early arrivals.
A comparison of my data with those recently published by Just
(1929) and included in the table shows agreement in one essential point,
i.e., in the average time taken after insemination for the sperm to
enter the cone, viz., 120 seconds. The disagreement lies in the time
taken for the sperm to arrive on the cone. Although Just states that
he made his observations both on living and fixed eggs, careful perusal
of his paper suggests that he depended more on data obtained from
fixed and sectioned material than from observations on the living egg.
According to my observations, the spermatozoa were never observed
to reach the surface of the egg in less than 45 seconds. I cannot ex-
plain Just's statement that this occurs within 5 seconds except on the
assumption that throwing the eggs into a fixative might possibly induce
a sudden contraction of materials so as to bring the sperm on the
cone before the fixing agent had time to exert its preservative action.
B. MlCRODISSECTION STUDIES
6. Physical Properties of the Cone and of the Insemination Filament
The entrance cone possesses a surprising stiffness somewhat at vari-
ance with the impression it gives to the eye from its ever-changing
contour.
A cone, Fig. 14, A, into which a spermatozoon had just entered, was
pushed inwards by means of the tip of a microneedle bearing down on
the fertilization membrane, B. The relative stiffness of the cone was
indicated by the fact that the general contour of the egg about the cone
was carried in while the cone persisted in its original form within the
SPERM ENTRY IN THE STARFISH EGG
359
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ROBERT CHAMBERS
resulting recess. The microneedle was then passed through the fer-
tilization membrane and the surface of the rounded cone was seized and
deformed by pulling, C. After removal of the needle, the dragged-out
part of the cone slowly and gradually withdrew, D-E.
In another case the fertilization membrane was first removed by
tearing and the cone pulled out into a long tapering strand. While held
FIG. 14. Micromanipulation of an entrance cone. For description see text.
in this position, the strand became lumpy as if it were breaking into
beads. The contour of the cone at its base kept changing, while the
lumpiness of the strand progressively disappeared and reappeared.
Finally the strand broke into beads. The basal position of the strand,
thus freed from the needle, gradually sank into the cone, which ulti-
mately flattened out and disappeared. This phenomenon is similar
FIG. 15. Effect of removing spermatozoon from the insemination filament.
to what occasionally occurs when the lifting of a fertilization membrane,
due to insemination elsewhere on the egg, drags out a retracting filament
attached to a spermatozoon outside the membrane.
SPERM ENTRY IN THE STARFISH EGG
361
With a microneedle a spermatozoon was removed from its filament
while the sperm was moving through the jelly, Fig. 15. The tip of
the needle was raised and moved against the spermatozoon, A. In the
process the cone became stretched as the filament was pushed to one side.
B. Eventually the spermatozoon became dislodged. C, whereupon the
filament retracted and beaded, D, while the freed spermatozoon re-
mained motionless in the jelly. In other cases I have tried without
success to separate the filament from its cone by manipulating the
needle-tip where the filament joins the cone. The filament continues
retracting and the spermatozoon moves steadily to the cone except when
the operation becomes so brutal as to disrupt the cone.
7. The Effect of Removing the Vitelline Membrane before Insemination
I have already described the fertilization of eggs previously deprived
of their vitelline membranes, (Chambers, 1923). The jelly adheres to
the membrane which in its turn is closely adherent to the egg. While
tearing the membrane the egg is usually injured. Occasionally, how-
ever, one is able to insert a fine needle under the membrane, Fig. 16.
and lift it off while delicate strands of protoplasm which appear, stretch
and break. The following experiment indicates that this membrane is
the same structure which lifts off as the fertilization membrane. The
FIG. 16
FIG. 17
FIG. 16. Operation of tearing the vitelline membrane of an unfertilized egg.
FIG. 17. An egg inseminated after partially tearing off the vitelline membrane.
A. Spermatozoon lying in space between vitelline membrane and egg. B. Fer-
tilization membrane lifted owing to insemination by a spermatozoon not shown
in figure.
membrane was partially torn from the surface of an egg which was then
inseminated, Fig. 17. A spermatozoon happened to find its way into the
space under the torn membrane, A, while the egg was fertilized by an-
other spermatozoon in a region not shown in the figure. The lifting of
the fertilization membrane spread over the egg until it reached the torn
region, where the presence of the horizontally stationed spermatozoon
362
ROBERT CHAMBERS
showed that the fertilization membrane was identical with the membrane
which previously had been torn, B. The spermatozoon in the figure
advanced somewhat within the space between the egg and the membrane.
Fig. 18 shows the way in which the jelly can be removed from an
unfertilized mature egg. After tearing the jelly, the exposed part of
the egg is seized with one needle while the jelly at the other end of
FTG. 18. Method of removing an egg from its investing vitelline membrane
jelly.
the egg is caught by a second needle. By gentle manipulation, the egg
can be drawn completely out of its jelly. Such an egg at the outset
is very sticky. However, by rolling it about, the adhesiveness diminishes
and the egg rounds up and cannot be distinguished from untreated
eggs except for the lack of an investing jelly.
F G H I
FIG. 19. Several steps in an unsuccessful insemination of a naked egg.
SPERM ENTRY IN THE STARFISH EGG
363
Fig. 19 represents an unsuccessful attempt at fertilizing a naked
starfish egg. An entrance cone developed at the spot where a sper-
matozoon touched it, A, B. The head of the spermatozoon was en-
gulfed by the cone, C and D. However, the sperm-head did not move
inward, E. Instead, the cone spread out at its base, became irregular,
F, and then diminished in size, G and H. Finally the spermatozoon
was expelled, 7, four minutes after it had arrived on the surface of
the egg.
Fig. 20 represents the stages of a successful sperm entry in another
naked egg. The entrance cone formed as before, A, B. It engulfed
'
,
0 i
. •
:V 5 '
B/ C 0 E F
FIG. 20. Several steps in the successful insemination of a naked egg.
the sperm-head and then receded as the sperm-head rapidly moved in-
ward along an ever-deepening hyaline pathway within the egg. C, D
and E. The sperm-head produced a typical sperm-aster, F, and the
egg segmented in a normal manner.
The striking features which are brought out in the behavior of
the naked egg are as follows : First, the spermatozoon touches the sur-
FIG. 21. Production of an endoplasmic exovate by cutting a gash in the
cortex of an unfertilized egg and causing the interior to flow out. Ectoplasmic
remnant, x, is fertilizable. Endoplasmic sphere, y, is unfertilizable. For de-
scription see text.
face of the egg before there is any evidence of a cone. Second, a cone
forms after the sperm is in contact with the egg. Third, the cone
forms no filamentous process such as is seen when a mass of jelly in-
364 ROBERT CHAMBERS
tervenes between the cone and the spermatozoon. Fourth, no fertiliza-
tion membrane whatever is produced.
The insemination of these naked eggs also bears on the question
of the existence of a specific attraction of the egg to spermatozoa.
Spermatozoa frequently swim up to a naked, unfertilized egg, wander
along its surface and then swim away. Apparently the formation of
an entrance cone is dependent on something more than the mere pres-
ence of a spermatozoon on its surface.
8. Insemination of Squashed Eggs
These experiments show the behavior, toward spermatozoa, of the
egg-cortex as contrasted with that of the extruded interior.
Fig. 21 represents the artificial production of an endoplasmic exovate
and the behavior of the isolated exovate and of the ectoplasmic remnant
to insemination. A deep gash was first made with a needle in one
side of an unfertilized, mature starfish egg. With a second needle the
other side of the egg was seized and pulled to the shallow edge of a
hanging drop, A. The interior of the egg flowed out at the spot where
the gash was made. The fluid exovate rounded up as its connection with
the more solid, cortical remnant of the egg became constricted. By
gentle manipulation, B, the neck pinched off so that the egg was thus
divided, C ' , into an ectoplasmic remnant still maintaining its jelly invest-
ment, x, and a naked endoplasmic sphere, y. As already described
(Chambers, 1923), the endoplasmic spheres are unfertilizable. On the
other hand, the ectoplasmic remnant is readily fertilizable and may de-
velop into a swimming larva. The difference in behavior of the two
pieces when inseminated is shown in D. The ectoplasmic remnant pro-
duced an entrance cone with its filament and the attached spermatozoon
readily entered, s, in D, and was followed by the lifting of a typical,
though collapsed, fertilization membrane. The endoplasmic sphere
showed no reaction to the presence of the spermatozoa. Some hit it
head on, others wandered over its surface, sometimes remaining motion-
less for a few seconds, only to swim away. No cones formed on the
sphere and no spermatozoon was ever observed to enter. In a previous
communication (Chambers, 1921) 1 stated that the endoplasmic spheres
never segment, although I assumed that spermatozoa may enter. This
assumption was based on the sections of several endoplasmic spheres
which contained numerous small chromatic bodies which I took to be
unaltered sperm-heads. In the light of more recent results I re-
examined the slides containing these sections and found that the chro-
matic bodies are far too small to be sperm-heads; they also differ in
being rod-shaped and are probably bacterial organisms. They certainly
SPERM ENTRY IN THE STARFISH EGG
365
are not spermatozoa. All the other endoplasmic spheres (eighteen in
all ) which were sectioned and stained showed no bodies even remotely
resembling sperm-heads, although they had been heavily inseminated
before fixing.
Fig. 22. A shows an egg which was torn and squashed. The original
V-1
FII;. 22. Insemination of a torn and squashed egg. For description see text.
cortex was maintained on the part still covered by the jelly. Upon the
addition of spermatozoa, cones formed on the original cortex at x and
y. Two minutes later, B, the sperm at x, had entered while the two
sperm at 3.' were discarded. Note that the fertilization membrane
formed only on the original cortex.
DISCUSSION
Filamentous structures have been known to develop on the surface
of Echinoderm ova. Some of them are delicate, wavy, cylindrical bodies
which often appear when the eggs are placed under abnormal conditions
of pressure, temperature, hypertonicity of their environment, etc. They
are probably degeneration phenomena.
Other filamentous structures of quite a different sort have been
noted on eggs after exposure to spermatozoa. Such are the structures
described by Seifriz (1926) and Hobson (1927), which are identical
with the flame-like processes which Fol long ago described as growing
out from the " cones d'exudation " at the site of sperm-penetration. In
immature eggs these flame-like processes attain considerable lengths.
They slowly change in shape and size although Seifriz found them to
be extraordinarily stiff when manipulated with microneedles. The in-
366 ROBERT CHAMBERS
semination filaments described in this paper resemble the flame-like
processes of the exudation cones except that they are extremely tenuous
and usually are single instead of multiple.. They also possess a stiffness
which is quite at variance with the limp, filamentous outgrowths on
degenerating eggs.
The results described in this paper indicate that the insemination
filaments of mature eggs develop with extraordinary rapidity, but when
they retract the process is a gradual one. Because of this, it has been
impossible to determine directly whether the insemination filament is
an outgrowth from the sperm-head to the cone or whether it emanates
from the cone itself. Fol argues against the former possibility, because
there is no apparent decrease in volume of the sperm-head. Another
case in point is the relatively weak attachment of the filament to the
sperm-head, for, whenever the filament is broken, either mechanically
or spontaneously (e.g., in the case of incomplete polyspermy), the
separation occurs at the head of the sperm and not at the cone. The
main argument in favor of the filament being an outgrowth of the
entrance cone is that it has been actually observed to develop from the
cone in immature and in old, mature eggs in which all the other steps of
the insemination process are identical with those of freshly matured
eggs. Occasionally an abortive filament has been 'observed to arise
from a cone without encountering a spermatozoon, and later to recede.
The development of the typical insemination filament appears to be
a peculiar adaptation to the presence of the radially structured jelly
about the eggs, because, when the jelly is completely removed, no fila-
ments develop and insemination occurs by the elevation of an ovoid
cone which engulfs the spermatozoon.
An extraordinary feature in the insemination process of the starfish
egg is the apparently passive role which the spermatozoon plays in its
migration through the jelly to the entrance cone. All the evidence in-
dicates that the movement of the spermatozoon is due to the progressive
shortening of the insemination filament. In this regard it is significant
that occasionally the connection of the filament with the head of the
spermatozoon is at such an angle that the spermatozoon moves as if
it were actually being dragged backward to the cone. The spermatozoon
in such a position could hardly be moving under its own motive power.
The main evidence for concluding that the insemination process de-
scribed in this paper is normal, is the fact that the fertilization membrane
always first rises over the cone at the base of the filament to which
the approaching spermatozoon is attached and its elevation then spreads
progressively from this site over the entire surface of the egg.
In the presence of too many sperm an egg frequently responds by
SPERM ENTRY IN THE STARFISH EGG 367
developing more than one filament with the result that several sper-
matozoa begin to migrate through the jelly. As the eggs age there
is an increased production of filaments. The successful penetration
into the egg of one spermatozoon and the failure of another to do so
is conditioned by a definite time relation. It is possible for all of several
spermatozoa to penetrate the egg if they begin migrating through the
jelly within a few seconds of one another. Their success in entering
the egg bears no relation to their distance from one another on the
surface of the egg but to the time when the filaments begin to draw
them in. In freshly matured eggs polyspermy tends to be prevented
because of the paucity of insemination filaments. If, out of several
migrating inward, one spermatozoon is sufficiently ahead of the others,
polyspermy may be prevented by- a gradual attenuation of the delayed
filaments which finally break loose from the spermatozoa attached to
them. Sometimes a delayed filament does not lose its spermatozoon,
but continues retracting until the spermatozoon arrives on the cone.
The spermatozoon, however, fails to enter the cone because of the
elevating fertilization membrane which has already begun to spread
from the region of another more success fully functioning cone. In such
a case the spermatozoon is definitely discarded by a peculiar process
which Just evidently saw when he described a spermatozoon being
" pushed off from the egg, a delicate strand connecting the tip with
the apex of the cone."
Just (1929) claims that filaments which are formed as a response
to insemination occur only on abnormal ova and are exaggerated en-
trance cones. The only observation which he records of a strand con-
necting the sperm with the cone is one which he states occurred when
the sperm was " pushed off from the egg." Such a case I have also
frequently observed on abnormal eggs. My crucial observations of
the true insemination filament were on fresh maturing eggs, from lots
of which over 95 per cent segmented and developed normally. Fixed
material is not suitable for a study of the movement of spermatozoa
to the surface of the egg. Our difference of opinion on living eggs
is one of observation, the methods we both used being presumably the
same.
Quoting from Just, the " spermatozoa rush toward the jelly hull ;
of these, one, rapidly moving through it, reached the egg within 5
seconds." Although this rapidity of the movement is greater than any
which I have observed, it is to be noted that Just admits the passage,
through the jelly, of only one out of many ; the others remain outside.
I have shown the phenomenon to several competent cytologists at
Woods Hole during the past summer. They agreed with me in the
368 ROBERT CHAMBERS
observation that the one migrating spermatozoon, during its passage
through the jelly to the egg, is connected by an ever-shortening, straight
filament to the entrance cone into which the head of the sperm finally
disappeared. Moreover, the elevation of the fertilization membrane
was observed to start over the base of this particular cone.
SUMMARY
1. Evidence is given to indicate that the formation of insemination
filaments is the normal procedure of fecundation in the starfish egg.
These filaments extend from the egg's surface to the spermatozoa lying
on the outer borders of the jelly surrounding the egg.
2. The spermatozoon on the end of an insemination filament moves
to the egg through the jelly by no apparent motive power of its own.
This movement is accompanied by a progressive shortening and thick-
ening of the filament.
3. The fertilization membrane begins to rise off the cone by the
time the spermatozoon has migrated about halfway through the jelly.
The elevation of the membrane spreads from this region.
4. The filament is a peculiar adaptation to the presence of the rela-
tively dense jelly surrounding the egg and to the inability of the blunt-
headed spermatozoa to reach the egg. In the absence of the jelly only
an ovoid entrance cone develops to receive the spermatozoon.
5. Polyspermy can be prevented by the breaking loose of super-
numerary insemination filaments from their attached spermatozoa. The
discarded spermatozoa remain motionless in the jelly while the filaments
are completely withdrawn.
6. There is a definite relation between the time that two or more
spermatozoa become attached to insemination filaments and the success
of one or all to enter the egg. This bears no relation to the distance
of their places of attachment on the surface of the egg but to the time
when the filaments begin to retract.
7. The original cortex is the only part of the starfish egg which re-
sponds to insemination. Endoplasmic exovates do not become insem-
inated.
BIBLIOGRAPHY
CHAMBERS, R., 1923. Studies on the Organization of the Starfish Egg. Jour.
Gen. Physio!.. 4: 41.
LILLIE, F. R., AND JUST, E. E., 1924. Fertilization, Section VIII in General
Cytology. University of Chicago Press.
JUST, E. E., 1929. The Production of Filaments by Echinoderm Ova as a Re-
sponse to Insemination, with Special Reference to the Phenomenon as
Exhibited by Ova of the Genus Asterias. Bio!. Bull., 57: 311.
FOL, H., 1877. Sur le premier developpement d'une fitoile de mer. Compt.
Rend. Acad. Sci., 84: 357.
SPERM ENTRY IN THE STARFISH EGG 369
FOL, H., 1879. Rccherches sur la fecondation ct la commencement de 1'henogenie
chez divers animaux. Mem. dc la Soc. de phys. ct d'liist. nat. de Geneve,
26: 89.
CHAMBERS, R., 1921. Microdissection Studies, III. Some problems in the matura-
tion and fertilization of the Echinoderm Egg. Biol. Bull., 41: 318.
SEIFRIZ, W., 1926. Protoplasmic Papilla; of Echinarachnhis Oocytes. Proto-
plasma, 1: 1.
HOBSON, A. D., 1927. A Study of the Fertilization Membrane in the Echinoderms.
Proc. Roy. Soc. Edin., 47: 94.
INDEX
A BSORPTION spectra of some
bloods and solutions containing
hemocyanin, 150.
ALPATOV, W. W. Phenotypical variation
in body and cell size of Drosophila
melanogaster, 85.
Ambystoma maculatum, growth of
larvae under natural conditions, 182.
Anoplophrya marylandensis, new spe-
cies, 176.
Arbacia punctulata, effect of oxygen
lack on sperm and unfertilized eggs
and on fertilization, 288.
— , effects of HgCl, on fertilized and
unfertilized eggs, 123.
Autocatalytic equation and question of
an autocatalyst in growth of
Euglena, 281.
tJLOOD sugar and activity in fishes
with notes on the action of insulin,
217.
BLUM, HAROLD F. Studies of photo-
dynamic action : I. Hemolysis by
previously irradiated fluorescein
dyes, 224.
Body temperature, influence of humid-
ity in certain poikilotherms, 52.
Busycon canaliculatum, copper content
and minimal molecular weight of
hemocyanin, 18.
(CASTRATION changes, prevention
of, by testis extract injections, 322.
Chaetopterus, cleavage of polar and
antipolar halves of the egg, 145.
CHAMBERS, ROBERT. The manner of
sperm entry in the starfish egg, 344.
COE, WESLEY R. Unusual types of
nephridia in nemerteans, 203.
Conjugation, effect within a clone of
Paramecium aurelia, 293.
Crustaceans, effects of temperature
changes on chromatophores of, 193.
Cleavage of polar and antipolar halves
of the egg of Chsetopterus, 145.
Conch, copper content and minimal
molecular weight of hemocyanin of,
18.
CONKLIN, CECILE. Anoplophrya mary-
landensis n. sp., a ciliate from the
intestine of earthworms of the fam-
ily Lumbricidae, 176.
J)EMPSTER, W. T. The growth of
larvae of Ambystoma maculatum
under natural conditions, 182.
Distomum vibex Linton, systematic
position, 336.
Drosophila melanogaster, phenotypical
variation in body and cell size, 85.
"RMBRYO, orientation in eggs with
spiral cleavage, 59.
Erythrocyte, osmotic properties of, 104.
Euglenoid flagellates, studies on physi-
ology of, 281.
pAURfi-FREMIET, E. Growth and
differentiation of the colonies of
Zoothamnium alternans (Clap, and
Lachm.), 28.
Fertilization of Arbacia punctulata,
effect of oxygen lack, 288.
(^OLDFISH, intake and expulsion of
colored fluids by lateral line canals
in, 313.
GRAY, I. E. and F. G. HALL. Blood
sugar and activity in fishes with
notes on the action of insulin, 217.
Growth of larvae of Ambystoma macu-
latum under natural conditions,
182.
UALL, F. G. Sec Gray and Hall,
217.
HALL, F. G. and R. W. ROOT. The in-
fluence of humidity on the body
temperature of certain poikilo-
therms, 52.
HARVEY, ETHEL BROWNE. The effect
of lack of oxygen on the sperm and
370
INDEX
371
unfertilized eggs of Arbacia punc-
tulata, and on fertilization, 288.
Hemocyanin-containing bloods and so-
lutions, absorption spectra of, 150.
Hemocyanin of Limulus polyphemus,
its equilibrium with oxygen deter-
mined by a spectrophotometric
method, 238.
Hemocyanins of Busycon canaliculatum
and Loligo pealei, copper content
and minimal molecular weight, 18.
Hemolysis by previously irradiated
fluorescein dyes, 224.
— , method for studying rate of, 104.
HgCU, some of its effects on ferti-
lized and unfertilized eggs of Ar-
bacia punctulata, 123.
HOADLEY, LEIGH. Polocyte formation
and the cleavage of the polar body
in Loligo and Chaetopterus, 256.
— . Some effects of HgCL on fer-
tilized and unfertilized eggs of
Arbacia punctulata, 123.
HOBER, RUDOLPH. The First Reynold
A. Spaeth Memorial Lecture. The
present conception of the structure
of the plasma membrane, 1.
Holothurian cloaca, effect of low oxy-
gen tension on its pulsations, 74.
Humidity, influence on body tempera-
ture of certain poikilotherms, 52.
T NSULIN, notes on action of, in
fishes, 217.
Intake and expulsion of colored fluids
by lateral line canals of goldfish,
313.
JACOBS, M. H. Osmotic properties
of the erythrocyte : I. Introduc-
tion. A simple method for study-
ing the rate of hemolysis, 104.
JAHN, THEODORE L. Studies on the
physiology of the Euglenoid flagel-
lates : II. The autocatalytic equa-
tion and the question of an auto-
catalyst in growth of Euglena, 281.
of Ambystoma maculatum,
growth under natural conditions,
182.
Lateral line canals, intake and expul-
sion of colored fluids by, in gold-
fish, 313.
Loligo and Chstopterus, polocyte for-
mation and the cleavage of the
polar body, 256.
Loligo pealei, copper content and mini-
mal molecular weight of hemocya-
nin of, 18.
LUTZ, BRENTON R. The effect of low
oxygen tension on the pulsations of
the isolated holothurian cloaca, 74.
MERCURIC CHLORIDE, some ef-
fects on fertilized and unfertilized
eggs of Arbacia punctulata, 123.
Metabolic gradient in Oligochasts, dis-
tribution of pigment and other
morphological concomitants of, 265.
Method for studying the rate of hemol-
ysis, 104.
MONTGOMERY, HUGH. The copper con-
tent and the minimal molecular
weight of the hemocyanins of
Busycon canaliculatum and of Lo-
ligo pealei, 18.
MORGAN, T. H. Sec Whitaker and
Morgan, 145.
MORGAN, T. H. and ALBERT TYLER.
The point of entrance of the sper-
matozoon in relation to the orienta-
tion of the embryo in eggs with
spiral cleavage, 59.
^EPHRIDIA, unusual types in
nemerteans, 203.
QLIGOCH^ETS, distribution of pig-
ment and other morphological con-
comitants of the metabolic gradient
in, 265.
Orientation of embryo in eggs with
spiral cleavage, as affected by point
of entrance of spermatozoon, 59.
Oxygen, its equilibrium with hemocya-
nin of Limulus polyphemus deter-
mined by a spectrophotometric
method, 238.
- lack, effect on sperm and unfer-
tilized eggs and on fertilization of
Arbacia punctulata, 288.
PARAMECIUM AURELIA, effect
of conjugation within a clone, 293.
Pheretima benguetensis Beddard, dis-
tribution of setae, 274.
Photodynamic action, studies of, I, 224.
PICKFORD, GRACE EVELYN. The distri-
bution of pigment and other mor-
phological concomitants of the
372
INDEX
metabolic gradient in Oligochaets,
265.
Plasma membrane, present conception
of its structure, 1.
Poikilotherms, influence of humidity on
body temperature, 52.
Polar body, polocyte formation and
cleavage of, in Loligo and Chsetop-
terus, 256.
Prevention of castration changes by
testis extract injections, 322.
Pulsations of isolated holothurian
cloaca, effect of low oxygen ten-
sion, 74.
J^AFFEL, DANIEL. The effect of
conjugation within a clone of Para-
mecium aurelia, 293.
REDFIELD, ALFRED C. The absorption
spectra of some bloods and solu-
tions containing hemocyanin, 150.
— . The equilibrium of oxygen
with the hemocyanin of Limulus
polyphemus determined by a spec-
trophotometric method, 238.
ROOT, R. W. Sec Hall and Root, 52.
gIVICKIS, P. B. Distribution of
setae in the earthworm, Pheretima
benguetensis Beddard, 274.
SMITH, DIETRICH C. The effects of
temperature changes upon the
chromatophores of crustaceans, 193.
SMITH, GEORGE MILTON. A mechanism
of intake and expulsion of colored
fluids by the lateral line canals as
seen experimentally in the gold-
fish (Carassius auratus), 313.
Sperm entry in the starfish egg, 344.
Squid, copper content and minimal
molecular weight of hemocyanin
of, 18.
Starfish egg, manner of sperm entry
in, 344.
Structure of the plasma membrane.
present conception of, 1.
STUNKARD, H. W. and R. F. NIGRELI.I.
On Distomum vibex Linton, with
special reference to its systematic
position, 336.
TEMPERATURE CHANGES, ef-
fects upon chromatophores of crus-
taceans, 193.
Temperature of certain poikilotherms,
as influenced by humidity, 52.
TYLER, ALBERT. See Morgan and
Tyler, 59.
VARIATION, in body and cell size
of Drosophila melanogaster, 85.
Vatna, Sup. Rat vas deferens cytology
as a testis hormone indicator and
the prevention of castration changes
by testis extract injections, 322.
^/"HITAKER, DOUGLAS and T.
H. MORGAN. The cleavage of
polar and antipolar halves of the
egg of Chfetopterus, 145.
£OOTHAMNIUM ALTERNANS,
growth and differentiation of colon-
ies, 28.
Volume LVIII Number 1
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Woods Hole, Massachusetts
•
Editorial Board
GARY N. CALKINS, Columbia University FRANK R. LlLLIE, University of Chicago
E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago
E. N. HARVEY, Princeton University GEORGE T. MOORE, Missouri Botanical Garden
SELIG HECHT, Columbia University T. H. MORGAN, California Institute of Technology
M. H. JACOBS, University of Pennsylvania G. H. PARKER, Harvard University
H. S. JENNINGS, Johns Hopkins University W. M. WHEELER, Harvard University
E. E. JUST, Howard University EDMUND B. WILSON, Columbia University
ALFRED C. REDFIELD, Harvard University
Managing Editor
FEBRUARY, 1930
Printed and Issued by
LANCASTER PRESS, inc.
LANCASTER, PA.
THE BIOLOGICAL BULLETIN is issued six times a year. Single
numbers, $1.75. Subscription per volume (3 numbers), $4.50.
Subscriptions should be addressed to the Biological Bulletin,
Prince and Lemon Streets, Lancaster, Pa. Agent for Great
Britain: Wheldon & Wesley, Limited, 2, 3 and 4 Arthur Street,
New Oxford Street, London, W.C. 2.
All communications and manuscripts should be sent to the
Managing Editor, 240 Longwood Avenue, Boston, Mass.
Entered October 10, 1902, at Lancaster, Pa., as second-class matter under
Act of Congress of July 16, 1894.
CONTENTS
Page
HOBER, RUDOLPH
The First Reynold A. Spaeth Memorial Lecture. The Pres-
ent Conception of the Structure of the Plasma Membrane . . 1
MONTGOMERY, HUGH
The Copper Content and the Minimal Molecular Weight of
the Hemocyanins of Busycon Canaliculatum and of Loligo
Pealei 18
FAURE-FREMIET, E.
Growth and Differentiation of the Colonies of Zoothamnium
Alternans (Clap, and Lachm.) 28
HALL, F. G., and ROOT, R. W.
The Influence of Humidity on the Body Temperature of Cer-
tain Poikilotherms 52
MORGAN, T. H., and TYLER, ALBERT
The Point of Entrance of the Spermatozoon in Relation to the
Orientation of the Embryo in Eggs with Spiral Cleavage ... 59
LUTZ, BRENTON R.
The Effect of Low Oxygen ^Tension on the'Pulsations of the
Isolated Holothurian Cloaca 74
ALPATOV, W. W.
Phenotypical Variation in Body and Cell Size of Drosophila
Melanogaster 85
JACOBS, M. H.
Osmotic Properties of the Erythrocyte, I. Introduction. A
Simple Method for Studying the Rate of Hemolysis 104
Volume LVIII Number 2
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
£v> "»*
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
GARY N. CALKINS, Columbia University FRANK R. LlLLIE, University of Chicago
E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago
E. N. HARVEY, Princeton University GEORGE T. MOORE, Missouri Botanical Garden
SELIG HECHT, Columbia University T. H. MORGAN, California Institute of Technology
M. H. JACOBS, University of Pennsylvania G. H. PARKER, Harvard University
H. S. JENNINGS, Johns Hopkins University W. M. WHEELER, Harvard University
E. E. JUST, Howard University EDMUND B. WILSON, Columbia University
ALFRED C. REDFIELD, Harvard University
Managing Editor
APRIL, 1930
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.
THE BIOLOGICAL BULLETIN is issued six times a year. Single
numbers, $1.75. Subscription per volume (3 numbers), $4.50.
Subscriptions and other matter should be addressed to the
Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa.
Agent for Great Britain: Wheldon & Wesley, Limited, 2, 3 and
4 Arthur Street, New Oxford Street, London, W.C. 2.
Communications relative to manuscripts should be sent to the
Managing Editor, 240 Longwood Avenue, Boston, Mass.
Entered October 10, 1902, at Lancaster, Pa., as second-class matter under
Act of Congress of July 1 6, 1894.
CONTENTS
Page
HOADLEY, LEIGH
Some Effects of HgCl2 on Fertilized and Unfertilized Eggs
of Arbacia punctulata 123
WHITAKER, DOUGLAS, and MORGAN, T. H.
The Cleavage of Polar and Antipolar Halves of the Egg of
Chaetopterus 145
REDFIELD, ALFRED C.
The Absorption Spectra of Some Bloods and Solutions Con-
taining Hemocyanin 150
CONKLIN, CECILE
Anoplophrya marylandensis n. sp., a Ciliate from the Intes-
tine of Earthworms of the Family Lumbric dae 176
DEMPSTER, W. T.
The Growth of Larvae of Ambystoma maculatum under Nat-
ural Conditions 182
SMITH, DIETRICH C.
The Effects of Temperature Changes upon the Chromato-
phores of Crustaceans 193
Ol
Volume LVIII Number 3
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
GARY N. CALKINS, Columbia University FRANK R. LlLLIE, University of Chicago
E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago
E. N. HARVEY, Princeton University GEORGE T. MOORE, Missouri Botanical Garden
SELIG HECHT, Columbia University T. H. MORGAN, California Institute of Technology
M. H. JACOBS, University of Pennsylvania G. H. PARKER, Harvard University
H. S. JENNINGS, Johns Hopkins University W. M. WHEELER, Harvard University
E. E. JUST, Howard University EDMUND B. WILSON, Columbia University
ALFRED C. REDFIELD, Harvard University
Managing Editor
JUNE, 1930
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.
THE BIOLOGICAL BULLETIN is issued six times a year. Single
numbers, $1.75. Subscription per volume (3 numbers), $4.50.
Subscriptions and other matter should be addressed to the
Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa.
Agent for Great Britain: Wheldon & Wesley, Limited, 2, 3 and
4 Arthur Street, New Oxford Street, London, W.C. 2.
Communications relative to manuscripts should be sent to the
Managing Editor, 240 Longwood Avenue, Boston, Mass.
Entered October 10, 1902, at Lancaster, Pa., as second-class matter under
Act of Congress of July 16, 1894.
CONTENTS
Page
COE, WESLEY R.
Unusual Types of Nephridia in Nemerteans 203
GRAY, I. E., and HALL, F. G.
Blood Sugar and Activity in Fishes with Notes on the Action
of Insulin 217
BLUM, HAROLD F.
Studies of Photodynamic Action. I. Hemolysis by Previously
Irradiated Fluorescein Dyes 224
REDFIELD, ALFRED C.
The Equilibrium of Oxygen with the Hemocyanin of Limulus
polyphemus determined by a Spectrophotometric Method . . 238
HOADLEY, LEIGH
Polocyte Formation and the Cleavage of the Polar Body in
Loligo and Chaetopterus 256
PICKFORD, GRACE EVELYN
The Distribution of Pigment and other Morphological Con-
comitants of the Metabolic Gradient in Oligochaets 265
SlVICKIS, P. B.
Distribution of Setae in the Earthworm, Pheretima ben-
guetensis Beddard 274
JAHN, THEODORE L.
Studies on the Physiology of the Euglenoid Flagellates.
II. The Autocatalytic Equation and the Question of an Auto-
catalyst in Growth of Euglena 287
HARVEY, ETHEL BROWNE
The Effect of Lack of Oxygen on the Sperm and Unfertilized
Eggs of Arbacia punctulata, and on Fertilization 288
RAFFEL, DANIEL
The Effect of Conjugation within a Clone of Paramecium
aurelia 293
SMITH, GEORGE MILTON
A Mechanism of Intake and Expulsion of Colored Fluids by
the Lateral Line Canals as Seen Experimentally in the
Goldfish (Carassius auratus) 313
VATNA, SUP
Rat Vas Defer ens Cytology as a Testis Hormone Indicator
and the Prevention of Castration Changes by Testis Extract
Injectims ' 322
W., and NiGRELLl, R. F.
OiDistomum vibex Linton, with Special Reference to its
Systematic Position 336
CHAMBERS, ROBERT
The Manner of Sperm Entry in the Starfish Egg 344
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