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THE JOURNAL
OF ‘
EXPERIMENTAL ZOOLOGY
EDITED BY
WILLIAM E. CastTLE Jacques LoEB
Harvard University The Rockefeller Institute
Epmunp B. WILSON
Columbia University
Epwin G. CONKLIN
Princeton University
THomas H. MorGan
CHARLES B. DAVENPORT
Columbia University
Carnegie Institution
GEORGE H. PARKER
HERBERT S. JENNINGS es Una ceveiee
Johns Hopkins University
RAYMOND PEARL
FRANK R. LILLIE Maine Agricultural
University of Chicago Experiment Station
and
Ross G. HARRISON, Yale University
Managing Editor
VOLUME 25
1918
THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY
PHILADELPHIA, PA.
CONTENTS
No. 1. FEBRUARY
Auicre M. Bortne AND RayMonpD PEARL. Sex studies. XI. Hermaphrodite
birds.) Nine text figuressandenine plates. =... J.tetcs so. see eee =
Rogsert StantEY McEwen. The reactions to light and to gravity in Droso-
philagand its mutantseeebnree fpures! 2 o-is2.. 2. qe meric ges selene tetas
C. V. Morrity. Some experiments on regeneration after exarticulation in
Diemyctylus viridescens. Ten figures (three plates)...................
Epvuarp Usuenuutn. Is the influence of thymus feeding upon develop-
ment, metamorphosis and growth due to a specific action of that gland?
J. M.D. Oumstep. The regeneration of triangular pieces of Planaria macu-
lata: Avstudysin:polarity.. Fourteen: figures... .. 2-22 det. 05 gece opens
Manton Corpetanp. The olfactory reactions of the marine snails Alectrion
obsoleta (Say) and Busycon canaliculatum (Linn.)....................
Sexic Hecut. The physiology of Ascidia atra Lesueur. I. General physi-
Olo py APU Cenet UTES hc. sire tree tare ai eg RNs ep ave Seacoast gers oe
Seria Hecut. The physiology of Ascidia atra Lesueur. II. Sensory physi-
LO my enor HPULE Ba. © oc ies aie, <3. 2,0,8 6s ele otoea ea atone aacapetol> etete otnw a, Re ears
INOsg2. eee wets
Cuester A. STEWART. Changes in the relative weights of the various parts,
systems and organs of young albino rats underfed for various periods.
(CYNE: Thea Tiel e S aas ecctea cece BG SN Ven Sans Ss Cover SAR Aerts See oa
Expon W. Sanrorp. Experiments on the physiology of digestion in the
Blatihidvess lowenty-oneslouresn... 10s sscncs aoa am oe de care ae era
Ross G. Harrison. Experiments on the development of the fore limb of
Amblystoma, a self-differentiating equipotential system. Forty-five
OrREN Lioyp-JoNES AND F. A. Hays. The influence of excessive sexual ac-
tivity of male rabbits. I. On the properties of the seminal discharge. .
S. R. DetwiterR. Experiments on the development of the shoulder girdle
and the anterior limb of Amblystoma punctatum. Thirty-three figures.
P. W. Wurrtine. Inheritance of coat-color in cats. Two plates (eight
fT UTES): ety eR PE er Aare ME Pn Ar ales ste ncrs lieu evcts ck do Tavera a eterauetors
Frank A. Hays. The influence of excessive sexual activity of male rab-
bits. II. On the nature of their offspring. Twenty-two charts.......
301
355
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AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY
THE BIBLIOGRAPHIC SERVICE, DECEMBER 22
SEX STUDIES
XI. HERMAPHRODITE BIRDS!
ALICE M. BORING AND RAYMOND PEARL
NINE TEXT FIGURES AND NINE PLATES
1. INTRODUCTION
Studies IX and X of this series have dealt with the normal
structure of the reproductive organs in the domestic chicken.
That matter was undertaken as a preliminary to the study of
certain hermaphrodite birds which have come into the posses-
sion of the Maine Agricultural Experiment Station. It is only
possible to interpret abnormal conditions in the light of and by
comparison with the normal. The present study undertakes a
description of the external sex characters and internal reproduc-
tive structures or these abnormal birds. The general bearing of
both normal and abnormal structures in the reproductive sys-
tems of the chicken on theories of secondary sex characters will
be discussed in still another paper, which has to be delayed for
some time as one of the authors (R. P.) has been called by the
government to turn his attention to war time problems.
Il. MATERIAL AND METHODS
The material for this study comprises a large number of birds
belonging to the poultry plant of the Maine Agricultural experi-
ment Station. The work has centered around eight hermaphro-
dite birds. Five of these birds were bought from Herr Houwink,
a well known writer on problems of heredity and evolution in
poultry, in Meppel, Holland. They are Drentisch fowls, the
common breed of North Holland. He states that “these fowls
1 Papers from the Biological Laboratory of the Maine Agricultural Experiment
Station No. 116.
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 1
FEBRUARY, 1918
eZ ALICE M. BORING AND RAYMOND PEARL
are cultivated by inbreeding.”” In 1909, he started this particu-
lar stock with one cock and six hens. From then on, he has
inbred them. In the F, generation in 1911 there appeared two
hermaphrodites out of 80 birds, and in F; in 1912, there were
three more hermaphrodites out of 80 birds. These are the five
birds sent to Maine. These were by no means all the birds of
the sort which Herr Houwink had produced. When one of the
writers (R. P.) visited his breeding plant in Meppel in 1910 there
were then on hand a considerable number of these supposed
hermaphrodite birds. Herr Houwink was of the opinion, as
already stated, that inbreeding was the cause of these abnor-
malities, but without producing evidence which was conclusive
to this effect.
These Holland birds were numbered 1425, 1426, 1427, 1428 and
1429 on the Experiment Station poultry plant, and will go by
these numbers in this paper. One of the other three hermaphro-
dites was a small black bird sent to the Experiment Station by
Prof. Horace Atwood of West Virginia. This will be referred to
as Atwood’s black hermaphrodite. The seventh hermaphrodite
appeared in the Station flock of pure Barred Plymouth Rock
ancestry. The number of this bird is 1349. The eighth bird,
1616, was sent from Michigan by Professor Dexter. These eight
birds vary in the proportion of male and female in their make-up,
both as to external and internal structure, and behavior. It
seems clearest to arrange them in a series from the most female
at one end to the most male at the other end as the various
features of structure and behavior are taken up, using the
structure of the prmary sex organs as the basis for this serial
arrangement.
This study has been carried a little further and included other
birds with abnormal sex behavior, but normal structure. That
is, to complete the more female end of the series, we have studied
several apparently normal hens that tread other hens. Of these,
1422 was sent from West Virginia by Professor Atwood as a pos-
sible hermaphrodite but has behaved normally in Maine. K134
and M408 were two such birds reared at the Station plant and
finally killed to study their anatomy and histology.
SEX STUDIES. XI 3
To complete the more male end of the series we have studied
two guinea-chicken hybrids, one raised on the Maine poultry
plant and the other sent in for examination from the country.
Their gonads appeared to be testes, but their behavior was
indifferent.
This series of birds has been studied from as many aspects as
possible. The external secondary sex characters have been ob-
served and many of the birds photographed to show any devia-
tions from the normal. The behavior of the entire series has
been carefully watched under various conditions and recorded.
The birds were finally killed to study the anatomy and histology
of the reproductive organs. The alimentary tract and associated
organs were removed and a piece of the back with the entire
urogenital system was put in McClendon’s fluid (10 per cent
formalin, sugar, lime) to fix and harden. Then a careful dis-
section was made of the urogenital system, and this was photo-
graphed. The reproductive organs and ducts were then sec-
tioned and studied histologically. Delafield’s haematoxylin and
orange G were used for the general histological condition of the
glands. The more special staining methods for studying inter-
stitial cells have been considered in detail in study IX of this
series.
III EXTERNAL SEX CHARACTERS AND SEX BEHAVIOR
Secondary sexual characters are very distinct and attain a
relatively high degree of development in the domestic fowl.
The cock bird is distinguished from the hen in a whole series of
obvious respects. Of these the best known and most apparent
are; (1) greater body size; (2) different shape of body and car-
riage; (3) large comb and wattles; (4) presence of spurs; (5)
presence of differentiated feathers in hackle, saddle and tail, (6)
in some breeds dimorphism in color and pattern. There are
many finer differences which can be recognized by the poultry
expert.
Presence of testes is supposed to cause the appearance of one
set of these characters; presence of ovary, the appearance of the
other. Actually in some ten years study of these characters in
4 ALICE M. BORING AND RAYMOND PEARL
poultry at the Maine Station it has been found that there is no
single one of the so-called male secondary sex characters which
has not at some time been seen well developed in an otherwise
perfectly normal female. Laying females with spurs, or with
large, male-like comb and wattles, while not common, still appear
every year in small numbers amongst the large numbers of birds
in the whole flock. Females with male secondary plumage are
rarer but still occur. Putting all our experience together we
have come finally to the view that there is no distinct secondary
sexual character for which the correlation with the primary or-
gan is perfect and unvarying.
Taking a general survey of the external characters of the birds
in this study, and taking especially into account the shape of
body and body carriage, they would fall into line from most
female to most male in the same order as mentioned above,
that is, the sum total of external characters, and particularly
the body shape and carriage, would correspond roughly to the
internal anatomy. They cannot be compared as to body size
as they were not all of the same breed nor were they of the same
age when killed for anatomical study.
Various female birds in the Station flock, which are normal
laying birds at times tread the other hens. Two such birds,
K134 and M408 were studied for comparison with the abnormal
birds. These were entirely normal females as to all external
structural characters. 1134 was a Barred Plymouth Rock and
Black Hamburg cross. She laid 184 eggs in 1913-14, and 84
from October to June of 1914-15, at which time she was killed.
M408 was a Rhode Island Red and had laid 96 eggs up to the
time of her death which occurred before she was a year old.
No. 1422 was a White Leghorn sent to the Station by Professor
Atwood of West Virginia, because he thought she might be a her-
maphrodite. Her behavior, however, at Maine was that of a
perfectly normal female. Her egg record shows 25 eggs during
the two months of September and October while she was at
Maine. During the winter she laid no more and died in Febru-
ary. There were no male second sex characters present in any
of these birds. We shall see later in detail that this habit of
SEX STUDIES. XI 5
treading hens has no evident basis in the structure of the repro-
ductive apparatus.
The external characters of four of the Holland birds is shown
in their photographs (figs. 1 to 4). Unfortunately 1425 died
before she was photographed. None of these birds look like
entirely normal females or males. No. 1429 (fig. 1) appears the
most female and 1426 (fig. 4) the most male. .They all have
large combs, 1426 the largest, and 1428 the smallest. The wattles
on 1426 and 1428 are larger than on the other two. The spurs
are large on 1427 and 1429, and very small on 1426 and 1428.
Quite evidently these three so-called secondary sex characters
do not correspond with body shape and carriage, as the most
male has small spurs and the most female large comb and spurs.
None of these five birds ever laid an egg or showed any sex
behavior. They stood around the pens in a perfectly indifferent
manner and never offered to fight either pullets or cockerels.
Atwood’s hermaphrodite was also unfortunately not photo-
graphed. The records show that she had a small comb, large
spurs, male carriage, female body shape, and that she was henny-
feathered. She had been presented to the Station by Mr.
Atwood of West Virginia because of her hermaphrodite character-
istics. Her behavior was watched while she was here at the poul-
try plant, and that proved to be also hermaphrodite in that
the bird fought both males and females. Normally in the fowl
females fight only with females and males only with males.
She is recorded as ‘‘a great fighter.” In fact, she met her death
as a result of a fight after she had been transferred from one pen
to another. The detailed account of structure in a later section
of this paper shows that this bird had a hermaphrodite gonad,
but that the germ cells were immature.
Bird 1349 was raised on the Station poultry plant, a cross
between a Barred Plymouth Rock and a Game. It was clearly
a female when it first reached maturity, but gradually developed
more and more male characters, until at the time of its death,
it was rather strongly male of the game type. Figure 5 was
photographed during the moult, but shows the male carriage.
The size of comb and spurs was typically male at the time of
6 - ALICE M. BORING AND .RAYMOND PEARL
death. There were no wattles, the head being typically female
in this regard. There were no true sickle feathers in the tail,
although the tail was better developed, i.e., more towards the
male type than in the normal female. There were no typical
male hackle or saddle feathers. This bird was completely a her-
maphrodite as to behavior, as it was observed to act alternately
as a male and as a female in copulation. It never laid an egg.
The bird was generally kept in a coop by itself except when being
watched under special conditions to record its behavior. When
put in a pen with pullets, it would act as a male, but as a female
when put in with the cockerels. Finally one day when it was
put down in a yard with some other hens to observe its behavior,
it started to fight and was dead in five minutes. Reference in
the later sections of this paper will show that the structure of
the gonad at the time of death indicated that it was changing
over from ovary to testis.
Bird 1616 (fig. 6) was sent to the Station as an abnormal cock-
erel by Dr. Dexter from Michigan in May, 1915. The bird was
a cross between a Rhode Island Red male and a Plymouth Rock
female. He was a year old when sent to Maine. For the Michi-
gan period of his life, he is described as entirely hen-feathered
and except for the head which is that of a rooster, he looked like
a hen. He was very active and crowed a great deal more than
usual. The other roosters chased him all over the farm, and
the hens would not permit him to copulate with them, although
he made frequent attempts to do so. After he reached Maine
the female characters became more pronounced and the bird
was recorded as a crowing hen. On July 31 she laid an egg, and
between that time and August 25 she laid 12 eggs in all and
nested twice. From then on she laid no more eggs and began
to become more malelike in appearance. She was killed on March
15, 1916, and at that time her external characters were a mix-
ture of male and-female. She had no spurs, merely slight knobs
such as all females have. She was very fat. She had no saddle
feathers, but the hackle feathers were partly male. These her-
maphrodite external characters ,correspond to the anatomical
conditions described later—that is, the bird has an ovotestis, with
SEX STUDIES. XI ae
indications of recent or present activity in both the male and
female parts of the gonad.
The study of the external characters and behavior of these
hermaphrodite birds shows a great variety of combinations. Evi-
dently the body shape and carriage and the plumage hang to-
gether more consistently as secondary sex characters than the
spurs, comb and: wattles. The latter group vary too much to be
considered as proofs of maleness or femaleness. Sex behavior
varies all the way from complete indifference to active reproduc-
tion. Three of the birds show double sex behavior acting as a
male or female under different conditions at the same general
period, or showing a gradual change from the behavior of one sex
to that of the other. A case somewhat similar to that of 1616
has been described by O. N. Eastman in the Poultry Advocate
for September, 1916. At first he did not know whether to class
this bird as a pullet or a cockerel, but as she became more mature
she looked like a pullet with a head like a cockerel. She began
to lay in November, 1915, while housed in a pen with pullets
and one cockerel. This one cockerel chased her so incessantly,
as one male bird does another, that she had to be removed to a
pen with only pullets. In August, 1916, she was seen to chase
and mate with a pullet, and she repeated this behavior several
times. This comparison will be taken up again after the full
description of the anatomy and histology of the sex organs of
these birds.
The two guinea chicken hybrids were entirely male in external
characters, but absolutely indifferent as to behavior. They stood
around the pens in much the same way as the Holland birds,
taking no interest in either males or females. We shall see later
this indifferent behavior is not accompanied by any gross ab-
normalities in the form of the reproductive organs—that is, not
to such anomalies as an oviduct or oocytes, but to a lack of dif-
ferentiation in the testis tissue.
oe)
ALICE M. BORING AND RAYMOND PEARL
IV. WOLFFIAN DUCTS IN NORMAL FEMALE BIRDS
Before describing the internal structures of this series of ab-
normal birds, it is necessary to mention certain points of normal
bird anatomy which have been observed in this connection.
Many of these points have already been described by Goodale.
Our own observations are recorded here simply in corroboration
of his, and because in certain particulars our evidence is more
detailed. The normal reproductive system of a male bird com-
prises two testes and two vasa deferentia. The normal repro-
ductive system of a female bird includes a left ovary and a left
oviduct. The right ovary and oviduct start to develop in the
embryo, but stop before long so that they do not function in the
adult. By the fifth or six day, according to Semon, the right
ovary is already smaller than the left. The right oviduct forms
as a right Mullerian duct, and usually degenerates along with
the ovary. However, this duct may sometimes continue to
grow and persist in the adult as a non-functioning oviduct.
There are fairly frequent cases of this recorded among the
autopsies of the birds of the Maine Experiment Station poultry
plant.
Every embryo female chick has besides its two gonads and two
Mullerian ducts, two Wolffian ducts which have been supposed
to degenerate in the female as the Mullerian ducts do in the
male. The statement in Lillie’s ‘‘ Development of the Chick”’
reads as follows: ‘‘In embryos that become females, the gonad
develops into an ovary, the Wolffian duct disappears or becomes
rudimentary, the Mullerian duct develops into the oviduct on
the left side and disappears on the right side.”’
In studying the anatomy of the hermaphrodite birds, the kind
of ducts present was at first taken as an indication of sex—that
is, the presence of vasa deferentia was regarded as a sign of male-
ness, and the presence of an oviduct as the corresponding sign
of femaleness. According to this criterion, all these birds were
hermaphroditic as they had a left oviduct and two vasa. The
vasa were small ducts which had to be searched for in the peri-
toneum but sections showed them to be tubes lined with columnar
SEX STUDIES. XI 9
epithelium, surrounding a distinct lumen, and therefore impos-
sible to be confused with either bloodvessels or nerves.
The same condition of ducts was found in bird No. 1422, the
bird sent from West Virginia with a record of treading hens.
Everything else about the anatomy of this bird was that of a
normal female—she laid eggs and showed no abnormal behavior
after reaching Maine. The suspicion arose that these small
non-functional vasa deferentia of the hermaphrodites might
signify simply an embryonic condition,—that is, persisting
Wolffian ducts—rather than maleness. Further, the fact of
finding them present in 1422, otherwise a normal female, sug-
gested the possibility of their beimg a normal feature of the
anatomy of an adult female bird. In Lillie’s ‘‘ Development of
the Chick” there occurs the following statement: ‘“‘In the
female, the Wolffian duct degenerates; at what time is not
stated in the literature, but presumably along with the Wolffian
body.” The persistence of the ducts of the other sex in adult
vertebrates is not an. unheard of phenomenon,—in fact, it is
the normal condition in the common leopard frog for the Mul-
lerian ducts to persist in the adult male.
To work out this point, as to how long the Wolffian ducts
persist in the female bird, dissections were made of a number of
just hatched chicks and chicks from pipped eggs, seven of which
proved to be females, and of five laying hens. All of them had
Wolffian ducts. They were not as large as in the male—in
fact, sometimes they looked like white threads along the peri-
toneum lateral to the ureter at the posterior end, crossing it
about half way between cloaca and gonad, and extending further
anterior than the ureter near the midline to the remnants of the
mesonephros. Sometimes they were as large as normal, but
never had as many coils at the posterior end. Figure 7 is
a dissection of one of the laying hens. ‘To be sure that this
white line was not a nerve or bloodvessel, parts of it were sec-
tioned in each bird. The columnar epithelial lining identified
it unmistakably (figs. 8 and 9). Figure 8 shows the vas along-
side of an artery, a vein, and the ureter. Each is easily identi-
fied. These ducts show variation in structure. Sometimes
10 ALICE M. BORING AND RAYMOND PEARL
they are single straight tubes, sometimes they show the char-
acteristic coils of a vas. Always toward the anterior end, and
sometimes posteriorly also they have branches. These are
probably remnants of the connection with the mesonephric
tubules in the embryo.
In the laying hens, it is sometimes hard to identify the Wolf-
fian duct on the left side on account of the coils of the large
oviduct. That it persists on the left as well as on the right
side, however, is established beyond a doubt by the fact that it
was found on some of the laying hens, and also by the fact that
all seven of the young female chicks had two Wolffian ducts.
We agree then with Goodale that the presence of Wolffian ducts
in an adult bird is not necessarily a sign of maleness.
V. ANATOMY AND HISTOLOGY OF ABNORMAL BIRDS
The internal structure of this series of abnormal birds shows
varying degrees of abnormality, and the interest of the study
lies chiefly in seeing whether there is a close correspondence
between these and the abnormalities of external structure and
behavior. We have seen that the normal female has two Wolf-
fian ducts of varying sizes, besides the left ovary and oviduct,
so that the presence of Wolffian ducts is not a sign of maleness.
No oviduct has, however, been found in any male, so its pres-
sence may be considered a sign of femaleness of internal struc-
ture. The external appearance of the reproductive organs has
proved to be insufficient to distinguish between an ovary and
a testis. An organ with a few projecting oocytes may be partly
testis, and an organ without any visible oocytes may be ovary,
testis or both. In deciding whether certain tissue is ovary or
testis, the only indisputable criterion is when it has oocytes or
spermatozoa. The general structure of the organ is, however,
usually sufficient to show the difference in sex even when in
an inactive condition, the testis being composed of tubules
with a small quantity of connective tissue between them, and
the ovary being largely stroma. However, there are some in-
termediate conditions found when it is difficult to sex the organ
as they both develop from a stage when the sex cords grow
SEX STUDIES. XI 11
out from the mesonephros into the germinal epithelium. The
interstitial cells might be used as an index of sex, because of
their consistent absence in Barred Plymouth Rock males over
six months of age, if it were not for the fact that the gonads
in some of these hermaphrodites seem to be cases of arrested
development, and interstitial cells have been found in young
just hatched males. The nests or groups of clear cells which
normally fill up the discharged follicles and form the ‘corpus
luteum’ are never found in the normal male. So their presence
may be counted for femaleness.
Many cases of hermaphrodite birds have been eased by
various authors, with varying degrees of maleness and female-
ness combined. More of them seem to be females which have
developed some male characters than vice versa. Some of these
abnormal combinations of external characters are associated
with corresponding internal abnormalities and some are not.
We shall discuss these cases after we have described the anatom-
ical and histological conditions found in the hermaphrodites
with which the present study is concerned.
In the description of their anatomy and histology the birds
will be taken up in the order stated above—that is, beginning
at the more female end of the series. Three of the birds with
normal external characters and abnormal behavior were killed
and dissected. No. 1482 was an entirely normal female with
ovary and oviduct on the left, and persistent Wolffian ducts.
K134 and M408 were killed and dissected and portions of the
ovary preserved and sectioned. Both of these also proved to
be normal females in structure. The ovaries were large with
many protruding oocytes of varying ages. The sections showed
also many small oocytes embedded in the surface layer of the
ovary. Both lutear cells and interstitial cells were present, in
the same general arrangement and number as in the normal
laying hen. It would seem then that the abnormal maleness of
behavior in these three birds does not depend on any abnormal-
ities of structure, there being no male organs present, or any
male cells in the ovary, and there is present the full quota of
female organs and cells.
12 ALICE M. BORING AND RAYMOND PEARL
We shall consider next the Holland birds, and begin with
the most female of them. No. 1429 is shown in dissection in
figure 10. This is a photograph of a part of the back of the
bird with all viscera removed except the urogenital system.
The posterior end can be distinguished by the small piece of
rectum remaining where the digestive tract was cut off slightly
above the cloaca. The ducts can be seen, all connecting with
the cloaca, and extending anteriorly to the region of the repro-
ductive organ which lies at the most anterior end of the dissec-
tion. This one reproductive organ lies to the left and is an
ovary with many oocytes visible to the naked eye, and a few
very small orange spots like remnants of corpora lutea. The
large round dark object to the left of the ovary is a tumor more
than twice the size of the ovary. There is a normal oviduct
with coils but the bird never laid an egg while in the Station
flock. The two median ducts are the ureters. The right Wolf-
fian duct shows its entire length plainly from cloaca to a spot
opposite the ovary. It is somewhat coiled at the posterior
end. At the anterior end, there is a slight enlargement, which
proves in section to be a mass of tubules, resembling an epididy-
mis. This is probably the remains of the mesonephric tubules,
sometimes spoken of as a parovarium. A small portion of the
left Wolffian duct shows in the photograph median to the ovi-
duct. This is also somewhat coiled. There seems to be nothing
male about the anatomy of this bird. It has an ovary and ovi-
duct on the left and two Wolffian ducts. The only abnormal
feature is the large tumor, which, of course, shows a diseased
condition. The anatomy of the bird is, in short, that of a fe-
male in the non-laying condition with a large tumor. The
histological study of the ovary shows it to differ in some points
from both the old Campine past the laying condition and the
actively laying birds, described in studies [IX and X. In general
it resembles the Campine more closely; that is, there is a large
relative amount of stroma and there are no very small develop-
ing oocytes (fig. 18). The oocytes present are of medium size
and lie in normal follicles. In addition there are a number of
cystic follicles filled with a watery fluid. These are visible to
SEX STUDIESS | Su 13
the naked eye, and some show in section in figure 18. There
is the usual large number of nests of lutear cells in the theca
interna, as shown in figure A. Some of these lutear cells
present a feature not seen in any other bird, they contain a large
number of acidophile granules. That these cells with granules
are not the interstitial cells can be seen by comparing figures
A and B. Figure B is a group of interstitial cells from the
stroma of this same ovary. The interstitial cells are smaller
and more closely packed with granules. The only difference
among the cells in figure A is that some are clear and some have
granules. They are the same as to size and nucleus. The
number of real interstitial cells is small (fig. C). In various
parts of the gonad, there are older lutear cells with the yellow
pigment. Whether these corpora lutea represent discharged
or atretic follicles it is impossible to decide, as the involution
process has proceeded beyond the stage where this distinction
can be made. The microscopical structure together with the
eross anatomy show 1439 to be a female.
There is not much choice as to the order in which the next
three birds shall be described, as the ducts of all three are of
the female type, and the reproductive organs of all are indiffer-
ent enough to make it somewhat difficult to sex them.
The dissection of 1428 is shown in figure 11. The two Wolf-
fian ducts are easily seen in this photograph, being of consider-
able size. The bird has a larger coiled oviduct than 1429, and
a lobulated reproductive organ on the left. This organ has
a large watery tumor to the left of it, showing that it is also
in an abnormal physiological condition. It is exceedingly
difficult to sex this organ. It is largely composed of tubules,
which radiate toward the periphery from a central connective
tissue core. But the entire surface looks like an ovarian stroma
(fig. 19) and all this connective tissue at the periphery and con-
tinuing down between the tubules contains many masses of
the lutear cells normally found in the theca interna (figs. 19 ¢
and 20 t). The tubules are in some places lined with character-
istic columnar epithelium cells (fig. 20), but in most the cells
appear to be breaking down (fig. 19). In the central core of
14 ALICE M. BORING AND RAYMOND PEARL
this organ, there are some interstitial cells loaded with se-
cretion granules (fig. D) also many streaks of tumor-like
material. This bird is probably a female arrested in the de-
velopment of its gonad earlier than 1429. To be sure
weaeM
C
Fig. A Portion of follicle wall from ovary of 1429 (x 570). G, epithelial
layer; N, nest of lutear cells in theca interna; A, lutear cells containing acido-
phile granules.
Fig. B- Portion of stroma of ovary from 1429 showing a few interstitial cells
loaded with granules (X 570).
Fig. C Portion of periphery of ovary from 1429, showing the small number
of interstitial cells present (X 264).
SEX STUDIES. XI 15
the tubules look like testicular structures, but they may in-
stead be mesonephric, a condition which might be found in
an embryonic ovary. In fact, it looks much like the ovary of
the just-hatched chick shown in figure 32. The presence
of the lutear cells in groups in the stroma also suggest arrested
development, as in the normal development of the egg follicle,
there is a stage before the theca layers are added when only the
granulosa is present, but the nests of lutear cells are conspicuous
in the stroma near the young follicles. Although the lutear cells
are present they have not gone through their full development,
Pes Fut eek 1
ha
'
is Re eye dey fi
2h wy ep tile = Bay
rs b> ¥.
- ee
righ tan a eee
Fig. D Portion of connective tissue core of gonad from. 1428, showing num-
erous, interstitial cells (* 264).
as there is no trace of the yellow pigment in any part of the
gonad. The dissection of 1427 is shown in figure 12. The
two Wolffian ducts are shown in the photograph, the right one
with a distinctly enlarged anterior end, which is of the nature
of a mass of mesonephric tubules or parovarium, as shown by
section. This bird has also a coiled oviduct and a lobulated
reproductive organ on the left. Here again is a large dark
tumor posterior to the reproductive organ as in 1429. Figure
21 is a section of this organ. It seems largely composed of
solid cords of cells. There are no oocytes and no hollow tubes,
16 ALICE M. BORING AND RAYMOND PEARL
so it is difficult to be sure of the sex. A comparison with sec-
tions of just-hatched ovaries and testes seems to throw a little
light on its nature. Even at this stage the testis has distinct
tubules, but in the ovary, the oocytes are not yet enclosed in
follicles, but the germinal epithelium has grown down into the
stroma in solid cords (fig. 32). The appearance of the cortex
is not unlike that of 1427. Sections of 1427 stained with Mal-
lory’s connective tissue stain show no interstitial cells present,
and also no lutear cells. ‘The probable conclusion then as to the
internal structure of 1427 is that it is a female with an inactive
gonad even less differentiated than in 1428. Development was
probably checked by some pathological condition, of which the
large tumor may be an index.
The dissection of 1425 (fig. 18) is not very different from that
of 1428 or 1427 just described, except for the absence of a vis-
ible tumor. Sections show the posterior portion of the gonad
to be filled with streaks of a secretion which resembles the
substance of the tumor in 1429. This would indicate that it
is in a similar pathological condition, although no separate
tumor has been formed. Externally the reproductive organs of
these three birds could scarcely be distinguished. Internally,
however, the gonad of 1425 is more like that of 1428, in that
the central portion is composed of tubules with distinct lumena,
as shown in figure 22. There is no sign of any mitoses in any
of the tubule cells, so they are probably of mesonephric origin, —
that is, undifferentiated sex cords without any primitive germ
cells. The peripheral portion has probably as in 1428 origin-
ated from the germinal epithelium. There are no interstitial
cells present in the stroma, and the number of groups of lutear
cells between the tubules is less than in 1428. The presence
of any of the latter clinches the diagnosis of this bird as a female
arrested in development. The fewness of these cells may place
it as more primitive than 1428,—that is, in between 1428 and
1427. Unfortunately there is no record or photograph of ex-
ternal characters to compare with the other two.
The last of the Holland birds in the series, 1426, is distinctly
different from the others in its structure. It has two repro-
SEX STUDIES. XI 17;
ductive organs, and the large coiled oviduct of a laying bird
(fig. 14). At the posterior end, to the right of the cloaca is a
crumpled mass, which is apparently a partly developed right
oviduct. No Wolffian duct was found on the left side, but the
immense size of the oviduct made it difficult to dissect on that
side. The organs of the two sides are very different in external
appearance, as can easily be seen in the photograph. The right
one is an active testis and the left one an inactive ovary. Fig-
ure 23 is a photograph of a section of the testis, a large mass of
tubules with very little connective tissue between them. Part
of this same section is shown at greater magnification in figure
24. This resembles a section of a normal active testis. The
black threads are fully formed spermatozoa, and they are
bunched into groups for each Sertoli cell in normal manner. The
vas, strange to say, is no larger than in the birds with no organ
on this side, and not as much coiled as the one in 1429. The
left reproductive organ closely resembles the ones in 1428, 1425
and 1427 in external appearance. On the surface are a couple
of depressions which might be degenerate oocytes or discharged
follicles. One place shows an orange mass like a corpus luteum.
Histologically the main substance is like that of 1427, and re-
sembles the stroma of an ovary with solid cords of cells in it,
but no oocytes (fig. 25). The peripheral tissue, however, looks
like a thickened germinal epithelium and contains several large
spaces which may have been oocytes. In the place with the
orange spot on the surface there is a mass of tissue containing
groups of cells with yellow pigment material, as described for
discharged or atretie follicles in study X. The entire stroma
shows great streaklike masses of secretion taking acid stains, as
in the organs of 1425 and 1428. In one limited area of the ovary,
there are a few interstitial cells filled with granules. In bothovary
and testis there are a few nests of lutear cells near the surface.
This condition in the testis is shown in figure E, as it is unusual
to find them in a testis, and probably indicates the generally
unbalanced sex condition of the animal which looks as though
it might be changing from female to male. This is the most
interesting of the Holland birds, absolutely indifferent as to
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, No. 1
18 ALICE M. BORING AND RAYMOND PEARL
sex behavior and yet with sperm in the testis, and at least one
corpus luteum remnant on the ovary, and the oviduct of a
laying hen. In external appearance, it is more like a male than
the others, which fact correlates well with the active condition
of the testis and inactive diseased ovary with only one corpus
luteum scar. The interstitial cells can scarcely be held account-
able for the male secondary sex characters, as the only ones in
Fig. E Portion of testis from 1426, showing some lutear cells between the
seminal tubules at the periphery (X 570).
an active secreting condition are a few in the ovary. This
bird is the most balanced hermaphrodite of the series, and close-
ly resembles bird 16, described by Pearl and Curtis, as to its
internal structure, except for the active condition of the testis.
A comparison of these five Holland birds shows a general
correspondence between the degree of femaleness of their ex-
ternal characters and their internal structure. They are all
fundamentally females in which the ovary for some reason has
failed to reach complete development. In all but 1429 and 1426
the lutear cells are immature. In 1426, a testis has developed
SEX STUDIES. XI 19
on the right side. They are all of them, however, entirely in-
different as to sex behavior, and such so-called secondary sex
characters as comb, wattles, and spurs vary regardless of in-
ternal structure. The interstitial cells vary too much in their
distribution to be considered the cause of the general male-
ness or femaleness of the external characters, and they cannot
have anything to do with the large combs and spurs, as the
distribution of the two does not correspond. For example,
1427 has no interstitial cells and a very well developed comb
and spurs. No. 1428 with the most interstitial cells has the
smallest spurs. No. 1429 in spite of having the most female
carriage and most normal ovary has longer spurs than 1426,
which is the most male of the series. Spurs and combs are too
variable to be considered distinctively male secondary sex
characters. On the other hand, the distribution of the inter-
stitial cells does not correspond with either the maleness or
femaleness of body shape and carriage. However, the only
one of these five birds with any lutear pigment worth mention-
ing is 1429, the one at the more female end of the series. This
may be significant. The cause back of the lack of develop-
ment of oocytes and lutear secretion may be the general ab-
normal physiological conditions indicated by the tumors and
pus present.
We shall consider next the anatomy and histology of the
three birds with hermaphrodite behavior. Atwood’s black
hermaphrodite has two Wolffian ducts and an infantile oviduct
(fig. 15). On the left is a large irregular organ. The left half
of this lefthand reproductive organ is not unlike the organs
found in 1427, 1425 and 1428,—that is, it is irregularly lobed,
but the one largest posterior lobe looks more like a testis in
the smoothness of its surface than any of the other organs re-
ferred to. The right half of the left hand organ appears like an
ovary with small oocytes all over the surface and two small
orange spots like corpus luteum remains. Sections show this
organ to be an ovotestis. The part which appears externally
like a testis is composed of tubules. In none of them are any
advanced stages of spermatogenesis; the majority of the cells
20 ALICE M. BORING AND RAYMOND PEARL
are spermatogonial cells or spermatocytes in synizesis. ‘The
portion that resembles an ovary in external appearance has
distinct ovarian tissue on the periphery, but the center is filled
with tubules in an even less developed condition than those of
the part already described (fig. 26), but distinctly testicular as
they are filled solid with cells, not hollow like mesonephric
tubules. On the right side of the body there is an enlargement
of the anterior end of the Wolffian duct and this, upon being
sectioned, appears like the center of the ovarian portion, a
mass of small tubules with inactive cells (fig. 27). The ovarian
portion of this organ contains oocytes of many sizes. Some
of them are contained in normal follicles with the characteristic
nests of lutear cells in the theca interna, but the majority are
beginning to degenerate. Many groups of these lutear cells
lie in the connective tissue of the stroma between the follicles
and a few among the testis tubules at the center of the ovary
and among the tubules of the small testis on the right. In three
places the sections passed through atretic follicles packed with
these cells here containing clumps of the yellow lutear pigment.
This histological structure represents the orange spots visible
on the surface to the naked eye. No interstitial cells were
found in any portion of the organ. This bird is a potential
hermaphrodite in its internal structure, a fact of especial in-
terest in view of its hermaphroditic behavior. The structure
looks as though it were changing from female to male. The
oocytes are mostly starting to degenerate and some atretic
follicles are already filled with lutear cells containing the char-
acteristic pigment. The presence of lutear cells among the
testis tubules looks as though the tubules were growing from
the center outward and forcing their way into the ovarian tissue
at the surface. But neither the ovarian or testicular tissue
was in active condition when the bird was killed.
No. 1349 is the second bird with hermaphrodite behavior.
Figure 16 is a dissection of the reproductive organs. In this
bird, they were dissected out of the body and preserved as pho-
tographed before the Wolffian duct situation had been worked
out, so that it is possible that the bird possessed the normal
SEX STUDIES. XI PA
two ducts. The organs shown in the photograph are a coiled
oviduct and a gland which proves to be mostly a testis (fig. 28).
Some of the tubules show spermatids and developing sperm
(fig. 29). This organ was sectioned in eleven different regions
and only three showed any structures other than testis tubules.
One of these parts is shown in figure 30. This strongly resembles
the indifferent ovary of 1426 and 1427, so we are probably justi-
fied in calling this an ovotestis. One other portion which was
not testicular is most remarkable in structure (fig. F). It isa
large solid collection of smaller masses of lutear cells partly de-
generated and containing a few yellow pigment granules (fig. G).
There is enough of this pigment to give the mass a yellowish
_ tinge to the naked eye. There are also nests of lutear cells
in normal undegenerated condition between the tubules of the
testicular portion, as shown in figure H. This organ looks as
though it had been an ovary and was largely changed over toa
testis. It was certainly mostly testis when the bird was killed.
But the yellow pigment must represent either discharged or
atretic egg follicles and the groups of lutear cells between the
tubules suggest that these tubules have somewhat recently
invaded ovarian tissue. It is especially significant that these
groups of lutear cells lie mostly toward the surface of the gonad.
These two birds with active sex behavior have reproductive
organs in a less active condition than 1426, with absolutely
indifferent behavior. In fact, 1426 has mature sperm in the
testes, while 1349 has either immature or degenerate sperm,
and Atwood’s bird had cells ‘no further developed than the
synizesis stage. On the female side, the oviduct of 1426 is much
larger and more coiled than that of either of the other two
birds, but they all have signs of having had active ovaries,—
that is, they have several degenerating oocytes, many immature
lutear cells, and several lutear pigment masses. No. 1426 has
less of the ovarian tissue remnants, just as it has a more ad-
vanced testicular structure. There are a few interstitial cells
in 1426, but none in either of the active birds, so that active
sex behavior can scarcely be based on these. Also the differ-
‘ ences cannot be laid to the lutear cells, as they are present in
all three.
bo
bo
ALICE M. BORING AND RAYMOND PEARL
Fig. F Section of old ovarian tissue from gonad of 1349, showing stroma
filled with groups of lutear cells resembling those in discharged or atretic fol-
licles (X 100).
Fig. G Group of lutear cells from figure F, showing masses of yellow secre-
tions (X 950).
SEX STUDIES. XI oe
The third of these genuine hermaphrodites is 1616, the bird
sent by Dr. Dexter from Michigan. The anatomy of its re-
productive organs is shown in figure 17. There is a large coiled
oviduct and two Wolffian ducts. There is one large lobulated
reproductive organ on the left. ‘This is larger than in any
of the other birds studied. The surface of this organ has oocytes
of varying sizes scattered at various places, but the whole tex-
ture of the lobes is more solid than in a normal ovary. There
are fourteen corpora lutea remnants. The egg record of this
bird shows that she laid 12 eggs and nested twice, so that in
this case, the orange spots just represent discharged instead
Fig. H_ Section of testicular tissue from gonad of 1349, showing a group of
lutear cells in a space between seminal tubules (X 570).
of atretic follicles. At the anterior end of the right Wolffian
duct is the same sort of enlargement as has been mentioned for
Atwood’s bird. The microscopical study of the large organ
shows it to be an active ovotestis with the testis portion in
the more active condition when the bird was killed. ‘The main
body of the organ is testis tubules with sperm in the lumens.
The small organ on the right is also active testis with sperm.
But the peripheral portion of the large organ is distinctly ovar-
ian. It consists of thickened stroma packed with interstitial
cells like the old Campine (fig. I). It contains oocytes of all
sizes, small ones with only a granulosa layer to the follicle and
large ones with nests of lutear cells in the theca interna. There
24 ALICE M. BORING AND RAYMOND PEARL
are not nearly as many oocytes as in a normal ovary. Finally
it contains a few discharged follicles; one recently discharged
with the cavity still large and the granulosa sloughing off into
it, a second with the cavity just obliterated by the shrinking of
the walls, and a number with the degenerating lutear cells in
the center containing the yellow pigment, exactly as in normal
birds. This is normal ovarian tissue, but there is not so much
of it as there is of the testicular tissue. The composition of this
organ is more like Atwood’s bird than any other, in the propor-
tion of male and female parts. The point in which this bird
7
Fig. I Portion of periphery of gonad from 1616, showing numerous inter-
stitial cells (X 264).
differs from all others studied is that both the ovary and the
testis show signs of very recent or present activity.
It is interesting to compare the structures found in these
birds with those of some of the hermaphrodite birds previously
described by other authors. In 1889, Brandt described eight
hermaphrodites of varying structure, seven of which he con-
siders modified females. In 1906, Shattock and Seligmann
described a two year old Leghorn with an ovotestis. Also of
interest are the hermaphrodites with ovary and testis described
by Pearl and Curtis, the four mule pheasants, which are sterile
females with some male secondary sex characters, described by
SEX STUDIES. XI 25
Smith and Thomas, and a gynondromorph pheasant described
by Bond in 1914. The oviduct is an almost constant feature in
these birds. The only bird without an oviduct is one described
in Brandt’s paper. Shattock and Seligmann’s Leghorn had two
oviducts. Five of Brandt’s birds had abnormal oviducts, either
anterior or posterior end being closed. In the rest, the female
ducts were normal, just as in the birds described in this paper.
In most of these papers, nothing is said about the vasa def-
erentia. Brandt shows one picture of sections through two
persistent Wolffian ducts, the one on the right side being larger
than the one on the left. In the bird which he claims is a male
with female characters developed, he says there are no vasa
deferentia. Pearl and Curtis’s bird 16 had an oviduct on the
left for the ovary, and a vas on the right for the testis. Shattock
and Seligmann’s bird had two vasa.
In comparing the structure of the reproductive organs, there
again seems to be a preponderance of female over male. Four
of Brandt’s birds had ovaries in more or less embryonic or de-
generate condition. The four mule pheasants of Smith and
Thomas had ovaries composed of stroma and interstitial cells,
with no oocytes. Nos. 1427, 1425 and 1428 of our birds belong
to this type. One of Brandt’s birds and 1429 of this paper
had normal ovaries with oocytes. An ovotestis or mixed gland
was present in two of Brandt’s birds, in Shattock’s Leghorn,
in Bond’s pheasant, and in Atwood’s Hermaphrodite, 1349, and
the Michigan bird of the present study. No. 1426 and Pearl
and Curtis’s bird No. 16 both had an ovary on the left and a
testis on the right, with the difference that while in both, the
ovaries were inactive, the testis in 1426 was active and that in
16 was not. One bird described by Brandt had two testes, one
on each side. Hammond Smith is also quoted by Bond as
describing three birds with some female secondary sex char-
acters with normal testes present. In most of these birds,
previously described, the gonad of whichever sex showed no
signs of activity of the germ cells The first of Brandt’s birds
is the one possible exception on the female side. Several of
the birds in this study, however, show signs of past or present
26 ALICE M. BORING AND RAYMOND PEARL
activity of the ovary. No. 1429, Atwood’s bird, and the Michi-
gan bird have many oocytes of varying sizes on the periphery.
No. 1349 and 1426 have some cystic oocytes, while there are
corpora lutea representing discharged or atretic follic'es on
four birds, 1426, Atwood’s bird, 1349, and the Michigan bird.
No. 1429 has no discharged follicles, nor any very small oocytes.
No signs of ovarian activity, either past or present could be
discovered in the other three birds, 1428, 1427, 1425.
The only birds with active testis in which sperm were
observed are Bond’s pheasant, 1426, 1349, and 1616 described
in this paper, possibly also the three birds of Hammond Smith,
although we do not know how carefully the histology of these
was studied. It should be noted that all eight birds described
by the present authors may be interpreted as fundamentally
females, some of them checked in the embryonic condition of
the gonads, and some of them changing over to a male condition.
Next in the series of abnormal birds we have placed the
guinea chicken hybrids. The anatomy of these is apparently
that of perfectly normal males with two testes and two vasa
deferentia. These were of normal size in one, and much en-
larged in the other. That these testes, however, are not normal,
is clearly shown in microscopic sections (fig. 31). There is no
sign of tubules or any cells distinguishable as germ cells. The
structure looks more like ovarian stroma than part of a testis,
that is absolutely indifferent, whether ovarian or testicular in
nature. In fact, it is probably neither, but simply an undif-
ferentiated gonad, as in the early embryo. Neither are there
any cells with the distinguishing marks of interstitial cells or
lutear cells. This is, of course, an. entirely different condition
from that found in Guyer’s guinea chicken hybrids, where there
were many tubules and an abnormal synapsis either stopped
the process of sperm formation or else resulted in abnormal
spermatozoa. Poll has worked out the theory from his hybrid
birds that the more closely related two crossed birds are, the
more normal will be the spermatogenesis. This, however, does
not explain how some male guinea chickens can have abnormal
SEX STUDIES. XI 27
sperm formed and others have no trace even of seminal tubules
in their structure,
VI. DISCUSSION
The study of the anatomy and histology of this whole series
of birds somewhat abnormal as to sex shows that they are all
fundamentally female, except the guinea chickens. These
are merely sterile males with consequent indifferent behavior.
The hens with a tendency to tread other hens are normal active
females. The eight other birds are fundamentally female,
either undeveloped or degenerating. Every bird has in its
embryonic development an undifferentiated sex stage as far
as organs are concerned. It has the ducts for both sexes, and
the gonad has the same early development regardless of whether
it develops later into an ovary or testis. This undifferentiated
gonad consists of a mass of sex cords growing out from the mes-
onephros covered over by a thickened germinal epithelium. If
the bird becomes a female, the left Millerian duct enlarges and
the germinal epithelum proliferates and forms most of the
reproductive organ. If the bird becomes a male, the Miillerian
ducts degenerate, the Wolffian ducts become larger and coiled,
and the sex cords become the main part of the reproductive
organ. One of the Holland birds, 1429, is nearly a normal fe-
male. Three of the Holland birds, 1428, 1425, 1427, are evi-
dently undeveloped females. They have infantile oviducts
and embryonic ovaries. The other four birds are also fun-
damentally female, but show that the reproductive apparatus,
has passed through a female stage and has become partly or
largely male. In 1426, the ovary is partly embryonic, and part-
ly degenerating, and a testis with active sperm has formed on
the right side of the body. It has the oviduct of a laying hen.
The other three: birds have large gonads on the left side only
and in all three cases the ovarian portion shows signs of degen-
eration and the testis portion signs of development.
UB a
Pas. nt
= aes
ee
ioe
43
26
(X 40).
27
80).
28
29
176).
PLATE 8
EXPLANATION OF FIGURES
Section of ovarian part of gonad on left side of Atwood’s hermaphrodite
ts, the testis tubules in the center of the organ.
Section of very small testis on right side of Atwood’s hermaphrodite (X
Section of testis part of gonad of 1349 (X 40).
Part of figure 28, showing seminal tubules with spermatids in center (X
+t
PLATE 8
XI
AND RAYMO
SEX STUDIES.
BORING
ND PEARL
ALICE M.
PLATE 9
EXPLANATION OF FIGURES
30 Section of an ovarian part of gonad of 1349, showing stroma (X 80).
31 Section of gonad of a guinea chicken hybrid, showing that organ is com-
posed entirely of stroma (X 176).
32 Section of ovary of a just-hatched chick (x 40). c, part composed of sex
cords; e, germinal epithelium. mm is mesonephros.
46
PLATE 9
XI
SEX STUDIES.
BORING AND RAYMOND PEARL
ALICE M.
47
AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY
THE BIBLIOGRAPHIC SERVICE, DECEMBER 22
Le REACTIONS TO LIGHT AND ,TO GRAVITY. IN
DROSOPHILA AND ITS MUTANTS
ROBERT STANLEY McEWEN
Columbia University, New York City
THREE FIGURES
INTRODUCTION
The presence in the Columbia laboratory of a great variety of
mutant stocks of Drosophila ampelophila involving differences
in the size of wings and color of eyes has offered an exceptional
opportunity to investigate the relation of these variations to the
responses of the fly to light and to gravity. The wing mutations,
moreover, have been useful in checking up results obtained by
removal of part of the normal wings, while the differences in eye
color have made possible a novel series of experiments on the
effects of lights of varying wave lengths.
Finally, in one case it has been possible to study the direct
hereditary modification of a reaction system apart from any sig-
nificant morphological variation.
Although a considerable amount of work on the behavior of
Drosophila toward light and gravity has already been done,
notably by F. W. Carpenter, W. H. Cole, Fernandus Payne, A.
O. Gross and F. E. Lutz, it was nevertheless found necessary to
try out a number of preliminary experiments in order to deter-
mine the source of certain discrepancies in the results of these
investigators. This was particularly true in regard to the effect
of the factors of age and sex on normal flies.
I wish at this point to express my sincere thanks to Dr. T. H.
Morgan for his many helpful suggestions in the course of my ex-
periments. I also wish to express my indebtedness to Dr. Fran-
cis H. Herrick for allowing me the use of the laboratory at
Western Reserve University during the summers of 1915 and
49
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 1
50 ROBERT STANLEY McEWEN
1916. Finally Iam under great obligation to Dr. W. L. Severing-
haus and Mr. E. C. Unnewehr, who, under the direction of Dr.
Trowbridge determined for me the wave lengths and energy trans-
mitted by my colored filters. This work was done in the Ernest
Kempton Adams Precision Laboratory of Columbia University.
METHOD AND APPARATUS
A variety of methods were employed in the course of the work,
and it therefore seems unprofitable at this point to do more than
give a general description of the apparatus and the chief systems
used in making the tests.
A glass tube 26 mm. in internal diameter and 91.4 em. long
contained the flies. This tube was graduated in inches because
at the beginning this seemed to be about the smallest unit in
which it was possible to observe rapidly moving flies with any
degree of accuracy. One end of the tube was closed by a piece
of isinglass, while the other was stopped with a cork covered
with dead-black paper. The flies were introduced into the tube
by means of a cardboard funnel from small vials in which they
were kept. The tube was then placed with the open end toward
a window in a room which was otherwise darkened. Since it
was early discovered in accordance with the results of Carpenter
(American Naturalist, 05) that the insects were much more
phototropic when mechanically stimulated, the tube was held in
the hands during the earlier trials and gently agitated.
After a short series of experiments under these conditions it
became evident that a more refined system must be used in order
to get results in any way comparable or consistent. The two
chief factors which demanded stancardization were the means of
mechanical agitation and the source of light. The first problem
was solved as follows. The tube was fastened in a horizontal
position on a board by means of rubber bands and nails padded
by thick felt. The four nails were placed in pairs, each pair
about 22 em. from the end of the tube, in such a way that the
latter could vibrate between the nails in a direction at right an-
gles to its length and through a distance of about 5mm. A pen-
dulum was now constructed from a piece of wood a meter in
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA ol
length, to the end of which was fastened an oblong piece of lead
weighing 410 grams and heavily padded with felt. This pendu-
lum was then suspended in such a manner that at the end of each
complete vibration the padded end would strike the tube at its
middle point. The length of the swing was limited by the tube,
upon the one hand, and a shelf against which the pendulum hit
on its backward stroke. This shelf was back of the tube a hori-
- gontal distance of 50 em. and 22 cm. above it. When the tube
was placed in position for operation, it was held in place by the
rubber bands mentioned above. These bands were fastened to
a couple of tacks on the side of the tube toward the pendulum
and at equal distances from its center. These were then passed
under the tube toward the operator, and then over it to the edge
of the board toward the pendulum, where they were again fas-
tened. Thus when the pendulum was set in motion it. would
strike the tube and push it a slight distance away from the nails
against which the bands tightly held it. These would immediately
snap it back into position ready to be again displaced by the
next swing of the pendulum. Thus the tube underwent a con-
stant jarring of a fixed degree of violence and at regular inter-
vals. The pendulum was kept in motion by a slight pressure
of the hand on its upper end, delivered just at the beginning of
each backward swing. While it might be objected that this pres-
sure would vary, thus varying the length of the stroke and the
force of the blow, this variation in practice was found to be very
slight indeed. This was made possible by the fact that great
care was used to give exactly the amount of pressure necessary
to make the pendulum just reach the shelf on its backward
swing. With a little-practice this action became mechanical and
exceedingly constant. The degree of accuracy was in fact de-
termined thus. An assistant counted the number of strokes
per minute for five separate trials with an interval of a minute
and a half between each trial. The count each time was 40
strokes. The strokes were then counted for three consecutive
minutes, and the count was 118. According to the other test it
should, of course, have been 120, thus showing the very small
error of 0.6 of a stroke per minute. No greater care was exer-
cised during these tests than was normally the case in the experi-
52 ROBERT STANLEY McEWEN
ments, and while it is possible that the desire for a certain
outcome might cause an unconscious variation at times, I think
it very unlikely that this was of sufficient magnitude to seriously
alter the results.
For the purpose of providing a constant standard source of
light a 200 watt nitrogen filled Mazda lamp was suspended within
a tin box 24 em. high by 14 em. wide and 14 deep. Small holes
were made in the bottom and the top of this box to allow for
ventilation, while in its front, just opposite to the concentrated
filament of the lamp, was cut an aperture 3.5 cm. in diameter.
In front of this openingjwas dastened a flat flask containing water.
A second box of the same dimensions was now fitted to the
front of the first and in its front a-hole was cut equal in size to the
hole in box number one and exactly on a line with it. This aper-
ture was fitted with clamps for holding the flasks which were to
contain colored liquids when such were desired. If only white
light was wanted the outer as well as the inner flask contained
clear water. This arrangement of two flasks not only shielded
the insects from heat rays, but also prevented the colored liquids
from becoming hot. The flasks used were about 1.5 em. thick,
and identical flasks were employed in the same positions through-
out the experiments.
This apparatus was set up at the end of the table on which
the testing tube was fastened, in such a manner that the center
of the lamp filament was exactly opposite to the end of the tube
and about 24 em. away from it. This made the end of the tube
about 3 cm. from the outer flask. Finally a blackened screen
was set up between the end of the tube and the light boxes, with
an aperture in it opposite to the end of the tube and about the
size of the flasks. The purpose of the screen was to shade the
tube from the slightly diffused light which escaped from cracks
in the boxes and through the holes above the lamp. The room
where most of the experiments were performed had its walls
painted a dead black, and the windows were provided with black
opaque shades. All timing was done with a good watch placed
in such a position that the face was illuminated by light issuing
from the aperture. A diagram of this apparatus is shown in
figure 1.
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 53
METHODS USED FOR MEASURING REACTIONS
The following were the chief systems devised for measuring
the phototropism of flies with the above apparatus. The in-
sects to be tested were taken from the breeding bottle and ether-
ized. ‘Those to be tested were then isolated and placed in small
vials with or without food, according to the detailed conditions
of the particular experiment. When about to be tested, unless
otherwise indicated, the flies were always light adapted, after
which they were introduced into the large testing tube. At first
this was done by means of a funnel, but later on the vials were
Fig. 1. A, First box containing 200 watt lamp. B, Second box to the front
of which is fastened the outer flask D, containing either clear water or colored
liquids. C, Inner flask fastened to front of first box, and always containing clear
water. #, Tube divided into fifths valued as indicated for the purpose of cal-
culating the tropic indices. /', Black screen to shield tube from stray light rays.
padded so that the mouths would just fit into that of the tube,
into which the insects were allowed to crawl with as little shaking
as possible. When the flies had been attracted into the proper
end the tube was slipped into position and the test begun. If
agitation was desired, a lever which released the pendulum was
struck the instant that the tube was in place. When the test
in question was completed the flies were replaced in their origi-
nal position as follows. The tube was taken before a window or
bright lamp and gently tapped with the fingers or twirled be-
tween them. If this method failed, as in the case of non-photo-
tropic flies, shaking was resorted to. Variations of this sort
will be mentioned in connection with the experiment under
discussion.
‘
54 ROBERT STANLEY McEWEN
There were two chief methods used for measuring the re-
sponse. In the first of these, one fly was tested at a time. It
was placed in the end of a tube away from the light, and allowed
to remain for one minute. During that minute a record was
taken of the furthest distance which it crawled toward the light,
expressed in inches. If it had not reached the light end, it was
now put there by the method described above, and the test re-
peated. This time, however, the furthest inch which it crawled
away from the light was recorded. Each of these tests was re-
peated three times, the ends of the tube in which the insects
started being alternated. An average for the inches crawled
toward the light in each trial was then taken and an average of
the inches crawled away from it.! For convenience, the for-
mer will be referred to as the t. 1. (toward light) average and the
latter as the f. 1. (from light) average. The algebraic sum of
these two averages was taken as the t. |. or f. 1. index of the fly
in question. In any given experiment as many flies as possible
were thus tested and the sums of the t. |. indices algebraically
added to the sums of the f. 1. indices. Thus, let us suppose that
there were four flies in a group. The index of fly 1 was t. 1. 20,
that of fly 25 %t- 1 30, that’ of fly. 8; tf). 10) and ‘that“of thy 4
1.5. The index for the group would be expressed as t. 1. 35.
This method will be designated as method I.
After using this method for some time it became evident that
a plan must be devised for testing more flies at a time. This
seemed desirable because of the extreme variability of individuals,
despite all efforts to keep environmental factors constant. The
following plan was worked out for obtaining the phototropic in-
dex of insects by groups. This system proved far more satis-
factory than the one described above and has been used in all
the later and more critical work. It will be designated as
method II.
The tube in this case was divided into fifths, and to each fifth
was assigned an arbitrary value as follows. The division fur-
1 This means of expression is necessary in order to avoid the use of the term
negative. The fact that the insects crawl away from the light end does not prove
them negative, since when placed in that end they have no other choice. Further-
more, other results make it unlikely that the flies ever give a truly negative
reaction.
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 55
thest from the light counted zero, the second 25, the third 50, the
fourth 75 and the last which came nearest the light, 100. PB) 24° 24.1° 2am | 24° | Zao
Hours Days
Age in hours or days.... 4 12 15 18 21 24 2 3 4
Group
MNS Sa ome ee a S220) | 6025s | oreo) | G2E0N G9n5n (225al 7120) S4a0n esan0
BE ie Sekt a a le 1356) |). || So. |) ED |) ea) ESA) |) EO) | Silt |) 77/6
Cees i ee 83.5 | 51.5 | 62.5 | 63.0 | 87.5 | 89.5 | 95.0 | 96.0 | 92.0
INGEN ood cose oa) Wash |) Doll SOs ic PRS NPC as || GG G8 I Sil || SALI
Temperature..... 21208 | 21.6° 23254 24.6° 22.8° PAB? 22.5° 20.0° 23.0° 24.5°
Days
Age in days..... 5 6 7 8 9 10 11 12 131 14
Group:
Wonsdsovecsl| Gedy | 7sei | GO Freer 73.7) |) ea | RB ere | Sil || 333.0
Bee eeeee | olieos|) COLON al SOM GS251 Gos on MOOR On ROMO Ni n4 450m aoe om mom)
Cree ea) Soe 0) Oe al OIA 07 89054 eS220N TS >xOMeSseon (OL onl Slm On msone
Average...... 79.6 | 81.9 | 76.2 181.9 | 73.5") 72.0 | 64.4 | 56-8 | 7325) a 26
1On this day one fly from the A group of males was lost. As the records
indicate that very probably the fly lost was a slow one, a new calculation was
made on the assumption that had this fly been present it would always have
remained in the zero section. The figures resulting from this calculation are
72.1 for the thirteenth day and 73.8 for the fourteenth. This changes the aver-
ages for these days to 70.5 and 69.6 respectively.
REACTIONS TO LIGHT AND GRAVITY
TABLE 2—Continued.
IN DROSOPHILA 61
Females
Temperature..... 222 20° Pim PE p> 24.0° 24.1° PBL 2 24.0° Donde 21.0°
Hours Days
Age in hours or
Cb apoaneead 12 15 18 a1 24 2 3 4 5
Groups
AS: 9225) 1169-0, | GOON 765: | 8410) |F84209| 75.5) | 7625") 70-5, | 72.0
13 a ee OSORIO oe eaaaO) ede | SOLOS STeOrleSacOn |e SonOn|| Oo) 1655
Ce CE I 0) |) CAO 7eha5s Oro I 0) || SoG |) aly || Why |) Lolo)
Average...... 94.0 | 73.8 | 79.1 | 76.8 | 90.5 | 87.6 | 81.6 | 80.3 | 74.1 | 69.6
Temperature............. 21.6° oroe 24.6° 22.8° PANS 22-20% 20.0° 23.0° 24.0°
Days
Avevin dave: ss.40s5.n oe 6 7 8 9 10 ll 12 13 14
Groups
AV eee LA tan ae GoLONPOSeon | oGron | Ole On|PoseOl|| soco) |Rooeon |e 44n0n ea 3n0
DESPRE srs y cle Hoon 4s OMe O) |PoSe oe (od Onl ooron lea (non losron lowe O
(Ces eee eee or GosON 4 oco 450) | 32-5) (R2Osonlom_ OnlmlSaor 2oeOne29eo
PAVETARE. 28) f5 2. ok S G18 1) GEO) 29.01) ZG) |! SB3LIL 4) SHG |) ABSky |) aio |) alot)
Showing change in phototropism of wild flies when fed daily with fresh food
Per cent of phototropism
Hours ° 9 /827 3645
Days d a 3
Tem pereture dt each
test also indicated,
é 7 8 q
Age of flies
Graph 2
62
ROBERT STANLEY McEWEN
TABLE 3
Males
Temperaturg....| 22.7° 2150 DH lap aS Diy 22322 24.5° 2325° 21.6° mele | PPA
Hours Days
Age in hours or
PATS édooeoouae 4 12 15 18 21 24 2 3 4 5t
Group A:
Of UC tran 91.4 | 80.6 |_69.9 | Slee 20 | 82a Gxommsoroule Ose Ome
emperature-c eee elo: Oe 22.6° 21.8° 23.0° 23.0° 22.6° PALES 212° 24.7°
Days SY
Aveunidayst oe seen. 6 7 8 92 10 11 12 13 14
Group A:
TELTES eee ee Pena He Se || Daal 93.3 | 94.9 | 92.4 | 82.4 | 88.3 | 80.1
Females
Temperature.....| 23.5° 22.4° PRIN 22eie 22.0e 23.0° 23.0° Zonk 23.0° PME
Hours Days
Age in hours or
BVS sera acl 4 12 15 18 21 24 2 3 4 51
6 flies A .| 80.8) 70.8 | 76.6 | 72.4 | 64.9 | 68.3 | 85.8 | 96.6 | 93.3 | 97.4
9 flies B.....| 99.4) 96.6 | 85.5 | 92.1 | 77.4 | 93.8 | 97.7 | 98.8 | 94.9 | 97.7
10 flies C.....} 100.0) 96.5 | 95.5 | 98.0 | 95.3 | 98.0 | 87.5 | 96.0 | 94.0 | died
Average...... 9374) 87-9) 85583) 87-5 || 7922) 8823y | S90RS O97 | 940) 89725
Temperature. ............ 21.2° (for B and C females only).
Days
IAPOITNIGAYS Sc. hecoe.cse le 6 7 8 9 10 11 12 13 14
() OWES ae so eel Sl 7353 |) 25 | 27/28 | eal |) Sl |) S28) SHS |) cliee!
Otiess Dee eee | O670nl mated
PTT Asa ag Goo oo oe alletctesers:
‘Indicates that food was changed at this point to prevent flies from dying.
The drop on the sixth day is probably accounted for by this fact.
* Indicates only six flies in this group from the ninth day on.
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 63
Y id 6 Jey
a
ped “0
3 iON
2 elenging t \
Q. es
° from this 7
~
2
5 3
rol at pg | ee OO ea = ?
fo} ‘
—) 65 ‘
io} 1
a=) 6o :
D H
(5) a
‘
i \
po ; Y 4
— fo ; - \
‘
4S i .
| v y
40 \
} Oied
P rs =
ORE me el 3 4 S or 3 J TOON toe sat Saas +
Age in hours Age in days
Graph 3
to flies of different ages, as well as on the problem of time of
maximum activity.
The first of these experiments was designed to show the effect
of testing flies aged 18 to 24 hours three times with an interval
of 2 hours between the tests. Seven groups of insects were
thus tested, each group save three containing sixteen flies, eight
male and eight female. Groups A and B only contained six
flies of each sex, and Group E only five. The usual three trials
constituted a test for each group, the sexes as always being
tested separately. The average of the three trials is given as the
index for the test in question.2. The following summary of the
results was obtained by averaging the indices of the seven groups
(table 4).
Along with this series of tests there was run a parallel series
similar in every respect except that the insects were 9 days
old instead of 18 hours. Only six groups were used in this case,
but there were eight flies in every group. A separate single
2 It is to be noted that in this experiment as well as in the one on fatigue,
the testing tube was only divided into four sections instead of five. The sec-
tions were then valued as follows: 100, 75, 25 and 0.
With this variation calcula-
tions were made as described under method II.
64. ROBERT STANLEY McEWEN
TABLE 4
MALE FEMALE TEMPERATU RE
SUES EAUCS UNEP ere ie © coh eee et 68.9 SiG DAR Se
PeECOMAETES tata asan facto ee aes 59.9 85.3 24.4°
4 BY Uti XG | HSS gee eae OPER stat Gap ain acetieg iy 5s 83.5 DAS ie
ANCL eee ee INS PE RT a Og NR 61.4 85.5
Difference between males and females............................ 24.1
Rotalemumberkotemalesyuce csi eee 47
horalsnumberiotetemalesaused s.-eria tana eee eee 47
fey) at pe 8.) NRA Uae REE PEN UEL WR. Ss dks dS teed ees 94
group was put through the series at 6 days, and the results
were practically similar to those obtained from the 9 day flies.
For the sake of uniformity, however, they are not included in
the following summary (table 5).
TABLE 5
MALE FEMALE TEMPERATURE
Ruins Gt OSitd eee ec one hace or oe iene T6 78.3 Dhyailte
SECON. CES THM et i Dict Seen eee re one te. 74.8 83.9 254°
Suir tester nme oe eh cre eee ee ee aane 76.8 83.3 DABS. 5)°
DANN RUT ahs oh 5 siete Be Nee ARES ene aan ts Be 74.4 81.8
Ditierence between! males and: females: sss.44..64.0cee eee eee Cae:
Rotalenumbernohummalessusedsss see eaeeee eerie 48
otal mumberotaemalessused eases eee eee 48
Totalag eer TTA Saeko ake edo eee 96
|
A glance at tables 4 and 5 is sufficient to indicate the general
results. It again appears that the younger flies, both male and
female, are fatigued by successive tests, whereas the older in-
sects actually improve. Also it is clear that although the fe-
males are more active than the males in both cases, they are
relatively less so in the older groups. Not only this, but the
older females are absolutely slightly less active than are the
females of the younger group. In this particular instance, there-
fore, 18 hours is actually the maximum age of activity. It may
be added, however, that were the results from the 6 day flies
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 65
TABLE 6
MALE | FEMALE
TENTED: UIVESSH pene eI Sa aRE aa eae he e aiea Simei tec a) Bie an ae 71.8 97.8
SGT | ETS yee ener ern aha MLC URN NMP iia te A pane bg a. ee Comte eo 90.0 100.0
IM vitiexa | HeYEA et ae aS COG A Aa ae Pe eer as Gee AU rae 93.1 97.8
included in this average this statement would no longer hold.
The indices for these insects were as follows (table 6): The
average male index is 84.9 and the female, 98.5, with a difference
of 13.6. Thus while the males have again gained relatively, the
females are also absolutely much faster than are younger females.
Finally, a series of tests were run on flies 4 to 6 days old as fol-
lows. An initial test was run at the same time of day at which
it had been the custom to remove newly hatched flies from their
bottles. They were then tested at the same relative intervals
as the newly hatched insects had been in the experiments de-
scribed above. In this case ten males and ten females were
used in each group, and the number of trials constituting a test
was raised to 5. The results were as follows (table 7):
TABLE 7
Male indices
0 HRS. 4 HRs. 12 HRS. 15 HRs. 18 HRs. 21 HRS. 24 HRs.
83.6 86.5 88.5 86.6 86.8 96.6 98.8
Female indices
96.8 98.3 | 97.6 Sigel 96.8 96.1 | 99.0
Temperatures
| |
22° oe | Ie alae 2 | Pale 2G. 22
The main feature of this series is that there is no drop occur-
ring in the middle of the series, such as was the case with the
young flies tested at similar intervals. It is thus made more
likely that the falling off in question was due, as suggested, to
the more rapid fatigue of newly hatched insects. Incidentally,
it will be noted that the males are not far behind the females,
and that they gain on them during the series.
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 1
66 ROBERT STANLEY McEWEN
In conclusion, it may be said that females are never twice as
active as males. They are, however, somewhat more active,
particularly when only 1 or 2 days old. As age advances the
difference between the sexes decreases until in some cases at 8 or
10 days the males actually surpass the females. Moreover, in-
stead of the maximum period of phototropic response occurring
at 18 hours, it would seem rather that both males and females,
if not too heavily fed, increase their response with age, reaching
a maximum in the neighborhood of 4 or 5 days. After this
point both sexes tend to become less active, the females more
rapidly than the males. It may also be added that the young
flies fatigue much more rapidly than do older insects.
EFFECTS OF OPERATIONS ON THE REACTIONS TO LIGHT
a. Removal of wings
The operation of removal of the wings was suggested by Dr.
T. H. Morgan as a laboratory experiment for one of his classes.
Mr. 8. Safir was the first to try the experiment, and obtained
the rather surprising result that flies so treated no longer showed
any response to light. This effect was so unexpected that it
was determined by the writer to investigate the matter as thor-
oughly as possible.
The first experiments performed in-this connection were under-
taken with a view to determining whether the insects would
recover their normal response if kept a sufficient length of time
after the operation. As these tests were made at the beginning
of the work no apparatus was employed except the tube and
light from the north window. One fly was tested at a time and
its record calculated according to method I, for instance, the fly
was placed alternately in the end of the tube toward the light
and in the end away from the light, and the algebraic sum of
the average of the two sets of records in inches crawled was
taken as the index of the fly in question. Five groups of animals
were tested, in which the number of insects varied from one to
five. When there was more than one fly in a group, the index
of the group as a whole was computed by adding algebraically
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 67
the indices of the individuals constituting the group. It is the
indices thus obtained that are set down in table 8. From these
results it would appear that there is a slight recovery of photo-
tropism. Nevertheless, in view of what occurs in normal flies
TABLE 8!
DAYS
GROUP 1 2 3 4 5
tl. fie tle fl. ll: fl. tl. fl. tl. fl.
Tey in te see 3 24.0 17.6 So 8.6 4.3
JUL). Mee tek ee ee ee 00.7 15},.83 12.8 2.0 Dee
TELE Pas ea tN Oe 10.0 8.9 3 18.5 9.9
Vite boy ikryard-ns Ay 1.@ 00.7 00.9 3.0
Vai a 2 hwy) ieee 00.4 00.3 2.0 \ 4.7 0.4
otal See eee 00.7| 38.7 43 1). 42.1 PAAD Bp 7 19.9
Average........ 00.7} 9.6 8.6 8.4 2 OR Set 3.9
Differences..... 8.9 8.6 8.4 6.1 3.9
DAYS
GROUP 6 7 8 9 10
tle fl. tl. fl. tl. fl. tl. fl. tle fl.
DiS ee lid 4 io (0 15.0 18.4 10.0
NW eres, Sere 10.3 3.6 16.0 4.3 137
Tig Soe See 3.9 4-2 145 Qe Sal 00.7
VERE eet 10.3 2.9 Bei 4.1 18.6
Ve ee 4.4 ee 4.3 Sno)
Rotalsiee eee 24.5) 6.1 10.6) yas) |) eel) |) TA || etssec) 423) || 00.7
Average........ eles seal 2.9) | ION eae de 14.1 | 00.7
Differences.....}| 5.1 2.4 4.0 Hall 13.4
' No temperature was taken in these early experiments, but later tests made
in the same room under similar conditions showed a variation of less than a
degree during a period of two weeks.
tl. indicates excess of inches crawled toward light, resulting from the alge-
braic sum of indices in the group. fl. indicates excess of inches crawled away
from the light, calculated in the same manner. Diff’s. indicates the algebraic
sum of the fl and tl averages.
68 ROBERT STANLEY McEWEN
TABLE 8—(Concluded)
DAYS
GROUP 11 12 13
tl fl tl. fi tl fl
Pe pat ce Sere SI es od ee 113e3 22.9 27:3
TUE, el a ae eerie es ean ar 25.0 22.6
TEE eeu 58 ae Oe fa ai Re ge ea ES ee CO (TAR 4.3
WB aed ci Se Rai a aii Pou aaicaee eae REA
AVON ee ee ed Ca DOORS E SAME RA cork ee NP
MOGAIS te tates a aii t escent 38.3] 4.3 | 45.5 27.3
AN ETA GESTS kar: a4 i..c soa ato ean men van he oie 19.1) 4.3 | 22.7 27.3
DifeETENCESaeen crxssieeen ie emer 14.8 220) 21nd
with advancing age, it seems likely that this recovery represents
nothing more than the usual increase. It should be noted that
the usual index for unmutilated flies under this system of record-
ing is from 30 to 36. This will appear in the next experiment.
In this experiment, also under method I, five groups of flies
were used. In the first group the wings were not removed until
the sixth day after hatching, while in the fifth group they were
removed on the day of hatching. The results were the same in
every case. The insects showed the usual positive reaction to
light until the day the wings were removed. At this point the
positive reaction disappeared, the insects being indifferent to
light and remaining substantially so for the rest of the tests.
This occurrence was perfectly regular and very striking. It will
therefore suffice to give only a couple of illustrations. In Group
I there was only one fly. On the day after hatching, a t. |. in-
dex of 30.4 was recorded. Its record was about this each day
until the sixth, when the wings were removed. For the subse-
quent five tests its average was f. 1. 1.7. On the sixth test it
went up to t. 1. 4.7, but on the seventh it dropped to t. 1. 2 and-on
the eighth and last the record was f. 1. 1. In Group IV there
were five flies. On the day after hatching they averaged t. 1.
33.4. The day following their wings were removed, one insect
having been lost in the meantime. On this, the third day after
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 69
hatching, they now averaged f. 1. 3.1. Thus, it does not ap-
pear that the age at which the wings are cut off has anything to
do with the effect.
It should be added that the slight f. 1. excesses recorded in
the above experiments are probably. not significant. From
watching the actual behavior of the flies it did not appear to me
that the operation did any more than to render them practi-
cally indifferent to light. Indeed, I have never observed a
clearly negative reaction in Drosophila.
So far the apparent loss of phototropism might mean merely
that the operation had made the insects inactive. However,
since Drosophila is strongly negatively geotropic it was possible
to use this reaction as a measure of general activity.’ ~For
this purpose the system of testing several insects at a time, known
as method II was used. The flies were introduced into the
usual testing tube and given one trial for the reaction to light
in the regular manner, except that no agitation was employed.
- Following this the tube was fastened in a vertical position with
the flies at the bottom, and at a distance of 41 cms. from a 100
watt tungsten lamp hung so that its tip just touched the table.
Three such tests were given, alternating with three light tests,
and the indices for the two sets calculated as usual for the above
method. The elimination of agitation in these tests was made
necessary in order to make comparable the records of the flies
with and without wings. When agitated the former move to-
3 Regarding the relative strength of the two stimuli, ight and gravity, Cole
decides in favor of the latter. He found that when flies were placed in a ver-
tical cylinder illuminated from below the larger per cent went to the upper-
most third. Carpenter, on the other hand, was able to attract the insects to
the bottom of a similar cylinder without using as strong a light as did Cole.
On account of the great variability of Drosophila, I suggest.that this discrep-
ancy may be due to the small number of flies used, Cole employing only twenty-
one and Carpenter only six.
My own results are not strictly comparable with those of either of these
authors, because I used a type of apparatus which did not directly oppose the
two stimuli, but such evidence as I have agrees with that of Carpenter. Thus,
a reference to any of the tables where the light and gravity indices of normal
insects are recorded will show that the light index in any given case always ex-
ceeds that for gravity. In any event, the matter of which stimulus is the stronger
is not one of any great significance.
70 ROBERT STANLEY McEWEN
ward the light both by crawling and flying, whereas the latter
can only crawl. When not agitated, however, the response even
of winged insects is almost purely a crawling reaction.
Three groups of flies containing 20 insects each were now se-
lected at random from the stock bottles. They were placed in
vials in the morning and tested according to the above plan in
the afternoon. After this first test all the insects were etherized
and the wings removed from the two groups which had made
the best record. These groups will be designated as B and C.
The following afternoon all three groups were tested again.
Following are the records of Groups B and C before and after
the wings were removed, and also the two records of the control
Group A (table 9).
TABLE 9
Before removal. Temperature 24°
A B Cc
Gravity , Light Gravity Light Gravity Light
(First test)
Males Males Males Males Males Males
29.1 57.6 28.3 75.0 MN 65.0
Females Females Females Females Females Females
31.6 81.6 45.0 90.8 a1) 5 85.0
After removal
‘(Second test)
Males Males Males Males Males Males
50.0 86.6 35.0 5.8 39.1 20.8
Females Females Females Females Females Females
41.6 90.8 30.0 Ud) PASO) By lh
As a further. check, Group A, the control, was tested a third
time 48 hours after the second test. The wings were then re-
moved and eight hours later a fourth test was given (table 10).
It is evident from this data that the removal of the wings affects
the light reaction specifically, and does not merely reduce the
activity of the insects.
The next point investigated was the effect of the removal of
parts of these appendages. The first experiment was done un-
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA vl
TABLE 10
Group A. Temperature 23°
GRAVITY LIGHT
Males Females Males Females
Third test, before wing removal........ 46.6 30.0 88.3 84.1
Hourchtest-patuern removal. s-..cs-e eee - 43.3 40.0 116 6.6
der method I, the age of the insects being unknown. The appa-
ratus was exactly the same as that described in connection with
the effect of removing the wings at different times after hatching.
Five groups of flies were employed in the first series of tests.
In the case of the first two groups only half of the wings were
removed throughout the tests, while in the case of the last three
groups one or two tests were given with half the wing removed,
and then a second operation was performed in which three-fourths
of the total wing was taken. Table 11 summarizes the results.
It appears that though the insects are very erratic and vary
much from time to time, those animals which had had the wing
completely removed, with one single exception, made lower rec-
ords than any made by flies with only half of the wings removed.
These experiments are unsatisfactory, however, in failing to show
what, if any, effect the removal of half the wing has. Later on,
therefore, another set of experiments was devised to answer this
question. In this case method II was used with the improved
apparatus. Likewise, the alternating gravity trials were intro-
duced as a control. In short, the general method was precisely
similar to that employed in the proof that wing removal has
a specific effect on the reaction to light (table 9).
Four groups of flies were selected at random from the bottles
and run through the tests. The flies were then etherized and
treated in the following way. Group I, which contained nine
males and ten females, had made the lowest record and was re-
stored to the vials without operation. Group II, which con-
tained ten males and eleven females, and had made next to the
lowest record, had only the tip ends of the wings removed.
Group III, which contained ten males and nine females and had
the second best record, had one-half of the wings cut off, and
2 ROBERT STANLEY McEWEN
TABLE i1
ONE-HALF WING REMOVED ANSI OE OA NO ORES WAMSTE,
pater GROUP TESTS AEE AON BSD
t.1. {iackbesl, t.1. f.1.
6 I 1 144.4
6 I 2 28.8
6 I 3 129.1
6 i, 4 61.3
IAWICT AD Cl cso. Menta cic ine pecs cisgepeh ets 90.9
6 II 1 57.0
6 1g 2 8.9
6 iM 3 52.3
AViGTAIC ON enc © entation 39.4
6 Iil 1 104.3
6 10. 2 38.6
6 JH HE 3 8.8
6 Ill 4 24.2
Awieragem serena okisc clan oie: 104.3 8.8 31.4
6 IV 69.0
5 IV 2 20.3
5 IV 3 32.3
JAS Sie a A IR a 69.0 i 26.3
6 V 1 48.1
6 Vv 2 46.4
6 V 3 orl
6 V 4 10.2
4 ‘ Vv 5 Pei
IAVETAL CLS Aciuatnns oo RCE mee 47.2 6.8
Averagetor one-half wigs. ...< snas.0 dem = © lee eee © ines rolls CADp A
Averagzenror three-fourths winlfS... «5s «fc scseeetr es es ook ells, 13 7
Group IV, which had made the best record and contained ten
males and six females had three-fourths of every wing removed. .
The next day all groups were retested with the following results
(table 12):
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 73
TABLE 12 .
GROUP II.- WINGS CUT
: ‘ if
GROUP I WINGS NOT CUT ONE-FOURTH
Light Gravity Light Gravity
Male |'Female| Male | Female} Male | Female} Male | Female
Before cutting...... S54 call (anc ‘82.5 | 2a We26eGr | SOLOF | S8ron |) 20.0) || 24:9
MEMpPeravUres a... peau «4. « 23.9 23 .5°
Wrrereutting......2: 0:5. o1.6 | 92.5 | 48.1 | 48.3 | 76.6 | 72.5 | 40.0 | 36.6
semmperatune: senses ae 24° 24°
GROUP IV. WINGS CUT
GROUP III. WINGS CUT HALF THREE-FOURTHS
Berar catia dy eee 80.0 | 95.3 | 41.6 | 42.5 | 90.8 | 95.8 40.8 | 38.3
EMpPCratunes issn cscs ae 23.5° 23.5°
After cutting..............] 19.1 | 21.2 | 65.0 | 49.0 | 25.8 | 26.3 | 61.6 | 49.9
Memperatunenssce- eo ee oe 24° 24°
It is evident from these figures that with a single exception,
such as the case of the groups in which three-fourths of the wing
was removed, these results support the conclusion that the de-
crease in phototropism is directly proportional to the amount of
the wings removed. Moreover, in considering this result and
particularly the one exception, it must be remembered that the
amount of wing cut off in each group was directly proportional to
the height of the index originally scored by that group in the
initial tests. Thus, the group having three-fourths of the wing
removed was originally the fastest of all, and this may account
for its still retaining enough speed to win out over the group
with only one-half of the wing removed. ‘This seems particu-
larly probable when this experiment is considered together with
the previous one. Taking the two together, I believe we are
justified in the conclusion that at least roughly speaking the pro-
portion between phototropism and wing length holds good.
74 ROBERT STANLEY McEWEN
b. Gluing the wings
Several attempts were made to glue the wings in such a way
that though uninjured, the insect could not use them. These at-
tempts were made fruitless, however, by the fact that a fly whose
wings are stuck thoroughly enough so that they can not be freed,
will spend all its time in an effort to do so, and will scarcely re-
spond to any other stimulus during the process. This experiment,
therefore, had to be given up.
There remained two other possibilities. First, the effect of
operations as such could be determined, by operating on other
parts of the insect. Secondly, the existence in this laboratory
of mutations of all degrees of winglessness made it possible to
discover the effect of the absence, or partial absence, of wings in
Drosophila upon which no operation had been performed. The
effects of other operations will be considered first.
c. Cutting off legs
For this purpose eleven males and ten females, newly hatched,
were selected and kept in vials until five days old, this being the
usual procedure when records comparable with those made by
other groups were desired. Before placing in the vials each in-
sect was operated on, and the tarsus and tibia of the middle
pair of legs cut off. It was thought that removal of the middle
pair in this manner would interfere least with the animal’s
balance and ability to crawl. On the fifth day these flies were
tested with a resulting index of 53.1 for the males and of 86.3
for the females. Under similar conditions it will be recalled that
a normal index would be approximately 95 and 97, though I
have cages where it was considerably lower. ‘Thus, though there
may be a slight effect from this operation, it is obviously not
very great. Furthermore, it must be remembered that however
quickly and accurately these flies might orient, they were neces-
sarily handicapped in their speed of movement except when they
took to wing. Since this experiment was performed under agi-
tation this was frequently the case. As a matter of fact, orien-
tation and movement toward the light was perfectly constant
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 79
with these insects, a statement that does not hold at all for flies
without wings.
d. Removal of antennae
The other type of operation which was attempted was the
removal of the antennae. Inasmuch as these organs in Dro-
sophila lie close against the head, considerable difficulty was ex-
perienced in cutting them off. At length, however, a technique
was developed which though tedious was successful in almost every
case in removing the entire organ close up to the head. In this
experiment four sets of tests were run which may be designated
as A, B, C, and D. Since the method varied slightly in each of
TABLE 13
Controls without antennas removed
LIGHT GRAVITY
eau, Vertical Horizontal
Male | Female
Male |Female| Male |Female
( 1 a0)! Sle) 27.0) 23.0
SIAR Oto c rh barbs detrh hy = Sao wk 4 2
[ 3
Me Tacicci (ails eee eae ee 23° 23°
Set B 1 100 | 97.5 20.0) 4820) 20:5) 35.0
RG Rieke aaa wibst el a)ie\je| «| «| © \p ofc) e) oe (0.0 © = je wine “o 1 4] 5 48 | 0 99 0 27 } 5
AVEO CT AUT OG iy apu aly cek ned ncest ce aieksesesye ers tonsa 23° 23°
‘ ‘ f 1 50.5) 54.0) 33.0) 24.5
Syenh (Chess oo eee Re ee are \ 9
Memperature (GROUP a). ..0.- 2-2: 2B BB}
1 36.6 olan
Seta) AHMAR asen See srs k, { 9 99.7 ©10.5
Teo ee 100 | 97.5 | 166.8| 135.5| 105.3| 82.5
Pe cristae) 100 | 97.5 | 33.3] 44.5] 21.0] 27.5
Merticalvexcesssammrallenarws 1 :pcth std Bases eo ain Hoos aay ee 12) 33
Wertiealsexcess) shemallenee 6 5c .75..\ 10!) 6:0), 272510 1620
{ 3 0 a0)
Memperatures pase a. cay eee eo es Dae 99°
BS) Cay Piles Ol Dr
ieee SOs Neale Mae es aa ie | 92-9) (80:9) © 20a eta eo ae
(
MemMperatUreser 2k. s.ik tt eee eae oe 24° 24°
; f 1 77.5) 100.0 Ol ieloe 1025) L230
SICEAGe cb ean sah nase Bi asia 82 2 | 71.0] 90.1] 15.5] 16.5] 14.0] 13.0
Temperature (Group b)....... 23° 23°
1 | Also wings 6.0 13.5
Sains Daan ste eR Oates 9 slipped 12.1 5 5
Mem peravUnTe Mey eee ne et eee oe 23°
Motaleseaccccee ate ae ae cee oe) 418 OA2 26! 42a O4RO ee ORO esha
VAGV CLAD OS = rpS ayant eyeah ph cite een eer tn ee 83:6) 85:5) 1557) dids3) 15s5) Ae
Wertieal excess! male an tcf ec os oon Re enh cee Seer ce OFZ
Vertical excess) females saa ee a een eee 0.2
these tests, each will have to be described separately. The re-
sults, however, will be found summarized in table 13.
In Set A, two groups of twenty flies were removed from the
bottle shortly after hatching. One group was operated on at
once, while the other was kept as a control. Five days later the
group from which the antennae had been removed was tested
for light reaction. Three hours later both groups were given a
gravity test. In this case this test consisted of two series of five
trials each. In the first series the tube was held vertically, and
the index calculated as usual, the result being designated as the
vertical index. The second series acted as a control, the tube
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA vi)
being placed horizontally and the result designated as the hori-
zontal index. The difference between these indices may thus be
taken as the index of the animal’s reaction to gravity. After
these tests the flies with the antennae removed were given fresh
food and kept for 3 days. The light and gravity tests were
then repeated. On the following day, for instance, 9 days
after hatching, the. males of this group were given one more
gravity test. At this point the alternating system for the ver-
tical and horizontal positions of the tube was introduced, and
used in all the subsequent tests of this experiment.
In Set B three groups of flies were taken and from one group the
antennae were removed. The other two were used as controls.
Five days later one control group and the one from which the
antennae had been cut were given the light and gravity tests as
in Set A. The second control group was given only a gravity
test.
In Set C two groups of twenty insects were taken and from one
group the antennae were removed as usual. At 5 days the
animals which had been operated on were tested for light and for
gravity. The control was tested for gravity only. Three days
later the former group was again subjected to both light and
gravity tests. 3
Set D consisted of two groups of male flies only, each having
been used previously in tests on the effect of wing removal. One
group, which we will designate as a, had been used as a control
and had not had the wings removed. In the other group, b, the
wings had been cut off. This latter group was now etherized
and the antennae as well as the wings were taken off. The con-
trol group was etherized at the same time but no operation per-
formed. Four hours later a gravity test was given to each
group, and 3 days later these tests were repeated.
From the results of this experiment it appears that there has
possibly been a very slight reduction in phototropic response.
However, it is certainly in no way comparable with the reduction
which occurs regularly as the result of wing removal. Further-
more, a study of the light responses of normal insects contained
in other tables, shows such variation that it is extremely doubtful
78 ROBERT STANLEY McEWEN
if the slight falling-off of some of the antennaeless groups in
table 13 is of any significance at all. From this result, therefore,
as well as that obtained by the removal of legs we are led to con-
clude that any operation as such is not sufficient to cause a loss
of phototropism. Incidentally, however, a rather interesting re-
sult does appear here as to the specific effect of the removal of
the antennae and reaction to gravity. From the small amount
of data on hand, it appears that the loss of these organs greatly
reduces a fly’s negative geotropism. It also seems to produce a
slight reduction in general activity. There are not, however,
sufficient data collected on these particular points to do more
than suggest a line for further investigation.
EXPERIMENTS ON MUTANT WING-CHARACTERS
We are now in a position to attempt the second method of
analyzing our problem by testing the various sorts of wing mu-
tants which have arisen in this laboratory. These mutants vary
all the way from vestigial, in which the wings are mere stubs to
curled, in which the wings though of normal length are turned
upward at the end and are not very effective in flying. There
are many other variants between these two, one of which is desig-
nated as strap. Strap has wings almost as long as normal, but
they are narrow, often cleft at the end, held off from the body
at a peculiar angle and are useless for flight. These three mu-
tants therefore were selected as bearing the closest approximation
to insects with one-fourth, one-half and three fourths of the wing
removed. The flies in question are represented in figure 2. A
normal insect is also included for the sake of comparison.
The tests on the above mutants have all been made ac-
cording to the plan already outlined in one of the experiments
for testing the effect of the removal of wings on light and gravity
(p. 69). In brief, three tests were made for light, alternating
with three tests for gravity, with the tube in the vertical posi-
tion only. No agitation was used during any of these tests.
Only one new point needs to be mentioned and that is in regard
to an improvement in the apparatus. Under the former system
of testing for geotropism the lamp whose rays struck the tube at
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 79
right angles was nevertheless near its foot. It was recognized
that this method was unreliable when we attempted to compare
the gravity reaction of flies which were phototropic with that
of those which were not. Thus, two sets of flies, one photo-
tropic and the other not, but possessing an equal amount of
Fig. 2. A, Normal insect. B, Specimen of ‘curled’ stock. C, Specimen of ‘vestigial’
stock. D, Specimen of ‘strap’ stock.
80 ROBERT STANLEY McEWEN
negative geotropism would show the latter more negatively
geotropic than the former. This would result from the fact
that though equally impelled to move upward, the phototropie
animals would be constantly handicapped by the attraction of
the light from below. In order to remedy this difficulty, there-
fore, three lamps of the same candle power were arranged in a
vertical line, so that one came opposite the foot of the tube, one
opposite the center, and one opposite the top. The end of each
bulb was a distance of 41 cm. from the tube. The latter, more-
over, was now held in its vertical position by a wire support, so
that there was no danger of its wabbling. With these improve-
ments, the following experiments were undertaken.
In the first place, it was decided to run a test on some wild
flies in order to get some data which should be comparable with
that obtained by the same system for the mutants. Also in mak-
ing these tests it was decided incidentally to run a few checks
on the effect of wing removal, in order to make sure that the
former tests were not invalidated by the position of the single
lamp. For this purpose three groups of insects, each contain-
ing ten males and ten females, were selected, and kept in the
usual manner until 5 days old. They were then tested as de-
scribed above. After the test, the males in the groups which
we shall designate as A and B were etherized and in the case of
group B the wings were removed. Eight hours later both groups
were retested. The control males in Group A were now also
operated on, and tested for the third time 10 hours later. In
the case of the females, Group A was operated on after the first
test and Group B kept as a control. Twenty-four hours later
both these female groups were tested again. Group B wasnow
operated on and tested after eight hours. The results of these
tests are summarized below (table 14).
This experiment confirms the conclusions already set forth re-
garding the effect of wing removal. In other words, the re-
arrangement of the lights has produced no significant effect. The
only thing of note in the record is the extremely high gravity index
registered by the males of Group B after being operated on. To
determine whether this is of any significance or not will require
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 81
TABLE 14
BEFORE WING REMOVAL AFTER WING REMOVAL
id Light Gear tice) bight (vertical)
Male | Female; Male | Female} Male | Female} Male | Female
1 96.6 | 98.3 | 68.3 | 77.5
Groupee see 2 | 96.6 66.6 41.6 50.0
ie eas 5.8 45.0
Memperagunrese ese ashe 23 .5° 230 Ow
1 98.3] 99.1] 67.5) 64.1
Croupsbe eee. 2 97.5 55.0} 52.5 91.6
3 35.8 | 10.1 | 80.0) 42.5
Memperaturesseeesse sear 2B) ay 23nou
Groupe.) he | 1 | 84.1] 94.1] 35.01 25.8 | |
Memperature.. st se. 7456 24° 24°
otal seas eee ee ee | etn Gl oso) Ol) 2374) 222.4 OAR Ie oleic GAG 92zo)
AWVETAPES eas eee Oon9| 90.2! (S98) 5 Gales 2.8) Waree2 | 40 ne
more data. It is true that a somewhat similar tendency is man-
ifest among the males in table 9, but the poor light arrangement
in the earlier experiments makes the records of doubtful validity
on this particular point.
Let us now consider the reactions of vestigial flies. Three
groups of insects, half male and half female, were kept for the
usual length of time after hatching, and then subjected to the
test just described for wild flies. The only difference was that
in this case the single lamp was used in the gravity trials. As
will appear from the results, however, this feature was of no
consequence in this instance because the insects were only very
slightly phototropic. Also since the wings were already only
stubs, nothing was cut off. Table 15 summarizes the results.
Strap stock was next tested. Three groups, constituted asin
vestigial and wild were kept as usual till 5 days old. The only
irregularity in this connection was the use of only nine instead
of ten females in Group A. As to the apparatus, the single
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 1
82 ; ROBERT STANLEY McEWEN
TABLE 15
Temperature 24°
LIGHT GRAVITY (VERTICAL)
Male | Female | Male | Female
(C6) £0) 5 an as eee ane wee OER SERS ARE ere 3 20.0 7.5 31.6 24.1
(GEOUDB ee ose nee ae eect re 13.3 10.0 24.1 20.8
Group Giza ce eee ae eRe Re PoE 10.0 1G; 42.5 35.0
OGAIS (tba eye ata tare eieea, Cre ate 43.3 29.1 98.2 79.9
VET AG Cee tyres ous tbe en recs eee Pes 14.4 9.7 32.7 26.6
lamp in the gravity trials was used in the first tests of the males
in Groups A and B. After the first test the males of Group A
were kept as a control, while those of Group B suffered the re-
moval of the rather poorly developed wings which they possessed.
Both sets were retested 8 hours later. Table 16 gives the
results:
The results from this experiment are enough to suggest strongly.
the slight increase in phototropic response which might be ex-
pected to distinguish these flies from the vestigials. The most
TABLE 16
BEFORE WING REMOVAL AFTER WING REMOVAL
ees ign) |) Gas a ||) Vea
Male | Female O Male Female] Male | Female} Male | Female
Temperature 24° es:
Group Ar «4: { ; =e ret ee Wee
Group) Be oe { ; 2223 000 63.8 68.4
Temperature 23°
Group C5728 1 10.0| 24.1 | 41.6} 48.3
Motalecwpcens- ce as 147.0) 41.7 | 202.8} 79.1 63.8. 68.4
PAY ORAS ES He mares O74) 2083) || S07) sy || Oss 68.4
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 83
striking point which distinguishes these data, however, is the
failure of wing removal to affect light reaction in Group B.
Indeed, the test after the operation shows an actual increase in
the reaction to light. In view of the rather low light indices in
all the flies of this variety, I am inclined to explain this as fol-
lows: Even normal insects whose wings have been removed,
vary a good deal from time to time in their degree of response
to light. Also the general variability is such that a fly with
three-fourths of its wings gone will not always show a lower re-
sponse than one with only one-half gone. I therefore suggest
that since these flies already have a low index on account of their
imperfect wings, the removal of the remainder of the wing might
not have sufficient additional effect to counterbalance some un-
known change in the physiological condition of the animal.
This statement is partially borne out by the fact that the males
in Group A which were not operated on, also showed a markedly
higher index for both light and gravity in the second test.
Further experiments now under way will serve to show whether
this is the true explanation or not. If it is not, we should have
to accept the rather astonishing hypothesis that a fly with short
wings and a low index to begin with, actually has its photo-
tropism increased instead of diminished by the removal of such
Wings as it has.
Finally, we have to consider the reaction of the flies desig-
nated as curled. As usual, three groups of insects, constituted
as in the previous tests of this series, were tested when 5 days
old. As this particular group was really the first of the series
the use of the single lamp in the gravity test was still customary.
The change to the new system was, indeed, made during the
work on this group, which accounts for the fact that only the
males in Group A were subjected to the improved treatment.
That this feature was really of no great significance, at least in
the case of these flies can be told by comparison of Group For a full discussion of Mendelism in Drosophila, see the Mechanism of
Mendelian Heredity by Morgan, Sturtevant, Muller and Bridges.
90 ROBERT STANLEY McEWEN
TABLE 21
LIGHT GRAVITY (VERTICAL)
Male Female Male Female
Temperature 24°
GroupeAss:20 sara oes Sepa 16.6 7.5 03.3 41.6
Temperature 23°
Group saat wre te ee Sh Rene tee 7.5 5.8 78.3 44.1
Groupe @uce. ee Sears Sere xe oe see 18.3 20.8 81.6 70.8
Motels eae eek ace eer ere eee 42.4 34.1 213.2 | 156.5
AVERT AD ESE esas eat ey emer reore ere 14.1 11.3 71.0 62.1
further borne out by observation of the insects. They are fully
as active as are those from normal stock.
Finally, a number of normal, white and vermillion eyed and
tan flies have been sectioned and examined, both with and with-
out staining. So far, no histological abnormality has been dis-
covered in the eyes of the tan insects, to account for their peculiar
lack of response to light.
EFFECT OF COLORED LIGHTS ON NORMAL AND MUTANT
EYE COLORS
A very considerable amount of work has been done by various
investigators upon the effects of different wave lengths on or-
ganisms which respond either positively or negatively to light.
Though it has generally been found that animals as well as plants
respond more readily to the more refrangible rays of the spec-
trum, such is by no means invariably the case. In Daphnia,
for instance, Lubbock (Journ. of the Linnean Society, 1881) and
others have found that the green and yellow rays are more effec-
tive than any of the others, including the blue and violet. Also
in the case of simpler organisms, it has been shown by Engel-
mann (Mast’s account, Mast ‘‘Light and the Behavior of the
Lower Organisms’’) that Bacterium photometricium tends to
form aggregations in the infra red.
The general apparatus used in this work has already been de-
scribed. The composition of the liquids used, together with the
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 91
wave lengths transmitted by each, were as follows (table 22).
The spectrum tests were made often enough to make sure that
no fading of the colors was taking place.
TABLE 22
FORMULA WAVE LENGTHS
\VVEHIGITC: conn oupennctewnl) Ge. 5380 A° (green)—4240 A° (violet
Violet (Ammonia ton mace Green strong at 4950 A°
Eee Copper sulphate Violet strong at 4510 A°
(tiliten) heererrrce 7.5 grams | Blue, weak
Wiaiheritncs tyakicy SUOROMCcE 5660 A°-5050 A°
Gr Licht griin........ 0.03 grams | Strongest at 5320 A°®
goer ts Napthol yellow... 0.25 grams
Napthol green.... 0.03 gram
Red WENGE codecsness a0) CoE 7200 A°-6325 A°
Paeei eee Ponceau Red..... 3.0 grams | Strongest at 6570 A°
The above formulae were only selected after a long series of
experiments, and are for the most part modifications of formulae
contained in the ‘Methods of Studying Vision in Animals” by
R. M. Yerkes and John B. Watson, Behavior Monographs,
1911. The red and the green are very satisfactory for colors
obtained by ray filters, while the so-called violet is evidently
not so good. It is, as a matter of fact, continuous from green to
violet. The blue, however, is very weak, the green moderate
and the violet band very strong and wide. The results of the
experiments show that it is probably not the green to which the
effectiveness of this filter is due, and since the blue band is so
slight, the probabilities are that violet is the effective stimulus.
It is practically impossible to get a strictly violet filter. We
find, however, that blue is obtainable, and it is intended to use
such a filter in analyzing our results further at the earliest
opportunity.
Besides the wave lengths, the relative energy transmitted by
the filters was also measured by means of a thermocouple, using
the same source of light employed during the experiments. The
results are indicated in graph 4. From this it appears that if the
energy transmitted by the colorless flasks be represented by 100
92 ROBERT STANLEY McEWEN
per cent, then the red flask transmits 103 per cent, the green 64
per cent and the violet 51 per cent. The fact that the red flask
actually transmits more than the white is explained by the fact
that the layer of clear water was slightly thicker than the red
solution. It will be noted, however, that in the visible spectrum
the red is somewhat less than the white, while the green and the
violet are approximately equal.
The first colored light experiments performed were under-
taken in the Zoological Laboratory of Western Reserve Univer-
sity, Cleveland, Ohio, during the summer of 1916. After some
preliminary experimentation it was decided to make use of
wo Curves showing relation between galvanometer
deflections and scale readings
a
>
\eRed Bottle, Area—65.4 cm
\
UJ
eet te Light, Area—Sl.7. e177}
420| Areasinyisible Spectra
White Lignt—is crm
Red Bottle — 9.8cm*
Grecia Bottle—/.7 om
Blue Bott/e—/-4em*
Def lectiovns
7ra
Spectr
Vis/ ble
Ey 6 4}
Seale readings
Graph 4
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 93
method II. Four sets of flies were employed, each set consist-
ing of six males and six females, the sexes being tested separately
as usual. Every test consisted of three trials which were aver-
aged to obtain the index for that test. Each of the four sets of
flies was tested once for each of the three colors, the successive
tests coming at intervals of 2 hours. For each set, however, the
arrangement of the colors in the series was varied. ‘Thus for set
A it was red, green, violet; for set B, red, violet, green; for set C,
violet, green; red; and for set D, green, red, violet. The results
of these tests when averaged together for the four groups were as
follows: males—violet, 64, red, 29.8, green, 24.5; females—violet,
81.8, red, 64.6, green, 57.2. In every one of the four sets violet
was first in each complete test for males and females. As be-
tween green and red, red won in three out of the four sets for
both males and females. Thus it would appear from this ex-
periment that the colors are effective in the order—violet, red,
green.
A further and better test was later made according to the fol-
lowing method. Two groups of insects each consisting of ten
males and ten females, were picked out and designated as Group
A and Group B. Each group was now tested six times at two
hour intervals, with three trials to a test. In this case, however,
the three trials constituting a test were not all of one color.
Instead, there was a single trial for each of the three colors in
every test. Furthermore, in each of the six tests the arrange-
ment of the colors was altered according to a set plan. Group
B was treated in exactly the same manner, except that the se-
quence of the color arrangements for each test in the series was
reversed.. Thus for test one the Group A arrangement was V,
Gal fortest2ev,k, G: for test's R, GY Vetor test 4 KR, V~G;
for test 5 G, V, R; and for test 6 G, R, V. For Group B, the
series began G, R, V and ended with V, G, R. At the end of
the tests the indices for all the trials of a given color were aver-
aged together in Group A and Group B. Finally the averages
thus obtained for Group A and for Group B were averaged.
* The tube in this experiment was only divided into four sections instead of
the usual five..
94 ROBERT STANLEY McEWEN
Furthermore a record was kept in such a way that it was possible
to see in how many trials a given color came out first, second,
or third. It is evident that in this scheme, since every color was
used in every test, the effect of previous tests would not change
the relative value for any color in any given test. On the other
hand the possible effect of the arrangement of colors in any given
test is overcome by altering the arrangement every test. Finally,
the method of recording gives not only the average of all the trials,
but an analysis of individual trials. It, therefore, seems that a
tolerably clear-cut result obtained in this way may reasonably
be supposed of some significance.
This method was now applied in testing a series of mutant eye
colors as well as the normal stock. The eye colors were as fol-
lows: white, an eye entirely lacking in pigment; tinged, almost
white, but containing the lightest shade of red; eosin, a reddish
yellow somewhat darker in the females; vermilion, a very good
sample of this color; normal; and sepia. The last named color
is virtually maroon on hatching, but grows darker with age until
at five days it is practically black. The results from the tests
on these stocks are summarized in tables 23 to 28. Graph 5 is
based on the results for each eye color as indicated in groups A
and B combined. Since there is no apparent sex differentiation
as regards reaction to varied wave lengths, the graph has been
constructed from the average male and female indices in every
case.
It is evident from these tables that in the case of all colors
lighter than normal, the general tendency is for the order of ef-
fectiveness to be violet, green, red. There are, however, three
exceptions. First in group A of white eyed males, the red is
ahead of the green both in the average index and in the number
of tests in which this occurs. This case is more than oftset,
however, by group B, so that in the average of the two the order
of colors is as stated above. It should be mentioned, moreover,
that white eyed insects are extremely erratic even for Dro-
sophila. It is quite usual for them after making a few con-
sistent trips up the tube to become very much excited and to
simply buzz about convulsively. The second exception is that
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA
95
TABLE 23
White eyed flies
Group A
AVERAGE
Sex Violet Green Red
Malev. 72.9 49.9 QED
Female... 48.7 38.7 29.1
meals a. Tied once
b. Tied once
Group B
Male... 61.3 Preven ils), 11
Female... 64.7 47.1 43.2
a. Tied twice. a. Tied once
Male i Tied twice emols te Tied once
Groups A and B combined
SEX Vv G R
Ist | 3 1 2
Male..... Yel || a3 Dy 1
S¥ol || Os eas. 83
Stale 1 1
Female. . 2d | 3a | 4b] 0
3d | 0 1 5
Ist | 6 0 0
Male..... 2d | 0 4 0
3d | 0 | 2a} 6b
| ston On el
Female. . 2d | 0 4 1
3d | 1 2a | 4b
Ist | 9 1 2
Male..... Zanes 6 1
3d | 0 5a | 9b
Ist | 8 1 72
Female. . Dae) 3a, sip) |i
Cel 3e | 9d
Mallen. 5: 67.1 39.0 33.8
Female... 56.7 42.9 36.1
Ie Tied once
, a. Tied twice b. Tied once
Male a Tied twice Hemel ec. Tied once
d. Tied once
of group A tinged females.
slightly exceeds the violet.
Here the average green index very
The table indicating the number of
times each color won, however, shows that from this standpoint
violet is still well in the lead. The third exception is found
in group B vermilion females where red very slightly exceeds
green both in the average index and in the number of times
which it was ahead. ‘There is no special explanation for this ex-
cept the fact that the eye color is approaching that of normal.
In any case group A more than overbalances it.
96 ROBERT STANLEY McEWEN
TABLE 24
Tinged eyed flies
Group A
AVERAGE
SEX V G R
Sex Violet z Green | Red
f st Ga Ona Ols | Mlaleteee.. 100.0 100.0 79.1
Male.....;|2d|0 | 6 | 0 | Female... 98.3 99.1 97.0
isd O70) 186
Teste gk ae Ne @ (a. Tied twice
Female. . 2d | 0 2 1 Female 4 b. Tied twice
|| 3d | 2a] 3b] 5e (ce. Tied three
Group B
Ist | 4 0 0 IMiaileyenee 100.0 98.7 70.1
Male..... ;| 2d | 2a| 6b] 0 | Female... 100.0 97.29 97.5
[uieesclin | OWe6
(| 1st] 6 | 0 E - :
Female. . Ze Omnle2aa|| a Male e ae ane Female ‘ BS as
llsa|o | 4a| 5b b. Tied tw Paltre ree
Groups A and B combined
ist (10.10 0) Male...) eto000 99.3 | 74.6
Male..... 2a | 2d |12b]| 0 Female... 99 1 9875 97 .2
34/0 |0 {12 |
1st 10% ele |"0 Ta ; 4 a. Tied twice
Female..{/2d]0 |4 |2 | Male in Tied tnien Pemale 4 b. Tied five
3d | 2a | 7b |10c Roatan es ce. Tied six
Turning now to the results obtained from the tests on normal
and sepia, we find that the early records for normal made during
the summer of 1916 have been confirmed. The order of effec-
tiveness is not violet, green, red, but violet, red, green. There is
one exception to this in the group A males where the green
slightly exceeds the red. Finally in the case of sepia, there is
the same reversal of the relative effectiveness of red and green.
This instance, however, is more clear-cut than is the case with
normal, for with sepia the red exceeds the green in all respects
with absolutely no exceptions.
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 97
TABLE 25
Hosin eyed flies
Group A
AVERAGE
SEX V G R
[ Sex Violet Green Red
Ist | 4 1 0 | Male..... ; 93 .5 87 9 70 .0
Male..... 2de| Zar db 0 Female... 89 .5 85 .7 70.0
3d | 0 0 6
ise |S 0 0
Hemalene< | 20) dat | 6b .0 Male
30810 0 6
fa. Tied once Feimale a. Tied once
\b. Tied once ~~ \b. Tied once
Group B
lat} WhO OMUMele..... |. 2400.0 0 || Waodecen) |) 71.8
Male..... 2de)|2a7| 6b) |) OF Hemale..- 96 .2 92.0 70.8
Sada Olen Om
Ist 5 1 0 . P
Female..{|2d]}1 | 5 | 0 Malle {ies ae
Sr Ome Ol 6
Groups A and B combined
Isr| 8 1 0 Male..... : 96.7 91.3 70.9
Male..... 2d | 4a j1lb | 0 Female... 92.8 88.8 70.4
sd |0 0 12
(Gets ta |lOnmlp a 0
Female ..<| 2d | 2a |11b| 0 Male
3d | 0 QO |12
fa. Tied three
\b. Tied three
fa. Tied once
\b. Tied once
Female
In order to discover a possible cause for the phenomenon just
described, sections of the eye were made and examined micro-
scopically. As expected, these sections showed that the pigment
which imparts to the organ its color, is simply the pigment
usually found surrounding the rhabdomes in the compound eye
of arthropods. This pigment so far as its function is known, is
supposed to be of a protective nature, placed so as to absorb all
rays of light which do not fall directly parallel to the axis of the
rhabdome in question. Of course, in cases such as we have un-
der consideration the pigment is not black but colored, and: will
consequently reflect light of a certain wave length. At first,
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, No. 1
98 ROBERT STANLEY McEWEN
TABLE 26
Vermilion eyed flies
Group A
AVERAGE
SsEX iV; G R
Sex Violet Green Red
St) nOun | OM sIEON a eIViialetecre ‘ 90 .4 72.0 59 .6
Male..... Axel || @ || 4! 1 Female... 94.1 94.1 60.8
[3d | 0 | 2a) 5b
Mee @ || @ | O :
Female..<|2d|0 |6 | 0 Male ti ses ere
24/0 10 16 . Tied once
Group B
Tet e3)|'0. MOU Mate =e 60.0 97 5 90.0
Malewee- 2d | 3a | 3b| Id} Female... 95 .0 72.0 76 .6
3d | 0 | 3c} 5e
AS tHlGn OO (a. Tied three
Female..{|2d | 0 | 2 | 3 |b. Tied twice Kattied
3d |0 | 4a| 3b} Male 4 ce. Tied twice Female eileen ae oe
| d. Tied once \b. Tied once
|e. Tied twice
Groups A and B combined
(St \59 Om) On| Males. : 95 .2 84.7 74.8
Males.... 2d | 3a | 7b | 2d} Female... 94.5 78 .0 68 .7
|| 3d | 0 | 5c |10e
Ist |12 OF a0 a. Tied three
Female..<| 2d |0 | 8 | 3 b. Tied twice fa Tied
|| 3d | 0 | 4a | 9b] Male ;c. Tied three Female ; |~ a eae
at aped mare \b. Tied once
ie Tied three
therefore, when normal eye color was thought to be the only one
with an increased effectiveness for red, it seemed possible that
this might be explained by assuming the red of the light to be
exactly the same shade as that of the eye. This would then
mean that a larger percentage of the red light entering the eye
would be reflected and therefore effective, than would be the
case with any other color. When it was discovered, however,
that a still darker shade of red still further increased the effect- -
REACTIONS TO LIGHT AND GRAVITY
IN DROSOPHILA
ue)
TABLE 27
Normal eyed flies
Group A
AVERAGE
SEX Vv R C |
Sex Violet Red Green
Stal ete Olen | Olen Vial emer J 100 .0 96 .2 97 .0
Male..... 2d | 2a | 2b] 1d} Female... 100 .0 99 .5 95 .4
[ axel |} (0) 4e | 5c
Tstielen OM 0 (a. Tied twice a. Tied three
Female.. { |-2d | 3a | 4d | 0 b. Tied once b. Tied twice
3d | 2b] 2e | 6c Male < ec. Tied three Female < c. Tied twice
d. Tied once | d. Tied three
le. Tied three \e. Tied twice
Group B
St e2ele2ee Ome elViales a=. - 93.7 93.3 86.6
Male..... 2d | 3a | 4d! 0 Female... 97.0 96.6 83.7
3d {1b.| 0 | 6c
ist 2. | 0 (a. Tied twice
Female.. | 2d | 4a | 5b} 0 Male! b. Tied once Female / 2 Tied three
3d 10 |0 |6 | e. Tied once b. Tied three
| | d. Tied twice
Groups A and B combined
StileGenlecen | NOleVlale. 96.8 94.7 91.8
Male..... 2d | 5a} 6e | Ic | Female... 98.5 98 .0 89.5
3d | 1b} 4£ |11ld
tstaleoe eye sO fa. Tied four Be iiedi si
Female.. ¢| 2d | 7a | 9d] 0 b. Tied once sala
q 5 b. Tied two
|| 3d | 2b | 2e |12e c. Tied once 5
Male : Female |ulal 0 Female... 95.2 88.8 61.5
Sol CO) = ah (8). |
Ist | 9 3 0 ;
Female... <|2d |2 |9 | 0 Female ‘e He at
3d | 1la|0 |12b ;
SUMMARY AND CONCLUSIONS
1. Females of Drosophila ampelophila react to light somewhat
more readily than do the males. This difference is most marked
in young insects and steadily decreases with age, until at 8 or 9
days it has almost vanished. The time of maximum activity for
both sexes does not seem to come at 18 hours, but more probably
at from 3 to 5 days.
2. The removal of the wings causes the fly to lose most of its
phototropism. The effect is specifically on the tendency to re-
act to light, as is shown by the fact that such an operation
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 101
affects little if at all the response to gravity. The effect is
roughly proportional to the amount of the wing cut off. It is
not a result of the operation as such, since other operations do
not produce it, and because wingless flies and flies of other
stocks with defective wings show the same deficiency of response.
Certain organs (fig. 3) occur on the wings of Drosophila, but
operations fail to show that they are connected with the response
Showing relative tropism of flies of varied eye colors toward lights of different
wave lengths
White Eyed Flies,
lt _ i
(+n A
Red Ps) es
dammed Eyed ties
| a ae
ES
Red
Eosin Eyed Flies
| FERS See
_ a
Red SEMRAE eA
Vermilion Eyed Flies
|. a
5 <<
eC IE 7
Normal Eyed Flies
| eo aa
i RE aS
“| SARS ee ea
Sepia Eyed Flies
|. AGES
Cc
\ | RRS!
Graph 5
- 102 ROBERT STANLEY McEWEN
to light. It appears fairly certain on the other hand that the
chief light receiving organs are the compound eyes as shown by
experiments with eyeless stock.
3. Operations on the antennae may produce a weakening of
the response to gravity, though they have little effect on the
reaction to light.
4. In a mutant stock of flies known as tan, there 1s clear-cut
evidence for the sex linked inheritance of a character which may
be described as indifference to light. It is apparently not due
to any structural defect in the eye.
5. Colored lights which may be conveniently described as vio-
let, green and red, are effective in the order named upon insects
whose eye color is lighter than the red eye of the wild fly. In
the case of wild flies, and flies whose eyes are of a still darker
shade called sepia, red is more effective than green.
GENERAL DISCUSSION
In most of the earlier work on various organisms, both animals
and plants, the conclusion generally reached was that the blue
and violet rays possessed much more stimulating value than
aid those which are less refrangible, particularly the red and
orange. Thus Payer (42) using both the solar spectrum and col-
ored media found that seedlings turned toward blue and violet
light but not toward red, yellow, orange or green. Sachs in
1864 obtained similar results, using colored solutions and glass.
Also in the case of animals Engelmann (’82) found that Euglena
viridis collected in the blue of a solar and gas spectrum, while
KE. B. Wilson (91) working on Hydra viridis with colored glasses
again found the maximum effect in blue. Finally, Loeb’s ear-
lier work (90-93) led him to conclude that as between red and
blue, the latter color was the more effective for fly larvae, plant
lice, caterpillars of Porthesia chrysorrhoea, moths of Sphinx
euphorbia, Geometra piniaria, various copepeds, the meal worm
Tenebrio molitor, the larvae of Polygordius, Limulus polyphemus,
and the June bug Melolontha vulgaris.
Even some of the early investigators, however, found cases in
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 103
which the above condition did not hold. Thus Kraus (’76) using
colored media, discovered that in Claviceps, a fungus, red light
was nearly as effective as blue, while Engelmann (’82) showed
by the use of a solar and gas spectrum that Bacterium photo-
metricum actually collects most readily in the infrared. Further-
more, the study of various other animals with more refined
methods began to show that many forms were most affected by
intermediate points in the spectrum. Thus Yerkes (99) has
shown that for Simocephalus the point of maximum efficiency
in a Welsbach gas prismatic spectrum is in the yellow. Bert
(69) and Lubbock (’81) located this point for Daphnia in the
green, while recently Loeb and Wasteneys (’16) using the spec-
trum from a carbon arc, have found the most effective point for
Balanus larvae in the yellow and that for Chlamydomonas
pisiformis in the yellow-green. Likewise, Hess, an opthalmolo-
gist, (10) using the spectrum from a Nernst glower has fixed
green or yellow-green as the maximum stimulating point for a
variety of forms, including ichneumon flies, Culex pipiens, adults
and larvae, Coccinella septenpunctata, Dasychira fascelina and
cephalopods. In the last instance the reaction of the pupil of
the eye is taken as a criterion of response. Lastly, 8. O. Mast
has recently given an excellent summary of work previously
done and the results of a recent series of experiments of his own
on Arenicola larvae, blowfly larvae and a number of unicellular
forms. For the blow-fly larvae the maximum is in the green,
while for Arenicola it is in the blue.
From these results it is apparent that the variation in the
point of maximum response for different animals and plants is
very wide. ‘To explain this. divergence, the existence in differ-
ent organisms of different chemical compounds varying respec-
tively in the degree to which they are altered by light of differ-
ent wave lengths has been suggested. That there are com-
pounds of this kind we know, but their presence in phototropic
organisms has not yet been proved. Aside from this view Hess
believes that phototropic animals are all color-blind, and that
they go to the part of the spectrum which seems to them bright-
est. He apparently gets this idea from the fact that he found
104 ROBERT STANLEY McEWEN
so many organisms for which yellow-green is the most effective
part of the spectrum, this being also the brightest part for color-
blind men. This notion, has been criticized. by Ewald (’15)
and Loeb (16).
The peculiar fact about Drosophila is the reversal in the ef-
fectiveness of red and green as the insects’ eye color grows darker.
Thus for eye colors lighter than normal the order of effectiveness
is violet, green, red, while probably for normal, and certainly for
sepia, the order is violet, red, green. This case besides showing
the peculiar reversal is remarkable as being the only instance so
far discovered among the lower animals in which red is more
effective than green, with the possible exceptions of Daphnia
(Frisch and Kupelwieser, 713; Ewald, ’14), and paramoecium
bursaria (Engelmann, ’82). How to account for this phenomenon
of reversal it is difficult to say. Were it not for the case of sepia
it might be explained on the basis of the changed amount of red
reflected by the normal colored rhabdomes as compared with that
reflected by those of lighter shades. When two shades produce
the same effect, however, it is difficult to see how this will suffice.
It would thus seem as though we must fall back on the assump-
tion that as the eye grows darker, the supposed sensitive chemi-
cal substances on which the light has its effect change also.
_ What this change could be, it is hard to imagine from what we
now know of photo-chemical reactions. I am inclined to think,
therefore that the explanation may yet be found in connection
with some sort of differential absorption.
It may be noted that my results with colored lights do not
agree in one respect with those of Dr. A. O. Gross who also
worked on Drosophila This writer found green more effective
than red for flies with normal eyes, while my experiments re-
versed this order. I suggest, however, that this descrepancy is
due to the fact that Dr. Gross used lights which were equated
in energy, whereas in the case of my filters, as is also true for the
normal spectrum, the energy of the red is much greater than
that of the green. This fact, nevertheless, does not invalidate
or make less interesting the very evident increase in the effective-
ness of red in the case of the darker eye colors, since whatever
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 105
the relative difference in energy content, that difference remained
constant for all the eye colors tested in my experiments.
Lastly, it may be well to emphasize the peculiar relation
which exists in Drosophila between general activity and photo-
tropism This phenomenon has been clearly recognized by
Carpenter and in general I agree with this author’s conclusions.
The fact seems to be that this insect is not phototropic unless
it is in a certain physiological state brought on by, or at least
accompanied by, activity. When the fly reaches a certain de-
gree of activity, induced by various means, it suddenly becomes
phototropic. When it quiets down, however, it may still crawl
about but ceases to be phototropic. Thus, when an insect has
been exposed to constant illumination for some time, it no
longer orients to light but wanders aimlessly up and down the
tube. Eventually such an animal may even come to rest with
its head away from the source of light. This phenomenon, Car-
penter suggests, is probably due to slight fatigue. However
this may be, it is certain that without a continuance of the me-
chanical agitation or sudden increases in light intensity, the ani-
mal’s general activity soon falls to the point where phototropic
response ceases.
106 ROBERT STANLEY McEWEN
BIBLIOGRAPHY
CaRPENTER, F. W. 1905 The reactions of the pomace fly (Drosophila ampelo-
phila Loew) to light, gravity and mechanical stimulation. Amer.
Nat., vol. 39, pp. 157-171.
1908 Some reactions of Drosophila, with special reference to con-
vulsive reflexes. Jour. Comp. Neur. and Psych., vol. 18, pp. 483-491.
Coir, W. H. 1917 The reactions of Drosophila ampelophila Loew to gravity
centrifugation and air currents. Jour. Animal Behav., vol. 7, no. 1,
pp. 71-80.
Gross, A. O. 1913 The reactions of arthropods to monchromatie lights of
equal intensities. Jour. Exp. Zoél., vol. 14, pp. 467-514.
Hess, C. 1910 Neue Untersuchungen iiber den Lichtsinn bei wirbellosen
Tieren. Arch. f. d. ges. Physiol., Bd. 136, pp. 282-367.
Loxrs, J. 1906 The dynamics of living matter. New York, 233 pp.
Lozrs, J. anD WastreNrys, H. 1915 The relative efficiency of various parts of
the spectrum for the heliotropic reactions of animals and plants. Jour.
Exp. Zoél., vol. 19, pp. 23-35.
1916 The relative efficiency of various parts of the spectrum for
the heliotropic reactions of animals and plants. Jour. Exp. Zodl.,
vol. 20, pp. 217-236.
Lurz, F. E. 1914 Biological notes concerning Drosophila ampelophila. Jour.
New York Entomol. Soc., vol. 22, no. 2.
Mast, 8S. O. 1911 Light and the behavior of organisms, New York, 410 pp.
1917 The relation between spectral color and stimulation in the
lower organisms. Jour. Exp. Zodl., vol. 22, no. 3, pp. 471-528.
McInpoo, N. E. 1914 The olfactory sense of the honey bee. Jour. Exp.
Zool., vol. 16, no. 3, April, pp. 265-346.
1916 The sense organs on the mouth parts of the honey bee. Smith-
sonian Miscell. Coll., vol. 65, no. 14, Jan.
Payne, F. 1911 Drosophila ampelophila loew bred in the dark forsixty nine
Payne, F. 1911 Drosophila ampelophila loew bred in the dark for sixty-nine
generations. Biol. Bull., vol. 21, pp. 297-301.
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY
THE BIBLIOGRAPHIC SERVICE, DECEMBER 22
SOME EXPERIMENTS ON REGENERATION AFTER
EXARTICULATION IN DIEMYCTYLUS
VIRIDESCENS
C. V. MORRILL
Department of Anatomy, Cornell University Medical College, New York City
TEN FIGURES (THREE PLATES)
The earlier writers (Phillipeaux and Fraisse)! on regeneration
in urodeles seem to have held the opinion that the extremities of
adult animals are completely replaced only when one or more
bones are injured in the amputation, that is to say, not after
total extirpation (exarticulation). Wound-irritation from an
injured bone was considered necessary as a stimulus to the re-
placement of a missing part. Also the bone supplied a ‘tissue-
rest’? to serve as a matrix. However in young salamanders and
especially in their larvae, it was found that the regeneration. of
extremities takes place very readily since here the joints are
only partially formed and wounding of the bones always occurs
in amputations. This general conclusion, that in adults re-
generation does not occur after complete extirpation, seems to
have been shared by a number of the more recent investigators,
some, Kochs (97) and Wendelstadt (01 and ’04) expressly
confirming it, others, Towle (’01), Morgan (’03), Reed (03) and
Glaeser (10), while not putting it to the test, seem to have taken
care in their experiments to amputate through a bone.
Kurz (12) in the course of his experiments on transplantation
of entire imbs in Triton, found that if the limb is completely
extirpated (exarticulated) at the hip- or shoulder-joint, a new
limb regenerates. Presumably no wounding of the bones of the
hip- or shoulder-girdle took place although Kurz does not state
The works of Phillipeaux and Fraisse were not accessible to the writer.
Their conclusions were obtained from Barfurth’s review in Merkel and Bonnet’s
Ergebnisse, vol. 1, 1891.
107
108 Cc. V. MORRILL
what precautions were taken to avoid this. The writer using
the American salamander, Diemyctylus viridescens, obtained
similar results some years previous to Kurz’s report but for
various reasons they were not published.? Recently a new series
of experiments were made to work out the histological details
of the process and to determine how it differs, if at all, from’
regeneration after injury to remaining bones or cartilages. In
addition, a number of more complicated operations were made
to analyze further, if possible, the extent and power of regenera-
tion after losses not usually met with in nature.
MATERIAL AND METHODS
A large supply of adult Diemyctylus was obtained through the
kindly assistance of Prof. A. Treadwell, of Vassar College. Since
many of these animals were in a weak, semi-starved condition
when brought to the laboratory, they were kept for a month in
glass aquaria before using and were fed on fresh liver. Under
these conditions the animals became very vigorous, and with-
stood the operations well. All operations were done under
narcosis. At first ether was used, but this, owing to its irritating
effect on the skin and to a certain percentage of mortality which
followed its use, was soon discarded. Much better results were
obtained by using a solution of chloretone, of 1: 2000, in which
the animals were immersed. This acts very gently. After
swimming around rapidly for a few minutes, the salamanders
slowly come to rest and in about ten minutes are completely
nareotized. The animals recover readily, though sometimes
slowly after this treatment. There is no irritation of the skin
and no mortality.
After the amputations, to be described in detail beyond, the
best results were obtained by closing the wounds with a stitch
or two of fine silk thread. Although this is not absolutely
necessary to the success of the experiment, healing then takes
place more rapidly and there is less danger of fungoid growths.
Immediately after operation, the animals were placed in adark
2 The experiment was made at the suggestion of Prof. T. H. Morgan, in 1907.
REGENERATION AFTER EXARTICULATION 109
chamber lined with moist filter paper for two days as recom-
mended by Reed (’03). During this period the operated ex-
tremity was moistened from time to time with a solution of
permanganate of potash 1:1000. The animals were then
returned to the aquaria. The above precautions almost entirely
prevented the growth of fungus and consequent failure of the
experiments.
For microscopic study, the regenerating regions were removed
and fixed for the most part in sublimate acetic or Gilson’s mer-
curo-nitric fluid. Other fixatives, such as Zenker’s fluid, Bouin’s
fluid and ten per cent formalin were occasionally used but on
the whole the sublimate mixtures proved the most satisfactory.
After hardening in alcohol for a few days, the objects were
decalcified in a mixture of four per cent nitric acid in seventy
per cent alcohol for three or four days. They were then imbedded
in paraffin and sectioned. As a rule, good series were obtained,
seven or eight micra thick, although the rather tough bone and
cartilage from large specimens sometimes gave trouble. For
staining Mayer’s haemalum followed by picro-acid fuchsin was
most frequently employed. ‘This gives a brilliant differentiation
of tissues but is not always permanent. Other stains such as
Mallory’s connective tissue stain, borax carmine and Lyons blue,
haemalum and congo red were also used but none proved as
satisfactory for most purposes as the haemalum and_ picro-
acid fuchsin combination.
EXPERIMENTS
Pari. I
The fact that regeneration does occur after complete extirpa-
tion (exarticulation) has been established by the observations
of Kurz and the writer as stated above. In order to work out
the detail of this process, two sets of operations were made, the
hind limbs being used in both cases. In the first set the limb
was amputated at the hip-joint, in the second at the knee-joint.
Great care was exercised in making these amputations. The
skin and muscles were first carefully divided with a small sharp
110 C. V. MORRILL
scalpel. Then the part to be removed was grasped with the
forceps and slight traction employed to draw the joint surfaces
apart. The capsular ligaments were then divided with the
scalpel and the limb removed, care being taken not to touch the
skeletal parts remaining (hip bones or femur according to the site
of operation). i
ae 2
7 wy aN Tet
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, JANUARY 19
THE REGENERATION OF TRIANGULAR PIECES OF
PLANARTA MACULATA. “AvSRUDY IN: POLARTTY!
J. M. D. OLMSTED
FOURTEEN FIGURES
Morgan (’98), in his studies on the regeneration of Planaria
maculata, describes two types of operation by which he was abie
to obtain regenerated pieces in which “‘the long axis of the new
head” was “at right angles to the long axis of the original worm.”’
When he cut narrow strips from the side of a planarian, he found
that the piece, through contraction, assumed the shape of a
crescent, the cut edge forming the concave margin. In certain
cases all the new tissue which formed in the concavity of the
crescent was used in the production of a head. A similarly
shaped worm was formed in several cases when he cut from the
side of a planarian a triangle the apex of which lay within the
body. Both these methods of cutting, however, produced other
pieces, which upon regeneration nearly or quite retained their
original polarity. Morgan remarks (p. 373) ‘‘The experiments
do not show clearly, why, at one time pieces cut from the side
give rise to new worms having the long axis in the direction of
the original long axis, and at other times at right angles to the
original long axis.’
Child (15, p. 165) states that in triangular pieces cut from the
side of Planaria dorotocephala the regenerated “‘head often devel-
ops nearly or quite in the direction of the transverse axis.”’
The possibility of producing regenerated planarians whose polar-
ity has apparently been so changed that their chief axis is at right
angles to the chief axis of the worm from which they were taken
having been demonstrated, at Dr. H. W. Rand’s suggestion a
more detailed study of the regeneration of such pieces was under-
‘taken, the results of which are given in this paper.
1 Contributions from the Zoological Laboratory of the Museum of Compara-
tive Zoology at Harvard College, No. 302.
157
‘
158 J. M. D. OLMSTED
The species of planarian used in these experiments was Plan-
aria maculata Leidy, and the specimens were taken from Fresh
Pond near Cambridge, Mass. Worms of various sizes, from 12
to 5 millimeters in length, were used. Some specimens, after
being brought into the laboratory, were fed on liver until at the
time of operation they were of the maximum size. Others,
medium and small worms, were kept without food for several
weeks. Neither the condition of satiety nor of starvation
noticeably influenced regeneration. At one time the mortality
of one lot would be greater, at another time, that of the other.
In the fed worms, however, it was found best to allow one week
to elapse after the last feeding before the operation was performed.
To prepare the planarians for operation, they were narcotized
in a 0.1 per cent solution of chloretone until they ceased to move.
Cuts were then made with a sharp scalpel, care being taken to
have the cut edges as nearly straight as possible. Triangular
pieces were taken from all regions of the body, each triangle
having for one of its sides a portion of the original uncut right
or left margin of the worm, and, for the other two sides, cut edges
which intersected near the original median axis of the worm (fig.
2a, 5a, 7a). The two cut edges, intersecting at a point which
I shall refer to as the vertex of the triangle, are distinguished in the
following account as the anterior and posterior edges.
It was only towards the end of experimentation that the im-
portance of fairly exact measurements of the lengths of the cut
and uncut edges, the angle where the cut edges meet, the distance
of the vertex of this angle from the median axis of the worm
from which the piece is taken, and the size of the piece, was
realized. In the earlier part of the work, no camera drawings.
were made until the day following the operation. Because of
the decided contraction of the pieces at this time and the con-
sequent distortion of their original shape, it was possible to
estimate only rather roughly their original measurements. Later
in the work, however, camera drawings were made immediately
after operation while the pieces were still in chloretone, the very
slight contraction in this condition being negligible; a second
drawing of each piece was made on the day following, when they
A STUDY IN POLARITY 159
were in the contracted state. The drawings made while the
pieces were still in chloretone formed the basis for classification
into groups, according to the relative lengths of the cut edges,
the size of the angle at the vertex, etcetera. The drawings
made on the day after the operation during the earlier experi-
ments were compared with those of the later work and each of
the earlier ones was placed in that group which it most resembled.
One may fairly assume that pieces which resemble one another
on the day after operation would also have been similar immedi-
ately after the operation. Thus it was possible to estimate with
some degree of accuracy the measurements which the triangular
pieces in the earlier work had immediately after operation.
In the following account it was thought best, however, to enumer-
ate the cases separately; hence the earlier experiments, in which
the original measurements are estimated merely, are referred to
as Series I, whereas the later ones, in which the pieces were
drawn while still in chloretone, are designated as Series II.
The mortality of such triangular pieces is very great. Less
than one-fifth of them survive the operation and regenerate.
Pieces taken from the region of the pharynx (fig. 5a) had the
greatest vitality, though regeneration of pieces from other regions
of the body, if accomplished, proceeded along exactly the same
lines as in the pieces from near the pharynx. Bardeen (03)
found that in Planaria maculata he could more frequently obtain
double-headed worms from cross-pieces when they were taken
from the pharyngeal region than when from any other region
of the body. Morgan (’04) was also more successful in getting
pieces from this same region to regenerate, but he remarks,
“Whether this is only because shorter pieces are more easily
obtained here, or because the very short pieces from this region
survive the operation, remains an open question.” The latter
explanation seems to be the true one, since in many cases in my
experiments the same sized pieces were taken from all regions
of the body and only those from near the pharynx survived.
When, in the operation of cutting, the epidermal layer -is
broken, a great mass of loose parenchyma cells flows out from
the wound, and if the two cuts form a very acute angle, the
160 yo J. M. D. OLMSTED
projecting point on the triangular piece becomes rounded off by
loss of material (cf. Morgan, ’98, p. 393). Immediately after
the operation there is always a very slight contraction of the cut
edges, even though the piece is still immersed in chloretone.
This, no doubt, is due to the direct stimulation of the muscle
fibers. As soon as the effect of the narcotic is gone, the piece
contracts greatly, often assuming the form of a hollow cone, the
apex of which lies approximately at the center of the dorsal
surface of the piece. Epithelial cells soon cover the wound
(Lang, 712, p. 272), and after twenty-four hours new white tissue
can be seen along the cut edges. This new material is never
evenly distributed along the cut edges, but (figs. 2b, 5c, 6c)
a greater amount of it appears near the center of the anterior
edge, a less amount along the posterior edge, and very little at
the vertex (cf. Morgan, 798, p. 378). os Sereetverenenaterierie kara ake 2262
DRAIN GIA KOU 1R5 MAO odd aoe dep tees pote Cacordtoon acco D.coNadoDeuO Code 266
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GOPVEPEALeG SUIMULAETON +5 cs -t.c ses ae dhs cteysucl als acs + oo cudlotaly va dobehauerele) siete 273
PD Tse ieee e A ese ees ake se bnad a 2. paietaetarerayeietete Os = Srailoel taney otarnomesieals 277
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Wane hem iC alnstimil agin ys os obo cle Sonmee pone boocEUCac domes 296
Wile. Bibliognap liye seasistec scree nae cera ec = cberckecelete setae hoa av ciotel sorsiete:» MA tetate 298
I. THE RESPONSES OF ASCIDIA AND THEIR NERVOUS RELATIONS
The assumption of a sessile mode of life involves a sacrifice
in the number and kinds of responses of which an animal is
capable. The comparatively few reactions exhibited by the
sessile tunicates are undoubtedly accountable for the almost
complete absence of our knowledge of their sensory physiology.
The common European ascidian, Ciona intestinalis, is the only
one in which anything is known of the behavior under stimulation.
1 Contributions from the Bermuda Biological Station for Research, No. 79,
and contributions from the Zodlogical Laboratory of the Museum of Compara-
tive Zodlogy at Harvard College, No. 304.
261
262 SELIG HECHT
Even here, however, the data are meager and scattered, and con-
sist largely of incidental observations. This lack of knowledge
and the abundant presence of Ascidia atra at Bermuda served
as incentives for the following investigation of the sensory re-
actions of this species.
1. Description of reactions
Jordan (’07) on the basis of his observations has called the
ascidian Ciona, an animal poorinreflexes (reflexarmes Tier). With-
out subscribing to any of his theoretical generalizations, which
Baglioni (13) has justly criticized, I have no hesitation in simi-
larly describing the behavior of Ascidia atra. Tests with a
variety of conditions of stimulation have revealed very definite
activities, by means of which the animals respond to changes
in the environment. The number of these activities, however,
is small.
The structures and movements which are involved in their
execution have already been described (Hecht, 717). It remains
to explain their relation to one another and to the source of
stimulation. The presence of the open siphons and of the water
current makes it possible for Ascidia to receive indications of
changes of the environment not only on its exterior, but also on
its interior surfaces. This distinction is of fundamental im-
portance, because the place of reception of the stimulus deter-
mines the kind of movement which the animal executes. As a
result there are manifested two qualitatively distinct groups of
reactions. Each of the groups consists of three responses, which
involve the use of different combinations of muscles.
The group of direct responses depends for its origin on a source
of stimulation which affects the external surface of the animal.
This group of responses is concerned mainly with mechanical
stimuli. Although the three reactions included under this head
result from different intensities of the same outside disturbance,
the reactions themselves involve an activity of different muscles,
and not a different degree of activity of the same effectors.
1) If the test of Ascidia be touched very lightly, the siphon
nearer the point of stimulation will contract. The extent of the
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 263
resulting closure depends on the intensity of the stimulus and on
its distance from the siphon. After a short interval the siphon
rim opens and the animal is normal again.
2) If, however, the stimulus has been stronger, not only does
the siphon rim nearer the stimulated area close, but the other
siphon rim also closes. A new set of muscles has been called into
play.
This reaction is to be differentiated from the one in which both
siphons are stimulated, such as when a drop of water is allowed to
fall on the surface of the water in the aquarium. In this case
each siphon is independently stimulated by the same disturbance.
Such a reaction persists when all nervous connections between
the two siphons have been cut (cf. Loeb, ’92 and Magnus, ’02).
It is otherwise with the response which I have described. Nor-
mally, if one siphon is touched so carefully that the animal is
not jarred, both siphons will close provided the proper intensity
of stimulus is used. When, however, the nervous connections
between the two siphons is severed, only the stimulated siphon
rim contracts.
3) In response to an ordinarily vigorous mechanical stimulus,
A. atra reacts by the employment of still an additional set of
effectors, the longitudinal muscles of the body. Not only do
both siphons close, but the body bends on its long axis toward
the right side.
This bending toward a structurely determined side is of signifi-
cance in the ecology of Ascidia. Most individuals of the species
are attached with the body projecting at any angle, but mainly
in a nearly horizontal plane. All such animals which I examined,
were found with the left side of the body uppermost. Conse-
quently, the curving toward the right side results in bringing the
siphons into such a position, that a disturbing body on the outside
will roll off, and one on the inside will fall out.
In the previous work on ascidians the reaction which involves
the bending of the body has been the only one which has received
any adequate attention. It has been generally regarded as the
only reflex of which this group of animals is capable, and there-
264 SELIG HECHT
fore called ‘the reflex’ (Loeb, ’02). Jordan (’07) has more appro-
priately called it the protective reflex (Schutzreflex).
The reactions which are comprised in this direct group show
individual variations depending upon the intensity of the stimulus
which sets them off. They can, however, be very definitely
separated from one another when the animal is observed with
any degree of care, or when graphic methods are employed. (See
for example, figure 8 of the first paper of this series: Hecht, 717.)
All these reactions are to be kept apart from those which are
in the group of crossed responses. The stimuli which result in
the reactions of this second type are all localized on the interior
surfaces of the siphons, of the atrial cavity, and of the branchial
sac. They include changes in the environment not only of a
mechanical nature, but of a thermal, photic, and chemical kind
as well.
Although they may be produced by the same kind of stimulus
varying in intensity, the three reactions included in this group
are, nevertheless, the result of different combinations of effectors.
As in the case of the direct reflexes, they must, therefore, be
sharply distinguished one from another. To make this clearer,
I may refer to the behavior of a human being suddenly exposed to
a bright light. The person will reflexly close his eyes. If, how-
ever, the light be made excessively bright, he will not only close
his eyes but also place his hand over them. The nature of the
stimulus is the same, but the greater intensity of the stimulus
brings forward a new activity superimposed upon the simple eye
closure. The same is true of the following reactions of Ascidia.
1) An exceedingly delicate stimulus on the inside of one siphon
results in a closure of the other siphon. The stimulated siphon
remains wide open, while the sphincter of the other siphon is
called into play. This kind of response can be secured only
under very carefully controlled conditions. The animal must
not be jarred and the stimulus must be a delicate one. It is
best to use large animals because they are not as sensitive as the
smaller ones.
2) An increase in the intensity of the stimulus produces a re-
action which does more than merely stop the water current; in
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 265
addition, it brings about a discharge of the water present in the
branchial sac. The stimulated siphon remains open, the other
siphon closes tightly, and the animal contracts vigorously along
its dorso-ventral axis, resulting in a sudden decrease in the ca-
pacity of the respiratory chamber. Occasionally the stimulated
siphon may also contract partially so as to decrease the size of
its opening. This gives the ejected water a greater momentum.
3) The last reaction of this group combines a bending of the
body on its long axis with the movements of the previous response.
This is the usual reaction which A. atra gives under ordinary
conditions of stimulation of its internal surfaces.
The last two reactions probably correspond to what Jordan
(07) has described in Ciona as the ‘Ejektionsreflex’: ‘Closure
of one siphon, rapid contraction of all muscles, other siphon
(most frequently, but not always, the anal siphon) remaining
open” (’07, p. 98). This description is repeated by Polimanti
(11), who, however, added nothing to it. Jordan did not study
this reflex at all, but contented himself with the statement that
it serves to throw out foreign bodies, and that the causes for its
appearance are not clear.
In Ascidia there is no doubt about the nature of the aeaite
which will produce any of these three crossed reactions. It is
always a disturbance on the interior surfaces of the body. Ihave
observed the same ‘Ejektionsreflex’ in the common Ecteinascidia
turbinata of Bermuda under the same conditions of stimulation
as in Ascidia atra. Jordan’s statement of its function is correct;
it must, however, be broadened to include not only the ejection
of foreign particles, but also the response to any internal irrita-
tion, such as strong light or chemicals.
The point of special significance is the crossed behavior of the
siphon rims. Stimulation of the outside of a siphon causes that
siphon rim to close. Stimulation of the inside of a siphon results
in that siphon remaining open while the other siphon rim con-
tracts. This points to the presence of a complexity of innervation
in ascidians of which there has previously been no suspicion.
The one factor which the six reactions of Ascidia possess in
common is their negative character. A source of stimulation
266 SELIG HECHT
is either excluded by the closing of the entrances to the body, or
it is thrown out by a discharge of water. I have never observed
any positive response to a stimulus in this species. This is not
unexpected from its mode of existence. The animals are entirely
dependent for their supply of energy on what is brought in by the
water current, and they merely exercise a choice by rejecting
anything which acts as a stimulus.
2. Nervous relations
Among higher animals, the tunicates are peculiar in the
concentration of the entire central nervous system into a single
inter-siphonal ganglion. In Ascidia atra, according to Hilton
(13),? this is a roughly cylindrical mass, on one side of which is
to be found a rather unusual neural gland (Metcalf, 00). It
gives off many more nerve trunks than are usually described for
this genus of ascidians. From the oral end therg arise three large
nerves, which go to the region of the oral siphon. Several nerves
leave the atrial end, while from the middle of the ganglion there
emerge three large nerves, four smaller ones, and many minute
ones. It is significant that all the nerves contain both afferent
and efferent fibers (Hilton, 13, p. 116).
Practically nothing is known of the nerve endings in ascidians.
The same may be said of the presence of sense cells. Hilton
describes the fibers of the oral nerves as ending in the oral tenta-
cles, but fails to state whether they form free nerve terminations
or arise from sense cells. Lorleberg (’07), after prolonged in-
vestigation of the nervous system of Styelopsis, concludes that
there is a complete lack of sense cells, but that there are un-
doubted free nerve terminations present.
In relation to the reactions of ascidians, one point is clear:
the only demonstrated means of direct nervous communication
between the siphons is by way of the ganglion. The ganglion,
however, has more than the mere conducting function supposed
2 This author refers to the species as Tunica nigra. I have it from Professor
Mark that Hilton’s work was done on Ascidia atra. Moreover, his description
of the species as the “ascidian very abundant on Agar’s Island” leaves no doubt
as to its identity.
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 267
by Loeb (92). Although some of the results of Frohlich (03)
on the removal of the ganglion of Ciona have been questioned by
later authors (Jordan ’07, and Kinoshita 710), the combined
work of all the investigators on Ciona proves that ganglion re-
moval affects at least the threshold sensitivity, the tonus, and the
rate of recovery after stimulation.
Ascidia does not remain normal in the laboratory long enough
to permit of a study of the quantitative effects of ganglion re-
moval. I had, therefore, to content myself with a determination
of the qualitative results produced by the mere nervous isolation
of the two siphon regions from each other. This was accom-
plished by means of a rapid incision into the test and mantle so
directed as to result in the severing of the nervous mass into two
parts. The animal recovered from this slight operation in a
few minutes.
The behavior of individuals under such nervous conditions was
very instructive. Of the group of direct reactions, the first
persisted, and seemed, qualitatively at least, to be normal. The
second reaction, that is, the closure of both siphons, disappeared
at once. As long as the whole animal was not jarred, no amount
of contraction of one siphon called forth a similar response of the
other siphon. The reaction involving the body flexure depends
mainly on the bending of the oral siphon. Therefore when
this siphon was stimulated the bending occurred, but the atrial
siphon still remained unaffected.
The essential element of the group of crossed responses is the
closure of the siphon which is not stimulated. This element
completely disappears after the operation. Stimulation of the
inside of the oral siphon, frequently even when strong enough to
involve the dorso-ventral contraction and the body bending, fails
to affect the atrial siphon, and only causes a partial contraction
of the oral one. Irritation of the inside of the atrial siphon
brings about no change at all in the oral.
These experiments leave no doubt of the ability of each portion
of the animal to perform its part of a reaction even though it is
isolated nervously from the rest. The reaction of the animal
as a whole, however, depends on its nervous system being intact.
268 SELIG HECHT
II. MECHANICAL STIMULATION
1. Touch
Ascidia atra is an animal that under normal conditions is
stimulated preéminently by mechanical means. This is the only
variety of stimulus which is capable of calling forth all the possible
responses of the species. The selection of its food—if mere ex-
clusion may be called selection—is made on the basis of size, and
rejection depends on the mechanical stimulation by the larger
particles. ‘The remarkable sensitivity to touch was known to
even the oldest zodlogists who concerned themselves with the
study of the large monascidians. Its very delicacy in Ascidia
atra was a stumbling block to locating precisely the sensitive
regions.
The presence of a heavy cellulose test would suggest an in-
sensitivity of the exterior to any stimulation. Yet, even a gentle
touch on the surface of the body results in a reaction of the direct
type. Careful experimentation has convinced me that this is
not due mainly to an irritability of the test to mechanical stimula-
tion. An individual normally attached to a rock, and removed
to the laboratory with its attachment intact, serves best for this
type of experimentation. Moreover, if the substrate be securely
clamped in the aquarium, the accidental jarring of the animal
may be almost completely eliminated. Under these conditions
a gentle touch with a glass rod on the test surface leaves the
animal undisturbed. A coarser application at once stimulates.
I am not prepared to deny the presence of touch receptors on
the surface of the test. But I am convinced that most of the
results of mechanical stimulation of the test are not due to
sense organs within it, but to the passage of the stimulus through
the elastic material to the more sensitive region of the siphon rim.
In favor of this view is the lack of any demonstrable nerve con-
nection between the test and the tissue underneath it. More-
over, sources of stimulation, such as light, heat, and chemicals,
which cannot easily be transmitted along the test substance, fail
to be effective when applied to the outside of the test; whereas
in all other regions, they are just as effective as touch.
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 269
The reaction to mechanical stimulation of the test is not due
to an irritation of the underlying mantle tissue. Individuals
whose tests have been removed from a portion of the body show
that the mantle is insensitive to touch. It is interesting to ex-
plore the sensitivity of such an animal. Even vigorous poking
of the mantle (the animal must be rigidly clamped, of course)
is followed by no effect. One may approach to within one
millimeter of the cut test and produce no stimulation. But
once the test is touched, the animal immediately gives its char-
acteristic response. An animal wholly denuded of its test is
insensitive to touch on the outside except near the rims of the
siphons.
In the normal animal as one approaches the. region of the
siphons the sensitivity to mechanical stimulation rises rapidly,
and at the rim of the siphons the irritability is very great. The
rim of the oral siphon is usually divided into eight lobes, and the
atrial into six lobes. These thin lobes are the most sensitive
portions of the outside of the body. By using large animals
that have been a few days in the laboratory, and stimulating
the individual lobes with a fine glass rod, I have secured local
contractions of the portion of the rim contiguous to the stimu-
lated lobe. The folds between the lobes are only slightly less
sensitive than the lobes themselves.
On theinside of the siphons below the lobes, a similar degree of
sensitivity exists. Inside the atrial siphon the irritability is
greatest near the rim, but the entire atrial cavity is also sensitive
to touch. Within the oral siphon the surface is extremely sen-
sitive, and remains so as far down as the ring of oral tentacles.
Beyond this the sensitivity falls off rapidly.
Of the surfaces which produce the group of crossed reactions,
the tentacles are probably the most sensitive. The prettiest
automatic response of Ascidia results from their stimulation.
By illuminating the inside of the oral siphon it is possible to touch
a single tentacle with a fine glass rod. If a delicate stimulus be
applied carefully, it is most interesting to see the atrial rim close
quietly while the oral siphon remains undisturbed. If the stim-
ulus is more intense the ‘Ejektionsreflex’ is produced. When a
270 SELIG HECHT
small particle of sand is dropped carefully upon the tentacles,
the slight back pressure produced by the closing of the atrial
rim at once squirts the particle out of the oral siphon.
In view of the certainty and ease with which these reactions
may be demonstrated, not only in A. atra, but also in Hcteinas-
cidia turbinata and in another unidentified species, it is dificult
to understand why some authors have reported that the tenta-
cles are practically insensitive to mechanical stimulation. Thus
Roule (’84, p. 37), who studied Phallusia, and Lacaze-Duthiers
et Delage (’99), who observed Cynthia, state that no noteworthy
reaction occurs when the tentacles are touched in this way.
This is all the more strange because it is precisely here that See-
liger (189311, p. 323) has found most of the bristle cells to which
he rather doubtfully ascribed the réle of touch receptors.
The perception of mechanical irritation by the internal surface
of the atrial siphon is of significance in the daily routine of the
species. A decidedly sensitive area is at the bottom of the atrial
cavity near the anus. The feces are discharged into this cavity.
Here they furnish the mechanical stimulus for a reflex of the
crossed type: the oral siphon closes and the body contracts,
squirting the water and the feces out through the atrial siphon.
To one unacquainted with the presence of the group of crossed
reflexes, the defecation of Ascidia seems almost a conscious pro-
cedure. It ‘tries’ to force out the feces, and if a piece becomes
caught in the siphon rim or in the atrial cavity, it ‘tries’ again
to dislodge it by means of the ejection reflex, until finally it
succeeds. The whole process can, however, be called forth by
placing a glass bead ora pebble in the atrial cavity, or by repeat-
edly stimulating it with a glass rod.
2. Vibration
The extreme sensitivity of Ascidia to mechanical stimulation
is manifested in its ability to respond to vibrations (compare
Marage, 05). Ascidia lives in shallow water, and if the rocks
within two or three meters of an individual are stamped upon
with even a modicum of vigor, it closes its siphons. NH,>Na
This parallels the stimulating strengths of these cations found by
Cole (10, p. 607) for the common chemical sense in the frog.
In order to determine the effects of a group of anions, the
following salts were used: KCl, KBr, KNO;, KI, CH;COOK,
and KSCN. The first experiments were made on small, and
consequently very sensitive, animals. By this means large
differences in stimulating power became evident; this is typified
by Exp. VIII.3 of which the following table is a summary (table6).
Later, in order to separate KCl, CH;COOK and KSCN,
larger, and therefore less sensitive, animals were used. Exp.
VIII.4 was of this type and gave the results shown in table 7.
THE JOURNAL OF EXPERIMENTAL ZOOLOGY. VOL. 25. NO. 1
290 SELIG HECHT
TABLE 6
Liminal concentrations of a series of potassium salts
SALT CONCENTRATION
KCl 0.075 N
KBr 0.050 N
KI 0.010 N
KNO; 0.15 N
CH;COOK , 0.075 N
KSCN 0.075 N
TABLE 7
SALT CONCENTRATION
KCl 0.20 N
CH;COOK 0.15 N
KSCN 0.10 N
A combination of the two tables gives an anion series of
stimulating power as follows:
I>Br>SCN >CH;COO >Cl>NO;
Excepting SCN and NOs, which are not in the usual positions,
this order agrees with the familiar Hofmeister series (Hoéber, ’14,
p. 309). An absolutely complete agreement is hardly to be
expected, because my tests were made in seawater. Hober (’14,
p. 323) has constructed ‘Uebergangsreihen,’ in which he has been
able to change the position of some members of this lyotropic
series by altering the milieu in which the experiments were per-
formed. Analogous to this is Cole’s (10) observation for the
stimulation of the frog foot, in which the positions of NH, and K
were reversed by an increase in the concentration of the solutions.
Acids. Seawater to which acid is added, gradually returns
to its normal hydrogen-ion concentration. Therefore, the solu-
tions to be tested were freshly made up immediately before being
applied to the animal. This was accomplished by having a
stock 0.1 N solution made up in rain water, and diluting it to the
desired concentrations with seawater. The effect of the dilution
of the seawater is insignificant. Three acids, hydrochloric, formic
and acetic, were tested. The following table gives the values
which were obtained in Exp. VIII.9, typical of the others (table 8)-
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 291
TABLE 8
Liminal strengths of acids for the stimulation of Ascidia
ACID CONCENTRATION
HCl 0.0016 N
HCOOH 0.0018 N
CH;COOH 0.010 N
The order of the stimulating efficiency of the acids is, therefore,
HCl > formic > acetic
Bases. As representatives of this group of substances, I used
NH,OH and NaOH. Exp. VIII.9.1 gave the liminal values shown
in table 9. This places them in the order,
NaOH >NH,OH
TABLE 9
BASE CONCENTRATION
NaOH 0.010 N
NH,OH 0.015 N
Sugars. Both glycerin and sucrose did not stimulate until
they reached a concentration of | M. This quantity of solute,
plus the salts of the seawater in which these substances were
dissolved, brought the concentration of the stimulating solution
to just that equivalent of concentrated seawater which irritated
Ascidia osmotically. We can, therefore, conclude that Ascidia
is not sensitive to these two substances.
This has been found to be generally true for aquatic animals
(Parker, 712). Crozier (15a), however, has shown that glycerin
and maltose can stimulate Holothuria.
Alkaloids. The sulphates of quinine, strychnine and morphine
were tested. The order of their effectiveness,
strychnine > quinine >morphine
was found in Exp. VIII.1, the results of which are given in
table 10.
These values show a surprising sensitivity of the species to
alkaloids. A bitter taste in man may be secured from 0.00004 M
quinine sulphate. This amounts to one-tenth of the uncorrected
concentration to which Ascidia reacts.
292 SELIG HECHT
TABLE 10
ALKALOID CONCENTRATION
Strychnine 0.00005 M
Quinine 0.0004 M
Morphine 0.001 M
Anesthetics. Ether, chloral hydrated, ethyl aleohol and amyl
aleohol all caused reactions which were very pronounced. The
order of their effectiveness, taken from the values obtained in
Exp. VIII.1.1 and VIII.2.2 and given in the accompanying table
(table 11), is
amyl ale. >chloral, ether >ethyl alc.
TABLE 11
ANESTHETIC CONCENTRATION
Ether 0.02 M
Chloral hydrated 0.02 M
Ethyl aleohol 0.75 M
Amyl alcohol 0.001 M
6. Nature of the sense organs
The morphological nature of the chemical, and indeed of any
other kind of receptors in ascidians, is practically unknown.
Seeliger (93-11, p. 323) has described, rather doubtfully and
with much reserve, the presence of bristle cells on the tentacles
of Ciona. Lorleberg (’07), however, failed to secure any trace
of such structures in Styelopsis; although he found many regions
richly supplied with nerve endings. It may then be that the
organs of chemical sense in Ascidia are similar to those which
underlie the common chemical sense of vertebrates (Parker, ’12).
The physiology of the receptors would seem to favor such an
assumption. The problem of the physiological nature of the
chemical sense organs is simplified in Ascidia atra by the mo-
notony of response to all classes of substances. This negative
reaction of the crossed type and its variation with the intensity
of the stimulus have already been made clear. We are, there-
fore, dealing apparently with an automatic reflex, of which the
receptor and effector mechanisms are all set, and the conduction
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 293
provided for. The application of the stimulating substance to
the sense organ merely starts a prearranged process of response.
In order to understand the nature of the process set up in the
receptor, it will be necessary to consider more closely the phys-
iological effects of the substances used in the stimulation of
Ascidia. It was on the basis of the action of the salts of the alkali
metals that Héber (14) first pointed out the relation between
irritability and colloidal constitution of the plasma membrane.
Since then the ubiquity of the cation and anion series has been
demonstrated for such diverse processes ‘as melanophore con-
traction (Spaeth, *13), hemolysis (Héber, 714) and rhythmic
pulsation (Crozier, ’16a). The presence of these ionic series in
the sensory stimulation of Ascidia indicates that the significant
process which underlies it, resembles, if it is not identical with,
the determining reactions of the other physiological phenomena.
The acids have already received attention in regard to their ©
sensory effects (Richards, ’00; Kahlbaum, ’00). The anomalies
which are exhibited by the acid taste in man are typified in the
behavior of the three acids which were used in these experiments.
Although HCl is more effective than formic acid, the difference
between them is not great. They both, however, are much more
powerful than acetic. In the penetration of cells by acids
(Crozier, ’16b), we find the same order of effectiveness. The
anomalies which were referred to are as follows. When the dis-
sociation constants of the acids are taken into account, it is found
that the same effect is produced by acetic acid with a lesser
quantity of hydrogen ions than by formic acid; and less in turn
by formie than by hydrochloric acid. In Ascidia the liminal
concentrations of the acids contain the following quantities of
hydrogen ions: acetic, 4.1x10-4N; formic, 6.0x10-‘N; and
hydrochloric, 1.6 10-3N.
An analogous difficulty exists in the effects of NaOH and
NH,OH. Experiments on penetration have shown that NH:OH
enters tissue rapidly, whereas NaOH may hardly be said to pene-
trate living tissue at all. Still, NaOH is more toxic than NH,OH
(Harvey, 13). Similarly it is a more effective sensory stimulant
than NH,OH.
294 SELIG HECHT
The physiological inertness of the sugars is known only too
well to require more than mention. Their ineffectiveness has
made their use possible in experiments where the effect of osmotic
pressure only is desired (Hoéber, 714, p. 496). It is therefore
altogether in keeping with the parallelism between genera! phys-
iological activity and sensory stimulation that Ascidia fails to
be stimulated by even high concentrations of glycerin and sucrose.
It has been suggested that the sensory inactivity of the sugars
may be due to the lack of these substances in an aquatic environ-
ment (Parker, °12). The improbability of the occurrence of
saccharin in the seawater, however, does not prevent its chemical
stimulation of Ascidia. The liminal concentration of a commer-
cial preparation was 0.025 M, to which the usual negative re-
sponse was given.
It is necessary, similarly, to look in a different direction for the
explanation of the sensitivity of Ascidia to alkaloids and anes-
_thetics. The minute quantities of alkaloids which are effective
in stimulation find their counterpart in the extremely low con-
centrations in which they penetrate cells (Overton, ’97).
As a consequence of these results there can be no doubt of the
essential similarity between the general physioiogical reactions
of chemical substances and their effects on the sensory processes
in Ascidia. This indicates that the action of the stimulating agent
on the sense organ involves an effect of the same nature as the
action of these substances on other cells and tissues. Moreover,
it shows that the effect of a chemical on the receptor concerns
that structure primarily as a cell, and only secondarily as an
organ for receiving stimuli.
It must be emphasized that these generalizations are not
intended for the sense of taste in vertebrates, but solely for the
sensitivity of animals, like Ascidia and Holothuria, which possess
a general chemical sense. This type of irritability corresponds
in many ways to the common chemical sense of vertebrates
(Parker, ’12), although the two need not necessarily be homolo-
gous. ‘The problems involved in the higher organs of taste, par-
ticularly the sweet taste, do not concern us here. They represent
specialization for certain needs; and in the present condition |
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 295
of our knowledge it is futile to attempt an explanation of their
physiology.
It has been tacitly assumed that chemical sense organs are
capable of detecting substances in concentrations which fail to
affect the ordinary cells of the body. This is largely because
the effects on the sense. organs become evident through certain
effectors, whereas the action on other tissues must be noted by
special, indirect means on the cells themselves. When, however
other tissues are studied, it is seen that they are influenced
by concentrations of the same magnitude as sense cells. The
effect of minute changes of the hydrogen and hydroxyl ions on
the permeability of eggs and blood corpuscles need only be men-
tioned. Acids and bases enter cells.in concentrations like those
which stimulate animals. The poisoning effects of extremely
low concentrations of alkaloids are also familiar.
The modifications produced by these various substances are
more or less the same for all cells and tissues: witness the simi-
arity of effects produced on egg cells, sperm cells, fronds of algae,
blood corpuscles, chromatophores, hearts, medusa bells and a
host of others too numerous to mention. The concepts of ionic
antagonism and salt balance apply not only to these tissues, but
to sensory stimulation as well (Crozier, ’15b). It is therefore
clear that chemical sensitivity is merely one of a large number of
similar manifestations of the fundamental nature of cells
The explanation which seems to me to account for all the
phenomena o! this sensory activity, in Ascidia at least, is that the
factor which primarily converts a group of cells nto chemical
sense organs is not any special modification of their structure or
sensitivity, but rather their connection, directly or indirectly,
with an effector system.
In this way the problem of the chemical sense of such aquatic
forms is linked with the general problems of the physical chemis-
try of cells and tissues. Our present knowledge, in this respect,
of the chemical senses is, however, extremely meager. The time
is therefore not ripe for any adequate explanation of the process
in the receptor cell which results from the contact with a sub-
stance in solution.
296 SELIG HECHT
One such attempt has been made. On the basis of his work
on echinoderm eggs and Arenicola larvae, Lillie (11) has proposed
an explanation for general irritability. It is, that sensory stimula-
tion means an increase in the permeability of the irritable element.
Lillie’s explanation is based on the assumption that the de-
marcation current and kindred phenomena are functions of the
differential permeability of the cell membrane to certain sub-
stances, notably H and OH ions. The work of Loeb and Beutner
(14) has, however, shown that this bioelectric potential is due
on the contrary to the presence of certain lipoid materials in the
protoplasm. It is still uncertain to what extent differential
solubility and the effect of interphase boundaries are concerned
in the interpretation of these results. It is much to be regretted
that the experiments were discontinued.
There is, moreover, another and more significant objection to
Lillie’s idea. All the substances which increase permeability
undoubtedly do stimulate. But many substances, like Ca and
the anesthetics in general, all of which have a decreasing action
on permeability (Osterhout, ’16), also serve as vigorous stimu-
lants to Ascidia and other aquatic organisms.
The theory in its present form can therefore not be accepted
as an adequate explanation. However, the attempt at an inter-
pretation along the lines of permeability and similar concepts
is entirely in the right direction.
VI. SUMMARY
+
1. Ascidia possesses six distinct reactions to stimuli, all of
them negative in character. They may be divided into two
groups of three each: the direct reflexes, which depend upon a
stimulation of the exterior of the body, and the crossed reflexes,
which depend upon a stimulation of the interior of the body.
2. Theintersiphonal ganglion connects the two siphons. Sev-
ering this nervous mass completely abolishes the crossed reactions,
and interferes with the direct ones. Nevertheless, each portion
of the animal is able to perform its part of a reaction, even though
nervously isolated from the rest.
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 297
3. Ascidia is sensitive to tactile stimulation. The regions of
greatest sensitivity are the siphon rims and the oral tentacles.
4. Vibrations through solid and liquid media affect Ascidia,
although transmission through the seawater is the normal method
of stimulation. The receptors are located in the lobes of the
siphon rims.
5. The records of the amplitude of contraction to regularly
repeated mechanical stimulation show that the cessation of
response after a time is due mainly to a fatigue of the receptor
mechanism.
6. The ‘ocelli’ of Ascidia are not organs for photo-reception.
The animals are sensitive to light of very high intensity only,
and the sense organs are located within the siphon near the
oral tentacles.
7. Ascidia is thermosensitive. It reacts to temperatures above
32°C. and below 20°C.
8. Its test is insensitive to light, heat and chemicals.
9. The animals react to large changes in osmotic pressure,
and to the presence of the following classes of substances in solu-
tion: salts, acids, bases, alkaloids and anesthetics. Solutions
of sugars do not stimulate, but saccharin gives a decided reaction.
10. The liminal concentrations and the relative effectiveness
of all these stimulating substances are very similar to those which
have been demonstrated for other physiological activities. It is,
therefore, suggested that the primary factor which converts a
group of cells of Ascidia into chemical sense organs is their con-
nection with an effector system.
298° SELIG HECHT
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AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY
THE BIBLIOGRAPHIC SERVICE, FEBRUARY 16
EXPERIMENTS ON THE PHYSIOLOGY OF DIGESTION
IN THE BLATTIDAE
ELDON W. SANFORD
Osborn Zoological Laboratory, Yale University, New Haven, Conn.
TWENTY-ONE FIGURES
INTRODUCTION
The following experiments were undertaken to explain certain
phases of digestion in cockroaches, and especially the processes
concerned in digestion of fat, as well as other related problems.
The subject is not a new one; many investigators have studied
various phases of the problem, but they have in several cases
arrived at very different conclusions, One of these workers was
Prof. Alexander Petrunkevitch (’00), who suggested the problem
to me.!
The real object of my investigation has been to attack the
problem from a new point of view, besides repeating to a greater
or less degree the experiments of previous workers. I have en-
deavored to amplify considerably the scope of previous work
through the use of newer stains and the physiological and ex-
perimental methods.
The main problems which I hoped to investigate and elucidate
were concerned with the rdle of the crop in digesting and ab-
1 Prof. Petrunkevitch’s paper appeared in 1900, and since then several inves-
tigators have come to results distinctly at variance with his. This disagreement
is explained in part by the fact that Professor Petrunkevitch gave almost no
data concerning the details of his experiments. Most of the data were lost in
consequence of political disturbances, and without the data the work could not
be defended. Moreover, lack of these data prevented repetition and verification
of the experiments. After many trials I arrived at methods of experimentation
which tended to verify Petrunkevitch’s results, and to explain the disagreement
of other authors. From this beginning I proceeded to further and more varied
experiments, always using groups of animals in each experiment. More than six
hundred cockroaches were used in all.
355
356 ELDON W. SANFORD
sorbing food, the various functions of the gizzard, the details
and significance of digestive processes in the stomach and coeca,
and the explanation of the presence of certain substances and
leucocytes in the tracheal tubes.
MATERIAL AND METHODS
The material first used consisted in the species of cockroach
previously used by most earlier investigators in the field of fat
digestion in insects, namely, Blatta orientalis Linnaeus (Peri-
planeta orientalis), the common black cockroach, and Blatella
(Philodromia) germanica, the croton bug. The latter species
was found rather small for convenient dissection and observa-
tion. Later on I used Periplaneta americana, the American
cockroach, almost exclusively. In this species, which is very
hardy and much larger, it seemed hopeful that some processes
which had been obscure or disputed in the smaller species might
be seen more plainly, and indeed this proved to be the case.
One especially fortunate fact was discovered, namely, that the
body cavity in the abdominal region could be entered in the
living animal by turning back a flap of the body wall. After the
flap was replaced, the animal experienced no serious incon-
venience. It was found possible thus to make ligations of the
alimentary canal just after food was taken, thus isolating certain
digestive organs from connection with others. This eliminated
the uncertainty of previous work as to the flowing of digestive
juices from one part to another. The digestion which occurred
in a part tightly ligated from other parts was considered to be
surely produced by digestive fluids and enzymes secreted in
that part.
Another fortunate feature about Periplaneta americana is its
tolerance of sunlight. Unlike Blatta orientalis, which is very
sensitive to light and avoids it whenever possible, my animals
walked around contentedly even in bright light and took food
normally.
The tissues were usually fixed in Flemming’s strong solution,
which contains osmic acid and stains fats black by an oxidative
process. This fixing fluid is especially effective in work with
PHYSIOLOGY OF DIGESTION IN BLATTIDAE aI
olive oil, for the pure oil consists mostly of the glyceride of oleic
acid, and oleic acid is the only one of the four common fatty
acids of ordinary fats which is blackened well with osmic acid,
this being due to its unsaturated molecular structure. In work
with tissues containing blackened fat it seemed inadvisable to
stain with any haematoxylin stains, for these stain so darkly and
tend to obscure appearances of the darkly stained fat. As a gen-
eral stain acid fuchsin was used successfully; it stains nuclear
and cytoplasmic structures sufficiently distinctly. For obtaining
the best histological details Perenyi’s fluid was much used, and
also a mixture of 5 per cent formol and 50 per cent aleohol. ‘The
best combination of stains for accurate. definition of structures
was acid fuchsin, followed by Ehrlich’s haematoxylin. In
working with the very compact and hard tissues of the gizzard,
Flemming’s killing fluid was very satisfactory when it penetrated
well, which was not always the case. Perenyi’s fluid was also
satisfactory. In order to bring out the muscle structures of the
gizzard, careful staining with iron haematoxylin and picro-
fuchsin is necessary. When the animals had eaten fat stained
with Sudan III or Nile blue sulphate, sections were made in a
freezing microtome.
Most of the tissues contain so much water that extra care is
necessary in running them through a series of reagents. In
running ordinary tissues from absolute alcohol through xylol to
melted paraffin, it is very desirable to interpose between the
alcohol and xylol two appropriate mixtures of these reagents.
In running from xylol to melted paraffin, it is necessary to inter-
pose a cold and a warm mixture of the two. Unless this is
done, the nuclei and cytoplasm shrivel and the fat globules be-
come distorted and liable to fade rapidly.
The fat used as food in the experiments was pure olive oil.
The animals refused to take pieces of solid fat, but would take
bread soaked in olive oil. But the ideal food seemed to be a
paste of pulverized sugar and olive oil. This was taken greedily,
even when stained brightly with Sudan III, Nile blue sulphate,
or litmus powder. The organs chiefly concerned in recognizing
the food semed to be the maxillary and labial palpi, not the eyes
358 ELDON W. SANFORD
or antennae. The feeding was done in covered glass jars, in
one part of which some of the paste was placed. The animals,
when put into the jars, walked or ran about until the mouth
parts and palpi happened to touch the food; then stopped,
moved the palpi over the food and began to eat by opening and
closing the mandibles. After feeding was completed the ani-
mals were removed to a clean dish. When colored foods had
been eaten, it was always possible to decide in a general way
how much a given animal had eaten, for the color of the food in
the crop showed through the transparent crop wall and the
semitransparent region of the body wall between the third pair
of legs. When an animal had eaten a moderate or very generous
amount of the paste, it stopped to clean itself carefully, going
over the whole of the anterior legs with the mouth parts, and
drawing the antennae through the mouth parts by aid of the
anterior legs, meanwhile apparently licking off any oil or other
extraneous matter on them. The fat-feeding methods of
Schliiter were also employed; that is, the smearing of the head
and anterior body with olive oil with a brush and then relying
on the cleaning habits of the animals to get the food into the
mouth and crop. This method was considered inadequate and
not practiced much, because it was so unnatural, and because
experience showed that fat smeared on the thorax entered the
thoracic spiracles readily, and thus gave false pictures of fat
distribution in subsequent preparations.
ANATOMY AND HISTOLOGY OF THE ALIMENTARY CANAL
In view of the fact that there is a discrepancy of opinion as to
the structure of the alimentary canal, it seemed desirable to in-
vestigate it once more without going into unnecessary detail.
This was undertaken as a basis for physiological work. The
comparative lengths of the various parts in a specimen where the
total length of the alimentary canal was 6.7 were as follows:
oesophagus 0.25 em., crop 2.5 em., gizzard 0.25 cm., stomach
1.3 em., small intestine 0.25 em., large intestine 1.6 cm., rectum
0.5em. It will be noted that the crop possesses by far the largest
PHYSIOLOGY OF DIGESTION IN BLATTIDAE 359
surface of any single organ, being 2.5 em. in length and on the
average 0.6 cm. in diameter when distended with food, thus
having a surface of about 3 sq. em. as against a corresponding
surface of 0.7 sq. em. in the stomach, which is 1.3 em. in length
and 0.25 em. in diameter.
The stomodeum, or that part of the anterior alimentary canal
which is turned in during the embryonic period, is of considerable
size in the American cockroach, and may be divided into six
parts: mouth cavity, pharynx, oesophagus, crop, anterior giz-
zard, and posterior gizzard. The mouth cavity and pharynx
were not studied, for it is most improbable that any important
digestive changes occur there. The salivary glands are paired,
one lying on each side of the crop along about half its length.
The ducts run forward into the mouth and are strengthened
by a tough spiral structure similar to that of the tracheal tubes.
The reservoir of the salivary secretion may be very large, it
may be almost as long as the crop when it is dilated with fluid.
Oesophagus. The oesophagus begins where the dilation of the
pharynx ends and varies much in size according as it is dilated
with food or not. When empty its cavity is nearly obliterated
by closely appressed folds. These folds are continuous with
those of the crop to such an extent that it is impossible to say
where one ends and the other begins. The oesophagus wall
consists of a single layer of nearly cubical epithelial cells (fig. 1),
between which are seen muscle processes and tracheal end cells
similar to those described by Petrunkevitch. Within the layer
of cells and secreted by it is the chitinous intima, of about the
same thickness as the cells. This is raised into projections, on
the summits of which long bristle-like processes are situated.
Around these structures is a layer of circular muscles.
Crop. The crop is by far the largest part of the alimentary
canal and no doubt has an important function, though Schliiter
considers it as a mere storage organ and conducting tube to
the stomach. The wall consists of three layers, muscular, epi-
thelial, and chitinous. The muscular layer consists of two
layers of striated fibers at right angles to each other. One layer
consists of circular muscles and serves to contract the crop walls
360 ELDON W. SANFORD
tightly over any food within. The other layer consists of lon-
gitudinal muscles. The muscles form a loose network, and
through the meshes the general body cavity connects with spaces
under the epithelial cells. These spaces are under folds in the
epithelial layer in the usual state, but are nearly or quite oblit-
erated when the crop is distended with food. -In these folds
are found muscle fibers and processes; which run from the mus-
cular layer to the epithelial and chitinous layers, and may be
considered as radial muscles, as shown in figure 2.. Wandering
blood cells and tracheal branches are also found in these spaces
(fig. 2). The latter extend on one side to larger tracheal branches
and on the inner side to tracheal end cells.
The epithelial cells have the shape of hexagonal prisms twice
to four times as high as broad. The nuclei are situated near the
bases of the cells; that is, toward the body cavity, as shown in
figures 2, 5, 17. The cytoplasm of the nuclear region usually
stains more deeply than the cytoplasm of the other end of the
cell, and is apparently more specialized. Between the cells and
the lumen of the crop is the rather thin chitinous intima.
Petrunkevitch has demonstrated that this intima is porous, and
I have verified his work, using his methods. Some earlier
workers used this porosity to account for the passage of fat
globules into the cells from the lumen, but it is now known that
fats cannot be absorbed as such, but must be split to fatty acid
and glycerol, both of which, being soluble, may be absorbed
into the cells.
The muscular layer may be separated to a greater or less extent
from the epithelial layer by teasing with needles under a bin-
ocular microscope. This applies for the oriental cockroach, and
not for the American cockroach. Flat preparations of the
epithelium may be made by holding a piece of the crop’s wall
tightly down and flattening out folds under a coverglass, then
running in Flemming’s fluid, or absolute alcohol, as recom-
mended by Petrunkevitch. A much better method of getting
flat preparations consists in feeding an animal until its crop is
distended and therefore all the folds of the surface are smoothed;
the crop is then removed without puncturing and preserved in
PHYSIOLOGY OF DIGESTION IN BLATTIDAE 361
Perenyi’s fluid. In the American cockroach there is a definite
region of the anterior crop where there are very few muscles,
this region is especially favorable for study. In these prepara-
tions the epithelial cells appear mostly as hexagons.
Petrunkevitch has described certain unusual conditions in
some of these cells, and has figured them as seen when flat and in
cross sections. In some cases the cell walls of a group of cells
degenerate and the cells fuse. The cytoplasm appears vacuo-
lated and abnormal and the nuclei begin to degenerate. Finally
the whole mass of protoplasm seems to be discharged and the
gap filled by the growth and cell divisions of adjacent. cells.
He also describes cells which contain two or more nuclei, but do
not appear abnormal otherwise; these represent small syncytia
resulting from nuclear divisions with suppressed cytoplasmic
divisions.
Among my preparations some slides show stages which may be
likened to these. The cells show great inequality of size, the
very small ones appear to be latent, but ready to replace by
growth any gaps in the epithelium. Binucleate and trinucleate
cells often appear, but I cannot explain their significance, per-
haps they represent small syncytia. Some of them are shown
in figure 18. Another unusual condition is shown in figures 17
and 18. In such cases the cytoplasm appears abnormal and
numerous nuclei are present, some of them appearing degenerate
or as mere vesicles. No traces of cell walls are present in these
regions, and the cytoplasm seems dead. I have been unable to
.find a definite explanation of such pictures; perhaps they repre-
sent a sort of lesion which results from injury to the cells through
mechanical damage by sharp pieces of food or through the ac-
tion of poisonous matter in the food. The process cannot, I
think, be compared with the normal casting of cells of the
stomach epithelium after they have become worn out by continual
functioning.
Petrunkevitch has also figured ring cells; that is, cells which
contain a very large vacuole surrounded by a thin layer of cyto-
plasm containing the nucleus. He believed that these cells
represented a stage in the fusion and casting of cells, especially
362 ELDON W. SANFORD
as two or three cells might combine to one ring cell. In the
species I have studied this appears not to be the case. I have
never found any such appearances except in preparations which
were fixed in absolute alcohol, and even then the appearances
were not distinct, and probably represented artifacts due to too
rapid dehydration.
Gizzard. The gizzard consists of two distinct parts, an anterior
and a posterior. Its shape is in general that of a cone whose
blunt end is anterior and adjoins the crop and whose pointed
end extends into the stomach. Its altitude about equals the
diameter of the base. The hinder; protruding part is of about
the same length as the anterior part. The two parts are entirely
different histologically. In the anterior part the chitin forms
six heavy teeth; the chitin has no distinguishable structure for
the most part, but may be shown by staining with erythrosin
to consist of three homogeneous layers, an outer and an inner
layer which take no stain and a median layer between them
which stains bright red.
The six thick teeth fill most of the cavity of the anterior part.
They are large projections, triangular in cross section and roughly
rectangular in longitudinal section, which nearly fill the space
within, leaving only a narrow lumen in the middle and small
spaces between their sides (fig. 8). The outlines of the teeth
are not quite triangular, but often have swellings or concavities
on the sides and somewhat flattened points. Usually a swelling
of one tooth lies opposite a cavity on the adjacent tooth, and
flattened and pointed ends do not sharply oppose each other.
Ramme has found that in cockroaches the six teeth fit tightly
and perfectly and are so well held together by the thick circular
muscles that a tight fastening may be made.
Under the chitinous intima lies a single layer of long cylindrical
epithelial cells, and below them a mass of connective tissue,
through which run many tracheal tubes. Just under the epi-
thelial cells these tubes end in tracheal end cells, whose proc-
esses run up between the cells above. Outside of the con-
nective-tissue layer is a broad layer of circular striated muscles.
PHYSIOLOGY OF DIGESTION IN BLATTIDAE 363
Between the main teeth are secondary and tertiary ones.
These, unlike the primary ones, are covered with short spines.
Between each pair of large teeth are three secondary ones, evenly
spaced (fig. 9). Between each primary tooth and its adjacent
secondary tooth are three or five tertiary teeth, and between
adjacent secondary teeth are two or three tertiary ones. So
the gizzard has six primary teeth, eighteen secondary ones, and
sixty or ninety-six tertiary ones.
The teeth extend through about half the length of the anterior
gizzard.. The region between their posterior ends and the pas-
sage from the gizzard into the stomach is provided with smaller
rounded projections which are set with long yellow spines or
needles. Just behind the primary teeth are considerable folds,
which Miall and Denny have called cushions for the teeth. These
are shown in figures 8 and 9. The spines here are large and uni-
form, and are situated in sockets on the summits of dome-shaped
chitinous projections. The structure and relations here will be
discussed later. Posterior to the cushions les a region of many
smaller lobes and folds, the walls of which merge into the duct
leading to the stomach. ‘These lobes are thickly set with simply-
structured bristles. Adjacent and opposite lobes are usually
closely opposed to each other, so that little or no passage is left
(figs. 8 and 15). They may thus effectually block the passage
of food from the crop through the gizzard to the stomach.
The epithelium of the anterior part of the gizzard merges into
that of the posterior. This posterior part projects far into the
stomach and consists of one epithelial layer bounding the lumen
and another which turns back from the apex of the projection
parallel with the first and outside of it. This epithelium merges
with the extreme anterior end of the stomach wall, in the region
where the coeca originate. The lumen of the projecting part
of the gizzard is small and is almost obliterated, being reduced
to a star shape by the projecting rounded folds of epithelium,
which are surrounded by muscles. Occasionally I have found
small black bodies in the cells here in preparations fixed in
Flemming. The epithelium of the outer side bears small rounded
chitinous bodies to which short spines are attached.
364 ELDON W. SANFORD
Stomach and coeca. The stomach extends from the gizzard to
the point of entrance of the Malphigian tubules. In its normal
position it is curved, and bent so that its posterior end is almost
directly below its anterior end. It is crowded in the abdominal
cavity, and is small in proportion to the size of the animal and
in proportion to the crop’s size. From the anterior end arise
eight coeca, each of which is of about a third the diameter of the
stomach and of about half the stomach’s length. The variation
in size is very great, especially depending on whether the coeca
are filled with food or not. Sometimes the coeca are as long as
the stomach. The outer wall of the stomach is composed of a |
loose meshwork of muscular fibers, among which tracheae
ramify. The same can be said for the outer wall of the coeca.
The assertion of Jordan that the fibers of the muscle meshes
only pass under unspecialized cells seems to apply in the Ameri-
ean cockroach. In this way much of the surface is left free of
striated muscle, which would, if present, hinder the passage of
food materials from the epithelial cells into the body cavity.
The epithelial cells are in general long and narrow as seen in
either transverse or longitudinal section, and are similar through-
out the whole length of the stomach. As in most insects, the
inner edge is set closely with fine filaments which serve to pro-
tect the epithelial surface (fig. 21). At very frequent intervals
groups of immature cells are seen between the true epithelial cells
and the muscular layer. These cells are small and closely packed.
In many places all intermediate stages between them and adult
cells are seen, as shown in figure 21, so it seems certain that there
is a continual replacement of the mature cells, which often die
and are cast, as I shall later describe. The epithelium usually
shows but few adult cells in a group, and these groups more or
less widely separated by the intermediate cells which take their
places later. The surface of the epithelium may be flat or undu-
lating, but is often raised into small villi with U-shaped spaces
between them, the so-called crypts of Frenzel. The ends of the
villi may be smoothly rounded or bulged out considerably.
The arrangement in villi allows a much greater surface for secre-
tion and absorption. I agree with Biedermann that the crypts
OLE
PHYSIOLOGY OF DIGESTION IN BLATTIDAE 365
are not glandular in function, but are adapted for efficient re-
generation of cells. The membrane lining the wall of the epi-
thelium is evidently cast at intervals, for it may be seen at
various stages of disintegration. Such stages correspond to
those described by Schroder. The spheres of secretion which
appear on the surfaces of the cells will be discussed in a later
section on the functions of the stomach.
The structure of the epithelium of the coeca is the same as
that of the stomach in every detail. This shows plainly in sec-
tions which show the branching of the coeca from the stomach.
In these cases it cannot be determined just where the coeca
originate. I have seen practically no evidence of more secre-
tion globules on the coeca epithelium, an observation which is
surprising in view of the fact that several authors have de-
scribed the coeca as preeminently secretory organs, capable of
producing most of the secretion found in the stomach. Later I
shall show that not only are the histological structures of the
stomach and coeca alike, but also the digestive functions.
At the hinder end of the stomach the epithelium makes a cir-
cular fold which partially constricts the passage into the small
intestine. This may be considered as a valve. Schroder sug-
gests that the purpose of it is to help roll up boluses of food
matter and pass these on to the small intestine, instead of a
constant stream. Backflow is somewhat prevented, too.
Small intestine. The small intestine is very short and has
asmalllumen. Just behind the fold or valve at the hinder end of
the stomach the high epithelium of this region gradually becomes
lower and merges into the thin epithelium of the small intestine.
At this point the Malphigian tubules enter in six great groups.
The epithelium in this section consists of nearly cubical cells
whose cell boundaries are hard to make out. The layer of cells
is very much folded. The chitinous intima bears small swellings
surmounted by very short spines. The cell structure resembles
that of the oesophagus. Surrounding the epithelium is a thick
layer of apparently confused muscular fibers. The function of
the small intestine is not absorptive, and it has never been so
considered except by Frenzel and Deegener. The explanation of
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 2
366 ELDON W. SANFORD
their findings may le in their considering cells immediately
behind the stomach valve as intestinal cells, while they really
belong to the stomach.
Colon. The large intestine or colon is long and coiled in its
position in the body. - Its epithelium consists of long, narrow
cells which are thrown into rather regular folds. Under the
folds tracheae and tracheal end cells may be seen. In the
American cockroach the histological details may be seen well
after using Flemming’s strong killing fluid. On the contrary,
in the oriental cockroach, the structure is spoiled by Flemming’s
fluid, as Petrunkevitch and Schliiter agree. In any case Pe-
renyi’s fluid is satisfactory. The colon is divided from the small
intestine by a fold or valve of the epithelium. The intima is
raised into significant mounds over each of the cells, but no
hairs are evident, as described for Blatta orientalis. The func-
tion of the colon is not absorptive, at least I have not found
it so, nor have many other authors except Frenzel, and his. ob-
servations have since been disproved by later investigators.
Simroth and Mingazzini have stated that certain sacs of the
colon were absorptive in Lamellicorn larvae, but their deserip-
tions indicate that there was no real absorption, but mere passage
through pores. Berlese has described a similar case.
Rectum. ‘The rectum is the last part of the digestive canal
and terminates at the anus. It is surounded by a thin muscular
layer. The so-called rectal glands are six lobes of very long cells
which bulge far out into the lumen. Their cells do not quite
reach the muscular layer, but leave small interspaces, in which
tracheae ramify. The cells of the glands have a distinct intima,
but no chitinous filaments. This intima is continuous between
the glands and dips down almost to the muscular layer,. there
being almost no cells between:
EXPERIMENTS IN PHYSIOLOGY
Oesophagus. The function of the oesophagus is merely to
conduct food into the crop or to retain it for a time if the crop
is quite full. The thick intima evidently precludes all secretion
and absorption here. No globules of absorbed fat were ever seen
in the cells.
PHYSIOLOGY OF DIGESTION IN BLATTIDAE 367
Salivary glands. Various investigators have found that the
reaction of the saliva in Orthoptera is never acid, but may be
neutral, as Plateau found in Blatta orientalis, or alkaline, as in
Blattella germanica. Jordan states that the saliva has about the
same digestive power on carbohydrates that human saliva has.
Crop. If we consider the function of the crop, there are two
principal possibilities, 1) secretion and 2) absorption.
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PLATE 3
EXPLANATION OF FIGURES
10 Transverse section of the gizzard in the region of the six cushions, showing
the passage of the lower ends of the six muscle bands through the cushions to the
needles on their surfaces. Flemming. Iron haematoxylin and picro-fuchsin.
35 diam.
11 Detail of the connection between the muscles and the needles of the
cushions. It is shown that the striated fibers divide to fibrils, themselves stri-
ated. These pass through the epithelium and become tendinous. The tendons
cross the matrix of the intima and insert in the bases of the needles. Flemming.
Tron haematoxylin and picro-fuchsin. 200 diam.
12 Section through intima of a cushion, nearly parallel to surface, showing
in transverse and diagonal section the tendons which traverse the intima.
Zenker. Iron haematoxylin and picro-fuchsin. 90 diam.
13. Surface view of a group of bristles of the cushions, showing the chitinous
projections to which they are movably attached. 200 diam.
14 Section through a tracheal tube which is filled with coagulated deposit,
in which leucocytes may be seen. Flemming. Acid fuchsin. 350 diam.
408
PHYSIOLOGY OF DIGESTION IN BLATTIDAE PLATE 3
ELDON W. SANFORD
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PLATE 4
EXPLANATION OF FIGURES
15 Longitudinal section through hinder end of the gizzard, showing the
closely appressed folds of the inner wall and the thick surrounding sphincter
muscle, which seems to make a tight closure. Flemming. Acid fuchsin. 35
diam.
16 Transverse section of the same region, showing the folds almost closing
the passage, and the surrounding sphincter. Flemming. Acid fuchsin. 60
diam.
17 Transverse section of the wall of the crop, showing in one place an ab-
normal appearance or ‘lesion’ of the epithelium. Flemming. Acid fuchsin.
325 diam.
1S Surface view of epithelium of the crop, showing unusual conditions.
Several binucleate cells are present, and also a region of cytoplasm where cell
walls are lacking. Perenyi. Ehrlich’s haematoxylin. 200 diam.
19 Transverse section of stomach two days after ingestion of fat. The
epithelium shows frequent dark regions which indicate absorption of fat here.
The lumen of the stomach contains oil drops, pieces of chitin, and other sub-
stances. Flemming. Acid fuchsin. 35 diam.
20 Transverse section of a caecum, prepared in the same way as the stomach
oi figure 19, and showing a similar condition of fat absorption. 35 diam.
21 Transverse section of epithelium of stomach’s wall after ingestion of fat,
showing in more detail the groups of cells which absorb fat, and also the em-
bryonic cells which continually make new cells to replace the lost ones. Flem-
ming. Acid fuchsin. 200 diam.
410
PHYSIOLOGY OF DIGESTION IN BLATTIDAE
ELDON W. SANFORD
PLATE 4
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AUTHORS’ ABSTRACT OF THIS PAPER ISSUED BY
THE BIBLIOGRAPHIC SERVICE, FEBRUARY 16
THE INFLUENCE OF EXCESSIVE SEXUAL ACTIVITY
OF MALE RABBITS
1. ON THE PROPERTIES OF THE SEMINAL DISCHARGE
ORREN LLOYD-JONES AND F. A. HAYS
Iowa State College
CONTENTS
EATEN ROK HOTA Sata ats aint gee ae mic or AIS COE eee EE eens Petibint.c oondo nanan 463
Generalaplanvof @xperiment,, csc vs ois aeae ee hoe ind ees eee aes elses 464
VE COMETyg Ole SC IMG M te ere ai. Nos MS cncrk ena ooo ess neers ae ghee yete ca ee Pouce cera 466
BY OTe ON NIE en a ao Sita bie cio eta aS ERR ISI Seo He Dieticians Bsitiao Ga ane aioM 468
Nolumeromsemen mecovieredia..
10-22 24 0 37 22 90 20 83
44 5 30 0 16
10- 1 20 0 72
10-15 28 0 0
11- 9 20 58 75
24 21 29
However, we feel that a study of the data as above suggested
will leave one with a decided opinion that there is a well-marked
tendency for the sperm in semen from the higher-service groups to
show less ‘vitality’ or ‘potential energy,’ as measured by the
duration of motion which they will display.
Table 5 is inserted here to compare the duration of motion of
sperm in semen as it was recovered from the female with sperm in
semen diluted with ten volumes of an isotonic solution. The
counts of ‘per cent active’ are much less accurate with undi-
luted semen than with diluted because of the crowding of the
sperm. Moreover, they are so close together that the agita-
tion set up by one active individual may cause several others to
appear active, whereas they are not actually of themselves motile.
But the point at which all sperm are inactive may of course be
determined with as great accuracy in one case as in the other.
Though the table is not extensive, the deduction to be made
from these figures is plain; namely, that sperm in diluted semen
display a longer duration of motion than sperm in ‘natural’
semen. The probable cause of this phenomenon is that in the
492 ORREN LLOYD-JONES AND F. A. HAYS
TABLE 6
, Matings and resulting pregnancies from the various end services
ist 5TH 10rH 15TH 20TH
a | & er |b] a RR Vale Ob ie | enw seoea carla anes s
Sie. |) SR RS BT =n T= fe = Daa lites ese |||
Sieteeees | 8 og | Seles le eae | 28) | Saale
2 Se || JE Spi ar SW SS Sie. oan |e = g
Simon es | a | euieers Ps) ae sali Mica Og |S aes
= 3 2 55 | 6 z 58 ° z 58 iS z 54 ° 2 58
Seeley | 6 B.| & | Be | a | a eB | a | a Be | & | &
1 17 | 11 |64.70) 9 TANTS CA) 74 6 |50.00) 12 | 6 |50.00) 26 | 10 |38.46
: 14 | 12 /85.71| 15 | 9 |60.00} 11 6 |54.54; 5 | 2 |40.00
4 12 | 8 66.66) 12 | 6 |50.00 6 150.00) 12) 4 |33.33) 19 | 6 131.57
Total] 43 | 31 |72.09) 36 | 22 |61.11} 35 | 18 |51.42) 29 | 12 |41.37| 45 | 16 |385.55
natural semen the by-products of the metabolism of the sperm,
and also of the developing bacteria more quickly reach a con-
centration that is deleterious or even fatal to the sperm.
Whether the fertilizing power of the sperm is diminished by this
dilution is a matter for further study. Certainly it is not com-
pletely destroyed, for Iwanoff reports inseminating rabbits with
semen diluted with a weak Na,CO; solution, with resulting
pregnancy.
CERTAINTY OF PREGNANCY
In the previous sections of this paper we have shown that ex-
cessive sexual service causes decrease in amount of ejaculated
semen, decrease in number of sperm cells per cubic millimeter,
decrease in the proportion of sperm that show progressive mo-
tion, and decrease in duration of motion. All these changes are
such as to reduce the likelihood of such semen causing pregnancy
of the female. In other words, we should expect the percentage
of pregnancies induced by the copulations to become less as the
number of preliminary services increases. This is in truth found
to be the case as shown in table 6.
The decline proceeds with fair regularity whether we consider
the males separately or together. This reduction in the per
cent of effective matings when the male is sexually overworked is
recognized by those engaged in animal breeding as one of the
SEXUAL ACTIVITY OF MALE RABBITS 493
TABLE 7
Size of litters at different services
Ist 5TH 10TH 157TH 20TH
MALE NUMBER | Num- | Aver- | Num-| Aver- | Num-| Aver- | Num-| Aver- | Num- | Aver-
ber age ber age ber age ber age ber age
litters size litters size litters size litters size litters size
1 6 6.66 6 6.00 1 (All 7 Hess], dul 4.27
3 11 6.50 8 5) (43) 7 Uf fB0/ Pe 10.5
4 8 Ye 6 6.16 6 nas) 4 5.9 6 5.00
Aerial] GSteaes|ezo: 6.92 20 5.95 20 6.95 13 6.46} 17 4.53
most noticeable and universal concomitants of heavy sexual serv-
ice, but there is great divergency of opinion as to how frequent
or how many copulations constitute ‘heavy service’ with vari-
ous classes of domestic animals. Apparently in bringing about
twenty services in three or four hours we have approacheda point
which for the rabbit seriously curtails his ability to produce off-
spring, and furthermore we have demonstrated in large part
what is the direct basis for this curtailment.
SIZE OF LITTER
Having observed the described changes in the vital properties
of the sperm in semen from advanced services, and also what is
probably in large part a result of the same, namely, the reduced
likelihood of pregnancy resulting from the more advanced serv-
ices, 1t would seem a priori to follow that the number of young
per litter would likewise undergo a reduction as the number of
copulations increases. Table 7 presents the facts in regard to
this matter.
Inspecting the columns up and down reveals no superiority of
one male over another. Inspecting the horizontal rows reveals
no marked effect of number of copulations on litter size; in fact,
it may be questioned if it reveals any significant reduction.
Certainly any falling off in litter size up to the 15th service is
impossible to detect by inspection, but a very perceptible drop
occurs between the 15th and 20th service.
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, No. 2
494 ORREN LLOYD-JONES AND F. A. HAYS
TABLE 8
Relation between size of litter and number of previous services
|
NUMBER OF SERVICES
SIZE OF LITTERS
Ist 5th 10th 15th 20th Total
1 1 1 2 1 5
2 1 1 2 4 8
3 2, 1 4 7
4 2 2
5 3 3 2 1 1 10
6 7 6 3 16
Uf 6 2 4 1 2 15
8 5 3 4 2 1 15
9 1 4 2 7
10 3 1 2, 1 | a
11 1 1 1 3
12 1 1
26 20 20 13 17 96
Correlation coefficient = — 0.2207 + 0.06526.
Table 8 shows the complete distribution of the litters on the
basis of size arranged as a correlation table. The coefficient of
correlation calculated from this table is — 0.2297 — 0.0653, a
figure which, though over three times its probable error, is yet
of rather questionable significance. An inspection of the table,
however, shows up a marked preponderance of small litters in
the 20th service. If-it is true that the critical number of serv-
ices, so far as decreasing the number in the litter is concerned,
is between the 15th and the 20th service, then of course the above
method of determining the correlation between frequency of
copulation and number in litter is not valid, for in this case the
20th, as one group, should be compared with all others as another
group, and not be considered as one of five coordinate groups.
In table 9 the first four columns of table 8 are combined and the
percentage of the total litters in each group which are of a given
size calculated.
The preponderance of small litters in the 20th-service group is
thus revealed. If we condense the data still further into a four-
fold classification, we find that in the 1 to 15 group 25.32 per cent
SEXUAL ACTIVITY OF MALE RABBITS 495
TABLE 9
Size of litter in 1st to 15th service group compared with 20th
1-15 20
81ZE OF LITTER
Number of litters Per cent of litters Number of litters Per cent of litters
of given size of given size of given size of given size
1 4 5.06 1 5.88
2, 4 5.06 4 23.53
3 3: 3.80 4 23.58
4 0 0 2 11.76
5 9 11.39 1 5.88
6 16 20.25 0
i 13 16.45 2 11.76
8 14 Vi 72 1 5.88
9 7 8.86 0 0
10 6 7.59 1 5.88
11 2 2.52 1 5.88
12 1 1.28 0 0
Totaless.= 79 Ad,
of the litters were five or less in size, whereas in the 20th-service
group 70.58 per cent of the litters were five or less in size. In
spite of the fewness of the 20th-service litters, there seems to be
a presumption in support of the idea, that by the time a male
rabbit has performed twenty copulations within the space of
three or four hours he is less able to beget large litters than
when he has performed fewer than fifteen services within the
same space of time.
When we consider in relation to each other the facts shown by
tables 6 and 7 we are confronted by something of a puzzle. We
have interpreted the facts shown in table 6, in part at least, as a
direct consequence of the facts shown in the previous tables,
i.e., that when the volume of semen, the number of sperm per
cubic millimeter, the amount of progressive activity and the
potential duration of motion of the sperm are all reduced, then
the likelihood of spermatozoa reaching and penetrating the ova
is reduced, and therefore the per cent of pregnancies is dimin-
ished. Table 6 shows a fairly regular and consistent decrease
in certainty of pregnancy as sexual service increases. Now, as
mentioned above, those circumstances which decrease the like-
496 ORREN LLOYD-JONES AND F. A. HAYS
lihood of pregnancies should a priori reduce in almost like degree
the size of those litters which are produced. But table 7 does
not by any means indicate such a regular and consistent decrease
in size of litter. In fact, up to the 15th service there is no per-
ceptible falling off in litter size whatever. It is possible of
course to form hypotheses to account for this condition of affairs,
but at this time we know of no sound basis on which this dis-
crepancy between tables 6 and 7 may be satisfactorily dealt
with.
There exists considerable discussion, but so far as the writers
know, no carefully controlled experimentation to the effect that
in multiparous animals the male is without influence on litter
size; the millions of sperm supplied by any ‘normal’ male at a
single ejaculation will, it is said, be more than ample to impreg-
nate the ova liberated. Our own work indicates that for rabbits
there is at least one condition, 1.e., performing twenty services in a
short time, under which ‘normal’ males may be unable to bring
about complete development of the full quota of ova liberated
by the female. It is not inconceivable that the reproductive
system of supposedly and apparently ‘normal’ males may be
chronically in a condition analogous to that brought about in
this experiment by excessive sexual service.
SEXUAL ACTIVITY OF MALE RABBITS 497
LITERATURE CITED
Biscuorr, W. 1842 Entwicklungeschichte des Kaninchen-Eies. Pp. 1-154,
Braunschweig, Vieweg und Sohn.
DetLersEen, J. A. 1914 Genetic studies on a Cavy species cross. Carnegie
Institute of Washington. Pub. No. 205, pp. 1-183.
Heart, WALTER. 1905 Ovulation and degeneration of the ova in the rabbit.
Proc. Royal Society of London, vol. 76, pp. 260-267.
Hensen, V. 1875 Beobachtungen iiber die Befruchtung und Entwicklung des
Kaninchens und Meerschweinchens. Zeit. fiir Anat. und Entwick.,
vol. 1, pp. 212-273.
Iwanorr, Evie. 1907 De la fécondation artificielle chez les mammiféres.
Arch. des Sci. Biol., vol. 12, pp. 377-511.
K6uurkerR, A. 1856 Physiologiche Studien tiber die Samenfliissigkeit. Zeit.
fiir wiss. Zool., vol. 7, pp. 201-272.
Krart, H. 1890 Zur Physiologie des Plimmerepithils bei Wirbelthieren.
Pfliiger’s Arch., vol. 47, p. 216.
LesPiInassE, V. D. 1917 Sterility studies. Jour. Amer. Med. Ass., vol. 68,
No. 5, pp. 346-348.
Lewis, L. L. 1911 The vitality of reproductive cells. Okla. Station Bulletin,
96, p. 30.
Lopr, Auots. 1891 Untersuchungen iiber die Zahlen- und Regenerations-
verhiltnisse der Spermatozoiden bei Hund and Mensch. Pfliiger’s
Archiv, vol. 50, pp. 278-292.
Lort, Gustav. 1872 Anatomie und Physiologie des Cervix Uteri. Pp. 1-147,
Ferdinand Enke, Erlangen.
Payne, Loyau F. 1914 Preliminary report of vitality and activity of sperm
cells and artificial insemination of the chicken. Oklahoma Circular
30, pp. 3-8.
Pierson. 1893 Duration of human spermatozoa. Anat. Anz., p. 299.
Reynoups, Epwarp. 1916 Fertility and sterility. Jour. Am. Med. Ass.,
vol. 47.
Rota, A. 1893 Uber das Verhalten beweglicher Mikroorganismen in stré-
mender Fliissigkeit. Deutsche Medicinische Wochenschrift, vol. 19,
p. 352.
StiaterR, R. 1914 Warmelihmung und Wirmestarre der menchlichen Sperma-
tozoen. Pfliiger’s Arch., vol. 155, pp. 201-230.
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AUTHOR’S ABSTRACI OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MARCH 30
EXPERIMENTS ON THE DEVELOPMENT OF THE
SHOULDER GIRDLE AND THE ANTERIOR LIMB
OF AMBLYSTOMA PUNCTATUM
S. R. DETWILER
From the Osborn Zoélogical Laboratory, Yale University
THIRTY-THREE FIGURES
1. INTRODUCTION
Experiments on the transplantation of limbs have been ecar-
ried on in connection with various problems: a) on the develop-
ment of nerves (Banchi, ’05; Braus, ’05, and Harrison, ’07);
b) on the question of orientation and laterality (Harrison, ’17);
c) on the question of the development of the shoulder girdle
(Braus, ’09), the rudiment of which is intimately bound up with
that of the limb. It is with these last experiments that we are
particularly concerned, since they have a more specific bearing
on the results of the investigations set forth in this paper.
Although the intimacy of these two systems—shoulder girdle
and limb—during development and differentiation led Wieder-
sheim (’92) to conclude. that girdle formation is possible only
under the formative influence of the free extremity, the experi-
ments of Braus showed, in part, the lack of interdependence of
these systems. Braus found that the removal of the fore-limb
buds of Bombinator included the tissue from which the central
or glenoid portion of the girdle develops, and that only the
distal parts—suprascapula and epicoracoid—were formed fol-
lowing such an operation. The differentiation of these isolated
girdle elements from unremoved blastema in the absence of the
developing appendage demonstrated their independence of the
free extremity. Confirmation of this observation was made on
Amblystoma by Harrison (718), who found, however, that as
development proceeded these two separate distal elements gradu-
499
500 S. R. DETWILER
ally approximated each other until, in a larva which was kept
alive eighty-five days after operation, they had become united
into a single cartilage.
The formation of the suprascapula and coracoid in the absence
of the glenoid portion of the girdle demonstrates that their rudi-
ments are already determined at the time of the operation, and
that, while they eventually grow together, their unremoved rudi-
ments are nevertheless not capable of restoring the missing parts,
viz., the scapula, all or only a portion of the procoracoid, and the
shoulder joint, the rudiments of which are removed in a typical
limb-bud extirpation.
Braus (’09) further found that when a limb bud is transplanted
to a heterotopic position, a complete shoulder girdle of one-third
to two-thirds the size of the normal develops at the place of
implantation. From this he concluded (page 271) that the
shoulder-girdle rudiment constitutes an Suiniotoimernic) restitu-
tion system.
According to this conclusion, totipotency is restricted to those
girdle-forming cells which become implanted along with the limb
bud, for, as has already been pointed out, the unremoved blas-
tema can develop only into those parts the rudiments of which
are already determined at the time of operation. The forma-
tion of a reduced girdle, with all its components, from cells
which, in the normal environment, give rise to only the more
central parts, would show that in their normal surroundings their
prospective potency is greater than their prospective significance.
The results of the experiments set forth in this paper seem
to necessitate for Amblystoma, however, an interpretation dif-
ferent from that which Braus placed on the results of his
experiments.
This investigation was taken up at the suggestion of Prof.
R. G. Harrison. It gives me pleasure to express here my thanks
to Dr. Harrison for the guidance he has given me during its
completion.
SHOULDER GIRDLE AND ANTERIOR LIMB 501
2. NORMAL ANATOMY
In order that the experiments may be more fully understood,
a description of the normal girdle will first be given. Chondri-
fication of the girdle is practically complete in a larva about
twenty days after the closure of the medullary folds. The girdle
then consists of a cartilaginous structure lying within the body
wall and extending from the lateral aspect of the third myo-
tome almost to the mid-ventral line (fig. 6). It is made up
of the following components: a) the suprascapula, which con-
sists of a rod-shaped element lying external to the pronephros
and constituting the greater portion of the dorsal zone (fig. 23,
s.sc); b) the scapula, which lies just dorsal and anterior to the
glenoid cavity and which, in the cartilaginous state, is continu-
ous with the suprascapula (fig. 28, sc); ¢) the procoracoid lying
immediately anterior and slightly ventral to the glenoid cavity
(fig. 23, p.cor), and d) the coracoid, a relatively broad expanse
of cartilage, constituting nearly the entire ventral zone of the
girdle and reaching close to the mid-ventral line (fig. 7, cor. and
fig. 23, cor.). The scapula, procoracoid, and coracoid are con-
tinuous proximally and enter into the formation of the glenoid
cavity which receives the head of the humerus (fig. 23, ge).
Chondrification
Chondrification of the girdle proceeds gradually from the cen-
tral portion towards the periphery. ‘There are three centers, one
for the scapula, one for the coracoid, and one for the procoracoid.
The center for the scapula is first to appear. This is followed by
the center for the coracoid and finally by the procoracoid center.
This was found by Wiedersheim (’89) to be the case with Triton,
Siredon, and Salamandra. The same observation was also made
by Braus (09) on Bombinator.
The union of these three centers completes the chondrification
of the central portion of the girdle. The suprascapula has no
separate center and chondrification of this element proceeds
gradually from the region of the scapula in a dorsal direction.
502 S. R. DETWILER
While the central part of the girdle is chondrified before the first
two digits of the fore limb are fully formed and before the elbow
joint becomes visible, the dorsal portion of the suprascapula is
not entirely chondrified until the fourth digit makes its ap-
pearance. The chondrification of the coracoid likewise proceeds
gradually from its center towards the mid-ventral. line.
These observations agree with those of Braus (’09) on Bombi-
nator. In this form there are no chondrification centers for the
suprascapula and the epicoracoid. The epicoracoid of Bom-
binator is homologous with the ventral portion of the coracoid in
Amblystoma, which, as has been pointed out, chondrifies gradu-
ally from the proximal part towards the periphery.
The cartilage center for the humerus appears somewhat earlier
than do those for the girdle. Considering the Amphibia as a
whole, it can be said that in most cases this is true (Wiedersheim,
’89, ’90, 92; Lignitz, ’97, and Braus, ’09). From Strasser’s (’79)
description of Triton, one would assume, however, that in this
form initial chondrification of the humerus and the girdle takes
place simultaneously.
The centers for the ulna and radius appear slightly later than
those for the girdle, but they are completely chondrified before
chondrification of the suprascapula and the coracoid have been
completed.
The greater part of the girdle remains cartilaginous throughout
life, but the entire scapula and those portions of the procoracoid
and coracoid which enter into the formation of the glenoid cavity
become ossified. The cartilaginous suprascapula which, in the
larva, is a long slender rod-shaped structure, elongates in an
antero-posterior direction so as to become a broad flat plate.
The procoracoid grows out in an antero-ventral direction and
becomes a structure very similar in shape to the procoracoid of
Necturus. The coracoid, which comprises the greater part of
the ventral zone of the girdle, is a large flat rounded plate of
cartilage lying ventral and posterior to the procoracoid. The
two coracoids overlap in the mid-ventral line. The shape of the
ventral portion of the adult girdle is very similar to that figured
by Firbringer (73) for Salamandra maculata.
SHOULDER GIRDLE AND ANTERIOR LIMB 503
No description of the shoulder muscles of Amblystoma could
be found in the literature. The musculature, however, so far as
has been studied, closely resembles that of Salamandra macu-
lata, a European tailed Amphibian described by Firbringer (op.
cit.). In referring to the musculature, Firbringer’s nomencla-
ture will be employed.
3. EXPERIMENTAL
The experiments were carried out upon embryos in two dif-
ferent stages: a) the so-called tail-bud stage, and b) the stage of
open medullary folds.
Fig. 1 Camera-lucida drawing of an embryo of Amblystoma in the tail-bud
stage. The larger of the two circles represents the typical limb dise. X 15.
pn, = pronephros.
1. EXPERIMENTS ON EMBRYOS IN THE TAIL-BUD STAGE (STAGE 29)
A. Extirpation expervments
1. Removal of the suprascapula rudiment. As has already
been pointed out (Harrison, 715), the fore-limb rudiment of an
embryo in the tail-bud stage consists of a somatopleural thick-
ening just ventral to the pronephros, centering in the region of
the fourth myotome and extending over into that of the third and
fifth. The formation of a suprascapula following the extirpation
of the limb rudiment in this stage shows that its rudiment is not
included with the limb mesoderm. Although there is no visible
suprascapula rudiment, nevertheless, extirpation of the region
a-e X 1-3 (text fig. 1), including the outer or cutis layer of the
504 S. R. DETWILER
TABLE 1
Showing the results following the removal of the area a-e X 1-3 (text fig. 1) including
the outer cell layer of the somites and the pronephros
2m CONDITION OF THE GIRDLE AND THE EXTREMITY
INDIVIDUAL} AFTER
al Supraseapula Scapula Procoracoid Coracoid Humerus
FSi 26 absent present present present present
BaslO a) 24 absent* present present present present
Rea es 26 absent present present present present
15; (O55 seree 26 absent present present present present
* Only dorsal half wanting. ‘
ventral halves of the somites and the pronephros, results in the
formation of a girdle without a suprascapula (fig. 24 and table 1).
The removal of this area has practically no effect on the de-
velopment of the extremity itself and of the remainder of the
girdle. Abnormalities in the limb sometimes occur if its rudi-
ment is disturbed to any great extent during the operation.
2. Removal of the suprascapula rudiment and of the myotomes.
This type of experiment consisted in the entire removal of the
third, fourth, and fifth somites and the pronephros. In these
experiments the wound was usually covered with ectoderm taken
from a second embryo, since complete exposure of the notochord
and the medullary tube frequently results in a disintegration
of the embryo. Removal of the entire somites produces the same
effect on the girdle as that described for the first type of experi-
ment, viz., the formation of a girdle without a suprascapula
(figs. 8 and 25 and table 2). In the absence of the somites, the
limb and the girdle undergo a dorsal shifting (fig. 8). This is
apparently due to a release of pressure from that direction.
Complete removal of the third, fourth, and fifth somites,
while leading to no defects in the limb musculature, brings
about marked deficiencies in the ventro-lateral musculature, ob-
servations corroborating those previously made by Miss Byrnes
(98) and Lewis (10). Miss Byrnes (op. cit.) produced the first
experimental evidence to show that the musculature of the an-
terior limb of Amblystoma develops in situ and that it is in no
way derived from the myotomes or their ventral processes.
SHOULDER GIRDLE AND ANTERIOR LIMB 505
TABLE 2
Showing the results following complete removal of the third, fourth, and fifth somites
and the pronephros
ee CONDITION OF THE GIRDLE AND THE EXTREMITY
INDIVIDUAL eres
iecey Supraseapula Scapula Procoracoid Coracoid Humerus
1B. 1183.,..5el) 9 283 absent present present present present
iBall anion || Pabsent present present present undifferen-
tiated
[Eb eee 26 | absent present present present absent
1oy Ce eee 27 | dorsal half | present present present present
absent
The absence of the limb in case Ex 1 is no doubt due to the destruction of its
rudiment in the removal of the pronephros.
Lewis (op. cit.), however, in addition to demonstrating this same
fact, showed that definite defects in the ventro-lateral muscu-
lature follow the removal of the myotomes of the limb region.
An examination of figure 8 will show that, in this case, there is
complete absence of the ventro-lateral musculature on the oper-
ated side. This is, however, not always the case, for in others in
which these same myotomes were completely excised, the ventro-
lateral musculature is partly filled in by a compensatory elonga-
tion of the derivatives of intact myotomes. This same observa-
tion was also made by Lewis.
In the absence of the suprascapula, the m. dorsalis seapulae,
which normally runs from the proximal end of the humerus over
the external surface of the suprascapula (fig. 7, m.dsc), now
attaches to the scapula (fig. 8).
It is essential, in order to remove successfully the supra-
scapula rudiment to include the pronephros. Its removal fa-
cilitates cleaning of the wound and thus affects indirectly the
results of the experiment even though it exerts no direct influ-
ence. ‘This applies as well to removal of the limb bud as a whole
(Harrison, ’15).
Since removal of the outer portions of the third, fourth, and
fifth somites suppresses development of the suprascapula and
since there is no visible rudiment for this element at the time
of the operation, the experiments show that the suprascapula is
506 S. R. DETWILER
derived from tissue other than that which gives rise to the limb
and the remainder of the girdle. Sections of older embryos
show that dorsal to the pronephros scattered mesenchyme cells
gradually appear and become continuous, external to the pro-
nephros, with the limb-forming cells. It is evident that this
mesenchyme, which later forms the suprascapula, is segregated
from the outer or cutis layer of the somites in this region and
that the suprascapula is formed in situ. Such a conclusion is
strengthened by the fact that after complete removal of the
limb rudiment and the pronephros, the suprascapula develops in
its normal place, provided the third, fourth, and fifth somites
are undisturbed. ;
Not only do these experiments show that the suprascapula is
already determined at the time of the operation and that it is
formed in situ, but they demonstrate as well the impotency of
the unremoved girdle tissue to replace the missing part.
3. Removal of the dorsal zone rudiment of the girdle and the
limb mesoderm. ‘This series of experiments consisted of the re-
moval of the area a-e X 1-5 (text fig. 1). This included the
outer portion of the ventral halves of the somites, the pronephros,
and the limb mesoderm. The wounds were cleaned and covered.
The removal of this area suppresses development of the supra-
scapula, scapula, and the free extremity, and only the ventral
half of the girdle develops, no glenoid cavity being formed in
any of these cases (figs. 9 and 26 and table 3). The formation
in situ of the procoracoid and coracoid is evidence that they, too,
are already determined at the time of the operation and are not
dependent for differentiation on the remainder of the girdle and
the limb, the rudiments of which were removed in this type of
experiment. Although the myotomes proper were left intact,
several larvae showed slight defects in the ventro-lateral mus-
culature. This is no doubt due to a partial injury of the ventral
portions which furnish the muscle buds.
The rudiments of practically all the shoulder musculature are
included in these extirpations. In one case a few partially de-
veloped muscle fibers were present just external to the unre-
moved girdle elements. They probably represent the m. supra-
SHOULDER GIRDLE AND ANTERIOR LIMB 507
TABLE 3
Showing the results following the removal of the area a-e X 1-8 (text fig. 1) including
the outer portion of the somites, the pronephros, and the limb mesoderm
CONDITION OF THE GIRDLE AND THE LIMB
INDIVIDUAL AGE 7 =
Suprascapula Scapula Procoracoid Coracoid Humerus
& |
days
RRO neers 26 | absent absent present present absent
1S, Pee 26 | absent absent present present absent
late eae 26 | dorsal por- absent present present absent
tion |
present
BONS. 28 | absent absent present present absent
Bis $0): 28 | absent absent | present present absent
IRAE Perak 26 | small nod- absent | present present absent
ule of
cartilage
Incomplete absence of the suprascapula in cases R 1 and R 2 is apparently
due to imperfect removal of the rudiment.
coracoideus, a muscle which normally runs from the proximal
end of the humerus to the external surface of the coracoid (fig.
7, m.spc).
4. Limb-bud extirpations. The effects of the removal of a
typical limb dise on the girdle (text fig. 1) are in accord with
those described by Harrison (18). In individual H 2 sectioned
twenty-two days after the operation, only the suprascapula, a
very small procoracoid, and the coracoid were present (fig. 10
and table 4). In another, H 5, only the dorsal part of the
suprascapula was present in addition to a fragmentary procor-
acoid and the coracoid (fig. 27 and table 4). In these cases the
pronephros was removed with the limb bud, and the incomplete-
ness of the suprascapula in the second case is no doubt due to a
partial destruction of its rudiment in the removal of the pro-
nephros.
Since no limbs developed in experiment 3 after the removal of
the area a-e X 1-5, it is obvious that the ventral portion of a
typical limb dise (Harrison, 715 and ’18) which is shown in
text figure 1 contains only girdle-forming cells.
The formation of only a portion of the ventral zone of the
508 Ss. R. DETWILER
TABLE 4
Showing the effects on the girdle of removal of the limb dise
CONDITION OF THE GIRDLE
INDIVIDUAL AGE |
Suprascapula Seapula Procoracoid Coracoid Shoulder joint
days
1 WPA 5 20 present absent |present present absent *
Ho 20 present* absent |fragmentary| present absent
* Dorsal half only.
girdle following a typical limb-bud extirpation not only indicates
that part of its rudiment is removed with the limb bud, but that
the part which is unremoved is already determined at the time
of the operation. Further, when a limb bud is transplanted to a
heterotopic position, the development of a girdle with a ventral
zone of reduced size (fig. 28) also serves to indicate that only a
portion of the rudiment is transferred with the limb cells.
If localization of the cells which are to form the ventral zone
of the girdle is complete at the time of the operation and they
can be successfully extirpated, then only the dorsal zone should
develop following their excision. x<-9 (bb. ¥y) = 2 (Bb. YY):
558 P. W. WHITING
c. The number of loci involved. The facts thus far collected,
then, are consistent with the assumption of two loci, one for the
banding factors and one for the ticking factors. Since, however,
all variations from very light to very dark ticking occur and since
dark-ticked cats heterozygous for black may produce kittens of
somewhat lighter grade than themselves, it is probable that fac-
tors at other loci recombine to modify the ticking. Tests are now
being made which, it is believed, will determine definitely whether
light ticking and dark ticking are both allelomorphic with the
same factor for black; that is, whether they form a triple allelo-
morphie series.
The three types of banding, lined, striped, and blotched, are
each entirely distinct. No intergrades have been observed.
The natural assumption is to suppose that they form a triple
allelomorphiec series, B’, B, and b, as I have tentatively assumed.
But if two loci are involved the conditions might be expressed
as follows: A lined cat might be LLBB, LLBb, LLbb, LIBB,
LIBb, or Libb. A striped cat might be JBB or IlBb. A blotched
cat would then be the double recessive, llbb. This scheme ap-
parently fits the genetic data thus far collected. Striped and
blotched would act as a pair of simple allelomorphs, B and b.
The crosses involving the lined cats would be expressed by
supposing that they are both of formula L/bb. Bred together
they produced lined, LLbb or Libb, and blotched, llbb. Crossed
with blotched, either black or ticked, llbb, they give lined, Llbb,
and blotched, llbb. Crossed with homoyzgous striped, lIBB,
they give lined, LIB}, and striped I1/Bb.
The same crosses for testing the agouti factors will also test
the allelomorphism of the banding factors. Other combinations
that are being made with lined should give yellow and maltese
lined.
d. Physiology of color-production. Reference should now be
made to Wright’s (17) papers on color inheritance in mammals.
Wright classifies color factors according to their effects on either
one of two enzymes. Enzyme 1 is the basic enzyme for color
production which, acting alone on chromogem, produces yellow.
Enzyme 2 is supplementary to enzyme 1. It has no effect alone
INHERITANCE OF COAT-COLOR IN CATS 559
either on chromogen or on yellow pigment. Combined with
enzyme | it oxidizes chromogen to sepia.
The agouti factors are considered as determining an inhibi-
tor of enzyme 2. ‘Factor A determines the production of an
inhibitor with the same subtraction effect on enzyme 2 every-
where.” This inhibitor acts in waves along the individual hairs.
The regions of greatest concentration determine the yellow
bands, while those of less concentration are black.
In yellow eats it is seén that banding occurs over the surface
of the body, straw-color alternating with orange. Banding,
therefore, affects enzyme 1. In black cats the bands are almost
indiscernible. There is, then, enough of enzymes | and 2 gen-
erally distributed to produce a uniform black. In the presence
of the agouti factor, however, yellow bands appear in the indi-
vidual hairs. These bands are much wider in the areas corre-
sponding to the straw-colored bands of yellow cats. In fact,
the black may be here entirely obliterated. The hairs in the
areas corresponding to the orange bands of yellow cats are much
darker and may be without apparent ticking. It therefore ap-
pears that the banding factors affect enzyme 2, for if enzyme 2
were uniformly distributed as in rodents, the agouti factor should
cause a uniform ticking over the body surface, not an alternation
of dark and light bands.
The banding factors may be thought of, then, as determining
waves of general metabolic activity affecting both enzyme | and
enzyme 2. In the black cat the regions corresponding to the
orange bands in the yellow cat would be a dense black, a sort of
black dominant to agouti, comparable to Punnett’s (12 and 715)
dominant black in rabbits, while the regions corresponding to the
straw-colored bands would be comparable to ordinary black in
being recessive to agouti.
For helpful criticism and discussion of these matters I am
much indebted to Dr. Wright, whose papers I have already
mentioned.
560 P. W. WHITING
III. THE ORIGIN OF COLOR VARIETIES OF THE CAT
It is generally assumed that the domestic cat is polyphyletic
in origin. Darwin considered this to be the case. Keller (02)
discusses the matter and agrees with Darwin on this point.
Elhot (83) believes that the cat is descended from a number of
wild species and supposes that it has crossed at various times
with small wild cats in different countries. He attempts to
trace the well-known color variations as well as variations in
form to such hybridizing.
Rope (81) and Pocock (07) both recognize the characters
blotched and striped and believe that all cats, whatever their
color, fall in one or the other of these two classes. Pocock
States:
It is needless to say more in support of the contention that if a de-
cided difference in the ‘pattern’ of Domestic Cats exists, it must be
regarded as furnishing a surer basis for their classification than the
length of hair, the tint of the coat, or the stunting of the tail. It may
also be claimed with assurance that the pattern supplies a more im-
portant clue to the ancestry of the Domestic Cat than the features
just mentioned. . . . Frequently at all events the so-called
‘blotched’ pattern can be detected in certain lights even in ‘Whites’
and ‘Blacks.
Pocock also recognized the lined variation, called by. him
. . \
Abyssinian.
;
Cats of the so-called ‘Abyssinian’ breed may be descended, for any-
thing I know to the contrary, from specimens of F. ocreata directly
exported from Abyssinia, They are certainly not unlike some self-
coloured examples of that species. On the other hand, it would, I
imagine, be difficult to separate them from fulvescent ‘Ticked’ Cats,
which appear to me to be nothing but examples of the torquata-type
in which the pattern is broken up and evanescent.
The torquata type is what I have called striped. Pocock dis-
cusses the synonymy of wildeats and of the domestic cat. The
whole matter appears to be much confused.*
* The wildeat of Europe is usually called Felis catus L., but inasmuch as
Linnaeus’ description agrees with the blotched pattern while the European wild-
cat is striped, it is considered by Pocock and others that Linnaeus was referring
to the domestic cat. Felis sylvestris Schreber is therefore chosen as the name
for the European wildeat. Of the striped form Pocock says: ‘To feral or do-
INHERITANCE OF COAT-COLOR IN CATS 561
Dr. A. Nehring (’87) believed that the cat has a dual origin,
being descended from a domestic Chinese cat and from the
Egyptian cat, Felis maniculata. The origin of the striped pat-
tern is easily traced to the European wildcat or to the African
wildeat. Of the blotched type Richard Lydekker (Encyclo-
paedia Brittanica) says:
It may be suggested that the blotched tabby type represents Dr.
Nehring’s presumed Chinese element in the cat’s parentage, and that
the missing wild stock may be one of the numerous phases of the
leopard-cat (F. bengalensis), in some of which an incipient spiral
arrangement of the markings may be noticed on the shoulder.
The attempt is made by many authors to trace all variations in
morphology and color to some wild ancestor. To do so appears
to me unnecessary, as all such variations might occur under
domestication. The strictly domestic color variations in the
cat may be considered maltese, white, white-spotting, yellow,
and Siamese dilution. Such variations, if they occur in nature,
appear to be blotted out, as they are certainly not characteristic
of any wild species. They occur in numerous domestic ani-
mals and may be said to be variations by which domestic species
‘mimic’ each other.‘
mesticated examples of this cat have been given many names, of which torquata
is the best known and angorensis or striata possibly the oldest. . . . . It
closely resembles in pattern two existing species, namely, the so-called Egyptian
cat (F. ocreata) and the European wildcat (F. sylvestris).”
Pocock thinks that the blotched or catus type is derived from some extinct,
probably Pleistocene cat of western Europe.
Pocock uses the term torquata for striped and catus for blotched, which is
just the reverse of many other authors.
There is, further, a so-called Felis torquata of India that is considered by
some to be related to the spotted desert cat (F. ornata).
Inasmuch as I have wished to name genetic factors rather than species of
cats, I have discarded the Latin names and have adopted the English words
blotched and striped, in regard to which there can be no confusion.
4 By the use of the term ‘mimic’ I wish merely to denote what inmy estima-
tion underlies many at least of the phenomena which biologists have attempted
to explain by the mimicry hypothesis. There are only a limited number of ways
in which an organism may vary. Thus a mammalian coat may vary in dilution
and distribution of the pigments black, brown, and yellow. No other pigments
can be developed.
Numerous cases of resemblance, moreover, are in all probability due to
homologous factorial differences, even in widely separated species. Metz (’16)
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 2
562 P. W. WHITING
On the other hand, variations in the ticking factors and in the
banding or pattern factors occur both in wild and in domestic
mammals. Such variations in the domestic cat produce color
patterns closely similar to numerous wild species. Variations
from red to silver occur in wild cats. The tiger has a high de-
gree of red with a moderate amount of ticking. Thus the pat-
tern is very well marked. In the lion and the puma as well as
in the jungle cat and others, the red is reduced to yellow while
the ticking is very intense. Hence the pattern appears only in
young animals and is obliterated by the increase of ticking inci-
dent with maturity. Other cats like the ounce or snow-leopard
and Pallas’ cat represent an extreme reduction of yellow pigment
comparable with silvering in domestic tabbies. Loss of agouti
producing black varieties of leopards and others are well known.
Small species of African and Asiatic cats vary so in color that
much confusion has resulted in taxonomy. All of this diversity
may apparently be reduced to variations in ticking, in banding,
and in the red-silver series. Spots, I believe, are produced by
crossing of longitudinal and transverse waves of pigment-form-
ing metabolic activity. In these respects the domestic cat tends
to ‘mimic’ its wild relatives, but whether the variations have
originated by crossing or by mutation is an open question.
IV. THE SUMMARY
The inheritance of color variations in the domestic cat has
been investigated at the Zoological Laboratory of the University
of Pennsylvania.
Maltese dilution segregates distinctly from intense color and
is probably recessive.
Solid white is a simple and complete dominant over color.
White-spotting is very irregular in inheritance. There is partial
correlation between dominant white, blue eyes, and deafness.
has shown that mutations have occurred in Drosophila virilis (species B) produc-
ing characters similar to mutant characters in D. ampelophila. Such varia-
tions are inherited according to a similar mechanism and show comparable link-
age relationships. I am of the opinion that resemblances in the colors and
patterns of different mammals are often due to such genetic homology,
INHERITANCE OF COAT-COLOR IN CATS 563
It is suggested that irregularities in inheritance of blue eyes and
deafness may be explained by correlation with white-spotting.
Yellow is determined by a sex-linked factorial difference from
other colors. The heterozygous female is usually yellow-spotted
or tortoiseshell, but ranges to solid black or tabby and to solid
yellow. It is suggested that yellow-spotting in the male de-
pends upon extreme selection or segregation of other factors.
Kittens resembling adult Siamese cats have been produced
from common cats.
Banding cleanly segregates in three different widths. It is
probable that the factorial differences involved act as triple
allelomorphs.
Much-ticking, little-ticking, and black probably constitute a
triple allelomorphic series. Intergrades occur between much-
ticking and little-ticking.
Ticking follows banding in its distribution.
Black-spotting in wild Felidae and in domestic tabbies is
explained by crossing of transverse and longitudinal bands.
Color varieties are classified as those that ‘mimic’ other do-
mestic animals, and those that ‘mimic’ wild species.
564 P. W. WHITING
V. LITERATURE CITED
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Davenport, C. B. 1905 Details in regard to cats. Report on the work of the
Station for Experimental Evolution, Cold Spring Harbor.
Doncaster, L. 1905 On the inheritance of tortoiseshell and related colors in
eats. Proceedings of the Cambridge Philosophical Society, 18, pt. 1,
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1912 Sex-limited inheritance in cats. Science, N.S8., vol. 36, no. 918,
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1913 On sex-limited inheritance in cats, and its bearing on the sex-
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1914 Chromosomes, heredity, and sex. Quarterly Journal of Micro-
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Eviiot, Dante, Grrarp. 1883 A Monograph of the Felidae. Published by
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Issen, Heman L. 1916 Tricolor inheritance. III. Tortoiseshell cats. Ge-
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Ketter, Conrap. 1902 Die Abstammung der iltesten Haustiere. Zirich.
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Merz, C.W. 1916 Linked Mendelian characters ina new species of Drosophila.
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NeHRING, A. 1887 Ueber die Sohlenfiirbung am Hinterfusse von Felis catus,
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XXV, S. 260.
Punnett, R.C. 1912 and 1915 Inheritance of coat-colour in rabbits. Journal
of Genetics.
Roper, G. T. 1881 On the colour and disposition of markings in the domestic
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Wetr, Harrison. 1889 Our eats and all about them. Houghton, Mifflin &
Company, Boston and New York.
Wuitina, P. W. 1915 The tortoiseshell cat. The American Naturalist, vol.
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Wriacut, Sewatt. 1917 Color inheritance in mammals. The Journal of He-
redity, vol. 8.
PLATES
565
PLATE 1
EXPLANATION OF FIGURES
A. Skin of silver-striped tabby, probably little-ticked, but with reduction of
pigment due to silvering.
B. Skin of much-ticked striped tabby. The longitudinal bands are obvious
as well as the tendency of the transverse bands to become broken into spots.
C. Skin of little-ticked lined cat.
A and C are from sibs from cross of dark-ticked lined by black.
D. Skin of little-ticked striped cat.
B and D are from sibs.
566
INHERITANCE OF COAT-COLOR IN CATS PLATE 2
P. W. WHITING
567
PLATE 2
EXPLANATION OF FIGURES
E. Skin of much-ticked lined kitten at birth.
F. Skin of much-ticked blotched kitten at birth.
E and F are from sibs from cross of little-ticked lined by much-ticked blotched.
G. Skin of little-ticked lined kitten one week old.
H. Skin of little-ticked blotched kitten one week old.
G and H are from sibs from cross of little-ticked lined by much-ticked lined.
The animals, the skins of which are shown in figures A, C, E, F, G, and H,
had the same male parent, a little-ticked lined or Caffre cat owned by the
Zoological Society of Philadelphia.
568
INHERITANCE OF COAT-COLOR IN CATS PLATE 2
P. W. WHITING
569
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MARCH 30
THE INFLUENCE OF EXCESSIVE SEXUAL ACTIVITY
OF MALE RABBITS
ie ON THE NATURE OF THEIR OFFSPRING
FRANK A. HAYS
Towa State College
TWENTY-TWO CHARTS
CONTENTS
MrT OCCT LOM Here ey crocs sire ee ekens ois 6 Fe sie He ane NACE RM e ee ara 571
MUsiteriallPantderne GHOCUS ety a hiers see cece <5 e-3 ar voe-s. 4,4) ois epsusiotal’ SEAS CEPR os «Soar sa 573
J Agaboa EN ITAUESTEXG| 4 eS olenes Gules Cid ao ce ERED eee hicaIE ie rafts ob. 6.5 5 o oer 573
RRECONG SRN DURA ee ata ee ss cists ids aa Ae eas BER Oe esis ones 575
NV ENN Over IR CMe aS UT eeere's ss obo. chs siereozen Sete os Dake ae OM se eee 575
Methods of interpreting weights and measurements..................5- 579
Dy Aa FATT ESUI See ee RR Sete Ses i's cia Sal Marek ane dards at or fh 582
Growth in weight of young as related to frequency of copulation of sire. 582
Mipter COEGIeM Is Ol VATIADINIGY: «2, ce eveure cose «3 st etn oS 2 594
DEeLviceeLOMpicoeiicientssot varia bilityires..1.o-2 2. 5 cea eee e- . 597
Growth by measurements as related to frequency of copulation........ 599
Percentage mortality in offspring during the first five days of life and
between the fifth and the ninetieth day of life....................... 605
The Relation of number of services made to sex of offspring............ 606
Sexeaoonel aed: tO maOnualliitives sa\.cu sivas seueteckstnie sie ol eT Rees sce 608
AS THTATVOMATAY/ CLT MTTOLICIS Gat ie Ge ee ERE IED eR Dene tohs Meare Rn cin o.5 bo o 4 pote ieee 608
LOSE RSS Ot AOE Re Sieg ne age eR CS co ccc 610
Nel ea waynidK2xo lan SAVE A WES tateys Beate EO See eae oe aE ed. 0\.c 5, /0.0,9 0. eae 612
LOU. NDT. 7 Se Rolo oe, 6A ee Store co: ee ‘613
INTRODUCTION
Too frequent copulation of males is often given as an impor-
tant cause of weak and inferior offspring from the standpoint of
growth and thriftiness. This idea seems to be rather universal,
though evidence of such being the case is difficult to obtain.
571
.
52 FRANK A. HAYS
Wright (p. 306) states that in his poultry-breeding operations he
does not expect normal size or vigor in offspring from cocks used
on too many hens, and further (p. 131), that cocks that are used
on too many hens show the effect in that the eggs fertilized by
them show signs of hatching but do not hatch because the em-
bryos fail in many cases to reach full development. Pusch (15,
p. 182) expresses the almost universal belief in this matter,
though he does not consider the idea well grounded when he
writes: “Braucht man die’ Deckhengste, wie tberhaupt jedes
miénnliche Zuchttier, zu stark, so schidigt man nicht nur deren
Begattungs- und Befruchtungsvermégen, sondern auch die
Qualitat ihrer Nachzucht; deshalb wird die Stutenzahl fiir wert-
volle Vollbluthengste auch nur auf 30-40 Stiick bemessen und
die zu bedeckende Stute erst durch den Probierhengst auf ihre
Rassigkeit hin gepriift.”’ Day (18, p. 219) expresses the belief
that excessive use of the boar is likely to result in small, weak
litters of pigs.
Just why sperm cells that are produced by a male in heavy
sexual service should produce inferior offspring when they take
part in fertilization is not clear. Can it be possible that the
genetic makeup of the spermatozoa is changed by heavy service?
Is it not possible that any sperm cell possessing life, however
depleted and weak it may be, will carry into the egg a potentiality
of full vigor? Or, on the other hand, can we conceive of differ-
ent degrees of vital force in a sperm cell? Since all of the activi-
ties of the animal body are so beautifully coérdinated, it would
appear very rash to assume without conclusive evidence that
males under natural breeding conditions would derange any vital
function, such as reproduction, by continuing to copulate after
the reproductive system was producing an abnormal product.
As has been pointed out in our first paper (Lloyd-Jones and
Hays, 717), there appears to be a relation between the number of
services performed by the male and the fertilizing power of his
semen, but as far as we have been able to measure, we are led to
believe that only a slight change can Be brought about by this
treatment. Pusch (15, p. 182) states that in Oldenburg, stal-
lions are often allowed to make from four to six or even eight
SEXUAL ACTIVITY OF MALE RABBITS Sie
services daily. He also states that bulls have been used on 400
cows in a year, and that even poorly fed bulls will make from
four to eight copulations daily, and this without bad effect.
Strictly speaking, ‘vitality’ of individuals cannot be measured,
for the vitality of any individual really means the sum total of
life force within every living cell of the organism; vitality, as
used in speaking of animals, may, however, in part be measured
by the rate of growth in weight, the skeletal development, and
the ability of the individual to live to a good age. Such factors
as body weight and the others mentioned above are measurable.
The purpose of this investigation has been to study the effects of
heavy service of males on the nature of their offspring, as far as
we could measure the effect.
MATERIAL AND METHODS
1. Animals used
The character of the animals used in this investigation has
been discussed to some extent in the first paper of this series.
Stocks of the European domestic rabbit, Lepus cuniculus, secured
from six different breeders, were used and no inbreeding was
practiced at any time. The weight! and age of the females is an
important factor in that it affects both the number in a litter and
the individual weights of the offspring. Likewise the weight of
the male probably is of much importance in affecting the weight
of the young. The maturity of the male is a factor that should
not be lost sight of, because all three of the males used were fully
mature and were in their prime of life—about two years old.
The average weights of the males are as follows: No. 1, 2850
grams; No. 3, 2575 grams; and No. 4, 2200 grams.
Shy breeders sometimes occur in rabbits, but most of these
females proved to be regular breeders. No. 25, however, was
barren and was discarded; No. 12 produced young three times,
the last time August 5, 1916, after which time she appeared
never to come in heat again and continually refused to copulate
1 Prof. H. W. Vaughan has found that Large Type Poland-China Swine pro-
duce larger litters than the Small Type.
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 2
574 FRANK A. HAYS
and was discarded; both No. 22 and No. 29 died after having
given birth to but one litter upon which we secured data; and
No. 18 died after she had given three litters to the experiment.
Age of the dam is an important factor as affecting the number
in the litter and probably to some extent the weight of the in-
dividuals of the litter. For this reason the approximate ages of
the breeding animals is here given in order that the reader may
understand fully how much error may have been introduced
through immaturity in the breeding females. One female gave
birth to young when six months old; two, when seven; four,
when nine; one, at ten, and one, at eleven months old. The
remainder of the females was fully mature, that is, fifteen months
old or over, at the time they gave birth to the first litter used in
this experiment.
The fact should be noted that the three females that died
during the experiment were all immature at the time they first
reproduced and that only one of them (No. 18) had more than
one litter upon which we secured data. Two of the three lit-
ters from No. 18 are 5th-service litters by male No. 3 and the
other is a Ist-service litter by the same male. Female No. 22
gave a single litter from the 5th-service by male No. 1; and
female No. 29, one litter from the Ist-service by male No. 4;
female No. 12 has contributed but two litters to the records;
namely a Ist- and a 10th-service litter by male No. 1.
It may appear to the reader that considerable error, resulting
from the use of these immature females, was overlooked in mak-
ing up our records of growth, but this has not been the case;
therefore, a brief consideration of the system of matings used to
overcome this error is not out of place here.
The system of matings was arranged so that each female was
mated to at least two of the males and many to all three males,
and where possible each female produced litters from all differ-
ent services from each male, thus reducing parental variability
to the males alone. By making the three breeding groups of
females as nearly equal as possible in age and weight; by distrib-
uting the heavy service among the females in such a way as to
secure all types of litters from both mature and immature fe-
~
SEXUAL ACTIVITY OF MALE RABBITS Dio
males, and by making matings at such a time as to secure all
types of litters at the samme season of the year, as far as pos-
sible, we hoped to overcome many possible sources of error.
However, as the experiment proceeded it was found impossible
to apply these corrections absolutely and we are thus not fully
justified in comparing litters in growth in body weight and in
mean dimension and assuming that any consistent differences
are due to the number of services performed by the males. We
should not overlook the slightly better opportunities offered the
15th- and 20th-service litters, a considerable proportion of
which were born during the latter part of the experiment and
were produced when the females were all mature.
2. Records kept
The following records were kept: Date of breeding, pedigree,
date of the next probable heat period—fifteen days after breed-
ing; actual date of parturition; number in litter; number born
dead; sex of offspring; individual weight of offgpring on day of
birth and for each five days thereafter up to ninety days; head
length and breadth through extremes of ilium, taken at the
same time as the weights; date of weaning; color, and mortality
record.
3. Weighing and measuring
Breeding records were kept for each female so that it was pos-
sible to weigh each litter on the actual day of birth. At this
time each litter was given a number which was the same as the
number of the matings, and each individual was given an indi-
vidual number and marked in such a way as to be easily dis-
tinguished from litter mates by color description or by clipping
ears and tail. The individual weight records were kept each
five days until the litter reached the age of ninety days.
The desirability of continuing all records to the full maturity
of the progeny is very apparent. As the work was handled, forty
or fifty animals were often weighed and measured on a single day
and, with the other routine work of the experiment, entailed a
576 FRANK A. HAYS
very large amount of labor. Such extensive records were im-
possible for reasons that need not be discussed here. Weights
were secured on a sensitive torsion balance and variations of 0.5
gram were recorded. Great errors may be introduced by a
‘fill’ if the records are not made at the proper times; therefore
the records were secured at about the same hour each day before
feeding, which was done once daily. However, there are cer-
tain errors in weight records which cannot be avoided by the ex-
perimenter. The general degree of health of the animals has
much to do with fluctuations in weight as MacDowell (14)
found in growth studies of rabbits; nevertheless, as with other
animals, weight seems to be the best available index of growth.
Two methods for studying the growth of the progeny produced
were chosen, namely, growth in body weight and growth in body
measurements. The first will be discussed here.
Body weight, according to Minot (’08, p. 87), represents the
total mass of the living body, while body measurements are only
partial indices of growth. That individuals show wide fluctua-
tions in weight Has been pointed out by MacDowell (14, p. 191)
in his studies on the rabbit. Although growing rabbits show
marked variability in weight on different days, it was thought
possible by the use of large numbers to secure growth curves
that would fairly represent a race of rabbits kept under uniform
conditions. There is a possibility that these growth curves
would diverge more as the animals grow older, because Mac-
Dowell has shown that though most rabbits apparently make a
a. normal growth to maturity, others fall much below the normal
and do not reach the average weight in what is considered the
normal period. But complete records were out of the question
as indicated above. Even though this is the case, it is very 1m-
portant to ascertain if this reputed inferiority of progeny which
is supposed to result from the weaker sperm cells of the over-
worked male is going to be apparent when his progeny are in the
most active stage of growth, i.e., during the first ninety days of
postnatal life. If progeny from the advanced services of males
are more poorly equipped with the necessary something to en-
able them to make normal growth, would this not be apparent
SEXUAL ACTIVITY OF MALE RABBITS Had
when the young rabbits are thrown upon their own resources,
as was done during the sixty days following weaning time that
the records were kept?
Concerning the second method of studying growth, namely,
by body measurements, it is important to discover whether body
development follows apace with body weight and to check one
against the other. For this reason, all litters born up to August
9, 1916 (45 in number), were measured as well as weighed, at five-
day intervals up to the age of ninety days.
A measure of head length was considered valuable, as measure-
ments of the skull have been found to be less variable than meas-
urements of long bones. MacDowell (14, p. 38) found this to
be true in rabbits. Hatai (’08) observed the same thing in the
albino rat, and Quetelet (’71) likewise found the same in man.
The head length as here reported was measured by the use of
calipers and represents the distance obtained by placing the sta-
tionary arm at the crest of the occipital bone allowing the beam
of the calipers to extend sagitally downward parallel with the face.
The movable arm was then brought up until it rested snugly
against the end of the nose and down over themouth. The lower
arm of the instrument was then just beneath the inferior extremity
of the premaxiliae and the upper arm was just above the superior
region of the occipital bone. There is very little flesh or soft
tissue covering the bones in this region, about the only structures
obscuring the bones are the skin and the hair coat. It is ap-
parent, that a head measurement in this particular region
approximates rather closely the actual skull size.
Since there is the possibility that some other body measure-
ment would make an entirely different growth curve from that
of the above-described head measurement, it was considered de-
sirable to secure one other measurement that could be taken with
considerable accuracy on the live animals. Moreover, we wished
to obtain a ‘mean dimension’ from the average of two measure-
ments, therefore some easy body measurement was searched for.
There is no little difficulty in securing external méasurements of
the body with accuracy, as the writer has learned from much ex-
perience with cattle and swine. A measure of the breadth be-
578 FRANK A. HAYS
tween the extremes of the ilia was thought to be as easily determined
as any of the possible body measurements and would represent
a dimension of breadth in contrast to head length, which might
be considered a dimension of depth. The iliac expanse was
therefore used as the second measurement.
Both measurements were taken Just after weighing on the
five-day periods beginning at birth and continuing to the ag. of
ninety days. Steel calipers were used with vernier graduated to
hundredths of a centimeter. Three independent readings of
each dimension were taken by removing the calipers and shifting
the arm after each reading. Readings were put down just as
read, and care was taken to avoid any tendency on the part of
the observer to modify readings to make them check with others.
As a rule, it was possible to obtain readings that varied less than
0.1 centimeter from each other. An average of the three read-
ings was taken as the correct reading for each measurement.
Little difficulty was experienced in securing what was consid-
ered a correct reading on head length. This was not always
true for the other dimension. ‘Three factors probably. enter to
modify this reading: 1, the amount of ‘fill; 2, the degree of
fatness; 3, the position of the hind limbs. Food and water in the
alimentary tract seem to bulge the walls of the abdomen to such
an extent as to often obscure the points of the ium and make
their exact location difficult. The variability of the feeding
habits of the rabbit is thus a factor of no little importance in
connection with the measurement of iliac extremes. Some in-
dividuals carry much more fat over the ilium bones than others.
In fat individuals the points of the bones are often greatly ob-
scured, especially in the younger rabbits. This condition is much
more common among the smaller and better nourished individuals
and may prevail to some extent throughout the period of ob-
servation. There is considerable flexibility in the pelvic girdle
before the symphisis pelvis becomes bony and firm as the ani-
mals approach maturity. The ilum, ischium, and pubis are
also distinct ind more or less flexible in early life. This great
flexibility causes the position of the hind limbs to be an impor-
tant factor in modifying the position and the breadth of the ex-
SEXUAL ACTIVITY OF MALE RABBITS 579
tremes of the ilium as determined by the calipers. In so far as
possible an effort was made to have the animals sit with the
limbs in the natural position while being measured. ‘The hair
was also clipped from this region of the body in order that it
might not obscure the point of the bones.
4. Methods of interpreting weights and measurements
In order to make the data for different-sized litters more nearly
comparable, all weight and measurement records are reduced to
an ‘individual mean’ for each litter for each of the nineteen periods
of observation. The individual mean for each litter was cal-
culated by dividing the total weight or total measurements of
each litter by the number of individuals for each of the nineteen
periods. From these individual litter means the series of cumu-
lative growth graphs are constructed. :
In attempting to compare the growth graphs of rabbits in the
different service groups, a very perplexing problem arose as to
how to best compare results in litters that vary so much in num-
ber of individuals. The number of individuals born in a litter
is an intensely important factor in influencing the weight of the
young. Our observations have shown this as did also observa-
tions of Minot (91, p. 111) on guinea-pigs. His results, based
upon 351 observations, show that the average birth weight is
85.5 grams in litters of one, the weight gradually decreased with
the increase in number of individuals to as low as 52.2 grams
in litters of eight.
Another item that makes comparisons of litters in different
service groups difficult is the fact that litters in the Ist and 5th
service groups are likely to contain more individuals than those
from the 15th and 20th services. For this reason the individual
mean of these advanced service litters is greater and they grew
faster because of a more generous supply of milk from the mother.
In this connection we find that King (16, p. 51) discovered that
in rats ‘“‘body weight at birth indicates the probable capacity of
the individual for subsequent growth.’ This being the case,
small litters from the advanced service should grow more rapidly
than the larger litters from the Ist and 5th services.
580 FRANK A. HAYS
In order to make the litters in the different service groups
comparable with each other, whatever their number, it was
thought first that litters of different numbers of individuals
could be standardized to a mean litter number. Jackson (13,
p. 17) in comparing the standard deviation of individual rate
with the standard deviation of the entire race, reduced all indi-
viduals to a common basis by multiplying the body weight of
each rat by a factor obtained by dividing the mean of the total
population at a given age by the mean of the given litter. Since
the object we have in view is not the study of individuals,
this formula cannot be used. Further attempts were made to
obtain a factor for reducing large and small litters to a compar-
able basis, but so far with no success. Again, it was thought
possible that the coefficient of correjiation between number of
individuals in the litter and average weight per individual might
be made use of to reduce the litters to a comparable basis,
but without any satisfactory results. Again, a comparison of
different-sized litters in the several service groups by con-
structing graphs upon a base line representing the different
litter numbers and the vertical line representing the variable
weights at birth, a second chart to show the time required to
make eight times the birth weight, and a third chart to show the
time required to make twenty-four times the birth weight were
attempted. By this means the data could be much condensed,
but such a system proved to be impracticable and was discarded.
Finally, it was deemed best to compare litters of the same num-
ber of individuals. Accordingly, the growth rate in the different
service groups must be shown by a whole series of charts, the
graphs on each chart representing a certain litter number. In
each case the chart shows the number of litters which are lumped
in each graph. Thus each chart gives a direct comparison of
the growth rate of the five service groups, namely, Ist, 5th,
10th, 15th, and 20th, the comparison being always between
litters of the same number.
As a further measure of divergence in rate of growth between
the service groups, the coefficient of variability of weight for all
litters in each of the five service groups is valuable, presented
SEXUAL ACTIVITY OF MALE RABBITS 581
at birth, at weaning time or thirty days, and at ninety days.
The object here sought is to find out if there is a greater varia-
bility in any one of the service groups, which might be expected
if any of the service groups contain weak offspring. This study
will also reveal if heavy service tends to produce a wide range
of variability in birth weight or a wide range in the weights of
individuals at the time that they are thrown upon their own re-
sources at weaning time, and it will show further if the individuals
tend to deviate more from the mean as they grow older. Devia-
tions, if they are going to occur, might be expected to occur,
more strikingly at these three periods than at any other time
during the observations. This coefficient of variability was de-
termined by the following formula:
q
(Sum of deviations of litters from mean)? x (frequency of class)
For each Sa Oa
litter in Number of individuals. x 100 )
ates | Mean of respective litters. j
Number of litters in service group.
The above formula is used-for the birth weights, the thirty-
day weights, and the ninety-day weights.
The measurement data secured were combined into one general
expression, the ‘mean dimension.’ The advantage of using one
expression to stand for body measurements les in the fact that
we have a mathematical expression for the cross section of the
animal. Graphs expressing cumulatively the percentage increase
in head length and iliac extremes are found to cross between the
thirty-fifth and fortieth day of postnatal development; but pre-
vious to this date and later, up to the time of the conclusion of °
the observations at ninety days, the graphs bear a close relation
to each other, therefore it was deemed correct to combine the
two measurements to obtain the mean dimension.
The mean dimension was obtained by the following formula:
Mean head length + mean iliac extremes.
9
582 FRANK A. HAYS
Mean head length is the sum of the averages of the three read
ings for each individual in a litter divided by the number of
individuals in the litter. Mean iliac extremes represents the
sum of the average of the three measurements divided by the
number in litter. By using the above formula for mean dimen-
sion, the calculation was made for each litter for each of the
nineteen periods of observation. Graphs presented on the
measurement data are made up in exactly the same way as has
been described for making the graphs for weight, comparing
only litters of the same number of individuals. A grand average
graph is likewise made up regardless of litter size. The graphs
based upon identical litter size are considered reliable for pur-
poses of comparing the offspring in the different service groups,
but the grand average graph is subject to considerable error.
DATA AND RESULTS
1. Growth in weight of young as related to frequency of copulation
of sure
For studying the offspring with a view of determining if there
is any relation between the number of services made by the males
and the rate of growth, there appears to be no better measure
than body weight. Body weight measures the animals as a
whole and should thus reveal any inherent weakness that retards
their growth. -
Below are presented charts 1 to 12 taken from the composite
weight records of the young of all three males. Each chart rep-
resents a single litter number at birth, all litters of one size, in
each of the five service groups being grouped together and the
' same grouping being followed for all litter numbers as described
on page 580.' Each service group is represented by a different
line, as is disclosed by the legend on the charts. The number
of litters represented by each curve is given in each case. Chart
13 shows the weighed grand average for all five service groups.
It was obtained by adding together the individual mean of each
litter and dividing by the total number of litters at each obser-
vation period. We have already shown that such a chart com-
paring directly the growth ratio of the different service groups
SEXUAL ACTIVITY OF MALE RABBITS 583
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SEXUAL ACTIVITY OF MALE RABBITS
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WEIGHT IN GRAMS
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SEXUAL ACTIVITY OF MALE RABBITS 587
CHart NOIL
Ground GUreves IN Service Grovrs
Litre R Si ZE 9
WEIGHT IN GRAMS
ANGE IN DAYS
el ae SL e aa T lose gle T learn sanera
1225, Guart NO. X
Z :
/600 (sRowTH (Guan IN Service Groves
/475 Litter Sizeld.
1350, ! 3
é |
z
1225 (Oe _6210.
iG ee
Th
1100 20 == eee /
ey
: ee
4 ge
2 oA
950.2 D227 C4
rE Za ime
13 a 7
229-G - Za
7 a
s y =—
A a
600 ae LE
- % a
——- ae a
475 — S
Z pee
350 = pe
2275) ee Gn —
eee
2 as
/00 ESS AGE /n Days
s ——
=
)
588
WEIGHT IN GRAMS
WEIGHTIN GRAMS
FRANK A. HAYS
Cuart NOX
Growrn Guaves in Senvice:'G roves
Livres Oize Il
[a
CHarr NO _XIL
Grow tn Gurves in Service Groves
Litrer Size 12
lie
67 ae
_————
Ace in DAYS
AGES in DAYS
SEXUAL ACTIVITY OF MALE RABBITS 589
Rares T T T al T T saat en oe u T =e T T toa
nas CHarat NO XML |
an Grano Averace Grown Coeves =}
Act Service Groves
=
#70 | ——_—_—_ =
»
=
x
ig
o
-
3£
2)
iw
Ss
may not be made with absolute justice because, as will be shown
later, the litters in the advanced service groups tend to be
smaller. A rough comparison of all progeny may be made in
this way, however.
Charts 1 to 12, inclusive, present the results in a form that
may be easily grasped by the reader, but there are a few points
revealed by a study of these graphs that require some discussion.
With but few exceptions, the 20th-service graph lies above all
other graphs. This is a striking and surprising result and the
question at once arises as to the cause of the almost uniform
heavier birth weight and more rapid growth of the 15th- and
20th-service litters compared with litters of the same size from
less advanced services. The results are in direct contrast to
what, according to the traditions of breeders, would be expected.
On their face they actually show that the heavier the service of
the male, the more thrifty the offspring. It seems best to here
consider the possible factors that may play a part in causing the
superiority of these advanced service litters over litters from the
Ist, 5th, and 10th service.
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 2
590 FRANK A. HAYS
During the production of the majority of the Ist- and 5th-
service litters the breeding animals were housed in somewhat
cramped quarters. Conditions there were not conducive to the
most rapid growth of the young and were not as favorable for the
breeding females because of small space and rather poor ventila-
tion and poor light. Furthermore, the progeny were crowded
into rather limited exercising pens, and probably for this reason
they did not develop at so rapid a rate as would have been the
case under the more favorable quarters used later. The ma-
jority of the 10th-service litters, on the other hand, and about
half of the 15th-service litters were produced while the stock was
housed in more ample quarters where the space was large, the
ventilation good, and everything was conducive to health and
thriftiness. In fact, the quarters used at that time were prac-
tically as good as the present permanent and excellent quarters
where the 20th-service litters were produced. The superior en-
vironment of the advanced service litters is no doubt partly re-
sponsible for the greater growth of the advanced service litters
compared with the moderate service litters; but environment
cannot be entirely the cause of the superiority of the 20th-service
litters over the 15th, and the 15th-service litters over the 10th-
service litters. Let us therefore seek a further explanation.
Parentage may be an important factor affecting the weight.
As has been previously noted, the variability of the female breed-
ing stock is considerable, the range of weight was from 2500 to
3250 grams, averaging 3050 grams, but the females have been
so distributed among the three breeding males as to make three
groups of practically uniform weight and variability in size.
Nevertheless, lack of uniform weights in the progeny may still
be partly due to variability of the female breeding stock.
The size of the sire may also be a factor in controlling individual
mean weight. The three sires used were quite different in weight;
their weights are as follows: No. 1, 2850 grams; No. 3, 2575 grams,
and No. 4, 2225 grams in ordinary breeding condition. Male
No. 1 sired eleven of the seventeen litters included in the 20th-
service group. He, being the largest of the three males, would be
expected to sire the heaviest offspring at birth, and such offspring
SEXUAL ACTIVITY OF MALE RABBITS 591
TABLE 1
Average birth weight of litters sired by the three different males used, by service
groups
Ist 5TH 10TH 15TH 207TH
MALE
se a ues Weight Bs Weight oe Weight Blame Weight Sony Weight
1 6 46.7 6 45.2 7 45.2 7 ono) 1 59.7
3 12 50.9 8 45.0 2 45.0 2, 42.7| 00 00
4 8 42.4 6 58.8 4 58.8 4 49.6 6 DOeO
Weight at ninety days
1 5 {1209.5 3 /1236.6 @ 123821 7 {1262.0 6 |1299.3
3 11 /1170.0 6 127.8 7 (1095.3 2 {1270.7} 00
4 7 {1054.9} 6 |1096.1 5 (1424.3 2 {1199.7 3 {1009.0
could be expected to keep ahead of the other classes of offspring
at least for ninety days. This way of explaining the position
of the 20th-service graph above the others is called in questions
by chart 3 and also by table 1. The graph of the 20th-service
litter lies below the others. This graph represents the growth
of a smgle 20th-service litter (after the first weight) also by
Male No. 1 and out of the heaviest female in the breeding stock
(No. 15). Therefore, the fact that this litter lies below 5th-
and 10th-service litters on this chart cannot be explained as the
result of small ancestry.
Table 1 shows that the size of the male ancestor is not a
very important factor in relation to the size of the young at birth.
At the age of ninety days, however, the effect of the heavier sire
becomes more important, but nevertheless is probably not as
important as some other factors concerned as will be pointed
out later.
When we consider the 15th-service group, we find that seven
litters were sired by Male No. 1, two by No. 3, and four by
No. 4. Again we should expect a more uniformly heavy progeny
than if all males had contributed an equal number of litters to
the data. Chart 8 shows the superiority of the 10th-service
group over the 15th-service group up to the fifty-fifth day,
after which time the graph rises above all others.
/
592 FRANK A. HAYS
Thus far we have attempted to account for the heavier weights
and the greater rate of growth of the advanced service litters as
due entirely to factors other than the nature of the spermatozoa
and not to any inherited superiority. The effect of such factors
does not seem adequate to explain the apparent superiority of
the advanced service litters, therefore there is good evidence that
a real superiority exists among the advanced service litters as
compared with the light service litters. The female ancestors in
both service groups were practically equal in weight. One of the
15th-service litters represented in chart 9 was sired by Male
No. 1, the other by No. 3. Two of the four litters combined in
the 10th-service graph were sired by No. 1 and two by No. 3.
The smaller weights of the 15th-service litters during the early
part of the observations cannot for the above reasons be explained
by male ancestry of different weights.
One other hypothesis may be proposed to account for the prob-
able superiority of the advanced service progeny over those from
very moderate service. Pearl (’17, p. 296) treated both cocks and
hens with ethyl aleohol, methyl alcohol, and ether at different
times during the breeding season in order to study the effects
on their progeny. He found the offspring from treated parents
in every way superior to those from untreated parents. Pearl
assumes that alcohol and other poisons act as selective agents
upon the germ cells of treated animals. It is possible that se-
lective action might be brought about by heavy sexual service
of the male. We have previously shown that heavy sexual
service induces the liberation of sperm which often show no pro-
gressive motion and are short-lived. Some few of the sperm
from these advanced services do exhibit the physical properties
that indicate high vital force. The possibility exists then that
what few spermatozoa do take part in fertilization are superior
to the average in the light service groups because the bulk of the
spermatozoa in the advanced service groups are not equipped to
take part in fertilization, while this is probably not true in the
light service groups. Such a hypothesis as the above will thus
account for the superiority of the advanced service progeny.
SEXUAL ACTIVITY OF MALE RABBITS 593
TABLE 2
Number of litters included in graphs of charts 1 to 12, inclusive, and the male
, ancestry
SERVICE GROUP
MALE
MEMBER :
Ist 5th 10th 15th 20th
1 6 6 7 7 11
3 12 8 7 2
4 8 6 6 4 6
Concerning the graphs for the 10th-, 5th-, and 1st-service
litters, we note that as a rule the Ist-service litters are inferior
in weight to either the 5th- or 10th-service litters and that the
10th-service litters are for the most part superior to the 5th-
service litters. As previously noted, less favorable environment
and greater immaturity of some of the female animals are thought
to be the chief factors entering here. The male ancestry is
almost uniformly distributed among the three males. Below
we note from the table just how the ancestry is distributed.
Table 2 shows us that the three males are about equally dis-
tributed in the progeny groups from the 5th and 10th services.
In the Ist-service group, however, No. 3 has sired twice as many
litters as No. 1 and 50 per cent more than No. 4. Since Male
No. 3 is a smaller animal than No. 1, we have here a partial
explanation for the apparent inferiority of the 1st-service litters
over all others. In the 15th- and 20th-service groups the prog-
eny of Male No. 1 predominate, and Male No. 3 sired no litters
in the 20th-service group.
A word of explanation in regard to a few remarkable features
of some of the charts may be of value at this point. On chart 3
the depression in the 5th-service graph at sixty days is due to a
failure to obtain data on.the heavier of the two litters making
this graph. This particular litter was unintentionally over-
looked for four weighings. On chart 5, the drop in the 10th-
service graph at sixty-five days is due to the incomplete record on
one litter at the time the graphs were constructed and this litter
was made up of very heavy individuals.
594 FRANK A. HAYS
Chart 13 represents the grand average growth of all litters in
the five service groups as explained on page 582. Each graph
thus represents the individual mean for the combined litters in
each service group. ‘These composite service group graphs bear
out the general deductions that we have madefrom a study of the
graphs taken one by one comparing litters of a given number
with each other in the five service groups. There is one out-
standing objection to the use of such graphs as are shown on
chart 18. There is a perceptible negative correlation between
number of services of the sire and the number of offspring in
litters resulting (Lloyd-Jones and Hays, p.492). In other words,
heavy service does reduce the size of litters, especially in the two
most advanced service groups used here. Consequently the
greater supply of nutrients furnished by the mother in utero as
well as the greater supply of milk available after birth will
enable the advanced service litters to outstrip the other litters
during the periods of observation in this experiment. This con-
dition would hold if all litters were equally fit genetically; and
we have no evidence that any class of offspring is rendered [ess
fit by heavy service of their sire.
To recapitulate, certain errors have been introduced into the
growth studies in body weight, chief among which are environ-
mental factors, the age and weight of the dam and the weight of
the sire. These errors have been partially corrected, and the
conclusion seems justified that there is no evidence in this data
to show that the amount of sexual service that the male has been
required to perform in any way affects the rate of growth of his
offspring in body weight for the first ninety days of postnatal
life.
2. Litter coefficient of variability
The coefficients of variability in table 3 presented below were
obtained in the following manner: The coefficient of variability
for each litter in each of the five service groups was determined
at birth, at thirty days, and at ninety days by the formula:
Standard deviation of each litter.
Mean of the litter.
SEXUAL ACTIVITY OF MALE RABBITS
TABLE 3
595
2. Coefficient of variability of individuals within the litters at three different periods
SERVICE
Ist 5th 10th 15th 20th
AGE zS me = a le
} iS 5 i) S)
52 52 52 52 Cm
as Percent /|29 Percent |93 Percent |= 2 Percent |22| Percent
= a eee ee ee
days
Birth 2610.81=1.01) 2010.78=1.14) 1912.52=1.37) 11/11.48+1.64) 16,8.80+1.05
30 23)/10.72=1.07) 17) 8.27+=0.96) 19,10.05=1.10) 11) 9.56=1.29) 11/7.89+1.11
90 23/10.10+1.02) 14) 6.77+0.86| 17| 7.55+0.87| 810.70+1.80) 9/8.94+1.42
|
Average. 10.55 8.82 TORTS 10.55 8.56
The coefficients of variability for all Ist-service litters at birth
were then added together and this sum was divided by the
number of litters concerned to secure the coefficient as given in
table 3. Likewise the coefficients of variability for all 1st-serv-
ice litters at thirty days were added together and this sum
divided by the number of litters concerned to obtain the co-
efficient as given in table 3. This method was used on the weights
at ninety days to get the coefficient, and a similar procedure
used on the weights in the other four service groups to obtain
their respective coefficients. For the information of the reader
the number of litters concerned in each case is presented in the
table. MacDowell (14, p. 44) shows in studies on weight of
adult rabbits that there is less variability within the litters than
between individuals of different litters. For this reason and be-
cause we wish to compare progeny of different ancestry, the
method of expressing the coefficient of variation of the popula-
tions as the average of the individual litter coefficients of that
population is considered accurate.
Table 3 shows that the coefficient of variation in rabbits is
greater at birth than at any other time during our observations.
This fact holds good in all service groups. While the coefficient
on the average is small, it serves to indicate that prenatal nutri-
tion must be subject to wide variations, otherwise greater uni-
596 FRANK A. HAYS
formity in weight at birth should be expected. The thirty-day
period is the weaning time for all of the litters studied in this
experiment. We note from the table that the coefficient of
variation falls below what it was at birth in all service groups.
- Here again there is no evidence of an increased percentage of
‘weak’ offspring in advanced service groups, for if such were the
case we should expect the coefficient to increase when the ani-
mals were thrown into competition for nutrition during the first
thirty days of postnatal life, and even one inferior individual
' would alter the coefficient for the litter. At the ninety-day
period there is again a decrease in the coefficient of variation in
all service groups, except the 15th- and 20th-service groups.
The large size of the probable error here indicates that the
15th-and -20th-service groups cannot safely be assumed to be
exceptions.
Taking up a comparison of the coefficients for the different
service groups, there appears to be slightly less variability in
the offspring as the number of services increases, but this de-
crease is not universal. Since the probable error is rather large,
this difference is no way significant. As has been previously
stated, there is also a slight reduction in the number in the
litters in the same direction. Our data show us further that
there is less variability in the smaller than in the larger litters.
This fact affords us an explanation for the slight reduction in the
coefficient of variation as the number of services increases.
In table 3 a further fact seems apparent that occasional
genetically weak offspring do not occur in any one of the service
group more frequently than in any other service group. The
table also shows us that for the first ninety days of postnatal
growth there is a tendency for individuals of the same litter to
approach nearer to a mean weight than was the case either at
birth or at thirty days of age. Fetal nutrition is thus more vari-
able than either the nutrition furnished by the mother during the
first thirty days after birth or the ordinary food supply fur-
nished from thirty days to ninety days.
SEXUAL ACTIVITY OF MALE RABBITS 597
TABLE 4
Service-group coefficients of variability at three different periods
, i
SERVICE
Ist 5th 10th 15th 20th
AGE — -_— Lond _ s
iS) 3 iS) iS) re)
a] nl a] il ‘
Oo” on on o@ on
26 Per cent 26 Percent | s Per cent 26 Percent |28 Per cent
§ ~~ Sip g » S| » &S
S5 B35 l= a Beet
|4 a a Z Z
days
Birth | 26)17.62+1.65
30 23)19.70+1.96
bo
(=)
28.10+3.10) 20,23.03+2.46) 13/15.59+3.39| 16:21.94+2.62
42.65+4.80) 21/35.53+3.69) 13/52.13+6.91| 11/46.37+6.66
_
(e/2)
90 23/24.25+2.41| 17/23.89+2.76| 21/19.63+2.04| 11)24.97+3.53] 9/30.91+4.92
Weighted
average.. |24.40 31.56 26.11 34.73 31.65.
3. Service group coefficients of variability
_ In table 4 are presented the service-group coefficients of vari-
ability for all of the progeny studied in the experiment. These
coefficients are obtained in the following manner: The sum of
the mean individual weights of each litter in the Ist-service
group was divided by the number of litters, to get an average
at birth, at thirty days, and at ninety days. The standard
deviation of this average was then calculated and the coefficient
of variability (e) obtained by the formula:
Standard deviation of the average _
Average zt
C
The same method was used for all five service groups.
The service-group coefficient of variability differs from the
litter coefficient of variability given in table 3 in that the former
measures the range in weight between the individual litter means
of the different service groups, while the latter is a measure of
the range in weight between individuals of the same litter.
The service-group coefficient of variability is valuable in study-
ing the effects of heavy service of males upon the growth in body
598 FRANK A. HAYS
weight of their offspring because it will bring to light occasional
litters in which every individual is inferior. For example, table
3 shows that there is not an occasional inferior individual in the
advanced service progeny. This fact does not remove the pos-
sibility of some entire litters being inferior because it is possible
to conceive that at one time a male rabbit might sire an excep-
tionally good litter on the 15th or the 20th service because of
extra high reserve, but the majority of his progeny might be
inferior in growth as entire litters. By table 4 we shall attempt
to discover if litters as a whole are inclined to be more variable
in any particular service group.
Table 4 shows that at birth there is less variability in the 1st-
service progeny than in any other progeny. This implies that
the individual mean weight of the Ist-service litters more nearly
represents the mean of every litter in the service group than is
the case in any of the other four service groups. There appears
to be little tendency for variability to increase as the amount of
service increases as shown in the other four service groups at
birth.
Concerning the variability between litters at thirty days of
age, practically the same relationship exists between the progeny
of the different service groups as has been already noted in con-
sidering the progeny at birth. The table shows us one additional
fact at the thirty-day age; namely, that the greatest variability
in weight during the ninety days of the observation exists at
weaning time or thirty days. This fact is additional evidence
that the nutrition furnished by the mother while suckling the
young may vary in absolute amount or may be distributed in
limited quantities because of the large number of individuals that
she may suckle.
At the age of ninety days there is a striking uniformity in the
coefficients for all five service groups. Only in the case of the
20th-service group is there any noticeable digression, and this is
probably due to the small number of litters concerned.
Table 4 as a whole does not in any way indicate that inferior
litters exist more frequently in any one service group than in any
other, and the fact has already been pointed out in connection
SEXUAL ACTIVITY OF MALE RABBITS 599
with the study of the weight graphs than in average body weight
the advanced service litters are equal and in some cases superior
to that of the litters in the light service groups. The fact that
variability within litters is small compared with the variability in
service groups is well illustrated by a comparison of the coeffi-
cients in tables 3 and 4.
4. Growth by measurements as related to frequency of copulation
Charts 14 to 21 are presented to show the growth in the mean
dimension as obtained on forty-five litters. The method of mak-
ing measurement and the determination of the mean dimension
have been already explained, pp. 581-582. Each graph repre-
sents averages of the mean dimension for all litters of the same
size in the respective service groups. The mean dimension for a
litter is obtained by adding all head measurements to all meas-
urements of ilial extremes and dividing the sum by the total
number of readings included in the sum. The expression thus
obtained is the average individual mean dimension for the re-
spective litters and may be compared with the average individual
weights used in the previous charts.
These charts of body development show that there is a maxi-
mum increase in the mean dimension up to about the twentieth
day, after which there is a very noticeable flattening of the
graphs. From about the twentieth day on to the end of the
observations at ninety days the progressive increase in the mean
dimension is about constant. The increase in the mean dimen-
sion is thus in marked contrast to the increase in body weight
previously illustrated by charts 1 to 138. Body weight has been
shown to make a rather constant increase up to the end of ninety
days, and this is well illustrated by the fact that the weight
graphs show little if any tendency to flatten out.
Though the number of litters making up a mean dimension
graph is in most cases small, they serve to illustrate the same
principle as the weight graphs, namely, that the advanced serv-
ice progeny are fully equal to the Ist- or 5th-service progeny
at all times during the ninety days of the observation. On
600
435
39
w
FRANK A. HAYS
Cuart No XLY
MEASUREMENT Curves in Service Groups Z
Litter Oize.2
MEAN DIMENSION—CLENI \METLAS
AGE IN DAYS
CuHart No XW L
Measurement Curves IN Service G ROUPS
Litter Size §&
es 2
5
jo Daas
|| ge: «== (0)
é 20_
:
|
E
: Ua
//
Z
ae
1
Yi
Z,
YY
AGE IN DAYS
(o) os 70 “5 20 25 30 9°35 FO ST 50 &5 JO Seer ES FO
SEXUAL ACTIVITY OF MALE RABBITS
Cuart No XVL
Measurement Curvesin Service Grours
Litter Size 6
37? 5
I 5
a4 4
5/ SS 4
4S =|
#5] 4
MEAN DiMENSION—CENTIMETERS
AG@E IN DAYS
Cuarr No XVIL
Measurement Curves \N Service Groues
Litter Size 7
y
DEN Ui 7
Glee ok | ak 4
Ca] 4
4
MEAN DIMENSION—CENTIMETERS
3.3 4
3-0 aI
a7 4
oe
AGE IN DAYS 7
Aa
601
60
“ek
t
N
T
MEAN DIMENSION— CENTIMETERS
MEAN Di MENSION —CENTI ME TERS
FRANK A. HAYS
a> ae eo Un leeiaeel T a5 T SSS T
CHart No XVID f
: Ve
Measurement Guaves in Service Groups ee
MATTER Size 3 |
ie a se |
o = 5 A
(er See
Gs ee FO) ye
BLO) oi eee O ZA
PE
AGE IN DAYS
TO Vig Om Ome OS Om ON Se ao OM OSp > OM onc OM Se Oo
T i ' Fae | Ti T a] T T T Si
CHarnt No XIX
MeasSuREMENT CURVES IN GOERVICE GROUPS
Litter Size 9 ee ee
sr ZF
| _. -! Lo
AGE IN DAYS
SEXUAL ACTIVITY OF MALE RABBITS 603
T T
sr sz,
al CHART Nowak
ele MeasUREMENT Curves in Service Grours
Litter Size 10
h ane eS i)
Ey M ~
ya eee
MEAN DIMENS|ON—CENTIMETERS
)
Wy wy
uw S ~
AGE in PAYS
ae) aes) = =O Ye 35 So ZO ae 5 43 oO 3s vO
CHart No. XXL
6 Measurement Gurves in Service Grours 4
5 Litter Size // zi
ce =
or
ie en ae |
d OG? ET IAS a
MEAN DIMENSION— CENTIMETERS
-
J
AGE /N DAYS
SDS Sn -O ne UO ae) te ta 77] Fee
i) bs) 40 YSZ SO
604 FRANK A. HAYS
03 Cuart No XX
Bi Grano Averase Measurement Curves oe
=
Saf) Au Service Groves
>
\
SSS ———— ee
YQ
4271p yl
a “4
1 ‘7
Z,
3.62 Gy
F
=
336
te h
3 ofS y
jus ty
27
24 :
AGE IN DAYS
sa) 10; al See 3 50 ao 75
charts where but a single litter makes up a graph a rather sudden
break may sometimes be noted in the graph. This, in our
opinion, is the result of error in measurement, and for this reason
the graphs made up of several litters will be less influenced by
minor errors and hence should be more representative of actual
dimensions.
In chart 22 are presented grand average graphs made up as
the average of twenty-one Ist-service litters, fifteen 5th-service
litters, eight 10th-service litters, and one 15th-service litter.
Here the coincidence of the Ist-, 5th-, and 10th-service graphs
is very striking. This fact bears out our previous conclusions
from body weight studies that heavy sexual service of the male
has no effect upon the growth of his offspring. Our evidence in
studying the increase in the mean dimension does not show any
effect on the progeny, from the heavy service of the male. The
15th-service graph is made up of but one litter of two individuals
sired by Male No. 1 and out of an average sized female. The
fact that this litter is few in numbers and has as a sire the larg-
est of the males will probably account for their larger mean
dimension.
SEXUAL ACTIVITY OF MALE RABBITS 605
TABLE 5
Percentage mortality in offspring during the first five days of life and between the
fifth and the ninetieth day of life
SERVICE
ist + 5th | toth | 15th | 20th
Numberof animals |borniess.... sites. alee 180 119 /|139 84 77
Number dying first five days.................. 16 15 16 11 7
Rericent dying first five days:....4.+...0..04- 8.89) 12.61} 11.51) 13.09) 9.09
Number dying between 5 and 90 daa ,:| 2 36 17 9 19
Per cent dying between 5 and 90 fee rasta aracrene 11.67| 30.25) 12.23) 10.71) 24.68
Summarizing the results of the measurement studies, we note
that there is very close proximity of the graphs for the different
service groups. This points very strikingly to the probable fact
that heavy service of males has no effect upon the growth of their
offspring in the length of head and in the breadth of ilial expanse.
In table 5 the progeny are grouped by services and the num-
ber and the percentage mortality is given for each service group.
Under the row marked ‘‘Number dying first five days” are in-
cluded all animals dead at birth as well as those that died dur-
ing the first five days of life. The other row of the table in-
cludes only animals actually dying between the fifth and the
ninetieth day of postnatal life.
The percentage of mortality during the first five days shows
a slight increase as the number of services increases up to the
15th-service group. Comparing the Ist-service group with the
20th-service group, we note that the percentage mortality in the
first five days is practically the same in both groups. Since the
environment has been more favorable for the 20th-service litters
than for the 1st-service litters, as previously pointed out, there
is no indication that twenty copulations by a male do in any way
tend to reduce the percentage of his progeny that will survive the
first five days of postnatal life. The table shows practically the
same percentage of mortality during the first five days in both the
5th- and the 15th-service groups. The explanation for the
rather high percentage of mortality in the 5th-service group is
that two litters were destroyed outright by the mother and a
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 2
606 FRANK A. HAYS
number of the other 5th-service litters were born during ex-
tremely hot weather when the mortality was very high even
among the older animals. The 10th-service group shows a
higher death rate than the Ist-service group. In all these cases
the percentage of mortality during the first five days does not
seem to depend upon the number of services that the male is
required to make.
Table 5 shows that there is very little consistency between the
mortality percentages as revealed in the first part of the table
and between the percentages of deaths that occurred between
five and ninety days. The first five days is a very critical
time in the life of the young rabbit and very slight exposure may
bring disaster. When this period is over the deaths usually re-
sult from bowel disorders or from septicaemia. Bowel disorders
are most common during the very hot weather of summer in the
stock, and it is very unfortunate that a large number of the ani-
mals in the 5th-service groups should have been so attacked.
The 10th-service progeny also show a higher death rate than the
Ist-service progeny, even though these 10th-service litters were
housed under more favorable conditions than were the majority
of the Ist-service litters. The mortality percentage of the 15th-
service offspring is the highest of any of the service groups dur-
ing the first five days of life, but it falls below that of all other
service groups between the age of five and ninety days. Practi-
cally one-fourth of the 20th-service rabbits died between ‘the
fifth and the ninetieth day of postnatal life. An outbreak of
septicaemia happened to occur among a number of these litters.
This being the case, we are inclined to believe that this sudden
outbreak of disease rather than any inherent weakness of the
progeny resulting from heavy sexual service of the sire is here
operating to cause the high percentage of mortalities.
6G. Relation of number of services made to sex of offspring .
A study of the relation of sex of the offspring to the amount
of sexual service the male is required to perform is important
because such data will show if either male or female producing
SEXUAL ACTIVITY OF MALE RABBITS 607
TABLE 6
Sex ratios in service groups. Males to 100 females
SERVICE
Ist 5th 10th 15th 20th
Number of individuals concerned............. 78 76 117 84 UT
EGA OMS oo EI EEE. Aho Sd Spa S aeaon 129 | Tel 80 | 53 28
He
sperm (Bachhuber, ’16) is weakened by excessive functioning of
the male reproductive organs. Table 6 presented below shows
the sex ratio of the offspring in the different service groups.
Table 6 shows that in the Ist-service group there are 129
males to every 100 females. After the Ist-service group there is
a regular decline in the number of males produced, with the ex-
ception of the 10th-service group. There is apparently some
underlying cause to bring about the high percentage of females
to males in all the advanced service groups, and there is a direct
relation between the amount of service previously performed by
the male and the proportion of female offspring that he will sire.
The properties of the spermatozoa are perceptibly modified by
heavy sexual service of males (Lloyd-Jones and Hays, ’17), there
being a larger percentage of weak sperm in the advanced service
sperm.
Two possibilities exist: either female-producing spermatozoa
are formed more. largely than male-producing spermatozoa as
the amount of service of the male increases or the male-producing
sperm are in themselves weaker than the female-producing
sperm and consequently fewer of them survive to take part in
fertilization. On the first point there is no evidence available.
Concerning the second point, Stockard (13) offers the hypothesis
that in the case of guinea-pigs the larger female-producing sperm
are more affected by alcoholization of the male than the smaller
male-sperm producing. In the case of excessive sexual service,
however, the large female sperm may be more vigorous because
of their size or their greater chromatin content and thus out-
distance the male-producing sperm in the struggle of fertiliza-
tion, thus giving a higher percentage of female progeny in the
heavy service groups as compared with the light service groups.
608 FRANK A. HAYS
TABLE 7
Sex as related to mortality. Percentage mortality of the sexes
SERVICE
PERIOD SEX:
5th 10th 15th 20th
BITS MivienteeyeRene ss. cic ss Sec wisgttdaces aoclaeks of 8.69 7.14 0 9.09
9 6.85 | 11.90] 17.74] -9.09
Between five and ninety days............| o& 6.52 8.93 9.09 | 27.27
Q 12.33 | 15.47 | 11.29 | 24.24
In table 6 we considered the relation of sexual service to the sex
of the offspring and found that .a predominance of females to
males is the rule in the heavy service groups. In table 7 we
shall consider sex of the offspring dying before the close of the
observation period at ninety days. |
Table 7 shows that up to the 15th-service group there is a
higher death rate among the female offspring than among the
male offspring. In the 20th-service group, however, the fact
will be noted that females are just as likely to survive as males
for the first five days of postnatal life. Between the fifth and
the ninetieth day there‘is a slightly lower death rate of females
than males in the 20th-service group. These facts seem to
indicate that in comparison with males of the same class, female
offspring from the 20th-service are in respect of their ability so
survive superior to ordinary offspring from the less advanced
service groups. The fact still seems evident that these female
offspring in the 20th-service group are slightly more likely to
die than ordinary offspring.
SUMMARY OF FACTS
1. Body weight of the rabbit is a measure of growth that is
subject to considerable variations largely brought about by slight
changes in the environment.
2. The rate of increase in body weight continues at a uniformly
rapid rate for the first ninety days of the rabbit’s life.
3. The factors that appear to govern the weight of the young
at birth are age of mother, state of health of mother, weight of
SEXUAL ACTIVITY OF MALE RABBITS 609
mother, weight of sire, character of food of mother, and num-
ber of individuals born in the litter.
4. The factors that govern the rate of postnatal growth of the
young for the first ninety days are weight at birth, number in
litter, milk supply furnished by the mother, and, after weaning,
the character of the food supplied to the young and general
character of the quarters.
5. No inferiority in the offspring from the heavy service groups
is revealed by comparing the body weights with those of the
light service groups.
6. The average litter coefficient of variability in body weight
at birth at thirty days and at ninety days is no greater in the
progeny in the heavy service groups than in the light service
groups. Greater variability might be expected if a part of the
offspring are made genetically inferior by inferiority of the male
element in the advanced service groups.
7. The service group coefficients of variability indicate greater
variability in the weight of the general population than within
the litters, but do not indicate that heavy service produces ‘weak’
litters.
8. Body development seems to progress at the maximum rate
during the first twenty days of postnatal life, after. which time
there is a rather marked decline in the rate of increase in head
length and breadth of ilial expanse.
9. No inferiority in the offspring from the advanced services
is revealed from a study of body growth by measurement.
10. Offspring in the more advanced service groups do not
show a significantly higher percentage of mortality during the
first five days of life than do the offspring in the light service
groups.
11. A higher mortality does not seem to exist in offspring from
the advanced service groups as compared with the light service
groups between the ages of five and ninety days.
12. Heavy sexual service of males gives a decrease in the
proportion of male to female offspring that is very perceptible.
13. Female offspring are to some degree more likely to suc-
cumb than male offspring in all service groups except the
twentieth.
\
610 FRANK A. HAYS
14. The high percentage of deaths of female progeny is largely
due to the predominance of females to males in the litters.
15. By no means thus far used has any inferiority of progeny
from heavy sexual service been discovered. They are fully
equal if not superior to progeny from very light service of male.
DISCUSSION
The amount of sexual service that the male performs has a
marked effect upon the physical properties of his spermatozoa
(Lloyd-Jones and Hays, ’17); the whole basis of this work is to
discover if these effects are in any way made manifest in the
offspring.
Growth in body weight must be assumed to be due to a com-
plex of stimuli acting upon every living cell of the organism. If
it were possible to modify the contribution of growth stimuli
from the male germ cell by extreme sexual use of the male, an
effect should be produced upon every cell of the body in his
offspring and a reduction of these stimuli would thus result in a
decreased body growth. The sum total of the body increase in
the offspring from the heavy service series is fully equal and
even superior to the increase in the offspring in the light service
groups. This apparent superiority has been attributed to vari-
ous factors, largely environmental and possibly to superior male
reproductive cell. After these factors are corrected for, which
we have found impossible to do, we believe that the rate of growth
in body weight would be identical in all five service groups. A
study of body weight as reported here will only reveal the char-
acter of the total population and will not reveal the occurrence
of an occasional inferior individual.
The coefficient of variability of litters, on the other hand, is
valuable in that it will reveal the occasional inferior individual
in the litter. If only a part of the offspring in the heavy serv-
ice groups are inferior as far as rate of growth is concerned,
there should be a greater coefficient of variability in the litters
from heavy service than among the light service litters. No
such evidence appears in our data, and this fact we feel warrants
the assumption that not even a part of the offspring in the
SEXUAL ACTIVITY OF MALE RABBITS 611
heavy service group are more inferior as far as ability to in-
crease in body weight is concerned than the offspring in the light
service groups.
The service group coefticient of variability does not reveal
that any inferiority of entire litters is brought about by heavy
sexual service of males. This coefficient does show that the
largest coefficient of the first ninety days of postnatal life is
found just at the close of the suckling period at thirty days.
The coefficient further shows that the variability in weight of
the general population is much greater than within the litters.
Body measurements furnish us with further material for the
study of the offspring in the different service groups. These data
do not reveal any new facts to indicate any greater inferiority of
offspring in any one of the five service groups. Here again the
same modifying factors have been in operation that have af-
fected the body-weight data, and a correction, if possible, for
these we think would show that the offspring in all five of the
service groups are identical in body dimensions.
Concerning the question of rate of mortality in progeny from
light and heavy service, we have no evidence that there is a
higher death rate in the advanced service groups over that ob-
served in the light service groups.
A direct relation apparently exists between the amount of
sexual service of males and the percentage of females that they
will sire. The ratio of males to females is highest in the Ist-
service group and progressively decreases up to the 20th-service
group. There is a possibility that heavy service exerts a selec-
tive action upon the sperm cells and may eliminate from fertili-
zation the majority of the male-producing spermatozoa. The
large female-producing sperm cells may show a greater rate of
motility, greater endurance, or for some other cause out-distance
the male-producing spermatozoa, thus resulting In a prepon-
derance of female offspring in advanced service groups.
A possible explanation for the high percentage of deaths
among females lies in evidence showing that the percentage of
female offspring is increased by heavy service of the male as
shown on page 607. The weight (Minot, Jackson, King) of
612 FRANK A. HAYS
female offspring in multiparous animals at birth is slightly less
than that of the males. If this is true for the rabbit, it may
render the females less able to compete with the male offspring
for nourishment during their early life when food supply is of
such vital importance in determining the survival of the young.
The fact that the great majority of the offspring dying in early
life have been females seems to warrant the assumption that
females are actually less able to compete with the males during
the early part of life. The data do not justify the conclusion
that there is any higher rate of mortality in the advanced service
groups than in the lighter service groups after the first five days
of postnatal life. If inferiority of offspring exists in the advanced
service groups because of the predominance of females, which we
may assume under all ordinary conditions are less able to survive
than males, it is apparent that no real inferiority exists, but
that the mortality is greater because the percentage of females
is greater in the heavy service groups.
In conclusion, it may be noted 1) that the methods used for
measuring the character of offspring from different degrees of
sexual service of sires fail to show that any inferiority of the
offspring can be induced by using a male excessively; 2) that the
male in heavy sexual service furnishes germ cells that are fully
the equal in their contribution to his offspring of those elaborated
by a male in very moderate sexual service.
ACKNOWLEDGMENTS
The writer wishes to express his high appreciation to Dr.
Orren Lloyd-Jones for his constant codperation and helpful ad-
vice, to Dr. H. 8S. Murphey for assistance in making a study of
the male and female genitalia, and to Prof. G. M. Turpin and
Prof. H. D. Hughes for furnishing quarters for this work for a
time.
; SEXUAL ACTIVITY OF MALE RABBITS 613
BIBLIOGRAPHY
Bacuuuser, L. J. 1916 The behavior of the accessory chromosomes and of
the chromatoid body in the spermatogenesis of the rabbit. Biol.
Bul., April.
Day, C. E. 1913 Productive swine husbandry. Philadelphia.
Harar, S. 1908 Studies on the variation and correlation of skull measurements
in both sexes of mature albino rats. Amer. Jour. Anat., vol. 7.
Jackson, C.M. 1913 Postnatal growth in the albino rat. Amer. Jour. Anat.,
vol. 15, No. 1.
Kine, H. D. 1916 On the postnatal growth of the body and of the central
nervous system of albino rats that are undersized at birth. Anat.
Rec., vol. 11.
Lioyp-JONES, ORREN, AND Hays, F. A. 1918 The effects of frequency of copu-
; lation on the properties of the seminal discharge. Journ, Exp. Zodl.,
vol. 25.
MacDowe Lt, E. C. 1914 Size inheritance in rabbits. Carnegie Inst., Pub.
No. 196.
MacDowe tt, E. C. 1914 Multiple factors in mendelian inheritance. Jour.
Exp. Zodl., vol, 16.
Minot, C. S. 1891 Senescence and rejuvenation. Jour. Phys., vol. 12.
Minot, C. 8S. 1908 Age, growth, and death. New York.
Peart, Raymond. 1917 The experimental modification of germ cells. Jour,
Exp. Zodél., vol. 22, No. 2.
Puscu, G. 1915 Algemeine Tierzucht. Dritte Auflage, Stuttgart.
QUETELET, A. 1871 Anthropometrie, On Measure des differentes Facultes de
Vhomme.
Srockarp, C. R., AnD PapanicoLaAou, G. 1916 A further analysis of the he-
reditary transmission of degeneracy and deformities by the descend-
ants of aleoholized mammals. Amer. Nat., vol. 50.
Vaucuan, H. W. 1916 Pedigree studies. (Unpublished.)
Wrieut, L. The illustrated poultry book.
&,
Sri
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a
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ae
4) =
SUBJECT AND AUTHOR INDEX
¥ CTIVITY of male rabbits: I. On the
Lr properties of the seminal discharge. .
| , The influence of excessive sexual....... 463
Activity of male rabbits. II. On the nature
of their offspring. The influence of exces-
BLYGIBOxIIal tare Amery etinicaeroemiewisoacbeaeys 571
Albino rats underfed for various periods.
Changes in the relative weights of the
various parts, systems and organs of
VOU tee ee cerns eanrate Cre ieeal gus chavs ereeeuaaeete 301
Alectrion obsoleta (Say) and Busycon cana-
liculatum (Linn.). The olfactory reac-
tions of the marine snails................. 177
Amblystoma, a self-differentiating equipo-
tentialsystem. Experiments on the devel-
opment of the fore limb of... ............. 413
Amblystoma punctatum. Experiments on
the development of the shoulder girdle
and the anterior limb of............... --. 499
Ascidia atra Lesueur. I. General physiol-
ogy. The physiology of................. _. 229
Asecidia atra Lesueur. II. Sensory physi-
ology. The physiology of................ 261
IRDS. Sex studies. XI. Hermaphro-
GUT Sense n oie ost eek aimee cmeeiees 1
Blattidae. Experiments on the physiology
odigestionimithers- ene aces. eee 355
Borine, Avice M., anp PEARL, RAYMOND.
Sex studies. XI. Hermaphrodite birds... 1
Busycon ecanaliculatum (Linn.). The olfac-
tory reactions of the marine snails Alec-
trion obsoleta (Say) and.................. 177
ANALICULATUM (Linn.). The olfac-
tory reactions of the marine snails
Alectrion obsoleta (Say) and Busycon.. 177
Cats. Inheritance of coat-color in............ 539
Coat-color in cats. Inheritance of....... Basan OOo)
Color in cats. Inheritance of coat............ 539
CoprLanp, Manton. The olfactory reac-
tions of the marine snails Alectrion obso-
leta (Say) and Busycon canaliculatum
(Gir) ee ee eo, eee Be nlZ(¢/
Dee S. R. Experiments on the
development of the shoulder girdle and
the anterior limb of Amblystoma punc-
UPDLITUD co haere ean “cies Ciera cin caacieaenreciey ote ter 499
Development, metamorphosis and growth due
to a specific action of that gland? Is the
influence of thymus feeding upon.. 135
Development of the fore limb of Ambly-
stoma, a self-differentiating equipotential
system. Experiments on the............. 413
Development of the shoulder girdle and the
anterior limb of Amblystoma punctatum.
Hxperiments Onibhes vases ease cee 499
Diemyctylus viridescens. Some experiments
on regeneration after exarticulation in... 107
Digestion in the Blattidae. Experiments on
thepiysioloey Oley eee eee ee eee 355
Drosophila and its mutants. The reactions
tolightand! toeravity ine ecce. o.salcoenee
QUIPOTENTIAL system. Experiments
on the development of the fore limb of
Amblystoma, a self-differentiating..... 413
Exarticulation in Diemyctylus viridescens.
Some experiments on regeneration after... 107
EEDING upon development, metamor-
phosis and growth due to a specific
action of that gland? Is the influence
OfsthyMUss see Oe. ove «ahi otednte 135
Fore limb of Amblystoma, a self-differentiat-
ing equipotential system. Experiments
on the development of the................ 413
IRDLE and the anterior limb of Ambly-
stoma punctatum. Experiments on the
development of the shoulder............ 499
Gravity in Drosophila and its mutants.
reactions to lightand tO;e.c.--..-.2...-+-
FABER: Ross G. Experiments on
the development of the fore limb of
Amblystoma, a self-differentiating equi-
potential system.
Hays, Frank A. The influence of excessive
sexual activity of male rabbits. II. On
the nature of their offspring............... 571
Hecut, Seria. The physiology of Ascidia
atra Lesueur. I. General physiology..... 229
Hecut, Serie. The physiology of Ascidia
atra Lesueur. II. Sensory physiology.... 261
Hermaphrodite birds. Sex studies. Xen 1
| ee RITANCE of coat-color in cats........ 539
IGHT and to gravity in Drosophila and
its mutants. The reactions to.......... 49
Limb of Amblystoma, a self-differentiating
equipotential system. peuerumente on
the development of the fore.. A . 413
Limb of Amblystoma punctatum. Experi-
ments on the development of the shoulder
girdle'and thejanterionss-ree. .~-..-.0250.- 499
Lioyp-JONES, ORREN AND Hays, F. A.
The influence of excessive sexual activity
of male rabbits. I. On the properties
of the seminal discharge................-- 463
cEWEN, Rosert STranuey. The reac- +
M tions to light and to gravity in Droso-
philaanditsmmutants ees oo. sessceune 49
Maculata. A study in polarity. The re-
generation of triangular pieces of Planaria. 157
Male rabbits. II. On the nature of their off-
spring. The influence of excessive sexual
EHR AO os cidtiono \'0Gnts SOR RES MEOnc 571
Metamorphosis and growth due to a specific
action of that gland? Is the influence of
thymus feeding upon development....... 135
Morritut, C. V. Some experiments on re-
generation after exarticulation in Di-
emyctylus viridescens................... 107
Mutants. The reactions to light and to
gravity in Drosophila and its............ 49
615
616
Oe (Say) and Busycon canalicu-
latum (Linn.). The olfactory reactions
of the marine snails Alectrion.......... 177
Offspring. The influence of excessive sexual
activity of male rabbits. II. On the na-
turevoLr thew ene see eee 571
Olfactory reactions of the marine snails Alec-
trion obsoleta (Say) and Busyecon canali-
Culatom (inns) hehe | )s =. oo ater oe 177
Outmstep, J. M. D. The regeneration of tri-
angular pieces of Planaria maculata. A
SEU yw OO LALIEV ec soe ous oe tbe rete 157
Organs of young albino rats underfed for
various periods. Changes in the relative
weights of the various parts, systems
TA Pee es wicle viene’ Sivare oe esbarede oie eboees 301
HYSIOLOGY of Ascidia atra Lesueur.
I. General physiology. The........... 229
Physiology of Ascidia atra Lesueur. II. Sen-
sory splysiolozy.: “het: -4hetis. soto 261
Physiology of digestion in the Blattidae.
Hperiments on thes. .2.40-ce-.0+-55--2*-
Planaria maculata. A study in poplarity.
The regeneration of triangular pieces of.. 157
Polarity. The regeneration of triangular
pieces of Planaria maculata. A study in. 157
Punctatum. Experiments on the develop-
ment of the shoulder girdle and the an-
terior limb of Amblystoma.............. 499
ABBITS. I. On the properties of the
seminal discharge. The influence of ex-
cessive sexual activity of male......... 463
Rabbits. II. On the nature of their off-
spring. The influence of excessive sexual
aetivaty on male... 1. 6. eek oe ete ee 571
Rats underfed for various periods. Changes
in the relative weights of the various
parts, systems and organs of young
alDINOMEE ES... Aa A tone geet 301
Reactions of the marine snails Alectrion obso-
jeta (Say) and Busyeon canaliculatum
(Emmns)n Che olfactory... sence sce 177
Reactions to light and to gravity in Drosophila
andiatsamutants. Theioe.2.. .t.. shee- + 49
Regeneration after exarticulation in Di-
emyctylus viridescens. Some _ experi-
TCEMLSIEO LIE... =:<).,> 0 Oa re ee ee 107
Regeneration of triangular pieces of Pla-
naria maculata. A study in polarity.
FICS |... = Jibei SUR, «fale pee nemeerertete '< 157
ANFORD, Etpon W. Experiments on
the physiology of digestion in the
LBIEKHAG io: 35 eae emi Mtoe MAResid 335
INDEX
Self-differentiating equipotential syster
Experiments on the development of ths
fore limb of Amblystoma,a............ 413
Seminal discharge. The influence of excessiy
sexual activity of male rabbits. I. O
the propertiesiot theses pase ee eee 463
Sensory physiology. The physiology o \
Ascidia) atrasbesueur ss Liew ace see eee 261
Sex studies. XI. Hermaphrodite birds...... 1
Sexual activity of male rabbits. I. On the |
properties of the seminal discharge.
The influence of excessive................
Sexual activity of male rabbits. II. On the
nature of their offspring. The influ-
Once (Of excessive aa: cece eee: ie eos 71
Shoulder girdle and the anterior limb of Am-
blystoma punctatum. Experiments on
the development of the....,............. .
Snails alectrion obsoleta (Say) and Busycon
ecanaliculatum (Linn.). The _ olfactory
reactions, of ‘the’ marine’.c7 2 = neces 177
STEWART, CHESTER A. Changes in the
relative weight of the various parts, sys-
tems and organs of young albino rats un-
derfed for various periods............... 301
System. Experiments on the development
of the fore limb of Amblystoma, a self-
differentiating equipotential............. 413
Systems and organs of young albino rats un-
derfed for various periods. Changes in the
relative weights of the various parts.... 301
HYMUS feeding upon development, me-
tamorphosis and growth due to a specific
action of that gland? Is the influence
(6) ENE AAR nee ern he Shasta Goh Sousa 135
HLENHUTH, Epwarp. Is the influence
of thymus feeding upon development,
metamorphosis and growth due to a spe-
Underfed for various periods. Changes in
the relative weights of the various parts,
systems and organs of young albino rats.. 301
TIRIDESCENS. Some experiments on
regeneration after exarticulation in
Diemyctylus!: S67. sen a. ete ee oe 107
EIGHTS of the various parts, systems
and organs of young albino rats un-
derfed for various periods. Changes
AGhEe LEAVE so. Se sretave ce oe hoe eee ees
Uy Cate saa si os sulodss scetees te ee 539
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